New prenylated flavonoid glycosides derived from Epimedium wushanense by ß-glucosidase hydrolysis and their testosterone production-promoting effects.

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3ß-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.

Steroidal saponins from Trillium tschonoskii rhizomes and their cytotoxicity against HepG2 cells.

A phytochemical study on the rhizomes of Trillium tschonoskii led to the isolation of fourteen new steroidal saponins, trillitschosides S1-S14 (1-14), along with ten known analogues (15-24). Their structures were established mainly by spectroscopic analyses as well as necessary chemical evidence. All isolated compounds were screened for the cytotoxicity against HepG2 cells, and the results demonstrated that only the known compounds 21-24 exhibited the remarkable cytotoxic activity against HepG2 cells which is much better than the positive control of 5-FU.

[Change of icariin content in five Epimedium species by heating process].

The change of icariin( ICA) content in thirty-three samples of five Epimedium species listed in the Chinese Pharmacopoeia( 2015 edition),including E. brevicornu,E. sagittatum,E. pubescens,E. koreanum,and E. wushanense has been investigated in this study. The results indicated that the optimized process procedure was baking at 150 ℃ for 30 min,and 3'''-carbonyl-2″-ß-L-quinovosyl icariin( CQICA) could not be translated into ICA and ICA could be converted under this heating process condition. ICA increased remarkably after the heating process by 1-3 times in E. brevicornu,E. wushanense and E. koreanum,and increased lightly in E. brevicornum and E. pubescens,while ICA slightly increased or decreased in E. sagittatum and E. wushanense.

[Analysis of chemical markers in sulfur-fumigated Anemarrhenae Rhizoma].

Sulfur-containing Anemarrhenae Rhizoma decoction pieces were prepared by using sulfur-fumigating procedure. The difference components before and after sulfur fumigation in Anemarrhenae Rhizoma were analyzed and on-line identified by UPLC-Q-TOF-MSE combined with UNIFI informatics platform, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) respectively. As a result, 16 major differences components were identified, and among them, 9 components were mainly from sulfur-fumigated samples. The main chemical markers in sulfur-fumigated Anemarrhenae Rhizoma were identified as the sulfite derivatives newly produced after sulfur-fumigating. Meanwhile, UPLC-Q-TOF-MSE was used to find the main chemical marker anemarrhena saponin BⅡ sulfite (m/z 983). By using this method, a rapid screening method for sulfur-fumigated Anemarrhenae Rhizoma was established. This was a convenient and accurate detection method for sulfur dioxide residue, and it can be used as an effective assistant method to control the quality of Anemarrhenae Rhizoma. At the same time, it was the first time to identify sulfited derivatives of steroidal saponins, and screen the sulfur-fumigated Anemarrhenae Rhizoma.

[Determination of three saponins in rhizoma and fibrous root of Trillium tschonoskii and Trillium kamtschaticum].

To compare the differences of main components between in rhizoma and fibrous root of Trillium tschonoskii and T. kamtschaticum, a simple, accurate and reliable high performance liquid chromatography coupled with the charged aerosol detector (HPLC-CAD) method was developed and then successfully applied for simultaneous quantitative analysis of three compounds, including polyphyllin Ⅶ (T1),pennogenin 3-O-α-L-rhamnopyranosyl-(1→2) [α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranoside (T2),polyphyllin Ⅵ (T3), in 16 batches of rhizome and 14 batches of fibrous root. The analytes were well separated from other constituents on TSK gel ODS (4.6 mm×250 mm, 5 µm) with acetonitrile-water (43∶57) at a flow rate of 1.0 mL•min⁻¹. The injection volume was 20 µL. The nitrogen inlet pressure for the CAD system was 35 psi and the nebulizer chamber temperature was 35 ℃.The method was validated for linearity (r>0.999 0), intra and inter-day precision (0.29%-3.0%), repeatability (0.45%-1.4%), stability (1.9%-2.6%), recovery (100.1%-100.2%, 1.2%-1.8%), limits of detection (0.002 g•L⁻¹), and limits of quantification (0.005 g•L⁻¹).The obtained datasets were processed by principal component analysis (PCA) and it showed that there was almost no difference in rhizoma of T. tschonoskii and T. kamtschaticum from different areas of China. However, the 3 major compounds existed in rhizoma were different from those in fibrous root of T. tschonoskii and T. kamtschaticum.

Three New Sesquiterpene Glycosides from the Rhizomes of Trillium tschonoskii.

Three new sesquiterpene glycosides, possessing a rare aglycone with a sulfonyl between C-1 and C-15 positions, named 3-(3'E-7'R,8'-dihydroxy-4',8'-dimethyl-3'-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7'-O-ß-d-glucopyranosyl-(1→4)-O-ß-d-glucopyranosyl-(1→4)-O-ß-d-glucopyranoside (1), 3-(3'E-7'R,8'-dihydroxy-4',8'-dimethyl-3'-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7'-O-ß-d-glucopyranosyl-(1→4)-O-ß-d-glucopyranoside (2), and 3-(3'E-7'R,8'-dihydroxy-4',8'-dimethyl-3'-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7'-O-ß-d-glucopyranosyl-6'-O-acetyl-(1→4)-O-ß-d-glucopyranosyl-(1→4)-O-ß-d-glucopyranoside (3), respectively, were isolated from the rhizomes of Trillium tschonoskii. Their structures were established on the basis of spectroscopic data, including HR-ESI-MS, IR, 1D and 2D NMR. The cytotoxic properties of the three compounds were investigated using human hepatic L02 cells.

[Chromatographic fingerprint analysis of Toosendan Fructus by HPLC-CAD coupled with chemometrics methods].

A new method was developed for the chromatographic fingerprint analysis of Toosendan Fructus by HPLC coupled with the charged aerosol detector (CAD) in the present study. Samples were well separated on an Agilent ZOBAX SB C18 column (4.6 mm × 250 mm, 5 µm) by gradient elution using acetonitrile and water containing 0.1 % formic acid (v/v) at the flow rate of 1.0 mL·min−1. The column temperature was 30 ℃ and the injection volume was 5 µL. The nitrogen inlet pressure of the charged aerosol detector (CAD) was 35 psi, and the nebulizer chamber temperature was 35 ℃. In addition, the method of the chromatographic fingerprints combined with multivariate statistical analysis was effective and reasonable lead to an accurate classification of 20 batches of samples from different locations. The results showed that 28 common peaks were observed in the fingerprint and the samples were classified into three clusters. The established method was well validated, and showed high precision, good repeatability, and satisfactory stability. It may serve in the quality control and evaluation of Toosendan Fructus.

An efficient approach to identify different chemical markers between fibrous root and rhizome of Anemarrhena asphodeloides by ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry with multivariate statistical analysis.

An ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry approach coupled with multivariate statistical analysis was established and applied to rapidly distinguish the chemical differences between fibrous root and rhizome of Anemarrhena asphodeloides. The datasets of tR-m/z pairs, ion intensity and sample code were processed by principal component analysis and orthogonal partial least squares discriminant analysis. Chemical markers could be identified based on their exact mass data, fragmentation characteristics, and retention times. And the new compounds among chemical markers could be isolated rapidly guided by the ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry and their definitive structures would be further elucidated by NMR spectra. Using this approach, twenty-four markers were identified on line including nine new saponins and five new steroidal saponins of them were obtained in pure form. The study validated this proposed approach as a suitable method for identification of the chemical differences between various medicinal parts in order to expand medicinal parts and increase the utilization rate of resources.

Furostanol and Spirostanol Saponins from Tribulus terrestris.

Twelve new steroidal saponins, including eleven furostanol saponins, terrestrinin J-T (1-11), and one spirostanol saponin, terrestrinin U (12), together with seven known steroidal saponins 13-19 were isolated from T. terrestris. The structures of the new compounds were established on the basis of spectroscopic data, including 1D and 2D NMR and HRESIMS, and comparisons with published data.

Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the separation of spirostanol saponins.

Spirostanol saponins are important active components of some herb medicines, and their isolation and purification are crucial for the research and development of traditional Chinese medicines. We aimed to compare the separation of spirostanol saponins by ultra-high performance supercritical fluid chromatography (UHPSFC) and ultra-high performance liquid chromatography (UHPLC). Four groups of spirostanol saponins were separated respectively by UHPSFC and UHPLC. After optimization, UHPSFC was performed with a HSS C18 SB column or a Diol column and with methanol as the co-solvent. A BEH C18 column and mobile phase containing water (with 0.1% formic acid) and acetonitrile were used in UHPLC. We found that UHPSFC could be performed automatically and quickly. It is effective in separating the spirostanol saponins which share the same aglycone and vary in sugar chains, and is very sensitive to the number and the position of hydroxyl groups in aglycones. However, the resolution of spirostanol saponins with different aglycones and the same sugar moiety by UHPSFC was not ideal and could be resolved by UHPLC instead. UHPLC is good at differentiating the variation in aglycones, and is influenced by double bonds in aglycones. Therefore, UHPLC and UHPSFC are complementary in separating spirostanol saponins. Considering the naturally produced spirostanol saponins in herb medicines are different both in aglycones and in sugar chains, a better separation can be achieved by combination of UHPLC and UHPSFC. UHPSFC is a powerful technique for improving the resolution when UHPLC cannot resolve a mixture of spirostanol saponins and vice versa.