【作者】 廖婉；

【导师】 傅超美；

【作者基本信息】 成都中医药大学 ， 中药学， 2015， 博士

【摘要】 莪术为姜科植物蓬莪术Curcuma phaeocaulis Val.、广西莪术Curcuma kwangsiensS.G Lee et C.F.Liang 和温郁金 Curcuma wenyujin Y.H.Chen et C.Ling的干燥根茎,收载于现行版《中国药典》,具有行气破血、消积止痛之功效。蓬莪术作为川产道地药材的代表性品种之一,其生品及醋制品均是中医临床常用的破血消癥要药。目的:道地性是中药独有的特色,也是评价药材质量的重要标准。本论文以川产道地药材蓬莪术为主要研究对象,比较不同道地产区的共51批次的3种姜黄属药材的化学成分差异,开展药材挥发油类化合物(有机成分)的定性、定量研究及微量元素(无机成分)与道地性的相关性研究,寻找蓬莪术的道地性特征成分,以期为道地药材的道地性评价研究提供一种新的研究方法及模式。中药“随方炮制”的理论和经验技术已有上千年的历史。炮制工艺亦是中药独有的特色,醋制法是中药炮制的常用方法之一,多用于疏肝解郁、散瘀止痛、攻下逐水类中药的炮制。莪术醋制后既能增强“破积消坚,去积聚癖块”之效,又可缓其峻猛药性。本论文通过研究蓬莪术醋制前后化学成分、肿瘤细胞毒活性、抑制模式生物斑马鱼血管生成等的异同,进一步阐释蓬莪术醋制机理,证明传统醋制理论的科学性,为蓬莪术及其炮制品的合理应用打下坚实的基础。全文通过开展川产道地药材蓬莪术道地性特征成分及醋制机理的研究,为今后川产道地药材蓬莪术的临床合理应用及综合开发利用提供科学依据。方法与结果:1.姜黄属中药中挥发油类化合物的定性、定量研究:挥发油被认为是莪术的主要药理活性成分,也是其发挥临床疗效的重要物质基础。采用GC-MS-AMDIS定性、定量气相色谱-质谱联用分析技术,通过NIST11.L标准质谱库检索并结合程序升温保留指数PTRI辅助定性,对比分析鉴别了现行版《中国药典》收载的蓬莪术、广西莪术、温郁金、姜黄、印尼姜黄、黄丝郁金、绿丝郁金总挥发油的含量及主要有效成分,从莪术、姜黄、郁金挥发油中一共鉴定了约133个成分,占总成分的80%以上,比较化合物种类和相对含量的变化以及特征性成分含量。结果发现不同来源姜黄属类药材挥发油成分种类和含量存在不同程度的差异。在道地产区四川种植的蓬莪术药材挥发油中,共鉴定出36种化合物,占挥发油总含量的百分比为98.37%,且主要特征性成分的相对百分含量显著高于其他产区。其中2,3,5,6-四甲基吡嗪、萜品油烯、D大牻牛儿烯、β-愈创木烯、莪术醇、匙叶桉油烯醇是蓬莪术中鉴定出的独有成分。并采用GC-MS多成分定量技术同时测定了蓬莪术中的特征性成分吉马酮、β-榄香烯、莪术醇和新莪术二酮的含量。不同产地莪术挥发油中4种特征性成分含量差异较大,β-榄香烯和吉马酮的含量普遍高于莪术醇和新莪术二酮。产于四川的蓬莪术挥发油中β-榄香烯和吉马酮含量明显高于其他产地的莪术。蓬莪术与温郁金挥发油主要成分种类及相对含量有较大差异,与广西莪术具有更高的相似性。2.姜黄属中药重金属元素与道地性的相关性研究:采用ICP-MS技术研究不同产地、不同基原的莪术、姜黄和郁金药材中微量元素含量的差异以及传统水煎法对重金属元素的影响。聚类分析数据显示,具有相同基原,不同入药部位的3种姜黄属药材能明显的区分开,同一产地的药材亦可较好地聚为一类;不同产地、不同基原药材中各重金属元素含量均存在Cu>Pb>Cd>As>Hg的趋势,说明不同品种对重金属元素选择性富集能力不同,产地环境对其重金属元素富集具有显著影响;3种不同基原的莪术药材,蓬莪术与广西莪术虽然产地不同,但具有更相似的特征,对于重金属元素的富集也更为相似。元素溶出率显示,中药材重金属在传统水煎过程中不易溶出,且经过煎煮各元素含量发生较大改变,溶出的元素含量存在As>Pb>Hg>Cu>Cd的趋势,表明Cu元素虽然在原饮片中含量远大于其他元素,但溶出率低。3.蓬莪术醋制前后挥发油类化合物的定性、定量研究:采用GC-MS技术对采集于四川道地产区的10批蓬莪术及其醋制品的挥发油进行比较分析,构建蓬莪术醋制前后挥发油的GC-MS特征指纹图谱。从蓬莪术生品挥发油中共鉴定出26个共有化合物,占挥发油总含量的百分比为71.72%,从对应的醋制品挥发油中共鉴定出24种共有化合物,占挥发油总含量的百分比为81.5%。两者共有成分22种,占其已鉴定化合物种类的78.57%,主要为萜类及倍半萜衍生物。醋制后新增γ-芹子烯和荜澄茄油烯等成分,萜品油烯、δ-榄香烯、吉马烯和γ-荜澄茄烯等成分消失,且各成分相对百分含量有增有减,由此可推测这是蓬莪术醋制后破血消癥之效增强的物质基础。并采用GC-MS多成分定量技术测定蓬莪术醋制品4个特征性成分β-榄香烯、莪术醇、吉马酮和新莪术二酮的含量,结果显示不同产地莪术醋制之后挥发油中主要特征性成分β-榄香烯、吉马酮、新莪术二酮含量均增加,而莪术醇含量降低。表明醋制对蓬莪术挥发油特征性成分含量影响较大。炮制后挥发油中化合物种类、相对百分含量及特征性成分的含量均产生较大变化,可作为阐释蓬莪术醋制机理的重要指标之一。4.蓬莪术醋制前后微量元素对比研究:采用ICP-MS法测定不同产地莪术醋制前后微量元素含量的变化。莪术中含有Zn、Ti、Cu、B、V、Ni、Co等微量必需元素,其中Zn元素居首位,其次为Ti、Cu。经炮制后Zn、Ti、B、Ni等微量必需元素的含量有所升高,Cu、V、Co的含量有所降低;Li、As、Cd、Pb等潜在有毒元素的含量有所下降。结果显示中药炮制前后药材中元素含量发生了变化,表明炮制过程对中药中微量元素含量有较大影响。5.蓬莪术醋制前后抗肿瘤作用及其机制的研究:采用CCK-8法、Edu细胞增殖检测、qPCR荧光实时定量技术研究蓬莪术醋制前后抗肿瘤作用及其机制。用CCK-8法检测了蓬莪术炮制前后挥发油对人乳腺癌MCF-7、MDA-MB-231细胞株和人结肠癌SW620、HT-29、HCT-116细胞株的半数抑制浓度IC50值,结果表明蓬莪术炮制之后对细胞毒活性具有降低的趋势。从细胞的生长速度层面分析蓬莪术醋制品对肿瘤细胞的生长影响,通过绘制蓬莪术醋制后对人乳腺癌SW620细胞、人宫颈癌Hela细胞株、人肝癌HepG2细胞株的生长曲线图,直观地显示蓬莪术醋制品挥发油能明显减慢肿瘤细胞的生长速度,其中对SW620和Hela细胞的抑制作用尤为明显;进行Edu细胞增殖检测,以荧光染色细胞照片定性显示蓬莪术醋制品挥发油对上述三种肿瘤细胞生长的抑制作用,结果显示处理后处于分裂期的细胞比例明显降低。qPCR荧光实时定量技术检测蓬莪术生品和醋制品在不同时间点对TP53、CyclinB1、H-P21和CyclinD1基因表达活性具有差异。低浓度和高浓度基因表达亦不同,显示蓬莪术醋制后有上调基因表达的趋势。蓬莪术炮制之后对肿瘤细胞活性抑制强弱有所改变。6.蓬莪术炮制前后抑制模式生物斑马鱼血管生成的研究:以模式生物斑马鱼胚胎、荧光血管斑马鱼(flk-GLP)为研究模型,探讨炮制前后的蓬莪术挥发油及水煎液对斑马鱼胚胎和心脏及抗血管生成的影响及作用机制。通过对斑马鱼胚胎形态学观察、胚胎死亡率LC50值的计算、心脏形态学观察、心率测定、荧光节间血管变化评价蓬莪术醋制前后对斑马鱼的胚胎急性毒性、心脏毒性、抑制血管生成作用。从形态学水平观察醋制前后的蓬莪术对斑马鱼胚胎发育和心脏的影响。结果显示蓬莪术挥发油对斑马鱼胚胎有致畸作用,出现了不同程度的脊柱侧弯,心包水肿,黑色素少的现象,且同浓度下生品挥发油较之醋制品对胚胎的致畸效果有更严重的趋势;蓬莪术生品挥发油对斑马鱼心脏毒性的趋势最为明显,出现了心膜出血,血细胞在心区堆积的现象,心率随浓度升高和作用时间的延长均有明显下降的趋势,醋制品挥发油对心脏无明显毒性作用;采用荧光血管斑马鱼品系(flk-GLP)研究了醋制前后的蓬莪术挥发油对斑马鱼抗血管生成的影响。醋制前后的莪术油在不会造成胚胎发育畸形的低浓度下,能使斑马鱼节间血管生成变得缺失,且杂乱,有明显抗血管生成作用。蓬莪术生品挥发油对斑马鱼节间血管生成无明显影响,但主血管生成呈现断裂趋势,有抑制作用。且醋制莪术油有比生品效果更佳的趋势。蓬莪术油可以保证斑马鱼胚胎正常发育却又能抑制血管生成。蓬莪术生品及醋制品水煎液组却对斑马鱼胚胎无明显致畸作用,对血管生成无明显抑制作用。结论:本文通过开展不同产地、不同来源的姜黄属类药材中挥发油类化合物的定性、定量分析以及微量元素与道地性成分的相关性研究,发现了姜黄属类药材挥发油类化合物及微量元素种类及含量存在差异,证明了其挥发油类化合物的种类和含量与道地性之间的相关性。不同产地姜黄属药材微量元素含量存在明显差异,与药材品种和产地分布呈一定规律性。莪术中重金属元素在煎煮过程中由饮片到汤液的分布变化显示饮片中重金属含量与患者口服的汤液中重金属元素含量不一定成正相关,各元素溶出率都较低,揭示了中药入中医临床传统汤剂的科学性和合理性,并可为重金属限量标准提供依据。表明挥发油化合物及微量元素能在一定程度上作为判断蓬莪术道地性特征成分的重要物质指标,可为全面了解道地性特征提供重要的参考依据,为评价“同名多基原”与“同种异名”类中药质量的优劣提供一种研究思路,也证明了定性分析与定量研究的结合是中药质量控制的较好途径。通过开展蓬莪术醋制前后化学成分(挥发油类成分、微量元素)的对比研究,发现了炮制前后蓬莪术物质基础的改变。蓬莪术醋制前后挥发油指纹图谱的建立从整体角度表征其化学成分变化的复杂性和整体性,醋制增效与挥发油类化合物种类和含量的变化密切相关。对莪术及其炮制品中无机元素含量的研究,能在一定程度上能更全面地反映莪术的性能,推测醋制品破血消癥止痛等功效增强可能与微量元素含量的变化紧密相关。提示炮制不仅改变了元素的含量,而且可能改变了元素的存在形态,对进一步探讨微量元素的具体存在形态,揭示蓬莪术炮制机理奠定基础;通过蓬莪术醋制前后抗肿瘤作用及其机制研究。表明炮制之后的药材对肿瘤细胞毒活性具有降低的趋势,能调控参与癌症发生的关键基因的表达水平,从分子层面阐释了炮制理论的科学性。首次运用模式生物斑马鱼研究蓬莪术的炮制机理,醋莪术对斑马鱼胚胎致畸、急性毒性及心脏毒性有减小趋势,并有明显抑制血管生成作用。进一步揭示蓬莪术孕妇禁用的科学内涵,阐释炮制前后蓬莪术“所增之效”及“所减之毒”,丰富了蓬莪术醋制的炮制内涵,为莪术及其炮制品的质量控制、质量检测及临床合理应用提供一定的科学依据。更多还原

【Abstract】 Curcumae Rhizoma derives from Curcuma phaeocaulis Val.(Pengezhu),Curcuma kwangsiensis S.G Lee et C.F.Liang(guangxiezhu)and Curcuma wenyujin Y.H.Chen et C.(wenyujin).As the current version of Chinese Pharmacopoeia recorded,Curcumae Rhizoma,one of the Sichuan geo-authentic crud drug,have the efficacy of qi-moving blood-breaking and pain-alleviating.Both its crud drug and vinegar-prepared drug serve as one of the commonly used medicine for blood-breaking and disorder-eliminating in the clinical practice of Traditional Chinese Medicine(TCM).Objectives:Genuineness is not only one of the specialty of Traditional Chinese Medicines(TCMs),but also the vital criteria for the evaluation of drug quality.With main research target of Pengezhu,3 kinds of curcuma medicinal materials gathered from authentic region,totally 51 batches,werecompared by the chemical compounds differences.The research including qualitative and quantitative measurement of the volatile compounds(organic part),inner relationship studying between microelement(inorganic part)and the genuineness and defining the specific compounds of the genuineness of Curcuma phaeocaulis Val..Thus a new researching method and model for the genuineness evaluation of the geo-authentic drug was expected to prove.The drug preparing theory and experience have been using for thousands of years.Vinegar preparing method is one of the commonly used preparing craft which is unique characteristic of TCM.The vinegar preparing method is mostly applied in the TCMs with such characteristics of soothing liver-qi stagnation,Scattered stasis pain and wiping out edema.After preparing with vinegar,not only can the efficacy of breaking-mass and eliminating-lump of Curcumae Rhizoma be improved,but also the fierce property is decreased.This dissertation is aim at the comparing the similarities and differences between crud drug and prepared drug of Pengezhu on the basis of chemical compound,tumor cytotoxic activity and anti-angiogenesis of zebrafish model.Therefore,we can make a further step to illustrate the preparing mechanism of Pengezhu,show more proof of the scientific of preparing theory and lay a solid foundation for the reasonable application of Pengezhu and its prepared drug.Through the research of the genuineness and preparing mechanism of Pengezhu,the main purpose of this dissertation is to put forward the scientific proof for its reasonable application in clinical practice and comprehensive development and utilization.Methods and Results:1.The qualitative and quantitative research of volatile oil consisted in Curcuma genus medicinal material:The volatile oil is believed to be both the main pharmacological active ingredients and clinical effectiveness of the important material base of Curcumae Rhizoma.By GC-MS-AMDIS qualitative and quantitative gas chromatography-mass spectrometry analysis technology,through NIST 11 standard MS library retrieval and combined with temperature programmed retention index PTRI auxiliary qualitative,comparative analysis to identify the total content of volatile oil and the main effective components consisted in Pengezhu,guangxiezhu,wenyujin,Curcumae Longae Rhizoma,Indonesia Curcumae Longae Rhizoma,Rithoma Curcuma and Radix Curcumae,recorded in the current edition of Chinese pharmacopoeia.Analyzed from Curcumae Rhizoma,Curcumae Longae Rhizoma and Curcumae Radix,133 constituents of volatile oil were identified,accounting for more than 80%of the total composition.And the change of the compound type and relative content and the characteristic component content were also analyzed.The results showed that different sources of Curcuma genus medicinal material component types and content of volatile oil of different level differences.Form Pengezhu planted in the original production areas of Sichuan,36 compounds were identified,accounting for 94.5%of the total amount of volatile oil and the contents of the main characteristic ix components were significantly higher than other regions.For example,2,3,5,6-tetramethyl pyrazine,terpinol ene,germacrene-D,β-guaiene,curcumol and spathulenol were the unique compounds identified in the Pengezhu.At the same time using GC-MS multicomponent quantitative technology,the content of the characteristic composition in Pengezhu,germacrone,β-guaiene,curcumol and curdione were detected.The four kinds of characteristic compounds content in volatile oil were different amount in different regions Curcumae Rhizoma:germacrone and β-guaiene content is generally higher than curcumol and curdione.The volatile oil of Pengezhu deriving from Sichuan areas had significantly higher content of germacrone and P-guaiene than other origins of Curcumae Rhizoma.Wenyujin differed from Pengezhu in main component type and relative content of volatile oil but showed a higher similarity in Guangxiezhu.2.The research of relationship between the heavy metal elements and genuineness of Curcuma genus:ICP-MS technology was used for the research of heavy metal elements determination of different regions,different original Curcumae Rhizoma,Curcumae Longae Rhizoma and Radix Curcumae medicinal materials and the influence of traditional decoction method.Clustering analysis indicate that,according to data with the same base,different section of three kinds of curcuma herbs can clearly distinguish,the same origin of medicinal materials can also get together better;Different regions,different base each heavy metal element content in the original medicinal materials have a certain trend:Cu>Cd>Pb>As>Hg,showing different varieties can selectively enrich different heavy metal elements and origin environment has a significant influence on its enrichment of heavy metals;Among the 3 different base raw Curcumae Rhizoma medicinal herbs,Pengezhu has the same characteristics with Guangxiezhu though with different origin,for the enrichment feature of heavy metals more similar.Dissolution rate showed that an element in the process of heavy metals in traditional Chinese medicinal materials decoction of difficult dissolution,and after decoction in great changes of content of each element,the element content of dissolution exists As>Pb>Hg>Cu>Cd,showed that although Cu element content is greater than the other elements in the original slices,but the dissolution rate is low.3.Comparative study on the qualitative and quantitative analysis of volatile oil consisting in Pengezhu:Through the study on the GC-MS fingerprints of 10 batches of raw products and 10 batches of the vinegar products of Pengezhu,we established the fingerprint of the volatile oil of the raw and vinegar products.Comparative analysis found that 26 common compounds in the raw products volatile oil,which occupied 71.72%of the total content of volatile oil,and 24 common compounds in the vinegar products volatile oil occupied 81.5%.There are 22 common compounds between the two products,which occupied 78.57%of total identified compounds and most of them are terpenes and sesquiterpenes.γ-selinene and cubebene etc.are the newly added compunds of the vinegar products,terpinolene、δ-elemene、germacrenes、γ-cubebene etc.are vanished,and relative percentage content of the compounds are changed,so we conduct that it is the material basis of the enhancement of blood-breaking and disorder-eliminating after vinegar prosessing.We used the GC-MS total constituents quantitative analysis technique determined 4 characteristic compounds content as β-elemene、germacrone、curdione and curcumol,then we founded out that the characteristic compounds content as β-elemene、germacrone、curdione were increased and the content of curcumol were decreased.This showed that the volatile oil characteristic compounds content of Pengezhu can be greatly influenced after processing with vinegar.The volatile oil of vinegar products compounds variety、relative percentage content and characteristic compounds content were all changed,this could be used as one of an effective quota for describing the vinegar products of Pengezhu mechanism.4.Comparative study of the trace element of Pengezhu before and after the vinegar processing:ICP-MS method was used for the evaluation of the change of trace elements before and after the vinegar processing of different regions of Pengezhu.Pengezhu contains Zn,Ti,Cu,B,V,Ni,Co and other essential trace elements,with Zn element in the first place,followed by Ti,Cu.After processing,the content of essential trace elements,such as Zn,Ti,B and Ni,increased and the content of Cu,V,Co decreased;the content of Li,As,Cd,Pb and other potentially toxic element decreased.Results showed that the element content in TCMs has changed before and after processing and processing has a great influence on the trace elements in TCMs.5.The research of antitumor effect and mechanism of the raw products and vinegar products of Pengezhu:CCK-8、Edu cell proliferation detection、qPCR fluorescence quantitative technique were adopted to found out the antitumor effect and mechanism of the raw products and vinegar products of Pengezhu.The half-inhibition concentration of the raw products and vinegar products volatile oil of Pengezhu to human breast cancer MCF-7、MDA-MB-231 cell line and human colon cancer cell line SW620‘HT-29’HCT-116 were detected by CCK-8 method and the results showed that Pengezhu after the processing had a lower trend of cytotoxic activity.We analyzed the effect of vinegar products of Pengezhu on tumor cells’ growth rates level,through drawing the growth chart of SW620 human breast cancer cells,human cervical cancer Hela cell line,HepG2 human hepatoma cell line,visually display the vinegar products volatile oil of Pengezhu can significantly slow down the growth of tumor cells,which the SW620 cells and Hela cells inhibition was particularly evident.Through the Edu cell proliferation detection,fluorescence staining cell images showed the qualitatively the inhibition of volatile oil of vinegar products to three tumor cell growth and the results showed that in the split phase cell ratio was significantly decreased.qPCR fluorescence quantitative technique was used to test the influence on gene expression activity of TP53,Cyclin B1,H-P21 and Cyclin D1 by the raw products and the vinegar products at different time points.Gene expression of low and high concentrations is also different,regulation of gene expression showed a trend on the vinegar products of Pengezhu.The effect of tumor cells inhibition of Pengezhu was changed after processing.6.The comparative analysis of the angiogenesis effect of raw products and vinegar products of Pengezhu to the model organism zebrafish:model biological zebrafish embryos and fluorescence blood vessels zebrafish(flk-GLP)as the research model,discussing the raw products and vinegar products volatile oil of Pengezhu and the water decoction of zebrafish embryos and heart the influence and function mechanism of anti-angiogenesis.Through analysis of zebrafish embryos morphology,calculation of LC50 of embryonic mortality,observation of cardiac morphology,measurement of heart rate and vascular changes between fluorescence section to evaluate the raw products and vinegar products effects on zebrafish embryos acute toxicity,cardiac toxicity and inhibition of angiogenesis action.Based on the morphological level,we investigated the influence of the raw products and vinegar products on the zebrafish embryo and heart.The results showed that the volatile oil of Pengezhu have teratogenic effects on zebrafish embryos,there have been varying degrees of scoliosis,pericardial edema,less melanin phenomenon,and the similar concentrations of raw products volatile oil have more serious trend on teratogenic effects embryos than the vinegar products.The raw products volatile oil of Pengezhu trend on zebrafish cardiac toxicity was the most obvious,emerging the heart membrane bleeding,blood cells accumulate in the heart area phenomenon,the trend of the increase of the heart rate and the time of the function of the time were obviously decreased,the volatile oil of vinegar products has no obvious toxic effect on the heart;We studied the raw products volatile oil and vinegar products volatile oil of Pengezhu effect on zebrafish antiangiogenic by using the fluorescein angiography zebrafish strains(flk-GLP).The volatile oil of raw products and vinegar products volatile oil of Pengezhu does not cause embryonic malformations in low concentrations,which could make zebrafish angiogenic become the missing section and messy,with a clear anti-angiogenic effect.The raw products volatile oil has no obvious effect on zebrafish intemode angiogenesis,but the main angiogenesis shows a trend of fracture and has inhibitory effect.And the vinegar products volatile oil has a tendency to than raw products effect is better.Volatile oil of Pengezhu can ensure normal embryonic development in zebrafish and also can inhibit angiogenesis.The raw products and vinegar products of Pengezhu water decoction group has no obvious teratogenic effect on zebrafish embryos,and no significant inhibition of angiogenesis.Conclusions:Through the qualitative and quantitative analysis of volatile compounds in Curcuma genus medicinal materials from different regions and different sources we founded that correlation between trace element and authentic ingredients,discovered the variation of type and content of volatile compounds and trace elements in Curcuma,prove that the correlation between type and content of the volatile compounds and authentic.The content of trace elements consisting in Curcuma medicinal materials different regions varied to a certain degree and there existing certain regularity between the varieties and producing areas.The translation changes of heavy metal elements in Curcumae Rhizoma from decoction pieces to the soup indicated that the content of heavy metals in decoction pieces did not has the positive correlation with the content of heavy metal element through oral asministration.And the low dissolution rate of each element revealed the scientific and rational of soup dosage form of TCMs and provided basis for the limit standard of heavy metals.In addition,it showed that volatile oil compounds and trace elements can be certainly applied to the indication of vital materials for the genuineness of Pengezhu,can be provided as an important reference basis for the comprehensive understanding of the feature of genuineness,provided a research idea to evaluate "the base of the original"and "the same vision of" the quality of TCMs and proved that the combination of qualitative and quantitative analysis is good way for the quality control of TCMs.By comparing the essential oils and microelement of Pengezhu before and after vinegar processing,we found that its material foundation has changed after processing.By establishing the fingerprints of essential oils before and after vinegar-process,we integrally showed the changes of chemical compounds were complicated,and those changes were closely related to synergia after processing.And the research of the inorganic element of Pengezhu and its processed products,to a certain extent,can reflect the performance of Ezhu more comprehensively,speculated that effect enhancement of the vinegar products’ may closely related to the change of trace element.And processing not only changed the content of element,but may also change the existence form,this had lay a foundation to further explore the concrete existence form of microelement and reveal the processing mechanism.According to the anti-tumor effect and its mechanism of Pengezhu and its products,we found that the activity and toxic of vinegar-products to tumor cells showed decreasing tendency than Pengezhu,meanwhile the products can control the key tumor genes expression level,explained the scientificity of processing theory from the molecular level.Using zebrafish to research the processing mechanism of Pengezhu for the first time,the vinegar products’ embryo teratogenic,acute and heart toxicity has a decreasing trend,and also had obvious inhibitory effect of angiogenesis.And it revealed the scientific connotation that Pengezhu must be avoided by pregnant women.We also interpreted the effect and toxic changes of Pengezhu after processing,enriched the processing connotation and provided scientific basis for the quality inspection,quality control and rational clinical application of Pengezhu as well as its vinegar products.更多还原