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1.
J Sep Sci ; 47(5): e2300647, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38466162

ABSTRACT

Accurate screening and targeted preparative isolation of active substances in natural medicines have long been two technical challenges in natural medicine research. This study outlines a new approach to improve the efficiency of natural product preparation, focusing on rapidly and accurately screening potential active ingredients in Inonotus obliquus as well as efficiently preparing 5-lipoxidase (5-LOX) inhibitors, to provide new ideas for the treatment of asthma with Inonotus obliquus. First, we used ultrafiltration (UF) mass spectrometry to screen for three potential inhibitors of 5-LOX in Inonotus obliquus. Subsequently, the inhibitory effect of the active ingredients screened in the UF assay on 5-LOX was verified using the molecular docking technique, and the potential role of the active compounds in Inonotus obliquus for the treatment of asthma was analyzed by network pharmacology. Finally, based on the above activity screening guidelines, we used semi-preparative liquid chromatography and consecutive high-speed countercurrent chromatography to isolate three high-purity 5-LOX inhibitors such as betulin, lanosterol, and quercetin. Obviously, through the above approach, we have seamlessly combined rapid discovery, screening, and centralized preparation of the active ingredient with molecular-level interactions between the active ingredient and the protease.


Subject(s)
Asthma , Lipoxygenase Inhibitors , Lipoxygenase Inhibitors/pharmacology , Molecular Docking Simulation , Inonotus , Asthma/drug therapy
2.
J Sep Sci ; 47(1): e2300505, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38135883

ABSTRACT

Poria Cum Radix Pini is a rare medicinal fungus that contains several potential therapeutic ingredients. On this basis, a particle swarm mathematical model was used to optimize the extraction process of total triterpenes from P. Cum Radix Pini, and xanthine oxidase inhibitors were screened using affinity ultrafiltration mass spectrometry. Meanwhile, the accuracy of the ultrafiltration assay was verified by molecular docking experiments and molecular dynamics analysis, and the mechanism of action of the active compounds for the treatment of gout was analyzed by enzymatic reaction kinetics and network pharmacology. A high-speed countercurrent chromatography method combined with the consecutive injection and the economical two-phase solvent system preparation using functional activity coefficient of universal quasichemical model (UNIFAC) mathematical model was developed for increasing the yield of target compound. In addition, dehydropachymic acid and pachymic acid were used as competitive inhibitors, and 3-O-acetyl-16alpha-hydroxydehydrotrametenolic acid and dehydrotrametenolic acid were used as mixed inhibitors. Then, activity-oriented separation and purification were performed by high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography and the purity of the four compounds isolated was higher than 90%. It will help to provide more opportunities to discover and develop new potential therapeutic remedies from health care food resources.


Subject(s)
Gout , Poria , Poria/chemistry , Xanthine Oxidase , Molecular Docking Simulation , Chromatography, High Pressure Liquid/methods , Enzyme Inhibitors/pharmacology , Countercurrent Distribution , Gout/drug therapy
3.
Phytochem Anal ; 34(4): 431-442, 2023 Jun.
Article in English | MEDLINE | ID: mdl-36958357

ABSTRACT

INTRODUCTION: The spores of the medicinal fungus Ganoderma lucidum possess hepatoprotective properties. The main components, triterpenes, are particularly beneficial, making the screening and preparation of active triterpenes from Ganoderma lucidum significant. OBJECTIVES: We aimed to screen and verify cyclooxygenase-2 inhibitors from G. lucidum spores, establish a rapid online hyphenated technique for the preparation of active ingredients, and analyze the structures of the active ingredients. METHODS: Ultrafiltration LC combined with an enzyme inhibition assay and molecular docking was employed to screen and evaluate cyclooxygenase-2 ligands, which were prepared by pressurized liquid extraction coupled online with countercurrent chromatography and semi-preparative LC. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS: Six cyclooxygenase-2 inhibitors, namely, ganoderic acids I, C2 , G, B, and A and ganoderenic acid A, were screened and evaluated. They were prepared using the online hyphenated technique, following which their structures were identified. CONCLUSION: This study provides opportunities for the discovery and development of new therapeutic drugs from other natural resources, as the present instrumental setup achieved efficient and systematic extraction and isolation of natural products compared with reference separation methods, thus exhibiting significant potential for industrial applications.


Subject(s)
Reishi , Triterpenes , Reishi/chemistry , Cyclooxygenase 2 Inhibitors/pharmacology , Cyclooxygenase 2 Inhibitors/analysis , Molecular Docking Simulation , Spores, Fungal/chemistry , Triterpenes/analysis , Countercurrent Distribution
4.
J Sep Sci ; 45(2): 492-506, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34799974

ABSTRACT

Black bean, in which isoflavones are the main active constituent, also contains saponins and monoterpenes. Soybean isoflavone is a secondary metabolite that is formed during the growth of soybean; it exhibits antioxidant and cardiovascular activities and traces estrogen-like effects. In this study, black bean isoflavones were extracted with n-butanol, and ultrafiltration-liquid chromatography-mass spectrometry was used to screen their activity. Subsequently, the inhibitors were isolated and purified using semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography. Thereafter, five active compounds were identified using mass spectrometry and nuclear magnetic resonance experiments. Finally, the inhibition types of the xanthine oxidase inhibitors were determined using enzymatic kinetic studies. The IC50 values of daidzin, glycitein-7-O-glucoside, genistin, daidzein, and genistein were determined to be 35.08, 56.22, 30.76, 68.79, and 95.37 µg/mL, respectively. Daidzin, genistin, and daidzein exhibited reversible inhibition, whereas glycitein-7-O-glucoside and genistein presented irreversible inhibition. This novel approach, which was based on ultrafiltration-liquid chromatography-mass spectrometry and stepwise flow rate countercurrent chromatography, is a powerful method for screening and isolating xanthine oxidase inhibitors from complex matrices. The study of enzyme inhibition types is helpful for understanding the underlying inhibition mechanism. Therefore, a beneficial platform was developed for the large-scale production of bioactive and nutraceutical ingredients.


Subject(s)
Countercurrent Distribution , Isoflavones , Xanthine Oxidase , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid , Isoflavones/chemistry , Kinetics , Phaseolus/chemistry , Plant Proteins/chemistry , Xanthine Oxidase/antagonists & inhibitors
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