The specific embodiment
Below in conjunction with the preparation embodiment and the test of pesticide effectiveness Chinese medicine composition of the present invention is described in further detail.
Embodiment 1:
Radix Puerariae 85 weight portions; Radix Isatidis 70 weight portions; Rhizoma Menispermi 60 weight portions;
Rhizoma Phragmitis 70 weight portions; Rhizoma Imperatae 65 weight portions; Radix agastaches 75 weight portions;
Flos Carthami 70 weight portions; Radix Et Rhizoma Rhei 15 weight portions.
More than eight flavor medical materials, pulverize, make powder, packing, promptly.
Embodiment 2:
Radix Puerariae 125 weight portion Radix Isatidiss 80 weight portion Rhizoma Menispermis 105 weight portions
Rhizoma Phragmitis 95 weight portion Rhizoma Imperataes 100 weight portion Herba Pogostemonis 110 weight portions
Flos Carthami 85 weight portion Radix Et Rhizoma Rhei 20 weight portions.
More than eight flavor medical materials, decoct with water 2 times, add 12 times in water amount at every turn and decocted 2.5 hours, filter; Merging filtrate is concentrated into the about 1.0-1.2 of relative density, adds ethanol and makes and contain alcohol amount and reach 60%; Get supernatant, reclaim ethanol, concentrate, make syrup, packing, promptly.
Embodiment 3:
Radix Puerariae 100 weight portions; Radix Isatidis 80 weight portions; Rhizoma Menispermi 80 weight portions;
Rhizoma Phragmitis 77 weight portions; Rhizoma Imperatae 75 weight portions; Herba Pogostemonis 80 weight portions;
Flos Carthami 85 weight portions; Radix Et Rhizoma Rhei 15 weight portions.
Get Herba Pogostemonis earlier and add 10 times of water logging bubbles 2 hours, distill 3 hours extraction volatile oil, volatile oil adds 7 times of water saturation solution that the beta cyclodextrin enclose is made, and 30 ℃ are stirred 1.5 hours enclose down, through cold preservation 24 hours, filter, and 60 ℃ of dryings get clathrate.Other collects by device respectively for aqueous solution after the distillation and medicinal residues; Radix Puerariae adds 8 times of amounts of ethanol at every turn and refluxed 2 hours with 85% alcohol reflux 3 times, filters, and is standby.Radix Et Rhizoma Rhei adds 10 times of amounts of ethanol at every turn and refluxed 1 hour with 60% alcohol reflux 1 time, filters, and is standby.Above-mentioned medicinal residues and all the other five kinds of Chinese medicine decoct with water 2 times, add 8 times of amounts of water at every turn and decoct filtered while hot 2 hours; Aqueous solution after the distillation of merging filtrate and above-mentioned Herba Pogostemonis is concentrated into relative density 1.15, adds ethanol and makes and contain the alcohol amount and reach 60%; Get supernatant and reclaim ethanol, concentrated, dry, pulverizing add the volatile oil beta cyclodextrin clathrate, make pill.
Embodiment 4:
Radix Puerariae 100 weight portion Radix Isatidiss 90 weight portion Rhizoma Menispermis 95 weight portions
Rhizoma Phragmitis 90 weight portion Rhizoma Imperataes 75 weight portion Herba Pogostemoniss 85 weight portions
Flos Carthami 80 weight portion Radix Et Rhizoma Rhei 20 weight portions.
Get Herba Pogostemonis and add 6 times of water logging bubbles 3 hours, distill and extracted volatile oil, the airtight preservation of volatile oil in 5 hours.Other collects by device respectively for aqueous solution after the distillation and medicinal residues; Radix Puerariae adds 6 times of amounts of ethanol (v/w) with 50% alcohol reflux 2 times at every turn, refluxes 1.5 hours at every turn, filters, and is standby.Radix Et Rhizoma Rhei adds 6 times of amounts of ethanol (v/w) with 80% alcohol reflux 2 times at every turn, refluxes 1 hour at every turn, filters, and is standby.Above-mentioned medicinal residues and all the other five kinds of Chinese medicine decoct with water 3 times, add 6 times of amounts of water at every turn and decoct filtered while hot 2 hours; Aqueous solution after the distillation of merging filtrate and above-mentioned Herba Pogostemonis is concentrated into relative density 1.20, adds ethanol and makes and contain the alcohol amount and reach 65%; Get supernatant and reclaim ethanol, concentrate, add volatile oil, make spray.
Embodiment 5:
Radix Puerariae 85 weight portion Radix Isatidiss 95 weight portion Rhizoma Menispermis 85 weight portions
Rhizoma Phragmitis 85 weight portion Rhizoma Imperataes 80 weight portion Herba Pogostemoniss 90 weight portions
Flos Carthami 95 weight portion Radix Et Rhizoma Rhei 30 weight portions.
Get Herba Pogostemonis earlier and add 8 times of water gagings immersions 2 hours, distill and extracted volatile oil in 3 hours, other collects by device respectively for aqueous solution after volatile oil, the distillation and medicinal residues; Radix Puerariae adds 8 times of amounts of ethanol (v/w) with 90% alcohol reflux 2 times at every turn, refluxes 2 hours at every turn, filters, and is standby.Radix Et Rhizoma Rhei is with 70% alcohol reflux 2 times, and 1 hour for the first time, 0.5 hour for the second time, merge extracted twice liquid, decompression recycling ethanol, medicinal liquid is standby.Above-mentioned medicinal residues and all the other five kinds of Chinese medicine decoct with water 3 times, add 6 times of amounts of water at every turn and decoct filtered while hot 1 hour; Aqueous solution after the distillation of merging filtrate and above-mentioned Herba Pogostemonis, it is about 1.2 to be concentrated into relative density, adds ethanol and makes and contain the alcohol amount and reach 60%; Get supernatant, reclaim ethanol, add volatile oil, make injection.
Embodiment 6
Radix Puerariae 90kg Radix Isatidis 90kg Rhizoma Menispermi 90kg
Rhizoma Phragmitis 90kg Rhizoma Imperatae 90kg Herba Pogostemonis 90kg
Flos Carthami 90kg Radix Et Rhizoma Rhei 45kg.
Get Herba Pogostemonis earlier and add 6 times of water logging bubbles 3 hours, distill 6 hours extraction volatile oil, volatile oil adds 15 times of water saturation solution that the beta cyclodextrin enclose is made, and 40 ℃ are stirred 2 hours enclose down, through cold preservation 24 hours, filter, and 40 ℃ of dryings get clathrate.Other collects by device respectively for aqueous solution after the distillation and medicinal residues; Radix Puerariae adds 6 times of amounts of ethanol (v/w) with 50% alcohol reflux 2 times at every turn, refluxes 2.5 hours at every turn, filters, and filtrate and medicinal residues are standby.Radix Et Rhizoma Rhei adds 6 times of amounts of ethanol (v/w) with 70% alcohol reflux 2 times at every turn, refluxes 1 hour at every turn, filters, and filtrate and medicinal residues are standby.Above-mentioned medicinal residues and all the other five kinds of Chinese medicine decoct with water 2 times, add 6 times of amounts of water at every turn and decoct filtered while hot 2 hours; Aqueous solution after the distillation of merging filtrate and above-mentioned Herba Pogostemonis, it is about 1.2 to be concentrated into relative density, adds ethanol and makes and contain the alcohol amount and reach 65%; Get supernatant and reclaim ethanol, concentrate, drying adds the volatile oil beta cyclodextrin clathrate, makes soft capsule.
Embodiment 7:
Radix Puerariae 5.45kg Radix Isatidis 5.45kg Rhizoma Menispermi 5.451kg
Rhizoma Phragmitis 5.45kg Rhizoma Imperatae 5.45kg Herba Pogostemonis 5.45kg
Flos Carthami 5.45kg Radix Et Rhizoma Rhei 1.85kg.
More than eight flavors, Radix Puerariae is with 8 times of amount 90% ethanol, reflux twice, each 2 hours, merging extracted twice liquid, decompression recycling ethanol, medicinal liquid is standby; Radix Et Rhizoma Rhei merges extracted twice liquid with 8 times of amount 70% alcohol heating reflux twice 1 hour first time 0.5 hour second time, decompression recycling ethanol, and medicinal liquid is standby; Volatile oil was extracted in the Herba Pogostemonis distillation in 3 hours, separated volatile oil, and volatile oil injects the β-CD saturated solution (oil: water: β-CD=1: 15: 8) that adopts the solwution method preparation, stirred 4 hours, and airtight preservation, the aqueous solution after distillation device is in addition collected, and medicinal residues are standby; The five tastes and the merging of above-mentioned medicinal residues such as Radix Isatidis, Rhizoma Phragmitis, Rhizoma Imperatae, Rhizoma Menispermi, Flos Carthami decoct with water twice, add 12 times of amounts of water for the first time, decocted 1 hour, for the second time add 8 times of amounts of water, decocted 0.5 hour, merge twice decocting liquid and distill after the Herba Pogostemonis aqueous solution, be concentrated into 1: 1 (every ml medicinal liquid is equivalent to the 1g crude drug), put cold back and add ethanol, make to contain the alcohol amount and reach 60%, leave standstill 48 hours after-filtration of cold preservation, decompression filtrate recycling ethanol, medicinal liquid is standby.Merge above medicinal liquid, it is about 1.1 to be evaporated to relative density, adds the dextrin of above-mentioned beta-CD inclusion and output 1/3rd, and spray drying (the control inlet temperature is 210 ℃, and outlet temperature is 90 ℃) becomes granule, packing, promptly.
Embodiment 8:
Radix Puerariae 90kg Radix Isatidis 90kg Rhizoma Menispermi 90kg
Rhizoma Phragmitis 90kg Rhizoma Imperatae 90kg Herba Pogostemonis 90kg
Flos Carthami 90kg Radix Et Rhizoma Rhei 30kg.
More than eight flavors, Radix Puerariae is with 8 times of amount 90% ethanol, reflux twice, each 2 hours, merging extracted twice liquid, decompression recycling ethanol, medicinal liquid is standby; Radix Et Rhizoma Rhei is measured 70% alcohol heating reflux 2 times with 8 times, and each 1 hour, merge extracted twice liquid, decompression recycling ethanol, medicinal liquid is standby; Volatile oil was extracted in the Herba Pogostemonis distillation in 3 hours, separated volatile oil, airtight preservation, and the aqueous solution after distillation device is in addition collected.The five tastes such as all the other Radix Isatidiss, decoct with water twice, each 1 hour, add 12 times of amounts of water for the first time, add 8 times of amounts of water for the second time, merge the Herba Pogostemonis aqueous solution after twice decocting liquid and the distillation, be concentrated into relative density and be about 1.0-1.2, put cold back and add ethanol, make to contain the alcohol amount and reach 60%, leave standstill 48 hours after-filtration of cold preservation, merge with above-mentioned medicinal liquid behind the filtrate recycling ethanol.Add water to 1000mL and stir evenly, cold preservation (2~8 ℃) is more than 48 hours, filter pressing (inner membrance is a polysulfones, and the aperture is 1 μ m), filtrate for later use.Get stevioside (0.6%) 6g and make syrup, filtering the back merges with above-mentioned medicinal liquid, other gets the fully mixed evenly back of an amount of tween 80 (about 0.5%) and volatile oil and is heated in the medicinal liquid about 70 ℃, after treating fully to dissolve, supplies the water yield to 1000ml, cold preservation, filter fill, sterilization (115 ℃ of flowing steams, 30 minutes), make oral liquid.
Embodiment 9:
Radix Puerariae 90kg Radix Isatidis 90kg Rhizoma Menispermi 90kg
Rhizoma Phragmitis 90kg Rhizoma Imperatae 90kg Herba Pogostemonis 90kg
Flos Carthami 90kg Radix Et Rhizoma Rhei 30kg.
More than eight flavors, Radix Puerariae is with 8 times of amount 90% ethanol, reflux twice, each 2 hours, merging extracted twice liquid, decompression recycling ethanol, medicinal liquid is standby; Radix Et Rhizoma Rhei merges extracted twice liquid with 8 times of amount 70% alcohol heating reflux twice 1 hour first time 0.5 hour second time, decompression recycling ethanol, and medicinal liquid is standby; Volatile oil was extracted in the Herba Pogostemonis distillation in 3 hours, separated volatile oil, and volatile oil injects the β-CD saturated solution (oil: water: β-CD=1: 15: 8) that adopts the solwution method preparation, stirred 4 hours, and airtight preservation, the aqueous solution after distillation device is in addition collected, and medicinal residues are standby; The five tastes and the merging of above-mentioned medicinal residues such as Radix Isatidis, Rhizoma Phragmitis, Rhizoma Imperatae, Rhizoma Menispermi, Flos Carthami decoct with water twice, add 12 times of amounts of water for the first time, decocted 1 hour, for the second time add 8 times of amounts of water, decocted 0.5 hour, merge twice decocting liquid and distill after the Herba Pogostemonis aqueous solution, be concentrated into 1: 1 (every ml medicinal liquid is equivalent to the 1g crude drug), put cold back and add ethanol, make to contain the alcohol amount and reach 60%, leave standstill 48 hours after-filtration of cold preservation, decompression filtrate recycling ethanol, medicinal liquid is standby.Merge above medicinal liquid, it is about 1.1 to be evaporated to relative density, adds above-mentioned beta-CD inclusion, makes effervescent tablet, packing, promptly.
Embodiment 10:
Radix Puerariae 5.45kg Radix Isatidis 5.45kg Rhizoma Menispermi 5.451kg
Rhizoma Phragmitis 5.45kg Rhizoma Imperatae 5.45kg Herba Pogostemonis 5.45kg
Flos Carthami 5.45kg Radix Et Rhizoma Rhei 1.85kg.
More than eight flavors, Radix Puerariae is with 8 times of amount 90% ethanol, reflux twice, each 2 hours, merging extracted twice liquid, decompression recycling ethanol, medicinal liquid is standby; Radix Et Rhizoma Rhei merges extracted twice liquid with 8 times of amount 70% alcohol heating reflux twice 1 hour first time 0.5 hour second time, decompression recycling ethanol, and medicinal liquid is standby; Volatile oil was extracted in the Herba Pogostemonis distillation in 3 hours, separated volatile oil, and volatile oil injects the β-CD saturated solution (oil: water: β-CD=1: 15: 8) that adopts the solwution method preparation, stirred 4 hours, and airtight preservation, the aqueous solution after distillation device is in addition collected, and medicinal residues are standby; The five tastes and the merging of above-mentioned medicinal residues such as Radix Isatidis, Rhizoma Phragmitis, Rhizoma Imperatae, Rhizoma Menispermi, Flos Carthami decoct with water twice, add 12 times of amounts of water for the first time, decocted 1 hour, for the second time add 8 times of amounts of water, decocted 0.5 hour, merge twice decocting liquid and distill after the Herba Pogostemonis aqueous solution, be concentrated into 1: 1 (every ml medicinal liquid is equivalent to the 1g crude drug), put cold back and add ethanol, make to contain the alcohol amount and reach 60%, leave standstill 48 hours after-filtration of cold preservation, decompression filtrate recycling ethanol, medicinal liquid is standby.Merge above medicinal liquid, it is about 1.1 to be evaporated to relative density, adds above-mentioned beta-CD inclusion, makes concentrated pill, packing, promptly.
Test of pesticide effectiveness embodiment
Make Chinese medicine composition of the present invention by crude drug by prescription and the method for making of embodiment 2, can obviously alleviate the mice auricle swelling degree, obviously suppress capillary permeability.Obviously suppress yeast and cause the rat temperature rising, the rabbit body temperature rising that triple vaccine is caused has the obvious suppression effect, and effect was kept more than 3 hours.Beta hemolytic streptococcus, staphylococcus aureus, pneumobacillus are had stronger inhibitory action, are candida albicans, bacillus pyocyaneus and Diplococcus pneumoniae secondly.It is dead that single administration can not stop the chmice acute of pathogenic bacterium such as streptococcus pneumoniae, group B streptococcus, Klebsiella pneumonia, hemophilus influenza to infect, but successive administration two days, then can obviously reduce its death toll at every day three times.At the external cytopathic effect that can suppress or delay some relevant virus with respiratory tract infection, can significantly alleviate the pneumonia degree of influenza virus induced mice in vivo, and can suppress the proliferative amount of influenza virus in the Mus lung by specificity, for sense and influenza on this Drug therapy provide the foundation of pharmacodynamics.
Experiment material:
1, animal: Kunming kind healthy mice, body weight 18-22g, the male and female dual-purpose, (cleaning level, the quality certification number be<word is moved in the capital〉8806M35 number 〉.Wistar-Imamich is a rat, about body weight 200g, (cleaning level, the quality certification number be<word is moved in the capital〉the 8806th R011 number 〉.Large ear rabbit: body weight 1.7-2.2kg, more than provide by University of the Inner Mongol's Experimental Animal Center.
2, medicine and reagent: the used Chinese medicine composition of the present invention of following effect experiment is the syrup of embodiment 2, tests used dosage and refers to that all every kilogram is tried the used crude drug amount of body;
Hydrocortisone sodium succinate: commercially available.
Indometacin: produce 0.3g/ grain, lot number: 960410 by the Yellow River pharmaceutical factory.
Beer yeast: produce by BJ Beer Factory.
Aspirin: by Chifeng Pharmaceutics Factory production, 25mg/ sheet, lot number: 980602-4.
Typhoid fever pays typhoid fever first and second triple vaccinies: Beijing Biological Product Inst. produces, injection, lot number 961-6.
The Folium Isatidis mixture: commercially available, produce lot number by Zibo, Shandong Province people pharmaceutical factory: 970308,10ml/ props up, 0.9g crude drug/ml.
Strain: group B streptococcus 111, staphylococcus aureus 107, streptococcus pneumoniae 01-16, klebsiella pneumoniae 75-14, (above antibacterial is clinical separation).Hemophilus influenza 530, Bacillus typhi 50094, dysentery bacterium 51582, staphylococcus aureus 26003, bacillus pyocyaneus 10104, beta hemolytic streptococcus 32210, candida albicans 10231,8 strain bacterium such as Diplococcus pneumoniae 31002 and pneumobacillus 46114 are provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.Get above-mentioned bacterial strains and, get 1ml strain cultured solution+meat soup and cultivate NS 9ml after 18-24 hour through 37 ℃ of cultivations, 10 times are diluted to 10 then
-5, 10
-6And 10
-7Standby, different strain is selected different concentration for use, and the amount of bacteria under this concentration is different, so represent with bacterial population at last.
Broth bouillon: produce the dehydrated medium of preparation by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, standby with distilled water diluting to suitable concn.
Virus influenza virus A-prime Mus lung adapted strain FM
1, herpes simplex virus I-type (HSV-I) and II type (HSV-II), enterovirus ECHO
11, CoxB
4, B
5, B
6, viral seed culture of viruses is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and laboratory is stored in-60 ℃ of cryogenic refrigerators.
Cell Hep-2 passage cell.Provide by the Ministry of Public Health biological products assay institute.
Immune serum: the FM that goes down to posterity with chick embryo allantoic liquid
1The virus immunity rabbit obtains high immune serum of tiring, and is frozen in-40 ℃ of refrigerators.
Traget antibody goat-anti body IgG-FITC, the Shanghai life is ground institute and is provided.4 ℃ of preservations are standby.
Erythromycin (pure product are provided by Chinese Academy of Medical Sciences institute of antibiotics)
Ampicillin (Taiyuan pharmaceutical factory, lot number 990804)
Gentamycin (Jinan No. 2 Pharmaceutical Factory Shandong Prov., lot number 991007)
3, experimental technique and result:
(1) antiinflammatory action
1. Oleum Tiglii is caused the influence of mice auricle swelling:
Get 40 of Kunming mouses, about body weight 22g, male and female are not limit, be divided into 4 groups at random, irritate stomach (ig.) normal saline (NS.) respectively, Chinese medicine composition 10g/kg of the present invention and 20g/kg, hydrocortisone sodium succinate 20mg/kg, continuous three times (first the sky, afternoon and the next morning each 1 time), after the last administration 1 hour, the Oleum Tiglii diethyl ether solution 0.05ml that the two sides of every Mus left side auricle is coated with 2% caused inflammation.White mice cervical vertebra dislocation caused death in 4 hours behind the Yu Zhiyan, cut two ears to the auricle baseline, lay the both sides auricle at same position respectively with the cornea card punch of diameter 9mm, (mg) weighs.The difference of two ear weight is auricle swelling degree, compares the difference of swelling degree between administration group and the matched group by the t check.Simultaneously count 100%, calculate the suppression ratio of medicine the Mus ear swelling with the matched group auricle edema.
The result is as shown in table 1.After giving Chinese medicine composition 10g/kg of the present invention, the mice auricle swelling degree obviously alleviates, and with matched group significant differences is arranged relatively, and its suppression ratio reaches 18.9%.
Table 1. Chinese medicine composition of the present invention causes influence (n=10, the X ± SD) of scorching mice auricle swelling to Oleum Tiglii
Annotate: compare * P<0.05 with matched group, * * P<0.01, * * * P<0.001 (down together)
2. to the influence of capillary permeability:
Get 32 of male mices, body weight 18-22g, be divided into 4 groups at random, behind ig.NS., Chinese medicine composition of the present invention 10 and 20g/kg and the indometacin 30mg/kg 40 minutes respectively, the blue 0.2ml/20g body weight of tail vein injection 0.5% ivens, and the acetum 0.8ml/20g body weight of ip.0.25% immediately.Put to death animal after 30 minutes, extract 10ml NS. flushing abdominal cavity, it is centrifugal to collect flushing liquor, gets supernatant colorimetric under 590nm.Read absorbance (A), compare, survey its significant difference with matched group.
The result as seen, behind the mice ig. Chinese medicine composition 10g/kg of the present invention, A value and matched group relatively have significant difference, illustrate that the ivens orchid obviously reduces by the amount that blood capillary enters the abdominal cavity.Thereby find out that Chinese medicine composition 10g/kg of the present invention has the effect of obvious inhibition capillary permeability.Its suppression ratio reaches 32.2%.The 30mg/kg indometacin also has obvious inhibitory action, and suppression ratio is 29.6%.As seen the capillary permeability that Chinese medicine composition Dichlorodiphenyl Acetate of the present invention causes has obvious suppression effect (table 2).
Table 2. Chinese medicine composition Dichlorodiphenyl Acetate of the present invention causes the influence that capillary permeability increases (n=8, X ± SD)
(2) refrigeration function
1. beer yeast is caused the influence that rat temperature raises:
Get the Wistar rat about 200g, male and female half and half, be divided into 4 groups at random by body weight and body temperature, after measuring twice normal body temperature, all back subcutaneous injection 10% yeast suspension 10ml/kg is with pyrogenicity, difference ig.1%CMC (carboxymethyl cellulose), Chinese medicine composition of the present invention 10 and 20g/kg after 3 hours, and aspirin 0.15g/kg.The body temperature of 1,2,3,4 and 5 hour (promptly to the 4th, 5,6,7 and 8 hour behind the yeast) after the mensuration administration.Compare with the basal body temperature before the administration, the changing value of different time body temperature is measured matched group and the body temperature changing value difference of corresponding time of administration group after the calculating administration.
Found that after the administration 2 hours, the body temperature of ig. Chinese medicine composition rat of the present invention was starkly lower than matched group, effect was kept about 3 hours, and 0.15g/kg aspirin also has obvious inhibitory action, both action intensities and hold time similar (table 3).
Table 3. Chinese medicine composition of the present invention causes the influence that rat temperature raises (n=8, X ± SD) (℃) to yeast
2. triple vaccine is caused the influence of rabbit fervescence:
Get 32 of the large ear rabbits (testing the body temperature eligible through in advance) of body weight 1.7-2.2kg, 13-4 is divided into 4 groups at random by table, respectively ig. with volume NS., aspirin 60mg/kg, Chinese medicine composition of the present invention 10 and 20g/kg after, immediately through ear vein injection triple vaccine 1.2ml/kg, 1,2,3,4,5,6 and 7 hour body temperature after the record administration, the changing value of body temperature is compared with the body temperature changing value of matched group after basal body temperature that calculating administration (or NS.) is preceding and the administration, does the t check.
Found that after ig. Chinese medicine composition 10g/kg2 of the present invention hour, the rabbit body temperature rising that triple vaccine is caused has the obvious suppression effect, effect was kept more than 3 hours.Aspirin 60mg/kg also has the obvious suppression effect, and is similar to the action intensity of 10g/kg Chinese medicine composition of the present invention, but holds time not as the Chinese medicine composition of the present invention strong (table 4) of 20g/kg.
Table 4 Chinese medicine composition of the present invention causes the influence that rabbit body temperature raises (n=8, X ± SD) to triple vaccine
(3) inside and outside bacteriostatic test:
1. bacteriostatic test in the body
Pathogenic bacterium are injected the Mus abdominal cavity with minimum lethal dose (1MLD), it is infected after, adopts once oral and repeatedly oral two kinds of methods, observe antibacterial situation in the body of Chinese medicine composition of the present invention to mice, matched group gives normal saline 1ml/.
Seek the maximum tolerated dose of mice to Chinese medicine composition of the present invention:
The oral administration maximum tolerated dose: by The acute toxicity tests as can be known, the once oral maximum tolerated dose of mice is 40g crude drug/kg.
Intraperitoneal administration maximum tolerated dose: get 30 of mices, divide 6 groups, 5 every group, medicinal liquid is 6 kinds of concentration: 1ml//times, 0.8ml//time, and 0.6ml//time, 0.4ml/ only/time, 0.2ml//time, 0.1ml//time, after 24 hours, observe result: 1ml//time of existence, 0.8ml//time, 0.6ml/ only/time, 0.4ml/ only/time, behind the injection drug 30 minutes ~ 5 hours, most dead.0.2ml/ only/time, 0.1ml//time, 90% and 100% survival was arranged after 24 hours.Determine: lumbar injection medicinal liquid mice maximum tolerated dose is 0.2ml//time, i.e. 10g crude drug/kg.
The preparation of bacterium liquid: the bacterium of choosing is inoculated in the culture medium that contains 10% horse serum.Adopt beef extract soup culture medium (contain Carnis Bovis seu Bubali cream 0.3% and albumen 1%, transfer ph to be 7.4, be standby), as the usefulness of dilution medicine through autoclaving.Behind the bacterium inoculation medium, add 10% horse serum, hatched 18 hours for 37 ℃, as original bacteria liquid, be diluted to 1: 200 with meat soup and be test organisms liquid, this test organisms liquid is put 4 ℃ of refrigerators and is preserved.Before facing infection, preparation infectious bacteria liquid: test organisms liquid is inoculated in 10ml contains in the fresh bovine meat soup culture medium of 10% horse serum, put 37 ℃ and increased bacterium 6 hours, get a small amount of bacterium liquid transferred species then in 20ml 10% horse serum meat soup, hatched 18 hours, as infectious bacteria stock solution, except streptococcus pneumoniae, group B streptococcus is with outside the stock solution, and it is infectious bacteria liquid that Klebsiella pneumonia, hemophilus influenza, staphylococcus aureus all use 5% gastric Mucin (mucin) to be diluted to make the lethal bacteria concentration of 90 ~ 100% mices.
Each bacterium minimum lethal dose (1MLD):
Group B streptococcus: cultivated back stock solution and do not dilute 0.35ml/ in 18 hours.
Streptococcus pneumoniae: cultivated back stock solution and do not dilute 0.4ml/ in 18 hours.
Hemophilus influenza, staphylococcus aureus, Klebsiella pneumonia: after the cultivation in 18 hours, add the dilution of 5% gastric Mucin, 0.5ml/ only.
Method is: group B streptococcus, streptococcus pneumoniae, with 8 concentration, 1ml/ only, 0.8ml/ only, 0.6ml/ only, 0.5ml/ only, 0.4ml/ only, 0.3ml/ only, 0.2ml/ only, 0.1ml/ only, directly inject the abdominal cavity, observe the death toll of surviving after 24 hours.
Hemophilus influenza, staphylococcus aureus, Klebsiella pneumonia are to be 10 with meat field culture medium with 10 times of dilutions with bacterium liquid
-1, 10
-2, 10
-3, adding 5% gastric Mucin 9ml with bacterium liquid 1ml again, dilution is 10
-2, 10
-3, 10
-4As uniform bacteria suspension.Different bacteria suspensions abdominal cavity is injected mice, 5 of every concentration, every 0.5ml observes the death toll of surviving after 24 hours, 48 hours then, finds out minimum lethal dose (1MLD), causes the bacteria suspension concentration of 80~100% death after promptly infecting.
Experimental therapy: each pathogenic bacterium is injected mouse peritoneal with minimum lethal dose (1MLD), make its infection, that adopt the back is once oral, oral two kinds of methods repeatedly, observes the antibacterial situation of the peaceful granule of sense to mice.
Method one: mice is divided four groups at random, and 10 every group, with two kinds of oral doses, after every Mus microbiological contamination, oral administration is once observed 24 hours, 48 hours death toll simultaneously, the results are shown in Table 5.
Bacteriostasis in mice body of table 5 oral administration
Method two: mice is divided four groups at random, and 10 every group, after every group of mice microbiological contamination, with two kinds of oral doses while oral administrations once, 3 times/day, once successive administration was after two days in 8 hours at interval, and dead mouse number after 24 and 48 hours is observed in drug withdrawal, the results are shown in Table 6.
Table 6 oral administration pressed down the mattress effect in the mice body on 2nd
Conclusion: the bacteriostatic test result shows in the mice body: it is dead that Chinese medicine composition single administration of the present invention can not stop the chmice acute of pathogenic bacterium such as streptococcus pneumoniae, group B streptococcus, Klebsiella pneumonia, hemophilus influenza to infect, but successive administration two days, every day three times, then can obviously reduce its death toll.Each group of Chinese medicine composition experiment of the present invention that infected back 24 hours, 48 hours more all has significant difference (most P<0.01, P<0.001, minority P<0.05) with matched group.Wherein the effect of suppression sense bacillus is similar to ampicillin in the body, and the effect that presses down Klebsiella pneumonia in the body is similar to gentamycin.
2. extracorporeal bacteria inhibitor test
Adopt the experiment of test tube broth bouillon method, Chinese medicine composition of the present invention is made into the solution of 1g crude drug/ml, after (the bacterium liquid 6ml+ broth bouillon 6ml) dilution in 1: 1 of meat soup do, be diluted to 0.55,0.275,0.138,0.0688 and the concentration of 0.0344g crude drug/ml by 2 times of pipes, cumulative volume 6ml, add each bacterial strain diluent 0.1ml respectively, incubated 18 hours together with control tube temperature in 37 ℃ of calorstats that adds culture medium behind the mixing, by perusal and with plate scarification inoculated bacteria, see if there is the bacterial population that antibacterial is inoculated with final decision after the cultivation.
The result is as shown in table 5, and Chinese medicine composition of the present invention has stronger inhibitory action to beta hemolytic streptococcus, staphylococcus aureus and pneumobacillus, is candida albicans, bacillus pyocyaneus and Diplococcus pneumoniae secondly.Effect to Bacillus typhi, dysentery bacterium is the most weak.Under same concentrations, the effect of Folium Isatidis and Chinese medicine composition of the present invention similar (table 7).
The vitro inhibition effect of table 7 Chinese medicine composition of the present invention
Annotate: (+) for antibacterial is arranged, (-) confirms that for last plate method of scoring antibacterial A is a Chinese medicine composition of the present invention for no bacterial growth bacterium amount
(4) antivirus action test:
The antivirus action of Chinese medicine composition of the present invention is selected the Strain relevant with respiratory tract infection, has carried out animal experiment in vitro tissue cultivation and the body, now experimental result is reported as follows.
1. in vitro tests:
Toxicity test to the Hep-2 cultured cell:
Chinese medicine composition of the present invention is made into the medicinal liquid of 1g crude drug/ml, makes 1: 2~1: 512 doubling dilution with the Eagles culture fluid again.The culture fluid of the Hep-2 cell monolayer that 96 hole microtest plates have been grown up to is outwelled, and adds different dilution medicinal liquid 50 μ l, and each dilution factor medicinal liquid respectively adds four porocytes, adds Eagles culture fluid 50 μ l again.Establish the normal cell contrast simultaneously.Culture plate is put 37 ℃ of 5%CO
2Cultivated in the incubator four days, observe the influence of medicinal liquid pair cell with inverted microscope every day, and the minimum dilution factor that regression do not occur with cell is judged to the nontoxic boundary of this medicine pair cell.Observe by toxicity test, 1: 128,1: 256,1: 512 (being 8mg, 4mg, 2mg/ml) three kinds of dilution medicinal liquid pair cells do not have influence, make antivirus test usefulness so get these three kinds of dilution factor medicinal liquids.
Influence to the pathological changes caused by virus effect:
The cell culture fluid that grows up to monolayer is outwelled, got 20TCID
50Different virus liquid 50 μ l be inoculated on every porocyte, each dilution viral liquid is inoculated 4 holes, puts 37 ℃ of 5%CO
2Absorption is 1 hour in the incubator, and viral liquid is outwelled, and keeps liquid with the Eagles that does not contain calf serum and washes the cell face three times, adds 1: 128,1: 256,1: 512 dilution factor medicinal liquid 50 μ l, and every dilution factor is inoculated four holes, adds 50 μ l again and keeps liquid.Establish virus, medicinal liquid, normal cell contrast simultaneously.Put 37 ℃ of 5%CO
2Cultivated in the incubator four days, every day under inverted microscope microscopy once, observation of cell pathological changes, continuous four days.Cytopathy person did not appear on the 4th day by test hole, be judged to inhibitory action (+), test hole pathological changes person occurs and prolongs the cytopathy time than virus control hole is late more than 48 hours, be judged to the generation (±) that can delay pathological changes, otherwise be judged to the unrestraint cytopathic effect.
The result as seen, during this medicine 8mg/ml, to enterovirus ECHO
11Institute's cytopathogenic effect has inhibitory action.To CoxB
4, CoxB
5, CoxB
6Institute's cytopathogenic effect has retarding action, to herpesvirus HSV-I, HSV-II unrestraint effect.2,4mg/ml is to used 6 kinds of equal unrestraint effects of virus (table 8).
Table 8. Chinese medicine composition of the present invention is to the cytopathogenic influence of virus
Annotate: "+" has inhibitory action "-" unrestraint effect " ± " that retarding action is arranged
2. in vivo test:
Effect to mice influenza virus property pneumonia:
With 50 of mices, be divided into five groups at random by body weight, three groups is test group, (middle dosage group is equivalent to clinical equivalent dosage by what the body weight ratio of people and animal was converted into to give Chinese medicine composition 8,16 of the present invention, 32g crude drug/kg respectively, heavy dose be one doubly, low dose be its 1/2), one group is the viral infection matched group, give with the volume distilled water one group of positive contrast medicine virazole group.With mice with the slight anesthesia of ether after, with 15 LD
50Influenza virus Mus lung adapted strain FM
1Virus drop nose infects every Mus 0.05ml.From infecting administration the previous day or feedwater, every day 2 times, each 0.5ml for three days on end, dissects after taking by weighing the mice body weight on the 4th day, the perusal pulmonary lesion, and the degree of record pulmonary liver sample consolidation is won all and is weighed, and calculates lung exponential sum suppression ratio one by one.
Heavy (the g)/body weight (g) * 100 of lung index=lung
The lung exponential quantity is big, and expression lung weight is also big, and pneumonopathy range degree is just serious.Each group lung index is organized a t check.
Table 9 Chinese medicine composition of the present invention is to the exponential influence of mice influenza infection lung (n=10)
The result shows, the lung exponential quantity of three dosage treated animals of Chinese medicine composition of the present invention all is lower than contrast (middle dosage group P<0.01, heavy dose of group p<0.001), and with the increase of dosage, the corresponding minimizing of lung exponential quantity.
Influence to proliferation of influenza virus amount in the Mus lung:
Removing the viral infection amount is 1000LD
50Outward, animal grouping, medication, drug dose etc. are all with 1.Put to death mice in 48 hours behind the infective virus, dissect and get lung, win left side middle period lung and fix, press that pathological section is conventionally fixed, dehydration, embedding, making paraffin section.With indirect immunofluorescence dyeing, it is former to do spike with fluorescein-labeled resisiting influenza virus serum, by the indirect immunofluorescence combination, observe special virus antigen in the infected mice lung, judge the effect of medicine to virus multiplication, the fluorescence number positive is many, show that virion propagation is many, and compare between the work group.
Table 10. Chinese medicine composition of the present invention is to the influence of influenza virus value added on products in the mouse lung
The result shows that three dosage groups of Chinese medicine composition of the present invention all have significant inhibitory effect to proliferation of influenza virus amount in the mouse lung, and the action intensity of heavy dose of group 32g/kg is close with positive control drug virazole 70mg/kg group.
The in-vivo tissue culture experiment shows that Chinese medicine composition of the present invention can suppress 20TCID
50Enterovirus ECHO
11, delay CoxB
4, CoxB
5, CoxB
6The cytopathic effect of relevant virus.
Influenza virus is behind the respiratory tract infection mice, cause the mice viral pneumonia, along with the progress of inflammation, increase is oozed out in pulmonary, lung weight increases, heavy divided by body weight with lung, promptly the lung exponential quantity can be reacted the order of severity that pneumonopathy becomes, and the lung exponential quantity is big more, expression pneumonopathy range degree is serious more, concerning the administration group, the lung exponential quantity is more little, shows that medicine is strong more to the inhibitory action of viral pneumonia.But the lung exponential quantity also is subjected to the influence of other factors, and therefore, we further observe the virion content that duplicates in the Mus lung with the specific immunity fluorescence method again, and the reaction medicine is to the inhibition degree of virus multiplication.The specificity fluorescent ratio is high more, represents that then the virus multiplication amount is big more, and the low more efficacy strength that then illustrates of administration group-specific fluorescence ratio is big more.
Date processing adopts t check and variance analysis (q check).
Conclusion: above-mentioned pharmacodynamic experiment result shows that Chinese medicine composition of the present invention can obviously alleviate the mice auricle swelling degree, suppresses capillary permeability, suppresses yeast and causes the rat temperature rising; Beta hemolytic streptococcus, staphylococcus aureus, pneumobacillus, candida albicans and Diplococcus pneumoniae there are strong inhibitory action, can improve anti-infection ability; The effect of the interior suppression sense bacillus of body is similar to ampicillin, and the effect that presses down Klebsiella pneumonia in the body is similar to gentamycin.At the external cytopathic effect that can suppress or delay some relevant virus, can significantly alleviate the pneumonia degree of influenza virus induced mice in vivo, and can suppress the proliferative amount of influenza virus in the Mus lung by specificity with respiratory tract infection.For sense and influenza in this medicine treatment provide the pharmacodynamics foundation.