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Hepatoprotective Activity | The Basic Guide


Introduction:

Hepatoprotective Activity, Hepatoprotection or Antihepatotoxicity is the ability to prevent damage to the liver.

There are numerous plants and traditional formulations available for the treatment of liver diseases1,2. About 600 commercial herbal formulations with claimed hepatoprotective activity are being sold all over the world. Around 170 phytoconstituents isolated from 110 plants belonging to 55 families have been reported to possess hepatoprotective activity. In India, more than 93 medicinal plants are used in different combinations in the preparations of 40 patented herbal formulations3. However, only a small proportion of hepatoprotective plants as well as formulations used in traditional medicine are pharmacologically evaluated for their safety and effi cacy4. Some herbal preparations exist as standardized extracts with major known ingredients or even pure compounds which are being evaluated2.
Liver is a vital organ play a major role in metabolism and excretion of xenobiotics from the body. Liver injury or liver dysfunction is a major health problem that challenges not only health care professionals but also the pharmaceutical industry and drug regulatory agencies. Liver cell injury caused by various toxic chemicals (certain anti-biotic, chemotherapeutic agents, carbon tetrachloride (CCL4), thioacetamide (TAA) etc.), excessive alcohol consumption and microbes is well-studied. Herbal medicines have been used in the treatment of liver diseases for a long time. A number of herbal preparations are available in the market. The present review is aimed at compiling data on promising phytochemicals from medicinal plants that have been tested in hepatotoxicity models using modern scientific system.

The liver plays an astonishing array of vital functions 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 reproduction. And it functions as a centre of metabolism of nutrients such as carbohydrates, proteins and lipids and excretion of waste metabolites. The bile secreted by the liver has, among other things, plays an important role in digestion. Therefore, maintenance of a healthy liver is essential for the overall well being of an individual1. Liver cell injury caused by various toxicants such as certain chemotherapeutic agents, carbon tetrachloride, thioacetamide, chronic alcohol consumption and microbes are common. Enhanced lipid per oxidation during metabolism of ethanol may result in development of hepatitis leading to cirrhosis3. Herbal drugs have gained importance and popularity in recent years because of their safety, efficacy and cost effectiveness. The IndianTraditional Medicine like Ayurveda, Siddha and Unani are predominantly based on the use of plant materials. The association of medical plants with other plants in their habitat also influences their medicinal values in some cases. One of the important and well documented uses of plantproducts is their use as hepatoprotective agents. Hence, there is an ever increasing need for safe hepatoprotective agent2. In spite of tremendous strides in modern medicine, there are hardly any drugs that stimulate liver function, offer protection to the liver from damage or help regeneration of hepatic cell. Many formulations containing herbal extracts are sold in the Indian market for liver disorders4. But management of liver disorders by a simple and precise herbal drug is still an intriguing problem. Several Indian medicinal plants have been extensively used in the Indian traditional system of medicine for the management of liver disorder. Some of these plants have already been reported to posse’s strong antioxidant activity5, 6.

Types of  liver disease:     

a)      Necrosis
b)      Cirrhosis
c)      Hepatitis – may be viral, toxic or deficiency type.
d)     Hepatic failure
e)      Chemical / Drug induced Hepatotoxicity: Generally may be hepatitis, jaundice and carcinogenesis.
f)       Liver disorders due to impaired metabolic function. Generally the disorders associated with fat (liposis) and bilirubin (Jaundice) metabolisms are very commonly seen.

1. Disorders associated with fat metabolism : Fatty Liver
2. Disorders associated with bilirubin metabolism – Jaundice or Icterus which may be of different Types based open mechanisms of action and etiology.
  • Hemolytic / prehapatic jaundice.
  • Obstructive (Post – Hepatic / Cholestatic Jaundice).
  • Hepatogenous jaundice
  • Hereditary Jaundice: Syndrome or familial hyper bilirubinaemia, Dubin – Johnson syndrome and Crigler – Najjar syndrome etc, are some of the hereditary jaundice types usually observe. 

  • Cirrhosis, which is a serious condition that causes tissues and cells in the liver to be replaced by scar tissue.
  • Type I glycogen storage disease, which causes problems in controlling blood sugars when a person fasts
  • Porphyria, a condition that causes a malfunction in how the body uses porphyrins.
  • Porphyrins are important in making hemoglobin in red blood cells, to carry oxygen throughout the body.
  • Hemochromatosis:, a condition which causes the body to absorb and store too much iron. The iron buildup causes damage to the liver and other organs.
  • Primary sclerosing cholangitis, a condition that causes the bile ducts of the liver to narrow due to inflammation and scarring
  • Sarcoidosis:, a disease that causes a buildup of lesions within the liver and other organs of the body
  • Gallstones, which may block the bile duct
  • Hepatitis:, an inflammation and infection of the liver caused by any of several viruses
  • Cystic disease of the liver, which causes lesions and fluid-filled masses in the liver

Signs and symptoms of the liver disease:


Symptoms partly depend on the type and the extent of liver disease. In many cases, there may be no symptoms. Signs and symptoms that are common to a number of different types of liver disease include: 
• Jaundice, or yellowing of the skin 
  • Darkened urine 
  • Nausea 
  • Loss of appetite 
  • Unusual weight loss or weight gain 
  • Vomiting 
  • Diarrhea 
  • Light-colored stools 
  Abdominal pain in the upper right part of the stomach 
  • Malaise, or a vague feeling of illness 
  • Generalized itching 
  Varicose veins (enlarged blood vessels) 
  • Fatigue 
  • hypoglycemia (low blood sugar
  • Low grade fever 
  • Muscle aches and pains 
  • Loss of sex drive 
  • Depression

Causes of liver disease:

Liver disease can be caused by a variety of factors. Causes include: 
• Congenital birth defects, or abnormalities of the liver present at birth 
• Metabolic disorders, or defects in basic body processes 
• Viral or bacterial infections 
• Alcohol or poisoning by toxins 
• Certain medications that are toxic to the liver 
• Nutritional deficiencies 
• Trauma, or injury

Prevention from Liver Diasease: 

Some, but not all, liver diseases can be prevented. For example, hepatitis A and hepatitis B can be prevented with vaccines. 

►Other ways to decrease the risk of infectious liver disease include:
• Practicing good hygiene, such as washing hands well after using the restroom or changing diapers.
• Avoiding drinking or using tap water when traveling internationally.
• Avoiding illegal drug use, especially sharing injection equipment. 
• Practicing safest sex. Practicing safer sex provides less protection. 
• Avoiding the sharing of personal hygiene items, such as razors or nail clippers. 
• Avoiding toxic substances and excess alcohol consumption.
• Using medications only as directed. 
• Using caution around industrial chemicals. 
• Eating a well balanced diet following the food guide pyramid. 
• Getting an injection of immune globulin after exposure to hepatitis A. 
• Using recommended safety precautions in healthcare and day care work.

Diagnosis for Liver Diseases:

A healthcare professional can determine whether a person's symptoms, medical history, and physical exam are consistent with liver disease. Hepatomegaly, an enlarged, firm liver, and other signs of liver disease may be found on examination.

Many further tests may also be used to support the diagnosis. These include blood tests, such as: 
• Liver function tests, which are blood tests that check a wide variety of liver enzymes and byproducts 
• A complete blood count (CBC), which looks at the type and number of blood cells in the body 
• Abdominal X-rays 
• Ultrasounds, to show size of abdominal organs and the presence of masses 
• An upper GI study, which can detect abnormalities in the esophagus caused by liver disease 
• Liver scans with radiotagged substances to show changes in the liver structure 
• ERCP, or endoscopic retrograde cholangiopancreatography. A thin tube called an endoscope is used to view various structures in and around the liver. 
• Abdominal CT scan or abdominal MRI, which provide more information about the liver structure and function 

In some cases, the only way to definitively diagnose the presence of certain liver diseases is by a liver biopsy. This procedure involves the removal of a tiny piece of liver tissue for examination under a microscope. Liver biopsies may have to be done repeatedly to see how the disease is progressing or responding to treatment.

Long term effects of the disease:

Long- term effects depend on the type of liver disease present. For example, chronic hepatitis can lead to: 
• Cirrhosis of the liver 
• Liver failure 
• Illnesses in other parts of the body, such as kidney damage or low blood counts
►Other long-term effects of liver disease may include: 
• Gastrointestinal bleeding. This includes bleeding esophageal varices, which are abnormally enlarged veins in the esophagus and/or the stomach. 
• Encephalopathy, which is deteriorating brain function that may progress to a coma 
• Peptic ulcers, which erode the stomach lining 
• Liver cancer

Liver Diseases and Medicinal Plants:

Liver has a pivotal role in regulation of physiological processes. It is involved in several vital functions such as metabolism, secretion and storage. Furthermore, detoxification of a variety of drugs and xenobiotics occurs in liver. The bile secreted by the liver has, among other things, an important role in digestion. Liver diseases are among the most serious ailment7. They may be classified as acute or chronic hepatitis (inflammatory liver diseases), hepatosis (non inflammatory diseases) and cirrhosis (degenerative disorder resulting in fibrosis of the liver). Liver diseases are mainly caused by toxic chemicals (certain antibiotics, chemotherapeutics, peroxidised oil, aflatoxin, carbon-tetrachloride, chlorinated hydrocarbons, etc.), excess consumption of alcohol, infections and autoimmune/disorder11. Most of the hepatotoxic chemicals damage liver cells mainly by inducing lipid peroxidation and other oxidative damages in liver. Enhanced lipid peroxidation produced during the liver microsomal metabolism of ethanol may result in hepatitis and cirrhosis

Treatment:

Treatment for  liver disease include allopathic and herbal drug treatment: 

A. Hepatoprotective allopathic treatment:


DRUGS:
Specific drugs used in the management of liver disease:

1) Ursodeoxycholic acid (Ursodiol):
Mechanism of action: 
Ursodeoxycholic acid decreases intestinal absorption and suppresses hepatic synthesis and storage of cholesterol. This is believed to reduce cholesterol saturation of bile and thereby allowing solubilization of cholesterol-containing gall stones. It has little effect on calcified gallstones or on radiolucent bile pigment stones and therapy is only successful in patients with a functional gall bladder. Ursodeoxycholic acid, a relatively hydrophilic bile acid, is also believed to protect the liver from the damaging effects of hydrophobic bile acids, which are retained in cholestatic disorders. The hepatoprotective effect may however, be less in cats and dogs than in humans as the major circulating bile acid in dogs and cats is taurocholate. This is more hydrophilic and less hepatotoxic than the major circulating bile acids in humans. The immunomodulatory effects of ursodeoxycholic acid are believed to involve decreased immunoglobulin production by B lymphocytes, decreased interleukin-1 and interleukin-2 production by T lymphocytes, decreased expression of hepatocyte cell surface membrane HLA class I molecules and possibly stimulation of the hepatocyte glucocorticoid receptor.
Clinical applications:
ursodeoxycholic acid has been used in the management of chronic hepatic diseases in humans such as primary biliary cirrhosis, biliary disease secondary to cystic fibrosis, nonalcoholic steatohepatitis, idiopathic chronic hepatitis, autoimmune hepatitis, primary sclerosing cholangitis, and alcoholic hepatitis. However, its therapeutic efficacy in some of these disorders has not been firmly established. 

Dose rate:
Dogs and cats: 10–15 mg/kg q24h or divided and given q12h. It is recommended that ursodeoxycholic acid be administered for 3–4 months after which the patient should be reassessed for improvement in biochemical markers of hepatocellular pathology. If there has been improvement, treatment is continued, but if there has been no improvement or progression, either treatment should be terminated or additional therapies such as glucocorticoids or colchicine added.
Adverse effects:
• Ursodeoxycholic acid appears to be well tolerated by dogs and cats; vomiting and diarrhoea are reported rarely. There is some concern in human patients that taurine depletion may be potentiated by chronic treatment with ursodeoxycholic acid.
• This may be important in cats that are obligate taurine conjugators. This potential for taurine depletion may be exacerbated in some cats with hepatobiliary disease that have increased urinary excretion of taurine-conjugated bile acids. 
• Dogs are less likely to become taurine depleted by this mechanism as they can shift to glycine conjugation. Ursodeoxycholic acid should not be used in patients with extra-hepatic biliary obstruction, biliary fistulas, cholecystitis or pancreatitis.
2) Penicillamine:
Penicillamine is a degradation product of penicillin but has no antimicrobial activity. It was first isolated in 1953 from the urine of a patient with liver disease who was receiving penicillin
Mechanism of action:
Penicillamine chelates several metals including copper, lead, iron, and mercury, forming stable water soluble complexes that are renally excreted. It also combines chemically with cystine to form a stable, soluble, readily excreted complex. Although it usually takes months to years for hepatic copper levels to decrease, clinical improvement is often seen in Bedlington Terriers after only a few weeks suggesting the drug has other beneficial effects other than copper depletion. Penicillamine induces hepatic metallothionein, which may bind and sequester copper in a nontoxic form. It may also have antifibrotic effects as it inhibits lysyl oxidase, an enzyme necessary for collagen synthesis and directly binds to collagen fibrils, preventing cross-linking into stable collagen fibres. However, its efficacy as an antifibrotic agent in humans is doubtful and it has not been evaluated in veterinary medicine. Penicillamine may have immunomodulatory effects and has been demonstrated to reduce IgM rheumatoid factor in humans with rheumatoid arthritis. However, its mechanism of action in this disease remains uncertain.

Clinical applications:

Penicillamine is a monothiol chelating agent which is used in veterinary medicine in the treatment of copper-storage hepatopathy (e.g., Bedlington Terriers), lead toxicity, and cystine urolithiasis. It has also been used in the management of rheumatoid arthritis in humans. Wilson’disease is treated by trientine or penicillamine.

Dosage and formulations:
For management of copper-associated hepatopathy, a dose of 10–15 mg/kg q12h PO is given on an empty stomach. However, if GIT adverse effects are experienced, these may be reduced if it is given with food, although absorption may be reduced. Alternatively, reduce dose and gradually build up to full dos
Adverse effects:
• GIT adverse effects are common resulting in nausea and vomiting. Smaller doses on a more frequent basis may alleviate adverse effects. Alternatively, the drug can be given with food although this will reduce absorp
• Other adverse effects observed infrequently or rarely include:
Fever.
Lymphadenopathy.
Skin hypersensitivity reactions.
Immune-complex glomerulonephropathy.

Other drugs includes:
Liver disease treatment will depend on the type and the extent of disease. For example, treating hepatitis B, hepatitis C, and hepatitis D may involve the use of medications such as the antiviral medication alpha interferon. Other medications used to treat liver disease may include ribavirin, lamivudine, steroids, and antibiotics.Wilson’disease is treated by trientine or penicillamine.
Other drugs are: 
Alphamethyldopa, halothane, INH (isoniazid), rifampicin, pyrazinamide,phenylbutazone allopurinol, chlorpromazine, methyltestosterone, erythromycin, glibenclamide.



SIDE EFFECTS OF DRUGS USED IN LIVER DISEASES:

It will depend on the treatments used for the liver disease. Antibiotics may cause stomach upset or allergic reactions. Side effects of interferon include a flu-like illness with fever, and body aches.

A liver transplant can cause many complications, including failure or rejection of the new liver. After a liver transplant, a person will need to take powerful anti-rejection medications for the rest of his or her life. Because these medications interfere with normal immunes system functioning, they increase the person's risk for infections and certain types of cancer. 

A person with hepatitis B, hepatitis C, or hepatitis D needs to be monitored for side effects and benefits during and after interferon treatment. Alpha interferon treatment might be repeated if the disease flares up again. A person who has received a liver transplant is checked for further disease, as well as for function of the new liver. 

Cirrhosis can lead to a number of complications, including liver cancer. In some people, the symptoms of cirrhosis may be the first signs of liver disease.

B. Herbal treatment:

CLASSIFICATION OF HERBAL AGENTS:

These are generally classified into 3 categories without any strict delineation amongst them. 

1.  Anti hepatotoxic agents:
These generally antagonise the effects of any hepatotoxin causing hepatitis or any liver disorder or disease.

2.  Hepatotropic agents:
These generally support or promote the healing process of the liver. In practice these two activities can not be easily distinguished from each other.

3.  Hepatoprotective agents:
These generally prevent various types of liver affections prophilactically.
In general any hepatoprotective agent can act as an antihepatotoxic or hepatotropic agent but the vice versa is always not true.

PLANTS USED IN THE TREATMENTT OF LIVER DISEASE (HEPATOPROTECTIVE NATURAL PLANTS):
Some of the crude drugs with activity against liver diseases are:


  • Eclipta alba (Asteraceae),
  • Glycyrrhiza glabra (Leguminosae),
  • Boerhaavia diffusa (Nyctaginaceae), 
  • Phyllanthus amarus (Euphorbiaceae),
  • Silybum marianum (Compositeae), 
  • Uncaria gamber (Rubiaceae), 
  • Andrograhis paniculata (Acanthaceae)

  •  Nelumbo nucifera
Nelumbo nucifera

►Some of the reported constituents with pharmacologically/ therapeutically proved claims may be enlisted as, was also reported for its hepatoprotective properties.

• Silymarin
• Glycyrrhizin
• (+) –Catechin 
• Saikosaponins
• Curcumin 
• Picrolive I and II
• Gomosin(Wagner et al.,1998)
• Acetylbergenin (Lim et al.,2000)
• Kolaviron (Oluwatosin and Edward, 2006) 

1). Silybum marianum: 
Synonyms: Carduus marianus, mariane thistle.
Common name:  Milk thistle
Family: Asteraceae
Origin:  indigenous to the Mediterranean region, North Africa & Western Asia.
Parts used:  Aerial parts
Chemical constituents:
• The active constituents of milk thistle are flavonolignans including silybin, silydianin, and silychristine, collectively known as silymarin.
• Silybin  is the component with the greatest degree of biological activity, and milk thistle extracts are usually standardized to contain 70-80 percent silybin. Silymarin is found in the entire plant but is concentrated in the fruit and seeds. 
• Silybum seeds also contain betaine (a proven hepatoprotector) and essential fatty acids, which may contribute to silymarin's anti-inflammatory effect.
• Active ingredients:  Silymarin – a flavolignin (hepatoprotective), lipids, proteins.
• Milk seeds  seeds also contain other flavonolignans namely dehydrosilybin, desocysilycristin, desoxysilydianin, silyhermin, neosilyhermi, silybinome, and silandrin.

Use: 
• Silybum marianum is currently the most well researched plant in the treatment of liver disease.
• Also use in the dyspepsia, disorders of biliary system, liver disorder.

• It is used as hepatoprotective and in chronic inflammatory hepatic disorders including hepatitis, cirrohis and fatty infiltration which occur due to industrial pollutants and alcohol.
• It has also been found to be effective against liver poisioning due to alpha-galactosamine, carbontetrachloride and tioacetamide.
• It has reported that therapeutic utility of silymarin is due to stabilization of cell membrane, stimulation of protein synthesis and accelerating the process of regeneration of hepatic cells.
• The michanism of hepatoprotective effect of silymarin has been suggested variously like antioxidant activity by trapping superoxide anions, stimulation of RNA synthesis and in case of amanita phalloides poisioning, blocking the receptor sites of outer liver cell membranes
• Silymarin is preferably given by parantral route, due to low water solubility of flavonoligans if taken orally, only 20-50% is absorbed. 


2). Taraxacum officinale:
Synonyms: Dandelion
Family: Asteraceae
Origin: All parts of the northern hemisphere.
Parts used:  Leaves & roots.
Chemical constituents:

• Bitter constituents like taraxecerin and taraxcin are active constituents of the medicinal herb.
• Other Active ingredients:  sesquiterpene lactones, phenolic  acid, inulin, K.

Use:
• Hepatic & biliary disorders, kidney stones.
• Traditionally taraxacum officinale has been used as a remedy for jaundice and other disorders of the liver and gallbladder, and as a remedy for counteracting water retention.
• Generally, the rrots of the plants have the most activity regarding the liver and gallbladder.
• Oral administration of extracts from the roots of taraxacum officinale has been shown to act as a cholagogue, increasing the flow of bile.
• Action: diuretic, tonic.

3). Cichorium intybus: 
Synonyms: Cichory.
Common name: Kasni
Family: Compositae(asteraceae)
Chemical constituents:
• A bitter glucoside, Cichorin has been reported to be the active constituent of the herb.
Use:  
• Use for the treatment of liver diseases. 
• It is commonly known as kasni and is part of polyherbal formulations used in the treatment of liver diseases. 
• In mice, liver protection was observed at various doses of Cichorium intybus but optimum protection was seen with a dose of 75 mg/kg given 30 minutes after CCl4 intoxication. 
• In preclinical studies an alcoholic extract of the Cichorium intybus was found to be effective against chlorpromazine-induced hepatic damage in adult albino rats.

4). Solanum nigrum:
Synonyms: Black nightshade.
Ayurvedic name: Kakamachi.
Family: Solanaceae. 
Chemical constituents: 
• Main active constituents are solamargine, andsolasonine.

Use:
• Aromatic water extracted from the drug is widely prescribed by herbal vendors for liver disorders. 
• Although clinical documentation is scare as far as hepatoprotective activity is concerned, but some traditional practitioners have reported favorable results with powdered extract of the plant. 
• It is in treatment of cirrhosis of the liver.
• Also used  as a emollient, diuretic, antiseptic, and laxative properties.
• Antimicrobial, antioxidants, cytotoxic properties.
• It is also have antiulcerogenic activity and hepatoprotective activity.

5). Glychyrrhiza glabra:
Synonyms: Liquorice
Family: Leguminosae
Chemical constituents:
• Licorice contains triterpene saponin, known as glycyrrhizin, which has potential hepatotprotective activity.
• It belongs to a group of compounds known as sulfated polysaccharides.
• Glycyrrhizin is potassium and calcium salt of Glycyrrhizinic acid.
• Glycyrrhizinic acid is a glycoside and on hydrolysis yields glycyrrhetinic acid which has a triterpenoid structure.
• Other constituents are glucose, sucrose, bitter principle glycyramarin resin, aspargin and fat.
• Another chemical aspects of liquorice is prencence of flavonoids(liquiritin and isoliquiritin) which cauce antigastric effect and are useful in peptic ulcer treatment.

Use: 
• Glycyrrhizin use for anti-viral. 
• It has potential for therapeutic use in liver disease.
• Experimental hepatitis and cirrhosis studies on rats found that it can promote the regeneration of liver cells and at the same time inhibit fibrosis. 
• Glycyrrhizin can alleviate histological disorder due to inflammation and restore the liver structure and function from the damage due to carbon tetrachloride.
• The effects including: lowering the SGPT, reducing the degeneration and necrosis and recovering the glycogen and RNA of liver cells. 
• Effects of glycyrrhizin has been studied on free radical generation and lipid peroxidation in primary cultured rat hepatocytes.
• Favorable results have been reported in children suffering from cytomegalovirus aftrer treating with glyrcyrrhizin.

6). Wilkstroemia indica:
Synonyms: Aradon indica, wilkstromia.
Family: Thymelaeaceae
Chemical constituents: 
• A dicoumarin, daphnoretin is the active constituent of the herb.
• The drug has shown to suppress HbsAG in Hep3B cells. 

Use: 
• W. indica is a Chinese herb and has been evaluated in patients suffering from hepatitis B.
• It is said to activator of protein kinase C.

7). Curcuma longa:
Synonyms: Curucuma, turmeric, Indian saffron
Family: Zingiberaceae
Chemical constituents:

• Diarylhepatonoids including Curcumin is the active constituent of the plant.
• It contains yellow colour substances known as curcuminoids.
• Curcuminoids is responsible for yellow colour.
• Curcuma species contain volatile oil, starch etc.
Use: 
• Like silymarin, turmeric has been found to protect animal livers from a variety of hepatotoxic substances, including carbon tetrachloride,galactosamine, pentobarbitol, 1-chloro-2,4-dinitrobenzene,7 4-hydroxy-nonenal,1and paracetamol. Diarylhepatonoids.
• Curcumin has been proved as anti-inflamatory drug.

8). Tephrosia purpurea:
Synonyms: basterd indigo, hoary pea.
Ayurvedicname:  sharpunkha
Family: Fabaceae.
Chemical constituents:
• The roots, leaves and seeds contain tephrosin, deguelin and quercetin.
• The hepatotprotective constituent of the drug is still to be proved.
Use: 
• Alkali preparation of the drug is commonly used in treatment of liver and spleen diseases. 
• In animal models, it offered protective action against carbon tetrachloride and D-galalactosamine poisoning.

9). Fumaria officinalis:
Synonyms: Fumatory
Family: Papaveraceae
Chemical constituents: Alkaloids, flavonoids
Origin:  Europe, Mediterranean, Middle East, but has now become a weed all over the world.
Parts used:  aerial parts
Actions:  Cholagogue, anti-spasmodic
Uses:  
• Biliary & dyspeptic disorders, especially spastic discomfort of the GIT, the gall-bladder & bile ducts


10). Peumus boldus: 
Synonyms: Boldo
Family: Monimiacee
Parts used:Leaf
Chemical constituents: 
• Alkaloids, volatile oils, flavonols and their glycosides.   
Origin: Chile and other south American regions.
Actions: Choleretic, diuretic, stomachic, mild sedative.
Use: 
• Dyspepsia, spastic complaints.  It is the traditional anthemintic in Chile. 
• It is also used in pharmaceutical slimming mixtures.

11). Chionanthus virginicus:
Synonyms: Fringe tree, old man’s beard.
Family: Oleaceae
Parts used:Dried root bark
Chemical constituents: Saponins, lignin glycoside.
Origin: Southern parts of Northern America.
Actions: Cholagogue, liver tonic, bitter tonic,  anti-emetic, laxative.
Use :
• liver & gall bladder ailments (gall stones, hepatitis, jaundice & other ailments associated with poor liver function).  
• It is also thought to be useful as a general tonic, diuretic & febrifuge. 
• It is also used for minor wounds, sores, bruises, inflammation, & infected wounds.

• Traditional uses:  American Indians used the herb for malaria & wound healing.
Homeopathic uses:  migraine, headache, liver & gall bladder disorders & symptoms of depression.


12). Andrograhis paniculata:
Synonyms: Kalmeg. 
Family: Acanthaceae. 
Parta used: 
• Andrographis leaves, as well as the fresh juice of the whole andrographis plant, have been used in a variety of cultures.  
Chemical constituents: 
•  Kalmegh contains bitter principles andrographolide, a bicyclic diterpenoid   lactone and Kalmeghin (upto 2.5%).
•  Andrographis contains andrographolide, deoxyandrographolide and neoandrographolide. Andrographis contains many flavonoids.
Use: 
• Andrographis dispels heat i.e., is antipyretic.
•  It  removes toxins, which makes it a good treatment for infectious fevercausing diseases. 
• It has been used in bacterial dysentery, arresting diarrhea and in upper respiratory infections, tonsillitis, pharyngitis, laryngitis, pneumonia, tuberculosis, and pyelonephritis. It has also been used in herpes, skin infections, and in helminthic (parasitic) infections. 
• Finally, it has been used for conditions as diverse and unrelated as snakebites and diabetes, as well as terminating pregnancies.

13). Elipta alba:
Synonyms: Eclipta arecta, eclipta prostata. 
Family: Compositae(asteraceae). 
Chemical constituents:
• It contain resins and a alkaloid known as ecliptin, nicotin, glucoside, alkaloids 

Use: 
• Viral hepatitis, liver disorders, skin- and hair care, improves complexion, calm the mind, memory disorders, swollen glands, due to upper respiratory viral infection, strengthen spleen, general tonic
• The tincture has a neutralizing effect on the venom of South America rattle snakes.
• The alcoholic extracts of the entire plant has been reported to have antiviral activity against Ranikhet disease virus.
• Aqueous extracts of the plant showed subjective improvement of vision in the cases of refractive errors. 
• The alcoholic extract of the plant shows protective effects on experimental liver damage in rats and mice.

14). Phyllanthus niruri/amarus:
Synonyms: Phyllanthus emblica, jonesiansoca.
Family: Euphorbiaceae
Chemical constituents: 
• Constituents of this plant are numerous, and include flavonoids and alkaloids.
Use:
• liver & gall bladder ailments (gall stones, hepatitis, jaundice & other ailments associated with poor liver function).  
• It is also thought to be useful as a general tonic, diuretic & febrifuge. 


15). Picrorrhiza kurrora:
Synonyms: Indian gentian, kutki.
Family: Scrophulariaceae 
Chemical constituents: 
• It is found to contain irridoid bitter substances picroside, picroside and kutkoside.
• Picroside and kutkosides are C9 monoterpene glycosides with an epoxy oxides in ring.  
Use: 
• Picrorrhiza is used as valuable bitter tonic, antiperiodic, febrifuge and stomachic and laxative in large doses. 
• Alcoholic extract of root is found to have antibacterial effect.
• The drug is found to useful in treatment of jaundice
• Kutkoside has been found to be a potential hapatoprotectant.

SOME OF HERBAL FORMULATIONS USED IN LIVER DISORDER:
1) Liv-52: It is non-toxic hepatoprotective substance from The Himalaya Drug Co. Liv.52 can improve the subjective condition and clinical parameters in patients with liver damage, in particular in alcoholic liver damage.

2) LIMARIN®: Capsules and Suspension : It has a potent hepatoprotective and free radical scavenging (antioxidant) action. LIMARIN® is developed from the active extract of the fruit of silybum marianum, or the milk thistle. Basically a European herbal product.

3) Cirrhitin: Cirrhitin is a natural medicine formulated specifically to treat Cirrhosis of the liver. Marketed by CCNOW.   Some other polyherbal preparations such as Livex, HD-03, Hepatomed, Live 100 and Hepatoguard with proven efficacy are also use in different types of liver disorders.

Evaluation of Hepatoprotective Activity:
►Investigation of Liver Function:
The liver function tests are employed for accurate diagnosis, to assess the severity of the
damage, to judge the prognosis and to evaluate the therapy.  The routinely performed liver function tests (LFTS) are as follows :

A. ABNORMALITIES OF BILE PIGMENTS AND BILE SALTS EXCRETION TESTS 
• Serum total direct and indirect bilirubin.
• Urine bile salts, bile pigments and urobilinogen.

B. SERUM ENZYMES ASSYAS 
• SGOT (AST )
• SGPT  (AST )
• Alkaline phosphatase (ALP) and if necessary
• γ – Glutamyl transpeptidase (γ-GT) 
• Other enzymes 

C.  CHANGES IN PLASMA PROTEIN TESTS
• Thymol turbidity test.
• Determination of total proteins, albumin globulins.

►Generally direct toxins injure many tissues including liver (eg. CCl4), an indirect affects particular metabolic pathway of the liver (eg. galactosamine). Thus the hepatotoxins affect the liver in a number of ways as: 

1. Interference with hepatic bilirubin uptake, conjugation and excretion eg. Rifampicin.
2. Dose and time dependant reactions.
• Acute toxic hepatitis eg. Paracetamol
• Fatty liver eg. Tetracyclin
3. Dose independent reaction.
• Diffuse hepatocellular damage eg. Isoniazid
• Cholastatic hepatitis eg. Chlorpromazine
• Granulomatous infiltration eg. Phenytoin, Chlorpropamide.
►Hepatoprotective activity can be most easily evaluated / screened with the aid of several model systems of liver damage in experimental animals. In all test model systems conditions for liver damage are implemented and an attempt is made to counteract this toxicosis with the substance / preparation under test. The magnitude of the protective effect can be measured by estimating the enzymes and the rate of survival and can be verified histologically. The available methods are invivo, exvivo & invitro methods. 

A. IN VITRO METHOD: 

• Hepatocytes are isolated by using in-situ under aseptic condition and placed in chilled HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid).

• These isolated hepatocytes than exposed to test samples and toxins like CCl4, thioacetamide, ethanol and paracetamol etc. 

• After a specified time period the degree of toxicity or protection is assessed by viability tests (Trypan blue dye exclusion method) and enzyme levels such as SGOT and SGPT. 


Advantages of in vitro models:

• More rapid and requires small quantities of test substances and fewer animals, where as in vivo studies require a large number of animals (six per group), 
• In Vivo study need up to 3-7 days of drug administration for a significant effect to produced and thus requires large quantities of drugs but In vitro method require 3 days study and less quantities of drugs. 
• Ability to dispose numerous samples at a time. 
• Low cost with a small size 
• Little variation and reproducibility of results. 
• The major disadvantage is that sometimes it may not reflect the events which occur in animals.

B. EX VIVO METHOD:

• In this method after completion of preselected in vivo test protocol hepatocytes are isolated and the percentage of viable cells and biochemical parameters are determined as liver function tests. 
• These methods are somewhat better correlated to clinical models than in vitro or in vivo methods.


C. IN VIVO METHOD:

These are of two types.     

1) Based on bile parameters: 

• The compounds having hepatoprotective claims are evaluated in general for their choleretic or anticholeretic activity in order to know whether the liver disorder is due to an abnormality of bilirubin metabolism or not. 

2) Based on serum parameters:

• Hepatotoxicity is produced in experimental animals by the administration of known dose of hepatotoxin like carbon tetrachloride, paracetamol, D-galactosamine, thioacetamide, ethyl alcohol etc., which produce marked measurable effects, the magnitude of which can be measured by carrying out various liver function tests. 
• It is very convenient laboratory method, reproducibility of results is rather poor.


Experimental models for Hepatoprotective screening:

►Several chemical reagents and drugs which induce liposis, necrosis, cirrhosis, carcinogenesis and hepatobiliary dysfunctions in experimental animals are classified as hepatotoxins. The most important ones used are carbon tetrachloride (CCl4), thioacetamide (TAA). D-galactosamine, Paracetamol, chloroform, ethyl alcohol and Pyridine. The following are some of the experimental rat models employing these hepatotoxins:



1. CCl4 model :
A number of CCl4 models are devised depending upon its dosage through different routes of administration.

A.  ACUTE HEPATIC DAMAGE:
Acute liver damage, characterized by ischemia, hydropic degeneration and central necrosis is caused by oral or subcutaneous administration of CCl4 (1.25ml / kg). The biochemical parameters elevated are found to be maximum after 24 hours of CCl4 administration. Normally administered as 50 % V/V solution in liquid paraffin or olive oil.


B. CHRONIC REVERSIBLE HEPATIC DAMAGE:
Administration of CCl4 (1ml/kg s.c) twice weekly for 8 weeks produces chronic, reversible liver damage.


C. CHRONIC, IRREVERSIBLE HEPATIC DAMAGE:
Administration of CCl4 (1ml/kg s.c) twice weekly for 12 weeks simulates chronic, irreversible liver damage.

2. Thioacetamide model :
Thioacetamide (100 mg / kg s.c) induces acute hepatic damage after 48 hrs of administration by causing sinusoidal congestion and hydropic swelling with increased mitosis.

3. D-Galactosamine model :
D-galactosamine 800 mg /kg i.p induces acute hepatotoxicity after 48 hrs of administration with diffused necrosis and steatosis.

4. Paracetamol model :
Paracetamol induces acute hepatotoxicity depending upon its dosage through different routes of administration, such as-

A. Paracetamol 800 mg/kg i.p. induces centrilobular necrosis without steatosis.
B. Paracetamol at a dose of 3 g/kg p.o stimulates acute hepatic damage.

5. Chloroform model: 
It produces hepatotoxicity with extensive central necrosis, fatty metamorphosis, hepatic cell degeneration and necrosis either by inhalation (for 1hrin atmosphere) or by subcutaneous administration. (0.4-0.5 ml/kg). 

6. Ethanol model :
Ethanol induces liposis to a different degree depending upon its dose, route and period of administration as follows-
A. A single dose of ethanol 1 ml/kg induces fatty degeneration.

B. Administration of 40 % (v/v) ethanol 2 ml/100g/day p.o for 21 days produces fatty liver.
C. Administration of country made liquor 3ml/100g/day p.o for 21 days produces liposis.

Mechanism of Action of Some selected Hepatotoxins

1. Carbon tetrachloride (CCl4):
The hepatotoxicity of CCl4 is due to the metabolic formation of the highly reactive trichloromethyl free radical which attacks the polyunsaturated fatty acids of the membrane of the endoplasmic reticulum and initiates a chain reaction. It is enhanced by induction of hepatic microsomal enzyme systems and vice versa by antioxidants which mop up the free radicals. The first cells to be damaged are those in the centrilobular region where microsomal enzyme activity is the greatest. The initial damage produced is highly localised in the endoplasmic reticulum which results in loss of cytocrome p450 leading to its functional failure with a decrease in protein synthesis and accumulation of triglycerides leading to fatty liver, a characteristic of CCl4 poisoning. If the damage is severe, it leads to disturbances in the water and electrolyte balance of hepatocytes leading to an abnormal increase in liver enzymes in plasma, there by impairing mitochondrial functions, followed by hepatocellular necrosis.

2. Paracetamol:
Paracetamol an analgesic and antipyretic is assumed to be safe in recommended doses, overdoses however taken with suicidal intent, produce hepatic necrosis- small doses are eliminated by conjugation followed by excretion, but when the conjugation enzymes are saturated the drug is diverted to an alternative metabolic pathway, resulting in the formation of a hydroxylamine derivative by cytochrome P450 enzyme. The hydroxylamine derivative, a reactive electrophillic agent, reacts non-enzymatically with glutathione and detoxifies. When the hepatic reserves of glutathione depletes, the hydroxylamine reacts with macromolecules and disrupts their structure and function. Extensive liver damage by paracetamol it self decreases its rate of metabolism and other substrates for hepatic microsomal enzymes.

3.  Thioacetamide:
Thioacetamide, a substitute for H2S with less toxicity and obnoxious smell, on repeated exposure produces cirrhosis by inhibiting the respiratory metabolism of the liver due to the uncontrolled entry of Ca+2 ions into hepatocytes; resulting in inhibition of oxidative phosphorylation. Early metabolic disturbances increase the RNA and protein content of the nuclear fraction of hepatocytes leading to varying graded liver damage including nodular cirrhosis, live cell proliferation, production of pseudolobules and parenchymal cell necrosis. The serum levels of glutamic dehydrogenase are also found to increase, indicative of mitochondrial injury, which plays an important role in thioacetamide induced hepatotoxicity.



Herbs with potentially hepatoprotective constituents

    Almond oil
    Amomum xanthoides
    Arctium lappa
    Astragalus membranaceus
    Cichorium intybus
    Curcuma longa
    Cajanus indicus,
    Centella asiatica
    Coccinia indica'
    Brassica, cum Crucifera.
    Eclipta
    Flickingeria fimbriata
    Ganoderma lucidum
    Glycyrrhiza glabra
    Halenia elliptica (a medicinal herb of Tibetan folk medicine used to treat hepatitis.)
    Kalopanax pictus
    Murraya koenigii
    Nymphaea stellata
    Ocimum sanctum
    Paeonia lactiflora
    Pergularia daemia
    Picrorhiza kurrooa
    Phyllanthus amarus
    Plumbago zeylanica
    Silybum marianum
    Scoparia dulcis
    Salvia miltiorrhiza
    Scutellaria baicalensis
    Schisandra chinensis
    Terminalia catappa
    Tinospora cordifolia
    Zizyphus mauritiana
    Vitis thunbergii, V. flexuosa, and V. kelungensis - three common wild grapes from Taiwan
    Paeonia lactiflora and Astragalus membranaceus:- The extract from the roots of P. lactiflora and A. membranaceus demonstrated better hepatoprotective activity than the herbs used individually.
    Chinese traditional prescriptions:-. Simo Yin, Guizhi Fuling Wan, Xieqing Wan, and Sini San.
    Ayurvedic herbs

Hepatoprotective Activity and the Mechanisms of Action of Ganoderma lucidum (Curt.:Fr.) P. Karst. (Ling Zhi, Reishi Mushroom)

Herbal medicines are always considered to be a safe and useful approach for the treatment of chronic hepatopathy. Ganoderma luciudm (Curt.:Fr.) P. Karst. [(Ling Zhi, Reishi mushroom) (Aphyllophoromycetideae)], a highly ranked medicinal mushroom in Oriental traditional medicine, has been widely used for the treatment of chronic hepatopathy of various etiologies. Data from in vitro and animal studies indicate that G. lucidum extracts (mainly polysaccharides or triterpenoids) exhibit protective activities against liver injury induced by toxic chemicals (e.g., CCl4) and Bacillus Calmette-Guerin (BCG) plus lipopolysaccharide (LPS). G. lucidum also showed anti–hepatitis B virus (HBV) activity in a duckling study. Recently, a randomized placebo-controlled clinical study showed that treatment with G. lucidum polysaccharides for 12 weeks reduced hepatitis B e antigen (HBeAg) and HBV DNA in 25% (13/52) patients with HBV infection. The mechanisms of the hepatoprotective effects of G. lucidum have been largely undefined. However, accumulating evidence suggests several possible mechanisms. These include antioxidant and radical-scavenging activity, modulation of hepatic Phase I and II enzymes, inhibition of b-glucuronidase, antifibrotic and antiviral activity, modulation of nitric oxide production, maintenance of hepatocellular calcium homeostasis, and immunomodulating effects. G. lucidum could represent a promising approach for the management of various chronic hepatopathies. Further studies are needed to explore the kinetics and mechanisms of action of G. lucidum constituents with hepatoprotective activities.



Hepatoprotective activities of picroliv, curcumin, and ellagic acid compared to silymarin on carbon-tetrachloride-induced liver toxicity in mice
To evaluate the hepatoprotective activity of active phytochemicals, picroliv, curcumin, and ellagic acid in comparison to silymarin in the mice model of carbon tetrachloride (CCl 4 ) induced liver toxicity. In addition, attempts were made to elucidate their possible mechanism(s) of action. Materials and Methods: Oxidative stress was induced in Swiss albino mice by a single injection (s.c.) of CCl 4 , 1 ml/kg body weight, diluted with arachis oil at a 1:1 ratio. The phytochemicals were administered once a day for 7& days (p.o.) as pretreatment at two dose levels (50 and 100 mg/kg/day). Results: CCl 4 -induced hepatotoxicity was manifested by an increase in the activities of liver enzymes (alanine transaminase, P < 0.001, aspartate transaminase, P < 0.001 and alkaline phosphatase, P < 0.001), malondialdehyde (MDA, P < 0.001)) levels and a decrease in activity of reduced glutathione (P < 0.001) and catalase in liver tissues. The histopathological examination of liver sections revealed centrizonal necrosis, fatty changes, and inflammatory reactions. The pretreatment with picroliv, curcumin, and ellagic acid normalized serum aminotransferase activities (P < 0.001), decreased levels of MDA (P < 0.001), improved the antioxidant status, and normalized the hepatic histo-architecture. The restoration of phenobarbitone-induced sleeping time also suggested the normalization of liver cytochrome P450 enzymes. Conclusion: This study supports the use of these active phytochemicals against toxic liver injury, which may act by preventing lipid peroxidation, augmenting the antioxidant defense system or by regenerating the hepatocytes.
 Introduction

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Liver diseases are one of the major causes of mortality and morbidity worldwide. Drug-induced liver toxicity is a major cause of hepatic dysfunction. An estimated 1000 drugs have been implicated in causing liver injury, and it is the most frequent reason for withdrawing approved drugs from the market. It accounts for 50% of the cases of acute liver failure. Drug-induced liver failure can mimic all forms of naturally occurring acute and chronic hepatobiliary diseases.

Oxidative stress is considered as a mechanism in contributing to the initiation and progression of hepatic damage in a variety of liver disorders. Cell damage occurs when there is an excess of reactive species derived from oxygen and nitrogen or deficiency of antioxidants.


Antioxidants obtained from plants represent a logical therapeutic strategy for treatment of liver diseases. There are many plant derived chemicals with potent antioxidant properties which can serve as primary compounds for development as hepatoprotective drugs.
Picroliv is a standardized iridoid glycoside mixture isolated from the roots and rhizomes of the plant Picrorrhiza kurroa. It contains at least 60% of a 1:1.5 mixture of picroside I and kutkoside; the remainder (40%) is a mixture of iridoid as well as cucurbitacin glycosides. Picroliv was reported to possess hepatoprotective activities by their anti-lipid peroxidative and free radical scavenging properties. Curcumin is the major biologically active phenolic compound from Curcuma longa with strong antioxidant, anti-inflammatory, and hepatoprotective activities. Ellagic acid is a polyphenolic compound found in grapes, strawberries, black currants, and raspberries, which have potent antioxidant property. Ellagic acid has been reported to reduce the production of hydroxyproline and fibrous connective tissue formation indicating its antifibrotic activity. Ellagic acid also slowed down the conversion of hepatic stellate cells (HSC) into their activated forms, which produce extracellular matrix and result in liver fibrosis. Although individual reports are available on the hepatoprotective activities of picroliv, curcumin, and ellagic acid, their relative efficacy is unknown. This study will enable us to select the most effective lead compound for further development as hepatoprotective drugs. Silymarin, a flavonolignan obtained from Silybum marianum, the most researched hepatoprotective agent, is used as the comparator drug. 

Oxidative stress caused by the highly reactive metabolite produces depletion of glutathione, and through nuclear factor-kappa B (NF-κB) induced proinflammatory cytokines and chemokines, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Inflammatory liver injury is caused by tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2). TNF-α in turn leads to apoptosis and cell death. It is reported that ellagic acid can inhibit NADPH oxidase-induced overproduction of superoxide, suppress the release of nitric oxide by down-regulating iNOS, enhance cellular antioxidant defences, and attenuate oxidized LDL-induced lipoxygenase-1 up-regulation and endothelial nitric oxide synthase down-regulation.­ Therefore, it is speculated that phytochemicals such as curcumin, ellagic acid, and picroliv can act at one or several sites of these cascade of events.

In this study, we have attempted to evaluate the hepatoprotective activity of picroliv, curcumin, and ellagic acid in comparison to silymarin, in carbon tetrachloride (CCl 4 ) induced acute liver toxicity in mice. We also aimed to study whether the antioxidant properties of these phytochemicals have a role in their hepatoprotective actions.
Materials and Methods

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Drugs and chemicals

All the chemicals and reagents used for this study were of analytical grade. Silymarin was purchased from Microlabs (Pondicherry, India), curcumin from Sigma (St. Louis, MO, USA), ellagic acid from Fluka (USA), and picroliv were kindly gifted by Dr. S. Singh, CDRI, Lucknow, India. CCl 4 was obtained from Merck Ltd (Mumbai, India).

Animals

Swiss albino mice (20-30 g) of either sex, bred in Central Animal House, JIPMER, Pondicherry, were used for the study. The animals were allowed standard food pellets (Hindustan Lever Ltd., Mumbai) and water ad libitum and were maintained in standard laboratory conditions (12 h:12 h dark and light cycle and 25±2 °C temperature). The study was approved by 'The Institute Animal Ethics Committee', JIPMER, Pondicherry and was done according to the ethical guidelines laid down by Committee for the Purpose of Control and Supervision of Experiment on Animals.

Treatment groups

The animals were divided into 30 groups (n = 6/group) with 10 groups for biochemical (estimation of serum levels of liver enzymes, namely alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP)) and histopathological parameters, 10 groups for pharmacological parameters (phenobarbitone-induced sleeping time) and another 10 for oxidative stress parameters (malondialdehyde (MDA), reduced glutathione (GSH), and catalase).

Group 1: Animals received distilled water for 7 days and served as normal control.

Group 2: The animals in this group received distilled water as in the previous group and given CCl 4 , 1 ml/kg body weight diluted with arachis oil at 1: 1 ratio, s.c., once on day 8.

Groups 3-6: Four phytochemicals, picroliv, curcumin, ellagic acid, and silymarin were administered once daily for 7 days p.o. (50 mg/kg body weight/day) followed by a single s.c. dose of CCl 4 (1 ml/kg body weight) on day 8.

Groups 7-10: A double dose pretreatment (p.o. 100 mg/kg body weight/day) with the phytochemicals were administered like the previous groups (3-6) followed by a single s.c. dose of CCl 4 (1 ml/kg body weight) on day 8.

Groups 11-20: As stated earlier, 10 groups of animals were used for phenobarbitone-induced sleeping time.

Groups 21-30: Another 10 groups of animals were used for the study of oxidative stress parameters as stated earlier.

The dose of the hepatoprotective drugs were chosen according to the previous studies and the responses shown by these drugs in our study. It was observed that the protection was improved further by increasing the dose to twice (100 mg/kg). Therefore, we chose the 50 and 100 mg/ kg BW dose of phytochemicals for our study. We used the same dose for all the four phytochemicals, so as to make the comparison easy.

Estimation of biochemical and histopathological parameters

After 24 h of CCl 4 administration, animals were anesthetized using ether and 1 ml of blood was collected by cardiac puncture. The blood was allowed to clot and centrifuged (Remi, Mumbai) at 350 g for 10 min. The serum was separated and used for assay of ALT, AST, and ALP by standard methods using enzyme assay kits (Span Diagnostics Limited, India) adapted to Microlab 200 semi-auto analyzer (E. Merck, Germany). The animals were killed by cervical dislocation and livers excised, washed in phosphate buffer and dried using a tissue paper. The liver was weighed by using electronic balance (Sartorius, Germany) and transferred to a 10% formalin fixative solution for 48 h. The liver tissues were processed for paraffin embedding and sections of 5 μm thickness were taken in a microtome. After staining with hematoxylin and eosin, slides were examined under a microscope (Olympus, Japan) at 100 × magnification for histopathological changes.

Phenobarbitone-induced sleeping time

After 24 h of CCl 4 administration, phenobarbitone was administered at a dose of 40 mg/kg body weight i.p., and sleeping time was recorded in minutes from onset of sleep to its natural arousal, i.e. loss of righting reflex to its recovery.­ We modified the method by using phenobarbitone instead of pentobarbitone.

Estimation of MDA, GSH, and catalase in liver tissue

After 24 h of CCl 4 injection, the liver tissues were removed and immediately transferred into cold phosphate buffer, blotted free of blood and tissue fluids and then weighed on an electronic balance. The tissues were chopped into small pieces with scissors and homogenized in ice-cold phosphate buffer (pH 8) at a concentration of 15% (weight by volume). They were then centrifuged in cooling centrifuge (Hettich Zentrifugen, Germany) at 960 g for 5 min. The supernatant was separated and further centrifuged at 7840 g for 45 min at 4°C. The final clear supernatant was used for evaluation of MDA, GSH, and catalase by standard methods.

Estimation of proteins

Proteins in the liver tissue homogenate were estimated by the method of Lowry.

Statistical analysis

Results were expressed as mean ± SEM. The data were analyzed by one way analysis of variance followed by the Student-Newman-Keuls test using Graphpad Instat version 3.06. A P value of less than 0.05 was considered as statistically significant.
 Results

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The hepatic injury induced by CCl 4 (1 ml/kg, s.c.) resulted in an increase in serum ALT, AST, and ALP levels as compared to the normal control group. The pretreatment with picroliv, curcumin, and ellagic acid (50 and 100 mg/kg) significantly reduced serum transaminase levels as compared to the CCl 4 -treated group. This observation was comparable to that of the standard drug, silymarin.

The administration of phenobarbitone in CCl 4 -treated animals caused an enhancement in the mean duration of sleeping time as compared to that of normal control. Pretreatment with hepatoprotective phytochemicals (50 mg/kg) reduced and restored the phenobarbitone-induced sleeping time in CCl 4 -induced hepatotoxicity except in the curcumin pretreatment group. When a dose was increased to 100 mg/kg, all the active phytochemicals significantly reduced and restored the prolonged sleeping time. The dose-dependent effect of curcumin was also observed at higher doses.

CCl 4 -induced hepatotoxicity resulted in oxidative stress and lipid peroxidation. This was reflected by an increase in the MDA levels from that of normal control. The pretreatment with the phytochemicals (50 and 100 mg/kg) effectively restored the elevated levels of MDA, which was comparable to silymarin. The GSH and catalase concentrations were significantly reduced with the administration of CCl 4 . A significant increase in GSH levels was observed with 50 and 100 mg/kg dose of picroliv, curcumin, ellagic acid, and silymarin pretreatment. The catalase activity was improved only at 100 mg/kg/day dose of the phytochemicals.
Conclusions

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In this study, the phytochemicals picroliv, curcumin, and ellagic acid showed hepatoprotective activities comparable to silymarin in CCl 4 -induced hepatotoxicity in mice. The protective action was improved further by doubling the dose of the phytochemicals. Apart from the anti-lipidperoxidative and antioxidant actions, these active phytochemicals might have played a role in restoring the cytochrome P450 enzyme system or promoted the liver regenerative activity. In future, the derivatives of these phytochemicals or their combinations may show efficacy in various experimental toxic models. They may be developed as future drugs for use in human liver diseases with antioxidant, antifibrotic, immunomodulatory, antiviral, and regenerative properties.
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