Hepatoprotective Effect of Zanthoxylum armatum DC

The liver is the prime organ concerned with various states of metabolic and physiologic homeostasis of organism and is a key organ of metabolism and excretion playing an important role in the maintenance of internal environment of the body through its multiple and diverse functions. It is continuously exposed to a variety of xenobiotics and therapeutic agents exposing the organ to numerous and varied disorders. There is a progressive increase in the incidence of hepatic damage mainly due to the viral infection, hepatotoxic chemicals (alcohol), toxin in food (especially aflotoxins), peroxides (particularly peroxidized edible oil), pharmaceuticals (antibiotics, chemotherapeutics, and CNS active agents), environment pollutants and xenobiotics (Hikino et al 1988).


Introduction
The liver is the prime organ concerned with various states of metabolic and physiologic homeostasis of organism and is a key organ of metabolism and excretion playing an important role in the maintenance of internal environment of the body through its multiple and diverse functions. It is continuously exposed to a variety of xenobiotics and therapeutic agents exposing the organ to numerous and varied disorders. There is a progressive increase in the incidence of hepatic damage mainly due to the viral infection, hepatotoxic chemicals (alcohol), toxin in food (especially aflotoxins), peroxides (particularly peroxidized edible oil), pharmaceuticals (antibiotics, chemotherapeutics, and CNS active agents), environment pollutants and xenobiotics (Hikino et al 1988).
Though liver disease are among the most important disease affecting mankind , no remedy is available at present in the modern system of medicine which include corticosteroids and immunosuppressive agents which bring about symptomatic relief supporting only the process of healing of liver regeneration and in most cases have no influence on the disease process. Further, their use is associated with the risk of relapses and danger of side effects. An actual curative therapeutic agent has not yet been found and thus management of liver disease is still a challenge to the modern scientific community. Hence increasing attention is being given to plants recommended for the treatment of hepatic disorders in traditional system of medicine. A number of medicinal preparations have been advocated especially in Ayurvedic system of Indian medicine, for the treatment of liver disorders. Their usage is in vogue since centuries and are quite often claimed to offer significant relief. In addition, the use of many folklore remedies mainly plant products are also common throughout India.
About 600 commercial preparations with claimed liver protecting activity are available all over the globe. In India about 33 patent herbal formulations are available for liver ailments and these preparations are a variety of combinations out of 100 Indian medicinal plants belonging to about 40 families (Handa et al 1986). Only a few scientific data with regard to their hepatoprotective action are on record. Phytoconstituents remains to be a major contributor in the treatment of liver disorders. The growing concern for the identification of novel hepatoprotective agents from natural sources is evident from literature available on the same.

Distribution
A large genus of aromatic, prickly, dioecious or rarely monoecious tree or shrubs, mainly pantropical, through also distributed in the subtropics. The genus as dealt with in the article includes species of Fagara, through some authors treat them as two distinct genera. About 13 species are recorded from India.
Zanthoxylum armatum DC found in the hot valleys of the Himalayas from Jammu to Bhutan at altitudes of 1,000-2100 m and in Eastern Ghats in Orissa and Andhra Pradesh at 1,200 m., in India. It is also sometimes planted for hedges in Assam.

Description
Zanthoxylum armatum DC, is an armed scandent or erect shrub or a small tree , 6 m tall or more with dense foliage, branches armed, the prickles flattened, up to 2 cm. long, bark pale brown, deep-furrowed, leaves imparipinnate or trifoliolate, 5-23 cm. long, often with flattened prickles , leaflets up to 5 pairs, opposite, ovate to lanceolate , entire to glandular crenate, acute to obtusely acuminate, flower green or yellow, in dense terminal, and occasionally axillary sparse panicles, follicles generally reddish, sub-globose, glabrous, seeds solitary in a fruit, globose, shining black. (Kirti & Basu 1975).

Phytochemical review
Several alkaloids have been isolated from the stem-bark and root-bark. The fruits of a number of species yielded essential oils. The oils from Z. acanthopodium, Z. armatum, and Z. nitidum are potential source of linalool, an important perfumery material; the first two species are also employed on a limited scale for the production of wartara oil. Z. americanum Mill. , a shrub of eastern North America, is used in its native country for toothache and rheumatism.

Pharmacological review
The bark, fruits, and seeds of Z. armatum are extensively used in indigenous system of medicine as a carminative, stomachic and anthelmintic. The stem has exhibited hypoglycemic activity in the preliminary trials. The bark is pungent and used to clean teeth. The fruits and seeds are employed as an aromatic tonic in fever and dyspepsia. An extract of the fruits is reported to be effective in expelling round worms. Because of their deodorant, disinfectant and antiseptic properties, the fruits are used in dental troubles, and their lotion for scabies.
Zanthobungeanine, found in stems and roots shows inhibitory activity to platelet aggregation, L-plananin is the most active compound.
The bark is used in India, for treating diarrhea and cholera. The fruits are analgesic and anodyne and used in tooth powder.

The bark of several Indian species of Zanthoxylum is medicinally active and noted for febrifugal, sudorific and diuretic properties (Wealth of India 2005).
The essential oil is said to possess antiseptic, disinfectant and deodorant properties. The freshly distilled essential oil from the seeds exhibited strong antibacterial activity against Escherichia coli, Vibro cholera, Micrococcus pyrogens var. aureus, Shigella dysenteriae and Salmonella typhi. The seed oil possesses ascaricidal, antibacterial, anthelmintic and antifungal properties. The oil on account of high percentage of linalool is highly fragrant and attractive and can be commercialized on this account.
The oil obtained by steam-distillation of the fresh plant showed antifungal activity against a number of fungi.
It is established that plants which having antioxidant property also exert hepatoprotective action (Fehar et al 1986). As Z. armatum has shown significant antioxidant activity  and also contain phenolic compounds, therefore, we have to investigate the hepatoprotective activity of ethanolic extract of Z. armatum in rats.

Plant material
The

Preliminary phytochemical screening
A preliminary phytochemical screening was carried out for the extracts employing the standard procedure revealed the presence of various phytoconstituents viz. alkaloids, steroids, terpenes, flavonoids, saponins, tannins, glycosides, carbohydrates and proteins (Harborne 1998).

Chromatographic studies
Thin layer chromatography (TLC) and High Performance Thin Layer Chromatography (HPTLC), of the alcoholic extract of Costus speciosus was done.

Animals
The Institutional Animal Ethics Committee, (IAEC) review the protocol and approved the use of animals for the studies, (Ethical clearance number: 711/02/a/CPCSEA).
Wistar albino rats of both sexes (weighing 130-170 g) were used in the present study. They were housed in clean polypropylene cages (38X23X10 cm) with not more than three animals per cage and maintained under standard laboratory condition (temperature 25 ± 2°C) with dark and light cycle (12/12 h) and provided standard pellet diet (Hindustan Lever, Kolkata, India) and water ad libidum.

Acute toxicity studies
Acute toxicity study was performed for the extract according to the acute toxic classic methods as per OECD guidelines (OECD Guidelines 1996). Wister albino rats were used for acute toxicity study. The animals were kept fasting for overnight providing only water, after which the extract was administered orally 500 mg/kg b. w. and observed for 14 days. The animals were observed If mortality was observed in 2 out of 3 animals, then the dose administered was assigned as toxic dose. If mortality was observed in 1 animal, then the dose administered was repeated again to confirm the toxic dose. If mortility was not observed, the procedure was repeated for further higher dose i.e. 2000 mg/kg.

Assessment of hepatoprotective activity
The rats were divided in to four groups of six rats each. The animals of group A and group B served as control and carbon tetrachloride (CCl 4 ) control received vehicle (0.1% tween 80, 10 ml/kg b. w.). Group C served as standard and received silymarin (100 mg/kg b. w. in 0.1% tween 80), and group D was given EEZA (500 mg/kg b. w. in 0.1% tween 80). All administration of doses was made by gastric intubations once daily for 7 days.
On the 8 th day 1 h after the administration of last dose, the animals of group B; C and D were given an intraperitoneal injection of CCl 4 with an equal quantity of liquid paraffin (0.5 ml/kg b. w.). All the animals were then fasted for 24 h and anaesthetized and the blood was collected by cardiac puncture. The liver was quickly dissected, washed with ice-cold saline and stored in freezer. The blood samples were allowed to coagulate at room temperature for 1 h. Serum was separated by centrifugation at 12,000 rpm at 4 0 C for 5 min (Verma et al 2007).

Histopathological studies
The hepatoprotective activity was confirmed through histopathological studies on liver of rats. Slices of liver were cut and washed in Ringer's solution soaked with filter paper for 1.5 min, then liver slices were fixed in Carnay's fluid I (Ethanol: chloroform: Glacial acetic acid-6:3:1) and processed for paraffin embedding following the standard microtechniques. Sections of liver, stained with aqueous haematoxylein and alcoholic eosin were observed microscopically for histopathological changes (Galigher et al 1971).

Statistical analysis
The data represent M ± S.E.M. Results were analyzed statistically by one-way ANOVA, followed by Students't' test. The minimum level of significance was set at P<0.001 compared to control. The entire statistics were estimated by using Sigma Stat 3.5™, statistical software.

Phytochemical screening
Phytochemical screening for the ethanolic extract of Zanthoxylum armatum revealed the presence of phytoconstituents like sterols, alkaloids, phenolic, flavonoids and reducing sugars. The ethanolic extract did not cause any mortality up to 2000 mg/kg and considered as safe.

CCl 4 induced Hepatotoxicity
The results of CCl 4 -induced hepatotoxicity are represented in Table 1. CCl 4 intoxication in normal rats elevated the levels of SGOT, SGPT, ALKP, SBLN and liver inflammation were observed significantly indicating acute hepatocellular damage and billiary obstruction. The rats that received 500 mg/kg of EEZA showed a significant (P<0.001) decrease in all the SGOT, SGPT, ALKP, SBLN levels and liver inflammation, compared to induced control group.
Normal histology of rat liver showed sinusoidal degeneration (Fig. 2a). The liver sections of the rats treated with CCl 4 showed cellular degeneration hydropic changes which were more around the central vein and fatty changes with wide spread hepatocellular necrosis and centrolobular necrosis (Fig. 2b). The liver section of EEZA treated showed micro fatty changes with dense collection of lymphoid cells, suggesting evidence of very little necrosis or degeneration. There was no hepatocellular damage, except small arrears of focal degeneration and sinusoidal dilation in treated rat livers ( Fig. 2c and d).
CCl 4 is biotransformed in to cytochrome P450 in the liver endoplasmic reticulum to the highly reactive trichloromethyl free radical which in turn reacts with oxygen to form a trichloromethyl peroxyradical, which may attack lipids on the membrane of endoplasmic  reticulum more readily than trichloromethyl free radical. The trichloromethylperoxy radical leads to elicit lipid peroxidation, the disruption of Ca 2+ homeostasis, elevation of hepatic enzymes and finally results in cell death (Clawson et al 1989).
The results obtained from the present study indicated that the EEZA exhibited hepatoprotective effect against CCl 4 -induced liver damage by normalizing the elevated levels of the hepatic enzymes. This suggested the possibility that EEZA is able to condition the hepatocytes, so as to cause accelerated regeneration of parenchyma cells, thus protecting against membrane fragility and decreases of leakage of the marker enzymes into the circulation as compared to silymarin, reported to have protective effect on the plasma membrane of hepatocytes (Ramellini et al 1976). The results supported the use of this plant for the treatment of hepatitis in oriental traditional medicine. Flavonoids have been reported as active substances for the treatment of hepatitis induced by chemicals (Khalid et al 2002) and virus (Kang et al 2006) in vitro and in vivo. Ethanolic extract of Z. armatum showed positive results for the presence of phenolics and flavonoids during preliminary phytochemical screening. The possible mechanism may be that the antioxidant potentiality of flavonoids can scavenge free radicals and protect the cell membrane from destruction. Hence, the transaminases (ALT/AST) may not leak into blood from the necrotic hepatocytes.