nuzhat sultana et al. irjp 2012, 3 (10) · nuzhat sultana et al. irjp 2012, 3 ... metabolizes the...

Nuzhat Sultana et al. IRJP 2012, 3 (10)

06

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

GROSS TOXICITIES AND HEPATOPROTECTIVE EFFECT OF ALOE VERA (L) BURM.F

Nuzhat Sultana*, Rahila Najam Department of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan

Article Received on: 17/08/12 Revised on: 13/09/12 Approved for publication: 12/10/12

*E- mail: [email protected] ABSTRACT Traditional herbal drugs have a great demand in under developed countries due to their efficacy, low cost and lesser adverse effects, and they are considered to be “natural. Aloe vera at the dose of 500 mg body weight per oral was studied for the gross toxicities and hepatoprotective effect and observed the level of liver biochemical parameters in rabbits. Aloe vera showed highly significant (p<0.001) hepatoprotective effect by lowering the serum levels of serum glutamic oxaloacetate transaminase (SGOT), serum glutamic pyruvates transaminase (SGPT) and direct bilirubin. The overall experimental results suggests that Aloe vera protects the liver from oxidative stress and inhibits the excessive free radicals accumulation and possessing many hepatoprotective phytoconstituents which are biologically active such as flavonoids, alkaloids, they may be responsible for the significant hepatoprotective activity and the results justify the use of Aloe vera as a hepatoprotective agent. Keywords: Aloe vera, gross toxicities, liver profile, rabbits. INTRODUCTION Liver plays a vital role in clearing and transforming the chemicals but some medicinal agents can damage the organ if given in therapeutic or high dose. Non-medicinal agents including industrial and environmental chemicals can also lead to hepatotoxicity, and are called as hepatotoxins1. Liver metabolizes the xenobiotics by the reduction in fat solubility and alters the biological activity after chemical transformation 2. Some biochemical markers such as bilirubin, alanine transferase and alkaline phosphatase indicate the normal function of hepatocytes or liver damage3. Liver injury is present when bilirubin and alkaline phosphatase is more than twice of upper limit of normal or alanine transferase is more than three times of upper limit of normal4. Liver normal function can be changed by the action of some toxins or due to infections 5. Some agents such as carbon tetrachloride and paracetamol elevate the level of alkaline phosphatase and induce the liver injuries. Damage to liver is identified with the help of the level of aspartate aminotransferase and alanine transaminase 6, 7. Traditional herbal drugs have a great demand in under developed countries due to their efficacy, low cost and lesser adverse effects, and they are considered to be “natural” 8 and for biological and pharmacological assessment at least 20% of plants are used 9. Due to the lack of hepatoprotective drugs in allopathic medical practice 5, herbal drugs play an important role in the cure of liver diseases but clinical trials are required to assess the role of medicinal herbs.10 Aloe vera (L) Burm.f family Liliaceae is green, fleshy, spiny and cactus like plant. Leaf filled with clear viscous gel 11 has high concentration of water (99.3%) and can survive in dry and harsh climate. It contains different nutrients including minerals, vitamins, sugars, saponins, enzyme (Carboxypeptidase, amylase and lipase), phenolic compounds, amino acid, lignin and sterol. Aloe vera contains many vitamins including vitamin B (thiamine), B2 (Riboflavin), B3 (Niacin) and important antioxidant vitamins (A, C and F) with traces of vitamin B12

12. Folic acid and choline are also present. For the reduction of inflammation and stress Aloe vitamin B and C play an important role. Lipase takes part in digestion of sugars and fats whereas amylase hydrolyses the starch. Zinc found in Aloe gel acts as

an anti-inflammatory agent by inactivation of bradykinin 13

through pancreatic carboxypeptidase (a Zinc-dependant metalloenzyme). Aloe vera contains Aspirin like compound such as lupeol, campesterol and sitosterol possessing anti-inflammatory properties14. Aloe vera contains monosaccharide and polysaccharide responsible for the enhancement of immune response. Due to the presence of anthraquinones derivatives such as Barbaloin and Isobarbaloin, it has purgative action 15. Approx. 3% saponins found in Aloe vera, has a cleansing action also possess anticarcinogenic and antiseptic activity 16 .Calcium, potassium, manganese, zinc, copper and iron are found in Aloe vera plant and these are required for the regulation of body functions, in which calcium is required for the bone formation and bone growth, potassium for regulation of blood pressure and water balance, manganese for the protein metabolism, zinc for the wound healing and reproduction, copper for the iron absorption, storage and metabolism and for the haemoglobin formation 17. It has 22 amino acids in which eight are essential amino acids and body cannot synthesize these amino acids. Aloe vera may have anxiolytic potential with sedative action because it showed decrease in traction in an inclined plane test and significant reduction of locomotor and exploratory activities 18. MATERIALS AND METHOD Selection of Animals Long-term administration of Aloe vera was done on rabbits. Average weight of animals of either sex is ranging from 1000-1790 gm. All rabbits were randomly assigned into two groups (n=10), one group served as control group, while other group of rabbits served as treated group (Aloe vera). All procedures and protocols were in accordance with guiding principles in the care and use of animals, Helsinki declaration19. Housing Rabbits were kept in 22 ± 1 oC room temperature with 12/12 hours light - dark cycle i.e. light on from 07.00 a.m to 07.00 p.m at the Department of Pharmacology, University of Karachi and had access to water and food ad labitum. They were housed two per cage under standard conditions and kept at least 1 week before start of dosing.


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Experimental Protocol Aloe vera was purchased from the market in the form of capsules and was used for experiment. The daily dosing of Aloe vera for the determination of liver function parameters was done for a period of 30 days in the dose of 500 mg / 70 kg orally 20 according to the body weight of animal. This dose is in accordance to control adult dose of Aloe vera capsule of 500 mg. Assessment of Gross Toxicities The gross toxicities of rabbits were assessed on weekly basis for 30 days and observing righting reflex, skin ulceration, anxiety, hematuria, passivity, loss of hair, loss of activity, tremor, salivation, vomiting, diarrhea, change in pupil size, edema and aggressive behavior. Mortality Rate Mortality Rate was observed in animals receiving Aloe vera and note the number of animals died during the total period of experiment. Assessment of liver function Blood samples were collected after 7, 15 and 30 day of dosing of Aloe vera in vacuum blood collection tubes. Centrifuging blood samples at 3000 rpm for 15 minutes immediately separated serum out. The serum was used for estimation of biochemical parameters to determine the functional state of the liver within 3 hours of sample collection and analyzed on Humalyzer 3000, Semi-automatic chemistry analyzer, Model # 16700 (Human Germany), using standard kits supplied by Human (HUMALYZER 3000). Total and Direct bilirubin were estimated by photometric test 21. Serum glutamic pyruvic transaminase (SGPT) was estimated by kinetic method with reference to the International Federation of Clinical Chemistry 22. Alkaline phosphatase (ALP) was estimated by method described by Mac Comb and Bowers 23. γ -GT was estimated by colorimetric method 24, 25. Serum glutamic oxalo-acetic transaminase (SGOT) or Aspartate amino-transferase (ASAT) was estimated by a UV kinetic method based on the reference method of International federation of Clinical Chemistry 26. Statistical Analysis All results were expressed as average value ± standard deviation (St.Dev). The significance of difference between averages was determined 27,28 and the data obtained from present study was analyzed for P-value. P-value < 0.01 was considered significant and P-value < 0.001 was considered highly significant, following the one way ANOVA. RESULTS Assessment of Gross Toxicities The gross toxicity was observed in animals receiving Aloe vera and no significant change was observed in animals during the total period of experiment. Mortality Rate Mortality Rate was observed in animals receiving Aloe vera and no death occurred during the total period of experiment. Assessment of liver function Figures 1, 2 , 3 , 4 , 5 and 6 show the statistical analysis of total bilirubin , direct bilirubin , SGPT , alkaline phosphatase , γ-GT, SGOT after 07 , 15 and 30 day of dosing of Aloe vera and data analyzed by One Way ANOVA show a significant effect of drug Aloe vera. Effect on Total Bilirubin In Figure 1, Post-hoc analysis by Newman – Keuls test shows that after 15 and 30 days of dosing of Aloe vera there was

significant increase in total bilirubin, i.e. 0.22±0.02 and 0.19±0.005 (mg/dl) respectively and after 07 day of dosing of Aloe vera showed non - significant rise in total bilirubin, i.e. 0.12±0.03 (mg/dl) in comparison to control animals group, i.e. 0.08±0.03 (mg/dl). Results showed that the total bilirubin level of Aloe vera animals group after 15 and 30 day was increased much significantly than after 7 day of dosing. Effect on Direct Bilirubin In Figure 2, Post-hoc analysis by Newman – Keuls test shows that animals at 07, 15 and 30 day of dosing of Aloe vera showed highly significant decrease in direct bilirubin, i.e. 0.01±0.004, 0.01±0.005 and 0.03±0.005 (mg/dl) respectively in comparison to control animals group, i.e. 0.06±0.004 (mg/dl). Results showed that the direct bilirubin level of Aloe vera animals group after 7, 15 and 30 day was decreased much significantly. Effect on SGPT In Figure 3, Post-hoc analysis by Newman – Keuls test shows that after 07 day of dosing of Aloe vera showed there was significant decrease in SGPT, i.e. 39±1.5 U/l and after 15 and 30 days of dosing showed non - significant rise in SGPT, i.e. 136±37 U/l and 144±88.5 U/l respectively in comparison to control animals group, i.e. 100±0.4 U/l. Results showed that the SGPT level of Aloe vera animals group after 7 day was decreased much significantly than after 15 and 30 days of dosing. Effect on Alkaline Phosphatase In Figure 4, Post-hoc analysis by Newman – Keuls test shows that animals after 07 and 15day of dosing of Aloe vera showed significant rise in alkaline phosphatase (ALP), i.e. 27±14.6 U/l, and 24±5.5 U/l and after 30 day of dosing of Aloe vera showed decrease in levels of Alkaline Phosphatase, i.e. 9±1.0 U/l in comparison to control animals group, i.e. 9±0.5 U/l. Results showed that the alkaline phosphatase level of Aloe vera treated animals group was decreased much significantly after 30 days of dosing. Effect on γ GT In Figure 5, Post-hoc analysis by Newman – Keuls test shows that animals after 07 and 15 day of dosing of Aloe vera showed highly significant increase in γ GT i.e. 8±0.5 and 24±4.5 U/l respectively and after 30 day of dosing showed significant increase in γ GT i.e. 9±2.5 U/l in comparison to control animals group, i.e. 5±0.5 U/l. Results showed that the γ GT level of Aloe vera animals group after 7 and 15 days was increased much significantly than after 30 day of dosing. Effect on SGOT In Figure 6, Post-hoc analysis by Newman – Keuls test shows that animals after 07 and 15 days of dosing of Aloe vera showed highly significant decrease in SGOT, i.e. 27±1.0 and 28±1.0 U/l respectively and after 30 day of dosing showed significant rise in SGOT, i.e. 143±90.6 U/l in comparison to control animals group, i.e. 42±0.4 U/l. Results showed that the SGOT level of Aloe vera animals group after 7 and 15 days was decreased much significantly than after 30 day of dosing. DISCUSSION Direct bilirubin is water soluble 29 and is a constituent of bile. Bile duct open into the small intestine and then bile is released into the intestine after the action of GI bacteria, bilirubin then metabolizes to urobilinogen and ultimately into stercobilin. This stercobilin provides the brown color of the feces. In urine some of urobilinogen is excreted out with urobilin, which is oxidized state of urobilinogen 30. Elevated direct bilirubin indicates the liver injury 31 but in current


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study Aloe vera slightly decreases the level of direct Bilirubin. In the blood plasma total bilirubin is measured with the direct bilirubin. Elevated bilirubin indicates the liver disease. In present study Aloe vera slightly increases the level of total bilirubin as compared to control group animals but it is within the reference range. Increase level of total bilirubin may be damaging of plasma membrane of hepatocytes 32 or due to the liver cell cytolysis33. Due to the structural damage of liver, the level of liver enzymes are increased in serum because liver enzymes are located in cell cytoplasm after damaging or injury they are released into the blood circulation and raises the level of enzymes in serum 34. Alanine transaminase (ALT) or Serum glutamic pyruvic transaminase (SGPT) is only present in hepatocytes and in blood6. Hepatocellular injury produces some health problems such as congestive heart failure, diabetes, bile duct problems and viral hepatitis. Aloe vera possesses hepatoprotective activity and reduces the level of SGPT 20. In current study Aloe vera also decreases the level of Alanine transaminase (ALT) or Serum glutamic pyruvic transaminase (SGPT) after 7day administration .For the detection of liver damage ALT is a biomarker to evaluate the liver disease 35, 31. Reduction of SGPT indicates the restoration of normal functioning of liver 36. Aloe vera protects the liver from oxidative stress and inhibits the excessive free radicals accumulation 37. In conclusion Aloe vera has hepatoprotective 38 and antioxidative property through oxidative stress suppression 39, Aloe vera has liver protective effect against hepatotoxic agent by restoration of glutathione, glucose-6-phosphatase, lipid peroxidation, amidopyrine N-demethylase and microsomal aniline hydroxylase, and showing the normal histopathology of liver tissues. Acute liver toxicity strongly blocked by the use of Aloe vera and it reduces the liver enzymes 34. Some bioactive compounds of Aloe vera are very effective such as tannins 40, steroids and alkaloids 41. Specific steroids and flavonoids are responsible to protect the liver from oxidative stress and play a key role in hepatoprotection 42. Aloe vera slightly increases the level of alkaline phosphatase (ALP) after 7 and 15 days of dosing and it could be due to the presence of alkaline phosphatase which is a constituent of leaf pulp and exudates of Aloe vera 43 but after long – term administration, it does not increase the level of alkaline phosphatase indicating the hepatoprotective effect. In current study Aloe vera slightly increases the level of alkaline phosphatase (ALP), indicating the infiltrative liver disease or bile duct obstruction. Alkaline phosphatase involved for the removal of phosphate substrate present in the protein 44 leads to decrease amount of total protein, albumin and globulin, it could be due to the increase level of enzyme alkaline phosphatase (ALP). Aloe vera has hepatoprotective effect in chronic and acute liver injuries 45 through oxidative stress suppression. It has liver protective effect against hepatotoxic agent such as carbon tetrachloride by restoration of normal hepatocytes. Carbon tetrachloride is a potent hepatotoxic agent, after administration it metabolizes by the liver enzyme cytochrome P450. It produces oxidative free radical capable to liver injury and inducing oxidative effect but Aloe vera does not act as carbon tetrachloride and restore the action of hepatotoxic agent by restoration of glutathione, lipid peroxidation and microsomal aniline hydroxylase. Acute liver toxicity strongly

blocked by the use of Aloe vera which reduces the liver enzymes in serum 34. In current study Aloe vera increases the level of Gamma-glutamyl-transferase (γ-GT), it could be due to the more generation of γ-GT by the body cell. Γ-GT present in cell membrane of hepatocytes , kidneys , prostate , heart and in blood6. Elevated level of γ-GT is observed in cardiovascular disease46 and in pathogenesis47. Reduced form of glutathione is used for the measurement of cell toxicity in oxidative stress.48 γ-GT is responsible for the degradation and biosynthesis of glutathione. Generally the level of γ-GT is slightly higher than the level of SGPT and SGOT, it could be due to the more generation of γ-GT by the body cell but it also indicate that if Aloe is used for short term then the levels will be in normal limits. Aloe vera possesses hepatoprotective activity and reduces the level of SGOT 20 but increased after 30 days. Aspartate aminotransferase or SGOT is not only present in hepatocytes but also found in skeletal, cardiac muscles and blood cells. The level is only increased after 30 days of dosing indicating that the use of Aloe should be short term. Aloe vera protects the liver from oxidative stress and inhibits the excessive free radicals accumulation37. In Ayurvedic formulation Aloe vera used for the protection of hepatocytes and it possesses many hepatoprotective constituents 41. Specific steroids and flavonoids are responsible to protect the liver from oxidative stress and play a key role as hepatoprotective agent42. In conclusion the plant Aloe vera has hepatoprotective 49 and antioxidative 50 properties through oxidative stress suppression and has liver protective effect against hepatotoxic agent such as carbon tetrachloride by restoration of the normal hepatocytes and normal appearance of liver cells. REFERENCES 1. Friedman, Scott E. Grendell, James H. McQuaid, Kenneth R. Current

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Figure 1: Effect of Aloe vera on Total Bilirubin

n = 10, Average value ± St.Dev, Significant difference by Newman keuls

test, **p < 0.001 as compared to control rabbits, following one way ANOVA

Figure 2: Effect of Aloe vera on Direct Bilirubin



Figure 3: Effect of Aloe vera on SGPT



Figure 4: Effect of Aloe vera on ALP

n = 10, Average value ± St.Dev, Significant difference by Newman keuls test, *p < 0.01 as compared to control rabbits, **p < 0.001 as compared to

control rabbits, following one way ANOVA

Figure 5: Effect of Aloe vera on γ GT

n = 10, Average value ± St.Dev, Significant difference by Newman keuls test, *p < 0.01 as compared to control, **p < 0.001 as compared to control

rabbits, following one way ANOVA

Figure 6: Effect of Aloe vera on SGOT

n = 10, Average value ± St.Dev, Significant difference by Newman keuls test, *p < 0.01 as compared to control rabbits, **p < 0.001 as compared to

control rabbits, following one way ANOVA

Source of support: Nil, Conflict of interest: None Declared IRJP is an official publication of Moksha Publishing House. Website: www.mokshaph.com. All rights reserved.

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