Screening of bioactive compounds in Rhizoma Polygoni Cuspidati with hepatocyte membranes by HPLC and LC–MS

A new method employing HPLC, LC–MS, and hepatocyte membranes for the screening of bioactive compounds in traditional Chinese medicines (TCMs) has been proposed. We hypothesized that exposure of the TCM extracts to hepatocyte membranes should decrease the concentration of membrane‐permeable compounds in the solution. Using this approach, the permeability of the compounds in Rhizoma Polygoni Cuspidati was investigated. By comparing chromatograms of samples prepared both before and after interaction with hepatocyte membranes, seven permeable compounds of Rhizoma Polygoni Cuspidati were identified. Additionally, it was found that piceid, resveratrol, emodin‐8‐β‐d‐glucoside, physcion‐8‐β‐d‐glucoside, aloe‐emodin, emodin, and physcion combined specifically with hepatocyte membranes, which might indicate a useful approach for revealing the antiatherosclerotic effects of Rhizoma Polygoni Cuspidati. Therefore, the proposed method could be a good approach to predict the potential bioactivities of multiple compounds in TCMs simultaneously. Based on the significance of these results, this method could be a novel approach for identifying potentially bioactive components in other TCMs.     

Simultaneous determination of anthraquinones in radix Polygoni multiflori by capillary gas chromatography coupled with flame ionization and mass spectrometric detection

A rapid, accurate and reliable analytical method was developed for the simultaneous determination of five major anthraquinones, aloe-emodin, chrysophanol, emodin, physcion, and rhein, in radix Polygoni multiflori, a traditional Chinese herbal medicine. The method comprises a fast ultrasonic extraction with methanol and derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+1% trimethylchlorosilane (TMCS) followed by capillary gas chromatographic (GC) separation. The effect of reaction time on the derivatization of anthraquinones was examined. A baseline separation of the anthraquinone and internal standard derivatives was achieved in 15min. The detection limits range from 0.22 to 0.60microg/mL for the five anthraquinones. The calibration curves are linear over the concentration range studied (from the detection limits to 40.0microg/mL) with the squares of correlation coefficients, R2, greater than 0.998. The developed method was successfully applied to the simultaneous determination of anthraquinones in radix P. multiflori samples. The peak identification was confirmed using GC-MS. The contents of anthraquinones in radix P. multiflori samples studied were 27.41, 289.6, 64.22, 202.1, 288.6microg/g for chrysophanol, emodin, aloe-emodin, physcion, rhein, respectively. All relative standard deviations are less than 3.2%. The recoveries range from 80.2% to 119.3% for the five analytes. To the authors' best knowledge, this is the first GC method reported for the simultaneous determination of the five anthraquinones in radix P. multiflori.     

微波辅助提取石蒜和虎杖中有效成分的动力学机理

以鳞茎药材石蒜(Lycoris radiata)和根茎药材虎杖(Rhizma Polygoni Cuspidati)作为研究对象,采用微波辅助提取(MAE)石蒜中的石蒜碱、力可拉敏和加兰他敏以及虎杖中的大黄素和白藜芦醇,研究了提取过程的动力学机理。分别对这5种组分在提取温度、提取时间、搅拌速度和颗粒度等因素影响下MAE过程的动力学行为和特征进行了探讨,并与常规的溶剂回流提取法(SRE)进行对比研究,采用扫描电镜观察了MAE和SRE提取后样品的表面细胞结构。结果表明,石蒜和虎杖的MAE提取过程具有明显不同的动力学特征,其机理分别基于内部扩散传质控制和细胞破壁引起的界面反应控制。与SRE相比,MAE能引起石蒜和虎杖的细胞结构发生显著变化,降低了表观活化能Ea,组分的表观速率常数明显增大。     

油菜蜂花粉黄酮含量的HPLC测定

以95％乙醇为溶剂,采用索氏提取器提取青海产油菜蜂花粉中的黄酮类化合物,将黄酮提取物中的黄酮甙水解为黄酮甙元后,利用HPLC法测定其中槲皮素、山萘酚、异鼠李素含量。结果表明,青海油菜蜂花粉中槲皮素、山萘酚、异鼠李素的平均含量分别为0．928％、0．295％、0．0834％,换算成总黄酮含量为3．28％。     

Chinese hamster ovary‐sphingomyelin synthase2 biospecific extraction and liquid chromatography with tandem mass spectrometry analysis for the prediction of bioactive components of Rhizoma Polygoni Cuspidati

A novel strategy for predicting bioactive components in traditional Chinese medicines using Chinese hamster ovary‐sphingomyelin synthase2 (CHO‐SMS₂) cell biospecific extraction and high‐performance liquid chromatography with diode array detection and tandem mass spectrometry analysis was proposed. The hypothesis is that when cells are incubated with the extract of traditional Chinese medicines, the potential bioactive components in the traditional Chinese medicines should selectively combine with the cells, while the cell‐combining components would be detectable in the extract of denatured cells. The identities of the cell‐combining components could be determined by liquid chromatography with tandem mass spectrometry. Using the proposed approach, the potential bioactive components of Rhizoma Polygoni Cuspidati, a commonly used traditional Chinese medicine for atherosclerosis, were detected and identified. Eight compounds in the extract of Rhizoma Polygoni Cuspidati were detected as the components selectively combined with CHO‐SMS₂ cells, which is a stable cell line that highly expresses sphingomyelin synthases, it was found that piceid, resveratrol, emodin‐8‐β‐d‐ glucoside, physcion‐8‐β‐d‐ glucoside, emodin, physcion, 3,5,4‘‐trihydroxystilbene‐3‐O‐(6“‐galloyl)‐glucoside, and emodin‐1‐O‐glucoside combined specifically with CHO‐SMS₂ cells. The results indicate that the proposed approach may be applied to predict the bioactive candidates in traditional Chinese medicines.     

Intestinal metabolism of Polygonum cuspidatum in vitro and in vivo

Rhizoma et Radix Polygoni Cuspidati (RRPC) is commonly prescribed for the treatment of amenorrhea, arthralgia, jaundice and abscess in traditional Chinese medicine. Previous pharmacological studies have indicated that polyphenols are the main pharmacological active ingredients in RRPC. Meanwhile, the poor bioavailability of polyphenols in RRPC implies that those components are probably metabolized by intestinal bacteria before absorption. However, there is rather limited information about RRPC''s metabolites produced by intestinal bacteria and the intestinal absorbed constituents. In the present study, the metabolites were characterized after the aqueous extract of RRPC was incubated with the crude enzyme of human intestinal bacteria in vitro. The metabolic characteristics of glycosides in RRPC were figured out by comparing the metabolic profiles of emodin‐8‐O‐β‐d ‐glucopyranoside and polydatin between aqueous extract of RRPC and equivalent amounts of these two glycosides. The transitional constituents absorbed into blood were investigated in rats via intraduodental administration and portal vein intubation. A total of 38 prototype components and 43 metabolites were detected and characterized in vivo. The overall results demonstrated that the intestinal bacteria played an important role in the metabolism of RRPC, and the main metabolic pathways were hydrolysis in vitro, glucuronidation and sulfation in vivo.     

凝胶渗透色谱净化超高效液相色谱-串联质谱法检测甘草及其提取物中的11种氨基甲酸酯类农药残留

建立了甘草及其提取物中11种氨基甲酸酯类农药多残留的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。以11种氨基甲酸酯类农药为目标分析物,样品经乙腈超声提取、凝胶渗透色谱(GPC)净化后,用UPLC-MS/MS检测。以甘草及其提取物为例,分别进行了0.02、0.04和0.1 mg/kg 3个添加浓度的11种目标分析物的加标回收率实验,甘草中11种目标分析物的回收率为72.2%～94.0%,相对标准偏差为0.7%～7.8%;甘草提取物中11种目标分析物的回收率为73.8%～94.7%,相对标准偏差为1.5%～12.7%。该方法灵敏度高、准确度好,符合农药多残留检测的技术要求,适用于甘草、黄芪等中药材及其提取物中氨基甲酸酯类农药残留的检测。     

Studies of the Synergistic Actions of Three Herbs in Tong-Mai Formula

Tong-Mai oral liquid, containing three herbs (Pueraria radix, radix Salviae miltiorrhizae and rhizoma Chuanxiong), is a formula created under the theory of traditional Chinese medicine (TCM) to invigorate the ‘blood’ and dispel ‘blood stasis’, which arises from poor blood circulation. In Tong-Mai formula, Pueraria radix is used for improved blood circulation and to treat cardiovascular diseases and arrhythmia. Radix Salviae miltiorrhizae was widely used in TCMs for treatment of coronary heart disease, blood circulation diseases, and other cardiovascular diseases; rhizoma Chuanxiong is used to treat thrombosis and platelet aggregation. Clinically, combining the use of these herbs was therapeutically valuable, which suggests that they might synergistically prevent thrombosis. In this study, the pharmacokinetic behaviors of puerarin and daidzein in rat plasma were studied after orally administrated to four kinds of extracts. Puerarin and daidzein were set as index components. In the Tong-Mai oral liquid group, puerarin and daidzein showed the maximum plasma concentration and the lowest clearance rate of plasma. The results indicated that the herbal constituents in Tong-Mai formula showed synergism, and it may be helpful to certificate the rationality of the matching in Tong-Mai formula.

     

Monitoring Volatile and Non-Volatile Salicylates in Filipendula ulmaria by Different Chromatographic Techniques

The use of salicylate containing herbs as analgesic and antirheumatic remedies can be traced back to ancient times. While salicaceen contains only derivatives of salicylalcohol, Filipendula ulmaria is characteristic for various forms of volatile and non-volatile salicylates. The main pharmacological effect of Filipendula ulmaria is due to its salicylic acid component, which is released via oxidation from various aglycones (e.g. salicylaldehyde, methylsalicylate) developed from glycosides through hydrolysis in the digestive system. Our current study is focussed on the determination of volatile and non-volatile salicylates in various parts and extracts of the plant, using different chromatographic techniques.     

Studies of the Synergistic Actions of Three Herbs in Tong-Mai Formula

Tong-Mai oral liquid, containing three herbs (Pueraria radix, radix Salviae miltiorrhizae and rhizoma Chuanxiong), is a formula created under the theory of traditional Chinese medicine (TCM) to invigorate the ‘blood’ and dispel ‘blood stasis’, which arises from poor blood circulation. In Tong-Mai formula, Pueraria radix is used for improved blood circulation and to treat cardiovascular diseases and arrhythmia. Radix Salviae miltiorrhizae was widely used in TCMs for treatment of coronary heart disease, blood circulation diseases, and other cardiovascular diseases; rhizoma Chuanxiong is used to treat thrombosis and platelet aggregation. Clinically, combining the use of these herbs was therapeutically valuable, which suggests that they might synergistically prevent thrombosis. In this study, the pharmacokinetic behaviors of puerarin and daidzein in rat plasma were studied after orally administrated to four kinds of extracts. Puerarin and daidzein were set as index components. In the Tong-Mai oral liquid group, puerarin and daidzein showed the maximum plasma concentration and the lowest clearance rate of plasma. The results indicated that the herbal constituents in Tong-Mai formula showed synergism, and it may be helpful to certificate the rationality of the matching in Tong-Mai formula.     

Analysis of resveratrol in wine by capillary electrophoresis

Capillary electrophoresis (CE) is a new analytical technique that has recently been reported as a method for analysis of resveratrol in wine. Several different separation approaches have been taken in these reports. In comparison with high-performance liquid chromatography (HPLC), CE methods have similar sensitivity and can discriminate between trans- and cis-isomers of resveratrol. CE methods also show promise for analysis of other flavonoid antioxidants (glycosides and aglycones) in wine.     

微波辅助提取石蒜和虎杖中有效成分的热力学机理研究

采用微波辅助提取(MAE)技术研究了石蒜和虎杖两种不同植物中石蒜碱、力可拉敏和加兰他敏以及白藜芦醇和大黄素提取过程的热力学机理. 以溶剂回流提取方法(SRE)作为对比, 采用一种简单的测定提取分配系数的方法, 计算了这些组分在两种提取过程中的热力学函数ΔH0, ΔS0和ΔG0, 对其化学结构与极性以及在MAE过程中的热力学行为进行了讨论, 并用扫描电镜法观察了MAE和SRE提取后样品的细胞结构. 结果表明, 石蒜和虎杖的提取是一个吸热熵增的过程, 微波的作用导致石蒜和虎杖细胞结构发生显著变化, 使MAE热力学函数变化较大, 其提取过程的热力学行为特征与SRE明显不同, 但提取效率提高.     

A Reliable Gas Capillary Chromatographic Determination of Lactulose in Dairy Samples

The rhizome of Polygonum cuspidatum is an important Chinese medicine used against infectious hepatitis, leucorrhagia, pruritus vulvae of the dampness-heat type, burns, snake bite, carbunculosis, amenorrhea, dysmenorrhea, trauma with blood stasis, and rheumatism, etc. Emodin, resveratrol, and polydatin are main active components of the rhizome. We report a simple densitometric HPTLC method for quantification of these compounds. The method was validated for precision, repeatability, and accuracy. The method was found to be precise, with RSD of 0.23, 0.25, and 0.32 (interday) and 0.45, 0.57, and 0.48 (intraday) for different concentrations of emodin, resveratrol, and polydatin, respectively. Instrument precision was 0.25, 0.23, and 0.34 (%CV) for emodin, resveratrol, and polydatin, respectively. The accuracy of the method was checked by measuring the recovery of the three compounds at three different levels; the average recoveries were 102.56%, 100.21%, and 100.27%, respectively. The amounts of emodin, resveratrol and polydatin in Polygonum cuspidatum, as estimated by the proposed method, were 4.96 mg g^–1, 1.81 mg g^–1, and 13.02 mg g^–1. The HPTLC method proposed for estimation of emodin, resveratrol and polydatin was found to be simple, precise, specific, sensitive, and accurate and can be used for quality control of Polygonum cuspidatum.     

Rapid microwave-assisted hydrolysis for determination of ginkgo flavonol glycosides in extracts of Ginkgo biloba leaves

A rapid microwave-assisted hydrolysis (MAH) method is presented for the sample pretreatment of the determination of ginkgo flavonol glycosides in extracts of Ginkgo biloba L. (EGb). By this method, flavonol glycosides can be completely hydrolyzed within 2 min. After investigating the effects of solvents, acidity, microwave power, and microwave radiation time on hydrolysis, the optimal hydrolysis conditions are as follows: 300 W of microwave power, 2 min of hydrolysis time, 5.7% of hydrochloric acid in the hydrolysis solution, and n-propanol as the hydrolysis solvent. After MAH of the samples, three flavonol aglycones, such as quercetin, kaempferol, and isorhamnetin are analyzed by high-performance liquid chromatography. Compared with conventional reflux hydrolysis, this method owns offers several advantages: it saves time, costs less, and is environmentally friendly.     

Traditional processing strongly affects metabolite composition by hydrolysis in Rehmannia glutinosa roots

The processing of biological raw materials is considered to have an important role in the therapeutic application in Traditional Chinese Medicine. The root of Rehmannia glutinosa has to be processed by nine cycles of rice wine immersing, steaming and drying before using in clinical applications. In order to understand the chemical changes resulting from the processing, a comprehensive analysis of Rehmanniae radix was made using (1)H-NMR and Fourier transform (FT)-mass spectrometry in combination with multivariate data analysis. After (1)H-NMR and principle component analysis, hydrolysis was found to be the major chemical process during the treatments. Catalpol, raffinose and stachyose levels gradually decreased during processing, whereas monosaccharides including galactose and glucose were found to be higher in processed roots. The metabolic profile changed gradually through the processing cycles although the differences became smaller after the fifth processing cycle. The positive and negative ion-mode mass spectra by high resolution FT-MS revealed several series of ion clusters with mass differences of 162.053 Da, indicating a difference of a hexose moiety. During the processing, the number and signal intensity of the smaller glycosides were increased. Therefore, these results indicate that the fresh Rehmanniae radix is rich in polysaccharides, which are hydrolyzed during the processing.     

Selective separation of flavonoid glycosides in Dalbergia odorifera by matrix solid-phase dispersion using titania

Dalbergia odorifera contains high concentrations of flavonoid aglycones and trace flavonoid glycosides. In this study, trace flavonoid glycosides were separated from D. odorifera by titania with matrix solid-phase dispersion (MSPD). Before the MSPD experiment, four standards, including two isoflavone glycosides (genistin and formononetin-8-C-apiosyl (1-6)-glucoside) and their aglycones (genistein and formononetin), were used to compare their retention on a titania column. The effect of acetonitrile concentration and pH on their retention was investigated and a conclusion was drawn that high acetonitrile concentration and pH lead to the greatest difference in the retention of flavonoid as glycosides and aglycones. Besides hydrophilic interaction and ligand-exchange interaction may exist between sugar moiety of flavonoid glycoside and titania, so that flavonoid glycosides have stronger retention than that of aglycones. Based on the chromatographic rule of flavonoid as glycosides and aglycones on the titania column, the MSPD method was optimized to elute high concentration flavonoid aglycones first with 90% acetonitrile and 10% water containing 100 mM ammonium acetate buffer, and then to elute trace flavonoid glycosides with 20% acetonitrile and 80% water containing 1% trifluoroacetate (TFA). Isolated flavonoid glycosides were further analyzed by UPLC-MS/MS, and their fragmentation in MS(2) showed they are C-glycosyl flavonoids.     

Medicinal foodstuffs. XVIII. Phytoestrogens from the aerial part of Petroselinum crispum MIll. (Parsley) and structures of 6"-acetylapiin and a new monoterpene glycoside, petroside

In the course of our screening for natural estrogenic compounds from Occidental medicinal herbs, the extracts of several herbs were found to show proliferative activity in MCF-7 (an estrogen-sensitive breast cancer cell line). Among these active herbs, the methanolic extract from the aerial parts of Petroselinum crispum (parsley) showed potent estrogenic activity, which was equal to that of isoflavone glycosides from soybean. Through bioassay-guided separation, we isolated several flavone glycosides and a new flavone glycoside, 6"-acetylapiin, with estrogenic activity together with a new monoterpene glucoside, petroside. The structures of 6"-acetylapiin and petroside were characterized by the chemical and physicochemical evidence. Estrogenic activities of these flavone glycosides were found to be enhanced by removal of their glycoside moieties. The EC50 values (concentration needed to enhance the MCF-7 proliferation 50% compared to non-estrogen treated cell) of their aglycones are as follows, apigenin (1.0 microM), diosmetin (2.9 microM), and kaempferol (0.56 microM). The estrogenic activities of these flavones are nearly equal to those of the isoflavones, daidzein (0.61 microM) and genistein (0.60 microM). The methanolic extract of parsley, apiin, and apigenin restored the uterus weight in ovariectomized mice when orally administered for consecutive 7 days.     

Die Cardenolide und Pregnan-Glykoside der Wurzeln von Asclepias lilacina WEIMARCK. I. Isolierungen Glykoside und Aglykone, 290. Mitteilung

The dried roots of Asclepias lilacina contain ca. 14% glykosides, in which both cardenolides and ester glycosides of pregnane derivatives are present, the latter predominating. Uzarigenin, coroglaucigenin, and their corresponding glycosides ascleposide and frugoside, resp., were shown to be present; of these four, uzarigenin was isolated in crystalline form. The ester glycosides, some of which could be isolated in a crystalline state, are predominantly composed of the aglycones 20-O-acetyl-12-O-benzoyl-sarcostin and 12,20-di-O-benzoyl-sarcostin, besides which esters of desacetyl-metaplexigenin, lineolon, and dihydrosarcostin are also present. These aglycones are mostly attached to various trisaccharides which are composed of at least two 2-deoxy-sugar residues. Mild acid hydrolysis gave, besides the genins, 7 sugars, namely cymarose, oleandrose, digitoxose, 3-O-methyl-6-deoxy-D-allose (U3), as well as 3 unknown sugars U1, U2 and U5 which are probably disaccharides. U1 and U2 were isolated in crystalline form; they are isomeric and have been named asclepobiose and lilacinobiose; they are probably composed of units similar to those in the isomeric pachybiose.     

Simultaneous quantification of terpenelactones and flavonol aglycones in hydrolyzed ginkgo biloba extract by liquid chromatography with inline ultraviolet and evaporative light scattering detection

We report here a liquid chromatography (LC) method with inline ultraviolet/evaporative light scattering (UV/ELS) detection for the simultaneous quantification of the terpenelactones and flavonol aglycones in a single sample of hydrolyzed Ginkgo biloba extract (GBE). The sample is hydrolyzed by a rapid and convenient oven heating method for 1 h at 90 degrees C with 10% hydrochloric acid. The 1 h hydrolysis was found to be equivalent to the 2.25 h reflux treatment for dry powder extract, where total flavonol glycosides were 28.4 and 28.1%, respectively. Acceptable precision was achieved for total terpenelactones [relative standard deviation (RSD) = 4.8%] by ELS detection, and total flavonol aglycones (RSD = 2.3%) by UV detection. The analytical range was 1.5 to 7.3% (w/w) for the individual terpenelactones (ELS) and 2.5 to 15.0% (w/w) for the individual glycosides (UV) calculated from the aglycones quercetin, kaempferol, and isorhamnetin. This improved method allows for the first time high throughput sample preparation coupled with the quantification of the predominant compounds generally used for quality control of GBE in a single assay.     

Detection of peonidin and pelargonidin glycosides in black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) by high-performance liquid chromatography/electrospray ionization mass spectrometry

Anthocyanins of black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) were investigated by high-performance liquid chromatography/electrospray ionization mass spectrometry. Besides the main compounds which have already been characterized in detail as cyanidin glycosides, six anthocyanins with m/z 757, 565, 595, 903, 933 and 903 were detected and unambiguously characterized as peonidin and pelargonidin glycosides, based on their fragmentation patterns. Peonidin and pelargonidin were identified after acid hydrolysis by comparison of the fragmentation patterns and retention times of the released aglycones with those of standard compounds. To the best of our knowledge this is the first report on anthocyanins in black carrots consisting of aglycones other than cyanidin.