Abstract: Background: Aloe vera, is a natural hepatoprotective agent used alone or an ingredient of number of medicinal preparations in Indian traditional medicine (Ayurveda) and in folk medicine across the globe for different liver ailments. However, considerable number of them lacks scientific proof for their claims. Hence, Aloe vera was evaluated for its protective effect against carbon tetrachloride (CCl4) induced liver toxicity in wistar albino mice and rabbits. Materials and methods: Two species of the animals (rabbit and mice) were used to evaluate Aloe vera for its effects on histopathology, biochemistry and mortality induced by carbon tetrachloride in experimental setup. The animals were divided into main three groups (GI, GII and GIII) of eighteen animals each. Each group was further divided into three subgroups of six animals each. Depending upon the treatment protocol, subgroup ‘a’ received linseed oil as control. Subgroup ‘b’ received CCl4. While as subgroup ‘c’ received extract of Aloe vera prior to CCl4 administration. Results: Animals pretreated with Aloe vera and subsequently treated with CCl4 showed significant reduction in histopathological changes (p<0.05), biochemical changes (p<0.05) and mortality rate (p<0.05) in comparison to the animals treated with CCl4 alone. Conclusion: This study reveals the hepatoprotective effect of Aloe vera against CCl4 induced liver toxicity.
INTRODUCTION
Liver plays a significant role not only in metabolism and detoxification of exogenous toxins and therapeutic agents, but also in the bio-regulation of fats, carbohydrates, amino acids and proteins (Alqasoumi et al., 2008). A number of pharmacological and chemical agents act as hepatotoxins and produce a variety of liver ailments (Ram, 2001). Though tremendous strides have been made in the last four decades in modern medicine, yet there is no drug that protects liver from damage or helps in the regeneration of hepatic cells. Corticosteroids are used empirically to prevent further fibrosis and check antigen antibody reaction but no specific action on the liver has been demonstrated.
Numberless drugs of indigenous origin were investigated by variety of animal experiments to see if they could help in the protection of regeneration of the hepatic parenchyma. Aloe vera is one such preparation used alone as a sole agent or one of the ingredients in many preparations in Indian traditional medicine (Ayurveda) and in other corners of the world as hepatic stimulant and in liver enlargement (Mossa et al., 1987; Vazquez et al., 1996; Reynolds and Dweck, 1999; Can et al., 2004; Boudreau and Beland, 2006; Miladi and Damak, 2008). Moreover, Aloe vera is used and has been evaluated for its potential role in many other clinical conditions (Langmead et al., 2004; Vinson et al., 2005; Esua and Rauwald, 2006; Hamman, 2008; Ramachandra and Rao, 2008). Though numerous herbal extracts are used for liver problems, however, considerable number of them lacks scientific proof for their claims. Hence, it was found worthwhile to evaluate the hepatoprotective effect of Aloe vera against carbon tetrachloride induced hepatotoxicity in experimental animal models.
MATERIALS AND METHODS
Animals: Healthy Wistar albino mice of either sex weighing between 20-30 g and wistar albino rabbits weighing between 1-2 kg were used for histopathological, survival and biochemical study to evaluate the hepatoprotective effect of Aloe vera. The animals were housed within the departmental animal house and the room temperature was maintained at 27°C. They were provided with Purina chow and free access to water ad libitum (Abdel-Kader and Alqasoumi, 2008). The protocol was approved and carried out after the permission of Institutional Animal Ethics Committee (IAEC).
Investigational drugs and dosage preparations: The appropriate body weight adjusted doses of extract of Aloe vera as extrapolated from doses used in similar studies conducted previously to be 0.2 mL kg-1 for mice and 2.5 mL kg-1 for rabbit were used (Alqasoumi et al., 2008). The formulations were fed to the animals through gastric tube (18 to 22 mm) for rabbit and 2-3 mm polythene tubing sleeved on an 18-20 gauge blunted hypodermic needle for mice (Ahmed et al., 2010). The extract of Aloe vera (L) Burm. f. (Asphodeclaceae) was obtained from E. Merck.
Experimental protocol: Animals (n = 54) consisting of mice (n = 36) and rabbits (n = 18) were allocated to three main groups of 18 animals each. Depending upon the treatment design, animals were further subdivided into three groups of six animals each for different experimental tests. The linseed oil alone was used as control in the subgroups Ia, IIa and IIIa. Carbon tetrachloride (CCl4) mixed with equal volume of linseed oil was used as hepatotoxic agent in the subgroups Ib, IIb and IIIb. While as the extract of Aloe vera was used in subgroups Ic, IIc and IIIc prior to CCl4 administration. Mice were used for histopathology and survival tests. However, rabbits were used for biochemical tests.
Histoprotective test: Three groups (GIa, Gib and GIc) of six mice each were used. GIa received linseed oil as control. GIb received 2.5 mL kg-1 of 50% CCl4 solution in linseed oil orally. However, GIc received 0.2 mL kg-1 of extracts of Aloe vera daily for seven days and on the eighth day 2.5 mL kg-1 of 50% CCl4 solution was given orally. After 48 h following CCl4 administration the animals were sacrificed using ether anesthesia. The liver was immediately removed and a small piece was fixed in 10% formalin and subsequently stained with hematoxilene and eosin for histopathological assessment.
Histopathology examination: Under aseptic procedure the liver of all the treated animals in group GI were removed and a piece of tissue was fixed in 10% formalin for histological assessment. Following the procedure the sections were ultimately placed onto the clean slides that were drained vertically for several minutes before placing them onto a warming table at 37-40°C (Prophet et al., 1994). The slides were dehydrated and cleared and finally mounted with resinous medium. While assessing the liver biopsies the control group (GIa) showed normal histological appearance. The architecture of liver was maintained in CCl4 treated group (GIb) of mice. Hepatocytes showed cloudy and fatty degeneration, central veins and sinusoids were dilated with infiltration of polymorphonuclear cells and few mononuclear cells. Necrosis of hepatocytes were seen which was mostly of centrilobular type. However, in group (GIc) of mice which were pretreated with Aloe vera and susequently with CCl4 the architecture of liver was maintained. Hepatocytes showed little cloudy and fatty degeneration, central veins and sinusoids were not dilated. Most importantly areas of necrosis was not seen.
Survival test (Mortality scores): Three groups (GIIa, GIIb and GIIc) of six mice each were used. GIIa received linseed oil as control. GIIb received 2.5 mL kg-1 of 50% CCl4 solution in linseed oil. GIIc received 0.2 mL kg-1 of extracts of Aloe vera daily for seven days and on the eighth day 2.5 mL kg-1 of 50% CCl4 solution was given orally. All the animals in each group were observed for one week and the mortality rate of animals in each group was recorded and compared among the treatment groups.
Biochemical test: Three groups (GIIIa, GIIIb and GIIIc) of six rabbits each were used. GIIIa received linseed oil as control. GIIIb received 2.5 mL kg-1 of 50% CCl4 solution in linseed oil. GIIIc received 2.5 mL kg-1 of extracts of Aloe vera daily for seven days and on the eighth day 2.5 mL kg-1 of 50% CCl4 solution in linseed oil was given orally. After 24 h of CCl4 administration, 2 mL of blood sample was collected from marginal ear vain of all the animals for biochemical parameter (SGOT, SGPT and serum protein) estimation (Boon et al., 2006). The enzyme activities were estimated by employing diagnostic strips (Reflotron®, ROCHE) and were read on a Reflotron® Plus instrument (ROCHE).
Statistical analysis: The statistical analysis was done by using Chi-square test for analysis of the results of histopathological and survival tests. However, Student’s ‘t’ test was employed for analysis of the results of biochemical test. A probability value of less than 0.05 (p<0.05) was considered to be statistically significant. The results are expressed as Mean±SEM.
RESULTS AND DISCUSSION
While assessing the results of this study for evaluation of hepatoprotective action of Aloe vera, a natural remedy used in various medicinal preparations in Indian traditional medicine (Ayurveda) and folk medicine in other corners across the globe (Grindlay and Reynolds, 1986; Davis et al., 1989; Rajasekaran et al., 2005; Madan et al., 2008). The present study was designed to test the Aloe vera for its role as hepatoprotective agent against carbon tetrachloride induced hepatic injury and histological changes. Two species of the animals (rabbit and mice) were used to evaluate Aloe vera for its effects on histopathology, biochemistry and mortality induced by carbon tetrachloride in experimental setup.
Mice were used to study histopathological changes, mortality scores and rabbits for biochemical changes induced by carbon tetrachloride.
In histopathological examination, the liver biopsies of control group showed normal histological appearance. The animals treated with CCl4 showed centrilobular hepatocyte necrosis, extensive fatty degeneration and infiltration by polymorphonuclear cells. Exposure to CCl4 leads to histopathological changes from the normal histological appearance (Alqasoumi et al., 2008). However, the degree of histopathological changes has significantly reduced (p<0.05) in animals pretreated with Aloe vera and subsequently treated with CCl4 in comparison to the animals treated with CCl4 alone. The architecture of liver was maintained, hepatocytes showed little cloudy and fatty degeneration. Moreover areas of necrosis were not seen, central veins and sinusoids were not dilated (Fig. 1).
Assessing the mortality score, no mortality was recorded in control group. 100% mortality occurred in CCl4 treated group within 48 h after oral administration of CCl4. However, this mortality rate significantly reduced (p<0.05) in a group pretreated with Aloe vera and subsequently treated with CCl4 (Fig. 2) as only 33% mortality was recorded in this group.
While assessing the biochemical parameters, an increase in SGOT, SGPT and serum protein was recorded in a group treated with CCl4 as compared to control and Aloe vera treated groups. Treatment of animals with the hepatotoxic agent CCl4 resulted in significant increase of transaminases (SGOT and SGPT) and alkaline phosphate levels (ALP) due to hepatocyte damage (Zafar and Ali, 1998). Severe jaundice was reflected by increased level of serum bilrubin (Lin et al., 1997). However, this increase of transaminases (SGOT and SGPT) and serum protein was significantly inhibited (p<0.05) in a group pretreated with Aloe vera and subsequently treated with CCl4 (Fig. 3). Our results are in accordance with the study stating animals (rats) treated with Aloe vera extract exihibited a significant reduction only in SGOT level at the lower dose. Where as the higher dose significantly lowered the levels of SGOT, SGPT, ALP and bilrubin, respectively, indicating a good level of protection against the toxicity of CCl4 (Alqasoumi et al., 2008).
CONCLUSION
This study reveals hepatoprotective effect of Aloe vera in terms of its hepatoprotective effect, no significant increase in biochemical parameters and significant reduction in mortality rate against CCl4 challenge as hepatotoxic agent.