peroxidation is probably the most explored area of research when it comes to ROS (Steinberg, 1997; Yla-Hertuala, 1999). It is commonly considered as a deleterious process (Benzie, 1996; Sevanian and Ursini, 2000) leading to structural modification of lipid/protein-assemblies, such as bio membranes and lipoproteins, and is usually associated with cellular modification. Cell membranes, which are structurally made up of large number of polyunsaturated fatty acids (PUFA), are highly susceptible to oxidative damage, resulting in membrane fluidity, permeability, and cellular metabolic functions (Bandhopadhyaya et al., 1999). All biological material contains a variety of PUFA, which are predominantly located in the membrane lipids. The spatial arrangement and interrelationships of these amphipathic lipids is thought to contribute to the overall susceptibility of cell membranes to free radical attack. PUFA have a characteristic methylene interrupted double bond structure. Because of double bond PUFA is susceptible to abstraction of hydrogen mediated by OH. The process of lipid peroxidation is a free radical chain reaction and involves three distinct steps; initiation, propagation and termination. PUFA reacts with a free radical to form a carbon centred radical, which further reacts with the molecular oxygen to form a lipid peroxyl radical. This is the initiation stage of lipid peroxidation process shown in Fig.5. In the propagation step the peroxyl radical reacts with another PUFA moiety, resulting in the formation of lipid hydro peroxides and lipid radicals. Lipid hydroperoxide is the first comparatively stable, product of the lipid peroxidation chain reaction (Halliwell and Gutteridge, 1985; Fuller et al., 1988). In the termination stage, these lipid or lipid peroxy radicals are either consumed or they interact with each other to form stable compound. Thus, lipid peroxidation products include lipid peroxides, hydro peroxides, epoxy alcohols, Malondialdehyde, ethane, pentane and 4-hydroxy alkenes (Bendetti et al., 1980; Mead et al., 1980; De Zwart, et al., 1998).
Collection of Plant material The medicinal plant material was collected. The plant material was Andrographis paniculata (Burm.f) Nees. Common name : Nelavemu, King of Bitters. Family : Acanthaceae. Fig.7. Morphology of Andrographis paniculata (field trip conducted for the collection of plant material) Overview Andrographis is shrub that is found throughout India and other Asian countries. It is sometimes referred to as “Indian Echinacea”. It contains as its primary chemical constituents, diterpenoid lactones (andrographolides), paniculides, farnesols and flavonoids. It was used historically in the Indian flu epidemic in 1919 during which it was credited with reversing the spread of the disease. Over the last decade, Andrographis has become popular, particularly in Scandinavia, as a treatment for colds, and is now available in the United States. Herb leaves and roots are used in different additional applications. In vitro and in vivo studies suggest that andrographis has antiinfective, antiviral,
antidiarrheal, antipyretic and analgesic activities. In addition, immunostimulant and activities have been observed in chick embryo. Preparation of Andrographis paniculata extract Powdered plant material was soaked in 95% ethanol for 48 h. The extract was distilled and dark green residue was obtained. The remaining ethanol was removed by evaporating the residue under reduced pressure. Toxicity studies of Andrographis paniculata AP extracts at a dose of 1.0, 2.0, 3.0, 4.0, 5.0 mg / egg was given to chick embryo on 12th day. The changes in body weight, liver, heart and brain weight were recorded. Levels of glucose, urea, protein, cholesterol and the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to ensure the non-toxic nature of the extract. Fixation of optimum dosage for the plant extract AP extract was given different doses 1.0, 2.0, 3.0, 4.0, and 5.0 mg / egg wt / day on 12th day of embryonic development. The dosage which gives maximum protection (as elicited by the activity of ALT and AST) against CP induced hepatic damage was fixed as the optimum dosages. Collection of embryonic tissues After collection of chick embryonic tissues of liver, heart and brain, the tissues were immediately stored in chilled cold saline at -200C. Preparation of embryonic tissue 10% homogenate (pH 7.4) 10% homogenate of the embryonic brain and heart was prepared by grinding it in Tris HCl buffer, pH 7.4. The homogenate was centrifuged at 3000 rpm Remi inflammatory
100, 200, 300, 400 and 500 µg at 12th day exposure resulted in 14%, 24%, 42%, 56% and 100% at 15th day with 100, 200, 300, 400 and 500µg exposure it was 26%, 34%, 44%, 62% and 100% respectively. The percentage of mortality was increased with proportionate of the drug concentration in all the stages of embryonic development. The lethal dose (LD50) was calculated directly from the graph drawn between percentage of mortality and concentration of CP in µg. The embryos treated on 3rd day, LD50 was assessed as a dose close to 30 µg while dose of 32µg corresponded to LD50 value of 4th day, while the values 5th , 7th and 12th and 15th day were 44, 79, 172, 320, 376 µg respectively. (Fig. 2.4-2.10). Table. 1.1: Effect of CP on Mortality of chick embryo Day CP Concentration in µg Mortality LD50 (µg) 3 day 10 32 30 20 44 40 56 80 84 100 100 4 day 10 30 32 20 40 40 52 80 76 100 100 5 day 10 28 44 20 38 40 48 80 56 100 100 7 day 10 18 79 20 32 40 46 80 50 100 100 50 16
OJECTIVE - II 2.1. CYCLOPHOSPHAMIDE INDUCED BIOCHEMICAL CHANGES IN 15 DAY – OLD CHICK EMBRYO Table 2.1 represents the biochemical parameters in chick amniotic fluid in controls and CP treated embryos. CP treatment was given on the 15th day of incubation and biochemical analysis was done after 24hrs of treatment. Significant increase (p<0.001) in the levels of glucose, uric acid, creatinine, inorganic phosphorous levels were observed with 100 µg, 200 µg and 300 µg of CP treatment, where as CP, 200 µg caused no significant increase in urea which showed an increased trend (p<0.001) with 300 µg compared with control. Protein levels were significantly increased with 100 µg (p<0.05) and with 200 and 300 µg (p<0.001) of CP treatment. Ascorbic acid, calcium and cholesterol levels were significantly decreased (p<0.001) in a dose dependent manner with CP treatment in AF.
Significant increase (p<0.001) in the levels of glucose, uric acid, Creatinine, inorganic phosphorous levels were observed with 100, 200, 300µg of CP treatment, where as CP, 200µg caused no significant increase in urea which showed an increased trend (p<0.001) with 300µg compared with control. Protein levels were significantly increased with 100 µg (p<0.05) and with 200 µg and 300 µg (p<0.001) of CP treatment. Ascorbic acid, calcium and cholesterol levels were significantly decreased (p<0.00) in a dose dependant manner with CP treatment in AF. Whereas with CP + AP treatment a significant decrease (p<0.001) in glucose, protein, urea, uric acid, Creatinine and inorganic phosphorous were observed. Glucose levels were significantly decreased (p<0.05) with 6.0mg AP treatment. Ascorbic acid levels were significantly decreased (p<0.001) with CP treatment. Whereas CP+AP caused significant (p<0.001) increase in ascorbic acid levels. The calcium levels were significantly decreased (p<0.001) with CP treatment. The cholesterol, sodium and potassium significantly decreased with CP treatment, whereas increased AP treated embryos. The enzymatic activity of alkaline Phosphatase (ALP), serum Alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) were increased considerably (p<0.001) with CP treatment in AF compared with to controls, whereas decreased with CP + AP treatment. The ionic balance was significantly decreased with CP treatment compared with to controls. Table 3.2 shows the effect of CP biochemical parameters in chick embryonic liver. Liver protein, inorganic phosphorous and ascorbic acid levels were found to significant decrease (p<0.001) with CP treatment. Increased activity of ALP, ALT, AST, LDH and MDH