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ORIGINAL ARTICLE
Year : 2011  |  Volume : 3  |  Issue : 1  |  Page : 40-43  

Evaluation of acute toxicity and hepatoprotective activity of the methanolic extract of Dichrostachys cinerea (Wight and Arn.) leaves


1 Phytochemistry and Pharmacology Laboratory, Department of Post Graduate Studies and Research in Biotechnology and Bioinformatics, School of Biological Sciences, Kuvempu University, Shankaraghatta - 577 451, India
2 Department of Post Graduate Studies and Research in Biotechnology, SDM College (Autonomous), Ujire - 574 240, India
3 Department of Pathology, Shivamogga Institution of Medical Sciences Shivamogga - 577 201, Karnataka, India

Date of Submission03-Jun-2010
Date of Decision22-Jun-2010
Date of Web Publication7-Apr-2011

Correspondence Address:
P Suresh Babu
Phytochemistry and Pharmacology Laboratory, Department of Post Graduate Studies and Research in Biotechnology and Bioinformatics, School of Biological Sciences, Kuvempu University, Shankaraghatta - 577 451, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-8490.79114

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   Abstract 

Background: D. cinerea are the chief source of drug compounds that are active against various ailments such as jaundice, inflammations rheumatism, fever, asthma, body ache, chest problems, toothache, ulcers, wounds, eye diseases and have an aphrodisiac property. In present study, It was aimed to test the hepatoprotective activity of the plant. Material and Methods: The methanolic extract of Dichrostachys cinerea (Mimoseae) leaves was subjected to evaluation of acute toxicity and hepatoprotective property, using albino mice and rats. The parameters for estimation of liver function, based on serum markers such as total bilirubin, total protein, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase with histopathological profile of the liver tissue, were studied consequently. Results: The methanolic extract did not show any mortality up to a dose of 3500 mg/kg body weight. The methanolic extract showed significant hepatoprotectivity. The histopathological profile of the drug-treated liver tissue demonstrated similar morphology as that of controls. Conclusions: Methanolic extract of Dichrostachys cinerea was found to have significant hepatoprotective activity.

Keywords: Acute toxicity, carbon tetrachloride induced, Dichrostachys cinerea, hepatoprotective


How to cite this article:
Babu P S, Krishna V, Maruthi K R, Shankarmurthy K, Babu RK. Evaluation of acute toxicity and hepatoprotective activity of the methanolic extract of Dichrostachys cinerea (Wight and Arn.) leaves. Phcog Res 2011;3:40-3

How to cite this URL:
Babu P S, Krishna V, Maruthi K R, Shankarmurthy K, Babu RK. Evaluation of acute toxicity and hepatoprotective activity of the methanolic extract of Dichrostachys cinerea (Wight and Arn.) leaves. Phcog Res [serial online] 2011 [cited 2019 Aug 24];3:40-3. Available from: http://www.phcogres.com/text.asp?2011/3/1/40/79114


   Introduction Top


Plants possess broadest spectrum of synthetic activities and are the chief source of many useful compounds. Dichrostachys cinerea (Mimosae) is a medium-sized tree commonly distributed in the forests of Africa, Australia, India and parts of Southeast Asia. It is commonly known as "sickle pod", "acacia Saint Domingue", "aroma", in English and its vernacular name is "Vada" in Karnataka, South India. As per the traditional claims, bark and leaves of D. cinerea are the chief source of drug compounds that are active against various ailments such as jaundice, inflammations rheumatism, fever, asthma, body ache, chest problems, toothache, ulcers, wounds, eye diseases and have an aphrodisiac property. [1],[2] Preliminary phytochemical analysis of the plant, especially leaves and bark extract, proved that it contains flavonoids, tannins, triterpenes, saponins and steroids. [3] The heartwood of D. cinerea contains aliphatics and triterpenoids. [4] Antibacterial activity of the tannins isolated from the D. cinerea is also well established. In the present investigation, acute toxicity and hepatoprotective property of methanol extract of D. cinerea leaves was screened.


   Materials and Methods Top


Plant collection and authentication

The fresh leaves of D. cinerea were collected from the Malebennur reserve forest range of Karnataka, India. Taxonomic authenticity was confirmed by referring to herbarium specimen at Madras herbarium, Botanical Survey of India, Southern Circle, Coimbatore, and a voucher specimen (FDD-53) is deposited at Kuvempu University herbaria, Shankaraghatta.

Preparation of extracts

The fresh leaves of D. cinerea were shade dried and powdered mechanically. The powdered material was subjected to extraction using soxhlet apparatus with methanol for about 48 hours (200 g Χ 5). The extract was filtered and concentrated in vacuum under reduced pressure using rotary flash evaporator (Buchi, Flawil, Switzerland) and allowed for complete evaporation of the solvent. The yield of the crude extract crude was 3.16% (w/w).

Preliminary phytochemical investigation

Crude methanolic extract was subjected to preliminary phytochemical qualitative analysis to detect the major chemical groups. [5],[6],[7]

Experimental animals

Albino mice of either sex weighing 20-25 g and male Wistar albino rats weighing 180-200 g were procured from Central Animal House, National College of Pharmacy, Shivamogga, and were maintained in standard housing conditions. The animals were fed with commercial diet (Pranav Agro Industries Ltd., Sangli, India) and water ad libitum during the experiment. The Institutional Animal Ethical Committee (Reg.No.144/NCP/IAEC/CLEAR/P.COL.3/2006-07) permitted the study to be conducted.

Acute toxicity studies

Per oral (p.o.) acute toxicity of the extract of D. cinerea was evaluated in Swiss albino mice, weighing about 20-25 g, by modifying the method of Lorke and Ghosh. [8],[9] This method involved the determination of LD 50 value in a biphasic manner. The animals were starved but allowed access to water 24 hours prior to the study. In the initial investigatory step (phase I), a range of doses of the extract producing the toxic effects was established. This was done by oral administration of widely differing doses of the extract (500, 1000, 2000, 3000 and 4000 mg/kg p.o.) to five groups of mice (four mice in each group). The result showed that there was no mortality up to the dose of 3000 mg/kg b.wt., but maximum mortality was observed at a dose of 4000 mg/kg b.wt. So, a phase II investigatory step was done by giving more specific doses like (3200, 3300, 3400, 3500, 3600 and 3700 mg/ kg p.o.) to five other groups of mice. The mice were observed for 24-48 hours for behavioral signs such as nervousness, excitement, dullness, ataxia or death. Thus, maximum nonlethal dose and the minimum lethal dose were determined as 3500 and 3600 mg/kg b.wt., respectively. One-tenth of the minimum lethal dose at which a minimum 50% of the animal group was mortal (LD-50) was selected as the maximum dose for the evaluation of pharmacological activity of the extracts. [10]

Drug formulations for animal dosage

One-tenth of the minimum lethal dose, i.e., 350 mg/kg b.wt., p.o., was selected as the therapeutic dose for the evaluation of hepatoprotective activity. Drug was prepared in gum tragacanth (1% w/v) in distilled water.

Evaluation of hepatoprotective activity

Animals were divided into four groups with six rats in each group. The animals of group I received the vehicle gum tragacanth (1 ml/kg/day; 1% w/v) and served as control. Carbon tetrachloride with olive oil (1:1) was administered to all the animals of groups II-V in a dose of 0.1 ml/kg/day, intraperitoneally for 14 days. [11] The group III animals treated with the standard drug silymarin (Ranbaxy Lab, Dewas, India; 100 mg/kg/day, p.o.) served as standard. Animals of group IV received methanolic extract (350 mg/kg/day, p.o.). The drugs were administered concomitantly for 14 days. Animals of all the groups were sacrificed on 14 th day under light ether anesthesia. The blood sample of each animal was collected separately by carotid bleeding into sterilized dry centrifuge tubes and allowed to coagulate for 30 minutes at 37ºC. The clear serum was separated by centrifugation at 2500 rpm for 10 minutes and subjected to biochemical investigations for liver function parameters including total bilirubin, total protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP). [12]

Statistical analysis

Results of biochemical estimations were expressed as mean ± SEM of all six animals in each group under study. The statistical analysis was carried out using one-way analysis of variance (ANOVA) on the data, followed by Dunnett's t-test. The difference in values at P ? 0.01 was considered as statistically significant.

Histopathologic study

The liver was excised from all the six animals of each group separately after draining the blood and was washed using normal saline. Initially, the liver of each animal was fixed in 10% buffered neutral formalin for 48 hours. Further, the liver of each animal was processed by paraffin embedding. Sections of 5 ΅m thickness were taken, processed in alcohol-xylene series and stained with alum-hematoxylin and eosin. The sections were examined microscopically for the evaluation of histopathologic changes. [13],[14]


   Results Top


The qualitative phytochemical analysis of methanolic extract of D. cinerea showed the presence of alkaloids, sterols, triterpenes, glycosides, tannins and proteins. Methanolic extract did not show any sign or symptom of toxicity and mortality up to a dose of 3500 mg/kg.

Evaluation of hepatoprotective activity

After 14 days treatment, biochemical analysis of blood samples of CCl 4 treated animals showed significant increase in the levels of total bilirubin (6.22-fold), AST (5.71-fold), ALT (4.22-fold) and ALP (3.47-fold), as compared to the controls. In addition, the total protein level (44.86%) was decreased, reflecting the liver injury due to the toxic effect of CCl 4 . The blood samples of the animals treated with methanolic extract showed significant reduction in the levels of liver function serum markers. The effect was more pronounced in the animals treated with methanol extract, thus proving the significant hepatoprotective action of the extract [Table 1].
Table 1: Hepatoprotective activity of the methanolic extract of the D. cinerea leaves

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Histopathologic observations

The histopathologic profile of control animals showed a normal architecture of hepatocytes [[Figure 1]a]. The histological observations of liver sections of animals treated with CCl 4 revealed intense centrilobular necrosis, vacuolization and macrovesicular fatty changes [[Figure 1]b]. The liver sections of silymarin treated animals showed normal tissue architecture [[Figure 1]c]. Normal hepatic cords with moderate fatty infiltration was observed in the liver sections of methanolic extract treated animals, demonstrating significant liver protection against CCl 4 -induced liver damage, as is evident by the presence of normal hepatic cords, absence of necrosis and fatty infiltration with reformation of tissue architecture [[Figure 1]d].
Figure 1: (a) Histology of the liver sections of control animals (Group I) showed normal hepatic cells with well-preserved cytoplasm, prominent nucleus, nucleolus and visible central veins; (b) the liver sections of CCl4-intoxicated rats exhibited intense centrilobular necrosis, vacuolization, macrovesicular fatty changes showing massive fatty accumulation in the hepatocytes, and broad infi ltration of the lymphocytes and the loss of cellular boundaries; (c) the histopathologic observations of liver tissue section of silymarin treated group showed regeneration of cells with compact arrangement and lack of fatty lobulation; (d) the histological architecture of liver sections of the rats treated with methanol extract of D. cinerea exhibited significant liver protection against CCl4 injury as evident by the presence of normal hepatic cords, absence of necrosis, fatty infi ltration

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   Discussion Top


Medicinal plants are an integral component of research and development in pharmaceuticals. Furthermore, an increasing reliance on the use of medicinal plants in the industrialized societies has been traced to the extraction and development of several drugs from plants as well as from traditionally used rural herbal remedies. The present investigation revealed the hepatoprotective efficacy methanolic extract of the D. cinerea leaves in CCl 4 -induced hepatic damage in experimental rat models. It is well established that CCl 4 induces hepatotoxicity by metabolic activation. Therefore, it selectively causes toxicity in liver cells, maintaining semi-normal metabolic function. [15] CCl 4 is bio-transformed by the cytochrome P450 system in the endoplasmic reticulum to produce trichloromethyl free radical (•CCl 3 ). Trichloromethyl free radical, when combined with cellular lipids and proteins in the presence of oxygen, forms trichloromethyl peroxyl radical which may degrade lipids on the membrane of endoplasmic reticulum, and thus elicits lipid peroxidation and finally results in cell death. [16] In this present study, it was observed that the administration of CCl 4 decreased the levels of total protein and increased the levels of marker enzymes. The estimation of total bilirubin depicts the depth of jaundice and the elevated level of liver function serum marker enzymes, viz., ALT, AST and ALP, indicates the intensity of liver damage. Marked elevation in the levels of total bilirubin and marker enzymes in the serum of CCl 4 -intoxicated rats was observed. But measurable decrease of the serum marker levels was observed in the serum of animals treated with methanolic extract. The protection against the injurious effects of CCl 4, which may result from the interference with cytochrome P450, leads to the hindrance of the formation of hepatotoxic free radicals. The site-specific oxidative damage in some susceptible amino acids of proteins may be regarded as the major cause of metabolic dysfunction of liver during pathogenesis (Uday et al., 1999). The restoration of bilirubin levels may be due to the inhibitory effects on cytochrome P450 or/and promotion of its glucuronidation. [17] The attainment of these marker enzymes toward a near-normalcy in the animals treated with methanol extract proved the hepatoprotective effect of the extract.


   Conclusion Top


It can be said that the methanolic extract of D. cinerea has afforded protection against CCl 4 -induced liver damage. The mechanism by means of which the extract may protect the liver from CCl 4 toxicity or may act as a free radical scavenger intercepting those radicals involved in CCl 4 metabolism is by its action on microsomal enzymes. By trapping oxygen-related free radicals, the compound would hinder their interaction with polyunsaturated fatty acids and abolish the enhancement of lipid peroxidative processes. The present investigation also supports the ethnomedical uses of D. cinerea and encourages further research on this plant.


   Acknowledgment Top


The author is grateful to the Principal and Management, National Pharmacy College, Shivomogga, for extending laboratory facilities to carry out animal experiment. The author is also grateful to Management, Arogyadhama Institute of Clinical Research, Shankaraghatta, for extending the support to carry out biochemical analysis for this work.

 
   References Top

1.Igoli JO, Igwue IC, Igoli NP. Traditional medicinal practices among the Igede people of Nigeria. J Herbs Spices Med Plants 2004;10:1049.   Back to cited text no. 1
    
2.Bako SP, Bakfur MJ, John I, Bala EI. Ethnomedical and phytochemical profile of some savanna plant species in Nigeria. Int J Bot 2005;1:147-50.  Back to cited text no. 2
    
3.Banso A, Adeyemo SO. Evaluation of antibacterial properties of tannins isolated from Dichrostachys cinerea. Afr J Biotechnol 2007;6:1785-7.   Back to cited text no. 3
    
4.Jain R, Saxena U. Aliphatics and triterpenoid's from the heartwood of Dichrostachys cinerea. J Indian Chem Soc 2003;80:656-8.  Back to cited text no. 4
    
5.Harborne JB. Phytochemical methods: A guide to modern techniques of plant analysis. London, England: Chapman and Hall; 1984.  Back to cited text no. 5
    
6.Evans W. Trease and Evans, Pharmacognosy. Harcourt Brace and Company, 1989.  Back to cited text no. 6
    
7.Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 23 rd ed. Pune: Nirali Prakashan; 1998. p. 106-14.   Back to cited text no. 7
    
8.Dietrich Lorke. A new approach to practical acute toxicity testing. Arch Toxicol 1983;54:275-87.   Back to cited text no. 8
    
9.Ghosh MN. Fundamentals of Experimental Pharmacology. 2 nd ed. Kolkata: Scientific Book Agency; 1984. p. 154.  Back to cited text no. 9
    
10.Jalalpure SS, Patil MB, Pai A, Shah BN, Salahuddin MD. Antidiabetic activity of Cassia auriculata seeds in alloxan-induced diabetic rats. Nig J Nat Prod Med 2004;8:22-3.  Back to cited text no. 10
    
11.Mankani KL, Krishna V, Manjunatha BK, Vidya SM, Jagadeesh SD, Manohara YN, et al. Evaluation of Hepatoprotective activity of stem bark of Pterocarpus marsupium Roxb. Indian J Pharmacol 2005;37:165-8.  Back to cited text no. 11
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12.Kumaravelu P, Subramaniyam S, Dakshinamoorthy DP, Devaraj NS. The antioxidant effect of eugenol on CCl 4 -induced erythrocyte damage in rats. J Nutr Biochem 1995;7:23-8.  Back to cited text no. 12
    
13.Saraswath B, Visen PK, Patnaik GK, Dhawan BN. Anticholestic effect of picroliv, active hepatoprotective principle of Picrorhiza kurrooa, against carbon tetrachloride induced cholestatis. Indian J Exp Biol 1993;31:316-8.  Back to cited text no. 13
    
14.Singh B, Chandan BK, Prabhakar A, Taneja SC, Singh J, Qazi GN. Chemistry and hepatoprotective activity of an active fraction from Barleria prionitis Linn., in experimental animals. Phytother Res 2005;19:391-404   Back to cited text no. 14
    
15.Mujumddar AK, Upadhye AS, Pradhan AM. Effect of Azadtrachta indica leaf extract on CCL 4 Induced hepatic damage in albino rats. Indian J Pharmacol Sci 1998;60:363.  Back to cited text no. 15
    
16.Opoku AR, Ndlovu IM, Terblanche SE, Hutchings AH. In vivo hepatoprotective effects of Rhoicissus tridentata sub sp. cuneifolia, a traditional Zulu medicinal plant, against CCl 4 -induced acute liver injury in rats. S Afr J Bot 2007;73:372-7.  Back to cited text no. 16
    
17.Cavin C, Mace K, Offord EA, Schilter B. Protective effects of coffee diterpenes against aflatoxin B1-induced genotoxicity: Mechanisms in rat and human cells. Food Chem Toxicol 2001;39:549-56.  Back to cited text no. 17
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