Study Of Adverse Reaction Material In Xueshuantong Injection

Background:Xueshuantong injection is widely used for cardiovascular disease in recent vasodilator agents,which is made from the panax notoginseng saponins(PNS) in radix notoginseng.It can dilation the blood vessels, improve the blood circulation, increase the cerebral blood flow, decrease the peripheral vascular resistance, dilatate the coronary artery, significantly reduce myocardial oxygen consumption by lowering cardiac after-load, direct dissolution of blood stasis in the body, dredge the blocked vessels, improve the function of vascular permeability. So it has good effect on the disease such as the cardiovascular system, endocrine system, respiratory system and it also is used for the surgery, the orthopedics, the ophthalmology in clinical.With the increasing application in clinical, the adverse reaction (ADR) increased gradually and public is concern about the safety of the Traditional Chinese Medicine injection(TCMI).The main adverse reactions of Xueshuantong injection is allergic.The most common is rash in allergic, and the allergic shock, exfoliative dermatitis eruption, bullous epidermolytic epispasis are rare but very serious. Other adverse reactions include the swelling and pain, subcutaneous bleeding, fever, arthritis, muscle pain and low blood potassiums and so on.Unlike the allergic reaction of other chemical drug, such as the spenicillin, most The allergic reactions of TCMI in clinical is a similar allergy but not the true allergy, Which is a seriously threat to the life safety of the user. There are many kinds of reasons about the allergies of TCMI. The studies of the safety evaluation focus on the overall of TCMI in modern research and There are no further study in revealing its allergic component or the quality standards of TCMI. So far, the animal model is one of the most important ways to detective the allergic component of drug. The vivo experimental models that the guidelines of drug safety research in our country recommended not only can not predict potential allergic of TCMI accurately, but also lack the correlation between animal experiments and clinical. The new research reported that the cell model can be used in TCMI anaphylactic research in some literature. The current cell model that established with various cells sensitive and repeatable, the result also has certain correlation with clinical reports.It suggest that the cell model has the potential application value in evaluating the anaphylactoid reaction of TCMIThe main effective component of Xueshuantong injection is total saponins, which may mostly caused the allergy and hemolysis in clinical due to its irritation. Most studies of ADR about Xueshuantong injection only focus on the overall level and do not indicate the specific internal components that cause the ADR in Xueshuantong injection. According to the good clinical efficacy of Xueshuantong injection in cardiovascular disease and its ADR in clinical, we try to find out the component that cause the ADR in the separated parts of Xueshuantong injection.Objectives1、To improve the P815cell degranulation model; 2、To screen the allergic material in the separated parts and components of PNS in Xueshuantong injection;3、To screen the hemolytic component in the separated parts and components of PNS in Xueshuantong injection;4、Based on the last two test,we try to explore ADR material in the Xueshuantong injection.Methods1、The separation of PNS in Xueshuantong injection. With the PNS in Xueshuantong injection as raw material, using acetonitrile-water solution of different concentration as separation mobile phase,the PNS was separated into different parts and component with preparative liquid chromatography. At last,the separated things was in quantitative analysis By high performance liquid chromatography.2、A high-performance liquid chromatograph tandem mass spectrometric (LC-MS/MS) method was developed for the determination of histamine in the supernatant after degranulation of P815cells. After pretreatment procedure, the sample was separated on a C18(4.6mm×100mm,3.5um) column using acetonitrile-0.5mmol/L ammonium acetate solution with0.3%formic acid (60:40) as mobile phase at a flow rate of0.6mL/min. The target compound was detected by positive ion ionization mass spectrometry(ESI MS) under multiple reaction monitoring (MRM) mode using m/z112>95and m/z112>68as detective ion. Several TCMI commonly used effect on degranulation of P815cells. Histamine were determined by LC-MS/MS method and ELISA method, and compare the difference between the result;3、Perfect the in vitro model of P815cell Degranulation. The enzyme of the aminoglycoside net release determination, and degranulation cell rate of neutral red staining determination. Finally, The in vitro cytotoxicity comparison will do among the L929, P815cell and RAW264.73cell.4、Screen the allergic material in the separated parts and components of PNS in Xueshuantong injection:The separated parts are screening on P815cells in vitro allergy model that established. First the IC50value of separation of PNS will be determined. And secondly the IC50value will be the determination concentration. The histamine is analyzed by ELISA method; The aminoglycoside net release is detective as the substrate. The number of degranulation cells is measure by neutral red. At last the result will be judged by three item data.5、The in vitro hemolysis test about separated parts of PNS in Xueshuantong injection. Spectrophotometry is applicated in the PNS that preparatived into different parts after with rabbit red blood cells, the hemolysis rate of supernatant is detection in575nm.Results1、The separation of PNS in Xueshuantong injection:The PNS is separated into2parts and1monomer the first step The monomers is Ginsenoside R1. The4monomers and4fractions were obtained in the second step. The monomer respectivelyis Ginsenoside Rbl, Ginsenoside Rd, monomer Rgl and monomer t=35.35min.2、Under the optimal condition, the analysis of sample could be completed within6min. A good linear relationship was obtained for histamine in the range of4.0-128.0μg/L with a correlation coefficient of0.9979. The limit of quantitation (S/N>10) and limit of detection (S/N=3) were1.2μg/L and0.3μg/L, respectively. The mean recoveries for histamine in the supernatant of cells at three spiked concentration levels of16、64、128μg/L were in the range of80%-94%with relative standard deviations (RSDs)(n=6) of4.1%-5.9%. The proposed method was simple, sensitive and reproducible, and was suitable for the determination of histamin in the supernatant of Traditional Chinese Medicine Injection on degranulation in P815cells.3、Toxicity test among the P815cells、L929、RAW264.7. The proliferation rate between P815cell and L929cell、RAW264.7cell is significant difference (P<0.01); Compared to. The cell proliferation rate is no statistical significance between L929cells and RAW264.7cells. At the same time, there is less influence and toxicity after TCMI on non-mast cells (L929cells and RAW264.7cells) cell, while the result TCMI on mast cell line (P815cell) on the contrary.Filter out the P815cell sensitization of three methods of detection of several commonly used commercial TCMI in twelve kinds of injections were detected, three methods of measuring results are in line. Which the trichosanthin, gastrodin, red fennel injection three indicators with the negative control group were significant differences, namely (P<0.01), prompted more than Chinese medicine injection of P815cells have a strong allergenic; The three indexes of breviscapine, Noki Uri and ligustrazine hydrochlori with the control group compared to the significant difference (P<0.01), and the other compared to with the control group show difference (P <0.05), suggesting that breviscapine, Noki Uri and ligustrazine hydrochloride injection sensitization of P815cells exist. Angelica compound released in beta hexosamine ratio and cell degranulation rate with the blank control group compare to the significance of difference (0.01<P<0.05), suggesting that three injections may be sensitized to P815cells; relatively Shenmai, Shenqi Injection in the three indexes with the control group is no significant differences suggesting that two injections on P815cells sensitization is relatively weak. Filter out the P815cell sensitization of three methods of detection of separated parts of PNS:three methods of measuring results are in line. Which the part2and3、 RB1PNS teams three indicators with the negative control group were significant differences, namely (P<0.01), prompted these material on P815cells have a strong allergenic. The released in beta hexosamine ratio and histamine of part4with the blank control group compare to the significance of difference (P<0.01), suggesting that it may be sensitized to P815cells; The released in beta hexosamine ratio of RG1with the blank control group compare to the significance of difference (P<0.01), and histamine of RG1with the blank control group compare to the significance of difference (0.05<P<0.01), suggesting that it may be sensitized to P815cells; Part1and6and Monomer2and RSD in the three indexes with the control group is no significant differences suggesting that these material on P815cells sensitization is relatively weak.4、 Parts5, Xueshuantong Injection, and the ingredients in vitro hemolytic experiment:PNS, part1,2, and5of the hemolytic is not obvious, only under high concentration (>4mg/ml) showed mild hemolytic, while low concentration (<basic no hemolytic under4mg/ml). And PNS and parts2,3,4,6, under the concentration (>1mg/ml) has certain hemolytic, only in the concentration of1mg/ml) hemolytic is not obvious.Conclusion1、The proposed method was simple, sensitive and reproducible, and was suitable for the determination of histamin in the supernatant of Traditional Chinese Medicine Injection on degranulation in P815cells.2、P815may be a new detection exact effect for the class of traditional Chinese medicine injection allergic reactions fast and stable allergens in vitro screening model.3、PNS and parts2,3,4, and is also likely has certain promoting cell degranulation of material, probably because of its smaller polarity, poorly soluble in water, large proportion of the PNS and so on; At the same time, it contains high content of Ginsenoside Rbl and other Ginsenoside Rbl and Rgl that may exist to promote cell degranulation of material. Ginsenoside Rbl belongs to the Ginsenoside glycol type, Rgl belong to three glycol type saponins of ginseng, they is the largest contents in PNS. Parts1and1(unknown), Ginsenoside Rd and Ginsenoside Re prompted the less allergenic substances, including1part of polarity, soluble in water, as a proportion of the PNS is less; Ginsenoside Re content is less, and larger polarity, soluble in water. To sum up:The P815degranulation of xueshuantong injection may be related to the content and its polarity monomer.4、PNS and parts of1at high concentration have hemolytic, and parts of the2,3,4and6in addition to higher concentrations with hemolytic, at low concentrations also showed hemolytic strong. At the same time, it shows that hemolytic depends on the polarity. The stronger polarity,the smaller hemolytic.