| Objective:Dragon's blood is a traditional precious Chinese medicine and included in the Chinese Pharmacopoeia of the 2015 edition,which has "blood rejuvenation" reputation.Dragon's blood is mainly processed by the resin obtained from fruit of Daemonorops draco Bl.It is produced in Indonesia,also known as the imported dragon's blood.At present,the domestic dragon's blood is mainly derived from the ethanol extracted from the fat wood of the Dracaena,including the dragon's blood produced in Guangxi and Yunnan("the Drug Standard of Yunnan Province","Standards Issued by the Ministry of Chinese Medicine")and the Hainan dragon's blood("the Drug Standards of Hainan Province")produced in Hainan.The study shows that both of them have similar efficacy and different chemical components.However,the domestic dragon's blood cannot completely replace the imported dragon's blood.Due to the scarcity of resources,there are more and more counterfeit medicines,dammarane resin and other materials in the market.Or domestic dragon's blood,which is relatively low in price,impersonate the expensive import of the imported blood.At present,the quality control of dragon's blood is mainly based on modern physical and chemical analysis technology,such as thin layer chromatography,ultraviolet visible spectrophotometry and HPLC content determination.These analytical methods are mature,but the pretreatment of samples is tedious,time-consuming and labor-intensive,and the cost of identification is high.The purpose of this study is to identify the adulteration of imported dragon's blood and domestic dragon's blood by infrared spectroscopy and thermal analysis,in order to achieve the purpose of fast,accurate,simple,economic adulteration identification.Flavonoids are main chemical constituents of imported dragon's blood and domestic dragon's blood.They have important biological activities,but the flavones are complex.Volatile components are the common features of resin herbs,contained in imported dragon's blood and domestic dragon's blood.Most scholars also believe that these compounds have some potential biological activity,clarify the two essential characteristics and differences in oil composition,which is of great significance for the resource utilization of dragon's blood and drug development.In addition,elemental analysis is also the important content of quality analysis of traditional Chinese Medicine.Many experimental results demonstrate that the effect of activating blood and removing stasis was related with the distribution of trace elements in Fe,Cu,Zn,Mg,Ca,Sr,K,Pb,Cd,Cr and interaction.There is no report on the determination and comparative analysis of trace elements in the dragon's blood.To solve the above problems,this study will focus on the flavonoids,volatile oils,trace elements of the analysis and research are carried out on the components of imported dragon's blood and domestic dragon's blood,in order to clarify the main chemical components of the dragon's blood and provide a scientific basis for the basic research of drug effect substances and clinical rational use of drugs.Methods:there were 31 batches of experimental samples from different producing areas,drug markets,pharmaceutical companies and pharmacies respectively,including 13 batches of imported dragon's blood and 18 batches of domestic dragon's blood(capsules).The experimental study on the identification and analysis of adulteration of imported dragon's blood and domestic dragon's blood,including:1.Identification and analysis of the samples:on the basis of the 2015 version of "China Pharmacopoeia" and "Chinese standard" provisions,reference to the relevant literature,using character identification method,chemical reaction method,microscopic identification method,UV Vis spectrophotometry,TLC,HPLC,alcohol insoluble detection method,rosin detection method,experimental samples were collected for analysis of comprehensive identification,and comprehensive advantages and disadvantages of current methods are compared.2.Authenticity and adulteration analysis on the imported dragon's blood and domestic dragon's blood by Fourier transform infrared spectroscopy:?Attenuated total reflection infrared spectroscopy using Fourier transform infrared spectroscopy(FTIR-ATR)were measured in 18 batches of samples of domestic dragon's blood range of 4000?650cm-1,the establishment of the FTIR-IR fingerprint discrimination model,the rapid identification of the authenticity of sample by spectral correlation coefficient,and the supervision of the PCA-X model to verify the results;?The basic spectra of 13 batches of imported dragon's blood samples were measured by Fourier transform infrared spectroscopy Spotlight 400 microscope,and Spectrum I MAGE V1.7 software for data processing,adulteration analysis according to the characteristics of infrared spectra of dragon's blood control medicine particles and dammarane resin spectral data.3.Qualitative and quantitative analysis of imported dragon's blood and domestic dragon's blood by TG-DTA thermal analysis:26 batches of imported dragon's blood and domestic dragon's blood samples were used in the experiment.The effects of temperature range,heating rate,powder particle size and other important factors on the TG-DTA experiment of dragon's blood were investigated.The TG-DTA characteristic map was used to discriminate rapidly imported dragon's blood and domestic dragon's blood,and counterfeits were identified.Then the TG-DTG method was used to do thermo gravimetric analysis and enthalpy calculation,and quantitatively discriminate the medicinal samples.4.Study on HPLC-PDA fingerprint of imported dragon's blood and domestic dragon's blood:Imported dragon's blood and domestic dragon's blood were researched respectively by the method of HPLC-PDA the fingerprints.Effects of extraction solvent,extraction method,chromatographic conditions(column temperature,detection wavelength,mobile phase,gradient elution)factors were considered.Through the investigation of methodology(precision,repeatability,stability),the methods of HPLC-PDA fingerprint and the fingerprint similarity evaluation were established.5.Analysis of the flavonoids in the imported dragon's blood and domestic dragon's blood based on HPLC-LTQ-Orbitrap-MS:The method of isolation and identification of Flavonoids from imported dragon's blood and domestic dragon's blood was established by HPLC-LTQ-Orbitrap-MS combined technology.Compounds were identified by control articles and references,and the rule of mass spectrometry fragmentation was summarized.The characteristics of the flavonoids in the imported dragon's blood and domestic dragon's blood were analyzed and compared.6.Analysis of the essential oil in the imported dragon's blood and domestic dragon's blood based on GC-MS:The method of separation and analysis of volatile oil from imported dragon's blood and domestic dragon's blood was established by the GC-MS technology.Through the analysis of the NIST database and Mass Hunter platform for mass spectrometry analysis of volatile oil samples qualitatively and quantitatively,the differences components of volatile oil from different areas of dragon's blood were compared.7.Distribution and correlation analysis of trace elements in imported dragon's blood and domestic dragon's blood:Samples of imported dragon's blood and domestic dragon's blood were processed by microwave digestion.The content of 10 trace elements(Cu,Zn,Fe Mg,Ca,Sr,K,Pb,Cd and Cr)in 20 batches of medicinal materials were determined by flame atomic absorption spectrometry and graphite furnace atomic absorption spectrometry.To research the distribution and correlation of elements in two kinds of "dragon's blood" through analysis of Radar chart and Spearman rank correlation coefficient.Result:1.The results of traditional method identification of samples:Combined with a variety of methods,the comprehensive results showed that:of the 31 samples collected,the DS-7,DR-5,DR-6 and DR-10 were the counterfeit products.2.The results of false adulteration analysis by infrared spectroscopy:?Validation results show that 5 infrared spectra of domestic dragon's blood and fingerprint samples were similar,the correlation coefficient is greater than the threshold and discrimination for 3 counterfeit,which's correlation coefficient is less than threshold and nondiscrimination for domestic dragon's blood.The results of PCA-X analysis are in agreement with the model discriminant results of infrared spectrum fingerprints.? FTIR spectroscopy adulteration analysis results,DS-1,DS-5,DS-9 doped with domestic dragon's blood,and DS-10 doped with dammarane resin impurity.The results of HPLC chromatography analysis showed that DS-1,DS-5 and DS-9 were mixed with domestic dragon's blood.3.The results of authenticity analysis by TG-DTA:Through the analysis of TG-DTA,it was found that the characteristic peaks of DTA curve in the imported dragon's blood were found:T1=400±5?,T2=508±5 ?,T3=541±5 ?.The characteristic peak of DTA curve identification for the domestic dragon's blood is T1=449±5?,T2=540±5?,T3=612±5?.There were no above characteristics in DS-7,DR-5,DR-6 and DR-10 of counterfeit.The quantitative analysis of TG-DTG showed that the Tv-max of the imported dragon's blood should be 298±5 ? and 477±5 ?,?W2+?W3>80%,and the Tv-max of domestic dragon's blood should be 535±5 ?,?W2+?W3<80%.According to this,domestic dragon's blood and imported dragon's blood can be distinguished quickly and accurately.4.Research results of HPLC-PDA fingerprint:12 batches of imported dragon's blood samples were identified and 37 common peaks were determined,and the chromatographic peak of the index component of Dracorhodin perchlorate was identified.Similarity evaluation results showed that 9 batches of similarity was higher than 0.9;15 batches of imported dragon's blood sample fingerprint,identified 43 common peaks,identified the index component of resveratrol,Loureirin A,Loureirin B and pterostilbene.The similarity evaluation showed that the similarity of the 15 batch of domestic dragon's blood was more than 0.9,indicating that the method was stable and reliable,and could be used for the quality control of the dragon's blood.5.Components analysis results of HPLC-LTQ-Orbitrap-MS technical:19 compounds have been identified from the imported dragon's blood,mainly for simple flavonoids,chalcone and two hydrogen chalcone,anthocyans,flavanol compounds and phenolic compounds;42 compounds have been identified from the domestic dragon's blood,mainly for flavonoids,chalcone and two hydrogen chalcone,flavanone class two,hydrogen flavones,flavanones and other phenolic compounds.On the basis of separation and identification of compounds,the mass spectrometric fragmentation of 6 kinds of flavonoids was further summarized.6.Components analysis results of GC-MS technical:51,56,42 and 48 compounds were qualitatively and quantitatively identified from the volatile oil in the dragon's blood of Indonesia,Guangxi dragon's blood,Yunnan dragon's blood,and Hainan dragon's blood.The composition of the volatile oil from the different origin of the dragon's blood was analyzed,and it was found that the composition of the essential oil in the dragon's blood of Indonesia was different from domestic dragon's blood.Further,through identification analysis of common peaks,it is clarified that volatile oil from Guangxi dragon's blood and Yunnan dragon's blood are similar,which are different from those of in Hainan dragon's blood.7.The results of distribution law and correlation research of elements:Radar analysis showed that Fe,K and Ca were most widely distributed in two kinds of "dragon's blood"(0?4000 ?g/g),followed by Cu,Mg,Sr and Zn(0?140 ?g/g).The contents of K,Ca,Fe,Mg and Cu in the imported dragon's blood were higher than those of domestic dragon's blood,while the contents of Sr and Zn were lower.There was a higher content of heavy metal elements Cr in domestic dragon's blood,and heavy metal element Cd,Pb were distributed equivalently in two "dragon's blood"(0?0.10 ?g/g);Spearman correlation analysis showed that the correlation coefficients of Cu-Fe,Cu-Zn and Fe-Zn in domestic dragon's blood were more than 0.9(P<0.01),while Cr-Fe?Cr-Mg and Cr-Zn in imported dragon's blood showed significant negative correlations(P<0.01).Conclusion:In view of the difficulties in the authenticity and adulteration of the imported dragon's blood and domestic dragon's blood,two kinds of differential analysis methods are proposed.The FTIR-ATR fingerprint model can be used to quickly,accurately and nondestructively analyze the authenticity of imported dragon's blood and domestic dragon's blood,and infrared microscopy can also be used to identify the adulteration of the imported dragon's blood.TG-DTA thermal analysis method is easy to operate and the amount of sample is very small.It can be used for qualitative and quantitative analysis of imported dragon's blood and domestic dragon's blood,so as to distinguish and distinguish authenticity.For mainly flavonoid compounds,volatile oil components and potential trace elements in the imported dragon's blood and domestic dragon's blood,HPLC-PDA fingerprints,LC-MS,GC-MS and elemental analysis methods were used respectively in this research.The results reflect the chemical components types,and the characteristics,generality and difference of flavonoids and volatile oils composition in the imported dragon's blood and domestic dragon's blood.It also clearly reveals the distribution rule and correlation of 10 trace elements in the imported dragon's blood and domestic dragon's blood.This study can provide important evidence for the clinical use and pharmacodynamic comparative study of dragon's blood. |
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