| OBJECTIVES:To screen a sensitive anaphylactoid reaction experimental method with high accuracy by the anaphylactoid reaction experiment of traditional Chinese medicine injections on mice, guinea pigs and dogs, to investigate the addition of Tween80 in the traditional Chinese medicine injections by qualitative and quantitative determination of the Tween80, then study the anaphylactoid reaction of the injections containing Tween80 on the Beagle dogs and export the effect of Tween80 on the sensitization of traditional Chinese medicine injections, to find one of solutions to solve the sensitization of traditional Chinese medicine injections.METHODS:1. Anaphylactoid reaction experiment on mice. The mice were allergized by tail intravenously injection, the dosage was 15 times as the adult dosage, the delivery volume was 0.5ml every mouse, then behavior changes of the mice were observed and the contents of serum Histamine were detected. The result was determined according to the synthetic index of changes of behavior and the serum Histamine.2. Anaphylactoid reaction experiment on guinea pigs. The guinea pigs were allergized by intravenously injection, the delivery volume was 2ml every guinea pig, the dosage was 6 times as the adult dosage , then behavior changes of the guinea pigs were observed and the contents of serum Histamine were detected. The result was determined according to the synthetic index of changes of behavior and the serum Histamine.3. Anaphylactoid and allergical reaction experiments on Beagle dogs. The Beagle dogs were allergized by forelimb injecting constantly( at a speed of 5ml/min with an injection pump). On the first, third, fifth and the fourteenth day, the delivery volume was 5ml/kg, the dosage was 2 times as the adult dosage, the forme three times were allergic steps, the last time was exciting step, the dosage of the last time was 2 times as the first, behavior changes of the dogs were observed , blood was drawn before and after the drug delivery, the contents of serum Histamine, immunoglobulin E , immunoglobulin G and immunoglobulin E were detected. The result was determined according to the synthetic index of changesof the behavior and the serum Histamine.4. The qualitative and quantitative determination methods of Tween80.4.1 Qualitative determination of Tween80.4.1.1 Cobalt thiocyanate ammonium color-developing method: The tested material were added in the 2ml cobalt thiocyanate ammonium color-developings in a tube, shaken up, then 1ml Chloroform was added in the tube , shaken up and standed for 1.5h, the colour of Chloroform layer were observed. Result determinning: the blue colour of Chloroform layer was determined positive, light blue was weak positive, green was susipicious, the others were negative. 4.1.2 Silicon thin-layer chromatography method.4.1.2.1 The condition of thin-layer chromatograph. Benthing with a mixed solvent of MeoH-H2O-HCOOH(8:5:1) as the developer, the test material can be developed well.4.1.2.2 Spotting and developing. Taking some tested material, spotting about 0.5cm from the bottom of the thin-layer plate, finished, blowing dry, putting the plate in the iodine jar, taking out when the developing length was 8cm, blowing dry the solvent, 15min later, observing the colour and position of the dots. The result was determined as positive when the dot was yellow and clear and the position was on the same line with the positive spot, weak positive when the colour was not clear, negative when there is no yellow spot in the corresponding position.4.2 Quantitative determination of Tween80: colorimetic method was used to determine the content of Tween80. The method was studied and the sample were determined.RESULTS:1. Results of the anaphylactoid reaction experiment on mice. According to the synthetic index of behavior changes and the serum Histamine, Double Coptis and Mailuoning were determined as positive, the Houttuynia cordata , astragalus and shenmai were susipicious, which were not conform with the clinical export.2. Results of the anaphylactoid reaction experiment of 10 traditonal Chinese medicine on guinea pigs. According to the synthetic index of behavior changes and the serum Histamine, Double Coptis was determined as susipicious, the others were negative, which were not conform with the clinical export. 3. Results of the anaphylactoid reaction experiment of 10 traditonal Chinese medicine injections on Beagle dogs. According to the synthetic index of changes of the behavior and serum Histamine, the 10 traditonal chinese medicine injections were determined as positive. There were no false negative, which were conform with the clinical export.4. Forty-three traditional Chinese medicine injections were determined. Fifteen species including the Houttuynia cordata injection and so on were checked out containing Tween80, the content were from 0.72±0.00 to 6.85±0.02 mg.ml-1 respectively. The others were not checked out.5. Among the 15 traditional Chinese medicines injections, there were 8 species which illustrating the addition of Tween80 openly, Delsheng, kang,ai and Xuebijing were not founded in the experiments. So the species which have no illustrations about the additions of Tween 80 but checked out in the experiment were Shengmai, Fufang kushen and Zhengqingfentongning.6. Results of the anaphylactoid reaction experiment of 5 traditonal Chinese medicine injections including Tween 80 on Beagle dogs. The dogs showed serious dystrophy, according to the synthetic index of changes of the behavior and serum Histamine, the 5 injections were determined as positive or strong positive .7. Results of the allergical reaction experiment of 5 traditonal Chinese medicine injections containing Tween 80 on Beagle dogs. The dogs showed serious dystrophy . According to the synthetic index of changes of the behavior and serum Histamine, the 5 injections containing Tween 80 were determined as positive or strong positive .CONCLUSIONS: 1. Anaphylactoid reaction experiment of the traditional Chinese medicine injections which rank the tops in the anaphylactic shock clinical reports on the mice, guinea-pigs and Beagle dogs were carried. The results show that dog is the best experiment animal , the result determination depend on the synthetic index of changes of the behavior and serum Histamine, IgE is the assistant determination index. The experiment result are accuate , and conform with the clinical report. There are no false negative.2. The results are accurate when the content determination of Tween 80 in the traditional chinese injections by the Cobalt thiocyanate ammonium color-developing method and silicon thin-layer chromatography.3. The additions of Tween80 of traditional Chinese medicine injections should be supervised strongly and standarded. The detect and the security evalution of excipient in the traditional Chinese medicine injections should be enhanced, especially the species which do not illustrate the addition of the Tween80. The qualitative and quantitative determination of Tween80 should be carried to study to evaluate and enhance the security of the traditional Chinese medicine, which offer the evidence for clinic medication.4. The serious adverse reaction of traditional Chinese medicine injections containing Tween80 is relate to the Tween80 . So Tween80 should be banned to add in the tradictional Chinese medicine injections step by step. Securial and reasonable medical excipient should be found to subsititute Tween80.5. A lot of experimental studies and clinical useness showed that some traditional Chinese medicine injections existed some hidden danger, especially the species rank in the tops of clinical report of adverse faction. The questions of them are more sierous. A system study shoule be carried as soon as quickly to solve this problem fundamentally from the source. Only the medicine quality ,security and validity can increase by this. |
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