Hepatoprotective potential of ether insoluble phenolic components of n-butanol fraction (EPC-BF) of flaxseed against CCl(4) -induced liver damage in rats.

OBJECTIVE: to investigate the hepatoprotective potential of ether insoluble phenolic components of n-butanol fraction (EPC-BF) of flaxseed against CCl(4) -induced liver damage in rats.
MATERIALS AND METHODS: Hepatotoxicity was induced to Wistar rats by administration of 0.2% CCl(4) in olive oil (8 mL/kg, i.p.) on the seventh day of treatment. Hepatoprotective potential of EPC-BF at doses, 250 and 500 mg/kg, p.o. was assessed through biochemical and histological parameters.
RESULTS: EPC-BF and silymarin pretreated animal groups showed significantly decreased activities of Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and level of total bilirubin, elevated by CCl(4) intoxication. Hepatic lipid peroxidation elevated by CCl(4) intoxication were also found to be alleviated at almost normal level in the EPC-BF and silymarin pretreated groups. Histological studies supported the biochemical findings and treatment of EPC-BF at doses 250 and 500 mg/kg, p.o. was found to be effective in restoring CCl(4) -induced hepatic damage. However, EPC-BF did not show dose-dependent hepatoprotective potential. EPC-BF depicted maximum protection against CCl(4) -induced hepatic damage at lower dose 250 mg/kg than higher dose (500 mg/ kg).
CONCLUSION: EPC-BF possesses the significant hepatoprotective activity against CCl(4) induced liver damage, which could be mediated through increase in antioxidant defenses.

Linum usitatissimum L. belongs to the family Linaceae, is commonly known as a “Flaxseed” or “Linseed.” Since, antiquity it has been grown for oil and fiber.[1] Flaxseed is the richest source of omega-3 fatty acid, alpha-linolenic acid (ALA), and mammalian lignan.[2] In India, flaxseed has been used as both food and medicine. In some parts of India, especially south India, it has been consumed as flaxseed chutney. In northern America and Europe flaxseed received at low levels as a component in specially breads, as a seed dressing on buns and various other bakery products.[3] Flaxseed was traditionally used in human health for constipation.[4] However, recent investigations were reported broad spectrum of bioactivities to flaxseed such as cardioprotective, immunomodulatory, and antidiabetic.[256] In addition, anticancer, estrogenic, antioxidant, and hepatoprotective activities were attributed to the flaxseed lignan content.[7-9]

Most of the liver diseases generally are associated with oxidative stress caused by free radicals. It is well known that antioxidants counteract with free radicals; reactive oxygen species (ROS) and nullify their pathological effects. Free radicals are generated continuously inside the human body as a result of the exposure to exogenous chemicals in our environment and/or to a number of endogenous metabolic processes involving redox enzymes and bioenergetic electron transfer. The free radicals readily attack and induce oxidative damage to several important biomolecules such as proteins, lipids, and nucleic acids, which contributing to the development of the several diseases such as atherosclerosis, diabetes, cancer, neurodegenerative diseases, hepatic diseases, and ageing.[10-12]

Flaxseed besides ALA is the valuable source of bioactive phenolic components. Majority of health beneficial effects shown by flaxseed phenolic components are associated with its antioxidant potential. Antioxidant potential of flaxseed phenolic components such as lignan secoisolariciresinol diglucoside (SDG) and its metabolites along with p-coumaric acid and ferulic acid glucosides were reported within in vitro models.[13] Moreover, the free radical scavenging activity of flaxseed phenolic components of n-butanol fraction such as herbacetin 3, 8-O-diglucopynanoside, herbacetin 3, 7-O-dimethyl ether, kaempferol 3, 7-O-diglucopyranoside, and (–)-pinoresinol diglucoside were also reported.[14]

In the absence of reliable liver protective drug in allopathic practices, plants play a vital role in management of liver disorders. In India, use of medicinal plants and their formulations are common for the treatment of liver diseases.[15] Earlier research worker reported that rat feeding with whole flaxseed containing diet, helps to the restoration of activities of hepatic enzymes such as catalase, superoxide dismutase (SOD), and peroxide dismutase, increased due to CCl4 intoxication.[9] Recently, hepatoprotective potential of hull fraction from Indian flaxseed cultivar was also studied and which was attributed to flaxseed lignan SDG.[16] However, to the best of our knowledge, the role of phenols other than lignan SDG in earlier observed hepatoprotection remains unclear.

In previous study, we selectively isolated ether insoluble phenolic components such as tannins, caffeic acid, and phenolic glycosides from the n-butanol fraction of flaxseed,[17] free from ether soluble phenolic components such as flavonoids, lignans etc. and reported their antioxidant potential in the in vitro models.[18] This study was undertaken to evaluate the role of these ether insoluble phenolic components of n-butanol fraction (EPC-BF) of flaxseed in alleviation of hepatotoxicity resulting from carbon tetrachloride (CCl4) intoxication through biochemical and histological parameters.

Materials and Methods

Chemicals

Trichloroacetic acid (TCA), thiobarbituric acid and 1, 1, 3, 3-tetramethoxypropane was obtained from Hi-media, Mumbai, India. Carbon tetrachloride (CCl 4 ) and gum acacia were obtained from molychem, Mumbai, India. Silymarin tablets (silybon-140) were purchased from Micro Lab Ltd. India. Olive oil was purchased from Eden Roc cosmetics, Mumbai, India. BCA protein assay kit was purchased from Novagen, U.S.A. All other reagents used were of analytical grade.

Sample collection and extraction

Double-pressed defatted flaxseed powder of brown colored flaxseed was obtained from omega-3 oil unit, established under project NAIP-ICAR Component-3, Sangamner, MS, India. The ether insoluble phenolic components from n-butanol fraction (EPC-BF) of defatted flaxseed powder were extracted as per our previously described method.[18]

Experimental Animals

The study was carried out on mixed sex of Wistar rats (150–250 g). Animals were maintained under standard husbandry conditions (temperature 25±2°C, 12-h light: 12-h dark cycle) and fed with standard pellet diet (Amrut, Sangali, M.S., India) and tap water ad libitum. Throughout the experiments, all animal were handled and used according to the international guide for the care and use of laboratory animals (National Research Council, 1996). Experimental protocol was approved by Institutional Ethical Committee (IAEC approval No. IAEC/ac/2011/11) of Amrutvahini College of Pharmacy, Sangamner.

Carbon tetrachloride-induced hepatotoxicity in rats

The hepatotoxicity was induced by CCl4.[19] Rats were divided into five groups (n = 6). Group I (normal control) animals were administered a single dose of water (25 ml/kg, p.o.) daily for 7 days and received olive oil (8 mL/kg, i.p.) on day 7. Group II (CCl4 control) animals also received single dose water (25 mL/ kg, p.o.) once daily for 7 days and received 0.2% CCl4 in olive oil (8 mL/kg, i.p.) on day 7. Animals of Group III received standard drug silymarin (100 mg/kg, p.o.) once daily for 7 days. Groups IV and V animals were administered 250 and 500 mg/kg, p.o. EPC-BF (dissolved in 2% gum acacia) once daily for 7 days, respectively. The Groups three to five animals were administered simultaneously 0.2% CCl4 in olive oil (8 mL/kg, i.p.) on day 7 after 1 h of administration of the silymarin and the EPC-BF.

After 24 h of the treatment, blood from all animals was collected by retro-orbital puncture and then animals were sacrificed. Blood was allowed to clot and serum was separated at 3500 rpm for 15 min and used for assessment of different enzyme activities. Liver tissue samples were taken from the left liver lobe, and cut into two pieces. One piece was fixed in 10% formalin for pathological examination; the other piece was utilized for the lipid peroxidation assay.

Blood biochemical markers assay

Activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin were estimated using standard kits (Merck Specialties Pvt. Ltd. India), according to the instruction of manufacturer with an autoanalyzer (Nihon Kohden, Japan).[12]

Preparation of liver homogenates and lipid peroxidation assay

Liver was excised from each rat, rinsed in ice cold saline. Liver homogenate (10%, w/v) was prepared with cold 0.15 M Tris-HCl (pH - 7.5) and centrifuged at 2000 rpm for 10 min. Cell free supernatant was used for lipid peroxidation assay.

Thiobarbituric acid reactive substances (TBARS) assay was used for the assessment of lipid peroxidation in hepatic tissue.[20] Briefly, to precipitate the serum proteins, 2.5 mL of 20% TCA (w/v) was added into 0.5 mL of the sample, which was then centrifuged at 1500 rpm for 10 min. Then 2.5 mL of sulfuric acid (0.05 M) and 2 mL TBA (0.2%) was added to the sediment, shaken and incubated in a boiling water bath for 30 min. Then, 4 mL n-butanol was added, and the solution was centrifuged, cooled. The absorption of supernatant was recorded at 532 nm using a UV-Visible spectrophotometer (Chemito UV2100, India). The calibration curve was obtained using different concentrations of 1, 1, 3, 3-tetramethoxypropane as standard to determine the concentration of TBA-MDA adducts in samples.

Histopathological Studies

For histopathological analysis, liver specimens fixed in 10% formalin were embedded in paraffin, sliced 5-μm thick, and stained with hematoxylin and eosin (H and E). The liver sections then assessed for pathological changes.[21]

Statistical Analysis

All values were expressed as mean ± S.E.M. Statistical analysis was carried out by one way ANOVA followed by Bonferroni test performed using GraphPad Prism 5.00.288, GraphPad Software Inc., San Diego, California, USA. P < 0.05 was considered statistically significant.

Results

Effect of EPC-BF on blood biochemical markers AST, ALT, ALP, and total bilirubin

Pretreatment of rats with 250 and 500 mg/kg p.o. of EPC-BF and silymarin (100 mg/kg, p.o.) exhibited a significant (P < 0.05) reduction in the carbon tetrachloride intoxicated increased levels of ALT, AST, ALP, and total bilirubin, compared with CCl 4 control group [Table 1].

Table 1

Effectiveness of EPC-BF and silymarin against carbon tetrachloride (CCl4) induced blood biochemical alterations.

Effect of EPC-BF on lipid peroxidation

Figure 1 shows TBARS levels in all studied groups. In all groups except normal control, hepatic lipid peroxidation levels were found to be increased due to the CCl4 toxicity. The groups treated with silymarin, 250 and 500 mg/kg, p.o. of EPC-BF showed significantly (P < 0.0001) decreased levels of lipid peroxidation; when compared with CCl4 control group.

Figure 1

TBARS levels in all groups intoxicated with CCl4. Normal control: group treated with olive oil (8 mL/kg, i.p.); CCl4 control: group treated with 0.2% CCl4 in olive oil (8 mL/kg); Silymarin+CCl4: group treated with silymarin (100 mg/kg, p.o.) and 0.2% CCl4 in olive oil; 250 EPC-BF+CCl4: group treated with ether insoluble phenolic components of n-butanol fraction of defatted flaxseed meal at dose 250 mg/kg, p.o. and 0.2% CCl4 in olive oil; 500 EPC-BF+CCl4: group treated with ether insoluble phenolic components of n-butanol fraction of defatted flaxseed meal at dose 500 mg/kg, p.o. and 0.2% CCl4 in olive oil. #significantly different from CCl4 (P < 0.0001)

Histopathological observations

The microscopic examination of liver section of normal control group animals shows normal liver architecture. The liver sections of CCl4 control group rats exhibited necrosis and ballooning degeneration. Liver sections of rats treated with silymarin showed normal liver architecture. Treatment of EPC-BF at doses 250 and 500 mg/kg, p.o. was found to be effective in restoring CCl4 induced hepatic damage when compared with normal control [Figure 2].

Figure 2

Histology of liver tissues (a) Normal control group shows normal lobular architecture. (b) CCl4 intoxicated group shows tissue ballooning degeneration and necrosis. (c) The group treatment with silymarin show almost normal histology. (d) Treatment of EPC-BF (250 mg/kg, p.o.) improves almost near normal cellular architecture. (e) Treatment of EPC-BF (500 mg/kg, p.o.) show normal cellular architecture

Discussion

Liver is the vital organ of the human body, play important role in synthesis, storage of biomolecules and detoxification of toxic metabolites. Liver injuries can be caused by toxic chemicals, drugs, and virus infiltration via ingestion or infection.[22] Most of the liver damages are associated with redox imbalance and oxidative stress.[23] The antioxidant capacity of phenolic compounds is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators.[24] Plant phenols counteract with redox imbalance and oxidative stress and neutralize its pathological effects. In the present study hepatoprotective potential of EPC-BF was assessed against carbon tetrachloride (CCl4) intoxication in rats. Hepatotoxin CCl4 is converted to trichloromethyl (-CCl3-) and then peroxy-radicals (CCl3OO) by chytochrome P450 enzymes in liver. These free radicals and reactive oxygen species (ROS) then initiate lipid peroxidation and resulted into damage of liver.[25]

The liver function is assessed by estimation of serum bilirubin. Bilirubin is yellow colored pigment produced after metabolism of heme. To take up and process bilirubin is associated normal liver function. On other hand, estimation of serum enzymes AST, ALT and ALP is the quantitative marker for the determination of type of liver diseases. The level of AST is an indicator of mitochondrial damage.[26] The increased activity of AST indicates that the mitochondria were destroyed. The plasma level of ALT is an indicator of the degree of the cell membrane damage. Liver injury leads to increase in levels of transaminases such as AST, ALT, and total bilirubin in the plasma. In our experiment, increased activities of enzymes AST, ALT, ALP, and total bilirubin level were observed in CCl4 control group, which could be due to the CCl4 intoxicated increased oxidative stress. The pretreatment of EPC-BF to rats at doses 250 and 500 mg/ kg, p.o. resulted into restoration of increased activities AST, ALT, ALP, and total bilirubin at the normal level, which could be mediated through neutralizing free radicals induced by CCl4 toxicity. Interestingly, EPC-BF depicted maximum protection against CCl4 -induced hepatic damage at lower dose 250 mg/kg than higher dose (500 mg/ kg). The observed results could be due to the prooxidant activity of some phenolic components of EPC-BF or increased concentration of glycosylated phenolic components in EPC-BF at its high dose (500 mg/kg), as most of the phenol compounds had prooxidant activity at low concentrations and antioxidant activity usually decreases with glycosylation.[27] However, further details studies are warranted prove our hypothesis. Finally, result of liver biochemical markers confirmed hepatoprotective potential of EPC-BF at doses 250 and 500 mg/kg, p.o., which is linked to its earlier reported antioxidant potential.

The TBARS assay is method of measuring the extent of lipid peroxidation in terms of thiobarbituric acid reactive substances, that is, TBARS produced by the liver tissue. The lipid peroxidation is accelerated when free radicals are formed as the results of losing a hydrogen atom from the double bond in the structure of unsaturated fatty acids. Scavenging of free radicals is one of the major antioxidant mechanisms to inhibit the chain reaction of lipid peroxidation. In CCl4 intoxicated group, TBARS level is increased due to tissue damage and failure of antioxidant defense mechanism. The animals treated with EPC-BF shows reduced levels of CCl4 -induced lipid peroxidation, which could be mediated through antioxidant defenses of EPC-BF.

The histological data of this study also support to the results of biochemical markers. Rats treated with EPC-BF showed almost normal hepatic cellular architecture similar to control and silymarin treated rat groups. This confirmed the hepatic structural integrity maintenance property of EPC-BF, as shown by the hepatoprotective drugs.

In conclusion, Results of current investigation first time demonstrated that EPC-BF has significant hepatoprotective potential, which is likely related to its antioxidant potential. Furthermore, result this study also suggested that EPC-BF could be largely contributed in hepatoprotective activity observed by flaxseed supplementation and it may have potential clinical application in therapy for liver diseases.