Background and Objective: Environmental pollutants and toxic chemicals are widely spread and can cause hepatotoxicity. In developing countries plant extracts are the main source for treatment of many ailments. The objective of this study is to investigate the protective effect of methanolic extracts of Prosopis farcta and Lycium shawii against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Material and Methods: The two plants were collected, identified and extracts were prepared. Phytochemical analysis for total phenolics, total tannins content, non-tannins phenolics and total flavonoids was performed. Liver damage was induced in rats by repeated administration of CCl4 (30% in olive oil (3 mL kg1 b.wt., i.p.). Liver protection was accomplished in 2 groups of rats by daily oral administration of extract of Prosopis farcta and Lycium shawii (250 mg kg1 b.wt.,) for 15 days prior to CCl4 administration. Liver marker enzymes, serum total bilirubin, malondialdehyde (MDA), reduced glutathione (GSH), glutathione-S-transferase (GST), lipid profile, total antioxidant capacity were determined together with histopathological damage evaluation. Results: The CCl4 treatment increased significantly (p<0.05) liver marker enzymes, total bilirubin, MDA, GST, lipid profile and decreased significantly total serum protein, total antioxidant capacity, high density lipoprotein cholesterol (HDL-C) and GSH. Prosopis farcta and Lycium shawii treatment significantly reversed the CCl4-induced changes, towards normalcy. Conclusion: Liver protection capability of these two plants may be due to their high contents of various flavonoids that act as powerful antioxidants and free radical scavengers.
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Environmental pollutants, toxic chemicals and some drugs can cause cellular injuries through various mechanisms1. Carbon tetrachloride (CCl4) has been used to study liver damage in animal models because it generates lipid peroxides that can cause injuries to the liver and various other organs2,3. Metabolism of CCl4 releases metabolites that can cause tissue injuries particularly in the liver. Exposure to CCl4-induces an increase in free radicals concentrations that are highly reactive and cause injury4.
Plant materials are used since ancient time for treatment of various diseases5-7 . The plant Prosopis farcta (PF) from Leguminosae and sub-family Mimosoideae is indigenous to dry and semi-dry areas of Asia, America and Africa8. Previous studies on this plant have reported some medicinal properties9. Asadollahi et al.10 reported that Prosopis farcta beans extracts have protective effects against induced hepatotoxicity in Wistar rats. However, the mechanism by which Prosopis farcta beans extracts exhibited protection is not clear. Lycium shawii, desert thorn or Arabian boxthorn is a species of thorny shrub adapted to desert environments. Lycium is a genus of flowering plants in the nightshade family, Solanaceae. They are xerophytic plants. Genus Lycium was reported to have some medicinal properties11-14.
Studies on the hepatprotective effects of phytochemicals are scarce. In this study, the role of Prosopis farcta and Lycium shawii in protection against induced hepatotoxicity in Wistar rats was evaluated.
MATERIALS AND METHODS
Animals and diet: Twenty four adult male Wistar rats weighing between 150-180 g were purchased from the College of Pharmacy, King Saud University, Saudi Arabia. Rats were housed in polyacrylate cages. They were provided with standard commercial rat pellets (No. 648-General Organization for Grain Silos and Flour Mills, Riyadh and water ad libitum. All experiments were approved by the Institutional Animal Ethics Committee (IAEC) of Qassim University, Saudi Arabia.
Preparation of plant material: Two plants Lycium shawii and Prosopis farcta were collected at the flowering stage from Qassim region (KSA). The collected plant species were identified and confirmed by the Department of Plant Production and Plant Protection, College of Agriculture and Veterinary Medicine, Qassim University.
Shade dried and powdered plant materials were successively extracted by methanol. The extracts were concentrated until obtaining paste under vacuum. The extract was used for evaluation of phytochemical constituents and for the treatment of rats.
Experiment design: Rats were segregated randomly into 4 groups of 6 rats each:
|Group 1:||Animals of this group (Negative control) were fed standard pellet diet and received water ad libitum.|
|Group 2:||The positive control, received 30% CCl4 in olive oil (3 mL kg1 b.wt., i. p.) after every 72 h for 3 days (3 doses).|
|Group 3:||Animals in this group were given daily an oral aqueous extract dose of Lycium shawii (250 mg kg1 b.wt.) for 2 weeks followed by CCl4 in olive oil (3 mL kg1 b.wt., i.p.) after every 72 h for 3 days (3 doses).|
|Group 4:||Animals in this group were given daily an oral aqueous extracts dose of 250 mg of Prosopis farcta for 2 weeks followed by CCl4 in olive oil (3 mL kg1 b.wt., i.p.) after every 72 h for 3 days (3 doses).|
Treatment with Lycium shawii and Prosopis farcta extracts continued for another 7 days for group 3 and 4, respectively. After that all animals were kept starved for 24 h. On the following day the animals were sacrificed by decapitation and blood was collected from the jugular vein. Serum was collected by centrifugation and stored at -20°C.
Following necropsy, liver specimens were harvested, preserved in 10% neutral buffered formalin and stored.
Determination of total phenolics and total tannins content: Total phenolics and total tannins contents of Prosopis farcta and Lycium shawii were determined as described by Singleton and Rossi Jr.15.
Determination of total flavonoids: The flavonoid content was determined as described by Zhishen et al.16 expressed as mg quercetin equivalents g1 residue.
Determination of malondialdehyde (MDA): Lipid peroxidation was determined in serum according to Ohkawa et al.17.
Determination of glutathione-S-transferase (GST) activity: The GST activity was assayed as described by Habig et al.18.
Evaluation of liver function: The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined spectrophotometrically according to the method of Schumann and Klauke19. While, the activities of alkaline phosphatase (ALP) was carried out according to the method of Tietz20. Total serum bilirubin concentration was estimated using commercially available kits (Boehringer, Mannheim, Germany).
Determination of reduced glutathione: Reduced glutathione was determined according to the method of Moron et al.21.
Determination of serum total protein: Total serum proteins was determined by the method of Bradford22.
Determination of serum lipid profile: Total Cholesterol (TC) was determined according to Allain et al.23, triglycerides (TG) according to Stein and Myers24, high density lipoprotein cholesterol (HDL-C) according to Sugiuchi et al.25. While low density lipoprotein (LDL-C) was calculated by Friedwald et al.26 formula:
Determination of total antioxidant capacity: Total antioxidant capacity was determined according to the method of Koracevic et al.27.
Histopathology: Liver specimens were processed via paraffin embedding technique (dehydration in ethanol, clearing in xylene and impregnation in melted wax). Five micrometers thick sections were prepared and stained with hematoxylin and eosin (H and E) according to Suvarna et al.28. The sections were examined for pathological findings of hepatotoxicity.
Statistical analysis: Statistical analysis was performed using the statistical package (SPSS-16, Chicago, USA). The results were expressed as Mean±Standard Deviation (SD).
The phytochemical contents of Lycium shawii and Prosopis farcta are presented in Table 1. Prosopis farcta contain more total phenolics, tannins, non-tannins phenolics and total flavonoids (271.92, 81.24, 190.68 and 135.0 mg g1), respectively when compared with Lycium shawii (101.7, 47.66, 54.04 and 59.8 mg g1), respectively.
The levels of total protein, total cholesterol, triglycerides, HDL-C, LDL-C in sera of the experimental rats are shown in Table 2. Treatment with CCl4 alone significantly (p<0.05) decreased the level of total protein. Administration of Prosopis farcta and Lycium shawii increased the levels of serum protein.
Table 2 shows the effect of Prosopis farcta and Lycium shawii on the serum lipid profile in CCL4 treated rats. There was a significant (p<0.05) elevation of serum TC, TG and LDL-C with concomitant significant (p<0.05) reduction in the level of HDL-C. Pre-treatment with Prosopis farcta decreased significantly (p<0.05) the level of TC, TG and LDL-C and increased significantly (p<0.05) the level of HDL-C.
While treatment with Lycium shawii resulted in a non-significant decrease in the level of serum TC, TG and LDL-C and a non-significant increase in the level of HDL-C.
The effect of Prosopis farcta and Lycium shawii on liver marker enzymes and bilirubin level in the serum of CCL4 treated rats is shown in Table 3.
|Table 1:||Quantitative analysis of methanolic extracts of Prosopis farcta and Lycium shawii (mg g1)|
|Table 2:||Effect of Prosopis farcta and Lycium shawii on serum total protein, total cholesterol, triglycerides, HDL-C, LDL-C in rats|
All values are expressed as Means±SD. Means on the same column with different letters are significantly different at p<0.05, HDL-C: High density lipoprotein cholestrol, LDL-C: Low density density lipoprotein cholestrol
|Table 3:||Effect of Prosopis farcta and Lycium shawii on liver enzymes and serum bilirubin concentration in rats treated with CCl4|
All values are expressed as Means±SD. Means on the same column with different letters are significantly different at p<0.05, ALP: Alkaline phosphatase, AST: Aspartate amino transferase, ALT: Alanine aminotransferase and CCl4: Carbon tetrachlodie
|Table 4:||Effect of Prosopis fracta and Lycium shawii on serum oxidative stress parameters in rats treated with CCl4|
|All values are expressed as Means±SD. Means on the same column with different letters are significantly different at p<0.05, MDA: Malondialdehyde, GST: Glutathione-S-transferase|
|Fig. 1:||Liver from rats given CCl4 alone (group 2) showing mild fatty change, H and E X100|
The increased activities of ALP, AST and ALT and bilirubin level in the serum of hepatotoxic rats were found to decrease upon treatment with Prosopis farcta and Lycium shawii. It can be observed that treatment with Prosopis farcta was more effective in reducing the hepatic marker enzyme activities and bilirubin compared to Lycium shawii.
The effect of Prosopis fracta and Lycium shawii on serum oxidative stress parameters in rats treated with CCl4 is shown in Table 4. Administration of CCl4 increased significantly (p<0.05) the level of malondialdehyde (MDA) and the activity of GST and reduced significantly (p<0.05) the level of the total antioxidant capacity and reduced glutathione (GSH). Treatment with Prosopis farcta and Lycium shawii resulted in improvement in the level of these parameters .In this study treatment with Prosopis farcta is more effective in improving the studied parameters when compared with Lycium shawii.
Histopathologic findings: The microscopic examination of livers of rats treated with (CCl4) revealed multifocal mild fatty changes characterized by minute sharp cytoplasmic vacuolation of the hepatocytes (Fig. 1).
|Fig. 2(a-b):||Liver from rats given CCl4 alone (group 2) showing small necrotic foci, H and E X160|
Moreover, few minute foci of necrosis were evident (Fig. 2). Upon administration of Prosopis farcta extract (group 4), there was marked improvement of the histopathologic picture, severity and distribution of the degenerative lesions were obviously reduced with disappearance of the necrotic lesions in addition to regenerative trials of the hepatic cells in the form of binucleation and nuclear hypertrophy (Fig. 3).
|Fig. 3(a-b):|| |
Liver from rats given CCl4 alone (group 2) showing nuclear hypertrophy and binucleation of some hepatocytes, H and E X160
Regarding rats that received Lycium shawii extract, both the dengenrative and necrotic changes were considerably diminished as seen in rats of group 3 but without hepatic cell regenerative capabilities.
The present study was carried out to investigate the protective effect of aqueous extract of Prosopis farcta and Lycium shawii against CCl4-induced liver damage in rats.
Few plants were used for the treatment of hepatotoxicity9. Many plants are used as a folk remedy without academic confirmation of its effects. However, Prosopis farcta and Lycium shawii are used for the treatment of various ailments10,29,30.
The present investigation showed that CCl4 administration in rats significantly increased the activities of serum ALT, AST and ALP indicating hepatocyte injuries via altering membrane integrity by covalent binding of CCl4 metabolites (i.e., CCl3, CCl3OO) to membranes. Similar findings, were reported by Weber et al.31, Miyazaki et al.32 and Preethi and Kuttan3.
However, after treatment with Prosopis farcta and Lycium shawii, the observed elevation in ALT, AST and ALP were significantly restored indicating that Prosopis farcta and Lycium shawii have the ability to protect against hepatocytes injuries induced by CCl4. The primary cause for hepatocytes injury is lipid peroxidation. Indications of lipid peroxidation in the present study included increases in the levels of MDA, GST and a decrease in the total antioxidant capacity and GSH in serum. Malondialdehyde (MDA) is an indicator of lipid peroxidation. An increase in free radicals causes over production of MDA. It is well known marker of oxidative stress33. Reduced glutathione (GSH) is a strong scavenger of free Radical Oxygen Species (ROS). The decreased concentration is an indication of GSH utilization in the neutralization of free radicals34. Administration of Prosopis farcta and Lycium shawii restored GSH and MDA levels to those in the control group.
Administration of CCl4 increased serum TC, TG and LDL and decreased the level of serum HDL, GSH and total antioxidant capacity. Administration of Prosopis farcta and Lycium shawii significantly decreased serum TC, TG and LDL-C levels. This indicates that these two plant extracts have lipid-lowering effect and have antioxidant properties.
As presented in Table 1 these plants are very rich in phenolic compounds, mainly tannins and flavonoids that are major group of antioxidants acting efficiently as scavengers of free radicals35. Flavonoids are natural substances with variable phenolic structures. It was found that consumption of flavonoid is associated with a decreased risk of several types of diseases36. Hepatoprotective effects of various flavonoids were directly related to their antioxidant activities and reduction of lipid peroxidation37,38. It can be noticed in Table 2, that both Prosopis farcta and Lycium shawii contain phenolic compounds with strong antioxidant activity. However, the level of these phenolic compounds is higher in Prosopis farcta compared with Lycium shawii.
From pathologic point of view, it was clear that both extracts alleviated the toxic potentials of CCl4 with additional hepatocytic regenerative capabilities for Prosopis farcta extract.
It can be concluded that Prosopis farcta and Lycium shawii have hepatoprotective and antihyperlipidaemic effect, which is evidenced by the decreased levels of TC, TG, LDL-C, MDA and elevated levels of HDL-C, total antioxidant capacity and GSH in the serum and in this respect, Prosopis farcta is more efficient .
The use of herbs to treat disease is widespread especially in poor societies. The study of plants chemical compounds is an effective way to discover future medicines:
|•||Prosopis farcta and Lycium shawii were found to contain high contents of various flavonoids well known as potent antioxidants and free radical scavengers that protected the liver from damage induced by CCl4|
|•||The study may provide baseline for use of these plants in food and in drugs manufacturing|
The authors would like to thank King Abdulaziz city for Science and Technology (KACST) for the financial support of this study, which is a part of a research grant No. AT-32-1.
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