GJRMI - Volume 3, Issue 7, July 2014 by Dr. Hari Venkatesh.K.Rajaraman

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INDEX – GJRMI - Volume 3, Issue 7, July 2014 MEDICINAL PLANTS RESEARCH Micro-biology & Bio-Chemistry PHYTOCHEMICAL ANALYSIS AND ANTI-LIPID PEROXIDATION ACTIVITY OF TAMARIX AFRICANA L. EXTRACTS BENABDALLAH Hassiba, GHARZOULI Kamel, KHENNOUF Seddik, AMIRA Smain, SOUFANE Sihem 278–285

Bio-Technology DIRECT SOMATIC EMBRYOGENESIS FROM MATURE LEAVES OF PIGEON PEA (CAJANUS CAJAN L. MILL SP) Pagadala Vijaya kumari

286–293

INDIGENOUS MEDICINE Ayurveda - Kaumarabhritya A COMPARATIVE STUDY OF BHASMAKNASHAK YOGA WITH EXERCISE AND DIET RESTRICTIONS IN OVERWEIGHT CHILDREN Renu B Rathi, Bharat Rathi

294–302

Ayurveda - Review Article HEPATOPROTECTIVE HERBS USED IN AYURVEDA - A REVIEW Giby Abraham

303–311

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF SAPTALA – ACACIA CONCINNA (WILLD.) DC., OF THE FAMILY MIMOSACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

Research Article PHYTOCHEMICAL ANALYSIS AND ANTI-LIPID PEROXIDATION ACTIVITY OF TAMARIX AFRICANA L. EXTRACTS BENABDALLAH Hassiba1*, GHARZOULI Kamel2, KHENNOUF Seddik3, AMIRA Smain4, SOUFANE Sihem5 1

Département de Microbiologie et Biochimie, Faculté des Sciences. Université de M’sila. Algérie. Département de Biologie, Faculté des Sciences de la Nature et de la Vie. Université de Sétif 1. Algérie. 5 Département de Biologie, Université de B.B. Arreridj. Algérie. *Corresponding Author: E-mail: benabdallahhassiba@yahoo.com 2,3,4

Received: 17/06/2014; Revised: 05/07/2014; Accepted: 06/07/2014

ABSTRACT The homogenate from rabbit brain represents an important source of lipids used directly in the study of peroxidation. The ratio of the peroxides is usually expressed as equivalent of malondialdehyde and determined by using 1,1,3,3-tetramethoxypropane as standard. Tamarix africana L. is widely used as a medicinal plant in Algeria. Polyphenols present in this plant are considered active compounds. The extraction of the flavonoids of Tamarix africana L. allowed their separation into two fractions (ethyl acetate extract and aqueous extract) containing flavonoids. The effect of extracts of Tamarix africana L. was studied in vitro. Examination of the data showed a significant inhibition of the relative rate of peroxidation by the ethyl acetate extract and the aqueous extract in comparison with the control representing 100% of peroxidation. Since there was no significant difference between these two extracts, the average rate is 53.8% inhibition. The ability of extracts to reduce the rate of lipid peroxidation resulted mainly because of the presence of flavonoids and phenolic acids. KEYWORDS: flavonoids, lipid peroxidation, malondialdehyde, Tamarix africana L., thiobarbituric acid.

Cite this article: BENABDALLAH Hassiba, GHARZOULI Kamel, KHENNOUF Seddik, AMIRA Smain, SOUFANE Sihem (2014), PHYTOCHEMICAL ANALYSIS AND ANTI-LIPID PEROXIDATION ACTIVITY OF TAMARIX AFRICANA L. EXTRACTS, Global J Res. Med. Plants & Indigen. Med., Volume 3(7): 278–285

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

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 7 | July 2014 | 278–285

INTRODUCTION

MATERIALS AND METHODS

In addition to conventional drugs used in the treatment of several diseases, traditional medicine practiced in the world makes use of plants to high levels of protective substances. Among these plants, Artemisia herba alba L., Punica granatum L., Quercus ilex L. and Tamarix africana L. which are widely used in Algerian traditional medicine in the treatment of gastroduodenal diseases. These plants are rich in polyphenols (phenolic acids, tannins and flavonoids) (Khennouf et al., 2003). Phenolic compounds are secondary metabolites widely distributed in the plant kingdom. Phenolic acids, tannins and flavonoids are the major classes of polyphenols. These compounds have remarkable biochemical and pharmacological activities such as antibacterial, antiviral and anti-inflammatory activities (Kim et al., 1998; Di Pietro et al., 2002). These activities are mainly due to their antioxidant power (De Whalley et al., 1990; Morton et al., 2000). Lipid peroxidation leads to oxidative degradation of unsaturated fatty acids. It involves the reaction of the latter with molecular oxygen to form a lipid radical and semi-stable hydroperoxides (Tapel, 1973; Barber and Bernheim, 1976). Many phenolic compounds react with free radicals (Lonchampt et al., 1989; Bagchi et al., 1998) to prevent the degradation of membrane phospholipids which is due to intense reactivity of free radicals (Halliwell et al., 1992). Lipid peroxidation can be enzymatic or non-enzymatic and occurs in three stages: initiation, propagation and termination (Halliwell and Gutteridge, 1984).

The materials for the study were collected during June in the region of Bordj Bou Arreridj, Algeria during the fruiting period. The plant was taxonomically identified using flora of Quézel and Santa (1962–1963), Ozenda (1983) and Maire (1952–1987); verified, characterized and confirmed by professional botanists of the department. Voucher specimens were deposited in the Herbarium. The samples of plant (leaves) were cleaned and allowed to dry at room temperature. The dried material is ground for use in the extraction of flavonoids.

In Algeria, leaves of Tamarix africana L. are traditionally used in decoction and infusion in the treatment of disorders of the digestive tract. In order to find principle compounds responsible for this effect and to search the mechanisms involved in this treatment, the present study was conducted to extract the flavonoids of this plant and to study their effects on lipid peroxidation.

Extraction of flavonoids The extraction of flavonoids was performed according to the method recommended by Markham (1982). It was based on the degree of solubility of these compounds in organic solvents. This method has two major steps: the first is with methanol to dissolve the flavonoids and the second is with chloroform and ethyl acetate to separate aglycones and glycosylated fractions of flavonoids. The extraction of flavonoids of Tamarix africana L. is made from the finely ground dry matter. After two successive extractions with 85% and 50% methanol, the filtrates were subjected to evaporation at low pressure (35°C Vapor Rota, Büchi 461, Germany). The filtrate was freed of waxes, fats and chlorophyll by successive washings with n-hexane to give an aqueous phase. To separate aglycones flavonoids and glycosylated flavonoids, the aqueous phase was mixed with chloroform to obtain an organic phase containing the flavonoid aglycones and aglycones methoxylated. The remaining aqueous phase underwent a series of extractions with ethyl acetate to recover the organic phase which contained some flavonoid aglycones, but especially mono- and diglycosides flavonoids. The remaining aqueous phase contained more polar glycosylated flavonoids such as di-, tri- and tetraglycosides flavonoids. In this study, two extracts were used: ethyl acetate extract and aqueous extract. The collected fractions were submitted to a concentration at low pressure at

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 7 | July 2014 | 278–285

35°C and then lyophilized for 24 hours. Each lyophilisate was weighed to calculate the yield of the extraction, expressed in grams per 100 grams of freeze-dried matter. Determination of total polyphenols The determination of total polyphenols was performed according to the method of Prussian blue and Price Butler (1977), modified by Graham (1992). The concentration of total polyphenols was derived from a standard curve prepared with gallic acid as the standard. The assay results were expressed as milligrams equivalent of gallic acid per gram of freezedried matter. Determination of total flavonoids The quantitative determination of flavonoids was performed according to the method of aluminium trichloride (Bahorun et al., 1996). A standard curve was set separately with quercetin to calculate the concentration of flavonoids in each extract. Assay results were expressed in milligrams equivalent of quercetin per gram of freeze-dried matter. Lipid peroxidation Both extracts of Tamarix africana L. (ethyl acetate extract and aqueous extract) were suspended in the 0.5% carboxymethylcellulose (CMC) at final concentrations 10, 25 and 50 μg/ml. The CMC solution alone was used as a control solution. The measure of the level of lipid peroxidation was carried out on a rabbit brain homogenate. Animals were anesthetized by intraperitoneal injection of urethane (25%). Cold saline (0.9% NaCl) was infused through the jugular vein to the brain to rid the blood of the tissue. The brain was removed and homogenized in 1.15% KCl. The rate of peroxide was measured by the method described by Ohkawa et al., (1979). This method was based on the reaction between peroxides and thiobarbituric acid (TBA) which lead to the formation of a pink complex indicator of lipid peroxidation. The brain

homogenate were incubated at 37°C in a water bath for one hour in the presence or absence of the test solutions. At the end of the incubation period, the mixture was centrifuged at 2700 g for 10 minutes (Rotina 35R, Hettich, Germany) and the supernatant were added to 8.1% sodium dodecyl sulfate (SDS) and 0.8% TBA prepared in acetic acid. The mixture was heated in a water bath at 100°C for one hour. After cooling, the samples were subjected to a second centrifugation to eliminate proteins and the optical density of the supernatant was read at 532 nm. The results are expressed as relative rate of peroxidation in relation to peroxidation of the homogenate untreated (control). Relative rate of peroxidation (%) = (absorbance of sample/absorbance of control) × 100. Chemicals Aluminium trichloride, acetic acid, gallic acid, CMC, quercetin, TBA, MDA, SDS were of analytical grade (Fluka, Merck, Prolabo, Sigma). Statistical Analysis The results of different experiments are expressed as mean ± SEM. The calibration curves were calculated by the method of linear regression. The significant difference between control and treated groups was determined by analysis of variance on ranks followed by Dunn's test for multiple comparisons with α=5%. RESULTS Extraction and determination phenolic compounds

total

Extraction of flavonoids by organic solvents from dry weight of Tamarix africana L. showed that the aqueous extract is the highest (19–22%) compared with ethyl acetate extract (3–4.5%) (Table 1). The determination of total polyphenols by the modified Prussian blue method showed the sensitivity and the reproducibility of this method. The amounts of total phenolic

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 7 | July 2014 | 278–285

compounds in the two extracts of Tamarix Africana L. are shown in Table 1. A high content was observed in ethyl acetate extract (500–546 mg/g) in comparison with the aqueous extract (140–170 mg/g). Aluminium trichloride is a method used for the determination of total flavonoids. Content of flavonoids is expressed as mg quercetin per g of freeze-dried matter. The amount of flavonoids is higher in ethyl acetate extract (332.2 mg/g) in comparison with the aqueous extract (14.2 mg/g).

Effect of extracts on lipid peroxidation Incubation of the homogenate of rabbit brain for an hour in the presence of extracts of Tamarix africana L. (ethyl acetate extract and aqueous extract) showed that these extracts (10, 25 and 50 µg/ml) inhibited lipid peroxidation. The presence of one of the two extracts in the incubation medium induced a significant decrease in the relative rate of peroxidation. Since there is no significant difference between these two extracts, which give 53.8% of inhibition of lipid peroxidation (Fig. 1).

Table 1. Determination of the rate of different classes of phenolic compounds of Tamarix africana L. extracts. Extracts Ethyl acetate Aqueous

Yield (%) 3.0–4.5

Total polyphenols* (mg/g) 500–546

Total flavonoids** (mg/g) 332

19.0–22.0

140–170

* Modified Prussian blue method. ** Aluminium trichloride method.

Figure 1. Effect of ethyl acetate and aqueous extracts of Tamarix africana L. on the rate of lipid peroxidation.

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DISCUSSION Folk medicine practiced throughout the world relies heavily on the use of plants as a source of natural active substances. Among these substances, plants containing phenolic compounds and flavonoids in particular are highly beneficial in therapeutics. Starting from the observation that Tamarix africana L. is widely used in Algerian traditional medicine and to obtain a good separation of their active principles responsible for its gastroprotective activity, the mining method adopted is based on differences in the degree of solubility of flavonoids in organic solvents. A relatively high yield (19–22%) was obtained with the aqueous extract of Tamarix africana L. containing the most polar flavonoids (di-, triand tetraglycosides). In contrast, the performance of the ethyl acetate extract containing some flavonoid aglycones including mono- and diglycosides is five times lower than that of the aqueous extract. Several methods are applied to extracts for the quantitative determination of different classes of phenolic compounds of Tamarix africana L. (total polyphenols and flavonoids). The modified Prussian blue method was effective for the determination of total polyphenols in the extracts. The quantitative determination of phenolic compounds showed that the amount of polyphenols in the ethyl acetate extract is relatively high in comparison with the aqueous extract. Quercetin is widely used as a standard for determining the content of flavonoids in a sample. In comparison with the ethyl acetate extract, the aqueous extract appears poor in flavonoids. Analysis of extracts of Tamarix africana L. with HPLC (unpublished results) showed the presence of quercetin, kaempferol, luteolin and isorhamnetin in the ethyl acetate extract. In addition, the aqueous extract contains mainly flavonoid glycosides or rutinosides and some flavonoid aglycones which isoquercetin and luteolin are identified by this method. In addition to flavonoids, phenolic compounds identification by HPLC showed that the extracts contain procyanidins and some

phenolic acids such as ellagic acid, gallic acid and vanillic acid. These data confirm that the extraction process adopted is acceptable to some extent to separate flavonoid glycoside and aglycone. The richness of Tamarix africana L. on active compounds such as flavonoids, phenolic acids and tannins can be one of the principles of its use in traditional medicine in the treatment of diseases of the digestive tract. Lipid peroxidation leads to oxidative degradation of unsaturated fatty acids and leads to the alteration of the structural integrity of membranes and their permeability. However, the conversion of Thiobarbituric Acid Reactive Substances (TBARS) equivalent of MDA is widely used to assess the importance of lipid peroxidation (Wills, 1987; Minamiyama et al., 1994). In the present study based on the extinction coefficient of MDA, the incubation of tissue for an hour results in the formation of 11.6 ± 1.5 nmol MDA equivalents per gram of fresh tissue. This rate is not far from that found in the liver homogenate (11.9 ± 1.1 nmol/g) and rat stomach (20.0 ± 2.5 nmol/g) (Yegen et al., 1990). Flavonoids, phenolic acids and tannins inhibit mechanisms of enzymatic and non-enzymatic initiation of lipid peroxidation (Nakayama et al., 1992; Galvez et al., 1995; Morton et al., 2000). Incubation of rabbit brain homogenate in the presence of caffeic acid, gallic acid and ellagic acid showed inhibition of lipid peroxidation. This result is consistent with that of Okuda et al., (1992) and Nardini et al., (1998) who found that phenolic acids are powerful antioxidants, including those with a catechol -type structure such as caffeic acid. The gallic acid and caffeic acid have the same antiradical efficiency (Sanchez –Moreno et al., 1998). The esterification of caffeic acid and gallic acid increases their antiperoxidation activity (Nakayama et al., 1992). The application of two extracts of Tamarix africana L. in peroxidation test revealed their antioxidant potential. The ethyl acetate extract and the aqueous extract have a similar effect. As mentioned before, the two extracts contain some aglycones but mostly glycosylated flavonoids. The presence of flavonoids and

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phenolic acids in Tamarix africana L. extracts suggests that the mechanisms involved in the antioxidant activity of these extracts are the same as those of the pure phenolic compounds (scavengers of free radicals, chelating metals). CONCLUSION Extraction protocol applied for the separation of flavonoids of Tamarix africana L. into two fractions is acceptable and it has achieved in all quite acceptable extraction yields. Moreover, the quantitative determination of different classes of phenolic compounds showed that the ethyl acetate extract is rich in flavonoids. Ethyl acetate extract and aqueous extract inhibited lipid peroxidation in vitro. Although the two

fractions are quite complex in their composition, they exert a similar effect. As the phenolic compounds have other activities in addition to their antioxidant effect, they can replace the classic antioxidant, ascorbic acid. The fact that the composition of each extract is complex, it is necessary to isolate and assess the major active principles of this plant to test their effect against several diseases. ACKNOWLEDGEMENT(S) The National Agency for the Development of Health Research and the Ministry of Higher Education and Research Scientist of Algeria are thanked for financial support for research projects (07/01/01/03/06/97, F/1901-04-95).

REFERENCES Bagchi D, Kuszynski C, Balmoori J, Bagchi M, Stohs SJ. (1998). Hydrogen peroxideinduced modulation of intracellular oxidased states in cultured macrophage J774A.1 and neuroactive PC-12 cells, and protection by a novel grape seed proanthocyanidin extract. Phytother. Res. 12: 568–571. Bahorun T, Gressier B, Trotin F, Brunet C, Dine T, Vasseur J, Gazin JC, Pinkas M, Luyckx M, Gazin M. (1996). Oxygen species scavenging activity of phenolic extract from Hawthorn fresh plant organs and pharmaceutical preparations. Arzneim-Forsch/Drug. Res. 1–6. Barber AA, Bernheim F. (1976). Lipid peroxidation: its measurement, occurence, and significance in animal tissues. In: "Advances in Gerontological Research". Eds. Strehler BL. Academic Press (New York); Chap. 2: 403.

De Whalley CV, Rankin SM, Hoult JRS, Jessup W, Leake DS. (1990). Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages. Biochem. Pharmacol. 39: 1743–1750. Di Pietro A, Conseil G, Pérez-Victoria JM, Dayan G, Baubichon-Cortay H, Trompier D, Stenfels E, Jault JM, de Wet H, Maitrejean M, Comte G, Boumendjel A, Mariotte AM, Dumontet C, McIntosh DB, Goffeau A, Castanys S, Gamarro F, Barron D. (2002). Modulation by flavonoids of cell multidrug resistance mediated by Pglycoprotein and related ABC transporters. Cell. Mol. Life Sci. 59: 307–322. Galvez J, De la Cruz JP, Zarzuelo A, Sanchez de la Casta F. (1995). Flavonoid inhibition of enzymic and nonenzymic lipid peroxidation in rat liver differs from its influence on the glutathionerelated enzymes. Pharmacol. 51 (2): 127–133.

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Graham HD. (1992). Modified Prussian Blue assay for total phenols. J. Agr. Food. Chem. 40: 801–805. Halliwell B, Gutteridge JMC. (1984). Oxygen toxicity, oxygen radicals, transition metals and disease. J. Biochem. 219: 1– 14. Halliwell B, Gutteridge JMC, Cross CE. (1992). Free radicals, antioxidants, and human disease: where are we now? Lab Clin Med. 119: 598–620. Khennouf S, Benabdallah H, Gharzouli K, Amira S, Ito H, Kim TH, Yoshida T, Gharzouli A. (2003). Effect of tannins Quercus suber and Quercus coccifera leaves on ethanol-induced gastric lesions in mice. Agric. Food. Chem. 51, 1469–1473. Kim HP, Mani I, Iversen L, Ziboh VA. (1998). Effects of naturally-occurring flavonoids and biflavonoids on epidermal cyclooxygenase and lipoxygenase from guinea-pigs. Prostaglandins, Leukotrienes and Essential Fatty Acids. 58:17–24. Lonchampt M, Guardiola B, Sicot N, Bertrand M, Perdrix L, Duhault J. (1989). Protective effect of a purified flavonoid fraction against reactive oxygen radicals in vivo and in vitro study. ArzneimForsch/Drug Res. 39 (II): 882–885. Markham KR. (1982). Techniques of flavonoid identification. Academic Press (London). Chap. 1 & 2: 1–113. Minamiyama Y, Yoshikawa T, Tanigawa T, Takahashi S, Naito Y, Ichikawa H, Kondo M. (1994). Antioxidative effects of a processed grain food. J. Nutr. Sci. Vitaminol. 40 (5): 467–477.

Morton LW, Abu-Amsha Caccetta R, Puddey IB, Croft KD. (2000). Chemistry and biological effects of dietary phenolic compounds: Relevance to cardiovascular disease. Clin. Exp. Pharmacol. Physiol. 27: 152–159. Nakayama T, Niimi T, Osawa T, Kawakishi S. (1992). The protective role of polyphenols in cytotoxicity of hydrogen peroxide. Mutat. Res. 281: 77–80. Nardini M, Pisu P, Gentili V, Natella F, Di Felice M, Piccolella E, Scaccini C. (1998). Effect of caffeic acid on tertbutyl hydroperoxide-induced oxidative stress in U937. Free. Radical. Bio. Med. 25 (9): 1098–1105. Ohkawa H, Ohishi N, Yagi K. (1979). Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95: 351–358. Okuda T, Yoshida T, Hatano T. (1992). Antioxidant effects of tannins and related polyphenols. In: "Phenolic compounds in food and their effects on health II". ACS Symposium Series N° 507. Eds. Huang MJ, Ho CT,Lee C. Am. Chem. Soc. Chap. 7: 87–97. Price MP, Butler LG. (1977). Rapid visual estimation and spectrophotometric determination of tannin content of sorghum grain. J. Agr. Food. Chem. 25: 1268–1273. Sanchez-Moreno C, Larrauri JA, Saura-Calixto S. (1998). A procedure to mesure the antiradical efficiency of polyphenols. J. Sci. Food. Agr. 76: 270–276. Tappel AL. (1973). Lipid peroxidation damage to cell components. Fed. Proc. 32: 1870–1874.

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Wills

ED. (1987). Evaluation of lipid peroxidation in lipids and biological membranes. In: "Biochemical Toxicology a practical approach". Eds. Smell K, Mullock B. IRL Press (Washington). Chap. 6: 127–151.

Source of Support: National Agency for the Development of Health Research and the Ministry of Higher Education and Research Scientist of Algeria

Yegen B, De deoglu A, Aykac I, Oktay S, Yalcin S. (1990). Effect of coldrestraint stress on glutathione and lipid peroxide levels in the liver and glandular stomach of rats. Pharmacol. Res. 22 (1): 45–48.

Conflict of Interest: None Declared

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Research Article DIRECT SOMATIC EMBRYOGENESIS FROM MATURE LEAVES OF PIGEON PEA (CAJANUS CAJAN L. MILL SP) Pagadala Vijaya Kumari1* !

Department of Botany, Cytogenetics and Plant Biotechnology Laboratory, Osmania University, HYDERABAD - 500 007, India. Present Address: Biotechnology, Department of Biology, Ambo University , AMBO – Ethiopia. *Corresponding Author: Email: vkpagadala@rocketmail.com

Received: 16/05/2014; Revised: 20/06/2014; Accepted: 30/06/2014

ABSTRACT Protocols were standardized for plant regeneration via direct somatic embryogenesis from 35day-old leaf explants of three cultivars of pigeonpea [Cajanus cajan] (ICPH-8, ICPL-87 and ICPL7295). Frequency of Somatic embryo induction was dependent on the age of the leaves. Leaves isolated from in vitro and glass house grown plants responded well when compared to field grown plants. Leaves produced embryogenic calli from cut ends and somatic embryos appeared directly from leaf margins when cultured on MS medium supplemented with 5 mg/l naphthalene acetic acid (NAA), 1 mg/l, 6-benzylaminopurine (BAP) and 6% sucrose. Dark incubation of cultures for 30 days showed a remarkable increase in frequency (50) of somatic embryogenesis. Somatic embryos at various developmental stages matured upon transfer to 0.2 mg/l BAP and 0.1 mg/l NAA with 4% sucrose in half-strength MS medium. Plantlets obtained from somatic embryos were transferred to pots for acclimatization and grown to maturity with 70–80% frequency. KEY WORDS: Somatic embryogenesis; Mature leaf; Pigeon pea; Cajanus cajan; grain legume.

Cite this article: Pagadala Vijaya kumari (2014), DIRECT SOMATIC EMBRYOGENESIS FROM MATURE LEAVES OF PIGEON PEA (CAJANUS CAJAN L. MILL SP), Global J Res. Med. Plants & Indigen. Med., Volume 3(7): 286–293

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 7 | July 2014 | 286–293

INTRODUCTION Pigeon pea (Cajanus cajan (L Mill sp) is one of the major grain legume (pulse) crops of the tropics and subtropics. Direct somatic embryogenesis is the formation of somatic embryos or embryo-genic tissue directly from the explant without the formation of an intermediate callus phase (Raghavan 1986). In embryogenesis systems, this is almost always what happens (Merkle et al., 1990; Finer 1994). Unfortunately, in most other plants somatic embryogenesis is more difficult to obtain, but progress has been made in some grain legumes. Grain Direct embryogenesis occurs when embryos are started directly from explant tissue creating an identical clone while indirectly occurs from unorganized tissue (callus). Plant transformations and Mass propagation are the two important methods which can make use of these Somatic Embryogenesis. Agrobacterium mediated transformation is the easiest and most simple plant transformation can be done by using these somatic embryogenesis. legumes are difficult to regenerate, however different few reports are available from different explants where the concept of regeneration was observed. Anbazhagan and Ganapathi (1999) described a protocol for initiation of cell suspension cultures from leaflet explants and subsequent plant regeneration via somatic embryogenesis in Cajanus cajan. haploid plant production by anther culture (Bajaj et al.,1980). An efficient and reproducible protocol for regeneration is an urgent need to develop transgenics and genetic variations in Pigeon pea. Few reports on direct somatic embryogenesis in Pigeon pea from different explants and genotypes are available. Regeneration of shoot buds from excised cotyledons of Pigeon pea with BAP was reported earlier (Kumar et al., 1983; Mehta U & Mohan Ram., 1980). Plant regeneration was obtained on MS medium with IAA, Kinetin and coconut mild in Pigeon pea (Patel et al., 1982). The frequency of success on in vitro plant regeneration via somatic embryogenesis is very low (Seenivasu et al.,1998) All the protocols reported till date were callus

mediated, and the present report mainly deals with direct somatic embryogenesis from leaves. For the first time, high frequency plant regeneration via direct somatic embryogenesis in Pigeon pea from mature leaf margins was accomplished. This finding is of great significance in developing the transformation protocols, which may be exploited in Pigeon pea crop improvement. MATERIALS AND METHODS Plant material - Seeds of three pigeonpea genotypes ICPH-8, ICPL-87 and ICPL-7295 were procured from Genetic Resources Unit, International Crops Research Institute for Semi - arid Tropics, Patancheru, and Hyderabad, India. Seeds were surface sterilized with 70% ethanol for 5 minutes followed by 0.1% aqueous mercuric chloride for 10 min. They were washed thoroughly with sterile water and germinated aseptically on hormone free Murashige and Skoog’s (1962) (MS) medium containing 3% sucrose and 0.8% difco bacto agar. MS medium containing 4–6% sucrose, BAP, NAA, indole-3-acetic acid (IAA), 2, 4dichlorophenoxyacetic acid (2, 4-D), zeatin (ZEA) and kinetin (KN) either alone or in combination was used for induction of somatic embryos and maturation. The pH of all the media was adjusted to 5.8 before autoclaving. The media were dispensed into culture tubes of 25×150 mm and sterilized at 1.4 Kg cm2 for 20 minutes. Experimental design / Methodology - The effect of age of the leaf on somatic embryo induction was investigated by collecting them from 5 to 40 day-old seedlings grown in field (29–35°C), in vitro (25°C) and glass house (30°C). Leaf number was counted from the top after the initiation of first two pair of leaves from the seedlings and the age from the date of emergence was noted. In all the cases, 25 explants from each set were inoculated. The effect of growth regulators such as BAP, NAA, 2,4-D, ZEA, IAA and KN (0.5–10.0 mg/l) either alone or in combinations was studied on the induction of somatic embryos. All the experiments were repeated twice with ten

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replicates, frequency of somatic embryogenesis and standard deviation were calculated. Induction of somatic embryos - Mature leaves aged 35-days were cut vertically into two pieces and inoculated onto MS medium supplemented with 6% sucrose, 5 mg/l NAA and 1.0 mg/l BAP. The cultures were incubated for three weeks at 25 2 C under light (60 Em-2s-1) with 16 / 8 light/dark photoperiods. The calli along with proembryos on the leaf margins were subcultured into the same media and incubated for 30 days in dark. Globular and heart shaped embryos were separated and transferred onto half-strength MS medium supplemented with 4% sucrose, 0.2 mg/l BAP and 0.1 mg/l NAA. Plantlets with welldeveloped roots were transferred to experimental pots with 75% humidity for hardening and then to greenhouse for acclimatization. Histological studies - For histological observations, the leaf along with callus and somatic embryos was fixed in acetic acid: ethanol (1:3) for 72 hours, dehydrated in an ethanol and butanol series followed by paraffin embedding as described by Sharma & Sharma (1980). The embedded tissue was cut with microtome (Reichert-Jung, No 2030 Supercut, Germany) into 10 m thick sections, stained with hematoxylin eosin, mounted with DPX and observed microscopically. RESULTS AND DISCUSSION The frequency of somatic embryogenesis was highest (70%) in 35-day-old leaf explants with an average of 52 somatic embryos per explant in presence of 5 mg/l NAA and 1 mg/l BAP only when incubated in dark for 30 days (Fig. 1A). Prolonged incubation in dark for 35 and 42 days decreased the frequency of embryogenesis to 20% and 10% respectively. Thus, it appeared that light may not be necessary for the induction of somatic embryogenesis. Similarly, in Triticale, (1) reported 2.25-fold increase in embryoid induction in dark as compared to light incubation at (3000 lux). At the end of third

week, the leaf margins showed tiny, globular, smooth, greenish as well as golden yellow coloured proembryos in bunches. Embryogenic callus was also induced from the cut ends of the leaf. Culture of 40 day-old leaf resulted only in callus initiation without somatic embryogenesis. This implies that age of the leaf is critical for embryo induction. Explants collected from in vitro grown seedlings responded well compared to leaf of other seedlings (Table 1). In the presence of different concentrations of auxins and cytokinins, callus was induced without somatic embryogenesis when cultures were incubated in dark for 30-days. At the concentrations tested, 2, 4-D yielded golden yellow, KN brownish yellow and ZEA green calli (Table 2). The effect of auxin along with cytokinin at various concentrations was tested for the induction of somatic embryos from the cultivar ICPH-8 (Table 3). With a NAA (5 mg/l), BAP (1 mg/l) and 6% sucrose the frequency of somatic embryogenesis was 70% only when incubated in dark for 30-days. Increasing concentrations of sucrose increased the frequency of response until 6% and declined thereafter (Fig. 1B). After three – four weeks, calli along with proembryos on the leaf margins were subcultured into the same media and incubated for 28–30 days in dark. Different stages of somatic embryos appeared only during this second subculture (Fig. 2A). Histological sections of these cultures revealed the presence of embryos at different stages (all the pictures of the embryos not shown)(Fig. 2B&C). They were isolated and transferred to half-strength MS medium fortified with 0.1 mg/l NAA, 0.2 mg/l BAP and 4% sucrose for maturation. Embryos developed into plants between 3–4 weeks on this medium (Fig. 2 D & E). Plantlets with well-developed roots were transferred into experimental small pots for 10 days for hardening at 75% humidity and 26 1 C and subsequently to green house for acclimatization (Fig F). Induction of somatic embryos in pigeonpea via callus from different explants with NAA and BAP, failed to develop further into complete plants (Nalini et al.,1996).

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Table 1. Effect of source on leaf Somatic embryogenesis in Pigeonpea Cv.ICPH-8. Age of leaf(D) 10 25 35 40

Field grown plants 0 0 0 0

Glass house grown plants 0 C C+S.E C

In vitro grown plants 0 C+S.E C+S.E C+S.E

D-Days, C-Callus, S.E-Somatic Embryogenesis

Table 2. Effect of Auxins and Cytokinins on the induction of callus in Pigeonpea Cv. ICPH-8.

2,4-D (mg/l) 0.5 1.0 2.0 2.5 3.0 4.0 5.0 6.0 7.5 10.0

% 50 10 15 17 20 25 30 29 40 20

Auxins IAA (mg/l) 0.5 1.0 2.5 2.5 3.0 4.0 5.0 7.5 0 0

% 60 0 20 0 0 0 0 0 0 10

NAA (mg/l) 1.0 2.0 2.5 5.0 6.0 7.5 10.0 0 0 0

% 20 0 0 0 0 0 0 0 0 0

BAP (mg/l) 0.5 1.0 2.0 2.5 3.0 3.5 5.0 6.0 7.5 10.0

Cytokinins KN % (mg/l) 30 1.0 0 2.0 0 2.5 0 5.0 10 6.0 15 7.5 0 10.0 0 0 0 0 5 0

% 20 0 0 0 0 0 20 0 0 0

ZEA mg/l) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 5.0 6.0 10.0

% 20 0 0 0 0 20 0 25 40 0

% = % Frequency of callus induction

Table 3. Effect of auxin and cytokinin on the induction of somatic embryogenesis from leaf margins of Pigeonpea Cv.ICPH-8 Conc.growth Regulators (mg/I) 2,4-D 2.0 + KN 0.5 2,4-D 5.0 + KN 0.5 2,4-D 7.5 + KN 1.0 2,4-D 2.0 +BAP 1.0 2,4-D 5.0 + BAP0.5 2,4-D 7.5 +BAP 1.0 NAA 1.0 + BAP 1.0 NAA 2.0 + BAP 1.0 NAA 3.0 + BAP 1.0 NAA 4.0 + BAP 1.0 NAA 5.0 + BAP 1.0 NAA 6.0 + BAP 1.0

Type of Response C C C C C C C C C C + S.E C + S.E C + S.E

Values represent mean

% of Somatic embryogenesis 0 0 0 0 0 0 0 0 0 25 ± 1.5 70 ± 2.1 30 ± 0.9

Average No. of somatic embryos 0 0 0 0 0 0 0 0 0 15 ± 0.7 52 ± 1.5 20 ± 1.2

SD ( C-Callus, S.E - Somatic embryos )sucrose 4%

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Table 4. Effect of BAP and NAA on maturation of Somatic embryos of Pigeonpea CV. ICPH 8 Hormones (mg/l) BAP 0.5 + 0.1 NAA BAP 1.0 + 0.5 NAA BAP 0.5 + 0.5 NAA BAP 1.0 +1.0 NAA BAP 0.2 + 0.1 NAA BAP 0.2 + 0.5 NAA

% of S.E. matured 0 0 10 ± 0.5 0 65 ± 1.2 15 ± 0.9 Values represent mean

No.of S.E/explants 0 0 5 ± 0.6 0 39 ±1.3 10 ±0.2

SD, S.E Somatic embryos

1A - Age of the leaf corossponding with frequency of somatic embryos. 1B – Sucrose Concentration showing the percentage of Somatic Embrygenesis. 1C - Genotype Variation with percentage of Somatic Embryos. Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

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In the present study, some of the embryos germinated on the leaf explant giving rise to shoots with two small leaflets with out roots. Anatomical studies in pigeonpea, made from different explants (Prakash et al., 1994) showed the initiation of shoot buds from subepidermal tissues. In the present study also, epidermal tissue of the leaf margins might have become competent for embryo induction. Different concentrations of BAP and NAA were tried for maturation of embryos and their subsequent germination into plantlets. Mature embryos were separated and transferred onto half-strength MS with 0.2 mg/l BAP and 0.1 mg/l NAA (Table 4). The frequency of maturation of embryos and subsequent conversion into plantlets was 65% with shoot

and root average of (39) somatic embryos per explant. The response of other two cultivars ICPL-87 and ICPL-7295 were also evaluated for the induction of somatic embryos (Fig. 1C) and the frequency if somatic embryogenesis was similar. These observations suggest that induction of embryogenesis from leaf explants may be genotype independent (Hazra et al., 1989). Induction of somatic embryogenesis in groundnut was reported by (Bernard., 1980) with 2,4-D alone or 2,4-D plus NAA. Somatic embryogenesis in pigeonpea was observed earlier by (George & Eapen., 1994), while they used 2,4-D, NAA or picloram. On the other hand (Seenivasu et al., 1998) used thidiazuran, but in all the above reports somatic embryogensis were observed in callus cultures.

Figure 2:

2 A. Mature leaf margins showing bunch of tiny globular stage somatic embryos. 2 B. Histological section of a globular embryo. 2 C. Histological section of globular embryo with suspensor from the margins of mature leaf. 2 D. Young plantlet germinated from bipolar embryo with shoot and root systems 2 E. Plants with well-established shoot and roof system derived from somatic embryos. 2 F. Regenerated Plant in the Experimental Pot. Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

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CONCLUSION

ACKNOWLEDGEMENTS

The present study clearly suggests that direct somatic embryogenesis from matured leaf in all the three cultivars of Pigeonpea were induced with high frequency (60–70%) for the first time. The Plantlets were regenerated and transferred to pots and were grown to maturity with high frequency 70–80%. The cultivar ICPH–8 with MS medium, 6% sucrose gave the maximum frequency of embryogenesis and also regeneration. Further standardization for the plantlet survival to soil is under experimentation. This finding has great significance and can be successfully exploited in developing genetic variations including transgenics of this economically important crop.

The author is grateful to Dr. Ramanandam, ICRISAT, Patanchuru for providing the seeds and to Dr. Shashikaran, National Institute of Nutrition (NIN), Hyderabad for carrying out histology (microtomy) work. Senior Research Fellowship awarded by the Council of Scientific and Industrial Research - CSIR, New Delhi, to Ms Pagadala Vijaya Kumari is gratefully acknowledged. Heartful thanks to Prof J.K.Bhalla (Ex- Head Dept of Botany) for constant encouragement and Supervision in Manuscript preparation. Together with Department of Biology - AMBO University Ethiopia for their constant Co operation.

REFERENCES Anbazhagan, V.R., Ganapati, A. (1999). Somatic embryogenesis in cell suspension cultures of pigeon pea (Cajanus cajan). Plant Cell Tiss. Org. Cult.56:179–184. Bajaj, Y.P.S., Singh, H., Gosal, S.S. (1980). Haploid embryogenesis in cultures of Pigeon pea ( Cajanus cajan) Theor.Appl. Genet. 58: 157–159. Bernard S, (1980). Invitro androgenesis in hexaploid triticale: determination of physical consitions increasing embryoid and green plant production. Z.Pflanzenzuchtung, 85 : 308−321. Finer JJ (1994) Plant regeneration via embryogenic suspension cultures. In: Dixon RA, Gonzales RA (eds) Plant cell culture: a practical approach. Oxford University Press, Oxford, pp 67–102 George L. & Eapen S, Organogenesis and embryogenesis from diverse explants in pigeon pea. Plant Cell Rep, 13 (1994) 417.

Hazra S, Sathaye SS & Mascarenhas, BioTech, 7(1989) 949. Kumar AS, Reddy TP & Reddy GM, Plant regeneration from different callus cultures of pigeonpea (Cajanus cajan L).Plant Sci. Lett , 32 (1983) 271. Mehta U & Mohan Ram HY, Regeneration of plants from cotyledons of cajanus cajan. Indian J. Exp. Biol , 8 (1980) 800. Merkle SA, Parrott WA, Williams EG (1990) Applications of somatic embryogenesis and embryo cloning. In: Bhojwani SS (ed) Plant tissue culture: applications and limitations. Elsevier, Amsterdam, pp 67–102 Murashige T & Skoog F, A revised mediun for rapid growth and bioassay with tobacco tissue culture Physiol. Plant, 15 (1962) 473. Nalini Mllikarjuna, Reena MJT, Sastri DC & Moss JP, Somatic embryogenesis in pigeon pea (cajanus cajan L ) Indian J. Exp. Biol,4 (1996) 282.

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Patel, D.B., Barve, D.M., Nagar, N., Mehta, A.R. In vitro development of immature and hybrid embryos of cajanus cajan L) (1994). Indian J. Exp. Biol. 32: 740– 744.

Seenivasu K.,Malik SK, Ananda Kumar P& Sharma RP, Plant regenratin via somatic embryogenesis in pigeon pea ( Cajanus cajan ( L.) Mill sp). Plant Cell Reports, 17 (1998) 294–297.

Prakash NS, Pental D & SarinNB, Regeneration of pigeon peas ( Cajajus cajan) from cotyledonary node via multiple shoot formation. Plant Cell Rep, 13 (1994) 623–627.

Sharma AK & Sharma A, Chromosome techniques: Theory and practice (Text Book), Butterworths London (1980).

Raghavan V (1986) Embryogenesis in angiosperms: a developmental and experimental study. Cambridge University Press, New York

Source of Support: NIL

Conflict of Interest: None Declared

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Research Article A COMPARATIVE STUDY OF BHASMAKNASHAK YOGA WITH EXERCISE AND DIET RESTRICTIONS IN OVERWEIGHT CHILDREN Renu B Rathi1*, Bharat Rathi2 1

Professor & Head, Kaumarbhritya Department, MGACHRC, Salod, Wardha, Maharashtra, India Dr. Bharat Rathi, M.D. Professor & Head, Rasshastra Bhaishajya Kalpana Department, MGACHRC, Salod, Wardha, Maharashtra, India * Corresponding Author: E-Mail: rbr.226@gmail.com; 919011058302 2

Received: 29/05/2014; Revised: 25/06/2014; Accepted: 01/07/2014

ABSTRACT Due to modern lifestyle, the rate of developing overweight in children is also increasing tremendously. It is a proved fact that exercise and diet restriction of specially sweet and fatty foodstuffs help to reduce weight, maintain fitness and can have the direct effect to prevent systemic illness. In overweight individuals, there will be increased hunger and thirst which again facilitates weight gain. In Rasatantrasaara and Siddhaprayoga Sangraha, it has been mentioned that Apamarga seeds (Achyranthus aspera Linn.) are useful in Bhasmaka Vyadhi (Voracious appetite). The action of Apamarga seeds was considered to be hard to digest creates sense of fullness and enables to cope with overweight. The study aims at evaluating the efficacy of Bhasmaknashak yoga in overweight children as compare to exercise and diet restrictions. Total 30 patients of age 8–15 years were divided into 3 groups of each 10. Subjects of Group A were put on exercise and diet restriction, Group B was administered with trial drug and Group C received both. It shows that the statistical effect on clinical features in group C was highly significant than group A and B (P valuesBMI <0.0005, Abdominal circumference <0.0005, climbing time <0.0001). The study revealed that awareness of overweight problems and adaptation of change in lifestyle along with remedies are much important in treating overweight as compare to remedy or regimen alone. KEY WORDS: Bhasmaknashak yoga, overweight, exercise and diet restrictions.

Cite this article: Renu B Rathi, Bharat Rathi (2014), A COMPARATIVE STUDY OF BHASMAKNASHAK YOGA WITH EXERCISE AND DIET RESTRICTIONS IN OVERWEIGHT CHILDREN, Global J Res. Med. Plants & Indigen. Med., Volume 3(7): 294–302

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INTRODUCTION In recent years, there is an increasing trend of developing overweight in children. There are lots of reasons behind it like more consumption of fast food, bakery items, sedentary life style and lack of outdoor playing. There is 16.9% prevalence of obesity in children and adolescents aged between 2–15 years (Ogden et al., 2012). Obesity is associated with a multitude of adverse health effects. Central or visceral fat in obesity pours out free fatty acids and increases insulin resistance. The adipose cells secrete multiple hormones, known as ‘adipokines,’ and markers of inflammation (H. M. Chandola, H. Sharma, 2013). According to WHO, a BMI greater than or equal to 25 is overweight and a BMI equal or more than 30 is obese (Centers for Disease control & prevention, 2014). The term most commonly used to quantify overweight is Body Mass Index or BMI calculated as Wt in kg / Ht in m2. It is a proved fact that exercise and diet restrictions play a key role in reducing overweight (Thomas A. et al., 2005), as ‘exercise’ means = designed, repetitive for the rationale of training any part of the body, hence taken for the study in group A. Drug intervention used in present study ‘Bhasmaknashak yoga’ (Krishnanand & Badrinarayan Shastri, 1991) is widely used in clinics to treat voracious appetite. Seeds of Achyranthus aspera are proved to have properties like anti-obesity potential (Neerja Rani et al., 2012) hepatoprotective (Manjunatha BK, et al., 2012; Kokila Parmar, et al., 2013) etc. Till date there is no documented clinical study on trial drug, as per author’s knowledge, hence the study was taken up with an aim of evaluating the therapeutic efficacy of ‘Bhasmakanashka Yoga’ in overweight children. The study was also planned in order to evaluate the role of diet restrictions specially fried, sweet food and

adoption of regular exercise or outdoor playing in reducing the overweight in children and to see the combined effect of remedy and regime. MATERIALS AND METHODS: Study design and duration It is an open ended comparative clinical pilot study.

randomized

Total 30 patients were divided into 3 groups randomly as per liking and consent. 30 patients of Sthaulya (overweight) attending the OPD of Balrog, Mahatma Gandhi Ayurveda College, Hospital and Research Centre, Wardha, Maharashtra, India were registered for this study. Present study was a pilot study with small sample size, further in continuation, a project with large sample size has been submitted for ethical approval. Group A – All 10 overweight children receiving diet instructions (Diet chart not to eat sweets, fried food stuffs was given to them) and at least half an hour daily exercise or outdoor play. Group B - All 10 overweight children receiving trial drug Bhasmaknashak yoga in a dose of 3gm with hot water twice a day post meal up to 1 month. Group C - All 10 overweight children were instructed for taking orally Bhasmaknashak yoga with diet restrictions and exercise. Drug Review: The prime ingredients of this yoga have been enumerated in Table I. All drugs were taken in equal proportion, powdered and mixed together to prepare the trial formulation. Dose and Duration: 3 gm twice a day, with hot water, post meal for a period of one month Follow-up has been taken up to 1 month and found that drug effect existed in.

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Table I showing ingredients of the Trial drug Bhasmaknashak yoga with Sanskrit and Botanical names with citation. Sr. No.

Sanskrit Name

Botanical Name

Useful part

Amalki

Phyllanthus emblica Linn.

Fruit

Bibhitaki

Terminalia belerica Roxb.

Fruit

Haritaki

Terminalia chebula Retz.

Fruit

Musta

Cyperus rotundus Linn.

Rhizome

Apamarga

Achyranthus aspera Linn

Seeds

Pippali

Piper longum linn

Fruit

Vidanga

Embelia ribes Burm

Fruit

Sita

Sugar

Inclusion Criteria: All overweight children of 8 to 15 years age group having > 25 BMI Child who was not taking weight-affecting medications directly or indirectly. Child not having a medical condition for which a weight loss program would be contraindicated. Exclusion Criteria: Overweight children having hormonal disorders like hypothyroidism and Diabetes mellitus. Genetic disorders like Downâ&#x20AC;&#x2122;s syndrome, Turner syndrome. Kwashiorkar, Nephrotic syndrome, Congestive Cardiac Failure in which there is anasarca, overviewed as overweight (Lily John, D.B. Tripathi, 1994). Withdrawal Criteria: The children who had left the treatment modality during the course or being irregular. Assessment Criteria: 1. Weight, height, BMI were recorded before starting the treatment and later on every week of the study. Weight was also recorded for all

the patients who have come for the follow-up study. 2. Abdominal circumferences at umbilicus level were recorded before and thereafter every week, till the completion of the course of treatment, to assess the effect of therapy. 3. Climbing time: The time taken to climb fixed number of stairs ten continuously without taking rest in seconds was taken as climbing time to assess the clinical features like Guruta, Ayasen Shwas, Daurbalya. No investigations had been studied like Lipid profile, etc. due to fund constraint as it was a pilot study. Statistical Analysis-was done by Sigma state software, paired t test RESULTS : It was observed that maximum patients were belonging from the age group of 10 to 15 years. Male patients were dominant total 24 boys (80%) and 6 girls in this study in all the 3 groups. The main cause of overweight was over eating in 73.33%, very less physical exertion in 60% and heredity cause was observed in 90% (At least 1 parent was overweight) in all the 3 groups. The common clinical features were similar to medoroga like desire for more food,

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water, cool air, sleep etc. (KR Srikantha Murthy, 2002).

in Utsahahani (30.58%), Daurbalyata (28.41), Anga Guruta (25.67%), Nidradhikya (23.88%) and Aayasena Shvasa (19.4%), Atiswedpravruti with statistically highly significant (p<0.0001 in all features) in group C children. Along with clinical features the results were drawn by using paired t test to judge the effect by different parameters like BMI, Abdominal circumference and climbing time which has been depicted in the following tables (Tables II to XIII).

The symptoms found in all the children enrolled for the study had Guruta (90%), Nidraadhikya-oversleep (86.34%), Atipipasa â&#x20AC;&#x201C; over thirst (93.97%), AtiKshudhavoracious appetite (100%). Other signs and symptoms observed were Daurbalya (92.77%), Ayasena shvasa (91.6%), Utsahahani (92.77%), Atisvedapravr uti (82.15%). It provided significant relief

Table-II, showing statistical efficacy on clinical features in Group A Clinical Feature*

Mean

Atikshudha

3.26

2.57

Atitrushna

3.090

Atinidra

Std. Error

t value

p value

0.69

0.070

0.068

10.24

<0.0001

2.500

0.59

0.057

0.073

7.42

<0.0001

3.378

2.689

0.69

0.040

0.061

14.2

<0.0001

Guruta

3.220

2.350

0.87

0.119

0.096

5.54

<0.0005

Utsahhani

3.470

2.320

1.15

0.076

0.116

7.01

=0.0001

Aayasen Shwas

3.350

2.590

0.76

0.054

0.087

8.86

<0.0001

Daurbalya

3.370

2.610

0.76

0.068

0.092

9.28

<0.0001

Atisweda

3.390

2.840

0.55

0.071

0.044

5.83

=0.0003

* Atikshudha-Voracious appetite, Atitrushna-over thirst, Atinidra-Over sleep, Gurutaheaviness, Utsah hani-lethargic, Aayasen shwas- dyspnea/palpitation, Daurbalya- Weakness, Atisweda- over sweating

Table-III, showing statistical efficacy on clinical features in Group B Clinical Feature

Mean BT AT

Std. Error BT AT

t value

p value

Atikshudha

3.05

2.52

0.53

0.060

0.065

12.2

<0.0001

Atitrushna

3.040

2.520

0.52

0.068

0.093

9.390

<0.0001

Atinidra

3.311

2.900

0.41

0.067

0.041

10.65

<0.0001

Guruta

3.280

2.410

0.87

0.120

0.056

7.292

<0.0001

Utsahhani

2.850

2.250

0.60

0.0764

0.099

9.000

<0.0001

AayasenShwas

3.350

2.8300

0.52

0.054

0.059

6.868

=0.0001

Daurbalya

3.230

2.760

0.47

0.055

0.045

10.48

<0.0001

Atisweda

3.180

2.821

0.36

0.059

0.062

7.962

<0.0001

P value<0.0001-highly significant

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Table -IVshowing statistical efficacy on clinical features in Group C Clinical Feature

Mean BT

Std. Error BT AT

Atikshudha

3.54

Atitrushna

t value

p value

1.620

1.92

0.091 0.093 18.63

<0.0001

3.760

2.180

1.58

0.077 0.063 15.66

<0.0001

Atinidra

3.34

2.47

0.87

0.05

0.047 21.34

<0.0001

Guruta

3.400

1.400

2.00

0.047 0.058 24.91

<0.0001

Utsahhani

3.170

1.530

1.64

0.059 0.083 15.68

<0.0001

AayasenShwas

3.71

2.35

1.36

0.038 0.070 26.12

<0.0001

Daurbalya

3.760

1.690

2.07

0.035 0.127 15.62

<0.0001

Atisweda

3.530

1.432

0.21

0.049 0.029 19.23

<0.0001

P value<0.0001-highly significant

Table V, showing effect on BMI in all groupsGroup Mean BT

D AT

Std. Error BT

0.0957 0.216

t value

p value

4.824

<0.001 <0.001

20.55 19.39

1.16

21.39 19.40

1.950 0.192

0.339

5.802

24.04 20.39

3.65

0.434

12.632 <0.001

0.297

P value<0.0001-highly significant

Table VI, showing Effect on Abdominal circumference in all groupsGroup

Mean BT AT

Std. Error BT AT

t value

p value

84.530 81.870

2.260 0.409

0.265 5.854

<0.001

85.27

82.36

2.910 0.146

0.475 7.472

<0.001

90.09

84.80

5.29

0.342 7.952

<0.001

0.736

P value<0.001-highly significant

Table VII, showing Effect on climbing time in all groupsGroup

Mean

Std. Error BT

t value

p value

17.8

16.4

1.32

0.218 0.243 5.381

<0.0001

18.88

17.86 1.00

0.322 0.395 3.348

<0.0001

19.38

14.87 4.51

0.228 0.241 21.31

<0.0001

P value<0.001-highly significant

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Table-VIII, showing comparison of means in Clinical features of group A Versus B Clinical Feature

Atikshudha Atitrushna Atinidra Guruta Utsahhani AayasenShwas Daurbalya Atisweda

Std. Error

t value

p value

−0.05 18

0.09402

−0.532

=0.6014

−0.25 −0.48 0.06 −0.07 0.24 −0.48 −0.48

0.11812 0.8898 0.11127 0.1528 0.10274 0.08898 0.08898

−2.117 −2.117 0.539 −0.458 2.336 −5.394 −5.394

<0.0005 <0.0005 =0.5963 =0.6523 =0.0313 <0.0001 <0.0001

18 18 18 18 18 18 18

Table-IX, showing comparison of means in objective parameters in group A versus B Objective Criteria

Std. Error

t value

p value

BMI

0.1

0.4015

0.249

0.8061

Abd. Circumference Climbing Time

0.49 0.30

18 18

0.5443 0.305

0.900 0.767

0.3799 0.453

Table-X, showing comparison of means in clinical features of group B Vs C Clinical Feature

Std. Error

t value

p value

Atikshudha

0.9

0.11306

7.961

<0.0001

Atitrushna Atinidra Guruta Utsahhani AayasenShwas Daurbalya Atisweda

0.34 0.17 1.01 0.72 0.48 0.48 0.48

18 18 18 18 18 18 18

0.11214 0.0611 0.0809 0.12937 0.08898 0.08898 0.08898

3.032 2.783 12.484 5.566 5.394 5.394 5.394

=0.0072 =0.0123 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

P value<0.0001-highly significant

Table XI, showing comparison of means in objective parameters in group B versus C Objective Criteria

Std. Error

t value

p value

BMI

0.99

0.5502

1.799

<0.0005

Abd. Circumference

2.44

0.5853

4.169

<0.0005

Climbing Time

3.49

0.093

9.408

<0.0001

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Table -XII, showing comparison of means in group C versus A Clinical Feature

Std. Error

t value

p value

Atikshudha

0.95

0.11532

8.238

<0.0001

Atitrushna

0.32

0.09637

3.320

<0.0005

Atinidra

0.219 18

0.07478

2.929

<0.0005

Guruta

1.01

0.0809

8.496

<0.0001

Utsahhani

0.95

0.11182

5.529

<0.0001

AayasenShwas

0.48

0.08898

5.394

<0.0001

Daurabalya

0.48

0.05838

5.134

<0.0001

Atisweda

0.48

0.08898

5.394

<0.0001

P value<0.0001-highly significant

Table-XIII, showing comparison of means in objective parameters in group C versus A Objective Criteria

Std. Error

t value

p value

BMI

1.09

0.4842

2.251

<0.0005

Abd. Circumference

2.93

0.4325

6.775

Climbing Time

3.19

0.033

9.847

<0.0001 <0.001

P value<0.0001-highly significant

DISCUSSION: Individually all the three modalities of overweight treatment were significant in lowering chief complaints as well as objective parameters but integrative approach has more quick and long lasting efficacy over separate regimen or remedy as seen in follow-up period. It was found that individually every group has statistically significant in clinical features but while comparing the effect of exercise and diet restrictions versus trial drug between Group A and B, it was observed that Group B was effective than group A in decreasing some chief complaints like Daurbalya and Atisweda which proving that Group B had good impact over Group A as a treatment modality of overweight children.

While comparing the effect of trial drug versus integrative approach between Group B and C, it was observed that Group C was highly effective than group B in decreasing almost all chief complaints except Atitrushna and Atinidra. Also C group has statistical significance on objective criteria on climbing time which proving that Group C has good impact over Group B as a treatment modality of overweight children. While comparing the effect of therapy between Group C and A, it was observed that Group C was highly effective than group A in decreasing almost all chief complaints like Guruta, Utsah hani, Aayasen Shwas, Daurbalya and Atisweda. Also C group had statistical significance on objective criteria like abdominal circumference and climbing time which proves that Group C had an edge over Group A as a treatment

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modality of overweight children. The mean BMI, Abdominal circumference and climbing time of group C children were less as compare to group A and B. All children felt easiness in routine activities with increased strength, decrease in dyspnea post treatment intervention. Ingredients of Bhasmaknashakyoga have effect on overweight due to their Kaphavathara (normalizing body humour) lekhana, (scrapping of body fat) deepana, pachana (normalizing metabolism) properties (Priyavrat Sharma, 2013; G. Pande, 2013). Mishri/sita provides palatability to the formulation. It was observed that the formulation was safe and had no any untoward effect in group B and C children. Group B and C children had their meal as routine. After study, children were advised for Psychologist’s counseling to decrease the affinity for diet. In this study, 4 patients were withdrawn due to irregularity in treatment modalities and replaced with other 4 overweight children.

to report for follow-up study for a period of 1 month. In follow-up study significant changes in body weight, abdomen circumference were observed. CONCLUSION: Overweight is becoming a burning problem in society. Above study reveals that to overcome it, multi-dimensional approach is essential. It’s not so easy and instant to change ones personality that too in children. The study has shown that awareness of overweight and adaptation of change in lifestyle along with remedies are much important in its treatment. Here as compare to remedy or regimen alone, the combination of both as in group C children shown more benefits when compared to group A and B. From the study, it can be concluded that trial medicine having encouraging weight reducing effect but it can be enhanced by adding diet restrictions and exercises. This was a pilot study, but it’s encouraging results indicate the need of further extensive research with large sample and long term follow-up.

Follow-up study- After completion of due course of treatment, all the children were asked REFERENCES Centers for Disease control & prevention (2014), http://www.cdc.gov/healthweight/assess ing/bmi/index.html?s_cid=tw_obo64, last retrieved on Jan 14

K.R.Srikantha Murthy (2002), Astanga Hrdaya, Uttarasthana, Chapter19th Page No.173– 175,sloka no.1–15 and 20th chapter Page No.179–182,sloka no.1-17, Reprint edition.

G. Pande (2013), Vimarshkar K.C. Chunekar, Bhavprakash Nighantu, Chaukhambha Bharati Academy, Reprint edition, pg no 400

Kashinath Shastri and Gorakhnath Chaturvedi, (1993) Charak Samhita, Uttarardha, chapter-15/217–220, Chaukhambha Bharati Academy, 19th edition, page481

H. M. Chandola, H. Sharma, (2013) Obesity in Ayurveda: Dietary, Lifestyle, and Herbal Considerations, Center for Integrative Medicine, the Ohio State University, Columbus, OH, USA†, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University Jamnagar, India

Kokila A Parmar, Sarju N Prajapati, Vaishali V Chauhan, Chetan R Patel, (2013)(1); 13–17 Scholars Research Library, Preliminary Phytochemical Pharmacognostical and Microbial Screening of Achyranthus aspera (Amaranthaceae), http://scholarsresearchlibrary.com, last retrieved on April14

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Krishnanand, Badrinarayan Shastri, (1991) Rastantrasar and Siddha prayogsangrah, 1st Volume, Krishna Gopal Ayurveda Bhavan, Kaleda, 13th edition, pg no.685

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Ogden CL, Guoss, Kuezmarski RJ, (2012) Prevalence of childhood and adult obesity in U.S., National Center for Health Statistics, National Health and Nutrition Examination Survey, JAMA,articleid=1832542,www.cdc.gov /nchs/databriefs/db82.pdf

John, D.B. Tripathi (1994), All Practitioner’s Of India-API Text book of Medicine, 5th edition, editor in Chief- G.S. Sainani, Obesity.

Manjunatha BK, Abhilash N, Vinay Hegde, Suchitra MN, Vidya SM (2012), Hepatoprotective potency of Achyranthus aspera: An invivo study, International Jr of Pharmaceuticals and phytopharmacological Research, 1(6):387–390 Neerja Rani, Surendrakumar Sharma, Neelu Vasudeva (2012), Assessment of antiobesity potential of Achyranthus aspera Linn. Seeds, Evidence based Complimentary and Alternative medicine, ID-715912

Source of Support: NIL

Choices (2014), http://www.nhs.uk/conditions/obesity/p ages/introduction.aspx).last retrieved on Jan 14

Priyavrat Sharma (2013) Dravyaguna Vigyan, volume 2nd, Reprint edition, Chaukhambha Bharati Academy, pg no 542 Thomas A. Wadden, Robert I. Berkowitz (2005), The new England journal of medicine, Randomized Trial of Lifestyle Modification and Pharmacotherapy for Obesity, November17, vol. 353 , pg no.20

Conflict of Interest: None Declared

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Review Article A REVIEW ON HEPATO-PROTECTIVE HERBS USED IN AYURVEDA Giby Abraham1* Research and Development, Confederation for Ayurvedic Renaissance – Keralam Limited (CARe Keralam Ltd.), KINFRA Small Industries Park, Nalukettu Road, Koratty, Thrissur – 680 309, Kerala, India *Corresponding Author: E-mail:dr.gibyabraham@gmail.com; Mob: 09995215790 1

Received: 10/06/2014; Revised: 20/06/2014; Accepted: 25/06/2014

ABSTRACT Liver is considered to be one of the vital organs which helps in maintaining the health of body. Yakrit (liver) is being described right from the vedic period. Modern lifestyles can overstress the liver and make it malfunctioning. No significant and safe hepato-protective drugs are available in modern therapeutics. The nature has bestowed some plants with the property to prevent, treat and cure hepatic disturbances with interception of fewer side effects. The focus of this review is to elucidate the importance of liver and aimed at compiling data based on reported works on medicinal plants that have been tested in hepato-toxicity models and proved as hepato-protective. Also the probable mode of action of a few herbs has been discussed in Ayurvedic and modern aspect. KEY WORDS: Liver, Yakrit, Hepato-protective, Medicinal plants.

Cite this article: Giby Abraham (2014), A REVIEW ON HEPATO-PROTECTIVE HERBS USED IN AYURVEDA, Global J Res. Med. Plants & Indigen. Med., Volume 3(7): 303–311

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INTRODUCTION "Is your life worth living”? It depends on the liver as it is the largest glandular organ in the body which works all the time to keep the body healthy. The liver is important because a person’s nutritional level is not only determined by what he or she eats, but by what the liver processes. The incredible complexity of liver chemistry and its fundamental role in human physiology is so daunting to researchers that the thought that simple plant remedies might have something to offer is astonishing and incredible! Liver is considered to be one of the most vital organs that functions as a centre of metabolism of nutrients such as carbohydrates, proteins and lipids and excretion of waste metabolites. Additionally, it is also handling the metabolism and excretion of drugs and other xenobiotics from the body thereby providing protection against foreign substances by detoxifying and eliminating them. In Ayurvedic literature, yakrit (liver) is considered as an important anga (organ) of the human body right from the vedic period. Bhavamisra (16th Century) has described that it is situated right and below to the hridaya (heart) and is the sthana (seat) of pitta and sonitha (blood) (Srikantha Murthy, 2002). Susrutha (500 BC) mentioned yakrit (liver) as

the abode of ranjaka pitta (Srikantha Murthy, 2004). Susrutha (500 BC) describes yakrit (liver) as the sthana (seat) of rakta (blood) (Srikantha Murthy, 2005). Charaka (1000 BC) while describing the srotas (body channels), mentioned yakrit (liver) and pleeha (spleen) as the moola (root) of raktavaha Srotas (blood carrying channels) (Sharma and Dash, 2007). But it is Bhavamisra who for the first time introduced the term ‘yakrit vikara’ (liver disorders). Madhavanidana, in parishista prakarana, explains yakrit roga (liver disease) as a separate entity (Yadunandan Upadhyaya, 2000). The etio-pathogenesis of Yakrit roga has been described in Figure 1. In dealing with problems of the liver, the primary goal is to enhance liver detoxification processes and to help protect against further liver damage. Significant and safe hepatoprotective agents are unavailable in modern therapeutics. Therefore, due importance has been given globally to develop plant-based hepato-protective drugs effective against a variety of liver disorders. The present review is aimed at compiling data based on reported works on promising phytochemicals from medicinal plants that have been tested on hepato-toxicity models.

Figure 1: Etio-pathogenesis of yakrit roga (liver disease) Vidahi annapanam (food and drinking that cause burning sensation, Madya sevana (alcohol intake), Teekshna padartha (strong/ sharp substance)

Pitta prakopa (aggrevation of pitta)

Dushita rakta dhatu (vitiated blood tissue)

Rakta pradoshaja roga (disease caused by vitiated blood tissue)

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yakritpleehakamala roga (diseases of liver, spleen, jaundice etc.)

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Table 1. showing some hepato-protective herbs with their pharmacological properties (Sharma P.V, 2009) Dosakarma S.N Plant Rasa Guna Virya Vipaka Guduchi (Tinospora cordifolia (Willd.) Miers.) Pippali (Piper longum Linn.)

Tikta, Kashaya

Guru, Snigdha

Ushna

Madhura

Tridoshahara

Katu

Ushna

Madhura

Tridoshahara

Punarnava (Boerhavia diffusa Linn.)

Ushna

Madhura

Tridoshahara

Kalamegha (Andrographis paniculata Nees.) Bhumyamalaki (Phyllanthus niruri Linn.)

Madhura, Tikta, Kashaya Tikta

Laghu, Snigdha, Teekshna Laghu, Ruksha Laghu, Ruksha

Ushna

Katu

Kaphapittahara

Tikta, Kashaya, Madhura Tikta, Kashaya Tikta

Laghu, Ruksha

Seeta

Madhura

Kaphapittahara

Laghu, Ruksha Laghu, Ruksha

Ushna

Katu

Kaphapittahara

Seeta

Katu

Kaphapittahara

Katu, Tikta, Kashaya Tikta, Kashaya Tikta, Kashaya

Laghu, Ruksha

Seeta

Katu

Kaphapittahara

Laghu, Ruksha Laghu, Ruksha, Teekshna

Ushna

Katu

Kaphavatahara

Ushna

Katu

Kaphavatahara

6. 7.

9. 10.

Daruharidra (Berberis aristata DC.) Katuki (Picrorhiza kurroa Royle ex Benth.) Rohitaka (Techoma undulata G. Don.) Bhringaraja (Eclipta alba Hassk.) Sharapunkha (Tephrosa purpurea Pers.)

[Rasa (taste) – Katu (pungent), Tikta (bitter), Kashaya (astringent) Guna (quality) – Guru (difficult to digest), Snigdha (unctous) Laghu (easily digestible), Ruksha (dry), Teeksha (sharp) Virya (potency) – Seeta (cold), Ushna (hot) Vipaka (post metabolic effect) Dosa karma (action on functional entites of the body), hara (pacifies)]

Hepatoprotective Drugs The important herbs used in Ayurveda to treat Liver diseases have been described in Table 1. Tinospora (Guduchi)

cordifolia

(Willd.)

Miers.

Tinospora cordifolia (Willd.) Miers., known as Guduchi, Amrita is one of the most valuable medicinal herbs of Ayurveda. The

term 'Amrita' is attributed to this herb in recognition of its ability to impart youthfulness, vitality and longevity to its patron. In modern medicine, it is well known for its hepatoprotective, adaptogenic, immunomodulatory activities and anti-fibrolytic activity. The active principle Tinosporin corrects immunosuppression associated with deranged hepatic function (Varsha et al., 2011).

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Kupffer cells are major determinants of outcome of liver injury. The effect of Tinospora cordifolia (Willd.) Miers. was evaluated on Kupffer cell function, using carbon clearance test as a parameter. Antihepatotoxic activity of Tinospora cordifolia (Willd.) Miers. was studied in albino rats intoxicated with Carbon tetrachloride (CCl4). Liver function was assessed based on morphological, biochemical (SGPT, SGOT, Serum alkaline phosphatase, Serum bilirubin) and functional (Pentobarbitone sleep time) tests. A study conducted by Nagarkatti et al., (1994) on Tinospora cordifolia (Willd.) Miers. indicates that it had decreased fibrosis in rats, induced by CCl4 and significantly improved the suppressed Kupffer cell function in another rat model of chronic liver damage induced by heterologous serum. This raises the possibility that anti-fibrotic effect of Tinospora cordifolia is mediated through activation of kupffer cells.

Boerhavia diffusa Linn. (Punarnava) The roots of Boerhavia diffusa Linn., commonly known as 'Punarnava', are used by a large number of tribes in India for the treatment of various hepatic disorders and for internal inflammation. Clinical data has also reported effectiveness of Boerhavia diffusa Linn. in cases of oedema and ascites resulting from early cirrhosis of the liver and chronic peritonitis (Varsha et al., 2011). The effect of ethanolic extract of roots of Boerhavia diffusa Linn. on country made liquor induced hepatotoxicity was studied in albino rats by Agarwal et al.(1991). Histo-pathological studies showed marked reduction in fat deposits in animals receiving Boehavia diffusa Linn. along with country made liquor. The plant protected the rats from hepatotoxic action by decreasing the serum alanine amino transferase (ALT), triglycerides, cholesterol and total lipid levels in both serum and tissues.

Piper longum Linn. (Pippali) Piper longum Linn. belongs to the family Piperaceae, is a common Indian dietary spice which has been shown to possess a wide range of therapeutic utilities. It has been reported to possess antiasthmatic, antiinflammatory, hepatoprotective, hypocholestremic and immunomodulatory activities. It contains various alkaloids like piperine, piperlongumine, piperlonguminine, etc. which helps in the regeneration of hepatocytes (Gupta AK, 2003). A study conducted by Jagruti and Urvi (2009) showed a significant hepatoprotective effect on Piper longum Linn. milk extract treatment in CCl4 induced hepatic damage. An evident decrease in level of serum enzymes, total bilirubin and direct bilirubin was observed. Histo-pathological findings indicated that administration of Piper longum Linn. milk extract offered protection to the hepatocytes from damage induced by CCl4, with mild fatty changes in the hepatic parenchymal cells, which corroborated the changes observed in the hepatic enzymes. It also showed regenerating liver cells around the necrotic area.

Punarnava contains alkaloids named as punarnavine and punarnavoside which shows anti-fibrinolytic activity but the hepatoprotective activity has been attributed to ursolic acid. Keppler and co-workers demonstrated that ursolic acid isolated from the leaves showed a dose dependent (5â&#x20AC;&#x201C;20 mg/kg) hepatoprotective activity (21â&#x20AC;&#x201C;l00%) in rats against thioacetamide, galactosamine and carbon tetrachloride induced hepatotoxicity in rats. These hepatotoxins decreased the viability of hepatocytes as assessed by trypan blue exclusion and rate of oxygen uptake tests and decreased the volume of bile as well as the level of its contents. Pretreatment with ursolic acid increased the viability of rat hepatocytes significantly (Keppler et al., 1968). Andrographis paniculata Nees. (Kalamegha) Andrographis paniculata Nees. is an ancient Indian medicinal herb, which has been used for centuries in Asia for its effects on various bodily functions and ailments, ranging from degenerative diseases to the common cold. The plant is known as King of Bitters. Andrographolide is an active constituent

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extracted and isolated from Andrographis paniculata Nees which is very bitter in taste (Anil Kumar et al., 2012). A study conducted by Visen et al. (1993) on andrographolide showed a significant dose dependent protective activity against paracetamol-induced toxicity on ex vivo preparation of isolated rat hepatocytes. It significantly increased the percent viability of the hepatocytes as tested by trypan blue exclusion and oxygen uptake tests. It blocked the toxic effects of paracetamol on certain enzymes (GOT, GPT and alkaline phosphatase) in serum as well as in isolated hepatic cells. The bioactive constituent also antagonizes toxic effects of CCl4 and acetaminophen on certain enzymes (GOT, GPT and alkaline phosphates) in serum as well as in isolated hepatic cells. The results clearly depicted the plant extract to exert a choleretic effect that reduces the cholestasis and diminishes retention as well as increase the excretion of toxic xenobiotics from liver. Further, it also stimulated immune system to fight against inflammation, is mediated from the release of cytokinin from immunomodulators (Varsha et al., 2011). Phyllanthus niruri Linn. (Bhumyamalaki) Phyllanthus niruri Linn. is a medicinal herb used in connection with secondary hepatitis and other ailments, in ayurvedic medicine for over 2000 years. It is a proved antiviral drug in Hepatitis-B in human subjects. In the preliminary study, carriers of Hepatitis-B virus were treated with a preparation of the plant 200 mg for 30 days. 22 of the 37(59%) treated patients had lost Hepatitis-B surface antigen, when tested 15– 20days after the end of the treatment, compared with only 1 out of 23 (4%) placebo treated controls. It has exhibited an inhibition of DNA polymerase on Hepatitis–B virus which is responsible for the replication of virus (Mehrotra et al., 1991). In a study, phyllanthin, hypophyllanthin and tricotanol were isolated from petroleum ether extract of Phyllanthus niruri Linn. shows

significant results on rat hepatocytes. Preclinical studies demonstrate that an extract of the Phyllanthus niruri Linn. plant inhibits endogenous DNA polymerase of hepatitis B virus and binds to the surface antigen of Hepatitis B virus. Extracts of Phyllanthus niruri Linn. have been shown to exert hepatoprotective effect against CCl4 induced HepG2 cell damage in rabbits. Pre-treatment with extract of Phyllanthus niruri Linn., reduced paracetamol-induced acute liver damage in rats as monitored by estimating the SGOT. In the in vitro-study, it decreased the release of AST and ALT in rat primary cultured hepatocytes being treated with ethanol (Tabassum et al., 2005). Berberis aristata DC. (Daruharidra) Berberis asiatica DC. being an important medicinal plant is used extensively for treating variety of ailments like infection of eyes, skin diseases, jaundice and rheumatism (Kirtikar and Basu, 1933). The major alkaloid of this plant is reported to be berberin which possess anti-oxidant property (Brijesh and Khosa, 2010). The roots of Berberis aristata DC. possess more effective hepatoprotective activity against CCl4 intoxication in rats because of its antioxidant bearing capacity. Acute CCl4 administration increased serum and liver lipid peroxides significantly. Berberine treatment could reduce these elevated levels. Pathological analysis showed degeneration and necrosis after CCl4 administration. Berberine treatment could minimize these effects to a certain extent. (Brijesh and Khosa, 2010) Picrorhiza kurroa Royle ex. Benth (Katuki) Picrorhiza kurroa Royle ex Benth. is a renowned herb in the Ayurvedic system of medicine and has traditionally been used to treat disorders of the liver, upper respiratory tract, reduce fevers, treat dyspepsia, chronic diarrhoea, and scorpion sting. Kutkin, the active principal of Picrorhiza kurroa Royle ex. Benth is comprised of kutkoside and iridoid

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glycosides like picrosides I, II, and III (Chaturvedi and Singh, 1996). The hepato-protective action of Picrorhiza kurroa Royle ex Benth. may be attributed to its ability to inhibit the generation of oxygen anions and to scavenge free radicals. Picrorhiza’s antioxidant effect has been shown to be similar to that of superoxide dismutase, metal-ion chelators and xanthine oxidase inhibitors. Animal studies indicate that Picrorhiza’s constituents exhibit a strong anticholestatic activity against a variety of livertoxic substances, appearing to be even more potent than silymarin (Chander et al., 1992). Picrorhiza also exhibits a dose-dependent choleretic activity, evidenced by an increase in bile salts and acids, and bile flow (Shukla et al., 1991). Techoma undulata G. Don (Rohitaka) Techoma undulata G. Don is a tropical coastal shrub that grows up to 1 m in height. It occurs throughout the Indian subcontinent. Techoma undulata G. Don leaves were tested against liver damage of albino rats. Loss of membrane structure and integrity because of lipid peroxidation was accompanied with the elevated levels of marker enzymes like SGOT, SGPT and total bilirubin. This shows that the plant has got membrane stabilizing function. Techoma undulata G. Don was potentially effective in blunting lipid peroxidation, suggesting that the extract possibly has antioxidant property to reduce ethanol-induced membrane lipid peroxidation and thereby to preserve membrane structure and might be due to the presence of glycosides, flavonoids, proteins, amino acids, tannins, saponins and triterpenoids (Singh D. et al., 2011). Eclipta alba Hassk. (Bhrngaraja) Eclipta alba Hassk. known as Bhringraja, is a plant belonging to the family Asteraceae. In ayurvedic medicine, the leaf extract is considered a powerful liver tonic. It possesses a wide range of biological activities and is used for the treatment of hepatitis and cirrhosis

(Wagner, H. 1986). A study conducted by Murugaian P et al., 2008 on the whole plant extract of Eclipta alba Hassk. exhibited the protective activity against CCl4 induced liver injury. The plant contains an alkaloid Ecliptine which has got choleretic action. The extract augmented the bile flow in rats suggesting a stimulation of liver secretory capacity. Tephrosia purpurea Pers. (Sharapunkha) Tephrosia purpurea Pers. known as Sharapunkha, forms one of the most effective ingredients of formulations available in Indian market for liver ailments. In the traditional Indian medicine it is famous for its effectiveness in bilious febrile attacks, obstruction of liver and spleen apart. Especially, it has shown good results in cirrhosis and viral hepatitis in clinical trials (human studies). Dried ethanolic extract of Tephrosia purpurea Pers. was studied for its efficacy using both acute and chronic models CCl4 of experimentally induced hepatotoxicity. In vitro studies exploiting trypan blue exclusion assay revealed that the alcoholic extract exerted a significant hydroxyl radical scavenging activity (Sree Rama Murthy and Srinivasan, 1993). Hepato-protective effect of aerial parts was evaluated against CCl4 induced hepatotoxicity in rats. An oral dose of powdered aerial parts to rats prevented the elevation of SGOT, SGPT, Bilirubin levels caused by CCl4. The mechanism of hepato-protection by Tephrosia purpurea Pers. mainly involves membrane stabilization of liver cells as indicated by decrease in levels of SGOT, SGPT and bilirubin levels, wherein it prevents cellular leakage and loss of functional integrity of the liver cell membranes caused by various hepatotoxic agents. Tephrosia purpurea Pers. also leads to increase in hepatic regeneration, which again contributes to its hepatoprotective efficacy (Jain, A. et al., 2006). DISCUSSION Yakrit (liver) is the sthana (seat) of pitta dosha (functional entity of the body), rakta

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dhatu (blood tissue) and agni (power of digestion). Treatment of all liver diseases in Ayurveda concentrates mainly on pitta dosha rather than the organ itself. Most of the hepatoprotective drugs are kapha pitta samaka (pacifies pitta & kapha entities). The medicines and diets that normalise pitta are commonly used for all types of liver diseases. Most of the hepato-protective herbs are having predominantly Tikta- Katu rasa (bitter and pungent taste) and deepana- pachana karma (digestive stimulant and carminative). These herbs are mainly agni vardhaka (increases fire entity in the body) and act on jatharagni (digestive fire) as well as dhatwagni (fire residing in tissues). These rasas (taste) have the property of increasing metabolism (mainly enhancing catabolism), thereby these herbs help in digestion of nitrogenous waste products collected in body, due to disturbed metabolism. Most of the hepato-protective herbs possess laghu (easy to digest) and ruksha (dry) gunas (quality). Laghu guna (easy to digest quality) helps in increasing jatharagni (digestive fire) as they are easily digestible and they form less nitrogenous waste products. Ushna virya (hot potency) help in enhancing the Jatharagni (digestive fire) as well dhatwagni because ushna virya (hot potency) increases metabolism (catabolism). According to modern pharmacology, the main mechanism involved in the protection of liver could be associated with the strong capability of hepato-protective drugs to reduce the intracellular levels of reactive oxygen species by enhancing the level of both enzymatic and non-enzymatic antioxidants. These drugs protect liver tissues against oxidative damage and somehow help in stimulating the repair mechanism of liver. The mode of action of hepato-protective herbs varies from herb to herb. Hepatocyte membrane stabilizing capacity is shown by Techoma undulata G. Don., thereby preventing toxins from entering the cell through enterohepatic recirculation. Berberis aristata DC., Tephrosa purpurea Pers. and Piper longum

Linn. help in regeneration of liver cells by stimulating nuclear polymerase A and increasing ribosomal protein synthesis. Tinospora cordifolia (Willd.) Miers. enhance the activity of Kuffer cells which is involved in the production of substances like interleukins and tumour necrosis factors which activate the immune system of the body and act as immuno-modulatory. Phyllanthus niruri Linn. possess antiviral property and help in microsomal induction or inhibition. Boerhavia diffusa Linn possess antifibrinolytic activity. Eclipta alba Hassk., Andrographis paniculata Nees. and Picrorhiza kurroa Royle ex Benth. increase the choleretic activity. Different single herbs are very much useful in liver disorders as shown by research studies. A few Ayurvedic compound formulations such as Phalatrikadi kwatha, Vasa guduchyadi kashaya, Patola katurohinyadi kashaya, Guda pippali, Arogyavardhini vati, Rohitakarista mentioned in Sharangadhara Samhitha (13th Century) are also found to be promising in hepatopathy. CONCLUSION The challenge that modern medical system face with liver disorders is that such drugs would have to be metabolized in the liver. Since the liver itself is in disorder, the problem is how to ensure effective metabolism of the drugs that have been prescribed. In this context, Ayurveda sages have used their genius, to formulate such herbal formulations that can be metabolized even by a sluggish liver. The logic on which such formulations work is that they first heal and reinvigorate the liver and thus contribute to the restoration of its normal functions. Preserving health of the liver means adding healthier years to oneâ&#x20AC;&#x2122;s life. Be polite to your liver & Keep it Living and Lively!! ACKNOWLEDGEMENT Author is thankful to Dr. Rajasekhara N, Head & Professor and Dr. Vijayalakshmi P.B, Lecturer, Dept. of Dravyaguna, KVG Ayurveda College, Sullia for all the help and guidance in writing this article.

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Source of Support: NIL

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Conflict of Interest: None Declared

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