鄂西北白及产地适宜性与品质评价研究
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摘要
目的
考察白及野生资源分布区域及产地生态适宜性,探讨白及组织培养的条件和方法、对白及质量评价方法进行研究,为鄂西北地区白及的规模化种植、资源开发与利用提供参考依据。
方法
1.白及的品种整理与鉴别研究
1.1白及产地适宜性分析
在对中国植物数字标本馆(CVH)收载的近600份白及与近缘物种小白及、黄花白及、华白及的标本数据进行整理、分析的基础上,利用中药材产地适宜性分析地理信息系统(TCMGIS)技术,对适宜白及生长的环境及其影响因子进行分析,得出相似度在95～100%的适宜白及规模化种植生长环境因素。
1.2白及的DNA条形码鉴别研究
提取白及药材及射干、黄精、玉竹、知母等混伪品共8个物种39余份样品DNA, PCR扩增其ITS2序列并双向测序,应用CodonCode Alignerv4.25对测序峰图进行校对拼接,并去除低质量序列及引物区,得到ITS2序列。用MEGA6.0软件计算物种种内和种间Kimura2-parameter(K2P)遗传距离,分析变异位点并构建NJ系统发育树,综合应用相似性搜索法、最近距离法以及NJ系统发育树等进行鉴定分析。
1.3鄂西北白及品种鉴别研究
建立白及与黄花白及的药材性状、显微、理化、TLC等特征鉴别方法。对白及与黄花白及根茎横切面进行显微鉴定、理化鉴定、薄层色谱鉴别研究。
2.白及组织培养技术研究
考察不同激素浓度和配比对白及不同外植体诱导分化、增殖及生根的影响。通过对白及外植体诱导苗的分化、丛生芽的增殖以及生根培养,建立了白及组培快繁体系；研究白及组培扩繁的方法,探讨白及组培最佳部位与最佳培养基配比。
3.鄂西北白及的品质评价研究
3.1白及的质量评价研究
探讨蒽酮-硫酸法测定多糖含量的影响因素,优选白及多糖含量测定的最优条件。对鄂西北地区不同产地的白及中所含的白及多糖含量进行测定,采用水提醇沉法提取白及多糖,以蒽酮-硫酸试剂为显色剂,采用紫外分光光度法测定多糖含量。测定鄂西北地区白及块茎中总酚的含量,以没食子酸为对照品,采用Folin-酚比色法,运用可见-紫外分光光度法测定不同产地白及块茎中总酚的含量,并进行了水分、灰分、重金属的测定。采用HPLC法,建立测定不同产地白及药材中1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯含量的HPLC方法,对其含量采用均数法进行聚类分析。通过对鄂西北地区19批次的白及进行梯度洗脱,建立鄂西北地区白及化学成分的HPLC指纹特征图谱。
3.2白及主要成分研究
采用顶空固相微萃取法(HS-SPME)萃取白及中挥发性成分,并用气相色谱-质谱(GC-MS)联用技术对白及的挥发性成分进行分析。
采用AB Sciex4000Q-TRAP型质谱仪多重反应监测(M R M)扫描模式对白及对照药材和白及样品分别进行检测。测定白及中1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯、原儿茶酸和咖啡酸的含量,建立了基于Q-TRAP LC-MS的白及活性成分的检测方法。
4.白及药理作用研究
分别对白及乙酸乙酯、正丁醇及水部位进行出、凝血时间测定,初步筛选出白及止血、活血部位；再通过大鼠血瘀模型和小鼠胃溃疡模型,进一步论证EtOAc部位对血瘀模型大鼠血液流变学各项指标的影响以及n-BuOH部位对小鼠胃溃疡模型的影响,探讨白及不同提取部位对血液流变学的影响与药理活性。
结果
1.白及品种整理与鉴别研究
1.1白及产地适宜性分析
通过对中国数字植物标本馆白及项下的近600份白及及其近缘种小白及、黄花白及、华白及的标本进行数据的录入,内容包括标本馆名称、条形码、采集人、采集时间、采集地、坐标、鉴定人、鉴定时间、鉴定结果等内容,通过对白及的采集地点进行整理、汇总、分析,基于“中药材产地适宜性分析地理信息系统”(TCMGIS)中基础地理信息数据库、气候因子数据库、土壤数据库和中药材分布空间数据库四个数据库中的图表索引和空间分析功能,提取白及标本点处的上述数据库中的气候数据和土壤数据,然后对数据进行系统分析,得出适宜白及规模化种植生长环境因素,对白及的规模化种植提供科学依据。
1.2白及的DNA条形码鉴别研究
白及的最大种内K2P遗传距离为0.035,远小于其与混伪品的种间最小遗传距离为0.31；NJ树结果显示,14条白及序列单独聚为一枝,且与混伪品可明显区分。以上鉴定方法分析结果表明,ITS2序列能将白及药材与混伪品鉴别开
1.3鄂西北白及品种鉴别研究
通过对白及根茎横切面进行显微鉴定、理化鉴定、薄层色谱鉴别,鄂西北地区的白及经鉴定主要为兰科白及属植物白及Bletilla striata、黄花白及Bletilla ochracea两个来源,白及与黄花白及薄层色谱鉴别,无明显区别。
2.白及幼根组织培养技术研究
通过研究不同浓度激素和不同激素配比对白及不同外植体诱导分化、增殖及生根的影响。结果表明,幼根最容易诱导出丛生芽,其最佳培养基为1/2MS+1.0mg/L6-BA+2.0mg/L NAA;丛生芽增殖最佳培养基为MS+1.0mg/L6-BA+0.05mg/L NAA;生根培养基以1/2MS+0.5mg/L NAA为最佳。白及叶片丛生芽在本实验中没有诱导出来,鳞茎丛生芽诱导率也比较低且后期生长缓慢。
3.鄂西北白及的品质评价研究
3.1白及多糖的含量测定研究
在单因素试验的基础上,采用L9(34)正交试验,以加热时间、加热温度、蒽酮-硫酸试液的用量作为考察因素,以吸光度值为评价指标,优选蒽酮-硫酸法测定白及多糖含量的最佳条件。最佳测定条件是蒽酮-硫酸试液用量为4.5mL,加热温度为100℃,加热时间为3min,显色剂浓度0.2%,测定波长625nm。精制白及多糖换算因子f为1.69。
对鄂西北地区不同产地的白及多糖含量进行测定,采用水提醇沉法提取白及多糖,以蒽酮-硫酸试剂为显色剂,采用紫外分光光度法测定多糖含量。测定结果显示,鄂西北地区不同产地的白及药材中多糖的含量相差较大。
3.2白及总酚的含量测定研究
以没食子酸为对照品,采用Folin-酚比色法,运用可见-紫外分光光度法测定不同产地白及块茎中总酚的含量。不同产地的白及药材中总酚含量差异较大,说明生长环境对白及总酚含量有明显影响。
3.3白及中苹果酸酯的含量测定研究
采用HPLC法,测定不同产地的白及药材,1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯的含量差别明显,对其含量采用均数法进行聚类分析,结果显示,1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯含量高低有明显的地域性。
3.4白及水分、灰分、重金属测定
对不同产地的白及进行水分、灰分检测,结果表明,不同产地的白及样品中总灰分、水分均未超标,不同产地白及之间的总灰分含量有一定的差异。
针对不同产地白及中的铅、镉、铜等重金属,通过原子吸收光谱法(AAS)进行测定,结果表明,鄂西北不同产地的白及样品中重金属铅、镉、铜的含量,仅有个别品种铜的含量超标,其余均在合格范围内。
3.5白及指纹图谱研究
建立鄂西北地区白及化学成分的HPLC指纹特征图谱。用乙腈和水为流动相,进行梯度洗脱,不同产地的15批白及药材化学成分特征图谱共检出9个分离度良好的共有峰。通过聚类分析,白及药材产地与特征图谱相似度差异不大。
3.6白及主要化学成分研究
采用顶空固相微萃取法(HS-SPME)萃取白及中挥发性成分,并用气相色谱-质谱(GC-MS)联用技术对白及的挥发性成分进行分析,从白及中共分离出27个色谱峰,经化学工作站数据处理系统及用面积归一化法从其总离子流图中计算了各组分的百分含量,按各峰的质谱图经计算机质谱数据库检索,鉴定出其中11种化合物,占总量的91.25%。其中主要成分为对甲酚(47.22%)、已醛(43.33%)等。
测定白及中活性成分1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯、原儿茶酸和咖啡酸的含量,建立了基于Q-TRAP LC-MS/MS的检测方法。采用AB Sciex4000Q-TRAP型质谱仪多重反应监测(M R M)扫描模式对白及对照药材和白及样品分别进行检测。白及中1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯、原儿茶酸和咖啡酸的含量分别为0.7088mg/g、0.0011mg/g和0.0004mg/g。
4.白及药理作用研究
通过对白及乙酸乙酯、正丁醇及水部位不同提取部位进行出、凝血时间测定,结果表明,EtOAc部位具有活血作用,n-BuOH及水部位具有止血作用；EtOAc部位高、中、低剂量均可显著地降低血瘀模型大鼠的血液流变学各项指标,n-BuOH部位有显著改善胃溃疡的作用。
结论
1.通过对白及样本的分析,提取了适宜白及生长的温度、气候、土壤条件,鄂西北地区在白及产地适宜性相似度100%的区域内,适宜白及的规模化种植。
2.ITS2序列能准确鉴别白及药材与混伪品,为避免白及药材混用及保障临床安全用药提供了新的技术手段。
3.通过研究不同激素浓度和配比对白及不同外植体诱导分化、增殖及生根的影响,建立了白及幼根组织培养体系。
4.对鄂西北地区不同产地的白及中多糖、多酚、1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯的含量进行测定,建立了白及药材多指标成分质量控制体系,为保证白及的质量提供了依据。
5.建立了白及的指纹图谱质量评价体系,可以全面评价白及的质量。
6.采用气相色谱-质谱(GC-MS)联用技术对白及的挥发性成分进行分析,并采用液相色谱-质谱(HPLC-MS)联用技术对白及中主要化学成分进行了分析,全面而客观的分析了白及的化学成分。
7.通过对白及不同提取部位的药理活性研究,白及乙酸乙酯部位具有活血作用,白及正丁醇部位可以显著改善出血症状。
Objective
Through 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.
Method
1The Studies of Bletilla Striata on Resource Survey and Species Identification
1.1The Analysis of the Bestilla Striata on ecological suitability
Through 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 Studies
Extraction 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 Striata
Establishment 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 Research
Hormone 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 Hubei
3.1The Studies of Quality Evaluation of Bletilla Striata
Discussion 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 Striata
Headspace 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 Striata
Through 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.
Results
1Resource Investigation and Identification of Bletilla Striata Varieties
1.1Distribution of Resources and Ecological Suitability Analysis of Bletilla Striata
Through 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 Studies
K2P 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 Striata
Microscopic 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 Technology
Hormone 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 Hubei
3.1Research on Content Determination of Polysaccharide of Bletilla Striata
On 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 Striata
Bletilla 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 Striata
The 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 Determination
Bletilla 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 Striata
Northwest 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 Striata
Volatile 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 Striata
Through 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.
Conclusion
1Through 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.
考察白及野生资源分布区域及产地生态适宜性,探讨白及组织培养的条件和方法、对白及质量评价方法进行研究,为鄂西北地区白及的规模化种植、资源开发与利用提供参考依据。
方法
1.白及的品种整理与鉴别研究
1.1白及产地适宜性分析
在对中国植物数字标本馆(CVH)收载的近600份白及与近缘物种小白及、黄花白及、华白及的标本数据进行整理、分析的基础上,利用中药材产地适宜性分析地理信息系统(TCMGIS)技术,对适宜白及生长的环境及其影响因子进行分析,得出相似度在95～100%的适宜白及规模化种植生长环境因素。
1.2白及的DNA条形码鉴别研究
提取白及药材及射干、黄精、玉竹、知母等混伪品共8个物种39余份样品DNA, PCR扩增其ITS2序列并双向测序,应用CodonCode Alignerv4.25对测序峰图进行校对拼接,并去除低质量序列及引物区,得到ITS2序列。用MEGA6.0软件计算物种种内和种间Kimura2-parameter(K2P)遗传距离,分析变异位点并构建NJ系统发育树,综合应用相似性搜索法、最近距离法以及NJ系统发育树等进行鉴定分析。
1.3鄂西北白及品种鉴别研究
建立白及与黄花白及的药材性状、显微、理化、TLC等特征鉴别方法。对白及与黄花白及根茎横切面进行显微鉴定、理化鉴定、薄层色谱鉴别研究。
2.白及组织培养技术研究
考察不同激素浓度和配比对白及不同外植体诱导分化、增殖及生根的影响。通过对白及外植体诱导苗的分化、丛生芽的增殖以及生根培养,建立了白及组培快繁体系；研究白及组培扩繁的方法,探讨白及组培最佳部位与最佳培养基配比。
3.鄂西北白及的品质评价研究
3.1白及的质量评价研究
探讨蒽酮-硫酸法测定多糖含量的影响因素,优选白及多糖含量测定的最优条件。对鄂西北地区不同产地的白及中所含的白及多糖含量进行测定,采用水提醇沉法提取白及多糖,以蒽酮-硫酸试剂为显色剂,采用紫外分光光度法测定多糖含量。测定鄂西北地区白及块茎中总酚的含量,以没食子酸为对照品,采用Folin-酚比色法,运用可见-紫外分光光度法测定不同产地白及块茎中总酚的含量,并进行了水分、灰分、重金属的测定。采用HPLC法,建立测定不同产地白及药材中1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯含量的HPLC方法,对其含量采用均数法进行聚类分析。通过对鄂西北地区19批次的白及进行梯度洗脱,建立鄂西北地区白及化学成分的HPLC指纹特征图谱。
3.2白及主要成分研究
采用顶空固相微萃取法(HS-SPME)萃取白及中挥发性成分,并用气相色谱-质谱(GC-MS)联用技术对白及的挥发性成分进行分析。
采用AB Sciex4000Q-TRAP型质谱仪多重反应监测(M R M)扫描模式对白及对照药材和白及样品分别进行检测。测定白及中1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯、原儿茶酸和咖啡酸的含量,建立了基于Q-TRAP LC-MS的白及活性成分的检测方法。
4.白及药理作用研究
分别对白及乙酸乙酯、正丁醇及水部位进行出、凝血时间测定,初步筛选出白及止血、活血部位；再通过大鼠血瘀模型和小鼠胃溃疡模型,进一步论证EtOAc部位对血瘀模型大鼠血液流变学各项指标的影响以及n-BuOH部位对小鼠胃溃疡模型的影响,探讨白及不同提取部位对血液流变学的影响与药理活性。
结果
1.白及品种整理与鉴别研究
1.1白及产地适宜性分析
通过对中国数字植物标本馆白及项下的近600份白及及其近缘种小白及、黄花白及、华白及的标本进行数据的录入,内容包括标本馆名称、条形码、采集人、采集时间、采集地、坐标、鉴定人、鉴定时间、鉴定结果等内容,通过对白及的采集地点进行整理、汇总、分析,基于“中药材产地适宜性分析地理信息系统”(TCMGIS)中基础地理信息数据库、气候因子数据库、土壤数据库和中药材分布空间数据库四个数据库中的图表索引和空间分析功能,提取白及标本点处的上述数据库中的气候数据和土壤数据,然后对数据进行系统分析,得出适宜白及规模化种植生长环境因素,对白及的规模化种植提供科学依据。
1.2白及的DNA条形码鉴别研究
白及的最大种内K2P遗传距离为0.035,远小于其与混伪品的种间最小遗传距离为0.31；NJ树结果显示,14条白及序列单独聚为一枝,且与混伪品可明显区分。以上鉴定方法分析结果表明,ITS2序列能将白及药材与混伪品鉴别开
1.3鄂西北白及品种鉴别研究
通过对白及根茎横切面进行显微鉴定、理化鉴定、薄层色谱鉴别,鄂西北地区的白及经鉴定主要为兰科白及属植物白及Bletilla striata、黄花白及Bletilla ochracea两个来源,白及与黄花白及薄层色谱鉴别,无明显区别。
2.白及幼根组织培养技术研究
通过研究不同浓度激素和不同激素配比对白及不同外植体诱导分化、增殖及生根的影响。结果表明,幼根最容易诱导出丛生芽,其最佳培养基为1/2MS+1.0mg/L6-BA+2.0mg/L NAA;丛生芽增殖最佳培养基为MS+1.0mg/L6-BA+0.05mg/L NAA;生根培养基以1/2MS+0.5mg/L NAA为最佳。白及叶片丛生芽在本实验中没有诱导出来,鳞茎丛生芽诱导率也比较低且后期生长缓慢。
3.鄂西北白及的品质评价研究
3.1白及多糖的含量测定研究
在单因素试验的基础上,采用L9(34)正交试验,以加热时间、加热温度、蒽酮-硫酸试液的用量作为考察因素,以吸光度值为评价指标,优选蒽酮-硫酸法测定白及多糖含量的最佳条件。最佳测定条件是蒽酮-硫酸试液用量为4.5mL,加热温度为100℃,加热时间为3min,显色剂浓度0.2%,测定波长625nm。精制白及多糖换算因子f为1.69。
对鄂西北地区不同产地的白及多糖含量进行测定,采用水提醇沉法提取白及多糖,以蒽酮-硫酸试剂为显色剂,采用紫外分光光度法测定多糖含量。测定结果显示,鄂西北地区不同产地的白及药材中多糖的含量相差较大。
3.2白及总酚的含量测定研究
以没食子酸为对照品,采用Folin-酚比色法,运用可见-紫外分光光度法测定不同产地白及块茎中总酚的含量。不同产地的白及药材中总酚含量差异较大,说明生长环境对白及总酚含量有明显影响。
3.3白及中苹果酸酯的含量测定研究
采用HPLC法,测定不同产地的白及药材,1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯的含量差别明显,对其含量采用均数法进行聚类分析,结果显示,1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯含量高低有明显的地域性。
3.4白及水分、灰分、重金属测定
对不同产地的白及进行水分、灰分检测,结果表明,不同产地的白及样品中总灰分、水分均未超标,不同产地白及之间的总灰分含量有一定的差异。
针对不同产地白及中的铅、镉、铜等重金属,通过原子吸收光谱法(AAS)进行测定,结果表明,鄂西北不同产地的白及样品中重金属铅、镉、铜的含量,仅有个别品种铜的含量超标,其余均在合格范围内。
3.5白及指纹图谱研究
建立鄂西北地区白及化学成分的HPLC指纹特征图谱。用乙腈和水为流动相,进行梯度洗脱,不同产地的15批白及药材化学成分特征图谱共检出9个分离度良好的共有峰。通过聚类分析,白及药材产地与特征图谱相似度差异不大。
3.6白及主要化学成分研究
采用顶空固相微萃取法(HS-SPME)萃取白及中挥发性成分,并用气相色谱-质谱(GC-MS)联用技术对白及的挥发性成分进行分析,从白及中共分离出27个色谱峰,经化学工作站数据处理系统及用面积归一化法从其总离子流图中计算了各组分的百分含量,按各峰的质谱图经计算机质谱数据库检索,鉴定出其中11种化合物,占总量的91.25%。其中主要成分为对甲酚(47.22%)、已醛(43.33%)等。
测定白及中活性成分1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯、原儿茶酸和咖啡酸的含量,建立了基于Q-TRAP LC-MS/MS的检测方法。采用AB Sciex4000Q-TRAP型质谱仪多重反应监测(M R M)扫描模式对白及对照药材和白及样品分别进行检测。白及中1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯、原儿茶酸和咖啡酸的含量分别为0.7088mg/g、0.0011mg/g和0.0004mg/g。
4.白及药理作用研究
通过对白及乙酸乙酯、正丁醇及水部位不同提取部位进行出、凝血时间测定,结果表明,EtOAc部位具有活血作用,n-BuOH及水部位具有止血作用；EtOAc部位高、中、低剂量均可显著地降低血瘀模型大鼠的血液流变学各项指标,n-BuOH部位有显著改善胃溃疡的作用。
结论
1.通过对白及样本的分析,提取了适宜白及生长的温度、气候、土壤条件,鄂西北地区在白及产地适宜性相似度100%的区域内,适宜白及的规模化种植。
2.ITS2序列能准确鉴别白及药材与混伪品,为避免白及药材混用及保障临床安全用药提供了新的技术手段。
3.通过研究不同激素浓度和配比对白及不同外植体诱导分化、增殖及生根的影响,建立了白及幼根组织培养体系。
4.对鄂西北地区不同产地的白及中多糖、多酚、1,4-二[4-(葡萄糖氧)苄基]-2-异丁基苹果酸酯的含量进行测定,建立了白及药材多指标成分质量控制体系,为保证白及的质量提供了依据。
5.建立了白及的指纹图谱质量评价体系,可以全面评价白及的质量。
6.采用气相色谱-质谱(GC-MS)联用技术对白及的挥发性成分进行分析,并采用液相色谱-质谱(HPLC-MS)联用技术对白及中主要化学成分进行了分析,全面而客观的分析了白及的化学成分。
7.通过对白及不同提取部位的药理活性研究,白及乙酸乙酯部位具有活血作用,白及正丁醇部位可以显著改善出血症状。
Objective
Through 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.
Method
1The Studies of Bletilla Striata on Resource Survey and Species Identification
1.1The Analysis of the Bestilla Striata on ecological suitability
Through 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 Studies
Extraction 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 Striata
Establishment 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 Research
Hormone 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 Hubei
3.1The Studies of Quality Evaluation of Bletilla Striata
Discussion 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 Striata
Headspace 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 Striata
Through 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.
Results
1Resource Investigation and Identification of Bletilla Striata Varieties
1.1Distribution of Resources and Ecological Suitability Analysis of Bletilla Striata
Through 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 Studies
K2P 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 Striata
Microscopic 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 Technology
Hormone 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 Hubei
3.1Research on Content Determination of Polysaccharide of Bletilla Striata
On 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 Striata
Bletilla 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 Striata
The 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 Determination
Bletilla 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 Striata
Northwest 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 Striata
Volatile 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 Striata
Through 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.
Conclusion
1Through 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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