| Hexue Zhiyang Formula(HXZYF)is a clinically empirical formula,based on Siwu Decoction,Zhiyang San and Huangqi Fangfeng Decoction,and based on the rule of"Treatment of wind first treats blood,and blood goes through the wind by itself".It is composed of nine herbs of Ligusticum chuanxiong,Radix Paeoniae Alba,Radix Rehmanniae,Radix Rehmanniae,Salvia Miltiorrhiza,Sophora flavescens and Kochia Kochia,used to treat skin pruritus caused by blood deficiency and wind dryness.In this thesis,the efficacy of HXZYF was determined through four experiments on improving blood deficiency,antipruritic,anti-inflammatory and immunosuppressive;UPLC-MS/MS high-resolution mass spectrometry technology was used to identify the main chemical components of HXZYF and its Into the blood components in the body;quantify the main components based on the analysis of the blood components,determine the quality control indicators and standards,and identify the taste of each medicine in a thin layer.1.Pharmacodynamic evaluation of HXZYFA mouse model of blood deficiency was prepared by subcutaneous injection of acetophenazine.Continuous administration for 15 days.30 minutes after the last administration,blood was collected from the orbit to measure the RBC,HCT and HGB values of whole blood.The results showed that the levels of HCT,RBC and HGB in the peripheral blood of the model group animals were significantly reduced,compared with the blank group,there was a very significant difference(P<0.01).And after subcutaneous injection of acetophenazine,the basic physical signs of the mice showed symptoms of blood deficiency syndrome,indicating that the model of blood deficiency disease was successful.Compared with the model group,the low-dose group significantly increased the contents of RBC,HCT and HGB in the peripheral blood of mice(P<0.01).It shows that HXZYF has a certain effect on improving blood deficiency in mice.Itching in mice was induced by injecting 0.05%dextran through the tail vein,and the number and duration of itching in mice within 30 min were recorded.The results showed that tail vein injection of dextran can significantly increase the number of itching in mice(P<0.01)and the total time of itching(P<0.05).It shows that the pruritus model is successful.At the same time,it was found that the three dose groups can alleviate the pruritus in mice.Among them,the number of pruritus and the total time of pruritus in the low-dose group were significantly different from those of the model control group(P<0.05).The medium-dose group and the high-dose group Compared with the model group,the number of pruritus,pruritus time and total pruritus time of the group were significantly different(P<0.01),and the high-dose group had the best effect.Capillary permeability test was used for anti-inflammatory,0.5%Evans Blue solution was injected into the tail vein,and then 0.1%histamine phosphate was subcutaneously injected into the abdomen of each mouse.Cut the blue skin of the abdomen and cut it into small pieces,soak it in 5ml of acetone:normal saline 7:3 liquid,take it out and centrifuge(3000rpm,15min)after 24h.Take the supernatant and measure its absorbance at 610nm.The results showed that the low-dose group could significantly inhibit the increase in mouse capillary permeability caused by histamine phosphate(P<0.05),while the middledose group and the high-dose group had extremely significant inhibitory effects(P<0.01).The middle-dose group has the best effect.For the immunosuppression experiment,delayed-type hypersensitivity and carbon particle clearance experiments were selected.Delayed hypersensitivity:Before administration,except for the normal control group,40 μL of 0.5%2,4dinitrofluorobenzene DNFB was evenly smeared on the abdomen of mice in each group before administration/Sensitization only.The administration is strengthened once every three days.1h after the last administration,apply 0.2%DNFB acetone olive oil solution evenly to the right ear of the mouse for stimulation,and apply acetone olive oil to the left ear for 24～48h.The results showed that compared with the blank group,the swelling degree of the right ear of the model group mice was significantly increased(P<0.01),and the spleen index was significantly increased(P<0.05).Compared with the model group,the ear swelling of the mice in each administration group was significantly reduced.Among them,the low-dose group and the model group had extremely significant differences(P<0.01),indicating that Hexue Zhiyang Formula was effective against 2,4dinitrofluoride the delayed hypersensitivity model caused by benzene DNFB has a significant inhibitory effect.But it has little effect on the organ index of DTH mice;carbon particle clearance experiment:14 consecutive days.Thirty minutes after the last administration,0.05ml/10g body weight of Indian ink was injected into the tail vein.At 2min and 20min after injection,use a quantitative blood collection tube to take 20μl of blood,dissolve it in 2ml 0.1%Na2CO3,and shake it at 640nm to measure the OD value.The results showed that compared with the blank group,each administration group could reduce the carbon particle clearance index and phagocytosis index,and the middle dose group had extremely significant differences(P<0.01).2.Study on the Main Chemical Components and Blood Components of HXZYF Based on LC-MSUPLC-Q-Exactive orbital hydrazine high-resolution mass spectrometry technology is used to identify the main chemical components of HXZYF.By optimizing liquid phase conditions and chromatographic columns,the separation and analysis of chemical components in the complex system of traditional Chinese medicine compounds are realized.In order to obtain more comprehensive compound information,the positive and negative ion mode was used for detection,combined with database and literature reports,and based on the primary and secondary MS/MS high resolution data,a total of 160 chromatographic peaks were identified,mainly phenylpropanoids,Phthalides,alkaloids,terpenes,glycosides,flavonoids,and organic acid compounds fully explain the chemical components of HXZYF.Due to the unknown origin of some components,chemical composition analysis of each single drug was carried out under the same conditions to determine the source of each component.At the same time,the blood components of the oral administration rat plasma were analyzed by UPLC-Q-Exactive orbital hydrazine highresolution mass spectrometry technology,and 42 prototype components of HXZYF were found.The above experimental results provide the basis for the study of the mechanism and quality control of HXZYF.3.Quantitative Analysis of Six Components of HXZYF and thin layer identificationQuantitative analysis of the content of six main components of ferulic acid,albiflorin,cimifugin,Prim-O-glucosylcimifugin,ligustalide H and calycosin in HXZYF by ultra-high phase liquid phase(UPLC).The linear relationship of the components determined in the investigated concentration range is good(R2>0.9958),the precision RSD<2.98%,the repeatability RSD<2.93%,the recovery rate(n=9)are all 92.06%～110.58%,the RSD is all less than 3.91%,the results show that the method has high sensitivity,good reproducibility,rapid,accurate and reliable analysis,and can be used for qualitative and quantitative detection and the main chemical components of HXZYF;using TLC method,established HXZYF.The qualitative identification method of nine medicines have laid the foundation for effective quality control. |
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