http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–6 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.941506
SHORT COMMUNICATION
Hepatoprotective effect of ethanol extract from Berchemia lineate against CCl4-induced acute hepatotoxicity in mice Cong Li, Li-Tao Yi, Di Geng, Yuan-Yuan Han, and Lian-jin Weng
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Department of Chemical and Pharmaceutical Engineering, College of Chemical Engineering, Huaqiao University, Xiamen, Fujian Province, PR China
Abstract
Keywords
Context: The roots of Berchemia lineate (L.) DC. (Rhamnaceae) have been long used as a remedy for the treatment of some diseases in Guangxi Province, China. Objective: The present study investigates the hepatoprotective effect of Berchemia lineate ethanol extract (BELE) on CCl4-induced acute liver damage in mice. Materials and methods: Effect of BELE administrated for 7 consecutive days was evaluated in mice by the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), albulin (ALB), globulin (GLB), and total protein (TP) levels, as well as liver superoxide dismutase (SOD) activity and malondialdehyde (MDA) level. Moreover, histopathological examinations were also taken. Results: Compared with the model group, administration of 400 mg/kg BELE for 7 d in mice significantly decreased the serum ALT (56.25 U/L), AST (297.67 U/L), ALP (188.20 U/L), and TBIL (17.90 mol/L), along with the elevation of TP (64.67 g/L). In addition, BELE (100, 200, and 400 mg/kg, i.g.) treated mice recorded a dose-dependent increment of SOD (291.17, 310.32, and 325.67 U/mg prot) and reduction of MDA (7.27, 6.77, and 5.33 nmol/mg prot) levels. Histopathological examinations also confirmed that BELE can ameliorate CCl4-induced liver injuries, characterized by extensive hepatocellular degeneration/necrosis, inflammatory cell infiltration, congestion, and sinusoidal dilatation. Discussion and conclusion: The results indicated that BELE possessed remarkable protective effect against acute hepatotoxicity and oxidative injuries induced by CCl4, and that the hepatoprotective effects of BELE may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity.
Antioxidant, hepatoprotection, serum biomarkers
Introduction There is vast evidence indicating that herbal extracts from edible and medicinal plants exhibit strong antioxidant activity that could act against hepatic toxicity caused by various toxicants (Harish & Shivanandappa, 2006; Madureira et al., 2011; Upur et al., 2009; Yeh et al., 2012; Zeashan et al., 2008). The roots of Berchemia lineate (L.) DC. (Rhamnaceae) have been long used as a remedy for the treatment of stomachache, gall stones, lumbago, and rheumatic arthritis in folk medicine in Guangxi Province, China (Teng, 2010). Previous phytochemical investigation revealed that lignans, quinones, flavones, and polyphenolic compounds largely found in this genus (Zhang et al., 2011). According to the research, Berchemia lineate exhibits anticancerous, antiviral, antifungal, and analgesic activities (Chen, 2011; Mukhtar, 2004; Sun & Li, 2009; Wu, et al., 2008). However, there is little Correspondence: Lian-jin Weng, Department of Chemical and Pharmaceutical Engineering, College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian Province, PR China. Tel: +86 592 6162286. Fax: +86 592 6162300. E-mail: ljwenghquxm@ 163.com
History Received 9 November 2013 Revised 31 March 2014 Accepted 28 June 2014 Published online 27 November 2014
research reported about hepatoprotective on Berchemia lineate which comes from Anxi, Fujian province, China. The liver is an important organ for the detoxification and deposition of endogenous and exogenous substances and hence it is susceptible to almost as different disease. Hepatotoxins, such as ethanol, acetaminophen, and carbon tetrachloride (CCl4), trigger liver injury which is characterized by varying degrees of hepatocyte degeneration and cell death (Wu et al., 1999). CCl4 is one of the oldest and most widely used toxins for experimental induction of liver injury in laboratory animals (Brautbar & Williams, 2002). Hepatotoxicity has been reported as one of the damages caused by free radicals (Plummer, 1982). Terminal-deoxynucleoitidyl transferasemediated nick end labeling (TUNEL) assay showed that CCl4-induced apoptosis in mouse liver was remarkable (Ma et al., 2014) and in vitro study indicated that CCl4 treatment caused a significant decrease in human liver hepatoma cells’ viability (Pareek et al., 2013). Therefore, CCl4, a potent hepatotoxin metabolized by cytochrome P450 to yield toxic intermediate, trichloromethyl radicals which cause increase in hepatic lipid peroxidation and consequently liver damage and oxidative stress (Shah, 1979), was utilized
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in this study. A multitude of studies suggested that herbal extracts from edible and medicinal plants exhibited strong antioxidant activity that could against CCl4-induced liver damage (Desai et al., 2012; Sreelatha et al,. 2009; Yang et al., 2010; Yeh et al., 2012). Hence, considering that quinines and flavones posses anticancer and hepatoprotective activities (Chen et al., 2011), we used a model of liver damage induced by CCl4 in mice to study the protective effect by using ethanolic extract (BELE) Berchemia lineate (L.) DC.
Group 1: CMC + Olive oil
Group 4: BELE 100 mg/kg + CCl4
Group 2: CMC + CCl4
Group 5: BELE 200 mg/kg + CCl4
Group 3: SL 200 mg/kg+ CCl4
Group 6: BELE 400 mg/kg + CCl4
Materials and methods
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Chemical and reagents
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The diagnostic kits for superoxide dismutase (SOD), malondialdehyde (MDA), and total protein (TP) are from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Silymarin (SL) is from Aladdin, Shanghai Jingchun Reagent Co., Ltd. (Shanghai, China). CCl4, olive oil, petroleum ether, CCl3, n-butyl alcohol, picric acid, and sodium carboxymethylcellulose (CMC) are from Xilong Chemical Industry Co., Ltd. (Shantou, China). According to the MSDS (Material Safety Data Sheet), the LD50 of CCl4 in rat is 2350 mg/kg body weight via per os and 5070 mg/kg body weight via per cutem. Animals Male Kunming mice (weighing 18–22 g), procured from the Wushi Experimental Animal Center (Fuzhou, China) were used. We used only male mice because of their constant metabolism compared with the variations in the female physiology. All animals were housed in standard environmental conditions with eight animals per polyacrylic cage and had free access to distilled water and a standard rodent diet which was purchased from Wushi Experimental Animal Co., Ltd. (Fuzhou, China). The mice were acclimatized to laboratory condition for a week prior to experiment. All procedures were performed in compliance with the published guidelines for the care of experimental animals and with internationally accepted principles for the use and care of laboratory animals by China Scientific and Technological Committee (approval 2/1988/10/31). Preparation of BELE The Berchemia lineate was provided from Anxi, Fujian province (Anxi, China). It was authenticated by Xue-hua Song, the herbarium curator of China Pharmaceutical University (Nanjing, China). The voucher specimen (No. 12-101507) was deposited in Department of Natural Medicinal Chemistry, China Pharmaceutical University. The sample was extracted three-times with 95% (v/v) ethanol for 2 h under reflux followed by rotary steaming at 60 C. After that, the extract was freeze dried and stored in a vacuum desiccator. Prior to the experiment, Berchemia lineate ethanol extract (BELE) and silymarin (SL) were dissolved in 0.5% CMC.
CCl4/Olive oil BELE/Silymarin/CMC
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Figure 1. Experimental design scheme.
was maintained as control and was given 10% CMC. They were observed for any gross behavioral changes and death after dosing with BELE which was continuous for 2 weeks (Sreelatha et al., 2009) as per guideline OECD-425 (2001). Experimental groups and dose selection Mice were divided into six groups of eight animals each. Figure 1 shows the experimental design scheme. The control group (group 1) and the model group (group 2) were given 0.5% CMC once daily for 7 d, intragastrically (i.g.). Group 3 was administered by SL once daily for 7 consecutive days. BELE was given i.g. at dose of 100, 200, and 400 mg/kg (group 4 to group 6) (Yeh et al., 2012). On the third and fifth days, the group 1 was administered by olive oil, intraperitoneally (i.p.) after 30 min of i.g. CCl4, which was dissolved in olive oil (1%, v/v) was given i.p. to the rest groups after 30 min of i.g. Autopsy and serum biochemistry Following BELE treatment for 7 d, food, but not water, was withdrawn from the animals 24 h prior to decapitation. Blood was gained from the eyeballs and serum was separated by centrifugation at 10 000 g at 4 C for 10 min. The serum was stored in the 80 C freezer until they were analyzed (Sreelatha et al., 2009). The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), albulin (ALB), globulin (GLB), and TP values were evaluated by an autoanalyzer (Beckmann, America). Histopathological examinations The livers were excised immediately after sacrifice. A portion of the median lobe of liver was dissected and fixed in 10% neutral buffered formalin solution for 24 h. The rest of the livers was washed with saline, blotted dry, divided into samples, frozen quickly, and stored at 80 C. The fixed tissues were processed routinely, embedded in paraffin, sectioned to 3–5 mm thickness, deparaffinized, and rehydrated using standard techniques. Sections were taken and stained with hematoxylin and eosin (H&E).
Behavioral and toxic effects
Preparation of hepatic homogenate
Different groups of mice were treated with graded doses of BELE (100, 200, 400, and 5000 mg/kg, i.g.). One group
Thawed tissue samples were homogenized in a glass-Teflon homogenizer (XHF-D, Haimen, China) with nine volumes
Hepatoprotective effect of ethanol extract from Berchemia lineate
DOI: 10.3109/13880209.2014.941506
of ice cold 0.86% saline, centrifuged at 12 000 g for 15 min at 4 C (L-800 R, Frankfurt, Germany). The supernatant was used for determination of SOD, MDA, and protein concentration by a spectrophotometer (MAPADA UV-1800, Shanghai Mapada Instruments Co., Shanghai, China) using commercially available diagnostic kits. The levels of SOD and MDA were normalized with protein.
Lipid peroxidation was assayed by the thiobarbituric acid (TBA) reaction method. MDA, a measure of lipid peroxidation, was calculated using an extinction coefficient. The results were expressed as nmol/mg protein. The activity of SOD was evaluated according to the method of hydroxylamine. The concentration of TP was measured using Coomassie brilliant blue (CBB). Statistical analysis All the tests were expressed as mean ± S.E.M (n ¼ 6). To compare experimental and control groups, we used oneway ANOVA, followed by post hoc Tukey test using the SPSS
software (SPSS Inc., Chicago, IL) 20.0. A value of p50.05 was considered statistically significant for analysis.
Results Toxicity studies No mortality was observed up to a dose level of 5000 mg/kg body weight. Physically, the mice appeared normal and no signs of changes were observed in their skins, furs and eyes. Tremor, sleep, and behavior patterns were similar to the normal group. Their food intakes were normal and neither diarrhea nor vomiting was noticed. Moreover, dissection results showed there was no damage in liver and kidney. Serum markers of liver damage The activities of various biochemical enzymes in normal, CCl4 control and BELE treated groups are presented in Figure 2. The activities of ALT, AST, and ALP are significantly increased in CCl4 control group with a significant decrease in total protein levels compared with the normal control group. Administration with BELE at a dose of 400 mg/kg can reverse the levels of the above enzymes. 400
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Figure 2. Effects of BELE on the serum ALT (a), AST (b), ALP (c), TBIL (d), ALB (e), GLB (f), and TP (g). ##p50.01, ###p50.001 versus the vehicle-control group (control); *p50.05, **p50.01, and ***p50.001 versus vehicle with the CCl4 group (model).
TP levels (g/L)
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Determination of lipid peroxidation and antioxidant enzymes
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C. Li et al.
Pharm Biol, Early Online: 1–6
Compared with the model group, administration of 400 mg/kg BELE for 7 d in mice significantly decreased the serum ALT, AST, ALP, and TBIL levels by 52.5, 17.3, 12.3, and 18.5%, along with the elevation of TP by 25.0%. The levels are similar to that treated with silymarin. However, the levels of ALB and GLB almost have no difference among the normal, CCl4 control, and BELE-treated groups.
As depicted in Figure 3, the hepatic antioxidant enzyme activities of SOD were decreased in the liver of mice treated with CCl4. Consistent with the serum ALT, AST, and ALP activities; pretreatment with BELE groups indicated a significant dose-dependent increase in the activities of SOD. The concentrations of MDA, which is an end product of lipid peroxidation, in mice treated with CCl4 distinctly Figure 3. Effects of BELE on the hepatic SOD (a) content and MDA (b) activity. ###p50.001 versus the vehicle-control group (control); *p50.05, **p50.01, and ***p50.001 versus vehicle with the CCl4 group (model).
Histopathology The results of hepatic histopathological examinations are shown in Figure 4. Compared with the normal liver tissues, liver tissues in the mice treated with CCl4 revealed extensive liver injuries, characterized by moderate to severe hepatocellular degeneration/necrosis around the central vein, inflammatory cell infiltration, congestion, and sinusoidal dilatation. However, pretreatment with BELE (400 mg/kg, Figure 4e) can remarkably ameliorate the histopathological hepatic lesions induced by administration of CCl4. The silymarintreated group demonstrated almost normalization of fatty accumulation and necrosis. The maximum of protection against hepatic injury was achieved with BELE at dose of 400 mg/kg (Figure 4e).
(a) SOD levels (U/mg prot)
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Hepatic antioxidant enzyme activity and lipid peroxidation
increased when compared with the vehicle control mice. However, the level of MDA in drug treated groups was significantly decreased.
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Figure 4. Effects of BELE on hepatic morphological analysis ( 400 H&E): control group (a), CCl4-model group (b), Silymarin + CCl4 group (c), BELE 100 mg/kg + CCl4 group (d), BELE 200 mg/kg + CCl4 group (e), and BELE 400 mg/kg + CCl4 group (f) (*inflammatory infiltration; #dilated sinusoidal spaces).
DOI: 10.3109/13880209.2014.941506
Hepatoprotective effect of ethanol extract from Berchemia lineate
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Discussion The present study reports the potential hepatoprotective activity of BELE against hepatic injury produced by CCl4 in mice. CCl4 is a well-known hepatotoxic agent and widely used that hepatoxicity derives from its metabolites (highly reactive trichloromethyl free radicals). This radical could react with oxygen to form the trichloromethylperoxy radical CCl3OO, which is a highly reactive species that initiate the chain reaction of lipid peroxidation, attack, and destroy polyunsaturated fatty acid, especially those associated with phospholipids (Weber et al., 2003). These processes affect the permeabilities of mitochondrial, endoplasmic reticulum, and plasma membranes, finally in leakage of liver enzymes into the blood. Therefore, estimation of enzymes in the serum is useful quantitative markers of the extent and types of hepatocellular damage (Jandon et al., 2007). As the results shown, administration of BELE to mice up to the dose of 5000 mg/kg was observed no mortality and intraperitoneal administration of CCl4 presented significant hepatotoxity, as evidenced by a dramatic elevation in the serum ALT, AST, and ALP activities and increased incidence and severity of histopathological hepatic damages in mice, along with a reduction of TP. Serum aminotransferase activities are indicative of cellular leakage and loss of functional integrity of cell membranes in liver (Rajesh & Lathe, 2004). Injury to the hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells (Zimmerman & Aeeff, 1970). Total protein is measured as a routine test to evaluate the toxicological nature of various chemicals (Nevin & Vijayammal, 2005). In chronic damaged liver, hepatic fibrosis is a common symptom, the level of GLB, ALB, and the ratio of ALB/GLB could be affected (Jiang et al., 2013). CCl4 is having an acute toxicity, according to the results, ALB and GLB have no difference among the normal, CCl4 control, and treated groups in this research. Moreover, the significant decrease in SOD activity and remarkable elevation in hepatic MDA level suggest a role of oxidative stress in CCl4 hepatotoxicity (Sreelatha et al., 2009). While, treated with BELE in mice indicated a dramatical protective effect against CCl4-induced acute hepatotoxicity and oxidative stress which were also confirmed by the hepatic histopathological examinations. The stimulation of hepatic regeneration makes the liver more resistant to damage by the toxin (Sadasivan et al., 2006). BELE inhibited liver lipid peroxidation and resultant tissue degeneration, thus acting as an effective antioxidant. Previous studies showed that Berchemia lineate largely possessed lignans, quinones, flavones, and polyphenolic compounds (Zhang et al., 2011), which have potent antioxidant activity (Aejaz & Neelakantan, 2013; Mitra et al., 2000; Urszul et al., 2009; Xin et al., 2008). MDA is a major reactive aldehyde that appears during the peroxidation of biological membrane polyunsaturated fatty acid (Vaca et al., 1988). An increase in MDA level in the liver suggests enhanced peroxidation leading to tissue damage and failure of the antioxidant-defense mechanisms to prevent the formation of excessive free radicals (Nail, 2003). Therefore, the concentration of MDA in liver tissue is used
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as an indicator of liver injury involving a series of chain reactions (Ohkawa et al., 1979). SOD is an effective defense enzyme that catalyzes the dismutation of superoxide anions into hydrogen peroxide (H2O2) (Reiter et al., 2000). Lipid peroxidant or reactive oxygen species could easily inactivate these antioxidant enzymes in toxicity (Polavarapu et al., 1998; Yang et al., 2008). The results revealed that BELE can store the activity of SOD and inhibit the lipid peroxidation so as to stabilizing the plasma membrane. The phenolic hydroxyl on the benzene ring is an antioxidant activity group to antagonize tissue damages, which was caused by free radicals, by eliminating free radicals, inhibiting oxidation reactions, and combining with biological membrane phospholipids to protect membrane lipid (Yu, 2009). Previous phytochemical investigation revealed that polyphenolic compounds largely existed in this genus (Zhang et al., 2011). In addition, chrysophanol, physcion, emodin, and aloe-emodin have the ability of scavenging free radicals (Luo et al., 2009) and these constituents exist in Berchemia lineata. So, BELE possessed a certain extent of antioxidant activity. In conclusion, the results of this study demonstrate that BELE can effectively prevent the CCl4-induced hepatic damage in mice and, therefore, it can be used as a hepatoprotective agent. The hepatoprotective effects of BELE may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity. This is the primary report about the hepatoprotective of BELE, further studies with individual active compounds existed in BELE are in proceeding, which will enable us to understand the exact mechanism of hepatoprotective action by BELE and it will help us to further exploit traditional medicine on this plant.
Declaration of interest The authors declare that there are no conflicts of interest. The project was supported by the Natural Science foundation of China (No. 81202940) and the Fundamental Research Funds for the Central Universities of Huaqiao University of China (Nos. JB-ZR1152 and JB-ZR1226).
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SHORT COMMUNICATION
Hepatoprotective effect of ethanol extract from Berchemia lineate against CCl4-induced acute hepatotoxicity in mice Cong Li, Li-Tao Yi, Di Geng, Yuan-Yuan Han, and Lian-jin Weng
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Department of Chemical and Pharmaceutical Engineering, College of Chemical Engineering, Huaqiao University, Xiamen, Fujian Province, PR China
Abstract
Keywords
Context: The roots of Berchemia lineate (L.) DC. (Rhamnaceae) have been long used as a remedy for the treatment of some diseases in Guangxi Province, China. Objective: The present study investigates the hepatoprotective effect of Berchemia lineate ethanol extract (BELE) on CCl4-induced acute liver damage in mice. Materials and methods: Effect of BELE administrated for 7 consecutive days was evaluated in mice by the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), albulin (ALB), globulin (GLB), and total protein (TP) levels, as well as liver superoxide dismutase (SOD) activity and malondialdehyde (MDA) level. Moreover, histopathological examinations were also taken. Results: Compared with the model group, administration of 400 mg/kg BELE for 7 d in mice significantly decreased the serum ALT (56.25 U/L), AST (297.67 U/L), ALP (188.20 U/L), and TBIL (17.90 mol/L), along with the elevation of TP (64.67 g/L). In addition, BELE (100, 200, and 400 mg/kg, i.g.) treated mice recorded a dose-dependent increment of SOD (291.17, 310.32, and 325.67 U/mg prot) and reduction of MDA (7.27, 6.77, and 5.33 nmol/mg prot) levels. Histopathological examinations also confirmed that BELE can ameliorate CCl4-induced liver injuries, characterized by extensive hepatocellular degeneration/necrosis, inflammatory cell infiltration, congestion, and sinusoidal dilatation. Discussion and conclusion: The results indicated that BELE possessed remarkable protective effect against acute hepatotoxicity and oxidative injuries induced by CCl4, and that the hepatoprotective effects of BELE may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity.
Antioxidant, hepatoprotection, serum biomarkers
Introduction There is vast evidence indicating that herbal extracts from edible and medicinal plants exhibit strong antioxidant activity that could act against hepatic toxicity caused by various toxicants (Harish & Shivanandappa, 2006; Madureira et al., 2011; Upur et al., 2009; Yeh et al., 2012; Zeashan et al., 2008). The roots of Berchemia lineate (L.) DC. (Rhamnaceae) have been long used as a remedy for the treatment of stomachache, gall stones, lumbago, and rheumatic arthritis in folk medicine in Guangxi Province, China (Teng, 2010). Previous phytochemical investigation revealed that lignans, quinones, flavones, and polyphenolic compounds largely found in this genus (Zhang et al., 2011). According to the research, Berchemia lineate exhibits anticancerous, antiviral, antifungal, and analgesic activities (Chen, 2011; Mukhtar, 2004; Sun & Li, 2009; Wu, et al., 2008). However, there is little Correspondence: Lian-jin Weng, Department of Chemical and Pharmaceutical Engineering, College of Chemical Engineering, Huaqiao University, Xiamen 361021, Fujian Province, PR China. Tel: +86 592 6162286. Fax: +86 592 6162300. E-mail: ljwenghquxm@ 163.com
History Received 9 November 2013 Revised 31 March 2014 Accepted 28 June 2014 Published online 27 November 2014
research reported about hepatoprotective on Berchemia lineate which comes from Anxi, Fujian province, China. The liver is an important organ for the detoxification and deposition of endogenous and exogenous substances and hence it is susceptible to almost as different disease. Hepatotoxins, such as ethanol, acetaminophen, and carbon tetrachloride (CCl4), trigger liver injury which is characterized by varying degrees of hepatocyte degeneration and cell death (Wu et al., 1999). CCl4 is one of the oldest and most widely used toxins for experimental induction of liver injury in laboratory animals (Brautbar & Williams, 2002). Hepatotoxicity has been reported as one of the damages caused by free radicals (Plummer, 1982). Terminal-deoxynucleoitidyl transferasemediated nick end labeling (TUNEL) assay showed that CCl4-induced apoptosis in mouse liver was remarkable (Ma et al., 2014) and in vitro study indicated that CCl4 treatment caused a significant decrease in human liver hepatoma cells’ viability (Pareek et al., 2013). Therefore, CCl4, a potent hepatotoxin metabolized by cytochrome P450 to yield toxic intermediate, trichloromethyl radicals which cause increase in hepatic lipid peroxidation and consequently liver damage and oxidative stress (Shah, 1979), was utilized
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in this study. A multitude of studies suggested that herbal extracts from edible and medicinal plants exhibited strong antioxidant activity that could against CCl4-induced liver damage (Desai et al., 2012; Sreelatha et al,. 2009; Yang et al., 2010; Yeh et al., 2012). Hence, considering that quinines and flavones posses anticancer and hepatoprotective activities (Chen et al., 2011), we used a model of liver damage induced by CCl4 in mice to study the protective effect by using ethanolic extract (BELE) Berchemia lineate (L.) DC.
Group 1: CMC + Olive oil
Group 4: BELE 100 mg/kg + CCl4
Group 2: CMC + CCl4
Group 5: BELE 200 mg/kg + CCl4
Group 3: SL 200 mg/kg+ CCl4
Group 6: BELE 400 mg/kg + CCl4
Materials and methods
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Chemical and reagents
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The diagnostic kits for superoxide dismutase (SOD), malondialdehyde (MDA), and total protein (TP) are from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Silymarin (SL) is from Aladdin, Shanghai Jingchun Reagent Co., Ltd. (Shanghai, China). CCl4, olive oil, petroleum ether, CCl3, n-butyl alcohol, picric acid, and sodium carboxymethylcellulose (CMC) are from Xilong Chemical Industry Co., Ltd. (Shantou, China). According to the MSDS (Material Safety Data Sheet), the LD50 of CCl4 in rat is 2350 mg/kg body weight via per os and 5070 mg/kg body weight via per cutem. Animals Male Kunming mice (weighing 18–22 g), procured from the Wushi Experimental Animal Center (Fuzhou, China) were used. We used only male mice because of their constant metabolism compared with the variations in the female physiology. All animals were housed in standard environmental conditions with eight animals per polyacrylic cage and had free access to distilled water and a standard rodent diet which was purchased from Wushi Experimental Animal Co., Ltd. (Fuzhou, China). The mice were acclimatized to laboratory condition for a week prior to experiment. All procedures were performed in compliance with the published guidelines for the care of experimental animals and with internationally accepted principles for the use and care of laboratory animals by China Scientific and Technological Committee (approval 2/1988/10/31). Preparation of BELE The Berchemia lineate was provided from Anxi, Fujian province (Anxi, China). It was authenticated by Xue-hua Song, the herbarium curator of China Pharmaceutical University (Nanjing, China). The voucher specimen (No. 12-101507) was deposited in Department of Natural Medicinal Chemistry, China Pharmaceutical University. The sample was extracted three-times with 95% (v/v) ethanol for 2 h under reflux followed by rotary steaming at 60 C. After that, the extract was freeze dried and stored in a vacuum desiccator. Prior to the experiment, Berchemia lineate ethanol extract (BELE) and silymarin (SL) were dissolved in 0.5% CMC.
CCl4/Olive oil BELE/Silymarin/CMC
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Figure 1. Experimental design scheme.
was maintained as control and was given 10% CMC. They were observed for any gross behavioral changes and death after dosing with BELE which was continuous for 2 weeks (Sreelatha et al., 2009) as per guideline OECD-425 (2001). Experimental groups and dose selection Mice were divided into six groups of eight animals each. Figure 1 shows the experimental design scheme. The control group (group 1) and the model group (group 2) were given 0.5% CMC once daily for 7 d, intragastrically (i.g.). Group 3 was administered by SL once daily for 7 consecutive days. BELE was given i.g. at dose of 100, 200, and 400 mg/kg (group 4 to group 6) (Yeh et al., 2012). On the third and fifth days, the group 1 was administered by olive oil, intraperitoneally (i.p.) after 30 min of i.g. CCl4, which was dissolved in olive oil (1%, v/v) was given i.p. to the rest groups after 30 min of i.g. Autopsy and serum biochemistry Following BELE treatment for 7 d, food, but not water, was withdrawn from the animals 24 h prior to decapitation. Blood was gained from the eyeballs and serum was separated by centrifugation at 10 000 g at 4 C for 10 min. The serum was stored in the 80 C freezer until they were analyzed (Sreelatha et al., 2009). The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), albulin (ALB), globulin (GLB), and TP values were evaluated by an autoanalyzer (Beckmann, America). Histopathological examinations The livers were excised immediately after sacrifice. A portion of the median lobe of liver was dissected and fixed in 10% neutral buffered formalin solution for 24 h. The rest of the livers was washed with saline, blotted dry, divided into samples, frozen quickly, and stored at 80 C. The fixed tissues were processed routinely, embedded in paraffin, sectioned to 3–5 mm thickness, deparaffinized, and rehydrated using standard techniques. Sections were taken and stained with hematoxylin and eosin (H&E).
Behavioral and toxic effects
Preparation of hepatic homogenate
Different groups of mice were treated with graded doses of BELE (100, 200, 400, and 5000 mg/kg, i.g.). One group
Thawed tissue samples were homogenized in a glass-Teflon homogenizer (XHF-D, Haimen, China) with nine volumes
Hepatoprotective effect of ethanol extract from Berchemia lineate
DOI: 10.3109/13880209.2014.941506
of ice cold 0.86% saline, centrifuged at 12 000 g for 15 min at 4 C (L-800 R, Frankfurt, Germany). The supernatant was used for determination of SOD, MDA, and protein concentration by a spectrophotometer (MAPADA UV-1800, Shanghai Mapada Instruments Co., Shanghai, China) using commercially available diagnostic kits. The levels of SOD and MDA were normalized with protein.
Lipid peroxidation was assayed by the thiobarbituric acid (TBA) reaction method. MDA, a measure of lipid peroxidation, was calculated using an extinction coefficient. The results were expressed as nmol/mg protein. The activity of SOD was evaluated according to the method of hydroxylamine. The concentration of TP was measured using Coomassie brilliant blue (CBB). Statistical analysis All the tests were expressed as mean ± S.E.M (n ¼ 6). To compare experimental and control groups, we used oneway ANOVA, followed by post hoc Tukey test using the SPSS
software (SPSS Inc., Chicago, IL) 20.0. A value of p50.05 was considered statistically significant for analysis.
Results Toxicity studies No mortality was observed up to a dose level of 5000 mg/kg body weight. Physically, the mice appeared normal and no signs of changes were observed in their skins, furs and eyes. Tremor, sleep, and behavior patterns were similar to the normal group. Their food intakes were normal and neither diarrhea nor vomiting was noticed. Moreover, dissection results showed there was no damage in liver and kidney. Serum markers of liver damage The activities of various biochemical enzymes in normal, CCl4 control and BELE treated groups are presented in Figure 2. The activities of ALT, AST, and ALP are significantly increased in CCl4 control group with a significant decrease in total protein levels compared with the normal control group. Administration with BELE at a dose of 400 mg/kg can reverse the levels of the above enzymes. 400
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Figure 2. Effects of BELE on the serum ALT (a), AST (b), ALP (c), TBIL (d), ALB (e), GLB (f), and TP (g). ##p50.01, ###p50.001 versus the vehicle-control group (control); *p50.05, **p50.01, and ***p50.001 versus vehicle with the CCl4 group (model).
TP levels (g/L)
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Determination of lipid peroxidation and antioxidant enzymes
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Compared with the model group, administration of 400 mg/kg BELE for 7 d in mice significantly decreased the serum ALT, AST, ALP, and TBIL levels by 52.5, 17.3, 12.3, and 18.5%, along with the elevation of TP by 25.0%. The levels are similar to that treated with silymarin. However, the levels of ALB and GLB almost have no difference among the normal, CCl4 control, and BELE-treated groups.
As depicted in Figure 3, the hepatic antioxidant enzyme activities of SOD were decreased in the liver of mice treated with CCl4. Consistent with the serum ALT, AST, and ALP activities; pretreatment with BELE groups indicated a significant dose-dependent increase in the activities of SOD. The concentrations of MDA, which is an end product of lipid peroxidation, in mice treated with CCl4 distinctly Figure 3. Effects of BELE on the hepatic SOD (a) content and MDA (b) activity. ###p50.001 versus the vehicle-control group (control); *p50.05, **p50.01, and ***p50.001 versus vehicle with the CCl4 group (model).
Histopathology The results of hepatic histopathological examinations are shown in Figure 4. Compared with the normal liver tissues, liver tissues in the mice treated with CCl4 revealed extensive liver injuries, characterized by moderate to severe hepatocellular degeneration/necrosis around the central vein, inflammatory cell infiltration, congestion, and sinusoidal dilatation. However, pretreatment with BELE (400 mg/kg, Figure 4e) can remarkably ameliorate the histopathological hepatic lesions induced by administration of CCl4. The silymarintreated group demonstrated almost normalization of fatty accumulation and necrosis. The maximum of protection against hepatic injury was achieved with BELE at dose of 400 mg/kg (Figure 4e).
(a) SOD levels (U/mg prot)
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Hepatic antioxidant enzyme activity and lipid peroxidation
increased when compared with the vehicle control mice. However, the level of MDA in drug treated groups was significantly decreased.
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Figure 4. Effects of BELE on hepatic morphological analysis ( 400 H&E): control group (a), CCl4-model group (b), Silymarin + CCl4 group (c), BELE 100 mg/kg + CCl4 group (d), BELE 200 mg/kg + CCl4 group (e), and BELE 400 mg/kg + CCl4 group (f) (*inflammatory infiltration; #dilated sinusoidal spaces).
DOI: 10.3109/13880209.2014.941506
Hepatoprotective effect of ethanol extract from Berchemia lineate
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Discussion The present study reports the potential hepatoprotective activity of BELE against hepatic injury produced by CCl4 in mice. CCl4 is a well-known hepatotoxic agent and widely used that hepatoxicity derives from its metabolites (highly reactive trichloromethyl free radicals). This radical could react with oxygen to form the trichloromethylperoxy radical CCl3OO, which is a highly reactive species that initiate the chain reaction of lipid peroxidation, attack, and destroy polyunsaturated fatty acid, especially those associated with phospholipids (Weber et al., 2003). These processes affect the permeabilities of mitochondrial, endoplasmic reticulum, and plasma membranes, finally in leakage of liver enzymes into the blood. Therefore, estimation of enzymes in the serum is useful quantitative markers of the extent and types of hepatocellular damage (Jandon et al., 2007). As the results shown, administration of BELE to mice up to the dose of 5000 mg/kg was observed no mortality and intraperitoneal administration of CCl4 presented significant hepatotoxity, as evidenced by a dramatic elevation in the serum ALT, AST, and ALP activities and increased incidence and severity of histopathological hepatic damages in mice, along with a reduction of TP. Serum aminotransferase activities are indicative of cellular leakage and loss of functional integrity of cell membranes in liver (Rajesh & Lathe, 2004). Injury to the hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells (Zimmerman & Aeeff, 1970). Total protein is measured as a routine test to evaluate the toxicological nature of various chemicals (Nevin & Vijayammal, 2005). In chronic damaged liver, hepatic fibrosis is a common symptom, the level of GLB, ALB, and the ratio of ALB/GLB could be affected (Jiang et al., 2013). CCl4 is having an acute toxicity, according to the results, ALB and GLB have no difference among the normal, CCl4 control, and treated groups in this research. Moreover, the significant decrease in SOD activity and remarkable elevation in hepatic MDA level suggest a role of oxidative stress in CCl4 hepatotoxicity (Sreelatha et al., 2009). While, treated with BELE in mice indicated a dramatical protective effect against CCl4-induced acute hepatotoxicity and oxidative stress which were also confirmed by the hepatic histopathological examinations. The stimulation of hepatic regeneration makes the liver more resistant to damage by the toxin (Sadasivan et al., 2006). BELE inhibited liver lipid peroxidation and resultant tissue degeneration, thus acting as an effective antioxidant. Previous studies showed that Berchemia lineate largely possessed lignans, quinones, flavones, and polyphenolic compounds (Zhang et al., 2011), which have potent antioxidant activity (Aejaz & Neelakantan, 2013; Mitra et al., 2000; Urszul et al., 2009; Xin et al., 2008). MDA is a major reactive aldehyde that appears during the peroxidation of biological membrane polyunsaturated fatty acid (Vaca et al., 1988). An increase in MDA level in the liver suggests enhanced peroxidation leading to tissue damage and failure of the antioxidant-defense mechanisms to prevent the formation of excessive free radicals (Nail, 2003). Therefore, the concentration of MDA in liver tissue is used
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as an indicator of liver injury involving a series of chain reactions (Ohkawa et al., 1979). SOD is an effective defense enzyme that catalyzes the dismutation of superoxide anions into hydrogen peroxide (H2O2) (Reiter et al., 2000). Lipid peroxidant or reactive oxygen species could easily inactivate these antioxidant enzymes in toxicity (Polavarapu et al., 1998; Yang et al., 2008). The results revealed that BELE can store the activity of SOD and inhibit the lipid peroxidation so as to stabilizing the plasma membrane. The phenolic hydroxyl on the benzene ring is an antioxidant activity group to antagonize tissue damages, which was caused by free radicals, by eliminating free radicals, inhibiting oxidation reactions, and combining with biological membrane phospholipids to protect membrane lipid (Yu, 2009). Previous phytochemical investigation revealed that polyphenolic compounds largely existed in this genus (Zhang et al., 2011). In addition, chrysophanol, physcion, emodin, and aloe-emodin have the ability of scavenging free radicals (Luo et al., 2009) and these constituents exist in Berchemia lineata. So, BELE possessed a certain extent of antioxidant activity. In conclusion, the results of this study demonstrate that BELE can effectively prevent the CCl4-induced hepatic damage in mice and, therefore, it can be used as a hepatoprotective agent. The hepatoprotective effects of BELE may be due to both the inhibition of lipid peroxidation and the increase of antioxidant activity. This is the primary report about the hepatoprotective of BELE, further studies with individual active compounds existed in BELE are in proceeding, which will enable us to understand the exact mechanism of hepatoprotective action by BELE and it will help us to further exploit traditional medicine on this plant.
Declaration of interest The authors declare that there are no conflicts of interest. The project was supported by the Natural Science foundation of China (No. 81202940) and the Fundamental Research Funds for the Central Universities of Huaqiao University of China (Nos. JB-ZR1152 and JB-ZR1226).
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