Research J. Pharm. and Tech.

5(5): May2012

ISSN 0974-3618 RESEARCH ARTICLE

www.rjptonline.org

Hepatoprotective effect of Gallic acid and Gallic acid Phytosome against Carbon Tetrachloride induced damage in albino rats
Radhey Shyam Kuamwat1*, K. Mruthunjaya2, Manish Kumar Gupta3
2

Bhagwant University, Ajmer, Rajasthan, India JSS College of Pharmacy, JSS University, Mysore, Karnataka, India 3 Sri Balaji College of Pharmacy, Jaipur, Rajasthan, India *Corresponding Author E-mail: radhey4183@rediffmail.com

ABSTRACT:
Phytoconstituents like many polyphenols are poorly absorbed either due to their multiple-ring large size molecules which cannot be absorbed by simple diffusion, or due to their poor miscibility with oils and other lipids, severely limiting their ability to pass across the lipid-rich outer membranes of the enterocytes of the small intestine. Watersoluble phytoconstituent molecules (mainly polyphenols) can be converted into lipid-compatible molecular complexes, which are called Phytosomes. Gallic acid (GA, 3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol. So the following study was undertaken to evaluate the protective effects of gallic acid and gallic acid Phytosomes (GAP) at different doses against CCl4 induced hepatic and renal damage in albino rats. Liver damage was induced in Wister albino rats by administering CCl4 (1.5 ml/kg, i.p) once only. Simultaneously, GAP (40, 60 mg/kg, p.o.), GA (100 and 200 mg/kg, p.o.), and the reference drug silymarin (50 mg/kg b.w.).were administered orally. Levels of marker enzymes (SGOT, SGPT and SALP), albumin (Alb) and total protein (TP) were assessed in serum. Treatment with gallic acid (100 and 200 mg/kg, p.o.) and gallic acid-phospholipids complex (40, 60 mg/kg, p.o.) showed dose-dependent recovery in all these biochemical parameters but the effect was more pronounced with gallic acid Phytosomes. Thus it may be concluded that 45mg/kg dose of gallic acid-phospholipids was found to be most effective against carbon tetrachloride induced liver and kidney damage.

KEYWORDS: Gallic acid, Hepatprotective, Phospholipids, Phytosomes, CCl4 INTRODUCTION:

Liver is one of the largest organs in human body and the chief site for intense metabolism and excretion. So it has a surprising role in the maintenance, performance and regulating homeostasis of the body. It is involved with almost all the biochemical pathways to growth, fight against disease, nutrient supply, energy provision and reproduction1. Liver diseases are a leading health problem after CVD, cancer and AIDS. Medicinal plants play a key role in the human health care. About 80% of the world populations rely on the use of traditional medicine which is predominantly based on plant materials2. Most of the bioactive constituents of herbal drugs are water soluble molecules membranes of the enterocytes of the small intestine4. However, water soluble phytoconstituent like many polyphenols are poorly absorbed 3 either due to their multiple-ring large size molecules which cannot be absorbed by simple diffusion, or due to their poor miscibility with oils and other lipids, severely limiting their ability to pass across the lipid-rich outer membranes of the enterocytes of the small intestine4.Plant Emblica officinalis Gaertn (commonly known in India as Amla, Syn. Phyllanthus emblica L.; Family: Euphorbiaceae) is available in the Indian market for the treatment of digestion and liver disorders2. Chemically, the presence of vitamin C, tannins viz., gallic acid, ellagic acid, phyllemblic acid and emblicol. In minor the presence of alkaloids viz., phyllantidine and phyllantine; pectin and minerals in the fruit of Emblica officinalis have also been reported.5 Gallic acid (GA, 3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol and its derivatives have been in use in various industries as antioxidant, photographic developer, in tanning and in the testing of free mineral acids, di-hydroxy acetone and alkaloids.6 Gallic acid possesses cytotoxicity against cancer cells7, anti-

Received on 29.03.2012 Accepted on 06.04.2012

Modified on 01.04.2012 RJPT All right reserved

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inflammatory8, antimutagenic9, hepatprotective10, 11 neuroprotective effect , anti-tumor potential12 and analgesic activity13 . It is also used in the pharmaceutical industry as a styptic agent and as a remote astringent in cases of internal hemorrhage. Some ointments to treat psoriasis and external hemorrhoids contain gallic acid.

Acute toxicity studies The acute toxicity (LD50 ) of GAP was evaluated using the oral route. GAP were prepared in distilled water and administered orally at the doses of 0.5, 1, 2, 4, 8 g/kg to 5 groups of 6 mice each. The animals were observed for clinical signs and symptoms of toxicity every 30 min up to 6 h on the first day and thereafter, everyday up to 7 days. Water-soluble phytoconstituent molecules (mainly The mortality occurring in each group was recorded. polyphenols) can be converted into lipid-compatible molecular complexes, which are called Phytosomes. Toxicant Phytosomes are more bioavailable as compared to simple Toxicity was induced by carbon tetrachloride (1.5 ml/kg, herbal extracts owing to their enhanced capacity to cross the i.p.)17. Equal amount of liquid paraffin was administered as lipid rich bio membranes and finally reaching the blood4. So vehicle. the following study was undertaken to evaluate the protective effects of gallic acid (3, 4, 5-trihydroxybenzoic Drug treatment and experimental design acid) and its comparison with gallic acid- phospholipids The rats of all groups except group 1 received CCl4 once complex (GAP) at different doses against CCl4 induced only, intraperitonially in liquid paraffin (1:1, v/v). In this hepatic and renal damage in albino rats. Carbon curative study first toxicant was administered as a bolus tetrachloride, which induces toxicity in rats closely, dose (single administration). After 24 h of toxicant resembles human cirrhosis14. It also induces sub lethal administration the gallic acid and gallic acid-phospholipids proximal tubular injury in the kidney and focal alterations complex was administered as a single dose, orally. The animals were divided into seven groups of six animals each in granular pneumatocytes15. and were treated as follows: Group 1: Normal control (vehicle only). MATERIALS AND METHODS: Group 2: Toxicant (CCl4 1.5 ml/kg, i.p. single Material administration). The phospholipids, hydrogenated soy Phosphatidyl choline (HSPC) was purchased from Lipoid, Ludwigshafen, Group 3: CC14 + silymarin (50 mg/kg b.w.). Germany. Gallic acid was purchased from Sigma (Sigma Group 4: CC14 +GA (100 mg/kg b.w.). Chemical, St. Louis, MO, USA); carbon tetra chloride was Group 5: CC14 +GA (200 mg/kg b.w.). purchased from SRL chemicals, Mumbai, India. Other Group 6: CC14 + GAP (40 mg/kg b.w.). chemical were of analytical grade. Group 7: CC14 + GAP (60 mg/kg b.w.). Preparation of Gallic acid-Phytosomes (GAP) The complex was prepared with phospholipids and gallic acid as a molar ratio of 1:1, 1.5:1, 2:1, 2.5:1and 3:1 respectively. Weight amount of gallic acid and phospholipids were placed in a 100ml round-bottom flask and 50ml of methanol was added as reaction medium. The mixture was refluxed and the reaction temperature of the complex was controlled to 50C for 3 h. The resultant clear mixture was evaporated and 20 ml of n-hexane was added to it with stirring. The precipitated was filtered and dried under vacuum to remove the traces amount of solvents. The dried residues were gathered and placed in desiccators overnight and stored at room temperature in an amber colored glass bottle16. An aqueous suspension was prepared in 2% gum acacia and administered to the animals orally. Animals Wister albino rats and mice of either sex were used for this study. Animals were maintained under uniform husbandry conditions of light (14 L: 10 D), temperature (242 C) and relative humidity (6070%). They were fed on pellet diet and water ad libitum. 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. Experimental protocol was approved by departmental ethical committee (Animal House Registration No 778/03/C/CPCSEA) The animals were sacrificed 24 h after therapy of gallic acid and gallic acid-phospholipids complex. Collection of serum and tissue samples Blood was collected by puncturing the retro-orbital venous sinus (in heparinized tubes). It was allowed to clot and then centrifuged at 3000 rpm for 15 min. The serum samples were collected and left standing at 20 C until required. Tissues (liver and kidney) were excised and transferred into ice cold containers for biochemical estimations. Biochemical evaluation Standard methods were employed for estimation of Estimation of SGPT, SGOT18, total bilirubin19, activity of superoxide dismutase20and catalase (CAT) activity.21 The measurement of lipid peroxidation22 was done by measuring the concentration of thiobarbituric acid reactive substances (TBARS) in liver. The reaction of malondialdehyde (MDA), a degradation product of per oxidized lipids with thiobarbituric acid (TBA) to produce TBA malondialdehyde chromophores has been taken as the index of lipid peroxidation. Estimation of glutathione (GSH) concentration.23

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Table 1.Effect of GA and GAP on various biochemical parameters in toxicity induced rat liver Bilirubin in mg/dl 1.00 0.31 *** 2.78 0.44 0.90 0.13a SGOT in U/l 71.58 4.82* ** 168.31 4.62 71.88 4.93* **a SGPT in U/l 74.38 TBARS in mol/mg 139.16 27.90 *** 446.94 55.84 141.48 31.29 ***a 297.13 47.00 *** 189.30 18.23 ***a 172.97 76.87 ***a 147.67 74.10 ***a Catalase in U/mg 5.15 0.86 *** 1.78 1.05 5.00 1.21 ***a 4.33 1.66 ** a 4.57 0.90 ** a 4.89 1.06 ***a 4.93 0.35 ***a SOD in U/mg 22.89 5.49* ** 4.78 4.51 22.69 6.88* **a 16.91 5.04* *a 6.69* **a 4.54* **a 3.43* **a GSH in mol/mg 11.41 2.46 *** 3.16 2.05 11.08 2.23 ***a 7.81 1.38 ** a 2.59 ***a 1.17 ***a 2.06 ***a

Normal

182.11 Control 72.616 CCl4 + a sily (50 mg/kg) 1.72 0.35 126.16 7.78* 156.83 8.11 CCl4 *** ** *** +GA (100 mg/kg) 1.43 0.18 124.2 2.71* 135.33 10.8 CCl4 ** ** *** +GA (200 mg/ kg) 0.97 0.14 72.96 3.19* 119.16 9.59 CCl4 *** **a *** +GAP (40 mg/ kg) 1.07 0.29 72.7 4.50* 75.733 5.85 CCl4 a *** **a +GAP (60 mg/ kg) *** - p<0.001 Highly significant when compared to Control ** - p<0.01 Significant when compared to Control * - p<0.05 Significant when compared to Control a - Non significant when compared to Normal

4.18 *** 6.72 7.79

20.69

8.76

22.21

10.17

22.39

10.77

Statistical analysis All the data were expressed as mean SD. Statistical analysis by using one-way ANOVA followed by post hoc analysis with Tukey test.

RESULTS:
The LD50 value by the oral route could not be determined as no mortality was observed until a dose of 8 g/kg of gallic acid Phytosomes. In this experiment, on the basis of biochemical evaluation shows in table no. 1, we find that CCl4 induced toxicity has increased the serum Bilirubin, GOT,GPT level to significantly higher level when compared to normal (P<0.001) as the table no. 1 show . The selected gallic acid phytosmes were able to reduce the increased bilirubin, SGOT and SGPT to highly significant level (P<0.001). Silymarin, GAP 60mg and GAP 40 mg when compared to normal, found to be non significant. This shows that bilirubin, SGOT and SGPT level of normal Silymarin, GAP 60mg and GAP 40 mg were similar indicating reversal of liver injury caused by CCl4. Lipid peroxidation, measured in terms of Malondialdehyde (MDA) in rat liver homogenate was significantly increased (P<0.001) in CCl4 group (Control) as compared to Normal group. MDA level of groups treated with gallic acid, gallic acid Phytosomes and Silymarin significantly decreased the MDA content as compared to Control. when compared to Normal, Silymarin, GA 100mg, GA 200mg, GAP40mg and GAP60mg were found to be insignificant (P>0.05). This indicates that liver injury caused by CCl4 was almost reversed by Silymarin, GA 100mg, GA 200mg, GAP 40mg and GAP 60mg.

levels of GA 200mg, GAP 40mg and GAP 60mg were significant to the level of P<0.00, whereas SOD levels of GA 100mg was found less significant with( P<0.01 ) when compared to Control. Silymarin at 50 mg/kg completely restored the enzyme activity (22.69 U/mg proteins) to the normal level. GAP 60 mg restored the normal enzyme level equally significant to the Silymarin. i.e when compared to the Normal level of SOD, both Silymarin and GAP 60 mg were found to be insignificant (P<0.05). This shows that Normal group and groups treated with Silymarin and GAP 60 mg are close to each other. Catalase activity in CCl4 group (Control - 1.78 U/mg protein) was observed to be strikingly lower than the Normal group (5.15 U/mg protein, P<0.001). In case of Silymarin, GAP 60 mg and GAP 40 mg CAT activity when compared to Control was found to be highly significant (P<0.001). GA 100mg and GA 200mg also increased the CAT level when compared to Control but less significantly (P<0.01). Silymarin at 50 mg/kg completely restored the enzyme activity (5.00 U/mg protein) to the normal level. GAP 60 mg also restored the normal enzyme level equally significant to the Silymarin. When compared to the Normal group Silymarin and GAP 60 mg showed no significant difference indicating no difference between Normal, GAP 60 mg and Silymarin.

GSH level in the liver homogenate of Normal and Control group were found to be 11.41 and 3.16 nmol/mg of protein. GAP 60 mg, GAP 40 mg and GA 200 mg were highly significant (P<0.001), Ga 100mg was less significant (P<0.01) when compared to Control. But when compared to Normal GAP 60 mg GAP 40 mg ,GA 200 mg and GA SOD activity in CCl4 treated group (Control - 4.78 U/mg 100mg were found to be insignificant indicating that the protein) was found significantly low when compared with results obtained were very close to Normal. Also, Silymarin the Normal group (22.89 U/mg protein, P<0.001). SOD almost completely restored the glutathione level in CCl4

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treated groups to the normal level. Over all the plant heapatotoxicity in rats, which is known model for both extracts showed hepatoprotective activity in CCl4 induced hepatic GSH depletion and injury. liver toxicity. But among the five plant extracts GAP 60 mg and GAP 40 mg were found to be very potent. The SOD converts superoxide radicals (O2-) into H2O2 plus O2, thus participating in the enzymatic defense against oxygen toxicity. In this study, SOD plays an important role DISCUSSION: The determination of enzyme levels such as SGPT and in the elimination of ROS derived from the peroxidative SGOT is largely used in the assessment of liver damage process of xenobiotics in liver tissues. The observed caused by CCl4 hepatotoxin. Necrosis or membrane damage increase of SOD activity suggests that the all the releases the enzyme into circulation; therefore, it can be Phytosomes that were selected have an efficient protective measured in serum. High levels of SGOT indicate liver mechanism in response to ROS. damage, such as that due to viral hepatitis as well as cardiac infarction and muscle injury. SGPT catalyses the CAT is a key component of the antioxidant defense system. conversion of alanine to pyruvate and glutamate and is Inhibition of these protective mechanisms results in released in a similar manner. GPT or ALT is located in the enhanced sensitivity to free radical induced cellular cytosol of the liver cell. Whereas GOT is located in the damage. Administration of GA 100mg, GA 200mg, GAP cytosol and also found in the mitochondria. Therefore, 40mg and GAP 60mg increased the activities of catalase in SGPT is more specific to the liver, and is thus a better CCl4 induced liver damage in rats to prevent the parameter for detecting liver injury. Our results using the accumulation of excessive free radicals and protects the CCl4-induced hepatotoxicity in the rats demonstrated that liver from CCl4. GA 100mg, GA 200mg, GAP 40mg and GAP 60mg/kg b.wt dose caused significant inhibition of SGPT and SGOT To conclude, our studies have shown that all the selected levels. Serum bilirubin levels on the other hand, are related Phytosomes possess marked hepatoprotective activity with to the function of hepatic cell. Our results demonstrated minimal toxicity and thus have a promising role in the GAP 40mg and GAP 60mg /kg body wt. caused significant treatment of acute hepatic injury induced by Hepatotoxins. inhibition bilirubin levels. Effective control of bilirubin level points towards an early improvement in the secretory REFERENCES: 1. Ward FM and Daly MJ. Hepatic Disease In Clinical Pharmacy mechanism of the hepatic cell. Cells have a number of mechanisms to protect themselves from the toxic effects of ROS. SOD removes superoxide (O2) by converting it to H2O2, which can be rapidly converted to water by CAT and glutathione peroxide (GPx). Lipid peroxidation is an autocatalytic process, which is a common consequence of cell death. This process may cause peroxidative tissue damage in inflammation, cancer and toxicity of xenobiotics and aging In our study, elevation in the levels of end products of lipid peroxidation in liver of rat treated with CCl4 were observed. The increase in MDA level in liver suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with GA 100mg, GA 200mg, GAP 40mg and GAP 60mg significantly reversed these changes. Hence it may be possible that the mechanism of hepatoprotection is due to their antioxidant effect. GSH is widely distributed in cells. GSH is an intracellular reductant and plays a major role in catalysis, metabolism and transport. It protects cells against free radicals, peroxides and other toxic compounds. GSH is a naturally occurring substance that is abundant in many living creatures. It is well known that a deficiency of GSH within living organisms can lead to tissue disorder and injury. For example, liver injury included by consuming alcohol or by taking drugs like acetaminophen, lung injury by smoking and muscle injury by intense physical activity, all are known to be correlated with low tissue levels of GSH. In the present study, we have demonstrated the effectiveness of phytosomes that were selected in CCl4 induced
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