Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 43, Num. 3, 2011, pp. 254-257

Indian Journal of Pharmacology, Vol. 43, No. 3, May-June, 2011, pp. 254-257

Research Article

Hepatoprotective activity of Leptadenia reticulata stems against carbon tetrachloride-induced hepatotoxicity in rats

Amit Kumar Nema¹, Abhinav Agarwal², Varsha Kashaw¹

¹ Pharmacognosy Research Laboratory, Sagar Institute of Pharmaceutical Sciences, Sagar, Madhya Pradesh, India
² Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, Madhya Pradesh, India

Correspondence Address: Amit Kumar Nema, Pharmacognosy Research Laboratory, Sagar Institute of Pharmaceutical Sciences, Sagar, Madhya Pradesh, India, nema.amit66@gmail.com

Date of Submission: 30-Jul-2010
Date of Decision: 05-Oct-2010
Date of Acceptance: 23-Feb-2011

Code Number: ph11070

DOI: 10.4103/0253-7613.81507

Abstract

Keywords: Carbon tetrachloride, hepatoprotective activity, Leptadenia reticulata, silymarin

Introduction

The liver is the second largest organ in the body, and is often seen as the most important one. The liver receives a dual blood supply with about 20% of blood coming from the hepatic artery and 80% from the portal circulation. The blood flow to the liver is around 20 to 25% of the total cardiac output. Toxins, infectious agents, medications, and serum inflammatory mediators may result in a diverse range of disease processes, leading to loss of normal histological architecture, reduced cell mass, and loss of blood flow. Consequently, functional liver capacity may be lost. Efforts have been made to search for effective hepatoprotective agents. However, no effective hepatoprotective therapies are available until now. Therefore, the prevention of liver diseases has a great significance both in theory and in practice. ^[1] Herbal drugs play a major role in the treatment of hepatic disorders. In the absence of reliable hepatoprotective drugs in modern medicine, a number of medicinal plants and their formulations are used to cure hepatic disorders in traditional systems of medicine in India. ^[2]

Several plants of the family Asclepiadaceae consisting of flavonoids like rutin, silymarin, and quercetin have been reported to possess a hepatoprotective potential. ^[3],[4],[5] Hence, it was decided to evaluate whether this activity could be related to this family. In this study, stems of Leptadenia reticulata (Retz.) Wight. and Arn. (Jivanti) were investigated for the hepatoprotective activity against carbon tetrachloride (CCl ₄ )-induced toxicity.

L. reticulata plant is a twining shrub that grows in sub-himalayan tracts of Punjab, Gujarat, Uttar Pradesh, and throughout peninsular India, ascending up to an altitude of 900 m. According to Ayurveda, jivanti is jeevana tonic that boost energy level of the body. It is mainly indicated for those who suffer from weak debility or a lack of energy. It is a cooling, mucilaginous, demulcent herb with light strengthening and tonic properties traditionally used in the treatment of seminal discharges and snake bite. ^[6],[7],[8] A number of studies have been carried out on the plant for its antioxidant, galactagogue, antibacterial, lactogenic, antifungal, hypotensive, restorative, tonic, and stimulant activities. ^[9],[10]

The plant contains a triterpenoid, leptadenol, α-amyrin, β-amyrin, ferulic acid, luteolin, diosmetin, rutin, β-sitosterol, stigmasterol, hentriacontanol, a triterpene alcohol simiarenol, and apigenin in significant amounts. ^[11],[12] It also contains pregnane glycosides such as reticulin, deniculatin, and leptaculatin isolated from aerial parts which on hydrolysis give calogenin tocopherols. ^[13],[14] The antioxidant activity of L. reticulata is due to its flavonoid contents like apigenin, rutin, and luteolin. Hence, it is implicit that they might be used as hepatoprotective agents also.

Materials and Methods

Chemicals

All chemicals used were of analytical grade. The kits for the estimation of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), total bilirubin (TB), and total protein were purchased from Beacon Diagnostic Pvt. Ltd. Kabilpore and Loba Chemicals, Mumbai.

Plant Collection and Extraction

The stems of L. reticulata (Retz.) were purchased from the local market of Sagar (M.P.) in the month of November 2009 and were identified and authenticated in Department of Botany, Dr. H. S. Gour University, Sagar. A voucher specimen (MPY-LR01) was deposited in the Pharmacognosy Division, Sagar Institute of Pharmaceutical Sciences, Sagar (M.P.).

Stems of L. reticulata were dried under shade, powdered with a mechanical grinder, and passed through sieve no. 40. The sieved powder was stored in airtight container and kept in room temperature. Dried plant material (500 g) was extracted with Soxhlet apparatus using petroleum ether for about 48 hours. After defatting, the marc was dried in hot air oven at 50°C, packed in Soxhlet apparatus, and further extracted with 95% ethanol exhaustively. The aqueous extract was prepared by cold maceration (72 hours). The solvents were removed from the extracts under reduced pressure by using rotary vacuum evaporator.

Animal Studies

Acute toxicity studies were performed according to Organization for Economic Co-Operation and Development (OECD)-423 guidelines. ^[15] Male Swiss mice selected by random sampling technique were used. The animals were fasted for 3 hours with free access to water only. L. reticulata extracts were administered orally at a dose of 5 mg/kg initially. Mortality was observed for 2 days. If mortality was observed in two of three animals, then the dose administered was considered as toxic dose. However, if no mortality was observed, the procedure was repeated for further higher dose such as 50, 300, and 2 000 mg / kg body weight. The animals were observed for toxic symptoms such as behavioral changes, locomotion, convulsion, and mortality for 24 hours. Two sub-maximal doses 1/10 ^th cut-off dose for extracts 250 and 500 mg/kg, p.o. were found to be safe (1/10 ^th of LD ₅₀ ) and used for further pharmacological investigations.

Wistar albino rats (150-200 g) were used for all other studies, and they were housed at a temperature of 24 ± 2°C at a relative humidity of 50% maintained on 12-hour light/dark cycle and allowed food and water ad libitum. Study was conducted after obtaining a clearance from the Institutional Animal Ethics Committee (Reg. No. 1252/AC/09/CPCSEA) of Bansal College of Pharmacy, Bhopal (M.P.).

Animals were randomly divided into seven groups of six animals each. Group I served as control and received normal saline. Group II served as toxic control and received 1 : 1 (v/v) mixture of CCl ₄ in olive oil at a dose of 1.25 ml/kg, p.o (for 7 days). Group III served as standard group and received silymarin (50 mg/kg), while Groups IV and V were treated with ethanolic extract of L. reticulata at the dose of 250 and 500 mg/kg/day, p.o. for 7 days, respectively. Groups VI and VII were treated with aqueous extract of L. reticulata at the dose of 250 and 500 mg/kg/day, p.o. for 7 days, respectively.

Twenty-four hours after CCl ₄ treatment (Day 8), all the animals were sacrificed under light ether anesthesia. Blood was collected from the retro-orbital plexus of all the rats and allowed to coagulate for 30 minutes at 37°C. Serum was separated by centrifugation at 2 500 rpm at 37°C for 15 minutes and analyzed for SGOT, SGPT, ALP, TB, and total protein. ^{[16],[17],[18],[19],[20]}

Histopathological Studies

After draining the blood, liver samples were excised, washed with normal saline, and processed separately for histological observations. Initially, the material was fixed in 10% buffered neutral formalin for 48 hours. The sections were cut in 5-mm thickness, stained with hematoxylin and eosin, and examined microscopically for histopathological changes.

Statistical Analysis

All the results were expressed as mean ± SEM. One-way Analysis of Variance (ANOVA) was used for the statistical analysis of data. Dunnett′s multiple comparison test ^[21] was used for determining the significance. A probability value of P<0.05 was considered as significant.

Results

The administration of CCl ₄ to the animals resulted in a marked increase (P<0.01) in TB, serum amino transaminases (SGOT and SGPT), and serum ALP. The serum total protein level was decreased when compared with Group I (vehicle control) [Table - 1]. The oral administration of alcoholic and aqueous extracts of L. reticulata and silymarin reduced the CCl ₄ -induced increase in the SGOT, SGPT, and TB levels (P<0.01). The extracts also reversed the depletion of total protein significantly (P<0.05 and P<0.01, respectively) when compared with Group II (CCl ₄ -treated group).

The toxic effect of CCl ₄ was controlled in the animals treated with the extracts by way of restoration of the levels of the liver function biochemistry similar to that of the standard drug silymarin. Among the extract-treated groups, significant hepatoprotective activity was observed.

In the histopathological studies, the liver sections of rats treated with vehicle showed normal hepatic architecture [Figure - 1], whereas that of CCl ₄ -treated group showed total loss of hepatic architecture with intense peripheral central vein necrosis, fatty changes, congestion of sinusoids, Kupffer cell hyperplasia, crowding of the central vein, and apoptosis [Figure - 2]. In case of rats treated with silymarin [Figure - 3] and L. reticulata alcoholic extract 250 mg/kg [Figure - 4] and 500 mg/kg [Figure - 5], respectively, a normal hepatic architecture was seen with only moderate accumulation of fatty lobules and mild degree of cell necrosis, clearly indicating the protection offered by standard drug silymarin and the plant extract.

Discussion

CCl ₄ is commonly used for induction of experimental liver toxicity. This toxic chemical causes peroxidative degradation in the adipose tissue, resulting in fatty infiltration of the hepatocytes. Its metabolites such as trichloromethyl radical (CCl ₃ ) and trichloromethyl peroxy radical (CCl ₃ O ₂ ) are involved in the pathogenesis of liver. As shown in [Figure - 2], CCl ₄ causes change around the central vein in the liver and other oxidative damages with the leakage of marker enzymes like SGOT, SGPT, and ALP in the serum, increase in serum TB levels and decrease in serum total protein. ^[22],[23]

Administration of ethanolic and aqueous extracts of L. reticulata stems showed significant hepatoprotective activity, which was comparable with the standard drug silymarin. The effect was more pronounced with ethanolic extract. The qualitative phytochemical investigations on the ethanolic extract of L. reticulata also showed positive for flavonoids by ferric chloride, alkaline reagent, and Shinoda tests. Furthermore, it has been reported that the flavonoid constituents of plant possess antioxidant properties ^[24] and was found to be useful in the treatment of liver damage.

Earlier works have reported the presence of flavonoids, alkaloids, saponins, and glycosides in the alcoholic extract. The results indicate that the ethanolic extract of L. reticulata has significant hepatoprotective activity. This may be probably due to the higher content of flavonoids like luteolin, rutin, and apigenin. The earlier investigators ^[25] have screened the hepatoprotective activity of the flavonoid compound, rutin, which is also claimed to have free radical scavenging property and it inhibits the lipid peroxidation against CCl ₄ -induced hepatic toxicity. The isolation and characterization of the flavonoids from the stem bark of L. reticulata and screening of the pharmacological action against the liver damage is being investigated.

The results of the present study suggest that alcoholic and aqueous extracts of L. reticulata stems have significant hepatoprotective activity and alcoholic extract offers a greater hepatoprotection than the aqueous.

References

1.	Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115:209-18.   Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.	Subramoniam A, Pushpangadan P. Development of phytomedicines for liver diseases. Indian J Pharmacol 1999;31:166-75.  Back to cited text no. 2
3.	Kshirsagar A, Purnima A, Ingawale D, Vyawahare N, Ingale K, Hadambar A. Antioxidant and Hepatoprotective activity of ethanolic extract of Calotropis gigantea against paracetamol induced liver damage in mice. J Cell Tissue Res 2009;9:1859-64.  Back to cited text no. 3
4.	Sethuraman MG, Lalitha KG, KapoorBR. Hepatoprotective activity of Sarcostemma brevistigma against carbon tetra chloride - induced hepatic damage in rats, Curr Sci 2003;84,1186.  Back to cited text no. 4
5.	Bhaskar VH, Balakrishnan N. Hepatoprotective activity of laticiferous plant species (Pergularia daemia and Carissa carandas) from Western Ghats, Tamilnadu, India. Der Pharmacia Lettre 2009;1:130-42.  Back to cited text no. 5
6.	Srivastav S, Deepak D, Khare A. Three novel pregnane glycosides from Leptadenia reticulata. Tetrahedron 1994;50:789-98.  Back to cited text no. 6
7.	Anjaria JV, Mankad BN, Gulati OD. Isolation of stigmasterols and tocoferols from Leptadenia reticulata by a shortcut method. Indian J Pharmacol 1974;36:148.  Back to cited text no. 7
8.	Agarwal SL, Deshmankar BS, Verma SCL, Saxena SP. Studies on Leptadenia reticulata. Part I. Pharmacological actions of aqueous extract. Indian J Med Res 1960;48:457-64.  Back to cited text no. 8
9.	Anjaria JV, Varia MR, Jarakiraman VN, Gulati OD. Studies on Leptadenia reticulata: Lactogenic effects on rats. Indian J Exp Biol 1975;13:448-9.  Back to cited text no. 9
10.	Patel RP, Dantwala AS. Antimicrobial activity of Leptadenia reticulata. Indian J Pharm 1958;20:241-4.  Back to cited text no. 10
11.	Jaytilak PG. Supplement to Glossary of Indian Plants. Indian J Exp Biol 1976;14:170.  Back to cited text no. 11
12.	Krishna PV, Venkata Rao E, Venkata Rao D. Crystalline principles from the leaves and twigs of Leptadenia reticulata. Planta Med 1975;27:395-400.  Back to cited text no. 12
13.	Subramanium PS, Lakshamanan AJ. On the constituents of Leptadenia reticulata Wright and Arn. Occurrence of simiarenol. Indian J Chem 1977;15:180.  Back to cited text no. 13
14.	Sastry BS, Vijayalakhami T, Venkata Rao E. Chemical constituents of the stems bark of Leptadenia reticulata. Indian Drugs 1985;22:611-2.  Back to cited text no. 14
15.	Ecobichon DJ. The Basis of Toxicology Testing. New York: CRC Press; 1997. p. 43-86.  Back to cited text no. 15
16.	Bergmeyer HU, Scheibe P, Wahlefeld AW. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin Chem 1978;24:58-73.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.	Bergmeyer HU. IFCC methods for the measurement of catalytic concentrations of enzymes Part 3. IFCC method for alanine aminotransferase (1-alanine:2oxoglutarate aminotransferase). Clin Chem Acta 1980;105:147.  Back to cited text no. 17
18.	Rick W. Klinische Chemie und Microskopie. Hepatoprotective activity of Capparis sepiaria against carbon tetrachloride induced hepatotoxicity. Berlin, Germany: Springer Verlag; 1990. p. 294.  Back to cited text no. 18
19.	Perry B, Doumas BT, Buffone G, Glick M, Ryder K. Measurement of total bilirubin by use of bilirubin oxidase. Clin Chem 1986;32:329-32.  Back to cited text no. 19  [PUBMED]  [FULLTEXT]
20.	Lowery OH, Rosenbrough NJ, Forr AL, Ramdall RJ. Protein measurement with Folin's phenol reagent. J Biol Chem 1951;193:265-75.  Back to cited text no. 20
21.	Steel RG, Torrie JH, Dickey DA. Principles and procedures of statistics, abiometrical approach. 3 rd ed. New York: McGraw-Hill Co. Inc.; 1997.  Back to cited text no. 21
22.	Recnagel RO. Carbon tetrachloride hepatotoxicity status and future prospects. Pharmacol Sci 1983;4:129-31.  Back to cited text no. 22
23.	Okuno H, Hazama H, Muraze T, Shiozaki Y, Sameshima Y. Drug metabolizing activity in rats with chronic liver injury induced by carbon tetrachloride: Relationship with the content of hydroxyproline in the liver. Jpn J Pharmacol 1986;41:363-71.  Back to cited text no. 23
24.	Hesham RE, Shgeru N. Chemistry of Bioflavonoids. Indian J Pharm Educ 2002;36:191-4.   Back to cited text no. 24
25.	Khalid HJ, Sheikh AS, Anwar HG. Protective effect of rutin on paracetamol and CCl4 induced hepatotoxicity in rodents. Fitoterapia 2002;73:557-63.  Back to cited text no. 25

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