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Hepatoprotective potential of Aloe barbadensis Mill. against carbon tetrachloride induced hepatotoxicity
Journal of Ethnopharmacology 111 (2007) 560–566
Hepatoprotective potential of Aloe barbadensis Mill. against carbon tetrachloride induced hepatotoxicity B.K. Chandan a,∗ , A.K. Saxena a , Sangeeta Shukla c , Neelam Sharma a , D.K. Gupta b , K.A. Suri b , Jyotsna Suri d , M. Bhadauria c , B. Singh a b
a Department of Pharmacology, Regional Research Laboratory, Canal Road, Jammu-Tawi 180 016, India Department of Natural Products Chemistry, Regional Research Laboratory, Canal Road, Jammu-Tawi 180 016, India c School of Studies in Zoology, Jiwaji University, Gwalior (MP), India d Department of Pathology, Government Medical College, Jammu 180001 (J&K), India
Received 27 February 2006; received in revised form 13 December 2006; accepted 4 January 2007 Available online 14 January 2007
Abstract Aloe barbadensis Mill. Syn. Aloe vera Tourn. ex Linn.(Liliaceae) has been used in variety of diseases in traditional Indian system of medicine in India and its use for hepatic ailments is also documented. In the present study an attempt has been made to validate its hepatoprotective activity. The shade dried aerial parts of Aloe barbadensis were extracted with petroleum ether (AB-1), chloroform (AB-2) and methanol (AB-3). The plant marc was extracted with distilled water (AB-4). All the extracts were evaluated for hepatoprotective activity on limited test models as hexobarbitone sleep time, zoxazolamine paralysis time and marker biochemical parameters. AB-1 and AB-2 were observed to be devoid of any hepatoprotective activity. Out of two active extracts (AB-3 and AB-4), the most active AB-4 was studied in detail. AB-4 showed significant hepatoprotective activity against CCl4 induced hepatotoxicity as evident by restoration of serum transaminases, alkaline phosphatase, bilirubin and triglycerides. Hepatoprotective potential was confirmed by the restoration of lipid peroxidation, glutathione, glucose-6-phosphatase and microsomal aniline hydroxylase and amidopyrine N-demethylase towards near normal. Histopathology of the liver tissue further supports the biochemical findings confirming the hepatoprotective potential of AB-4. The present study shows that the aqueous extract of Aloe barbadensis is significantly capable of restoring integrity of hepatocytes indicated by improvement in physiological parameters, excretory capacity (BSP retention) of hepatocytes and also by stimulation of bile flow secretion. AB-4 did not show any sign of toxicity up to oral dose of 2 g/kg in mice © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Hepatoprotective activity; Carbon tetrachloride (CCl4 ); Aloe barbadensis
1. Introduction Aloe barbadensis Mill. Syn. Aloe vera Tourn. ex Linn. (Liliaceae) commonly known as Ghee kanwar (Hindi) found in semi wild state and can be cultivated in all parts of India (Mhaskar et al., 2000). In “Indian System of Medicine” fresh
Abbreviations: AB-1, petroleum ether extract; AB-2, chloroform extract; AB-3, methanol extract; AB-4, aqueous extract; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; TG, triglycerides; GSH, glutathione; LP, lipid peroxidation; G-6-P, glucose-6-phosphatase; AH, aniline hydroxylase; N-de, amidopyrine-Ndemethylase ∗ Corresponding author. Tel.: +91 191 2544382; fax: +91 191 2543829. E-mail address: [email protected] (B.K. Chandan). 0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.01.008
juice of leaves is used in diseases of eye and enlargements of spleen and liver (The wealth of India, 2000). The ethanolic extract from stem of the plant is reported for antibacterial activity against Escherichia coli (Singh et al., 1974) whereas leaf extract is active against Mycobacterium tuberculosis. Pulp of the leaves is reported for anti-fertility and oxytocic activities (Bradbury et al., 1967). It is also useful in X-ray burns, dermatitis, cutaneous leishmaniasis and other disorders of skin (Mhaskar et al., 2000). It has also been reported for antiviral (Saoo et al., 1996) and anti-inflammatory activities (Beatriz et al., 1996). It is also an important constituent of large number of ayurvedic preparations/formulations. Chemically, the presence of barbaloin, chrysophanol, glycoside aloe-emodin, glucose, galactose, mannose and galacturonic acid in addition to aldopentose and proteins with 18 amino acids have been reported. It
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also contains aloesone and aloesin (The Wealth of India, 2000). Beatrice et al., 2000 have reported that intraperitoneal injection of aloe emodin exhibited some protective effect against CCl4 on limited number of parameters but there is no report indicating hepatoprotective activity in aqueous extract containing number of other compounds which may be responsible for the activity individually or in combination. Based on its diversified pharmacological properties and its use in liver diseases in traditional Indian System of Medicine, an attempt has been made to validate the plant for its hepatoprotective potential against most widely used hepatotoxin CCl4 in experimental studies. Human dose of, one of the constituent (barbaloin) of aloe barbadensis is approximately 30 mg/kg, po and the doses of AB-4 used in the study are 125, 250, and 500 mg/kg, po which comes to be 21, 43, and 86 mg/kg approximately in human with respect to the presence of barbaloin in AB-4. 2. Materials and methods 2.1. Plant material, extraction and chromatography The plant was collected from the botanical garden of Regional Research Laboratory, Jammu and was authenticated at source by Dr. B.K. Kaphai taxonomist of the institute. A voucher specimen has been deposited at the herbarium of the Institute vide RRL collection No. 17875, Acc. No. 19650. The authenticated and freshly collected aerial parts of the plant were chopped and dried in shade. The coarse plant material (185 g) was extracted successively with petroleum ether (4× 1000 ml), chloroform (4× 900 ml) and methanol (5× 900 ml) at room temperature. During extraction with solvents, the solvent was changed after every 24 h. The extracts with each solvent after pooling were dried under reduced pressure to afford petroleum ether (AB-1 2 g; yield 1.08%, w/w), chloroform (AB-2, 1.9 g; yield 1.03%, w/w) and methanol extract (AB-3, 10 g; yield 5.40%, w/w). The plant marc was finally extracted with distilled water (3× 1000 ml) and lyophilised in freeze drier to furnish aqueous extract (AB-4, 31 g; yield 16.75%, w/w). Quantification of the barbaloin in the extracts was carried by HPLC (Shimadzu make HPLC system with LC-10A TVP pumps, DGU-14A degassing unit, SIL-10A PDA detector and CTO-10AS column oven. Data was acquired and analyzed by using Shimadzu Class-VP software version 6.10) using mobile phase, methanol:water (45:55); column: RP-8 (5 m), flow rate: 1 ml/min, detection: 254 nm., injection volume: 50 and 100 l, Barbaloin was used as standard for quantification of barbaloin in the extracts. Methanolic and aqueous extracts were found to contain 0.0127% (w/w) and 2.09% (w/w) barbaloin, respectively. While barbaloin in petroleum ether (AB-1) and chloroform (AB-2) extracts were below detection limits. 2.2. Chemicals Carbon tetrachloride (CCl4 ) was obtained from S.D. FineChem. Ltd., Mumbai. All other chemicals used were of analytical grade.
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2.3. Experimental animals Male Wistar rats (150–200 g) or Swiss albino mice (20–25 g) were used in all the studies except for bile flow and bile solids where rats of same strain but of higher body weight (200–250 g) were used. Animals were housed under standard conditions (23.0 ± 2 ◦ C, 60–70% humidity, and 12 h photo period) and allowed free access to food (Lipton India Ltd., Bombay, India) and water. The experiments were conducted according to the ethical norms approved by Institutional Animal Ethics Committee guide lines for animal care and were adhered to as recommended by the Indian National Science Academy, New Delhi (1992). 2.3.1. Induction of hepatic injury Hepatic injury was produced by oral administration of CCl4 diluted with liquid paraffin in all the studies. 2.4. Hexobarbitone induced narcosis and zoxazolamine induced paralysis time Six groups of mice with six mice in each group were used for each study. Three groups (groups 1–3) were fed orally once with AB-4 125, 250 and 500 mg/kg, respectively. Silymarin (50 mg/kg, po) was administered to fourth group and last two groups (groups 5 and 6) were given vehicle only and served as control. CCl4 (0.1 ml/kg po) was given to animals of first five groups treated with AB-4, silymarin and one group of controls (group 5) that served as CCl4 control, 1 h after feeding test material. The second control group (group 6) was given vehicle only and served as normal control. Hexobarbitone (60 mg/kg, ip) sleep time were monitored individually 2 h after CCl4 administration in all the animals. To study zoxazolamine induced paralysis time grouping of the animals and treatment is same except for the dose of zoxazolamine, which was 70 mg/kg, ip. The time to onset of loss of the righting reflex or paralysis to the recovery in minutes was taken as duration of sleep or paralysis time respectively (Singh et al., 2005). 2.5. Effect on biochemical parameters 2.5.1. Prophylactic studies Six groups with six rats in each were used for this study. First three groups were fed orally AB-4 125, 250 and 500 mg/kg, respectively, 48, 24 and 2 h before and 6 h after CCl4 (1 ml/kg, po) administration (Bramanti et al., 1978). Similarly fourth group was fed with silymarin (50 mg/kg, po). Out of remaining two groups one was given the same dose of CCl4 which served as CCl4 control group and to other proportionate volume of vehicle was given and served as vehicle control. Liver and blood samples were collected 24 h after CCl4 administration. 2.5.2. Curative studies The grouping of animals and doses of AB-4, silymarin and CCl4 are the same as described for prophylactic studies except that AB-4 and silymarin were given exactly 2, 6, 24, and 48 h after CCl4 administration (Singh et al., 1998). Liver and blood samples were collected 50 h after CCl4 administration.
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2.5.3. Blood biochemistry Blood samples were collected in glass tubes from orbital sinus to obtain haemolysis free clear serum for the analysis of ALT and AST (Reitman and Frankel, 1957), ALP (Walter and Schutt, 1974), LDH (Wroblewski and La Due, 1964), bilirubin (Malloy and Evelyn, 1937) and TG (Neri and Frings, 1973) by standard methods.
treatment) to 1 h before treatment. The choleretic activity as percent increase of bile flow was determined (Chandan et al., 1991). Bile collected from the animals as mentioned above was dried and the percent increase in bile solids was calculated as in case of bile flow.
2.5.4. Preparation of liver homogenate and microsomes All the animals were sacrificed by decapitation and livers were quickly excised freed from any adhering tissues, washed and perfused with chilled normal saline, minced and homogenized in ice bath using Potter-S-homogenizer (B. Braun, Melsungen AG, Germany, 1100 rpm for 2 min) in chilled 10 mM Tris–HCl buffer (pH 7.4) to obtain 10% liver homogenate for the estimation of (GSH) glutathione (Ellman, 1959), (LP) lipid peroxidation (Buege and Aust, 1978), (G-6-P) glucose6-phosphatase (Baginski et al., 1974) and (TG) triglycerides (Neri and Frings, 1973) by using standard methods. Liver microsomes were prepared by the method as described by Schenkman and Cinti, 1978. Stored in liquid nitrogen for the estimation of (AH) aniline hydroxylase (Kato and Gillette, 1965) and (N-de) amidopyrine-N-demethylase (Chochin and Axelrod, 1959).
The safety study was carried out using OECD guide lines No. 423. Three female mice of same age group and weight were taken in a single dose up to the highest dose of 2000 mg/kg orally. The animals were observed for 1 h continuously and then hourly for 4 h and finally after every 24 h up to 15 days for any mortality or gross behavioural changes.
2.5.5. Histopathology From all the animals used for curative study small portion of liver tissue was fixed in 10% formaline saline, processed and embedded in paraffin wax to obtain 5–6 m thick hematoxylin and eosin stained sections (Krajian, 1963). 2.6. Hepatoprotective activity The hepatoprotective activity, expressed as hepatoprotective percentage (H), was calculated as follows: T −V H = 1− × 100 C−V where T is mean value of drug and CCl4 , C mean value of CCl4 alone and V is the mean value of normal control animals. 2.7. Effect on bile flow and bile solids Male rats were divided into four groups of six rats in each and their food was withdrawn 18 h before experiment but allowed free access to water. To determine the choleretic activity bile duct was cannulated with PE-10 tubing under urethane anesthesia (6.0 ml/kg ip of 25% solution) and bile was collected individually in a small test tube initially for 1 h (which served as control) and at the end of the first hour all the animals of first two groups were administered with test material (AB-4, 250 or 500 mg/kg, id) and group three with dehydrocholic acid (DHC, 20 mg/kg, iv) as reference standard. The collection of the bile continued hourly for next 4 h. The last group was not given any treatment and served as normal control. The criterion for choleretic activity is based on the increase in bile volume in 4 h cumulated (after
2.8. Safety evaluation study
2.9. Statistical analysis The data obtained were analyzed by One Way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using computerized program. P-value < 0.05 or was taken as the criterion of significance. 3. Results 3.1. Hexobarbitone induced narcosis and zoxazolamine induced paralysis time in mice Hexobarbitone sleep time and zoxazolamine induced paralysis time were significantly (P < 0.001) raised in CCl4 treated animals. AB-4 counteracted CCl4 enhanced hexabarbitone induced sleep time and Zoxazolamine paralysis time and thus showing hepatoprotective activity of different magnitude depending upon dose (Table 1). 3.2. Effect on biochemical parameters (Prophylactic studies) Carbon tetrachloride significantly (P < 0.001) raised ALT, AST, LDH, ALP, bilirubin, TG, and LP. It also decreased the levels of GSH, G-6-P, N-de, AH and protein significantly (P < 0.001). Treatment with different doses of AB-4 reversed these parameters significantly in a dose dependant manner. AB-4 (500 mg/kg, po) exhibited hepatoprotection more or less equivalent to silymarin 50 mg/kg, po (Figs. 1 and 2). 3.3. Effect on biochemical parameters (Curative studies) A significant increase (P < 0.001) in the levels of ALT, AST, LDH, ALP, bilirubin, TG and LP and decrease in the levels of GSH, G-6-P, N-de, AH and protein were observed even after 50 h of CCl4 intoxication in rats. Curative treatment with different doses of AB-4 reversed these biochemical parameters significantly towards normal in a dose dependant manner. AB-4 (500 mg/kg, po) exhibited hepatoprotection almost equivalent to silymarin 50 mg/kg, po and in some parameters slightly better than silymarin (Figs. 1 and 2).
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Table 1 Effect of AB-4 on hexobarbitone sleep time and zoxazolamine induced paralysis time against CCl4 (0.1 ml/kg) induced liver damage in mice Treatments
Dose AB-4 (mg/kg)
Sleep time (in min)
% Hepatoprotection
Paralysis time (in min)
% Hepatoprotection
Vehicle Vehicle + CCl4 AB-4 + CCl4 AB-4 + CCl4 AB-4 + CCl4 Sily + CCl4 ANOVA
– – 125 250 500 50 F P
23.00 ± 1.59 84.50 ± 6.31 C 68.83 ± 3.38 a 55.00 ± 1.82 cf 46.66 ± 1.92 cgi 45.16 ± 2.93 cgip 38.754 <0.0001
– – 25.47 47.96 61.52 63.96
21.33 ± 1.33 63.00 ± 3.46 C 54.33 ± 3.99 d 47.66 ± 2.64 bh 37.00 ± 1.75 cfj 35.83 ± 2.53 cfjp 28.731 <0.0001
– – 20.8 36.81 62.39 65.2
Values represent the mean ± S.E. of six animals per group. P-value CCl4 vs. vehicle—C < 0.001. P-value treatments vs. CCl4 —a < 0.05, b < 0.01, c < 0.001, d not significant. P-value AB-4 125 vs. AB-4 250, 500 and Sily—f < 0.01, g < 0.001, h not significant. P-value AB-4 250 vs. AB-4 500 and Sily—i < 0.05, j < 0.01, l not significant. P-value AB-4 500 vs. Sily—p not significant.
3.4. Histopathology Microscopic examination of the animals treated with CCl4 alone showed centrilobular necrosis, macro-vesicular fatty changes (steatosis) and scattered lymphomononuclear (LMN) cell infiltrate in hepatic parenchyma. AB-4 showed dose dependent reversal of these changes induced by CCl4 . The hepatoprotective effect of AB-4 500 mg/kg was well compa-
rable to silymarin as evident by the reversal of centrilobular necrosis, macro-vesicular fatty changes (steatosis) and scattered lymphomononuclear cell infiltrate in hepatic parenchyma. 3.5. Bile flow and bile solids The ratio between first and second to fifth hour (culminated) bile volume and bile solids in control group was
Fig. 1. Effect of AB-4 on serum biochemical parameters against CCl4 (1 ml/kg, po) induced liver damage (prophylactic and curative study). (A) Representation of alanine aminotransferase. (B) Representation of aspartate aminotransferase. (C) Representation of alkaline phosphatase. (D) Representation of bilirubin. (E) Representation of triglycerides. (F) Representation of lactate dehydrogenase. Values represent the mean ± S.E. of six animals in each group. P-value vs. vehicleC < 0.001. P-value vs. CCl4 —a < 0.05, b < 0.01, c < 0.001, d not significant. P-value vs. AB-4 125 mg/kg—e < 0.05, f < 0.01, g < 0.001, h not significant. P-value vs. AB-4 250 mg/kg—i < 0.05, j < 0.01, k < 0.001, l not significant. P-value vs. AB-4 500 mg/kg—m < 0.05, p not significant.
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Fig. 2. Effect of AB-4 on hepatic parameters against CCl4 (1 ml/kg, po) induced liver damage (prophylactic and curative study). (A) Representation of total protein. (B) Representation of triglycerides. (C) Representation of glucose-6-phosphatase. (D) Representation of amidopyrine-N-demethylase. (E) Representation of aniline hydroxylase. (F) Representation of lipid peroxidation. (G) Representation of glutathione. Values represent the mean ± S.E. of six animals in each group. P-value vs. vehicle—C < 0.001. P-value vs. CCl4 —a < 0.05, b < 0.01, c < 0.001, d not significant. P-value vs. AB-4 125 mg/kg—e < 0.05, f < 0.01, g < 0.001, h not significant. P-value vs. AB-4 250 mg/kg—i < 0.05, j < 0.01, k < 0.001, l not significant. P-value vs. AB-4 500 mg/kg—p not significant.
1:2.66 and 1:2.52, respectively. Treatment with 250 and 500 mg/kg of AB-4 raised bile flow and bile solids. The percent increase in bile volume and bile solids exhibited by AB-4 (500 mg/kg) and DHC (50 mg/kg, id) was almost similar (Table 2).
3.6. Safety evaluation study Mice when fed with AB-4 up to 2000 mg/kg, po exhibited no mortality or any sign of gross behavioural changes when observed initially for 72 h and finally up to 15 days.
Table 2 Effect of AB-4 on bile flow and bile solids in rats Treatments
Dose (mg/kg)
Bile collected during (in ml) First hour
Vehicle AB-4 AB-4 DHC
– 250 500 50
0.71 0.60 0.62 0.66
± ± ± ±
0.04 0.06 0.02 0.04
Second to fifth hour 1.88 1.85 2.20 2.39
± ± ± ±
0.95 0.20 0.07 0.18
Values represents the mean ± S.E. of six animals in each group. a Ratio between first hour to second to fifth hour.
Solids collected during (in mg) %
Increase/ratioa
1:2.66a 13.92 ± 1.63 33.83 ± 3.73 35.49 ± 1.70
First hour 18.83 17.66 17.00 20.16
± ± ± ±
1.49 1.45 1.03 2.25
Second to fifth hour 48.00 53.50 57.66 67.00
± ± ± ±
4.86 5.25 4.07 7.86
% Increase/ratioa 1:2.52a 19.57 ± 3.09 34.26 ± 2.80 31.39 ± 1.60
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4. Discussion
Acknowledgements
Present study reports the hepatoprotective potential of aqueous extracts (AB-4) of Aloe barbadensis against extensively studied hepatotoxin CCl4 (Sambath et al., 2005). Our results correlates with the findings of previous authors that the time of lost reflex induced by short acting barbiturates is significantly prolonged in the event of any hepatic damage (Anand et al., 1997). AB-4 significantly shortened the hexobarbitone “sleep time” and zoxazolamine “paralysis time” as compared to CCl4 alone treated animals. This may be due to ability of AB-4 to protect hepatic drug metabolising enzymes. Prophylactic and curative treatment with AB-4 counteracts the CCl4 induced reduction of microsomal aniline hydroxylase and amidopyrine-N-demethylase activities indicates functional integrity of cells. CCl4 damage to liver raises the serum level of enzymes such as ALT, AST, ALP and LDH by releasing them in the blood stream (Asha, 2001). Our experiments with CCl4 elevated all these enzymes significantly (P < 0.0001) indicating severe hepatic cell necrosis. Hyperbilirubinaemia which indicates the severity of necrosis (Singh et al., 2005) raised significantly (P < 0.0001) with CCl4 . Prophylactic and curative treatment with AB-4 restore all the enzymes studied and bilirubin in a dose dependent manner showing its potential to maintain the normal functional status of the liver. Treatment with AB-4 significantly reversed CCl4 induced increase in triglycerides, accumulation of which leads to fatty liver (Seakins and Robinson, 1963). An accelerated lipid peroxidation and drastic fall in hepatic GSH contents by toxicants has been demonstrated (Brien et al., 2000). The damage of the cellular membrane due to lipid peroxidation also leads to decrease in the activity of membrane bound enzymes such as glucose-6-phosphatase (De Groot et al., 1985). In our study both prophylactic and curative treatment with AB-4 significantly reversed increased lipid peroxidation, maintained hepatic GSH and glucose-6-phosphatase levels. Histopathological studies under light microscope confirms the curative efficacy of AB-4 against CCl4 induced liver damage as evident by the reversal of centrilobular necrosis, macro-vesicular fatty changes (steatosis) and scattered lymphomononuclear cell infiltrate in hepatic parenchyma after AB-4 administration. Increase in bile flow and bile solids under the influence of AB-4 is suggestive of a strong stimulating action on the secretary activity of the liver cells. In conclusion, the possible mechanism of hepatoprotective action of aqueous extracts (AB-4) of Aloe barbadensis may be due to its antioxidant activity as indicated by protection against increased lipid peroxidation and maintained glutathione contents. Rest of the biochemical and pharmacological parameters studied indicate the status of structural and functional integrity of the cells and provide further support to the suggestive mechanism of action. Since AB-4 does not reveal any gross behavioural changes or mortality even at a dose of 2 g/kg po in mice and therefore it can be considered relatively safe. Further studies are in progress to identify active principle(s) responsible for hepatoprotective activity and to find out synergy among different compounds present in AB-4.
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Hepatoprotective potential of Aloe barbadensis Mill. against carbon tetrachloride induced hepatotoxicity B.K. Chandan a,∗ , A.K. Saxena a , Sangeeta Shukla c , Neelam Sharma a , D.K. Gupta b , K.A. Suri b , Jyotsna Suri d , M. Bhadauria c , B. Singh a b
a Department of Pharmacology, Regional Research Laboratory, Canal Road, Jammu-Tawi 180 016, India Department of Natural Products Chemistry, Regional Research Laboratory, Canal Road, Jammu-Tawi 180 016, India c School of Studies in Zoology, Jiwaji University, Gwalior (MP), India d Department of Pathology, Government Medical College, Jammu 180001 (J&K), India
Received 27 February 2006; received in revised form 13 December 2006; accepted 4 January 2007 Available online 14 January 2007
Abstract Aloe barbadensis Mill. Syn. Aloe vera Tourn. ex Linn.(Liliaceae) has been used in variety of diseases in traditional Indian system of medicine in India and its use for hepatic ailments is also documented. In the present study an attempt has been made to validate its hepatoprotective activity. The shade dried aerial parts of Aloe barbadensis were extracted with petroleum ether (AB-1), chloroform (AB-2) and methanol (AB-3). The plant marc was extracted with distilled water (AB-4). All the extracts were evaluated for hepatoprotective activity on limited test models as hexobarbitone sleep time, zoxazolamine paralysis time and marker biochemical parameters. AB-1 and AB-2 were observed to be devoid of any hepatoprotective activity. Out of two active extracts (AB-3 and AB-4), the most active AB-4 was studied in detail. AB-4 showed significant hepatoprotective activity against CCl4 induced hepatotoxicity as evident by restoration of serum transaminases, alkaline phosphatase, bilirubin and triglycerides. Hepatoprotective potential was confirmed by the restoration of lipid peroxidation, glutathione, glucose-6-phosphatase and microsomal aniline hydroxylase and amidopyrine N-demethylase towards near normal. Histopathology of the liver tissue further supports the biochemical findings confirming the hepatoprotective potential of AB-4. The present study shows that the aqueous extract of Aloe barbadensis is significantly capable of restoring integrity of hepatocytes indicated by improvement in physiological parameters, excretory capacity (BSP retention) of hepatocytes and also by stimulation of bile flow secretion. AB-4 did not show any sign of toxicity up to oral dose of 2 g/kg in mice © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Hepatoprotective activity; Carbon tetrachloride (CCl4 ); Aloe barbadensis
1. Introduction Aloe barbadensis Mill. Syn. Aloe vera Tourn. ex Linn. (Liliaceae) commonly known as Ghee kanwar (Hindi) found in semi wild state and can be cultivated in all parts of India (Mhaskar et al., 2000). In “Indian System of Medicine” fresh
Abbreviations: AB-1, petroleum ether extract; AB-2, chloroform extract; AB-3, methanol extract; AB-4, aqueous extract; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; TG, triglycerides; GSH, glutathione; LP, lipid peroxidation; G-6-P, glucose-6-phosphatase; AH, aniline hydroxylase; N-de, amidopyrine-Ndemethylase ∗ Corresponding author. Tel.: +91 191 2544382; fax: +91 191 2543829. E-mail address: [email protected] (B.K. Chandan). 0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.01.008
juice of leaves is used in diseases of eye and enlargements of spleen and liver (The wealth of India, 2000). The ethanolic extract from stem of the plant is reported for antibacterial activity against Escherichia coli (Singh et al., 1974) whereas leaf extract is active against Mycobacterium tuberculosis. Pulp of the leaves is reported for anti-fertility and oxytocic activities (Bradbury et al., 1967). It is also useful in X-ray burns, dermatitis, cutaneous leishmaniasis and other disorders of skin (Mhaskar et al., 2000). It has also been reported for antiviral (Saoo et al., 1996) and anti-inflammatory activities (Beatriz et al., 1996). It is also an important constituent of large number of ayurvedic preparations/formulations. Chemically, the presence of barbaloin, chrysophanol, glycoside aloe-emodin, glucose, galactose, mannose and galacturonic acid in addition to aldopentose and proteins with 18 amino acids have been reported. It
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also contains aloesone and aloesin (The Wealth of India, 2000). Beatrice et al., 2000 have reported that intraperitoneal injection of aloe emodin exhibited some protective effect against CCl4 on limited number of parameters but there is no report indicating hepatoprotective activity in aqueous extract containing number of other compounds which may be responsible for the activity individually or in combination. Based on its diversified pharmacological properties and its use in liver diseases in traditional Indian System of Medicine, an attempt has been made to validate the plant for its hepatoprotective potential against most widely used hepatotoxin CCl4 in experimental studies. Human dose of, one of the constituent (barbaloin) of aloe barbadensis is approximately 30 mg/kg, po and the doses of AB-4 used in the study are 125, 250, and 500 mg/kg, po which comes to be 21, 43, and 86 mg/kg approximately in human with respect to the presence of barbaloin in AB-4. 2. Materials and methods 2.1. Plant material, extraction and chromatography The plant was collected from the botanical garden of Regional Research Laboratory, Jammu and was authenticated at source by Dr. B.K. Kaphai taxonomist of the institute. A voucher specimen has been deposited at the herbarium of the Institute vide RRL collection No. 17875, Acc. No. 19650. The authenticated and freshly collected aerial parts of the plant were chopped and dried in shade. The coarse plant material (185 g) was extracted successively with petroleum ether (4× 1000 ml), chloroform (4× 900 ml) and methanol (5× 900 ml) at room temperature. During extraction with solvents, the solvent was changed after every 24 h. The extracts with each solvent after pooling were dried under reduced pressure to afford petroleum ether (AB-1 2 g; yield 1.08%, w/w), chloroform (AB-2, 1.9 g; yield 1.03%, w/w) and methanol extract (AB-3, 10 g; yield 5.40%, w/w). The plant marc was finally extracted with distilled water (3× 1000 ml) and lyophilised in freeze drier to furnish aqueous extract (AB-4, 31 g; yield 16.75%, w/w). Quantification of the barbaloin in the extracts was carried by HPLC (Shimadzu make HPLC system with LC-10A TVP pumps, DGU-14A degassing unit, SIL-10A PDA detector and CTO-10AS column oven. Data was acquired and analyzed by using Shimadzu Class-VP software version 6.10) using mobile phase, methanol:water (45:55); column: RP-8 (5 m), flow rate: 1 ml/min, detection: 254 nm., injection volume: 50 and 100 l, Barbaloin was used as standard for quantification of barbaloin in the extracts. Methanolic and aqueous extracts were found to contain 0.0127% (w/w) and 2.09% (w/w) barbaloin, respectively. While barbaloin in petroleum ether (AB-1) and chloroform (AB-2) extracts were below detection limits. 2.2. Chemicals Carbon tetrachloride (CCl4 ) was obtained from S.D. FineChem. Ltd., Mumbai. All other chemicals used were of analytical grade.
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2.3. Experimental animals Male Wistar rats (150–200 g) or Swiss albino mice (20–25 g) were used in all the studies except for bile flow and bile solids where rats of same strain but of higher body weight (200–250 g) were used. Animals were housed under standard conditions (23.0 ± 2 ◦ C, 60–70% humidity, and 12 h photo period) and allowed free access to food (Lipton India Ltd., Bombay, India) and water. The experiments were conducted according to the ethical norms approved by Institutional Animal Ethics Committee guide lines for animal care and were adhered to as recommended by the Indian National Science Academy, New Delhi (1992). 2.3.1. Induction of hepatic injury Hepatic injury was produced by oral administration of CCl4 diluted with liquid paraffin in all the studies. 2.4. Hexobarbitone induced narcosis and zoxazolamine induced paralysis time Six groups of mice with six mice in each group were used for each study. Three groups (groups 1–3) were fed orally once with AB-4 125, 250 and 500 mg/kg, respectively. Silymarin (50 mg/kg, po) was administered to fourth group and last two groups (groups 5 and 6) were given vehicle only and served as control. CCl4 (0.1 ml/kg po) was given to animals of first five groups treated with AB-4, silymarin and one group of controls (group 5) that served as CCl4 control, 1 h after feeding test material. The second control group (group 6) was given vehicle only and served as normal control. Hexobarbitone (60 mg/kg, ip) sleep time were monitored individually 2 h after CCl4 administration in all the animals. To study zoxazolamine induced paralysis time grouping of the animals and treatment is same except for the dose of zoxazolamine, which was 70 mg/kg, ip. The time to onset of loss of the righting reflex or paralysis to the recovery in minutes was taken as duration of sleep or paralysis time respectively (Singh et al., 2005). 2.5. Effect on biochemical parameters 2.5.1. Prophylactic studies Six groups with six rats in each were used for this study. First three groups were fed orally AB-4 125, 250 and 500 mg/kg, respectively, 48, 24 and 2 h before and 6 h after CCl4 (1 ml/kg, po) administration (Bramanti et al., 1978). Similarly fourth group was fed with silymarin (50 mg/kg, po). Out of remaining two groups one was given the same dose of CCl4 which served as CCl4 control group and to other proportionate volume of vehicle was given and served as vehicle control. Liver and blood samples were collected 24 h after CCl4 administration. 2.5.2. Curative studies The grouping of animals and doses of AB-4, silymarin and CCl4 are the same as described for prophylactic studies except that AB-4 and silymarin were given exactly 2, 6, 24, and 48 h after CCl4 administration (Singh et al., 1998). Liver and blood samples were collected 50 h after CCl4 administration.
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2.5.3. Blood biochemistry Blood samples were collected in glass tubes from orbital sinus to obtain haemolysis free clear serum for the analysis of ALT and AST (Reitman and Frankel, 1957), ALP (Walter and Schutt, 1974), LDH (Wroblewski and La Due, 1964), bilirubin (Malloy and Evelyn, 1937) and TG (Neri and Frings, 1973) by standard methods.
treatment) to 1 h before treatment. The choleretic activity as percent increase of bile flow was determined (Chandan et al., 1991). Bile collected from the animals as mentioned above was dried and the percent increase in bile solids was calculated as in case of bile flow.
2.5.4. Preparation of liver homogenate and microsomes All the animals were sacrificed by decapitation and livers were quickly excised freed from any adhering tissues, washed and perfused with chilled normal saline, minced and homogenized in ice bath using Potter-S-homogenizer (B. Braun, Melsungen AG, Germany, 1100 rpm for 2 min) in chilled 10 mM Tris–HCl buffer (pH 7.4) to obtain 10% liver homogenate for the estimation of (GSH) glutathione (Ellman, 1959), (LP) lipid peroxidation (Buege and Aust, 1978), (G-6-P) glucose6-phosphatase (Baginski et al., 1974) and (TG) triglycerides (Neri and Frings, 1973) by using standard methods. Liver microsomes were prepared by the method as described by Schenkman and Cinti, 1978. Stored in liquid nitrogen for the estimation of (AH) aniline hydroxylase (Kato and Gillette, 1965) and (N-de) amidopyrine-N-demethylase (Chochin and Axelrod, 1959).
The safety study was carried out using OECD guide lines No. 423. Three female mice of same age group and weight were taken in a single dose up to the highest dose of 2000 mg/kg orally. The animals were observed for 1 h continuously and then hourly for 4 h and finally after every 24 h up to 15 days for any mortality or gross behavioural changes.
2.5.5. Histopathology From all the animals used for curative study small portion of liver tissue was fixed in 10% formaline saline, processed and embedded in paraffin wax to obtain 5–6 m thick hematoxylin and eosin stained sections (Krajian, 1963). 2.6. Hepatoprotective activity The hepatoprotective activity, expressed as hepatoprotective percentage (H), was calculated as follows: T −V H = 1− × 100 C−V where T is mean value of drug and CCl4 , C mean value of CCl4 alone and V is the mean value of normal control animals. 2.7. Effect on bile flow and bile solids Male rats were divided into four groups of six rats in each and their food was withdrawn 18 h before experiment but allowed free access to water. To determine the choleretic activity bile duct was cannulated with PE-10 tubing under urethane anesthesia (6.0 ml/kg ip of 25% solution) and bile was collected individually in a small test tube initially for 1 h (which served as control) and at the end of the first hour all the animals of first two groups were administered with test material (AB-4, 250 or 500 mg/kg, id) and group three with dehydrocholic acid (DHC, 20 mg/kg, iv) as reference standard. The collection of the bile continued hourly for next 4 h. The last group was not given any treatment and served as normal control. The criterion for choleretic activity is based on the increase in bile volume in 4 h cumulated (after
2.8. Safety evaluation study
2.9. Statistical analysis The data obtained were analyzed by One Way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using computerized program. P-value < 0.05 or was taken as the criterion of significance. 3. Results 3.1. Hexobarbitone induced narcosis and zoxazolamine induced paralysis time in mice Hexobarbitone sleep time and zoxazolamine induced paralysis time were significantly (P < 0.001) raised in CCl4 treated animals. AB-4 counteracted CCl4 enhanced hexabarbitone induced sleep time and Zoxazolamine paralysis time and thus showing hepatoprotective activity of different magnitude depending upon dose (Table 1). 3.2. Effect on biochemical parameters (Prophylactic studies) Carbon tetrachloride significantly (P < 0.001) raised ALT, AST, LDH, ALP, bilirubin, TG, and LP. It also decreased the levels of GSH, G-6-P, N-de, AH and protein significantly (P < 0.001). Treatment with different doses of AB-4 reversed these parameters significantly in a dose dependant manner. AB-4 (500 mg/kg, po) exhibited hepatoprotection more or less equivalent to silymarin 50 mg/kg, po (Figs. 1 and 2). 3.3. Effect on biochemical parameters (Curative studies) A significant increase (P < 0.001) in the levels of ALT, AST, LDH, ALP, bilirubin, TG and LP and decrease in the levels of GSH, G-6-P, N-de, AH and protein were observed even after 50 h of CCl4 intoxication in rats. Curative treatment with different doses of AB-4 reversed these biochemical parameters significantly towards normal in a dose dependant manner. AB-4 (500 mg/kg, po) exhibited hepatoprotection almost equivalent to silymarin 50 mg/kg, po and in some parameters slightly better than silymarin (Figs. 1 and 2).
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Table 1 Effect of AB-4 on hexobarbitone sleep time and zoxazolamine induced paralysis time against CCl4 (0.1 ml/kg) induced liver damage in mice Treatments
Dose AB-4 (mg/kg)
Sleep time (in min)
% Hepatoprotection
Paralysis time (in min)
% Hepatoprotection
Vehicle Vehicle + CCl4 AB-4 + CCl4 AB-4 + CCl4 AB-4 + CCl4 Sily + CCl4 ANOVA
– – 125 250 500 50 F P
23.00 ± 1.59 84.50 ± 6.31 C 68.83 ± 3.38 a 55.00 ± 1.82 cf 46.66 ± 1.92 cgi 45.16 ± 2.93 cgip 38.754 <0.0001
– – 25.47 47.96 61.52 63.96
21.33 ± 1.33 63.00 ± 3.46 C 54.33 ± 3.99 d 47.66 ± 2.64 bh 37.00 ± 1.75 cfj 35.83 ± 2.53 cfjp 28.731 <0.0001
– – 20.8 36.81 62.39 65.2
Values represent the mean ± S.E. of six animals per group. P-value CCl4 vs. vehicle—C < 0.001. P-value treatments vs. CCl4 —a < 0.05, b < 0.01, c < 0.001, d not significant. P-value AB-4 125 vs. AB-4 250, 500 and Sily—f < 0.01, g < 0.001, h not significant. P-value AB-4 250 vs. AB-4 500 and Sily—i < 0.05, j < 0.01, l not significant. P-value AB-4 500 vs. Sily—p not significant.
3.4. Histopathology Microscopic examination of the animals treated with CCl4 alone showed centrilobular necrosis, macro-vesicular fatty changes (steatosis) and scattered lymphomononuclear (LMN) cell infiltrate in hepatic parenchyma. AB-4 showed dose dependent reversal of these changes induced by CCl4 . The hepatoprotective effect of AB-4 500 mg/kg was well compa-
rable to silymarin as evident by the reversal of centrilobular necrosis, macro-vesicular fatty changes (steatosis) and scattered lymphomononuclear cell infiltrate in hepatic parenchyma. 3.5. Bile flow and bile solids The ratio between first and second to fifth hour (culminated) bile volume and bile solids in control group was
Fig. 1. Effect of AB-4 on serum biochemical parameters against CCl4 (1 ml/kg, po) induced liver damage (prophylactic and curative study). (A) Representation of alanine aminotransferase. (B) Representation of aspartate aminotransferase. (C) Representation of alkaline phosphatase. (D) Representation of bilirubin. (E) Representation of triglycerides. (F) Representation of lactate dehydrogenase. Values represent the mean ± S.E. of six animals in each group. P-value vs. vehicleC < 0.001. P-value vs. CCl4 —a < 0.05, b < 0.01, c < 0.001, d not significant. P-value vs. AB-4 125 mg/kg—e < 0.05, f < 0.01, g < 0.001, h not significant. P-value vs. AB-4 250 mg/kg—i < 0.05, j < 0.01, k < 0.001, l not significant. P-value vs. AB-4 500 mg/kg—m < 0.05, p not significant.
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Fig. 2. Effect of AB-4 on hepatic parameters against CCl4 (1 ml/kg, po) induced liver damage (prophylactic and curative study). (A) Representation of total protein. (B) Representation of triglycerides. (C) Representation of glucose-6-phosphatase. (D) Representation of amidopyrine-N-demethylase. (E) Representation of aniline hydroxylase. (F) Representation of lipid peroxidation. (G) Representation of glutathione. Values represent the mean ± S.E. of six animals in each group. P-value vs. vehicle—C < 0.001. P-value vs. CCl4 —a < 0.05, b < 0.01, c < 0.001, d not significant. P-value vs. AB-4 125 mg/kg—e < 0.05, f < 0.01, g < 0.001, h not significant. P-value vs. AB-4 250 mg/kg—i < 0.05, j < 0.01, k < 0.001, l not significant. P-value vs. AB-4 500 mg/kg—p not significant.
1:2.66 and 1:2.52, respectively. Treatment with 250 and 500 mg/kg of AB-4 raised bile flow and bile solids. The percent increase in bile volume and bile solids exhibited by AB-4 (500 mg/kg) and DHC (50 mg/kg, id) was almost similar (Table 2).
3.6. Safety evaluation study Mice when fed with AB-4 up to 2000 mg/kg, po exhibited no mortality or any sign of gross behavioural changes when observed initially for 72 h and finally up to 15 days.
Table 2 Effect of AB-4 on bile flow and bile solids in rats Treatments
Dose (mg/kg)
Bile collected during (in ml) First hour
Vehicle AB-4 AB-4 DHC
– 250 500 50
0.71 0.60 0.62 0.66
± ± ± ±
0.04 0.06 0.02 0.04
Second to fifth hour 1.88 1.85 2.20 2.39
± ± ± ±
0.95 0.20 0.07 0.18
Values represents the mean ± S.E. of six animals in each group. a Ratio between first hour to second to fifth hour.
Solids collected during (in mg) %
Increase/ratioa
1:2.66a 13.92 ± 1.63 33.83 ± 3.73 35.49 ± 1.70
First hour 18.83 17.66 17.00 20.16
± ± ± ±
1.49 1.45 1.03 2.25
Second to fifth hour 48.00 53.50 57.66 67.00
± ± ± ±
4.86 5.25 4.07 7.86
% Increase/ratioa 1:2.52a 19.57 ± 3.09 34.26 ± 2.80 31.39 ± 1.60
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4. Discussion
Acknowledgements
Present study reports the hepatoprotective potential of aqueous extracts (AB-4) of Aloe barbadensis against extensively studied hepatotoxin CCl4 (Sambath et al., 2005). Our results correlates with the findings of previous authors that the time of lost reflex induced by short acting barbiturates is significantly prolonged in the event of any hepatic damage (Anand et al., 1997). AB-4 significantly shortened the hexobarbitone “sleep time” and zoxazolamine “paralysis time” as compared to CCl4 alone treated animals. This may be due to ability of AB-4 to protect hepatic drug metabolising enzymes. Prophylactic and curative treatment with AB-4 counteracts the CCl4 induced reduction of microsomal aniline hydroxylase and amidopyrine-N-demethylase activities indicates functional integrity of cells. CCl4 damage to liver raises the serum level of enzymes such as ALT, AST, ALP and LDH by releasing them in the blood stream (Asha, 2001). Our experiments with CCl4 elevated all these enzymes significantly (P < 0.0001) indicating severe hepatic cell necrosis. Hyperbilirubinaemia which indicates the severity of necrosis (Singh et al., 2005) raised significantly (P < 0.0001) with CCl4 . Prophylactic and curative treatment with AB-4 restore all the enzymes studied and bilirubin in a dose dependent manner showing its potential to maintain the normal functional status of the liver. Treatment with AB-4 significantly reversed CCl4 induced increase in triglycerides, accumulation of which leads to fatty liver (Seakins and Robinson, 1963). An accelerated lipid peroxidation and drastic fall in hepatic GSH contents by toxicants has been demonstrated (Brien et al., 2000). The damage of the cellular membrane due to lipid peroxidation also leads to decrease in the activity of membrane bound enzymes such as glucose-6-phosphatase (De Groot et al., 1985). In our study both prophylactic and curative treatment with AB-4 significantly reversed increased lipid peroxidation, maintained hepatic GSH and glucose-6-phosphatase levels. Histopathological studies under light microscope confirms the curative efficacy of AB-4 against CCl4 induced liver damage as evident by the reversal of centrilobular necrosis, macro-vesicular fatty changes (steatosis) and scattered lymphomononuclear cell infiltrate in hepatic parenchyma after AB-4 administration. Increase in bile flow and bile solids under the influence of AB-4 is suggestive of a strong stimulating action on the secretary activity of the liver cells. In conclusion, the possible mechanism of hepatoprotective action of aqueous extracts (AB-4) of Aloe barbadensis may be due to its antioxidant activity as indicated by protection against increased lipid peroxidation and maintained glutathione contents. Rest of the biochemical and pharmacological parameters studied indicate the status of structural and functional integrity of the cells and provide further support to the suggestive mechanism of action. Since AB-4 does not reveal any gross behavioural changes or mortality even at a dose of 2 g/kg po in mice and therefore it can be considered relatively safe. Further studies are in progress to identify active principle(s) responsible for hepatoprotective activity and to find out synergy among different compounds present in AB-4.
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