Global Profiling and Novel Structure Discovery Using Multiple Neutral Loss/Precursor Ion Scanning Combined with Substructure Recognition and Statistical Analysis (MNPSS): Characterization of Terpene-Conjugated Curcuminoids in Curcuma longa as a Case Study.

To fully understand the chemical diversity of an herbal medicine is challenging. In this work, we describe a new approach to globally profile and discover novel compounds from an herbal extract using multiple neutral loss/precursor ion scanning combined with substructure recognition and statistical analysis. Turmeric (the rhizomes of Curcuma longa L.) was used as an example. This approach consists of three steps: (i) multiple neutral loss/precursor ion scanning to obtain substructure information; (ii) targeted identification of new compounds by extracted ion current and substructure recognition; and (iii) untargeted identification using total ion current and multivariate statistical analysis to discover novel structures. Using this approach, 846 terpecurcumins (terpene-conjugated curcuminoids) were discovered from turmeric, including a number of potentially novel compounds. Furthermore, two unprecedented compounds (terpecurcumins X and Y) were purified, and their structures were identified by NMR spectroscopy. This study extended the application of mass spectrometry to global profiling of natural products in herbal medicines and could help chemists to rapidly discover novel compounds from a complex matrix.

Biotransformation of 20(R)-panaxadiol by the fungus Rhizopus chinensis.

Microbial transformation of 20(R)-panaxadiol by the fungus Rhizopus chinensis CICC 3043 yielded seven metabolites. Their structures were elucidated on the basis of extensive spectroscopic analyses. R. chinensis could catalyze hydroxylation and further dehydrogenation at C-24 of 20(R)-panaxadiol, as well as hydroxylation at C-7, C-15, C-16, and C-29. Three of these compounds at 10µM could moderately inhibit growth of HepG2 human hepatocellular carcinoma cells with an inhibition rate of about 40%. Three compounds (also at 10µM) showed approximately 30% inhibition on NF-κB transcriptional activity in SW480 human colon carcinoma cells stably transfected with NF-κB luciferase reporter and induced by LPS.

Smith degradation, an efficient method for the preparation of cycloastragenol from astragaloside IV.

Cycloastragenol (CA) is the genuine sapogenin of astragaloside IV (ASI). This study focuses on the preparation of CA from ASI. Five hydrolysis methods were compared including H2SO4 hydrolysis, HCl hydrolysis, two-phase acid hydrolysis, mild acid hydrolysis, and Smith degradation. Seven hydrolysis products were purified, and five of them were identified as new compounds. The results indicated that Smith degradation was the most effective approach to prepare CA. In contrast, mild acid hydrolysis produced CA at a low yield, accompanied with the artificial sapogenin astragenol. The other three acid hydrolysis methods mainly produced astragenol. Furthermore, the reaction conditions for Smith degradation were optimized as follows: ASI was dissolved in 60% MeOH-H2O solution, oxidized with 5 equiv. NaIO4 for 12h, followed by reduction with 3 equiv. NaBH4 for 4h, and finally acidified with 1M H2SO4 at pH2 for 24h. Under the optimal conditions, CA could be prepared from ASI at a yield of 84.4%.

Comprehensive chemical analysis of the rhizomes of Drynaria fortunei by orthogonal pre-separation and liquid chromatography mass spectrometry.

The chemical composition of Drynaria fortunei, a traditional Chinese herbal medicine, is very complicated. In order to separate these chemicals to obtain their structural information, an orthogonal sample enrichment procedure was established. The ethyl acetate extract of D. fortunei was pre-separated by Sephadex LH-20 × polyamide columns to yield 15 fractions. These fractions were analyzed successively using a reversed-phase Agilent Zorbax SB-C18 column, coupled with diode array detection and electrospray ionization tandem mass spectrometry. The method reduced co-elution and enriched minor compounds on the basis of their chemical features. A total of 369 compounds were detected by LC/MSn, compared to less than 50 compounds without pre-separation. The pretreatment facilitated the analytical separation of flavonoids, proanthocyanidins, triterpenoids, phenolic acids, and lignans in D. fortunei, and allowed a comprehensive chemical profiling of these constituents. This method could be applied to other multicomponent herbal extracts.

Simultaneous determination of five minor coumarins and flavonoids in Glycyrrhiza uralensis by solid-phase extraction and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

Minor phenolic compounds in licorice (Glycyrrhiza uralensis) have recently been proved for diverse bioactivities and favorable bioavailability, indicating that they may play an important role in the therapeutic effects or herb-drug interactions of licorice. However, so far, their abundance in licorice remains unknown. In this study, a reliable solid-phase extraction coupled with a high-performance liquid chromatography and diode array detection method was established to determine the minor phenolic compounds in licorice. The analytes were enriched by a three-step solid-phase extraction method, and then separated on a YMC ODS-A column by gradient elution. Five coumarins and flavonoids were identified by electrospray ionization tandem mass spectrometry, and then quantified using high-performance liquid chromatography and diode array detection. The amounts of glycycoumarin, dehydroglyasperin C, glycyrol, licoflavonol, and glycyrin in G. uralensis were 0.81 ± 0.28, 1.25 ± 0.59, 0.20 ± 0.08, 0.12 ± 0.04, and 0.17 ± 0.08 mg/g, respectively. Abundances of these compounds in other Glycyrrhiza species (G. glabra, G. inflata, and G. yunnanesis) were remarkably lower than G. uralensis.

Identification of key licorice constituents which interact with cytochrome P450: evaluation by LC/MS/MS cocktail assay and metabolic profiling.

Licorice has been shown to affect the activities of several cytochrome P450 enzymes. This study aims to identify the key constituents in licorice which may affect these activities. Bioactivity assay was combined with metabolic profiling to identify these compounds in several complex licorice extracts. Firstly, the inhibition potencies of 40 pure licorice compounds were tested using an liquid chromatography/tandem mass spectrometry cocktail method. Significant inhibitors of human P450 isozymes 1A2, 2C9, 2C19, 2D6, and 3A4 were then selected for examination of their structural features by molecular docking to determine their molecular interaction with several P450 isozymes. Based on the present in vitro inhibition findings, along with our previous in vivo metabolic studies and the prevalence of individual compounds in licorice extract, we identified several licorice constituents, viz., liquiritigenin, isoliquiritigenin, together with seven isoprenylated flavonoids and arylcoumarins, which could be key components responsible for the herb-drug interaction between cytochrome P450 and licorice. In addition, hydrophilic flavonoid glycosides and saponins may be converted into these P450 inhibitors in vivo. These studies represent a comprehensive examination of the potential effects of licorice components on the metabolic activities of P450 enzymes.

Three new phenolic compounds from the roots of Glycyrrhiza yunnanensis.

Three new phenolic compounds (1-3), together with 16 known compounds (4-19), were isolated from the roots of Glycyrrhiza yunnanensis Cheng f. et L. K. Dai. On the basis of 1D and 2D NMR, HR-ESI-MS, and circular dichroism (CD) spectroscopic analysis, structures of the new compounds were elucidated as 2-(2'-methoxy-4'-hydroxy)-aryl-3-methy-6-hydroxy-benzofuran (1), (2S)-6,7-(2,2-dimethyldihydropyrano)-8-prenyl-4'-hydroxyflavanone (2), and 6-prenyl-7,3',4'-trihydroxyflavone (3). Compounds 1, 3, 5, 12, 14, 15 and 16 showed antioxidant activity by an ABTS-based assay.

Two new oxidation products obtained from the biotransformation of asiatic acid by the fungus Fusarium avenaceum AS 3.4594.

Asiatic acid (AA) is a natural triterpenoid possessing anti-inflammatory, anticancer, neuroprotective, and hepatoprotective activities. Structural modification of AA may provide valuable information for further structure-activity relationship analysis. Biotransformation is an efficient, specific, and environment friendly technology for structural modification of complicated natural products. In this study, the capabilities of twenty-five strains of filamentous fungi to transform AA were screened. Two new and one known oxidation products metabolized by Fusarium avenaceum AS 3.4594 were isolated. Their chemical structures were characterized as 2-oxo-3ß,15α,23-trihydroxyurs-12-en-28-oic acid (1), 3-oxo-2,15α,23-trihydroxyurs-1,12-dien-28-oic acid (2), and 2-oxo-3ß,23-dihydroxyurs-12-en-28-oic-acid (3) by extensive analysis of spectroscopic data.

Effects of volatile components and ethanolic extract from Eclipta prostrata on proliferation and differentiation of primary osteoblasts.

Eclipta prostrata, an aromatic plant, is known in Chinese herbal medicine for the treatment of various kidney diseases. In the present study, the volatile components were isolated from the aerial parts of this plant by hydrodistillation and analysed by GC-MS. A total of 55 compounds, which were the major part (91.7%) of the volatiles, were identified by matching mass spectra with a mass spectrum library (NIST 05.L). The main components were as follows: heptadecane (14.78%), 6,10,14-trimethyl-2-pentadecanone (12.80%), n-hexadecanoic acid (8.98%), pentadecane (8.68%), eudesma-4(14),11-diene (5.86%), phytol (3.77%), octadec-9-enoic acid (3.35%), 1,2-benzenedicarboxylic acid diisooctyl ester (2.74%), (Z,Z)-9,12-octadecadienoic acid (2.36%), (Z)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene (2.08%) and (Z,Z,Z)-1,5,9,9-tetramethyl-1,4,7-cycloundecatriene (2.07%). The effects of volatile components and ethanolic extract from the aerial parts of this plant on the proliferation and differentiation of primary osteoblasts were evaluated by the MTT method and measuring the activity of alkaline phosphatase (ALP activity). Both volatile components and ethanolic extract (1 microg/mL to 100 microg/mL) significantly (p < 0.01) stimulated the proliferation and increased the ALP activity of primary osteoblasts. These results propose that E. prostrata can play an important role in osteoblastic bone formation, and may possibly lead to the development of bone-forming drugs.