CHEMICALS
Kits for the analyses of blood serum enzymes (AST, ALT, ALP and LDH), Glucose, Cholesterol, Triglycerides, Urea, Creatinine, Bilirubin (total & direct) were purchased from Merck (private) Limited, France, and Ecoline, Germany. Acetaminophen was purchased from Sigma Aldrich, U.S.A. Other chemicals and reagents were used in the analyses of reduced Glutathione and Lipid peroxidation are also of diagnostic rank.
COLLECTION AND IDENTIFICATION OF SEAWEEDS
Seaweeds are largely available on substratum in shallow coastal water were collected in plastic bags at low tide from Paradise point and Buleji beach at Karachi coast, Pakistan and identified as Ulva fascicata, Soleria robusta, Sptoglossum varibalie according to the different pigmentation pattern and features. These seaweeds were washed with tap water thoroughly in order to remove salts, debris and epiphytes. For experimental use seaweeds were dried under shade, milled in an electrical grinder and fine powder was stored at room temperature. Voucher specimens and herbarium sheets of the seaweeds were prepared and submitted in the Seaweed Herbarium, MAH Qadri Biological Research Center, University of Karachi.
PREPARATION OF CRUDE WATER EXTRACTS
Dry powders (100g) of Ulva fascicata, Soleria robusta, Sptoglossum varibalie seaweeds were soaked separately in distilled water and homogenized by homogenizer. Separately each seaweed extracts were filtered through filter paper and the residues were re-extracted until the colorless filtrate appeared. The filtrates were stored at -20oC for freezing. The freeze samples were freeze dried on Eyela freeze dryer FD-I and powdered forms were stored at -20 C until used.
TEST ANIMALS
Healthy male albino rats of Wister strain (130-200g) were purchased from the animal house of Dow University of Health Sciences, Karachi, Pakistan. Animals were kept in an environmentally controlled temperature (25±2°C) with 12/12h light and dark cycle and nourished with free attain of standard laboratory diet and water. Experiments were held according to the policy of Institutional animal Ethical Committee, University of Karachi for care and make use of experimental animals.
EXPERIMENTAL Design:
Heptoprotective activity of seaweeds were performed in two models
• Normal model
• Acetaminophen intoxicated model
Normal model
In this model rats were comprised into two groups
Group I: Normal (Control) group:
Rats were treated with distilled water orally for 14 days
Group II: Seaweeds treated groups: were separated into three further subgroups:
Group 1: Soleria robusta treated group
Group 2: Ulva fascicata treated group
Group 3: Sptoglossum varibalie treated group
Rats were orally treated with water extract (200mg/kg) of Soleria robusta, Ulva fascicata, and Sptoglossum varibalie separately for 14 days.
Acetaminophen model
In this model rats were comprised into three groups
Group I: Normal (Control) group:
Rats were treated with distilled water orally for 14 days
Group II: Group 2: acetaminophen control group
In this group rats were lso orally treated with distilled water for 14 days daily. On day 14 rats were intraperitoneally injected with acetaminophen (1g/kg) prepared in 40% polyethylene glycol (400) in 0.9% NaCl solution.
Group III: Seaweeds treated groups: Rats were separated into three further subgroups:
Group 1: Soleria robusta pretreated group
Group 2: Ulva fascicata pretreated group
Group 3: Sptoglossum varibalie pretreated group
Rats were orally treated with water extract (200mg/kg) of Soleria robusta, Ulva fascicata, and Sptoglossum varibalie separately for 14 days and intraperitoneally injected with acetaminophen (1gm/kg) on day 14. Before decapitation both model of rats were fasted for 12 hour. On day 15th rats were weighed and decapitated by cervical dislocation. Blood samples were collected and Serums were alienated after centrifugation for 10 minutes at 3000 rpm and stored at -20°C until used. Liver and kidney were rapidly dismembered, rinsed with ice-cold normal saline (0.9%), blotted dry, weighed and saved at -20°C for analysis.
BIOCHEMICAL ANALYSIS
Blood serum enzymes (Alanine transaminase (ALT), Alkaline phosphatase (ALP), Aspartate transaminase (ASAT), Lactate dehydrogenase (LDH) and other biochemical parameter including (glucose, urea, creatinine, cholesterol, triglycerides, Bilirubin (total & direct) were estimated on Microlab analyzer 300 (semi-automated), Merck (private) limited by using commercially available Kitts. All analysis was completed according to the standard procedures specified along with the kits purchased.
Measurement of liver Malondialdehyde (MDA) and reduced glutathione
Lipid peroxidation was determined by quantifying the concentration of malondialdehyde (MDA) as described by Ohkawa et al., (1979). Reduced glutathione was estimated according to the method of Samarth et al., (2008) with slight modifications. Liver homogenate (0.1ml) was mixed with 100 μl of 25% trichloroacetic cid (TCA) and kept for 5 minutes at room temperature in order to precipitate protein. The mixture was centrifuged at 3000 rpm for 10 minutes. 0.2ml of the supernatant was mixed with 1.8ml of 0.1mM DTNB (5, 5′-Dithiobis-2-nitrobenoic acid in 0.3M sodium phosphate buffer PH 8). The tubes were incubated for 10 minutes and absorbance was recorded against reagent blank at 412 nm.