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Research J. Pharm. and Tech. 5(5): May2012
677
ISSN 0974-3618 www.rjptonline.org
RESEARCH ARTICLE
Hepatoprotective effect of Gallic acid and Gallic acid Phytosome against
Carbon Tetrachloride induced damage in albino rats
Radhey Shyam Kuamwat
1*, K. Mruthunjaya
2, Manish Kumar Gupta
3
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: [email protected]
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. 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
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.
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
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-
Research J. Pharm. and Tech. 5(5): May2012
678
inflammatory8, antimutagenic
9, hepatprotective
10,
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
.
MATERIALS AND METHODS: Material
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.
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 (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.
Toxicant
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 dismutase20
and 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
Research J. Pharm. and Tech. 5(5): May2012
679
Table 1.Effect of GA and GAP on various biochemical parameters in toxicity induced rat liver
Bilirubin
in mg/dl
SGOT
in U/l
SGPT
in U/l
TBARS
in mol/mg
Catalase
in U/mg
SOD
in U/mg
GSH
in mol/mg
Normal 1.00 ±0.31
***
71.58 ±4.82*
**
74.38 ±4.18
***
139.16 ±27.90
***
5.15 ±0.86
***
22.89 ±5.49*
**
11.41 ±2.46
***
Control 2.78 ±0.44 168.31 ±4.62 182.11 ±6.72 446.94 ±55.84 1.78 ±1.05 4.78 ±4.51 3.16 ±2.05
CCl4 +
sily (50
mg/kg)
0.90 ±0.13a 71.88 ±4.93*
**a
72.616 ±7.79
a
141.48 ±31.29
***a
5.00 ±1.21
***a
22.69 ±6.88*
**a
11.08 ±2.23
***a
CCl4
+GA (100
mg/kg)
1.72 ±0.35
***
126.16 ±7.78*
**
156.83 ±8.11
***
297.13 ±47.00
***
4.33 ±1.66
** a
16.91 ±5.04*
* a
7.81 ±1.38
** a
CCl4
+GA (200
mg/ kg)
1.43 ±0.18
**
124.2 ±2.71*
**
135.33 ±10.8
***
189.30 ±18.23
***a
4.57 ±0.90
** a
20.69 ±6.69*
**a
8.76 ±2.59
***a
CCl4
+GAP (40
mg/ kg)
0.97 ±0.14
***
72.96 ±3.19*
**a
119.16 ±9.59
***
172.97 ±76.87
***a
4.89 ±1.06
***a
22.21 ±4.54*
**a
10.17 ±1.17
***a
CCl4
+GAP (60
mg/ kg)
1.07 ±0.29
***
72.7 ±4.50*
**a
75.733 ±5.85
a
147.67 ±74.10
***a
4.93 ±0.35
***a
22.39 ±3.43*
**a
10.77 ±2.06
***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.
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.
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
Research J. Pharm. and Tech. 5(5): May2012
680
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.
DISCUSSION: 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.
REFERENCES: 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.
Research J. Pharm. and Tech. 5(5): May2012
681
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.
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