GJRMI - Volume 1, Issue 10, October 2012 by Dr. Hari Venkatesh.K.Rajaraman

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ISSN 2277 â&#x20AC;&#x201C; 4289 www.gjrmi.com Editor-in-chief Dr Hari Venkatesh K Rajaraman

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Honorary Members - Editorial Board Dr Farhad Mirzaei Mr. Harshal Ashok Pawar

INDEX – GJRMI, Vol.1, Iss. 10, October 2012 MEDICINAL PLANT RESEARCH Ethno- Linguistics ETHNO-NOMENCLATURE OF THE SHEA TREE (VITELLARIA PARADOXA C.F. GAERTN.) AND ITS PRODUCTS IN THE SHEA ZONES OF UGANDA Okullo JBL, Omujal F, Agea JG, Mulugo LW, Vuzi PC, Namutebi A, Okello JBA, Okonye G, Nyanzi SA …………………………………………………………………………………….477–484 Bio-Technology A NOVEL REGENERATION SYSTEM FOR A WILD PASSION FRUIT SPECIES (PASSIFLORA FOETIDA L.) BASED ON DIRECT SOMATIC EMBRYOGENESIS FROM LEAF EXPLANT Patil Anita S, Paikrao Hariprasd M..................................................................................... .485–495 Bio-Technology EVALUATION OF EFFECT OF METHANOLIC AND AQUEOUS EXTRACTS OF PUNICA GRANATUM L. AGAINST BACTERIAL PATHOGENS CAUSING BOVINE MASTITIS Gopinath S M, Suneetha T B, Singh Sumer………………………………………………..496–502 Bio-Technology SAPONIN: A WONDER DRUG FROM CHLOROPHYTUM SPECIES Sharma Rohit, Thakur Gulab S, Sanodiya Bhagwan S, Pandey Mukeshwar, Bisen Prakash S………………………………………………………………………………503–515 Pharmacology STANDARDIZATION OF POLYHERBAL FORMULATION – ARSHONYT FORTE Agrawal SS, Ghorpade SS, Gurjar PN……………………………………………………...516–523 Agriculture STUDIES ON SEED GERMINATION AND GROWTH IN GLORIOSA SUPERBA Anandhi S, Rajamani K……………………………………………………………………...524–528 Bio-Technology MASS PROPAGATION AND IN VITRO CONSERVATION OF INDIAN GINSENG (WITHANIA SOMNIFERA). Chatterjee Tuhin, Ghosh Biswajit…………………………………………………. ………529–538 Indigenous Medicine Ayurveda A COMPARATIVE PHARMACOGNOSTICAL EVALUATION OF RAW AND TRADITIONALLY SHODHITA VACHA (ACORUS CALAMUS LINN.) RHIZOMES Bhat Savitha D, Ashok B K, Harisha C R, Acharya Rabinarayan, ShuklaV J................539–550 STUDY OF UNIDENTIFIED PLANTS FROM RASA RATNA SAMUCCHAYA Pampattiwar S P, Bulusu Sitaram, Paramkusa Rao M……………………………………551-556

HERBAL DRUG SWIETENIA MAHAGONI JACQ. - A REVIEW Khare Divya, Pradeep H R, Kumar Krishna Kishore, Hari Venkatesh K R, Jyothi T…557–567

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FLOWERS OF ASCLEPIAS CURASSAVICA L., APOCYNACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

Research article ETHNO-NOMENCLATURE OF THE SHEA TREE (VITELLARIA PARADOXA C.F. GAERTN.) AND ITS PRODUCTS IN THE SHEA ZONES OF UGANDA Okullo John Bosco Lamoris1, Omujal Francis2, Agea Jacob Godfrey1*, Mulugo Lucy Were1, Vuzi Peter California3, Namutebi Agnes1, Okello John Bosco Acot1, Okonye Godman4, Nyanzi Steven Allan3 1

College of Agricultural and Environmental Sciences, Makerere University, P. O. Box 7062, Kampala, Uganda. 2 Natural Chemotherapeutic Laboratory, Wandegeya, Kampala, Uganda 3 College of Natural Sciences, Makerere University, P. O. Box 7062, Kampala, Uganda. 4 Department of English, College of Education, University of Juba, Government of South Sudan (GOSS) *Corresponding Author’s Email: agea@forest.mak.ac.ug/jgagea@gmail.com

Received: 04/09/2012; Revised: 30/09/2012; Accepted: 01/10/2012

ABSTRACT A cross sectional survey was conducted in north-eastern Shea zones of Uganda to assess ethnonomenclature of the Shea tree (Vitellaria paradoxa C.F.Gaertn.) and products. The largely qualitative study that involved a total of six different ethnic groups was analyzed using emerging themes and patterns. Findings collected through individual and group interviews revealed variations and similarities in the ethno names. There was a wide variation in ethno-names of the Shea tree/products across and within the ethnic groups. The variations are explained by differences in languages spoken as well as dialects across the ethnic groups. It could also be a reflection of extensive range of occurrence of the Shea trees. Some ethnic groups e.g. Acholi and Langi; Madi and Lugbara had some similarities in the ethno-names. The similarity seemed to be explained by shared historical background and frequent interactions. Migration, intermarriages and frequent trade interactions had a contribution to the similarities between the ethnic groups. This study, however, did not investigate into the meanings of the ethno names, an area that should be taken up for further research. KEY WORDS: ethno-nomenclature, Shea tree, Vitellaria paradoxa, parklands, Uganda.

Cite this article: Okullo JBL, Omujal F, Agea JG, Mulugo LW, Vuzi PC, Namutebi A, Okello JBA, Okonye G, Nyanzi SA (2012), ETHNO-NOMENCLATURE OF THE SHEA TREE (VITELLARIA PARADOXA C.F. GAERTN.) AND IT’S PRODUCTS IN THE SHEA ZONES OF UGANDA, Global J Res. Med. Plants & Indigen. Med., Volume 1(10): 477–484

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 10 | October 2012 | 477–484

INTRODUCTION

1994; Steiner and Scheidegger, 1994; Warren and MacKiernan, 1995). It should have however be noted that both local communities’ and scientific knowledge have strengths and weaknesses. This is the case, for instance, if local communities’ knowledge that was valid in the past fails to adapt to the rapidly changing environment.

Small-scale farmers in sub-Saharan Africa depend heavily on natural resources for food security and other socio-economic needs. Knowledge systems of these people that relate to the natural resources and non-timber harvested products for improved livelihood have never been well documented. Although such knowledge systems remain invisible to the developing communities and are not easily accessible to development practitioners operating in rural communities, they are vital in the search for solutions to community problems (Warren & Rajasekaran, 1993). The Shea tree (Vitellaria paradoxa C.F.Gaertn.) is one of such natural resources mankind has been endowed with in Uganda. This tree is a dominant species in agro-forestry parkland systems (Lovett and Haq, 2000; Okullo et al., 2004; Okia et al., 2005). The tree has been described as a “Green gift from God to mankind” (Guru, 2007), “sacred tree” for super natural powers and “Miracle tree” (CFC and FAO, 2005) because of its multiple uses. Bouvet et al., (2004) describes V. paradoxa as economically and socially important plant species. In Uganda V. paradoxa is found mainly in northern, northeastern and West Nile regions (Katende et al., 1995). The Shea tree is one of the most important sources of vegetable oil whose seeds are used for oil processing for home consumption and trading (PROTA, 2007).

More than Western scientists, local people are aware of the weaknesses that may exist in their knowledge base (Warren, 1991). Eliciting these drawbacks can be imperative for the proper identification and definition of problems and for effective research and extension. Further, inputs targeting specific knowledge gaps can render information transfer more efficient, acceptable, and practicable for local people especially farmers (Bentley 1992; Sherwood, 1997). However, information transfer should occur in both directions. For most natural phenomena, local people have their own frameworks within which they interpret and explain observations and facts. Former extension approaches (Transfer of Technology, Training and Visit System), building on one-sided information transfer from the extension agent to the farmer, failed to recognize, acknowledge, and incorporate farmers’ concepts. This often resulted in negative self-esteem patterns for the farmers, though their knowledge and role as research partners are increasingly gaining recognition (Haverkort and Hiemstra, 1999).

Although the Shea tree is a nutritional and economic resource of great importance in these regions of Uganda, little has been systematically documented on the local community knowledge about this tree (tree characteristic, harvest preferences) and its harvested products (fruit, nuts, kernels, oil). Local knowledge and perception of Shea tree and its products is an important issue for rural development programs and ample experience has shown that communities’ local knowledge can differ profoundly from scientific knowledge in terms of significance for development (Chambers, 1997; Horton and Ewell, 1991; Nazarea-Sandoval and Rhoades,

It is hoped that the information contained in this paper will contribute to an understanding of local community knowledge on folk nomenclature about the Shea tree (tree characteristic, harvest preferences) and its harvested products (fruit, nuts, kernels, oil) in Uganda. The information were gathered based on local communities’ views and concepts based on their experience taking into account the ethnic variability in qualitative knowledge. Qualitative knowledge is a composite knowledge based on amalgamation of individual knowledge. As such the information presented here exceeds the individual knowledge by far.

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 10 | October 2012 | 477–484

STUDY AREA AND METHOD

METHODS

Study Area

Several data-gathering methods were applied to gain a comprehensive picture of the local community knowledge system of the Shea tree and its harvest to validate information. The research approach combined methods including interviews, semi-structured questionnaires and free-listing. A total of 275 questionnaires were administered. For analysis, the data was transferred to a spreadsheet. The frequency of items mentioned across the lists and in the questionnaires was calculated by counting the total number of reports of each item among the respondents. It is important to note that the frequency of mention is a good measure for salience, although it does not consider the item’s position within the list.

This study was conducted in the Acholi, Lango, Teso, Acholi and West Nile sub regions, Uganda. Specifically this was carried out in Pader, Lira, Katakwi and Arua districts respectively. These districts have got well established, reliable Shea stand populations and the community highly depends on Shea butter oil/fat for both food uses and other benefits. Fieldwork was conducted during several visits between July 2007 and January 2008 in the districts of Lira, Pader, Katakwi, Nebbi, Arua and Moyo. These sampled districts are in the Shea producing zones of Uganda. In these districts, there is high dependence on Shea butter oil/fat and other related products by the local communities. These districts are also the ones where there are well established, reliable Shea stand populations and varied Shea butter processing technologies and processing practices in Uganda. As the local communities in these Shea parkland areas have not been adequately involved in any research or development activities targeting the Shea so far, they were very interested and eager to join exercises and discussions. Their willingness and curiosity to participate made the research an extremely pleasant task.

RESULTS Socio-demographic characteristics respondents from the Shea zones

The socio-demographic characteristics of the respondents are presented in Table 1. Majority of the respondents among Acholi, Lango, Madi and Lugbara ethnic groups were men as opposed to the Alur and Iteso ethnic groups. Most respondents interviewed were aged between 19 and 60 years and their main (90 %) occupation was subsistence farming. Very few respondents engaged in trade.

Table 1: Socio-demographic characteristics of respondents from the Shea producing zones % Response

Variable Sex Male Female Age < 18years 19–37 years 38–56 years >56 years Occupation Subsistence farming Trade

Acholi

Lango

Iteso

Madi

Alur

Lugbara

72 28

69 31

47 53

02 38

37 63

63 37

00 43 42 15

09 37 46 08

03 44 44 09

13 53 34 00

00 50 42 08

04 38 52 06

89 11

85 15

87 13

85 15

95 05

90 10

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however, not sought in this study.

Ethno-names of the Shea tree in the Shea parkland areas of Uganda

Ethno-names of the Shea tree products in the Shea parkland areas of Uganda

The ethno-naming of the Shea tree varied widely among the studied ethnic communities in the Shea parklands (Table 2). The Acholi ethnic group called the Shea tree yaa, yao; the Alur called it yen yao, danyu, awa; the Lango ethnic group called it, yao; the Iteso refer to it as ekungur while the Lugbara called it awa and the Madi ethnic group called it awa, awa pati and kiwee. The ethno-name yao was common to Acholi, Alur and Lango while awa was common to the Lugbara and the Madi ethnic groups. The meanings behind such naming was

Just like ethno-names, the naming of the Shea tree products varied widely among the ethnic groups. For example among the Acholi ethnic group, the Shea fruit was called by different names such as odua, eduu, kitigu and kiduu. The Iteso called it akungur, adanyoi and odu, the Lango people called the fruit adu, adanyo, kom yao while the the Madi called it as Awa udu, awa adu, aweki, awasodi (Table 3).

Table 2: Ethno-names of the Shea tree in the Shea parkland areas of Uganda Ethnic groups

Ethno-names of Shea tree

Acholi Alur Iteso Lango Lugbara Madi

Yaa, yao Yen yao, awa, danyu Ekungur Yao Awa Awa pati, Awa kwee

Table 3: Ethno-names of the Shea tree products in the Shea parkland areas of Uganda Ethno-names of Shea tree products Ethnic groups

Acholi

Alur Iteso Lango

Lugbara

Madi

Shea fresh fruit

Shea nut

Odu, odua, eduu, kitigu, kiduu.

Yao magolo, yaa magolo.

Yaa/yao nyinge, nying yaa/yao, magolo yaa/yao koro.

Moo yaa, moo yao.

Awakorongo, dend yao, pok yao.

Nyinge yao, aweki.

Moo yao, awa odu, odu omoo.

Akungur.

Elemut, akungur Kiwee.

Akungur, alinyo moo yaa.

Yao, yao agulu.

Yao koro, yao.

Moo yao.

Awodu, aswadi, awadu, odu, owodu, aweki.

Aweki, awasodri, awa ongolo awaongorobo awakorongo, awakini, iki ikiya.

Sundri, nyinge, den yao, awa gili,awa ogiri, awaikiki awasodi.

Odu, oduni, omo, ikuya awadu, awaa adu, ikiya.

Awa udu, awa adu, aweki, awasodi.

Awa echwi, awa ekwi, awa gili awa boroso awa obo.

Awa boroso, awa ekwi, ugalera, awa opalarekwi, awaikiki, awa gili, awa ogiri, awa opkolo, aweki.

Awa odu, awa adu.

Dany yao, danyo, odanyo, adu, awa adu. Akungur, adanyoi, odu. Adu, adanyo, kom yao.

Shea seed kernel

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Shea oil

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 10 | October 2012 | 477–484

The Shea nut was also known by various names. The Acholi for instance called it yao magolo or yaa magolo and the Lango called it yao agulu while the Alur ethnic group called the nut as pok yao, apoka yao, awakorongo or dend yao (Table 3). The ethno-names of other Shea tree products such as Shea kernel and the Shea oil are also presented in Table 3. What is common is that there is immense variation among these ethno-names across the ethnic groups (Acholi, Alur, Iteso, Lango, Lugbara and Madi) in the Shea parklands of Uganda.

products (Table 3). The various ethnic communities in the Shea parklands of Uganda can, with no hesitation, name a Shea tree and even its products like fruits, nuts, oil and charcoal at a distance without any difficulty. We have tested the perspicacity of the Shea communities in different tribes and have rarely found them hesitant, doubtful or in error. And the same ethnic group living at appreciable distances from one another and in different tribes will give these many names with amazing consistency.

DISCUSSION

The variation in the ethno-names reported in this study could perhaps, be due to the differences in languages spoken by these ethnic groups or dialectical differences within an ethnic group. For instance the Acholi, Lango and Alur people speak the Western Nilotic languages which are found in the sub-sub phylum of Luo, closely related to the language of the Luo society in Kenya while the Iteso speak the Eastern Nilotic language called the Ateso (Byrnes, 1992; Nyeko, 1996). The Lugbara and Madi, however, speak the Central Sudanic languages (ITA, 2008). Many vernacular names used for Shea tree has previously been reported as a reflection of its extensive range of occurrence– nearly 5,000 Km from Senegal (west) to Uganda (east) across the African Continent (Shea butter, 2008). The historic-nomenclature and synonymy of this tree is said to have followed a very tortuous evolution since the oldest specimen was first collected by Mungo Park on May 26, 1797 (Shea butter, 2008)

Given that Shea tree and its products are very important in the livelihood of the rural poor, an understanding of the ethno-knowledge about the tree and its products is essential for its continued use and conservation. The findings presented in this study indicate that ethno-naming of the Shea tree and its products varied widely among the studied ethnic communities in the Shea zones of Uganda. Sometime during the previous century it was unfashionable to use vernacular names of plants. This happened (and still does) in the applied fields of plant ecology and botany. The rationale for this seemingly ‘reversexenophobic’ decision was that local people name and classifies plants differently from ecologists/botanists (Hashim, 2007). Nevertheless, it is still widely believed that vernacular names of plants could productively inform research on the conceptual categories of plants and their classifications, thus benefiting all of mankind- academic as well as practical use of the world's flora (Richard, 1994). There are real enigmas which botanists cannot easily explain in the Ugandan recognition of "kinds" or "names" of Shea tree species which offer no morphological or otherwise tangible differences but which are well established and named in the native classifications. And this skill on the part of the Shea communities is manifested not only to the name of Shea tree and Shea products but to other wild plants alike. In some ethnic groups, there is more than one name of Shea tree or its

The few similarities in ethno-names of the Shea tree and its products in the Shea parkland areas especially among the Luo speakers, and those among Lugbara and Madi ethnic groups could perhaps be attributed to the shared historical background, movement of these people, intermarriages or trade among them (Nzita and Niwampa, 1998). The Luo migration for instance, brought changes during the 15th Century. The Lango got mixed with the Acholi people and subsequent intermarriage resulted in the Lango losing their Ateker language and later migrating closer to Lake

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 10 | October 2012 | 477–484

Kyoga in the 18th Century after living for more than two hundred years in the Acholi region (Nzita and Niwampa, 1998). They lost their traditional lifestyle of pastoralism; started subsistence farming and began to speak Luo. The Luo migration also had influence on ethnic groups that already settled in the West Nile, northern and eastern regions of Uganda. They introduced their language and culture (Nzita and Niwampa, 1998). Therefore, these ethnic movements, intermarriages and modification of tribal languages could greatly account for the similarities in the ethno-names of the Shea tree and its products. However, there is still a big gap in our knowledge and understanding especially to the meaning of the various ethnonames of Shea tree and/or its products among the different ethnic groups as these were not sought in the study. CONCLUSION & RECOMMENDATIONS Based on the findings discussed above, it can be concluded that there was a wide inter and intra variability in ethno-names of the Shea

tree and its products among ethnic groups living in the Shea parklands of Uganda. This diversity of ethno-names is perhaps a reflection of the extensive range of occurrence of the Shea trees including ethnic movements, intermarriages and modification of tribal languages. There is, however, a need to investigate whether the meaning of the various ethno-nomenclatures are in anywhere linked to prototypes or conservation issues that can be used to enhance conservation of Shea trees in Uganda’s parklands or beyond. ACKNOWLEDGEMENTS The authors of this paper are grateful to the ethnic communities in north and north-eastern Uganda particularly the Langi, Acholi, Iteso, Alur, Lugbara and Madi for their participation in the study that culminated into this paper. Special thanks also go to the Carnegie Corporation of New York for providing the financial support through the School of Graduate Studies, Makerere University Kampala, Uganda.

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Source of Support: Nil

Conflict of Interest: None Declared

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Research article A NOVEL REGENERATION SYSTEM FOR A WILD PASSION FRUIT SPECIES (PASSIFLORA FOETIDA L.) BASED ON DIRECT SOMATIC EMBRYOGENESIS FROM LEAF EXPLANT Patil Anita S1*, Paikrao Hariprasd M2 1

Associate Professor, Department of Bio-technology, SGB Amravati University, Amravati, Maharashtra, India Junior Research fellow, Department of Bio-technology, SGB Amravati University, Amravati. * Corresponding Author: Email: anitapatil@sgbau.ac.in; Mobile: +919881735354 2

Received: 17/08/2012; Revised: 22/09/2012; Accepted: 26/09/2012

ABSTRACT Direct somatic embryogenesis in leaf explants of Passiflora foetida L. a rare and endangered medicinal plant under threat of extinction has been studied. A detailed study of embryo formation would provide information onto their subsequent development, its germination events and also aid in propagation of this species. Somatic embryo induction in leaf explant was favored by the addition of 2, 4-Dichlorophenoxy acetic acid (2 mg l-1)and lower cytokinin 6-Benzylaminopurine (0.5 mg l-1 ) along with the highest micro salt concentration (9X) in the induction media, with average induction of 11.0 ± 0.1 globular embryos/1 cm of explant surface. In vitro propagation of P. foetida via somatic embryogenesis was more effective to overcome the chimeral plants with ethylene accumulation by explant in a medium. KEY WORDS: Passiflora foetida L., somatic embryogenesis, embryogenic callus, embryo developmental stages, histology ABBREVIATIONS: 2, 4-D = 2, 4-Dichlorophenoxyacetic acid; BAP = 6-Benzylaminopurine; S.E = Somatic embryo; PGRs = Plant growth regulators; D.W = Distilled water

Cite this article: Patil Anita S, Paikrao Hariprasd M (2012), A NOVEL REGENERATION SYSTEM FOR A WILD PASSION FRUIT SPECIES (PASSIFLORA FOETIDA L.) BASED ON DIRECT SOMATIC EMBRYOGENESIS FROM LEAF EXPLANT, Global J Res. Med. Plants & Indigen. Med., Volume 1(10), 485–495

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INTRODUCTION Passiflora foetida L. (wild water lemon, stinking passion flower, love in the mist or running pop) is a species of the passion flowers that is native to the south western United states, southern Texas and Arizona, Mexico, the Caribbean, Central America, and much of South America. It has been introduced to tropical regions around the world, such as Southeast Asia and Hawaii. It is a creeping vine like other members of the genus, and yields an edible fruit. Passiflora foetida L. commonly known as Passion fruit, it is an exotic fastgrowing perennial vine occurring in USA and extended to India. The species name, foetida, means “stinking” in Latin and refers to the strong aroma emitted by the damaged foliage (Nellis, 1997). Traditionally, the fresh or dried whole plant and their preparations are accepted for medicinal use in European countries for their utility in nervous anxiety (Blumenthal, 1997; Felter and Lloyd 1898). Various phytochemicals are reported in this plant, ranging from alkaloids, phenols, glycoside flavonoids and cynogenic compounds (Dhawan et al., 2004). Compounds like passifloricins polyketides and alpha- pyrones are investigated and reported to be not present in other species (Echeverri et al., 2001). P. foetida supposed to be the enormous source of chrysoeriol, apigenin, isovitexin, vitexin, 2”-xylosylvitexin, lutelin7-β-d-glucoside, kaempferol and contains few more important constituents are hydrocyninic acid, harmane, harmalol, harmine (Pongpan et al., 2007). Somatic embryogenesis, too, might not only allow the clonal propagation of valuable genotypes, but also facilitate genetic engineering. In spite of this, to the best of our knowledge there have been no studies of the capacity of Passiflora tissue to form somatic embryos. Present study on, the species of Passiflora foetida will show the induction of direct somatic embryos in three weeks and its subsequent maturation in 8–9 weeks. However, embryogenesis occurrence in P. foetida had

been clearly demonstrated by anatomical evidence. Thus, the study has been focused on the sequences of events, leading to the process of somatic embryo formation and its regeneration. This investigation also deals with the effect of different hormone combinations as well as elimination of ethylene blocking agent in culture media. MATERIALS AND METHODS Plant material cultures:

and

establishment

P. foetida L. was collected from the Melghat forest area, Amravati, India. The plant was authenticated using standard flora and cross-checked with herbarium records at the NBRI, Lucknow, India as Passiflora foetida L. with an accession number- 98181. Healthy leaves were removed from the plant and initially washed under running tap water to remove phenolics, again with 5% (v/v) laboline detergent for 5 min, followed by repeated washing under running tap water, until all traces of detergent were removed and then rinsed 4–5 times in sterile distilled water (D.W.). The explant was surface sterilized by 70% ethanol for 30 sec followed by rinsing twice with sterile D.W. Finally explants were treated with 0.2% HgCl2 for 1 to 1.5 min, again washed thrice with double DW. The surface sterilized explants were cut into 1 cm × 1.5 cm size and blotted onto filter paper folds. Induction of somatic embryos on leaf explants: The surface sterilized explants were inoculated onto Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with auxin 2, 4Dichlorophenoxyaceticacid (2, 4-D) and cytokinin 6-Benzylaminopurine (BAP), in combination. In the first set of an experiment explants were initially cultured on induction medium consisting of the basal media with 1, 2 and 3 mg l-1 (2, 4-D) in combination with 0.5 mg l-1 (BAP) with variable salt concentrations.

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The salt content of the medium was kept higher as compare to MS basal medium, the stock A (Macro nutrient) was same of the original while stock B (Micronutrients), C (Iron source) and D (Vitamins) was taken in high concentration (5X, 7X and 9X) compare to 1X in original media. All PGRs and salt stock solutions were added to the medium before autoclaving. The pH of the medium was adjusted to 5.8 with either 1M NaOH or 1M HCl and 0.25% Phytagel w/v was added and autoclaved at 121oC at 15 lb for 15 min. Cultures were maintained by incubating in the culture room at 26 ± 1°C under cool, white fluorescent light (1000 lux) for 12 h/day, with relative humidity of 70–80%. Embryogenic response was observed on the cultured explant after 15 days of incubation. The variable combinations of 2, 4-D and 6Benzylaminopurine (BAP) , with variable in salt content, in 16 different combinations with 1X salt content as Set I, 5X – Set II, 7X – Set III and 9X –Set IV. The total 30 leaf explants were inoculated for a single set (duplicate) giving 960 explants. After 25–28 days of culture in induction media, the numbers of explants showing positive embryogenic response were recorded. For each treatment, 30 explants were employed and the experiments were repeated at least twice, and the data was statistically analyzed. Proliferation of somatic embryos: To evaluate the embryogenic potential of the induced response of explants (181 no.) were transferred to fresh medium with similar composition of plant growth regulators (PGR) with its corresponding salt content of a medium. The medium was prepared as described above and cultures were maintained under the same conditions used for induction. The number of explants with embryogenic response and number of embryos per explant was recorded after 20 days of incubation. Occasionally, some explant tended to turn brown and had to be transferred to a fresh

medium again, subcultured.

otherwise

Effect of BAP levels on embryogenic response:

was

not

frequency of

For this experiment, the levels of BAP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/ l-1) were included into the multiplication medium with constant 2, 4-D (2 mg/lit) along with 1X salt conc. The 64 explants with the positive embryonic response from the proliferating stage from set IV, were inoculated with 10 explants each in media containing constant 2, 4-D (2 mg/l-1) and variable BAP (0.5, 1.0, 1.5, 2.5 and 3.0 mg l-1) in MS basal medium with normal salt concentration. The cultures were maintained under the same conditions used for determination of embryogenic callus frequency. The percentage of explants with well developed somatic embryos formation was assayed after 28–30 days. Somatic embryo germination: S.E from this study was derived from explants cultured and proliferated from set IV. About, 30-day old embryos were pooled and used for germination studies. The attempt was made to identify the explant origin, whether these embryogenic cell lines were induced directly or indirectly. The embryogenic callus induced in few experimental sets on original leaf explants were also maintained by sub culturing after 28-day interval. The MS culture medium in full or half strength, without PGRs, solidified with 0.25% phytagel. The pH of the medium was adjusted to 5.8, sterilized and dispensed in test tubes. The excised embryos had been placed for germination, and cultures were incubated at 25oC and at a photoperiod of 16 h. After 20 days of culturing the percent germination was recorded.

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Histo microscopical analysis: For histological studies explants were fixed in FAA (Formalin 5% (v/v): Acetic acid 5% (v/v): Ethanol 90% (v/v), dehydrated in ethanol series and embedded in paraffin wax. About 10–15 um thick sections were cut and stained with (2%) safranine and observed under the compound microscope (Nikon ECLISPSEE100 with camera COOL -PIX –MDC lens). The histological analysis was carried out in terms of developing stages of a somatic embryo along with type of supporting cell. RESULTS Initiation of embryogenic culture from Passiflora foetida leaf disc: A simple and effective protocol had been developed for in vitro direct somatic embryogenesis and subsequent germination of S.E in of Passiflora foetida L. The leaf explant was cultured onto solid MS basal medium containing various combinations of 2, 4-D (mg l-1) and BAP (mg l-1) along with variable high salt content (Micro, iron and vitamins) for the induction of embryogenic response. In the first step of the experiment, P. foetida leaf explant proved to be amenable to induction of somatic embryogenesis via the direct procedure of culturing leaf disc as explant. Auxins are critical for the induction of somatic embryos (Jimenez 2005). This study, investigates the influence of BAP and 2, 4-D on the induction of embryogenic response, with variable high salt content in a medium. Initial experiments were designed to assess induction of embryogenic cells in response to combinations of PGRs and variable levels of high salt content in the basal medium. Although embryogenic cells which developed in the presence of high salt content in four different sets viz. Set, I, II, III and IV had a distinctly nodular appearance.

At the levels of 2, 4-D (2 mg l-1) and BAP (0.5 mg l-1) i.e. set IV with 9X salt concentration, resulted in faster and higher frequency of embryo induction in 20 days, then 28 days in other sets, as compared to other combinations and salt content assessed for the induction experiment (Table 1). It is proving that, the presence of 2, 4-D alone in induction media was less effective than a combination of 2, 4-D and BAP for the induction of embryogenic callus and somatic embryo induction. It is observed that, there was not a very distinctive difference in appearance of embryogenic masses of other sets as compare to set VI with 9X salt concentration. The explants with positive response 181 no. are transferred to the multiplication medium, for the conversion of embryogenic mass to embryoid development. It was observed that, it is faster in explant selected from set IV as compared to Set I, II and III. Further, the maximally embryogenic masses from set II and III were turning brown, with slow and few abnormal embryo formations, and on subculture unable to retain in their normal form. Developing S.E appeared as greenish structures on the surface of explant and some had cotyledons. S.E was also induced on a medium in Set, I, II and III in combination with PGRs, but at a lower frequency ranging from 30–53.33, with some developing embryos were normal, but were unable to further develop into a complete embryo. Further 'well-develop' somatic embryo with the normal cotyledons and growth frequency of 60–86.66%, were observed in set IV, which suggests for the presence of BAP in low concentration along with 2, 4-D with salt content (9X) in the induction medium is crucial for high frequency and fast induction of direct embryogenesis in P. foetida L.

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Table 1 :The frequency of embryogenic response induction of P. foetida leaf explant in response to PGRs combinations and high salt conc. Micronutrient Salt Concentration

Growth regulators (mg/l) 2, 4-D

MS (1X) Control

MS (5X) Control

MS (7X) Control

MS (9X) Control

Frequency of induction % of

Embryogenesis

BAP

0.0

1 2 3 -1 2 3 -1 2 3 --

0.5 0.5 0.5 -0.5 0.5 0.5 -0.5 0.5 0.5 --

10 ± 2.3 12 ± 1.5 11 ± 3.3 NR 12 ± 2.1 15 ± 1.2 14 ± 1.5 NR 13 ± 2.1 16 ± 1.1 14 ± 1.4 NR

33.33 40.00 36.66 0.0 40.00 30.00 46.66 0.0 43.33 53.33 46.66 0.0

1 2 3

0.5 0.5 0.5

18 ± 2.1 26 ± 0.54 20 ± 0.83

Proliferation of somatic embryos: It has been found out that, transfer of embryogenic culture from the induction media to multiplication medium accelerates the embryo growth. The culture system after further incubation under light conditions, acquired a yellow to green colour. As the culture progressed, some embryogenic calli started differentiating into yellowish-white nodules, which then finally produced embryogenic mass after 20 days of incubation. Somatic embryos at different stages of development, from globular to cotyledonary shape, had arisen by this time. The frequency of embryogenic culture to S.E development was higher in multiplication media with 2, 4-D (2 mg l-1) and BAP (0.5 mg l-1) with normal 1X salt conc. It has been seen that highest response of embryo formation (100%) and well developed somatic embryo

60.00 86.66 66.66

(19 ± 0.41) formation was observed in similar media combinations. Furthermore, other combinations show 90–70% embryogenic response with lower S.E (16 ± 0.68 to 11 ± 2.3) formation. It has been seen that, the lowest embryogenic response with (10 ± 2.3) S. E formation was observed in a medium with 2, 4D (2 mg l-1) and BAP (3.0 mg l-1) (Table 2). The embryogenic callus induced in few experimental sets on original leaf explants were also maintained by subculturing after four week intervals. This was to check whether these embryogenic cell lines were induced directly or indirectly. The initiated embryogenic calli and globular embryos were cultured on the multiplication media. They showed a positive response after 7 days of incubation. However, this confirms, that subcultured embryogenic callus had retained their embryogenic competence.

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Table 2. The effect of BAP on embryo formation frequency of leaf explants of P. foetida L. Growth regulator (mg/l) 2, 4-D 2

BAP

Frequency of embryogenic response

0.5

10 ± 0.23

100

19 ± 0.41

1.0

9 ± 0.54

16 ± 0.68

1.5

7 ± 0.64

15 ± 0.7

2.0

7 ± 0.68

13 ± 1.9

2.5

7 ± 0.72

11 ± 2.3

3.0

4 ± 0.72

10 ± 2.3

At the subculture, explants with friable nodules consisting of pre-embryogenic masses, most of which later showed surface cell proliferation and differentiation of globular structures. It has been seen that, the embryogenic capacity differs from line to line, as some lines exhibit the high capacity for embryo proliferation while other lines show only pre embryogenic callus and further fail to develop into somatic embryos. Absence of any callus formation indicated that embryo development was direct, with the appearance of globular stage embryos (Fig 1A and1B). Development of embryogenic calli with some of their structures was bordered by a distinct protoderm, an indication of the presence of globular or pre-globular embryos. The transition between globular to cotyledonary stage was very rapid and also the intermediate stages of embryo development such as late heart stage and torpedo stage (Fig. 1C) and cotyledonary stage (Fig. 1D) were observed.

% response

No. of well developed S.E

The explants with globular embryos on multiplication media followed all the developing stages. It has been observed that the surrounding cell mass started to produce embryogenic calli, with some filamentous embryos, this might provide the embryogenic environment for the competence of induced embryos. Germination of SE: Subculture of cotyledonary embryos on the fresh full strength and half strength MS medium, after 20 days showed the germination of excised embryos. The regeneration medium without PGRs showed the highest germination response 60% in half strength MS basal medium as compared to 43.33 % in a fullstrength medium (Table 3). The initiation into regeneration begins with a small shoot like greenish appearance (Fig. 1E) and direct shoot regeneration (Fig. 1F). The induction of rooting to somatic embryos was recorded after sub culturing (Fig. 1G).

Table 3 Effects of media composition on germination and survival of P. foetida somatic embryos Medium MS Half strength MS

No. of embryos Germinated/inoculated 13 (30) 18 (30)

% Germination

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43 60.00

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Fig. 1.

The leaf explant of 1–1.5 cm size showed swelling of the explant and initiation of small embryo-like embryo structures in 25 days of culture with globular stage embryo (GE) with few embryogenic calli (EC) (Figure A and B) on induction medium. In the next 20 days, embryogenic clumps were visible and appeared morphologically prominent on the surface of explants, showing heart shape embryo (HE) (arrow) and torpedo shape embryo (TE) (Figure C) on multiplication medium. Well-developed developed cotyledonary embryos (CE) were observed all over the cultured explants within 28-30 28 days of culture incubation (Fig D)) on multiplication medium. On the regeneration medium, explant show's initiation of regeneration showing greenish colour leaf primordia (LP) (Fig E). Direct regeneration of induced somatic embryos from leaf disc explant (Fig. F.) Root induction from the somatic embryos (Fig.G)

Developmental stages of somatic embryos and their histological study: The initiation of direct somatic embryos was observed from the lower layer of epidermis, with initiation of procambium. The globular lobular stage embryos on multiplication medium responded well for its developing organization. After 15 days of a subculture, the globular cells modified in the heart shaped embryo, a bipolar structure with axis in the centre of the body,, were as after 20 days symmetrical axis elongated to form the torpedo shape embryo, which further modified itself into cotyledonary stage. Embryonic cells observed with dense cytoplasm and prominent nuclei in the centre originated from the proliferation tissue mass without previous callus formation leading to form the embryonic mbryonic buds. Histological analysis revealed the presence of protuberances with the

linings of columnar cells at the margin. The inner mass is about to differentiate in the vascular tissue to support the shoot regeneration, appearing as the early vascular organization having primary phloem and xylem with the presence of vascular cambium at the intermediate lining (Fig. 2A). The epidermal layers of explants acquired origination of globular structure proving the anatomical evidence of direct embryogenesis. The globular stage embryo was prominent (Fig. 2B). An early heart stage somatic embryo with distinct protoderm from the epidermis of the explant with a bipolar organization (Fig. 2C). Since the fourth week of culture, this structure appeared lengthened, became bulbous at its base, bas and its top was tapered red transforming to form the torpedo stage embryo (Fig. 2D). The histological analysis confirms the presence of early apical meristem, showing the tunica – corpus zonation, with conical nature (Fig. 2E).

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The cells of the central mother cell zone were observed, ved, formation of new leaf primordia and associated immature internodal tissue also observed as the result of the highly

meristematic proximal region, embryos showing no connective tissue with callus cells provides the evidence for direct somatic embryogenesis (Fig.2F).

Fig. 2. Somatic embryogenesis in Passiflora foetida L.

Fig 2: A, vascular tissue (VT) (arrow) about to organize in the center and proembryos are at the surroundings. B, Globular stage embryo (G.E) surrounded by endospermic nuclei. C, Transition stage from globular to heart shape (HE) showing vascular standardisation. D, Torpedo shape embryo (TE) with lengthened symmetrical axis. E, Regeneration of Apical meristem (AM) with surrounding leaf primordia. F, Globular embryos with direct origin, arrows showing no connective tissue between embryo and callus cells. Scale bar=15 µm.

DISCUSSION In vitro techniques are now successfully applied to a range of threatened endangered medicinal and aromatic plant species for their multiplication and conservation. No efficient, reproducible somatic embryogenesis regeneration system exists for this species; impeding ng rare regeneration from type of explants in the other species of this family has been reported. Induction of direct somatic embryo formation in P. foetida L. species is reported here for the first time. A direct somatic embryogenesis protocol is developed using leaf explant, which would be certainly helpful in the efficient multiplication and conservation system for P. foetida L. using in vitro techniques. Although in this system leaf explants of P. foetida has shown high contamination levels and difficulty in direct shoot and root development due to

accumulation of ethylene harmony (Reis et al., 2003). In the present investigation leaf explants were treated with high HgCl2 conc. (0.2%) and proved satisfactory after disinfection. The somatic embryo induction started after 20–28 days of incubation of leaf explant on the induction medium. As an elimaeteric fruit Passiflora species shows high rates of ethylene production (Shiomi et al., 1996a; b), which limits the in vitro morphogenic potential of the explanted cultures. Ethylene plays a role in several in vitro developments’ processes (Kumar et al., 1998) and also effects of ethylene in vitro morphogenesis in passionflower had been reported earlier in Passiflora species (Barbosa et al., 2001). In the present study, growth regulators in the medium, especially auxin 2, 4-D (2 mg l-1) in combination with cytokinin BAP (0.5 mg l-1) with high salt concentration in micronutrient

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stock (9X) appears to be essential for the onset of growth and the induction of embryogenesis (Chawla 2009) without addition of ethylene inhibitors in the medium. These results are also supported by the fact that high salt conc. Viz MgSO4 and K2SO4 in the culture media induced direct somatic embryogenesis in Theobroma cacao L. (Minyaka et al., 2008). Furthermore, it has been found out that, somatic embryogenesis from chili pepper leaves is favored by the addition of nicotinic acid to the culture medium and the increase of copper concentration (Kintzios et al., 2001). It has been also observed that medium containing the auxin 2, 4-D (2 mg l-1) and BAP (0.5 mg l-1) showed the induction of embryogenic callus, while other combinations in media did not produce callus. The formation of callus may be due to the ratio of cytokinin to auxin as mentioned by Skoog and Miller (1957) and Gaspar et al., (2003). At this concentration highest 86.66% induction was observed and only the creamy white friable embryogenic callus formation was observed. A similar result on embryogenic callus induction was reported on Camellia spp. (Wachira and Ogada, 1995). Commonly, embryogenic callus induction has been effectively achieved by the combined treatment with auxin and cytokinins (Hernandez et al., 2003; Aly et al., 2002). In the present study, individual effect of 2, 4-D and BAP were also tested, and they showed a poor response for the induction of embryogenic callus. Again, the embryogenic callus induced in few experimental sets in original explants were also maintained by subculture after 4 week intervals, this to check whether the embryogenic cell lines induce directly or indirectly, this confirming its embryogenic competence (Corredoira et al., 2002). It is clear from the histological studies, that the organogenesis often involves more than one cell that acts in coordinating manner (Brown and Thorpe 1986). In this study, the embryoid are derived from the single cell, although the evidence for a multicellular origin has also been reported (Dornels et al., 1992). Some authors argued about the high mutational rate in

plants regenerated from callus phase, unlike in the present study fewer mutations may be expected due to regeneration via direct somatic embryogenesis. Lack of reproducibility of propagation protocol is reported for many species of Passiflora. Although in vitro propagation by mature endosperm culture was reported for P. foetida (Mohamed et al., 1996), induced embryo germination (Guzzo et al., 2004) and callus induction (Rasool et al., 2011). Attempt of somatic embryogenesis from zygotic embryos was reported for P. cincinnatas Mast. Also the ploidy stability of somatic embryogenesis-derived P. cincinnata Mast. was assessed by flow cytometry (Silva et al., 2009; Pinto et al., 2010a).Similarly direct organogenesis for P. alata (Pinto et al., 2010b), and Direct and Indirect in vitro organogenesis for P. cincinnatas Mast. was observed. (Lombardi et al., 2007). In the present investigation, the efforts in establishing embryogenic system indicate that multiplication via somatic embryos was more effective to overcome chimeral plants. Furthermore, it has been found out that ethylene production by explant has no effect on direct somatic embryogenesis in P. foetida L. CONCLUSIONS Although the induction of somatic embryoâ&#x20AC;&#x2122;s and its regeneration from Passiflora foetida leaf explant had been achieved; further research is still required regarding their development into complete plantlet and their acclimatization. The use of S.E in biotechnology, including disease-free root stock production, clonal propagation and genetic improvement programmes could be highly beneficial (Lombardi et al., 2007). In the present investigation, the efforts in establishing embryogenic system indicate that multiplication via S.E was more effective to overcome chimeral plants. Furthermore, it has been found out that ethylene production by explant has no effect on direct somatic embryogenesis in P. foetida L.

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ACKNOWLEDGEMENT The authors wish to acknowledge the grants from Department of Science and Technology (DST) New Delhi, INDIA under

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Kumar P.P.; Lakshmanan P.; Thorpe T.A. (1998). Regulation of morphogenesis in plant tissue culture by ethylene. In vitro Cell Dev Biol Plant 34:94–103. Lombardi S.P.; da Silva Passos I.R.; Nogueira M.C.S.; Appezzato-da-Glória B. (2007). In Vitro Shoot Regeneration From Roots And Leaf Discs Of Passiflora Cincinnata Mast. Braz arch biol technol 50:239–247. Minyaka E.; Niemenak N.; Fosto.; Sangare A.; Omokolo D.N (2008). Effect of MgSO4 and K2SO4 on somatic embryo differentiation in Theobroma cacao L. Plant Cell Tiss Organ Cult 94:149–160. Mohamed M.E.; Hicks G.T.; Blakesley D. (1996). Shoot regeneration from mature endosperm of Passiflora foetida. Plant Cell Tiss Organ Culture 46:161– 164. Murashige, T.; Skoog, F. (1985). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 79: 988-991. Nellis D.W. (1997). Poisonous Plants and Animals of Florida and the Caribbean. Pineapple Press Inc. p. 224. ISBN 9781561641116. Pinto A.P.C.; Critina A.B.A.; Monteiro H.; Stipp L.C.L.; Mendes B.M.J. (2010). In vitro organogenesis of Passiflora alata. In vitro Cell Dev Biol Plant 46: 28–33 Pinto D.L.P., Barros B.D.A.; Viccini L.F.; Campus J.M.S.; Silva M.L.; Otoni W. (2010). Ploidy stability of somatic embryogenesis-derived Passiflora cincinnata Mast. plants as assessed by flow cytometry. Plant Cell Tiss Organ Cult. doi:10.1007/s11240-010-9756-y.0. Source of Support: Nil

Pongpan N.; Luanratana O.; Suntorusuk L (2007). Rapid reversed phase high performance liquid chromatography for vitexin analysis and fingerprint of Passiflora foetida. Curr Sci 93:378– 382. Rasool S.N.; Jaheerunisa S.; Jayveera K.N.; Suresh Kumar C. (2011). In vitro callus induction and in vivo antioxidant activity of Passiflora foetida L. leaves. IJARNP 4:1–10. Reis L.B.; Paiva Neto V.B.; Picoli E.A.T. (2003). Axillary bud development of passion fruit as affected by ethylene precursor and inhibitors. In Vitro Cell Dev Biol Plant 39: 618– 622. Shiomi S.; Wamocho L.S.; Agong S.G. (1996a). Ripening characteristics of purple passion fruit on and off the vine. Postharvest Biol Technol 7:161–170. Shiomi S.; Kubo Y.; Wamocho L.S.; Koaze H.; Nakamura R.; Inaba (1996b). A. Post harvest ripening and ethylene biosynthesis in purple passion fruit. Postharvest Biol Technol 8:199–207 Silva M.L.; Pinto D.L.; Guerra M.P.; Floh E.I.S.; Bruckner C.H.; Otoni W.C. (2009). A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos. Plant Cell Tiss Organ Cult 99:47–54. Skoog F.; Miller C.O. (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11:118–131. Wachira F.; Ogada J. (1995) In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis. Plant Cell Rep 14: 463–464. Conflict of Interest: None Declared

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Research article EVALUATION OF EFFECT OF METHANOLIC AND AQUEOUS EXTRACTS OF PUNICA GRANATUM L. AGAINST BACTERIAL PATHOGENS CAUSING BOVINE MASTITIS Gopinath S M1, Suneetha T B2, Singh Sumer3 1

Acharya Institute of Technology, Soldevanahalli, Hesaraghatta Road, Bangalore-560 090 Dept of Biotechnology, Singhania University, Rajasthan. * Corresponding Author: Email: acharyadrgopinath@gmaiL.com; Mobile: +919738888095 2, 3

Received: 01/09/2012; Revised: 22/09/2012; Accepted: 27/09/2012

ABSTRACT Bovine Mastitis is an intra-mammary infection which is most common among the dairy cattle and continues to be the most costly disease to the dairy farmers. Presently, antibiotics are used for treatment of mastitis leading to the development of antibiotic resistant strains and consumer health problem. The ethno veterinary information about plants in Karnataka region to control Bovine mastitis was collected and the effect of different solvent extracts of Punica granatum L was investigated. Phytochemical analysis revealed the presence of bioactive compounds such as alkaloids, flavonoids, saponins, tannins, phenols, terpenoids etc. Each of the bioactive compounds were estimated and isolated separately by solvent-solvent extraction of Punica granatum. Saponins content was higher followed by flavonoids. All these bioactive compounds isolated from crude extracts were tested for antibacterial activity. Flavonoids of Methanolic extracts inhibits remarkable Zone against S. uberis, S. aureus, E. coli and Coagulase negative S. aureus was 11 mm, 12 mm, 14 mm, 16 mm and for water extracts it was 16 mm, 12 mm, 15 mm and 13 mm respectively.

KEYWORDS: Punica granatum, Phyto-cehmical analysis, Antibacterial activity, flavonoids, methanolic extracts, aqueous extracts

Cite this article: Gopinath S M, Suneetha T B, Singh Sumer (2012), EVALUATION OF EFFECT OF METHANOLIC AND AQUEOUS EXTRACTS OF PUNICA GRANATUM L. AGAINST BACTERIAL PATHOGENS CAUSING BOVINE MASTITIS, Global J Res. Med. Plants & Indigen. Med., Volume 1(10): 496–502

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INTRODUCTION Mastitis is a persistent, inflammatory reaction of the udder tissue in cows. Bacteria secrete toxins which damage the milk-secreting tissue and various ducts throughout the mammary gland. Bovine mastitis may also be indicated by abnormalities in milk such as watery appearance, flakes, clots, or pus. An increased somatic cell count is observed in cows suffering from bovine mastitis. It is considered and continues to be the costliest disease in the dairy industry all over the world (Adaobi 2011). The repeated use of antibiotics to treat Mastitis for a long period may cause multidrug resistivity in causative organisms which requires high doses of antibiotics, which may leads to accumulation of large amount of antibiotics in milk and its products, again a potential hazard (Annapoorani Chockalingam 2007). Knowledge of medicinal plants has been accumulated in course of many centuries. Even today, 85% of Indians use higher plants as effective anti-microbials for the treatment of various diseases. The aim of this work was to collect ethno veterinary information about plants used in the prevention and control of Bovine mastitis in Karnataka region. There were no reports available relating to In-vitro applications of P. granatum extracts in Bovine mastitis studies. Therefore, the present study was designed to investigate antibacterial activity of the leaves of P. granatum and identification of particular bioactive compound as potential drug for the treatment of Bovine Mastitis. About the plant Punica granatum L. commonly known as Pomegranate belongs to the Family Punicaceae. Punica granatum is a shrub or small tree with several upright, thorny stems, the leaves are elliptic, roughly 2 x 1 inches. In the Indian subcontinent's ancient Ayurveda system of medicine, the pomegranate has extensively been used as a source of traditional remedies for thousands of years. The plant has also been used as an antispasmodic and antihelmintic. Pomegranate juice (of specific fruit strains is

also used as eye drops as it is believed to slow the development of cataracts (Vasant Lad 2002). Pomegranate has been used as a contraceptive and abortifiacient by means of consuming the seeds, or rind, as well as by using the rind as a vaginal suppository. MATERIAL AND METHODS All the solvents and reagents used in the study were analog grade sourced from Hi media. Collection and Extraction of plant material The plant was collected in the month of March-2011 from Acharya Institute of technology campus, Soladevanhalli, Bangalore. The plant with leaves was rinsed with sterilized water and leaves were removed and separated. The leaves were air dried for 3 weeks and then crushed with mortar and pestle and kept in air tight glass container at 4°C until further use (Harborne JB, 1973; Jamine. R Daisy, 2007) Preparation of crude extracts Aqueous extract was prepared by using 50 g of crushed leaves and 500 ml of distilled water in soxhlet apparatus and the apparatus was allowed to run for 10 h. Similarly the methanol extract was prepared (C.K. Hindumathy, 2011). Bacterial strains Bacterial strains used in this study were isolated from clinical cases of Bovine mastitis namely Staphylococcus aureus, Streptococcus uberis, Escherichia coli and coagulase negative Staphylococcus aureus. All the strains were confirmed by cultural and biochemical studies (Gopinath. S. M., 2011) and maintained in nutrient agar slants at 4ºC for further use. Antibacterial activity The antibacterial assay of aqueous and methanolic extracts was performed by agar disc diffusion method (Harborne JB., 1973; Jamine.R.Daisy, 2007). The molten Mueller

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Hinton agar was inoculated with 100µl of the inoculums (1*106 CFU/ml) and poured into the petriplate (Himedia). For agar disc diffusion method, the disc (0.7 cm), (Himedia) was saturated with 100 µl of the test compound, allowed to dry and was introduced on the upper layer of the seeded agar plate. The plates were incubated overnight at 37ºC. Microbial growth was determined by measuring the diameter of the zone of inhibition of each bacterial strain. Phytochemical analysis Phytochemical analysis for major phytoconstituents of the plant extracts was undertaken using standard qualitative methods as described by various authors (Leite JR, 1986; Parekh, J, 2007). The plants extracts were screened for the presence of biologically active compounds like glycosides, alkaloids, phenolics, tannins, flavonoids, saponins and steroids. Estimation compounds

and

extraction

phyto-

Alkaloids Isolated crude sample was extracted with solvent ether and alcohol mixture (4:1) and ammonia solution (5% v/v). To, this 1N H2SO4 followed by 0.5N H2SO4 and alcohol mixture (3:1) was added and the acid layers were separated until the aqueous layer is colorless. This acid layer was then washed with chloroform. Further this chloroform layer was washed with acid alcohol mixture. This layer was then added with 5% v/v ammonia solution in excess. This was then extracted with chloroform and washed with water. The chloroform layer was filtered through a layer of anhydrous sodium sulfate in pre-weighed beaker. The chloroform was allowed to evaporate followed by addition of alcohol which was then dried at 105°C in hot air oven, with alkaloids been left in the beaker. The beaker was then weighed to know the content alkaloids isolated.

Flavonoids Isolated crude sample was dissolved in water washed with hexane to remove oil content. The aqueous layer was washed with chloroform followed by warming the aqueous layer. This warmed aqueous layer was extracted with ethyl-acetate into pre-weighed beaker. The ethylacetate extracted layer was concentrated and dried at 105°C in hot air oven and the beaker was weighed again. (Wynn GS. 2001; Prasad, N.R., 2008) Saponins Isolated crude sample was extracted with 90% methanol and further concentrated to more than half of the original. This concentrated extract was then extracted with petroleum ether followed by chloroform. The obtained aqueous layer was washed with 90% methanol and again allowed to concentrate. This was then added into pre-weighed beaker containing acetone drop by drop to form saponin precipitates. This was then filtered through preweighed filter paper. The pre-weighed beaker and filter paper were then allowed to dry at 105° C in hot air oven. Tannins The material was extracted with mixture of distilled water and 8% Sodium carbonate in a boiling flask under reflux for two hours having used a liquor / crude extract ratio of 15:1. This was repeated again and again to produce more of tannins. After extraction, the material was filtered under vacuum using a Büchner funnel. Finally the filtrate in pre-weighed beaker was dried in hot air oven at 105° C (Williamson G., 2005; Klastrup O, 1975) Phenolic compounds Isolated crude sample was extracted with 20 mL of the extracting ethanol in a conical flask. Conical flask was covered with parafilm and aluminium foil to prevent light exposure. The mixture was shaken at constant rate using a water bath shaker for 2 h at 50°C. The ethanol

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extracted was then filtered through a Whatman No. 1 filter paper into a pre-weighed beaker, and the filtrate was evaporated at 105° C (Williamson G., 2005; Klastrup O., 1975). Terpenoids Terpenoids were isolated in the form of essential oils. Isolated crude sample was extracted with solvent- hexane. This was then washed with alcohol and the hexane layer was evaporated in water bath to concentrate and then evaporated in hot air oven at 105°C RESULTS AND DISCUSSION Crude methanolic and aqueous extracts of leaves of plant P. granatum was prepared and then analyzed for phyto-compounds present in them. Most of the secondary metabolites were identified in the polar extracts (Table-1) Alkaloids are one of the characteristic secondary metabolite in leaves of this genus found in aqueous extract. Tannins are water soluble polyphenols known as tannic acid which acts as antimicrobial agents. Presence of tannins is to prevent the development of microorganism by precipitating microbial proteins. Phyto-therapeutically, Flavonoids are known to be synthesized by plants in response to microbial infection. Hence it should not be

surprising that they have been found to be effective as antibacterial substances against a wide array of infectious agents (Tyler V., 1994). Alkaloids, Flavonoids, Saponins, Tannins, Phenols, Terpenoids were isolated separately and the content of each was found by Content, Content (%) = (Weight of phytocompounds) / (weight of crude extract) * 100. Antibacterial activity was performed for these isolated samples such as alkaloids, flavonoids, saponins, tannins, phenols (Table-3; Fig. 2). Flavonoids isolated from methanolic and aqueous extracts of P. granatum showed antibacterial activity against the causative organisms of Bovine mastitis. The highest inhibition zone was observed by methanolic extracts of P. granatum against Coagulase negative Staphylococcus aureus (CONS) and S. uberis (16 mm) and the least was observed by methanolic extracts of P. granatum (11 mm). Phyto-compounds isolated from water extracts have shown higher inhibition zones than methanolic extracts. Saponin was found to be higher in content with highest in aqueous extracts (16%). (Table – 2; Fig. 1) Followed by this is flavonoids, tannins and phenols were higher in content. The highest content of flavonoids was found in aqueous extracts of P. granatum (13.83%). The least is alkaloids in water extracts of P. granatum (0.162%).

Table-1 Phytochemical analysis of Punica granatum in Methanol and Water extracts. Compound Steroids Terpenoids Alkaloids Flavonoids Saponins Tannins Phenolic compounds Catechin Anthraquinone quinone

Methanol Water − + + + + + + − − +

− + + + + + + − − +

+ indicates the presence of Phytocompound − Indicates the absence of phytocompound

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Table – 2. Phyto-compounds present in methanol and aqueous extracts of Punica granatum PHYTOCOMPOUNDS

CONTENT (%)

PLANTS

PUNICA GRANATUM

SOLVENTS

METHANOL WATER

Alkaloids

0.181

0.162

Flavonoids

13.55

13.83

Saponins

14.7

Tannins

10.1

10.4

Phenolic compounds

11.2

10.5

Terpenoids

0.178

0.198

+ indicates the presence of Phytocompound − Indicates the absence of phytocompound

Table-3Antibacterial activity of different phytocompounds of Punica granatum Causative organisms

Alkaloids M 0 0 0 0

S. uberis S. aureus E.coli CONS

W 0 0 0 0

Flavanoids M 11 12 14 16

W 16 12 15 13

Saponins M 0 0 0 0

W 0 0 0 0

Tannins M 0 0 0 0

W 0 0 0 0

Terpenoids M 0 0 0 0

W 0 0 0 0

Fig-1. Phyto-compounds present in methanol and aqueous extracts of Punica granatum 18 16 14 12 10 8 6

methanol

water

2 0

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Fig-2 Antibacterial activity of different phyto-compounds of Punica granatum

CONCLUSION Methanol and water extracts of flavonoids from Punica granatum have antibacterial potential against the causative organism of Bovine mastitis. Water as solvent is better for

extraction of bioactive compounds. Further, flavonoids can be studied to find specific spec compound which can further act as a drug and solve the problem of antibiotic drug resistance resistan by causative organisms of Bovine mastitis. mastitis

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Harborne JB (1973). Phytochemical methods, London Chapman and Hall, Ltd, pp. 49–88. Jamine. R, Daisy. P and Selvekumar.B.N., 2007. Research Journal of Microbiology.2 (4):369–374

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Mattila P, Hellström J (2007). Phenolic acids in potatoes, vegetand some of their products. J. Food Compos Anal., 20: 152–60. Parekh, J., Chanda, S., 2007b. Afr. J.Biol. Res. 10: 171–181 Prasad, N.R., Viswanathan.S., Renuka Devi, J., Vijayashree Nayak., Sweth,V.C., Archana parathasarathy, N and Johana Rajkumar., Journal of Medicinal Plants Research.2; 2008; 268–270.

Source of Support: Nil

Tyler V (1994). Phytomedicines in Western Europe: their potential impact on herbal medicine in the United States Herbalgram; 30: 24–3

Vasant Lad (2002), Textbook of Ayurveda, Volume 1. Ayurvedic Press. ISBN 1883725-07-0, Williamson G, Manach C (2005). Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am. J. Clin. Nutr., 81(Suppl 1): S 243–55 Wynn G S. (2001). Herbs in Veterinary Medicine. Alternative Veterinary Medicine. http://www.altvetmed.com/articles/herbs.ht ml as retrieved on 9 Oct 2001 21(47):38.

Conflict of Interest: None Declared

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Review article SAPONIN: A WONDER DRUG FROM CHLOROPHYTUM SPECIES Sharma Rohit1*, Thakur Gulab S2, Sanodiya Bhagwan S3, Pandey Mukeshwar4, Bisen Prakash S5 1

Department of Post Graduate Studies and Research in Biological Sciences, Rani Durgavati Vishwavidyalaya, Jabalpur – 482004 (M.P), India 1, 2, 3, 5 Plant Biotechnology Laboratory, R&D Division, Tropilite Foods Pvt Ltd, Davars Campus, Tansen Road, Gwalior- 474002 , India 4 Xcelris Genomics, Old Prem Chand Nagar Road, Opp Satyagarh Chhavani, Bodakdev, Ahmedabad380054 , India *Corresponding Author: Email: accessrohit25@gmail.com

Received: 27/08/2012; Revised: 27/09/2012; Accepted: 30/09/2012

ABSTRACT A wide range of herbs from the genus Chlorophytum (Asparagaceae) are known for their therapeutic potential with a vast range of pharmacologically important saponins. The important plants of the genus like C. borivilianum, C.malayense, C. comosum, and C. arundinaceum have steroidal saponins which has attracted much attention due to their structural diversity and therapeutic capability. The saponins from C. borivilianum have aphrodisiac property and popularly used as a safe alternative for Viagra while saponins from C. malayanese and C. comosum have anti-tumor properties and cytotoxicity against cancerous cell line. The review presents an approach to different chemical constituents and gives a brief outline of the various therapeutic properties showed by the saponins from the genera Chlorophytum. KEY WORDS: Saponins, Chlorophytum, steroid, anti-cancer, phyto-nutrients, anti-edemic, flavonone glycoside.

To Cite this article: Sharma Rohit, Thakur Gulab, Sanodiya Bhagwan S, Pandey Mukeshwar, Bisen Prakash S (2012), SAPONIN: A WONDER DRUG FROM CHLOROPHYTUM SPECIES, Global J Res. Med. Plants & Indigen. Med., Volume 1(10), 503–515

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INTRODUCTION Saponins are a vast group of structurally diverse glycosides of the plant kingdom widely distributed in nature; their surfaceactive properties distinguish them from other glycosides (Lasztity et al., 1998). They are non-volatile primary compounds, when dissolved in aqueous solution they form soap like foaming on shaking. They are referred to as steroidal glycosides and triterpenes consisting nonpolar aglycones coupled with one or more monosaccharide moieties (Oleszek, 2002). This combination of polar and non-polar structural elements in their molecules explains their soap-like behavior in aqueous solutions. Saponins are the important chemical compounds from tubers of this plant. They are used in the indigenous systems of medicine as a well known health tonic, aphrodisiac and galactogogue (Chopra et al., 1956; Marais and Reilly, 1978; Nadkarni, 1996; Oudhia, 2001b). Pharmaceutical industries buy saponins in large quantities because of their use for the semisynthesis of steroidal drugs for phyto-therapy and in cosmetic industry (Haque et al., 2011; Ksouri et al., 2011). They are believed to form the main constituents of many plant drugs and folk medicines responsible for numerous pharmacological properties (Marais and Reilly, 1978; Estrada et al., 2000; Debnath et al., 2006; Katoch et al., 2010). Therefore, it is a category of phyto-nutrients (plant nutrients) found abundantly in many beans, and other plants such as Ginseng, Alfalfa, Yucca, Aloe, Quinoa seed and also in Safed Musli (Chopra et al., 1956; Nadkarni, 1996). Saponins have a diverse range of properties from sweetness to bitterness (Grenby, 1991; Kitagawa, 2002; Heng et al., 2006; Thakur et al., 2009), foaming and emulsification (Price et al., 1987), pharmacological and medicinal (Attele et al., 1999; Debnath et al., 2007; Rajeev et al., 2012), haemolytic (Oda et al., 2000; Sparg et al., 2004), and antimicrobial, insecticidal, and molluscicidal activities (Sparg et al., 2004; Sundaram et al., 2011) and finds some place in beverages, confectionery and cosmetic industry

(Price et al., 1987; Petit et al., 1995; Uematsu et al., 2000). (Fig. 1) Saponins consist of a sugar moiety, usually containing glucose, galactose, glucuronic acid, xylose, rhamnose or methylpentose, glycosidically linked to a hydrophobic aglycone (sapogenin) which may be triterpenoid or steroid (Abe et al., 1993; Haralampidis et al., 2002); derived from the 30 carbon atoms containing precursor oxidosqualene (Haralampidis et al., 2002). The difference between the two classes lies in the fact that the steroidal saponins have three methyl groups removed (i.e. they are molecules with 27 C-atoms), whereas in the triterpenoid saponins all 30 C-atoms are retained. Saponins were classified into three classes, namely, the triterpenoid saponins, the spirostanol saponins and the furostanol saponins. However, due to secondary biotransformation such a classification emphasizes incidental structural elements and does not reflect the main biosynthetic pathways (Sparg et al., 2004). There are some other classes of compounds that have been considered as saponins, such as the glycosteroid alkaloids (Haralampidis et al., 2002). Baumann et al., (2000) reported that saponins have hemolytic properties that generally are attributed to the interaction between the saponins and the sterols of the erythrocyte membrane. As a result erythrocyte membrane bursts, causing an increase in permeability and a loss of haemoglobin. A study was made to establish the relationship between the adjuvant and haemolytic activity of saponins derived and purified from 47 different food and medicinal plants. However, the results indicated that the adjuvant activity does not relate with haemolytic activity (Oda et al., 2000). The toxicity towards coldblooded species has lead to the use of saponin containing drugs to catch fish. Saponins are also highly toxic to molluscs and have been investigated as molluscicides in the control of schistosomiasis (Sindambiwe et al., 1998; Abdel-Gawad et al., 1999). Itabashi et al., (1999) isolated furcreastatin, a steroidal

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saponin from ethanolic extract of the leaves of Furcraea foetida (L.) Haw (Agavaceae) and screened for its selective cytotoxicity towards mutant p53-expressing mouse fibroblasts.

Their finding suggests that saponins have weak toxicity if taken orally by warm-blooded species which is probably attributed to low absorption rates.

The genus Chlorophytum includes 300 species, which are distributed throughout the tropical and subtropical parts of the world with 85% species reported from tropical and subtropical Africa. There are 17 species of Chlorophytum recorded in India out of these 11 species of Chlorophytum are found to be

growing in Maharashtra (Patil and Deokule, 2010). It is being widely cultivated in different parts of India on commercial basis. This review discusses about the major species of Chlorophytum, their major component saponins and their therapeutic values in Indian system of Medicine.

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Chlorophytum borivilianum Santapau & R. R. Fern. Chlorophytum borivilianum also known as Safed Musli is a traditional herbal plant with assorted Ayurvedic relevance. It has therapeutic application in Ayurvedic system of medicine (Purohit et al., 1994). The species was first described from India in 1954 and reached rare status in nature due to over exploitation. The National Medicinal Plant Board (NMPB) of Government of India has recognized Safed Musli as sixth among the 28 selected priority medicinal plants to be protected and promoted. In India C. borivilianum is mainly distributed in Southern Rajasthan, North Gujarat and West Madhya Pradesh. The plants grow in a wide variety of places in nature, starting from open rocky places to shady and highly humus rich soil in the forest (Thakur et al., 2009). It is considered as an excellent herb to increase general body immunity. Its aphrodisiac properties have proved very much useful for the people suffering from Erectile Dysfunction and to increase male potency. It has spermatogenic property and helpful in curing impotency as they are rich in glycosides. Roots are widely used for various therapeutic applications in the Ayurvedic and Unani systems of medicine. It is known to cure many physical illness and weaknesses. It is also reported to cure diabetes, arthritis (Oudhia, 2001b). However, in recent years its effectiveness in increasing male potency has become very popular and is now considered as an alternative to ‘Viagra’ (Thakur et al., 2009). Saponins and therapeutic value of C. borivilianum Among all the species of Chlorophytum present in India, C. borivilianum produces the maximum root tuber along with the highest saponin content (Attele et al., 1999). Traditionally, roots of these species are reputed to posses various pharmacological utilities having saponins as one of the important phyto-chemical constituents (Marais and Reilly, 1978). Four new spirostane-type

saponins named borivilianosides E-H (1–4) were isolated from an ethanol extract of the roots of C. borivilianum together with two known steroidal saponins (5 and 6). The structures of 1–4 were elucidated using mainly 2D NMR spectroscopic techniques and mass spectrometry. The cytotoxicity of borivilianosides F (2), G (3), and H (4) and three known compounds were evaluated using two human colon cancer cell lines (HT-29 and HCT 116) (Acharya et al., 2009). Compounds 1–4 had been isolated as white, amorphous powders. The sugars obtained by aqueous acid hydrolysis of each compound have been identified by comparison on TLC with authentic samples as glucose, galactose, xylose, arabinose, and rhamnose (in the case of 1 and 2), glucose and galactose (in the case of 3), and glucose, galactose, and xylose (in the case of 4) (Acharya et al., 2009) [Fig 2(A, B)]. The plant yields a flavonone glycoside, which is a powerful uterine stimulant, steroidal saponins having muscle building properties and their structure is similar to male anabolic hormones testesterone. Roots of Chlorophytum contain 42% carbohydrate, 80– 89% protein, 3–4% fiber and 2–17% saponin (Wagle et al., 2000; Jat and Bordia, 2003). Apart from biologically effective steroidal and triterpenoidal saponins, sapogenins and fructans are reported to have prebiotic importance (Devon, 1975). The other phytoconstituents from the plant contain high quantities of simple sugars mainly sucrose, glucose, fructose, galactose, mannose and xylose (Sreevidya et al., 2006). Proteins, phenolics, triterpenoids, gallo-tannins and mucilage have also been reported from C. borivilianum (Thakur and Dixit, 2005). Several medicinally important attributes have been assigned to the plant because of its multi-pharmacological aspect which includes aphrodisiac, immuno-modulatory, antidiabetic, antioxidant, anti-stress, antimicrobial, anti-aging, antitumor and antiinflammatory activities (Jat and Bordia, 2003) (Fig 3). Aqueous extract of dried roots of C. borivilianum displayed enhanced sexual

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behaviour with increased potential of spermatogenesis in albino rats (Kenjale et al., 2008). The plant has also been acclaimed for its anti-diabetic activity against streptozotocin induced diabetes (Mujeeb et al., 2009). The anti-hyperglycemic activity of the aqueous extract of C. borivilianum roots was comparable with glibenclamide, a standard hypoglycemic drug (Govindarajan et al., 2005a; 2005b). The herb is found to be significantly effective in ameliorating the lipid metabolism in hyper-cholestremic animals and the presence of fructans are reported to be the major contributing factor in better management of hypercholestramia (Sreevidya

et al., 2006; Visavadiya and Narsimhacharya, 2007; Kenjale et al., 2007; Deore and Khadabadi, 2009a). It also increased the HDLcholesterol levels having a protective role in cardiovascular diseases (Deore and Khadabadi, 2009a; Loo et al., 2005). Tuber extracts of C. borivilianum have been proved as anti-stress agent (Loo et al., 2005; Mimaki et al., 1996; Deore and Khadabadi, 2009b). Their study is based on the traditional claim of utilization of this herb against rheumatoid arthritis (Panda et al., 2007). This activity could in part be attributed to the steroidal components in the plant.

Fig. 3- Therapeutic applications of Saponins

Chlorophytum arundinaceum Baker. Chlorophytum arundinaceum (Asparagaceae) a tuberous angiosperm, commonly also taken as â&#x20AC;&#x2DC;Safed Musliâ&#x20AC;&#x2122; is indigenous to India and distributed in Eastern Himalayas, Eastern Ghats, Assam, Bihar and Andhra Pradesh. Due to excessive harvesting and poor ways of germination and vegetative propagation, this plant is now standing between one of the endangered species of Chlorophytum (Samantaray and Maiti, 2011). It is a plant of repute as its fasciculated roots are reported to be used as a tonic and

constitute an important ingredient of more than 20 Ayurvedic and Unani preparations with active constituents, especially steroidal sapogenins known to possess adoptogenic and aphrodisiac attributes. Owing to its therapeutic properties, it has been exclusively cannibalized from its wild habitats. According to one survey, the species has been placed in the endangered category in Eastern Ghats of India and figures prominently among the rare medicinal herbs of India (Panda et al., 2007). The major reported constituents in the roots of C. arundinaceum include 4 hydroxy8, 11 oxidoheniconesol and pentacosanol,

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docasonoic acid, pentacosonyl docosanoate, nnonacosane, tetracosanoic acid, stigmasterol and stigmasterol β-d-glucopyranoside. Arundinoside A and B have also been reported as major glycosidic portions from C. arundinaceum. Presence of such constituents as straight chain alcohols with tetrahydrofuran moiety in saponin containing drugs are a rarity (Sreevidya et al., 2003; Tandon and Shukla, 1995). Saponins and therapeutic value of C. arundinaceum Compounds isolated and identified from C. arundinaceum are; nonacosane, tetracosanoinc, triacontanoc, 4-hydroxyl- 8, 11-oxidoheneicosanol and pentacosyl docosanoate 2, 2ʹ, 4, 4ʹ-tetrahydrobibenzyl xyloside and tokorogenin based saponin arundinoside-A (Tandon and Shukla, 1997); four sapogenins - stigmasterol, tigogenin, neogitogenin, and tokorogenin (Tandon and Shukla, 1992) and glucopyranoside from the fruits of C. arundinaceum (Tandon and Shukla 1993) [Fig 4(A)]. However, no reports are available on the pharmacological assays of the compounds isolated. The tuberous roots of the plant are specially used for the treatment of rheumatism, antiulcer activity and strengthening of the gastric mucosal barrier (Jackson et al., 1999). Moreover, its active constituents’ especially steroidal sapogenins are known to possess adoptogenic and aphrodisiac attributes (Chopra et al., 1956). The root extract is considered as a potent antioxidant as it could render effective protection against the hemolysis and disruption and stress induced elevated plasma corticosterone (Ghosal, 2006). Chlorophytum malayense Ridl. Chlorophytum malayense is another important plant group evaluated extensively for various medicinal properties. Chromaloside A, isolated from this plant, is reported as a major cytotoxic agent and is being explored for its

potential as an anticancer agent. Chlorophytum malayense is also known as spider lily plant. Saponins and therapeutic value of C. malayense C. malayense Ridl. is indigenous to southeast Asia and south-west of Yunnan province of China. Four steroidal saponins (1–4) were isolated from C. malayense rhizome. These Four saponins, termed as chloromaloside A, B, C and D (1–4,) [Fig. 4(B)] have neohecogenin and neotigogenin as the aglycone moiety with various substitutions of sugar moiety. Chloromaloside - A, C and D belong to 25 (S) spirostane series, while Chloromaloside-B (4) is found to be furostane type. Chloromaloiside A (1), isolated as colorless needles, is also the major saponin of C. malayense with 0.49% yield while yield of compound 2, 3, and 4 was 0.025, 0.074, and 0.018%, respectively (Qui et al., 2000). In a bioassay guided fractionation, compound 1 showed broad cytotoxicity against various human cancer cell lines (Qui et al., 2000). The ED50 values varied from 1.4 to 5 -l g/ml to diﬀerent cell lines indicating moderate toxicity when compared to positive control colchicine and ellipticine; while, other compounds are still to be investigated for their pharmacological activity. A new steroidal saponin named as chloromaloside E having neohecogenin as aglycone (5) has been isolated (Yang and Yang, 2000). So far, activity of this compound has not been tested. Chlorophytum comosum (Thunb.) Jacques Chlorophytum comosum is another medicinal plant which has got maximum demand and commercial value today. This plant is one of the fast growing ever green plants of Chlorophytum species reaching up to 1–1.5 ft tall with a spread of 2 feet, popularly growing for its attractive foliage. Some other common names for the plant are “Ribbon plant or Spider plant”. The plant is native to South Africa having the tendency to grow in dry and humid conditions (Kaushik, 2005). It also produces branched stolons with small white flowers and baby plantlets. It has fleshy

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tuberous roots that store reserve food. These spider plants are excellent house plants or indoor plants as they are not only easygrowing plants but have air purifying abilities by cleansing electronic air pollutants emitted by artificial lighting especially formaldehyde and carbon monoxide. They are ideally able to tolerate artificial lighting very well with air purifying abilities in office environment where electronic pollutants are emitted (Charlton, 1990). Saponins and therapeutic Chlorophytum comosum

value

Seven anti-tumour promoter crude steroidal saponins have been isolated by silicagel, reverse phase RP18, and Diaion HP20 chromatography and by partitioning of methanol extract with n-butanol from this species (Mimaki et al., 1996). Compound 6–9 are known spirostanol saponins while compound 10, 11 and 12 are new spirostanol pentaglycosides embracing b-D-apiofuranose. The saponins of C. comosum are different from saponins of C. malayense having aglycone based on (25R)-Spirostan series as tigogenin, gitogenin and hecogenin, while saponins of C. malayense are based on (25S) spirostan series as neotigogenin and neohecogenin. The isolated saponins have been evaluated for in vitro anti tumor promoter activity by measurement of the inhibitory activity on TPA stimulated 32P-incorporation into phospholipids of HeLa cells. Compounds 7, 8, 10, 11 and 12 are found cytotoxic to HeLa cells at 50 µg/ml concentrations (Mimaki et al., 1996) [Fig. 4(C)]. Compounds 6 and 9 exhibited 23.1% at 57.8% inhibition at 50 µg/ml without cytotoxicity towards HeLa cells. However, more investigations are required against various other human cancer cell lines (Ahmad and Basha, 2007). C. comosum is traditionally known to be used against bronchitis; however, the active principle responsible for the cure of bronchitis is yet to be investigated. In China, these species has been traditionally used as a folk

medicine for cough, fracture, burns and treatment of bronchitis. There are only few reports on the biological behavior of C. comosum and its specific component so far (Matsushita et al., 2005). Chlorophytum orchidastrum Lindl. The plant Chlorophytum orchidastrum can generally be seen growing on the mountain grassy slopes in India especially in the rainy season. As, like other Chlorophytum species, this plant also grows gaining its nutrition by tuberous roots. The tuberous roots are long, slender 15–34 cm long and each about 1 cm in diameter. This plant can be distinguished from other spider plants because of its different flower spike producing many fertile seeds. C. orchidastrum is found to have various nutritional properties along with immuno enhancing and hepato-protective properties (Nergard et al., 2004; Patil and Deokule, 2010). Saponins and therapeutic Chlorophytum orchidastrum

value

Six new spirostane-type saponins (1–6), named orchidastrosides A–F, and chloromaloside D were isolated from an ethanol extract of the roots of Chlorophytum orchidastrum. The saponins have neotigogenin or neogitogenin as the aglycon and oligosaccharidic chains possessing seven to nine sugar units. Their structures were elucidated mainly by 2D NMR spectroscopic analyses COSY (Correlation spectroscopy), TOCSY (Total correlation spectroscopy), NOESY (Nuclear overhauser effect spectroscopy), HSQC (Heteronuclear singlequantum correlation spectroscopy), HMBC (Heteronuclear multi-bond correlation spectroscopy) and FABMS (Fast atom bombardment mass spectroscopy) and HRESIMS (High resolution electron spray ionization mass spectroscopy) [Fig. 4(D)]. Compounds 1–6 were tested for cytotoxicity against two human colon cancer cell lines, HCT 116 and HT-29 (Acharya et al., 2010).

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Fig. 4-(A, B, C, D):

Saponins from different species of Chlorophytum (A) Structure of saponin from C. arundinaceum, (B) Structure of saponin from C. malayense, (C) Structure of saponin from C. comosum, (D) Structure of saponin from C. orchidastrum

CONCLUSION PROSPECTS

AND

FUTURE

Chlorophytum species grows wild in thick forests and are traditional medicinal plants. Because of its significant medicinal properties, some varieties got maximum demand and commercial value which is increasing day by day. There are around 256 varieties of Chlorophytum in the world; in India we have around 17 of them, of which, C. borivilianum has got a good market all over the world, especially in the Gulf countries and the West. Presently production is not even 5% of the

estimated demand because of its use in more than a hundred Ayurvedic, Allopathic, Homoeopathic and Unani medical preparations. Due to its vast demand its retail price in India is 1500 INR per Kg. or (US$ 30) which is very high if compared to other plants with medical applications (Garima and Shruthi, 2012). The work on production of secondary metabolites and their pharmacological investigations should be on momentum by the pharmaceuticals and neutraceuticals sectors on C. borivilianum which can play a vital role in human welfare.

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Saponins are found in wide varieties of foods such as asparagus, beans, blackberries, peas, potatoes, sugar beet and tea etc. The isolation, purification and formulations of the phytochemicals of this plant viz. steroidal saponins, β-sitosterol, stigmasterol and hecogenin, fructans and fructooligosaccharides which is reported for various therapeutic applications, viz. aphrodisiac, adaptogen, antidiabetic, antimicrobials, anti-inflammatory effective against lipid metabolism, analgesic etc, definitely provide effective drugs against these destructive diseases. Eventhough, a number of pharmacological studies have been performed, the phyto-chemistry of the tuber and leaf is not clearly understood. Similarly a major limitation in this species appears to be a poor knowledge about various physiological

and biochemical processes. Saponin from Chlorophytum is a hidden gift from nature which is now proving its efficacy and potential as a miracle herb for biopharmaceutical and neutraceutical attention for human welfare. It appears that there are still a number of biologically active compounds to be explored in this genus and the future research may be oriented in that direction along with evaluation of the remedial properties. ACKNOWLEDGEMENT Authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for the award of Emeritus Scientist to Prof. P.S. Bisen and to Mr. Avinash Dubey for preparing the images.

Glycosides: Spirostanes, Bufanolides, Cardenolides. Biomed. Life Sci. 3:1802-1803.

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Source of Support: Nil

Conflict of Interest: None Declared

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Research article STANDARDIZATION OF POLYHERBAL FORMULATION – ARSHONYT FORTE Agrawal SS1*, Ghorpade SS2, Gurjar PN3 1, 2

SVKM’S, NMIMS, SPTM, Shirpur, Dist: Dhule, 425 405, Maharashtra, India Sharadchandra Pawar College of Pharmacy, Otur, Dist: Pune, 412 409, India *Corresponding Author: E-mail: sagrawal80@gmail.com; Mobile: +919923694748

Received: 23/08/2012; Revised: 25/09/2012; Accepted: 30/09/2012

ABSTRACT Indian system of Medicine comprises of Ayurveda, Unani, and Siddha. In all the systems, maximum drugs are made up of poly-herbs. The World health organization (WHO) had given a detailed protocol for standardization of herbal drugs which mostly consists of single herbs. A detailed protocol is given by the WHO to avoid any adulteration in the formulation and to maintain its quality, safety and efficacy. Objective of this work was to standardize a polyherbal formulation available in the market for quality and efficacy. Arshonyt forte formulation was selected for carrying out standardization, it is a polyherbal drug comprising of complex mixture of different herbal substances. A pack of 80 tablets of Arshonyt forte 650 mg had been taken from Charak Pharma Himachal Pradesh outlet; batch no.AR 055 Exp.03/2012. Arshonyt forte is a mixture of 5 herbs. The formulation was subjected to preliminary phytochemical test, colour test for pesticides, colour test for heavy metals, estimation of active constituents by UV spectrophotometer, chromatographic studies like TLC, HPTLC, HPLC and microbial load test. The results obtained indicated proper extraction of polyherbal drugs which yields some active constituents which are identified by high performance thin layer chromatography, high performance liquid chromatography, ultraviolet spectroscopy determination. KEYWORDS: Polyherbal formulation, Standardization, Arshonyt Forte

Cite this Article Agrawal S S, Ghorpade S S, Gurjar P N (2012), STANDARDIZATION OF POLYHERBAL FORMULATION – ARSHONYT FORTE, Global J Res. Med. Plants & Indigen. Med., Volume 1(10), 516–523

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INTRODUCTION Medicinal plants constitute a source of raw material for both traditional systems of medicine (e.g. Ayurvedic, Chinese, Unani, Homeopathy, and Siddha) and modern medicine. Nowadays, plant materials are employed throughout the industrialized and developing world as home remedies, over-thecounter drugs, and ingredients for the pharmaceutical industry. As such, they represent a substantial proportion of the global drug market (Bhanu et al., 2005; Bhutani K, 2003). Most rural populations, especially in the developing world, depend on medicinal herbs as their main source of primary healthcare. Although most medicinal herbs are not in their natural state, fit for administration, preparations suitable for administration are made according to pharmacopoeial directions. So standardization of poly-herbal formulation is necessary. It involves adjusting the herbal drug preparation to a defined content of a constituent or a group of substances with known therapeutic activity by adding excipients or by mixing herbal drugs or herbal drug preparations. Botanical extracts made directly from crude plant material show substantial variation in composition, quality, and therapeutic effects. Standardization is done by determining the extractive value, ash value, heavy metal content, pesticide residue, microbial contamination and active content by chromatographic methods (Mukherjee et al., 1998; Mukherjee PK, 2008). Standardized extracts are high quality extracts containing consistent levels of specified compounds, and they are subjected to rigorous quality controls during all phases of the growing, harvesting, and manufacturing processes (Gokhale & Surana, 2006; Kokate et al., 2005; Lazarowych & Pekos, 1998). So Objective of this work was to standardize a polyherbal formulation (Arshonyt Forte), available in the market for quality and efficacy. MATERIALS AND METHODS A pack of 80 tablets of Arshonyt forte 650 mg had been taken from Charak Pharma, Himachal Pradesh outlet; batch no. AR 055

Exp.03/2012.Arshonyt forte is a mixture of 5 herbal drugs. Arshonyt forte is a mixture of the following 5 polyherbal materials. Cyamopsis tetragonoloba (L.)Taub Acacia catechu.(L.f.) Willd. Aloe vera (L.) Burm.f. Terminalia chebula.Retz. Plumbago zeylanica.L Steps for Standardization of herbal medicine Step 1: Preliminary phytochemical test. Hydro alcoholic solution was used for extraction of various constituents from Arshonyt forte sample powder. Then test for identification of alkaloids, flavonoids, tannins, saponin were carried out. Step 2: Extraction and authentication of the active therapeutic constituent from the extract. Isolation of total glycoside contents: Weight of 5 g of the Arshonyt forte sample powder was taken into 100 ml volumetric flask and was made acidic with dilute HCl (5%). Then we took the sample in separating funnel with chloroform and then chloroform layer was separated and evaporated on water bath. The residue was collected and was used for further studies. Isolation of total tannin contents: Extract of 400 mg was weighed accurately in 100 ml volumetric flask. 50 ml of hot water was added to it, pH was above 7, the temperature was maintained at above 40oC and it was shaken well. The aqueous fraction was used for the estimation of tannin content. Step 3: Estimation of active chemical constituents by UV spectrophotometer (Rubesh et al., 2010). Standard solution: 10 mg of all standard was dissolved in 100 ml of methanol to give 100 Âľg/ml. Test solution: 100 mg of all test extract prepared as mentioned earlier was dissolved in

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100 ml of methanol. From that 10 ml was taken and diluted up to 100 ml with methanol to give 100 µg/ml. Preparation of calibration curve: 1. For Estimation of total tannin: concentrations of 10, 20, 30, 40, 50 ppm were prepared. 2. For Estimation of total polyphenolics: concentrations of 20, 40, 60, 80, 100 ppm were prepared. 3. For Estimation of total saponin: concentrations of 25, 50, 75, 100, 125, 150, 200, 250 ppm were prepared. Step 4: Estimation and quantification of active constituents from the extract by HPTLC (Sanjeeth et al., 2010). Standard solution: 2 mg of all standard was dissolved in 500 ml of methanol to give 100 µg/ml. Test solution: 10 mg of extract was dissolved in 20 ml of methanol to give 500 µg/ml. Scanning: Absorption mode from 200 to 800 nm. Step 5: Identification and estimation of active chemical constituents by HPLC. Standard solution: 10 mg of all standard was dissolved in 100 ml of methanol to give 100 µg/ml. Test solution: 100 mg of all test was dissolved in 100 ml of methanol. From that 10 ml was taken and diluted up to 100 ml with methanol to give 100 µg/ml. Step 6: Extraction, Identification and quantification of pesticides from the finished product (Shrikumar et al., 2006). Extraction of pesticides from material: 5 gm sample was taken in a round bottomed flask and added sodium sulphide with 50 ml nHexane. It was refluxed for 1 h. The filtrate was taken in a separating funnel and extracted with 25 ml and 12.5 ml Acetonitrile. The acetonitrile layer was mixed with 250 ml

demineralized water with 1.5 ml saturated sodium sulphide and again shaken in a separating funnel with n-Hexane layer and evaporated on a water bath. The residue obtained was used for analysis of organochloro, organophosphate and carbamate pesticides. Step 7: Extraction, Identification and quantification of heavy metals from the finished product. Extraction of pesticides from material: 5 g sample was taken in a silica crucible and heated to remove the moisture. It was kept in a muffle furnace at 600°C, for 3 h to remove organic material. The crucible was cooled down and was examined for any colour change. The change in colour reveals the presence of copper and zinc. Next the residue was boiled with 10 ml of dilute HCl and filtered. This filtrate may contain metals like arsenic, mercury, lead, cadmium and zinc. Materials: 1. Copper wire wound tightly around glass rod. 2. Nitric acid 2.5 N. Procedure for the test of heavy metals: A copper wire was washed with 2.5 N nitric acid and then it was rinsed with 95% ethanol and dried. Then 20 ml of residue was placed and dissolved in water into a small flask to which 4 ml of conc. HCl was added. Then freshly treated copper wires were added to flask. Then the solution was heated for about 1 h. Next the copper wire was removed and examined for any colour change. Step 8: Microbiological load test of finished product (The Ayurvedic Pharmacopoeia of India, 2006). Sample of 0.5 g sample was taken and poured onto a nutrient agar media and incubated in appropriate condition for microbial testing. RESULTS Results of Preliminary phytochemical test and pharmacognostic investigation are shown below in table I

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Table I: Results of preliminary Pharmacognostic investigation Sr. No.

Tests

Observation

Extractive value 5g Total ash 0.4 g Acid insoluble ash 10 mg Water soluble ash 0.2 g Dragondroff reagent Reddish brown colour precipitate Mayer’s reagent Cream colour precipitate Wagner’s reagent Reddish brown colour precipitate Hager’s reagent Gives yellow colour precipitate Shinoda test crimson red colour + indicates Present; − indicates absent

1. 2. 3. 4. 5. 6. 7. 8. 9.

Result for extraction and authentication of the active therapeutic constituent from the extract. i.

Arshonyt forte tablet

ii.

For Estimation of total polyphenolics: Maximum absorbance was found at 775 nm. The equation of calibration curve was found to be Abs = 0.0723x + 0.0231 with R2 = 0.9984. The percentage estimation of gallic acid is is given below in table III.

For Estimation of total tannin: Maximum absorbance was found at 775 nm. The equation of calibration curve was found to be Abs = 0.0059x + 0.0032 with R2 = 0.9965. The percentage estimation of tannic acid is given below in table II

− − − − + + + + +

iii.

For Estimation of total saponin: Maximum absorbance found at 775 nm. The equation of calibration curve was found to be Abs = 0.0027x - 0.0049 with R2 = 0.9973. The percentage estimation of saponin is given below in table IV.

Table II: Total Tannin estimation Sr. No

Extract

Absorbance at 775 nm

1 2 3

sample

0.041 0.046 0.043

% Estimation 7.49 8.34 7.83

Mean ± S.D. 7.89 ± 0.42

Table III: Total polyphenolics estimation Sr. No. Extract Absorbance 765 nm % Estimation 1

sample

1.384 1.378 1.383

18.59 18.50 18.57

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Mean ± S.D. 18.54 ± 0.072

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Table IV: Total saponin estimation Sr. No. Extract Absorbance 472 nm % Estimation Mean ± S.D. sample 0.017 8.11 7.61 ± 0.567 1 0.014 7.00 0.016 7.74 Result for extraction and authentication of the active therapeutic constituent from the extract by HPTLC. Figure 1:

Figure 2:

Track 1 for Gallic acid

Figure 3:

Track 1 for Gallic acid

Track 3 for extract

Table V: HPTLC study on the extract Tr. Pk No. 1 2 3 3 3 3 3 3 3

1 1 1 2 3 4 5 6 7

AUC

Content in mg

0.72 0.26 0.02 0.12 0.24 0.26 0.73 0.91 0.97

1544.7 1274.1 1165.7 2035.4 878.1 749.3 1753.4 2265.7 1842.9

− − − − 29.40 mg − 56.75 mg − −

The Rf value of standard catechin, gallic acid was found to be 0.72 and 0.26 respectively.

Result for extraction and authentication of the active therapeutic constituent from the extract by HPLC. Figure 4: Figure 5: Figure 6:

Chromatogram for Epicatechin

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Chromatogram for sample

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Figure 7:

Figure 8:

Chromatogram for Lupeol

Chromatogram for sample

Result for Colour test for pesticides: Table VI: Qualitative determination of the pesticides Sr. no

Test performed

Observations

Results

1. 2.

Organo chloro Organo phospho

No colour observed No colour observed

Dichloropropane absent Phosphate absent

Carbamate

No colour observed

Amide group absent

Result for Extraction, Identification and quantification of heavy metals from the finished product: No change was observed in the colour of copper wire which revealed that metals are absent in the formulation. Result for Microbiological load test of finished product: No evidence of the colony formation and no turbidity in the nutrient broth suggested the absence of microbial load in the sample. DISCUSSION Arshonyt forte powder extract shows the presence of Alkaloids, Flavonoids, Tannins, Saponins and Glycosides. The extractive value denotes the presence of active constituents present in formulation. As extractive value of the formulation is more it can help to carry out all tests neatly and contents can be determined. Total ash usually consists of carbonates, phosphates, silicates and silica which include

both physiological ash and non-physiological ash. So a lower total ash value indicates minimal presence of the above mentioned contents. Water soluble ash is that part of total ash content which is soluble in water. It is good indicator of either previous extraction of the water soluble salts in the drug or incorrect preparation. The value for water soluble ash revealed that the formulation is free from other foreign matter. Tannins and polyphenolics are complex substances. The relatively fair amount of tannins, poly-phenolics and saponins in formulation indicates presence of higher amount of active constituents which helps in playing a good therapeutic activity of formulation. Content of catechin and gallic acid from extract was found to be 56.75 mg and 29.40 mg in sample extract respectively by HPTLC. In HPLC determination, the RT value of sample gallic acid, epicatechin, lupeol was found to be 3.3667, 2.55, 6.66 and peak area of

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sample gallic acid, epicatechin, lupeol 557.679, 1809.335, 1340.61. Content of gallic acid, epicatechin, lupeol was determined to be 21.25 mg, 18.25 mg and 100 mg in sample extract respectively.

causes serious infections which can enter through oral and ophthalmic formulations. So absence of microbes in formulation indicates the safety of formulation. CONCLUSION

Organo-chloro pesticides like DDT cause poisoning and potential hazards to animal and human beings. Aldrin, dieldrin and endrin are considered to be compounds that cause poisoning. Organo-phosphorous compounds are potent cholinesterase inhibitors and can be very toxic. It also acts on CNS and causes depression. So absence of pesticide in the formulation indicates its safety. Presence of metal in formulation causes severe diseases on consumption. As due to negative results for colour test of metal, it indicates that the formulation is suitable for consumption (Table VI). The presence of microbes like salmonella and pseudomonas

It can be concluded that the marketed formulation (Arshonyt forte tablet) has been standardized by intervention of modern quality control measures. Pharmacognostic characters established for the raw materials could be employed as quality control standards for evaluating its identity and can be used for routine analysis. The results obtained could be used to set new Pharmacopoeial limits for optimal efficacy of the medicine. ACKNOWLEDGEMENT Authors are thankful to SPTM, SVKM’s NMIMS for providing necessary facility for carrying out the reported work.

REFERENCES: Bhanu PS, Zafar R, Pawar R (2005). Herbal drug standardization. The Indian Pharmacist, 4 (35): 19–22. Bhutani K (2003). Herbal medicines an enigma and challenge to science and directions for new initiatives. Journal of Natural Products, 19 (1): 3–8. Gokhale SB, Surana SJ (2006), Fluorescence quenching as a tool for identification and quality control of crude drug. Planta indica, 2 (3): 47. Kokate CK, Purohit AP, Gokhale SB (2005). Analytical pharmacognosy. pp 1, 99. Lazarowych NJ, Pekos P (1998). Use of fingerprinting and marker compound for identification and standardization of botanical medicines strains: Strategies for applying pharmaceutical HPLC analysis to herbal. Drug Information Journal, 32: 497–512.

Mukherjee PK, Rai S, Bhattacharya S, Wahile A, Saha BP (2008). Marker analysis of Polyherbal formulation, Triphala- A well known Indian traditional medicine. Indian journal of Traditional Knowledge, 7 (3): 379–383. Mukherjee PK (2008). Quality Control of Herbal Drugs, An approach to evaluation of Botanicals. Pharmaceutical Publishers, I edition: pp 426–517. Rubesh KS, Kishan RJ, Venkateshwar KN, Duganath N, Kumanam R (2010). Simultaneous spectrophotometric estimation of Curcuminoids and Gallic Acid in Bulk Drug and Ayurvedic Polyherbal Tablet Dosage Form. International journal of Pharmaceutical Quality Assurance, 2 (1): 56–59.

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Sanjeeth SI, MannaPK, Manavalan R, Jolly CI (2010). Quantitative estimation of Gallic Acid, Rutin and Quercetin in certain herbal plants by HPTLC method. Pelagia Research Library, 1 (2): 80–85. Sawant L, Pandita N, Prabhakar B (2010). Determination of Gallic acid in Phyllanthus emblica Linn. Dried fruit powder by HPTLC. Journal of Pharmacy & Bioallied Sciences, 2 (2): 105–108.

random primed polymerase chain reaction. Planta Medica, 61 (5): 466– 469. Shrikumar S, Maheshwari U, Sughanti A, Ravi TK (2006). WHO guidelines for herbal drug standardization. The Ayurvedic Pharmacopoeia of India (2006), Government of India Ministry of Health & Family Welfare, Part-1, volume 1, 5– 8.

Shaw PC, Butt P (1995). Authentication of Panax species and their adulterants by

Source of Support: Nil

Conflict of Interest: None Declared

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Research article STUDIES ON SEED GERMINATION AND GROWTH IN GLORIOSA SUPERBA L. Anandhi S1*, Rajamani K2 1, 2

Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India *Corresponding Author: sanandhijasmin@gmail.com; Mobile: +919842392444

Received: 02/08/2012; Revised: 25/09/2012; Accepted: 02/10/2012

ABSTRACT Studies on imposing seed germination in glory lily revealed that seeds soaked in hot water for one hour was recorded to be the best treatment with maximum germination of 32.75 % and vigor index (565.92). So, the seed treatment can be recommended as a nursery practice. Earlier germination (48.35 days) was observed for the seeds soaked in hot water, when compared to other chemical treatments. The maximum number of leaves and root length was recorded for the seeds soaked in GA3 at a concentration of 250 ppm. KEY WORDS: Glory lily, seed, germination, hard seed coat, growth regulators

Cite this Article Anandhi S, Rajamani K (2012), STUDIES ON SEED GERMINATION AND GROWTH IN GLORIOSA SUPERBA L., Global J Res. Med. Plants & Indigen. Med., Volume 1(10): 524â&#x20AC;&#x201C;528

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INTRODUCTION

RESULTS

Gloriosa superba L. (Colchicaceae) is an important medicinal plant, native to Tropical Asia and Africa. It is highly valued in modern medicine due to the presence of colchicines and colchicoside which are used in treatment of gout and rheumatism. It is commercially propagated by tubers which are ‘V’ or ‘L’ shaped sourced from the wild especially from the forest areas and hillocks. It has been reported that more than 500 tonnes of wild tubers are collected every year and used for planting in Tamil Nadu alone. About 800 Kg of tubers are required to be planted in one acre. The cost involved towards planting material (Rs. 250 to 300/Kg of tubers), alone accounts to 2.0 lakhs at the rate of Rs. 250/- per kg of tuber prevailing for the last three years. Seed germination is erratic and takes three weeks to three months (Azhar and Sreeramu, 2004). Seedlings grow and produce micro tubers which can also be used as planting material. Hence, the present investigation was contemplated with the objective of standardizing seed treatment methods to induce better germination of Gloriosa superba seeds.

Statistically significant differences were observed among the various dormancy breaking seed treatments for the characters observed (Table 1). Soaking the seeds in hot water for one hour resulted in higher seed germination percentage (32.75 per cent) and was significantly superior to all other treatments. Soaking seeds in GA3 250 ppm for one hour (19.50 per cent) and KNO3 1.0 % recorded 16.25 per cent. The lowest germination percentage was observed in thiourea 1.0 % for one hour (7.50 per cent). No seeds germinated in control.

MATERIALS AND METHODS Dried seeds of Gloriosa superba collected from Mulanur of Thirupur district were used for the experiment. To test the effect of hot water soaking on seed germination, 250 ml of water was heated up to 100oC and then taken away from the heat source (Hartmann et al., 1997). The seeds were immersed in hot water. After an hour, seeds were taken out and soaked in cold water overnight. Similarly, seeds were soaked in chemicals viz., GA3 (100 and 250 ppm), Thiourea and Potassium nitrate at various concentrations (0.5, 1.0 and 1.5 %) for an hour. Treated seeds were sown in the raised beds at a distance of 10 cm between the lines in the beds. Seeds of 100 numbers were sown in each treatment with the replication of four.

The earliest germination (48.35 days) was observed in the seeds soaked in hot water, followed by soaking in GA3 250 ppm which took 48.45 days. Seeds soaked in thiourea 1.5 % (T5) showed the most delayed germination (60.80 days) among all the treatments. The highest number of leaves per seedling (5.05) was observed in the treatment viz., GA3 250 ppm as against the lowest number of leaves per seedling (3.80) observed in seeds treated with KNO3 1.5 %. The treatment GA3 250 ppm recorded the highest shoot length (11.78cm) as well as root length (6.94 cm). GA3 100 ppm and hot water soaking recorded higher shoot length (11.05 and 11.12 cm) and root length (6.45 and 6.15 cm). Seeds treatment with thiourea 0.5 % recorded the lowest shoot length (10.51 cm). The vigour index was calculated from the mean seedling length and germination percentage of each treatment. The vigour index was maximum for hot water treatment (565.92), which was significantly superior over other treatments. This was followed by GA3 250 ppm, which recorded 365.23. The lowest vigour index (123.67) was observed in T4 (thiourea 1.0 %).

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Table 1. Effect of seed treatments on germination percentage (%), and days for germination

Treatments

Seed treatments (one hour soaking)

Control

T2 T3 T4 T5 T6 T7 T8 T9 T10

Hot water soaking Thiourea 0.5 % Thiourea – 1.0 % Thiourea – 1.5 % KNO3 – 0.5 % KNO3 – 1.0 % KNO3 – 1.5 % GA3 – 100 ppm GA3 – 250 ppm Mean

Germination percentage (%) 0.00 (0.45) 32.75 (34.88) 11.00 (19.17) 7.50 (15.88) 8.00 (16.16) 13.75 (21.72) 16.25 (23.57) 15.25 (22.75) 12.20 (20.47) 19.50 (24.93) 13.47 (20.00)

Days for germination (days)

Shoot length (cm)

Root length (cm)

Vigour index

0.00

48.35

11.12

6.15

565.92

52.95

10.51

5.48

175.94

57.55

10.68

5.80

123.67

60.80

10.76

5.78

132.32

55.00

10.62

5.78

225.63

56.75

10.65

5.63

264.71

49.80

10.84

5.43

248.27

50.15

11.05

6.45

214.43

48.45

11.78

6.94

365.23

47.99

9.80

5.34

231.42

SE(d)

1.9905

0.9920

0.2186

0.1959

40.7236

CD(0.05)

4.0842

2.0356

0.4486

0.4020

83.5600

Values in parenthesis are arcsine transformed harsh condition until the rainy season. Soaking DISCUSSION of seeds in hot water could have helped in Dormancy is a condition where seeds will enhancing the seed germination by softening not germinate even when the environmental the hard seed coat and facilitating leaching out conditions (water, temperature and aeration) of the germination inhibitors (Azhar and are favourable for germination. Poor and Sreeramu, 2004). delayed seed germination in G. superba was reported and the germination was erratic which The results are similar to the observation took three weeks to three months In the present made in Leucaena glauca, where the boiling study, various dormancy breaking treatments water significantly reduced the percentage of like soaking in hot water and various chemicals abnormal seedlings and dead seeds were tried and the results revealed that hot (Venkatratnam, 1948). Sarker et al. (2000) also water treatment imposed for an hour recorded reported that steeping Sesbania rostrata seeds the higher germination percentage (32.75 %), in boiling water for one minute showed the earlier germination (48.35 days), and vigor highest percentage of germination (62.63 %). index (565.92). The water impervious seed coat Singh et al., (1984) found that water soaking protects the plant from germination during the

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enhanced the seed germination in Tephrosia purpurea and Abrus precatorius. Eventhough significant improvement in seed germination through different treatments, other than hot water soaking treatment could not be achieved. GA3 250 ppm showed a positive response which induced germination upto 19.50 per cent. Probably, still more higher germination per cent could have been achieved if the duration of soaking could be increased or by altering the concentration of GA3. The involvement of GA3 on the activation of cytological enzymes have been reported for better seed germination. GA3 is involved in the photomechanism, where it may activate the intermediaries of phytochrome interconvertable step(s) changing it into an active form in initiating germination as reported by Choudhary and Gupta (1995). Similar results was reported by Habibah et al. (2007) in Argania spinosa, who reported that soaking of seeds in GA3 500 ppm resulted in highest germination (30 %) which was followed by 100 ppm and 250 ppm (27 and 26 % respectively). Kandari et al. (2008) also reported that seeds of Arnebia benthamii when soaked in GA3 100 ppm for 24 hours and incubation of 25oC in 12 hours light photoperiod conditions resulted in maximum germination (100 %). The results of Ferraz and Takaki (1992) in Phyllanthus corcaradensis, Ponnuswamy (1993) and Fahmy et al. (1987) in Kenaf, and Dhankhar and Santhosh (1996) in Phyllanthus species are also on line with the findings of the present study.

(16.25 %). The enhancement in germination due to KNO3 treatment could be attributed to cytochrome oxidase activity. According to Copeland (1988), KNO3 breaks dormancy by acting as a substitute for light. Similar results were reported by Harakumar (1997) who found that Gymnema seeds soaked in 0.2%. KNO3 solution for 6 hrs enhanced germination percentage upto 75 %. Kevseroglue (1993) reported that seed treatment with KNO3 for 15 min increased the germination percentage in Datura stromanium. In the present study, thiourea 0.5 % treatment also exhibited a positive response towards better seed germination (11.00 %) which might be ascribed to the deactivating capacity of thiourea to certain inhibitors present in the seed thus helping in germination enhancement. Thiourea probably inhibits the breakdown of RNA in the seed and also inhibits denaturation of enzymes (Mayer, 1957). Similar results were reported by Choudhary and Kaul (1973) in Atropa belladonna in which seeds soaked in thiourea (2 %) induced the germination. CONCLUSION Treatment with growth regulators and other chemicals on induction of better seed germination would have been due to the antagonistic effect on growth inhibitors and also enhancement of the rate of metabolism during germination (Verma and Tondon, 1988). Thus, among all the seed treatments, soaking the seeds in hot water for one hour was the most effective for inducing better germination of seeds in Glory lily.

In the present study, KNO3 (1.0 %) solution also increased the germination percentage REFERENCES Azhar Ali Farooqi and BS Sreeramu (2004). Glory lily. Cultivation of medicinal and aromatic crops. Universities Press Private Ltd., Hyderabad. 131–138.

Choudary S and Gupta (1995). Studies on the germination of Catharanthus roseus (L.) seeds. Effect of temperature and promoters. Seed Sci. and Technol., 23: 831–841.

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Choudhary DK and BK Kaul (1973). Note on the effect of thiourea on the germination of Atropa belladonna. Indian J. Agric. Sci., 43(10): 967–968. Copeland L.O (1988). Principles of seed science and technology. Surjeet Publisher, New Delhi. Dhankhar DD and M Santhosh (1996). Seed germination and seedling growth in Anola (Phyllanthus emblica Linn.) as influenced by GA3 and Thiourea. Crop Res., 12(3): 363–366. Fahmy R, SA Abd-El-Daimen, S Abd-ElHafeez and MAA Rady (1987). The effect of GA3 on the germination rate and seedlings properties of Kenaf and Roselle. Agricultural Res. Rev., 61(8): 137–150. Ferraz FGA and MM Takaki (1992). Seed germination of invader species of crops. In: Phyllanthus corcovadensis Muell.Arquivosde Biologiaa e Technologia., 35(1): 53–62. Habibah S, Al-Menaie, NR Bhat, M Abo ElNil, P Gamalin and N Suresh (2007). Seed germination of argan (Argania spinosa). America-Eurasian J. Sci. Res., 2(1): 1–4. Harakumar C (1997). Seed technological studies in Gymnema sylvestre R.BR. M.Sc. Thesis, Tamil Nadu Agricultural University, Coimbatore. Hartmann HT, DE Kester, FT Jr Davies RL Geneve (1997). PlantPropagation, Principles and Practices. Sixth Ed. PrenticeHall, Inc. Upper SaddleRiver, New Jersey , USA. 770pp

Source of Support: Nil

Kandari LS, KS Rao, RK Maihkuri and Kusum Chauhan (2008). Effect of pre sowing, temperature and light on the seed germination of Arnebia benthamii (Wall.Ex G.Don). An endangered medicinal plant of central Himalaya, India. Kevseroglue K (1993). The effects of some physical and chemical treatments on germination of Datura. Seeds collected from natural vegetation. Doya Turk Tarum Ve Ormancilit, 17(3): 727– 735. Mayer AM (1957). The formation of a yellow pigment in lettuce seedling roots treated with thiourea. J. Exp. Bot., 8: 125–126. Ponnuswamy P (1993) Seed technological studies in Neem. Ph.d Thesis, Tamil Nadu Agricultural University, Coimbatore. Sarker PC, SMA Hossain, MSU Bhuija and M Salim (2000). Breaking seed dormancy in Sesbania rostrata. Pakistan J. Biol. Sci., 3(11): 1801–1802. Singh K, KP Singh and Susheel Kumar (1984). Seedling growth and vigour responses of some Indian medicinal plants to certain physical and chemical treatments. Indian J. Plant Physiol., 27(3): 295–299. Venkatratnam L (1948). Effect of heat and cold treatment on germination of Leucaena glauca. The Madras J.Agril., 35(8): 179–184. Verma AN and P Tondon (1988). Effect of growth regulator on germination and seedling growth of Pinus kesiya and Schima khasiana. Indian J. Forestry, 11(1): 32–36.

Conflict of Interest: None Declared

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Research article MASS PROPAGATION AND IN VITRO CONSERVATION OF INDIAN GINSENG - WITHANIA SOMNIFERA (L.) DUNAL Chatterjee Tuhin1, Ghosh Biswajit2* 1, 2

Plant Biotechnology Laboratory, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata- 700118, India. *Corresponding Author: E-mail: ghosh_b2000@yahoo.co.in

Received: 24/08/2012; Revised: 30/09/2012; Accepted: 03/10/2012

ABSTRACT Withania somnifera (L.) Dunal. (Solanaceae) commonly known as Ashwagandha or Indian Ginseng is an important medicinal plant with excellent export potential in herbal drug trade. The present work was aimed to develop a protocol for rapid propagation by using tissue culture technique and for in vitro conservation of elite genotype of this species, ultimately enable to keep pace with commercial needs. A successful micropropagation system had been developed by in vitro culture of nodal segments from 30 days old seedlings. A maximum of 21.0 ± 0.1 mean number of axillary shoots were obtained after two subcultures in presence of BA (1.0 mgl-1) and Kn (1.0 mgl-1) in MS medium. Individual shoot (1.0 cm) was elongated in MS medium fortified with GA3 (0.5 mgl-1). The elongated shoots were rooted in MS medium supplemented with IBA (1.0 mgl-1). Rooted plants were acclimatized and established in soil with survival rate of about 94%. For in vitro conservation of the species by slow growth technique, 2% sorbitol and 2% mannitol showed best performance for reducing growth. The established plants were uniform in respect to morphological as well as flowering characters compared to mother plant. Thus this study elucidated an efficient method for mass propagation and ex-situ conservation of elite germplasm through slow growth technique and sustainable management of this high demanding medicinal plant. LIST OF ABBREVIATIONS: BA - 6 benzyladenine, GA3 -Gibberellic acid, IAA - Indole-3-acetic acid , IBA - Indole-3-butyric acid, Kn- 6- furfurylaminopurine, 2iP – isopentenyl adenine, MS Murashige and Skoog’s medium (1962), NAA - α-naphthalene acetic acid. KEY WORDS: Withania somnifera, micropropagation, in vitro conservation, field evaluation.

Cite this article: Chatterjee Tuhin, Ghosh Biswajit (2012), MASS PROPAGATION AND IN VITRO CONSERVATION OF INDIAN GINSENG - WITHANIA SOMNIFERA (L.) DUNAL, Global J Res. Med. Plants & Indigen. Med., Volume 1(10), 529–538

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INTRODUCTION Withania somnifera (L.) Dunal. of Solanaceae, is a valuable herb used in traditional Ayurvedic medicine and is often taken for its nervous sedative, hypnotic, tonic, astringent and aphrodisiac properties (Matsuda, 2000; Winters, 2006). Ashwagandha roots are a constituent of over 200 formulations in Ayurveda, Siddha and Unani medicine, which are used in the treatment of various physiological disorders. Some of the commercially available formulations are ‘Ashwagandha Capsule’ by Sriram Herbals, ‘Ashwagandha Anti-Stress & Energy’ by Himalaya Herbal Healthcare, ‘Stresscom’ (Dabur) etc. It is an official drug mentioned in the Indian Pharmacopoeia of 1985. It has received much attention in recent years due to the presence of a large number of steroidal alkaloids and lactones known as Withanolides. This drug is known to have anti-inflammatory, antitumor, antioxidant, anticonvulsive, and immunosuppressive properties (Baldi et al., 2008). Presently, withanolides have been commercially obtained by solvent extraction of roots and leaves of the plant. Low yield from the natural source, genotypic and chemotypic variations, heterogeneity in content, long gestation period (4–5 years) between planting and harvesting, and uneconomical chemical synthesis are major constrains in industrial withanolide production. Multiple uses of the plant have necessitated its large-scale collection as raw material to the medicine industry, leading to over exploitation and making it an endangered plant species. Commonly Withania propagated commercially by the means of seeds because of the lack of natural ability for vegetative propagation (Sen and Sharma, 1991) but the seed viability is limited to one year (Rani and Grover, 1999), making the long duration seed storage futile (Farooqi and Sreeramu, 2004). Due to poor viability of stored seed, alternative procedure of propagation is essential for constant supply for industrial level. In vitro technology can be used as an alternative because the advantage of tissue culture

technology lies in the production of high quality planting material on a year- round under disease-free condition anywhere irrespective of the season and weather. In addition micropropagation helps to obtain a high degree of crop uniformity to overcome complex dormancy problem, seedling growth, low seed viability and difficulty in procedures dependent upon micropropagation. In vitro culture methods through axillary bud multiplication using nodal segment have proved successful for quick propagation of number of medicinally important species such as Santolina canescens (Casado et al,. 2002), Bupleurum fruticosum (Fraternale et al., 2002), Rauvolfia tetraphylla (Faisal et al., 2005). In vitro propagation via nodal segments are also reported from India (Rao et al., 2012; Fatima & Anis, 2011). But not same genotype and chemotype of Withania which is important aspect for withanolide accumulation as well as their rate of multiplication. In modern conservation biotechnology, elite and over exploited plant germplasm conservation by in vitro method has been done using slow growth procedures or cryopreservation (Withers, 1986; Tripathi and Tripathi, 2003). Slow growth is usually achieved by reducing the culture temperature, by modifying culture media with supplements of osmotic agents, growth inhibitors, or by removing growth promoters (Dodds and Roberts, 1995). The objective of the present study based on (i) to develop a simple efficient protocol via node culture for large scale uniform plant production and (ii) to develop a simple in vitro conservation protocol for future exploitation. MATERIALS AND METHODS Plant material Seeds of Ashwagandha (Withania somnifera) were collected from ripe fruits of elite germplasm. For in vitro seed germination, surface sterilization was done by treating the seeds with 4% (v/v) Teepol (Rickett & Colemann, India Ltd., Kolkata, India) detergent

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solution for 15 min followed by freshly prepared HgCl2 (0.1%) treatment for 15 min on continuous shaking under laminar hood. Finally, the seeds were washed four or five times with sterile distilled water and the disinfected seeds (five per test tube) were inoculated on semi-solid gel consist of water and 0.6% agar (Merck, India) only without any nutrient media for germination purpose. Epicotyledonary nodes obtained from 30d old seedlings and were placed vertically on to culture tubes containing 20 ml semi-solid medium per tube. Media and culture conditions The nutrient basal medium used in all the experiments consisted of MS salts and vitamins. The basal medium was supplemented with different cytokinins- BA, Kn, 2iP or auxins- IAA, IBA, NAA at varying concentrations and 3% (w/v) sucrose (Merck, India) as a sole carbon source. All the salts used were of analytical grade. The medium was solidified with 0.7% (w/v) bacteriological grade agar (Merck, India) and the pH of the medium was adjusted to 5.8 before autoclaving at 121°C for 15 min. All the culture vials were placed in plant growth room at 25 ± 2°C under 16/8 h (light/ dark) photoperiod with a light intensity of 50 µmol m-2 s-1 supplied by cool white fluorescent lamps (2 tubes 40 W, Philips, India) and with 60– 65% relative humidity. Multiplication of shoots Single nodal explants containing axillary bud derived from primary in vitro regenerated shoots were cultured in MS medium augmented with different concentrations of cytokinins alone or in combinations for further multiplication. All cultures were transferred to fresh medium after 4 weeks interval. The mean number of shoots and their lengths were evaluated after 6 weeks of inoculation. Shoot elongation Proliferated multiple shoots with an average height of 1.0 cm were carefully excised and transferred to shoot elongation medium containing different concentrations of

Gibberellic acid (GA3: 0.1, 0.2, 0.5, 0.8, and 1.0 mgl-1). The cultures were maintained at 25 ± 2°C and 16 h photoperiod with light intensity of 30 µmol m-2s-1. After 63 weeks, shoots longer than 2.5–3.0 cm were selected and transferred to rooting medium. Rooting, acclimatization and transfer of plantlets to soil Elongated shoots were transferred to full strength, half strength of MS media and MS medium containing IAA, IBA and NAA (0.2, 0.5, 1.0, 1.5, 2.0 mgl-1). The cultures were maintained as described for shoot elongation. After 2 weeks the rooted plants were transplanted to paper cups containing soilrite. The plantlets were initially covered with a transparent plastic bag (Fig.5) to maintain high humidity and were placed in polygreen house. The plants were watered daily with Hoagland’s nutrient solution. After 4 weeks the plants were transplanted to earthen pots and were grown in garden under full sun for developing into mature plants. In vitro conservation through slow- growth treatments To investigate the slow- growth treatments, the effect of osmotic agents and temperature on the survival and re-growth of the in vitro cultures of Withania somnifera, MS media were supplemented with mannitol (1–3% w/v), sorbitol (1–3% w/v) with 3% sucrose (w/v) and 0.7% (w/v) agar. Shoot tips and nodes were dissected from aseptically grown cultures and inoculated onto the slow growing media in order to increase subcultural intervals. Cultures were maintained at 4°C to 18°C for 8 months into growth chambers under a 16 h photoperiod with fluorescent light at 25°C. Re-growth and establishment of plantlets Cultures were monitored during and after storage for survival and subsequently transferring on to the shoot multiplication medium (MS media with PGR) under culture room conditions at 25°C. The number of new buds and shoots induced on multiplication media was counted 30 days after transfer. Data

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collected reflect the rate of plant conservation from storage buds to proliferating buds, shoots and plantlets. Proliferated shoots were rooted on MS medium containing IBA. RESULTS AND DISCUSSION Shoot Multiplication In order to establish an efficient in vitro micropropagation system for Withania somnifera from nodal explants, seedling nodal segments were incubated on MS (Murashige and Skoog, 1962) solid medium supplemented with varying levels of either BA or Kn alone or in combination (Fig.1). Nodal segments cultured on MS basal medium without growth regulator did not show any response up to six weeks but 1–2 axillary shoots developed after 10 weeks of culture without any subculture. However, on MS basal medium supplemented with various concentration of cytokinin (BA, 2iP or Kn) alone or their combination swelled in their size after 2 weeks of culture and differentiated axillary shoots in another 4 weeks (Table 1). Among the single cytokinin, irrespective of the concentration, BA supplemented in MS medium induced multiple axillary shooting and a maximum of 16 shoots per explants were produced at 2.0 mgl-1 concentration while 1.0 mgl-1 Kn induced 10 shoots (Table-1). The positive effect of BA on bud proliferation and multiple shoot formation has been reported for several medicinal and aromatic plant species such as Chlorophytum borivilianum (Purohit et al., 1994), Eclipta alba (Franca et al., 1995), Ocimum sp. (Patnaik and Chand, 1996). By increasing the concentration of BA beyond the optimal level, a gradual reduction in the number of shoots was also reported for several medicinal plants including Withania somnifera

(Sen and Sharma, 1991), Tylophora indica (Faisal and Anis, 2003). Further BA and Kn combination with them was more effective for shoot multiplication than BA or Kn individual treatment. A higher degree of shoot bud differentiation (21.0 ± 0.1) was observed at 1.0 mgl-1 concentrations of BA in combination with 1.0 mgl-1 Kn. (Table 1) (Fig.2). Shoot elongation For shoot elongation GA3 were treated of in vitro produced shoots. GA3 at 0.5 mgl-1 induced the maximum shoot length of 7.6 ± 2.5 cm after 3 weeks of culture. However, increase in the concentration of GA3 (0.8 and 1.0 mgl-1) not trigger enough for further shoot elongation (Table- 2). Gibberrellic acid (GA3) appears to be effective in shoot elongation in cucumber (Selvaraj et al., 2006) and also in Withania somnifera (Sivanesan, 2007). Slow- growth storage Over 8 month’s culture growth period, the percentage increase in shoot length showed that Withania somnifera cultures in control grew healthy and vigorously up to 55 days after subculture. At low temperature regime (4°C), culture showed poor performance as the shoots degenerated after 45 days and culture did not survive as in case of control. In moderate temperature regime (20ºC) the cultures grew healthy with reduced growth in comparisons to control. This experiment suggests that culture growth could be reduced at 6°C, but storage period could not be increased to maintain healthy cultures. This aspect needs further experimentation to prolong the subculture period. It also suggests that low temperature (4ºC) is not suitable for W. somnifera in vitro storage.

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Table: 1 Effect of Cytokinin on shoot multiplication from nodal explants of Withania somnifera (L.) Dunal after 6 weeks. Treatments (mg/l) MS (Full strength) 0.5 1.0 2.0 3.0 5.0 0.5 1.0 2.0 3.0 5.0 0.5 1.0 2.0 3.0 5.0 0.5 + 0.5 1.0 + 1.0 2.0 + 1.0 3.0+ 1.0 5.0+ 1.0

Mean No. of shoots/ nodal explant MS + BAP 11.1 ± 0.1 14.2 ± 0.2 16.3 ± 0.2 12.2 ± 0.1 12.3 ± 0.2 MS + Kn 6.2 ± 0.2 10.2 ± 0.2 9.1 ± 0.2 7.9 ± 0.1 6.8 ± 0.1 MS + 2ip 3.7 ± 0.3 6.4 ± 0.6 5.6 ± 0.3 3.4 ± 0.3 1.3 ± 0.3 MS + BAP + Kn 12.0 ± 0.1 21.0 ± 0.1 18.3 ± 0.2 14.8 ± 0.2 14.2 ± 0.2

Mean shoot length (cm) 2.3 ± 0.2 2.8 ± 0.1 3.1 ± 0.1 2.9 ± 0.2 2.8 ± 0.1 2.3 ± 0.2 2.4 ± 0.2 2.2 ± 0.2 2.1 ± 0.2 2.1 ± 0.1 2.1 ± 0.1 2.8 ± 0.03 2.33 ± 0.1 1.3 ± 0.06 1.2 ± 0.04 3.5 ± 0.2 3.9 ± 0.2 4.2 ± 0.2 3.8 ± 0.2 3.7 ± 0.2

(Each value represents the mean ± SD of 10 replicates and each experiment was repeated thrice)

Table 2: Effect of GA3 on shoot elongation from regenerated shoots cultured on MS medium supplemented with GA3 (mgl-1) after 3 weeks. GA3 (mgl-1)

Shoot elongation response (%)

Mean shoot length (cm)

0.1

77.6 ± 2.5

3.0 ± 2.6

0.2

82.2 ± 2.3

5.0 ± 0.5

0.5

99.6 ± 3.1

7.6 ± 2.5

0.8

85.6 ± 2.6

7.3 ± 1.5

1.0

64.6 ± 2.5

6.3 ± 2.7

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Table-3. Effect of different concentration of IBA on rooting of in vitro regenerated shoots of Withania somnifera (L.) Dunal. After 3 weeks. Treatments (mgl-1) No. of roots per shoots Rooting (%) 3.4 ± 0.3 24.5 MS 2.1 ± 0.1 12.7 Half-strength MS MS + IBA (0.2) MS + IBA (0.5) MS + IBA (1.0) MS + IBA (1.5) MS + IBA (2.0)

5.8 ± 0.2 7.6 ± 0.1 10.4 ± 0.1 9.3 ± 0.2 9.2 ± 0.2

62.3 66.7 86. 8 78.7 72.3

MS + NAA (0.2) MS + NAA (0.5) MS + NAA (1.0) MS + NAA (1.5) MS + NAA (2.0)

2.02 ± 0.06 2.46 ± 0.2 4.46 ± 0.5 5.82 ± 0.14 4.02 ± 0.9

38.5 40.4 51.6 60.5 56.4

MS + IAA (0.2) MS + IAA (0.5) MS + IAA (1.0) MS + IAA (1.5) MS + IAA (2.0)

2.77 ± 0.15 5.37 ± 0.6 8.48 ± 0.06 6.1 ± 2.1 5.7 ± 1.5

48.5 62.3 73.5 53.0 45.6

(Each value represents the mean ± SD of 10 replicates and each experiment was repeated at least thrice)

The growth suppression approach using osmotic agents was attempted in this study and proved to be very useful. The influences of various osmotic agents on this species showed different results. The growth of shoot cultured in medium supplemented with 3% sucrose was controlled for our experiment. The results showed that the addition of sorbitol (w/v) and mannitol (w/v) in MS media, at different concentrations, was more effective for in vitro storage of this important medicinal plant than the storage at low temperature (4°C). The addition of osmotic agents 2% sorbitol (w/v) and 2% mannitol (w/v), to each of the media has increased survival rate 85%. A declination in survival rate and re-growth occurred when the cultures were stored also at higher concentrations of osmotic agents i.e., with 3% mannitol (w/v) and with 3% sorbitol each and with a combination of 2% sorbitol (w/v) and 2% mannitol (w/v). Our results also showed that 20°C and 16 h photoperiod were better than 4°C during slow-growth storage condition.

The shoots survived after slow-growth storage had longer shoot height than those not maintained in slow-growth condition. In case of 4°C temperature, the leaves of some plants were curled and withered. Re-growth and establishment of plantlets After 8 months, these shoots were transferred onto fresh MS medium supplemented with different concentration of BAP (0.5 to 5.0 mg/l), IAA (0.5 to 5.0 mg/l) and IBA (0.5 to 5.0 mg/l) for in vitro multiplication and in vitro rooting and cultured for 6 weeks. Growth suppression had positively reduced the labor during culture maintenance in the tissue culture laboratory and also promoted uniformity of growth among the converted plantlets. No signs of shoot or root growth was noticed during the 8 months of storage. Adding sucrose to the media has prevented dehydration in storage but did not improve shelf-life of germplasm, while frequency of plantlet

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conservation was higher on shoot tips stored on 2% (w/v) sucrose/agar support with roots breaking during low temperature (4ºC) storage condition. All the cultures in storage condition were able to form roots during re-growth and successfully acclimatized in soil rite. After low temperature storage, plantlets improved their survival during acclimatization and more vigorous in field plantings was observed. Similar reports have documented post–storage beneficial effect in apricot by Koubouris and Vasilakakis (2006), Lata et al. (2010), Kanchanapoom and Promsorn (2012). Our studies provided an effective protocol for storage of medicinal plants under slow growth conditions. Germplasm can be stored effectively for 8 month without subcultures, alleviating maintenance labor in the laboratory. Rooting and acclimatization Induction of rooting is an important step for in vitro plant propagation. Microshoots (3-4 cm) excised from cytokinin containing medium were individually transferred to root induction medium both basal medium and medim containing various concentrations of auxins. The in vitro-regenerated shoot induced roots when transferred to full and half-strength MS medium. Full-strength growth regulator-free MS medium was found superior to halfstrength MS medium for root development (Table-3). The incidence of root formation in auxin-free medium may be due to the presence of endogenous auxin in in vitro shootlets (Minocha, 1987). The presence of IBA (0.2, 0.5, 1.0, 1.5 and 2.0 mgl-1) facilitated better rhizogenesis (Table 3). The maximum frequency of root formation was achieved on full-strength MS medium supplemented with

1.0 mgl-1 IBA (Fig. 3&4). The success of IBA for efficient root induction is also reported in Swaisona formosa (Jusaitis, 1997), Cunila galoides (Fracro and Echeverrigaray, 2001), and Rauvolfia tetraphylla (Faisal et al., 2005). Acclimatization is the final step in a successful micropropagation system. Successful establishment of in vitro regenerated plantlets in field conditions requires great care (Hoagland and Arnon, 1950). During this stage plants have to adapt to the new environment of greenhouse or field. The plantlets usually need some weeks of acclimatization in shade with the gradual lowering of air humidity (Pospíšilová et al., 1998). After 8–10 months old all field growing regenerated plants (R0) produce flower as well as fertile seeds (Fig.-6). No apparent variation was detected between in vitro generated clones and they were as good as their mother plant. The morphological efficiency was not hampered even after longterm sustained culture of 24 months. Similar observation was noted in Aloe (Gantait et al., 2010). CONCLUSION To conclude, the successful tissue culture of W. somnifera provides a system that is efficient for propagation of this valuable medicinal plant en masse. It could support conservation and ultimately enable to keep pace with commercial needs and keep off the species from indiscriminate exploitation from the natural resources. The described protocol could be worked as a useful tool for adapting in vitro culture strategies to increase the biomass of tissue production.

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Conflict of Interest: None Declared

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Research article A COMPARATIVE PHARMACOGNOSTICAL EVALUATION OF RAW AND TRADITIONALLY SHODHITA VACHA (ACORUS CALAMUS LINN.) RHIZOMES Bhat Savitha D1*, Ashok B K2, Harisha C R3, Acharya Rabinarayan4, ShuklaV J5 1

Lecturer, Department of Dravyaguna, Muniyal Institute of Ayurveda Medical Sciences, Manipal, Karnataka, India 2 Research Assistant, Pharmacology laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar, India 3 Head, Pharmacognosy laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India 4 Associate professor, Department of Dravyaguna, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India 5 Head, Pharmaceutical chemistry lab, IPGT&RA, Gujarat Ayurved University, Jamnagar, India *Corresponding Author: E-Mail: anudivas@yahoo.co.in; Mobile: +919483128930 Received: 28/08/2012; Revised: 19/09/2012; Accepted: 25/09/2012

ABSTRACT Acorus calamus Linn. (Vacha) is a highly valued medicinal plant not only in traditional system, but also in western medicine. Though it is a non poisonous drug, Shodhana (purificatory procedure) has been indicated in Ayurveda prior to its use. Even in folklore practice, the rhizomes are processed in media like milk (Ksheera) and whey of curd (Mastu) prior to medicinal use. In the present study, pharmacognostical and preliminary phytochemical analysis of A. calamus was carried out in comparison with Ksheera and Mastu processed A. calamus rhizomes in order to know if there are any gross differences occurring after Shodhana. Few changes in oil globules were observed in the transverse sections of Shodhita (processed) samples in comparison to non-processed. Further thin layer chromatography revealed that Shodhana procedure did not affect the β-asarone qualitatively. Various parameters like macro, micro and physiochemical standards of this study will be helpful in authenticating Shodhita Vacha and will also serve as reference material for further scientific investigations. KEY

WORDS:

Vacha,

Acorus

calamus,

Shodhana,

β-asarone,

Mastu,

Ksheera.

Cite this article: Bhat Savitha D, Ashok B K, Harisha C R, Acharya Rabinarayan, ShuklaV J (2012), A COMPARATIVE PHARMACOGNOSTICAL EVALUATION OF RAW AND TRADITIONALLY SHODHITA VACHA (ACORUS CALAMUS LINN.) RHIZOMES, Global J Res. Med. Plants & Indigen. Med., Volume 1(10), 539–551 Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 10 | October 2012 | 539–550

INTRODUCTION Vacha (Acorus calamus Linn.) is a semiaquatic, perennial and aromatic herb which is found ascending up to an altitude of 2,200 m in the Himalayas. It is commonly known as sweet flag and is a valued medicinal plant in Ayurveda (Gupta, 2004) and as well as other traditional systems (Sharma, 2000; Savitha Bhat et al., 2011). In the classical literature the morphological characteristics of Vacha are described by the synonyms like Ugragandha, Golomi, Shadgrantha, Shataparvika etc., owing to its aromatic and rhizomatous nature (Shastry, 2001). The rhizome is the main useful part and is the genuine source of ‘Vacha’ in commerce [Figure-1] (Khare, 2007). Major chemical constituents of the rhizome are Asarone, Calamene, Calamenenol, Calameone, α – pinene, Camphene, Eugenol etc., among which β-asarone is the most researched one (Ernest Guenther, 1976). Acorus has shown significant effects on CNS and other systems which proves its utility in diseases like Apasmara (epilepsy), Unmada (schizophrenia), Vibandha (constipation), Adhmana (Tympanitis), Shoola (Colic), Karnasrava (otitis media) etc (Yende et al., 2008; Sharma PV, 2004). In Ayurveda, Shodhana has been advocated not only for poisonous drugs but also for non poisonous drugs like Haridra (Curcuma longa Linn.), Hingu (Ferula narthex Boiss.), Chitraka (Plumbago zeylanica Linn.) and Lashuna (Allium sativum Linn.) using different media like Gomutra (Cow’s urine), Godugdha (Cow’s milk) etc., as per the nature of the drugs (Ramnarayan Vaidya, 1982; Ramachandra Reddy, 2005). It seems that Shodhana was carried out with an intension of not only purifying the drugs but also to alter their pharmacological effects (Kamble et al., 2008). This was also evidenced in one of our pharmacological study in which Shodhita Vacha samples showed better anti-convulsant activity than non-purified one (Savitha Bhat et al., 2012). Similarly, even though Vacha is not considered as a poisonous drug, classical texts

like Chakradatta advises Shodhana of Vacha using Gomutra, Mundi kwatha (Decoction of Sphaeranthus indicus Linn.), Panchapallava kwatha (Decoction of a group of five tender leaves) and Gandhodaka (Decoction of group of aromatic herbs) (Ramanath Dwivedi, 2005). Ayurvedic pharmacopeia of India also recommends the use of Vacha after Shodhana (Anonymous, 2007).Other than the classical method; there are other folklore methods which are simple and economically viable. One of the methods practised in certain parts of Kerala is soaking rhizomes of Vacha in Dadhi Mastu (whey) overnight. Another method seen practised in certain coastal regions of Karnataka is soaking of Vacha in Goksheera (cow’s milk) overnight. Many research works have been carried out on pharmacognosy of Acorus calamus (Datta, 1950; Dipali Dey et al., 2005; Narayana Aiyar, 1957) but no work has been reported on the pharmacognostical aspects of Shodhita Vacha (processed Vacha). Hence this study intends to explore the pharmacognostical differences present in raw (unpurified) Vacha and Ksheera and Mastu Shodhita Vacha in order to lay certain standards which can serve as a future reference material. MATERIALS AND METHODS: Collection of plant material: Fresh rhizomes of Acorus calamus were collected from the forest areas of Yelagiri Hills, Tamil Nadu, India in matured condition, in the month of November as per Ayurvedic criteria for collecting rhizomes (Indradeo Tripathi, 2003). After proper identification and authentication by Dr. Harisha C. R., Head, Pharmacognosy Laboratory, IPGT & RA, Jamnagar, the voucher specimen was deposited in the institute’s Pharmacognosy department vide voucher specimen No. PhM. 6002. The leaves attached to the rhizomes were cut and separated. It was then rubbed by a gunny cloth to remove the roots and old leaf scars. Later the rhizomes were washed thoroughly in water to remove the soil adhered to it and dried in partial shade for 10 days.

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 10 | October 2012 | 539–550

Fig – 1: Photograph showing plant profile of Vacha

Method of Shodhana: Mature and long rhizomes of Acorus were selected, cut into pieces of one inch length and equally partitioned into three groups of 200 g each. The first group consisted of raw Vacha and was marked as sample RV. The second group was soaked in 2 litres of pasteurised Goksheera overnight (9 hours), washed with warm water (47°C) the next day and dried in sunlight for 6 days and marked as sample KV. The third group was soaked in 2 litres of Dadhi Mastu overnight (9hours), later washed in warm water (50°C) and dried in sunlight for 6 days. It was marked as sample MV. Macroscopic and microscopic evaluation: Macroscopical evaluation of raw Vacha rhizomes in both fresh as well as dry state in comparison with Shodhita samples was carried out as per standard procedure (Evans WC, 2002). Thin free hand transverse sections of dry rhizomes of raw and Shodhita samples were taken to evaluate both microscopical characteristics and histochemical reactions (Khandelwal KR, 2000). Further the samples were coarsely powdered with the help of a pulveriser, passed through sieve no 60 and were used for powder microscopy (Kokate,

Fig – 2: Rhizome of Vacha showing numerous round root scars on the ventral surface

2003). Both stained and unstained specimens were used to identify and confirm the microscopic structures (Anonymous, 2008a). Photomicrographs were taken using Carl Zeiss binocular microscope. Phytochemical evaluation: Physicochemical analysis, namely loss on drying at 105°C, ash value, acid insoluble ash, water soluble extractive value, alcohol soluble extractive value, pH value as well as qualitative test for various functional groups like alkaloids, glycosides etc were also carried out for all the three samples. Heavy metal analysis and pesticide residue analysis was done only for raw Vacha to check the contamination (Anonymous, 2008b). Histochemical tests were carried out by treating the transverse sections of all the samples using specific reagent to detect the colour changes and localization of chemicals. Fluorescence analysis was carried out with the powder of the rhizome sieved through 60 mesh and treated with various reagents. The supernatants were examined under day light and ultraviolet light (234 nm and 366 nm) (Krishnamurthy, 1988; Anonymous, 1998; Maluventhan, 2010).

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Thin layer chromatography: Methanol extracts of RV, KV and MV were subjected to thin layer chromatography and compared with β-asarone (1 mg dissolved in 2 ml of methanol) standard marker compound. Silica gel G plate of thickness 0.3 mm activated at 105°C for 30 minutes was used as stationary phase and Toluene:Ethyl acetate (9:1) as mobile phase (Anonymous, 2007). 10 µl of test solution and 5 µl of standard solution were applied on the TLC plate and the plates were developed in the solvent phase till the solvent front run was 9.6 cm. They were visualized under UV light at 254 nm and 366 nm, also after derivatization with Vanillin – Sulphuric acid reagent followed by heating for ten minutes at 105°C. RESULTS AND DISCUSSION: Macroscopic characters: Raw Vacha (RV): The fresh rhizome was woody, horizontal, creeping partially underground, varying in length from 25 cm–30 cm, vertically slightly compressed from 1.8–2.5 cm in diameter. It was rarely straight, much branched with thick long adventitious roots arising from the lower side. The dried rhizome was brownish in colour, tortuous, branched, sub cylindrical, 1.2– 1.8 cm in thickness, having distinct nodes and internodes. The nodes were broad with dry, fibrous, persistent, triangular, transverse leaf scars often attached to the upper side. The internodes were ridged, 7–10 mm in diameter. The under surface of the rhizomes are provided with irregularly arranged, slightly elevated Fig – 3: Transverse section of raw Vacha (RV) (20X) En. – Endodermis; Ph. – Phloem; Xy. – Xylem; OC. – Oil cell; OG. – Oil globule; S. – Starch grain

round root scars and short fragment of roots [Figure 2]. Fracture short, granular and porous, emitted strong aromatic odour and had a pungent taste. The fracture surface exhibited cream coloured interior with a central and peripheral region marked by a faint endodermal line. Ksheera Shodhita Vacha (KV): KV was pale brownish in colour, with an average diameter of 1.8–2.5 cm, had a pleasing aromatic odour with a reduction in its pungent taste. Fracture short, granular, porous and the fractured surface was whitish cream in colour. No gross changes were observed in other macroscopical characteristics. Mastu Shodhita Vacha (MV): MV was also pale brown in colour, pungent and slightly sour in taste, with an average diameter of 2–2.5cm and had mixed odour of both raw Vacha and sour whey. Fracture short, granular and porous and the exposed surface exhibited a dull white interior. Other macroscopical features were similar to raw Vacha. Microscopic features: Important microscopic characteristics observed in the transverse sections of RV, KV, and MV rhizomes [Figures 3, 4 & 5] have been given in table - 1. The few changes observed in the structures, taste and oil globules may be due to the method of soaking in different media where the rhizomes tend to acquire the properties of the media used. Fig – 4: Transverse section of Ksheera Shodhita Vacha (KV) (20X)Ph. – Phloem; Xy. – Xylem; OC. – Oil cell; Ct Vb. – Cortical vascular bundle; St Vb. – Stelar Vascular bundle

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Table â&#x20AC;&#x201C; 1: Important microscopic characteristics observed in the rhizomes of both raw and Shodhita Vacha Microscopic Sample RV Sample KV Sample MV characters Transverse Oval to round More round More round section Outline/ Faintly wavy Faintly wavy Faintly wavy margin The rhizome is differentiated into cortical region and stelar The differentiation of The size of cortex and region. Cortical region areas and the extent of stellar region is similar Region constitutes around 1/3rd of the cortex and stellar region to raw Vacha area whereas stellar region is same as raw Vacha constitutes around 2/3rd The periphery of the cortex consists of a single layered dark brown corky tissue. The cork layer is followed by a single layered epidermis having Brown coloured, single Pale brown, single radially elongated cells with layered cork tissue layered coloured cork thickened walls. Under the forms the outermost tissue outlines the epidermis there are 2 to 3 layers part of the cortex cortex followed by the of closely arranged followed by epidermal epidermis having oval collenchymatous cells forming cells which are more or to round cells. The the hypodermis. It is followed less round in shape. The collenchymatous cells by spherical to oblong collenchymatous cells are more compact than Cork, Cortex moderately thick walled are same as in RV. The RV. The parenchyma and parenchymatous cells which parenchyma cells are cells are more Stele covers the rest of the cortex. more compact, round spherical with reduced These cells are arranged to and are arranged in a intercellular spaces. form a network leaving large similar manner in The endodermis forms intercellular spaces. The lower cortex as well as in the lower boundary of boundary of the cortex is stellar region. The the cortex and other characterised by a distinct endodermis is similar to features are same as endodermis which separates it raw Vacha raw Vacha from the stellar region. The stellar region also consists of parenchymatous cells similar to cortex. The rhizomes of KV The endodermal cells are barrel also exhibit similar There is no visible shaped, thin walled, arranged in structure and difference in the Endodermis a single layer and shows arrangement of features of endodermis casparian thickening endodermal cells as in as compared to RV. sample RV The vascular bundles are The vascular bundles in The structure and of sheathed, collateral having cortex as well as stellar the vascular bundles Vascular large air spaces and found region are similar to RV both in the cortex and bundles scattered in the cortex. The in the architecture and stele is similar to RV.

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Oil cells and globules

Oleoresin content

Starch grains

vessels are fairly large and have arrangement, while the spiral and annular, scalariform air spaces inside the thickening. The fibres are very bundles are few, thin walled and pitted. comparatively smaller. Stele also consists of vascular There are few pale bundles in large numbers yellow and white especially arranged closely near coloured oil globules the endodermis. They form seen inside both the almost complete rings under the vascular bundle. epidermis and also seen scattered throughout the ground tissue. They are mostly leptocentric (amphivasal) but irregular types of bundles are also found to occur. Few yellow to brown oil globules are also seen scattered in both cortical and stellar vascular bundle. Root trace bundles are also seen occasionally. The oil cells are globose / spheroidal found amongst the parenchyma cells devoid of There are no gross starch grains. They are slightly changes seen in the larger and thin walled than the shape of the oil cells in surrounding cells. They often comparison to RV. The contain yellowish brown oil. oil cells contain pale Many broken oil cells are also yellow oil which observed with spilled out oil appeared crimson red droplets. The oil droplets upon staining with appear crimson red and the Sudan red III and walls of the oil cell appear light safranin pink upon staining with Sudan red III and safranin Dark brown coloured Oleoresin deposits are found scattered Dark brown oleoresin throughout the cortex and the deposits are scattered stellar region but found more in throughout. sub epidermal region. The starch grains are abundant and often seen clustered in the parenchyma cells and sometimes arranged in the form There are no changes in of a string of beads. They are the shape, colour or mostly round with occasionally nature of the starch oval shaped, simple, single or grain in comparison to in aggregation. The grains are RV translucent white in colour, highly refractive and blackish blue upon iodine staining.

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The inner air spaces are shrunken while pale yellow coloured oil globules are rarely seen inside the vascular bundle

Most of the oil cells are found ruptured and the larger oil globules seem to be split into smaller ones in comparison to RV. The oil globules appear to be pale yellow in colour and turned crimson red upon staining with Sudan red III and safranin Few dark brown oleoresin patches are seen when compared to RV

There are no changes in the shape, colour or nature of the starch grain in comparison to RV

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 10 | October 2012 | 539–550 Fig – 5: Transverse section of Mastu Shodhita Vacha (MV) (20X) Ph. – Phloem; Xy. – Xylem; OC. – Oil cell; S. – Starch grain; OR. – Oleoresin

Fig – 6: TLC profile of raw and Shodhita Vacha samples under 254 nm; Track 1 - [RV]: raw Vacha; Track 2 - [KV]: Ksheera Shodhita Vacha; Track 3 - [MV]: Mastu Shodhita Vacha; Track 4 - [S]: Standard marker compound β-asarone.

Powder microscopy:

seen scattered but were comparatively less in number when compared to raw Vacha. Other characteristics observed were similar to raw Vacha.

The powder of sample RV was brown in colour with strong aromatic odour and pungent taste. Patches of parenchyma cells, ruptured spheroid oil cells, scattered pale yellow oil globules and oleoresin content were seen in addition to abundant simple, spherical to ovoid starch granules. Also lignified, scalariform and pitted vessels, fibres of fibrovascular bundles, occasional fragments of the epidermis and cork tissue and occasional hairs of the leaf scars in case of unpeeled rhizome were also observed. The powder of sample KV was pale brownish in colour, having mild aromatic odour with both pungent and astringent taste. Abundant starch grains, patches of parenchyma cells and pale yellow oil globules similar to sample RV were observed. In addition, small translucent white coloured oil globules were also seen which might be acquired by the sample from the milk used as medium. Other characteristics were same as sample RV. The powder of sample MV was also pale brownish in colour, having mixed aroma of raw Vacha and whey along with pungent and sour taste. Occasional dull brown coloured cork tissue patches, oleoresin content and oil globules were

Phytochemical evaluation: Extractive values of raw and Shodhita Vacha samples have been tabulated in table - 2. Higher percentage of loss on drying as seen in sample MV showed that Vacha after Mastu Shodhana contained components having more moisture holding capacity. The ash value of sample MV was comparatively more indicating inorganic residue remaining after incineration. There were also indications of presence of acid insoluble particles like silica in sample MV. Water soluble extractive values showed an increase in both Shodhita samples indicating the presence of polar constituents like sugars, acids, glycosides acquired by the media used in Shodhana (Yogesh Patel et al., 2010). Functional groups like carbohydrates, flavonoids, steroids, glycosides, alkaloids and tannins were present in all the three samples. Saponin content which was absent in raw Vacha was observed in Mastu and Ksheera Shodhita Vacha [Table -3]. Occasionally saponin content was detected in cow milk

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based on the type of its feed. Since the source of the milk was same for both groups, saponins might have been imbibed by the drug from the milk during the soaking process. Histochemical tests also showed the presence of starch, tannin, glycosides etc [Table – 4]. Heavy metals like mercury, lead, arsenic, cadmium and pesticides like lindane, aldrin, hexa-chlorobenzene and endosulfan were not detected indicating the safety of the drug. Fluorescence analysis of the sample powder

showed the presence of florescence compounds and specific colour variations with various reagents which are tabulated in tables-5, 6 & 7. In comparison to RV there was a slight variation in colour pattern of KV and MV, which may be due to imbibing of media components during processing. Since no reported data regarding florescence analysis of processed Acorus is available, florescence analysis of present study will serve as reference value for future studies.

Table - 2: Physicochemical parameters Parameters

RV 14.8 Loss on drying at 105°C 6.2 Ash value 0.56 Acid insoluble Ash 21.02 Water soluble extractive Methanol soluble extractive 15.54 4.23 pH

Results KV 16.12 10.57 0.48 14.69 11.49 5.19

MV 21.2 10.63 0.98 15.10 13.38 4.40

Table - 3: Phytochemical tests for various functional groups Functional groups

Tests performed

Carbohydrates Flavonoids Steroids Glycoside

Molisch’s test Shinoda test LB reagent Molisch’s test Dragendroff’s test Mayer’s reagent test Distilled water Neutral FeCl3

Alkaloids Saponin Tannin

Results RV KV MV + + + + + + + + + + + + +

− +

+ +

Table - 4: Histochemical tests Test

Reagent

Colour observed

RV KV MV Iodine Dark blue Dark blue Dark blue Starch Ferric chloride Brown Brown Yellow Tannin Conc. H2SO4 Light yellow Light yellow Light yellow Saponin Sudan III Crimson red Crimson red Pink Fat 20%aq NAOH Yellow Yellow Light yellow Sugar Orange Orange Orange Alkaloids Dragendroff’s reagent

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Table - 5: Fluorescence analysis of rhizome powders of different Acorus samples in daylight Acorus samples Sl. Treatment category no RV KV MV Powder + 1N NaOH Fossil Milk toffee Golden aura 1. Peach rose Peach melba Butter scotch 2. Powder + 1N NaOH (alcohol) Powder + 1N Hcl Pale Dawn Yellow iris Candle wick 3. Powder + 1:1 H2SO4 Hip purple Earth song Olive grove 4. Powder + 1:1 HNO3 Wild yellow Fairy glitter Yellow scoop 5. Powder + Acetone Cool grey Mellow orange Sky mimic 6. Powder + Alcohol (ethanol) Cool grey Blue harmony Tear drop 7. Powder + Benzene Cool grey Camphor Camphor 8. Powder + Chloroform Cool grey Water Water 9. Powder + Ammonia Majestic purple Spice tree Mahogany 10. NOTE: The colour mentioned in the table are based on the “Asian paints” colour spectra, Asian paints limited, Mumbai (www. asianpaints.com)

Table - 6: Fluorescence analysis of rhizome powders of different Acorus samples in Short UV (254nm) Acorus samples Sl. Treatment category no RV KV MV Powder + 1N NaOH Divine pink First blush First blush 1. Green wisp Firefly flicker 2. Powder + 1N NaOH (alcohol) Misty meadow Powder + 1N Hcl Sky pink Pale ivory Wheat spring 3. Powder + 1:1 H2SO4 Passion fruit Majestic purple Hip purple 4. Powder + 1:1 HNO3 Soft breeze Peach organza Touch of paprika 5. Powder + Acetone Twilight sky Sassy violet Frosted rose 6. Powder + Alcohol (ethanol) Twilight sky Washout Twilight sky 7. Powder + Benzene Twilight sky Twilight sky Washout 8. Powder + Chloroform Twilight sky Pale blush Pink seduction 9. Powder + Ammonia Hip purple Dusky beauty Black grape 10.

Table - 7: Fluorescence analysis of rhizome powders of different Acorus samples in Long UV (366nm) Acorus samples Sl. Treatment category no RV KV MV Powder + 1N NaOH Soft focus Evening moon Soft honey 1. Sand bed Sunny sands 2. Powder + 1N NaOH (alcohol) Burst of spring Powder + 1N Hcl Ivory coast Mist Blank canvas 3. Powder + 1:1 H2SO4 Dynamic Lakeside Blue lake 4. Powder + 1:1 HNO3 Malabar hills Deep sea Blue weed 5. Powder + Acetone Angel harp Peach blossom Mellow orange 6. Powder + Alcohol (ethanol) Blank canvas Crescent Crescent 7. Powder + Benzene Soft whisper Fringe green Meadow mist 8. Powder + Chloroform Soft breeze Soft breeze Touch of fuschia 9. Powder + Ammonia Red wood Berry bloom Berry bloom 10.

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 10 | October 2012 | 539–550

Shodhana procedure did not affect the βasarone qualitatively as revealed by thin layer chromatography in which all the three samples showed similar spots with Rf value 0.44 which was the only spot obtained from TLC of marker β-asarone. Under short UV, both MV and KV showed extra spots with different Rf values [Figure – 6], this may be due to some active principle acquired by the sample during Shodhana. Similarly under long UV, sample

MV showed one spot and KV showed two spots lesser than that of RV [Figure- 7], the reason for this might be movement of some active principles from the drug into media during Shodhana [Table 8]. The derivatization with vanillin-sulphuric acid exhibited same number of spots in all the three samples indicating that the steroidal compounds were not altered by Shodhana procedure [Figure- 8].

Table - 8: TLC analysis of methanolic extract of different Acorus samples Conditions RV Short UV (254 nm) Long UV (366 nm)

0.05, 0.19, 0.44 [3]*

0.05, 0.10, 0.14, 0.22, 0.27, 0.31, 0.41, 0.46 [8] 0.05, 0.1, 0.25, 0.44, Vanillin 0.52 [5] sulphuric reagent * Parenthesis shows total number of spots Fig – 7: TLC profile of raw and Shodhita Vacha Vacha samples under 366 nm

Rf values KV 0.05, 0.15, 0.20, 0.44 [4] 0.05, 0.10, 0.15, 0.19, 0.21, 0.31, 0.41 [7] 0.06, 0.12, 0.27, 0.40, 0.44 [5]

MV 0.05, 0.20, 0.23, 0.44 [4] 0.05, 0.10, 0.15, 0.20, 0.25, 0.43 [6] 0.05, 0.11, 0.26, 0.41, 0.44 [5]

βasarone 0.44 -

0.44

Fig – 8: TLC profile of raw and Shodhita samples after derivatization

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CONCLUSION In the present investigation, there were no marked changes in the anatomy between raw Vacha and Ksheera or Mastu Shodhita Vacha rhizomes except for a slight reduction in the number of oil globules in Mastu Shodhita

Vacha. There were only few variations in organoleptic characters which were implied to the process of Shodhana. Various parameters like macro, micro and physiochemical standards observed in this article will be helpful in authenticating Shodhita Vacha and will also serve as reference material in future research.

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Ayurvedic drugs (Kerala). Series 1, No 3. The central research institute, University of Travancore, Trivandrum. pp. 43–46. Ramachandra Reddy K (2005). Bhaishajya Kalpana Vijnanam, Chaukambha Sanskrit Bhawan, Varanasi. pp. 535– 542. Ramanath Dwivedi (2005), editor, Chakradatta of Chakrapanidatta, Chaukamba Sanskrit Samsthan Varanasi. pp. 155. Ramnarayan Vaidya (1982). Ayurveda Sara Sangraha, 12th ed. Baidyanath Ayurved Bhavan, Jhansi. pp. 237–240. Savitha Bhat, Ashok BK, Bhat DV, Rabinarayan Acharya, Shukla VJ (2011). A Comparative Phytochemical Evaluation of Wild and Cultivated Acorus calamus Linn (Vacha) with Special Reference to β-Asarone Content. Inventi Rapid Pharm Ana and Qual Assur. 2(1):1–4. Savitha D Bhat, Ashok BK, Rabinarayan Acharya, Ravishankar B (2012). Anticonvulsant activity of raw and classically processed Vacha (Acorus

Source of Support: Nil

calamus Linn.) rhizomes. AYU. Article accepted and under Issue preparation. Sharma PC, Yelne MB, Dennis TJ (2000). Database on Medicinal Plants Used in Ayurveda, volume 1. Central Council for Research in Ayurveda and Siddha, New Delhi, pp. 469–475. Sharma PV (2004). Classical uses of medicinal plants, Chaukhambha Visvabharati, Varanasi. pp. 334–336. Shastry JLN (2001). Ayurvedokta Oushadha Niruktamala, 1st ed. Chaukambha orientalia, Varanasi. pp.15. Yende SR, Harle UN, Rajgure DT, Tuse TA, Vyawahare NS (2008). Pharmacological profile of Acorus calamus: An Overview. Pharmacognosy Reviews. 2(4): 22–26. Yogesh Patel, Savitha Bhat, Ashok BK, Rabinarayan Acharya, Shukla VJ (2010). Role of Shodhana on analytical parameters of Datura Innoxia Mill and Datura metel Linn seeds. International Journal of research in Ayurveda and Pharmacy. 1(2): 249–254.

Conflict of Interest: None Declared

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Review article STUDY OF UNIDENTIFIED PLANTS FROM RASA RATNA SAMUCCHAYA Pampattiwar S P1*, Bulusu Sitaram2, Paramkusa Rao M3 1

P.G. Scholar – final Year, P.G. Dept. of Dravyaguna, T.T.D’S S.V. Ayurveda College, Tirupati, Andhra Pradesh, India 2 Professor, Dept. of Dravyaguna, T.T.D’S S.V. Ayurveda College, Tirupati, Andhra Pradesh, India 3 P.G. Dept. of Dravyaguna, T.T.D’S S.V. Ayurveda College, Tirupati, Andhra Pradesh, India *Corresponding Author: Email: vd.yash.728@gmail.com; Mobile: +91 9700307493

Received: 24/08/2012; Revised: 30/09/2012; Accepted: 02/10/2012

ABSTRACT Ayurveda as a whole utilizes many plants in clinical practice. Some plants are used also for processing, purification and calcination of minerals, metals and gems. Mainly the purpose of plants in Rasa-shastra is to obtain absorbable metallic molecules in their maximum possible nano form. Recent studies reveal that Tulasi (Ocimum sanctum Linn) , the famous sacred basil helped in the formation of nano molecules of silver. But some plants mentioned in lexicons of Rasa-shastra have not been identified properly, because Rasa-Siddhas like Nagarjuna and Rasa-Vagbhata have used some rare synonyms for these plant drugs. At every twelve miles the names of plants are changed. And this has resulted and contributed to many controversial plants. But at present, it is necessary to identify them properly, to use the plants for precise calcination and purification for the benefit of the Mankind . In the present study, Rakta Agastya, Sthula Kumbhi Phala, Vajrakanda, Nagini, Sarpakshi, Mahakali were selected from Rasa-Ratnasamucchaya written by Rasa-Vagbhata in the 13thAD. This article will discuss about the identity of such plant drugs used in Indian Metallurgical processing’s in detail. This paper may help the Rasa-shastra people in the modern days. KEYWORDS: Nagarjuna, Rasa-Vagbhata, Rasa-Ratna Samucchaya, Patangi, Rakta agastya, Vasubhallaka, Rudanti ABBREVIATIONS: RRS – Rasa Ratna Samucchaya

To Cite this article: Pampattiwar S P, Bulusu Sitaram, Paramkusa Rao M (2012), STUDY OF UNIDENTIFIED PLANTS FROM RASA RATNA SAMUCCHAYA, Global J Res. Med. Plants & Indigen. Med., Volume 1(10), 551–556

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INTRODUCTION Ayurveda as a whole utilizes many plants in clinical practice. Some plants are used also for processing, colouring (coating), purification and calcinations of minerals, metals and gems. The main purpose of utilization of plants in the field of Rasa-Shasta is to obtain quickly absorbable nano-metallic molecules to the maximum possible extent to make their bioavailability more viable. Recent studies revealed that ‘Tulasi’ (Ocimum sanctum Linn), the famous sacred basil helped in the formation of nano molecules of silver. In the lexicons of Rasa shastra, few hundred plants are used for the above said purpose. Rasa siddhas like Nagarjuna and Vagbhata have used some rare synonyms for these plant drugs. At every twelve miles the names of plants are changed. And this has resulted and contributed to many controversial plants (Vaidya Bapalal, 2010). In the present study the famous lexicon Rasa-Ratna Samucchaya, written by RasaVagbhata in 13th A.D has been considered for researching medicinal plants used in the Rasa shastra. In this treatise, nearly 200 plants are used in the first part i.e. from 1st to 11th chapters. Out of them, maximum numbers of plants are commonly found and available in our vicinity or in the nearby markets. But to our dismay, around 17 plants described in the text are of doubtful identity. They are botanically not identified well and the synonyms used are leading us to a state of confusion, which is a real handicap to the workers and students of Rasa shastra. A review has been made to work out such unidentified plants in the full length, depending upon synonyms, utility and combination to bring them into the streamline which definitely helps the enthusiastic workers in processing metals and minerals. MATERIALS AND METHODS Out of 200 plants, we have found that nearly 16 to 18 plant names are confusing, misidentified or wrongly interpreted. Therefore in this review those synonyms have

been discussed one after the other as by available literature, taking help from the commentators of other works on Rasa shastra. Wherever necessary, the opinions of commentators like Chakrapani and Dalhana along with works of modern commentators like Bapalal vaidya, Sharma P.V. and Chunekar K.C. have also been considered. Some other Nighantus (lexicon) have also mentioned plant drugs in a similar fashion as that of Rasa-Ratna-Samucchaya but in different context. Clarification is sought, after duly regarding the opinions of the contemporary works. Rakta-Agastya [(RRS - 3rd chapter 97th verse) (Rasa Vagbhata, 13th Cent. AD)] In the context of purification of Manashila (Arsenic sulphide) (3/97), leaves of red flowered variety of Agastya (Sesbania grandiflora Linn.) are advised in the form of juice along with four other drugs. Usually we come across only white flowered variety of Agastya (Sesbania grandiflora Linn.) and very rarely we can find red flowered variety. This type grows as an eco-variety in some provinces of Bengal and Western part of U.P (fig.1). It also grows well in the countries like Thailand (Pade 2009) belonging to family Fabaceae. In the purification of any mineral or metal, it is always ideal to consider red flowered variety so that the processing time can be minimized (Kulkarni, 2010). Sthula Kumbhiphala [(RRS 4th chapter 64th verse); (Rasa Vagbhata, 13th Cent. AD)] The fruits of Kumbhi which are grown to larger size are advised for utilization in Ratna dravana (liquefaction of gems). The word Kumbhi is interpreted by some commentators as fruit of Katphala i.e. Myrica nagi Thumb. belonging to family Myricaceae. (Singh Thakur Balwant, 1999) fruits of which are known as ‘Beberi’ and are red in colour which are edible. Some other physicians are using the fruits of Careya arborea Roxb. belonging to family

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Lecythidaceae, the fruits of which are not edible which contain resinous gum (Desai V. G., 2009) (fig.2). Here the prefix sthula is used which leads us to its large variety and is used in processing when it is completely ripened. Fruits of this Fig: 1 Sesbania grandiflora Linn (Rakta-agastya)

plant contain some saponins in addition to its strong acid like Carayagenol – E. (Kokate, 2008). This is quite helpful in the ‘Ratnadravana’ to break up stronger molecular attachments. So it is advisable that the fruits of Careya arborea Roxb may be used as it is easily available.

Fig: 2 Careya arborea Roxb (Sthula Kumbhiphala)

Fig. 3 Urginia indica kunth (Vajrakanda)

Vajrakanda [(RRS 2nd chapter 66th verse, 96th verse); (Rasa Vagbhata, 13th Cent. AD)]

bulbs of Urginia in this type of processing. (fig.3)

Vajrakanda juice or paste is used in process of Satvapatana (extraction of metallic content)of Vaikrant (calcium fluoride),Vimala (cubic sulphide of iron) and other uparasas (2/66, 2/96, 3/119, 11/54).

Nagini [(RRS 11th chapter 89th verse); (Rasa Vagbhata, 13th Cent. AD)]

Two species of plants are utilized by the physicians for this purpose. Some commentators have suggested ‘Vanasurana’ which is a wild variety of Corm (Kulkarni, 2010). But Dalhana in his commentary on Sushruta samhita equated it with ‘Snuhi’ (Sharma P. V., 1985). Sushruta advised use of oil prepared of its root in sinus diseases (Sharma P. V., 1985). But in southern part of India ‘Vanapalandu’ is used in place of Vanasurana’. Some commentators also suggests Vajrakanda as Vanapalandu i.e. Urginia indica kunth. belonging to family Liliaceae (Sharma P. V., 1985). This plant is known as ‘Vajjurkanda’ in Madhya Pradesh (Sharma P. V., 1985). It would be better to use

Tuber of a plant Nagini is used along with other plants in the Murcchana (swooning) of Parada (mercury). Commentators of RRS have equated this plant with ‘Lakshmana’ which seems to be incorrect, as again ‘Lakshmana’ is of controversial identity. In some parts of Maharashtra and Andhra Pradesh, the roots of Arisaema murrayi (J Graham) Hook. of the family Araceae also known as ‘Cobra Lilly’ in English and ‘Sapacha kanda’ in Marathi is used as its source. (Pade, 2009). This Nagini and ‘Nagapushpi’ mentioned by Bhavmishra appears to be one and the same. Though this plant is limited to certain geographical area it is abundantly available in the west coast. But the plant ‘Lakshmana’ has got only a long root but not a tuber. So, it is ideal to use Arisaema murrayi (J Graham) Hook. as Nagini kanda.

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Plant appears like a snake holding its hood up in an attacking position. (fig.4) Sarpakshi – [(RRS 11th chapter 53rd verse); (Rasa Vagbhata, 13th Cent. AD)] Another plant drug named ‘Sarpakshi’ is included as one among the plants or plant parts used in processing of metals and minerals. In that list nearly 45 plants are present. Many of the previous commentators like Dalhana, Arundatta could not clarify the plant and opined conflictingly. Singh Thakur Balwant, (1999) have equated this plant with Ophiorrhiza mungos Linn. or some Rouvolfia species (Singh Thakur Balwant, 1999) Morphologically some parts of this plant should resemble snake or snake with projecting eyes or tongue. But neither of the two above species is fulfilling the said features. Hence there is a need to go on for another plant which is also used in Rasa-shastra. In another context of the same work, Arisaema murrayi (J. Graham) Hook., is equated with Nagini kanda. Similarly it is ideal to consider another plant of the same genera whose projecting spathe appears like snake’s hood with a scaly appearance and two prominently growing red or white coloured oil glands on both sides looking like eyes (fig 5). Arisaema candidissimus W. W. Sm. var. alba

Fig.4 Arisaema murrayi (Nagini)

grows in Western Himalayas and is preferred in these regions. It is also known for its antipoisonous actions in local areas. Mahakali – [(RRS 10th chapter 71st verse); (Rasa Vagbhata, 13th Cent. AD)] `Mahakali is one among the seeds used for extracting oils and its utilization is indicated either individually or in combination with other oils in the processing of mercury. This plant is identified by many commentators as Krishna rajika (Black mustard seeds) which appears to be somewhat confusing. Nowhere in the synonyms of Rajika, is the word ‘Mahakali’ used. There is a tradition in Bengal and Maharashtra to utilize seeds of ‘Vishala’ i.e. Trichosanthes tricuspidata Lour. Var. tricuspidata (fig.6) to extract oil and use it in some skin diseases and also in processing of minerals. (Vaidya Bapalal, 2010) It bears red fruits when ripe, when we break open a dried fruit from inside a black smoky powder (dried embryo) comes out like a black dust. Due to this black dust it has been named as Mahakala meaning “great blackness” inside. It is known as Makali in Bengal and Makel in Maharasthra (Vaidya Bapalal, 2010).

Fig.5 A. candidissimus (Sarpakshi)

Fig.6.Trichosanthes tricuspidata Lour (Mahakali)

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Rest of the Plant drugs in Rasa Ratna Samucchaya There are some more plants which are either controversial or non-available. They are Kshir-Kakoli, Rudanti, Payasya, Patangi, Kakajangha, Grishma sundari, Vasubhallaka and Kambuki. From this list Patangi may be the extract of Caesalpinia sappan i.e. Patanga. Kshirakakoli though identified, it is very difficult to procure the plant. Payasya is likely to be a plant of either Convolvulaceae or Apocynaceae which has got nutritive or nourishing values. The word ‘Vasubhallaka’ may be Bhallataka but its identity is yet to be searched for. Grishmasundari is likely to be ‘Vasanthi’ which is Jasminum arborescens Roxb. Kambuki is another plant in processing of mercury. The name of the plant suggests that some part of it should resemble a conch. The flowers of Trichodesma indicum Lour. are pure white in colour. And its corolla look likes a curved conch which is accepted by many commentators also. This plant is commonly known as ‘Adhahpushpi’ or ‘Andhahuli’. DISCUSSION Plants constitute a valuable source in the purification and processing of metals and minerals. From among the numerous number of plants used for this purpose for centuries are still unidentified or under controversy. Rasa Ratna Samucchaya being a standard work has been considered for the study of such plants. Six plant drugs which are frequently used are discussed here. Rakta Agastya is indentified as an eco-type of Sesbania grandiflora Linn. and is used by traditional vaidyas of Andhra Pradesh, till to date. Regarding sthula-kumbhiphala, fruits of Myrica nagi Thumb. are suggested because these fruits yields sufficient quantity of juice where as the fruits of the other plant known with the name of Kumbhi , i.e. Careya arborea

Roxb. do not contain juice at all. Though many plants are equated with Vajrakanda , bulbs of urginea indica, Kunth. may be used , as it is observed that regional names of this plant are mimicking Sanskrit terminologies. The plant drug Nagini is confused with Lakshmana by commentators in its place, an endemic plant Arisaema murrayi (J. Graham) Hook., known as Nagini kanda which appears like a snake holding its hood up in an attacking position. Regarding the plant drug Sarpakshi, another variety of the same genera i.e. Arisaema candidissimus W W Sm. var. alba may be considered, based on its morphological characters suggesting the synonyms. Seed oil of the plant drug Mahakali is used in the processing of mercury. This plant is identified as Trichosanthes tricuspidata Lour. var tricuspidata. Depending on the inner structure of the fruits and traditions of the physicians of Maharashtra and West Bengal. CONCLUSION Rasa Ratna Samucchaya is traditionally accepted as a standard work in the field of ‘Rasa-shastra’. It is both a work on processing of metals and also their utilization in treatment. Some plants mentioned in this work have become unidentified and posing a problem in the field of Rasa-shastra. Therefore a literary review work has been taken up by Dept. of Dravya-Guna, TTD’s S.V. Ayurveda College, Tirupati, to clarify at least some names of unknown origin. Nearly six plants are discussed in this review and their identity is remarkably proved. Rest of the work will be taken up in the due course. ACKNOWLEDGEMENT We sincerely acknowledge our gratitude to the management of TTD (Tirumala Tirupathi Devasthanams) for the constant encouragement. In submitting this research paper, we pray to Lord Venkateswara to shower his blessings on all of us.

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REFERENCES Vaidya Bapalal, (2010), Some Controversial drugs of Indian medicine 3rd edn, Chaukhamba Orientalia, Varanasi, Pg83, 148, 320.

Sharma P.V. (1985), Dravyaguna Vigyan Vol.5, Chaukhamba Bharathi Academy, Varanasi, Pg-170, 258, 271, 291, 321, 323, 336

Sitaram Bulusu, (2006), Bhavaprakash Nighantu 1st edn, Chaukhamba Orientalia, Varanasi, Pg-170, 297, 302, 333.

Pade Shankardaji Shastri, (2009), Vanaushadhi Gunadarsha vol-1-7, Pg- 5,105, 258, 417. Dole

Kulkarni Dattatreya Ananta, (2010), Rasa Ratna Samucchaya vol-1 Chs-1-11, Meharachand Laxmandas, New Delhi, Pg- 28, 32, 57, 63, 64, 84. Tripati

Indradeva, (2009), Rasa Ratna Samucchaya, Chaukhamba Sanskrit Samsthan, Pg-16, 19, 35, 37, 49, 124, 128.

Chunekar K.C., (2009), Bhavaprakash Nighantu, Chaukhamba Bharathi Academy, Varanasi, Pg100,136,192,193, 489, 508, 543, 686, 810.

Source of Support: Nil

A Vilas, (2006), Rasashastra, Chaukhamba Sanskrit Pratisthana, Pg97

Desai V. G., (2009), Aushadhi Samgraha, Rajesh Prakashan, 2nd edn. Pg-256 Singh Thakur Balwant, (1999) Glossary of Vegetable Drugs in Brihatrayi, Chaukhambha Amar Bharathi Prakashan 2nd edn. Pg- 66, 426 Kokate

C.K., (1990), Textbook Pharmacognosy, Nirali Prakashan edn. Pg- 8.46

of 1st

Conflict of Interest: None Declared

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Review article HERBAL DRUG SWIETENIA MAHAGONI JACQ. - A REVIEW Khare Divya1*, Pradeep H R2, Kumar K K 3, Hari Venkatesh K.R4, Jyothi T5 1

PG Scholar, Department of Dravyaguna, ALN Rao Memorial Ayurvedic Medical College, Koppa, Karnataka, India 2 Professor, Department of Dravyaguna, ALN Rao Memorial Ayurvedic Medical College, Koppa 3, 4 Lecturer, Department of Dravyaguna, ALN Rao Memorial Ayurvedic Medical College, Koppa 5 Research Assistant, ALN Rao Memorial Ayurvedic Medical College, Koppa *Corresponding Author: divyakhare2008@gmail.com; Mobile: +919731068468

Received: 02/08/2012; Revised: 25/09/2012; Accepted: 05/10/2012

ABSTRACT Swietenia mahagoni Jacq. commonly known as West Indian Mahogany belongs to the family Meliaceae and is a valuable tree of commercial and ethno pharmacological importance. The present review aims to compile the scattered information regarding the morphological features, chemical constituents and medicinal importance of the plant. The different parts of S. mahagoni Jacq. (Leaves, bark, fruits) are having both ethnobotanical and medicinal significance. Biological activities of the plant are due to the abundance of phenolic compounds including different terpenoids and limonoids. The dire need for such a review arises as the plant is included in the list of endangered species due to its high exploitation for timber utilization. Key words: Swietenia mahagoni, morphological features, chemical constituents, ethnobotanical, phenolic compounds, terpenoids, limonoids, endangered species.

Cite this article: Khare Divya1, Pradeep H R2, Kumar K K 3, Hari Venkatesh K R4, Jyothi T5 (2012), HERBAL DRUG SWIETENIA MAHAGONI JACQ. - A REVIEW, Global J Res. Med. Plants & Indigen. Med., Volume 1(10); 557â&#x20AC;&#x201C;567

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INTRODUCTION: Swietenia mahagoni Jacq. (Meliaceae) is a large, deciduous, and economically important timber tree native to the West Indies (Ref) and is commonly known as “Mahogany”. This tree is mainly cultivated at tropical zones, such as India, Malaysia, and Southern China. It is a valuable species closely related to the African genus Khaya and the source of one of the most popular traditional medicines in Africa (Sahgal G. et al. 2009) History (George Watt. 1972): Mahogany was brought to India by the British. In 1795, for the first time, several Mahogany trees were introduced as seedlings from Jamaica into the Botanic Gardens at Calcutta. In 1796 Dr. Roxburgh, in a letter to the sub-secretary to the Government of Bengal, mentions among other things that “the Mahogany plants sent out by the Court of Directors in 1794–95 thrive very well”. By 1799, the plant got established in India. The trees continued to flourish but several trees were destroyed in the great cyclone of 1864. The trees were about 71 years of age, about 12 ft in girth at 4 ft above the ground. A log taken from them, after squaring and removal of sapwood, gave 169 cubic feet of timber. In 1865, 183 pods, containing 8235 seeds were received from Jamaica by the Superintendent of the Government Botanical Gardens, Calcutta. From these, only 460 plants were produced, 338 were sent to Darjeeling to be planted, remaining 112 were kept in the botanical gardens. The plantations in Darjeeling proved to be a failure but the trees throve well in Bengal, from where it was sent to other places in India, Europe and Africa. From Bengal, the plant was propagated to Saharanpur gardens, Bombay, Yellapur and Madras. Botanical classification (Wikipedia): Kingdom: Plantae (unranked): Angiosperms (unranked): Eudicots (unranked): Rosids

Order: Sapindales Family: Meliaceae Genus: Swietenia Species: Swietenia mahagoni Synonyms (life.ku.dk): Swietenia mahogoni (L.) Lam., Swietenia fabrilis Salisbury, Cedrus mahogany (L.) Miller. Vernacular/common names (life.ku.dk): English - Small leaved, West Indian, Spanish or Cuban mahogany Spanish - Caoba Bahamas - Madeira Cuba - Coabilla Dom.Rep. - Caoba dominicana Fr., Haiti - Acajou Bengali - Mehgoni Kannada - Hebbevu, Hiribevu, Davala, Mahaagani Tamil - Mahaagoni, Seemainukku Telugu - Maaghani, Mahaagani Habitat (life.ku.dk and www.dfsc.dk): S. mahagoni Jacq. is a humid zone species, with natural distribution in the Caribbean region (S. Florida, Bahamas, Antilles, Haiti and Jamaica). It has been extensively planted mainly in southern Asia (India, Sri Lanka, Bangladesh) and in the Pacific (Malaysia, Philippines, Indonesia and Fiji), and has been introduced into cultivation in West Africa. Morphology of Swietenia mahagoni Jacq. (Anonymous 1976): Habit: a medium or large, evergreen tree, native to Central America, with a handsome spreading habit. But in India it is entirely deciduous or semi-deciduous. It has a buttressed base and in its native country, the tree reaches a height of 30 m and a girth of 4.5 m, but in India it attains a height of 18– 24 m only. Bark: rugose, grey-black or dark brown, flaked. Leaves: alternate, exstipulate, clustered young leaves are of emerald shade, drying coppery

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brown, 12–15 cm long, paripinnate; leaflets 2– 4 pairs, opposite, very oblique, subfalcate, 5– 6 cm long, 2–3 cm wide, lanceolate or ovate, apex acuminate, venation reticulate. Inflorescence: axillary, 8–15 cm long, slender, pendulous panicles, shorter than leaves. Fruit: capsule, 5–10 cm long, 3–6 cm in diameter, ovoid or oblong, 5-celled, splits from base to apex, valves thick, woody, surface coriaceous when mature. Seed: 35–45 to each capsule, brownish, 4–5 cm long, compressed, crested and extended into a wing at the point of attachment. Different species (Wikipedia): Swietenia humilis, Swietenia macrophylla, Swietenia mahagoni, Swietenia aubrevilleana Among these, the first 3 species in the genus Swietenia are said to be important. They occur from Mexico to Brazil, and in the Caribbean region. The three species are poorly defined biologically, in part because they hybridize freely when grown in proximity (life.ku.dk). •

Swietenia humilis: Pacific Coast Mahogany - Pacific coast of Central America and Mexico; medium sized trees found at higher elevations (Anonymous 1976).

•

Swietenia macrophylla: Honduras Mahogany - Atlantic coast of Central America, South America, south to Bolivia; leaves 3–8 pairs (usually), ovate-lanceolate, young leaves red or pink; flowers greenish in supra-axillary panicles; capsule shape inverted club; bark greyish brown, smooth or sometimes rough, flakes into patches (Anonymous 1976).

•

Swietenia mahagoni: West Indian Mahogany - Southern Florida, Cuba, Jamaica, Hispaniola; leaves 2–4 pairs, very oblique, subfalcate, old leaves coppery brown, young leaves emerald shade; flowers greenish yellow in axillary

pendulous panicles; capsule ovoid; bark rough, grey black (Anonymous 1976). •

Swietenia aubrevilleana Stehle. & Cusin. is a putative hybrid between S. macrophylla and S. mahagoni (life.ku.dk).

Phenology (life.ku.dk): Pollination occurs by insects. Hybridisation is frequent, especially with S. macrophylla wherever the species grow together. Usually only one flower of the inflorescence develops into a fruit, the other flowers being aborted, even if fertilization has taken place. Development from flower to mature fruit takes from 8–10 months. Due to the long development time for the fruit, crop assessment can usually be undertaken several months before harvest. Flowering varies according to climate i.e. geographical site; it usually takes place shortly before the rainy season. S. mahagoni flowers in the Caribbean Islands between April and July and the fruits are mature 8–10 months later, between January and March. Mahoganies usually have regular annual flowering and fruiting from about 10–15 years of age. Cultivation and propagation (life.ku.dk): S. mahagoni is difficult to start from cuttings, and usually is grown from seed. Mahogany's little winged seeds are spread by the wind and often give rise to numerous seedlings in the vicinity of mature trees. Pretreatment is generally not necessary but germination of stored low moisture content seed may be enhanced by soaking in water for 12 h. The seeds are sown in a bed of light sand in 3–7 cm deep furrows or holes or directly in containers. Germinating seeds should be under shade and kept moist. Seeds will germinate in 10–21 days. Germination is hypogenous. The seedlings are kept under shade until outplanting. The seedlings can be planted in the field when they are about 50–100 cm tall.

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Collection/Harvest (life.ku.dk): The fruits are preferably collected from the trees just before opening or from the ground immediately after seed fall. Seed production varies according to site and year. A crucial factor for seed production is pollination efficiency, which may be erratic especially outside the natural area of distribution. Threat status: Under IUCN Redlist of Threatened species, Swietenia humilis (Pacific coast mahogany) is listed as Vulnerable species (Status Vulnerable A1cd ver 2.3), S. macrophylla (Large leaved mahogany) as Status Vulnerable A1cd+2cd ver 2.3, S. mahagoni (Small leaved mahogany) as Status Endangered A1ch ver 2.3 (iucnredlist.org) Ethnomedicinal uses: •

•

In India, traditionally it is used for several medicinal purposes. The seeds and bark are used for the treatment of Hypertension, Diabetes, Malaria (Nagalakshmi MAH. et al., 2001), and in Epilepsy as a folk medicine in Indonesia and India. (Kadota S et al.,1990) The bark is considered as an astringent and is taken orally as a decoction for diarrhoea, as a source of vitamins and iron, and as haemostyptic. The bark serves as antipyretic and tonic (Khare CP. 2007). Traditionally the bark decoction is used orally to increase appetite, to restore strength in cases of tuberculosis, to treat Anaemia, Diarrhoea, Dysentery, Fever and Toothache (Anonymous 1986). The leaf decoction is used against Nerve disorders, the seed infusion against Chest pain and a leaf or root poultice against bleeding. (Miroslav MG. et al., 2005). The local people of East Medinipur (West Bengal), Balasore (Orissa) traditionally use the aqueous extract of its seed and bark for

•

curing Psoriasis, Diabetes, Diarrhea and also used as an antiseptic in cuts and wounds (Pallab K et al., 2011). Mahogany seeds have also been reported to have medicinal value for treatment of Cancer, Amoebiasis, Coughs and intestinal parasitism (Bacsal K et al., 1997).

Other uses (life.ku.dk): S. mahagoni has potential use for large scale timber production plantations, especially in dry areas, due to the excellent timber quality. The wood density is 560–850 kg/m3 at 15% moisture content. It is also used in agroforestry, for soil improvement and as an ornamental tree. It also yields a gum. Chemical constituents The proximate nutritional compositions of S. mahagoni Jacq. seed cake and the fatty acids present in the seed oil were investigated. The proximate nutritional composition of the seed cake were analyzed by the standard methods and it was found to contain moisture (14.37%), minerals (16.36%), fats (19.42%), crude fiber (19.60%), protein (8.76%) and carbohydrate (21.49%). The fatty acid composition of the oil was analyzed by Gas Chromatography and a total of 48 compounds were identified. The major constituents of the methylated fatty esters were linoleic acid (26.00%), elaidic acid (24.39%), stearic acid (14.32%), palmitic acid (12.97%), 10-methyl-10-nonadecanol (5.24%), ecosanoic acid (2.48%), 3-heptyne-2,5-diol, 6methyl-5-(1-methylethyl) (2.03%) octadecanoic acid, 9,10,12-trimethoxy (1.90%); 1,3dioxalane, 4 ethyl-4-methyl-2-pentadecyl (1.89%) and 2-furapentanoic acid (1.03%). It is evident from this study that the oil can be considered as a good source of unsaturated fatty acids. The oil is bitter in taste and considered as a moderate drying oil, which can be useful in different chemical industries for soap and dying (M. Mostafa et al., 2011).

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Physico-chemical characters of the seed oil were also determined and are as follows: Parameters Result Brown Colour 24.60 % Moisture 0.9334 Specific gravity at 30° C 10.92 Acid value 5.49 % (as oleic acid) Free fatty acid (FFA) 191.27 Saponification value 94.4 Iodine value 1.49 % Unsaponifiable matter 53.75 % Oil (dry basis ) 0.35 Polenske value

Solvent partitioning followed by column chromatography of the Methanolic extract of the seeds of S. mahagoni Jacq. afforded two limonoids, swietenolide (Fig. 1) and 2hydroxy-3-O-tigloylswietenolide (Fig. 2). The compounds were identified by spectroscopic means. The antibacterial activity of these compounds was assessed against eight multiple-drug-resistant bacterial strains (clinical isolates) by the conventional disc diffusion method. While both compounds were active against all test organisms, compound (Fig. 2) displayed overall more

potent activity than compound (Fig. 1.) (A.K.M. Shahidur Rahman et al., 2009) Two novel limonoids, swiemahogins A (Fig. 1) and B (Fig. 2) isolated from the twigs and leaves of S. mahagoni Jacq. are the first examples of andirobin and phragmalin types of limonoids, of which the D-ring δ-lactone is demolished and a rare γ-lactone is fused to the C-ring at C-8 and C-14. Their structures were elucidated by extensive spectroscopic means, and that of Fig. 1 was confirmed by singlecrystal X-ray diffraction. (Yu-Yu Chen et al., 2007)

Fig. 1 Swietenolide & Fig. 2. 2-hydroxy-3-O-tigloylswietenolide

Pharmacological activity Acute Toxicity Studies: Methanolic extract of S. mahagoni Jacq. seed (SMSE) was injected i.p in increasing doses to mice. The LD50 (24 h) was calculated according to Ghosh M.N. It was found that

SMSE was non-toxic up to 1.2 g/kg, i.p. body weight up to 24 h. The two doses of SMSE used in the study were 50 and 100 mg/kg i.p. (Ghosh S et al., 2009) According to another study conducted by the method of brine shrimp lethality assay, LD50 of oral acute toxicity for S. mahagoni

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Jacq. seed methanolic extract (SMCM) is more than 2500 mg/kg. The oral LD50 value in this study suggests that the SMCM seed extract is a relatively nontoxic plant. The results of the study concur with the use of this plant by traditional healers as traditional medicine.(Geethaa Sahgal et al., 2010) Anti-microbial activity (Sahgal G et al., 2009): The study was designed to evaluate the antibacterial activities of S. mahagoni Jacq.crude methanolic (SMCM) seed extract. The antimicrobial activity of the oily extract against Gram-positive, Gram-negative, yeast and fungus strains was evaluated based on the inhibition zone using disc diffusion assay, minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) values. The crude extract was subjected to various phytochemical analyses. The demonstrated qualitative phytochemical tests exhibited the presences of common phytocompounds including alkaloids, terpenoids, antraquinones, cardiac glycosides, saponins, and volatile oils as major active constituents, while test for tannins, flavonoids and steroids demonstrated negative responses. The SMCM seed extract had inhibitory effects on the growth of Candida albicans, Staphylococcus aureus, Pseudomonas aeroginosa, Streptococcus faecalis and Proteus mirabillase and illustrated MIC and MBC values ranging from 25 mg/ml to 50 mg/ml. Anti-diabetic activity (SMM Mahid-Al-Hasan et al., 2011): The study was performed to investigate the blood glucose lowering effect of S. mahagoni Jacq. seeds in experimentally induced diabetic rats. Administration of ethanolic extract of S. mahagoni Jacq. seeds to normal rats produced no significant change in the blood glucose. Administration of ethanolic extract of Swietenia mahagoni seeds in alloxan induced Diabetic rats (120 mg/kg body weight) produced a significant reduction in blood glucose level as compared to diabetic control. Histological examination of pancreas showed destruction of beta cells in Islets of pancreas in control group whereas retaining of islets and few degranulations of beta cells of pancreas was found in the group treated with

S.mahagoni Jacq. seed extract. These observations and results provided the information that ethanolic extract of S. mahagoni Jacq. seeds has hypoglycemic effect in experimentally induced diabetic rats. Another comparative clinical study on the seeds of S. mahagoni had shown promising results with the seed powder encapsulated into 500 mg capsules and administered as 1 capsule twice a day after food for 60 days. The study was in comparison with another Ayurvedic classical herb Syzygium cumini. S. cumini and S. mahagoni showed definite demonstrable Madhumehahara (anti-diabetic) action as observed by clinical study. The drug S. mahagoni was more effective in all the parameters except in Pipasa (Polydipsia) where S. cumini showed better results. (Khare Divya et al., 2012) Antidiabetic, antioxidative (Geethaa Sahgal et al., 2009), and antihyperlipidemic activities of aqueous-methanolic (2 : 3) extract of S. mahagoni Jacq. seed was studied in streptozotocin-induced diabetic rats. Feeding with seed extract (25 mg in 0.25 ml distilled waterâ&#x2C6;&#x2019;1100 gm b.w./1rat/1 day) for 21 days to diabetic rat lowered the blood glucose level as well as the glycogen level in liver. Moreover, activities of antioxidant enzymes like catalase, peroxidase, and levels of the products of free radicals like conjugated diene and thiobarbituric acid reactive substances in liver, kidney, and skeletal muscles were corrected towards the control after this extract treatment in this model. Furthermore, the seed extract corrected the levels of serum urea, uric acid, creatinine, cholesterol, triglyceride, and lipoproteins towards the control level in this experimental diabetic model. The results indicated the potentiality of the extract of S. mahagoni seed for the correction of diabetes and its related complications like oxidative stress and hyperlipidemia.(Debasis De et. al 2011) Antioxidant and Antidiabetic activity (Subhadip Hajra et al., 2011 and Siva Prasad Panda et al., 2010): The ethanolic extract of Swietenia mahagoni seeds showed DPPH

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radical scavenging activity at concentrations of 10, 50, 100, 250 and 400 µg/ml. The extract also showed significant hydroxyl radical scavenging activity. It significantly inhibited nitric oxide radical and ferric reducing power in a concentration dependent manner. All the results were compared with that of standard drug Butylated Hydroxyl Anisole (BHA). The total phenolic content of seeds extract was found to be 1µg/mg of catechol equivalent when measured by Folin- Ciocalteau reagent. The extract showed relatively better antidiabetic activity of 72.53, 70.33 and 70.33% with respective concentration of 2, 20 and 200 µg/ml when measured by amylase inhibition assay. Amylase catalyses the hydrolysis of α -1, 4-glucosidic linkages of starch, glycogen and various oligosaccharides and glucosidase further breaks down the disaccharides into simpler sugars. The assay showed that the extract contains amylase inhibitory compounds. This inhibition of the amylase activity, in the digestive tract of humans, might be effective in controlling diabetes by diminishing the absorption of glucose. These observations support the use of S. mahagoni Jacq. seeds as a natural antioxidant and antidiabetic agent. PPARγ agonistic activity (Li DD et al., 2006): The seed of S. mahagoni Jacq. is a natural agonist of peroxisome-proliferator activated receptor (PPARγ). The functions of these PPARγ receptors after activation by drugs include an increase in lipid and cholesterol metabolism, adipocyte differentiation, and improvement in insulin sensitivity. It has been demonstrated that PPARγ is the receptor of the thiazolidinedione (TZD) class ligands. Among the TZD type antidiabetic drugs, Rosiglitazone and Troglitazone are potent adiopocytedifferentiating agents, which activate ap-2 gene expression in a PPARγ- dependent manner. Cytotoxic effect (Mohammad Ahsanul Akbar et al., 2009): The seed extract and its dichloromethane and pet-ether fractions exhibited the most significant cytotoxic properties. The moderate

cytotoxic activities were showed by bark extract, methanol fraction of bark extract, leaf extract and pet-ether fraction of bark extract. Anti-inflammatory, Analgesic Antipyretic study (Ghosh S et al., 2009)

and

S.mahagoni Jacq. seed methanolic extract (SMSE) showed significant anti-inflammatory and analgesic activity in experimental animals at doses of 50 and 100 mg/kg i.p. The antiinflammatory effect of SMSE was observed in acute (carrageenan and arachidonic acidinduced paw edema in rat and croton oilinduced ear inflammation in mice), sub-chronic (cotton pellet-induced granuloma in rat) and chronic (Freund's complete adjuvant-induced polyarthritis in rat) models of inflammation. Since SMSE inhibited edema similar to that of the dual-blocker BW755C in arachidonic acid induced-paw edema in rat and since indomethacin failed to show any significant inhibitory effect in this model, it is plausible that SMSE reduced inflammation by blocking both the lipo-oxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism. The observation that SMSE significantly reduced inflammation in the Freund's adjuvant-induced polyarthritis in rat reveals that SMSE possesses anti-arthritic activity as well. It is interesting to note that in all models of inflammation, the effect produced by 100 mg/kg i.p. of SMSE was either more than or comparable to that produced by 100 mg/kg i.p. of ibuprofen, the standard NSAID. While SMSE reduced acetic acid-induced writhing significantly it also showed analgesic activity in tail clip and tail flick models of analgesia in a time and dose-dependent manner in comparison to ibuprofen, the reference antiinflammatory agent. The extract did not possess significant antipyretic activity. Effect on normal peritoneal cell: (Ghosh S et al., 2009) It was observed that the average number of macrophages was increased after S. mahagoni seed methanolic extract treatment in a dosedependent manner as compared to the control.

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The linear increase was effective up to 24 hours and then on the 48 th hour the count came down. Though the actual role of SMSE in the enhancement of peritoneal cell count and macrophage count cannot be explained at the present juncture, it is possible that SMSE may also alter the immune response along with the anti-inflammatory effect.

activity against the groundnut rust Puccinia arachidis. 6-acetylswietenine and 6-acetyl-3tigloylswietenolide from S. mahogany Jacq. effectively reduced the number of rust pustules on detached groundnut leaves. (T. R. Govindachari et al., 1999)

Anti-tumour activity: (Ghosh S et al., 2009)

A study was performed to evaluate the antiulcer activity of S. mahagoni Jacq. ethanol leaf extract against ethanol-induced gastric ulcer. Results showed that rats pre-treated with leaf extract of S. mahagoni Jacq. before being given absolute alcohol had significantly reduced areas of gastric ulcer formation compared to rats pretreated with only Carboxy Methyl Cellulose (ulcer control group). Moreover, the leaf extract significantly suppressed the formation of the ulcers and it was interesting to note the flattening of gastric mucosal folds in rats pretreated with S. mahagoni Jacq. extract. It was also observed that protection of gastric mucosa was more prominent in rats pre-treated with 500 mg/kg plant extract. Ethanol-induced mucosal damage was significantly and dose dependently reduced in the size and severity by pretreatment of the animals with S. mahagoni Jacq. leaf extract. (Salmah Al-Radahe1 et al., 2012)

There is a close relationship between inflammation and cancer. It has been reported that tumor promoters recruit inflammatory cells to the application site and cancer development may also act by aggravating inflammation in the tissue and vice versa and that inflammatory cells are capable of inducing genotoxic effects. So it is likely that S. mahagoni Jacq. methanolic extract possesses anti-tumor activity as well. Anti-fungal activity: (Sahgal, G et al., 2012) S. mahagoni Jacq. crude methanolic (SMCM) seed extract was investigated for the antifungal activity against Candida albicans. The antifungal activity was evaluated against C. albicans via disk diffusion, minimum inhibition concentration (MIC), scanning electron microscope (SEM), transmission electron microscope (TEM) and time killing profile. The SEM and TEM findings showed that there are morphological changes and cytological destruction of C. albicans at the MIC value. Animal model was used to evaluate the in vivo antifungal activity of SMCM seed extract. The colony forming unit (CFU) was calculated per gram of kidney sample and per ml of blood sample respectively for control, curative and ketaconazole treated groups. There was significant reduction in the CFU/ml of blood and CFU/g of kidney in the SMCM treated group. This indicated that the extract is effective against C. albicans in vitro and in vivo conditions. In another study, Isolation and characterization of B,D-seco limonoids from S. mahagoni Jacq. was done. Seven limonoids from S. mahogani were tested for antifungal

Anti-ulcer activity:

PAF inhibition activity: The ether extract from the seeds of S. mahagoni Jacq. was found to inhibit plateletactivating factor (PAF)-induced platelet aggregation. Systematic separation of the extract afforded twenty eight tetranortriterpenoids related to swietenine and swietenolide. Among them, several new compounds, named swietemahonin A, D, E, and G and 3-O-acetylswietenolide and 6-Oacetylswietenolide, showed a strong inhibition against PAF-induced aggregation in vitro and in vivo assays. (Ekimoto H et al 1991) Swietemahonins and Swietenolide inhibited blood platelet aggregation, Swietemahonin A showed most potent (97.4% inhibition) antiPAF activity (Kadota S et al., 1990).

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CONCLUSION S. mahagoni Jacq. is a commonly used herb in Folklore medicine. This review supports all updated information on its botanical aspects, phytochemistry, pharmacological activities and traditional uses. Its chemical markers or target molecules have been identified and separated. The chemical entities of this plant have been proved for their Anti-bacterial activity, Antimicrobial Activity, Anti-oxidant activity, Antiulcer activity, Anti-fungal activity, Antiinflammatory, Analgesic activity, Hypoglcemic activity, Platelet Aggregation Inhibitors activity etc. These scientifically proved activities can be related with the traditional usage of the plant.

Thus S. mahagoni Jacq. is one of the most important plants that has a tremendous scope for research in future. The novelty and applicability of this valuable species are hidden. Such things should be overcome through extensive scientific research. The drug may be a good candidate for developing a safe, tolerable, and promising neutraceutical treatment for the management of many diseases. Though the plant is widely used for the treatment of a large number of human ailments, being an endangered species, our prime motive is to conserve such valuable plant species from going extinct.

REFERENCES A.K.M. Shahidur Rahman, A.K.Azad Chowdhury, Husne-Ara Ali, Sheikh Z. Raihan, Mohammad S.Ali, Lutfun Nahar and Satyajit D. Sarker. (2009). Antibacterial activity of two limonoids from Swietenia mahagoni against multiple-drug-resistant (MDR) bacterial strains. J Nat Med, Volume 63, 41–45. Anonymous (1976). The Wealth of India – Raw Materials, Volume 10 (Sp-W). Publications and Information Directorate, Council of Scientific and Industrial Research, New Delhi. Reprint edition 1982, pg 100. Anonymous. (1986). The Useful Plants of India. New Delhi: Publications & Information Directorate, Council of Scientific and Industrial Research, 967– 968. Bacsal K, Chavez L, Diaz I, Espina S, Javillo J, Manzanilla H, Motalban J, Panganiban C, Rodriguez A, Sumpaico C, Talip B, Yap S. (1997). The Effect of Swietenia Mahogani (Mahogany) Seed Extract On Indomethacin-Induced Gastric Ulcers In Female Sprague- Dawley Rats. Acta Medica Philippina 3, 127–139.

Debasis De, Kausik Chatterjee, KaziMonjur Ali, Tushar Kanti Bera, and Debidas Ghosh. (2011). Antidiabetic Potentiality of the Aqueous-Methanolic Extract of Seed of Swietenia mahagoni (L.) Jacq. in Streptozotocin-Induced Diabetic Male Albino Rat: A Correlative and Evidence-Based Approach with Antioxidative and Antihyperlipidemic Activities. Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine, Article ID 892807, 11 pages, doi:10.1155/2011/892807 Ekimoto H, Irie Y, Araki Y, Han GQ, Kadota S, Kikuchi T. (Feb 1991). Platelet aggregation inhibitors from the seeds of Swietenia mahagoni: inhibition of in vitro and in vivo platelet-activating factor-induced effects of tetranortriterpenoids related to swietenine and swietenolide. Planta Med.;57(1):56–8. Geethaa Sahgal, Surash Ramanathan, Sreenivasan Sasidharan, Mohd Nizam Mordi, Sabariah Ismail and Sharif Mahsufi Mansor. (2009). In Vitro Antioxidant and Xanthine Oxidase

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Inhibitory Activities of Methanolic Swietenia mahagoni Seed Extracts, Molecules 2009, 14, 4476–4485

Khare CP. (2007). Indian Medicinal Plants - an Illustrated Dictionary. Springer, New Delhi, 633–634.

Geethaa Sahgal, Surash Ramanathan, Sreenivasan Sasidharan, Mohd. Nizam Mordi, Sabariah Ismail, and Sharif Mahsufi Mansor. (2010 Jul-Aug). Brine shrimp lethality and acute oral toxicity studies on Swietenia mahagoni (Linn.) Jacq. seed methanolic extract, Pharmacogn Res. 2(4): 215–220.

Khare Divya, Pradeep H.R. and Kumar Krishna K (2012), Therapeutical Evaluation of Swietenia mahagoni Jacq. seeds and Syzygium cumini (Linn.) Skeels. Seeds in Madhumeha with special reference to Diabetes mellitus - A comparative study, Rasamruta, 4:12, 1–15

George Watt. (1972). A Dictionary of the economic products of India, Volume 6, Part 3 (Silk to Tea). Gordhan and Company, Delhi. pg 393. Ghosh S, Besra SE, Roy K, Gupta JK, Vedasiromoni JR. (2009). Pharmacological effects of methanolic extract of Swietenia mahagoni Jacq (Meliaceae) seeds; Int J Green Pharm; 3:206–10. http://en.wikipedia.org/wiki/Swietenia http://en.wikipedia.org/wiki/Swietenia_mahago ni http://www.iucnredlist.org/search?page=105 http://www2.sl.life.ku.dk/dfsc/pdf/Seedleaflets/ Swietenia%20mahagoni_int.pdf Magadi.R.Gurudeva. (2001). Botanical and Vernacular names of South Indian Plants. Divyachandra Prakashana Publishers and Book sellers, Bangalore. pg 382. Kadota S, Marpaaung L, Kikuchi T, Ekimoto H. (1990). Constituents of the seeds of Swietenia mahagoni JACQ. III. Structures of mahonin and secomahoganin. Chem Pharm Bull. 38: 1495–1500. AND Pullaiah T. (2006) Encyclopedia of World Medicinal Plants. Vol.4. Regency Publications, New Delhi. 453–454.

Li DD, Chen JH, Chen Q, Li GW, Chen J, Yue JM, Chen ML, Wang XP, Shen JH, Shen X, Jiang HL. (2005). Swietenia Mahagony extract shows agonistic activity to PPARg and gives ameliorative effects on diabetic db/db mice. Acta Pharmacol Sinica. 26(2):220–22. M. Mostafa, Ismet Ara Jahan, M. Riaz, Hemayet Hossain, Ishrat Nimmi, A. Sattar Miah and J. U. Chowdhury. (2011, June). Comprehensive Analysis of the Composition of Seed Cake and its Fatty Oil from Swietenia mahagoni Jacq. Growing in Bangladesh. Dhaka Univ. J. Pharm. Sci. 10(1): 49–52. Miroslav MG. (2005). Elsevier's Dictionary of Trees. Vol I. London: Elsevier Inc., 381. Mohammad Ahsanul Akbar, Rubina Ahamed, Khondoker Dedarul Alam, Mohammad Shawkat Ali. (2009). In Vitro Cytotoxic Properties of Ethanolic Extracts of Various Parts of Swietenia Mahagoni. European Journal of Scientific Research, ISSN 1450-216X Vol.32 No.4, pp.541–544. Nagalakshmi MAH, Thangadurai D, Muralidara D. & Pullaiah RT. (2001). Phytochemical and antimicrobial study of Chukrasia tabularis leaves. Fitoterapia 72, 62–64.

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Pallab K. Haldar, Soumitra Adhikari, Samit Bera, Sanjib Bhattacharya, Siva P. Panda, Chandi C. Kandar. (Apr-Jun, 2011). Hepatoprotective Efficacy of Swietenia Mahagoni L. Jacq. (Meliaceae) Bark against Paracetamolinduced Hepatic Damage in Rats. Ind J Pharm Edu Res. Vol 45/ Issue 2, pg 108–113. Sahgal G, Ramanathan S, Sasidharan S, Mordi M.N, Ismail S. and Mansor S.M. (2009), Phytochemical and antimicrobial activity of Swietenia mahagoni crude methanolic seed extract. Tropical Biomedicine 26(3): 274–279 Sahgal, G., Ramanathan, S., Sasidharan, S., Mordi, M.N., Ismail, S. and Mansor, S.M. (2011). In vitro and in vivo anticandidal activity of Swietenia mahogani methanolic seed extract, Tropical Biomedicine 28(1): 132–137. Salmah

Al-Radahe1, Khaled Abdul-Aziz Ahmed, Suzy Salama, Mahmood Ameen Abdulla, Zahra A. Amin, Saad Al-Jassabi and Harita Hashim. (30 March 2012). Anti-ulcer activity of Swietenia mahagoni leaf extract in ethanol-induced gastric mucosal damage in rats, J. Med. Plants Res. Vol. 6(12), pg. 2266–2275.

Seed Leaflet, Danida forest seed centre, www.dfsc.dk.

Source of Support: Nil

Siva Prasad Panda, Pallab Kanti Haldar, Samit Bera, Soumitra Adhikary, Chandi Charan Kandar. (2010). Antidiabetic and antioxidant activity of Swietenia mahagoni in streptozotocin-induced diabetic rats. Pharm Biol; 48 (9): 974– 979. SMM Mahid-Al-Hasan, MI Khan, BU Umar. (2011). Effect of Ethanolic Extract of Swietenia mahagoni Seeds on Experimentally Induced Diabetes Mellitus in Rats. Faridpur Med. Coll. J.;6(2):70–73 Subhadip Hajra, Archana Mehta, Pinkee Pandey and Suresh Prasad Vyas. (2011 May). Antioxidant and Anti-Diabetic Potential of ethanolic extract of Swietenia mahagoni (Linn.) seeds, IJPRD; Vol 3/Issue 4/Article 25: 180– 186 T. R. Govindachari, G. Suresh, B. Banumathy, S. Masilamani, Geetha Gopalakrishnan and G. N. Krishna Kumari. (1999). Antifungal Activity of Some B,D-Seco Limonoids from Two Meliaceous Plants, Journal of Chemical Ecology, Volume 25, Number 4, 923–933. Yu-Yu Chen, Xiao-Ning Wang, Cheng-Qi Fan, Sheng Yin, Jian-Min Yue. (15 October 2007) Swiemahogins A and B, two novel limonoids from Swietenia mahogany, Tetrahedron Letters, Volume 48, Issue 42, Pages 7480–7484.

Conflict of Interest: None Declared

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