Screening Methods for Hepatoprotective Agents in Experimental Animal’s

 

Nimbalkar V.V.*, Pansare P.M., Nishane B.B.

Department of Pharmacology, P.D.V.V.P.F’s College of Pharmacy, Vilad Ghat, Post: MIDC, Ahmednagar, Maharashtra, India.414111

*Corresponding Author E-mail:  rajevikram@gmail.com

 

ABSTRACT:

Hepatic disease is term for a collection of conditions disease and infection that affect the cells, tissues, structure or functions of the liver.liver has a wide range of function ,including detoxification, protein  synthesis , and production of biochemical necessary for digestion and synthesis as well as breakdown of  small and complex, many of which are necessary for normal vital function .

 

Both in-vitro and in-vivo liver models have been develops in the past year two study the hepatoprotective   agents. These system majors the ability of the test drug to prevent or cure liver toxicity (Induced by hepatotoxin) in experimental animals. In in-vitro model fresh hepatocytes are treated with hepatotoxin and the effect of the test drug on the same in evaluation .In in –vivo models, a toxic dose or repeated doses of a known hepatotoxinare administrated to induce liver damage in experimental animals. The test substance administrated along, with prior to and/ or after the toxin treatment. Various chemical agents normally used to induce hepatotoxicity in experimental animal for the evaluation hepatoprotective agents include carbon tetrachloride, paracetamol, Acryilamide, adrimacyin, alcohol antituburcularetc. These explain the mechanism of action of various hepatotoxic chemical drugs, their doses and routes of administration.

 

KEYWORDS: Silymarin, Paracetamol, Feroussulphate, Ethanol etc.

 

 


INTRODUCTION :( 1)

Herbal medicines have been of tremendous interest since the beginning of human civilization for the treatment of all kinds of ailments. Following the advent of modern medicines herbal preparations suffered a serious setback. During the last couple of decades, however, advances in photochemistry and in identification of plant products-effective against certain diseases-have rekindled the interest. Indeed the plant kingdom has been the therapeutic arsenal of all documented traditional systems of medicine. Liver diseases remain one of the serious health problems. In the absence of reliable liver protective drugs in allopathic medicinal practices, herbs play a role in the management of various liver disorders.

 

Numerous medicinal plants and their formulations are used for liver disorders in ethno medical practices as well as in traditional systems of medicine in India. Liver disease appears to be on the increase. Part of this increase may be due to our frequent contact with chemicals and other environment pollutants. The amount of medicine consumed has increased greatly with resulting dangers to the liver.

 

The liver, the detoxifying factory in the body, has become an increasingly overworked organ. While those who smoke, abuse alcohol and drugs, and live in severely polluted environments are at greatest risk, we all suffer from threat of liver. Reducing the consumption of alcohol, mixing drugs or taking unnecessary drugs and consulting a physician if there are sign or symptoms of liver disease can prevent damage to the liver. Prevention also includes maintaining a balanced diet that includes nutrients and herbs that support a healthy liver. Problems associated with liver dysfunction can ultimately lead to serious illness such as hepatitis, cirrhosis, fatty liver, alcoholic liver disease, and biliary cirrhosis. Millions of Americans are afflicted with liver disease, with over 43,000 deaths each year and hospitalization costs greater than 7 billion dollars.

 

EXPERIMENTAL MODELS:

Carbon tetrachloride induced hepatotoxicity in rats:

Mechanism of action:

CCl4 is widely used experimentally as hepatotoxic which is bio transformed by the cytochrome p-450 system to produce the trichloormethyl free radical which in turn covalently binds to cell membrane and organelles to elicit lipid peroxidation disturbs calcium hemostasis and finally results in death of cell. Lipid peroxidation is a complex and natural deleterious process. The significant increase observed in levels of lipid peroxides in liver of CCL4 intoxicated shows free radical induced liver damage.(2) The hepatotoxic effect of CCl4 results in intense centrilobular necrosis and vacuolization with significant no of swollen hepatocytes which ultimately leads to accumulation of fat in liver and kidney. Fatty degeneration was also observed in centrilobular areas. The AST, ALT and ALP are found higher in concentration in cytoplasm, when the liver cell membrane damaged by CCl4 administration, these enzymes which are normally located in cytosols’ are released into the blood stream. AS Twill be released into the cytosol also by the injury of the organelles such as mitochondria. The activity of these enzymes in serum is useful quantitative marker of the extent and type of hepatocellular damage. The elevation of alkaline phosphatase in serum due to toxic effect of CCl4 is the result of defective excretion of bile by the liver. This ALP activity is related to functioning of hepatocytes. Increase in its activity is due to the increase synthesis in presence of increase biliary pressure. (3) The increased level of serum bilirubin is conventional indicator of liver injury. One of the most important distinguishing features observed in experimental hepatic damage is the pronounced depression in the level of serum total protein. It is evident that CCL4 poisoning leads to cessation of movement of large amount of triglycerides from the liver to the plasma. So the lipid profile of CCl4 intoxicated group showed considerable degree of elevation in the concentration of serum, total lipid, cholesterol and triglycerides. Also the above parameters were increased in the tissue such liver, kidney, heart and lungs.

 

The injury and dysfunction of liver caused by the toxic effect of CCl4 in experimental animals simulated the human viral hepatitis model. In CCl4 induced toxic hepatitis a toxic reactive metabolite trichloro methyl radical was produced by the microsomal oxidase system. These activated radicals bind covalently to macromolecules of the lipid membrane of endoplasmic reticulum and causes peroxidative degradation of lipids. As a result fats from the adipose tissues weretranslocated and accumulated in the liver. The estimation of total bilirubin confirms the intensity of jaundice. In viral and toxic hepatitis the degree of excretion of bilirubin from the intestine is very less and bilirubin present in the liver is excreted into the canaliculated and then Regurgitated into the blood stream. Hence hyperbilirubinemia is most common in hepatitis patients. It is also known that liver synthesizes number of proteins. The change in serum protein level forms the bases for important laboratory aids to diagnose the depth of jaundice. (4) Hepatocellular necrosis leads to very high level of AST and ALT released from the liver in blood. Between the two ALT is better index of liver injury as live ALT activity represents 90% of total enzyme present in the body.

 


 

 

Table No.1. Experimental Protocol (Grouping, Treatment and Observations)

Sr.No

Group (N=6)

Treatment and Dose\Day

Observation

         I.             

Normal control

Control (Distilled water p.o)

1) Biochemical parameter on 8 th Day.

2) Histopathological examination on 8th day.

       II.   

Negative control

CCl4 (0.7ml\kgsc. Alternate days)

     III.             

Positive control

CCl4 (0.7ml\kgsc. Alternate days  )+ Siymarin (100mg\kg) p.o from day 1to day 7 o.d

     IV.             

Test Drug (25 mg/kg).

CCl4 (0.7ml\kg sc. Alternate days) + Test Drugp.o from day 1 to day 7 o.d

       V.             

Test Drug (50 mg/kg).

CCl4 (0.7ml\kg sc. Alternate days) + Test Drugp.o from day 1 to day 7 o.d

     VI.             

Test Drug (100mg/kg)

CCl4 (0.7ml\kg sc. Alternate days) + Test drug (100 ml\kg) p.o from day 1 to day 7 o.d

 


Paracetamol induced hepatotoxicity in rats:

Mechanism of action:

Paracetamol (N-acetyl p-amino phenol or acetaminophen) is well known antipyretic and analgesic which produces hepatic necrosis at higher doses. Indiscriminate ingestion can lead to accidental poisoning and potentially lethal hepatotoxicity. Paracetamol is mainly metabolizes by glucoronide and sulphate conjugation. A small amount is metabolized by the cytochrome P-450 system toa toxic metabolite. The cell is normally protected from injury by conjugation of this toxic metabolite with glutathione. As a dose increases the glutathione content of hepatocytes available for detoxification of the toxic metabolite is exhausted and the hepatocytes become vulnerable to the noxious effects of the metabolite resulting in liver cell necrosis.(5) Liver is the organ highly affected primarily by toxic agents. Paracetamol produces wide areas of frank necrosis of liver parenchyma and a picture of dilated vasculature and sinusoids around the necrotic zones.(6) Paracetamol produces hepatic necrosis in high doses by covalent binding of its toxic metabolite N-acetyl-p-benzoquinone imine to sulphadryl groups of protein resulting in cell necrosis through lipid peroxidation induced by decreasing glutathione in the liver. Damage to the structural integrity of liver is reflected by an increasing in levels of serum transaminases because they are cytoplasmic in location and are release into the circulation after cellular damage. Free radicals cause damage in biological systems this in turn cause cellular damage that may lead to cancer, liver injury, heart disease etc.(7) the toxicity is medicated by a metabolite imidazole which binds covalently in endoplasmic and in protein of cytoplasm.

 

 

Fig. 1. Mode of action of hepatotoxicity caused by paracetamol


 

 

Table No.2. Experimental Protocol (Grouping, Treatment and Observations)

Sr.no

Group (N=6)

Treatment and Dose/Day

Observation

         I.             

Normal control

Control (distilled water p.o

1)Biochemical parameter on 3rd and 14th day.

2)Function parameter on 14th day.

3)Morphological parameter on 14th day.

4)Histopathological examination on 14th day.

       II.             

Negative control

Paracetmol (1g/kg) p.o till day 8

     III.             

Positive control

Paracetmol (1g/kg) p.o till day 8 +Silymarin (100mg/kg) p.o from day 4 to day 13 o.d

     IV.             

Test Drug (25 mg/ml)

Paracetmol (1g/kg) p.o till day 8 + Test Drug p. o from day to day 13 o.d

 

 

 


Ferrous sulphate induced hepatotoxicity in rats:

Mechanism of action:

Iron toxicity results when too much iron is injected or less often when too much is given orally. Iron overload is associated with liver damage, characterized by massive Iron deposition in hepatic parenchymal cells, leading fibrosis and eventually, to Hepatic necrosis. (8) Lipid peroxidation (LPO) had been proposed to be the major factor in iron Toxicity, including iron induced hepatotoxicity.

 

A ferrous salt reacts with hydrogen peroxide derived by the action of the Superoxide anion radical, to form the highly reactive radical hydroxyl (Fenton Reaction) hydroxyl ion attacks all biological molecules, including cell membrane Lipids, to initiate LPO. The highly toxic per oxidative metabolite induces widespread cellular injury .and the leakage of cellular enzymes into the Bloodstream, results in the augmented damage and dysfunction.(9) Histologicallythe iron produced perioral necrosis.


 

Fig.2Mode of action ferrous sulphate induced hepatoxicity

 

 


 


Table No.3. Experimental Protocol (Grouping, Treatment and Observations)

Sr.no

Group

( N=6)

Treatment and Dose/Day

Observation

      I.             

Normal control

Control (distilled water p.o)

1) Biochemical parameter on 10th day.

2) Histopathological examination on 10th day.

    II.             

Negative control

Control (distilled water p.o) for 9 days )+Ferroussulphate (30mg/kg) i.p. on day 10

  III.             

Positive control

Ferroussulphate (30mg/kg) i.p. on day 10+ Silymarin (100mg/kg) p.o from day1 to day 9o.d

  IV.             

Test Drug

(25 mg/kg)

Ferroussulphate (30mg/kg) i.p. on day 10+ Test Drugp.o from day 1 to day 9 o.d

 

 

 


Ethanol induced hepatotoxicity in rat:

Mechanism of action:

Liver being the major site for detoxification is the primary target for environmental or occupational toxic exposure. The alcoholic liver injury appears to be generated by the effects of ethanol metabolism and toxic effect of acetaldehyde, which may be mediated by acetaldehyde, altered protein. (10)

 

The oxidation of ethanol via the alcohol dehydrogenase pathway results in the production of acetaldehyde with loss of hydrogen ions. NAD (Nicotinamide Adenine dinucleotide) is reduced to NADH. The large amounts of reducing equivalents generated the hepatocytes ability to maintain redox homeostasis and number of disorders as hyper uremia, hyper lipemia, and rise in HDL. The rise in NADH promotes fatty acid synthesis as a net result hepatic fat accumulation. An ethanol inducible form of cytochrome P-450 called 2E1 not only catalyzes ethanol oxidation but also capable of activating various other compounds to highly toxic metabolites. The proliferation of endoplasmic reticulum associated with P-450 E1induction is also accompanied by enhance activity of other cytochrome P-450s, resulting in accelerated metabolism and tolerance to other drugs as well as increase degradation of retinal and its hepatic depletion. Also chronic ethanol consumption results in hepatic vitamin A depletion. Ethanol oxidation, whether by the ADH or the microsomal path way resulting acetaldehyde, which may cause ubiquitous damage including in the mitochondria. Chronic ethanol treatment causes 10-fold increase in splanchnic acetaldehyde release in the hepatic vein, associated with a raking leakage of mitochondrial enzyme glutamic dehydrogenase in to the hepatic venous blood.

 

One mechanism of hepatotoxicity of acetaldehyde is its high chemical reactivityAnd formation of adducts with various protein

 

Fig.3.Interaction of direct toxicity of ethanol.

 

Acetaldehyde binding shown to impair microtubule polymerization and hepatic protein secretion together with an increase in constituent protein resulted in protein accumulation and hepatocytes swelling explaining the ballooning of the hepatocytes and the hepatomegaly, to characteristic features of alcohol induced liver injury. Acetaldehyde also contributes the depletion of glutathione and its potentiation of lipid peroxidation. After chronic alcohol consumption the fibroblasts identified in normal liver proliferates and stellate cells (also called lymphocytes or fat storing cells) where found to be transformed or activated to “transitional” my fibroblast like cells associated with active fibro genesis. Chronic alcohol intake is known to produce hypercholesterolemia, hyperlipidemia, and hypertriglyceridemia, (11)in chronic lipid accumulation the liver cells become fibrotic and lead to impaired liver function. Enhanced lipid peroxidation had been reported in hyperlipidemia induced by ethanol. Ethanol increases triglycerides and cholesterol levels thus inducing imbalance in lipid metabolism in liver, heart, kidney and other organs and this could explain the reason for the increase in lipid peroxidation in these organs. It has been proven that hyperlipidemia and elevated lipid peroxidation are interrelated. Increase in serum triglycerides in alcohol treated rats may be due to decrease activity of lipoprotein lipase, which is involved, in the uptake of triglycerides rich lipoprotein by extra hepatic tissue. Increase the synthesis or decreased lipid deposition or both resulted in simultaneous accumulation of lipids in blood and liver. Ethanol induces hyperlipidemia and hyperlipidemia enhances lipid peroxidation causing hepatotoxicity by increasing free radical formation which in turn increases the level of lipid peroxides in hepatic tissue. Glutathione protects the hepatocytes by combining with the reactive metabolites and preventing their covalent binding to liver protein. Liver glutathione after alcohol administration was found to decrease due to increase \ utilization by the hepatocytes because GSH seems to act as scavengers for toxic chemical agents. The non-availability of glutathione decreases the activity of glutathione peroxidase and glutathione transferees. Glutathione act as substrate for both GSH-Px and GST. Depletion of glutathione will render the enzymes (GSH-Px and GST) in active or less active.


 

 

 


Fig.3 Hepatic, nutritional and metabolic abnormalities after ethanol abuse.


 

Ethanol is currently recognized as the most prevalent known cause of abnormal human development. Chronic alcohol intake is known to produce hepatocellular damage. Recently it was reported that high dose of ethanol impair hepatic microcirculation by producing endothelin-1. Ethanol induced hepatic hypoxia also been involved as a possible cause of the potentiation of hepatotoxicity. (12) Chronic ethanol ingestion produces fatty liver, hepatomegaly, alcoholic hepatitis, Fibrosis and cirrhosis. Indeed, steatosis due to ethanol exposure, which was localized in cetrilobuler areas in males and pent lobular in females. Ethanol elevates the serum AST levels and steatosis, inflammation, necrosis assessed histologically develop more rapidly and more severe in females than males. One mechanism of ethanol hepatotoxicity is free radical formation. Four week of eternal ethanol treatment increases plasma endotoxin level in portal vein significantly.

 

 


 

Table No.4.Experimental Protocol (Grouping, Treatment andObservations)

Sr.No

Group (N=6)

Treatment and Dose\day.

Observation

1

Normal control

1% Gum acacia in water

1) Biochemical parameter on 22 day.

2) Morphological parameter on 22 day.

3) Histopathological examination on 22 day.

2

Negative control

CML 3ml\100gm bd.wt/day in two divided doses for 21 days.

3

Positive control

CML 3ml\100gm bd.wt/day in two divided doses for 21 days. +Silymarin (100mg/kg) p.o for 21 days o.d

4

Test Drug) (25 mg/kg)

CML 3ml\100gm bd.wt./day in two divided doses for 21 days + Test Drugp.o for 21 days o.d

 

 


D-galactosamine induced hepatotoxicity in mice:

Mechanism of action:

The histopathology of the liver gives the evidence for the protection imparted by the herbal mixture and Silymarin. The metabolites of -Dgalactosamine (GaIN), uridiophosphogalactos amine may deplete several uracil nucleotides such as UDP-lactose, UDP-glucose and UTP, causing reduction of mRNA and glycoprotein synthesis (i.e. reduction of ATP and glycogen synthesis), which leads to cellular membranes alteration. (13) Nevertheless there is increasing evidence that GaIN causes production of free hydroxyl radical leading to lipid peroxidation. Also the levels of SOD, CAT, GPx are also concomitantly reduced. (14)Ultimately the cellular damage and the inflammation caused by GaIN are similar to the histopathological features of viral hepatitis in humans. (15) This phenomenon may lead to cellular damage and cellular inflammation resulting in histological and biochemical picture closely resembling viral hepatitis. The Ca++homeostasis perturbation, inhibition of oxidative of NADPH and FADH substrate at the dehydrogenase co enzyme level(16) are also considered to be responsible for pathogenesis of GaIN induced hepatitis. PS is co administered with GaIN by certain investigators. These leads to the release of TNF from the macrophages and Kuffer cells. This cytokine has been firmly implicated as an important causative mediator in the pathogenesis of alcoholic liver diseases and hepatitis. Nevertheless TNF too has positive roles it is responsible for the normal proliferation of the hepatocytes, but in pathological condition it acts as panoptic agent.

 

Gain caused hepatitis can be recovered by;

1      Protein synthesis enhancement

2      Galactosamine liver uptake inhibition

3      RES activation

4      Renormalization of the changed Ca++homeostasis

 


 

 

Table No 5.  Experimental Protocol (Grouping, Treatment and Observations)

Sr.No

Group (N=6)

Treatment and Dose\day.

Observations

1

Normal control

Control (distilled water p.o)

1)Biochemical parameter on 2th and 9th day.

2) Histopathological examination on 9th day.

2

Negative control

D-galactosamine (800mg/kg) i.p on day 1 o.d +Distilled Water p.o form day 2 to day 8.

3

Positive control

D-galactosamine (800mg/kg) i.p on day 1 o.d + Livfit(50mg/kg) p.o. form day 2 to day 8 o.d.

4

Test Drug (25 mg/kg)

D-galactosamine (800mg/kg) i.p on day 1 o.d + Test Drug form day 2 to day 8 o.d.

 


Doxorubicin induced hepatotoxicity in mice.

Mechanism of action:

The animals treated with doxorubicin resulted in a significant hepatic damage. (17) Doxorubicin drastically decreases the levels of Cytochrome P-450 in liver. Deficiency in these drug metabolizing reactions often results in slower clearance and potentially deleterious side effects and toxicities related to accumulation in the body. So the decrease in xenobiotic metabolizing proteins could have resulted into slow clearance of doxorubicin and exacerbation on normal cells.

 

Doxorubicin can stimulate NADPH dependent microsomal lipid peroxidation apparently by generating reactive species Superoxide ions, hydroxyl radical and electronically excited singlet oxygenic believed to initiate and propagate peroxidation of unsaturated membrane lipids. The reduced oxygen species, particular OH- (hydroxyl) radical interacts with lipids, proteins and nucleic acids resulting in loss of membrane integrity, structural or functional changes in proteins and genetic mutation Carbon tetrachloride induced hepatotoxicity in rats. (18)

 


 

 

 

Table No.5. Experimental Protocol (Grouping, Treatment and Observations)

Sr.No

Group (N=6)

Treatment and Dose\day.

Observations

            I.             

Normal control

Control (distilled water p.o

1)Biochemical parameter on 11th day.

 

 

2) Histopathological examination on 11th day.

          II.             

Negative control

Doxorubicin (2mg/kg) i.p on day 1,3,5,7  o.d.

        III.             

Positive control

Doxorubicin (2mg/kg) i.p on day 1,3,5,7  o.d. +Silymarin (100mg/kg) p.o from day2 till  day 10 o.d

        IV.             

Test Drug (25 mg/kg)

Doxorubicin (2mg/kg) i.p on day 1,3,5,7 o.d. + Test Drugp.o. form day 2 to day 8 o.d

 


Ethynyl estradiol induced cholestasis in rats

Mechanism of action:

The formation of bile is a vital function, and its impairment by drugs or infectious, autoimmune, metabolic, or genetic disorders results in the syndrome commonly known as cholestasis. (19) The secretion of bile normally depends on the function of a number of membrane transport systems in hepatocytes and bile-duct epithelial cells (cholangiocytes) and on the structural and functional integrity of the bile-secretory apparatus. This review summarizes the molecular defects in hepatocellular membrane transporters that are associated with various forms of cholestasis liver disease inhumans.Several animal models of intrahepatic and obstructive cholestasis simulate human cholestasis diseases. These disorders include sepsis-induced cholestasis (in endotoxin-treated rats), oral-contraceptive –induced cholestasis and cholestasis of pregnancy (in ethyl estradiol–treated rats), and extra hepatic biliary obstruction induced by ligation of the common bile duct. The cholestatic effects of endotoxin and endotoxin-induced cytokines not only have a role in the pathogenesis of sepsis-induced cholestasis, but also may explain defects inhepatobiliary excretory function during total parenteral nutrition and in alcoholic and viral hepatitis.(20)Many drugs (e.g., cyclosporine A and chlorpromazine) also cause intrahepatic cholestasis at the level of the bilecanaliculus in both humans and animals despite their different causes, each of these diseases results in marked functional impairment of hepatocellular uptake and canalicular excretion of bile salts and various other organic anions. (21) Cholestasis results from impaired transport of these compounds into bile and the loss of osmotic driving forces for bile secretion.

 


 

 

 

Table No.6. Experimental Protocol (Grouping, Treatment and Observations)

Sr.No

Group (N=6)

Treatment and Dose\day.

Observations

         I.             

Normal control

Control (distilled water p.o)

1) Biochemical parameter on 14th day.

 

2) Function parameter on 14th day.

 

3) Histopathological examination on 14th day.

       II.             

Negative control

Ethynyl estradiol 5mg/kg) from day 1 to day 5 o.d.

     III.             

Positive control

Ethynyl estradiol 5mg/kg) from day 1 to day 5 o.d. Picroliv (12mg/kg) p.o. form day 6 till day 13 o.d.

     IV.             

Test Drug (25 mg/kg)

Ethynyl estradiol 5mg/kg) from day 1 to day 5 o.d+ Test Drugp.o. form  day 6 till  day 13 o.d.

 

 

 


SUMMARY AND CONCLUSION:

Summary:

The present study demonstrated that Test drug showed significant activity in all the model of hepatotoxicity employed for the study. The activity of Test drugs when compared with standard like Silymarin, Livfit and Picroliv showed similar or increased hepatoprotective and regenerative activity. In models which cause hepatotoxicity by generation of free radicals like incase of carbon tetrachloride toxicity which causes the loss of cell membrane integrity or in case of the heavy metal toxicity like iron overload which generates free radicals which causes the lipid peroxidation of the cell membrane, the Test drugs showed better results than that of Silymarin.

 

In the model which causes the hepatotoxicity by fatty accumulation like incase of the alcohol, the herbal mixture showed better results than that of Silymarin.

 

In drug induced hepatotoxicity like in case of paracetamol and doxorubicin whose toxic metabolite causes the lipid peroxidation, the Test drugs  showed similar effects to that of Silymarin. While in case drugs that causes cholestasis by impaired membrane transport system of bile like in case of ethynyl estradiol, the effects shown by Test drugs were better as compared to that of Picroliv. In viral hepatitis resembling model i.e. in case of D-galactosamine induced hepatotoxicity which cause the reduction in the ATP and glycogen synthesis

 

CONCLUSION:

Taking in to consideration the results obtained in the present investigation, it can be concluded that Test Drug has a definite hepatoprotective and regenerative activity; hence it could be used in the treatment of liver disorders like liver dysfunction, hepatomegaly, viral hepatitis and various alcoholic live disorders.

 

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Received on 07.09.2015             Modified on 25.09.2015

Accepted on 28.09.2015           © RJPT All right reserved

Research J. Pharm. and Tech. 8(12): Dec., 2015; Page 1725-1732

DOI: 10.5958/0974-360X.2015.00310.8