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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., (© 2024 Wiley-VCH GmbH.)

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., (© 2022 Wiley-VCH GmbH.)

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., (© 2021 Wiley-VCH GmbH.)

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., (© 2021 Wiley-VCH GmbH.)

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., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simple and efficient method based on ultrafiltration with liquid chromatography and mass spectrometry was used for the rapid screening and identification of ligands in the extracts of Stellera chamaejasme. The bound ligands, i.e. daphnoretin, isopimpinellin, chamaechromone, neochamaejasmin A, and chamaejasmine (purity of 96.8, 90.75, 91.41, 93.98, and 98.91%, respectively), were separated by semi-preparative high-performance liquid chromatography combined with high-speed counter-current chromatography. To the best of our knowledge, this is the first study to report the detection of potent lipoxidase and lactate dehydrogenase inhibitors in Stellera chamaejasme extracts. The results demonstrate that our method of ultrafiltration with liquid chromatography and mass spectrometry combined with mixed chromatography can be used to screen and confirm the bioactivity of all isolated compounds. This method also eliminates the need for separation of inactive compounds, thereby improving efficiency when studying bioactive substances. For some complex mixtures, neither semi-preparative high-performance liquid chromatography nor high-speed counter-current chromatography can purify all the target active compounds with high purity in a one-step separation. The combination of the two methods allow for efficient purification of target bioactive compounds with different polarities and physicochemical properties based on their complementary properties., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nonsteroidal anti-inflammatory drugs appear to reduce the risk of developing cancer. One mechanism through which nonsteroidal anti-inflammatory drugs act to prevent carcinogenesis is inhibition of the activity of the enzyme cyclooxygenase-2. The cyclooxygenase-2 inhibitors are widely used to reduce the risk of developing cancer. Natural products are considered to be a promising source of several novel cyclooxygenase-2 inhibitors. Ultrafiltration with liquid chromatography and mass spectrometry is an efficient method that can be applied to rapidly screen and identify the ligands from the barks of Phellodendron amurense Ruprecht. A continuous online method comprised of pressurized liquid extraction, countercurrent chromatography, and semi-preparative liquid chromatography was developed for the efficient scaled-up production of eight compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of bioactivity screening method combined with preparation method of bioactive compounds and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of the cyclooxygenase-2 inhibitors from complex samples. This could be used as an efficient method for the large-scale production of functional ingredients., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nonsteroidal anti-inflammatory drugs reportedly reduce the risk of developing cancer. One mechanism by which they reduce carcinogenesis involves the inhibition of the activity of cyclooxygenase-2, an enzyme that is overexpressed in various cancer tissues. Its overexpression increases cell proliferation and inhibits apoptosis. However, selected cyclooxygenase-2 inhibitors can also act through cyclooxygenase-independent mechanisms. In this study, using ultrafiltration, enzyme-immobilized magnetic beads, high-performance liquid chromatography, and electrospray-ionization mass spectrometry, several isoflavonoids in Trifolium pratense L. extracts were screened and identified. Semi-preparative high-performance liquid chromatography and high-speed counter-current chromatography were then applied to separate the active constituents. Using these methods, seven major compounds were identified in Trifolium pratense L. As cyclooxygenase-2 inhibitors: rothindin, ononin, daidzein, trifoside, pseudobaptigenin, formononetin, and biochanin A, which were then isolated with >92% purity. This is the first report of the presence of potent cyclooxygenase-2 inhibitors in Trifolium pratense L. extracts. The results of this study demonstrate that the systematic isolation of bioactive components from Trifolium pratense L., by using ultrafiltration, enzyme-immobilized magnetic beads, semi-preparative high-performance liquid chromatography, and high-speed counter-current chromatography, represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)