Rapid screening and isolation of 5-lipoxygenase inhibitors in Inonotus obliquus and mechanism of action in the treatment of asthma.

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.

In-depth analysis of the xanthine oxidase inhibitors of Cicer arietinum L.-based receptor-ligand affinity coupled with complex chromatography.

INTRODUCTION: Cicer arietinum L. is the choice of health food for people with diabetes, hypertension, and hyperlipidemia. As an essential source of high-nutrition legumes, it is also an important source of dietary isoflavones. OBJECTIVES: In order to improve the preparation efficiency of natural plants, a rapid biological activity screening and preparation of xanthine oxidase inhibitors from C. arietinum L. was established. METHODS: Xanthine oxidase (XOD) inhibitors were rapidly screened using ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) based on receptor-ligand affinity. The change in XOD activity was evaluated by enzymatic reaction kinetics measurement. The potential bioactive compounds were verified through molecular docking. In addition, the biological activity of ligands screened was separated and purified by complex chromatography. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS: Three active ingredients, namely daidzin, daidzein, calycosin with XOD binding affinities were identified and isolated from the raw plant materials via semi-preparative high-performance liquid chromatography (HPLC), 0-60 min, 5-50% B and countercurrent chromatography (CCC) (ethyl acetate/acetic acid/water [5:0.8:10, v/v/v]). CONCLUSION: This study will help to elucidate the mechanisms of action of natural plants of interest at the molecular level and could also provide more opportunities for the discovery and development of new nutritional value from other natural resources.

Modeling and optimization of the protocol of complex chromatography separation of cyclooxygenase-2 inhibitors from Ganoderma lucidum spore.

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.

Efficient Combination of Complex Chromatography, Molecular Docking and Enzyme Kinetics for Exploration of Acetylcholinesterase Inhibitors from Poria cocos.

Poria cocos (P. cocos) is a traditional Chinese medicinal product with the same origin as medicine and food. It has diuretic, anti-inflammatory and liver protection properties, and has been widely used in a Chinese medicine in the treatment of Alzheimer's disease (AD). This study was conducted to explore the activity screening, isolation of acetylcholinesterase inhibitors (AChEIs), and in vitro inhibiting effect of P. cocos. The aim was to develop a new extraction process optimization method based on the Matlab genetic algorithm combined with a traditional orthogonal experiment. Moreover, bio-affinity ultrafiltration combined with molecular docking was used to screen and evaluate the activity of the AChEIs, which were subsequently isolated and purified using high-speed counter-current chromatography (HSCCC) and semi-preparative high-performance liquid chromatography (semi-preparative HPLC). The change in acetylcholinesterase (AChE) activity was tested using an enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on AChE and explore its mechanism of action. Five potential AChEIs were screened via bio-affinity ultrafiltration. Molecular docking results showed that they had good binding affinity for the active site of AChE. Meanwhile, the five active compounds had reversible inhibitory effects on AChE: Polyporenic acid C and Tumulosic acid were non-competitive inhibitors; 3-Epidehydrotumulosic acid was a mixed inhibitor; and Pachymic acid and Dehydrotrametenolic acid were competitive inhibitors. This study provided a basis for the comprehensive utilization of P. cocos and drug development for the treatment of AD.

An efficient strategy based on two-stage chromatography and in vitro evaluation for rapid screening and isolation of acetylcholinesterase inhibitors from Scutellaria baicalensis Georgi.

The extraction of Scutellaria baicalensis Georgi was investigated using the response surface methodology-genetic algorithm mathematical regression model, and the extraction variables were optimized to maximize the flavonoid yield. Furthermore, a simple and efficient ultrafiltration-liquid chromatography-mass spectrometry and molecular docking methods were developed for the rapid screening and identification of acetylcholinesterase inhibitors present in Scutellaria baicalensis Georgi. Subsequently, four major chemical constituents, namely baicalein, norwogonin, wogonin, and oroxylin A, were identified as potent acetylcholinesterase inhibitors. This novel approach, involving the use of ultrafiltration-liquid chromatography-mass spectrometry and molecular docking methods combined with stepwise flow rate counter-current chromatography and semi-preparative high-performance liquid chromatography, could potentially provide a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices and be a useful platform for the production of bioactive and nutraceutical ingredients.

Development of a method to screen and isolate xanthine oxidase inhibitors from black bean in a single step: Hyphenation of semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography.

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.

Development of ultrasound-assisted centrifugal extraction combined with two countercurrent chromatography systems for the simultaneous extraction and isolation of phytochemicals.

We proposed a method for the extraction of medicinal herbs, called ultrasound-assisted centrifugal extraction, and an online solvent concentration method. These techniques were coupled with two countercurrent chromatography systems and applied to the continuous extraction and online isolation of chemical constituents from Inonotus obliquus. Raw plants were extracted using a two-phase petroleum-ethanol-water (2.0:1.0:2.0, v/v/v) process, and then the aqueous and organic phases were concentrated using the proposed online solvent concentrator. The countercurrent chromatography preparation prior to separation includes pumping of the two-phase solution, rotating column, and equilibrium column. Following online concentration, the extracted solution was pumped into a second countercurrent chromatography process for separation. During separation, the extraction solution and concentrated extract were prepared automatically. Upon completion of the first cycle of ultrasound-assisted centrifugal extraction/two countercurrent chromatography, the second cycle experiment starts. This process can be indefinitely repeated. In this study, six target compounds with purities above 97.71% were successfully extracted and isolated online using a two-phase solvent system consisting of n-hexane-ethyl acetate-acetonitrile (4.5:1.5:5.5, v/v/v) and n-hexane-ethyl acetate-methanol-water (0.4:3.0:1.5:2.5, v/v/v/v). Compared to conventional extraction methods, the instrumental setup of the proposed method provides enhanced automation, efficiency, purity, and systematic extraction and isolation of natural products.

Screening and isolation of potential lipoxidase and superoxide dismutase inhibitors from Scutellaria baicalensis Georgi using high-speed countercurrent chromatography target-guided by ultrafiltration-liquid chromatography-mass spectrometry.

We present a simple and efficient method based on ultrafiltration high-performance liquid chromatography coupled with a photodiode array detector and electrospray ionization mass spectrometry for the rapid screening and identification of ligands obtainable from the extract of Scutellaria baicalensis. Five major compounds (chrysin-6-C-arabinosyl-8-C-glucoside, chrysin-6-C-glucosyl-8-C-arabinoside, baicalin, oroxylin A-7-O-glucuronide, and wogonoside) were identified as potentially effective inhibitors of lipoxidase and superoxide dismutase. Subsequently, specific binding ligands were separated by high-speed countercurrent chromatography, using ethyl acetate/ethyl alcohol/water acetate (0.1%) (1.0:0.1:1.0, v/v/v) as the solvent system. To the best of our knowledge, this is the first report of S. baicalensis extracts containing potent lipoxidase and superoxide dismutase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from the n-butyl alcohol layer of S. baicalensis guided by ultrafiltration high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could also be employed for the identification and isolation of other enzyme inhibitors.

Rapid screening and purification of potential inhibitors from Medicago sativa by ultrafiltration-liquid chromatography combined with stepwise flow rate counter-current chromatography.

INTRODUCTION: Medicago sativa contains flavonoids, saponins, coumarins, sterols, monoterpenes, and organic acids, with flavonoids being the main active constituents. Flavonoids naturally contain a 2-phenylchromone structure with antioxidant, free radical scavenging, cardiovascular, and trace estrogen-like effects. OBJECTIVE: Screening and isolation of neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors from M. sativa via ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) combined with stepwise flow rate counter-current chromatography (CCC). METHOD: Utilising the medicinal plants M. sativa as the research objects and UF-LC-MS was used for activity screening followed by isolation and purification of the inhibitors by stepwise flow rate CCC. Finally, identification of the three active compounds was achieved by MS and nuclear magnetic resonance. RESULTS: Three major compounds, viz. quercetin, genistein, and formononetin, were identified as potent neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors, respectively. A two-phase solvent system of ethyl acetate/methanol/n-butanol/water (5.0:1.5:5.0:10; v/v/v/v) was subsequently selected for separation by stepwise flow rate CCC. CONCLUSION: This novel approach based on UF-LC-MS and stepwise flow rate CCC represents a powerful tool for the screening and isolation of neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors from complex matrices. Therefore, a useful platform for the large-scale production of bioactive and nutraceutical ingredients was developed herein.

Extraction and isolation of acetylcholinesterase inhibitors from Citrus limon peel using an in vitro method.

A simple and efficient ultrafiltration-liquid chromatography-mass spectrometry-based method was developed for the rapid screening and identification of ligands from Citrus limon peel, which are suitable acetylcholinesterase inhibitors. Subsequently, the anti-Alzheimer's activity of these compounds was assessed using a PC12 cell model. Six major compounds, viz. neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin, were identified as potent acetylcholinesterase inhibitors. A continuous and efficient online method, which involved the use of a microwave-assisted extraction device, solvent concentration tank, and centrifugal partition chromatography column, was developed for the scale-up of these compounds, and the obtained compounds presented high purity. Next, their bioactivity was evaluated using a PC12 cell model. This novel approach, which was based on ultrafiltration-liquid chromatography-mass spectrometry, microwave-assisted extraction online coupled with solvent concentration tank, and centrifugal partition chromatography along with in vitro evaluation, could represent a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices, and could be a useful platform for the large-scale production of bioactive and nutraceutical ingredients.