From The Study Of Hemostatic Effect And Mechanism Of Phellodendron Chinense, The Research On "burning Charcoal To Stop Bleeding" And "burning Ash"

BackgroundThere is a long history of clinical application of traditional Chinese medicine for charcoal,and there are several prescriptions related to charcoal making in Zhongjing's prescriptions.For example,"burning the three herbs into ashes with mulberry root skin" in Wangbu Liuxing formula,and "burning the Immature Bitter Orange to char" in Zhishi Shaoyao powder,his idea of "burning to ash and preserving property" about charcoal drug is also considered to be a clear proposal for the ancient charcoal drug processing standards.At present,the procedure of carbonization is often judged by experience,which makes the clinical efficacy uneven.charcoal drug used for hemostasis has occupied a very important position in medical application of traditional Chinese medicine in ancient times,and exploring the material basis of"carbonization to get hemostatic effect" is the important and difficult points of charcoal drug study.Phellodendri Cortex Carbonisatus(PCC)is the product after high temperature carbonization of Phellodendri Cortex,with largely decreased bitter cold nature.It also has astringent properties in clearing heat and drying dampness,clinically used to treat bleeding disorders such as collapse.As a prodrug of PCC,Phellodendri Cortex contains alkaloids,limonoids and other active ingredients,after Phellodendri Cortex was heated to high temperature,these main components were almost completely disappeared,but it began to have hemostatic effect.Although the hemostatic effect of PCC has been supported by a considerable amount of clinical evidence,the material basis and mechanism of its hemostatic effect are still unknown.ObjectiveIn this study,PCC was studied with the characterization technology of nanomaterials to study the preparation technology standards of the newly discovered PCC-CDs in PCC decoction,and to evaluate the hemostatic effect and its mechanism of PCC-CDs based on multiple bleeding models,to confirm that PCC-CDs are one of the key material basis for the hemostatic effect of Phellodendri Cortex after high temperature carbonization,and to provide some experimental evidence for elucidating the material basis of "carbonization to get hemostatic effect" and the idea of "burning to ash and preserving property" about charcoal medicine of Zhang Zhongjing.Methods1.Establish the preparation,separation,and purification method of PCC-CDs.The Hemocoagulase(HC)was used as control drug,and mouse tail bleeding model was used to preliminarily evaluate the hemostatic effect of PCC-CDs.2.Investigate the calcination temperature and time of the muffle furnace,transmission electron microscopy characterization techniques(TEM,HRTEM)were used,and the electron microscopy results and hemostatic effects as evaluation indicators to optimize the preparation conditions of PCC-CDs.3.The components of PCC-CDs were analyzed by high performance liquid chromatography(HPLC);Fourier transform infrared spectroscopy(FTIR),ultraviolet spectroscopy(UV-Vis),fluorescence spectroscopy(FL)and other commonly used modern Chinese medicine research techniques were combined with nanotechnology such as TEM,HRTEM,EDS,and XPS to characterize the physicochemical properties of the selected PCC-CDs and to calculate their quantum yields.4.The effect of different concentrations of PCC-CDs on cell viability was evaluated by cell counting kit(CCK-8),and cytotoxicity was evaluated to obtain the safety parameters of PCC-CDs.5.The hemostasis effect of subcutaneous injection(sc),intragastric administration(ig),and intraperitoneal injection(ip)three administration methods of PCC-CDs were examined using tail bleeding model.The capillary clotting method was used to determine the clotting time at 10 min,45 min,90 min,120 min,180 min,300 min and 480 min after single administration of PCC-CDs.Tail bleeding model and hepatic injury bleeding model were used to examine the dose-effect relationship of the hemostasis of PCC-CDs.PCC-CDs heparin sodium antagonism test and its effect on mouse PLT and plasma coagulation parameters(PT,APTT,TT,FIB)were used to explore the possible ways for PCC-CDs to play hemostatic effect.6.Establish the agkistrodon acutus venom(AAV)acute hemorrhage model,set the control group,model group,high,middle,and low dose of PCC-CDs groups,with six time points(1 h,3 h,12 h,1 d,2 d,and 5d)in each group.Blood samples were taken from each group at each time points for PLT detection.Plasma levels of 6-keto-PGF1?,TXB2,t-PA,and PAI-1 were measured.The hemostatic effect of PCC-CDs on AAV acute hemorrhage models and their mechanism were explored.The serum levels of blood urea nitrogen(BUN)and serum creatinine(CRE)were measured at each time point in each group.HE staining was performed on the kidneys to observe pathological changes.ELISA was used to detect the levels of IL-10,IL-1?and MCP-1 in the kidney,to explore the protective effect and mechanism of PCC-CDs on acute renal injury induced by AAV hemorrhage in mice.Results1.The PCC-CDs prepared by muffle furnace calcining at 300? for 1 h exhibited a spherical shape with uniform dispersion.The particle size was less than 10 nm and the lattice spacing was about 0.24 nm.There was no difference in the hemostasis time between the PCC group and the PCC-CDs group(P>0.05).It was significantly shorter in the PCC group and the PCC-CDs group than that in the NS group and the Phellodendri Cortex group(P<0.05),but without statistical difference compared with HC group(P>0.05).2.When the muffle furnace calcining temperature were 250?,300?,350?,400? and 450?,respectively,the yield of PCC-CDs and the particle size and shape of PCC-CDs under electron microscope were different,and the effect of hemostasis was different.The 350? group was choosed considering the results of electron microscopy and hemostasis;the calcining time in the muffle furnace at 350? was 15 min,30 min,45 min,and 60 min,respectively,and 60 min group was chosen considering electron microscopy and hemostasis.The final optimized PCC-CDs preparing parameter calcined using the muffle furnace was at 350? for 1 h.3.PCC-CDs components analysis and physicochemical properties characterization:(1)The purified PCC aqueous solution was detected by HPLC,and no active ingredients such as alkaloids and limonoids originally in Phellodendri Cortex were detected.(2)PCC-CDs morphology:PCC-CDs were spherical and uniformly distributed under TEM,with a diameter range of 1.2-4.8 nm and a particle size distribution of 2.640±0.694 nm.The lattice spacing of PCC-CDs was 0.232 nm measured by HRTEM diffraction.(3)Spectral properties of PCC-CDs:(A)UV-Vis showed that PCC-CDs had a weak absorption peak at 270 nm.(B)FL results showed that the strongest emission wavelength of PCC-CDs was 438 nm,and the strongest excitation wavelength was 363 nm.(C)FTIR results showed that PCC-CDs showed characteristic absorption peaks at 3385,3130,2925,2860,1566,1401 and 1121 cm-1.The-N-H group was located at 3385 cm-1;the presence of-O-H groups was observed at 3130 cm-1;the weak absorption at 2925 and 2860 cm-1 was attributed to the stretching of-C-H;in addition,the absorption peak at 1566 cm-1 was considered as the characteristic absorption peak of-C=O;the absorption peak at 1401 cm-1 might be-C-N,-N-H or COO-group;the absorption peak at 1121 cm-1 was-C-O.(4)PCC-CDs elements and surface structure analysis:EDS results showed that PCC-CDs were mainly composed of C and O,with content ratio 575:175,close to 3,and the results of EDS were consistent with the results of XPS.XPS results indicated that PCC-CDs mainly consisted of carbon(71.64%,atomic percent),nitrogen(1.30%,atomic percent),and oxygen(23.26%,atomic percent).Analysis of XPS peaks on the surface of PCC-CDs might indicate C-C/C=C,C=O,C-O,C=N,O-C=O,C-OH,C=O,N=C,C-N-C,and N-(C)3,and other functional groups.The XPS analysis results were consistent with the FTIR results.(5)The extraction rate of PCC-CDs was 0.26%and the relative quantum yield was 9.62%.4.In the CCK-8 cytotoxicity experiment,when the PCC-CDs concentrations were 0,0.01,0.1,1,10,100 and 1000 ?g/mL,the cell viability was about 100%,and the cells viability began to decline at a concentration of 5000 ?g/mL,and gradually decreased to 52.78%at a concentration of 10,000 ?g/mL.5.Results of the hemostatic effect of PCC-CDs and its mechanism:(1)The different administration method of sc,ig,and ip all showed hemostatic effect,and there was a statistically significant difference compared with NS group(P<0.05).The time of hemostasis in the sc and ip groups were not different from that in the HC group(P>0.05).(2)After single administration of PCC-CDs,procoagulant effect was exhibited at 10 min,and the coagulation time of administration group was significantly different from NS group at 10 min,45 min,90 min,120 min,180 min and 300 min time points(P<0.05).From 300 min to 480 min,the clotting time in the administration group gradually extended to the same level as the NS group.(3)In the mouse model of tail bleeding and hepatic injury bleeding,the time of hemostasis was significantly shorter in the PCC-CDs group compared with the NS group(P<0.05)and not different with the positive drug(P>0.05).(4)In the heparin sodium antagonism experiment,there was no significant difference between the time of hemostasis of PCC-CDs group and HC group(P>0.05);compared with PCC-CDs group,the time of hemostasis was significantly prolonged in HS+PCC-CDs group(P<0.05).(5)Results of PLT and plasma coagulation parameters compared with the NS group,PLT and FIB in PCC-CDs group increased(P<0.05),and PT decreased(P<0.05).6.Effect and mechanism of PCC-CDs on AAV acute hemorrhage model:(1)In the AAV modeling dose experiment,4 mice died in the LD50 group(2 mg/kg),and none of the other mice died;1/2 LD50 group and 1/4 LD50 group mice appeared poisoning symptoms.PLT in LD50 group decreased to(134.00±42.81)×109/L,PLT in 1/2 LD50 group decreased to(343.80±63.41)×109/L,and 1/4 LD50 group decreased to(751.30±70.64)×109/L,compared with NS group[(1160.10±77.38)×109/L],the difference was statistically significant(P<0.01).The AAV dose for establishing an AAV acute hemorrhage model was 1 mg/kg.(2)In the AAV acute hemorrhage experiment,the PLT of the model group was lower than that in the control group at 1 h,3 h,12 h,1 d,and 2 d(P<0.05).The PLT decreasing caused by AAV was inhibited in the treatment group,among which the high dose group was significantly different the model group at 3 hours,12 hours,1 day,and 2 days(P<0.05).Compared with the control group,the 6-keto-PGF1? and t-PA in the model group increased(P<0.05),TXB2 and PAI-1 decreased(P<0.05),while the treatment group could significantly suppress the decrease of TXB2 and PAI-1 caused by AAV,and relieve the increase of 6-keto-PGF1? and t-PA,with a statistically significant difference(P<0.05).(3)In the experiment of exploring the protective effect of PCC-CDs on AAV bleeding-induced acute kidney injury in mice,the BUN and CRE in model group were higher than those in control group at 1 d,2 d and 5 d(P<0.05),while treatment group could inhibit the increase of BUN and CRE caused by AAV,and the difference was statistically significant(P<0.05).Kidney pathological findings in the model group:glomerular hyperemia,tubular congestion around the renal tubules,swelling and dilation of tubular tubules,edema,and degeneration of tubule epithelial cells could be seen at 1 h.Tubular swelling and hyperemia in the treatment group were less than those in the model group.The levels of IL-10,IL-1?,and MCP-1 in the model group were significantly higher than those in the control group(P<0.05),while the treatment group could inhibit the increase of IL-10,IL-1?,and MCP-1 caused by AAV,and the difference was statistically significant(P<0.05).Conclusions1.In this study,PCC-CDs was first found,the optimized preparation conditions of PCC-CDs were calcined in a muffle furnace at 350? for 1 h.The PCC-CDs were 1.2-4.8 nm in diameter,0.232 nm in lattice spacing,39.28%in carbon yield,mainly composed of C,O,and N elements,with active functional groups such as carbonyl groups,carboxyl groups,hydroxyl groups,ether groups,and amino groups on the surface.There were no traditional Chinese medicine active ingredients such as small molecules,metal ions or salts in the aqueous solution of PCC-CDs.2.There was no cytotoxicity of PCC-CDs below 1000 ?g/mL(equivalent to about 3 g/mL of PCC),and the hemostatic effect was equivalent to the Hemocoagulase in vitro and in vivo.It maintained at a high level of hemostatic effect from 10 min to 5 h,and gradually decreased to normal after 5-8 h.The recommended administration method was subcutaneous injection and intraperitoneal injection.3.PCC-CDs could activate the endogenous coagulation pathway,promote the fibrous protein,increase the PLT,accelerate TXB2 production to promote the platelet aggregation,and increase the PAI-1 to inhibit the fibrinolytic system to play a role in hemostasis.4.Taking PCC as an example,the material basis of "carbonization to get hemostatic effect"and the idea of "burning to ash and preserving property" about charcoal medicine of ZhangZhongjing could be interpreted as "PCC-CDs with a large number of plastic reactive groups on the surface are one of the key materials for the hemostatic effect of PCC.When preparing PCC,calcining at 350? for 1 h could prepare PCC-CDs with the best hemostatic effect,and too much or too little burning could not produce hemostatic effect or produce weak hemostatic effect".