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

1Bhagwant University, Ajmer, Rajasthan, India

2JSS College of Pharmacy, JSS University, Mysore, Karnataka, India

3Sri Balaji College of Pharmacy, Jaipur, Rajasthan, India

*Corresponding Author E-mail: radhey4183@rediffmail.com



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. Water-soluble 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



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-inflammatory8, antimutagenic9, hepatprotective10, neuroprotective effect11, 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.


Water-soluble phytoconstituent molecules (mainly polyphenols) can be converted into lipid-compatible molecular complexes, which are called Phytosomes. Phytosomes are more bioavailable as compared to simple herbal extracts owing to their enhanced capacity to cross the lipid rich bio membranes and finally reaching the blood4. So the following study was undertaken to evaluate the protective effects of gallic acid (3, 4, 5-trihydroxybenzoic acid) and its comparison with gallic acid- phospholipids complex (GAP) at different doses against CCl4 induced hepatic and renal damage in albino rats. Carbon tetrachloride, which induces toxicity in rats closely, resembles human cirrhosis14. It also induces sub lethal proximal tubular injury in the kidney and focal alterations in granular pneumatocytes15.




The phospholipids, hydrogenated soy Phosphatidyl choline (HSPC) was purchased from Lipoid, Ludwigshafen, Germany. Gallic acid was purchased from Sigma (Sigma Chemical, St. Louis, MO, USA); carbon tetra chloride was purchased from SRL chemicals, Mumbai, India. Other chemical were of analytical grade.


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 50°C 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.



 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 (24±2 ◦C) and relative humidity (60–70%). 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)

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. The mortality occurring in each group was recorded.



Toxicity was induced by carbon tetrachloride (1.5 ml/kg, i.p.)17. Equal amount of liquid paraffin was administered as vehicle.


Drug treatment and experimental design

The rats of all groups except group 1 received CCl4 once only, intraperitonially in liquid paraffin (1:1, v/v). In this curative study first toxicant was administered as a bolus dose (single administration). After 24 h of toxicant administration the gallic acid and gallic acid-phospholipids complex was administered as a single dose, orally. The animals were divided into seven groups of six animals each and were treated as follows:

     Group 1: Normal control (vehicle only).

     Group 2: Toxicant (CCl4 1.5 ml/kg, i.p. single administration).

     Group 3: CC14 + silymarin (50 mg/kg b.w.).

     Group 4: CC14 +GA (100 mg/kg b.w.).

     Group 5: CC14 +GA (200 mg/kg b.w.).

     Group 6: CC14 + GAP (40 mg/kg b.w.).

     Group 7: CC14 + GAP (60 mg/kg b.w.).


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


Table 1.Effect of GA and GAP on various biochemical parameters in toxicity induced rat liver



in mg/dl


in U/l


in U/l


in mol/mg


in U/mg


in U/mg


in mol/mg































CCl4 + sily (50 mg/kg)






±7.79 a









CCl4 +GA (100 mg/kg)










±1.66** a


±5.04** a


±1.38** a

CCl4 +GA (200 mg/ kg)










±0.90** a





CCl4 +GAP (40 mg/ kg)















CCl4 +GAP (60 mg/ kg)






±5.85 a









*** - 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


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.



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.


SOD activity in CCl4 treated group (Control - 4.78 U/mg protein) was found significantly low when compared with the Normal group (22.89 U/mg protein, P<0.001). SOD 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 100mg were found to be insignificant indicating that the results obtained were very close to Normal. Also, Silymarin almost completely restored the glutathione level in CCl4 treated groups to the normal level. Over all the plant extracts showed hepatoprotective activity in CCl4 induced liver toxicity. But among the five plant extracts GAP 60 mg and GAP 40 mg were found to be very potent.



The determination of enzyme levels such as SGPT and SGOT is largely used in the assessment of liver damage caused by CCl4 hepatotoxin. Necrosis or membrane damage releases the enzyme into circulation; therefore, it can be measured in serum. High levels of SGOT indicate liver damage, such as that due to viral hepatitis as well as cardiac infarction and muscle injury. SGPT catalyses the conversion of alanine to pyruvate and glutamate and is released in a similar manner. GPT or ALT is located in the cytosol of the liver cell.   Whereas GOT is located in the cytosol and also found in the mitochondria. Therefore, SGPT is more specific to the liver, and is thus a better parameter for detecting liver injury. Our results using the CCl4-induced hepatotoxicity in the rats demonstrated that GA 100mg, GA 200mg, GAP 40mg and GAP 60mg/kg b.wt dose caused significant inhibition of SGPT and SGOT levels.  Serum bilirubin levels on the other hand, are related to the function of hepatic cell. Our results demonstrated GAP 40mg and GAP 60mg /kg body wt. caused significant inhibition bilirubin levels. Effective control of bilirubin level points towards an early improvement in the secretory 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 heapatotoxicity in rats, which is known model for both hepatic GSH depletion and injury.


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 in the elimination of ROS derived from the peroxidative process of xenobiotics in liver tissues. The observed increase of SOD activity suggests that the all the Phytosomes that were selected have an efficient protective mechanism in response to ROS.


CAT is a key component of the antioxidant defense system. Inhibition of these protective mechanisms results in enhanced sensitivity to free radical induced cellular damage. Administration of GA 100mg, GA 200mg, GAP 40mg and GAP 60mg increased the activities of catalase in CCl4 induced liver damage in rats to prevent the accumulation of excessive free radicals and protects the liver from CCl4.


To conclude, our studies have shown that all the selected Phytosomes possess marked hepatoprotective activity with minimal toxicity and thus have a promising role in the treatment of acute hepatic injury induced by Hepatotoxins.



1.     Ward FM and Daly MJ. Hepatic Disease In Clinical Pharmacy and Therapeutics. Churchill Livingstone, New York.1999.

2.     Kirtikar KR and Basu BD. Indian Medicinal Plants. Lalit Mohan Basu, India.1993.

3.     Manach C, Scalbert A, and Morand C. Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 79; 2004: 727-47.

4.     Bombardelli E, Curri SB and Della R. Complexes between phospholipids and vegetal derivatives of biological interest. Fitoterapia. 60; 1989:1-9.

5.     Indian Herbal Pharmacopoeia .Revised new edition, Indian Drug Manufacturers

association, Mumbai.2002.

6.     Madsen HL and Bertelsen G. Spices as antioxidants. Trends in Food Science and Technology. 1995.

7.     Gali HU et al. Antitumor-promoting activities of hydrolysable tannins in mouse skin. Carcinogenesis.13; 1992:715-18.

8.     Stich HF, Rosin MP and Brison L. Inhibition of mutagenicity of a model nitrosation reaction by naturally occurring phenolics, coffee and tea. Mutation Research.95; 1982: 119-28.

9.     Ohno Y et all. Induction of apoptosis by gallic acid in lung cancer cells. Anticancer Drugs.10;1999: 845–851.

10.   Anjana J et al. Protective effect of Terminalia belerica Roxb. and gallic acid against carbon tetrachloride induced damage in albino rats. Journal of Ethnopharmacology.109; 2007: 214–218.

11.   Zhongbing L and Guangjun N. Structure–activity relationship analysis of antioxidant ability and neuroprotective effect of gallic acid derivatives. Neurochemistry International. 48;2006: 263–274.

12.   Chiara D et al. Anti-tumour potential of a gallic acid-containing phenolic fraction from Oenothera biennis. Cancer Letters.26; 2005: 17–25.

13.   Krogh R and Yunes R. Structure–activity relationships for the analgesic activity of gallic acid derivatives. Farmaco.55; 2000: 730–735.

14.   Shabanah Al et al. Protective effect of aminoguanidine, a nitric oxide synthetase inhibiter against CCl4 induced hepatotoxicity in mice. Life Sciences. 66; 2000: 265– 270.

15.   Rajesh MG and Latha MS. Preliminary evaluation of the antihepatotoxic effect of Kamilari, a polyherbal formulation. Journal of Ethnopharmacology. 91; 2004: 99–104.

16.   Kuntal M et al. Curcumin–phospholipid complex: Preparation, therapeutic evaluation and pharmacokinetic study in rats. International Journal of Pharmaceutics.330; 2007: 155–163.

17.   Janbaz KH and Gilani AH. Evaluation of protective potential of Artemisia maritime extract on acetaminophen and CCl4 induced liver damage. Journal of Ethnopharmacology. 47;1995: 43–47.

18.   Moudgil  KD and Narang  B S. The liver and biliary system. In: Textbook of Biochemistry and Human Biology. Prentice-Hall of India. Private Ltd. 1989.

19.   Malloy HJ and Evelyn KA. The determination of bilirubin with the photoelectric colorimeter. J Biol Chem . 122(3); 1937: 597-603.

20.   Misra HP and Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem.247; 1972: 3170-75.

21.   Fiske CH and Subbarow Y. The colorimetric determination of phosphates. Journal of Biological Chemistry. 66;1925: 375–400.

22.   Sharma SK and Krishnamurthy CR. Production of lipid peroxides of brain. Journal of Neurochemistry. 15;1968: 147–149.

23.   Moran MA et al. Levels of glutathione, glutathione reductase, glutathione-S-transferase activities in rat liver. Biochimica Biophysica  Acta. 582; 1979:67-68.





Received on 29.03.2012          Modified on 01.04.2012

Accepted on 06.04.2012         © RJPT All right reserved

Research J. Pharm. and Tech. 5(5): May2012; Page 677-681