| The promotion of bone healing is one of the most important applications in traditional chinese medicine(TCM),and the Huo-Xue-Hua-Yu decoction(HXHYD)prescription possesses good clinical practice for the treatment of fracture,which is made up of 12 kinds of TCMs.According to the own drug efficacy of TCM,the HXHYD can consists of four drug-group prescriptions,which are described as follow:Huo-Xue-Zhi-Tong drug group decoction(HXZTDGD),Hua-Yu-Sheng-Xin drug group decoction(HYSXDGD),Tiao-He drug group decoction(THDGD)and Bu-Qi drug group decoction(BQDGD).The surface-enhanced Raman scattering(SERS)spectroscopy has demonstrated great potential in the analysis of TCM prescription and detection of biomedical sample.In this thesis,we first introduced the SERS technique,combining the current research situation and clinical demand to develop detection methods for drug groups in HXHYD and their biological activity.That is,based on the SERS,on the one hand,in order to reveal the relationship between the single decoction and prescription,the drug group rale of prescription,and the relationship between drug groups,spectral characteristics of drug groups in HXHYD were detected.On the other hand,in order to reveal what kind of active mechanism in drug groups and what kind of interaction between themselves in HXHYD,and state molecular mechanism of HXHYD in improving fracture healing,the biochemical composition changes of callus tissue from the fracture healing site were monitored in the process of improving fracture healing,with the aim of new assessment of biological activity and optimization of prescriptions.The main research results as follow:1)The preliminary work:The molecular mechanisms of cytotoxicity induced by Houttuynia cordata Thunb decoction(HCTD)in nasopharyngeal carcinoma cells was first investigated by Raman spectroscopy(RS)combined with principal component analysis(PCA)and linear discriminate analysis(LD).Compared to control group,the intensities of selected bands(1002,1338 and 1448 cm-1)related to protein,DNA,and lipid in the treatment groups decreased obviously as the concentration of HCT and drug treatment time increased.Both cell groups treated with 250 and 500 ug/ml of HCT could be differentiated from control group by the PCA-LDA with diagnostic accuracy of 100%,suggesting that cyotoxicity occurred and 250 ug/ml was the proper dose for treatment.In addition,The conventional Raman spectrum and SERS spectrum of HCTD were first tested and analyzed.There was no Raman signal in conventional Raman spectrum of HCTD.However we observed that the SERS spectrum of HCTD had primary thirteen SERS peaks.The results showed that the SERS spectroscopy might provide a new kind of high-sensitive,accurate,easy,and rapid detecting method for TCM.2)Firstly,we have evaluated the capabilities of SERS technology for analyzing two Huo-Xue-Hua-Yu decoctions(HXHYDs)prepared according to different prescriptions.HXHYD I was prepared according to the original prescription;The same preparation method was given for the HXHYDⅡ except the crudeweight ratio described in original prescription.High quality SERS spectra were obtained.However,significant differences in SERS spectra between HXHYD Ⅰ and Ⅱ can be observed,which showed special changes in the percentage of biochemical constituents in different decoctions.PCA combined with LDA were employed to generate diagnostic algorithms for classification of SERS spectra of two HXHYDs,and a diagnostic accuracy of 100%can be achieved.This work demonstrated that the SERS technique has potential for spectral characteristic detection for decoction of TCM with high sensitivity,and this technique combined with PCA-LDA can be used for quality control of the extracted decoction of TCM and production management of chinese herba preparations.Then,the SERS spectroscopy of Sheng-Di-Dang-Gui decoction(SDDGD),Sheng-Di decoction(SDD)and Dang-Gui decoction(DGD)were tested and analyzed.The characteristic Raman bands of three decoctions were tentatively assigned.The medical ingredients in the SDDGD were not the simple addition of SDD and DGD,which revealed the relationship between the single decoction and prescription.Finally,to reveal the rule of drug group in prescription,the HXHYD was divided into four drug-group prescriptions according to the own drug efficacy of TCM,which was described as follow:Tiao-He drug group decoction(THDGD),Hua-Yu-Sheng-Xin drug group decoction(HYSXDGD),Huo-Xue-Zhi-Tong drug group decoction(HXZTDGD)and Bu-Qi drug group decoction(BQDGD).The SERS spectroscopy of HXHYD,THDGD,HYSXDGD,HXZTDGD and BQDGD were tested and analyzed,and the characteristic Raman bands of five decoctions were tentatively assigned.Some Raman bands in SERS spectra of drug group decoctions were retained in the HXHYD.However some Raman bands never appeared in the HXHYD.And new Raman bands were generated in the SERS spectra of HXHYD,such as 523 and 977cm-1,which meant new created chemical compositions.The results showed that medical ingredients in the HXHYD were not the simple addition of THDGD,HYSXDGD,HXZTDGD and BQDGD,and SERS technique has great potential for providing a novel method for effectively and accurately studying the drug group rule of prescription.3)Raman parameters used included the deposition content of calcium hydroxyapatite(CHA～960 cm-1)and mineral-to-matrix ratio.This study aimed to assess,through SERS spectroscopy,the incorporation of CHA and other biochemical substances on the repair of complete radial fractures in rabbits treated with or without HXHYD therapy.Rabbits with complete radial fractures were randomly divided into two groups,one treated HXHYD therapy and the other one without therapy acted as control.The animals were sacrificed at 15,30,and 45 days after surgery.Specimens were routinely prepared for SERS measurement,and high quality SERS spectra from a mixture of bone tissues and silver nanoparticles were obtained.The ratio of mineral-to-matrix from the control and treated groups was calculated in this study.Results showed that both deposition content of CHA measured by SERS spectroscopy and mineral-to-matrix ratio in the treated group were always greater than that in the control group during the experimental time,demonstrating that HXHYD therapy was effective in improving fracture healing and SERS spectroscopy might be a novel tool to assess fracture healing.4)The HXHYD prescription,which possesses good clinical practice for healing fractures,is made up of 12 kinds of TCMs.According to the own drug efficacy of TCM,the HXHYD can consists of four drug-group prescriptions,which are described as follow:HXZTDGD,HYSXDGD,THDGD and BQDGD.This study focused on what kind of active mechanism in drug groups and what kind of interaction between themselves in HXHYD to reveal the drug efficacy rule of drug groups in prescription.A total of 210 rats were divided into seven groups each of which was subdivided into three subgroups(evaluated on days 15,21 and 30 after surgery).Six treated groups were respectively treated HXHYD,HXZTDGD,HYSXDGD,THDGD,BQDGD and Gu-Zhe-Cuo-Shang capsules therapy(treated control).SERS readings were taken at the fractured sites.Compared with fractured control group(no treatment),no statistically significant difference was found in the CHA peak,carbonate-to-phosphate and phosphate-to-phenylalanine ratio among the THDGD and BQDGD therapy at all experimental time-points,indicating that the two drugs is invalid.In comparison with fractured control group,the more content of CHA,higher carbonate-to-phosphate and phosphate-to-phenylalanine ratio were observed in HXHYD,HXZTDGD,HYSXDGD and Gu-Zhe-Cuo-Shang capsules therapy at all time-points,indicating the effective efficacy in improving fracture healing.The SERS imaging analysis on CHA peaks and support vector machine(SVM)algorithm analysis of the two ratios were first used to prove those results.The results showed that efficacy of HXHYD was stronger than each of drug group as a result of the the most content of CHA and highest carbonate-to-phosphate and phosphate-to-phenylalanine ratio among treated groups at all time-points,which was in conformity with the concept of TCM treatment,that is,"prescriptions are used as carriers,and attention is given to the integration",and SERS technique has great potential for providing a novel method for effectively and accurately studying efficacy rule of drug groups within one prescription with the aim of optimization of prescriptions. |
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