In a project to search for new natural hepatroprotective agents 3 plant extracts; Aloe vera, Clematis hirsute and Cucumis prophetarum, in addition to Bee Propolis were studied. The ethanol extracts of the 3 plants and propolis were subjected to hepatoprotective assay using Wistar albino rats. Liver injury induced in rats using carbon tetrachloride. The biochemical parameters; serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and total bilirubin were estimated as reflection of the liver condition. Based on the good results of the biochemical parameters measurements, histopathological study was performed on the liver of rats treated with Propolis and Aloe vera. The livers of rats treated with Propolis showed good protection against the toxic effect of carbon tetrachloride. On the other hand treatment with Aloe vera extract failed to restore the normal appearance of hepatocytes. All the results were compared with silymarin, as a reference hepatoprotective drug.
PDF Abstract XML References Citation
How to cite this article
The liver is an organ of prime importance and plays a significant role not only in metabolism and detoxification of exogenous toxins and therapeutic agents, but also in the bioregulation of fats, carbohydrates, amino acids and proteins. A number of pharmacological and chemical agents act as hepatotoxins and produce a variety of liver ailments (Ram, 2001). Numerous herbal extracts are used for liver problems, however, considerable number of them lack the scientific prove for these claims. In our search for hepatoprotective natural remedies three plant extracts; Aloe vera, Clematis hirsute and Cucumis prophetarum in addition to Bee Propolis were tested against liver injury induced in rats using carbon tetrachloride. In Saudi folk medicine, the leaves of Aloe vera are used as hepatic stimulant and in liver enlargement (Mossa et al., 1987). The aerial parts of Cucumis prophetarum, known locally as Shari-al-deeb is used by traditional medicine practitioners as remedy for liver disorders (Al-Yahya et al., 1990). Clematis hirsuta known locally as Ghashoua and Shajarat Al-Riyah (Chaudhary, 1998) is used as other Clematis species members as anti-inflammatory (Morgan, 1981; Xu et al., 1996). Natural medicine practitioners often use propolis for the relief of various conditions, including inflammations and viral diseases. Propolis also exhibits immunomodulatory effects (Brätter et al., 1999; Ansorge et al., 2003).
MATERIALS AND METHODS
Test material: The fruits of Cucumis prophetarum L. (Cucurbitaceae) were collected from the Northern part of Saudi Arabia in March 2003. The aerial parts of Clemati hirsuta Guillemin and Perr (Ranunculaceae) were collected from Tanuma region in March 2000. The plants were identified by Dr. Mohammad Atiqur Rahman, taxonomist of the Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Voucher specimens (#12465 and 11411) were deposited at the herbarium of the center.
The extract of Aloe vera (L.) Burm. f. (Asphodelaceae) was obtained from E. Merck. Extracted Bees Propolis (98% purity, Flavonoids: 14%) was obtained from Guangzhou Longstone Import and Export Trading Co., Ltd., China.
Extraction: Fifty grams of the dried ground aerial parts of Clemati hirsute and the dried fruits of Cucumis prophetarum were extracted to exhaustion by percolationn at room temperature with 90% ethanol, the extracts were evaporated in vacuo to leave 4.25 and 2.41 g extracts, respectively.
Animals: Wistar albino rats (150-200 g) of either sex roughly the same age (8-10 weeks), obtained from the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh were used. The animals were housed under constant temperature (22±2°C), humidity (55%) and light/dark conditions (12/12 h). They were provided with Purina chow and free access to drinking water ad libitum (Abdel-Kader and Alqasoumi, 2008). The experiments and procedures used in this study were approved by the Ethical Committee of the College of Pharmacy, King Saud University.
Chemicals: Silymarin (Sigma Chemical Company, USA).
Hepatoprotective activity: Male Wistar rats were divided into 5 groups 6 animals each. Group 1 was kept as a control group. Groups 2, 3, 4 and 5 received 0.125 mL of CC14 in liquid paraffin (1:1) per 100 g body weight intraperitoneally. Group 2 received only CCl4 treatment. Group 3 was administered silymarin at a dose of 10 mg kg-1 p.o. Groups 4 and 5 were treated with 250 and 500 mg kg-1 of extracts respectively. Drug treatment was started 5 days prior to CCl4 administration and continued till the end of the experiment. After 48 h, following CC14 administration the animals were sacrificed using ether anesthesia. Blood samples were collected by heart puncture and the serum was separated for evaluating the biochemical parameters. The liver was immediately removed and a small piece was fixed in 10% formalin for histopathological assessment.
Determination of the enzyme levels: The biochemical parameters such as Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP) and total bilirubin were estimated by reported methods (Boon et al., 2006). The enzyme activities were measured using diagnostic strips (Reflotron®, ROCHE) and were read on a Reflotron® Plus instrument (ROCHE).
Statistical analysis: For each set of experiments where two or more than two groups were compared, an analysis of variance (ANOVA) test was used to determine the significance of the differences. Differences between the control and CCl4-treated group were compared for significance using student`s t-test for non paired samples (Woolson and Clarke, 2002). All the values shown are the mean±SE.
Histopathology: The livers of treated animals were immediately removed and a small piece was fixed in 10% formalin for histopathological assessment. All specimens were placed in cassettes and loaded into tissue baskets. The specimens were subjected to dehydration, clearing and inflatration by immersion in different conc of ethanol (70-100%), xylene (3 times, 1 h each) and finally paraffin wax (4 times, 1 h each). The tissues were then transferred into moulds filled with paraffin wax. After orienting the tissues by hot forceps the moulds were chilled on cold plates and excess wax were trimmed off using a knife. The rotary microtome (Leitz, 1512) was used for making thin sections (3 μm). The sections were placed onto clean slides that were drained vertically for several min. before placing them onto a warming table at 37-40°C (Prophet et al., 1994). The slides were then deparaffinized, hydrated and to stained in Mayer`s hematoxylin solution for 15 min The slides were then washed in lukewarm running tap water for 15 min, placed in distilled water, 80% ethyl alcohol for 1 to 2 min then counterstained in eosin-phloxine solution for 2 min. The slides were then dehydrated and cleared through 2 changes each of 95% ethyl alcohol, absolute ethyl alcohol and xylene, 2 min each and finally mounting with resinous medium.
RESULTS AND DISCUSSION
Treatment of animals with the hepatotoxic agent carbon tetrarchloride resulted in Significant Increase of Transaminases (SGOT and SGPT) and Alkaline Phosphate Levels (ALP) due to hepatocytes damage (Zafar and Ali, 1998). Sever jaundice was reflected by increase level of serum bilirubin (Lin et al., 1997) (Table 1).
Treatment of animals with the known hepatoprotective agents silymarin resulted in significant decrease in the elevated levels of SGOT, SGPT, ALP and bilirubin (p<0.001) (Table 1). Silymarin act as an antioxidant by scavenging prooxidant free radicals and by increasing the intracellular concentration of GSH. It also exhibits a regulatory action of cellular membrane permeability and increase in its stability against xenobiotics injury; increasing the synthesis of ribosomal RNA by stimulating DNA polymerase-I, exerting a steroid like regulatory action on DNA transcription and stimulation of protein synthesis and regeneration of liver cells (Dehmlow et al., 1996a, b; Gakova et al., 1992; Saller et al., 2007). Silymarin efficacy is not limited to the treatment of toxic and metabolic liver damage; it is also effective in acute, chronic hepatitis and in inhibiting fibrotic activity (Saller et al., 2007).
|Table 1:||Effects of ethanolic extracts of Clematis hirsute, propolis powder, Aloe vera and Cucumis prophetarum on serum biochemical parameters|
|*p<0.05; **p<0.01; ***p<0.001; aAs compared with the normal saline (control) group; bAs compared with the CCl4 only group|
Treatment of animals with crude extract of Clematis hirsuta (Aerial parts) at doses (250 and 500 mg kg-1) showed little decreases in all the parameters estimated (Table 1). In addition, the decreases were not statistically significant.
The SGOT, SGPT, ALP and bilirubin levels in Propolis treated rats (500 mg kg-1) were significantly decreased when compared with CCl4 treated group (p<0.05-p<0.001) (Table 1). The reduction in the biomarkers levels with the 250 mg kg-1 doses were significant (p<0.05 and p<0.01) except for that of SGPT which resulted in a 21.5% statistically insignificant reduction. These results revealed a good degree of protection against the toxic substance CCl4.
The group of animals treatment of Aloe vera extract exhibited a significant reduction only in SGPT level (22%, p<0.05) at the lower dose (250 mg kg-1). Where as the higher dose (500 mg kg-1) significantly lowered the levels of SGOT, SGPT, ALP and bilirubin (p<0.05, p<0.01, p<0.01 and p<0.01), respectively, indicating a good level of protection against the toxicity of CCl4.
The crude extracts of Cucumis prophetarum at the lower (250 mg kg-1 body weight) dose failed to protect against elevated levels of all biomarkers. Whereas, only SGPT and bilirubin levels were found to be reduced significantly (26.5 and 37.0% reduction; p<0.05 and p<0.05, respectively) at higher dose in C. prophetarum crude extract treated animals (Table 1).
Based on the above results the livers of rats treated with Propolis and Aloe vera were subjected to histopathological study to evaluate the extent of protection of hepatocytes. The histological appearance of the hepatocyte reflects their conditions (Prophet et al., 1994). Exposure of hepatocytes to toxic agents such as CCl4 leads to histopathological changes from the normal histological appearance (Fig. 1A). The hepatocytes of rat livers treated with a single dose of 1.25 mL CC14 kg-1, showed centrilobular hepatocyte necrosis and extensive fatty change were observed on the midzonal or entire lobe at 24 h (Fig. 1B). Liver tissue of rats treated with CCl4 and silymarin showed good recovery with absence of necrosis and fatty depositions (Fig. 1C).
Histological appearance of rat liver treated with Aloe vera (500 mg kg-1) and CCl4. Effects showed extensive inflammation around central veins, less apoptosis, microvesicular fatty changes and ballooning degeneration. This appearance indicates poor protection of the hepatocyte against the hepatotoxic agent.
|Fig. 1:||Histopathological appearance of liver cells; (A): Normal cells; (B): Liver cells of rats treated with CCl4; (C): Liver cells of rats treated with CCl4 and silymarin; (D): Liver cells of rats treated with CCl4 and Aloe vera; (E): Liver cells of rats treated with CCl4 and Propolis powder|
Liver tissue of rats treated with CCl4 and Propolis powder (500 mg kg-1) showed mild to moderate portal inflammation (Fig. 1E). It displayed a good progress with disappearance of fatty changes and necrosis. This appearance of the hepatocytes indicates the effectiveness of Propolis powder to express significant protective effect of the liver cells against CCl4.
The present study proves that Propolis has some protective effect against the hepatotoxic agent CCl4. Although there are some claims for the benefits of Cucumis prophetarum and Aloe vera for liver problems (Mossa et al., 1987; Al-Yahya et al., 1990), the obtained results could not prove their effectiveness as hepatoprotective agents. No significant results were obtained from the use of Clematis hirsute indicating the lack of any protective effect for the liver.
The authors are grateful to Dr. Abdulmalik M. Al-Sheikh for help in the histopathological study, Mr. Ali D. Al-Sawadi and Mr. Malik Sawood for technical assistance. The research was supported by grants from King Abdul Aziz City of Science and Technology (KACST), Riyadh, Saudi Arabia to Dr. Alqasoumi.
- Ansorge, S., D. Reinhold and U. Lendeckel, 2003. Propolis and some of its constituents down-regulate DNA synthesis and inflammatory cytokine production but induce TGF-beta1 production of human immune cells. Z. Naturforsch., 58: 580-589.
- Brätter, C., M. Tregel, C. Liebenthal and H.D. Volk, 1999. Prophylactic effectiveness of propolis for immunostimulation: A clinical pilot study. Forsch Komplementarmed., 6: 256-260.
- Dehmlow, C., J. Erhard and H. de Groot, 1996. Inhibition of Kupffer cell functions as an explanation for the hepatoprotective properties of silibinin. Hepatology, 23: 749-754.
- Dehmlow, C., N. Murawski and H. de Groot, 1996. Scavenging of reactive oxygen species and inhibition of arachidonic acid metabolism by silibinin in human cells. Life Sci., 52: 1591-1600.
- Gakova, N., E. Mishurova and K. Kropachova, 1992. Effects of flavobion on nucleic acids in tissues of rats irradiated with gamma rays. Biull. Eksp. Biol. Med., 113: 275-277.
- Xu, R., W. Zhao, J. Xu, B. Shao and G. Qin, 1996. Studies on bioactive saponins from Chinese medicinal plants. Adv. Exp. Med. Biol., 404: 371-382.