GJRMI - Volume 1, Issue 9, September 2012

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INDEX MEDICINAL PLANT RESEARCH Bio-chemistry APHRODISIAC STUDIES OF DIHERBAL MIXTURE OF ZANTHOXYLUM LEPRIEURII GUILL. & PERR. AND PIPER GUINEENSE SCHUMACH. & THONN. ON MALE WISTAR RATS Kpomah E D, Uwakwe A A, Abbey B W……………………………………………………..381–390 Veterinary Medicine EFFICACY OF PHYTO MEDICINES AS SUPPLEMENT IN FEEDING PRACTICES ON RUMINANT’S PERFORMANCE: A REVIEW Mirzaei F, Hari Venkatesh K R………………………………………………………………..391–403 Pharmacology A COMPREHENSIVE REVIEW ON SPHAERANTHUS INDICUS LINN. Pawar Harshal A, Therani Deepika……………………………………………………………404–410 Nature & Life sciences ETHNOBOTANICAL STUDY OF THERAPEUTIC PLANTS USED TO TREAT ARTERIAL HYPERTENSION IN THE HODNA REGION OF ALGERIA SARI Madani, SARRI Djamel, HENDEL Noui and BOUDJELAL Amel…………………...411–417 Bio-technology ISOLATION AND PARTIAL CHARACTERIZATION OF UREASE INHIBITOR FROM AGAVE SPECIES Latha K, Manasa C……………………………………………………………………………418–426 Biotechnology EFFICACY OF AYURVEDIC / HERBAL PATENT MEDICINES IN TYPE 2 DIABETES MELLITUS AS PER THE CLAIM Mishra Subhransu Sekhar, Mishra Amarendra Narayan……………………………………...427–439

INDIGENOUS MEDICINE Ayurveda ANTIVIRAL ACTIVITY OF LATHAKARANJA (CAESALPINIA CRISTA L.) CRUDE EXTRACTS ON SELECTED ANIMAL VIRUSES Patil Usha, Sharma M C……………………………………………………………………….440–447 A COMPARATIVE DERMAL TOXICITY EVALUATION OF RASAKARPURA AND MERCURIC CHLORIDE IN RATS Mehta N J, Ashok B K, Ravishankar B, Prajapati P K………………………………………..448–456 ACTION OF SHIRODHARA – A HYPOTHETICAL REVIEW Ajanal Manjunath, Chougale Arun……………………………………………………………457–463

HARAMEKHALA – A MASTERPIECE ON INDIAN COSMETICS Archana I, Praveen, Vyas Mahesh, Bhat Jeddu Ganapathi………………………………….464–469 Ayurveda & Siddha COMPARATIVE ANALYSIS OF AYURVEDIC AND SIDDHA SYSTEM OF INDIAN MEDICINE Panja Asit Kumar, Gahunge Pankaj, Chattopadhyaya Abichal……………………………..470–476

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FLOWERS OF SHWETHA DHATAKI, GETONIA FLORIBUNDA, [SYN: CALYCOPTERIS FLORIBUNDA (ROXB) LAM EX.POIR] COMBRETACEAE

PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 381–390 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article APHRODISIAC STUDIES OF DIHERBAL MIXTURE OF ZANTHOXYLUM LEPRIEURII GUILL. & PERR. AND PIPER GUINEENSE SCHUMACH. & THONN. ON MALE WISTAR RATS Kpomah E D1*, Uwakwe A A2, Abbey B W3 1, 2, 3

*

Department of Biochemistry, University of Portharcourt, Rivers State, Nigeria.

Corresponding Author: Ph: +2348034173608; E-mail: denniskpomah@yahoo.com

Received: 09/08/2012; Revised: 24/08/2012; Accepted: 29/08/2012

ABSTRACT The effects of 50, 100 and 150 mg/kg body weight of ethanol extract of diherbal mixture of Zanthoxylum leprieurii and Piper guineense on aphrodisiac potentials and hormonal levels was investigated. A total of twenty healthy, sexually experienced Albino male rats (Rattus norvergicus), weighing between 190–230g were randomly divided into four groups (A–D) of five rats each, group A were orally administered once daily with 1 ml of distilled water (vehicle), while groups B, C and D received 50, 100 and 150 mg/kg body weight of the extract in 1ml of the vehicle. After 21 days of treatment the animals were studied for mating behaviour and hormonal assay, the mating behaviour monitoring showed that mount latency, intromission latency and post ejaculatory intervals were significantly decreased (P < 0.05), mount frequency, intromission frequency, ejaculatory frequency and ejaculatory latency were all significantly increased (P < 0.05), however, only the 150 mg/kg body weight dosage produced significant increase in copulatory efficiency. Hormonal assay showed that all doses produced significant increase (P < 0.05) in testosterone, luteinizing hormone and follicle stimulating hormone, similarly all doses produced significant increase (P < 0.05) in progesterone except 50 mg/kg, prolactin was decreased significantly (P < 0.05) by all dose level. The effects of these extracts on the mating and hormonal studies were dose dependent and the findings validated the acclaimed use of this herbal product as an aphrodisiac in men.

KEYWORDS: Aphrodisiac, Diherbal mixture, Zanthoxylum leprieurii and Piper guineense

Cite this Article Kpomah E D, Uwakwe A A, Abbey B W (2012), Aphrodisiac studies of diherbal mixture of Zanthoxylum leprieurii Guill. & Perr. and Piper guineense Schumach. & Thonn. on Male wistar rats, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 381–390

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

Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 381–390

INTRODUCTION The incidence of male sexual dysfunction is on the increase globally (Kaiser, 1999). It is reported as having a prevalent rate of 10% across all ages (Lauman et al., 1999), and because sexual dysfunction is an inevitable process of aging, the prevalence is over 50% in men between 50 and 70 years of age (Rendell et al., 1999).

Male sexual dysfunction is classified as a disorder of desire that is persistent absence of sexual fantasy and desire for sexual activity (Kandeel et al., 2001), erectile dysfunction that is persistent inability to develop and maintain a penile erection that is for intercourse and ejaculation in 50% or more of attempts (Kandeel et al., 2001), disorder of ejaculation that is problem with expulsion of semen at the climax of the sexual act, disorder of orgasm that is delay in or absence of orgasm after a normal sexual excitement phase during sexual activity (Rosen and Lieblum, 1995), failure to detumescence that is prolonged (> 4 hours duration) and extreme painful erection unaccompanied by sexual desire (Kandeel et al., 2001).

The causes of male sexual dysfunction include factors like psychological disorders (performance anxiety, strained relationships, depression, stress, guilt, fear of sexual failure), hormonal condition (androgen deficiency, hyperprolactinemia), chronic medical conditions (diabetes, hypertension), penile disease (priapism, smooth muscle dysfunction), neurological disorder (Parkinson disease, stroke, cerebral trauma, alzheimer’s disease, spinal cord or nerve injury), drug side effect (antihypertensive, psychiatric medications, antiulcer and antidepressants), lifestyle (chronic alcohol abuse, cigarette smoking), aging and systemic disease (cardiac, hepatic, renal, pulmonary, metabolic), post organ transplant (Kandeel et al., 2001; Guay et al., 2003; Yakubu et al., 2007).

Despite the medical advancements in treatment and treatment facilities for male sexual dysfunction (Lim et al., 2005), most

sufferers often shy away from these treatment options and these could be attributed to its sensitivity and social stigma attached to male sexual dysfunction in the African context (Lim et al., 2005), these treatment options are equally very expensive, not easily accessible to the poor and rural dwellers and are often associated with some serious side effects, consequently medicinal plants with marked pharmacological activities are readily available all year round, cheap and accessible and often with minimal side effects (Tilburt and Kaptchuk, 2008; Ufelle et al., 2011; Yadav and Agarwala, 2011), and are being explored globally as panacea. All through history many preparation from plants have been used and reputed to have sex invigorating (aphrodisiac) properties and these include Yohimbine, Gingseng, Massularia acuminata, Montanoa tomentosa (Ang et al., 1997; Baljinder et al., 2010; Yakubu and Akanji, 2011).

The composite extract of the bark of Zanthoxylum leprieurii Guill. & Perr. and the seeds of Piper guineense Schumach. & Thonn. are two of such sex tonics that is largely used by the people of Niger Delta region of Nigeria, but with no scientific footing. Z. leprieurii belong to the family Rutaceae and locally called Prickly ash or toothache tree, it is an aromatic, spiny, thicket forming deciduous shrub or tree. The alternate branches are armed with strong brown prickles about 2–3 cm long, cone shaped with a broad base and found irregularly throughout the tree (Todd, 2008).

Ethnomedically, it is used in the treatment and management of muscle spasm, varicose vein, raynauld disease, arthritis, rheumatism, neuraligia, flu, fever, toothache and gum diseases (Tilloston, 2011). P. guineense commonly called climbing pepper is a slender climber up to 12 m high with prominent nodes and clasping roots, the leaves are elliptic in shape about 15 cm long and 7 cm broad, the flowers are small, borne on common stalk as cluster opposite to the leaves. The fruits are red and turns black when dry (Iwu, 1988).

Traditionally the seeds are used as spices and as sex invigorator in men.

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 381–390

The present study was therefore undertaken to evaluate the aphrodisiac property of the composite ethanol extract of the stem bark of Zanthoxylum leprieurii and the seeds of Piper guineense using a dose level of 50, 100 and 150 mg/kg body weight with a view to validate the acclaimed use of this sexual invigorator.

MATERIALS AND METHODS The plant samples were bought in a local herb market in Warri, Delta State, Nigeria.

Both the species were identified and confirmed at the Herbarium of the Department of Plant Science and Biotechnology of the University of Portharcourt, Rivers State.

Preparation of Plant Extract The bark Z. leprieurii and the seeds of P. guineense were thoroughly washed with distilled water to remove debris and contaminants, they were then dried in an oven at 40℃ until a constant weight was reached, and then pulverized using an electric blender (Blender, 462 Nakai Japan). 200 g of the powdered mixture (i.e 100 g each of Z. leprieurii and P. guineense) was extracted in 600 ml of absolute ethanol for 24 h at room temperature with constant shaking using a flask shaker (Model, Denly A - 500). The extract was filtered with Whatman No. 1 filter paper and the resulting filtrate was evaporated to dryness using a rotatory evaporator at 40℃ to give 5.74 g, the resultant concentrate was reconstituted in distilled water to give the required doses used in the study.

Experimental Animals A total of twenty healthy sexually experienced Albino male rats (Rattus norvergicus), 2.5–3 months old, weighing between 190–230 g and thirty female rats between 2–2.5 months old and weighing between 150–180 g were obtained from the animal house unit of the Department of Biochemistry, University of Portharcourt, Rivers State. The animals were kept in a clean cage and housed in a well ventilated room at temperature 28–30℃ under natural light and

dark cycle with free access to grower’s mash and water.

Experimental Design The twenty male rats were randomly divided into four groups (A–D), consisting of five rats each. Group A were orally administered once daily with 1 ml of distilled water (vehicle), groups B, C, and D were orally administered with 50, 100 and 150 mg/kg body weight of the extract in 1 ml of the vehicle for 21 days.

Mating Behaviour Test Procedure The investigation was carried out on the 22nd day after the commencement of extract administration by adopting the methods described by (Gauthaman et al., 2002; Yakubu and Akanji, 2011). The investigation was conducted between 19 and 22 h in the same laboratory and under a dim light. The female rats were artificially brought to oestrus by administering estradiol benzoate 10 µg/100 g body weight orally 48 h prior to mating and progesterone injected subcutaneously at a dose of 0.5 mg/100 g 6 h prior to the mating (Szectman et al., 1991; Yakubu et al., 2005).

The artificially warmed female was introduced to the cage of the male. The male and female were observed from the cage side for proceptive, precopulatory and copulatory behaviours. The occurrence of events and phases of mating was monitored for 30 min observatory period and the following male sexual behavior indices recorded/calculated.

Mount latency: the time interval between the introduction of the female to the first mount by the male; Mount frequency: the number of times the male assumed copulatory position but failed to achieve intromission; Intromission latency: the time interval from the introduction of the female until the first intromission of the female (vaginal penetration) by the male; Intromission frequency: the number of intromissions (vaginal penetration) made by the male from the first time of introduction of the female; Ejaculatory latency: the time interval between the first intromission and ejaculation, it is usually characterized by longer, deeper

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pelvic thrusting and slow dismount, followed by a period of reduced activity; Ejaculation frequency: The number of times there was expulsion of semen by the males after vaginal penetration characterized by rhythmic contraction of the posterior abdomen; Post ejaculatory interval (PEI): the time interval from ejaculation to intromission of the next mating series; Copulatory efficiency: number(s) of intromission divide by number(s) of mount multiplied by 100 %. (Yakubu et al., 2005, and Yakubu and Akanji, 2011).

Method of Collection and Handling of Serum At the end of the treatment period, the animals were anaesthetized in a chloroform chamber and blood samples collected from the jugular vein into sample bottles, the blood samples were allowed to clot for 10 min at room temperature and subsequently centrifuged to obtain serum for hormonal assay.

Assay Kits Testosterone, follicle stimulating hormone (FSH), luteinizing hormone, prolactin and progesterone radioimmunoassay test kits are products of BYK-Sangtic Diagnostica, GmbH and Co. KG, while estradiol benzoate and progesterone are products of Sigma Chemicals, St. Louis, USA and Shalina Laboratories, Mumbai, India, respectively.

Hormonal Assay Serum samples were assayed for the following hormones testosterone, follicle stimulating hormone, luteinizing hormone, prolactin and progesterone by using the procedure described by BYK-Sangtic Diagnostica. This was based on the principle of radioimmunoassay of competitive binding between the sample serum and the standards for a constant amount of the antisera. (Tietz, 1995).

Statistical Analysis The results were expressed as the mean of five replicates Âą standard deviation (S.D),

means were analysed using one way analysis of variance (ANOVA) followed by Posthoc (Turkey). P < 0.05 was regarded as significant.

The Statistical Package for Social Sciences (SPSS) Computer software version 16 was used for data analysis.

RESULTS Effects of the Composite Mixture of Z. leprieurii and P. guineense on Mating Behaviour Upon the introduction of the female to the male cage, the male responded with immediate advances towards the female and displayed precopulatory behaviours such as chasing, anogenital sniffing which eventually culminated into mounting, the female also displayed receptivity that allowed mating to occur. The ethanol extract of the diherbal mixture of Z. leprieurii and P. guineense after 21 days of treatment at a dosage of 50, 100 and 150 mg/kg body weight was able to influence significantly (P < 0.05) on the mount latency, intromission latency, mount frequency, intromission frequency, ejaculatory latency, ejaculatory frequency and post ejaculatory interval, however, only the 150 mg/kg body weight of the extract had significant effect (P < 0.05) on the copulatory efficiency (Table 1.0).

Effects of the Composite Mixture of Z. leprieurii and P. guineense on Hormonal Levels.

The ethanol extract of the composite mixture of Z. leprieurii and P. guineense after 21 days of treatment at 50, 100 and 150 mg/kg body weight dose was able to significantly increase (P < 0.05) the levels of testosterone, luteinizing hormone and follicle stimulating hormone, however, only 100 and 150 mg/kg body weight was able to significantly (P < 0.05) increase progesterone. All doses of the extract studied were also able to significantly decrease (P < 0.05) the level of prolactin (Table 2.0).

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 381–390

Table 1.0 Effect of 50, 100 and 150 mg/kg Body Weight of Ethanol Extract of Composite of Zanthoxylum leprieurii and Piper guineense on Mating Behaviour Parameters.

S/N

1.

2.

MATING PARAMETERS Mount Latency (Seconds) Intromission Latency (Seconds)

C

D

100 mg/kg

150 mg/kg

B.W

B.W

813.00 ± 4.95 a 746.00 ± 6.67 b

610.00 ± 7.21 c

493.00 ± 9.76 d

820.00 ± 7.91 a 750.00 ± 6.63 b

618.00 ± 5.70 c

499.00 ± 8.63 d

A CONTROL

B 50 mg/kg B.W

3.

Mount Frequency

5.20 ± 0.84 a

10.40 ± 2.07 b

14.20 ± 1.92 c

18.80 ± 1.48 d

4.

Intromission frequency

3.00 ± 0.71 a

6.80 ± 1.64 b

9.60 ± 1.52 c

13.00 ± 1.58 d

5.

6.

7.

8.

Ejaculatory Latency (Seconds)

234.00 ± 6.52 a 274.00 ± 9.19 b 310.00 ± 11.81c 409.00 ± 8.54 d 1.00 ± 0.00 a

3.20 ± 0.45 c

4.20 ± 0.84 d

400.00 ± 7.91 a 353.40 ± 8.44 b

308.60 ± 6.84 c

241.20 ± 7.01 d

57.33 ± 7.23 a

67.49 ± 2.46 a

69.00 ± 360 b

Ejaculatory Frequency Post Ejaculatory Interval (Seconds) Copulatory Efficiency (%)

2.00 ± 0.00 b

65.01 ± 3.70 a

Values are means of five replicates ± standard deviation, values with different superscript letters b, c and d are significantly different (P < 0.05) from the control ‘a’. BW: body weight.

Table 2.0 Effect of 50, 100 and 150 mg/kg Body Weight of Ethanol Extract of Composite of Z. leprieurii and P. guineense on Hormonal Levels.

S/N

A HORMONE

CONTROL

B

C

D

50 mg/kg

100 mg/kg

150 mg/kg

B.W

B.W

B.W

3.10 ± 0.16 a 4.22 ± 0.19 b 7.94 ± 0.21 c 10.62 ± 0.22 d

1.

Testosterone (nmol/L)

2.

Luteinizing hormone (IU/L)

2.86 ± 0.19 a 3.64 ± 0.38 b 4.98 ± 0.29 c

5.30 ± 0.31 a

3.

Follicle stimulating hormone

2.92 ± 0.23 a 4.12 ± 0.33 b 4.98 ± 0.15 c

6.08 ± 0.28 d

(IU/L) 4.

Progesterone (nmol/L)

0.98 ± 0.24 a 1.34 ± 0.24 a 1.44 ± 0.18 b

1.64 ± 0.18 c

5.

Prolactin (ng/ml)

6.82 ± 0.37 a 6.14 ± 0.32 b 5.60 ± 0.46 c

5.04 ± 0.19 d

Values are means of five replicates ± standard deviation, values with different superscript letters b, c and d are significantly different (P<0.05) from the control ‘a’. B.W: body weight.

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DISCUSSION Aphrodisiacs can be defined as substances which are ingested, applied topically, smoked/snorted or otherwise delivered into the body to induce sexual arousal, heighten sexual experience and to improve sexual performance.

History had it that many preparations from plants and animals have been used for this purpose e.g. Yohimbine, the mandrake plant, Gingseng, ground rhinocerous horn, sheep and bull testicle and the Spanish fly (Ang et al.,1997; Baljinder et al., 2010). This present study was therefore designed to investigate the aphrodisiac properties of the composite mixture of Zanthoxylum leprieurii and Piper guineense.

Mount latency and intromission latency are indices of sexual motivation and there is an inverse relationship between mount latency, intromission latency and sexual motivation (Yakubu and Akanji, 2011). The extract was able to significantly decrease (P < 0.05) the mount latency and intromission latency and this might imply stimulation of sexual appetite and arousal, thus lending credence to the sexual improving effect of the extract under study just like other aphrodisiac plants that have been studied e.g. Lepidium myenii (Cicero et al., 2001), Montanoa tomentosa (Carro-Juarez et al., 2004) and Microdemis kenyana (Zamble et al., 2008).

Mount and intromission frequencies are indicators of libido, sexual vigour, strength, power and energy (Yakubu et al., 2005). The extract was able to significantly increase (P < 0.05) the Mount and intromission frequencies, thus supporting the sexual improving effect of the extract as in the case of other aphrodisiac plants that have been investigated e.g. Triholepis glaberrima (Padashetty and Mishra, 2007), Mucuna pruriens (Amin et al., 1994) and Terminalia catappa (Ratnasooriya and Dharmasiri, 2000).

Increased intromission frequency is also a function of erection efficiency, penile orientation and the ease by which ejaculatory reflexes are activated (Agmo, 1997; Yakubu and Akanji, 2011).

Ejaculatory latency and ejaculatory frequency are pointers of enhanced copulatory performance, the extract was also able to significantly increase ejaculatory latency and ejaculatory frequency (P < 0.05). Ejaculatory latency also implies prolonged coitus duration which translates into increased staying power, strength and vigour thus validating its aphrodisiac properties just other aphrodisiac plants that have been investigated e.g. Vanda tsellata (Suresh et al., 2000), Dactylorhiza hatagirea (Thakur and Dixit, 2007) and Mondia whitel (Watcho et al., 2007).

Post ejaculatory interval is a positive marker of sexual potency, libido and a fast pace of recovery from exhaustion after the first series of mating, all doses of the extract significantly decreased the post ejaculatory interval (P < 0.05) and compared favourably with other aphrodisiac plants that have been studied e.g. Chlorophytum boriviliaunum (Thakar and Dixit, 2006) and Syzygium aromaticum (Tajuddin et al., 2004).

Copulatory efficiency is an indication that the copulatory action of the male was well enhanced with well coordinated pelvic thrusting and this further indicates sustained increase in interest, focus, agility and stamina in the sexual act. Only the 150 mg/kg body weight of the extract was able to increase the copulatory efficiency significantly (P < 0.05).

Thus lending credence its aphrodisiac potency which compares to other aphrodisiac plants that have been studied e.g. Alpinia calcarata (Ratnasooriya and Tayakody, 2006) and Withania somnifera (Illayperuma et al., 2002).

The precopulatory and copulatory behaviours of the extract treated rats showed that the rats were extremely aroused and the effects of the extract on the mating behaviour were dose dependent with the 150 mg/kg body weight been more potent.

Sexual behaviour and erection are largely dependent on androgen which may act through central and peripheral mechanism (Mill et al.,

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1996; Yakubu et al., 2011). All doses of the extract significantly increased (P < 0.05) testosterone, luteinizing hormone and follicle stimulating hormone. Luteinizing hormone and follicle stimulating hormone are produced by the anterior pituitary lobe and are needed for maintaining testosterone levels, hence an increase in luteinizing hormone and follicle stimulating hormone automatically triggers an increase in testosterone levels (Yakubu et al., 2007). Studies have shown that testosterone supplementation helps to improve sexual function and libido (Aversa and Fabiri, 2001; Grahl et al., 2007), in addition to the intensity of orgasm and ejaculation (Morales, 1996). All doses of the extract except 50mg/kg body weight was able to increase the progesterone level significantly (P < 0.05), optimal level of progesterone have associated with improved sexual function (Andersen and Tufik, 2006; Andersen et al., 2007). High levels of prolactin in men (hyperprolactinemia) have been associated with hypogonadism, decreased sperm count and motility, erectile dysfunction and decreased libido (Kruger et al., 2003; Paick

et al., 2006), all doses of the extract was able to decrease significantly the levels prolactin in a manner that was dose dependent.

The observed improvement in mating behaviour seen between the control and the extract treated groups may be due to plant chemicals present in the extract (alkaloid, saponin, tannin, flavonoid, sterols), these plant chemicals are able to exert their effect through elevation of androgens and gonadotropins, vasodilation and generation of nitric oxide (Yakubu and Akanji, 2011), which are key factors in the initiation and sustenance of erection, libido and other sexual factors.

CONCLUSION The systemic use of the ethanol extract of Zanthoxylum leprieurii and Piper guineense have marked enhancement on mating behaviour parameters and the sex hormones of male rats, thus corroborating the acclaimed use of this product as an aphrodisiac by the people of the Niger Delta region of Nigeria.

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Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 391–403 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article EFFICACY OF PHYTO MEDICINES AS SUPPLEMENT IN FEEDING PRACTICES ON RUMINANT’S PERFORMANCE: A REVIEW Mirzaei F1*, Hari Venkatesh K R2 1

Seniour Scientist, Department of Livestock Production and Management, Animal Science Research Institute of Iran, Karaj, 31585, Iran.

2

Lecturer/ Scientist Incharge - Animal House, A.L.N.Rao Memorial Ayurvedic Medical College, Koppa - 577126, Chikkamagalur District, Karnataka, India

*

Corresponding Author: E-mail address: farmir2003203@yahoo.com

Received: 10/07/2012; Revised: 11/08/2012; Accepted: 20/08/2012

ABSTRACT This article summarizes the experimental knowledge on efficacy, possible modes of action, and aspects of application of phyto medicine as feed additives for ruminant. Phytogenic feed additives comprise a wide variety of herbs, spices, and products derived thereof, and are mainly essential oils. The assumption that phyto medicine compounds might improve the palatability of feed has not yet been confirmed by choice-feeding studies. In total, available evidence indicates that phytogenic feed additives may add to the set of non-antibiotic growth promoters for use in livestock, such as organic acids and probiotics. However, a systematic approach toward the efficacy and safety of phytogenic compounds used as feed additives for livestock is still missing.

KEYWORDS: Phyto medicine, ruminant, feed additive

Cite this article: Mirzaei F and Hari Venkatesh K R (2012), Efficacy of phyto medicines as supplement in feeding practices on ruminant’s performance: A review, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 391–403

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1. INTRODUCTION Phytogenic feed additives are plant-derived products used in animal feeding to improve the performance of agricultural livestock. This class of feed additives has recently gained increasing interest, especially for use in livestock sector. This appears to be strongly driven by the ban on most of the antibiotic feed additives within the European Union in 1999, a complete ban enforced in 2006, and ongoing discussions to restrict their use outside the European Union because of speculated risk for generating antibiotic resistance in pathogenic microbiota. In this context, phytogenic feed additives are discussed possibly to add to the set of non-antibiotic growth promoters, such as organic acids and probiotics, which are already well established in animal nutrition. Phytogenics, however, are a relatively new class of feed additives and our knowledge is still rather limited regarding their modes of action and aspects of their application. Further complications arise because phytogenic feed additives may vary widely with respect to botanical origin, processing, and composition.

Most studies investigate blends of various active compounds and report the effects on production performance rather than the physiological impacts. In this context, the following provides an overview of recent knowledge on the use of phytogenic feed additives in piglet and poultry diets, possible modes of action, and safety implications.

Phytogenic feed additives (often also called phytobiotics or botanicals) are commonly defined as plant-derived compounds incorporated into diets to improve the productivity of livestock through amelioration of feed properties, promotion of the animals’ production performance, and improving the quality of food derived from those animals.

(Windisch et al., 2008). The use of feed additives is usually subject to restrictive regulations. In general, they are considered as products applied by the farmer to healthy animals for a nutritional purpose on a permanent basis (i.e., during the entire production period of the respective species and category), in contrast to veterinary drugs (applied for prophylaxis and therapy of diagnosed health problems under veterinarian control for a limited time period, partially associated with a waiting period). In the European Union, for example, feed additives need to demonstrate the

identity and traceability of the entire commercial product, the efficacy of the claimed nutritional effects, including the absence of possible interactions with other feed additives, and the safety to the animal (e.g., tolerance), to the user (e.g., farmer, worker in feed mills), to the consumer of animal-derived products, and to the environment (EU commission, 2003) A knowledge of the chemical constituents of plants is desirable, not only for the discovery of therapeutic agents, but also because such information may be of value in disclosing new sources of such economic materials as tannins, oils, gums, precursors for the synthesis of complex chemical substances, etc.

Therefore keeping in mind aforesaid constraints and possible benefits, present study was planned with the following objectives: To study the effect of herbal supplementation on feed intake and feed conversion efficiency of ruminant production management.

To study the effect of medicinal plants supplementation on production performance of ruminant production management.

2. Effect of polyherbal supplementation on: 2.1. Milk production Leptaden® tablet containing Shatavari (Asparagus racemosus Willd.) as a major component enhances milk production significantly in buffaloes and cows but not in goat and sheep (Anjaria and Gupta, 1967). Result happened in crossbred cows (Chauhan et al., 1971). Supplementation of ‘Galog®’ an herbal preparation having Shatavari, enhanced milk production and persistency throughout lactation in the treatment group. Similarly, supplementation of Shatavari based herbal formulation Payapro® enhanced milk production (Arora et al., 1978). The increase in milk production was on an average 30.9%. It has been concluded that herbal preparation showed galactopoietic activity and can be considered as an alternative for lactogenic hormones for inducing and enhancing milk yield in crossbred cows (Singhal, 1995). Supplementation of ‘Galactin® (50 g

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/d/animal), a Shatavari based polyherbal galactogogue, in lactating crossbred cows had improvement in milk production over control group (Ramesh et al., 2000).

A. racemosus in combination with other herbal substances in the form of 'Ricalex®' tablets (Aphali pharmaceutical Ltd. Ahmednagar) has been shown to increase milk production in females complaining of deficient milk secretion.

Gradual decrease in milk secretion, on withdrawal of the drug suggested that the increase in milk secretion was due to drug therapy only and not due to any psychological effect (Joglekar et al., 1967).

Galactogogue effect of roots of A. racemosus has been shown in buffaloes (Patel and Kanitkar, 1969). However, no increase in prolactin levels in females complaining of secondary lactational failure with A. racemosus suggesting that it has no lactogenic effect (Sharma et al., 1996).

It was assessed the effect of some herbal biostimulators of Galog®, Hbstrong® and Livol® (trade marks of polyherbal medicines in India) in optimizing ruminal digestion and milk production and also their effect on various biochemical constituents in dairy cows. There was significant increase in milk yield in the three treatment groups with maximal increase being recorded in Galog® group followed by Hbstrong® and Livol® groups as compared to control group. The average milk yield in all the treatment groups even at the termination of the experimental study was still higher than the base value. Further, the milk yield declined in control group by 18.65 percent with the progress in the process of lactation and in normal lactation curve, whereas the treated groups showed steady increase in milk yield. Results showed gross over activity of rumen microflora particularly of the predominance of cellulolytic bacteria with increased total volatile fatty acids and normal pH of rumen liquor. There was marked increase in protozoal concentration and motility in rumen liquor due to herbal feed additives especially Hbstrong® which helped better digestion of the cellulose matter of the feed and thus renders energy supply for milk production followed by Livol® and Galog®. There was also increase in molar proportion of propionic acid which is glucogenic. In the rumen there was faster

rate of ammonia metabolism into microbial proteins (Randhawa et al., 1995).

The efficacy of the herbal galactagogue Payapro® in lactating buffaloes was conducted at an organised farm. Ten healthy Murrah buffaloes, calved 35–74 days earlier were fed Payapro® at the rate of 4 bolus daily for 16 days. The effect of increased milk yield was manifested within 7 days of treatment, reached its maximum level at day 21 and remained above pre-treated level till day 35.

However, the beneficial effect in arresting the decline in milk yield vis-à-vis untreated animals was seen till the last observation at day 49. The cumulative gain in milk yield in treated animals over the control animals during the 7 weeks observation worked out to 35.5 Kg per animal. The economics of medication was highly favourable. The influence of the Payapro® was applied in twenty five crossbred cows, calved 60–89 days earlier were divided into 2 groups viz., treated (15 animals) and control group (10 animals), near identical in terms of body weight., calving and lactation status. The animals in treated group were administered Payapro® at the rate of 4 bolus per animal daily along with feed consecutively for 15 days while the control animals were given feed without Payapro®. The daily milk yield was recorded at the start of trial and thereafter for each animal in both the groups for 49 days. The result indicated that Payapro® administered in the declining phase of lactation resulted in both increased milk yield compared to pre-treatment levels and persistence of lactation levels compared to the control groups.

This was evident by extra milk yield of 30.4 l/ animal over a Seven weeks period in treated animals along with sustenance of increased milk yield for as long as 7 weeks. The treatment was also found highly cost effective with a cost benefit ratio of 1 : 3 (Khurana et al., 1996).

2.2. Colostrum and milk composition Goat milk has less fat, higher vitamin A content, less lactose, tuberculosis virus resistant than cow’s milk. The reaction of goat milk is alkaline, the same as Mother’s milk. Cow milk produces an acid reaction. Normal milk from high producing Holstein or Friesian dairy cows is composed of water (87%), fat (3.8%), protein (3.4%), sugars

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(i.e., lactose, 4.5%) and other solids such as minerals (1.3%). Milk also contains a number of minor components including sloughed epithelial cells and white blood cells. High quality milk should be white in appearance, have no objectionable odors and be free of abnormal substances such as pesticides, added water or antibiotic and antiseptic residues. In most developed dairy countries milk quality is defined by the somatic cell count and the bacterial count (standard plate count) in pre-pasteurized bulk tank milk. Somatic cells are composed of white blood cells and occasional sloughed epithelial cells. Most cells found in normal bovine milk are a type of white blood cells (macrophages) that function as early warning signals when bacteria invade the udder.

Effect of 10 herbal feed supplementations was studied on the performance of lactating cows. Results showed that the herbal feed supplement did not have any significant effect on dry matter intake, digestibility of nutrition, live weight changes, whereas milk and fat corrected milk yield was improved without affecting its composition. It was concluded that dietary supplementation of a commercial herbal feed additive 10 g/d to lactating crossbred cow, increased the milk yield (Thakur et al., 2006). Supplementing of lactating goatâ&#x20AC;&#x2122;s diet with fenugreek and Nigella sativa seeds galactogogue on milk yield, composition and its effect on Domiati cheese have been studied, fenugreek increased milk yield and total nitrogen soluble nitrogen and salt contents. The acidity tyrosine and tryptophan in cheese increased significantly compared with the control.

However, a decrease in total solids, fat and fat/total solids and total nitrogen/soluble nitrogen was also observed in cheese over the control.

However, N. sativa decreased total solids, fat and total nitrogen soluble nitrogen and pH of milk.

The organoleptic properties of treated cheese were better than the control, except in colour, storage period significantly affected organoleptic properties and cheese composition. It is concluded that fenugreek was economically better than the other groups (Kholif et al., 2001).

2.3. Somatic cell and immunoglobulin G concentrations in milk The white blood cells in milk, together with a relatively small number of epithelial cells from milk-secreting tissues, are known as somatic cells.

These cells are an important part of the goat's natural defence mechanism. When udder tissue is injured or becomes infected, significant numbers of white blood cells accumulate in the milk. Normal goat milk has a higher cell count than normal milk from cows. This has long been a concern of goat owners because of regulatory standards and marketing problems. Current Grade A standards require that milk contain no more than 4,000,000 cells/ml. Despite this reduction for cow milk, regulatory standards for goat milk will remain at 1,000,000/ml. This is because somatic cell counts in goat milk may easily approach 750,000/ml and still be normal.

The influence of different somatic cell count on the average daily milk production, composition and some properties of goat milk were evaluated.

110 lactating dairy goats (Bulgarian Dairy White) were investigated during the lactation period. No statistical differences were found in the average daily milk production at 3.2% fat corrected milk and at 3.0% protein corrected milk between the number of somatic cell count less than 400,000/ml (275,000/ml), SCC from 400,000 to 1,000,000/ml (652,000/ml) and somatic cell count over 1,000,000/ml (3,150,000/ml), (Petrova et al., 2008).

The synergistic effect of polyherbal immunomodulator along with the antibiotic therapy was assessed in the treatment of subclinical mastitis in cows. The immuno-modulatory effect was evaluated based on the increase in absolute lymphocyte count and immunoglobulin G level along with clinical recovery. The curative effect was assessed based on the increase in milk yield and reduction in somatic cell count below 0.5 million cells/ml.

Significant increases in absolute lymphocyte count and immunoglobulin G were observed in individual cows treated with polyherbal immunomodulators alone and in antibiotic combination.

The immuno-modulatory effect of the herbal immuno-modulator was at par with that of levami-

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sole. Polyherbal immuno-modulator alone was found to be effective in 60% of subclinical mastitis cases and it was effective in 100% of the cases when used with antibiotics. Recently, the efficacy of a commercial herbal immuno-stimulant product (ImmuPlus®) for prevention and effective treatment of clinical mastitis in bovines was evaluated. The different groups of cows suffering from clinical mastitis and those which were more vulnerable to this disease were administered Immu-21® alone and in combination with antibiotics, to assess their comparative clinical and immunological benefits. When animals affected with clinical mastitis were treated with lmmu-21® alone they showed 20% clinical recovery along with significant rise in immunoglobulin G levels (Das et al., 2003).

The effect of Immu-21® (a herbal immunomodulator manufactured by Indian herbs containing Withania somnifera, Sphaeranthus indicus, Loranthus falcata, Scrophularia lielzzi, Panax ginseng, Nyctanthes arbor-tristis, Phyllanthus emblica, Mimosa tenuiflora, Ocimum tenuiflorum and/or Tinospora cordifolia were applied to Black Bengal goat in the last month of the pregnancy and their kids. They reported significantly higher birth weight and absence of kid mortality respectively. They concluded that the supplementation of polyherbal preparation during the later part of pregnancy in goats and to kids during the growth period is much more beneficial than administration at either stage alone (Sahoo et al., 2001).

Efficacy of supplementation of immu-21® to Black Bengal goats showed, in the last month of the pregnancy and /or kids and reported significantly higher birth weights, increased concentrations of blood protein and colostrum immunoglobulin and absence of kid mortality respectively. They concluded that the supplementation of immu-21® during the later part of pregnancy in goats and to kids during the growth period is much more beneficial than administration at either stage alone (Sahoo et al., 2001).

Influence of intramammary application of Tilox® (ampicillin puls cloxacillin; compared to 4 cows with 11 positive quarters) with the topical application of a paste containing roots of Withania

somnifera, Asparagus racemosus and Curcuma amada and leaves of Ocimum sanctum (5 animals with 12 positive quarters) for treatment of subclinical mastitis and found both the preparations effective, as assessed by a return to normal biochemical milk profiles, but the plant preparation acted more slowly (Kolte et al., 1999). Effect of anti-histaminic and anti-anaphylactic property of PulmoFlex® (polyherbal supplementation) was studied in mice. The results indicated that PulmoFlex® protected histamine aerosol induced collapse in guinea pig and compound 48/80 induced histamine release from guinea pig chopped lung preparation. PulmoFlex® also exhibited significant protection in passive foot anaphylaxis and passive cutaneous anaphylaxis in albino mice.

Bovine serum albumin induced anaphylactic shock in male albino mice was also antagonised

by PulmoFlex® (Gomes and Dasgupta, 2000).

2.4. Blood glucose and total leucocyte oncentrations After calving, the initiation of milk synthesis and rapidly increasing milk production greatly increases demands for glucose for milk lactose synthesis, at a time when feed intake has not reached its maximum. Because much of the dietary carbohydrate is fermented in the rumen, little glucose is absorbed directly from the digestive tract. Consequently, dairy cows rely almost exclusively on gluco-neogenesis from propionate in the liver to meet their glucose requirements. Limited feed intake during the early postpartum period means that supply of propionate for glucose synthesis also is limited. Amino acids from the diet or from skeletal muscle breakdown as well as glycerol from mobilized body fat contribute to glucose synthesis. Glucose supply to the mammary gland is also enhanced by the decreased oxidative use of glucose that accompanies the initiation of lactation (Drackley et al., 2001). Negative energy balance during early phase of lactation is more intense in cattle than buffalo (Drackley et al., 1999).

Plasma glucose concentrations are lower during the catabolic phase of lactation, and are higher during the anabolic phase of lactation when energy intake is equal or superior to the energy release (Goff, 1999).

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Plasma glucose concentration and hepatic glycogen decreased, while the lipid was increased during the transition period (Grummer, 1995). In addition, the effect of Leptadenia reticulata powder and Leptaden® tablets on lactogenic property of goats, sheep, cows and buffaloes was evaluated; the experiment was conducted on identical sets of animals in each group. Leptaden® tablets, 4 twice daily in goats and sheep, 10 twice daily in cows and buffaloes; and L. reticulata powder, 536 mg, (equivalent to the L, reticulata content of 4 Leptaden® tablets) in goats and sheep, 1340 mg (equivalent to the L. reticulata content of 10 Leptaden® tablets) in cows and buffaloes were given for 12 days. Both drugs produced galactopoletic response in most of the experiments. No changes were produced in the contents of milk and blood of goats as shown by their analysis during the experimental period (Anjaria and Gupta, 1967).

There was gradual increase of blood glucose concentration with feeding Livol® associated with marked increase in milk yield due to the stimulatory effect on liver functions and thus enhanced gluconeogenesis. The increase in blood glucose concentration was found steadily high due to increased synthesis even though there was increased demand for lactose synthesis to meet requirement for increased milk yield. An interesting finding was that with increased demand for conversion of blood glucose into lactose due to increased milk yield in all three feed additives groups, the plasma ketone bodies levels were within the normal range and even below base value throughout the period of study. There was no adverse effect due to regular feeding of the herbal products and there were also no alterations in blood biochemical profile.

The decline in electrical conductivity of the milk and lower somatic cell counts due to feeding of herbal products showed positive effects of the products in maintaining the integrity and health of the udder. The increased sustained milk production in all the three treatment groups was due to the galactopoietic effect of the feed additives which helped in better utilization of absorbed nutrients in the body tissues and lactopoietic system (Randhawa et al., 1995).

2.5. Ketosis The product herbal Nebsui as growth promoter feed additive was non-toxic, safe and without any residual or other side effect in dairy cows (Wheeler and Agrawal, 1999).The response of different therapeutic combinations against ketosis were studied. 42 ketotic cows were selected at random and were divided into 7 groups (Gr- 1, 2, 3, 4, 5, 6 and 7) having 6 cows in each. Gr-1 was kept as non-treated control and the remaining (Gr2–7) were utilized to study the efficacy, safely and cost-economics of different treatments. Before the commencement of therapy there was fall in serum glucose, insulin, cholesterol, plasma calcium phosphorus and magnesium and rise in serum ketone bodies in ketotic cows. Rothra's test and Ross test were also positive for ketone bodies in urine and milk respectively. Six different therapeutic combinations were tried against ketosis. On 7th day post treatment all the parameters like blood metabolites returned to normal and ketone bodies in urine and milk were found negative. There was general clinical recovery and improvement in appetite and digestion, the milk yield increased by 30–50 % in different treated groups. It is observed that the therapeutic regimen applied for the treatment of ketotic cows of Gr-3 with herbal antistress and metabolic and liver tonic products and MeboLiv® orally once daily for 7 days was found to be more scientific, rational, efficient, safe, economical and comparatively quicker in recovery without relapse of ketosis. There was steady increase in milk yield of the treated cows (Nayak and Nayak, 2001).

2.6. Some physiological parameters Three trials with Brown Swiss and aged Holstein cows with Leptaden (extracts of plants -L. reticulata and Breynia patens) added to a standard ration. The cows were in late, early, and middle stages of lactation in Trials 1, 2 and 3, respectively. Following three weeks pre-treatment, cows in the Leptaden® group were administered 10 Leptaden® tablets (268 mg/tablet) twice daily in the first trial and 15 tablets twice daily in the second and third trials for 21 days. This period was succeeded by a three week post-treatment period.

Leptaden® feeding did not produce any significant

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effect on average feed intake. Both control and experimental groups lost body weight during the trials, but there was no significant differences in weight change between the groups. Leptaden®-fed cows did not increase in milk production or change in the content of fat or total solids in their milk compared to the controls. Heart rates, respiratory rates, and rectal temperatures of all cows were within normal ranges in all periods. The serum protein-bound iodine and blood glucose indicated that Leptaden® did not impair thyroid activities of the cows. No significant differences were noted in other health related data between the Leptaden®-fed and control cows (Dash et al., 1992).

Effect of herbal ART-400® powder 20 g orally mixed in treacle daily to the male calves as III group, group II treated by diclofenac sodium and group I served as control, The elevated respiration and heart rate on 5th post-induction day, started declining and by 24th post-induction day, these parameters reached to near normal base values, however the rectal temperature remained within normal range in all the animals of different group (Kanwar and Varshney, 2003). Therapeutic efficacy of Caflon®, an herbal product of Indian herbs, Saharanpur, against non-specific respiratory disorders of goats. Treatment with Caflon® at the rate 10 g/animal, twice daily orally for 5 days had given the best results (100%cure on 5th day of treatment), treatment with Caflon® only at the rate 10 g/animal, twice daily, also had a remarkable positive response against the non-specific respiratory disorders in goats in comparison to the unmedicated group (Ghosh et al., 1994).

2.7. Reproductive performance Influence of herbal Prajana SS® Premix on synchronisation of oestrus and conception rate was evaluated in cows. In experiment 1, all cows and heifers came in oestrus and were served by all. Out of 9 cows and 5 heifers 86 % became pregnant on first oestrus. The remaining two cows repeated 21 days later and became pregnant by natural service. In experiment 2, all the 20 cows came in oestrus, within 10 h of each other, after 6 days of last Prajana SS® administration. It was noteworthy that unlike with the use of Pros-

taglandins, the length of oestrus period was not reduced in the Prajana SS® Premix treated cows, which made it possible to easily complete Al in the whole herd (Wheeler and Agrawal, 1999).

The efficacy of "Replanta®" an herbal product for improving breeding efficiency was assessed in cows; it improved uterine tone when used for resolving reproduction problems like retention of placenta and other postpartum complications in cows. Replanta® being purely herbal and biodegradable and with no problem of withdrawal time has big advantage over chemical treatments. Further, with the use of Replanta® it is also observed that the service period and the interval of insemination can also be reduced along with faster uterine involution. Replanta also ensured early start of the ovarian cycle and insemination (Koutecka, 1997).

Drug trial on 60 cows and 10 buffaloes was conducted which were divided randomly into five groups with equal number in each group including cows and buffaloes, out of those five groups, four groups were experimental and one served as control. Experimental groups were separately dosed with Replanta® powder, Replanta® liquid, Uterotone liquid and a cleansing drench containing ergot immediately after parturition and observations recorded. The cows and buffaloes treated with Replanta® powder and Replanta® liquid came early in first heat after calving and reproductive cycle was found to be more regularised as compared to other drugs tested. Replanta® treated animals come to heat second time also (Pandey and Raghuvanshi, 1992). The efficacy of Replanta® treatment on placental expulsion, lochial cessation, uterine involution and post partum oestrus was assessed in Murrah buffaloes. In all, 92.0% buffaloes of Replanta® treated group expelled their foetal membranes within a mean duration of 6.30 h as compared to 84.0% buffaloes of control group within 7.4 h of parturition. Earlier cessation of lochia and uterine involution was observed in Replanta® treated group than the untreated control group of buffaloes. 88% buffaloes of Replanta® treated group expressed first post partum oestrus with a mean duration of 78.2 d as compared to eighty per cent buffaloes of control group within 87.0 d post partum (Paul et al., 1995).

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2.8. Ruminal fermentation and methane emission Livestock contribute both directly and indirectly to climate change through the emissions of greenhouse gases such as carbon dioxide, methane and nitrous oxide. Globally, the sector contributes 18 percent (7.1 billion tonnes CO2 equivalent) of global greenhouse gas emissions. Although it accounts for only nine percent of global CO2, emissions it generates 65% of human-related N2O emissions and 35 percent of CH4 emissions, which have 296 times and 23 times the Global Warming Potential of CO2 respectively. CH4 emissions mostly occur as part of the natural digestive process of animals (enteric fermentation) and manure management in livestock operations.

CH4 emissions from livestock are estimated at about 2.2 billion tonnes of CO2 equivalent, accounting for about 80% of agricultural CH4 and 35% of the total anthropogenic methane emissions. N2O emissions are associated with manure management and the application and deposition of manure. Indirect N2O emissions from livestock production include emissions from fertilizer use for feed production, emissions from leguminous feed crops and emissions from aquatic sources following fertilizer application. The livestock sector contributes about 75 percent of the agricultural N2O emissions (2.2 billion tonnes of CO2 equivalent). CO2 emissions from the livestock sector are related to fossil fuel burning during production of fertiliser for feed production, the livestock production process, processing and transportation of refrigerated products. Furthermore, livestock are a major driver of the global trends in land-use and land-use change including deforestation (conversion of forest to pasture and cropland), desertification, as well as the release of carbon from cultivated soils. The overall contribution of CO2 emissions from the livestock sector are estimated at 2.7 billion tonnes of CO2 (Mirzaei, 2010). Feed additives that manipulate the microorganisms living in the rumen to quicken microbial fermentation (Mirzaei and Hari, 2012).

There is currently interest in the role of plant secondary metabolites such as saponins and tannins in reducing CH4 emissions (Wallac, 2004).

Saponins have been shown to possess strong de-

faunating properties both in vitro and in vivo which could reduce CH4 emissions (Wallace and McPherson, 1994). Scientists recently reviewed literature related to their effect on CH4 and concluded that there is evidence for a reduction in CH4 from at least some sources of saponins, but that not all are effective. Likewise they reported that there is evidence that some condensed tannins can reduce CH4 emissions. Some legumes contain condensed tannins, but unfortunately these may reduce forage digestibility and the condensed tannins containing varieties tend to have weak agronomic performance. Extracts from plants such as rhubarb and garlic could decrease CH4 emissions, while there is insufficient evidence to conclude on the potential of plant secondary compounds or extracts as mitigation strategies, this is likely to be an area of significant research over the coming years (BSAS, 2008).

An experiment tested on combination of certain herbs having galactogogue and stomachic actions to test its efficiency on milk production in jersey crossbred cows, fed diets according to NRC standard computed on fortnightly basis. The mean dry matter intakes cow/ day were similar in both the groups for the full lactation. It was observed that the cows showed an increase in milk production by 74.0 Kg given this combination of herbs over the control group; the effect was noticed mainly in the mid trimester. The feed efficiency for milk production was 1.4 Kg DM/kg milk in herbal supplemented group (Tiwari et al., 1993).

The effect of supplementing four medicinal herbs and plants (garlic, fenugreck, Nigella sativa and camomile) assigned to Zaraibi goat ration on their performance. Results showed improvement in digestibility coefficients of dry matter, organic matter, and crude protein. Ration with non- supplement showed the highest nutritive values, but milk yield was increased in all treatment groups fed by supplementation compare to control group.

It was concluded that medicinal herbs and plants improved feed conversion, economic return and decreased feed cost of 1 Kg milk compared with control group (Allam et al., 1999). Clinical efficacy of some herbal preparations (The combination of Leptadenia reticulata, Nigella sativa,

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Foeniculum vulgare, Pueraria tuberosa and Asparagus racemosus) was evaluated and reported that, this supplementation was effective in curing digestive disorders and early restoration of normal milk production in lactating Buffaloes (Kumari and Akbar, 2006).

The data further suggested that 4 % tannins through babul pods in the diet of lactating goats can be incorporated without affecting the production performance of animals.

2.10. Growth rate, daily weight gain) in growing fattening ruminants

2.9. Feed intake and feed conversion ratio Smith et al., (2005) compared the nutritional values of Acacia nilotica and Dichrostachys cinerea fruits treated with NaOH and Polyethylene Glycol. After 48 h incubation, in vitro organic matter digestibilities of both species were increased by Polyethylene Glycol and NaOH treatment. Dry matter (DM) intake and DM digestibility were lowest and N-retention negative in goats fed. Farmantan, a natural extract from Chestnuts tannins improved body weight gain and feed conversion efficiency in lactating cow (Dumanovski and sotoseki, 1998). Similarly, Errante et al., (1998) reported farmantan at 120 g/day increased daily weight gain and feed conversion efficiency by 33% and 7%, respectively in dairy cow. Similarly, Bensalem et al., (2002) reported that supplementation of lambs feeding on spineless cactus pods with 100 g of air- dried Acacia leaves and SBM increased growth rate to 102 g/d from 75 g/d of control diet. Bayssa, (2006) studied the effects of feeding untreated and treated Acacia nilotica pods on lactating goats. Four diets were prepared. The diet consisted of green maize fodder and concentrate in 50: 50 ratio (TMR I) where as in TMR II untreated babul pods (4% tannins) was added to the extent of 22.2% while, in TMR III, the babul pods(6% tannin) to the extent of 33.3% treated with 3% Ca(OH)2 and TMR IV, the babul pods (6 % tannins) to the extent of (33.3%) treated with 3% Ca(OH)2 + PEG (0.10 Polyethylene glycol : Tannin). He found non-significant difference among all treatment groups in terms of DMI, DOMI, CPI and TDNI. The feed conversion efficiency (DMI, kg/kg milk production) also showed similar trend and was better than control (1.75 versus 1.39 kg DMI/kg MY). Similarly the gross energetic efficiency (%) was comparatively higher in treated group than control diet but the gross protein efficiency remained similar across the dietary treatments.

Wina et al., (2005) reported large increase in average daily gain in goats fed diet containing water soaked Acacia villosa leaves and cassava flour (71 g/day) compared to those fed diet containing unsoaked leaves and water soaked leaves (38.9 and 44.7 g/day) (P < 0.05). Tanner et al.

(1990) reported that supplementation of Acacia nilotica pods with seeds to growing rams reduced growth rate significantly (P < 0.05) as compared to control. Rate of gain in growing animals reflects total intake and availability of nutrients in the diet. Low total intake and low growth rates were also observed in animals eating A. sieberiana pods and leaves of A. cyanophylla (Reed et al., 1990). Rubanza et al., (2007) reported that supplementation with browse tree leaves resulted to improved weight gains (P < 0.05) of animals fed on Leucaena leucocephala than those supplemented with A. nilotica and A. polyacantha, compared to the control group, which lost weight.

Improved weight gains were mainly due to the corrected dietary crude protein (CP) in the basal diet from browse supplements and probably due to improved feed digestibility and nutrients supply to the animals. Supplementation of animals with A.nilotica and L. leucocephala leaves at 20% of the expected dry matter intake (DMI) optimized weight gains. Dubey (2007) reported that In all the treatment groups, body weight did not differ significantly showing that 16.7% babul pods equivalent to 3% tannins in the diet may be considered safe level of untreated babul pods whereas 27.8% babul pods equivalent to 5% tannins in the diet treated with either calcium hydroxide or polyethylene glycol may be considered safe level in diet of growing crossbred calves without affecting body weight gain. Supplementation of 5 g Polyethylene Glycol -6000 to kids grazing on khejri (Prosopis cineraria) containing 11.6% condensed tannin, increased body weight gain by 2.5% with increased DMI and CP digestibility as

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compared to condensed tannin acting group (Bhatta et al., 2002).

CONCLUSION Ethno-veterinary alternatives (based on medicinal plants) are an option for small-scale livestock farmers who cannot use allopathic drugs or for those larger conventional farmers whose economic circumstances prevent the use of veterinary services for minor health problems of livestock.

Current scientific evidence suggests there is significant potential to use plants to enhance animal health in general and that of ruminants.

Active areas of research for plant bioactives

(particularly saponin and tannin containing plants) include reproductive efficiency, milk and meat quality improvement, foam production/bloat control and methane production.

Nematode control is also a significant area of research and the evidence suggests a much broader range of phytochemicals may be effective. This review presents a summary of the literature and examines international research efforts towards the development of plant bioactives for animal health This review suggests that plants may indeed be beneficial for animal health, whilst at the same time, highlights the need for more controlled in vivo research to validate plant bioactivity.

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Kanwar M.S., Varshney A. C., 2003. Evaluation of therapeutic efficacy of herbal Art 400® in induced traumatic arthritis in calves: Clinical and radiological observations, Phytomedica. 4, 23–28.

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Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 404â&#x20AC;&#x201C;410 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article A COMPREHENSIVE REVIEW ON SPHAERANTHUS INDICUS LINN. Pawar Harshal A1*, Therani Deepika2 1,2

Dr.L.H.Hiranandani College of Pharmacy, CHM Campus, Opp. Railway Station, Ulhasnagar, Maharashtra, India * Corresponding Author: Email: hapkmk@rediffmail.com

Received: 17/07/2012; Revised: 20/08/2012; Accepted: 26/08/2012

ABSTRACT Sphaeranthus indicus Linn belongs to the family Asteraceae. It grows in rice fields, dry waste places and cultivated lands in tropical parts of India. All the parts of the plant have medicinal uses. A wide range of phytochemical constituents have been reported in S. indicus which include eudesmanoids, sesquiterpenes, stigmasterol, sitosterol, geraniol, methyl chavicol. It is widely used in Ayurvedic system to treat CNS disorders like epilepsy and mental diseases and also useful in the treatment of diabetes, jaundice and leprosy. The objective of the present review was to compile an up-to-date and comprehensive information on S. indicus that cover its pharmacognosy, phytochemistry and biopotential. The presented review may give a direction for further research and may provide strong evidence to use S. indicus in development of new herbal formulations with better therapeutic activity.

KEY WORDS: Sphaeranthus indicus, Asteraceae, Eudesmanoids, Tonic, Tuberculosis.

Cite this article Pawar Harshal A & Therani Deepika (2012), A comprehensive review on Sphaeranthus indicus Linn., Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 404â&#x20AC;&#x201C;410

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INTRODUCTION:

Morphology:

Herbal medicine is the oldest form of healthcare known to mankind. Herbs had been used by all cultures throughout history. It is an integral part of the development of modern civilization. Now a day, the use of herbal medicine is becoming more prevalent in both developing and developed countries. It is becoming more mainstream as improvements in analysis and quality control along with advances in clinical research show the value of herbal medicine in the treating and preventing disease. Herbal drugs have lesser side effects and are largely replacing synthetic drugs [Izzo et al.,2009].

Sphaeranthus indicus Linn. (Photo slide.1) is an annual herb with sessile, decurrent, obovate, bristly serrate, downy, glutinous leaves and globular heads of purple flowers. It is a branched herb distributed in wet places [Bafna, A.R. et al., 2006]. The stem is greenish in colour; roots are brown externally and internally light brown, tuberous with 10–15 cm in length and 0.1–0.4 cm in diameter with longitudinal striations and transverse scars seen at regular intervals. Odour is characteristic [Galani, V.J. et al., 2009, Bafna, A.R. et al., 2006, Cooper, E.L. et al., 2004].

Sphaeranthus indicus Linn. (HindiGorakhmundi) is a much branched herb belonging to family Asteraceae. All the parts of the plant were reported to possess medicinal value. It is widely used in Ayurvedic system to treat CNS disorders like epilepsy and mental diseases and also useful in the treatment of diabetes, jaundice and leprosy [Kirtikar KR et al., 1981]. Other species are Sphaeranthus africans Linn. and Sphaeranthus alatus. It grows plentifully in rice fields [Kirtikar KR et al., 1981, Sadaf , F et al., 2006] and is distributed throughout India, Ceylon, Malay, China and Africa.

The objective of the present review was to compile an up-to-date and comprehensive information on Sphaeranthus indicus that cover its pharmacognosy, phytochemistry and biopotential.

Taxonomy: The taxonomical classification of Sphaeranthus indicus [Kirtikar et al., 1981] is mentioned below: •

Kingdom: Plantae

•

Subkingdom: Viridaeplantae

•

Phyllum: Tracheophyta

•

Subphyllum: Euphyllophytina

•

Infraphyllum: Radiatopses

•

Class: Magnoliopsida

•

Subclass: Asteridae

•

Superorder: Asteranae

•

Order: Asterales

•

Family: Asteraceae

•

Genus: Sphaeranthus

•

Species: indicus

Photo slide 1: Sphaeranthus indicus Linn.

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Climatic Conditions for Cultivation: The temperature range for cultivation of S. indicus is 10°C to 40°C. It requires an altitude of about 500–1500m. It grows well in hot and humid climate. Alluvial soil, red soil or lateritic soil is found to be more ideal for its cultivation.

The pH of soil should be in the range of 6.3– 7.3. It is rainfed type of plant and is propagated during monsoon [Galani, V.J. et al., 2009].

Microscopy: The root showed secondary characters and has a circular outline. Open vascular bundle are observed. Secondary phloem consisted of sieve tubes, companion cells and phloem parenchyma, while secondary xylem consisted of tracheids, vessels, fibers and xylem parenchyma.

The leaf is dorsiventral and shows abundant trichomes of varying types on both the epidermis. Simple trichomes are three to four celled, thick walled and measure 130.8– 145.2 µm in length and 29.0–43.5 µm in width.

Trichomes are straight/knee shaped, with a swollen base and with collapsed cell at the middle or at the apex.

The stem shows cork with two to three layers of parenchymatous cells covered with papillose cuticle having trichomes. Medullary rays are pitted, lignified and about unitetraseriate [Vikani KV et al., 2008].

General Methods Phytoconstituents:

for

Extraction

of

1) Soxhlet method The plant material should be powdered and extracted in Soxhlet apparatus for 48 h using ethanol. The extracts obtained should be filtered through Whatman filter paper and Fig. 1: Methyl chavicol

concentrated by vacuum evaporation [A K Nandkarni 2002 and J B Harborne 1998] .

2) Maceration method The flowers of S. indicus should be powdered and soaked in 50% ethanol with occasional shaking for 10 days and then concentrated using a rotary vacuum evaporator under reducing pressure and controlled temperature at 40°C. A semi-solid mass of dark brown colour will be obtained, which is then subjected to lyophillization and finally brown powder will be obtained [Farzana Sadaf et al., 2006 and J B Harborne 1998] .

Phytochemistry Eudesmanoids such as 11α, 13-dihydro-3α, 7α-dihydroxy-4,5-epoxy-6β,7-eudesmanolide, 11α,13-dihydro-7α-acetoxy-3β-hydroxy-6β, 7eudesm-4-enolide and 3-keto-β-eudesmol have been isolated from S . indicus [Pujar PP et al., 2007]. The plant is reported to contain deep cherry colored essential oil having methyl chavicol (Fig.1), d-cadinene (Fig.2), α-ionone (Fig.3), p-methoxycinnamaldehyde (Fig. 4), αterpinene (Fig.5), citral (Fig.6), geraniol (Fig.7), geranyl acetate, β-ionone (Fig.8), oscimene (Fig.9), eugenol (Fig.10), sphaeranthene, sphaeranthol, Indicusene [Dhar ML et al., 1968, Farzana Sadaf et al., 2006] and alkaloid sphaeranthine [Basu NK et al., 2006].

Carbohydrates such as arabinose, galactose, glucose, fructose, lactose, maltose, raffinose and rhamnose have been reported from leaves of S. indicus [Yadava RN et al., 1998].

A sesquiterpene lactone, 7hydroxyeudesm-4-en-6, 12-olide, and a sesquiterpene acid, 2-hydroxycostic acid, along with the known compounds, β-eudesmol and ilicic acid, have been isolated from the acetone extract of S. indicus.

Fig. 2: d-Cadinene

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Fig.3: Alpha-ionone

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Fig.4: p-methoxycinnamaldehyde

Fig.7: Geraniol

Fig.5: Alpha-terpinene

Fig.8: Beta-ionone

Biopotential of S. indicus: 1) Analgesic activity The analgesic activity was evaluated by two methods-Acetic acid induced writhing method in albino mice and tail flick latency method in albino rats respectively. The ethanolic extract of S. indicus in different doses (100, 200 and 400 mg/kg) exhibited dose dependent and significant analgesic activity in both models of pain [Nanda et al., 2009].

2) Hepatoprotective activity The hepatoprotective effect of aqueous and methanolic extracts of flower heads of S. indicus on Acetaminophen-induced heptotoxicity was studied in rats. As compared to aqueous extract, significant decrease in liver function markers such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), acid phosphatase (ACP) and alkaline phosphatase (ALP), bilirubin and total protein, was observed while using methanolic extract of S. indicus with same dose [Tiwari et al., 2009, Nayak et al., 2007] .

3) Anticonvulsant activity Different extracts of the whole parts of plant of S. indicus like benzene, chloroform, ethanol and water extract was studied for its anticonvulsant effect on maximal electroshockinduced seizures and pentylenetetrazole, picrotoxin, bicuculline and N-methyl-dl-

Fig.9: Oscimene

Fig.6: Citral

Fig.10: Eugenol

aspartic acid-induced seizures in mice. Ethanol extract (200â&#x20AC;&#x201C;400 mg/kg) significantly reduced the duration of seizures induced by maximal electroshock (MES). The aqueous extract (400 mg/kg) significantly reduced the latency, but did not alter the incidence of seizures elicited by maximal electroshock to any significant extent [Sander et al., 1996, Harborne 1998].

4) Anthelmintic activity The ethanolic and aqueous extracts of the whole plant of S. indicus Linn. were used against Pheretima posthuma and Ascardia galli. The extract was used in following concentrations-10, 50, 100 mg/ml to determine time of paralysis and time of death of worms.

The most significant activity was observed at the highest concentration of 100 mg/mL against both types of worms. Albendazole (10 mg/ml) and distilled water were included as standard reference and control, respectively [Lal et al., 1976] .

5) Antimicrobial activity It was found that aqueous extracts of flowers of S. indicus exhibited antibacterial activity. Antimicrobial Activity of S. indicus L. was studied by V. Duraipandiyan et al., [Sohoni JS et al., 1988]. Hexane extracts of flower and aerial parts of S.indicus showed good antibacterial activity against gram positive organisms. Also coli forms were used to study the antimicrobial activity of S. indicus flower extracts. This investigation revealed that

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the aqueous extract of S. indicus flower produced good inhibitory zones against test organism.

6) Anti hyperlipidemic activity Antihyperlipidemic activity of alcoholic extract of S. indicus Linn. flower heads was studied in atherogenic diet induced hyperlipidemia in rats. S. indicus extract (500 mg/kg/day, p.o. for 8 days) was found to cause a marked decrease in body weight, total cholesterol, triglyceride, and low density lipoprotein (LDL) and very low density lipoprotein (VLDL). Also a significant rise in high-density lipoprotein (HDL) was observed after treatment with S. indicus extract [Pande VV et al., 2009].

7) Anti inflammatory activity Ethanolic extract of S. indicus was used in albino rats for this study. The antiinflammatory activity was evaluated using acute inflammatory model like carrageenan induced paw edema and chronic inflammatory model like cotton pellet induced granuloma respectively. The ethanolic extract in different doses (100, 200, and 400 mg/kg) exhibited dose dependent and significant antiinflammatory activity in acute (carageenan induced hind paw edema, p < 0.05) and chronic (cotton pellet granuloma formation, p < 0.05) model of inflammation [A K Nandkarni 2002, Winter CA et al., 1957].

8) Antioxidant activity The free radical scavenging activity of the plant S. indicus was studied by using different antioxidants models of screening. The ethanolic extract at 1000 microgram/ml showed maximum scavenging of the radical cation, 2,2-azinobis-(3-ethylbenzothiazoline-6sulphonate) (ABTS) observed upto 41.99 % followed by scavenging of the stable radical 1,1-diphenyl, 2-picryl hydrazyl (DPPH) (33.27%), superoxide dismutase (25.14 %) and nitric oxide radical (22.36%) at the same

concentration. Total antioxidant capacity of the extract was found to be 160.85 nmol/g against Ascorbic acid as standard indicating good antioxidant potential of plant. [Tiwari Bk et al., 2009, Shiwarkar A et al., 2006].

9) Antihyperglycemic activity The 50% ethanolic extract of plant was reported to possess hypoglycemic activity.

Antihyperglycemic effect of alcoholic extract of S. indicus was evaluated in the nicotinamide (120 mg/kg, i.p.) and streptozotocin (60 mg/kg, i.p.) induced diabetes in rats. Fasting normal rats treated with the alcoholic extract of S. indicus showed significant improvement in oral glucose tolerance test. Oral administration of S. indicus for 15 days resulted in a significant decrease in blood glucose levels and increase in hepatic glycogen and plasma insulin levels [Prabhu KS et al., 2008].

10) Neuroleptic activity Neuroleptic activity of hydroalcoholic extract of whole plant of S. indicus has been reported. Hydroalcoholic extract of whole herb of S. indicus (100, 200 and 500 mg/kg, p.o.) produced catalepsy, potentiated haloperidolinduced catalepsy and antagonized apomorphine-induced stereotypy [Galani, V.J. et al., 2009].

CONCLUSION: The scientific studies and research data available on Sphaeranthus indicus indicated enormous biopotential of this plant. It is reported to contain eudesmanoids, eudesmanolides, sesquiterpene lactone, sterol glycoside, flavanoids, and essential oil. There is no doubt that this plant is a reservoir of potentially useful phyto-constituents which can serve as a lead for the synthesis of newer class of synthetic medicines. Clinical studies with standardized extract and isolated constituents need to be performed to find out unexploited use of this plant.

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REFERENCES: Ambavade, S.D., N.A. Mhetre, V.D. Tate and S.L. Bodhankar, (2006).

Pharmacological evaluation of the extracts of Sphaeranthus indicus flowers on anxiolytic activity in mice.

Indian J.Pharmacol.,38:254–259.

Bafna,

Baslas

A.R. and S.H. Mishra, (2006).

Protective effect of bioactive fraction of Sphaeranthus indicus L. against cyclophosphamide induced suppression of humoral immunity in mice. J. Ethnopharmacol.,104:426–429.

KK. (1959) Essential oil from Spharanthus indicus. Perf Essent Oil Rec. 50:7658.

Basu NK, Lamsal PP. (1946) A chemical investigation of Sphaeranthus indicus Linn. J Am Pharmaceut Asso Sci Ed.35:274–5.

Chatterjee A, Pakrashi SC. (2003).1st ed. Vol5. New Delhi: National Institute of Science communication and information resources; The treatise on Indian medicinal plants p. 177.

Chopra RN, Chopra IC, Honda KL, Kapur LD (1958). Indigenous drugs of India, 2 edn.,UNDhur and Sons (P) Ltd, Calcutta Cooper, E.L., 2004. Complementary and alternative medicine, when rigorous, can be science.

Evid.

Based Complement. Alternat. Med., 1: 104– 114.

Dhar ML, Dhar MM, Dhawan BN, Mehrotra BN, Ray C. (1968),Screening of Indian plants for biological activity. Indian J Exp Biol. 6:232–47.

Farzana Sadaf, Saima Hashmi, and Tariq Shrafatullah.(2006). Anti Inflammatory and antinociceptive activity in herbal drug-Aujaie. Pak. J. Pharmcol., 23: 1– 5.

Galani,

V.J. and B.G. Patel, (2009).

Psychotropic activity of Sphaeranthus indicus Linn. in experimental animals.

Phcog. Res., 1: 307–313.

Gupta RK, Chandra S, Mahadevan V. (1967), Chemical composition of Sphaeranthus indicus Linn. Indian J Pharm. 29:47–8.

Harborne J B. (1998), Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis, 3rd Edition, New Delhi: Springer, Rajkamal Electric Press, 1– 32, 40–227.

Izzo AA, Ernst E. Interactionsbetween herbal medicines and prescribed drugs: an updated systematic review.Drugs 2009;69(13):1777-98.

Kirtikar KR, Basu BD, (1981). ICS Indian medicinal plants. In: Blatter E, Caius JF, Mhaskar KS, editors. 2nd ed. Vol-2.

Allahabad India: Lalit Mohan Basu; pp.

1346–8.

Lal J, Chandra S ,Raviprakash V and Sabi M, (1976),In vitro anthelmintic action of some indigenous medicinal plants on Ascardia galli worms, Indian. J. Physiol.Pharmacol , 20(2), 64–68.

Lodha V. (2003), Chemical analysis of the essential oil of Sphaeranthus indicus an Ayurvedic plant of India. Indian Perfumer. 47:29–30.

Mhetre NA, Ambavade SD, Bodhankar SL. (2006), Neuroleptic activity of extract of Sphaeranthus indicus in mice. Indian J Nat Prod. 22:24–7.

Nanda BK, Jena J, Rath B, Behera BR. (2009), Analgesic and Antipyretic activity of whole parts of Sphaeranthus indicus Linn. J Chem Pharma Res. 1:207–12.

Nadkarni A K (2002), Indian Materia Medica, 3rd revised ed., Vol. I, Popular Prakashan , Mumbai.

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Nayak SS, Maity TK, Maiti BC. (2007), Hepatoprotective activity of Sphaeranthus indicus Linn. Int J Green Pharm. 1:32–6.

Shirwaikar A, Prabhu KS, Punitha IS. (2006), In vitro antioxidant studies of Sphaeranthus indicus (Linn)Indian J Exp Biol. 44:993–6.

Pande

Sohoni JS, Rojatkar SR, Kulkarni MM, Dhaneshwar NN, Tavale SS, Gururow TN, et al. (1988), A new eudesmenolide and 2-hydroxycostic acid from Spharanthus indicus Linn.x-ray molecular structure of 4-alpha, 5-alphaepoxy-7-alpha-hydroxyeudesmanolide.

J Chem Soc Perkin 1. 2:157–60.

VV, Dubey S. (2009), Antihyperlipidemic activity of Sphaeranthus indicus on atherogenic diet induced hyperlipidemia in rats. Int J Green Pharm. 3:159–61.

Prabhu KS, Lobo R, Shirwaikar A. (2008), Antidiabetic properties of the alcoholic extract of Sphaeranthus indicus in streptozotocin-nicotinamide diabetic rats. J Pharm Pharmacol. 60:909–16.

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PP, Sawaikar DD, Rojatkar SR, Nagasampagi BA. (2000), Eudesmanoids from Sphaeranthus indicus. Fitoterapia. 71:264–8

Sadaf, F.; Saleem, R.; Ahmed, M.; Ahmad, S. I.; Zafar, N. (2006),Healing potential of cream containing extract of Sphaeranthus indicus on dermal wounds in Guinea pigs.

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Bk, Khosa RL. (2009), Hepatoprotective and antioxidant effects of Sphaeranthus indicus against acetaminophen induced hepatotoxicity in rats. J Pharma Sci Res. 1:26–30.

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8:61–7.

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Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 411–417 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article ETHNOBOTANICAL STUDY OF THERAPEUTIC PLANTS USED TO TREAT ARTERIAL HYPERTENSION IN THE HODNA REGION OF ALGERIA SARI Madani1,2*, SARRI Djamel3, HENDEL Noui4 and BOUDJELAL Amel5 1, 3, 4, 5

Department of Natural Sciences and Life, Faculty of Science, M'sila University, 28000 M’sila (Algeria) Laboratory of Phytotherapy, Applied to Chronic Diseases, Faculty of Natural Sciences and Life, Ferhat Abbas University, 19000 Sétif (Algeria).

2

*Corresponding Author E-mail: Mad_sari@yahoo.fr

Received: 30/07/2012; Revised: 24/08/2012; Accepted; 29/08/2012

ABSTRACT Ethnobotanical investigations were conducted from February 2006 to June 2010 in the Hodna region of Algeria to identify different medicinal plants used in the traditional local pharmacopoeia for the treatment of arterial hypertension. Information was collected through questionnaires and personal interviews (77 males and 8 female). Thirty-five species belonging to 21 families (with a dominance of especially Lamiaceae and Asteraceae) were encountered during the study. The modes of herbal drug preparation were decoction (48%) and infusion (25%). The most frequently used plant parts were the aerial parts (49%).

Keywords: Arterial hypertension (AHT), ethnobotanical uses, ethnomedicinal survey, medicinal plants.

Cite this article: SARI Madani, SARRI Djamel, HENDEL Noui and BOUDJELAL Amel (2012), ETHNOBOTANICAL STUDY OF THERAPEUTIC PLANTS USED TO TREAT ARTERIAL HYPERTENSION IN THE HODNA REGION OF ALGERIA, Global J Res. Med. Plants & Indigen.

Med., Volume 1(9), 411–417

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INTRODUCTION

MATERIAL AND METHODS

Arterial hypertension (AHT) is a prevalence major chronic disease among the Algerian population. The high prevalence of AHT among Algerian adults is due to the fact that this disease represents a real public health problem (Temmar et al., 2010). Several scientific studies, conducted both by the Ministry of Health and by academic researchers, to show that this condition affects nearly 35% of adults (Benkhedda et al., 2004).

Several endogenous and exogenous factors contribute to the increased incidence of AHT in Algerian society. These include new eating habits (including the excessive consumption of salty and sweet foods, settlement, social environment, genetics and stress (Benkhedda et al., 2004). The goal of this study was to identify medicinal plants used in the treatment of AHT grown in the Hodna region.

Study area

The Hodna region (M’sila), occupies a position in the central part of Northern Algeria (Figure 1). As a whole, it is part of the central highlands. It covers an area of 18,718 Km2 and it is located at an altitude of approximately 500 m, situated between 35°42'07" N 4°32'49" E (Moreau et al., 2005). The climate of the investigation area is continental, due in part to the Saharan influences. Summer is hot and dry while winter is very cold, with low and irregular rainfall in the order of 100– 250 mm/year (Le Houerou, 1995). Its geographical position gives this region a unified ecological aspect represented by the predominance of the steppe, which covers 1.2 million hectare (63% of the total area) of the state. The areas used for agriculture accounts 20% of the total area devoted mainly to cereals, arboriculture and market gardening (FAO, 1966).

Figure 1: Location map of the study area (sitesatlas.com)

H

Ethnobotanical survey The aimed survey was conducted from February 2006 June 2010 in the Hodna region of Algeria. The plants were collected, dried and

preserved for identification. They were identified using flora of Maire 1952–1987, Quezel and Santa, 1962–1963, Ozenda, 1983 and Babba Aïssa, 1999; verified, characterized and confirmed by professional botanists of the

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plant, dosage, how to use it (decoction, infusion, etc.), usage period of the plant and side effects of the plant. Information regarding the medicinal uses of the plants was obtained from local people through questionnaires (Figure 3).

department. Voucher specimens were deposited in the Herbarium of the department. All investigations described the information about (Figure 2): date, research area (district/village), informants (name/age/sex/educational level), scientific name of plant, local name of plant, part of the plant used, usage purpose of the

Figure 2: Questionnaire card of survey QUESTIONNAIRE CARD N°………..

SECTION A Date

Area

…

…....

Sexe M

Age

W

…

…

Educational level

Informants

Illiterate

Primary

Intermediary

Secondary

Academic

Herbalist

Healer

Villager

…

…

…

…

…

…

…

…

…..

SECTION B

Utilization (Type of disease) NB : No recipes

Botanical name

Scientific name

Names : Arab / Amazigh / Targui or other

………………..

………………. …

…………………………….………. …

…………………………. ……………………………. ……………………………. ……… ……………………. ……………………………. ……………………………. …………… ………………. ……………………………………………………………………………… ……………………. ……………………………. ……………………………. …………… ………………. ………………………………………………………………………………

Infusion

Decoction

Fumigation

Maceration

Powder

Cream

Bath

Plaster

Other

Mode of use

…..

…..

…..

…..

…..

…..

…..

…..

…..

Part (s)

Root

Leaf

Fruit

Flower

Seed

Flowering tops

Aerial parts.

Plant Whole

Other

used (es)

…..

…..

…..

…..

…..

…..

…..

…..

…..

SECTION C

Plants associated Utilization (Type of disease ) NB : Recipes (mode, period, amount, nature….)

Botanical name

Common name

Names : Arab / Amazigh / Targui or other

………………..……

…………..………

…………………………………. ………

…………………………. ……………………………. ……………………………. ……… ……………………. ……………………………. ……………………………. …………… ………………. ……………………………. ……………………………. ………………… …………. ……………………………. …………………….………. ……………………… ……. ……………………………. ……………………………. ……………….……………. …. ……………….……………………………………………………………………………. ……. ……………………………. ……………………………. ……………….……………. …. ……………….…………………………………………………………………………….

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Figure 3: Methodological approach of the survey Material (Plant species)

Questionnaire Card

Meeting aleatory

Meeting organized

Villagers

Herbalists

Herbalists

FINAL RESULTS OF SURVEY

RESULTS AND DISCUSSION During the survey, a total of 35 different species of medicinal plant belonging to 21 families and 28 genera were reported to be used for treating AHT disease (Table 1). In terms of number of medicinal plants, the family

Lamiaceae was the highest with 9 species followed by Asteraceae with 5 species. A total of 85 personal interviews, comprising of 77 men and 8 women appeared in the present study (Table 2). The age of the informants ranged from 22–92 with an average of 35.20 years for men and 50.54 years for women.

Table 1: Plants traditionally used to treat Arterial Hypertension in the Hodna region of Algeria Scientific name / Family name

Local (Vernacular) Name

Part used / use citations

Preparation

Ajuga iva (L.) Schreb.

(Lamiaceae) Allium sativum L.

Chendgoura

Aerial parts (10)

Infusion – decoction

Thoum

Fruits (2)

Maceration

Vervene

Aerial parts (1)

Decoction

Diss

Aerial parts (1)

Decoction

Landj

Leaves (1)

Decoction

Beroustoum

Roots (1)

Powder

Chih

Aerial parts (3)

Infusion - decoction – maceration

Chadjaret meriem

Aerial parts (2)

Infusion

(Liliaceae)

Aloysia triphylla (L'Hér.) Britton (Verbenaceae)

Ampelodesma mauritanicum (Poiret) Dur. and Sch. (Poaceae)

Arbutus unedo L. (Ericaceae)

Aristolochia glauca Desf.

(Aristolochiaceae)

Artemisia herba alba Asso.

(Asteraceae)

Artemisia absinthium L. (Asteraceae)

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Table 1 (Continued…)

Scientific name / Family name Artemisia campestris L.

Local (Vernacular) Name Tgoufet

Part used / use citations Aerial parts (3)

Preparation

Elhadj

Fruits (1)

Bath

Zaarour

Fruits- leaves (7)

Decoction

Khorchef

Aerial parts (2)

Decoction

Fegous el hamir

Fruits (1)

Fumigation

Tesselgha

Aerial parts (3)

Decoction

Megramène

Aerial parts (2)

Decoction

Araar

Leaves (1)

Decoction – powder

Rand

Leaves (10)

Infusion- decoction

Meriouet

Leaves (3)

Infusion - decoction – maceration

Naanaa

Flowers (5)

Infusion – decoction

Etoute

Leaves (2)

Infusion-decoction

Rayhane

Leaves (1)

Infusion

Habek

Aerial parts (2)

Decoction

Ezeboudj

Leaves (6)

Decoction

Zitoune

Leaves (4)

Decoction

Zaater

Aerial parts (1)

Decoction

Bardkouche

Flowers (1)

Decoction

Harmel

Seeds(1)

Unction – powder

Esenouber

Fruit (1)

Infusion

Dharou

Aerial parts (1)

Decoction

Balout

Leaves – roots (1)

Decoction – powder

Eklil el djabel

Aerial parts (6)

Infusion – decoction

Fidjel

Leaves (9)

Infusion- decoction – powder

Feliou

Aerial parts (1)

Infusion – decoction

Souak enebi

Aerial parts (5)

Infusion – decoction

Djertil

Aerial parts (10)

Infusion – decoction

Decoction

(Asteraceae)

Citrullus colocynthis (L.) Schrad.

(Cucurbitaceae)

Crataegus oxyacantha L. (Rosaceae)

Cynara scolymis L. (Asteraceae)

Ecballium elaterium Rich.

(Cucurbitaceae)

Globularia alypum L. (Globulariaceae)

Inula viscosa (L.) Ait. (Asteraceae)

Juniperus phoenicea L. (Cupressaceae)

Laurus nobilis L. (Lauraceae)

Marrubium vulgare L. (Lamiaceae) Mentha spicata L. em. Huds.

(Lamiaceae) Morus nigra L. (Moraceae)

Myrtus communis L. (Myrtaceae)

Ocimum basilicum L. (Lamiaceae) Olea laperrini B. and T. (Oleaceae)

Olea europaea L. (Oleaceae)

Origanum glandulosum Desf.

(Lamiaceae) Origanum majorana L. (Lamiaceae) Peganum harmala L. (Zygophyllaceae)

Pinus halepensis L. (Pinaceae)

Pistacia lentiscus L. (Anacardiaceae)

Quercus ilex L. (Fagaceae)

Rosmarinus officinalis L. (Lamiaceae) Ruta chalepensis L. (Rutaceae)

Ruta montana L. (Rutaceae)

Salvia officinalis L. (Lamiaceae) Thymus ciliatus (Desf.) Benth.

(Lamiaceae)

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Table 2 : Percentages of respondents in Hodna region according to their age group Number of respondents

Percentage of respondents

Age group (Years) Men

Women

Men

Women

Youths (20–40)

60

5

70.59%

5.88%

Adults (41–60)

14

2

16.47%

2.35%

Elderly (61–above)

3

1

3.53%

1.18%

Different plant parts (both underground and above ground) such as leaf, root, flowers, fruits and whole plant were determined to be used to treat AHT. The plant parts most commonly used were the aerial parts (49%), leaves (18.37%) and flowers (12.24%). The local people employed a variety of methods including decoction or infusion to prepare remedies for treating different diseases.

Decoction and infusion were the predominant methods of preparation. The most frequently used form of preparations were decoction (48%) followed by infusion (25%) and others (23%).

to make an inventory as complete as possible with medicinal plants used to treat arterial hypertension in the Hodna region of Algeria.

The results showed 35 species recorded, with maximum representation from the family Lamiaceae with nine species. In the viewpoint of ethno-botany and pharmacology, the aerial parts were the most used while decoction and infusion were the most practiced. Furthermore, this study was used to assess knowledge of traditional practices used by the population of Hodna. But it is important to experimentally validate the remedies identified by rigorous scientific protocols.

CONCLUSION

ACKNOWLEDGEMENTS

In conclusion, this area was the subject of several studies into the valorization of traditional pharmacopoeia (Sari et al., 2011, Sari et al. 2012, Hendel et al., 2012) and this study was an addition to previous studies.

Thus, the present work was undertaken in order

We are very much grateful to all the local herbalists, healers and villagers who shared their knowledge on the use of medicinal plants with us. Without their contribution, this study would have been impossible.

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Benkhedda S., Chibane H., Temmar M, Ziari D. (2004) Hypertension in Algeria an epidemiological overview. Fourteen European meeting on Hypertension.13– 17.

F.A.O (1966). Study agropedological, district of Constantine, Roma, 146–150.

Hendel N., Larous L., Sari M., Boudjelal A., Sarri Dj; (2012) Place of Labiates in folk medicine of the area of M’sila (Algeria). Global J. Res. Med. Plants & Indigen. Med., Volume 1, Issue 8, 315– 322.

Le Houerou H.N. (1995) Bioclimatology and Biogeography of the arid steppes of North Africa. biodiversity, sustainable

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development and desertisation.

Mediterranean optional, Serial B, 10.

Maire R. (1952-1987) Flora of North Africa (Morocco, Algeria, Tunisia, Tripolitania, Cyrenaica and the Sahara).

16 vol., Le Chevalier Publisher, Paris.

Moreau S., Benziene A.S., Boudjadja A., Gaouar A., Kaabeche M., Moali A., Sellami D. (2005) Plan of management of site of Mergueb. Wilaya of M’sila (Algeria). PROJECT DGF/PNUDALG/G35/2005.

Ozenda P. (1983) Flora of the Northern Sahara.

CNRS, Paris.

Quezel P. and Santa S. (1962-1963) New flora of Algeria and Southern desert regions.

2 Tomes, CNRS, Paris.

and floristic analysis of medicinal plants used in the Southeast region of M'sila.

International Workshop, The Knowledge, the Valuation and Sustainable Management of Natural Resources in Arid Zones; Nov. 16-17: Biskra, DZ. University of Biskra.

Sari M., Hendel N., Boudjelal A., Sarri Dj. (2012) Inventory of medicinal plants used for traditional treatment of Eczema in the region of Hodna (M'sila Algeria). Global J. Res. Med. Plants & Indigen. Med., Volume 1, Issue 4, 97– 100.

Temmar. M., Chibane A.,.Guendouz N.¸ Bouamra A., Bouafia M.T., Hamida F. (2010) Prevalence of hypertension and cardiovascular risk factors. Study El Menia. 1–18.

Sari M., Hendel N., Sarri Dj., Boudjelal A., Benkhaled A. (2011) Ethnobotanical

Source of Support: Nil

Conflict of Interest: None Declared

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Research article ISOLATION AND PARTIAL CHARACTERIZATION OF UREASE INHIBITOR FROM AGAVE SPECIES Latha K1*, Manasa C2 1 2

*

Department of Biotechnology, Government Science College, Hassan-573201, Karnataka, India Department of Bioscience, Hema Gangothri P G Centre, Hassan-573201, Karnataka, India Corresponding Author: Email: latha.gsch@gmail.com

Received: 08/07/2012; Revised: 25/08/2012; Accepted: 31/08/2012

ABSTRACT Enzymes are biological catalysts ubiquitously distributed in nature. The activity can be modulated by small organic molecules, inorganic ions. There are 3 types of inhibition competitive, uncompetitive, and non competitive. In the present study it has been identified and partially purified competitive Urease inhibitor from members of the Agave family. (Agave americana L., Agave cantala Roxb. and Agave sisalana Perrine.). Urease inhibitor was fractionated on activated charcoal.

In fraction-1 total extract was eluted, fraction-2 eluted with water, fraction-3 eluted with 50% Acetone, Fraction-4 eluted with 100% acetone. Total extract of Agave cantala, Agave sisalana showed inhibitor activity, Agave americana showed the presence of an activator, the activator was not further characterized. Acetone extract of Agave cantala showed inhibitor activity; Agave americana showed the presence of an activator, Agave sisalana had an activator. On Thin Layer Chromatography, single band was observed in the extracts which showed inhibitor activity. The Ki value of inhibitor was found to be 9.73 µg of protein. The fluorescence characterization of 4 fractions was studied.

KEY WORDS: Urease, Agave, isolation, partial characterization

Cite this article: Latha K and Manasa C (2012), ISOLATION AND PARTIAL CHARACTERIZATION OF UREASE INHIBITOR FROM AGAVE SPECIES, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 411–419

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INTRODUCTION

MATERIALS & METHODS

Enzymes are biological catalysis ubiquitously distributed in nature. All the reaction of a living cell is catalyzed by enzymes, which can increase the rate of reaction to an extent of up to 1019 times than uncatalyzed reaction. Enzymes not only catalyze individual reactions but also are capable of regulating entire metabolic pathways. Urease (EC 3.5.1.5) is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. Urease is an enzyme found in microorganisms, plants and animals, involved in the metabolism of urea and Arginine. It belongs to the class-3 Hydrolasess.

The reaction catalyzed by Urease is as follows (NH2)2CO + H2O → CO2 + 2NH3. The incorporation of urease inhibitors, and nitrification inhibitors into urea and ammonium containing fertilizers should be recommended as a best management practice. Diterpene esters were isolated from Euphorbia decipiens expressed inhibitory activity against urease (Ahmad VU et al., 2003). Bacillus pasteurii urease inhibitor was isolated from Euphorbia decipiens (Muhammad Arif Lodhi.and et al., 2006). Potent Urease inhibitors were isolated from Hypericum oblongifolium (Arfan M, and et al., 2010). Urease inhibitor was also isolated from Cucumis melo seeds which showed inhibitor activity in-vitro the inhibitor was susceptible to protease (Makkar H P S, and et al., 1980). Urease was isolated from Aspergillus niger and reported hydroxyurea is competitive inhibitor of urease activity, and Nmethylurea is weak uncompetitive inhibitor (Patrickt, Smit and et al., 1993). Tight-binding inhibitors of urease were isolated from jack bean (Stephen W Faraci, et al., 1995).

Methanolic extracts of edible plants and seaweeds were tested for their inhibitory activity against Jack bean urease (Samah Shabana and et al., 2010)

Materials

In our study we have identified Urease inhibitor from the members of Agave family and we have partially purified and characterized Urease inhibitor from Agave plants.

Soyabean (Glycine max) was purchased locally. Agave americana L., Agave cantala Roxb. and Agave sisalana Perrine., plants were identified by Dr Cleatus D Souza Professor Dept of Biochemistry University of Mysore and myself. Plants were collected from the wild in and around Hassan District, Karnataka, India. All the Chemicals and solvents were purchased locally and were of laboratory reagent grade.

Method [a] Extraction of Urease and Urease assay Seeds of soybean were powdered using a pestle and mortar; Powdered Seeds were suspended in phosphate buffer 50 ml, pH 7.4 and stored for 30 min. It was allowed to settle down and the decanted solution was further filtered using a filter paper and the filtrate was used as a source of Urease enzyme (Ainsworth, 2008). Urease was assayed using urea as the substrate. Urease converts urea to carbon dioxide and ammonia. The ammonium ion reacts with phenol in the presence of Hypochlorite to give blue dye indophenols, the presence of nitroprusside acts as a catalyst and increases the yield of indophenols, which is blue in color with a λmax of 600 nm.

(Weatherburn, M.W.1967) Urea standards Urea standard (66.7 mM) was prepared by dissolving 20 mg of urea in 50 ml of distilled water Assay mixture • To 0.2 ml of 66.7 mM urea 0.7 ml of phosphate buffer (pH 7.4) was added. The reaction was started by the addition of 0.1 ml of enzyme extract. The reaction was carried out at 37°C for 3 min. It was stopped by the addition of 2 ml of phenol nitroprusside reagent (4.7 g phenol in 100 ml water containing 6 mg of sodium nitro prusside. Phenol nitro prusside mixture was mixed with 2 ml of alkaline

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hypochlorite solution that is 2 gm of NaOH was dissolved in 92.5 ml of water. To this 7.5 ml of 5% sodium hypochlorite solution was added, mixed & stored cold).The reaction mixture was warmed at 50°C for 10 min. and then allowed to cool to room temperature. The optical density was measured at 600 nm. The color obtained was compared with that obtained using a calibration curve made with ammonium sulphate solution from 0.03 µm to 0.15 µm. One unit of enzyme activity was defined as that amount of enzyme which gives one ammonium ion at 37°C in min (McGee H 2004). Protein in the enzyme extracts was determined by using Lowry’s Method (Lowry OH et al., 1951) [b] Characterization of Urease pH optimum and Temperature optimum The pH Optimum of Urease was obtained using phosphate buffer from pH 5.8–8.0 the

entire buffer was of 0.1M ionic strength. Effect of temperatures was assayed using Urea as a substrate over a temperature range of 5–100°C at the optimum pH. (Joseph C et al., 1984) Vmax and Km Vmax and Km for Urease was determined using urea as substrate from 1–6 mM with all other conditions maintained as described for assay of Urease. Vmax and Km were then calculated from the Line weaver Burk plot. [c] Isolation of inhibitor Inhibitor activity of Agave extract was determined by mixing 0.1 ml of the extract with Urease enzyme. (Rao. D.L.N et al., 1986). The activity of enzyme in the absence of inhibitor was used as 100% activity, the activity in the presence of inhibitor was determined and the percentage of the activity inhibited was calculated as follows’

% inhibition= (urease activity without inhibition – urease activity with inhibition)*100 Urease activity without inhibition

1 unit of inhibitor activity was defined as that amount of inhibitor, which decreased the activity of Urease by 1%

Fractionation of Inhibitor Activity The Urease inhibitor was fractioned on activated charcoal as follows; Activated Charcoal (1 g) was suspended in water and passed in a 5 ml syringe plugged with a cotton plug. After the water completely drained, 2 ml of Agave extract was added and the elute was collected as fraction I, the column was washed with 2 ml of water that eluted was collected as fraction II, Then the column was eluted with 2 ml of 50% acetone and collected as fraction III and finally with 100% of 2 ml acetone and collected as fraction IV. The Urease inhibitor

activity of all the fractions were determined as described above Determination of IC-50 Isolated inhibitor was mixed in increasing concentration with the enzyme prior to determine its activity. The percent inhibition was plotted against log of inhibitor concentration.

From the graph the concentration required to inhibit the enzyme by 50% was calculated.

Determination of Ki and Type of Inhibition Michaelis Menten kinetics was determined for the Urease in the presence of 20 µl, 40 µl

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and 70 µl partially purified A. sisalana inhibitor, Line weaver Burk plot for each of the experiment was plotted.

Estimation of Phenolics in the Agave extraction Phenolics were estimated in the extract sample of Agave by the modified method of phenol estimation (Bolanle A et al., 2004).

Thin Layer Chromatography Separation of Inhibitor

(TLC)

The water extracts from A. americana, A. cantala and A. sisalana was spotted on TLC plate and developed using the solvent system Chloroform: acetic Acid 99:1 (1%). The plates visualized in an iodine chamber, one spot was obtained. This was scrapped and the fraction was extracted in 3 ml water. The inhibitor activity of the extracted sample was tested as described earlier.

RESULTS AND DISCUSSION The relative activity of Urease in different seeds was tested. Soybean had far greater activity hence it was used as a source of enzyme in all our studies [Fig-1]. The pH activity profile of the Soybean Urease is shown in the optimum pH was found 7.2 [Fig-2]. The temperature activity profile is shown in [Fig3]. The optimum temperature was found 70°C with rapid loss of activity at 90°C. The Arrhenius plot for energy of activation is shown in [Fig-4]. The ∆E was found to be 4.0 Kcal/mol, which was well within the range of energy of activation of enzymes. The Mechaelis Menton plot for the Urease enzyme is shown in [Fig-5] and the Line weaver Burk plot is shown in [Fig-6]. The Vmax of enzyme is 1.02 µm and Km was 2.0 mM. The initial velocity of the enzyme was calculated from a

plot of activity Vs time [Fig-7] and it was17.4 nmol/min. The activity of Urease in the presence of Agave extracts is shown in Table 1, when total extract was considered: Agave sisalana appeared to have maximum inhibitor activity. On Fractionation of the inhibitor activity on activated charcoal column. The water extract of A. cantala and A.sisalana showed inhibitor activity whereas A. americana showed the presence of an activator. However acetone extract of A. americana and A. cantala had inhibitor activity whereas A. sisalana had an activator. The activator was not further characterized. Antiurease activity reported in Et-OH and five Me-OH extracts of medicinal plants (D.L.N. Rao, and S.K. Ghai 1986).

Found potent inhibitor of urease, in unripe fruits of Aegle marmelos, Rutaceae (Muhammad Shaiq and et al., 2011). Cucurbit seeds exhibit enzyme inhibitors (S K Samant, and D V Rege 1989).

The result of thin layer chromatography showed only one band, which had inhibitor activity [Fig-8]. The kinetics of inhibition by A. sisalana inhibitor is shown in [Fig-9]. The Michaelis Menten plot shown in figure -9A and Line weaver Burk plot is shown in Figure-9B indicate that the inhibition is competitive. The Ki was calculated by plotting the log of inhibitor Vs percent activity [Fig-10]. The IC50 was 9.73 µg of protein. However whether the estimated component by Biuret reaction was protein, peptide or some other component is not known whether the protein if any is the inhibitor is also not known. For want of a better reference standard, the protein in the extract was used as a reference standard. The phenol content in the Agave species was estimated and plotted along with the inhibitor activity [Fig11] from which it appears that there is no correlation between the Phenolic content and inhibition.

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[Fig-1] Activity of Urease on Different seeds

[Fig-2] pH activity profile

[Fig-3] Temperature Activity

[Fig-4] Energy of Activation

[Fig-5] Michaelis Menten Plot

[Fig-6] Line Weaver Burk Plot

[Fig-7] Time Kinetics

[Fig-8] Thin Layer Chromatography

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TABLE -1 FRACTIO ONS

ACTIVITY IN N %OF CONTROL

Agave americana

Agave cantala

Agave sisalana

Urease alone

100

100

100

Urease+ Total extract

44

53.3

12,7

Urease +column unbound fraction

108

88

67

Urease +water wash from column

108

92

78

Urease +50%acetone extract

96.4

101.3

100

Urease +100%acetone extract

81.1

88.8

127

[Fig-9A] 9A] Kinetics of Inhibition by Agave sisalana (Michaelis Menten Plot)

Fluorescence Characterization The fluorescence excitation and emission spectra are shown Fraction 1 of A. americana did not show any characteristic excitation pattern. However it had a distinct emission spectrum when exited at 232 nm with Îťmax values one at 351 nm and other at 475 nm [Fig [Fig12]. Fraction 1 of A. cantala did not show any characteristic excitation pattern, where it had

[Fig-9B] 9B] Kinetics of Inhibition by Agave sisalana Linewear Burk Plot

two distinct emission bands when excited at 272 nm, one at 306 nm and other at 395 nm [Fig-13].

13]. Fraction I of A. sisalana had an excitation maximum at 605 nm and an excitation at 470 nm [Fig-14].

[Fig The excitation and emission characteristics of fraction II were significantly different. The fraction III had an excitation maximum at 305 nm and strong emission maximum at 670 nm [Fig-15].

[Fig Fraction IV showed an emission maximum at 428 nm when exited at 260 nm and had an excitation maximum at 614 nm [Fig-16]. [

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[Fig-10] IC50 Plot

[Fig-11] 11] Phenolic content and Inhibitor Activity Plot

[Fig-12] Exited at 232 nm

[Fig-13] 13] Excited at 270nm

[Fig-14] Excited at 405 nm

[Fig-15] 15] Excited at 335nm

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[Fig-16] Excited at 260 nm

CONCLUSION The study on enzyme inhibition is a vital area of research. The present study has identified Urease inhibitor from the members of Agave family which reports partial purification and characterization of Urease inhibitor from Agave plants. A. sisalana had maximum inhibitor activity. The estimated compound needs to be studied further with better reference standards for characterization.

where to prevent ammonia loss, fertilizer manufacturers routinely add urease inhibitors, the actual chemical relative of urea to block hydrolysis, thus preserving nitrogen until it’s taken up by the plants. It is studied that inhibition of ammonia production in the cattle sheds have been known to increase milk production and also egg production in poultry farms. Most of the loss occurs through a process called hydrolysis. It is suggested that future studies should be done in this regard.

The present study may be helpful in the field of urea based commercial fertilizers, REFERENCES Ahmad VU, (2003) Chem Pharm Bull (Tokyo).

First natural urease inhibitor from Euphorbia decipiens. 51(6):719–23.

Arfan M, (2010) Ali M,Enzyme Inhib Med Chem.

Urease inhibitors from Hypericum oblongifolium 25(2):296–9.

Ainsworth C, (2008), Locking the cradle.

Science in School 8:2528W

Joseph C. (1984) Polacco and Rodney G. Winkler Soybean Leaf Urease: A Seed Enzyme? Plant Physiol. 74(4): 800– 803.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein Measurement with the Folin phenol reagent. J. Biol. Chem.

193 (1):265–75.

Makkar H.P.S, (1980) i In VitroInhibition of Rumen Urease by Melon (Cucumis

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melo) Seed Urease Inhibitor ,Journal of Dairy Science Volume 63,Issue 5,pg 785–788.

guajavaSamah Bioscience, Biotechnology, and Biochemistry Vol. 74, No. 4 pp.878–880

McGee H (2004) on food and cooking.

London, UK: Hodder & Stoughton.

ISBN: 0340831499

Stephen Faraci, W (1995) Inhibition of Helicobacter pylori Urease by Phenyl Phosphorodiamidates: Mechanism of Action Bioorganic & Medicinal Chemistry Volume 3, Issue 5, Pages 605–610

Muhammad Arif Lodhi, (2006) Journal of Enzyme Inhibition and Medicinal Chemistry Muhammad Shaiq Ali, (2011) A Potent Urease inhibitor from Fresh Unripe Fruits of Aegle marmelos (Rutaceae) Journal of the Chemical Society of Pakistan, Vol 33, No 6 Patrick

T Smith (1993) Isolation and characterization of urease from Aspergillus niger Journal of General Microbiology, 139, 957–962.

Samant S K, (1989) Some enzymes and enzyme inhibitors from cucurbit seeds, ournal of the Science of Food and Agriculture. Volume 47, Issue 3, pages 383–385, .

Shabana (2010) Inhibitory Activity against Urease of Quercetin Glycosides Isolated from Allium cepa and Psidium

Source of Support: Nil

Tarek M. Mohamed, (1999) Purification of urease from water melon seeds for clinical diagnostic kits Bio-resource Technology, Volume 68, Issue 3, Pages 215–223 Tahseen Ghous, (2010). Screening of selected medicinal plants for urease inhibitory activity. Biology and Medicine, Vol.2 (4): 64–69.

Weatherburn, M.W. (1967).

Phenolhypochlorite reaction for determination of ammonia. Analytical Chemistry 39, 971–974.

Wilfred Vermerris, Phenolic BiochemistryBy ISBN: 5163-0. 4.152–153.

Compound 978-14020-

Conflict of Interest: None Declared

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Research article EFFICACY OF AYURVEDIC / HERBAL PATENT MEDICINES IN TYPE 2 DIABETES MELLITUS AS PER THE CLAIM Mishra Subhransu Sekhar1*, Mishra Amarendra Narayan2 1

Ph.D research fellow, Fakir Mohan University / Incharge R & D, Biotechayur Pvt.Ltd. Sergarh, Odisha

2

H.O.D. school of biotechnology Fakir Mohan University, Balasore, Odisha

*

Corresponding Author: E-mail: subhransusekhar4@gmail.com; Mob: +919040593475

Received: 15/07/2012; Revised: 25/08/2012; Accepted: 30/08/2012

ABSTRACT The study was designed to evaluate the comparative efficacy of a commercially available polyherbal drug in Indian market to that of a modern sulfonylurea - Gliclazide. Currently, there are many herbal drugs available in market claiming promising results in managing type-2 diabetes mellitus.

The aim of this study was to evaluate the efficacy of one among them in type-2 diabetes mellitus along with Gliclazide as control. The present drug being called as Diazen comprising Gymnema sylvestre (Retz.) Schult, Momordia charantia L, Eugenia jambolana Lam., Tinospora cordifolia (Willd.), Trigonella foenogricum L, Withania somnifera (L.) Dunal, Cassia auriculata L., Aegle marmelos (L.) Correa, Azadirachta indica A. Juss, Curcuma longa L. For the clinical study, type -2 diabetic patients were selected voluntarily and divided in to 3 groups, each comprising 10 patients.

One group supplied only with the sulfonylurea drug Gliclazide, another group supplied only with the herbal drug Diazen whereas the last group supplemented with both the herbal drug Diazen and Gliclazide .The patients were observed for a period of one month. The herbal drug was found to be effective in bringing normoglycemia as per the claim. A review of possible mechanism of anti diabetic activity of the ingredients of Diazen was done.

KEY WORDS: Sulfonylurea, Gliclazide, Diazen, Hypoglycaemia,

Cite this article: Mishra Subhransu Sekhar, Mishra Amarendra Narayan (2012), EFFICACY OF AYURVEDIC / HERBAL PATENT MEDICINES IN TYPE 2 DIABETES MELLITUS AS PER THE CLAIM, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 427â&#x20AC;&#x201C;439

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INTRODUCTION About Diabetes mellitus Diabetes mellitus: often referred to simply as diabetes is a condition in which the body either does not produce enough, or does not properly respond to, insulin, a hormone produced in the pancreas. Insulin enables cells to absorb glucose in order to turn it into energy. In diabetes, the body either fails to properly respond to its own insulin, does not make enough insulin, or both. This causes glucose to accumulate in the blood, often leading to various complications1.

Treatment and drugs: Several groups of medicines are available in allopathic system of medicine e.g. Sulfonylureas, Biguanides, Thiazolidinediones, Alpha-glucosidase inhibitors, Peptide analogues etc. but herbal extracts are also occupied a category among these groups. Different patent medicines of different manufacturers composed of various combinations of multiple herbs have widely occupied recent pharmaceutical market.

Status of diabetes in India - India has become the diabetic capital of the world with 50.8 million (7.1%) of its people suffering from diabetes 2, 3.

From the available region wise population based studies it is clear that in the last two decades, there has been a marked increase in the prevalence of diabetes among both urban as well as the rural Indians4. Out of the total diabetics the total figure of Type-1/insulin dependent diabetes mellitus is 1–5% and the rest 95–99% are of type -2 insulin independent Diabetic mellitus patients 3, 4 .

In a study over one year, it was observed that mortality amongst hospitalised patients with non insulin dependent diabetes mellitus (NIDDM) was nearly 20% and the mean age of death in these patients is 61 year. Ischemic heart disease and cerebro-vascular accident accounted for 80% of deaths in this group5.

Age group/ Male and Female Out of total the maximum number of diabetic population comes under the age of 40– 50years of both male and female. Maximum diabetic patients are of age ≥ 50years 6.

This paper discusses about a trial conducted regarding the efficacy of such herbal formulated drug available in current market.

Table no-1 Statistical data of diabetic peoples suffering type-2 in rural India 6 SEX

DIABETICS PRE DIABETICS 6.20% 13.50% MALE 9.60% FEMALE 4.40% Table-2 Age groups (from total diabetic population) suffering with Type 2 diabetes in rural India6 40–50 years ≥50 years ≤40years

The crude prevalence rate of diabetes in urban areas is about 9% and that the prevalence in rural areas has also increased to around 3% of the total population7.

43.30% 50% 0.70% Use of Allopathic and Ayurvedic drugs in Diabetes mellitus type-2 According to World Health Organization in India 80% of population directly or indirectly use herbal drugs8. Although people use

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allopathic drugs as principal and emergency medicine they also take some form of herbal/Ayurvedic drugs as an adjuvant therapy8. In traditional system of medicine and the tribal populations in India so many different herbs are used in practice for the treatment of diabetes 9. Most of them are not tested yet for their significant hypoglycaemic properties. In current pharmaceutical market there are a number of herbal drugs available for the treatment of Diabetes mellitus. So it is necessary to take the independent clinical trials out of these available drugs to get confirm and to make the people aware about the true efficacy of these drugs10.

MATERIALS AND METHODS A herbal drug with the following composition (DIAZEN from the manufacturer Green milk health products, Apex herbal division), which is already available in market was selected and it’s efficacy was compared with the standard allopathic drug Gliclazide (Sulfonylurea group) to prove the efficacy.

The safety of this product and the adverse reactions were also studied in this trial. The herbal drug composed of the multiple herbal extracts composing of following herbs. Each soft gelatine capsule of 200 mg (total weight with all the ingredients mentioned in Table-3), Diazen (the trade name) was selected because it’s composition is only of herbs and no metallic drugs.

Table-3 List of Ingredients in the Herbal formulation ‘Diazen’ Herbs Common name Part Used Mg/200 mg cap.

Gudmari Leaf 15 Gymnema sylvestre (Retz.) Schult.

Kalera Whole fruit pulp 30 Momordia charantia L. Jamu Seed 18.75 Eugenia jambolana Lam. Guduchi Stem 15 Tinospora cordifolia (Willd.) Miers Methi Seed 10 Trigonella foenogricum L. Aswagandha Root 20 Withania somnifera (L.) Dunal Avartaki Flowers & Roots 25 Cassia auriculata L. Bael Dried fruit pulp 18.75 Aegle marmelos (L.) Correa Neem Leaf 7.5 Azadirachta indica A. Juss Haldi Rhizome 3.5 Curcuma longa L. The study duration was one month in which the hypoglycaemic activity was studied. The included subjects were divided into 3 groups each consisting of 10 individuals. The control group was supplied with Gliclazide 80 mg/24hr. The doses /24 hr were given as per the Hyperglycemic condition of the patients.

The trial group was administered with soft gelatine Capsules of Diazen. Third group patients were administered with the combination of medication composed of the herbal drug with the Sulfonyleurea drug Gliclazide.

In every 15 days interval the blood sugar test for both fasting and postprandial of each

patient were repeated. Dietary restrictions were also applied to all these patients.

Observation: All the symptoms were noted prior to the trial. Very common among those were constipation, fatigueness, polyurea, polydipsia. The plasma glucose level (both fasting and post prandial) were also recorded.

Out of 10 patients 9 patients feeling normal and the complications like constipation, fatigueness, muscle weakness, palpitation, polyurea, polydipsia were also absent. Other symptoms like recurrent urine infection, blurring vision, dyspepsia, were also found in the diabetic patients

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Table-4 Results for patients using both herbal drug and Allopathy drug gliclazide-80Mg. (For a duration of one month) Sl No

Age

Blood glucose

After 15days

22 days

30days

Doses of medicines

F.B.S.

P.P.B.S

F.B.S.

P.P.B.S.

F.B.S.

P.P.B.S.

F.B.S.

P.P.B.S.

(Mg/Dl)

Mg/Dl

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

DIAZEN

GLICLAZIDE

419

260

340

199

301

145

178

2CAPS B.D.

80Mg B.D.

1

40

341

2

44

306

386

208

269

188

208

108

133

2CAPS B.D.

80Mg B.D.

3

41

290

370

190

250

112

140

79

96

2CAPS B.D.

80Mg O.D

4

45

285

355

170

230

108

126

85

99

2CAPS B.D.

80Mg O.D

5

48

310

365

215

290

171

200

110

156

2CAPS B.D.

80Mg B.D.

6

55

320

380

225

285

182

225

109

129

2CAPS B.D.

80Mg B.D.

7

54

286

330

180

240

126

142

103

134

2CAPS B.D.

80Mg O.D

48 8 Table-5

330

410

265

332

173

290

111

136

2CAPS B.D.

80Mg B.D.

9

52

322

385

256

310

166

275

98

120

2CAPS B.D.

80Mg B.D.

10

42

260

325

156

236

98

123

66

110

2CAPS/B.D.

80Mg O.D.

Abreviation –B.D.- bis die (BD), a latin term meaning twice per day .O.D.-Once Daily , Mg/Dl- Milligrams per Deciliter.

Table -5 Days after treatment with both Herbal & Allopathic drugs Herbal + Glidazide 400 Blood glucose (ppbs), mg/dl

Blood glucose (fbs), mg/dl

400

300

200

100

0 0

10

20

30

Days after treatm ent

In this group patients were required low dose of gliclazide. For all patients in this group the blood sugar level came down to normal range with an average of 21 days.

Second group of patients who were receiving herbal therapy only, got the normal blood glucose level but it took time longer

300

200

100

0 0

10

20

30

Days after treatm e nt

duration than patients consuming both herbal therapy and Gliclazide .Within first 15 days the blood sugar decreased but not up to the normal range of (Fasting blood sugar) 70– 110 mg/Dl and Post Prandial blood sugar 90– 140 mg/Dl. Out of 10 patients in 7 patients the response was up to the desired as the drug able to bring normoglycemic stage.

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TABLE NO. 6 RESULT WITH IN ONE MONTH OF ONLY HERBAL THERAPY Sl. no

Age

Blood glucose

After 15days

22 days

Doses of medicines

30days

F.B.S.

P.P.B.S.

F.B.S.

P.P.B.S.

F.B.S.

P.P.B.S.

F.B.S.

P.P.B.S.

(Mg/Dl)

Mg/Dl

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

DIAZEN

1

42

268

345

210

250

179

202

126

167

2CAPS B.D.

2

45

305

375

280

330

285

355

200

260

2CAPS B.D.

3

43

190

230

115

136

86

105

79

96

2CAPS B.D.

4

49

220

265

170

230

129

158

103

124

2CAPS B.D.

5

55

258

325

205

230

171

200

115

175

2CAPS B.D.

6

58

210

280

160

204

118

130

96

129

2CAPS B.D.

7

62

176

205

168

198

126

142

105

134

2CAPS B.D.

8

48

196

278

130

167

100

140

99

112

2CAPS B.D.

9

52

290

370

210

270

137

176

98

130

2CAPS B.D.

10

54

285

315

255

290

205

245

125

157

2CAPS/B.D.

Abreviation –B.D.- bis die (BD), a latin term meaning twice per day .O.D.-Once Daily , Mg/Dl- Milligrams per Deciliter.

Table – 7 Days after treatment with Diazen

Herbal 400 Blood glucose (ppbs), mg/dl

Blood glucose (fbg), mg/dl

400

300

200

100

0

0

10

20

Days after treatm ent

30

300

200

100

0 0

10

20

Days after treatm ent

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It took more than 1 month to get the glucose level up to the normal range. The Control group or the patients who were supplied with the only sulfonylurea group of medicine Gliclazide got their sugar level normal. Out of 10 patients 8 got their sugar

level normal with in the period 30 days from the hyperglycemic condition (Table-No-8 Patient No-3 -10). After the sugar level got to normal range the dose reduced to 80mg per day. The initial doses of medication were Gliclazide 80 mg twice daily.

TABLE NO. 8 Patients with the standard drug Gliclazide (BD) Sl. no

Age

Blood glucose

Sex

After 15days

22 days

Doses of medicines

30days

F.B.S.

P.P.B.S.

F.B.S.

P.P.B.S.

F.B.S.

P.P.B.S.

F.B.S.

P.P.B.S.

(Mg/Dl)

Mg/Dl

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

(Mg/Dl)

GLICLAZIDE

1

44

M

306

376

245

310

200

281

145

210

80Mg B.D.

2

42

F

267

320

196

259

176

255

143

190

80Mg B.D.

3

56

M

256

336

176

250

145

196

109

142

80Mg B.D.

4

67

M

246

316

170

230

108

126

82

99

80Mg B.D.

5

45

F

290

340

210

270

174

200

101

130

80Mg B.D.

6

56

M

210

280

165

245

132

225

106

132

80Mg B.D.

7

49

F

190

269

108

185

86

142

67

90

80Mg O.D.

8

47

F

230

310

155

225

112

290

86

119

80Mg B.D.

9

53

M

259

339

175

235

139

275

93

122

80Mg B.D.

10

59

M

180

225

136

186

88

123

69

89

80Mg O.D.

Abbreviation â&#x20AC;&#x201C; B.D.- bis die (BD), a latin term meaning twice per day .O.D.-Once Daily , Mg/Dl- Milligrams per Deciliter.

Table-9 Glidazide 400 Bloodglucose(ppbs), m/dl

Blood glucose(fbs), mg/dl

400

300

200

100

0 0

10

20

Days afte r tre atm e nt

30

300

200

100

0 0

10

20

Days afte r tre atm e nt

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After the glucose level came down to normal state the doses were reduced to 80 mg once/Day from the initial 80mg twice a day dose.

RESULTS AND DISCUSSION Taking the standard normal blood glucose range (F.B.S.-70 mg/Dl-110 mg/Dl and P.P.B.S.-90 mg/Dl-140 mg/Dl) the comparative study of the herbal drug with the standard drug Gliclazide shows that the herbal drug is effective at least in lowering the blood glucose level up to normal range. No case of severe hypoglycemia has been found in any of the patients. In case where both herbal drug and Gliclazide simultaneously used condition of hypoglycemia may arise (Table-4 patient no10). In case of high doses of Gliclazide may also cause severe hypoglycemia. The group using both herbal drug and Gliclazide; in case of 1 patient (Table-4 patient no.1) the glucose control was not so effective and took longer time to make the glucose level to normal in spite of in an increased doses .The main possible cause is the Obesity and distorted lipid profiles Regarding the efficacy of the herbal drug ; there was no contra indicatory undesired effects like nausea, vomiting, body rashes or belching dyspepsia etc. were observed by any of the patients. From this trial it is established that the drug “Diazen” has the desired efficacy to be used in the treatment of diabetes mellitus type2(NIDDM). The patients who were receiving the only herbal therapy, 6 out of 10 patients got their glucose level normal in one month, 2 out of 10 patients have got moderate result near to normoglycemic state. One out of ten patients was observed not to be up to satisfactory result.

There were no such adverse effects noticed in case of patients who used this drug during the trial study Mechanism of action of Gliclazide Gliclazide is a sulphonylurea drug with an intermediate half-life of around 11 hours11. It is extensively metabolized. The molecule contains an azabicyclo-octyl group which

confers special properties on the basic sulphonylurea moiety. Gliclazide stimulates insulin secretion through the beta cell sulphonylurea receptor, and possibly through a direct effect on intracellular calcium transport.

It specifically improves the abnormal first phase insulin release in type 2 diabetes, and also has an effect on the second phase. This pattern of insulin release is thought to explain the lower incidence of hypoglycaemic episodes and weight gain compared with some other sulphonylurea. There is also a reduction in hepatic glucose production and improvement in glucose clearance, without changes in insulin receptors. This suggests a possible postreceptor effect on insulin action, perhaps by stimulation of hepatic fructose-2,6bisphosphatase and muscle glycogen synthesis 12 .

Possible mechanism of anti diabetic action of the herbal drug Gymnema sylvestre (Retz.) Schult. - Sanskrit name: Meshasringi, Madhunasini; FamillyAsclepiadacae 13 Hypoglycemic action mechanisms Research shows that a water-soluble extract of Gymnema sylvestre (Retz.) Schult., causes reversible increases in intracellular calcium and insulin secretion in mouse and human β-cells when used at a concentration (0.125 mg/ml) without compromising cell viability. Hence forth these data suggest that extracts derived from Gymnema sylvestre (Retz.) Schult . may be useful as therapeutic agents for the stimulation of insulin secretion in individuals with Type 2 Diabetes. Gymnema leaves raise the production of insulin by regeneration of the cells in the pancreas that produce insulin 14.

Research has shown that Gymnema also improves glucose uptake by cells by increasing the activity of the glucose utilizing enzymes, and stops adrenaline15 from stimulating the liver to produce extra glucose, thereby controlling blood sugar levels16.

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Momordia charantia L .-Sanskrit name: Karavellaka, Family – Cucurbitaceae 17 Antidiabetic mechanism – Bitter melon contains a lectin that has insulin-like activity18.

The insulin-like bioactivity of this lectin is due to its linking together 2 insulin receptors19. This lectin lowers blood glucose concentrations by acting on peripheral tissues and, similar to insulin's effects in the brain, suppressing appetite 20, 21.

The proven hypoglycaemic properties through animal trial experiments can be summarized as follows:-

indicating that it may modulate insulin release which have observed with an increase in insulin level with EJ water ext. (E. jambolana Lam. water extracts) treatment31. Further, the flavonoids also stimulate 16% increase in insulin release in vitro from pancreatic islets32.

All previous animal model trials are in concordance with earlier reports where EJ was found to increase insulin secretion 33.

Tinospora cordifolia (Willd.) Miers; Sanskrit name-Guduchi, Amrita; Family 34 Menispermaceae Anti diabetic mechanism

4. Inhibition of glucose-6-phosphatase and fructose-1, 6-bisphosphatase in the liver 26

Aqueous extract causes a reduction in blood sugar in alloxan induced hyperglycaemias in rats and rabbits in the dose of 400 mg/kg. The aqueous extract also exhibits some inhibitory effect on adrenaline-induced hyperglycemias.

Ethyl acetate extract of its roots has afforded a pyrrolidine derivative with hypoglycaemic activity in rabbits35. The Water extract of Guduchi has hypoglycaemic properties and used to treat diabetes mellitus. It has been estimated in animal model that 400 mg/Kg is equivalent to the action of 1unit/kg insulin36.

5. Stimulation of red-cell and hepatic glucose6-phosphate dehydrogenase activities 27

Trigonella foenogricum L . , Sanskrit name – Methika, Family- Fabaceae 37

6. Inhibition of glucose transport at the brush border of the small intestine 28

Antidiabetic mechanism

Pancreatic activities1. Insulin promoting or mimetic 22.

2. Increased GLUT4 transporter protein of muscles 23-24.

3. Increased glucose utilization in liver and muscle tissues 25.

Eugenia jambolana Lam. , Sanskrit name: Jambu, Family-Myrtaceae 29.

Possible Mechanism of hypoglycaemic action- A study in vitro model systems shows aqueous extracts from Eugenia jambolana Lam. (Myrtaceae) seeds have an inhibitory action on carbohydrate hydrolyzing enzymes, namely, porcine pancreatic á-amylase, rat intestinal α-glucosidase, and sucrose. These findings emphasize that inhibition of carbohydrate hydrolyzing enzymes is one of the mechanisms through which E. jambolana exerts its hypoglycemic effect in vivo 30. EJ has been reported to show significant antihyperglycaemic activity in mild diabetes rats which have functioning pancreatic β cells

The possible Mechanism behind the hypoglycaemic property is Fenugreek may increase the number of insulin receptors in red blood cells and improve glucose utilization in peripheral tissues, thus demonstrating potential anti-diabetes effects both in the pancreas and other sites38.

The amino acid 4hydroxyisoleucine, contained in the seeds, may also directly stimulate insulin secretion.

Fenugreek seed has remarkable power to reduce blood sugar level hence used in diabetes39 .Fenugreek seeds contain alkaloids, including trigonelline, gentianine and carpaine compounds, fibers, 4-hydroxyisoleucine and Fenugreekine, a component that may have hypoglycemic activity40.

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Withania somnifera (L.) Dunnal , Sanskrit name-Aswagandha/Ajagandha, Family – Solanaceae41 Antidiabetic mechanism Possible mechanism of Hypoglycaemic action as revealed from different animal model trials are that it has (The root extract and leave extract) got antioxidant properties and free radical scavenging activities42.

Centuries of Ayurvedic medical experience using Withania somnifera (L.) Dunal have revealed it to have pharmacological value as an adaptogenic.

The activities of liver G6P (glucose-6phosphatase) and serum enzymes like AST (aspartate transaminase), ALT (alanine transaminase), ACP (acid phosphatase) and ALP (alkaline phosphatase) when assayed (method of King) significantly found increase in the diabetic rats when compared to those of normal control rats43. But the activities of liver G6P and serum AST , ALT , ACP and ALP significantly decreases in diabetic rats treated with WSREt (Withania somnifera (L.) Dunal root extract). WSREt and WSLEt(leaf extract) replenishes liver glycogen stores and suppresses the hepatic gluconeogenesis by decreasing activity of G6P44 .

Cassia auriculata L., Sanskrit Avartaki, Family -Fabaceae

name-

The dried flowers and flower buds are used as a substitute for tea in case of diabetes patients. How does it work? Cassia auriculata L. might increase the body's production of insulin. This property is confirmed through a laboratory trial upon animal models in the Department of Biochemistry Faculty of Science, Annamalai University.

The possible mechanism by which CFEt (Cassia auriculata L. flower extract) brings about its anti-hyperglycemic action may be by potentiating the pancreatic secretion of insulin from β-cell of islets or due to enhanced transport of blood glucose to peripheral tissue.

This was clearly evidenced by the increased

level of insulin in diabetic rats treated with CFEt.45-46 Aegle marmelos (L.) Correa, Sanskrit nameBilwa, Family-Rutacaeae47 Possible mechanism of Antidiabetic properties of Aegle marmelos (L.) Correa extract.

Aegle marmelos contain the minerals like Cu, Ni, Zn, K, and Na were found to be in trace amounts, whereas Fe, Cr, and V levels were found in marginal levels. These minerals play a role to maintain normoglycemia in blood by stimulating pancreatic beta cells to secret insulin48.

Azadirachta indica A. Juss , Sanskrit nameNimba, Family –Meliaceae49 Possible mechanism of antihyperglycemic effect Effect of Azadirachta indica A.Juss leaf extract on serotonin inhibition in glucose mediated insulin release in rat pancreas was studied in vitro to elucidate the possible mechanism of antihyperglycemic effect of A. indica leaf extract. A. indica leaf extract blocks significantly the inhibitory effect of serotonin on insulin secretion mediated by glucose50.

In the animal model experimental trial (tudied in normal and streptozotocin-induced diabetic rabbits) studies it has been proved that, “A. indica leaf extract, in itself, was found to have no action on peripheral utilization of glucose or on hepatic glycogen. The reduction in peripheral utilization of glucose and glycogenolytic effect is due to the complete block of epinephrine action by A. indica leaf extract. It almost completely block the action of epinephrine (the insulin antagonistic hormone) in diabetic rabbits and to a certain extent in normal ones 50-51 Aqueous leaf extract also reduces hyperglycaemia in streptozotocin diabetes and the effect is possibly due to presence of a flavonoid, Quercetin 52-53.

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Curcuma longa L. , Sanskrit name-Haridra, Family: Zingiberaceae54

antioxidant properties55.

Anti diabetic potentials

CONCLUSION

The hypoglycaemic property is due to the Curcumin, or diferuloyl methane, is the yellow pigment extracted from turmeric. Curcumin exhibits an even more pronounced anti diabetic action.

As the results observed from all the patients based upon the result of fasting blood sugar and postprandial sugar the herbal drug which was chosen out of many herbal Ayurvedic drugs has proved out to have the desired antihyperglycemic efficacy.

Also it has the composition of standardized herbal extracts to combat day to day diabetic complications like constipation, fatigues, polydipsia, polyurea etc. .Also it has the property to resurrect the lipid profile of blood. So as per the claim the drug has the desired efficacy for use in the treatment of Diabetes mellitus type -2 as per the claim.

The study (in animal models) reveals that curcumin feeding improves the metabolic status in diabetic conditions, despite no effect on hyperglycaemic status or body weight. The mechanism by which curcumin improves this situation is probably by virtue of its hypocholesterolemic influence and its

and

free-radical-scavenging

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54. API VOL-1 Part-1 Page-60–61 55. L.A. Usman, A.A. Hamid, O.C. George, O.M. Ameen, N.O. Muhammad, M.F. Zubair and ,A. Lawal World Journal of Chemistry 4 (2): 178-181, 2009

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 440â&#x20AC;&#x201C;447 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article ANTIVIRAL ACTIVITY OF LATHAKARANJA (CAESALPINIA CRISTA L.) CRUDE EXTRACTS ON SELECTED ANIMAL VIRUSES Patil Usha1*, Sharma M C2 1

Assistant Professor / Reader, Department of Dravyaguna Vigyan, Sri Jayendra Saraswati Ayurveda College, Nazarathpet, Chennai, Tamil Nadu, India 2 Director (Retd.) National Institute of Ayurveda (NIA), Jaipur, Rajasthan, India *

Corresponding Author: Mob: +919094948098; Email: ushapatildg@gmail.com

Received: 20/07/2012; Revised: 24/08/2012; Accepted: 26/08/2012

ABSTRACT Viruses cause economically important diseases to animals and antiviral drugs are not commonly used in veterinary practice. Present study reports the antiviral effect of Caesalpinia crista (Lathakaranja) crude extracts the drug mentioned in Ayurvedic literature for krimighna activity. The drug was evaluated using battery of phyto-chemical tests and crude extracts of aqueous, methanol, ethanol and chloroform was prepared. Antiviral activity against paramyxovirus and orthomyxovirus isolates recovered from disease outbreaks in poultry birds was tested. Aqueous, ethanol and methanolic extracts of lathakaranja showed complete inhibition on paramyxovirus while showing highly significant inhibitory activity on orthomyxovirus. Results of the study conclude that the medicinal plant C.crista might be useful against economically important viral pathogens of poultry birds.

KEY WORDS: Antiviral Activity, Lathakaranja, Caesalpinia crista, crude extracts, phyto-chemical study

Cite this article: Patil Usha and Sharma M C (2012), Antiviral activity of lathakaranja (Caesalpinia crista L.) crude extracts on selected animal viruses, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 440â&#x20AC;&#x201C;447

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INTRODUCTION Viruses are obligate intracellular parasites, which contain little more than bundles of gene strands of either RNA or DNA, and some are surrounded by a lipid-containing envelope.

Unlike bacteria, fungus and parasites, viruses are not autonomous organisms and therefore, require living cells in which they replicate.

Consequently, most of the steps in their replication involve normal cellular metabolic pathways, and this makes it difficult to design a treatment to attack the virion directly or its replication, without accompanying adverse effects on the infected cells (Wagner and Hewlett, 1999). Today viral diseases cause devastating endemic diseases both in animals and humans. Anti-virals are used clinically to limited extent in human medicine while these expensive antiviral drugs are not economically viable options in veterinary practice. The development of new medicinal plant products is vital in controlling the threats posed by pathogenic viruses. In this context various research groups in Asia, Far East, Europe and America have given particular attention to develop antiviral agents from their native traditional plant medicines.

Drugs acting on microbial agents have been mentioned in Ayurvedic texts as Krimighna Dravyas. The drug is known to act in Kushta (Sushruta Samhita Suthra Sthana 38/5, 38/10; Bhavaprakasha Nighantu; Saligrama Nighantu), Krimigna (Kayadeva Nighantu; Bhavaprakasha Nighantu). In addition, a special chapter has been mentioned about krimiprakarana in Charaka samhita, vimanasthana chapter 7. In Sushruta Samhita Uttaratantra chapter 54, krimi and its treatment protocol has been mentioned. Seed kernel of Lathakaranja with sour gruel elevates dysentery with mucous blood and gripping pain (Vrindha Madhava 6/6).

Many traditional medicinal plants have been reported to have strong antimicrobial activity (Makhloufi et al., 2012) and some of them have already been used to treat animals and people who suffer from viral infection

(Hudson, 1990; Venkateswaran et al., 1987).

Several hundred plant and herbs species that have potential as novel antiviral agents have been studied and wide variety of active phytochemicals, including the flavonoids, terpenoids, lignans, sulphides, polyphenolics, coumarins, saponins, furyl compounds, alkaloids, thiophenes, proteins and peptides have been identified. Some volatile essential oils of commonly used culinary herbs, spices and herbal teas have also exhibited a high level of antiviral activity (Cowan, 1999).

Lathakaranja (Caesalpinia crista L.) is one of the herbs mentioned in all Ayurvedic texts and has been generously used all over India since centuries. Traditionally, it is used in post partum period, as it is a uterine stimulant, to cleanse the uterus. It also alleviates fever, oedema and abdominal pain during this period.

In the seeds of C. crista, bitter principles like bonducin and natin have been found in addition to other constituents like linolic acid, fatty acids, seta sitosterol, linolenic acids and 13 types of diterpenoids. Caesapin is the major constituent extensively studied for therapeutic effects. Percentage of nut-oil yield is 60â&#x20AC;&#x201C;80%, kernel yield is 20%, bonducellin has been isolated and its structure established recently (Satnami and Yadava 2011). Studies have shown the antiascaridal (Javed et al., 1994), antimalarial (Linn et al., 2005) and anthelmintic (Jabbar et al., 2007) activity of crude extracts of C crista. However, literature is silent on antimicrobial activity especially the antiviral activity of the crude drug extracts.

Myxoviruses and paramyxoviruses cause economically important disease both in humans and animals (Beare, 1975). For the purpose of present study myxovirus isolates causing influenza and paramyxovirus isolates causing Newcastle disease in poultry birds have been selected to study the antiviral activity. The study results regarding the evaluation of antiviral activity of lathakaranja crude extract on orthomyxo and paramyxo viruses are presented here.

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MATERIALS AND METHODS Collection material

and

identification

of

plant

Based on the textual references in Ayurveda and the available recent literatures Lathakaranja was considered for its antimicrobial activity in the present study. Its authenticity was identified and confirmed using morphological and anatomical features by Professor N.P. Kaur, Department of Botany, College of Basic Sciences, Punjab Agriculture University, Ludhiana, Punjab.

A voucher specimen was deposited at the Herbarium of the Babe Ke Ayurvedic College and Hospital, Daudhar, Moga, Punjab.

Preliminary phytochemical analysis Preliminary phytochemical analysis was done for qualitative assessment of phytoconstituents as per the standard protocols mentioned in Trease and Evans (1983).

Grounded dried powder was subjected to tests for detection of tannins, alkaloids, saponins, cardiac glycosides, anthroquinone glycosides, steroids: (terpenoids and flavonoids), resins and volatile oils.

of muslin cloth and then with Whatman No.1 filter paper. The filtrate was air dried under low heat of 50°C and stored at −20°C till further use.

Cell culture, embryonated hen’s egg and virus isolates (Saif, 2003) Bovine kidney cells (MDBK) cell line was grown as a monolayer culture in Eagles minimum essential medium (MEM) supplemented with 10% foetus bovine serum (FBS), 100 units/ml penicillin and100 µg/ml streptomycin, 20 mg/ml. The cultures were maintained at 37°C in a humidified 5% CO2 incubator. For the growth and culturing of viruses used in the study, embryonated hen’s eggs were obtained from a reputed poultry breeding company (Venkateswara Hatcheries Ltd. Ludhiana). Viruses were maintained in 10–11 days old embryos by inoculating via all allantoic route of inoculation. Standard laboratory and clinical viral isolates of Orthomyxovirus and Paramyxovirus were kindly provided by Department of Animal Biotechnology, Guru Anged Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana.

Cytotoxicity assay (Weyermann et al., 2005)

Preparation of crude drug extract from plant material Plant extracts known to have antimicrobial activity was done as per the standard procedures published elsewhere. Aqueous extraction of the drug was done by subjecting 20 g of air dried powder for soxhlet extraction continuously for 12 h. The extract was air dried under mild heat of 50°C till moisture completely evaporated. Ethanol, methanol and chloroform extractions were accomplished, following the procedures reported elsewhere with minor modification suiting to the laboratory conditions. For each extraction, the ground powder was weighed 100 g and immersed in ten times of 80% ethanol or methanol or chloroform and allowed for cold percolation on magnetic stirrer for 24 h. The extracts were first filtered through double layer

Cytotoxicity assays were conducted in vitro using cell cultures. The dry crude extracts were re-dissolved in dimethyl sulfoxide (DMSO) and 10 fold dilutions were made in cell culture medium.

For tetrazolium-dye (MTT) cytotoxicity assay, adherent cell monolayers in cultures were trypsinized and washed with culture medium. The cells were plated at 15,000 cells/well in 96-well flat-bottomed plates. After a 24 h pre-incubation period, extract dilutions were added to the appropriate wells and the plates were incubated for 24 and 48 h at 37°C in a humidified CO2 incubator.

Untreated cells were used as controls. After the incubation, the supernatants were removed from all the wells and 25 µl of MTT (2 mg/ml) solution in phosphate buffer saline (PBS) was added and the plates were incubated for 2 hr at 37°C. Then 125 µl of DMSO was added to the

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wells to stabilize the MTT crystals. The plates were placed in shaker for 15 min and absorbance was read at 492 nm on multiwell spectrophotometer. Control cells lysed with DMSO 2 h previous to evaluation of cellular viability by the MTT assay were used as blank the spectrophotometer. The percentage of cytotoxicity was calculated as (A−B)/A × 100, where A is the mean optical density of untreated wells and B is the optical density of the wells with plant extract.

bottomed plates; first 25 µl of PBS was added to all the wells. Allantoic fluids harvested from the inoculated embryonated hen’s eggs were serially diluted at 2 fold dilution. Equal quantity of 1% chicken RBCs were added to all the wells and incubated at room temperature for 20–30 min and reading was taken as mat formation and button formation. Titer of the virus was calculated as reciprocal of the highest dilution of the virus showing the haemagglutination.

Antiviral assay

RESULTS

To screen the antiviral activity, 10–11 days old embryonated hen’s eggs were used.

Dilutions of the plant extracts were preincubated with the standard concentration of virus for one hr in shaker incubator at 37°C. After the incubation 0.1 ml of mixtures were inoculated to eggs through intra-allantoic route, holes were sealed and incubated for 48 h in humidified incubator of 37°C. Controls consisted of only virus, only plant extract and the DMSO. The observations for antiviral activity were recorded after 48 h by observing the survivability of embryos in the inoculated eggs and checking the embryo fluid by haemagglutination test using 1% chicken red blood cells.

Qualitative phytochemical analysis for tannins, alkaloids, saponins, cardiac glycosides, steroids: terpenoids and flavonoids were conducted to confirm the authenticity of the drug collected for the present study. All the results were in accordance with the previously published standard observations. Percentage of crude extracts in various solvents are presented in Table 1.

Micro-haemagglutination assay (HA) Haemagglutination assay was performed as per the standard procedure (Swayne et al., 1998). The test was conducted in 96-well V-

The in vitro cytotoxicity of the crude plant extract was evaluated using MDBK cells and embryonated hens eggs thorough allantoic route of inoculation. . The IC50s to the mammalian cells indicate the general cytotoxic effect of these compounds. In the present study antiviral drugs evaluated did not show any cytotoxicity to MDBK cells and embryonated hens egg. Hence might be safe for use in vitro studies (Table 2)

Table 1: Yield of crude extract of Caesalpinia crista Solvent used for drug extract Aqueous

Yield of crude extract (%)

Ethanol

4.5

Methanol

5.8

Chloroform

3.8

6.9

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Table: 2. Cytoxicity assay results (percentage toxicity) of different crude extracts of Lathakaranja (Caesalpinia crista) on MDBK cells.

-1

10

-2

Dilution Plant crude extract Aqueous Ethanol Methanol Chloroform Virus control DMSO control

10

36 34 36 35 100 0

35 34 36 35 100 0

Aqueous Ethanol Methanol Chloroform Virus control DMSO control

48 47 48 45 100 0

48 47 47 45 100 0

24 hr post inoculation 10-3 10-4 10-5 10-6 35 35 34 32 34 33 33 33 36 34 34 33 36 34 34 34 100 100 100 100 0 0 0 0 48 hr post inoculation 47 47 45 45 43 45 46 45 47 48 46 46 45 44 44 43 100 100 100 100 0 0 0 0

10-7

10-8

32 32 33 32 100 0

31 32 30 30 100 0

45 44 46 43 100 0

42 44 45 43 100 0

Table 3. Antiviral activity (percentage inhibition) of different crude extracts of Lathakaranja (Caesalpinia crista)on some standard animal viruses (values are means of five observations) Plant material Aqueous Ethanol Methanol Chloroform Virus control DMSO control Aqueous extract of lathakaranja showed complete inhibition on paramyxovirus while showing highly significant inhibitory activity on orthomyxovirus tested in the present study.

Similarly, ethanol extract showed complete inhibitory activity on paramyxovirus while showing highly significant inhibitory activity on orthomyxoviruses. Methanol extract showed significant inhibition of paramyxovirus while showing highly significant inhibition of orthomyxovirus. Chloroform extract showed no inhibition paramyxovirus while moderate inhibition was observed on orthomyxoviurs (Table 3).

Paramyxovirus 100% 100% 75% 0% 100% 0%

Orthomyxovirus 87.5% 87.5% 96.25% 50% 100% 0%

DISCUSSION In the present study plant Lathakaranja (Caesalpinia crista) mentioned in Ayurvedic literatures was selected for evaluating antiviral activity. Their authenticity was established by phytochemcial analysis and crude extracts of drug was prepared with different solvents using standard established extraction protocols. Their activity against economically important veterinary viral pathogens was evaluated.

It has been reported that the methanol extract of C. crista seed and seed kernel possess antifeedant and anthelmintic (Javed et al., 1994; HĂśrdegen et al., 2006; Jabbar et al.,

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2007) property. In Ayurveda, the seeds, rootbark and the leaves of C. crista are used for medicinal purpose. Externally, the paste of leaves gives great relief from the pain and oedema. Internally, it is the best panacea of abdominal pain, diarrhoea, dysentery and colitis. Recent studies have demonstrated a potent antioxidant activity and ROS scavenging activity of methanolic extract of the plant (Mandal et al., 2009). Based on the medicinal usage and the phyto-constituents identified in the drug, the literatures on anti-malarial activity are numerous (Lin et al., 2005). However there are no reports on the antiviral activity of the plant material.

In the present study aqueous and ethanol extract showed complete inhibitory effect on the growth of paramyxovirus tested. Though significant effect was observed in methanol extract, chloroform extract did not show any inhibitory effect. Against orthomyxovirus all the extracts showed significant inhibitory activity except the chloroform extract which showed moderate effect.

It has been found that C. crista plant extract shows significant amount of flavonoid and phenolic content. Detailed phytochemical investigation of this plant species revealed bioactive compounds as diterpenes, like methyl migrated cassane-type furanoditerpenes and normal cassane- type furanoditerpenes (Kalauni et al., 2005), neocaesalpins H (Kinoshita et al., 2005) and caesalpinins and norcaesalpinin (Awale et al., 2006). This furano-diterpenes are phytochemically classified as cassanediterpenes and otherwise known as caesalpins.

The observed antiviral activity in the study may be attributed to these bioactive compounds.

This is the first report on antiviral activity of C. crista plant. However, further research work is needed to identify the specific constituent in the plant extract exhibiting the antiviral activity.

CONCLUSION In the present study Orthomyxovirus and Paramyxovirus candidate animal viruses were selected since, they represent important group of viruses causing economically important and zoonotic diseases of domestic animals and poultry. Crude extracts of antiviral drugs were confirmed safe by testing for their cytotoxicity in MDBK cell lines and by inoculating embryonated chicken eggs. Many of the plants are being used individually or in formulations for treatment of infectious diseases and as germicidal agents. One of the major problems with these herbal formulations is that the active ingredients are not well defined (Cowan 1999).

It is important to know the active component and their molecular interaction, which will help to analyse therapeutic efficacy of the product and also to standardize the product. Efforts need to focus on investigating mechanism of action of some of these plants using model systems.

Despite the lack of scientific understanding behind the mechanism of action of medicinal plant extracts, present investigation showed that the Ayurvedic medicinal plant C. crista has immense potential to be developed as antiviral agent. Ayurvedic medicines do have antiviral drugs for use in animal and human practice as to date no antiviral therapy is effectively followed for the reasons inherent to the biology of the virus. However it should be noted that these in vitro results may not translate into clinical effectiveness. Further, studies are needed to understand the phyto-constituents responsible for the observed pharmacological activity and to standardize the dosage and administration schedules for successful clinical applications both in medical and veterinary practice.

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REFERENCES Awale S, Linn TZ, TezukaY, Kaluauni SK, Banskota AH, Attamimi F, Ueda J, Kadota S (2006). Constituents of Caesalpinia crista from Indonesia, Chem Pharm Bull.54:213–218.

Beare AS (1975). Myxoviruses. Developments in biological standardization. 28:3–17.

Cowan

MM (1999). Plant Products as Antimicrobial Agents.

Clinical Microbiological Reviews. 12 (4) 564– 582.

Makhloufi A, L.Benlarbi, L.Mebarki, Akermi M.M (2012). Antimicrobial activities of essential oil and crude extracts from Artmisla herba-alba asoo, growing wild in Bechar, South West of Algeria.

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Med. 1 (1) 7–13.

Jabbar A, Zaman MA, Iqbal Z, Yaseen M, Shamim A (2007). Anthelmintic activity of Chenopodium album (L) and Caesalpinia crista (L) against trichostrongylid nematodes of sheep.

Journal of Ethnopharmacology. 114:86– 91.

Javed I, Akhtar MS, Rahman ZU, Khaliq T, Ahmad M (1994). Comparative anthelminthic efficacy and safety of Caesalpinia crista seed and piperazineadipate in chickens with artificially induced Ascaridia galli infection. Acta Veterinaria Hungarica, 42:103–109.

Hördegen P, Cabaret J, Hertzberg H, Langhans W,Maurer V (2006). In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyltetrazolium reduction assay. Journal of Ethnopharmacology, 108:85–89.

Kalauni SK, Awale S, Tezuka Y, Banskota AH, Linn TZ, Kadota S (2005). Methyl Migrated Cassane-Type Furanoditerpenes of Caesalpinia cristafrom Myanmar. Chem. Pharm.

Bull.53: 1300–1304.

Kinoshita T, Haga Y, Narimatsu S, Shimada M,Goda Y (2005).The Isolation and Structure Elucidation of New Cassane Diterpene-Acids from Caesalpinia crista L. (Fabaceae) and Review on the Nomenclature of some Caesalpinia Species. Chem Pharm Bull.53:717–720.

Linn TZ, Awale S, Tezuka Y, Banskota AH, Kalauni SK, Attamimi F, Ueda JY, Asih PB, Syafruddin D, Tanaka K andKadota S (2005). Cassane- and norcassane-type diterpenes from Caesalpinia crista of Indonesia and their antimalarial activity against the growth of Plasmodium falciparum, J Nat Prod.68:706–710.

Mandal S, Hazra B, Sarkar R, Biswas S, Mandal N (2009). Assessment of the antioxidant and reactive oxygen species scavenging activity of methanolic extractof Caesalpinia crista Leaf. eCAM:1–11.

Saif YM (2003). Diseases of poultry. 11th Edition. A Blackwell Publishing Company, Iowa State Press, USA pp 63–100.

Satnami DK and Yadava RN (2011). Potential Phytochemical from Caesalpinia Crista Linn. Research Journal of Phytochemistry. 22–31.

Swayne DE, Glisson JR, Jackwood MW, Pearson JE, Reed WM (1998). A laboratory manual for the isolation and identification of avian pathogens. 4th Edition, Published by American Association of Avian Pathologist.

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Trease GE, Evans WC (1983). Pharmacognosy, Edition.

Published by 12th BailliereTindall, London.

Weyermanna J, Lochmanna D and Zimmerb A (2005). A practical note on the use of cytotoxicity assays.

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Wagner EK, Hewlett MJ (1999). Basic Virology, Malden, MA, USA Blackwell Science.

Source of Support: Nil

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 448–456 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Research article A COMPARATIVE DERMAL TOXICITY EVALUATION OF RASAKARPURA AND MERCURIC CHLORIDE IN RATS Mehta N J1*, Ashok B K2, Ravishankar B3, Prajapati P K4 1

Lecturer, BAMS Foreigner’s Course, Dept of Rasashastra and Bhaishajya Kalpana, Shree Gulabkunvarba Ayurved Mahavidyalaya, Gujarat Ayurved University, Jamnagar 2 Research Assistant, Pharmacology laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar.

3 Director, SDM Centre for Research in Ayurveda and Allied Sciences, Laxminarayana Nagar, Kuthpady, Udupi-574118., Karnataka, India 4 Professor and Head, Dept of Rasashastra and Bhaishajya Kalpana, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India *Corresponding Author: Email: nekymehta@rediffmail.com; Mobile: 09724348145

Received: 24/07/2012; Revised: 26/08/2012; Accepted: 31/08/2012

ABSTRACT Rasakarpura is a mercurial preparation which is used to treat the diseases of skin. It is a well known fact that Rasakarpura, prepared by Rasa Tarangini method, is chemically mercuric chloride (98%) with some trace elements. Metallic drugs have the potential to produce many side effects if the drugs are not prepared or used in proper manner. Hence dermal toxicity of Rasakarpura Drava (solution form of Rasakarpura) was carried out in albino rats and compared with solution of mercuric chloride. Results show that Rasakarpura is relatively safe on sub-acute dermal application in terms of haematological, serum biochemical and histopathological parameters, while mercuric chloride is apparently toxic. Further chronic toxicity studies are needed to provide further safety information on this formulation.

KEY WORDS Parada, Gandhakamla, Saindhava Lavana, Rasakarpura Drava, Mercuric chloride.

Cite this article: Mehta N J, Ashok B K, Ravishankar B, Prajapati P K (2012), A COMPARATIVE DERMAL TOXICITY EVALUATION OF RASAKARPURA AND MERCURIC CHLORIDE IN RATS, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 448–456

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INTRODUCTION Rasakarpura is a mercurial preparation and chemically very similar to mercuric chloride (HgCl2). It is used in the treatment of diseases like Phiranga (syphilis), Atisara (diarrhea) and various Twak Vikaras (skin diseases) (Sharma S.N., 2004a) internally. It is also used externally for local application in the form of Rasakarpura Drava (Sharma S.N., 2004b) in various skin diseases and considered as one of the best remedies.

Rasakarpura and other Rasashatriya preparations (metal and mineral preparations) are very toxic if they are not prepared properly or given in higher dose level. All the scholars of Ayurveda know about the adverse effect of metal and mineral preparations, which are prepared improperly. Now a day, a drug is allowed to be marketed only after a detailed toxicity and pharmacological evaluation to be assured about safety and efficacy aspects.

Taking into consideration the toxic potential of the preparations and the present day scientific requirement in the present study a repeated dose dermal toxicological evaluation was carried out to find safety margin and toxic Fig: 1 Image of mercuric chloride

Animals: Charles Foster strain albino rats of either sex; weighing 280 ± 20 g were used for the study. The animals were obtained from the animal house attached to pharmacology lab, IPGT & RA, Gujarat Ayurved University, Jamnagar. Animals were housed individually in

effect of Rasakarpura vis-à-vis Mercuric chloride. Rasakarpura, prepared by Rasa Tarangini method, was found 98% water soluble (Mehta NJ, 2007), hence the solution of the Rasakarpura was prepared and used for the experimental evaluation.

MATERIALS AND METHODS Test formulations: Rasakarpura solution was prepared in the department of R.S. & B.K., I.P.G.T. & R.A. of Gujarat Ayurved University, Jamnagar. As per the reference of Rasa Tarangini (Sharma S.N., 2004c), 1 part of purified Parada (mercury) and 1.5 part of Gandhakamla (conc. Sulphuric acid) were taken in a glass vessel and heated till the mixture becomes moisture less powder.

Then equal amount of Saindhava Lavana (rock salt) was mixed by grinding and this mixture was sublimed in Valuka Yantra to collect Rasakarpura. The Rasakarpura Drava was prepared by using Rasakarpura and distilled water. Mercuric chloride (Ranbaxy laboratories batch no. P049D02) was procured from local market. Percentage of test drugs in both solutions was selected as 0.1% in final product.

Fig :2 Image of prepared Rasakarpura

cages made up of poly-propylene with stainless steel top grill. The dry wheat (post hulled) waste was used as bedding material and was changed every morning. The animals were exposed to 12 h light and 12 h dark cycle with the relative humidity of 50–70% and the ambient temperature during the period of experimentation was 22 ± 03ºC. Animals were

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fed with Amrut brand rat pellet feed supplied

by Pranav Agro Mills Pvt. Limited, Vadodara, Gujarat. For their drinking purpose tap water ad libitum was used. The experiment was carried out in conformity with the guidelines of Institutional Animal Ethics Committee (IAEC) after obtaining its permission.

Dermal toxicity study: The selected animals were acclimatized in laboratory conditions for seven days. Twenty four hours prior to the application of test drug, fur at the back of the each animal was shaved off from about 10% of total body surface area and they were randomly divided into three groups with six animals in each group. To the first group distilled water was applied and served as control. To second and third groups 0.1% Rasakarpura solution and Mercuric chloride solution was applied respectively for 14 consecutive days, twice daily by using sterile cotton swab. Animals were kept individually in separate cages. All animals were observed daily for signs of toxicity and also changes in skin, mucous membrane, fur, behavioral pattern. On 15th day, the overnight fasted animals, were weighed and blood was collected from retro-orbital plexus under ether anesthesia and collected in two different types of tubes, one containing anticoagulant fluid for haematological parameters and another plain tube for serum biochemical investigations.

Further, all the rats were sacrificed by overdose of ether anesthesia and from the sacrificed animals, organs like brain, heart, liver, lung, kidney, thymus, spleen, lymph nodes, jejunum, testicles, uterus and skin were dissected out.

The vital organs were weighed and all the organs were transferred to a glass bottle containing 10% formalin for histo-pathological studies.

To estimate haematological parameters 0.08 ml blood was mixed with 0.02 ml of EDTA (33.33 mg/ml) and fed to the auto analyzer (MS-9 Veterinary Melet Schloesing haematology cell counter, France). The parameters measured were total WBC count, neutrophil percentage, lymphocyte count, lymphocyte percentage, monocyte percentage,

haemoglobin content, total RBC, MRBC percentage, Red cell distribution width (RDW), haematocrit percentage, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). For estimation of biochemical parameters, serum was separated from collected blood and requisite quantity of serum was fed to the auto analyzer (Fully automated Biochemical Random Access Analyzer, BS-200; Lilac Medicare Pvt. Ltd., Mumbai, India) which was automatically drawn in to the instrument for estimating different parameters. Biochemical parameters like blood sugar (Pennock, 1973), serum total cholesterol (Roeschlau, 1974), serum triglyceride (Fossati, 1982), serum HDL cholesterol (Dominiczak, 2000), serum urea (Tiffany et al., 1972), serum creatinine (Slot, 1965), serum glutamic oxaloacetic transaminase (SGOT) (Tietz, 1995), serum glutamic pyruvic transaminase (SGPT) (Burtis and Ashwood, 1999a), total protein (Tietz, 1986), serum albumin and serum globulin (Doumas et al., 1972), AG ratio, serum alkaline phosphatase (Wilkinson, 1969) and serum total bilirubin (Burtis and Ashwood, 1999b) were estimated.

The histopathological slides of different organs like heart, liver, lung, kidney, thymus, spleen, lymph node, intestine, adrenal, testis, prostate, seminal vesicle, uterus, ovary and skin were prepared by referring to standard procedure of Raghuramulu et al., (1983). The slides were viewed under trinocular research Carl-Zeiss’s microscope at various magnifications to note down the changes in the microscopic features of the tissues studied.

Statistical analysis: The obtained data have been presented as Mean ± SEM. The difference between the groups was statistically determined by student’s t test for unpaired data with the level of significance set at P < 0.05.

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RESULTS No behavioral changes were observed in both Rasakarpura and mercuric chloride treated rats on dermal application. No mortality was observed during the course of study.

Normal progressive gain in body weight occurred in control group in comparison to initial body weight (Table – 1). In both the test formulations applied group also gain in body weight was observed, however the magnitude of gain in body weight is comparatively less.

Further application of test drugs did not affect the weight of any of the organ to significant extent in comparison to control group.

Application of Rasakarpura did not affect any of the thirteen heaematological parameters in comparison to control group. In contrast, application of mercuric chloride significantly decreased neutrophil percentage and MCH, also significantly increased lymphocyte percentage in comparison to control group (Table – 2).

Among fourteen serum biochemical parameters only one parameter (increase in blood urea) was affected by application of Rasakarpura solution, while mercuric chloride did not affect any of these parameters in comparison to control group (Table – 3).

Table-1: Effect of Rasakarpura and Mercuric chloride on body weight and relative organ weight Parameters

Control

Rasakarpura

Mercuric chloride

Gain in body weight (g)

22.32 ± 4.26

15.33 ± 4.55

13.33 ± 5.58

Liver (g/100 g)

02.27 ± 00.08

02.27 ± 00.11

02.20 ± 00.06

Spleen (g/100 g)

00.22 ± 00.01

00.23 ± 00.01

00.22 ± 00.01

Heart (g/100 g)

00.29 ± 00.01

00.30 ± 00.01

00.31 ± 00.01

Kidney (g/100 g)

00.58 ± 00.03

00.63 ± 00.02

00.66 ± 00.02

Thymus (g/100 g)

00.17 ± 00.01

00.17 ± 00.02

00.15 ± 00.01

Testis (g/100 g)

00.89 ± 00.09

00.86 ± 00.02

00.87 ± 00.05

Uterus (g/100 g)

00.18 ± 00.02

00.24 ± 00.02

00.25 ± 00.04

Data: Mean ± SEM DISCUSSION Out of twelve organs screened histopathologically, application of Rasakarpura showed pathological changes in only one organ, that is moderate decrease in spermatogenesis, while mercuric chloride application lead to cell depletion in lymph node, extensive fatty changes in liver, cell infiltration and fluid effusion in lung and decrease in spermatogenesis (Plates 1, 2 & 3).

Albino rats are among the commonest and most standardized of all laboratory animals suitable for experimental work, because of their small size and greater sensitivity to most drugs.

Pharmacological experiments on rats are among the most necessary preliminary steps towards discoveries of new drugs. Its skin is relatively sensitive to irritants, so a great deal of experimentation on topical agents is conducted on rats (Jun Sekizawa et al., 1994).

The drug, Rasakarpura is mainly intended for

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local application and it is chemically equivalent to mercuric chloride. To ascertain whether Rasakarpura, which is almost similar to mercuric chloride, has similar toxicity potential or not, a comparative evaluation was carried

out for dermal toxicity following standard protocol. The implications of the changes observed would be discussed in the following paragraphs.

Table-2: Effect of Rasakarpura and Mercuric chloride on various haematological parameters Parameters

Control

Rasakarpura

Mercuric chloride

WBC (103/µl)

1.40 ± 0.31

1.45 ± 0. 40

1.06 ± 0.20

Neutrophil (%)

5.08 ± 0.55

3.73 ± 0.97

2.65 ± 0.33**

Lymphocyte (%)

93.35 ± 0.69

95.42 ± 1.25

96.15 ± 0.37**

Lymphocyte count (10e3/ µl)

1.37 ± 0.30

1.40 ± 0.40

1.00 ± 0.20

Monocyte (%)

1.55 ± 0.24

0.87 ± 0.31

1.23 ± 0.11

Haemoglobin (g/dl)

09.62 ± 0.43

10.32 ± 0.78

10.17 ± 0.35

RBC (106/µl)

06.33 ± 0.37

07.10 ± 0.58

07.07 ± 0.25

MCV(fl)

77.28 ± 0.89

75.92 ± 0.80

75.95 ± 0.62

MCH (Pg)

15.25 ± 0.22

14.57 ± 0.36

14.38 ± 0.14**

MCHC(g/dl)

19.77 ± 0.34

19.13 ± 0.34

18.95 ± 0.23

MRBC (%)

00.95 ± 0.11

01.03 ± 0.07

01.15 ± 0.05

RDW (%)

07.63 ± 0.29

07.15 ± 0.32

07.47 ± 0.13

Haematocrit (%)

48.83 ± 3.04

53.88 ± 4.41

53.68 ± 1.76

Data: Mean ± SEM, **P < 0.01 Results related to body weight changes showed moderate decrease in body weight gain in rats treated with test drugs in comparison to control rats. This indicates some interference with nutritional metabolism. The fact that though at reduced rate body weight gain was observed indicates that there is no serious degeneration producing potential as observed decrease is non-significant and also the weight of important organs was not affected.

Analysis of the results obtained with hematological parameters shows that local application of Rasakarpura did not produce any significant alteration in any of the

parameters studied whereas three parameters were found to be altered in mercuric chloride applied rats. The affected parameters are increase in the lymphocyte percentage, decrease in the granulocyte percentage and decrease in MCH. Thus mercuric chloride has higher hematological parameters disturbing proclivity in comparison to Rasakarpura.

Analysis of the results of biochemical parameters showed that, out of fourteen parameters only one parameter (increase in urea level) was found to be significantly affected in Rasakarpura treated group and none of these parameters were affected in mercuric

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chloride treated group. The increase in urea level indicates higher turnover of nitrogenous material. The increase was only 26% and is not remarkable. However it does indicate that the mercury is being absorbed in to systemic circulation. It should be noted that on systemic

administration, blood urea lowering was observed,which may be due to drastic effect on the liver. When administered locally only small amount may be absorbed in to systemic circulation and may have pre-renal, renal or post renal effects that lead to elevation of urea.

Table-3: Effect of Rasakarpura and Mercuric chloride on various biochemical parameters Parameters

Control

Rasakarpura

Mercuric chloride

Blood sugar (mg/dl)

76.00 ± 5.22

70.00 ± 4.26

72.17 ± 1.62

Cholesterol (mg/dl)

57.67 ± 7.54

68.67 ± 6.08

65.50 ± 6.69

Triglyceride (mg/dl)

124.83 ± 18.48

130.83 ± 18.26

137.50 ± 24.34

HDL cholesterol (mg/dl)

28.00 ± 5.03

31.33 ± 3.34

27.83 ± 3.96

Blood urea (mg/dl)

41.17 ± 2.36

52.00 ± 2.50**

41.83 ± 1.14

Creatinine (mg/dl)

0.92 ± 0.03

0.90 ± 0.03

0.95 ± 0.06

SGPT (IU/L) activity

085.00 ± 06.28

112.00 ± 34.57

074.00 ± 05.66

SGOT (IU/L) activity

276.67 ± 20.77

343.00 ± 63.55

286.33 ± 20.52

Total protein (g/dl)

7.50 ± 0.29

7.68 ± 0.28

7.47 ± 0.36

Albumin (g/dl)

4.12 ± 0.10

4.05 ± 0.19

4.30 ± 0.19

Globulin (g/dl)

3.38 ± 0.25

3.63 ± 0.16

3.17 ± 0.22

A:G ratio

1.22 ± 0.10

1.13 ± 0.06

1.35 ± 0.08

Alkaline phosphatase (IU/L) activity

154.67 ± 25.85

136.33 ± 12.51

188.83 ± 27.45

Billirubin total (mg/dl)

0.55 ± 0.06

0.57 ± 0.04

0.60 ± 0.05

Data: Mean ± SEM, **P < 0.01 Plate – 1: PHOTOMICROGRAPHS OF LYMPH NODE (×400 MAGNIFICATION)

Control

Rasakarpura Cp-Capsule, C-Cortex, M- Medulla

Mercuric chloride

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PLATE – 2: PHOTOMICROGRAPHS OF LIVER (×400 MAGNIFICATION)

Control Rasakarpura Mercuric chloride Kc-Kupffer cell, Hc-Hepatic cells, S- Sinusoid, Cv-Central vein, Fc-Fatty changes PLATE – 3: PHOTOMICROGRAPHS OF TESTIS (×400 MAGNIFICATION)

Control

Rasakarpura

The toxicity of mercury to animals and man is well established and this depends greatly on the form of the mercury compounds. Oral absorption of organic mercury is nearly complete (Patrick, et al., 2002). In most animals' species, including man, the kidney and liver are the main sites of deposition of inorganic mercury and target organ for its toxicity (Kojima, 1989). In the present study, even on dermal application, mercuric chloride not only showed pathological changes in liver, but also in other organs like testicles, lymph nodes and lungs. In contrast, classically prepared Rasakarpura did not show any apparent toxicity in terms of haematological, serum biochemical and histopathological parameters. This provides explicit evidence on safety of the classical formulation Rasakarpura, on dermal application. Thus the results obtained clearly indicate that, though

Mercuric chloride

Rasakarpura and mercuric chloride are chemically similar – there is definite difference in their toxicity potential, at least on dermal application. The latter bears higher toxicity potential in comparison to Rasakarpura. It may be due to the presence of other inorganic ingredients in Rasakarpura in addition to mercuric chloride which may be retarding the absorption of mercury in to the systemic circulation. It is also possible that they may be interfering with biological effects of mercuric chloride there by reducing its toxicity potential.

CONCLUSION Rasakarpura is relatively safe on sub-acute dermal application in comparison to mercuric chloride. Chronic toxicity studies would be required to further substantiate the relative reduced toxicity potential of Rasakarpura vis a vis mercuric chloride.

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REFERENCES Burtis CA and Ashwood ER, (1999a.) Tietz textbook of Clinical Chemistry, 3rd ed., (Philadelphia, PA: WB Saunders); pp 652.

Burtis CA, Ashwood ER, editors (1999b.), Tietz Textbook of Clinical Chemistry, 3rd ed. Philadelphia, PA: WB Saunders; pp1136.

Dominiczak M, Mc Namara J. (2000), The system of cardiovascular prevention 103–125; Nauk M, Wiebe D, Warnick G, Measurement of High-DensityLipoprotein Cholesterol, 221–244. In: Handbook of Lipoprotein testing, 2nd edition, edited by Rifai, Warnick and Dominiczak, (AACC Press, Washington DC); pp 819.

Doumas BT, Arends RL, Pinto PC. (1972), Standard methods of clinical chemistry.

Academic Press Chicago; Volume 7. pp. 175–89.

Fossati

P, Prencipe L. (1982), Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem; pp 2077–80.

Jun Sekizawa, Kazuo Yashuhara, Yuji Suyama, Sumie Yamanaka, Masuo Tobe, Masao Nishimura. (1994), A simple method for screening assessment of skin and eye irritation. The journal of Toxicological sciences;19:25-35.

Kojima S, Shimada H, Kiyozumi M. (1989), Comparative effects of chelating agents on distribution, excretion and renal toxicity of inorganic mercury in rats.

Res Commun Chem. Pathol Pharmacol; pp 471–484.

Mehta

NJ. (2007), Pharmaceutical standardisation of Rasakarpura & Rasakarpura Drava, its safety profile & therapeutic effect on Kshudra Kustha, M.D. thesis, Gujarat Ayurved University, Jamnagar.

Patrick L. (2002), Mercury toxicity and antioxidants: Part I: Role of glutathione and alpha-lipoic acid in the treatment of mercury toxicity. Altern Med Rev; 7: pp 456–471.

Pennock CA, Murphy D, Sellers J, Longdon KJ. (1973), A comparison auto analyzer method for the estimation of glucose in blood. Clin Chim Acta; pp 193–201.

Raghuramulu N, Nair KM and Kalyanasundaram S. (1983) A manual of laboratory techniques, (National Institute of Nutrition, Hyderabad); pp 246–53.

Roeschlau P, Bernt E, Gruber WA. (1974), Enzymatic determination of total cholesterol in serum. Jour clinical Chem clinical Biochem; pp 226.

Sharma S. N. (2004a), Rasa Tarangini, Shastri K. N., Hindi commentary, Motilal Banarasi Das, Delhi; pp 117.

Sharma S. N. (2004b), Rasa Tarangini, Shastri K. N., Hindi commentary, Motilal Banarasi Das, Delhi; pp 123.

Sharma S. N. (2004c), Rasa Tarangini, Shastri K. N., Hindi commentary, Motilal Banarasi Das, Delhi; pp115.

Slot

C. (1965), Plasma creatinine determination. A new and specific Jaffe reaction method. Scand J Clin Lab Invest; pp 381–87.

Tietz NW (1995), Clinical guide to laboratory tests, 3rd ed., (Philadelphia, PA: WB Saunders); pp 76.

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Tietz, N.W. (ed) (1986), Text book of Clinical Chemistry, W.B. Saunders pp 579.

by use of a GeMSAEC fast analyzer.

Clinical Chemistry, pp 829–840.

Tiffany TD, Janseen JM, Burtis CA, Overton JB, Scott TD (1972), Enzymatic kinetic rate and end point analysis of substrate

Wilkinson JH, Boutwell JH, Winsten S. (1969), Evaluation of a new system for kinetic measurement of serum alkaline phosphatase. Clin Chem; pp 487–95.

Source of Support: Nil

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 457–463 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article ACTION OF SHIRODHARA – A HYPOTHETICAL REVIEW Ajanal Manjunath1*, Chougale Arun2 1 2 *

PG Scholar, Department of Dravyaguna, KLEU, Shri BMK Ayurved Mahavidhyalaya, Belgaum, Karnataka.

Asst. Professor, Dept of Dravyaguna, KLEU Shri BMK Ayurved Mahavidhyalaya, Belgaum, Karnataka.

Corresponding Author: Mob: +919036616510; Email – manju.ajanal@gmail.com

Received: 21/07/2012; Revised: 18/08/2012; Accepted: 25/08/2012

ABSTRACT Shirodhara is one of the Bahya snehana (external Oleation therapy) method mentioned in Ayurveda. Shira (head) is the Seat of Mind. Shirodhara is mainly indicated in Manasa bhava pradhan vikaras (mental disorders) i.e Raja & Tama Dosha Pradhana (predominated). The apparent mode of action of this procedure is not understood. Ayurveda extensively highlighted the outcome of the procedure rather than its action. To influence & enhance the perception, it is needed to have the hypothetical views regarding the apparent mode of action of any treatment procedures. Hence, present work was planned to foot forth a hypothetical view of its action, since the site of procedure which performing is Sthapani marma (level of pituitary gland), Ajnya chakra (Penial gland and Medulla oblongata in brain), Shavasana (Yogic corpus posture) is the posture in which therapy is performing and Manomaya kosha (mental sheath) which yogic science believed that which responsible for mental status of an individual. We hypothesize the action of Shirodhara might be stimulation of Sthapani marma and motivation of Ajnya chakra and organizing different phases of Manomaya kosha and added benefit of posture Shavasana with auto body suggestion therapy facilitates to counterbalance the Manasika and shareerika doshas which occurs simultaneously and this is potentiated by Psychoneuroimmunology (PNI) mechanism, hence the effect may be seen instantly.

Key Words: Shirodhara, Marma, Chakra, Shavasana, Manomaya kosha, Psychoneuroimmunology

To Cite this article: Ajanal M and Chougale A (2012), Action of Shirodhara – A hypothetical review, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 457–463

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INTRODUCTION Shirodhara is one of the Bahya snehana (external Oleation therapy) procedures being practiced in Ayurveda. The author Vagbhata grouped under group of Murdni taila1 (process of anointing the head with oil). Technically this procedure is defined as a stream of continuous pouring of liquid over forehead or scalp with specified time is known as the Shirodhara2. It can be done by different medicaments like Taila (oil), Takra (medicated butter milk), Kshira (alkali), Kwatha (decoction) etc3, and Shirasheka, Shirodhara, Pariseka, Sechana are synonyms of Shirodhara3. Due to synergetic action over Kapha, it is contraindicated in Kaphavikaras (diseases of Kapha).

Therapy extensively indicated in Ardhavabhedaka (migraine) 4, 5, Suryavarta6 (type of headache), Ardita (facial palsy), Pakshaghata (stroke), Hanugraha (loc jaw), Nidranasha (insomnia), Shirogata Vata (diseases of vata in head region), Shirahkampa (head tremors) 6, 7. Other than these indications, present day it has extended its utility in diseases like Diabetes, Hypertension, Psychosomatic diseases, pre mature graying of hairs, fatigue, and infertility etc8. Ayurveda extensively highlighted the outcome of the procedure rather than its action. This therapy appears to be simple but essentially complex to understand its mode action. The apparent mode of action of this therapy is not critically understood. Hence present study was planned to generate a hypothesis to predict the apparent action of Shirodhara. The classical literatures of Ayurveda, Yoga and recently published research updates were reviewed to generate the possible mode of action of this therapy.

METHODOLOGY Procedure of Shirodhara9 The procedure of this therapy includes 3stages for the descriptive purpose as Purvakarma (pre procedural), Pradanakarma (actual procedure) and Paschat karma (post procedural).

Purva Karma: It includes preparation of drug and patient for the procedure. Drug is selected based on the disease and patient comfort. As the routine check up patient must undergo physical examinations to evaluate fitness, then patient must have pass stool and urine before the procedure. Recommended position of patient is supine position for this procedure and Dharapatra (vessel) should be fixed 4-angula (inches) above the fore head and eyes and ears are covered with cotton to prevent the entry of liquid into eyes.

Pradhana Karma: room must be cleaned and well ventilated and procedure is done in empty stomach preferably after Abhyanga (massage).

The selected warm liquid is kept in Dharapatra (vessel) and poured continuously, neither to fast nor to slow on the fore head of the patient for about 45-minutes to one hour.

Pashchat Karma: after completing the procedure the head of the patient is wiped out and dried. This followed a short duration of rest and later massage of the body and head with oil. Then lukewarm bath is advised.

Mode of action: The mode of action of Shirodhara is hypothesized by incorporating Marma concept (Vital Spots), Chakra Concept (Energy Vertex), Manomaya Kosha (Mental sheath) and Asana (Yogic Posture) â&#x20AC;&#x201C; involved in the procedure.

Action of Shirodhara based on Marma concept: Marmas (vital Spots) are the critical spots in our body, any injury to these spots will lead to deformities or even fatality. Overall 107 Marmas are present in our body10. Out of which shiras (Head region) is having 37marmas11 (Shown in Fig No.1), in which 15 are Sadhyopranahara (injury to which results in instant death), 5-Kalantara Pranahara (injury to which results in delayed death), 3Vishalyagna (death after removing foreign body), and 14-Vaikalyakara (injury to which results in physical impairment)12. Most of marmas (vital Spots) of shiras (head) are Agni

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(fire) and Vayu (air) Mahabuta pradhana (predominate) 13.

The site of Sthapani marma (Sthapani vital spot) and the site of pituitary gland and pineal gland exist at the same level. The function of pituitary gland is controlled by hormones or nerve signals this gland is activated whenever the stimulus from CNS or hormones. It has the capacity to maintain all the endocrine systems

of human body14. Mental stress and irritability were the causative factors were endocrine system gets imbalance of its normal function15.

Hence present therapy helps in relieving the mental stress by regulating the normal function of this gland. This can be achieved by Stimulating Sthapani marma and indirect stimulation of the master gland pituitary this in turn brings ultimatum in psycho-somatic level.

Fig No.1 Positions of Marma

Action of Shirodhara based on Chakra concept: This is also a distinctive notion of the Yogic science; Chakras (nerve plexuses) means nerve centers the fly wheels in the machine that is the human body. These are the regions situated in the spinal column where the Nadi (nerve) cross each other16. It is believed that these are in a state of subtle and not easily cognizable. There are seven chakras located in our body (shown in Fig No.2), which are Muladhara (pelvic plexus), Swadistana (Hypogastric plexus), Manipura (Solar plexus), Anahata (Cardiac plexus), Vishuddha (Pharyngeal plexus), Ajna (the plexus of command between the two eye brows) and 7th one Sahastrara (the thousand petal lotus, the upper cerebral centre) it as capacity to control all other six chakras17.

Shiras (head) is the seat of both Ajnya chakra (6th chakra) & Sahastrara (7th Chakra).

Ajnya (6th chakra) is located at the level of external point of the eye brow center corresponds to Penial gland and Medulla oblongata in brain, and it is considered as the third eye because of its position. This point is the trigger point of concentration. Hence it is considered as centre of consciousness. From this centre the bodily systems and states of awareness are controlled.18 Hence Shirodhara treatment over Ajnya chakra (Penial gland and Medulla oblongata in brain) would stimulates Sahastrara chakra (the thousand petal lotus, the upper cerebral centre) which controls the mechanism of all chakras to generate preferred results.

Penial gland one of the endocrine gland situated at the roof of third ventricle under posterior part of corpus calosum and considered as seat of soul19. It is responsible for

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rhythmic changes in body, mood changes, sleep promotion and sexual behavior. More importantly actual function of this gland is still mysterious. But many believed that it got stimulations from sympathetic supply from CNS20. Medulla oblongata or brain stem is also Fig No.2 Positions of Chakras

Action of Shirodhara based on Manomaya Kosha: Ayurveda defines Ayu (life) is the combined state of Sharira (body), Indriya (senses), Sattva (psyche) and Atma (Soul) 22. In this way, Manasa is chiefly responsible for perceiving qualitative healthy life. Signs of good health are Samadosha (Balance state of Dosha), Samagni, (Balance state of digestion) Samadathu (tissues) and Mala (by products), Prasanna (healthy state) of Atma (soul) and Manas (mind) 23. Manas and its pathogenesis can be perceived at intellectual or body level.

"Rajas" and "Tamas" are considered as "Manas doshas" and having capacity to generate Samprapti (i.e. etiopathogenesis).

Any imbalance in body level would certainly cause disease of mind and vice-versa. Tripod methods of treatment which includes Daiva vyaparsraya (Magico-religious practice), Yukti vyapasraya (Physio-pharmacological procedure) and Satvavajaya (Psychological methods) 24 were followed. In that Satvavajaya chikitsa indicated specially for Mano nigraha (control over

one of the main sites where equilibrium of body is controlled21. Hence corresponding stimulation of pineal gland along with medulla oblongata both synergistically helps to produce the desired effects of Shirodhara.

Fig No.3 Koshas of atman

mind). Since Shiras seat of mind25 treatment which provided at the level of Shiras (head) would certainly helps in controlling Manasa vikaras (diseases of mind).

According to Yogic philosophy there are Kosha (sheaths) around atman (body). The nature of human being encompasses corporal and emotional phase that functions as one holistic organism. The Kosha system refers to these diverse phases as Layers of subjective experience. Layers array from the opaque corporal body to the more subtle points of emotions, mind and spirit. In another sense unfolding one by one, of five koshas leads to manifestation of a progressive expression of life.26 these are five in number which are 1.

Annamaya (Physical), 2. Pranamaya (vital), 3.

Manomaya (mental), 4. Vijnanamaya (intellect) and 5. Anandamaya (blissful) 27 (Shown in Fig no.3).

Manomaya Kosha refers to the emotional, mental and spiritual aspects of human being collectively frame our subjective experience of

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being alive which is composed of manas (mind). Shiras is the seat of Manas25. The Manas (mind) in conjunction with the five sensory appendages holds to represent the manomaya kosha. It is the root of diversity, it has four purposes: a) remembrance b) reasonableness c) knowledge of bliss and ache based on responses from past deeds, d) vision28.

Body and mind both are innately interconnected hence healing process to mind indirectly heals the body also. So dhara therapy hypothetically heals the mind in turn heals the body.

Action of Shirodhara based on Asana â&#x20AC;&#x201C; involved in the procedure:

may act as mechanical stimulus to the receptor which counterbalances the pain regulating centre32.

Psychoneuroimmunology (PNI): The study of relationship that exists between the central nervous system, autonomous nervous system, endocrine system and immune system is called as Psychoneuroimmunology (PNI)33. It says that the states of anything which influence any one system among nervous system, immune system and endocrine system the effect would seen all the three systems since they are mutual interrelated between them 34, 35 .

The posture of Shirodhara is similar as that of Shavasana (corpses pose). This is one of the complete relaxation procedure mentioned Yogashastra (texts of Yoga). Complete relaxation can be experienced only when both physical and mental relaxation is attained29. In medical science, it is generally believed that complete mental relaxation decreases the somatic symptom because mind and body are inter-connected22 & 36. Shavasana is one of the very important yogic practice, provides total relaxation. Some recent researches suggested that Shavasana is significantly reduces the subjective improvement in the symptoms like Headache, giddiness, nervousness, irritability and diminished sleep30. In Shirodhara, Shavasana like posture is followed for about 45-minutes. Hence this posture may add the benefit to the procedure out come.

This shows the relation between stress and the emotional state of an individual, this relation play a significant role in making vulnerable to diseases. This relation exists by connection between mind-body by the interactions of the endocrine, nervous, and immune systems36. The core dictum of this course of action is to encourage relaxation mentally followed physically. Hence this information helps to hypothesize that stimulation of Sthapani marma, motivation of Ajnya chakra, organizing different phases of manomaya kosha and added benefit of Shavasana would helps to counterbalance the mansika and shareerika doshas. Thus synchronization of Metabolism, Immunity and nervous system, so that the deepest level of healing can be achieved.

Action over local site:

Action of Shirodhara has been hypothesized on the base of stimulation of Pituitary gland, pineal gland and nerve endings of skin which in turn stimulates the CNS system. The incorporation of hypothetical aspects of Manomaya kosha, Chakra, Marma and Shavasana posture would certainly help to predict hypothetical views of mode of action of Shirodhara with subjective outcomes.

Skin and other its appendages are having nerve endings. They are wide spread in the superficial layers of the skin as well as certain internal tissues and are rich with pain receptors such as Bradykinin, Serotonin, histamine, potassium ions, acetylcholine etc. and these are known to get stimulated by mechanical, thermal and chemical stimuli31. Present therapy

CONCLUSION:

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12. Sushruta, Sushruta Samhita Shareera sthana (Prathyeka marma nirdesha shareera) verse- 6/9 Nibandha Sangraha commentary

by Dalhana, In: Acharya YT. 8th ed.

Varanasi: Choukhamba orientalia; 2005. p.370.

13. Sushruta, Sushruta Samhita Shareera sthana (Prathyeka marma nirdesha shareera) verse6/16 Nibandha Sangraha commentary by Dalhana, In: Acharya YT. 8th ed. Varanasi: Choukhamba orientalia; 2005. p.371.

14. Guyton, Hall. Pituitary hormone and their control by the Hypothalamus. 11th edi.

Textbook of Medical physiology. Noida; Elesvier. Reprint 2008.p.919–20.

15. Anthony S. Fauci, Eugene Braunwald, Dennis L. kasper, Stephen L. Hauser, Dan L. Longo, Larry Jameson L. Mental disorders. Harrison’s Principles of Internal medicine. 2nd vol. 17th edi. New Delhi; McGraw-Hill companies.p.2710.

16. Iyangar B K S. Light on Yoga, Ygasanas Banda and kriya, 2nd part. New Delhi; Harper Collins Publisher: 2003. P379–80 17. Jelusich A Richard, Psychology of the Chakras Eye of the Lotus. 1st ed, Lotus press; Wisconsin: 2004.pp.11

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18. Swatmarama, Hatayoga pradeepika, 4th chapter verse 48, English commentary by Mukthi bodhananda, Munger; Yoga publication trust, reprinted 201. P538–9 19. Guyton, Hall.

Endocrinology and th reproduction. 11 edi. Textbook of Medical physiology. Noida; Elesvier. Reprint 2008.p.1007.

20. Chaudhuri K. Sujit, Autacoids, Concise Medical physiology, 4th Edi, Calcutta; New central book agency.2002.p306.

21. Guyton, Hall. Cortical and brain stem control of motor function. 11th edi.

Textbook of Medical physiology. Noida; Elesvier. Reprint 2008.p.691.

22. Agnivesh, Charaka Samhita – Sutra sthana – (Dirghamjivitiya) verse-1/42, Ayurveda dipika Sanskrit commentary by Chakrapani. In: Acharya YT, editor.

Varanasi: Krishnadas academy; 2006.p.8.

23. Sushruta, Sushruta Samhita Sutra sthana (Dosha datu mala kshaya vruddhi vijnaniya Adhyaya) verse- 15/41 Nibandha Sangraha commentary by Dalhana, In: Acharya YT. 8th ed. Varanasi: Choukhamba orientalia; 2005. p.75.

27. Shumsky G Susan, Exploring ChakrasAwaken Your Untapped Energy, Book mart press; USA: 2003.p.76.

28. Nagarathna R, Nagendra H.R, Integrative approach of Yoga therapy for Positive health. 4th Edi. Bangalore; Swami vivekanda yoga prakashana. Reprint 2007.p.24.

29. Swatmarama, Hatayoga pradeepika, 1th chapter verse 32, English commentary by Mukthi bodhananda, Munger; Yoga publication trust, reprinted 201. P98–99 30. Jaggi O.P, Yogic and Tantric medicine, 4th chapter, 5th Vol, New Delhi; Atmaram and sons. 1990.p. 106–11 31. Guyton, Hall. Somatic sensation, 2nd Unit.

11th edi. Textbook of Medical physiology.

Noida; Elesvier. Reprint 2008.p.598.

32. Guyton, Hall. Somatic sensation, 2nd Unit.

11th edi. Textbook of Medical physiology.

Noida; Elesvier. Reprint 2008.p.600.

33. Thomas L. Clayton, Taber’s Cyclopedic Medical dictionary, 1st Vol, Philadelphia; F.A. Davis company 1993.p.1630

24. Agnivesh, Charaka Samhita – Sutra sthana – (Stisreshaniya) verse-11/54, Ayurveda dipika Sanskrit commentary by Chakrapani. In: Acharya YT, editor.

Varanasi: Krishnadas academy; 2006.p.77.

34. Michael Irwin, Kavita Vedhara (2005). Human Psychoneuroimmunology.

Oxford University Press. ISBN 978-0-19856884-1.

25. Gauda Shivkumar, Ayurvediya shareera kriya vijnana: 9th edn, 4th part. Rohatak (Hariyan); Naya pustaka Bandar; 1990.

Chapter I, pp.259.

35. Chrousos, G. P. and Gold, P. W. (1992).

The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis. JAMA 267(Mar 4), 1244–52.

26. Yogananda Paramahansa, God Talks With Arjuna. Yogada satsanga society of India; Kolkatta: 2005.p.63.

Source of Support: Nil

36. Madeline M. Lorentz, RN, MSN. Stress and Psychoneuroimmunology Revisited: Using mind-body interventions to reduce stress.

Alterna J of Nursing Switzerland 2006; 11:1–11.

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 464–469 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article HARAMEKHALA – A MASTERPIECE ON INDIAN COSMETICS Archana I1*, Praveen2, Vyas Mahesh 3 , Bhat Jeddu Ganapathi4 1

Ph.D Scholar, Dept. of Basic Principles, I.P.G.T & R.A., Gujarat Ayurveda University, Jamnagar.

Lecturer, Dept. of Drvavyaguna,, K.P.S.V.S. Ayurvedic Medical College, Manvi, Karnataka.

3 Associate Professor, Dept. of Basic Principles, IPGT & RA, Gujarat Ayurveda University, Jamnagar.

4 Professor, Dept. of Ayurveda Siddhanta, Alvas Ayurveda medical College, Moodbidri, Karnataka.

2

*

Corresponding author: Email: drarubhat@gmail.com; Mob. No. +919408564467

Received: 16/07/2012; Revised: 29/08/2012; Accepted: 02/09/2012

ABSTRACT The concept of beauty and cosmetics is as old as civilization. Each and every individual wants to look beautiful. This is the reason we find the usage of cosmetic preparations since Vedic period.

Later in post Vedic period, Kavya, Natya and also in the treatises of Ayurveda we find references of various preparations of cosmetics. Haramekhala is one such treatise of humanities explaining various preparations of cosmetics using simple herbal drugs.

KEY WORDS: Haramekhala, Māhuka, Cosmetics

To Cite this article Archana I, Praveen, Vyas Mahesh, Bhat Jeddu Ganapathi (2012), HARAMEKHALA – A MASTERPIECE ON INDIAN COSMETICS, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 464–469

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INTRODUCTION: Haramekhala is a medieval work on Humanities especially documenting the cosmetical preparations prevalent in medieval India. Māhuka who lived during 9th century A.D. in central India authored this work in Prakrit language supported by Sanskrit [1]. The only original manuscript that forms the basis of the publication of Haramekhala was obtained from Brahmashree C. Narayanan Bhattatiri Avarugal of Paramburillom of Tiruvella near Kottayam. Later this Manuscript was published

by Dept. of Publications, Oriental Manuscript’s library, Trivandrum by the authority of his Highness Maharaja of Travancore in the year 1938. The manuscript was very old written about 800–900 years before and was deplorably worn and unreadable. Shri K. Sambashiva Shastri edited this book in two parts. The first part contains 2nd, 3rd, 4th chapters and the second part contains 5th chapter. The manuscript of 1st chapter is not obtained by him. There is information that P.V Sharma wrote an article named “Haramekhala Tantra (the first chapter on medicine)” in 1986 which is published in Ancient Science of Life 5, 3 which contains a translation of first chapter of Haramekhala Tantra according to the text manuscript P.G.I.6 of the collection of B.H.U. The concept of beauty and cosmetics is as old as race or civilization. It may also vary among people belonging to different continents or cultures, but the desire to look more attractive, young and seductive is a common factor. The earliest references for cosmetics are traced in Rig-Veda. Later the Vedas, Brahmanas and Upanishads give ample references for cosmetics used in India. In later works like Puranas, Kavyas and Ayurvedic Samhitas various cosmoceuetical preparations are specifically mentioned. Māhuka carried further the concept and application of ancient Indian cosmetology into a more organized form. Though Haramekhala seems to be a complete hand book on ancient Indian cosmetology, Māhuka honestly agrees that he just followed the earlier Acharyas in this regard. He calls them as Mahabuddhi or Mahamathi – the enlightened elders. These

original cosmetic preparations were further developed into precise branch by Māhuka.

Varieties of cosmetical preparations such as Adhara Prasādhana Yogas (Lip Balm), Netra Prasadhana Yogas (Eye care preparations), Mukhavasana Gulika (Mouth fresheners), Snana Choorna (Bathing powders), Udvartana Choorna (Massaging powders), Sugandhi Lepa Yogas (Anointments), Romashatana Yogas (Depilatory agents), Various Varnya Yogas (Whitening preparations), Hair care preparations, Foot care preparations etc, are found in Haramekhala in systematically arranged form.

Cosmetical preparations in Haramekhala Some of the simpler and special, elsewhere not found, easy to prepare cosmetical preparations of Haramekhala are enumerated here under. They can be developed into special formulations using modern technology.

Adhara Prasadhana Yogas Cracked lips, besides being painful, spoil the beauty of the face. Māhuka recommended certain remedies in such cases along with coloring of the lips. There are 4 various formulations mentioned by Māhuka for healthy and colorful lips. [2] For e.g. Paste is prepared from Sarjarasa (Resin of Shorea robusta , Tila taila (oil of Sesamum indicum) and Saindhava (Sodium chloridum). If this paste is applied to the lips it cures cracking.

Tilaka Yogas: Māhuka mentions certain “Tilaka - Bindi” which are mainly used for Vashikarana purpose. He mentioned these for both man and woman and also for the kings. Drugs like Gorocana (Benzoar), Manahshila (Realgar), Kumkuma (Crocus sativus Linn.), Tamala patra (Cinnamomum tamala), Bhringaraja (Eclipta alba), Aparajita (Clitoria ternata), Sahadevi (Vernonia cinerea Lese.), Vishnukranta (Evolvulus alsinoides Linn.), Kumari (Aloe barbadensis) and Rakta Karavira (Nerium odorum soland.) are used for this purpose. Gorocana (Bezoar) is the main

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ingredient in every preparation which induces color to the formula. [3] Netra Prasadhana preparations:

Yogas/

Eye

care

The beauty of face mainly depends on the eyes. If eyes are healthy and beautiful person will catch others eyes. Māhuka incorporated various Anjana preparations in Haramekhala which not only beautifies the eye but also protects it. Many Vashikarana Anjana preparations are also mentioned by Māhuka which can be integrated in the preparations for beautification of eyes as they are mainly used for attraction purposes. Totally 6 such Yogas are mentioned. [4] For e.g.

Souviranjana/Nilanjana prepared from the fine powder of Candana (Santalum album), Karpura (Borneo camphor), Truti (Elettaria cardamomum), Gandhapatra (Cinnamomum cassia) , Kushta (Saussurea lappa), Yashtimadhu (Glycyrrhiza glabra) and Nagakesara (Mesua ferrea Linn) which are taken in equal part is very aromatic, auspicious and used to beautify the eyes.

Romashatana Yogas/ Depilatory agents: The presence of hair on arm, face, legs and in pubic area is considered as uncomfortable.

Certain paste, oil preparations are mentioned to get rid of this. There are 3 references of depilatory agents in Haramekhala. [5] For e.g.

Oil extracted from seeds of Koushambi/Koshataki (Luffa aegyptiaca) is applied over pubic area after shaving. Then there will not be again growth of the hair.

Skin lightening: A fair skin and thin body has always been an attraction. Everyone wants to look fair and slim. Māhuka mentions various paste and powder preparations for the purpose of Lepana (anointing), Udvartana (massaging) and Snana (bathing) which fulfill the above criteria. While signifying the Udvartana Yogas, Māhuka

describes that a proper Udvartana procedure can even slim up Kumbhadasi, means stout body servants doing hard physical activities. He describes, it also cleanses and enhances the texture of skin.

Udvartanam Kumbhadasinam Karmakarinam Kleshakarkasha Shariranamapi Gatramatigouram Swamodam Cha Sampadayatiti || (H.M.5/59) Māhuka mentioned totally 28 Yogas in the form of massaging and bathing which helps in enhancing the skin tone.[6] For e.g.

Paste prepared from Tila (Sesamum indicum), Sarshapa (Brassica campestris), Haridra (Curcuma longa), Daruharidra (Berberis species) and Kushta (Saussurea lappa) is applied to the body and then bathing is done. It makes the body fairer.

Hair care preparations: Many remedies for hair fall, premature graying of hair, dandruff, boils in the scalp etc are mentioned by Māhuka. Preparations for hair wash, growth of hair and to get rid of hair lice are integrated by him. Fumigation to protect the hair and to produce aroma is also mentioned. In 4th chapter he mentioned totally 27 Yogas which help in hair care. There are 10 preparations which are used as fumigation for hair.[7] For e.g.

Juice of Phanivalli (Piper betel) is mixed with Parada/Mercury and applied to the comb. If this is used for combing the lice will stick to it.

Application of paste of Amalaki (Embelica officinalis), Kushta (Saussurea lappa), Nilotpala/Indivara (Nelumbium speciosum Willd), Nalada/Ushira (Vetiveria zizanioides) and Bala (Seda cordifolia Linn) prevents falling of hair and make it thick and beautiful.

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Preparations that increase the size of the Ear pinnae (Karnapali Vriddhikara Yogas): There are totally 8 formulations mentioned

by Māhuka which helps in increasing the ear pinna.[8] For e.g.

Massaging the ear pinna with a medicated ghee processed with powder of Gunja (Abrus precatorius) and milk of buffalo increases its size.

Mouth fresheners: Many preparations for Kavala and Gandusha which eradicates the bad odor of the mouth are mentioned by Māhuka. Pills in the name of Mukhavasana Gulika are also cited in Haramekhala. Totally 12 Yogas which are used in the form of mouth fresheners as they are prepared by using various drugs having aroma are mentioned. [9] For e.g.

Drugs like Aguru (Aquilaria agallocha), Candana (Santalum album), Sitasharkara (Cane Sugar), Harenu (Vitex agnus-castus Linn.), Jati phala (Myristica fragrans), Kasturi (Moschus), Kumkuma (Crocus sativus Linn), Jati patra (Myristica fragrans Houtt), Tugakshiri (Bambusa arundinacea) and Lavanga (Caryophyllus aromaticus Linn) – all taken in equal quantity, Karpura (Borneo camphor) and Kolaphala (Ziziphus jejuba) – 4 parts and 16 parts of Khadira Kashaya (Decoction of Acasia catechu) are triturated with Sahakara Taila (oil prepared from Mangifera indica Linn) to prepare pills. This is a good mouth freshener.

Facials skin care preparations: Preparations which remove the unwanted discoloration of the face and make it fair are stated by Māhuka. Some of the preparations are very simpler and are according to the seasonal availability. He used medicated oil, ghee and ointment preparations for this purpose. Totally 11 different Yogas mentioned which beautify the facial skin. [10]

For e.g.

Application of paste of Ingudi phala Majja (fruit pulp of Balanitis aegyptiaca) cures Vyanga.

Application of pulp of Badara (Ziziphus jejuba) along with honey and butter cures the Vyanga.

Stana Vardhaka Yogas/Breast developers: Many formulations used for Abhyanga (massaging), Lepa (Application), Nasya and Udvartana which increase in size of the breast and make them firm and beautiful are mentioned in Haramekhala. Sthana vardhaka Yogas are the special contribution of Haramekhalakara in the field of cosmetic by using herbal drugs. Māhuka mentioned totally 10 preparations for increasing the size of breast.[11] For e.g Nasya with Tandulajala (rice washed water) along with Strotonjana makes the breast firm, large and beautiful.

Application of paste of Kushta (Saussurea lappa), Vacha (Acorus calamus) and Nagabala (Sida veronicaefolia) triturated with fresh butter of buffalo makes the breast large and beautiful.

Foot care preparations: As like other parts of body, foot is also an important part to be considered as per cosmetology. A cracked unhealthy foot always spoils the dignity and personality of a person who looks perfect in every other sense. There are totally 4 preparations which help in maintaining the beauty of foot.[12] For e.g.

Application of paste prepared from Gula/Guda/Jaggery, Gairika (Red ochre), Guggulu (Balsamodendron mukul), Rala (Resin of Shorea robusta), Saindhava (Sodium chloridum), Honey and Ghee cures the cracking of foot.

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Rejuvenation enhancers:

process/General

body

Māhuka explains some of the rejuvenation preparations by using which a person can become a best orator (Vageeshwara), his perception power of ear, nose, eye, mind etc get enhanced. They also prevent aging, falling and graying of hair. In total they help in the maintenance and enhancement of complete health of a person making him look younger.

Totally 15 various Rasayana Yogas have been mentioned by him. [13] For e.g.

If powder of Amalaki (Phyllanthus emblica) is licked with water or honey and ghee during evening time it gives strength to mind, eyes, nose and ears and makes the person look like young.

General Body care deodorants: Māhuka mentioned a variety of Sugandha Taila (aromatic oils), Vilepana Yogas (anointing preparations), Snana choornas (bathing powders) and Udvartana choornas (massaging powder) which act as deodorants by their regular use. Season wise usage of these with particular drug combination is also found in the book. Apart from deodorizing the body they also help in preventing skin ailments and keep the body healthy. Explanation of preparing various Gandha (pastes) which are having the aroma of fragrant drugs like Bakula (Mimusops elengi), Nagakesara (Mesua ferrea), Kuvalaya (Nymphaea nouchali), Campaka (Santalum album), Indivara

REFERENCES

(Nelumbium speciosum Willd), Karavira (Nerium odorum), Ketaki (Pandanus odoratissimus), Mallika (Jasminum sambac) are found in Haramekhala. These are used to apply over body as well as to scent the daily belongings. [14] For e.g.

Oil prepared from equal quantities of Truti/Ela (Elettaria cardamomum), Dala/Tamala Patra (Cinnamomum tamala), Jala/Hribera (Pavonia odorata Willd), Kusha/Vishana (Eragrostis cynosuroides), Nakha (Helix aspera), Sillhaka (Liquidamber orientalis) – one part each and Shilapushpa/Shaileya (Parmelia perlata) – double the quantity of all above drugs is beneficial for massaging in Sharad Rutu.

CONCLUSION A thorough review of Haramekhala leads to an assumption that the cosmetical preparations were made easily available with simpler procedures. Māhuka covered all aspects of cosmetology prevalent during his period. More than three hundred preparations are discussed in Haramekhala making the work which can be called as a master piece in Indian Cosmetology. It is astonishing to note that Māhuka has classified the subject matter in a scientific way in different headings covering all aspects of modern cosmetology. Many of the preparations are worth trying and hence relevant in modern times. Most of the formulations are made from easily available drugs and they are simpler in form. On this ground we can rightly call Māhuka as the “Father of Indian Cosmetology” Trivandrum, chapter 2nd verse 68, chapter 4/71, 72; chapter 5/13, 14

1. I. Archana, Jeddu Ganapathy Bhat, (Oct-Dec 2011) A critical review on antiquity, authorship 3. Ibid, chapter 3, verse 5-14 and content of Haramekhala – A medieval 4. Ibid, Chapter 3, verse 16-20; chapter 5 verse 15 work on humanities.

2. K. Sambashiva Shatry, Editor (1938), 5. Ibid, chapter 2, verse 70, 71; chapter 4, verse 334,335,336 Haramekhala of Māhuka, printed by the superintendent, Government press, 6. Ibid, chapter 4, verse 143, 147,148; chapter 5, verse 59-85

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7. Ibid, chapter 4, verse 2-28, chapter 5 verse 91- 11. Ibid, chapter 4, verse 38-40, 111-118 101 12. Ibid, chapter 4, verse 206-209 8. Ibid, chapter 4, verse 35-40,112,113,115 13. Ibid, chapter 4, verses 21-28,100-101, 142, 9. Ibid, chapter 4, verse 75-77, chapter 5 verse 147-148, 163-164, 358-367 12, 16-32 14. Ibid, chapter 4, verses 143-146, 5/33-57, chapter 5, verses 59-85, 125-130, 133-158 10. Ibid, chapter 4 verse 87-103

Source of Support: Nil

Conflict of Interest: None Declared

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Global J Res. Med. Plants & Indigen. Med. | Volume 1, Issue 9 | September 2012 | 470–476 ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

Review article COMPARATIVE ANALYSIS OF AYURVEDIC AND SIDDHA SYSTEM OF INDIAN MEDICINE Panja Asit Kumar1*, Gahunge Pankaj2, Chattopadhyaya Abichal3 1

Lecturer, PG Dept of Maulik Siddhanta, NIA Jaipur, Rajasthan, India PG Scholar, Dept of Maulik Siddhanta, NIA Jaipur.

3 Head, PG Dept of Sarir & Samhita, IPGT RA, Kolkata, West Bengal, India 2

*

Corresponding author: E-mail: gahungepankaj@gmail.com, Mob: +917737039665

Received: 10/07/2012; Revised: 20/08/2012; Accepted: 01/09/2012

ABSTRACT Ayurveda and Siddha systems of medicine have taken their origin from the vedic and post-vedic medical wisdom. Initially both were united and gradually they inculcated slight different angle of approach in treatment and is being practiced separately for the last 1500 years. Siddha has been mainly popular in Tamil Nadu and Kerala in comparison to nationwide popularity of Ayurveda.

Though from various aspects both seem to be different systems but owing to their same origin they have ample number of similarities. Scientific and rational analysis of similarities and dissimilarities between the two systems is the main focus of this review.

KEY WORDS: Ayurveda, Siddha,

To Cite this article Panja Asit Kumar, Gahunge Pankaj, Chattopadhyaya Abichal (2012), COMPARATIVE ANALYSIS OF AYURVEDIC AND SIDDHA SYSTEM OF INDIAN MEDICINE, Global J Res. Med. Plants & Indigen. Med., Volume 1(9), 470–476

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INTRODUCTION: Linguistically Ayurveda is not only Sanskrit based, but also extends in various Indian languages like Tamil, Malayalam, Telugu, Bengali, Marathi (including works up to later 18th century) whereas Siddha reference is found mostly in Tamil.1 Geographically Siddha is being practiced in Tamil speaking regions and Ayurvedis being practiced in almost all of the Indian states including Tamil Nadu and in major parts of Sri Lanka and Nepal2.

History: References of Ayurveda and like concepts are also found in early Tamil literature. In Silappatikaram3, a mid-fifth century C.E. text, description of Ayurveda (Tamil: āyulvetar) is available. The three humours (Tamil: tiritocam, Sanskrit. tridosha) are also documented in the Tirukural,4 a collection of poems that dates from around 450–550 C.E. The first Tamil Siddha text is the Tirumandiram5 written by Tirumular probably around the 6th or 7th century C.E. Alchemy used to transform iron into gold is mentioned in the said text; the major sources of Siddha medicine belong to a religious group who call themselves KayaSiddhas. They emphasize the “perfection Lineage:

of the body” by means of yoga, alchemy, medicine and certain types of Tantric rituals.

Their works date from about 13th to 14th century C.E., and are attributed to numerous (Sanskrit: authors including Akattiyar6 Agastya), the traditional founder of Siddha medicine and Teraiyar7 (late 17th century), who is said to have written twelve works on medicine, and whose famous disciple Iramatevar travelled to Mecca in the late 17th or early 18th century where he studied, converted to Islam, and took on the name Yakkopu 8 (i.e., Jacob). Most critical scholars of Siddha agree that on the basis of their language, the numerous texts on Siddha medicine, which present it as a system of healing, cannot be older than the 16th century.

We must, therefore, acknowledge that Tamil Siddha medicine, as it is now exists in both academic and practical field began in Tamil Nadu around 16th century, but elements of healing practices which became part of Siddha medicine, including those held in common with Ayurveda, came from an earlier period9.

Origin: Like all systems of Hindu knowledge, Siddha medicine attributes its origin to a divine source; hence its knowledge is sacred and eternal, passed down to humans for the benefit of all humanity.

AYURVEDA10: According to Charak samhita BRAMHA

Daksa

Ashwinikumaras

Bharadvaja

Punarvasu atreya,

Indra

Agnivesha.Etc.

According to Sushruta samhita BRAMHA

Daksa

Ashwinikumaras

Bharadvaja

Dhanwantari

Sushruta Etc.

SIDDHA11: Shiva

Parvati

Nandi

18 Siddhars.

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Indra

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Tradition lists a total number of eighteen Siddhars, beginning with Nandi and the semilegendary Agattiyar through to the final Siddhar, Kudhambai.

They are the acknowledged transmitters of medical doctrines and practices.According to one tradition, the Siddhars are Nandi, Agasthiyar, Tirumular, Punnakkeesar, Pulasthiyar, Poonaikannar, Idaikkadar, Bogar, Pulikaisar, Karuvurar, Konkanavar, Kalangi, Sattainathar, Azhuganni, Agappai, Pumbatti, Theraiyar and Kudhambai12.

Basic principles: Like Ayurveda, Siddha medicine mentions that the three humours predominate in humans in accordance with the nature and stage of life and that they vary with the seasons. But the assortment of the humours according to stages of life and seasons in Siddha differs from that of Ayurveda. In the case of the seasons, the variation is attributed simply to the different climatic conditions that occur in the different periods of the year in the northern inland areas and the southern, Tamil coastal and inland environments. According to Siddha, VATA predominates in the first third of life, PITTA in the second third, and KAPHA in the last third of life13; while in Ayurveda, kapha dominates the first third and vata in the last third of life14.

In terms of climate, the north is colder in the winter (Dec.- Jan.) than is the south and the west coast has rain in June and July, when the east coast is extremely hot. A dry, cold climate is rare in the south but it is precisely that climate which increases VATA. pitta and kapha, on the other hand, are increased when it is hot and wet .

Pancha vata & dasha vata: In Siddha system there are five secondary VATAs apart from the major and those mentioned in Ayurveda15. These are:1) Pranan 2) Apan 3) vyanan 4) samana 5) uthanan 6) nagam, the air for higher intellectual functions; 7) kurmam, the air of yawning; 8) kirukaram, the air of salivation; 9) devadhattham, the air of laziness; and 10) dhananjayam, the air that acts on death.16

Diagnostic tools: The diagnosis of disease in Siddha medicine relies on the examination of eight anatomical features (envagai thaervu), which are evaluated in terms of the three humors.

They are; 1) Tongue, 2) complexion, 3) voice, 4) eyes, 5) touch, 6) stool, 7) pulse and 8) urine17. Most modern Siddha doctors pay the greatest emphasis on the examination of the pulse and believe to obtain both diagnosis and prognosis through one process. This method of diagnosis also is available in Ayurveda in later works mainly after the 14th century. Prior to that the Ayurvedic diagnosis of a disease was purely based on examination of vitiation of one or more humours, dasavidha pariksa and precisely on observation, touch and interrogation18.

Branches: Siddha has developed expertise in five particular branches of medicine: 1) General medicine, 2) Paediatrics, 3) Toxicology, 4) Ophthalmology, and 5) Rejuvenation19, while traditional Ayurveda has 8 branches - 1) Kayachikitsa - general medicine 2) Bala pediatrics 3) Graham - exogenous demonology 4) Urdhwanga - diseases above clavicle 5) Shalya – the surgery , 6) Damstra - the toxicology, 7) Jara - rejuvenation and 8) Vrsa - aphrodisiac therapy20.

Shodana karma: Ayurveda prescribes a therapeutic regimen consisting of the “five purifying actions” emetics, purgatives, enemas, bloodletting and errhines21, on the contrary, Siddha employs only purgation22.

Surgery23: Unlike Ayurveda, in which it forms a separate school of medicine, surgery per se is not a significant part of Siddha medicine.

Medicated oils and pastes are applied to treat wounds and ulcers, but the use of a knife (reference of surgical instruments) is hardly found in Siddha medicine24.

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Varmam-the martial art: Closely connected with the tradition of the martial arts in South India, there developed a type of acupressure treatment based on the vital points in the human body, known as varmam (Sanskrit: marma). There are 108 vital points in the body as mentioned in the Ayurveda classics, which identify them and explain25. In Siddha medicine the number of important varmam points is also 108 (some say 107) out of a total of 40026. Siddha doctors developed techniques of applying pressure to special points, called Varmakkalai, to remove certain ailments and of massaging the points to cure diseases. They are also specialized in bone-setting and they often practiced an Indian form of the martial arts, called cilampam or silambattam, which involved a kind of dueling with staffs. Varmam is particularly wide spread among the hereditary Siddha practitioners belonging to the Nadar caste in the district of Kanyakumari in Tamil Nadu.27 Rejuvenation therapy: Closely connected with Siddha yoga, the Siddha system of rejuvenation- therapy, known as Kayakalpa28 (from Sanskrit, meaning “making the body competent for long life”), marks the most distinctive feature of Siddha medicine. It involves a five step process for rejuvenating the body and prolonging life29.

1. Preservation of vital energy via breathcontrol (Tamil: vasiyogam or Sanskrit. pranayama) and Yoga.

2. Conservation of “muppu” (A Union of Three Salts) 4. Use of calcinated powders (Tamil: chunnam, Sanskrit: bhasma) prepared from metals and minerals, and 5. Use of drugs prepared from plants special to each Siddha doctor.

The esoteric substance called “muppu” is particular to Siddha medicine and may be considered as Siddha’s equivalent of the “philosopher’s stone.” It is generally thought to

consist of three salts (moo-uppu) called puniru, kallupu, and vediyuppu, which correspond respectively to the sun, moon, and fire.30 The alchemy: Although mercury plays a key role in both Ayurveda and Siddha forms of medical alchemy, mercury in its pure form is not found in India and, therefore, must be imported, often from Italy. The core of Siddha medicine is its alchemy, whose fundamental principles conform to the alchemical traditions of ancient Greece and China, and of Arabic alchemy. It would, therefore, seem possible that both Siddha and Ayurveda alchemy might well have derived from one or a combination of these older traditions. Further investigation into each system in relationship to Indian alchemy could reveal important connections between Indian and other systems of alchemy and medicine.

The alchemical part of Siddha is present from at least the time of Tirumular’s Tirumandiram (6th or 7th cent. C.E.) in which various alchemical preparations are mentioned.

Alchemy is also found in Sanskrit texts from North India, but only from about 6th–7th Century C.E., and later became integral part of Ayurvedic medicine called Rasashastra (knowledge about mercury and allied matter) In the classical treatises of Ayurveda, however, mention of alchemy isdesiring and only certain metals and minerals are mentioned in late classical texts of 7th century C.E. by the author Vagbhata. Since alchemy had reached a far greater level of development in Siddha medicine than in Ayurveda, it is believed that medical alchemy may well have begun in South India among the Siddha yogis and ascetics and was later assimilated into Ayurveda.31 The facts like installing Rasalinga, name of Nandideva in Rasa Siddhas in Rasaratnasamucchaya supports this view32.

Bhasma-chunnam: Both Ayurveda’s Rasashastra and Siddha’s alchemy have devised slightly different methods for purifying or detoxifying metals and minerals, called suddhi murai in Tamil and

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shodhana in Sanskrit, before they are reduced to ash (Tamil: chunnam, Sanskrit: bhasma).

2. There is no fixation without alkali.

Siddha practitioners add the esoteric substance muppu34, which seems to vary in composition from one Siddha doctor to the other. Other ingredients added to increase a chunnam’s potency are healthy human urine (amuri) or urine salts (amuriuppu) obtained from the evaporation of large quantities of urine. Neither of these additives are found in Ayurveda’s Rasashastra.

3. There is no colouring without sulphur.

Research at present:

4. There is no quintessence without copper sulphate.

Research in the field of Siddha medicine in Tamil Nadu has revealed certain difficulties which must be overcome in order to understand properly this medical system and its history.

The central problem lies with the reliability of the secondary sources, which are written primarily by Tamil Siddha doctors.35

There are nine principles that must be followed in the calcination of metals and minerals33: 1. There is no alchemical process without mercury.

5. There is no animation without conflagration.

6. There is no calcination without corrosive lime.

7. There is no compound without correct blowing.

8. There is no fusion without suitable flux.

9. There is salammoniac.

no

strong

fluid

without

In the calcination process explained in traditional Ayurveda, the duration and intensity of the heat is regulated by the size of the pile of dung cakes called puta in Sanskrit. Siddha medicine has devised a method with a special substance made of inorganic salts, in Tamil called jayani, which reduces the number of burnings to only three or four. In order to increase the potency of the ash (chunnam),

CONCLUSION: Unlike Ayurveda, which has a long and detailed textual tradition in Sanskrit from around the beginning of the Common Era, Most of the knowledge about Siddha medicine comes from modern-day practitioners, who often maintain a historically unverified development of their own tradition and who because of the upsurge of Tamil pride tend to make fantastic claims about the age and importance of Siddha medicine vis-à-vis its closest counterpart in India, Ayurveda.

REFERENCES: 1. Iyer, T.G.Ramamurti: The Gems of Siddha System: Ist Edition 2005 Choukhamba Sanskrit Pratisthan, Delhi, Chapter- XVII, Urine- Page228–230, Eye-226, Ear-226, Body-226, Tongue-226–227, Mouth -227, Motions(Stool) 227–228, Pulse-219–225& in Chapter I pg.no3

3.

SilappatikaraSilambu, Indiravila Eduthakada 43– 44, page-12 (in evolution & development of Siddha Medicine)

2. Pillai, Kandaswamy. N., 1979., History of Siddha medicine., Government of tamilnadu publication,Madras; pages 428–429

5.

TirumandiramSubramaniam S.V. ,Madhavan V.R. : Ist Edition March 1983 Heritage of Tamil -Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-17–18

4. Kenneth G. Zysk nr. 4, 2008 Siddha Medicine in Tamil Nadu pg.no. 2

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6. Akattiyar=Agasthiar- Subramaniam S.V. ,Madhavan V.R. : Ist Edition March 1983 Heritage of Tamil -Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-16 7. Theraiyar- Subramaniam S.V. ,Madhavan V.R. : Ist Edition March 1983 Heritage of Tamil -Siddha Medicine: International Institute of Tamil Studies, Madras-113, , , Page-19 8. Kenneth G. Zysk nr. 4, 2008 Siddha Medicine in Tamil Nadu pg.no. 3 9. Kenneth G. Zysk nr. 4, 2008 Siddha Medicine in Tamil Nadu pg.no. 3 10. Agnivesh, Sutrasthan, 1st Chapter, 4-5th Shloka , Caraka Samhita, Ayurveda Depika Commentry by Cakrapanidutta, Pt. Yadavji Trikamji Acharya editor,Rastriya Sanskrit Samsasthan , Reprint 2006 Page .5 11. Subramaniam S.V. ,Madhavan V.R. : Ist Edition March 1983 Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-6,15,51 12. Iyer, Dr. T.G.Ramamurti: The Gems of Siddha System: Ist Edition 2005 Choukhamba Sanskrit Pratisthan, Delhi, , Chapter-VI,Page15 13. Subramaniam S.V. ,Madhavan V.R. : Ist Edition March 1983 Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-550–567 14. Vagbhat , Sutrasthan, 1st chapter, 8th verse, Ashtanga Hridaya, Sarvangasudara and Ayurveda Rasayan Commentary of Arundutta and Hemadri,Bhisagacharya Harishastri Paradakara Vaidya editor, Chaukhamba Orientalia, 8th edition,1998 page -7

Urine- Page-228–230, Eye-226, Ear-226, Body-226, Tongue-226–227, Mouth -227, Motions(Stool) 227–228, Pulse-219–225& in Chapter-v pg.no. 76 17. Iyer, Dr. T.G.Ramamurti: The Gems of Siddha System: Ist Edition 2005 Choukhamba Sanskrit Pratisthan, Delhi, , Chapter- XVII, Urine- Page-228–230, Eye-226, Ear-226, Body-226, Tongue-226–227, Mouth -227, Motions(Stool) 227–228, Pulse-219–225& in Chapter-X-141–155.

18. Agnivesh, vimansthana, 8th Chapter, 84th Shloka , Caraka Samhita, Ayurveda Depika Commentry by Cakrapanidutta, Pt. Yadavji Trikamji Acharya editor,Rastriya Sanskrit Samsasthan , Reprint 2006 Page .274 19. Subramaniam S.V. ,Madhavan V.R. : Ist Edition March 1983 Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-51 20. Vagbhat , Sutrasthan, 1st chapter, 5th verse, Ashtanga Hridaya, Sarvangasudara and Ayurveda Rasayan Commentary of Arundutta and Hemadri,Bhisagacharya Harishastri Paradakara Vaidya editor, Chaukhamba Orientalia, 8th edition,1998 page 5 21. Agnivesh, Sutrasthan, 2nd Chapter, 15th Shloka , Caraka Samhita, Ayurveda Depika Commentry by Cakrapanidutta, Pt. Yadavji Trikamji Acharya editor,Rastriya Sanskrit Samsasthan , Reprint 2006 Page 25 22. Subramaniam S.V. ,Madhavan V.R. : , Ist Edition, March 1983 Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-62 23. Subramaniam S.V. ,Madhavan V.R. : Ist Edition, March 1983 Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, , Page-461–471.

15. Iyer, Dr. T.G.Ramamurti: The Gems of Siddha System: Ist Edition 2005 Choukhamba Sanskrit Pratisthan, Delhi, , Chapter-VI, Page68–80

24. Kenneth G. Zysk nr. 4, 2008 Siddha Medicine in Tamil Nadu pg.no 10

16. Iyer, Dr. T.G.Ramamurti: The Gems of Siddha System: Ist Edition 2005 Choukhamba Sanskrit Pratisthan, Delhi, , Chapter- XVII,

25. Sushruta, sarirashana , 6th Chapter, 3 shloka, Sushruta Samhita, Nibandhasamgraha commentary by Dalhana, Yadavji Trikamji

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Acharya editor,Chaukhamba Orientalia, 6th edition,1997; page 369 26. Subramaniam S.V. ,Madhavan V.R. : Ist Edition, March 1983, Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-472–483.

27. Subramanian. S.V & Madhavan.V.R., 1983, heritage of the tamils Siddha medicine, English with tamil verses, international institute of tamil studies, madras pages 29–57 28. Subramaniam S.V., Madhavan V.R. : Ist Edition, March 1983, Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-132–145.

29. Kayakalpa Mooligai in Siddha Medicine : Ist Edition, March 1983, Heritage of Tamil Siddha Medicine EditedBy: S.V. Subramaniam & V.R. Madhavan, International Institute of Tamil Studies, Madras-113, By:G.Geetha,Page13.

Source of Support: Nil

30. Pillai, Kandaswamy.N .,1979., History of Siddha medicine., Government of Tamilnadu publication, Madras; pages 676–677 ) 31. Kenneth G. Zysk nr. 4, 2008 Siddha Medicine in Tamil Nadu Pg.no 13 32. Subramaniam S.V., Madhavan V.R. : Ist Edition, March 1983, Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-277 33. Subramaniam S.V. ,Madhavan V.R. : Ist Edition, March 1983, Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page-130 34. Subramaniam S.V. ,Madhavan V.R. : Ist Edition, March 1983, Heritage of Tamil Siddha Medicine: International Institute of Tamil Studies, Madras-113, Page27,30,31,133,146 35. Kenneth G. Zysk nr. 4, 2008 Siddha Medicine in Tamil Nadu pg.no. 2

Conflict of Interest: None Declared

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