VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY INTERNATIONAL UNIVERSITY - TRAN THI KIM NGAN Student ID: PBTIU14001 SUMMARY OF DISSERTATION Investigation on Euphorbia hirta Linn extracts for drug development against type diabetes mellitus Doctor of Philosophy in Biotechnology HO CHI MINH CITY – June 2022 Abstract Investigation on Euphorbia hirta Linn extracts for drug development against type diabetes mellitus Tran Thi Kim Ngan School of Biotechnology International University, VNU-HCM Vietnam is a country with high biodiversity, with about 4000 species of plants used for medicine Many medicinal plants are used in folk medicine to treat diabetes mellitus, including Euphoria hirta Linn (E hirta), which belongs to the family Euphorbiaceae Previous studies show that E hirta has potential for the future production of antidiabetic drugs The overall objectives of the present study were to investigate, determine, and quantify the phytochemicals, mainly phenolic compounds, from E hirta extracts and to evaluate their antidiabetic activities in in vitro, in vivo, and in silico approaches In in silico study, flavonoids containing in E hirta gave good interactions with two digestive enzymes including alpha-amylase and alpha-glucosidase Due to variations in pharmacophore characteristics, the flavonoid family has an advantage in binding to these receptors due to relatively strong hydrogen bonds including myricetin, afzelin, kaempferol, quercetin, isoquercitrin, rutin, quercitrin, and myricitrin Using binding energy calculations and pharmacophore modelling, it was shown that there are numerous interesting chemicals in E hirta that exhibit affinity for carbohydrate hydrolysing enzymes connected to type diabetes mellitus Five fractional extracts of E hirta were obtained after the extraction process, including EM-1, EP-1, EC-1, EE-1, and EB-1 The value of the phenolic content was 109.86 ± 1.38, 90.89 ± 1.45, 55.86 ± 0.66, 254.96 ± 10.05 and 70.90 ± 0.65 mg of GAE/g extract for EM1, EP-1, EC-1, EE-1, and EB-1, respectively EE-1 had the highest total phenol content, followed by EM-1, EP-1, EB-1, EC-1 The values of the total flavonoid content were 18.92 ± 1.33, 8.48 ± 1.16, 16.41 ± 1.44, 27.66 ± 0.73 and 12.43 ± 1.66 mg of quercetin/g extract for EM-1, EP-1, EC-1, EE-1, and EB-1, respectively EE-1 had the highest total flavonoid content Ten pure compounds were isolated and identified from EE-1, including EA01 (scopoletin), EA02 (methyl gallate), EA03 (gallic acid), EA04 (kaempferol), EA05 (quercetin), EA06 (myricetin), EA07 (afzelin), EA08 (quercitrin), EA09 (myricitrin) and EA10 (rutin) To investigate the antioxidant activity of E hirta extracts, the DPPH radical scavenging assay of E hirta was carried out Our results revealed that EM-1 and EE-1 extracts had stronger free radical scavenging activity than other extracts For lipid peroxidation inhibitory activity, IC50 values of EM-1, EP-1, EC-1, EE-1, and EB-1 were also measured EE-1 extract appeared to be 18.74-fold as potent as Trolox Anticancer activities of those extracts were examined by sulforhodamine B (SRB) in vitro cytotoxicity assay on two cancer cell lines, including lung cancer cells NCI-H460 and liver cancer cells Hep G2 EE-1 at a concentration of 100 μg/mL has significant inhibitory activity in the growth of lung cancer cells NCI-H460 and liver cancer cells Hep G2 compared to all other extracts Therefore, our results are consistent with the earlier research showing the beneficial effects of phenolic and flavonoid content on free radical scavenging activity To elucidate the antidiabetic activity of those extracts, we investigated the therapeutic effects of five extracts of E hirta in AA and AG inhibitory activity (in vitro) and hypoglycemic activity in normal mice by evaluating the samples on plasma glucose in oral glucose tolerance test (in vivo) In in vitro study, E hirta extracts had strong inhibitory activity against AG and relatively mild AA inhibitory activity EE-1 had the stronger inhibitory activity of two enzymes than other extracts Besides, EE-1 expressed a higher hypoglycemic effect in oral glucose tolerance tests in normal mice than in the remaining extracts Our results suggest that ehtyl acetate extract has the most potent activities and should be used to determine phytochemicals and mechanisms of their antidiabetic activity EA extract was mixed with 20% maltodextrin in a ratio of 1:10 to spray-dry microencapsulation The spray powder reduced 51% fast blood glucose (FBG) after 4hrs treatments Furthermore, administration of spray powder for 15 days significantly lowered fasting blood glucose level in streptozotocin-diabetic mice by 23.32%, whereas, acarbose - a standard antidiabetic drug and distilled water reduced fasting blood glucose level by 30.87% and 16.89% Our results show that obtained E hirta powder has potential antidiabetic activity Among ten isolated compounds, five compounds, including EA02 (methyl gallate), EA03 (gallic acid), EA04 (kaempferol), EA05 (quercetin), and EA08 (quercitrin) were carried out the AA and AG inhibitory activity Those compounds gave weaker AA inhibitory activity than that of acarbose but it proved phenolic compounds could inhibit carbohydrate hydrolyzing enzyme and has potential antidiabetic activity The AG inhibitory action of all five compounds was stronger than acarbose Moreover, EA08 (quercitrin) was subjected to evaluate the effect in fasting plasma glucose level in normal mice This study suggests that EA extract might be employed as a functional food for type DM treatment, as well as by the nutraceutical sector as a source of quercetin and quercitrin It is clear that there are certain similarities in the results of investigating the activities of the compounds in vitro and in silico These results give us a theoretical basis for future research in the development of drugs for type diabetes mellitus Keywords: Euphorbia hirta Linn.; bioactive compounds; antidiabetic activity, alpha-amylase inhibition, alphaglucosidase inhibition, in silico approach SUMMARY OF PHD DISSERTATION The dissertation was structured into chapters Chapter 1: General introduction The first chapter of the dissertation provided as a broad introduction The purpose of the study and its contributions were also explained in Chapter Chapter 2: In silico study on antidiabetic activity of bioactive compounds Euphorbia hirta Linn Chapter presented the in silico study on antidiabetic activity of bioactive compounds in the objective of the dissertation to predict interactions of compounds with diabetes-associated proteins on antidiabetic activity by computer simulations such as molecular docking, and pharmacophore modelling Chapter 3: Preparation of Euphorbia hirta Linn extracts and investigation of bioactive compounds from ethyl acetate extract Chapter described in detail the preparation of E hirta extracts and techniques in isolation and identification of bioactive compounds from EA extract Chapter 4: Evaluation of antioxidant activity and cytotoxic activity of Euphorbia hirta Linn extracts Chapter displayed the study on the biological activity of E hirta extracts, including antioxidant activity and cytotoxic activity to find out the potential extract having higher biological activity Chapter 5: In vitro and in vivo experiments of Euphorbia hirta Linn extracts against type diabetes mellitus Chapter described the in vitro and in vivo experiments against type diabetes of E hirta extracts to elucidate the antidiabetic activity of those extracts and isolated compounds in ethyl acetate extract Chapter 6: GENERAL CONCLUSIONS AND RECOMMENDATIONS The last chapter provided conclusions about the main results, contributions, and implications of the dissertation This chapter also discussed future research directions ...Abstract Investigation on Euphorbia hirta Linn extracts for drug development against type diabetes mellitus Tran Thi Kim Ngan School of Biotechnology International University, VNU-HCM... theoretical basis for future research in the development of drugs for type diabetes mellitus Keywords: Euphorbia hirta Linn.; bioactive compounds; antidiabetic activity, alpha-amylase inhibition, alphaglucosidase... experiments of Euphorbia hirta Linn extracts against type diabetes mellitus Chapter described the in vitro and in vivo experiments against type diabetes of E hirta extracts to elucidate the antidiabetic