Antihepatotoxic effect of isolated chitin from Rhizopus oryzae against paracetamol-induced hepatotoxicity

The present study was aimed at investigating the antihepatotoxic activity of isolated fungal chitin, a natural polysaccharide from Rhizopus oryzae NCIM1009 against paracetamol-induced toxicity in rats. Changes in the levels of biochemical markers of hepatic injury such as  serum glutamate oxaloacetic transaminase (SGOT), serum glutamic pyuruvic transaminase (SGPT), alkaline phosphatase, bilirubin and total protein were determined in both treated and control group of rats. The effect of fungal chitin was compared with that of silymarin (25 mg/kg). The results showed that paracetamol (750 mg/kg) elevated the levels of biomarkers enzymes. Treatment with fungal chitin (200 mg/kg) brought the marker level near to normal and showed significant hepatoprotective activity. Acute toxicity studies of fungal chitin was carried out and found safe up to 2,000 mg/kg. The isolated fungal chitin was characterized by IR spectroscopy and compared with commercial chitin.

problems of seasonal and limited supply in some countries and environmental pollution while collecting large amounts of shell waste (White et al., 1979) .
Owing to unique biological properties of chitin, much attention has been given to the study of its applications. However, most of its biological properties remain to be studied. To overcome the above disadvantages in traditional production of chitin and in the study of its biological properties, As an alternative, an attempt has been made in the present work, to obtain fungal chitin isolated from Rhizopus oryzae NCIM 1009 and its hepatoprotective activity was studied.

Materials and Methods
Culture and its maintenance: The microorganism used in this study was Rhizopus oryzae NCIM1009 obtained from National chemical laboratory, Pune, India.The fungus was maintained at 4°C on PDF slant and transferred on a monthly basis.
Cultivation conditions: The standard inoculum was prepared by the spores of Rhizopus oryzae NCIM1009, harvested from 7 day petriplate culture on PDA medium at 28°C. The spore suspension was prepared and adjusted to10 8 spores/mL using a hematocytometer for counting (Thayza et al., 2007).
For submerged cultivation 10% v/v of the standard inocula was inoculated to 300 mL of fermentation broth in the seven 500 mL capacity Erlenmeyer flasks and incubated at 28°C for 7 days in a rotary shaker at 200 rpm. The experiment was carried out in triplicate and the results are expressed as mean ± S.D.
Chitin isolation: After cultivation of culture in fermentation broth, the fungal mycelia was harvested for every 24 hours by filtration (No.1; Whatman) washed with distilled water until a clear filtrate was obtained and then dried at 60°C to a constant weight. The chitin isolation was carried out by suspending the fungal mycelia with 1M NaOH solution (1:30 w/v) and autoclaved at 121°C for 15 min. The alkali insoluble fractions were collected after centrifugation at 12,000x g for 15 min. The residues were further extracted using 10%acetic acid (1:40% w/v) at 60°C for 6 hours. The resulting slurry was isolated by filtration containing acid insoluble material chitin. The isolated fungal chitin was then washed with distilled water, 95%ethanol (1:20 w/v), acetone (1:20 w/v) and dried at 60°C to a constant weight (Pochanavanich and Suntornsuk, 2002).
Chitin characterization: The IR spectra of the KBr discs containing chitin and compared with commercial chitin (Cochin Fisheries, Cochin) were measured from 4000 cm -1 to 400 cm -1 with a JASCO FT-IR spectrophotometer.
Chemicals: All chemicals were of high quality from HI-Media Laboratories, Mumbai.
Experimental animals: Male Swiss albino mice weighing between 20-25 g and male Wistar albino rats weighing between 150-220 g were used. The animals were placed at random and allocated to treatment groups in polypropylene cages with paddy husk as bedding. Animals were housed at a temperature of 24 ± 2°C and relative humidity of 30-70%. A 12:12-hours light:dark cycle was followed. All animals were allowed to free access to water and fed with standard commercial pelleted rat chaw (M/s. Hindustan Lever Ltd, Mumbai). All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics Committee and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA).
Acute toxicity study: A safe oral dose of the extract was determined by acute oral toxic class method of of Economic Co-operation and Development (OECD) as per 425 guidelines (OECD 2000). Swiss albino mice were kept overnight fasting and food was with held for 3-4 hours prior to isolated chitosan administration. A total of five animals were used which received a single oral dose (2000mg/kg b.w.) of isolated chitosan. Animals were observed individually at least once during the first 30 min after dosing, periodically during the first 24 h (with special attention during the first 24h) and daily thereafter for a period of 14 days. No mortality was found even at 2000 mg/kg. The dose of 200mg/kg, p.o. was selected for the further activity.
Anti hepatotoxic activity in paracetomol induced liver toxicity rat model: The experiment was carried out after obtaining clearance from Institutional Animal Ethics Committee. The animals were divided in to 5 groups of 6 animals each. Animals were divided into five groups of 6 animals each. Group I served as control, which received normal saline 5 mL/kg orally for 7 days. Group II received paracaetmol 750 mg/kg, Group III and IV received isolated chitin (200 mg/kg, 400 mg/kg respectively) for 7 days and Group V received standard drug silymarin 25 mg/kg. On the 7 th day, after 1 hour of administration of various doses of isolated chitin (200 mg/kg and 400 mg/kg) to group III and IV and silymarin to Group V, paracetamol suspension was given orally to all groups of rats except the rats in Group I. On the 8 th day, the blood sample was collected by cardiac puncture after euthanasia with diethyl ether and centrifuged at 200 rpm for 15 min to separate the serum for various biochemical estimations. The serum activities of serum hepatic marker enzymes namely SGOT, SGPT, ALP (Reitman and Frankel 1957), bilirubin (Kind and King, 1954;Jendrassik and Grof, 1938) and total protein were assayed.

Statistical analysis:
The values were expressed as mean ± SEM. The statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Dunnet's t-test. P values <0.01 were considered significant.

Results and Discussion
The chitin was isolated from fungus Rhizopus oryzae NCIM 1009, characterized by IR (Figure 1 and Figure 2) and screened for antihepatotoxic activity. Acute toxicity study was first determined and found safe up to 2,000 mg/kg body weight. Hence, the antiulcer activity was studied at 200 mg/kg and at 400 mg/kg. The levels of hepatic marker enzymes SGOPT, SGPT, ALP, bilirubin and total protein was elevated in paracetamol treated animals when compared to control groups ( Table I).
The isolated fungal chitin and silymarin treatment decreases the levels of SGOT, SGPT, ALP and total protein when compared to paracetamol treated groups. As chitin is made up of N-acetyl D-glucosamine units its anti hepatotoxic activity may be due to the presence of glucosamine units as they play an important role in detoxification of liver by strengthening the antioxidant defensive system in the body to counteract any oxidative damage caused to the liver (Zupanets et al., 1990;Yan et al., 2006). The results of this study clearly demonstrated that the isolated fungal chitin at a dose of 200 mg/kg body weight, prevents liver damage against paracetamol induced toxicity.