| 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. |
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