Characterization and protein engineering of glycosyltransferases for the biosynthesis of diverse hepatoprotective cycloartane-type saponins in Astragalus membranaceus.

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1G146V/I variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1G146V/S and the reported AmGT8 and AmGT8A394F . The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

Expanding the Scope of Targeted Metabolomics by One-pot Microscale Synthesis and Tailored Metabolite Profiling: Investigation of Bile Acid-Amino Acid Conjugates.

Accurate metabolite characterization plays a vital role in targeted metabolomics. Nonetheless, the library of metabolites is still limited, especially for downstream conjugates, and it is time-consuming to synthesize each of these compounds due to high structural diversity. Herein, a green and smart strategy was developed to expand the scope of targeted metabolomics. The reference standards were synthesized in a one-pot microscale reaction, and the analytical method was tailored using the synthetic products. A group of new metabolites, namely bile acid-amino acid conjugates (BA-AAs), was studied as a proof-of-concept. First, in total 160 BA-AAs were synthesized using a small amount (2 mg each) of bile acids and low-toxic reagents within 4 h. Then, an ultra-high-performance liquid chromatography /Orbitrap-MS method was established to comprehensively profile 202 bile acid derivatives in 20 min. Finally, the method was applied to mice with inflammatory bowel disease (IBD) to discover the accumulation of 70 rare BA-AAs in small intestine and liver, where 55 were first reported from biosamples. These BA-AAs are farnesoid X receptor modulators and might contribute to the development of IBD. Our study demonstrated a feasible approach for the broad-spectrum targeted metabolomics of bile acids.

AmAT19, an acetyltransferase from Astragalus membranaceus, catalyses specific 6α-OH acetylation for tetracyclic triterpenes and steroids.

Tetracyclic triterpenes and steroids are pharmacologically important molecules, and acetylation could improve their bioactivities. In this study, a highly regio- and stereo-specific acetyltransferase, AmAT19, was discovered from Astragalus membranaceus. AmAT19 could selectively catalyze the 6α-OH acetylation of four tetracyclic triterpenes and steroids. The strict selectivity is associated with different orientations of the 6α/ß-OH as indicated by molecular docking. Acetylated products 1a, 3a and 4a remarkably increased the inhibitory activity against the 3-chymotrypsin-like protease (3CLpro) of SARS-CoV-2, compared to 1, 3, and 4. AmAT19 could be a promising catalyst for specific 6α-OH acetylation to expand the molecular diversity of triterpenes and steroids.

Diterpenoids from Euphorbia glomerulans with potential reversal activities against P-glycoprotein-mediated multidrug resistance.

The development of collateral sensitivity agents that are able to modulate P-glycoprotein (P-gp) is the most promising approaches to overcome multidrug resistance (MDR) in cancer. In this study, eight new diterpenoids of jatrophane and ingenane type, 1-8, and three known ones (9-11) were isolated from Euphorbia glomerulans. Their structures were elucidated by spectroscopic analysis and electronic circular dichroism (ECD) calculations. The MDR reversal activity evaluation of these isolates on breast cancer MCF-7/ADR cells demonstrated the four potent MDR modulators (3, 4, 5, and 9) with great chemoreversal ability and low cytotoxicity. The structure-activity relationship (SAR) analysis indicated that the presence of isobutanoyloxy group at C-8 significantly enhance reversal efficiency. Compound 5 exhibited high efficacy (EC50 = 159.5 nM) in reversing MDR resistance, being stronger than verapamil (EC50 = 302.9 nM). The MDR reversal mechanism assays revealed that 5 could promote the accumulation of Rh123 and DOX in drug-resistant cells in a certain dose-dependent manner, and inhibit P-gp transport function. In addition, the possible recognition mechanism of compound 5 and verapamil (VRP) with P-gp was predicted by molecular docking.

Jatrophane Diterpenoids from Euphorbia glomerulans.

In a phytochemical investigation of the whole plant of Euphorbia glomerulans, 17 new (1-17) and five known jatrophane diterpenoids (18-22) were identified. The X-ray crystallographic data of compounds 1, 4, and 21 permitted the definition of the absolute configurations of these compounds. The cytotoxicity and multidrug resistance reversal activities of the 17 new compounds were evaluated on multidrug-resistant MCF-7/ADR cells overexpressing P-glycoprotein. Several compounds showed different chemoreversal activities and considerably decreased cytotoxicity. Compounds 11 (IC50 value of 5.0 ± 0.8 µM) and 12 (IC50 value of 5.2 ± 2.0 µM) possessed MDR reversal activities that were as good as that of verapamil (IC50 value of 4.7 ± 0.6 µM).

A new anti-vitiligo unsaturated fatty glycoside from Euphorbia humifusa Willd.

A new unsaturated fatty glycoside Humionoactosides A, together with four known compounds were isolated from the whole plant of Euphorbia humifusa Willd. Their structures were identified by MS/MS, NMR and ECD spectroscopic analyses, and comparison with the literature data. All compounds were evaluated for the effect on melanogenesis and tyrosinase activity use B16 cells in vitro, and the results showed that compound 1 could significantly improve the formation of melanin (138.7%, 50 µM) and tyrosinase activity (137.2%, 50 µM) compared with the positive control 8-MOP (125.2%, 138.9%, 50 µM) in a dose-dependent manner. This is the first time that those compounds reported from Euphorbia humifusa Willd. with potential anti-vitiligo activity.

A pharmacokinetic-pharmacodynamic study to elucidate the cardiovascular protective constituents in Danhong Injection.

Danhong Injection (DHI) is one of the most popular Chinese medicine formulations to treat cardiovascular diseases. However, the effective components of DHI have not been well addressed. In the present study, a pharmacokinetics-pharmacodynamics (PK-PD) approach was employed to elucidate the effective compounds of DHI for the first time. Firstly, the cardiovascular protective effect of DHI was demonstrated on an adrenaline-induced acute blood stasis rat model by echocardiography and histopathology. Secondly, the levels of four blood stasis-related cytokines in plasma were examined by ELISA. Thirdly, the plasma concentrations of 10 compounds in DHI were determined using UHPLC-Q-Orbitrap-MS. Finally, PK-PD profiles were established to describe the relationship between compound concentrations and cytokine levels in plasma at 0-12 h following DHI administration. The results showed that DHI attenuated cardiovascular injury and regulated IL-2, cTnT, VEGF, and VEGFR-1. Except for the endogenous metabolites cytidine and uridine, danshensu, rosmarinic acid, and salvianolic acid B exhibited the highest plasma exposure. PK-PD correlation analysis indicated that concentrations of salvianolic acid A, caffeic acid, and ferulic acid were negatively correlated with the level of cTnT, while the concentration of salvianolic acid A was negatively correlated with the level of IL-2. These compounds may contribute to the cardiovascular protective effect of DHI.

Bioactive prenylated phenolic compounds from the aerial parts of Glycyrrhiza uralensis.

In this work, a bioassay-guided fractionation strategy was used to isolate 26 phenolic compounds from the ethyl acetate partition of an ethanol extract of the aerial parts of Glycyrrhiza uralensis Fisch. ex DC. Among them, 8 prenylated phenolic compounds (glycyuralins Q-X) were described for the first time. The two enantiomers of glycyuralin Q were purified and their absolute configurations were established by ECD spectral calculations. (1″R, 2″S)-glycyuralin Q and (1″S, 2″R)-glycyuralin Q showed significant inhibitory activities against SARS-CoV-2 virus proteases 3CLpro with IC50 values of 1.5 ± 1.0 and 4.0 ± 0.3 µM, and PLpro with IC50 values of 2.4 ± 0.2 and 1.9 ± 0.1 µM, respectively. Four compounds showed potent cytotoxic activities against A549, Huh-7, and HepG2 human cancer cells with IC50 values ranging from 0.5 to 2.5 µM.

GuRhaGT, a highly specific saponin 2''-O-rhamnosyltransferase from Glycyrrhiza uralensis.

A highly regio- and donor-specific 2''-O-rhamnosyltransferase GuRhaGT was characterised from the medicinal plant Glycyrrhiza uralensis. GuRhaGT could efficiently catalyse rhamnosylation at 2''-OH of the C-3 glycosyl moiety of triterpenoid saponins.

A highly selective 2''-O-glycosyltransferase from Ziziphus jujuba and De novo biosynthesis of isovitexin 2''-O-glucoside.

A novel and efficient 2''-O-glycosyltransferase ZjOGT38 was identified from Ziziphus jujuba. It could regio-selectively glycosylate 2-hydroxyflavanone C-glycosides. ZjOGT38 allowed de novo biosynthesis of isovitexin 2''-O-glucoside in E. coli.

Spectrum-effect relationship between UPLC fingerprints and antidiabetic and antioxidant activities of Rosa rugosa.

In this study, the antidiabetic and antioxidant properties of the chemical constituents of Rosa rugosa Thunb. (R. rugosa) was evaluated through analysis of spectrum-effect relationship. The ultra-performance liquid chromatography (UPLC) fingerprints of 21 batches of R. rugosa were evaluated by similarity analysis (SA) and hierarchical clustering analysis (HCA). The 28 common components were identified by ultra-high-performance liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-orbitrap-HRMS/MS). Meanwhile, the antidiabetic activities and antioxidant activities of 21 batches of R. rugosa were estimated in vitro. Besides, four chemometrics named principal component analysis (PCA), grey correlation analysis (GRA), partial least squares regression (PLSR) and the bivariate correlations analysis (BCA) were applied to construct spectrum-effect relationship between the UPLC fingerprints and biological activities of R. rugosa. The spectrum-effect relationship study revealed that di-O-galloyl-HHDP-glucoside, galloyl-HHDP-glucoside and avicularin were more relevant to antidiabetic activity. Di-O-galloyl-HHDP-glucoside, galloyl-HHDP-glucoside and ellagic acid were the main antioxidant components of R. rugosa. The current bioassay and spectrum-effect relationships are proper for associating sample quality with the active ingredient, and our finding would provide foundation and further understanding of the quality evaluation and quality control of R. rugosa.