| Background Baweiguanxinning capsule is a self-made preparation from the second people's Hospital in Wen County,Henan Province.It contains Safflower,Trichosanthis,Aloes,Chuanxiong,Dalbergia odorifera,Salvia miltiorrhiza,Red peony root and Radix notoginseng.It has the function of easing chest and regulating Qi,promoting blood circulation and removing blood stasis.It is mainly used in the treatment of Qi stagnation and blood stasis due to chest obstruction,chest-yang obstruction type 1.Commonly known as coronary heart disease.Approved and sold since 2004,the treatment is effective and has a good local market.Compared with common drugs,hospital-made preparations have small market and limited use scope,but due to their moderate price,high reimbursement rate of New Rural Cooperative Medical Insurance,relatively small side effects of traditional Chinese medicine and convenient use,etc.it has a relatively large market in the local area.Compared with the well-marketed drugs,the current standards for hospital preparations were formulated in the last century.The methods are simple and the projects are relatively few,which are not enough to meet the current situation of strict control of drug quality advocated by the state.In order to improve the quality standard of Baweiguanxinning capsule,Hydroxysafflor yellow A was investigated,in order to control the quality of Baweiguanxinning capsule,it is proposed to use the more mature and widely used high-performance liquid chromatography to quantitative analysis the capsule.On this basis,the partial pharmacological effects of the drug were investigated.Objective In this study,the method of determination of hydroxysafflor yellow A in Baweiguanxinning capsule was studied,and the method of determination of hydroxysafflor yellow A in High-performance liquid chromatography was established,to control the quality of the drug.In addition,by measuring the changes of serum total cholesterol(TC)before and after administration in SD rats,the effect of Baweiguanxinning Capsules on serum total cholesterol(TC)was explored.Method 1.The determination methods of hydroxysafflor yellow A in Carthamus Tinctorius L.were studied from three aspects of precision,repeatability and stability.2.The content of hydroxysafflor yellow A in Baweiguanxinning capsule was established by High-performance liquid chromatography method:(1)UV photometer method was used to determine the final detection wavelength.(2)The best sample processing method was determined by orthogonal test of different extraction solvent,extraction method and extraction time.(3)The specificity,linearity,precision,repeatability,stability and sample recovery of the method were investigated.3.To investigate the effect of Baweiguanxinning capsule on serum total cholesterol level:(1)To establish the model: Twenty four SD rats were randomly divided into four groups: control group,low-dose group,medium-dose group,and high-dose group,with six in each group.The rats were stained and numbered with monochromatic picric acid solution.Approximately 0.5ml of blood was collected from the tail vein after 3 days of normal feed feeding,placed in a heparinized centrifuge tube,centrifuged at 2000 r / min for 10 min,and the supernatant was taken.The total cholesterol(TC)in the serum was determined by the iron sesquichloride-acetic acid-sulphuric acid coloring.On the fourth day,they was fed with a cholesterol-rich diet every day at 6 pm.Blood was collected from the tail vein for one week,the serum was separated,the TC value in the serum of each group of rats was measured in the same way,and the data results were analyzed.(2)Administer the medicine:normal rat feed in the control group;0.6 g of Baweiguanxinning capsule mixed in the low-dose group feed;1.2 g of Baweiguanxinning capsules in the medium-dose group feed;1.8 g of Baweiguanxinning capsules in the high-dose group.Use a small amount of rat feed to dip the contents of the Baweiguanxinning capsules to ensure that each rat ingests a certain amount of drug before returning to the cage to eat freely.(3)Take a blood sample: blood was taken before the administration,once every 3 days,the serum was separated,and the TC value was measured.Repeated for 15 days,the SD rats in each group were measured on the 3rd,6th,9th,12 th,and 15 th days.Total serum cholesterol(TC)was analyzed using SPSS 25.0 statistical software.Result 1.Methodological investigation on determination of hydroxysafflor yellow A in safflower: for the High-performance liquid chromatography used,the results showed that the theoretical plate number of hydroxysafflor yellow A was more than 3000 and the resolution was more than 1.5,which met the systematic test requirements of Pharmacopoeia.The average peak area of 6 hydroxysafflor yellow A was 2922268,RSD was 0.55%,the precision was good.The average retention time of 6 samples was 8.984,RSD was 0.22%,and the repeatability was good.The average retention time was 8.991,RSD was 0.46%,the average peak area was 2914772,RSD was 0.30%,the result showed that the method was stable in 24 hours.2.To establish a new method for determination of Baweiguanxinning capsule:(1)Chromatographic conditions: Thermoc 18 column(4.6 mm 250 mm,5 m),mobile phase methanol-acetonitrile-0.7% phosphoric acid solution(26:2:72,V / V),detection wavelength: 403 nm,column temperature: 30 °C,flow rate: 1.00 ml / min,UV detector.(2)Sample preparation method: 25% methanol solution was used as solvent and 30 min ultrasonic treatment was used as sample extraction method.(3)Methodological investigation: A good linear relationship(r=0.99999)of hydroxysafflor yellow A was found in the range of 0.02625~0.4200 g for the established HPLC method.The average peak area of 6 hydroxysafflor yellow A was 823.7,RSD was 0.47%,the precision was good.The average of the contents of six samples was 0.4371mg/g,RSD was 0.58%,and the repeatability was good.The average peak area of the sample was 833.7,and the RSD was 1.2%.The results showed that the sample was stable in 24 hours.The average recovery of hydroxysafflor yellow A was calculated to be 98.78% with RSD of 0.59%.(4)Through the determination of five batches of Baweiguanxinning capsules,the results showed that the content of hydroxysafflor yellow A,the main component in the capsules was stable.3.Effect of Baweiguanxinning capsule on serum total cholesterol level:(1)The serum total cholesterol(TC)level of rats was significantly increased after high-fat diet.Paired sample t test was performed,t =-33.415,P = 0.000,and there were statistically significant differences.The modeling was successful.(2)The multivariate test results of changes in serum TC values at 6 time points before and after 15 days in the four groups of SD rats showed F = 3.235,P = 0.001 <0.05,indicating that the interaction between time points and groups was significant.Conclusion The method for determination of hydroxysafflor yellow A in Carthamus Tinctorius L.has good reproducibility and stability.It can provide technical support for determination of Baweiguanxinning capsule.The content of hydroxysafflor yellow A in Baweiguanxinning capsule was determined by High-performance liquid chromatography.The method has good reproducibility,strong stability,and is simple,accurate and convenient to use,determination of hydroxysafflor yellow A in Baweiguanxinning capsule.Through animal experiments,we will find the contents of Baweiguanxinning Capsule can significantly reduce the serum total cholesterol(TC)level of SD rats in a dose-dependent manner. |
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