CN106176877A - A kind of compositions with enhancing immunity function and preparation method thereof - Google Patents
A kind of compositions with enhancing immunity function and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of compositions and preparation method thereof, said composition is to be prepared from according to a certain weight ratio by Radix Ginseng, Radix Codonopsis, Radix Glehniae, and it can be made into any common dosage forms, preferred oral preparation.The present composition has the function strengthening body immunity.
Description
Technical field
The present invention relates to a kind of compositions with enhancing immunity function and preparation method thereof, belong to the technical field of health food.
Background technology
Immune system is to cover the defence network of whole body, the first line of defence of protection health.Immune system (immunologicfunction) is the protection capability that living organism progressively obtains in phyletic evolution and ontogenetic process.Mainly include three aspects: body resists the immune protection function of extraneous infectious；Body removes damage and dead cell, maintains the homeostatic function of own physiological balance；Body is monitored, finds and remove the immune surveillance function of mutant cell.Under normal physiological conditions, the immune system of body relies on its congenital immunocompetence having and acquired immunocompetence to play co-immunization effect, and the physiological function keeping body is the most stable.If immunologic function occurs abnormal, inevitably result in the imbalance of body physiological function, thus occur that pathologic changes.And theory of Chinese medical science thinks that all paathogenic factors are pathogen, all resistances against diseases are healthy energy.Healthy energy, is divided into again and defends gas, vigour and the qi of zang-fu viscera etc. of Main Tissues organ.It is to play defence exopathogen to invade the healthy energy of human body that what is called defends gas.If healthy energy is vigorous, body just can eliminating evil be gone out, and avoids sick, i.e. so-called " healthy energy internal memory, heresy can not be done ".If the positive deficiency of vital energy declines, then pathogen easily invades body and causes disease, and i.e. so-called causing a disease is due to " being beaten of heresy, its gas must be empty ".Therefore the strengthening vital QI to eliminate pathogenic factors that the traditional Chinese medical science proposes during preventing and curing diseases, with the basic principle consolidated, is consistent with the health-care method of enhancing human body immunity power.
Summary of the invention
It is an object of the present invention to provide a kind of compositions with enhancing immunity function, said composition has benefiting QI for activating blood circulation, nourishing the liver and kidney, spleen reinforcing stomach function regulating, effect of nourishing YIN and moistening the lung, by three burnt logical benefits, reaches to strengthen the purpose of body immunity.
Another object of the present invention there is provided the preparation method of said composition.
The present invention is achieved by the following technical solutions:
The present invention is made up of the raw material of following weight parts:
Radix Ginseng 0.5-10 part, Radix Codonopsis 0.5-10 part, Radix Glehniae 0.5-10 part.
It is preferably:
Radix Ginseng 1-5 part, Radix Codonopsis 1-5 part, Radix Glehniae 1-5 part.
More preferably:
Radix Ginseng 2 parts, Radix Codonopsis 2 parts, Radix Glehniae 2 parts.
In the present invention, the mechanism of action of each component is as follows:
Radix Ginseng: sweet in the mouth, micro-hardship is warm in nature, flat.Enter spleen, lung meridian.Having strongly invigorating primordial QI, multiple arteries and veins takes off admittedly, and invigorating the spleen to benefit the lung promotes the production of body fluid, the effect calmed the nerves, and can be used for weak body and prostration, cold extremities faint pulse, insufficiency of the spleen lack of appetite, and the deficiency of the lung is breathed with cough, and Tianjin wound is thirsty, and interior-heat is quenched one's thirst, prolonged illness weakness with emaciation, palpitation with fear insomnia, sexual impotence cold womb；Heart failure, cardiogenic shock.For dyspnea with shortness of breath, palpitation and amnesia, thirsty hyperhidrosis, lack of appetite is unable, all acute and chronic diseases and the shock caused after losing blood, collapse.Strongly invigorating primordial QI, promotes the production of body fluid Gu de-, calms the nerves.Controlling impairment caused by overstrain deficient, lack of appetite, asthenia, regurgitation is told food, stool lingering diarrhea, empty cough with asthma rush, spontaneous perspiration sudden collapse, is palpitated with fear, forgetful, dizziness and headache, sexual impotence, and frequent micturition is quenched one's thirst, woman uterine bleeding, children's's intermittent infantile convulsion, and prolonged deficiency is multiple, the card of all qi-blood-body fluid deficiencies.Containing ginsenoside, nitrogen-containing compound, organic acid, polysaccharide and other multiple active components, wherein ginsenoside and ginseng polysaccharide can significantly improve body immunity.Research finds; the most protected effect of stress that (cold, overheated, aggravating activities, the lonizing radiation), (foreign serum, antibacterial, the transplantation tumor) of property biology of physical property, (poisonous substance, anesthetics, hormone, anticarcinogen etc.) all stimulations of chemical are caused by stem and leaf of Radix Ginseng saponin and root Saponin; the functional recovery that can make disorder is normal, and someone is called adaptation immunogenic substance (a kind of material strengthening the non-specific resistivity of human body).
Radix Codonopsis: property is put down, sweet in the mouth, there is invigorating the spleen and replenishing QI, strengthening spleen and tonifying lung, anticancer, blood pressure lowering, anti-hypoxia, effect of defying age, body immunity can also be strengthened, improve the activity of superoxide dismutase, strengthen the ability eliminating free radical, there is Gastrointestinal motility adjustment, antiulcer, gastric acid secretion inhibiting, the effect of reduction pepsin activity.It is applicable to the diseases such as deficiency of the spleen and lung, cardiopalmus of breathing hard, anorexia and loose stool, ulcer, anemia, dyspnea due to deficiency is coughed, interior-heat is quenched one's thirst.Research shows, Radix Codonopsis extract can strengthen the ability of Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell.Mouse peritoneal, intramuscular, intravenous injection asiabell preparation all can make Turnover of Mouse Peritoneal Macrophages number substantially increase, and cell volume increases, and pseudopodium increases, and the multiple enzymatic activity such as born of the same parents' nucleic acid in vivo, saccharide, ATP enzyme, succinate dehydrogenase strengthens, thus strengthens its phagocytosis.Radix Codonopsis decocting liquid low concentration can promote the mitosis of In vitro culture lymphocyte, and promotes that the mouse spleen lymphocyte DNA that COnA activates synthesizes.The humoral immune function of normal mouse is affected inconspicuous by Radix Codonopsis, but the immunosuppressed mice causing cyclophosphamide then can be obviously promoted the conversion of its lymphocyte, strengthens the function of antibody produced cell, improves antibody titer.Radix Codonopsis polysaccharide is principle active component.
Radix Glehniae: cold nature, sweet in the mouth, micro-hardship.Enter lung, spleen channel.There is nourishing YIN and clearing away lung-heat, effect of expelling phlegm for arresting cough, can be used for controlling lung-heat type cough, chronic consumptive disease chronic cough, the moon hinders dry pharynx, the disease such as thirsty.Radix Glehniae contains the compositions such as volatile oil, coumarin, starch, alkaloid, triterpenic acid, stigmasterol, each sterol, Radix Adenophorae (Radix Glehniae) element.It is demonstrated experimentally that Radix Glehniae can improve T cell ratio, improve lymhocyte transformation rate, leukocyte increasing, strengthen macrophage function, extend antibody and there is the time, improve B cell, promote immunologic function.Radix Glehniae can strengthen healthy energy, reduces disease, the generation of prophylaxis of cancer.
The present composition can use the conventional method of Chinese medicine preparation to be prepared as the preparation of any routine, including oral formulations, external preparation, injection.These crude drug such as can be ground into powder mix homogeneously；The crude drug of described weight portion can be added water respectively or the ethanol extraction of variable concentrations, extracting solution concentrate drying obtains crude extract, or employing process for purification, such as, decoction and alcohol sedimentation technique, organic solvent extractionprocess, column chromatography, carbon dioxide supercritical extraction method, steam distillation carry out being refining to obtain extract.
When using said medicine, both can use and clean respectively for raw material with the medicine being equivalent to described weight proportion, and be dried, pulverize, be mixed to get and meet the granule of formulation requirements granularity or powder is directly taken.Can also use and after suitably processing, add pharmaceutic adjuvant with the medicine being equivalent to described weight proportion relation for raw material, be made into various preparation as required.During being prepared as preparation by above-mentioned raw materials medicine, above-mentioned raw materials medicine can be adopted and process with the following method: add water respectively or the ethanol extraction of variable concentrations, and extracting solution concentrate drying obtains crude extract；Or use further decoction and alcohol sedimentation technique, water back dissolution method, organic solvent extractionprocess, flocculent precipitation, column chromatography, carbon dioxide supercritical extraction method, steam distillation one or more be used in combination suitably refine after extract；Adoptable concrete operations and/or using method when above-mentioned effective medicinal ingredient is extracted, both can be with the ingredient of described each proportional quantities as raw material, extract decomposite mode after its effective medicinal ingredient respectively, it would however also be possible to employ the mode of common extraction again after mixing by each medicine material of described proportional quantities.The actual conditionses such as ideal required when using different extraction means, equipment and extract or optimal Extracting temperature, solvent load, extraction time, extraction time, then can be screened by test according to practical situation and find.
In order to make each raw material in said composition preferably play drug effect, preferably the present composition is used following extracting method, but cannot be used for limiting the scope of the invention.
The preparation method of the present composition is as follows:
By the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 4-20 times amount 20-95% ethanol extraction 1-4 time, each 1-3 hour, extracting solution merges, and filters, and decompression filtrate recycling ethanol is to without alcohol taste, drying under reduced pressure, is ground into fine powder, obtains the active component of the present composition.
This preparation method is preferably:
By the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 6-12 times amount 50-95% ethanol extraction 1-4 time, each 1-3 hour, extracting solution merges, and filters, and decompression filtrate recycling ethanol is to without alcohol taste, drying under reduced pressure, is ground into fine powder, obtains the active component of the present composition.
Or, the preparation method of the present composition can also be:
By the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, adding 4-20 times amount water extraction 1-4 time, each 1-3 hour, extracting solution merged, and filters, filtrate reduced in volume to thick paste, drying under reduced pressure, is ground into fine powder, obtains the active component of the present composition.
This preparation method is preferably:
By the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, adding 6-12 times amount water extraction 1-4 time, each 1-3 hour, extracting solution merged, and filters, filtrate reduced in volume to thick paste, drying under reduced pressure, is ground into fine powder, obtains the active component of the present composition.
The active component of the present composition prepared can directly be used as medicine and take or add acceptable adjuvant on pharmaceutics required preparation is produced in conventional fashion into.As the oral drugs of the solid dosage form such as conventional tablet (dispersible tablet, effervescent tablet, oral cavity disintegration tablet, buccal tablet, chewable tablet, effervescent tablet), capsule (hard capsule, soft capsule), granule, pill (drop pill), powder can be made, it is also possible to make the oral drugs of the liquid forms such as syrup, oral liquid, packed medicinal tea, bag tea agent；Can be to make the external used medicine of the external preparation forms such as unguentum, gel, ointment, cataplasma, emplastrum, liniment, lotion, liniment.Therefore, in this pharmaceutical composition in addition to effective ingredient, it is also possible to containing pharmaceutically acceptable adjuvant.
Adjuvant described here, can be different according to different preparations, such as diluent conventional in the solid preparations such as tablet, capsule, granule, disintegrating agent, excipient, binding agent, lubricant, surfactant, filler etc.；Surfactant conventional in the liquid forms such as syrup, oral liquid, diluent, preservative, stabilizer, correctives, thickening agent, fluidizer etc.；Acceptable oil base conventional in the external preparation form such as gel, ointment, aqueous matrix, preservative, antioxidant, wetting agent, Percutaneous absorption enhancer, surfactant etc..
nullIts conventional adjuvant such as starch、Lactose、Dextrin、Icing Sugar、Microcrystalline Cellulose、Mannitol、Xylitol、Polyethylene Glycol、Calcium sulfate、Calcium hydrogen phosphate、Calcium carbonate、Modified starch、Sorbitol、Polyvinylpyrrolidone、Heavy Magnesium Carbonate、Sodium carboxymethyl cellulose、Hydroxypropyl methylcellulose、Methylcellulose、Ethyl cellulose、Carboxymethylstach sodium、Hydroxypropyl cellulose、PVP K30、Kaolin、Pregelatinized Starch、Magnesium stearate、Pulvis Talci、Micropowder silica gel、Stevioside、Glycine betaine、Aspartame、Glycyrrhizin、Saccharin sodium、Citric acid、Sodium bicarbonate、Sodium carbonate、Carrageenan、Agar、Gelatin、Sodium alginate、Xanthan gum、Guar gum、Tragcanth、Arabic gum、Locust bean gum、POLY-karaya、Stearic acid、Glyceryl monostearate、Polyacrylamide、Cross linked sodium polyacrylate、Polyvinyl alcohol、Carbomer、Sorbic acid、Potassium sorbate、Ethyl hydroxybenzoate、Benzyl alcohol、Nipalgin、Thimerosal、Dimethyl sulfoxide、Azone、Triethanolamine、Sodium hydroxide、Glycerol、Propylene glycol、BHT、BHA、Sodium lauryl sulphate、Tweens、Spans etc..
The present invention relates to a kind of compositions for enhancing human body immunity power, there are nourishing the liver and kidney, invigorating the spleen and regulating the stomach, effect of nourishing YIN and clearing away lung-heat, three burnt logical benefits, be the nutrient substance having no side effect of a kind of pure natural, be different from the like product made by chemical composition.
Detailed description of the invention
Embodiment 1
Radix Ginseng 20g, Radix Codonopsis 20g, Radix Glehniae 20g
Weigh the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 8 times amount 70% ethanol extraction 3 times, each 1.5 hours, extracting solution merges, and filters, and decompression recycling ethanol is to without alcohol taste, drying under reduced pressure, is ground into fine powder, adds sucrose, aspartame, dextrin is appropriate, mixing, with alcohol granulation, is dried, granulate, obtains granule.
Embodiment 2
Radix Ginseng 10g, Radix Codonopsis 0.5g, Radix Glehniae 10g
Weigh the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 12 times amount 50% ethanol extraction 2 times, each 2 hours, extracting solution merges, and filters, and decompression recycling ethanol is to without alcohol taste, drying under reduced pressure, it is ground into fine powder, adds lactose, mannitol in right amount, mixing, with 85% alcohol granulation, it is dried, granulate, obtains granule.
Embodiment 3
Radix Ginseng 0.5g, Radix Codonopsis 10g, Radix Glehniae 0.5g
Weighing the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 6 times amount 95% ethanol extraction 4 times, each 1 hour, extracting solution merged, and filtered, decompression recycling ethanol, to without alcohol taste, drying under reduced pressure, is ground into fine powder, adds microcrystalline Cellulose, lactose in right amount, mixing, pelletize, be dried, granulate, obtain granule.
Embodiment 4
Radix Ginseng 10g, Radix Codonopsis 50g, Radix Glehniae 10g
Weigh the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 10 times amount 80% ethanol extraction 3 times, each 1 hour, extracting solution merges, and filters, and decompression recycling ethanol is to without alcohol taste, drying under reduced pressure, is ground into fine powder, and carboxymethyl starch sodium, micropowder silica gel, microcrystalline Cellulose, polyvinylpolypyrrolidone are appropriate, mixing, pelletizes, and is dried, add magnesium stearate, mixing, tabletting, coating or not coating, obtain tablet.
Embodiment 5
Radix Ginseng 50g, Radix Codonopsis 10g, Radix Glehniae 50g
Weighing the Radix Ginseng of described weight proportion, Radix Codonopsis, Radix Glehniae, add 15 times amount 60% ethanol extraction 1 time, each 3 hours, extracting solution filtered, and decompression recycling ethanol is to without alcohol taste, drying under reduced pressure, is ground into fine powder, adds appropriate amount of auxiliary materials, mixing, pelletize, be dried, encapsulated, obtain capsule.
Embodiment 6
Radix Ginseng 30g, Radix Codonopsis 7g, Radix Glehniae 15g
Weighing the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 8 times amount water extraction 3 times, each 2 hours, extracting solution filtered, and is evaporated to thick paste, drying under reduced pressure, was ground into fine powder, adds right amount of auxiliary materials, mixing, makes pill.
Embodiment 7
Radix Ginseng 15g, Radix Codonopsis 20g, Radix Glehniae 40g
Weighing the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 8 times amount 60% ethanol extraction 3 times, each 2 hours, extracting solution filtered, reclaim ethanol to without alcohol taste, suitably concentrate, add appropriate suspensoid, flavoring agent, preservative, mix, add water constant volume, filters, subpackage, obtains oral liquid.
Embodiment 8
Radix Ginseng 25g, Radix Codonopsis 10g, Radix Glehniae 8g
Weighing the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 12 times amount water extraction 2 times, each 3 hours, extracting solution filtered, and is evaporated to thick paste, drying under reduced pressure, is ground into fine powder, makes powder.
Embodiment 9
Radix Ginseng 20g, Radix Codonopsis 20g, Radix Glehniae 20g
Weighing the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 10 times amount water extraction 2 times, each 2 hours, extracting solution filtered, and is evaporated to thick paste, drying under reduced pressure, was ground into fine powder, added appropriate amount of auxiliary materials, pelletized, and is dried, granulate, obtains granule.
Embodiment 10
Radix Ginseng 20g, Radix Codonopsis 20g, Radix Glehniae 20g
Weigh the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, be ground into coarse powder, conventionally make packed medicinal tea.
Embodiment 11
Radix Ginseng 20g, Radix Codonopsis 20g, Radix Glehniae 20g
Weigh the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, be ground into coarse powder, conventionally make bag tea agent.
Embodiment 12
Radix Ginseng 20g, Radix Codonopsis 20g, Radix Glehniae 20g
Weigh the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 4 times amount 40% ethanol extraction 3 times, each 2 hours, extracting solution merges, and filters, and decompression recycling ethanol is to without alcohol taste, drying under reduced pressure, is ground into fine powder, adds sucrose, aspartame, dextrin is appropriate, mixing, with alcohol granulation, is dried, granulate, obtains granule.
Embodiment 13
Radix Ginseng 10g, Radix Codonopsis 0.5g, Radix Glehniae 10g
Weigh the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 20 times amount 20% ethanol extraction 2 times, each 1.5 hours, extracting solution merges, and filters, and decompression recycling ethanol is to without alcohol taste, drying under reduced pressure, it is ground into fine powder, adds lactose, mannitol in right amount, mixing, with 85% alcohol granulation, it is dried, granulate, obtains granule.
Embodiment 14
Radix Ginseng 0.5g, Radix Codonopsis 10g, Radix Glehniae 0.5g
Weighing the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 20 times amount water extraction 3 times, each 1 hour, extracting solution filtered, and is evaporated to thick paste, drying under reduced pressure, was ground into fine powder, added appropriate amount of auxiliary materials, pelletized, and is dried, granulate, obtains granule.
Embodiment 15
Radix Ginseng 10g, Radix Codonopsis 50g, Radix Glehniae 10g
Weigh the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 4 times amount water extraction 3 times, each 1 hour, extracting solution filters, and is evaporated to thick paste, drying under reduced pressure, it is ground into fine powder, adds carboxymethyl starch sodium, micropowder silica gel, microcrystalline Cellulose, polyvinylpolypyrrolidone in right amount, mixing, pelletize, be dried, add magnesium stearate, mixing, tabletting, coating or not coating, obtain tablet.
Embodiment 16
Radix Ginseng 50g, Radix Codonopsis 10g, Radix Glehniae 50g
Weighing the Radix Ginseng of described weight proportion, Radix Codonopsis, Radix Glehniae, add 15 times amount water extraction 2 times, each 2 hours, extracting solution filtered, and is evaporated to thick paste, drying under reduced pressure, was ground into fine powder, adds appropriate amount of auxiliary materials, mixing, pelletizes, is dried, encapsulated, obtained capsule.
Embodiment 17
Radix Ginseng 20g, Radix Codonopsis 40g, Radix Glehniae 60g
Weighing the Radix Ginseng of described weight proportion, Radix Codonopsis, Radix Glehniae, add 18 times amount water extraction 2 times, each 1.5 hours, extracting solution filtered, and is evaporated to thick paste, drying under reduced pressure, was ground into fine powder, adds appropriate amount of auxiliary materials, mixing, pelletizes, is dried, encapsulated, obtained capsule.
The beneficial effect of of the present invention compositions be expanded on further by following experiment:
1, material and method
1) sample: the sample prepared according to embodiment 1-3.
2) laboratory animal: 18-22g Kunming kind health cleaning grade female mice 192, is divided into four batches and tests, and every batch is randomly divided into 4 groups, often group 12.
Test and a collection of carry out internal organs/weight ratio pH-value determination pH, delayed allergy experiment, the mensuration of half hemolysis value (HC50) and the mensuration of antibody-producting cell number；
Test two batches and carry out carbonic clearance experiment；
Test three batches and carry out Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell experiment；
Test four batches of mouse lymphocyte transformation experiments carrying out ConA induction and NK cytoactive detection.
3) dosage:
Tested material is prepared with sterilized water, and per os gives once a day, and continuous gavage surveys indices after 33 days.Mouse stomach volume is 0.1mL/10g Mus weight.
Setting a blank group (0g/kgBW) simultaneously, substitute tested material with sterilized water, every day, gavage volume was identical with each tested material group.
4) key instrument and reagent:
ES-2100A electronic balance, BS223S electronic balance, 755 spectrophotometers, microplate reader, CO2 gas incubator, low speed centrifuge, water bath with thermostatic control, microscope, inverted microscope, spiral micrometer.
Clean bench, sterile surgical instrument, microsyringe (25 μ L), cell counter, the flat Tissue Culture Plate in 24 holes and 96 holes, the 96 U-shaped Tissue Culture Plates in hole, glass dish, gauze, test tube, slide frame, 200 eye mesh screens, timer, hemoglobin pipet, microscope slide etc..
nullSheep red blood cell (SRBC) (SRBC)、Normal saline、Hank's liquid (pH 7.2-7.4)、RPMIl640 culture fluid、Calf serum、Penicillin、Streptomycin、Concanavalin A, Con A (ConA)、1% glacial acetic acid、The HCl solution of 1mol/L、The acid isopropyl alcohol HCl solution 4mL of 1mol/L (the 96mL isopropanol add)、MTT、PBS (pH 7.2-7.4)、Complement (guinea pig serum)、SA buffer、Agarose、Dou Shi reagent (sodium bicarbonate 1.0g，High-potassium ferricyanide 0.2g，Potassium cyanide 0.05g，Add distilled water to 1000mL)、YAC-1 cell、EINECS 212-761-8、Nitro tetrazolium chloride、PMS、Oxidized form of nicotinamide-adenine dinucleotide、The Tris-HCl buffer (pH 8.2) of 0.2mol/L、1%NP40、India ink、0.1%Na2CO3、Chicken red blood cell、Methanol、Giemsa dye liquor etc..
5) experimental technique:
A. the mensuration of organ weight ratio value
After mouse weights, cervical dislocation is put to death, and takes spleen and thymus, removes most fascia, blots organ surface blood stains with filter paper, weigh, and calculates spleen weight ratio and thymus body weight ratio.
B. delayed allergy (DTH) experiment (the foot sole of the foot thickens method)
Taking Sanguis caprae seu ovis, brine 3 times, every Mus is through lumbar injection 2% (v/v, with normal saline) hematocrit SRBC (2000r/min, 10min) 0.2mL, 4d after sensitization, measuring left back sufficient sole of the foot portion thickness, same position is measured three times, averages.Then at measuring point subcutaneous injection 20% (v/v, with normal saline) hematocrit SRBC 20 μ L, after injection, 24h measures left back sufficient sole of the foot portion thickness, represents the degree of DTH with the difference of foot sole of the foot thickness before and after attacking.The difference of given the test agent group is significantly higher than the difference of matched group, can determine that this experimental result positive.
The mouse lymphocyte transformation experiment (mtt assay) of C.ConA induction
Aseptic take spleen, be placed in the little plate filling appropriate aseptic Hank ' s liquid, gently spleen ground with tweezers, make individual cells suspension.Filter through 200 eye mesh screens, make cell suspension.Wash 3 times with Hank ' s liquid, be centrifuged 10min (1000r/min) every time.Then by cell suspension in the complete culture solution of 1mL, microscopy counts, and adjusting cell concentration is 3 × 106/mL.Adding in 24 well culture plates by splenocyte suspension point holes again, every hole 1mL, a hole adds 75 μ LConA liquid (being equivalent to 7.5 μ g/mL) wherein, and 5%CO2, as comparison, is put in another hole, cultivates 72h in 37 DEG C of CO2 incubators.Cultivation terminates front 4h, and every hole sucks supernatant 0.7mL gently, adds the 0.7mL RPMI1640 culture fluid without calf serum, is simultaneously introduced MTT (5mg/mL) 50 μ L/ hole, continues to cultivate 4h.After cultivation terminates, every hole adds 1mL acid isopropyl alcohol, piping and druming mixing, makes purple crystal be completely dissolved.Then this liquid is moved in cuvette, colorimetric determination on 755 spectrophotometers, wavelength 570nm.The multiplication capacity of lymphocyte deducts by the optical density value adding ConA hole and is not added with the optical density value in ConA hole and represents the competence for added value of lymphocyte.The optical density difference of given the test agent group is significantly higher than the optical density difference of matched group, can determine that this experimental result positive.
D. the mensuration (Jerne improves slide method) of antibody-producting cell number
Taking Sanguis caprae seu ovis, brine 3 times, every Mus is through lumbar injection 2% (v/v, with normal saline) hematocrit SRBC 0.2mL.By SRBC immunity 5 days after mice cervical dislocation put to death, take out spleen, grind spleen gently, make cell suspension.Centrifugal (1000r/min) 10min, washes 2 times with Hank ' s liquid, finally by cell suspension in 8mL Hank ' s liquid.After agarose heating for dissolving, Hank ' s liquid double with equivalent mixes, subpackage small test tube, often pipe 0.5mL, (the v/v that adds 10% in pipe again, prepare with SA liquid) hematocrit SRBC 50 μ L, splenocyte suspension 8 μ L, rapidly after mixing, it is poured on the slide of brush agarose thin layer, does parallel plate, after agar solidification, slide level is buckled and is placed on horse, put into 37 DEG C of incubation 1h in CO2 gas incubator, then join in slide frame groove with the complement (1: 8) of SA buffer dilution, after continuing incubation 1.5h, count hemolysis plaque number.Represent with plaque number/full splenocyte.The plaque number of given the test agent group is significantly higher than the plaque number of matched group, can determine that this experimental result positive.
E. the mensuration of half hemolysis value (HC50)
Taking Sanguis caprae seu ovis, brine 3 times, every Mus carries out immunity through lumbar injection 2% (v/v, with normal saline) hematocrit SRBC 0.2mL.After 5 days, extracing eyeball and take blood in centrifuge tube, place about 1h, peeled off with tube wall by solidification blood, make serum fully separate out, 2000r/min is centrifuged 10min, collects serum.It is 300 times with SA buffer by serum-dilution, takes 1mL and put in test tube, be sequentially added into 10% (v/v uses SA buffer) hematocrit SRBC 0.5mL, complement 1mL (with SA buffer by 1: 8 dilution).Separately set the control tube (replacing with SA buffer) of not increase serum.Putting after being incubated 15min in 37 DEG C of waters bath with thermostatic control, ice bath terminates reaction.2000r/min is centrifuged 10min, takes supernatant 1mL, adds Dou Shi reagent to 3mL.Take the hematocrit SRBC 0.25mL of 10% (v/v uses SA buffer) simultaneously, add Dou Shi reagent to 4mL in another test tube, fully mix, after placing 10min, sentence control tube in 540nm and make blank, measure each pipe optical density value respectively.The amount of hemolysin represents with half hemolysis value (HC50), it is calculated as follows: the HC50 of optical density value during sample half hemolysis value=sample optical density value/SRBC HD50 × extension rate given the test agent group is significantly higher than the HC50 of matched group, can determine that this experimental result positive.
F. mice carbonic clearance experiment
Dilute the india ink (0.05mL/10g) of 4 times from mouse tail vein injection by body weight.Treat that prepared Chinese ink injects, immediately timing.Inject after prepared Chinese ink 2,10min, take blood 20 μ L from angular vein clump respectively, and be added in 2mL 0.1%Na2CO3 solution.With 755 spectrophotometers densitometric value (OD) at 600nm wavelength, make blank with Na2CO3 solution.By sacrifice, take liver and spleen is weighed.Represent the ability of mice carbonic clearance with carbonic clearance index (a), be calculated as follows a:
K=(lgOD1-lgOD2)/(t2-t1)
A=body weight ÷ (liver weight+spleen weight) × k1/3
The carbonic clearance index of given the test agent group is significantly higher than the carbonic clearance index of matched group, can determine that this experimental result positive.
G. Turnover of Mouse Peritoneal Macrophages phagocytosis chicken red blood cell experiment (half intracorporal method)
Mouse peritoneal injection 20% (v/v, with normal saline) chicken red blood cell (2000r/min, 10min) suspension 1mL, interval 30min, cervical dislocation is put to death, is faced upward position and be fixed on Mus plate, through abdominal cavity saline injection 2mL, rotate Mus plate 1min.Take peritoneal macrophage washing liquid 1mL, drip respectively on 2 microscope slides, put into the enamel box being lined with wet gauze, 37 DEG C of incubator incubation 30min of dislocation.Incubate complete, rinse in normal saline, to remove non-paster cell.Drying, fix with 1: 1 acetone methanol solution, Gicmsa-phosphate buffer dyes, then dries with distilled water rinsing.Count under oil mirror, 100 macrophages of every counting, be calculated as follows phagocytic rate and phagocytic index:
Macrophage number × 100 of the macrophage number/counting of phagocytic percentage (%)=phagocytosis chicken red blood cell
The macrophage number of the chicken red blood cell sum/counting of phagocytic index=swallowed
The phagocytic percentage drawn carries out data conversion, X=Sin the most as the following formula-1 , in formula, P is phagocytic percentage, represents decimally.The data obtained is measurement data, and the phagocytic percentage of given the test agent group and phagocytic index are all remarkably higher than phagocytic percentage and the phagocytic index of matched group, can determine that this experimental result positive.
The mensuration (lactate dehydrogenase L DH algoscopy) of H.NK cytoactive
Before experiment, target cell YAC-1 is carried out Secondary Culture by 24h, washes 3 times with Hank ' s liquid before application, and adjusting cell concentration with the RPMI1640 complete culture solution containing 10% calf serum is 4 × 105Individual/mL.Test mice cervical dislocation is put to death, and aseptic takes spleen, makes splenocyte suspension, washes 2 times with Hank ' s liquid, is centrifuged 10min (1000r/min) every time.Abandon supernatant cytoplasm to be upspring, add 0.5mL aquesterilisa 20 seconds, 2 times of Hank ' s liquid of 0.5mL and 8mLHank ' s liquid is added after splitting erythrocyte, 1000r/min, 10min is centrifuged, resuspended with the 1mL RPMI1640 complete culture solution containing 10% calf serum, microscopy counts, and adjusting cell concentration with RPMI1640 complete culture solution is 2 × 107Individual/mL.Making effect target ratio is 50: 1.Take target cell and each 100 μ L of effector lymphocyte, add in U-shaped 96 well culture plate；Target cell Spontaneous release hole adds target cell and each 100 μ L of culture fluid, and target cell maximum release aperture adds target cell and each 100 μ L of 1%NP40；Above-mentioned every it is all provided with three parallel holes, 37 DEG C, 5%CO2 incubator is cultivated 4h, 96 orifice plates are centrifuged 5min with 1500r/min, every hole is drawn in 96 well culture plates at the bottom of supernatant 100 μ L horizontalization, adding LDH matrix liquid 100 μ L, react 3-10min, then every hole adds the HCl solution 30 μ L termination reaction of 1mol/L, densitometric value (OD) at microplate reader 490nm, calculating NK cytoactive:
NK cytoactive (%)=(reacting hole OD-Spontaneous release hole OD)/(maximum release aperture OD-Spontaneous release hole OD) × 100
The NK cytoactive drawn carries out data conversion, X=Sin as the following formula-1 , in formula, P is NK cytoactive, represents decimally.The data obtained is measurement data, and the NK cytoactive of given the test agent group is significantly higher than the NK cytoactive of matched group, can determine that this experimental result positive.
6) data process
Data process is carried out with SPSS software.Using variance analysis, but need to first carry out homogeneity test of variance by the program of variance analysis, variance is neat, calculates F value, F value ＜ F0.05, conclusion: no significant difference between each group mean, F value >=F0.05, P≤0.05, add up with the comparative approach two-by-two of mean between multiple experimental grouies and a matched group；The data of abnormal or heterogeneity of variance are carried out suitable variable conversion, after meeting normal state or variance requires together, adds up by the data after conversion；If being still not up to normal state or the neat purpose of variance after variable conversion, using rank test instead and adding up.
7) result judgment basis
" health food inspection and assessment technique specification) " (2003 editions) regulation: in cellular immune function, humoral immune function, monocytes/macrophages function, any two aspect results positives of four aspects of NK cytoactive, can determine that this given the test agent has enhancing immunity function.Wherein two experimental results in cellular immune function assay project are the positive, or two dosage group results of arbitrary experiment are positive, can determine that cellular immune function assay result is positive.Two experimental results in humoral immune function mensuration project are the positive, or two dosage group results of arbitrary experiment are positive, can determine that humoral immune function measurement result is positive.Two experimental results in monocytes/macrophages functional examination project are the positive, or two dosage group results of arbitrary experiment are positive, can determine that monocytes/macrophages function result is positive.More than one dosage group result of NK cytoactive detection experiment is positive, can determine that NK cytoactive result is positive.
2, result
1) present composition impact on Mouse Weight
By weighing, the original body mass of mice compares with between blank group four batches of laboratory animal each sample groups, and there are no significant for difference (P ＞ 0.05).The i.e. original body mass of mice more equalizes between each group.
Table
1
The present composition impact on Mouse Weight
From table 1, after per os gives the mice present composition 33 days, the body weight of mice compares with between blank group four batches of each sample groups, and there are no significant for difference (P ＞ 0.05).Mouse Weight is had no adverse effects by the i.e. present composition.
2) present composition impact on mice organs/body weight ratio
The impact on mouse spleen/body weight ratio of table 2 present compositionTable 2 The present composition is to mouse spleen / The impact of body weight ratio
| Group | Number of animals (only) | Spleen/body weight ratio (mg/g) | P value |
| Blank group | 12 | 5.5±1.0 | —— |
| Sample sets 1 | 12 | 5.2±0.8 | 0.695 |
| Sample sets 2 | 12 | 5.3±0.5 | 0.728 |
| Sample sets 3 | 12 | 5.6±0.4 | 0.899 |
From table 2, after per os gives the mice present composition, each sample group spleen/body weight ratio compares with blank group, and there are no significant for difference (P ＞ 0.05).Spleen/body weight the ratio of mice is affected by the i.e. present composition without special.
The impact on mouse thymus/body weight ratio of table 3 present compositionTable 3 The present composition is to mouse thymus / The impact of body weight ratio
| Group | Number of animals (only) | Thymus/body weight ratio (mg/g) | P value |
| Blank group | 12 | 2.7±0.5 | —— |
| Sample sets 1 | 12 | 2.6±0.8 | 0.946 |
| Sample sets 2 | 12 | 2.4±0.6 | 0.853 |
| Sample sets 3 | 12 | 2.6±0.5 | 0.951 |
From table 3, after per os gives the mice present composition 33 days, each dosage group thymus/body weight ratio compares with blank group, and there are no significant for difference (P ＞ 0.05).Thymus/body weight the ratio of mice is affected by the i.e. present composition without special.
3) present composition impact on mouse cell immunologic function
The impact on mice delayed allergy (DTH) of table 4 present compositionTable 4 The present composition is to mice delayed allergy (DTH) Impact
| Group | Number of animals (only) | Swelling degree of the paw (mm) | P value |
| Blank group | 12 | 0.54±0.18 | —— |
| Sample sets 1 | 12 | 0.85±0.12* | 0.012 |
| Sample sets 2 | 12 | 0.83±0.21* | 0.025 |
| Sample sets 3 | 12 | 0.82±0.25* | 0.030 |
* compare with blank group, P ＜ 0.05
From table 4, after per os gives the mice present composition 33 days, each sample group compares with blank group, and the swelling degree of the paw of mice has significant difference (P ＜ 0.05).The i.e. present composition can significantly improve mice swelling degree of the paw.
The impact on mouse lymphocyte transformation experiment of table 5 present compositionTable 5 The present composition impact on mouse lymphocyte transformation experiment
| Group | Number of animals (only) | Lymphopoiesis ability (OD difference) | P value |
| Blank group | 12 | 0.26±0.15 | —— |
| Sample sets 1 | 12 | 0.44±0.15* | 0.011 |
| Sample sets 2 | 12 | 0.38±0.18* | 0.035 |
| Sample sets 3 | 12 | 0.40±0.17* | 0.024 |
* compare with blank group, P ＜ 0.05
From table 5, after per os gives the mice present composition 33 days, each sample group compares with blank group, and the lymphopoiesis ability of mice has significant difference (P ＜ 0.05).The i.e. present composition can significantly improve the mouse lymphocyte conversion capability of ConA induction.
4) present composition impact on humoral immunization
The impact on mouse antibodies cellulation number of table 6 present compositionTable 6 The present composition impact on mouse antibodies cellulation number
| Group | Number of animals (only) | Hemolysis plaque number (× 103/ full spleen) | P value |
| Blank group | 12 | 150±44 | —— |
| Sample sets 1 | 12 | 195±47* | 0.041 |
| Sample sets 2 | 12 | 199±52* | 0.029 |
| Sample sets 3 | 12 | 196±35* | 0.037 |
* compare with blank group, P ＜ 0.05
From table 6, after per os gives the mice present composition 33 days, each sample group compares with blank group, and mouse antibodies cellulation number has significant difference (P ＜ 0.05).The i.e. present composition can significantly improve mouse antibodies cellulation number.
The impact on mice half hemolysis value (HC50) of table 7 present compositionTable 7 The present composition is to mice half hemolysis value (HC50) Impact
| Group | Number of animals (only) | Sample half hemolysis value | P value |
| Blank group | 12 | 175±42 | —— |
| Sample sets 1 | 12 | 215±37* | 0.014 |
| Sample sets 2 | 12 | 210±48* | 0.026 |
| Sample sets 3 | 12 | 216±26* | 0.010 |
* compare with blank group, P ＜ 0.05
From table 7, after per os gives the mice present composition 33 days, each sample group compares with blank group, and the half hemolysis value of mice has significant difference (P ＜ 0.05).The i.e. present composition can significantly improve mice half hemolysis value.
5) present composition impact on mouse monokaryon-macrophage phagocytic function
The impact on mice carbonic clearance ability of table 8 present compositionTable 8 The present composition impact on mice carbonic clearance ability
| Group | Number of animals (only) | Phagocytic index (a) | P value |
| Blank group | 12 | 5.14±0.59 | —— |
| Sample sets 1 | 12 | 6.17±0.33* | 0.008 |
| Sample sets 2 | 12 | 6.04±0.46* | 0.016 |
| Sample sets 3 | 12 | 5.98±0.61* | 0.020 |
* compare with blank group, P ＜ 0.05
From table 8, after per os gives the mice present composition 33 days, each sample group compares with blank group, and the carbonic clearance ability of mice has significant difference (P ＜ 0.05).The i.e. present composition can significantly improve the carbonic clearance ability of mice.
The impact on mouse macrophage phagocytosis chicken red blood cell phagocytic rate of table 9 present compositionTable 9 The present composition impact on mouse macrophage phagocytosis chicken red blood cell phagocytic rate
| Group | Number of animals (only) | Phagocytic rate (%) | Phagocytic rate square root arcsine conversion value | P value |
| Blank group | 12 | 23±10 | 0.50±0.10 | —— |
| Sample sets 1 | 12 | 35±11* | 0.67±0.14 | 0.022 |
| Sample sets 2 | 12 | 37±5* | 0.71±0.11 | 0.014 |
| Sample sets 3 | 12 | 35±8* | 0.67±0.08 | 0.023 |
* compare with blank group, P ＜ 0.05
From table 9, after per os gives the mice present composition 33 days, each sample group compares with blank group, and phagocytic rate has significant difference (P ＜ 0.05).The i.e. present composition can significantly improve mouse macrophage phagocytosis chicken red blood cell phagocytic rate.
The impact on mouse macrophage phagocytosis chicken red blood cell phagocytic index of table 10 present compositionTable 10 The present composition impact on mouse macrophage phagocytosis chicken red blood cell phagocytic index
| Group | Number of animals (only) | Phagocytic index | P value |
| Blank group | 12 | 0.38±0.19 | —— |
| Sample sets 1 | 12 | 0.62±0.13* | 0.026 |
| Sample sets 2 | 12 | 0.58±0.26* | 0.047 |
| Sample sets 3 | 12 | 0.60±0.21* | 0.034 |
* compare with blank group, P ＜ 0.05
From table 10, after per os gives the mice present composition 33 days, each sample group compares with blank group, and phagocytic index has significant difference (P ＜ 0.05).The i.e. present composition can significantly improve mouse macrophage phagocytosis chicken red blood cell phagocytic index.
6) present composition impact on NK cells in mice activity
The impact on NK cells in mice activity of table 11 present compositionTable 11 The present composition is to mice NK The impact of cytoactive
| Group | Number of animals (only) | NK cytoactive (%) | NK cytoactive square root arcsine conversion value | P value |
| Blank group | 12 | 23.4±6.2 | 0.50±0.10 | —— |
| Sample sets 1 | 12 | 36.7±8.1* | 0.71±0.08 | 0.022 |
| Sample sets 2 | 12 | 33.5±6.9* | 0.64±0.12 | 0.038 |
| Sample sets 3 | 12 | 32.9±8.4* | 0.60±0.09 | 0.045 |
* compare with blank group, P ＜ 0.05
From table 11, after per os gives the mice present composition 33 days, each sample group compares with blank group, and NK cells in mice activity has significant difference (P ＜ 0.05).The i.e. present composition can significantly improve NK cells in mice activity.
Embodiment of the present invention 4-17 has carried out similar test the most according to the method described above, and result is identical with the above results.
3, conclusion
After per os gives the mice present composition 33 days, compare with blank group, the present composition can significantly improve mice swelling degree of the paw (P ＜ 0.05), improve the mouse lymphocyte conversion capability (P ＜ 0.05) of ConA induction, improve mouse antibodies cellulation number (P ＜ 0.05), improve mice half hemolysis value (P ＜ 0.05), improve the carbonic clearance ability (P ＜ 0.05) of mice, improve NK cells in mice activity (P ＜ 0.05), and Mouse Weight growth is had no adverse effects by the present composition.According to " health food inspection and assessment technique specification " (003 edition) judgment criteria to enhancing immunity health food, the present composition has the function of enhancing immunity.
Claims (9)
1. a compositions with enhancing immunity function, it is characterised in that by weight, the crude drug making said composition is: Radix Ginseng 0.5-10 part, Radix Codonopsis 0.5-10 part, Radix Glehniae 0.5-10 part.
Compositions the most according to claim 1, it is characterised in that by weight, the crude drug making said composition is: Radix Ginseng 1-5 part, Radix Codonopsis 1-5 part, Radix Glehniae 1-5 part.
3. the preparation method of compositions described in claim 1 or 2, it is characterised in that it is prepared:
The crude drug of described weight portion is directly ground to coarse powder；Or add water or the ethanol extraction of variable concentrations, extracting solution concentrate drying obtains crude extract；Or use decoction and alcohol sedimentation technique, organic solvent extractionprocess, column chromatography, steam distillation to carry out being refining to obtain extract further；Above-mentioned medicinal material coarse powder or crude extract or extract are the active component of the present composition.
The preparation method of compositions the most according to claim 3, it is characterised in that it is prepared:
By the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, add 4-20 times amount 20-95% ethanol extraction 1-4 time, each 1-3 hour, extracting solution merges, and filters, and decompression filtrate recycling ethanol is to without alcohol taste, drying under reduced pressure, is ground into fine powder, obtains the active component of the present composition.
The preparation method of compositions the most according to claim 3, it is characterised in that it is prepared:
By the Radix Ginseng of described weight portion, Radix Codonopsis, Radix Glehniae, adding 4-20 times amount water extraction 1-4 time, each 1-3 hour, extracting solution merged, and filters, filtrate reduced in volume to thick paste, drying under reduced pressure, is ground into fine powder, obtains the active component of the present composition.
6. according to the preparation method of compositions described in claim 4 or 5, it is characterised in that the active component of prepared compositions adds or is not added with adjuvant technique routinely makes the preparation accepted on pharmaceutics.
The preparation method of compositions the most according to claim 6, it is characterised in that described preparation is oral formulations.
The preparation method of compositions the most according to claim 7, it is characterised in that described oral formulations refers to tablet, granule, capsule, pill, powder, oral liquid, packed medicinal tea, bag tea agent.
9. compositions described in claim 1 or 2 has the application in the health food of enhancing immunity function for preparation.
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| KR20150037109A (en) * | 2013-09-30 | 2015-04-08 | 주식회사 함양산양삼 | Manufacturing method of composition of health food containing wild ginseng and herb extracts and composition of health food containing wild ginseng and herb extracts manufactured by the same |
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| KR20150037109A (en) * | 2013-09-30 | 2015-04-08 | 주식회사 함양산양삼 | Manufacturing method of composition of health food containing wild ginseng and herb extracts and composition of health food containing wild ginseng and herb extracts manufactured by the same |
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