Extraction and separation of lactate dehydrogenase inhibitors from Poria cocos (Schw.) Wolf based on a hyphenated technique and in vitro methods

Stroke is one of the most common diseases worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke, with natural products considered a promising source of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Poria cocos . Five lactate dehydrogenase inhibitors were selected: dehydropachymic acid, pachymic acid, dehydrotrametenolic acid, trametenolic acid, and eburicoic acid. The inhibitors were extracted and isolated with purities of 96.75, 98.15, 97.25, 95.46, and 94.88%, respectively, by using a new “hyphenated” strategy of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography by a two‐phase solvent system of n‐hexane/ethyl acetate/ethanol/water at the volume ratio 0.965:1.000:0.936:0.826 v/v/v/v. The bioactivity of the isolated compounds was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay in PC12 cells. The results also showed that the hyphenated technique of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography was an efficient method for the continuous extraction and online isolation of chemical constituents from medicinal herbs. Furthermore, the research route based on the activity screening, extraction, separation, and activity verification of the compounds offered advantages of efficiency, orientation, and objectivity.     

Bioactivity screening,extraction, and separation of lactate dehydrogenase inhibitors from Polygala tenuifolia Willd. based on a hyphenated strategy

Stroke is the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are currently widely used in the treatment of ischemic stroke, and natural products are considered promising sources of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Polygala tenuifolia . Furthermore, five lactate dehydrogenase inhibitors, sibiricose A5, 3,6′‐di‐O‐sinapoyl‐sucrose, glomeratose A, tenuifoliside B, and tenuifoliside C, were selected as target lactate dehydrogenase inhibitors. In addition, the five target compounds with purities of 96.45, 97.65, 96.38, 94.34, and 93.29% were extracted and isolated using a new hyphenated strategy of microwave‐assisted extraction coupled with countercurrent chromatography with a two‐phase solvent system of n‐hexane/n‐butanol/ethanol/water (5.321:1.00:1.664:6.647). The bioactivities of the isolated compounds were analyzed using PC12 cells and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. The results also demonstrated that microwave‐assisted extraction coupled with countercurrent chromatography is an efficient method of isolating chemical constituents from medicinal herbs. Moreover, the research route consisting of activity screening, extraction, separation, and activity verification of the compounds has the advantages of being efficient, orientated, and objective.     

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.     

Ionic‐liquid‐based ultrasound‐assisted extraction of isoflavones from Belamcanda chinensis and subsequent screening and isolation of potential α‐glucosidase inhibitors by ultrafiltration and semipreparative high‐performance liquid chromatography

The separation of a compound of interest from its structurally similar homologues to produce high‐purity natural products is a challenging problem. This work proposes a novel method for the separation of iristectorigenin A from its structurally similar homologues by ionic‐liquid‐based ultrasound‐assisted extraction and the subsequent screening and isolation of potential α‐glucosidase inhibitors via ultrafiltration and semipreparative high‐performance liquid chromatography. Ionic‐liquid‐based ultrasound‐assisted extraction was successfully applied to the extraction of tectorigenin, iristectorigenin A, irigenin, and irisflorentin from Belamcanda chinensis . The optimum conditions for the efficient extraction of isoflavones were determined as 1.0 M 1‐ethyl‐3‐methylimidazolium tetrafluoroborate with extraction time of 30 min and a solvent to solid ratio of 30 mL/g. Ultrafiltration with liquid chromatography and mass spectrometry was applied to screen and identify α‐glucosidase inhibitors from B. chinensis , followed by the application of semipreparative high‐performance liquid chromatography to separate and isolate the active constituents. Four major compounds including tectorigenin, iristectorigenin A, irigenin, and irisflorentin were screened and identified as α‐glucosidase inhibitors, and then the four active compounds abovementioned were subsequently isolated by semipreparative high‐performance liquid chromatography (99.89, 88.97, 99.79, and 99.97% purity, respectively). The results demonstrate that ionic liquid extraction can be successfully applied to the extraction of isoflavones from B. chinensis .     

Centrifugal partition chromatography – A review of recent applications and some classic references

Countercurrent chromatography, based on liquid–liquid partitioning, has many technological variants. One of them is centrifugal partition chromatography, introduced by Wataru Murayama and Kanichi Nunogaki in 1982. This technique, like other countercurrent chromatography techniques, is based on the phenomenon of liquid–liquid partitioning between two immiscible liquid phases that stay at equilibrium. But the significant difference between this technique and others is the retention mechanism of stationary phase. In the case of centrifugal partition chromatography, this mechanism is based on hydrostatic force, formed by the centrifugal field in the rotor in one‐axis centrifuge. Sometimes that allows more control of stationary phase, for example, when aqueous two‐phase and other difficult solvent systems are used. However, the efficiency of the separation in centrifugal partition chromatography is also affected by a variety of parameters dependent on the sample properties in the solvent system, physical properties of the solvent system, parameters of the instrument, and the method. This article includes also recent ideas for improvements to the technique and broadening its application (e.g., (multiple) dual‐mode or elution–extrusion procedure, pH‐zone‐refining centrifugal partition chromatography, ion‐exchange centrifugal partition chromatography, online and offline coupling of centrifugal partition chromatography).     

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.     

In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large‐scale production of functional food and nutraceutical ingredients.     

Optimization of extraction for Anemarrhena asphodeloides Bge. using silica gel‐based vortex‐homogenized matrix solid‐phase dispersion and rapid identification of antioxidant substances

A novel and simple method was established for the extraction and determination of seven compounds in Anemarrhena asphodeloides Bge. using silica gel‐based vortex‐homogenized matrix solid‐phase dispersion and ultra‐high performance liquid chromatography quadrupole‐time of‐flight mass spectrometer. The conditions for the extraction were optimized. Silica gel was used as the dispersant, 50% methanol–water was selected as an elution solvent and the grinding time was 3 min. Compared with the traditional ultrasonic‐assisted extraction, the developed method was rapid and efficient. In order to screen potential antioxidants, extract dealing with the optimized method was applied to a polyamide chromatography column and a D‐101 macroporous resin column. Fr.2.2 showed the highest antioxidant activities with the most content of flavonoid. A total of 25 peaks were identified from the active fraction. A 2,2′‐diphenyl‐1‐picrylhydrazyl ultra‐high performance liquid chromatography coupled with mass spectrometry approach was adopted for the rapid and exact screening and identification of antioxidant compounds. It indicated that flavonoids exhibited potential antioxidant activities. The antioxidant activities of nine monomeric compounds in vivo were tested. Structure–activity relationships were discussed. Five flavonoids with the concentration of 500 µg/mL would reduce the oxidative stress of PC12 cells that were induced with 2,2′‐azobis[2‐methylpropionamidine] dihydrochloride.     

Development of a method to screen and isolate potential α‐glucosidase inhibitors from Panax japonicus C.A. Meyer by ultrafiltration,liquid chromatography,and counter‐current chromatography

A new assay based on ultrafiltration, liquid chromatography and mass spectrometry was developed for the rapid screening and identification of the ligands for α‐glucosidase from the extract of Panax japonicus. Six saponins were identified as α‐glucosidase inhibitors. Subsequently, the specific binding ligands, namely, notoginsenoside R₁, ginsenoside Rb₁, chikusetsusaponin V, chikusetsusaponin IV, chikusetsusaponin IVa, and ginsenoside Rd (the purities were 94.18, 95.43, 96.09, 93.26, 94.50, 93.86%, respectively) were separated by counter‐current chromatography using two‐phase solvent systems composed of tert‐butyl methyl ether, acetonitrile, 0.1% aqueous formic acid (3.8:1.0:4.4, v/v/v) and the solvent system composed of methylene chloride, isopropanol, methanol, 0.1% aqueous formic acid (5.8:1.0:6.0:2.2, v/v/v). The results demonstrate that ultrafiltration, liquid chromatography and mass spectrometry combined with high‐speed counter‐current chromatography might provide not only a powerful tool for screening and isolating α‐glucosidase inhibitors in complex samples but also a useful platform for discovering bioactive compounds for the prevention and treatment of diabetes mellitus.     

Screening of drugs of abuse and toxic compounds in human whole blood using online solid‐phase extraction and high‐performance liquid chromatography with time‐of‐flight mass spectrometry

A novel method for the screening of 151 drugs of abuse and toxic compounds in human whole blood has been developed and validated by online solid‐phase extraction with liquid chromatography coupled to time‐of‐flight mass spectrometry. Analytes were extracted and separated by using a fully automated online solid‐phase extraction liquid chromatography system with total chromatographic run time of 26 min. Time‐of‐flight mass spectrometry screening of 151 drugs of abuse and toxic compounds was performed in a full‐scan (m/z 50–800) mode using an MS^E acquisition of molecular ions and fragment ions data at two collision energies (one was 6 eV and another one was in the range of 5–45 eV). The compounds were identified based on retention times and exact mass of molecular ions and fragment ions. The limit of detection ranged from 1 to 100 ng/mL and the recovery of the method ranged from 6.3 to 163.5%. This method is proved to be a valuable screening method allowing fast and specific identification of drugs in human whole blood.     

Microwave‐assisted extraction versus Soxhlet extraction to determine triterpene acids in olive skins

Microwave‐assisted extraction is compared with a more classical technique, Soxhlet extraction, to determine the content of triterpene acids in olive skins. The samples used in their original unmilled state and milled were extracted with ethyl acetate or methanol as solvents. The optimized operating conditions (e.g., amount and type of solvent, and time and temperature of extractions) to attain the better extraction yields have been established. For the identification and quantitation of the target compounds, an ultra high performance liquid chromatography with tandem mass spectrometry method was employed. The best results were achieved using the microwave‐assisted extraction technique, which was much faster than the Soxhlet extraction method, and showed higher efficiency in the extraction of the triterpenic acids (oleanolic and maslinic).     

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin ( 1 ), isoliquiritin ( 2 ), and liquiritigenin ( 3 ) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC₅₀ of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.     

Separation of cucurbitane triterpenoids from bitter melon drinks and determination of partition coefficients using vortex‐assisted dispersive liquid‐phase microextraction followed by UHPLC analysis

A rapid, effective method applying vortex‐assisted liquid–liquid microextraction before ultra‐high performance liquid chromatography with mass spectrometry and evaporative light scattering detection was developed for the analysis of four cucurbitane triterpenoids (momordicoside L, momordicoside K, momordicoside F₂, and 3β,7β,25‐trihydroxy cucurbita‐5,23(E )‐dien‐19‐al) in bitter melon juices. Variables affecting the extraction efficiency including different extraction solvents, volume of extraction solvent, salt amount, acid condition, vortex speed and time were optimized thoroughly. Under the optimum conditions, precision was determined by the intra‐ and inter‐day tests in a range of 1.1–5.7% and 2.9–4.0% (RSD), respectively, with recoveries between 95.7 and 106.1%. The calibration curves showed good linearity with square correlation coefficient of 0.9936–0.9991 (evaporative light scattering detection) and 0.9858–0.9989 (MS). The detection limits ranged from 0.8–1.9 ng/mL (MS) to 3–10 ng/mL (evaporative light scattering detection) for these compounds. Enrichment factors of four target compounds were between 27 and 63 times. The proposed method was also used to determine the apparent solvent/water partition coefficients of analytes within the range of 53–120. The developed method can effectively enrich and quantify cucurbitane triterpenoids from bitter melon drinks.     

Large‐scale separation of acetylcholinesterase inhibitors from Zanthoxylum nitidum by pH‐zone‐refining counter‐current chromatography target‐guided by ultrafiltration high‐performance liquid chromatography with ultraviolet and mass spectrometry screening

A new strategy by converging ultrafiltration high‐performance liquid chromatography with ultraviolet and mass spectrometry and pH‐zone‐refining counter‐current chromatography was developed for the rapid screening and separation of potential acetylcholinesterase inhibitors from the crude alkaloidals extract of Zanthoxylum nitidum. An optimized two‐phase solvent system composed of chloroform/methanol/water (4:3:3, v/v) was used in this study. And, in the optimal solvent system, 45 mM hydrochloric acid was added to the aqueous stationary phase as the retainer, while 5 mM triethylamine was added to the organic mobile phase as the eluter. As a result, with the purity of over 95%, five alkaloids including jatrorrhizine ( 1 , 340 mg), columbamine ( 2 , 112 mg), skimmianine ( 3 , 154 mg), palmatine ( 4 , 226 mg), and epiberberine ( 5 , 132 mg) were successfully purified in one step from 3.0 g crude alkaloidals extract. And their structures were identified by ultraviolet, mass spectrometry, ¹H and ¹³C NMR spectroscopy. Notably, compounds 2 , 4 and 5 were firstly reported in Z. nitidum. In addition, acetylcholinesterase inhibitory activities of compounds 1–5 were evaluated, and compounds 3, 4 and 5 exhibited stronger acetylcholinesterase inhibitory activity (IC₅₀ values at 8.52 ± 0.64, 14.82 ± 1.21 and 3.12 ± 0.32 μg/mL, respectively) than berberine (IC₅₀ value at 32.86 ± 2.14 μg/mL, positive control). The results indicated that the proposed method is an efficient technique to rapidly screen acetylcholinesterase inhibitors from complex samples, and could be served as a large‐scale preparative technique for separating ionizable active compounds.     

Ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography for screening and isolating potential α‐glucosidase and xanthine oxidase inhibitors from Cortex Phellodendri

Cortex Phellodendri is a typical Chinese herb with a large number of alkaloids existing in all parts of it. The most common methods for screening and isolating alkaloids are mostly labor intensive and time consuming. In this study, a new assay based upon ultrafiltration liquid chromatography was developed for the rapid screening of ligands for α‐glucosidase and xanthine oxidase. The C. Phellodendri extract was found to contain two alkaloids with both α‐glucosidase‐ and xanthine oxidase binding activities and one lactone with α‐glucosidase‐binding activity. Subsequently, with the help of high‐speed countercurrent chromatography, the specific binding ligands including palmatine, berberine, and obaculactone with purities of 97.38, 96.12, and 96.08%, respectively, were successfully separated. An optimized low‐toxicity two‐phase solvent system composed of ethyl acetate/n‐butanol/ethanol/water (3.5:1.7:0.5:5, v/v/v/v) was used to isolate the three compounds mentioned above from C. Phellodendri. The targeted compounds were identified by liquid chromatography coupled with mass spectrometry and NMR spectroscopy. Therefore, ultrafiltration liquid chromatography combined with high‐speed countercurrent chromatography is not only a powerful tool for screening and isolating α‐glucosidase and xanthine oxidase inhibitors in complex samples but is also a useful platform for discovering bioactive compounds for the prevention and treatment of diabetes mellitus and gout.     

Ultrasound/microwave‐assisted solid–liquid–solid dispersive extraction with high‐performance liquid chromatography coupled to tandem mass spectrometry for the determination of neonicotinoid insecticides in Dendrobium officinale

A one‐step ultrasound/microwave‐assisted solid–liquid–solid dispersive extraction procedure was used for the simultaneous determination of eight neonicotinoids (dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, acetamiprid, thiacloprid, imidaclothiz) in dried Dendrobium officinale by liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode. The samples were quickly extracted by acetonitrile and cleaned up by the mixed dispersing sorbents including primary secondary amine, C₁₈, and carbon‐GCB. Parameters that could influence the ultrasound/microwave‐assisted extraction efficiency such as microwave irradiation power, ultrasound irradiation power, temperature, and solvent were investigated. Recovery studies were performing well (70.4–113.7%) at three examined spiking levels (10, 50, and 100 μg/kg). Meanwhile, the limits of quantification for the neonicotinoids ranged from 0.87 to 1.92 μg/kg. The method showed good linearity in the concentration range of 1–100 μg/L with correlation coefficients >0.99. This quick and useful analytical method could provide a basis for monitoring neonicotinoid insecticide residues in herbs.     

Design of guanidinium ionic liquid based microwave‐assisted extraction for the efficient extraction of Praeruptorin A from Radix peucedani

A series of novel tetramethylguanidinium ionic liquids and hexaalkylguanidinium ionic liquids have been synthesized based on 1,1,3,3‐tetramethylguanidine. The structures of the ionic liquids were confirmed by ¹H NMR spectroscopy and mass spectrometry. A green guanidinium ionic liquid based microwave‐assisted extraction method has been developed with these guanidinium ionic liquids for the effective extraction of Praeruptorin A from Radix peucedani. After extraction, reversed‐phase high‐performance liquid chromatography with UV detection was employed for the analysis of Praeruptorin A. Several significant operating parameters were systematically optimized by single‐factor and L₉ (3⁴) orthogonal array experiments. The amount of Praeruptorin A extracted by [1,1,3,3‐tetramethylguanidine]CH₂CH(OH)COOH is the highest, reaching 11.05 ± 0.13 mg/g. Guanidinium ionic liquid based microwave‐assisted extraction presents unique advantages in Praeruptorin A extraction compared with guanidinium ionic liquid based maceration extraction, guanidinium ionic liquid based heat reflux extraction and guanidinium ionic liquid based ultrasound‐assisted extraction. The precision, stability, and repeatability of the process were investigated. The mechanisms of guanidinium ionic liquid based microwave‐assisted extraction were researched by scanning electron microscopy and IR spectroscopy. All the results show that guanidinium ionic liquid based microwave‐assisted extraction has a huge potential in the extraction of bioactive compounds from complex samples.     

Development of a method to screen and isolate bioactive constituents from Stellera chamaejasme by ultrafiltration and liquid chromatography combined with semi‐preparative high‐performance liquid chromatography and high‐speed counter current chromatography

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.     

Screening,separation, and evaluation of xanthine oxidase inhibitors from Paeonia lactiflora using chromatography combined with a multi‐mode microplate reader

Natural products have become one of the most important resources for discovering novel xanthine oxidase inhibitors, which are commonly employed in the treatment of hyperuricemia and gout. However, to date, few reports exist regarding the use of monoterpene glycosides as xanthine oxidase inhibitors. Thus, we herein report the use of ultrafiltration coupled with liquid chromatography in the screening of monoterpene glycoside xanthine oxidase inhibitors from the extract of Paeonia lactiflora (P. lactiflora ), and both high‐performance counter‐current chromatography and medium‐pressure liquid chromatography were employed to separate the main constituents. Furthermore, the xanthine oxidase inhibitory activities and the mechanisms of inhibition of the isolated compounds were evaluated using a multi‐mode microplate reader by Molecular Devices. As a result, three monoterpene glycosides were separated by combined high‐performance counter‐current chromatography and medium‐pressure liquid chromatography in purities of 90.4, 98.0, and 86.3%, as determined by liquid chromatography. These three compounds were identified as albiflorin, paeoniflorin, and 1‐O‐β‐ᴅ‐glucopyranosyl‐8‐O‐benzoylpaeonisuffrone by electrospray ionization tandem mass spectrometry, and albiflorin and paeoniflorin were screened as potential xanthine oxidase inhibitors by ultrafiltration with liquid chromatography. The evaluation results of xanthine oxidase inhibitory activity corresponded with the screening results, as only albiflorin and paeoniflorin exhibited xanthine oxidase inhibitory activity.     

Ultrasound‐assisted,hybrid ionic liquid,dispersive liquid–liquid microextraction for the determination of insecticides in fruit juices based on partition coefficients

An ultrasound‐assisted, hybrid ionic liquid, dispersive liquid–liquid microextraction method coupled to high‐performance liquid chromatography with a variable‐wavelength detector was developed to detect ten insecticides, including diflubenzuron, triflumuron, hexaflumuron, flufenoxuron, lufenuron, diafenthiuron, transfluthrin, fenpropathrin, γ‐cyhalothrin and deltamethrin, in fruit juices. In this method, an appropriate extraction solvent was chosen based on the partition coefficient of the target compounds. A mixture of 1‐octyl‐2,3‐dimethylimidazolium bis(trifluoromethylsulfonyl)imide and 1‐hexyl‐3‐methylimidazolium bis(trifluoromethylsulfonyl)imide was used as the extractant. The extraction efficiency was screened using Plackett–Burman design and optimized using central composite design. Under the optimal conditions, good linearity was obtained for all the analytes in the pure water model and the fruit juice samples. In pure water, the recoveries of the ten insecticides ranged from 85.7 to 108.9%, with relative standard deviations for one day ranging from 1.24 to 2.64%. The limits of detection were in the range of 0.19–0.69 μg/L, and the enrichment factors were in the range of 123–160. The logarithm of the n‐octanol/water partition coefficient in this experiment is a useful reference to select a suitable extraction solvent, and the proposed technique was applied for the analysis of ten insecticides in fruit juice with satisfactory results.