Biological investigation of Caryota urens by zareen a.abedin

Biological investigation of Caryota urens 1.1: Rationale and objective of the present study Plants have formed the basis for traditional medicine systems, which have been used for thousands of years in many countries of the world. These plant-based systems continue to play an essential role in health care, and it has been estimated by the World Health Organization that approximately 80% of the worldâ&#x20AC;&#x2122;s inhabitants rely mainly on traditional medicines for their primary health care (Schultes et al.,1972 ). Plants have provided a source of inspiration for novel drug compounds, as plant-derived medicines have made large contributions to human health and well-being (Nelson 1982). Approximately 119 pure chemical substances extracted from higher plants are used in medicine throughout the world (Farnsworth et al., 1985). More than 30% of the pharmaceutical preparations are based on plants (Shinvari and Khan, 2003). The plant is a biosynthetic laboratory and the remedial phytoelements produced inside a plant through a cascade of biochemical reactions significantly contribute to the traditional and modern medicines. Chemical diversity of secondary plant metabolites that results from plant evolution may be equal or superior to that found in synthetic combinatorial chemical libraries (Vagelos, 1991). The number of higher plant species ( angiosperms and gymnosperms) on this planet is estimated at 250,000 (atensu E.S. et al,1978), with a lower level at 215,000 (Cronquist,1981; Cronquist,1988) and an upper level as high as 500,000 (Tippo et al.,1977; Schultes,1972). Of these only about 6% have been screened for biologic activity, and a reported 15% have been evaluated phytochemically (Verpoorte, 2000) About 2.5 million species of higher plants and the majority of these have not been investigated in detail for their pharmacological activities (Ram et al., 2003) The goals of using plants as sources of therapeutic agents are (Daniel et al., 2001) a) to isolate bioactive compounds for direct use as drugs, e.g. digoxin, digitoxin, morphine, reserpine, taxol, vinblastine, vincristine; b)

to produce bioactive compounds of novel or known structures as lead compounds for semisynthesis to produce patentable entities of higher activity and/or lower toxicity, e.g., metformin, nabilone, oxycodon (and other narcotic analgesics), taxotere, teniposide, verapamil, and miodarone, which are based, respectively, on galegine, Î&#x201D;9- tetrahydrocannabinol, morphine, taxol, podophyllotoxin, khellin, and khellin; c) to use agents as pharmacologic tools, e.g., lysergic acid diethylamide, mescaline, yohimbine; and d) to use the whole plant or part of it as an herbal remedy, e.g., cranberry, echinacea, feverfew, garlic, etc. There are several familiar approaches for lead searching from the plants and the isolated bioactive compounds are utilized in three basic ways (Cox, P.A., 1994): 1. Unmodified natural plant products where ethno medical uses suggested clinical efficacy, e.g.,digoxin, digitoxin (1), morphine (2) . O O

OH O

CH3

HO OH

(1)

(2)

2. Unmodified natural products of which the therapeutic efficacy was only remotely suggested by indigenous plant use, e.g., vincristine 3. Modified natural or synthetic substances based on a natural product used in folk medicine, e.g., aspirin

With high throughput screening methods becoming more advanced and available, these numbers will change, but the primary discriminator in evaluating one plant species versus another is the matter of approach to finding leads. There are some broad starting points to selecting and obtaining plant material of potential therapeutic interest. However, the goals of such an endeavour are straightforward. Chemical diversity of secondary plant metabolites that results from plant evolution may be equal or superior to that found in synthetic combinatorial chemical libraries. It was estimated that in 1991 in the United States, for every 10,000 pure compounds (most likely those based on synthesis) that are biologically evaluated (primarily in vitro), 20 would be tested in animal models, and 10 of these would be clinically evaluated, and only one would reach U.S. Food and Drug Administration approval for marketing. The time required for this process was estimated as 10 years at a cost of $231 million (U.S.) (Vagelos, 1991). Most large pharmaceutical manufacturers and some small biotechnology firms have the ability to screen 1,000 or more substances per week using high throughput in vitro assays. In addition to synthetic compounds from their own programs, some of these companies screen plant, microbial, and marine organisms. The work described in this dissertation is an attempt to isolate and characterize the chemical constituents of indigenous medicinal plants,Caryota urens (Fam. Arecaceae)to evaluate the possible pharmacological, toxicological and microbiological profiles of the crude extracts as well as purified compounds.

2.1: The plant family: The plant under investigation Caryota urens belongs to the family Arecaceae or Palmae.Arecaceae are a family of flowering plants, the only family in the monocot order Arecales. Over 230 genera with around 3000 species(real palm trees.com2012)are currently known, most of which are restricted to tropical, subtropical, and warm temperate climates. Most palms are distinguished by their large, compound, evergreen leaves arranged at the top of an unbranched stem. However, many palms are exceptions, as palms in fact exhibit an enormous diversity in physical characteristics. As well as being morphologically diverse, palms also inhabit nearly every type of habitat within their range, from rainforests to deserts. Palms are among the best-known and most extensively cultivated plant families. They have been important to humans throughout much of history. Many common products and foods are derived from palms, and palms are widely used in landscaping for their exotic appearance, making them one of the most economically important plants Scientific classification: Kingdom : Plantae Clade : Angiosperms Clade : Monocots Clade : Commelinids Order : Arecales Family: Arecaceae Palms range from tiny understory plants to towering trees, and are found throughout the tropics and subtropics. Some commercially important palms include coconut (Cocos nucifera), date (Phoenix dactylifera) and oil palm (Elaeis guineensis)

2.2: Properties Palms, considered in a dietetical and medicinal point of view, are of the highest importance to the inhabitants of tropical regions. Their stems yield starch (sago) sugar, and wax; their terminal leaf buds are boiled and eaten as a kind of cabbage; their fruits yield oil, sugar, and resins; and their seeds form articles of food, and yield, by pressure, fixed oil. In the abundance of sugar and starch which the palms yield, this family resembles the grasses. However, they are distinguished from the latter in containing, in some cases, a large quantity of fixed oil. To these three principles are chiefly due the nutritive qualities of palms. However, these substances being non-nitrogenized, are merely fat making and heat yielding, and without the addition of proteine compounds (found in the seeds, and probably in other edible parts of palms), would be insufficient to support life.

Wax, astringent matter (tannin), and resinous principles, are useful products obtained from palms, the ashes obtained by the combustion of palm leaves yield potash. The only resinous substance used in medicine and the arts, and which is obtained from the palms, is Dragon's blood, the produce of Calamus Draco.(henriettasherbal.com)

2.3 Habitate and Distribution : Most palms grow in the tropics. They are abundant throughout the tropics, and thrive in almost every habitat therein. Their diversity is highest in wet, lowland tropical forests, especially in ecological "hotspots" Only an estimated 130 palm species grow naturally beyond the tropics, mostly in the subtropics. Palms inhabit a variety of ecosystems. More than two-thirds of palm species live in tropical forests, where some species grow tall enough to form part of the canopy and shorter ones form part of the understory. Some species form pure stands in areas with poor drainage or regular flooding, including Raphia hookeri which is common in coastal freshwater swamps in West Africa. Other palms live in tropical mountain habitats above 1000 meters,. Palms may also live in grasslands and scrublands, usually associated with a water source, and in desert oases such as the date palm. A few palms are adapted to extremely basic lime soils, while others are similarly adapted to very acidic serpentine soils.

Figure 2.1: Distribution of plants of Arecaceae family 2.4: Taxonomy: Palms are a monophyletic group of plants, meaning the group consists of a common ancestor and all its descendants. Extensive taxonomic research on palms began with botanist H.E. Moore, who organized palms into 15 major groups based mostly on general morphological characteristics. The following classification, proposed by N.W. Uhl and J. Dransfield in 1987, is a revision of Moore's classification that organizes palms into six subfamilies. A few general traits of each subfamily are listed. Coryphoideae are the most diverse subfamily, and are a paraphyletic group, meaning all members of the group share a common ancestor, but the group does not include all the ancestor's descendants. Most palms in this subfamily have palmately lobed leaves and solitary flowers with three, or sometimes four carpels. The fruit normally develops from only one carpel. Subfamily Calamoideae includes the climbing palms, such as rattans. The leaves are usually pinnate; derived characters (synapomorphies) include spines on various organs, organs specialized for climbing, an extension of the main stem of the leaf-bearing reflexed spines, and overlapping scales covering the fruit and ovary. Subfamily Nypoideae contains only one one species, Nypa fruticans], which has large, pinnate leaves. The fruit is unusual in that it floats, and the stem is dichotomously branched, also unusual in palms. Subfamily Ceroxyloideae has small to medium-sized flowers, spirally arranged, with a gynoecium of three joined carpels.

The Arecoideae are the largest subfamily, with six diverse tribes containing over 100 genera. All tribes have pinnate or bipinnate leaves and flowers arranged in groups of three, with a central pistillate and two staminate flowers. The Phytelephantoideae are a monoecious subfamily. Members of this group have distinct monopodial flower clusters. Other distinct features include a gynoecium with five to ten joined carpels, and flowers with more than three parts per whorl. Fruits are multiseeded and have multiple parts. Currently, few extensive phylogenetic studies of Arecaceae exist. In 1997, Baker et al. explored subfamily and tribe relationships using chloroplast DNA from 60 genera from all subfamilies and tribes. The results strongly showed the Calamoideae are monophyletic, and Ceroxyloideae and Coryphoideae are paraphyletic. The relationships of Arecoideae are uncertain, but they are possibly related to Ceroxyloideae and Phytelephantoideae. Studies have suggested that the lack of a fully resolved hypothesis for the relationships within the family is due to a variety of factors including difficulties in selecting appropriate outgroups, homoplasy in morphological character states, slow rates of molecular evolution important for the use of standard DNA markers, and character polarization. However, hybridization has been observed among Orbignya and Phoenix species, and using chloroplast DNA in cladistic studies may produce inaccurate results due to maternal inheritance of the chloroplast DNA. Chemical and molecular data from non-organelle DNA, for example, could be more effective for studying palm phylogeny.

Table2.1:Subfamily and Genus of Arecaceae family Subfamily

Tribe

Subtribe

Genus

Coryphoideae

Corypheae

Thrinacinae

Thrinax, Chelyocarpus, Crysophila, Itaya, Schippia, Thrinax, Coccothrinax, Zombia, Trachycarpus, Guihaia, Rhapis, Rhapidophyllum, Chamaerops, Maxburrietia

Livistoninae

Livistona, Pholidocarpus, Brahea, Johannesteijsmannia, Licuala, Pritchardia, Pritchardiopsis, Serenoa, Copernicia, Colpothrinax, Acoelorraphe, Washingtonia

Coryphinae

Corypha, Nannorrhops, Chuniophoenix, Kerriodoxa

Sabalinae

Sabal

Phoeniceae Borasseae Calamoideae

Calameae

Lepidocaryeae Nyphoideae Ceroxyloideae

Phoenix Lataniinae

Borassodendron, Latania, Borassus, Lodoicea

Hyphaeninae

Hyphaene, Medemia, Bismarckia

Ancistrophyllinae

Laccosperma, Eremospatha

Eugeissoninae

Eugeissona

Metroxylinae

Metroxylon, Korthalsia

Calaminae

Eleiodoxa, Salacca, Daemonorops, Pogonotium, Ceratolobus, Retispatha

Plectocomiinae

Myrialepsis, Plectocomiopsis, Plectocomia

Pigafettinae

Pigafetta

Raphiinae

Raphia

Oncocalaminae

Oncocalamus

Calamus,

Calospatha,

Mauritia, Mauritiella, Lepidocarym Nypa

Cyclospaeae

Pseudophoenix

Ceroxyleae

Ceroxylon, Oraniopsis, Juania, Louvelia, Ravenea

Hyophorbeae

Gaussia, Hyophorbe, Wendlandiella

Synecanthus,

Chamaedorea,

Arecoideae

Caryoteae Iriarteae

Arenga, Caryota, Wallichia Iriarteinae

Podococceae Areceae

Dictyocaryum, Iriartella, Iriartea, Socratea Podococcus

Oraniinae

Halmoorea, Orania

Manicariinae

Manicaria

Leopoldiniinae

Leopoldinia

Malortieinae

Reinhardtia

Dypsidinae

Vonitra, [Chrysalidocarpus: now Dypsis], [Neodypsis: now Dypsis], Phloga, Dypsis

Euterpeinae

Euterpe, Prestoea, Neonicholsonia, Oecocarpus, Jessenia, Hyospathe

Roystoneinae

Roystonea

Neophloga,

Archontophoenicinae Archontophoenix, Chambeyronia, Hedyscepe, Rhopalostylis, Kentiopsis, Mackeea, Actinokentia

Cocoeae

Geonomeae Phytelephantoideae

Cyrtostachydinae

Cyrtostachys

Linospadicinae

Calyptrocalyx, Linospadix, Howea, Laccospadix

Ptychospermatinae

Drymophloeus, Carpentaria, Veitchia, Normanbya, Wodyetia, Ptychosperma, Ptychococcus, Brassiophoenix, Balaka

Areninae

Loxococcus, Gronophyllum, Areca, Siphokentia, Hydriastele, Gulubia, Nenga, Pinanga

Oncospermatinae

Deckenia, Acanthophoenix, Rocheria, Oncosperma, Tectiphiala, Verscheffeltia,Phoenicophorium, Nephrosperma

Sclerospermatinae

Sclerosperma, Marojejya

Beccariophoenicinae Beccariophoenix Butiinae

Butia, Jubaea, Jubaeopsis, Cocos, Syagrus, Lytocaryum, Parajubaea, Allagoptera, Polyandrococos

Attaleinae

Attalea [Scheelea, Orbignya, Maximiliana: these three genera are now included in Attalea]

Elaeidinae

Barcella, Elaeis

Bactridinae

Acrocomia, Gastrococos, Astrocaryum Pholidostachys, Welfia, Asterogyne, Geonoma

Aiphanes,

Bactris,

Calyptronoma,

Desmoncus, Calyptrogyne,

Palandra, Phytelephas, Ammandra

2.4 Evolution Arecaceae is the first modern family of monocots that is clearly represented in the fossil record. Palms first appear in the fossil record around 80 million years ago, during the late Cretaceous Period. The first modern species, such as Nypa fruticans and Acrocomia aculeata, appeared 69-70 million years ago, confirmed by fossil Nypa pollen dated to 70 million years ago. Palms appear to have undergone an early period of adaptive radiation.

Figure 1: Fossil of Permineralized Nypa prop roots. By 60 million years ago, many of the modern, specialized genera of palms appeared and became widespread and common, much more widespread than their range today. Because palms separated from the monocots earlier than other families, they developed more interfamilial specialization and diversity. By tracing back these diverse characteristics of palms to the basic structures of monocots, palms may be valuable in studying monocot evolution. Several species of palms have been identified from flowers preserved in amber including Palaeoraphe dominicana and Roystonea palaea. Evidence can also be found in samples of petrified palmwood.(Wikipedia 2012)

2.5: Characteristics of palmae family Leaves clustered, terminal, very large, pinnate, or flabelliform, plaited in vernation. Spadix terminal, often branched, or enclosed in a one or many-valved spathe. Flowers small, greenish ,with bractlets. Perianth six-parted, in two series, persistent; the three outer segments often smaller, the inner sometimes deeply connate. Ovary one, three-celled, or deeply three-lobed; the lobes or cells one-seeded, with an erect ovule, rarely one-seeded. Fruit occasionally very large. (R. Brown, 1810.)Fruit baccate or drupaceous, with fibrous flesh. Albumen cartilaginous, and either ruminate or furnished with a central or ventral cavity; embryo lodged in a particular cavity of the albumen, usually at a distance from the hilum, dorsal, and indicated by a little nipple, taper or pulley-shaped; Trunk arborescent, simple, occasionally shrubby and branched, rough, with the dilated half-sheathing bases of the leaves or their scars.

Figure 1.2: Patterns of the Arecaceae or Palmae Family

2.6: Uses Palms represent the third most important plant family with respect to human use (Johnson, 1998). Numerous edible products are obtained from palms, including the familiar date palm fruits, coconut palm nuts, and various palm oils. Some less well-known edible palm products include palm “cabbage” or “heart-of-palm”, immature inflorescences, and sap from mature inflorescences.

Arecaceae has great economic importance including coconut products, oils, dates, palm syrup, ivory nuts, carnauba wax, rattan cane, raffia and palm wood The members of the Palm Family with human uses are numerous. • • • • • • • • • •

• •

The type member of Arecaceae is the Areca palm, the fruit of which, the betel nut, is chewed with the betel leaf for intoxicating effects (Areca catechu). Rattans, whose stems are used extensively in furniture and baskets are in the genus Calamus. Palm oil is an edible vegetable oil produced by the oil palms in the genus Elaeis. Palm sap is sometimes fermented to produce palm wine or toddy, an alcoholic beverage common in parts of Africa, India, and the Philippines. Dragon's blood, a red resin used traditionally in medicine, varnish, and dyes,. Coir is a coarse water-resistant fiber extracted from the outer shell of coconuts, used in doormats, brushes, mattresses, and ropes. Some indigenous groups living in palm-rich areas use palms to make many of their necessary items and food. Sago, for example, a starch made from the sago palm is a major staple food for lowland peoples of New Guinea and the Moluccas. Recently the fruit of the açaí palm Euterpe has been used for its reputed healthful benefits. Saw palmetto (Serenoa repens) is under investigation as a drug for treating enlarged prostates. Palm leaves are also valuable to some peoples as a material for thatching, basketry, clothing, and in religious ceremonies .

Ornamental Uses. Today, palms are valuable as ornamental plants Few palms tolerate severe cold, however, and the majority of the species are tropical or subtropical. The three most cold-tolerant species are Trachycarpus fortunei, Rhapidophyllum hystrix and Sabal minor,

2.7: Arecaceae species available in Bangladesh Table2.1 Arecaceae species available in Bangladesh (Bangladesh National Herbarium, 2005) Scientific name Local name Distribution of the plant 1.Borasses xlabelliser 2.Cocos nucifera 3.Areca catechu 4.Calamus tenuis 5.Nipa fruticans 6.Caryota urens 7.Oreodoya redia

Dhaka,Mymensingh, Kishoreganj, Jamalpur, Barisal,Noakhali, Mymensingh Narical Kishoreganj. Sherpur, Sylhet, Jamalpur, Dhaka, Chittagong, Coxbazar,Rajshai, Tangail. Supari Noakhali,Chittagong, Gajni, Kishoreganj, Rajshai, Faridpur, Sylhet,Netrokona, Mymensingh Bet Sylhet, Habigang Nipa Sundarban Chaur(Fishtail palm) Cultivated in the garden Royal palm Cultivated in the garden

8.Phoenies sylvespris

2.8: Description of Caryota urens: 2.8.1:Taxonomic hierarchy (Zipcodezoo.com) Kingdom: Plantae – Plants Subkingdom: viridaeplantae Phylum: Tracheophyta Subphylum: Euphyllophytina

Tal

Khejor

Jessore,Comilla

Class: Magnoliopsida Subclass: Arecidae Order: Arecales Family:Palmae Subfamily: Coryphoideae Genus: Caryota urens Species: Caryota urens(L.) Botanical name: - Caryota urens L.

Common names: (Bengali):Bonsopari,Golsagu,Chaur (English) : fishtail palm, Indian sago palm, kitul palm, toddy palm, wine palm (Hindi): mari (Sanskrit): dirgha, mada (Sinhala): kitul (Tamil) : konda panna, koondalpanai, kundal panai, thippali, tippili

2.9.2:General botanical data: Habit: tree Description: Caryota urens is an unarmed, hapaxanthic, solitary or clustered, medium-sized palm up to 20 or 30 m tall; bole straight, unbranched palm tree. Leaves 5-6 m long, bipinnate, drooping; leaflets are fish tail shaped. Flowers seen in peculiar pendular spadix, inflorescence is long, resembling a women's hair. Flowers in a group of three, central female guarded by two male flowers. Fruits globose, reddish when ripe.of three, central female guarded by two male flowers.

2.9.3:Photographs of Caryota urens:

(a) Whole Plants with fruits

(b) Leaves

Figure2.9.3: a) Whole Plants with fruites (b) Leaves c) fruites

2.8.4 Ecology and distribution Geographic distribution Native : India, Myanmar, Nepal, Sri Lanka,Bangladesh Exotic : Papua New Guinea, Thailand, Vietnam Culture Light: Toddy palm thrives in full sun to part shade. Moisture: This palm prefers a rich, moist, but well drained soil. Hardiness: USDA Zone 9 to 10. A mature toddy palm can handle temperatures as low as 26째F (-3째C) without damage, but young palms must be protected from frost. Seeds obtained from populations living at higher altitudes are colder hardy and more frost resistant.

Reproductive Biology C. urens is monoecious, flowering and leaf flushing continues throughout the year. Since the plants have a determinate growth habit, no new leaves originate after emergence of the 1st terminal inflorescence, which signals the start of the plantâ&#x20AC;&#x2122;s reproductive phase. Flowering begins at the top of the trunk and often continues downwards for several years. Individual staminate flowers remain open for 16-20 days, while a single inflorescence has flowers opening for about 6 weeks. The pistillate flowers open for 2-3 weeks after all the staminate flowers have bloomed and remain receptive for 313 days. C. urens is an obligate out breeder. Fruit development takes 32-38 weeks.

2.8.5:Propagation and management Propagation methods C. urens can be propagated by seed with direct sowing being a viable method. Exposure of seeds to direct sunlight for 6 hours prior to sowing inhibits germination. Therefore, satisfactory germination could be obtained by placing seeds in a moist, dark environment. Seeds germinate in 18-30 days. Germplasm Management At room temperature the seeds remain viable for 30-90 days, depending on storage conditions.

2.8.6:Medicinal uses: Local physicians to treat gastric ulcers, migraine headaches, snakebite poisoning and rheumatic swellings, prescribe a porridge prepared from C. urens flour. Plant pacifies vitiated pita, hyperpiesia, burning sensation, and general weakness. The root is used for tooth ailments, the bark and seed to treat boils, and the tender flowers for promoting hair growth. (Anon. 1986).

2.8.7:Chemistry of Caryota urens: It contains three types of Catechin(flavan-3-ol, a type of natural phenol and antioxidant namely 1-epicatechin, lgallocatechin and l-epicatechingaliate.(Center of Excellence for Medicinal Plants in Sri Lanka:2006).Catechin is a plant secondary metabolite. Catechin exists in the form of a glycoside.

Figure2.5: Catechin Catechin has four diastereoisomers. Two of the isomers are in trans configuration and are called catechin and the other two are in cis configuration and are called epicatechin. 1-epicatechin(also known under the names L-epicatechin, epicatechol, (-)-epicatechol, l-acacatechin, l-epicatechol, 2,3cis-epicatechin or (2R,3R)-(-)-epicatechin)is the most common epicatechin isomer.

(-)Figure2.5:(-)-Epicatechin (2R,3R)

Caryota urens contain calcium oxalate(skin irritant toxic substance)and possibly irritant proteins. 3.1 GENERAL APPROACHES TO DRUG DISCOVERY FROM NATURAL SOURCES

Despite the progress of science during the past four centuries, Shakespeare’s words “There are more things between heaven and earth…” did not lose their actuality. For many life-threatening illnesses, no effective treatment exists and knowledge about the etiology of diseases is still limited Traditional, empirical and molecular approaches have been utilized to discover new medicines (Harvey, 1999). The traditional approach provides drugs that have been found by trial and error over many years in different cultures and systems of medicine (Cotton, 1996). Examples include drugs like morphine, quinine and ephedrine that have been in widespread use for a long time. The empirical approach develops a therapeutic agent from a naturally occurring lead molecule and builds on an understanding of a relevant physiological process (Verpoorte, 1989, 2000). Examples include tubocurarine and other muscle relaxants, propranolol and other β-adrenoceptor antagonists, and cimetidine and other H 2 receptor blockers. The molecular approach is based on the availability or understanding of a molecular target for the medicinal agent (Harvey, 1999). With the development of molecular biological techniques and the advances in genomics, the majority of drug discovery is currently based on the molecular approach. Natural products have advantages over synthetic drug design in that it provides lead compounds having new structural features with novel biological activity. One-half of the medicines we use today, has been derived from natural sources. Virtually every pharmacological class of drugs includes a natural product prototype. The future of plants as sources of medicinal agents for use in investigation, prevention, and treatment of diseases is very promising. . Nature has been a valuable source of drugs and will always continue to deliver lead compounds (Martin and Lars, 2004) 3.2 GENERAL APPROACHES TO DRUG DISCOVERY FROM NATURAL SOURCES

Natural products have been the most successful source of drugs ever (Newman et al, 2003). Research progressed along two major lines: ethno pharmacology (medicinal herbs, substances of abuse, ordeal poisons) and toxicology (poisonous plants, venomous animals, arrow and fish poisons) (Heinrich, M., and Gibbons, S., 2001). These strategies have already produced many valuable drugs and are likely to continue to produce lead compounds (Tulp and Bohlin, 2002). Approximately 60% of the world’s population relies entirely on plants for medication (Farnsworth, 1994). Of the 520 new drugs approved between 1983 and 1994, 39% were natural products or derived from natural products and 60–80% of antibacterial and anticancer drugs were derived from natural products (Cragg et al, 1997). Thirteen natural product related drugs were approved from 2005 to 2007 (Butler, 2008), and five of these represented the first members of new classes of drugs: the peptides exenatide and ziconotide, and the small molecules ixabepilone, retapamulin. Current commercial evidence also supports the case for natural products. Of the 20 bestselling non-protein drugs in 1999, nine were either derived from or developed as the result of leads generated by natural products — simvastatin, lovastatin, enalapril, pravastatin, atorvastatin, augmentin, ciprofloxacin, clarithromycin and cyclosporin — with combined annual sales of >US$16 billion. Newer developments based on natural products include the antimalarial drug artemisinin and the anticancer agents taxol, docetaxel and camptothecin (Harvey and Waterman, 1998), (Verpoorte, 1998), (Grabley and Thiericke, 1999). Today, many new chemotherapeutic agents are synthetically derived, based on "rational" drug design. The study of natural products has advantages over synthetic drug design in that it leads optimally to materials having new structural features with novel biological activity. Not only do plants continue to serve as important sources of new drugs, but phytochemicals derived from them are also extremely useful as lead structures for synthetic modification and optimization of bioactivity. Natural products are naturally derived metabolites and/or by products from microorganisms, plants, or animals (Baker et al., 2000). The major advantage of natural products for random screening is the structural diversity. Bioactive natural products often occur as a part of a family of related molecules so that it is possible to isolate a number of homologues and obtain structure-activity relationship. Of course, lead compounds found from screening of natural products can be optimized by traditional medicinal chemistry or by application of combinatorial approaches. Overall, when faced with molecular targets in screening assays for which there is no information about low molecular weight leads, use of a natural products library seems more likely to provide the chemical diversity to yield a hit than a library of similar numbers of

compounds made by combinatorial synthesis. Since only a small fraction of the world’s biodiversity has been tested for biological activity, it can be assumed that natural products will continue to offer novel leads for novel therapeutic agents. 3.3: Experimental design 3.3.1 Evaluation of antioxidant activity:

The medicinal properties of plants have been investigated in the recent scientific developments throughout the world due to their potent antioxidant activities  no side effects and  economic viability. The antioxidant activity in terms of  DPPH free radical scavenging capability  Antioxidant activity assay by the Phosphomolybdenum method  Determination of total phenolic content  Determination of reducing power by potassium ferricyanide and trichloro acetate was evaluated to explore the potent antioxidant components from the investigated plants. 3.3.2 Brine shrimp lethality test: A rapid bioassay:

Brine shrimp lethality bioassay (Mclaughlin et al., 1976; Meyer et al., 1986) has been suggested for screening pharmacological activities in plant extracts. It is considered as a useful tool for preliminary assessment of toxicity and is a rapid and comprehensive bioassay for the bioactive compounds of natural and synthetic origin . A simple zoological organism (Brine shrimp nauplii) is utilized in this method to conveniently monitor in vivo lethality for screening and fractionation in the discovery of new bioactive natural products. The brine shrimp assay has several advantages of being rapid (24 hours),  inexpensive,  simple (e.g., no aseptic techniques are required).  It easily utilizes a large number of organisms for statistical validation  requires no special equipment and a relatively small amount of sample (2-20 mg or less).  Furthermore, it does not require animal serum as is needed for cytotoxicities. Brine shrimp toxicity is closely correlated with 9KB (human nasopharyngeal carcinoma) cytotoxicity (p=0.036 and kappa = 0.56). For cytotoxicities ED50 values are generally about one-tenth the LC 50 values found in the brine shrimp test. Thus, it is possible to detect and then monitor the fractionation of cytotoxic, as well as 3PS (P388) (in vivo murine leukaemia) active extracts using the brine shrimp lethality bioassay. 3.3.3 Microbiological investigations:

The antibacterial as well as antifungal spectrum of the crude extracts can be ascertained by observing the growth response with the help of in vitro antimicrobial study. These experiments are rationalized on the fact that many infectious diseases are caused by bacteria and fungi and if the test materials inhibit bacterial or fungal growth then they may be used in those particular diseases. However, a number of factors can influence the results like the extraction method  inocula volume,  culture medium composition,  pH and  incubation temperature. 3.3.4 Thrombolytic activity investigation

Cerebral venous sinus thrombosis (CVST) is a common disorder that is often accompanied by significant morbidity and mortality. In anticoagulation therapy the intravenous heparin is the first line of treatment for CVST, because of its efficacy, safety and feasibility. However, thrombolytic therapy with its ability to produce rapid clot lysis has long been considered as an attractive alternative.Thrombolytic drugs like tissue plasminogen activator(t-PA) ,urokinase, streptokinase etc. play a crucial role in the management of patients with CVST. 3.3.5 Membrane stabilizing activity investigation:

Inflammatory cells produce a complex mixture of growth differentiation cytokines as well as physiologically active arachidonate metabolites .In addition they possess the ability to generate reactive oxygen species(ROS) that can damage cellular biomolecules which in return augement the stae of inflammation.(Cochrane, 1991)The erythrocyte membrane resembles to lysosomal membrane and such as the effect of drugs on the stabilization of erythrocytes can be extrapolated to the stabilization of lysosomal membrane(Omale 2008).Therefore when membrane stabilizes they interfare in the release and in the action of mediators like histamine, serotonin, prostaglandine, leukotrines etc. (Shinde et al., 1999)

4.1 Rationale and objective Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many of which are based on their uses in traditional medicine. Plants produce a diverse array of bioactive molecules that are particularly important in the treatment of life-threatening conditions. Oxidation reactions initiated by excess free radicals have been shown to lead to the formation of tumors, damage of DNA ,mRNA , proteins, enzymes; cause cancer, cardiovascular diseases, nervous disorders, premature ageing, Parkinsonâ&#x20AC;&#x2122;s and Alzheimer's diseases, rheumatic and pulmonary disorders. Therefore, the need for systematic screening of medicinal plants for antioxidant activity cannot be overemphasized. Free radicals are atoms or group of atoms that have at least one unpaired electron, making them highly reactive. The potentially reactive derivatives of oxygen are known as reactive oxygen species (ROS) (e.g. superoxide anions, hydrogen peroxide and hydroxyl, nitric oxide radicals), and play an important role in oxidative damage to various biomolecules including proteins, lipids, lipoproteins and DNA, related to the pathogenesis of various important diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis, and neurodegenerative diseases and also in the ageing process. Antioxidants are the substance that when present in low concentrations compared to those of an oxidisable substrate significantly delays or prevents oxidation of that substance. Antioxidants prevent the oxidative damage by directly reacting with ROS, quenching them and/or chelating catalytic metal ions and also by scavenging free oxygen. Since ancient times, many herbs have been potentially used as an alternative remedies for treatment of many infections, diseases and as food preservatives suggesting the presence of antimicrobial and antioxidant constituents (Tatjana et al., 2005). There is an increasing interest in the antioxidants effects of compounds derived from plants, which could be relevant in relations to their nutritional incidence and their role in health and diseases. Plants are the potential source of natural antioxidants. Natural antioxidants or phytochemical antioxidants are the secondary metabolites of plants (Walton and Brown, 1999). Carotenoids, flavonoids, cinnamic acids, benzoic acids, folic acid, ascorbic acid, tocopherols, tocotrienols etc., are some of the antioxidants produced by the plant for their sustenance. Beta-carotene, ascorbic acid and alpha tocopherol are the widely used antioxidants (Mccall and Frei, 1999). Different synthetic antioxidant such as tert-butyl-1-hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) used as food additives to increase shelf life are known to have not only toxic and carcinogenic effects in humans (Ito et al.,1986; Wichi,1988), but abnormal effects on enzyme systems (Inatani et al., 1983). Therefore, the interest in natural antioxidant, especially of plant origin, has greatly increased in recent years (Jayaprakasha & Jaganmohan Rao, 2000). Plant polyphenols have been studied largely because of the possibility that they might underlie the protective effects afforded by fruit and vegetable intake against cancer and other chronic diseases (Elena et al., 2006). Because of the complex nature of phytochemicals, the antioxidant activities of plant extracts must be evaluated by combining two or more different in vitro assays. A number of reports on the isolation and testing of plant derived antioxidants have been described during the past decade. The purpose of this study was to evaluate different extractives of Albizia chinensis stem bark and Picrasma javanica leaves as new potential sources of natural antioxidants and phenolic compounds.

4.2: Assays for total phenolics The antioxidative effect is mainly due to phenolic components, such as flavonoids (Pietta, 1998), phenolic acids, and phenolic diterpenes (Shahidi, Janitha, & Wanasundara, 1992). The phenolic compounds exert their antioxidant properties by redox reaction, which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Osawa, 1994). Many phytochemicals possess significant antioxidant capacities that may be associated with lower incidence

and lower mortality rates of cancer in several human populations (Velioglu et al., 1998). Phenolic compounds are secondary metabolites of plants and can act as antioxidants by many potential pathways such as free radical-scavenging, oxygen radical absorbance, and chelating of metal ions

4.2.1 Principle: In the alkaline condition phenols ionize completely. When Folin-Ciocalteu reagent is used in this ionized phenolic solution the reagent will readily oxidize the phenols. Usual color of Folin-Ciocalteu reagent is yellow and after the oxidation process the solution becomes blue. The intensity of the color change is measured in a spectrophotometer at 760 nm. The absorbance value will reflect the total phenolic content of the compound (Harbertson and Spayd, 2006).

Phenols + Na2CO3

Ionized phenols + Folin-Ciocalteu reagent (Yellow)

Complete ionization

Ionized phenols

Oxidation

Folin-Ciocalteu reagent complex (Blue)

4.2.2 Materials & Methods: Total phenolic content of A. chinensis and P. javanica extractives was measured employing the method as described by Skerget et al., (2005) involving Folin-Ciocalteu reagent as oxidizing agent and gallic acid as standard (Majhenic et al., 2007).

4.2.2.1 Materials: 



UV-spectrophotometer



Folin-Ciocalteu reagent (10 fold diluted) Na2CO3 solution (7.5 %)



Vial



tert-butyl-1-hydroxytoluene (BHT)



Beaker (100 & 200ml)



Ascorbic acid



Test tube



Methanol



Pipette (1ml)



Chloroform



Pipette (5ml)



Carbon tetra chloride



Micropipette (50-200 µl)



n-hexane



Distilled water

4.2.2.2 Composition of Folin-Ciocalteu reagent: SL. No. Component 1 2 3 4 5 6

Water Lithium Sulfate Sodium Tungstate Dihydrate Hydrochloric Acid>=25% Phosphoric Acid 85 % solution in water Molybdic Acid Sodium Dihydrate

Percent 57.5 15.0 10.0 10.0 5.0 2.5

4.2.3 Standard curve preparation: Gallic acid was used here as standard. Different gallic acid solution were prepared having a concentration ranging from 100 µg / ml to 0 µg / ml. 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times with water) and 2.0 ml of Na 2CO3 (7.5 % w/v) solution was added to 0.5 ml of gallic acid solution. The mixture was incubated for 20 minutes at room temperature. After 20 minutes the absorbance was measured at 760 nm. After plotting the absorbance in ordinate against the concentration in abscissa a linear relationship was obtained which was used as a standard curve for the determination of the total phenolic content of the test samples.

4.2.4 Sample preparation: 2 mg of the extractives was taken and dissolved in the distilled water to get a sample concentration of 2 mg / ml in every case. The samples along with their concentration for the total phenolic content measurement are given in the Table 6.

Table 4.1: Test samples for total phenolic content determination

Plant part

Sample code

Test Sample

MESAC Fruit of C.urens

HXSF CTCSF AQSF

Calculated amount (mg/ml)

Methanolic extract of fruit of C.urens

2.0

Hexane soluble partitionate

2.0

Carbon tetrachloride soluble partitionate Aqueous soluble partitionate

2.0 2.0

4.2.5 Total phenolic compound analysis To 0.5 ml of extract solution (conc. 2 mg/ml), 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times with water) and 2.0 ml of Na 2CO3 (7.5 % w/v) solution was added. The mixture was incubated for 20 minutes at room temperature. After 20 minutes the absorbance was measured at 760 nm by UV-spectrophotometer and using the standard curve prepared from gallic acid solution with different concentration, the total phenols content of the sample was measured. The phenolic contents of the sample were expressed as mg of GAE (gallic acid equivalent) / gm of the extract. 2.5 ml Folin-Ciocalteu reagent (10 times diluted)

2.0 ml Na2CO3 (7.5 % w/v) solution

0.5 ml of diluted extract (Conc. 2.0 mg/ml) Incubated for 20 minutes at room temperature Absorbance measured at 760 nm

Figure 4.1: Schematic representation of the total phenolic content determination 4.3 Antioxidant activity assay by the Phosphomolybdenum method The Phosphomolybdenum method was based on the reduction of Molybdenum, Mo (VI) to Mo (V) by the antioxidant compound and the formation of a green phosphate-Mo (V) complex with a maximal absorption at 695 nm. The assay is successfully used to quantify vitamin E in Whole plant, roots and trunks. As it being simple and independent of other antioxidant measurements commonly employed, it was decided to extend its application to plant extracts (Prieto et al., 1999). Moreover, it is a quantitative one, since the antioxidant activity is expressed as the number of equivalents of ascorbic acid. The study reveals that the antioxidant activity of the extract exhibits increasing trend with the increasing concentration of the plant extract.

4.3.1 Materials and Method: The antioxidant activity of the extract was evaluated by the phosphomolybdenum method according to the procedure described by Prieto et al. (1999). The assay is based on the reduction of Mo (VI)â&#x20AC;&#x201C;Mo (V) by the extract and subsequent formation of a green phosphate-Mo (V) complex at acid pH. A 0.4 ml extract was combined with 4 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction mixture were incubated at 95 0C for 90 minutes. After cooling to room temperature, the absorbance of the solution was measured at 695 nm using a UV-spectrophotometer against blank. 0.4 ml of methanol and 4 ml reagent solution was added in the blank instead of extract solution. 4.3.1.1 Materials: Ammonium Beaker molybdate Sodium phosphate Pipette

4.3.1.2 Method:

Sulfuric acid

Amber reagent bottle

Methanol

Micropipette

Ferric chloride

UV-spectrophotometer

An aliquot of 0.4 ml of sample solution (1000 Âľg/ml in methanol) was combined in test tube with 4 ml of reagent solution (0.6 M Sulfuric acid, 28 mM sodium phosphate and 4 mM of ammonium molybdate). The blanks solution contained 4 ml of reagent solution and 1 ml of methanol. These test tubes were cotton plugged and incubated at 95 oC for 90 minutes. The absorbance of the mixture was measured at 695 mm against a blank after cooling at room temperature. The antioxidant activity was expressed relative to that ascorbic acid.

Reagent solution were prepared mixing 0.6 M H2SO4 with 28 mM Sodium Phosphate and 4 mM Ammonium Molybdate

0.4 ml sample solution were combined with 4 ml reagent Blank is prepared by mixing 4ml reagent solution & 0.4 ml methanol methanol

All test tubes are cotton pluged and incubated at 950 C for 90 minutes

Test tubes are cooled at room temperature

Absorbanc were measured at 695 nm against blank

Figure 4.2: Schematic representation of antioxidant capacitydetermination by

Phosphomolybdenum method

4.4 Antioxidant activity: DPPH assay 4.4.1 Principle: The free radical scavenging activities (antioxidant capacity) of the plant extracts on the stable radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were estimated by the method of Brand-Williams et al., 1995. 2.0 ml of a methanol solution of the extract at different concentration were mixed with 3.0 ml of a DPPH methanol solution (20Îźg/ml). The antioxidant potential was assayed from the bleaching of purple colored methanol solution of DPPH radical by the plant extract as compared to that of tert-butyl-1-hydroxytoluene (BHT) and ascorbic acid (ASA) by UV spectrophotometer.

N N

+ RH Antioxidant

O2N

NO2

*DPPH (oxidized) DPPH (reduced) *DPPH=1, 1-diphenyl-2-picrylhydrazyl

4.4.2 Materials & Methods:

DPPH was used to evaluate the free radical scavenging activity (antioxidant potential) of various compounds and medicinal plants (Choi et al., 2000; Desmarchelier et al., 1997).

4.4.2.1 Materials: 1,1-diphenyl-2-picrylhydrazyl tert-butyl-1-hydroxytoluene (BHT) Ascorbic acid Distilled water Methanol Chloroform Carbon tetra chloride n-hexane

UV-spectrophotometer Beaker (100 & 200ml) Amber reagent bottle Test tube Light-proof box Pipette (5ml) Micropipette (50-200 µl)

4.4.2.2 Control preparation for antioxidant activity measurement: Ascorbic acid (ASA) and tert-butyl-1-hydroxytoluene (BHT) was used as positive control. Calculated amount of ASA and BHT were dissolved in methanol to get a mother solution having a concentration 1000 μg/ml. Serial dilution was made using the mother solution to get different concentration ranging from 500.0 to 0.977 μg /ml.

4.4.2.3 Test sample preparation: Calculated amount of different extractives were measured and dissolved in methanol to get the mother solution (Conc. 1000 μg/ml). Serial dilution of the mother solution gave different concentration ranging from 500.0 to 0.977 μg /ml which were kept in the marked flasks.

4.4.2.4 DPPH solution preparation: 20 mg DPPH powder was weighed and dissolved in methanol to get a DPPH solution having a concentration 20 μg/ml. The solution was prepared in the amber reagent bottle and kept in the light proof box. ]

4.4.2.5 Assay of free radical scavenging activity: 2.0 ml of a methanol solution of the sample (extractives/ control) at different concentration (500 μg/ml to 0.977 μg/ml) were mixed with 3.0 ml of a DPPH methanol solution (20 μg/ml). After 30 min reaction period at room temperature in dark place the absorbance was measured at 517 nm against methanol as blank by UV spetrophotometer. Inhibition of free radical DPPH in percent (I%) was calculated as follows: (I%) = (1 – Asample/Ablank) X 100

Where Ablank is the absorbance of the control reaction (containing all reagents except the test material). Extract concentration providing 50% inhibition (IC50) was calculated from the graph plotted inhibition percentage against extract concentration.

Fig 4.3: Antioxidant activity DPPH assay

DPPH in methanol-3.0 ml

Extract in methanol-2.0 ml

(conc.- 20 μg/ml)

(conc.- 500 to 0.977 μg/ml )

Purple colour Incubated for 30 minutes in absence of light at room temperature

Decolorization of purple colour of DPPH

Absorbance measured at 517 nm using methanol as blank

Calcualtiion of IC50 value from the graph plotted inhibition percentage against extract concentration

Figure 4.4: Schematic representation of the method of assaying free radical scavenging activity The experiments were performed thrice and the the results were expressed as mean ± SD in every cases.

4.5 Results and discussion of the test samples of fruit of Caryota urens 4.5.1 Total phenolic content (TPC): The methanolic extract of C.urens (MESF) and different partitionates i.e. hexane (HXSF), carbon tetrachloride (CTCSF), and aqueous (AQSF) soluble partitionates of the methanolic extract of fruit of C.urens were subjected to total phenolic content determination. Based on the absorbance values of the various extract solutions, reacted with Folin-Ciocalteu reagent and compared with the standard

solutions of gallic acid (table 6.2) equivalents, results of the colorimetric analysis of the total phenolics are given in table 6.3. Total phenolic content of the samples are expressed as mg of GAE (gallic acid equivalent)/ gm of extractives. The amount of total phenolic content differs in different extractives and ranged from 1.45mg of GAE / gm of extractives to 106.88mg of GAE / gm of extractives of C.urens fruits. Among all extractives, the highest phenolic content was found in MESF 106.88mg of GAE / gm of extractives) followed by AQSF (41.82 mg of GAE / gm of extractives,CTC (7.44mg of GAE / gm of extractives and HXSF(1.45mg of GAE / gm of extractives,)

Table 6.2 Standard curve preparation by using gallic acid SL. No. 1 2 3 4 5 6 7 8 9 10

Conc. Of the Standard (Âľg / ml) 100 50 25 12.5 6.25 3.125 1.5625 0.78125 0.3906 0

Absorbance 1.620 0.866 0.450 0.253 0.120 0.059 0.034 0.022 0.020 0.011

Regression line

0.9985 y = 0.0162x + 0.0215

Fig:6.5: Standard curve of gallic acid for total phenolic determination Table 6.3: Test samples for total phenolic content determination

Plant part

Sample code

Fruit of C.urens

MESAC HXSF CTCSF

Test Sample

Methanolic extract of fruit of C.urens

Total phenolic content (mg of GAE / gm of extractives 106.88

Hexane soluble partitionate

1.45

Carbon tetrachloride soluble partitionate

7.48

AQSF

Aqueous soluble partitionate

41.82

Fig:4.6: Total phenolic content (mg of GAE / gm of extractives) of different extractives of fruit of C.urens

4.5.2 Antioxidant activity assay by the Phosphomolybdenum method: Total antioxidants capacity in terms of absorbance values of methanolic extract and different solvent partitionates of methanolic extract of stem bark of Albizia chinensis were determined by Phosphomolybdenum method and were expressed as equivalents of ascorbic acid and BHT (standard) (table 6.4). The highest antioxidant capacity was found in carbon tetrachloride soluble partitionate of stem bark of Albizia chinensis (MESAC) and the activity was decreased in the following order: CTCSF and MESAC>AQSF > HXSF (table 6.4)

Plant part

Fruit of C.urens

Table 4.4: Total antioxidant capacity in terms of absorbance of different extracts Sample code Test Sample Absorbance

MESAC

Methanolic extract fruit of C.urens

4.00

CTCSF

Carbon tetrachloride soluble partitionate

4.00

AQSF

Aqueous soluble partitionate

3.61

HXSF

Hexane soluble partitionate

1.12

Ascorbic acid

3.000

BHT

1.268

Fig. 4.7 : Total antioxidant capacities of different extracts of fruit of C.urens determined by phosphomolybdenum method

4.5.3 Free radical scavenging activity (DPPH): The Methanolic extract of fruit of C.urens (MESSF), and different partitionates i.e. hexane(HXSF), carbon tetrachloride (CTCSF), and aqueous (AQSF) soluble partitionate of the methanolic extract of fruit of C.urens were subjected to free radical scavenging activity by the method of Brand-Williams et al., 1995. Here, tert-butyl-1-hydroxytoluene (BHT) and ascorbic acid (ASA) was used as reference standard. In this investigation, MESF showed the highest free radical scavenging activity with IC 50 value 102.74 μg/ml . At the same time the CTCSF and HESF also exhibited antioxidant potential having IC 50 value 120.26 and 387.74 μg/ml respectively.

Table 4.5: IC 50 values of the standard and partitionates of fruit of C.urens Plant part

Fruit of C.urens

Sample code

Test Sample

IC50 (μg/ml)

MESF

Methanolic extract of fruit of C.urens

102.74

CTCSF

Carbon tetrachloride soluble partitionate

120.26

HXSF

Hexane soluble partitionate

387.74

BHT (tert-butyl-1-hydroxytoluene ) (standard)

27.50

ASA (Ascorbic acid) (standard)

5.80

Fig: 4.8: IC 50 values of the standard and partitionates of fruit of C urens

Table 4.6: IC50 value of tert-butyl-1-hydroxytoluene (BHT) Absorb -ance of the blank

0.324

Conc (Âľgm/ml)

Absor -bance of the extract

% Inhibi -tion

500 250 125 62.5 31.25 15.625 7.813 3.906 1.953

0.018 0.068 0.097 0.135 0.159 0.175 0.206 0.225 0.238

94.46 79.07 70.15 58.46 51.07 46.15 36.61 30.76 26.76

0.977

0.287

IC50

27.5

11.69

Figure 4.9: IC50 value of tert-butyl-1-hydroxytolune(BHT)

Table 4.7: IC50 value of Ascorbic acid (ASA) Absorb -ance of the blank

Conc (µgm/ml)

0.324

500 250 125 62.5 31.25 15.625 7.813 3.906 1.953

Absor -bance of the extrac t

% Inhibi -tion

0.005 0.006 0.015 0.024 0.068 0.098 0.139 0.186 0.175

98.46 98.15 95.38 92.61 79.07 69.84 57.23 42.76 46.15

IC50

5.8

Figure 4.10: IC50 value of Ascorbic acid 0.977

0.193

40.61

Table 4.8: IC50 value of methanolic extract (MESF) of fruit of C.urens Absor -bance of the blank

0.324

Conc (µgm/ml)

500 250 125 62.5 31.25 15.625

Absor -bance of the Extract 0.106 0.104 0.090 0.067 0.061 0.138

% Inhibi -tion

IC50

70.55 71.11 75.00 81.38 83.05 61.67

102.4 7

7.813 3.906 1.953 0.977

0.248 0.307 0.345 0.345

31.11 14.72 4.17 4.44

Fig 4.11:IC50 value of methanolic extract of fruit of C.urens Table 4.9: IC50 value of carbon tetrachloride soluble partitionate (CTCSF) of methanolic extract of fruit of C.urens Absor -bance of the blank

0.324

Conc (Âľgm/ ml)

500 250 125 62.5 31.25 15.625 7.813 3.906 1.953 0.977

Absor -bance of the Extract CTCS F 0.112

% Inhibi -tion

0.128 0.205 0.246 0.260 0.304 0.325 0.338 0.348 0.358

64.44 54.16 34.40 30.60 18.93 13.53 9.85 7.20 4.50

IC50

68.88

120.26

Fig 4.12 :IC50 value of carbon tetrachloride fraction of fruit of C.urens Table 4.11: IC50 value of hexane soluble partitionate (HXSF) of methanolic extract of fruit of C.urens

Absor -bance of the blank

Conc (µgm/ ml)

Absor -bance of the Extract HXSF

% Inhib i -tion

0.171

52.5

0.186

48.3

0.248

31.11

0.256

28.88

0.267

26.00

0.307

15.00

0.326 0.329

12.01 9.00

0.338

6.20

0.345

4.16

IC50

500 0.324

250 125 62.5 31.25 15.625 7.813

Fig 4.14:IC50 value of hexane soluble fraction of fruit of C.urens 387.74

3.906 1.953 0.977

5.1: Introduction

World wide, infectious disease is one of main causes of death accounting for approximately one-half of all deaths in tropical countries. Perhaps it is not surprising to see these statistics in developing nations, but what may be remarkable is that infectious disease mortality rates are actually increasing in developed countries, such as the United States. Death from infectious disease, ranked 5th in 1981, has become the 3rd leading cause of death in 1992, an increase of 58% .It is estimated that infectious disease is the underlying cause of death in 8% of the deaths occurring in the US (Pinner et al., 1996). This is alarming given that it was once believed that we would eliminate infectious disease by the end of the millenium. The increases are attributed to increases in respiratory tract infections and HIV/AIDS. Other contributing factors are an increase in antibiotic resistance in nosicomial and community acquired infections. Furthermore, the most dramatic increases are occurring in the 25–44 year old age group (Pinner et al., 1996). These negative health trends call for a renewed interest in infectious disease in the medical and public health communities and renewed strategies on treatment and prevention. It is this last solution that would encompass the development of new antimicrobials (Fauci, 1998). The antimicrobial screening which is the first stage of antimicrobial drug research is performed to ascertain the susceptibility of various fungi and bacteria to any agent. This test measures the ability of each test sample to inhibit the in vitro fungal and bacterial growth. This ability may be estimated by any of the following three methods (Ayaforet al., 1982).  Disc diffusion method  Serial dilution method  Bioautographic method

But there is no standardized method for expressing the results of antimicrobial screening (Ayaforet al., 1982). Some investigators use the diameter of zone of inhibition and/or the minimum weight of extract to inhibit the growth of microorganisms. However, a great number of factors viz., the extraction methods, inoculum volume, culture medium composition (Bayer et al., 1966), pH, and incubation temperature can influence the results. Among the above mentioned techniques the disc diffusion (Bayer et al., 1966) is a widely accepted in vitro investigation for preliminary screening of test agents which may possess antimicrobial activity. It is essentially a quantitative or qualitative test indicating the sensitivity or resistance of the microorganisms to the test materials. However, no distinction between bacteriostatic and bactericidal activity can be made by this method (Roland R, 1982). 5.2: Principle of disc diffusion method

In this classical method, antibiotics diffuse from a confined source through the nutrient agar gel and create a concentration gradient. Dried and sterilized filter paper discs (6 mm diameter) containing the test samples of known amounts are placed on nutrient agar medium uniformly seeded with the test microorganisms. Standard antibiotic (kanamycin) discs and blank discs are used as positive and negative control. These plates are kept at low temperature (4째C) for 24 hours to allow maximum diffusion of the test materials to the surrounding media (Barry, 1976). The plates are then inverted and incubated at 37째C for 24 hours for optimum growth of the organisms. The test materials having antimicrobial property inhibit microbial growth in the media surrounding the discs and thereby yield a clear, distinct area defined as zone of inhibition. The antimicrobial activity of the test agent is then determined by measuring the diameter of zone of inhibition expressed in millimeter (Barry, 1976; Bayer et al., 1966.) In the present study the crude extracts as well as fractions were tested for antimicrobial activity by disc diffusion method. The experiment is carried out more than once and the mean of the readings is required (Bayer et al., 1966). 5.3: Experimental

5.3.1: Apparatus and reagents Filter paper discs Nutrient Agar Medium Petridishes Sterile cotton Micropipette Inoculating loop Sterile forceps Screw cap test tubes

Autoclave Laminar air flow hood Spirit burner Refrigerator Incubator Chloroform Ethanol Nosemask and Hand gloves

5.3.2: Test organisms

The bacterial and fungal strains used for the experiment were collected as pure cultures from the Institute of Nutrition and Food Science (INFS), University of Dhaka. Both gram positive and gram-negative organisms were taken for the test and they are listed in the Table 5.1 Gram positive Bacteria Bacillus cereus

Gram negative Bacteria Escherichia coli

Fungi Aspergillusniger

Bacillus megaterium

Salmonella paratyphi

Candida albicans

Bacillus subtilis

Salmonella typhi

Sacharomycescerevacae

Sarcinalutea Staphylococcus aureus

Shigellaboydii Shigelladysenteriae

Pseudomonas aeruginosa Vibrio mimicus Vibrio parahemolyticus 5.3.3: Test materials

Table 5.2: List of Test materials Plant part

Fruits of Caryotaurens

Sample code

Sample Name

MESCU

Methanolic extract of leaves of C.urens

HEXCU

Hexane soluble partitionate

CTCCU CSFCU

Carbon tetrachloride soluble partitionate Chloroform soluble partitionate

5.3.4: Composition of culture medium The following media was used normally to demonstrate the antimicrobial activity and to make subculture of the test organisms. a) Nutrient agar medium Ingredients

Amount

Bacto peptone

0.5 gm

Sodium chloride

0.5 gm

Bacto yeast extract

1.0 gm

Bacto agar

2.0 gm

Distilled water q.s.

100 ml

7.2 + 0.1 at 250C

b) Nutrient broth medium Ingredients

Amount

Bacto beef extract

0.3 gm

Bacto peptone

0.5 gm

Distilled water q.s.

100 ml

7.2 + 0.1 at 250C

c) Muller â&#x20AC;&#x201C; Hunton medium Ingredients

Amount

Beef infusion

30 gm

Casamino acid

1.75 gm

Starch

0.15 gm

Bacto agar

1.70 gm

Distilled water q.s.

100 ml

7.3 + 0.2 at 250C

d) Tryptic soya broth medium (TSB)

Ingredients

Amount

Bactotryptone

1.70 gm

Bactosoytone Bacto dextrose Sodium chloride Di potassium hydrogen Phosphate Distilled water q.s pH

0.30 gm 0.25 gm 0.50 gm 0.25 gm 100 ml 7.3 + 0.2 at 250C

Nutrient agar medium is the most frequently used and also used in the present study for testing the sensitivity of the organisms to the test materials and to prepare fresh cultures. 5.3.5: Preparation of the medium

To prepare required volume of this medium, calculated amount of each of the constituents was taken in a conical flask and distilled water was added to it to make the required volume. The contents were heated in a water bath to make a clear solution. The pH (at 250C) was adjusted at 7.2-7.6 using NaOH or HCl. 10 ml and 5 ml of the medium was then transferred in screw cap test tubes to prepare plates and slants respectively. The test tubes were then capped and sterilized by autoclaving at 15-lbs. pressure at 121 0C for 20 minutes. The slants were used for making fresh culture of bacteria and fungi that were in turn used for sensitivity study. 5.3.6: Sterilization procedure

In order to avoid any type of contamination and cross contamination by the test organisms the antimicrobial screening was done in Laminar Hood and all types of precautions were highly maintained. UV light was switched on one hour before working in the Laminar Hood. Petridishes and other glassware were sterilized by autoclaving at a temperature of 1210C and a pressure of 15-lbs/sq. inch for 20 minutes. Micropipette tips, cotton, forceps, blank discs etc. were also sterilized by UV light. 5.3.7: Preparation of subculture

In an aseptic condition under laminar air cabinet, the test organisms were transferred from the pure cultures to the agar slants with the help of a transfer loop to have fresh pure cultures. The inoculated strains were then incubated for 24 hours at 370C for their optimum growth. These fresh cultures were used for the sensitivity test 5.3.8: Preparation of the test plate

The test organisms were transferred from the subculture to the test tubes containing about 10 ml of melted and sterilized agar medium with the help of a sterilized transfer loop in an aseptic area. The test tubes were shaken by rotation to get a uniform suspension of the organisms. The bacterial and fungal suspension was immediately transferred to the sterilized petridishes. The petridishes were rotated several times clockwise and anticlockwise to assure homogenous distribution of the test organisms in the media. 5.3.9: Preparation of discs

Measured amount of each test sample (specified in table 5.3) was dissolved in specific volume of solvent (Chloroform or methanol) to obtain the desired concentrations in an aseptic condition. Sterilized metrical (BBL, Cocksville, USA) filter paper discs were taken in a blank petridish under the laminar hood. Then discs were soaked with solutions of test samples and dried. Table 5.3: Preparation of sample Discs Test Sample

Dose μg/disc

Methanolic extract of fruits of C.urens Hexane soluble partitionate Carbon tetrachloride soluble partitionate Chloroform soluble partitionate

400 400 400 400

Plant part

Fruits of C.urens

Required amount for 20 disc(mg) 8.0 8.0 8.0 8.0

Standard Ciprofloxacin (30 µg/disc) discs were used as positive control to ensure the activity of standard antibiotic against the test organisms as well as for comparison of the response produced by the known antimicrobial agent with that of produced by the test sample. Blank discs were used as negative controls which ensure that the residual solvents (left over the discs even after air-drying) and the filter paper were not active themselves. 5.3.10: Diffusion and incubation

The sample discs, the standard antibiotic discs and the control discs were placed gently on the previously marked zones in the agar plates pre-inoculated with test bacteria and fungi. The plates were then kept in a refrigerator at 40C for about 24 hours upside down to allow sufficient diffusion of the materials from the discs to the surrounding agar medium. The plates were then inverted and kept in an incubator at 370C for 24 hours. 5.3.11: Determination of the zone of inhibition

The antimicrobial potency of the test agents are measured by their activity to prevent the growth of the microorganisms surrounding the discs which gives clear zone of inhibition. After incubation, the Antimicrobial activities of the test materials were determined by measuring the diameter of the zones of inhibition in millimeter with a transparent scale .

Figure 5.1: Clear zone of inhibition

Figure5.2: Determination of clear zone of inhibition

5.4: Results and discussion of In vitro antimicrobial screening of C.urens

The Methanolic extract of fruits of Caryotaurens(MESCU) and different partitionates i.e. carbon tetrachloride (CTCSF),hexane(HEXCU) and chloroform (CSFCU) soluble partitionate of the methanolic extract of fruits of C.urenswere subjected to antimicrobial screening with a concentration of 400 µg/disc in every case.The results are given in the Table 7.4. Some of the fractions showed zone of inhibition .The plant have anti microbial action against somel bacteria and fungi. Table 5.4: Antimicrobial activity of test samples of C. urens

Diameter of zone of inhibition (mm)

Test microorganisms MESCU

Hexane

CTCSF

CHCl

Ciprofloxacin

Gram positive bacteria Bacillus cereus Bacillus megaterium

Bacillus subtilis

Staphylococcus aureus Sarcina lutea

40 42 38 40 37

Gram negative bacteria Escherichia coli Pseudomonas aeruginosa

Salmonella paratyphi

Salmonella typhi

Shigellaboydii

Shigelladysenteriae

Vibrio mimicus

Vibrio parahemolyticus

Candida albicans

Aspergillusniger

Sacharomycescereva cae

41 43 36 35 41 40 38 37

Fungi

40 42 38

6.1 Introduction

Bioactive compounds are always toxic to living body at some higher doses and it justifies the statement that 'Pharmacology is simply toxicology at higher doses and toxicology is simply pharmacology at lower doses'. Brine shrimp lethality bioassay (McLaughlin, 1990; Persoone, 1980) is a rapid and comprehensive bioassay for the bioactive compound of the natural and synthetic origin. By this method, natural product extarcts, fractions as well as the pure compounds can be tested for their bioactivity. In this method, in vivo lethality in a simple zoological organism (Brine shrimp nauplii) is used as a favorable monitor for screening and fractionation in the discovery of new bioactive natural products.

This bioassay indicates cytotoxicity as well as a wide range of pharmacological activities such as antimicrobial, antiviral, pesticidal & anti-tumor etc. of the compounds (Meyer, 1982; McLaughlin, 1988). Brine shrimp lethality bioassay technique stands superior to other cytotoxicity testing procedures because it is rapid in process, inexpensive and requires no special equipment or aseptic technique. It utilizes a large number of organisms for statistical validation and a relatively small amount of sample. Furthermore, unlike other methods, it does not require animal serum. 6.2 Principle (Meyer et al., 1982)

Brine shrimp eggs are hatched in simulated sea water to get nauplii. By the addition of calculated amount of dimethylsulphoxide(DMSO), desired concentration of the test sample is prepared. The nauplii are counted by visual inspection and are taken in vials containing 5 ml of simulated sea water. Then samples of different concentrations are added to the premarked vials through micropipette. The vials are then left for 24 hours. Survivors are counted after 24 hours.

6.3 Materials Artemia salina leach (brine shrimp eggs) Sea salt (NaCl) Small tank with perforated dividing dam Micropipette Glass vials Magnifying glass

Test samples of experimental plants Lamp to attract shrimpsMicropipette Test tubes Pipettes

Table 6.1:Test samples of C.urens for brine shrimp bioassay Sample code

Test Sample

MESF HESF

Methanolic extract Hexane partitionate

Calculated (mg) 4 4

CTCF

Carbon tetrachloride soluble partitionate

CSF

Chloroform soluble partitionate

AQSF

Aqueous soluble partitionate

amount

6.4 Experimental Procedure 6.4.1 Preparation of seawater

38 gm sea salt (pure NaCl) was weighed, dissolved in one litre of distilled water and filtered off to get clear solution. 6.4.2 Hatching of brine shrimps

Artemia salina leach (brine shrimp eggs) collected from pet shops was used as the test organism. Seawater was taken in the small tank and shrimp eggs were added to one side of the tank and then this side was covered. One day was allowed to hatch the shrimp and to be matured as nauplii. Constant oxygen supply was carried out through the hatching time. The hatched shrimps were attracted to the lamp through the perforated dam and they were taken for experiment. With the help of a Pasteur pipette 10 living shrimps were added to each of the test tubes containing 5 ml of seawater.

Figure 6.1 Brine Shrimp Hatchery 6.4.3 Preparation of test samples of the experimental plant

All the test samples (MESF, HESF, CTCF, CSF, and AQSF) of the plant was taken in vials and dissolved in 200 µl of pure dimethyl sulfoxide (DMSO) to get stock solutions. Then 100 µl of solution was taken in the first test tube containing 5ml of simulated seawater and 10 shrimp nauplii. Thus, final concentration of the prepared solution in the first test tube was 400 µg/ml. Then a series of solutions of varying concentrations were prepared from the stock solution by serial dilution method. In every case, 100 µl samples were added to test tube and fresh 100µl DMSO was added to vial. Thus different concentrations were found in the different test tubes (Table 6.2). Table 6.2: Test samples with concentration values after serial dilution Test Tube No Concentration (µg/ml) 1

400.0

200 .0

100 .0

50 .0

25 .0

12.5

6.25

3.125

1.5625

0.78125

6.4.4 Preparation of control group

Control groups are used in cytotoxicity study to validate the test method and ensure that the results obtained are only due to the activity of the test agent and the effects of the other possible factors are nullified. Usually two types of control groups are usedi) Positive control ii) Negative control 6.4.4.1 Preparation of the positive control group

Positive control in a cytotoxicty study is a widely accepted cytotoxic agent and the result of the test agent is compared with the result obtained for the positive control. In the present study vincristine sulphate was used as the positive control. Measured amount of the vincristine sulphate was dissolved in DMSO to get an initial concentration of 20 µg/ml from which serial dilutions are made using DMSO to get 10 µg/ml, 5 µg/ml, 2.5µg/ml, 1.25 µg/ml, 0.625 µg/ml, 0.3125 µg/ml, 0.15625 µg/ml, 0.078125 µg/ml, 0.0390 µg/ml. Then the positive control solutions were added to the premarked vials containing ten living brine shrimp nauplii in 5 ml simulated sea water to get the positive control groups. 6.4.4.2 Preparation of the negative control group

100 µl of DMSO was added to each of three premarked glass vials containing 5 ml of simulated sea water and 10 shrimp nauplii to use as control groups. If the brine shrimps in these vials show a rapid mortality rate, then the test is considered as invalid as the nauplii died due to some reason other than the cytotoxicity of the compounds. 6.4.5 Counting of nauplii

After 24 hours, the vials were inspected using a magnifying glass and the number of survivors were counted. The percent (%) mortality was calculated for each dilution. The concentration-mortality data were analysed statistically by using linear regression using a simple IBM-PC program. The effectiveness or the concentration-mortality relationship of plant product is usually expressed as a median lethal concentration (LC 50) value. This represents the concentration of the chemical that produces death in half of the test subjects after a certain exposure period. 6.5 Results and Discussions 6.5.1 Results of C.urens

The methanolic extract of fruits(MEF), hexane soluble partitionate (HESF), carbon tetrachloride soluble partitionate (CTSF), chloroform soluble partitionate (CSF) and aqueous soluble partitionate (AQSF) of C. urens were subjected to brine shrimp lethality bioassay following the procedure of Meyer et al., (1982). The lethality of the extractives to brine shrimp was determined and the results are given in Table 6.9. The lethal concentration LC50 of the test samples after 24 hr. was obtained by a plot of percentage of the shrimps died against the logarithm of the sample concentration (toxicant concentration) and the best-fit line was obtained from the curve data by means of regression analysis. Vincristine sulfate (VS) was used as positive control and the LC 50 was found 0.451 µg/ml for VS. Compared with the negative control VS (positive control) gave significant mortality and the LC 50 values of the different extractives were compared to this positive control. The LC50 values of MEF, HESF, CTCF, CSF, AQSF were found to be 3.59, 0.59, 3.11, 0.20, 0.068µg/ml respectively (Table 6.9).AQSF, CTSF showed significant lethality whereas HESF, MES and CTCF showed moderate activity. However, varying degree of lethality to Artemia salina was observed with exposure to different dose levels of the test samples ranging from 0- 400 µg/ml. Table 7.9: LC50 values of the test samples of C.urens Test samples

Regression line

LC50 (µg/ml)

y = 30.799x + 60.645

0.9720

0.451

HESF

y = 5.55x+51.273

0.3813

0.59

CTCF

y = 27.5x+36.446

0.8589

3.11

CSF

y = 17.279x+62.173

0.7591

0.20

AQSF

y = -13.029x+96.95

0.5286

0.068

MEF

y = 6x+46.727

0.4296

3.51

VS HESF

= =

Vincristine sulphate Hexane soluble fraction of the methanolic extract of C. urens

CTSF CSF AQSF MEF

= = = =

Carbon tetrachloride soluble fraction of the methanolic extract of C. urens Chloroform soluble fraction of the methanolic extract of C. urens Aqueous soluble fraction of the methanolic extract of C. urens Methanolic extract of fruits of C. urens

Figure 7.7: LC50 values of the different extractives of C.urens. From the results of the brine shrimp lethality bioassay, it can be well predicted that the crude extract and partionate fractions possess cytotoxic principles. Compared with positive control (VS) the cytotoxicity exhibited by the extractives indicated that further bioactivity guided investigation can be done to find out potent antitumor and pesticidal compounds from this plant Table 6.3: Effect of Vincristine sulphate (positive control) on shrimp nauplii

LC 50 Conc. Âľ/ml 0

Log10 -

% Mortality 0

0.039 0.07812 5 0.15625

-1.40894

-1.10721

-0.80618

0.3125

-0.50515

0.625

-0.20142

1.25

0.09691

2.5

0.39794

0.69897

1.30103

100

Conc.

0.451

Figure 6.1 Plot of % mortality and predicted regression line of VS

Table 6.4: Effect of methanolic extract of fruits (MEF) of C. urens on shrimp nauplii

Conc. (mg/mL) 0 0.78125

Log10 Conc. -1.1072

% Mortality LC50 3.51 0 60

0.1938 2 0.4948 5 0.7958 8 1.0969 1 1.3979 4 1.6989 7 2 2.3010 3 2.6020 6

1.5625 3.125 6.25 12.5 25 50 100 200 400

100 100 80 80 100 90 100 100 Figure 6.2 Plot of % morality and predicted regression line of MEF of C.urens

100

Table 6.5:Effect of carbon tetrachloride soluble fraction of the methanolic extract (CTCF) of C. urens on shrimp nauplii

Conc. (mg/mL)

Log10 Conc.

% Mortality

0.78125 1.5625

-1.1072 0.19382

10 20

3.125

0.49485

6.25 12.5

0.79588 1.09691

70 60

1.39794

100

LC50

3.11

50 1.69897 80 100 2 90 200 2.30103 100 Figure 6.3 Plot of % mortality and predicted 400 2.60206 100 regression line of CTSF of C.urens. Table 6.6: Effect of chloroform soluble fraction of the methanolic extract (CSF) of C. urens on shrimp nauplii

Conc.

Log10

(mg/m)

Conc.

Mortality

LC50

0.20

0.78125

-1.1072

1.5625

0.19382

3.125

0.49485

6.25

0.79588

12.5

1.09691

100

25 50

1.39794 1.69897

90 100

Figure 6.4 Plot of % mortality and predicted

100

200 400

2.30103 2.60206

100 100

regession line of CSF of C.urens

Table 6.7:Effect of aqueous soluble fraction of the methanolic extract (AQSF) of C. urens on shrimp nauplii

Conc.

Log10

(mg/mL)

Conc.

Mortality

0.78125

-1.1072

1.5625

0.19382

3.125

0.49485

100

6.25

0.79588

12.5

1.09691

1.39794

1.69897

100

200

2.30103

400

2.6020 6

100

LC50

0.068

Figure 6.5 Plot of % mortality and predicted regression line of AQSF of C.urens

Table 6.8: Effect of Hexane soluble fraction of the methanolic extract (HESF) of C. urens. on shrimp nauplii

Conc. (mg/mL) 0 0.78125 1.5625 3.125 6.25 12.5 25 50 100 200 400

Log10 Conc. -1.1072 0.1938 2 0.4948 5 0.7958 8 1.0969 1 1.3979 4 1.6989 7 2 2.3010 3 2.6020 6

% LC50 Mortality 0 70 100 100 90 80

0.59

100 90 100 100 100

Figure 6.6 Plot of % mortality and predicted regression line of HESF of C.urens

7.1: Introduction Since ancient times, herbal preparations have been used for the treatment of several diseases. The leaves and/or twigs, stem, bark and underground parts of plants are most often used for traditional medicines. Herbal products are often perceived as safe because they are â&#x20AC;&#x153;naturalâ&#x20AC;? (Gesler, 1992). Cerebral venous sinus thrombosis (CVST) is a common disorder which accompanied by significant

morbidity and mortality (Watson et al., 2002). Heparin, an anticoagulating agent, is the first line of treatment for CVST, because of its efficacy, safety and feasibility (Biousse and Newman, 2004). Thrombolytic drugs like tissue plasminogen activator (t-PA), urokinase, streptokinase etc. play a crucial role in the management of patients with CVST (Baruah, 2006). Thus, the aim of the present study was to investigate the thrombolytic activity of methanolic extracts and its different fractions of fruits of Caryota urens.

7.2 Materials & Methods 7.2.1 Preparation of sample: The thrombolytic activity of all extractives was evaluated by a method using streptokinase (SK) as standard substance. 10 mg of methanolic extracts and its different fractions of fruits of Caryota urens were taken in different vials to which 1ml distilled water was added.

7.2.2 Streptokinase (SK): Commercially available lyophilized Altepase (Streptokinase) vial (Beacon pharmaceutical Ltd) of 15, 00,000 I.U., was collected and 5 ml sterile distilled water was added and mixed properly. This suspension was used as a stock from which 100μl (30,000 I.U) was used for in vitro thrombolysis.

7.2.3 Blood Sample: Whole blood (n=10) was drawn from healthy human volunteers without a history of oral contraceptive or anticoagulant therapy and 1ml of blood was transferred to the previously weighed sterile vials and was allowed to form clots.

7.2.4 Thrombolytic activity: Aliquots (5 ml) of venous blood were drawn from healthy volunteers who were distributed in ten different pre weighed sterile vials (1 ml/tube) and incubated at 37 °C for 45 minutes. After clot formation, the serum was completely removed without disturbing the clot and each vial having clot was again weighed to determine the clot weight (clot weight = weight of clot containing tube – weight of tube alone). To each vial containing pre-weighed clot, 100 μl aqueous solutions of different partitionates along with the crude extracts was added separately. As a positive control, 100 μl of streptokinase (SK) and as a negative non thrombolytic control, 100 μl of distilled water were separately added to the control vials. All the vials were then incubated at 37 °C for 90 minutes and observed for clot lysis. After incubation, the released of fluid was removed and vials were again weighed to observe the difference in weight after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis as shown below: % of clot lysis = (wt of released clot /clot wt) × 100

7.3 Results and Discussion of thrombolytic activity of Caryota urens As a part of discovery of cardio protective drugs from natural sources, the extractives fruits of C.urens either assess or not for thrombolytic activity and the results are presented in Table 9.1. Addition of 100μl SK, a positive control (30,000 I.U.), to the clots and subsequent incubation for 90 minutes at 37°C, showed 61.50% lysis of clot. On the other hand, distilled water was treated as negative control which exhibited a negligible percentage of lysis of clot (8.12%). The mean difference in clot lysis percentage between positive and negative control was found very significant. In this study,the methanolic extract of C. urens(MESF),hexane soluble fraction (HXSF), aqueous soluble fraction (AQSF) exhibited no thrombolytic activity except carbon tetrachloride soluble fraction (CTCSF) exhibited 20.48% thrombolytic activity.

Table 7.1: Thrombolytic Activity (in terms of % of clot lysis) of the extractives of C.urens Fractions

Weight of empty vial W1 g

Weight of clot containing vial before clot disruption W2 g

W3 Weight of clot containing vial after clot disruption W3 g

Weight of clot before clot disruption clot W4=W2-W1 g

Weight of clot after clot disruption clot W5=W3-W1 g

% of lysis

( W4-W5)/ X100% W4 %

MESAC CTCSF HXSF Blank SK

4.81 5.17 4.82 4.72 4.65

5.46 6.00 5.60 5.09 5.05

5.46 5.95 5.48 5.06 4.79

0.62 0.83 0.78 0.37 0.4

0.65 0.66 0.78 0.34 0.14

2.40 20.48 0 8.12 65

Fig. 7.1: Thrombolytic activity of the extractives of C. urens

From this experiment, it can be concluded that the extractives of C. urens showed no clot lysis activity than the standard substance streptokinase (SK) except the Carbontetrachloride soluble fraction (CTCSF), exhibiting very poor (20.48%) thrombolytic activity compared to Sterptokinase (65%)

8.1: Introduction: In many of the pathological disorders, inflammation is the one of the important processes. Inflammatory cells produce a complex mixture of growth and differentiation of cytokines as well as physiologically active arachidonate metabolites. In addition they possess the ability to generate reactive oxygen species (ROS) that can damage cellular biomolecules which in turn augment the state of inflammation (Cochrane, 1991). Compounds that possess radical scavenging ability may therefore expect to have the therapeutic potentials for inflammatory disease (Trenam et al., 1992). The erythrocyte membrane resembles to lysosomal membrane and as such, the effect of drugs on the stabilization of erythrocyte could be extrapolated to the stabilization of lysosomal membrane (Omale, 2008). Therefore, as membrane stabilizes that interfere in the release and or action of mediators like histamine, serotonin, prostaglandins, leukotrienes etc. (Shinde et al., 1999). Thus, the aim of the present study was to investigate the anti-inflammatory activity of methanolic extract and its different fraction of fruits of C.urens 8.2: Materials and method: 8.2.1: Preparation of the extract: Table 8.1: Preparation of different extracts of fruits of C.urens Sample code Concentration Hypotonic medium

50 mM

MEF

2 mg/mL

HESF

2 mg/mL

CTCSF

2 mg/mL

Acetyl salicylic acid

0.10 mg/mL

Solvent used: Methanol analytical grade 8.2.2: Drug: Standard Acetyl Salicylic Acid (ASA) or Aspirin was used as standard drug for comparison with different methanolic extracts of fruits of C.urens. 8.2.3: Red Blood Cells (RBC) collection: Human RBCs were collected for the study. RBCs collected from the human was male, 70 kg, fare complexion and free from diseases. The collected RBCs were kept in a test tube with an anticoagulant EDTA under standard conditions of temperature 23±2°C and relative humidity 55±10%. 8.2.4: Preparation of Phosphate buffer solution: A buffer is an aqueous solution that has a highly stable pH.The buffer was prepared at pH 7 using monosodium phosphate and its conjugate base, disodium phosphate. 8.2.4.1: Phosphate buffer materials: • Monosodium phosphate • Disodium phosphate • Water • pH meter • Glassware • Stirring bar

8.2.4.2: Calculation of Phosphate buffer: A pH of about 7.4 with buffer strength of 10 mM was obtained using 0.0352% monosodium phosphate dehydrate and 0.1099% disodium phosphate anhydrate. The buffer was made by adding 0.352 gm monosodium phosphate dehydrate and 1.099 gm disodium phosphate anhydrate to 1000 mL water. • pH: 7.4 • Buffer strength: 10.00 mM • Monosodium phosphate, dehydrate: 0.0352% • Disodium phosphate, anhydrate: 0.1099% 8.2.5: Preparation of isotonic solution A solution that has a concentration of electrolytes, nonelectrolytes or a combination of the two that will exert equivalent osmotic pressure as that solution with which it is being compared. Either 0.16M sodium chloride (NaCl) solution (approximately 0.95% salt in water) or 0.3M nonelectrolyte solution is approximately isotonic with human red blood cells. For the preparation of 500 ml isotonic solution of 154 mM strength, 4.5045 gm NaCl was added and mixed.

8.2.5.1: Material for isotonic solution • • • •

Sodium chloride (NaCl) Water Glassware Stirring bar

8.2.5.2: Calculation for isotonic solution: 1000 ml solution of strength 1 M contain = 58.5 gm NaCl 500 ml solution of strength 1 M contain = 58.5/2 gm NaCl 500 ml solution of strength 1000 mM contain = 58.5/2 gm NaCl 500 ml solution of strength 154 mM contain = 58.5 × 154/2 × 1000 gm NaCl = 4.5045 gm NaCl 8.2.6: Preparation of hypotonic solution A solution of lower osmotic pressure than that of a reference solution or of an isotonic solution is called hypotonic solution. For the preparation of 500 ml hypotonic solution, having strength of 50 mM, 1.4625 gm NaCl was added and mixed.

8.2.6.1: Materials for hypotonic solution • • • •

Sodium chloride (NaCl) Water Glassware Stirring bar

8.2.6.2: Calculation for hypotonic solution: 1000 ml solution of strength 1 M contain = 58.5 gm NaCl 500 ml solution of strength 1 M contain = 58.5/2 gm NaCl 500 ml solution of strength 1000 mM contain = 58.5/2 gm NaCl 500 ml solution of strength 50 mM contain = 58.5 × 50/2 × 1000 gm NaCl = 1.4625 gm NaCl

8.2.7: Effect on haemolysis: 8.2.7.1: Erythrocyte suspension:

Whole blood was collected from male human under standard condition. EDTA was used to prevent clotting. The blood was washed three times with isotonic solution (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4) through centrifuge action for 10 min at 3000 g. Thus, the suspension finally collected was the stock erythrocyte (RBC) suspension.

8.2.7.2: Hypotonic solution- induced haemolysis: The experiments were carried out with hypotonic solution. The test sample consisted of stock erythrocyte (RBC) suspension (0.50 mL) with 5 ml of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffer saline (pH 7.4) containing either the different methanolic extract (2.0 mg/mL) or Acetyl Salicylic Acid (0.10 mg/mL). The Acetyl Salicylic Acid was used as a reference standard. The mixtures were incubated for 10 min at room temperature, centrifuged for 10 min at 3000 g and the absorbance (O.D.) of the supernated was measured at 540 nm using Shimadzu UV spectrophotometer. The percentage inhibition of either haemolysis or membrane stabilization was calculated using the following equation: % inhibition of haemolysis = 100 Ă&#x2014; {(OD1- OD2)/ OD1} Where, OD1 = Optical density of hypotonic-buffered saline solution alone (control) and OD2 = Optical density of test sample in hypotonic solution. 8.2.7.3 Heat- induced haemolysis Aliquots (5 ml) of the isotonic buffer containing 1.0 mg/mL of different extractives of plants were put into two duplicate sets of centrifuge tubes (Shinde et al., 1999). The vehicle, in the same amount, was added to another tube as control. Erythrocyte suspension (30 ÂľL) was added to each tube and mixed gently by inversion. One pair of the tubes was incubated at 54 oC for 20 min in a water bath. The other pair was maintained at 0-5oC in an ice bath. The reaction mixture was centrifuged for 3 min at 1300 g and the absorbance of the supernatant was measured at 540 nm. The percentage inhibition or acceleration of hemolysis in tests and was calculated according to the equation: % Inhibition of hemolysis = 100 x [1- (OD2-OD1/ OD3-OD1)] Where, OD1 = test sample unheated, OD2 = test sample heated and OD3 = control sample heated 8.3: Results and discussion of the test samples of C.urens The fruits of C.urens were mild effective in the membrane stabilizing activity as the extractives prevented the lyses of erythrocytes induced by hypotonic solution. During hypotonic condition the different fractions of C.urens i.e. MEF, HESF and CTCSF showed inhibition.19.00%, 9.67% and 10.01% respectively. During hypotonic condition, the percent inhibition of ASA was about 84.444%. The different methanolic extracts of fruits of C.urens at concentration 2.0 mg/mL significantly protected the lysis of human erythrocyte membrane heat induced by isotonic solution, as compared to the standard acetyl salicylic acid (0.10 mg/mL) (Table-8.2). During heat induced condition, the metahanolic extract (MEF) inhibited 94.17%. hexane soluble partitionate inhibited (HESF) 93.28% and carbon tetrachloride soluble partitionate (CTCSF) inhibited 90.91% of hemolysis of RBC. During heat, induced condition the percent inhibition of ASA was about 42.12%. Table-8.1: Effect of different extractives of fruit of C.urens on hypotonic solution-induced haemolysis of erythrocyte membrane. Sample code

Concentration

Absorbance

% inhibition of haemolysis

Hypotonic medium

50 mM

3.225

MEF

2 mg/mL

2.612

19.00

HESF

2 mg/mL

2.913

9.67

CTCSF

2 mg/mL

2.902

10.01

Acetyl salicylic acid

0.10 mg/mL

0.014

84.444

Figure-8.1: % inhibition of haemolysis of different extractives of fruits of C.urens at hypotonic condition. Table-8.2: Effect of different extractives of fruits of C.urens on heat induced haemolysis of erythrocyte membrane. Sample code

Concentration

Absorbance

% inhibition of haemolysis

Hypotonic medium

50 mM

MEF

2 mg/mL

0.801

94.17

HESF

2 mg/mL

0.615

93.28

CTCSF

2 mg/mL

0.852

90.91

Acetyl salicylic acid

0.10 mg/mL

0.014

42.12

Figure 8.2: % inhibition of haemolysis of different extractives of fruits of C.urens at heat induced condition. The effect of synthetic and herbal anti-inflammatory agents on the stabilization of erythrocyte membrane exposed to isotonic solution has been studied extensively. The erythrocyte membrane resembles to lysosomal membrane and as such, the effect of drugs on the stabilization of erythrocyte could be extrapolated to the stabilization of lysosomal membrane (Omale, 2008). The results showed that the extracts were potent on human erythrocyte adequately protecting it against heat-induced lyses. The activity was comparable to that of standard anti-inflammatory drug (Acetyl Salicylic Acid). It has been reported that flavorous exert profound stabilizing effects on lysosomes both in vitro and in vivo experimental animals (Van-Cangeghem, 1972; Sadique et al., 1989; Middleton, 1996) while tannin and saponins have the ability to bind cations and other biomolecules, and are able to stabilize erythrocyte membrane (Oyedapo, 2001; El-Shanbrany et al., 1997). The present investigation suggests that the potent membrane stabilizing activity of the fruits of C.urens plays a significant role in its anti-inflammatory activity may be due to its high flavonoids and tannin content.

Conclusion Caryota urens test materials were involved in several biological screening which include antioxidant activity screening, brine shrimp lethality bioassay, antimicrobial screening, thrombolytic activity, membrane stabilizing activity where different fractionates showed biological activities that where statistically evaluated. Of which the anti-oxidant,anti-microbial, and anti- inflammatory activities of test materials of the plant extract were highly significant. Therefore, considering the potential bioactivity, the plant materials can be further studied extensively to find out their unexplored efficacy and to rationalize their uses as traditional medicines.