Study for the beneficial effect and application of phytochemicals

Abstract

To characterize inhibitory activities of phenolic compounds against NS2B-NS3 protease of ZIKA virus (ZIKV NS2B-NS3pro) and their structure activity relationship, in this study, ZIKV NS2B-NS3pro was expressed in E. coli BL21(DE3) as 35 kDa protein. The enzyme was displayed a Km of 26.3 µM with fluorogenic peptide Dabcyl-KTSAVLQSGFRKME-Edan, and purified ZIKV NS2B-NS3pro was used for inhibition and kinetic assays with twenty-two phenolic compounds. These phenolic compounds inhibited the activity of ZIKV NS2B-NS3pro by 6.2% to 87.9%. The IC50 of seven phenolic compounds ranged from 22.2 ± 0.2 to 112.0 ± 5.5 µM. Myricetin showed mixed type inhibitory pattern against ZIKV NS2B-NS3pro protease with IC50 values of 22.2 ± 0.2 µM and Ki value of 8.9 ± 1.9 µM. Relationships between chemical structures and inhibitory activities against ZIKV NS2B-NS3pro can be further explored to develop highly selective inhibitors against ZIKV NS2B-NS3pro. Solubility plays a key role of bioavailability in human body. Rubusoside is a steviol glycoside sweetener in herbal tea and widely known for enhancing solubility in water. Stevioside, also a steviol glycoside sweetener, was commonly cultivated in many countries compared with rubusoside. β-galactosidase from Thermus thermophilus presented hydrolytic activity on β-1,2 linkage of stevioside, and can convert from stevioside to rubusoside. In this study, to industrialize production of rubusoside, firstly, β-galactosidase from T. thermophilus was expressed in E. coli BL21(DE3)plysS using lactose induction. Enzyme immobilization has several advantages for enzyme reaction. Immobilized enzyme could be repeatedly used without another method of separation and the decrease in its activity was not largely observed. Sodium alginate was used for enzyme immobilization. Stevioside conversion for free enzyme and immobilized enzyme were shown over 77%. Enzyme reaction was carried out in the vertical double-jacket glass column reactors thermostatted at 70°C for continuous reaction. Immobilized enzyme was stable for 31 days in double-jacket glass column reactors and activity of immobilized enzyme was remained 51.4% after 31 days.