Comparison of antioxidant activities of different parts from snow chrysanthemum (Coreopsis tinctoria Nutt.) and identification of their natural antioxidants using high performance liquid chromatography coupled with diode array detection and mass spectrometry and 2,2'-azinobis(3-ethylbenzthiazoline-sulfonic acid)diammonium salt-based assay.

Snow chrysanthemum (Coreopsis tinctoria Nutt.), a world-widely well-known flower tea material, has attracted more and more attention because of its beneficial health effects such as antioxidant activity and special flavor. In this study, a high performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) and 2,2'-azinobis(3-ethylbenzthiazoline-sulfonic acid)diammonium salt (ABTS) based assay was employed for comparison and identification of antioxidants in different samples of snow chrysanthemum. The results showed that snow chrysanthemum flowers possessed the highest while stems presented the lowest antioxidant capacities. Fourteen detected peaks with antioxidant activity were temporarily identified as 3,4',5,6,7-pentahydroxyflavanone-O-hexoside, chlorogenic acid, 2R-3',4',8-trihydroxyflavanone-7-O-glucoside, flavanomarein, flavanocorepsin, flavanokanin, quercetagitin-7-O-glucoside, 3',5,5',7-tetrahydroxyflavanone-O-hexoside, marein, maritimein, 1,3-dicaffeoylquinic acid, coreopsin, okanin and acetyl-marein by comparing their UV spectra, retention times and MS data with standards or literature data. Antioxidants existed in snow chrysanthemum are quite different from those reported in Chrysanthemum morifolium, a well-known traditional beverage in China, which indicated that snow chrysanthemum may be a promising herbal tea material with obvious antioxidant activity.

Effect of sample preparation on components and liver toxicity of Polygonum multiflorum.

It was shown that different extracts had significant differences in the toxicity of Polygonum multiflorum. In this study, the effect of sample preparation on components and liver toxicity of different extracts from P. multiflorum were determined. Hepatoxic components were discovered based on biomembrane extraction. Comparative chemistry and toxicology between ethanol and water extracts were also performed. The results showed that ethanol extract had much stronger hepatotoxicity, the content of emodin-8-O-ß-d-glucopyranoside, physcion-8-O-ß-d-glucopyranoside, emodin and physcion was significantly higher in ethanol extract than in water extract, while the human hepatocytes extraction showed that 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucopyranoside, emodin-8-O-ß-d-glucopyranoside, physcion-8-O-ß-d-glucopyranoside, emodin and physcion had interaction with human hepatocytes. The hepatotoxic effect of these components was investigated on human hepatocytes LO2 cells and emodin-8-O-ß-d-glucopyranoside, physcion-8-O-ß-d-glucopyranoside, emodin and physcion were finally confirmed to be, at least partial, hepatotoxic components. The results showed that sample preparation has significant effect on components in extracts of P. multiflorum especially the components related to hepatotoxicity. Water extract, the conventional administration form of Chinese herbs, is prefer for phytotherapy before well understanding their chemistry and biological activities.

High-performance liquid chromatography coupled with post-column dual-bioactivity assay for simultaneous screening of xanthine oxidase inhibitors and free radical scavengers from complex mixture.

Xanthine oxidase (XO) can catalyze hypoxanthine and xanthine to generate uric acid and reactive oxygen species (ROS), including superoxide anion radical (O2(•-)) and hydrogen peroxide. XO inhibitors and free radical scavengers are beneficial to the treatment of gout and many related diseases. In the present study, an on-line high-performance liquid chromatography (HPLC) coupled with post-column dual-bioactivity assay was established and successfully applied to simultaneously screening of XO inhibitors and free radical scavengers from a complex mixture, Oroxylum indicum extract. The integrated system of HPLC separation, bioactivity screening and mass spectrometry identification was proved to be simple and effective for rapid and sensitive screening of individual bioactive compounds in complex mixtures.

Advanced development in chemical analysis of Cordyceps.

Cordyceps sinensis, also called DongChongXiaCao (winter worm summer grass) in Chinese, is a well-known and valued traditional Chinese medicine. In 2006, we wrote a review for discussing the markers and analytical methods in quality control of Cordyceps (J. Pharm. Biomed. Anal. 41 (2006) 1571-1584). Since then this review has been cited by others for more than 60 times, which suggested that scientists have great interest in this special herbal material. Actually, the number of publications related to Cordyceps after 2006 is about 2-fold of that in two decades before 2006 according to the data from Web of Science. Therefore, it is necessary to review and discuss the advanced development in chemical analysis of Cordyceps since then.

A novel sample preparation and on-line HPLC-DAD-MS/MS-BCD analysis for rapid screening and characterization of specific enzyme inhibitors in herbal extracts: case study of α-glucosidase.

Drug discovery from complex mixture like Chinese herbs is a challenge and extensive false positives make the obtainment of specific bioactive compounds difficult. In the present study, a novel sample preparation method was proposed to rapidly reveal the specific bioactive compounds from complex mixtures using α-glucosidase as a case. Firstly, aqueous and methanol extracts of 500 traditional Chinese medicines were carried out with the aim of finding new sources of α-glucosidase inhibitors. As a result, the extracts of fruit of Terminalia chebula (FTC), flowers of Rosa rugosa (FRR) and Eugenia caryophyllata (FEC) as well as husk of Punica granatum (HPG) showed high inhibition on α-glucosidase. On-line liquid chromatography-diode array detection-tandem mass spectrometry and biochemical detection (HPLC-DAD-MS/MS-BCD) was performed to rapidly screen and characterize α-glucosidase inhibitors in these four extracts. After tentative identification, most of compounds with inhibitory activity in the investigated crude extracts were found to be tannins commonly recognized as non-specific enzyme inhibitors in vitro. Subsequently, the four extracts were treated with gelatin to improve specificity of the on-line system. Finally, two compounds with specific α-glucosidase inhibition were identified as corilagin and ellagic acid. The developed method could discover specific α-glucosidase inhibitors in complex mixtures such as plant extracts, which could also be used for discovery of specific inhibitors of other enzymes.

Preparation of inulin-type fructooligosaccharides using fast protein liquid chromatography coupled with refractive index detection.

A fast protein liquid chromatography coupled with refractive index detection (FPLC-RID) method was firstly developed for preparation and purification of fructooligosaccharides with different degree of polymerization from burdock, Arctium lappa. After extraction with 60% ethanol and decolorization with MCI gel CHP20P, total fructooligosaccharides were purified on Bio-Gel P-2 column eluted with water at the flow rate of 0.3 ml/min, which was the optimized conditions. The obtained fructooligosaccharides with degree of polymerization of 3-9 were identified based on their methylation analysis, MS and NMR data. This method has the advantages of high automation, good recovery and easy performance, which could be used for preparation of FOS from other sources, as well as other targeted compounds without UV absorbance.

A novel strategy with standardized reference extract qualification and single compound quantitative evaluation for quality control of Panax notoginseng used as a functional food.

Root of Panax notoginseng (Burk.) F.H. Chen (Sanqi in Chinese) is one of traditional Chinese medicines (TCMs) based functional food. Saponins are the major bioactive components. The shortage of reference compounds or chemical standards is one of the main bottlenecks for quality control of TCMs. A novel strategy, i.e. standardized reference extract based qualification and single calibrated components directly quantitative estimation of multiple analytes, was proposed to easily and effectively control the quality of natural functional foods such as Sanqi. The feasibility and credibility of this methodology were also assessed with a developed fast HPLC method. Five saponins, including ginsenoside Rg1, Re, Rb1, Rd and notoginsenoside R1 were rapidly separated using a conventional HPLC in 20 min. The quantification method was also compared with individual calibration curve method. The strategy is feasible and credible, which is easily and effectively adapted for improving the quality control of natural functional foods such as Sanqi.

Advanced phytochemical analysis of herbal tea in China.

Herbal tea is a commonly consumed beverage brewed from the leaves, flowers, seeds, fruits, stems and roots of plants species rather than Camellia sinensis L., which has been widely used for health care and diseases prevention for centuries. With the increasing consumption of herbal tea, a number of public health issues e.g., efficacy, safety and quality assurance have attracted concern. However, to date, there is no a review focus on herbal tea. Phytochemical analysis, as a key step to investigate the chemical composition of herbal tea and ensure the quality, is very important. In this review, we summarized and discussed the recent development (2005-2012) in phytochemical analysis of herbal tea commonly used in China.

Complementary laboratory indices for predicting the disease status of patients with hepatitis B virus infection.

To identify complementary laboratory indices for determining the disease status of patients with hepatitis B virus. Subjects were divided into six groups: hepatitis B virus carrier, mild chronic hepatitis B, moderate chronic hepatitis B, severe chronic hepatitis B, fulminant hepatitis B and healthy controls. Serum alanine aminotransferase, total bilirubin and direct bilirubin were measured by an automatic analyser. The levels of T-cell immunoglobulin domain and mucin-domain-containing molecule-3, macrophage inflammatory protein 2, neutrophil gelatinase-associated lipocalin and inducible nitric oxide synthase were measured by ELISA. T-cell immunoglobulin domain, mucin-domain-containing molecule-3, macrophage inflammatory protein 2 and inducible nitric oxide synthase levels were significantly higher in patients with severe chronic hepatitis B compared with those in patients with mild and moderate chronic hepatitis B or fulminant hepatitis B (P < 0.05). When normal or abnormal alanine aminotransferase was present, significant differences between macrophage inflammatory protein 2 and T-cell immunoglobulin domain and mucin-domain-containing molecule-3 levels between patients with mild, moderate, severe chronic hepatitis B or fulminant hepatitis B were observed (P < 0.05). Our results suggest that T-cell immunoglobulin domain and mucin-domain-containing molecule-3 and macrophage inflammatory protein 2 could serve as alanine aminotransferase, direct bilirubin or total bilirubin complementary indices for determining the status of patients with hepatitis B.

Quality evaluation of Polygonum multiflorum in China based on HPLC analysis of hydrophilic bioactive compounds and chemometrics.

Polygonum multiflorum is one of the most commonly used Chinese medicines. In this study, an effective pressurized water extraction and HPLC method was developed for first simultaneous determination of 8 hydrophilic compounds, including gallic acid, Hypaphorine, Catechin, Proanthocyanidin B1, Epicatechin, Proanthocyanidin B2, Emodin-8-O-ß-d-glucopyranoside, stilbene glycosides, in P. multiflorum. The analysis was performed on a Zorbax SB-AQ column with gradient elution of 0.05% phosphoric acid aqueous solution and acetonitrile in 45 min. All calibration curves showed good linearity (R(2)>0.9994) within test ranges. The LOD and LOQ were lower than 0.2 and 1.0 µg/mL on column, respectively. RSD for intra- and inter-day of 8 analytes were less than 4.1% and 4.0%, respectively, and the overall recovery was 96.0-100.7%. The validated method was successfully applied to quantification of 8 hydrophilic compounds in samples of P. multiflorum from different locations of China. Chemometrics such as principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used to evaluate homogeneity of P. multiflorum in China, which suggested that their quality homogeneity was good.

Cloning and characterization of a ß-amyrin synthase gene from the medicinal tree Aralia elata (Araliaceae).

Aralia elata is an important medicinal plant in China; it produces large amounts of oleanane type triterpene saponins. A full-length cDNA encoding ß-amyrin synthase (designated as AeAS) was isolated from young leaves of A. elata by reverse transcription-PCR. The full-length cDNA of AeAS was found to have a 2292-bp open reading frame, encoding a protein with 763 amino acid residues. The deduced amino acid sequence of AeAS showed the highest identity (97%) to Panax ginseng ß-amyrin synthase. When AeAS cDNA was expressed in Escherichia coli, an 87.8-kDa recombinant protein was detected by SDS-PAGE and Western blotting. The sequence was also heterologously expressed in the yeast Pichia pastoris, and production of ß-amyrin was detected by HPLC. Tissue expression pattern analysis by real-time reverse transcription-PCR revealed that AeAS is strongly expressed in leaves and stems, and weakly expressed in roots and flowers.

Comparison of polysaccharides from different Dendrobium using saccharide mapping.

Multiple species of Dendrobium are widely used as Shihu, a well known Chinese herb, for medicinal purpose in China. Small molecules such as phenols, alkaloids and coumarins are obviously varied in different species of Dendrobium. But there are few reports on polysaccharides, one of major active components, from Dendrobium. In this study, polysaccharides from different species or locations of Dendrobium were compared using saccharide mapping. The results showed that polysaccharides of Dendrobium from different species or locations were obviously varied in spite of they had some similar characters, which is helpful to control the quality of Dendrobium.

Strategies for quality control of Chinese medicines.

Chinese medicines (CM) have been attracting interest and acceptance in many countries. Quality control is vital for ensuring the safety and efficacy of CM. Usually, CM are used as whole plant and/or combination of several herbs, and multiple constituents are responsible for the therapeutic effects. Therefore, quality control of CM is very difficult. To date, the valid method for quantitatively evaluating the quality of CM is poor. In this article, the strategies for quantification, related to the markers, reference compounds and approaches, in quality control of CM were reviewed and discussed.

Discrimination of polysaccharides from traditional Chinese medicines using saccharide mapping--enzymatic digestion followed by chromatographic analysis.

Polysaccharides isolated from traditional Chinese medicines (TCMs) exhibit multiple pharmacological activities. However, quality control of polysaccharides is a challenge because of their complicate structure and macro-molecular mass. In this study, saccharide mapping based on specific enzymatic digestion of polysaccharides and chromatographic analysis was proposed to discriminate the polysaccharides from different TCMs. Endo-carbohydrase such as glucanase, arabinanase, xylanase, galactanase, cellulase, amylase and pectinase were used for enzymatic digestion of polysaccharides from 9 TCMs namely Panax ginseng, P. notoginseng, P. quinquefolium, Cordyceps sinensis, C. militaris, Ganoderma lucidum, G. sinense, Astragalus membranaceus and Angelica sinensis. By using high performance size exclusion chromatography (HPSEC) as well as derivatization with 1-Phenyl-3-methyl-5-pyrazolone (PMP) and HPLC analysis, the enzymatic hydrolysis properties of polysaccharides and their saccharide mapping were determined. The polysaccharides from 9 TCMs were firstly successfully distinguished based on their characteristic saccharide maps, which is helpful to improve the quality control of polysaccharides.

Simultaneous determination of five flavonoids in licorice using pressurized liquid extraction and capillary electrochromatography coupled with peak suppression diode array detection.

Pressurized liquid extraction (PLE) and capillary electrochromatography (CEC) methods were developed for the simultaneous determination of five flavonoids, namely liquiritin, isoliquiritin, ononin, liquiritigenin and isoliquiritigenin, in licorice using baicalein as internal standard (IS). Peak suppression technique was used for the quantification of ononin because of its poor resolution with isoliquiritin. The analysis was performed on a Hypersil C(18) capillary (3 microm, 100 microm/25 cm) with a mixture of 10mM phosphate buffer (pH 3.0)/ACN (65:35, v/v) as mobile phase running at 25 kV and 30 degrees C. The detection wavelengths were set at 275 nm (without reference wavelength for liquiritin and liquiritigenin), 360 nm (without reference wavelength for isoliquiritin and isoliquiritigenin) and 254 nm (with reference wavelength of 405 nm for ononin). All calibration curves showed good linearity (R(2)>0.9993) within the test ranges. The LOD and LOQ were lower than 2.1 and 8.3 microg/mL, respectively. The RSDs of intra- and interday for relative peak areas of five analytes to IS were less than 3.8 and 4.7%, respectively, and the recoveries were 98.2-103.8%. The validated method was successfully applied to the quantitative analysis of five flavonoids in licorice, which is helpful to its quality control.

Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis.

Random amplified polymorphic DNA (RAPD) and high performance liquid chromatography (HPLC) were applied to investigate genetic and chemical variations of 2 natural C. sinensis, 16 fungal strains isolated from C. sinensis, and 2 fungal strains of C. militaris. Five of the 68 arbitrary decamer primers were available for discrimination of the investigated samples. As a result, 20 investigated samples were divided into three main clusters according to the genetic distance, and some fungal strains isolated from natural C. sinensis were obviously different. But according to the contents of nucleosides, including uracil, uridine, hypoxanthine, inosine, guanosine, adenosine, adenine, and cordycepin, natural and cultured Cordyceps were in two individual sub-groups, which suggested that chemical characteristics among cultured mycelia of different fungal strains isolated from natural C. sinensis were similar, but they were different from natural one.

Rapid method for simultaneous determination of flavonoid, saponins and polyacetylenes in folium ginseng and radix ginseng by pressurized liquid extraction and high-performance liquid chromatography coupled with diode array detection and mass spectrometry.

A rapid pressurized liquid extraction (PLE) and high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) method for the simultaneous determination of one flavonoid (panasenoside), nine saponins (ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3 and Rd) and two polyacetylenes (panaxydol and panaxynol) in folium ginseng and radix ginseng was developed. A Prevail C(18) rocket column (33 mm x 7 mm, 3.0 microm) and gradient elution were used during the analysis. Flavonoid was quantified at 355 nm, and saponins and polyacetylenes were determined at 203 nm. The chromatographic peaks of 12 investigated compounds in samples were unambiguously identified by compared their UV spectra and/or MS data with the related reference compounds. All calibration curves showed good linearity (r>0.999) within the test ranges. The intra- and inter-day variations for 12 analytes were less than 1.17% and 2.17%, respectively. The developed method was successfully applied to determine the investigated compounds in 10 samples of radix ginseng and folium ginseng, respectively. The result showed that PLE combined with rocket column HPLC analysis could provide a rapid method for analysis of compounds in traditional Chinese medicines (TCMs), which is helpful to comprehensive evaluation of quality of radix ginseng and folium ginseng.

Differentiation of Herba Cistanches by fingerprint with high-performance liquid chromatography-diode array detection-mass spectrometry.

Herba Cistanche (Rou Cong Rong in Chinese), dried succulent stems of Cistanche deserticola or C. tubulosa, is a famous Chinese herbal medicine and has been recorded in the Chinese Pharmacopoeia. In recent years, another two non-official species, C. salsa and C. sinensis have also been used as Herba Cistanche in some regions of China. To investigate the possibility of using these two non-official species as alternatives to the official species, a high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS) fingerprint method was developed to comparatively analyze the crude herbs of these four species. The fingerprint of C. deserticola, a historically certified species of Herba Cistanche, serves as 'standard pattern' for comparing the similarities with the other species by means of similarity and Principle Component Analysis. Additionally, 18 characteristic peaks in the fingerprints were identified by comparing their retention times, UV spectra and ESI-MS data with those of the reference substances and/or the data in the literatures. The comparative results demonstrate that the fingerprints of C. tubulosa and C. salsa possess high similarity to the standard pattern, suggesting that these two species may be used as alternative species; while that of C. sinensis has low similarity (0.053 correlation coefficient) to the standard pattern, indicating that it cannot be used as the substitute of the official herb. However, the varying fingerprint patterns among the samples of C. deserticola collected from various habitats illustrate that the quality consistency of crude herbs is still a problem worthy of serious concern.

Simultaneous determination of nucleobases, nucleosides and saponins in Panax notoginseng using multiple columns high performance liquid chromatography.

A new multiple columns HPLC method for simultaneous determination of 16 characteristic components, 5 nucleobases and nucleosides (uracil, cytidine, uridine, guanosine and adenosine), and 11 saponins (notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, notoginsenoside R4, notoginsenoside Fa, ginsenoside Rb1, notoginsenoside R2, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rd and notoginsenoside K), in the root of Panax notoginseng, a valued traditional Chinese medicinal herb, were developed. Notoginsenoside R4, Fa and K were first quantitatively determined in P. notoginseng. The 5 nucleobases and nucleosides compounds were separated on a Zorbax SB-Aq column (150 x 4.6 mm, 5.0 microm) and 11 saponins were analyzed using a Zorbax Bonus-RP column (150 x 4.6 mm, 5.0 microm) with column switching. The column temperature was set at 30 degrees C. Mobile phase was composed of 5mM ammonium acetate aqueous (A), water (B) and acetonitrile (C) using a gradient elution. The flow rate was 1.5 mL/min and detection wavelengths were set at 260 nm for nucleobases and nucleosides, and 203 nm for saponins. The developed method had good repeatability and sensitivity for quantification of 16 analytes with overall precision (including intra- and inter-day) less than 3% (RSD), and LOD and LOQ were less than 1.33 microg/mL and 5.12 microg/mL, respectively. The method was successfully applied to the simultaneous determination of 16 analytes in 15 samples of P. notoginseng collected from different places of China, which indicated that multiple columns HPLC can be used for comprehensive quality control of P. notoginseng.

Qualitative and quantitative analysis of four species of Curcuma rhizomes using twice development thin layer chromatography.

The rhizomes of Curcuma phaeocaulis, Curcuma kwangsiensis, Curcuma wenyujin and Curcuma longa are used as Ezhu or Jianghuang in traditional Chinese medicine for a long time. Due to their similar morphological characters, it is difficult to distinguish their origins of raw materials used in clinic. In this study, a simple, rapid and reliable twice development TLC method was developed for qualitative and quantitative analysis of the four species of Curcuma rhizomes. The chromatography was performed on silica gel 60F(254) plate with chloroform-methanol-formic acid (80:4:0.8, v/v/v) and petroleum ether-ethyl acetate (90:10, v/v) as mobile phase for twice development. The TLC markers were colorized with 1% vanillin-H(2)SO(4) solution. The four species of Curcuma were easily discriminated based on their characteristic TLC profiles, and simultaneous quantification of eight compounds, including bisdemethoxycurcumin, demethoxycurcumin, curcumine, curcumenol, curcumol, curdione, furanodienone and curzerene, in Curcuma were also performed densitometrically at lambda(scan)=518nm and lambda(reference)=800 nm. The investigated compounds had good linearity (r(2)>0.9905) within test ranges. Therefore, the developed TLC method can be used for quality control of Curcuma rhizomes.