Ameliorative effect of methanol extract of Rumex vesicarius on CCl₄-induced liver damage in Wistar albino rats

Abstract

Context: Rumex vesicarius L. (Polygonaceae), an edible plant, is reported to have many bioactive phytochemicals, especially flavonoids and anthraquinones with antioxidant and detoxifying properties.

Objective: This study evaluated the methanolic extract of R. vasicarius (MERV) for hepatoprotective activity in rats against CCl₄-induced liver damage.

Materials and methods: The whole plant extract was prepared and investigated for its hepatoprotective activity. Rats were pretreated with MERV (100 and 200 mg/kg, p.o.) for 7 d prior to the induction of liver damage by CCl₄. Animals were then sacrificed 24 h after CCl₄ administration for the biochemical (AST, ALT, and ALP activity in serum; lipid peroxidation (LPO) and glutathione (GSH) levels in liver tissue) and histological analyses.

Results: CCl₄-induced hepatotoxicity was confirmed by an increase (p < 0.05) in serum AST (4.55-fold), ALT (3.51-fold), and ALP (1.82-fold) activities. CCl₄-induced hepatotoxicity was also manifested by an increase (p < 0.05) in LPO (3.88-fold) and depletion of reduced glutathione (3.14-fold) activity in liver tissue. The multiple dose MERV administration at 200 mg/kg showed promising hepatoprotective activity as evident from significant decrease levels of serum AST (230.01 ± 13.21), serum ALT (82.15 ± 5.01), serum ALP (504.75 ± 19.72), hepatic LPO (3.38 ± 0.33), and increased levels of hepatic glutathione (0.34 ± 0.04) towards near normal. Further, biochemical results were confirmed by histopathological changes as compared with CCl₄-intoxicated rats.

Discussion and conclusion: The results obtained from this study indicate hepatoprotective activity of Rumex plant against CCl₄-induced liver toxicity; hence, it can be used as a hepatoprotective agent.

• Antioxidant
• aspartate transaminase
• hepatotoxicity
• liver damage

Introduction

The liver is a vital organ and plays a very important role in the maintenance of metabolic function and detoxification from the exogenous and endogenous challenges, such as xenobiotic, drugs, viral infection, and chronic alcoholism. Every year about 20 000 deaths are due to liver disorders (Sharma & Sharma, 2010). Liver damage is always associated with cellular necrosis, increase in tissue liquid peroxidation, and depletion in the glutathione (GSH) levels in tissue. In addition, serum levels of many biochemical markers such as AST, ALT, ALP, and billirubin are elevated (Mascolo et al., 1998; Mossa et al., 1991). Drug-induced liver injury is one of the most common causative factors that poses a major clinical and regulatory challenge (Russmann et al., 2009).

Recently there has been a large volume of work aimed at scientific validation of the efficacy of herbal drugs used in the traditional medicine. Modern medicine does not have suitable answers for many health conditions such as liver disorder, asthma, cardiovascular disorder, etc. Many active plant extracts are frequently utilized to treat a wide variety of clinical diseases including liver disease (Chattopadhyay, 2003). Therefore, searching for effective and safe drugs for liver disorders continues to be an area of interest. In our field survey, we found that a widely grown plant Rumex vesicarius L. (Polygonaceae), an annual herb, is a wild edible plant eaten fresh or cooked (Mostafa et al., 2011). It grows throughout desert and semi-desert areas of North Africa, Asia, and Australia (Rechinger, 1984). In Saudi Arabia, R. vesicarius is widely used as food, as a medicinal herb, and as an antidote to scorpion stings (Al-Yahya et al., 1990). The leaves are used as asperient, diuretic, and considered as an antidote for snake venom; the seeds are considered as an antidote for scorpion venom (Joshi, 2000). Antibacterial and antioxidant activities of R. vesicarius have been performed (Mostafa et al., 2011; Panduraju et al., 2009). Despite its importance, only few studies have been conducted on R. vesicarius. However, there are no scientific bases or reports in the modern literature regarding its usefulness as a hepatoprotective agent. Thus, the present study was conducted to evaluate the hepatoprotective activity of the methanol extract of R. vesicarius (MERV) by using CCl₄-induced hepatic damage in male Wistar albino rats.

Materials and methods

Chemicals

All the chemicals and solvents used were of highest purity, commercially available, and analytical grade.

Plant material

Whole plants of R. vesicarius were collected in the Spring of 2012 from the Al-Kharj area, Kingdom of Saudi Arabia. The plants were cleaned, dried under shade, ground to a coarse powder, and stored in an air-tight container at 25 °C for further use. Plant specimens were botanically identified and authenticated by expert taxonomist Mr. Osman Ali Elmakki. A voucher specimen was deposited at the herbarium of the Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

Extraction

Air-dried and fine powder of R. vesicarius was subjected to extraction with methanol by using the percolation method, three times, with an interval of 24 h each. The container with its contents was sealed and kept for a period of 4 d accompanying occasional shaking and stirring. The extract was filtered in the Buchner funnel and the filtrate was concentrated with a rotary evaporator at bath temperature not exceeding 40 °C to have a gummy concentrate of greenish black extract (yield approx. 16.55%).

Animals

Eight week old adult male Wistar rats (150–200 g) were obtained from the Animal House Facility of the College of Pharmacy, Salman Bin Abdulaziz University, Al-Kharj and were housed in a ventilated room at 25 ± 2 °C under a 12-h light/dark cycle. The animals were acclimatized for 1 week before the study and had free access to standard laboratory feed and water ad libitum.

Hepatoprotective activity

Rats were randomly divided into six groups (N = 6 animals/group). Groups I (normal control) and II (toxic control) were given normal saline orally (2 ml/kg) for 7 d. Groups III, IV, and V were treated with R. vesicarius in a dose of 100, 200, and 200 mg/kg body weight, respectively, orally for 7 d. Group VI was administered with silymarin (100 mg/kg) for 7 d. On the 7th day, 30-min post-dose of normal saline, extract, and silymarin administration, animals of groups II, III, IV, and VI received CCl₄ at a dose of 1 ml/kg (1:1 v/v of CCl₄ in olive oil) orally. Group V was not treated with CCl₄, and serves as the Rumex per se group. On the 8th day, after an overnight fasting, the rats were anesthetized with ether and blood samples were collected in tubes for biochemical analysis by the cardiac puncture method. Blood samples were centrifuged for 10 min at 3000 rpm to separate the serum. After blood collection, the animals were sacrificed using ether anesthesia and the liver tissue was harvested for biochemical and histopathological studies.

Serum AST and ALT levels were determined by the method of Reitman and Frankel (1957). Each substrate (0.5 ml) (2 mM α-ketoglutarate and either 200 mM α-l-alanine or l-aspartate) was incubated for 5 min at 37 °C in a water bath. Serum (0.1 ml) was then added and the volume was adjusted to 1.0 ml with sodium phosphate buffer. The reaction mixture was incubated for exactly 30 min and 60 min for ALT and AST, respectively. Then, to the reaction mixture, 0.5 ml of DNPH (1 mM) was added and left for another 30 min at room temperature. Finally, the color was developed by the addition of 5.0 ml of NaOH (0.4 N) and the product was read at 505 nm. Serum alkaline phosphatase was estimated following the method of Bessey et al. (1976).

Liver tissue (1 g) was homogenized in nine volumes of phosphate buffer and centrifuged at 12 000 × g for 30 min at 4 °C. The supernatant was collected and used for the estimation of lipid peroxidation (LPO) (Esterbauer & Cheeseman, 1990) and GSH (Jollow et al., 1974).

The liver samples from different groups were preserved in 10% buffered formalin and processed for routine paraffin block preparation. Sections of a thickness of about 5 μm were cut and stained with hematoxylin and eosin. Then, the samples were observed under microscope for degeneration, fatty changes, necrotic changes, and evidence of hepatotoxicity if any, by an experienced histopathologist.

Statistical analysis

All values were expressed as mean ± SE (n = 6 in each group). One-way ANOVA was applied to test for the significance of biochemical data of the different groups. Significance is set at p < 0.05.

Results

Administration of CCl₄ (1 ml/kg, p.o.) induced a marked increase in the serum hepatic enzyme levels, AST, ALT and ALP, as compared with normal controls indicating liver damage. Pretreatment of the rats with MERV for 7 d prior to CCl₄ administration caused a significant reduction in the levels of AST, ALT, and ALP in serum (p < 0.01), which were almost comparable with silymarin (Table 1).

Table 1. Effect of the methanolic extract of R. vesicarius on AST, ALT, and ALP levels in serum.

Treatment	AST (IU/l)	ALT (IU/l)	ALP (IU/l)
I (normal control)	146.50 ± 7.57	62.08 ± 1.96	386.25 ± 11.55
II (CCl4 only)	667.50 ± 19.89*	217.40 ± 18.66*	702.25 ± 27.80*
III (MERV-100 mg/kg + CCl4)	275.25 ± 15.04#	110.60 ± 10.65#	536.75 ± 15.32#
IV(MERV-200 mg/kg + CCl4)	230.01 ± 13.21#	82.15 ± 5.01#	504.75 ± 19.72#
V (MERV-200 mg/kg)	139.54 ± 9.62	57.92 ± 2.13	378.74 ± 21.28
VI (silymarin-100 mg/kg + CCl4)	225.75 ± 17.15#	80.70 ± 3.68#	465.75 ± 8.91#

AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase.

*p < 0.01 as compared with the normal control group.

#p < 0.01 as compared with the CCl₄ only treated group.

Table 2 shows that administration of CCl₄ results in a marked increased in LPO levels and a decrease in the GSH level in liver homogenate. The toxic effect of CCl₄ was controlled in the animals treated with MERV (100 and 200 mg/kg, p.o.) by restorating LPO and GSH levels in liver.

Table 2. Effect of the methanolic extract of R. vesicarius on LPO and GSH levels in liver.

Treatment	LPO (nmol of MDA formed/ min/g tissue)	GSH (µmoles/g tissue)
I (normal control)	1.91 ± 0.06	0.22 ± 0.02
II (CCl4 only)	7.41 ± 0.30*	0.07 ± 0.01*
III (MERV-100 mg/kg + CCl4)	3.61 ± 0.38#	0.30 ± 0.02#
IV (MERV-200 mg/kg + CCl4)	3.38 ± 0.33#	0.34 ± 0.04#
V (MERV-200 mg/kg)	1.94 ± 0.13	0.23 ± 0.02
VI (silymarin-100 mg/kg + CCl4)	3.12 ± 0.22#	0.35 ± 0.05#

LPO, lipid peroxidation; GSH, total glutathione.

*p < 0.01 as compared with the normal control group.

#p < 0.01 as compared with the CCl₄ only treated group.

Histological studies of the liver tissue of control and treated animals also confirmed the hepatoprotective effect of the MERV. The histological architecture of liver sections of normal rats showed normal cellular architecture with distinct hepatic cells and sinusoidal space (Figure 1). In the liver section of rats intoxicated with CCl₄, there was disarrangement and degeneration of normal hepatic cells with intense centrilobular necrosis, cloudy swelling, and fatty degeneration of hepatocytes (Figure 2). The histopathological profile of the rats treated with MERV showed no visible changes, confirming the safety of the extract at selected dose (Figures 3 and 4). Rats treated with MERV only in a dose of 200 mg/kg showed normal pattern of histological architecture (Figure 5). While silymarin treatment in CCl₄-intoxicated rats showed less disarrangement and degeneration of hepatocytes, indicating marked regeneration activity (Figure 6).

Figure 1. Normal control group rat liver section showing normal cellular architecture with distinct hepatic cells and sinusoidal space.

Figure 2. Toxic control group rat liver section showing disarrangement and degeneration of normal hepatic cells with intense centrilobular necrosis.

Figure 3. CCl₄ + MERV-100 mg/kg-treated group rat liver section showing moderate hepatoprotective activity.

Figure 4. CCl₄ + MERV-200 mg/kg-treated group rat liver section showing typical lobular arrangement.

Figure 5. MERV per se group rat liver section showing normal pattern of histological architecture.

Figure 6. CCl₄ + silymarin-100 mg/kg-treated group rat liver section showing less disarrangement and degeneration of hepatocytes.

Discussion

In general, CCl₄ has been extensively used as a model to study hepatic damage and used as an indicator of protective activity of newly discovered drugs (Clawson, 1989). In the present study, CCl₄ was used as a toxic agent and the hepatoprotective effect of MERV was studied against the CCl₄-induced hepatotoxicity. The extent of toxicity was estimated by biochemical markers like AST, ALT, and ALP levels in serum; LPO and GSH levels in liver; and histopathological studies of liver tissues. The present study reports the potential hepatoprotective activity of rumex against hepatic injury produced by CCl₄ in Wistar albino rats.

It is well known that CCl₄ is biotransformed by the Cyt P450 system to produce a trichloromethyl-free radical, which further undergoes reduction to form a trichloromethylperoxyl radical, which leads to LPO and finally leads to cell death (Clawson, 1989; Recknagel et al., 1989). Therefore, leakage of large quantities of enzymes into the blood stream is often associated with massive necrosis of the liver (Ha et al., 2005; Rees & Spector, 1961). Our study corroborates previous findings and elicited a significant increase in the activities of serum enzymes (AST, ALT, and ALP in serum); hepatic LPO levels and decreased levels of antioxidant marker (GSH) on exposure of rats to CCl₄, indicating considerable hepatocellular injury (Ha et al., 2005; Rawat et al., 1997). GSH is an important endogenous antioxidant system, known to have key functions in protective mechanism. Excessive production of CCl₃ radical results in the oxidation of reduced glutathione GSSG and, therefore, the GSH levels in CCl₄-treated groups are decreased as compared with the normal group.

Treatment with MERV reversed the changes produced by CCl₄. MERV significantly decreased hepatic LPO levels and increased the levels of above-mentioned biochemical markers to near normal levels. Pretreatment with MERV also increased the levels of GSH. Hence, the hepatoprotective effect of MERV may be due to the prevention of depletion in the GSH levels in tissue.

As expected, silymarin maintained normal architecture with minimal injuries and better protection than Rumex extract alone. Chronic treatment with silymarin also illustrated marked recovery in serum enzyme levels and oxidative parameters in liver tissue. Photomicrograph clearly reveals that the MERV works as a hepatoprotectant. The histopathological profile reveals a major damage in the toxic control group. Thus, it clearly states that, toxicity is due to either of the above mechanisms such as depletion of glutathione store or free radical generation or LPO. Simultaneous treatment of the extract and silymarin with CCl₄ exhibits less damage to the hepatic cells as compared to the rats treated with the toxic group.

The MERV showed a significant result. Previous reports show flavonoids and steroids are responsible for hepatoprotective effect (Khatri et al., 2009; Panigrahi et al., 2010; Pattanayak et al., 2011; Singh & Gupta, 2011). The plant contains many bioactive substances such as flavonoids (vitexin, isovitexin, orientin, and isorientin), anthraquinones, particularly in roots (emodin and chrysophanol), carotenoids, vitamins (especially vitamin C), proteins, lipids, and organic acids. Flavonoids present in the plant may be responsible for the marked hepatoprotective effects, as observed in the present study.

Conclusion

With the aid of enzyme levels and histopathological studies of rat liver, we can conclude that MERV has potent hepatoprotective action against CCl₄-induced hepatic damage in rats. The present study, thus, justifies the traditional use of R. vesicarius in the treatment of liver diseases and also points out that R. vesicarius warrants future detailed investigation as a promising hepatoprotective agent. However, the exact mechanism(s) and the active compound(s) involved in these effects are still not clear and need to be clarified in future studies.

Declaration of interest

The authors declare that they have no conflict of interest. The authors are thankful to Deanship of Scientific Research, Salman bin Abdulaziz University, Al-Kharj Saudi Arabia, for providing the funds to carry out this study under research Grants no. 4/H/1432.