CN103070949B - Traditional Chinese medicament composition and preparation and preparation method thereof - Google Patents
Traditional Chinese medicament composition and preparation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicament composition, which consists of the following components in parts by weight: 50 to 100 parts of rhizoma atractylodis macrocephalae, 50 to 150 parts of radix scutellariae, 20 to 40 parts of sophora flavescens, 60 to 150 parts of patchouli, 60 to 120 parts of astragalus, 60 to 100 parts of Chinese pulsatilla root, 25 to 60 parts of gardenia and 60 to 140 parts of lonicera japonica. The traditional Chinese medicament composition has reasonable proportion, and the effects of strengthening body resistance and eliminating evil, invigorating the spleen and eliminating dampness and clearing heat and stopping dysentery. The invention further discloses a preparation containing the traditional Chinese medicament composition, and the preparation is convenient to use and simple to prepare, and can be used for treating moist heat dysentery of swine, transmissible gastroenteritis of swine, weakened immune system and other diseases, thereby having a good application prospect.
Description
Technical field
The invention belongs to field of medicaments, relate to a kind of Chinese medicine composition, also relate to preparation and the preparation method prepared by this Chinese medicine composition.
Background technology
Pig lets out dysentery, and Chinese veterinarian thinks it is mainly the disease of damp-heat in the spleen and stomach, how invades in the intestines and stomach and forms because of wind, cold, summer-heat, wet, heat, poison, can be divided into four types such as following heat-toxin type, damp-heat type, cold-dampness type and asthenic cold type.Wherein, the damp and hot dysentery of letting out is called again acute enteritis, is by caused animal body diseases of infected by microbes such as antibacterial and viruses, is commonly encountered diseases, frequently-occurring disease.Primary disease is mainly in summer and autumn, is mainly caused by spleen and stomach function obstacle and gastrointestinal dysfunction, and clinical manifestation is diarrhea, vomiting, heating etc., and severe patient can cause dehydration, electrolyte disturbance etc.
Transmissible gastroenteritis of swine is the acute height contact of the one pig infectious intestinal disease being caused by virus.In recent years, in I ground piglet and Adult Pig, have popular, particularly the farrow early spring initial phase of popular primary disease in season, can cause large quantities of piglet death, 5 week age, above piglet mortality rate was low, but the pig fertility performance of resistance to mistake declines after infecting, price of deed rate declines, and has caused larger economic loss to pig industry.The pig infecting is take hyperemesis, water sample diarrhea and serious dehydration as principal character.
Piglet pujos blancos causes by escherichia coli, is a kind of infectious intestinal diseases that 10 ~ 30 age in days piglets often send out, abrupt change of climate, overcast and rainy moist, column home is polluted, sow feed mismate, in milk the too high or lactation amount of fat content not enough be all the key factor that inspires primary disease.Clinical symptom: dysentery, feces gray or pistac, contain mucus and in the pasty state, diarrhea increased frequency subsequently, feces is stench and thin.Piglet becomes thin gradually, thick disorderly unglazed by hair, tail and hind leg faecal contamination.Down in spirits, aversion to cold, sucks the breast and reduces, and dehydration, eventually because stupor collapse is dead.
Due to above-mentioned disease incidence reason difference, existing nothing is treated damp and hot dysentery, transmissible gastroenteritis of swine and the piglet pujos blancos let out of pig simultaneously, allows to treatment, in the time for the treatment of, can not improve the immunologic function of pig simultaneously.Therefore, be badly in need of a kind of Chinese medicine composition, said composition can be treated the damp and hot diseases such as dysentery, transmissible gastroenteritis of swine and piglet pujos blancos of letting out of pig, can also improve the immunologic function of pig simultaneously.
Summary of the invention
In view of this, one of object of the present invention is to provide a kind of Chinese medicine composition, and formula is unique, can treat the damp and hot diseases such as dysentery, transmissible gastroenteritis of swine and piglet pujos blancos of letting out of pig simultaneously, can also improve the immunologic function of pig.
For achieving the above object, the invention provides following technical scheme:
A kind of Chinese medicine composition, is made up of 60 ~ 140 parts of 50 ~ 100 parts of the Rhizoma Atractylodis Macrocephalaes, 50 ~ 150 parts of Radix Scutellariaes, 20 ~ 40 parts of Radix Sophorae Flavescentiss, 60 ~ 150 parts of Herba Pogostemonis, 60 ~ 120 parts of the Radixs Astragali, 60 ~ 100 parts of the Radix Pulsatillaes, 25 ~ 60 parts of Fructus Gardeniaes and Flos Loniceraes by weight.
Preferably, formed by 100 parts of 80 parts of the Rhizoma Atractylodis Macrocephalaes, 100 parts of Radix Scutellariaes, 30 parts of Radix Sophorae Flavescentiss, 70 parts of Herba Pogostemonis, 100 parts of the Radixs Astragali, 80 parts of the Radix Pulsatillaes, 40 parts of Fructus Gardeniaes, Flos Lonicerae by weight.
Each raw material of Chinese medicine composition of the present invention has following character and effect:
The Rhizoma Atractylodis Macrocephalae be the feverfew Rhizoma Atractylodis Macrocephalae (
atractylodes macrocephalakoidz.) dry rhizome; Bitter in the mouth, sweet, warm in nature; Return spleen, stomach warp; Function invigorating the spleen and benefiting QI, dampness diuretic, hidroschesis, antiabortive; Cure mainly insufficiency of the spleen lack of appetite, abdominal distention is let out, phlegm retention vertigo and palpitation, edema, spontaneous perspiration, frequent fetal movement.
Radix Scutellariae be labiate Radix Scutellariae (
scutellaria baicalensisgeorgi) dry root; Bitter in the mouth, cold in nature; Return lung, gallbladder, spleen, large intestine, small intestine meridian; Function heat clearing and damp drying, lets out fire removing toxic substances, and hemostasis is antiabortive; Cure mainly hygropyrexia, fever disease in summer vomiting and nausea uncomfortable in chest, damp and hot feeling of fullness, lets out dysentery, jaundice, and cough due to lung-heat, high hot excessive thirst, heat in blood is told nosebleed, carbuncle sore tumefacting virus, frequent fetal movement.
Radix Sophorae Flavescentis be leguminous plant Radix Sophorae Flavescentis (
sophora flavescensait.) dry root; Bitter in the mouth, cold in nature; GUIXIN, liver, stomach, large intestine, urinary bladder channel; Function heat clearing and damp drying, parasite killing, diuresis; Cure mainly hematodiarrhoea, have blood in stool, jaundice urine retention, leucorrhea with red and white discharge, swelling of the vulva pudendal pruritus, eczema, eczema, skin pruritus, scabies leprosy.
Herba Pogostemonis be labiate Herba Pogostemonis (
pogostemon cablin(Blanco) Benth.) dry aerial parts; Acrid in the mouth, slightly warm in nature; Return spleen, stomach, lung meridian; Function eliminating turbid pathogen with aromatics, appetizing preventing or arresting vomiting, delivers expelling summer-heat; Cure mainly turbid damp obstructing in middle-JIAO, the vomiting of gastral cavity painful abdominal mass, heat-damp in summer asthenia, uncomfortable in chest not easypro, cold-damp is closed summer-heat, and stomachache is told and is let out, nasosinusitis headache.
Astragalus polysaccharides be leguminous plant Radix Astagali (
astragalus memeranaceus(Fisch.) Bge. var.
mongholicus(Bge.) Hsiao) or Radix Astragali (
astragalus membranaceus(Fisch.) Bge.) the polyoses extract of dry root; Radix Astragali sweet in the mouth, slightly warm in nature; Return lung, spleen channel; Function invigorating QI to consolidate the body surface resistance, diuresis poison holding, evacuation of pus, expelling pus and promoting granulation.Modern pharmacological research result shows: astragalus polysaccharides is the main effective site of the Radix Astragali; have the immunologic function of enhancing, induction body generation interferon, protection cardiovascular system, regulate the multiple pharmacologically actives such as blood glucose, blood pressure lowering, wherein the promotion to immunologic function and regulating action are particularly evident.
The Radix Pulsatillae be the ranunculaceae plant Radix Pulsatillae (
pulsatilla chinensis(Bge.) Regel) dry root.Nature and flavor hardship, cold; Return stomach warp, there is heat-clearing and toxic substances removing, eliminating pathogenic heat from blood to cure dysentery.Show according to the study, the Radix Pulsatillae contains the multiple effective active compositions such as anemoside, betulic acid, daucosterol, protoanemonin and Anemonin, protoanemonin and the Radix Pulsatillae have anti-various bacteria and viral effect, wherein diphtheria corynebacterium, staphylococcus, streptococcus, escherichia coli, tubercule bacillus, clostridieum welchii etc. are had to obvious inhibitory action; Simultaneously the Radix Pulsatillae has calmness, analgesia and spasmolytic, to having good therapeutical effect due to bacterial intestinal bleeding dysentery and enterospasm dysentery.
Fructus Gardeniae be Maguireothamnus speciosus Fructus Gardeniae (
gardenia jasminoidesellis) dry mature fruit; Bitter in the mouth, cold in nature; GUIXIN, lung, tri-jiao channel; Function is let out fiery relieving restlessness, clearing away heat-damp and promoting diuresis, removing pathogenic heat from blood and toxic substance from the body; Cure mainly calentura vexed, jaundice dark coloured urine, blood strangury and dry pain, heat in blood is told nosebleed, conjunctival congestion and swelling pain, pathogenic fire,toxin and furuncles.
Flos Lonicerae be caprifoliaceae plant Radix Ophiopogonis (
lonicera japonicathund.) flower that dry flower or band are just opened; Sweet in the mouth, cold in nature; Return lung, the heart, stomach warp; Function heat-clearing and toxic substances removing, wind-heat dissipating; Cure mainly carbuncle furuncle, sore throat, erysipelas, toxic-heat and blood stasis, anemopyretic cold, epidemic febrile disease heating.
Two of object of the present invention is to provide the application of Chinese medicine composition in the damp and hot medicine of letting out dysentery of preparation treatment pig, and technical scheme is:
The application of described Chinese medicine composition in the damp and hot medicine of letting out dysentery of preparation treatment pig.
Three of object of the present invention is to provide the application of Chinese medicine composition in the medicine of preparation treatment transmissible gastroenteritis of swine, and technical scheme is:
The application of described Chinese medicine composition in the medicine of preparation treatment transmissible gastroenteritis of swine.
Four of object of the present invention is to provide the application of Chinese medicine composition in the medicine of preparation treatment immunologic hypofunction, and technical scheme is:
The application of described Chinese medicine composition in the medicine of preparation treatment immunologic hypofunction.
Five of object of the present invention is to provide the preparation of Chinese medicine composition, and technical scheme is:
The preparation of being prepared by described Chinese medicine composition.
Preferably, described preparation is granule.
Six of object of the present invention is to provide the preparation method of above-mentioned preparation, and technical scheme is:
The preparation method of described preparation, concrete steps are as follows:
A. get the Rhizoma Atractylodis Macrocephalae, Herba Pogostemonis and Flos Lonicerae by formula, extract its volatile oil, then use beta-cyclodextrin inclusion compound, obtain volatile oil clathrate compound;
B. get Radix Scutellariae, Radix Sophorae Flavescentis, the Radix Pulsatillae, Fructus Gardeniae, the Radix Astragali by formula, first use alcohol reflux, obtain extracting solution A, residue medicinal residues decoct with water extraction again, collect extracting solution and are designated as extracting solution B, after extracting solution A and extracting solution B are mixed and be condensed into clear paste; Simultaneously that medicinal residues are dry, then pulverize, be designated as medicinal residues A;
C. step a gained volatile oil clathrate compound, step b gained clear paste and medicinal residues A are mixed, then add adjuvant according to the agent of conventional method granulation.
Preferably, described adjuvant comprises one or more in binding agent, filler and correctives.
Preferred, described filler be in dicalcium phosphate, corn starch, Icing Sugar and lactose one or more; Described binding agent is one or both in corn starch, dextrin and cellulose; Described correctives is one or more of sucrose, saccharin sodium and grass essence.
Beneficial effect of the present invention is: the invention discloses Chinese medicine composition, it fills a prescription unique, and raw material is easy to get, and cost is low, has effect of strengthening vital QI to eliminate pathogenic factors, the spleen strengthening and damp drying, clearing away heat to cure dysentery; The invention also discloses preparation prepared by Chinese medicine composition, its preparation method is simple, Chinese medicine composition is extracted according to a conventional method to the adjuvants such as rear and filler, binding agent and correctives and be mixed and made into granule, after adding adjuvant, make solid dispersible carrier in high degree of dispersion state, increase the bioavailability of Chinese medicine composition; The granule of preparation has absorption to transmissible gastroenteritis of swine (TGEV) virus to be suppressed, suppresses to copy and effect of deactivation, and wherein directly deactivation effect is best, can improve the protective capability of the cell damaging because of transmissible gastro-enteritis virus; Preparation prepared by In Vitro Bacteriostasis experiment demonstration Chinese medicine composition can suppress the growth of the antibacterials such as escherichia coli, staphylococcus aureus, pig source clostridieum welchii, Salmonella and Candida albicans, therefore can be used in treatment piglet pujos blancos; The damp and hot dysentery clinical test results of letting out of pig is shown, Chinese medicinal composition granules reaches 85% to the damp and hot cure rate of letting out dysentery of pig; Chinese medicinal composition granules disclosed by the invention also has the effect that improves immunologic function simultaneously.
The specific embodiment
Below with reference to embodiment, the present invention is described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, conventionally according to normal condition.
Embodiment 1
A kind of Chinese medicine composition, fill a prescription as follows:
Formula 1: Rhizoma Atractylodis Macrocephalae 60g, Radix Scutellariae 50g, Radix Sophorae Flavescentis 20g, Herba Pogostemonis 60g, Radix Astragali 60g, Radix Pulsatillae 60g, Fructus Gardeniae 25g, Flos Lonicerae 60g.
Formula 2: Rhizoma Atractylodis Macrocephalae 50g, Radix Scutellariae 60g, Radix Sophorae Flavescentis 25g, Herba Pogostemonis 60g, Radix Astragali 60g, Radix Pulsatillae 70g, Fructus Gardeniae 30g, Flos Lonicerae 60g.
Formula 3: Rhizoma Atractylodis Macrocephalae 80g, Radix Scutellariae 100g, Radix Sophorae Flavescentis 30g, Herba Pogostemonis 70g, Radix Astragali 100g, Radix Pulsatillae 80g, Fructus Gardeniae 40g, Flos Lonicerae 100g.
Formula 4: Rhizoma Atractylodis Macrocephalae 90g, Radix Scutellariae 120g, Radix Sophorae Flavescentis 35g, Herba Pogostemonis 150g, Radix Astragali 120g, Radix Pulsatillae 90g, Fructus Gardeniae 45g, Flos Lonicerae 120g.
Formula 5: Rhizoma Atractylodis Macrocephalae 100g, Radix Scutellariae 150g, Radix Sophorae Flavescentis 40g, Herba Pogostemonis 80g, Radix Astragali 100g, Radix Pulsatillae 100g, Fructus Gardeniae 60g, Flos Lonicerae 140g.
Embodiment 2
In order to improve therapeutic effect, by herbal composite can water or alcohol equal solvent effective component extracting make extract, its preparation method is:
A. extract volatile oil and coated:
Get the Rhizoma Atractylodis Macrocephalae, Herba Pogostemonis and Flos Lonicerae by above-mentioned formula, add in extraction pot, add again and be equivalent to raw material gross weight 5-8 water doubly, heating recovery, reclaim distillate, distillate is carried out to double evaporation-cooling backflow, liquid to be distilled stops distillation while being equivalent to raw material weight 1/10-2/10, reclaim distillate, obtain volatile oil.
Take the beta-schardinger dextrin-that is equivalent to volatile oil weight 1/5, then add the distilled water that is equivalent to 15 times of powder-beta-dextrin weight, after heating for dissolving, add gained volatile oil, under 40 ℃ of conditions, stir 1 hour with 600rpm speed with blender, cold preservation 24 hours under 4 ℃ of conditions again, filters collecting precipitation, to be deposited under 50 ℃ of conditions and dry, obtain volatile oil clathrate compound;
B. the extraction of Radix Scutellariae, Radix Sophorae Flavescentis, the Radix Pulsatillae, Fructus Gardeniae, the Radix Astragali:
Get Radix Scutellariae, Radix Sophorae Flavescentis, the Radix Pulsatillae, Fructus Gardeniae, the Radix Astragali by said ratio, add in extraction pot, then add and be equivalent to the ethanol that raw material gross weight 5-8 volume fraction is doubly 50%, evaporation backflow 2-3 hour, collect and extract filtrate, filtrate is reclaimed ethanol with ethanol recovery tower, and remaining liq is designated as extracting solution A;
Continue to add in extraction pot to be equivalent to raw material gross weight 3-5 water doubly, decoct and extract 2-3 hour, collect and extract filtrate, must extract B, medicinal residues are dried, for subsequent use after pulverizing, and are designated as medicinal residues A;
C. step b gained extracting solution A and extracting solution B are mixed, be then condensed into clear paste shape, add step a gained volatile oil clathrate compound after cooling, mix homogeneously, is designated as mixture C.
The effective ingredient that the present embodiment gained mixture C is Chinese medicine composition.
Embodiment 3
For the ease of storing and carrying, taking convenience, makes the various preparations that clinical treatment needs that are applicable to by Chinese medicine composition or its extract, in preparation, can add acceptable carrier on pharmaceutics, and the method for preparation is conventional method.For example, the various Chinese crude drugs in herbal composite directly can be ground into fine powder, make powder, or add binding agent to make pill; Acceptable carrier on the extract of herbal composite and pharmaceutics can be made to capsule, tablet, granule etc.These extracts and preparation have identical therapeutical effect with herbal composite.
The mixture C making take embodiment 2 below and medicinal residues A are raw material, prepare granule, and concrete formula is:
Formula 1: mixture C 150g, medicinal residues A150g, dicalcium phosphate 100g, corn starch 200g, saccharin sodium 1.5g.
Formula 2: mixture C 80g, medicinal residues A100g, dicalcium phosphate 80g, corn starch 150g, saccharin sodium 1g.
Formula 3: mixture C 140g, medicinal residues A140g, dicalcium phosphate 120g, corn starch 250g, saccharin sodium 2g.
Formula 4: mixture C 200g, medicinal residues A180g, dicalcium phosphate 140g, corn starch 300g, saccharin sodium 2.5g.
Concrete preparation method is:
(1) corn starch of formula ratio 2/5 is made to starch slurry in jacketed pan, for subsequent use;
(2) saccharin sodium of formula ratio is dissolved in mixture C, then adds the medicinal residues A, dicalcium phosphate of formula ratio and remaining corn starch, mix homogeneously, obtains mixture D;
(3) by adding in (1) described starch slurry in mixture step (2) Suo Shu, after stirring, forward on granulator and granulate, after granulation, under 60 ℃ of conditions, dry 10-12 hour, be then placed in granulate in pelletizing machine, obtain Chinese medicinal composition granules.
In the present embodiment, dicalcium phosphate is filler, can replace with the corn starch of identical weight, Icing Sugar or lactose, also can replace with the mixture of dicalcium phosphate, corn starch, Icing Sugar and the lactose of identical weight; Corn starch is binding agent, can replace with the dextrin of identical weight or cellulose, also can replace with the corn starch of identical weight, dextrin and cellulosic mixture; Saccharin sodium is correctives, can replace with the sucrose of identical weight or grass essence, also can replace with the sucrose of identical weight, saccharin sodium and grass essence mixture.
embodiment 4, Chinese medicinal composition granules are to directly inactivation test of TGEV virus
1. cultivate PK-15 porcine kidney cell line and preparation TGEV virus liquid
Cultivate PK-15 porcine kidney cell line according to prior art, then cultivate transmissible gastroenteritis of swine (TGEV) virus, prepare virus liquid, for subsequent use.
2.TGEV half histiocyte infective dose (TCID
50) and the concentration determination of medicine maximal non-toxic
Reference literature (Jiang Chenggang. Chinese herbal medicine anti-swine infectious enterogastritis virus activity and Study on mechanism [D]. Northeast Agricultural University, 2003:14-15) method measure TCID
50, adopt Reed-Muench method to calculate TCID
50.
Reference literature (Jiang Chenggang, Zhang Xiuying, imperial official loyal to his sovereign. the research [J] of compound Chinese medicinal preparation anti-swine infectious enterogastritis virus activity. journal of animal science and veterinary medicine, 2004, (5): 587-589; Wei Wenqiang. screening and the mechanism of action preliminary study [D] of anti-swine infectious enterogastritis virus Chinese herbal medicine. Gansu Agriculture University, 2009:24-25.) method is measured medicine maximal non-toxic concentration, each test group result and blank group result are carried out T check, and the inapparent maximum drug level of difference is maximal non-toxic concentration.
3. cell survival rate is measured
Adopt the tetramethyl azo blue micro-enzyme reaction colorimetry of azoles (MTT) to measure cell survival rate, be specially: take out 96 orifice plates, abandon cell culture fluid, then adding 20 μ L concentration is the MTT solution of 5 mg/mL, puts 37 ℃, 5% CO
2in incubator, cultivate after 4 hours and to take out, liquid in hole is inhaled and abandoned, then add 100 μ L DMSO, put 10 min that vibrate on micro oscillator, take off photometry density value (OD) under microplate reader 490 nm wavelength, result represents by cytoprotective rate.
Cytoprotective rate=(test group OD value-model group OD value)/(blank group OD value-model group OD value) × 100%.
Measure directly inactivation test of TGEV virus by the Chinese medicinal composition granules of formula 1-5 according to the method described above, concrete grammar is: 96 well culture plates of the monolayer PK-15 porcine kidney cell covering with are divided into blank group (not doing any processing), model group, and (PK-15 porcine kidney cell infects TGEV virus, not administration processing), (PK-15 porcine kidney cell infects TGEV virus afterwards with containing 10 to astragalus polysaccharides matched group
-5the processing of g/mL astragalus polysaccharides), formula 1 ~ 5(PK-15 porcine kidney cell is 10 by the concentration of formula 1-5 respectively after infecting TGEV virus
-5the Chinese medicinal composition granules aqueous solution processing of g/mL), every group is repeated 6 holes, and result is as shown in table 1.
The Chinese medicinal composition granules of table 1, different formulations is to directly inactivation test of TGEV virus
Note: in same column, do not represent significant difference containing same letter shoulder motes, represent not remarkable (lower case represents P > 0.05, and capitalization represents P > 0.01) of difference containing same letter shoulder motes in same column.
As shown in Table 1, the Chinese medicine composition of formula 1-5 all can directly suppress TGEV virus, but granule effect prepared by 3 the Chinese medicine composition of filling a prescription is best.
96 well culture plates of the monolayer PK-15 porcine kidney cell covering with are divided into blank group (not doing any processing), model group, and (PK-15 porcine kidney cell infects TGEV virus, not administration processing), (PK-15 porcine kidney cell infects TGEV virus afterwards with containing 10 to astragalus polysaccharides matched group
-5the processing of g/mL astragalus polysaccharides), test group 1 ~ 5(PK-15 porcine kidney cell is 10 by the concentration of formula 3 respectively after infecting TGEV virus
-4g/mL, 10
-5g/mL, 10
-6g/mL, 10
-7g/mL and 10
-8the Chinese medicinal composition granules aqueous solution processing of g/mL), every group is repeated 6 holes, then measures the direct inactivating efficacy of Chinese medicinal composition granules of formula 3 preparations, and result is as shown in table 2.
Table 2, Chinese medicinal composition granules are to directly inactivation test of TGEV virus
Note: in same column, do not represent significant difference containing same letter shoulder motes, represent not remarkable (lower case represents P > 0.05, and capitalization represents P > 0.01) of difference containing same letter shoulder motes in same column.
As shown in Table 2, Chinese medicinal composition granules is inactivation of viruses directly, reduces the damage of virus to cell, thus protection PK-15 cell.1 ~ 4 pair of cytoprotection of experimental group is obvious, compared with model group difference extremely significantly (
p< 0.01), and between experimental group 2 ~ 4, there is obvious dose-effect relationship, the effect of experimental group 2 is the strongest, and protective rate reaches 29.62%.3 effects of filling a prescription are carried out.
Then calculate result according to Reed-Muench method and show, it is 1TCID that TGEV infects half cell infection amount
50be 0.2 mL 10
-3.654dilution virus liquid.
Medicine maximal non-toxic concentration determination demonstration, Chinese medicinal composition granules is in concentration c>=10
-3when g/mL cell survival rate reduce, with blank group relatively have utmost point significant difference (
p< 0.01); In concentration c≤10
-4when g/mL cell survival rate and blank group comparing difference not significantly (
p> 0.05), determine that maximal non-toxic concentration is 10
-4g/mL.
embodiment 5, Chinese medicinal composition granules are to TGEV viruses adsorption inhibition test
1. the Chinese medicinal composition granules of different formulations is to TGEV viruses adsorption inhibition test
Covering with in 96 well culture plates of monolayer PK-15 porcine kidney cell, the concentration that every hole adds 200 μ L to be prepared by the 1-5 that fills a prescription is respectively 10
-5the Chinese medicinal composition granules aqueous solution (being followed successively by formula 1 ~ 5) of g/mL, hatches 2 hours at 37 ℃, abandons medicinal liquid; Access again 200 μ L 100TCID
50virus liquid, hatches after 2 hours for 37 ℃ and abandons virus liquid, then adds 200 μ L cell maintenance mediums (DMEM containing 2% FBS cultivates), puts 37 ℃, 5%CO
2in incubator, continue to cultivate 72 hours.Simultaneously, using the PK-15 porcine kidney cell of not doing any processing as blank group, PK-15 porcine kidney cell is infected to the post processing of TGEV virus, not administration is model group, and PK-15 porcine kidney cell is infected to TGEV virus afterwards with containing 10
-5g/mL astragalus polysaccharides is processed as astragalus polysaccharides group, and above every group is repeated 6 holes.Then measure the Chinese medicinal composition granules of different formulations to TGEV viruses adsorption inhibition, result is as shown in table 3.
The Chinese medicinal composition granules of table 3, different formulations suppresses TGEV viruses adsorption
Note: in same column, do not represent significant difference containing same letter shoulder motes, represent not remarkable (lower case represents P > 0.05, and capitalization represents P > 0.01) of difference containing same letter shoulder motes in same column.
As shown in Table 3, the Chinese medicine composition granular preparation of formula 1-5 all has absorption to suppress to TGEV virus, but 3 the Chinese medicinal composition granules effect of filling a prescription is best, and the protective rate of cell is the highest.
2. the Chinese medicinal composition granules of variable concentrations is to TGEV viruses adsorption inhibition test
Covering with in 96 well culture plates of monolayer PK-15 porcine kidney cell, every hole adds 200 μ L concentration to be respectively 10
-4g/mL, 10
-5g/mL, 10
-6g/mL, 10
-7g/mL and 10
-8chinese medicinal composition granules aqueous solution (being followed successively by test group 1 ~ 5), hatch 2 hours at 37 ℃, abandon medicinal liquid; Access again 200 μ L 100TCID
50virus liquid, hatches after 2 hours for 37 ℃ and abandons virus liquid, then adds 200 μ L cell maintenance mediums (DMEM containing 2% FBS cultivates), puts 37 ℃, 5%CO
2in incubator, continue to cultivate 72 hours.Simultaneously, using the PK-15 porcine kidney cell of not doing any processing as blank group, PK-15 porcine kidney cell is infected to the post processing of TGEV virus, not administration is model group, and PK-15 porcine kidney cell is infected to TGEV virus afterwards with containing 10
-5g/mL astragalus polysaccharides is processed as astragalus polysaccharides group, and above every group is repeated 6 holes.Then measure Chinese medicinal composition granules to TGEV viruses adsorption inhibition, result is as shown in table 4.
The Chinese medicinal composition granules of table 4, variable concentrations suppresses TGEV viruses adsorption
Note: in same column, do not represent significant difference containing same letter shoulder motes, represent not remarkable (lower case represents P > 0.05, and capitalization represents P > 0.01) of difference containing same letter shoulder motes in same column.
As shown in Table 4, Chinese medicinal composition granules can suppress viruses adsorption, and In vitro culture PK-15 cell is all had to protective effect in various degree.Compared with model group, 1～4 pair of cytoprotection of test group obviously (
p< 0.01), and at test group 2(10
-5g/mL) ~ test group 4(10
-7g/mL) between, there is obvious dose-effect relationship.Wherein test group 2 activity are the highest, and protective rate is 24.79%.3 effects of filling a prescription are carried out.
embodiment 6, Chinese medicinal composition granules are to TGEV virus replication inhibition test
1. the Chinese medicinal composition granules of different formulations is to TGEV virus replication inhibition test
First 100TCID50 virus liquid access is covered with in 96 well culture plates of monolayer PK-15 porcine kidney cell, then to add respectively 200 μ L concentration be 10 in every hole
-5the Chinese medicinal composition granules aqueous solution of being prepared by formula 1-5 (being followed successively by formula 1 ~ 5) of g/mL, puts under 37 ℃ of conditions and hatches after 2 hours and abandon virus liquid, adds 200 μ L cell maintenance mediums, puts 37 ℃, 5%CO
2in incubator, continue to cultivate 72 hours.Simultaneously, using the PK-15 porcine kidney cell of not doing any processing as blank group, PK-15 porcine kidney cell is infected to the post processing of TGEV virus, not administration is model group, and PK-15 porcine kidney cell is infected to TGEV virus afterwards with containing 10
-5g/mL astragalus polysaccharides is processed as astragalus polysaccharides group, and above every group is repeated 6 holes.Then detect TGEV viral load, calculate cytoprotective rate, result is as shown in table 5.
The Chinese medicinal composition granules of table 5, different formulations suppresses TGEV virus replication
Note: in same column, do not represent significant difference containing same letter shoulder motes, represent not remarkable (lower case represents P > 0.05, and capitalization represents P > 0.01) of difference containing same letter shoulder motes in same column.
2. the Chinese medicinal composition granules of variable concentrations suppresses TGEV virus replication
First 100TCID50 virus liquid access is covered with in 96 well culture plates of monolayer PK-15 porcine kidney cell, every hole adds 200 μ L, under 37 ℃ of conditions, hatches 2 hours, abandons virus liquid, and then to add respectively 200 μ L concentration be 10 in every hole
-4g/mL, 10
-5g/mL, 10
-6g/mL, 10
-7g/mL and 10
-8chinese medicinal composition granules aqueous solution (being followed successively by test group 1 ~ 5), put under 37 ℃ of conditions and hatch after 2 hours and abandon virus liquid, add 200 μ L cell maintenance mediums, put and in 37 ℃, 5%CO2 incubator, continue to cultivate 72 h.Simultaneously, using the PK-15 porcine kidney cell of not doing any processing as blank group, PK-15 porcine kidney cell is infected to the post processing of TGEV virus, not administration is model group, and PK-15 porcine kidney cell is infected to TGEV virus afterwards with containing 10
-5g/mL astragalus polysaccharides is processed as astragalus polysaccharides group, and above every group is repeated 6 holes.Then detect TGEV viral load, calculate cytoprotective rate, result is as shown in table 6.
The Chinese medicinal composition granules of table 6, variable concentrations suppresses TGEV virus replication
Note: in same column, do not represent significant difference containing same letter shoulder motes, represent not remarkable (lower case represents P > 0.05, and capitalization represents P > 0.01) of difference containing same letter shoulder motes in same column.
As shown in Table 6, Chinese medicinal composition granules can suppress virus replication, and In vitro culture PK-15 cell is all had to protective effect in various degree.Compared with model group, test group 1-3 to cultured cells have obvious protective effect (
p< 0.01), test group 1(10
-4g/mL) ~ test group 3(10
-6g/mL) between, have obvious dose-effect relationship, test group 1 protective rate is the highest, is 12.69%.
embodiment 7
Chinese medicinal composition granules In vitro Bactericidal Experiments
1, doubling dilution
Chinese medicinal composition granules is soluble in water, make the aqueous solution that concentration is equivalent to crude drug 0.24g/mL, be then diluted to variable concentrations according to the method for table 7, then in each test tube, inoculate 0.1mL test antibacterial, in the calorstat of 37 ℃, cultivate 6 ~ 8 hours.
The operating process of table 7, doubling dilution
| Test tube numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| Meat soup (ml) | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 |
| Bacterium liquid (ml) | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | ? |
| Medicine dilution factor | 1:2 | 1:4 | 1:8 | 1:16 | 1:32 | 1:64 | 1:128 | 1:256 | 1:512 | ? | 1:2 |
The fungistatic effect of the Chinese medicinal composition granules of detection formula 3 to escherichia coli, staphylococcus aureus, pig source clostridieum welchii, Salmonella and Candida albicans, micrococcus luteus, Bacillus pumilus and bacillus subtilis according to the method described above, find out the lowest concentration of drug to various bacterial growths, this concentration is the minimum inhibitory concentration (MIC) of Chinese medicinal composition granules, the results are shown in Table shown in 8.
The external minimum inhibitory concentration of Chinese medicinal composition granules (MIC) of table 8, variable concentrations
| Pipe number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | Without medicine | Aseptic |
| Medicinal liquid extension rate | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | 0 | 1 |
| Escherichia coli | ― | ― | ― | ― | ― | ― | ― | + | + | +++++ | ― |
| Staphylococcus aureus | － | － | － | － | － | － | － | － | + | +++++ | － |
| Pig source clostridieum welchii | － | － | ― | ― | ― | + | + | + | ++ | +++++ | － |
| Salmonella | － | ― | ― | ― | ― | ― | + | + | + | +++++ | － |
| Candida albicans | － | ― | ― | ― | ― | ― | + | + | + | +++++ | － |
| Bacillus pumilus | + | + | + | ++ | ++ | +++ | ++++ | ++++ | ++++ | +++++ | － |
| Bacillus subtilis | + | + | ++ | ++ | ++ | ++ | +++ | +++ | ++++ | +++++ | － |
| Micrococcus luteus | + | + | + | ++ | ++ | ++ | +++ | +++ | ++++ | +++++ | ― |
Note: "+" indicates bacterial growth, "-" indicates without bacterial growth.With "+", " ++ ", the growth pressure of expression antibacterial
As shown in Table 8, Chinese medicinal composition granules is respectively the MIC of each bacterial strain: escherichia coli are 3.75mg/mL, staphylococcus aureus 1.875mg/mL, pig source clostridieum welchii 15mg/mL, Salmonella and Candida albicans 7.5mg/mL, and to micrococcus luteus, Bacillus pumilus, bacillus subtilis fungistatic effect is not good, does not measure its MIC.
According to method same as described above, detect Chinese medicinal composition granules antibacterial situation to escherichia coli, staphylococcus aureus, pig source clostridieum welchii, Salmonella and Candida albicans under 16 times of conditions of dilution of formula 1-5, antibacterial the results are shown in Table shown in 9.
The external minimum inhibitory concentration of Chinese medicinal composition granules (MIC) of table 9, different formulations
| Pipe number | Formula 1 | Formula 2 | Formula 3 | Formula 4 | Formula 5 | Without medicine | Aseptic |
| Medicinal liquid extension rate | 16 | 16 | 16 | 16 | 16 | 0 | 16 |
| Escherichia coli | ― | ― | ― | ― | ― | +++++ | ― |
| Staphylococcus aureus | - | － | － | － | － | +++++ | － |
| Pig source clostridieum welchii | － | － | ― | + | + | +++++ | － |
| Salmonella | － | + | ― | ― | + | +++++ | － |
| Candida albicans | + | ― | ― | ― | + | +++++ | － |
Note: "+" indicates bacterial growth, "-" indicates without bacterial growth.With "+", " ++ ", the growth pressure of expression antibacterial
As shown in Table 9, under the condition of 16 times of dilutions, formula 3 can suppress escherichia coli, staphylococcus aureus, pig source clostridieum welchii, Salmonella and Candida albicans simultaneously; Formula 1 can not suppress Candida albicans, and formula 2 can not suppress Salmonella, and formula 4 can not suppress pig source clostridieum welchii, and formula 5 can only suppress escherichia coli and staphylococcus aureus, 3 the fungistatic effect the best of therefore filling a prescription.
2, cup pipe method
First 10mL is poured in culture dish as bottom through autoclaved agar culture medium, the preparation method that spreads the agar culture medium 5mL(bacterium liquid that approximately 40 ~ 50 ℃ of one decks contain 0.1mL bacterium liquid after solidifying is in the above for by after antibacterial activation, be put in the calorstat of 37 ℃ and cultivate 12 ~ 14 hours, turbidity is about 400,000,000/mL), after solidifying, upper strata places 4 Oxford cup (external diameter 7.8mm in each culture dish, internal diameter 6.8mm), in the cup of Oxford, add the Chinese medicinal composition granules aqueous solution of the formula 3 of variable concentrations, finally put into 37 ℃ of calorstat incubated overnight.Measure the inhibition zone size of the medicinal liquid generation of variable concentrations next day, observe its antibacterial situation, result is as shown in table 10.
The Chinese medicinal composition granules of table 10, variable concentrations is to sensitive organism inhibition zone size (unit: mm)
As shown in Table 10, Chinese medicinal composition granules has good fungistatic effect to escherichia coli, staphylococcus aureus, Salmonella, Candida albicans, 5 kinds of bacterial strains of pig source clostridieum welchii, and in the time that concentration is 5%, the concentration of inhibition zone is larger.Therefore, Chinese medicinal composition granules disclosed by the invention has good fungistatic effect to escherichia coli, staphylococcus aureus, Salmonella, Candida albicans, 5 kinds of bacterial strains of pig source clostridieum welchii.
Then the Chinese medicinal composition granules prepared of formula 1-5 is made to mass fraction and is 5% aqueous solution, the fungistatic effect of the granule of preparing with the Chinese medicine composition of cup pipe method detection different formulations to escherichia coli, staphylococcus aureus, Salmonella, Candida albicans, pig source clostridieum welchii, result is as shown in table 11.
The Chinese medicinal composition granules of table 11, different formulations is to sensitive organism inhibition zone size (unit: mm)
As shown in Table 11, the Chinese medicinal composition granules of formula 3 is antibacterial best.
embodiment 8
The impact of the mice of Chinese medicinal composition granules on immunologic hypofunction
1. the impact of the mice of different formulations Chinese medicinal composition granules on immunologic hypofunction
Choose 160 of healthy mices, be divided at random 8 groups, 20 every group.Get 140 mices for building immunosuppression mouse model, concrete grammar is: lumbar injection 0.2mL cyclophosphamide, and 1 times/day, injection volume is pressed 80mg/kg, totally 3 days; The distilled water of remaining 20 mouse peritoneals injection same amount, as blank group.The immunosuppression mouse model of structure is divided into model group, positive drug matched group (astragalin injection, 0.15g/kg), formula 1-5 group (every dosage is: 1.4g crude drug/kg), 1 times/day, successive administration 3 days at random; Blank group, model group gives equivalent distilled water.After treatment, respectively organize the blood sampling of half mice eye frame, blood is for carbon clearance assessment of indices; Get each group of second half mouse spleen, thymus for index of immunity mensuration (mg/g) and T, bone-marrow-derived lymphocyte conversion test.
(1) mensuration of Immune Organs Index
Method: claim weight, get spleen and thymus, claim wet quality, and calculate spleen index and thymus index by following formula.
Spleen index=spleen quality/body weight (mg/g); Thymus index=thymus quality/body weight (mg/g).
Spleen index and thymus index measurement result are as shown in table 12:
Table 12, the impact of Chinese medicinal composition granules on mouse immune index
| Group | Dosage (g/kg) | Index and spleen index (mg/g) | Thymus index (mg/g) |
| Blank group | -- | 4.706±0.24 | 1.156±0.01 |
| Model group | -- | 3.215±0.57 △ | 0.856±0.14 △△ |
| Positive drug matched group | 0.15 | 5.323±0.53** | 1.880±0.05** |
| Formula 1 | 1.4 | 3.832±0.83** | 1.380±0.32** |
| Formula 2 | 1.4 | 3.380±0.60 | 1.230±0.15* |
| Formula 3 | 1.4 | 4.832±0.83** | 1.389±0.01** |
| Formula 4 | 1.4 | 4.832±0.83** | 1.580±0.21** |
| Formula 5 | 1.4 | 5.250±0.82 | 1.839±0.18* |
As shown in Table 12, the mice of formula 1-5 group index and spleen index and thymus index increase after administration.Therefore, the Chinese medicinal composition granules of formula 1-5 can be treated the mice of immunocompromised, and formula 5 effects are best.
(2) carbon clearance assessment of indices
?each group mice, in the last administration india ink 10mL/kg that after 1 hour, tail vein injection volume fraction is 25%, was then got blood 20 μ L at 2 minutes and 10 minutes in every rathole ball rear vein beard respectively, was blown at once 2mL mass fraction and is 0.1% Na
2cO
3in solution, get after blood to survey absorbance (OD) in 570nm, school zero solution is that 2mL mass fraction is 0.1% Na
2cO
3.Be calculated as follows and clean up index K and engulf coefficient (correction phagocytic index) α, result is as shown in table 13.
Carbon clearance index K=(logOD
2-logOD
10)/(T
10-t
2) (OD
2, OD
10be respectively 2, absorbance when 10min)
K value, after weight and liver spleen mass conversion, obtains phagocytic index α value: phagocytic index α=weight/(liver quality+spleen quality) × K
1/3(T
10=10min, T
2=2min).
(3) T lymphocyte, bone-marrow-derived lymphocyte conversion test are measured
?under aseptic condition, get spleen, then rinse twice with PBS buffer.After grinding with aseptic dismembyator, get serosity, then under 1000 r/min conditions centrifugal 5 minutes, abandon supernatant; Adding 5mL pH is 7.2 Tris NH again
4cl, gently piping and druming, erythrocyte is broken completely, then under 1000 r/min conditions centrifugal 5 min, abandon supernatant; Precipitation is rinsed 1 time with PBS buffer, then with centrifugal 5 min of 1000r/min, abandons supernatant; The full culture fluid re-suspended cell of RPM II640 of the gentamycin that is finally 2% with the new-born calf serum that is 10% containing volume fraction and mass fraction, adjusting splenocyte concentration is 3 × 10
6individual/mL, for subsequent use.
T lymphocyte transformation test: with reference to improved MTT colorimetric analysis, get 96 porocyte culture plates, every hole adds prepares splenoblast suspension 200 μ L, and concanavalin A, Con A 15 μ L(final concentrations are 6mg/L), 8 multiple holes, in the CO of 37 ℃ 5%
2in incubator, cultivate 72 hours.Within 4 hours before cultivation finishes, abandon supernatant 100 μ L, add people MTT liquid 20 μ L and continue to cultivate 4h, suck supernatant, every hole adds DMSO150 μ L cessation reaction, 96 orifice plates are moved into dull and stereotyped oscillator, and level concussion 10min, dissolves MTT reduzate completely, measure every hole absorbance (OD value) in 630nm wavelength place with enzyme-linked immunosorbent assay instrument, result is as shown in table 13.
Bone-marrow-derived lymphocyte conversion test: its method is identical with T lymphocyte transformation test, difference is to change 15 μ L canavalines into 20 μ L lipopolysaccharide, and adding rear final concentration is 50 mg/L.Measure every hole absorbance (OD value) in 630nm wavelength place with enzyme-linked immunosorbent assay instrument, result is as shown in table 13.
The impact on mice carbon clearance and T, bone-marrow-derived lymphocyte propagation of table 13, Chinese medicinal composition granules
| Group | Dosage (g/kg) | T lymphocyte OD 630Value | Bone-marrow-derived lymphocyte OD 630Value | Carbon is worth clearly α |
| Blank group | -- | ?0.421±0.05 | 0.391±0.04 | 5.125±0.08 |
| Model group | -- | 0.251±0.07 △ | 0.127±0.02 △ | 3.420±0.52 △△ |
| Positive drug matched group | 0.15 | ?0.403±0.02* | 0.364±0.04** | 4.892±0.44* |
| Formula 1 | 1.4 | 0.320±0.16* | 0.282±0.02** | 4.520±0.20* |
| Formula 2 | 1.4 | 0.427±0.12* | 0.235±0.02** | 4.530±0.28* |
| Formula 3 | 1.4 | ? 0.520±0.02** | 0.371±0.04** | 4.571±0.03* |
| Formula 4 | 1.4 | 0.480±0.16* | 0.332±0.03** | 4.225±0.38* |
| Formula 5 | 1.4 | 0.467±0.15* | 0.330±0.12** | 4.420±0.28* |
As shown in Table 13, conversion ratio and carbon that the Chinese medicinal composition granules of formula 1-5 all can increase T, bone-marrow-derived lymphocyte are worth clearly α, but 3 effects of filling a prescription are best.
2. the impact of the mice of variable concentrations Chinese medicinal composition granules on immunologic hypofunction
Choose 120 of healthy mices, be divided at random 6 groups, 20 every group.Get 100 mices for building immunosuppression mouse model, concrete grammar is: lumbar injection 0.2mL cyclophosphamide, and 1 times/day, injection volume is pressed 80mg/kg, totally 3 days; The distilled water of remaining 20 mouse peritoneals injection same amount, as blank group.The immunosuppression mouse model of structure is divided into model group, positive drug matched group (astragalin injection at random, 0.15g/kg), Chinese medicinal composition granules high dose group (formula 3,1.4g crude drug/kg), dosage group (formula 3 in Chinese medicinal composition granules, 1.0g crude drug/kg), Chinese medicinal composition granules low dose group (formula 3,0.6g crude drug/kg), 1 times/day, successive administration 3 days; Blank group, model group gives equivalent distilled water.After treatment, respectively organize the blood sampling of half mice eye frame, blood is for carbon clearance assessment of indices; Get each group of second half mouse spleen, thymus for index of immunity mensuration (mg/g) and T, bone-marrow-derived lymphocyte conversion test, the spleen index and the thymus index that record are as shown in table 14.
Table 14, the impact of Chinese medicinal composition granules on mouse immune index
| Group | Dosage (g/kg) | Index and spleen index (mg/g) | Thymus index (mg/g) |
| Blank group | -- | 4.706±0.24 | 1.156±0.01 |
| Model group | -- | 3.215±0.57 △ | 0.856±0.14 △△ |
| Positive drug matched group | 0.15 | 5.323±0.53** | 1.880±0.05** |
| Chinese medicinal composition granules high dose group | 1.4 | 4.832±0.83** | 1.389±0.01** |
| Dosage group in Chinese medicinal composition granules | 1.0 | 3.351±0.53 | 1.239±0.16* |
| Chinese medicinal composition granules low dose group | 0.6 | 3.285±0.39 | 1.192±0.13 |
Note: represent P<0.05 with model control group comparison " * ", " * * " represents P<0.01; Represent P<0.01 with blank group comparison " △ △ ".
As shown in Table 14, model group mouse spleen exponential sum thymus index reduces, with significantly (P<0.01) of blank group ratio heteropole, after Chinese medicinal composition granules treatment, especially the mouse spleen index of Chinese medicinal composition granules high dose group, thymus index are more not remarkable than difference with blank group, trend towards normal; Each dosage group has a certain amount of effect relationship.
Carbon clearance assessment of indices
?each group mice, in the last administration india ink 10mL/kg that after 1 hour, tail vein injection volume fraction is 25%, was then got blood 20 μ L at 2 minutes and 10 minutes in every rathole ball rear vein beard respectively, was blown at once 2mL mass fraction and is 0.1% Na
2cO
3in solution, get after blood to survey absorbance (OD) in 570nm, school zero solution is that 2mL mass fraction is 0.1% Na
2cO
3.Be calculated as follows and clean up index K and engulf coefficient (correction phagocytic index) α, result is as shown in Table 15.
Carbon clearance index K=(logOD
2-logOD
10)/(T
10-t
2) (OD
2, OD
10be respectively 2, absorbance when 10min)
K value, after weight and liver spleen mass conversion, obtains phagocytic index α value: phagocytic index α=weight/(liver quality+spleen quality) × K
1/3(T
10=10min, T
2=2min).
3.T lymphocyte, bone-marrow-derived lymphocyte conversion test are measured
?under aseptic condition, get spleen, then rinse twice with PBS buffer.After grinding with aseptic dismembyator, get serosity, then under 1000 r/min conditions centrifugal 5 minutes, abandon supernatant; Adding 5mL pH is 7.2 Tris NH again
4cl, gently piping and druming, erythrocyte is broken completely, then under 1000 r/min conditions centrifugal 5 min, abandon supernatant; Precipitation is rinsed 1 time with PBS buffer, then with centrifugal 5 min of 1000r/min, abandons supernatant; The full culture fluid re-suspended cell of RPM II640 of the gentamycin that is finally 2% with the new-born calf serum that is 10% containing volume fraction and mass fraction, adjusting splenocyte concentration is 3 × 10
6individual/mL, for subsequent use.
T lymphocyte transformation test: with reference to improved MTT colorimetric analysis, get 96 porocyte culture plates, every hole adds prepares splenoblast suspension 200 μ L, and concanavalin A, Con A 15 μ L(final concentrations are 6mg/L), 8 multiple holes, in the CO of 37 ℃ 5%
2in incubator, cultivate 72 hours.Within 4 hours before cultivation finishes, abandon supernatant 100 μ L, add people MTT liquid 20 μ L and continue to cultivate 4h, suck supernatant, every hole adds DMSO150 μ L cessation reaction, 96 orifice plates are moved into dull and stereotyped oscillator, and level concussion 10min, dissolves MTT reduzate completely, measure every hole absorbance (OD value) in 630nm wavelength place with enzyme-linked immunosorbent assay instrument, result is as shown in Table 15.
Bone-marrow-derived lymphocyte conversion test: its method is identical with T lymphocyte transformation test, difference is to change 15 μ L canavalines into 20 μ L lipopolysaccharide, and adding rear final concentration is 50 mg/L.Measure every hole absorbance (OD value) in 630nm wavelength place with enzyme-linked immunosorbent assay instrument, result is as shown in Table 15.
The impact on mice carbon clearance and T, bone-marrow-derived lymphocyte propagation of table 15, Chinese medicinal composition granules
| Group | Dosage (g/kg) | T lymphocyte OD 630Value | Bone-marrow-derived lymphocyte OD 630Value | Carbon is worth clearly α |
| Blank group | -- | ?0.421±0.05 | 0.391±0.04 | 5.125±0.08 |
| Model group | -- | 0.251±0.07 △ | 0.127±0.02 △ | 3.420±0.52 △△ |
| Positive drug matched group | 0.15 | ?0.403±0.02* | 0.364±0.04** | 4.892±0.44* |
| Chinese medicinal composition granules high dose group | 1.4 | ? 0.520±0.02** | 0.371±0.04** | 4.571±0.03* |
| Dosage group in Chinese medicinal composition granules | 1.0 | 0.427±0.12* | 0.235±0.02** | 4.529±0.18* |
| Chinese medicinal composition granules low dose group | 0.6 | 0.313±0.03 | 0.218±0.01* | 4.002±0.16* |
Represent P<0.05 with model control group comparison " * ", " * * " represents P<0.01; Represent P<0.01 with blank group comparison " △ △ ".
As shown in Table 15, model group mouse T lymphocyte and bone-marrow-derived lymphocyte conversion ratio cell transformation rate reduce, with blank group than significant difference (P<0.05); Model group mice carbon is worth clearly α to be reduced, with significantly (P<0.01) of blank group ratio heteropole; Therefore, conversion ratio and carbon that Chinese medicinal composition granules obviously increases T, bone-marrow-derived lymphocyte are worth clearly α, and each dosage group has a certain amount of effect relationship, show that Chinese medicinal composition granules can improve the immunologic function of mice.
embodiment 9
chinese medicinal composition granules is let out dysentery clinical trial to pig is damp and hot
In March, 2012, not eating, vomitting, suffering from diarrhoea appears in pig farm, Rongchang County, Chongqing City piglet, the clinical symptoms of some serious dehydration, totally 120 of morbidity piglets.Diagnosis, this disease belongs to the damp and hot dysentery of letting out, suitable strengthening vital QI to eliminate pathogenic factors, the spleen strengthening and damp drying, clearing away heat to cure dysentery.
Therapeutic scheme: morbidity piglet is divided into 6 groups at random, and 20 every group, test group adopts Chinese medicinal composition granules spice to feed, and dosage is 2g/kg body weight, another group adopts the loose spice of the Radix Pulsatillae to feed, and dosage is 2g/kg body weight.Test period is one week,
Therapeutic effect: the clinical symptoms of the 2nd day part piglet after medication is significantly taken a turn for the better, and result is shown in table 16.
Table 16, Chinese medicinal composition granules are let out dysentery clinical trial to pig is damp and hot
As shown in Table 16, after medication the 3rd day, the 4th day, the 5th day, the 6th day, formula 1-5 group all had a piglet to be cured, and the loose group of the Radix Pulsatillae has respectively 4,8,13,15 piglets to be cured.By after medication the 6th day, the loose group of experimental group and the Radix Pulsatillae had respectively 15,15,17,16,15 and 15 piglets to be cured, and cure rate reaches respectively 75.5,75.5%, 85%, 80% and 75.5%.Therefore, Chinese medicinal composition granules has therapeutic effect to the damp and hot dysentery of letting out of pig, and formula 3 effects are best, and cure rate reaches 85%.
In above-described embodiment, the function of checking Chinese medicine composition as an example of the granule of Chinese medicine composition example, owing to containing whole effective ingredient of granule in Chinese medicine composition.Therefore, Chinese medicine composition has the function identical with granule.
Finally explanation is, above preferred embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is described in detail by above preferred embodiment, but those skilled in the art are to be understood that, can make various changes to it in the form and details, and not depart from the claims in the present invention book limited range.
Claims (2)
1. the application of Chinese medicine composition in the medicine of preparing anti-swine infectious enterogastritis virus, described Chinese medicine composition is made up of 60 ~ 140 parts of 25 ~ 60 parts of 50 ~ 100 parts of the Rhizoma Atractylodis Macrocephalaes, 50 ~ 150 parts of Radix Scutellariaes, 20 ~ 40 parts of Radix Sophorae Flavescentiss, 60 ~ 150 parts of Herba Pogostemonis, 60 ~ 120 parts of the Radixs Astragali, 60 ~ 100 parts of the Radix Pulsatillaes, Fructus Gardeniae and Flos Loniceraes by weight; And preparation as follows:
A. get the Rhizoma Atractylodis Macrocephalae, Herba Pogostemonis and Flos Lonicerae by formula, extract its volatile oil, then use beta-cyclodextrin inclusion compound, obtain volatile oil clathrate compound;
B. get Radix Scutellariae, Radix Sophorae Flavescentis, the Radix Pulsatillae, Fructus Gardeniae, the Radix Astragali by formula, first use alcohol reflux, obtain extracting solution A, residue medicinal residues decoct with water extraction again, collect extracting solution and are designated as extracting solution B, after extracting solution A and extracting solution B are mixed and be condensed into clear paste; Simultaneously that medicinal residues are dry, then pulverize, be designated as medicinal residues A;
C. by step a gained volatile oil clathrate compound and step b gained clear paste and medicinal residues A mixing, then add adjuvant according to the agent of conventional method granulation.
2. application according to claim 1, is characterized in that: be made up of 100 parts of 80 parts of the Rhizoma Atractylodis Macrocephalaes, 100 parts of Radix Scutellariaes, 30 parts of Radix Sophorae Flavescentiss, 70 parts of Herba Pogostemonis, 100 parts of the Radixs Astragali, 80 parts of the Radix Pulsatillaes, 40 parts of Fructus Gardeniaes, Flos Lonicerae by weight.
3. application according to claim 1, is characterized in that: described adjuvant comprises one or more in binding agent, filler and correctives.
4. application according to claim 3, is characterized in that: described filler be in dicalcium phosphate, corn starch, Icing Sugar and lactose one or more; Described binding agent is one or more in corn starch, dextrin and cellulose; Described correctives is one or more of sucrose, saccharin sodium and grass essence.
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| US5980870A (en) * | 1994-07-26 | 1999-11-09 | Aekyung Industrial Co., Ltd. | Herb medicine extract-containing non-bleeding striped dentifrice composition |
| CN101537059A (en) * | 2009-04-20 | 2009-09-23 | 西南大学 | Anti-enteritis canine parvovirus Chinese medicinal composition and preparation method and applications thereof |
| CN102342998A (en) * | 2010-07-28 | 2012-02-08 | 丰建萍 | Traditional Chinese drug composition for treating chronic enteritis |
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