CN103070949A - 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 preparation method by this Chinese medicine composition preparation.
Background technology
Pig lets out dysentery, and Chinese veterinarian thinks it mainly is 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 a kind of acute height contact Intestinum Sus domestica road transmission disease that is 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 piglets dead, 5 ages in week, above piglet mortality rate was low, but the pig fertility performance of anti-mistake descends after infecting, and price of deed rate descends, and has caused larger economic loss to pig industry.The pig that infects 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, the sow feed mismate, in the milk the too high or lactation amount of fat content not enough all be 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, and is thick disorderly unglazed by hair, tail and hind leg faecal contamination.Down in spirits, aversion to cold is sucked the breast and is reduced, and dehydration is dead because of the stupor collapse eventually.
Owing to above-mentioned disease incidence reason is different, existing nothing is treated damp and hot dysentery, transmissible gastroenteritis of swine and the piglet pujos blancos let out of pig simultaneously, namely allows to treatment, can not improve simultaneously the immunologic function of pig when treatment.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 purpose of the present invention is to provide a kind of Chinese medicine composition, and prescription is unique, can treat simultaneously 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.
For achieving the above object, the invention provides following technical scheme:
A kind of Chinese medicine composition is comprised 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.
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; The function invigorating the spleen and benefiting QI, the 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 the labiate Radix Scutellariae (
Scutellaria baicalensisGeorgi) dry root; Bitter in the mouth, cold in nature; Return lung, gallbladder, spleen, large intestine, small intestine meridian; The function heat clearing and damp drying is let out the fire detoxifcation, and hemostasis is antiabortive; Cure mainly hygropyrexia, fever disease in summer vomiting and nausea uncomfortable in chest, damp and hot feeling of fullness is let out dysentery, jaundice, and the 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 the leguminous plant Radix Sophorae Flavescentis (
Sophora flavescensAit.) dry root; Bitter in the mouth, cold in nature; GUIXIN, liver, stomach, large intestine, urinary bladder channel; The 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 the labiate Herba Pogostemonis (
Pogostemon cablin(Blanco) dry aerial parts Benth.); Acrid in the mouth, slightly warm in nature; Return spleen, stomach, lung meridian; The function eliminating turbid pathogen with aromatics, the appetizing preventing or arresting vomiting is delivered expelling summer-heat; Cure mainly turbid damp obstructing in middle-JIAO, the vomiting of gastral cavity painful abdominal mass, the heat-damp in summer asthenia, uncomfortable in chest easypro, cold-damp is closed summer-heat, and stomachache is told and is let out nasosinusitis headache.
Astragalus polysaccharides be the leguminous plant Radix Astagali (
Astragalus memeranaceus(Fisch.) Bge. var.
Mongholicus(Bge.) Hsiao) or Radix Astragali (
Astragalus membranaceusThe polyoses extract of dry root (Fisch.) Bge.); Radix Astragali sweet in the mouth, slightly warm in nature; Return lung, spleen channel; The function invigorating QI to consolidate the body surface resistance, diuresis poison holding, evacuation of pus, expelling pus and promoting granulation.The modern pharmacological research result shows: astragalus polysaccharides is the main effective site of the Radix Astragali; have the immunologic function of enhancing, induce body to produce interferon, protection cardiovascular system, regulate the multiple pharmacologically actives such as blood glucose, blood pressure lowering, wherein particularly evident to promotion and the regulating action of immunologic function.
The Radix Pulsatillae be the ranunculaceae plant Radix Pulsatillae (
Pulsatilla chinensis(Bge.) dry root Regel).Nature and flavor are bitter, and are cold; Return the stomach warp, have 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 the effect of anti-various bacteria and virus, wherein diphtheria corynebacterium, staphylococcus, streptococcus, escherichia coli, tubercule bacillus, clostridieum welchii etc. are had obvious inhibitory action; Simultaneously the Radix Pulsatillae has calmness, analgesia and spasmolytic, to because bacterial intestinal bleeding dysentery and enterospasm dysentery have good therapeutical effect.
Fructus Gardeniae be the 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; It is vexed to cure mainly calentura, the jaundice dark coloured urine, and 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; The function heat-clearing and toxic substances removing, wind-heat dissipating; Cure mainly the carbuncle furuncle, sore throat, erysipelas, toxic-heat and blood stasis, anemopyretic cold, epidemic febrile disease heating.
Two of purpose 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 purpose 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 purpose 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 purpose of the present invention is to provide the preparation of Chinese medicine composition, and technical scheme is:
Preparation by described Chinese medicine composition preparation.
Preferably, described preparation is granule.
Six of purpose 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 prescription, extract its volatile oil, then use beta-cyclodextrin inclusion compound, get volatile oil clathrate compound;
B. get Radix Scutellariae, Radix Sophorae Flavescentis, the Radix Pulsatillae, Fructus Gardeniae, the Radix Astragali by prescription, use first alcohol reflux, get extracting solution A, the 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 the correctives.
Preferred, described filler be in dicalcium phosphate, corn starch, Icing Sugar and the lactose one or more; Described binding agent is one or both in corn starch, dextrin and the 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 the 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 the preparation of Chinese medicine composition preparation, its preparation method is simple, with Chinese medicine composition extract according to a conventional method after and the adjuvants such as filler, binding agent and correctives be mixed and made into granule, make the solid dispersible carrier be in the high degree of dispersion state after adding adjuvant, 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 the effect of deactivation, and wherein directly the deactivation effect is best, can improve the protective capability of the cell that damages because of transmissible gastro-enteritis virus; The In Vitro Bacteriostasis experiment shows that the preparation of Chinese medicine composition preparation 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 the treatment piglet pujos blancos; The damp and hot dysentery clinical test results of letting out of pig is shown that 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 the preferred embodiment is usually according to normal condition.
Embodiment 1
A kind of Chinese medicine composition, it is as follows to fill a prescription:
Prescription 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.
Prescription 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.
Prescription 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.
Prescription 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.
Prescription 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, with herbal composite can water or pure 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 prescription, add in the extraction pot, add again and be equivalent to raw material gross weight 5-8 water doubly, heating recovery, reclaim distillate, distillate is carried out double evaporation-cooling reflux, liquid to be distilled stops distillation when being equivalent to raw material weight 1/10-2/10, reclaim distillate, get volatile oil.
Take by weighing 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, add gained volatile oil after the heating for dissolving, under 40 ℃ of conditions, stirred 1 hour with 600rpm speed with blender, collecting precipitation is filtered in again cold preservation 24 hours under 4 ℃ of conditions, to be deposited under 50 ℃ of conditions and dry, get 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 the extraction pot, then adding and being equivalent to raw material gross weight 5-8 volume fraction doubly is 50% ethanol, evaporation backflow 2-3 hour, collect and extract filtrate, filtrate is used ethanol recovery tower Recycled ethanol, and remaining liq is designated as extracting solution A;
Continue in extraction pot, to add and be equivalent to raw material gross weight 3-5 water doubly, decoct and extracted 2-3 hour, collect and extract filtrate, must extract B, the medicinal residues oven dry, pulverizing is rear for subsequent use, is designated as medicinal residues A;
C. step b gained extracting solution A and extracting solution B are mixed, then be condensed into the clear paste shape, add step a gained volatile oil clathrate compound after the cooling, mix homogeneously is designated as mixture C.
Present embodiment gained mixture C is the effective ingredient of Chinese medicine composition.
Embodiment 3
For the ease of storing and carrying, taking convenience is made the various preparations that clinical treatment needs that are applicable to Chinese medicine composition or its extract, can add acceptable carrier on the pharmaceutics in preparation, and the method for preparation is conventional method.For example, the various Chinese crude drugs in the herbal composite directly can be ground into fine powder, make powder, perhaps add binding agent and make pill; Acceptable carrier on the extract of herbal composite and the pharmaceutics can be made capsule, tablet, granule etc.These extracts have identical therapeutical effect with preparation and herbal composite.
Below the mixture C that makes take embodiment 2 and medicinal residues A as raw material, the preparation granule, concrete prescription is:
Prescription 1: mixture C 150g, medicinal residues A150g, dicalcium phosphate 100g, corn starch 200g, saccharin sodium 1.5g.
Prescription 2: mixture C 80g, medicinal residues A100g, dicalcium phosphate 80g, corn starch 150g, saccharin sodium 1g.
Prescription 3: mixture C 140g, medicinal residues A140g, dicalcium phosphate 120g, corn starch 250g, saccharin sodium 2g.
Prescription 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 starch slurry in jacketed pan, for subsequent use;
(2) saccharin sodium with formula ratio is dissolved in the mixture C, then adds the corn starch of medicinal residues A, dicalcium phosphate and the remainder of formula ratio, and mix homogeneously gets mixture D;
(3) with adding in (1) described starch slurry in the described mixture of step (2), after stirring, forward on the granulator and granulate, under 60 ℃ of conditions, dried by the fire 10-12 hour after granulating, then place granulate in the pelletizing machine, namely get Chinese medicinal composition granules.
In the present embodiment, dicalcium phosphate is filler, can replace with corn starch, Icing Sugar or the lactose of identical weight, also can use the mixture of dicalcium phosphate, corn starch, Icing Sugar and the lactose of identical weight to replace; Corn starch is binding agent, can replace with dextrin or the cellulose of identical weight, also can replace with corn starch, dextrin and the cellulosic mixture of identical weight; Saccharin sodium is correctives, can replace with sucrose or the grass essence of identical weight, also can replace with sucrose, saccharin sodium and the grass essence mixture of identical weight.
Embodiment 4, Chinese medicinal composition granules are to the direct inactivation test of TGEV virus
1. cultivate PK-15 porcine kidney cell line and preparation TGEV virus liquid
Cultivate the PK-15 porcine kidney cell line according to prior art, then cultivate transmissible gastroenteritis of swine (TGEV) virus, the preparation 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, method 2003:14-15) is measured TCID
50, adopt the 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 mensuration medicine maximal non-toxic concentration, each test group result and blank group result carry out the 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
2Cultivate after 4 hours in the incubator and take out, liquid in the hole is inhaled abandoned, add 100 μ L DMSO again, put vibration 10 min on the micro oscillator, take off photometry density value (OD) under microplate reader 490 nm wavelength, the result represents with the 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 with the Chinese medicinal composition granules of prescription 1-5 according to the method described above, concrete grammar is: 96 well culture plates of the monolayer PK-15 porcine kidney cell that covers with are divided into blank group (not doing any processing), model group, and (the PK-15 porcine kidney cell infects TGEV virus, not administration is processed), (the PK-15 porcine kidney cell infects TGEV virus afterwards with containing 10 to the astragalus polysaccharides matched group
-5The g/mL astragalus polysaccharides is processed), prescription 1 ~ 5(PK-15 porcine kidney cell infects after the TGEV virus respectively that the concentration with the 1-5 that fills a prescription is 10
-5The Chinese medicinal composition granules aqueous solution of g/mL is processed), 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 the direct inactivation test of TGEV virus
Annotate: in the same column, do not contain the same letter shoulder motes and represent significant difference, contain the same letter shoulder motes in the same column and represent difference not significantly (lower case represents P＞0.05, and capitalization represents P＞0.01).
As shown in Table 1, the Chinese medicine composition of prescription 1-5 all can directly suppress TGEV virus, but the granule effect of 3 the Chinese medicine composition preparation of filling a prescription is best.
96 well culture plates of the monolayer PK-15 porcine kidney cell that covers with are divided into blank group (not doing any processing), model group, and (the PK-15 porcine kidney cell infects TGEV virus, not administration is processed), (the PK-15 porcine kidney cell infects TGEV virus afterwards with containing 10 to the astragalus polysaccharides matched group
-5The g/mL astragalus polysaccharides is processed), it is 10 that test group 1 ~ 5(PK-15 porcine kidney cell infects after the TGEV virus respectively with 3 the concentration of filling a prescription
-4G/mL, 10
-5G/mL, 10
-6G/mL, 10
-7G/mL and 10
-8The Chinese medicinal composition granules aqueous solution of g/mL is processed), every group is repeated 6 holes, then measures the direct inactivating efficacy of Chinese medicinal composition granules of prescription 3 preparations, and the result is as shown in table 2.
Table 2, Chinese medicinal composition granules are to the direct inactivation test of TGEV virus
Annotate: in the same column, do not contain the same letter shoulder motes and represent significant difference, contain the same letter shoulder motes in the same column and represent difference not significantly (lower case represents P＞0.05, and capitalization represents P＞0.01).
As shown in Table 2, Chinese medicinal composition granules is inactivation of viruses directly, reduces virus to the damage of cell, thus protection PK-15 cell.1 ~ 4 pair of cytoprotection of experimental group is obvious, compare with model group difference extremely significantly (
P＜0.01), and have obvious dose-effect relationship between experimental group 2 ~ 4, 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 the result according to the Reed-Muench method and show, it is 1TCID that TGEV infects half cell infection amount
50Be 0.2 mL 10
-3.654Dilution virus liquid.
The concentration determination of medicine maximal non-toxic shows that Chinese medicinal composition granules is in concentration c 〉=10
-3Cell survival rate reduces during g/mL, with the blank group relatively have utmost point significant difference (
P＜0.01); In concentration c≤10
-4During g/mL cell survival rate and blank group comparing difference not significantly (
P＞0.05), determines 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
In 96 well culture plates that cover with monolayer PK-15 porcine kidney cell, every hole adds 200 μ L and is respectively 10 by the concentration that prescription 1-5 prepares
-5The Chinese medicinal composition granules aqueous solution of g/mL (being followed successively by prescription 1 ~ 5) was hatched 2 hours at 37 ℃, abandoned medicinal liquid; Access again 200 μ L 100TCID
50Virus liquid is hatched after 2 hours for 37 ℃ and is abandoned virus liquid, then adds 200 μ L cell maintenance mediums (DMEM that contains 2% FBS cultivates), puts 37 ℃, 5%CO
2Continue in the incubator to cultivate 72 hours.Organize as blank with the PK-15 porcine kidney cell of not doing any processing simultaneously, the PK-15 porcine kidney cell is infected the post processing of TGEV virus, not administration is model group, and the PK-15 porcine kidney cell is infected TGEV virus afterwards with containing 10
-5The g/mL astragalus polysaccharides is processed as the astragalus polysaccharides group, more than every group repeat 6 holes.Then measure the Chinese medicinal composition granules of different formulations to TGEV viruses adsorption inhibition, the result is as shown in table 3.
The Chinese medicinal composition granules of table 3, different formulations suppresses the TGEV viruses adsorption
Annotate: in the same column, do not contain the same letter shoulder motes and represent significant difference, contain the same letter shoulder motes in the same column and represent difference not significantly (lower case represents P＞0.05, and capitalization represents P＞0.01).
As shown in Table 3, the Chinese medicine composition granular preparation of prescription 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
In 96 well culture plates that cover with monolayer PK-15 porcine kidney cell, every hole adds 200 μ L concentration and is 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), hatched 2 hours at 37 ℃, abandon medicinal liquid; Access again 200 μ L 100TCID
50Virus liquid is hatched after 2 hours for 37 ℃ and is abandoned virus liquid, then adds 200 μ L cell maintenance mediums (DMEM that contains 2% FBS cultivates), puts 37 ℃, 5%CO
2Continue in the incubator to cultivate 72 hours.Organize as blank with the PK-15 porcine kidney cell of not doing any processing simultaneously, the PK-15 porcine kidney cell is infected the post processing of TGEV virus, not administration is model group, and the PK-15 porcine kidney cell is infected TGEV virus afterwards with containing 10
-5The g/mL astragalus polysaccharides is processed as the astragalus polysaccharides group, more than every group repeat 6 holes.Then measure Chinese medicinal composition granules to TGEV viruses adsorption inhibition, the result is as shown in table 4.
The Chinese medicinal composition granules of table 4, variable concentrations suppresses the TGEV viruses adsorption
Annotate: in the same column, do not contain the same letter shoulder motes and represent significant difference, contain the same letter shoulder motes in the same column and represent difference not significantly (lower case represents P＞0.05, and capitalization represents P＞0.01).
As shown in Table 4, Chinese medicinal composition granules can suppress viruses adsorption, and In vitro culture PK-15 cell is all had in various degree protective effect.Compare 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) there is obvious dose-effect relationship between.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 by prescription 1-5 preparation of g/mL (being followed successively by prescription 1 ~ 5) is put to hatch after 2 hours under 37 ℃ of conditions and is abandoned virus liquid, adds 200 μ L cell maintenance mediums, puts 37 ℃, 5%CO
2Continue in the incubator to cultivate 72 hours.Organize as blank with the PK-15 porcine kidney cell of not doing any processing simultaneously, the PK-15 porcine kidney cell is infected the post processing of TGEV virus, not administration is model group, and the PK-15 porcine kidney cell is infected TGEV virus afterwards with containing 10
-5The g/mL astragalus polysaccharides is processed as the astragalus polysaccharides group, more than every group repeat 6 holes.Then detect the TGEV viral load, calculate the cytoprotective rate, the result is as shown in table 5.
The Chinese medicinal composition granules of table 5, different formulations suppresses the TGEV virus replication
Annotate: in the same column, do not contain the same letter shoulder motes and represent significant difference, contain the same letter shoulder motes in the same column and represent difference not significantly (lower case represents P＞0.05, and capitalization represents P＞0.01).
2. the Chinese medicinal composition granules of variable concentrations suppresses the 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, hatches 2 hours under 37 ℃ of conditions, 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 to hatch after 2 hours under 37 ℃ of conditions and abandon virus liquid, add 200 μ L cell maintenance mediums, put and continue to cultivate 72 h in 37 ℃, 5%CO2 incubator.Organize as blank with the PK-15 porcine kidney cell of not doing any processing simultaneously, the PK-15 porcine kidney cell is infected the post processing of TGEV virus, not administration is model group, and the PK-15 porcine kidney cell is infected TGEV virus afterwards with containing 10
-5The g/mL astragalus polysaccharides is processed as the astragalus polysaccharides group, more than every group repeat 6 holes.Then detect the TGEV viral load, calculate the cytoprotective rate, the result is as shown in table 6.
The Chinese medicinal composition granules of table 6, variable concentrations suppresses the TGEV virus replication
Annotate: in the same column, do not contain the same letter shoulder motes and represent significant difference, contain the same letter shoulder motes in the same column and represent difference not significantly (lower case represents P＞0.05, and capitalization represents P＞0.01).
By shown in the table 6, Chinese medicinal composition granules can suppress virus replication, and In vitro culture PK-15 cell is all had in various degree protective effect.Compare 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) have obvious dose-effect relationship between, test group 1 protective rate is the highest, is 12.69%.
Embodiment 7
The 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, then the method according to table 7 is diluted to variable concentrations, and inoculation 0.1mL test antibacterial in each test tube was cultivated in 37 ℃ the calorstat 6 ~ 8 hours again.
The operating process of table 7, doubling dilution
| The 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 | ? |
| The medicine dilution factor | 1:2 | 1:4 | 1:8 | 1:16 | 1:32 | 1:64 | 1:128 | 1:256 | 1:512 | ? | 1:2 |
Detect according to the method described above the Chinese medicinal composition granules of prescription 3 to the fungistatic effect of escherichia coli, staphylococcus aureus, pig source clostridieum welchii, Salmonella and Candida albicans, micrococcus luteus, Bacillus pumilus and bacillus subtilis, 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 the Chinese medicinal composition granules of table 8, variable concentrations (MIC)
| The pipe number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | Without medicine | Aseptic |
| The 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 | + | + | + | ++ | ++ | ++ | +++ | +++ | ++++ | +++++ | ― |
Annotate: "+" expression has bacterial growth, and "-" represents 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, the bacillus subtilis fungistatic effect is not good, does not measure its MIC.
According to method same as described above, the Chinese medicinal composition granules that detects prescription 1-5 under 16 times of conditions of dilution to the antibacterial situation of escherichia coli, staphylococcus aureus, pig source clostridieum welchii, Salmonella and Candida albicans, antibacterial the results are shown in Table shown in 9.
The external minimum inhibitory concentration of the Chinese medicinal composition granules of table 9, different formulations (MIC)
| The pipe number | Prescription 1 | Prescription 2 | Prescription 3 | Prescription 4 | Prescription 5 | Without medicine | Aseptic |
| The medicinal liquid extension rate | 16 | 16 | 16 | 16 | 16 | 0 | 16 |
| Escherichia coli | ― | ― | ― | ― | ― | +++++ | ― |
| Staphylococcus aureus | - | － | － | － | － | +++++ | － |
| Pig source clostridieum welchii | － | － | ― | + | + | +++++ | － |
| Salmonella | － | + | ― | ― | + | +++++ | － |
| Candida albicans | + | ― | ― | ― | + | +++++ | － |
Annotate: "+" expression has bacterial growth, and "-" represents without bacterial growth.With "+", " ++ ", the growth pressure of expression antibacterial
As shown in Table 9, under the condition of 16 times of dilutions, prescription 3 can suppress escherichia coli, staphylococcus aureus, pig source clostridieum welchii, Salmonella and Candida albicans simultaneously; Prescription 1 can not suppress Candida albicans, and prescription 2 can not suppress Salmonella, and prescription 4 can not suppress pig source clostridieum welchii, and prescription 5 can only suppress escherichia coli and staphylococcus aureus, and 3 the fungistatic effect of therefore filling a prescription is best.
2, cup pipe method
At first 10mL is poured in the culture dish as bottom through autoclaved agar culture medium, spreading about 40 ~ 50 ℃ of preparation methoies that contain the agar culture medium 5mL(bacterium liquid of 0.1mL bacterium liquid of one deck after solidifying in the above is with after the antibacterial activation, be put in 37 ℃ the calorstat and cultivated 12 ~ 14 hours, turbidity is about 400,000,000/mL), after solidifying, the upper strata places 4 Oxford cup (external diameter 7.8mm in each culture dish, internal diameter 6.8mm), add the Chinese medicinal composition granules aqueous solution of the prescription 3 of variable concentrations in the cup of Oxford, put at last 37 ℃ of calorstat incubated overnight.Measure the inhibition zone size of the medicinal liquid generation of variable concentrations next day, observe its antibacterial situation, the 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 preferably fungistatic effect to escherichia coli, staphylococcus aureus, Salmonella, Candida albicans, 5 kinds of bacterial strains of pig source clostridieum welchii, and the concentration of inhibition zone is larger when concentration is 5%.Therefore, Chinese medicinal composition granules disclosed by the invention has preferably fungistatic effect to escherichia coli, staphylococcus aureus, Salmonella, Candida albicans, 5 kinds of bacterial strains of pig source clostridieum welchii.
It is 5% aqueous solution that the Chinese medicinal composition granules of the 1-5 that then will fill a prescription preparation is made mass fraction, detect the granule of Chinese medicine composition preparation of different formulations with cup pipe method to the fungistatic effect of escherichia coli, staphylococcus aureus, Salmonella, Candida albicans, pig source clostridieum welchii, the 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 prescription 3 is antibacterial best.
Embodiment 8
Chinese medicinal composition granules is on the impact of the mice of immunologic hypofunction
1. the different formulations Chinese medicinal composition granules is on the impact of the mice of immunologic hypofunction
Choose 160 of healthy mices, be divided at random 8 groups, 20 every group.Get 140 mices and be used for making up 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 is as the blank group.With the immunosuppression mouse model that makes up be divided at random model group, positive drug matched group (astragalin injection, 0.15g/kg), (every dosage is prescription 1-5 group: 1.4g crude drug/kg), 1 times/day, successive administration 3 days; Blank group, model group give the equivalent distilled water.After the treatment, each organizes half mice eye frame blood sampling, and blood is used for the carbon clearance assessment of indices; Get and respectively organize 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, Chinese medicinal composition granules are on the impact of mouse immune index
| Group | Dosage (g/kg) | Index and spleen index (mg/g) | Thymus index (mg/g) |
| The blank group | -- | 4.706±0.24 | 1.156±0.01 |
| Model group | -- | 3.215±0.57 △ | 0.856±0.14 △△ |
| The positive drug matched group | 0.15 | 5.323±0.53** | 1.880±0.05** |
| Prescription 1 | 1.4 | 3.832±0.83** | 1.380±0.32** |
| Prescription 2 | 1.4 | 3.380±0.60 | 1.230±0.15* |
| Prescription 3 | 1.4 | 4.832±0.83** | 1.389±0.01** |
| Prescription 4 | 1.4 | 4.832±0.83** | 1.580±0.21** |
| Prescription 5 | 1.4 | 5.250±0.82 | 1.839±0.18* |
As shown in Table 12, the mice of prescription 1-5 group index and spleen index and thymus index increase after administration.Therefore, the Chinese medicinal composition granules of prescription 1-5 can be treated the mice of immunocompromised, and 5 effects of filling a prescription are best.
(2) carbon clearance assessment of indices
Each organize mice in the last administration after 1 hour the tail vein injection volume fraction be 25% india ink 10mL/kg, then get blood 20 μ L 2 minutes and 10 minutes in every rathole ball rear vein beard respectively, be blown at once the 2mL mass fraction and be 0.1% Na
2CO
3In the solution, it is complete rear to survey absorbance (OD) in 570nm to get blood, and school zero solution is that the 2mL mass fraction is 0.1% Na
2CO
3Be calculated as follows and clean up index K and engulf coefficient (correction phagocytic index) α, the 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, the absorbance during 10min)
The K value gets phagocytic index α value behind weight and liver spleen mass conversion: 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 with twice of PBS buffer flushing.Get serosity after grinding with aseptic dismembyator, then under 1000 r/min conditions centrifugal 5 minutes, abandon supernatant; Add again 5mL pH and be 7.2 Tris NH
4Cl, piping and druming is broken erythrocyte fully gently, and centrifugal 5 min under 1000 r/min conditions abandon supernatant again; Precipitation with centrifugal 5 min of 1000r/min, is abandoned supernatant again with PBS buffer flushing 1 time; Be that 10% new-born calf serum and mass fraction are the full culture fluid re-suspended cell of RPM II640 of 2% gentamycin with containing volume fraction at last, adjusting splenocyte concentration is 3 * 10
6Individual/mL, for subsequent use.
The T lymphocyte transformation test: with reference to improved MTT colorimetric analysis, get 96 porocyte culture plates, every hole adds preparation splenoblast suspension 200 μ L, and concanavalin A, Con A 15 μ L(final concentrations are 6mg/L), 8 multiple holes are in 37 ℃ 5% CO
2Cultivated 72 hours in the incubator.Abandoned supernatant 100 μ L in front 4 hours in the cultivation end, 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 the MTT reduzate fully, measure every hole absorbance (OD value) in 630nm wavelength place with enzyme-linked immunosorbent assay instrument, the result is as shown in table 13.
The bone-marrow-derived lymphocyte conversion test: its method is identical with the T lymphocyte transformation test, and difference is to change 15 μ L canavalines into 20 μ L lipopolysaccharide, and final concentration is 50 mg/L after adding.Measure every hole absorbance (OD value) in 630nm wavelength place with enzyme-linked immunosorbent assay instrument, the result is as shown in table 13.
Table 13, Chinese medicinal composition granules are on the impact of mice carbon clearance and T, bone-marrow-derived lymphocyte propagation
| Group | Dosage (g/kg) | T lymphocyte OD 630Value | Bone-marrow-derived lymphocyte OD 630Value | Carbon is worth clearly α |
| The 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 △△ |
| The positive drug matched group | 0.15 | ?0.403±0.02* | 0.364±0.04** | 4.892±0.44* |
| Prescription 1 | 1.4 | 0.320±0.16* | 0.282±0.02** | 4.520±0.20* |
| Prescription 2 | 1.4 | 0.427±0.12* | 0.235±0.02** | 4.530±0.28* |
| Prescription 3 | 1.4 | ? 0.520±0.02** | 0.371±0.04** | 4.571±0.03* |
| Prescription 4 | 1.4 | 0.480±0.16* | 0.332±0.03** | 4.225±0.38* |
| Prescription 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 prescription 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 variable concentrations Chinese medicinal composition granules is on the impact of the mice of immunologic hypofunction
Choose 120 of healthy mices, be divided at random 6 groups, 20 every group.Get 100 mices and be used for making up 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 is as the blank group.The immunosuppression mouse model that makes up is divided into model group, positive drug matched group (astragalin injection at random, 0.15g/kg), the Chinese medicinal composition granules high dose group (prescription 3,1.4g (the prescription 3 of dosage group in crude drug/kg), the Chinese medicinal composition granules, 1.0g crude drug/kg), Chinese medicinal composition granules low dose group (prescription 3,0.6g crude drug/kg), 1 times/day, successive administration 3 days; Blank group, model group give the equivalent distilled water.After the treatment, each organizes half mice eye frame blood sampling, and blood is used for the carbon clearance assessment of indices; Get and respectively organize 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, Chinese medicinal composition granules are on the impact of mouse immune index
| Group | Dosage (g/kg) | Index and spleen index (mg/g) | Thymus index (mg/g) |
| The blank group | -- | 4.706±0.24 | 1.156±0.01 |
| Model group | -- | 3.215±0.57 △ | 0.856±0.14 △△ |
| The positive drug matched group | 0.15 | 5.323±0.53** | 1.880±0.05** |
| The Chinese medicinal composition granules high dose group | 1.4 | 4.832±0.83** | 1.389±0.01** |
| Dosage group in the Chinese medicinal composition granules | 1.0 | 3.351±0.53 | 1.239±0.16* |
| The Chinese medicinal composition granules low dose group | 0.6 | 3.285±0.39 | 1.192±0.13 |
Annotate: compare " * " expression P＜0.05 with model control group, " * * " represents P＜0.01; Compare " △ △ " expression P＜0.01 with the blank group.
By shown in the table 14, model group mouse spleen exponential sum thymus index reduces, with blank group ratio heteropole remarkable (P＜0.01), after the 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 the blank group, trend towards normal; Each dosage group has a certain amount of effect relationship.
The carbon clearance assessment of indices
Each organize mice in the last administration after 1 hour the tail vein injection volume fraction be 25% india ink 10mL/kg, then get blood 20 μ L 2 minutes and 10 minutes in every rathole ball rear vein beard respectively, be blown at once the 2mL mass fraction and be 0.1% Na
2CO
3In the solution, it is complete rear to survey absorbance (OD) in 570nm to get blood, and school zero solution is that the 2mL mass fraction is 0.1% Na
2CO
3Be calculated as follows and clean up index K and engulf coefficient (correction phagocytic index) α, the 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, the absorbance during 10min)
The K value gets phagocytic index α value behind weight and liver spleen mass conversion: 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 with twice of PBS buffer flushing.Get serosity after grinding with aseptic dismembyator, then under 1000 r/min conditions centrifugal 5 minutes, abandon supernatant; Add again 5mL pH and be 7.2 Tris NH
4Cl, piping and druming is broken erythrocyte fully gently, and centrifugal 5 min under 1000 r/min conditions abandon supernatant again; Precipitation with centrifugal 5 min of 1000r/min, is abandoned supernatant again with PBS buffer flushing 1 time; Be that 10% new-born calf serum and mass fraction are the full culture fluid re-suspended cell of RPM II640 of 2% gentamycin with containing volume fraction at last, adjusting splenocyte concentration is 3 * 10
6Individual/mL, for subsequent use.
The T lymphocyte transformation test: with reference to improved MTT colorimetric analysis, get 96 porocyte culture plates, every hole adds preparation splenoblast suspension 200 μ L, and concanavalin A, Con A 15 μ L(final concentrations are 6mg/L), 8 multiple holes are in 37 ℃ 5% CO
2Cultivated 72 hours in the incubator.Abandoned supernatant 100 μ L in front 4 hours in the cultivation end, 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 the MTT reduzate fully, measure every hole absorbance (OD value) in 630nm wavelength place with enzyme-linked immunosorbent assay instrument, the result is as shown in Table 15.
The bone-marrow-derived lymphocyte conversion test: its method is identical with the T lymphocyte transformation test, and difference is to change 15 μ L canavalines into 20 μ L lipopolysaccharide, and final concentration is 50 mg/L after adding.Measure every hole absorbance (OD value) in 630nm wavelength place with enzyme-linked immunosorbent assay instrument, the result is as shown in Table 15.
Table 15, Chinese medicinal composition granules are on the impact of mice carbon clearance and T, bone-marrow-derived lymphocyte propagation
| Group | Dosage (g/kg) | T lymphocyte OD 630Value | Bone-marrow-derived lymphocyte OD 630Value | Carbon is worth clearly α |
| The 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 △△ |
| The positive drug matched group | 0.15 | ?0.403±0.02* | 0.364±0.04** | 4.892±0.44* |
| The Chinese medicinal composition granules high dose group | 1.4 | ? 0.520±0.02** | 0.371±0.04** | 4.571±0.03* |
| Dosage group in the Chinese medicinal composition granules | 1.0 | 0.427±0.12* | 0.235±0.02** | 4.529±0.18* |
| The Chinese medicinal composition granules low dose group | 0.6 | 0.313±0.03 | 0.218±0.01* | 4.002±0.16* |
Compare " * " expression P＜0.05 with model control group, " * * " represents P＜0.01; Compare " △ △ " expression P＜0.01 with the blank group.
By shown in the table 15, model group mouse T lymphocyte and bone-marrow-derived lymphocyte conversion ratio cell transformation rate reduce, with the blank group than difference remarkable (P＜0.05); Model group mice carbon is worth clearly α to be reduced, with blank group ratio heteropole remarkable (P＜0.01); 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 the 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: the piglet that will fall ill is divided into 6 groups at random, and 20 every group, test group adopts the Chinese medicinal composition granules spice to feed, and dosage is the 2g/kg body weight, and another group adopts the loose spice of the Radix Pulsatillae to feed, and dosage is the 2g/kg body weight.Test period is a week,
Therapeutic effect: the clinical symptoms of the 2nd day part piglet after medication is significantly taken a turn for the better, and the result is shown in table 16.
Table 16, Chinese medicinal composition granules are let out the 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, prescription 1-5 group all had a piglet to obtain curing, and the loose group of the Radix Pulsatillae has respectively 4,8,13,15 piglets to obtain curing.After the 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 3 effects of filling a prescription 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 is owing to contain whole effective ingredient of granule in the Chinese medicine composition.Therefore, Chinese medicine composition has the function identical with granule.
Explanation is at last, 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 claims limited range of the present invention.
Claims (10)
1. Chinese medicine composition is characterized in that: be comprised 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.
2. Chinese medicine composition according to claim 1 is characterized in that: be comprised 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. claim 1 or the 2 described Chinese medicine compositions application in the damp and hot medicine of letting out dysentery of preparation treatment pig.
4. claim 1 or the 2 described Chinese medicine compositions application in the medicine of preparation treatment transmissible gastroenteritis of swine.
5. claim 1 or the 2 described Chinese medicine compositions application in the medicine of preparation treatment immunologic hypofunction.
6. the preparation that is prepared by claim 1 or 2 described Chinese medicine compositions.
7. preparation according to claim 6, it is characterized in that: described preparation is granule.
8. the preparation method of preparation claimed in claim 7 is characterized in that, concrete steps are as follows:
A. get the Rhizoma Atractylodis Macrocephalae, Herba Pogostemonis and Flos Lonicerae by prescription, extract its volatile oil, then use beta-cyclodextrin inclusion compound, get volatile oil clathrate compound;
B. get Radix Scutellariae, Radix Sophorae Flavescentis, the Radix Pulsatillae, Fructus Gardeniae, the Radix Astragali by prescription, use first alcohol reflux, get extracting solution A, the 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. with 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.
9. preparation method according to claim 8, it is characterized in that: described adjuvant comprises one or more in binding agent, filler and the correctives.
10. preparation method according to claim 9 is characterized in that: described filler be in dicalcium phosphate, corn starch, Icing Sugar and the lactose one or more; Described binding agent is one or more in corn starch, dextrin and the cellulose; Described correctives is one or more of sucrose, saccharin sodium and grass essence.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103386113A (en) * | 2013-08-08 | 2013-11-13 | 西南大学 | Traditional Chinese medicine composition, extractive and preparation for treating swine transmissible gastroenteritis and applications of Chinese medicine composition, extractive and preparation |
| CN103705820A (en) * | 2013-12-23 | 2014-04-09 | 山东新希望六和集团有限公司 | Medicine for treating yellow scours of newborn piglets and preparation method thereof |
| CN106666100A (en) * | 2017-02-09 | 2017-05-17 | 合肥隆扬农业科技有限公司 | Feed additive for preventing and treating swine tuberculosis |
| CN108478652A (en) * | 2018-06-13 | 2018-09-04 | 西安默瑞柯生物科技有限公司 | A kind of Chinese medicine preparation and preparation method thereof preventing transmissible gastroenteritis of swine |
| CN114470117A (en) * | 2020-10-26 | 2022-05-13 | 广东诸阁靓动物药业有限公司 | Medicine auxiliary method for enhancing survival rate of pigs |
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| CN103386113A (en) * | 2013-08-08 | 2013-11-13 | 西南大学 | Traditional Chinese medicine composition, extractive and preparation for treating swine transmissible gastroenteritis and applications of Chinese medicine composition, extractive and preparation |
| CN103705820A (en) * | 2013-12-23 | 2014-04-09 | 山东新希望六和集团有限公司 | Medicine for treating yellow scours of newborn piglets and preparation method thereof |
| CN103705820B (en) * | 2013-12-23 | 2016-04-20 | 山东新希望六和集团有限公司 | Medicine being used for the treatment of yellow scour of piglet and preparation method thereof |
| CN106666100A (en) * | 2017-02-09 | 2017-05-17 | 合肥隆扬农业科技有限公司 | Feed additive for preventing and treating swine tuberculosis |
| CN108478652A (en) * | 2018-06-13 | 2018-09-04 | 西安默瑞柯生物科技有限公司 | A kind of Chinese medicine preparation and preparation method thereof preventing transmissible gastroenteritis of swine |
| CN114470117A (en) * | 2020-10-26 | 2022-05-13 | 广东诸阁靓动物药业有限公司 | Medicine auxiliary method for enhancing survival rate of pigs |
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| CN103070949B (en) | 2014-05-28 |
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