Yuchi Zhang

Comprehensive separation and identification of chemical constituents from Apocynum venetum leaves by high-performance counter-current chromatography and high performance liquid chromatography coupled with mass spectrometry.

High-performance counter-current chromatography (HPCCC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) was efficiently utilized for the separation and identification of the chemical components with a wide range of polarity from the mixed extract of Chinese medicinal herb Apocynum venetum. For HPCCC separation, four sets of solvent systems, n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:4.5, v:v:v:v), ethyl acetate-methanol-water (5:2:5, v:v:v) and n-butanol-methanol-water (5:1:5, v:v:v) were used for the one-step separation by four stages. The HPCCC separation was initiated by filling the column with the lower phase of n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:5, v:v:v:v) as a stationary phase followed by elution with the upper phase of n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:5, v:v:v:v) to separate the hydrophobic compounds (tail to head). Then the mobile phase was switched to the upper phase of ethyl acetate-acetonitrile-water (5:3:7, v:v:v) to eluted the moderate hydrophobic compounds, then the mobile phase was switched to the upper phase of ethyl acetate-methanol-water (5:2:5, v:v:v) to eluted the moderate hydrophilic compounds, and finally the hydrophilic compounds still retained in the column was eluted by the upper phase of n-butanol-methanol-water (5:1:5, v:v:v). A total of 16 named compounds including adhyperforin, hyperforin, amentoflavone, biapigenin, quercetin, avicularin, acetylated isoquercetin, acetylated hyperoside, astragalin, trifolin, isoquercetin, hyperoside, querciturone, rutin, chlorogenic acid and quercetin-3-O-β-D-glucosyl-β-D-glucopyranoside were successfully separated via the four sets of solvent systems in one step operation for 130 min. The compounds separated by HPCCC were identified by comparing with mixed standards data of HPLC-MS as well as NMR data.

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.

Application of accelerated solvent extraction coupled with high-performance counter-current chromatography to extraction and online isolation of chemical constituents from Hypericum perforatum L.

Accelerated solvent extraction (ASE) coupled with high-performance counter-current chromatography (HPCCC) was successfully used for the extraction and online isolation of five chemical constituents from the plant Hypericum perforatum L. The upper phase of the solvent system of ethyl acetate-methanol-water (5:2:5, v:v:v) was used as both the ASE solvent and the HPCCC stationary phase. Two hydrophobic compounds including 28.4 mg of hyperforin with a HPLC purity of 97.28% and 32.7 mg of adhyperforin with a HPLC purity of 97.81% were isolated. The lower phase of ethyl acetate-methanol-n-butanol-water (5:2:2.5:12, v:v:v:v) was used as both the ASE solvent and CCC stationary phase. Three hydrophilic compounds of 12.7 mg of 3,4,5-O-tricaffeoylquinic acid with a HPLC purity of 98.82%, 15.2 mg of 1,3,5-O-tricaffeoylquinic acid with a HPLC purity of 99.46% and 42.5mg of 3-O-caffeoylquinic acid with a HPLC purity of 96.90%, were obtained in a one-step extraction-separation process with less than 3h from 10.02 g of raw material of H. perforatum. The targeted compounds isolated, collected and purified by HPCCC were analyzed by high performance liquid chromatography (HPLC), the chemical structures of all five compounds above mentioned were identified by UV, MS and NMR.

Development of a method to screen and isolate potential xanthine oxidase inhibitors from Panax japlcus var via ultrafiltration liquid chromatography combined with counter-current chromatography

Panax japlcus var is a typical Chinese herb with a large number of saponins existing in all parts of it. The common methods of screening and isolating saponins are mostly labor-intensive and time-consuming. In this study, a new assay based on ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) was developed for the rapid screening and identifying of the ligands for xanthine oxidase from the extract of P. japlcus. Six saponins were identified as xanthine oxidase inhibitors from the extract. Subsequently, the specific binding ligands, namely, 24 (R)-majoroside R1, chikusetsusaponin IVa, oleanolic acid-28-O-β-D-glucopyranoside, notoginsenoside Fe, ginsenoside Rb2 and ginsenoside Rd (the purities of them were 95.74%, 96.12%, 93.19%, 94.83%, 95.07% and 94.62%, respectively) were separated by high-speed counter-current chromatography (HSCCC). The component ratio of the solvent system of HSCCC was calculated with the help of a multiexponential function model was optimized. The partition coefficient (K) values of the target compounds and resolutions of peaks were employed as the research indicators, and exponential function and binomial formulas were used to optimize the solvent system and flow rate of the mobile phases in a two-stage separation. An optimized two-phase solvent system composed of ethyl acetate, isopropanol, 0.1% aqueous formic acid (1.9:1.0:1.3, v/v/v, for the first-stage) and that composed of methylene chloride, acetonitrile, isopropanol, 0.1% aqueous formic acid (5.6:1.0:2.4:5.2, v/v/v/v, for the second-stage) were used to isolate the six compounds from P. japlcus. The targeted compounds isolated, collected and purified by HSCCC were analyzed by high performance liquid chromatography (UPLC), and the chemical structures of all the six compounds were identified by UV, MS and NMR. The results demonstrate that UF-LC-MS combined with HSCCC might provide not only a powerful tool for screening and isolating xanthine oxidase inhibitors in complex samples but also a useful platform for discovering bioactive compounds for the prevention and treatment of gout.

Solvent gradient elution for comprehensive separation of constituents with wide range of polarity in Apocynum venetum leaves by high-speed counter-current chromatography

A novel gradient elution was efficiently utilized for the separation of the chemical components with a wide range of polarity from the mixed extract of the Chinese medicinal herb Apocynum venetum or mixed standards by high-speed counter-current chromatography. Three sets of solvent systems, n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v), ethyl acetate-methanol-water (5:2:5 v/v/v) and n-butanol-methanol-water (5:1:5 v/v/v) were used for the one-step elution. The separation was initiated by filling the column with the lower phase of n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v) as a stationary phase followed by elution with the upper phase of n-hexane-ethyl acetate-methanol-water (1.5:3.5:2:4.5 v/v/v/v) to separate the hydrophobic compounds (tail to head). Then the mobile phase was switched to the upper phase of ethyl acetate-methanol-water (5:2:5 v/v/v) to elute the moderate hydrophobic compounds, and finally the hydrophilic compounds still retained in the column were fractionated by eluting the column with the upper phase of n-butanol-methanol-water (5:1:5 v/v/v). A total of 13 compounds including adhyperforin, hyperforin, amentoflavone, biapigenin, quercetin, astragalin, trifolin, isoquercetin, hyperside, acetyled hyperside, rutin, chlorogenic acid and quercetin-3-O-β-D-glucosyl-β-D-glucopyranoside were successfully separated via the three sets of solvent systems in one-step operation for 90 min.

Circulating ultrasound-assisted extraction, countercurrent chromatography, and liquid chromatography for the simultaneous extraction, isolation, and analysis of the constituents of Uncaria tomentosa

A hyphenated automated technique for the online extraction, isolation, analysis, and identification of natural organic compounds was established. Circulating ultrasound-assisted extraction (CUAE) was coupled with countercurrent chromatography (CCC), high performance liquid chromatography (HPLC), and a diode array detector (DAD). This approach was applied to the fractionation and purification of alkaloids from Uncaria tomentosa. A biphasic solvent system of chloroform-methanol-water (6:4:5, v:v:v) was used for the CUAE and CCC separation of compounds from 500 g of U. tomentosa. Two CUAE/CCC/HPLC/DAD modes were established. Either the upper aqueous phase or the lower organic phase of the solvent system could be used as the extraction solvent. The target compounds were extracted by CUAE, and the extract was pumped into a sample loop before being directly injected into the CCC column, or pre-purified using a flash chromatography column before injection. The target compounds were eluted using either the organic or aqueous phase of the solvent system and the fractions were monitored using a UV detector. The target fractions were collected by a sample loop via a six-port valve, and analyzed by HPLC/DAD for purity and structural identification. This system isolated of 8.2mg, 7.4 mg, and 12.9 mg of rhynchophylline, corynoxine, and corynoxine B with HPLC purities of 96.15%, 95.34%, and 95.49%, respectively via the first mode; and isolated 26.6 mg, 24.6 mg, and 45.3mg of rhynchophylline, corynoxine, and corynoxine B with a HPLC purities of 98.22%, 97.18%, and 97.93% via the second mode.

Ultrafiltration LC-PDA-ESI/MS combined with reverse phase-medium pressure liquid chromatography for screening and isolation potential α-glucosidase inhibitors from Scutellaria baicalensis Georgi

Diabetes mellitus (DM) has become a worldwide health problem. α-Glucosidase inhibitors are widely used in the treatment of type 2 diabetes, and natural products are considered as the promising sources of α-glucosidase inhibitors. In this research, ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (Ultrafiltration LC-PDA-ESI/MS) were applied to screen and identify α-glucosidase inhibitors from Scutellaria baicalensis. Following this, reverse-phase medium-pressure liquid chromatography (RP-MPLC) was applied to separate and isolate the active constituents. As a result, eleven major compounds in S. baicalensis were screened and identified as α-glucosidase inhibitors by Ultrafiltration LC-PDA-ESI/MS and six ligands, baicalin, wogonoside, 5,7,3,2′,6′-pentahydroxy flavanone, chrysin-6-C-arabinosyl-8-C-glucoside, chrysin-6-C-glucosyl-8-C-arabinoside and wogonin, were isolated by RP-MPLC. The purities of the six compounds were 89.7%, 83.5%, 76.2%, 93.7%, 75.1% and 96.2%, respectively, as determined by high performance liquid chromatography. The results demonstrate that the method for systematic isolation of bioactive components in S. baicalensis guided by Ultrafiltration LC-PDA-ESI/MS is a feasible and efficient technique and could be expected to extend for the separation of other enzyme inhibitors.

Neuroprotection by Paeoniflorin in the MPTP mouse model of Parkinson's disease.

&NA; Paeoniflorin (PF) is a major bioactive ingredient in Radix Paeonia alba roots that has low toxicity and has been shown to have neuroprotective effects. Our in vitro experiments suggested that PF affords a significant neuroprotective effect against MPP+‐induced damage and apoptosis in PC12 cells through Bcl‐2/Bax/caspase‐3 pathway. The objectives of the present study were to explore the potential neuroprotective effect of PF in 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP)‐treated mouse model of Parkinsons disease (PD). Our results demonstrated that PF treatment ameliorated the behavioral deficits of “in spontaneous motor activity and latency to fall of the rotarod test”, and reduced dopaminergic cell loss that were induced by MPTP in a dose‐dependent manner in an in vivo model of PD. In addition, we found that treatment of PF protected dopaminergic neurons by preventing MPTP‐induced decreases in striatal and substantia nigra dopaminergic transporter (DAT) and tyrosine hydroxylase (TH) protein levels, and by changing dopamine catabolism and inhibiting dopamine turnover. Furthermore, it was also associated with up‐regulation of the Bcl‐2/BAD ratio, and inhibition of the activation of caspase‐9 and caspase‐3. These results showed that PF promoted dopamine neuron survival in vivo due to the MAO‐B inhibition, and the PI3K/Akt signaling pathway may have mediated the protection of PF against MPTP, suggesting that PF treatment might represent a neuroprotective treatment for PD. Graphical abstract The objectives of the present study were to explore the potential neuroprotective effect of Paeoniflorin (PF) in 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐ tetrahydropyridine (MPTP)‐treated mouse model of Parkinsons disease (PD). The results showed that PF treatment ameliorated the behavioral deficits of “in spontaneous motor activity and latency to fall of the rotarod test”, and reduced dopaminergic cell loss that were induced by MPTP in a dose‐dependent manner in an in vivo model of PD. In addition, we found that treatment of PF protected dopaminergic neurons by preventing MPTP‐induced decreases in striatal and substantia nigra dopaminergic transporter (DAT) and tyrosine hydroxylase (TH) protein levels dopaminergic transporter (DAT) protein levels and by changing dopamine catabolism and inhibiting dopamine turnover. Furthermore, it was also associated with up‐regulation of the Bcl‐2/BAD ratio, and inhibition of the activation of caspase‐9 and caspase‐3. These results showed that PF promoted dopamine neuron survival in vivo due to the MAO‐B inhibition, and the PI3K/Akt signaling pathway may have mediated the protection of PF against MPTP, suggesting that PF treatment might represent a neuroprotective treatment for PD. Figure. No caption available. HighlightsPaeoniflorinin (PF) has a protective against MPTP‐induced neuronal damage.The protective effect of PF appears to be related to DA system and PI3K/Akt signaling pathway.Treatment with PF returned both TH and DAT to normal levels.PF has potential as a naturally derived treatment to halt the progression of PD.

Screening and isolation of potential lactate dehydrogenase inhibitors from five Chinese medicinal herbs: Soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali.

Stroke is among the leading causes of death and severe disability worldwide. Flavonoids have been extensively used in the treatment of ischemic stroke by reducing lactate dehydrogenase levels and thereby enhancing blood perfusion to the ischemic region. Here, we used ultrafiltration high-performance liquid chromatography coupled with diode array detection and mass spectrometry for the rapid screening and identification of flavonoids from five Chinese medicinal herbs: soybean, Radix pueraria, Flos pueraria, Rhizoma belamcandae, and Radix astragali. Using PC12 cells as a suitable in vitro model of toxicity, cell viability was quantitated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that the extracts of soybean and the six major components, namely, acetyldaidzin, malonylgenistin, daidiain, glycitin, genistin, and acetylcitin; the extract of R. pueraria and its main component daidzein; the extract of F. pueraria and its three major components, tectorigenin, tectoridin, and tectorigenin-7-O-xylosylglucosid; and the extract of R. belamcandae and its main component, tectoridin, were strong lactate dehydrogenase inhibitors. Also, the components of R. astragali showed no bioactivity. These findings indicate that the ultrafltration high-performance liquid chromatography coupled with diode array detection and mass spectrometry method could be utilized in rapid screening and separation of bioactive compounds from a complex matrix.

Ultrafiltration-LC–MS combined with semi-preparative high-performance liquid chromatography for the simultaneous screening and isolation of lactate dehydrogenase inhibitors from Belamcanda chinensis

Stroke represents the fourth leading cause of death in the USA and the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke and natural products are considered a promising source of novel lactate dehydrogenase inhibitors. In this study, we used PC12 cells to determine the protective effect of extracts from the herb Belamcanda chinensis following toxic challenge. Using ultrafiltration high-performance liquid chromatography coupled with photo-diode array detection and electrospray ionization mass spectrometry, we screened and identified isoflavonoids from Belamcanda chinensis extracts. Semi-preparative high-performance liquid chromatography was then applied to separate and isolate the active constituents. Using these methods, we identified six major compounds in Belamcanda chinensis as lactate dehydrogenase inhibitors: tectoridin, iristectorin A, iridin, tectorigenin, irigenin, and irisflorentin, which were then isolated to >92% purity. This is the first report that Belamcanda chinensis extracts contain potent lactate dehydrogenase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from Belamcanda chinensis guided by ultrafiltration high-performance liquid chromatography coupled with photo-diode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could be extended for the identification and isolation of other enzyme inhibitors.