Summary of the invention
The shortcoming that the objective of the invention is above-mentioned in order to overcome " YINQIAO Lasiosphaera Seu Calvatia loose " Chinese medicine name side decoction provides a kind of Chinese patent medicine preparation of safe and effective, quality controllable, taking convenience for larynx resistance pharyngalgia patient.Use the method for pharmacological evaluation simultaneously, prove that medical instrument of the present invention has " antiviral " effect, clearly increases " antiviral " clinical scope of application of medicine of the present invention.Another purpose provides the preparation method and the new purposes thereof of medicine of the present invention.
The present invention is achieved in that preparation of the present invention is prepared from by following raw medicaments in portion by weight:
1~10 part of 3~15 parts of Lasiosphaera Seu Calvatia of 5~25 parts of Rhizoma Belamcandae of 6~30 portions of Flos Loniceraes of 10~50 parts of Fructus Arctiis of Fructus Forsythiae
In preparation of the present invention, its crude drug also can add 5~20 parts of Rhizoma Phragmitiss.
The preferred weight ratio scope of above-mentioned raw materials medicine is:
7~15 parts of 3~8 parts of Rhizoma Phragmitiss of 5~12 parts of Lasiosphaera Seu Calvatia of 10~20 parts of Rhizoma Belamcandae of 9~24 portions of Flos Loniceraes of 15~40 parts of Fructus Arctiis of Fructus Forsythiae
The weight ratio scope of above-mentioned raw materials medicine is preferred:
7.52 parts of 7.52 parts of Lasiosphaera Seu Calvatia of 12.48 parts of Rhizoma Belamcandae of 15 portions of Flos Loniceraes of 25 parts of Fructus Arctiis of Fructus Forsythiae
The weight ratio scope of above-mentioned raw materials medicine is also preferred:
10 parts of 6 parts of Rhizoma Phragmitiss of 9 parts of Lasiosphaera Seu Calvatia of 15 parts of Rhizoma Belamcandae of 18 portions of Flos Loniceraes of 30 parts of Fructus Arctiis of Fructus Forsythiae
The preparation method of preparation of the present invention is as follows:
(1), get each crude drug, add 1~20 times of water gaging, heating extraction 3 times, each 0.5~3 hour, merge 3 times the water extract, filter, filtrate concentrates;
(2), adding 95% ethanol, to transfer pure content be 30~75%, places, centrifugal;
(3), get supernatant and add the beta-schardinger dextrin-inclusion, stirring evenly the back decompression and solvent recovery and being concentrated into relative density is 1.04~1.60 clear paste, centrifugally goes out beta-cyclo dextrin included compound, drying is pulverized, and crosses 80 mesh sieves;
(4), with above-mentioned dry thing, make acceptable various dosage forms on the pharmaceutics according to a conventional method.
The dosage form of preparation of the present invention comprises for example granule, capsule, tablet, oral liquid, pill, suspensoid, drop pill, pellet, buccal tablet, soft gelatin capsule, soft capsule, dispersible tablet, solution, aerosol, spray, cataplasma or patch etc.
In above-mentioned preparation technology:
Method for concentration can be to concentrate under the normal pressure, also can be concentrating under reduced pressure, and the concentrating under reduced pressure condition is: 0.01~0.09MPA, 40~90 ℃;
Reclaiming solvent method can be that normal pressure reclaims, and also can be reclaim under reduced pressure, and the condition of decompression and solvent recovery is: 0.01~0.09MPA, 30~80 ℃;
Drying means can be: contact drying, pneumatic conveying drying, tunnel type oven drying, vacuum (decompression) drying, airpillow-dry, spray drying, lyophilization, far-infrared ray drying, microwave drying.
The preparation method of preparation of the present invention is preferred: get crude drug Rhizoma Belamcandae, Fructus Arctii, Lasiosphaera Seu Calvatia, Flos Lonicerae and Fructus Forsythiae, add 10 times of water gagings, 90-95 ℃ of heating extraction three times, each 1h, merge the water extract, filter, filtrate concentrates below 80 ℃ and is cooled to 40 ℃ must relative density be the clear paste of 1.06-1.07; Add ethanol and transfer and to contain the alcohol amount and reach 50%, stir evenly, placement is spent the night, and is centrifugal; Get supernatant and add beta-schardinger dextrin-, stir evenly, 70 ℃, decompression recycling ethanol under the 0.07Mpa condition, and be concentrated into the medicinal liquid that relative density is 1.10-1.15, and standing over night, centrifugal, supernatant is stand-by; Be deposited in 70-80 ℃ of oven dry, pulverize, cross 80 mesh sieves, add cane sugar powder, mixing is made granule with one step of supernatant, and making particulate technological parameter is 80 ℃ of intake air temperatures, 50 ℃ of air outlet temperature, and granulate, packing, promptly.
We's five kinds of Chinese medicine, Fructus Forsythiae, Flos Lonicerae are monarch drug, and Fructus Arctii, Lasiosphaera Seu Calvatia are ministerial drug, and Rhizoma Belamcandae is a messenger drug, and wherein Lasiosphaera Seu Calvatia is the fungus medical material, is used as medicine with sporinite, character is different with other five tastes.Therefore, consider that the Lasiosphaera Seu Calvatia list carries, because Lasiosphaera Seu Calvatia does not have characteristic component, mainly adopting dried cream yield is index, in conjunction with definite optimum conditions such as the color and luster of dried cream, abnormal smells from the patient, meltings.All the other five tastes, one of Fructus Forsythiae effective ingredient are phillyrin, and the main effective ingredient of Flos Lonicerae is a chlorogenic acid, and Fructus Arctii contains arctiin, and Rhizoma Belamcandae contains flavone compound etc.By measuring index components such as volatilization oil mass, total flavones, chlorogenic acid, phillyrin, arctiin, calculate the rate of transform, compare extracting method such as steam distillation, ethanol refluxing process, alcohol percolation method, water extraction method, in conjunction with factors such as the yield of dried cream, color and luster, meltings, overall merit is selected optimum extraction process.
Steam distillation: Fructus Forsythiae 60g, Flos Lonicerae 30g press Chinese Pharmacopoeia version essential oil extraction method in 2000 and extract volatile oil, and extraction time 5-10 hour, filter, medicinal liquid is standby.Medicinal residues and Fructus Arctii 36g, Rhizoma Belamcandae 18g, Lasiosphaera Seu Calvatia 18g, 6-14 times of decocting boils 3 times, each 0.5-2h, filter, merging filtrate was concentrated to 1: 1, add 95% ethanol, accent contains alcohol to be measured to 50-70%, stirs evenly, standing over night, centrifugal, filter, get filtrate, merge extractive liquid,, decompression recycling ethanol, drying under reduced pressure, powder 20.95g gets dry extract.
Ethanol refluxing process: take by weighing Fructus Forsythiae 30g, Flos Lonicerae 15g, Fructus Arctii 18g, Rhizoma Belamcandae 9g, Lasiosphaera Seu Calvatia 18g mixing, totally three parts, add the 50%-70% alcohol reflux 3 times that 6-14 doubly measures respectively, each 0.5-2h, merge three times filtrate, drying under reduced pressure behind the decompression and solvent recovery, pulverize the powder that gets dry extract, weigh.Take by weighing an amount of dried cream powder respectively, measure These parameters in accordance with the law, the results are shown in Table 1.
Alcohol percolation method: take by weighing Fructus Forsythiae 30g, Flos Lonicerae 15g, Fructus Arctii 18g, Rhizoma Belamcandae 9g, Lasiosphaera Seu Calvatia 18g coarse powder, mixing, pack in the percolator, add 50%-70% ethanol, carry out percolation in accordance with the law, collect percolate to color light (volume is about heavy 15 times of medical material), reclaim ethanol respectively, drying under reduced pressure is pulverized the powder that gets dry extract.Take out an amount of dried cream powder respectively and carry out These parameters mensuration in accordance with the law,
Extraction process of the present invention: take by weighing Fructus Forsythiae 30g, Flos Lonicerae 15g, Fructus Arctii 18g, Rhizoma Belamcandae 9g, add 6-14 times of water gaging, 60-95 ℃ of heating extraction three times, each 0.5-2h merges the water extract, filters, it is centrifugal that filtrate concentrates, extracting centrifugal liquid and the Lasiosphaera Seu Calvatia leaching liquid merging drying under reduced pressure powder that gets dry extract.
The selection process of preparation of the present invention is: add 10 times of water gagings, 90-95 ℃ of heating extraction three times, each 1h merges the water extract, filter, filtrate concentrate below 80 ℃ be cooled to 40 ℃ relative density is the clear paste of 1.06-1.07; Add ethanol and transfer and to contain the alcohol amount and reach 50%, stir evenly, placement is spent the night, and is centrifugal; It is an amount of to get supernatant adding beta-schardinger dextrin-, stir evenly, and 70 ℃, decompression recycling ethanol under the 0.07Mpa condition, and be concentrated into the medicinal liquid that relative density is 1.10-1.15, and standing over night, centrifugal, supernatant is stand-by; Be deposited in 70-80 ℃ of oven dry, pulverize, cross 80 mesh sieves, the adding Icing Sugar is an amount of, and mixing is made granule with one step of supernatant, and making particulate technological parameter is 80 ℃ of intake air temperatures, 50 ℃ of air outlet temperature, and granulate, packing, promptly.
Each extracting method of table 1 relatively
|
Volatilization oil mass ml (%) |
Dried cream g (yield) |
Chlorogenic acid mg (rate of transform) |
Arctiin mg (rate of transform) |
Phillyrin mg (rate of transform) |
Flavone g (rate of transform) |
Color and luster |
Melting |
Medicinal material (182g) steam distillation ethanol refluxing process alcohol percolation method |
0.50 (0.55) |
20.97 (11.52%) ) 18.20 (20.00% ) 16.12 (17.71% ) |
579.00 (100%) 177.9 0 (30.72%) 179.84 (62.12%) 218.86 (75.60%) |
2066.40 (100%) 1293.51 (62.60%) 730.90 (70.74%) 856.42 (82.89%) |
114.00 (100%) 43.14 (31.84%) 35.06 (61.50%) 39.27 (68.89%) |
9.6g (100%) 4.77 (49.69% ) 4.34 (90.41% ) 4.52 (94.42% ) |
Sepia dark brown dark brown |
+ ++ + |
Extraction process of the present invention |
15.2 (16.70% ) |
189.68 (65.52%) |
774.08 (74.92%) |
38.82 (68.10%) |
4.37 (91.04% ) |
Brown |
- |
Annotate :-: no breeze dissolved; +: dissolve micro-breeze; ++: dissolve a small amount of breeze.
The every index of overall merit determines that extraction process of the present invention is an optimum process.
The isolation and purification method
At present Chinese medicine preparation preparation process isolation and purification method commonly used for filter, methods such as centrifugal, ultrafiltration, natural precipitant method, macroporous resin adsorption desorption method, alcohol precipitation.For obtaining the comparatively separation purifying technique of science, the system of selection 1 pure sedimentation method, method 2 natural precipitant methods, method 3 ultrafiltrations, these 4 kinds of methods of method 4 centrifuging compare research, with the isolation and purification method of selecting to be fit to.
Branch is got the about 0.2g of dried cream powder that above 4 kinds of technologies obtain as a result, carries out every index determining, the results are shown in Table 2
The different separation purifying technique results of table 2
Method |
Dried cream g (yield) |
Chlorogenic acid mg (rate of transform) |
Arctiin mg (rate of transform) |
Phillyrin mg (rate of transform) |
Total flavones (g) is (rate of transform) (n=3-5) |
Appearance character |
Medical material (91g) 1234 |
10.46 (11.49%) 15.50 (17.03%) 13.19 (14.49%) 15.60 (17.14%) |
289.50 (100%) 171.90 (59.38%) 197.70 (68.29%) 185.19 (63.97%) 191.70 (66.22%) |
1033.20 (100%) 590.35 (57.14%) 647.45 (62.66%) 633.47 (61.31%) 77 0.50 (74.57%) |
57.00 (100%) 30.33 (53.21%) 35.69 (62.61%) 34.16 (59.9 3%) 36.63 (64.26%) |
4.8 (100%) 3.64 (75.83%) 4.19 (87.29%) 4.17 (86.88%) 4.37 (91.04%) |
Brown, it is brown to be difficult for the moisture absorption, very easily the moisture absorption is brown, very easily the moisture absorption is brown, very easily the moisture absorption |
Separation, purification process result of study: the pure sedimentation method reduce dried cream yield, but the active ingredient rate of transform is lower slightly, consider the industrialized suitability, so method for selecting 1.
By extracting method with separate, purification process research more finally determines method for making of the present invention, repeats second trial by the technology of this method for making, the results are shown in Table 3.
Table 3 separates, purification process research is compared
Technology |
Dried cream gram (yield) |
Chlorogenic acid milligram (rate of transform) |
Arctiin milligram (rate of transform) |
Phillyrin milligram (rate of transform) |
Total flavones gram (rate of transform) |
Medical material sample 1 sample 2 is average |
13.64(14.99% ) 13.73(15.09% ) 13.68(15.04% ) |
289.50(100%) 187.07(64.62% ) 191.65(66.20% ) 188.87(65.24) |
1033.20(100%) 746.07(72.21% ) 738.74(71.50% ) 742.35(71.85% ) |
57.00(100%) 29.36(51.50% ) 28.44(49.90% ) 28.90(50.70% ) |
4.80(100%) 3.58(74.58%) 3.49(72.71%) 3.54(73.75%) |
By table as seen, method for making favorable reproducibility of the present invention can adapt to suitability for industrialized production.
The beneficial effect of medicine of the present invention shows:
Prove that through pharmacodynamics test medicine of the present invention has tangible antiviral, antiinflammatory, bacteriostasis.Be particularly useful for being used for anti-respirovirus and SARS virus.Concrete test is as follows:
1, antivirus action:
(mice) and external (cell) campaign proves that silver-colored horse antiviral drugs has the effect of tangible preventing respiratory viruses in body, and is approximate with positive control drug SHUANGHUANGLIAN KOUFUYE and ribavirin result of the test.Through the medicine efficacy screening test of vitro inhibition SARS virus, adopt the CPE method, its therapeutic index is 2.
(1), the external preventing respiratory viruses test of pesticide effectiveness of medicine of the present invention:
1., influenza first 3 types virus test: in Testis et Pentis Canis passage cell (mdck cell), drug toxicity is tested positive control drug SHUANGHUANGLIAN KOUFUYE and ribavirin.The results are shown in Table 4.
Table 4 silver horse antiviral drugs influenza first 3 types virus result of the test
Medicine |
Method |
The single administration group |
Three administration groups |
IC
50μg/ml |
MIC
μg/ml |
TI |
IC
50μg/ml |
MIC
μg/ml |
TI |
Silver horse antiviral drugs |
CPE method mtt assay |
19±0.6 22±2.6 |
46.9±0 46.9±0 |
115 94.9 |
17±0.9 20±3.8 |
46.9±0 46.9±0 |
127 102 |
SHUANGHUANGLIAN KOUFUYE |
CPE method mtt assay |
18±4.8 17.7±4 |
46.9±0 46.9±0 |
136 117 |
>11.7±0 >11.7±0 |
23.4±0 23.4±0 |
<202 <172 |
Ribavirin |
CPE method mtt assay |
17±0.9 18.6±5.3 |
46.9±0 46.9±0 |
>176 >173 |
15±0.6 17±0.3 |
46.9±0 46.9±0 |
>196 >175 |
2., anti-Ad3 virus test: in people's pulmonary carcinoma passage cell (293 cell) and human cervical carcinoma cell (Hela cell), drug toxicity is tested positive control drug SHUANGHUANGLIAN KOUFUYE and ribavirin.The results are shown in Table 5.
The anti-Ad3 virus of table 5 silver horse antiviral drugs result of the test
Medicine |
Cell |
Method |
The single administration group |
Three administration groups |
IC
50μg/ml |
MIC
μg/ml |
TI |
IC
50μg/ml |
MIC
μg/ml |
TI |
Silver horse antiviral drugs |
293 cells |
CPE method mtt assay |
17.7±1.9 18.8±0.8 |
46.9±0 46.9±0 |
112 108 |
15.9±0.6 18±0.3 |
46.9±0 46.9±0 |
134 110 |
SHUANGHUANGLIAN KOUFUYE |
CPE method mtt assay |
14.6±0.7 21±1.4 |
46.9±0 46.9±0 |
179 81 |
<11.7±0 <14±0.6 |
46.9±0 23.4±0 |
224 126 |
Ribavirin |
CPE method mtt assay |
15.8±1.4 19±0.9 |
46.9±0 46.9±0 |
190 158 |
15±0.3 19±3.9 |
46.9±0 46.9±0 |
198 159 |
Silver horse antiviral drugs |
The Hela cell |
CPE method mtt assay |
25.5±2 28.9±9.7 |
93.8±0 78.1±27 |
84 78.2 |
18±1.4 19±3.9 |
62.5±27 31.3±13.5 |
118 109 |
SHUANGHUANGLIAN KOUFUYE |
CPE method mtt assay |
14±0.3 22±1.1 |
46.9±0 31.3±13. 5 |
173 77 |
<11.7±0 <11.7±0 |
23.4±0 23.4±0 |
213 146 |
Ribavirin |
CPE method mtt assay |
17.8±0.6 23.8±0.7 |
78.1±27 46.9±0 |
168 126 |
14.8±2.6 17.7±0.6 |
46.9±0 46.9±0 |
207 170 |
3., anti-RSV virus test: in African green monkey kidney cell (Vero cell) and people's pharyngeal cancer epithelial cell (Hep-2 cell), drug toxicity is tested positive control drug SHUANGHUANGLIAN KOUFUYE and ribavirin.The results are shown in Table 6.
The anti-RSV virus of table 6 silver horse antiviral drugs result of the test
Medicine |
Cell |
Method |
The single administration group |
Three administration groups |
IC
50μg/ml |
MIC
μg/ml |
TI |
IC
50μg/ml |
MIC
μg/ml |
TI |
Silver horse antiviral drugs |
The Vero cell |
CPE method mtt assay |
18±1.6 20±0.8 |
46.9±0 23.4±0 |
119 106 |
16±1.9 22.5±0.5 |
46.9±0 23.4±0 |
132 94 |
SHUANGHUANGLIAN KOUFUYE |
CPE method mtt assay |
12±0.5 21±1.4 |
46.9±0 23.4±0 |
246 86 |
<11.7±0 <11.7±0 |
23.4±0 23.4±0 |
>258 >153 |
Ribavirin |
CPE method mtt assay |
16±0.5 21±0.8 |
46.9±0 23.4±0 |
184 140 |
12.9±1.2 23±0.8 |
39±14 23.4±0 |
232 129 |
Silver horse antiviral drugs |
The Hep-2 cell |
CPE method mtt assay |
23.7±5.2 25.8±8.6 |
93.8±0 78.1±27 |
94 84 |
16.9±1 18.7±0 |
46.9±0 23.4±0 |
127 109 |
SHUANGHUANGLIAN KOUFUYE |
CPE method mtt assay |
12.8±0 21.3±1.2 |
46.9±0 31.3±13. 5 |
225 82 |
<11.7±0 <11.7±0 |
23.4±0 11.7±0 |
>246 >149 |
Ribavirin |
CPE method mtt assay |
19.6±3.8 21±1.8 |
78.1±27 54.7±35. 8 |
158 144 |
18±1.9 17.5±0.5 |
78.1±27 39±13.5 |
167 171 |
(2), the resisiting influenza virus test of pesticide effectiveness in the medicine body of the present invention
1., give influenza virus after, observe 14 days according to each the group dead mouse count statistical result, positive control drug SHUANGHUANGLIAN KOUFUYE and ribavirin.The results are shown in Table 7.
Resisiting influenza virus result of the test (is index with the mortality rate) in the table 7 silver horse antiviral agents object
Medicine |
Dosage (g/kg) |
Number of mice (only) |
Survival number (only) |
Death toll (only) |
Mortality rate (%) |
Protective rate (%) |
Statistical procedures |
Interpretation of result |
* median effective dose (ED
50)
|
X
2 |
P |
Silver horse antiviral drugs shuanghuanglian mixture, shuanghuanglian koufuye Ribavirin |
4 2 1 0.5 4 2 1 0.5 0.1 0.05 0.025 |
30 30 30 30 30 30 30 30 30 30 30 |
30 29 23 17 23 19 17 10 30 30 24 |
0 1 7 13 7 11 13 20 0 0 6 |
0 3 23 43 23 37 43 67 0 0 20 |
100 97 75 54 75 60 54 28 100 100 78 |
52.50 48.65 30.24 17.33 30.24 21.17 17.33 6.66 52.50 52.50 33.85 |
<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 |
There is remarkable result to have remarkable result to have remarkable result to have remarkable result to have remarkable result to have remarkable result to have the remarkable result remarkable result that produced effect to have remarkable result that remarkable result is arranged |
<0.5 g/kg 0.93 g/kg <0.025 g/kg |
Virus control |
10 LD
50 |
30 |
2 |
28 |
93 |
|
|
|
|
|
*Calculate according to protective rate
2., give influenza virus after, observing 14 days is the indicator-specific statistics result according to each group life of mouse rate, positive control drug SHUANGHUANGLIAN KOUFUYE and ribavirin.The results are shown in Table 8
Resisiting influenza virus result of the test in the table 8 silver horse antiviral agents object (is index to prolong vital rates)
Medicine |
Dosage (g/kg) |
Number of mice (only) |
Mortality rate (%) |
Protective rate (%) |
The average survival time day (my god) |
Prolong vital rates (%) |
*Median effective dose (ED
50)
|
Silver horse antiviral drugs SHUANGHUANGLIAN KOUFUYE ribavirin virus control |
4 2 1 0.5 4 2 1 0.5 0.1 0.05 0.025 10LD
50 |
30 30 30 30 30 30 30 30 30 30 30 30 |
0 1 7 13 7 11 13 20 0 0 6 28 |
100 97 75 54 75 60 54 28 100 100 78 |
14.0±0.00 13.7±1.46 12.5±2.82 10.5±4.17 12.17±3.4 11.13±3.91 10.33±4.31 8.73±4.20 14±0.00 14±0.00 12.97±2.84 6.23±2.36 |
125 120 101 69 95 79 66 40 125 125 108 |
<0.5 g/kg 0.76 g/kg <0.025 g/kg |
*Calculate according to protective rate
3. after giving influenza virus, with pathological changes Mus lung suspension inoculated into chick embryo, detecting viral pneumonia is the indicator-specific statistics result, positive control drug SHUANGHUANGLIAN KOUFUYE and ribavirin.The results are shown in Table 9.
Resisiting influenza virus result of the test in the table 9 silver horse antiviral agents object
(with pathological changes Mus lung suspension inoculated into chick embryo, detecting viral pneumonia is index)
Medicine |
Dosage (g/kg) |
Number of mice (only) |
Viral pneumonia |
Viral pneumonia incidence rate (%) |
Viral pneumonia slip (%) |
Statistical procedures |
Interpretation of result |
* median effective dose (ED
50)
|
Have |
Do not have |
X
2 |
P |
Silver horse antiviral drugs shuanghuanglian mixture, shuanghuanglian koufuye Ribavirin virus control |
4 2 1 0.5 4 2 1 0.5 0.1 0.05 0.025 10LD
50 |
30 30 30 30 30 30 30 30 30 30 30 30 |
0 1 9 20 9 11 13 21 0 2 15 27 |
30 29 21 10 21 19 17 9 30 28 15 3 |
0 3.3 30 67 30 37 43 70 0 7 50 90 |
100 96 67 26 67 59 52 22 100 92 44 |
52.50 49.09 22.50 4.81 22.50 18.37 14.70 3.75 52.50 41.71 11.42 |
<0.01 <0.01 <0.01 <0.05 <0.01 <0.01 <0.01 >0.05 <0.01 <0.01 <0.01 |
Having obvious resisiting influenza virus effect to have obvious resisiting influenza virus effect to have obvious resisiting influenza virus effect to have the resisiting influenza virus effect to have obvious resisiting influenza virus effect to have obvious resisiting influenza virus effect to have obvious resisiting influenza virus effect not have the resisiting influenza virus effect has obvious resisiting influenza virus effect to have obvious resisiting influenza virus effect that the resisiting influenza virus effect is arranged |
0.83 g/kg 0.97 g/kg 0.027 g/kg |
*Calculate according to the viral pneumonia slip
(3), the test of the medicine efficacy screening of vitro inhibition SARS virus, carried out the inhibiting experiment to coronavirus No. 04 in African green monkey kidney passage cell (VERO E6 cell) is cultivated of silver-colored horse antiviral drugs, method and result are as follows:
Test method: pilot study: 1. silver-colored horse antiviral drugs is to the toxicity test of VERO E6 cell: VERO E6 cell is inoculated 96 well culture plates with 400,000/ml concentration, and 37 ℃, 5%CO
2Cultivated 24 hours, add the checking medicine, being tried thing silver horse antiviral drugs doubling dilution is 6000 μ g/ml ∽, 11.7 μ g/ml, the dilution of positive control medicine ganciclovir is 6000 μ g/ml ∽, 11.7 μ g/ml, every concentration is inoculated 3 holes, and every hole 100 μ l establish the normal cell contrast simultaneously, 37 ℃, 5%CO
2Cultivated 6 days, every day is observation of cell metamorphosis (CPE) under inverted microscope, is (+) with 25% following metamorphosis, 26 ∽ 50% are (++), 51 ∽ 75% are (+++), and 76 ∽ 100% are (++ ++), calculate medicine median toxic concentration (TD with the Reed-Muench method
50) and maximal non-toxic concentration (TD
0).2. in VERO E6 cell culture separation and the virulence of coronavirus No. 04 (No. 04 specimen of SARS patients serum is provided by You An hospital) are measured: VERO E6 cell is inoculated 96 test tubes with every milliliter 400,000 concentration, and 37 ℃, 5%CO
2Cultivated 24 hours, and discarded culture fluid, every pipe adds SARS patients serum 0.2ml, and 37 ℃ of rotary drums were cultivated 5 ∽ 7 days. and after the CPE variation appears in cell, adopt the method for PCR to detect coronavirus, No. 04 specimen PCR positive is defined as the coronavirus separated strain.With whole last dilution method purified virus 2 times, it is still positive that PCR detects coronavirus, through determination of immunofluorescence method double SARS patients serum, and the IgM positive, IgG4 doubly raises, and is defined as coronavirus.Adopting viral CPE method to measure it tires.Virus CPE method: VERO E6 cell is inoculated 96 culture plates with every milliliter 400,000 concentration, and 37 ℃, 5%CO
2Cultivated 24 hours, and added viral liquid, viral dilution 10
-1∽ 10
-6, 6 concentration, every concentration is inoculated 3 holes, and every hole 100 μ l establish the normal cell contrast, and 37 ℃, 5%CO
2Cultivated 5 ∽ 7 days, per 24 hours under inverted microscope the observed and recorded cellular morphology change (CPE): with 25% following metamorphosis is (+), and 26 ∽ 50% are (++), and 51 ∽ 75% are (+++), 76 ∽ 100% are (++ ++), calculate viral half with the Reed-Muench method and infect concentration TCID
50
Formal experiment: silver-colored horse antiviral drugs in VERO E6 cell culture to the inhibitory action of coronavirus No. 04:
VERO E6 cell is inoculated 96 well culture plates with every milliliter 400,000 concentration, and 37 ℃, 5%CO
2Cultivated 24 hours, cell culture discards culture fluid to monolayer, adds 100TCID
50Coronavirus liquid, establish normal cell contrast and virus control simultaneously.Put 37 ℃, 5%CO
2Adsorb after 2 hours, abandon viral liquid, add the silver-colored horse antiviral drugs of variable concentrations, the maximal non-toxic concentration (TD of choice of drug pair cell
0) i.e. 1500 μ g/ml ∽, the 2.93 μ g/ml of 10 concentration of 2 times of dilutions, positive control medicine ganciclovir is i.e. 6000 μ g/ml ∽, the 11.7 μ g/ml of 10 concentration of 2 times of dilutions, the medicine of dilution is added respectively in the cell hole, and every concentration 3 holes are established normal cell contrast and virus control simultaneously.Put 37 ℃, 5%CO
2Incubator was cultivated 5 ∽ 7 days, observed viral CPR day by day under inverted microscope, occurred with virus control +++-++ ++ in time, finish to test, and calculates the medium effective concentration (IC of medicine with the Reed-Muench method
50) and therapeutic index (TI), the test triplicate.
The result: silver-colored horse antiviral drugs is to the toxic action of VERO E6 cell, maximal non-toxic concentration (TD
0) be 1500 ± 0 μ g/ml, median toxic concentration (TD
50) be 3000 ± 0 μ g/ml; Contrast medicine ganciclovir maximal non-toxic concentration (TD
0) be>6000 ± 0 μ g/ml, median toxic concentration (TD
50) be>6000 ± 0 μ g/ml.
Silver horse antiviral drugs is cultivated in VERO E6 cell, to the inhibitory action of coronavirus No. 04, CPE method: medicine medium effective concentration (IC
50) be 375 ± 0 μ g/ml, minimum effective drug concentration (MIC) is 750 ± 0 μ g/ml; Contrast medicine ganciclovir CPE method: medicine medium effective concentration (IC
50) be 11.7 ± 0 μ g/ml, minimum effective drug concentration (MIC) is 23.44 ± 0 μ g/ml.
Anti-SARS virus result of the test shows, a little less than in VERO E6 cell, the inhibitory action comparison of coronavirus No. 04 being wanted according to the medicine ganciclovir of silver horse antiviral drugs, has only 1/32nd of contrast medicine, but, the treatment SARS there is assosting effect because it also has pharmacological actions such as heat-clearing and toxic substances removing, antiinflammatory be antibacterial.
2, antiinflammatory action:
(1), silver-colored horse antiviral drugs is to the influence of auricle inflammation due to the mice dimethylbenzene
All can be significantly after silver horse antiviral drugs 9.1,18.2g/kg and the administration of SHUANGHUANGLIAN KOUFUYE 12.0ml/kg group and highly significant suppress have significance meaning and highly significant meaning (P<0.05 or P<0.01) to see Table 10 with water group comparing difference by auricle inflammation due to the dimethylbenzene.
Table 10 silver horse antiviral drugs is to the influence of auricle inflammation due to the mice dimethylbenzene (X ± SD)
Group |
Dosage (g/kg) |
Number of animals (only) |
Inflammation swelling degree (mg) |
Inhibitory rate of intumesce (%) |
Water group silver horse antiviral drugs silver horse antiviral drugs silver horse antiviral drugs SHUANGHUANGLIAN KOUFUYE |
9.1 18.2 36.4 12.0ml/kg |
10 10 10 10 10 |
8.2±3.3 4.0±3.7
* 3.2±2.1
** 5.4±3.4 4.0±3.1
** |
51.2 61.0 34.1 51.2 |
Compare * P<0.05, * * P<0.01 with the water group.
(2), silver-colored horse antiviral drugs is to the granulomatous influence of rat agar
Silver horse antiviral drugs 4.55 and 9.1g/kg and SHUANGHUANGLIAN KOUFUYE 6ml/kg can be significantly and the highly significant inhibition by the granuloma due to the agar, itself and water group relatively have significant difference and significant differences (P<0.05 and P<0.01) sees Table 11.
Table 11 silver horse antiviral granule is to the granulomatous effect of rat agar (X ± SD)
Group |
Number of animals (only) |
Dosage (g/kg) |
Agar granuloma weight in wet base (g) |
Water group silver horse antiviral drugs silver horse antiviral drugs silver horse antiviral drugs SHUANGHUANGLIAN KOUFUYE |
10 10 10 10 10 |
4.55 9.10 18.2 6ml/kg |
1.47±0.23 1.23±0.26
* 1.13±0.25
** 1.32±0.36 1.13±0.38
* |
Compare * P<0.05, * * P<0.01 with the normal saline group.
3, silver-colored horse antiviral drugs causes the influence of fever in rabbit body temperature to endotoxin
Only when 0.5h, significantly can suppress endotoxin after the silver horse antiviral drugs 3.64g/kg medication and cause fever in rabbits, compare (P<0.05) with the water group; 7.28g/kg then 0.5,1,1.5,2h four time periods all can be significantly and highly significant suppress to cause fever in rabbits by endotoxin, with water group relatively (P<0.05 and P<0.01).And the aspirin group also can significantly suppress to cause fever in rabbits by endotoxin at 2.5h except that above-mentioned four time periods, compared (P<0.05) with the water group and saw Table 12.
Table 12 silver horse antiviral granule is to influence (X ± SD) (n=7) of endotoxin pyrogenic rabbit body temperature
Group |
Dosage (g/kg) |
Different time body temperature changing value after the administration (℃) |
0.5h |
1h |
1.5h |
2h |
2.5h |
3h |
Water group silver horse antiviral drugs silver horse antiviral drugs aspirin |
3.64 7.28 0.1 |
1.16± 0.38 0.58± 0.45
* 0.59± 0.28
** 0.59± 0.49
* |
1.49± 0.50 1.16± 0.54 0.93± 0.38- 0.91± 0.33
* |
1.48± 0.50 1.04± 0.33 0.83± 0.28
** 0.79± 0.23
** |
1.14± 0.34 0.59± 0.58 0.52± 0.29
** 0.56± 0.23
** |
0.75± 0.48 0.59± 0.36 0.33± 0.33 0.34± 0.11
* |
0.66± 0.44 0.61± 0.58 0.27± 0.39 0.31± 0.31 |
Compare * P<0.05, * * P<0.01 with the normal saline group.
4, extracorporeal bacteria inhibitor test
Silver horse antiviral drugs all has in various degree bacteriostasis to the examination strain, is 0.125g/ml to the minimal inhibitory concentration (MIC) of staphylococcus aureus, beta hemolytic streptococcus, alpha streptococcus.Minimal inhibitory concentration (MIC) to bacillus pyocyaneus, Diplococcus pneumoniae sees Table 13 for 0.25g/ml.
The minimal inhibitory concentration (g/ml) of table 13 silver horse antiviral granule
Test organisms |
Silver horse antiviral drugs |
Extension rate |
Golden yellow staphylococcus beta hemolytic streptococcus alpha streptococcus pyocyanic pneumonia diplococcus |
0.125 0.125 0.125 0.25 0.25 |
1∶8 1∶8 1∶8 1∶4 1∶4 |
5, bacteriostatic test in the silver-colored horse antiviral agents object
Silver horse antiviral drugs 18.2g/kg and cefradine 0.30g/kg have stronger protective effect to the clinical bacteria of staphylococcus aureus, relatively have significant difference (P<0.05) with the water group and see Table 14.
Bacteriostatic test in the table 14 silver horse antiviral agents object
Group |
Number of animals (only) |
Dosage (g/kg) |
Survival (only) |
Dead (only) |
Mortality rate (%) |
Water group silver horse antiviral drugs silver horse antiviral drugs silver horse antiviral drugs cefradine |
20 20 20 20 20 |
9.10 18.2 36.4 0.30 |
1 5 7 3 9 |
19 15 13 17 11 |
95 75 65
* 85 55
* |
Compare * P<0.05 with the water group.
6, silver-colored horse antiviral drugs is to the influence of mice delayed hypersensitivity
Remarkable and the highly significant inhibition mice ear of silver horse antiviral drugs 18.2g/kg and 36.4g/kg and SHUANGHUANLIAN 6.0ml/kg energy relatively has significant difference with model group and significant differences (P<0.05 and P<0.01) sees Table 15.
Table 15 silver horse antiviral drugs is to the influence of mice delayed hypersensitivity (X ± SD)
Group |
Dosage (g/kg) |
Number of animals (only) |
Two ear weight differences (mg) |
Normal group model group silver horse antiviral drugs silver horse antiviral drugs silver horse antiviral drugs SHUANGHUANGLIAN KOUFUYE |
9.1 18.2 36.4 6.0ml/kg |
10 10 10 10 10 10 |
1.3±1.16 8.4±4.22
△△△ 7.1±2.38 4.7±3.33
* 3.3±2.58
** 4.6±3.20
* |
Compare with normal group
△ △ △* P<0.05, * * P<0.01 are compared with model group in P<0.01.
7, the analgesic activity of silver-colored horse antiviral drugs
(1) mice hot plate method test
Silver horse antiviral drugs 36.4 gram kilograms
-1In the time of 30 minutes, can obviously improve the effect of the mice hot plate method threshold of pain, relatively have significant difference (P<0.05) to see Table 16 with the water group.
Table 16 silver horse antiviral granule is to the influence of mice hot plate method pain threshold (X ± SD)
Group |
Number of animals (only) |
Dosage (g/kg) |
Before the administration |
Pain threshold after the administration |
30 |
60 |
90 |
120 |
Physiological saline silver horse antiviral drugs silver horse antiviral drugs silver horse antiviral drugs aspirin |
10 10 10 10 10 |
9.10 18.2 36.4 0.30 |
15.7± 5.02 16.2± 5.2 0 16.2± 484 16.5± 7.51 15.7± 5.56 |
11.5± 2.95 13.7± 4.20 13.4± 3.37 17.9± 9.42
* 19.1± 6.26
** |
13.3± 6.20 13.8± 6.26 13.3± 7.18 15.4± 11.9 19.6± 4.24
* |
16.5± 8.45 13.9± 10.2 15.7± 6.90 18.3± 12.8 20.8± 5.27 |
17.5± 12.9 13.5± 6.25 15.4± 7.26 16.1± 8.68 21.5± 4.70 |
Compare with the normal saline group
*P<0.05,
*P<0.01.
(2) writhing method test
Lack than the water group though turn round the body number of times after the administration of silver horse antiviral drugs three dosage groups, relatively there are no significant that difference (P>0.05) sees Table 17 with the water group.
Table 17 silver horse antiviral drugs Dichlorodiphenyl Acetate causes the influence (X ± SD) of mouse writhing reaction
Group |
Dosage (g/kg) |
Number of animals (only) |
Turn round body number of times (inferior/15 minute) |
Water group silver horse antiviral drugs silver horse antiviral drugs silver horse antiviral drugs aspirin |
9.10 18.2 36.4 0.30 |
11 11 11 11 11 |
15.1±11.3 10.6±8.87 11.9±9.99 12.2±9.53 6.27±5.12
* |
Compare * P<0.05 with the water group.