GJRMI - Volume 1, Issue 10, October 2012

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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

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

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

ISSN 2277-4289 |www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

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 Francis

2, Agea Jacob Godfrey

1*,

Mulugo Lucy Were1, Vuzi Peter California

3, Namutebi Agnes

1, Okello John Bosco Acot

1,

Okonye Godman4, Nyanzi Steven Allan

3

1 College of Agricultural and Environmental Sciences, Makerere University, P. O. Box 7062, Kampala,

Uganda. 2 Natural Chemotherapeutic Laboratory, Wandegeya, Kampala, Uganda

3College of Natural Sciences, Makerere University, P. O. Box 7062, Kampala, Uganda.

4Department of English, College of Education, University of Juba, Government of South Sudan (GOSS)

*Corresponding Author’s Email: [email protected]/[email protected]

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 ethno-

nomenclature 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.

Research article

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



INTRODUCTION

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).

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,

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.

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).

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.



STUDY AREA AND METHOD

Study Area

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.

METHODS

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.

RESULTS

Socio-demographic characteristics of

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

Variable % Response

Acholi Lango Iteso Madi Alur Lugbara

Sex

Male 72 69 47 02 37 63

Female 28 31 53 38 63 37

Age

< 18years 00 09 03 13 00 04

19–37 years 43 37 44 53 50 38

38–56 years 42 46 44 34 42 52

>56 years 15 08 09 00 08 06

Occupation

Subsistence farming 89 85 87 85 95 90

Trade 11 15 13 15 05 10



Ethno-names of the Shea tree 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

however, not sought in this study.

Ethno-names of the Shea tree products in

the Shea parkland areas of Uganda

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

Yaa, yao

Alur Yen yao, awa, danyu

Iteso Ekungur

Lango Yao

Lugbara Awa

Madi Awa pati, Awa kwee

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

Ethnic groups Ethno-names of Shea tree products

Shea fresh fruit Shea nut Shea seed kernel Shea oil

Acholi Odu, odua, eduu,

kitigu, kiduu.

Yao magolo, yaa

magolo.

Yaa/yao nyinge, nying

yaa/yao, magolo yaa/yao

koro.

Moo yaa, moo

yao.

Alur

Dany yao, danyo,

odanyo, adu, awa

adu.

Awakorongo, dend yao,

pok yao. Nyinge yao, aweki.

Moo yao, awa

odu, odu omoo.

Iteso

Akungur, adanyoi,

odu. Akungur.

Elemut, akungur

Kiwee.

Akungur, alinyo

moo yaa.

Lango

Adu, adanyo,

kom yao. Yao, yao agulu. Yao koro, yao. Moo yao.

Lugbara

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.

Madi

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.



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.

DISCUSSION

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 ‘reverse-

xenophobic’ 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

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.

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)

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



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 ethno-

names 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



ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

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 M

2

1Associate Professor, Department of Bio-technology, SGB Amravati University, Amravati, Maharashtra, India

2Junior Research fellow, Department of Bio-technology, SGB Amravati University, Amravati.

*Corresponding Author: Email: [email protected]; Mobile: +919881735354


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

Research article

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



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 fast-

growing 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 phyto-

chemicals 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 and establishment of

cultures:

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, 4-

Dichlorophenoxyaceticacid (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.



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 6-

Benzylaminopurine (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, otherwise it was not

subcultured.

Effect of BAP levels on frequency of

embryogenic response:

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.



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 ECLISPSE-

E100 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.



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)

Frequency of

induction %

of

Embryogenesis

2, 4-D BAP

MS (1X)

Control

-- -- NR 0.0

1 0.5 10 ± 2.3 33.33

2 0.5 12 ± 1.5 40.00

3 0.5 11 ± 3.3 36.66

MS (5X)

Control

-- -- NR 0.0

1 0.5 12 ± 2.1 40.00

2 0.5 15 ± 1.2 30.00

3 0.5 14 ± 1.5 46.66

MS (7X)

Control

-- -- NR 0.0

1 0.5 13 ± 2.1 43.33

2 0.5 16 ± 1.1 53.33

3 0.5 14 ± 1.4 46.66

MS (9X)

Control

-- -- NR 0.0

1 0.5 18 ± 2.1 60.00

2 0.5 26 ± 0.54 86.66

3 0.5 20 ± 0.83 66.66

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

(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, 4-

D (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.



Table 2. The effect of BAP on embryo formation frequency of leaf explants of P. foetida L.

Growth

regulator

(mg/l)

2, 4-D

BAP Frequency of

embryogenic

response

% response

No. of well

developed

S.E

2 0.5 10 ± 0.23 100 19 ± 0.41

2 1.0 9 ± 0.54 90 16 ± 0.68

2 1.5 7 ± 0.64 70 15 ± 0.7

2 2.0 7 ± 0.68 70 13 ± 1.9

2 2.5 7 ± 0.72 70 11 ± 2.3

2 3.0 4 ± 0.72 40 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.

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 full-

strength 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 No. of embryos

Germinated/inoculated % Germination

MS 13 (30) 43

Half strength MS 18 (30) 60.00

Global J Res. Med. Plants & Indigen. Med.

Global Journal of Research on Medicinal Plants & Indigenous Medicine

The leaf explant of 1–1.5 cm size showed swelling of the explant and initiation of small embryo

days of culture with globular stage embryo (GE)

medium. In the next 20 days, embryogenic clumps were visible and appeared morphologically prominent on

of explants, showing heart shape embryo (HE)

medium. Well-developed cotyledonary embryos (CE) were observed all over the cultured

culture incubation (Fig D) on multiplication medium.

regeneration showing greenish colour leaf primordia

leaf disc explant (Fig. F.) Root induction from the somatic embryos (Fig.G)

Developmental stages of somatic

and their histological study:

The initiation of direct somatic embryos

was observed from the lower layer of

epidermis, with initiation of procambium. The

globular stage embryos on multiplication

medium responded well for its developing

organization. After 15 days of a subculture,

globular cells modified in the heart

embryo, a bipolar structure with axis in

centre of the body, were as after 20

symmetrical axis elongated to form the torpedo

shape embryo, which further modified

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 buds. Histological analysis

revealed the presence of protuberances with the



Fig. 1.

cm size showed swelling of the explant and initiation of small embryo

with globular stage embryo (GE) with few embryogenic calli (EC) (Figure A

embryogenic clumps were visible and appeared morphologically prominent on

of explants, showing heart shape embryo (HE) (arrow) and torpedo shape embryo (TE) (Figure

developed cotyledonary embryos (CE) were observed all over the cultured explants within 28

) on multiplication medium. On the regeneration medium, explant

regeneration showing greenish colour leaf primordia (LP) (Fig E). Direct regeneration of induced somatic embryos from

Root induction from the somatic embryos (Fig.G)

Developmental stages of somatic embryos

The initiation of direct somatic embryos

observed from the lower layer of

epidermis, with initiation of procambium. The

lobular stage embryos on multiplication

medium responded well for its developing

subculture, the

the heart shaped

embryo, a bipolar structure with axis in the

, were as after 20 days

axis elongated to form the torpedo

shape embryo, which further modified itself

Embryonic cells observed with dense

cytoplasm and prominent nuclei in the centre

originated from the proliferation tissue mass

without previous callus formation leading to

mbryonic buds. Histological analysis

the presence of protuberances with the

linings of columnar cells at the margin.

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

with the presence of vascular cambium at the

intermediate lining (Fig. 2A).

layers of explants acquired origination of

globular structure proving the anatomical

evidence of direct embryogenesis.

stage embryo was prominent (Fig.

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 bas

top was tapered 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).

cm size showed swelling of the explant and initiation of small embryo-like structures in 25

(Figure A and B) on induction

embryogenic clumps were visible and appeared morphologically prominent on the surface

(TE) (Figure C) on multiplication

explants within 28-30 days of

explant show's initiation of

Direct regeneration of induced somatic embryos from

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

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

heart stage somatic embryo with distinct

from the epidermis of the explant

organization (Fig. 2C). Since the

fourth week of culture, this structure appeared

lengthened, became bulbous at its base, and its

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).



The cells of the central mother cell zone were

observed, formation of new leaf primordia and

associated immature internodal tissue also

observed as the result of

Fig. 2. Somatic embryogenesis in

Fig 2: A, vascular tissue (VT) (arrow)

Globular stage embryo (G.E) surrounded by endospermic nuclei.

showing vascular standardisation. D, Torpedo

Apical meristem (AM) with surrounding leaf primordia.

connective tissue between embryo and callus cells. Scale bar=15

DISCUSSION

In vitro techniques are now successfully

applied to a range of threatened endangered

medicinal and aromatic plant species for

multiplication and conservation. No efficient,

reproducible somatic embryogenesis

regeneration system exists for this

impeding rare regeneration from type of

explants in the other species of this family

been reported.

Induction of direct somatic

formation in P. foetida L. species is reported

here for the first time. A direct

embryogenesis protocol is developed using leaf

explant, which would be certainly helpful

efficient multiplication and conservation

system for P. foetida L. using

techniques. Although in this system leaf

explants of P. foetida has shown

contamination levels and difficulty in direct

shoot and root development due to



mother cell zone were

ved, formation of new leaf primordia and

associated immature internodal tissue also

observed as the result of the highly

meristematic proximal

showing no connective tissue with callus cells

provides the evidence for direct somatic

embryogenesis (Fig.2F).

Fig. 2. Somatic embryogenesis in Passiflora foetida L.

(VT) (arrow) about to organize in the center and proembryos are at the surroundings.

Globular stage embryo (G.E) surrounded by endospermic nuclei. C, Transition stage from globular to heart shape

Torpedo shape embryo (TE) with lengthened symmetrical axis.

surrounding leaf primordia. F, Globular embryos with direct origin, arrows showing no

connective tissue between embryo and callus cells. Scale bar=15 µm.

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;

ng rare regeneration from type of

explants in the other species of this family has

somatic embryo

L. species is reported

direct somatic

developed using leaf

be certainly helpful in the

multiplication and conservation

L. using in vitro

this system leaf

has shown high

difficulty in direct

shoot and root development due to

accumulation of ethylene harmony

2003).

In the present investigation leaf explants

were treated with high HgCl

proved satisfactory after

somatic embryo induction started

days of incubation of

induction medium. As

Passiflora species shows high rates of ethylene

production (Shiomi et al.,

limits the in vitro morphogenic potential of the

explanted cultures. Ethylene plays a role in

several in vitro developments’

(Kumar et al., 1998)

ethylene in vitro

passionflower had been reported

Passiflora species (Barbosa

In the present study,

the medium, especially auxin 2,

in combination with cytokinin

with high salt concentration in micronutrient

meristematic proximal region, embryos

tissue with callus cells

provides the evidence for direct somatic

L.

about to organize in the center and proembryos are at the surroundings. B,

Transition stage from globular to heart shape (HE)

symmetrical axis. E, Regeneration of

Globular embryos with direct origin, arrows showing no

accumulation of ethylene harmony (Reis et al.,

the present investigation leaf explants

were treated with high HgCl2 conc. (0.2%) and

after disinfection. The

induction started after 20–28

of incubation of leaf explant on the

As an elimaeteric fruit

Passiflora species shows high rates of ethylene

et al., 1996a; b), which

morphogenic potential of the

explanted cultures. Ethylene plays a role in

developments’ processes

and also effects of

morphogenesis in

been reported earlier in

(Barbosa et al., 2001).

study, growth regulators in

especially auxin 2, 4-D (2 mg l-1

)

cytokinin BAP (0.5 mg l-1

)

with high salt concentration in micronutrient



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

embryo’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.



ACKNOWLEDGEMENT

The authors wish to acknowledge the

grants from Department of Science and

Technology (DST) New Delhi, INDIA under

Fast Track Young Scientist scheme [SR/FT/LS-

127/2008] and to Head and staff, Department

of Biotechnology, SGB Amravati University,

Amravati (M. S) India for providing necessary

facilities and help during the investigation.

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EVALUATION OF EFFECT OF METHANOLIC AND AQUEOUS

EXTRACTS OF PUNICA GRANATUM L. AGAINST BACTERIAL

PATHOGENS CAUSING BOVINE MASTITIS

Gopinath S M1, Suneetha T B

2, Singh Sumer

3

1Acharya Institute of Technology, Soldevanahalli, Hesaraghatta Road, Bangalore-560 090

2, 3 Dept of Biotechnology, Singhania University, Rajasthan.

*Corresponding Author: Email: [email protected]; Mobile: +919738888095


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

Research article

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



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



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 phyto-

constituents 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 and extraction of phyto-

compounds

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


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 pre-

weighed 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


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



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 phyto-

compounds) / (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.

+ indicates the presence of Phytocompound

− Indicates the absence of phytocompound

Compound Methanol

Water

Steroids − −

Terpenoids + +

Alkaloids + +

Flavonoids + +

Saponins + +

Tannins + +

Phenolic compounds + +

Catechin − −

Anthraquinone − −

quinone + +



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 16

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 Flavanoids Saponins Tannins Terpenoids

M W M W M W M W M W

S. uberis 0 0 11 16 0 0 0 0 0 0

S. aureus 0 0 12 12 0 0 0 0 0 0

E.coli 0 0 14 15 0 0 0 0 0 0

CONS 0 0 16 13 0 0 0 0 0 0

Fig-1. Phyto-compounds present in methanol and aqueous extracts of Punica granatum

0

2

4

6

8

10

12

14

16

18

methanol

water



Fig-2 Antibacterial activity of

CONCLUSION

Methanol and water extracts of flavonoids

from Punica granatum have

potential against the causative organism of

Bovine mastitis. Water as solvent is better for

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ml as retrieved on 9 Oct 2001 21(47):38.





SAPONIN: A WONDER DRUG FROM CHLOROPHYTUM SPECIES

Sharma Rohit1*, Thakur Gulab S

2, Sanodiya Bhagwan S

3, Pandey Mukeshwar

4,

Bisen Prakash S5

1Department 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 4Xcelris Genomics, Old Prem Chand Nagar Road, Opp Satyagarh Chhavani, Bodakdev, Ahmedabad-

380054 , India

*Corresponding Author: Email: [email protected]


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.

Review article

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

mailto:[email protected]



INTRODUCTION

Saponins are a vast group of structurally

diverse glycosides of the plant kingdom

widely distributed in nature; their surface-

active 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 semi-

synthesis 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 cold-

blooded 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



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.



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 phyto-

constituents 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, anti-

diabetic, antioxidant, anti-stress,

antimicrobial, anti-aging, antitumor and anti-

inflammatory activities (Jat and Bordia, 2003)

(Fig 3). Aqueous extract of dried roots of C.

borivilianum displayed enhanced sexual



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 HDL-

cholesterol 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 ‘Safed Musli’ 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 hydroxy-

8, 11 oxidoheniconesol and pentacosanol,



docasonoic acid, pentacosonyl docosanoate, n-

nonacosane, 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).


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.


malayense

C. malayense Ridl. is indigenous to south-

east 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 neo-

hecogenin 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 furos-

tane 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



tuberous roots that store reserve food. These

spider plants are excellent house plants or

indoor plants as they are not only easy-

growing 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 value of

Chlorophytum comosum

Seven anti-tumour promoter crude

steroidal saponins have been isolated by

silicagel, reverse phase RP18, and Diaion HP-

20 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 neo-

hecogenin. 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 value of

Chlorophytum orchidastrum

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 single-

quantum 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).



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 AND FUTURE

PROSPECTS

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.



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.

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STANDARDIZATION OF POLYHERBAL FORMULATION –

ARSHONYT FORTE

Agrawal SS1*, Ghorpade SS

2, Gurjar PN

3

1, 2 SVKM’S, NMIMS, SPTM, Shirpur, Dist: Dhule, 425 405, Maharashtra, India

3 Sharadchandra Pawar College of Pharmacy, Otur, Dist: Pune, 412 409, India

*Corresponding Author: E-mail: [email protected]; Mobile: +919923694748


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

Research article

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



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-the-

counter 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.


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 µg/ml.

Test solution: 100 mg of all test extract

prepared as mentioned earlier 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.

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).



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.



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 n-

Hexane. 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



Table I: Results of preliminary Pharmacognostic investigation

Sr. No. Tests Observation Arshonyt forte

tablet

1. Extractive value 5 g −

2. Total ash 0.4 g −

3. Acid insoluble ash 10 mg −

4. Water soluble ash 0.2 g −

5. Dragondroff reagent Reddish brown colour precipitate +

6. Mayer’s reagent Cream colour precipitate +

7. Wagner’s reagent Reddish brown colour precipitate +

8. Hager’s reagent Gives yellow colour precipitate +

9. Shinoda test crimson red colour +

+ indicates Present; − indicates absent

Result for extraction and authentication of

the active therapeutic constituent from the

extract.

i. 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

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.

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 % Estimation Mean ± S.D.

1 sample 0.041 7.49 7.89 ± 0.42

2 0.046 8.34

3 0.043 7.83

Table III: Total polyphenolics estimation

Sr. No. Extract Absorbance 765 nm % Estimation Mean ± S.D.

1 sample 1.384 18.59 18.54 ± 0.072

1.378 18.50

1.383 18.57



Table IV: Total saponin estimation

Sr. No. Extract Absorbance 472 nm % Estimation Mean ± S.D.

1 sample 0.017 8.11 7.61 ± 0.567

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: Figure 3:

Track 1 for Gallic acid Track 1 for Gallic acid Track 3 for extract

Table V: HPTLC study on the extract

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 Chromatogram for Epicatechin Chromatogram for sample

Tr. Pk No. Rf AUC Content in mg

1 1 0.72 1544.7 −

2 1 0.26 1274.1 −

3 1 0.02 1165.7 −

3 2 0.12 2035.4 −

3 3 0.24 878.1 29.40 mg

3 4 0.26 749.3 −

3 5 0.73 1753.4 56.75 mg

3 6 0.91 2265.7 −

3 7 0.97 1842.9 −



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. Organo chloro No colour observed Dichloropropane absent

2. Organo phospho No colour observed Phosphate absent

3. 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



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.

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

causes serious infections which can enter

through oral and ophthalmic formulations. So

absence of microbes in formulation indicates

the safety of formulation.

CONCLUSION

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.

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& Family Welfare, Part-1, volume 1, 5–

8.





STUDIES ON SEED GERMINATION AND GROWTH IN

GLORIOSA SUPERBA L.

Anandhi S1*, Rajamani K

2

1, 2

Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore-641 003,

Tamil Nadu, India

*Corresponding Author: [email protected]; Mobile: +919842392444


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

Research article

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): 524–528



INTRODUCTION

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.


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.

RESULTS

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.

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 %).



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

Treatments

Seed

treatments

(one hour

soaking)

Germination

percentage (%)

Days for

germination

(days)

Shoot

length

(cm)

Root

length

(cm)

Vigour

index

T1 Control 0.00

(0.45) 0.00 0.00 0.00 0.00

T2 Hot water

soaking

32.75

(34.88) 48.35 11.12 6.15 565.92

T3 Thiourea -

0.5 %

11.00

(19.17) 52.95 10.51 5.48 175.94

T4 Thiourea –

1.0 %

7.50

(15.88) 57.55 10.68 5.80 123.67

T5 Thiourea –

1.5 %

8.00

(16.16) 60.80 10.76 5.78 132.32

T6 KNO3 – 0.5

%

13.75

(21.72) 55.00 10.62 5.78 225.63

T7 KNO3 – 1.0

%

16.25

(23.57) 56.75 10.65 5.63 264.71

T8 KNO3 – 1.5

%

15.25

(22.75) 49.80 10.84 5.43 248.27

T9 GA3 – 100

ppm

12.20

(20.47) 50.15 11.05 6.45 214.43

T10 GA3 – 250

ppm

19.50

(24.93) 48.45 11.78 6.94 365.23

Mean 13.47

(20.00) 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

DISCUSSION

Dormancy is a condition where seeds will

not germinate even when the environmental

conditions (water, temperature and aeration)

are favourable for germination. Poor and

delayed seed germination in G. superba was

reported and the germination was erratic which

took three weeks to three months In the present

study, various dormancy breaking treatments

like soaking in hot water and various chemicals

were tried and the results revealed that hot

water treatment imposed for an hour recorded

the higher germination percentage (32.75 %),

earlier germination (48.35 days), and vigor

index (565.92). The water impervious seed coat

protects the plant from germination during the

harsh condition until the rainy season. Soaking

of seeds in hot water could have helped in

enhancing the seed germination by softening

the hard seed coat and facilitating leaching out

of the germination inhibitors (Azhar and

Sreeramu, 2004).

The results are similar to the observation

made in Leucaena glauca, where the boiling

water significantly reduced the percentage of

abnormal seedlings and dead seeds

(Venkatratnam, 1948). Sarker et al. (2000) also

reported that steeping Sesbania rostrata seeds

in boiling water for one minute showed the

highest percentage of germination (62.63 %).

Singh et al., (1984) found that water soaking



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.

In the present study, KNO3 (1.0 %) solution

also increased the germination percentage

(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.

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.



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-El-

Hafeez 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 El-

Nil, 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. Prentice-

Hall, Inc. Upper SaddleRiver, New Jersey ,

USA. 770pp

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.

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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.

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Schima khasiana. Indian J. Forestry,

11(1): 32–36.





MASS PROPAGATION AND IN VITRO CONSERVATION OF

INDIAN GINSENG - WITHANIA SOMNIFERA (L.) DUNAL

Chatterjee Tuhin1, Ghosh Biswajit

2*

1, 2 Plant Biotechnology Laboratory, Ramakrishna Mission Vivekananda Centenary College, Rahara,

Kolkata- 700118, India.

*Corresponding Author: E-mail: [email protected]


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.

Research article

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



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.


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



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



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.



Table: 1 Effect of Cytokinin on shoot multiplication from nodal explants of Withania somnifera

(L.) Dunal after 6 weeks.

(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.

Treatments (mg/l) Mean No. of shoots/

nodal explant

Mean shoot length

(cm)

MS (Full strength) - - MS + BAP

0.5 11.1 ± 0.1 2.3 ± 0.2 1.0 14.2 ± 0.2 2.8 ± 0.1 2.0 16.3 ± 0.2 3.1 ± 0.1 3.0 12.2 ± 0.1 2.9 ± 0.2 5.0 12.3 ± 0.2 2.8 ± 0.1

MS + Kn

0.5 6.2 ± 0.2 2.3 ± 0.2 1.0 10.2 ± 0.2 2.4 ± 0.2 2.0 9.1 ± 0.2 2.2 ± 0.2 3.0 7.9 ± 0.1 2.1 ± 0.2 5.0 6.8 ± 0.1 2.1 ± 0.1

MS + 2ip

0.5 3.7 ± 0.3 2.1 ± 0.1 1.0 6.4 ± 0.6 2.8 ± 0.03 2.0 5.6 ± 0.3 2.33 ± 0.1 3.0 3.4 ± 0.3 1.3 ± 0.06 5.0 1.3 ± 0.3 1.2 ± 0.04

MS + BAP + Kn

0.5 + 0.5 12.0 ± 0.1 3.5 ± 0.2 1.0 + 1.0 21.0 ± 0.1 3.9 ± 0.2 2.0 + 1.0 18.3 ± 0.2 4.2 ± 0.2 3.0+ 1.0 14.8 ± 0.2 3.8 ± 0.2 5.0+ 1.0 14.2 ± 0.2 3.7 ± 0.2

GA3

(mgl-1)

Shoot elongation

response (%)

Mean shoot

length (cm)

0.1

0.2

0.5

0.8

1.0

77.6 ± 2.5

82.2 ± 2.3

99.6 ± 3.1

85.6 ± 2.6

64.6 ± 2.5

3.0 ± 2.6

5.0 ± 0.5

7.6 ± 2.5

7.3 ± 1.5

6.3 ± 2.7



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 (%)

MS 3.4 ± 0.3 24.5 Half-strength MS 2.1 ± 0.1 12.7

MS + IBA (0.2) 5.8 ± 0.2 62.3 MS + IBA (0.5) 7.6 ± 0.1 66.7 MS + IBA (1.0) 10.4 ± 0.1 86. 8 MS + IBA (1.5) 9.3 ± 0.2 78.7 MS + IBA (2.0) 9.2 ± 0.2 72.3

MS + NAA (0.2) 2.02 ± 0.06 38.5 MS + NAA (0.5) 2.46 ± 0.2 40.4 MS + NAA (1.0) 4.46 ± 0.5 51.6 MS + NAA (1.5) 5.82 ± 0.14 60.5 MS + NAA (2.0) 4.02 ± 0.9 56.4

MS + IAA (0.2) 2.77 ± 0.15 48.5 MS + IAA (0.5) 5.37 ± 0.6 62.3 MS + IAA (1.0) 8.48 ± 0.06 73.5 MS + IAA (1.5) 6.1 ± 2.1 53.0 MS + IAA (2.0) 5.7 ± 1.5 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



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 half-strength 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 long-term 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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A COMPARATIVE PHARMACOGNOSTICAL EVALUATION OF RAW AND

TRADITIONALLY SHODHITA VACHA (ACORUS CALAMUS LINN.)

RHIZOMES

Bhat Savitha D1*, Ashok B K

2, Harisha C R

3, Acharya Rabinarayan

4, ShuklaV J

5

1Lecturer, Department of Dravyaguna, Muniyal Institute of Ayurveda Medical Sciences, Manipal, Karnataka,

India

2Research Assistant, Pharmacology laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar, India

3 Head, Pharmacognosy laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India 4Associate 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: [email protected]; Mobile: +919483128930


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

Research article



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.



Fig – 1: Photograph showing plant profile Fig – 2: Rhizome of Vacha showing

of Vacha numerous round root scars on the

ventral surface

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,

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).



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

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 – 3: Transverse section of raw Vacha (RV) Fig – 4: Transverse section of

(20X) En. – Endodermis; Ph. – Phloem; Xy. – Xylem; Ksheera Shodhita Vacha (KV) (20X)Ph. – Phloem;

OC. – Oil cell; OG. – Oil globule; S. – Starch grain Xy. – Xylem; OC. – Oil cell; Ct Vb. – Cortical vascular

bundle; St Vb. – Stelar Vascular bundle



Table – 1: Important microscopic characteristics observed in the rhizomes of both raw and

Shodhita Vacha

Microscopic

characters Sample RV Sample KV Sample MV

Transverse

section Oval to round More round More round

Outline/

margin Faintly wavy Faintly wavy Faintly wavy

Region

The rhizome is differentiated

into cortical region and stelar

region. Cortical region

constitutes around 1/3rd

of the

area whereas stellar region

constitutes around 2/3rd

The differentiation of

areas and the extent of

cortex and stellar region

is same as raw Vacha

The size of cortex and

stellar region is similar

to raw Vacha

Cork, Cortex

and

Stele

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

radially elongated cells with

thickened walls. Under the

epidermis there are 2 to 3 layers

of closely arranged

collenchymatous cells forming

the hypodermis. It is followed

by spherical to oblong

moderately thick walled

parenchymatous cells which

covers the rest of the cortex.

These cells are arranged to

form a network leaving large

intercellular spaces. The lower

boundary of the cortex is

characterised by a distinct

endodermis which separates it

from the stellar region. The

stellar region also consists of

parenchymatous cells similar to

cortex.

Brown coloured, single

layered cork tissue

forms the outermost

part of the cortex

followed by epidermal

cells which are more or

less round in shape. The

collenchymatous cells

are same as in RV. The

parenchyma cells are

more compact, round

and are arranged in a

similar manner in

cortex as well as in

stellar region. The

endodermis is similar to

raw Vacha

Pale brown, single

layered coloured cork

tissue outlines the

cortex followed by the

epidermis having oval

to round cells. The

collenchymatous cells

are more compact than

RV. The parenchyma

cells are more

spherical with reduced

intercellular spaces.

The endodermis forms

the lower boundary of

the cortex and other

features are same as

raw Vacha

Endodermis

The endodermal cells are barrel

shaped, thin walled, arranged in

a single layer and shows

casparian thickening

The rhizomes of KV

also exhibit similar

structure and

arrangement of

endodermal cells as in

sample RV

There is no visible

difference in the

features of endodermis

as compared to RV.

Vascular

bundles

The vascular bundles are

sheathed, collateral having

large air spaces and found

scattered in the cortex. The

The vascular bundles in

cortex as well as stellar

region are similar to RV

in the architecture and

The structure and of

the vascular bundles

both in the cortex and

stele is similar to RV.



vessels are fairly large and have

spiral and annular, scalariform

thickening. The fibres are very

few, thin walled and pitted.

Stele also consists of vascular

bundles in large numbers

especially arranged closely near

the endodermis. They form

almost complete rings under the

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.

arrangement, while the

air spaces inside the

bundles are

comparatively smaller.

There are few pale

yellow and white

coloured oil globules

seen inside both the

vascular bundle.

The inner air spaces

are shrunken while

pale yellow coloured

oil globules are rarely

seen inside the

vascular bundle

Oil cells and

globules

The oil cells are globose /

spheroidal found amongst the

parenchyma cells devoid of

starch grains. They are slightly

larger and thin walled than the

surrounding cells. They often

contain yellowish brown oil.

Many broken oil cells are also

observed with spilled out oil

droplets. The oil droplets

appear crimson red and the

walls of the oil cell appear light

pink upon staining with Sudan

red III and safranin

There are no gross

changes seen in the

shape of the oil cells in

comparison to RV. The

oil cells contain pale

yellow oil which

appeared crimson red

upon staining with

Sudan red III and

safranin

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

Oleoresin

content

Dark brown coloured Oleoresin

deposits are found scattered

throughout the cortex and the

stellar region but found more in

sub epidermal region.

Dark brown oleoresin

deposits are scattered

throughout.

Few dark brown

oleoresin patches are

seen when compared

to RV

Starch grains

The starch grains are abundant

and often seen clustered in the

parenchyma cells and

sometimes arranged in the form

of a string of beads. They are

mostly round with occasionally

oval shaped, simple, single or

in aggregation. The grains are

translucent white in colour,

highly refractive and blackish

blue upon iodine staining.

There are no changes in

the shape, colour or

nature of the starch

grain in comparison to

RV

There are no changes

in the shape, colour or

nature of the starch

grain in comparison to

RV



Fig – 5: Transverse section of Mastu Fig – 6: TLC profile of raw and Shodhita

Shodhita Vacha (MV) (20X) Vacha samples under 254 nm;

Ph. – Phloem; Xy. – Xylem; Track 1 - [RV]: raw Vacha;

OC. – Oil cell; S. – Starch grain; Track 2 - [KV]: Ksheera Shodhita Vacha;

OR. – Oleoresin Track 3 - [MV]: Mastu Shodhita Vacha;

Track 4 - [S]: Standard marker compound β-asarone.

Powder microscopy:

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

seen scattered but were comparatively less in

number when compared to raw Vacha. Other

characteristics observed were similar to raw

Vacha.

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



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. Histo-

chemical 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 Results

RV KV MV

Loss on drying at 105°C 14.8 16.12 21.2

Ash value 6.2 10.57 10.63

Acid insoluble Ash 0.56 0.48 0.98

Water soluble extractive 21.02 14.69 15.10

Methanol soluble extractive 15.54 11.49 13.38

pH 4.23 5.19 4.40

Table - 3: Phytochemical tests for various functional groups

Functional groups Tests performed Results

RV KV MV

Carbohydrates Molisch’s test + + +

Flavonoids Shinoda test + + +

Steroids LB reagent + + +

Glycoside Molisch’s test + + +

Alkaloids Dragendroff’s test

Mayer’s reagent test + + +

Saponin Distilled water − + +

Tannin Neutral FeCl3 + + +

Table - 4: Histochemical tests

Test Reagent Colour observed

RV KV MV

Starch Iodine Dark blue Dark blue Dark blue

Tannin Ferric chloride Brown Brown Yellow

Saponin Conc. H2SO4 Light yellow Light yellow Light yellow

Fat Sudan III Crimson red Crimson red Pink

Sugar 20%aq NAOH Yellow Yellow Light yellow

Alkaloids Dragendroff’s reagent Orange Orange Orange



Table - 5: Fluorescence analysis of rhizome powders of different Acorus samples in daylight

Sl.

no Treatment category

Acorus samples

RV KV MV

1. Powder + 1N NaOH Fossil Milk toffee Golden aura

2. Powder + 1N NaOH (alcohol) Peach rose Peach melba Butter scotch

3. Powder + 1N Hcl Pale Dawn Yellow iris Candle wick

4. Powder + 1:1 H2SO4 Hip purple Earth song Olive grove

5. Powder + 1:1 HNO3 Wild yellow Fairy glitter Yellow scoop

6. Powder + Acetone Cool grey Mellow orange Sky mimic

7. Powder + Alcohol (ethanol) Cool grey Blue harmony Tear drop

8. Powder + Benzene Cool grey Camphor Camphor

9. Powder + Chloroform Cool grey Water Water

10. Powder + Ammonia Majestic purple Spice tree Mahogany 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)

Sl.


Acorus samples

RV KV MV

1. Powder + 1N NaOH Divine pink First blush First blush

2. Powder + 1N NaOH (alcohol) Misty meadow Green wisp Firefly flicker

3. Powder + 1N Hcl Sky pink Pale ivory Wheat spring

4. Powder + 1:1 H2SO4 Passion fruit Majestic purple Hip purple

5. Powder + 1:1 HNO3 Soft breeze Peach organza Touch of paprika

6. Powder + Acetone Twilight sky Sassy violet Frosted rose

7. Powder + Alcohol (ethanol) Twilight sky Washout Twilight sky

8. Powder + Benzene Twilight sky Twilight sky Washout

9. Powder + Chloroform Twilight sky Pale blush Pink seduction

10. Powder + Ammonia Hip purple Dusky beauty Black grape

Table - 7: Fluorescence analysis of rhizome powders of different Acorus samples in Long UV

(366nm)

Sl.


Acorus samples

RV KV MV

1. Powder + 1N NaOH Soft focus Evening moon Soft honey

2. Powder + 1N NaOH (alcohol) Burst of spring Sand bed Sunny sands

3. Powder + 1N Hcl Ivory coast Mist Blank canvas

4. Powder + 1:1 H2SO4 Dynamic Lakeside Blue lake

5. Powder + 1:1 HNO3 Malabar hills Deep sea Blue weed

6. Powder + Acetone Angel harp Peach blossom Mellow orange

7. Powder + Alcohol (ethanol) Blank canvas Crescent Crescent

8. Powder + Benzene Soft whisper Fringe green Meadow mist

9. Powder + Chloroform Soft breeze Soft breeze Touch of fuschia

10. Powder + Ammonia Red wood Berry bloom Berry bloom



Shodhana procedure did not affect the β-

asarone 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 Rf values

RV KV MV β-

asarone

Short UV

(254 nm)

0.05, 0.19, 0.44 [3]* 0.05, 0.15, 0.20, 0.44

[4]

0.05, 0.20, 0.23,

0.44 [4]

0.44

Long UV

(366 nm)

0.05, 0.10, 0.14, 0.22,

0.27, 0.31, 0.41, 0.46

[8]

0.05, 0.10, 0.15,

0.19, 0.21, 0.31, 0.41

[7]

0.05, 0.10, 0.15,

0.20, 0.25, 0.43

[6]

-

Vanillin

sulphuric reagent

0.05, 0.1, 0.25, 0.44,

0.52 [5]

0.06, 0.12, 0.27,

0.40, 0.44 [5]

0.05, 0.11, 0.26,

0.41, 0.44 [5]

0.44

* Parenthesis shows total number of spots

Fig – 7: TLC profile of raw and Shodhita Fig – 8: TLC profile of raw and Shodhita

Vacha Vacha samples under 366 nm samples after derivatization



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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STUDY OF UNIDENTIFIED PLANTS FROM RASA RATNA SAMUCCHAYA

Pampattiwar S P1*, Bulusu Sitaram

2, Paramkusa Rao M

3

1P.G. Scholar – final Year, P.G. Dept. of Dravyaguna, T.T.D’S S.V. Ayurveda College, Tirupati, Andhra

Pradesh, India 2Professor, Dept. of Dravyaguna, T.T.D’S S.V. Ayurveda College, Tirupati, Andhra Pradesh, India 3P.G. Dept. of Dravyaguna, T.T.D’S S.V. Ayurveda College, Tirupati, Andhra Pradesh, India

*Corresponding Author: Email: [email protected]; Mobile: +91 9700307493


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

Review article

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



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 bio-

availability 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 Rasa-

Vagbhata 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 11

th

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.


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 97

th

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 64

th

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



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

plant contain some saponins in addition to its

strong acid like Carayagenol – E. (Kokate,

2008). This is quite helpful in the ‘Ratna-

dravana’ 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: 1 Sesbania grandiflora Linn Fig: 2 Careya arborea Roxb Fig. 3 Urginia indica kunth

(Rakta-agastya) (Sthula Kumbhiphala) (Vajrakanda)

Vajrakanda [(RRS 2nd

chapter 66th verse,

96th verse); (Rasa Vagbhata, 13

th Cent. AD)]

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).

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

bulbs of Urginia in this type of processing.

(fig.3)

Nagini [(RRS 11th chapter 89

th verse); (Rasa

Vagbhata, 13th Cent. AD)]

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.



Plant appears like a snake holding its hood up

in an attacking position. (fig.4)

Sarpakshi – [(RRS 11th chapter 53

rd 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

grows in Western Himalayas and is preferred in

these regions. It is also known for its anti-

poisonous actions in local areas.

Mahakali – [(RRS 10th chapter 71

st 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.4 Arisaema murrayi Fig.5 A. candidissimus Fig.6.Trichosanthes tricuspidata Lour

(Nagini) (Sarpakshi) (Mahakali)



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.



REFERENCES

Vaidya Bapalal, (2010), Some Controversial

drugs of Indian medicine 3rd edn,

Chaukhamba Orientalia, Varanasi, Pg-

83, 148, 320.

Sitaram Bulusu, (2006), Bhavaprakash

Nighantu 1st edn, Chaukhamba

Orientalia, Varanasi, Pg-170, 297, 302,

333.

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, Pg-

100,136,192,193, 489, 508, 543, 686,

810.

Sharma P.V. (1985), Dravyaguna Vigyan

Vol.5, Chaukhamba Bharathi Academy,

Varanasi, Pg-170, 258, 271, 291, 321,

323, 336

Pade Shankardaji Shastri, (2009), Vanaushadhi

Gunadarsha vol-1-7, Pg- 5,105, 258,

417.

Dole A Vilas, (2006), Rasashastra,

Chaukhamba Sanskrit Pratisthana, Pg-

97

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 of

Pharmacognosy, Nirali Prakashan 1st

edn. Pg- 8.46





HERBAL DRUG SWIETENIA MAHAGONI JACQ. - A REVIEW

Khare Divya1*, Pradeep H R

2, Kumar K K

3, Hari Venkatesh K.R

4, Jyothi T

5

1PG Scholar, Department of Dravyaguna, ALN Rao Memorial Ayurvedic Medical College, Koppa,

Karnataka, India 2Professor, Department of Dravyaguna, ALN Rao Memorial Ayurvedic Medical College, Koppa

3, 4Lecturer, Department of Dravyaguna, ALN Rao Memorial Ayurvedic Medical College, Koppa

5Research Assistant, ALN Rao Memorial Ayurvedic Medical College, Koppa

*Corresponding Author: [email protected]; Mobile: +919731068468


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.

Review article

Cite this article:

Khare Divya1, Pradeep H R

2, Kumar K K

3, Hari Venkatesh K R

4, Jyothi T

5 (2012),

HERBAL DRUG SWIETENIA MAHAGONI JACQ. - A REVIEW,

Global J Res. Med. Plants & Indigen. Med., Volume 1(10); 557–567



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



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.



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, 6-

methyl-5-(1-methylethyl) (2.03%) octadecanoic

acid, 9,10,12-trimethoxy (1.90%); 1,3-

dioxalane, 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).



Physico-chemical characters of the seed oil were also determined and are as follows:

Parameters Result

Colour Brown

Moisture 24.60 %

Specific gravity at 30° C 0.9334

Acid value 10.92

Free fatty acid (FFA) 5.49 % (as oleic acid)

Saponification value 191.27

Iodine value 94.4

Unsaponifiable matter 1.49 %

Oil (dry basis ) 53.75 %

Polenske value 0.35

Solvent partitioning followed by column

chromatography of the Methanolic extract of

the seeds of S. mahagoni Jacq. afforded two

limonoids, swietenolide (Fig. 1) and 2-

hydroxy-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 single-

crystal 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



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

water−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



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 adiopocyte-

differentiating 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 and

Antipyretic study (Ghosh S et al., 2009)

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 anti-

inflammatory effect of SMSE was observed in

acute (carrageenan and arachidonic acid-

induced paw edema in rat and croton oil-

induced 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 anti-

inflammatory 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 dose-

dependent manner as compared to the control.



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.

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

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

activity against the groundnut rust Puccinia

arachidis. 6-acetylswietenine and 6-acetyl-3-

tigloylswietenolide from S. mahogany Jacq.

effectively reduced the number of rust pustules

on detached groundnut leaves. (T. R.

Govindachari et al., 1999)

Anti-ulcer activity:

A study was performed to evaluate the anti-

ulcer 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 pre-

treated 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 pre-

treated 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)

PAF inhibition activity:

The ether extract from the seeds of S.

mahagoni Jacq. was found to inhibit platelet-

activating 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-O-

acetylswietenolide, 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) anti-

PAF activity (Kadota S et al., 1990).



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, Anti-

microbial Activity, Anti-oxidant activity, Anti-

ulcer activity, Anti-fungal activity, Anti-

inflammatory, 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.

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