Protective Effect of Ricinus communis Leaves Extract on Carbon Tetrachloride Induced Hepatotoxicity in Albino Rats

Document Type: Research Paper

Abstract

       Ricinus communis Linn. (Euphorbiaceae) is a soft wooded tree widely prevalent throughout tropics regions of the world which have a warm temperature. In the Indian system of medicine, the leaves, roots and seed oil of this plant have been used for the treatment of inflammation and liver disorders for a long time. In the present study, the protective effects of ethanol extract of Ricinus communis L. leaves on carbon tetrachloride-induced liver damage were investigated in rats. Results were compared with those of silymarin, a standard hepatoprotective drug. It was found that an increase in the activities of serum transaminases and the level of liver lipid peroxidation, protein, glycogen and the activities of acid and alkaline phosphatase in liver induced by CCl4 were significantly inhibited by treatment with Ricinus communis ethanolic extract (250/500mg/kg b.wt). In addition, the depletion of glutathione level and adenosine triphosphatase activity observed in the CCl4-induced rat liver were effectively prevented by treatment with Ricinus communis ethanolic extract (250/500mg/kg b.wt). Histopathological examination further confirmed the hepatoprotective activity of Ricinus communis ethanol extract when compared with the CCl4-induced control rats. In conclusion, these results indicate that the ethanol extract of Ricinus communis ethanolic extract exhibits hepatoprotective action.

Keywords


 

1. Introduction

            Hepatitis associated with liver cirrhosis has become one of the most prevalent diseases in the world, which can be induced by virus, alcohol or other toxic chemicals [1]. Carbon tetrachloride, an established hepatotoxin, induces toxicity in rats which closely resembles human cirrhosis [2]. Experimentally, liver diseases have been shown to be produced by the administration of carbon tetrachloride (CCl4), thioacetamide, paracetamol, etc. CCl4-induced hepatotoxicity model is frequently used for the investigation of hepatoprotective effect of drugs and plant extracts on experimental animals. CCl4 hepatotoxicity depends on the reductive dehalogenation of CCl4 catalyzed by CYP450 in the liver cell endoplasmic reticulum leading to the generation of an unstable complex of CCl3 radical. This trichloromethyl radical reacts rapidly with O2 to yield tricholomethyl peroxy radical which is reported as a highly reactive species. This free radical attacks microsomal lipids leading to their peroxidation and also covalently binds to microsomal lipids and proteins ultimately initiating a site of secondary biochemical processes which is the ultimate cause of pathological consequences of CCl4 metabolism [3]. Despite extensive research in the medicine, no drug in the modern system of medicine can be claimed to be effective to cure liver disorders, which in many times become fatal, however, the search for new medicines is still going on [4, 5]. Therefore, many remedies of folk tradition from plant origins are evaluated for its possible antioxidant and hepatoprotective effects against different chemical-induced liver damage in experimental animals.

            Ricinus communis Linn. (Euphorbiaceae) is a soft wooded tree widely prevalent throughout tropics regions of the world which have a warm temperature [6]. In the Indian system of medicine, the leaves, roots and seed oil of this plant have been used for the treatment of inflammation and liver disorders for a long time [7]. They have been found to be hepatoprotective against galactosamine-induced hepatic damage [8], hypoglycemic [9], laxative [10], diuretic [11] and antibacterial effects [12]. The leaves of Ricinus communis found to contain flavanoids like kaempferol-3-0-beta-D-rutinoside and kaempferol-3-0-beta-D-xylopyranoid and tannins [13, 14]. However, scientific studies on its utility in damaged liver are few and the aim of the present study was to confirm the hepatoprotective effect of Ricinus communis extract against CCl4-induced hepatic injury in rats. In addition, its hepatoprotective effect was compared to the effect of silymarin which is known to be effective against CCl4-or acetaminophen-induced liver damages [15].

 

Figure 1. Photomicrographies of paraffin-embedded rat liver. A: Normal Architecture of the liver cell was seen in olive oil alone treated (normal) rats; B: Few hepatocytes showing increased cytoplasmic staining and infiltration of mono nuclear cells around the portal triad and in the lobule were observed in liver of CCl4 -induced rats; C: Liver showing minimal inflammatory cellular filtration was observed in Ricinus communis ethanol extract (250 mg/kg b.wt) treated CCl4-induced rats; D: Liver showing marked improvement to normal architecture was observed in Ricinus communis ethanol extract (500 mg/kg b.wt) treated CCl4-induced rats; E: Liver returned to almost normal architecture was observed in silymarin (50 mg/kg b.wt) treated CCl4-induced rats. F: Normal Architecture of the liver cell was seen in Ricinus communis ethanol extract (500mg/kg b.wt) alone treated rats.


Figure 1A
.

 

Table 1. Activities of aspartate transaminase (AST) and alanine transaminase (ALT) in serum of normal and experimental rats.

 

Parameters

(IU/L)

Group –I (Control)

Group –II(CCl4-induced)

Group –III (CCl4-induced +Ricinus communis extract 250mg/kg/b.wt)

Group -IV(CCl4-induced +Ricinus communis extract 500mg/kg/b.wt)

Group-V(CCl4-induced + silymarin 50mg/kg/b.wt)

Group -VI(Ricinus communis extract 500mg/kg/b.wt)

 

Serum AST

 

44.4 ± 5.77

237.0± 4.64 a*

60± 5.21 b*

55± 5.21 b*

54.9± 5.2 b*

46.3± 3.94

Serum ALT

 

55.0 ± 7.94

120.4 ± 21.85 a*

65.2±  6.91 b*

60.5±  6.91 b*

59.5 ± 7.64 b*

57.2 ±6.75

The values are expressed as mean ± S.D. Each group consists of six animals Comparisons were made as follows: a: Group II (CCl4-induced) vs. Group I (control); b: Group II vs. Group III (CCl4-induced +Ricinus communis extract 250mg/kg/b.wt); Group IV (CCl4-induced +Ricinus communis extract 500mg/kg/b.wt); Group-V (CCl4-induced + silymarin 50mg/kg/b.wt); The symbol represents the stastical significance at:  *p < 0.05.


2.  Experimental

2.1. Animals

            Male albino rats of Wistar strain (140±10 g b.w.) were obtained from the Tamil Nadu Veterinary College, Chennai, India. They were acclimatized for a week in a light and temperature-controlled room with a 12 hr dark-light cycle and fed with commercial pelleted feed (Water 8.9%, Protein 25.4%, Lipid 4.4%, Carbohydrate 50.3%, Ash 6.9%, and Crude fiber 4.1%) from Hindustan Lever Ltd. (Mumbai, India) and water was made freely available. The animals used in this study were treated and cared for in accordance with the guidelines recommended by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, Ministry of Culture, Chennai, India. Experimental protocol was approved by the departmental ethical committee.

2.2. Preparation of the extract

            Fresh leaves of Ricinus communis were collected from rural areas around Chennai, India, and it was authenticated by Chief Botanist, VIT University, Vellore, India. The leaves were dried at the room temperature. The dried and powdered leaves of Ricinus communis (1 kg) were extracted with 80% ethanol at room temperature for 24 hr. It was then filtered and the filtrate was evaporated and dried in a vacuum dessicator. The percentage yield of the dried extract was 15% from the initial raw material. This dried extract and a reference drug, silymarin, Micro Labs, Tamil Nadu, India were dissolved in water and administered to the animals by oral intubation method, respectively. 

 Fig 1B

 

 

Table 2. Effect of Ricinus communis extract on CCl4-induced liver damage in rats.

Parameters

Group -I(Control)

Group -II(CCl4-induced)

Group -III(CCl4-induced +Ricinus communis extract 250mg/kg/b.wt)

Group -IV(CCl4-induced +Ricinus communis extract 500mg/kg/b.wt)

Group -V(CCl4-induced + silymarin 50mg/kg/b.wt)

Group –VI

(Ricinus communis extract 500mg/kg/b.wt)

ALP (μ moles of phenol / min/ mg protein

9.56± 0.33

24.81± 1.02a*

9.35± 0.55 b*

10.0± 1.01 b*

11.2 ±1.5 b*

10.4± 1.05

ACP (μ moles of phenol / min/ mg of protein)

5.12± 0.32

9.37± 0.56 a*

6.32± 0.45 b*

6.21± 0.39 b*

5.8 ±0.41 b*

5.4 ±0.37

Total ATPase (mg Pi/100g/min)

2.9± 0.12

1.35± 0.10 a*

2.6 ±0.09 b*

2.1 ±0.22 b*

2.45± 0.19 b*

2.32± 0.15

Glycogen (mg/100g)

 

11.00± 1.26

7.50 ±0.51 a*

10.00± 0.63 b*

10.33 ±1.03 b*

10.50 ±0.83 b*

9.83± 1.32

Protein (mg/100 g)

 

185.1± 19.2

155.2 ±10.9 a*

190.5± 14.25b*

192.5± 18.9 b*

189.2± 18.5 b*

191.2 18.2

Lipid peroxidation (nmol MDA/mg protein)

164.82 ±8.15

237.33± 9.11 a*

172.28± 7,89 b*

186.66± 16.32 b*

169.21± 11.63 b*

166.83± 5.84

Glutathione (μmol/g)

 

9.57 ± 0.17

7.52 ± 0.44 a*

9.82±  0.19 b*

9.90±  0.21 b*

8.90 ± 0.16 b*

9.01± 0.17

 

2.3. Experimental Design

            In this study, all group of rats except group I and group VI (negative control) received CCl4 once only. The animals were divided into six groups of six animals each and were treated as follows:

  • Group I: normal control (vehicle olive oil only)
  • Group II: CCl4 (3 ml/kg/b.wt i.p. in olive oil (1:1, v/v), single administration).
  • Group III:  CCl4 + Ricinus communis ethanol extract (250 mg/kg/b.wt, p.o. for five days).
  • Group IV: CCl4 + Ricinus communis ethanol extract (500 mg/kg/b.wt, p.o. for five days).
  • Group V: CCl4 + silymarin (50 mg/kg/b.wt, p.o. for five days).
  • Group VI: negative control (Ricinus communis ethanol extract 500 mg/kg/b.wt, p.o. for five days).

At the end of the experimental period, all the animals were sacrificed under ether anesthesia. Blood and liver samples were collected. The blood was allowed to clot for 30 min; serum was separated by centrifuging at 37 °C which was used for biochemical estimations.

 

Fig 1C.

 

 

 

 Fig 1D.

 

 

 

2.4. Assessment of hepatoprotective activity

            The activities of serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT) were assayed by the method of Reitman and Frankel [16]. Estimation of activities of alkaline phosphatase (ALP), acid phosphatase (ACP) by the method of King [17], and total ATPases [18], protein [19], and glycogen content [20], lipid peroxide [21] and glutathione level [22] were also carried out in liver tissue to assess the damage caused by CCl4.


2.5. Histopathological studies

            Immediately after sacrifice, a portion of the liver was fixed in 10% formalin. The washed tissue was dehydrated in descending grades of isopropanol and finally cleared in xylene. The tissue was then embedded in molten paraffin wax and cut into 5 μm thick sections in a rotary microtome. The sections were then stained with haematoxylin and were studied for histopathological changes, i.e. necrosis, fatty changes and lymphocyte infiltration. Histological damages were scored as follows: 0; absent; +: mild; ++: moderate; +++: severe.  

 

Fig 1E.


2.6. Statistical analysis

            The results were expressed as mean±S.D. and statistical analysis was performed using ANOVA to determine significant differences between the groups, followed by Student’s Newman-Keul’s test. p< 0.05 was the implied significance.

 

3. Results

            Administration of CCl4 to rats caused a significant elevation in serum aspartate, and alanine aminotransmainases (Table 1). However, the elevated SGOT and SGPT activity observed in CCl4-treated rats was found to be significantly decreased by prior administration of Ricinus communis ethanol extract.

            Alterations in the activities of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, glycogen and protein content, lipid peroxidation and glutathione level of liver after single exposure to CCl4 were expressed in Table 2. Reduced glutathione and enhanced lipid peroxide level were seen in the CCl4-treated  group, whereas the drug treated groups showed a significant rise in glutathione level with the reduction in lipid peroxidation level, when compared to the  CCl4-treated group. In CCl4-treated rats, the activities of acid phosphatase and alkaline phosphatase were found to be increased, whereas a decreased glycogen and protein content and adenosine triphosphatase activity was observed when compared to the normal control groups. Administration of Ricinus communis ethanol extract to the CCl4-treated rats modulates the above alterations to near normal levels.

            As shown in Figure 1A-F and Table 3, liver sections from the control group (Figure 1A) and Ricinus communis ethanol extract (500 mg/kg. b.wt) alone treated rats (Figure 1F) showed normal lobular architecture and normal hepatic cells.  The liver section from animals given CCl4 showed increased cytoplasmic staining and infiltration of mono nuclear cells around the portal triad and in the lobule (Figure 1B). The histological pattern of the liver of the CCl4-induced rats treated with Ricinus communis ethanol extract (Figure 1C and 1D) and silymarin (Figure 1E) showed marked improvement and return to normal architecture, respectively.

 

Fig 1F.

 

 

Table 3. Effect of the Ricinus communis ethanol extract on histopathological damages induced by CCl4 injection in rats.

Microscopic observation

 

Groups

Necrosis

Fatty changes

Infiltration of lymphocyte

Control

 

0

0

0

CCl4 (3ml/kg/b.wt, i.p.)

 

+++

+++

+++

CCl4 (3ml/kg/b.wt, i.p.) + Ricinus communis ethanol extract (250mg/kg/b.wt, p. o)

++

++

++

CCl4 (3ml/kg/b.wt, i.p.) + Ricinus communis ethanol extract (500mg/kg/b.wt, p. o)

+

+

+

CCl4 (3ml/kg/b.wt, i.p.) + silymarin (50mg/kg/b.wt, p. o.)

+

0

+

Negative control (Ricinus communis ethanol extract 500mg/kg/b.wt, p. o

+

0

+

0; absent; +: mild; ++: moderate; +++: severe; rats were injected (i.p.) with Ricinus communis ethanol extract for five days after the injection of CCl4 (3ml/kg/b.wt, i.p.). Histopathological damages were assessed as explained under materials and methods.


4. Discussion

            CCl4-induced hepatic injury is often used as a model for hepatoprotective drug screening and the extent of the hepatic damage is assessed by the level of release cytoplasmic transaminases (SGOT and SGPT) in circulation [23]. The present study revealed a significant increase in the activities of SGOT and SGPT in serum (Table 1) and elevated acid and alkaline phosphatases (ALP) (Table 2) in liver on exposure to CCl4, indicating considerable hepatocellular injury. Administration of Ricinus communis ethanol extract at two different dose levels attenuated the increased levels of marker enzymes, observed in CCl4-induced rats. The hepatoprotective effect of the Ricinus communis ethanol extract was further supported by the limited extent of histological changes (Figure 1), in Ricinus communis treated CCl4-induced rats. This is in agreement with the commonly accepted view that serum levels of transaminases return to normal with healing of hepatic parenchyma and the regeneration of hepatocytes [24].

            CCl4 is believed to be metabolized by microsomal CYP450 in the liver to a highly reactive trichloromethyl free radical, which can start a chain of reactive free radical formation resulting in peroxidation of lipids and damage to proteins and cell components [5]. The level of lipid peroxide is a measure of membrane damage and alterations in structure and function of cellular membranes. Glutathione protect cells against electrophilic attacks provided by xenobiotics such as free radicals and peroxides. The elevation of MDA levels, which is one of the end products of lipid peroxidation in the liver tissue, and reduction of hepatic GSH levels are important indicators in CCl4-intoxicated rats [25]. In this study, enhanced liver lipid peroxidation and reduced glutathione level were seen in Group II CCl4 treated rats (Table 2). However, Ricinus communis extract treatment, prevented glutathione depletion with reduction in lipid peroxidation level in CCl4-induced rats when compared to CCl4-treated control group. The Ricinus communis extract with the potentiality to scavenge the free radicals contains flavanoids and tannins. Flavanoids and tannins have been reported to have anti-peroxidative effects [26]. Moreover recently Ricinus communis root extract was also found to have free radical scavenging activity [27]. Thus, this finding suggests that the Ricinus communis extract was effective in bringing about functional improvement of hepatocytes.

            The decreased hepatic glycogen content, and adenosine triphosphatase activity observed in CCl4 induced rats (Table 2) in our study agrees with previous reports [28]. Glycogen is the main source of energy in the liver [29], so the reduced glycogen content observed in CCl4 induced rats may be due to excess requirement of energy in liver. CCl4 intoxication is capable of initiating cell injury and cellular sites for free radical generation include mitochondria, endoplasmic reticulum and plasma membrane. A significant fall in the activity of adenosine triphosphatase observed in CCl4 induced rats may be due to the structural and functional disorganization of mitochondria assembly. Upon Ricinus communis extract treatment to CCl4 induced rats, the above said changes were found to be restored significantly when compared to CCl4 induced rats.

            The ability of a hepatoprotective drug to reduce the injurious effects or to preserve the normal hepatic physiological mechanisms, which have been disturbed by a hepatotoxin, is the index of its protective effects [30]. It is reported earlier that fresh leaves and aqueous extract of Ricinus communis offered protection against CCl4-induced hepatic damage in albino rats [31]. In our study, our results demonstrated that the possible hepatoprotective mechanisms of the ethanol extract of Ricinus communis leaves on CCl4-induced hepatic damage in rats. Histopathological examinations also show that Ricinus communis ethanol extract (250/500mg/kg/b.wt) at both levels of dosage offer hepatoprotection. In addition, this plant extract up to an oral dose of 1 g/kg was found to be devoid of any lethal effects and no apparent behavioral change was observed.

 

5. Conclusions

            In conclusion, the present study has demonstrated that the ethanolic extract of Ricinus communis has hepatoprotective effect against CCl4-induced hepatotoxicity in rats. The beneficial effect of the Ricinus communis ethanol extract may be due to the presence of some flavanoids that may have membrane stabilizing and antiperoxidative effects. Thus, this result suggests that the flavanoids and tannins present in the Ricinus communis ethanol extract might efficiently increase the regenerative and reparative capacity of the liver. Although Ricinus communis ethanol extract has comparable hepatoprotective effect with silymarin in our study, clarification of the hepatoprotective mechanism and the active components of the Ricinus communis extract need further investigation.

 

[1]     Wang N, Li P, Wang Y, Peng W, Wu Z, Tan S, Liang S, Shen X,  Su WW. Hepatoprotective effect of Hypericum japonicum extract and its fractions. J Ethanopharmacal 2008; 116: 1-6.

[2]     Al-Shabanah OA, Alam K, Nagi MN, Al-Rikabi AC, Al-Bekairi AM. Protective effect of aminoguanidine, a nitric oxide synthetase inhibitor against CCl4-induced hepatotoxicity in mice. Life Sci 2000; 66: 265-70.

[3]     Olantunde Farombi E. Mechanisms for the hepatoprotective action of kolaviron: studies on hepatic enzymes, microsomal lipids and lipid peroxidation in carbontetra chloride-treated rats. Pharmacol Res 2000; 42: 75-80.

[4]     Agarwal M, Srivastava VK, Saxena KK, Kumar A. Hepatoprotective activity of Beta vulgaris against CCl4-induced hepatic injury in rats. Fitoterapia 2006; 77: 91-3.

[5]     Jamshidzadeh A, Fereidooni F, Salehi Z, Niknahad H. Hepatoprotective activity of Gundelia tourenfortii. J Ethanopharmacol 2005; 101: 233-7.

[6]     Ivan A. Chemical constituents, traditional and modern uses. In: Medicinal plants of the world. Totowa, NJ: Ross Humana Press Inc., 1998; pp. 375-95.

[7]     Kiritikar KR, Basu BA. Indian medicinal plants. Shiva Publishers: Dehradun, India 1991; 3: 2274-7.

[8]     Visen P, Shukla B, Patnaik G, Tripathi S, Kulshreshtha D, Srimal R,  Dhawan B. Hepatoprotective activity of Ricinus communis leaves. Intl J Pharmacog 1992; 30: 241-50.

[9]     Dhar ML, Dhar MM, Dhawan BN, Mehrotra BN, Ray C. Screening of Indian plants for biological activity. Part I. Indian J Exp Biol 1968; 6: 232-47.

[10]   Capasso F, Mascolo N, Izzo AA,  Gaginella TS. Dissociation of castor oil induced diarrhea and intetinal mucosal injury in rat: effect of NG-nitro-L-arginine methyl ester. Br J Pharmacol 1994; 113: 1127-30.

[11]   Abraham Z, Bhakuni SD, Garg HS, Goel AK, Meharotra BN, Patnaik GK. Screening of Indian plants for biological activity. Part XII. Indian J Exp Biol 1986; 24: 48-68.

[12]   Verpoorte R, Dihal PP. Medicinal plants of the Surinam. IV. Antimicrobial activity of some medicinal plants. J Ethanopharmacol 1987; 21: 315-8.

[13]   Kang SS, Cordell A, Soejarto DD, Fong HHS. Alkaloids and flavanoids from Ricinus commmunis. J Nat Prod 1985; 48: 155-6.

[14]   Khogali A, Barakat S, Abou-Zeid H. Isolation and identification of the phenolics from Ricinus communis L. Delta J Sci 1992; 16: 198-211.

[15]   Chrungoo VJ, Singh K, Singh J. Silymarin mediated differential modulation of toxicity induced by carbontetra chloride, paracetamol and D-galactosamine in freshly isolated rat hepatocytes. Indian J Exp Biol 1997; 35: 611-7.

[16]   Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxaloacetatic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28: 56-63.

[17]   King J. The hydrolases-acid and alkaline phosphatases, In: Van D, (editor). Practical clinical enzymology. London: Nostrand Company Limited, 1958; pp.191-208.

[18]   Seth PK, Tangari KK. Biochemical effects of newer salicylic acid congenesis. J Pharm Pharmacol 1966; 18: 831-3.

[19]   Lowry OH, Rosebrough NJ, Farr AI, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-75.

[20]   Hassid WZ, Abraham S. Chemical procedures for analysis of polysaccharides. Methods Enzymol 1957; 3: 34-50.

[21]   Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem1997; 95: 351-8.

 [22]  Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 1980; 106: 207-12.

[23]   Janbaz KH, Saeed SA, Gilani AH. Protective effect of rutin on paracetamol and CCl4 induced hepatotoxicity in rodents. Fitoterapia 2002; 73: 557-63.

[24]   Thabrew M, Joice P, Rajatissa W. A comparative study of the efficacy of Pavetta indica and Osbeckia octandra in the treatment of liver dysfunction. Planta Medica 1987; 53: 239-41.

[25]   Souza MF, Rao VSN, Siliveira ER. Inhibition of lipid peroxidation by ternatin, a tetramethoxyflavone from Egletes viscose L.. Phytomedicine 1997; 4: 25-9.

[26]   Yokozawa T, Chen CP, Dong E, Tanaka T, Nonaka GI, Nishioka I. Study on the inhibitory effect of tannins and flavanoids against the 1,1-diphenyl-2-picrylhydrazyl radical. Biochem Pharmacol 1998; 56: 213-22.

[27]   Ilavarasan R, Mallika M, Venkataraman S. Anti-inflammatory and free radical scavenging activity of Ricinus communis root extract. J Ethanopharmacol 2006; 103: 478-80.

[28]   Jadon A, Bhadauria M, Shukla S. Protective effect of Terminalia belerica Roxb. and gallic acid against carbon tetrachloride induced damage in albino rats. J Ethanopharmacol 2007; 109: 214-18.

[29]   Krehenbuhl S, Weber Jr FL, Brans E. Decreased hepatic glycogen content and acclerated response to starvation in rats with carbon tetrachloride induced cirrhosis. Hepatology 1991; 14: 1189-95.

[30]   Yadav NP, Dixit VK. Hepatoprotective activity of leaves of Kalanchoe pinnata Pers. J Ethanopharmacol 2003; 86: 197-202.

[31]   Natu MV, Agarwal S, Agarwal SL, Agarwal S. Protective effect of Ricinus communis leaves in experimental liver injury. Ind J Pharmac 1977; 9: 265-8.