Comparison of contents of three active ingredients in Bletilla striata from different sources / 中国中药杂志

In order to understand the difference of contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ in 60 different sources of Bletilla striata planted under the same conditions. UPLC method was used and the analysis was performed on a Waters ACQUITY UPLC BEH C18 column( 2. 1 mm×100 mm,1. 7 μm),eluted with acetonitril-0. 1% formic acid solution by gradient. The flow rate was 0. 208 m L·min-1,the detection wavelength was 270 nm,the column temperature was 35 ℃ and the injection volume was 4μL. Under the above chromatographic conditions,the three components can be separated well with good linearity in the range of 0. 156-5. 000 mg·L-1. The average contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ were( 0. 116 ± 0. 071) %,( 0. 386 ±0. 185) % and( 0. 086±0. 034) %,respectively. After planting for two years under the same conditions,there was no significant difference in chemical composition among different sources and varieties,but the contents of the three components had some regional differences,which indicated that the western region was higher than the eastern region,while the contents of coelonin and batatasin Ⅲ in B.sinensis were slightly higher than those in B. striata. The chromatographic method above is simple,stable and reproducible,and can be used for quantitative analysis of three components. The content analysis of different sources of B. striata can provide reference for future B. striata breeding and quality control.

Effect of pinostrobin chalcone on adipogenic differentiation of mesenchymal stem cell C3H10T1/2 / 中国中药杂志

Chalcones is a flavonoid wildly presented in many herbs. It has the effect to inhibit cells adipogenic differentiation. In order to study the effect of pinostrobin chalcone extracted and isolated from leaves of hickoryes on the adipogenic differentiation of murine embryonic mesenchymal stem cell (C3H10T1/2), MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium] method was used to detect the cell proliferation; adipogenic differentiation was characterized by oil red O staining and isopropanol extraction; the triglyceride content was detected by GAP-PAP enzyme method; and the C3H10T1/2 cell differentiation into adipocytes was also examined by the mRNA and protein expression of PPARγ, C/EBPα and FABP4 by RT-PCR and Western blot respectively. Results indicated that pinostrobin chalcone almost had no effect on cell proliferation activity when the concentration was less than or equal to 50 μmol•L⁻¹; the oil red O staining, isopropanol extraction and GAP-PAP enzyme method showed that pinostrobin chalcone significantly decreased the C3H10T1/2 adipogenic differentiation and triglyceride content in the cytoplasm of adipocytes; the RT-PCR and Western blot analysis showed that pinostrobin chalcone can down-regulate the mRNA and protein levels of FABP4, PPARγ and C/EBPα in C3H10T1/2 cells(P<0.05 or P<0.01). The experiment results suggest that pinostrobin chalcone can inhibit C3H10T1/2 adipogenic differentiation.

Chrysin inhibits adipogenic differentiation of mouse embryonic fibroblasts / 中国中药杂志

Chrysin is an active flavonoid wildly presented in many herbs. It has the effect to reduce serum lipid. To investigate the effect of chrysin on the adipogenic differentiation of mouse embryonic fibroblasts, methyl thiazolyl tetrazolium (MTT) and crystal violet were used to detect the cytotoxic effect of chrysin on Immortalized mouse embryonic fibroblasts (iMEFs). Propidium iodide (PI) staining combined with flow cytometry (FCM) was employed to detect the effects of different concentrations of chrysin on iMEFs cell cycle. The effect of chrysin on adipogenic differentiation ability of iMEFs was determined by oil red O staining. Semi-quantitative PCR was employed to detect the effect of chrysin on mRNA transcriptional levels of adipogenic differentiation markers, including perilipin 2, adiponectin (adipoq), Fabp4, LPL, MCP-1 and adipogenic differentiation key transcription factor peroxisome proliferators-actiated receptor-gamma 2(PPAR-γ2). Results indicated that chrysin had certain cytotoxic effect for iMEFs in a dose-dependent manner, and the IC₅₀ was identified nearly to 30 μmol•L⁻¹. FCM analysis showed that chrysin could affect the cell-cycle distribution of iMEFs, increasing the ratio of cells in G1 phase. Adipogenic differentiation inducing experiment showed that 30 μmol•L⁻¹ chrysin significantly reduced lipid drops accumulation induced by insulin and dexamethasone. In addition, the mRNA transcriptional levels of PPAR-γ2 and LPL were significantly decreased and mRNA levels of fabp 4, MCP-1, adipoq were also affected after chrysin treatment. The experiment results suggest that chrysin attenuates the adipogenic differentiation capacity of mesenchymal stem cells.

Study on anti-angiogenesis effect of three curcumin pigments and expression of their relevant factors / 中国中药杂志

To study the in vitro anti-angiogenesis effect of three curcumin pigments (curcumin, demethoxycurcumin, bisdemethoxycurcumin). In the study, the inhibitory effect of the three curcumin pigments on proliferation of HUVEC cells induced by OX-LDL and the effect on migration of HUVEC cells were detected. The effect on neovascularization was observed by chorioallantoic membrane (CAM) test. The effect on cell adhesion factors ICAM-1 and VCAM-1 of HUVECs were tested by Real-time RT-PCR. It was found that the three curcumins could inhibit the proliferation of HUVEC cells induced by OX-LDL within the dosage range 4, 8, 16 mg x L(-1), with a dose-dependence. The proliferative effect of curcumins on HUVECs was greater than the other two derivatives (P < 0.01). All of the three curcumin pigments inhibited the migration of HUVEC cells and the angiogenesis of chick chorioallantoic membrane (CAM). The migration inhibition rate of curcumins at middle and high concentrations was greater than the other two (P < 0.01). All of the three curcumin could down-regulate the expression of VEGF and ICAM-1, and curcumins showed more obvious effect in down-regulating VEGF than demethoxycurcumin and bisdemethoxycurcumin(P < 0.01); Bisdemethoxycurcumin showed the most significant effect in down-regulating ICAM-1 (P < 0.01). All of the three showed no remarkable effect on expression of VCAM-1, and only bisdemethoxycurcumin showed the down-regulating effect (P < 0.05). According to the findings, all of the three curcumin pigments could resist angiogenesis by inhibiting proliferation and migration of endothelial cells and down-regulating the expression of VEGF and adhesion molecules ICAM-1.

Protoplasts isolation, purification and plant regeneration of Pinellia cordata / 中国中药杂志

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.

Differences of fungal diversity and structure in rhizosphere of Fritillaria thunbergii from different provenances / 中国中药杂志

To explore the mechanism of soil microbial ecology, the differences of fungal diversities in rhizosphere of different provenances of Fritillaria thunbergii were analyzed. The diversities and compositions of rhizo-fungi of the samples were analyzed by using DGGE and 454 pyrosequencing. DGGE results showed the Shannon index of Ninbo provenance planted in Ninbo was the highest one. And its dominant fungi were Ascomycota, Deuteromycota and Zygomycota. Except the same fungi, every provenance planted in Ninbo had its own special ones. From the 454 pyrosequencing, the fungal diversity in Panan producing was the highest which was similar with DGGE result. Among the ten phylum detected in its rhizosoil, Fungi_incertae_sedis, Ascomycota, Mucoromycotina, Basidiomycota and Chytridiomycota almost amounted to 90% of the whole community. The fungal types and amounts in Panan were more than those in Ninbo indicating the differences between producing areas and the advantage of macro genome sequencing. There were 10 phyla, 29 families, 28 genus and 159 species of fungi in Panan provenance, 6 phyla, 20 families, 19 genus, 136 species in Ninbo provenance, 8 phyla, 37 families, 47 genus, 289 species in Nantong provenance and 7 phyla, 25 families, 24 genus, 102 species in the bulk soil. Some genus such as Dothidea, Capnobotryella and Conidiobolus were only existed in Nantong provenance, while Pyrenochae- ta, Glomus and Pseudonectria were only in Panan provenance, which implied these species could grew because F. thunbergii influenced the existence of fungi. Experiments of provenance and producing area of F. thunbergii showed that the fungal diversity of indigenous provenance was higher than that of exotic provenance and each provenance had unique fungal species in the rhizosphere, which indicated that the diversity and structure was shaped cooperatively by the species and soil type. These fungal species are interacted with the soil-rhizhosphere-microbe microecological system, which in turn influence the growth of F. thunbergii.

Effect of different treatment on endophytic bacterial communities in continuous cropping of Chrysanthemum morifoliu / 中国中药杂志

To reveal the effect of rotation cropping and bacterial manure on the growth of Chrysanthemum morifolium and screen the beneficial endophytic, the diversity of endophytic and dominant genera of different treatment groups were analyzed. Four different treatments were continuous cropping, rotation, self-made organic fertilizer and commercially available fertilizer, respectively. Endophytic bacterial diversity and dominant genera in different organs were examined using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results showed that enzyme Hae III was more appropriate than enzyme Hinfl because the number of TRFs digested by enzyme Hae III was more than that of enzyme Hinfl. In comparison of diversity, the endophytic bacterial communities' diversity index in group of cropping rotation and fertilizer was higher than that of continuous cropping which indicated that the addition of exogenous microorganism in soil could increase the diversity of plant endophyte. 18 dominant species were selected, including 3 kinds of Firmicutes, 4 kinds of Actinomycetes and 11 kinds of Proteobacteria. The results of dominant species comparison showed that the number of dominant species in continuous cropping of Ch. morifolium was significantly less than that of the rotation group. Some dominant bacteria in rotation group and fertilizer group such as Arthrobacter, Streptomyces, Streptomyces, Flavobacterium and Mycobacterium were not found in the continuous cropping of Ch. mortfolium group. Dominant species of fertilizer treatment group was similar with the rotation group, and the continuous cropping group's dominant species was more abundant. It indicates that these bacteria may be able to mitigate hindrance in continuous cropping, especially the Flavobacterium which can decompose the pathogenic fungi is worthy of further attention. Compared with leaves, there are more dominant species in roots and stems. The diversity of edophytic bacterial communities in continuous cropping of Ch. morifolium stays below than that in the rotation of Ch. morifolium, and fertilizer treatment can increase the diversity of continuous cropping so that it could mitigate hindrance in continuous cropping.

Effects of growth years of Paeonia lactiflora on bacterial community in rhizosphere soil and paeoniflorin content / 中国中药杂志

To explore the relationship between microecological environment and Paeonia lactiflora the effects of growth years of P. lactillora on rhizosphere bacterial communities were studied by PCR-DGGE and the paeoniflorin content determined by HPLC. Results showed that the soil pH increased with growing years of P. lactillora. In the fourth year, soil pH and enzyme activity reached the highest level, while organic matter content was the lowest. The bacterial diversity had a positive correlation with growing years varied from 3.38 to 3.61. Sequencing results demonstrated that Gammaproteobacteria, llphaproteobacteria, Actinobacteria, Acidobacte- ria and Firmicutes were predominant bacteria kinds in the soil of P. lactillora. Gammaproteobacteria was only detected in the bulk soil, while llphaproteobacteria, Acidobacteria G1l, Actinobacteria were only in the rhizosphere soil and the bacterial community among different growing years were similar except few species. HLPC results showed that paeoniflorin content was 3.26%, 3.30%, 3.36%, 3.41% separately from one to four-year-old P. lactiflora with an upward trend. The correlation analysis indicated that the paeoniflorin content had a positive correlation with soil pH and bacterial diversity, conversely, had a negative correlation with organic matter con- tent. During the growth years the rhizosphere bacterial diversity increased without changes of predominant bacteria and the paeoniflorin content increased without significant differences while its production increased significantly, which was different from the plants showing replanting diseases. This is in line with the farming practice choosing 4-year-old P. lactllora, but not the 1-3 year old one. In addition, the accumulation of paeoniflorin is closely related to soil pH, organic matter content and bacteria diversity, confirming that the geoherblism of P. lactiflora is closely related with microbial environment in the soil.

Extraction, purification and identification of type II collagen from Agkistrodon acutus / 中国中药杂志

The object of the research was to extract, purify and identify the type II collagen of Agkistrodon acutus. Type II collagen of A. acutus was extracted by enzyme decomposition method, and purified by ion exchange column chromatography. It was characterized by SDS-PAGE gel electrophoresis, ultraviolet spectrophotometry, infrared absorption spectroscopy and mass spectroscopy. The results showed that the size of C II was about 130 kDa. It absorbed at 223 nm. IR spectrum obtained showed that the triple helical domains of amino-acid sequences were characterized by the repetition of triplets Gly-X-Y. The MS spectrum graphically stated that C II extracted from cow and A. acutus have the similar peptides. The C II of A. acutus was obtained by extraction and purification. Appraisal analysis by SDS-PAGE, UV, IR and MS, C II of A. acutus was consistent with the standard C II of cow. It was proved that the extracted protein was C II.

Study on intervention effect of Jieduquyuziyin prescription systemic lupus erythematosus by HPLC-Q-TOF/MS / 中国中药杂志

To establish a metabonomic method based on high performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-Q-TOF/MS), in order to study the changes in serum metabolites of systemic lupus erythematosus (SLE) mice after treatment of Jieduquyuziyin prescription, the pathogenesis of SLE and mechanism of drug action. The orthogonal partial least squares (OPLS) was applied for the pattern recognition of experimental data, finding a significant difference in the control group, the SLE model group, the Jieduquyuziyin prescription-treated group and the prednisone acetate-treated group. According to the OPLS load diagram, 12 differential metabolites, including traumatic acid, PAF, 12 (S)-HEPE, 15(S)-HETrE and Hepoxilin B3 were identified by using accurate mass combined with MS/MS data After treatment with Jieduquyuziyin prescription, the relative contents of PAF, 12 (S)-HETE were close to the level of the control group. According to the analysis on metabolic pathway, SLE could cause significant changes in unsaturated fatty acid and amino acid metabolism pathway, while Jieduquyuziyin prescription has a effect in regulating disorder of unsaturated fatty acid metabolism pathway.

Rapid propagation of Bletilla striata by synthetic seeds technology / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a new manufacturing method for Bletilla striata synthetic seeds, and provided a new way for rapid propagation of B. striata, the correlated influential factors were studied.</p><p><b>METHOD</b>The synthetic seeds were manufactured by taking seeds of B. striata as materials which were beforehand germinated in 1/2 MS medium for 10 days, and the influential factors such as artificial endosperm components, episperm substances, storage conditions and germination groundmass impact on the germination rate and seedling rate of the synthetic seeds were evaluated.</p><p><b>RESULT</b>Compound 4.0% sodium alginate + 0.2 mol x L(-1) CaCl2 + 0.4 mg x L(-1) penicillin + 0.3% carbendazim powder + 0.2% sodium benzoate served as the best episperm substances while MS + 1.0 mg x L(-1) NAA + 2.0 mg x L(-1) KT as the best endosperm components, in which, high germination rate and seedling rate were obtained. The synthetic seeds storing in the 4 degrees C for a long time was able to have still high vitality.</p><p><b>CONCLUSION</b>The B. striata synthetic seeds manufacturing system was established successfully, while efforts should be taken to improve the sowing technique of the synthetic seeds in non-sterile conditions.</p>

Application of SCAR molecular marker technology in identification of Monascus strains / 中国中药杂志

<p><b>OBJECTIVE</b>To establish an effective way for rapid identification of Monascus strains based on DNA molecular marker.</p><p><b>METHOD</b>A random amplified polymorphic DNA (RAPD) marker named F421 in genomic DNA of Monascus F strain was observed during a comparison of DNA fingerprints derived from 10 cultivated strains of Monascus. F421 was cloned and sequenced. Comparing the sequence of F421 (GenBank accession number EF063107) with other relative sequences in the GenBank databases, no distinct comparability was found. A pair of sequence characterized amplified region (SCAR) primers were designed based on the sequence of the cloned fragment and tested for the specific detection of Monascus F.</p><p><b>RESULT</b>The results of polymerase chain reaction showed that only a 421bp segment of Monascus F strain was amplified compared with other 9 cultivated strains of Monascus. And the acquired SCAR marker of strain F could be used as a specific DNA fingerprint to identify Monascus strain F within one day.</p><p><b>CONCLUSION</b>SCAR molecular marker technology is an effective new way to identify Monascus strains more rapidly. And also is an assistant tool to identify Monascus strains more accurately when disagreements come out using traditional classification. It could be applied widely to the protection of germ plasm resources, classification and identification distinguishing false strains of pharmaceutical fungi.</p>

Study function of endophytic fungus in parasitism process of mistletoe / 中国中药杂志

<p><b>OBJECTIVE</b>To research the function of endophytes of mistletoe in parasitism process of mistletoe in Pterocarya stenoptera.</p><p><b>METHOD</b>Endophytes from eight different parts of the mistletoe were separated by explant culture, and further screened by different CMC plates culture and DNS method to get cellulase high productive strains. The distribution of the endophytic fungus parasitized in mistletoe were prepared and stained to demonstrate by histological section of the intumescentia part of the P. stenoptera.</p><p><b>RESULT</b>The histological section indicated that aboundent of hyphasma were distributed around the haustorium of the mistletoe. Eighty three strains of endophytic fungus were separated, 38 of them were able to degrade cellulose, 19 strains showed high cellulase activity and 10 of which were separated from the parasitic position.</p><p><b>CONCLUSION</b>Endophytic fungus of mistletoe can secrete cellulase and assist the haustorium of mistletoe to breakthrough the cell walls as well as intercellular space tissues of the P. stenoptera, thus, the endophytic fungus plays an important role in the parasitism process of mistletoe in P. stenoptera.</p>