Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris.

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K ( m ) of 15 ± 2 µM with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoid compounds were 47-381 µM. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47 µM, respectively. GCG showed a competitive inhibition pattern with K ( i ) value of 25 ± 1.7 µM. In molecular docking experiments, GCG displayed a binding energy of -14 kcal mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.

Variation of Triterpenic Acids in 12 Wild Syzygium formosum and Anti-Inflammation Activity on Human Keratinocyte HaCaT.

Syzygium formosum (Wall.) Masam leaf is known as a Vietnamese traditional herbal medicine used to treat atopic dermatitis and stomach ulcers. Recently, its potent anti-allergic effects were reported with possible active compounds analysis. Here, we collected S. formosum leaves from 12 wild trees and compared compositions of triterpenic acids (TA) with Centella asiatica. Anti-inflammatory activities of S. formosum leaf extract (SFLE) was compared with C. asiatica extract (CAE) using human keratinocyte, HaCaT. In this study, up to seven TAs were identified in SFLE, while only madecassic and asiatic acids were detected in the CAE. Total TA content varied among SFLE, but asiatic, corosolic, and betulinic acids were the major components. Surprisingly, wild tree sample 12 (S12) contained total TA of 27.2 mg/g dry-leaves that was 5-fold greater than that in the C. asiatica sample, and S4 had the highest content of asiatic acid (12.6 mg/g dry-leaves) that accounted for 50% of the total TA. S4 and S12 showed more than 3-fold higher anti-oxidative power than the CAE. In the UVB irradiation model, S4 and S12 (5 µg/mL) strongly repressed mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-6, and IL-8) and COX-2, while the CAE at the same condition showed moderate or weak repression. The difference in anti-inflammation effects between the SFLE and the CAE was also confirmed by protein quantifications. Taken together, SFLE has great potentials as a new cosmeceutical ingredient with a higher amount of skin-active phytochemicals.

Identification of a naturally-occurring 8-[alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]daidzein from cultivated kudzu root.

INTRODUCTION: Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one-year-old kudzu root cultivated in Vietnam. OBJECTIVE: To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI(+) TOF MS-MS, (1)H-, (13)C-NMR and enzymatic hydrolysis. METHODOLOGY: Samples were prepared by extraction of one-year-old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV-light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the (13)C and (1)H NMR spectra recorded at 100.40 and 400.0 MHz, respectively. RESULTS: ESI(+) TOF MS-MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by (1)H-, (13)C-NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an alpha-1,6-glycosidic bond. This compound is the first naturally-occurring 8-[alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl-alpha-1,6-puerarin in kudzu root was 2.3 mg/g as determined by HPLC. CONCLUSION: The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water-soluble glycosylated puerarin.