Advanced Development And Initial Safety Evaluation Of The Catalpol And Puerarin For Injection

CPI researched by my own laboratory was a new type of Chinese medicine monomer compound preparations for the prevention and treatment of ischemic cerebrovascular disease, it was original and could win the independent intellectual property rights. This subject was supported by the research fund of the Ministry of Education project (XDJK2009C084. XDJK2010C059).Objective:According to the relevant requirements of "Provisions for Drug Registration" which was promulgated by the State Food and Drug Administration, determined the prescription, preparation process of the CPI. Then, established its quality standards, studied the stability and initial safety of the CPI.Methods:1. Prescription of the CPI:SBE-β-CD was used as solubilizer to puerarin. The solubilization plot was drawed. and then the feed ratio of SBE-β-CD and puerarin was determined by the grad concentration method. The appearance and moisture of CPI with different concentrations of glucose, lactose, mannitol. and blank fluid was investigated by grouping score method to select the appropriate filler in order to determine the prescription of the CPI.2. Preparation process of the CPI:The impact of the different order of feeding and heating conditions on the dissolution rate of puerarin was investigated to determine the preparation process of CPI. The effects of different pre-freezing time and temperature, sublimation drying time and temperature, resolve drying time and temperature on the freeze-dried products was investigated by grouping score method to determine the freeze-dry technology of CPI.3. Quality control and standards of the CPI:To establish a method to determinate the concent and its related substances by RP-HPLC. We investigated the contents. related substances, moisture content. PH value.etc of the products to control the quality of the products. Then, establish the quality standards of CP1.4. Stability of the CPI:Three batches of CPI by enlarge experiment were produced and taken under the factor experiment, the accelerated experiment and the long-term experiment to study the stability of the product.5. Initial safety evaluation of the CPI:The safety inspection was divided into the allergic experiment, the hemolytic experiment, the stimulant experiment, the acute toxicity experiment and the general pharmacology experiment.(1)The allergic experiment:the body active allergy test was exploited to observe cavia cobayas allergic reactions after administration and to determine the allergic reaction extent;the passive skin allergy test was exploited to observe SD rats locus coeruleus response after administration and to record the diameter of the locus coeruleus.(2) The hemolytic experiment:to observe the hemolytic and agglomeration reaction of2%rabbit erythrocytes after adding CPI with clinically concentration.(3) The stimulant experiment:the corresponding pathological slicing of rabbit ears and muscle tissue was investigated to determine whether occurrence process or necrosis on medication side.(4) The acute toxicity experiment:to observe and record the reaction and death of KM mice after being given different concentrations of CPI.(5) The general pharmacology experiment:to compare the effects on the nervous system, respiratory and cardiovascular system of animals after being given CPI between the medication group and the physiological saline control group.Results:1. Prescription of the CPI:SBE-β-CD was a suitable solubilizer to puerarin and the linear relationship of solubilization curve was qualified(r=0.9997). The feed ratio of SBE-β-CD and puerarin was determined. The best prescription of CPI was X mg puerarin. Y mg catalpol and Z mg SBE-β-CD. prepared for the solution of2mL to be freeze-dried.2. Preparation process of the CPI:(1) Preparation of CPI solution:weigh each component by the best prescription, the order of feeding and heating temperature was determined.(2)Freeze-dry technology:①Pre-freezing:the and holding time was determined;②Sublimation drying:the vacuity and holding time was determined;③Resolve drying:the vacuity and temperature was determined.3. Quality control and standards of the CPI:(1)Contents and related substances determination methods:The RP-HPLC method was used, and the mobile phase was water-acetonitrile. gradient elution. The detection wavelength was210nm, and both catalpol and puerarin had a good linear in the range of5～200mg/L (r=0.9999). The contents was controlled within90%～110%, and related substances was controlled<2%.(2) The moisture content was controlled<3%.(3) The pH was controlled within4.5-6.5.4. Stability of the CPI:The result of stability of the CPI displaied:In the factor experiment, high temperature condition was a stronger factor to products than the conditions of high humidity and light. The results of the accelerated experiment and the long-term experiment showed that the contents, related substances, moisture content. pH value was relatively stable.5. Initial safety evaluation of the CPI:(1) The allergic experiment:treatment group of CPI did not appear body active allergy reaction; treatment group of did not cause obvious passive skin allergy in SD rats.(2) The hemolytic experiment:every tube of CPI did not appear hemolysis and agglomeration.(3) The stimulant experiment:medication side pathological slicing of rabbit ears and muscle tissue showed no significant process or necrosis.(4) The acute toxicity experiment:the LD50of the CPI in KM mice was1584.5mg/kg.(5) The general pharmacology experiment:there were no significant difference (P>0.05) between animals in the CPI group and the physiological saline negative control group.Conclusions:1. The CPI was a product with clear prescription ingredients and well stability. Its preparation method and process was simple, and its quality was easy to control:2. The CPI did not cause the allergic, irritant, hemolytic reactions, and the effect on animal general physiological functions was not obvious. Therefore, the toxicity of the product with a large range of security was low. This product was worth further developing and researching.