Molecular diversity and hypoglycemic polypeptide-P content of Momordica charantia in different accessions and different seasons.

BACKGROUND: Momordica charantia (MC) has been used for treating diabetes mellitus from ancient times in Asia, Africa and South America. There are many MC accessions in local markets. Polypeptide-P as a main hypoglycemic component in MC was first studied in this experiment to illustrate the different contents in MC of different accessions and different harvesting times. RESULTS: Nineteen MC accessions collected from different regions were clustered into three groups using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers. Content of polypeptide-P in the tested MC accessions was detected by western blot (WB) method. The WB results revealed that polypeptide-P was detected in MC accessions harvested in June and July but not in September and October. Furthermore, Polypeptide-P content corresponded well with the MC accessions. CONCLUSION: Our results suggest that the MC accessions and the harvesting times or the weather during harvest play significant roles in high content of polypeptide-P.

[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.

An efficient way from naringenin to carthamidine and isocarthamidine by Aspergillus niger.

Biotransformation of naringenin with Aspergillus niger CGMCC 3.4628 yielded two hydroxylation products which were identified unambiguously as 6-hydroxylnaringenin (carthamidin) and 8-hydroxylnaringenin (isocarthamidin) by ESI-MS and (1)H-NMR. Both products simultaneously arrived at high level after 48 h in the biotransformation process. The highest conversion efficiency of carthamidin was 0.38 mg/mg of naringenin and that of isocarthamidin was 0.43 mg/mg of naringenin. Antioxidant property assay using a thin layer chromatography-bioautographic-based DPPH scavenging method demonstrated that both hydroxylation metabolites exhibited much stronger activity than naringenin. The high efficiency and convenient procedure makes the biotransformation with A. niger described in current work a potential way to produce carthamidin and isocarthamidin.

[Expression of human estrogen receptor alpha and beta in Escherichia coli].

Estrogen participates in many life activities through combination with estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) in the body. In order to establish an in vitro estrogen-like compound screening model, the coding region of human ERalpha and ERbeta was separately constructed into pET32-ERalpha and pET43-ERbeta prokaryotic expression vector and water-soluble recombinant ERalpha and ERbeta proteins were expressed in Escherichia coli strain BL21. Western blotting revealed that both recombinant proteins have estrogen receptor binding sites. The proteins were purified using S-Tag affinity Purification Kit and digested with enterokinase to get the ERalpha and ERbeta proteins. About 0.90 mg of ERalpha and 0.65 mg of ERbeta were obtained at the concentration of 0.181 and 0.131 mg x mL(-1), respectively.

[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.

Gene cloning and expression of a novel hypoglycaemic peptide from Momordica charantia.

BACKGROUND: Momordica charantia (MC) is used in many Asian countries as a traditional functional food and medicine. Polypeptide-P, a 166 amino acid (AA) polypeptide isolated from MC seeds, has been reported to show hypoglycaemic effects in patients with type I or type II diabetes. The AA sequence of this peptide has been determined, but its gene sequence has yet to be published. RESULTS: In this study a gene-cloning strategy was employed to obtain the polypeptide-P gene sequence using degenerate reverse transcription polymer chain reaction and genome-walking methods. A complete 498 bp sequence encoding the polypeptide-P protein was cloned from MC seeds. Subsequent assays of the bioactivity of the expressed recombinant protein revealed that it had significant hypoglycaemic activity in alloxan-induced diabetic mice. This result suggests that recombinant polypeptide-P has hypoglycaemic effects. CONCLUSION: This is the first report of cloning and expression of the MC polypeptide-P gene. The cloned gene could be helpful for exploring the mechanisms of polypeptide-P gene expression and regulation in MC. Furthermore, this gene could be used as a potential tool both for screening MC varieties with high hypoglycaemically active substance content and for breeding new varieties of MC with high economic value, which could in turn be beneficial to farmers.

[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.

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.

[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.

Expression and characterization of Momordica Chanrantia anti-hyperglycaemic peptide in Escherichia coli.

A nucleic acid sequence MC, encoding Momordica Chanrantia anti-hyperglycaemic peptide MC6 (accession: AAX06814) synthesized according to Escherichia coli preferred codons, was cloned and expressed in E. coli. Recombinant protein pQE8-MC (about 3.5 kDa) was purified and analyzed by 20% SDS-PAGE and western blot. It revealed that the expressed pQE8-MC had good solubility in aqueous media. An HPLC assay was used to confirm the expression of pQE8-MC. Subsequent pharmacological activity assay revealed a significant hypoglycemic effect of low dose treatments of pQE8-MC on male kunming mice. Four hours after an intravenous tail injection, the blood sugar levels of mice treated with pQE8-MC saline solution A3 (1 mg/kg BW) decreased greatly (P < 0.01) relative to the levels of a control group. This suggests that pQE8-MC, expressed in bioengineered E. coli, has a similar hypoglycemic function to the natural protein MC6 from M. Chanrantia. These results reveal the possibility of using bio-engineered bacteria as an anti-diabetic agent.

[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.

Sulfated beta-glucan derived from oat bran with potent anti-HIV activity.

China is a major producer of oats; the annual harvested area of 350,000 ha yields approximately 465,000 tons, giving an average yield of 1.33 tons/ha. The bran is not used for animal feed as it is of poor digestibility and low nutritive content and is considered a waste byproduct. Therefore, it is advantageous to produce a value-added product from the bran. We extracted the native polysaccharide, a linear (1-3)-, (1-4)-linked beta-glucan (OBG) from the oat bran and synthesized a sulfated derivative OBGS containing 36.5% sulfate. OBGS had potent activity against a primary isolate of human immunodeficiency virus (HIV-1) in peripheral blood mononuclear cells at a concentration (EC(50)=5.98 x 10(-4) microM) approximately 15,000 times below its cytotoxic concentration. OBGS was also active postinfection (EC(50)=5.3 x 10(-4) microM) and protected pretreated peripheral mononuclear cells (EC(50)=5.2 x 10(-2) microM) washed free of the compounds prior to infection. Thus, OBGS has potential as a vaginal microbicide and is the first such report for oat bran derived sulfated beta-glucan.

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.

Astragaloside IV improves lipid metabolism in obese mice by alleviation of leptin resistance and regulation of thermogenic network.

Obesity is a worldwide threat to public health in modern society, which may result from leptin resistance and disorder of thermogenesis. The present study investigated whether astragaloside IV (ASI) could prevent obesity in high-fat diet (HFD)-fed and db/db mice. In HFD-fed mice, ASI prevented body weight gain, lowered serum triglyceride and total cholesterol levels, mitigated liver lipid accumulation, reduced fat tissues and decreased the enlargement of adipose cells. In metabolic chambers, ASI lessened appetite of the mice, decreased their respiratory exchange ratio and elevated VCO2 and VO2 without altering circadian motor activity. Moreover, ASI modulated thermogenesis associated gene expressions in liver and brawn fat tissues, as well as leptin resistance evidenced by altered expressions of leptin, leptin receptor (ObR) or appetite associated genes. In SH-SY5Y cells, ASI enhanced leptin signaling transduction. However, in db/db mice, ASI did not change body weight gain and appetite associated genes. But it decreased serum triglyceride and total cholesterol levels as well as liver triglyceride. Meanwhile, it significantly modulated gene expressions of PPARα, PGC1-α, UCP2, ACC, SCD1, LPL, AP2, CD36 and SREBP-1c. Collectively, our study suggested that ASI could efficiently improve lipid metabolism in obese mice probably through enhancing leptin sensitivity and modulating thermogenic network.

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.

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.

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.

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.