Screening Methods for Hepatoprotective Agents

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Chapter: Pharmacognosy and Phytochemistry : Biological Screening of Herbal Drugs

A toxic or repeated dose of a known hepatotoxin is admin-istered to induce liver damage in experimental animals. The test substance is administered before or after the toxin treatment. If the hepatotoxicity is prevented or reduced, the test substance is effective. There are various models of inducing hepatotoxicity in rodents (rats and mice).


SCREENING METHODS FOR HEPATOPROTECTIVE AGENTS

 

 

A toxic or repeated dose of a known hepatotoxin is admin-istered to induce liver damage in experimental animals. The test substance is administered before or after the toxin treatment. If the hepatotoxicity is prevented or reduced, the test substance is effective. There are various models of inducing hepatotoxicity in rodents (rats and mice).

 

Hepatitis in Long-Evans Cinnamon Rats

 

The Long-Evans Cinnamon strain of rats has been recom-mended as a useful model to study genetically transmitted hepatitis and chronic liver disease. It has been speculated that this strain of rats is prone to liver diseases due to excessive copper accumulation in the liver.

 

Long-Evans Cinnamon rats are housed in temperature and humidity controlled rooms at a 12:12 light/dark cycle. Groups of 6–10 rats are given different diets based on a 15% purified egg protein diet and supplemented with vitamins or drugs. Drugs are applied via mini-pumps intraperito-neally implanted under ether anaesthesia. The occurrence of jaundice is easily observable as the time when the ears and tail turn yellow and the urine becomes bright orange, staining the fur in the lower abdominal region. Usually, the jaundice progressively worsens, ending in death of the animal within about a week. Incidence of jaundice and mortality versus time are used as parameters to measure the extent of hepatoprotective activity.

 

Allyl Alcohol-induced Liver Necrosis in Rats

 

In this method allyl alcohol is used as a liver necrosis-inducing agent in rats.

 

Albino rats weighing 120–150 g are used. On the first day, food but not water is withdrawn. After 6 h, the com-pounds to be tested for protective activity are administered i.p or orally. After 1 h, the animals are dosed orally with 0.4 ml/kg of a 1.25% solution of allyl alcohol in water. Next morning, the treatment with the potentially protective drugs is repeated. Food but not water is withheld until the third day. Next morning, the animals are sacrificed and the liver is removed. The parietal sides of the liver are checked using a stereomicroscope with 25 times magnifica-tion. Focal necrosis is observed as white-green or yellowish hemorrhagic areas clearly separated from unaffected tissue. The diameter of the necrotic areas is determined using an ocularmicrometer. These values are added for each animal to obtain an index for necrosis.

 

Carbon Tetrachloride-induced Liver Fibrosis in Rats

 

Chronic administration of carbon tetrachloride to rats induces severe disturbances of hepatic function together with histologically observable liver fibrosis. This model is used for the screening of hepatoprotective agents.

 

Albino rats are treated orally twice a week with 1 mg/ kg carbon tetrachloride, dissolved in olive oil 1:1, over a period of 8 weeks. The animals are kept under standard conditions (day/night rhythm: 8:00 AM to 8:00 PM; 22°C room temperature; standard diet; water ad libitum). Twenty animals serve as controls receiving only olive oil; 40–60 animals receive only the carbon tetrachloride. Groups of 20 rats receive in addition to carbon tetrachloride, the com-pound under investigation in various doses by gavage twice daily (with the exception of the weekends, when only one dose is given) on the basis of the actual body weight. The animals are weighed weekly. At the end of the experiment (8 weeks), the animals are anaesthetized and exsanguinated through the caval vein.

 

The serum is analysed for parameters like total bilirubin, total bile acids, 7 S fragment of type IV collagen, procollagen III N-peptide. The liver, kidney, aortic wall, and tail tendons are prepared for determination of hydroxyproline. They are weighed and completely hydrolysed in 6 N HCl. Hydroxyproline is measured by HPLC and expressed as mg/mg wet weight of the organs.

 

For histological analysis, three to five pieces of the liver weighing about 1 g are fixed in formalin and Carnoy solution.

 

Three to five sections of each liver are embedded, cut, and stained with azocarmine aniline blue (AZAN) and evaluated for the development of fibrosis using a score of 0–IV.

 


 

The values of all the parameters of the test group are compared with the control group using suitable statistical methods.

 

Bile Duct Ligation-induced Liver Fibrosis in Rats

 

Ligation of the bile duct in rats induces liver fibrosis, which can be evaluated histologically and by determination of serum collagen parameters. This model is used for the screening of hepatoprotective agents.

 

Albino rats are anaesthetised and laparotomy is performed under aseptic conditions. A midline incision in the abdomen is made from the xiphosternum to the pubis, exposing the muscle layers and the linea alba, which is then incised over a length corresponding to the skin incision. The edge of the liver is then raised and the duodenum pulled down to expose the common bile duct, which pursues an almost straight course of about 3 cm from the hilum of the liver to its opening into the duodenum. There is no gall bladder, and the duct is embedded for the greater part of its length in the pancreas, which opens into it by numerous small ducts. A blunt aneurysm needle is passed under the part of the duct selected, the pancreas is stripped away with care, and the duct is double ligatured with cotton thread.

 

The peritoneum and the muscle layers as well as the skin wound are closed with cotton stitches. The animals receive normal diet and water ad libitum throughout the experiment. Groups of 5–10 animals receive the test com-pound in various doses or the vehicle twice daily for 6 weeks. They are then sacrificed and the blood harvested for determination of bile acids. 7 S fragment of type IV collagen, and procoilagen III N-peptide. The liver is used for histological studies and for hydroxyproline determinations. Control animals show excessive bile duct proliferation as well as formation of fibrous septa. This is consistent with complete biliary cirrhosis. The value of the test is compared with the control using suitable statistical analysis.

 

Galactosamine-induced Liver Necrosis

 

A single dose or a few repeated doses of D-galactosamine causes acute hepatic necrosis in rats. Prolonged administra-tion leads to cirrhosis. This model is used for the screening of hepatoprotective agents.

 

Adult albino rats weighing 110–180 g are injected intra-peritoneally three times weekly with 500 mg/kg D-galac-tosamine over a period of one to three months. The test substances are administered orally with food or by gavage. The control group receives only vehicle or food without drugs. The rats are sacrificed at various time intervals and the livers excised and evaluated by light microscopy and immunohistology using antibodies against macrophages, lymphocytes, and the extracellular matrix component.

 

Country-made Liquor Model

 

Country-made liquor (CML, containing 28.5% alcohol) is used to produce hepatotoxicity in this model. CML is administered orally at a dose of 3 ml/100 g/day for 30 days, which results in severe fatty changes in liver.

 

Rats are divided into groups of eight each. The control group receives 1% gum acacia as vehicle, corn oil (1 ml/100 g/day), and glucose isocaloric to the amount of alcohol. The positive control group receives CML (3 ml/100 mg/day) in two divided doses and corn oil (1 ml/100 g/day) in a single dose. Other test groups receive drugs in respective doses along with CML (3 ml/100 g/day) and corn oil.

 

After 21 days, the blood is withdrawn for analysis of SGOT, SGPT, alkaline phosphatase, serum cholesterol, albumin, total proteins, bilirubin, glucose, and creatinine. The rats are sacrificed and the livers dissected out for his-topathological analysis. The value of the test is compared with the control using suitable statistical analysis.

 

Paracetamol Model

 

This model is used to produce experimental liver damage only in mice, since rats are resistant to paracetamol-induced hepatotoxicitiy. Paracetamol administered orally as a single dose of 500 mg/kg in mice produces hepatotoxicity.

 

Adult albino mice are used for the study. Paracetamol is administered as a single dose of 500 mg/kg. After 48 h, they are treated with the test drugs for 5 days. At the end of the experiment, blood is withdrawn for biochemical analysis of SGOT, SGPT, alkaline phosphatase, serum cholesterol, albumin, total proteins, bilirubin, glucose, and creatinine. The liver is subjected to histopathological studies. The value of the test is compared with the control using suit-able statistical analysis.

 

Partial Hepatectomy Model

 

In this method, partial hepatectomy (removal of 70% of liver mass) is done and the action of drugs on the regeneration of liver cells studied. Hepatoprotective agents improve the regeneration capability of liver.

 

Rats are used for this study as they can withstand surgical infections better than mice. They are anaesthetized using light ether anaesthesia. A median line incision reaching 3–4 mm posteriorly from the xiphoid process of the sternum is done and the large median lobe of the liver with the left lateral lobe taken out. These lobes are ligated by coarse linen and excised. Around 68 ± 2% of the total hepatic parenchyma is also removed. The peritoneum is closed using absorbable suture and the integument closed using nonabsorbable surgical suture. Various hepatoprotective drugs can be screened for their activity using these hepatectomized rats.

 

At the end of the screening experiments, the blood is col-lected for analysis of the serum. The following parameters are determined: SGOT, SGPT, alkaline phosphatase, serum cholesterol, albumin, total proteins, bilirubin, glucose, and creatinine. The animals are sacrificed. The liver is excised out, weighed, and subjected to histopathological evaluation. The value of the test is compared with the control using suitable statistical analysis.

 

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