Studies on the Chemical Constituents of Carum roxburghianum Benth. (Radhuni) Seeds for its Essential by zareen a.abedin

Studies on the Chemical Constituents of Carum roxburghianum Benth. (Radhuni) Seeds for its Essential Oil, Fatty Oil, Minerals and other Valuable Nutrients. 1.1 General discussion: From the prehistoric period the practical aspects of food, shelter, clothing and heat were of primary importance for these men depends on green plants. So man migrated to those areas where such facilities could be obtained readily. Plants are generally differentiated by human by their ability to manufacture food, the presence of cell walls, and their unlimited type of growth. The basic food for all organisms is produced by green plants. In this process of food manufacture, oxygen gas is liberated. This oxygen, which we obtain from air as we breathe in, is essential to life. The only source of food and oxygen is plant; no animal or man alone can supply these. Natural products are traditionally the cornerstones of drug discovery and research in this field continues to provide tremendous variety of lead structures, which are used as templates for the development of new drugs. Despite tremendous advances witnessed in modern medicine through synthesis, similarly aromatic plants are continually getting importance in healing of diseases and comfort of man. Medicinal and aromatic plants continue to hold an important part of pharmacopoeias in both the developed as well as developing countries. Medicine is a very ancient art and drugs have been used in days of antiquity as far back as history can take us. In particular higher plants have been the source of medicinal agents since earliest times and today they continue to play a dominant role in the primary health care of about 80% of more than 6 billion in habitants of the world rely chiefly on traditional medicines for their primary health care needs and it can be safely be presumed that a major part of the traditional therapy involves the use of plants, extracts or their active principles. It is impossible to think of medicine as something not connected with treatment and drugs have formed an integral part of treatment from the commencement of human memory. Increasing human population and increasing pressures on plants are leading to shrinkage in the worldâ&#x20AC;&#x2122;s natural floral resources and having deleterious impacts on species and ecosystems. Recent estimates from the International Union for Conservation of Nature and Natural Resources (IUCN) has given grim statistics that over 12.5% of the worlds. Vascular plants are threatened at the global scale (Walter and Gillett 1998). Natural products and medicinal agents derived there from are also an essential feature in the health care systems of the remaining 20% of the population residing mainly in developed

countries, with more than 50% of all drugs. Clinical use having natural product origin 1. This is because of various reasons: 1) There is historical legacy of folklore, ethno medicinal uses and well- documented indigenous system of medicine using natural resources for the treatment of diseases; 2) They are believed to be comparatively safer than drugs modern medicine; 3) They are economical; 4) They are environ mentally better suitable to local conditions; 5) They possess curatives/preventives for certain disorders for which modern medicine has nothing to provide 2. Bangladesh is endowed with rich plant resources on which we depend directly or indirectly not only for food and essential requirements but also for our very existence. This plant resource is impoverished by continuous loss and degradation of the plant diversity. To halt the decline in this priceless heritage, urgent action is needed at local, national and international levels. No concrete steps have been taken to arrest the process. The first and foremost step in this direction is to identify the threatened species and to assess their status in the wild.

1.2 Medicinal importance of the plant materials: From ancient times plants have been used as a source of medicines that form the backbone of human health care. The use of medicinal plants as a source for relief from illnesses can be traced back over five millennia to written documents of the early civilization in China, India and the nearest but it is doubtless an art as old as mankind 3. In the modern age, the scientists are endeavoring to isolate different chemical compounds from plants having biological properties. On the other hand while hunting well known lead compounds, many useful chemicals can be isolated from plant which are important for human comfort and health care 4, 7.

1.3 Chemical constituents of medicinal plants6: Plants produce within their cells and tissues, through metabolic activities, not only food materials but also the life sustaining oxygen gas and other substances, such as glycosides, steroids, terpenoids, alkaloids, flavones, tannins, pigments etc. There are usually called secondary metabolites that are responsible for their various, therapeutic properties and pharmacological actions. The compounds stored in their bodies may exert both harmful and useful effects on human beings. Traditional healers have been using toxic as well as nontoxic materials for treatment of various diseases. But unfortunately the traditional practitioners did not document the active principles of these natural products, so researchers have been trying to find out the active ingredient(s) of toxic and nontoxic plants 6. The active principles of both the medicinal and poisonous plants are often definite chemical substances but in other cases. They are complicated mixtures.

Briefly the following group of substances occurs in plants and is responsible for their medicinal as well as their toxic properties: 1) The first classes of these substances with medicinal and toxic properties are vegetable basis which include amines and alkaloids. As a class these bodies are characterized by their profound physiological action and in many case their intensely poisonous nature. Some of the amines give a footed odor to some weeds and to some mushrooms their poisonous characters. The alkaloids as a rule give a bitter taste to a plant in which. They naturally occur and that in itself is frequently a sufficient protection against livestock eating it, except in unusual cases of hunger. Examples of alkaloids are strychnine from nix, vomica, aconitum from aconites, atropine and allied alkaloids from belladonna, nicotine from tobacco, morphine from poppy, etc. 2) Another Class of these substances are represented by glycosides which form a large group and are much wider in occurrence than alkaloids many are non-toxic but quite a large number of them are intensely poisonous. They have generally a bitter taste and occur in many of the plant extracts used in medicine. Well known examples of toxic glycosides are those occurring in the oleander family (Apocynaceae) and Digitalis (Serophulariaceae). 3) The third group of active substances is furnished by essential or volatile oils which give characteristic odors to plants. These bodies are characterized by their insecticidal and insect-repellent properties, while in man and livestock they produce toxic effects by gastrointestinal irritation. Common examples are those occurring in eucalyptus, in absinth which produces convulsions by its action on the nervous system. 4) The fourth group of toxic substances is known as to albumins which occur in castor, croton and abrus seeds; these are essentially blood-poisons and are responsible for frequent losses among livestock. 5) The fifth group of substances are called resins such as those occurring in podophyllum, bitters such as are found in wild members of the cucumber family for example colocynth, phenolic compounds such as those found in many members of the cashew family. 6) The sixth group of active substances occurring in plants is the antibiotics. It is wellknown that some of the most powerful antibiotics now in use such as penicillin, streptomycin, etc are derived from the lower from of vegetable life, i.e. fungi 5.

1.4 Herbaceous medicinal plant is extensively used as Spices and Condiments: Most of the medicinal plants are still a minor forest products and their population is decreasing day by day. Urbanization and deforestation, has led to alteration in the ecosystem with considerably decreased forest area. The only alternative appears to be large scale commercial cultivation of medicinal plants, particularly of those which are in high demand. It is rather significant that the intensive development on the earth of annual herbs that is of

small, no woody plants that flower and produce seed in one reason had its beginnings in this early period and that during this same epoch man was emerging from the primitive stage. In fact this epoch has been called the age of man and herbs. Herbaceous annual plants were an important factor in human development, not only in the early period but also throughout man’s entire history. In ancient times, spices ranked with precious stones in the inventory of royal possessions and were monopolized by the few. The determined the wealth and policies of nations and also played an important role in ancient medicine. Besides, they also provided an incentive for the discovery of new water ways and new continents. The term ‘spices and condiments’ applies to “Such natural plant or vegetable products or mixtures thereof, in whole or ground form as are used for imparting flavor aroma and piquancy to and for seasoning of foods”. There are over 80 spices grown in different parts of the world and 50 spices are grown in India and Bangladesh. It constitutes an important group of agricultural commodities which are virtually indispensable in the culinary art. They also play a significant role in our national economy and so also in the national economics of several spice producing, exporting and importing countries. The people of those times used spices, as we do today, to enhance or vary the flavors of their foods, spices were also flavor disguisers, masking the taste of the fainted food that was still nutritious but would if unspiced have to be thrown away. Some spices were also used for preserving food. Such was the economic importance of spices in those days 8−10.

1.5 General Description of Carum roxburghianum. Benth. (Radhuni): The family umbelliferae or Apiaceae consists of about 270 genera and 3,000 species which are cosmopolitan but chiefly temperate, especially in the northern hemisphere.

Characteristic of the family11: This family of nearly exclusively herbaceous species is characterized by hermaphrodite flowers in a double umbel (Figure-1.1); note that the closely related Araliaceous have a simple umbel. Typical for the family are the furrowed stems and hollow internodes, leaves Figure-1.1: Double Umble with a sheathing base and generally a much divided lamina. The flowers are relatively inconspicuous, with two pistils, an inferior gynaecium with two carpels, a small calyx and generally a white to greenish corolla, with free petals and sepals.

Chemical characteristics of the family11: Unlike the Araliaceae, members of this family are often rich in essential oil, which is one of the main reasons for the pharmaceutical importance for many of the apiaceous drugs. Also common are 17-carbon skeleton polyacetylenes, which are sometimes poisonous, and coumarins, which are responsible for phototoxic effects (e.g. in Heracleum mantegazzianum, Sommier and Levier, hogweed). Some species accumulate alkaloids (e.g. the toxic coniine from hemlock, Conium maculatum).

 Genus Carum having three species: 1) Carum roxburghianum Benth. (syn. Trachyspermum roxburghianum (DC.) 2) Carum copticum Heirn (syn. Trachyspermum ammi Linn.) 3) Carum carvi Carum roxburghianum Benth. (Syn. Trachyspermum roxburghianum (DC.) Craib.; Athamantha roxburghianum (Benth.) Wall. T. involucratum Marie) belongs to the family Apiaceae native to tropical Asia and is cultivated in Bangladesh, India and Indo-China.

1.5.1

Scientific Classification of Carum roxburghianum. Benth. Kingdom

Division

Plantae Magnoliophyta

Class

Magnoliopsida

Order

Apiales

Family

Apiaceae (Umbelliferae)

Genus

Trachyspermum (Carum)

Species

T. roxburghianum (C. roxburghianum)

Binomial name

: Trachyspermum roxburghianum (DC.) Craib

Synonyms: T. involucratum Wolff non Marie, Carum roxburghianum Benth Seeds of Trachyspermum roxburghianum (also known as Carum roxburghianum)

1.5.2 Various names12, 13: Local name name Other name

: Radhuni, Randhuni, Randhuni Soj, Choto Soj, Choto Dhonia English : Celery, Bishops weed168 : Marathi: Ova, Gujrati: Ajamoda, Hindi: Ajamoda, Tamila:

Ashamtavoram, Punjabi: Kernauli, Tamil: Ajmoda, Bengali: Bandhuri, Chanur, Malayalam: Ayamodakam, Nepali Name: Ajmoda, Spanish : Aipo, French : Celeri, German : Sellerie, Swedish : Selleri, Arabic : Karafs, Dutch : Selderij, Italian : Sedano, Portuguese : Apio, Russian : Syel’derey, Japanese : Serorii, Chinese : Chin, Thai : Phak chi lom.

1.5.3 Description of the herb Carum roxburghianum. Benth12: It is an annual or biennial herb. It is often cultivated as cold weather crop. Cultivated, adventives on forest margins and in rural areas. And also cultivated as a spice throughout the Indian subcontinent, SE Asia, and Indonesia.

Stem: Plants annual, 20.100 cm. Leaves petiolate, petioles slen-der, 1.2 cm; blade ovate in outline, 3.8 × 2.12 cm, 2-pinnate or ternate-pinnate; ultimate segments narrowly oblong, 5.20 × 2.3 mm, base cuneate. Stem 1-3 feet (90 cm) high, erect, slightly branched, cylindrical, finely striated, smooth, pale green.

Leaves: Leaves reduced upwards, ultimate seg-ments becoming linear-lanceolate. Umbels 2.4 cm across; peduncles 5.9 cm; bracts and bracteoles few, linear-subulate or ciliate, 3.5 mm; rays 4.12, 1.3 cm, filiform, unequal, hirsutu-lous or glabrescent; umbellules 12.20-flowered; pedicels 1.5 mm, unequal, hirsutulous.

Flower: Flowers rather small, in terminal or axillary, compounds umbles; white or greenish white; numerous. On longish slender Pedicels. Calyx, teeth wanting. Petals, roundish, entire, with an involute obtuse apex, shining. Filaments, short, in curved stylopod−flat, over lapping the base of the petals; somewhat to bed. Styles are very short.

Fruit: Fruit ovoid, Fruit about 1/5 inch (1.5-3.0 mm) long, broadly oval (ovid) in outline, apex contracted forming a very short neck, densely hirsutulous or glabrescent. Flower and fruits at Feb-July, rounded at both ends, smooth, tipped with the small stylopod; mericarps readily separable, much dorsal ally compressed, yellow to yellowish green when ripe; vittae solitary in each groove, broad, and two in the commissural.

Root: Tap roof branched

Used part: Seeds are mainly used as spice but it has a great use in medicine and its Herb can also use as same.

 The different parts of Carum roxburghianum (Radhuni) are shown in Figure–1.2, 1.3 & 1.4

Figure–1.2: View of the leaf and blooming flowering plant of Carum roxburghianum Benth. (Radhuni)

Figure–1.3: View of the leaf of Carum roxburghianum Benth. (Radhuni)

Figureâ&#x20AC;&#x201C;1.4: View of the seeds of Carum roxburghianum Benth. (Radhuni)

1.5.4

Habitat and range 5, 11, 14â&#x2C6;&#x2019;16:

Carum roxburghianum Benth. (Radhuni) is one of the 3 species of the genus Carum belonging to the family umbelliferae or Apiaceae. It is an annual herb, as a wild plant this is not unfrequented among corn and other crops throughout southern Europe, extending from Spain to the Caucasus and Persia and southward into Egypt and Ethiopia. It also occurs more rarely as a cornfield weed or casual straggles in Northern Europe, It is indigenous to the countries bordering the Mediterranean Sea, but also cultivated in the south of France, Xony and Russia. It has long been cultivated as a garden plant, and was grown by the Greeks and Romans. It was introduced to England in 1570 12, 13, 17, 18. It is also found to grow in the sub tropical region of India and Bangladesh. The plant is cultivated faintly extensively in the northern part of Bangladesh. In Bangladesh radhuni is grown in Dhaka, Natore, Rajshahi, Naogaon, Bogra, Dinajpur, Rangpur, Gaibandha, Jaipurhat, Sirajgonj, and middle part in Dhaka (Keranigonj) and Faridpur. Gajipur. And it is also grown in experimental field of Spice research center (BARI) 20.

1.5.5 Plant History: C. roxburghianum regarded as a Radhuni or Randhuni soj in Bangladesh, Indian counterparts of is a cold weather crop, largely cultivated for culinary and medicinal purposes and also for fodder C. roxburghianum is grown mostly for its herb oil and for its seed oil. This species very closely resembles and is probably a cultivated from of T. stictocarpum (C.B. Clarke) wolff syn. C. stictocarpum C.B. Clarke, Which is found wild from the lower Himalayas to

South India. There are, minute difference in the fruit due to cultivation 12, 21. The fruit ajmod like those of ajowan are subjected to the attack of spice-beetle or drugstore beetle in store house22. C. roxburghianum, often used in Indian cuisine, are a kind of very strong spice with characteristic smell similar to parsley. A couple of pinches can easily overpower a curry 23. In Bengali cuisine the seeds are used in whole, quickly fried in very hot oil until they crackle. They are part of a local panch phoran (Bengali five spice) mixture, where they replace the more commonly used mustard seed; the other ingredients are white cumin seed, fenugreek seed, fennel seed and kalongi24. The main component of seed oil was limonene. Other notable constituents were sabinene, terpinen-4-ol, (Z) - ligustilide and γ-terpinene 26. Fruit contains Bergapten, essential oil comprises of α and β- pinenes, sabinene, terpenine, α and βphellandrene, linalool, α- terpineol, thymol, carvacrol, β- cyclovandulic acid and serelin11.

1.5.6 Planting and cultivation: Radhuni is a cold-season (Rabi) crop and is fairly tolerant to frost and very low temperature. Under irrigation, the crop can be grown as pot-herb throughout the year. It is best suited to tracts of moderate or low rain fall20. Radhuni grows well in any good garden soil, a fertile, prepared, sandy loam being most suitable, light sand or heavy clay should be avoided. It seeds are small size; the soil must be smooth and free of clods. Care should be taken not to fertilize directly beneath radhuni, because this would favor uneven ripening and development of weeds. Although a native of Mediterranean countries, Radhuni is quite a hardy plant and may thrive in much cooler climates, provided it finds a warm situation and well-drained soil12, 20. For good early growth of the crop and better yield the field should be well prepared. The land should be plowed in the fall, or as early in the winter as the weather permits. The usual procedure is to sow the seed, November-December (very early in winter), about 1/2 inch deep, with specially constructed drills. The rows are spaced at 14 to 18 inches the drills 1ft apart. Depending on the method of cultivation for the control of weeds, the rows are sometimes spaced as far as 3ft apart. The planted seed needs but a thin covering of soil. It has been claimed that the seed crop is better when the plants are not crowded too much. For the same reason, the plants should be thinned at the proper time, so that their distance in the rows is not less than 6 to 15 inchs20. One–half ounce of seed suffices for 150ft of drill; at this rate, 1 lb. should sow one acre. Goods result has been obtained by sowing late in the fall; the seed then germinates in the winter as soon as conditions become favorable. This is often much earlier than the ground can be prepared and the seed planted, in the case of winter sowing.

Radhuni herb seems to be more susceptible to weather hazards than most crops. If heavy hail hits the plants during the flowering period, they may be injured so much that the yield of oil is practically nil. The same might happen through damage by strong winds or driving rains. Extreme heat during the critical period of maturity is apt to blight the herb, with similar results in regard to yield of oil. Nitrogen and phosphorus as urea and superphosphate was applied uniformly in the plots for yield eve crop 80kg N/ha and 30kg P/ha were found best for fruit yield and 40kg N/ha and 30kg N/ha and 30kg P/ha for essential oil. The combined application of same doses of N and P gave much more pronounce results. The combination of N 80 P30 and N40 P45 gave highest fruit yield and essential oil content; respectively 80kgN, 30kg P and The combination of N80P30 gave 53, 20.2 and 96.8% more fruit yield over the respective controls8.

1.5.7 Harvesting: Proper timing of the harvest is very important, the quality of oil depending mainly upon the state of maturity of herb and seed the harvest takes place in early fall. For the production of Radhuni herb oil, the plant should be harvested immediately after the blooming period, when the seed has just started to ripen but is not yet fully developed. This period is very short, lasts not longer than two weeks, and usually falls sometime between the 1st week of March and the end of April 20. To obtain good, typical herb oil, distillation has to be speeded up and a great amount of plant material must be distilled within a short period of time. It is advisable to harvest only as much as can be processed in the distillery during one day. After very short drying in the fields, the herb is hauled to the distillery. Drying for many hours in the field would result in considerable loss of oil by evaporation, especially of the more volatile terpenes consequently, the oil distilled from dried herb would be relatively high in carvone. The herb should be distilled as fresh as possible; otherwise the seed attached to the stacked up plant material continues to ripen, and the oil thus obtained approaches the undesired seed oil character. From the middle of April to mid May the seeds mature fully and must be harvested very carefully when the seeds have lost their green colour. For this purpose the plants are cut with scythes or machines, then bundled and stacked up in the field until all seeds become fully ripe and dry. Subsequently the threshed seed is dried in barns in order to prevent the formation of mold. Finally the seed is winnowed. The main November−December sown crop is ready for harvesting during March–April and January–sown crop is ready in April−May.

The herb is not affected by many diseases and pests. However, collar and root- pot, caused by Sclerotium rolfsii Sacc has been recorded. It has been also reported that Radhuni treated with a number of common fungicides18, 20, and 25.

1.5.8 Yield: Yields are variable, depending upon whether the crop is grown as a dry crop or as an irrigated crop, mixed or pure and on the number of cuttings before seed-setting. The yield of seeds varies considerably. The yield of seed per acre in North America ranges from 500-700 Ib and herb oil varies from 20-30 Ib per acre; it may be higher or lower in exceptional years. In India Radhuni is grown in about 10,000 hectors producing nearly 5000 MT. of seeds 19. In Hungary produces 1000 to 2000 kg of green Radhuni herb per 1.422acres and yield of herb oil ranges from 0.29 to 1.50 percent, yield of Radhuni seed 1.422 acres produces about 300 to 700 kg and yields oil from 2.30 to 2.50 percent 12. In England one acre yields about 700 Ib of Radhuni seed. In Bangladesh Radhuni is grown in about 10000 hac producing nearly 5000Mt of seeds and yield of essential oil from seeds ranges from 1.20 to 2.5 percent20.

1.5.9 Storage: Fresh Radhuni for vegetable does not store well and should, therefore be disposed of soon after harvesting. The well-dried leaves however can be stored for one year the seed can be stored for two years10, 12.

1.5.10 Use of Carum roxburghianum herb and its seeds: In Medicine: C. roxburghianum plant oils and extracts have been used for a wide variety of purposes for many thousands of years27. The seeds are well known for their medicinal properties, mostly due to the essential oil in them both seeds and oil enter into the composition of various indigenous medicinal preparations. The essential oil, Radhuni oil, or its emulsion in water, are the main constituents of â&#x20AC;&#x153; grip water â&#x20AC;? and considered to be aromatic, carminative specially useful in flatulence, collie pain, Anti-diarrheal, Anti-tumor, Anti-oxidant, CNS, Diuretic, Cardiovascular, Hypotensive, vomiting, Spasmolytic and hiccups of infants and children the seeds are useful in hiccup, vomiting and pain in the bladder 13. They form ingredients of carminative and stimulant preparations and are very useful in dyspepsia and flatulence28. Seeds ketonic compound showed antispasmodic activity particularly on smooth muscle of rabbit gut 29. Essential oil and crystalline substance lowered blood pressure in dogs and rats due to direct action on blood vessels30. Fruits left after extraction of essential oil showed marked cardio tonic activity30 and it also shows antibacterial activity31. In particular,

the antimicrobial activity of plant oils and extracts has formed the basis of many applications, e.g. in raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies32, 33. Pharmacological studies have been carried out on different fractions of drug. The crystalline ketonic substance (C13H12O3;

m.p., 117-18˚C) exhibited powerful

antispasmodic activity34: the action was particularly marked on the smooth muscle of the rabbit’s gut. The essential oil and crystalline substance were found to lower blood pressure in dogs and rats35, 36; the effects were due to the direct action on the blood–vessel. The oil produced marked diuretic effect in rabbits. The fruits left after the extraction of the essential oil showed pronounced cardio tonic activity12.

As a food flavoring37: Radhuni seeds are used, both whole and ground, as a condiment in soups, salads, processed meats, sausages, spicy, table sauces, salads, sauerkraut and particularly in Radhuni pickling. Radhuni stems and blossom heads are used for Radhuni pickles and for flavoring soups, Ground seed is an ingredient of seasonings. Sometimes, it is used as a substitute for caraway12. Radhuni oils are used as soap perfume and n food industry for flavoring and seasoning8, 10. The oil has a strong odor, and an unctuous farinaceous taste with slight bitterness38, 39. Herb is also used as a fodder for cattle40, 41.

Other uses: The essential oil of the seed and its fractions possess toxicity and insect repellent properties against pesticides of stored grain insect, especially during the rainy season. The insect repellent property investigated against Tribolium castaneum herbs38,

. The oil and its

fractions, applied topically or impregnated on filter papers, were shown highly toxic to larvae and adult beetles of Tribolium castaneum. At the highest dosage of 100µl/ml killed all the adult beetles and larvae within 48h the overall repellency in the range 45-95%. The seeds extracts of Radhuni products can improve glucose metabolism and the overall condition of persons with diabetes not only by direct hypoglycemic effects but also by improving lipid metabolism, antioxidant status and capillary function 40, 41. Use of essential oils is not under regulatory control in many countries, although very little is known about their acute toxicity. Only a few papers contain data on their “latent” toxicities such as teratogenesis, carcinogenesis and mutagenesis8, 10.

1.6 Objective of the Research Program: Bangladesh is flourished with plants, herbs and trees. Various herbaceous medicinal and aromatic plant have been used for medicinal, spices and flavoring purpose in Bangladesh.

Carum roxburghianum (Radhuni) is a popular pot herb. It has been cultivated and consumed mostly in the northern part of Bangladesh. The composition of plant based product depends on the agro climatic condition (climate and soil) of the country. The chemical composition of the Radhuni herbs varies with the agro climatic conditions of the part of the country where it is grown. Many Investigations have been carried out but no systematic research has been carried out on Carum roxburghianum (Radhuni) in Bangladesh. Some disagreement about the presence of its constituents was observed. Therefore the present work was undertaken to carry out a complete investigation of the Carum roxburghianum (Radhuni) seeds.

The followings are the aims and objective of the study: 1) To carry out the complete study and to get information for consumption and utilization as well as industrial application of plant and its product. 2) To observe its popularity consumption and availability as a pot herb and condiment (its seed) as well as to explore export market to the foreign countries. 3) Characterization quantification and identification of the constituent compounds of the essential oil isolated by steam distillation from its seed by GC-MS (gas chromatography and mass spectroscopy). 4) Characterization of the fatty oil isolated from the seeds by TLC and GLC. 5) To investigate the mineral contents including toxic heavy metals and nutrients which remained after extraction of essential oil as well as fatty oil of seeds by XRF Spectrometer and Atomic absorption spectrometer and flame photometer (AAS). 6) A comparative study on the composition of Carum roxburghianum (Radhuni) seeds of different region of Bangladesh. 7) To carry out the complete study on the nutrients of Carum roxburghianum (Radhuni) seeds.

1.7 Scheme of the work: Carum roxburghianum (Radhuni) seeds were collected from different parts of Bangladesh. Following experiment were undertaken for complete investigation on Radhuni seeds. 1.

Essential oil part: a.

Extraction of Essential oil by means of steam distillation.

Determination of Physical and Chemical properties of the essential oils.

Analysis of the oils by GCâ&#x2C6;&#x2019;MS for identification and quantification of its constituent compounds as well as their structures.

Fatty oil part: a.

Extraction of fatty oil.

Determination of Physical and Chemical properties of the fatty oils.

Methylation of the extracted fatty oil for having methyl ester of fatty acids for TLC and GLC experiment.

Determination of approximate number of fractions of methylated fatty oils by TLC.

Determination of fatty acids methylated constituents of the fatty oils for identified and quantified by GLC.

Residual part: a.

Determination of moisture, ash, crude fiber, nitrogen, i.e., protein carbohydrates content and food energy of the seed and flower.

Determination of mineral content of seeds of the herb by XRF spectrometry and AAS and flame photometry.

Identification of toxic and useful trace elements contained in the seeds.

Collected seeds

Dirt free seeds

Powder

Elemental analysis by AAS

Proximate analysis

Steam distillation

Essential oil

Sample residue after steam distillation

Essential oil were taken for Determination of PhysicoChemical properties

Fatty oil with some impurities

Pure fatty oil

Pure fatty oil were taken for Determination of PhysicoChemical properties

Sample residue after sox let extraction

Ash to determine Minerals by XRF

Schemeâ&#x20AC;&#x201C;1.1: Scheme of the research work

1.5 Collection of sample: Carum roxburghianum regarded as a Radhuni or Randhuni soj in Bangladesh, Indian counterparts of is a cold weather crop, largely cultivated for culinary and medicinal purposes in Bangladesh. According to the aim of our research work we collected the sample from three different district of Bangladesh. These are the following region of sample collection: Sample-1: Mature seeds are collected from Regional Spice Research Center Joydebpur, Gajipur, in the month of March, 2009 which mentioned as sample ID: C. ROX-1. Sample-2: Mature seeds are collected from field of cultivation of Keranigonj, Dhaka, in the month of March, 2009 which mentioned as sample ID: C. ROXâ&#x2C6;&#x2019;2.

Sample-3: Mature seeds are collected from Regional Spice Research Center Faridpur, in the month of August, 2008 which mentioned as sample ID: C. ROX−3. The collected sample were kept in airtight plastic bags, sealed and transported to the laboratory. The sample was cleaned to separate dirt and sun dried.

Figure–1.5: Area of sample collection from different parts of Bangladesh

Literature survey Carum roxburghianum Benth. is a species of umbelliferae or Apiaceae and genus carum. Genus carum having three species: 1) Carum roxburghianum Benth. (Syn. Trachyspermum roxburghianum (DC.) 2) Carum copticum Heirn (Syn. Trachyspermum ammi Linn.) 3) Carum carvi Carum roxburghianum is grown is the sub-tropical and temperate regions of India and Bangladesh. It is a popular herb in Bangladesh mainly in the North–Eastern part of Bangladesh. People of this region use the green herb as a pot–herb and as a flavoring agent

especially in the winter season. The green herb is also used as a flavoring agent in soups, table sauces and salads. Seed has got a very limited use as condiment but it has got an export possibility. The herb and its seeds are used as folkloric medicine e.g. aromatic carminative especially useful in flatulence, colic and hiccups of infants and children. The popularity of the herb is primarily due to the characteristic flavor, which is responsible to the essential oil present in the herb as well as in its seed. Many important elements which are present in the herb and its seed may have important metabolic and nutritional activity for human being. No systematic study has been carried out so far in Bangladesh to ascertain the usefulness of the herb and its seed. The following are the reported information found in the literature. Thomas, Allahad Fmr22 reported, this species very closely resembles and is probably a cultivated from of T. stictocarpum (C.B. Clarke) wolff syn. C. stictocarpum C.B. Clarke, Which is found wild from the lower Himalayas to South India. There are, minute difference in the fruit due to cultivation. The fruit ajmod like those of ajowan are subjected to the attack of spice-beetle or drugstore beetle in store house. Ashraf, M.; Aziz, J.; Bhatty, M.K42 investigated on the essential oils of the Pakistani species of the family Umbelliferae.V. Carum roxburghianum seed oil. And reported that Yield, physico-chemical properties and chemical composition of essential oil from fresh mature and 1 yr-old unripe seeds of C. roxburghianum were compared. Tabulated results showed that compared with 1 yr old unripe seeds, fresh mature seed oil had lower optical rotation (+19 degree 12' vs. +35 degree 14') and higher acid value (3.1-3.8 vs. 1.42-1.50) lower total hydrocarbons (44.2 vs. 55.0%), monoterpenes (20.9 vs. 26.0%), limonene (15.1 vs. 20.8%), terpinene (1.9 vs. 2.6%) and sesquiterpene (23.3 vs. 29.0%) contents, and higher oxygenated compounds (55.8 vs. 45.0%) and a novel unidentified ketonic acid (mol. wt. 168, C10H16O2, previously found in Carum roxburghianum essential oil). K. M. Braj and D. Sikhibhushan34 & S. Choudhury, A. Rajkhowa, S. Dutta, et al26 reported that Terpinen-4-ol, (Z)-ligustilide and Îł-terpinene, which have been reported as major constituents in the fruits essential oil of Indian origin.

Chowdhury, Bhuiyan, and Begum43, reported that Essential oil from the leaves and fruits of Carum roxburghianum Benth. is analyzed by gas chromatography-mass spectrometry (GCMS). The oil leaf is dominated by apiol (20.81%), 2(3H)-furanone,dihydro-5-pentyl(17.21%), 1,3-benzodioxole, 4-methoxy-6-(2-propenyl)- (9.51%) and citronellol (6.62%). The fruit oil is rich in 2-cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)- (40.03%), and other components that follow are apiol (18.71%), limonene (17.11%), myristicin (12.30%), dihydrocarvone (7.89%) and eugenol (1.68%). The compositions of both oils vary qualitatively and quantitatively. Malavya & Dutta45 reported that the Carum roxburghianum fruits yield an essential oil (up to 2.5 %), a fixed oil (4.5 %). The essential oil obtained by steam distillation is greenish yellow in colour and has following characteristics sp. Gr. 20˚C, 0.9488; [α]D33˚,+25.5˚; n20˚C,1.4880; acid value, 4.9; sap. val., 49.1; sap. value after acetylation, 74.2. The oil contains:

d-linalool,

4.7; d-limonene,

35.1; α- terpinene,

19.4; dl-pipertone,5.7;

thymohydroquinone 0.2: thymol 1.7; dl- piperitone, 13.6; cuminic acid,0.4; cumminaldehyde, traces; an unidentified ketone (C10H14O3).1.0; unidentified esters ,5.9; And d- limonene and dipentene mixture, 2.5 %. M.L. Gujral et al.46 reported that Seeds of C. roxburghianum yielded 1.8-2.0% of an essential oil, 4.4-4.5% of fatty oil (n2D 1.4914, I.no. 98.4, Sapon. no. 189.5), and 0.08-0.09 % of a ketonic cryst. Compd.(Ι); 2,4 dinitro phenyl hydrazone m. 95-6˚C, p-nitro phenylhydrazone m. 204-5˚C,has spasmolytic activity. Mahmud, Shahid ; Saleem, Muhammad ; Yamin, Muhammad ; Khan, Muhammad Naeem47

reported that Total lipids extracted from the powdered seeds of Carum

roxburghianum were fractionated into hydrocarbons (0.30%), wax esters (0.30%), sterol esters (1.35%), triacylglycerols (72.41%), free fatty acids (6.06%), 1,3-diacylglycerols (4.60%),

1,2- diacylglycerols

(0.64%), glycolipids

(5.10%),

sterols

(1.20%),

monoacylgylcerols (3.18%), 1-monoacylglycerols (1.46%), phosphatidylethanolamines (1.08%)

phosphatidylcholines

(0.40%),

lysophosphatidylethanolamines

(1.48%)

and

phosphatidylinositols (0.44%) with the help of TLC. The fatty acid composition of all the lipid fractions was determined after converting them into their methyl esters with BF 3– methanol reagent and then analyzing them by GC. Oleic acid was found as a major component in all the lipid classes, whereas palmitic, linoleic and linolenic acids were present

in lesser quantities. Arachidic acid was identified as a minor component in only seven out of twelve lipid classes. Tanveer Ahmad Chaudri, Ijaz Ahmad, M. Yamin and Shahid Mahmud52 reported that the triacylglycerols were first sepd. from the seed oil of Carum roxburghianum by plain TLC and then fractioned into groups differing in unsatn. by argentation. The position of fatty acids at 1, 2 and 3 carbons of triacylglycerol mols. in each group were detd. by lipolytic hydrolysis with pancreatic lipase and GLC by methyl esters. The position of these glycerol’s was found to be occupied by the unsatd. acids preferentially. The level of unsatn. detn. the distribution of the fatty acids. The results of the study allow possibility of predicting the distribution pattern of fatty acids in different triacylglycerol fractions. Shiojir;MasatoshI; Ito; Hisatomi53 reported that Carum plant seed exts. From Carum copticam, C. bulbocastum and C. roxburghianum are claimed as NGF formation promoters and health foods for treatment and prevention of memory and learning disorders Alzheimer’s disease and diabetic neuropathy. Formulation examples of health foods and health drinks were given. Mahmud, Shahid; Wahid, Amran; Khnum Razia 54 were reported that the lipase and phospholipase activities of C. roxburghianum were studied at different temps. Solvents and pH. Both the enzymes showed the max. activities at 40˚C and in n-heptane used as solvent. However the max. activities of two different pH one at pH 5 (acidic) and other at pH 8 (alk). Whereas phospholipase showed only one pH optimum at pH 8. During the course of germination the lipase showed an increase whereas reverse was the case with phospholipase. Chandhuri. Tanweer Ahmed; Ahmed, Ijaj; Muhammad, Din; Ahmad, Manjoor55 reported in the family Umbelliferae octadecenoic acid (C18:1) is a phylogenitic character having distribution on the different positions of triacylglycerols mols. It has a role as a key intermediate in fat metab. during seed germination which created an interest to investigate further to find out its positional isomers in the oils of Carum species. Therefore, the octadecenoic acid sepd. from the seed oil of Carum copticum, Carum carvi and Carum roxburghianum of umbelliferae family was oxidized septd. By von Rudloff’s, reagents. The liberated mono and difunctional fatty acids were sepd. and identifiedby the application of thin layer and gas liq. Chromatog. to det. positional isomers. The positional isomers detd. among

these three species were cis-6-octadecenoic acid, cis-9-octadecenoic acid. The percentage of these isomers varied from 4.5 to 46.0%.

Proximate Analysis 3.1 Determination of moisture content and dry matter 56−59, 155: Procedure112: At first Petri dish was dried and weighed in an electronic balance. Then definite amount of sample was taken in the Petri dish and weighed accurately. The Petri dish with sample was placed in an oven at 110°C for 6 hrs. The oven dried Petri dish was taken in desiccators. After 30 min the Petri dish was removed from desiccators and weighed. In this way the Petri dish was weighed repeatedly after heating and cooling in the same way until the constant weight was obtained. The percentage of moisture content was calculated by the following formula: weight of moisture

% of moisture content = weight of sample ×100 Where, Weight of moisture = the wt. of sample before drying – the wt. of sample after drying. % of dry matter = 100−moisture %

Calculation method: Before drying, Weight of Petri dish = w1 gm Weight of Petri dish + sample = w2 gm Weight of sample = (w2−w1) gm After drying, Weight of Petri dish + sample = w3 gm Weight of moisture = (w3−w2) gm weight of moisture

% of moisture = weight of sample ×100 =

w3 − w2 ×100 w2 − w1

% of dry matter = 100 − moisture %

Table–3.1: Moisture and dry mater contents of C. roxburghianum seeds:

 Each value represents the average value from three experiments .

3.2 Determination of ash content (the destruction of organic matter) 56-59: Plant sample contains organic matter which must be destroyed prior to the estimation of minerals. Dry ashing is generally used for the destruction of organic matter. The choice of procedure depends on the nature of organic material, the nature of any inorganic constituent present, the metal to be determined and the sensitivity of the method used for the determination. The ash remaining following ignition of medicinal plant materials is determined by three different methods which measure total ash, acid-insoluble ash and watersoluble ash. The total ash method is designed to measure the total amount of material remaining after ignition. This includes both “physiological ash”, which is derived from the plant tissue itself, and “non-physiological” ash, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the plant surface. Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth. Water-soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water.

Apparatus:    

Crucible (porcelain or nickel) Bunsen burner Muffle furnace Ash less filter-paper

Reagents:  Potassium dichromate  Sulphuric acid  Dil. HCl Sample collected from

Weight of Sample, (w2w1) gm

Weight of moisture, (w3- w2) gm

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

7.2428 7.5459 5.8469

0.7099 0.8081 0.6783

Moisture %, Dry matter w3 − w2 %, 100 – ×100 moisture %. w2 − w1 9.8014 10.7091 11.6010

90.1985 89.2909 88.3990

3.2.1 Cleaning the crucible and it prepared for ash62: At first nickel or porcelain crucible were taken. For cleaning the crucible chromic acid was prepared. For this purposes potassium dichromate was taken in a beaker and sulfuric acid was poured to the beaker. For better cleaning chromic acid was kept on crucibles for several hours. Washing the crucibles with clean water and finally rinsed with distilled water. Crucibles were kept in oven at 110˚C for drying. Then blank crucibles were heated in a muffle furnace at 650˚C for 6 hrs and cooled in desiccators and weighed. The process of heating and cooling was continued till a constant weight was obtained.

3.2.2 Determination of Total ash 62, 121: A certain amount of moisture less sample was taken in a crucible and it was weighed (which was previously been cleaned, heated at 650˚C and cooled and weighed). To remove the organic matter as much as possible, the crucible with sample was fired with Bunsen burner. Then the crucible heated to a temperature controlled muffle furnace at 650˚C for 6 hrs. Then the crucible was removed from the furnace and allowed to cool in desiccators and weighed. If charred matter still remains, add small amount of ethanol, break up the ash with a glass rod, wash the glass rod with small amount of ethanol, and evaporate carefully the ethanol. Then, proceed as directed previously and weigh accurately. The process of heating and cooling was repeated till a constant weight was obtained and the ash is almost white or grayish white in colour.

Calculation method: Before ignition, Weight of crucible = w1 gm Weight of crucible + sample = w2 gm Weight of sample = (w2 – w1) gm

After ignition, Weight of crucible + ash = w3 gm

Weight of ash = (w3- w1) gm % of ash =

w − w1 weight of the ash ×100 = 3 ×100 weight of the sample w2 − w1

% of organic matter = 100 – ash %

Table−3.2: Total ash contents of Carum roxburghianum (Radhuni) seeds:  Each value represents the average value from three experiments .

3.2.3 Determination of Acid soluble and insoluble ash100, 121: The Acid-insoluble ash limit Test is designed to measure the amount of ash insoluble to diluted hydrochloric acid.

Procedure: Add carefully 25 ml of dilute hydrochloric acid to the 1 gm of ash (obtained as directed under the Ash Limit Test or total ash), boil gently for 5 minutes, collect the insoluble matter on an ash less filter-paper for quantitative analysis, wash thoroughly with hot water, and dry the residue together with the filter paper. Ignite it for 1 hour in a crucible of platinum, quartz, or porcelain, which has been prepared as directed in the Ash Limit Test and whose weight is already known. Cool it in desiccators (silica gel) and weigh accurately. If the measured amount is larger than the specified value, ignite until a constant weight is obtained.

Calculation method: Sample collected from

Weight of Sample, (w1- w2) gm

Weight of Ash, (w3- w1) gm

Joydebpur, Gajipur

5.0277

0.4521

8.9920

91.008

Keranigonj, Dhaka

5.3340

0.4807

9.0124

90.9876

Faridpur

3.7628

0.3102

8.2431

91.7569

Weight of ash sample = w gm Weight of crucible = w1 gm

% of ash ,

% of organic matter, (100 w3 − w1 × 100 – ash %) w2 − w1

Weight of crucible + insoluble ash = w2 gm Weight of acid insoluble ash = (w2 – w1) gm % of acid insoluble ash =

w − w1 weight of acid in − so lub le ash ×100 = 2 ×100 weight of ash sample w

% of acid soluble ash = 100 – % of acid insoluble ash

Table−3.3: Acid soluble and insoluble ash content of C. roxburghianum seeds:  Each value represents the average value from three experiments .

3.2.4 Determination of Water soluble and insoluble ash 91, 100: The water soluble ash limit Test is designed to measure the amount of ash soluble to hot water.

Procedure: Add carefully 25 ml of distilled water to the 1 gm of ash (obtained as directed under the Ash Limit Test or total ash), and boil for 5 minutes. Collect the insoluble matter in a sintered-glass crucible or on an ash less filter-paper. Wash with hot water and ignite in a crucible for 15 minutes at a temperature not exceeding 450°C. Subtract the weight of this residue in gm from the weight of total ash. Calculate the content of water soluble ash in per gm of air dried material.

Calculation method: Weight of ash sample = w gm Weight of crucible = w1 gm Sample collected from

Weight of ash sample, w gm

Weight of acid insoluble ash, (w2 – w1) gm

% of acid insoluble ash, w2 − w1 ×100 w

% of acid soluble ash, 100 – % of acid insoluble ash

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

1.1344

0.4534

39.9682

60.0318

1.1514

0.5236

45.4750

54.5249

1.1929

0.6035

50.5924

49.409

Weight of crucible + insoluble ash = w2 gm Weight of water insoluble ash = (w2 – w1) gm

% of water insoluble ash =

w − w1 weight of water inso lub le ash ×100 = 2 ×100 weight of ash sample w

% of water soluble ash = 100 – % of water insoluble ash

Table−3.4: Water soluble and insoluble ash contents of Carum roxburghianum (Radhuni) seeds:  Each value represents the average value from three experiments .

3.3 Estimation of nitrogen content: Kjeldahl’s method 57, 65, 100: This process is widely employed in industrial and research laboratories. The principle of the method is the conversion of the nitrogen of the nitrogenous substances into ammonia by boiling with concentrated sulfuric acid which was fixed by the excess of acid as ammonium sulfate. The latter is determined by adding an excess of caustic alkali to the solution after digestion with the acid and distilling off the liberated ammonia into standard acid. Simple digestion with concentrated sulfuric acid is, however, a slow process and various modifications have been suggested to increase the speed of the reaction. This includes the addition of potassium sulfate, which raises the boiling point of the acid (Kjeldahl-Gunning process), and of catalysts, such as mercury, mercuric oxide etc. the simple process works well

Sample collected from

Weight of ash sample, w gm

Weight of water insoluble ash, (w2 – w1) gm

% of water insoluble ash, w2 − w1 ×100 w

% of water soluble ash, 100 – % of water insoluble ash

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

1.0860

0.8748

80.5525

19.4475

1.0461

0.8027

76.7326

23.2674

1.0006

0.7028

70.2345

29.7655

for nitrogen determinations in proteins and also in amines and amides, but is not applicable to nitro, azo, hydrazo and cyano compounds without modification. In the presence of nitrates there is a danger of loss of nitric acid, salicylic acid is then added to the sulfuric acid, which fixes the nitric acid as nitro-salicylic acid. Upon the addition of zinc dust or of anhydrous

sodium thiosulfate, the nitro-salicylic acid is reduced to the amino compound, which can then be estimated by the Kjeldahl process.

Reagents:  0.1 N H2SO4solution  0.1N Na2CO3 solution  60% NaOH solution  2% Boric acid solution  Digestion mixture  Concentrated sulfuric acid A. R.  Methyl red indicator  Methylene blue indicator  Methyl orange indicator (0.1%)  Phenolphthalein indicator (1%)

Figure –3.1: Kjeldahl’s distillation unit

3.3.1 Reagent Preparation: 1) 0.1 N H2SO4 Solution : 0.7 ml conc. H2SO4 (36N) was taken in 250 ml volumetric flask and it was diluted up to the mark with distilled water. 2) 0.1 N Na2CO3 solution: 2.65 g Na2CO3 was taken in 50 ml volumetric flask and it was diluted up to the mark. The strength of Na2CO3 =

2.666 ×0.1 = 0.10006 N 2.65

3) 60% NaOH solution: 60g of NaOH was taken in 100 ml water. 4) Digestion mixture: The mixture of 98 parts K2SO4 and 2 parts CuSO4. 5) Methyl red indicator: 0.1g of Methyl red dissolved in 60 ml of alcohol and water added to make to 100 ml. 6) Methyl orange (0.1%):

0.1g Methyl orange dissolves in 100 ml distilled water. 7) Phenolphthalein indicator (1%): 1g Phenolphthalein dissolves in 100ml alcohol 8) Methylene blue indicator: 1 gm Methylene blue in100ml water 9) Preparation of 2% Boric acid solution: 5g of Boric acid was taken in 250 ml volumetric flash and diluted up to the mark with distilled water. 10) Preparation of mixed indicator: Mixed indicator was prepared freshly by mixing 2 parts of Methyl red and 1 part of Methylene blue in a Stoppard test tube.

3.3.2 Procedure for determination of nitrogen content: A) Digestion of the residue: 0.05–0.5 g of the sample was taken into dry Kjeldahl flask, (a round bottomed flask with a long narrow neck) and 5ml concentrated H2SO4 and 0.5g of the digestion mixture were taken into it. Glass beads ware added to prevent bumping. Two Kjeldahl flasks were taken. One of them for sample and another one were for blank determination. In the blank determination flask samples were absent the flasks were shaking for well mixing. The flasks were supported on a hole (4.5 cm in diameter) in a piece of asbestos board and the necks were inclined at an angle of 60º. The flasks were closed with loosely fitting glass stopper elongated to a point and having a balloon-shaped top. The flasks were heated with a small flame in a fume cup-board. When foaming had ceased the flame was increased until the mixture boiled gently. The mixture digested by heating for 4 to 5 hours. The heating was continued for 30/60 minutes after the solution became colourless or clear (Usually 90-120 minutes) and the solutions were allowed to cool.

B) Standardization of H2SO4 with 0.1N Na2CO3 solution: 10 ml of Na2CO3 was taken in a conical flask with the help of pipette, a few drops of methyl orange were added to the solution and the solution becomes pink colour. H 2SO4 solution was taken in a burette. H2SO4 solution was added drop wise into Na2CO3 solution. At the end of titration the colour of the solution became yellowish. Titration was carried out exactly three times the results are tabulated as follows:

Table–3.5: Titration data for determining the strength of H 2SO4 acid Solution: Observe. no. 1. 2. 3.

Na2CO3 solution (ml) 10 10 10

Burette reading , Volume of H2SO4 solution (ml) IBR FBR Difference Average volume 0.0 9.5 9.5 9.5 9.7 19.2 9.5 0.0 9.5 9.5

Calculation: As we know, V1S1 = V2S2 Here,

V1 = Volume of Na2CO3 solution = 10 ml S1 = Strength of Na2CO3 solution = 0.10006 N V2 = Volume of H2SO4 solution

Strength of H2SO4 solution,

S2 = =

= 9.5ml

9.5 Ă&#x2014;0.10006 10

= 0.1059 N

C) Distillation of ammonia in Kjeldahl distillation unit: 5 ml of 2% boric acid solution was taken in a 100ml conical flask and it was placed in such a way that the tip of the condenser outlet dipped below the surface of the boric acid solution. 2 to 3 drops of mixed indicator were added to the conical flask. The digested sample was transferred completely by means of rapid ringing to chamber of steam distillation apparatus. Which was previously been cleared of any contaminating ammonia by repeated washing. The enough 60% NaOH (nearly 11-16 ml) was added to the digest in the chamber to more than neutralized the amount of acid present, which is ensure by means of phenolphthalein indicator. Than the distillation set was made air tight and steam generation in the boiler was started. The sample was steam distilled until 20-40 ml of distillate collect in the receiving flask (in about 20 minutes of distillation). Than the receiving flask is lowered and the steam was stopped side of the condenser outlet tube was rinsed into the receiving flask with a little water. The colour of the content of the receiving flask would have changed during the distillation.

D) Titration: Ammonia in the receiving flask was titrated against 0.1 N Sulfuric acids, the end point being the reversion to the original greenish blue colour.

E) Blank determination: Blank was determined by digesting only the sulfuric acid and distilling the same after addition of 60% NaOH and enough water and titrated the ammonia liberated against the standard acid.

The titrate value of blank was subtracted from the titter value of the sample, which gives the amount of 0.1N H2SO4 needed to neutralize the ammonia evolved from 0.05g sample. 1 ml of 0.1N H2SO4 ≡ 0.014 g Nitrogen ≡ 14 mg Nitrogen.

Calculation method: The percentage of nitrogen in the solid residue was calculated as flows: % of Nitrogen =

( s − b) × N ×14 ×100 W ×1000

Where, s = Volume (ml) of standard acid used in the sample titration. b = Volume (ml) of standard acid used in the blank titration. N = Strength of the H2SO4 acid (N). W = Weight (gm) of the sample. Strength of H2SO4, N = 0.1059 N

Table – 3.6: (Blank Titration with 0.1 N H2SO4 solution) Observe no

Weight of

Vol. of Conc.

Vol. of standard

Digestion mixture,

H2SO4,ml

acid used in blank

gm 1. 2.

0.1896 0.1998

titration, s ml 5 5

0.05 0.05

So, the Volume of standard acid used in the blank titration, b = 0.05 ml

Table-3.7: Titration data for determination of nitrogen content of Carum roxburghianum seeds:

a) For the sample of Joydebpur, Gajipur: Observe.

Wt. of

Vol. of

Vol. of acid

% of

Mean %

Digestion

sample,

Conc.

used in

Nitrogen,

mixture,

w gm

H2SO4,

sample

titration, s ml

5 5 5

2.85 2.85 2.80

gm 1. 2. 3.

0.1938 1.8560 1.8350

0.1160 0.1159 0.1139

Nitrogen

( s −b) × N ×14 ×100 W ×1000

3.5787 3.5786 3.5788

3.5787

Result: The Nitrogen content of Radhuni seeds from Joydebpur, Gajipur 3.5787 Observe

Wt. of

Vol. of

Vol. of acid

% of

Mean %

. no

Digestion

sample,

Conc.

used in

Nitrogen,

mixture,

w gm

H2SO4,

sample

titration, s ml

5 5 5

2.80 2.85 2.85

gm 1. 2. 3.

0.1720 0.1640 0.1865

0.1360 0.1390 0.1419

Nitrogen

( s −b) × N ×14 ×100 W ×1000

2.9978 2.9856 2.926

2.9698

b) For the sample of Keranigonj, Dhaka: Result: The Nitrogen content of Radhuni seeds from Keranigonj, Dhaka 2.9698

c) For the sample of Faridpur: Observe.

Wt. of

Vol. of

Vol. of acid

% of

Mean %

Digestion

sample

Conc.

used in

Nitrogen,

mixture,

w mg

H2SO4,

sample

0.1227 0.1212 0.1242

ml 5 5 5

titration, s ml 2.75 2.70 2.8

1. 2. 3.

gm 0.1942 0.1356 0.1455

( s − b) × N ×14Nitrogen ×100 W ×1000

3.2624 3.2424 3.2829

Result: The Nitrogen content of Radhuni seeds from Faridpur 3.2624

3.4 Determination of protein content 57, 65, and 93:

3.2624

Proteins (also known as polypeptides) are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. The amino acids in a polymer chain are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism. The protein content of a foodstuff is obtained by estimating the nitrogen content of the material and multiplying the nitrogen value by 6.25(a factor) this referred to as crude protein content since the non protein nitrogen(NPN)present in the material is not taken into consideration57. So, the protein content is = N2 content × 6.25

Table –3.8: Protein content of Carum roxburghinum (Radhuni) seeds:

Sample

% of

% of Protein,

collected

Nitrogen

% of Nitrogen × 6.25

Joydebpur, Gajipur

3.5787

22.3663

Keranigonj, Dhaka

2.9698

18.5618

Faridpur

3.2624

20.3899

from

 Each value represents the average value from three experiments .

3.5 Determination of crude fiber 57, 66, 70: Crude fiber is made up primarily of plant structural carbohydrates such as cellulose and hemicelluloses and lignin, which is a highly indigestible material. Often there is much confusion about the difference between dietary fiber (soluble fiber) and crude fiber, or what is now referred to as insoluble fiber. Most crude fiber contains one-seventh to one-half dietary fiber.

Crude fiber is determined by laboratory analysis and is mainly composed of lignin, which is found in the tissues of plants and cellulose basically a plant's skeleton. In layman's terms, analysis of crude fiber in the laboratory involves oven-drying the fiber to be analyzed after exposing it to a series of sulfuric acid and sodium hydroxide solutions. Crude fiber left a mixture of insoluble fibers that have no nutritional value.

Insoluble or crude fiber is expelled by the body and aids in maintaining regular intestinal peristalsis (bowel) movements. In short, most people need some crude fiber in their diets. Excellent sources of crude fiber, or insoluble fiber, include: vegetables like leafy greens, whole grains like whole wheat and rye, and beans such as kidney beans and black beans. While most doctors and nutritionists recommend eating a diet that is high in both soluble and insoluble fiber, many suggest that those suffering from irritable bowel syndrome not eat insoluble fiber on an empty stomach.

Reagents:  0.255 N H2SO4  0.313 N NaOH Solutions.  Distilled water  Alcohol and diethyl ether.  BaCl2 solution  Phenolphthalein indicator

3.5.1 Reagent Preparation: 1) 0.255 N H2SO4 Solution: 3.6 ml Concentrated H2SO4 (36N) was taken in 500ml volumetric flask and flask was made up to the mark with distilled water. 2) 0.313 N NaOH Solution: 6.26 g NaOH pellet was taken in 500ml volumetric flask and flask was made up to the mark with distilled water.

3.5.2 Procedure for determination of crude fiber 57: An amount of 2-5 g moisture and fat free sample was weight into a 500ml R.B. flask than 200ml boiling 0.255 N H2SO4 was added. The mixture was heated exactly 30 minutes. A water condenser attached with R.B flask for keeping the volume constant. The mixture was filtered through linen cloth. The residue was washed repeatedly with boiled water to free the residue form acid. 3 or 4 drops of the filtrate collected in a test tube and a drop or two of BaCl2 solution was added for testing the presence SO 42– ion. The process was continued precipitate of SO42– appeared. Then the residue has free from acid. The charge was again started in another R.B. flask with 200ml 0.313 N NaOH solution after boiling for 30 minutes (keeping the volume constant as before). The mixture was again filtered through linen cloth. The residue is washed with hot water till free from alkali. 2/3 drops of filtrate taken in a test tube and 1 drop of phenolphthalein solution was added to it. If there was no pink colour appeared. It can be conclude the alkali was removed from the residue. Then filtrate was washed initially with alcohol and secondly with diethyl ether. The residue over the cloth was then transferred in porcelain crucible which is previously cleaned and weighted. It was dried at 1100c in oven and weighted until a constant weight. The difference of weight of the crucible gave the weight of crude fiber. The crucible was than heated in a muffle furnace at 6000C for 3 hour. The crucible was allowed to cool and weighted again. The difference in the weights present the weight would gave the amount of ash present in crude fiber. Thus the crude fiber percentage can be calculate with this formula, % of crude Fiber =

Weight of dried fibre −Weight of ash ×{100 − (moisture + fat )} Moisture and fat free sample (taken)

Calculation method: Weight of moisture & fat free sample (taken)

=m1 gm

Weight of empty crucible

= m2 gm

Weight of dried fiber + crucible Weight of dried fiber, Weight of crucible with ash after firing at 600˚C Weight of ash,

= m3 gm w1 = (m3 − m2) gm = m4 w2 = (m4 − m2) gm

Moisture content = M Fat content

% of crude Fiber =

( m3 − m2 ) − ( m4 − m2 ) × {100 − ( M + F )} m1

w1 − w2 × { 100 − ( M + F )} m1

Table–3.9: Crude fiber content of Carum roxburghianum (Radhuni) seeds: a) For the sample of Joydebpur, Gajipur:  Moisture content, M = 9.8014 %  Fat content, F = 15.3137 % No of observe.

Weight of sample, m1 gm

1 2 3

5.5695 5.5240 7.0457

Weight of dried fiber, w1 gm 1.6769 1.7778 2.0050

Weight of ash, W2 gm 0.1224 0.1342 0.1683

% of Fiber,

w1 − w2 × { 100 − ( M + F )} m1

20.9011 22.2810 19.5212

Mean % of fiber

20.9011

Result: The crude fiber content of Radhuni seeds from Joydebpur, Gajipur 20.9011 %

b) For the sample of Keranigonj, Dhaka:  Moisture content, M = 10.7091  Fat content, F = 20.3176 % No of observe.

Weight of sample, m1 gm

1 2 3

5.2449 5.7287 5.6259

Weight of dried fiber, w1 gm 1.7495 1.8292 2.0015

Weight of ash, W2 gm 0.1012 0.1132 0.1532

% of Fiber,

w1 − w2 × { 100 − ( M + F )} m1

21.6602 20.6605 22.6599

Mean % of fiber

21.6602

Result: The crude fiber content of Radhuni seeds from Keranigonj, Dhaka 21.6602 % c) For the sample of Faridpur:  Moisture content, M =11.6010 %  Fat content, F = 20.2304 % No of observe.

Weight of sample, m1 gm

1 2 3

5.3364 7.4124 7.0029

Weight of dried fiber, w1 gm 2.0328 3.0280 2.4508

Weight of ash, W2 gm 0.1608 0.208 0.1992

% of Fiber, w1 − w2 × { 100 − ( M + F )} m1 23.9262 25.9345 21.9179

Mean % of fiber

23.9262

Result: The crude fiber content of Radhuni seeds from Faridpur 23.9262 %

3.6 Determination of Carbohydrates content 57: Carbohydrates or saccharides are the most abundant of the four major classes of biomolecules. They fill numerous roles in living things, such as the storage and transport of energy (e.g., starch, glycogen) and structural components (e.g., cellulose in plants and chitin

in arthropods). In addition, carbohydrates and their derivatives play major roles in the working process of the immune system, fertilization, pathogenesis, blood clotting, and development60. Carbohydrates are simple organic compounds that are aldehydes or ketones with many hydroxyl groups added, usually one on each carbon atom that is not part of the aldehyde or ketone functional group. The basic carbohydrate units are called monosaccharides; examples are glucose, galactose, and fructose. The general stoichiometric formula of an unmodified monosaccharide is (CH2O)n, where n is any number of three or greater; however, not all carbohydrates conform to this precise stoichiometric definition (e.g., uronic acids, deoxysugars such as fructose), nor are all chemicals that do conform to this definition automatically classified as carbohydrates 61. The content of the available carbohydrates is determined by difference, i.e., by subtracting from the sum of the values (per 100 gms) for moisture, protein, ether extractives, ash, and crude fiber. So, the carbohydrates content = 100 â&#x20AC;&#x201C; (moisture % + ash % + protein % + Crude fiber % + ether extractives %)

Table â&#x20AC;&#x201C; 3.10: Carbohydrates content of Carum roxburghianum seeds: ď&#x192;&#x2DC; Each value represents the average value from three experiments.

3.7 Estimation of food energy 71, 72: Food energy is the amount of energy in food that is available through digestion. Like other Sample collected from

Moisture content %

Ash content %

Protein content %

Crude fiber %

Ether extract. %

Carbohydrates content %

Joydebpur, Gajipur Keranigonj, Dhaka

9.8014

8.9920

22.3663

20.9011

15.3137

22.6255

10.7091

9.0124

18.5618

21.6602

20.3176

19.7389

11.6010

8.2431

20.3898

23.9262

20.2304

15.6095

Faridpur

forms of energy, food energy is expressed in calories or joules. The kilo joule is the unit officially recommended by the World Health Organization and other international organizations. Fiber, fats, proteins, organic acids, polyols, and ethanol contain food energy. All foods are made up of a combination of these six caloric nutrients and non-caloric

nutrients. Non-caloric food includes (but not limited to) water, vitamins, minerals, antioxidants, caffeine, spices and natural flavors. Tea and coffee also have no calories without sugar or milk added. Nutritionists usually talk about the number of calories in a gram of a nutrient. Fats and ethanol have the greatest amount of food energy per gram, 9 and 7 kcal/g (38 and 30 kJ/g), respectively. Proteins and most carbohydrates have about 4 kcal/g (17 kJ/g). Carbohydrates that are not easily absorbed, such as fiber or lactose in lactoseintolerant individuals, contribute less food energy. Polyols (including sugar alcohols) and organic acids have fewer than 4 kcal/g. The food energy were estimated using the equation71, 72:

Food energy (FE) = (% of crude protein × 4) + (% of lipids ×9) + (% of Carbohydrate ×4) calories/gm.

Table–3.11: Estimation of food energy of Carum roxburghianum seeds: Sample collected from

% of Protein

% of lipids (ether extract.)

% of Carbohydrates

Food energy (FE), (CP×4+lipids×9+CHO×4) calories/gm

Joydebpur,

22.3663

15.3137

22.6255

317.7909

Gajipur Keranigonj,

18.5618

20.3176

19.7389

336.0612

Dhaka Faridpur

20.3898

20.2304

15.6095

326.0708

 Each value represents the average value from three experiments.

3.8 Physical characteristics of Carum roxburghianum (Radhuni) seeds: Following physical characteristics parameter were determined73, 74.

3.8.1 Determination of seed volume73, 74: Seed volume were determined by transferring 1 gm Radhuni seeds sample in a 10ml measuring cylinder, and recorded the volume. Then 5 ml distilled water were added to the cylinder and recorded the volume. The gained volume divided by weight of seeds was taken as seed volume. Seed volume = =

volume of sample and added water −volume of added water weight of seed

ml/gm

V2 −V1 ml/gm W

Table –3.12: Determination of seed volume Radhuni seeds.

Sample collected from

Weight of sample,W gm

Volume of added water and sample,V2 ml

V2 −V1 ml/gm W

1.0083

Volume of added water, V1 ml 5

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

Seed volume,

0.9918

1.0093

0.9908

1.0641

6.5

1.4096

 Each value represents the average value from three experiments.

3.8.2 Determination of seed density73, 74: Seeds density was calculated as seed weight divided by seed volume. seed weight

Seed density = seed volume gm/ml

Table –3.13: Determination of seed density Radhuni seeds. Sample collected from Joydebpur, Gajipur

Seed weight, gm 1.0083

Seed volume, ml 0.9918

Seed density, gm/ml 1.0166

Keranigonj, Dhaka Faridpur

1.0093

0.99078

1.0187

1.0641

1.4096

1.0138

 Each value represents the average value from three experiments.

3.8.3 Determination of seed hydration capacity73, 74: 1 gm Radhuni seeds sample transferring in 10ml measuring cylinder 5 ml distilled water were added to the cylinder and allowed it for overnight soaking. After then seeds were made dry by using tissue or filter paper. The gained weight was taken as seed hydration capacity. Hydration capacity = weight of seeds after overnight – weight of seeds

Table –3.14: Determination of hydration capacity Radhuni seeds. Sample collected from Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

weight of

weight of seeds after

Hydration capacity,

seeds, gm

overnight, gm

1.0083

1.7109

0.7026

1.0093

1.6108

0.6015

1.0640

1.8395

0.7755

 Each value represents the average value from three experiments.

3.8.4 Determination of seed hydration index73, 74: Hydration index was calculated as seed hydration capacity divided by original seed weight. Hydration index =

seed hydration capacity seed weight

Table –3.15: Determination of hydration index Radhuni seeds.

Sample collected from

Seed weight, gm

Seed Hydration capacity, gm

Hydration index

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

1.0083

0.7026

0.6969

1.0093

0.6015

0.5960

1.0640

0.7755

0.7289

 Each value represents the average value from three experiments.

3.8.5 Determination of seeds swelling capacity73, 74: Seed swelling capacity were determined by transferring 1 gm Radhuni seeds sample in a 10ml measuring cylinder, and recorded the volume. Then 5 ml distilled water were added to the cylinder and allowed it for overnight soaking. Then recorded the volume. The gained volume was taken as seed swelling capacity. Swelling capacity =

seed volume after overnight − seed volume after added water seed weight

Table –3.16: Determination of swelling capacity Radhuni seeds Sample collected from

Seed weight, gm

Volume of

Volume of seeds

Swelling

seeds before,

after overnight,

capacity,

1.0083

ml 6

ml 6.4

ml/gm 0.3967

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

1.0093

6.5

0.4953

1.0640

6.5

0.4699

 Each value represents the average value from three experiments.

3.8.6 Determination of seeds swelling index73, 74: Swelling index was calculated as seed swelling capacity divided by original seed volume. Swelling index =

seed swelling capacity seed volume

Table –3.17: Determination of swelling index Radhuni seeds. Sample collected from

Seed volume, ml

Seed Swelling capacity, ml

Swelling index

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

0.9918

0.3967

0.3999

0.9908

0.4953

0.4999

1.4096

0.4699

0.3333

 Each value represents the average value from three experiments.

Essential Oil

4.1 Volatile oils, “Essential oil” or “Ethereal oil”: The term “Essential oil” or “Ethereal oil” defined as the volatile oil obtained by the steam distillation of plants. With such a definition it is clearly intended to make a distinction between the fatty oils and the oils which are easily volatile. Their volatility and plant origin are the characteristic properties of these oils. Essential oil is any of a class of volatile oils composed of a mixture of complex hydrocarbon (usually terpenes) and other chemicals

extracted from a plant. Essential oils are characterized by their capacity to generate a flavor or aroma and taste75, 78. All distinctly aromatic plants contain essential oils and these plants are found in some sixty families, which are distributed throughout the Plant Kingdom. Certain families, however, are noted for their aromatic members and, in fact, the possession of aromatic oils is a characteristic of some families. Such additional families as the laurels, wax myrtle’s and the daisy family also contain numbers of aromatic members77. Essential or volatile oils are by products of metabolism, which have become useful to the plant only secondarily. The oils are never in Free State in living cells but are deposited in various types of cavities throughout the plants body. These Storage spaces may consist of dead cells, cavities formed by the disintegration or shrinkage of calls or especially formed capsules, and they are found in any or all parts of the plant, such as root (ginger), wood (cedar), bark (cinnamon), leaves (mint), petals (rose), fruit (orange), or seeds (cardamom). They are generally liquids, but may be semi solids or solids77. Gradually with the advance of science came improvement in the methods of preparing the oils, and parallel with this development a better knowledge of the constituents of the oils was gained. If was found that the oils contain chiefly liquid and more or less volatile compounds of many classes of organic substances. Thus we find acyclic and csocyclic hydrocarbons and their oxygenated derivatives. Some of the compounds contain nitrogen and sulfur. Although a list of all the known oil components would include a variety of chemically unrelated compounds, it is possible to classify a large number of these into four main groups, which are characteristic of the majority of the essential oils i.e.: •

Terpenes, related to isoprene or isoterpenes;

•

Straight-chain compounds, not containing any side branches;

•

Benzene derivatives;

•

Miscellaneous.

Representatives of this last group are incidental and often. Rather specific for a few species (or genera) 75.

Most volatile oils are lighter than water and dissolve in it to slight extent. Some are heavier than water, e.g. oils of wintergreen, sassafras, cinnamon, mustard, clove, etc.  Use of some essential oils and their important constituents are given Table − 4.1

Table â&#x20AC;&#x201C; 4.1: Use of some essential oils and their important constituents 5: Name of oil

Constituents

Uses

Bergamot Clove

Linalyl acetate (36% ester) Eugenol (82%)

Perfume, Flavor. Flavor, perfume, manufacture of vanillin. Flavor, Perfume.

Cinnamon

Cinnamaldehyde, total aldehydes 80%

Turpentine

Pinene

Winter green

Methyl salicylate

Pine Myristica Orange Rose

Terpene alcohols Terpenes; Eugenol D-Limonene; linalol Geraniol; gerany-lacetate; citral

Solvent; Synthetic camphor; Paints; Lubricant. Flavor; Perfume; Anti rheumatic. Antiseptic Perfume; carminative Flavor; Perfume Perfume

Eucalyptus

Eucalyptol (70%)

Antiseptic

4.2 Extraction of essential oils75, 79: There are a number of methods are employed for the extraction of essential oil or Volatile oils from plant and the common ones are: i) Hydro-distillation method, e. g. turpentine oil; ii) Expression method, e.g. orange oil; iii) Effleurage method and iv) Solvent extraction method, e. g. rose oil, All these processes consist of removing the odoriferous substances present in the aromatic plants.

4.2.1 Hydro-distillation method75, 78, 79: This method not only produces the best yields but also enables the operator to attain the end sought most cheaply and with the simplest apparatus. Further, larger quantities of oil can thus be produces without much human labor. Hydro-distillation method is three types: i) Water distillation ii) Water and steam and distillation iii) Steam distillation. The majority of essential oils have always been obtained by steam distillation or by hydro distillation or by solvent extraction. Essential, volatile or ethereal oils are the mixture composed of volatile substances like hydrocarbon, ether, alcohols, esters etc.

Distillation may be defined as the separation of the components of mixture of two or more liquids by virtue of the difference in their vapor pressure occurred when steam depresses the vapor pressure of essential oil. So extraction takes palace when the mixed vapor was condensed, then system of water & essential oil forms a two phases liquid; therefore this type of distillation is of primary importance to the essential oil producer.

4.2.2 Principle of steam distillation75: A liquid boils when its vapor pressure is equal to the atmospheric pressure. In steam distillation, a mixture of water and an organic liquid is heated. The mixture boils when the combined vapor pressure of water (p1) and that of the organic liquid (p 2) is equal to the atmospheric pressure (P) i.e. P = p 1 + p2 The oils were isolated by hydro-distillation using Clevengerâ&#x20AC;&#x2122;s apparatus for 4 hrs

and then

dried over anhydrous sodium sulphate.

4.3 Steam distillation for extraction of essential oil of Carum

roxburghianum (Radhuni) seeds: The essential oils were obtained by steam distillation from the fresh plant materials. For this purposes it were collected from the different region of Bangladesh such as, Joydebpur, Gajipur; Keranigonj, Dhaka and Faridpur. After blending definite amount (100gm) of sample (dirt free fresh seeds) were taken in the distillation flask (Clevengerâ&#x20AC;&#x2122;s apparatus). Then water were added in the flask two third of its volume. The flask with crushed sample and water heated by means of electric heating mental for 4 hours. Volatile substances of Radhuni and steam that produce in the flask were condensed by water condenser. The essential oil was lighter than water and separated. The steam distilled essential oil layer, which was colleted over water, was extracted and washed with analytical grade ether or chloroform. The ether extract of the oil was dried over anhydrous Na2SO4 the filtered. It was collected in vial. The ether or chloroform was removed in vacuum environment and weighted refrigerated at 40C70. The percentage of the essential oil content was calculated by the following formula:

weight of essential oil

% of essential oil = weight of fresh sample taken ×100

Table – 4.2: Essential oil contents of C. roxburghianum (Radhuni) seeds:  Each value represents the average value from three experiments.

Sample collected from

Weight of Sample,w1 gm

Weight of essential oil collected, w2 gm

% of essential oil,

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

100

1.6

100

2.5665

100

2.265

w2 ×100 w1

Figure – 4.1: Steam distillation for extraction of essential oil.

Collected seeds Wash with water to remove dirt, mud And other impurities and then sun dried Dirt free seeds

Crush with blender Powder

Hydro-distillation using Clevenger’s apparatus for 4 hrs Steam distillation

Sample residue after steam distillation

Essential oil

Dried over anhydrous sodium sulphate.

Pure Essential oil were taken for GCMS analysis

Anhydrous Essential oil

Pure Essential oil were taken for Determination of physical properties

Pure Essential oil were taken for Determination of Chemical properties

Scheme – 4.1: Extraction & analysis of essential oil

4.4 Determination of Physical Properties of the essential oil: 4.4.1 Determination of Specific Gravity or Density of essential oil 83: 4.4.1.1 Principal: Density is defined as mass per unit volume (d =

m ) of the liquid, the unit volume being the v

cubic centimeter (cm3) or the milliliter (ml). The term ‘specific gravity’ is the ratio of the density of a substance to the density of a reference substance 84. The reference substance for solids and liquids is usually water.

density of a substance

Specific gravity = density of reference substance It is one of the most important criteria of the quality and purity of essential oil and fatty oil of all the physico-chemical properties, the specific gravity has been reported most frequently in the literature.  Density or Sp. gr. of a substance is a temperature dependent property or factor. So it is generally increases or decreases with rise or fall of each degree of temperature.  The specific gravity of fats or fatty acids at a given temperature in the solid state is higher than in the liquid state.  The specific gravity of a saturated fatty acid is lower than that of the corresponding glyceride.  The hydrogenation of a fat or fatty acid lowers their specific gravity. 

The density and specific gravity of fats and oils varies directly with molecular weight and unsaturation.

Most of the conventional methods for measuring the density or specific gravity of liquids utilizing specific gravity bottle, pycnometer or the westphal balance are applicable to oils and liquid fats. When only small quantities of liquid are available, or where greater accuracy is required, the density of liquids is best determined by means of vessels of accurately defined volume, called pycnometer85, 86. The procedure described here for measuring the density of oils and liquid fats employed a pycnometer2.

4.4.1.2 Procedure for determination of sp. Gravity (Pycnometer method) 85: The pycnometer was cleaned by filling it with a saturated solution of chromium trioxide in Conc. sulphuric acid (H2SO4) and allowed it to stand for at least three hours. The solution was removed from the pycnometer and rinsed with distilled water. Then it was dried completely and it was permitted to stand for 30 minutes. Then the empty pycnometer was weighed accurately by an electronic balance and it was filled with distilled water up to the mark and weighed. The pycnometer was then dried and filled with sample oil to the previous mark and weighed again. The specific gravity of the essential oil was calculated by the following formula.

Calculation method: Weight of empty pycnometer

Weight of pycnometer + water

=W1

Weight of water

=W1 − W

Weight of pycnometer + sample oil Weight of sample oil Room temperature

=W2 = W2 – W

θ˚C = 30˚C

Density of water at 30˚C temp., Dθ˚C = 0.99567 Specific Gravity = =

Wt. of the definite amount of oil (gm) × density of water Wt. of the definite amount of water (gm) W2 − W × Dθ C W1 −W

Table –4.3: Specific Gravity of Essential oil of Carum roxburghianum (Radhuni) seeds:

 Each value represents the average value from three experiments. Sample collected from

Weight of Sample oil, (W2 – W) Gm

Weight of water, (W1 – W) gm

Specific Gravity, W2 − W × Dθ C W1 − W

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

0.9283

1.0679

0.8654

0.9628

1.0679

0.8977

0.9241

1.0679

0.8616

4.4.2 Determination of Refractive Index [η t˚C]:

The refractive index [η] of a substance is the ratio of the speed of light in a vacuum to the speed of light in the substance. The index of refraction of oils is characteristics within certain limits for each kind of oil. It is related to the degree of saturation but is affected by other factors such as free fatty acid content oxidation and heat treatment. This method is applicable to all normal oils and liquid fats86. The following general rules can be outlined regarding the index of refraction:  It increases together with the increase of molecular weight of the fatty acid.  It increases together with the increase of number of existing double bonds.  For any simple glyceride it is higher than that of the corresponding fatty acids or oil.  It increases when temperature increases by about 0.00038/ 0 C.

4.4.2.1 Principle: The refractive index [η] of a substance is defined as the ratio of the velocity of light in vacuum or air to that in the substance84: η=

Medium (I)

veiocity of light in substance veiocity of light in air

i>r

When a ray of light passes from air into a liquid its

direction is changed. This change of direction is called

refraction. The refractive index of a solid and liquid is conveniently

determined

when

ray

Medium (II) Figure–4.2: Refraction of light through Denser a denser liquid medium.

monochromatic light passes from a less dense to a

Medium II denser medium; it is bent or refracted towards the normal. Thus in figure if,(I) is the less dense and (II) the more dense medium, a ray of light passing from (I) to (II) will be bent so that the angle of refraction ‘r’ will be less than the angle of incidence ‘i’ ( i > r). And according to the law of refraction, the relation between these two angles will be such Sin i

that 59, 85 : Sin r = Where ‘n’ is the index of refraction of the less dense, and ‘N’ the index of refraction of the more dense medium. As the angle ‘i’ increases the angle ‘r’ also increases, and reaches its maximum volume ‘r’ when ‘i’ becomes equal to a right angle. That is, when the incident light is horizontal; since i =900 then, Sin i = Sin 900 = 1 The above equation becomes,

1 Sin r

Or, Sin r =

If i > 900 the ray is totally internally reflected in total internal reflection occurred in the medium concerned.

4.4.2.2 Instrument85:

Various Refractometers are used for determination of refractive index. Such as Abbe Refractometer,

Immersion

Refractometer,

Pulfrich

Refractometer,

Hilgerchange

Refractometer, Differential Refractometer, etc. But the best determination of refractive index of oils and fats by Refractometer, the Abbe Refractometer is one of them, this instrument required only a few drops of liquid, and the refractive index can be read very quickly. The usual prism covers the range 1.30 to 1.70 with an accuracy of ± 0.0002

Model of the instrument: ATAGO DR-A-1, Made in Japan 4.4.2.3 Procedure for determination of refractive index 100, 101: The instrument was placed in such a position that diffused day light or some form of artificial light (such as sodium light) can readily be obtained for illumination. The prism was cleaned carefully with alcohol and then with ether. The double prism was opened by means of the screw head and few drops of fatty oil placed on the prism. For solid fat the temperature should be suitably adjusted by circulating hot water. The prism was closed firmly by tightening the screw head. The instrument was allowed stand for few minutes before the reading was noted so that the sample and the instrument attained the same temperature. The alidade backward or forward was moved until the field of vision was divided into a light and dark portion by a line and this line appeared in the form of a band of colour. The screw head was rotated in such way that the colour band disappeared while a sharp colourless line was obtained. The reading was taken from the scale a second reading was taken a few minutes later to assure that temperature equilibrium was attained.

Table – 4.4: Refractive index [η]t˚C of Essential oil of Carum roxburghianum (Radhuni) seeds.

 Each value represents the average value from three experiments. 4.4.3 Determination of Optical rotation, [α]tD 76, 77, 84:

Sample collected from

Refractive index of Essential oil at 30˚C, [η]30˚C

Joydebpur, Gajipur Keranigonj, Dhaka

1.4885 1.5001

Faridpur

1.4930

The emerging beam of light having oscillation in a single plane is said to be plane polarized. When plane-polarized light is passed through certain solution of optical active substance, the plane of polarized light is rotated; the field of view appears alternately light and dark. A compound that can rotate the plane of polarized light is called optically active. This property of a compound is called optical activity. The prism, of which the light is polarized, is called the polarizer and the second prism by which the light is examined is called the analyzer. In order to obtain darkness, the analyzer has to be turned to the right, i.e. clock wise, the optically active substance is said to be dextrorotatory, and levorotatory when the analyzer must be turned to the left. By convention, rotation to the left is given a minus sign (–) and rotation to the right is given a plus sign (+). One complete rotation of the prism through 360 0, there are two positions of the analyzer, 1800 apart, at which the field is dark, and similarly, two positions at which there is a maximum of brightness. In determining the sign of the activity of a substance, one takes the direction in which the rotation required to give extinction is less than 900.  The angle of rotation depends on:  The nature of substance,  The length of the layer through which the light passes.  The wavelength of the light employed (The shorter the wavelength, the greater the angle of rotation).  The temperature. In order to obtain a measure of the rotator power of a substance, these factors must be taken into account and one then obtains the specific rotation. Most essential oil when placed in a beam of polarized light rotates the plane of polarized light to the right (dextrotatory) or to the left (laevorotatory). The extent of the optical activity of oil is determined by a polarimeter.

4.4.3.1 Specific Rotation: Specific rotation is defined as the angle of rotation produced by a liquid which in the volume of 1ml contains 1g of active substance, when the length of the column through which the light passes is 1dcm. The specific rotation is represented by (α) the observed angle of rotation being represented simply by ‘α’ conventionally a specific rotation is reported as [α] where‘t’ stands for temperature and for ‘D’ line of sodium used for determination.

t D

When the active substance is examined in solution, the concentration must be taken into account, is accordance with the expressions: α ×100 [α] tD = l ×c

Where, [α] = Specific rotation in degree α = Observed angle of rotation in degrees. l = Length of the sample solution in decimeters c = concentration of the sample solution in g/ml or, the number of grams of active substance in 100ml of solution,

Instrument model: WXG – 4, Made in: China 4.4.3.2 Procedure for determination of optical rotation: About 0.5g oil or fat materials was dissolved in 50ml chloroform to prepare 1% solution. The solution was put to a polarimeter tube so that no bubble remained in it. The light source was so adjusted that when the analyzer is at zero position the field appears bright. The analyzer was then rotated until the field was uniformly bright. This was the zero point of the instrument. This was determined by approaching from either side several times and the means of the reading were taken. The tube was then emptied cleaned and tilled with chloroform. Another set of reading with the chloroform was taken. The difference between the two readings gave the optical rotation. By this procedure, the value of optical rotation of sample essential oil was found:

Table – 4.5: Optical rotation of Essential oil of Carum roxburghianum (Radhuni) seeds.

 Each value represents the average value from three experiments. 4.4.4 Determination of solubility in different Strength of alcohol and other solvents 75, 86: Sample collected from

Optical rotation of Essential oil at 26˚C,

Joydebpur, Gajipur Keranigonj, Dhaka

+28˚ +25˚

Faridpur

+26˚

[α]D26˚C

As several industrial processes are based on the property that fatty acids and fats have of

dissolving property in some solvents, it is important to know how these oily substances to behave when they come into contact with these solvents. The most widely used solvents are hexane, isopropyl alcohol, acetone and water.

4.4.4.1 Procedure for determination of solubility: Introduce Exactly 1 cc of the oil into a test-tube, and was added slowly in small portions of alcohol (10 drops = 1 volume) of proper strength. The cylinder was shaking thoroughly after each addition. When a clear solution is first obtained, recorded the strength and the number of volumes of alcohol required was recorded. The addition of alcohol until 10cc has been added. If opalescent or cloudiness occurs during these subsequent additions of alcohol, record the point at which this phenomenon occurs. In the event that a clear solution is not obtained at any point during the addition of the alcohol, the determination was repeated, using an alcohol of higher strength.

Table–4.6: Solubility test of essential oil of Carum roxburghianum (Radhuni) Solubility in 60% alcohol 70% alcohol 80% alcohol 90% alcohol 100% alcohol Distilled water Chloroform CCl4 Pet-ether Diethyl ether

n – Hexane

Sample collected from Joydebpur, Gajipur Not soluble

Keranigonj, Dhaka Not soluble

Faridpur Not soluble

Cloudy up to 20 volume Soluble in 8.5 volume

Cloudy up to 20 volume soluble in 8.5 volume

Cloudy up to 20 volume soluble in 9 volume

soluble in 5 volume

soluble in 4.5 volume

Soluble at any volume

Not soluble

Soluble at any volume Soluble at any volume Soluble at any volume Soluble at any volume Soluble at any volume

in different (%) of alcoholic solution other solvent:  Each value represents the average value from three experiments

The solubility test of essential oil was carried out in a test tube with different percentage of alcoholic solvent such as: 90% alcohol, 80% alcohol, 70% alcohol & 60% alcohol. And other solvent like Chloroform, CCl4, Pet-ether, n- Hexane, Diethyl ether of the essential oil was determined at the same procedure. Solubility in alcohol of different strength of sample essential oil was

mentioned in

Table−4.6.

Remarks:  Insoluble in 60%, 70% alcohol and Distilled water at any volume.  Soluble in 80%, 90% alcohol at different volume.  Soluble in Chloroform, CCl4, Pet-ether, n- Hexane, Diethyl ether at any volume.

4.4.5 Colour Test59: The characteristic colour of most oils is predominantly a mixture of yellow and red and it is due primarily to the presence of pigments of a carotenoid type. Other colour like blue, green and brown are also not uncommon.

Table -4.7: Colour observation test for essential oil of C. roxburghianum (Radhuni) seeds: Sample collected from

Colour

Joydebpur, Gajipur

Slight yellowish

Keranigonj, Dhaka Faridpur

Slight yellowish Slight yellowish

4.4.6 Appearance at room temperature:

Essential oil of Carum roxburghianum (Radhuni) seeds is:

 Homogeneous,  Slight yellowish transparent liquid and  Lighter than water.  Bitter in taste

 Spicy odor - at room temperature (30˚C)

4.5 Determination of Chemical Properties of Essential oil: 4.5.1 Determination of Acid Value 83, 102: Most essential oil contain only small amount of free acids. Acid value denotes the number of mg of potassium hydroxide (KOH) needed to neutralize the free acids in 1 gm of fat or oil. Acid value indicates the proportion of free fatty acid in the oil or fat. The free fatty acid is produced by the hydrolytic decomposition of the oil. The low acid value is an indication of freshness of the oil and order the materials higher is the amount of the free acid.

•

Reagents: 

95 % aqueous neutral ethanol.

 0.1N (aq) (aq) NaOH solution  0.1N (aq) Na2CO3 solution  0.1N HCl  0.1%Methyl orange indicator  1% phenolphthalein solution.

4.5.1.1 Procedure for the Normality determination of 0.1N NaOH solution: a) Preparation of 0.1 N Na2CO3 Solution, b) Preparation of 0.1 N HCl solution, c) Standardization of HCl by primary standard Na2CO3 solution, d) Preparation of 0.1 N NaOH Solution, e) Standardization of NaOH by HCl,

4.5.1.1.a Preparation of 0.1 N Na2CO3 Solution: Analytical reagent quality sodium Carbonate (Na 2CO3.10 H2O) of 99.9% purity is obtained commercially. This contains a little moisture and must be dehydrated by heating at 260˚−270˚C for half an hour and allowed to cool in desiccators before use. Equivalent weight of Na2CO3 is 53.

1.325 gm of Na2CO3 was needed to prepare 250 ml of 0.1 N Na2CO3 solution. For this purposes 1.3463 g Na2CO3 was taken in 250 ml volumetric flask and it was diluted up to the mark. The strength of Na2CO3 =

1.3463 Ă&#x2014;0.1 = 0.1016N 1.325

4.5.1.1.b Preparation of 0.1 N HCl solution: 2.08 ml conc. HCl (12N) was taken from Burette in 250 ml volumetric flask and it was diluted up to the mark with distilled water.

4.5.1.1.c Standardization of HCl by 0.1 N Na2CO3 solution: 10 ml of 0.1N Na2CO3 solution was taken in a conical flask with the help of pipette. 50 ml distilled water and few drops of methyl orange added to the solution and the solution became yellowish colour. HCl was taken in a burette. HCl added drop wise into Na 2CO3 solution. At the end point of titration the colour of the solution became rosy colour. Titration was carried out exactly three times. The results are tabulated as follows. Reaction: Na2CO3 + 2HCl = 2NaCl + CO2 + H2O The results are tabulated as follows:

Table-4.8: Titration Data (Standardization of HCl by 0.1 N Na2CO3 solution): Observe. no.

Na2CO3 solution (ml)

Burette reading , Volume of HCl solution (ml) IBR FBR Difference Average volume

0.0

10.9

21.7

10.8

21.7

32.6

10.9

Calculation: As we know, V1S1 = V2S2 Here, V1 = Volume of Na2CO3 solution

= 10 ml

10.9

S1 V2

= Strength of Na2CO3 solution = Volume of HCl solution

= 0.1016 = 10.90

The strength of HCl solution,

S2 = =

10 Ă&#x2014; 0.1016 = 0.0932N 10.9

4.5.1.1.d Preparation of 0.1 N NaOH Solution: Molecular weight of NaOH = 23+16+1= 40 2.0 g of NaOH was taken in 500 ml volumetric flask and it was diluted up to the mark.

4.5.1.1.e Standardization of NaOH by HCl: 10 ml of 0.1N HCl was taken in a conical flask with the help of pipette. Few drops of 1% phenolphthalein added. The solution became colourless. NaOH solution was taken in a burette. NaOH added drop wise in to the HCl solution at the end point of titration the colour of the solution became pink. Titration was carried out exactly three times. The results are tabulated as follows. Reaction: HCl + NaOH = NaCl + H2O

Table-4.9: Titration data (Standardization of NaOH by HCl): Observe. no.

HCl solution (ml)

Burette reading , Volume of NaOH solution (ml) IBR FBR Difference Average volume

0.0

6.5

13.1

6.6

13.2

19.7

6.5

As we know, V1S1 = V2S2 Here, V1

= Volume of HCl

= Strength of HCl

= Volume of NaOH

The strength of NaOH solution,

= 10 ml = 0.0932 N = 6.5 ml

6.5

S2 = =

10 × 0.0932 = 0.14338 N 6.5

4.5.1.2 Neutralization of 95% alcohol: A few drops of 1% phenolphthalein added to the 95% alcohol. Then 0.1N NaOH solution added drop wise from burette until pink colour appeared. Then the alcohol was neutralized.

4.5.1.3 Procedure for the determination of acid value: About 0.5 g of Oil was taken in a conical flask. 50 ml of neutral 95% alcohol was added to the flask. Then the solution was warmed in a water bath and shacked in intervals until the oil substance has completely dissolved. The solution was titrated as hot as possible; titration was carried out quickly with 0.1 N aqueous alkali solutions (NaOH) by using 0.5 ml 1% phenolphthalein solution as indicator. The alkali was added slowly at a rate of about 30 drops per minute form a burette. During the titration, the contents of the flask were continuously agitated. The first appearance of red colouration that did not fade within 10 second’s was considered the end point and the volume of alkali required was recorded.

Calculation method: X ml of 0.1N NaOH ≡ X ml of 0.1 N KOH. 1000 ml of 1N KOH contains 56.1g of KOH Acid value

56.1×V NaOH × S NaOH Weight of sample, w gm

Table-4.10: Titration data for acid value of C. roxburghianum (Radhuni) Seeds essential oil: a) Joydebpur, Gajipur: Observ e no.

Weight of sample, w gm

1. 2. 3.

0.9886 0.7740 0.6315

Burette reading , Volume of NaOH Solution, VNaOH (ml) IBR FBR Diff. 0.1 0.50 1.0

0.6 0.95 1.40

0.50 0.45 0.40

Acid value,

Average

56.1 ×V NaOH × S NaOH Acid Weight of sample, wvalue, gm

4.3521 5.0021 5.4502

4.9148

Result: The acid value of C. roxburghianum (Radhuni), Joydebpur, Gajipur 4.9148

b) Keranigonj, Dhaka: Observe no.

Weight of sample, w gm

Burette reading , Volume of NaOH Solution, VNaOH (ml) IBR FBR Diff.

0.6545

2.00

2.55

0.55

2. 3.

0.6990 0.7080

3.0 3.70

3.65 4.35

0.65 0.65

Acid value,

Average

56.1 ×V NaOH × S NaOH Acid Weight of sample, wvalue, gm

7.2310 8.0012

7.7108

7.9001

Result: The acid value of C. roxburghianum (Radhuni), Keranigonj, Dhaka 7.7108

c) Faridpur: Observe no.

Weight of sample, w gm

0.9161

2. 3.

1.1852 0.7742

Burette reading , Volume of NaOH Solution, VNaOH (ml) IBR FBR Diff. 5.0 5.50 6.10

5.50 6.05 6.55

0.50 0.55 0.45

Acid value,

Average

56.1 ×V NaOH × S NaOH Acid value, Weight of sample, w gm

4.3901 4.4614 3.9931 5.0012

Result: The acid value of C. roxburghianum (Radhuni), Faridpur 4.4614

4.5.2 Determination of Ester value75: The esters value is a relative measure of the amount of ester present. The ester in the essential oil is expressed by the ester number. It is defined as the number of milligrams of KOH, required to saponify the ester present in 1 gm of the oil. The determination of the ester value is of great importance in the evaluation of many essential oil. The processes of saponification of ester may be represented by the following reaction. RCOOR' + NaOH RCOONa + R'OH Where R and R' may be an aliphatic, aromatic or alicyclic radical. â&#x20AC;&#x2DC;Râ&#x20AC;&#x2122;may also is a hydrogen atom.

Reagents: a) 95% alcohol. b) 0.l (N) aqueous KOH solution. c) 0.5 (N) alcoholic KOH solution. d) 0.5 (N) aqueous HCl solution. e) 1% alcoholic phenolphthalein indicator. d) 0.5 N Na2CO3 solution

4.5.2.1 Standardization of HCl by 0.5 N Na2CO3 solution: 10 ml of 0.5 N Na2CO3 solutions was taken in a conical flask with the help of pipette. 50 ml distilled water and few drops of methyl orange added to the solution and the solution became yellowish colour. 0.5N HCl was taken in a burette. HCl added drop wise into Na 2CO3 solution. At the end point of titration the colour of the solution became rosy colour. Titration was carried out exactly three times. The results are tabulated as follows. Reaction: Na2CO3 + 2HCl = 2NaCl + CO2 + H2O The results are tabulated as follows:

Table-4.11: Titration Data (Standardization of HCl by 0.5 N Na2CO3 solution):

Observe. no.

Na2CO3 solution (ml)

Burette reading , Volume of HCl solution (ml) IBR FBR Difference Average volume

0.0

9.1

9.10

18.2

9.1

18.2

27.3

9.1

Calculation: As we know, V1S1 = V2S2 Here, V1 = Volume of Na2CO3 solution = 10 ml S1 = Strength of Na2CO3 solution = 0.5050 N (see section-4.5.7.1.a) V2 = Volume of HCl solution = 9.1 ml The strength of HCl solution, S2 = =

10 Ă&#x2014; 0.5050 = 0.55801 N 9.1

So the strength of HCl acid solution was 0.55801 N

4.5.2.2 Procedure for determination of ester value: 1.5303 gm of the oil was weighed accurately into a 100 ml alkali resistant saponification flask. 5 ml of neutral 95% ethanol and 3 drops of 1% alcoholic phenolphthalein were added to the saponification flask. The free acids were neutralized with standard 0.1 N aqueous KOH solutions. Then 10 ml of 0.5 N alcoholic KOH was added which was measured accurately from a pipette. An air cooled condenser of 1 m in length and 1 cm in diameter was attached to the flask. The contents of the flask were refluxed for 1 hr on a steam bath. The flask was removed and permitted to cool at room temperature. The excess alkali was titrated with standard 0.5 N aqueous solution of HCl. In order to determine the amount of alkali consumed, a blank determination was carried out at the same condition in different experiments with different amounts of oil. The difference in the amounts of acid used in titrating the actual determination and the blank gave the amount of alkali used for the saponification of the esters.

Calculation method: The ester value of the oil was found calculated by the following formula: 28.05 × (B - S) × SHCl Ester value of essential oil = W Where, B = Blank determination (Volume of 0.5 N HCl in ml) S = Sample determination (Volume of 0.5 N HCl in ml) SHCl = Strength of HCl W= Weight of the sample taken in gm.

Table - 4.12: (Blank Titration with 0.5 N alcoholic HCl solution) Observe. no.

Burette reading ,Volume of 0.5 N HCl solution (ml) IBR FBR Difference Average volume

1. 0.0 11.25 11.25 2. 10.50 22.0 11.50 3. 22.0 33.25 11.25 Result: the blank determination of 0.5 N HCl, B = 11.25ml

11.25

Table-4.13: Titration data for Ester value of Carum roxburghianum (Radhuni) seeds essential oil: a) Joydebpur, Gajipur: Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

1. 2. 3.

0.8866 0.7678 0.6094

9.1 18.2 27.5

18.2 27.5 37.2

9.1 9.3 9.7

Ester value of essential oil 28.05 × (B - S) × SHCl W 37.9564 39.7531 39.8093

Average Ester value

39.1729

Result: The ester value of C. roxburghianum (Radhuni), Joydebpur, Gajipur 39.1729

b) Keranigonj, Dhaka. Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

1. 2. 3.

0.8328 0.7432 1.0159

0.0 9.3 19.0

9.3 19.0 28.1

9.3 9.7 9.1

Ester value of essential oil 28.05 × (B - S) × SHCl W 36.6496 32.6434 33.1256

Average Ester value

34.1395

Result: The ester value of C. roxburghianum (Radhuni), Keranigonj, Dhaka 34.1395

c) Faridpur: Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

1. 2. 3.

0.8299 0.8618 0.8161

0.0 8.9 20.0

8.9 17.7 28.8

Ester value of essential oil 28.05 × (B - S) × SHCl W 44.3210 46.3149 46.9912

8.9 8.7 8.8

Average Ester value

45.8757

Result: The ester value of Carum roxburghianum (Radhuni), Faridpur 45.8757

4.5.3 Determination of Alcohol and Ester Number after acetylation75: The alcoholic constituents of an essential oil were determined by acetylation. The oil is acetylized usually with acetic anhydride and the ester content of the resulting oil was determined from this value. The ester number after acetylation is numerically equal to the number of milligrams of KOH required to saponify, the ester present in 1 g of the acetylized oil75. The basic chemical reactions were involved in the determination may be summarized below:

R R'

R COOCH3 + NaOH

R''

COH + (CH3 CO)2O

R''

C–OH + CH3COONa

COOCH3 + CH3 COOH

R''

Where R, R' and R" may be a hydrogen atom, an aliphatic, aromatic or alicyclic radical. •

Reagents required:  Anhydrous sodium sulphate  Acetic anhydride  Distilled water  Anhydrous sodium acetate,

 95% alcohol  0.1N aqueous NaOH solution  0.5 N alcoholic NaOH solution  0.5 N aqueous HC1 solution  1% alcoholic phenolphthalein indicator.

4.5.3.1 Procedure of acetylation of essential oil75: 1.5588g ml of oil (Measured from a graduated cylinder), 10 ml of acetic anhydride (similarly measured) and 2 g of anhydrous sodium acetate were introduced into a 100 ml of acetylation flask. An air condenser was attached and the contents of the flask were boiled for 1 h on a steam bath. The flak was then permitted to cool for 15 min. and 50 ml of distilled water was introduced through the top of condenser. The flask was heated on a steam bath for several minute with frequent shaking to remove the excess of acetic anhydride. The contents of the flask were transferred to a separator funnel and the flask was thrice rinsed with 10 ml distilled water each time. Shaking was done thoroughly to assure good contact of the aqueous layer with the oil. When the liquid layers separated completely, the aqueous layer was removed and remaining organic oil layer was washed with 100 ml. portions of saturated salt solution repeatedly, until the oil was dried with anhydrous sodium sulphate and titration was done. Then the acetylized oil was saponified by using the procedure of the determination of esters.  The technique of determination of ester value has already described early in the Section- 4.5.2.2. So, in this section calculation data only have shown.

Calculation method:  Calculation for ester content of essential oil after acetylation: The ester content of the oil after acetylation can be calculated by the following formula: Ester content after acetylation = Where,

28.05 × (B - S) × SHCl W

B = Blank determination (Volume of 0.5 N HCl in ml) S = Sample determination (Volume of 0.5 N HCl in ml) SHCl = Strength of HCl W= Weight of the sample taken in gm.

Strength of HCl, SHCl =0.55801 N (see Section-4.5.2.1) Blank determination: Volume of 0.5 N HCl =11.25 ml (see section-4.5.2.2, table-4.12)

Table-4.14: Titration data for Ester value after acetylation of Carum roxburghianum (Radhuni) essential oil: a) Joydebpur, Gajipur. Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

1. 2. 3.

0.3644 0.3133 0.3579

0.0 10.3 20.7

10.25 20.65 30.9

10.25 10.35 10.2

Ester value of essential oil 28.05 × (B - S) × SHCl W 42.9545 44.9641 45.9121

Average Ester value

44.6102

Result: The ester value after acetylation of Carum roxburghianum (Radhuni), Joydebpur, Gajipur 44.6102.

b) Keranigonj, Dhaka: Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

1. 2. 3.

0.3826 0.4021 0.5401

0.0 10.3 20.7

10.25 20.55 30.6

10.25 10.25 9.9

Ester value of essential oil 28.05 × (B - S) × SHCl W 40.9121 38.9241 39.1205

Average Ester value

39.6522

Result: The ester value after acetylation of Carum roxburghianum (Radhuni), Keranigonj, Dhaka 39.6522.

c) Faridpur: Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

1. 2. 3.

0.5447 0.5845 0.4685

0.0 9.5 20.0

9.5 18.8 29.6

9.5 9.3 9.6

Ester value of essential oil 28.05 × (B - S) × SHCl W 50.2915 52.2150 55.1253

Average Ester value

52.5439

Result: The ester value after acetylation of Carum roxburghianum (Radhuni), Faridpur 52.5439.

4.5.3.2 Calculation for alcohol content75: If the original oil contains an appreciable amount of esters (as indicated by the ester number), the percentage of free alcohol in the original oil may be estimated or calculated by the following formula: % of alcohol =

Where,

( D - E) ×m 561.04 − 0.42 × ( D - E )

D = Ester number after acetylation E= Ester number m = Molecular wt. of the major alcohol =128 (as 5-Octen-1-ol, found in GCMS)

Table-4.15: Calculation data, for alcohol content of Carum roxburghianum (Radhuni) essential oil: Sample collected

Ester number, E

Ester number after acetylation, D

( D - E) ×m 561.04 − 0.42 × ( D - E )

Joydebpur,

39.1729

44.6102

1.2456

Gajipur Keranigonj,

34.1395

39.6522

1.2630

Dhaka Faridpur

45.8757

52.5439

1.5290

from

4.5.4 Determination of Aldehyde value75:

% of alcohol

Of the many procedures which have been suggested for the determination of aldehyde and ketones, only four general methods have attained practical significance. These are bisulfite method, the neutral sulfite method, the phenylhydrazine method, and the hydroxylamine methods75.

4.5.4.1 Hydroxylamine hydrochloride method: Two important techniques have been developed, both based up on the use of hydroxylamine for the determination of aldehydes and ketones. The first makes use of a solution of hydroxylamine hydrochloride and subsequent neutralization with standardize alkali of the hydrochloric acid liberated by the reaction. The second technique makes use of a solution of hydroxylamine; after the reaction with the aldehydes or ketones the mixture is titrated with standardize acid. The later procedure is known as Stillman-Reed method. Both modifications are based upon the fundamental reaction: Reaction: RCHO + NH2OH.HCI

RCH = NOH + H2O + HCl

R''

R'' C=O + NH2OH.HCI

C= NOH + H2O+ HCl R'

Reagents required:  0.5 N alcoholic KOH solution  0.5 N hydroxylamine hydrochloride solution  0.5 N HCl solution

4.5.4.2 Preparation of reagents: 4.5.4.2.a Preparation of bromophenol blue indicator: 44 mg of bromophenol blue indicator was dissolved in 96% ethanol.

4.5.4.2.b Preparation of 0.5 N Alcoholic KOH solutions: Molecular weight of KOH=39.1+16+1=56.1g. 15g of KOH pellets was dissolved in 8ml of distilled water and the solution was mixed with 500 ml of 96% (v/v) ethanol. The solution was allowed to stand for several hours. The clear supernatant liquid was filtered off and the filtered solution was kept in amber coloured bottle at dark place for further use. Using phenolphthalein indicator, the strength of KOH was determined by titrating it (KOH) by 0.5 N HCl Solution.

4.5.4.2.c Preparation of 0.5 N HCl solution: 20.82 ml conc. HCl (12N) was taken from Burette in 500 ml volumetric flask and it was diluted up to the mark with distilled water.

4.5.4.2.d Recrystallization of NH2OH.HCl: NH2OH.HCI crystals were dissolved in a minimum amount of rectified sprit by heating over steam bath and heating was continued for 2h and the solution allowed to cooling. The solid crystals of NH2OH.HCl formed were then filtered off by means of a suction pump. The residue was taken in a watch glass and then it was dried by inserting it in desiccators. Thus recrystallized NH2OH.HCl was obtained.

4.5.4.2.e Preparation of 0.5 (N) Hydroxylamine hydrochloride (NH2OH.HCl) Solution: About 9.0 g of recrystallized NH 2OH.HC1 was taken in a 250 ml volumetric flask and was dissolved in 219 ml of 60% ethanol. About 2â&#x20AC;&#x201C;3 ml of bromophenol blue indicator was added to the solution. Then 0.5 N KOH alcoholic solution was added drop wise to make the solution greenish in colour, such that one drop of 0.5 (N) HCl solution would change the colour in to yellow. Then the resulting solution was made up to the mark of the volumetric flask with distilled water.

4.5.4.3 Standardization of alch. KOH by 0.5 (N) HCl: 10 ml of HCl was taken in a conical flask with the help of pipette. Few drops of 1% phenolphthalein added. The solution become colourless KOH solution was taken in a burette. KOH was added drop wish into the HCl solution. At the end of the titration the colourless solution became pink. The result is tabulated as follows:

Observe. no.

HCl solution (ml)

Burette reading , Volume of KOH solution (ml) IBR FBR Difference Average volume

0.0

11.1

22.2

11.1

22.2

33.4

11.2

11.1

Table-4.16: Titration data (Standardization of alch. KOH by 0.5 (N) HCl): As we know, V1S1 = V2S2 Here, V1 = Volume of HCl

= 10 ml

S1 = Strength of HCl V2 = Volume of KOH

= 0.55801 N (see section-4.5.2.1) = 11.1 ml

The strength of alcoholic KOH solution, S2 = =

10 ×0.55801 = 0.50271 N 11.1

4.5.4.4 Procedure for the Determination of Aldehyde Value75: Accurately 0.5001 g of oil was taken in a conical flask. Then 35 ml of 0.5 N NH 2OH.HCI solutions was added to it. The mixture was kept for 24 hour then the mixture was titrated by means of alcoholic 0.5 N KOH solutions until the greenish shade appeared. To another 250 ml of conical flask, 35 ml of 0.5 N NH 20H.HCl was taken for colour match by the blank titration.

Calculation method: The Aldehyde value was calculated by the following formula: % of aldehyde =

Where,

(SKOH − BKOH ) × N KOH × M 20 × W

SKOH = Vol. of alch.KOH solution required for titration of the sample BKOH = Vol. of alch.KOH solution required for titration of the blank NKOH = Strength of alcoholic KOH. M= Molecular weight of Aldehyde (the major % of aldehyde)

W = Weight of the sample taken in gm. Calculation: Strength of alcoholic KOH, N = 0.5027 N Molecular weight of major aldehyde or ketone, M = 190 (As Dihydrocoumarin- 5, 7, 8-trimethyl- found in GCMS)

Table-4.17: Titration data, of Aldehyde Value of Carum roxburghianum

(Radhuni) essential oil: a) Joydebpur, Gajipur. Observe no.

Weight of sample, w gm

Burette reading , Volume of alcoholic KOH Solution, V(ml) IBR FBR Diff.

% of Aldehyde, (SKOH

Average

− BKOH ) × N KOH ×Aldehyde M 20 × W

value, %

5.7688

0.5381

0.0

1.45

0.5786

1.45

2.95

1.5

5.9867

0.5584

2.95

4.45

1.5

5.9867

5.8444

Result: The essential oil of Carum roxburghianum (Radhuni), Joydebpur, Gajipur contained Aldehyde value 5.8444.

b) Keranigonj, Dhaka. Observe no.

Weight of sample, w gm

Burette reading , Volume of alcoholic KOH Solution, V(ml) IBR FBR Diff.

% of Aldehyde, (SKOH

Average

− BKOH ) × N KOH ×Aldehyde M 20 × W

value, %

7.08031

0.5396

5.0

6.6

1.6

0.6001

6.6

8.2

1.6

6.3665

0.5896

8.5

10.15

1.65

6.8848

6.7772

Result: The essential oil of Carum roxburghianum (Radhuni), Keranigonj, Dhaka contained Aldehyde value 6.7772

c) Faridpur. Observe no.

Weight of sample, w gm

0.5399

11.0

12.75

1.75

0.6001

12.8

14.5

1.70

7.1623

0.5299

14.5

16.25

1.75

8.5617

Burette reading , Volume of alcoholic KOH Solution, V(ml) IBR FBR Diff.

Average − BKOH ) × N KOH ×Aldehyde M value, 20 × W %

% of Aldehyde, (SKOH

8.4032 8.0424

Result: The essential oil of Carum roxburghianum (Radhuni), Faridpur contained Aldehyde value 8.0424

4.5.5 Determination of Phenol content75:

Phenols, as we know, contain one or more than one (OH) groups attached directly to the benzene ring, therefore, differ from alcohols in many respects. Phenols are weak acids forming salts with bases and are thus distinct from aliphatic alcohols which are neutral. But the aromatic alcohols which have the (OH) group in the side chain like benzyl alcohol arc neutral. Phenols react with the alkali hydroxides giving rise to water-soluble phenolates. This is the basis of the classical method for the estimation of phenols in essential oils. Since the potassium salts of many phenols are more soluble than the corresponding sodium salts, the use of potassium hydroxide is preferred75.

4.5.5.1 Procedure for determination of phenol content: Into a well cleaned Cassia flask (capacity 150 ml) 5 ml of the essential oil sample was introduced. To it 37.50 ml of 1 N KOH was introduced by a measuring cylinder. The flask was then Stoppard and shaken well for 5 minutes and kept aside for one hour. The unreacted oil was forced into the graduated portion of the neck of the Cassia flask through displacement effected by the addition of excess K.OH solution, scrupulously avoiding disturbing the layer of the separated oil. This was facilitated by keeping the Cassia flask in a inclined position in a clamp during the slow flow down of the alkali along the inner wall of the neck of the flask. One drop/sec of the alkali flow ensure a clear sharp line separation of oil. Occasionally droplets of oil adhered to the sides of the flask. These need to be dislodged and driven to the above column of oil in the neck by gently tapping the flask and revolving it rapidly between the palms of hands. The volume of unreacted oil is noted and the results calculated like in eugenol determination75.

Calculation method: % of Phenol = 10 (10 â&#x20AC;&#x201C; no of ml of undissolved oil)

Table-4.18: Calculation data for phenol content of Carum roxburghianum (Radhuni) essential oil:

a) Joydebpur, Gajipur. Observe no.

ml of undissolved oil

% of Phenol, 10 (10 â&#x20AC;&#x201C; no of ml of undissolved oil)

Average

phenol content, %

1. 2. 3.

4.5 4.3 4.35

55 57 56.5

56.1667

Result: The phenol content of Carum roxburghianum (Radhuni), Joydebpur, Gajipur 56.1667 %.

b) Keranigonj, Dhaka: Observe no.

ml of undissolved oil

% of Phenol, 10 (10 â&#x20AC;&#x201C; no of ml of undissolved oil)

Average

phenol content, %

1. 2. 3.

4.65 4.55 4.55

53.5 54.5 54.5

54.1667

Result: The phenol content of Carum roxburghianum (Radhuni), Keranigonj, Dhaka 54.1667 %.

c) Faridpur:

Observe no.

ml of undissolved oil

% of Phenol, 10 (10 – no of ml of undissolved oil)

Average

phenol content, %

3.9

61.0

3.85

61.5

3.8

62.0

61.5

Result: The phenol content of Carum roxburghianum (Radhuni), Faridpur 61.5 %.

4.5.6 Determination of Iodine Value75, 83, 84, 86: Iodine value is expressed in gm of iodine absorbed by 100 gms of oil or fat. It gives the indication of degree of unsaturation of the constituent oil and is thus a relative measure of the unsaturated bonds present in the oil. The iodine value is a characteristic of oil. Unsaturated compounds absorb iodine (in soluble form) and form saturated compounds. One double bond absorbs one molecule of iodine. The amount of iodine absorbed in percentage is the measure of unsaturation in the oil. Oils are classified as drying, semi-drying and non-drying on the basis of iodine value. The use of iodine numbers for the evaluation of essential oil has never attained practical significance. It has been shown frequently that the iodine numbers of many essential oil nary with the size of sample as well as with the period of contact with the reagent. Unsaturated compounds absorb iodine as flows. Reaction: CH2–COO(CH2)7CH=CH–(CH2)7CH3

CH2–COO(CH2)7CHI−CHI–(CH2)7CH3

CH–COO(CH2)7CH=CH–(CH2)7CH3 + 3I2 = CH–COO(CH2)7CHI–CHI–(CH2)7CH3 CH2–COO(CH2)7CH=CH–(CH2)7CH3

Or, C57H104O6

3I2

57×12+1×104+16×6

3×127×2

=884

=762

CH2–COO(CH2)7CHI−CHI–(CH2)7CH3 =

C57H104I6O6

So, 884gms oleic acid reacts with 762 gm iodine. Iodine value of oleic acid =

762 ×100 = 86.2 884

Of the many procedure that have been proposed for determination the Iodine value four methods are better known than all others. These are the methods of Wijs and Hanus, Hubl and Rosenmund, Kuhnuhenn method. The Wijs and Hanus method, especially the former, is the most widely used of all. The International union of pure and applied chemistry approved wijs, Hanus and Hubl methods for the iodine value determination. The iodine value of sample oil was determined here by using Hanus method.

• Reagents required:            

15 % KI Solution. 0.1 N Na2S2O3 Solution 0.1N K2Cr2O7 Solution Starch indicator Hanus solution Pure iodine. Glacial acetic acid Solid sodium bicarbonate (NaHCO3) Bromine Chloroform (CHCl3) Solid KI Conc. HCl

4.5.6.1 Preparation of Reagents for iodine value: 4.5.6.1.a O.1 N Na2S2O3 solution preparation: Sodium thiosulphate (Na2S2O3.5H2O) is readily obtainable in a state of high purity, but there is always some uncertainty as to the exact water content because of the efflorescent nature of the salt and for other reasons. The substance is therefore unsuitable as primary standard. It is a reducing agent by virtue of the half-cell reaction: 2S2O32− S4O6 2− + 2e− The equivalent weight of sodium thiosulphate penta hydrate (Na2S2O3.5H2O) is 248. So, 24.8gm of A.R. crystallized sodium thiosulphate pentahydrate (Na2S2O3. 5H2O) was taken in 1liter of volumetric flask and it was diluted up to the mark with distilled water. Two or three drops of chloroform added to it. The solution must be stored in amber or yellow glass Stoppard bottle. As Na2S2O3 is not a primary standard substance. It must be standardized by potassium dichromate (K2Cr2O7) solution that is a primary standard substance.

4.5.6.1.b Preparation of 0.1N K2Cr2O7 solution: Molecular weight of K2Cr2O7 = (39 × 2 + 52×2+16×7) gm = 294g Oxidation Number of K2Cr2O7 = 6

Equivalent weight of K2Cr2O7 = 294/6 = 49 1.225g of K2Cr2O7 is required to prepare 0.1N K2Cr2O7 Solution in 250ml water. So, 1.2487 gm of K2Cr2O7 was taken in 250ml volumetric flask and it was diluted up to the mark with distilled water. So, the strength of K2Cr2O7 Solution =

1.2487 ×0.1 = 0.10193 N 1.225

4.5.6.1.c 15% KI solution preparation: About 15 gm of dry A.R. potassium iodide was weighed out and then it was dissolved in 100ml of distilled water in the titration vessel. It was stored in amber colour glass Stoppard bottle.

4.5.6.1.d Preparation of starch solution: 1 gm of soluble starch was poured in 100ml of distilled water and stirred rapidly. It was heated till boiling and kept on boiling 1 minute. The solution was allowed to cool and 2-3 gm of potassium iodide (K1) was added. For long storage 1 gm of boric acid was added and taken a clear solution from it. The solution was kept in a Stoppard bottle and preserved in a refrigerator at 4˚ to 10˚C.

4.5.6.1.e Preparation of Hanus solution: 13 gm of pure resublimed iodine was added to glacial acetic acid by warming over water bath. When the iodine was completely dissolved the solution was cooled and diluted to 1000ml with glacial acetic acid. Now 3ml of pure (sulfur free) Br2 was added to the solutionstored it in amber colour Stoppard bottle. The whole operation was conducted in a fume hood.

4.5.6.2 Precaution: Acetic acid has a relatively high temperature coefficient of expansion so that blank titration made at different periods of the day may vary significantly due to changes in temperature within the laboratory.

4.5.6.3 Standardization of Na2S2O3 solution by 0.1 N K2Cr2O7 solutions: 10 ml 15% potassium iodide (KI) solution, 2 gm of sodium bicarbonate (NaHCO3) were taken in a 500ml Stoppard conical flask and 100ml of distilled water was added in it to dissolve the solids. Then 6ml of concentrated HCl added. 10 ml of 0.1 N K 2Cr2O7 added to the conical flask. The conical flask was kept in a dark room for fifteen minutes. The solution became deep brown in colour. Then 200ml of the distilled water was added to the solution. Earlier sodium thiosulfate (Na2S2O3) solution was taken in a burette. Titration was carried out by adding sodium thiosulfate (Na2S2O3) solution from burette to the solution kept in conical flasks. When deep brown colour changed into yellowish green colour, then 1 ml starch solution was added. The colour quickly changed into deep blue, at this stage titration was carried out with special care by adding sodium Thiosulfate (Na 2S2O3) solution drop by drop. At the end point the colour of the solution changed greenish blue to light green.

Table-4.19: Titration data (Standardization of Na2S2O3 solution): Observe No.

Volume of K2Cr2O7 solution (ml)

1. 2. 3.

10 10 10

Burette reading ,volume of Na2S2O3 solution (ml) IBR FBR Diff. Average 0.0 6.35 12.65

6.35 12.65 19.0

6.35 6.30 6.35

6.35

Calculation: As we know, V1S1 = V2 S2 Here, V1 = Volume of K2Cr2O7 solution = 10 ml V2 = Volume of Na2S2O3 solution = 6.35 ml S1 = Strength of K2Cr2O7 solution = 0.10193 N The strength of Na2S2O3 solution, S2 = =

10 ×0.10193 = 0.1605 N 6.35

4.5.6.4 Procedure for the determination of Iodine value: About 0.5 gm of fatty oil was taken in a well Stoppard conical flask. The oil was dissolved in 10 ml of chloroform. 25 ml of Hanus solution was added to the Stoppard conical flask from a pipette and the solution was allowed to stand for half an hour in dark place with occasional shaking. At the end of this period, 100 ml of distilled water was added followed by 10ml of 15% KI solution. The solution was titrated with standard 0.1 N Na 2S2O3 solutions. The sodium thiosulphate was gradually added with constant shaking, until yellow colour of the solution was almost disappeared. Few drops of starch solution were added and the titration was continued until the dark violate colour was entirely disappeared. The blank determination was also carried out observing the same condition but omitting the oil.

Calculation method: Iodine value was determined by following formula: (B −S) × N ×12.69 Iodine value = W Where, B = Volume of NaS2O3 required for the blank titration S=

Volume of Na2S2O3 required for the sample

Strength of the Na2S203 solution

Wt of the fatty oil taken in gm

Table-4.20: (Blank Titration with Na2S2O3 solution) Observe. no.

Volume of Hanus soln. (ml)

Burette reading , Volume of 0.1 N Na2S2O3 solution (ml) IBR FBR Difference Average volume

0.0

49.7

0.0

49.75

0.0

49.7

Result: The blank determination of 0.1 N Na2S2O3 solution, B = 49.7 ml

Table-4.21: Titration data of iodine value of Carum roxburghianum (Radhuni) essential oil: a) Joydebpur, Gajipur: Observe no.

1. 2. 3.

Weight of sample, w gm 0.4433 0.4637 0.4717

Burette reading , Volume of 0.1 N Na2S2O3 solution, S(ml) IBR

FBR

Diff.

0.0 0.0 0.0

37.25 36.5 36.3

Iodine value, Average Iodine (B − S) × N ×12.69 value W 57.2016 47.9825 57.8596

57.6812

Result: The iodine value of Carum roxburghianum (Radhuni), essential oil, Joydebpur, Gajipur 57.6812

b) Keranigonj, Dhaka: Observe no.

Weight of sample, w gm 0.4805

Burette reading , Volume of 0.1 N Na2S2O3 solution, S(ml) IBR

FBR

Diff.

0.0

37.2

Iodine value, Average Iodine (B − S) × N ×12.69 value W 52.9850

2. 3.

0.4918 0.4725

0.0 0.0

37.4 38.1

50.9354 50.0012

51.3072

Result: The iodine value of Carum roxburghianum (Radhuni), essential oil, Keranigonj, Dhaka 51.3072

c) Faridpur: Observe no.

1. 2. 3.

Weight of sample, w gm 0.5035 0.5305 .5263

Burette reading , Volume of 0.1 N Na2S2O3 solution, S(ml) IBR

FBR

Diff.

0.0 0.0 0.0

38.3 38.7 38.85

Iodine value, Average Iodine (B − S) × N ×12.69 value W 45.9326 42.2354 41.9926

43.3869

Result: The iodine value of Carum roxburghianum (Radhuni), essential oil, Faridpur 43.3869

4.5.7 Determination of the Saponification value 83, 84, 86, 91: Saponification is the conversion of glycerol or ester into soap. This term is often used to describe the hydrolysis of any ester by alkali. From the study of the composition of oils and fats, it becomes apparent that proportion and nature of different fatty acids differ in various oils and fats. As the amount of alkali required to saponify a given amount of oil depends upon the molecular weights of the fatty acids present in the oil or the fat. The saponification value tells us whether and oil of fat contains lower of higher proportion of same fatty acids and also whether it contains high proportion of lower fatty acids or higher fatty acids. Saponification value is expressed in number of milligrams of caustic potash (KOH) required to saponify one gram of oil or fat. This and the neutralization value are measures for determining the average molecular weights of fatty substances. This method depends on the reaction expressed by this equation. CH2COOR

CH2COOR

CHOOOR + 3 KOH

CHOOOR

CH2COOR

+ 3RCOOK

Saponification is done by excess of alcoholic potash on a given weight of oil or fat boiling under reflux and the excess of KOH neutralized by standard acid. The amount of potash necessary to saponify the given amount of oil was thus determined.

•

Reagents required: a) 0.5N Alcoholic KOH solution b) 0.5N HCl solution c) 0.5N Na2CO3 solution d) 1 % Phenolphthalein e) 0.1 % Methyl orange indicator.

4.5.7.1 Procedure for the Normality determination of 0.5 N HCl Solutions: a) Preparation of 0.5 Na2CO3 Solution b) Preparation of 0.5 N HCl solution c) Standardization of HCl by primary standard Na2CO3 solution

4.5.7.1. a Preparation of 0.5 N Na2CO3 Solution: Analytical reagent quality sodium Carbonate (Na 2CO3.10 H2O) of 99.9% purity is obtained commercially. This contains a little moisture and must be dehydrated by heating at 260˚– 270˚C for half an hour and allowed to cool in desiccators before use. Equivalent weight of Na2CO3 is 53. 2.65 gm of Na2CO3 needed to prepare 100 ml of 0.5 N Na 2CO3 solution. For this purposes 2.6494 g Na2CO3 was taken in 100 ml volumetric flask and it was diluted up to the mark. The strength of Na2CO3 =

2.6494 ×0.5 2.65

= 0.49989 N

4.5.7.1.b Preparation of 0.5 N HCl solution: 20.82 ml conc. HCl (12N) was taken from Burette in 500 ml volumetric flask and it was diluted up to the mark with distilled water.

4.5.7.1.c Standardization of HCl by 0.5 N Na2CO3 solution: 10 ml of 0.5 N Na2CO3 solutions was taken in a conical flask with the help of pipette. 50 ml distilled water and few drops of methyl orange added to the solution and the solution became yellowish colour. 0.5N HCl was taken in a burette. HCl added drop wise into Na 2CO3 solution. At the end point of titration the colour of the solution became rosy colour. Titration was carried out exactly three times. The results are tabulated as follows. Reaction: Na2CO3 + 2HCl = 2NaCl + CO2 + H2O The results are tabulated as follows:

Table-4.22: Titration Data (Standardization of HCl by 0.5 N Na2CO3 solutions):

Observe. no.

Na2CO3 solution (ml)

1. 2. 3.

10 10 10

Burette reading , Volume of HCl solution (ml) IBR FBR Difference Average volume 0.0 9.0 9.0 9.0 18.1 9.1 9.0 18.1 27.1 9.0

Calculation: As we know, V1S1 = V2S2 Here, V1 = Volume of Na2CO3 solution = 10 ml S1 = Strength of Na2CO3 solution = 0.49989 N V2 = Volume of HCl solution = 9.0 ml The strength of HCl solution, S2 = =

10 Ă&#x2014; 0.49989 = 0.55542 N 9.0

4.5.7.2 Preparation of 0.5 N Alcoholic KOH solution: Molecular weight of KOH=39.1+16+1=56.1 15 gm of KOH pellets was dissolved in 8ml of distilled water and the solution was mixed with 500 ml of 96% (v/v) ethanol. The solution was allowed to stand for several hours. The clear supernatant liquid was filtered off and the filtered solution was kept in amber coloured bottle at dark place for further use. Using phenolphthalein indicator, the strength of KOH was determined by titrating it (KOH) by 0.5 N HCl Solution.

4.5.7.3 Procedure for the determination of saponification value: About 0.5 g of Oil or fat was taken into a 250 ml quick fit round bottom flask of Pyrex glass (alkali resistant). Exactly 25 ml of 0.4606 N alcoholic KOH solutions added into the flask and the reflux condenser was connected to the R.B flask the flask kept in a boiling water bath and boiled until the oil was completely saponified. The saponification was carried out for 1 hour with occasional shaking. The condenser was washed down with a little water 0.5 ml of 1% alcoholic phenolphthalein indicator was added and the warm soap solution was titrated with 0.5 N aqueous HCl acid solution. The blank titration of the alcohol KOH solution was carried out at the same condition. The difference between the volumes of HCl required in the two titration will correspond to the amount of alkali used in the saponification.

Calculation method: Saponification value =

56.1 × N × (B - S) W

Where, B = ml of 0.5 N HCl required for blank. S = ml of 0.5N HCl required for Sample N = Strength of HCl (N) W = Wt. of Sample (gm)

Table - 4.23: (Blank Titration with 0.5 N HCl solution) Observe. no. 1. 2. 3.

Volume of KOH, (ml) 25 25 25

Burette reading , Volume of 0.5N HCl solution (ml) IBR FBR Difference Average volume 0.0 28.6 28.6 28.6 0.0 28.5 28.5 0.0 28.6 28.6

Result: the blank determination of 0.5 N HCl, B = 28.6 ml

Table-4.24: Titration data for saponification value of Carum roxburghianum (Radhuni),essential oil: a) Joydebpur, Gajipur. Observe no.

Weight of sample, w gm

0.6042

0.0

26.5

0.7345

0.0

26.0

110.2956

0.8266

0.0

25.7

109.3124

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff.

Saponification Average value, Saponification 56.1 × N × (B - S) value, W

108.2986 109.3022

Result: The saponification value of Carum roxburghianum (Radhuni), essential oil, Joydebpur, Gajipur 109.3124.

b) Keranigonj, Dhaka: Observe no.

Weight of sample, w gm

0.6208

0.0

26.5

0.9431

0.0

25.5

102.4213

1.1172

0.0

24.9

103.1932

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff.

Saponification Average value, Saponification 56.1 × N × (B - S) value, W

105.3927 103.6690

Result: The saponification value of Carum roxburghianum (Radhuni), essential oil, Keranigonj, Dhaka 103.6690.

c) Faridpur: Observe no.

Weight of sample, w gm

1.7456

0.0

24.1

1.0235

0.0

25.9

82.1954

1.2078

0.0

25.3

85.1341

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff.

Saponification Average value, Saponification 56.1 × N × (B - S) value, W

80.3219 82.5505

Result: The saponification value of Carum roxburghianum (Radhuni), essential oil, Faridpur 82.5505.

4.5.8 Determination of saponification value of essential oil after Acetylation75, 91: The procedure of acetylation has already described early in the Section-4.5.3.1 and same procedure has also followed in this section. The technique of determination of saponification value has described in the section-4.5.7.3. So, in this section calculation data only have shown.

Calculation method: Saponification value =

56.1 × N × (B - S) W

Where, B = ml of 0.5 N HCl required for blank = 29.1 ml (see as table – 4.23) S = ml of 0.5 N HCl required for Sample N = Strength of HCl (N) = 0.55801 N (see section – 4.5.2.1) W = Wt. of Sample (gm)

Table-4.25: Titration data for saponification value after acetylation of Carum roxburghianum (Radhuni) essential oil: a) Joydebpur, Gajipur. Observe no.

Weight of sample, w gm

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff.

Saponification Average value, Saponification 56.1 × N × (B - S) value, W

1. 0.2449 0.0 27.6 27.6 2. 0.2654 0.0 27.5 27.5 3. 0.3027 0.0 27.3 27.3 Result: The saponification value after acetylation of essential oil, Joydebpur, Gajipur 188.8751.

191.7376 188.8751 188.7345 186.1532 Carum roxburghianum (Radhuni),

b) Keranigonj, Dhaka. Observe no.

Weight of sample, w gm

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff.

1. 0.2232 0.0 28.1 28.1 2. 0.1758 0.0 28.3 28.3 3. 0.2470 0.0 28.0 28.0 Result: The saponification value after acetylation essential oil, Keranigonj, Dhaka 140.6926.

c) Faridpur.

Saponification Average value, Saponification 56.1 × N × (B - S) value, W

140.2345 140.6926 142.4219 139.4215 of Carum roxburghianum (Radhuni),

Result: The saponification value after acetylation of Carum roxburghianum (Radhuni), essential oil, Faridpur 123.4121.

4.5.9 Determination of Unsaponifiable Matter59, 66, 86: Oil derived from natural sources contain small amount of dissolved Unsaponifiable matter. Mineral oil contamination, higher aliphatic alcohols, sterols, pigments and hydrocarbons are the materials of this nature. The determination of unsaponifiable matter is important to know the purity and quality of the sample. The term ‘‘unsaponifiable matter’’ is now generally understood to indicate that material present in oils and fats which after saponification of the oil or fat by caustic alkali and extraction by the solvent specified (under the conditions detailed in the description of the method of the society of public analysis and other analytical chemists) remains non volatile on drying at 80˚C unsaponifiable matter as defined above includes, interalia, hydrocarbons and higher alcohol, cholesterol and phytosterol. The method of determination aims at the exclusion of free fatty acids, soap free fat, mineral matter and glycerol and readily volatile substances. The methods are generally based on the preliminary saponification of the oil with caustic alkali and subsequent extraction of the soap so formed by means of solvent. Later on aqueous alcoholic solution of the soap was extracted with light petroleum; the extract was washed with water and alkali to remove soap and evaporated to yield the unsaponifiable matter.

• Reagents required:  0.5N Alcoholic KOH solution, colour not darker than pale yellow.  Diethyl ether (Sp.gr. - 0.720-0.724 at 15.5° C)  1% Phenolphthalein indicator

4.5.9.1 Procedure for the determination of Unsaponifiable Matter 86, 104: An accurately weighed 1.0 gm of oil or fat was taken into a 250 ml R.B. flask and 25 ml of alcoholic KOH was added to it. The flask was attached to a refluxing condenser and heated on a boiling water bath for an hour. The contents of flask were occasionally stirred to mix the solution properly and to ensure complete saponification. After completion of saponification, the flask was removed from the bath, the condenser was detached and the content of the flask was transferred to a 250 ml separating funnel. The solution was washed with 50 ml of distilled water. Then the flask was rinsed with 50 ml of diethyl ether and this ether was poured cautiously into the separating funnel. The funnel was covered and shaken vigorously while the contents were still slightly warm. After shaking for about 30 seconds the funnel was then suspended and left stationary till there appeared two distinct layers in the liquid mixture. The upper ethereal layer was collected in a R.B. flask by transferring the lower aqueous layer of the solution into another flask. The aqueous layer of Observe no.

1. 2. 3.

Weight of sample, w gm 0.2746 0.2537 0.3094

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff.

Saponification Average value, Saponification 56.1 × N × (B - S) value, W

0.0 0.0 0.0

28.0 28.1 27.9

125.4219 123.3925 121.4220

123.4121

the soap solution was extracted twice more with ether in the similar way. The ether extract were then combined 30 ml of distilled water and then 20 ml of 0.5 N aqueous KOH solutions. After one or other of these preliminary treatments, the ethereal solution was washed twice with 20 ml of water. It was shacked vigorously on each occasion. And then successively washed with 20 ml of 0.5 N aqueous KOH solutions, 20 ml of distilled water and again with 20 ml of 0.5 N aqueous KOH solution and at least twice more with 20 ml of distilled water. Washing with water was continued until the wash water no longer turned pink, on addition of phenolphthalein indicator. The ethereal solution was transferred to a sample flask and ether was evaporated from the solution. Then the flask being almost entirely immersed, hold obliquely and rotated in a boiling water bath. When the flasks become dried and weight (with its contents) was constant at 80°C. The extract was dissolved in 10 ml of freshly boiled and neutralized 95% ethanol and titrated with the 0.1 N alcoholic sodium hydroxide solutions, using phenolphthalein indicator.

Calculation method: The Unsaponified matter was found by the following formula. 100 × W1 Unsaponified matter (% by weight) = W Where,W1 = Weight of residue (gm). W = Weight of sample taken (gm)

Table-4.26: Calculation table for Unsaponifiable matter of Carum roxburghianum (Radhuni) essential oil: a) Joydebpur, Gajipur. Observe no.

Weight of sample, w gm

Weight of residue, W1,gm

1 1.0852 0.0112 2 1.1035 0.0115 3 1.3328 0.0143 Result: The Unsaponifiable matter of Joydebpur, Gajipur 1.0490 %.

Unsaponified matter, %

Average

Unsaponified matter, %

100 × W1 W 1.0322 1.0421 1.0490 1.0729 Carum roxburghianum (Radhuni), essential oil,

b) Keranigonj, Dhaka. Observe no.

Weight of sample, w gm

Weight of residue, W1,gm

Unsaponified matter, %

Average

Unsaponified matter, %

100 × W1 W 1 1.1740 0.0226 1.9251 2 0.0321 1.6947 1.8941 1.8971 3 1.8802 0.0352 1.8721 Result: The Unsaponifiable matter Carum roxburghianum (Radhuni), essential oil, Keranigonj, Dhaka 1.8971 %.

c) Faridpur. Observe no.

Weight of sample, w gm

Weight of residue, W1,gm

Unsaponified matter, % 100 × W1 W

1 2 3

1.8235 1.8232 1.6997

0.0192 0.0188 0.0182

1.0529 1.0311 1.1032

Average

Unsaponified matter, %

1.0624

Result: The Unsaponifiable matter of Carum roxburghianum (Radhuni), essential oil, Faridpur 1.0624 %.

4.5.10 Determination of Peroxide Value 56, 66, 86, 87: The peroxide value is the number of mili-equivalent of active oxygen that expresses the amount of peroxide contained in 1000g of the substance or peroxide oxygen per 1 kilogram of fat or oil. The sample is treated with potassium iodide and the iodine which was liberated by the peroxides was treated with sodium thiosulphate solution. The Peroxide value of an oil or fat is used as a measurement of the extent to which rancidity reactions have occurred during storage. Other methods are available but peroxide value is the most widely used. The double bonds found in fats and oils play a role in autoxidation. Oils with a high degree of unsaturation are most susceptible to autoxidation. The best test for autoxidation (oxidative rancidity) is determination of the peroxide value. Peroxides are intermediates in the autoxidation reaction. Autoxidation is a free radical reaction involving oxygen that leads to deterioration of fats and oils which form off-flavours and off-odours. Peroxide value, concentration of peroxide in an oil or fat, is useful for assessing the extent to which spoilage has advanced87. The peroxide value is determined by measuring the amount of iodine which is formed by the reaction of peroxides (formed in fat or oil) with iodide ion. 2 I− + H2O + ROOH = ROH + 20H− + I2

Note that the base produced in this reaction is taken up by the excess of acetic acid present. The iodine liberated is titrated with sodium thiosulphate. 2S2O32− + I2 = S4O62− + 2 I− The indicator used in this reaction is a starch solution where amylose forms a blue to black solution with iodine and is colourless where iodine is titrated. Correlation of rancid taste and peroxide value depends on the type of oil and is best tested with taste panels. The odours and flavours associated with typical oxidative rancidity are mostly due to carbonyl type compounds. The shorter-chain aldehydes and ketones isolated from rancid fats are due to oxidative fission and are associated with advanced stages of oxidation. The carbonyl-type compounds develop in low concentrations early in the oxidative process87. Traditionally this was expressed in units of milliequivalents, although if we are using SI units then the appropriate option would be in millimoles per kilogram (N.B. 1 millimole = 2 milliequivalents). Note also that the unit of milliequivalent has been commonly abbreviated as mequiv or even as meq87. •

Reagents used:        

0.1N K2Cr2O7 solution 15 % KI solution Chloroform-Acetic acid solution with the ratio of 2: 3. Saturated KI solution Conc. HCl Starch solution Na2S2O3 solution (a few strength) Solid sodium bicarbonate (NaHCO3)

4.5.10.1 Preparation of reagents: 4.5.10.1.a O.05 N Na2S2O3 solution preparation: See Section 4.5.6.1.a 4.5.10.1.b Preparation of 0.1N K2Cr2O7 solution: The strength of K2Cr2O7 Solution =

1.225 ×0.1 = 0.1 N (see Section−4.5.6.1.b) 1.225

4.5.10.1.c 15% KI solution preparation: See Section 4.5.6.1.c 4.5.10.1.d Preparation of starch solution: See Section 4.5.6.1.d 4.5.10.1.e Preparation of Chloroform-Acetic acid 2:3 solution: 30 ml acetic acid and 20 ml chloroform was added in a 100 ml cylinder.

4.5.10.1.f Preparation of Saturated KI solution preparation: 100 ml distilled water was taken in a clean beaker. Gradually KI was added. At this time, heat was applied for dissolving. As long as KI was dissolved in water further KI was added. Soon it was seen that no potassium iodide was dissolving in water any way. This was saturation point of KI then KI addition was stopped.

4.5.10.2 Procedure for the determination of Peroxide Value: 0.5 gm of sample was placed in a 250 ml glass Stoppard conical flask. 7.5 ml of ChloroformAcetic acid 2:3 solution added and shaken until the sample was dissolved. Then 0.25 ml of saturated Potassium iodide (KI) solution and few drops of conc. HCl also added to it. Then the solution had been shaken for at least 1 minute. 15 ml distilled water was added to the solution and it was titrated carefully with 0.055 N Sodium thiosulphate solutions until the brown colour was faded to pale yellow. 0.5 ml of starch solution was then added and the titration was completed with continuous shaking when the blue colour just disappeared at the end point.

Calculation method: The peroxide value of the fatty oil was calculated by the following formula: VNa 2 S2O3 × S Na 2 S2O3 ×1000 Peroxide value = Weight of sample, W(gm) The strength of Na2S2O3 solution, S Na

2 S 2 O3

= 0.0870 N (see as section–4.5.6.3)

Table-4.27: Titration data for peroxide value of Carum roxburghianum (Radhuni) essential oil: a) Joydebpur, Gajipur: Observe no.

1. 2. 3.

Weight of sample, w gm 0.7903 0.8352 0.8289

Burette reading , Volume of Na2SO3 Solution, VNa S O (ml)

Average Peroxide value, VNa 2 S 2O3 × S Na 2 S 2O3 ×1Peroxide 000 value, W(gm)

IBR 0.0 2.4 5.0

264.3249 260.4259 262.5244

FBR 2.4 4.9 7.5

Diff. 2.4 2.5 2.5

262.4251

Result: The Peroxide value of Carum roxburghianum (Radhuni) seeds essential oil, Joydebpur, Gajipur 262.4251.

b) Keranigonj, Dhaka: Observe no.

1. 2. 3.

Weight of sample, w gm 0.7466 0.7773 0.8104

Burette reading , Volume of Na2SO3 Solution, VNa S O (ml) 2

IBR 0.0 2.0 4.1

FBR 2.0 4.1 6.3

Peroxide value,

VNa 2 S 2O3 × S Na 2 S 2O3 W(gm)

Diff. 2.0 2.1 2.2

233.1638 235.1639 236.2941

Average ×1Peroxide 000 value,

234.8740

Result: The Peroxide value of Carum roxburghianum (Radhuni) seeds essential oil, Keranigonj, Dhaka 234.8740.

c) Faridpur: Observe no.

1. 2. 3.

Weight of sample, w gm 0.9090 0.8510 0.8775

Burette reading , Volume of Na2SO3 Solution, VNa S O (ml) 2

IBR 7.0 9.3 12.0

FBR 9.3 11.6 14.2

Peroxide value,

VNa 2 S 2O3 × S Na 2 S 2O3

value,

W(gm)

Diff. 2.3 2.3 2.2

Average

×1Peroxide 000

220.2221 225.2310 218.2215

221.2249

Result: The Peroxide value of Carum roxburghianum (Radhuni) seeds essential oil, Faridpur 221.2249.

4.6 Essential Oil Analysis by Gas Chromatography and Mass Spectrometer (GC-MS) 89: Gas chromatograph–Mass Spectrometer (GS-MS) is an integrated composite analysis instrument combine GC, which is able to separate and quantify the components, with MS which is also able to identify each component. It has made remarkable progress and is commonly used in the fields of organic chemistry, medical science, pharmacy etc. It has come into use and also for analysis in the environmental field. GC-MS–QP 5050 A is a high performance quadruple mass spectrometer. Two systems, column and direct inlet are available with this instrument to enter the samples into the ion box. It has two methods for

sample analysis such as electron impact ionization (EI) and chemical ionization (CI). Conventional EI method is thought of as a hard ionization and chemical ionization as thought of as a soft ionization. Negative chemical ionization (NCI) is an especially sensitive method for substances with high electro negativity, such as halogenated compounds. A library having 1, 07,000 spectra of organic and organometalic compounds has been integrated with PC to identify the selected compounds. Name, molecular weight, formula and structure of compounds can be established by matching and comparing the mass fragment patterns of their mass spectrums with those of the library enlisted compounds.

4.6.1 Apparatus and Operating conditions for GC-MS analysis98: a) Gas chromatograph: The analysis was carried out by GC-MS electron impact ionization (EI) method on GC-17A gas chromatograph (Shimadzu, Japan) coupled to a GC-MS-QP 5050A. Mass-Spectrometer (Shimadzu). b) Name of column: fused silica capillary column (30m × 0.25mm), coated with DB-5 ms (J & W), 0.25 µm film thickness were utilized. c) Temperature of column: programming 40-280°C, held at 40°C for 2min, rate 3°C/min and held at 170°C for 10 min. d) Detector: Flame Ionization Detector (FID) e) Detector temperature: 300°C. f) Injector temperature: Injection port temperature 290°C g) Column Packing : Column packing was done with 10% diethylene glycol succinate on 100 – 120 mesh diatomic CAW h) Syringe: 10 µ L Hemilton Syringe, 0.2 µ L Injector. i) Splitting: Samples were injected by splitting with the split ratio 1:20. j) Integrator: Integrator, LKB Bromma; (2220 Recording Integrator). k) Speed of the chromatogram: 0.5 mm/min. l) Searched library: NIST 107 Library, Shimadzu Corporation. m) Acquisition parameters: Full scan; scan range 40- 350 amu. n) Mass analyzer: Quadrupole with prerod mass filter. o) Carrier gas: Helium gas at constant pressure 90 kPa. p) Flow rate of carrier gas: 20 ml per min. q) Sample dissolved: In chloroform. r) Reference standards: Known mixtures of essential oil. s) Cooling speed: 300°C down to 50°C - 6 min (50°C/min)

t) Range of linear temperature increase: 40°C /min up to 200°C, 15°C /min up to 350°C , 7°C /min up to 450°C u) Operating voltage: 230V v) Temperature range: +4°C to 450°C.

4.6.2 Identification of the compounds: Identification of compounds was done by

comparing the NIST library data of the peaks and

mass spectra of the peaks with those reported in literature. Percentage composition was computed from GC peak areas on DB–5 ms column without applying correction factors.

4.6.3 GCMS analysis of essential oil of Carum roxburghianum seeds from different region of Bangladesh: The essential oils Carum roxburghianum (Radhuni) seeds were analyzed by Electron Impact Ionization (EI) method on GC-17A Shimadzu Gas Chromatograph, coupled to a GC-MS QP 5050A Shimadzu Mass Spectrometer; fused silica capillary column (30m x 2.5mm; 0.25 μm film thickness), coated with DB-5 ms (J&W) were utilized. Column temperature of 40 oC (2 min) to 170oC at the rate of 3oC/min was maintained with carrier gas helium at a constant pressure of 90 kPa (Acquisition parameters full scan; scan range 40-350 amu). Samples were injected by splitting with the split ratio 1:20. Essential oil sample was dissolved in chloroform. The GC-MS report of the essential oil was given in Table–4.28 & 4.30 and the structure of compounds are given in Table–4.29 & 4.31. The GC-MS chromatogram of the essential oil were shown in Figure–4.4 & 4.6 and the spectrum for compound information of GC-MS peaks collected from GC-MS NIST 107 library were shown to be Figure-4.5 & 4.7.

Figure−4.3: Shimadzu GC-17A, QP 5050A GC-MS instrument.

Table –4.28: Constituent of the essential oil of Carum roxburghianum seeds from Joydebpur, Gajipur, analysed by GC-MS. (Sample ID: C. ROX 1).

Peak no.

R.Time

% Total

1. 2.

2.325 2.433

1.69 0.30

3. 4. 5. 6.

2.750 2.858 3.142 3.283

7. 8.

3.442 3.883

4.125

10. 11. 12.

4.375 4.633 4.717

13. 14.

5.158 5.483

15. 16. 17.

5.750 5.817 6.242

18.

6.317

19.

6.508

20.

6.558

21.

6.717

22.

6.767

Name of Compound

alpha.- Thujene (1R)-2,6,6Trimethylbicyclo[3.1.1]hept-2-ene 17.13 Sabinene 1.15 beta.-Pinene 0.05 alpha.-Phellandrene 0.94 1,4-Cyclohexadiene, 1-methyl-4-(1methylethyl)33.46 Limonene 3.01 1,4-Cyclohexadiene, 1-methyl-4-(1methylethyl)2.22 Bicyclo[3.1.0]hexen-2ol, 2-methyl5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)1.09 Terpinolene 0.04 5-Octen-1-ol 1.79 Bicyclo [3.1.0] hexen-2ol, 2methyl-5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)0.30 Sabinene 0.15 1,4-Cyclohexadiene, 1-methyl-4-(1methylethyl)0.31 6-butyl-1,4-cycloheptadiene 0.04 9-Methylbicyclo[3.3.1]nonane 2.92 3-Cyclohexen-1-ol, 4-methyl-1-(1methylethyl)0.06 Alpha, alpha,4-trimethylbenzyl carbonate 0.14 Bicycle[2.2.1]heptane,2,2-dimethyl3-methylene-,(1S)0.15 Bicyclo[3.1.0]hexen-2ol, 2-methyl5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)0.03 Cyclohexanone, 2-methyl-5-(1methylethenyl)0.07 2-Cyclohexen-1-ol, 3-methyl-6-(1methylethyl)-,trans-

M.W.

M.F

136 136

C10H16 C10H16

136 136 136 136

C10H16 C10H16 C10H16 C10H16

136 136

C10H16 C10H16

154

C10H18O

136 128 154

C10H16 C8H16O C10H18O

136 136

C10H16 C10H16

150 138 154

C11H18 C10H18 C10H18O

269

C17H19NO2

136

C10H16

154

C10H18O

152

C10H16O

154

C10H18O

Tableâ&#x20AC;&#x201C;4.28: (Continued) Peak no.

R.Time

% Total

Name of Compound

M.W.

M.F

23.

7.442

0.38

2-Cyclohexen-1-ol, 2-methyl-5-(1methylethyl)-,(S)-

150

C10H14O

24. 25. 26.

8.192 9.025 9.242

0.23 0.03 0.17

27. 28. 29.

9.425 9.800 10.550

0.05 0.03 2.07

30. 31. 32.

11.117 11.700 12.017

0.12 0.21 0.28

33.

12.900

0.20

34. 35. 36.

13.000 13.383 13.642

0.25 0.50 0.03

37. 38.

13.775 14.142

0.05 1.18

39. 40.

15.100 16.017

26.69 0.28

41.

16.208

0.04

Total:

99.83

Anisole, p-allylgamma.-Elemene 1,4-Cyclohexadine-1,2dicarboxylic anhydride Acetophenone Germacrene D Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-, [1R-(1R@,4Z,9S@)]alpha.-Caryophyllene gamma.-Elemene 1,3-Benzodioxole, 4-methoxy-6-(2propenyl)1H-Cycloprop(e) azulen-7ol,decahydro-1,1,7-trimethyl-4methylene-,[1ar(1a.alpha.,4a.alpha.,7.beta., 7a.beta.,7b.alpha.)]Caryophyllene oxide Apoil 4,4-Dimethyl-3-(3-methylbut-3enylidene)-2methylenebicyclo[4.1.0]heptane Seychellene 1(3H)-Isobenzofuranone, 3butylidinedihydrocoumarin,5,7,8-trimethylOxalic Acid, monoamide, monohydrazide, N-(2,5dimethylphenyl)-N2-(4methylbenzylideno)1,4-methanoazulene-9-methanol, decahydro-4,8,8-trimethyl-,[1S(1.alpha.,3a.beta.,4.alpha.,8a.beta.,9 R@)]-

148 204 150

C10H12O C15H24 C8H6O3

120 204 204

C8H8O C15H24 C15H24

204 204 192

C15H24 C15H24 C11H12O3

220

C15H24O

220 222 202

C15H24O C12H14O4 C15H22

204 188

C15H24 C12H12O2

190 309

C12H14O2 C18H19N3O2

222

C15H26O

Tableâ&#x20AC;&#x201C;4.29: The name of the compounds indentified by GCMS and their respective structure of essential oil of Carum

roxburghianum (Radhuni) seeds from Joydebpur, Gajipur. Sl. no.

Total %

Name of Compound

M.F.

1.69

alpha- Thujene

C10H16

0.30

C10H16

17.13

(1R)-2,6,6Trimethylbicyclo[3.1.1]hept-2ene Sabinene

1.15

beta.-Pinene

C10H16

0.05

alpha.-Phellandrene

C10H16

4.10

C10H16

33.46

1,4-Cyclohexadiene, 1-methyl4-(1-methylethyl)Limonene

4.16

C10H18O

1.09

Bicyclo[3.1.0] hexen-2ol, 2methyl-5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)Terpinolene

10.

0.04

5-Octen-1-ol

C8H16O

11.

0.30

Sabinene

C10H16

12.

0.31

6-butyl-1,4-cycloheptadiene

C11H18

13.

0.04

9-Methylbicyclo[3.3.1]nonane

C10H18

14.

2.92

C10H18O

15.

0.06

3-Cyclohexen-1-ol, 4-methyl-1(1-methylethyl)Alpha, alpha,4-trimethylbenzyl carbonate

16.

0.14

Bicycle[2.2.1]heptane,2,2dimethyl-3-methylene-,(1S)-

C10H16

17.

0.03

C10H16O

18.

0.07

Cyclohexanone, 2-methyl-5-(1methylethenyl)2-Cyclohexen-1-ol, 3-methyl-6(1-methylethyl)-,trans-

C10H16

C17H19NO2

C10H18O

Structure

Table - 4.29: (Continued): 19. 0.38 2-Cyclohexen-1-ol, 2-methyl-5-

C10H14O

20.

0.23

(1- methylethyl)-,(S)Anisole, p-allyl-

21.

0.24

gamma.-Elemene

C15H24

22.

0.17

C8H6O3

23.

0.05

1,4-Cyclohexadine-1,2dicarboxylic anhydride Acetophenone

24.

0.03

Germacrene D

C15H24

25.

2.07

C15H24

26.

0.12

Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-, [1R-(1R@,4Z,9S@)]alpha.-Caryophyllene

27.

0.28

C11H12O3

28.

0.20

29.

0.25

1,3-Benzodioxole, 4-methoxy6-(2-propenyl)1H-Cycloprop(e) azulen-7ol,decahydro-1,1,7-trimethyl-4methylene-,[1ar(1a.alpha.,4a.alpha.,7.beta., 7a.beta.,7b.alpha.)]Caryophyllene oxide

30.

0.50

Apoil

C12H14O4

31.

0.03

4,4-Dimethyl-3-(3-methylbut-3enylidene)-2methylenebicyclo[4.1.0]heptane

C15H22

32.

0.05

Seychellene

C15H24

33.

1.18

34. 35.

36.

1(3H)-Isobenzofuranone, 3butylidine26.69 dihydrocoumarin,5,7,8trimethyl0.28 Oxalic Acid, monoamide, monohydrazide, N-(2,5dimethylphenyl)-N2-(4methylbenzylideno)0.04 1,4-methanoazulene-9methanol, decahydro-4,8,8trimethyl-,[1S(1.alpha.,3a.beta.,4.alpha.,8a.bet a.,9R@)]-

C10H12O

C8H8O

C15H24

C15H24O

C12H12O2 C12H14O2 C18H19N3O2

C15H26O

Figure â&#x20AC;&#x201C; 4.4: GC-MS chromatogram of Carum roxburghianum (Radhuni) seeds essential oil from Joydebpur, Gajipur. Figure - 4.5: Mass Spectrum for the compound of Joydebpur, Gajipur

Figure -4.5.1: Mass spectrum for compound SL. no-1, peak no-1

Figure -4.5.2: Mass spectrum for compound SL. no-2, peak no-2

Figure -4.5.3: Mass spectrum for compound SL. no-3, peak no-3

Figure -4.5.4: Mass spectrum for compound SL. no-4, peak no-4

Figure -4.5.5: Mass spectrum for compound SL. no-5, peak no-5

Figure -4.5.6: Mass spectrum for compound SL. no-6, peak no-6, 8

Figure -4.5.7: Mass spectrum for compound SL. no-7, peak no-7

Figure -4.5.8: Mass spectrum for compound SL. no-8, peak no-9, 12, 20

Figure -4.5.9: Mass spectrum for compound SL. no-9, peak no-10

Figure -4.5.10: Mass spectrum for compound SL. no-10, peak no-11

Figure -4.5.11: Mass spectrum for compound SL. no-11, peak no-13

Figure -4.5.12: Mass spectrum for compound SL. no-12, peak no-15

Figure -4.5.13: Mass spectrum for compound SL. no-13, peak no-16

Figure -4.5.14: Mass spectrum for compound SL. no-14, peak no-17

Figure -4.5.15: Mass spectrum for compound SL. no-15, peak no-18

Figure -4.5.16: Mass spectrum for compound SL. no-16, peak no-19

Figure -4.5.17: Mass spectrum for compound SL. no-17, peak no-21

Figure -4.5.18: Mass spectrum for compound SL. no-18, peak no-22

Figure -4.5.19: Mass spectrum for compound SL. no-19, peak no-23

Figure -4.5.20: Mass spectrum for compound SL. no-20, peak no-24

Figure -4.5.21: Mass spectrum for compound SL. no-21, peak no-25, 31

Figure -4.5.22: Mass spectrum for compound SL. no-22, peak no-26

Figure -4.5.23: Mass spectrum for compound SL. no-23, peak no-27

Figure -4.5.24: Mass spectrum for compound SL. no-24, peak no-28

Figure -4.5.25: Mass spectrum for compound SL. no-25, peak no-29

Figure -4.5.26: Mass spectrum for compound SL. no-26, peak no-30

Figure -4.5.27: Mass spectrum for compound SL. no-27, peak no-32

Figure -4.5.28: Mass spectrum for compound SL. no-28, peak no-33

Figure -4.5.29: Mass spectrum for compound SL. no-29, peak no-34

Figure -4.5.30: Mass spectrum for compound SL. no-30, peak no-35

Figure -4.5.31: Mass spectrum for compound SL. no-31, peak no-36

Figure -4.5.32: Mass spectrum for compound SL. no-32, peak no-37

Figure -4.5.33: Mass spectrum for compound SL. no-33, peak no-38

Figure -4.5.34: Mass spectrum for compound SL. no-34, peak no-39

Figure -4.5.35: Mass spectrum for compound SL. no-35, peak no-40

Figure -4.5.36: Mass spectrum for compound SL. no-36, peak no-41

Table –4.30: Constituent of the essential oil of Carum roxburghianum seeds, From Keranigonj, Dhaka analysed by GC-MS. (Sample ID: C. ROX 2). Peak no.

R.Time

Total %

Name of Compound

M.W.

M.F

1. 2.

2.447 2.541

2.69 0.19

136 136

C10H16 C10H16

3. 4. 5. 6.

2.891 2.979 3.289 3.424

2.49 0.69 0.85 4.86

alpha.-Phellandrene Bicyclo[3.1.1]hept-2-ene, 2,6,6trimethyl-, Sabinene beta.-Pinene alpha.-Phellandrene Terpinolene

136 136 136 136

C10H16 C10H16 C10H16 C10H16

3.531

7.67

134

C10H14

3.623

59.37

Benzene,1-methyl-4-(1methylethyl)Limonene

136

C10H16

4.060

10.13

1,4-Cyclohexadiene, 1-methyl-4(1-methylethyl)-

136

C10H16

10.

4.317

1.36

154

C10H18O

11. 12.

4.609 4.997

2.17 1.53

136 154

C10H16 C10H18O

13.

5.605

0.54

154

C10H18O

14.

6.117

0.35

154

C10H18O

15.

7.492

5.11

Bicyclo[3.1.0]hexen-2ol, 2methyl-5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)Terpinolene Bicyclo(3.1.0)hexen-2ol, 2methyl-5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)Bicyclo(3.1.0)hexen-2ol, 2methyl-5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)Bicyclo[3.1.0]hexen-2ol, 2methyl-5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)3-Cyclohexen-1-ol, 4-methyl-1(1-methylethyl)-

154

C10H18O

100

Table–4.31: The name of the compounds indentified by GCMS and their respective structure of essential oil of Carum

roxburghianum (Radhuni) seeds from Keranigonj, Dhaka . Sl. no.

Total %

Name of Compound

M.F.

3.54

alpha.-Phellandrene

C10H16

0.19

C10H16

2.49

Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, Sabinene

0.69

beta.-Pinene

C10H16

7.03

Terpinolene

C10H16

7.67

Benzene,1-methyl-4-(1methylethyl)-

C10H16

59.37

Limonene

C10H16

10.13

C10H16

3.78

10.

5.11

1,4-Cyclohexadiene, 1methyl-4-(1-methylethyl)Bicyclo[3.1.0]hexen-2ol, 2methyl-5-(1-methylethyl)(1.alpha.,2.alpha.,5.alpha.)3-Cyclohexen-1-ol, 4methyl-1-(1-methylethyl)-

100

C10H16

C10H18O

Structure

Figure â&#x20AC;&#x201C; 4.6: GC-MS chromatogram of Carum roxburghianum (Radhuni) seeds essential oil from Keranigonj, Dhaka.

Figure - 4.7: Mass Spectrum for the compound of Keranigonj, Dhaka.

Figure -4.7.1: Mass spectrum for compound SL. no-1, peak no-1, 5

Figure -4.7.2: Mass spectrum for compound SL. no-2, peak no-2

Figure -4.7.3: Mass spectrum for compound SL. no-3, peak no-3

Figure -4.7.4: Mass spectrum for compound SL. no-4, peak no-4

Figure -4.7.5: Mass spectrum for compound SL. no-5, peak no-6, 11

Figure -4.7.6: Mass spectrum for compound SL. no-6, peak no-7

Figure -4.7.7: Mass spectrum for compound SL. no-7, peak no-8

Figure -4.7.8: Mass spectrum for compound SL. no-8, peak no-9

Figure -4.7.9: Mass spectrum for compound SL. no-9, peak no-10, 12, 13, 14

Figure -4.7.10: Mass spectrum for compound SL. no-10, peak no-15

Fatty Oil 5.1 Fats and fatty acid and its relation to the plant materials: A non volatile oil composed of fatty acids, usually of animal or vegetable origin is called fatty oil or fixed oil. Nature has endowed the world with a variety of important oil bearing plants and animals. Vegetable oils and fats are essential items of human consumption either as such or in refined or hydrogenated form. The fatty acids that occur in nature usually have straight chains and usually contain even number of carbon atoms. They are esters of glycerol. These compounds are known as glycerol esters. They are either oily liquids or waxy solids and contain the element carbon,

hydrogen and oxygen only Individual fat is usually a mixture of esters of glycerol and two or three different fatty acids. Fatty acid is also found free and in lore molecular complexes in protein105. Every fat molecule has essentially four parts: The core of the molecule is glycerol, a three carbon compound that is related to the alcohol. Three fatty acids combined with the glycerol molecule to form a fat. The nature of a fat depends on what kinds of fatty acids are linked to the glycerol core, the number of carbon atoms of the fatty acids, and the degree of saturation or unsaturation of the fatty acids70. A fat molecule may be composed of three identical fatty acids, three different ones, or a combination of two alike and one different, when all of the fatty acids in a fat molecule are identical. The molecule is called a simple triglyceride; a molecule with different fatty acids is called a mixed triglyceride. The mixed triglycerides are found in both animal and vegetable foods106, 107. Fatty acids are long chain carboxylic acids, it contain only one carboxylic group and thus monobasic. They fall roughly into three groups; saturated, unsaturated and those, which contain branches or rings. Although numerous fatty acids are now known in plants, the palmitic acid (C-16) is the major saturated acid in leaf lipids and also occur in varying quantities in some seeds oils. Stearic acid (C-18) is the major saturated acid in seed fats of a number of plant families108; unsaturated acids mainly C-16 and C-18 are widespread in both leaf and seed oil. A number of rare fatty acids 108 (e.g. erucic and sterculic acid) are found in

seed oils of a few plants. The plant glycerides have a relatively higher proportion of the more unsaturated acids109. Although alkaloids, terpenoids, steroids, flavones and their glycosides, phenols and phenolic acids constitute a major portion of non â&#x20AC;&#x201C; carbohydrate materials of a plant, fatty acids are always present in varying amounts in all plant materials. They occur in plants primarily in bonds form as fats or lipids. These lipids comprise up to 7% of the dry weight in leaves in higher plants, about 1-5% in stems of green plants. These are important as membrane constituent in the chloroplasts and mitochondria. These substances also occur in considerable amounts in the seeds of fruits of a number of plants and act as a storage form of energy to be used during germination. These fatty materials influence the handling of the plant tissues as well as any chemical treatment done in it 108. Seed oils from plants such as olive, palm, coconut etc. are exploited commercially and are used as edible oils. For soap manufacture and in the paint industry therefore the study of fatty acids. The major constituent of all fatty matters is important. A fatty acid is a carboxylic acid often with a long unbranched aliphatic tail (chain), which is either saturated or unsaturated. Fatty acids derived from natural fats and oils may be assumed to have at least 8 carbon atoms, e.g. caprylic acid (octanoic acid). Most of the natural fatty acids have an even number of carbon atoms, because their biosynthesis involves acetyl-CoA, a coenzyme carrying a two-carbon-atom group. In nature they are found as glyceryl esters 112, 86

5.1.1 Classification of fatty acids110: Fatty acids are two types on the basis of the nature of the bonds in the acid molecule. They are â&#x20AC;&#x201C; 1) Unsaturated fatty acid 2) Saturated fatty acid Examples: Some examples of saturated and unsaturated fatty acids are given below.

1) Unsaturated fatty acid: Name of the acid Myristoleic acid: Palmitoleic acid: Oleic acid: Linoleic acid: Alpha-linolenic acid: Erucic acid:

Chemical formula CH3(CH2)3CH=CH(CH2)7COOH CH3(CH2)5CH=CH(CH2)7COOH CH3(CH2)7CH=CH(CH2)7COOH CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH CH3(CH2)7CH=CH(CH2)11COOH

2) Saturated fatty acid: Name of the acid

Chemical formula

Butyric Caproic Caprylic Capric Lauric Myristic Palmitic Stearic Arachidic Behenic Lignoceric

CH3(CH2)2COOH CH3(CH2)4COOH CH3(CH2)6COOH CH3(CH2)8COOH CH3(CH2)10COOH CH3(CH2)12COOH CH3(CH2)14COOH CH3(CH2)16COOH CH3(CH2)18COOH CH3(CH2)20COOH CH3(CH2)22COOH

5.1.2 Necessary Fatty Acids112: The human body can produce all but two of the fatty acids it needs. These two are called essential fatty acid. They are linoleic acid (LA) and alpha-linolenic acid (LNA) and are widely distributed in plant oils. In addition, fish oils contain the longer chain omega-3 fatty acids ecosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Essential fatty acids are poly unsaturated fatty acids and are the parent compounds of the omega-6 and omega-3 fatty acid series respectively.

5.2 Extraction of fatty oil: The fatty oil sample was extracted by using extraction method by using 40- 60 0 C. pet ether as solvent from crushed (essential oil and moisture free) sample in a glass Sox let apparatus as following.

5.2.1 Sox let extraction for collection of fatty oil 105−107: The residue obtained after steam distillation of sample taken in a thimble, which was prepared by filter paper. Then the definite amount of moisture and essential oil free sample in thimble was placed in the Sox let apparatus unit and extraction was carried out with 40˚- 60 ˚ C. petroleum ether for 30 hrs. in a water bath at 80°-90°C.

5.2.2 Purification of fatty oil112: The fatty oil in the solvent obtained from the sox let unit was filtered to remove impure materials. The filtrate containing the fatty oil was distilled at low temperature on the water both. Thus the solvent was removed from the mixture. The trace amount of solvent remained in the fatty oil was eliminated by using high vacuum. Then dried in an oven at 80˚C. And cooled in desiccators and weighed. By this process the purified fatty oil was obtained for further characterization.

Calculation method: The percentage of the fatty oil content was calculated by the following formula: The percentage of the fatty oil obtained =

Fatty oil collected (gm) ×100 Wt. of sample (gm)

Weight of sample taken Weight of fatty oil extracted % of fatty oil

= w1 gm = w2 gm w2 ×100 = w1

Table – 5.1: Fatty oil contents of Carum roxburghianum (Radhuni):

Sample collected

Weight of

Weight of fatty

from

Sample,w1

oil collected, w2 gm

% of fatty oil, w2 ×100 w1

Joydebpur, Gajipur Keranigonj, Dhaka

255.8695 118.0823

39.1831 23.9915

15.3137 20.3176

Faridpur

120.3559

24.3485

20.2304

 Each value represents the average value from three experiments .

Figure–5.1: Sox let Extractor

Sample residue after steam distillation Crush with blender

Powder Extraction with pet

ether 40˚-60˚C Residue

Fatty oil layer with pet ether 40˚-60˚C. Filtered then evaporated to Remove n-hexane

Crude fiber and ash content

Fatty oil with some impurities Evaporated to dryness using Rotary vacuum evaporator Pure fatty oil

Pure fatty oil were taken for Determination of physical properties

Pure Fatty oil were taken for GLC analysis

Pure fatty oil were taken for Determination of Chemical properties

Scheme–5.1: Extraction & analysis of fatty oil

5.3 Determination of Physical Properties of fatty oil: 5.3.1 Determination of Specific Gravity or Density of fatty oil85: The technique of determination of Specific Gravity or Density has already described early in the Section-4.4.1. So, in this section calculation data only have shown. Sample collected from

Weight of Sample oil, (W2 – W) gm

Weight of water, (W1 – W) gm

Specific Gravity, W2 − W × Dθ C W1 − W

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

0.9356 0.9847 0.9636

1.0679 1.0679 1.0679

0.8723 0.9180 0.8984

Room temperature: 30˚C Density of water at 30˚C temp., Dθ˚C = 0.99567

Table - 5.2: Specific Gravity of fatty oil:

 Each value represents the average value from three experiments

5.3.2 Determination of Refractive Index [η] of fatty oil 86: The technique of determination of Refractive Index [η] has already described early in the Section-4.4.2. So, in this section calculation data only have shown.

Table –5.3: Refractive index [η] of fatty oil:

 Each value represents the average value from three experiments

5.3.3 Determination of Optical rotation, [α]tD of fatty oil 76, 77, 84: The technique of determination of Optical rotation, [α]tD has already described early in the Section-4.4.3. So, in this section calculation data only have shown.

Table –5.4 : Optical rotation of fatty oil:

 Each value represents the average value from three experiments.

Sample collected from

Refractive index of Essential oil at 30˚C, [η]30˚C

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

1.4702 1.4651 1.4701

Sample collected from

Optical rotation of Essential oil at 26˚C, [α]D26˚C

Joydebpur, Gajipur Keranigonj, Dhaka Faridpur

+9.03˚ +8.54˚ +9.56˚

5.3.4 Determination of Solubility of fatty oil in Alcohol of Different Strength and other solvents 75, 86: The technique of determination of Solubility has already described early in the Solubility in

Joydebpur, Gajipur

90% alcohol 100% alcohol Distilled water Chloroform CCl4 Pet-ether Diethyl ether n – Hexane 4.4.4.

So,

Not soluble Soluble at any volume Not soluble

Sample collected from Keranigonj, Dhaka Not soluble Soluble at any volume Not soluble

Section-

Faridpur Not soluble Soluble at any volume Not soluble

Soluble at any Soluble at any volume volume Soluble at any Soluble at any volume volume Soluble at any Soluble at any volume volume Soluble at any Soluble at any volume volume Soluble at any Soluble at any volume volume this section calculation data only

Soluble at any volume Soluble at any volume Soluble at any volume Soluble at any volume Soluble at any volume have shown.

Table–5.5: Solubility test of fatty oil in of alcoholic solution other solvent:   Each value represents the average value from three experiments

Remarks: Sample collected from

Colour

Joydebpur, Gajipur Dark green or greenish black Keranigonj, Dhaka Greenish black Faridpur Dark green  Insoluble in 90% alcohol and Distilled water at any volume.  Soluble in Chloroform, 100% alcohol, CCl4, Pet-ether, n- Hexane, Diethyl ether at any volume.

5.3.5 Colour Test of fatty oil59: Table -5.6: Colour observation test for fatty oil of Carum roxburghianum:

5.3.6 Appearance of fatty oil at room temperature: Fatty oil of Carum roxburghianum (Radhuni) seeds is:

   

Homogeneous, Dark green or greenish black viscous liquid and Lighter than water. Bitter in taste,

 Spicy odor

- at room temperature (30˚C)

5.4 Determination of Chemical properties of Fatty oil: 5.4.1 Determination of Acid Value of fatty oil 83, 102: Acid value denotes the number of mg of potassium hydroxide (KOH) needed to neutralize the free acids in 1 gm of fat or oil. The technique of determination of acid value has already described early in the Section4.5.1. So, in this section calculation data only have shown.

Calculation method: X ml of 0.1N NaOH ≡ X ml of 0.1 N KOH. 1000 ml of 1N KOH contains 56.1g of KOH Acid value

56.1×V NaOH × S NaOH Weight of sample, w gm

Strength of NaOH, SNaOH = 0.1165 N (see as Section-4.5.1)

Table-5.7: Titration data for acid value of Carum roxburghianum fatty oil: a) Joydebpur, Gajipur: Observe no.

Weight of sample, w gm

Burette reading , Volume of NaOH Solution, VNaOH (ml) IBR FBR Diff.

Acid value,

Average

56.1 ×V NaOH × S NaOH Acid value, Weight of sample, w gm

1. 1.0185 0.0 25.0 25.0 160.4243 162.9871 2. 0.9521 25.0 49.1 24.1 165.4250 3. 0.9697 0.0 24.2 24.2 163.1120 Result: The acid value of C. roxburghianum (Radhuni), Joydebpur, Gajipur 162.9871. b) Keranigonj, Dhaka. Observe Weight of Burette reading , no. sample, w Volume of NaOH gm Solution, VNaOH (ml) IBR FBR Diff. 1. 2.

0.7875 0.8410

0.0 18.0

18.0 38.0

18.0 20.0

Acid value,

Average

56.1 ×V NaOH × S NaOH Acid value, Weight of sample, w gm

150.4400 155.4320

154.1015

3. 0.9191 20.0 42.0 22.0 156.4325 Result: The acid value of C. roxburghianum (Radhuni), Keranigonj, Dhaka 154.1015.

c) Faridpur. Observe no.

Weight of sample, w gm

Burette reading , Volume of NaOH Solution, VNaOH (ml) IBR FBR Diff.

Acid value,

Average

56.1 ×V NaOH × S NaOH Acid value, Weight of sample, w gm

1. 0.7878 25.1 42.4 17.3 143.5221 143.8384 2. 0.7920 0.8 18.1 17.3 142.7610 3. 0.7875 17.5 35.0 17.5 145.2320 Result: The acid value of Carum roxburghianum (Radhuni), Faridpur 143.8384.

5.4.2 Determination of Ester value of fatty oil75, 86, 94: The esters value is a relative measure of the amount of ester present. The ester in the fatty oil is expressed by the ester number. It is defined as the number of milligrams of KOH, required to saponify the ester present in 1 gm of the oil. The technique of determination of ester value has already described early in the Section4.5.2. So, in this section calculation data only have shown.

Table-5.8: Titration data for Ester value of Carum roxburghianum fatty oil: a) Joydebpur, Gajipur: Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

1. 2. 3.

0.5907 0.4037 0.6163

6.5 9.5 19.5

16.0 19.5 28.8

9.5 10.0 9.3

Ester value of Fatty oil 28.05 × (B - S) × SHCl W 46.3709 48.4705 49.5231

Average Ester value

48.1215

Result: The ester value of C. roxburghianum (Radhuni), Joydebpur, Gajipur 48.1215.

b) Keranigonj, Dhaka: Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

Ester value of Fatty oil 28.05 × (B - S) × SHCl W

0.7796

0.0

8.5

55.2131

0.7250

8.5

17.1

8.6

57.2132

0.6253

17.1

26.1

9.0

56.3132

Average Ester value

56.2465

Result: The ester value of C. roxburghianum (Radhuni), Keranigonj, Dhaka 56.2465

c) Faridpur: Observe no.

Weight of sample, W gm

Burette reading , Volume of 0.5 N HCl Solution, S (ml) IBR FBR Diff.

Ester value of Fatty oil 28.05 × (B - S) × SHCl W

Average Ester value

1. 1.0957 26.1 33.1 7.0 60.7132 62.0342 2. 1.0021 33.1 40.4 7.3 61.6945 3. 0.8232 40.4 48.3 7.9 63.6950 Result: The ester value of Carum roxburghianum (Radhuni), Faridpur 62.0342.

5.4.3 Determination of Iodine Value of fatty oil 75, 83, 84, 86: Iodine value is expressed in gm of iodine absorbed by 100 gms of oil or fat. It gives the indication of degree of unsaturation of oil and is thus a relative measure of the unsaturated bonds present in the oil. The technique of determination of iodine value has already described early in the Section4.5.6. So, in this section calculation data only have shown.

Calculation method: Iodine value was determined by following formula: (B −S) × N ×12.69 Iodine value = W Where, B = Volume of NaS2O3 required for the blank titration S = Volume of Na2S2O3 required for the sample N = Strength of the Na2S203 solution W = Wt of the fatty oil taken in gm

Vol. of NaS2O3 required for the blank titration, B =54.8 ml (see Section-4.5.6.4) Strength of the Na2S203 solution, N = 0.1508 N (See Section-4.5.6.3)

Table-5.9: Titration data of iodine value of Carum roxburghianum fatty oil: a) Joydebpur, Gajipur. Observe no.

Weight of sample, w gm

Burette reading , Volume of 0.1 N Na2S2O3 solution, S(ml) IBR

FBR

Diff.

Iodine value,

Average

(B − S) × N ×12.69 Iodine value W

1. 0.4188 0.0 51.65 51.65 15.0801 15.8974 2. 0.5246 0.0 50.60 50.60 16.0521 3. 0.3874 0.0 51.6 51.6 16.5599 Result: The iodine value of C. roxburghianum fatty oil, Joydebpur, Gajipur 15.8974

b) Keranigonj, Dhaka. Observe no. 1. 2. 3.

Weight of sample, w gm 0.4128 0.4022 0.4201

Burette reading , Volume of 0.1 N Na2S2O3 solution, S(ml) IBR

FBR

Diff.

0.0 0.0 0.0

52.9 52.0 52.5

Iodine value, Average (B − S) × N ×12.69 Iodine value W 9.2285 13.9584 10.9773

11.3881

Result: The iodine value of C. roxburghianum, fatty oil, Keranigonj, Dhaka 13.3881

c) Faridpur. Observe no. 1. 2. 3.

Weight of sample, w gm 0.5172 0.5070 0.5155

Burette reading , Volume of 0.1 N Na2S2O3 solution, S(ml) IBR

FBR

Diff.

0.0 0.0 0.0

52.7 52.5 52.3

Iodine value,

Average

8.1410 9.0958 9.7237

8.9868

(B − S) × N ×12.69 Iodine value W

Result: The iodine value of C. roxburghianum fatty oil, Faridpur 8.9868

5.4.4 Determination of the Saponification value of fatty oil 83, 84, 86, 91: Saponification value is expressed in number of milligrams of caustic potash (KOH) required to saponify one gram of oil or fat. The technique of determination of Saponification value has already described early in the Section-4.5.7. So, in this section calculation data only have shown.

Calculation method: Saponification value =

56.1 × N × (B - S) W

Where, B = ml of 0.5 N HCl required for blank. S = ml of 0.5N HCl required for Sample N = Strength of HCl (N) W = Wt. of Sample (gm) Vol. of 0.5 N HCl required for blank, B = 28.6 ml (see Section-4.5.7.3 & Table-4.23)

Strength of HCl, N = 0.5554 N

(see Section-4.5.7.1.c)

Table-5.10: Titration data for Sap. value of C. roxburghianum fatty oil: a) Joydebpur, Gajipur. Observe no.

Weight of sample, w gm

1. 2. 3.

0.8807 0.9108 0.8865

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff. 0.0 21.1 20.9

21.1 42.0 42.1

21.1 20.9 21.2

Saponification value,

56.1 × N × (B - S) W

Average Saponification value,

265.3541 263.4219 260.1029

262.9596

Result: The sap. value of C. roxburghianum fatty oil, Joydebpur, Gajipur 262.9596.

a) Keranigonj, Dhaka. Observe no.

Weight of sample, w gm

1. 2. 3.

1.0695 0.7026 0.3302

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff. 10.0 0.0 0.0

30.0 23.0 26.0

20.0 23.0 26.0

Saponification value, 56.1 × N × (B - S) W

Average Saponification value,

250.5354 248.3340 245.3369

248.0688

Result: The sap. value of C. roxburghianum fatty oil, Keranigonj, Dhaka 248.0688.

a) Faridpur. Observe no.

Weight of sample, w gm

1. 2. 3.

0.7807 0.7768 0.7385

Burette reading , Volume of HCl Solution, S(ml) IBR FBR Diff. 0.0 21.9 0.0

21.9 42.6 22.0

21.9 21.7 22.0

Saponification value, 56.1 × N × (B - S) W

Average Saponification value,

276.3808 276.7633 278.4435

277.1959

Result: The sap. value of C. roxburghianum fatty oil, Faridpur 277.1959.

5.4.5 Determination of Unsaponifiable Matter of fatty oil 59, 66, 86: Fatty acids derived from natural sources contain small amount of dissolved unsaponifiable matter. Mineral oil contamination, higher aliphatic alcohols, sterols, pigments and hydrocarbons are the materials of this nature. The technique of determination of unsaponifiable matter has already described early in the Section-4.5.9. So, in this section calculation data only have shown.

Calculation method: The Unsaponified matter was found by the following formula.

100 × W1 W Where,W1 = Weight of residue (gm).

Unsaponified matter (% by weight) =

W = Weight of sample taken (gm)

Table-5.11: Calculation table for Unsaponifiable matter of Carum roxburghianum (Radhuni) fatty oil: a) Joydebpur, Gajipur: Observe no.

Weight of sample, w gm

Weight of residue, W1,gm

Unsaponified matter, % 100 × W1 W

Unsaponified matter, %

Average

1 2 3

1.2127 1.0400 1.2446

0.0728 0.0729 0.0813

6.0030 7.0093 6.5323

6.5149

Result: The Unsap. matter of C. roxburghianum fatty oil, Joydebpur, Gajipur 6.5149%

b) Keranigonj, Dhaka: Observe no.

Weight of sample, w gm

Weight of residue, W1,gm

% Unsaponified 100 × W1 matter, W

Unsaponified matter, %

Average

1 2 3

1.2568 0.0403 1.3709

0.0503 0.9474 0.0609

4.0023 4.2533 4.4433

4.2326

Result: The Unsap. matter of C. roxburghianum fatty oil, Keranigonj, Dhaka 4.2326%

c) Faridpur.

Observe no.

Weight of sample, w gm

Weight of residue, W1,gm

Unsaponified matter, % 100 × W1 W

Unsaponified matter, %

Average

1 2 3

1.0977 1.0502 1.0523

0.0373 0.0361 0.0402

3.3980 3.4774 3.8202

3.5637

Result: The Unsap. matter of C. roxburghianum fatty oil, Faridpur 3.5637%.

5.4.6 Determination of Peroxide Value of fatty oil 56, 66, 86, 87: The peroxide value is the number of mili-equivalent of active Oxygen that expresses the amount of peroxide contained in 1000g of the substance 87. The technique of determination of peroxide value has already described early in the Section4.5.10. So, in this section calculation data only have shown.

Calculation method: The peroxide value of the fatty oil was calculated by the following formula: VNa 2 S2O3 × S Na 2 S2O3 ×1000 Peroxide value = Weight of sample, W(gm) Strength of Na2S2O3, S Na 2 S2O3 = 0.0870 N (see section- as 4.5.6.3 )

Table-5.12: Titration data for peroxide value of C. roxburghianum fatty oil: a) Joydebpur, Gajipur. Observe no.

Weight of sample, w gm

Burette reading , Volume of Na2SO3 Solution, VNa S O (ml) IBR FBR Diff. 2

Average Peroxide value, Peroxide VNa 2 S 2O3 × S Na 2 S 2O3 ×1000 value W(gm)

1. 0.7494 0.0 0.2 0.2 23.2292 23.2499 25.2219 2. 0.8623 0.2 0.45 0.25 21.2987 3. 1.2254 0.25 0.55 0.3 Result: The Peroxide value of C. roxburghianum fatty oil, Joydebpur, Gajipur 23.2499.

b) Keranigonj, Dhaka Observe no.

1. 2. 3.

Weight of sample, w gm 1.0203 0.7227 0.7960

Burette reading , Volume of Na2SO3 Solution, VNa S O (ml) IBR FBR Diff. 2

0.0 0.4 0.7

0.4 0.7 1.05

0.4 0.3 0.35

Peroxide value,

Average

VNa 2 S 2O3 × S Na 2 S 2O3 ×Peroxide 1000 value W(gm)

34.1066 36.1129 38.2539

36.1578

Result: The Peroxide value of C. roxburghianum fatty oil, Keranigonj, Dhaka 36.1578.

c) Faridpur. Observe no.

1. 2. 3.

Weight of sample, w gm

0.7728 0.9990 1.1311

Burette reading , Volume of Na2SO3 Solution, VNa S O (ml) 2

IBR

FBR

Diff.

0.0 0.25 0.3

0.25 0.55 0.65

0.25 0.3 0.35

Average Peroxide value, Peroxide VNa 2 S 2O3 × S Na 2 S 2O3 ×1 000 value W(gm)

28.1573 26.1243 26.9215

27.0677

Result: The Peroxide value of C. roxburghianum fatty oil, Faridpur 26.0677.

5.4.7 Determination of Reichert−Meissl (RM) value of fatty oil 103, 110: The Reichert–Meissl value is the number of milliliters of 0.1 N sodium hydroxide required to neutralize the volatile and water soluble fatty acids components distilled from 5 gm of the fat or oil under the specific condition of the method 88, 110. This value represents the amount of volatile and water soluble acid of oil.

Reagent required:  0.1N NaOH  1:4 H2SO4  Glycerol-soda solution  Phenolphthalein as an indicator.

Procedure: Weight accurately 5 gm of sample in to a clean 300 ml quick fit flask; add 20 ml of glycerol soda solution, prepared by adding 20 ml (1:1) sodium hydroxide to 180 ml pure concentrated glycerol. Heat the solution for completely saponification. If foaming occurs, shake flask gently and add 135 ml dist. Water to prevent foaming then add 6 ml of (1:4) sulfuric acid as a result of which the soap is converted into sodium sulfate and fatty acids the aqueous mixture then distilled to collect 110 ml distillate and filtered. 100 ml of filtered distillate is titrated against 0.1 N NaOH, using phenolphthalein as an indicator. The blank determination was also carried out observing the same condition but omitting the oil.

Calculation method: If V ml of 0.1 N NaOH are required for the neutralization if 100 ml of the distillate, then R.M. value can be determined by following formula: R.M. value

= (V−B) ×1.1

Where, B = Volume of NaOH required for the blank titration

Table-5.13: (Blank Titration with NaOH solution) Observe. no.

Volume of NaOH ,ml

1.6 1.6

1. 2.

Average, ml

1.6

Result: The blank determination of 0.1 N NaOH, B = 1.6 ml

Table-5.14: Titration data for R.M.value of Carum roxburghianum (Radhuni) fatty oil: a) Joydebpur, Gajipur. Observe. no.

Volume of

R.M. value,

NaOH

(V−B)×1.1

3.9 4.0 3.95

2.53 2.64 2.585

1. 2. 3.

Average R.M. Value

2.585

Result: The R.M.value of Carum roxburghianum (Radhuni) fatty oil, Joydebpur, Gajipur is 2.585

b) Keranigonj, Dhaka. Observe. no. 1. 2. 3.

Volume of

R.M. value,

NaOH

(V−B)×1.1

4.5 4.45 4.35

3.19 3.135 3.025

Average R.M. Value

3.1167

Result: The R.M.value of Carum roxburghianum (Radhuni) fatty oil, Keranigonj, Dhaka is 3.1167

c) Faridpur. Observe. no.

Volume of

R.M. value,

NaOH

(Vâ&#x2C6;&#x2019;B)Ă&#x2014;1.1

3.5 3.4 3.35

2.09 1.98 1.925

1. 2. 3.

Average R.M. Value

1.9983

Result: The R.M.value of Carum roxburghianum (Radhuni) fatty oil, Faridpur is 1.9983

5.4.8 Determination of Poleneske value of fatty oil 103, 110: The Poleneske value is the number of milliliters of 0.1 N sodium hydroxide required to neutralize the water insoluble, but alcohol soluble fatty acids distilled from 5 gm of the fat or oil under the condition of method110.

Procedure: The material left on the filter paper after filtering the distillate during R.M. value determination that is insoluble acids of fatty acids sample. Wash the acids upon the filter paper with three successive 15 ml of distilled water, and then dissolve the insoluble acids by passing three successive 15 ml of neutral 95% alcohol, through the filter paper. Titrate the combined alcoholic washing with 0.1 N NaOH using the using phenolphthalein as an indicator. The blank determination was also carried out observing the same condition but omitting the oil.

Calculation method: If V ml of 0.1 N NaOH is required for the neutralization of the washing combined with alcohol and B is for blank titration, then the Poleneske value can be determined by following formula: The Poleneske value = (V-B) Blank determination for 0.1 N NaOH = 0.8 ml Table-5.15: Titration data for Poleneske value of Carum roxburghianum (Radhuni) fatty oil: a) Joydebpur, Gajipur. Observe. no.

Volume of NaOH

Poleneske value, (V-B)

Average Poleneske value

1. 2.

5.9 5.85

5.1 5.05

5.0667

Result: The Poleneske value of C. roxburghianum fatty oil, Joydebpur, Gajipur 5.0667 b) Keranigonj, Dhaka. Observe. no.

Volume of NaOH

Poleneske value, (V-B)

Average Poleneske value

1. 2.

7.05 6.95

6.25 6.15

6.20

Result: The Poleneske value of C. roxburghianum fatty oil, Keranigonj, Dhaka is 6.20 c) Faridpur. Observe. no.

Volume of NaOH

Poleneske value, (V-B)

Average Poleneske value

1. 2.

4.9 4.95

4.1 4.15

4.1167

Result: The Poleneske value of Carum roxburghianum fatty oil, Faridpur is 4.1167

5.4.9 Determination of Henher Value of fatty oil 103, 110: The amount of insoluble fatty acids plus unsaponifiable matter present in oil or fat is determined by measuring Henher value, which is expressed as percentage of the weight of oil or fat110.

Procedure: The material left in the flask after removing the distillate during R.M. value determination that is insoluble acids of fatty acids sample, is vigorously shaken with water to dissolve the soluble matter and then filtered in a previously weighted filter paper. The residue is dried and weighted.

Calculation method: The Henher value can be determined by following formula: Wieght of the residue

Henher value = Weight of sample(= 5 gm) Ă&#x2014;100

Table-5.16: Titration data for Henher value of Carum roxburghianum (Radhuni) fatty oil: a) Joydebpur, Gajipur: Observe. no.

Weight of sample

Weight of residue

1. 2.

5.0804 5.0001

4.8029 4.6425

Henher value

Average

Wieght of the residue ×100Henher Weight of sample(= 5 gm) value

94.5378 92.8314

93.6846

Result: The Henher value of C. roxburghianum fatty oil, Joydebpur, Gajipur is 93.6846 b) Keranigonj, Dhaka: Observe. no.

Weight of sample

Weight of residue

1. 2.

5.0992 5.0872

4.8821 4.8525

Henher value

Average

Wieght of the residue ×100Henher Weight of sample(= 5 gm) value

95.7425 95.3865

95.5645

Result: The Henher value of C. roxburghianum fatty oil, Keranigonj, Dhaka is 95.5645 c) Faridpur: Observe. no.

Weight of sample

Weight of residue

1. 2.

4.8820 4.5039

3.9920 3.9850

Henher value

Average

Wieght of the residue ×100Henher Weight of sample(= 5 gm) value

81.7697 88.4789

85.1243

Result: The Henher value of C. roxburghianum fatty oil, Faridpur is 85.1243

5.5 Analysis of fatty acids: 5.5.1 Esterification of fatty acids 86, 89: The fatty oil was analyzed as the methyl ester of their fatty acid contents by GLC. During esterification of fatty oil, glycerides and phospholipids were saponified and fatty acids were liberated and esterified in presence of sodium methoxide. Production of fatty acids is the most important phase in the industrial chemistry of acids. The esters produced are of several types and include that resulting from reaction with monohydric alcohols, polyhydric alcohols and ethylene or propylene oxide and acetylene or vinyl acetate. Glycerides are saponified and fatty acids are liberated and esterified in presence of BF 3 catalyst for further analysis by GLC. Method is applicable to common animal and vegetable

oils and fats and fatty acids. Unsaponifiable matter is not removed and if present in large amounts may interfere with subsequent anlalysis.

5.5.1.1 Reagents required for esterification:      

Boron trifluoride –MeOH complex Methanolic sodium hydroxide solution 0.5 N 0.1% Benzoic acid solution Hexane Anhydrous Sodium sulphate Nitrogen

5.5.1.2 Preparation of reagent: a) Boron trifluoride reagent: BF3–MeOH complex of analytical grade. b) 0.5 N Methanolic sodium hydroxide solution: 2.0 gm NaOH was dissolved in 100ml of MeOH containing less than 0.5% water. c) 0.1 % Benzoic acid solution: 0.05g of benzoic acid was dissolved in 50ml of distilled water to prepare 0.1 % benzoic acid solution. d) Standard fatty acids sample: Lauric, myristic, palmitic, Stearic, Oleic, Linoleic and other standard fatty acids from E. Merk 99.5 % pure.

5.5.1.3 Precautions on esterification: The methyl ester preparation was done in fume hood. The glass ware was cleaned after use. A stream of N2 was passed through flask for removing air. Hexane solution was kept under N2 in refrigerator.

5.5.1.4 Preparation of methyl esters of reference fatty acids 66, 113: Reference fatty acids (Lauric, myristic, palmitic, stearic, oleic, linoleic and other standard fatty acids 100mg of each) were taken in a pear-shaped flask. 4ml of 0.5N methanolic sodium hydroxide (NaOH) and some boiling clip was added to the sample. A condenser was attached with it and reflux for 10 minutes. Then 5 ml BF3-CH3OH complex was added from a bulb pipette through condenser and continued boiling for 2 minutes. 5 ml of hexane was added through condenser and boiled for another one minute. Then hot mixture was poured into small separator funnel with 10ml of hexane, 5 ml of distilled water added to it. The funnel was shaken vigorously and layers were allowed to separate. The aqueous methanol layer was drained off and discarded. The hexane layer was dried by treating with anhydrous sodium sulfate, filtered into a quick-fit flask, concentrated to a small volume by using vacuum evaporator and transferred to a vial. Finally a hexane containing mixture of methyl ester of

acids was concentrated to a few drops by blowing N 2 gas and stored in a refrigerator before analysis by GLC.

5.5.1.5 Preparation of methyl esters of sample fatty acid fraction66, 113: 0.1044g of sample fatty acid fraction was taken in a pear shaped flask. 5 ml of 0.5N methanolic NaOH and some boiling chips were added to the sample. A Condenser was attached with the flask and refluxed for 1 hour. The sodium salt of fatty acid was formed. Then the resultant solution was evaporate and 5 ml distilled water, few drops of Conc. HCl (1:1) (When pH was maintain nearly 2.5) 10 ml of hexane and 0.1% 1 ml benzoic acid (as a internal standard) was added to the solution. Then the mixture was poured into a small separator funnel. The funnel was shaken vigorously and the layer was allowed to separate. The aqueous layer was drained off and discarded the hexane layer was collected and dried by treating with anhydrous sodium sulfate (Na 2SO4) filtered into a quick fit flask. Then 2 ml of BF3â&#x2C6;&#x2019;CH3OH complex was added to the flask and continued boiling for 10 minutes. Again 10 ml of hexane was added to the flask and boiled for another 1 minute. Then the hot mixture were concentrated to a sample volume by using thin layer evaporator and transferred to a vial. Finally the hexane containing mixture of methyl ester of acids was concentrated to few drops by blowing N2 gas. The mixture was stored in a refrigerator for TLC analysis and further for GLC experiment.

5.5.2 Determination of Composition of Fatty Oil by Thin Layer Chromatography (TLC) 66, 114â&#x2C6;&#x2019;119: TLC is an instrumental technique as other forms of chromatography.

It was done for

observing the purity of organic compounds. It has several advantages over other forms of chromatography: (a) Sample preparation is usually relatively simple. (b) Sample may be directly compared, often as they are running. (c) Parallel development of related and unrelated samples can be carried out simultaneously. (d) A range of detection procedures can be applied, often to the sane plate. (e) The separation can be followed throughout the whole process and stopped when desired or when the solvent systems are changed. (f) Solvents and other reagents are required in very small volumes.

 Two types of thin layer chromatography (TLC) plates were used: 1) Pre- coated TLC plates: 0.2mm thin coating on glass and aluminum sheets. 2) Manually prepared the glass plates.  The sample fatty acid methyl ester of seed was analyzed initially with the help of thin layer chromatography(TLC) as follows:  Stationary phase: Silica–gel G.  Development: Ascending one dimension.  Thickness of plates layer: 0.2mm.

5.5.2.1 The Technique of TLC: 1) Preparation of TLC plate66, 114−119: The silica gel G was taken with distilled water (50:50 ratios) to produce slurry. The slurry was uniformly spread over the entire surfaces of the glass plates (16x16 cm, 12x12 cm) with the help of spreader. The coated plates were first dried in air then activated by heating at 110°C in an oven for 30 minutes. After activation the plates were kept in a moisture free chamber for further analysis.  Two points of practical importance:

a) TLC plates should never be handled or touched on the surface, but carefully held only by the edges this will avoid possible contamination due to perspiration. b) Precleansing of the plate is advisable in order to remove extraneous material that might be contained in the layer. This may be carried out by running the development solvent to the top of the plate, and then re drying it before use.

2) Preparation of sample solution66, 114−119: The fatty oil of sample was dissolved in n–hexane to make 1% solution.

3) Technique of sample spotting66, 114−119: Small spot of the solution was applied at 2 cm above the lower edge of the activate silica gel G plates with the help of capillary tube. The diameter of the spot was kept below 2 mm. The plate was then dried with a blow drier and a straight line was drawn 2 cm below the upper edge of the activated plates which the solvent front was allowed to travel.

 Precaution :

Care must be taken to avoid disturbing the surface of the adsorbent as these causes. Distorted shapes of the spots on the subsequently developed chromatogram and so hinders quantitative measurement.

4) Selection of the solvent system:

Solvent system Chloroform : methanol

Properties of solvent 4.5 : 0.5

Methanol : Chloroform : acetic acid : water

4.5 : 4: 0.4: 0.1

Chloroform : acetic acid

4.8 : 0.2

Pet-ether : ether : acetic acid

4.5 : 0.5 : 0.05

Hexane : ether : acetic acid

9 :10 : 1

The initial choice of solvent was influenced by the nature and solubility of the mixture. Different solvent systems were employed for the analysis of the compounds of methyl ester on TLC plate. Among the solvent systems Pet. ether (bp 60˚-80˚C) and acetic acid was the only appropriate solvent system. Because, by this system four distinct spots of the methyl ester of sample fatty acid were obtained.

Table –5.17: Different solvent system for TLC. 5) Development of plate66, 114−119: The spotted plate was placed gently in to the chromatographic tank containing the selective solvent system and was allowed to travel up to the mark. While the developing mobile phase reached the pre-marked straight line, the plate was taken out and was dried by air blow. 6) Chamber for saturation66, 114−119:

Cylindrical shaped glass chamber with air tight glass lid was used for the development of plates. The chamber was saturated with the vapours of the respective solvent system in the additional manner.

7) Development of plates66, 114−119: The spotted plate was placed gently in to the chromatographic tank containing the selective solvent system and was allowed to travel up to the mark. While the developing mobile phase reached the pre- marked straight line, the plate was taken out and was dried by air blow.

8) Detection of compounds66, 114−119: Irrigated plates were developed by the following methods to detect the portion of the components.

a) Chemical method: i. Iodine vapours: The developed chromatograph was exposed to iodine vapour for five minutes in a closed jar containing few crystals iodine resolution behavior of the compounds was indicated be the development of brownish spots. ii. By spraying vanillin-H2SO4: The plates were sprayed with 1% vanillin in concentrated H2S04 acid followed by heating in an oven at 120˚C for 10 minutes.

b) Physical methods: The development plates were examined under UV lamp at wavelengths 360nm.

9) Calculation of Rf. value of developed plate: Under constant conditions of temperature, solvent system and adsorbent, any individual solute will move by a Constant ratio with respect to the solvent front. This is known as the Rf. value (relative front or retardation factor) where, Rf. value =

5.5.2.2 TLC for methyl ester fatty acids of seeds:  Stationary phase: Silica gel G.  Solvent system: Hexane : ether : acetic acid = 9 :10 : 1

A B C

Origin

Figure-5.2: Measurement of Rf value Plate-1. Joydebpur, Gajipur. Four sports were found from the development plate. The Rf value were A=0.04, B=0.17, C=0.26, D=0.67 respectively. Plate- 2. Keranigonj, Dhaka. Three spots were found from the development plate. The Rf value, were A=0.108, B=0.60, C = 0.94 respectively. Plate-3.Faridpur. Three sports were found from the development plate. The Rf value were A=0.108, B=0.60, C=0.94 respectively.

A= Fatty acids sample of Carum roxburghianum B= Methyl ester of fatty acids of Carum roxburghianum

Figure -5.3: Thin layer chromatogram of fatty acids and its methyl esters.

5.5.3 Determination of fatty acid Composition of the Fatty oil by Gas Liquid Chromatography 89: Gas-Liquid Chromatography accomplishes a separation by partitioning sample between a mobile gas and a thin layer of nonvolatile liquid held on a solid support. Gas Chromatography analysis is used to identify the basic components of a sample and it gives the information about of each components. Therefore, the fatty oil of sample was analyzed with the help of Gas chromatography for determination of the composition of the extracted fatty oil after proper treatment i.e. methylation. ď ś Usually a Gas chromatograph consists of six parts: 1) A supply of carrier gas in a high pressure cylinder with attendant pressures regulators and flow meters. 2) A sample injection column. 3) The separation column

4) The detector 5) An electrometer and strip chart recorder 6) Separate thermostat compartments for housing the column and detector so as to regulate the temperature. Detector is the most important part of the Gas chromatography. It is a device which measures the concentration of the sample component by generating an electrical signal proportional to the sample concentration. The most important and popular detector is Flame Ionization Detector (FID). Which is useful for detection of most of the sample organic substances. Flame Ionization Detector operates on the principle that the flame is burnt with the separated substance contained in the sample carried by the carrier gas. Depending on the flame temperature ionization takes place so potential difference occurs which is directly electrical proportional to the concentration of charge particles within the gas. The electric signals are amplified and recorded.  The optimum condition of the Gas chromatography analysis depends upon the following variables89: 1) Length and diameter of column. 2) Temperature of column 3) Injector 4) Detector 5) Carrier gas flow rate 6) Speed of the chromatogram 7) Resolution required 8) Size of the sample 9) Time of analysis 10) Flow rate of carrier gas.

5.5.3.1 Apparatus and Operating conditions for Gas Chromatography 133: 1) Gas chromatograph: Trace GC ULTRA, Thermo Electron Corp., Japan. 2) Name of column: SE –54, Capillary column (30 m × 0.25 mm) temperature maintained at 100˚–260˚C 3) Detector: Flame Ionization Detector (FID) 4) Detector temperature: 260˚C 5) Injector temperature: 150˚C 6) Temperature of column: 150˚C (1min) to 180˚C (10min) to 220˚C (1.5min) to 260˚C in total 30 min at the rate of 7˚C/min. 7) Flow rate of carrier gas: 1.3 ml/min. 8) Column Packing : Column packing was done with 10% diethylene glycol

succinate on 100 – 120 mesh diatomic CAW 9) Syringe: 10 µ L Hemilton Syringe, 0.2 µ L Injector. 10) Splitting : 80% 11) Integrator: Integrator, LKB Bromma ; (2220 Recording Integrator). 12) Speed of the chromatogram: 0.5 mm/min.

5.5.3.2 Reagents of GLC: The following reagents are used: a) Carrier gas: Dried N2 gas. b) Reference standards: Known mixtures of methyl esters of fatty acids.

Figure –5.4: Trace GC ULTRA instrument. 5.5.3.3 Procedure for GLC: Methyl ester of fatty oil was diluted to 7% with n–hexane solvent. An inert carrier gas (e.g. N2) was introduced from a large gas cylinder through the port, the column and the detector. The flow rate of the carrier gas was adjusted to insure reproducible retention times and to minimize detector drift. The sample was injected with the help of a micro syringe through a heated injection part. The injected sample was vaporized and carried into the column. The long tube of the column was tightly packed with solid particles. The solid support was uniformly covered with a thin film of a high boiling liquid (the stationary phase). The mobile

and stationary phase was then portioned by the sample and it was separated into its individual’s components. The carrier gas and the sample components were then emerged from the column and passed through the detector. The amount of each component was measured on the basis of its concentration by this device and generated a signal which was registered electrically. This signal was passed to an integrator. The standard reference sample was analyzed following the same procedure.

5.5.3.4 Identification of fatty acids: The fatty acids methyl esters of sample were identified by comparing the retention time of authentic sample as palmitic, Stearic, oleic, linoleic, linolenic, myristic and other fatty acids. Further the peaks on the chart were identified with the help of respective chain lengths of different fatty acids. By measuring the retention times of the fatty acids in the experimental sample and comparing them with those of the standard of the pure substance. The percentage composition was expressed as weight percentage. The fatty acid composition was computed by dividing the corrected peak areas of each peak with sum of the corrected peak areas and multiplying with 100.

Calculation method: The relative percentage of the individual peak calculated as, % of individual component = × 100

5.5.4 Gas chromatographic analysis of fatty acid ester of Carum roxburghianum seeds from different region of Bangladesh: The fatty acid methyl esters of Carum roxburghianum seeds from different region of Bangladesh were analyzed on a Trace GC ULTRA, Thermo Electron Corp., Gas Chromatograph fitted with a flame ionization detector (FID) and an electronic integrator equipped with SE-54 quartz capillary column (30 m × 0.25 mm i .d. and 0.25µm film thickness). Carrier gas nitrogen (N2) at a flow rate of 1.5 ml/min. The separation was affected at 100˚C–220˚C. The following temperature program carried out the GC analysis: Initial temperature 150˚C, increases at 7˚C/min to 260˚C at 30 min. The oven, injection and detection temperatures were fixed at 150˚, 180˚and 220˚C respectively. Splitting 80 %, speed of the chromatogram was 0.5 mm/min. The fatty acids were identified by comparison of relative retention times and peak positions of the chromatogram with that for the standard fatty acids. The amounts of fatty acids were calculated from the peak areas computed by LKB 2220 electronic recording integrator. The fatty acid composition of the extracted oil obtained from this study is represented in Table-5.18 to 5.20. The chromatograms of sample ester of esterified fatty acids are shown in Figure–5.5 to 5.10 respectively and structures of fatty acids found from GLC analysis are shown in Section-5.5.3.6.

Table –5.18: GLC report of Carum roxburghianum seeds, from Joydebpur, Gajipur (dry matter basis): Sample ID: C.ROX-1

a) Percentage of different component in the sample: Peak no.

Name of the component

Retention time

Peak Area (.1*uV*sec)

Relative percentage

Palmitic acid

3.785

49473

5.490

Unknown

3.908

1650

0.183

Unknown

3.982

2903

0.322

Unknown

4.042

1438

0.160

Stearic acid

5.115

527

0.058

Oleic acid

5.480

675340

74.940

Linoleic acid

5.630

18550

2.058

Unknown

5.673

1620

0.180

Unknown

5.803

1151

0.128

10.

Linolenic acid

6.018

136311

15.126

11.

Ecosenoic acid

6.255

4718

0.524

12.

Unknown

6.752

1583

0.176

13.

Unknown

6.910

1427

0.158

14.

Unknown

7.268

1221

0.135

15.

Unknown

7.365

2982

0.331

16.

Unknown

7.708

284

0.031

901178

100.00

ď ś % of Unidentified component: 1.804 b) Percentage of different fatty acids in the sample: (Chromatogram has been subjected to manual integration) Sl. No.

Name of the component

Retention

Peak Area

Relative

time

(.1*uV*sec)

percentage

Palmitic acid

3.785

49208

5.573

Stearic acid

5.112

6188

0.701

Oleic acid

5.482

674907

76.436

Linoleic acid

5.630

12201

1.382

Linolenic acid

6.018

135995

15.402

Ecosenoic acid

6.253

4473

0.506

882972

100.00

Figure â&#x20AC;&#x201C; 5.5: GLC Chromatogram of Carum roxburghianum (Radhuni)

seeds fatty oil from Joydebpur, Gajipur.

Figure â&#x20AC;&#x201C; 5.6: GLC Chromatogram of Carum roxburghianum (Radhuni)

seeds fatty oil from Joydebpur,Gajipur. (Chromatogram has been subjected to manual integration)

Tableâ&#x20AC;&#x201C;5.19: GLC report of Carum roxburghianum seeds, from Keranigonj, Dhaka (dry matter basis): Sample ID: C.ROX-2

a) Peak Name of the component no.

Retention time

Peak Area (.1*uV*sec)

Relative percentage

Unknown

3.367

0.023

Unknown

3.863

396

0.110

Palmitic acid

3.977

17782

4.944

Unknown

4.088

790

0.220

Unknown

4.178

385

0.107

Unknown

4.245

505

0.140

Unknown

4.602

150

0.042

Unknown

4.672

188

0.052

Unknown

4.867

1169

0.325

10. 11. 12. 13. 14. 15. 16. 17.

Unknown Oleic acid Unknown Unknown Unknown Ecosenoic acid Unknown Unknown

5.353 5.695 5.723 5.768 5.998 6.290 7.088 7.325

2551 274095 4786 1468 240 54253 466 269 359575

0.709 76.214 1.331 0.408 0.067 15.085 0.130 0.093 100.00

b) Percentage of different component in the sample: ď ś % of Unidentified component: 3.757 c) Percentage of different fatty acids in the sample:

(Chromatogram has been subjected to manual integration) Sl. No.

Name of the component

Retention time

Peak Area (.1*uV*sec)

Relative percentage

1. 2. 3.

Palmitic acid Oleic acid Ecosenoic acid

3.977 5.695 6.290

17495 271145 53891 342531

5.108 79.159 15.733 100

Fi gureâ&#x20AC;&#x201C; 5.7: GLC Chromatogram of Carum roxburghianum (Radhuni) seeds fatty oil from Keranigonj, Dhaka.

Figureâ&#x20AC;&#x201C; 5.8: GLC Chromatogram of Carum roxburghianum (Radhuni)

seeds fatty oil from Keranigonj, Dhaka. (Chromatogram has been subjected to manual integration)

Tableâ&#x20AC;&#x201C;5.20: GLC report of Carum roxburghianum seeds, from Faridpur (dry matter basis): Sample ID: C.ROX-3

a) Percentage of different component in the sample: Peak no.

Name of the component

Retention time

Peak Area (.1*uV*sec)

Relative percentage

Unknown

3.360

179

0.028

Palmitic acid

3.975

31644

4.917

Unknown

4.087

2347

0.365

Unknown

4.177

630

0.098

Unknown

4.240

828

0.129

Unknown

4.665

232

0.036

Unknown

4.860

2080

0.323

Oleic acid

5.740

495656

77.015

Unknown

5.765

5596

0.870

10.

Unknown

6.008

627

0.097

11.

Linoleic acid

6.315

97404

15.135

12.

Unknown

7.088

771

0.120

13.

Unknown

7.337

568

0.088

14.

Unknown

13.775

212

0.033

638774

99.254

ď ś % of Unidentified component: 2.187

b) Percentage of different fatty acids in the sample: (Chromatogram has been subjected to manual integration) Sl. Name of the component Retention time Peak Area No. (.1*uV*sec) 1.

Palmitic acid

3.975

31382

Relative percentage 4.948

2. 3.

Oleic acid Linoleic acid

5.738 6.315

502044 96906 630332

79.151 15.278 99.377

Fi gure â&#x20AC;&#x201C; 5.9: GLC Chromatogram of Carum roxburghianum (Radhuni) seeds fatty oil from Faridpur.

Fi gure â&#x20AC;&#x201C; 5.10: GLC Chromatogram of Carum roxburghianum (Radhuni) seeds fatty oil from Faridpur. (Chromatogram has been subjected to manual integration)

5.5.5 Structure of fatty acids found from GLC analysis:

Oleic acid IUPAC name: (9Z)-Octadec-9-enoic acid, Molecular formula: C18H34O2 O

Palmitic acid

IUPAC name: Hexadecanoic acid, Molecular formula: C16H32O2 O

Stearic acid

IUPAC name Octadecanoic acid, Molecular formula: C18H36O2,

Ecosenoic acid Linolenic acid Linoleic acid IUPAC name: IUPAC name: IUPAC name: [Z]-Eicos-11-enoic acid, 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-, 9,12-Octadecadienoic acid (Z,Z)-, formula: CMolecular 20H38O2, Molecular formula: C18Molecular H30O2, formula: C18H32O2,

Elemental analysis 6.1 Introduction:

During the past decades, spice and medicinal plants gained a more important role in agronomy production, pharmacy, and exportation because of their increased use as a raw material for the pharmaceutical industry and pharmaceutical preparations and in the everyday life of the general population. In recent years the cultivation of medicinal and aromatic plants has been achieved with increasing interest in many countries of the world. The interest in our country for this plant is much greater because of the possibility of exportation and domestic use.

The knowledge of the trace element content of these plants is important because it may influence the production of its active constituents, and its pharmacological action. Active constituents of medicinal plants are metabolic products of plant cells and a number of trace elements play an important role in the metabolism. It is now well recognized that more than 25 elements are considered to be essential for human metabolism.123 From plant nutrition studies, it is known that plants require a certain amount of trace elements, that they respond differently to an enhanced or lowered trace element supply, and that in some cases, agricultural products may be contaminated with toxic heavy metals.124 There are two major reasons to monitor levels of toxic metals in medicinal plants, the first reason; contamination of the general environment with toxic metals has increased. The sources of this environmental pollution are quite varied, ranging from industrial traffic emissions to the use of purification mud and agricultural expedients. Such as cadmium-containing dung, organic mercury fungicides and the insecticide lead arsenate the second reason, exotic herbal remedies, particularly those of Asian origin have been repeatedly reported to contain toxic levels of heavy metals and/or arsenic125. Several investigators have performed several studies on the residual levels of toxic metals in medicinal herbs. Most studies on residual levels of toxic metals in medicinal herbs have focused on lead, cadmium and mercury. The term heavy metals â&#x20AC;&#x153;vigorously defined but most authors use it to describe metallic elements126 having density greater than 6000 kg/m3. Some essential micro nutrients or trace

elements such as Co, Cu, Fe, Mn, Zn, etc are also termed as heavy metals according to the definition of heavy-metals.” They are essential for animal and plant growth. These micro nutrients and other non-essential trace elements are known to have undesirable effect on plant and animal growth if present in excess concentration both in soil and vegetable. The nonessential heavy metals such as Pb, Cd, Hg and As have prove to be highly toxic. Ni, Mo and Al are moderately and B, Cu, Mn and Zn are relatively feebly toxic. Different source of heavy metals and their cycling in the soil–water–air –organism ecosystem 127

have been shown in the Figure−6.1

It is a matter of interest to analysis of Carum roxburghianum (Radhuni) for its major, minor and trace elemental content because of its great economical importance due to use of a culinary herbs condiments and play a significant role in human health and diseases and day to days use for the same our research program is centered on the quantitative estimation of different of heavy metals present in our daily different plant intake.

6.2 Minerals present in living organisms 128, 129: There is no universally accepted of mineral. As applied to food and nutrition, the term usually refers to elements other than C, H, O and N that are present in foods. These four non mineral elements present primarily in organic molecules and water and constitute about 99% of the total number of concentrations in living systems. Thus mineral elements are present in relatively low concentrations in foods nevertheless, the plays key functional roles in both living system and foods. Historically, minerals have been classified as major or trace, depending on their concentrations in plants and animals. This classification arose at the time when analytical methods were not accomplished for measuring small concentrations of elements with much precision. Thus the term ‘trace’ was used to indicate the present of an element that could not be measured precisely. Today, modern methods and instruction allow for extremely precise and accurate measurement of virtually of all the elements in the periodic table. Nevertheless, the terms major and trace continue to be used to describe mineral elements respectively in higher and lower concentrations in biological system. In our earth’s crust ninety chemical elements occur. Of them about twenty five are known to be essential for life and thus present in living cells. Living cells also contain other elements as they can accumulate nonessential elements from their environment. Living organism contain at least thirty elements of which thirteen are not metals (C, H, O, N, S, P, Cl, F, Br, I, B, Si, As) and the rest metals (Ca, Mg, K, Na, Fe, Cu, Zn, Ni, Co, Mn, Al, Pb, Sn, Mo, N, Cr and Ti). These elements are classified into five groups 128, 129:

The first group includes carbon, hydrogen, oxygen, nitrogen, and sulphur, which are the major compounds of body molecules. These are obtained through intake of water, fats, carbohydrates and proteins. Calcium, potassium, phosphorus, magnesium, sodium and chlorine, which are known to be nutritionally important minerals, are positioned in the second group. These are required in diet in amount greater than 100 mg per day. The third group contains the elements, which are required in very small amount. These are iron, copper, chromium, cobalt, manganese, molybdenum and iodine. The fourth group contains additional elements required for animal nutrition but they have no essential functions in human body. These are arsenic, cadmium, nickel, silicon, tin, and vanadium. The fifth group elements are found in most plants, foods, vegetables, meat, and fishes, dairy food etc. but generally occur in these plants, foods only in trace amounts. Thus, it is necessary to consume a sufficient quantity of variety of plants to meet the requirements of these nutritional elements. Different living organism contain such elements (essential and trace) in different concentrations. An adult human body includes these minerals in a composition mentioned in Table−6.1. Industrial products, burned fuel, fertilizers, pesticides

Rocks in Earth’s Crust

Air

Water Soil

Birds

Fish Plants Domestic animals

Humans Human and animal wastes

Figure−6.1: Sources of heavy metals and their cycling in the soil–water– air organism ecosystem Table−6.1: Mineral composition of an adult human body per day:

Element

Percent of total ash

g/70 kg man

Na K Ca Mg Fe

2 5 39 0.7 0.15

63 150 11600 21 4.5

6.3 Elemental Analysis by WD XRF: 6.3.1 XRF analysis 114, 130−133: Wavelength dispersive X-ray fluorescence (WDXRF) is a technique that has become indispensable when fast, accurate elemental analysis is needed, as when controlling a melt in a steel works or the raw mix at a cement plant. One reason for its popularity in these applications is that its ease of use, and the ruggedness of the equipment, allows quality results to be obtained in plant conditions by operators without advanced analytical skills. The inherent precision, speed, and sample preparation simplicity of WDXRF can often eliminate many of the problems encountered with solution-based methods like ICP and atomic absorption (AA) spectroscopy. WDXRF technology provides freedom from interferences, high sample throughput with superior precision and low detection limits.

Typical field of analysis: • • • • • •

Cement Environmental Geological Metals and alloys Mining Paper

• • • • • •

Petroleum Catalysts Mapping Research and development Plant minerals Forensics

Other analyses include oils and fuel, plastics, rubber and textiles, pharmaceutical products, foodstuffs, cosmetics and body care products, fertilizers, minerals, ores, rocks, sands, slags, cements, heat-resistant materials, glass, ceramics, semiconductor wafers; the determination of coatings on paper, film, polyester and metals; the sorting or compositional analysis of metal alloys, glass and polymeric materials; and the monitoring of soil contamination, solid waste, effluent, cleaning fluids, sediments and air filters.

X-ray fluorescence spectrometry, XRF method inherently very precise, rivals the accuracy of chemical technique used to identify and determine the concentrations of minor elements present in solid powdered and liquid samples. The main advantage of the XRF technique is its very wide dynamic range in elemental concentration that can be measured from beryllium (Be) to uranium (U) in a several percent down to a few ppm at relatively very high speed. It is a variant of the energy dispersive X-ray fluorescence (EDXRF) and wave length dispersive X-ray fluorescence Spectrometer (WDXRF) analysis with special excitation geometry of the primary X-ray beam based on the total reflection phenomenon of X-rays. This new mode of excitation was obtained through the attachment of a module with conventional system. This module is known as the XRF module the EDXRF and WDXRF technique with the XRF module is called the XRF system. The sensitivity of the methods is much higher than that of the conventional XRF method due to lower spectral background leading to and increased signal to background ratio. This technique is one of the most sensitive and very specific analysis tools for multi-element analysis in metallurgical laboratories in the processing of Metallic ores in the cement industry and other research purpose. Modern computer controlled system, operation is fully automatic and result is typically delivered within minutes or even second.

6.3.2 Theory and principle of XRF analysis 130, 131: X-ray is produced when high-speed electrons collide with a solid target (Which can be the material under investigation) these X-rays are often known as primary X-rays. They arise because the high-energy electron beam may penetrate the outer orbital of the atom and collide with electrons in the inner orbitals. These inner orbital electrons may be removed completely from the atom leaving it in an unstable state. To restore stability electron re-arrangement takes place within the atom and energy can be released as X-ray. It is possible to identify certain emission peaks which are characteristic of elements contained in the target. The discreet wavelengths of the peaks which are related to the atomic number of the elements producing them. They are called characteristic X-rays. The detection and measurement of characteristic X-rays are the basis of X-ray spectrometry.

When a beam of short-wavelength primary X-rays (white radiation) strikes the surface of a sample a similar mechanism as described above will cause the target material to emit X-rays at wavelength characteristic of the atoms involved this is called secondary of fluorescence radiation. The term “X-ray fluorescence” should strictly only be used to describe the process of exciting characteristic X-ray from the sample with a beam of primary X-ray; the higher the current the more electrons are released and the more X-rays are produced. By examining the peak heights of the fluorescence radiation it is possible to get an indication of the sample composition and presence of element. The intensity is proportional to the quantity of the corresponding element in the sample.

Figure–6.2: X-ray fluorescence, the photo electric effect

Figure–6.3: 14 K yellow gold karat alloy X-ray spectrum

6.3.3 Characteristic lines130, 131: The production of characteristic X-ray line spectra can be satisfactorily explained in terms of the Bohr Concept of the atom. The nucleus of the atom is surrounded by shells of electrons, each shell having its own principal energy level and within a shell each electron having its own discrete energy. The shells have the nomenclature K, L, M, N and soon depending on the size (atomic number) of the atom. It an incident photon has sufficient energy then it may eject an electron from the innermost (k) shell. This leaves the atom unstable and the “hole” in the K shell is filled by internal rearrangement of the electrons characteristic X-rays generated as a result of a transition into the k shell are called the K series. L-K transitions give rise to Kα lines, and M-K transition to Kβ lines, in the same way transitions into the L shall give rise to lines designated as the L series and so on. It is clear that once a vacancy has been created in the K shell, then transition of an L electron to the K shell causes a vacancy in the L shell and, provided that the atom is large enough, then the L series lines will also be emitted, the same principal applies to the M series lines. If an incident photon has insufficient energy to eject an electron form the K shell but sufficient for ejection from L shell, then the L series (and upwards if the atom is large enough) will be emitted, but not the K series. Elements in the 1st period of the periodic table (H and He) only have a K shell so they cannot, and do not, have a characteristic X-ray spectrum. Similarly, elements in the 2 nd period (Li to F) only have K and L shells so they emit kα lines (L-k) but not Kβ (M-K). The relative intensity of the characteristic lines is a function of the transition probability, which is an exponential function of the difference in the energy states of the electrons in the different orbitals. Although the intensity ratios of the characteristic lines are constant for a given element they change with atomic number. As for example kα : kβ is about 5:1 for Cu, about 3:1 for Sn, and about 25:1 for Al. This rules falls down when the electrons involved in transitions are also involved in valency, such as the M electrons (M-K, Kβ line) in the 3rd period e.g. Sulfur. Indeed X-ray line ratios

and small wavelength shifts are successfully used for X-RF determination of concentrations of different compounds of the same element in the some sample (e.g. S

-2

and SO42-) and in

the study of valency state and compound formation. In general, the K spectrum is more intense than the L spectrum and so for reasons of sensitivity and precision, K lines are preferred for X-ray fluorescence analysis. However, for the larger atoms (Z > ~ 55) the excitation potential for the K spectrum may be too high to achieve optimum excitation and so mostly L lines are used. As a rough guide, k spectra are used in the determination of elements from Z=4-53 (Be-I) and L spectra for Z=55-92 (Cs-U). For the elements with Z from 37 (Rb) to about 60 (Nd) either the K or the L spectrum can be used successfully and the choice often depends on the nature of the sample (Solid, liquid, fusion) and other elements present (spectral interferences). In fact for the lighter elements Z=5(B) to Z=17 (S) it is the Rh L spectrum which is the main contribution to the excitation of the K spectra of these low atomic elements.

K In

Kα1 Kα2 Kβ1

Xray

LI LII LIII Lα1 Lα2 Lβ1

MI MII MIII K lines

fluorescence

L lines

Figure−6.4: Characteristic Lines

photoelectric

MIV MV

analysis, absorption of the

incident radiation yields the characteristic lines from the sample (The peaks) whiles the scattered tube spectrum in the source of the background.

6.3.4 Instrument of X-RF analysis: For measuring the absorbency of the sample a wave length-dispersive X-ray fluorescence spectrometer (WDXRF) was used133. Model: Rigaku, ZSX Primus, Made by: Rigaku, USA.

6.3.4.1 Instrumentation information of Rigaku, ZSX Primus XRF133: The component of an energy dispersive X-ray spectrometer that is Rigaku, ZSX Primus XRF instrument developed in this laboratory consists of:

1) A Primary X-ray source: In an X-ray spectrometer there are two sources of X-ray: a) The X-ray tube. In the X-ray tube the excitation is provided by electrons from the tube Filament, it consists of : ♦ ♦ ♦ ♦ ♦

Filament, Acceleration voltage, Current, Vacuum, Anode (target) material: 4 KW Rh anode X-ray tube,

b) The Sample : In the sample the excitation is provided by photons of the X-ray tube. 2) Sample holder: Manual 3) An X-ray detector : Two detector automatically run, i.

SC detector (scintillation detector): for wave length shorter than 0.336 nm (Xenon filled detector for mid range in tendon with flow counter.

ii.

F-PC detector (Follow proportion detector): for wave length longer than 0.154 nm 4) A multi channel analyzer (MMCA): Manual

5) Crystals for determination of elements: a) Lif1− crystals, b) Lif 200 − crystals, c) PET − crystals d) Ge − crystals,

e) RX25, RX35, RX40, RX45, RX60, RX61, RX61F, RX70, RX75 crystals

6) Operating software: Optional 7) Monitor & operating system: A windows vista base computer is dedicated to this system for online X-RF data analysis.

Filter: Five type of filter automatically run to detect different element, a) Zr–filter, b) Cu−filter, c) Ti–filter, d) Al−filter, e) Be–filter (optional).

9) Slit: Three slit automatically run, a) S2: standard slit for detector SC. High resolution is obtained b) S4: standard slit for detector PC c) S8: Resolution lowers, but high X-ray intensities are obtained

10) Diaphragm: Different diameter’s Diaphragm automatically run, a) Standard type : 35 mm, 30 mm, 20 mm, 10 mm, 1.0 mm, 0.5 mm in diameter. b) USA type

: 35 mm, 28 mm, 20 mm, 10 mm, 1.0mm, 0.5 mm in diameter.

11) Gas use: a) P-10 (90%Argon+10% methane) gas: For solid or powder type sample.

b) 96% He + 4% iso-C4H10 gas: For liquid sample.

Figure–6.5: Rigaku, ZSX primus XRF instrument 6.3.5 Technique of XRF analysis 131−133: Moisture and organic maters free dried sample were made into small disc, and then it is ready for x-ray analysis. These are the procedure of XRF analysis.

6.3.5.1 Chemicals for preparation of tablet or disc:  Base powder - Boric acid, (A.R. grade Sigma/ E. Merk),  Binder - Stearic acid, (A.R. grade Sigma/ E. Merk).

6.3.5.2 Technique of sample preparation131−133: The biological materials burned to ashes at 400±50˚C temperature for approximately 5 hours for burning the organic maters present in the sample. The ash found is cooled and crushed for 20 minutes in a plenetary ball mill (PM-200, Retsch, Germany) to make powder from in well mixing conditions. The powder sample then pulverized in a pulverized machine. The finely

ground powder (< 75 µm) was then put in a porcelain crucible and dried at 110˚C in an oven for 6 hours to remove moisture. The dried powder samples were mixed with binder (stearic acid: sample at a ratio of 1:10) and pulverized in a pulverized machine for 2 minutes. The resulting mixture was spooned into an aluminum cap (30mm height and 3 cm diameter in inner side) with base powder, boric acid (if necessary). The cap was sandwiched between two tungsten carbide pellets using a manual hydraulic press with 10 tons/sq. inch. for 2 minutes and finally pressure was released slowly. The desired tablet or pellet was prepared for XRF analysis. The moisture content (at 110˚C) of the ash powder was recorded for XRF data imputation.

6.3.5.3 Precaution of XRF analysis131−133: X-RF is a surface analysis technique even small amount of contamination such as fingerprint, deposits, dust and moisture may thus lead to significant increases in the measured values. So contamination is to be avoided during the sample preparation.

Figure−6.6: Making of disc for XRF analysis.

Figure−6.7: Pulverize machine.

Figure−6.8: A

window of

Spectrometer status while analysis running

6.4 Determination of the elements and their respective Concentration of Carum roxburghianum seeds by X-RF analysis: 6.4.1 X-RF analysis of Carum roxburghianum (Radhuni) seeds of Joydebpur, Gajipur: Carum roxburghianum seeds sample burned to ashes at 400±50˚C temperature for approximately 5 hours for burning the organic maters present in the sample then the ash sample was made to the desired tablet or pellets by the method describe in Section−6.3.5 Thus the desired tablet or pellet was prepared for XRF analysis. The moisture content (at 110˚C) of the ash powder was recorded for XRF data imputation.

Table-6.2: Analytical information of Carum roxburghianum seeds of Joydebpur, Gajipur. Elemental analysis of seeds sample was carried out by WDXRF in which calculate and fixed calculation methods were used in X-ray fluorescence spectrometer (Rigaku, ZSX Primus XRF instrument). R.M.S. Sum before normalization Normalized to Sample type Initial Sample Weight (g) Weight after pressing(g) Correction applied for medium Correction applied for film Used Compound list Results database

0.000 50.6% 99.99% Pressed powder 1.5 3.1818 No None Oxides iq+

Analyte

Filter

Crystal

Slit

Detector

Voltage

current

Na Mg Al Si P S Cl K Ca Ti Cr Mn Fe Ni Cu Zn Br Rb Sr Zr Nb Ba

F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE F–BE

RX25 RX25 PET PET Ge Ge Ge Ge LiF1 LiF1 LiF1 LiF1 LiF1 LiF1 LiF1 LiF1 LiF1 LiF1 LiF1 LiF1 LiF1 LiF1

S4 S4 S4 S4 S4 S4 S2 S2 S4 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2 S2

PC PHA 100-250 PC PHA 100-250 PC PHA 100-30 PC PHA 100-300 PC PHA 100-300 PC PHA 150-300 PC PHA 150-300 PC PHA 100-300 PC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300 SC PHA 100-300

30 KV 3O KV 3O KV 3O KV 3O KV 3O KV 3O KV 4O KV 4O KV 5O KV 5O KV 5O KV 5O KV 5O KV 5O KV 5O KV 5O KV 5O KV 5O KV 5O KV 5O KV 5O KV

100 mA 100 mA 100 mA 100 mA 100 mA 100 mA 100 mA 75 mA 67 mA 60 mA 60 mA 60 mA 60 mA 60 mA 60 mA 60 mA 60 mA 60 mA 60 mA 60 mA 60 mA 60 mA

Table-6.3: Analytical condition of Rigaku, ZSX Primus XRF instrument to Carum roxburghianum seeds of Joydebpur, Gajipur.  Others common features of analysis condition:

X-ray (Anode) Diaphragm Attenuator Temperature Atmosphere PR Gas Water Outer pressure Inner pressure

On (Rh 4.0KW) 30 mm 1/1 36.6Ë&#x161; C Vacuum APC off Normal (out put pressure 80kpa) Normal 12.5 Pa 5.5 Pa

Table−6.4: Peak Identification Result of Carum roxburghianum seeds of Joydebpur, Gajipur: Sample ID: C.ROX-1

Application: EZS001XNV

Spectrum No.

Peak position (deg)

Peak int. (kcps)

BG int. (kcps)

Element line

15.345 15.605 16.524 17.592 18.524 21.361 22.536 23.793 25.173 26.646 29.980 37.560 41.824 45.055 48.695 51.784 56.744 57.553 62.422 63.011 69.384 77.299 79.264 86.175 113.235 136.796 92.941 110.833 139.140 141.167 109.133 142.322 144.858 38.683 47.004 48.223

3.003 17.489 35.016 102.324 110.718 1.782 13.362 4.619 24.340 16.897 2.034 3.395 17.097 3.750 3.929 41.562 1.072 210.243 1.409 5.297 8.586 0.612 0.212 2.515

26.423 27.568 31.606 36.600 56.475 15.164 12.807 10.915 8.835 7.172 5.047 2.512 1.715 1.279 0.966 1.016 0.829 1.020 0.412 0.420 0.237 0.146 0.109 0.095 4.508 5.865 0.696 0.904 5.889 8.526 2.716 2.885 1.469 0.526 0.106 0.089

Rh – KB2 Rh – KB1 Rh – KB1– Compton Rh – KA

Heavy

Ca – KA K– KA Cl– KA S– KA P– KA Si– KA Al– KA Mg– KA Na– KA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 1 1 1 1 2 1 1 2 1 1 2

1185.432

624.573 11.339 102.720 34.443 639.857 395.251 6.417 103.764 39.767 2.281 0.127

Model: Bulk

Rh – KA – Compton Nb – KB1

Nb – KA Zr– KA

Sr – KB1

Rb– KB1 Sr– KA Rb– KA Br – KB1 Br– KA Zn– KB1 Zn– KA Cu– KA Ni– KA Fe– KB1 Mn– KB1 Fe– KA Cr– KB1 Mn– KA Cr– KA Ti– KB1 Ba– LB1 Ti– KA Ba–LA Ca– KA K– KA Cl– KA S– KA P– SKA3 P– KA Si– KA Al– SKA3 Al– KA Mg– KA Na– KA P – KA – 2nd

Figureâ&#x2C6;&#x2019;6.9: XRF spectrum for heavy metals of Carum roxburghianum seeds of Joydebpur, Gajipur.

Figureâ&#x2C6;&#x2019;6.10: XRF spectrum for minor elements of Carum roxburghianum seeds sample of Joydebpur, Gajipur.

Table−6.5: SQX Calculation Result of Carum roxburghianum seeds sample as oxide of Joydebpur, Gajipur. Sample ID: C.ROX – 1 No. Component Result

Application: EZS001XNV Unit

Det. limit

El. Line

Model: Bulk Intensity

w/o normal

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

]

Na2O MgO Al2O3 SiO2 P2O5 SO3 Cl K2O CaO TiO2 Cr2O3 MnO Fe2O3 NiO CuO ZnO Br Rb2O SrO ZrO2 Nb2O5 BaO

0.717 5.11 5.52 22.6 16.3 3.97 0.668 9.55 29.3 0.399 0.461 0.164 4.74 0.0477 0.0349 0.120 0.0051 0.0452 0.0496 0.0155 0.0032 0.0854

mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass% mass%

0.01816 0.01080 0.00801 0.01245 0.00933 0.00479 0.00555 0.00627 0.00852 0.07822 0.00677 0.00509 0.00581 0.00302 0.00271 0.00234 0.00144 0.00830 0.00156 0.00183 0.00175 0.03436

Na – KA Mg – KA Al – KA Si – KA P – KA S – KA Cl – KA K – KA Ca – KA Ti – KB1 Cr – KA Mn – KA Fe – KA Ni – KA Cu – KA Zn – KA Br – KA Rb –KB1 Sr– KA Zr – KA Nb – KA Ba – LB1

2.2806 0.5151 39.7665 3.6691 103.7636 3.9610 395.2514 16.2500 639.8567 11.7216 102.7203 2.8501 11.3388 0.4796 624.5725 6.8595 1185.432 21.0573 0.6116 0.2867 8.5865 0.3308 5.2975 0.1176 210.2426 3.4056 3.9293 0.0342 3.7502 0.0251 17.0971 0.0860 2.0342 0.0037 4.6191 0.0324 24.3398 0.0356 13.3616 0.0111 1.7815 0.0023 0.2121 0.0613

Tableâ&#x2C6;&#x2019;6.6: Chemical conversions chart (Oxide to Element and Element to oxide)134:

% of Element × factor =% of Oxide Al 1.8899 Al2O3 Ba 1.1165 BaO C 3.6633 CO2 Ca 1.3992 Cao Co 1.2715 CoO Cr 1.4615 Cr2O3 Cu 1.2513 CuO Fe 1.2865 FeO Fe 1.4297 Fe2O3 H2 9.0000 H2O K 1.2046 K2O Li 2.1526 Li2O Mg 1.6579 MgO Mn 1.2913 MnO Na 1.3480 Na2O Nb 1.4305 Nb2O5 Ni 1.2725 NiO P 2.2912 P2O5 Rb 1.0936 Rb2O S 2.4953 SO2 Si 2.1392 SiO2 Sr 1.1820 SrO Th 1.1379 ThO2 Ti 1.6681 TiO2 U 1.1793 U3O8 Zn 1.2447 ZnO Zr 1.3508 ZrO2  Carbonates CaO 1.7848 CaCO3 MgO 2.0915 MgCO3  Units of Concentration • 1%=10000 ppm • 1ppm=1000 ppb • 1ppm=1 g/t (gram per metric tonns)

× factor 0.5291 0.8957 0.2730 0.7147 0.7864 0.6842 0.7988 0.7773 0.6994 0.1111 0.8302 0.4645 0.6032 0.7744 0.7919 0.6991 0.7858 0.4365 0.9144 0.4008 0.4675 0.9450 0.8788 0.5995 0.8480 0.8033 0.7403

= % of Element Al Ba C Ca Co Cr Cu Fe Fe H2 K Li Mg Mn Na Nb Ni P Rb S Si Sr Th Ti U Zn Zr

0.5603 0.4781

CaO MgO

Example:  If you wants to convert K2O wt% to K in ppm you have to: K (ppm) = K2O wt% × 0.8302 × 1000 (or simply the oxide by 8302).  If you wants to convert K wt% to K2O in ppm you have to: K2O (ppm) = K wt% ×1.2046 × 1000 (or simply the oxide by 12046).

Table−6.7: Element identified by X-RF and their respective concentration found in Carum roxburghianum seeds of Joydebpur, Gajipur with the method: Identified element concentration as Oxide was converting to element percent by chemical conversion process as describe in Table−6.6. No.

Component as Oxide

% as Oxide

× factor

Component as Element

% as Element

1 2 3 4 5 6 7

Na2O MgO Al2O3 SiO2 P2O5 SO3 Cl

0.717 5.11 5.52 22.6 16.3 3.97 0.668

0.7919 0.6032 0.5291 0.4675 0.4365 0.4008 ---

Na Mg Al Si P S Cl

0.5678 3.0824 2.9206 10.5655 7.1150 1.5912 0.668

8 9 10 11 12 13 14 15 16 17 18 9 20 21 22 23

K2O CaO TiO2 Cr2O3 MnO Fe2O3 NiO CuO ZnO Br Rb2O SrO ZrO2 Nb2O5 BaO

9.55 29.3 0.399 0.461 0.164 4.74 0.0477 0.0349 0.120 0.0051 0.0452 0.0496 0.0155 0.0032 0.0854

0.8302 0.7147 0.5995 0.6842 0.7744 0.6994 0.7858 0.7988 0.8033 --0.9144 0.9450 0.7403 0.6991 0.8957

Total

K Ca Ti Cr Mn Fe Ni Cu Zn Br Rb Sr Zr Nb Ba O 99.9056

7.9284 20.9407 0.2392 0.3154 0.1270 3.3152 0.0375 0.0279 0.0964 0.0051 0.0413 0.0469 0.0115 0.0022 0.0765 40.2717

 Normalized up to : 99.99%  Where minimum detection limit is 0.01%.  Each value represents the average value from three experiments.

6.4.2 X-RF analysis of Carum roxburghianum (Radhuni) seeds of Keranigonj, Dhaka: Carum roxburghianum seeds sample burned to ashes at 400±50˚C temperature for approximately 5 hours for burning the organic maters present in the sample then the ash sample was made to the desired tablet or pellets by the method describe in Section−6.3.5 Thus the desired tablet or pellet was prepared for XRF analysis. The moisture content (at 110˚C) of the ash powder was recorded for XRF data imputation. Analytical condition was as same as to the condition of Joydebpur, Gajipur sample in the Section−6.4.1 & Table−6.3

Table−6.8: Analytical information of Carum roxburghianum seeds of Keranigonj, Dhaka:

Elemental analysis of seeds sample was carried out by WDXRF in which calculate and fixed calculation methods were used in X-ray fluorescence spectrometer (Rigaku, ZSX primus XRF instrument). R.M.S. Sum before normalization Normalized to Sample type Initial Sample Weight (g) Weight after pressing(g) Correction applied for medium Correction applied for film Used Compound list Results database

0.000 60.60% 99.99% Pressed powder 1.5020 2.7848 No None Oxides iq+

Table−6.9: Peak Identification Result of Carum roxburghianum seeds of Keranigonj, Dhaka. Sample ID: C.ROX-2

Application: EZS001XNV

Spectrum No.

Peak position (deg)

Peak int. (kcps)

BG int. (kcps)

Element line

15.606 16.528 17.588 18.519 22.532 23.786 25.166 26.643 29.980

18.146 30.701 102.650 120.844 12.321 4.156 23.698 15.172 2.004

24.771 30.967 30.874 55.939 14.279 12.127 10.268 8.622 5.847

Rh – KB1 Rh – KB1– Compton Rh – KA Rh – KA– Compton Zr – KA Sr – KB1 Rb – KB1 Sr– KA Rb– KA Br – KB1 Br– KA

Heavy

1 2 3 4 5 6 7 8 9

Model: Bulk

Ca – KA K– KA Cl– KA S– KA P– KA Si– KA Al– KA Mg– KA Na– KA

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 1 1 1 1 2 1 1 2 1 1 2

37.562 40.492 41.821 43.777 45.056 48.694 51.783 56.733 57.553 62.433 63.008 69.386 77.305 86.173 87.178 113.240 136.801 92.941 110.845 139.136 141.169 109.144 142.346 144.887 38.682 44.457 46.993

3.603 0.943 17.152 0.903 3.911 3.981 43.739 1.255 223.810 1.816 5.768 9.374 0.489 2.541 0.234 1127.074

580.142 9.884 99.543 30.043 566.614 342.444 5.333 87.040 34.961 0.029 2.007

2.734 2.031 1.843 1.567 1.386 1.012 1.402 0.954 1.074 0.453 0.440 0.264 0.176 0.082 0.099 4.437 6.071 0.720 0.852 5.334 7.632 2.331 2.467 1.313 0.478 0.126 0.105

Zn– KB1 Cu– KB1 Zn– KA Ni– KB1 Cu– KA Ni– KA Fe– KB1 Mn– KB1 Fe– KA Cr– KB1 Mn– KA Cr– KA Ti– KB1 Ti– KA Ba–LA* Ca– KA K– KA Cl– KA S– KA P– SKA3 P– KA Si– KA Al– SKA3 Al– KA Mg– KA Na– KA

Figureâ&#x2C6;&#x2019;6.11: XRF Spectrum for heavy metals of Carum roxburghianum seeds of Keranigonj, Dhaka.

Figureâ&#x2C6;&#x2019;6.12: XRF Spectrum for minor elements of Carum roxburghianum seeds of Keranigonj, Dhaka.

Table−6.10: SQX Calculation Result of Carum roxburghianum seeds sample as oxide of Keranigonj, Dhaka. Sample ID: C.ROX−2 No. Component Result

Application: EZS001XNV Unit

Det. limit

El. Line

Model: Bulk Intensity

w/o normal

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Na2O MgO Al2O3 SiO2 P2O5 SO3 Cl K2O CaO TiO2 Cr2O3 MnO Fe2O3 NiO CuO ZnO Br Rb2O SrO ZrO2

0.686 mass% mass% 4.98 mass% 5.11 mass% 21.5 mass% 15.7 mass% 4.16 0.631 mass% mass% 9.62 mass% 30.5 0.403 mass% 0.557 mass% 0.198 mass% mass% 5.62 0.0546 mass% 0.0412 mass% 0.136 mass% 0.0057 mass% 0.0461 mass% 0.0549 mass% 0.0153 mass%

0.02047 0.01136 0.00828 0.01272 0.00955 0.00509 0.00611 0.00665 0.00917 0.01323 0.00808 0.00625 0.00659 0.00358 0.00318 0.00276 0.00177 0.00985 0.00189 0.00224

Na − KA 2.0069 Mg – KA 34.9612 Al – KA 87.0403 Si – KA 342.4443 P – KA 566.6138 S – KA 99.5426 Cl – KA 9.8843 K – KA 580.1423 Ca – KA 1127.0735 Ti – KA 2.5406 Cr – KA 9.3735 Mn – KA 5.7676 Fe – KA 223.8098 Ni – KA 3.9806 Cu – KA 3.9115 Zn – KA 17.1521 Br – KA 2.0042 Rb –KB1 4.1565 Sr – KA 23.6976 Zr – KA 12.3214

0.4497 3.2652 3.3500 14.0993 10.2823 2.7258 0.4133 6.3066 19.9632 0.2643 0.3649 0.1296 3.6843 0.0358 0.0270 0.0892 0.0037 0.0302 0.0359 0.0100

Table−6.11: Element identified by X-RF and their respective concentration found in Carum roxburghianum seeds of Keranigonj, Dhaka with the method. Identified element concentration as oxide was converting to element percent by chemical conversion process as describe in Table−6.6. No.

Component as Oxide

% as Oxide

× factor

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 9 20 21

Na2O MgO Al2O3 SiO2 P2O5 SO3 Cl K2O CaO TiO2 Cr2O3 MnO Fe2O3 NiO CuO ZnO Br Rb2O SrO ZrO2

0.686 4.98 5.11 21.5 15.7 4.16 0.631 9.62 30.5 0.403 0.557 0.198 5.62 0.0546 0.0412 0.136 0.0057 0.0461 0.0549 0.0153

0.7919 0.6032 0.5291 0.4675 0.4365 0.4008 --0.8302 0.7147 0.5995 0.6842 0.7744 0.6994 0.7858 0.7988 0.8033 --0.9144 0.9450 0.7403

Total

Component as Element Na Mg Al Si P S Cl K Ca Ti Cr Mn Fe Ni Cu Zn Br Rb Sr Zr O 100.0188

% as Element 0.5432 3.0040 2.7037 10.0513 6.8531 1.6673 0.631 7.9865 21.7984 0.2416 0.3811 0.1533 3.9306 0.0429 0.0329 0.1092 0.0057 0.0422 0.0519 0.0113 39.7588

 Normalized up to : 99.99%  Where minimum detection limit is 0.01%.  Each value represents the average value from three experiments.

6.4.3 X-RF analysis of Carum roxburghianum seeds of Faridpur: Carum roxburghianum seeds sample burned to ashes at 400±50˚C temperature for approximately 5 hours for burning the organic maters present in the sample then the ash sample was made to the desired tablet or pellets by the method describe in Section−6.3.5. Thus the desired tablet or pellet was prepared for XRF analysis. The moisture content (at 110˚C) of the ash powder was recorded for XRF data imputation. Analytical condition was as same as to the condition of Joydebpur, Gajipur sample in the Section−6.4.1 & Table−6.3

Table−6.12: Analytical information of Carum roxburghianum seeds sample from Faridpur: Elemental analysis of seeds sample was carried out by WDXRF in which calculate and fixed calculation methods were used in X-ray fluorescence spectrometer (Rigaku, ZSX primus XRF instrument). R.M.S. Sum before normalization Normalized to Sample type Initial Sample Weight (g) Weight after pressing(g) Correction applied for medium Correction applied for film Used Compound list Results database

0.000 60.60% 99.99% Pressed powder 1.5023 3.0972 No None Oxides iq+

Table−6.13: Peak Identification Result of Carum roxburghianum seeds sample from Faridpur. Sample ID: C.ROX-3

Application: EZS001XNV

Spectrum No.

Peak position (deg)

Peak int. (kcps)

BG int. (kcps)

Element line

15.615 16.520 17.592 18.518 22.537 23.784 25.175 26.642 29.962 37.563 41.825 45.048 48.705 51.786 56.712 57.554 62.425 62.999 69.388 77.273 79.272 86.178 87.179 113.244 136.804 92.946 110.838 139.156 141.170 109.149 142.360 144.900 38.683 47.001 48.038

16.120 29.406 98.855 126.727 12.696 4.314 24.201 15.414 1.986 3.788 16.366 3.716 1.266 40.575 1.089 212.881 1.660 5.137 8.406 0.483 0.207 2.698 0.191

26.719 32.424 37.164 55.718 13.693 11.819 9.696 8.327 5.614 2.766 1.731 1.440 1.000 1.262 0.983 1.086 0.484 0.474 0.306 0.190 0.117 0.098 0.091 4.445 5.847 0.696 0.783 5.195 7.396 2.526 2.628 1.443 0.505 0.104 0.090

Rh – KB1 Rh – KB1– Compton Rh – KA Rh – KA– Compton Zr– KA Sr – KB1 Rb– KB1 Sr – KA Rb– KA Br– KB1 Br– KA Zn– KB1 Zn– KA Cu– KA Ni– KA Fe– KB1 Mn– KB1 Fe– KA Cr– KB1 Mn– KA Cr– KA Ti– KB1 Ba– LB1 Ti– KA Ba– LA Ca– KA K– KA Cl– KA S– KA P– SKA3 P– KA Si– KA Al– SKA3 Al– KA Mg– KA Na– KA Zn – LA

Heavy

Ca – KA K– KA Cl– KA S– KA P– KA Si– KA Al– KA Mg– KA Na– KA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1 1 1 1 1 2 1 1 2 1 1 2

1146.386

573.286 10.271 86.380 29.565 556.552 358.946 5.991 94.260 36.290 2.147 0.117

Model: Bulk

Figureâ&#x2C6;&#x2019;6.13: XRF Spectrum for heavy metals of Carum roxburghianum seeds of Faridpur.

Figureâ&#x2C6;&#x2019;6.14: XRF Spectrum for minor elements of Carum roxburghianum

seeds sample from Faridpur. Table−6.14: SQX Calculation Result of Carum roxburghianum seeds sample as oxide from Faridpur: No. Component Result

Unit

Det. limit

El. Line

Intensity

w/o normal

1 Na2O 0.732 mass% 0.01995 Na – KA mass% 2 MgO 5.10 0.01157 Mg – KA mass% 3 Al2O3 5.47 0.00868 Al – KA mass% 4 SiO2 22.4 0.01307 Si – KA mass% 5 P2O5 15.4 0.00942 P – KA mass% 6 SO3 3.59 0.00484 S – KA 7 Cl 0.649 mass% 0.00593 Cl – KA mass% 8 K2O 9.41 0.00649 K – KA mass% 9 CaO 30.5 0.00899 Ca – KA 10 TiO2 0.424 mass% 0.01294 Ti – KA mass% 11 Cr2O3 0.494 0.00849 Cr – KA 12 MnO 0.174 mass% 0.00626 Mn – KA mass% 13 Fe2O3 5.28 0.00657 Fe – KA 14 NiO 0.0170 mass% 0.00351 Ni – KA 15 CuO 0.0384 mass% 0.00318 Cu – KA 16 ZnO 0.127 mass% 0.00265 Zn – KA 17 Br 0.0055 mass% 0.00169 Br – KA mass% 18 Rb2O 0.0469 0.00952 Rb –KB1 19 SrO 0.0548 mass% 0.00180 Sr– KA 20 ZrO2 0.0154 mass% 0.00213 Zr – KA 21 BaO 0.0679 mass% 0.02908 Ba – LA Sample ID: C.ROX – 3 Application: EZS001XNV

2.1473 0.4844 36.2899 3.3741 94.2604 3.6201 358.9459 14.8138 556.5524 10.1895 86.3802 2.3765 10.2707 0.4295 573.2861 6.2294 1146.3856 20.2260 2.6977 0.2804 8.4062 0.3273 5.1367 0.1154 212.8811 3.4968 1.2661 0.0113 3.7164 0.0254 16.3660 0.0842 1.9857 0.0037 4.3139 0.0310 24.2008 0.0363 12.6960 0.0102 0.1911 0.0450 Model: Bulk

Table−6.15: Element identified by X-RF and their respective concentration found in Carum roxburghianum seeds of Faridpur with the method:

Identified element concentration as oxide was converting to element percent by chemical conversion process as describe in Table−6.6 No.

Component as Oxide

% as Oxide

× factor

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Na2O MgO Al2O3 SiO2 P2O5 SO3 Cl K2O CaO TiO2 Cr2O3 MnO Fe2O3 NiO CuO ZnO Br Rb2O SrO ZrO2 BaO

0.732 5.10 5.47 22.4 15.4 3.59 0.649 9.41 30.5 0.424 0.494 0.174 5.28 0.0170 0.0384 0.127 0.0055 0.0469 0.0548 0.0154 0.0679

0.7919 0.6032 0.5291 0.4675 0.4365 0.4008 --0.8302 0.7147 0.5995 0.6842 0.7744 0.6994 0.7858 0.7988 0.8033 --0.9144 0.9450 0.7403 0.8957

Total

Component as Element

% as Element

Na Mg Al Si P S Cl K Ca Ti Cr Mn Fe Ni Cu Zn Br Rb Sr Zr Ba O 99.9959

0.5797 3.0763 2.8942 10.472 6.7221 1.4389 0.649 7.8122 21.7984 0.2542 0.3380 0.1347 3.6929 0.0134 0.0307 0.1020 0.0055 0.0429 0.0518 0.0114 0.0608 39.8189

 Normalized up to : 99.99% 

Where minimum detection limit is 0.01%.



Each value represents the average value from three experiments.

6.5 Elemental analysis comparatively on fresh weight basis (mg/100g): Human and other animals take plants materials as fresh not ash, so it is necessary to calculate the percentage of element in Carum roxburghianum (Radhuni) seeds as fresh weight basis 134, 135 . Element percentage on Fresh Wt. Basis can be calculated by following formula: Element percentage on Fresh Wt. Basis = E×A×D×10÷ N mg/100g Where, D = Dry Matter (g/100g) A = Ash (g/100g) N = Normalized up to (%)

Element percentage of dry value (gm/100gm)

Table−6.16: Elemental analysis comparatively on fresh weight basis (mg/100g) calculated from their dry values in Carum roxburghianum seeds of different places in Bangladesh. No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Component as Element

Name of the region Joydebpur, Gajipur Keranigonj, mg/100g Dhaka, mg/100g

Na Mg Al Si P S Cl K Ca Ti Cr Mn Fe Ni Cu Zn Br Rb Sr Zr Nb Ba O Total

46.05687 250.0276 236.9033 857.0163 577.1304 129.0696 54.18455 643.109 1698.597 19.40261 25.58354 10.30155 268.9111 3.041798 2.263097 7.819447 0.413684 3.350033 3.804275 0.932818 0.178452 6.205267 3267.158 8111.460291

43.71706 241.7637 217.5954 808.9347 551.5416 134.1853 50.78326 642.7583 1754.348 19.44411 30.67116 12.33768 316.337 3.452618 2.647812 8.788482 0.458739 3.396281 4.176943 0.909431 0 0 3199.812 8048.059576

Faridpur, mg/100g 42.24593 224.1869 210.9163 763.1522 489.8764 104.8606 47.2962 569.318 1588.569 18.52495 24.63192 9.81633 269.1219 0.976532 2.237278 7.433301 0.400815 3.126359 3.774951 0.830781 0 4.43083 2901.822 7287.549477

6.6 Determination of the elements and their respective Concentration of Carum roxburghianum seeds by AAS analysis: Model of the AAS133:  Varian Cary 50 Conc UV-Visible Spectrophotometer  Chemito AA 203  Varian Spectra AA 55B Atomic absorption Spectrophotometer Flow gas: N2O Flame: Oxy acetylene flame

Minimum detection Limit: ppb for Varian Spectra AA 55B Atomic absorption Spectrophotomete & ppm for Chemito AA 203 and Varian Cary 50 Conc UV-Visible Spectrophotometer. Lamps Use: Hollow Cathode Lamps Digestion mixture: Nitric acid: Perchloric acid (5:1) A) Digestion of the plant material for mineral analysis 165: 0.5 g of the plant sample and 5 ml acid mixture (Nitric/Perchloric acidâ&#x2C6;&#x2019;5:1) was taken into a 250 ml Kjeletec Auto Plus II mineralization set. The treated samples were left until the 4 to 5 hours when complete mineralization was carried out. After the solution became colourless or clear the solutions were allowed to cool. The mineralized sample was diluted with water to a volume of 15 ml and filtered into a dry flusk71, 165. B) Elemental analysis by AAS Technique166, 71, 74: From the digest sample aliquots was used in different volume to estimate different element content. The minerals such as P, S, and B were determined with an Spectrophotometer, Model: Varian Cary 50 Conc UV-Visible Spectrophotometer with respective hollow cathode lamps. K, Ca, Mg, Fe, Mn, and Zn were determined with an Atomic absorption Spectrophotometer, Model: Chemito AA 203 with respective hollow cathode lamps. Pb, Cd, Ni and Cr were determined with an Atomic absorption Spectrophotometer, Model: Varian Spectra AA 55B Atomic absorption Spectrophotometer with respective hollow cathode lamps. The Percentages of different elements in these samples were determined by the corresponding standard calibration curves obtained by using Standard AR grade solutions of the elements of interest152.

Tableâ&#x2C6;&#x2019;6.17: Elemental analysis by AAS comparatively on fresh weight basis in Carum roxburghianum seeds of different region in Bangladesh.

No.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Component as Element Na (%) Mg (%) P (%) S (%) K (%) Ca (%) Zn (µg/g) Mn (µg/g) Fe(µg/g) B (µg/g) Cr (mg/L) Ni (mg/L) Pb (mg/L) Co (mg/L)

Joydebpur, Gajipur

Name of the region Keranigonj, Dhaka

Faridpur

0.18 0.69 0.55 0.24 0.43 1.89 115 31 105 42 0.561 0.450 0.013 0.029

0.20 0.69 0.43 0.29 0.34 1.70 110 20 96 41 0.290 0.464 0.020 0.033

0.17 0.68 0.35 0.21 0.50 1.60 96 25 71 43 0.030 0.090 0.042 0.025

 Each value represents the average value from three experiments.

Figure−6.15: AAS Spectrum for heavy elements of Carum roxburghianum seeds sample from different region in Bangladesh.

Figure- 6.15 (a): AAS spectrum of Cobalt analysis

Figure- 6.15 (b): AAS spectrum of Chromium analysis

Figure- 6.15 (c): AAS spectrum of Nickel analysis

Figure- 6.15 (d): AAS spectrum of Lead analysis

Results and discussion On the basis of information found in the literature, we have planned and formulated the schemes of the research works (Scheme-1.1) on Carum roxburghianum (Radhuni) seeds. The experimental results, data, charts, graphs found on the basis of the conducted experiments are tabulated and discussed as follows in the comparison with the previous reported values and different regions of Bangladesh.

7.1 Proximate Analysis of Carum roxburghianum (Radhuni) seeds: The proximate analysis like moisture, dry matter, ash, crude fiber, protein, carbohydrates, food energy, seed volume, seed density, hydration capacity, hydration index, swelling capacity, swelling index of Carum roxburghianum (Radhuni) seeds of different zones of Bangladesh were determined by the conventional methods described in Section−3.1 to 3.8.

7.1.1 Discussion on proximate analysis of C. roxburghianum seeds of different regions of Bangladesh The proximate analysis of Carum roxburghianum (Radhuni) seeds can be compared with different regions. The comparative result of the proximate analysis of seeds of different regions of Bangladesh appears in Table –7.1 and comparative studies of graphical chart are shown in Figure–7.7. And graphical chart of the individual proximate analysis of different regions of Bangladesh are shown in Figure –7.1 to 7.6.

In our study some variation is observed in our data. These variation may be due to on such factors as type of genetic variety, maturity, collection time, climatic condition in geographical location, composition of the soil, water, fertilizer used as well as permissibility, selectivity and absorbility of plants for the uptake of these parameter. All the effects caused the final level of proximate analysis in a plant. So far we aware till now the proximate analysis of Carum roxburghianum (Radhuni) seeds have not been investigated in Bangladesh by using modern analytical techniques and anywhere and were not reported earlier.

Tableâ&#x2C6;&#x2019;7.1: Comparative study on the Proximate Analysis of Carum roxburghianum (Radhuni) seeds of different regions of Bangladesh Parameter of proximate analysis (g/100g)

Sample collected from Joydebpur, Gajipur March, 2009

Keranigonj, Dhaka December,2008

August, 2008

mature 9.8014

mature 10.7091

mature 11.6010

Dry Matter

90.1985

89.2909

88.3990

Total ash

8.9920

9.0124

8.2431

Acid soluble ash

60.0318

55.524

49.409

Acid insoluble ash

39.9682

45.4750

50.5924

Water soluble ash

19.4475

23.2674

29.7655

Water insoluble ash

80.5525

76.7326

70.2345

Organic matter

91.9920

90.9876

91.7569

Crude fiber

20.9011

21.6602

23.9262

Nitrogen

3.5787

2.9698

3.2624

Protein

22.3663

18.5618

20.3899

Carbohydrates

22.6256 317.7909

19.7389

15.6095

336.0612

326.0708

Month of collection Maturity Moisture

Food energy,(cal/gm)

Faridpur

336.0612

340 335 330 325 320 315 310 305

326.0708 317.7909

Food energy Joydebpur, Gajipur

Keranigonj, Dhaka

Faridpur

Figure -7.1: Food energy of Carum roxburghinum (Radhuni) seeds.

9.8014

22.6256

15.3137

8.992

20.9011

22.3663

Moisture Protien

Total ash Ether extractives

Crude fiber Carbohydrates

Figureâ&#x20AC;&#x201C;7.2: Proximate analysis on Joydebpur, Gajipur 10.7091 19.7389

9.0124

21.6602

20.3176 18.5618

Moisture Protien

Total ash Ether extractives

Crude fiber Carbohydrates

Figureâ&#x20AC;&#x201C;7.3: Proximate analysis on Keranigonj, Dhaka 15.6095

11.601 8.2431

20.2304

23.9262 20.3899

Moisture Protien

Total ash Ether extractives

Crude fiber Carbohydrates

Figure–7.4: Proximate analysis on Faridpur 120

100

40 20 0

Joydebpur, Gajipur

Faridpur

Acid soluble ash

Acid insoluble ash

Joydebpur, Gajipur

Water soluble ash

Figure−7.5: Acid soluble & in soluble ash Figure−7.6: Water soluble & in soluble ash

Faridpur

Water insoluble ash

100

Mo Dr As h ym i st ur a t e t er

Joydebpur, Gajipur

Cr Ni Pr Ca tro oti ud rb en ef ge oh n ud ibe rat r es

Keranigonj, Dhaka

Faridpur

Figure -7.7: Comparative study of the proximate analysis of Carum roxburghianum (Radhuni) seeds of different regions of Bangladesh.

7.1.2 Discussion on Physical characteristics of Carum roxburghianum (Radhuni) seeds of different regions of Bangladesh:

The Physical characteristics like seed volume, seed density, hydration capacity, hydration index, swelling capacity, swelling index of Carum roxburghianum (Radhuni) seeds of different zones of Bangladesh were determined by the conventional methods described in Section–3.8. And comparative result of physical characteristics of seeds of different regions of Bangladesh appeared in Table –7.2 and comparative studies of graphical chart are shown in Figure –7.8.

Table –7.2: Physical characteristics of Carum roxburghianum (Radhuni) seeds of different regions of Bangladesh. Sample collected

Joydebpur,

Keranigonj, Dhaka

Faridpur

from Seed volume (ml/gm) Seed density (gm/ml) Hydration capacity (gm) Hydration index Swelling capacity (ml/gm) Swelling index

Gajipur 0.9918 1.0166 0.7026 0.6969 0.3967 0.3999

0.9908 1.0187 0.6015 0.5960 0.4953 0.4999

1.4096 1.0138 0.7755 0.7289 0.4699 0.3333

 Each value represents the average value from three experiments 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

See d

See Hy Hy Sw Sw dr dr ell ell dd vo a a i in g n t t e i i gc lum ns o o ind n n it y ap cap ind e ex a ci t ex aci y ty

Joydebpur, Gajipur

Keranigonj, Dhaka

Faridpur

Figure−7.8: Physical characteristics of Carum roxburghianum (Radhuni) seeds of different regions of Bangladesh.

7.1.3 Discussion on proximate analysis and Physical characteristics of C. roxburghianum seeds: The nutritive and proximate compositions determined in the studied sample of C. roxburghianum seed and a comparative studies on the proximate analyses of Carum

roxburghianum (Radhuni) seeds of different places of Bangladesh were presented in Table−7.1 and the comparative studies on the physical properties were presented in Table−7.2 respectively. The average results of proximate compositions (Table−7.1) of showed that the moisture content (9.8014 to 11.6010) are found in the highest amount in Faridpur sample (11.6010) and ash content (8.2431 to 9.0124) are found in the highest amount in Keranigonj, Dhaka sample, but both of the moisture & ash content are very similar in the three region of Bangladesh s (g/100 g on fresh weight basis) and their corresponding dry matter values were vise versa. The ash value implies that Carum roxburghianum (Radhuni) seeds is a fairly good source of minerals. The moisture content of the seeds is in line with definitions of vegetables, because they are characterized by high water content136. Crude fiber, protein and total carbohydrates were found to be 20.9011 to 23.9362%, 18.5618 to 22.3663% and 15.6095 to 22.6256% respectively. It was observed that plants with relatively high fiber content had low percentage of ash content. The Radhuni seed should fairly high food energy value 317.7909 to 33.60612 (gm/calories) which might be due to the high lipid content (15.3137 to 20.3176%). The high content of carbohydrate, protein as well as crude fiber is noticeable characteristics of the seed. The high fiber content of the C. roxburghianum seed may possess positive effect in human diet. Besides, it may increase feacal bulk and lowers gastric cholesterol 71, 137. Physical characteristics of seeds sample (Table−7.2) show considerable variation and each sample excelled over other sample in one or other aspect. Faridpur sample had the largest seed volume (1.4096) & other two samples had similar (0.9908-0.9918). All other physical properties like seed density, hydration capacity, hydration index, swelling capacity, swelling index had not showed any considerable difference. Which were not reported earlier. The crude protein of Radhuni can be used to supplement other sources of protein in human and animal diets. However, plant protein might be consumed as whole plant or leaves, raw or cocked 137.

7.2 Discussion on the essential oil of C. roxburghianum (Radhuni): The essential oil of Carum roxburghianum (Radhuni) was extracted by steam distillation according to the procedures described in Section-4.2 And Scheme-4.1. It was found that the Radhuni contained 1.6% to 2.6% of essential oil was determined

(Table-4.2) on the fresh

weight basis (g/100g), whereas Guenther found no reports of essential oil from the Carum roxburghianum (Radhuni)75 but in Wealth of India found up to 2.5% of essential oil 40.

7.2.1 Discussion on the physical properties of the essential oil: The physical characteristics such as colour, appearance, specific gravity, optical rotation, solubility, refractive index of the essential oil were determined by conventional methods described in Section-4.4. The result of the physical properties of Carum roxburghianum. (Radhuni) seeds essential oil of different zones of Bangladesh appeared in Table-7.3 and comparative studies of graphical chart are shown in Figure-7.9. The slight variation of this oil content and the composition of the essential oil depend on several factors such as genotype, stage of maturity, cultivation peculiarities, soil composition and climatic differences in various geographical locations. Fluctuation of the oil composition can impart change in the organoleptic properties of the plant belonging to the botanical species and variety. So far we aware till now no systematic investigation on the Chemical composition of the essential oil of Carum roxburghianum (Radhuni) seeds have not been investigated in Bangladesh by using modern analytical techniques.

Table -7.3: Comparative studies on Physical properties of essential oil from different of Bangladesh :

Sample collected from Physical Characteristics Oil yield (%) g/100g OrganolTaste Odor eptic Colour Appearance at room temperature 30˚C Specific gravity at 30˚C Refractive index [η]30˚C Optical rotation [α]D26˚C Solubility 60% alcohol 70% alcohol in 80% alcohol 90% alcohol 100% alcohol Distilled water Chloroform CCl4 Pet-ether Diethyl ether n – Hexane

Joydebpur, Gajipur 1.60 Bitter in taste Spicy Slight yellowish Homogeneous, transparent liquid, lighter than water 0.8654 1.4885 +28˚ Not soluble Cloudy up to 20 volume Soluble in 8.5 volume soluble in 5 volume Soluble at any volume Not soluble

Keranigonj, Dhaka 2.5665 Bitter in taste Spicy Slight yellowish Homogeneous, transparent liquid, lighter than water 0.8977 1.5001 +25˚ Not soluble Cloudy up to 20 volume soluble in 8.5 volume soluble in 4.5 volume Soluble at any volume Not soluble

Faridpur 2.265 Bitter in taste Spicy Slight yellowish Homogeneous, transparent liquid, lighter than water 0.8616 1.4930 +26˚ Not soluble Cloudy up to 20 volume soluble in 9 volume soluble in 4.5 volume Soluble at any volume Not soluble

Soluble at any volume Soluble at any volume Soluble at any volume Soluble at any volume Soluble at any volume

 Each value represents the average value from three experiments.

28 27.5

2.5

2 1.5 1

26.5

25.5

–

25 24.5

0.5

24 0

23.5

O il yeild % (g/100g) Joydebpur, Gajipur Faridpur

Keranigonj,Dhaka

7.9(a): Oil yield

O ptical rotation Joyde bpur, Gajipur Faridpur

Keranigonj,Dhaka

Figure–7.9(b): Optical rotation

0.9

1.502 1.5

0.89

1.498 1.496

0.88

1.494 0.87

1.492

0.86

1.488

1.49 1.486

0.85 0.84

1.484 1.482 Spe cific gravity Joyde bpur, Gajipur Faridpur

Keranigonj,Dhaka

Figure–7.9(c):Specific graviety

Re fractive inde x

Joyde bpur, Gajipur Faridpur

Keranigonj,Dhaka

Figure–7.9(d): Refractive index

Figure -7.9: Comparative studies on physical properties of Carum roxburghianum (Radhuni) seeds essential oil of different regions of Bangladesh.

7.2.2 Comparative study of the physical properties of essential oil with Author and other researchers:

Comparative studies of physical characteristics with other authors of the essential oil of radhuni have been shown in Table−7.4. And graphical chart are shown in Figure−7.10.

Table -7.4: Comparative study of the physical characteristics of essential oil with Author and other researchers: Name of the Researchers Author

Yield of essential 1.6-2.6 %

Specific gravity 0.8616-0.8977 at 30˚C

Refractive index 1.4885-1.5001 at 30˚C

Optical rotation +25˚-28˚ at 26˚C

Ashraf, M., et al.42

……

…….

+35˚ 14'

Malavya & Dutta45 Chowdhury, et al.43 M.L. Gujral et al.

2.5 % 1.15%,

0.9488 at 20˚C ……

1.4880 at 20˚C ……

+25.5˚ at 33˚C ......

1.8-2.0%

…….

……..

…….

(different region basis)

F 3 2.5 2

1.5 1

0.5 0

r O il yeild % (g/100g)

Authors Malavya. e tal. M.L. Gujral e t al

7.10(a): Oil yield

Ashraf. e t al C howdhury

e –

1.5 0.96 0.94 1 0.92

0.9 0.5 0.88

10 0

O ptical0.86 rotation 0 Authors Malavya. e tal.

Figure–7.10(b): Optical

Refractive inde x Spe cific gravity Ashraf. et al Chowdhury Authors Ashraf. e t al Malavya. e tal. Chowdhury

rotation Figure–7.10(c): Specific graviety

Figure–7.10(d): Refractive index

Figure−7.10: Comparative study of the physical characteristics of essential oil with Author and other researchers

7.2.3 Comparative study of the chemical characteristics of Carum roxburghianum. (Radhuni) seeds essential oil of different regions of Bangladesh:

Chemical characteristics of the oil such as acid value, Aldehyde value, ester value, ester number, alcohol value, phenol content, ester number after acetylation, saponification value, saponification value after acetylation, iodine value, Unsaponifiable matter and peroxide value were determined by the conventional methods described in Section - 4.5. The result of the chemical characteristics of Carum roxburghianum (Radhuni) seeds essential oil of different zones of Bangladesh appeared in Table -7.5 and comparative studies of graphical chart are shown in Figure -7.11

Tableâ&#x2C6;&#x2019;7.5: Chemical characteristics of essential oil:

Chemical

Sample collected from

Characteristics

Joydebpur,

Keranigonj,

Faridpur

Acid value Aldehyde value Phenol content Ester value Ester value after

Gajipur 4.9148 5.8444 56.1667 39.1729 44.6102

Dhaka 7.7108 6.7772 54.1667 34.1395 39.6522

4.4614 8.0424 61.5 45.8757 52.5439

acetylation Alcohol content Saponification value Saponification value

1.2456 109.3022 188.8751

1.2630 103.6690 140.6926

1.5290 82.5505 123.4121

after acetylation Unsaponifiable matter Iodine value Peroxide value

1.0490 57.6812 262.4251

1.8971 51.3072 234.8740

1.0624 43.3869 221.2249

ď&#x192;&#x2DC; Each value represents the average value from three experiments.

300

250

200

150

100

e lu va e xid ro ue n Pe al io v lat ne t er ty di t a ce Io m ra p. te sa af U n lue va p. n e Sa u io l lat va ty t. p. ce Sa l con ra ho a fte co Al ue al rv te Es ue al nt rv te te Es on c t. ol on en Ph de c hy de e Al lu va

id Ac

Faridpur

Keranigonj, Dhaka

Joydebpur, Gajipur

Figureâ&#x2C6;&#x2019;7.11: Comparative study of the chemical characteristics of essential

oil of different regions of Bangladesh.

7.2.4 Comparative study of the chemical characteristics of essential oil With Author and other researchers: Comparative studies of chemical characteristics with other authors of the essential oil of Radhuni have been shown in Table -7.6. And graphical chart are shown in Figure-7.12.

Table -7.6: Comparative study of the chemical characteristics of essential oil with Author and other researchers: Name of the Researchers Author

Acid value

Sap. value

4.4614 - 7.7108

82.5505-109.3022

Sap. value after acetylation 123.4121-188.8751

Ashraf, M., et al.42

3.1- 3.8

…….

….....

Malavya & Dutta45 Chowdhury, et al.43

4.9 …….

49.1 ……..

74.2 ……..

(different region basis)

7 6 5 4 3 2 1 0

100 80 60 40 20 0

Acid value Authors Malavya e t al.

Ashraf. et al Chowdhury

Figure−7.12 (a): Acid value

Sap. Value Authors Malavya e t al.

Figure−7.12 (b): Sap. value

150 100 50 0

Ashraf e t al Chowdhury

Sap. Value afte r acetylation Authors Malavya et al.

Ashraf et al Chowdhury

Figure-7.12(c): Sap. Value after acetylation

Figure -7.12: Comparative study of the chemical characteristics of essential oil with Author and other researchers

7.2.5 Discussion on the Physico-Chemical characteristics of essential oil of Carum roxburghianum (Radhuni) seeds. Carum roxburghianum seeds contain 1.6-2.6 % essential oil where Malavya & Dutta45 reported 2.5 % was very close. The comparative physico-chemical properties of the oil are shown in Table−7.3 & 7.5. The oil was Homogeneous, transparent liquid and lighter than water at room tempt. (30˚C), Slight yellowish in colour, with a spicy odor and bitter taste. It was freely miscible in chloroform, carbon tetrachloride, petroleum ether, di-ethylether and Soluble in 8.5 volumes in 80% alcohol & in 5 volumes in 90 % alcohol. The optical rotation was found to be +25˚ to +28˚ and highest optical rotation in Joydebpur, Gajipur (+28˚) sample and lowest at Keranigonj, Dhaka (+25˚) sample which very close to Malavya & Dutta45 (+25.5˚ at 33˚C). The refractive index (RI) of the oil was 1.4445 to 1.5001 at 30˚C and highest refractive index in Keranigonj, Dhaka (1.5001) sample and lowest at Joydebpur, Gajipur (1.4885) sample which very close to Malavya & Dutta45 (1.4880 at 20˚C). The refractive index indicated that the oil contains fairly large amount of long chain unsaturated compound. The specific gravity of the oil was found 0.8615 to 0.8977 at 30˚C which is very close to that of to Malavya & Dutta45 0.9488 at 20˚C. The extracted essential oil had the iodine value 57.2016-43.3869, acid value of Radhuni essential oil was found to be 4.4614 to 7.7108, whereas Ashraf, M., et al.42 reported 3.1- 3.8 & Malavya & Dutta45 reported 4.9. It indicates that the essential oil of Radhuni has a smaller free acid. The saponification value of Radhuni essential oil was found to be 82.5505109.3022 whereas Malavya & Dutta45 reported 49.1. The saponification value after acetylation of Radhuni essential oil was found 123.4121-188.8751 whereas Malavya & Dutta45 reported 74.2. The saponification Properties of the oil such as acid value, iodine value and saponification value gave structural, stability and quality information value indicated the presence of lower proportion of acids and iodine value also suggested the oil to be an unsaturated. Peroxide value was found 221.2249 to 262.4251which were not reported earlier. Peroxide value shows that the oil has much free active oxygen. Aldehyde value of the oil was 5.8444 to 8.0424. The alcohol value of this oil was found 1.2456 to 1.5290, ester number was found 34.1395to 45.8757 and the ester number after acetylation was found 39.6522 to 52.5439. Phenol content was found 54.1667 to 61.5.

Besides these parameters other characteristics parameters are showed in Table-7.5, which was not reported earlier. The variation of this oil content may be due to soil composition, maturity of seeds and climate difference in various geographical locations.

7.2.6 Discussion on GC-MS analysis of the essential oil: In the present study, essential oil has been extracted from Carum roxburghianum (Radhuni) seeds and isolated 38 compounds from the essential oil of two different regions in Bangladesh and they were identified and quantified by GC-MS were represented in Table−4.28 & 4.30. Structures of the identified compounds obtained from the library of GCMS instrument were given in Table−4.29 & 4.31. The main GC-MS spectrum of the essential oil from Radhuni was shown in Figure−4.4 & 4.6. Information regarding the main peak analysis collected from GC-MS NIST 107 library was shown to be in the Figure-4.5 & 4.7. The chemical constituents of the essential oil of Radhuni seeds were identified by the above mentioned techniques consist of 11 oxygenated (36.72%), 4 hydrocarbon (0.55%), 12 monoterpene (59.51%), 7 sesquiterpene (2.96%), 1 kitonic (0.05%) and 1 alcoholic (0.04%) compounds in the sample of Joydebpur, Gajipur and 2 oxygenated (8.89%), 1 hydrocarbon (7.67%), 7 monoterpene (83.44%) compounds in the sample of Keranigonj, Dhaka. This was not reported earlier. The components of the essential oil were identified by TLC and finally by GC-MS methods which were determined in Section-4.6. The GC-MS analysis was done to ensure better identification and quantification of different components of essential oil. The percentages of various components have been determined from their peak areas. In this investigation, 41 peaks were found for 36 compounds in Joydebpur, Gajipur sample &15 peaks were found for 10 compounds in Keranigonj, Dhaka sample. The major components of Carum roxburghianum essential oil were found as limonene 33.46%, Dihydrocoumarine, 5,7,8-trimethyl- 26.69%, sabinen 17.13% in Joydebpur, Gajipur sample & limonene 59.37%, 1,4-Cyclohexadiene, 1-methyl-4-(1-methylethyl)- 10.13 %, Terpinolene 7.03% in Keranigonj, Dhaka sample. Results show that both the two regions oils are a complex mixture of numerous compounds, many of which are found in trace amounts. It is worth mentioning that there is a great variation in the chemical composition of these two regions oil of Carum roxburghianum. This confirms that the reported variation in oil is due to geographic divergence and ecological conditions.

The result of the GCMS analysis report of C. roxburghianum (Radhuni) seeds essential oil of different zones of Bangladesh appeared comparatively in Tableâ&#x20AC;&#x201C;7.7

Tableâ&#x20AC;&#x201C;7.7: The name of the compounds indentified by GCMS and their respective structure of essential oil of Carum roxburghianum (Radhuni) seeds.

Sl. no.

Name of Compound

1. 2. 3. 4. 5. 6.

.alpha.- Thujene (1R)-2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene Sabinene beta.-Pinene alpha.-Phellandrene 1,4-Cyclohexadiene, 1-methyl-4-(1methylethyl)Limonene Bicyclo[3.1.0]hexen-2ol, 2-methyl-5-(1methylethyl)-(1.alpha.,2.alpha.,5.alpha.)Terpinolene 5-Octen-1-ol Sabinene 6-butyl-1,4-cycloheptadiene 9-Methylbicyclo[3.3.1]nonane 3-Cyclohexen-1-ol, 4-methyl-1-(1methylethyl)Alpha, alpha,4-trimethylbenzyl carbonate

1.69 0.30 17.13 1.15 0.05 4.10

Not detected Not detected 2.49 0.69 3.54 10.13

33.46 4.16

59.37 3.78

1.09 0.04 0.30 0.31 0.04 2.92

7.03 Not detected Not detected Not detected Not detected 5.11

0.06

Not detected

Bicycle[2.2.1]heptane,2,2-dimethyl-3methylene-,(1S)Cyclohexanone, 2-methyl-5-(1methylethenyl)2-Cyclohexen-1-ol, 3-methyl-6-(1methylethyl)-,trans2-Cyclohexen-1-ol, 2-methyl-5-(1methylethyl)-,(S)Anisole, p-allylgamma.-Elemene 1,4-Cyclohexadine-1,2-dicarboxylic anhydride

0.14

Not detected

0.03

Not detected

0.07

Not detected

0.38

Not detected

0.23 0.24 0.17

Not detected Not detected Not detected

Acetophenone Germacrene D Bicyclo[7.2.0]undec-4-ene, 4,11,11-trimethyl8-methylene-,[1R-(1R@,4Z,9S@)]alpha.-Caryophyllene

0.05 0.03 2.07

Not detected Not detected Not detected

0.12

Not detected

7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

26. Tableâ&#x20AC;&#x201C;7.7: (Continued)

Total % Joydebpur, Keranigonj, Gajipur Dhaka

Sl. no.

Name of Compound

27. 28.

1,3-Benzodioxole, 4-methoxy-6-(2-propenyl)1H-Cycloprop(e) azulen-7-ol,decahydro-1,1,7trimethyl-4-methylene-,[1ar(1a.alpha.,4a.alpha.,7.beta., 7a.beta.,7b.alpha.)]Caryophyllene oxide Apoil 4,4-Dimethyl-3-(3-methylbut-3-enylidene)-2methylenebicyclo[4.1.0]heptane Seychellene 1(3H)-Isobenzofuranone, 3-butylidinedihydrocoumarin,5,7,8-trimethylOxalic Acid, monoamide, monohydrazide, N(2,5-dimethylphenyl)-N2-(4methylbenzylideno)1,4-methanoazulene-9-methanol, decahydro4,8,8-trimethyl-,[1S(1.alpha.,3a.beta.,4.alpha.,8a.beta.,9R@)]Benzene,1-methyl-4-(1-methylethyl)Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-,

29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

% Total Joydebpur, Keranigonj, Gajipur Dhaka 0.28 0.20

Not detected Not detected

0.25 0.50 0.03

Not detected Not detected Not detected

0.05 1.18 26.69 0.28

Not detected Not detected Not detected Not detected

0.04

Not detected

Not detected Not detected

7.67 0.19

On the basis of the above fact it may be concluded that C. roxburghianum, grown widely in Bangladesh, may be utilized as a source for the isolation of natural limonene 33.46%, Dihydrocoumarine, 5,7,8-trimethyl- 26.69%, sabinen 17.13%% in Joydebpur, Gajipur sample & limonene 59.37%, 1,4-Cyclohexadiene, 1-methyl-4-(1-methylethyl)- 10.13 %, Terpinolene 7.03% in Keranigonj, Dhaka sample. Their high concentration in seeds oil makes it potentially useful in medicine because they exhibit antibacterial activities31.The oil has been known to be used in folk medicine in the treatment of dyspepsia, hiccough, vomiting and pain in bladder 5. C. roxburghianum plant oils and extracts have been used for a wide variety of purposes for many thousands of years 27. In particular, the antimicrobial activity of plant oils and extracts has formed the basis of many applications, e.g. in raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies32, 33.

7.2.7 Comparative study on the essential oil composition of Carum roxburghianum with Author and other researchers:

The major components of Carum roxburghianum essential oil were found as limonene 33.46%, Dihydrocoumarine, 5,7,8-trimethyl- 26.69%, sabinen 17.13% in Joydebpur, Gajipur sample & limonene 59.37%, 1,4-Cyclohexadiene, 1-methyl-4-(1-methylethyl)- 10.13 %, Terpinolene 7.03% in Keranigonj, Dhaka

sample. And 11 oxygenated (36.72%), 4

hydrocarbon (0.55%), 12 monoterpene (59.51%), 7 sesquiterpene (2.96%), 1 kitonic (0.05%) and 1 alcoholic (0.04%) compounds in the sample of Joydebpur, Gajipur and 2 oxygenated (8.89%), 1 hydrocarbon (7.67%), 7 monoterpene (83.44%) compounds in the sample of Keranigonj, Dhaka . Which is similar to that of Ashraf, M.; Aziz, J.; Bhatty, M.K 42 reported seeds oil consists of lower total hydrocarbons (44.2 vs. 55.0%), monoterpenes (20.9 vs. 26.0%), limonene (15.1 vs. 20.8%), terpinene (1.9 vs. 2.6%) and sesquiterpene (23.3 vs. 29.0%) contents, and higher oxygenated compounds (55.8 vs. 45.0%) and a novel unidentified ketonic acid (mol. wt. 168, C10H16O2, previously found in Carum roxburghianum essential oil). And that of Malavya & Dutta45 reported the oil contains: d-linalool, 4.7; d-limonene, 35.1; Îą- terpinene, 19.4; dlpipertone,5.7; thymohydroquinone 0.2: thymol 1.7; dl- piperitone, 13.6; cuminic acid,0.4; cumminaldehyde, traces; an unidentified ketone (C10H14O3) 1.0; unidentified esters, 5.9; And d- limonene and dipentene mixture, 2.5 %. K. M. Braj and D. Sikhibhushan34 & S. Choudhury, A. Rajkhowa, S. Dutta, et al

reported

that Terpinen-4-ol, (Z)-ligustilide and Îł-terpinene, which have been reported as major constituents in the fruits essential oil of Indian origin were totally not found in our present study. Chowdhury, Bhuiyan, and Begum43 reported that Carum roxburghianum seeds essential oil is rich in 2-cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)- (40.03%), and other components that follow are apiol (18.71%), limonene (17.11%), myristicin (12.30%), dihydrocarvone (7.89%) and eugenol (1.68%). In that study it has shown that the Radhuni oil is a rich source of undesirable toxic constituent apiol which brings down its quality. But in present study apoil was found in Joydebpur, Gajipur sample only 0.50% and Keranigonj, Dhaka sample is totally free of toxic compound apoil. In view of their broad activity, these essential oils may find industrial applications as natural preservatives and conservation agents in the cosmetic and /or food industries and as active ingredients in medical preparations.

7.3 Discussion on the fatty oil from Carum roxburghianum seeds:

The fatty oil extracted from residue obtained after steam distillation by successive solvent extraction method using light pet-ether (40º-60ºC) as an extracting solvent in a Sox let apparatus Section-5.2. So far we aware till now the chemical composition of the fatty oil of Carum roxburghianum (Radhuni) seeds have not been investigated in Bangladesh by using modern analytical techniques.

7.3.1 Discussion on the physical characteristics of the fatty oil: The physical characteristics such as colour, appearance, specific gravity, optical rotation, solubility, refractive index of the fatty oil were determined by conventional methods described in Section-5.3. The result of the physical characteristics of Carum roxburghianum (Radhuni) seeds fatty oil of different zones of Bangladesh appeared in Table-7.8 and comparative studies of graphical chart are shown in Figure-713.

Table−7.8: Comparative studies on Physical characteristics of fatty oil: Sample collected from Physical Characteristics

Joydebpur, Gajipur

Keranigonj, Dhaka

Faridpur

Oil yield (%) g/100g OrganTaste Odor oleptic Colour Appearance at Room temperature 30˚C Specific gravity at 30˚C Refractive index [η]30˚C Optical rotation [α]D26˚C Solubility 90% alcohol in 100% alcohol Distilled water Chloroform CCl4 Pet-ether

15.3137 Spicy bitter taste Spicy Dark green Homogeneous, opaque liquid, lighter than water 0.8723 1.4702 +9.03˚ Not soluble in any volume Soluble in any volume Not soluble

20.3176 Spicy bitter taste Spicy Dark green Homogeneous, opaque liquid, lighter than water 0.9180 1.4651 +8.54˚ Not soluble in any volume Soluble in any volume Not soluble

20.2304 Spicy bitter taste Spicy Dark green Homogeneous, opaque liquid, lighter than water 0.8984 1.4701 +9.56˚ Not soluble in any volume Soluble in any volume Not soluble

soluble soluble soluble soluble

Diethyl ether

Fi 25

g 9.6

r 9.2

e–

9.4

8.8 10

8.6 8.4

8.2 0

O il ye ild % (g/100g) Joyde bpur, Gaji pur Faridpur

Ke ranigonj,Dhaka

7.13(a): Oil yield 0.92

O ptical rotation Joydebpur, Gajipur Faridpur

Figure–7.13(b): Optical rotation 1.471

0.91

1.47

0.9

1.469 1.468

0.89

1.467

0.88

1.466

0.87

1.465

0.86

1.464

0.85

1.463

0.84

Ke ranigonj,Dhaka

1.462 Spe cific graviety Joydebpur, Gajipur Faridpur

Keranigonj,Dhaka

Figure–7.13(c): Specific graviety

Re fractive inde x

Joydebpur, Gaji pur Faridpur

Kerani gonj,Dhaka

Figure–7.13(d): Refractive index

Figure -7.13: Comparative studies on physical characteristics of Carum roxburghianum (Radhuni) seeds fatty oil of different regions of Bangladesh. The yield of Carum roxburghianum (Radhuni) seeds fatty oil was found to be 15.31 % to 20.23 % was determined (Table-5.1) on the fresh weight basis (g/100g), whereas Malavya & Dutta45 got about (4.5 %) of fatty oil from the seeds. The slight variation of this oil content and the composition of the fatty oil depend on several factors such as genotype, stage of maturity, cultivation peculiarities, soil composition and climatic differences in various geographical locations. Fluctuation of the oil composition can impart change in the organoleptic properties of the plant belonging to the botanical species and variety.

7.3.2 Comparative study of the physical characteristics of fatty oil

with Author and other researchers: Comparative studies of physical characteristics with other authors of the fatty oil of Radhuni have been shown in Table−7.9. And graphical chart are shown in Fig-7.14.

Table -7.9: Comparative study of the physical characteristics of essential oil with Author and other researchers: Name of the Researchers Author

Yield of fatty oil

Refractive index

15.3137-20.31766 %

1.4651-1.4702 at 30˚C 1.4914 at 20˚C

(different region basis)

M.L. Gujral et al.46

4.4-4.5%

25 20 15 10 5 0

Oil yeild % (g/100g) Authors

M.L. Gujral et al

Figure–7.14(a): Oil yield 1.495 1.49 1.485 1.48 1.475 1.47 1.465 1.46 1.455

Refractive index Authors

M.L. Gujral et al

Chemical

Sample collected from

Characteristics

Joydebpur,

Keranigonj,

Faridpur

Acid value Ester value Saponification value Unsaponifiable matter Iodine value Peroxide value Reichert-Miessel

Gajipur 162.9871 48.1215 262.9596 6.5149 15.8974 23.2499 2.585

Dhaka 154.1015 56.2465 248.0688 4.2326 11.3881 36.1578 3.1167

143.8384 62.0342 277.1959 3.5637 8.9868 27.0677 1.9983

(R.M.) value Poleneske value Henher value

5.0667 93.6846

6.20 95.5645

4.1167 85.1243

Figure-7.14(b): Refractive index Figure -7.14: Comparative study of the physical characteristics of essential oil with Author and other researchers

7.3.3 Comparative study of the chemical characteristics of Carum roxburghianum (Radhuni) seeds fatty oil of different regions of Bangladesh:

Chemical characteristics of the fatty oil such as acid value, ester value, saponification value, iodine value, unsaponifable matter, peroxide value, Reichert-Miessel (R.M.) value, polenesky value, Hen her value were determined by the conventional methods described in Section-5.4. The result of the chemical characteristics of Carum roxburghianum (Radhuni) seeds fatty oil of different zones of Bangladesh appeared in Table-7.10 and comparative studies of graphical chart are shown in Figure -7.15.

Tableâ&#x2C6;&#x2019;7.10: Chemical characteristics of fatty oil:

300

250

200

150

100

cid

e lu va r e he lu en va H ky es len Po ue al .v e lu .M R va de xi ro e Pe lu va ne r di te Io at .M ap ns U e lu Va p. e Sa lu va r te Es e lu

Joydebpur, Gajipur

Keranigonj, Dhaka

Faridpur

Figure -7.15: Comparative study of the chemical characteristics of fatty oil of different regions of Bangladesh.

7.3.4 Comparative study of the chemical characteristics of fatty oil with Author and other researchers:

Comparative studies of chemical characteristics with other authors of the fatty oil of Radhuni have been shown in Table−7.11. And graphical chart are shown in Figure-7.16.

Table -7.11: Comparative study of the chemical characteristics of essential oil with Author and other researchers: Name of the Researchers Author

Iodine value

Sap. value

8.9868-15.8974

248.0688 - 277.1959

98.4

189.5

(different region basis)

M.L. Gujral et al.46

100 80 60 40 20

Figure-7.16

300

Iodine value Authors

(a): Acid value

M.L. Gujral e t al

250 200 150 100 50 0

Sap. Value Authors

M.L. Gujral et al.

Figure-7.16(b): Sap. value Figure–7.16: Comparative study of the chemical characteristics of fatty oil with Author and other researchers

7.3.5 Discussion on the Physico-Chemical characteristics of fatty oil of Carum roxburghianum (Radhuni) seeds. Carum roxburghianum seeds contain 15.3137-20.31766 % fatty oil where Malavya & Dutta45 and M.L. Gujral et al.46 reported 4.5 % which are very much different from the experimental results. The physico-chemical properties of the oil are shown in Table −7.8 & 7.10

The oil was Homogeneous, opaque liquid; lighter than water, Dark green in colour, with a spicy odor and unpleasant bitter taste at room temperature of 30˚C. It was freely miscible in chloroform, carbon tetrachloride, petroleum ether, di-ethylether and alcohol. The optical rotation was found to be +8.54˚ to +9.56˚ at 26˚C and highest optical rotation in Joydebpur, Gajipur (+9.56˚) sample and lowest at Keranigonj, Dhaka (+8.54˚) sample which was not reported earlier. The refractive index (RI) of the oil was 1.4651 to 1.4702 at 30˚C and highest refractive index in Joydebpur, Gajipur (1.4702) sample and lowest at Keranigonj, Dhaka (1.4651) sample which was very closest to M.L. Gujral et al.46 (1.4914 at 20˚C). The refractive index of the fats and oils depends to some extent on their unsaturation. The higher the unsaturation the greater is the refractive index. The refractive index indicated that the fatty oil contains fairly large amount of long chain unsaturated compounds. The specific gravity of the oil was found 0.8723 to 0.9180 at 30˚C which was not reported earlier. The specific gravity of the oil is a temperature dependent property. It depends on molecular weight and extent on their unsaturation. The extracted fatty oil had the iodine value 8.9868 to 15.8974 which was very close to coconut oil (8 to 12), whereas M.L. Gujral et al.46 reported 98.4 which was very much different from present experimental result. We know the iodine value measure the degree of unsaturation of fatty acids content in any fat or oil. Therefore the more the iodine value the more unsaturated the oil or fat will be and vice versa. Therefore the less the iodine values the low unsaturated the oil or fat will be. The result of iodine value indicates the fat contains very lower unsaturation. Acid value of Radhuni fatty oil was found to be 143.8384 To 162.9871, was not reported earlier. These values indicate the proportion of free fatty acid is very high than that of edible oil like soyabean, musterd (ghani), palm. If the concentration of free fatty acid content in a fat or oil reaches to an elevated level, elevated acid value of oil is hazardous for human health. This high acid value indicates that this oil cannot be used for edible purpose. The saponification value of Radhuni fatty oil was found 248.0688 to 277.1959 whereas M.L. Gujral et al.46 reported 189.5. High saponification value indicates the presence of higher molecular wt. fatty acids in a fats or oils and vice versa. High molecular wt. fatty acids are

not helpful for our health than fatty acids having low molecular wt. In that sense, fats or oils having low saponification number should be preferred. The saponification Properties of the oil such as acid value, iodine value and saponification value gave structural, stability and quality information value indicated the presence of lower proportion of acids and iodine value also suggested the oil to be an unsaturated. And it is also mentioned that the result of iodine value and refractive index of Carum roxburghianum (Radhuni) oil give this idea. Ester number was found 48.1215 to 62.0342 and Peroxide value was found 23.2499 to 36.1578 which was not reported earlier. Peroxide value shows that the oil has much free active oxygen. Unsaponifiable matter 3.5637 to 6.5149 which was very higher than any other edible or no edible fatty oil this indicates that the oil contaminated with Mineral oil, higher aliphatic alcohols, sterols, pigments and hydrocarbons. Besides these parameters other characteristics parameters such as Unsaponifiable matter, Reichert-Miessel (R.M.) value, Poleneske value, Henher value are showed in Table-7.10 which was not also reported earlier. Reichert-Miessel (R.M.) value 1.9983 to 3.1167 which are very much close to palm oil (0.91.9) & corn oil (15-2.8) and Poleneske value 4.1167 to 6.20 this indicates that the oil have not much volatile components and low chain fatty acids which is very close to palm carnal oil (4.2) & coconut oil (4.4). And Henher value 85.1243 to 93.6846 shows the oil’s unsaponifiable mater and non volatile component is very high and similar to palm oil (94-97) & corn oil (93-95). The variation of these oil properties may be due to soil composition, maturity of seeds and climate difference in various geographical locations.

7.3.6 Discussion on identification and quantification of fatty acids: Fatty acids of Carum roxburghianum (Radhuni) seed were identified and quantified from the pet-ether extract (40˚- 60˚C) as described in Section−5.5. For this study, TLC and GLC experiments were carried out by preparing methyl esters of the oil. The methyl esters were prepared by standard procedure (Section−5.5.1) using BF3–MeOH complex. The methylation

was monitored on TLC. For this purpose a small portion of the reaction mixture was taken and applied on TLC. Which was developed with hexane: diethyl ether: acetic acid (9:10:1) & Pet. ether: ether: acetic acid = 4.5:0.5:0.05 mixture. The fatty acid methyl esters of seeds gave 4 and 3 spots (Figure−5.2 & 5.3) respectively. So far, we aware, the identification and quantification of fatty acids from this plant were not reported earlier in Bangladesh.

7.3.7 Gas chromatographic analysis of Carum roxburghianum (Radhuni) seeds fatty oil: The fatty acid composition of Carum roxburghianum. (Radhuni) seeds extracts (methyl ester of seeds fatty oil) was determined by Gas liquid chromatography (GLC) as described in Section−5.5.3. The relative percentage of the individual fatty acids of different zones of Bangladesh appeared in Table-7.12 and comparative studies of graphical chart are shown in Figure -7.17

Table−7.12: The relative percentage of the individual fatty acids of different regions of Bangladesh in Radhuni seeds dry matter basis (g/100g). (Chromatogram has been subjected to manual integration) Name of the fatty

Name of the regions Joydebpur,

Keranigonj,

Gajipur

Dhaka

Palmitic acid (%)

5.573

5.108

4.948

Stearic acid (%) Oleic acid (%) Linoleic acid (%) Linolenic acid (%) Ecosenoic acid (%) Unidentified (%)

0.701 76.436 1.382 15.402 0.506

Not identified 79.159 Not identified Not identified 15.733

Not identified 79.151 Not identified 15.278 Not identified

1.804

3.757

2.187

acids

Faridpur

0 Radhuni, Joydebpur

Palmitic acid Linoleic acid

Radhuni, Keranigonj

Stearic acid Linolenic acid

Radhuni, Faridpur

Oleic acid Ecosenoic acod

Figure -7.17: The relative percentage of the individual fatty acids of different regions of Bangladesh in Radhuni seeds. From GLC analysis of the Carum roxburghianum (Radhuni) seeds chromatogram has approximately 18 peaks assigned in different regions sample to the above acids. The

unidentified peaks remained unidentified. From the chromatogram (Figure-5.5 to 5.10) retention time and peak area (Table â&#x20AC;&#x201C;5.18 to 5.20) six fatty acids were studied the relative percentage of fatty acids in Carum roxburghianum. (Radhuni) seeds fatty oil of was calculated, three in Keranigonj, Dhaka sample, three in Faridpur sample all six in Joydebpur, Gajipur sample. The six peaks were identified by comparing the peaks with those of standard or pure fatty acids peaks such as Palmitic acid, Stearic acid, Oleic acid, Linoleic acid, Linolenic acid, Ecosenoic acid. Gas liquid chromatographic analysis of the fatty acid showed that Oleic acid was the major fatty acid found in the extract and the saturated fatty acids present in the oil sample were mainly palmitic acid (4.948% to 5.573%), Stearic acid (0.701%) only found in Joydebpur, Gajipur sample. The unsaturated fatty acids present in the oil samples were mainly oleic acid (76.436% to 79.159%), Linolenic acid (1.382%) only found in Joydebpur, Gajipur sample. And Ecosenoic acid was identified 0.506% in Joydebpur, Gajipur sample, 15.733% in Keranigonj, Dhaka sample and not identified in Faridpur sample. The oleic acid percentage is comparable to the olive oil (65- 85%), safflower oil (79.7%) and Linolenic acid percentage is similar to the soybean oil (2-10%). On the other hand the saturated acid as palmitic is almost comparable to the grape seed oil (4-11%) and sunflower oil (4.29%) 86, 138. Healthful fats (i.e. lower saturates and higher mono -unsaturated) have led to meet various consumer demands: (i) increased monounsaturated fatty acids: oils with increased monounsaturated (such as the high oleic varieties of oil seed) are low in polyunsaturated (linolenic acid) which are pron to oxidation. (ii) A high oleic oil also may help to reduce raised levels of total plasma cholesterol without reducing the high density lipoprotein (HDL) cholesterol level 139, 140. Common vegetable oils usually contain 6-15% saturated fatty acids 141â&#x2C6;&#x2019;143. Radhuni seeds oil contain 4.948 to 6.454% saturated fatty acid of the total oil, although it is slightly higher in respect of the lower limit of the said percentage range but it is comparatively better for human consumption144. Owing to its high percentage of the unsaturated fatty acid (93.456%) especially the oleic acid (76.436% to 79.159%). It may be concluded that Carum roxburghianum (Radhuni) seeds of Bangladesh origin has suitable nutritional properties and therefore, may be considered as edible oil after proper refining. Otherwise the extracted oil may be tapped as a source of oleic acid. On the basis of above information the oil can also be used in the food and pharmaceutical industries as an important raw material for developing functional products.

7.3.8 Comparative study of Carum roxburghianum (Radhuni) seeds fatty oil composition with other different seeds fatty oil: Without determination of the composition of fatty acids and comparison with others, the actual nutritional quality of any kind of edible and non-edible fats or oils cannot be

determined. Fats or oils having lower proportion of saturated fatty acids ate more useful than fats having higher proportion of saturated fatty acids and vice versa. The fatty acid composition of Carum roxburghianum (Radhuni) seeds oil can also be compared with some edible and non-edible fats or oils. The results are enumerated Table – 7.13 & graphical chart are shown in Figure−7.18

Table–7.13: Percentage of fatty acids composition of different fats or oils: Name of the fats or

Palmitic

Stearic

Oleic acid

Linoleic

Linolenic

oils

acid (%)

(%)

acid (%)

Radhuni

Joydebpur,

5.573

0.701

76.436

1.382

15.402

Seeds

Gajipur Keranigonj,

5.108

Not

79.159

Not

4.948

identified Not

79.151

identified Not

identified 15.278

Soybean

12.69

identified 1.98

22.11

identified 54.44

---

Palm oil Tishi oil Til oil Neem oil

39.98 9.91 19.16 --

2.10 30.05 8.53 --

42.71 -37.11 8.29

15.20 16.90 33.09 48.74

---------

oil

Dhaka Faridpur

The fatty acids produced from any type of oil seed may vary with lipid class, geographical location, cultivar, soil type, climate, moisture, temperature, maturity of the seed and agricultural practice 138, 145−147.

0 Ra Ra Ra So Pa Tis Til ya lm dh dh dh hi o il be un un un o il oil a i, J i, K i, F no oy ari il era de dp nig bp ur on ur j

Palmitic acid

Stearic acid

Oleic acid

Ne em

Linoleic acid

oil

Linolenic acid

Figureâ&#x20AC;&#x201C;7.18: Comparative study of Carum roxburghianum (Radhuni) seeds fatty oil composition with other different seeds fatty oil.

7.3.9 Comparative study on chemical properties of Carum roxburghianum (Radhuni) seeds fatty oil with others edible & non-edible seeds fatty oil: Some characteristics of Carum roxburghianum. (Radhuni) seeds fatty oil can be compared with some other edible and non-edible fats and oils available in Bangladesh. In the comparison some parameters like iodine value, acid value, saponification value and R.M. value were used. The result of iodine value, acid value, saponification value and R.M. value of different oils were given in Table−7.14 and shown in Figure−7.19. Table−7.14: Iodine value, Saponification value and acid values of different oils. Name of the fats and oils Joydebpur, Gajipur Keranigonj, Dhaka

Iodine value 15.8974 11.3881

Saponification value 262.9596 248.0688

Acid value 162.9871 154.1015

R.M. value 2.585 3.1167

Faridpur

8.9868

277.1959

143.8384

1.9983

Soybean86 Mustard (ghani)86 Palm86 Tishi86 Til86 Neem86 Coconut oil110

138.52 97.18 61.21 137.35 98.268 88.75 5-12

258.27 229.89 233.58 265.54 220.75 227.06 255-260

0.383 3.657 0.175 1.592 28.32 2.53 2.5-10

0.5-2.8 ….. 0.9-1.9 …. … … 6.6-8.4

Radhuni Seeds oil

Carum roxburghianum (Radhuni) seed fatty oil has Iodine value of the oil was similar to the iodine value (Witz method) for the range of Coconut oil. The iodine value showed the presence of maximum unsaturation. Which was in agreement with physical state of the oil that means the oil is non-drying in nature. It is known that the oils having iodine value of below 100 generally known is non-drying oil, for example: olive oil, palm oil, coconut oil, etc. The saponification value was quite similar to the range of Coconut oil, Soybean, Mustard (ghani), Palm, Tishi, Til, Neem which were typical C16 and C18 oils 138. R.M. value is very much similar to palm oil, coconut oil which indicates the Carum roxburghianum (Radhuni) seeds fatty oil mixed with volatile oil. The other hand, Carum roxburghianum (Radhuni) seeds fatty oil has very higher acid value than Soyabean, Mustard, Palm Tishi and Neem and loest of Til oil. The higher acid value indicated that the grade of this extracted oil is not suitable for direct edible purposes. It may be used for consumption through refining or may be used for industrial purposes.

300

250

200

150

100

0 oi l

pu eb yd

il il to nu )o ni co ha co (g rd ta

us M

m ee

Acid value

, ni

hu ad

u dh

hu ad

il lo Ti il io sh Ti l oi lm l Pa oi r an be pu id ya ar So ,F j ni on ig an er r K R

Iodine value

Saponification value

R.M. value

Figureâ&#x20AC;&#x201C;7.19: Comparative study on chemical properties of Carum roxburghianum (Radhuni) seeds fatty oil with others edible & non-edible seed fatty oil.

7.4 Discussion on mineral elemental Analysis of Carum roxburghianum (Radhuni) seeds: The mineral content was determined by XRF Spectrometer. Model no: Rigaku Primus ZSX, made by USA. Instrument type: wave length dispersive XRF (WDXRF) with SC and PC type detector, XRF equipped with an X-ray tube anode Rh tube, and also five filters, the XRF instrument was fully automatic. And P10 gas was used and mineral element concentration was detected on dry matter basis. Which have described in Section−6.3

7.4.1 Mineral contents of Carum roxburghianum (Radhuni) seeds: Mineral content such as Na, Mg, Al, Si, P, S, Cl, K, Ca, Ti, Cr, Mn, Fe, Cu, Zn, Br, Rb, Sr, Zr, Nb, Ba etc. were determined in its seed residue. Plants and its seed were collected from different regions of Bangladesh, such as, Joydebpur, Gajipur; Keranigonj, Dhaka and Faridpur regions. The result of element analysis of its seed was determined by XRF (X-ray fluorescence) Spectrometry method in g/100g dry weight basis of the sample. The element analysis of Carum roxburghianum (Radhuni) seeds can be compared with different regions. The result of minerals content of Radhuni seeds of different zones of Bangladesh appeared as element and element oxide basis in Table−7.15 & 7.16 and comparative studies of graphical chart are shown in Figure–7.23. And graphical chart of the individual elemental analysis of different regions of Bangladesh are shown in Figure−7.20 to 7.22. In our study some variation is observed in our data. These variation may be due to on such factors as type of genetic variety, maturity, collection time, climatic condition in geographical location, composition of the soil, water, fertilizer used as well as permissibility, selectivity and absorbility of plants for the uptake of these elements. All the effects caused the final level of mineral components in a plant. Fluctuation of the element composition can impart change in the organoleptic properties of the plant belonging to the botanical species and variety. The total concentration of macro and micro elements were measured in Radhuni seeds from three different places of Bangladesh by XRF spectrometry for the first time in Bangladesh by the authors. Precision of the measurements was taken by analysis of three sub samples of each region. So far we aware till now the elemental composition of Carum roxburghianum (Radhuni) seeds have not been investigated on elemental analysis in Bangladesh by using modern analytical techniques such as XRF analysis anywhere and were not reported earlier.

Table–7.15: Elemental analysis as oxide comparatively (on dry matter basis) of Carum roxburghianum (Radhuni) seeds of different

regions of Bangladesh (g/100g). No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Component as oxide Na2O MgO Al2O3 SiO2 P2O5 SO3 Cl K2O CaO TiO2 Cr2O3 MnO Fe2O3 NiO CuO ZnO Br Rb2O SrO ZrO2 Nb2O5 BaO Total

Name of the region Joydebpur, Keranigonj, Gajipur Dhaka 0.717 5.11 5.52 22.6 16.3 3.97 0.668 9.55 29.3 0.399 0.461 0.164 4.74 0.0477 0.0349 0.120 0.0051 0.0452 0.0496 0.0155 0.0032 0.0854 99.9056

0.686 4.98 5.11 21.5 15.7 4.16 0.631 9.62 30.5 0.403 0.557 0.198 5.62 0.0546 0.0412 0.136 0.0057 0.0461 0.0549 0.0153 0 0 100.0188

Faridpur 0.732 5.10 5.47 22.4 15.4 3.59 0.649 9.41 30.5 0.424 0.494 0.174 5.28 0.0170 0.0384 0.127 0.0055 0.0469 0.0548 0.0154 0 0.0679 99.9959

 Normalized up to : 99.99%  Each value represents the average value from three experiments

Table–7.16: Elemental analysis in percent comparatively (on dry matter basis) of Carum roxburghianum (Radhuni) seeds of different regions of Bangladesh (g/100g).

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Component as Element Na Mg Al Si P S Cl K Ca Ti Cr Mn Fe Ni Cu Zn Br Rb Sr Zr Nb Ba O Total

Name of the region Joydebpur, Keranigonj, Gajipur Dhaka 0.5678 3.0824 2.9206 10.5655 7.1150 1.5912 0.668 7.9284 20.9407 0.2392 0.3154 0.1270 3.3152 0.0375 0.0279 0.0964 0.0051 0.0413 0.0469 0.0115 0.0022 0.0765 40.2783 100

0.5432 3.0040 2.7037 10.0513 6.8531 1.6673 0.631 7.9865 21.7984 0.2416 0.3811 0.1533 3.9306 0.0429 0.0329 0.1092 0.0057 0.0422 0.0519 0.0113 0 0 39.7588 100

 Normalized up to : 99.99%  Each value represents the average value from three experiments

Faridpur 0.5797 3.0763 2.8942 10.472 6.7221 1.4389 0.649 7.8122 21.7984 0.2542 0.3380 0.1347 3.6929 0.0134 0.0307 0.1020 0.0055 0.0429 0.0518 0.0114 0 0.0608 39.8189 100

O, 40.2783 Nb, 0.0022

Al, 2.9206

Ba, 0.0765 Zr, 0.0115

Mg, 3.0824

S r, 0.0469

Na , 0.5678

Br, 0.0051 Rb, 0.0413 Zn, 0.0964 Cu, 0.0279

Mn, 0.127

Ni, 0.0375

Fe, 3.3152

Ti, 0.2392

Cr, 0.3154

S i, 10.5655 K, 7.9284 P, 7.115 S , 1.5912 Ca, 20.9407

Na Fe

Cl, 0.668

Mg Ni

Al Cu

Si Zn

P Br

S Rb

Cl Sr

K Zr

Ca Nb

Ti Ba

Cr O

Figureâ&#x2C6;&#x2019;7.20: Element identified in their respective concentration found in Carum roxburghianum seeds of Joydebpur, Gajipur (in g/100g).

O, 39.7588 Al, 2.7037 Mg, 3.004

Zr, 0.0113 S r, 0.0519

Na , 0.5432

Br, 0.0057 Rb, 0.0422 Zn, 0.1092

Cu, 0.0329

Mn, 0.1533

Ni, 0.0429, 0%

Cr, 0.3811

Fe, 3.9306

Ti, 0.2416

S i, 10.0513 P, 6.8531 S , 1.6673

K, 7.9865 Ca, 21.7984

Na Mn

Cl, 0.631

Mg Fe

Al Ni

Si Cu

P Zn

S Br

Cl Rb

K Sr

Ca Zr

Ti O

Figureâ&#x2C6;&#x2019;7.21: Element identified in their respective concentration found in Carum roxburghianum seeds of Keranigonj, Dhaka (in g/100g).

O, 39.8189 Ba, 0.0608 Al, 2.9842 Zr, 0.0114

Mg, 3.0763

S r, 0.0114

Na , 0.5797

Br, 0.0055 Rb, 0.0429 Zn, 0.102

Cu, 0.0307

Mn, 0.1347

Ni, 0.0375

Fe, 3.6929

Ti, 0.2542

Cr, 0.338

S i, 10.472 K, 7.8122

P, 6.7221 S , 1.4389

Ca, 21.9784

Na Fe

Mg Ni

Cl, 0.649

Al Cu

Si Zn

P Br

S Rb

Cl Sr

K Zr

Ca Ba

Ti O

Figureâ&#x2C6;&#x2019;7.22: Element identified in their respective concentration found in Carum roxburghianum seeds of Faridpur (in g/100g).

0 Na Mg Al

K Ca Ti Cr Mn Fe

Joydebpur, Gajipur

Ni Cu Zn Br Rb Sr Zr Nb Ba O

Keranigonj, Dhaka

Faridpur

Figureâ&#x2C6;&#x2019;7.23: Percent of mineral element comparatively of Carum roxburghianum (Radhuni) seeds of different regions of Bangladesh (on dry matter basis in g/100g)

Tableâ&#x2C6;&#x2019;7.17: Elemental analysis by AAS verses XRF comparatively on fresh matter basis in Carum roxburghianum seeds of different region

in Bangladesh. No Element

Joydebpur, Gajipur XRF

AAS

mg/100g 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Na Mg Al Si P S Cl K Ca Ti Cr Mn Fe Ni Cu Zn Br Rb Sr Zr Nb Ba B Pb Co O

Name of the region Keranigonj, Dhaka XRF

AAS

mg/100g 0.18 % 0.69 % ------0.55 % 0.24 % ---0.43 % 1.89 % ---0.561mg/L 31 µg/g 105 µg/g 0.450mg/L ---115 µg/g ------------42 µg/g 0.013mg/L 0.029mg/L ---

43.71706 241.7637 217.5954 808.9347 551.5416 134.1853 50.78326 642.7583 1754.348 19.44411 30.67116 12.33768 316.337 3.452618 2.64781 8.78848 0.45873 3.39628 4.17694 0.90943 0 0 0 0 0 3199.812

Faridpur XRF

AAS

mg/100g 0.20 % 0.69 % ------0.43 % 0.29 % ---0.34 % 1.70 % ---0.290 mg/L 20 µg/g 96 µg/g 0.464 mg/L ---110 µg/g ------------41 µg/g 0.020 mg/L 0.033 mg/L ---

42.24593 224.1869 210.9163 763.1522 489.8764 104.8606 47.2962 569.318 1588.569 18.52495 24.63192 9.81633 269.1219 0.976532 2.237278 7.433301 0.400815 3.126359 3.774951 0.830781 0 4.43083 0 0 0 2901.822

0.17 % 0.68 % ------0.35 % 0.21 % ---0.50 % 1.60 % ---0.030 mg/L 25 µg/g 71 µg/g 0.090 mg/L ---96 µg/g ---------------43 µg/g 0.042 mg/L 0.025 mg/L ---

7.4.2 Discussion on Comparative study on elements analysis of Carum roxburghianum (Radhuni) seeds of different regions of Bangladesh: The comparative result of the element of Radhuni seeds of different region of Bangladesh are presented in Table−7.15 &7.16 respectively on dry matter basis (in g/100g) and fresh Wt. basis in Table−6.16 (see Section−6.5). And comparative result of the element of Radhuni seeds of different region of Bangladesh by AAS at fresh wt. basis are presented in Table−6.17. And a Elemental analysis by AAS verses XRF comparatively on fresh matter basis in Carum roxburghianum seeds of different region in Bangladesh presented in Table−7.17. The Carum roxburghianum (Radhuni) seeds contained twenty two significant amounts of important mineral elements which were detected in present study. The elements presents in

order by quantity (g/100g on dry mater basis) were Ca, K, NA, S, Mg, Fe, P, AL, Ni, Si, Ti etc. The highest amount of Ca(21.7984), Si(10.5655), K(7.9865), P(7.1150), Fe(3.9306), Mg(3.0824), Al(2.9206), S(1.6673) were found significantly. Comparatively Ca (21.7944) was found in highest amount in Keranigonj, Dhaka; and Faridpur sample. Ba and Nb were both detected in Joydebpur, Gajipur sample and of them Ba was only detected in Faridpur sample, but none of them were detected in Keranigonj sample. Ba was detected highest amount (0.0765) in Joydebpur, Gajipur sample. Ni was detected highest amount (0.0429) in Keranigonj, Dhaka sample. And all other mineral element quantity was approximately same in the three different regions which were not reported earlier. The toxic elements like Co, Cd, As, Cr, Pb and Hg were not detected in the present XRF analysis investigation. In our study some variation is observed in data. These variation may be due to on such factors as type of genetic variety, maturity, collection time, climatic condition in geographical location, composition of the soil, water, fertilizer used as well as permissibility, selectivity and absorbility of plants for the uptake of these elements. All the effects caused the final level of mineral components in a plant80, 148. In the present study the highest concentration of Ca, Si, P, K and Fe were observed in all three region sample. In general, research findings have indicated that Radhuni seeds contain substantial amount of mineral element. The high amount of these mineral elements could provide alternative sources of these minerals, especially; ‘Phosphorus’ remains a problem as it more often than not in a complexed form135. Mineral elements play vital roles in much important process in the body, such as Ca is the major component of bone and assists in teeth development 150. Where low intake of Calcium can be one of the risk factors in the bone disease osteoporosis. Ca is required for the normal growth and development of the skeleton. Ca-ions increase the force of contraction of heart 151−155 . Iron is required for the synthesis of oxygen transport proteins, hemoglobin and myoglobin and for the formation of heam enzymes and Fe other containing enzymes, which are particularly important for energy production immune defence and thyroid function. Fe deficiency anemia can decrease mental and psychomotor development in children135. On the other hand, Na, K and Ca play an important role in the electrophysiology of cardiac tissue. Ca-ions increase the force of contraction of the heart151. Na maintains the osmotic equilibrium between the cellular fluid and the tissue cells and maintains the Ph of blood within normal limit. It is also concerned with the conduction of nervous impulses, muscle contractility and control of heart muscle conduction156. Mg, Fe, and P are essential for most metabolic process157. Zn is an important constituent of several enzymes and plays vital role in clinical, biochemical and immunological effects158. The important of these elements cannot be overemphasized because many enzymes require them as cofactors149. In the present communication, the mineral element composition of Radhuni seeds is an agreeable limit for our dietary allowance (Table−7.18) Table−7.18: The mineral elemental composition present in Carum

roxburghianum (Radhuni) seeds is compared with daily dietary allowance. Mineral elements (g/100g)

Daily dietary allowance for human adult160, mg

Daily dietary allowance for infants160 , mg

Amount present determined in the study by XRF(mg/100g on fresh weight basis)

Na Mg P Cl K Ca Fe Zn Cu Mn

500 320-420 800 750 2000 1200 10-18 15 1.5-3 2.5

120-200 75 275 300 700 270 10 5 0.6-0.7 0.6-1.0

42.24593 to 46.05687 224.1869 to 250.0276 489.8764 to 577.1304 47.2962 to 54.18455 569.318 to 643.109 1588.569 to 1754.348 268.9111 to 316.337 7.819447 to 7.819447 2.237278 to 2.647812 9.81633 to 12.33768

Dietary sources of essential elements are important for correct physiological functions of human body. The high quantity of K, Mg and Ca together with the quantity of Na plus the content of the essential elements Fe, Mn, Zn and Cu allow the seeds of Radhuni is to be considered as excellent sources of bioelements. The results indicate that analyzed Radhuni seeds should be considered as sufficient amount of mineral element supplement to meet the daily dietary allowance160. Inorganic elements remain complexes with organic ligands and made them bioavailable to the body system. At any rate, it is considered that Radhuni seeds play a meaningful role in human nutrition as useful mineral sources. The high level of these mineral elements in Radhuni seeds make it useful as supplements as human diets or livestock feed and also be as raw materials in pharmaceuticals and ayurvedic formulation.