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Evaluation of hepatoprotective activity of Syringa oblata leaves ethanol extract with the indicator of glutathione S-transferase A1

Abstract

The leaves of Syringa oblata Lindl., Oleaceae, had been extensively used as a folk medicine to treat various infections, heal inflammations, icteric hepatitis and acute mastitis. The study was designed to evaluate the hepatoprotective activity of S. oblata leaves ethanol extract against CCl4-induced hepatotoxicity in primary hepatocytes and mice with the indicator of glutathione S-transferase alpha 1. The hepatoprotective effects of S. oblata leaves ethanol extract were evaluated on the basis of liver histopathology and biochemical parameters as well as hepatic oxidative stress markers. The results showed that CCl4 negatively modulated biochemical parameters and liver antioxidant activities. However, the use of S. oblata leaves ethanol extract restored altered-serum biochemical parameters and liver antioxidant activities in a dose-dependent manner. Importantly, the trends in S-transferase alpha 1 were similar to alanine aminotransferase and aspartate aminotransferase level, and S-transferase alpha 1 was suggested to be a marker for the evaluation of hepatoprotective activity of S. oblata leaves ethanol extract. Histopathological examination showed that CCl4 causes significant hepatic injury relative to control group. The above findings suggested that S. oblata leaves ethanol extract has hepatoprotective effects against CCl4-induced hepatic injury and S-transferase alpha 1 may be an indicator to evaluate the protective effects of S. oblata leaves ethanol extract.

Keywords:
Carbon tetrachloride; Hepatic injury; Extraction; Glutathione S-transferase A1; Hepatoprotection

Introduction

Liver is an important digestive gland in the body, playing a major role in the metabolism, biotransformation and detoxification of toxins (Li et al., 2004Li, L.J., Wu, Z.W., Xiao, D.S., Sheng, J.F., 2004. Changes of gut flora and endotoxin in rats with d-galactosamine-induced acute liver failure. World J. Gastroenterol. 10, 2087-2090.). Meanwhile, liver is a susceptible organ to xenobiotic metabolisms, chemical substances, virus, alcohol and drugs (Zuo et al., 2014Zuo, A.R., Yu, Y.Y., Shu, Q.L., Zheng, L.X., Wang, X.M., Peng, S.H., Xie, Y.F., Cao, S.W., 2014. Hepatoprotective effects and antioxidant, antityrosinase activities of phloretin and phloretin isonicotinyl hydrazone. J. Chin. Med. Assoc. 77, 290-301.). Acute hepatic injury is very common induced by one of the several factors, including radical oxidative damage, viral exposure, alcohol consumption, drug and immune system issues (Andrade et al., 2015Andrade, K.Q.D., Moura, F.A., Santos, J.M.D., Santos, J.C.D.F., Goulart, M.O.F., 2015. Oxidative stress and inflammation in hepatic diseases: therapeutic oossibilities of N-acetylcysteine. Int. J. Mol. Sci. 16, 30269-30308.). Carbon tetrachloride (CCl4), a highly toxic chemical agent, is widely used for hepatotoxicity model in animals (Weber et al., 2003Weber, L.W., Boll, M., Stampfl, A., 2003. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit. Rev. Toxicol. 33, 105-136.). It is believed that CCl4 in liver could form metabolic trichloromethyl radicals (CCl3•) and react with excess O2 producing reactive oxygen species (ROS), which is the normal product of aerobic metabolic reaction (Zira et al., 2013Zira, A., Kostidis, S., Theocharis, S., Sigala, F., Engelsen, S.B., Andreadou, I., Mikros, E., 2013. 1H NMR-based metabonomics approach in a rat model of acute liver injury and regeneration induced by CCl4 administration. Toxicology 303, 115-124.). However, the high levels of ROS in the body could induce lipid peroxidation and protein oxidation in the cell membrane, causing liver enzyme leakage, mitochondrial injury and cellular disorders, as well as resulting in hepatic damage and apoptosis (Karakus et al., 2011Karakus, E., Karadeniz, A., Simsek, N., Can, I., Kara, A., Yildirim, S., Kalkan, Y., Kisa, F., 2011. Protective effect of Panax ginseng against serum biochemical changes and apoptosis in liver of rats treated with carbon tetrachloride (CCl4). J. Hazard. Mater. 195, 208-213.).

Glutathione S-transferases (GST), a large family of multifunctional proteins that share similar dimer interactions and glutathione-(GSH) binding sites, play a critical role in protecting cells from xenobiotics and therapeutic compounds (Hayes et al., 2005Hayes, J.D., Flanagan, J.U., Jowsey, I.R., 2005. Glutathione transferases. Annu. Rev. Pharmacol. Toxicol. 45, 51-88.). Glutathione S-transferase A1 (GSTA1), one isoenzyme of GST, is important in phase II metabolism by catalyzing the conjugation of reduced glutathione with a wide variety of electrophilic substances (Eaton and Bammler, 1999Eaton, D.L., Bammler, T.K., 1999. Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol. Sci. 49, 156-164.). Furthermore, the cytosolic concentration of GSTA1 is high and distribution of GSTA1 is uniform throughout the liver lobules. It is worth mentioning that GSTA1 could easily release to the blood due to its small molecular weight relative to alanine aminotransferase (ALT) and aspartate aminotransferase (AST) following liver damage. Consequently, GSTA1 is a more sensitive marker for monitoring the acute liver damage. (Bernuau et al., 2000Bernuau, J., Erlinger, S., Valla, D., 2000. Detection of hepatocellular damage. Lancet 356, 1029-1030.). The sensitivity and specificity of GSTA1 as a marker have been tested in various conditions of acute viral, toxic and drug induced hepatitis (Liu et al., 2014Liu, F., Lin, Y., Li, Z., Ma, X., Han, Q., Liu, Y., Zhou, Q., Liu, J., Li, R., Li, J., Gao, L., 2014. Glutathione S-transferase A1 (GSTA1) release, an early indicator of acute hepatic injury in mice. Food Chem. Toxicol. 71, 225-230.).

Syringa oblata Lindl., belongs to Oleaceae family and widely grows across China. The leaves of S. oblata have been extensively used as a folk medicine to treat various infections, inflammations, acute jaundiced hepatitis and acute mastitis. It is also used in China to treat vomiting, nausea, rheumatic pain and kidney deficiency (Xin et al., 2010Xin, L., Wang, J., Zhou, C., Gan, L., 2010. Preparative separation and enrichment of syringopicroside from Folium syringae leaves with macroporous resins. J. Biomed. Biotechnol. 2010, http://dx.doi.org/10.1155/2010/572570.
http://dx.doi.org/10.1155/2010/572570...
; Su et al., 2015Su, G., Cao, Y., Li, C., Yu, X., Gao, X., Tu, P., Chai, X., 2015. Phytochemical and pharmacological progress on the genusSyringa. Chem. Cent. J. 9, 1-12.). To date more than 140 chemical constituents have been isolated from the bark, leaves and flowers of Syringa including phenylpropanoid, lignans, iridoid, crack iridoid, phenethyl alcohol and flavonoids etc. (Eriksson et al., 2008Eriksson, C., Mansson, P.E., Sjodin, K., Schlyter, F., 2008. Antifeedants and feeding stimulants in bark extracts of ten woody non-host species of the pine weevil, Hylobius abietis. J. Chem. Ecol. 34, 1290-1297.; Zhao et al., 2016Zhao, M., Tang, W.X., Li, J., Bai, L.M., Wang, J.L., Zhang, W.Z., Zhang, S.J., 2016. Two new monoterpenoids from the fresh leaves of Syringa oblata. Chem. Nat. Compd. 52, 1023-1025.). Previous reports demonstrated that S. oblate Lindl., S. vulgaris L., and S. dilatata Nakai leaves, flowers or fruit extract have potential hepatoprotective activities (Oh et al., 2003Oh, H., Ko, E.K., Kim, D.H., Jang, K.K., Park, S.E., Lee, H.S., Kim, Y.C., 2003. Secoiridoid glucosides with free radical scavenging activity from the leaves of Syringa dilatata. Phytother. Res. 17, 417-419.; Toth et al., 2016Toth, G., Barabas, C., Toth, A., Kery, A., Beni, S., Boldizsar, I., Varga, E., Noszal, B., 2016. Characterization of antioxidant phenolics in Syringa vulgaris L. flowers and fruits by HPLC-DAD-ESI-MS. Biomed. Chromatogr. 30, 923-932.). There is evidence of hepatoprotective activity of S. oblata leaves due to the syringopicroside and flavonoids content, which have potent antioxidant properties (Erdogan et al., 2015Erdogan, E., Ilgaz, Y., Gurgor, P.N., Oztas, Y., Topal, T., Oztas, E., 2015. Rutin ameliorates methotrexate induced hepatic injury in rats. Acta Cir. Bras. 30, 778-784.). In addition, rutin has the effect of anti-lipid peroxidation, diastolic blood vessels as well as affects the activity of metabolic enzymes (Magalingam et al., 2013Magalingam, K.B., Radhakrishnan, A., Haleagrahara, N., 2013. Rutin, a bioflavonoid antioxidant protects rat pheochromocytoma (PC-12) cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. Int. J. Mol. Med. 32, 235-240.).

In the light of the traditional use of S. oblata leaves for the treatment of liver inflammation, this study explored the potential hepatoprotective activities of S. oblata leaves ethanol extract (SOLEE) against CCl4-induced hepatotoxicity both in vitro and in vivo, and evaluated the possibility of GSTA1 being used as an indicator of hepatic injury.

Materials and methods

Reagents

CCl4 was purchased from the Guoyao Chemical Reagent Co. LTD (Shanghai, China). Silymarin was purchased from Harbin Jiacheng Dispensary (Harbin, China). Type IV collagenase, dimethyl sulfoxide (DMSO), insulin, transferrin, heparin, dexamethasone, and trypan blue were bought from Sigma Chemical Co. (St. Louis, MO, USA). Mice feed (license no. SCXK (Liao) 2015-0003) was obtained from Changsheng Biotechnology (Liaoning, China). Rutin standard (lot no. 30225) was bought from Dr. Ehrenstorfer GmbH (Augsburg, Germany).

Plant material and preparation of extracts

Syringa oblata Lindl., Oleaceae, leaves were purchased from Harbin Jiacheng Dispensary (Harbin, China) and authenticated in Heilongjiang University of Chinese Medicine. The specimen (accession no. 2015052006) has been deposited at Herbarium in the College of Veterinary Medicine, Northeast Agricultural University. SOLEE was prepared as previously described (Shi et al., 2017Shi, C.X., Lin, Y.X., Liu, F.P., Chang, Y.C., Li, R., Li, C.W., Li, Y., He, J.S., Ma, X., Li, Z., 2017. Hepatoprotective effects of ethanol extracts from Folium Syringae against acetaminophen-induced hepatotoxicity in vitro and in vivo. J. Chin. Med. Assoc. 80, 623-629.). In brief, 2000 g of fresh leaves were dried at 50 °C and ground to crude powder with a universal grinder (AK-1000A, Wenling, China), then the powder (500 g) was dissolved in 5000 ml ethanol (50%, v/v) in ultrasonic waves (KQ3200 Kunshan Ultrasonic Instrument Co., Ltd., China) for 60 min. After extraction, the solvent was filtered and evaporated at 60 °C till the extracts concentrated to a paste and dried by vacuum drying method at 35 °C.

Quantification of rutin bioactive compound from SOLEE

SOLEE was analyzed by using high performance liquid chromatography (HPLC) method. Rutin, one of the principal constituents of S. oblata leaves, was used as an external standard. The analysis were performed using Waters 2695 HPLC system (Waters, USA) and Venusil XBP C18 (L) column (250 mm × 4.6 mm, 5 µm, Bonna-Agela Technologies, China). The mobile phase consisted of methanol and acetonitrile in 80:20 (v/v) at a flow rate of 1 ml/min with an injection volume of 10 µl. The compounds were detected at a wavelength of 256 nm at a column temperature of 25 °C.

Mice

Adult male Kunming mice having the body weight 20 ± 2 g were obtained from Harbin Pharmaceutical Group CO., Ltd. General Pharm. Factory, laboratory animal center. The License number of mice was SCXK (black) 2012-007. Mice were housed under standard conditions at a temperature (20 ± 2 °C) and light (12 h light/12 h dark cycles), a relative humidity (40–60%), and allowed free access to food and water. Mice were acclimatized for at least 1 week before experiments. All procedures involving animals were carried out in accordance with the guidelines and approved protocols of Northeast Agricultural University and Harbin Veterinary Research Institute Animal Ethics Committee (approval number: SYXK (Hei) 2012-2067).

Primary hepatocytes

Hepatocytes were isolated from mice with the improved method of Seglen's two-step perfusion in situ as previously described (Chang et al., 2017Chang, Y.C., Liu, F.P., Ma, X., Li, M.M., Li, R., Li, C.W., Shi, C.X., He, J.S., Li, Z., Lin, Y.X., Zhao, C.W., Han, Q., Zhao, Y.L., Wang, D.N., Liu, J.L., 2017. Glutathione S-transferase A1 – a sensitive marker of alcoholic injury on primary hepatocytes. Hum. Exp. Toxicol. 36, 386-394.). Briefly, the liver was perfused with preheated (37 °C) liver perfusion medium containing 1 ml heparin (2500 U/ml) and then digested by digesting medium including type IV collagenase (300 mg/ml). Primary hepatocytes were separated from liver tissue following centrifugation. The primary hepatocytes were seeded in 24 well plates for 6 h, and the cells were cultured in the growth medium without serum for 24 h. Then, the cells were again cultured at 37 °C under a humidified atmosphere of 5% (v/v) CO2 for subsequent experiments.

Experimental protocol

Primary hepatocytes treatment

The primary hepatocytes were treated with or without SOLEE concentration (mass/volume) high (200 µg/ml), medium (100 µg/ml) and low concentration (50 µg/ml) of SOLEE for 12 h, and then incubated with CCl4 (10 mmol/l) for 8 h. Silymarin was chosen as a positive control drug. Afterwards, the hepatocytes were centrifuged at 3000 × g for 10 min. Supernatant was collected for the detection of ALT, AST and GSTA1 enzyme activities, and the cells were tested for superoxide dismutase (SOD) and malonaldehyde (MDA) level.

Mice treatment

Forty eight mice were randomly assigned into six groups with eight animals per group. The animals of control group and model group (CCl4 alone group) received only isopyknic physiological saline; positive control group were administered with silymarin (200 mg/kg), while the three experimental groups were administered with 100 mg/kg, 200 mg/kg and 300 mg/kg of SOLEE daily by gavage for 7 successive days. The administration volume of each mouse was 10 ml/kg. During experiments, mice were provided with a standard diet (composition shown in Table 1) and water ad libitum. After receiving treatments for 7 days, all groups fasted for 16 h. All groups were given 0.35% CCl4 soya bean oil solution (10 ml/kg) to induce hepatic injury in mice except control group. The control group was only given soya bean oil (10 ml/kg). After 24 h of acute exposure, the mice were anesthetized (1.5% isoflurane), and the blood was collected from the retro-orbicular plexus. A piece of fresh liver was washed in ice-cold saline and dissected to prepare tissue homogenate. The remaining liver was stored at −80 °C for further analysis.

Table 1
The nutritive formula composition of the mice diet.

ALT, AST, MDA, SOD and GSTA1 assays for monitoring liver function

ALT (Kit No. C009-1), AST (Kit No. C010-1) activity in serum and MDA (Kit No. A003-1), SOD (Kit No. A001-1-1) concentration in tissue homogenates were detected according to the instructions of each kit. The kits were obtained from Jiancheng Institute of Biotechnology (Nanjing, China). GSTA1 levels in serum and tissue homogenates were determined with a mouse glutathione S-transferase Alpha1 (GSTA1) ELISA Kit (American Rapidbio company, America. Kit No. DRE30790). The basic procedure was performed in accordance with the manufacturer's recommendations.

Histopathological examination of liver

The liver tissues from each group were collected and fixed in 10% (v/v) formalin for 24 h, dehydrated in graded ethanol solution varied in between 50% to 100%, cleared in xylene, and embedded in paraffin. Afterwards, 5-µm-thick sections were prepared, stained with hematoxylin and eosin dye, and observed for histopathological changes under a microscope.

Statistical analysis

Data is represented as mean ± standard deviation (SD). Statistically significant differences among all groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test using the SPSS software 19.0 (SPSS, Inc., Chicago, IL, USA). A value of p < 0.01/p < 0.05 was considered as statistically significant.

Results

Validation of the extraction method

The chromatograms of [0] rutin standard and ethanol extract of S. oblata leaves are shown in Fig. 1. Rutin peaks appeared at a retention time of approximately 15 min.

Fig. 1
(A) Chromatogram of rutin standard and (B) ethanol extract of Syringa oblata leaves.

Protective effects of SOLEE in vitro

Compared to control group, ALT, AST, GSTA1 (p < 0.01) and MDA (p < 0.05) level significantly increased in model group. Meanwhile, SOD level significantly (p < 0.05) decreased in model group (Table 2). Pretreated with the high concentration (200 µg/ml) of SOLEE, transaminase (ALT and AST) activity and GSTA1 level in the supernatant were effectively decreased (p < 0.05). The same high concentration markedly increased (p < 0.05) SOD level and obviously decreased (p < 0.05) MDA level in cells. However, there were no significant changes in the parameters above mentioned in the groups treated with the medium concentration (100 µg/ml) and low concentration (50 µg/ml) of SOLEE.

Table 2
The changes of indicators in cell culture supernatant and in hepatocyte (x ± s , n = 6).

Effects of SOLEE on serum ALT and AST activities

Results of SOLEE on serum ALT and AST activities are shown in Fig. 2A. CCl4 significantly (p < 0.01) increased serum ALT and AST activity, while pretreatment with silymarin and SOLEE (300 mg/kg), serum ALT and AST activity extremely (p < 0.01) reduced relative to model group. Treatment with SOLEE (200 mg/kg) caused significant (p < 0.05) decrease in serum ALT and AST activity. However, there was no statistically significant difference between low dose SOLEE group (100 mg/kg) and model group.

Fig. 2
(A) The changes of ALT and AST activity in the serum of mice. (B) The changes of GSTA1 level in mice serum and liver tissue (n = 8).

GSTA1 concentration in serum and liver tissue

Variations of GSTA1 in serum and liver tissue are presented in Fig. 2B. In the control group, GSTA1 concentrations in the liver tissues were almost 2.42 times higher than GSTA1 in the serum. CCl4 treatment significantly (p < 0.01) increased GSTA1 concentration in serum and markedly decreased (p < 0.01) GSTA1 level in liver tissue. The ratio of GSTA1 concentrations in the liver and serum is 1:1. While, compared to model group, mice pre-treated with silymarin and SOLEE (300 mg/kg or 200 mg/kg) respectively, serum GSTA1 remarkably (p < 0.01) reduced and tissue GSTA1 significantly (p < 0.05 or p < 0.01) increased in a dose-dependent manner. Intriguingly, the GSTA1 levels of liver tissues were 2.40 and 2.27 times higher than the serum GSTA1 levels in silymarin group and SOLEE (300 mg/kg) group, respectively.

Effects of SOLEE on MDA and SOD levels

Compared to control group, the levels of MDA progressively increased to a high extent (p < 0.01), and the level of SOD obviously decreased (p < 0.01) in the model group (Fig. 3). The elevated level of MDA was significantly (p < 0.01–0.05) reduced and the depressed level of SOD was significantly (p < 0.01–0.05) increased in mice pretreated with silymarin and SOLEE (300 mg/kg, 200 mg/kg). The trend of MDA and SOD were similar in mice treated with silymarin and SOLEE (300 mg/kg). Furthermore, low dose SOLEE (100 mg/kg) showed no significant effect relative to model group.

Fig. 3
The changes of MDA level (A) and SOD level (B) in liver tissue of mice (n = 8).

Tissue sections analysis

As shown in Fig. 4, the photomicrographs from the control group (Fig. 4A) showed normal morphology, hepatic cords were neatly arranged and the structures of liver lobules were clearly seen. CCl4-induced histopathological changes in liver were observed, including the obscure edge of the hepatic lobule, mussy hepatic cord, cloudy swelling of the hepatic cells, congestion and necrosis, significant acidophilic changes and obvious inflammatory infiltrates in focal areas. Compared to CCl4 group (Fig. 4B), the sections from the groups treated with silymarin (Fig. 4C) and high dose (300 mg/kg) SOLEE (Fig. 4D) showed normal morphology. However, slight alteration and degeneration of the hepatocytes, mild inflammatory infiltration and congestion in the central retinal and hepatic sinusoids have been observed in the groups treated with silymarin (Fig. 4C).

Fig. 4
(A) Control group, (B) Model group, (C) Group with silymarin (200 mg/kg), (D) Group with Syringa oblata leaves extract (300 mg/kg). Inflammatory infiltration was marked out by blue arrows. Necrosis was pointed out by the red arrows.

Discussion

Oxidative stress is an important cause of liver dysfunction and also the pathophysiological basis of hepatic injury (Yang et al., 2015Yang, B.Y., Zhang, X.Y., Guan, S.W., Hua, Z.C., 2015. Protective effect of procyanidin B2 against CCl4-induced acute liver injury in mice. Molecules 20, 12250-12265.). Previous reports demonstrated that increase oxidative stress causes lipid peroxidation in cells, mitochondrial calcium overload, inflammatory reaction, abnormal function of organelles, oxidative DNA damage and even cancerization (Singh et al., 2017Singh, M.P., Kim, K.Y., Kim, H.Y., 2017. Methionine sulfoxide reductase A deficiency exacerbates acute liver injury induced by acetaminophen. Biochem. Biophys. Res. Commun. 484, 189-194.). The efficacy of any hepatoprotective drug is essentially dependent on its ability in reducing the harmful effects or maintaining the normal hepatic physiology that has been disturbed by a hepatotoxin. Numerous literatures demonstrated that S. oblata leaves had various therapeutic properties. Hyuncheol reported that activity-guided fractionation of the ethyl acetate and methyl alcohol extract of the leaves of S. dilatata could scavenge free radical (Oh et al., 2003Oh, H., Ko, E.K., Kim, D.H., Jang, K.K., Park, S.E., Lee, H.S., Kim, Y.C., 2003. Secoiridoid glucosides with free radical scavenging activity from the leaves of Syringa dilatata. Phytother. Res. 17, 417-419.). Gergő Tóth studied the polyphenolic composition of S. vulgaris flowers and fruits proving that the extracts and the constituents have radical scavenging activity (Toth et al., 2016Toth, G., Barabas, C., Toth, A., Kery, A., Beni, S., Boldizsar, I., Varga, E., Noszal, B., 2016. Characterization of antioxidant phenolics in Syringa vulgaris L. flowers and fruits by HPLC-DAD-ESI-MS. Biomed. Chromatogr. 30, 923-932.). Rutin is a flavonoid glycoside found in fruits, tea, vegetables and medicinal plants (Liu et al., 2017Liu, Q., Pan, R., Ding, L., Zhang, F., Hu, L., Ding, B., Zhu, L., Xia, Y., Dou, X., 2017. Rutin exhibits hepatoprotective effects in a mouse model of non-alcoholic fatty liver disease by reducing hepatic lipid levels and mitigating lipid-induced oxidative injuries. Int. Immunopharmacol. 49, 132-141.). Studies reported that rutin have many pharmacological and therapeutic properties including anticarcinogenic, neuroprotective, nephroprotective, hepatoprotective and anti-inflammatory activities (Ghorbani, 2017Ghorbani, A., 2017. Mechanisms of antidiabetic effects of flavonoid rutin. Biomed. Pharmacother. 96, 305-312.). Besides, Khan et al., demonstrated that rutin possess radical scavenging activity and antioxidant potential that is useful in preventing CCl4-induced liver damage (Khan et al., 2012Khan, R.A., Khan, M.R., Sahreen, S., 2012. CCl4- induced hepatotoxicity: protective effect of rutin on p53, CYP2E1 and the antioxidative status in rat. BMC Complement. Altern. Med. 12, http://dx.doi.org/10.1186/1472-6882-12-178.
http://dx.doi.org/10.1186/1472-6882-12-1...
). In the present study, the active ingredient of SOLEE is rutin that may be responsible for the prevention of CCl4 induced liver injury in mice hepatocytes. Intriguingly, we obtained reasonably resolved and good chromatographic peaks for standard rutin and SOLEE in HPLC analysis. In addition, silymarin is an important medicinal plant for alleviating hepatic damage, whose history of hepatoprotective activity could dates back to 2000 years ago and is used to treat jaundice and enlarged liver and spleen (Bahmani et al., 2015Bahmani, M., Shirzad, H., Rafieian, S., Rafieian-Kopaei, M., 2015. Silybum marianum: beyond hepatoprotection. J. Evid. Based Complement. Altern. Med. 20, 292-301.). Hence, we employed silymarin as a reference to study the hepatoprotective effects of SOLEE in vivo.

ALT and AST, as a traditional markers of hepatic damage, could reflect the effects of CCl4-induced hepatotoxicity in mice (Knapen et al., 2000Knapen, M.F., Steegers, E.A., Mulder, T.P., Peters, W.H., 2000. Detection of hepatocellular damage. Lancet 356, 1030.). Moreover, MDA is the byproduct of lipid peroxidation and the level of it in hepatic tissue is used as a biomarker of lipid peroxidation and a key feature in liver damage (Lin et al., 2006Lin, Y.L., Wu, C.H., Luo, M.H., Huang, Y.J., Wang, C.N., Shiao, M.S., Huang, Y.T., 2006. In vitro protective effects of salvianolic acid B on primary hepatocytes and hepatic stellate cells. J. Ethnopharmacol. 105, 215-222.). Furthermore, cells have enzymatic antioxidant system to protect itself against oxidative damage. SOD is a major antioxidant enzyme and plays a crucial role in the conversion of superoxide radicals into hydrogen peroxide. Increase of oxidative stress in the cells could inactivate SOD enzyme (Escobar et al., 1996Escobar, J.A., Rubio, M.A., Lissi, E.A., 1996. Sod and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radic. Biol. Med. 20, 285-290.). Therefore, detecting oxidative stress parameters, such as MDA, SOD and GSH-Px can monitor CCl4-induuced oxidative injury.

In our study, we found that CCl4 treatment (model group) caused oxidative damage in liver tissues and negatively altered serum biochemical parameters. Interestingly, SOLEE significantly reversed CCl4-induced changes in serum and liver biochemical parameters and antioxidant activities both in vitro and in vivo. The supernatant and serum levels of ALT, AST and GSTA1 as well as the cell and tissue level of MDA depressed significantly (p < 0.01 or p < 0.05) in the groups treated with high and medium dose of SOLEE. Meanwhile, the concentration of GSTA1 and SOD in liver tissues were elevated obviously (p < 0.01 or p < 0.05). We have noted similar trends in ALT, AST, GSTA1, MDA and SOD level in high dose SOLEE group and silymarin group, respectively. Hence, it is suggested that SOLEE had hepatoprotective activity, which was similar to silymarin, protecting the liver against CCl4-induced injury.

Our histological findings were in general agreement with previous studies that CCl4-induced liver injury in mice (Ma et al., 2017Ma, X., Liu, F., Li, M., Li, Z., Lin, Y., Li, R., Li, C., Chang, Y., Zhao, C., Han, Q., 2017. Expression of glutathione S-transferase A1, a phase II drug-metabolizing enzyme in acute hepatic injury on mice. Exp. Ther. Med. 14, 3798-3804.). We also noted that CCl4 caused hepatocytes vacuolization, partial disruption in radial arrangement, fatty degeneration and inflammatory infiltrates. The pathological changes had the obvious uniformity with the variations of aminotransferases, SOD and GSTA1, demonstrating that SOLEE was a powerful antioxidant and prevents lipid peroxidation.

Previous studies demonstrated that GSTA1 was a more suitable marker of liver damage than ALT in vivo and in vitro (Liu et al., 2016Liu, F.P., Ma, X., Li, M.M., Li, Z., Han, Q., Li, R., Li, C.W., Chang, Y.C., Zhao, C.W., Lin, Y.X., 2016. Hepatoprotective effects of Solanum nigrum against ethanol-induced injury in primary hepatocytes and mice with analysis of glutathione S-transferase A1. J. Chin. Med. Assoc. 79, 65-71.; Li et al., 2017Liu, Q., Pan, R., Ding, L., Zhang, F., Hu, L., Ding, B., Zhu, L., Xia, Y., Dou, X., 2017. Rutin exhibits hepatoprotective effects in a mouse model of non-alcoholic fatty liver disease by reducing hepatic lipid levels and mitigating lipid-induced oxidative injuries. Int. Immunopharmacol. 49, 132-141.). Our data showed that the levels of GSTA1 in serum showed similar trend with that of ALT and AST, which is in agreement with previous studies (Liu et al., 2014Liu, F., Lin, Y., Li, Z., Ma, X., Han, Q., Liu, Y., Zhou, Q., Liu, J., Li, R., Li, J., Gao, L., 2014. Glutathione S-transferase A1 (GSTA1) release, an early indicator of acute hepatic injury in mice. Food Chem. Toxicol. 71, 225-230.). Interestingly, the trends of GSTA1 level were consistent with that of ALT and AST level in the experimental groups treated with SOLEE. Thus, GSTA1 could be used as an indicator to evaluate the hepatoprotective activity of S. oblata leaves extract. Nevertheless, higher molecular studies are needed to know the effect of S. oblata leaves extract on the role of GSTA1 and the mechanism involved in CCl4-induced liver injury.

Conclusion

In summary, SOLEE had a free radical scavenging activity, prevented lipid peroxidation and enhanced antioxidant activities that prevented the hepatocytes from CCl4-induced liver injury.

Ethical disclosures

  • Protection of human and animal subjects
    The authors declare that the procedures were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).
    Confidentiality of data
    The authors declare that they have followed the protocols of their work center on the publication of patient data.
    Right to privacy and informed consent
    The authors have obtained the written informed consent of the patients or subjects mentioned in the article. The corresponding author is in possession of this document.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant number 31472241) and the Application Technology Research and Development Projects in Heilongjiang Province of China (grant number PC13S03).

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Publication Dates

  • Publication in this collection
    Jul-Aug 2018

History

  • Received
    13 Mar 2018
  • Accepted
    28 May 2018
Sociedade Brasileira de Farmacognosia Universidade Federal do Paraná, Laboratório de Farmacognosia, Rua Pref. Lothario Meissner, 632 - Jd. Botânico, 80210-170, Curitiba, PR, Brasil, Tel/FAX (41) 3360-4062 - Curitiba - PR - Brazil
E-mail: revista@sbfgnosia.org.br