New Glycosyltransferases in Panax notoginseng Perfect Main Ginsenosides Biosynthetic Pathways.

Ginsenosides, the main bioactive ingredients of the Panax genus, are dammarane or oleanane triterpenoids with glycosylated modifications at C3/C6/C20 hydroxyls or C28 carboxyl, and their diverse glycosylation pattern has attracted great attention. However, the biosynthesis of some important saponins is still unclear. In this study, six UGTs were characterized, two of which were novel. PnUGT71A3 catalyzes not only the C6 hydroxyl glycosylation of protopanaxatriol (PPT) and F1 to form Rh1 and Rg1, respectively, but also the C20 hydroxyl glycosylation of protopanaxadiol (PPD)-type Rg3 to generate Rd. Especially, PnUGT94M1 is UDP-ß-l-rhamnose (UDP-Rha)-dependent, regioselectively catalyzing the C2' hydroxyl rhamnosylation of C6 glucose of the PPT-type ginsenosides Rg1 and Rh1 to generate ginsenosides Re and Rg2, respectively. Site-directed mutagenesis showed that His21, Asp120, Ser363, and Pro372 are key residues, and the triple mutant (G344S/G345S/L346T) highly improved the activity toward Rg1 and Rh1. The findings in this study, perfect main ginsenosides biosynthetic pathways in the Panax genus, expand the biocatalyst toolbox for ginsenoside production and show that the PSPG motif is one of the options to modify UGTs to improve their activities.

An integrated method for optimized identification of effective natural inhibitors against SARS-CoV-2 3CLpro.

The current severe situation of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reversed and posed great threats to global health. Therefore, there is an urgent need to find out effective antiviral drugs. The 3-chymotrypsin-like protease (3CLpro) in SARS-CoV-2 serve as a promising anti-virus target due to its essential role in the regulation of virus reproduction. Here, we report an improved integrated approach to identify effective 3CLpro inhibitors from effective Chinese herbal formulas. With this approach, we identified the 5 natural products (NPs) including narcissoside, kaempferol-3-O-gentiobioside, rutin, vicenin-2 and isoschaftoside as potential anti-SARS-CoV-2 candidates. Subsequent molecular dynamics simulation additionally revealed that these molecules can be tightly bound to 3CLpro and confirmed effectiveness against COVID-19. Moreover, kaempferol-3-o-gentiobioside, vicenin-2 and isoschaftoside were first reported to have SARS-CoV-2 3CLpro inhibitory activity. In summary, this optimized integrated strategy for drug screening can be utilized in the discovery of antiviral drugs to achieve rapid acquisition of drugs with specific effects on antiviral targets.

[Chemical constituents from traditional Chinese medicine Siegesbeckia pubescens].

Column chromatography on silica gel was used to study the chemical constituents of traditional Chinese medicine Siegesbeckia pubescens. The chemical structures of the separated compounds were elucidated by spectroscopic data analyses. As a result, eighteen compounds were obtained and identified as 3, 4'-dimethoxy quercetin(1), 3, 3', 4'-trimethoxy quercetin(2), 3, 3'-dimethoxy quercetin(3), 7, 3', 4'-trimethoxy luteolin(4), ursolic acid(5), 2ß,19α-dihydroxyursolic acid(6), 2ß-hydroxyursolic acid (7), stigmasterol-7-one(8), 5α, 8α-epidioxy-24(R)-methyl-cholesta-6, 22-diene-3ß-ol(9), ß-sitosterol(10), 2, 6-di(3-hydroxy-4-methoxyphenyl)-3, 7-dioxacyclo [3. 3. 0] octane (11), aurantiamide acetate (12), 3-(m-hydroxyl-p-methoxy)-N-(2'-p-hydroxyl-phenethyl)-2E-acrylamide(13), p-hydroxy benzaldehyde (14), m-hydroxy-p-methoxy benzaldehyde (15), 3, 4, 5-trimethoxybenzoic acid(16), monoethyl malonate(17), and p-hydroxylcinnamic acid(18). Among them, compounds 1-9, 11-18 were isolated from this plant for the first time.

Application of LightCycler polymerase chain reaction and melting curve analysis to the authentication of the traditional Chinese medicinal plant Cimicifuga foetida.

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and fluorescence melting curve analysis of LightCycler real-time polymerase chain reaction products were exploited for their applications in the authentication of the traditional Chinese medicinal plant Cimicifuga foetida from four substitutes: C. heracleifolia, C. dahurica, C. acerina, and C. simplex. According to the melting temperature--which is a function of the GC/AT ratio, length, and nucleotide sequences of the amplified product--C. foetida was differentiated from C. heracleifolia, C. dahurica, C. acerina, and C. simplex. Melting curve analysis offers a rapid and reliable method for the authentication of the traditional Chinese medicinal plant C. foetida.

Identification and quantification of the traditional Chinese medicinal plant Gentiana macrophylla using Taqman real-time PCR.

Gentiana macrophylla Pall. is a commonly used antirheumatic herb. There are four species of Gentiana recorded as herbal drugs in the Chinese Pharmacopoeia. The other species are often marketed as G. macrophylla, and thus the therapeutic effects of G. macrophylla are not achieved. A novel one-step methodology based on real-time polymerase chain reaction (PCR) technology has been developed for the identification of G. macrophylla. This relative quantification methodology does not require a known amount of standard, allowing the analysis of many more samples together. The utilization of real-time PCR does not require sample handling, preventing contamination and resulting in much faster and higher throughput results.

Differentiation of the traditional Chinese medicinal plants Euphorbia humifusa and E. maculata from adulterants by TaqMan real-time polymerase chain reaction.

DNA sequence analysis of the rDNA internal transcribed spacer 1 (ITS1) and TaqMan real-time polymerase chain reaction were exploited for their applications in the differentiation of the traditional chinese medicinal plants euphorbia humifusa and e. maculata from three related adulterants e. hypericifolia, E. atoto and E. prostrata. The data demonstrated that variations in the ITS1 regions were very low at the intra-species level but extremely high at the inter-species level, so that they could be easily distinguished at the DNA level. The sequence difference allowed an effective and reliable differentiation of E. humifusa and E. maculata from the adulterants by TaqMan real-time PCR.