Hepatoprotective and Anti-inflammatory activities of Hydroalcoholic extract of Bark of Erythrina indica

 

Vrunda V. Shah¹*, Vipul K. Shah², N. R. Sheth³, M. M. Patel⁴

¹M. Pharm, Ph. D Running, Department of Pharmacognosy, Shree Swaminarayan Sanskar Pharmacy College, Zundal, Gandhinagar-382421, Gujarat, India

²M. Pharm, Ph. D Running, Troikaa Pharmaceuticals Ltd., Ahmedabad, Gujarat, India

³M. Pharm, Ph. D, Vice Chancellor of Gujarat Technological University, Ahmedabad, Gujarat, India.

⁴M. Pharm, Ph. D, Director, Shree Swaminarayan Sanskar Pharmacy College, Zundal, Gandhinagar, Gujarat, India.

 

ABSTRACT:

Background: Natural remedies from medicinal plants are considered to be effective and safe alternative treatment for liver toxicity and inflammation.

Aim: This present work was done to investigate the Hepatoprotective and Anti-inflammatory activities of Hydroalcoholic extract of Bark of Erythrina indica .

Materials and Methods: Drugs like Silymarin, Carbon tetrachloride, Liquid paraffin, carrageenan, Aspirin were used. For Hepatoprotective activity SGOT, SGPT, ALKP, Total cholesterol, Total bilirubin, Albumin, Total protein Estimation kit and for anti-inflammatory activity carrageenan-induced paw oedema method were used.

Result and discussion: The hepatoprotective activity of the Hydroalcoholic extract was assessed in CCl4 induced hepatotoxic Rats. The extract of plant (300mg/kg and 500mg/kg, orally) was given for 6 days, daily and CCl4 (1 ml/kg, 1:1 with liquid paraffin, s.c.) was given at 6^th day after dosing of plant extract. Alteration in the levels of biochemical markers of hepatic damage like SGOT, SGPT, ALKP, Bilirubin, Cholesterol, Total Protein and Albumin were tested in both CCl4 treated and untreated groups. CCl4 has enhanced the SGOT, SGPT, ALKP and Bilirubin in serum, while Total protein and Albumin were decreased. The study was also supported by histopathology of liver sections. Treatment of Hydroalcoholic extract of Bark of Erythrina indica has brought back the altered levels of biochemical markers to near about normal levels in the dose dependent manner. Anti-inflammatory activity was done in rats at a dose 300 and 500 mg/kg in carrageenan-induced paw oedema method. Hydroalcoholic extract of Erythrina indica bark significantly reduced the migration of neutrophils and monocytes. Results of in vivo activity lead to the conclusion that the hydroalcoholic extract of Erythrina indica bark showed predominantly significant activity in dose dependent manner, which is comparable to the standard drug Aspirin. The present study reveals that the Hydroalcoholic extract of Bark of Erythrina indica could afford a significant Hepatoprotective and Anti-inflammatory activity.

 

 

 

1.      INTRODUCTION:

The liver is a key organ involved in a large no. of metabolic function as well as in the detoxification of hazardous substances and is a target of number of toxins ^[1]. Liver disease is a worldwide health problem. Liver damage is always associated with cellular necrosis, increase in tissue lipid peroxidation and depletion in the tissue GSH (Gonedotropin Stimulating Hormone) levels. In addition serum levels of many biochemical markers like SGOT, SGPT, triglycerides, cholesterol, bilirubin, alkaline phosphatase, are elevated^{[2,3] .}

 

Most of the hepatotoxic chemicals damage liver cells mainly by inducing lipid peroxidation and other oxidative damages in liver^[4]. Hepatotoxicity implies chemical driven liver damage. The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Certain medicinal agents when taken in overdoses and sometimes even when introduced within therapeutic ranges may injure the organ.

 

It has been reported that the metabolism of carbon tetrachloride involves the production of free radicals through its activation by drug-metabolizing enzymes located in the endoplasmic reticulum^[5]. Carbon tetrachloride can cause liver lipid peroxidation, resulting in liver fibrosis^[6]. It is generally believed that the toxicity of carbon tetrachloride is the result of its reductive de halogenation in to the highly reactive free radical through the cytochrome P450 enzyme system^[7]. Liver injuries induced by carbon tetrachloride are the best characterized mechanism of xenobiotic-induced hepatotoxicity, and those models are commonly used to screen the antihepatotoxic and hepatoprotective activity of various drugs^[7]. Therefore, carbon tetrachloride induced rats are an animal model commonly used to evaluate the efficacy of potential Hepatoprotective substances.

 

Unfortunately, conventional or synthetic drugs used in the treatment of liver diseases are inadequate and sometimes can have serious side effects^[8-11]. More than 900 synthetic allopathic drugs have been implicated in causing liver injury and it is the most common reason for a drug to be withdrawn from the market^[12].

 

In the Indian system of medicine (ISM), the plants are classified in various groups on the basis of experience and ethnomedicinal uses. Hepatoprotective plants have an important place in the literature of ISM. Many plants mentioned in the ISM, like Andrographis paniculata, Berberis aristata, and Phyllanthus amarus are used either alone or in combination as hepatoprotectives[^{13, 14]}.

 

This is one of the reasons for many people worldwide including those in developed countries to return to complementary and alternative medicine. Many traditional remedies employ herbal drugs for the treatment of liver ailments^[8-11] .So the present study has been done to evaluate the use of Erythrina indica Bark as a Hepatoprotective agent.

 

Inflammation is a local response of living mammalian tissues to injury. It is a body defense reaction in order to eliminate or limit the spread of injurious agent. There are various components to an inflammatory reaction that can contribute to the associated symptoms and tissue injury. Edema formation, leukocyte infiltration and granuloma formation represent such components of       inflammation ^[15].

 

The barks of Erythrina indica are used traditionally as astringent, febrifuge and in leprosy and fever. Leaves are Anthelmintic, Laxative and Diuretic. Paste of leaves is applied externally to cure inflammations and to relieve pain in the joints. Juice is also used to relieve earache and toothache^[16,17]. Inflammation is considered as a primary physiologic defense mechanism that helps body to protect itself against infection, burn, toxic chemicals, allergens or other noxious stimuli, an uncontrolled and persistent inflammation may act as an etiologic factor for many of these chronic illnesses^[18]. During the last three decades, over 50 flavonoids have been obtained from 15 Erythrina species^[19], with prenylated flavanones, isoflavones and pterocarpans being the major nonalkaloid secondary metabolites isolated thus far^[20-23]. These compounds are of biological importance as they exhibit various pharmacological activities. Flavonoids are the chief chemical constituents present in Erythrina indica and are known to possess anti-inflammatory property.

 

2.      MATERIAL AND METHODS:

2.1   Collection and Authentication of plant:

The Bark of Erythrina indica Linn. Were collected from Saurashtra University Campus, Near Bioscience Department, Rajkot during the month of September- October (2008). The plant was authentified by Botanical Survey of India, Jodhpur and a voucher specimen no. SU/DPS/Herb/08 was deposited. Further, the plant was identified by comparing it morphologically and microscopically with the description given in different standard texts and floras^[24]. Fresh bark of plant were cleaned, dried at room temperature and powdered.

 

2.2 Preparation of plant extract:

The shade dried powder of bark of E. indica was subjected for size reduction to fine powder. The powder was macerate with Hydroalcoholic solution for 6-7 days on rotary shaker. Filter and concentrated under vacuum to get the residues. The extracts were suspended in 7% acacia solution for pharmacological study.

 

2.3 Drugs and Chemicals:

Silymarin (Cadila Healthcare Pvt. Ltd.), Carbon tetrachloride, Liquid paraffin (E. Merck (India) Ltd., Mumbai), Acetyl salicylic acid (Ozone International, Mumbai), λ-Carrageenan (Sigma Aldrich, Mumbai) were used.

 

SGOT, SGPT, ALKP, Total cholesterol, Total bilirubin, Albumin, Total protein Estimation kit (Span diagnostic, Surat).

 

2.4 Acute toxicity study:

Different groups of five mice each were given graded doses of the plant extract (0.5-3.0 g/kg, orally) and were kept under constant observation for 6 h to note any behavioral changes and mortality was recorded after 24 h of the drug administration^[25].

 

2.5 Animal selection and Ethical clearance:

Wistar albino male rats weighing 200-250 gm were acclimatized to the experimental room having temperature 23 ± 2°C, controlled humidity conditions (55±10 %), and 12:12 hour light and dark cycle. Animals were caged in polypropylene cages with maximum of five animals per cage. The rats were fed with standard food pellets and water ad libitum. Study was conducted after obtaining the approval from the Institutional Animal ethical committee as per the guidance of the committee for the purpose of control and supervision of experiments on animals (CPCSEA).

 

2.6 Protocol for Hepatoprotective activity:

Animals were divided into five groups of five rats in each for all the experiment. The first group served as vehicle control and received 7% gum acacia in distilled water (2ml/kg) only. The second group served as carbon tetra chloride (CCl4) intoxicated control and received by gavage vehicle (7% gum acacia in distilled water) and CCl4 diluted with liquid paraffin (1:1). Third group was given standard drug Silymarin at the dose of 100 mg/kg body weight and the remaining two groups were given EIHAE (Erythrina indica Hydroalcoholic extract) at the dose of 300 mg/kg and 500 mg/kg body weight respectively and CCl4. The vehicle (7% gum acacia in distilled water) or test drugs were administered orally for 6 days. CCl4 diluted with liquid paraffin (1:1) was administered in a dose of 1 ml/kg subcutaneously (s.c) on 6th day (After 2 hr. of previous administration).

Group I: Vehicle (7% gum acacia in distilled water, 2ml/kg. p.o.)

Group II: Vehicle for 6 days + CCl4 (1ml/kg, s.c.) 0n 6th day.

Group III: Silymarin (100mg/kg, p.o.) for 6 days + CCl4 (1ml/kg, s.c.) 0n 6th day.

Group IV: EIHAE (300 mg/kg, p.o.) for 6 days + CCl4 (1ml/kg, s.c.) 0n 6th day.

Group V: EIHAE (500 mg/kg, p.o.) for 6 days + CCl4 (1ml/kg, s.c.) 0n 6th day.

 

Food was withdrawn 12 hr. before CCl4 administration to enhance liver damage in animals of groups II, III, IV and V. Twenty four hours after CCl4 administration, blood was obtained from all groups of rats by puncturing retro-orbital plexus. The blood samples were allowed to clot for 45 min at room temperature. Serum was separated by centrifugation at 2500 rpm at 30°C for 15 min and analyzed for various biochemical    parameters^[26-28].

 

2.7 Parameters assessed for Hepatoprotective activity:

On the 7th day blood was collected from all groups of rats by puncturing retro-orbital plexus and animals were sacrificed. The following parameters were assessed.

 

2.8 Biochemical parameters:

Serum glutamate oxaloacetae transaminase (SGOT) Serum glutamate pyruvate transaminase (SGPT) Alkaline phasphatase (ALKP) Total Bilirubin (TB) Total Cholesterol (CHL) Total Protein (TP) Albumin (ALB)

 

2.9 Histopathological Studies:

One animal from each of the treated groups, showing maximum activity as indicated by improved biochemical parameters was used for this purpose. The animals were sacrificed and the abdomen was cut open to remove the liver. The liver was fixed in 10 % formalin solution. After 12 hours, liver was embedded in paraffin using conventional methods^[29] and cut in to 5μm thick section and stained using haematoxylin-eosin dye and finally mounted in di-phenyl xylene. Then the sections were observed under microscope for histopathological changes in liver architecture and their photomicrographs were taken.

 

2.10 Photomicrography:

The photomicrography of the sections at different magnifications as demanded by the anatomical details to be studied. The microphotographs were taken using Olympus CH20i microscope attached with Magnus MIPS camera.

 

2.11 Statistical analysis:

The mean values ± S.E.M. are calculated for each parameter. For determining the significant inter group difference, each parameter was analyzed separately and one way analysis of variance (ANOVA)^[30] was carried out.

 

2.12 Protocol for Carrageenan induced hind paw oedema:

Evaluation of anti-inflammatory activity Carrageenan-induced rat paw edema: Rats were divided into four groups of six animals each. The first group served as the control and received vehicle only (7% gum acacia in distilled water), second and third group was treated with EIHAE (300mg/kg, 500 mg/kg respectively) and the fourth group was administered with standard drug, Aspirin (200 mg/kg, orally). A mark was made on both the hind paws just below the tibiotarsal junction so that every time the paw could be dipped in the mercury column of plethysmograph up to the mark to ensure constant paw volume^[31].

 

Forty-five minutes after treatment, an inflammatory oedema was induced in the left hind paw by injection of 0.1 ml of Carrageenan (1% w/v) in the plantar tissue of all the animals. The paw volume was measured in control, standard and sample treated groups immediately before and after 3 h of Carrageenan injection^[32-35]. The degree of edema formation was assayed by the percentage increase in paw treated with standard drug and those treated with extract. These were compared with the increased paw volume of control animals. Thus, percentage inhibition of paw volume in treated animals i. e. edema rate (E) % = (Vt/Vc) x100,

 

Which was used for calculating the percentage inhibition rate % = 1-(Vt/Vc) x100, where Vt and Vc are the mean relative changes in the paw volume of the test and control respectively ^[35].

Group I: Vehicle (7% gum acacia in distilled water, 2ml/kg. p.o.) + 0.1 ml Carrageenan (1% w/v, s.c.)

Group II: EIHAE (300 mg/kg, p.o.)+0.1 ml Carrageenan (1% w/v, s.c.)

Group III: EIHAE (500 mg/kg, p.o.)+0.1 ml Carrageenan (1% w/v, s.c.)

Group IV: Aspirin (200 mg/kg, p.o.)+0.1 ml Carrageenan (1% w/v, s.c.)

 

2.13 Statistical analysis:

For determinations of significant inter group difference, mean paw oedema ± S.E.M. were analyzed by One way analysis of variance (ANNOVA) using Sigma Stat Software^[36].

 

3. RESULT AND DISCUSSION:

3.1 Acute Toxicity Study: LD50 was found to be 3000 mg/kg.

 

 

 

3.2 In vivo Hepatoprotective Study:

Administration of CCl4 in normal rat elevated the serum level of SGOT, SGPT, ALKP, TB, DB and CHL; quite decreased in TP and ALB were observed, significantly indicate Hepatocellular damage. It may be due to enzymatic activation CCl3 free radical which alters the structure and function of liver cell. The rat treated with Erythrina indica  Hydroalcoholic (50%) extract showed decreased in all elevated level of SGOT, SGPT, ALKP, TB, DB and CHL level and increased in TP and ALB levels. Pretreatment with Erythrina indica Hydro alcoholic (50%) extract showed dose dependent protection against injurious effect of CCl4 that may result from interfere with cytochrome P450 resulting in the hindrance of the formation of Hepatotoxic free radical. This biochemical restoration may be due to the inhibitory effect on cytochrome P450 and/or promotion of its glucuronidation. It also indicates stabilization of plasma membrane as well as repair of Hepatic tissue damage, caused by CCl4.

 

3.3 Histopathology of Liver:

Fig.1 Shows Histopathological studies which also provided supportive evidence for biochemical analysis. Histology of the liver section of Normal control group showed in Fig. 1.I showed distinct hepatic cells, sinusoidal spaces HC: Hepatocyte, SS: Sinusoidal space, VC: Vacuoles. Histology of the liver section of Standard group showed in Fig. 1.II showed regeneration of Hepatocyte, less vacuoles, reduced sinusoidal space compared to hepatotoxin. Histology of the liver section of hepatotoxic group showed in Fig. 1.III showed degeneration of normal hepatic cells with lobular necrosis, vacuole formation. Histology of the liver section of EIHAE (300mg/kg) showed in Fig. 1.IV showed less degeneration of Hepatocyte, less vacuoles, less sinusoidal space compared to hepatotoxin. Histology of the liver section of EIHAE (500mg/kg) showed in Fig. 1.V showed regeneration of Hepatocyte, less vacuoles, reduced sinusoidal space compared to hepatotoxin.

Table: 1 shows the effects of Erythrina indica Bark on different Biochemical parameters.

Groups (n=5)	Dose	(SGOT) (IU/l)	(SGPT) (IU/l)	(TB)  (mg/dl)	(ALKP)  (IU/l)	(DB) (mg/dl)	(CHL)  (mg/dl)	(TP) (mg/dl)	(ALB)  (g/dl)
Control (Vehicle)	2ml/kg	99.2±16.608	42±4.658	0.302±0.0332	5.878±0.0153	0.358±0.0248	114.932±9.218	5.810±0.376	3.202±0.238
Hepatotoxin (CCl4)	1ml/kg	178.8±34.852	109.6±9.042	1.418±0.0427	51.112±3.996	0.636±0.0339	210.130±7.808	3.120±0.397	1.908±0.0969
Standard  (Silymarin)	100mg/kg	116.6±7.580	68±7.273	0.296±0.0178	5.282±0.585	0.404±0.0293	117.6±10.402	5.300±0.414	2.720±0.116
EIHAE  (300mg/kg)	300mg/kg	160±4.94	70±10.139	0.896±0.0262	23.918±2.874	0.506±0.0353	127.864±0.997	3.890±0.214	2.2±0.0515
EIHAE (500mg/kg)	500mg/kg	119.6±11.994	48.4±15.052	0.528±0.0299	19.148±0.823	0.378±0.0183	90.794±7.476	4.640±0.0927	2.6±0.145

Value are mean ±SEM; n=5.   Compared to Control (P<0.001)

 

HC: Hepatocyte, SS: Sinusoidal space, VC: Vacuoles

Fig. 1: Histopathological of Liver

 

3.4 Anti-inflammatory activity:

Table 2 Shows that % Inhibition of paw edema is increased in dose dependent manner in groups treated with Erythrina indica  Hydroalcoholic (50%) extract as compared to control.

 

 

Table 2: Effects of Erythrina indica bark on carrageenan induced rat paw oedema

Treatment	Dose	Paw Oedema (ml) after 3 hr	% Inhibition
Control (Carrageenan)	Vehicle 2ml/kg	0.504±0.0807	-
Standard (Aspirin)	200mg/kg	0.0620±0.0203	87.698 %
EIHAE	300mg/kg	0.290±0.0321	42.460 %
EIHAE	500mg/kg	0.222±0.0512	55.952 %

 Value are mean ±SEM; n=5.     Compared to Control (P<0.001)

The preliminary Anti-inflammatory activity was evaluated by rat-paw edema model which showed dose dependent Anti-inflammatory activity in case of acute inflammation.

 

4. CONCLUSION:

From present study, results show that the bark of Erythrina indica has a strong Hepatoprotective and Anti-inflammatory activity.

 

HC

SS

HC

HC

5. ABBREVIATIONS:

(IL) Interleukins

(EIHAE) Erythrina indica Hydroalcoholic extract

(CCl₄ ) Carbon tetrachloride

(SGOT) Serum glutamate oxaloacetae transaminase

(SGPT) Serum glutamate pyruvate transaminase

(ALKP) Alkaline phosphatase

(TB) Total Bilirubin and (DB) Direct Billirubin

(CHL) Total Cholesterol

(TP) Total Protein

(ALB) Albumin

(AST) Aspartate Transaminase

(ALT) Alanine Transaminase

(S.E.M.) Standard Error Mean

(CE) Cholesterol Esterase

(POD) Peroxidase

(CHOD) Cholesterol Oxidase

 

6. REFERENCES:

1.     Meyer S.A., Kulkarni A.P., hepatotoxicity. In: Hodgson E., Smart R.C., Editors. Introduction to biochemical toxicology. New York: John Wiley and Sons .2001; 487-490.

2.     Mascolo N., Sharma R., Jain S.C., Capasso F., J Ethnopharmacol. 1994; 22: 211.

3.     Mossa J.S., Tariq M., Mohsin A., Aqeel A.M., al-Yahya M.A., al-Said M.S., Am J Chin Med.1991; 19: 223.

4.     Handa. S.S., Sharma. A., Chakraborty. K.K., Natural products and plants as liver protecting drugs; Fitoterapia.. 1989; 57: 307-351.

5.     Slater T.F., Sawyer B.C., The Stimulatory effect of carbon tetrachloride and other halogenoalkanes on peroxidative reactions in rat liver functions in vitro. Inhibitor effects of free radical scavengers and other agents. Biochem J. 1971; 123: 823-828.

6.     De Pomerai D. I., Pritchard D. J., Clayton R. M., Biochemical and immunological studies of Lentoid formation in cultures of embryonic chick neural retina and day-old chick lens epithelium. Dev Biol. 1977; 60: 416-427.

7.     Recknagel R.O., Glende E.A., Dolak J.A., Waller R.L., Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther. 1989; 43: 139-154.

8.     Venkateswaran S., Pari L., Viswanathan P., Menon V.P., Protective effect of herbal formulation against erythromycin estolate- induce hepatotoxicity in rats. J. Ethnopharmacol. 1997; 57: 161-167.

9.     Latha U., Rajesh M.G., Latha M.S., Hepatoprotective effect of an Ayurvedic medicine, Ind. Drugs. 1999; 36: 470-473.

10.   Mithra S.K., Seshadri S.J., Venkataranganna M.V., Gopumadhavan S., Venkatesh Udupa U., Sarma D.N.K., Effect of HD-03-a herbal formulation in Galactosamine induced hepatopathy in rats. Indian J. Physiol. Pharmacol. 2000; 44: 82-86.

11.   Dhuley G.P., Naik S.R., Protective effect if Rhinax, herbal formulation against CCL4 induced liver injury and survival in rats. J. Ethnopharmacol. 1997; 56: 159-164.

12.   Freidman. S.E., Grendell. J.H., McQuaid. K.R., Current diagnosis and treatment in gastroenterology. New York: Lang Medical Books/McGraw-Hill.   2003; 664-679.

13.   Mukherjee P.K., Quality Control of Herbal Drugs— An Approach to Evaluation of Botanicals. New Delhi: Business Horizons. 2002.

14.   Maity T.K., Mandal S.C., Mukherjee P.K., Saha K., Saha B.P., Das J., Pal M., Evaluation of Hepatoprotective potential of Cassia tora leaf extract. Nat Prod Sci. 1997; 3: 122–126.

15.   Mitchell R. N. and R.S. Cotran, In: Robinsons Basic Pathology, 7th Edn., Harcourt (India) Pvt Ltd., New Delhi. 2000; 33. 

16.   Haque R., Mohammad S., Achinto S., Alimuzzaman M.D., Analgesic Activity of Methanolic Extract of the Leaf of Erythrina variegate. J. Pharm. Sci. 2006; 5(1-2): 77-79.

17.   Ghani A., Medicinal Plants of Bangladesh with chemical constituents and uses. published by Asiatic Society of Bangladesh. 2^nd edition, 222-223.

18.   Kumar G., Banu G.S., Pappa P.V., Sundrarajan M., and Pandian M.R., Hepatoprotective activity of Trainthema portulacastrum L. against paracetamol and thioaacetamide intoxication in albino rats. J.Ethnopharmacol. 2004; 92(1): 37-40.

19.   Kamat V.S., Chuo F.Y., Kubo I., Nakanishi K.,. Anti-microbial agents from an East-African medicinal plant, Erythrina abyssinica. Heterocycles 1981; 15: 1163–1170.

20.   Fomum Z.T., Ayafor J.F., Erythrina studies: Part 2. Structures of the novel prenylated antibacterial flavanones, sigmoidins A–C from Erythrina sigmoidea. J. Chem. Soc., Perkin Trans. 1986; 1: 33–37.

21.   Fomum Z.T., Ayafor J.F., Mbafor J.T., Erythrina studies: Part 1. Novel antibacterial flavanones from Erythrina sigmoidea. Tetrahedron Lett. 1983; 24: 4127– 4130.

22.   Fomum Z.T., Nkenfack A.E., Wandji J., Erythrina studies: Part 15. Pharmacological screening of three esters isolated from Erythrina plants. Ann. Fac. Sci. Chim. 1988;2:79– 104.

23.   Wandji J., Fomum Z.T., Tillequin F., Seguin E., Koch M., Erythrina studies: Part 24. Two isoflavones from Erythrina senegalensis. Phytochemistry. 1994; 35: 245– 248.

24.   Kirtikar K.R., Basu B.D., Indian medicinal plants, International Book Distributors, Dehradun, 2^nd Ed. 1991; Vol-1, 781-784.

25.   Khalid H. J., Anwar H. G., Evaluation of the protective potential of Artemisia maritime extract on acetaminophen and CCl4 induced liver damage. Journal of Ethnopharmacology. 1995; 47: 43-47.

26.   Bahar A., Tanveer A., Shah A.K.., Hepatoprotective activity of Luffa echinata fruits. Journal of Ethnopharmacology. 2001; 76: 187–189.

27.   Porchezhian E., Ansari S.H., Hepatoprotective activity of Abutilon indicum on experimental liver damage in rats. Phytomedicine. 2005; 12: 62–64.

28.   Rao K.S., Mishra S.H., Anti-inflammatory and Hepatoprotective activities of fruits of Moringa pterygosperma Gaertn. Ind. J. Nat. Prod. 1998; 3: 14.

29.   Rao K.S., Mishra S.H., Anti-inflammatory and Hepatoprotective activities of fruits of Moringa pterygosperma Gaertn. Ind. J. Nat. Prod. 1998; 3: 14.

30.   Fakurazi S., Hairuszah I., Nanthini U., Moringa oleifera Lam prevents acetaminophen induced liver injury through restoration of glutathione level. Food and Chemical Toxicology. 2008; 46: 2611–2615.

31.   Vishnukanta, Rana A.C., Analgesic and Anti-Inflammatory Activity of Hydroalcoholic Extract of Leaves of Plumbago zeylanica. Phcog Mag. 2008; 4.

32.   Vendruscolo A., Takaki I., Amado L.E.B., Dantas J. A., Amado C.A.B., Suman R. K. N., Anti-inflammatory and antinociceptive activities of Zingiber officinale Roscoe essential oil in experimental animal models. Ind. J. Pharmacol. 2006; 38(1): 58-59.

33.   Balian S., Ahmad S., Zafar R., Anti-inflammatory activity of leaf and leaf callus of Silybum marianum (L.) Gaertn. In albino rats. Ind. J. Pharmacol. 2006; 38(3): 213-214.

34.   Sutradhar R.K., Rahman A. K. M. M., Ahmad M.U., Datta B.K., Bachar S.C., Saha A., Anti-inflammatory activity of Sida cordifolia Linn. Ind. J. Pharmacol. 2006; 38(3): 207-208.

35.   Ahmet C.O., Ufuk M., Hatice O., Nureddin C., Remzi E.,Hanefi O., Anti-inflammatory and Hepatoprotective Activities of Trigonella foenum-graecum L. Pharmacologyonline. 2008; 2: 126-132.

36.   Ratheesh M., Helen A., Anti-inflammatory activity of Ruta graveolens Linn on carrageenan induced paw edema in wistar male rats.  African Journal of Biotechnology. 2007; 6 (10): 1209-1211.