CN103484524A - Pharmacodynamic and toxicologic test methods for honeysuckle and aster immunoenhancer - Google Patents
Pharmacodynamic and toxicologic test methods for honeysuckle and aster immunoenhancer Download PDFInfo
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- CN103484524A CN103484524A CN201310442426.8A CN201310442426A CN103484524A CN 103484524 A CN103484524 A CN 103484524A CN 201310442426 A CN201310442426 A CN 201310442426A CN 103484524 A CN103484524 A CN 103484524A
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Abstract
The invention discloses pharmacodynamic and toxicologic test methods for a honeysuckle and aster immunoenhancer. A pharmacodynamic test method comprises the following steps: preparing an immunoenhancer, preparing common pathogenic bacteria in a farm, carrying out an immunoenhancer antibacterial test, carrying out a test of a fever abatement effect of the immunoenhancer on a rabbit model with a fever, and carrying out a test of an effect of the immunoenhancer on a small white mouse model with cough and asthma. A toxicologic test method comprises the following steps: carrying out a small mouse acute toxicity test, carrying out a rat chronic toxicity test, and analyzing test results. The test methods provided by the invention show that an immunoenhancer preparation has remarkable effects of resisting bacteria, abating the fever and relieving the cough and asthma; a hematological examination shows that various immunity indexes of a Chinese herbal medicine group are higher than those of a blank control group; the toxicity tests prove that body weights, important organ coefficients and visual inspection results of test animals eating the honeysuckle and aster immunoenhancer are not remarkably different or abnormal and the honeysuckle and aster immunoenhancer is an efficient and safe Chinese herbal medicine preparation.
Description
Technical field
The invention belongs to clinical medicine domain, relate in particular to a kind of pharmacodynamics and toxicology test method of Japanese Honeysuckle extraction chrysanthemum immunostimulant.
Background technology
Japanese Honeysuckle extraction chrysanthemum immunostimulant is basic theory and the diagnosis and treatment based on an overall analysis of the illness and the patient's condition principle according to motherland's medical science, pathophysiological features in conjunction with livestock and poultry, combine the efficient immunomodulator that some herbal medicine that can transfer the animal body systemic immune system develop, said preparation have significantly antibiotic, bring down a fever and function of relieving cough and calming asthma, in these years successively in a plurality of plants, carry out clinical trial and applied research, obtained good effect.In order to verify clinical drug effect and the security of this preparation, now need to be to Japanese Honeysuckle extraction chrysanthemum immunostimulant be carried out to pharmacodynamics and toxicology test research.
Summary of the invention
The object of the present invention is to provide a kind of pharmacodynamics and toxicology test method of Japanese Honeysuckle extraction chrysanthemum immunostimulant, be intended to verify clinical drug effect and the security of Japanese Honeysuckle extraction chrysanthemum immunostimulant.
The present invention is achieved in that the pharmacodynamic experiment method of Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises that the preparation, Japanese Honeysuckle extraction chrysanthemum immunostimulant bacteriostatic test, the Japanese Honeysuckle extraction chrysanthemum immunostimulant that prepare Japanese Honeysuckle extraction chrysanthemum immunostimulant, plant's common pathogen kind extract test, the interpretation of chrysanthemum immunostimulant on the impact of small white mouse IgM, C3 and C4 on experiment, the Japanese Honeysuckle extraction chrysanthemum immunostimulant of the antipyretic effect of pyrogenicity Rabbit Model to experiment, the Japanese Honeysuckle that coughs the effect of breathing heavily Mouse Model.Its concrete steps are as follows:
The first step, preparation Japanese Honeysuckle extraction chrysanthemum immunostimulant.The prescription of Japanese Honeysuckle extraction chrysanthemum is comprised of 10 Chinese herbal medicines such as Japanese Honeysuckle, extraction chrysanthemum, Herba Houttuyniae, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), the capsule of weeping forsythia, the root of large-flowered skullcap, Root of Indigowoad, the Radix Astragali, radix bupleuri, Radix Glycyrrhizaes, the herbal medicine of selected above-mentioned various genuine high-qualitys during preparation, after taking by a certain percentage, be ground into coarse meal, after being soaked in water 30 minutes, being placed in pot and decocting 3 times, merge filtrate three times, be condensed into 1:1 decoction (by every ml liquid, containing the former medicine of 1g), the bottling sterilizing is standby.
Second step, the preparation of plant's common pathogen kind.Select the common several pathogenic bacterium of plant (streptococcus aureus, intestinal bacteria, Pseudomonas aeruginosa, dysentery bacterium and paratyphosum Bacterium), bacterial strain obtains from certain plant's separation and Culture, identify and preserve and provide by transmissible disease teaching and research room of animal science institute of University Of Agriculture and Forestry In Fujian, the cultivation breeding of bacterium adopts the ordinary nutrient agar substratum, concrete preparation method is for after dissolving nutrient agar medium fully, after 121 ℃, 20min autoclaving, pour culture dish into, be laid into the substratum of 3～5mm, standby after cooled and solidified; Inoculation adopts transfering loop and method of scoring; Inoculation is placed on 37 ℃ of constant incubators and cultivates 24h, is placed in 4 ℃ of refrigerators and saves backup.
The 3rd step, Japanese Honeysuckle extraction chrysanthemum immunostimulant bacteriostatic test.In the Chinese herbal medicine decoction concentrated solution that the circular filter paper sheet autoclaving that is 6mm by diameter, drying are placed on 1:1 concentration, infiltrate standby, then in sterile cabinet, adopt aseptic method to spread upon equably on nutrient agar with different spreading rods by the fresh culture thing of above-mentioned bacterial strains respectively, media surface is close at the even interval of the scraps of paper that will infiltrate again herbal medicine, adjustment makes its contact well be placed on 37 ℃ of constant incubators by paper-pressing sheet and cultivates 24h, then take out and observe its inhibition zone, measure antibacterial circle diameter D (mm) and record its result.Criterion is according to " pharmacology of Chinese materia medica " and " antibacterials drug sensitive test criterion " regulation, and antibacterial circle diameter≤10mm is muting sensitive, and antibacterial circle diameter >=10～18mm is medium sensitivity, and antibacterial circle diameter >=18mm is extremely sensitive.
The 4th step, the experiment of Japanese Honeysuckle extraction chrysanthemum immunostimulant to the antipyretic effect of pyrogenicity Rabbit Model.Some for the examination large ear rabbit,, select 18 of healthy anosis, the qualified large ear rabbits of body temperature, the standard range that body temperature is qualified is 38.6-39.5 ℃, survey the anus temperature every day 3 times, after prerun 3d, then be divided at random 3 groups, test front fasting 12h but can't help water, 30min before on-test, survey the anus temperature 3 times, each 5min, get the basal body temperature of its mean value as this test rabbit again.Carry out Rabbit Model according to herbal medicine pharmacology test method inquiry and make disease, give every experimental rabbit auricular vein injection 2,30min after 2, 4-dinitrophenol 1ml/100g (15mg/kg) rabbit pyrogenicity to be tested, according to 3 groups of different grouping drug treatments, wherein A group employment catheter per os is to only pouring into Japanese Honeysuckle extraction chrysanthemum concentrated solution 10ml/kg/ in the rabbit stomach; The B group gives aspirin phenacetin caffeine 100mg/kg-1; C group control group only gives physiological saline 10mi/kg/; Laboratory animal all adopts the administration by gavage administration.After administration 30,60,90,120,180,240,300,360min is determination test rabbit anus temperature 3 times respectively, gets its mean value, and usings the difference of this test rabbit body temperature and basal body temperature and carry out the t check as observation index, and the comparison Chinese-western medicine is tested the antipyretic effect of rabbit to each group.
The 5th step, Japanese Honeysuckle extraction chrysanthemum immunostimulant is to coughing the experiment of the effect of breathing heavily Mouse Model.For 30 of examination small white mouses, be divided at random 3 groups, every heavy 25 ± 5g, male and female half and half dual-purpose.A group Japanese Honeysuckle extraction chrysanthemum oral liquid group 0.5ml (20g/kg)/only wherein; B group compound codeine syrup 30mg/kg; C group physiological saline control group 0.5ml (20g/kg)/only; Laboratory animal all adopts the administration by gavage administration, test each administration of upper and lower noon of first day 1 time, after testing the 2nd day administration in morning 1h, laboratory animal is put into to the boxes and baskets that the constant-pressure atomization device is housed, in under constant voltage, strong aqua evenly being entered to boxes and baskets with atomize, pressure on weather gage is advisable with 0.2 normal atmosphere or 18.7kPa (140mmHg), spray time is about 30min, observe the cough symptom time of occurrence of respectively organizing laboratory animal, typical case's cough is shunk for the small white mouse abdominal muscle, magnify mouth simultaneously, and s cough is arranged.Calculate latent period and the antibechic rate of the cough symptom of each treated animal.
The 6th step, the test of Japanese Honeysuckle extraction chrysanthemum immunostimulant on the impact of small white mouse IgM, c3 and c4.For 60 of examination small white mouses, every heavy 25 ± 5g, male and female half and half dual-purpose, be divided into 3 large groups at random, 20 of every large groups, every large component becomes two groups, and 10 of every groups, be divided into control group and experimental group.Every group experiment small white mouse feed corresponding Japanese Honeysuckle extraction chrysanthemum oral liquid 0.3ml/ only/d; Control group is filled with and is fed physiological saline, 0.3ml//d.Raise two weeks continuously, carry out blood sampling examination (eyeball blood sampling).Small white mouse is taken out from cage, and the right hand is mentioned tail and is rotated rapidly, makes it dizzy, then, on the ground, drag tail after the right hand, while leaping up before mouse, left index finger and thumb are clamped rapidly the skin after its ear, again tail is held in the palm, makes its eyeball outstanding, with tweezers, eyeball is extracted, the blood oozed is lived with little clear bottle graft, puts signs on.The blood of adopting is joined in the centrifuge tube of carrying out mark accordingly, and low-temperature centrifugation 8000rpm * 3min, get supernatant liquor, in-20 ℃ of preservations.The blood of adopting is joined in the centrifuge tube of carrying out mark accordingly, and low-temperature centrifugation 8000rpm * 3min, get supernatant liquor, in-20 ℃ of preservations.Do not add the centrifugal immediately of antithrombotics, get serum.The second the People's Hospital, Fujian Province is entrusted in the detection of immunoglobulin content, and detection method adopts immunoturbidimetry.
The 7th step, interpretation.
Further, the pharmacodynamic experiment animal of Japanese Honeysuckle extraction chrysanthemum immunostimulant is for supplying 90 of examination small white mouses, every heavy 25 ± 5g, 18 of large ear rabbits, body weight 1.5 ± 0.25kg.
Further, the pharmacodynamic experiment instrument of Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises constant-pressure atomization device (being comprised of glass atomizer, air compressor machine, tensimeter and animal container), whizzer (model, the FT120 type), microplate reader (model, the P-E4100 type), constant incubator (model, DU640).
The virusology experimental technique of Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises chmice acute toxicity test, rat long term toxicity test, interpretation.Concrete steps are as follows:
The first step, the chmice acute toxicity test.Comprise 1 maximum dosage-feeding determination experiment and 1d maximum dosage-feeding determination experiment.
1 time maximum dosage-feeding determination experiment step is as follows: by 40 mouse, male and female half and half, be divided into 2 groups at random, and control group and Japanese Honeysuckle extract chrysanthemum immunostimulant group, 20 every group, male and female half and half.Water 12h is prohibited in fasting, presses 40mL/kg dosage gavage, and control group is given with amount distilled water, observes 7d, records toxic reaction symptom and the death condition of mouse; Put to death after experiment finishes, dissect, the body weight of 7d before the administration of weighing mouse and after administration.Visual inspection mouse main organs lesion tissue situation.
1d maximum dosage-feeding determination experiment step is as follows: by 40 mouse, male and female half and half, be divided into 2 groups at random, and 20 every group, male and female half and half.Water 12h is prohibited in fasting, presses the 3 times/d of Japanese Honeysuckle extraction chrysanthemum immunostimulant gavage of 40mL/kg dosage, every minor tick 8h, and control group adopts with amount distilled water gavage, observes 7d, records toxic reaction symptom and the death condition of experiment mice; Experiment is put to death after finishing.Visual inspection mouse main organs lesion tissue situation.
Second step, the rat long term toxicity test.After rat prerun is observed to 7d, be divided at random 4 groups by sex and body weight, male and female half and half.The 1st group is that to give with amount distilled water, the 2nd, 3,4 groups be high, medium and low 3 the class dosage group (20.0g of Japanese Honeysuckle extraction chrysanthemum immunostimulant experimental group to the blank group, 10.0g, 5.0g crude drug/kg), every group 20, each group is all by gastric infusion, dosage is 20ml/kg.1 time/d, successive administration 2 months (8 weeks).Claim weekly the laboratory animal body weight 2 times before administration and during administration, and suitably adjust dosage according to body weight change.Experimental session is observed rat sign, general state, registers its food ration every day, drinking-water and ight soil situation.24h after the last administration, each group is all got 10 animals, male and female half and half; Cut open after execution to kill and get blood, check hematology and blood biochemical index.Get after blood and cut open immediately internal organs such as getting the animal heart, liver, spleen, lung, kidney, testis (epididymis), ovary, uterus, duodenum, brain, suprarenal gland, Tiroidina, thymus gland and weigh, according to weight calculator official weight coefficient (organ coefficient).By above-mentioned internal organs visual inspection, it changes, and then by 10% formaldehyde solution, fixes, and makes above-mentioned organs and tissues section, and its histopathology of microscopy changes, and observes the impact of immunostimulant on the rats'liver renal function.
The 3rd step, interpretation.
The present invention is by pharmacodynamics and toxicology test research to Japanese Honeysuckle extraction chrysanthemum immunostimulant, show, Japanese Honeysuckle extraction chrysanthemum immunostimulant agent formulation have significantly antibiotic, bring down a fever and function of relieving cough and calming asthma, during hematological examination also shows, every immune indexes of herbal medicine group is higher than the blank group; Toxicity test also proves that laboratory animal body weight, vitals organ coefficient and the visual inspection of using this medicine are all without obvious difference or abnormal, proves that thus Japanese Honeysuckle extraction chrysanthemum immunostimulant is a kind of efficient, safe Chinese herbal and crude drugs preparations.
The accompanying drawing explanation
Fig. 1 is the schema of the pharmacodynamic experiment method of Japanese Honeysuckle extraction chrysanthemum immunostimulant.
Fig. 2 is the schema of the virusology experimental technique of Japanese Honeysuckle extraction chrysanthemum immunostimulant.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Fig. 1 shows the flow process of the pharmacodynamic experiment method of Japanese Honeysuckle extraction chrysanthemum immunostimulant.As shown in the figure, the pharmacodynamic experiment method of Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises:
Preparation Japanese Honeysuckle extraction chrysanthemum immunostimulant S101;
The preparation S102 of plant's common pathogen kind;
Japanese Honeysuckle extraction chrysanthemum immunostimulant bacteriostatic test S103;
The experiment S104 of Japanese Honeysuckle extraction chrysanthemum immunostimulant to the antipyretic effect of pyrogenicity Rabbit Model;
Japanese Honeysuckle extraction chrysanthemum immunostimulant is to coughing the experiment S105 of the effect of breathing heavily Mouse Model;
The test S106 of Japanese Honeysuckle extraction chrysanthemum immunostimulant on the impact of small white mouse IgM, c3 and c4;
Interpretation S107.
Its concrete steps are as follows:
The first step, preparation Japanese Honeysuckle extraction chrysanthemum immunostimulant.The prescription of Japanese Honeysuckle extraction chrysanthemum is comprised of 10 Chinese herbal medicines such as Japanese Honeysuckle, extraction chrysanthemum, Herba Houttuyniae, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), the capsule of weeping forsythia, the root of large-flowered skullcap, Root of Indigowoad, the Radix Astragali, radix bupleuri, Radix Glycyrrhizaes, the herbal medicine of selected above-mentioned various genuine high-qualitys during preparation, after taking by a certain percentage, be ground into coarse meal, after being soaked in water 30 minutes, being placed in pot and decocting 3 times, merge filtrate three times, be condensed into 1:1 decoction (by every ml liquid, containing the former medicine of 1g), the bottling sterilizing is standby.
Second step, the preparation of plant's common pathogen kind.Select the common several pathogenic bacterium of plant (streptococcus aureus, intestinal bacteria, Pseudomonas aeruginosa, dysentery bacterium and paratyphosum Bacterium), bacterial strain obtains from certain plant's separation and Culture, identify and preserve and provide by transmissible disease teaching and research room of animal science institute of University Of Agriculture and Forestry In Fujian, the cultivation breeding of bacterium adopts the ordinary nutrient agar substratum, concrete preparation method is for after dissolving nutrient agar medium fully, after 121 ℃, 20min autoclaving, pour culture dish into, be laid into the substratum of 3～5mm, standby after cooled and solidified; Inoculation adopts transfering loop and method of scoring; Inoculation is placed on 37 ℃ of constant incubators and cultivates 24h, is placed in 4 ℃ of refrigerators and saves backup.
The 3rd step, Japanese Honeysuckle extraction chrysanthemum immunostimulant bacteriostatic test.In the Chinese herbal medicine decoction concentrated solution that the circular filter paper sheet autoclaving that is 6mm by diameter, drying are placed on 1:1 concentration, infiltrate standby, then in sterile cabinet, adopt aseptic method to spread upon equably on nutrient agar with different spreading rods by the fresh culture thing of above-mentioned bacterial strains respectively, media surface is close at the even interval of the scraps of paper that will infiltrate again herbal medicine, adjustment makes its contact well be placed on 37 ℃ of constant incubators by paper-pressing sheet and cultivates 24h, then take out and observe its inhibition zone, measure antibacterial circle diameter D (mm) and record its result.Criterion is according to " pharmacology of Chinese materia medica " and " antibacterials drug sensitive test criterion " regulation, and antibacterial circle diameter≤10mm is muting sensitive, and antibacterial circle diameter >=10～18mm is medium sensitivity, and antibacterial circle diameter >=18mm is extremely sensitive.
The 4th step, the experiment of Japanese Honeysuckle extraction chrysanthemum immunostimulant to the antipyretic effect of pyrogenicity Rabbit Model.Some for the examination large ear rabbit,, select 18 of healthy anosis, the qualified large ear rabbits of body temperature, the standard range that body temperature is qualified is 38.6-39.5 ℃, survey the anus temperature every day 3 times, after prerun 3d, then be divided at random 3 groups, test front fasting 12h but can't help water, 30min before on-test, survey the anus temperature 3 times, each 5min, get the basal body temperature of its mean value as this test rabbit again.Carry out Rabbit Model according to herbal medicine pharmacology test method inquiry and make disease, give every experimental rabbit auricular vein injection 2,30mi n after 2, 4-dinitrophenol 1ml/100g (15mg/kg) rabbit pyrogenicity to be tested, according to 3 groups of different grouping drug treatments, wherein A group employment catheter per os is to only pouring into Japanese Honeysuckle extraction chrysanthemum concentrated solution 10ml/kg/ in the rabbit stomach; The B group gives aspirin phenacetin caffeine 100mg/kg-1; C group control group only gives physiological saline 10ml/kg/; Laboratory animal all adopts the administration by gavage administration.After administration 30,60,90,120,180,240,300,360min is determination test rabbit anus temperature 3 times respectively, gets its mean value, and usings the difference of this test rabbit body temperature and basal body temperature and carry out the t check as observation index, and the comparison Chinese-western medicine is tested the antipyretic effect of rabbit to each group.
The 5th step, Japanese Honeysuckle extraction chrysanthemum immunostimulant is to coughing the experiment of the effect of breathing heavily Mouse Model.For 30 of examination small white mouses, be divided at random 3 groups, every heavy 25 ± 5g, male and female half and half dual-purpose.A group Japanese Honeysuckle extraction chrysanthemum oral liquid group 0.5ml (20g/kg)/only wherein; B group compound codeine syrup 30mg/kg; C group physiological saline control group 0.5ml (20g/kg)/only; Laboratory animal all adopts the administration by gavage administration, test each administration of upper and lower noon of first day 1 time, after testing the 2nd day administration in morning 1h, laboratory animal is put into to the boxes and baskets that the constant-pressure atomization device is housed, in under constant voltage, strong aqua evenly being entered to boxes and baskets with atomize, pressure on weather gage is advisable with 0.2 normal atmosphere or 18.7kPa (140mmHg), spray time is about 30min, observe the cough symptom time of occurrence of respectively organizing laboratory animal, typical case's cough is shunk for the small white mouse abdominal muscle, magnify mouth simultaneously, and s cough is arranged.Calculate latent period and the antibechic rate of the cough symptom of each treated animal.
The 6th step, the test of Japanese Honeysuckle extraction chrysanthemum immunostimulant on the impact of small white mouse IgM, c3 and c4.For 60 of examination small white mouses, every heavy 25 ± 5g, male and female half and half dual-purpose, be divided into 3 large groups at random, 20 of every large groups, every large component becomes two groups, and 10 of every groups, be divided into control group and experimental group.Every group experiment small white mouse feed corresponding Japanese Honeysuckle extraction chrysanthemum oral liquid 0.3ml/ only/d; Control group is filled with and is fed physiological saline, 0.3ml//d.Raise two weeks continuously, carry out blood sampling examination (eyeball blood sampling).Small white mouse is taken out from cage, and the right hand is mentioned tail and is rotated rapidly, makes it dizzy, then, on the ground, drag tail after the right hand, while leaping up before mouse, left index finger and thumb are clamped rapidly the skin after its ear, again tail is held in the palm, makes its eyeball outstanding, with tweezers, eyeball is extracted, the blood oozed is lived with little clear bottle graft, puts signs on.The blood of adopting is joined in the centrifuge tube of carrying out mark accordingly, and low-temperature centrifugation 8000rpm * 3mi n, get supernatant liquor, in-20 ℃ of preservations.The blood of adopting is joined in the centrifuge tube of carrying out mark accordingly, and low-temperature centrifugation 8000rpm * 3min, get supernatant liquor, in-20 ℃ of preservations.Do not add the centrifugal immediately of antithrombotics, get serum.The second the People's Hospital, Fujian Province is entrusted in the detection of immunoglobulin content, and detection method adopts immunoturbidimetry.
The 7th step, interpretation.
Further, the pharmacodynamic experiment animal of Japanese Honeysuckle extraction chrysanthemum immunostimulant is for supplying 90 of examination small white mouses, every heavy 25 ± 5g, 18 of large ear rabbits, body weight 1.5 ± 0.25kg.
Further, the pharmacodynamic experiment instrument of Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises constant-pressure atomization device (being comprised of glass atomizer, air compressor machine, tensimeter and animal container), whizzer (model, the FT120 type), microplate reader (model, the P-E4100 type), constant incubator (model, DU640).
Fig. 2 shows the schema of the virusology experimental technique of Japanese Honeysuckle extraction chrysanthemum immunostimulant, and as shown in the figure, the virusology experimental technique of Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises:
Chmice acute toxicity test S201
Rat long term toxicity test S202
Interpretation S203.
Concrete steps are as follows:
The first step, the chmice acute toxicity test.
Comprise 1 maximum dosage-feeding determination experiment and 1d maximum dosage-feeding determination experiment.
1 time maximum dosage-feeding determination experiment step is as follows: by 40 mouse, male and female half and half, be divided into 2 groups at random, and control group and Japanese Honeysuckle extract chrysanthemum immunostimulant group, 20 every group, male and female half and half.Water 12h is prohibited in fasting, presses 40mL/kg dosage gavage, and control group is given with amount distilled water, observes 7d, records toxic reaction symptom and the death condition of mouse; Put to death after experiment finishes, dissect, the body weight of 7d before the administration of weighing mouse and after administration.Visual inspection mouse main organs lesion tissue situation.
1d maximum dosage-feeding determination experiment step is as follows: by 40 mouse, male and female half and half, be divided into 2 groups at random, and 20 every group, male and female half and half.Water 12h is prohibited in fasting, presses the 3 times/d of Japanese Honeysuckle extraction chrysanthemum immunostimulant gavage of 40mL/kg dosage, every minor tick 8h, and control group adopts with amount distilled water gavage, observes 7d, records toxic reaction symptom and the death condition of experiment mice; Experiment is put to death after finishing.Visual inspection mouse main organs lesion tissue situation.
Second step, the rat long term toxicity test.After rat prerun is observed to 7d, be divided at random 4 groups by sex and body weight, male and female half and half.The 1st group is that to give with amount distilled water, the 2nd, 3,4 groups be high, medium and low 3 the class dosage group (20.0g of Japanese Honeysuckle extraction chrysanthemum immunostimulant experimental group to the blank group, 10.0g, 5.0g crude drug/kg), every group 20, each group is all by gastric infusion, dosage is 20ml/kg.1 time/d, successive administration 2 months (8 weeks).Claim weekly the laboratory animal body weight 2 times before administration and during administration, and suitably adjust dosage according to body weight change.Experimental session is observed rat sign, general state, registers its food ration every day, drinking-water and ight soil situation.24h after the last administration, each group is all got 10 animals, male and female half and half; Cut open after execution to kill and get blood, check hematology and blood biochemical index.Get after blood and cut open immediately internal organs such as getting the animal heart, liver, spleen, lung, kidney, testis (epididymis), ovary, uterus, duodenum, brain, suprarenal gland, Tiroidina, thymus gland and weigh, according to weight calculator official weight coefficient (organ coefficient).By above-mentioned internal organs visual inspection, it changes, and then by 10% formaldehyde solution, fixes, and makes above-mentioned organs and tissues section, and its histopathology of microscopy changes, and observes the impact of immunostimulant on the rats'liver renal function.
The 3rd step, interpretation.
Although above-mentioned, by reference to the accompanying drawings the specific embodiment of the present invention is described; but be not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various modifications that performing creative labour can make or distortion still within protection scope of the present invention.
Claims (4)
1. the pharmacodynamic experiment method of Japanese Honeysuckle extraction chrysanthemum immunostimulant, it is characterized in that, the pharmacodynamic experiment method of described Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises preparation Japanese Honeysuckle extraction chrysanthemum immunostimulant, the preparation of plant's common pathogen kind, Japanese Honeysuckle extraction chrysanthemum immunostimulant bacteriostatic test, the experiment of Japanese Honeysuckle extraction chrysanthemum immunostimulant to the antipyretic effect of pyrogenicity Rabbit Model, Japanese Honeysuckle extraction chrysanthemum immunostimulant is to coughing the experiment of the effect of breathing heavily Mouse Model, Japanese Honeysuckle extraction chrysanthemum immunostimulant is to small white mouse IgM, the test of the impact of c3 and c4, interpretation, concrete steps are as follows:
The first step, preparation Japanese Honeysuckle extraction chrysanthemum immunostimulant; The prescription of Japanese Honeysuckle extraction chrysanthemum is comprised of Japanese Honeysuckle, extraction chrysanthemum, Herba Houttuyniae, Herba ajugae ciliatae (Herba Ajugae Ajuga ciliata Bge.), the capsule of weeping forsythia, the root of large-flowered skullcap, Root of Indigowoad, the Radix Astragali, radix bupleuri, Radix Glycyrrhizae, the herbal medicine of selected above-mentioned various genuine high-qualitys during preparation, after taking in proportion, be ground into coarse meal, after being soaked in water 30 minutes, being placed in pot and decocting 3 times, merge three times filtrate, be condensed into the 1:1 decoction, by every ml liquid, containing the former medicine of 1g, the bottling sterilizing is standby;
Second step, the preparation of plant's common pathogen kind; Select the common pathogenic bacterium of plant, the cultivation breeding of bacterium adopts the ordinary nutrient agar substratum, and concrete preparation method is for after dissolving nutrient agar medium fully, after 121 ℃, 20min autoclaving, pour culture dish into, be laid into the substratum of 3～5mm, standby after cooled and solidified; Inoculation adopts transfering loop and method of scoring; Inoculation is placed on 37 ℃ of constant incubators and cultivates 24h, and be placed in 4 ℃ of refrigerators and save backup,
The 3rd step, Japanese Honeysuckle extraction chrysanthemum immunostimulant bacteriostatic test; In the Chinese herbal medicine decoction concentrated solution that the circular filter paper sheet autoclaving that is 6mm by diameter, drying are placed on 1:1 concentration, infiltrate standby, then in sterile cabinet, adopt aseptic method to spread upon equably on nutrient agar with different spreading rods by the fresh culture thing of above-mentioned bacterial strains respectively, media surface is close at the even interval of the scraps of paper that will infiltrate again herbal medicine, adjustment makes its contact well be placed on 37 ℃ of constant incubators by paper-pressing sheet and cultivates 24h, then take out and observe its inhibition zone, measure antibacterial circle diameter D (mm) and record its result; Criterion is according to " pharmacology of Chinese materia medica " and " antibacterials drug sensitive test criterion " regulation, and antibacterial circle diameter≤10mm is muting sensitive, and antibacterial circle diameter >=10～18mm is medium sensitivity, and antibacterial circle diameter >=18mm is extremely sensitive;
The 4th step, the experiment of Japanese Honeysuckle extraction chrysanthemum immunostimulant to the antipyretic effect of pyrogenicity Rabbit Model; Some for the examination large ear rabbit,, select 18 of healthy anosis, the qualified large ear rabbits of body temperature, the standard range that body temperature is qualified is 38.6-39.5 ℃, survey the anus temperature every day 3 times, after prerun 3d, then be divided at random 3 groups, test front fasting 12h but can't help water, 30min before on-test, survey the anus temperature 3 times, each 5min, get the basal body temperature of its mean value as this test rabbit again; Carry out Rabbit Model according to herbal medicine pharmacology test method inquiry and make disease, give every experimental rabbit auricular vein injection 2,30mi n after 2, 4-dinitrophenol 1ml/100g (15mg/kg) rabbit pyrogenicity to be tested, according to 3 groups of different grouping drug treatments, wherein A group employment catheter per os is to only pouring into Japanese Honeysuckle extraction chrysanthemum concentrated solution 10ml/kg/ in the rabbit stomach; The B group gives aspirin phenacetin caffeine 100mg/kg-1; C group control group only gives physiological saline 10ml/kg/; Laboratory animal all adopts the administration by gavage administration; After administration 30,60,90,120,180,240,300,360min is determination test rabbit anus temperature 3 times respectively, gets its mean value, and usings the difference of this test rabbit body temperature and basal body temperature and carry out the t check as observation index, and the comparison Chinese-western medicine is tested the antipyretic effect of rabbit to each group;
The 5th step, Japanese Honeysuckle extraction chrysanthemum immunostimulant is to coughing the experiment of the effect of breathing heavily Mouse Model; For 30 of examination small white mouses, be divided at random 3 groups, every heavy 25 ± 5g, male and female half and half dual-purpose; A group Japanese Honeysuckle extraction chrysanthemum oral liquid group 0.5ml (20g/kg)/only wherein; B group compound codeine syrup 30mg/kg; C group physiological saline control group 0.5ml (20g/kg)/only; Laboratory animal all adopts the administration by gavage administration, test each administration of upper and lower noon of first day 1 time, after testing the 2nd day administration in morning 1h, laboratory animal is put into to the boxes and baskets that the constant-pressure atomization device is housed, in under constant voltage, strong aqua evenly being entered to boxes and baskets with atomize, pressure on weather gage is advisable with 0.2 normal atmosphere or 18.7kPa (140mmHg), spray time is about 30min, observe the cough symptom time of occurrence of respectively organizing laboratory animal, typical case's cough is shunk for the small white mouse abdominal muscle, magnify mouth simultaneously, and s cough is arranged; Calculate latent period and the antibechic rate of the cough symptom of each treated animal;
The 6th step, the test of Japanese Honeysuckle extraction chrysanthemum immunostimulant on the impact of small white mouse IgM, c3 and c4; For 60 of examination small white mouses, every heavy 25 ± 5g, male and female half and half dual-purpose, be divided into 3 large groups at random, 20 of every large groups, every large component becomes two groups, and 10 of every groups, be divided into control group and experimental group; Every group experiment small white mouse feed corresponding Japanese Honeysuckle extraction chrysanthemum oral liquid 0.3ml/ only/d; Control group is filled with and is fed physiological saline, 0.3ml//d; Raise two weeks continuously, carry out blood sampling examination; Small white mouse is taken out from cage, and the right hand is mentioned tail and is rotated rapidly, makes it dizzy, then, on the ground, drag tail after the right hand, while leaping up before mouse, left index finger and thumb are clamped rapidly the skin after its ear, again tail is held in the palm, makes its eyeball outstanding, with tweezers, eyeball is extracted, the blood oozed is lived with little clear bottle graft, puts signs on; The blood of adopting is joined in the centrifuge tube of carrying out mark accordingly, and low-temperature centrifugation 8000rpm * 3min, get supernatant liquor, in-20 ℃ of preservations; The blood of adopting is joined in the centrifuge tube of carrying out mark accordingly, and low-temperature centrifugation 8000rpm * 3min, get supernatant liquor, in-20 ℃ of preservations; Do not add the centrifugal immediately of antithrombotics, get serum;
The 7th step, interpretation.
2. Japanese Honeysuckle as claimed in claim 1 extracts the pharmacodynamics test method of chrysanthemum immunostimulant, it is characterized in that, the pharmacodynamic experiment animal of described Japanese Honeysuckle extraction chrysanthemum immunostimulant is for supplying 90 of examination small white mouses, every heavy 25 ± 5g, 18 of large ear rabbits, body weight 1.5 ± 0.25kg.
3. Japanese Honeysuckle as claimed in claim 1 extracts the pharmacodynamics test method of chrysanthemum immunostimulant, it is characterized in that, the pharmacodynamic experiment instrument of described Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises constant-pressure atomization device, whizzer, microplate reader, the constant incubator be comprised of glass atomizer, air compressor machine, tensimeter and animal container.
4. the virusology experimental technique of a Japanese Honeysuckle extraction chrysanthemum immunostimulant, is characterized in that, the virusology experimental technique of described Japanese Honeysuckle extraction chrysanthemum immunostimulant comprises chmice acute toxicity test, rat long term toxicity test, interpretation; Concrete steps are as follows:
The first step, the chmice acute toxicity test;
Comprise 1 maximum dosage-feeding determination experiment and 1d maximum dosage-feeding determination experiment;
1 time maximum dosage-feeding determination experiment step is as follows: by 40 mouse, male and female half and half, be divided into 2 groups at random, and control group and Japanese Honeysuckle extract chrysanthemum immunostimulant group, 20 every group, male and female half and half; Water 12h is prohibited in fasting, presses 40mL/kg dosage gavage, and control group is given with amount distilled water, observes 7d, records toxic reaction symptom and the death condition of mouse; Put to death after experiment finishes, dissect, the body weight of 7d before the administration of weighing mouse and after administration, visual inspection mouse main organs lesion tissue situation;
1d maximum dosage-feeding determination experiment step is as follows: by 40 mouse, male and female half and half, be divided at random 2 groups, 20 every group, male and female half and half, water 12h is prohibited in fasting, press the 3 times/d of Japanese Honeysuckle extraction chrysanthemum immunostimulant gavage of 40mL/kg dosage, every minor tick 8h, control group adopts with amount distilled water gavage, observe 7d, record toxic reaction symptom and the death condition of experiment mice; Experiment is put to death after finishing, visual inspection mouse main organs lesion tissue situation;
Second step, the rat long term toxicity test; After rat prerun is observed to 7d, be divided at random 4 groups by sex and body weight, male and female half and half, the 1st group is that to give with amount distilled water, the 2nd, 3,4 groups be high, medium and low 3 the class dosage groups of Japanese Honeysuckle extraction chrysanthemum immunostimulant experimental group to the blank group, every group 20, each group is all by gastric infusion, and dosage is 20ml/kg.1 time/d, successive administration 2 months; Claim weekly the laboratory animal body weight 2 times before administration and during administration, and suitably adjust dosage according to body weight change, experimental session is observed rat sign, general state, registers its food ration every day, drinking-water and ight soil situation, 24h after the last administration, each group is all got 10 animals, male and female half and half; Cut open after execution to kill and get blood, check hematology and blood biochemical index, get after blood and cut open immediately internal organs such as getting the animal heart, liver, spleen, lung, kidney, testis, ovary, uterus, duodenum, brain, suprarenal gland, Tiroidina, thymus gland and weigh, according to weight calculator official weight coefficient (organ coefficient), by above-mentioned its variation of internal organs visual inspection, then by 10% formaldehyde solution, fix, make above-mentioned organs and tissues section, its histopathology of microscopy changes, and observes the impact of immunostimulant on the rats'liver renal function;
The 3rd step, interpretation.
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