Hepatoprotective effect of Rosa canina fruit extract against carbon tetrachloride induced hepatotoxicity in rat.

OBJECTIVE: The present study was conducted to investigate the hepatoprotective activity of hydro-ethanolic fruit extract of Rosa canina (R. canina) against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats.
MATERIALS AND METHODS: Male Wistar albino rats were randomly divided into six groups of 8 animals of each, including control, toxic (CCl4), R. canina 250, 500, and 750 mg/kg + CCl4 and R. canina 750 mg/kg alone. R. canina (p.o., daily) and CCl4 (1 ml/kg twice a week, 50% v/v in olive oil, i.p.) were administered to animals for six weeks. Serum analysis was performed to assay the levels of aspartate aminotransferase (AST), alanine amino transaminase (ALT), alkaline phosphatase (ALP), albumin (ALB), total protein (TP) and malondialdehyde (MDA). Biochemical observations were also supplemented with histopathological examination (haematoxylin and eosin staining) of liver section.
RESULTS: Hepatotoxicity was evidenced by considerable increase in serum levels of AST, ALT, ALP, and lipid peroxidation (MDA) and decrease in levels of ALB and TP. Injection of CCL4 also induced congestion in central vein, and lymphocyte infiltration. Treatment with hydro-alcoholic fruit extract of R. canina at doses of 500 and 750 mg/kg significantly reduced CCl4-elevated levels of ALT, AST, ALP and MDA (p<0.01). The extract also increased the serum levels of ALB and TP compared to CCl4 group (p<0.01) at the indicated dose Histopathological studies supported the biochemical finding.
CONCLUSION: Our finding indicated hepatoprotective effects of the hydro-alcoholic fruit extract of R. canina on CCl4-induced hepatic damage in rats and suggested that these effects may be produced through reducing oxidative stress.

Introduction

Liver is the most important metabolic organ in the body. It plays a major role in the metabolism, detoxification, storage and secretory functions in the body of animals (Swarnalatha and Reddy 2012). Various xenobiotics or oxidative stress can produce deleterious effects on these important functions of liver (Kale et al., 2012 ▶). Despite improvement in the pharmaceutical sciences, hepatic disease remains as an unsolved worldwide health problem. Thus searching for new treatment is still ongoing. Nowadays, herbal medicines are one of the main options for prevention or treatment of liver disorders because of their multiple beneficial properties such as antioxidant and anti-inflammatory activities (Jaishree and Badami 2010 ▶; Pareek et al., 2013).

Rosa canina L. is a shrub of the Rosaceae family that grows in Europe, western Asia and north-eastern Africa. Rose fruit is traditionally used as diuretic, laxative, and the management of gallstones, arthritis, gout, fever, colds (Orhan et al., 2007; Chrubasik et al., 2008 ▶; GHIORGHIŢĂ1 et al., 2012 ▶). Phytochemical analysis of the fruit extract of R. canina indicated the presence of flavonoids, phenolic acids, tannins, carotenoids, fatty acids, phospholipids, galactolipids, minerals, vitamins (particularly vitamin C and also vitamins B1, B2, K, PP, E) in the extract (Demir and Özcan 2001 ▶; Lattanzio et al., 2011 ▶). R. canina is well-known for its high phenolic contents (Roman et al., 2013). These compounds constitutes an important class of known natural antioxidants that exhibit various important physiological and biological properties in in-vivo and in-vitro (Campos-Vega et al., 2010 ▶). Antioxidant activity of the R. canina fruit extract has been documented in several studies (Kilicgun and Altiner 2010 ▶; Naser et al., 2011; Fattahi et al., 2012 ▶; Roman et al., 2013). Moreover, many natural agents with antioxidant activity have been proposed to prevent and cure hepatotoxicity induced by different toxins (Akindele et al., 2010 ▶, Murat Bilgin et al., 2011, Bachar et al., 2012, Ottu et al., 2013).Therefore, the present study was designed to examine the potential hepatoprotective effects of hydro-alcoholic extract of R. canina fruit on hepatotoxicity induced by carbon tetrachloride (CCl4) in rats.

Materials and Methods

Plant material

The fruit of R. canina was collected in October 2013 from Dena Mountains, Kohgilouyeh and Boyerahmad province, Iran. The collected parts were neatened and shade dried.

Extract preparation

At first, the dried fruits of R. canina have been grinded. The powdered R. canina fruit was macerated for 48 h in ethanol/water (50/50, v/v) at room temperature and then percolated. Next, for the second time, adequate 50% ethanol was added to residual crushed material for 24 h and subsequently re-percolated. The extract solution was collected and concentrated using a rotary evaporator (Hyedolph, type: HeizbadHei-VAP, Germany) at 40°C under reduced pressure. Finally, the extract was weighted and stored at -20°C (Sadeghi et al., 2014).

Chemicals

Carbon tetrachloride (CCl4), Trichloroacetic acid (TCA), thiobarbituric acid (TBA), diethyl ether and other solvents were obtained from Merck, Germany. The assay kits for the determination of ALT, AST, ALP, ALB and TP were purchased from Pars Azmun,Tehran, Iran.

Experimental animals

Forty eight male albino Wistar rats (180–220 g) were purchased from Razi Institute of Iran (Tehran, Iran). The animals were maintained under standard environmental conditions and were allowed to take standard laboratory feed, and tap water.

Hepato-protective activity

The rats were randomly divided into six groups of eight animals per each. Treatment was carried out according to following groups for six weeks:

Group I (control group) received olive oil.

Group II (hepatotoxic group) received i.p. injection of CCl4 (50% v/v in olive oil,1 ml/kg twice a week (AkbartabarToori et al., 2015 ▶).

Groups III–V (the extract groups) receivedCCl4plus R. canina (p.o., daily) extract at doses of 250, 500 and 750 mg/kg (Deliorman Orhan et al., 2007 ▶).

Group VI only received R. coninafruit extract at doses of 750 mg/kg

Assessment of liver function parameters

Twenty-four hours after the last dose of the CCl4 and R. canina extract, blood samples were collected by cardiac puncture. Then, the animals were euthanized by an overdose of diethyl ether. In order to serum separation the blood was allowed to coagulate at room temperature for 30 min and then centrifuged at 2000 rpm for 15 min. The serum was evaluated for levels of ALT, AST, ALP, ALB and TP using pars azmun diagnostic kits, Tehran, Iran, according to the method described by the manufacturers.

Determination of lipid peroxidation

The measurement of serum MDA level was performed according to the method previously described(Buege and Aust 1978 ▶). According to this method, 375 mg of TBA was dissolved in 2 mL of chlorhydric acid (HCl, 0.25 N), followed by 15 g of trichloroacetic acid (TCA) for a total volume of 100mL. The solution was heated in a water bath at 50 °C till TBA properly dissolved Then, 1 mL of serum was mixed with 2 mL of TCA—TBA—HCl. Next, the solution was heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation (1700×g,15 min). Finally the absorption of supernatant was determined at 535 nm against a blank that contained all reagents except the serum sample. Serum MDA concentration was expressed as nmol/ml (Sharma et al., 2006).

Histological studies

After euthanizing the animals, livers were carefully removed and fixed in 10% buffered formaldehyde solution for 1 week. Then, the fixed biopsies were embedded in paraffin and cut into 3–4 mm slices. The slices were mounted on glass slides and stained with hematoxylin and eosin for pathological analysis. The images were examined under light microscope (Sadeghi et al., 2014)

Statistical analysis

The data were expressed as the mean±SD. The differences between the control and treatment groups were tested by ANOVA followed by the Tamhane'spost-hoc test, using SPSS 16.0 software. p<0.05 was considered to show significant differences for all comparisons.

Results

Assessment of liver function parameters

As shown in Table 1, i.p. injection of CCl4 significantly (p<0.001) increased the serum levels of ALT, AST and ALP in toxic group, compared to the control group. The serum levels of ALB and TP was significantly decreased because of intoxication with CCl4 (p<0.01). R. canina fruit extract at doses of 500 and 750 mg/kg markedly inhibited the CCl4-induced liver injury according AST, ALT and ALP (p<0.01, p<0.001, respectively). R. canina considerably increased the diminished serum levels of ALB at doses of 500 and 750 mg/kg (p<0.05 and p<0.01 respectively).The extract also increased the serum levels of TP at the indicated doses compared to the toxic group, although this effect was statistically significant only at the dose of 750 mg/kg (p<0.001). Additionally, the administration of R. canina alone (750 mg/kg, Group VI) did not show any effect on the serum levels of ALT, AST, ALP, ALB and TP compared to control group.

Table1

Effect of Rosa canina fruit extract on serum biochemical parameters in CCl4-induced hepatotoxicity in rats.

Treatment group	Dose (per kg)	ALT (U/l)	AST (U/l)	ALP (U/l)	ALB(g/dl)	TP (g/dl)
Control	1 ml vehicle	72.4 ± 4.07	146.6 ± 18.73	259.4 ± 44.08	3.38 ± 0.14	7.06 ± 0.32
CCl 4	CCl4 in olive oil(1:1) 1 ml	167.3 ± 20.41***	252 ± 32.46 ***	591.4 ± 46.99 ***	2.72 ± 0.22 **	6.34 ± 0.20 **
RC + CCl 4	250 mg; 1 ml	129.9 ± 17.72 **	228.8 ± 32.66 *	513.7 ± 64.24 ***	2.88 ± 0.09 ***	6.35 ± 0.13 *
RC + CCl 4	500 mg; 1 ml	85.6 ± 9.75 c	177.7 ± 48.29	412.4 ± 72.29 **b	3.14 ± 0.05 a	6.87 ± 0.36
RC + CCl 4	750 mg; 1 ml	74.4 ± 8.38 c	166.7 ± 24.16 b	310.1 ± 59.32 c	3.45 ± 0.08 b	7.19 ± 0.29 c
RC only	750 mg	64.8 ± 3.87 c	152.2 ± 18.51 b	273.2 ± 41.15 c	3.41 ± 0.07 b	7.01 ± 0.30 a

The values are reported as the mean ± SD (n = 8) RC, hydro-alcoholic extract from Rosa canina fruit; ALT, alanine aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALB, albumin; TP, total protein; MDA, malondialdehyde.

p<0.05,

p<0.01,

p<0.001 vs. control group.

p<0.05,

p<0.01,

p<0.001 vs. CCl4 group

Determination of lipid peroxidation

Lipid peroxidation was determined by measurement of malondialdehyde (MDA) concentration in plasma. As shown in Fig 1, a significant increase in the serum level of MDA was observed in rats intoxicated with CCl4 when compared with the control group (p<0.001). Treatment of the animals with R. caninaat doses of 500 and 750 mg/kg inhibited the increase in serum levels of MDAdue to injection of CCl4 (p<0.001).There was no difference in MDA serum levels in the rats treated with R. canina alone (750mg/kg) and control group.

Figure 1

Effect of Rosa canina fruit on serum malondialdehyde (MDA) level in CCl4-treated rats. Data express the mean ± S.D.* p<0.05 and *** p<0.001 vs. control group. C, p<0.001 vs. CCl4 group

Histopathological studies

As illustrated in Fig.2, histological assessment of the liver sections revealed that injection of CCl4 induced pathological changes such as loss of cellular boundaries, congestion in central vein, destroyed lobular structure and lymphocyte infiltration. Treatment of the animals with R. canina at doses of 500 and 750 mg/kg reversed the hepatic damage induced by CCl4 towards normal. However, the extract of R. canina fruit at dose of 250 mg/kg did not show a considerable protective effect on the pathological changes induced by CCl4. Moreover, administration of the extract alone (750 mg/kg) did not produce any liver damage compared to control group.

Figure 2

Effect of R. canina fruit extract on CCl4 induced hepatotoxicity in rats. The liver slices of rats were mounted on glass slides and stained with hematoxylin and eosin. The images were examined under light microscope. Intoxication with CCl4 induced pathological changes such as loss of cellular boundaries, congestion in central vein, destroyed lobular structure and lymphocyte infiltration (B). Treatment of the animals with R. canina at doses of 500 and 750 mg/kg reversed the hepatic damage induced by CCl4 towards normal (D,F). A : Control group, B: CCl4 group, C: CCl4+ R. canina 250 mg/kg D: CCl4+ R. canina 500 mg/kg, E: CCl4 + R. canina 750 mg/kg, F: R. canina 750 mg/kg alone

Discussion

The results of the present study clearly indicated hepatoprotective effects of the ethanolic extract of R. canina fruit against CCl4-induced hepatic damage in rats. Hepatotoxicity induced by CCl4 is one of the best characterized systems of xenobiotic-induced hepatotoxicity in experimental animals. This method usually used to evaluation hepatoprotective properties of many bioactive substances and medicinal plants (Huang et al., 2013 ▶). It has been reported that lipid peroxidation, reducing activity of antioxidant enzymes and generation of free radicals are the primary reasons of CCl4-induced hepatic injury (Srivastava and Shivanandappa 2006 ▶). These changes are similar to those observed during cellular oxidative stress, which is considered to have an important role in the pathogenesis of many disorders such as hepatic injuries (Wu et al., 2007 ▶).

In this study, CCl4 intoxication considerably raised the serum levels of ALT, AST and ALP in the animals, a marker of cellular leakage and failure in activities of cell membrane in liver (Mujahid et al., 2013). These results are in good agreement with previous studies. Yang and co-workers showed that intoxication of rats with CCL4 increases levels of ALT, AST and ALP in serum indicate liver damage (Yang et al., 2011 ▶). Furthermore, reduction of ALB and TP by CCl4 is a further indication of liver injury. CCl4 causes disruption and disassociation of polyribosomes on endoplasmic reticulum which lead to reducing the biosynthesis of protein (Sathesh Kumar et al., 2009 ▶). Treatment with the fruit extract of R. canina at 500 and 750 mg/kg doses significantly inhibited the increase in the levels of these marker enzymes, implying protection against liver damage. Therefore, it is possible that the extract stabilizes the plasma membrane or repairs the hepatic tissue injuries induced by CCl4. Injection of CCl4 also caused a significant reduction in the serum levels of TP and ALB, further indicator of liver toxicity (Ravikumar and Gnanadesigan 2012 ▶). The extract reversed the CCl4-induced decrease of serum levels of TP and ALB towards normal.

As mentioned above, generation of reactive oxygen species and lipid peroxidation participate in the liver toxicity of CCl4.MDA is a secondary product of poly–unsaturated fatty acids peroxidation (Amat et al., 2010 ▶) and serves as a main marker to estimate the levels of lipid peroxidation (Cho et al., 2013 ▶). In addition, the level of lipid peroxidation is a measure of membrane damage and shows alteration in structure and function of cellular membrane (Kepekçi et al., 2013 ▶). In the present study the fruit extract of R. canina at the doses of 500 and 750 mg/kg significantly prevented the raise in serum levels of MDA due to intoxication with CCl4 in the rats. It is important to notice that the fruits of R. canina are rich in Phenolic compounds (Hvattum 2002 ▶, Fecka 2009 ▶) which exhibit a variety of biological and pharmacological activities, including anti-inflammatory and anti-bacterial activities (Zdařilová et al., 2009 ▶). Furthermore, antioxidant activity of R. canina has been reported in some studies (Daels‐Rakotoarison et al., 2002 ▶, Serteser et al., 2009 ▶, Kilicgun and Altiner 2010 ▶). Also, it has been revealed that radical scavenging capacities of R. canina extracts positively correlated with phenolic content (Wenzig et al., 2008 ▶, Fattahi et al., 2012 ▶). Hence, it is possible that the hepatoprotective activity of the R. canina fruit extract to some extent, is mediatedby the scavenging free radical activity of the extract.

Histopathological examination of liver sections confirmed our biochemical findings. Injection of CCl4 induced a variety of hepatic histological changes including congestion in central vein, destroyed lobular structure and leucocyte infiltration. These changes were significantly attenuated by the R. canina fruit extract.

In conclusion, the results of the present study demonstrate hepatoprotective effects of the ethanolic extract of R. canina fruit on CCl4-induced hepatic damage in rats. These protective effects may be, at least in part, related to antioxidant properties of the extract.

Conflict of interest

Authors declare no conflict of interest.