[Identification of Dendrobii Caulis basing on ITS sequence].

Isolation of high-quality genomic DNA from dried and processed crude drug is the key for the DNA identification of Dendrobii Caulis. The DNA extract of Dendrobii Caulis was firstly compared using different method to isolate genomic DNA from dried and processed crude drug, including commercial DNA extracted kit and CTAB method. Using modified CTAB method (extracted from large samples), the genomic DNA was successfully isolated from Dendrobii Caulis, including Huangcao and Fengdou. The ITS regions were amplified using the purified DNA as template, and then cloned and sequenced. These ITS sequences were compared with data from Genbank database and our lab, 14 Dendrobium species and five similar species were identified from "Huangcao" and "Huangcao" slice, while six species and three similar species from "Fengdou" according to their sequence similarity. The study demonstrated that the dried Dendrobii Caulis could be identified using DNA molecular method, which could overcome deficiencies and limitations of traditional identification method and has a certain application prospects.

[Screening of endophytic fungi from Huperzia serrata for acetylcholinesterase inhibitory activity and its taxonomic identification].

OBJECTIVE: To screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata. METHOD: Endophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics. RESULT: Fungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium. CONCLUSION: Endophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

[Regulation of Vitreoscilla hemoglobin on biosynthesis of astragaloside IV].

In the present study, the regulation of Vitreoscilla hemoglobin (VHb) on astragaloside IV biosynthesis was investigated. An intermediate expression vector consisting of the CaMV35S promoter fused to the vgb and nopaline synthase terminator was transferred into Astragalus membranaceus via Agrobacterium rhizogenes. The transgenic hairy roots were confirmed by PCR amplification and Southern blot hybridization. The expression of vgb in transgenic hairy roots was confirmed by RT-PCR. After 15 days cultivation, the dry weight and growth rate of transgenic hairy roots were higher than that of the non-transgenic hairy root. ELSD-HPLC analysis showed that astragaloside IV content of transgenic hairy roots was 5 to 6 times of non-transgenic hairy root control and 10 to 12 times of Radix Astragali from Shanxi Province. These results suggested that the expression of vgb promoted the growth of transgenic hairy roots, and increased the content of astragaloside IV.

[Evaluation on hepatotoxicity caused by Dioscorea bulbifera based on analysis of bile acids].

Metabolic profile of bile acids was used to evaluate hepatotoxicity of mice caused by ethanol extraction of Dioscorea bulbifera L. (ethanol extraction, ET) and diosbulbin B (DB), separately. Ultra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) was applied to determine the contents of all kinds of endogenous bile acids including free bile acids, taurine conjugates and glycine conjugates. Obvious liver injuries could be observed in mice after administrated with ET and DB. Based on the analysis using principle components analysis (PCA), toxic groups could be distinguished from their control groups, which suggested that the variance of the contents of bile acids could evaluate hepatotoxicity caused by ET and DB. Meanwhile, ET and DB toxic groups were classified in the same trends comparing to control groups in the loading plot, and difference between the two toxic groups could also be observed. DB proved to be one of the toxic components in Dioscorea bulbifera L. Bile acids of tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), cholic acid (CA) and others proved to be important corresponds to ET and DB induced liver injury according to analysis of partial least square-discriminant analysis (PLS-DA) and the statistical analysis showed that there were significant differences between the control groups and toxic groups (P < 0.01). Furthermore, good correlation could be revealed between the foregoing bile acids and ALT, AST. It indicated that taurine conjugated bile acids as TUDCA, TCDCA, TCA and TDCA along with CA could be considered as sensitive biomarkers of ET and DB induced liver injury. This work can provide the base for the further research on the evaluation and mechanism of hepatotoxicity caused by Dioscorea bulbifera L.

Determination and biosynthesis of multiple salvianolic acids in hairy roots of Salvia miltiorrhiza.

Danshen (Salvia miltiorrhiza Bunge) hairy roots were obtained by infecting Danshen leaves with Agrobacterium rhizogenes 9402. Besides rosmarinic acid (RA) and salvianolic acid B (SAB), the hairy root could also produce salvianolic acid K (SAK), salvianolic acid L, ethyl salvianolic acid B (ESAB), methyl salvianolic acid B (MSAB), and a compound with a molecular weight of 538 (compound 538) identified by using LC-MS. Effects of methyl jasmonate (MeJA) and yeast elicitor (YE) on the accumulation of these compounds had been investigated. MeJA increased the accumulation of SAB, RA, SAK, and compound 538 from 4.21%, 2.48%, 0.29%, and 0.01% of dry weight to 7.11%, 3.38%, 0.68%, and 0.04%, respectively. YE stimulated the biosynthesis of RA from 2.83% to 5.71%, but depressed the synthesis of SAB, SAK and compound 538. It was indicated in all the results that these Danshen hairy roots could be used as alternative resources to produce salvianolic acids. Analysis of the content variation of these compounds after elicitation suggested that SAK and compound 538 might be the intermediates in the biosynthesis from RA to SAB in Danshen hairy roots.

[The utility of trnH-psbA gene for phylogenetic analysis of Huperziaceae and plant barcoding].

OBJECTIVE: The purpose of study was to discover the phylogenetic relations and plant barcoding of 17 plants from Huperziaceae. METHODS: Phylogenetic tree of chloroplast trnH-psbA gene of 17 plants from Huperziaceae was constructed by software. RESULTS: It showed that Huperziaceae could be divided into two genera Huperzia and Phlegmariurus and bootstrap value reached 91%. CONCLUSIONS: Holub and Qing' taxonomy was supported and 17 species in Huperziaceae were monophyletic groups and it suggested that trnH-psbA could be used as a DNA barcode to identify plants.

Advancement in analysis of Salviae miltiorrhizae Radix et Rhizoma (Danshen).

This review summarizes the recent advances in the chemical analysis of Danshen and its finished products, including the introduction of the identified bioactive components, analytical methods for quantitative determination of target analytes and fingerprinting authentication, quality criteria of Danshen crude herb and its preparations, as well as the pharmacokinetic and pharmacodynamic studies on the active components of Danshen and its finished products. Danshen contains mainly two types of constituents, the hydrophilic depsides and lipophilic diterpenoidal quinones and both of them are responsible for the pharmacological activities of Danshen. In order to monitor simultaneously both types of components which have different physicochemical properties, numerous analytical methods have been reported using various chromatographic and spectrophotometric technologies. In this review, 110 papers on analysis of Danshen are discussed, various analytical methods and their chromatographic conditions are briefly described and their advantages/disadvantages are compared. For obtaining a quick, accurate and applicable analytical approach for quality evaluation and establishing a harmonized criteria of Danshen and its finished products, the authors' suggestion and opinions are given, including the reasonable selection of marker compounds with high concentration and commercial availability, a simple sample preparation procedure with high recoveries of both the hydrophilic phenols and lipophilic tanshinones, and an optimized chromatographic condition with ideal resolutions of all the target components. The chemical degradation and transformation of the predominant constituent salvianolic acid B in Danshen during processing and manufacturing are also emphasized in order to assure the quality consistency of Danshen containing products.

Chromatographic fingerprinting analysis of Danshen root (Salvia miltiorrhiza Radix et Rhizoma) and its preparations using high performance liquid chromatography with diode array detection and electrospray mass spectrometry (HPLC-DAD-ESI/MS).

Danshen root (Salvia miltiorrhiza Radix et Rhizoma) is a representative Chinese herb containing multiple components contributing to its polyvalent bioactivities. Advanced analysis approaches are needed to obtain a comprehensive picture of the targeting constituents in complete matrix. In this study, a chromatographic fingerprinting method to monitor simultaneously the hydrophilic and lipophilic constituents was developed for the quality evaluation of Danshen root and its preparations. Ten hydrophilic and nine lipophilic components were identified using HPLC-diode array detection-electrospray-MS (HPLC-DAD-ESI/MS) by comparing with the available references and reported data. Using the established method, 13 Danshen root samples collected from different sources, and 21 batches of Danshen preparations including tablets, injections, capsules, and dropping pills produced by different manufacturers were analyzed and their chromatographic fingerprints (CFP) were constructed. The results showed that the products of Danshen roots such as the tablets and capsules contained both the hydrophilic and lipophilic components, while the injections and dropping pills contained mainly the hydrophilic components. Principal components analysis (PCA) was applied for the statistical analysis of the fingerprinting data of crude herb and its preparations. The established CFPs demonstrate the representative chemical profiling of the existing components and can be applied to the authentication and quality assessment of Danshen roots and other Danshen containing formulated preparations.

Gastroprotective effect of fructus evodiae water extract on ethanol-induced gastric lesions in rats.

Fructus Evodiae is a widely used herbal medicine with anti-inflammatory and analgetic activities in China. The present study was designed to investigate the effect of Fructus Evodiae water extract (FE) on ethanol-induced gastric lesions in rats. Three hours before ethanol challenge, animals were intraperitoneally treated with FE (424.8 mg/kg, 141.6 mg/kg, and 47.6 mg/kg). Subsequently, we employed ex-vivo chamber technique to examine the effect of FE on gastric transmucosal potential difference (PD) changes. NO(x) (nitrate and nitrite) in gastric perfusate and gastric lesion index of whole glandular stomach were determined by intubation. The results showed that FE dose-dependently accelerated the recovery of PD reduction by ethanol, and increased NO(x) production in gastric perfusate. FE also inhibited gastric lesion formation in a dose-dependent manner. These results suggested that FE prevented ethanol-induced gastric mucosal lesions by strengthening the mucosal barrier integrity and increasing gastric mucosal nitric oxide (NO) synthesis.

[HPLC determination of acteoside in Radix Rehmanniae].

OBJECTIVE: To develop an HPLC method for the determination of acteoside in Radix Rehmanniae. METHOD: The chromatographic conditions were as follows: Polaris C18(4.6 mm x 250 mm, 5 microm) column, a mobile phase in gradient mode composed of acetonitrile 0.1% acetic acid solution, a flow rate of 1.0 mL x min(-1), and 334 nm as the detection wavelength. RESULT: Acteoside showed good linear relationship at the range of 10-500 microg x mL(-1) (r = 0.9990). The average recovery was 100.1%, RS D 3.7%. CONCLUSION: The proposed method promised to be applicable for the quality control of Radix Rehmanniae.

[Studies on chemical constituents in stem of Dendrobium chrysotoxum].

OBJECTIVE: To investigate the chemical constituents of Dendrobium chrysotoxum. METHOD: The chemical constituents were isolated by various column chromatographic methods and structurally elucidated by spectral evidences. RESULT: Ten compounds were obtained and identified as (+)-syringare sinol (1), 5alpha, 8alpha-epidioxy-24( R)-methycholesta-6, 22-dien-3beta-ol (2), trans-3-(4-hydroxy-3-methoxyphenyl)-acrylic acid octacosyl ester (3), defusin (4), 3, 4-dihydroxy benzoic acid (5), 3, 4-dimethoxy-benzoic acid (6), vanillic acid (7), 3, 4-dimethoxy-benzoic acid methyl ester (8), 3, 5-dibromo-2-aminobenzaldehyde (9), heptadecanoic acid 2, 3-dihydroxy-propyl ester (10). CONCLUSION: Compounds 1, 2 and 6-10 were isolated from this plant for the first time.

Simultaneous determination of six isoflavonoids in commercial Radix Astragali by HPLC-UV.

A HPLC-UV method for the quantification of six major isoflavonoids, calycosin 7-O-beta-D-glucoside (1), formononetin 7-O-beta-D-glucoside (2), (6alphaR, 11alphaR) 3-hydroxy-9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (3), 7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-beta-D-glucoside (4), calycosin (5) and formononetin (6), in Radix Astragali (Huangqi) was developed and validated. The method was proven to be sensitive, specific, accurate and precise, as well as effective and easy.

Ruggedness/robustness evaluation and system suitability test on United States Pharmacopoeia XXVI assay ginsenosides in Asian and American ginseng by high-performance liquid chromatography.

The work of the ruggedness/robustness evaluation and system suitability tests was oriented to profound understand the practicability of using assay methods issued by United States Pharmacopoeia (USP XXVI and XXVII) for ginsenosides in Asian ginseng and American ginseng. The items chosen for the method validation included quantitative related items such as recovery of Rg(1) and Rb(1), respectively, and qualitative related items such as resolution, theoretical plate number, relative retention time of two critical-band-pairs, Rg(1)/Re and Rb(1) with its neighboring peak, respectively. Totally, 16 column types were used for comparison of different vendors, different packing materials, different size, etc. and five sets of LC systems and two laboratories were involved in comparing the data of both quantitative and qualitative items. The results showed that different packing materials of columns used might significantly alters separation. The column packing material Hypersil afforded the preferable separating for the ginsenosides. No significant difference was observed from the different instrumentations and inter-laboratories. Our results suggest a modification of the system suitability test as given in USP26-NF21 and the latest version of USP27-NF22, which was not suitable for most systems. Using resolutions of Rg(1)/Re and Rb(1) with its neighboring peak as critical parameters for the ginsenosides assay and omitting the relative retention time of both Rg(1)/Re and Rb(1) with its neighboring peak is our suggestion for a more reasonable, yet practicable system suitability. Six typical chromatograms gain from different columns were figured out as well.

Identification and chemical profiling of monacolins in red yeast rice using high-performance liquid chromatography with photodiode array detector and mass spectrometry.

Monascus purpureus-fermented rice (red yeast rice) was one of the food supplements that had the ability of lowering the blood-lipid levels, and monacolins have been proved to be main active constituents. In total 14 monacolin compounds such as monacolin K (mevinolin), J, L, M, X, and their hydroxy acid form, as well as dehydromonacolin K, dihydromonacolin L, compactin, 3alpha-hydroxy-3,5-dihydromonacolin L, etc. were identified in red yeast rice, using high-performance liquid chromatography with photodiode array detector and tandem mass spectrometry. A chemical fingerprint profiling method to display bioactive monacolins in red yeast rice was established and could be used for the quality control of the target material and its related products. Ten finish products labeled as red yeast rice from different manufacturers in marketing were traced using the chromatographic chemical profiling method, and the results show that only two of them were similar while the other eight were significantly different from the reference red yeast rice. All of these materials including raw material powder and finished products available were quantified and the contents of monacolins were calculated with reference of monacolin K (mevinolin) as the standard.

Effects of calycosin on the impairment of barrier function induced by hypoxia in human umbilical vein endothelial cells.

The purpose of the present study was to examine the effects of calycosin, an isoflavonoid isolated from Astragali Radix, on the impairment of barrier function induced by hypoxia in cultured human umbilical vein endothelial cells. Hypoxia induced an increase in endothelial cell monolayer permeability, indicating endothelial cell barrier impairment. Endothelial barrier dysfunction induced by hypoxia was accompanied by decreases in cytosolic ATP concentration and cAMP level, the development of actin stress fibers and intercellular gap formation, suggesting that the decreases in cytosolic ATP and cAMP levels and rearrangements of F-actin could be associated with an increase in permeability of endothelial monolayers. Application of calycosin inhibited the hypoxia-induced increase in endothelial permeability in a dose-dependent fashion, which is compatible with inhibition of lactate dehydrogenase release, decrease of the fall in ATP and cAMP contents, and improvement of F-actin rearrangements. These findings indicate that calycosin protected endothelial cells from hypoxia-induced barrier impairment by increasing intracellular energetic sources and promoting regeneration of the cAMP level, as well as improving cytoskeleton remodeling.

[Determination of shionone in Radix Asteris by HPLC].

OBJECTIVE: To determine the content of shionone in Radix Aster from several different locations and markets. METHOD: The HPLC analysis was used to determine shionone directly, using Polaris C18 column and acetonitrile as the mobile phase with a flow rate of 1.0 mL.min-1, and the UV detection wavelength was 200 nm. RESULT AND CONCLUSION: The content of shionone was from 0.06% to 0.18%, depending on different locations and markets.

[Determination of calycosin-7-O-beta-D-glucopyranoside in radix astragali by HPLC].

OBJECTIVE: TO establish a RP-HPLC method for the determination of calycosin-7-O-beta-D-glucopyranoside in Radix Astragali, and to analyse the calycosin-7-O-beta-D-glucopyranoside content of ten samples of Radix Astragali, collected from different regions. METHOD: A Polaris C18(250 mm x 4.6 mm, 5 microns) column was used and a mixture of methanol-water (30:70) was used as the mobile phase at a flow rate of 1.0 mL.min-1. The column temperature was 25 degrees C and the UV detection wavelength was 254 nm. RESULT: The calibration curve was in good linearity over the range of 0.0106-2.12 micrograms with the regression equation Y = 3035. 97 X - 14.85(r = 0.9999). The average recovery was 95.8% (n = 5, RSD = 1.3%). CONCLUSION: The method is simple, quick, sensitive and reproducible. In all of the samples, the calycosin-7-O-beta-D-glucopyranoside contents differ markedly.

Effect of astragaloside IV on T, B lymphocyte proliferation and peritoneal macrophage function in mice.

AIM: To investigate the effect of astragaloside IV (ASI) on T, B lymphocyte proliferation, antibody production, and cytokines produced by murine peritoneal macrophages. METHODS: MTT assay was used to determine T, B lymphocyte proliferation and quantitative hemolysin spectrophotometry (QHS) assay was applied to test antibody production; IL-1 production was measured by thymocyte proliferation assay; TNF-alpha production was determined by the cytotoxicity assay against L929 cells. RESULTS: 1) In vivo, ASI 50-200 mg/kg ig for 7 d increased T lymphocyte proliferation and antibody production, and ASI 50-100 mg/kg ig for 7 d increased B lymphocyte proliferation but ASI 200 mg/kg had no effect on B lymphocyte proliferation; 2) In vitro, ASI increased T, B lymphocyte proliferation only at 100 nmol/L; 3) ASI increased IL-1 activity at 1 nmol/L in vitro, but decreased it at 100 and 1000 nmol/L; 4) ASI inhibited TNF-alpha activity with or without LPS-stimulation in vitro. CONCLUSION: ASI increased T, B lymphocyte proliferation and antibody production in vivo and in vitro; but inhibited productions of IL-1 and TNF-alpha from peritoneal macrophages in vitro.

[Effect of components of dang-gui-bu-xue decoction on hematopenia].

OBJECTIVE: To investigate the effects and the related mechanisms of the components of Dang-Gui-Bu-Xue decoction (DGBXD) on improving blood deficiency. METHOD: The effects of promoting hematopoietic function were observed with the blood difficient model mice, by giving components of DGBXD. RBC, WBC, reticulocytes and bone marrow nucleated cells (BMNC) were determined. The components of DGBXD on proliferation of BMNC and on clony forming unit (CFU) were also determined. RESULT: The components of DGBXD remarkably increased the quantity of RBC, WBC, and BMNC. Some of the components promoted the proliferation of BMNC and increased the quantity of CFU-Mix. Among them, polysaccharide of angelica was most potent. CONCLUSION: The studies show that the extracts and some components of DGBXD can promote the hemopoietic function system of the model mice, and they exert the effects in a comprehensive way.