| ObjectiveThrough the studies of distribution of resources and ecological suitability of Bletilla striata, and the studies of the tissue culture, the quality evaluation of Bletilla striata. They provide experimental evidence and theoretical foundation for the large-scale cultivation, resource development and utilization of Bletilla striata in Northwest Hubei.Method1The Studies of Bletilla Striata on Resource Survey and Species Identification1.1The Analysis of the Bestilla Striata on ecological suitabilityThrough the sorting and analysis of Bletilla striata and its related species like Bletilla formosana, Bletilla ochracea and the Chinese rhizoma bletillae in the China Digital Plant Herbarium(CVH) and the TCMGIS, and through suitable environment for the growth of Bletilla striata and its influence factors analysis, we derive growth environment of95to100%of a suitable large-scale cultivation of Bletilla striata.1.2Bletilla Striata DNA Bar-Code StudiesExtraction of Bletilla striata and other adulterants herbs of four species of more than39samples DNA, PCR amplification and direct sequencing of their ITS2sequences, application CodonCode Aligner v4.25sequencing peak figure proofread stitching and remove low quality sequences and primer area, get ITS2sequences. MEGA6.0calculated using various software objects within the distance and interspecific Kimura2-parameter (K2P) genetic analysis of variable sites and phylogenetic tree constructed NJ, integrated application similarity search, nearest distance method and NJ phylogenetic tree, etc. identification and analysis. 1.3The Species Identification of Bletilla StriataEstablishment of Bletilla striata and Bletilla ochracea medicinal properties, microstructure, chemical, TLC and other characteristics identification method. Bletilla striata and and Bletilla ochracea roots for microscopic identification of cross-section, the use of physical and chemical properties, TLC method, a comparative study of Bletilla striata and Bletilla ochracea.2Bletilla Striata Young Root Tissue Culture Technology ResearchHormone concentrations and ratios of different explants of Bletilla striata induced by different research differentiation, proliferation and rooting. By differentiation Bletilla striata seedlings, buds, and the proliferation of rooting culture, the establishment of a Tissue Culture System of Bletilla striata for Bletilla striata tissue culture propagation medium to find the best location and the best ratio.3The Studies of Quality Evaluation of Bletilla Striata in Northwest Hubei3.1The Studies of Quality Evaluation of Bletilla StriataDiscussion anthrone-Determination of polysaccharide content factors sulfuric acid method, determination of the polysaccharide content of Bletilla striata preferred optimum conditions. Bletilla striata polysaccharide content of different origin contained in northwest Hubei were determined using water extraction and alcohol precipitation polysaccharide extracted Bletilla striata to anthrone-sulfuric acid reagent as chromogenic reagent, using UV spectrophotometry polysaccharide content. Determination of the content of the Northwest Hubei Bletilla striata tubers total phenols, Bletilla striatae rence, using the Folin-phenol colorimetric method, using visible-ultraviolet spectrophotometry content from different areas of Bletilla striata tubers total phenols, and moisture, ash determination of heavy metals. HPLC was used to establish the origin of Bletilla striata of different herbs in the determination of1,4-bis [4-(glucose, oxygen) benzyl]-2-isobutyl malate content of the HPLC method, its content using mean clustering method analysis. By19batches of Bletilla striata gradient elution northwest Hubei Bletilla striata establish the chemical composition of the HPLC fingerprint feature map.3.2The Main Chemical Constituents of Bletilla StriataHeadspace solid phase micro extraction (HS-SPME) extraction of volatile components in Bletilla striata, and by gas chromatography-combined technique on Bletilla striata volatile components were analyzed mass spectrometry (GC-MS). Using AB Sciex4000Q-TRAP mass spectrometer by multiple reaction monitoring (MRM) scan mode for herbs of Bletilla striata were tested. Bletilla striata determination of active ingredients militarine, protocatechuic acid and caffeic acid content, the establishment of a detection method based on Q-TRAP LC-MS Bletilla striata active ingredients.4Pharmacological Effects of Bletilla StriataThrough the Bletilla striata ethyl acetate, n-butanol and water extracts of different parts of the site, clotting time was measured initially screened Bletilla striata bleeding, blood parts; then through a rat model of stasis and gastric ulcer model in mice, further proof EtOAc site hemorheology indexes stasis Rats and n-BuOH parts affect ulcer model in mice. Effects of Bletilla striata of different extraction sites on blood rheology, and further studies of pharmacological activity of each site.Results1Resource Investigation and Identification of Bletilla Striata Varieties1.1Distribution of Resources and Ecological Suitability Analysis of Bletilla StriataThrough the nearly600species of Bletilla striata and its related digital herbarium specimens of Chinese Bletilla striata under data entry, including herbarium name, bar code, gathering people, acquisition time, gathering places, coordinates, appraiser, appraisal time identification results, etc., through the Bletilla striata Locality organize, summarize, analyze, based on the "Origin of Chinese herbal medicines GIS suitability analysis"(TCMGIS) in basic geographic information database, the database climatic factors, soil and medicines distributed database four spatial database indexing and database diagrams spatial analysis to extract the sample database Bletilla striata at the point of climatic data and soil data, then the data system analysis, the large-scale cultivation of Bletilla striata suitable for the growth of environmental factors on Bletilla striata the large-scale cultivation and provide scientific basis.1.2Bletilla Striata DNA Bar-Code StudiesK2P largest species of Bletilla striata genetic distance is0.035, which is much smaller than between species and adulterants minimum genetic distance of0.31; NJ tree showed that14spieces Bletilla striata sequences clustered separately and with adulterants distinguishable. The above results show that identification methods, ITS2sequence herbs and Bletilla striata can adulterants Identification open.1.3The Studies of Identification of Bletilla StriataMicroscopic identification of cross-section through dialogue and rhizomes, chemical identification, TLC, Two sources were identified mainly Bletilla Striata and Bletilla ochracea in northwest Hubei.There is no significant difference between Bletilla Striata and Bletilla ochracea on TLC identification.2Research of Bletilla Striata Cultivation TechnologyHormone concentrations and ratios of different explants of Bletilla striata induced by different research differentiation, proliferation and rooting. The results showed that the most likely induced radicle buds, the best medium for1/2MS+1.0mg/L6-BA+2.0mg/LNAA; buds best medium for the proliferation of MS+1.0mg/L6-BA+0.05mg/LNAA; rooting medium to1/2MS+0.5 mg/L NAA is the best. Bletilla striata leaf buds in this experiment did not induce out bulb shoot induction rate is relatively low and late slow growth.3Quality Evaluation of Bletilla Striata in Northwest Hubei3.1Research on Content Determination of Polysaccharide of Bletilla StriataOn the basis of single factor experiments, using L9(34) orthogonal experiment, heating time, heating temperature, anthrone-sulfuric acid test solution investigated factors as the amount to the absorbance value evaluation, preferably anthrone-sulfuric acid method optimal conditions Bletilla striata polysaccharide content. Optimum measurement conditions are anthrone sulfuric acid test solution dosage4.5mL, the heating temperature is100℃, the heating time is3min, reagent concentration of0.2%, measured wavelength625nm. Refined Bletilla striata polysaccharide conversion factor f is1.69. Polysaccharide content of Bletilla striata from different areas of northwest Hubei were determined using water extraction and alcohol precipitation polysaccharide extracted Bletilla striata to anthrone-sulfuric acid reagent as chromogenic reagent, using UV spectrophotometry polysaccharide content. The measurement results show that the content of different origin in northwest Hubei Bletilla striata medicinal polysaccharides were quite different.3.2Determination of Total Phenolic Content of Tubers of Bletilla StriataBletilla Striata, using the Folin-phenol colorimetric method, using visible ultraviolet spectrophotometry content from different areas of Bletilla Striata tubers total phenols. Different origin of Bletilla striata herbs large differences in total phenol content, indicating that the environment has a significant effect on the growth of Bletilla striata total phenolic content.3.3Malate content in Determination of Bletilla StriataThe HPLC determination of different origin Bletilla Striata herbs,1,4-bis [4-(glucose oxidation) benzyl]-2-isobutyl malate content of clear differences between their content using mean clustering method analysis showed that1,4-bis [4-(glucose oxidation) benzyl]-2-isobutyl-malate regional significantly low levels.3.4Bletilla Striata Moisture, Ash, Heavy metals DeterminationBletilla striata of different origin carried moisture, ash, and the results showed that the Bletilla striata samples from different areas of the total ash, moisture were not exceeded, the total ash content between different origins of Bletilla striata have some differences.For different origin Bletilla striata lead, cadmium, copper and other heavy metals by atomic absorption spectrometry (AAS) were measured, the results show that Bletilla striata samples from different areas of northwestern Hubei Pb content of cadmium, copper, copper just individual species the content exceeded, the rest were within acceptable ranges.3.5Fingerprints of Bletilla StriataNorthwest establish the chemical composition of Bletilla striata HPLC fingerprint feature map. With acetonitrile and water as the mobile phase gradient elution,15batches of different origin characteristic patterns of Bletilla striata medicinal chemical composition were detected nine common peaks good separation. Through cluster analysis, the origin and characteristics of Bletilla striata, herbs map similarity little difference.3.6The Main Chemical Constituents of Bletilla StriataVolatile components using headspace solid phase micro-extraction (HS-SPME) extraction of Bletilla striata, and gas chromatography-mass spectrometry for analysis (GC-MS) combined technique on Bletilla striata volatile components were isolated from Bletilla striata27chromatography peak, after ChemStation data processing system and by area normalization method to calculate its total ion current in the percentage of each component, according to the peaks of mass spectra with a computer database searches identified11compounds 91.25%, accounting for the total. Where in the main components of p-cresol (47.22%), hexanal (43.33%) and so on.Determination of active ingredients in traditional Chinese medicine Bletilla striata militarine, protocatechuic acid and caffeic acid content, the establishment of a detection method based on Q-TRAP LC-MS. Using AB Sciex4000Q-TRAP mass spectrometer by multiple reaction monitoring (MRM) scan mode control medicine for Bletilla striata and Bletilla striata samples were tested. Bletilla striata herbs in militarine, protocatechuic acid and caffeic acid content were0.7088mg/g,0.0011mg/g and0.0004mg/g.4Pharmacological Effects of Bletilla StriataThrough the Bletilla striata ethyl acetate, n-butanol and water extracts of different parts of the site, clotting time was measured, the results show, EtOAc parts with blood effects, n-BuOH and water sites with hemostasis; high EtOAc parts, medium and low dose can significantly reduce the indices of blood stasis model rats, n-BuOH parts have significantly improved ulcer.Conclusion1Through the analysis of samples of Bletilla striata, Bletilla striata extract the appropriate growth temperature, climate, soil conditions, large-scale cultivation of Bletilla striata provides a scientific basis.2ITS2sequence can accurately identify adulterants Bletilla striata and herbs, medicinal herbs to avoid medication mix-clinical safety and security provides a new technical means.3Hormone concentrations and ratios of different explants of Bletilla striata induced differentiation through different studies, proliferation and rooting, the establishment of a Bletilla striata radicle tissue culture system.4Bletilla striata in northwest Hubei polysaccharides on different areas of origin, polyphenols,1,4-bis [4-(glucose oxidation) benzyl]-2-isobutyl malate content were determined to establish a multi-index components medicinal Bletilla striata quality control system to ensure the quality of the Bletilla striata provided.5The establishment of a fingerprint quality evaluation system of Bletilla striata, can fully evaluate the quality of Bletilla striata.6By gas chromatography-mass spectrometry (GC-MS) combined technique on Bletilla striata volatile components were analyzed using high performance liquid chromatography-mass spectrometry (HPLC-MS) combined technique on Bletilla striata, the main chemical components were analyzed, a comprehensive and objective analysis of the chemical composition of the Bletilla striata.7Bletilla striata ethyl acetate, n-butanol and water extracts of different parts of the site, clotting time was measured. |
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