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An International, Peer Reviewed, Open access, Monthly E-Journal

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INDEX – GJRMI, Vol.2, Iss. 5, May 2013

MEDICINAL PLANTS RESEARCH

Bio-Chemistry NATURAL PLANT PRODUCT BERBERINE/CISPLATIN BASED RADIOTHERAPY FOR

CERVICAL CANCER: THE NEW AND EFFECTIVE METHOD TO TREAT CERVICAL CANCER

Komal, Singh Mayank, Deshwal Vishal kumar 278–291

Bio-technology PHYTOCHEMICAL SCREENING OF SECONDARY METABOLITES OF EUPHORBIA

NERIIFOLIA LINN.

Chouhan Leela, Bhatt Shashank, Dhyani Suresh 292–297

Ethno-Medicine

TRADITIONAL MEDICINES OF HERBAL ORIGIN PRACTICE BY THE ADI TRIBE OF EAST

SIANG DISTRICT OF ARUNACHAL PRADESH, INDIA

Das Moushumi, Jaishi Anju, Sarma Hirendra N 298–310

Bio-technology PRELIMINARY PHYTOCHEMICAL SCREENING OF SECONDARY METABOLITES OF

ADHATODA VASICA NEES. FLOWERS

Chouhan Surksha, Bhatt Shashank, Dhyani Suresh 311–316

Review Article

HERBAL MEDICINES FOR DEPRESSION AND ANXIETY: A COMPREHENSIVE STATE OF

THE ART REVIEW

Patel Shanti, De Sousa Avinash 317–336

Review Article A BRIEF REVIEW ON NONI (MORINDA CITRIFOLIA L.) - A HERBAL REMEDY FOR BETTER

HEALTH

Patel Swetal, Krishanamurthy R 337–347

INDIGENOUS MEDICINE

Ayurveda – Dravya Guna DEVELOPMENT OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS FOR

AUTHENTICATION OF RIVEA HYPOCRATERIFORMIS (DESR.) CHOISY

Borkar Sneha D, Naik Raghavendra, Harisha C R, Acharya R N 348–356

Ayurveda – Dravya Guna

ANTIMICROBIAL EVALUATION OF CROTON ROXBURGHII BALAK. (EUPHORBIACEAE)

STEM BARK

Patel Esha, Padiya RH, Acharya RN 357–364

Ayurveda – Kaya Chikitsa

AN ESTIMATION OF HUMIC SUBSTANCES IN AN AYURVEDIC HERBOMINERAL DRUG

SHILAJATU (ASPHALTUM) AS PART OF PHYTO-PHARMACEUTICAL STANDARDIZATION

Akarshini A M, Renuka, Shukla V J, Baghel M S 365–373

Ayurveda – Shalya Tantra

MANAGEMENT OF ARBUDA (CANCER) WITH HERBOMINERAL FORMULATION - A PILOT

STUDY

Mahanta Vyasadeva, Dudhamal T S, Gupta S K 374–379

Ayurveda – Review Article - Moulika Siddhanta

IMPORTANCE OF UPAYOGASAMSTHA (DIETETIC RULES) IN RELATION TO DIGESTION OF

THE FOOD

Avhad Anil D, Vyas H A, Dwivedi R R 380–385

Ayurveda – Review Article - Moulika Siddhanta

CONCEPT OF VYADHIKSHAMATVA (IMMUNITY) AND ITS RELATIONSHIP WITH BALA

(VITAL STRENGTH)

Sharma Mahesh Kumar 386–391

Ayurveda – Review Article - Dravya Guna

A REVIEW ON VARIETIES OF ARKA - CALOTROPIS PROCERA (AITON) DRYAND. AND

CALOTROPIS GIGANTEA (L.) DRYAND.

Poonam, Gaurav Punia 392–400

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FLOWER & IMMATURE FRUIT OF NONI (MORINDA

CITRIFOLIA. L. ), OF THE FAMILY RUBIACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

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

NATURAL PLANT PRODUCT BERBERINE/CISPLATIN BASED

RADIOTHERAPY FOR CERVICAL CANCER: THE NEW AND EFFECTIVE

METHOD TO TREAT CERVICAL CANCER

Komal1, Singh Mayank

2, Deshwal Vishal kumar

3*

1, 2

Department of Biochemistry, All India Institute of Medical Science, New Delhi, India. 1, 3

School of Life Sciences, Singhania University, Pachri Bari, Rajasthan, India.

*Corresponding Author: E-mail: vishal_deshwal@rediffmail.com; Mobile: +919897538555

Received: 28/03/2013; Revised: 12/04/2013; Accepted: 15/04/2013

ABSTRACT

Cervical cancer is the site of excessive inflammation which leads to extensive DNA damage and

thus promotes carcinogenesis. Existing treatment regime for cervical cancer is radiotherapy along

with platinum based drugs like cisplatin and carboplatin but it is associated with various side effects

to normal cells and problem of radio-resistance. Berberine is a natural chemo-preventive agent

extracted from Berberis aristata that has been shown to suppress and retard carcinogenesis by

inhibiting inflammation. In this study we compared the cisplatin based radiotherapy with a

Berberine/cisplatin based radiotherapy in cervical cancer in vitro. Treatment of cervical cancer cell

lines SiHa and CaSki with Berberine/cisplatin combination followed by treatment by ionizing

radiation (IR) resulted in increased apoptosis in comparison to cisplatin based radiotherapy. The

combination therapy of Cisplatin/Berberine/IR resulted in upregulation of pro apoptotic proteins like

Bax, p73 and down regulation of anti apoptotic Bcl Xl, inflammatory Cox 2, Cyclin D1 accompanied

by increase in activity of Caspase -9 and -3. Reduction in Telomerase activity was also seen in all the

HPV positive cells.

KEYWORDS: Cervical cancer, Radiotherapy, Berberine, Cisplatin

Research article

Cite this article:

Komal, Singh M, Deshwal V K (2013), NATURAL PLANT PRODUCT BERBERINE/CISPLATIN

BASED RADIOTHERAPY FOR CERVICAL CANCER: THE NEW AND EFFECTIVE METHOD TO

TREAT CERVICAL CANCER, Global J Res. Med. Plants & Indigen. Med., Volume 2(5): 278–291

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

INTRODUCTION

Cervical cancer remains one of the major

cancers amongst women worldwide with a high

rate of mortality (Ciesielska et al., 2012).

Radiotherapy in combination with cisplatin

remains the treatment of choice in majority of

cases in which the cancer is locally advanced.

Currently the chemotherapeutic drugs cisplatin

and 5-Fluorouracil are used as radio-sensitizers

along with ionizing radiation (IR) for the

treatment of cervical cancer (Rosa et al., 2012).

Nevertheless, therapeutic results are far from

optimal, so new and safer therapeutic

combinations need to be investigated which

specifically target cervical cancer cells with

minimal toxicity to normal tissue. Although

chemo radiotherapy is more effective as

compared to radiotherapy alone, it is associated

with dose limiting toxicities like

gastrointestinal and hematological toxicities

(Tan et al., 2012). Natural products offer an

excellent alternative for therapeutic use as

opposed to synthetic compounds because of

their relatively well established safety profile

(Deshwal, 2012; Kumar et al., 2012; Makhloufi

et al., 2012). Several natural products are being

tested as potential radio sensitizers. Berberine

is a natural compound that allows prevention,

suppression and retardation of carcinogenesis.

Berberine [1, 7-bis- (4 –Hydroxy-3-

methoxyphenyl) -1, 6 heptadiene -3, 5-dione] is

a major constituent of product extracted from

the rhizome of the plant Berberis aristata found

in South and Southeast tropical Asia. Berberine

(Plate 1) has been shown to be a potent chemo-

preventive agent inhibiting tumor progression

against skin, oral, intestinal, breast, colon and

prostate cancer (Diogo et al., 2011). Berberine

has been shown to confer radiosensitizing

effect in prostate cancer cells, squamous cell

carcinomas (Vuddanda et al., 2010; Tillhon et

al., 2012) and recently in HeLa and SiHa,

human cervical cancer cells (Javvadi et al.,

2008). The major problem with cervical cancer

is that the cancer cells become increasingly

radioresistant due to activation of various

antiapoptotic genes/cascades resulting in

therapy failure, and the standard chemo-

radiotherapy regime is unable to address this

problem (Aggarwal et al., 2006). The goal of

the present study was to compare the standard

chemo-radiotherapy regime comprising of

cisplatin/IR with the cisplatin/Berberine/IR

combination therapy and assess its effect on

protein which confers radio resistance to

cervical cancer cells in vitro.

Plate 1: Chemical structure of Berberine

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

MATERIAL AND METHODS

Cell culture and chemicals

Human cervical cancer SiHa and Ca Ski

cells were obtained from National Centre for

Cell Sciences, Pune, India and were maintained

in either Dulbecco’s modified Eagle’s medium

or RPMI1640 (Sigma, USA) supplemented

with 10% (v/v) heat-inactivated fetal bovine

serum (Hyclone), antibiotics, in a humidified

atmosphere of 95% air and 5% CO2 at 37ºC.

Cells were exposed to varying Ionizing

radiation (IR). Antibodies against p73, Bcl xl,

Bax, Cyclin D1, AIF and Cox 2 as well as

secondary AP conjugated antibodies were

obtained from Santa Cruz, USA. Berberine was

obtained from Sigma, USA

Flow cytometry

Cells (1 × 104

cells) were treated with

50 μM and 75 μM Berberine for 24 hrs and

then harvested. Cells were fixed in 70% ethanol

and left overnight at −20ºC. Cells were then

washed with PBS and incubated in staining

solution (20 μg/ml propidium iodide, 50 μg/ml

RNAse, 0.1% Triton X-100 and 0.1 mM

EDTA) for 2 hrs at 4ºC in dark. The DNA

content of the cells was measured by flow

cytometer (Becton Dickenson, USA) using

Diva software.

Assay of telomerase activity

This was measured using the PCR-ELISA

kit based on TRAP (Telomerase repeat

amplification protocol) assay (Roche Molecular

Bio-chemicals, Germany). The samples were

lysed and an aliquot containing 2μg protein was

used for the assay. Telomerase positive

embryonic kidney cell line (HEK-293) was

used as positive control while heat inactivated

HeLa extract was used as negative control. The

telomerase activity was detected and expressed

as relative units (RU) (Khanna et al., 2003).

Western blot analysis

The level of expression of various proteins

was determined in control and treated cells by

Western blotting as described previously

(Singh et al., 2007). Briefly, cells were washed

twice in PBS (Phosphate buffered saline) and

lysed in RIPA lysis buffer. Total protein was

determined by the Bradford assay. Equal

amount of protein was loaded and run on 10–

15% SDS-polyacrylamide gel. The proteins

were transferred to a nitrocellulose membrane.

The membrane was blocked with 5% BSA

(Bovine serum albumin), followed by

hybridization with respective primary and

secondary antibody. Final detection was

performed with BCIP/NBT BCIP (5-bromo-4-

chloro-3'-indolyphosphate p-toluidine salt/

NBT (nitro-blue tetrazolium chloride),

substrate (Promega, USA). The bands were

analyzed and quantified using ά image scanner

densitometer and normalized with β actin

control. The density of control was taken as 1

and results of treatment were expressed in

relation to the control as relative unit (RU).

Assay of Caspase -3, -9 activities

Caspases-3 and -9 activity were measured

by the direct assay for Caspase enzyme activity

in the cell lysate using synthetic fluorogenic

substrate (Ac-DEVD-AFC; substrate for

caspase 3; MBL Bioscience, USA; Ac-LEHD-

AFC, substrate for Caspase 9; Genotech, USA)

as described by the manufacturer. Amount of

fluorogenic AMC/AFC moiety released was

measured using a spectro-fluorimeter

(ex.380 nm, em.420–460 nm for Caspase -3;

ex.400 nm, em.490–520 nm for Caspase-9).

The results are expressed in arbitrary

fluorescence units/mg protein (Singh and

Singh, 2008).

Statistical analysis

Results were expressed as mean of three

individual experiments ± standard deviation

which was calculated using Microsoft excel.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

RESULTS

Effect of ionizing radiation (IR) on cervical

cancer cells

For treatment with ionizing radiation HPV

positive cell lines were chosen i.e HPV 16

positive cell lines SiHa, CaSki which vary in

their HPV copy number. Cells were treated

with ionizing radiation doses ranging from 1Gy

to 3Gy, to assess their effect on these cell lines.

Gradual increase in apoptosis was found on

increasing radiation dose from 1Gy to 3Gy (Fig

1) but the sensitivity to ionizing radiation

varied from one cell line to another

demonstrating the variation of resistance to

radiation with cell type. Our data indicates that

Ionizing radiation dose of 1Gy results in 15.5%

apoptosis in SiHa and 18.5% apoptosis in

CaSki cells. Treatment with 2 Gy radiation

dose resulted in increase in apoptosis in both

the cell lines, in which SiHa showed 23.6% and

CaSki showed 19.08% apoptosis respectively,

hence demonstrating the radio-resistance.

Escalation of radiation dose to 3Gy resulted in

marginal increase in apoptosis 26.23% in SiHa

and 22.04% in CaSki cells. Since there was

only a marginal increase in apoptosis on

increasing radiation dose from 2Gy to the dose

of 3Gy, hence we settled for the radiation dose

of 2Gy for all the experiments. This dose was

used in combination treatments with cisplatin

and Berberine also.

Effect of Cisplatin and Cisplatin / IR on

cervical cancer cells

Platinum compounds like cisplatin in

combination with radiotherapy are used

extensively for treatment of cervical cancer so

we tried to mimic this situation in vitro on the

cell lines. First we assessed the effect of

cisplatin alone on these cell lines and in

combination with IR (2Gy). Treatment of SiHa

and CaSki with 5μg/ml cisplatin for 24 hrs

resulted in 19.4% and 12.95% apoptosis

respectively (Fig 2). Coupling this dose of

cisplatin with 2 Gy ionizing radiation (IR)

resulted in increase in apoptosis to 23.4% and

16.74% in SiHa and CaSki cells respectively.

Escalation of cisplatin dose to 10 µg/ml for 24

hrs resulted in increase in apoptosis in SiHa

and CaSki to 32.65% and 35.4%, whereas in

combination with 2Gy IR it increased to

35.25% and 38.42%. Thus a chemoradiation

dose of 10 μg/ml CP (cisplatin) / 2Gy IR was

selected for further experiments (Fig 2).

Effects of Berberine / CP / IR on cervical

cancer cells

Berberine has been established as a

potential chemotherapeutic agent in various

clinical trials and has been found to be well

tolerated at higher doses but its bio-availability

remains a major problem. Howell et al., (2007)

have summarized based on in vitro and in vivo

studies and clinical trials on natural products

like Curcumin and Berberine. It is shown that

the concentrations of natural products that were

achievable in the plasma of patients were only

at a lower micromolar range; hence, they have

suggested that for in vitro studies concentration

of berberine in less than 50 μM range do not

have any physiological relevance. The

significant radio-sensitization achieved by the

moderate dose of berberine at relevant doses in

vitro (2–6 Gy) has promising implications for

improving radiation therapy especially in radio

resistant tumors such as the tumors of the

uterine cervix. Hence we coupled the above

dose of 10 μg/ml CP with 50 μM berberine.

The cells were treated to this combination dose

for 24 hrs followed by 2Gy IR. An abrupt

increase in apoptosis was seen in the cell lines

with SiHa showing 49.8% apoptosis which was

higher than 35.25% apoptosis observed with

10 μg/ml CP/ 2Gy IR (Ionizing Radiation)

dose, used as standard therapy. Similarly,

CaSki showed an increase in apoptosis to

41.65% on treatment with the combination dose

against apoptosis achieved on treatment with

CP/ IR combination (Fig 2). Clearly our results

show that Berberine is selectively increasing

apoptosis in these cell lines. We then compared

the effect of Berberine/ CP combination with

CP/ IR, on various proteins involved in

apoptosis both pro-apoptotic and anti-apoptotic,

radio resistance and inflammatory response.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

Fig 1

Microscopic and Flow cytometric analysis of apoptosis in SiHa (Fig 1A) and Ca Ski (Fig 1B) cells on treatment with IR

(1-3Gy).

Fig 2

Microscopic and Flow cytometric analysis of apoptosis in SiHa (Fig 2A) and Ca Ski (Fig 2B) cells on treatment with

CP/IR and 75µM Berberine/10µgm Cisplatin/IR for 24 hrs . The percentage Apoptosis shown in the bar diagram is

mean ± SD of three individual experiments

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

Effects of berberine / Cisplatin / IR and

Cisplatin / IR on protein p73

The protein p53 is a well-known tumor

suppressor protein that functions primarily as a

transcription factor, initiates cell cycle arrest

and apoptosis after genotoxic stress. Both the

cell lines used in the current study are HPV

(Human Papilloma Virus) positive cell lines

which encodes for viral protein E6 which

causes ubiquitin mediated degradation of p53

so another member of p53 family p73 becomes

important in these cells. It has been shown that

p73 plays an important role in Hydrogen

peroxide induced apoptosis (Singh et al., 2007;

Singh and Singh 2008). Since radiation induced

apoptosis involves ROS (Reactive oxygen

species) so we determined the effect of

cisplatin/IR and cisplatin/Berberine/IR

combination on p73 in these cell lines. In SiHa

and CaSki cells there was 36% and 44%

increase in p73 expression on treatment with

cisplatin/IR but on treatment with

Berberine/cisplatin/IR the expression of p73

increased to 54%, 98% (Fig.3B) demonstrating

that p73 level changes in response to Berberine.

Javvadi et al., 2008 have shown that ROS plays

an important role in Berberine/IR mediated

apoptosis in SiHa cells. Our results are in

agreement with their findings and suggest that

cisplatin/ Berberine /IR induced activation of

p73 which may involve reactive oxygen species

(ROS).

Effects of Berberine / Cisplatin / IR

combination on cyclin D1

Cyclin D1 is involved in cell-cycle arrest in

DNA-damage response. Cyclin D1 has been

shown to be induced by low-dose ionizing

radiation in human keratinocytes with an

adaptive radio-resistance (Ahmed et al., 2008).

On exposure of SiHa and CaSki to Cisplatin/IR

there was a 22% and 30% decrease in level of

cyclin D1 which was reduced to 49% in case of

CaSki but remained unchanged in SiHa on

treatment by Berberine/cisplatin/IR, thereby

demonstrating variation from cell type to cell

type (Fig.3A)

Fig 3

Effect on the level of Cyclin D1 (Fig 3A) protein and apoptotic p73 (Fig 3B) protein in SiHa and Ca Ski cells by western

blotting .Lane 1 control, Lane 2 CP 10μg for 24 hrs followed by 2 Gy IR, Lane 3 CP 10μg/75µM Berberine for 24 hrs

followed by 2Gy IR (Relative unit, RU). The results shown are mean ± SD of three individual experiments in the bar

diagram.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

Berberine / Cisplatin / IR combination

treatment resulted in activation of

proapoptotic Bax and reduction in level of

Bcl XL

Cells on exposure to IR along with

chemotherapeutic agent results in DNA damage

and if this is severe, p53 and its counterparts

like p73 may trigger programmed cell death by

means of pro-apoptotic genes such as Bax and

inhibition of anti-apoptotic Bcl XL. It has been

demonstrated that radio resistant laryngeal

cancer was associated with increased Bcl-2 and

Bcl-XL expression and loss of Bax expression.

Bcl-2 family has been proposed to predict

radiotherapy outcome (Nix et al., 2005). The

association between expression of Bcl-2, Bcl-

XL and Bax with radio resistant cancer

suggests a potential mechanism by which

cancer cells avoid the destructive effects of

radiotherapy. We probed the effect of

Berberine/Cisplatin/IR combination on level of

Bax in SiHa and CaSki cells. The Bax

expression increased to 45% and 52% on

treatment with cisplatin/IR, but this increased

to 46% and 68% with respect to control on

treatment with Berberine/cisplatin/IR

combination.(Fig.4A) There was a marginal

increase in case of CaSki and nearly no change

in case of SiHa. However, in case of anti-

apoptotic Bcl XL we obtained a 7% decrease in

its level in case of SiHa and 15% decrease in its

level in case of CaSki cells on treatment with

cisplatin/IR (Fig.4B). In contrast, on treatment

with Berberine/cisplatin/IR we obtained a

substantial decrease in level of BclXL i.e. 22%

in SiHa and 26% in CaSki.

Berberine / Cisplatin / IR combination

treatment did not affect the expression of

COX 2 and AIF

COX-2 has been implicated in

carcinogenesis of systemic cancers. COX-2

inhibition has been shown to increase the radio-

sensitivity of various tumors. Results from the

present study demonstrate that on treatment of

SiHa and CaSki cells with cisplatin/IR there

was a 36% and 13% increase respectively in

expression of COX 2 but treatment of these

cells with Berberine/cisplatin/IR resulted in 8%

and 6% decrease respectively in level of

COX2(Fig.5A). These results indicate that

though Berberine/cisplatin/IR based therapy is

repressing COX 2 the effect is only marginal

and it varies with cell type. AIF expression was

unaltered remaining nearly the same on both

the treatments in both the cell lines (Fig 5B),

suggesting its noninvolvement.

Berberine / Cisplatin / IR treatment

enhances activation of both Caspase-3 and -9

Caspase -3 and -9 have been implicated to

play an important role in mitochondrial

mediated apoptosis by causing activation of

Caspase activated DNAse, finally causing

degradation of DNA. Hence we compared the

effect of Berberine/cisplatin/IR and cisplatin/IR

on Caspase -3 and -9 activities. The results

show that in SiHa and CaSki cells there was

1.19 and 1.23 fold increase in activity (Arbitary

fluroscence units, Afu converted to fold change

compared to control) in activity of Caspase -3

on treatment with cisplatin/IR, while there was

a 1.55 and 2.27 fold increase on treatment with

Berberine/cisplatin/IR (Fig 6A). Clearly

Berberine is causing activation of caspase -3.

To assess whether enhanced apoptosis was

being mediated through mitochondrial

pathway, we also studied the effect on caspase-

9 activity. SiHa and CaSki cells showed 0.29

and 0.11 fold increase in activity of Caspase -9

on treatment with cisplatin/IR while there was a

0.38 and 0.78 fold increase on treatment with

Berberine/cisplatin/IR. Thus Berberine appears

to be mediating apoptosis through

mitochondrial pathway (Fig 6B).

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

Fig 4

Effect on the level of antiapoptotic Bcl XL (Fig 4A) protein and apoptotic Bax (Fig 4B) protein in SiHa and CaSki cells

by western blotting .Lane 1 control, Lane 2 CP 10μg for 24 hrs followed by 2 Gy IR, Lane 3 CP 10μg/75µM Berberine

for 24 hrs followed by 2Gy IR (Relative unit, RU). The results shown are mean ± SD of three individual experiments in

the bar diagram.

Fig 5

Effect on the level of AIF (Apoptosis inducing factor) (Fig 5A) and inflammatory COX 2 (Fig 5B) protein in SiHa and

Ca Ski cells by western blotting .Lane 1 control, Lane 2 CP 10μg for 24 hrs followed by 2 Gy IR, Lane 3 CP

10μg/75µM Berberine for 24 hrs followed by 2Gy IR (Relative unit, RU). The results shown are mean ± SD of three

individual experiments in the bar diagram.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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

Fig 6

Caspase -3 (Fig 7A) and -9 (Fig 7B) activity (Arbitrary fluorescence unit, Afu) after treatment with CP 10μg for 24 hrs

followed by 2 Gy IR and 10μg/75µM Berberine for 24 hrs followed by 2Gy IR in SiHa and Ca Ski. The results are mean

± SD of three individual experiments.

Fig 7

Effect of 10 μg CP for 24 hrs followed by 2 Gy IR and CP 10 μg/75µM Berberine for 24 hrs followed by 2Gy IR on

telomerase activity in SiHa and Ca Ski cells (RU). The results shown are mean ± SD of three individual experiments

Berberine / Cisplatin / IR combination

causes reduction in activity of Telomerase

with respect to Cisplatin/IR

Telomerase activation plays a critical role

in tumor growth and progression, in part by

maintenance of telomere structure. Indeed, the

ubiquitous expression of telomerase in human

cancers makes telomerase a promising target

for cancer therapy. We assessed the effect of

Berberine/cisplatin/IR combination dose on

telomerase with respect to cisplatin/IR

combination dose by using PCR/ELISA

methodology which assesses activity of

hTERT. We observed that on treatment of SiHa

and CaSki there was 22% and 39% decrease in

telomerase with cisplatin/IR while there was an

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82% and 75% decrease in telomerase activity

on treatment with Berberine/cisplatin/IR

(Figure 7). This data suggests that berberine

based therapy causes substantial decrease in

telomerase activity and this therapy can be a

potent telomerase targeted approach for

treatment of cervical cancer.

DISCUSSION

Cervical cancer remains one of the major

killers amongst women worldwide. Chemo/

radiotherapy regime which is cisplatin based

radiotherapy is used for the treatment of

advanced cervical cancer in India. Evidence

shows that most of the chemotherapeutic drugs

used in current clinical practice are radio-

sensitizers. Several newer cytotoxic agents with

radio-sensitizing properties are being tried in

combination with cisplatin but their use is

generally limited by dose related toxicities

(Rosa et al., 2012). Natural products open a all

new avenue for treatment of cancer as they are

generally tolerated at high doses. Animal

studies have confirmed the anti-tumorigenic

activity of natural products like Berberine and

curcumin (Howells et al., 2007). Phase I

clinical trials on curcumin showed that it is safe

to humans up to 12,000 mg/day when taken

orally (Cheng et al., 2001; Sharma et al., 2001;

Lao et al., 2006) and caused histological

improvement of precancerous lesions in

patients, suggesting that it is biologically active

at these doses (Cheng et al., 2001). Previous

reports have indicated that Berberine confers

radio-sensitizing effects in prostate and

squamous cell carcinoma cell lines, and

recently in cervical cancer cell lines HeLa and

SiHa. Thus we evaluated the effect of

Berberine in combination with cisplatin/IR and

compared it with cisplatin/IR treatment. Our

results suggest that Berberine/cisplatin/IR

based therapy is likely to be more effective and

safer to treat cervical cancer, as Berberine is

well tolerated in humans, even at high doses.

We present in vitro evidence that this approach

targets radio-resistance and anti-apoptotic

proteins in a much more specific way compared

to the standard cisplatin/IR based therapy.

When cells are exposed to clinically

relevant doses of ionizing radiation it causes

DNA damage by generation of reactive oxygen

species (ROS). This DNA damage causes a

rapid ROS dependent activation of pro-

apoptotic and anti-apoptotic cascade which

involves activation of p53 family of genes

acting downstream to trigger apoptosis if the

damage is not repairable. The tumor cells are

dynamic with respect to their reliance on

specific cell signaling pathway to exist and

rapidly adapt to repeated toxic challenges in an

attempt to maintain tumor survival. Prolonged

inhibition of any one of these pathways

however, gives rise to lineage of cells which

become resistant to inhibitor drug, with point

mutations in the specific targeted proteins, or

by reprogramming of multiple signaling

processes within the cell (Valerie et al., 2007).

So the need for today is therapies which target

these multiple pathways and result in selective

apoptosis of cancer cells and thus deal with the

problem of radio-resistance. The cell lines used

in this study are HPV positive, in which wild

type p53 is targeted for ubiquitin mediated

degradation so other members of p53 family

like p73 become particularly important in these

cell lines (Singh et al., 2007; 11 Singh and

Singh 2008). We observed the activation of p73

in cisplatin/IR treated sample but this activation

was much more in berberine/cisplatin/IR

treated samples which indicates that

combination of Berberine is causing much

more activation of p73 probably through

mediation of ROS. This has been demonstrated

recently in some studies in which natural

products like Berberine were shown to be a

pro-oxidant in combination with ionizing

radiation, and its radio-sensitizing properties

were attributed to ROS mediated signaling

(Javvadi et al., 2008).

Bax and Bcl XL are members of Bcl 2

family of proteins which control apoptosis and

are associated with regulating the

mitochondrial membrane permeability. Pro-

apoptotic proteins like Bax by translocation

from the cytosol to the mitochondria, induce

cytochrome c release, whereas Bcl-XL exerts

its anti-apoptotic activity, at least in part by

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291

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inhibiting the translocation of Bax to the

mitochondria (Mohammad et al., 2008; Ow et

al., 2008). Our results show a slight increase in

Bax and a decrease in Bcl XL on Berberine

treatment in both the cell lines. AIF (Apoptosis

inducing factor) remained unchanged thereby

suggesting its non involvement.

Caspases, a family of aspartic acid-specific

proteases, are the major effectors of apoptosis.

Once activated, caspases preside over the

ordered dismantling of the cell through

restricted proteolysis of hundreds of substrate

proteins (Ow et al., 2008). Caspase -3 has been

implicated in both the extrinsic and intrinsic

pathway of apoptosis. Our findings show

activation of caspase -3 on both the treatments

but this activation was more marked on

Berberine/cisplatin/IR treatment implicating

that cells are undergoing apoptosis through

caspase-3 mediated pathway. Similarly,

caspase -9 was activated on both the treatments

in both the cell lines, but was more marked on

Berberine/cisplatin/IR treatment, implicating

involvement of mitochondrial mediated

apoptosis pathway. Next we assayed the effect

of Berberine/cisplatin/IR on proteins involved

in radioresistance like Telomerase, Cyclin D1

and Cox 2.

Cyclin D1 is involved in cell-cycle arrest in

DNA-damage response. Cyclin D1 contributes

to regulate G1 progression by forming a

complex with different cyclin-dependent

kinases. It has oncogenic properties and is

frequently overexpressed in several human

tumor types. Cyclin D1 has been shown to be

induced by low-dose ionizing radiation in

human keratinocytes with an adaptive radio-

resistance (Ahmed et al., 2008). Our results

show a similar reduction in level of cyclin D1

on berberine treatment in both cell lines, but to

different extents, demonstrating cell to cell type

variation.

Cyclooxygenase-2 (COX-2), an enzyme

induced by pro-inflammatory cytokines,

mitogenic substances, oncogenes, growth

factors, and hypoxia, among others, is involved

in the metabolic conversion of arachidonic acid

to prostaglandins in inflamed tissues and

neoplasia. COX-2 is often overexpressed in

malignant tumors and premalignant lesions.

Because COX-2 may also be a determinant of

tumor radio-resistance, its inhibition or

inhibition of its products (prostaglandins) may

improve tumor response to radiotherapy.

Analyses of the effect of cisplatin/IR show that

COX 2 was elevated in both the cells on

treatment with cisplatin/IR indicating that this

radio-resistant marker is elevated in all the cell

lines however on treatment with berberine

cisplatin/IR COX 2 was down-regulated in both

the cell lines.

Telomeres and telomerase play a role in

the regulation of the life span of the cell.

Human cells express low levels of telomerase;

however when telomere length reaches a

critical level, abnormal activation of telomerase

can lead to immortalization and uncontrolled

proliferation (Agarwal et al., 2008; Gandellini

et al., 2007). Our result shows that Berberine

treatment caused a substantial reduction in

telomerase activity in SiHa and CaSki cervical

carcinoma cell lines. Thus Berberine is

conferring a selective advantage over cisplatin

based radiotherapy by causing marked decrease

in telomerase activity.

In summary, our data provides in vitro

evidence that supports the clinical importance

of coupling Berberine with cisplatin as an

efficient radiosenitizer for treatment of cervical

cancer as it causes enhanced activation of p73

causing further down-regulation of anti-

apoptotic Bcl XL and activation of caspase -3

and -9, resulting in enhanced apoptosis. We

also provide evidence that Berberine/cisplatin

based radiotherapy causes substantial down-

regulation of telomerase activity, Cyclin D1

and COX 2, thus acting as a potent radio-

sensitizer. This data has clinical relevance as it

highlights the importance of overcoming the

problem of radio-resistance by specifically

targeting radio-resistant proteins using

Berberine which is well tolerated in human

beings even at high doses, and thus promises to

be effective drug in future clinical trials along

with cisplatin/IR.

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ACKNOWLEDGEMENT

We would like to acknowledge DBT for

financial support to Komal. This work was also

supported by SRF grant to Mayank Singh from

CSIR. We also acknowledge the technical help

from Jyotibasu for carrying out IR Treatment at

IRCH, AIIMS.

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Source of Support: Nil Conflict of Interest: None Declared

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

PHYTOCHEMICAL SCREENING OF SECONDARY METABOLITES OF

EUPHORBIA NERIIFOLIA LINN.

Chouhan Leela¹, Bhatt Shashank²*, Dhyani Suresh3

1Department of Biotechnology, Rajiv Gandhi Govt. P.G. College, Mandsaur, Madhya Pradesh, 458001,

India. 2,3

Department of Biotechnology, NIMS University, Jaipur, Rajasthan, 303121, India. *Corresponding author: Email: shashank_bhatt2003@yahoo.co.in; Mobile: +919826840428

Received: 02/04/2013; Revised: 25/04/2013; Accepted: 02/05/2013

ABSTRACT

Metabolites play an important role for the protection of plants against various diseases. Each

secondary metabolite has a specific activity. Euphorbia neriifolia is one such medicinal plant with

maximum secondary metabolites. These metabolites were extracted from different polar solvents -

petroleum ether, chloroform, methanol, 95% ethanol and distilled water. Alkaloids, flavonoids,

glycosides, phenols, saponins, sterols, lignins, and tannins were found in Euphorbia neriifolia’s

flowers with cold percolation method. Each metabolites show effective activities against diseases.

KEY WORDS: Alkaloids, flavonoids, sterols, Euphorbia neriifolia Linn.

Research article

Cite this article:

Chouhan Leela, Bhatt Shashank, Dhyani Suresh (2013), PHYTOCHEMICAL SCREENING OF

SECONDARY METABOLITES OF EUPHORBIA NERIIFOLIA LINN., Global J Res. Med. Plants &

Indigen. Med., Volume 2(5): 292–297

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 292–297

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

INTRODUCTION

Plants have different types of therapeutic

properties by which different ailments to

mankind are dealt with. Medicinal plants

naturally accumulate different types of primary

and secondary metabolites. Different types of

medicinal plants have been used for the

treatment of diseases. The quantity of

secondary metabolites vary in different parts of

a plant and is also influenced by environmental

conditions (Aanchal Jain et al., 2012; Surendra

K. Rathore et al., 2012; Priyanka Parmar et al.,

2012).

Plant species are widely used in Indian

systems of medicine since times immemorial

and one such ancient plant is Euphorbia

neriifolia Linn. The general name of Euphorbia

neriifolia Linn. is Sehund /Thohar in Hindi and

Dog’s Tongue in English. It is known as Indian

spurge tree, Oleander spurge, Hedge

Euphorbia, Snuhi (Sanskrit) and Ilachevikalli

(siddha). This plant belongs to the family of

Euphorbiaceae (Burkill IH; 1936, Ambasta SP;

1986).

Generally Euphorbia neriifolia grows

luxuriously around dry, rocky, North hilly

areas, Central and Southern parts of India. The

leaves are thick, greenish, 6–12 inches long and

ovular shape (Anonymous, 1952). Most of the

people know about it that it is very toxic and

waste plant but recent researches have shown

that it has medicinal uses in the treatment of

typical diseases. It is a bitter laxative,

carminative and useful in abdominal troubles,

bronchitis, tumors, leucoderma, piles,

enlargement of Spleen, anemia, ulcers, fever

and in chronic respiratory troubles (AK

Nadkarni, 1954).

MATERIAL AND METHODS

Collection of Plant Material

Euphorbia neriifolia Linn. is found all over

India. The plant flowers were collected from

Mandsaur district, Madhya Pradesh. Mandsaur

District forms the northern projection of

Madhya Pradesh (Fig.-1). It lies between the

parallels of latitude 23° 45' 50" North and 25°

2' 55" North, and between the meridians of

longitude 74° 42' 30" East and 75° 50' 20" East.

Fig.1- Map of Madhya Pradesh

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Fig.2- Euphorbia neriifolia Linn.

Preliminary Screening of Secondary

Metabolites

The flower of Euphorbia neriifolia (Fig.2)

were collected from Mandsaur Madhya

Pradesh.The plant was identified to Dr. S.N.

Mishra, Principal Scientist, All India

Coordinated Research Project on Medicinal and

Aromatic Plants, College of Horticulture,

Mandsaur, affiliated to R.V.S.K.V.

Vishwavidyalaya, Gwalior.

The flowers were kept in shade, dried and

powdered using mixer grinder, and subjected to

cold percolation process for 48 hours with

petroleum ether, chloroform, methanol, 95%

ethanol and distilled water. After this process,

the extracts were filtered and used for

preliminary phytochemical screening such as

alkaloids (Iodine, Wagner, and Dragendorff’s

test), flavonoids (Pew’s, Shinoda and NaOH

tests), glycosides (Keller-Killani, Conc. H2SO4,

and Molisch tests), lignin (Labat and Lignin

tests), phenols (Ellagic acid and Phenol tests),

saponins (Foam and Haemolysis test), sterols

(Libermann- Burchard, and Salkowski tests),

tannins (Gelatin and Lead acetate tests) were

carried out (Shashank Bhatt et al.,2011).

Preliminary Screening of Phytochemical

Test

Phytochemical Screening

The filtrate obtained was subjected to

preliminary phytochemical screening.

For identification of alkaloids Iodine test

(Khandelwal K.R., 2008), Wagner’s test

(Kokate C.K. et al., 2001) and Dragendorff’s

test (Kokate C.K. et al.,2001) were performed.

For identification of flavonoids Pew’s test

(Peach K., Tracey MV. 1956), Shinoda test

(Kokate C.K. et al., 2001) and NaOH test

(Khandelwal K.R., 2008) were performed.

For identification of glycosides Keller-Killani

test (Kokate C.K. et al., 2001), Glycoside test

(Treare GE, Evans WC. 1985), Conc. H2SO4

test (Khandewal K.R., 2008) and Molish test

(Kokate C. K. et. al., 2001) were performed.

For identification of Phenol Ellagic acid test

(Gibbs R.D., 1974) and Phenol test (Gibbs

R.D., 1974) were done.

For identification of lignin lignin test (Gibbs

R.D., 1974) and labat test (Gibbs R.D., 1974)

were performed.

For identification of saponins foam test

(Kokate C. K. et. al., 2001), and Haemolysis

test (Kokate C.K., 1994) were done.

For Identification of sterols Liebermann-

Burchard test (Kokate C. K. et. al., 2001) and

Salkowski’s test (Kokate C. K. et. al., 2001)

were done.

For identification of tannins gelatin test (Treare

GE, Evans WC. 1985) and lead acetate test

(Treare GE, Evans WC. 1985) were performed.

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TABLE-1: Phytochemical Screening of Euphorbia neriifolia Linn. Flowers

Test Petroleum

Ether

Chloroform Methanol 95% Ethanol Distilled water

Alkaloids

Iodine Test − − − − −

Wagners Test − − + + −

Dragendorff’s

Test

− − + + −

Flavonoids

Pews Test − − + + −

Shinoda Test − − + + −

NaOH Test − − + + −

Glycosides

Keller- Killani

Test

+ + + + +

Glycosides Test − + + + −

Conc. H2SO4 + + + + +

Molishs Test + + + + +

Phenol

Ellagic Test − − + + −

Phenol Test − − + + −

Lignin

Lignin Test − + + + −

Labat Test − + + + −

Saponins

Foam Test − − + + +

Haemolysis Test − − + + −

Sterols

Libermann-

Burchard Test

+ + + + +

Salkowski Test − + + + −

Tannins

Gelatin Test − − + + −

Lead Acetate Test − − + + +

(+)Presence, (−) Absent

RESULT AND DISCUSSION

The results of the phytochemical screening

of Euphorbia neriifolia’s flowers’ have been

presented in Table-1. It showed that different

types of secondary metabolites such as

alkaloids, flavonoids, glycosides, phenol,

lignins, saponins, sterols and tannins were

present. Most of the secondary metabolites

present in flowers show the good therapeutic

activity against diseases compared to other

parts. According to the guidelines of WHO &

Ayurveda, if a plant has anti-pathogenicity,

anti-cancer and different types of activities it

can be involved into medicinal plant category.

Secondary metabolites are these types of

compounds.

Flavonoids have inherent abilities to

modify the body’s reaction to allergen, virus

and carcinogens. Tannins have general

antimicrobial and antioxidant activities

(Rievere et al., 2009). Current reports show

that tannins may have potential values such as

cytotoxic and antineoplastic agents (Aguinaldo

et al., 2005). Plant steroids have cardiotonic

activity, possess insecticidal and antimicrobial

properties. It is generally used in herbal

medicines and cosmetic products (Callow;

1936).

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CONCLUSION

Euphorbia neriifolia Linn. has different

types of medicinal properties. It has different

types of secondary metabolites that have been

presented in phyto-chemical screening of

secondary metabolites study. These secondary

metabolites have anti-HIV, anti-bacterial, anti-

viral, anti-fever, anti-diabetic, anti-cancerous

activities etc. Therefore, it can be involved in

medicinal plant category.

ACKNOWLEDGEMENT

The authors are very thankful to Dr. S.N.

Mishra for assist in identification of plant and

kind assistance of my dear father Shri Krishna

Kumar Bhatt, Bharti, Smt. Subhadra bhatt and

brother Mr. Mayank Bhatt. Last but not least

the Almighty God is unforgettable without

whose kindness and grace, anything was not

possible.

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Source of Support: Nil Conflict of Interest: None Declared

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

TRADITIONAL MEDICINES OF HERBAL ORIGIN PRACTICE BY THE

ADI TRIBE OF EAST SIANG DISTRICT OF ARUNACHAL PRADESH, INDIA

Das Moushumi1*, Jaishi Anju

2, Sarma Hirendra N

3

1Assistant Professor, Department of Zoology, University of Science and Technology, Meghalaya, Ri-Bhoi

District, Meghalaya, India 2Research Scholar, Department of Zoology (Center with Potential for Excellence in Biodiversity), Rajiv Gandhi

University, Itanagar – 791 112, India 3Professor, Department of Zoology (Center with Potential for Excellence in Biodiversity), Rajiv Gandhi

University Itanagar – 791 112, India *Corresponding Author: Email: dasmoushumi092000@yahoo.co.in; Mob: +919508419900

Received: 01/03/2013; Revised: 17/04/2013; Accepted: 25/04/2013

ABSTRACT

A survey was conducted on the use of traditional medicine by the Adi tribe of East Siang district

of Arunachal Pradesh, a state of eastern Indian Himalayan Mountain range. Ten numbers of

Traditional Medicine Practitioners (TMP) belonging to the age group of 45–60 years were interacted.

Data were collected preparing semi structured questionnaire. Thirty three plant species were

identified as being used for Traditional Medicine preparation by the Adi people. Eighty one percent

of these plants were used for human, while 19% were used for animal diseases. Traditional medicines

were used for treatment of Jaundice, typhoid and malaria (9%), eye infection (6%) burn injuries

(6%), skin disease and repellant (6%), diarrhea (9%), toothache (9%), wound healing and infection

(15%), bone fracture (15%) and fertility control (24%). Some of these TM were prepared in a

composite form using multiple number of plants or part thereof. Use of TM is still prevailing among

the villagers as one of the cheapest and easily available medical practice. It has been speculated that

proper screening of these herbal medicines shall provide new insight on the hitherto unexplored

traditional medicines of Adi people of Arunachal Pradesh.

KEYWORDS: Abortifacient, Antifertility, Traditional medicine, Indigenous Knowledge

Research article

Cite this article:

Das M, Jaishi A, Sarma H N (2013), TRADITIONAL MEDICINES OF HERBAL ORIGIN PRACTICE

BY THE ADI TRIBE OF EAST SIANG DISTRICT OF ARUNACHAL PRADESH, INDIA,

Global J Res. Med. Plants & Indigen. Med., Volume 2(5): 298–310

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INTRODUCTION:

Arunachal Pradesh a state of the Eastern

Himalayan region of India is one of the Mega

Biodiversity hot spots of the world. This

Biodiversity hot spot zone is characterized by

wide variety of floral and faunal population.

Along with a variety of plants and animals this

state is enriched with a number of ethnic

groups of people. These people have an age-old

history and practice of using Traditional

Medicine (TM) of herbal origin for treatment of

various ailments/ conditions of both human and

animals. Arunachal Pradesh is sharing

international border with China and Myanmar.

Thus, it was earlier known as North East

Frontier Agency (NEFA) and governed by

Government of India as Union Territory till

20th

February 1987. The NEFA gets the

statehood on 20th

February, 1987 with its new

identity of Arunachal Pradesh (http://

arunachalpradesh.nic/people.htm.). Belonging

to the eastern Himalayan Biodiversity rich

zone, this hill state is the home land of more

than 25 tribal people groups

(http://WWW.indianetzone.com/8/tribes_aruna

chal_pradesh.htm). The „Adi‟ is one of the

major tribe among these groups of tribal

population of Arunachal Pradesh. The name

„Adi‟ was earlier known as „Abor‟, (Dalton

E.T, 1872). The transformation of the name

from „Abor‟ to „Adi‟ took place after the

development of disregard to the meaning of

„Abor‟. The use of the word „Abor‟ which

means „unruly‟ or „savage‟ has been traced

back to the first century A.D., that a great

valley of Himalayas called „Aborimon‟ was

inhabited by wild men (Hutton J.H, 1946).

However, the term „Abor‟ has been used with a

broader sense of meaning of „independence,

unruly, savage‟ and so on and applied to all the

hill tribes of valley of Assam (Mackenzie A,

1884). The original home of Adi people has not

yet been studied and ascertained. According to

some authors Adis are considered to be origin

in the lofty mountain ranges of Tibet and

Assam (Nyori Tai, 1993). Coming from the

northern and north-eastern part of greater

Himalayas, these Adi people moved

southwards and settled in the erstwhile „Siang

frontier division‟ of NEFA. This „Siang

Frontier Division‟ spreads from Subansiri

River in the west and Dibang River in the east.

With advancement of time the Siang Frontier

Division has been demarcated into three Siang

districts (East Siang, West Siang and Upper

Siang) and Dibang valley district of present day

Arunachal Pradesh. Damroh, a Padam village

in the East Siang district has been considered as

the epicenter of the dispersal of the Adi people

(Nyori Tai, 1993). In the course of time, Adis

are divided into different groups, such as

Padam, Minyong, Pangi, Shimong, Ashing,

Pasi, Karko, Bokar, Bori, Ramo, Pailibo,

Milan, Tangam and Tagin (Roy S 1966). The

Adis belong to the Mongoloid stock and thus

similar to those of the other of the same stock

(Chatterjee S.K, 1951). The history of the Adi

people‟s relationship to modern world dates

back to early 19th

century; when Captain

Bedford, the first European from Great Britain

visited the Adi inhabited area of Eastern

Himalayas in the year 1825–26. In the early

part of 20th century, the Adi villages came

under the British authority (Roy S, 1966).The

traditional Adi villages are organized under a

village council called „Kebang‟ in Adi

language (Rizvi et al., 2006). The „Kebang‟ is a

powerful body honored by all sections of Adi

people and involved in looking after over all

social development as well as settlement of

social dispute amicably. The “Adi language” is

the lingua franka of the Adi people. These

indigenous people are self-sufficient in most of

the spheres of life in their remote villages. They

collect their food and medicinal requirements

from the untouched natural resources of

neighboring forest. In the present investigation

such plants and part there off used for cure of

various ailments like diarrhea, fever, jaundice

etc., treatment for bone fracture and for

reproduction control especially of domesticated

animals practiced by the Adi tribe of East Siang

district have been documented through first

hand information collection. The quest for

development of novel drugs using herbs

mentioned in various folk literatures is in force

in the scientific community. In such herbal

preparation plants are used either singly (parts

thereof) or in composite form. It has been

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speculated that the present study shall be

helpful for preservation of indigenous

knowledge on TM, its preparation and

application, over & above the societal

development of these tribal communities in

India.

MATERIALS AND METHODS

Study area

The East Siang district is located in

between 27°43´ and 29°20´ North latitude and

94°42´ and 95°35´ East latitude (Fig.1). The

district has a total of area of 4005 sq.km

covering the other bank of Siang River. It

spreads over a wide range of altitudinal height

of mean sea level from a minimum of 133 ft

(Ruksin area) and maximum 752 ft (Riga).

Siang River flows from northern part of lofty

mountains of Arunachal Pradesh and merges

with the Brahmaputra River in plains of Assam.

The „Siyom‟ and „Siku‟ are two main

tributaries of Siang River, flowing across the

west and east bank of the mighty Siang

respectively. Thus, the Siang River flows

throughout the entire district creating the Siang

valley in Arunachal Pradesh. The entire district

is characterized by presence of series of spurs

of mountains. The outer range of the mountain

at the southern part of the district touches the

northern part of the Brahmaputra basin

bordering Assam. The northern mountain range

of the district is densely covered with forest.

The southern portion is comparatively flat and

densely wooded except the places having

human settlement. The climate of the district is

tropical humid in summer and sub temperate

during winter. It receives heavy rainfall every

year during monsoon season starting from May

till October. A cold dry seasonal wind blows

throughout the winter from November to

February. The soil texture varies from sandy to

loamy sand. Cultivation and rearing of

livestock is the main way of livelihood for

majority of the population. The dominant crop

of Adi people is the paddy followed by maize,

millet, ginger, mustard, potato, orange,

pineapple etc grown on the hill slopes.

Fig 1. Study Area: The East Siang District of AP

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Fig 2. Adi Female (A) and Adi male (B) Traditional Medicinal Practitioners (C) and an Adi

village (D).

Data collection

The data on medicinal use of plants practiced by the „Adi‟ peoples were collected primarily from rural villages of East Siang district. The villages namely Bogo Bango, Mikung, Ngorlung, Ledum, GTC Pashighat, Oyan, Mebo, Boleng and Pangin were selected for information collection. Villages were selected with a view to collect the information covering different circles of East Siang district. A „circle‟ consists of two or more villages within the district. Very often the names of the villages are found to be similar with the name of the circle it belongs to, e.g „Mebo‟ village is situated in the „Mebo‟ circle. Many of the villages are located in the remote areas accessible only on foot. During the survey, it was observed that a good number of aged people (both male and female folk) possess knowledge on traditional medicines. Among these villagers, the authors met the Traditional Medicine Practitioner (TMPs) who regularly prepare the TM and do practice and prescribe among the villagers. These people (TMP) belonging to age group of 40–65 years are locally known as „Miri‟. During the present investigation 10 numbers of „Miri‟s both male and female (Fig-2) were interacted to obtain information on the use of medicinal plants. In addition, 29 local informants of the villages were approached for information on the herbal medicines. The local informants neither practice nor prescribe the TM among the villagers, however, use this knowledge and

information for their own requirement whenever necessary.

The authors selected the „Miris‟ and local informants by approaching and interacting with the village heads, locally called as Gaon Burha of the surveyed villages. The Gaon Burha is the head of the village administration. The Gaon Burha or village head can take decisions/ order to settle any minor disputes within the The Gaon Burha also got the knowledge about the various activities going in and around the village. Some of the informants and „Miris‟ neither understands nor can speak the language other than „Adi language‟, In such situations local Adi people knowing both „Adi‟ and English languages were used as interpreters and mediators for data collection. Important to mention that many Adi people with higher education have been working as TMP and pursue the practice of TM among the villagers in East Siang district. Survey was carried out ten times in a time span of 23 months starting from the month of January 2005. The ethno medicinal data was collected following standard methods (Martin GJ., 1995; Jain SK., 1964) through general conversation with the informants, using semi structured questionnaire, interviews and the participants‟ observations.

The Questionnaire used for data collection 1. Name of the person 2. Sex 3. Age 4. Locality 5. Utilization of the plant for medicine

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(yes/No) 6. Plant medicine use for fever/bone fracture/sterility/abortion/other conditions 7. Description of plant 8. Mode of preparation/ parts used/composition of medicine 9. How much it is effective (temporary or permanent) 10. Mode of application/Procedure (eg., Dose, duration, time etc.) 11. Side effects, if any (whether vomiting, Nausea, weakness or any other adverse effect)

The Adi people have deep belief on the Sun and the Moon locally termed as “Donyi Polo” in Adi language and thus developed their own religion called “Donyi- Poloism”. Very often, preparation and practice of the traditional medicine is associated with worships and superstitions among these people. The data originated from the epistemology of indigenous knowledge on traditional medicines were reviewed and evaluated within epistemology arising from scientific reasoning and knowledge. The TMPs were interviewed with a view to exploring (1) sources of medicine, preparation and prescription, (2) TM associated with traditional belief associated with worships, (3) route of administration and application, (4) method of preparation, (5) Part used, (6) used for human and/ or animal. The authors accompanied the TMPs with the local interpreters to the field for observation and collection of plant species used for TM preparation. Plant species shown by the local practitioner were collected and stored in the form of herbarium in for identification.

Collection and preservation of the plant species were made following standard methods (Jain et al., 1976). Identification of the plant species was done using reference herbarium materials, various volumes of books on Indian medicinal plants (Kirtikar K.R et al., 1935; Hooker JD.1872–1897) and by taxonomist from Rajiv Gandhi University in Itanagar, Arunachal Pradesh.

RESULTS

The present study recorded a total of 33 plant species belong to 25 families (Table-1)

used as components of TM of Adi people. These herbal preparations were used for treatment of both animal and human being. Nineteen percent (19%) of the presently recorded plants have been used for treatment of animal diseases while remaining 81% are used for treatment of human diseases (Fig-4). Results showed that these TM are used for treatment of Jaundice, typhoid and malaria (9%), eye infection (6%) burn injuries (6%), skin disease and repellant (6%), diarrhea (9%), toothache (9%), wound healing and infection (15%), and bone fracture (15%). Twenty four percent (24%) of plants documented were used for preparation of TM for fertility control of both human and domestic animal (Fig-3). Mode of use of the plant parts varied according to the medicines prepared and its uses. Application of the fresh leaves was found to be the highest (51%) used among the collected information. 33% medicines were prepared from stems. Roots, tubers, rhizomes were used for preparing 18% of the traditional medicines recorded so far among the Adi people. Similarly, barks (9%), fruits and seeds (6%) were used for preparation of various traditional medicines by the Adi people. The medicines were prepared for both external (topical) application and through oral route depending upon the use of the medicines for ailments. Paste of freshly collected leaves were used for external application in wound healing and infection covering 52% of the application of the TM. Dry powder of the plants or parts thereof was used in 15% of the medicines taken along with food or water for treatment of diseases like malaria, diarrhea, jaundice, typhoid etc and for fertility control of animal and human. The crude juice and/ or sap and boiled residues of freshly collected plants (leaves, stems) were used in 33 % of medicines collected during the present investigation. While most of the medicinal plants were reported to be used in freshly collected form, some reported to be used in dried form. Plants belong to families Apocynaceae, Poaceae, Rutaceae, Rubiaceae, Scrophularaceae and Mimosaceae were reported to use for more than one TM preparation. The collected plant parts were stored in the museum of Life science Department, Rajiv Gandhi University, Itanagar.

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Table -1. Plants and its medicinal use prevail among the Adi people of East Siang district

Name of the plant (Family)&

No./vernacular name Part

used Use

Mode of preparation/use

Alstonia scholaris (L.) Br

(Apocynaceae) Adi/RGU/G-01/ Cingar

Leaves Wound healing,

malaria,

abortifacient in

pigs.

Paste of fresh leaves. Paste is mixed

with the meal of cattle to feed for

wound healing and same preparation

use in pigs for abortion.

Amphineuron extensus

(BI.)HOLTT (Thelypteridaceae) Adi/ RGU/G-02/ Rokji

Leaves Tick repellent in

chicken

Fresh leaves collected from the plant

are kept inside the poultry farm to

repel and/ or removes the tick from

the animal.

Ageratum conyzoidesL

(Asteraceae) Adi/ RGU/G-03/ Namsing

Leaves

Wound healing Paste is prepared from fresh leaves.

The paste is applied in the cut or

wound to stop bleeding and healing

of the wound. Doses depend on the

intensity of the wound.

Axonopus compressus

(SW)P.Beauv (Poaceae) Adi/ RGU/G-04/Bobosa

Roots Eye infection/

Disease Root is chewed by the patient to get

relief. Three times for three days.

Bannaya reptans Sprengel

(Scrophulariceae) Adi/RGU/G-05/Kat-Buk usueng

Leaves Nail infection

Freshly collected leaves are warmed

to make a paste. The paste is wrapped

with clean cloth at the site of

infection. Generally it is used at

night during sleeping hours.

Bryophyllum pinnatum. Kuntz.

(Crassulaceae.) Adi/ RGU/G-06/ Nebi – Nelum

Leaves Skin burn,

diarrhea, fracture

and Sciatica.

Pastes of fresh leaves are applied to

burnt area and use for fracture. For diarrhea,2–3 fresh leaves are

recommended to eat 4–5 times ina

day (for 4 days)

Clacaria macrophylla Wall

(Rubiaceae.) Adi/ RGU/G-07/ Pemi lagin

Sap of

the stem Conjunctivitis,

Eye infection

1–2 drops of the sap of the stem is

poured into the eye during infection

Clerodendrum spp (Nerbenaceae) Adi/ RGU/G-08/ Dumkar

Fresh

leaves Skin Burn Paste is prepared from fresh leaves to

apply on the burnt area.

Coptis teeta ( Ranunculaceae ) Adi/ RGU/G-09/Mishimi teeta

Roots,

leaves Malaria, Jaundice,

Typhoid and in

fertility control in

women

(abortifacient)

Powder is prepared from shade dried

Roots of Coptis teeta. The powder is

mixed with water and the mixture is

boiled for few minute to feed the

Malaria patient. For Abortive use

Preparation is made by mixing the

leaves with Caraca papaya, Moringa

oleifera (bark),Solanum spirale

(roots) and bark of Alstonia

schlolaris.

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Drymaria cordata Roem and

Schuletes (Caryophyllaceae) Adi/

RGU/G-10/ Taggom

Leaves

and stem Skin disease and

Bone fracture

Paste is prepared from the tender

branches of the plant. Applied on the

fractured area skin.

Laportia crenulata Gaud

(Urticaceae) Adi/RGU/G11/Matpepereng

Leaves

and stem Wound infection A paste is prepared from freshly

collected leaves and applied on the

wound of animals.

Melothria leucocarpa (BP) Cong

(Cucurbitaceae) Adi/ RGU/G-12/Pumroll

Leaves Wound infection

in animal Paste of Fresh leaves is mixed with

the meal of domestic animals.

Mikenia mirantha L. (Asteraceae) Adi/RGU/G13/Arunachal Eng

Leaves Cut, wound

healing Freshly collected leaves are grinded

to make paste and applied on wound/

cuts to heal the wound and for

prevent bleeding.

Oroxylum indicum L. Vent.

(Bignoniaceae) Adi/ RGU/G-14/Domir ettkung

Bark Tonic for malaria

patients Small pieces bark are sun dried and

then boiled with leaves of Ocimum

sanctum, Zingiber zerampet

(Rhizomes) and Mentha spicata. The

boiled residue is advised to use

during the whole treatment period.

Psidium guajava L. (Myrtaceae) Adi/ RGU/G-15/ Mudhuri anne

Leaves

/buds

Treatment of

diarrhea. Fresh tender leaves are advised to

chew by the person suffering from

diarrhea. Doses are 4–5 fresh twigs

takes twice or thrice a day for 3–4

days.

Paederia foetida (Rubiaceae.) Adi/RGU/G-16/Yepetare

Leaves Stomach disorder/

Diarrhea Fresh leaves are boiled with other

vegetables. The boiled residue is

given to eat to the patient to cure

fever and diarrhea. The leaves are

tied on head or hangs on neck to cure

headache and sinusitis.

Solanum nigrum L (Solanaceae)

Adi/ RGU/G-17/ Bangko

Leaves,

roots and

stem

Diarrhea, Malaria

and conjunctivitis Paste prepared from leaves is boiled

in water. Diarrhea and malaria

patient‟s treated with both the boiled

leaves paste and the extract for 4–5

days. The grinded roots are given to

the malaria patient to get rid of the

parasite. Mildly heated leaves are put

on the eye to cures conjunctivitis.

Zanthoxylum acanthopodium DC

(Rutaceae) Adi/ RGU/G-18/ Ombe

Stem

Tooth ach

The stem of plant is used. Chewing

or brush with it gives immediate

relief from the toothache and to

makes the teeth strong.

Zanthoxylum hamiltoniamun wall

(Rutaceae) Adi/ RGU/G-19/ Ongar

Leave,

fruit Tooth ache,

Abortifacient Leaves are dried on the sunlight or

smoke and then applied on the tooth.

Immediately it gives relief of the

toothache. The fruit is used to eat in

large quantity as abortifacient.

Zingiber cassumunar (Zingiberceae) Roxb.Adi/RGU/G-

20/ Kekir

Rhizome Tooth ache,

cough, post

delivery pain

relief

The rhizome is boiled with water;

this extract is given to sip by the

person for getting relief of cough.

Medicine is applied in 3–4 times a

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day for 2–3 days. During post

delivery pain; the cooked Rhizome is

given to mother in a low dose.

Raphidopora spp (Araceae) Adi/ RGU/BF-21/ Loma losut

Stem and

leaves Bone fracture The plants to be applied is collected

and processed in the crude form.

Leaves or stems are collected,

cleaned and crushed, making a paste

of the plant or part thereof. The paste

is applied to the specific part of the

bone fracture. At every 24 hours the

paste is reapplied replacing the old

one. The process is repeated for

specific period as suggested by the

“Miri” (the local healer).

Entada scadens (Mimosaceae)

Adi/ RGU/BF-22/ Taboh

leaves

Bone fracture

Phyllostachys assamica (Poaceae) Adi/RGU/BF-23/Tempor

Stem and

Bark Bone fracture

Equisetum arvernse/diffusum

(Equisetaceae) Adi/ RGU/BF-24/ Sisi dungki

Stem Bone fracture

Ricinus communis L

(Euphorbiaceae) Adi/ RGU/BF-25/ Aki-Rokmic

Bark and

leaves Bone fracture

Annanus comosus (Bromeliaceae) Adi/ RGU/RC-26 /Anaros, Tako

bela

Fruit Fertility control in

women. (abortifacient)

Fruit is used to induce abortion.

Dysoxylum alliarum (Maleaceae) Adi/ RGU/RC-27/ Situ paiyu

Bark Fertility control in Pigs and Dogs

(abortifacient)

The bark is dried in sunlight. The

dried bark is grinded and the fine

powdered form is given to the

domestic animals mixing with other

food.

Hemidesmus indicus

(Apocynaceae) Adi/ RGU/RC-28/

Stem Fertility control in

women. (abortifacient)

Stem portion is shade dried to make

powder. The powder id eaten with

water.

Mimosa pudica (Mimosaceae)

Adi/ RGU/RC-29/ Anying ing

Whole

plant Fertility control in

women Fresh juice extracted from the whole

plant taken orally with other food by

the desired woman to avoid

pregnancy.

Scoparia dulcis

(Scrophulariaceae) Adi/ RGU/RC30/Meetagoss.

Arial part Fertility control in

women Fresh juice of aerial part taken orally

by the desired women to prevent

pregnancy.

Calotropis gigantea (Apocynaceae) Adi/ RGU/RC-31 / Abok

Roots Fertility control in

women Calotropis gigantea, Carica papaya

(seeds), Coptis teeta are used in

composite form (paste). This

preparation is given to the desired

women for prevention of pregnancy Carica papaya (Cariaceae) Adi/ RGU/RC-32/ Omir

Mature

seeds Fertility control in

women

Dioscoria allata (Dioscoraceae) Adi/ RGU/RC-33/ Janghli allu.

Tuber Fertility control in

women The tuber is eaten in uncooked form.

It has been stated that eating

frequently by desired women to lose

fertility.

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Figure 3. Percentage of use of TM for various ailments and diseases

Figure 4. Percentage of TM use

DISCUSSION

During the last few years attention of the

world communities has been drawn on

conservation and protection of Biological

diversity of the world. Emphasis was put on the

conservation, protection and sustainable use of

TM in the Convention on Biological Diversity

(World Health Organization, 1999; Note by the

Executive secretary, Secretariat of the

Convention on Biological Diversity. Geneva,

November, 2000). India has an age old tradition

of medicinal practice especially of herbal origin

in Ayurveda. More than 6000 medicinal plant

species has been used in preparation of

medicines by the traditional medicine

practitioners in Indian subcontinent (Seth et al.,

2004). Arunachal Pradesh is one of the north

eastern provinces of India and recognized as the

eastern Himalayan Mega Biodiversity hotspot

(Myers N et al., 2000). The state is situated on

the top of eastern Himalayan Mountain range

having more than eighty percent forest coverage

of the total geographic area of the state. The

biodiversity of the state not fully explored due to

dense forest, very thin population distribution

and lack of infrastructure for surface

communication. Despite all the difficulties,

efforts have been made by some authors to

study and document the ethno medicinal

practice of these indigenous people. Till date

over 500 species of medicinal plant has been

reported from Arunachal Pradesh (Arunachal

Pradesh Information bulletin no.12, 2011).

Traditional medicines among the Chakma,

Singpho, Tangsa, Nishi, Hillmiri, Sulung and

Apatani (Nath et al., 1993; Pal GD, 1984) tribes

of Arunachal Pradesh were reported earlier in

different scientific studies. A few plants used

for traditional medicines among the Adi people

have been reported elsewhere (Gangwar et al.,

1990). These studies showed that plants like

Alstonia scholaris, Calotopris gigantea and

Dioscorea alata which are used by the Adi tribe

are also reported to use by Apatani tribe (Kala

CP, 2005) of Arunachal. On the contrary to the

use of Dioscorea alata as anti-fertility medicine

by the Adi tribe, the Monpas and Apatani use it

for gastritis and indigestion respectively (Nima

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D et al., 2011 and Kala CP., 2005). However,

species Psidium guajava is found to be used by

both the Adi and Monpas for the treatment of

diarrhea (Nima D et al., 2011). The study of the

present authors on traditional medicines is an

added information in wider areas of common

ailments like diarrhea, malaria, eye infection,

wound healing etc as well as joining of bone

fracture and reproduction control prevail among

the Adi people living in remote villages of East

Siang District. The present study revealed a

good number of plants used for fertility control

practice by Adi people of East Siang district. It

has been speculated that practice of herbal

medicines used for fertility control originated in

the population control requirement of domestic

animals. In fact, the Adi people use the anti-

fertility herbal preparation for control of

population growth of their domestic animals. In

this context it is to be mentioned that

maintaining domestic pets (dogs and pigs) is a

cultural entity of the Adi people. Bigger family

size of domestic animals leads to the

requirement of more feed and space. Very often,

owners are unable to provide necessary food or

space to the growing members of their domestic

animals. To control the population growth of

the domesticated animal they regularly practice

such use of plants. These antireproductive

medicines are used during the first trimester of

pregnancy. Once the owner observes mating of

animals; they fed the herbal formulation to the

female to avoid giving birth of new born. These

herbal formulations used during post coital

period may possess abortifacient / antizygotic,

and/ or anti implantation properties. The present

study revealed that these medicines are

successfully utilized by these folk people to

control the reproduction of their domestic pets

and very often to control the unwanted

pregnancies of women. The in-vivo testing of

Dysoxylum alliarium and Piper species showed

pregnancy disruption activity in albino rats

(Das M et al., 2013, Ali N & Ghosh B, 2007,

Mibang Tamo and Chowdhari SK, 2003).

During the survey 15% of total collected

plants have recorded for its use in treatment of

bone fracture. Discussion with the local people

revealed that bone fracture is a common

phenomenon among the villagers as because

the rural folk people have to walk in the steep

hills for livelihood earning. There is no modern

facility for treatment of fractured bone in the

remote areas. This leads to the use of traditional

medicines for recovery from these ailments in

the remote villages. Moreover, malaria,

diarrhea, typhoid are some of the common

health problems faced by these people who use

TM for disease cure and prevention. These

diseases are found among the people who

suffer from unavailability of pure drinking

water and live in unhygienic environment. It

has been found that a single plant is used for

more than one health problem or condition;

such as the Alstonia scholaris (malaria, wound

infection and abortion), Coptis teeta (malaria

and abortion) and Zanthoxylum hamiltoniamun

(toothache and abortion). Medicinal use of

Zanthoxylum spp. for various purpose e.g. use

in malaria (Singh HB & Singh TB., 2005),

relief of labor pain and safe childbirth by

women (Qureshi et al., 2006) as well as use in

toothache (Cheryl Lans, 2007) has been

documented earlier. The Carica papaya has

been reported to use in composite form with

other plant parts for abortion (Valsala S,

Varpagaganapathy P.R. 2002). Important to

note that some of the plant species used by the

Adis of East Siang are reported for its

medicinal use from other states of India as well

as other countries of the world. Mimosa pudica

and Scoparia dulcis which is widely used by

Adi people are also reported to use by the

people of Trinidad and Tobago for treatment of

reproductive disorders of both males and

females and during childbirth (Ignacimuthu et

al., 2006). These folk people of Trinidad and

Tobago reported to use the plant Mimosa

pudica for treatment of women‟s menstrual

cycle disorder (Cheryl Lans, 2007). The

medicinal properties of Solanum nigrum and

Alstonia scholaris is reported to be used by

other tribes of India. Solanum nigrum is used

for wound healing, stomachache and also as an

abortive (Ignacimuthu et al., 2006, Sen et al.,

2012) while, Alstonia scholaris is used for

increase lactation in women (Nain Jaspreet et

al., 2011). The Adi people developed the

practice of using composite medicines. More

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than one plant species combined together; e.g.

the combination Calotropis gigantea, Coptis

teeta, Dioscorea allata are used as abortifacient

drug. The justification for use of composite

medicines is not known by the TMPs. As

mentioned earlier, practice of these medicines

by the local TMPs is associated with

superstitions and worships of their deity. The

epistemology of the traditional practice of

preparation and use of these TM needs to be

reviewed by the epistemology of scientific

knowledge.

Some of the TMPs, the authors interacted

were well educated and aware of the need of

conservation of the traditional knowledge and

the conservation of plant species. Many of the

practitioners were maintaining their own

medicinal plant garden for conservation and

regular use for medicine formulation. Proper

laboratory screening of these traditional herbal

preparations may come across through

development of new lead, useful for cure of

various ailments/ conditions as well as for

fertility control. This approach shall help in the

development of pharmaceutical industry,

production of cheaper health friendly drugs for

the rural people especially of the developing

countries and rural economic empowerment.

ACKNOWLEDGEMENT

The authors acknowledge the funding

received under the scheme “Women Scientist

Scholarship Scheme for Societal Programme

(WOS-B), Department of Science and

Technology, Government of India” for

carrying out this research. The authors are also

grateful to the University Grants commission,

New Delhi for providing necessary support to

undertake this research under the Center with

Potential for Excellence in Biodiversity, Rajiv

Gandhi University. We extend our thanks to

Osiri Ratan, Nanku Gao,Y.J Lego and other

TMPs for sharing their knowledge on

traditional medicine and help in various ways.

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Source of Support: Women Scientist

Scholarship Scheme for Societal

Programme (WOS-B), Department of

Science and Technology, Government of

India

Conflict of Interest: None Declared

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

PRELIMINARY PHYTOCHEMICAL SCREENING OF SECONDARY

METABOLITES OF ADHATODA VASICA NEES. FLOWERS

Chouhan Surksha¹, Bhatt Shashank²*, Dhyani Suresh3

1Department of Biotechnology, Rajiv Gandhi Govt. P.G. College, Mandsaur, Madhya Pradesh, 458001,

India. 2,3

Department of Biotechnology, NIMS University, Jaipur, Rajasthan, 303121, India. *Corresponding author: Email: shashank_bhatt2003@yahoo.co.in; Mobile: +919826840428

Received: 02/04/2013; Revised: 25/04/2013; Accepted: 30/04/2013

ABSTRACT

Adhatoda vasica Nees. belongs to the family Acanthaceae. The work was done on Adhatoda

vasica Nees. flowers and different solvents were used for extraction of the phyto-constituents.

Solvents like petroleum ether, chloroform, methanol, 95% ethanol and distilled water were selected

for extraction of the flowers according to their polarity. Alkaloids, flavonoids, glycosides, saponin,

phenol, lignin, sterols and tannins were found in phyto-chemical screening. These metabolites have

different types of medicinal activities as antibacterial, antiviral, anti-diabetic etc. which could be

efficiently used in treating various ailments.

KEY WORDS: Adhatoda vasica Nees., Alkaloids, Glycosides, Sterols, Lignins.

Research article

Cite this article:

Chouhan Surksha, Bhatt Shashank, Dhyani Suresh (2013), PRELIMINARY PHYTOCHEMICAL

SCREENING OF SECONDARY METABOLITES OF ADHATODA VASICA NEES. FLOWERS,

Global J Res. Med. Plants & Indigen. Med., Volume 2(5): 311–316

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INTRODUCTION

Plants can be categorized according to their

properties as medicinal and wild. Near about

80% plant species’ compounds have medicinal

activities and used as medicine (WHO, 1993).

In India, 45,000 plant species are officially

recorded and 7500 medicinal plant species

grows in 16 agro-climatic zones under 63.7

million hectares of forest coverage (H. Tag,

2007). Most of the plants have been involved

into medicinal category.

Plants are the natural sources for the

development of medicines (Kumar, 2004).

Metabolites found in plants show an important

role in the treatment of diseases. Metabolites

are falls into two categories one of which is

primary and another is secondary metabolites.

Primary metabolites play important roles in

metabolic pathways while secondary

metabolites support it. These secondary

metabolites are alkaloids, flavonoids,

glycosides, saponins, lignin etc.

Adhatoda vasica Nees. commonly known

as Malabar nut belongs to the family

Acanthaceae. It is an evergreen shrub and

grows in waste places. It is distributed through

out in India and utilized in rural areas for

different ailments (K. Jayapaul et al., 2005).

The stem, leaf, flower, fruit and seeds have

drug properties (B.L. Manjunath, 1948, U.P.

Claeson et al., 2000). It has antispasmodic,

fever reducing, anti-inflammatory, anti-

bleeding, bronchodilator, anti-diabetic, anti-

helminthic, disinfectant, anti-jaundice,

antiseptic, expectorant and many other

medicinal uses (P.K. Patel et al., 1984, A.

Chakraborty et al., 2001, R. L. Wakhloo et al.,

1980).

MATERIAL AND METHODS

Collection of Plant Material

Adhatoda vasica Nees. is found all over the

world. The plant flowers were collected from

Mandsaur district, Madhya Pradesh. Mandsaur

District forms the northern projection of

Madhya Pradesh (Fig.-1). It lies between the

parallels of latitude 23° 45' 50" North and 25°

2' 55" North, and between the meridians of

longitude 74° 42' 30" East and 75° 50' 20" East.

Figure 1: Map of Madhya Pradesh.

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Figure 2: Adhatoda vasica Nees.

Preliminary Screening of Secondary

Metabolites

The flowers of Adhatoda vasica Nees. (Fig.

2) were collected from Mandsaur Madhya

Pradesh and plant identified to Dr. S.N. Mishra,

Principal Scientist, All India Coordinated

Research Project on Medicinal and Aromatic

Plants, College of Horticulture, Mandsaur,

affiliated to R.V.S.K.V. Vishwavidyalaya,

Gwalior.

The flowers were shade dried, powdered

using mixer grinder, and subjected to cold

percolation process for 48 hours with

petroleum ether, chloroform, methanol and

distilled water. After this process, the extracts

were filtered and used for preliminary

phytochemical screening such as alkaloids

(Iodine, Wagner, and Dragendorff’s test),

flavonoids (Pew’s, Shinoda and NaOH tests),

glycosides (Keller-Killani, Conc. H2SO4, and

Molisch tests), lignin (Labat and Lignin tests),

phenols (Ellagic acid and Phenol tests),

saponins (Foam and Haemolysis test), sterols

(Libermann- Burchard, and Salkowski tests),

tannins (Gelatin and Lead acetate tests) were

carried out (Shashank Bhatt et al.,2011).

Preliminary Screening of Phyto-chemical

Test

Phytochemical Screening

The filtrate obtained was subjected to

preliminary phyto-chemical screening.

For identification of alkaloids Iodine test

(Khandelwal K.R., 2008), Wagner’s test

(Kokate C.K. et al., 2001) and Dragendorff’s

test (Kokate C.K. et al., 2001) were done.

For identification of flavonoids Pew’s test

(Peach K., Tracey MV. 1956), Shinoda test

(Kokate C.K. et al., 2001) and NaOH test

(Khandelwal K.R., 2008) were performed.

For identification of glycosides Keller-Killani

test (Kokate C.K. et al., 2001), Glycoside test

(Treare GE, Evans WC. 1985), Conc. H2SO4

test (Khandewal K.R., 2008) and Molish test

(Kokate C. K. et. al., 2001) were done.

For identification of Phenol Ellagic acid test

(Gibbs R.D., 1974) and Phenol test (Gibbs

R.D., 1974) were done.

For identification of lignin lignin test (Gibbs

R.D., 1974) and labat test (Gibbs R.D., 1974)

were performed.

For identification of saponins foam test

(Kokate C. K. et al., 2001), and Haemolysis

test (Kokate C.K., 1994) were performed.

For Identification of sterols Liebermann-

Burchard test (Kokate C. K. et al., 2001) and

Salkowski’s test (Kokate C. K. et al., 2001)

were done.

For identification of tannins gelatin test (Trease

GE, Evans WC. 1985) and lead acetate test

(Trease GE, Evans WC. 1985) were done.

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TABLE-1: Phytochemical Study of Adhatoda vasica Nees. Flowers

Test Petroleum

Ether

Chloroform Methanol 95% Ethanol Distilled Water

Alkaloids

Iodine Test − − − − −

Wagners Test − − + + −

Dragendorff’s

Test

− − + + −

Flavonoids

Pews Test − − + + −

Shinoda Test − − + + +

NaOH Test − − + + +

Glycosides

Keller- Killani

Test

− − + + +

Glycosides Test − − + + +

Conc. H2SO4 − − + + +

Molishs Test − − + + +

Phenol

Ellagic Test − − + + −

Phenol Test − − + + −

Lignin

Lignin Test − − + + −

Labat Test − − + + −

Saponins

Foam Test − − + + −

Haemolysis Test − − + + −

Sterols

Libermann-

Burchard Test

− + + + +

Salkowski Test − + + + +

Tannins

Gelatin Test − − + + +

Lead Acetate Test − − + + +

RESULT & DISCUSSION

The flowers were powdered and subjected

to cold percolation with petroleum ether,

chloroform, methanol, 95% ethanol and

distilled water for 48 h. and found alkaloids,

flavonoids, glycosides, phenol, lignins,

saponins, sterols and tannins. Adhatoda

vasica’s flowers have most of the secondary

metabolites that mention in table- 1. Each

metabolite has specific activities. Therefore, it

may be effective.

Flavonoids, tannins, saponins, steroids and

phenols have been used in the treatment of

different types of diseases. These moieties are

isolated and extracted from medicinal plants’

parts and used for development of new drug.

If flowers are selected in research most of the

secondary metabolites can be found easily and

isolate it for development of new drugs. We

know that steroids are also involved into

hormonal category which is responsible for

changes in male and female characteristics.

Tannins have general antimicrobial and

antioxidant activities (Rievere et al., 2009).

Current reports show that tannins may have

potential value such as cytotoxic and anti-

neoplastic agents (Aguinaldo et al., 2005).

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CONCLUSION

I have concluded that Adhatoda vasica

Nees. flowers have most of the secondary

metabolites that have different activities by

which diseases treated. The specifications of

Siddha and Ayurveda show the limit of

medicinal plant categories. The Ayurveda and

Siddha have shown the uses of this plant parts

but not involve the flowers. Each part of plant

has not all secondary metabolic compounds

that have concluded but only flower have it.

Therefore, according to my results, I have

concluded that its flowers may be most useful

part to another part of plant.

ACKNOWLEDGEMENT

I am very thankful to Dr. S.N. Mishra for

assist in identification of plant and kind

assistance of my dear father Shri Krishna

Kumar Bhatt, Smt. Subhadra Bhatt and brother

Mr. Mayank Bhatt.

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Source of Support: Nil Conflict of Interest: None Declared

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

HERBAL MEDICINES FOR DEPRESSION AND ANXIETY: A

COMPREHENSIVE STATE OF THE ART REVIEW

Patel Shanti1, De Sousa Avinash

2*

1Medical Intern – Lokmanya Tilak Municipal Medical College and General Hospital, Mumbai, Maharashtra,

India 2Consultant Psychiatrist and Founder Trustee – De Sousa Foundation, Mumbai- 400054, Maharashtra, India

*Corresponding Author: E-mail – avinashdes999@yahoo.co.uk; TEL – 91-22-26460002

Received: 01/03/2013; Revised: 26/04/2013; Accepted: 30/04/2013

ABSTRACT

This review looks at all the herbal medicines and formulas in treating depression and anxiety

disorders. Pubmed and the Cochrane Library were searched for pharmacological and clinical

evidence of herbal medicines with antidepressant and anti-anxiety action. Good evidence exists for

the use of kava and St John‟s wort in the treatment of anxiety and depression respectively, while

there is insufficient clinical evidence for the use of many other herbal medicines in psychiatric

disorders. Newer herbal preparations that potentially have significant use in depression and anxiety

and urgently require more research are Rhodiola rosea (roseroot), Crocus sativus (saffron),

Passiflora incarnata (passion flower) and Piper methysticum (kava). They need further evidence

base via clinical studies. Depression and anxiety are commonly researched but the efficacy of herbal

medicines in these disorders requires attention. The review addresses all the current issues in herbal

therapy, safety issues and future areas of application in the field.

KEY WORDS: Herbal medications, depression, anxiety, kava, St John‟s wort, passion flower.

Review article

Cite this article:

Patel Shanti, De Sousa Avinash (2013), HERBAL MEDICINES FOR DEPRESSION AND ANXIETY: A

COMPREHENSIVE STATE OF THE ART REVIEW, Global J Res. Med. Plants & Indigen. Med.,

Volume 2(5): 317–336

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INTRODUCTION

Mood disorders, anxiety and sleep disorders

are largely prevalent and highly comorbid

psychiatric conditions (Kessler et al., 2005). It

is estimated that by 2020 depression will result

in 2nd

greatest increase in morbidity after

cardiovascular diseases, presenting a significant

socioeconomic burden (WHO, 2006). Since the

past decade, many herbal medicines have been

used in people with mood and anxiety disorders

(Schulz et al., 2001). Due to the increasing

popularity of herbal medications majority of

the patients are consulting herbalists,

naturopaths, and other healers, in addition to

physicians. A data from a nationally

representative sample of 2055 people

interviewed during 1977–1988 revealed that

57% of those suffering anxiety attacks, and

54% of those with severe depression reported

using herbal medicine during the previous 12

months to treat their disorder (Kessler et al.,

2001). Similarly interviews of 82 psychiatric

North American inpatients revealed that 44%

had used herbal medicine (mainly for

psychiatric purposes) during the previous 12

months (Elkins et al., 2005).

There is however, a limited data regarding

the benefits and liability of herbal remedies and

other natural remedies. There have been few

reports of serious adverse effects from these

medications and by and large these medications

have been considered safe and effective

(Schulz et al., 2001; Mischoulon, 2004). This

article reviews the literature on various herbal

medications in the treatment of depression and

anxiety.

MECHANISM OF ACTION OF HERBAL

MEDICATIONS

The primary mechanism of action involves

modulation of neuronal communication, via

specific plant metabolites binding to

neurotransmitter/neuromodulator receptors

(Spinella, 2011) and via alteration of

neurotransmitter synthesis and general function

(Sarris, 2007). Other mechanisms involve

stimulating or sedating CNS activity, and

regulating or supporting the healthy function of

endocrine system (Kumar, 2006; Sarris, 2007;

Spinella, 2011). The psycho-pharmacological

effects of herbal medicines and their clinical

validation can be explored by the use of “omic”

genetic technologies (Ulrich-Merezenich et al.,

2007).

HERBAL MEDICATIONS USED IN THE

MANAGEMENT OF DEPRESSION

Hypericum perforatum L. (St.John’s wort)

For centuries, hypericum an extract of the

flower of St. John‟s Wort (SJW) (Hypericum

perforatum L.) is used for the treatment of

depression (Schulz et al., 2001). Its use in the

United States has been dramatically increased

in the past decade. Polycylic phenols, hypericin

and pseudohypericin are the active compounds

in extract of St. John‟s Wort. Other compounds

include flavonoids (hyperoside, quercetin,

isoquercitrin, rutin), kaempferol, luteolin,

biapigenin and hyperforin (Muller-Kuhrt and

Boesel, 1993; Staffeldt et al., 1993; Wagner et

al., 1993). Out of all the active compounds

hypericin is the main active compound.

Hypericin decreases serotinin receptor

density (Muller-Kuhrt and Boesel, 1993). It

also inhibits monocyte production of

interleukin 6 and 1β resulting in a decrease in

corticotropin releasing hormone and thus

dampening cortisol production (Thiele et al.,

1993). It decreases expression of β

adrenoreceptors and increases density of

serotonin by nonselective inhibition of neuronal

reuptake of serotonin, dopamine,

norepinephrine, GABA and l-glutamate,

decreased degradation of neurochemicals, and a

sensitization of and increased binding to

various receptors (e.g. GABA, glutamate and

adenosine) (Butterweck, 2003; Mennini and

Gobbi, 2004; Zanoliu, 2004; Muller et al.,

1993; Teufel-Mayer et al., 1997). SJW

modulates salivary and serum cortisol levels,

and has a slight effect on growth hormone

(Franklin et al., 2006). Hyperforin, hypericin

and various flavonoids appear to be responsible

for the neurochemical modulation (Butterweck,

2003; Laakmann et al., 1998; Zanoliu, 2004).

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In a large number of clinical European

clinical trials hypericum has been compared

with low dose imipramine and maprotiline

(75 mg/day) (Varbach et al., 1994; Harrer et

al., 1993). Despite these low doses of active

controls, the response rates in these trials

seemed comparable to those in studies that use

higher doses of tricyclic antidepressant agents

(TCAs) (eg. Imipramine ≥ 150 mg/day). The

response rates for hypericum ranged from

35.3% to 81.8%, and the response to TCAs

ranged from 41.2% to 77.8%. In a meta-

analysis (Nirenberg et al., 2002), hypericum,

300 mg three times a day was judged to be

effective in 79 of 120 subjects (65.8%),

whereas placebo was considered effective in

only 36 of 125 subjects (28.8%). The placebo

response rate seemed comparable to that

observed in many outpatient studies of anti

depressants conducted in United States. A

recent meta-analysis conducted by Rahimi et

al., (2009) yielded a significant relative risk

(RR) for response in favour of the active of

1.22 (95%Cl :1.03, 1.45) and weighed a mean

difference between treatments of 1.33 points

(95%Cl: 1.15, 1.51) on the Hamilton

Depressing Rating Scale (HAM-D). Where as

comparison with SSRIs yielded a non

significant difference between treatments of

0.32 (95% Cl: - 1.28, 0.64) for mean reduction

in HAM-D score from baseline.

A meta-analysis (Linde et al., 1996)

examined 15 trials comparing Hypericum with

placebo and eight trials comparing Hypericum

with TCAs in 1757 patients who had mild to

moderate depression.In six trials that used

single preparation of Hypericum, (containing

only St.John‟s Wort), hypericum yielded

greater response rates than placebo (55.1% for

Hypericum versus 22.3% for placebo) and

comparable response rates to tricyclic

antidepressants (69.3% for Hypericum versus

58.5 & for tri-cyclic antidepressants). In two

trials that used combination preparations of

Hypericum (containing St.John‟s wort and

other herbal medications such as Kava,

Hypericum was found to be more effective than

TCAs (67.7% versus 50%).

In a 6 week trial with 375 patients,

Lecrubier and colleagues (Lecrubier et al.,

2002) found that St. John‟s Wort, 900 mg/day,

was significantly more effective than

placebo,especially who had higher base line

HAM-D scores. Shelton and colleagues (2001)

found that St.John‟s Wort (900–1200 mg/day)

was no more effective than placebo in the full

intent to treat analysis, although among

completers the remission rates were

significantly higher with St. John‟s Wort than

the placebo. A 2004 meta-analysis of SJW

(dosage 300–1200 mg/day) in the treatment of

mild to moderate depression (Roder et al.,

2004) reviewed 30 trials and concluded a

significant advantage of SJW over placebo

(n=2129, relative risk , RR = 0.66,95% Cl 3.0

to 6.6, mean response 53.2 SJW vs 51.3 %

synthetic antidepressants).A meta-analysis of

16 trials ,inspection of individual studies

showed that SJW was found to demonstrate

greater efficacy than synthetic

antidepressants.Six RCTs tested SJW against

placebo and fluoxetine in treating MDD, as

commonly assessed via the Hamilton rating

scale for depression (HAM-D) and clinical

global impression (CGI). Four studies

demonstrated that SJW had similar (Behnke et

al., 2002; Bjerkenstedt et al., 2005) or superior

(Fava et al., 2005; Schradar, 2000) effects to

fluoxetine. An analysis of the sub-sample of a

12-week 3-arm study discovered that SJW

(160- 900mg/day) ameliorated depression –

based vegetative presentations, while fluoxetine

(20mg/day) was statistically equivalent to

placebo (Murck et al., 2005).

In comparison to paroxetine, SJW was

statistically more effective in treating

moderate-severe depression (Szegedi et al.,

2005). Other comparative trials demonstrated

SJW‟s statistical ewuivalence to imipramine

(Woelk, 2000), citalopram (Gaspere et al.,

2006), Maprotiline (Harrer et al., 1994) and

amitriptyline (Wheatley, 1997) in treating

major depressive disorder. The Hypericum

Depression Study, the medicine is currently

used for the treatment of mild to moderate

depression (Clement et al., 2006; Lawvere and

Mahoney, 2005; Linde et al., 2005).

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A comparative analysis between paroxetine

and Hypericum extract WS 5570 revealed that

paroxetine had 10 to 38 times higher adverse

event rate.An increasing number of averse drug

reaction have been noted between St.Johns

wort and other medications.Majority of the

interactions are due to the liver enzyme CYP-

450-3A4 which results in the decreased activity

of several drugs, including warfarin,

phenprocoumon, digoxin, indinavir,and

irinotecan (Baede-van Dijk et al., 2000; Miller

et al., 1998; Moore et al., 2000; Miller et al.,

2000; Piscitelli et al., 2000). The interactions

are mainly due to high dose hyperforin extracts

(Izzo, 2004). Hyperforin increases the

expression of pregnane-X-receptor,which

increases P-glycoprotein expression (Dresser et

al., 2003; Izzo, 2004; Moore et al., 2000). Low

hyperforin preparations may not affect this

response and hence may be safer (Izzo 2004,

Muller et al., 2006). A systematic review of 19

studies showed that high dose hyperforin

extracts (>10mg/day) had outcomes consistent

with CYP3A induction while studies using low

dose hyperforin extracts (<4mg/day)

demonstrated no significant effects on CYP3A

(Whiten et al.,2004). Because of the

monoaminase inhibiting ativity of St.John‟s

Wort, its combination with SSRI‟s may result

in Serotonin syndrome, hence it should not be

combined with SSRI‟s (Hu et al., 2005). As

monotherapy adverse effects are mild (Schulz,

2005). Adverse events include dry mouth,

dizziness,constipation, other gastrointestinal

symptoms and allergic reactions.(Schulz,2001;

Schulz, 2005).

Phototoxicity has been found in animals

with hypericum but rarely in humans.

Hypericum at a dose of 1800mg caused minor

increase in sensitivity to uv light in humans but

no phototoxicity. It is recommended that

patients taking high dose of hypericum should

be isolated from UV radiation for 7 days

(Seigerse et al., 1993). At least 17 cases of

psychosis have been resulted from St, John‟s

Wort ,of which 12 comprised mania or

hypomania. Researchers compared St.John‟s

Wort, 900 to 1800mg/d with sertraline 50 to

100mg/d, in 12 community based primary care

offices. It was found that St John‟s Wort

resulted in significantly fewer adverse events

(Van Gurp et al., 2002). In a 2006 review of 16

post marketing surveillance studies (n=34834)

(Schulz, 2006), SJW was deemed to be 10 fold

safer than synthetic antidepressants (adverse

effects 0.1% to 2.4%). Overall SJW has

demonstrated equal efficacy to pharmaceutical

antidepressants with a more favourable side

effect profile and fewer dropouts than its

synthetic counterparts. SJW has been

recommended as a first line treatment in milder

forms of depression (Roder et al., 2004).

Crocus sativus L. (Saffron)

It increases the re-uptake inhibition of

monoamines (dopamine, norepinephrine and

serotonin). It is also a NMDA receptor

antagonist and a GABA-α receptor agonist

(Hosseinzadeh and Noraei, 2009; Lechtenberg

et al., 2008; Schmidt et al., 2007). There have

been two trials (Akhondzadeh et al., 2004;

Noorbala et al., 2005) comparing saffron with

imipramine and fluoxetine, it was found that

saffron demonstrated improvement of

depression. A similar response was

demonstrated in a study in which 30 mg saffron

was effective over placebo (Akhondzadeh et

al., 2004; Moshiri et al., 2006). Clinical trials

have detailed anxiety, tachycardia, nausea,

dyspepsia and changes in appetite as possible

side effects (Moshiri et al., 2006).

Lavendula spp. (Lavender)

It causes GABA modulation. In animal

studies it is effective in anxiety symptoms

(Atsumi and Tonosaki, 2007; Bradley et al.,

2007; Perry and Parry, 2006; Shaw et al., 2007;

Toda and Morimoto, 2008). In a 4 week RCT

comparing comparing lavender tincture (1:5

50% alcohol,60 drops) against imipramine in

patients (n=45) with a HAM-D rating of atleast

18 it was found that although lavender was less

effective than the synthetic counterpart ,the

combination of both was more effective than

Imipramine alone,indicating a synergistic effect

(Akhondzadeh et al., 2003).

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Rhodiola rosea L. (Rose root)

It causes inhibition of cortisol, stress

induced protein kinases, nitric oxides and

Monoamine oxidase A. In animal models it has

shown to cause normalization of 5-HT and anti

stress effects (Chen et al., 2009; Panossian et

al., 2007; Panossian et al., 2008; Mattioli et al.,

2009; Perfumi and Matticki, 2007; Van

Dierman et al., 2009). Authors (Schevtsov et

al., 2003) assessed the influence of roseroot on

various mental and biological parameters of

161 adults, it was found to have an ant fatigue

effect. This property along with the monoamine

modulation can be used in the treatment of

monopolar depression (Stancheva and

Mosharrof, 1987). A three-arm study using R.

rosea 5HR-5 standardised extract (340 mg and

680 mg/day) against placebo in the treatment of

mild-moderate depressive disorder revealed a

significant dose dependent improvement

occurred in the active groups compared with

placebo (Darbinyan et al., 2007).

S-Adenosyl Methionine (SAMe)

It is a methyl donor in the brain and is

involved in the pathways for synthesis of

hormones, neurotransmitters, nucleic acids,

proteins and phospholipids. Its potential role in

mood regulation was determines by its activity

as an intermediate in the synthesis of

norepinephrine, dopamine, and serotonin. It is

used in the treatment of major depression as

well as in other medical conditions (Spillmann

et al., 1996). Depression has been associated

with Folate and vitamin B12 deficiency and

about 10% to 30% of depressed patients have a

low folate and these patients respond less to

antidepressants (Alpert et al., 2000). Vitamin

B12 is converted to methylcobalamin which is

involved in the synthesis of various

neurotransmitters. Hence its deficiency may

result in earlier age of onset of depression

(Fava et al., 1997). SAMe is synthesized from

the amino acid l-methionine through the one

carbon cycle, a metabolic pathway involving

the vitamins folate and B12 (Spillmann et al.,

1996). Low SAMe levels have been found in

cerebrospinal fluid of depressed individuals

(Bottiglieri et al., 1990) and higher plasma

SAMe levels have been associated with

improvement in depressive symptoms (Bell et

al., 1994). It has been found that folate

augmentation in partial responders has

achieved good results (Coppen et al., 2000;

Alpert et al., 2002). In 8 placebo controlled

studies SAMe demonstrated superiority to

placebo in 6 studies and equivalency to placebo

in the other 2 studies (Spillmann et al., 1996;

Alpert et al., 2000; Coppen et al., 2000). In 6 of

the 8 comparison studies with TCAs, SAMe

was equivalent in efficavy to TCAs and was

more effect than imipramine in one study and

SAMe may have a relatively faster onset of

action than conventional depressants

(Spillmann et al., 1996; Alpert et al., 2000;

Coppen et al., 2000). In one study, some

patients improved within a few days,and most

did so within 2 weeks.(Fava et al., 1995). Two

studies have shown that combination of SAMe

and a low dose TCA resulted in earlier onset of

onset than a TCA alone (Alvarez et al., 1987;

Berlanga et al., 1992).

Researchers have examined the efficacy of

SAMe as an adjunctive treatment for partial

and nonresponders to SSRIs (Alpert et al.,

2004). Thirty subjects who had residual

depression despite SSRI or venlafaxine

treatment received a 6-week course of 800–

1600mg. Response and remission rates with

SAMe augmentation were 50% and 43%

respectively, and the treatment was well

tolerated. Besides depression, SAMe is

effective for dementia-related cognitive defects,

depression in patients who have parkinson‟s

disease or other medical illness, psychological

distress during the puerperium and opoid and

alcohol detoxification (Mischoulon et al.,

2002). SAMe is well tolerated and relatively

free of side effects. Side effects include mild

insomnia, lack of appetite, constipation, nausea,

dry mouth, sweating, dizziness and nervousness

(Spillmann et al., 1996). Cases of increased

anxiety, mania or hypomania in bipolar

depression have been reported (Spillmann et

al., 1996; Carney et al., 1987; Carney et al.,

1983) and therefore it should be used carfully

in patients with bipolar disorders.

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Omega-3 fatty acids

The intake of more and more processed

foods rich in omega-6 containing vegetable oils

has decreased the intake of omega- 3 fatty acids

in the Western diet. This has resulted in higher

physiologic ratio of omega-6:omega-3 fatty

acids in Western countries (Adams 1996;

Hibbeln, 1995; Cross-National Collaborative

Group, 1992; Hibbeln, 1998; Hibbeln, 1999). It

has been postulated that the modern western

diet and the additional stresses of twenty-first

century create a proinflammatory state in

humans that may contribute to cardiovascular

and also may play a role in the development of

mood disorders (Stoll and Lacke, 2002). So

administration of omega-3 supplements may

potentially reverse this proinflammatory state

by correcting the omega-6:omega-3 ratio. It has

an effect on membrane -bound receptors and

enzymes involves in the regulation of

neurotransmitter signaling,as well as regulation

of calcium ion influx through calcium channels

(Stoll and Lacke, 2002). Omega-3 fatty acids

cause decrease corticosteroid release and

dampen mood-altering effects associated with

cortisol by inhibiting secretion of inflammatory

cytokines. Eicosapentanoic acid resembles

amitriptyline in antidepressant action, it inhibits

the synthesis of prostaglandin E2, thus

dampening the synthesis of p-glycoprotein

(Murck et al., 2004). Peet and Horrobin (2002)

conducted a randomized, placebo-controlled,

dose finding study of ethyl-eicosapentaenoate

(EPA) as adjunctive therapy for 70 adults who

had persistent depression despite treatment with

a standard antidepressant. Subjects who

received 1 g/d EPA for 12 weeks showed

significantly higher response rates (53%) than

subjects receiving placebo (29%), with notable

improvement of depressed mood, anxiety, sleep

disturbance, libido, and suicidality. The 2 g/d

group showed little evidence for a drug-placebo

difference, and the 4 g/d group showed a

nonsignificant trend toward improvement.

These results suggest that there may be an

optimal dose of omega-3 that humans require

for maximum benefit, and it is possible that an

overcorrection of the omega-6:omega-3 ratio

with higher omega-3 doses may limit the

antidepressant effect of EPA.

Researchers (Su et al., 2002) conducted an

8-week, double-blind, placebo-controlled trial

comparing adjunctive omega-3 (6.6 g/d)

against placebo in 28 depressed patients.

Patients in the omega-3 group had a significant

decrease in HAM-D scores compared with

placebo. Nemets and colleagues found a

statistically significant benefit of adjunctive

EPA in 20 subjects who had major depressive

disorder and who were on antidepressant

therapy, 1 g/d, and a clinically important

difference in the mean reduction of the 24-item

HAM-D scale by the study endpoint at week 4

compared with placebo (12.4 versus 1.6). A

single placebo- controlled study with 36

subjects showed lack of efficacy of DHA, 2

g/d, for depression (Marangell et al., 2003).

Researchers (Osher et al., 2005) treated 12

bipolar I depressed subjects with open

adjunctive EPA, 1.5 to 2 g/d, for up to 6

months. Ten patients completed at least 1

month of follow-up, and eight achieved a 50%

or greater reduction in HAM-D scores. No

cycling occurred with any patients. Further

investigation is needed to determine whether

bipolar disorder actually requires higher doses

of omega-3 fatty acids than unipolar illness and

to unravel the respective contributions of EPA

and DHA. Omega-3 fatty acids are relatively

very safe. Side effects include gastrointestinal

upset and fishy aftertaste tends to occur with

higher doses (> 5 g/d) with less pure

prepararions. At doses of 1 g/d with highly

purified omega-3 preparations, these adverse

effects are less common. There is a

documented risk of bleeding but it is minimal

at doses less than 3 g/d. Hence individuals

taking warfarin should be cautious and should

use omega-3 fatty acids under a physician‟s

supervision. Also, there are few documented

cases of cycling in bipolar patients (Freeman et

al., 2006).

Hence it is recommended hat low doses of

omega-3 fatty acids may be effective and well

tolerated monotherpy or adjunctive therapy for

depressed adults. Freeman and colleagues

(2006) recommends that depressed individuals

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may safely use approximately 1 g/d of an EPA-

DHA mixture but should not substitute omega-

3s for conventional antidepressants at this time.

For, individuals who take more than 3 g/d of

omega-3 should do so under a physician‟s

supervision (Freeman et al., 2006). They can

also be used to treat specific populations (eg.

Pregnant or lactating women) for whom

antidepressants may be used with a caution

(Chiu et al., 2003), for elderly people and for

those with cardiovascular diseases.

Echium amoenum Fisch. & C.A.Mey (Borage)

Its antidepressant action is currently

unknown and anxiolytic activity is shown in

animal studies (Rabbani et al., 2004). In a RCT

single dose of Echium amoenum (375mg/day

for 6 weeks) was compared against placebo in

35 patients with depression and anxiety,

assessed via HAM-D and Hamilton anxiety

scale (HAM-A). It was found that the herbal

medication was superior than placebo in

reducing depressive symptoms with a effect

size d of 0.92 but this result was not maintained

at week 6 (Sayyah et al., 2006). It also had no

anxiolytic activity.

Dan Zhi Xiao Yao

It is a chinese preparation which contains

Dan Pi (Cortex Moutan), Zhi Zi (Fructus

Gardneiae), Chai Hu (Radix Bupleuri), Dang

Gui (Radix Angelicae Sinensis), Bai Shao

(Radix Alba Paeoniae), Bai Zhu (Rhizoma

Atractylodis Macrocephalae), Fu Ling

(Poria) Gan Cao (Radix Glycyrrhizae).It is

modified from Xiao yao san (Rambling

powder) herbal preparation which is used in the

treatment of depression by moving stasis

(Bensky and Gamble, 1991) and in addition it

has Dan zhi (Cortex Moutan). In a RCT, 63

patients with depression assessed via the HAM-

D, self rating depression scale (SDS), self

rating anxiety scale (SAS) and the scale for

TCM syndrome and symptom differentiation,

the formulation was compared with

maprotiline. It was found that maprotiline was

effective in 84% patients in reduction of

depression whereas Dan zhi xizo yao was

effective in 87% (Lou et al., 2006).

Banxia Houpu

It consists of Pinella ternata, Poria cocos,

Magnolia officinalis, Perilla frutescens and

Zingiber officinale. There is no human clinical

data to determine the efficacy of Banxia houpu

but large number of animal studies have

demonstrated its anti-depressant activity

comparable to fluoxetine in trail suspension

and forced swimming tests (Li et al., 2003; Luo

et al., 2000). It was found that an increase in

serotonin (5-HT) and 5-hydroxyindoleacetic

acid (5-HIAA) levels was found to occur in

mouse hippocampus and striatum. Researchers

(Wang et al., 2005) found that Banxia houpe

decoction decreased the level of triglycerides in

serum enhanced the activity of the natural killer

cellsin the spleen, decreased the activity of

superoxide dismutase in red blood cells and the

activity of the nitric oxide synthase in the

serum and the tissue, and reduced the content

of malondialdehyde in tissue via the effect on

lipid peroxidation.

HERBAL MEDICINES USED IN THE

MANAGEMENT OF ANXIETY

Piper methysticum L.f (Kava)

It causes GABA channel modulation (lipid

membrane structure and sodium channel

function) and weak GABA binding which

causes increased synergistic effect of [3H]

muscimol binding of GABA-α-receptors.It also

causes β-adrenergic downregulation and MAO-

B inhibition.It inhibits reuptake of

norepinephrine in prefrontal cortex (Bonon and

Haberlein, 1998; Dacies et al., 1992; Jussofie et

al., 1994; Magura et al., 1997; Uedelhack et

al., 1998). A 2003 cochrane review of

randomosid ,double blind ,controlled trials of

rigorous methodology using Kava mono

preparations (60-280 mg of kavalactones),

Pittler and Ernst found that Kava had a

stastically significant anxiolytic activity on

Hamilton Anxiety Scale (HAMA) compared

with placebo (95% Cl;0.1,7.7) but one trial

demonstrated that kava was effective in short

term treatment of anxiety. A meta analysis

(Sarris et al., 2010b) revealed a similar

conclusion A meta-analysis of 7 homogenous

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trials using HAM-A demonstrated that kava

reduced anxiety significantly than placebo

(weighted mean difference 3.9 over placebo on

the HAM –A;95% Cl:0.1 to 7.7 p=0.05; n-

380). A 4 week study by Connor and Davidson

(2002) found no significant difference between

a standardised Kava extract and placebo.

A meta-analysis beased on six placebo

controlled randomized trials using Kava extract

WS 1490 in anxiety demonstrated that kava

significantly reduced anxiety, with a mean

improvement of 5.94 better than placbo (Witte

et al., 2005). A 3-month randomized

prospective open study investigating kava in

peri menopausal women revealed that the

reduction in anxiety with kava was

significantly greater than in controls (on

calcium supplementation) as assessed via the

State trait anxiety index (STATI). It was also

observed that depression depression declined at

3 months (-5.03+/-1.4) as assessed via the

Zung‟s depression scale (Cagnacci et al.,

2005). A randomized controlled double blind,

multicenter clinical trial compared kava with

synthetic agents like busiprone or opramol

(Boerner et al., 2003). The outcomes were

measured using HAM-A, Boerner anxiety

scale, SAS, CGI, a self rating scale for well

being, a sleep questionnaire, a quality of life

questionnire (QOL) and global judgement by

investigator and patients.It was found there

there was no significant difference between

Kava and Busiprone or opipramol regarding all

efficacy and safety measures.75% of the patient

were classified as responders (50% reduction of

HAM-A score) in each treatment group with

60% achieving full remission. A novel study

involving 13 subjects evaluated kava‟s

potential in improving vagal control in suffers

of GAD (Watkins et al., 2001). It was observed

that significantly more patients treated with

kava showed improved BRC compared with

placebo group,reflecting a favourable effect on

reflex vagal contral of heart rate in patients

with GAD. Due to potential hazard of

hepatotoxicity, P.methysticum was withdrawn

from the European and UK markets in 2002. It

was found that the factors responsible for

hepatotoxicity included individuals hepatic

insufficiency to metabilise kavalactones

(cytochrome P-450 (CYP) 3A4 and 2D6),

incorrect cultivation (medicinal, tudie or

wichmanni varieties) being used, preparations

made using acetone or ethanolic media low in

glutathione, potentially contaminated or poorly

stored material, and use of ariel parts or root

peelings which are higher in alkaloids (Sarris et

al., 2010c). It is recommended that only peeled

roots from noble cultivers (cultivated species

that are traditionally considered safe and

therapeutic) using water soluble extraction

method is advised (Teschke et al., in press).

In a study of kava use( Av

118g/week,median duration of use=12 years) in

an Arnhem Land community in northern

territory of Australia it was found that liver

functions in users of aqueous kava at these

moderate levelsof consumption appears to be

reversible and began to return to baseline after

1–2 weeks abstinence from kava. No evidence

of irreversible liver damage has been found

(Clough et al., 2003). Kava has also been found

to cause significant drug interaction and

interactions with CYP 450 enzyme (Singh,

2005). One human pharmacokinetic trial

determined that kava caused CYP2E1

inhibition in approximately 40% (Gurley et al.,

2005). Whole kava extract (normalized to

100µm total kavalactones), caused

concentration dependent decreases in P450

activities, with significant inhibition of the

activities of CYP1A2 (56% inhibition), 2C9

(92%), 2C19 (86%), 2D6 (73%), 3A4 (78%)

and 4 A9/11(65%) following preincubation

(Mathews et al., 2002). Kava also interacts

with benzodiazepines and causes sedation

(Singh, 2005; Stevinson et al., 2002). However,

the risk-benefit ratio is highly favourable

towards kava due to respectable clinical

efficacy and relative low risk of potential liver

toxicity (1 case /million monthly doses (Bauer,

2003).

Passiflora incarnata L. (Passion flower)

It is a benzodiazepine receptor partial

agonist and causes GABA-system mediated

anxiolysis. Animal behavioural models have

shown non-sedative anxiolytic effect.In an in

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vivo study employing a methanol extract of

passion flower (125 mg/kg,orally) measured

anxiolytic activity in mice, using the elevated

plus-maze model,an increase in number of

entries in open arm was demonstrated (Dhawan

et al., 2001a; 2001b; 2002; Grundmann et al.,

2008; Sena et al., 2009). A 4 week RCT using

passion flower extract on patients with GAD

(n=36) showed that passion flower was as

effective as oxazepan (30 mg/day) in reducing

anxiety and it had less number of side effects

(Akhondzadek et al., 2001). In an acute study

RCT (n=60) using 500 mg of passion flower vs

placebo for presurgical anxiety (Movafegh et

al., 2008), it was demonstrated that anxiety

scores were significantly lower in the

passionflower group than in the control group

on a numerical rating scale.

Valeriana spp. (Valerian)

Felter and Lioyd demonstrated that species

of valerian officinalis and edulis have been

used in traditional American and European

medicine as a soporific and to treat various

nervous system disorders. It decreases the

degradation and simultaneously increases the

binding of GABA. Also, valerenic acid from

valerian has demonstrated GABA-A receptor

(β3 subunit) agonism and also 5-HT5a partial

agonism (Benke et al., 2009; Dietz et al., 2005;

Murphy et al., 2009; Oritz et al., 1999; Sichardt

et al., 2007; Trauner et al., 2008). A large 8

week internet based RCT (n=391) using a

valerian (6.4 valarenic acids/day) placebo, kava

(300 mg kavalactones/day) + placebo or double

placebo was conducted to determine the

efficacy in treating co morbid anxiety and

insomnia (Jacobs et al., 2005). The primary

outcome measure used in rating change in

anxiety state was STATI-State. The results

suggested that neither kava norvalerin relieved

anxiety and insomnia more than placebo. But

the design of this trial presents several potential

problems, with internet recruitment for trials

resulting in samples of questionable

representativeness, and the STATI-state having

the inadequate test-retest reliability to be a

sensitive measure of therapeutic change in

anxiety. In a systemic review and metaanalysis

of 18 RCTs (Fernandez-San Martin et al.,

2010) using Valerian vs placebo or active

controls,valerian reduced sleep latency over

placebo by only 0.70min (95% Cl-

3.44,4.83),with the standardized mean

difference between the groups measured being

stastically equivocal-0.02 (95% Cl-0.35, 0.31)

Scutellaria lateriflora L. (Skull cap)

It has a GABA-α binding affinity (Awad et

al., 2003). A double blind placebo controlled

cross over study of healhy individuals (n = 19)

revealed that skullcap dose-dependently

reduced symptoms of anxiety and tension after

acute administration compared to that with

control (Wolfson and Hoffmann, 2003). In

animal maze model test skullcap demonstrated

anxiolytic activity (Awad et al., 2003).

Melissa officinalis L. (Lemon balm)

It is shown to cause MAO-inhibition. Also

it is found to be a potent invitro inhibitor of rat

brain GABA transaminase (GABA-T) (Awad

et al., 2009; Lopez et al., 2009). An RCT with

20 participents who were given single doses of

300,600 and 900 mg of lemon balm or a

matching placebo at 7-day intervals revealed

that self rating calmness as assessed by Bond

Lader mood scales was elevated at the earliest

time points by the lowest dose, while alertness

was significantly redused at all time points

following the highest dose (Kennedy et al.,

2002). A double blind ,placebo controlled,

randomized, balanced cross over experiment

utilizing a standardized product containing

lemon balm and valerian extracts in healthy

volunteers (n=24) assessed mood and anxiety

via a DISS test (Kennedy et al., 2006).The

results demonstrated that a 600 mg dose of the

combination ameliorated the negative effects of

the DISS the level of anxiety. In a 4 week open,

multicenter study in children less than 12 years

(n=918) suffering from restlessness and

nervous dyskoimesis a combination of valerian

and lemon balm preparation (2×2 tablets /day

of 160 mg valerian root dry extract (4–5:1) and

80 mg lemon balm leaf dry extract (4-6:1) was

given.The primary symptoms of dyssomnia and

restlessness were reduced from

„moderate/severe‟ to „mild‟ or „absent „ in most

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of the children with 70.4% 0f the patients with

restlessness improving. Both parents and

investigators assessed efficacy as „very good‟

or „good‟ (65.5% and 67.7%, respectively)

(Muller and Klement, 2006).

Eschscholzia californica (DC.) Stapf. (Lemon

grass)

In 50 participants lemongrass infusion was

evaluated for hypnotic and anxiolytic activity

(Aleite et al., 1986), it was found that there was

no difference between lemon grass and

placebo.

Centella asciatica (L.) URB (Gotu Kola)

It is used in ayurvedic and traditional

pacific medicine for the tr atment of anxiety

and depression (Bone, 2003). In a double blind

placebo controlled study (Bradwejn et al.,

2000), 40 healthy participants were randomly

assigned to receive either a single 12 g orally

administered dose of gotu kola or placebo, it

was found that gotu kola significantly

attenuated peak ASR amplitude 30 and 60 min

after treatment indicating anxiolytic activity in

humans.

Withania somnifera (L.) Dona.

(Ashwagandha)

It is classified as rasayana in ayurvedic

medicine and it is used to enhance mental and

physical performance.It is widely used in the

western countries in various nervous system

disorders (Bone, 2003). In an animal study

(Bhattacharya and Muruganandam, 2003) it

was observed the adaptogenic behavior of

ashwagandha in stress –inducing procedure, via

the attenuation of stress related parameters

(cortisol levels, mental depression, sexual

dysfunction.

Bacopa monnieri (L.) Wettst. (Brahmi)

A 12 week RCT using 300 mg of brahmi

revealed that there was marked reduction in

anxiety by brahmi as compared to placebo

(Stough et al., 2001).

Ginkgo biloba L. (Maiden hair)

In a RCT using EGb 761 extract (480 mg or

240 mg per day) or placebo for 4 weeks in

adults with GAD or adjustment disorder with

anxious mood as assessed by DSM-III R using

HAM-A as the primary outcome measure and

CGI,Erlangen anxiety tension and aggression

scale (EAAS) as the secondary outcome

measure it was demonstrated that the HAM-A

total scores decresed by -14.3 ( 8.1),-12

( 9.1),and -7.8( 9.2) in the 480 mg per day

Ginkgo biloba group, the 240 mg per day

Ginkgo biloba group and the placebo group

respectively.It demonstrated specific dose

dependent anxiolysis compared with placebo in

both higher dose and lower dose group (Woelk

et al., 2007).

Crataegus spp. (Hawthorn berry/leaf)

In a RCT (Walkar et al., 2002)

administered 500 mg of hawthorn extract to

mildly hypertensive patients, there was a non

significant reduction in anxiety as compared to

placebo. A double blind ,randomized placebo

controlled trial involving adults presenting with

mild moderate GAD as assessed via DSM-III R

(n=264) were prescribes two tablets containing

fixed quantities of Crataegus oxycantha

(300mg),Eschscholtzia californica (80 mg) and

magnesium (300 mg elemental) twice daily for

3 months (Hanus et al., 2004), it was observed

that the formula was highly effective in

decreasing anxiety as compared to placebo

which was determined by HAM-A and

subjectively assessed anxiety.

CONCLUSIONS

Herbal medications in psychiatry are still

under researched. The present review looked at

various herbal preparations used in depression

and anxiety over the years. The preparations

excluding St Johns wort and kava have been

under used and need further clinical trials

including randomized double blind clinical

evidence and direct comparisons with

antidepressant drugs to help us understand their

efficacy. Most herbal medications may serve as

alternatives to traditional antidepressants in

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patients who do not tolerate them as they have

a favorable safety profile and are free from

major side effects. There is also a need for

research of herbal medication in the

management of various subtypes of depression,

bipolar disorder and anxiety disorders like post

traumatic stress disorder and obsessive

compulsive disorder. The use of these

medications in various age groups and diverse

clinical populations is warranted.

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Source of Support: Nil Conflict of Interest: None Declared

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

A BRIEF REVIEW ON NONI (MORINDA CITRIFOLIA L.) - A HERBAL

REMEDY FOR BETTER HEALTH

Patel Swetal1, Krishanamurthy R

2*

1M.Sc. final year project student, C.G Bhakta Institute of Biotecnology, Uka Tarsadia University, Mahuva

Road, Bardoli-394601, Surat(Dist. Surat), Gujarat, India. 2Director, C.G Bhakta Institute of Biotecnology, Uka Tarsadia University, Mahuva Road, Bardoli-394601,

Surat(Dist. Surat), Gujarat, India.

*Corresponding Author: Email: krishnashanti@gmail.com; krishnamurthy@utu.ac.in; Mob: +919825349279

Received: 02/04/2013; Revised: 18/04/2013; Accepted: 22/04/2013

ABSTRACT

Noni (Morinda citrifolia L.) is an important herb of tropical regions of the world. It has been

used for over 2000 years in Polynesia. All parts of this plant are useful for many purposes. It has

more than 160 phyto - chemicals which make Noni a wonderful herbal remedy for the treatment of

many disease and disorders, also a wide range of physiochemical compounds and essential elements

which make Noni an effective health enhancer. Noni fruit juice is recently accepted as a novel food

in the European Union. Now a day Noni juice is in high demand as an alternative medicine for

various illnesses and it helps to live a long and healthy life. This review presents some active phyto-

chemicals and therapeutic effects of Noni which was proved by various scientists earlier.

KEY WORDS: Morinda citrifolia, Noni, phyto chemicals, remedy, disease, disorders, enhancer.

Review article

Cite this article:

Patel Swetal, Krishanamurthy R (2013), A BRIEF REVIEW ON NONI (MORINDA CITRIFOLIA

L.) - A HERBAL REMEDY FOR BETTER HEALTH, Global J Res. Med. Plants & Indigen. Med.,

Volume 2(5): 337–347

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 337–347

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

INTRODUCTION

Herbal remedies refer to the use of plants

for the promotion of healing and maintenance

of health. Till date about 80 % of people in

developing countries still relays on traditional

medicine based largely on species of plants and

animals for their primary health care. Herbal

medicines are currently in demand and their

increasing day by day (Von Reis, 1977).

Noni, with the botanical name Morinda

Citrifolia L., is a medicinal plant which is used

as a natural nutritional food supplement

worldwide for centuries. Morton (1990)

reported that the fruit of Noni plant have a

history of use in the pharmacopoeias of pacific

islands and south East Asia. Noni is one of the

important traditional folk medicinal plants that

have been used for over 2000 years in

Polynesia. The Polynesians utilized the whole

Noni plant for herbal remedies. The species of

Morinda especially M. citrifolia has been

reported to have a broad range of health

benefits for cancer, infections, arthritis, asthma,

hypertension, and pain (Whistler, 1992). The

leaves, seeds, bark, fruits and roots of Noni

have been used in various topical remedies in

South Pacific Islands and South East Asia

(Wang et al., 2002; Fygh-Berman, 2003). The

various therapeutic benefits of Noni are due to

the enriched phyto constituents. The high

therapeutic profile and safety potential of Noni

has made it a popular health enhancer and food

supplement worldwide.

PLANT DESCRIPTION

Morinda citrifolia L., is a small tree with a

height of 3–10 meter. It has an abundant long

& broad elliptic leaves (5–17 cm length &10–

40 cm width). Flowers are small, tubular and

white in colour. They are arranged in group and

inserted in one peduncle. The petiole leave ring

like marks on stalks and the corolla is greenish

white. (Morton, 1992; Elkins, 1998; Dixon et

al., 1999; Ross, 2001; Cardon, 2003). Fruits of

Noni plant are oval and have an embossed

appearance. Fruits have a range of colour from

dark green to yellow with their different

maturity stages. (Morton, 1992; Dixon et al.,

1999). A mature fruit appear almost white in

colour and is covered with a small reddish

brown bud containing the seeds. The unripe

fruit is odourless but the ripe fruit has a strong

smell of butyric acid. The seeds are triangular

in shape and radish brown in colour (Dittmar,

1993).

Chemical constituents

About 160 phyto - chemical compounds

have been already identified in the Noni plant,

and the major micronutrients are Phenolics

compounds, organic acids and alkaloids (Wang

and Su, 2001). Several classes of compounds

have been isolated from M. citrifolia L.,

including amino acids, anthraquinones,

coumarins, fatty acids, flavonoids, iridoids,

lignans and polysaccharides (Chan-Blan-co et

al., 2006). However, chemical composition

differs significantly according to the part of the

plant. The complete physico - chemical

composition of the fruit has not yet been

reported, and only partial information is

available on M. citrifolia L. juice. The fruit

contains 90% water, and the main components

of the dry matter appear to be soluble solids,

dietary fibre and proteins (Chunhieng, 2003).

Xeronine system

Noni fruit contains a natural precursor for

Xeronine that Heinicke named Proxeronine.

Proxeronine is converted to the alkaloid,

Xeronine, in the body by an enzyme

Proxeroninase. A hypothesis is that Xeronine is

able to modify the molecular structure of

proteins. Thus Xeronine has a wide range of

biological activities. When a protein such as an

enzyme, receptor, or signal transducer is not in

the appropriate conformation, it will not work

properly. Xeronine will interact with the

protein and make it fold in to its proper

conformation. The result is a properly

functioning protein (Heinicke, 1985).

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Table: 1 Some active bio chemical component of Noni juice & leaf powder

Characteristics

Fruit juice Leaf powder

Chunhieng

(2003)

Shovic and

Whisler (2001)

European

commission (2002)

(Leung et al.,

1972)

Protein 2.5% 0.4g/100g 0.2–0.5% 1 g/100g

Lipid 0.30g/100g 0.1–0.2% − 0.2 g/100g

Glucose 11.9 ± 0.2 g/l − 3.0–4.0% −

Fructose 8.2 ± 0.2 g/l − 3.0–4.0% −

Potassium 3900 mg/l 188 mg/100g 30–150 mg/100g −

Sodium 214 mg/l 21 mg/100g 15–40 mg/100g −

Magnesium 14 mg/l 14.5 mg/100g 3–12 mg/100g −

Calcium 28 mg/l 41.7 mg/100g 20–25 mg/100g 58 g/100g

Vitamin C − 155 mg/100g 3–25 mg/100g 50 g/100g

Table: 2 Chemical constituent, there place and its chemical nature in different plant part

recognize by various authors:

Plant part

(place)

Chemical constituent Structure Reference

Flower 2-methyl-4-hydroxy-5,7-

dimethoxyanthraquinone

4-O-ß-D-glucopyranosyl

(1, 4)-a-L-rhamnopyranoside

Anthraquinone

Glycosides

Sang et al., (2002)

5,8-dimethyl-apigenin

4'0-O-ß-D-galactopyranoside

Flavonoids Sang et al., (2002), Elkins

(1998)

Fruit

2,6-di-O-(ß-D-glucopyranosyl

1-O-octanoylß- D

glucopyranose

Fatty acid ester Dittmar (1993)

6-O-(ß-D-glucopyranosyl-1-O-

octanoyl-ß-D glucopyranose

glycosides Wang et al., (1999)

Ascorbic acid Acid Liu et al., (2001)

Asperulosidic acid Flavonoids Morton (1992), Elkins (1998)

Aspuruloside tetraacetate Flavonoids Wang et al., (1999), Liu et

al., (2001) Cardon (2003)

Caproic acid Acid Elkins (1998)

Caprylic acid Acid Sang et al., (2002), Dittmar

(1993),Wang et al., (2002),

Elkins (1998), Liu et al.,

(2001)

Ethyl acetate Ester Elkins (1998)

Ethyl caprylate Ester Solomon (1999b),Elkins

(1998), Dittmar (1993), Wang

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et al.,(2002), Cardon (2003),

Liu et al.,(2001), Srivastava

& Singh (1993)

Ethyl caproate Ester Dittmar (1993)

Hexanoic acid Acid Dittmar (1993)

Quercetin

3-O-a-L-rhamnopyranosyl-

(1-6)-ß-D-glucopyranoside

Flavonoide Sang et al., (2002), Cardon

(2003), Wang & Su (2001),

Farine et al., (1996)

Leaves Quercetin

3-O-a-L-rhamnopyranosyl-

(1-6)-ß-D-glucopyranoside

Flavonoids Sang et al., (2002)

Alanine Amino acid Sang et al., (2002), Cardon

(2003), Srivastava & Singh

(1993)

Serine Amino acid Dittmar (1993), Elkins (1998)

Threonine Amino acid Dittmar (1993), Elkins (1998)

Tryptophan Amino acid Dittmar (1993), Elkins (1998)

Tyrosine Amino acid Dittmar (1993), Elkins (1998)

Urosolic acid Triterpenoids

and sterols

Sang et al., (2002), Elkins

(1998), Wang et al., (2002),

Cardon (2003)

Valine Amino acid Dittmar (1993), Elkins

(1998)

Arginine Amino acid Dittmar (1993)

Aspartic acid Amino acid Dittmar (1993)

ß-sitosterol

Sterols Sang et al., (2002), Wang et

al., (2002), Chunhieng (2003)

Citrifolinoside B Iridois Sang et al., (2002)

Cysteine Amino acid Elkins (1998)

Glutamic acid Amino acid Dittmar (1993)

Glycine Amino acid Dittmar (1993), Elkins (1998)

Histidine Amino acid Dittmar (1993), Elkins (1998)

Isolucine Amino acid Dittmar (1993), Elkins (1998)

Leucine Amino acid Dittmar (1993), Elkins (1998)

Phenylalanine Amino acid Dittmar (1993), Elkins (1998)

Methionine Amino acid Dittmar (1993), Elkins (1998)

Proline Amino acid Dittmar (1993), Elkins (1998)

Kaempferolm 3-O-ß-D-

glucopyranosyl-(1-2)- a-

Lrhamnopyranosyl-(1-6)- ß-D-

galactopyranoside

Chlorophyll

derivatives

Sang et al., (2002)

Quercetin 3-O-ß- D-

glucopyranoside

Flavonoids Sang et al., (2002)

Stem 2-hydroxyanthraquinone Anthraquinone Siddiqui et al., (2006)

2-methoxyanthraquinone Anthraquinone Siddiqui et al., (2006)

Morindicininone Anthraquinone Siddiqui et al., (2006)

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 337–347

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Seeds Ricinoleic acid Acid Solomon (1999b)

Root

8-hydroxy-8-methoxy-2-

methyl-anthraquinone

Anthraquinone Cardon (2003),

Solomon (1999b)

rubichloric acid Acid Elkins (1998)

1,3-dihydroxy-6-

methylAnthraquinone

Anthraquinone Morton (1992)

Morenone 1 Anthraquinone Solomon (1999b)

Morenone 2 Anthraquinone Solomon (1999b)

Ruberythric acid Acid Cardon (2003)

Rubiadin Anthraquinone Elkins (1998), Cardon (2003),

Inoue et al., (1981), Ross

(2001)

Root bark Chlororubin Chlorophyll

derivatives

Dittmar (1993)

Hexose Saccharide Dittmar (1993)

Morindadiol Anthaquinone Dittmar (1993)

Morindanidrine Anthraquinone Dittmar (1993)

Morindine Anthraquinone Elkins (1998), Cardon (2003)

Pentose Saccharides Dittmar (1993)

Physcion Anthraquinone Solomon (1999b)

Rubiadin monomethyl ether Phenol Dittmar (1993)

Soranjidiol Anthraquinone Dittmar (1993), Elkins

(1998), Ross (2001)

Trioxymethyl anthraquinone

monoethyl ether

Anthraquinone Dittmar (1993)

Hard wood Physcion 8-O-a-L

arabinopyranosyl-

(1-3)-ß-Dgalactopyranosyl-(1-

6)- ß -D-galactopyranoside

Anthraquinone

Glycoside

Wang & Su (2001), Wang et

al., (2002)

Root,

Hard

wood,

Root bark

Morindone Anthraquinone Sang et al., (2002), Dittmar

(1993), Wang et al., (2002),

Ross (2001)

Root,

Hard

wood,

Seeds

Damnacanthal Anthraquinone Sang et al., (2002), Cardon

(2003)

Root, Root

bark, Fruit

Alizarin Anthraquinone Elkins (1998), Dittmar

(1993), Wang et al., (2002),

Cardon (2003), Ross (2001),

Dittmar (1993)

Plant

2-methyl-3,5,6

Trihydroxyanthraquinone

Anthraquinone Cardon (2003)

Scopoletin Miscellaneous

compound

Wang et al., (2002), Farine et

al., (1996)

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2-methyl-3,5,6-

trihydroxyanthraquinone

6-O-ß-D-xylopyranosyl-(1-6)-

ß-D-glucopyranoside

Anthraquinone

glycoside

Cardon (2003), Inoue et al.,

(1981)

3-hydroxymorindone Anthaquinone Cardon (2003), Inoue et al.,

(1981)

3-hydroxymorindone

6-O-ß-D-xylopyranosyl- (1-6)-

ß-Dglucopyranoside

Anthraquinone

Glycoside

Cardon (2003), Inoue et al.,

(1981)

5,6-dihydorxylucidin 3-O-ß-D-

xylopyranosyl-(1-6)- ß-D –

glucopyranoside

Anthraquinone

glycoside

Cardon (2003), Inoue et al.,

(1981)

5,6-dihydroxylucidin Anthraquinone Cardon (2003), Inoue et al.,

(1981)

Aucubin Anthraquinone Elkins (1998), Wang et al.,

(2002)

Linoleic acid Acid Wang et al., (2002)

Lucidin Anthraquinone Cardon (2003), Inoue et al.,

(1981)

Lucidin 3-O-ß-Dxylopyranosyl-

(1-6)-ß-Dglucopyranoside

Anthraquinone

glycosides

Cardon (2003)

Some selective medicinal, biological and

therapeutic activity of noni

The Polynesians utilized the whole Noni

plant for herbal remedies. The fruit juice is in

high demand in alternative medicine for

different kind of illnesses such as arthritis,

diabetes, high blood pressure, muscle aches and

pains, menstrual irregularities, headache, heart

disease, AIDS, cancers, gastric ulcer, sprains,

mental depression, senility, poor digestion,

arteriosclerosis, blood vessel problems, and

drug addiction. Scientific evidence of the

benefits of the Noni fruit juice is limited but

there are some subjective evidences for

successful treatment of colds and influenza

(Solomon, 1999a). It is reported to have

antibacterial, anti-fungal, analgesic,

hypotensive, anti inflammatory and Immuno-

stimulatory effects (McClatchey W., 2002 a &

b; Wang et al., 2002; Mathivanan et al., 2005).

Noni has a broad range of therapeutic effects

such as analgesic, anti-inflammatory,

antihypertensive, immune enhancing,

anticancer, antibacterial, antiviral, antifungal,

antistress, antituberculous, antiprotozoal,

antioxidant, and also sedative properties, also

Noni is effective in cough, nausea, enlarged

spleen, joint disorders such as gout and

arthritis, senility, poor digestion, arthrosclerosis

and drug addiction. These beneficial effects of

Noni are strongly documented and well

authenticated by valid scientific literature

evidences. Noni also has a strong cancer

preventive effect (Murugesh, 2007).

a. Anti- cancer activity

Damnacanthal, an anthraquinone extracted

from Noni induces normal morphology in a

particular type of cell found in human

neoplasias (K-ras-NKR cells) that multiply

uncontrollably and are highly malignant

(Hiramatsu et al., 1993; Hirazumi et al.,

1996; Hirazumi & Furusawa, 1999).

b. Anti HIV activity

A compound isolated from Noni roots

named 1 - methoxy - 2 – formyl – 3 -

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 337–347

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

hydroxyanthraquinone suppresses the

cytopathic effect of HIV infected MT-4

cells without inhibiting cell growth

(Umezawa et al., 1992).

c. Anti tubercular activity

At the International Chemical Congress of

the Pacific Basin Societies meeting in

Honolulu, Saludes and colleagues from the

Philippines reported that Noni Kills

Mycobacterium tuberculosis. A

concentration of extracts from Noni leaves

killed 89% of the bacteria in a test tube,

almost as effectively as the leading anti-TB

drug Rifampicin, which has an inhibitory

rate of 97% at the same concentration.

d. Anti tumour activity

TNJ showed dose-dependent cytotoxicity

on cultured cancer cells by inducing cancer

cell necrosis at high doses and apoptosis at

lower doses. Synergistic effects of TNJ

with known anticancer drugs have been

found (Wang et al., 2002).

e. Immunological activity

An alcohol extract of Noni fruit at various

concentrations inhibits the production of

tumour necrosis factor-alpha (TNF-α),

which is an endogenous tumour promoter.

Therefore, the alcohol extract may inhibit

the tumour promoting effect of TNF-α

(Ashina et al., 1994). Noni is also capable

of stimulating the release of several

mediators from murine effector cells,

including TNF-α, interleukin-1 beta (IL-1),

IL-10, IL-12, interferon-gamma (IFN- α

and nitric oxide (NO) (Himzumi et al.,

1990).

f. Anti oxidant activity

The SAR scavenging activity of Noni juice

was shown to be 2.8 times higher than that

of vitamin C, 1.4 times that of pycnogenol

(PYC) and almost the same magnitude as

that of grape seed powder (Wang & Su,

2001).

g. Analgesic activity

The results showed that rats fed 10% or

20% Noni juice had greater pain tolerance

(162% or 212%, respectively) compared

with the placebo group (Wang et et al.,

2002). Noni root extract (1600 mg/kg)

showed significant analgesic activity in the

animals, similar to the effect of morphine

(75% and 81%) protection using Noni

extract and morphine, respectively), and it

also proved to be non-toxic (Younos et al.,

1990).

h. Anti inflammatory activity

Commercial M. citrifolia L. juice

selectively inhibits cyclo oxygenase

enzymes (COX-1 and COX-2) involved in

breast, colon and lung cancer and also has

anti-inflammatory activity (Su et al., 2001).

The ability of Noni juice to inhibit these

enzymes was compared to that of

traditional commercial non-steroidal

inflammatory drugs, such as aspirin,

Indomethacin and Celebrex.

i. Anti lithiatic effect

M. citrifolia. L has the anti Lithiatic effect

on Ethylene Glycol induced Lithiasis in

male albino rats. This observation provided

the basis for considering Noni for inhibiting

stone formation induced by ethylene glycol

(Murugesh and Christina, 2007).

j. Anthelmintic activity

An ethanol extract of tender Noni leaves

was found to induce paralysis and death in

the human parasitic nematode Ascaris

Lumbricoides within a day (Raj, 1975).

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 337–347

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k. Hypotensive activity

A Hawaiian physician reported Noni, fruit

juice to have a diuretic effect (Youngken et

al., 1960).

A hot water extract of Noni roots lowered

the blood pressure of an anesthetized dog

(Youngken et al., 1958, Davison C, 1927).

A Hawaiian physician reported that Noni

fruit juice had a diuretic effect (Asahina AY

et al., 1994).

l. Anti fungal activity

Recent research has demonstrated that it

contains a water-soluble component or

components that interfere with the

morphological conversion of Candida

albicans and may have potential therapeutic

value with regard to candidiasis (Banerjee

et al., 2006; Usha et al., 2010). Other

studies showed that methanol extract of the

dried fruit exhibited maximum percentage

of inhibition against Trichophyton

mentagrophytes (79.3%), while

approximately 50% activity was recorded

against Penicillium, Fusarium and Rhizopus

species (Jainkittivong et al., 2009).

m. Anti bacterial activity

It has been reported that M. citrifolia L.

inhibits the growth of certain bacteria, such

as Staphylococcus aureus, Pseudomonas

aeruginosa, Proteus morgaii, Bacillus

subtilis, Escherichia coli, Helicobacter

pylori, Salmonella and Shigella (Atkinson,

1956). Anti-microbial effect observed may

be due to the presence of phenolic

compounds such as acubin, l-asperuloside,

alizarin, scopoletin and other

anthraquinones. Another study showed that

an acetonitrile extract of the dried fruit

inhibits the growth of Pseudomonas

aeruginosa, Bacillus subtilis, Escherichia

coli, and Streptococcus pyrogene (Locher et

al., 1995).

n. Anxiolytic activity

Recent research has demonstrated the

effects of Noni fruit on preventing anxiety

disorders, affecting an estimated 25% of the

adult population at some point during their

lifetime (Kjernised and Bleau, 2004).

o. Cardio vascular activity

Recent research has demonstrated the

ability of Noni fruit to prevent

arteriosclerosis, a disease related to the

oxidation of low density lipoproteins

(LDLs). This beneficial effect could be due

to presence of lignans (Kamiya et al.,

2004).

p. Esrogenic activity

Recent research has demonstrated the

effects of Noni fruit on preventing anxiety

disorders, affecting an estimated 25% of the

adult population at some point during their

lifetime (Kjernised and Bleau, 2004).

CONCLUSION

Noni with a botanical name Morinda

citrifolia has a long history of widespread use

as a food in tropical regions from Indonesia to

the Hawaiian Islands, and it is used as an herbal

remedy for multiple diseases. It carry several

vitamins, minerals, micro and macro nutrients

that help the body in various ways from cellular

level to organ level. It also carries several phyto

- chemicals which help to prevent severe

infection and many diseases. It has been proved

by various researchers that, drinking TNJ was

beneficial for the prevention of heart, lung, and

brain diseases as well as delaying the ageing

processing, and maintaining overall good

health so now a day a wide range of

therapeutic effects of Noni make it a popular

herbal medicine for better health.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 337–347

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Source of Support: Nil Conflict of Interest: None Declared

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

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

DEVELOPMENT OF RANDOM AMPLIFIED

POLYMORPHIC DNA MARKERS FOR AUTHENTICATION OF

RIVEA HYPOCRATERIFORMIS (DESR.) CHOISY

Borkar Sneha D1, Naik Raghavendra

2, Harisha C R

3, Acharya R N

4

1,2P G Scholar, Dravyaguna Department, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, INDIA

3Head, Pharmacognosy Laboratory, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, INDIA

4Associate Professor, Dravyaguna Department, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat,

India

*Corresponding Author: E-mail: dr.sneha.borkar@gmail.com

Received: 26/03/2013; Revised: 17/04/2013; Accepted: 20/04/2013

ABSTRACT

Rivea hypocrateriformis (Convolvulaceae) is consumed as a leafy vegetable and also reported for

its ethno-medicinal uses in cough, headache, skin disease etc. The Pharmacognostic study of its leaf

and molecular characterization of the plant by Random Amplified Polymorphic DNA (RAPD)

markers was studied following standard parameters. Genomic isolation of DNA from fresh leaves

was amplified by RAPD markers. The unique bands obtained in Polymerase Chain Reaction (PCR)

amplification were clearly discriminated having, many bright and light bands indicating the

genuinity of the plant. The diagnostic characters of its leaf were presence of paracytic stomata, oil

globule, trichomes, rosette crystals and tannin. The findings of this study may provide useful

information with regards to its DNA, pharmacognostic and taxonomic species identification.

KEYWORDS: DNA finger prints, Fanji, Leaf drug, Pharmacognosy, Rivea hypocrateriformis,

Stomatal index

Research article

Cite this article:

Borkar S D, Naik R, Harisha C R, Acharya R N (2013), DEVELOPMENT OF RANDOM AMPLIFIED

POLYMORPHIC DNA MARKERS FOR AUTHENTIFICATION OF RIVEA

HYPOCRATERIFORMIS (DESR.) CHOISY, Global J Res. Med. Plants & Indigen. Med.,

Volume 2(5): 348–356

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 348–356

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

INTRODUCTION

The use of plants as source of food is as old

as humanity. Classical texts of Ayurveda

recommend medicine as well as dietetic items

(ahara dravyas) for the management of

different disease condition like Jwara (fever),

Atisara (diarrhea) Shwasa (asthama) etc.

Ayurveda delineates these types of ahara

dravyas under the heading shakavarga (group

of vegetables). Fanji, botanically identified as

Rivea hypocrateriformis (Desr.) Choisy

(Convolvulaceae), is used as leafy vegetable

and for medicinal purposes. Its leaves are taken

internally, to relieve cough and headache (A.

Sarvalingam et al., 2011); as food supplement

(Tribhuban Panda et al., 2007); in rheumatism

and skin disease, as an analgesic, anti

inflammatory (B. Swathy et al., 2010); piles

(H.M. Patil et al., 2006) and whole plant

powder is added to milk and taken in sexual

weakness (S.Y. Kamble et al., 2010).

Random amplified polymorphic DNA

(RAPD) analysis is powerful and convenient

molecular marker system, widely used for the

genetic mapping, taxonomic and polygenic

studies of many plants. Review of literature

reveals that leaves of Rivea hypocrateriformis

have not been studied, in detail, for molecular

characterization through RAPD analysis except

some pharmacognostical characters of its leaf.

(Rajiv Kukkar et al., 2011) Hence, the present

study was undertaken to establish certain

botanical standards for identification and

standardization of R. hypocrateriformis leaf.

MATERIALS AND METHODS:

Collection and preservation of the sample:

Leaves of Rivea hypocrateriformis, were

collected from its natural habitat, Rakha khatia

forest area, Jamnagar, Gujarat, during October

2012 and identified with the help of Forest

flora of Gujarat state (R I Patel, 1984). A

sample specimen was authenticated by expert

taxonomist and deposited to institutes

pharmacognosy museum (SPECIMEN NO-

PHM 6063/21/09/2012) for future references.

The leaves were washed, shade dried,

powdered, sieved through 80 mesh and

preserved in an air-tight glass vessel. For

microscopical evaluation, fresh samples were

preserved in a solution prepared from 70%

ethyl alcohol: glacial acetic acid: formalin

(AAF) in the ratio of 90:5:5 (Johnson

Alexander Donald, 1940). Fresh leaves of

Rivea hypocrateriformis were utilized for

RAPD analysis.

Pharmacognostic studies:

Morphological characters were studied by

observing the leaves as such and also with the

help of the dissecting microscope. For detailed

microscopical observation, free hand thin

transverse sections passing through the midrib

were taken. Observed sections were cleared

with chloral hydrate and observed as such for

the presence of any crystals, then were stained

with Phloroglucinol and Hydrochloric acid

(HCl) to notice the lignified elements like

fibers, vessels etc. of the meristele and other

parts (Khandelwal K.R., 2008). Photographs of

the section were taken with the help of Canon

digital camera attached to Zeiss microscope.

Powder characters were studied as per the

guidelines of Ayurvedic Pharmacopoeia of

India, (Anonymous, 1999). The histo-chemical

tests were carried out according to the standard

guidelines. (Krishnamurty K. V, 1988).

Surface study and micrometry

Surface study of epidermis was carried out

to determine type and distribution of stomata,

epidermal cell and trichomes. Quantitative

microscopy was carried out to determine

epidermal cell number, stomatal number,

stomatal index and size of the stomata etc.

Mean values are taken by five successive

readings. (Wallis, 1985).

The stomatal index was found by using

following formula

I = S x 100

E + S

(I = stomatal index, S = no. of stomata per unit

area, E = no. of epidermal cells in the same unit

area.)

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Molecular characterization (DNA

fingerprints)

Fresh leaves were used in molecular

characterization and DNA fingerprints were

obtained by standard and most convenient

RAPD method. The RAPD reaction was

performed following standard procedures

(Baum BR, Mechanda S., 2001) at Aristogene

Biosciences Pvt. Ltd, Bangalore.

DNA isolation:

Young leaves were selected, cut into small

pieces without cutting the veins. They were

washed with distilled water and ethanol. Frozen

with dry ice and crushed. To that, 2 ml of plant

DNA extraction buffer was added. The samples

were ground thoroughly, transferred into

centrifuge tube and added 10 ml plant DNA

extraction buffer. 50 µl of BME added, to each

tube, mixed well. Incubated at 65ºC for 1 hour

with intermittent mixing. Centrifuged for 15

minutes at 10 K (10000). Supernatant was

transferred carefully into fresh tube and added

equal volume of chloroform and mixed well.

Centrifuged for 15 minutes at 10 K (10000).

Aqueous layer carefully pipetted into fresh tube

and precipitated with isopropanol. DNA pellet

suspended in 300µl of TE and subjected to

column purification.

Column purification

Silica spin columns and buffers were from

Qiagen

The column was placed in collection tube,

400 µl of equilibration buffer was added to the

column and centrifuged at 10000 rpm for 1min.

Collected buffer was discarded. 400 µl of

equilibration buffer was added to the DNA

samples, mixed and loaded into the column

(This step was repeated till the DNA sample

was completed). Flow through was collected.

500 µl of wash buffer 1 was added, centrifuged

at 10000 rpm for 1minute and buffer was

collected. 500 µl of wash buffer 2 was added,

centrifuged at 10000 rpm for 1minute and

buffer was collected. The empty column was

centrifuged with collection tube to completely

remove the wash buffer for 2 minute. 50 µl of

elution buffer was added to the column placed

in new collection tube. Incubated at room

temperature for 2 minutes and centrifuge at

10000 rpm for 1minute and eluted sample was

saved (elution 1). Previous step was repeated

(elution 2). Quantization of eluted DNA

samples was done by loading into the agarose

gel (Table 1–3).

Table 1: RAPD PCR

Sequences of primers used:

OPA-02 TGCCGAGCTG

OPB-10 CTGCTGGGAC

OPC-06 GAACGGACTC

Table 2: Reactions were set up with PCR master mix and respective Random primer. OPA-02 OPC-06 OPB-10 Notes

Double Distilled

water

18 µl 18 µl 18 µl

2X PCR master mix 20 µl 20 µl 20 µl 1X Contains 100µM each of dATP,

dGTP, dCTP and dTTP. Assay

buffer with 15mM MgCl2,

3U/reaction Taq Polymerase.

DNA sample 1 µl 1 µl 1 µl 10 pM used for each reaction

Random primer 1 µl 1 µl 1 µl

Total Volume 40 µl 40 µl 40 µl

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Table 3: PCR Conditions:

Temperature Time No. of cycles

94˚C 2 minutes 1

94˚C 30 seconds 40

45˚C 1 minute

72˚C 1 minute 30 seconds

72˚C 7 minutes 1

RESULT AND DISCUSSION:

Morphological study:

Leaves were alternate, simple, ex-stipulate, orbicular, dark green above light greenish blue below, appressedly silky hairy beneath when young, petiolate, petiole 2–6.3cm long, silky, lamina 6–7 × 6–6.5 cm, contained 6 pairs of nerves, nerves and midrib surface were light purplish, margin entire, base chordate, apex obtuse, dark purple glands were present at the base where lamina is attached to the petiole. (PLATE A-1, 2)

Transverse section of petiole

T S showed an outermost single layer of epidermis covered with cuticle. Unicellular warty trichomes were present on the epidermis. A single layer of parenchymatous hypodermis present beneath the epidermis consisted of the rosette crystals at places extended upto 2–3 layers followed by chollenchymatous 4–5 layers. In cortical region some parenchyma cells consisted of yellowish brown content was observed, which may be tannin. Meristele with bi-collateral vascular bundles was present at the centre portion. Each vascular bundle consisted of phloem towards lower epidermis and xylem towards center region. The vascular bundles were separated by uniseriate to biseriate medullary rays. Xylem consisted of xylem parenchyma and fibers. Some of the parenchyma cells also consisted of oil globules. The xylem parenchyma cells and also medullary rays were filled with tannin material. (PLATE –A 3, 4)

Transverse section of leaf

Leaf was dorsi-ventral, T.S. of the leaf through mid rib showed distinguished upper

palisade and lower spongy parenchyma and centrally located large vascular bundles. Upper and lower epidermis single layered, barrel shaped and consisted of compactly arranged cells with thick cuticle. Some of the epidermal cells on both the epidermis were with unicellular warty trichomes. Stomata were present more on lower epidermis as compared to upper epidermis. (PLATE – A 9, 10, 11)

Mesophyll differentiated into upper palisade and lower spongy parenchyma. Palisade parenchyma were elongated, 2–3 layered, compactly arranged, heavily filled with chloroplast pigments. Some of the palisade parenchyma cells consisted of rosette crystals of calcium oxalate. Spongy parenchyma present at side of lower epidermis, rounded to oval in shape with large intercellular spaces filled with chlorophyll pigment and some rosette crystals. (PLATE – A 7, 8)

Through midrib

On either side of upper and lower epidermis 1–2 layers of collenchyma cells, thick compactly arranged give mechanical support to ground tissue. Underneath the collenchyma, parenchymatous ground tissue compactly arranged without any intercellular spaces, consisted of large number of rosette crystals of calcium oxalate and tannin material towards the lower side of epidermis. Large vascular bundle situated at centre of the section which was collateral and closed in type, surrounded by parenchymatous sheath. Phloem was towards lower epidermis with some sieve elements. Xylem was towards upper epidermis, meta-xylem facing phloem and proto-xylem towards centre. Xylem consisted of some xylem parenchyma cells with fibres (PLATE– A 5, 6).

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PLATE – A: Pharmacognostic characters of Rivea hypocrateriformis (Desr.) Choisy

1.Plant in natural Habitat

2.Leaf measurements

3.T.S. of petiole (unstained)

4. T.S. of petiole (stained)

5.T.S. through midrib

(unstained)

6.T.S. through midrib

(stained)

7.Pallisade with rosette crystal

8.Pallisade 2-3 layered

9.Stomata upper epidermis

10.Stomata lower epidermis

11.Stomata lower epidermis

with measurments

12.Simple warty trichomes

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13.Simple fibers

14.Trichome thick walled

15.Paracytic stomata

16.Rosette crystals

17.Spiral vessels

18.Annular vessels

19.Tannin containg cell

20. Histo – chemical test

Table 4: Quantitative microscopic analysis of leaf

Sr. No. Characters Results

Upper Epidermis Lower epidermis

1 Stomata length 9.0 µm 9.0µm

2 Stomata breadth 7.0 µm 7.0µm

3 Epidermal cell 8 × 7 µm2 9 × 9 µm

2

4 Stomatal circumference 21 µm 2 24 µm

2

5 Epidermal cell circumference 24 µm2 26 µm

2

6 No. of stomata 6 9

7 No. of epidermal cells 36 26

8 Stomatal index 15 24

9 Palisade ratio 2–3 Nil

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Table 5: Histo – chemical test of leaf of Rivea hypocrateriformis

Sr. no Reagent Observation Characteristics Result

1. Phloroglucinol+

Concentrated HCl

Red Lignified cells ++

2. Iodine Blue Starch grains −

3. Phloroglucinol+

Concentrated HCl

Dissolved Calcium oxalate

crystals

++

4. Fecl3 solution Dark blue to

black

Tannin cells ++

5. Sudan III Red Oil globules ++

PLATE - B

Surface study & micrometry

The stomatal length and breadth of upper

and lower epidermis measured 9.0 µm and

7.0 µm respectively. Epidermal cells of upper

epidermis measured about 8 × 7 µm2

and that of

lower epidermis measured 9 × 9 µm

2. Stomatal

circumference measured 21 µm2 and 24 µm

2

where as epidermal cell circumference

measured 24 µm2

and 26 µm2

of upper and

lower epidermises respectively. The study

showed number of stomata 6 & 9, numbers of

epidermal cells 36 & 26, and stomatal index 15

& 24 of upper and lower epidermis

respectively. The palisade ratio of upper

epidermis was 2–3. Upper and lower epidermis

of leaf showed paracytic stomata, oil globule

and epidermal cells (Table 4). The stomatal

index number and palisade ratio are constant in

all geographical area but this may change in

extreme environmental condition. Micrometric

evaluation is the only way to specify the

specific species.

Histo – chemical test

To confirm the presence and absence of the

chemical constituents the material was

subjected to various tests. Lignified cells,

calcium oxalate crystals, tannin, oil globule

were present in the leaf.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 348–356

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Powder microscopy

Organoleptic characters shows greenish

colour with characteristic odour, bitter taste,

coarse in touch. Diagnostic character of leaf

powder showed simple trichomes, warty

trichomes and stomata (Paracytic) of both the

epidermis. Rosette crystals of calcium oxalate,

tannin content of mesophyll. Spiral and annular

vessels of vascular bundles. Simple fibers and

fragments of spongy parenchyma. (PLATE –

12–19)

DNA finger printing:

All the primers gave good band pattern.

Very prominent band at ~1kb and ~0.65 kb was

obtained with OPB-10 and OPA-02

respectively (Plate – B).

CONCLUSION

The unique bands obtained in Polymerase

Chain Reaction (PCR) amplification are clearly

discriminated having, many bright and light

bands indicating the genuinity of the plant

Rivea hypocrateriformis (Desr.) Choisy. The

leaf of R. hypocrateriformis can be identified

on the basis of key microscopical and

histochemical tests which showed the presence

of characters like paracytic stomata, oil

globule, trichomes, rosette crystals and tannin.

The micrometric value such as the stomatal

index (15 upper & 24 lower); stomatal length

and breadth of upper and lower epidermis

(9.0 µm × 7.0 µm); palisade ratio of upper

(2−3) and absence in lower epidermis. The

observed DNA finger prints and

pharmacognostical characters like stomatal

type, statistical data of palisade ratio, stomatal

index, stomatal number may be useful to

establish the botanical standards for

identification and standardization of Rivea

hypocrateriformis leaf.

ACKNOWLEDGEMENT

The authors like to acknowledge the

administrative authorities of the institute IPGT

& RA, Jamnagar for providing facilities during

work. Authors also express their sincere thanks

to Dr. Sudha, Director, Aristogene Biosciences

Pvt Ltd, Bangalore for their co-operation for

DNA RAPD study of the plant.

REFERENCES

Anonymous (1999). The Ayurvedic

Pharmacopoeia of India. Govt. of India

publication, New Delhi. 1st edition. 1:

Appendix 2.

A.Sarvalingam, A. Rajendran, V. Aravindhan

(2011). Curative climbers of

maruthamalai hills in southern Western

Ghats of Tamilnadu. Int. J. Med. Arom.

Plants. 1(3):326–332.

Baum BR Mechanda S, Livesey JF, Binns SE,

Arnason JT. (2001) Predicting

quantitative phytochemical markers in

single Echinacea plants or clones from

their DNA fingerprints.

Phytochemistry. 56 (6):543–9

B. Swathy, S. Mohana lakshmi, A. Saravana

Kumar (2010). Review on herbal drugs

for analgesics and anti inflammatory

activities. IJBPR. 1(1):7–12.

Govinda das, Ambikadatta Shastri (2011)

Bhaishajya Ratnavali, Chaukhambha

Prakashana, Varanasi, pp no.201, 202,

237, 238, 473, 474.

H M Patil, V V Bhaskar (2006). Medicinal

knowledge system of tribals of Nandurbar

district Maharashtra. Indian journal of

traditional Knowledge. 5(3):327–330.

Johnson Alexander Donald (1940). Plant Micro

technique. Macgrow Hill Book Company,

New York, London. pp. 105.

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Khandelwal K.R. (2008), Practical

Pharmacognosy Techniques and

Experiments, 19th

Ed, Nirali Prakashan;

2008. pp no. 15– 18.

Krishnamurty K.V. (1988). Methods in the

plant histochemistry, Vishwanadhan Pvt

Limited, Madras, pp.1– 77.

R I Patel (1984). Forest flora of Gujarat state.

Forest department, Gujarat state, Baroda.

2nd

edition. pp. 218.

Rajiv Kukkar, Mona kukkar, A.K.Saluja (2011)

pharmacognostic studies on rivea

hypocrateriformis inventi rapid: planta

active publication 2011: 9/11

S Y Kamble, S R Patil, P S Sawant, Sangita

Sawant, S G Pawar, E A Singh (2010).

Studies on plants used in traditional

medicine by bhilla tribe of Maharashtra.

Indian journal of traditional Knowledge.

9(3):591-598.

Tribhubana Panda, Rabindra N Pandhy (2007)

Sustainable food habits of the hill

dwelling kandha tribe in Kalahandi

district of Orissa. Indian journal of

traditional medicine. 6(1):103–105.

Wallis TE (1985). Textbook of Pharmacognosy.

London Churchill Publication. pp. 572–

82.

Source of Support: Nil Conflict of Interest: None Declared

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

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

ANTIMICROBIAL EVALUATION OF CROTON ROXBURGHII BALAK.

(EUPHORBIACEAE) STEM BARK

Patel Esha1*, Padiya RH

2, Acharya RN

3

1Ph.D. Scholar, Dept. of Dravyaguna, Institute for Postgraduate Teaching and Research in Ayurveda, Gujarat

Ayurved University, Jamnagar, Gujarat – 361 008, India. 2Ph.D. Scholar, Dept. of Dravyaguna, Institute for Postgraduate Teaching and Research in Ayurveda, Gujarat

Ayurved University, Jamnagar, Gujarat – 361 008, India. 3Associate Professor, Dept. of Dravyaguna, Institute for Postgraduate Teaching and Research in Ayurveda,

Gujarat Ayurved University, Jamnagar, Gujarat – 361 008, India.

*Corresponding Author: Email: esha.ayupharma@gmail.com

Received: 26/02/2013; Revised: 10/04/2013; Accepted: 15/04/2013

ABSTRACT

Antibacterial and anti fungal activity of Croton roxburghii Balak. (Euphorbiaceae) stem bark

methanol extract against four pathogenic bacterial strains; two Gram positive (B.subtilis & S.aureus),

two Gram negative (E.coli and K. pneumoniae), and two fungal strains (S. flavus & C.albicans), in

different concentrations (5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, 250 μg/ml) was studied. A zone of

inhibition of extract was compared with that of different standards like Streptomycin, Cifpodoxime

and Gentamycin for antibacterial activity and Amphotericin, Fluconazole and Clotrimazole for

antifungal activity. The test drug extract showed remarkable inhibition antibacterial and antifungal

activities comparable with that of standard against the organisms tested.

KEYWORDS: Antibacterial activity, antifungal activity, Croton roxburghii stem bark, methanol

extract, Gandhamardana hills

Research article

Cite this article:

Patel Esha, Padiya RH, Acharya RN (2013), ANTIMICROBIAL EVALUATION OF CROTON

ROXBURGHII BALAK. (EUPHORBIACEAE) STEM BARK, Global J Res. Med. Plants & Indigen.

Med., Volume 2(5): 357–364

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 357–364

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

INTRODUCTION

Over the past few decades there has been

much interest in natural materials, as sources of

new antimicrobial agents. Different extracts

from traditional medicinal plants have been

tested. Many reports show the effectiveness of

traditional herbs against microorganisms and as

a result, plants have become one of the bases of

modern medicine (Evans et. al., 2002). Plants

have given the Western pharmacopoeia about

7,000 different pharmaceutically important

compounds and a number of top-selling drugs

of modern times, such as quinine, artemisinin,

shikonin and camptothecin (Tshibangu et al.,

2002). Recently much attention is being paid to

the biologically active compounds derived from

plants used in herbal medicine (Deshwal V K,

2012).

Tribal people in India used various parts of

Croton roxburghii Balak. (Euphorbiaceae)

against snake poisoning and to treat infertility,

fever and wounds (Gupta et al., 2004). In India,

only five species of Croton are used in ethno-

medicine for treatment of various diseases,

disorders and ailments like boils, bowel

complaints, chicken pox, cholera, cold and

cough, constipation, cuts and wounds,

diarrhoea, dysentery, eye diseases, epilepsy,

fever, gastric disorders, insanity, jaundice, liver

complaints, malaria, rheumatism, ringworms,

scurvy, spasmolytic agent, snake bite, sprains,

etc (Salatino et al., 2007). Its stem bark is used

as external application for sprains, bruises and

rheumatic swellings and internally in cholera,

diarrhoea, malaria, jaundice, stomach and urine

trouble, against snakebite, on inflammation etc.

(Anonymous, 2008) However no reports are

available on its antimicrobial activity except

Thatoi et Al., 2008 . Hence the study was

planned to evaluate the antimicrobial activity of

Croton roxburghii stem bark.

MATERIALS AND METHODS

Collection of plant materials

The plant Croton roxburghii Balak.

(Euphorbiaceae) was identified from its natural

habitat of Gandhamardana hill ranges,

Bolangir, Odisha, India; after studying its

morphological characters and comparing them

with the characters mentioned in various floras

(Gamble, 1993; Haines, 1988; Hooker, 1885;

Saxena, & Brahman, 1995; Shah, 1978). The

stem bark was collected, washed properly

under running water, to make them free from

foreign matter like sand, soil etc. and dried

under shade. Herbarium voucher No. 6047 was

also prepared and submitted to Pharmacognosy

museum of IPGT & RA, Jamnagar, for future

reference.

Sample preparation:

For the analysis, Croton roxburghii stem

bark was coarsely powdered to 60# and then

tests were performed.

Determination of microbial load for plant

material

Microbial load of the samples was done by

total viable aerobic count method as given in

WHO monograph (Anonymous, 1996;

Anonymous, 1998).

To 500 mg, accurately weighed sample, 1–

2 drops of Tween 80 and a homogeneous

suspension was prepared by slowly adding 5 ml

of sterile buffered sodium chloride peptone

(SBSCP) solution of pH 7.0. This suspension

was diluted 10-1

onwards as required in sterile

dilution blanks (SBSCP). One ml each from

these aliquots was added to sterile melted and

cooled top agar (Soyabean casein digest agar,

for fungal count Potato dextrose agar medium

used) tubes. These tubes were poured to sterile

petri dishes and allowed to solidify. These

plates were incubated at 30–35°C for 48 hours.

The numbers of colonies were counted and the

results were expressed in Cfu / g.

Cfu / g = Number of average colonies

Dilution × Volume plated

Antimicrobial activity of plant materials

(Anonymous, 1996)

Extract preparation: Plant extracts prepared in

methanol, 1 g of material was extracted in

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methanol by sonicating it for 10 min and then

kept overnight. Next day after filtration,

methanol was evaporated, and 5 different

concentrations i.e. 5 µg/ml, 25 µg/ml, 50

µg/ml, 100 µg/ml, 250 µg/ml were prepared

from the residue of each sample. These were

used for determination of antimicrobial

activity.

Culture conditions: The antimicrobial efficacy

of these plant materials tested on 6 different

strains, 2 Gram positive bacteria namely

Bacillus subtilis (NCIM 2063) &

Staphylococcus aureus (NCIM 2079), 2 Gram

negative bacteria namely Escherichia coli

NCIM 2065 and Klebsiella pneumoniae (NCIM

2719). Two Fungal strains namely Aspergillus

flavus (NCIM 1028) & Candida albicans

(NCIM 3471). All cultures were obtained from

NCL, Pune. 24 hour old cultures of all these

organisms were inoculated in sterile broths and

incubated till 0.5 Mcfarland standard turbidity

obtained, and then used for assay.

Antimicrobial assay (Doman & Deans, 2000)

Sterile soybean casein digest agar (25 ml/plate)

was used for antibacterial activity and sterile

sabouraud agar (25 ml per plate) was used for

antifungal activity. Medium obtained from

Himedia laboratories. 20 ml sterile medium

was poured in sterile plates aseptically and they

were kept until solidified. Then 0.5 ml of

culture is inoculated in 5 ml sterile melted and

cooled medium and poured on solidified agar

plates aseptically. After solidification well was

made with the help of cup borer and 0.3 ml of

each sample was inoculated in them. For

antibiotic discs there is no need to make wells,

disc were directly placed on agar surface

aseptically. For diffusion purpose plates were

placed in refrigerator for 20–25 minutes. Then

plates were incubated at 37°C for 24 hours

except sabouraud agar plates and plates

containing K. pneumoniae organism, they were

incubated at 30°C for 24–48 hours. After

incubation zone of inhibition was measured

with Himedia antibiotic zone scale- c.

Pathogen study (Anonymous, 1996)

Same extracts were used as for

antimicrobial activity assay. These extracts

were transferred to specialized mediums given

below and incubated at their optimum

temperature for growth, then after incubation

plates were observed and results were

concluded.

Selective differential mediums according

pathogens:

Pseudomonas aeruginosa – Citrimide agar

Salmonella typhi – TSI agar slant, XLD agar

Escherichia coli – EMB agar

Staphylococcus aureus – Mannitol salt agar

RESULTS AND DISCUSSION

Description: Croton roxburghii stem bark

powder is reddish coloured coarse powder.

Microbial load report:

The observations on the microbial load of

Croton roxburghii bark shows that the tested

samples, when collected from their natural

sources, were within prescribed limit of the

microbes. (Table 1).

Antimicrobial activity (Plate 1 & 2):

The antimicrobial activity of methanol

extracts of Croton roxburghii stem bark was

studied in different concentrations (5 μg/ml,

25 μg/ml, 50 μg/ml, 100 μg/ml, 250 μg/ml)

against four pathogenic bacterial strains two

Gram positive B. subtilis NCIM 2063 & S.

aureus NCIM 2079, two Gram negative (E. coli

NCIM 2065, K. pneumoniae NCIM 2719) and

two fungal strains (S. flavus NCIM 1028 and C.

albicans NCIM 3471). Antibacterial and

antifungal potential of extracts were assessed in

terms of zone of inhibition.

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Table 1 : Microbial load report

Parameter Sample Permissible Microbial

contamination limits

(Anonymous, 2001)

C. roxburghii stem bark

Total Viable Aerobic Count (Cfu/g)

a) Bacterial count 14.6 × 103 10

5/g

b) Fungal count 30 103/g

Pathogens (per gram)

a) S. aureus Absent Absent

b) E. coli Absent Absent

c) P. aeruginosa Absent Absent

d) S. typhi Absent Absent

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Table 2: Antibacterial activity of methanol extracts of Croton roxburghii stem bark and

standard drugs against Gram + ve and Gram -ve organisms

Sample Concentration Zone of inhibition (mm)

B. subtilis

(NCIM

2063)

S. aureus

(NCIM

2079)

E. coli

(NCIM

2065)

K.

pneumoniae

(NCIM

2719)

C. roxburghii stem bark

methanol extract

5 µg/ml 12 Nil 13 13

25 µg/ml 13 Nil 14 14

50 µg/ml 15 12 16 16

100 µg/ml 17 13 17 17

250 µg/ml 18 15 20 18

Methanol

(Control)

− Nil Nil Nil Nil

Gentamycin 10 µg 28 25 22 24

Cifpodoxime 10 µg 22 23 21 19

Streptomycin 10 µg 27 17 17 24

Figure 1: Effect of C. roxburghii stem bark methanol extract against Gram +ve and

Gram −ve strains

Figure 2: Effect of standard drug against Gram +ve and Gram −ve strains

0

5

10

15

20

25

B. subtilis (NCIM 2063)

S.aureus (NCIM 2079)

E. coli (NCIM 2065) K. pneumoniae

5

25

50

100

250

0

5

10

15

20

25

30

B. subtilis (NCIM 2063)

S.aureus (NCIM 2079)

E. coli (NCIM 2065)

K. pneumoniae

Gentamycin 10µg

Cifpodoxime 10µg

Streptomycin 10µg

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Table 3: Antifungal activity of methanol extracts of Croton roxburghii stem bark and standard

drugs

Sample Concentration Zone of inhibition (mm)

S. flavus

(NCIM 1028)

C. albicans

(NCIM 3471)

C. roxburghii

stem bark

methanol extract

5 µg/ml Nil 16

25 µg/ml 11 17

50 µg/ml 12 18

100 µg/ml 13 20

250 µg/ml 14 22

Methanol (Control) − Nil 11

Amphotericin B 50 µg 10 µg 14 19

Fluconazole 30 µg 10 µg 11 28

Clotrimazole 10 µg 10 µg 24 30

Figure 3: Effect of C. roxburghii stem bark methanol extract against two fungal strains

Figure 4: Effect of standard drugs against two fungal strains

The antibacterial and antifungal activity of

the methanol extracts of Croton roxburghii

stem bark, increased linearly with increase in

concentration of extracts (μg/ml). As compared

with standard drugs, the results revealed that in

the extracts for bacterial activity, E. coli and B.

subtilis were more sensitive when compared to

K. pneumoniae and S. aureus, and for fungal

activity C. albicans showed good results when

compared to S. flavus but S. flavus was more

0

5

10

15

20

25

S. flavus (NCIM 1028) C. albicans (NCIM 3471)

5

25

50

100

250

0

5

10

15

20

25

30

35

S. flavus (NCIM 1028) C. albicans (NCIM 3471)

Amphotericin B 50µg

Fluconazole 30µg

Clotrimazole 10 µg

Methanol (Control)

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sensitive. The growth inhibition zone measured

ranged from 12–20 mm for all the sensitive

bacteria, and ranged from11–22 mm for fungal

strains. (Table 2−3) (Fig 1−4)

The inhibitory effect of C. roxburghii stem

bark methanol extracts (5, 25, 50, 100,

250 μg/ml) showed (13, 14, 16, 17, 20 mm)

against E. coli; (13, 14, 16, 17, 18 mm) against

K. pneumonia; (00, 00, 12, 13, 15 mm) against

S. aureus; (12, 13, 15, 17, 18 mm) against B.

subtilis; (00, 11, 12, 13, 14 mm) against S.

flavus; (16, 17, 18, 20, 22 mm) against C.

albicans.

The result shows that the extracts of all

samples were found to be effective against all

the microbes tested.

CONCLUSION

In this screening work, the test drug extract

at different concentrations was found to be

effective against all organisms such as Gram

positive, Gram negative and fungal strains.

From the above results the activity of all

extracts shows significant antibacterial and

antifungal activity. The present study justified

the claimed ethnic uses of C. roxburghii stem

bark externally in ringworms, scurvy, bruises

and to treat various infectious diseases caused

by the microbes. However, further studies are

required to isolate the active compounds from

Croton roxburghii stem bark, responsible for

the antimicrobial property which may lead to

compounds in the field of antimicrobial.

REFERENCES

Anonymous, (1996). Indian Pharmacopoeia

Government of India, Ministry of

Health and Family Welfare, Controller

of Publications, Delhi; 1(1); p.no. 37–

49.

Anonymous, (2001). The Ayurvedic

Pharmacopoeia of India Part-I, Volume-

I, First edition, Ministry of Health and

Family Welfare, Government of India,

Department of Indian Systems of

Medicine & Homoepathy, New-Delhi;

p.no. 139.

Anonymous, (1998). Quality control methods

for herbal materials W.H.O Monograph

for limitation of microbes, WHO Press,

World Health Organization, 20-Avenue

Appia, 1211 Geneva 27, Switzerland;

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Anonymous, (2009). Review on Indian

Medicinal plants Vol. 8, ICMR, New

Delhi; p.no.145-147, 156–157.

Deshwal VK (2012). Antibacterial activity of

seeds of Mucuna pruriens L. against

Escherichia coli and Staphylococcus

aureus, GJRMI, 4(1); p.no. 109 – 113.

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agents from plants. Antimicrobial

activity of plant volatile oils, Journal of

Applied Microbiology; 88(2); p.no.

308–316.

Evans CE, Banso A, Samuel OA (2002).

Efficacy of some nupe medicinal plants

against Salmonella typhi: an in vitro

study, Journal of Ethnopharmacology,

80: p.no. 21–24.

Gamble JS (1993). Flora presidency of Madras,

vol. II, Bishen Singh Mahendra Pal

Singh, Dehradun, India; (London); p.no.

1010.

Gupta M, Mazumder UK, Vamsi MLM,

Sivakumar T, Kandar CC (2004).

Anti‐steroidogenic activity of the two

Indian medicinal plants in mice. J

Ethnopharmacol; 90 suppl 1; p.no.21–

25.

Haines HH (1988). The Botany of Bihar and

Orissa, part III-IV, Bishen Singh

Mahendra Pal Singh, Dehradun (India);

p.no.667.Hooker JD (1885). The flora

of British India, Vol. IV, Bishen Singh

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Mahendra Pal Singh, Dehradun, India,

(London); p.no. 394–395.

Salatino A, Salatino MLF, Negri G (2007).

Traditional uses, chemistry and

pharmacology of Croton species

(Euphorbiaceae). J Braz Chem Soc,18 ;

p.no.11–33.

Saxena HO, Brahman M (1995). The flora of

Orissa, vol. III, Regional Research

Laboratory, Orissa Forest Development

Corporation Ltd., Bhubaneswar,

Orissa.(India); p.no.1323, 1372–1374.

Shah GL (1978). Flora of Gujarat State, Part-I,

Shri. K. A. Amin, Registrar, Sardar

Patel University, Vallabh Vidyanagar;

p.no. 525-527, 549–550.

Thatoi HN, Panda SK, Rath SK, Dutta SK

(2008). Antimicrobial activity and

ethnomedicinal uses of some medicinal

plants from Similipal Biosphere

Reserve, Orissa. Asian J Plant Sci;7:

p.no.260–267.

Tshibangu JN, Chifundera K, Kaminsky R,

Wright AD, Konig GM (2002).

Screening of African medicinal plants

for antimicrobial and enzyme inhibitory

activity, Journal of Ethnopharmacology,

80; p.no. 25–35.

Source of Support: Nil Conflict of Interest: None Declared

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

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

AN ESTIMATION OF HUMIC SUBSTANCES IN AN AYURVEDIC

HERBOMINERAL DRUG SHILAJATU (ASPHALTUM) AS PART OF

PHYTO-PHARMACEUTICAL STANDARDIZATION

Akarshini A M1*, Renuka

2, Shukla V J

3, Baghel M S

4

1PhD scholar, Department of Kayachikitsa, IPGT &RA, Gujarat Ayurveda University, Jamnagar, Gujarat,

India 2PhD scholar, Department of Pharmaceutical chemistry, IPGT &RA, Gujarat Ayurveda University,

Jamnagar, Gujarat, India 3Department of pharmaceutical Chemistry, IPGT &RA , Gujarat Ayurveda University, Jamnagar, Gujarat,

India 4Director, IPGT &RA, Gujarat Ayurveda University, Jamnagar, Gujarat, India

*Corresponding Author: Email: drakarshini@ymail.com; Mob: +919737234034

Received: 28/03/2013; Revised: 28/04/2013; Accepted: 04/05/2013

ABSTRACT

Shilajatu an important Ayurvedic drug having several medicinal properties. If administered after

proper purificatory procedure (shodhana), it has miraculous benefits. Market products are subjected

to some processing, but the purity among samples vary, thereby therapeutic efficacy too. Sothere is

need for standardization of purity assessment strategies which could help to scrutinize the good

sample. Previous efforts towards standardization of the raw drug shilajatu aimed to identify plant

source in formation of shilajatu, along with basic phyto-pharmaceutical parameters assessment.

Present work attempts to see how different the data varies when the basic phyto-pharmaceutical

parameters performed on processed sample. Also an additional assay of shilajatu, to quantify fulvic

acid through UV spectrophotometry, and quantify humic acid through gravimetric method of

analysis was carried out. Lignin decomposition logic has been attempted using UV &

chromatographic assay. Chromatography was performed as per Certified Reference Material. The

data may be used as a reference parameter for purity analysis of processed shilajatu. Results showed

the shilajatu from Nepal had high purity and greater percentage of fulvic acid compared to that from

India. This work is unique among efforts towards establishment of universal strategies in purity

assessment of shilajatu.

KEYWORDS: Shilajatu, Chromatography, UV spectrophotometry, Gravimetric method, humic

acid, fulvic acid.

Research article

Cite this article:

Akarshini A M, Renuka, Shukla VJ, Baghel MS (2013), AN ESTIMATION OF HUMIC SUBSTANCES IN AN

AYURVEDIC HERBOMINERAL DRUG SHILAJATU (ASPHALTUM) AS PART OF PHYTO

PHARMACEUTICAL STANDARDIZATION, Global J Res. Med. Plants & Indigen. Med., Volume 2(5): 365–373

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 365–373

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

INTRODUCTION

Shilajatu (Mineral Pitch) is a herbo-mineral

drug formed out from fissures of iron rich rocks

during hot weather. Traditional uses of

Shilajatu indicate its efficacy in treating

diabetes and diseases of the urinary tract as

well as edema, tumors, diseases causing

emaciation, epilepsy and insanity. Modern

science extends its indications to all the

systems of the human body with a significant

number of additions in the reproductive and

nervous system (Robert, 2004). Lots of

controversy exists, regarding the sources and

availability of pure form of shilajatu. This

article aims to analyze the difference between

samples collected from Nepal and India in

comparison to synthetic fulvic acid. This work

is unique among efforts towards establishment

of universal strategies in purity assessment of

shilajatu.

Shilajatu in its raw form contains free

radicals and may also contain mycotoxins and

fungal toxins. The processing needs to remove

the free radicals, Polymeric Quinone radicals,

toxins, mycotoxins and inactive ingredients

(Shilajit, Ayurwiki.info). Only the purified

extract gives the desired benefits of shilajatu.

So for the present work, shilajatu which had

undergone processing with triphala qwatha and

cow’s milk following the method described in

Ayurvedic Texts was obtained (Shastri Ambika

datta, 2010). Samples collected were subjected

to phyto-pharmaceutical assay comprising

quantitative and qualitative estimation of

various constituents present therein, and the

obtained results were compared. Previous

efforts towards standardization of the raw drug

shilajatu aimed to identify plant source in the

formation of shilajatu, along with basic phyto-

pharmaceutical parameters assessment

(Saileshnath saxena, 1995). Present work

attempts to see how different the data varies

when the basic phyto-pharmaceutical

parameters were performed on processed

sample. The UV spectro-photometry and

Gravimetric Methods were specially designed

for the qualitative analysis of shilajatu. Method

used for estimation of maturity of organic

compost served as pedestal for application of

above two methods here; as shilajatu itself is a

complex mixture of organic humic substance.

MATERIALS AND METHODS

Phytochemical analysis

Powder microscopy

1. Sudda shilajatu of punjab, obtained from the

pharmacy of IPGT & RA, Jamnagar, India

(Sample I).

2. Sudda shilajatu from Singh Darbar

Dawakhana, Kathmandu, Nepal (Sample II).

Shilajatu found in Nepal is considered as the

best and is also acclaimed for a highly potential

medicinal herbo-mineral drug.

Phytochemical assay of Shilajatu

Shilajatu Sample I & II were analyzed by

using qualitative and quantitative parameters at

Pharmaceutical chemistry laboratory of IPGT

& RA, Gujarat Ayurveda University, Jamnagar.

RESULTS AND DISCUSSION

Organoleptic Parameters

The characters of the sample are tabulated

in Table 1.

Microscopy of Shilajatu (Sample I and II)

Samples were subjected to microscopic

analysis after staining the samples with and

without water. The Microscopic features are

tabulated in Table 2. Images of powder

microscopy of sample I & II are shown in

Figure 1, 2.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 365–373

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

TABLE 1. Organoleptic parameteres of Shilajatu sample I & II

Sl.No Parameters Suddha Shilajatu Sample I Suddha Shilajatu

Sample II

1 Colour Brownish black Black

2 Odour Smell of cows urine Smell of cows urine

3 Taste Bitter, Astringent Bitter, Astringent

4 Consistency Solid Thick semisolid

5 Solubility in

Water

Easily dissolves Easily soluble

In acid

(HCl)

Dissolves in HCl with blackish brown color

changing to light brown

Slightly soluble

Alcohol Slightly soluble Insoluble

Acetic acid Slightly soluble Soluble

TABLE 2. Microscopic study of samples

Reagents used Sample I Sample II

Water High amount of crystalline material Calcium oxalate crystals

Oil globules

Crystalline material

HCl - No crystalline material

Oil globules present

FIGURE 1-Powder microscopy of Suddha Shilajatu sample I

Crystalline material Prismatic crystal

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 365–373

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

FIGURE 2- Microscopy of Suddha Shilajatu sample II

Crystals in group Oil globule Prismatic crystal

Physico - chemical parameters

Sample I & II were evaluated for physico-

chemical parameters like water and methanol

soluble extractive and pH. The water and

methanol soluble extractive in sample II

measured 61.03% and 26.31% respectively and

that of sample I measured 25.2% and 18.2%

respectively. The pH was 5 in both, acidic by

nature.

Qualitative tests

The water extract of the samples were

analyzed for different functional groups.

Results of qualitative tests are shown in Table

3.

High Performance Thin layer

chromatographic study (HPTLC)

Methonolic extract of Sample I & II was

fixed through TLC. Findings of TLC shown in

Figure 3 Later HPTLC was carried out.

Methanolic extract of Sample I & II and Fulvic

acid (standard) were spotted on pre-coated

silica gel GF 60254 aluminium base plate by

Camag Linomate V sample applicator fitted

with a 100 L Hamilton syringe, 10 ml of 25%

ammonium Hydroxide : n-propanol (7:3v/v)

was used as a mobile phase. The development

distance was 6.4 cm (development time

30 min). After development, densitometric

scanning was performed with a camag TLC

scanner III in reflectance absorbance mode at

254 nm and 366 nm under control of win

CATS software (V 1.2.1 camag) (Figure 4).

The slit dimensions were 6 mm × 0.45 mm and

the scanning speed was 20 mm s-1

. Visual

observation under densitometer showed

2 spots. However the chromatogram showed

two prominent spots at hRf at 0 and 80 in

sample I and one spot at hRf 80 in sample II,

one prominent spot at hRf 80 in Fulvic acid

(standard), in short wave UV 254 nm. Result

implies both the samples contained fulvic acid.

Sample II showed high purity level whereas

sample I had constituents other than fulvic acid

too which impede its purity.

TABLE 3. Qualitative parameters for SRC for different functional groups

Sl.No Functional groups Name of the test/reagent Results

Sample I

Results

Sample II

1 Alkaloids Wagner’s Reagent Negative Negative

2 Tannin Lead acetate Positive Positive

3 Saponin Glycosides Foam test Negative Negative

4 Protein Biuret test Positive Positive

5 Steroids Libermann Burchard’s test Negative Negative

6 Flavonoids NaOH test Positive Positive

7 Reducing sugars Fehling’s test Absent Absent

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FIGURE 3. TLC of methanol extract of sample I & II

A - 260nm

B - 472nm

C - After spraying with anisaldehyde Sulphuric acid

FIGURE 4. Densitometer curve of methanolic extract of sample I, standard fulvic acid and

sample II at 254nm

UV spectrophotometry

There is no any accepted method to

quantify fulvic acid, which is practical and cost

effective. Hence it was aimed to evaluate and

develop a spectro-photometric fulvic acid

quantification protocol for future laboratory

analysis of shilajatu & other humic substances.

UV spectrophotometry was carried out to

evaluate the quantity of fulvic acid present in

the Sample I & II compared to the reference

standard fulvic acid.

Specificity to undertake UV

spectrophotometry:

Humic substances are formed by the

microbial degradation of dead plant matter,

such as lignin. These can be divided into three

main fractions: humic acids, fulvic acids, and

humin (Humate fertilizer) (Shilajatu has been

found to consist of a complex mixture of

organic humic substances and plant and

microbial metabolites come about in the rock

rhizospheres of its natural habitat. The active

principle of shilajatu is fulvic acid and it seems

to have that unique capacity to dilate and

permeate the thick cell walls to transmit the

minerals into the cells, thereby overcome

tiredness, lethargy, and chronic fatigue

(Rudramani Shilajatu). Moreover shilajatu is

not only about Fulvic acid. It contains more

than 85 minerals and nutrients which can be

instantly transported to the cells by fulvic acid

(Rudramani Shilajatu). Based on the ratio of

fulvic acid with humic acid, purity and action

potential of shilajatu can be ascertained.

Aimed to establish a quantitative

correlation between UV/viz absorption and the

concentrations of fulvic acid isolated from

different sources. Based on the assumption that

the overall optical properties of humic

substances are very similar regardless of their

C B A

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 365–373

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origins, this was undertaken. (Daqing Gan,

2007) An index was generated to quantify

fulvic acid in a sample with both humic acid

(HA) and fulvic acid (FA). If FA is more than

HA, then the sample is good; or vice versa. The

reactive moiety is equal to FA. This was based

on the principle that, in agricultural science

composting is believed to yield humus like

substances through various biochemical

process. This influences the quality of soil

organic matter (Carmen, 2004). Shilajatu is

also a complex form of organic humic

substances. As humic substances are formed by

the microbial degradation of dead plant matter,

such as lignin. Conversion of lignin and other

components into humic substances like humic

acids, fulvic acids, and humin is known by

Humification Index (HI). HI is used as process

controlled parameter, while in shilajatu the

humification process will be completed

naturally (not induced). Hence HI will predict

the quality of shilajatu. Thus parameter is used

to determine the quality of naturally collected

sample rather than only measuring selected

FA/HA from decomposed mass. Knowing the

ratios of HA to FA helps us predict HI, because

FAs (more aliphatic and richer in carboxiylic

acid, phenolic and quinone groups) are more

soluble and reactive than HAs (more aromatic

and insoluble when carboxylate groups are

protonated at low pH). (Carmen, 2004)

Quinones in FA have stronger antioxidant

capacity and contain higher levels of phenolics

than others which have potential antioxidant

and chemo-protective agents. (Hua-Bin- li et

al., 2011) This action is needed for

neurodegenerative diseases like DPN.

The alkaline extract of sample I & II was

prepared. After suitable dilution of one gram of

sample with 50ml of 0.5 Normal NaOH

(sodium hydroxide), it was kept overnight.

Next day the supernatant was collected, and

was scanned through 200–800 nm in a

Shimadzu UV-visible double beam recording

spectrophotometer (UV-160A) and the

absorbance in spectra were recorded based on

reference to standard range. Three standard

wavelengths 260 nm, 280 nm, 472 nm were

selected (Radosław Zbytniewski, 2005). The

UV-visible spectrums of the alkaline extract of

both samples were recorded Results observed

under UV–visible spectrum has been presented

in Table 4. It shows two absorption peaks at

472 nm and 664 nm, negative peaks at 280 nm,

260 nm. There is no absorption in the visible

region. The following absorbance ratios was

used for calculation (Kononova, M., 1968,

Gieguzy_nska E, 1998)-

Q2/6(HA) = Absorbance Ratio of 260/664,

denotes the relation between non-humified and

strongly humified material. No decay started.

Q4/6(FA) = Absorbance Ratio 472/664, is

often called the humification index. Typical

values of the Q4/6 ratio for humified material

are usually <5. (Gieguzy_nska E, 1998)

Q2/4(lignin & others) = Absorbance Ratio

280/472, reflects the proportion between the

lignins and other materials at the beginning of

humification, and the content of materials at the

beginning of transformation.

Low Q2/6 or Q4/6 ratios reflect a high degree

of aromatic condensation and indicate a higher

level of organic material humification.

(Carmen, 2004)

As per index sample II contained good

amount of FA (3.823) in comparison to sample

I (3.071). The standard sample showed only

1.666 of fulvic acid. Table 4.

The UV spectrum is much practical, rapid

means of estimating the concentration of a

fulvic acid solution. It estimates characteristic

pH independent, exponential spectrum of a

standard FA in the 200 – 800 nm wavelength

range. Given the length and complexity of FA

sample isolation and the likelihood of at least

some sample loss on the chromatography

columns involved, this simple spectro-

photometric approach described here (Elham A,

2009) merits application for the analysis of

fulvic acid solutions. The results of this work

have potential use for the routine analysis of

FA samples and for the certification and

regulation of commercial shilajatu and its

formulations.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 365–373

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TABLE 4 - Qualitative parameters for SRC for different functional groups

Ratio FA Sample I Sample II

Q1= HA −10.703 −16.357 −9.000

Q2= FA 1.666 3.071 3.823

Q3=lignin and others −5.288 −6.0697 −1.692

TABLE 5 - Showing the absorbance at different wavelengths

Sample 280 nm 472 nm 664 nm 260 nm

Shilajatu I −0.261 0.043 0.014 −0.229

Shilajatu II −0.220 0.130 0.034 −0.306

Fulvic acid −0.238 0.045 0.027 −0.289

Gravimetric method/Acid precipitation

method

The acid precipitation method has been

widely accepted for the separation and

subsequent quantification of humic acid

(Thurman EM, 1981). The quantification of

humic substances is important because humic

materials have a relatively high content of free

radicals which play important roles in

polymerization and redox reactions. This

affects the mobility of metals (both those with

nutrient value and those that are of concern

because they are pollutants (Carmen, 2004).The

humic acid (HA) precipitates at pH < 2 and

thus can be quantified by gravimetric

measurements. A comparison of appropriate

methods of HA analysis favors HA

precipitation from alkaline solution by addition

of concentrated HCl followed by washing of

the precipitate with water and oven drying at

110oC (Fataftah AK, 2001). The humic and

fulvic acids were extracted into a strongly basic

aqueous solution of sodium hydroxide. This

was made acidic by adding normal

hydrochloric acid (6N HCl) by adjusting the pH

up to 2 and kept overnight. Precipitate obtained

was filtered and was oven dried at 110 o C. HA

is precipitated from this solution, leaving the

fulvic acids in solution. This measure implies

to the weight of HA in sample I & II. This

measure should be low to say if good sample.

This is the operational distinction between

humic and fulvic acids HA in sample II (-

9.000) was less compared to sample I(-

16.357).The standard showed −10.703 of HA.

Table 5.

By this procedure, Sample I contained

0.320 gms, sample II 0.546 gms and standard

contained 0.094gms of humic acid out of 1 gm

of samples. This high amount of humic acid

compared to standard may be due to the

method followed for purification of sample I &

II. Subjection of drugs to processing might

have switched humification. Decomposition is

controlled by climatic condition (Choppin

1985), induced naturally. So based on the level

of humification the quantity of humic acid

could be varied.

CONCLUSION

Humic substances are used in medical

science with substantial benefits in improving

the health status. Shilajatu is one such complex

mixture of humic substance used widely for its

strengthening and rejuvenative qualities.

Market products are subjected to some

processing, but the purity among samples vary,

thereby therapeutic efficacy too. So there is

need for standardization of purity assessment

strategies, which could help to scrutinize the

good sample. There are no universal standards

established to quantify the humic substances in

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 365–373

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

shilajatu. Knowing the ratio of FA and HA

helps to predict the activity potential of

shilajatu and its purity. Keeping this in mind

the UV spectrophotometer and Gravimetric

methods were specially designed for easy and

cost effective qualitative analysis of shilajatu.

Method used for estimation of maturity of

organic compost served as pedestal for

application of above two methods here. It is

reported that both the samples meets the

minimum standards as reported in API at the

preliminary level. The shilajatu sample

collected from Nepal showed better level of

fulvic acid comparatively. The results of this

work have potential for the routine analysis of

FA samples and for the certification and

regulation of commercial shilajatu and other

fulvic acid containing drugs. Thus inference

from this study may be used as reference

method in further quality control researches.

ACKNOWLEDGEMENTS

Harisha CR, Head of Pharmacognosy

Laboratory, for carrying out pharmacognostical

works related to work and Prof. Prajapati PK,

Director of Pharmacy, Department of

Rasashastra, IPGT & RA, Gujarat Ayurveda

University, Jamnagar for his guidance during

the work.

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Rasaratnasamucchaya, Suratnojivala

Hindi commentory(2010), Shilajatu

shodhana prakarana. Chowkamba

Amarabharati Publications, Varanasi:

chap 2/111 pp. 53

Shilajit |Ayurwiki.info. Retrived from

http://www.ayurwiki.info/wiki/shilajit

Skogerboe, R.K., Wilson, S.A.,. Anal. Chem:

Vol.53, pp. 228–31.

Thurman EM, Malcolm RL (1981). Preparative

isolation of aquatic humic substances.

Environ. Sci. Technol: Vol 15.,pp. 463–

66.

Source of Support: Nil Conflict of Interest: None Declared

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 374–379

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

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

MANAGEMENT OF ARBUDA (CANCER) WITH HERBOMINERAL

FORMULATION - A PILOT STUDY

Mahanta Vyasadeva1*, Dudhamal T S

2, Gupta S K

3

1,2 Assistant Professor, Shalya - Tantra, IPGT & RA. Gujarat Ayurved University, Jamnagar.

Gujarat, India 3

Associate Professor & I/C HOD, Shalya - Tantra, IPGT & RA. Gujarat Ayurved University,

Jamnagar, Gujarat, India

*Corresponding Author: Email: drvyasayu@yahoo.in; Mobile: +91 9408323779

Received: 26/03/2013; Revised: 14/04/2013; Accepted: 25/04/2013

ABSTRACT

Arbudaharana rasayana is an anubhuta yoga (Self experienced compound medicine). It is a

herbo-mineral compound formulation, practicing by traditional healers of Orissa as a folklore

medicine, for the management of Arbuda. The formulation was studied clinically in 10 patients of

non operable malignancy of different regions of body like scalp, vagina, oesophagus, bladder and

oral cavity. The drug was given in powder form, 5 gm twice a day, orally with Luke warm water for

two months. Symptomatic relief was observed in treated patients with overall improvement in quality

of life. The drugs might have been effective due to anti-cancerous, antioxidant and immuno-

modulator activities. During follow up period there was no any adverse effect of drug observed.

KEYWORDS: Anti-cancerous, Anti-oxidant, Arbuda, Arbudaharana rasayana, Immuno-modulator

Research article

Cite this article:

Mahanta Vyasadeva, Dudhamal T S, Gupta S K (2013), MANAGEMENT OF ARBUDA (CANCER)

WITH HERBOMINERAL FORMULATION - A PILOT STUDY, Global J Res. Med. Plants &

Indigen. Med., Volume 2(5): 374–379

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 374–379

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

INTRODUCTION:

Globally, the burden of new cancer cases is increasing day by day even after several advances made in treatment field of cancer. As per the WHO data, about 70% of all cancer death occurs in low and middle income country (http://www.who.int). Worldwide death from cancer is projected to continue to raise over 11 million in 2030. Every year in India, about 11 lakh new cases are registered and approximately 5 lakh patients die due to cancer. In India, it has been estimated that about 71% of deaths occur due to cancer belongs to 30–69 years age groups of people (Dikshit R et al., 2012). As etiological factors, tobacco consumption, low fibre diet intake, lack of physical activities, eating much fast food and alcohol intake are usually linked with prevalence of the cancer directly or indirectly (http://www.cancerfoundationofindia.org, http://www.cancer.gov). In addition, viral infections of HBV/HCV and HPV are also held responsible for about 20% of cancer deaths.

To manage the excessive proliferation of cells and to control the metastasis of abnormal cells by mutation (RCG Russel et.al. 2004) is still remained as a challenge before physicians. Now a day, multi modality treatment is being practised even then the outcome and prognosis is not found satisfactory due to poor awareness and late detection of cancer. The contemporary treatment like chemotherapy, radiotherapy and surgical intervention are still not affordable by poor patients and those receiving such therapy, they also need some adjuvant therapy like immunotherapy to overcome untoward effects. At this juncture, Ayurveda can extend good support as a safe, effective and more affordable treatment to improve the overall quality of life (QOL).

As per Ayurveda, manifestation of various non inflammatory swelling occurs due to vitiation of Vata and Kapha Dosha and they are classified as Arbuda (tumour), Granthi (glandular swelling). Galaganda (cervical lymphadenopathy) etc. (Shastri A, 1995). One can correlate the Arbuda with cancer on the basis of its signs and symptoms. The definite aetiological factors of Arbuda are not defined in Ayurvedic classics but the causative factors defined for Granthi and Vranasopha have been

considered for management of Arbuda. Acharya Sushruta has given more emphasis for surgical management of Adhyarbuda (tumour grows over the pre existing one), Dwirbuda (tumour grows simultaneously or one after other) and Mamsarbuda (fleshy tumour) (Singhal G.D, 1972, Ghanekar B, 1977) to improve the quality of life. He has also advised to go for eshansi Samudharet (En-block resection) of Arbuda to prevent recurrence. Maggots’ therapy and Shodhana therapy have been advised in non operable conditions as palliative therapy.

“No disease is manifested without involvement of Tridosha” is the basic ideology of Ayurveda and one can control the vitiation of Tridosha with the help of herbal / herbomineral formulations. The formation of any Arbuda can occurs in any of dushya i.e. Rakta (blood), Mamsa (muscle) and Meda dhatu (fat) due to vitiation of Vata and Kapha dosha. The vitiated Vata and Kapha dosha produce Ama (autotoxin) by affecting the Jatharagni and Dhatwagni. That Ama along with apakwa dhatu produces various dhatugat vikara in the form of Arbuda, Shopha, Granthi etc. by obstructing the Srotasa (channels).

Hence, the involvement of Agni, Ama and Srotavarodha are to be considered during the management of Arbuda. On the basis of such concept Arbudaharana Rasayana, as a folklore medicine, is being practiced by traditional vaidyas of Orissa for the management of Arbuda (cancer). For present study, total 10 patients of non operable malignant cases of scalp, vagina, oesophagus, colon, bladder and oral cavity were selected from the OPD of Dept. of Shalya-Tantra.

MATERIALS AND METHODS:

Total 10 diagnosed cases of cancer of various parts of the body, were selected irrespective of age, sex and religion for this study. All the selected patients were having features of local and distant metastasis. Different types of cancer patients were included in this study as a pilot study.

The Arbudaharana Rasayana (anubhuta yoga) is the combination of following herbo-mineral drugs having anticancerous, antioxidant, immuno-modulator activities.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 374–379

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Method of drug administration to the patients

To

Sr.

No.

Name of Drug Ingredients Quantity

/ Dose

1. Haridrakhanda Haridra (Curcuma longa), Ghrita (Ghee), Godughdha (Cow

milk), Sarkara (Sugar), Trikatu (Combination of Zingiber

officinale, Piper logum, Piper nigrum), Trijataka

(Combination of Cinnamomum zeylanica, Cinnamomum

tamala, Elettaria cardamomum), Vidanga (Embelia ribes),

Trivrit (Operculina turpethum), Triphala (Combination of

Terminalia chebula, Terminalia bellerica, Phyllanthus

emblica), Keshar (Crocus sativus), Musta (Cyperus rotundus),

Louha (Iron)

4 g

2. Arogyavardhini Parada (Mercury), Gandhak (Sulphur), Louha (Iron), Tamra

(Copper), Abhrak (Mica), Triphala (Combination of

Terminalia chebula, Terminalia bellerica, Emblica

officinalis), Shilajatu (Asphatum panjabinum), Guggulu

(Commiphora mukul), Chitrak moola (Plumbago zeylanica),

Katuki (Pichrorhiza kurroa)

250 mg

3. Vydadhiharan

Rasa

Parada (Mercury), Gandhak (Sulphur), Somal (White

arsenic), Haratal (Yellow arsenic), Manashila (Red arsenic) ,

Rasakarpur (Per chloride of mercury)

150 mg

4. Guduchi satwa Guduchi (Tinospora cordifolia) 600 mg

Posology – 5 g powder twice a day (In morning, after breakfast and in evening after light snacks)

orally with Luke warm water.

RESULTS:

Demographic Analysis:

Out of 10 patients only 20% female patients

were found in age group of 30–40 years

whereas above 40 years age 80% patients were

recorded suffering from different types of

cancer. The data itself revealed that the cancer

is occurred in older age (Table No.1).

In case of female patients except cervix and

vaginal cancer 20% and breast cancer 10% no

other site of cancer like head & neck,

oesophagus, lungs, colon and urinary bladder

were found as reported in cases of male

patients (70%). This data show that there are

more chances of occurrence of these sites of

cancer in male population (Table No.2).

Incidences of squamous cell carcinoma

(60%) were found more in comparison to

adenocarcinoma (30%) and fibroadenoma

(10%). (Table No.3)

Maximum addiction of tobacco, smoking

and tea (70% each) were observed in male

patients and may be held responsible more

incidences of cancer in male population (Table

No.4).

The data of socio-economical status

revealed that maximum lower middle (50%)

and lower (20%) classes of patient were

suffering from different sites of cancer which

show that cancer may have relation up to some

extent to the socio-economical status of the

people (Table No.5).

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Table No.1 Age wise distribution n-10

Sr. No. Age (Years) Male % Female % Total %

1. 30–40 00 00% 02 20% 02 20%

2. 41–50 04 40% 01 10% 05 50%

3. 51–60 02 20% 00 00% 02 20%

4. >60 01 10% 00 00% 01 10%

Table No.- 2; Analysis of Cancer Sites n- 10

Sr. No. Sites of Cancer Male % Female % Total %

1. Head and neck 02 20% 00 00% 02 20%

2. Oesophagus 01 10% 00 00% 01 10%

3. Lungs 01 10% 00 00% 01 10%

4. Colon 01 10% 00 00% 01 10%

5. Urinary Bladder 02 20% 00 00% 02 20%

6. Cervix and Vagina 00 00% 02 20% 02 20%

7. Breast 00 00% 01 10% 01 10%

Table No.- 3 Histopathological Analysis n- 10

Sr. No. Tissue / Cell type Male % Female % Total %

1. Squamous cell

carcinoma

04 40% 02 20% 06 60%

2. Adeno carcinoma 03 30% 00 00% 03 30%

3. Fibroadenoma 00 00% 01 10% 01 10%

Table No.- 4 Addiction wise Analysis n-10

Sr. No. Habits Male % Female % Total %

1. Tobacco chewing 07 70% 01 10% 08 80%

2. Smoking 07 70% 00 00% 07 70%

3. Tea 07 70% 03 30% 10 10%

4. Alcohol 02 20% 00 00% 02 20%

Table No.- 5 Analysis of Socio-economical Status n-10

Sr. No. Social status Male % Female % Total %

1. Upper 01 10% 00 00% 01 10%

2. Upper middle 01 10% 01 10% 02 20%

3. Lower middle 03 30% 02 20% 05 50%

4. Lower 02 20% 00 00% 02 20%

DISCUSSION:

Outcome is the key of success in cancer

management. As cancer is a leading cause of

death recorded in the world. An integrative

approach of treatment is the need of the time to

improve the quality of life in non operable and

non tolerable cancer cases to radiotherapy /

chemotherapy. The Arbudaharana Rasayana

(anubhuta yoga) formulation contains

ingredients like Haratal (arsenic)

(http://www.wakehealth.edu), a good source of

anti-cancerous drug which helps to check the

growth of tumour by inhibiting the mutation of

the cells. Other ingredients like Guduchi

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 374–379

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(Tinospora cordifoloia) and Haridra (Curcuma

longa) (Ranjith M. S, 2008; Bharat B, 2003)

have antioxidant, immuno-modulator, analgesic

and anti inflammatory effects which might

have extended relief by reducing inflammation

and pain.

As it is considered that Amadosha and

Srotavorodha are the known factors for

causation of Shotha and Vedana (inflammation

and pain), the Vydadhiharan Rasa (Rasatantra

Sara, 1990)is capable to remove Srotavorodha

by digesting Amadosha with increasing

Dhatwagni (cellular metabolism) and rendered

relief in the features of Shotha and Vedana.

Due to dominance of Vata Dosha in older

age and various addiction like smoking,

tobacco chewing are the favourable factors for

developing cancer. In this study 70% patients

were older and addicted for smoking and

tobacco chewing and they were developed

cancers on different site. Socio-economical

status of people may be related with the

nutrition, immunity and overall health of a

person. The data of this study also goes in

favour of that and the causes of cancer may be

attributed to the status of nutrition and

immunity as it is obviously low in socio-

economically week patients.

Squamous cell carcinoma and

adenocarcinoma were found more in this study

which showed the involvement of the particular

organs like head & neck, oesophagus, lungs,

colon and urinary bladder, etc. It may have

some relation with food and addiction habit of

the persons. In Gujarat, tobacco in the form of

Mawa - Masala, spicy and fast foods are the

favourable items for routine consumption.

These may be held responsible for causation of

cancer by developing Amadosha and

Srotavorodha with help of vitiated Vata and

Kapha.

The overall effect of the selected

formulation in the cancer patients was found

satisfactory with no any untoward effect

observed.

CONCLUSION:

Cancer is a major and global health

problem and the outcome of any available

therapy is still under the question, particularly

in non operable cases of malignancies. The

Arbudaharana Rasayana (Anubhuta yoga) is a

potent formulation for providing symptomatic

relief from Arbuda (cancer). The formulation

was found effective to improve the quality of

life (QOL) by rendering anti inflammatory,

analgesic, anti oxidant and immuno-modulator

activities.

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

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and Singh Sidhu Harcharan, (2008)

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page no 169.

Source of Support: Nil Conflict of Interest: None Declared

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 380–385

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

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

IMPORTANCE OF UPAYOGASAMSTHA (DIETETIC RULES) IN RELATION

TO DIGESTION OF THE FOOD

Avhad Anil D1, Vyas H A

2, Dwivedi R R

3

1Ph.D. Scholar, Department of Basic Principles, Institute for Post Graduate Teaching & Research in

Ayurveda, Gujarat Ayurved University, Jamnagar - 361 008, Gujarat, India. 2Assi. Professor, Department of Basic Principles, Institute for Post Graduate Teaching & Research in

Ayurveda, Gujarat Ayurved University, Jamnagar - 361 008, Gujarat, India. 3Professor& HOD, Department of Basic Principles, Institute for Post Graduate Teaching & Research in

Ayurveda, Gujarat Ayurved University, Jamnagar - 361 008, Gujarat, India.

*Corresponding Author: E-mail: anilavhad4u@yahoo.com

Received: 14/03/2013; Revised: 17/04/2013; Accepted: 20/04/2013

ABSTRACT According to Ayurveda food is one of the important factor for health as well as source for

diseases i.e. wholesome and unwholesome food is responsible for happiness and misery

respectively.. Beside the quality and quantity of the food it is very much important that how it is

taken, because food taken in improper way can lead to various diseases. Keeping these things in

mind 8 factors (Aharavidhivisheshayatana) has been mentioned in Vimanasthana of Charak Samhita

which determines the utility of the wholesome food. ‘Upayogasamstha’ is the eighth factor among

these which deals with the dietetic rules. Further in the sixth chapter of Sharirasthana six factors

which are necessary for the transformation and digestion of the food (Aharaparinamakara bhava)

have been mentioned. The study was planned to evaluate the relation between ‘Upayogasamstha’

(dietetic rules) and digestion of the food. It is found that each and every rule mentioned in

Upayogasamstha (dietetic rules) has very essential role in the process of digestion. Conflictions of

these rules are directly mentioned as the causative factors for the production of Ama (undigested

food), which is the cause for several diseases. Along with balanced diet, incorporating the dietetic

rules in our daily routine can prevent many diseases. All the rules mentioned under

‘Upayogasamstha’ (dietetic rules) strengthen the Aharaparinamakar Bhavas (digestive factors) and

also governs the digestion process along with Agni.

KEY WORDS: Aharavidhivisheshayatana, Upayogasamstha, Aharaparinamakara bhava, Dietetic

rules

Review article

Cite this article:

Avhad Anil D, Vyas H A, Dwivedi R R (2013), IMPORTANCE OF UPAYOGASAMSTHA (DIETETIC

RULES) IN RELATION TO DIGESTION OF THE FOOD, Global J Res. Med. Plants & Indigen. Med.,

Volume 2(5): 380–385

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 380–385

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

INTRODUCTION

Ayurveda being the science of life deals

with several aspect of the human life and also

cares a lot about the factors responsible for its

maintenance and prosperity. Ahara (Food),

Nidra (Sleep) and Bramhacharya (Celibacy)

are said to be the tripods of human life and

proper balance of them is responsible for the

health of an individual (Tripathi Ravidutt,

2005). Among these also ‘Ahara’ is considered

as the chief factor since the complexion,

longevity, happiness, satisfaction, nourishment,

strength and intellect are all conditioned by it

(Tripathi Ravidutt, 2005). In the conceptual

part it has been found that all the authors of

Ayurveda have emphasized on Ahara (Food) as

well as its Matra (quantity), Kala (Time of

preparation), Samskar (processes of

preparation), Desha (place; including soil,

climate and surrounding conditions), Swabhava

(constitution) and Ashanavidhi (codes and

conducts of taking food). Modern science has

particularly described Ahara according to the

nutritional value of its components. They have

not described about Hita (wholesome), Ahita

(unwholesome), Pathya (healthy), Apathya

(unhealthy) Ahara etc. for each individual.

There is no constraint of diet imposed in the

Allopathic methods of treatment, where

medicines are given principal importance. But

it is not so in the case of Ayurveda where, on

the contrary, appropriately selected and

controlled diet is an integral part of treatment

so as to enhance vitality and regulate the

medicinal effects in natural harmony with the

entire system of the body and mind .

For the sustenance of an individual

digestion plays an important role. The good

quality food taken in appropriate amount will

nourish the tissue elements of the body along

with mind and senses only when it is properly

digested. ‘Agni’ is considered as the key factor

in the process of digestion and metabolism, and

all the diseases are said to be produced due to

the weakness of Agni (Atridev Gupt, 1997).

Along with ‘Agni’ there are many co-

supportive factors which also control the

digestive process as the same type of food

could have contrary effects, under different

circumstances or for different persons; most

important is what guidelines one follows in the

selection and preparation of food and in his

eating habits. In today’s fast paced life, many

norms regarding diet & regimen have been

compromised which is showing the overall

declining status of health in the society. Ahara

and its related codes and conducts have been

long forgotten by the human being; hence to

evaluate this ancient science of dietetics this

study was undertaken.

The present review deals with

‘Upayogasamstha’ (dietetic rules) which

governs the process of digestion as all these

rules can be included under the factors which

are necessary for the digestion. Also this

review explores the importance of

Upayogasamstha (dietetic rules) and evaluates

the relation between Upayogasamstha (dietetic

rules) and digestion.

MATERIALS

The data from various texts (Tripathi Ravidutt,

2005) along with Brihatrayee has been

collected, compiled and analyzed for the

discussion and attempt has been made to draw

some conclusions.

DISCUSSION

Ayurvedic nutrition is a vast topic that takes

into account the individual constitution, the

medicinal value of culinary spices, the theory

of shad rasa (or six tastes, which should all be

present for a meal to be balanced), and more.

The founders of Ayurveda had affirmed food as

the essential source of sustenance and

strengthening of health. However, they had also

alerted that "what one eats and how?" could be

the major factor for or cause of illness.

According to modern science, ‘people who are

eating according to the rules of a traditional

food culture are generally healthier than those

of eating a modern western diet of processed

foods.

Acharya Charaka has described ‘Ashta

Aharavidhivisheshayatana’ (eight factors

determining utility of the food) (Tripathi

Ravidutt, 2005). These eight factors are

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associated specifically with the useful and

harmful effects of the food and they are

conditioned by one another. ‘Upayogasamstha’

is the eighth factor among these which

indicates the dietetic rules. Healthy as well as

diseased individuals should follow these rules

even while using wholesome food by nature.

According to Charaka; Ushma (heat), Vayu,

Kleda (moist), Sneha (Unctuousness), Kala

(time) and Samayoga (appropriate intake) are

the ‘Aharaparinamakara bhavas’ (Digestive

factors) (Tripathi Ravidutt, 2005). The rules

mentioned under Upayogasamstha and their

relations with Aharaparinamakara bhava are as

follows

i. Ushnam Bhunjit (Food should be warm)

When the food is taken warm, it becomes

appetizing and after intake it provokes the

factors responsible for the digestion. It gets

digested quickly and helps in the downward

movement of Vata and alleviation of Kapha.

This warmness indicates the ‘Ushma’ (heat),

which is the most essential and vital factor

responsible for the digestion as it directly takes

part in the digestion (Tripathi Ravidutt, 2005).

On the other hand, when the food is kept for

longer time after preparation or when stale food

is consumed, it takes more time to digest or

sometimes may produce Ama (undigested food)

(Tripathi Ravidutt, 2005). In modern science

also it has been mentioned that stale food can

lead to food poisoning at times.

ii. Snigdham Bhunjit (Food should be

unctuous)

The unctuous food is said to be delicious

and after intake it provokes the subdued power

of digestion. It gets digested quickly, helps in

Vatanulomana (downward movement of Vata),

promotes growth of the body (Sharira

upachaya), strengthens the sense faculties, and

brings out the complexion.

It indicates the third and fourth factor of

digestion i.e. Sneha (unctuousness) and Kleda

(moist). Due to them the food particles become

soft and well dissolved, so that they can be

easily digested. On the contrary Ruksha ahara

(food having dry nature) is mentioned as one of

the causative factor for the production of Ama

(Tripathi Ravidutt, 2005) and also can lead to

Vatavyadhis (disorders caused by Vata dosha).

iii. Matravat Bhunjit (Food in proper

quantity)

When the food is taken in proper quantity,

it promotes the longevity of life without

afflicting Vata, Pitta and Kapha. It does not

impair the power of digestion and gets digested

without any difficulty. For the proper digestion

one part of the stomach should be left for the

free movement of Vata, Pitta and Kapha. In

modern science also it has been said that,

always leave the table a little hungry; many

cultures have rules that you stop eating before

you are full. In Japan, they say eat until you are

four-fifths full. Islamic culture has a similar

rule and in German culture they say, ‘tie off the

sack before it’s full’ (Michael Pollen, 2006).

Hence this rule can be correlated with the

second factor necessary for digestion i.e. Vayu.

Prana Vayu (one among the 5 subtypes of Vata

dosha) takes the food nearer to the site of Agni

and Samana Vayu (one among the 5 subtypes

of Vata dosha) stimulates Agni, facilitating the

process of digestion (Tripathi Ravidutt, 2005).

If one fills his stomach with excess of the food

then it leads to obstruction in free flow of the

‘tridoshas’ due to which the digestion becomes

uneasy. Atimatrashana (Excessive intake of

food) is mentioned as the major factor for the

production of Ama (Tripathi Ravidutt, 2005)

whereas Pramitashana (less intake of food) is

described to produce emaciation of the body.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 380–385

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Table 1: Relation between Upayogasamstha and digestion

Sr. no. Upayogasamstha Digestive factors

1 Ushnam Ushma

2 Snigdham Sneha, Kleda

3 Matravat Vayu

4 Jeerne, Natidrutam, Nativilambitam Kala

5 Veerya aviruddha, Ishta deshe Ishta sarvopakarane,

Tanmana bhunjit, Atmanamabhisamikshya

Samayoga

iv. Jeerne ashniyat (Intake after digestion of

previous meal)

According to Ayurveda, one should take the

food after the complete digestion of previous

food. Such food promotes the longevity of the

body and keeps the Doshas in balanced state.

On the other hand, when an individual takes

food before the digestion of previously taken

meal, then it gets mixed with Apakva

Ahararasa (partially digested food) of previous

meal; which leads to the vitiation of all the

three Doshas instantaneously.

This rule is nothing but the fifth factor of

the digestion process i.e. Kala (time). Even in

the presence of all other factors, digestion

requires time for completion of the process.

Hence Kala (time) is described as one of the

major factor as it brings the maturity of the

process of digestion. Vishamashana (intake of

food at wrong time), Adhyashana (intake of

excess food), Ajirnashana (intake of food

before the digestion of previous food) etc.

known causative factors can be included under

the title of Akalabhojan (intake of food at

wrong time) which is nothing but opposite of

Kalabhojana (intake of food at proper time).

v. Veerya aviruddha ashniyat (Intake of food

having no contradictory potencies)

One should take the food having no

contradictory potencies. By taking such food

one does not get afflicted with diseases and all

the attributes of the food are transformed into

the tissue elements. If the properties of food

articles are contradictory against each other,

one may suffer from the diseases like Kushtha

(skin disease), Visarpa (cellulitis) etc (Tripathi

Ravidutt, 2005), and also such food ruins the

body gradually (Tripathi Ravidutt, 2005).

vi. Ishta deshe, Ishta sarvopakarane (Intake

in proper place and with all accessories)

Desha is such a factor which is to be

understood in three ways in relation to food i.e.

Bhoomi (soil), Ahara Dravya Desha

(originating place of the food material) and

Atura Desha (eating place). Here in this context

it is mainly related with the latter one.

According to Ayurveda an individual should

take the food at proper place which is clean and

desired. Food consumed at unhygienic or

undesired place may disturb the mindset

leading to indigestion. In modern science also it

has been revealed that hundreds of millions of

people are affected by preventable diseases

originate in the environment in which they live.

Further one should be equipped with all the

necessary equipments while taking the food;

because in absence of these, an individual may

not be satisfied even with the good quality of

food.

vii. Natidrutam (Intake not in hurry)

One should not take food too speedily. If

food is taken too hurriedly it enters into a

wrong passage; it gets depressed and it does not

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 380–385

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

enter into the stomach properly. In this

situation one can never determine the taste of

food articles and will lead to improper mixing

of Bodhaka Kapha (one among the 5 subtypes

of Kapha dosha), which softens the food

particles and thereby facilitates the process of

digestion. Further, an individual taking food

speedily is unable to detect the foreign bodies

like hair etc., mixed with food.

viii. Nativilambitam (Intake not too slow)

One should not take food very slowly

because this will not give satisfaction to the

individual. In this situation he would take more

than what is required; the food would become

cold and there will be irregularity in the

digestion process. Also by taking food very

slowly, enzymes responsible for digestion will

come late in contact with food and in an

irregular manner and this will result in irregular

digestion.

The above mentioned two rules (seventh

and eighth) are also related with the factor Kala

(time). Hence they again emphasize the

importance of Kala (time) in digestion process.

ix. Tanmana Bhunjit (Intake with

concentration)

One should not talk or laugh or be

unmindful while taking food. An individual

taking food while talking, laughing or with

detracted mind, subjects himself to the same

trouble as one eating too hurriedly. In modern

dietetics also it is said that, ‘If we eat while we

are working or while watching TV or driving,

we eat mindlessly and as a result eat a lot more

than we would’ (Michael Pollen, 2006).

x. Atmanamabhisamikshya (Intake with self

confidence)

One should take food in a prescribed

manner; with due regard to his own self. The

knowledge of the usefulness of food articles is

the main thing for self-preservation.

Rule number five, six, nine and ten can be

understood under the broad heading of

‘Samayoga’ (appropriate intake) which is the

last factor necessary for digestion. Samayoga

means appropriate intake of the food which

brings the equilibrium of Dhatus. In absence of

this even the wholesome food is not digested

well.

CONCLUSION

In Ayurveda food plays a prominent role in

promoting health and is therefore considered

medicine. Ayurvedic concept of digestion takes

into account many things viz. food, its quantity,

place, time, factors affecting it, dietetic rules,

digestive factors, Agni etc.; which is the most

ideal than any other science. Along with Agni

there are many other factors which are found

useful for proper digestion and by taking care

of which one can eliminate the causes of

imbalance. Each and every rule mentioned in

Upayogasamstha has very essential role in the

perspective of digestion. Conflictions of these

rules are directly mentioned as the causative

factors for the production of Ama, which is the

cause for several diseases. Along with

balanced diet, incorporating the dietetic rules in

our daily routine can prevent many diseases.

All the rules mentioned under

‘Upayogasamstha’ (dietetic rules) strengthen

the ‘Aharaparinamakar Bhavas’ (digestive

factors). Hence these ten rules are directly

related with the ‘Aharaparinamakar Bhavas’

(digestive factors) and govern the digestion

process.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 380–385

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

REFERENCES

Atridev Gupt (1997), Ashtang Hriday with

Vidyotini hindi commentary by, twelth

edition, Chaukhamba Sanskrit

Bhawan, Varanasi, Nidanasthana 12.

Michael Pollen (2006), The Omnivore’s

dilemma: a natural history of four

meals, second edition, Penguin press,

New York.

Tripathi Ravidutt, (2005), Charaka Samhita

edited with Vaidyamanorama Hindi

commentary by Reprint edition,

Chaukhamba Sanskrit Pratisthana,

Delhi, Sutrasthana 11, 25, 26;

Vimanasthana 1, 2; Sharirasthana 6;

Chikitsasthana 15.

Source of Support: Nil Conflict of Interest: None Declared

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 386–391

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

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

CONCEPT OF VYADHIKSHAMATVA (IMMUNITY) AND ITS

RELATIONSHIP WITH BALA (VITAL STRENGTH)

Sharma Mahesh Kumar1*

1Assistant Professor, Department of Basic Principles, MSM Institute of Ayurveda, BPS Mahila

Vishwavidyalaya, Khanpur kalan, Haryana (131305)

*Corresponding Author: E-mail: ayurmahesh@gmail.com; Mob: +919468342229;

Received: 26/03/2013; Revised: 17/04/2013; Accepted: 20/04/2013

ABSTRACT

When a group of people exposed to a particular disease, the affect seen is categorized in four

categories mild, moderate, severe and unaffected. The pathogenic factors require some essential

favorable conditions to flourish and create the disease. Ayurveda finds Bala and Vyadhikshamatava

as profound explanations for this phenomenon. These are also necessary for prevention and rapid

recovery from illness. Bala is the cause for the good defense mechanism and to carry out all the

physical and psychological actions. The depreciation of bala is a constant sign observed in various

degenerative diseases and recurrent infections. It can be increased by intake of wholesome food,

immunomodulator drugs like Tinospora Cordifolia Willd. (family- Menispermaceae) etc., a regular

routine of seasonal and daily regimens along with mild physical workout. So, bala and

vyadhikshamatva have cause and effect relationship.

KEY WORDS: bala, immunity, vyadhikshamatva.

Review article

Cite this article:

Sharma Mahesh Kumar (2013), CONCEPT OF VYADHIKSHAMATVA (IMMUNITY) AND ITS

RELATIONSHIP WITH BALA (VITAL STRENGTH), Global J Res. Med. Plants & Indigen. Med.,

Volume 2(5): 386–391

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 386–391

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

INTRODUCTION

In our daily experiences with the

continuously changing environment (Shastri

Ambikadutta, 2003) we came across many

infecting agents. The external environment also

checks the adaptive power of humans (Trikamji

Yadavaji, 2005). Kala (time) causes the caya

(deposition), parakopa (vitation) and

prasamana (normalization) of the doshas to

affect the body and the parakopa make one to

suffer with the diseases. The nature has made

the natural safety measures to claim superiority

over such natural influences that hamper life.

The nature had given us power to adapt and

overcome these variations. This adaptability of

the body is termed as vyadhikshamatva

(Trikamji Yadavaji, 2005). In terms of medical

science it is called immunity. It is the diversity

of life that life exists in enormous forms

starting from the smallest viruses and bacteria

up to the largest one. All these creatures are the

miracles of nature that somehow affect each

other by causing them diseases and take their

life too. The body with adequate

vyadhikshamatva is capable to overcome the

effect of these pathogens to make oneself

disease free or with the mild prevalence. The

one who possess good vyadhikshmatva has a

good dehabala (Physical health) (Jangid

Chhagan et.al 2008). The good physical health

means healthy body metabolism etc. Hence this

review explores the relationship between

Vyadhikshmatva and Bala and the factors that

enhance both.

LITERARY REVIEW

The word „vyadhikshamatva’ is made of

two words- „vyadhi + kshamatva‟. Vyadhi is a

condition which comes into existence as

consequence of non-equilibrium of the doshas

(physiological factors i.e vata, pitta & kapha),

dhatus (tissues systems) and malas (excretory

products of body) which in their normal status

maintains the physical and psychological health

(Dass Ranjip Kumar, 2013). The other word

„kshamatva’ is derived from „kshamus sahane’

meaning there by to be patient or composed to

suppress anger, to keep quite or to resist. So,

vyadhikshamatwa is that factor which limits the

pathogenesis and opposes the strength of

disease.

There are nine factors in an individual

which promotes them towards the incapability

to resist the disease manifestation (Trikamji

Yadavaji, 2005).

1) Ati- Sthoola (Excessively obese persons)

2) Ati-Krisha (Excessively emaciated person)

3) Anivista-Mamsa (Individual having

improper musculature)

4) Anivista-Asthi (persons having defective

bone tissues)

5) Anivista-Shonita ( persons with defective

blood)

6) Durbala (Constantly weak person)

7) Asatmya-Aaharopachit (Those nourished

with unwholesome food)

8) Alpa-Aaharopachit (Those taking diet in

small quantity) 9) Alpa-Sattva (Individuals with

feeble mind)

Along with this, there are some parameters

which make the body resistant to the external

environmental changes. Individuals whose

bodies are neither too corpulent nor too

emaciated, who possess healthy and good

musculature, blood and bones are well

nourished with whole some and sufficient food

and who have strong manas are capable of

resisting diseases. Persons having proportionate

musculature, compactness of the body and

healthy sensory & motor organs are not

overruled by the onslaught of diseases. They

can withstand hunger, thirst and the heat of the

sun, cold weather and physical exertion. They

can also digest and assimilate food properly.

The factors which enhance the status of

vyadhikshamatva (Trikamji Yadavaji, 2005).

These are mentioned below-

1) Adequate Ojas

2) Adequate Bala

3) Follow of Seasonal Diet & regimen

4) Proper Anupana (post prandial drinks)

5) Consumption of six Rasa (type of foods)

6) Jatakarma (Birth ceremonial rituals)

7) Drugs (antioxidants)

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When etiological factors come in contact

with a person then they try to produce diseases.

Some persons remain unaffected even after

coming in contact with the relevant pathogenic

factors while some others become victims of

the disease. This capacity or power which

makes the body capable to fight against the

presentation of diseases in the body is said as

vyadhikshamatva. The reasons for the same is

given by Charaka (200 BC) that people have

different ability to resist the disease causing

factors. The reason is that when the resistance

power of the body is sufficiently strong it

destroys the causes. A conference titled

“factors which provoke the diseases” the

conclusion is that “apathya aahara (unhealthy

food) is the cause for diseases and pathya

aahara (healthy food) is cause for health”.

Here, the significance of favourable and non-

favourable diet in the development and

production of a disease is proved. (Trikamji

Yadavaji, 2005)

It is also known that many people with right

daily and seasonal habits also develop disease

and some though indulging in improper habits

hardly get any disease. Therefore a search for

the answer to above conditions lead to some

more factors involved in disease manifestation

and the individual‟s body provides a fertile bed

for the disease causing agents. The disease not

only depends upon the etiological factor but

also depends on the resistance of the individual.

So we can have a greater safety from the

disease if we possess a good health (Deha

bala).

The concept of vyadhikshamatva described

in charaka samhita (1500 BC) gives us a detail

account of immunology and gives us newer

thoughts to present day modern medical

immunology.

Caraka (200 B.C) quoted that all the

individuals have different resistance for

diseases. Vyadhikshamatva (the functional

ability of immune system) varies from

individual to individual in spite of their similar

nutritional, environmental, physical and mental

status.

Chakrapani (Ayurveda-Deepika, 1100

A.D) commented on the term vyadhikshamatva

and gave two opinions:

(I) Vyadhi-balavirodhitvam: It is the capacity

to restrain or withstand the strength

(severity) of the diseases i.e., strength to

arrest the progress of disease.

(II) Vyadhi-utpadapratibandhakatva: The

resisting power of the body competent

enough to arrest the occurrence and re-

occurrence of the disease.

These both sub-types of vyadhikshamatva

commutatively form the resistance which now

a day‟s called Immunity that is specific and non

specific resistance.

DISCUSSION

Excessively obese or emaciated persons are

very weak i.e. they are durbala. Similarly, the

strength of the body depends largely upon the

status of dhatus i.e. mamsa dhatu, asthi dhatu

and sonita. Hence persons in whom these three

dhatus have improper physiological functions

are naturally very weak. Caraka (200 BC) has

especially mentioned mamsa, asthi and sonita

have direct relation with healthy immune

response. This may be due to following

reasons.

A. The healthy condition of these three dhatus

will naturally maintained on equilibrium of

three doshas, which is also required for

resisting disease. This is because there is

relation of interdependence (ashraya-

ashrayee-bhava) between rakta and pitta,

mamsa and kapha and asthi and vata.

B. The cells; WBC of blood (rakta) fight

against infections and prevent the body

from foreign pathogenic substance.

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C. The red bone marrow in the asthi (bone) is

responsible for manufacture of the red and

white cells of the blood.

Persons involved with alpa (inadequate)

and ahita aahara (unfavourable food) are very

weak, as their dhatus (tissue system) are not

properly nourished.

Persons having feeble sattva (mind) are

also weak because sarirabala (Physical

strength) and sattvabala (Psychological

strength) are interdependent so, the weak

manas result in the weak sarira resulting poor

immunity.

Persons having good bala are found with

good defense mechanism. It is the bala which

makes the person capable for performing all the

physical and psychological actions. The proper

psychosomatic status of the body has adequate

agnibala (digestive capacity), dehabala

(physical strength) which manifests itself as

healthy immune system.

Kala (time) also affects the immunity the

visarga kala (southern solstice) increases the

bala of the body results in healthy immunity

Food taken in proper quality and quantity helps

in bringing the strength, complexion, happiness

and longevity. The habit of using all the six

rasa everyday is ideal for maintenance of

health. Intake of madhura (sweet) and amla

(sour) rasa (taste) produces more strength in

the body. To avoid the adverse affects of

dosha’s caya, prakop and prasamana one

should have to follow the seasonal regimens by

consumption of the rasa which are opposite to

kalakrat caya, prakop of doshas (Sharma

Shivprasad, 2008).

Licking of madhu (honey), ghrita (fat) in

unequal quantity with gold at the time of birth

to neonates act as an external nutrition for them

to activate their digestive system. Gold has a

property to enhance the immune system

(Shastri Ambikadutta, 2003).

Jatakarma Karnavedhana sanskara

(ceremonial rituals of ear puncturing) is done at

6th

or 7th

month of age by puncturing the ear

pinna for raksha (protection or resistance

power) and bhusan (cosmetic value). Several

drugs are claimed to enhance immunity i.e.

before gramya dharma (coupulation) tail

pradhana ahara for female and ghrita

pradhana ahara for male is prescribed.

Specific drugs for pregnant women are

described in different months of pregnancy for

better fetal growth. According to the age group

of children different aahara kalpana are

prescribed for enhancing bala (strength) of

children. Rasayana therapies which fortify

dhatus are described in all the texts for

enhancing bala and prevent old age and

disease. (Tripathi Brahmanand, 2010)

VYADHIKSHAMATVA & BALA

Bala is a multidimensional functional

identity of the body, which is the output of

proper equilibrium state of dosha, dhatu &

mala in the body. This comes in the form of

ojas which is considered as the supreme

essence of all the seven rasadi dhatus. It is also

stated as sarvadhatusara. The elementary

constituents of our body i.e. rasa, rakta,

mamsa, meda, asthi, majja & sukra contribute

their extreme purest extract in the formation of

ojas. In this manner ojas is formed in our body

which is the purest factor of all the dhatus

(Flow Chart 1).

The expression of this supreme essence in

the body results in the good musculature, stable

body, ability to perform activities, clarity of

voice, good complexion, healthy karmendriyas

(motor organs and nerves) & gyanendriyas

(sensory organs and nerves) and healthy

vyadhikshamatva (immunity power) of body.

All these features are related to bala. Hence, it

is to be said that bala is the karya (action) and

ojas is the karan (cause) for bala.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 386–391

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

FLOW CHART 1: Shows the relationship of Bala with other bodily factors

Bala is visualized as an identity which is

observed as the following:

1. Adaptability of the body to the climate with

adequate quantity & quality of rasadi

dhatus. Which are externally observed as

well built body, stable musculature and to

carry out physical activities in a normal

way.

2. Psychological stability in life which is

observed as the person endowed with

memory & devotion, grateful, learned,

pure, courageous, skill-full, resolute,

fighting in battles with prowess, free

from anxiety, intellectual engaged in

virtuous acts.

3. Specific resistance in comparison to

specific diseases which is otherwise called

as pratyanek bala or immunity.

CONCLUSION

A person with proper amount of bala is also

said to be complete with adequate amount of

saptdhatusara (tissue system). This provides

the capacity to resist the external disease

causing agents. It is concluded that

vyadhikshamatva is the external and internal

manifestation of bala (vital strength). The

kapha prakriti body constitution, possess the

best sahaja (natural) bala when compared to

pitta and vata body constitution persons.

REFERENCES

Shastri Ambikadutta (2003) - Sushruta

Samhita, Hindi Commentary

Ayurvedatatva-Sandeepika by

Ambikadutt Shastri 4th

edition,

Chowkhamba Sanskrit Sansthan,

Varanasi.

Trikamji Yadavaji (2005) – Charaka Samhita,

Sanskrit Commentary Ayurveda-Dipika

by Chakrapanidutta Edited by Vaidya

Yadavaji Trikamji Acharya,

Chaukhamba Surabharti Prakshan,

Varanasi.

Sharma Shivprasad (2008) - AstangaSamgraha,

with the Sanskrit Commentaries,

Sashilekha of Indui, Edited by

Dr.Shivprasad Sharma, Chaukhambha

Sanskrit Series Office, Varanasi.

Jangid Chhagan, Vyas H A, Dwivedi R.R.

(2008), “CONCEPTUAL AND

APPLIED STUDY ON EFFECT OF

RITUS ON BALA AS PER THE

SUTRA – Adavante Ca – Nirdiset”

IPGT & RA, Jamnagar.

RASA

RAKTA

MAMSA

MEDA

ASTHI

MAJJA

SUKRA

SUPREME

EXTRACT

OJAS

(karan)

BALA

HEALTHY PHYSIQUE

HEALTHY IMMUNITY HE

AL

TH

Y M

IND

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 386–391

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

Dass Ranjip Kumar (2013), THE ROLE OF

PANCHAKARMA THERAPY IN

MUSCULOSKELETAL DISORDERS

WITH SPECIAL REFERENCE TO

VATAVYADHI, Global J Res. Med.

Plants & Indigen. Med., Volume 2(1):

23–29

Tripathi Brahmanand (2010) – Sharangdhar

Samhita, Amatel with Dipika hindi

commentary by Brahmanand tripathi,

Chaukhamba Surbharti Prakashan,

Varanasi.

Source of Support: Nil Conflict of Interest: None Declared

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392–400

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

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

A REVIEW ON VARIETIES OF ARKA - CALOTROPIS PROCERA (AITON)

DRYAND. AND CALOTROPIS GIGANTEA (L.) DRYAND.

Poonam1*, Gaurav Punia

2

1Assistant Professor, Gaur Brahmin Ayurvedic College, Rohtak., India

2HCDS Dental Surgeon, Khanda kheri, Hisar.

*Corresponding Author: E-mail: drpoonamkaleramana@gmail.com, dr_pounia@yahoo.com,

drpoonamkaleramana@gmail.com; Mob: +918930123462, +9198930123430

Received: 14/03/2013; Revised: 14/04/2013; Accepted: 23/04/2013

ABSTRACT

Herbal medicines have been used from the earliest times to the present day. The ethno-botanical

pharmacology is as old as man himself. Herbal medicines exhibit a remarkable therapeutic diversity.

Arka is a plant which has been used in several traditional medicines to treat a variety of diseases.

Calotropis procera and Calotropis gigantea are two varieties of Arka described in Ayurveda by the

name of Rakta Arka and Shveta Arka. Both have almost similar properties but C. procera is

considered somewhat superior. In Ayurvedic texts also by the name of Arka, Acharyas mentioned

Rakta Arka (Calotropis procera) because of having more medicinal properties than C. gigantea. This

plant has been known to possess analgesic, antitumor, antihelmintic, antioxidant, hepatoprotective,

antidiarrhoeal, anticonvulsant, antimicrobial, oestrogenic, antinociceptive, and antimalarial activity.

KEY WORDS: Rakta Arka, Calotropis gigantia, Arka, antinociceptive.

Review article

Cite this article:

Poonam, Gaurav Punia (2013), A REVIEW ON VARIETIES OF ARKA - CALOTROPIS PROCERA

(AITON) DRYAND. AND CALOTROPIS GIGANTEA (L.) DRYAND., Global J Res. Med. Plants &

Indigen. Med., Volume 2(5): 392–400

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392–400

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

INTRODUCTION:

Arka (Rakta Arka) an important drug of

Ayurveda is known since Vedic period. In the

vedic literature Arka leaves were used in the

sacrifical rites (Sharma P.V., 2005). There are

two common species of Calotropis, viz

Calotropis gigantea (L.) Dryand nd C. procera

(Aiton) Dryand described in the classical

literature of Ayurveda by the name of Shveta

Arka and Rakta Arka. Calotropis is a genus of

plants that produce milky sap hence also

commonly called milkweed. The latex of

Calotropis procera is said to have a mercury-

like effects on the human body, and is

sometimes referred to as vegetable mercury and

is used in place of mercury in aphrodisiacs. C.

procera is used variously but sometimes leaves

are fried in oil for medicinal purposes. Cattle

often stay away from both the plant C. procera

and C. gigantea because of their unpleasant

taste and due to presence of Cardiac Glycosides

in its sap. Root bark of C. procera has Digitalis

like effect on the heart, but was earlier used as

a substitute of Ipecacuanha. It is a poisonous

plant; calotropin, a compound in the latex, is

more toxic than strychnine.

The Calotropis Shrub that produces white

or sometimes voilet flowers is called as

Swetarka. This is a rare shrub. Flowers of

Swetarka are considered to be favorite of Lord

Shiva.

C. gigantea as indicated by its name it is

much larger and coarse plant than C.procera.

Hindus obtain Ganapati from the root of C.

gigantea that sometimes takes the shape of

Lord Ganesh. The root of C. gigantea shrub is

invited on some auspicious day (in the Ravi-

Pushya Nakshatra ) and carved into the form of

Ganapati or Ganesh in some auspicious

muhurta. Hindus believe that those who

worship this idol of Ganesh enjoy the presence

of Mahadevi Laxmi and Lord Shiva. Most

recently C. gigantea is scientifically reported

for several medicinal properties viz. the flowers

are reported to possess analgesic activity,

antimicrobial and cytotoxic activity. Leaves

and aerial parts of the plant are reported for

anti-diarrhoeal activity, anti-Candida activity

and antibacterial activity, antioxidant activity.

Roots are reported to contain anti-pyretic

activity, cytotoxic activity. Although both

varieties of Arka have almost similar properties

yet C. procera is having comparatively more

medicinal properties.

Synonyms of Arka:

Shveta Arka Ganrupa, Mandar, Vasukh, Shvetapushpa Sadapushpa, Alarka,

Partapsh

Rakta Arka Arkaparna, Vikran, Raktapushpa, Sukhalphal, Ashphot. All

synonyms of Sun belong to Arka, because it has tikshnata like that

of Sun.

English Name Milk weed, Sodom Apple (Deseret), Madar, Giant milkweed

Marathi Rui, Akda

Bangla Akanda

Gujarati Akdo,

Due to its poisonous activities it is called as Darkhatae Jahnak.

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392–400

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

Sources of Arka

Raktaarka – mainly has two sources

Calotropis procera – smaller red flowers -

(Chief source of Raktaarka in N. India).

Calotropis gigantea (red variety) – larger red

flowers – (Chief source of Raktaarka in S.

India) (Bodhi Nighantu, 2010)

Shwetaarka – mainly has only one source

Calotropis gigantea (White variety) – larger

White flowers – (Chief source of Shwetaarka

all over the country). (Bodhi Nighantu, 2010).

Most of the Nighantus (Ayurvedic Materia

medica) have mentioned same properties of

both Arka (Sharma Ramprasad, 1990). Dalhana

in commentary Of Susruta Samhita mentioned

that by Arka we should take Rakta Arka. Rakta

Arka is having more tikshnata (having more

sharp in properties) compared to Shveta Arka

(Vyas Shiv Kumar, 1986).

Habitat

Calotropis procera is native to northern

Africa (i.e. Algeria, Egypt, Libya, Morocco,

Eritrea, Ethiopia, Somalia, Sudan, Kenya,

Tanzania, Uganda, Cameroon and Equatorial

Guinea)

Calotropis gigantea (Crown flower) is a

species of Calotropis native to Cambodia,

Indonesia, Malaysia, Philippines, Thailand, Sri

Lanka, India and China.

The second variety is of 2 types ie. C.

gigantea (Red) & C. gigantea (White).

C. procera Grown in Punjab, Bihar,

Mumbai. The white variety of Calotropis

gigantea is indigenous to Rajasthan & Gujarat

(which is taken as Shwetarka in Ayurveda)

The red variety of Calotropis gigantea is

common in South India.

So the white variety of C. gigantea has

been introduced into South India especially in

temples for its sanctity. (but when scaned

through the market samples in South India the

root of this variety is hardly available) (Bodhi

Nighantu, 2010)

Botanical Description:

Calotropis procera (Rakta Arka) is a soft-

wooded, evergreen, perennial shrub grows up

to height of 3–6 m. It has one or a few stems,

few branches, and relatively few leaves, mostly

concentrated near the growing tips. The bark is

corky, furrowed, and light grey. A copious

white sap flows whenever stems or leaves are

cut. Giant milkweed has a very deep, stout

taproot with few or no near-surface lateral

roots. Giant milkweed roots reach up to depths

of 1.7–3.0 m in Indian sandy desert soils

(Sharma B.M., 1968). The opposite leaves are

oblong-obovate to nearly orbicular, short-

pointed to blunt at the apex and have very short

petioles below a nearly clasping, heart-shaped

base (Nadkarni A.K., 2000). The leaf blades are

light to dark green with nearly white veins.

(Rastogi RP, 1999). They are 7–18 cm long and

5–13 cm broad, slightly leathery, and have a

fine coat of soft hairs that rub off.

inflorescenence is panicale with purple corolla

and erect lobes. (Anonymous, 2000). The

flowers are umbelliform cymes that grow at or

near the ends of twigs. Flowers are shallowly

campanulate with five sepals that are 4–5 mm

long, fleshy and variable in colour from white

to pink, often spotted or tinged with purple.

The fruits are inflated, obliquely ovoid follicles

that split and invert when mature to release flat,

brown seeds with a tuft of white hairs at one

end Sodom's apple milkweed produces a

simple, fleshy fruit in a grey-green inflated pod,

containing numerous flat, brown seeds with

tufts of long, white silky hair (‘pappus’) at one

end (Howard, R.A., 1989)

C. gigantea is a large shrub or small tree,

about 4–10 m tall. Its stem is erect, up to 20 cm

in diameter. The leaves are broadly elliptical to

oblong-obovate in shape, with the size of 9–20

cm × 6–12.5 cm but subsessile. The cymes are

5–12.5 cm in diameter. The inflorescence stalk

is 5–12 cm long, the stalk of an individual

flower is 2.5–4 cm long. Sepal lobes are

broadly egg-shaped with a size of 4–6 mm × 2–

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392–400

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

3 mm. Petal is 2.5–4 cm in diameter. It has

clusters of waxy flowers that are either white or

lavender in colour. Each flower consists of five

pointed petals and a small, elegant "crown"

rising from the centre, which holds the stamens.

The plant has oval, light green leaves and milky

stem. The petal lobes are broadly triangular

measuring 10–15 mm × 5–8 mm; they are pale

lilac and cream coloured towards the tips. The

outgrown like structure from the petal (corona)

has 5 narrow fleshy scales, connected to and

shorter than the staminal column, forming an

upturned horn with 2 obtuse auricles on either

side, cream coloured or lilac to purple, with a

dense longitudinal dorsal row of short white

hairs. The egg-shaped or boat-shaped fruits are

mostly in pairs, inflated, 6.5–10 cm × 3–5 cm.

The flowers last long. Taxonomical comparison

between C. procera & C. gigantea is given in

Table. No 1.

Table No 1- Comparison between C. procera and C. gigantea

Plant Origin Height Leaves Flower Petiole Fruits

Calotropis

gigantea/

Gigantic swallow

wort,

Madar/Shveta

Arka

India 8–10

m

Opposite White to purple,

rarely light

green yellow or

white. Flowers

not scented

Sessile Follicles recurved, 2

or 1 follicles,

second more often

suppressed, 3–4"

long

Calotropis

procera/Rakta

Arka

Swallow-

wort/Rakta Arka

India 3–6 m Opposite White to pink,

scented.

Sub-

sessile

Follicles 3–4",

recurved

The photosynthetic capacity of C.

procera is higher than that of C. gigantea

(Wilmer Tezara et al., 2011).

Properties of both arka according to

Bhavprakash (Chunekar K.C., 2007)

Shveta Arka Pushpa-Vrishya (potent), Laghu

(Light), Dipana (Appetizer), Pachna

(Digestive), Aruchi, Prasek (controls excessive

salivation), Svasa Kasa hara (cures asthma and

cough)

Rakta Arka: Madhura Tikta, krimi (removes

worms) kushta (cures all type skin disease)

kapha hara arsha (piles), visha (poison),

raktapitta (haemophillia), gulma, sopha hara

(removes inflammation).

Chemical compostion-

Calotropis procera plant latex contains the

cardenolide, proceraenin, while the root bark

contains benzoylinesolone and

benzoylisolinelone. The leaves and stalk

contain calotropin, and calotropagenin while

the flower contains calotropenyl acetate, and

multiflavenol and the latex contains uzarigenin,

and terpenol ester (Yoganarasimhan SN, 2000).

Chemical investigation of this plant has shown

the presence of triterpenoids, calotropursenyl

acetate and calopfriedelenyl, a norditerpenyl

ester, calotropternyl ester oleanene triterpenes

like calotropoleanyl ester, procerleanol A and B

(Ansari SH et al., 2001) and cardiac glycosides

calotropogenin, calotropin, uscharin, calotoxin

and calactin(Ahmed KKM et al., 2005). The

plant also has been investigated for the

presence of cardenolides (Seiber JN, 1982) and

anthocyanins. Phytochemical investigation of

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392–400

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

the roots of Calotropis procera yields two new

phytoconstituents, procerursenyl acetate and

proceranol, together with the known

compounds N-dotriacont-6-ene, glyceryl mono-

oleolyl-2-phosphate, methyl myrisate, methyl

behenate and glyceryl-1, 2-dicapriate-3-

phosphate. In the leaves, mudarine is the

principal active constituent as well as a bitter

yellow acid, resin and 3 toxic glycosides

calotropin, uscharin and calotoxin. The latex

contains a powerful bacteriolytic enzyme, a

very toxic glycoside calactin (the concentration

of which is increased following insect or

grasshopper attack as a defense mechanism),

calotropin D I, calotropin D II, calotropin F I,

calotropin F II and a non toxic protealytic

enzyme calotropin (2–3%). This calotropin is

more proteolytic than papain, and bromelain

coagulates milk, digests meat, gelatin and

casein. The whole plant of Calotropis gigantea

contains a- and b- amyrin, teraxasterol,

gigantin, giganteol. They are poisonous plants;

calotropin, a compound in the latex, is more

toxic than strychnine. Calotropin is similar in

structure to two cardiac glycosides which are

responsible for the cytotoxicity of Apocynum

cannabinum. Extracts from the flowers

of Rakta Arka (C. procera) have shown strong

cytotoxic activity in the patients of colorectal

cancer. Quercetin-3-rutinoside is identified in

the roots, stem, leaves, flowers and latex.

Voruscharin is isolated from African plant.

Cardenolides contents in leaf (2.04 mg/gm) and

in latex (162.0 mg/g), mostly calotropagenin –

derived cardenolides present from Calotropis

gigantea, two triterpene esters – 3

methylbutanoates of amyrin and taraxasterol

are isolated from latex of C. gigantea.

calotropins D1 and D2 had been isolated

from C. gigantea (Pal and Sinha, 1980). The

new oxiopregnane- oligoglycosides named

Calotropis A and B have been isolated from the

root of C. gigantea and their chemical structure

have been elucidated by chemical and

spectroscopy methods (Kitagawa Isao, 1992).

The cytotoxic principles of 'Akond mul' (Root

of C. gigantea) cardenoloids glycosides,

calotropin frugoside and 4-O-Beta-D-

glucopyranosyl frugoside were obtained as the

cytotoxic principles (Kiuchi, F., 1998).

Gigantin is a chemical extracted only from C.

gigantea.

Rasa Panchaka :

Rasa Katu, Tikta

Guna Laghu, Ruksha, Tikshna

Virya Ushna

Vipaka Katu

Doshakarma Kapha Vata hara

Uses: All the parts, viz, root, stem, leaf and

flowers of Calotropis are in common use in

indigenous system of medicine. Rakta Arka

(Ait.) R. Br., a wild growing plant of family

Apocynaceae is well known for its medicinal

properties. Different parts of this plant have

been reported to exhibit anti-inflammatory,

analgesic, and antioxidant properties. (Ahmed

UAM, 2006). Important factors of the various

parts of this plant have been widely reported.

Latex has been used in leprosy, eczema,

inflammation, cutaneous infections, syphilis,

malarial and low hectic fevers and as

abortifacient (Basu A, 1997). Rakta Arka latex

demonstrated strong inhibitory effect on E.

coli, S. aureus, S. pyogenes, S. pneumonia,

fungus like Aspergillus niger and yeast

Candida albicans. Efficacy of Rakta Arka in

the treatment of S. typhi and S. paratyphi has

been confirmed. C. gigantea's latex which is

highly poisonous is used as purgative. Also

when thorn entered to foot after removing the

thorn its milk applied to reduce the pain.

Ethanol extract of stems of C. gigantea was

reported for hepato-protective activity in male

Wistar rats against carbon tetrachloride induced

liver damage and showed marked hepato-

protective results. Proteins present in the latex

of C. gigantea are strongly proteolytic and

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392–400

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responsible for procoagulant activity of C.

gigantea (Rajesh R. 2005).

Leaves: In rheumatism, as an anti-

inflammatory and antimicrobial. The leaves

of Rakta Arka are said to be valuable as an

antidote for snake bite, sinus fistula,

rheumatism, mumps, burn injuries, and body

pain. The leaves of Rakta Arka are also used to

treat jaundice. In Egypt, the dried leaves are

smoked in pipes for relieving cough. A

decoction of the leaves is given to relieve

cough. For whooping cough the leaves are

boiled with Momordica charantia and the

decoction is drank a glassful each time. Leaves

of C. gigantea were reported to carry

antioxidant activity. (Singh N., 2010)

Roots: As hepatoprotective agents against

colds, coughs and elephantiasis, as an anti-

inflammatory, analgesic, anti-malarial and

antimicrobial. The root is also used in the

treatment of venereal diseases like gonorrhea

and syphilis. In these cases the root or root bark

is fermented in honey and given to the patient

to drink, reported that Rakta Arka has anti-

HIV-1. It is therefore a plant of interest for the

treatment of HIV-AIDS. (Remya Mohanraj,

2010) For poisonous snake bites, 2 to 4 leaves

of C. gigantea plant is chewed well by the

patient. Also fresh root of this plant are crushed

well and applied well by rubbing firmly over

the bitten area. The anti-pyretic activity of C.

gigantea roots in water : ethanol (50 : 50) has

been reported (Chitema et al., 2005). Anti-

pyretic activity was studied by using yeast and

TAB (Typhoid) vaccine induced pyrexia in

Albino Swiss rats and rabbits.

Flower: As cytostatic, abortifacient,

antimalarial, in asthma and piles and villagers

in Bikaner district ingest almost all plant parts

of Calotropis procera in various dietary

combinations for malarial fevers and pyrexias. In small doses, powdered flowers of Calotropis

gigantea are useful in the treatment of colds,

coughs, asthma, catarrh, indigestion and loss of

appetite (A. K. K. Mueen, 2005) The flowers

are bitter, digestive, astringent, stomachic,

anthelmintic, and tonic (Agharkar 1991;

Warrier et al., 1996). The alcoholic extract of

the flowers of C. gigantea was reported for

analgesic activity in chemical and thermal

models in mice (Pathak AK, 2007).

Latex: Spectrum of ocular toxicity following

accidental inoculation of latex of Calotropis

procera has been reported which leads to

diminish vision (Samar K Basak, 2009). The

latex also used to induce abortion, infanticide.

Latex also has wound healing properties

(Narendranalwaya, 2009). The anti-

inflammatory property of the latex of Rakta

Arka was studied on carrageenin- and formalin-

induced rat paw oedema model. A single dose

of the aqueous suspension of the dried latex

was effective to a significant level against the

acute inflammatory response. Dried latex and

chloroform extract of roots has been reported to

possess anti-inflammatory activity (Raman

Sehgal, 2005), (Kumar VL, 1994).

Calotropis gigantea yields a durable fibre

useful for ropes, carpets, fishing nets and

sewing.

Adverse effects:

The adverse effects Calotropis procera

consumption are reported to cause blisters,

lesions and eruptions when taken by patients

for the treatment of joint pains and

gastrointestinal problems and ocular toxicity.

Besides sometimes cardio-protective steroid

also show toxicity. The preparations of

Calotropis procera need to be used under the

supervision of a trained medical practitioner

(Lewis Nelson, 2007). Latex of C. gigantea

causes irritation to mucosa. An unidentified

allergen is found in the latex of C. gigantea.

CONCLUSION:

The World Health Organization has

estimated more than 80% of the world’s

population in developing countries depends

primarily on herbal medicines for their basic

healthcare needs. In recent years, traditional

uses of natural compounds, especially those of

plant origin, have received much attention of

the world as they are well known for their

efficacy and are generally believed to be safe

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392–400

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

for human use. It is best to use the classical

approach in the search for new molecules to

manage a variety of diseases. A thorough

review of the published literature on Calotropis

procera and C. gigantea shows that it is a

popular remedy in a variety of ethnic groups, as

well as Ayurvedic and traditional practitioners

for the treatment of a range of ailments.

Researchers are exploring the therapeutic

potential of Arka as it is likely to have more

therapeutic properties than known. As in

advanced researches on Calotropis procera has

been proven to be a good medicine in case of

HIV, breast cancer, syphilis etc. which are

becoming challenging to our society.

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Source of Support: Nil Conflict of Interest: None Declared

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