鄂西北地区柴胡与北柴胡品质的比较研究
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摘要
[目的]
通过对鄂西北地区所产柴胡的资源调查、品质评价以及与北柴胡的品质比较研究,为药用柴胡新资源的发现、开发与利用提供实验依据和理论基础。
[方法]
1.鄂西北地区柴胡的资源调查和品种鉴定研究
1.1鄂西北地区柴胡的资源调查
对鄂西北地区野生与栽培柴胡品种进行资源调查研究,采集并制作鄂西北地区原植物标本20余份,比较鉴别其根、茎、叶、花、果实、种子的异同,建立植物分类检索表。
1.2鄂西北地区竹叶柴胡品种鉴别研究
建立竹叶柴胡与北柴胡、三岛柴胡的药材及种子性状、显微、理化、TLC等特征鉴别方法。通过石蜡切片法观察竹叶柴胡、北柴胡和三岛柴胡根及种子的显微结构,并分别制作药材和种子的横切片进行显微鉴定；利用泡沫反应鉴别不同品种的柴胡；按照薄层色谱法,鉴别竹叶柴胡与北柴胡。
1.3鄂西北地区柴胡与北柴胡的遗传多样性ISSR分析
利用分子标记ISSR技术对鄂西北地区柴胡种群进行遗传多样性分析并建立其遗传多样性指纹图谱,从分子生物学的角度分析鄂西北地区竹叶柴胡与其他柴胡的主要差别及其亲缘关系。
1.4鄂西北地区竹叶柴胡野生变家种关键技术研究
通过对鄂西北地区柴胡生长的物候学观察,初步制定鄂西北地区竹叶柴胡种子的质量标准；徒手切片法观察竹叶柴胡种子特征,考察温度和不同培养介质对种子发芽率的影响；考察鄂西北地区竹叶柴胡的最佳采收期。
2.鄂西北地区竹叶柴胡与北柴胡的质量评价比较研究
2.1鄂西北地区竹叶柴胡与北柴胡的药用成分的比较分析
采用HPLC法测定柴胡药材中柴胡皂苷a(SSa)、柴胡皂苷d(SSd)的含量,比较鄂西北地区竹叶柴胡与北柴胡中柴胡总皂苷(SSt)的差异；采用蒽酮-硫酸比色法,测定柴胡中可溶性多糖和粗多糖含量,比较两者总多糖的含量差异；采用可见分光光度法测定总黄酮的含量；采用紫外分光光度法测定柴胡中挥发油含量,并进行醇溶性浸出物、总灰分、酸性不溶性灰分测定；使用紫外-可见分光光度法测量竹叶柴胡经蜜制、酒制、醋制前后挥发油和总皂苷的含量,比较鄂西北地区竹叶柴胡经不同方法炮制前后有效成分的含量；采用HPLC法比较分析不同产地的HPLC图谱,建立鄂西北地区竹叶柴胡HPLC指纹图谱；采用UPLC-MS联用技术,建立柴胡药材的特征图谱,鉴定分析柴胡中的主要成分,在此基础上对主要成分进行定量分析。
2.2鄂西北地区竹叶柴胡与北柴胡的药理活性比较研究
鄂西北地区竹叶柴胡与北柴胡的解热镇痛作用的比较：采用2,4-二硝基苯酚致热大鼠模型比较两种柴胡水煎液的解热作用；采用醋酸扭体实验及热板法比较两种柴胡水煎液及醇提液的镇痛作用。
鄂西北地区竹叶柴胡与北柴胡的抗炎、保肝作用的比较：采用二甲苯致耳壳肿胀及蛋清致足肿胀的小鼠模型比较两种柴胡水煎液的抗炎作用；采用四氯化氮的花生油溶液复制小鼠肝损伤模型,检测小鼠的血清生化指标ALT、AST、ALP;检测肝组织匀浆生化指标SOD、MDA、GSH,观察小鼠肝组织病理切片及免疫组化指标,比较鄂西北地区竹叶柴胡与北柴胡根的抗炎、保肝作用。
[结果]
1.鄂西北地区竹叶柴胡的资源调查和品种鉴定研究
1.1鄂西北地区竹叶柴胡的资源调查
本地区所产柴胡习称“北柴胡”,经鉴定多为伞形科植物竹叶柴胡Bupleurum. marginatum Wall. ex DC.的干燥根,少数为石竹科植物蝇子草Silene fortunei Vis.的干燥根。
1.2鄂西北地区竹叶柴胡的品种鉴别
1.2.1竹叶柴胡与北柴胡原植物性状鉴别特征
竹叶柴胡：小总苞片窄而小,披针形,叶较厚,近革质,有白色软骨质边缘。根粗厚,纺锤形,表面通常深红棕色或棕褐色。茎基部扭曲,少有分枝,上部叶长披针形,绿色,不向下反折。
北柴胡：叶较薄,无白色软骨质边缘。根通常有分支或不分枝,表面通常淡黄色。茎单一或丛生,枝条稍呈之字形弯曲。小总苞片较厚,三脉,边缘不透明；小伞形花序多而小。
1.2.2竹叶柴胡与北柴胡根的显微特征
竹叶柴胡根(2～3mm)横切面木栓细胞3-5列,皮层狭窄。韧皮部较发达,薄壁细胞35-45列。形成层成环,木质部宽广,约占直径的2/3,导管切向排列,直径10-50μ m,木纤维束和木薄壁细胞在木质部中部排列成续断的环状,木质部外侧无木纤维束和木薄壁细胞。
北柴胡根(2-3mm)横切面木栓细胞5-8列。皮层细胞8-15列,细胞呈切向延长,皮层内侧油管散在排列,油管由8-12个细胞组成,直径20～45μ m,内含淡黄色油状物。韧皮部宽广,约占直径的1/3,内含散在油管。形成层成环。木质部宽广,导管切向排列,多排列成行,直径10～53μ m,木纤维束和木薄壁细胞较多,排列成1至多个续断的环状,射线3～5列。
1.2.3竹叶柴胡与北柴胡的理化及薄层色谱鉴别
各柴胡样本均产生泡沫,山西省运城县种植的北柴胡和湖北省保康县种植的竹叶柴胡产生的泡沫少于野生竹叶柴胡,放置后消失较快,但在鄂西北地区竹山、竹溪、郧县等地作柴胡使用的柴胡伪品石竹科植物蝇子草根可产生非常明显的泡沫,且保持长时间不消失。
各药材样品与对照药材的主要斑点相同,在对照品SSa、SSd相应的位置均显相同颜色的斑点或荧光斑点。说明竹叶柴胡、北柴胡以及三岛柴胡的主要成分相近,利用TLC可以鉴别柴胡,但不能有效区分不同品种的柴胡。
1.3鄂西北地区竹叶柴胡和北柴胡的遗传多样性ISSR研究
7条ISSR引物共扩增出115个ISSR条带,其中多态性带59条,多态性比率高达51.4%；12个竹叶柴胡共扩增出81条ISSR条带,多态性带42条,多态性比率高达51.9%。12个竹叶柴胡样品聚为一类,另两个北柴胡样品聚为另一类,两者的相似系数为0.55,说明竹叶柴胡与北柴胡有较近的亲缘关系。
1.4鄂西北地区竹叶柴胡野生变家种关键技术研究
1.4.1竹叶柴胡种子萌发特性研究
不同浓度竹叶柴胡外种皮浸提液处理的柴胡种子萌发率、萌发势与对照组比较有差异显著,且随浓度升高抑制作用明显增强,表明种皮中存在抑制种子萌发抑制物质；竹叶柴胡种子培养床的选择以滤纸最优,40℃温水浸种能使竹叶柴胡的发芽率提高到64.8%；发芽介质对竹叶柴胡的种子无显著性的影响,但是30℃以上的萌发温度使种子发芽率显著降低。
1.4.2竹叶柴胡最佳采收期研究
竹叶柴胡SSa、SSd的含量从5月至7月呈递增趋势,到7月含量达最高,然后持续下降,到10月后地上部分枯萎进入休眠期含量达到最低,说明从SSa、SSd分析,本地区柴胡的采收应该在其地上部分枯萎前进行。
2.鄂西北地区竹叶柴胡与北柴胡的质量评价比较
2.1鄂西北地区竹叶柴胡与北柴胡的药用成分比较研究
鄂西北地区竹叶柴胡野生品、种植品的SSt含量相近,低于河南、甘肃的野生柴胡,但明显高于山西省万荣县的北柴胡和湖北省南漳县的三岛柴胡,结果表明鄂西北地区竹叶柴胡质量较好。
河南省内乡县和湖北省秭归县的竹叶柴胡总皂苷含量较高,湖北省房县野人谷镇野生竹叶柴胡、保康县栽培品竹叶柴胡和山西省运城县的北柴胡总皂苷含量相近,说明生长环境对柴胡有效成分含量有明显影响。
鄂西北地区种植的竹叶柴胡与野生竹叶柴胡相比,多糖含量稍低,竹叶柴胡地上部分也含有一定量的多糖成分,湖北省南漳县种植的三岛柴胡和山西省运城县种植的北柴胡中的多糖含量较高,结果显示不同品种、不同生长条件和部位柴胡中多糖含量有较大差异。
鄂西北地区竹叶柴胡野生品根中黄酮含量高于种植品,鄂西北地区竹叶柴胡地上部分总黄酮含量明显高于地下部分；湖北省房县野人谷镇野生柴胡根的黄酮含量高于山西省运城县、甘肃及湖北省南漳县的种植品；结果显示地上部分总黄酮含量明显高于柴胡根,说明柴胡的使用应根据其所发挥的药理作用选用合适的药用部位。
竹叶柴胡中挥发油含量高于北柴胡和三岛柴胡；鄂西北地区种植的柴胡挥发油含量高于野生柴胡挥发油含量,说明柴胡药材适于实施野生变家种。
炮制后,挥发油的含量明显高于生柴胡,挥发油的含量高低依次为蜜柴胡>酒柴胡>醋柴胡>生柴胡；柴胡总皂苷含量高低依次为蜜柴胡>生柴胡>醋柴胡>酒柴胡,即柴胡蜜制品皂苷含量高于生品,但酒制品和醋制品中总皂苷含量低于生品,运用SPSS16.0进行单因素方差分析表明,生柴胡与醋制品、酒制品总皂苷含量有显著性差异(P<0.05),其它各组之间均有极显著差异(P<0.01),说明柴胡炮制前后性味功效的变化是由于化学成分发生了转化,由此引起其药理作用的转变符合中药炮制理论。
不同柴胡品种的化学成分指纹图谱共检出8个分离度良好的共有峰。北柴胡、小柴胡指纹图谱相似度相差较小,竹叶柴胡地上部分、三岛柴胡指纹图谱相似度相差很大。
根据质谱信息共分离鉴定了7个皂苷类化合物并作为柴胡药材的特征图谱峰,各个柴胡样本其特征峰的相对峰面积有显著差异,柴胡皂苷C、A、D的含量也有明显差别。
2.2鄂西北地区竹叶柴胡与北柴胡的药理活性比较研究
竹叶柴胡和北柴胡的水煎液均具有明显的解热作用,两者解热作用没有显著性差异(P>0.05)；醋酸扭体实验结果表明竹叶柴胡和北柴胡水煎液、醇提液均有镇痛作用,且两者没有显著性差异；热板法实验结果表明两种柴胡水煎液有镇痛作用且无明显差异(P>0.05),但醇提液无明显镇痛作用。
耳肿胀度实验结果表明竹叶柴胡和北柴胡水煎液均具有明显抗炎作用,两者抗炎作用没有显著性差异(P>0.05)；足肿胀实验结果表明竹叶柴胡高剂量组和北柴胡高低剂量组在2h、4h、6h时均有抗炎作用,且与北柴胡抗炎作用比较没有显著性差异；竹叶柴胡低剂量组、北柴胡高低剂量组ALT、AST与模型组比较有统计学差异(P<0.05)；各治疗组ALP与模型组比较没有统计学差异(P>0.05)；竹叶柴胡高低剂量组和北柴胡高低剂量组SOD与模型组比较有统计学差异；竹叶柴胡10.Og/kg和北柴胡10.0g/kg剂量组的MDA、GSH与模型组比较有统计学差异。病理切片及免疫组化结果显示,竹叶柴胡和北柴胡均有保肝作用,且两者没有显著差异。
[结论]
(1)鄂西北地区所产柴胡习称为“北柴胡”品种实际为竹叶柴胡的干燥根,是鄂西北地区的主流柴胡品种。
(2)鄂西北地区竹叶柴胡生物多样性和种质资源较为丰富,有利于竹叶柴胡资源开发利用；ISSR分子标记特征能显著区别竹叶柴胡与其近缘种北柴胡等,可作为分子特征进行物种鉴别。
(3)竹叶柴胡种子中含有发芽抑制物质和胚后熟是影响其种子发芽率的重要原因。
(4)建立了鄂西北地区竹叶柴胡的指纹图谱和质量评价体系,可以作为竹叶柴胡药材质量评价的依据,从而保证其临床用药安全、质量可控。
(5)测定了不同产地野生和种植竹叶柴胡根的有效成分,通过与其它柴胡品种和中国药典收录的北柴胡标准药材相比较,发现其有效成分含量相当于北柴胡样品,高于药典的所规定的柴胡含量标准；其降热、镇痛、抗炎、保肝活性作用也接近或超过北柴胡样品；ISSR分子标记实验证实竹叶柴胡与北柴胡是亲缘关系紧密联系的两个种。
综上所述,竹叶柴胡是优质柴胡的药用植物资源之一,与北柴胡有较近的亲缘关系和相似的药用品质。基于竹叶柴胡长期作为柴胡药材应用的历史,且是我国三大柴胡主流商品之一,而鄂西地区竹叶柴胡一直被作为优质“柴胡药材”销向全国中药材市场,同时柴胡属多种植物的根曾经是中国药典中明确规定的基源植物,本文建议《中国药典》应考虑重新将竹叶柴胡作为中药柴胡的基源植物,收录进入《中国药典》,以使这一宝贵资源得到充分利用,扩大柴胡药用品种,缓解柴胡市场紧张局面。
Objective
Study on the quality evaluation and germplasm resources of Bupleurum in Northwest of Hubei Province, provided experimental and theoretical basis for the development and utilization of the Bupleurum in Northwest of Hubei Province
Methods
1. Study on the resource of Bupleurum in Northwest of Hubei Province1.1Investigation on Bupleurum resources in northwest area of Hubei Province
Through investigate the resources of the wild and cultivated bupleurum in northwest area of Hubei Province, acquisited and producted twenty plant specimens of bupleurum in northwest of HuBei province, compared and identified the difference of the root, stem, leaf, flower, fruit, seed and plant, to establish the retrieval table of plant classification, provide the basis for the identi of plant properties.
1.2Identification of Bupleurum marginatum Wall. ex DC. in northwest area of Hubei Province
Compared the root and seed of Bupleurum marginatum Wall. ex DC., Bupleurum chinese DC. and Bupleurum Falcatum Linne.by paraffin method. Observed the microstructure of the roots and identif icated the microscopic characteristics. Identificated the species of Bupleurum margina tum Wall. ex DC. and Bupleurum chinese DC. by foam reaction and TLC.
1.3Study on the kinship of Bupleurum marginatum Wall. ex DC. in northwest of HuBei province by ISSR molecular marker technology Analysised genetic diversity of Bupleurum marginatum Wall. ex DC. in northwest of HuBei by ISSR molecular marker technology and established the genetic diversity fingerprint, analysis the main difference and genetic relationship of Bupleurum marginatum Wall. ex DC. in northwest of HuBei at molecular biology
1.4Study on the key technology of home-grown research of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province Research on the growth phenology of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province, formulated the quality standards of the Seed of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province; and observed the relationship between the temperature, culture media and seed germination rate. Study the best harvest time of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province
2. The quality evaluation of Bupleurum marginatum Wall. ex DC. in Northwest of Hubei Province
2.1Compared the Chemical compositions between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC, establish the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province
Detected the content of the SSa、SSd in Bupleurum by HPLC, Compared the content of the SSt between Bupleurum margina tum Wall. ex DC. and Bupleurum chinese DC.. Detected soluble polysaccharide and polysaccharides of Bupleurum through the Anthrone-sulfuric acid colorimetric,compared the total polysaccharide content. Measured the absorbance by visible spectrophotometry, calculated the content of total flavonoid. Measured the content of the volatile oil of the Bupleurum by UV-visible spectrophotometry, and determinated the ash. Detected the volatile oil and total saponins of the Bupleurum by UV-visible spectrophotometry,which were made with Honey wine vinegar before and after. Compared the active ingredient of Bupleurum by the different concoction methods. Established the fingerprint of Bupleurum margina tum Wall. ex DC. in northwest of Hubei province by HPLC. Isolated and identif icated the constituents of Bupleurum compounds by UPLC-MS, established the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in the northwest of Hubei province,and comparing the characteristic spectrum peaks relative peak area.
2.2Compared the pharmacological activity between between Bupleurum margina tum Wall. ex DC. in the northwest of Hubei province and Bupleurum chinense
The antipyretic effects of Bupleurum decoction were monitored at the2,4-dinitrophenol-induced rat pyrexia. The analgesic effect was verified by the test of HAc-induced writhing response in mice and Hot-Plate. The anti-inflammatory of Bupleurum decoction were monitored by the test of ear and foot-swelling in rats. Induced Liver injury in rats with peanut oil of tetrachloride nitrogen, detected ALT, AST, ALP of Serum, and SOD, MDA, GSH of liver tissue, observed the Histopathology and immunohistochemical indicators of liver tissue, compared the anti-inflammatory and hepatoprotective effects between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC..
Result
1. Study on resource of Bupleurum in Northwest of Hubei Province
1.1Investigation on Bupleurum resources in northwest area of Hubei Province
The result show that:the Bupleurum around the region,known as the Bupleurum chinense DC,had been identified as the dried roots of Bupleurum margina tum Wall. ex DC. of Umbelliferae, a small amount of Bupleurum had been identified as the dried roots of Silene fortunei Vis.
1.2Identification of Bupleurum marginatum Wall. ex DC. in northwest area of Hubei Province
The characteristic of Bupleurum marginatum Wall. ex DC. and Bupleurum chinense DC. as follows:Bupleurum margina tum Wall. ex Dc.: The small bracts narrow and small, lanceolate; Leaf blade thick, nearly leathery, margin conspicuously white-cartilaginous; Root thick, spindle, the surface usually brown or dark brown; Stem base distortion, few branched, upper Leaves long-lanceolate to linear, green. Bupleurum chinese DC.:Leaves thin, no margin conspicuously white-cartilaginous; Root stout, elongate, brown, woody, usually branched; Stem solitary or several, dichotomously much-branched bove, rod abends into zigzag. Small total bracts thick, three veins, margin not transparent; Small umbellules much and small. Under the ultraviolet visible light, the result of TLC showed that different kinds of bupleurum under different growth conditions have the same color spots in the same position at TLC chromatogram controled with control medicinal materials and Bupleurum saponins reference substance.
1.3Study on the kinship of Bupleuruw marginatum Wall. ex DC. in northwest of HuBei province by ISSR molecular marker technology The results showed that7ISSR primers amplified115ISSR stripes,12samples of Bupleurum margina tum Wall, ex DC. amplified81ISSR bands, there were42polymorphic bands, the rate of polymorphic was51.9%. Twenty samples of Bupleurum marginatum Wall. ex DC. were clustered into one group, two samples of Bupleurum chinese DC. were clustered into another group, the coefficient of similarity was0.55, with close phylogenetic relationship.
1.4Study on the key technology of home-grown research of Bupleurum margina tum Wall. ex DC. in northwest of Hubei province
The germination rate of Bupleurum marginatum Wall. ex DC. could increase to64.8%at40℃water temperatrue; the germination medium have no significant influence on the germination rate of Bupleurum margina tum Wall. ex DC., but the germination rate of Bupleurum margina tum Wall. ex DC. significantly descended above30℃. The content of the saikosaponin show an increasing trend from May to July, arrived the peak at july, then show an downward trend, meet the minimum at October, the result show that Bupleurum marginatum Wall, ex DC. should be harvested before it wither up.
2. The quality evaluation of Bupleurum marginatum Wall. ex DC. in Northwest of Hubei Province
2.1Compared the Chemical compositions between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC., establish the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province
The content of SSt are similar between wild and, less than wild bupleurum in Henan and Gansu, much higher than Bupleurum chinese DC. of Shanxi and Mishima Shibako of Hubei, the results show that the quality of Bupleurum marginatum Wall. ex DC. of the northwest of Hubei province is good.
Bupleurum marginatum Wall. ex DC. in the Neixiang County of Henan province and zhigui County of Hebei province have more total saponins, the content of total saponins is similar between Bupleurum chinese DC. in Yunchen County of Shanxi province and Bupleurum margina tum Wall. ex DC. in Fangxian and Baokang County of Hubei province, the results show that growth environment is the important factor for the content of total saponins.
The results show that the content of polysaccharide of the cultivated Bupleurum marginatum Wall. ex DC. in northwest of Hubei province is slightly lower than the wild Bupleurum, the content of polysaccharide of Bupleurum overground part also contains a certain amount of polysaccharide, polysaccharide content is high in the Mishima Shibako of Nanzhang County of Hubei province and cultivated Bupleurum of Yunchen County of Shanxi. the results show that the content of polysaccharide were great difference in the different growth conditions, different varieties and different position of Bupleurum.
The content of flavonoids in the root of wild Bupleurum marginatum Wall. ex DC. in northwest of Hubei is higher than the cultivated Bupleurum. aboveground parts is higher than is higher than that of underground parts.
The content of flavonoids of Bupleurum in the savage Valley town of Nanzhang County of Hubei province is higher than Yunchen County of Shanxi province, Ganshu province and Nanzhang County of Hubei province. The results showed that the content of total flavonoid of the ground part of Bupleurum was significantly higher than that of root, so as to choose the right medicinal parts according to the selection of appropriate pharmacological effects.
The content of volatile oil of Bupleurum marginaturn Wall, ex DC. is higher than Bupleurum Chinese DC. and Mishima Shibako, the content of volatile oil of Bupleurum margina turn Wall, ex DC. in northwest of Hubei province is higher than the wild Bupleurum. The results show that it is suitable for implementation of wild Bupleurum sibiricum redoute.
The content of volatile oil was higher after processing, from high to low is honey, wine, vinegar. The contents of total saponin of Bupleurum from high to low is honey, Radix. vinegar, wine. The single factor variance analysis by SPSS16.0showed that, Radix Bupleuri and vinegar products, The contents of total saponin of radix Bupleuri, vinegar products and wine products have significant difference (P<0.05), other groups had extremely significant difference (P<0.01). The results show that, before and after processing. the effectiveness of taste changed is due to the conversion of chemical composition changed, that is consistent with the theory of traditional Chinese medicine processing purpose. There were8good separation of common peak in fingerprints spectrum, the difference of the fingerprint similarity was small between Bupleurum Chinese DC. and Bupleurum scorzonerifolium willd. the difference of the fingerprint similarity was big between aboveground parts of Bupleurum marginatum Wall, ex DC. and Bupleurum falea tum L.
Separated and identified7saponins according to the information by Mass, and as the fingerprint peak of bupleurum, there are significant differences between the relative peak area of characteristic peak of each sample, the content of saikosaponin A, D are also different.
2.2Compared the pharmacological activity between between Bupleurum margina tum Wall. ex DC. in northwest of Hubei province and Bupleurum chinense
The result Show that the decoction of Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant antipyretic effect, and there were no significant difference between them. Both the decoction and alcohol extract of Bupleurum chinense DC. and Bupleurum marginatum Wall. ex DC. possessed the analgesic effect in the test of HAc-induced writhing response, and there were no significant differences. The decoction of Bupleurum chinense DC. and Bupleurum marginatum Wall. ex DC. all showed the analgesic effect in the test of Hot-Plate, and there were no significant differences, but alcohol extract of them had no significant analgesic effect.
Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant anti-inflammatory effect in the experiment of ear swelling,and there were no significant difference between them. Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant anti-inflammatory function at2h,4h,6h in the test of foot swelling experiment, and there were no significant difference between them; Bupleurum marginatum Wall. ex DC.5.0g/kg dose group and Bupleurum chinese DC.10.0g/kg dose groups had significant difference compared with model group at the test of ALT,AST. All the treatment group had no significant difference compared with model group at the test of ALP. The decoction of Bupleurum chinese DC. and Bupleurum marginatum Wall, ex DC. all groups had significant difference compared with model group at the test of SOD. Bupleurum marginatum Wall, ex DC.10. Og/kg dose group and Bupleurum chinese DC.10.0g/kg dose groups had significant difference compared with model group at the test of MDA,GSH. The result of the Histopathology and immunohistochemical indicators of liver tissue Showed that the decoction of Bupleurum chinese DC. and Bupleurum marginatum all showed significant hepatoprotective effect, and there were no significant difference between them.
Conclusion
(1) Bupleurum marginatum Wall, ex DC. was the main kind of bupleurum in northwest of HuBei province, bupleurum in northwest of HuBei province, known as the " Bupleurum chinense DC.", and sold all over the country were the dry root of bupleurum marginatum in fact.
(2) The biodiversity and germplasm resources of Bupleurum marginatum Wall, ex DC. in northwest of Hubei Province is abundant, it is conducive to the development of production of Bupleurum marginatum Wall. ex DC.. The ISSR molecules technology can significantly distinguish Bupleurum marginatum Wall. ex DC. from its sibling species, it also can be used as the molecular characteristics of the species identification.
(3) Seeds contained germination inhibitors and embryo after-ripening are the important factors to affect the germination rate of Bupleurum seeds.
(4) Establish the fingerprint spectrum and quality evaluation system of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province. The fingerprint of Bupleurum marginatum Wall. ex DC. can be used as the basis of the overall quality establish system. and establish the "identity card" for bupleurum, to ensure safety and controllable of clinical medication, provide theoretical basis for the modernization of traditional Chinese medicine.
(5) Analysis a large number of wild and cultivated Bupleurum marginatum Wall. ex DC., compared with other samples of bupleurum chinense DC, which is recorded in the Chinese Pharmacopoeia, founded that the content of the effective components were similar or more than the bupleurum chinense
DC., which meet the standard of the Chinese Pharmacopoeia, or higher than that. The activity effect of antipyretic, analgesic, anti-inflammatory, and hepatoprotective were also close to or more-than the bupleurum chinense DC..Furthely, it was confirmed by ISSR that Bupleurum marginatum Wall, ex DC. and bupleurum chinense DC. were two species separately,but the relationship of them was closely. The root of Bupleurum marginatum Wall, ex DC. was used as bupleurum chinense in the history for long time,which was one of the mainstream commodity of three major bupleurum, Bupleurum marginatum Wall. ex DC. in northwest of Hubei Province has been sold all the country considered as the high quality of bupleurum chinense DC.. On the basis of the above, bupleurum chinense DC. is one of the medicinal plant resources of the high quality of bupleurum. the root of Bupleurum was specified as the original plant in Chinese Pharmacopoeia, therefore, we suggested to restore the original plant status of Bupleurum marginatum Wall. ex DC. in Chinese Pharmacopoeia, or add the Bupleurum marginatum Wall. ex DC. into Pharmacopoeia independently, in order to make full use of this valuable resource.
通过对鄂西北地区所产柴胡的资源调查、品质评价以及与北柴胡的品质比较研究,为药用柴胡新资源的发现、开发与利用提供实验依据和理论基础。
[方法]
1.鄂西北地区柴胡的资源调查和品种鉴定研究
1.1鄂西北地区柴胡的资源调查
对鄂西北地区野生与栽培柴胡品种进行资源调查研究,采集并制作鄂西北地区原植物标本20余份,比较鉴别其根、茎、叶、花、果实、种子的异同,建立植物分类检索表。
1.2鄂西北地区竹叶柴胡品种鉴别研究
建立竹叶柴胡与北柴胡、三岛柴胡的药材及种子性状、显微、理化、TLC等特征鉴别方法。通过石蜡切片法观察竹叶柴胡、北柴胡和三岛柴胡根及种子的显微结构,并分别制作药材和种子的横切片进行显微鉴定；利用泡沫反应鉴别不同品种的柴胡；按照薄层色谱法,鉴别竹叶柴胡与北柴胡。
1.3鄂西北地区柴胡与北柴胡的遗传多样性ISSR分析
利用分子标记ISSR技术对鄂西北地区柴胡种群进行遗传多样性分析并建立其遗传多样性指纹图谱,从分子生物学的角度分析鄂西北地区竹叶柴胡与其他柴胡的主要差别及其亲缘关系。
1.4鄂西北地区竹叶柴胡野生变家种关键技术研究
通过对鄂西北地区柴胡生长的物候学观察,初步制定鄂西北地区竹叶柴胡种子的质量标准；徒手切片法观察竹叶柴胡种子特征,考察温度和不同培养介质对种子发芽率的影响；考察鄂西北地区竹叶柴胡的最佳采收期。
2.鄂西北地区竹叶柴胡与北柴胡的质量评价比较研究
2.1鄂西北地区竹叶柴胡与北柴胡的药用成分的比较分析
采用HPLC法测定柴胡药材中柴胡皂苷a(SSa)、柴胡皂苷d(SSd)的含量,比较鄂西北地区竹叶柴胡与北柴胡中柴胡总皂苷(SSt)的差异；采用蒽酮-硫酸比色法,测定柴胡中可溶性多糖和粗多糖含量,比较两者总多糖的含量差异；采用可见分光光度法测定总黄酮的含量；采用紫外分光光度法测定柴胡中挥发油含量,并进行醇溶性浸出物、总灰分、酸性不溶性灰分测定；使用紫外-可见分光光度法测量竹叶柴胡经蜜制、酒制、醋制前后挥发油和总皂苷的含量,比较鄂西北地区竹叶柴胡经不同方法炮制前后有效成分的含量；采用HPLC法比较分析不同产地的HPLC图谱,建立鄂西北地区竹叶柴胡HPLC指纹图谱；采用UPLC-MS联用技术,建立柴胡药材的特征图谱,鉴定分析柴胡中的主要成分,在此基础上对主要成分进行定量分析。
2.2鄂西北地区竹叶柴胡与北柴胡的药理活性比较研究
鄂西北地区竹叶柴胡与北柴胡的解热镇痛作用的比较：采用2,4-二硝基苯酚致热大鼠模型比较两种柴胡水煎液的解热作用；采用醋酸扭体实验及热板法比较两种柴胡水煎液及醇提液的镇痛作用。
鄂西北地区竹叶柴胡与北柴胡的抗炎、保肝作用的比较：采用二甲苯致耳壳肿胀及蛋清致足肿胀的小鼠模型比较两种柴胡水煎液的抗炎作用；采用四氯化氮的花生油溶液复制小鼠肝损伤模型,检测小鼠的血清生化指标ALT、AST、ALP;检测肝组织匀浆生化指标SOD、MDA、GSH,观察小鼠肝组织病理切片及免疫组化指标,比较鄂西北地区竹叶柴胡与北柴胡根的抗炎、保肝作用。
[结果]
1.鄂西北地区竹叶柴胡的资源调查和品种鉴定研究
1.1鄂西北地区竹叶柴胡的资源调查
本地区所产柴胡习称“北柴胡”,经鉴定多为伞形科植物竹叶柴胡Bupleurum. marginatum Wall. ex DC.的干燥根,少数为石竹科植物蝇子草Silene fortunei Vis.的干燥根。
1.2鄂西北地区竹叶柴胡的品种鉴别
1.2.1竹叶柴胡与北柴胡原植物性状鉴别特征
竹叶柴胡：小总苞片窄而小,披针形,叶较厚,近革质,有白色软骨质边缘。根粗厚,纺锤形,表面通常深红棕色或棕褐色。茎基部扭曲,少有分枝,上部叶长披针形,绿色,不向下反折。
北柴胡：叶较薄,无白色软骨质边缘。根通常有分支或不分枝,表面通常淡黄色。茎单一或丛生,枝条稍呈之字形弯曲。小总苞片较厚,三脉,边缘不透明；小伞形花序多而小。
1.2.2竹叶柴胡与北柴胡根的显微特征
竹叶柴胡根(2～3mm)横切面木栓细胞3-5列,皮层狭窄。韧皮部较发达,薄壁细胞35-45列。形成层成环,木质部宽广,约占直径的2/3,导管切向排列,直径10-50μ m,木纤维束和木薄壁细胞在木质部中部排列成续断的环状,木质部外侧无木纤维束和木薄壁细胞。
北柴胡根(2-3mm)横切面木栓细胞5-8列。皮层细胞8-15列,细胞呈切向延长,皮层内侧油管散在排列,油管由8-12个细胞组成,直径20～45μ m,内含淡黄色油状物。韧皮部宽广,约占直径的1/3,内含散在油管。形成层成环。木质部宽广,导管切向排列,多排列成行,直径10～53μ m,木纤维束和木薄壁细胞较多,排列成1至多个续断的环状,射线3～5列。
1.2.3竹叶柴胡与北柴胡的理化及薄层色谱鉴别
各柴胡样本均产生泡沫,山西省运城县种植的北柴胡和湖北省保康县种植的竹叶柴胡产生的泡沫少于野生竹叶柴胡,放置后消失较快,但在鄂西北地区竹山、竹溪、郧县等地作柴胡使用的柴胡伪品石竹科植物蝇子草根可产生非常明显的泡沫,且保持长时间不消失。
各药材样品与对照药材的主要斑点相同,在对照品SSa、SSd相应的位置均显相同颜色的斑点或荧光斑点。说明竹叶柴胡、北柴胡以及三岛柴胡的主要成分相近,利用TLC可以鉴别柴胡,但不能有效区分不同品种的柴胡。
1.3鄂西北地区竹叶柴胡和北柴胡的遗传多样性ISSR研究
7条ISSR引物共扩增出115个ISSR条带,其中多态性带59条,多态性比率高达51.4%；12个竹叶柴胡共扩增出81条ISSR条带,多态性带42条,多态性比率高达51.9%。12个竹叶柴胡样品聚为一类,另两个北柴胡样品聚为另一类,两者的相似系数为0.55,说明竹叶柴胡与北柴胡有较近的亲缘关系。
1.4鄂西北地区竹叶柴胡野生变家种关键技术研究
1.4.1竹叶柴胡种子萌发特性研究
不同浓度竹叶柴胡外种皮浸提液处理的柴胡种子萌发率、萌发势与对照组比较有差异显著,且随浓度升高抑制作用明显增强,表明种皮中存在抑制种子萌发抑制物质；竹叶柴胡种子培养床的选择以滤纸最优,40℃温水浸种能使竹叶柴胡的发芽率提高到64.8%；发芽介质对竹叶柴胡的种子无显著性的影响,但是30℃以上的萌发温度使种子发芽率显著降低。
1.4.2竹叶柴胡最佳采收期研究
竹叶柴胡SSa、SSd的含量从5月至7月呈递增趋势,到7月含量达最高,然后持续下降,到10月后地上部分枯萎进入休眠期含量达到最低,说明从SSa、SSd分析,本地区柴胡的采收应该在其地上部分枯萎前进行。
2.鄂西北地区竹叶柴胡与北柴胡的质量评价比较
2.1鄂西北地区竹叶柴胡与北柴胡的药用成分比较研究
鄂西北地区竹叶柴胡野生品、种植品的SSt含量相近,低于河南、甘肃的野生柴胡,但明显高于山西省万荣县的北柴胡和湖北省南漳县的三岛柴胡,结果表明鄂西北地区竹叶柴胡质量较好。
河南省内乡县和湖北省秭归县的竹叶柴胡总皂苷含量较高,湖北省房县野人谷镇野生竹叶柴胡、保康县栽培品竹叶柴胡和山西省运城县的北柴胡总皂苷含量相近,说明生长环境对柴胡有效成分含量有明显影响。
鄂西北地区种植的竹叶柴胡与野生竹叶柴胡相比,多糖含量稍低,竹叶柴胡地上部分也含有一定量的多糖成分,湖北省南漳县种植的三岛柴胡和山西省运城县种植的北柴胡中的多糖含量较高,结果显示不同品种、不同生长条件和部位柴胡中多糖含量有较大差异。
鄂西北地区竹叶柴胡野生品根中黄酮含量高于种植品,鄂西北地区竹叶柴胡地上部分总黄酮含量明显高于地下部分；湖北省房县野人谷镇野生柴胡根的黄酮含量高于山西省运城县、甘肃及湖北省南漳县的种植品；结果显示地上部分总黄酮含量明显高于柴胡根,说明柴胡的使用应根据其所发挥的药理作用选用合适的药用部位。
竹叶柴胡中挥发油含量高于北柴胡和三岛柴胡；鄂西北地区种植的柴胡挥发油含量高于野生柴胡挥发油含量,说明柴胡药材适于实施野生变家种。
炮制后,挥发油的含量明显高于生柴胡,挥发油的含量高低依次为蜜柴胡>酒柴胡>醋柴胡>生柴胡；柴胡总皂苷含量高低依次为蜜柴胡>生柴胡>醋柴胡>酒柴胡,即柴胡蜜制品皂苷含量高于生品,但酒制品和醋制品中总皂苷含量低于生品,运用SPSS16.0进行单因素方差分析表明,生柴胡与醋制品、酒制品总皂苷含量有显著性差异(P<0.05),其它各组之间均有极显著差异(P<0.01),说明柴胡炮制前后性味功效的变化是由于化学成分发生了转化,由此引起其药理作用的转变符合中药炮制理论。
不同柴胡品种的化学成分指纹图谱共检出8个分离度良好的共有峰。北柴胡、小柴胡指纹图谱相似度相差较小,竹叶柴胡地上部分、三岛柴胡指纹图谱相似度相差很大。
根据质谱信息共分离鉴定了7个皂苷类化合物并作为柴胡药材的特征图谱峰,各个柴胡样本其特征峰的相对峰面积有显著差异,柴胡皂苷C、A、D的含量也有明显差别。
2.2鄂西北地区竹叶柴胡与北柴胡的药理活性比较研究
竹叶柴胡和北柴胡的水煎液均具有明显的解热作用,两者解热作用没有显著性差异(P>0.05)；醋酸扭体实验结果表明竹叶柴胡和北柴胡水煎液、醇提液均有镇痛作用,且两者没有显著性差异；热板法实验结果表明两种柴胡水煎液有镇痛作用且无明显差异(P>0.05),但醇提液无明显镇痛作用。
耳肿胀度实验结果表明竹叶柴胡和北柴胡水煎液均具有明显抗炎作用,两者抗炎作用没有显著性差异(P>0.05)；足肿胀实验结果表明竹叶柴胡高剂量组和北柴胡高低剂量组在2h、4h、6h时均有抗炎作用,且与北柴胡抗炎作用比较没有显著性差异；竹叶柴胡低剂量组、北柴胡高低剂量组ALT、AST与模型组比较有统计学差异(P<0.05)；各治疗组ALP与模型组比较没有统计学差异(P>0.05)；竹叶柴胡高低剂量组和北柴胡高低剂量组SOD与模型组比较有统计学差异；竹叶柴胡10.Og/kg和北柴胡10.0g/kg剂量组的MDA、GSH与模型组比较有统计学差异。病理切片及免疫组化结果显示,竹叶柴胡和北柴胡均有保肝作用,且两者没有显著差异。
[结论]
(1)鄂西北地区所产柴胡习称为“北柴胡”品种实际为竹叶柴胡的干燥根,是鄂西北地区的主流柴胡品种。
(2)鄂西北地区竹叶柴胡生物多样性和种质资源较为丰富,有利于竹叶柴胡资源开发利用；ISSR分子标记特征能显著区别竹叶柴胡与其近缘种北柴胡等,可作为分子特征进行物种鉴别。
(3)竹叶柴胡种子中含有发芽抑制物质和胚后熟是影响其种子发芽率的重要原因。
(4)建立了鄂西北地区竹叶柴胡的指纹图谱和质量评价体系,可以作为竹叶柴胡药材质量评价的依据,从而保证其临床用药安全、质量可控。
(5)测定了不同产地野生和种植竹叶柴胡根的有效成分,通过与其它柴胡品种和中国药典收录的北柴胡标准药材相比较,发现其有效成分含量相当于北柴胡样品,高于药典的所规定的柴胡含量标准；其降热、镇痛、抗炎、保肝活性作用也接近或超过北柴胡样品；ISSR分子标记实验证实竹叶柴胡与北柴胡是亲缘关系紧密联系的两个种。
综上所述,竹叶柴胡是优质柴胡的药用植物资源之一,与北柴胡有较近的亲缘关系和相似的药用品质。基于竹叶柴胡长期作为柴胡药材应用的历史,且是我国三大柴胡主流商品之一,而鄂西地区竹叶柴胡一直被作为优质“柴胡药材”销向全国中药材市场,同时柴胡属多种植物的根曾经是中国药典中明确规定的基源植物,本文建议《中国药典》应考虑重新将竹叶柴胡作为中药柴胡的基源植物,收录进入《中国药典》,以使这一宝贵资源得到充分利用,扩大柴胡药用品种,缓解柴胡市场紧张局面。
Objective
Study on the quality evaluation and germplasm resources of Bupleurum in Northwest of Hubei Province, provided experimental and theoretical basis for the development and utilization of the Bupleurum in Northwest of Hubei Province
Methods
1. Study on the resource of Bupleurum in Northwest of Hubei Province1.1Investigation on Bupleurum resources in northwest area of Hubei Province
Through investigate the resources of the wild and cultivated bupleurum in northwest area of Hubei Province, acquisited and producted twenty plant specimens of bupleurum in northwest of HuBei province, compared and identified the difference of the root, stem, leaf, flower, fruit, seed and plant, to establish the retrieval table of plant classification, provide the basis for the identi of plant properties.
1.2Identification of Bupleurum marginatum Wall. ex DC. in northwest area of Hubei Province
Compared the root and seed of Bupleurum marginatum Wall. ex DC., Bupleurum chinese DC. and Bupleurum Falcatum Linne.by paraffin method. Observed the microstructure of the roots and identif icated the microscopic characteristics. Identificated the species of Bupleurum margina tum Wall. ex DC. and Bupleurum chinese DC. by foam reaction and TLC.
1.3Study on the kinship of Bupleurum marginatum Wall. ex DC. in northwest of HuBei province by ISSR molecular marker technology Analysised genetic diversity of Bupleurum marginatum Wall. ex DC. in northwest of HuBei by ISSR molecular marker technology and established the genetic diversity fingerprint, analysis the main difference and genetic relationship of Bupleurum marginatum Wall. ex DC. in northwest of HuBei at molecular biology
1.4Study on the key technology of home-grown research of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province Research on the growth phenology of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province, formulated the quality standards of the Seed of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province; and observed the relationship between the temperature, culture media and seed germination rate. Study the best harvest time of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province
2. The quality evaluation of Bupleurum marginatum Wall. ex DC. in Northwest of Hubei Province
2.1Compared the Chemical compositions between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC, establish the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province
Detected the content of the SSa、SSd in Bupleurum by HPLC, Compared the content of the SSt between Bupleurum margina tum Wall. ex DC. and Bupleurum chinese DC.. Detected soluble polysaccharide and polysaccharides of Bupleurum through the Anthrone-sulfuric acid colorimetric,compared the total polysaccharide content. Measured the absorbance by visible spectrophotometry, calculated the content of total flavonoid. Measured the content of the volatile oil of the Bupleurum by UV-visible spectrophotometry, and determinated the ash. Detected the volatile oil and total saponins of the Bupleurum by UV-visible spectrophotometry,which were made with Honey wine vinegar before and after. Compared the active ingredient of Bupleurum by the different concoction methods. Established the fingerprint of Bupleurum margina tum Wall. ex DC. in northwest of Hubei province by HPLC. Isolated and identif icated the constituents of Bupleurum compounds by UPLC-MS, established the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in the northwest of Hubei province,and comparing the characteristic spectrum peaks relative peak area.
2.2Compared the pharmacological activity between between Bupleurum margina tum Wall. ex DC. in the northwest of Hubei province and Bupleurum chinense
The antipyretic effects of Bupleurum decoction were monitored at the2,4-dinitrophenol-induced rat pyrexia. The analgesic effect was verified by the test of HAc-induced writhing response in mice and Hot-Plate. The anti-inflammatory of Bupleurum decoction were monitored by the test of ear and foot-swelling in rats. Induced Liver injury in rats with peanut oil of tetrachloride nitrogen, detected ALT, AST, ALP of Serum, and SOD, MDA, GSH of liver tissue, observed the Histopathology and immunohistochemical indicators of liver tissue, compared the anti-inflammatory and hepatoprotective effects between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC..
Result
1. Study on resource of Bupleurum in Northwest of Hubei Province
1.1Investigation on Bupleurum resources in northwest area of Hubei Province
The result show that:the Bupleurum around the region,known as the Bupleurum chinense DC,had been identified as the dried roots of Bupleurum margina tum Wall. ex DC. of Umbelliferae, a small amount of Bupleurum had been identified as the dried roots of Silene fortunei Vis.
1.2Identification of Bupleurum marginatum Wall. ex DC. in northwest area of Hubei Province
The characteristic of Bupleurum marginatum Wall. ex DC. and Bupleurum chinense DC. as follows:Bupleurum margina tum Wall. ex Dc.: The small bracts narrow and small, lanceolate; Leaf blade thick, nearly leathery, margin conspicuously white-cartilaginous; Root thick, spindle, the surface usually brown or dark brown; Stem base distortion, few branched, upper Leaves long-lanceolate to linear, green. Bupleurum chinese DC.:Leaves thin, no margin conspicuously white-cartilaginous; Root stout, elongate, brown, woody, usually branched; Stem solitary or several, dichotomously much-branched bove, rod abends into zigzag. Small total bracts thick, three veins, margin not transparent; Small umbellules much and small. Under the ultraviolet visible light, the result of TLC showed that different kinds of bupleurum under different growth conditions have the same color spots in the same position at TLC chromatogram controled with control medicinal materials and Bupleurum saponins reference substance.
1.3Study on the kinship of Bupleuruw marginatum Wall. ex DC. in northwest of HuBei province by ISSR molecular marker technology The results showed that7ISSR primers amplified115ISSR stripes,12samples of Bupleurum margina tum Wall, ex DC. amplified81ISSR bands, there were42polymorphic bands, the rate of polymorphic was51.9%. Twenty samples of Bupleurum marginatum Wall. ex DC. were clustered into one group, two samples of Bupleurum chinese DC. were clustered into another group, the coefficient of similarity was0.55, with close phylogenetic relationship.
1.4Study on the key technology of home-grown research of Bupleurum margina tum Wall. ex DC. in northwest of Hubei province
The germination rate of Bupleurum marginatum Wall. ex DC. could increase to64.8%at40℃water temperatrue; the germination medium have no significant influence on the germination rate of Bupleurum margina tum Wall. ex DC., but the germination rate of Bupleurum margina tum Wall. ex DC. significantly descended above30℃. The content of the saikosaponin show an increasing trend from May to July, arrived the peak at july, then show an downward trend, meet the minimum at October, the result show that Bupleurum marginatum Wall, ex DC. should be harvested before it wither up.
2. The quality evaluation of Bupleurum marginatum Wall. ex DC. in Northwest of Hubei Province
2.1Compared the Chemical compositions between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC., establish the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province
The content of SSt are similar between wild and, less than wild bupleurum in Henan and Gansu, much higher than Bupleurum chinese DC. of Shanxi and Mishima Shibako of Hubei, the results show that the quality of Bupleurum marginatum Wall. ex DC. of the northwest of Hubei province is good.
Bupleurum marginatum Wall. ex DC. in the Neixiang County of Henan province and zhigui County of Hebei province have more total saponins, the content of total saponins is similar between Bupleurum chinese DC. in Yunchen County of Shanxi province and Bupleurum margina tum Wall. ex DC. in Fangxian and Baokang County of Hubei province, the results show that growth environment is the important factor for the content of total saponins.
The results show that the content of polysaccharide of the cultivated Bupleurum marginatum Wall. ex DC. in northwest of Hubei province is slightly lower than the wild Bupleurum, the content of polysaccharide of Bupleurum overground part also contains a certain amount of polysaccharide, polysaccharide content is high in the Mishima Shibako of Nanzhang County of Hubei province and cultivated Bupleurum of Yunchen County of Shanxi. the results show that the content of polysaccharide were great difference in the different growth conditions, different varieties and different position of Bupleurum.
The content of flavonoids in the root of wild Bupleurum marginatum Wall. ex DC. in northwest of Hubei is higher than the cultivated Bupleurum. aboveground parts is higher than is higher than that of underground parts.
The content of flavonoids of Bupleurum in the savage Valley town of Nanzhang County of Hubei province is higher than Yunchen County of Shanxi province, Ganshu province and Nanzhang County of Hubei province. The results showed that the content of total flavonoid of the ground part of Bupleurum was significantly higher than that of root, so as to choose the right medicinal parts according to the selection of appropriate pharmacological effects.
The content of volatile oil of Bupleurum marginaturn Wall, ex DC. is higher than Bupleurum Chinese DC. and Mishima Shibako, the content of volatile oil of Bupleurum margina turn Wall, ex DC. in northwest of Hubei province is higher than the wild Bupleurum. The results show that it is suitable for implementation of wild Bupleurum sibiricum redoute.
The content of volatile oil was higher after processing, from high to low is honey, wine, vinegar. The contents of total saponin of Bupleurum from high to low is honey, Radix. vinegar, wine. The single factor variance analysis by SPSS16.0showed that, Radix Bupleuri and vinegar products, The contents of total saponin of radix Bupleuri, vinegar products and wine products have significant difference (P<0.05), other groups had extremely significant difference (P<0.01). The results show that, before and after processing. the effectiveness of taste changed is due to the conversion of chemical composition changed, that is consistent with the theory of traditional Chinese medicine processing purpose. There were8good separation of common peak in fingerprints spectrum, the difference of the fingerprint similarity was small between Bupleurum Chinese DC. and Bupleurum scorzonerifolium willd. the difference of the fingerprint similarity was big between aboveground parts of Bupleurum marginatum Wall, ex DC. and Bupleurum falea tum L.
Separated and identified7saponins according to the information by Mass, and as the fingerprint peak of bupleurum, there are significant differences between the relative peak area of characteristic peak of each sample, the content of saikosaponin A, D are also different.
2.2Compared the pharmacological activity between between Bupleurum margina tum Wall. ex DC. in northwest of Hubei province and Bupleurum chinense
The result Show that the decoction of Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant antipyretic effect, and there were no significant difference between them. Both the decoction and alcohol extract of Bupleurum chinense DC. and Bupleurum marginatum Wall. ex DC. possessed the analgesic effect in the test of HAc-induced writhing response, and there were no significant differences. The decoction of Bupleurum chinense DC. and Bupleurum marginatum Wall. ex DC. all showed the analgesic effect in the test of Hot-Plate, and there were no significant differences, but alcohol extract of them had no significant analgesic effect.
Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant anti-inflammatory effect in the experiment of ear swelling,and there were no significant difference between them. Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant anti-inflammatory function at2h,4h,6h in the test of foot swelling experiment, and there were no significant difference between them; Bupleurum marginatum Wall. ex DC.5.0g/kg dose group and Bupleurum chinese DC.10.0g/kg dose groups had significant difference compared with model group at the test of ALT,AST. All the treatment group had no significant difference compared with model group at the test of ALP. The decoction of Bupleurum chinese DC. and Bupleurum marginatum Wall, ex DC. all groups had significant difference compared with model group at the test of SOD. Bupleurum marginatum Wall, ex DC.10. Og/kg dose group and Bupleurum chinese DC.10.0g/kg dose groups had significant difference compared with model group at the test of MDA,GSH. The result of the Histopathology and immunohistochemical indicators of liver tissue Showed that the decoction of Bupleurum chinese DC. and Bupleurum marginatum all showed significant hepatoprotective effect, and there were no significant difference between them.
Conclusion
(1) Bupleurum marginatum Wall, ex DC. was the main kind of bupleurum in northwest of HuBei province, bupleurum in northwest of HuBei province, known as the " Bupleurum chinense DC.", and sold all over the country were the dry root of bupleurum marginatum in fact.
(2) The biodiversity and germplasm resources of Bupleurum marginatum Wall, ex DC. in northwest of Hubei Province is abundant, it is conducive to the development of production of Bupleurum marginatum Wall. ex DC.. The ISSR molecules technology can significantly distinguish Bupleurum marginatum Wall. ex DC. from its sibling species, it also can be used as the molecular characteristics of the species identification.
(3) Seeds contained germination inhibitors and embryo after-ripening are the important factors to affect the germination rate of Bupleurum seeds.
(4) Establish the fingerprint spectrum and quality evaluation system of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province. The fingerprint of Bupleurum marginatum Wall. ex DC. can be used as the basis of the overall quality establish system. and establish the "identity card" for bupleurum, to ensure safety and controllable of clinical medication, provide theoretical basis for the modernization of traditional Chinese medicine.
(5) Analysis a large number of wild and cultivated Bupleurum marginatum Wall. ex DC., compared with other samples of bupleurum chinense DC, which is recorded in the Chinese Pharmacopoeia, founded that the content of the effective components were similar or more than the bupleurum chinense
DC., which meet the standard of the Chinese Pharmacopoeia, or higher than that. The activity effect of antipyretic, analgesic, anti-inflammatory, and hepatoprotective were also close to or more-than the bupleurum chinense DC..Furthely, it was confirmed by ISSR that Bupleurum marginatum Wall, ex DC. and bupleurum chinense DC. were two species separately,but the relationship of them was closely. The root of Bupleurum marginatum Wall, ex DC. was used as bupleurum chinense in the history for long time,which was one of the mainstream commodity of three major bupleurum, Bupleurum marginatum Wall. ex DC. in northwest of Hubei Province has been sold all the country considered as the high quality of bupleurum chinense DC.. On the basis of the above, bupleurum chinense DC. is one of the medicinal plant resources of the high quality of bupleurum. the root of Bupleurum was specified as the original plant in Chinese Pharmacopoeia, therefore, we suggested to restore the original plant status of Bupleurum marginatum Wall. ex DC. in Chinese Pharmacopoeia, or add the Bupleurum marginatum Wall. ex DC. into Pharmacopoeia independently, in order to make full use of this valuable resource.
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