Combining multidimensional chromatography-mass spectrometry and feature-based molecular networking methods for the systematic characterization of compounds in the supercritical fluid extract of Tripterygium wilfordii Hook F.

Tripterygium wilfordii Hook F from the family Celastraceae is a traditional Chinese medicine (TCM) whose principal chemical constituents are terpenoids, including sesquiterpene alkaloids and diterpenoids, which have unique and diverse structures and remarkable biological activities. In order to advance pharmacological research and guide the preparation of monomer compounds derived from T. wilfordii, a systematic approach to efficiently discover new compounds or their derivatives is needed. Herein, compound separation and identification were performed by offline reversed-phase × supercritical fluid chromatography coupled mass spectrometry (RP × SFC-Q-TOF-MS/MS) and Global Natural Product Social (GNPS) molecular networking. The 2D chromatography system exhibited a high degree of orthogonality and significant peak capacity, and SFC has an advantage during the separation of sesquiterpene alkaloid isomers. Feature-based molecular networking offers the great advantage of quickly detecting and clustering unknown compounds, which greatly assists in intuitively judging the type of compound, and this networking technique has the potential to dramatically accelerate the identification and characterization of compounds from natural sources. A total of 324 compounds were identified and quantitated, including 284 alkaloids, 22 diterpenoids and 18 triterpenoids, which means that there are numerous potential new compounds with novel structures to be further explored. Overall, feature-based molecular networking provides an effective method for discovering and characterizing novel compounds and guides the separation and preparation of targeted natural products.

Highly enhanced activity and stability via affinity induced immobilization ß-glucosidase from Aspergillus niger onto amino-based silica for the biotransformation of ginsenoside Rb1.

In this study, an enzyme immobilization method for the effective biotransformation of ginsenoside Rb1 to impart activity and stability was developed. Using a hydrolase enzyme model, ß-glucosidase from Aspergillus niger, immobilization within chemically affinity-linked amino-based silica provided an immobilization efficiency 5.86-fold higher than that of free enzyme. Compared with the free enzyme, the immobilized enzyme functioned optimally at a wider pH range and had higher thermostability. The optimum pH for the free and immobilized enzymes was 5.5. The optimal reaction temperature of the immobilized enzyme was 45 °C, which was 5 °C higher than that of the free enzyme. The Michaelis constant (Km) values before and after immobilization were 0.482 mmol•L-1 and 0.387 mmol•L-1, respectively. The catalytic rate (Kcat) for the immobilized and free enzymes was 22.269 mmol•L-1and 8.800 mmol•L-1, respectively, and the catalytic efficiency (Kcat/Km) activity of the immobilized enzyme was 3.30-fold higher than that of the free enzyme. The immobilized enzyme could preserve 97 % of the activity after 45 cycles of repeated use. The high catalytic activity and significant operational stability are beneficial for industrial applications.

Retention behavior of ginsenosides in a sulfo-based high performance liquid chromatography column.

We herein report the use of a sulfo-based column and hydrophilic interaction chromatography (HILIC) to separate 14 ginsenosides, namely Rb1, Rb2, Rb3, Rc, Rd, Rf, Re, Rg1, Rg2, Rg3, Rh1, Rh2, F2, and C-K. In addition to its rapid and efficient ability to separate these ginsenosides, the sulfo-based column exhibited a good relationship between the ginsenoside capacity factor (k') and molecular weight (Mw) and a strict elution order corresponding to the polarity (P) of the ginsenosides, as confirmed by thin layer chromatography.

Efficient separation determination of protopanaxatriol ginsenosides Rg1, Re, Rf, Rh1, Rg2 by HPLC.

An efficient and flexible protocol was developed for simultaneous detection of the ginsenosides Rg1, Re, Rf, Rh1, and Rg2 in ginseng extract. In the analysis of white ginseng that contains no ginsenoside Rf, separation of the remaining ginsenosides was achieved within just 10 min under isocratic chromatographic conditions. For the analysis of red ginseng that contains ginsenoside Rf as a characteristic constituent, the gradient elution conditions were optimized. The method is based on high-pressure liquid chromatography employing a Shiseido UG 80 Capcell Pak NH2(4.6 mm I.D. × 250 mm, 5 µm)column and isocratic elution using acetonitrile (A) and water(B) in a ratio of 76: 24 (v/v), The optimal gradient elution conditions are as follows: 0-3 min, 89% A, 3-25 min, 89-84% A, 25-30 min, 84-82% A, 30-35 min, 82-76% A, then returning to 89% solvent A in 5 min. The flow rate was 0.80 mL min-1. The column temperature was set at 25℃ and the detection wavelength was at 203 nm. The working concentration ranges for ginsenoside Re, Rh1, Rg2, Rg1, and Rf were 0.23-1450.0 mg• L-1, 0.05-1130.0 mg• L-1, 0.11-687.0 mg• L-1, 0.051-1325 mg• L-1, and 0.55-800.0 mg• L-1, respectively. The method was validated for linearity, precision, and accuracy. And the further confirmation of five ginsenosides was conducted by QTOF-MS. Analysis of raw extracts of ginseng, white ginseng, and red ginseng for the five components showed satisfactory recovery.