Study On The Functions And Mechanisms Of Platycladi Cacumen Carbonisatum In Cooling Blood And Stanching Bleeding

Platycladus orientalis belongs to the oriental arborvitae genus of Cupressaceae plants,mainly produced in southern Inner Mongolia,Jilin.Liaoning,Hebei.Shanxi,Shandong,Zhejiang,Fujian,Anhui,Jiangxi,Henan,Shaanxi,Gansu,Sichuan,Yunnan,Guizhou,Hubei,Hunan,northern part of Guangdong and Guangxi,and also cultivated in Deqing and Dazi of Tibet.Platycladi cacumen,the dry shoots and leaves of Platycladus orientalis(L.)Franco,have the effects of cooling blood to stanch bleeding,eliminating rheumatism and relieving pains.It is mainly used in the TCM clinical oral administrations to treat cough,hypertension,dysentery,rheumatoid,and a variety of bleeding disorders,such as vomiting blood,nose bleeding,uterine bleeding,and blood in the urine or stool.It can also be used externally in the treatments of erysipelas,mumps,burns,bone-setters,and some other traumas.After carbonized,its hemostatic efficacy can be enhanced and used in many Chinese herbal prescriptions,such as Shihui Powder treating hematemesis,hemoptysis and hematemesis(“Shi Yao Shen Shu"),Sijicebai Powder treating intestinal bleeding("Qi Xiao Liang Fang"),Zhixue Powder treating gastrointestinal bleeding with powder("TCM Treatments and Prescriptions''),Shenxiao Decoction treating hematuria("Jing Yan Liang Fang").Although Platycladi Cacu,men Carbonisatum was commonly used as hemostatic medicine in the TCM clinical treatment,the mechanism of action and material basis have not been clear elaborated.This paper mainly try to verify the hemostatic efficacy Platycladi Cacumen Carbonisatum and filter the effective hemostasis parts through two kinds of blood-heat and hemorrhage syndrome rat models.Then make a preliminary qualitative and quantitative analysis of the main chemical ingredients with HPLC to provide the basis for further reveal of the mechanism and material basis.The studies in this paper mainly include the following sections:Part one:Literature reviewAfter having reviewed relative books and literatures,we summarize a review of Platycladi Cacumen and Platycladi Cacumen Carbonisatum from the evolution of concocted history,the chemical composition,pharmacological effects and clinical applications to support the new research direction,the feasible experimental program and examine indicators.Part two:Establish two kinds of blood-heat and hemorrhage syndrome rat models base on the Characteristics of Traditional Chinese Medicine(TCM)syndrome medication1.Acute bleeding and fever rat model induced by the solutions of dry yeast.Rats were given with subcutaneous injections of 2 g·kg-1 dry yeast suspension and supplemented with 5%ethanol instead of water.After injection,monitor the rectal temperature within 12 hours,hemorheology parameters,endogenous and exogenous coagulation parameters,pathological changes of tongue,lung and stomach.Experimental results show that,the rectal temperature of model-group(M-group)was lower than the control-group(C-group)within 3 hours,then rose to the maximum gradually in the next 4 hours and kept the highest for 3 hours.Ten hours later,the rectal temperature of M-group began to decrease,while the C-group did not have large changes.Biochemical detections and comparisons show that,in the M-group,RBC,HGB,HCT,the whole blood viscosity and plasma viscosity were obviously increased(P<0.05,P<0.01),TT and APTT were significantly extended(P<0.01),PT was significantly shortened(P<0.01),and the concentration of FIB was significantly increased(P<0.01).There was no significant change of tongue and stomach,while the surface of the lung scattered some pink bleeding points.Microscope observations show that,the tongue and stomach had visible vascular dilatation and congestion,the lung had pathological changes such as fibroplasias,narrow alveolar cavity,interval congestive,interstitial hemorrhage and so on(P<0.01).2.Blood-heat and hemorrhage syndrome rat model induced by Rhizoma Zingiberis.Rats fed with water decoctions of Rhizoma Zingiberis in dose of 15 g·kg-1 and 5%ethanol instead of water for 14 days.Observe the general signs about body weight,food intake,water intake,urine output,weight of manure,fecal water content and rectal temperature.Then detect the hemorheology parameters,coagulation indexes,and the pathological examination of tongue,lung and stomach.Seven days after administration,some of the general signs of the model-group(M-group)had obviously changes except the food intake and water intake.Fourteen days later,the rectal temperature,dietary intake,water intake and urine output of the M-group were significantly increased(P<0.05,P<0.01),while the weight of manure and fecal water content reduced significantly(P<0.01).Compare the growth rate of urine output and the water intake,the former is higher.Biochemical detections showed that,the indexes of HGB,HCT,FIB,plasma viscosity and the whole blood viscosity were obviously upgraded(P<0.05,P<0.01),the TT and APTT were significantly extended(P<0.01),while the PT was significantly shorted(P<0.01).Visually observed,the tongue and stomach had no obvious changes,while the surface of the lung scattered some dark red bleeding points.Observed under the optical microscope,vascular dilatation and congestion can be seen in the tongue and stomach,while pathological changes such as interval hyperemia,fibrosis,alveolar stenosis and interstitial hemorrhage can be observed in the lung(P<0.05,P<0.01).These results indicated that this two blood-heat and hemorrhage models of rats have been established successfully.Part three:Preliminary study on the function and mechanism of Platycladi Cacumen Carbonisatum to the blood-heat and hemorrhage syndrome rat model caused by Rhizoma ZingiberisEstablish the blood-heat and hemorrhage syndrome rat model caused by Rhizoma Zingiberis,and give the test drugs of Platycladi Cacurmen Carbonisatum after modeling.General signs(body weight,food intake,water intake,urine output,manure weight,fecal water content and rectal temperature),the hemorheology and coagulation parameters,the thyroid function parameters and pathological examination of lung were detected to compare the effect of Platyclacdi Cacumen Carbonisatum in different doses.Experimental results show that:(1)The water decoction of Platycladi Cacumen Carbonisatum in the low dose effectively acted on the indexes including the rectal temperature,water intake,fecal water content and body weight(P<0.05,P<0.01).The contents of T4 and rT3 in plasma obviously reduced,and TSH obviously increased(P<0.05).But the blood hyperviscosity,coagulation dysfunction and hemorrhagic pathological changes in lung did not show obvious changes.(2)In the group given with the low dose of Platycladi Cacumen Carbonisatum,the rectal temperature,food intake,water intake,urine output,fecal water content and the growth rate of weight obviously improved(P<0.05,P<0.01).WBC,RBC,HGB and HCT in the blood were obviously reduced(P<0.05,P<0.01).T4 and T3 in the plasma were obviously decreased.TT and APTT were significantly shortened(P<0.01).Observed under the microscope,the pulmonary interstitial hemorrhage and inflammatory cell infiltration were obviously reduced(P<0.05,P<0.01).(3)The water decoction of Platycladi Cacumen Carbonisatum in the medium or high dose obviously improved the indicators of general signs(P<0.05,P<0.01).It also decreased the WBC,RBC,HGB,HCT,blood viscosity and plasma viscosity obviously(P<0.05,P<0.01).Meanwhile,the TT,APTT,FIB,T4,T3 and rT3were reduced when PT and TSH were obviously upgraded(P<0.05,P<0.01).Pathological examinations showed the pulmonary hemorrhage of rats fed with Platycladi Cacumen Carbonisatum were effectively treated,and the high dose was better than the medium dose by comparison.(4)The water decoction of Platycladi Cacumen Carbonisatum had weak effect to the platelets in model rats caused by Rhizoma Zingiberis.According to the results above,we compared the treatment effects of Platycladi Cacumen Carbonisatum in different doses and different administration forms to the blood-heat and hemorrhage model rats caused by Rhizoma Zingiberis,When given in the low dose,the powder was better than its water decoction.When given with the water decoction,the high dose was the best,and the medium dose was better than the low dose.We can speculate that,the effect and mechanism of Platycladi Cacumen Carbonisatum cooling blood to stanch bleeding were related to regulate the thyroid hormone level,correct the hemorheological abnormality and coagulation dysfunction.Part four:Study on the function of Platycladi Cacumen before and after carbonized to the acute fever and hemorrhage syndrome rat model caused by dry yeastWe gave rats subcutaneous injection of 20%dry yeast suspension,then gave 1 mL of ethanol 7 hours after the subcutaneous injection when body temperature rise to the maximum,to copy the acute fever and hemorrhage syndrome rat model.We select Yunnan Baiyao as a positive control to compare the hemostatic effect of Platycladi Cacumen in different forms before and after carbonized.Compared these indexes including the whole blood viscosity,plasma viscosity,TT,APTT,PT,FIB,ADP-induced platelet aggregation,RBC,HGB,HCT,PLT,PCT,MPV,PDW and the pathological examinations of lungs and stomachs among each group.The experimental results show that,Platycladi Cacumen and its product had some certain hemostatic effect,and this effect was enhanced after carbonized.In addition,we compared the therapeutic effects of Platycladi Cacumen Carbonisatum in different extract-methods to this acute fever and hemorrhage model rats.After the statistical analysis,the composite extracts of Platycladi Cacumen Carbonisatum showed better therapeutic effect than the others in some directions mainly as follows:decreased the whole blood viscosity,plasma viscosity,FIB,PLT,PCT,MPV,PDW and pulmonary hemorrhage obviously(P<0.05,P<0.01),it also significantly shortened TT and APTT(P<0.01).This part of experiment laid the foundation to the next program of screen the effective polarity fraction.Part five:Screen the hemostatic effective polarity fraction of Platycladi Cacumen CarbonisatumAccording to the previous experimental results,we chosen the chemical composition of the alcohol extracts and water extracts of Platycladi Cacumen Carbonisatum to prepare the different polarity fractions as the petroleum ether fraction,ethyl acetate fraction,n-BuOH fraction and the water fraction.Rats were injected subcutaneously with 20%dry yeast suspension,then given with 1 mL of ethanol intragastricly 7 hours after the subcutaneous injection.Yunnan Baiyao was selected as the positive control.We also chosen some relative indexes such as the whole blood viscosity,plasma viscosity,TT,APTT,PT,FIB,ADP-induced platelet aggregation rate,RBC,HGB.HCT,PLT,PCT,MPV,PDW,TXB2,6-keto-PGF1? and the pathological examination of the lung and stomach to inspect the cooling blood and hemostasis effects of the different polarity fractions from Platycladi Cacumen Carbonisatum.The statistics and analysis showed that:?The ethyl acetate extracts obviously decreased the whole blood viscosity,plasma viscosity,TT,APTT,FIB,PDW and 6-keto-PGF1?(P<0.05,P<0.01),and significantly increased the TXB2 and ADP-induced platelet aggregation rate(P<0.01).It also had obviously inhibited the alveolar wall thickening,interval tissue hyperemia and interstitial hemorrhage to rectify the pathological changes of lung in the model rats(P<0.05,P<0.01).?The n-BuOH extracts obviously increased the ADP-induced platelet aggregation rate and TXB2(P<0.05),while significantly reduced the 6-keto-PGF1?(P<0.01).?The water extracts could obviously reduce the plasma viscosity,FIB and 6-keto-PGF1?(P<0.05,P<0.01),and shortened the TT obviously(P<0.05).?The petroleum ether fraction had weak action to these model rats.The potency of the ethyl acetate fraction was better than the n-BuOH and water fractions.Thus,the preliminary study showed that the ethyl acetate fraction of Platycladi Cacumen Carbonisatum was the main effective part to cooling blood to stanch bleeding.Part six:Qualitative and quantitative analysis of Flavonoids to Platycladi Cacumen before and after carbonized in different extract-methods,and the different polarity fraction of Platycladi Cacumen CarbonisatumThe flavonoids of Platycladi Cacumen mainly include myricitrin,rutin,quercitrin,quercetin,kaempferol,apigenin,amentoflavone,hinokiflavone and so on.Choose the extracts of Platycladi Cacumen and Platycladi Cacumen Carbonisatum in different methods given to rats in chapter four to inspect the dissolution of the main flavonoids with HPLC.The experimental results showed the contents of these flavonoids changed after carbonized.For example,the contents of myricitrin,rutin,quercitrin,amentoflavone and hinokiflavone decreased,while the quercetin,kaempferol and apigenin increased.After comparison,the extraction rates of these flavonoids with alcohol and water is higher than the water only.Investigate the content disparities of six kinds of flavonoids including quercitrin,quercetin,kaempferol,apigenin,amentoflavone and hinokiflavone in the different polarity fractions of Platycladi Cacumen Carbonisatum given to rats in chapter five with HPLC.Experimental results showed that these six kinds of flavonoids were mainly distributed in the ethyl acetate fraction,and the n-BuOH fraction followed.In addition,a small amount of quercetin,amentoflavone and hinokiflavone could be detected in the water fraction,while in the petroleum ether fraction only little amentoflavone could be detected.