Hepatoprotective and antioxidant activity of Melaleuca styphelioides on carbon tetrachloride-induced hepatotoxicity in mice

Abstract

Context: Liver disease is a serious problem. Polyphenolic compounds have marked antioxidant effect and can prevent the liver damage caused by free radicals. In vitro studies have revealed the strong antioxidant activity of an ellagitannin-rich plant, namely, Melaleuca styphelioides Sm. (Myrtaceae).

Objective: In view of the limited therapeutic options available for the treatment of liver diseases, the hepatoprotective potential of the methanol extract of M. styphelioides leaves (MSE) was investigated against CCl₄-induced liver injury in mice.

Materials and methods: MSE was administered (500 and 1000 mg/kg/d p.o.) along with CCl₄ for 6 weeks. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were determined in the serum. Glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST), and malondialdehyde (MDA) were estimated in the liver homogenate. The bioactive components of MSE were identified by NMR, UV and HRESI-MS/MS data.

Results: MSE treatment (500 and 1000 mg/kg/d) markedly inhibited the CCl₄-induced increase in the levels of AST (31 and 38%), ALT (29 and 32%), ALP (13 and 19%), and MDA (22 and 37%) at the tested doses, respectively. MSE treatment markedly increased the levels of GSH (29 and 57%) and antioxidant enzymes compared with the CCl₄-treated group. MSE was more effective than silymarin in restoring the liver architecture and reducing the fatty changes, central vein congestion, Kupffer cell hyperplasia, inflammatory infiltration, and necrosis induced by CCl₄. The LD₅₀ of MSE was more than 5000 mg/kg.

Conclusion: MSE confers potent antioxidant and hepatoprotective effects against CCl₄-induced toxicity.

• Ellagitannins
• glutathione
• liver enzymes
• lipid peroxidation
• superoxide dismutase

Introduction

The liver is an important organ involved in the detoxification of xenobiotics (Ramachandra Setty et al., 2007). Excessive exposure to drugs and environmental pollutants overpower the detoxification mechanisms of the liver and lead to liver injury (Ramachandra Setty et al., 2007). Liver damage is associated with lipid peroxidation, enzyme leakage, and depletion in the glutathione level (GSH) (Ramachandra Setty et al., 2007). Chronic liver injury may develop into several liver diseases, including hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (Srivastava & Shivanandappa, 2010). There is now general agreement among hepatologists that the number of beneficial drugs currently available for the treatment of liver diseases is far from sufficient, and that there is an urgent need for safe and efficient therapeutic agents for the prevention and treatment of liver diseases (Muriel & Rivera-Espinoza, 2008). Corticosteroids, colchicines, and vaccines have been used for the treatment of several liver diseases; however, they have serious adverse side-effects and are of limited therapeutic benefits (Muriel & Rivera-Espinoza, 2008; Srivastava & Shivanandappa, 2010). Interferons have antifibrotic action besides their antiviral properties and can be utilized in patients having cirrhotic or fibrotic diseases. However, interferons are expensive and have serious side effects (Muriel & Rivera-Espinoza, 2008). Silymarin is a widely used hepatoprotective drug and has been used clinically for the treatment of acute and chronic hepatitis, alcoholic liver disease and toxin-induced hepatitis (Jacobs et al., 2002). Silymarin is a favored drug for liver diseases because of its oral effectiveness, low cost, and safety (Muriel & Rivera-Espinoza, 2008). Pharmacological studies have shown that silymarin exerts hepatoprotective, antioxidant, and antifibrotic properties (Abenavoli et al., 2010). Few well-designed clinical studies have indicated the efficacy of silymarin (at doses from 240 to 800 mg/d) to treat several liver disorders such as acute and chronic hepatitis, alcoholic liver disease, cirrhosis, and toxin-induced hepatitis (Abenavoli et al., 2010; Colturato et al., 2012; Pradhan & Girish, 2006). However, the results of some clinical trials were not in favor of silymarin use; although silymarin (given at a dose of 150 mg for 6 months in alcoholic cirrhosis) increased GSH and decreased lipid peroxidation; there were no concurrent amelioration of pathology indices (Abenavoli et al., 2010; Muriel & Rivera-Espinoza, 2008). The use of silymarin in patients with alcoholic cirrhosis has shown no beneficial effect in other studies (Singal et al., 2011). In other trials, silymarin, at daily doses of 210–800 mg, showed no effect on mortality or improvements in liver histology or liver functions in patients with chronic liver disease (Jacobs et al., 2002). In addition, serious adverse effects including gastroenteritis have been reported in patients using silymarin (Jacobs et al., 2002). Silymarin was also ineffective to treat CCl₄-cirrhosis, indicating that it is effective in preventing liver damage but not in reversing an established cirrhosis (Muriel & Rivera-Espinoza, 2008). Therefore, there is no conclusive evidence on the clinical efficacy of silymarin. The benefits of silymarin can be expanded if more controlled trials are performed (Muriel & Rivera-Espinoza, 2008). In view of the limited therapeutic options available for the treatment of liver diseases, the search for new, safe hepatoprotective candidates is quite apparent.

Natural products have received considerable attention in recent years because of their reduced side effects compared with synthetic drugs (Srivastava & Shivanandappa, 2010). Plant-derived phytochemicals represent a new therapeutic strategy in the search for new and effective hepatoprotective agents (Adewusi & Afolayan, 2010). Ellagitannins have attracted considerable attention for their marked pharmacological activities, including potent radical scavenging activity and inhibitory effect on lipid peroxidation (Okuda, 2005; Shimoda et al., 2008). Polyphenolic compounds can prevent the liver damage caused by free radicals through different mechanisms, including direct radical scavenging, metal chelation, and modulation of antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) (Han et al., 2007; Ramos, 2008). These attributes prompted us to investigate the hepatoprotective activity of an ellagitannin-rich plant belonging to the Myrtaceae family, namely Melaleuca styphelioides Sm. Previous studies have reported that various Melaleuca species are rich sources of bioactive compounds, such as volatile oils (Farag et al., 2004), ellagitannins (Yoshida et al., 2008), and flavonoids (Yoshimura et al., 2008). Different Melaleuca species are used in the traditional medicine for the treatment of several ailments (Southwell & Robert, 1999). Several studies have proved the strong in vitro antioxidant activity of various Melaleuca species (Farag et al., 2004; Kim et al., 2004). M. styphelioides, known as prickly-leaf tea tree, is a small to medium-sized ornamental tree (Byrnes, 1986). Despite the various medicinal properties of most Melaleuca species, very little is known about the chemical composition and pharmacological properties of the non-volatile constituents of M. styphelioides (Al-Sayed et al., 2014). Most studies have mainly focused on the chemical composition of the essential oil of M. styphelioides.

To the best of our knowledge, no studies have so far been reported on the hepatoprotective activity of M. styphelioides. Therefore, this study was designed to determine the hepatoprotective and antioxidant activities of the polyphenolic-rich extract obtained from M. styphelioides against CCl₄-induced hepatotoxicity in mice, with the aim to develop a safe, effective hepatoprotective agent. Hepatoprotection was determined by assaying the activities of serum markers of liver damage, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in the control and treated mice. Furthermore, the lipid peroxidation and antioxidant parameters (SOD, GSH, GPx, GR, and GST) were estimated in the liver homogenates in an attempt to determine the possible mechanisms of the hepatoprotective activity. A histopathological examination of the liver sections was conducted to confirm the hepatoprotective effect.

Materials and methods

General experimental procedures

The NMR spectra were measured by Bruker Avance 500 spectrometer (Bruker BioSpin Inc., Fällanden, Switzerland). CD₃OD was used as a solvent. ¹H NMR spectra were acquired with spectral width of 8 kHz consisting of 64 k data points. ¹³C NMR proton-decoupled spectra were acquired with a spectral width of 30 kHz consisting of 64 k data points. An exponential weighting of 1 Hz was applied prior to Fourier transformation. DQF-COSY, HSQC, HMBC, and selective 1D-TOCSY were all measured by the pulse programs originally installed by Bruker. The LC-HRESIMS was performed on a Bruker micrOTOF-Q Daltonics (API) Time-of-Flight Mass Spectrometer (Bremen, Germany) coupled to 1200 series HPLC system (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed on a Superspher 100 RP-18 (75 × 4 mm i.d.; 4 μm) column (Merck, Darmstadt, Germany). The ionization technique was electrospray. The mass spectrometer was operated in the negative mode. The following settings were applied to the instrument: capillary voltage, 4000 V; end plate offset, −500 V; drying gas (N₂) flow rate, 10 L/min, and temperature, 200 °C. For MS/MS measurements, argon was used as a collision gas and the voltage over the collision cell varied from 20 to 70 eV. Silica gel 60 (200–300 mesh) was obtained from Merck, Darmstadt, Germany. Sephadex LH-20 was purchased from Amersham Biosciences, Uppsala, Sweden. RP-C18 was obtained from Sigma-Aldrich GmbH, Munich, Germany and precoated silica gel TLC GF254 was purchased from Riedel-De Häen-AG, Seelze, Germany.

Chemicals

Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (), quercetin, sodium ascorbate, and AlCl₃ were obtained from Sigma-Aldrich GmbH, Munich, Germany. Carbon tetrachloride (CCl₄), ethylenediamine tetracetic acid (EDTA), hydrogen peroxide (H₂O₂), 5,5′-dithiobis-2-nitrobenzoic acid (DTNB, Ellman’s reagent), potassium dihydrogen phosphate (KH₂PO₄), reduced glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), nicotinamide adenine dinucleotide phosphate reduced form (NADPH), nicotinamide adenine dinucleotide reduced form (NADH), GR, oxidized glutathione (GSSG), nitroblue tetrazolium (NBT), phenazine methosulphate (PMT), trichloroacetic acid (TCA), thiobarbituric acid (TBA), and silymarin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). The assay kits were purchased from Spectrum, MDSS, GmbH, Hannover, Germany.

Plant material

The leaves of M. styphelioides were collected in July 2009 from Anshas Botanical Garden, El-sharkya, Egypt. The plant was botanically identified by Therese Labib, the taxonomy specialist at the herbarium of El-Orman Botanical Garden, Giza, Egypt. A voucher specimen of M. styphelioides was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ASU MSM2009).

Extraction and isolation

Air-dried powdered leaves of M. styphelioides (500 g) were extracted three-times with 80% MeOH. The total extract was concentrated and freeze-dried to obtain a dry powder, which was dissolved in absolute MeOH. The methanol-soluble portion was concentrated and freeze-dried to obtain a dry powder 80 g (MSE). Column fractionation was performed with a portion of MSE (30 g) using a Sephadex LH-20 column (5 × 50 cm) by eluting with H₂O followed by H₂O–MeOH mixtures to yield seven major fractions (I–VII). Compounds 1 (380 mg) was obtained by repeated column chromatography of fraction I (eluted with water) over a Sephadex LH-20 column (5 × 50 cm) using H₂O–MeOH gradient for elution. Fr II (eluted with 20% MeOH) was re-chromatographed over a Sephadex LH-20 column (5 × 50 cm), followed by fractionation over a RP-18 column (1 × 20 cm) with elution performed using H₂O–MeOH mixtures to yield compound 2 (73 mg). Compounds 3 (220 mg) and 4 (107 mg) were purified by repeated CC of fr III (eluted with 30% MeOH) over different Sephadex LH-20 columns using mixtures of H₂O and MeOH. Fr IV (eluted with 40% MeOH) was repeatedly chromatographed over Sephadex LH-20 columns to obtain compound 5 (103 mg). Fr V (eluted with 50% MeOH) was re-chromatographed over a Sephadex LH-20 column (5 × 50 cm) to yield compound 6 (1 g). Compound 7 (5.6 mg) was obtained by repeated CC of fr VI (eluted with 60% MeOH) over Sephadex LH-20. Fr VII (eluted with MeOH) was fractionated by Sephadex LH-20 CC, followed by purification over RP-18 CC to yield compounds 8 (1.5 mg) and 9 (4.5 mg).

radical-scavenging assay

The assay was carried out as reported before (Al-Sayed et al., 2012). The inhibition percentage of the radical scavenging activity was calculated using the equation: inhibition (%) = 100 − 100 (A_Sample/A_Blank). The antioxidant activity was expressed in terms of the IC₅₀. Quercetin and sodium ascorbate were used as the positive controls. The data are expressed as the mean ± SEM of three independent experiments.

Determination of total phenols

The sample methanol solution (50 μL) was added to 100 μL of methanol and mixed with 100 μL of Folin–Ciocalteu reagent. The mixture was shaken and allowed to stand at room temperature for 3 min before the addition of 500 μL of 20% Na₂CO₃. The solution was mixed thoroughly and the absorbance was measured at 730 nm after 2 h according to a previously described method (Velioglu et al., 1998). The assay was conducted in triplicates. Results were expressed as gallic acid equivalents per g dry weight of MSE from a calibration curve of gallic acid (0–500 μg/mL). The data are expressed as the mean ± SEM of three independent experiments.

Determination of total flavonoid content

An aliquot of either the methanol solution of the sample or the standard solution was mixed with an equal volume of AlCl₃·6H₂O (0.2%). Quercetin was used as a standard and the absorbance was measured at 367 nm (Dewanto et al., 2002). The assay was conducted in triplicates. Results were expressed as mg of quercetin equivalents per g dry weight of MSE from a calibration curve of the standard (0–500 μg/mL). The data are expressed as the mean ± SEM of three independent experiments.

In vivo experiments

Animals

Male Swiss albino mice (CD-1 strain) weighing 20–25 g were purchased from the Schistosome Biological Supply Center at the Theodor Bilharz Research Institute, Giza, Egypt. The animals were kept in a temperature and humidity-controlled room under 12 h light and dark cycles. The mice were fed a laboratory pellet chow and given water ad libitum. The animals were acclimatized for at least l week before use. All the animal experiments were performed in accordance with the internationally accepted guidelines for the care and the use of laboratory animals and were approved by the ethical committee of the Faculty of Pharmacy, Ain Shams University, Cairo, Egypt (ASU 2013-8 Research Article 3, approval date 5 August 2013).

Acute toxicity study

A group of 36 normal male mice weighing 20–25 g were used to determine the acute toxicity of MSE according to the previous method (Bruce, 1985). The mice were divided equally into six subgroups (n = 6 in each group). All subgroups were treated orally with rising doses (500, 1000, 2000, 3000, 4000, and 5000 mg/kg body weight p.o.) of MSE. The animals were kept under observation for 24 h after treatment to record toxicity symptoms and mortality rates.

Experimental protocol

The animals were divided into five groups (n = 9 in each group). Group (I) was given vehicle only (0.5%, w/v, carboxymethyl cellulose) and served as the normal control. Group (II) was treated intra-peritoneally with a sublethal dose of CCl₄ (20% CCl₄/olive oil, 3 d/week) for 6 weeks to induce chronic liver injury. Groups (III and IV) were treated orally with 500 and 1000 mg/kg body weight of MSE, respectively, along with CCl₄ for 6 weeks. MSE was given 5 d/week, while CCl₄ was administered 3 d/week. Group (V) was treated with silymarin at a daily dose of (500 mg/kg body weight p.o., 5 d/week) for 6 weeks. Similarly, CCl₄ was administered 3 d/week. The animals were anesthetized using diethyl ether and then sacrificed by decapitation 48 h after the last treatment dose. The blood was immediately collected and the liver samples were dissected (Rao et al., 2006). Each liver sample was then split in two parts. The first part was fixed in 10% formalin for the histopathological examination. The second part was washed immediately with 0.9% ice cold saline. A piece of 0.5 g was homogenized in 2.5 volumes (w/v) ice cold 0.1 M potassium phosphate buffer (pH 7.4). The homogenate was centrifuged at 600 g for 10 min to remove the cell debris. The supernatant was then centrifuged at 10 000 g and the pellet was discarded. The supernatant was collected and maintained at −70 °C for the estimation of liver content of GSH, antioxidant enzymes, and lipid peroxidation.

Hepatoprotective activity

The collected blood was allowed to clot; the serum was separated at 1800 g for 15 min and the biochemical markers of hepatic damage, including serum AST (U/L), ALT (U/L), and ALP (IU/L) according to previously reported methods (Kind & King, 1954; Reitman & Frankel, 1957).

Hepatic GSH content

Liver homogenate was deproteinized in 5% (w/v) TCA, centrifuged at 2000 g for 20 min, and the GSH content in the deproteinized supernatant was estimated by Ellman’s reagent using a standard curve (Ellman, 1959).

Antioxidant activity

The SOD assay was based on the inhibition of NBT reduction in the NADH-PMT-NBT reaction system. One unit of enzyme activity was defined as the amount of enzyme that causes half-maximal inhibition of NBT reduction. The SOD activity was expressed in µmole/min/g liver (Winterbourn et al., 1975). Hepatic GPx activity was determined by monitoring GSH oxidation (Paglia & Valentine, 1967). The enzyme reaction contained NADPH, GSH, and GR and was initiated by the addition of H₂O₂. The change in absorbance was monitored at 340 nm. One unit of GPx activity was defined as the amount of enzyme that catalyzes the oxidation of 1 µmole of NADPH/min/g liver. The GR activity was assayed by monitoring the oxidation of NADPH at 340 nm using GSSG as a substrate (Zanetti, 1979). The GST activity was measured based on the rate of increase in the conjugate formation between GSH and CDNB, and the absorbance was monitored at 340 nm. One unit of GST activity was defined as 1 μmole conjugate formation/min/g liver (Habig et al., 1974).

Lipid peroxidation

Malonyldialdehyde (MDA) is a biomarker of lipid peroxidation and reacts with TBA to form a pink chromogen (Ohkawa et al., 1979). About 1 mL of the liver homogenate was mixed with 1 mL of TCA (10% w/v) and was centrifuged at 1850 g for 15 min. About 1 mL of TBA solution (0.67% w/v) was added to 1 mL of supernatant and boiled for 45 min. Absorbance was read after cooling at 530 nm against a blank containing all the reagents except the liver homogenate. The content of thiobarbituric acid reactive substances (TBARS) in the samples was calculated using the extinction coefficient of MDA (1.56 × 10⁵M⁻¹cm⁻¹) and the results were expressed as MDA equivalents in nmole/g liver.

Histopathological examination

Liver sections were embedded in paraffin and sliced into 5 µm thick sections in a microtome (Leica, Allendale, NJ), and then stained with hematoxylin–eosin dye (Merck, Darmstadt, Germany). The histopathological examination of the slides was performed with × 200 magnification power. The parameters examined were as follows:

1. Hepatic architecture: preserved, partial loss, or complete loss.

2. Hydropic degeneration: absent, mild, moderate, or marked.

3. Fatty changes: absent or present.

4. Central vein congestion: present or absent.

5. Kupffer cell hyperplasia: absent or present.

6. Necrosis: absent or present.

7. Infiltration of portal tract by lymphocytes: absent or present.

Statistical test

All the data were expressed as the mean ± SEM. Statistical analysis of the data was performed by one-way ANOVA test followed by a post hoc test to determine the difference between the mean values of the different groups. All statistical analyses were performed using the SPSS 11.0 software (SPSS, Inc., Chicago, IL). A value of p < 0.05 was considered statistically significant.

Results

Fractionation of the methanol-soluble portion (MSE) obtained from the 80% methanol extract of M. styphelioides yielded compounds 1–9 (Figure 1). Notably, this is the first report of the isolation of compounds 1, 4, 5, and 7–9 from M. styphelioides. Compounds 2, 3, and 6 were previously isolated from M. styphelioides (Al-Sayed et al., 2014). All the compounds were identified based on their UV and HRESI-MS/MS data as well as by comparing these data with those previously reported in the literature (Table 1). The structures of compounds (2, 4, and 6–8) were also elucidated based on 1D and 2D NMR data (¹H, ¹³C, 1D-TOCSY, DQF-COSY, HSQC, and HMBC) (Supplementary data). The identification of compounds 1, 3, 5, and 9 was further confirmed by comparing their retention time, UV, and HRESI-MS/MS data with those of authentic samples in our lab.

Figure 1. Structures of the isolated compounds.

Table 1. Identification of the isolated compounds 1–9 using UV and HRESI/MS/MS.

No.	Isolated compounds	UV (nm)	(M-H)^- m/z	Fragments (MS/MS) m/z	References
1	Gallic acid	211.3, 260.5	169.0162	124.0134	Al-Sayed et al. (2012)
2	Kaempferol 3-O-α-l-rhamnopyranoside	210.0, 262.5, 341.1	431.0948	285.0348, 284.0286, 255.0255, 227.0305	Al-Sayed et al. (2014), Mabry et al. (1970), and Markham et al. (1978)
3	Pedunculagin: 2,3-4,6-di-O-HHDP-(α/β)-d-glucopyranose	218.0, 273.0 (sh)	783.0644	481.0589, 300.9946, 275.0147, 249.0364	Al-Sayed et al. (2014, 2012)
4	Pterocarinin A	216.6, 273.3	1067.1196 (M-2H)^2−:533.0863	300.9961, 275.0156, 249.0359, 169.0115	Nonaka et al. (1989)
5	Tellimagrandin I: 2,3-di-O-galloyl-4,6-O-HHDP- β-d-glucopyranose	214.8, 273.3	785.0837	300.9958, 275.0169, 249.0374, 169.0110	Al-Sayed et al. (2012)
6	Casuarinin	216.6, 271.0	935.0804 (M-2H)^2−:467.0349	783.0696, 633.0652, 481.0636, 300.9968, 275.0166, 249.0376, 169.0126	Al-Sayed et al. (2014) and Yoshida et al. (2008)
7	Tellimagrandin II: 1,2,3-tri-O-galloyl-4,6-O- HHDP-β-d-glucopyranose	214.8, 275.7	937.0964 (M-2H)^2−:468.0454	300.9998, 275.0225, 249.0407, 169.0124	Hatano et al. (1988)
8	1,2,3,6-tetra-O-galloyl-β-d-glucopyranose	214.8, 275.7	787.0992, (M-2H)^2−:393.0447	465.0689, 313.0570, 169.0149, 125.0239	Duan et al. (2004)
9	1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose	216.6, 275.7	939.1100, (M-2H)^2−:469.0504	617.0779, 465.0682, 313.0555, 169.0146, 125.0241	Al-Sayed et al. (2012) and Duan et al. (2004)

¹H and ¹³C NMR spectral data were in accordance with the previously reported data in the literature.

DPPH· radical-scavenging, total phenolic, and total flavonoid contents

MSE exhibited considerable inhibitory activity in the DPPH^· radical-scavenging assay, with an IC₅₀ value of 21.04 ± 0.06 µg/mL. The IC₅₀ values of sodium ascorbate and quercetin (positive controls) were 60.55 ± 0.03 and 20.71 ± 0.11 µM, respectively. The strong radical-scavenging activity is consistent with the results obtained from the determination of the total phenol content. MSE had considerably higher phenol content (320.77 ± 8.60 mg gallic acid-equivalents per g dry weight of MSE) and lower flavonoid content (20.03 ± 1.09 mg quercetin-equivalent per g of MSE).

Acute toxicity

The administration of MSE did not induce the death of mice in doses up to 5000 mg/kg (LD₅₀ > 5000 mg/kg). No adverse behavioral changes and toxicity symptoms were observed; therefore, MSE is considered safe in mice.

Hepatoprotective effect

Substantial increase in AST, ALT, and ALP levels were observed (p < 0.001) in the CCl₄-treated group compared with the negative control group (Table 2). The treatment of intoxicated mice with MSE produced a significant hepatoprotective effect and decreased the activity of serum ALT, AST, and ALP (Table 2). The percentage decrease in the liver marker enzymes at the treatment doses (500 or 1000 mg/kg/d) was 29 and 32% for ALT, 31 and 38% for AST and 13 and 19% for ALP, respectively, compared with the CCl₄-treated group. Treatment of intoxicated mice with silymarin at 500 mg/kg/d significantly improved all the serum markers of hepatic damage. Notably, treatment with MSE at a dose of 1000 mg/kg resulted in a substantial reduction in the serum ALT and AST levels, which was comparable with that produced by silymarin.

Table 2. Effect of MSE on hepatic marker enzymes after 6 weeks of CCl₄ intoxication in mice.

Animal groups	ALT (U/L)	AST (U/L)	ALP (IU/L)
Control	11.63 ± 0.63	113.38 ± 2.05	77.34 ± 5.51
CCl4	38.25 ± 2.17***	202.50 ± 5.35***	138.68 ± 8.73***
MSE (500 mg/kg/d)	27.33 ± 2.19***##$ (−29%)	139.11 ± 5.08***### (−31%)	120.97 ± 6.75***$$$ (−13%)
MSE (1000 mg/kg/d)	26.14 ± 2.94***## (−32%)	125.71 ± 3.17**### (−38%)	112.61 ± 6.29***#$$ (−19%)
Silymarin (500 mg/kg/d)	20.56 ± 0.92***### (−46%)	124.56 ± 4.64*### (−38%)	81.84 ± 4.70### (−41%)

Data are expressed as the means ± SEM (n = 9). The numbers in parentheses represent the percentage of reduction from the CCl₄-intoxicated group.

*p < 0.05, **p < 0.01, and ***p < 0.001; significantly different compared with the normal control group.

#p < 0.05, ##p < 0.01, and ###p < 0.001; significantly different compared with the CCl₄-intoxicated group.

$p < 0.05, $$p < 0.01, and $$$p < 0.001; significantly different compared with the silymarin-treated group.

Antioxidant activity

The levels of hepatic GSH and antioxidant enzymes (GPx, GR, GST, and SOD) were significantly decreased after the administration of CCl₄. In contrast, the hepatic MDA level was significantly increased compared with the normal control group (Table 3). Administration of MSE at the two treatment doses (500 and 1000 mg/kg) produced a significant increase in GSH (by 29 and 57%, respectively), and restored all the antioxidant enzymes close to their normal levels. The percentage increase in the antioxidant enzymes of the treated groups compared with the CCl₄-treated group is listed in Table 3. In addition, the elevated hepatic level of MDA was reduced by 22 and 37% at the tested doses, respectively, compared with the CCl₄-intoxicated group. The higher dose of MSE reduced the MDA level almost as effective as silymarin. Notably, the GSH, GST, and GR levels in the group treated with 1000 mg/kg of MSE were markedly higher compared with the silymarin-treated group. These results clearly indicate the potent in vivo antioxidant activity of MSE. The levels of the antioxidant parameters (except SOD) in the groups treated with MSE (500 and 1000 mg/kg) were not significantly different from the silymarin-treated group.

Table 3. Effect of MSE on antioxidant parameters after 6 weeks of CCl₄-intoxication in mice.

Animal groups	GSH (µmole/g liver)	GPx (µmole/min/g liver)	GR (µmole/min/g liver)	GST (µmole/min/g liver)	SOD (µmole/min/g liver)	MDA (nmole/g liver)
Control	3.93 ± 0.20	3.48 ± 0.24	2.47 ± 0.18	56.22 ± 6.26	366.27 ± 7.21	22.31 ± 3.16
CCl4	2.11 ± 0.17 ***	2.11 ± 0.32**	1.78 ± 0.13**	29.24 ± 3.84**	248.31 ± 7.40***	46.52 ± 6.18**
MSE (500 mg/kg/d)	2.73 ± 0.13***# (+29%)	2.62 ± 0.18* (+24%)	2.41 ± 0.11## (+35%)	36.31 ± 2.62* (+24%)	277.82 ± 8.53***#$$$ (+12%)	36.21 ± 5.12* (−22%)
MSE (1000 mg/kg/d)	3.31 ± 0.36# (+57%)	2.93 ± 0.16# (+39%)	2.47 ± 0.12## (+39%)	45.83 ± 3.65## (+57%)	292.70 ± 6.34***###$$$ (+18%)	29.30 ± 2.60# (−37%)
Silymarin (500 mg/kg/d)	2.97 ± 0.25**# (+41%)	3.11 ± 0.17# (+47%)	2.38 ± 0.16## (+34%)	44.26 ± 3.52# (+51%)	332.61 ± 7.53***### (+34%)	27.71 ± 3.37# (−40%)

Data are expressed as the means ± SEM (n = 9). The numbers in parentheses represent the percent change from the CCl₄-intoxicated group.

*p < 0.05, **p < 0.01, and ***p < 0.001; significantly different compared with the normal control group.

# p < 0.05, ##p < 0.01, and ###p < 0.001; significantly different compared with the CCl₄-intoxicated group.

$$$p < 0.001; significantly different compared with the silymarin-treated group.

Histopathological observations

The liver of the CCl₄-intoxicated group showed obvious loss of the hepatic architecture (in 62.5% of animals), marked hydropic degeneration (in 62.5% of animals), fatty changes (75% of animals), Kupffer cell hyperplasia (75% of animals), central vein congestion (62.5% of animals), lymphocytic infiltration (100% of animals), and necrosis (100% of animals). The histological changes induced by CCl₄ were markedly reduced in the groups treated with MSE at the two tested doses (Figure 2 and Supplementary data). It was clear that the liver architecture was markedly preserved in 87.5 and 100% of animals treated with 500 and 1000 mg/kg of MSE, respectively. Moreover, MSE conferred protection from liver damage as evidenced by the marked decrease in hydropic degeneration, fatty changes, central vein congestion, Kupffer cell hyperplasia, and necrosis, as well as minimal inflammatory infiltration, indicating potent hepatoprotective action of MSE (Figure 2 and Supplementary data). It was obvious that MSE, at the two treatment doses, is more effective than silymarin in restoring the liver architecture and reducing the fatty changes, central vein congestion, Kupffer cell hyperplasia, inflammatory infiltration, and necrosis induced by CCl₄. Notably, treatment with MSE at a dose of 1000 mg/kg resulted in complete preservation of liver architecture and prevention of fatty changes.

Figure 2. Hepatoprotective effect of MSE in CCl₄-intoxicated mice. (A) Group I (normal control): showing normal hepatic architecture and normal hepatocytes. (B) Group II (CCl₄ only): showing marked loss of hepatic architecture, central vein congestion, hydropic degeneration, scattered lymphocytes in between the hepatocytes and spotty necrosis in the sinusoids. (C and D) Groups III and IV (CCl₄ + 500 mg/kg or CCl₄ + 1000 mg/kg, respectively, of MSE): showing intact hepatic architecture and normal hepatocytes. (E) Group V (CCl₄ + 500 mg/kg of silymarin): showing normal hepatic architecture and mild hydropic degeneration of hepatocytes, with moderate scattered lymphocytes in between the hepatocytes and in the sinusoids (H&E, 200×). Hydropic degeneration

, central vein congestion

, scattered lymphocytes in between the hepatocytes and in the sinusoids

.

Discussion

Oxidative stress plays a pivotal role in the pathogenesis and progression of liver diseases, as well as in drug-induced hepatotoxicity (Singal et al., 2011). Many experimental animal models use carbon tetrachloride (CCl₄) to induce liver injury (Lee & Lim, 2008). The hepatotoxic trichloromethyl radicals (CCl₃O₂^• and CCl₃) are formed from ^•CCl₄ by cytochrome P-450 and they have a central role in the initiation of lipid peroxidation and oxidative stress (Lee & Lim, 2008; Srivastava & Shivanandappa, 2010). The trichloromethyl radicals change the antioxidant state by decreasing the activity of the defense antioxidant enzymes SOD, GPx, GR, and GST, as well as GSH (Srivastava & Shivanandappa, 2010).

In this study, CCl₄ treatment markedly increased the serum levels of AST, ALT, and ALP, which indicated severe liver injury (Wang et al., 2004). The leakage of the marker enzymes into the blood was associated with substantial necrosis, loss of hepatic architecture, hydropic degeneration, fatty changes, Kupffer cell hyperplasia, central vein congestion, and inflammatory infiltration of the liver. The MDA level in the liver tissue was increased in response to CCl₄ treatment. In contrast, the levels of GPx, SOD, GST, GSH, and GR were markedly decreased over those of the normal group, indicating oxidative damage of the liver. Preliminary antioxidant testing of MSE, using the DPPH^· assay, clearly indicated its strong radical-scavenging activity. The potent scavenging of the DPPH^· was closely correlated with the high phenol content of MSE. Based on these findings, the hepatoprotective and antioxidant effect of MSE were investigated against CCl₄-induced liver damage in mice. MSE was shown to have a significant hepatoprotective effect, by reducing the leakage of the hepatic enzymes and by preventing the lipid peroxidation. Treatment with MSE returned the increased MDA close to its normal level, and ameliorated the aforementioned histopathological changes of the liver. It was clear that MSE is more effective than silymarin in restoring the normal liver architecture and reducing the pathological changes induced by CCl₄. There is a growing body of evidence that hepatocellular damage and subsequent infiltration by inflammatory cells activate Kupffer cells to release different cytokines and free radicals, which in turn stimulate the transformation of hepatic stellate cells to myofibroblast-like cells, ultimately leading to hepatic fibrosis (Shin et al., 2010). The protective effect of MSE against Kupffer cell hyperplasia, inflammatory infiltration, and other CCl₄-induced histopathological changes in liver provided further evidence in favor of the therapeutic potential of this plant as a hepatoprotective and anti-fibrogenic agent. In addition, the levels of GSH and antioxidant enzymes, especially GR and GST, were markedly enhanced compared with the silymarin-treated group. Modulation of these antioxidant defenses clearly contributed to the strong antioxidant and hepatoprotective effect of MSE. The remarkable antioxidant effect of MSE may be related to the presence of ellagitannins and other phenoilc compounds, which can be synergistic in their action against lipid peroxidation (Haslam, 1996; Shimoda et al., 2008). Experimental evidence proved that whole plant extracts usually possess much better pharmacological activities than single isolated ingredients due to synergistic interactions between the individual components (Wagner & Ulrich-Merzenich, 2009; Williamson, 2001). It is also known that mixtures of antioxidant compounds are more active than the individual components of these mixtures (Procházková et al., 2011).

Ellagitannins are a unique group of phenolic metabolites that are widely distributed in plant foods. They have attracted considerable attention due to their potent antioxidant effects (Haslam, 1996; Okuda, 2005). Previous studies revealed that the ellagitannin-rich fraction of walnuts exhibited hepatoprotective activity against CCl₄-induced liver injury in mice and suppressed the elevated AST and ALT levels (Shimoda et al., 2008). Tellimagrandin I and II were shown to be the principal constituents responsible for the hepatoprotective effect of walnuts (Shimoda et al., 2008). In vitro studies revealed that ellagitannins having both hexahydroxydiphenoyl (HHDP) and galloyl groups, such as tellimagrandin I and II were more effective in protecting the hepatocytes from CCl₄-induced damage than other polyphenols. The mechanism of the hepatoprotective activity of tellimagrandin I and II was shown to be different from that of scavenging free radicals because other phenolic compounds, including pedunculagin, pentagalloylglucose, and casuarinin exhibited strong antioxidant activity but showed weak protective activity against CCl₄-induced damage (Shimoda et al., 2008). However, other studies indicated that pentagalloylglucose produced a strong inhibitory activity against lipid peroxidation and a cytoprotective effect (Okuda, 2005; Piao et al., 2009). Pentagalloylglucose also showed strong radical-scavenging activities and enhanced the levels of the antioxidant enzymes, including SOD and GPx (Piao et al., 2009), which might also contribute to the strong antioxidant activity of MSE. Therefore, the hepatoprotective activity of MSE may be related to the presence of tellimagrandin I, tellimagrandin II, and pentagalloylglucose. The presence of other phenolic compounds in MSE, including pedunculagin and casuarinin might contribute to the overall activity through their strong antioxidative effect (Okuda, 2005; Shimoda et al., 2008). Flavonoids, especially flavonols, possess various biological effects that contribute to health benefits including antioxidant and hepatoprotective effects (Han et al., 2007; Procházková et al., 2011). Flavonols prevent the oxidative stress by direct scavenging of free radicals, metal chelation, and induction of antioxidant enzymes as well as phase II detoxifying enzymes (Han et al., 2007; Kondratyuk & Pezzuto, 2004; Procházková et al., 2011; Ramos, 2008). Therefore, the presence of kaempferol rhamnopyranoside in MSE could contribute to its overall antioxidant and hepatoprotective activity.

Conclusions

The current study provides evidence that MSE confers potent antioxidant and hepatoprotective effects against the liver toxicity induced by CCl₄. The remarkable hepatoprotective effect of MSE is mediated, at least in part, by the potent antioxidant properties of its polyphenolic constituents, which may exert a synergistic action. Notably, this is the first report that has confirmed the antioxidant and hepatoprotective potentials of MSE through in vivo and in vitro experiments. These findings suggest that MSE could protect against liver diseases and xenobiotic-induced hepatotoxicity. Further research with the individual compounds of M. styphelioides is currently conducted to determine the mechanisms involved in the hepatoprotective effect and to elucidate the possible synergism from these components, which may potentiate the hepatoprotective activity at the molecular level.

Declaration of interest

The authors have declared no conflicts of interest.

Acknowledgements

We thank Professor Juha-Pekka Salminen, Department of Chemistry, University of Turku, Finland, for the use of the LC-MS instrument during this study.