Hepatoprotective effects of the decoction and macerated leaves of Rhamnus alaternus L . on rats exposed to carbon tetrachloride

The plant of Ramnus alaternus L. (Rhamnaceae) has been object of many therapeutic indications for traditional medicine. Many studies have showed a hepatoprotective activity of R. alaternus L. The objective of this study was to compare the hepatoprotective activities of decoction and macerated R. Alaternus L. leaves extracts on rats initially exposed to carbon tetrachloride CCl4 (1 ml/kg). The macerated leaves (250 mg/kg body weight) showed a highly significant hepatoprotective activity (p < 0.01) expressed by a significant decrease in enzymatic biochemical markers such as total bilirubin, alkaline phosphatase (ALP) and transaminases (GOT and GPT). Mean serum biochemical marker levels were 4.5 ± 0.9 mg/dL, 143 ± 3.7, 43.5 ± 9.2 and 32.2 ± 5.1 U/L, respectively. These results illustrate the dominance of the hepatoprotective pharmacological activity of macerated R. alaternus L. leaves.


INTRODUCTION
Liver diseases have a multifactorial etiology including infectious factor.High mortality rate has been correlated with two types of liver diseases namely, jaundice and hepatitis.(Pang et al., 1992).Hepatitis, has been associated with the drinking of contaminated water and poor hygiene (WHO, 2005).Jaundice which is common in children has been characterized by yellow skin because of an excess of bilirubin (Sourabie et al., 2012).Hepatoprotective effects of drugs and plant extracts have been studied by using carbon tetrachloride (CCl 4 ) induced hepatotoxicity in animal models (Suja et al., 2002).Studies have shown toxicity of CCl 4 which leads to free radicals in tissues such as liver, kidney, heart, lung, testis, brain and blood (Kumar et al., 2005;Khan and Ahmed, 2009).Research in herbal medicine has been an alternative therapy for liver disease (Pramyothin et al., 2005).Plant drugs have been known for their role in the management of hepatic diseases.Medicinal plants have been an important source of bioactive molecules, used in the food industry, cosmetics and pharmacy.
These molecules include coumarins, alkaloids, phenolic acids, tannins, flavonoids and terpenes (Bahorun et al., 1996).Polyphenols have therapeutic virtues, mainly against cancer and cardiovascular disease.These molecules are involved in the protection of plants against microbial attacks (Bruneton, 1999).Different medicinal plants species such as Tinospora bakis, Cochlospermum tinctoria, Nauclea latifolia and Argemone mexicana, have been used in traditional medicine for the therapy of jaundice (Sourabie et al., 2012).Through a rich literature on the phototherapy, it has been found that Rhamnus alaternus L. known by populations of different areas of Algeria for its therapeutic activities against jaundice (Figure 1).Important works on the chemistry of R. alaternus L. have been carried out and significant pharmacological properties have been reported by studies in Algeria and Tunisia (Ben Ammar et al., 2008;Chemli et al., 2006).Therapeutic properties of this species have been demonstrated in vitro because of their active compounds (Ben Ammar et al., 2008).The present study aimed to compare the hepatoprotective effects of the decoction and macerated leaves of R. alaternus L. on rats exposed to CCl 4 .

Description of plant
Leaves of R. alaternus have been collected during the month of October, 2013 in rural areas of Mohammedia, located in North-West of Algeria (Figure 2).R. alatenus L. is a sun-loving plant, growing in Mediterranean areas.It is a shrub of 5 m high with non spiny leaves and fruits (Chemli et al., 2006;Rameau et al., 2008).The leaves are oval or lance-shaped with slightly toothed edges, buckthorn, short-stalked, thick and leathery.The fruits appear in late fall.There are small red and black berries, grouped in compact clusters.Identification of the plant was made by M. Terras, researcher and team member of Laboratory of water sources and environment Biology Department at the University of Saida (Algeria).Dried leaves have been transformed into powder using an electric mill (Moulinex model D5001).Powders have been used to prepare aqueous decoction and macerated extract of the leaves.

Preparation of extract
To prepare an aqueous decoction, two hundred fifty grams (250 g) of powder leaves of R. alaternus have been used in a flask containing 1000 ml of distilled water.Decoction has been maintained under continuous reflux for 2 h at 80°C (Belhattab et al., 2004).Decoction has been filtered through a funnel containing cotton wool and then centrifuged at 2500 rpm for 5 min.Aqueous macerated extract of the leaves was obtained in the same conditions as the decoction with the only difference that it has made macerated extract (cold extraction) without any heating process of the test portion (250 g).

Preparation of animals
Forty (40) male adult Wistar rats, 2 months old and weighing about (180 to 200 g) from the central animal house of the Department of Biology of Saida University, were used in this study.They were kept under standard environmental conditions at 25°C with 12:12 h lightdark cycle in ventilated plastic cages.The rats were fed with standard feed livestock and water ad libitum.The animals were divided into 4 groups (10 rats per group) as follow: Group 1: served as normal controls (NC) and rats received a tap water daily for 7 days orally.Group 2: served as toxic control and the rats were injected intraperitoneal with carbon tetrachloride (CCl 4 ) (1 ml/kg body weight) daily for the same period (7 days).Group 3: (CCl 4 + decoction of R. alaternus leaves): the rats were injected intraperitoneal with CCl 4 (1 ml/kg body weight) and treated orally with the decoction of R. alaternus leaves (250 mg/kg body weight) daily for the same period.Group 4: (CCl 4 + macerated R. alaternus leaves): the rats were injected intraperitoneal with CCl 4 (1 ml/kg body weight) and treated orally with the macerated R. alaternus leaves (250 mg/kg body weight) daily for the same period (Sourabie et al., 2012).

Biochemical study
At the end of the experiments, all animals were sacrificed and the blood from each animal was taken into haemolysis tubes (5 mL).The blood samples were centrifuged at 2500 rpm for 10 to 15 min and the sera isolated were used for estimation of the serum biochemical markers of liver; as serum glutamic oxaloacetic transaminase (GOT), serum glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP) and total bilirubin.

Histopathology
The animals were dissected to isolate liver tissues.After draining the blood, liver samples were excised, washed with normal saline and processed separately for histological observations.Initially, the materials were fixed in 10% buffered neutral formalin for 48 h and then with bovine solution for 6 h.Paraffin sections were taken at 5 mm thickness, processed in alcohol-xylene series and were stained with alum haematoxylin and eosin (Galighter et Koyloff, 1971).The sections were examined microscopically for histopathological changes.

Statistical analysis
Data are expressed as mean ± SD, with a value of p < 0.05 considered statistically significant.Statistical evaluation was performed by one way analysis of variance (ANOVA) followed by the Tukey's t-test for multiple comparisons.All analysis was made with the statistical software Sigmaplot (version 11.0).

RESULTS
As shown in Table 1 and Figure 3, body weight of rats exposed to CCl 4 (Group 2) was highly decreased significantly (p** < 0.01) compared with normal control rats (Group 1).The median body weight in group of rats administered CCl 4 was lower (174.57± 5.2 g) compared to normal control rats (Group1) which showed a higher mean body weight (224.28 ± 6.6 g).Whereas the body weight of rats (Groups 3 and 4) has not showed a significant difference.Their mean body weights were 179.85 ± 1.68 and 196.28 ± 1.63 g, respectively.
The administration of carbon tetrachloride CCl 4 (1 ml/kg body weight) developed a significant increase in serum enzymatic markers (p** < 0.01), especially serum transaminase (GOT and GPT) and alkaline phosphatase (ALP).Similarly, it was observed concomitantly, a significant increase in serum total bilirubin (p** < 0.01) (Table 1).This phenomenon was particularly observed in rats of Group 2 which were administrated CCl 4 , in comparison with the normal control rats of Group 1 as shown in Figures 4, 5, 6, 7.
The aqueous extracts (decoction and macerated) administered at the same dose (250 mg/kg) developed a significant decrease in serum enzymes (GOT and GPT) (p** < 0.01) and a decrease in the serum total bilirubin concentration as compared to animals which were administered CCl 4 (Group 2) (Figures 4,5,6,7).The effect of different treatments (decoction and macerated) on serum enzyme markers and serum total bilirubin is reported in Figures 4, 5, 6, 7. From the perspective of pharmacological activity, the decrease of biological parameters observed in the test Groups 3 and 4 is a clear sign and an illustration of the ability of these extracts to reduce the hepatotoxicity induced by the administration of toxic organochlorine (CCl 4 , 1 ml/ kg B.W). Histopathologies of various groups are shown in Figure 8.In Group 1 (normal control rats), liver tissue section liver tissue section (Figure 8C) shows mild degree of liver necrosis.Hepatocytes are compact.Hepatic sinusoids appear normal.The Hepatocytes are well arranged like clusters.In Group 4 (rats treated with CCl 4 and the macerated R. alaternus leaves), liver tissue section (Figure 8D) shows that hepatocytes were regenerative and showed no visible changes and prominent nuclei, reduced score of necrosis and no fatty changes.Thus, confirming the safety of the extract.

DISCUSSION
CCl 4 has been used in this present study for its liver toxicity and tropism for hepatocytes.Cell mechanism of CCl 4 , has been previously described in different studies (Sarada et al., 2012;Letteron et al., 1990), showing alteration and necrosis of liver cells.Hepatic metabolism of CCl 4 has been initiated by transforming it into its primary metabolites (trichloromethyl and trichlorométhyl peroxyde) through the hepatic cytochrome P450 oxidase and main enzymatic system involved into redox reactions of xenobiotics in the liver.Trichloromethyl peroxide is highly reactive free radicals that will initiate a lipid peroxidation (Sourabie et al., 2012).Free radicals have caused an important flow of GOT and GPT from hepatocyte membranes into blood medium (Sourabie et al., 2012).Results of this present study showed that a dose of CCl 4 (250 ml/kg ) have caused severe hepatocellular injury as indicated by the massive elevations of GPT, GOT, ALP and total bilirubin levels in rats intoxicated with CCl 4 (Group 2) when compared with normal control animals (Group 1).Treated rats (Groups 3, 4) have preserved the integrity of liver cells and significantly decreased elevated enzymatic parameters (GOT, GPT and ALP) and total bilirubin levels.Dose (250 mg/kg) of aqueous extract R. alaternus leaves has been used in the present study based on some published studies demonstrating the hepatoprotective activity of this dose (Gopal et al., 2008).In this present study, decoction and specially macerated leaves treated groups (Groups 3 and 4) have exhibited lower levels of GPT, GOT, ALP and bilirubin as compared to CCl 4 treated group (Group 1).Reduction of these parameters is an indication of the stabilization of cell membranes as well as repair of hepatic tissue damage caused by CCl 4 (Lin et al., 2008).The stabilization of serum bilirubin, GPT, GOT, and ALP levels by decoction and macerated plant leaves is a clear indication of the improvement of the functional status of the liver cells.This indicates the anti-lipid peroxidation of aqueous extract of R. alaternus leaves which acted against the damaging effects of free radicals produced by CCl 4 .Histopathological examination clearly reveals that the hepatic cells, central vein and portal triad are almost normal in decoction and macerated R. alaternus leaves treated group (250 mg/kg) in contrast to group which received CCl 4 .Thus, decoction and macerated R. alaternus leaves can be considered to be an effective hepatoprotective agent as it ameliorated almost to normalcy the damage caused by CCl 4 to hepatic function.In literature, it has been suggested that administration of extract of R. alaternus increased enzymatic parameters (GOT, GPT and ALP) and total bilirubin level and it may be due to the presence of active constituents such as flavonoids and alkaloids (Ben Ammar et al., 2009).Studies have shown that phenolic compounds had an antioxidant activity as scavenging free radicals (Seyoum et al., 2006).
Phenolic compounds can also act as antioxidants by chelating metal ions, preventing radical formation and improving the antioxidant endogenous system (Al-Azzawie et al., 2006).These compounds are known to act as antioxidants not only because of their ability to donate hydrogen or electrons but also because they are stable radical intermediates.Probably the most important natural phenolics are flavonoids because of their broad spectrum of chemical and biological activities, including antioxidant and free radical scavenging properties (Kahkonen et al., 1999).In fact, flavonoids have been reported as antioxidants, scavengers of a wide range of reactive oxygen species and inhibitors of lipid peroxidation (Williams et al., 2004).

CONCLUSION
Both decoction and macerated R. alaternus L. leaves have exhibited a potent hepato-ameliorating and antioxidant effects in rats exposed to CCl 4 .But hepatoameliorating and antioxidant effects of macerated R. alaternus L. leaves were found to be better than those of decoction plant leaves.In two cases, the hepatoprorective qualities of aqueous extract of R. alaternus L. leaves need to be addressed by isolating and characterizing the active principle(s) responsible for hepatoprotective activity.

Figure 2 .
Figure 2. Geographical map of Mohammedia, located in North-West of Algeria.(Diagram tourism development of the department of Mascara, 2015).

Table 1 .
Effects of decoction and macerated Rhamnus alatenus leaves on serum hepatic parameters in groups of control rats and exposed to CCl 4 .