Bangladesh J Pharmacol. 2016; 11: 248-256

Available Online: 28 January 2016

DOI: 10.3329/bjp.v11i1.25289

Hepatoprotective effect of Monotheca buxifolia fruit against antitubercular drugs-induced hepatotoxicity in rats

Irfan Ullah¹, Jamshaid Ali Khan¹, Achyut Adhikari² and Muhammad Shahid¹

¹Department of Pharmacy, University of Peshawar, Peshawar, Pakistan; ²HEJ Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan.

Abstract

The present study investigates the hepatoprotective potential of Monotheca buxifolia fruit hydro-ethanolic extract, against isoniazid- and rifampicin- induced hepatotoxicity in rats. Phytochemical investigations lead to the isolation of oleanolic acid and isoquercetin. Pretreatment with M. buxifolia extract at doses of 150 and 300 mg/kg for 21 days, restored the isoniazid- and rifampicin-induced elevation of serum levels of alanine aminotransferase (p<0.001), aspartate aminotransferase (p<0.001 and p<0.05), alkaline phospha-tase (p<0.001), billirubin (p<0.001) and total proteins (p<0.001) as well as afforded significant protection against histopathological changes in the liver. From these results, it is conceivable that M. buxifolia exhibited selective protective effect against isoniazid- and rifampicin-induced hepatotoxicity, mediated through the presence of oleanolic acid and isoquercetin.

Introduction

Monotheca buxifolia (Falc.) is a member of the genus Monotheca and belongs to family Sapotaceae. Several members of this family including Bassia latifolia (Sheikh et al.), Chrysophyllum albidum (Adebayo et al., 2011), and Madhuca longifolia (Kumar et al., 2013) have been evaluated for hepatoprotective potential in various experimental models. Forlklorically, M. buxifolia fruit is used as hematinic, laxative, purgative, vermicidal, anti-pyretic and in the management of gastro-urinary disorders (Marwat et al., 2011; Rehman, 2013; Shah, 2013; Ullah et al., 2010). M. buxifolia contains flavonoids, terpenoids, cardiac glycosides, anthraquinones, saponins, reducing sugars, tannins and polyphenolic compounds. Flavonoids and polyphenolic compounds possess potent antioxidant and hepatoprotective potential. Previously in vitro anti-oxidant potential of its fruit has been investigated (Jan et al., 2013).

Keeping in view the strong in vitro anti-oxidant potential of M. buxifolia, the current study further investigated the hyrdo-ethanolic extract of M. buxifolia fruit for its hepatoprotective potential against isoniazid- and rifampicin-induced hepatotoxicity in rats. The extract was also subjected to phytochemical isolation for its active compounds.

Materials and Methods

Plant material

Fruit of M. buxifolia was collected from the northern areas of Pakistan in the month of August and authenticated by a taxonomist at the Department of Botany, University of Peshawar. A specimen was deposited in the herbarium of University of Peshawar for further reference (Bot. 20061-PUP).

Animals

Male Sprague Dawley rats (200 ± 20 g) were kept at standard conditions i.e. temperature 25 ± 2°C and 12 hours dark/light cycle throughout the experiment. The animals were fed with standard diet and tap water ad libitum.

Chemicals and drugs

Isoniazid and rifampicin were obtained from Schazo-Zaka while silymarin from Medicraft Pharmaceuticals. Formalin, xylene and normal phase silica were purchased from Merck, Germany. Serum diagnostic kits for biochemical assays were obtained from Chema Diagnostica (Italy). All solvents used were of analytical grade.

Preparation of extract

Fruit of M. buxifolia were collected and washed with distilled water to remove dust. Seeds were separated and fruit pulp was dried under shade in a well-ventilated place at ambient temperature. The dried pulp was crushed to powder, subjected for extraction with hydro-ethanolic (30:70) solvent, shaken occasionally for 15 days and filtered through Whatman No. 1 filter paper. The solvent was evaporated under reduced pressure in a rotary evaporator (BUCHI Rotavapor R-200, Switzerland) at 40°C. The semisolid mass (M. buxifolia hydro-ethanolic extract) was kept in refrigerator.

Gross phytochemical investigation

M. buxifolia extract was screened for glycosides (Okunlola et al., 2007), triterpenoids (Nayak and Pereira, 2006), tannins, flavonoids, saponins (Sofowora, 1996) and alkaloids (Nayak and Pereira, 2006; Oyedapo et al., 1999).

Isoniazid- and rifampicin-induced toxicity

Suspension of isoniazid and rifampicin were separately prepared and given to rats in dose of 50 mg/kg each for 21 days via oral gavage tube (Pal et al., 2006).

Experimental design

Animals were divided into five groups (n = 6) in following manner: Group 1: Saline as control, Group 2: Isoniazid plus rifampicin 50 mg/kg each, Group 3: M. buxifolia extract (150 mg/kg) one hour before isoniazid plus rifampicin (50 mg/kg each), Group 4: M. buxifolia extract (300 mg/kg) one hour before isoniazid plus rifampicin (50 mg/kg each), Group 5: (silymarin 100 mg/kg one hour before isoniazid plus rifampicin (50 mg/kg each).

Blood collection and serum preservation

At the end of experiment, animals were anesthetized with ketamine (i.p, 100 mg/kg). Blood was collected through cardiac puncture and immediately transferred to evacuated gel and clot activator centrifuge tubes (AST Diagnostics). Serum was separated by centrifugation at 3,000 rpm for 15 min (Centurion Scientific LTD, UK).

Biochemical assays

Serum was assayed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), billirubin and total proteins (TP) according to the standard diagnostic kits protocol supplied by the manufacturer. The analysis was performed using double beam UV/Visible spectrometer (Lambda 25, Perkin Elmer, USA).

Histology

After 21 days of treatment, liver of each animal was excised and fixed in 10% neutrally buffered formalin for 48 hours. The tissues were dehydrated in graded ethanol solutions (50, 70, 80, 90, two changes each of 100%), cleared in two changes each of 100% xylene, infiltrated and embedded in paraffin wax. Tissue blocks were sectioned at 4 μm through a rotary microtome (SLEE Mainz CUT 5062, Germany), stained with Harris hematoxylin and eosin for microscopic observation (Labomed Lx400 with digital camera iVu 3100, USA). Histopathological changes were scored as none (–), mild (+), moderate (++), or severe (+++) damage.

Isolation and structure elucidation of compounds

M. buxifolia extract (2.304 kg) was mixed with 2.5 L distilled water and soaked overnight, extracted thrice with 5 L each of n-hexanes, chloroform, ethyl acetate, and n-butanol to get n-hexane soluble (56 g), chloroform soluble (57.7 g), ethylacetate soluble (34.9 g) and n-butanol soluble (54.5 g) fractions respectively. Ethyl-acetate fraction (30 g) was subjected to vacuum liquid chromatography on normal phase silica gel and eluted using hexane, hexane-ethyl acetate, ethyl acetate, ethyl acetate-methanol and methanol with increasing polarity to yield 18 fractions (Fr. 1-18). Compound 1 (13 mg) was obtained from fraction 2 (1.2 g) through repeated column chromatography using hexanes-ethyl acetate (9:1–8:2) while fraction 15 (1.4 g) was re-chromatographed on silica gel column using ethyl acetate-hexanes (8:2) to get compound 2 (15 mg). Purity of the compounds was assessed using TLC followed by spraying with ceric sulfate and heating. Structures were elucidated using various techniques such as ¹H-NMR, ¹³C-NMR, 2D-NMR, EI-MS, FAB-MS, UV, and IR. All the data of the compounds were unambiguously matched with reported data from literature (Eldahshan, 2011; Seo et al., 1981).

Statistical analysis

Data were expressed as mean ± SD. Statistical analysis was done by one-way ANOVA followed by Tukey’s post hoc test using Graph pad Prism 5 (Graph pad Software Inc. San Diego CA, USA).

Results

Phytochemical analysis

The gross phytochemical analysis of hexane extract of M. buxifolia showed the presence of glycosides, triterpenoids, saponins, tannins, flavonoids, reducing sugars and alkaloids (Table I). Further chromatographic separation along with different analytical techniques lead to isolation of 2 compounds, namely oleanolic acid as compound 1 and isoquercetin as compound 2.

Hepatoprotective activity

Biochemical analysis

As shown in Table II, treatment with isoniazid plus rifampicin (50 mg/kg each) for 21 days significantly raised (p<0.001) the serum levels of ALT, AST, ALP, billirubin and TP as compared to saline treated animals. Pretreatment with M. buxifolia extract at 100 mg/kg (Group 3) restored the serum levels of ALT (p<0.001), AST (p<0.05), ALP (p<0.001), billirubin (p<0.001) and TP (p<0.001) to normal as compared to isoniazid plus rifampicin alone treated animals (Group 2). Likewise, pretreatment with M. buxifolia extract at 300 mg/kg (Group 4) inhibited (p<0.001) the isoniazid plus rifampicin induced elevation of serum ALT, AST, ALP, billirubin and total protein. Moreover, highly significant restorative effect (p<0.001) on serum levels of ALT, AST, ALP, billirubin and total proteins was noted in animals treated with silymarin at a dose of 100 mg/kg (Group 5) as compared to isoniazid plus rifampicin alone treated animals (Group 2).

Histopathological evaluation

After 21 days of treatment with isoniazid plus rifampicin, the central veins of the hepatic lobules were congested with red blood cells and their epithelium were disrupted (Figure 1). The hepatocytes were depleted of glycogen. The sinusoidal spaces were dilated and infiltrated by large number of lymphocytes. Increased numbers of focal aggregations of lymphocytes with necrotic hepatocytes were present around the central vein. Although the liver retained its characteristic lobular appearance, however the hepatocytes appeared necrotic and exhibited ballooning degeneration. Pre-treatment with M. buxifolia extract (150 and 300 mg/kg) or silymarin (100 mg/kg) for 21 days provided significant protection against isoniazid plus rifampicin induced hepatotoxicity as no significant histopathological changes were observed in the liver (Figure 1; Table III).

Discussion

Drug induced hepatotoxicity occurs in a variety of mechanisms such as membrane disruption or cellular necrosis, which may be resulted from binding of drug or its metabolite to cellular proteins, making new adducts that serves as targets for immune system and activates immunological reactions (Ramaiah et al., 2001). Other causes might be the inhibition of drug metabolism pathways. Interruption in the bile flow or disruption of filaments at sub-cellular level of bile duct causes abnormal or disturbed bile secretions, leading to jaundice and minimal cellular injury. Programmed cell death or apoptosis due to tissue necrosis factor or FAS pathways and inhibition of mitochondrial functions lead to accumulation of reactive oxygen species and subsequently lipid peroxidation and cell death (Girling, 1982).

Isoniazid and rifampicin are the first line antitubercular drugs and are used as standard hepatotoxic in various experiments. Administration of isoniazid and rifampicin causes changes in both morphology and cellular function of liver. In the current study, Sprague dawly rats were given isoniazid plus rifampicin (50 mg/kg per day orally for 21 days) to induce hepatotoxicity. Three folds rise in transaminases level in the serum of animals was a biochemical warning of hepatic injury. Isoniazid and rifampicin are potent hepatotoxic drugs shown by various studies but the exact mechanism of hepatotoxicity is still unclear. Isoniazid is converted to acetyl-isoniazid via hepatic N-acetyltransferase-2, which is then hydrolyzed to acetylhydrazine. Acetylhydrazine is oxidized by cytochrome P450 to form certain hepatotoxic intermediates. Hydrazine, either as direct (from isoniaizd) or indirect (from acetyl hydrazine) induces CYP2E1 (Poloyac et al., 2001). The best role of CYP2E1 is the production of reactive oxygen species which is a clear mechanism for the hepatotoxicity caused by isoniaizd (Yue et al., 2004). Rifampicin exaggerate the isoniazid hepatotoxicity possibly by increasing the production of hydrazine or inhibition of bile pathway (Dugasani et al., 2014; Rao et al., 2015). In this study, rifampicin and isoniazid significantly increased the serum levels of ALT, AST, ALP and billirubin while it decreased the level of TP. However, treatment with M. buxifolia extract at doses of 150 and 300 mg/kg significantly decreased the isoniazid and rifampicin induced elevated serum levels of ALT, AST, ALP, billirubin and total protein, and this protective effect was comparable to the standard hepatoprotective drug silymarin, thus proving the hepatoprotective effect of M. buxifolia. The biochemical investigation was corroborated by histopathological findings which showed that there are certain morphological changes which are typical of rifampicin and isoniazid induced hepatotoxicity. Treatment with M. buxifolia at both doses (150 and 300 mg/kg) significantly inhibited these morphological changes. These observations strongly supported the hepatoprotective potential of M. buxifolia fruit against isoniazid and rifampicin induced hepatotoxicity in rats.

M. buxifolia extract was fractioned and eluted through column chromatography. Two compounds were isolated which are characterized as isoquercetin and oleanolic acid. Isoquercetin is a potent anti-oxidant and is responsible for free radical scavenging activity while oleanolic acid is proved to regenerate glutathione and inhibit the induction of CYP2E1 (Delnavazi, 2015; Jeong, 1999; Yim et al., 2001). Due to the presence of these compounds it might be suggested that the possible mechanism of M. buxifolia fruit for the hepatoprotective effect is the glutathione regeneration and reduction of oxidative stress by preventing the induction of CYP2E1 and scavenging free radicals.

Conclusion

The hydroethanolic extract of M. buxifolia fruit possessed potent hepatoprotective activity as demonstrated by significant amelioration of isoniazid- and rifampicin- induced biochemical and histopathological changes in liver. The significant hepatoprotective activity of M. buxifolia might be due to the presence of isoquercetin and oleanolic acid.

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