ICAPS-2021
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Item In vitro antidiabetic activity of Spondias pinnata aqueous extract and encapsulated chitosan-TPP nanoparticles(Faculty of Science, University of Kelaniya, Sri Lanka., 2021) Wadasinghe, R. R.; Attanayake, A. P.; Kalansuriya, P.Spondias pinnata (L. f.) Kurz is a medicinal plant used in complementary medicine. Decoctions prepared using stem-bark of S. pinnata find applications in treating diabetes mellitus. However, low bioavailability of bioactive metabolites (polyphenols and flavonoids) and lack of appropriate release of metabolites delimit the antidiabetic activity of S. pinnata aqueous extract (SAE). Encapsulation of SAE with chitosan-tripolyphosphate (CS-TPP) could enhance its therapeutic potential and provide controlled release. The objective of this work to determine in vitro antidiabetic activity of S. pinnata stem-bark extracts and SAE-encapsulated CS-TPP nanoparticles using α-amylase inhibitory, α-glucosidase inhibitory, glucose uptake and glucose adsorption assay. The extracts were prepared by extracting dried and powdered stem-bark of S. pinnata into distilled water, acetone, ethyl acetate, dichloromethane under ultrasonication (40 kHz, 37 °C, 30 min) separately. The total phenol content (TPC) and flavonoid content (TFC) of the extracts were determined using Folin-Ciocalteu and aluminium chloride methods, respectively. Based on the results of α-amylase inhibitory assay, SAE was selected for the encapsulation with CS-TPP. The SAE had TPC of 4.18±0.02 mg gallic acid equivalents per gram of dry weight (GAE/g DW) and TFC of 0.37±0.01 mg quercetin equivalents per gram of dry weight (QE/g DW) and showed the highest α-amylase inhibitory activity (IC50 53.34±7.43 µg/mL). The acetone extract had TPC of 34.43±0.35 mg GAE/g DW and TFC of 4.06±0.05 mg QE/g DW and showed the highest α-glucosidase inhibitory activity (IC50 8.82±1.42 µg/mL). The highest glucose uptake and glucose adsorption were shown by acetone extract and aqueous extract, respectively. SAE-encapsulated nanoparticles were prepared from CS-TPP at varying concentrations (0.250, 0.375, 0.500 and 0.625% w/v) of SAE using ionic gelation method under magnetic stirring; the highest encapsulation efficiency (68.21% ± 0.66%) and loading capacity (0.79% ± 0.17%) were obtained at 0.625% w/v of SAE. Loaded nanoparticles were separated by centrifugation and free polyphenols were determined by Folin-Ciocalteu method. The Z-average particle diameter of SAE-encapsulated CS-TPP nanoformulations was 417±86 nm with polydispersity index of 0.574 and zeta potential of +20.63 mV. The IC50 values corresponding to α-amylase inhibitory activity and α-glucosidase inhibitory activity of SAE-encapsulated CS-TPP nanoparticles were 1.10±0.03 mg/mL and 3.16±0.15 mg/mL, respectively. Although the percentage of glucose uptake and adsorption in SAE encapsulated CS-TPP nanoparticles is lower than the crude extract, it had shown 11.59±1.03 % glucose uptake at 5 mM glucose concentration and 1.47 mmol/g glucose adsorption at 100 mM glucose concentration. The SAE, acetone extract and SAE-encapsulated CS-TPP nanoparticles showed higher antidiabetic activity than the positive control, acarbose. Further investigations on the releasing profiles of SAE-encapsulated CS-TPP nanoparticles would reveal the rates at which the active metabolites are released to the media during the timeframes of the conducted assays.Item Optimization of high-fat diet fed streptozotocin induced Wistar rat model for screening antidiabetic agents(Faculty of Science, University of Kelaniya, Sri Lanka., 2021) Wickramasinghe, A. S. D.; Attanayake, A. P.; Kalansuriya, P.High-fat diet (HFD) fed streptozotocin (STZ) induced Wistar rats are frequently used as animal models of type 2 diabetes mellitus for screening novel antidiabetic agents. As the composition of HFD, age and strain of rats, dose of STZ and the intended degree of pathophysiological changes vary among studies, the development of a model that best fits to a particular research setting is pivotal. Furthermore, ensuring the long-term stability and establishment of an adequate biochemical profile of the model are necessities which have been addressed by limited studies to date. This study attempted the development of a model which mimics type 2 diabetes mellitus for screening of novel antidiabetic drugs. Wistar rats were fed with a HFD (60% calories from fat) for four weeks, followed by STZ intraperitoneal injection (30, 40 and 50 mg/kg). Rats with fasting serum glucose >11.1 mmol/L were enrolled for the study. There were five groups (n=10/group); healthy rats, HFD fed rats, HFD+STZ (30 mg/kg) rats, HFD+STZ (40 mg/kg) rats, HFD+STZ (50 mg/kg) rats. The glycemic status of the rats was monitored weekly by the routine conduct of oral glucose tolerance tests. Experimental rats were euthanized after 28 days and blood samples were collected for biochemical investigations. Glycemic status of the model was assessed by determining fasting serum glucose, insulin, glycated hemoglobin (HbA1c) and homeostatic model assessment-insulin resistance (HOMA-IR). Lipid profiles were assessed by determining total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C) levels. STZ induced rats (30, 40 and 50 mg/kg) showed a significant dose dependent increase in fasting serum glucose (by 67, 61 and 136%) and insulin (by 19, 15 and 13%) concentrations (p<0.05). HOMA-IR was above 2.5 and increased in a dose dependent manner by 98, 108 and 176% in STZ induced rats (30, 40 and 50 mg/kg). However, only the STZ (50 mg/kg) induced group of rats showed fasting serum glucose concentration of 13.71 ± 1.01 (>11.1 mmol/L) and a significant increase in HbA1c by 66% compared to the healthy rats (p<0.05). Further, the STZ 50 mg/kg rats showed stable hyperglycemia throughout the study period. STZ induced rats (30, 40 and 50 mg/kg) also showed a significant dose dependent increase in TC (by 6, 7 and 9%), and TG (by 16, 15 and 23%) respectively (p<0.05). However, only the STZ induced (50 mg/kg) group of rats showed significant increase in serum concentrations of LDL-C (by 12%) and VLDL-C (by 16%) compared to the healthy rats (p<0.05). Only slight changes in HDL-C levels were observed in the STZ induced groups of rats however, the values were not significant (p>0.05). The results revealed that the Wistar rats fed with HFD rich in saturated fat for four weeks followed by a single intraperitoneal dose of STZ (50 mg/kg) would produce stable diabetic model which closely mimic pathophysiological features of type 2 DM characterized by insulin resistance and dyslipidemia.Item In vitro antidiabetic activity of fractionated extracts of Coccinia grandis (L.) Voigt(Faculty of Science, University of Kelaniya, Sri Lanka, 2021) Wasana, K. G. P.; Attanayake, A. P.; Jayasinghe, J. M. S.; Weeraratna, T. P.; Jayatilaka, K. A. P. W.The Paspanguwa herbal formulation is commonly consumed as a traditional medicine in Sri Lanka. Paspanguwa consists of five ingredients, namely the rhizome of Zingiber officinale (Inguru), leaves and stem of Hedyotis corymbosa (Pathpadagam), dried berries of Solanum xanthocarpum (Katuwalbatu), dried stem of Coscinium fenestratum (Venivalgata), and dried seeds of Coriandrum sativum (Koththamalli). The importance and objective of this study was to prove the antioxidant and anti-inflammatory properties of traditionally used decotion, Paspanguwa claimed to have. In the present study, water extracts of the individual ingredient and the Paspanguwa decoction were screened for their total soluble phenolic content (TPC), total soluble flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and their ability to inhibit protein denaturation (anti-inflammatory activity). The highest and the lowest TPC was seen in Coriander and ginger as 12.76 ± 1.00 and 7.89 ± 0.86 mg Gallic acid equivalent/g dry weight, respectively. The highest and the lowest TFC was seen in Katuwalbatu and Pathpadagam as 778.19 ± 1.40 and 282.14 ± 1.49 µg Catechin equivalent/g of dry weight, respectively. The lowest and the highest IC50 values for the DPPH assay was seen in Paspanguwa decoction and Katuwalbatu as 253.4 ± 8.2 and 609.7 ± 5.6 µg/mL, respectively, while the standard ascorbic acid showed 111.0 ± 6.1 µg/mL. The highest and lowest reducing power percentages were seen in Paspanguwa decoction and coriander as 94.74 ± 1.31 and 22.95 ± 0.96 while the standard ascorbic acid showed 109.89 ± 0.96. The ability to inhibit protein denaturation varied in the order of: Acetylsalicylic acid (standard) > Paspanguwa decoction > ginger > coriander > Venivalgata > Katuwalbatu > Pathpadagam at all the three concentrations (625, 1250, and 2500 µg/mL). These results suggest that Paspanguwa water extract is a good source of antioxidants with TFC and TPC with a higher ability to inhibit protein denaturation. Our findings corroborate with the previous in vitro studies of the antioxidant activity of Paspanguwa. However, our study is the first to reveal the anti-inflammatory action, total flavonoid content, and reducing power of the Paspanguwa herbal formula. Further, this study validated the use of Paspanguwa as a good source of antioxidants together with anti-inflammatory activity in traditional Ayurvedic medicine.