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ThesisItem Open Access BIOACTIVITY OF LEAF EXTRACTS OF SIMAROUBA GLAUCA AND ITS HEPATOPROTECTIVE EFFECT(AAU, Jorhat, 2021) Islam, Ashfeeka; Bhattacharyya, RumaThe present investigation entitled “Bioactivity of leaf extracts of Simarouba glauca and its hepatoprotective effect” was carried out in order to study the proximate composition, phytochemical estimation, antioxidant activities of Simarouba glauca leaf extracts and its in vivo study for hepatoprotective activity. The leaf was collected from Assam Agricultural University and thereafter a series of laboratory experiments were carried out to fulfill the objectives of the present study. The fresh leaves were dried and powdered to carry out different analytical procedures. The nutrient composition of the Simarouba glauca leaf powder was determined where the moisture content was 9.01 gm/100 gm of the sample. The protein content of the leaf powder was 12.42 gm/100 gm, the fat was 4.33 gm/100 gm, the fibre was 27.95 gm/100 gm, the ash content was 3.29 gm/100 gm, the carbohydrate content was 39.81 gm/100 gm and the dry matter was 90.99 gm/100 gm of sample. The vitamin content of the Simarouba glauca leaf powder was evaluated where the presence of thiamin was 0.71 mg/100 gm, riboflavin was 0.42 mg/100 gm, niacin was 1.59 mg/100 gm, ascorbic acid was 22.1 mg/100 gm and vitamin A was 5.01/100 gm of the sample. Fluorescence was estimated in Simarouba glauca leaf powder under visible and ultra violet light at 245 nm. The leaf powder when treated with different reagents such as sodium hydroxide, sodium chloride, potassium hydroxide, sulphuric acid, nitric acid, acetic acid, chloroform, ethanol, methanol, iodine and water exhibited bright green, dark brown, brown, pale green, yellowish green, black, reddish brown, red, light yellow and green colour under visible day light and ultra violet light. The extraction yield of Simarouba glauca leaf extracts ranged from 1.87-2.30 per cent where the water extracts of Simarouba glauca leaf was highest (2.30 per cent) and it was lowest in methanol extracts of Simarouba glauca leaf (1.87 per cent). The preliminary phytochemical screening showed presence of flavonoids, terpenoids, phenols, anthraquinones and glycosides in the chloroform extracts of the leaf. Presence of alkaloids, flavonoids, terpenoids, steroids, tannins, phenols and glycosides were found in the ethanol extracts of Simarouba glauca leaf. The methanolic extract of Simarouba glauca leaf had alkaloids, flavonoids, terpenoids, steroids, tannins, phenols and glycosides. In the water extracts of Simarouba glauca leaf, presence of flavonoids, tannins and phenols was found. Quantitative analysis of phytochemical constituents was done where the total phenols ranged from 0.29-1.67 mg GAE/100 gm which was highest in the ethanolic extract and lowest in methanolic extract. The total flavonoid ranged from 0.303-0.497 mg QE/gm with the highest value in chloroform extract and lowest in methanol extract. The percentage inhibition of DPPH free radical scavenging activity was determined which showed maximum inhibition at increased concentration of the Simarouba glauca leaf extract. The per cent inhibition in chloroform extracts of the leaf were in the range of 69.64-91.60 per cent according to the increase in level of concentration from 100 - 500 mg. It ranged from 64.22-73.30 per cent in ethanolic extract, 59.85 – 90.50 per cent in methanolic extract and 30.41-83.52 per cent in water extracts of Simarouba glauca leaf with its increasing level of concentration. Antibacterial activity of Simarouba glauca leaf extracts was studied on Pseudomonal aeruginosa, Selmonella typhi and Selmonella paratyphi. Among the different concentration viz., 50, 100, 150, 200 and 250 mg/ml of chloroform, ethanol, methanol and water extracts of Simarouba glauca leaf were tested of which 250 mg/ml produced highest inhibitory activity against the three gram negative pathogens. The antibacterial activity of the chloroform and ethanol extracts of Simarouba glauca leaf showed highest inhibition of 20.67 mm and 18.33 mm at 250 mg/ml concentration respectively in Salmonell typhi. The zone of inhibition of methanol and water extracts of Simarouba glauca leaf was 23.33 mm and 15.33 mm respectively in Pseudomonas aeruginosa at 250 ml concentration which was the highest. The viability of HCT 116 cell lines with extracts of Simarouba glauca leaf was examined where the cell viability decreased with increase in concentrations of leaf extracts of Simarouba glauca hence increasing the per cent inhibition. The inhibition of chloroform, ethanol, methanol and water extracts of Simarouba glauca was highest at 450 μg/ml concentration which was 98.77 per cent, 96.89 per cent, 98.93 per cent and 98.84 per cent respectively. Acute toxicity study on experimental animals showed no change in behavior, eating habit, sleep and mortality on administration of different dosage of chloroform, ethanol, methanol and water extracts of Simarouba glauca in two phases of the experiment. The triglyceride level in blood samples of experimental animals fed with 400 gm/kg b.w. of CHSG decreased significantly to 93.5 mg/dl from 102.313 mg/dl in the toxic group. The cholesterol level decreased significantly from 181.39 gm/dl to 140.39 gm/dl in the group fed with 400 gm/kg b.w. of CHSG. The alkaline phosphatase level decreased from 90.65 U/L in toxic group to 64.52 U/L in the group fed with 400 gm/kg b.w . of EHSG. The glucose level decreased significantly to 92.27 mg/dl in the group fed with 400 gm/kg b.w. of CHSG from 96.56 in the toxic group. Significant decrease was observed in the SGPT level of the group fed with 400 gm/kg b.w. of EHSG which decreased to 51.18 U/L from 82.06 in toxic group. The SGOT level decreased significantly from 115.24 U/L in toxic group to 96.41 U/L in the group fed with 400 gm/kg b.w. of EHSG. In vivo histopathological study on paracetamol induced changes in liver cured by Simarouba glauca leaf extracts was conducted. 42 male albino rats were sacrificed (7 groups, 6 rats each) by cervical dislocation and detail necropsy were performed. Representative tissue samples from liver were collected and stored in formal saline solution for histopathological evaluation. The sections of liver in control group showed normal hepatocytes with pink staining cytoplasm and blue staining vesicular nucleus with characteristic hexagonal shape. The central veins were clearly visible and were normal. The hepatocytes were arranged in the cord like fashion showing characteristic hexagonal shape of the hepatic cells which were surrounding the central vein. The microscopic sections of liver of the toxic group showed moderate to severe degeneration of hepatocytes with a condensed pyknotic nucleus with distortion in the architecture in hepatic lobules. The hepatic cords were distorted. Formations of pseudo-lobules were also observed in this group. Blood vessels were severely congested. Necrotic changes were also observed and mild to moderate fibrous tissue proliferation were recorded. Infiltrations of mononuclear cells were another characteristic observation recorded in this group. The severity of the lesions increased towards the central part of the lobule. The degenerated hepatocytes showed granular eosinophilic cytoplasm. The histopathological study in the standard group revealed normal hepatic architecture of the hepatocyte. Mild congestion of blood vessels were observed in scattered area. Also mild degree of cellular swelling was observed in few hepatocytes. This might be indicative of progressive healing of liver damage induced by paracetamol toxicity. Microscopic sections of liver of the group fed with 200 mg CTSG showed congestion of blood vessels. No degenerative changes were observed. Mild to moderate haemorrhagic changes were also observed. The histopathological sections of liver of the group fed with 400 mg CHSG showed normal hepatic architecture with clear pink cytoplasm and blue stain nucleus. The hepatocytes were arranged in the cord like fashion showing characteristic hexagonal shape of the hepatic cells. No degenerative changes were observed. Blood vessels were congested with mild haemorrhages in the organs and no infiltrating cells were observed. In the group fed with 200 mg/kg body weight of ETSG, no hepato-cellular degenerative changes were observed in the hepatocytes during the microscopic study. The liver sections were showed presence of moderate to severe haemorrhages and congestion in the blood vessels. Severe congestion and haemorrhages were invariably seen in the microscopic section of liver of group fed with 400 mg/kg body weight of ETSG. Sinusoidal spaces were dilated. Hepatocytes were showed normal architecture with pink stained cytoplasm and blue staining nucleus. Therefore it is evident from the present study that leaves of Simarouba glauca had theraputic and medicinal properties which would be beneficial in the cure of liver diseases.