CN103800390A - Health-care product with immunity-reinforcing and liver-protecting functions, preparation method and application thereof - Google Patents
Health-care product with immunity-reinforcing and liver-protecting functions, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a health-care product with immunity-reinforcing and liver-protecting functions, a preparation method and application thereof. The health-care product with immunity-reinforcing and liver-protecting functions is prepared from the following raw materials in parts by weight: 15-60 parts of curcumin, 60-240 parts of ganoderma extract, 255-500 parts of reishi shell-broken spore powder, and 2-13 parts of selenium-enriched yeast. Animal tests prove that the health-care product has a function of reinforcing immunity, and has the function of auxiliary protection to alcoholic liver injury of animals.
Description
Technical field
The present invention relates to preparation method and the application of health product, relate in particular to and there is enhancing immunity and protect the liver health product of function and preparation method thereof and application.
Background technology
Immunity generally also can be resistance.Conventionally the body immunity of saying be exactly health in the time being subject to external infringement, during such as antibacterial, poisoning intrusion human body, the ability of body resistance exotic invasive.The immunity of human body comes from immune system, immune system mainly contains the function of three aspects:, the firstth, immune defence, it to antigenic substance as: pathogenic microorganism, vaccine, toxoid, foreign protein etc. occur immunne response, finally eliminate antigenic substance, play the effect of infection immunity.If the defense function of body occurs when damaged, very easily to suffer from infectiousness or infectious disease; The secondth, immunological homeostasis, can remove damage, aging, dead cell, and effectively carry out immunoregulation effect, as from steady functional disorder, easily occurs autoimmune disease; It three is immune surveillance functions, its major function shows that body identifies the antigen on cancerous tumor cell by immunocyte, and by special or non-specific killer cell, cancerous cell is destroyed, emerging cancerous cell is eliminated before not causing a large amount of propagation, spreading, and plays antineoplastic action.As immune surveillance mistake department, the normal tumor that easily occurs.Along with social progress, the quickening of people's rhythm of life, the variation of living environment, and the pressure of each side is increasing, causes thus the crowd of immunity degradation also more and more.So enhancing human body immunity power, safeguards healthy most important.
Liver is important substance metabolism organ, plays an important role at aspects such as the absorption of the materials such as carbohydrate, lipid, protein, vitamin, hormone, bile, storage, biotransformation, secretion, excretions.Have removing toxic substances and phagocytic function, in participation blood, many thrombins is synthetic, participates in hematopoiesis, storage and release Hemopoietic factor simultaneously; Or the synthetic organ of many enzymes in human body, is guaranteeing the metabolism of human normal.Ethanol and many chemical toxicant are as hypertoxic class: phosphorus, trinitrotoluene, carbon tetrachloride, chloronaphthalene, acrylic aldehyde.Hypertoxic type: arsenic, hydrargyrum, antimony, aniline, chloroform, arsenic hydride, dimethyl formamide etc.The dinitrophenol of lower toxicity, acetaldehyde, organophosphor, propylene are fine, plumbous etc. all easily causes hepatic injury, and alcoholic hepatic injury is modal at present.These poisonous substances are general susceptible in crowd, and incubation period is short, can cause stem cell necrosis in various degree of liver, fatty distortion, liver cirrhosis, cholestasis, hepatic fibrosis, hepatocarcinoma even.For the patient of hepatic injury, modern medicine adopts the hepatics such as film protective agent, anti peroxidation of lipid agent, anti-immune response agent to treat, but toxic and side effects is larger, and curative effect is also not obvious.And herbal toxic effect is little, there is the feature of many target spots, too many levels comprehensive function, aspect treatment hepatic injury, there iing original curative effect.
Along with the number ubiquity of social development subhealth state and there is the trend increasing gradually.Subhealth state is a kind of chronic disease, and except the improvement of life style, the health food of taking enhancing immunity has become people's common recognition.Hepatic injury is the disease of clinical common harm humans health, except ethanol and chemical solvent, approximately has at present 500-1000 kind Western medicine can cause hepatic disease, and serious hepatic disease mortality rate is very high.Modern medicine there is no specific drug aspect the treatment of intervening hepatic injury, rest, reinforcement nutrition, vitimin supplement and the symptomatic treatments etc. of adopting more, and severe patient is even forced to discontinue medication.Chinese herbal medicine is concocted through strict processing, and toxic and side effects is very little.Aspect treatment hepatic injury, obtaining certain effect.
Rhizoma Curcumae Longae extract is that tuber or the rhizome of zingiberaceous plant Rhizoma Curcumae Longae refines through extracting the crocus crystallization or the powder that obtain, and its active component is mainly made up of curcumin, demethoxycurcumin, Bisdemethoxycurcumin.The pungent temperature of Rhizoma Curcumae Longae property, return spleen, Liver Channel, there is removing blood stasis circulation of qi promoting, the effect of inducing menstruation to relieve menalgia.Water insoluble and ether, is dissolved in ethanol, glacial acetic acid, propylene glycol, to thermally-stabilised but unstable in illumination and alkali liquor.There is antioxidation, infection, antiinflammatory, anticoagulant, blood fat reducing, atherosclerosis and suppress the effects such as tumor growth.The industry such as curcumin is widely used in food, article of everyday use and pharmacy, safety non-toxic, has no side effect, and is one of a kind of natural pigment that has DEVELOPMENT PROSPECT.
Ganoderma extract is the extract of Ganoderma through processing.Ganoderma is Basidiomycetes, Polyporaceae, Ganoderma fungus.Ancient Chinese medicine doctor thinks that Ganoderma is strengthening the body resistance, the treasure of strengthening by means of tonics.Its property is flat, sweet in the mouth, GUIXIN, lung, liver, kidney channel.Compendium of Material Medica is recorded, and Ganoderma can " be controlled knot in the heart, the motive of hanging ", and " enter the heart being responsible for production of blood, help the heart to fill arteries and veins ", " calming the nerves ", " protecting god ", " lung benefiting, spleen, vital essence ", " tonifying liver gas " etc., all has benefiting action to whole body QI of five ZANG-organs.Modern study shows that Ganoderma contains polysaccharide, aminoacid, protein, polypeptide, steroid class, terpenoid, organic acid and volatilization wet goods composition.Wherein ganoderan is main biological activity.There is adjusting immunologic function, suppress the growth of real tumor body in Mice Body.Stimulate insulin secretion, promote the effects such as synthesizing of nucleic acid, protein.
Ganoderma spore is the female and male gametophyte that Ganoderma produces offspring.Ganoderma spore contains triterpenes, Ganodenic acid, polysaccharide, enzyme, alkaloid, multivitamin etc.Ganodenic acid there is multiple pharmacologically active, as can be killed tumor cell; Polysaccharide, triterpenes, the tumoricidal telomerase of enzyme energy, make the cell of cancer lose energy for growth, promote macrophage growth, improve granulocytic function, the generation of stimulating cytokine is a kind of important cytokine that mediates body panimmunity and inflammatory process.The ability that improves the synthetic anti-tumor element I of cell and II, strengthens the generation of interferon, can remove rapidly Radiotherapy chemotherapy and tired free radical and the active oxygen producing containing the superoxide dismutase of volume.And by stimulating lymphocytic propagation, improve the activity of immunologically competent cell and the factor, thereby strengthen growth stimulator that immunologic function comes antagonism and anticancer to spread and reach anticancer object, Ganoderma spore also has the chemicals of elimination to be caused and the function of liver injury prevents liver cirrhosis and angiosclerotic effect.
Yeast rich in selenium is exactly in the process of culture yeasts, to add selenium element, when yeast growth, absorb selenium, the protein of selenium in yeast body and polysaccharide organic are combined and are converted into biological selenium, stimulate thereby eliminated the toxicity of chemical selenium (as sodium selenite) to human body and the intestines and stomach, make the selenium utilization that can more efficiently, more safely be absorbed by the body.
Summary of the invention
An object of the present invention is to provide a kind of health product that there is enhancing immunity and protect the liver function.
The health product that have enhancing immunity and protect the liver function provided by the present invention, is characterized in that: the active component of described health product is made up of the raw material of following weight portion: 2 parts-13 parts of 15 parts-60 parts of curcumins, 60 parts-240 parts of Ganoderma extracts, 255 parts-500 parts of Ganoderma spore powder with cellular wall brokens and yeast rich in seleniums.
The raw material of described weight portion is: 6 parts-10 parts of 5 parts-50 parts of bisdemethoxycurcumins, 60 parts-185 parts of Ganoderma extracts, 225 parts-360 parts of Ganoderma spore powder with cellular wall brokens and yeast rich in seleniums.
Described health product also comprise pharmaceutically acceptable adjuvant or complementary composition.
Described Ganoderma extract is Ganoderma water extract or Ganoderma extractive with organic solvent; Described curcumin is Rhizoma Curcumae Longae water extract or Rhizoma Curcumae Longae extractive with organic solvent.
Described pharmaceutically acceptable adjuvant or complementary composition are microcrystalline Cellulose and magnesium stearate.
The raw material of weight portion described in described health product is 15 parts-60 parts of curcumins, 60 parts-240 parts of Ganoderma extracts, 255 parts-500 parts of Ganoderma spore powder with cellular wall brokens, 2 parts-13 parts of yeast rich in seleniums; Described adjuvant or complementary composition and weight portion thereof are: 3 parts-6 parts of 81 parts-153 parts of microcrystalline Cellulose and magnesium stearate.
Described in described health product, the raw material of weight portion is: 5 parts-50 parts of bisdemethoxycurcumins, 60 parts-185 parts of Ganoderma extracts, 225 parts-360 parts of Ganoderma spore powder with cellular wall brokens, 6 parts-10 parts of yeast rich in seleniums; Described adjuvant or auxiliary element and weight portion thereof are: 3 parts-4 parts of 81 parts-106 parts of microcrystalline Cellulose and magnesium stearate.
The described health product that have enhancing immunity and protect the liver function also belong to protection scope of the present invention in the application of preparing in enhancing immunity and hepatic.
Prove through animal experiment, health product provided by the present invention have the function of enhancing immunity.Per os gives the tested material 30 days of mice various dose, can strengthen mice delayed allergy, can improve the carbon of mouse monokaryon-macrophage and clean up ability, can promote the lymphocyte proliferation ability of mice; Can promote the antibody-producting cell increment of mice; Can improve the serum hemolysin of mice; Can improve mice carbon single and macrophage and clean up ability; Promote macrophage phagocytosis of mice; On weight of mice, internal organs/body weight ratio, NK cytoactive without impact.
, prove through animal experiment, health product provided by the present invention have animal alcoholic liver injury auxiliary protection function function meanwhile.With the tested material gavage mice of 0.15g/kgBW, 0.3g/kgBW, 0.6g/kgBW dosage 30 days, each treated animal vegetative activity was good, and each treated animal increases weight compared with model control group, difference not statistically significant (P>0.05); In middle and high dosage group liver homogenate, MDA concentration and TG concentration are all lower than model control group; The steatosis of middle and high dosage group is marked lower than model control group, and difference has statistical significance (P<0.05).
The specific embodiment
Embodiment 1, preparation have enhancing immunity and protect the liver the health product of function
Raw material curcumin, Ganoderma extract, Ganoderma spore powder with cellular wall broken, yeast rich in selenium, microcrystalline Cellulose and magnesium stearate used in this embodiment all can be bought and obtain from commercial channels.
The health product that this embodiment prepares are as follows:
This embodiment has enhancing immunity and protects the liver the preparation method of health product of function as follows:
Curcumin, Ganoderma extract, Ganoderma spore powder with cellular wall broken and yeast rich in selenium in this embodiment can prepare by following method, also can buy and obtain from commercial channels.
One, raw material preparation
1, prepare curcumin
Get Rhizoma Curcumae Longae crude drug, with 70% ethanol of 9 times of volumes, with the speed percolation of 3mL/min, utilize D101 purification by macroporous resin to regulate loading material liquid pH is 7 simultaneously, and the speed eluting with 80% ethanol with 4mL/min, obtains concentrated solution; Concentrated solution, through drying under reduced pressure, is obtained to dry extract; Dry extract is pulverized, and crossed after 80 mesh sieves, obtain extract fine powder, be Rhizoma Curcumae Longae extract, curcumin.
2, prepare Ganoderma extract
Get Ganoderma crude drug, with the water extraction of 8 times of weight 2 times, each 2 hours, merge 2 times aqueous extract; At 60 ℃, under-0.08MPa condition, aqueous extract is concentrated into relative density 1.09(60 ℃), obtain concentrated solution; Concentrated solution, through drying under reduced pressure (0.09MPa), is obtained to dry extract; Dry extract is pulverized, and crossed after 80 mesh sieves, obtain extract fine powder, be Ganoderma extract.
3, prepare Ganoderma spore powder with cellular wall broken
Ganoderma is through hair tube reason and go out sesame and manage, then the added bullet powder spore powder of gathering.The spore powder of newly gathering through super-dry, sieve, after multiple sieve, breaking cellular wall, airtight preservation.
4, prepare yeast rich in selenium
Add appropriate selenium compound as culture medium take molasses, access barms after sterilization, add the nutritive salt such as nitrogen, phosphorus and carry out fermentation culture, 30 ℃ of temperature, aerlbic culture 24~48 hours under the condition of pH4.5~5.0, make inorganic selenium be combined into the selenium of organic, isolate yeast by centrifuging, after water rinses repeatedly, be dried and obtain flaxen yeast rich in selenium powder through concentrated and spraying.
Two, production technology
1. batching, pre-treatment: require to get all the ready according to formula the supplementary material conforming to quality requirements, check outer package, remove qualified outer package, proceed in 300,000 grades of clean areas, for subsequent use.Supplementary material is crossed respectively 60 mesh sieves, weighs, for subsequent use.
2. first yeast rich in selenium and microcrystalline Cellulose are pressed to equivalent incremental method mix homogeneously, must mix powder I, mixed powder I is mixed 30 minutes with Rhizoma Curcumae Longae extract, Ganoderma extract, Ganoderma spore powder with cellular wall broken again, makes evenly, to obtain mixed powder II.
3. granulate: in mixed powder II, add purified water soft material processed, soft material, with can be agglomerating and tack-free with hand-tight holding, splits for degree with pointing light pressure energy.Gained soft material is crossed 20 mesh sieve granule processed, and wet granular 50-55 ℃ dry, is dried to pellet moisture≤5%.20 mesh sieve granulate for the dry granule of gained, obtain granule.
4. always mixed, filling, polishing: gained granule mixes with magnesium stearate 10 minutes, makes evenly, obtains and always mixes material.Total mixed material filled capsules.Populated capsule polishing, checks and removes inferior capsule, obtains polishing capsule.
Health product prepared by embodiment 2, the embodiment of the present invention 1 are for the functional evaluation of enhancing immunity
1. materials and methods
1.1 samples: health product A, B, C, D and E that above-described embodiment 1 prepares.
1.2 laboratory animal
The clean level ICR male mice that the Bethune of Jilin University medical college zoopery center provides, production licence number: SCXK-(Ji) 2007-0003.Feedstuff is provided by Changchun hundred million these laboratory animal technology Co., Ltds, production licence number: SCXK-(Ji) 2010-0001.This laboratory animal environmental facility quality certification, lucky moving establish word 10-1005, laboratory animal occupancy permit number: SYXK-(Ji) 2010-0011.Select 48 of healthy male mices, body weight 18g-22g, is divided into 4 groups, 12 every group, as immune one group, carries out liver/weight ratio pH-value determination pH, half hemolysis value (HC
50) mensuration and antibody-producting cell detect; Select 48 of healthy male mices, body weight 18g-22g, is divided into 4 groups, 12 every group, as immune two groups, carries out carbon and cleans up experiment; Select 48 of healthy male mices, body weight 18g-22g, is divided into 4 groups, 12 every group, as immune three groups, carries out mouse lymphocyte transformation experiment and the NK cytoactive detection of ConA induction; Select 48 of healthy male mices, body weight 18g-22g, is divided into 4 groups, 12 every group, as immune four groups, carries out delayed allergy test; Select 48 of healthy male mices, body weight 18g-22g, is divided into 4 groups, 12 every group, as immune five groups, carries out Turnover of Mouse Peritoneal Macrophages and engulfs chicken red blood cell experiment.
1.3 dosage are selected and preparation
0.03,0.30,0.90g/kg BW composition capsule 1.8g/ day, that is: 1.8g/60kg BW, is equivalent to 0.03g/kg BW, with 1,10 and 30 times of the intake of being grown up, gavage dosage is set, and, each dosage group is with distilled water diluting.Make negative control group with pure water, respectively organize mouse stomach amount and be 0.2mL/10g BW, gavage is surveyed every immune indexes after 30 days continuously.
Sample thief 4.50g adds pure water and mixes standardize solution to 100mL and be high dose group tested material; Sample thief 1.5g adds pure water and mixes standardize solution to 100mL and be middle dosage group tested material; Sample thief 0.15g adds pure water and mixes standardize solution to 100mL and be low dose group tested material.The fresh preparation of each group tested material, every day, gavage gave tested material once.
1.4 instruments and reagent
Electronic balance (0.1g), analytical balance, clean bench, CO2 gas incubator, centrifuge, 722 spectrophotometers, water bath with thermostatic control, microplate reader, microscope etc., aseptic operation apparatus, slide gauge (precision 0.02mm), 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., sheep red blood cell (SRBC) (SRBC), normal saline, Hank ' s liquid (PH7.2-7.4), RPMI1640 culture fluid, calf serum, mycillin, concanavalin A, Con A (ConA), 1% glacial acetic acid, the HCl solution of 1mol/L, acid isopropyl alcohol (96mL isopropyl alcohol adds 4mL hydrochloric acid), MTT, PBS buffer (pH7.2-7.4), complement (guinea pig serum), SA buffer, agarose, Dou Shi reagent (sodium bicarbonate 1.0g, high-potassium ferricyanide 0.2g, potassium cyanide 0.05g, adding distil water is to 1000mL), YAC-1 cell, sodium lactate, nitro tetrazolium chloride, PMS, oxidized coenzyme I, the Tris-HCl buffer (Ph8.2) of 0.2mol/L, 1%NP40, india ink, Na
2cO
3, chicken red blood cell, methanol, Giemsa dye liquor etc.
1.5 experimental techniques and result
1.5.1 liver/weight ratio pH-value determination pH
Assay method: mice dislocates and puts to death after weighing, and gets spleen and thymus, removes most fascia, blots internal organs show blood stains with filter paper, weighs, and calculates spleen/body weight ratio and thymus/body weight ratio.
Result:
(1) impact of tested material on immune one group of Mouse Weight
Health product C prepared by embodiment 1 on the impact of immune one group of Mouse Weight in table 1.
Table 1. tested material is on immune one group of Mouse Weight impact
From table 1, to learn and process by statistics, per os gives tested material C30 days, and each dosage group Mouse Weight, weightening finish and negative control group comparing difference are without significance (P > 0.05), and tested material has no significant effect Mouse Weight, weightening finish.
The impact of health product A, B, D and E prepared by embodiment 1 on immune one group of Mouse Weight and table 1 are without significant difference.
(2) impact of tested material on immune two groups of Mouse Weights
Health product C prepared by embodiment 1 on the impact of immune two groups of Mouse Weights in table 2.
Table 2. tested material is on immune two groups of Mouse Weights impact
P value: each experimental group and negative control group comparison
From table 2, to learn and process by statistics, per os gives tested material C30 days, and each dosage group Mouse Weight, weightening finish and negative control group comparing difference are without significance (P > 0.05), and tested material has no significant effect Mouse Weight, weightening finish.
The impact of health product A, B, D and E prepared by embodiment 1 on immune two groups of Mouse Weights and table 2 are without significant difference.
(3) impact of tested material on immune three groups of Mouse Weights
Health product C prepared by embodiment 1 on the impact of immune three groups of Mouse Weights in table 3.
Table 3. tested material is on immune three groups of Mouse Weights impact
P value: each experimental group and negative control group comparison
From table 3, to learn and process by statistics, per os gives tested material C30 days, and between each dosage group Mouse Weight, weightening finish and negative control group, comparing difference is without significance (P > 0.05), and tested material has no significant effect Mouse Weight, weightening finish.
The impact of health product A, B, D and E prepared by embodiment 1 on immune three groups of Mouse Weights and table 3 are without significant difference.
(4) impact of tested material on mice organs/body weight ratio
Health product C prepared by embodiment 1 on the impact of mice organs/body weight ratio in table 4.
The impact of table 4. tested material on mice organs/body weight ratio
Spleen/body weight ratio P
1value: each experimental group and negative control group comparison;
Thymus/body weight ratio P
2value: each experimental group and negative control group comparison;
From table 4, per os gives the tested material C30 days of mice various dose, between each dosage group spleen/body weight ratio and thymus/body weight ratio and negative control group, comparing difference is without significance (P > 0.05), and tested material has no significant effect mice organs/body weight ratio.
The impact of health product A, B, D and E prepared by embodiment 1 on mice organs/body weight ratio and table 4 are without significant difference.
1.5.2 delayed allergy (the sufficient sole of the foot thickens method DTH)
Assay method: get Sanguis caprae seu ovis, normal saline washing, mice 2%(V/V) SRBC peritoneal immunity, every Mus injection 0.2mL/ is only.Latter 4 days of immunity, measures left back sufficient sole of the foot portion thickness, measuring point subcutaneous injection 20%(V/V) SRBC, only, after injection, 24h measures left back sufficient sole of the foot portion thickness three times to 20 μ L/, calculating mean value.
Result: the impact of tested material on mouse cell immunologic function
Health product C prepared by embodiment 1 on the impact of Mouse Weight and delayed allergy (DTH) in table 5.
The impact of table 5. tested material on Mouse Weight and delayed allergy (DTH)
P
1value: each experimental group and negative control group comparison.
P
2value: each experimental group and negative control comparison
*p < 0.05
From table 5, to learn and process by statistics, per os gives tested material C30 days, and between each dosage group Mouse Weight, weightening finish and negative control group, comparing difference is without significance (P > 0.05); Between each dosage group and negative control group, comparing difference all has significance (P < 0.05), and the tested material of each dosage all can strengthen the delayed allergy of mice.
The impact of health product A, B, D and E prepared by embodiment 1 on Mouse Weight and delayed allergy (DTH) and table 5 are without significant difference.
1.5.3ConA the mouse lymphocyte transformation experiment (mtt assay) of induction
The aseptic spleen of getting, grinds spleen to make single cell suspension in Hank ' s liquid, and Hank ' s liquid is washed 2 times, each centrifugal 10min(1000r/min).By cell suspension, in the complete culture solution of 1mL, the blue dyeing counting viable count of platform phenol (should more than 95%), adjusts cell concentration to 3 × 10
6individual/mL.Divide two holes to add in 24 well culture plates every part of cell suspension, every hole 1mL, a hole adds 75 μ L ConA liquid (being equivalent to 7.5 μ g/mL), and 5%CO in contrast, is put in another hole
237 ℃ of CO
2in incubator, cultivate 72h.Cultivation finishes front 4h, and every hole sucks supernatant 0.7mL not containing the RPMI1640 culture fluid of calf serum, adds MTT(5mg/mL simultaneously) 50mL/ hole, continue to cultivate 4h.After cultivation finishes, every hole adds 1mL acid isopropyl alcohol, and piping and druming mixes, and purple crystal is dissolved completely, determines OD afterwards
570nm.
Result:
The impact of health product C prepared by the embodiment 1 mouse lymphocyte transformation experiment on ConA induction is in table 6.
The impact of the mouse lymphocyte transformation experiment of table 6. tested material on ConA induction
P value: each experimental group and negative control group comparison
From table 6, per os gives the tested material C30 days of mice various dose, learn and process by statistics, between the lymphocytic multiplication capacity of high dose group and negative control group, comparing difference has significance (P < 0.05), and tested material can strengthen mouse lymphocyte increment, conversion capability.
The impact of health product A, B, D and E prepared by the embodiment 1 mouse lymphocyte transformation experiment on ConA induction and table 6 are without significant difference.
1.5.4 antibody-producting cell detects (Jerne improves slide method)
Sanguis caprae seu ovis is put into the sterilizing conical flask of bead, towards a direction shake, with defiber, 4 ℃ of Refrigerator stores (can preserve 2 weeks) for subsequent use.Hematocrit SRBC is made into 2%(V/V with normal saline) cell suspension, Mus lumbar injection 0.2mL/ is only.Mice dislocation by SRBC immunity after 4-5 days is put to death, get spleen, put into Hank ' s liquid, spleen is ground and makes cell suspension, filtered through gauze, Hank ' s liquid is washed 2 times, centrifugal (1000r/min) 10min at every turn, after splenocyte suspension is added in RPMI1640 culture fluid, counting cells, cell concentration is adjusted to 5 × 10
6individual/mL.After agarose heating for dissolving, 45-50 ℃ of water bath heat preservation, mixes subpackage small test tube with Hank ' the s liquid of equivalent pH7.2-7.42 times concentration, every pipe 0.5mL, in pipe, add 10%(V/V again, with the preparation of SA liquid) hematocrit SRBC 50 μ L, splenocyte suspension 20 μ L, after mixing, be poured on the slide that is brushed with agarose thin layer, do parallel plate, after agar solidifies, slide level is buckled and is placed on horse, CO
2incubation 1.5h in incubator, the rear complement (1:8) with the dilution of SA buffer joins in slide frame groove, continues after incubation 1.5h counting hemolysis plaque number.
Result: the impact of tested material on humoral immunization
Health product C prepared by embodiment 1 on the impact of mouse antibodies cellulation number in table 7.
The impact of table 7. tested material on mouse antibodies cellulation number
P value: each experimental group and negative control group comparison
From table 7, per os gives the tested material C30 days of mice various dose, learn and process by statistics, high dose group antibody-producting cell number and negative control comparing difference have significance (P < 0.05), and tested material can promote the antibody-producting cell number increment of mice.
The impact of health product A, B, D and E prepared by embodiment 1 on mouse antibodies cellulation number and table 7 are without significant difference.
1.5.5 half hemolysis value (HC
50) mensuration
Getting Sanguis caprae seu ovis, normal saline washing 3 times, centrifugal (2000r/min) 10min at every turn, lumbar injection 2%(V/V, normal saline preparation) hematocrit SRBC 0.2mL/ only carries out immunity.After 4 days, get blood in centrifuge tube, place about 1h, solidification blood is peeled off in tube wall, serum is fully separated out, the centrifugal 10min of 2000r/min, collects serum.With SA buffer, by serum dilution (400 times), the serum 1mL after dilution puts in vitro, adds successively 10%(V/V) SRBC0.5mL, complement 1mL(presses 1:8 dilution with SA buffer).Separately establish the not control tube of increase serum (replacing with SA buffer).In 37 ℃ of waters bath with thermostatic control, be incubated 15-30min, ice bath cessation reaction.The centrifugal 10min of 2000r/min, gets supernatant 1mL, adds Dou Shi reagent 3mL.Get 10%(V/V simultaneously) SRBC0.25mL, add Dou Shi reagent 4mL, fully mix, place after 10min, make blank to contrast, measure respectively each pipe OD
540nm, the amount of hemolysin is with half hemolysis value (HC
50) represent, calculate:
HC
50optical density value × extension rate when=sample optical density value/SRBC HD50
Result: tested material is to mice half hemolysis value (HC
50) impact
Health product C prepared by embodiment 1 is to mice half hemolysis value (HC
50) impact in table 8.
Table 8. tested material is to mice half hemolysis value (HC
50) impact
P value: each experimental group and negative control group comparison
From table 8, per os gives the tested material C30 days of mice various dose, learn and process by statistics, between high dose group half hemolysis value and negative control group, comparing difference has significance (P < 0.05), and tested material C can improve mice serum hemolysin level.
Health product A, B, D and E prepared by embodiment 1 is to mice half hemolysis value (HC
50) impact and table 8 without significant difference.
1.5.6 mice carbon is cleaned up experiment
The india ink that mouse tail vein injection 1:4 doubly dilutes, timing immediately, injects after prepared Chinese ink 2,10min, gets blood 20 μ L respectively, and be added at once 2mL0.1%Na from angular vein clump
2cO
3in solution, with Na
2cO
3for contrast, measure OD
600nm.Put to death mice, get liver and spleen, weigh.Calculate phagocytic index a:
Result:
The impact that health product C prepared by embodiment 1 cleans up function to mouse monokaryon-macrophage carbon is in table 9.
The impact that table 9. tested material is cleaned up function to mouse monokaryon-macrophage carbon
P value: each experimental group and negative control group comparison
*p < 0.05.
From table 9, per os gives the tested material C30 days of mice various dose, learn and process by statistics, each dosage group and negative control group comparing difference all have significance (P < 0.05), and each dosage group tested material C all can improve mouse monokaryon-macrophage carbon and cleans up ability.
Health product A, B, D and E prepared by embodiment 1 is to mice half hemolysis value (HC
50) impact and table 9 without significant difference.
1.5.7 Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell experiment
Mouse peritoneal injection 20%(V/V prepares with normal saline) hematocrit chicken red blood cell (2000r/min, 10min) suspension 1mL, interval 30min, dislocation is put to death, get peritoneal macrophage washing liquid 1mL, drip on microscope slide 37 ℃ of incubator incubation 20min, rinsing in normal saline, to remove not paster cell.Dry, methanol is fixed, 4%(V/V) dyeing of Giemsa-phosphate buffer, distilled water rinsing is dried.Under oil mirror, count, 100 macrophages of every counting, calculate phagocytic rate and phagocytic index:
Phagocytic rate %=engulfs macrophage number × 100 of the macrophage number/counting of chicken red blood cell
The macrophage number of the chicken red blood cell sum/counting of phagocytic index=engulfed
Result:
The impact of health product C prepared by embodiment 1 ability on Mouse Weight and macrophage phagocytic chicken red blood cell is in table 10.
The impact of the ability of table 10. tested material on Mouse Weight and macrophage phagocytic chicken red blood cell
P
1value: each experimental group and negative control group comparison
Phagocytic rate (%) P
2value: each experimental group and negative control group comparison
Phagocytic index P
3value: each experimental group and negative control group comparison
From table 10, to learn and process by statistics, per os gives tested material C30 days, and each dosage group Mouse Weight, weightening finish and negative control group comparing difference are without significance (P > 0.05); High dose group phagocytic rate and negative control group comparing difference have significance (P < 0.05); High dose group phagocytic index and negative control group comparing difference have significance (P < 0.05), and tested material has the ability that promotes that mouse macrophage is engulfed chicken red blood cell.
The impact of health product A, B, D and E prepared by embodiment 1 ability on Mouse Weight and macrophage phagocytic chicken red blood cell and table 10 are without significant difference.
1.5.8NK cytoactive detection (lactate dehydrogenase L DH algoscopy)
Before experiment, 24h goes down to posterity and cultivates target cell YAC-1, before application, washes 3 times with Hank ' s liquid, adjusts cell concentration to 4 × 10 containing the RPMI1640 complete culture solution of 10% calf serum
5individual/mL.Tested mice dislocation is put to death, and gets spleen, makes splenocyte suspension, Hank ' s liquid is washed 3 times, and each centrifugal 10min of 1500r/min is resuspended containing the RPMI1640 complete culture solution of 10% calf serum with 2mL, the blue dyeing counting of platform phenol (viable count should more than 95%), adjusts cell concentration to 2 × 10
7individual/mL, makes to imitate target than being 50:1.Get the each 100 μ L of target cell and effector lymphocyte, add in U-shaped 96 well culture plates; Target cell Spontaneous release hole adds target cell and a culture fluid 100 μ L, and the maximum release aperture of target cell adds target cell and the each 100 μ L of 1%NP40; Above-mentioned every three parallel holes, 37 ℃, 5%CO of all establishing
2in incubator, cultivate 4h, by 96 orifice plates, with the centrifugal 5min of 1500r/min, every hole is drawn supernatant 100 μ L and is put in ELISA Plate, adds LDH substrate liquid 100 μ L, reaction 10min, and then every hole adds the HCl solution 30 μ L cessation reactions of 1mol, measures OD
490nm, calculate NK activity:
NK cytoactive %=(reacting hole OD – Spontaneous release hole OD)/(maximum release aperture OD-Spontaneous release hole OD) × 100%
The preparation of LDH substrate liquid: sodium lactate 5 × 10
-2mol/L
Nitro tetrazolium chloride 6.6 × 10
-4mol/L
PMS 2.8 × 10
-4mol/L
Oxidized coenzyme I 1.3 × 10
-3mol/L
Mentioned reagent is dissolved in the Tris-HCl buffer of 0.2mol/L (pH8.2).
Result:
Health product C prepared by embodiment 1 on the impact of NK cells in mice activity in table 11.
The impact of table 11. tested material on NK cells in mice activity
P value: each experimental group and negative control group comparison
From table 11, per os gives the tested material C30 days of mice various dose, learn and process by statistics, each dosage NK cytoactive and negative control group comparing difference are without significance (P > 0.05), and tested material has no significant effect NK cells in mice activity.
The impact of health product A, B, D and E prepared by embodiment 1 on NK cells in mice activity and table 11 are without significant difference.
2. brief summary
Per os gives the tested material 30 days of mice various dose, can strengthen mice delayed allergy, can improve the carbon of mouse monokaryon-macrophage and clean up ability, can promote the lymphocyte proliferation ability of mice; Can promote the antibody-producting cell increment of mice; Can improve the serum hemolysin of mice; Can improve mice carbon single and macrophage and clean up ability; Promote macrophage phagocytosis of mice; On weight of mice, internal organs/body weight ratio, NK cytoactive without impact.Judge thus, health product prepared by the embodiment of the present invention 1 have the function of enhancing immunity.
Health product prepared by embodiment 3, the embodiment of the present invention 1 functional evaluation for protecting the liver
1 materials and methods
1.1 samples: health product A, B, C, D and E that above-described embodiment 1 prepares, people intend with dosage be 2.7g/60kgBW day.
1.2 laboratory animals: SPF level male mice in kunming, 18~22 grams of body weight, are provided by Inst. of Hygienics and Environmental Medical Science, Academy of Military Medici.The quality certification number is SCXK-(army) 2009-0030001082.
1.3 experimental animal feeding environment: SPF level experimental animal room, 20~25 ℃ of temperature, relative humidity 40~70%RH, the quality certification number: SYXK (Tianjin) 2008-0004.Feedstuff is provided by Tianjin Huarong Animal Science company limited, Feed Manufacturing credit number: SCXK(Tianjin) 2012-0001.
1.4 key instruments: TU-1810 ultraviolet-uisible spectrophotometer, TOSHIBA TBA-40FR automatic clinical chemistry analyzer; CM1900 freezing microtome (Leca), Olympus company microscope.
1.5 main agents: dehydrated alcohol (analytical pure) by Li Anlongbohua (Tianjin) medical chemistry company limited provide, malonaldehyde (MDA) test kit, reduced glutathion (GSH) test kit, histone measure test kit (biuret method) and build up Bioengineering Research Institute by Nanjing and provide; Triglyceride (TG) reagent is provided by Beijing Jiuqiang Biotechnology Co., Ltd..
1.6 experimental techniques: adopt alcoholic liver injury model, divide high, medium and low three dosage groups by experiment mice, a blank group and a model control group, three dosage group gavages every day give tested material, and dosage is respectively 0.15g/kgBW, 0.3g/kgBW, 0.6g/kgBW(is equivalent to people and intends 5,10,20 times with dosage).Take respectively tested material 1.5g, 3g, 6g, each adding distil water is to 200ml, gavage amount 0.2ml/10gBW, and blank group and model control group give equivalent distilled water.Every day 1 time, carry out continuously 30 days.Experiment gives 50% ethanol (with distilled water diluting) 0.14ml/10gBW by model control group and gavage of three dosage groups while end.After fasting 16h, put to death all animals and get liver, carry out every biochemical indicator detection and histopathological examination.
1.7 detect index
1.7.1 liver organization biochemical indicator detects
Get liver 0.3g and add normal saline 6ml, fully grind and make 5% liver homogenate.The method that in liver homogenate, the mensuration of lipid peroxide catabolite malonaldehyde (MDA), reduced glutathion (GSH), triglyceride (TG) and histone content all adopts test kit to provide is measured.
1.7.2 pathology of hepar inspection
1.7.2.1 pathological observation material: do from animal leftlobe of liver middle part to draw materials in cross section, frozen section, oil red dyeing, Microscopic observation fat drop in distribution, scope and area liver.
1.7.2.2 pathological observation method: 70 visuals field of 40 times of object lens continuous records, each visual field root for every routine animal liver tissue
How many according to positive cell (hepatocyte of dripping containing fat) and distribute scopes, marks by 0,1,2,3,4 point, the fat stains scoring using the meansigma methods of 70 visual field obatained scores as this example hepatic tissue.
1.7.2.3 pathological diagnosis standard: histopathology is observed using hepatocyte fat stains as observation index, and quantize according to pathological change degree " 0 ", " 1 ", " 2 ", " 3 ", " 4 ", carry out the evaluation of hepatic injury degree.Hepatocyte fat stains is divided into Pyatyi:
1.8 experimental data statistics: experimental data statistics adopts the processing of SPSS11.5FOR WINDOWS software kit.The data of model control group and dosage group are through homogeneity test of variance, and variance is neat, carry out variance analysis, as P value is less than 0.05, compare between two by LSD method; If heterogeneity of variance, carries out data transaction, still uneven, use rank test instead, as P value is less than 0.05, use Dunnett ' s T3 (Wilcoxon) method to compare between two.Blank group and model control group adopt T check.
2 results
The impact of 2.1 tested materials on Mouse Weight:
Health product C prepared by embodiment 1 on the impact of Mouse Weight in Table 2-1.
The impact (g, means standard deviation) of table 2-1. tested material on Mouse Weight
From table 2-1, with the tested material C gavage mice of various dose 30 days, each treated animal growth, movable normal.Each treated animal weightening finish and model control group comparison, difference not statistically significant (P>0.05).
Health product A, B, D and E prepared by embodiment 1 is on the impact of Mouse Weight and show 2-1 without significant difference.
The impact of 2.2 tested materials on MDA, GSH, TG content:
Health product C prepared by embodiment 1 on the impact of MDA, GSH, TG content in Table 2-2.
The impact (means standard deviation) of table 2-2. tested material on MDA, GSH, TG content
*with model control group comparison, difference has statistical significance (P<0.05)
From table 2-2, model control group and the comparison of blank group, MDA, TG be higher than matched group, and GSH is lower than matched group, and difference all has statistical significance (P<0.05), shows that model is successfully established.With the tested material C gavage mice of various dose 30 days, in middle and high dosage group liver homogenate, MDA concentration and TG concentration were all lower than model control group, and difference all has statistical significance (P<0.05).
Health product A, B, D and E prepared by embodiment 1 is on the impact of MDA, GSH, TG content and show 2-2 without significant difference.
2.3 pathology of hepar check results:
Health product C prepared by embodiment 1 to mouse liver steatosis appraisal result in Table 2-3.
Table 2-3. liver fat degeneration appraisal result (means standard deviation)
*with model control group comparison, difference has statistical significance (P<0.05)
From table 2-3, model control group steatosis oil red O stain is marked higher than blank group, and blank group and model control group steatosis oil red O stain diversity of values have statistical significance (P<0.05), show that model is successfully established.With the tested material C gavage mice of various dose 30 days, the steatosis oil red O stain of middle and high dosage group was marked all lower than model control group, and difference has statistical significance (P<0.05).
Health product A, B, D and E prepared by embodiment 1 is to mouse liver steatosis appraisal result and show 2-3 without significant difference.
3 brief summaries
With the tested material gavage mice of 0.23g/kgBW, 0.45g/kgBW, 0.90g/kgBW dosage 30 days, each treated animal vegetative activity is good, each treated animal increases weight compared with model control group, difference not statistically significant (P>0.05); In middle and high dosage group liver homogenate, MDA concentration and TG concentration are all lower than model control group; The steatosis of middle and high dosage group is marked lower than model control group, and difference has statistical significance (P<0.05).According to " health food check and assessment technique standard " (version in 2003) auxiliary protection chemical liver injury function result criterion, health product prepared by prompting the embodiment of the present application 1 have animal alcoholic liver injury auxiliary protection function function.
Claims (8)
1. there is enhancing immunity and protect the liver the health product of function, it is characterized in that: the active component of described health product is made up of the raw material of following weight portion: 2 parts-13 parts of 15 parts-60 parts of curcumins, 60 parts-240 parts of Ganoderma extracts, 255 parts-500 parts of Ganoderma spore powder with cellular wall brokens and yeast rich in seleniums.
2. the health product that have enhancing immunity and protect the liver function according to claim 1, is characterized in that: the raw material of described weight portion is: 6 parts-10 parts of 5 parts-50 parts of bisdemethoxycurcumins, 60 parts-185 parts of Ganoderma extracts, 225 parts-360 parts of Ganoderma spore powder with cellular wall brokens and yeast rich in seleniums.
3. the health product that have enhancing immunity and protect the liver function according to claim 1 and 2, is characterized in that: described health product also comprise pharmaceutically acceptable adjuvant or complementary composition.
4. the health product that have enhancing immunity and protect the liver function according to claim 3, is characterized in that: described Ganoderma extract is Ganoderma water extract or Ganoderma extractive with organic solvent; Described curcumin is Rhizoma Curcumae Longae water extract or Rhizoma Curcumae Longae extractive with organic solvent.
5. the health product that have enhancing immunity and protect the liver function according to claim 3, is characterized in that: described pharmaceutically acceptable adjuvant or complementary composition are microcrystalline Cellulose and magnesium stearate.
6. the health product that have enhancing immunity and protect the liver function according to claim 5, is characterized in that: the raw material of weight portion described in described health product is 15 parts-60 parts of curcumins, 60 parts-240 parts of Ganoderma extracts, 255 parts-500 parts of Ganoderma spore powder with cellular wall brokens, 2 parts-13 parts of yeast rich in seleniums; Described adjuvant or complementary composition and weight portion thereof are: 3 parts-6 parts of 81 parts-153 parts of microcrystalline Cellulose and magnesium stearate.
7. the health product that have enhancing immunity and protect the liver function according to claim 5, is characterized in that: described in described health product, the raw material of weight portion is: 5 parts-50 parts of bisdemethoxycurcumins, 60 parts-185 parts of Ganoderma extracts, 225 parts-360 parts of Ganoderma spore powder with cellular wall brokens, 6 parts-10 parts of yeast rich in seleniums; Described adjuvant or auxiliary element and weight portion thereof are: 3 parts-4 parts of 81 parts-106 parts of microcrystalline Cellulose and magnesium stearate.
In claim 1-7 arbitrary described health product that there is enhancing immunity and protect the liver function in the application of preparing in enhancing immunity and hepatic.
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