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.     

Systematic characterization of flavonoids from Siraitia grosvenorii leaf extract using an integrated strategy of high‐speed counter‐current chromatography combined with ultra high performance liquid chromatography and electrospray ionization quadrupole time‐of‐flight mass spectrometry

The chemical constituents of the Siraitia grosvenorii leaf extract were studied. Firstly, high‐speed counter‐current chromatography was applied to the one‐step separation of four compounds from S. grosvenorii leaf extract with the solvent system composed of 0.01% acetic acid water/n‐butanol/n‐hexane/methanol (5:3:1:1, v/v/v/v). In this work, 270 mg of crude sample yielded four compounds, a new kaempferol O‐glycoside derivative, kaempferol 3‐O‐α‐L‐[4‐O‐(4‐carboxy‐3‐hydroxy‐3‐methylbutanoyl)]‐rhamnopyranoside‐7‐O‐α‐L‐rhamnopyranoside, named kaempferitrin A (2.1 mg, 90%), and three known compounds, grosvenorine (3.4 mg, 93%), kaempferitrin (14.4 mg, 99%) and afzelin (4 mg, 98%), and the structures of these compounds were identified by NMR spectroscopy and mass spectrometry. Then, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry was used to illustrate the dominant flavonoids in S. grosvenorii leaf extract. 34 flavonoids including 19 kaempferol O‐glycosides, 4 quercetin O‐glycosides, 6 flavanone derivatives, and 5 polymethoxyflavones, were accurately or tentatively identified by carefully comparing their retention times, UV data, precise masses, the typical fragments of the standards and literature data. Most of these compounds were reported for the first time. This study establishes a foundation for the further development and utilization of S. grosvenorii leaves in future.     

Combinative application of pH‐zone‐refining and conventional high‐speed counter‐current chromatography for preparative separation of caged polyprenylated xanthones from gamboge

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography for the first time. pH‐zone‐refining counter‐current chromatography was performed with the solvent system composed of n‐hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high‐speed counter‐current chromatography with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n‐hexane/methyl tert‐butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β‐morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high‐performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.     

Separation of three phenolic high‐molecular‐weight compounds from the crude extract of Terminalia Chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography

This study presents an efficient strategy for separation of three phenolic compounds with high molecular weight from the crude extract of Terminalia chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography. The ultrasound‐assisted extraction conditions were optimized by response surface methodology and the results showed the target compounds could be well enriched under the optimized extraction conditions. Then the crude extract was directly separated by high‐speed counter‐current chromatography without any pretreatment using n‐hexane/ethyl acetate/methanol/water (1:7:0.5:3, v/v/v/v) as the solvent system. In 180 min, 13 mg of A, 18 mg of B, and 9 mg of C were obtained from 200 mg of crude sample. Their structures were identified as Chebulagic acid (A, 954 Da), Chebulinic acid (B, 956 Da), and Ellagic acid (C) by ¹H NMR spectroscopy.     

Isolation and purification of macrocyclic components from Penicillium fermentation broth by high‐speed counter‐current chromatography

In this paper, high‐speed counter‐current chromatography (HSCCC), assisted with ESI‐MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7′‐O‐formylbrefeldin A (6.5 mg, 95.0%) and 7′‐O‐acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK‐15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two‐step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two‐step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n‐hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X‐ray crystallography, ESI‐MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.     

A liquid chromatography–tandem mass spectrometry method for preclinical pharmacokinetics and tissue distribution of hydrolyzable tannins chebulinic acid and chebulagic acid in rats

A simple and sensitive liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of chebulinic acid and chebulagic acid in rat plasma and tissues and well used in the pharmacokinetic and tissue distribution studies after intraperitoneal injection administration. Samples were processed with methanol by protein precipitation, and chromatographic separation was performed on an Agilent Zorbax SB‐C₁₈ column (50 × 2.1 mm, 1.8 μm) with a mobile phase consisting of methanol and water containing 0.1% formic acid (60:40, v/v). Quantification was performed by selected reaction monitoring with m/z 977.1 → 806.8 for chebulagic acid, m/z 979.0 → 808.7 for chebulinic acid and m/z 851.2 → 704.9 for the internal standard. Good linearity was observed over their respective concentration range. The pharmacokinetic study showed that both compounds reached their peak concentration values (605.8 ± 35.6 ng/mL for chebulinic acid and 1327.1 ± 118.6 ng/mL for chebulagic acid) at the same time of 0.9 h following intraperitoneal injection administration. The two compounds could be detected in blood‐abundant tissues. The kidney had the highest concentrations (462.6 ± 138.5 ng/g for chebulinic acid and 1651.7 ± 167.7 ng/g for chebulagic acid) at 1 h post‐dose, followed by the heart, liver, spleen and lung.     

Characterization of protostane triterpenoids in Alisma orientalis by ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

A reliable and sensitive ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method has been optimized and established for analysis of protostane triterpenoids in a commonly used traditional Chinese herbal medicine Alisma orientalis (Sam.) Juzep. The separation of crude extract of A. orientalis was achieved on a Waters ACQUITY HSS T3 column (100 mm × 2.1 mm, 1.8 µm) eluting with 0.1% (v/v) formic acid/acetonitrile. A total of 20 protostane triterpenoids including 19 known compounds and a new one were well separated within 7 min. The collision‐induced dissociation (CID) tandem mass spectrometric (MS/MS) fragmentation patterns of protostane triterpenoids was firstly reported in this study. The hydrogen rearrangement at the C‐23‐OH leads to dissociation of the bond between C‐23 and C‐24 in the protostane triterpenoid skeleton during the CID process. This dissociation was the characteristic CID fragmentation pathway of this class of triterpenoids, and was useful for further differentiation of some positional isomers which contain an acetyl unit on the C‐23 or C‐24 position. The identities of isolated compounds were identified by comparing their retention times and CID fragmentation behaviors with those of reference standards or tentatively assigned by matching the empirical molecular formulae with those reported in the literature. It is concluded that this newly established UPLC/Q‐TOF‐MS method is a powerful approach for structural elucidation of protostane triterpenoids isolated from A. orientalis. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

High‐performance countercurrent chromatography separation of Peucedanum cervaria fruit extract for the isolation of rare coumarin derivatives

For the first time, rare major and minor compounds from fruits of Peucedanum cervaria were isolated. High‐performance countercurrent chromatography with two different solvent systems, heptane/ethyl acetate/methanol/water (3:2:3:2 and 2:1:2:1, v/v), was successfully used in the reversed‐phase mode. A scale‐up process from analytical to semipreparative in a very short time was developed. The structures of isolated compounds were evaluated by high‐performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry, gas chromatography with mass spectrometry, and one‐ and two‐dimensional NMR spectroscopy. (8S,9R)‐9‐(3‐Methylbutenoyloxy)‐O‐acetyl‐8,9‐dihydrooroselol (compound B), (8S,9R)‐9‐(2‐methyl‐Z‐butenoyloxy)‐O‐acetyl‐8,9‐dihydrooroselol (edultin, compound C), and (8S,9R)‐9‐acetoxy‐O‐(2α‐methylbutyryl)‐8,9‐dihydrooroselol (compound D) were obtained using heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) in <40 min. The method yielded 4.6 mg of a mixture of compounds B and C (11:89) and 3.7 mg of compound D. These amounts were obtained from the crude extract (0.5 g) in a single run. Although the compounds are known, their isolation by countercurrent chromatography and the analysis of their relative stereochemistry by two‐dimensional NMR spectroscopy have been performed for the first time. Additionally, heptane/ethyl acetate/methanol/water (3:2:3:2, v/v) led to the isolation of oxypeucedanin (1.2 mg; compound A). This is the first time that angular dihydrofuranocoumarin was isolated from plant extract by countercurrent chromatography.     

Separation of six compounds from pigeon pea leaves by elution–extrusion counter‐current chromatography

A valid and reliable method was established to separate six compounds from pigeon pea leaves via elution‐extrusion counter‐current chromatography. A solvent system composed of n‐hexane/methanol/formic acid aqueous solution with pH = 3 (10:6:4, v/v) was screened to achieve satisfactory isolation from the ethanol extract of pigeon pea leaves. Four compounds, 9.2 mg of compound 1 (96.8%), 3.2 mg of 2 (88.0%), 6.2 mg of 4 (94.2%) and 25.2 mg of 5 (94.2%), were obtained by conventional elution from 100 mg of the precipitation fraction, respectively. Two compounds, 14.4 mg of 3 (96.3%) and 28.1 mg of 6 (96.6%), with high K values were obtained by the subsequent extrusion procedure. The compounds 1 – 6 were identified as 3‐methoxy‐5‐(2‐phenylethenyl)‐phenol, pinostrobin chalcone, pinostrobin, 2‐hydroxy‐4‐methoxy‐6‐(2‐phenylvinyl)‐benzoic acid, longistylin C and cajaninstilbene acid by quadrupole time‐of‐flight mass spectrometry, and ¹H and ¹³C NMR spectroscopy. The in vitro antiproliferation activities of compounds 1 , 5 and 6 against human hepatoma cell were evaluated and the half‐maximum inhibitory concentrations were acquired.     

Separation and purification of antioxidants from Ampelopsis heterophylla by counter‐current chromatography

In this study, bioactive components from Ampelopsis heterophylla were separated by counter‐current chromatography (CCC). The antioxidant activity of the crude extract was initially evaluated by an online HPLC method. Five compounds in the crude extract exhibited good antioxidant activities, namely, hyperoside ( 1 ), isoquercitrin ( 2 ), rutin ( 3 ), kaempferol‐3‐rutinoside ( 4 ), and quercetin ( 5 ). These compounds were further separated by CCC with biphasic solvent systems and their structures were identified by MS and NMR spectroscopy. All the compounds exhibited significant 1,1‐diphenyl‐2‐picryl‐hydrazyl radical scavenging activities with IC₅₀ values at 18.2 ± 1.3, 17.0 ± 1.4, 24.2 ± 1.2, 38.1 ± 1.7, and 9.0 ± 1.2 μM, respectively. The scavenging ratios of the compounds against hydroxyl radicals were 65 ± 5, 68 ± 4, 96 ± 2, 70 ± 4, and 98 ± 2%, respectively.     

Optimization of microwave‐assisted extraction for six inorganic and organic arsenic species in chicken tissues using response surface methodology

Response surface methodology was applied to optimize the parameters for microwave‐assisted extraction of six major inorganic and organic arsenic species (As(III), As(V), dimethyl arsenic acid, monomethyl arsenic acid, p‐arsanilic acid, and roxarsone) from chicken tissues, followed by detection using a high‐performance liquid chromatography with inductively coupled mass spectrometry detection method, which allows the simultaneous analysis of both inorganic and organic arsenic species in the extract in a single run. Effects of extraction medium, solution pH, liquid‐to‐solid ratio, and the temperature and time of microwave‐assisted extraction on the extraction of the targeted arsenic species were studied. The optimum microwave‐assisted extraction conditions were: 100 mg of chicken tissue, extracted by 5 mL of 22% v/v methanol, 90 mmol/L (NH₄)₂HPO₄, and 0.07% v/v trifluoroacetic acid (with pH adjusted to 10.0 by ammonium hydroxide solution), ramping for 10 min to 71°C, and holding for 11 min. The method has good extraction performance for total arsenic in the spiked and nonspiked chicken tissues (104.0 ± 13.8% and 91.6 ± 7.8%, respectively), except for the ones with arsenic contents close to the quantitation limits. Limits of quantitation (S/N = 10) for As(III), As(V), dimethyl arsenic acid, monomethyl arsenic acid, p‐arsanilic acid, and roxarsone in chicken tissues using this method were 0.012, 0.058, 0.039, 0.061, 0.102, and 0.240 mg/kg (dry weight), respectively.     

Separation of betacyanins from flowers of Amaranthus cruentus L. in a polar solvent system by high‐speed counter‐current chromatography

Betacyanin extract of Amaranthus cruentus L. flowers was fractionated by semi‐preparative high‐speed counter‐current chromatography in a highly polar solvent system: propan‐1‐ol/acetonitrile/(NH₄)₂SO_{4satd. soln}/H₂O (1.0:0.5:1.2:1.0, v/v/v/v) in tail‐to‐head mode with 76% retention of the stationary phase. The crude extract as well as the fractions containing betacyanins were analyzed by liquid chromatography with tandem mass spectrometry as well as by high‐resolution ion‐trap time‐of‐flight mass spectrometry detection technique for the molecular formulae and multi‐step fragmentation pattern elucidation. Four betacyanins; namely, amaranthin, betanin, 6′‐O‐formyl‐amaranthin, and 6′‐O‐malonyl‐amaranthin as well as their diastereomeric forms differing in the configuration of the C‐15 carbon atom were identified in the fractions. Amaranthin was the dominant pigment in the extract and was additionally analyzed by nuclear magnetic resonance correlation techniques after the counter‐current chromatographic and high‐performance liquid chromatographic isolation. Betacyanins were highly enriched during a single high‐speed counter‐current chromatographic step; therefore, the tentative identification of new compounds for the whole Amaranthaceae family, 6′‐O‐formyl‐amaranthin and 6′‐O‐malonyl‐amaranthin was possible. Different elution profiles of the pigments observed in the counter‐current chromatographic system in comparison to high‐performance liquid chromatography system confirm a complementarity of both the techniques especially in the separation of diastereomeric pairs of betacyanins.     

Melodamide A from Melodorum fruticosum — Quantification using HPLC and one‐step‐isolation by centrifugal partition chromatography

Melodamide A, a phenolic amide from the leaves of Melodorum fruticosum Lour., has previously shown pronounced anti‐inflammatory activity. In order to rapidly isolate larger quantities for biological testing, a fast, one‐step isolation method by centrifugal partition chromatography was developed within this study. Fractionation of the dichloromethane extract was performed with a two‐phase solvent system consisting of n‐hexane, ethyl acetate, methanol, and water (3:7:5:5, v/v), leading to the isolation of melodamide A with a purity of >90% and a yield of 6.7 w% within 32 min. The developed method can also be used in dual mode for the enrichment of further constituents like flavonoids or chalcones. In order to support the centrifugal partition chromatography method development, additionally, a high‐performance liquid chromatography method was established and validated to determine quantities of melodamide A in plant material and crude extracts. Analysis of M. fruticosum leaves and a dichloromethane extract obtained from this plant material showed a total melodamide A content of 0.19 ± 0.008 and 8.9 ± 0.249 w%, respectively.     

An effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography for enrichment and separation of three minor components with similar polarity from Dracocephalum tanguticum

The separation of minor compounds, especially those with similar polarities from a complex sample, remains challenging. In the proposed study, an effective method based on medium‐pressure liquid chromatography and recycling high‐speed counter‐current chromatography was developed for the enrichment and separation of three minor components from Dracocephalum tanguticum. The crude extract was directly introduced to medium‐pressure liquid chromatography for the enrichment of the three minor components. Based on high‐performance liquid chromatography analysis, the total content of these three compounds increased from 0.48% in the crude extract to 85.3% in the medium‐pressure liquid chromatography fraction. In addition, high‐speed counter‐current chromatography was employed to separate the enriched compounds using the solvent system hexane/ethyl acetate/methanol/water (1.18:8.82:1.18:8.82, v/v/v/v). As a result, compound 3 and a mixture of compounds 1 and 2 were obtained. In order to improve the resolution of compounds 1 and 2 while saving separation time, a recycling and heart‐cut mode was used. Finally, compounds 1 and 2 were obtained after five cycles. These compounds were identified as 3‐phenylethyl β‐d ‐glucopyranoside ( 1 ), tazettoside E ( 2 ), and cirsiliol‐4′‐glucoside ( 3 ). Compounds 1 and 2 were primarily separated from D. tanguticum. Moreover, the developed method provided a reference for the separation of minor components from the complex sample.     

Tracing antibacterial compounds from Acalypha australis Linn. by spectrum‐effect relationships and semi‐preparative HPLC

Several Acalypha australis Linn. species are used in traditional medicine in Southeast Asia. In this work, the ultra‐performance liquid chromatography/mass spectrometry fingerprints and the antibacterial activities of A. australis Linn. were investigated. An in‐depth discussion on the reliability of identifying and obtaining potentially active compounds by spectrum‐effect relationship and semi‐preparative high performance liquid chromatography was conducted. The result shows that gallic acid and a compound with molecular weight of 634.1 in the fingerprints were the main antibacterial compounds. Compared to the crude extract of A. australis Linn., both compounds increase the antibacterial efficacy 10 to 20 times. Compounds with molecular weights of 154.0, 292.0, and 485.1 in the fingerprints were the auxiliary antibacterial compounds. Through the entire isolation procedure, we obtained these antibacterial compounds with purities of 92.53, 87.98, 90.73, 89.36, and 88.14%, respectively. This work provides a general model of the combination of ultra‐performance liquid chromatography/mass spectrometry fingerprinting and antibacterial test to study the spectrum‐effect relationships of A. australis Linn. This model can be used to discover further the active compounds of this herb.     

高速逆流色谱法分离制备母丁香和公丁香中的5种非挥发性化合物

应用高速逆流色谱法从母丁香和公丁香中快速分离了3种已知非挥发性化合物,并利用相同方法从公丁香中分离出2种色原酮类化合物。两相溶剂系统A为正己烷-乙酸乙酯-甲醇-水(5:8:6:13, v/v/v/v),系统B为正己烷-乙酸乙酯-甲醇-水(5:8:9:10, v/v/v/v),以系统A的上相为固定相,系统A和B的下相为流动相,利用梯度洗脱方式,在主机转速为880 r/min、流速1.2 mL/min条件下,成功地从70 mg母丁香粗提物中分离得到12.3 mg鞣花酸、9.6 mg鼠李素、17.2 mg槲皮素,从50 mg公丁香粗提物中分离得到5,7-二甲氧基-2-甲基色原酮10.2 mg、5,7-二甲氧基-2,6-二甲基色原酮8.6 mg,纯度均在96%以上。各化合物的结构均由质谱和核磁共振氢谱、碳谱鉴定。利用该方法可以对丁香不同药用部位中的非挥发性化合物进行有效的分离和纯化。     

Preparative isolation and purification of harpagoside and angroside C from the root of Scrophularia ningpoensis Hemsley by high‐speed counter‐current chromatography

In this study, the bioactive component harpagoside and angroside C in the root of Scrophularia ningpoensis Hemsley was simultaneously separated by high‐speed counter‐current chromatography (HSCCC). A two‐phase solvent system containing chloroform/n‐butanol/methanol/water (4:1:3:2, v/v/v/v) was selected following consideration of the partition coefficient of the target compound. The crude extract (200 mg) was loaded onto a 280‐mL HSCCC column and yielded 22 mg harpagoside and 31 mg angroside C with the purity of higher than 98 and 98.5%, respectively. It is feasible to isolate active compounds harpagoside and angroside C from S. ningpoensis using HSCCC.     

Single‐step isolation of embelin using high‐performance countercurrent chromatography and determination of the fatty acid composition of seeds of Embelia schimperi

Embelin (2,5‐dihydroxy‐3‐undecyl‐p ‐benzoquinone) is known for its potent anthelmintic activity, but also for wound‐healing, antidiabetic, anticonvulsant, antitumour, anti‐inflammatory, analgesic, hepatoprotective, antioxidant, antibacterial and antispermatogenic activities. A high‐performance countercurrent chromatography method was developed for the purification of embelin from an extract of the seeds of Embelia schimperi fruit. The optimized solvent system (n ‐hexane–ethylacetate–ethanol–water, 7:3:7:3) resulted in the isolation of 13.9 mg of embelin, directly from 100 mg of crude extract, in a single step within a short time (40 min). Although the compound appeared to be completely pure when analysed by ultra‐performance liquid chromatography (UPLC) with photo diode array detection, the purity was established as ~90% by UPLC–mass spectrometry. Furthermore, we report the fatty acid composition of the seeds of E. schimperi fruit. Nine fatty acids were quantified from the fruit seed extract by gas chromatography–mass spectrometry, with linoleic (46.4%), palmitic (21.5%) and oleic (19.6%) acids making up the largest proportions.     

A New Isoflavanol from the Fruits of Kotschya strigosa (Fabaceae)

The phytochemical investigation of the MeOH extract from fruits of Kotschya strigosa using repeated normal and reversed‐phase column chromatography and Sephadex LH‐20 chromatography led to the isolation and characterization of a new isoflavanol, named kotstrigoisoflavanol ( 1 ), together with three known compounds, diosmetin ( 2 ), β‐sitosterol ( 3 ), and the 3‐O‐β‐d‐glucopyranoside of β‐sitosterol ( 4 ). The antioxidant activity of crude extract, 1, and 2 was determined using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH˙) method. The crude extract (IC₅₀ 61.7 ± 0.2 μg/ml) and 2 (IC₅₀ 70.2 ± 0.1 μg/ml) showed moderate antioxidant activities, while 1 was weakly active (IC₅₀ 153.1 ± 0.1 μg/ml), as compared with the standard reference l ‐ascorbic acid (IC₅₀ 21.9 ± 0.0 μg/ml).