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Synergistic hepatoprotective effect of Schisandrae lignans with Astragalus polysaccharides on chronic liver injury in rats
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Phytomedicine 16 (2009) 805–813 www.elsevier.de/phymed
Synergistic hepatoprotective effect of Schisandrae lignans with Astragalus polysaccharides on chronic liver injury in rats Fei Yana,1, Qiao-Yan Zhanga,1, Lei Jiaoa, Ting Hana, Hong Zhanga, Lu-Ping Qina,, Rahman Khalidb a
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China Faculty of Sciences, School of Biomolecular Sciences, Liverpool John Moores University, Liverpool L33AF, UK
b
Abstract The aim of this study was to investigate the synergistic hepatoprotective effect of lignans from Fructus Schisandrae chinensis (LFS) with Astragalus polysaccharides (APS) on chronic liver injury in male Sprague-Dawley rats. Subcutaneous injection of 10% CCl4 twice a week for 3 months resulted in significantly (po0.001) elevated serum alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP) activities compared to controls. In the liver, significantly elevated levels (po0.001) of malondialdehyde (MDA), lowered levels of reduced glutathione (GSH) (po0.05) and catalase (CAT) (po0.001), superoxide dismutase (SOD) (po0.01)were observed following CCl4 administration. ‘LFS+ASP’ treatment of rats at doses of ‘LFS (45 mg/kg)+APS (150 mg/kg)’ and ‘LFS (135 mg/kg)+APS (450 mg/kg)’ displayed hepatoprotective and antioxidative effects than the administration of either LFS or APS, as evident by lower (po0.005 or 0.001) levels of serum ALT, AST, ALP and hepatic MDA (po0.001) concentration, as well as higher SOD (po0.05 or 0.005), CAT activities(po0.01 or 0.005), GSH concentration (po0.05 or 0.005) compared to the toxin treated group. Histopathological examinations revealed severe fatty degeneration in the toxin group, and mild damage in groups treated with ‘LFS+APS’ were observed. The coefficients drug interaction (CDI) between each individual drug and their combination (at the same dose of their single treatment) of these foregoing parameters were all less than 1, indicating that LFS and APS display hepatoprotective and antioxidant properties and act in a synergistic manner in CCl4 induced liver injury in rats. r 2009 Elsevier GmbH. All rights reserved. Keywords: Astragalus polysaccharides; Fructus Schisandrae chinensis; Hepatoprotective; Synergistic
Introduction Liver is considered to be the key organ in the metabolism, detoxification, and secretary functions of Abbreviations: APS, Astragalus polysaccharides; CDI, Coefficient of drug combination; LFS, lignans from Fructus Schisandrae chinensis. Corresponding author. Tel.: +86 21 25070394; fax: +86 21 25070394. E-mail address: [email protected] (L.-P. Qin). 1 Contributed equally to this work. 0944-7113/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.02.004
the body. Hepatic injury is a fundamental pathological process in most chronic hepatic diseases and longstanding hepatic injury leads to hepatic fibrosis, liver cirrhosis, and even hepatocellular carcinoma. Investigations have indicated that some herbal extracts and their chemical constituents can significantly inhibit these aforementioned pathologic processes and protect hepatocytes against the etiologies of chronic hepatic injury (Strader et al. 2002; Stickel and Schuppan 2007). Of all the hepatoprotective herbal medicines, Fructus Schisandrae and Radix Astragali are the most widely used in the
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prevention and treatment of liver injury, and these two herbal drugs have been developed into various healthy foods (Feng et al. 2006; Tian 2008). Fructus Schisandrae chinensis (Magnoliaceae), a commonly used traditional Chinese medicine for centuries, has a wide spectrum of pharmacological action, such as stimulating central nervous system, preventing cough and eliminating at phlegm, preventing liver injuries, anti-aging, anti-virus (Guo et al. 2006). The chemical constituents of Fructus Schisandrae chinensis include lignans, polyose, organic acid, triterpene, sesquiterpene and volatile oil (Yang et al. 2003). Investigations showed that dibenzocyclooctene lignans (schisandrin, deoxyschizandrin, Schisantherin, r-Schisandrin etc.), can inhibit the increase of serum transferase level induced by chemical toxin materials (Shen et al. 2005; Li X.G. et al. 2005; Li P.F. et al. 2005; Zhang et al. 2002), and are capable of eliminating of HBV, strengthening the immune system, stimulating liver cell regeneration, as well as suppress the proliferation of hepatocellular carcinoma cells (Adrian et al. 2007; Loo et al. 2007), these are also the main hepatoprotective chemical constituents. The Radix Astragalus mainly contains saponins, flavone, polysaccharides and other chemical constituents. Astragalus polysaccharides (APS) have been reported to be effective in modulating immune functions including promoting humoral immunity and cellular immunity, improving the immune functions of the immunosuppressive model mice, and modulating the production of cytokines (Shan et al. 2000; Li X.G. et al. 2005; Li P.F. et al. 2005). In addition, some investigations have reported that APS had hepatopretective effects on liver injury induced by CCl4 in mice. Biological factors (hepatitis virus, bacteria, parasite etc.), chemical factors (medicine, industrial poisons, alcohol etc.), hereditary factor, environmental factor can directly or indirectly induce liver injury. Causative agents such as virus, medicine, toxicant mainly through bring out free radicals include disruption of calcium homeostasis and cell membrance injury, canalicular and cholestatic injury, metabolic bioactivation by cytochrome P450 enzymes, autoimmunity, apoptosis and mitochondrial injury (Xu and Qu 2008). Then hepatocyte apoptosis, necrosis, gradually lead to hepatic fibrosis or hepatic cirrhosis even hepatoma, which is always accompanied with hypoimmunity (Bockhold et al. 2005; Nelson Fausto 2006). The outcome of hepatic injury chemopreventive strategies relies largely on the ability of chemopreventive agents to maximally exploit the intrinsic hepatoprotective potential without incurring undue toxicity. Targeting multiple pathways by a combinatorial approach using natural products would ideally empower the clinician to better delay the progression of liver injury. Emerging evidence suggest that liver injury is multi-
pathways and always accompanied by hypoimmunity, and therefore a multi-targeting agent or the combination of agents targeting multiple pathways or having immunomodulatory action without causing any toxicity would be required for the success of prevention and/or treatment of liver injury. Therefore, we believe that the combination of LFS and APS, or even other agents together would be highly effective in preventing and treating liver injury. The present investigation examined the ability of APS, LFS and their combinations to protect against carbon tetrachloride (CCl4)-induced hepatotoxicity. We found that the combination approach is better in protecting liver injury. This will be helpful for understanding the effect relationship between LFS and APS, exploring their synergistic effects on liver injury of CCl4-induced in rats, providing scientific basis for their application in dietary supplement and drugs of prevention and treatment liver disease.
Materials and methods Chemicals Ethanol (CP), CCl4 (AR), acetic acid (AR), dehydrated alcohol (AR), methanol (AR), NaCl (AR) was purchased from Sinopharm Chemical Reagent Co., Ltd. China. HPLC-grade acetonitrile were from Merck KGaA (Darmstadt,Germany). Water was deionized distilled water. Bifendate Pills (Zhejiang Medicine Co., LTD Xinchang Zhejiang, China.), Catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA) and protein quantization measurement kits were purchased form Nanjing Jiancheng Bioengineering Institute.
Preparation of the lignans from Fructus Schisandrae chinensis (LFS) and Astragalus Polysaccharides (APS) The dried Fructus Schisandrae chinensis collected from Jilin Province, and Radix Astragali purchased from Shanghai Hua Yu Chinese Herbs Co., Ltd., were identified by Prof. Lu-Ping Qin, Department of Pharmacognosy, Second Military Medical University. The Fructus Schisandrae chinensis was extracted with 95% ethanol by reflux twice, and each time for 2 hours. The extract was concentrated under reduced pressure, and then dissolved in 30% ethanol to give 1 g/ml (crude medicine/volume) sample liquor, and then this liquor was passed through AB-8 macroporous resin column with 1 time the volume which was comparable to the weight of macroporous resin. The lignans were adsorbed
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300 250
Schisandrin
150 150
Deoxyschizandirn
r-Schisandrin
32.698
100
40.284
MAU
200
50 0 0
10
20
30
40
50
Min
Fig. 1. HPLC chromatogram of lignans in Fructus Schisandrae chinensis.
onto macroporous resin column and eluted with 30%, 70%, and 95% ethanol, respectively. The combined elutes were concentrated under reduced pressure and 70% ethanol elutes was used as the total lignans from Fructus Schisandrae chinensis (LFS). The content determination of total LFS (including schisandrin, deoxyschizandrin and r-Schisandrin) was performed on Agilent 1200 series (HPLC) including a quaternary pump, vacuum degasser, thermostatic column compartment and a diode array detector (DAD). An Agilent Zorbax SB-C18 column (250 mm 4.6 mm I.D., 5 mm) with a Extend C-18 guard column (10 4.6 mm, 5 mm) was used. Gradient elution was employed using solvent systems C (acetonitrile) and B (aqueous) at ambient temperature. The gradient program used was as follows: initial 0–13 min, C–B (48:52, v/v); 13–30 min, linear change to C–B (52:48, v/v); 30–55 min, linear change to C–B (80:20, v/v); The flow rate was 1.0 ml min1 and column temperature was maintained at 30 1C. The detector wavelength was set at 254 nm and an aliquot of 20 ml solution was injected for acquiring chromatograms (Fig. 1). The content of LFS was 23.1% (W/W), including schisandrin 17.5% (W/W), deoxyschizandrin 2.7% (W/W) and r-Schisandrin 2.9% (W/W). The Astragali Radix was extracted 3 with 12 vol of distilled water at 75 1C for 1.5 h. The extracts was concentrated to 1.2 g crud drug/ml, and precipitated with 95% EtOH (1:4, v/v) at 4 1C for 12 h. The precipitating process was repeated for two times to obtain crud polysaccharides. The crud polysaccharide was suspended in distilled water, and was applied to a column of DEAE-Sepharose CL-6B, eluted with different concentrations of NaCl aqueous solution to obtain
APS.The glucose concentration in APS was 51% (w/w) which detected by the phenol–sulfuric acid standard curve method on UV Spectrophotometer. Polysaccharides compositional analysis by TLC and gas chromatography showed that it consisted of D-glucose, D-galactose, L-arabinose (1.75:1.63:1). The optical rotation was (a) D30.5+101.5 (H2O) detected by polarimeter. The average molecular of the extract was 35 800 as determined by gel filtration method.
Animals and experimental protocol Eighty male Sprague-Dawley rats (180-200 g) were purchased from SLACOM experimental animal company (Shanghai, China) and acclimated to conditions for 1 week before the experiment. The experimental animals were housed in an air-conditioned room with 12 h/12 h light-dark illumination cycles at constant temperature 2470.5 1C and humidity (45-50%). Food and drinking water were supplied ad labium and the rats were weighed weekly during the experimental period. Ten rats were treated with vehicle (deionized water) as normal control. The remaining 70 rats were administrated subcutaneous injections of CCl4 in arachis oil (1:9) at a dose of 5 ml/kg twice a week and equally randomized into seven groups. They were treated with vehicle (deionized water), Bifendate Pills (100 mg/kg once daily), APS (450 mg/kg once daily), LFS (135 mg/ kg once daily), LFS (15 mg/kg)+APS (50 mg/kg), LFS (45 mg/kg)+APS (150 mg/kg), LFS (135 mg/kg)+APS (450 mg/kg) (once daily), for 3 months respectively. At the end of the treatment, rats were fasted for 8 h after the last treatment. The blood was drawn from all groups
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via the abdominal artery for the assessment of biochemical parameters ALT, AST and ALP. The liver sample was collected, the left hepatic lobe was cut into 1.5 cm 1.5 cm 2 cm (length width height) slices, and fixed in the 10% formalin for the histopathological examinations, the right hepatic lobe was kept at 80 1C to measure the activity of catalase (CAT), superoxide dismutase (SOD), reduced glutathione hormone (GSH), malondialdehyde (MDA) content and total protein. This experiment was approved by the Bioethic Committee of the Second Military Medical University (Shanghai, China), and the procedures of the experiment were strictly according to the generally accepted international rules and regulations.
Determination of enzyme levels in serum Serum ALT, AST, and ALP were measured on an automatic analyzer (HITACHI 7600-020, Japan) using diagnostic reagent kit.
Determination of biological parameters of liver tissue Each right hepatic lobe sample was washed thoroughly in ice-cold saline to remove the blood after thawing, blotted the saline gently using filter paper. 0.15 g of each sample was placed in 1.35 ml saline and ten percent of homogenate in the vitric homogenizer at 20 1C was prepared. The homogenate was centrifuged at 3000 rpm for 10 min, the supernatant was used for the estimate of catalase (CAT), superoxide dismutase (SOD) activities, and reduced glutathione (GSH), malondialdehyde (MDA) and protein content. The CAT, SOD, GSH, MDA and proteins were estimated using kits according to the manufacturers’ instructions. Table 1.
Histopathological examinations Liver tissue sections was dissected and fixed in 10% formalin, then processed routinely, embedded in paraffin, sectioned to 4 mm thickness, deparaffinized, rehydrated using standard techniques, stained with hematoxylin and eosin (H&E). The extent of CC14induced liver necrosis and steatosis was evaluated by assessing morphological changes in liver sections.
Analysis of LFS and APS interaction The coefficient of drug interaction (CDI) was used to analyze the synergistically effect of drug combinations. (Cao and Zhen 1989; Wan et al. 2008) CDI is calculated as follows: CDI ¼ AB/(A B). AB is the ratio of the corresponding parameters of the combination groups to toxin control group; A or B is the ratio of these parameters in the single agent groups to toxin control group. Thus CDI value less than, equal to or greater than 1 indicates that the drugs are synergistic, additive or antagonistic, respectively. CDI less than 0.7 indicates that the drugs are significantly synergistic.
Statistical analysis Data was expressed as mean7SD. The differences among different groups were analyzed using one-way analysis of variance (ANOVA) with Student’s t-test.
Results Serum ALT, AST and ALP level Administration of CC14 caused significant elevation of serum ALT, AST and ALP activities in rats
Effects of APS and LFS on serum transaminase level.
Groups
ALT (u/l)
AST (u/l)
ALP (u/l)
Normal control Toxin control (CCl4) CCl4+bifendate (100 mg/kg) CCl4+APS (450 mg/kg) CCl4+LFS (135 mg/kg) CCl4+LFS (15 mg/kg)+APS (50 mg/kg) CCl4+LFS (45 mg/kg)+APS (150 mg/kg) CCl4+LFS (135 mg/kg)+APS (450 mg/kg)
33.676.9 202.2730.8DDDD 101.4718.0**** 175.7719.6 137.5738.8** 129.7714.1 120.4715.7*** 88.7715.2****(0.74)
103.8716.1 286.4727.0DDDD 176.7726.7**** 247.9724.3 189.5723.1*** 194.9725.6** 177.6722.5*** 153.2718.1****(0.93)
85.2714.0 195.3722.1DDDD 138.8714.9*** 181.0722.3 156.4727.7* 188.3740.0 142.1728.5*** 131.2726.6****(0.91)
Values are expressed as mean7SD of ten rats in each group. D po0.05, DDpo0.01, DDDpo0.005, DDDDpo0.001, compared with normal control group. *po0.05, **po0.01,***po0.005, ****po0.001, compared with toxin control group. Numbers in parentheses indicate the coefficient of drug interaction. ALT (alanine aminotransferase). AST (asparate aminotransferase). ALP (alkaline phosphatase).
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compared with normal group. The protective effects of LFS and APS on the CC14 induced elevation of serum ALT, AST and ALP activities are presented in Table 1. The activities of serum ALT, AST and ALP in the CC14 group were much higher than those in the normal group. Only administration of APS did not cause significant changes in the serum transaminase activities, whilst administration of LFS, serum ALT (po0.01), AST (po0.001) activities decreased in CC14 treated rats. However, the administration of a combination of LFS and APS significantly prevented the elevation of serum transaminase activities induced by CC14 treatment in a dose-dependent manner. At the dose of 135 mg/kg LFS combination with 450 mg/kg APS, Serum ALT, AST and ALP activities respectively decreased 56%, 47%,
809
and 33% compared with toxin control, and the CDIs of ALT, AST and ALP activities between their combination and the single treatment were all less than 1.
Biochemical parameters of liver tissue As shown in Fig. 2A, the hepatic superoxide dismutase (SOD) activity in the CC14 group was significantly reduced by 21% when compared with the vehicle control group. LFS or APS did not affect SOD activity in CC14 treatment rats. However, administration of ‘LFS(135 mg/ kg)+APS (450 mg/kg)’ significantly (po0.005) increased the SOD activity from 98.9 (u/mgprot) to 119.3 (u/mgprot). The CDI of SOD activity between the
Fig. 2. Dpo0.05, DDpo0.01, DDDpo0.05, DDDDpo0.001, compared with normal control group; *po0.05, **po0.01, ***po0.005, ****po0.001, compared with toxin control group; Group I: Normal Control. Group II: Toxin control CCl4. Group III: CCl4+bifendate (100 mg/kg). Group IV: CCl4+APS (450 mg/kg). Group V: CCl4+LFS (135 mg/kg). Group VI: CCl4+LFS (15 mg/kg)+APS (50 mg/kg). Group VII: CCl4+LFS (45 mg/kg)+APS (150 mg/kg). Group VIII: CCl4+LFS (135 mg/kg)+APS (450 mg/kg). CDI (coefficient of drug interaction) see Materials and methods.
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‘LFS(135 mg/kg)+APS (450 mg/kg)’ treatment group and their single treatment group was 0.86. The hepatic reduced glutathione (GSH) content significantly (po0.005) decreased in the CC14 group when compared with the vehicle control group. LFS or APS did not affect GSH con in CC14 treatment rats. However, the combination of LFS with APS at dose of ‘LFS(135 mg/kg)+APS (450 mg/kg)’ significantly (po0.005) increased the GSH concentration when compared with the CC14 group. The CDI of GSH activity between the ‘LFS(135 mg/kg)+APS (450 mg/kg)’ treatment group and their single treatment group was 0.84 (Fig. 2B). Malondialdehyde (MDA) concentration in the CC14 group was significantly increased to the 4-fold level when compared with the vehicle control group. LFS and APS significantly (po0.005) decreased MDA activity in CC14 treatment rats respectively at dose of 135 and 450 mg/kg. The combination of LFS with APS significantly (po0.001) decreased the MDA concentration when compared with the CC14 group in a dosedependent manner. The combination of LFS with APS is more effective than LFS or APS at the same dose in decreasing MDA level in liver tissue of CC14-treatment rats as shown in Fig. 2C. And the CDI of MDA content between the ‘LFS (135 mg/kg)+APS (450 mg/kg)’ treatment group and the single LFS and APS treatment group was 0.94. Catalase (CAT) activity in the CCl4 group was also found significantly (po0.001) depleted in CC14 treatment group as compared to the normal control group. However, the activity of CAT was significantly (po0.001) elevated in group which treated with 135 mg/kg LFS combination with 450 mg/kg APS. Administration of LFS or APS only did not display effect of increase the CAT activity. The CDI of CAT activity between the ‘LFS (135 mg/kg)+APS (450 mg/kg)’ treatment group and their single treatment group was 0.85 (Fig. 2D).
Histopathological change The normal liver histological sections showed that hepatocytes were well-preserved and uniform cytoplasm, prominent nucleus, nucleolus and central central veins were visible (Fig. 3A). Compared with the normal group, liver tissue in the rats treated with CC14 revealed extensive liver injuries, characterized by severe hepatocellular degeneration and necrosis around the central vein, fatty changes, inflammatory cell infiltration, congestion, sinusoidal dilatation, cytoplasmic vacuolation, massive fatty degeneration and the loss of cellular boundaries (Fig. 3B). However, the histopathological hepatic lesions induced by administration of CC14 were remarkably ameliorated by LFS, APS and
their combinations in a dose-dependent manner (Figs. 3C–H), and this was in good agreement with the results of serum aminotransferase activity and hepatic oxidative stress level. Administration combination of LFS with APS improved the hepatocellular injury induced by CC14 in rats better than that of only LPS or APS.
Discussion In the present study, we evaluated the effects of LFS, APS and their combination on serum ALT, AST and ALP level, antioxidant enzymes of liver tissue and liver histopathological change in CC14 treated rats. The results showed that LFS and their combination were effective in the prevention of CC14-induced liver injury, and the effects of the combination of LFS with APS are more significant than that of LFS or APS on their own. The CDIs of serum transaminase activities and hepatic antioxidant enzymes between the ‘LFS(135 mg/kg)+ APS (450 mg/kg)’ treatment group and their single treatment group were less than 1, which indicated that LFS and APS had synergistic effects on hepatoprotection and antioxidation in CC14-treated rats. Animal liver damage by CCl4 has been widely used to evaluate liver protective activity of drugs (Li 2006), and carbon tetrachloride (CCl4) is a well-known hepatotoxic agent. Toxicity begins with the change in endoplasmic reticulum, which result in the loss of metabolic enzymes located in the intracellular structures (Recnagal 1983). The concentration of transferase in hepatocyte is about 1000–5000 times higher than in serum normally. When liver was injured by CCl4, membrane permeability of liver parenchyma cell intensified, the activities of ALT and AST in serum increased sharply as a consequence and serum aminotransferase activities have long been considered as sensitive indicators of hepatic injury. In the present study, the 10% CC14 at dose of 5 ml/kg for 3 months caused a dramatic elevation in serum ALT, AST, ALP activities and liver tissue histopathological change, indicating an hepatotoxicity induced by administration of CC14. Administration of combinations of LFS and APS prevented the CC14-induced elevation of serum transaminase activities in a dose-dependent manner, indicating the hepatoprotective activity of these against the intoxication of CC14. This was also confirmed by the results of histopathological examination, as evidenced by a dose-related decrease in the incidence and severity of histopathological hepatic lesions. On the other hand, the basis of CC14 hepatotoxicity lies in its biotransformation through the cytochrome P450 system to two free radicals. During metabolic process of CC14, CC14 is firstly transferred into a trichloromethyl free radical, forming covalent adducts
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Fig. 3. Histopathology of the liver ( 100). A: Normal control. B: Toxin control (treated with CCl4). C: Treated with CCl4+bifendate (100 mg/kg). D: Treated with CCl4+APS (450 mg/kg). E: Treated with CCl4+LFS (135 mg/kg). F: Treated with CCl4+LFS (15 mg/kg)+APS (50 mg/kg). G: Treated with CCl4+LFS (45 mg/kg)+APS (150 mg/kg). H: Treated with CCl4+LFS (135 mg/kg)+APS (450 mg/kg).
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with lipids and proteins, which can interact with O2 to form a second metabolite, a trichloromethylperoxy free radical, or can remove hydrogen atoms to form chloroform. This sequence of events leads to lipid peroxidation of membranes and consequent liver injury (Sheweita et al. 2001; Brattin et al. 1985). In response to this hepatocellular injury, ‘activated’ hepatic Kupfer cells release increased quantities of active oxygen species and other bioactive agents (Eisisi et al. 1993). Malondialdehyde (MDA), a secondary product of lipid peroxidation, is used as an indicator of tissue damage involving a series of chain reactions. It reacts with thiobarbituricacid, producing red-colored products. Lipid peroxidation has been implicated in the pathogenesis of increased membrane rigidity, osmotic fragility, reduced erythrocyte survival and perturbations in lipid fluidity (Ohkawa et al. 1979). The observation of elevated levels of hepatic MDA in CCl4-treated rats (toxin group) in the present study is consistent with this hypothesis. Thus, the maintenance of near normal levels of hepatic MDA in rats administered combination of LFS and APS is of supporting evidence to suggest an antioxidant role for LFS and APS. A major defense mechanism involves the antioxidant enzymes, including SOD, CAT, and GSH which convert active oxygen molecules into non-toxic compounds. The enzymatic antioxidant defense systems are the natural protector against lipid peroxidation (Zimmermann et al. 1973). These enzymes prevent generation of hydroxyl radical and protect the cellular constituents from oxidative damage. A reduction in the activities of these enzymes is associated with the accumulation of highly reactive free radicals, leading to deleterious effects such as loss of integrity and function of cell membranes. Administration of CCl4 leads to generation of peroxy radical O2, which is associated with inactivation of CAT, GSH, SOD enzymes (Cheeseman 1995; Mates et al. 1999). This probably explains the significantly reduced activities of CAT, GSH, SOD observed in rats treated with CCl4. In CCl4-treated rats, administration of LFS, APS and their combination caused the increase of CAT, SOD activities and GSH content ,especially in the group of treated with 135 mg/kg LFS combination with 450 mg/kg APS. An interesting phenomenon in our experiment is that single administrated APS did not significantly change the serum ALT, AST, ALP and biochemical parameters of liver tissue in CCl4 treated rats, and single administrated LFS, the abovementioned indicator were improved to some extent, but when LFS was combined with APS, its effects on serum and liver tissue related parameters were significantly enhanced, and the calculate result of the coefficient of drug combination between the ‘LFS(135 mg/kg)+APS (450 mg/kg)’ treatment group and their single treatment group give a strong evidence of the synergistic protective effect of
LFS and APS on CCl4 treated rats. The synergistic mechanism may relate with the immunomodulatory effects of APS. Recent research has shown that components of the innate immune system are intimately associated with liver disease and hepatic regeneration. It is well known that TNF plays a major role in alcoholic liver disease, and that cytokines such as Il-6, may contribute to fibrotic processes. More surprising, however, was the realization that some components of the innate immune system, including TNF and IL-6 may participate in the initiation of liver regeneration after partial hepatectomy (Mei et al. 2007). It has been reported that the APS have significant immunomodulatory effects, including promoting humoral immunity and cellular immunity, improving the immune functions of the immunosuppressive model mice (Shao et al. 2004), and modulating the production of cytokines (Schepetkin and Quinn 2006). Therefore, APS may enhanced the hepatoprotective effects of LFS by modulating function of immune system. In conclusion, LFS and APS provide significant protective effects on CCl4-induced hepatic injury through decreasing the serum levels of ALT, AST and ALP, and enhancing SOD, CAT, GSH activities. The data from CDI result revealed that LFS combined with APS has a synergistic action in reducing ALT, AST and restoring SOD and GSH levels, and was superior to the administration of either LFS or APS. These synergy effects have been first described with examples in two review articles from Wiliamson (2001) and UlrichMerzenich et al. (2007). Therefore, dietary LFS and APS may be useful as a hepatoprotective agent again. However, the hepatoprotective mechanisms of these medicinal herb constituents remain to be elucidated.
Acknowledgements Authors are thankful to Hangzhou Chinese traditional medicine modernization research center for the financial assistance, and Inspection Branch of Shanghai Eastern Hepatobiliary Hospital for analyzing serum biochemical parameters.
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Phytomedicine 16 (2009) 805–813 www.elsevier.de/phymed
Synergistic hepatoprotective effect of Schisandrae lignans with Astragalus polysaccharides on chronic liver injury in rats Fei Yana,1, Qiao-Yan Zhanga,1, Lei Jiaoa, Ting Hana, Hong Zhanga, Lu-Ping Qina,, Rahman Khalidb a
Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China Faculty of Sciences, School of Biomolecular Sciences, Liverpool John Moores University, Liverpool L33AF, UK
b
Abstract The aim of this study was to investigate the synergistic hepatoprotective effect of lignans from Fructus Schisandrae chinensis (LFS) with Astragalus polysaccharides (APS) on chronic liver injury in male Sprague-Dawley rats. Subcutaneous injection of 10% CCl4 twice a week for 3 months resulted in significantly (po0.001) elevated serum alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP) activities compared to controls. In the liver, significantly elevated levels (po0.001) of malondialdehyde (MDA), lowered levels of reduced glutathione (GSH) (po0.05) and catalase (CAT) (po0.001), superoxide dismutase (SOD) (po0.01)were observed following CCl4 administration. ‘LFS+ASP’ treatment of rats at doses of ‘LFS (45 mg/kg)+APS (150 mg/kg)’ and ‘LFS (135 mg/kg)+APS (450 mg/kg)’ displayed hepatoprotective and antioxidative effects than the administration of either LFS or APS, as evident by lower (po0.005 or 0.001) levels of serum ALT, AST, ALP and hepatic MDA (po0.001) concentration, as well as higher SOD (po0.05 or 0.005), CAT activities(po0.01 or 0.005), GSH concentration (po0.05 or 0.005) compared to the toxin treated group. Histopathological examinations revealed severe fatty degeneration in the toxin group, and mild damage in groups treated with ‘LFS+APS’ were observed. The coefficients drug interaction (CDI) between each individual drug and their combination (at the same dose of their single treatment) of these foregoing parameters were all less than 1, indicating that LFS and APS display hepatoprotective and antioxidant properties and act in a synergistic manner in CCl4 induced liver injury in rats. r 2009 Elsevier GmbH. All rights reserved. Keywords: Astragalus polysaccharides; Fructus Schisandrae chinensis; Hepatoprotective; Synergistic
Introduction Liver is considered to be the key organ in the metabolism, detoxification, and secretary functions of Abbreviations: APS, Astragalus polysaccharides; CDI, Coefficient of drug combination; LFS, lignans from Fructus Schisandrae chinensis. Corresponding author. Tel.: +86 21 25070394; fax: +86 21 25070394. E-mail address: [email protected] (L.-P. Qin). 1 Contributed equally to this work. 0944-7113/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.02.004
the body. Hepatic injury is a fundamental pathological process in most chronic hepatic diseases and longstanding hepatic injury leads to hepatic fibrosis, liver cirrhosis, and even hepatocellular carcinoma. Investigations have indicated that some herbal extracts and their chemical constituents can significantly inhibit these aforementioned pathologic processes and protect hepatocytes against the etiologies of chronic hepatic injury (Strader et al. 2002; Stickel and Schuppan 2007). Of all the hepatoprotective herbal medicines, Fructus Schisandrae and Radix Astragali are the most widely used in the
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prevention and treatment of liver injury, and these two herbal drugs have been developed into various healthy foods (Feng et al. 2006; Tian 2008). Fructus Schisandrae chinensis (Magnoliaceae), a commonly used traditional Chinese medicine for centuries, has a wide spectrum of pharmacological action, such as stimulating central nervous system, preventing cough and eliminating at phlegm, preventing liver injuries, anti-aging, anti-virus (Guo et al. 2006). The chemical constituents of Fructus Schisandrae chinensis include lignans, polyose, organic acid, triterpene, sesquiterpene and volatile oil (Yang et al. 2003). Investigations showed that dibenzocyclooctene lignans (schisandrin, deoxyschizandrin, Schisantherin, r-Schisandrin etc.), can inhibit the increase of serum transferase level induced by chemical toxin materials (Shen et al. 2005; Li X.G. et al. 2005; Li P.F. et al. 2005; Zhang et al. 2002), and are capable of eliminating of HBV, strengthening the immune system, stimulating liver cell regeneration, as well as suppress the proliferation of hepatocellular carcinoma cells (Adrian et al. 2007; Loo et al. 2007), these are also the main hepatoprotective chemical constituents. The Radix Astragalus mainly contains saponins, flavone, polysaccharides and other chemical constituents. Astragalus polysaccharides (APS) have been reported to be effective in modulating immune functions including promoting humoral immunity and cellular immunity, improving the immune functions of the immunosuppressive model mice, and modulating the production of cytokines (Shan et al. 2000; Li X.G. et al. 2005; Li P.F. et al. 2005). In addition, some investigations have reported that APS had hepatopretective effects on liver injury induced by CCl4 in mice. Biological factors (hepatitis virus, bacteria, parasite etc.), chemical factors (medicine, industrial poisons, alcohol etc.), hereditary factor, environmental factor can directly or indirectly induce liver injury. Causative agents such as virus, medicine, toxicant mainly through bring out free radicals include disruption of calcium homeostasis and cell membrance injury, canalicular and cholestatic injury, metabolic bioactivation by cytochrome P450 enzymes, autoimmunity, apoptosis and mitochondrial injury (Xu and Qu 2008). Then hepatocyte apoptosis, necrosis, gradually lead to hepatic fibrosis or hepatic cirrhosis even hepatoma, which is always accompanied with hypoimmunity (Bockhold et al. 2005; Nelson Fausto 2006). The outcome of hepatic injury chemopreventive strategies relies largely on the ability of chemopreventive agents to maximally exploit the intrinsic hepatoprotective potential without incurring undue toxicity. Targeting multiple pathways by a combinatorial approach using natural products would ideally empower the clinician to better delay the progression of liver injury. Emerging evidence suggest that liver injury is multi-
pathways and always accompanied by hypoimmunity, and therefore a multi-targeting agent or the combination of agents targeting multiple pathways or having immunomodulatory action without causing any toxicity would be required for the success of prevention and/or treatment of liver injury. Therefore, we believe that the combination of LFS and APS, or even other agents together would be highly effective in preventing and treating liver injury. The present investigation examined the ability of APS, LFS and their combinations to protect against carbon tetrachloride (CCl4)-induced hepatotoxicity. We found that the combination approach is better in protecting liver injury. This will be helpful for understanding the effect relationship between LFS and APS, exploring their synergistic effects on liver injury of CCl4-induced in rats, providing scientific basis for their application in dietary supplement and drugs of prevention and treatment liver disease.
Materials and methods Chemicals Ethanol (CP), CCl4 (AR), acetic acid (AR), dehydrated alcohol (AR), methanol (AR), NaCl (AR) was purchased from Sinopharm Chemical Reagent Co., Ltd. China. HPLC-grade acetonitrile were from Merck KGaA (Darmstadt,Germany). Water was deionized distilled water. Bifendate Pills (Zhejiang Medicine Co., LTD Xinchang Zhejiang, China.), Catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA) and protein quantization measurement kits were purchased form Nanjing Jiancheng Bioengineering Institute.
Preparation of the lignans from Fructus Schisandrae chinensis (LFS) and Astragalus Polysaccharides (APS) The dried Fructus Schisandrae chinensis collected from Jilin Province, and Radix Astragali purchased from Shanghai Hua Yu Chinese Herbs Co., Ltd., were identified by Prof. Lu-Ping Qin, Department of Pharmacognosy, Second Military Medical University. The Fructus Schisandrae chinensis was extracted with 95% ethanol by reflux twice, and each time for 2 hours. The extract was concentrated under reduced pressure, and then dissolved in 30% ethanol to give 1 g/ml (crude medicine/volume) sample liquor, and then this liquor was passed through AB-8 macroporous resin column with 1 time the volume which was comparable to the weight of macroporous resin. The lignans were adsorbed
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300 250
Schisandrin
150 150
Deoxyschizandirn
r-Schisandrin
32.698
100
40.284
MAU
200
50 0 0
10
20
30
40
50
Min
Fig. 1. HPLC chromatogram of lignans in Fructus Schisandrae chinensis.
onto macroporous resin column and eluted with 30%, 70%, and 95% ethanol, respectively. The combined elutes were concentrated under reduced pressure and 70% ethanol elutes was used as the total lignans from Fructus Schisandrae chinensis (LFS). The content determination of total LFS (including schisandrin, deoxyschizandrin and r-Schisandrin) was performed on Agilent 1200 series (HPLC) including a quaternary pump, vacuum degasser, thermostatic column compartment and a diode array detector (DAD). An Agilent Zorbax SB-C18 column (250 mm 4.6 mm I.D., 5 mm) with a Extend C-18 guard column (10 4.6 mm, 5 mm) was used. Gradient elution was employed using solvent systems C (acetonitrile) and B (aqueous) at ambient temperature. The gradient program used was as follows: initial 0–13 min, C–B (48:52, v/v); 13–30 min, linear change to C–B (52:48, v/v); 30–55 min, linear change to C–B (80:20, v/v); The flow rate was 1.0 ml min1 and column temperature was maintained at 30 1C. The detector wavelength was set at 254 nm and an aliquot of 20 ml solution was injected for acquiring chromatograms (Fig. 1). The content of LFS was 23.1% (W/W), including schisandrin 17.5% (W/W), deoxyschizandrin 2.7% (W/W) and r-Schisandrin 2.9% (W/W). The Astragali Radix was extracted 3 with 12 vol of distilled water at 75 1C for 1.5 h. The extracts was concentrated to 1.2 g crud drug/ml, and precipitated with 95% EtOH (1:4, v/v) at 4 1C for 12 h. The precipitating process was repeated for two times to obtain crud polysaccharides. The crud polysaccharide was suspended in distilled water, and was applied to a column of DEAE-Sepharose CL-6B, eluted with different concentrations of NaCl aqueous solution to obtain
APS.The glucose concentration in APS was 51% (w/w) which detected by the phenol–sulfuric acid standard curve method on UV Spectrophotometer. Polysaccharides compositional analysis by TLC and gas chromatography showed that it consisted of D-glucose, D-galactose, L-arabinose (1.75:1.63:1). The optical rotation was (a) D30.5+101.5 (H2O) detected by polarimeter. The average molecular of the extract was 35 800 as determined by gel filtration method.
Animals and experimental protocol Eighty male Sprague-Dawley rats (180-200 g) were purchased from SLACOM experimental animal company (Shanghai, China) and acclimated to conditions for 1 week before the experiment. The experimental animals were housed in an air-conditioned room with 12 h/12 h light-dark illumination cycles at constant temperature 2470.5 1C and humidity (45-50%). Food and drinking water were supplied ad labium and the rats were weighed weekly during the experimental period. Ten rats were treated with vehicle (deionized water) as normal control. The remaining 70 rats were administrated subcutaneous injections of CCl4 in arachis oil (1:9) at a dose of 5 ml/kg twice a week and equally randomized into seven groups. They were treated with vehicle (deionized water), Bifendate Pills (100 mg/kg once daily), APS (450 mg/kg once daily), LFS (135 mg/ kg once daily), LFS (15 mg/kg)+APS (50 mg/kg), LFS (45 mg/kg)+APS (150 mg/kg), LFS (135 mg/kg)+APS (450 mg/kg) (once daily), for 3 months respectively. At the end of the treatment, rats were fasted for 8 h after the last treatment. The blood was drawn from all groups
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via the abdominal artery for the assessment of biochemical parameters ALT, AST and ALP. The liver sample was collected, the left hepatic lobe was cut into 1.5 cm 1.5 cm 2 cm (length width height) slices, and fixed in the 10% formalin for the histopathological examinations, the right hepatic lobe was kept at 80 1C to measure the activity of catalase (CAT), superoxide dismutase (SOD), reduced glutathione hormone (GSH), malondialdehyde (MDA) content and total protein. This experiment was approved by the Bioethic Committee of the Second Military Medical University (Shanghai, China), and the procedures of the experiment were strictly according to the generally accepted international rules and regulations.
Determination of enzyme levels in serum Serum ALT, AST, and ALP were measured on an automatic analyzer (HITACHI 7600-020, Japan) using diagnostic reagent kit.
Determination of biological parameters of liver tissue Each right hepatic lobe sample was washed thoroughly in ice-cold saline to remove the blood after thawing, blotted the saline gently using filter paper. 0.15 g of each sample was placed in 1.35 ml saline and ten percent of homogenate in the vitric homogenizer at 20 1C was prepared. The homogenate was centrifuged at 3000 rpm for 10 min, the supernatant was used for the estimate of catalase (CAT), superoxide dismutase (SOD) activities, and reduced glutathione (GSH), malondialdehyde (MDA) and protein content. The CAT, SOD, GSH, MDA and proteins were estimated using kits according to the manufacturers’ instructions. Table 1.
Histopathological examinations Liver tissue sections was dissected and fixed in 10% formalin, then processed routinely, embedded in paraffin, sectioned to 4 mm thickness, deparaffinized, rehydrated using standard techniques, stained with hematoxylin and eosin (H&E). The extent of CC14induced liver necrosis and steatosis was evaluated by assessing morphological changes in liver sections.
Analysis of LFS and APS interaction The coefficient of drug interaction (CDI) was used to analyze the synergistically effect of drug combinations. (Cao and Zhen 1989; Wan et al. 2008) CDI is calculated as follows: CDI ¼ AB/(A B). AB is the ratio of the corresponding parameters of the combination groups to toxin control group; A or B is the ratio of these parameters in the single agent groups to toxin control group. Thus CDI value less than, equal to or greater than 1 indicates that the drugs are synergistic, additive or antagonistic, respectively. CDI less than 0.7 indicates that the drugs are significantly synergistic.
Statistical analysis Data was expressed as mean7SD. The differences among different groups were analyzed using one-way analysis of variance (ANOVA) with Student’s t-test.
Results Serum ALT, AST and ALP level Administration of CC14 caused significant elevation of serum ALT, AST and ALP activities in rats
Effects of APS and LFS on serum transaminase level.
Groups
ALT (u/l)
AST (u/l)
ALP (u/l)
Normal control Toxin control (CCl4) CCl4+bifendate (100 mg/kg) CCl4+APS (450 mg/kg) CCl4+LFS (135 mg/kg) CCl4+LFS (15 mg/kg)+APS (50 mg/kg) CCl4+LFS (45 mg/kg)+APS (150 mg/kg) CCl4+LFS (135 mg/kg)+APS (450 mg/kg)
33.676.9 202.2730.8DDDD 101.4718.0**** 175.7719.6 137.5738.8** 129.7714.1 120.4715.7*** 88.7715.2****(0.74)
103.8716.1 286.4727.0DDDD 176.7726.7**** 247.9724.3 189.5723.1*** 194.9725.6** 177.6722.5*** 153.2718.1****(0.93)
85.2714.0 195.3722.1DDDD 138.8714.9*** 181.0722.3 156.4727.7* 188.3740.0 142.1728.5*** 131.2726.6****(0.91)
Values are expressed as mean7SD of ten rats in each group. D po0.05, DDpo0.01, DDDpo0.005, DDDDpo0.001, compared with normal control group. *po0.05, **po0.01,***po0.005, ****po0.001, compared with toxin control group. Numbers in parentheses indicate the coefficient of drug interaction. ALT (alanine aminotransferase). AST (asparate aminotransferase). ALP (alkaline phosphatase).
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compared with normal group. The protective effects of LFS and APS on the CC14 induced elevation of serum ALT, AST and ALP activities are presented in Table 1. The activities of serum ALT, AST and ALP in the CC14 group were much higher than those in the normal group. Only administration of APS did not cause significant changes in the serum transaminase activities, whilst administration of LFS, serum ALT (po0.01), AST (po0.001) activities decreased in CC14 treated rats. However, the administration of a combination of LFS and APS significantly prevented the elevation of serum transaminase activities induced by CC14 treatment in a dose-dependent manner. At the dose of 135 mg/kg LFS combination with 450 mg/kg APS, Serum ALT, AST and ALP activities respectively decreased 56%, 47%,
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and 33% compared with toxin control, and the CDIs of ALT, AST and ALP activities between their combination and the single treatment were all less than 1.
Biochemical parameters of liver tissue As shown in Fig. 2A, the hepatic superoxide dismutase (SOD) activity in the CC14 group was significantly reduced by 21% when compared with the vehicle control group. LFS or APS did not affect SOD activity in CC14 treatment rats. However, administration of ‘LFS(135 mg/ kg)+APS (450 mg/kg)’ significantly (po0.005) increased the SOD activity from 98.9 (u/mgprot) to 119.3 (u/mgprot). The CDI of SOD activity between the
Fig. 2. Dpo0.05, DDpo0.01, DDDpo0.05, DDDDpo0.001, compared with normal control group; *po0.05, **po0.01, ***po0.005, ****po0.001, compared with toxin control group; Group I: Normal Control. Group II: Toxin control CCl4. Group III: CCl4+bifendate (100 mg/kg). Group IV: CCl4+APS (450 mg/kg). Group V: CCl4+LFS (135 mg/kg). Group VI: CCl4+LFS (15 mg/kg)+APS (50 mg/kg). Group VII: CCl4+LFS (45 mg/kg)+APS (150 mg/kg). Group VIII: CCl4+LFS (135 mg/kg)+APS (450 mg/kg). CDI (coefficient of drug interaction) see Materials and methods.
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‘LFS(135 mg/kg)+APS (450 mg/kg)’ treatment group and their single treatment group was 0.86. The hepatic reduced glutathione (GSH) content significantly (po0.005) decreased in the CC14 group when compared with the vehicle control group. LFS or APS did not affect GSH con in CC14 treatment rats. However, the combination of LFS with APS at dose of ‘LFS(135 mg/kg)+APS (450 mg/kg)’ significantly (po0.005) increased the GSH concentration when compared with the CC14 group. The CDI of GSH activity between the ‘LFS(135 mg/kg)+APS (450 mg/kg)’ treatment group and their single treatment group was 0.84 (Fig. 2B). Malondialdehyde (MDA) concentration in the CC14 group was significantly increased to the 4-fold level when compared with the vehicle control group. LFS and APS significantly (po0.005) decreased MDA activity in CC14 treatment rats respectively at dose of 135 and 450 mg/kg. The combination of LFS with APS significantly (po0.001) decreased the MDA concentration when compared with the CC14 group in a dosedependent manner. The combination of LFS with APS is more effective than LFS or APS at the same dose in decreasing MDA level in liver tissue of CC14-treatment rats as shown in Fig. 2C. And the CDI of MDA content between the ‘LFS (135 mg/kg)+APS (450 mg/kg)’ treatment group and the single LFS and APS treatment group was 0.94. Catalase (CAT) activity in the CCl4 group was also found significantly (po0.001) depleted in CC14 treatment group as compared to the normal control group. However, the activity of CAT was significantly (po0.001) elevated in group which treated with 135 mg/kg LFS combination with 450 mg/kg APS. Administration of LFS or APS only did not display effect of increase the CAT activity. The CDI of CAT activity between the ‘LFS (135 mg/kg)+APS (450 mg/kg)’ treatment group and their single treatment group was 0.85 (Fig. 2D).
Histopathological change The normal liver histological sections showed that hepatocytes were well-preserved and uniform cytoplasm, prominent nucleus, nucleolus and central central veins were visible (Fig. 3A). Compared with the normal group, liver tissue in the rats treated with CC14 revealed extensive liver injuries, characterized by severe hepatocellular degeneration and necrosis around the central vein, fatty changes, inflammatory cell infiltration, congestion, sinusoidal dilatation, cytoplasmic vacuolation, massive fatty degeneration and the loss of cellular boundaries (Fig. 3B). However, the histopathological hepatic lesions induced by administration of CC14 were remarkably ameliorated by LFS, APS and
their combinations in a dose-dependent manner (Figs. 3C–H), and this was in good agreement with the results of serum aminotransferase activity and hepatic oxidative stress level. Administration combination of LFS with APS improved the hepatocellular injury induced by CC14 in rats better than that of only LPS or APS.
Discussion In the present study, we evaluated the effects of LFS, APS and their combination on serum ALT, AST and ALP level, antioxidant enzymes of liver tissue and liver histopathological change in CC14 treated rats. The results showed that LFS and their combination were effective in the prevention of CC14-induced liver injury, and the effects of the combination of LFS with APS are more significant than that of LFS or APS on their own. The CDIs of serum transaminase activities and hepatic antioxidant enzymes between the ‘LFS(135 mg/kg)+ APS (450 mg/kg)’ treatment group and their single treatment group were less than 1, which indicated that LFS and APS had synergistic effects on hepatoprotection and antioxidation in CC14-treated rats. Animal liver damage by CCl4 has been widely used to evaluate liver protective activity of drugs (Li 2006), and carbon tetrachloride (CCl4) is a well-known hepatotoxic agent. Toxicity begins with the change in endoplasmic reticulum, which result in the loss of metabolic enzymes located in the intracellular structures (Recnagal 1983). The concentration of transferase in hepatocyte is about 1000–5000 times higher than in serum normally. When liver was injured by CCl4, membrane permeability of liver parenchyma cell intensified, the activities of ALT and AST in serum increased sharply as a consequence and serum aminotransferase activities have long been considered as sensitive indicators of hepatic injury. In the present study, the 10% CC14 at dose of 5 ml/kg for 3 months caused a dramatic elevation in serum ALT, AST, ALP activities and liver tissue histopathological change, indicating an hepatotoxicity induced by administration of CC14. Administration of combinations of LFS and APS prevented the CC14-induced elevation of serum transaminase activities in a dose-dependent manner, indicating the hepatoprotective activity of these against the intoxication of CC14. This was also confirmed by the results of histopathological examination, as evidenced by a dose-related decrease in the incidence and severity of histopathological hepatic lesions. On the other hand, the basis of CC14 hepatotoxicity lies in its biotransformation through the cytochrome P450 system to two free radicals. During metabolic process of CC14, CC14 is firstly transferred into a trichloromethyl free radical, forming covalent adducts
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Fig. 3. Histopathology of the liver ( 100). A: Normal control. B: Toxin control (treated with CCl4). C: Treated with CCl4+bifendate (100 mg/kg). D: Treated with CCl4+APS (450 mg/kg). E: Treated with CCl4+LFS (135 mg/kg). F: Treated with CCl4+LFS (15 mg/kg)+APS (50 mg/kg). G: Treated with CCl4+LFS (45 mg/kg)+APS (150 mg/kg). H: Treated with CCl4+LFS (135 mg/kg)+APS (450 mg/kg).
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with lipids and proteins, which can interact with O2 to form a second metabolite, a trichloromethylperoxy free radical, or can remove hydrogen atoms to form chloroform. This sequence of events leads to lipid peroxidation of membranes and consequent liver injury (Sheweita et al. 2001; Brattin et al. 1985). In response to this hepatocellular injury, ‘activated’ hepatic Kupfer cells release increased quantities of active oxygen species and other bioactive agents (Eisisi et al. 1993). Malondialdehyde (MDA), a secondary product of lipid peroxidation, is used as an indicator of tissue damage involving a series of chain reactions. It reacts with thiobarbituricacid, producing red-colored products. Lipid peroxidation has been implicated in the pathogenesis of increased membrane rigidity, osmotic fragility, reduced erythrocyte survival and perturbations in lipid fluidity (Ohkawa et al. 1979). The observation of elevated levels of hepatic MDA in CCl4-treated rats (toxin group) in the present study is consistent with this hypothesis. Thus, the maintenance of near normal levels of hepatic MDA in rats administered combination of LFS and APS is of supporting evidence to suggest an antioxidant role for LFS and APS. A major defense mechanism involves the antioxidant enzymes, including SOD, CAT, and GSH which convert active oxygen molecules into non-toxic compounds. The enzymatic antioxidant defense systems are the natural protector against lipid peroxidation (Zimmermann et al. 1973). These enzymes prevent generation of hydroxyl radical and protect the cellular constituents from oxidative damage. A reduction in the activities of these enzymes is associated with the accumulation of highly reactive free radicals, leading to deleterious effects such as loss of integrity and function of cell membranes. Administration of CCl4 leads to generation of peroxy radical O2, which is associated with inactivation of CAT, GSH, SOD enzymes (Cheeseman 1995; Mates et al. 1999). This probably explains the significantly reduced activities of CAT, GSH, SOD observed in rats treated with CCl4. In CCl4-treated rats, administration of LFS, APS and their combination caused the increase of CAT, SOD activities and GSH content ,especially in the group of treated with 135 mg/kg LFS combination with 450 mg/kg APS. An interesting phenomenon in our experiment is that single administrated APS did not significantly change the serum ALT, AST, ALP and biochemical parameters of liver tissue in CCl4 treated rats, and single administrated LFS, the abovementioned indicator were improved to some extent, but when LFS was combined with APS, its effects on serum and liver tissue related parameters were significantly enhanced, and the calculate result of the coefficient of drug combination between the ‘LFS(135 mg/kg)+APS (450 mg/kg)’ treatment group and their single treatment group give a strong evidence of the synergistic protective effect of
LFS and APS on CCl4 treated rats. The synergistic mechanism may relate with the immunomodulatory effects of APS. Recent research has shown that components of the innate immune system are intimately associated with liver disease and hepatic regeneration. It is well known that TNF plays a major role in alcoholic liver disease, and that cytokines such as Il-6, may contribute to fibrotic processes. More surprising, however, was the realization that some components of the innate immune system, including TNF and IL-6 may participate in the initiation of liver regeneration after partial hepatectomy (Mei et al. 2007). It has been reported that the APS have significant immunomodulatory effects, including promoting humoral immunity and cellular immunity, improving the immune functions of the immunosuppressive model mice (Shao et al. 2004), and modulating the production of cytokines (Schepetkin and Quinn 2006). Therefore, APS may enhanced the hepatoprotective effects of LFS by modulating function of immune system. In conclusion, LFS and APS provide significant protective effects on CCl4-induced hepatic injury through decreasing the serum levels of ALT, AST and ALP, and enhancing SOD, CAT, GSH activities. The data from CDI result revealed that LFS combined with APS has a synergistic action in reducing ALT, AST and restoring SOD and GSH levels, and was superior to the administration of either LFS or APS. These synergy effects have been first described with examples in two review articles from Wiliamson (2001) and UlrichMerzenich et al. (2007). Therefore, dietary LFS and APS may be useful as a hepatoprotective agent again. However, the hepatoprotective mechanisms of these medicinal herb constituents remain to be elucidated.
Acknowledgements Authors are thankful to Hangzhou Chinese traditional medicine modernization research center for the financial assistance, and Inspection Branch of Shanghai Eastern Hepatobiliary Hospital for analyzing serum biochemical parameters.
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