Research Of Xiangdan Injection On Safety Evaluation Of Compatibility Medication

In this study, active systemic allergies, local passive allergy, vitro hemolysis, intravenous administration of local irritation, muscle administration of local irritation and other special safety tests was used,that can evaluate the interaction of the medication group combining Xiangdan injection with dexamethasone and 654-2, to determine the safety of three drugs'combination.1. Active systemic allergic guinea pigs (ASA) test1.3Testing GroupTotally 36 male healthy guinea pigs were divided into 6 groups randomly,6 guinea pigs per group. The first group is the negative control (supplying 5% glucose injection). The second group is the positive control (supplying Ovalbumin 2 mg/per guinea pig).The third is Xiangdan injection group (supplying Xiangdan injection 1.6mL/kg).The fourth is dexamethasone sodium phosphate injection group (supplying dexamethasone sodium phosphate injection 0.8mg/kg).The fifth is Raceanisodamine Hydrochloride injection (supplying the racemic Raceanisodamine Hydrochloride injection 0.8mg/kg) and the sixth is medication group which combined Xiangdan injection with dexamethasone and 654-2 (supplying xiangdan injection 1.6mL/kg, dexamethasone sodium phosphate injection 0.8mg/kg and racemic Raceanisodamine Hydrochloride injection 0.8mg/kg).1.4 Sensitization and excitationSensitization All the animals were tested once every other day by intraperitoneal injection the appropriate drugs, five times in all. The dosage was 0.5mL/per animal.Excitation After the first administration 14 days and 21 days later, all groups were excited by injecting 2ml drug per animal in penile vein.60min later, observe immediately if they apper shaking, scratching the nose, sneezing, coughing,convulsions, difficulty breathing, incontinence, shock, death and other allergic reactions, recording the time of symptoms appearance and disappearance.1.5 ResultsResults showed that the six guinea pigs of ovalbumin positive control group appeared different degrees of standing hair, shivering, sneezing, coughing, difficulty breathing, wheezing, convulsions, incontinence and other obvious symptoms of an allergic reaction, all six guinea pigs died. While 21 days later the 30 guinea pigs does not appear allergic symptoms, in 5% glucose injection negative control group, Xiangdan injection group, dexamethasone sodium phosphate injection group, Raceanisodamine Hydrochloride injection group and the medication group which combined Xiangdan injection with dexamethasone and 654-2, and none of the animals died. That indicating the guinea pigs ASA test are conformed to the regulations in Xiangdan injection group, dexamethasone sodium phosphate injection group, Raceanisodamine Hydrochloride injection group and the medication group combining Xiangdan injection with dexamethasone and 654-2.2 Rat passive cutaneous anaphylaxis (PCA) test2.3 Testing groupTotally 24 healthy Wistar rats were taken and divided into 6 groups randomly,4 guinea pigs per group. The first is the negative control group (supplying 5% glucose injection). The second is the positive control group (supplying Ovalbumin). The third is Xiangdan injection group (supplying Xiangdan injection 1.8mL/kg).The fourth is dexamethasone sodium phosphate injection group (supplying dexamethasone sodium phosphate injection 0.9mg/kg).The fifth is Raceanisodamine Hydrochloride injection group (supplying the racemic Raceanisodamine Hydrochloride injection 0.9mg/kg) and the sixth is medication group combining Xiangdan injection with dexamethasone and 654-2 (supplying xiangdan injection 1.8mL/kg, dexamethasone sodium phosphate injection 0.9mg/kg and racemic Raceanisodamine Hydrochloride injection 0.9mg/kg).2.4 Sensitization and excitationSensitizationTaking 4 rats weighed 130～150g/per animal, they are divided into the negative control group (supplying 5% glucose injection), positive control group (supplying Ovalbumin), the Xiangdan injection group (supplying Xiangdan injection 1.8mL/kg), the dexamethasone sodium phosphate injection group (supplying dexamethasone sodium phosphate injection to 0.9mg/kg), the Raceanisodamine Hydrochloride injection group (supplying the racemic Raceanisodamine Hydrochloride injection 0.9mg/kg), and the medication group combining Xiangdan injection with dexamethasone and 654-2 (supplying Xiangdan injection 1.8mL/kg, dexamethasone sodium phosphate injection 0.9mg/kg, and hydrochloric acid racemic Raceanisodamine Hydrochloride injection 0.9mg/kg), one animal per group. Every other day, the rats should be sensitized by intraperitoneal injecting,5 times in all. Collectting blood as ten days after the last sensitization, centrifuged 2000×rpm ten minutes, separated the serum, and stored at-20℃, standby within two weeks.ExcitationPre-epilating the back of the animals on both sides of the rats 3×4cm2,is injected with the corresponding group of anti-serum in intradermal (diluted by sodium chloride injection into 1:2,1:8,1:32). Each dilution injected two points, 0.1mL of each point. After 24 hours, the rats were injected intraperitoneally with corresponding drugs. After medicine was supplied 30min later, injected 0.5% Evans blue 1mL in intravenous each group. The rats were decapitated 30min later, removing the skin where was injected with anti-serum using 1 cm punch, cutting into pieces and putting into 5mL acetone saline solution. The OD value was measured at 590nm after 24 hours.2.5 ResultsThe results show that the ovalbumin group is significantly higher (p<0.05) compired with 5% glucose injection group. The OD value of Xiangdan injection group, dexamethasone sodium phosphate injection group, Raceanisodamine Hydrochloride injection group and the medication group that combined Xiangdan injection with dexamethasone and 654-2 had no significant difference(p>0.05).That Indicates that Xiangdan injection group, dexamethasone sodium phosphate injection group, Raceanisodamine Hydrochloride injection group and the medication group combining Xiangdan injection with dexamethasone and 654-2 are conformed to the regulations of rat PCA tests.3. In vitro hemolysis testing3.4 Preparation of 2% erythrocyte suspensionRabbit blood was collect about 10mL, and placed in beakerflask containing glass beads and shaked 10 min. That is to remove fibrinogen and obtain the defibrinated blood.10 times sodium chloride injection was added, shaking and putting into centrifuge tubes, and the tubes were centrifuged at 2500 xrpm for 5min, and the supernatant was removed. Precipitation of red blood cells and then using sodium chloride injection mixed and centrifuged. Repeating several times until the supernatant was colorless and transparent only for the test. According to the volume, the red blood dubbed suspension by 2% sodium chloride injection for testing.3.5 Testing processTests are divided into five batches.The first batch of tested drugs was 5% glucose solution. Taking 7 tubes and to number it, from the numberl to number 5, added into 5% glucose control injection0.1,0.2,0.3,0.4,0.5mL,0.9% sodium chloride injection 2.4,2.3, 2.2,2.1,2.0mL,2.5mL 2% of the red blood cell suspension (RBC)is added into each tube. The sixth tube add only 0.9% sodium chloride pipe injection and 2.5mL 2% of RBC suspension as the blank control of the tube. The seventh tube is added 2.5mL 2% of RBC and distilled water only as the positive control hemolysis. Gently shake the tube after the water bath at 37℃, recording the results of 30min and results of 1h,2h,3h,4 h. The second, the third, the fourth and the fifth batche of the testing process are same with the first batch. The second is instead with Xiangdan injection.The third is instead with dexamethasone sodium phosphate injection. The fourth is instead with the Raceanisodamine Hydrochloride injection and the fifth for the medication group combining Xiangdan injection with dexamethasone and 654-23.6 ResultsThe results showed that the distilled water group showed a diaphanous red indicating that hemolysis have happened.In 5% glucose injection group,Xiangdan injection group, dexamethasone sodium phosphate injection group, Raceanisodamine Hydrochloride injection group, the medication group combining Xiangdan injection with dexamethasone and 654-2, it is visible that all red blood cells were sinked. The upper liquid is colorless and transparent, no reddish brown flocculent precipitate, indicating that the 5% glucose injection, Xiangdan injection,dexamethasone phosphate sodium injection, Raceanisodamine Hydrochloride injection and the medication group combining Xiangdan injection with dexamethasone and 654-2 have no hemolysis within 4h and no red blood cells gathering. Not any change at room temperature for 24h, indicates that has no hemolysis of rabbit red blood cells.4 Vascular irritation test in rabbits4.3Testing GroupTotally 30 rabbits were divided into 5 groups randomly. Each group has 6 rabbits. The first group is the control group (supplying 5% glucose injection).The second group is Xiangdan injection group (supplying Xiangdan injection 0.9mL/kg).The third group is dexamethasone sodium phosphate injection (supplying dexamethasone sodium phosphate injection to 0.45mg/kg).The fourth group is Raceanisodamine Hydrochloride injection group (supplying the racemic Raceanisodamine Hydrochloride injection 0.45mg/kg).The fifth medication group combining Xiangdan injection with dexamethasone and 654-2 (supplying xiangdan injection 0.9mL/kg, dexamethasone sodium phosphate injection 0.45mg/kg and racemic Raceanisodamine Hydrochloride injection 0.45mg/kg). The subjects of drug dissolved into 5% glucose injection 100ml.4.4 Testing processTaking 30 rabbits of either sex (females were not pregnant). In drug tested group, the rabbits ear vein is injucted with tested drugs 100ml every day. In the control group, the rabbits ear vein is injucted with 5% glucose of equal volume, once one day for 7 days. In addition, every drug administration was observed during and outside the administration of local reaction. After 24hours late on last time injuction, cut off ear where tested with drug. After conventional fixed, from the static point of the needle in the proximal of 1cm, cut 0.5 cm wide samples every 1 cm, three samples were taken. In addition to obevering whether the congestion, edema and other phenomenon by naked eye, make tissue sections stained for microscopic pathology observation.4.5 ResultsThe results show that, the blood vessel dilatation is visual but the bleeding and swelling is invisible in rabbit ear, in Xiangdan injection group, Raceanisodamine Hydrochloride injection group, and Xiangdan injection combined with dexamethasone and injection of 654-2.In 5% glucose injection group and dexamethasone sodium phosphate injection group, we can't obverse the rabbit ear blood vessel dilatation, bleeding and swelling. Under the microscope,5% glucose injection group and each other drug group, we can't obverse the blood vessel swelling, infiltration of inflammatory cell, necrosis on injection site. The experiments show that, the Xiangdan Injection, dexamethasone sodium phosphate injection, Raceanisodamine Hydrochloride injection, and the medication group combine Xiangdan injection with dexamethasone and 654-2, all test of intravenous injection of vascular irritation are compliance.5. Rabbit muscle stimulation test5.3Testing GroupTotally 10 rabbits were randomly divided into 5 groups, each group 2 rabbits. The first group is the control group (supplying 5% glucose injection).The second group is Xiangdan injection (supplying Xiangdan injection).The third group is Dexamethasone sodium phosphate injection group (supplying Dexamethasone sodium phosphate injection).The fourth group is Raceanisodamine Hydrochloride injection group (supplying racemic hydrochloride injection).The fifth medication group combined Xiangdan injection with dexamethasone and 654-2 (all to Xiangdan Injection, dexamethasone sodium phosphate injection, and racemic Raceanisodamine Hydrochloride injection.)5.4 Testing processTaking 10 rabbits, an equal number of males and females (Females do not pregnant). The number1 and number 2 rabbits are injected respectively on quadriceps of left and right shares with 5% glucoseion lml after disinfected with iodophors.By the same way, the Number3 and number 4 rabbits are injected respectively with Xiangdan injection lml.The Number5 and number 6 rabbits are injected respectively with dexamethasone sodium phosphate injection 1ml.The number7 and number 8 rabbits are injected respectively with Raceanisodamine Hydrochloride injection 1ml.The Number9 and number 10 rabbits are injected respectively with the injection combine Xiangdan with dexamethasone and 654-2 1ml.48 hours later, animals were killed by injected into the ear vein with air. Then the quadriceps was removed and the changes are observed, converted into the corresponding response level. After fixed by 10% formaldehyde, the quadriceps was submissed for detected it pathology. Under the microscope, we observe the Situation of the quadriceps such as infiltration of inflammatory cells, proliferation, necrosis, and so on.5.5 ResultsThe results showed that, the total of response leve in quadriceps of trabbits are all between 0 and 2, in 5% glucose injection group, Xiangdan injection group, dexamethasone sodium phosphate injection group, Raceanisodamine Hydrochloride injection group, and the medication group combine Xiangdan injection with dexamethasone and 654-2. Pathological observing of the quadriceps indicating that it does not apper infiltration of inflammatory cells, proliferation, necrosis, and so on. The experiments show that, all of the Xiangdan Injection, dexamethasone sodium phosphate injection, Raceanisodamine Hydrochloride injection, and the medication group combine Xiangdan injection with dexamethasone and 654-2 have no significantly stimulate in injection site and all test of muscles irritation are compliance.