Investigation on Euphorbia hirta Linn. Extracts for Drug Development Against Type 2 Diabetes Mellitus

Abstract

Vietnam is a country with high biodiversity, with about 4000 species of plants used as medicine, in which 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 enzymes. Due to variations in pharmacophore characteristics, the flavonoid family has an advantage in binding to these receptors due to relatively strong hydrogen bonds. 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 EM-1, 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, and 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. To investigate the antioxidant activity of E. hirta extracts, a DPPH radical scavenging assay of E. hirta was carried out. Our results revealed that EM-1 and EE-1 extracts had more potent 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. Anticancerxx 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 on the growth of lung cancer cells NCI-H460 and liver cancer cells Hep G2 compared to all other extracts. 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 more potent inhibitory activity of two enzymes than other extracts. Besides, EE-1 expressed higher hypoglycemic effect in the oral glucose tolerance test in normal mice than the remaining extracts. Our results suggest that EA extract (EE-2) 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 levels streptozotocin-diabetic mice by 23.32%, whereas acarbose - a standard antidiabetic drug - and distilled water reduced fasting blood glucose levels by 30.87% and 16.89%. Our results show that obtained E. hirta powder has potential antidiabetic activity. 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). Among them, five isolated 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 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 on fasting plasma glucose level in normal mice. This study suggests that EA extract might be employed as a functional food for type 2 DM treatment and by the nutraceutical sector as a source of quercetinxxi and quercitrin. 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 2 DM