Extraction and preparation of 5-lipoxygenase and acetylcholinesterase inhibitors from Astragalus membranaceus stems and leaves.

In this study, an efficient method that employs 5-lipoxygenase and acetylcholinesterase as biological target molecules in receptor-ligand affinity ultrafiltration-liquid chromatography was developed for the screening of enzyme inhibitors derived from the Astragalus membranaceus stems and leaves. The effects of the extraction time, number of extraction cycles, ethanol concentration, and liquid-solid ratio on the total yield of the target compounds were investigated using response surface methodology, and the bioactive components were isolated using a combination of semi-preparative high-performance liquid chromatography and high-speed countercurrent chromatography via a two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (1:6:2:6, v/v/v/v). Subsequently, 10 naturally-occurring bioactive components in the Astragalus membranaceus stems and leaves, including wogonin, ononin, isoquercitrin, calycosin-7-glucoside, 3-hydroxy-9,10-dimethoxyptercarpan, hyperoside, 7,2'-dihydroxy-3',4'-dimethoxyisoflavan, baicalein, calycosin, and soyasaponin, were screened using affinity ultrafiltration to determine their potential effects against Alzheimer's disease. Consequently, all target compounds had purities higher than 95.0%, and the potential anti-Alzheimer's disease effect of the obtained bioactive compounds was verified using molecular docking analysis. Based on the results, the back-to-back screening of complex enzyme inhibitors and separation of the target bioactive compounds using complex chromatography could provide a new approach to the discovery and preparation of natural active ingredients.

Receptor-ligand affinity-based screening and isolation of water-soluble 5-lipoxygenase inhibitors from Phellinus igniarius.

We developed an efficient combination method for extraction, biological activity screening, and preparation of 5-lipoxygenase inhibitors from Phellinus igniarius. 5-Lipoxygenase inhibitors were rapidly screened using ultrafiltration-liquid chromatography based on the receptor-ligand affinity. Parameters such as extraction time, extraction times, and temperature as well as liquid-solid ratio were optimized using response surface methodology to maximize the total yield of the three target compounds. Next, bioactive ingredients were isolated using high-speed countercurrent chromatography and semi-preparative liquid chromatography. Three active ingredients, phellibaumin E, protocatechuic aldehyde, and osmundacetone, were obtained via ultrafiltration-liquid chromatography. Subsequently, the potential anti-dementia effects of the obtained bioactive compounds were verified using molecular docking assays. The above-mentioned target compounds, with purities of 98.82%, 98.89%, and 99.51%, respectively, were separated using a two-phase solvent system consisting of n-hexane-ethyl acetate-ethanol-water (2.5:2:0.75:3, v/v/v/v) coupled with semi-preparative liquid chromatography.

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.

Development of ultrasound-assisted mixture extraction and online extraction solution concentration coupled with countercurrent chromatography for the preparation of pure phytochemicals from Phellinus vaninii.

Herein, ultrasound-assisted mixture extraction (UAME) and online extraction solution concentration (OESC) were conducted to extract products from crops and plants. These techniques were coupled with parallel countercurrent chromatography (PCCC) and applied for continuous extraction and online isolation of chemical constituents from Phellinus vaninii. The UAME instrument comprises extraction and solution separation chambers. It provides higher extraction efficiency and fewer impurities and is suitable for processing various sample matrices. The OESC device comprises a spray nozzle, concentrating cylinder, and hot-blast air nozzle. The mechanical parameters for UAME and OESC were optimized, and the operation of online UAME and OESC coupled with PCCC was described. Raw plant materials were extracted using a two-phase extractant comprising petroleum-ethyl acetate-ethanol-water (0.5:2.0:0.5:2.0, v/v/v/v). The aqueous and organic phases were then concentrated using the OESC technique. Two CCC runs were conducted for preparatory work. After extraction and online concentration, the concentrate was pumped into the CCC for separation. During PCCC separation, continuous automated extraction and concentration were still conducted. When the first cycle of the UAME/OESC/PCCC was completed, followed by the initiation of the second cycle, and the process was continued. Six target compounds with purities exceeding 97.22% were successfully separated using the CCC solvent systems comprising n-hexane-ethyl acetate-acetonitrile-water (5.5:2.5:5.0:0.4, v/v/v/v) and n-butanol-ethanol-water (4.5:1.3:6.5, v/v/v). Compared with conventional extraction methods, the proposed UAME/OESC/PCCC method has higher efficiency, facilitates high-purity separation of analytes, and offers opportunity for automation and systematic preparation of natural products.