Study On The Material Basis And Mechanism Of Blood Residual Charcoal "stop Bleeding And Treat Epilepsy"

The application of Crinis Carbonisatus?CC?is originated from the ancient era.Before the pre-Qin period,it was initially recorded in Prescriptions for Fifty-two Diseases via bamboo slips.In the modern era,it has been recorded in Pharmacopoeia of the People's Republic of China?2015?and acknowledged by the Chinese statute book and scholars.Nevertheless,the scientific accuracy of CC's hemostatic bioactivities remains unclear.In particular,no progress has been made regarding the research on the hemostatic material basis.To break this deadlock,the following researches were conducted.Objective:?1?The hemostatic fraction?HF?of CC aqueous extracts was screened with the index of hemostatic efficacy and identified by high-performance liquid chromatography?HPLC?analysis method and nanotechnology.?2?The conventional charcoal process of CC was modified,and the morphology,optical properties and functional groups of CC's HF obtained under various conditions were characterized.Then,the charcoal conditions of CC HF were determined using the tail bleeding time as an assessment index.?3?The characterization information of the HF of CC prepared by optimal conditions was further enriched,and the safety in vitro and in vivo was explored.?4?The theory of"burning charcoal to stop bleeding"was proved by the investigation on the impacts of the dose-effect and time-effect relationship of CC's HF on hemostatic action and its mechanism.It attempts to take the mystery out of the hemostatic material base and provides a new research perspective and methods for the material basic study of other charcoal drugs.?5?The research on the analgesic,sedative,neuroprotective activities and corresponding mechanism of CC's HF will provide the experimental support to explain the scientificity of CC's HF on"stunning epilepsy".Methods:?1?The hemostatic fraction of CC aqueous solution divided by dialyzing and purifying processing was screened using the mouse's tail bleeding model.HPLC analysis was used to compare hair solution with HF.Further,the hemostatic fraction of CC was identified and analyzed by TEM,UV-Vis,FL,and FTIR.?2?Unlike the traditional preparation process of CC that was calcined in a pot sealed by the lid,CC was prepared by carbonizing hair in a muffle furnace at high temperature.The influence of charcoal temperature and time on the morphology,optical property,functional group distribution and compositional differences of the hemostasis effect of CC was analysed.Then the tail bleeding time was used as an evaluation index to find the optimal temperature and time conditions of the hemostatic fraction of CC.?3?The carbonization yield and water-soluble extract content of 3 batches of CC prepared by the modified method under optimal conditions were analysed to estimate the stability of this preparation method.Electron microscopy?high and low-resolution electron microscopy?,optical technology?UV-Vis,FL,FTIR?,HPLC,X-ray diffraction?XRD?and X-ray photoelectron spectroscopy?XPS?were used to further analyse and enrich the information,including morphology,particle size distribution,lattice spacing,optical property,functional group distribution,and components.?4?The in vitro impacts of CC's HF on the viability of RAW 264.7 cells were assessed using a CCK-8 assay in 12h,24h,48h,and 72h,respectively;while its toxicity in vivo was measured by acute toxicity test on mice.?5?Taking the tail bleeding time as the evaluation index,the dose-response relationship of the hemostatic activity of CC's hemostatic fraction was evaluated using the tail amputation model and the liver scratch model.The Capillary coagulation test on mice was used to examine the time-effect relationship of CC's hemostatic fraction with the target of clotting time.The mechanism underlying the possible activation of the coagulation pathway was evaluated through the measurement of the coagulation parameters?PT,APTT,TT,and FIB?using mouse plasma;the impacts on the platelet system were evaluated by measuring platelet count,and the levels of TXB₂ and 6-keto-PGF_1?.The concentration of tPA,PAI-1,and D2D in plasma was used to explore the impacts on the fibrinolytic system.?6?Through the prolonged mice's sleep time test by pentyl barbital sodium,the sedative effect of CC's HF was studied using latency to loss of righting reflex,total sleep time and the change of rectal temperature as the index.?7?The open field experiment and elevated labyrinth experiment were carried out to study the anxiolytic effect of HF;and the preliminary study on the mechanism of action was performed to detect the contents of 5-HT,NE,DA,and GABA in brain tissue.?8?Taking the change of pain threshold as the evaluation index,the two classical experiments?the hot plate experiment and the tail flick experiment?were used to examine the analgesic activity and the dose-effect relationship of CC's HF.The opioid receptor antagonist?naloxone?and the cholinergic receptor antagonist?atropine?were injected to explore the possible mechanism of this fraction.?9?Middle cerebral artery ischemia-reperfusion injury?MACO?model was constructed to investigate the protective effect of CC's hemostatic fraction on the neuroprotective function,blood-brain barrier permeability and cerebral infarction volume ratio among mice.The levels of TNF-?and IL-1?in the serum of mice were detected to preliminarily investigate the possible mechanism underlying the neuroprotective activity of CC's HF.Results:?1?The hemostatic active fraction screened by the mouse's tail bleeding model of CC demonstrates that the molecular weight is beyond 1000.By comparing HPLC fingerprint analysis of hair decoction,CC and the two parts derived from CC aqueous solution,no chromatographic absorption peak is found in the CC's effective part,which is in sharp contrast with the fingerprint of CC and hair decoction.In addition,the results of TEM scanning show that CC's effective part is composed of nearly spherical particles with good dispersion.The size distribution is in the range of 20⁵5 nm with the hydroxyl group,the ether group,and the carbonyl group.The maximum excitation(?_EX)and emission wavelength(?_EM)are 382 nm and451 nm,respectively.This finding confirms that the effective hemostatic part of CC consists of nano-components?CC-NCs?.?2?The CC-NCs obtained by the modified preparation process have the hemostatic effect,and the most active processing conditions are heating at 350°C for1 h.Under the same time of preparation,the temperature displays an impact on the apparent properties,appearance morphology,optical characteristics and functional group distribution characteristics of CC-NCs.To be more specific,the temperature difference shows an impact on the peak intensity of the infrared absorption spectrum of the prepared CC-NCs,resulting in an insignificant peak;as the temperature increases,the?_EM of CC-NCs presents a blue shift tendency.Under the same temperature of preparation,little difference is found in the infrared spectrum of CC-NCs;with the extension of carbonization time,the?_EM of CC-NCs has a blue shift.In addition,the HPLC fingerprints of CC-NCs prepared under different conditions appear no absorption peaks of small molecule compounds.?3?CC-NCs prepared by optimal conditions show good stability.The results of further analysis on CC-NCs by various characterization methods show that CC-NCs obtained by carbonization at 350°C for 1 h mainly contain C,O,and N.The content of the elements is 98.69%;the particle size distribution is between 1.5⁵.0nm;the shape is nearly spherical,and the dispersion is good in water.The lattice spacing is0.203 nm,in consistent with the XRD analysis result;its?EM and?EX are 435 nm and 361 nm respectively;the main functional groups include a hydroxyl group,a carboxyl group and the like.?4?The safe dose of CC-NCs at the cellular level is 1160 g/mL,and the LD50 of the mouse acute toxicity test reaches at least 100 mg/kg.?5?The results of mouse tail bleeding and liver hemorrhage experiments show that CC-NCs have significant hemostatic activity,lasting from10min to 6h.In addition,hemostasis studies show that CC-NCs show the ability to reduce APTT time,increase plasma PIB and PLT concentration in the whole blood,significantly reduce plasma 6-keto-PGF1?concentration,increase TXB2 concentration,and degrade tPA,elevate PAI-1 and D2D level.?6?In the pentobarbital-induced sleep experiments,CC-NCs prolong the sleep time of mice to a large extent and shorten the time that takes to reduce anal temperature,but exert little impact on sleep latency;?7?CC-NCs in open field experiments significantly expand the active distance of mice in the central zone,while CC-NCs reduce the total activity distance and the velocity of movement in the central zone;CC-NCs in the elevated maze experiment prolong the time and expand the active distance of the mice.In addition,the results of neurotransmitter levels in mouse brain tissue show that CC-NCs increase the content of GABA content and decrease the content of 5-HT,NE and DA.?8?The results of two models of thermal stimulation analgesia show that the three doses of CC-NCs increase the pain threshold of hot plate test mice.In addition,the analgesic effect of the nano-components can be antagonized by naloxone,rather than by atropine.?9?CC-NCs improve the neurological function score of the MACO mouse model,reduce the permeability of the blood-brain barrier and reduce the volume of cerebral infarction.In addition,the levels of IL-6 and TNF-?in the MACO model shows an upward trend.High phenomena can also be improved by CC-NCs.Conclusion:Blood charcoal is one of the representative drugs of"burning charcoal to stop bleeding".Its significant hemostatic activity has been confirmed by a large number of clinical practice and pharmacological experiments;while its hemostatic substance research has no progress.Herein,the high-temperature carbonization process was taken as the starting point,and the nanotechnology analysis was applied innovatively.It is found that the hemostatic material basis of blood charcoal is CC-NCs,and the optimal preparation conditions are improved and optimized to 350°C,1 h.This study demonstrates for the first time that CC-NCs exert the hemostatic activity by activating endogenous coagulation pathways and common pathways,increasing platelet counts,increasing their activation factor TXB2,lowering 6-keto-PGF1?,and inhibiting fibrinolysis systems.In addition,for the first time,this study examined the scientific nature regarding the impacts of sedation,anxiolytic,analgesic and neuroprotective activities of CC-NCs on the treatment of"scarring epilepsy"in the treatment of blood.The anti-anxiety activity of the active ingredient is associated with the increased GABA levels in the brain tissue of mice,the decreased levels of 5-HT,NE and DA;the analgesic activity is correlated to the activation of the opioid receptor system;neuroprotection is related to the reduction in the mouse cortex of the inflammatory factor.This study provides a new research perspective on the hemostasis of blood charcoal and the treatment of the"pseudo-epileptic"material basis and related mechanisms.It not only defines the preparation process with clear parameters and broadens the clinical application of residual carbon,but also proposes new research ideas and methods for basic research and process stability of other carbon drugs.