| Schizonepetae Herba Carbonisata(SHC)has a long history of application and e-xact clinical hemostatic efficacy.However,little information is available on its haemostatic components and mechanism.In order to explain this seemingly co-ntradictory question,we made some researches in this paper as follows.Objective:(1)The hemostatic fraction of SHC aqueous extracts was screen-ed by pharmacodynamic experiment,and was investigated the characteristic by the analysis method of high-performance liquid chromatography(HPLC)and mo-dern nanotechnology.(2)The preparation process of SHC's hemostatic fraction which has high yield and hemostatic activities was optimized and identified.(3)The analysis method of nanotechnology was utilized to obtain the structural c-haracterization data such as morphology,particle size,spectral properties and organic functional groups of hemostatic fraction in SHC.(4)The Cell viability,dose-efect and time-effect relationship of hemostatic effects of SHC's hemostatic fraction were investigated and the mechanism underlying the hemostatic activity was explored preliminarily,which not only formulated the material basis for t-he hemostatic effect of SHC,but give new insights into similar research of ot-her charcoal drugs from TCM.Methods:(1)The aqueous extracts of SHC was separated to two fractions by dialysis method,which included the fractions inside(IDB)and outside(ODB)the dialysis bag.Tail amputation model was utilized to screen the hemostatic f-raction of SHC.Comparing the chemial components among Schizonepetae He-rba,SHC,the fraction of SHC inside and outside the dialysis bag using an HPLC method.The analysis method of modern nanotechnology including transmission electron microscopy(TEM),Fourier transform infrared(FT-IR)and fluorescence spectroscopy(FL)was utilized to identify and analyze the hemostatic fraction(2)By examining the technical parameters such as carbonization time and temperature,hemostatic fraction in SHC was prepared by a modified pyrolysis method and was analyzed using TEM,FT-IR and FL,Components difference of SHC and SHC hemostatic fraction which prepared under different conditions were compared by HPLC method.Finally,mouse tail amputation model was utilized to screen the optimum conditions of preparing the hemostatic fraction of SHC.(3)The morphology,particle size and lattice spacing information of SHC's hemostatic fraction were characterized using TEM,high-resolution TEM(HRTEM)and X-ray diffraction(XRD);The organic functional groups and the elemental analysis of it were investigated by X-ray photoelection spectroscopy(XPS)and FTIR;The spectral properties of it were studied using utraviolet visible absorption spectra(UV-vis)and FL.(4)The effects of SHC's hemostatic fraction on the viability of RAW 264.7 cells was assessed using a CCK-8 assay.(5)Take hemostatic time as an indicator,tail amputation and liver scratch mouse model were established to assess the dose-effect relationship of hemostatic effect of SHC's hemostatic fraction.Capillary coagulation test was used to assess the time-effect relationship of hemostatic effect of SHC's hemostatic fraction.(6)The mechanism underlying possibly activating the coagulation pathway was evaluated by measured the coagulation parameters(PT,APTT,TT and FIB)using mouse plasma;the effects on platelet was evaluated by measured platelet count,TXB2 and 6-keto-PGF1?.In all experiments,normal saline was used as a controlResults:(1)Screened by pharmacodynamic experiment,IDB was the hemostatic fraction of SHC aqueous extracts which and the hemostatic effect was significantly different from those of the SH and ODB-treated group,but was not different from those of the SHC-treated group.The HPLC results of this study showed that no active small molecule compounds were detected in the prepared IDB.In contrast,the compounds of ODB was similar to SHC.The characterization of the hemostatic fraction showed that the size distribution of it was in the range of 30-60 nm and well separated from each other.The strongest emission of it was at approximately 467 nm with the strongest excitation at 371 nm and the organic functional groups on the surface of it include carboxyl?hydroxyl group,etc.Taken together,the hemostatic fraction was nanoparticles(NPs).(2)The large differences were performed among NPs prepared under different conditions.The largest size distribution was prepared under 250?-1h,which the smallest was 350?-1h.The strongest emission of NPs took blue shift with the rise of temperature,but there is little influence on the condition of time.The organic functional groups on the surface of NPsmainly included carboxyl?hydroxyl gl oup.The HPLC fingeiprint showed that there was difference of compounds among SHC prepared under different conditions,but all HPLC fingerprint of IDB revealed no small molecule components existed after charcoal processing and dialysis,which excluded the possible interference of active small molecule compounds.According the results of pharmacodynamic experiment,the optimal prepared condition of SHC-NPs was 350?-1h.(3)Under 350?-1h condition,The NPs were well separated from each other with an size distribution with an average range of 3.2 nm and a lattice spacing of 0.393 nm.The emission spectrum of SHC-NPs showed the strongest emission at approximately 454 nm with the strongest excitation at 355 nm.The quantum yield(QY)of the SHC-NPs was determined to be 2.26%using quinine sulfate as a reference QY.The NPs were mainly composed of the elements C,N,O and the organic functional groups on the surface of NPs mainly include carboxyl,hydroxyl group.(4)The NPs under 350?-1h condition exhibited no toxicity within approximately 0.84 mg/mL in the CCK-8 assay.(5)Two hemorrhaging experiments showed that SHC-NPs treated mice had significantly shorter bleeding time than did normal saline(NS)-treated control group and exhibited dose-dependent relationship.The capillary coagulation test showed that the coagulation effects could maintain 7h(6)The PT was decreased significantly by treatment with SHC-NPs at high-,medium-and low doses compared with the NS treatment(P<0.01).Moreover,the SHC-NPs at high and low doses showed a significant increase(p<0.05)in the FIB compared with that of the control group.Furthermore.the SHC-NPs had no effect on the APTT and TT.In addition,the SHC-NPs increased the platelet count and TXB2 and decreased 6-keto-PGF1? of plamsa,the low dose group had optimal hemostatic effect.Hemocoagulase was used as a positive control in all experiments.Conclusion:The exact hemostatic effect of SHC has been well known by many medical experts,while the investigation of SHC's hemostatic material basis and mechanism was still in a "b lank" status.This study breakthrough the current concept of the material basis of traditional Chinese medicine and for the first time,find and demonstrate that SHC-NPs are the hemostatic material basis by adopting a brand-new technology.Moreover,a optimal condition prepared SHC was obtained by modified pyrolysis method and we investigated morphology,optical properties and surface chemical groups of SHC-NPs.In this base,the cell and further pharmacodynamic experiments indicated that SHC-NPs had hypotoxicity,dose-dependent hemostatic effect and longer duration of action.The hemostatic effect of SHC-NPs relates to activate extrinsic and common pathways,promote platelet counts and modulate the level of TXB2 and 6-keto-PGFia This study explored the hemostatically active substance basis and hemostatic mechanism of SHC from a brand-new perspective,thus providing new ideas for the research of other charcoal drugs. |
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