GJRMI - Volume 2, Issue 5, May 2013

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ISSN 2277 – 4289 www.gjrmi.com Editor-in-chief Dr Hari Venkatesh K Rajaraman

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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 NERIIFOLIA LINN.

SCREENING

OF

SECONDARY

METABOLITES

Chouhan Leela, Bhatt Shashank, Dhyani Suresh

OF

EUPHORBIA 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 AUTHENTICATION OF RIVEA HYPOCRATERIFORMIS (DESR.) CHOISY Borkar Sneha D, Naik Raghavendra, Harisha C R, Acharya R N

MARKERS

FOR

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

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

392–400

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 278–291 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article NATURAL PLANT PRODUCT BERBERINE/CISPLATIN BASED RADIOTHERAPY FOR CERVICAL CANCER: THE NEW AND EFFECTIVE METHOD TO TREAT CERVICAL CANCER Komal1, Singh Mayank2, Deshwal Vishal kumar3* 1, 2

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

School of Life Sciences, Singhania University, Pachri Bari, Rajasthan, India. *Corresponding Author: E-mail: vishal_deshwal@rediffmail.com; Mobile: +919897538555 1, 3

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

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

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

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-3methoxyphenyl) -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 chemopreventive 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 chemoradiotherapy 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

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

MATERIAL AND METHODS

Western blot analysis

Cell culture and chemicals

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-4chloro-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).

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).

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-LEHDAFC, 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.

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

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.

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

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

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

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 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

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.

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

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, BclXL 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 antiapoptotic 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 radiosensitivity 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 caspase9 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).

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

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.

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

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

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 radiosensitizers. 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 proapoptotic 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. Proapoptotic 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

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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 radioresistance (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 antiapoptotic Bcl XL and activation of caspase -3 and -9, resulting in enhanced apoptosis. We also provide evidence that Berberine/cisplatin based radiotherapy causes substantial downregulation of telomerase activity, Cyclin D1 and COX 2, thus acting as a potent radiosensitizer. 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.

REFERENCES Aggarwal BB, Sethi G, Ahn KS, Sandur SK, Pandey MK, Kunnumakkara AB, Sung B and Ichikawa H (2006). Targeting signal-transducer-and-activator-oftranscription-3 for prevention and therapy of cancer: modern target but ancient solution. Ann. N.Y. Acad. Sci. 1091: 151–169.

Ahmed KM, Fan M, Nantajit D, Cao N and Li JJ (2008). Cyclin D1 in low-dose radiation-induced adaptive resistance.

Oncogene. 27: 6738–6748.

Agarwal M, Pandita S, Hunt CR, Gupta A, Yue X, Khan S, Pandita RK, Pratt D, Shay JW, Taylor JS and Pandita TK (2008).

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Cancer Res. 68(9):3370–3378.

Ciesielska U, Nowińska K, Podhorska-Okołów M and Dziegiel P (2012). The role of human papillomavirus in the malignant transformation of cervix epithelial cells and the importance of vaccination against this virus. Adv. Clin. Exp .Med. 21(2):235–244.

Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, Ko JY, Lin JT, Lin BR, MingShiang W, et al (2001). Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or premalignant lesions. Anticancer Res. 21:2895–2900.

Deshwal VK (2012). Antibacterial Activity of Seeds of Mucuna pruriens Against Escherichia coli and Staphylococcus aureus. Global J. Res. Medicinal Plants & Indigen. Med. 1(4):109–113.

Diogo CV, Machado NG, Barbosa IA, Serafim TL, Burgeiro A and Oliveira PJ (2011).

Berberine as a promising safe anticancer agent - is there a role for mitochondria?

Curr.

Drug Targets.12(6):850–859.

Gandellini P, Folini M, Bandiera R, De Cesare M, Binda M, Veronese S, Daidone MG, Zunino F and Zaffaroni N (2007) Down-regulation of human telomerase reverse transcriptase through specific activation of RNAi pathway quickly results in cancer cell growth impairment. Biochem. Pharmacol.

73(11):1703–1714.

Howells LM, Moiseeva EP, Neal CP, Foreman BE, Andreadi CK, Sun YY, Hudson EA and Manson MM (2007). Predicting the physiological relevance of in vitro cancer preventive activities of phytochemicals. Acta Pharmacol. Sin. 28:1274–1304.

Javvadi P, Andrew T, Tuttle S and Koumenis C (2008). The Chemopreventive Agent Curcumin Is a Potent Radiosensitizer of Human Cervical Tumor Cells via Increased Reactive Oxygen Species Production and Overactivation of the Mitogen-Activated Protein Kinase Pathway. Mol. Pharmacol. 73: 1491– 1501.

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Khanna N, Sen S, Sharma H and Singh N (2003). S29 ribosomal protein induces apoptosis in H520 cells and sensitizes them to chemotherapy. Biochem.

Biophys. Res Comm. 304: 26–35.

Kostanová-Poliaková D and Sabová L (2005).

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Kumari R, Tiwary BK, Prasad A and Ganguly S (2012).

Study on the immunomodulatory effect of herbal extract of Asparagus racemosus willd. in broiler chicks. Global J. Res. Medicinal Plants & Indigen. Med. 1(1): 1–6.

Lao CD, Ruffin MT, Normolle D, Heath DD, Murray SI, Bailey JM, Boggs ME, Crowell J, Rock CL and Brenner DE (2006).

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Mohammad R, Giri A and Goustin AS (2008).

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Makhloufi A, Benlarbi L, Mebarki L and Akermi MM (2012). Antimicrobial activities of essential oil and crude extracts from Artemisia herba-ALBA ASOO, growing wild in bechar, south west of Algeria. Global J. Res. Medicinal Plants & Indigen. Med. 1(1): 7–13.

Nix P, Cawkwell L, Patmore H, Greenman J and Stafford N (2005). Bcl-2 expression predicts radiotherapy failure in laryngeal cancer.Br. J. Cancer.

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Rosa DD, Medeiros LR, Edelweiss MI, Pohlmann PR and Stein AT (2012).

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Singh M and Singh N (2008). Induction of apoptosis in HPV 16 positive cervical cancer cell lines, Involvement of mitochondrial pathway. Mol. Cell Biochem. 310:57–65.

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Tan S, de Vries EG, van der Zee AG and de Jong S (2012). Anticancer drugs aimed at E6 and E7 activity in HPV-positive cervical cancer. Curr. Cancer Drug Targets. 12(2):170–184.

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Tillhon M, Guamán Ortiz LM, Lombardi P and Scovassi AI (2012). Berberine: new perspectives for old remedies. Biochem Pharmacol. 84(10):1260–1267.

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Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 292–297 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article PHYTOCHEMICAL SCREENING OF SECONDARY METABOLITES OF EUPHORBIA NERIIFOLIA LINN. Chouhan Leela¹, Bhatt Shashank²*, Dhyani Suresh3 1

Department 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.

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

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

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 Metabolites

Screening

of

Secondary

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 preliminary phytochemical screening.

to

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 LiebermannBurchard 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 LibermannBurchard 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

ACKNOWLEDGEMENT

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, antiviral, anti-fever, anti-diabetic, anti-cancerous activities etc. Therefore, it can be involved in medicinal plant category.

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.

REFERENCES Aanchal Jain, Shashank Bhatt, Dr. Suresh Dhyani.

(2012), Phytochemical Screening of Secondary Metabolites of Ziziphus mauritiana Lam. Bark. . Int. J. Curr. Pharma. Res. Vol. 4, Issue 3, 156–159.

Aguinaldo, A.M., El-Espeso, B.Q.Guovara, M.G.Nanoto (2005) Phytochemistry. In: Guevara B.Q (ed) A. Guide book to plant screening phytochemical and biological. University of Santo Tomas, Manila, Philippines.

Anonymous (1952), Raw material, In: The Wealth of India, Vol. 3 (D-E).CSIR Publication, New Delhi; 226.

Ambasta SP. (1986) The useful plants of India.

New Delhi: CSIR Publication; 213:270.

Burkill I H. (1936) A Dictionary of the Economic Products of the Malay Peninsula London: Crown Agents for the Colonies. Vol.1 and 2.

Callow, R.K. (1936) Steroids. Proc. Royal, Soc London Series. 157:194.

Gibbs

R.D., (1974) Chemotaxonomy of Flowering Plants. Vol.1, McGill Queen’s University Press, Montreal and London.

Khandelwal K.R., (2008) Pharmacognocy. Nirali Pune, edition: 19.

Kokate C.K., (1994) Practical Pharmacognosy, 4th ed., Vallabh Prakasan, Delhi, 107– 111.

Kokate C K, Purohit A P and Gokhale SB. (2001) Carbohydrate and derived Products, drugs containing glycosides, drugs containing tannins, lipids and protein alkaloids. Text book of Pharmacognosy, 7, edition: 133–166, 167–254, 255–269, 272–310, 428–523.

Nadkarni AK. (1954) Indian Material Medica.

Vol. 1, Bombay. Popular Prakashan, 424–426.

Peach K., Tracey MV. (1956) Modern methods of plant analysis. Vol. 3, Springer Verlag, Berlin.

Priyanka Parmar, Shashank Bhatt, Dr. Suresh Dhyani, Aanchal Jain.

(2012) Phytochemical Studies of the Secondary Metabolites of Ziziphus mauritiana Lam.Leaves . Int. J. Curr. Pharma. Res. Vol. 4, Issue 3, 153–155.

Rievere, C., J.H. Van Nguyen, L.Pieters, B. Dejaegher, Y.V. Heyden, C.V. Minh, J.Quetin-Leclercq. (2009) Polyphenols isolated from antiradical extracts of Mallotus metcalfianus. Phytochemistry., 70: 86–94.

Practical Prakashan,

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Shashank bhatt, Dr. Suresh Dhyani (2012).

Preliminary Phytochemical Screening of Ailanthus excelsa Roxb. Int. J.Curr.

Pharma Res. 2011; vol. 4, Issue 1, 87– 89.

Surendra K.Rathore, Shashank Bhatt, Dr. Suresh Dhyani, Aanchal Jain (2012).

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Source of Support: Nil

of Medicinal Plant of Ziziphus mauritiana Lam.Fruits. . Int. J. Curr.

Pharma. Res. Vol. 4, Issue 3, 160–162.

Tag H, Das AK, Loyi H. (2007) Natural Product Radiance; 6(4) 334–340, p.532.

Treare GE, Evans WC. (1985). Pharmacognosy 17th edn., Bahiv Tinal, London. P 149.

Conflict of Interest: None Declared

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Research article TRADITIONAL MEDICINES OF HERBAL ORIGIN PRACTICE BY THE ADI TRIBE OF EAST SIANG DISTRICT OF ARUNACHAL PRADESH, INDIA Das Moushumi1*, Jaishi Anju2, Sarma Hirendra N3 1

Assistant Professor, Department of Zoology, University of Science and Technology, Meghalaya, Ri-Bhoi District, Meghalaya, India 2 Research Scholar, Department of Zoology (Center with Potential for Excellence in Biodiversity), Rajiv Gandhi University, Itanagar – 791 112, India 3 Professor, 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

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 whenever necessary.

own

requirement

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

Leaves

Wound healing, malaria, abortifacient in pigs.

Tick repellent in chicken

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.

Fresh leaves collected from the plant are kept inside the poultry farm to repel and/ or removes the tick from the animal.

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.

Root is chewed by the patient to get relief. Three times for three days.

Alstonia scholaris (L.) Br (Apocynaceae) Adi/RGU/G-01/ Cingar Leaves Amphineuron extensus (BI.)HOLTT (Thelypteridaceae) Adi/ RGU/G-02/ Rokji Ageratum conyzoidesL (Asteraceae) Adi/ RGU/G-03/ Namsing

Wound healing Leaves

Axonopus compressus (SW)P.Beauv (Poaceae) Adi/ RGU/G-04/Bobosa Bannaya reptans Sprengel (Scrophulariceae) Adi/RGU/G-05/Kat-Buk usueng

Roots

Eye infection/ Disease

Leaves

Nail infection

Bryophyllum pinnatum. Kuntz.

(Crassulaceae.)

Adi/ RGU/G-06/ Nebi – Nelum

Leaves

Skin burn, diarrhea, fracture and Sciatica.

Clacaria macrophylla Wall (Rubiaceae.)

Adi/ RGU/G-07/ Pemi lagin Clerodendrum spp (Nerbenaceae) Adi/ RGU/G-08/ Dumkar

Sap of the stem

Conjunctivitis, Eye infection

1–2 drops of the sap of the stem is poured into the eye during infection

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.

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.

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)

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

Leaves

Wound infection Paste of Fresh leaves is mixed with in animal the meal of domestic animals.

Leaves

Cut, healing

wound Freshly collected leaves are grinded to make paste and applied on wound/ cuts to heal the wound and for prevent bleeding.

Bark Tonic for malaria Small pieces bark are sun dried and Oroxylum indicum L. Vent. patients then boiled with leaves of Ocimum (Bignoniaceae) sanctum, Zingiber zerampet Adi/ RGU/G-14/Domir ettkung (Rhizomes) and Mentha spicata. The boiled residue is advised to use during the whole treatment period.

Leaves Treatment of Fresh tender leaves are advised to Psidium guajava L. (Myrtaceae) /buds diarrhea. chew by the person suffering from Adi/ RGU/G-15/ Mudhuri anne diarrhea. Doses are 4–5 fresh twigs takes twice or thrice a day for 3–4 days.

Leaves Stomach disorder/ Fresh leaves are boiled with other Paederia foetida (Rubiaceae.)

Diarrhea vegetables. The boiled residue is Adi/RGU/G-16/Yepetare 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.

Leaves, Diarrhea, Malaria Paste prepared from leaves is boiled Solanum nigrum L (Solanaceae) roots and and conjunctivitis in water. Diarrhea and malaria stem patient‟s treated with both the boiled Adi/ RGU/G-17/ Bangko 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.

Tooth ach The stem of plant is used. Chewing Zanthoxylum acanthopodium DC Stem or brush with it gives immediate (Rutaceae) relief from the toothache and to Adi/ RGU/G-18/ Ombe makes the teeth strong.

Tooth ache, Leaves are dried on the sunlight or Zanthoxylum hamiltoniamun wall Leave, fruit Abortifacient smoke and then applied on the tooth.

(Rutaceae) Immediately it gives relief of the Adi/ RGU/G-19/ Ongar toothache. The fruit is used to eat in large quantity as abortifacient.

Rhizome Tooth ache, The rhizome is boiled with water; Zingiber cassumunar cough, post this extract is given to sip by the (Zingiberceae) Roxb.Adi/RGU/Gdelivery pain person for getting relief of cough.

20/ Kekir relief 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.

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).

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

Stem and leaves

Bone fracture

Entada scadens (Mimosaceae)

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 Ricinus communis L (Euphorbiaceae) Adi/ RGU/BF-25/ Aki-Rokmic Annanus comosus (Bromeliaceae) Adi/ RGU/RC-26 /Anaros, Tako bela

Stem

Bone fracture

Bark and leaves

Bone fracture

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)

Hemidesmus indicus (Apocynaceae) Adi/ RGU/RC-28/ Mimosa pudica (Mimosaceae) Adi/ RGU/RC-29/ Anying ing

Stem

Fertility control in women. (abortifacient) Fertility control in women

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.

Stem portion is shade dried to make powder. The powder id eaten with water.

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.

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

Arial part

Fertility control in women

Roots

Fertility control in women

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

Adi/ RGU/BF-22/ Taboh

Whole plant

Fresh juice of aerial part taken orally by the desired women to prevent pregnancy.

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

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 antifertility 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 womenâ€&#x;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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Martin, GJ (1995). Ethnobotany: a methods manual. London, UK: Chapman and Hall Mackenzie A (1884). The north east frontier of Bengal, Calcutta.

Meena, AK, Garg Nikita, Nain Jaspreet, Meena, RP & Rao, MM (2011).

Review on ethnobotany, phytochemical and Pharmacological profile of Alstonia scholaris. International Res. Journal of Pharmacy. Vol 2 (1): 49–54 Mibang T, Chowdhari, SK (2003). (eds) Ethno medicines of the tribes of Arunachal Pradesh.

Himalayan Publishers, Itanagar.

Myers N, Muttermeier RA, Muttermeir CG, Foneca GAB, Kents J (2000).

Biodiversity hotspots for conservation priorities. Nature ,403: 853–858.

Note by the Executive secretary, Secretariat of the Convention on Biological Diversity, UNCTAD, (2000) Expert meeting on Systems and National experience for protecting, traditional knowledge, Innovations and practices of Indigenous and local communities. Geneva, November, 2000.

(Document TD/B/COM.1/EM.13/2.)

Nath SC, Bordoloi DN (1993). Diversity of economic flora in Arunachal Pradesh: Plant folk medicines among the Chakma, Singpho and Tangsa tribals.

Himalayan Biodiversity: Conservation Strategies,179–189.

Nyori, Tai (1993). History& Culture of the Adis.

Official website of Government of Arunachal Pradesh.http:// arunachalpradesh.nic/people.htm.

Tribes of Arunachal Pradesh (last updated 28.01.2011) http://WWW.indianetzone.com/8/tribes_ arunachal_pradesh.htm.

Pal, GD (1984).Observation on Ethnobotany of the tribals of Subansiri, Arunachal Pradesh. Bull.Bot.Surv.India , 26: 26– 37 Qureshi, AA, Sanghai DB and Padgilwar SS. (2006).Herbal option for Contraception: A Review. Pharmacognosy Magazine 2006, 204–215 Roy S (1996). Aspects of Padam-Minyong culture. Itanagar.

Rizvi,SHM & Roy, Shibani (2006). Adi Tribes of Arunachal Pradesh.

Seth SD, Sharma B (2004). Medicinal plants in India. Indian.J.Med. Research,120:9–11 Sen, SK, Pradhan R, Pattnaik, MR, Behera, LM (2012). Traditional knowledge of Medicinal plants against birth control by the tribals and other rural people of Bargarh district in Western Odisha, India. Global Journal Res.Med.Plants & Indigen.Med. Vol 1 (2),670–677 Singh, HB and Singh TB (2005). Plants used for making traditional rosaries in Manipur. Indian Journal of Traditional Knowledge, 15–20.

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Valsala S, Varpagaganapathy, PR (2002).

Effect of Mimosa pudica root powder on oestrous cycle and ovulation in cycling female albino rat. Rattus norvegicus.Phytotherapy research, 16:190–192.

Source of Support: Women Scientist Scholarship Scheme for Societal Programme (WOS-B), Department of Science and Technology, Government of India

World

Health Organization: Regional consultation on development of Traditional medicine in South-East Asia Region. 1999, New Delhi. WHO Regional Office for South-East Asia, 1999 (document reference SEA/Trad.Med./80)

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 311–316 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article PRELIMINARY PHYTOCHEMICAL SCREENING OF SECONDARY METABOLITES OF ADHATODA VASICA NEES. FLOWERS Chouhan Surksha¹, Bhatt Shashank²*, Dhyani Suresh3 1

Department 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.

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. as

Adhatoda vasica Nees. commonly known 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, antibleeding, bronchodilator, anti-diabetic, antihelminthic, 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 Metabolites

Screening

of

Secondary

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 preliminary phyto-chemical screening.

to

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 LiebermannBurchard 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 LibermannBurchard 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 antineoplastic 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.

REFERENCES A. Chakraborty and A.H. Brantner, (2001) phytotherapy research, 15, pp. 532– 534.

Kokate C.K., (1994) Practical Pharmacognosy, 4th ed., Vallabh Prakasan, Delhi, 107– 111.

Aguinaldo, A.M., El-Espeso, B.Q.Guovara, M.G.Nanoto (2005). Phytochemistry.

In: Guevara B.Q (ed) A. Guide book to plant screening phytochemical and biological. University of Santo Tomas, Manila, Philippines.

Kumar P. (2004). Valuation of medicinal plants for pharmaceutical uses. Curr. Sci. 86 (7):930–937.

B.L. Manjunath, (1948) The wealth of India, CSIR Delhi, pp. 31–32.

R.L. Wakhloo, G. Kaul, O.P. Gupta and C.K. Atal, (1980) Indian Journal of pharmacology, 13, pp.129.

Gibbs

R.D., (1974) Chemotaxonomy of Flowering Plants. Vol.1, McGill Queen’s University Press, Montreal and London.

Khandelwal K.R., (2008) Pharmacognocy. Nirali Pune, edition: 19.

Practical Prakashan,

K. Jayapaul, K.Kishor and R.K. Janardhan, (1984) In Vitro Cell Dev. Biol.- Plant, 41,pp. 682–685.

Kokate C K, Purohit A P and Gokhale SB. (2001) Carbohydrate and derived Products, drugs containing glycosides, drugs containing tannins, lipids and protein alkaloids. Text book of Pharmacognosy, 7, edition: 133–166, 167–254, 255–269, 272–310, 428–523.

P.K. Patel and P.H. Bhatt, (1984) Ind. J. Med. Sci., 38,pp. 70–72.

Rievere, C., J.H. Van Nguyen, L.Pieters, B. Dejaegher, Y.V. Heyden, C.V. Minh, J.Quetin-Leclercq. (2009) Polyphenols isolated from antiradical extracts of Mallotus metcalfianus. Phytochemistry., 70: 86–94.

Shashank bhatt, Suresh Dhyani. (2011); Preliminary phytochemical screening of Ailanthus excelsa Roxb. International Journal of Current pharmaceutical research. vol. 4, Issue 1, 87–89.

Tag H, Das AK, Loyi H. (2007); Natural Product Radiance 6(4) 334–340.

Treare GE, Evans WC. (1985) Pharmacognosy 17th edn., Bahiv Tinal, London. P 149.

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U.P. Claeson, T. Malmfors, G. Wikman and J.G. Bruhn, (2000) Journal of Ethnopharmacology, 72, pp.1–20.

Source of Support: Nil

WHO (1993). Regional Office for Western Pacific, research guidelines for evaluating the safety and efficacy of herbal medicines. Manila.

Conflict of Interest: None Declared

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Review article HERBAL MEDICINES FOR DEPRESSION AND ANXIETY: A COMPREHENSIVE STATE OF THE ART REVIEW Patel Shanti1, De Sousa Avinash2* Medical Intern – Lokmanya Tilak Municipal Medical College and General Hospital, Mumbai, Maharashtra, India 2 Consultant Psychiatrist and Founder Trustee – De Sousa Foundation, Mumbai- 400054, Maharashtra, India *Corresponding Author: E-mail – avinashdes999@yahoo.co.uk; TEL – 91-22-26460002 1

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.

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 metaanalysis (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 CYP450-3A4 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 800â&#x20AC;&#x201C; 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 parkinsonâ&#x20AC;&#x;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 physicianâ&#x20AC;&#x;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 EPADHA mixture but should not substitute omega3s 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 HAMD, 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 MAOB 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; n380). 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% Cl3.44,4.83),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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Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 337â&#x20AC;&#x201C;347 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article A BRIEF REVIEW ON NONI (MORINDA CITRIFOLIA L.) - A HERBAL REMEDY FOR BETTER HEALTH Patel Swetal1, Krishanamurthy R2* 1

M.Sc. final year project student, C.G Bhakta Institute of Biotecnology, Uka Tarsadia University, Mahuva Road, Bardoli-394601, Surat(Dist. Surat), Gujarat, India.

2 Director, 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 phytochemicals and therapeutic effects of Noni which was proved by various scientists earlier.

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

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): 337â&#x20AC;&#x201C;347

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

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

Protein Lipid Glucose Fructose

Chunhieng (2003) 2.5% 0.30g/100g 11.9 ± 0.2 g/l 8.2 ± 0.2 g/l

Fruit juice Shovic and Whisler (2001) 0.4g/100g 0.1–0.2% − −

Leaf powder European (Leung et al., commission (2002) 1972) 0.2–0.5% 1 g/100g − 0.2 g/100g 3.0–4.0% − 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

Characteristics

Table: 2 Chemical constituent, there place and its chemical nature in different plant part recognize by various authors: Plant part Chemical constituent (place) 2-methyl-4-hydroxy-5,7Flower dimethoxyanthraquinone 4-O-ß-D-glucopyranosyl (1, 4)-a-L-rhamnopyranoside 5,8-dimethyl-apigenin 4'0-O-ß-D-galactopyranoside 2,6-di-O-(ß-D-glucopyranosyl Fruit 1-O-octanoylßD glucopyranose 6-O-(ß-D-glucopyranosyl-1-Ooctanoyl-ß-D glucopyranose Ascorbic acid Asperulosidic acid Aspuruloside tetraacetate

Structure

Reference

Anthraquinone Glycosides

Sang et al., (2002)

Flavonoids

Sang et al., (2002), Elkins (1998) Fatty acid ester Dittmar (1993)

glycosides

Wang et al., (1999)

Acid Flavonoids Flavonoids

Liu et al., (2001) Morton (1992), Elkins (1998) Wang et al., (1999), Liu et al., (2001) Cardon (2003) Elkins (1998) Sang et al., (2002), Dittmar (1993),Wang et al., (2002), Elkins (1998), Liu et al., (2001) Elkins (1998) Solomon (1999b),Elkins (1998), Dittmar (1993), Wang

Caproic acid Caprylic acid

Acid Acid

Ethyl acetate Ethyl caprylate

Ester Ester

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Leaves

Stem

Ethyl caproate Hexanoic acid Quercetin 3-O-a-L-rhamnopyranosyl(1-6)-ß-D-glucopyranoside Quercetin 3-O-a-L-rhamnopyranosyl(1-6)-ß-D-glucopyranoside Alanine

Ester Acid Flavonoide

Serine Threonine Tryptophan Tyrosine Urosolic acid

Amino acid Amino acid Amino acid Amino acid Triterpenoids and sterols

Valine

Amino acid

Arginine Aspartic acid ß-sitosterol

Amino acid Amino acid Sterols

Citrifolinoside B Cysteine Glutamic acid Glycine Histidine Isolucine Leucine Phenylalanine Methionine Proline Kaempferolm 3-O-ß-Dglucopyranosyl-(1-2)aLrhamnopyranosyl-(1-6)- ß-Dgalactopyranoside Quercetin 3-O-ßDglucopyranoside 2-hydroxyanthraquinone

Iridois Amino acid Amino acid Amino acid Amino acid Amino acid Amino acid Amino acid Amino acid Amino acid Chlorophyll derivatives

Flavonoids

Amino acid

et al.,(2002), Cardon (2003), Liu et al.,(2001), Srivastava & Singh (1993) Dittmar (1993) Dittmar (1993) Sang et al., (2002), Cardon (2003), Wang & Su (2001), Farine et al., (1996) Sang et al., (2002)

Sang et al., (2002), Cardon (2003), Srivastava & Singh (1993) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Sang et al., (2002), Elkins (1998), Wang et al., (2002), Cardon (2003) Dittmar (1993), Elkins (1998) Dittmar (1993) Dittmar (1993) Sang et al., (2002), Wang et al., (2002), Chunhieng (2003) Sang et al., (2002) Elkins (1998) Dittmar (1993) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Dittmar (1993), Elkins (1998) Sang et al., (2002)

Flavonoids

Sang et al., (2002)

Anthraquinone

Siddiqui et al., (2006)

2-methoxyanthraquinone

Anthraquinone

Siddiqui et al., (2006)

Morindicininone

Anthraquinone

Siddiqui et al., (2006)

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Seeds

Ricinoleic acid

Acid

Solomon (1999b)

Root

8-hydroxy-8-methoxy-2methyl-anthraquinone

Anthraquinone

Cardon (2003), Solomon (1999b)

rubichloric acid 1,3-dihydroxy-6methylAnthraquinone Morenone 1 Morenone 2 Ruberythric acid Rubiadin

Acid Anthraquinone

Elkins (1998) Morton (1992)

Anthraquinone Anthraquinone Acid Anthraquinone

Chlororubin

Chlorophyll derivatives Saccharide Anthaquinone Anthraquinone Anthraquinone Saccharides Anthraquinone Phenol Anthraquinone

Solomon (1999b) Solomon (1999b) Cardon (2003) Elkins (1998), Cardon (2003), Inoue et al., (1981), Ross (2001) Dittmar (1993)

Root bark

Hexose Morindadiol Morindanidrine Morindine Pentose Physcion Rubiadin monomethyl ether Soranjidiol Trioxymethyl anthraquinone monoethyl ether 8-O-a-L Hard wood Physcion arabinopyranosyl(1-3)-ß-Dgalactopyranosyl-(16)- ß -D-galactopyranoside Morindone Root, Hard wood, Root bark Root, Hard wood, Seeds

Damnacanthal

Root, Root Alizarin bark, Fruit

Plant

2-methyl-3,5,6 Trihydroxyanthraquinone Scopoletin

Anthraquinone

Dittmar (1993) Dittmar (1993) Dittmar (1993) Elkins (1998), Cardon (2003) Dittmar (1993) Solomon (1999b) Dittmar (1993) Dittmar (1993), Elkins (1998), Ross (2001) Dittmar (1993)

Anthraquinone Glycoside

Wang & Su (2001), Wang et al., (2002)

Anthraquinone

Sang et al., (2002), Dittmar (1993), Wang et al., (2002), Ross (2001)

Anthraquinone

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

Anthraquinone

Anthraquinone

Elkins (1998), Dittmar (1993), Wang et al., (2002), Cardon (2003), Ross (2001), Dittmar (1993) Cardon (2003)

Miscellaneous compound

Wang et al., (2002), Farine et al., (1996)

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2-methyl-3,5,6trihydroxyanthraquinone 6-O-ß-D-xylopyranosyl-(1-6)ß-D-glucopyranoside 3-hydroxymorindone

Anthraquinone glycoside

Cardon (2003), Inoue et al., (1981)

Anthaquinone

3-hydroxymorindone 6-O-ß-D-xylopyranosyl- (1-6)ß-Dglucopyranoside 5,6-dihydorxylucidin 3-O-ß-Dxylopyranosyl-(1-6)ß-D – glucopyranoside 5,6-dihydroxylucidin

Anthraquinone Glycoside

Cardon (2003), Inoue et al., (1981) Cardon (2003), Inoue et al., (1981)

Anthraquinone glycoside

Cardon (2003), Inoue et al., (1981)

Anthraquinone

Aucubin

Anthraquinone

Linoleic acid Lucidin

Acid Anthraquinone

Cardon (2003), Inoue et al., (1981) Elkins (1998), Wang et al., (2002) Wang et al., (2002) Cardon (2003), Inoue et al., (1981) Cardon (2003)

Lucidin 3-O-ß-Dxylopyranosyl- Anthraquinone (1-6)-ß-Dglucopyranoside glycosides

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 Immunostimulatory 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 -

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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).

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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.

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Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 348–356 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article DEVELOPMENT OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS FOR AUTHENTICATION OF RIVEA HYPOCRATERIFORMIS (DESR.) CHOISY Borkar Sneha D1, Naik Raghavendra2, Harisha C R3, Acharya R N4 1,2

P G Scholar, Dravyaguna Department, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, INDIA Head, Pharmacognosy Laboratory, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, INDIA 4 Associate Professor, Dravyaguna Department, IPGT&RA, Gujarat Ayurved University, Jamnagar, Gujarat, India *Corresponding Author: E-mail: dr.sneha.borkar@gmail.com 3

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

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

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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 NOPHM 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 fingerprints)

characterization

(DNA

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: TGCCGAGCTG OPA-02 OPB-10 OPC-06

CTGCTGGGAC GAACGGACTC

Table 2: Reactions were set up with PCR master mix and respective Random primer.

OPA-02 18 µl

OPC-06 18 µl

OPB-10 18 µl

Notes

20 µl

20 µl

20 µl

DNA sample

1 µl

1 µl

1 µl

1X Contains 100µM each of dATP, dGTP, dCTP and dTTP. Assay buffer with 15mM MgCl2, 3U/reaction Taq Polymerase.

10 pM used for each reaction

Random primer

1 µl

1 µl

1 µl

Total Volume

40 µl

40 µl

40 µl

Double Distilled water 2X PCR master mix

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Table 3: PCR Conditions: Temperature 94˚C 94˚C 45˚C 72˚C 72˚C

Time 2 minutes 30 seconds 1 minute 1 minute 30 seconds 7 minutes

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

No. of cycles 1 40

1

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, metaxylem 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 â&#x20AC;&#x201C; 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

1 2 3 4 5 6 7 8 9

Stomata length Stomata breadth Epidermal cell Stomatal circumference Epidermal cell circumference No. of stomata No. of epidermal cells Stomatal index Palisade ratio

Results Upper Epidermis 9.0 µm 7.0 µm 8 × 7 µm2 21 µm 2 24 µm2 6 36 15 2–3

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Lower epidermis 9.0µm 7.0µm 9 × 9 µm2 24 µm2 26 µm2 9 26 24 Nil

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Sr. no

1.

Table 5: Histo – chemical test of leaf of Rivea hypocrateriformis Reagent Observation Characteristics Result Phloroglucinol+ Red Lignified cells ++ Concentrated HCl

2.

Iodine

Blue

Starch grains

3.

Dissolved

4.

Phloroglucinol+ Concentrated HCl Fecl3 solution

5.

Sudan III

Dark blue black Red

−

Calcium oxalate ++ crystals to Tannin cells ++ 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 µm2. Stomatal circumference measured 21 µm2 and 24 µm2 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.

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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.

B. Swathy, S. Mohana lakshmi, A. Saravana Kumar (2010). Review on herbal drugs for analgesics and anti inflammatory activities. IJBPR. 1(1):7–12.

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.

Govinda das, Ambikadatta Shastri (2011) Bhaishajya Ratnavali, Chaukhambha Prakashana, Varanasi, pp no.201, 202, 237, 238, 473, 474.

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

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

Source of Support: Nil

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.

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 357–364 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article ANTIMICROBIAL EVALUATION OF CROTON ROXBURGHII BALAK. (EUPHORBIACEAE) STEM BARK Patel Esha1*, Padiya RH2, Acharya RN3 1

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

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

3 Associate 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

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

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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 ethnomedicine 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.

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 pathogens:

mediums

according

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.

Antimicrobial assay (Doman & Deans, 2000) Microbial load report: 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

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) 14.6 Ă&#x2014; 103 a) Bacterial count 30 b) Fungal count

105/g 103/g

Pathogens (per gram) a) S. aureus b) E. coli c) P. aeruginosa d) S. typhi

Absent Absent Absent Absent

Absent Absent 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 B. subtilis (NCIM 2063)

C. roxburghii stem bark methanol extract

Methanol (Control) Gentamycin Cifpodoxime Streptomycin

5 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 250 µg/ml −

12 13 15 17 18 Nil

10 µg 10 µg 10 µg

28 22 27

Zone of inhibition (mm) S. aureus E. coli K. (NCIM (NCIM pneumoniae 2079) 2065) (NCIM 2719) Nil 13 13 Nil 14 14 12 16 16 13 17 17 15 20 18 Nil Nil Nil 25 23 17

22 21 17

24 19 24

Figure 1: Effect of C. roxburghii stem bark methanol extract against Gram +ve and Gram −ve strains 25 20

5

15

25

10

50

5

100

0 B. subtilis (NCIM 2063)

S.aureus (NCIM 2079)

E. coli (NCIM 2065)

K. pneumoniae

250

Figure 2: Effect of standard drug against Gram +ve and Gram −ve strains 30 25 20 15

Gentamycin 10µg

10

Cifpodoxime 10µg

5

Streptomycin 10µg

0 B. subtilis (NCIM S.aureus (NCIM 2063) 2079)

E. coli (NCIM 2065)

K. pneumoniae

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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 C. albicans (NCIM 1028) (NCIM 3471) 5 µg/ml Nil 16 C. roxburghii stem bark 25 µg/ml 11 17 methanol extract 50 µg/ml 12 18 100 µg/ml 13 20 250 µg/ml 14 22 − Nil 11 Methanol (Control) 10 µg 14 19 Amphotericin B 50 µg 10 µg 11 28 Fluconazole 30 µg 10 µg 24 30 Clotrimazole 10 µg

Figure 3: Effect of C. roxburghii stem bark methanol extract against two fungal strains 25 20

5

15

25

10

50

5

100

0

250 S. flavus (NCIM 1028)

C. albicans (NCIM 3471)

Figure 4: Effect of standard drugs against two fungal strains 35 30 25 20 15 10 5 0

Amphotericin B 50µg Fluconazole 30µg Clotrimazole 10 µg Methanol (Control) S. flavus (NCIM 1028)

C. albicans (NCIM 3471)

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

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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.

Dorman HJD, Deans SG (2000). Antimicrobial agents from plants. Antimicrobial activity of plant volatile oils, Journal of Applied Microbiology; 88(2); p.no.

308–316.

Anonymous, (2001).

The Ayurvedic Pharmacopoeia of India Part-I, VolumeI, First edition, Ministry of Health and Family Welfare, Government of India, Department of Indian Systems of Medicine & Homoepathy, New-Delhi; p.no. 139.

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.

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; p.no. 75.

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.

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.

Patel University, Vallabh Vidyanagar; p.no. 525-527, 549–550.

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.

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.

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

Source of Support: Nil

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.

Conflict of Interest: None Declared

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Research article AN ESTIMATION OF HUMIC SUBSTANCES IN AN AYURVEDIC HERBOMINERAL DRUG SHILAJATU (ASPHALTUM) AS PART OF PHYTO-PHARMACEUTICAL STANDARDIZATION Akarshini A M1*, Renuka2, Shukla V J3, Baghel M S4 1

PhD scholar, Department of Kayachikitsa, IPGT &RA, Gujarat Ayurveda University, Jamnagar, Gujarat, India 2 PhD scholar, Department of Pharmaceutical chemistry, IPGT &RA, Gujarat Ayurveda University, Jamnagar, Gujarat, India 3 Department of pharmaceutical Chemistry, IPGT &RA , Gujarat Ayurveda University, Jamnagar, Gujarat, India 4 Director, 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.

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): 365â&#x20AC;&#x201C;373 Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

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

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 phytopharmaceutical 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.

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TABLE 1. Organoleptic parameteres of Shilajatu sample I & II Sl.No

Parameters

Suddha Shilajatu Sample I

1

Colour

Brownish black

Suddha Shilajatu Sample II 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

Easily dissolves

Easily soluble

Water In acid (HCl) Alcohol Acetic acid

Dissolves in HCl with blackish brown color changing to light brown Slightly soluble Slightly soluble

Slightly soluble Insoluble Soluble

TABLE 2. Microscopic study of samples Reagents used Sample I Sample II High amount of crystalline material Calcium oxalate crystals Water Oil globules Crystalline material No crystalline material HCl Oil globules present

FIGURE 1-Powder microscopy of Suddha Shilajatu sample I

Crystalline material

Prismatic crystal

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FIGURE 2- Microscopy of Suddha Shilajatu sample II

Crystals in group

Oil globule

Physico - chemical parameters Sample I & II were evaluated for physicochemical 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 chromatographic study (HPTLC)

layer

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

Prismatic crystal

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 1 2 3 4 5 6 7

Name of the test/reagent

Results Results Sample I Sample II Alkaloids Wagner’s Reagent Negative Negative Tannin Lead acetate Positive Positive Saponin Glycosides Foam test Negative Negative Protein Biuret test Positive Positive Steroids Libermann Burchard’s test Negative Negative Flavonoids NaOH test Positive Positive Reducing sugars Fehling’s test Absent Absent

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

B

C

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 spectrophotometry:

undertake

UV

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

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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 spectrophotometric 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.

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TABLE 4 - Qualitative parameters for SRC for different functional groups Ratio Q1= HA

FA Sample I Sample II −9.000 −10.703 −16.357

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

Gravimetric method

Sample Shilajatu I

280 nm 472 nm 664 nm 260 nm 0.043 0.014 −0.229 −0.261

Shilajatu II Fulvic acid

−0.220 −0.238

method/Acid

precipitation

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.

0.130 0.045

0.034 0.027

−0.306 −0.289

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

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

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.

REFERENCES Carmen Rivero, T. Chirenje, L.Q. Ma, G. Martinez (2004), Influence of compost on soil organic matter quality under tropical conditions. Geoderma. 123: pp.

355–61

Elham A. Ghabbour & Geoffrey Davies (2009), Spectrophotometric analysis of fulvic acid solutions – a second look. In: Annals of Environmental Science: Vol 3, pp. 131–8.

Choppin, G.R., Allard, B.,(1985), Handbook on the Physics and Chemistry of the Actinides. Amsterdam, The Netherland.

In: Freeman, S., Keller, B. (Eds.), Elsevier: pp. 407–29.

Fataftah AK, Walia DS, Gains B, Kotob SI(2001), A comparative evaluation of known liquid humic acid analysis methods. In: Ghabbour EA, Davies G. eds. Humic Substances: Structures, Models and Functions. Cambridge, UK: Royal Society of Chemistry: pp. 337– 42.

Daqing Gan, Shaban I. Kotob and Daman S (Received June 7, 2006, in final form November 12, 2006; Accepted January 2, 2007), Evaluation of a spectrophotometric method for practical and cost effective quantification of fulvic acid. D. Gan et al., Annals of Environmental Science (2007), USA. Walia ARCTECH, Inc. Chantilly, VA 20151: Vol 1, pp. 11–15

Gieguzy_nska, E., Ko_cmit, A, Gołezbiewska, D (1998), Studies on humic acids in eroded soils of Western Pomerania. In: Zaujec, A., Bielek, P., Gonet, S.S. (Eds.), Humic Substances.

Gieguzy_nska, E., Ko_cmit, A, Gołezbiewska, D (1998), Studies on humic acids in eroded soils of Western Pomerania. In: Zaujec, A., Bielek, P., Gonet, S.S. (Eds.), Humic Substances in Ecosystems, Slovak Agricultural University, Nitra: pp. 35–41.

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Hua-Bin- li et al, ( Copyright 2011), Reactive oxygen species and antioxidants in higher plants. Antioxidant properties of Chinese medicinal plants, edited by S dutta gupta, USA, Enfield, New Hamspshire science publications, CRC press Taylor and Francis group: pp.

337.

Humate fertilizer-60-80 mesh Humic Acid Powder humine powder. Retrieved from http://humatechina.en.alibaba.com/product/ 852809057-

214117043/Humate_fertilizer_60_80_m esh_Humic_Acid_Powder_humine_po wder.html

California College of Ayurveda 1117A East Main Street, Grass Valley, California.

Rudramani Shilajit - Special formula from the shilajit plant for anti-aging and energy.

Retrived from http://www.rudramani.com/ Saileshnath saxena, B Ravishankar, Subrata De, H C Jain (1995). Silajatu Viniscaya, Department of Rasashastra and Baishajya Kalpana, IPGT&RA, Gujarat Ayurveda University, Jamnagar.

Shastri

Ambika datta, Vagbhatacharya, Rasaratnasamucchaya, Suratnojivala Hindi commentory(2010), Shilajatu shodhana prakarana.

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Shilajit

|Ayurwiki.info.

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PWRiL, Warszawa (in Polish).

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Thurman EM, Malcolm RL (1981). Preparative isolation of aquatic humic substances.

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Conflict of Interest: None Declared

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Research article MANAGEMENT OF ARBUDA (CANCER) WITH HERBOMINERAL FORMULATION - A PILOT STUDY Mahanta Vyasadeva1*, Dudhamal T S2, Gupta S K3 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 immunomodulator activities. During follow up period there was no any adverse effect of drug observed.

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

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

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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 herbomineral drugs having anticancerous, antioxidant, immuno-modulator activities.

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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 4 g 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) 2. Arogyavardhini Parada (Mercury), Gandhak (Sulphur), Louha (Iron), Tamra 250 mg (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) Parada (Mercury), Gandhak (Sulphur), Somal (White 150 mg 3. Vydadhiharan Rasa arsenic), Haratal (Yellow arsenic), Manashila (Red arsenic) , Rasakarpur (Per chloride of mercury) 600 mg 4. Guduchi satwa Guduchi (Tinospora cordifolia) 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 Sr. No. Age (Years) Male 1.

2.

3.

4.

30–40 41–50 51–60 >60

00 04 02 01

%

Female

%

Total

n-10 %

00% 40% 20% 10%

02 01 00 00

20% 10% 00% 00%

02 05 02 01

20% 50% 20% 10%

Table No.- 2; Analysis of Cancer Sites Sr. No. Sites of Cancer Male Head and neck 02 1.

Oesophagus 01 2.

Lungs 01 3.

Colon 01 4.

Urinary Bladder 02 5.

Cervix and Vagina 00 6.

Breast 00 7.

Table No.- 3 Histopathological Analysis Sr. No. Tissue / Cell type Male Squamous cell 04 1. carcinoma Adeno carcinoma 03 2.

Fibroadenoma 00 3.

Table No.- 4 Sr. No. 1.

2.

3.

4.

Addiction wise Analysis Habits Tobacco chewing Smoking Tea Alcohol

% 20% 10% 10% 10% 20% 00% 00%

Female 00 00 00 00 00 02 01

% 00% 00% 00% 00% 00% 20% 10%

n- 10 Total % 02 20% 01 10% 01 10% 01 10% 02 20% 02 20% 01 10%

% 40%

Female 02

% 20%

Total 06

30% 00%

00 01

00% 10%

03 01

n- 10 % 60% 30% 10%

% 70% 70% 70% 20%

Female 01 00 03 00

% 10% 00% 30% 00%

Total 08 07 10 02

n-10 % 80% 70% 10% 20%

Table No.- 5 Analysis of Socio-economical Status Sr. No. Social status Male % Upper 01 10% 1.

Upper middle 01 10% 2.

Lower middle 03 30% 3.

Lower 02 20% 4.

Female 00 01 02 00

% 00% 10% 20% 00%

Total 01 02 05 02

n-10 % 10% 20% 50% 20%

Male 07 07 07 02

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

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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 socioeconomically 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.

REFERENCES: Bharat B, Agarwal Anushree Kumar and Alok C. Bharti, (2003) Anticancer research, Anticancer Potential of Curcumin: Preclinical and Clinical Studies 23: 363–398

Ghanekar Bhaskar Govind, (2008) Sushruta Samhita – with Ayurveda Rahasya Deepika Vyakhya, New Delhi, Meherchand laxman Das Publications, Nidansthan, page no 176

Dikshit Rajesh, Gupta Praksh C, Chinthanie Ramasundarahettige, (2012) Cancer mortality in India: a nationally representative survey ,The Lancet, volume no 379, issue, 9828,Pages 1807–1816.

http://www.who.int/mediacentre/factsheets/fs2 97/en/, Review on cancer, WHO Report accessed on February 18, 2013 http://www.cancerfoundationofindia.org, accessed on February 28, 2013

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http://www.cancer.gov/cancertopics/wyntk/can cer/page3, accessed on February 28, 2013 http://www.wakehealth.edu/ news, wake Forest University, 2007, June 4 Research Shows Survival Benefit for Leukemia Patients Treated With Arsenic Trioxide, accessed on February 20, 2013 Kaviraj Dr. Ambikadutta Shastri, (1995) Sushruta Samhita (Purvardha), Ayurveda Tatwa Snadipaika Hindi Vyakhya, 9th edition, Varanasi, Chaukhamba Sanskrit Sansthan, Nidansthan, page no 270 Ranjith M. S., Ranjitsingh A.J.A., Shankar S. Gokul, Vijayalaksmi G. S., K. Deepa and Singh Sidhu Harcharan, (2008) Enhanced Phagocytosis and Antibody

Source of Support: Nil

Production by Tinospora cordifolia A new dimension in Immunomodulation., African Journal of Biotechnology Vol. 7 (2), pp. 081–085.

Rasatantra Sara evam Sidhiproyoga Samgraha, Srikrishna Gopal Ayurvedashram, Ajmer 1990 vol-1, page no 283.

RCG Russel, Norman S Williams, Christopher JK (2004), Bailey & Love’s Short Practice of Surgery, 24th edition, London, page no 213 Singhal G.D., Suhruta Samhita English translation, (1972) Delhi, Chaukhamba Sanskrit Pratisthan, 1st edition, Vol 3, page no 169.

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 380–385 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article IMPORTANCE OF UPAYOGASAMSTHA (DIETETIC RULES) IN RELATION TO DIGESTION OF THE FOOD Avhad Anil D1, Vyas H A2, Dwivedi R R3 1

Ph.D. Scholar, Department of Basic Principles, Institute for Post Graduate Teaching & Research in Ayurveda, Gujarat Ayurved University, Jamnagar - 361 008, Gujarat, India.

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

3 Professor& 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

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

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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 cosupportive 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 unctuous)

should be

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 quantity)

(Food

in

proper

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.

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Table 1: Relation between Upayogasamstha and digestion

iv.

Sr. no.

1

Upayogasamstha Ushnam

Digestive factors Ushma

2

Snigdham

Sneha, Kleda

3

Matravat

Vayu

4

Jeerne, Natidrutam, Nativilambitam

Kala

5

Veerya aviruddha, Ishta deshe Ishta sarvopakarane, Samayoga Tanmana bhunjit, Atmanamabhisamikshya

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).

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)

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.

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

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

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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 concentration)

(Intake

with

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.

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REFERENCES Atridev Gupt (1997), Ashtang Hriday with Vidyotini hindi commentary by, twelth edition, Chaukhamba Sanskrit Bhawan, Varanasi, Nidanasthana 12.

Michael Pollen (2006), The Omnivoreâ&#x20AC;&#x2122;s dilemma: a natural history of four meals, second edition, Penguin press, New York.

Source of Support: Nil

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.

Conflict of Interest: None Declared

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Review article CONCEPT OF VYADHIKSHAMATVA (IMMUNITY) AND ITS RELATIONSHIP WITH BALA (VITAL STRENGTH) Sharma Mahesh Kumar1* 1

Assistant 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.

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

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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 below1) 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 nonfavourable diet in the development and production of a disease is proved. (Trikamji Yadavaji, 2005)

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:

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).

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.

(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 reoccurrence 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

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.

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 (ashrayaashrayee-bhava) between rakta and pitta, mamsa and kapha and asthi and vata.

Caraka (200 B.C) quoted that all the individuals have different resistance for diseases. Vyadhikshamatva (the functional

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 doshaâ&#x20AC;&#x2122;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.

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FLOW CHART 1: Shows the relationship of Bala with other bodily factors

RASA

HEALTHY IMMUNITY

MAMSA MEDA ASTHI

SUPREME EXTRACT

OJAS (karan)

BALA

MAJJA SUKRA

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.

HEALTHY MIND

RAKTA

HEALTHY PHYSIQUE

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.

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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

Source of Support: Nil

Tripathi Brahmanand (2010) – Sharangdhar Samhita, Amatel with Dipika hindi commentary by Brahmanand tripathi, Chaukhamba Surbharti Prakashan, Varanasi.

Conflict of Interest: None Declared

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Review article A REVIEW ON VARIETIES OF ARKA - CALOTROPIS PROCERA (AITON) DRYAND. AND CALOTROPIS GIGANTEA (L.) DRYAND. Poonam1*, Gaurav Punia2 1

Assistant Professor, Gaur Brahmin Ayurvedic College, Rohtak., India HCDS Dental Surgeon, Khanda kheri, Hisar. *Corresponding Author: E-mail: drpoonamkaleramana@gmail.com, drpoonamkaleramana@gmail.com; Mob: +918930123462, +9198930123430 2

dr_pounia@yahoo.com,

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.

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): 392â&#x20AC;&#x201C;400

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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 mercurylike 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.

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 RaviPushya 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.

The Calotropis Shrub that produces white or sometimes voilet flowers is called as 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.

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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 softwooded, 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, shortpointed 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–

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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

India Calotropis gigantea/ Gigantic swallow wort, Madar/Shveta Arka Calotropis procera/Rakta Arka Swallowwort/Rakta Arka

India

Flower

Petiole

Fruits

8–10 m

Opposite White to purple, Sessile rarely light green yellow or white. Flowers not scented

Follicles recurved, 2 or 1 follicles, second more often suppressed, 3–4" long

3–6 m

Opposite White to pink, Subscented. sessile

Follicles recurved

The photosynthetic capacity of C. procera is higher than that of C. gigantea (Wilmer Tezara et al., 2011).

Properties of both arka according Bhavprakash (Chunekar K.C., 2007)

to

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).

3–4",

Chemical compostionCalotropis 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

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the roots of Calotropis procera yields two new phytoconstituents, procerursenyl acetate and proceranol, together with the known compounds N-dotriacont-6-ene, glyceryl monooleolyl-2-phosphate, methyl myrisate, methyl behenate and glyceryl-1, 2-dicapriate-3phosphate. 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-Dglucopyranosyl frugoside were obtained as the cytotoxic principles (Kiuchi, F., 1998).

Gigantin is a chemical extracted only from C. gigantea.

Rasa Panchaka : Rasa Katu, Tikta Laghu, Ruksha, Tikshna Guna Ushna Virya Katu Vipaka 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 hepatoprotective results. Proteins present in the latex of C. gigantea are strongly proteolytic and

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

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392â&#x20AC;&#x201C;400

responsible for procoagulant activity of C. gigantea (Rajesh R. 2005).

Leaves: In rheumatism, as an antiinflammatory 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 antiinflammatory, 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 antiHIV-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). Antipyretic 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 antiinflammatory property of the latex of Rakta Arka was studied on carrageenin- and formalininduced 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 worldâ&#x20AC;&#x2122;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 Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 5 | May 2013 | 392–400

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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Conflict of Interest: None Declared

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