CN103800390B - 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, particularly relate to and there is enhancing immunity and the health product protecting the liver function and preparation method thereof and application.
Background technology
Immunity generally also can be resistance.The body immunity of usual theory be exactly health when being subject to external infringement, such as when 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, and first is immune defence, it to antigenic substance as: pathogenic microorganism, vaccine, toxoid, foreign protein etc. occur immunne response, final elimination antigenic substance, plays the effect of infection immunity.If during the defense function generation defect of body, very easily suffer from infectiousness or infectious disease; Second is immunological homeostasis, can remove damage, aging, dead cell, and effectively carry out immunoregulation effect, as from steady functional disorder, easily occur autoimmune disease; It three is immune surveillance functions, its major function shows that body is by the antigen on immunocyte identification cancerous tumor cell, and by special or non-specific killer cell by cancer cell destruction, namely emerging cancerous cell is eliminated before not causing a large amount of propagation, diffusion, and plays antineoplastic action.As immune surveillance mistake department, often easily there is tumor.Along with the progress of society, the quickening of people's rhythm of life, the change of living environment, and the increasing pressure of each side is large, causes the crowd of immunity degradation also to get more and more thus.So enhancing human body immunity power, safeguard healthy most important.
Liver is important substance metabolism organ, plays an important role in the absorption of the materials such as carbohydrate, lipid, protein, vitamin, hormone, bile, storage, biological conversion, secretion, excretion etc.There is removing toxic substances and phagocytic function simultaneously, participate in the synthesis of many thrombins in blood, participate in hematopoiesis, storage and release Hemopoietic factor; Or the synthetic organ of many enzymes, ensures the metabolism of human normal in human body.Ethanol and many chemical toxicants 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 downright bad, the fatty distortion of liver stem cell in various degree, liver cirrhosis, cholestasis, hepatic fibrosis, even hepatocarcinoma.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 comparatively large, and curative effect is also not obvious.And herbal toxic effect is little, there is the feature of Mutiple Targets, too many levels comprehensive function, in treatment hepatic injury, have original curative effect.
Along with the development subhealth state of society number ubiquity and have the trend increased gradually.Subhealth state is a kind of chronic disease, and except the improvement of life style, the health food taking enhancing immunity has become the common recognition of people.Hepatic injury is the disease of clinical common harm humans health, and except ethanol and chemical solvent, about have 500-1000 kind Western medicine to cause hepatic disease at present, serious hepatic disease mortality rate is very high.Modern medicine there is no specific drug in the treatment intervening hepatic injury, and adopt rest more, strengthen nutrition, vitimin supplement and symptomatic treatment etc., severe patient is even forced to discontinue medication.Chinese herbal medicine is through strict Processing methods, and toxic and side effects is very little.Certain effect has been achieved in treatment hepatic injury.
The Rhizoma Curcumae Longae extract crocus crystallization that to be the tuber of zingiberaceous plant Rhizoma Curcumae Longae or rhizome obtain through Hydrolysis kinetics or powder, its active component is primarily of curcumin, demethoxycurcumin, Bisdemethoxycurcumin composition.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 the effects such as antioxidation, infection, antiinflammatory, anticoagulant, blood fat reducing, atherosclerosis and Tumor suppression growth.Curcumin is widely used in food, the industry such as article of everyday use and pharmacy, and safety non-toxic, has no side effect, and is one of a kind of natural pigment having 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 and tie in the heart, the motive of hanging ", " enter the heart being responsible for production of blood, help the heart to fill arteries and veins ", " calming the nerves ", and " protecting god ", " lung benefiting, spleen, vital essence ", " tonifying liver gas " etc., all have 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 immunity moderation function, suppress the growth of real tumor body in Mice Body.Stimulate insulin secretion, promote the effects such as the synthesis 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.There is multiple pharmacologically active, as Ganodenic acid can kill tumor cell; The tumoricidal telomerase of polysaccharide, triterpenes, 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 mediating body panimmunity and inflammatory process.Improve the ability that cell synthesizes anti-tumor element I and II, strengthen the generation of interferon, the superoxide dismutase containing volume can remove rapidly Radiotherapy chemotherapy and the tired free radical that produces and active oxygen.And by stimulating lymphocytic propagation, improve the activity of immunologically competent cell and the factor, thus the growth stimulator that enhancing immunologic function comes antagonism and anticancer spreads and reaches anticancer object, Ganoderma spore also has eliminates the function that chemicals causes liver injury, prevents liver cirrhosis and angiosclerotic effect.
Yeast rich in selenium is exactly add selenium element in the process of culture yeasts, selenium has been absorbed during yeast growth, the protein of selenium in yeast body and polysaccharide organic are combined and are converted into biological selenium, thus eliminate chemical selenium (as sodium selenite) toxicity of human body and the intestines and stomach are stimulated, selenium more efficiently, is more safely absorbed by the body utilization.
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 having 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: curcumin 15 parts-60 parts, Ganoderma extract 60 parts-240 parts, Ganoderma spore powder with cellular wall broken 255 parts-500 parts and yeast rich in selenium 2 parts-13 parts.
The raw material of described weight portion is: bisdemethoxycurcumin 5 parts-50 parts, Ganoderma extract 60 parts-185 parts, Ganoderma spore powder with cellular wall broken 225 parts-360 parts and yeast rich in selenium 6 parts-10 parts.
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 curcumin 15 parts-60 parts, Ganoderma extract 60 parts-240 parts, Ganoderma spore powder with cellular wall broken 255 parts-500 parts, yeast rich in selenium 2 parts-13 parts; Described adjuvant or complementary composition and weight portion thereof are: microcrystalline Cellulose 81 parts-153 parts and magnesium stearate 3 parts-6 parts.
The raw material of weight portion described in described health product is: bisdemethoxycurcumin 5 parts-50 parts, Ganoderma extract 60 parts-185 parts, Ganoderma spore powder with cellular wall broken 225 parts-360 parts, yeast rich in selenium 6 parts-10 parts; Described adjuvant or auxiliary element and weight portion thereof are: microcrystalline Cellulose 81 parts-106 parts and magnesium stearate 3 parts-4 parts.
The described health product having enhancing immunity and protect the liver function also belong to protection scope of the present invention preparing the application 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 carbonic clearance ability of mouse monokaryon-macrophage, can promote the lymphocyte proliferation ability of mice; Can promote that the antibody-producting cell of mice rises in value; The serum hemolysin of mice can be improved; The carbonic clearance ability of mice list and macrophage can be improved; Promote macrophage phagocytosis of mice; On Mouse Weight growth, 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 30 days of 0.15g/kgBW, 0.3g/kgBW, 0.6g/kgBW dosage, each treated animal vegetative activity is good, each treated animal weightening finish compared with model control group, no significant difference (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 score of middle and high dosage group is lower than model control group, and difference has statistical significance (P<0.05).
Detailed description of the invention
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 from commercial channels and obtain.
The health product that this embodiment prepares are as follows:
The preparation method of the health product that this embodiment has enhancing immunity and protects the liver function is as follows:
Curcumin in this embodiment, Ganoderma extract, Ganoderma spore powder with cellular wall broken and yeast rich in selenium can prepare by following method, also can buy from commercial channels and obtain.
One, raw material preparation
1, curcumin is prepared
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 to be 7 simultaneously, with 80% ethanol with the speed eluting of 4mL/min, obtain concentrated solution; By concentrated solution through drying under reduced pressure, obtain dry extract; Dry extract is pulverized, and after crossing 80 mesh sieves, obtains extract fine powder, be Rhizoma Curcumae Longae extract, curcumin.
2, Ganoderma extract is prepared
Get Ganoderma crude drug, with the water extraction 2 times of 8 times of weight, each 2 hours, merge 2 aqueous extracts; At 60 DEG C, under-0.08MPa condition, aqueous extract is concentrated into relative density 1.09(60 DEG C), obtain concentrated solution; By concentrated solution through drying under reduced pressure (-0.09MPa), obtain dry extract; Dry extract is pulverized, and after crossing 80 mesh sieves, obtains extract fine powder, be Ganoderma extract.
3, Ganoderma spore powder with cellular wall broken is prepared
Ganoderma is through hair tube reason and go out sesame and manage, and then added bullet powder is gathered spore powder.The spore powder of newly gathering through super-dry, sieve, after multiple sieve, breaking cellular wall, airtight preservation.
4, yeast rich in selenium is prepared
Appropriate selenium compound is added for culture medium with molasses, barms is accessed after sterilization, add the nutritive salt such as nitrogen, phosphorus and carry out fermentation culture, temperature 30 DEG C, 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 repeatedly rinsing with water, namely obtain flaxen yeast rich in selenium powder through concentrated and spraying dry.
Two, production technology
1. batching, pre-treatment: get all the supplementary material conformed to quality requirements according to recipe requirements ready, checks outer package, removes qualified outer package, proceed in 300,000 grades of clean areas, for subsequent use.Supplementary material crosses 60 mesh sieves respectively, weighs, for subsequent use.
2. first yeast rich in selenium and microcrystalline Cellulose are pressed equal increments method mix homogeneously, obtain mixed powder I, mixed powder I mixes 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, soft material, with can be agglomerating and tack-free with hand-tight holding, splits for degree with the light pressure energy of finger.20 mesh sieve granules crossed by gained soft material, and wet granular 50-55 DEG C drying, is dried to pellet moisture≤5%.The dry granule of gained 20 mesh sieve granulate, obtain granule.
4. always mixed, fill, polishing: gained granule mixes 10 minutes with magnesium stearate, makes evenly, to obtain and always mix material.Total mixed material filled capsules.Populated capsule polishing, checks and removes inferior capsule, obtain polishing capsule.
The health product of embodiment 2, the embodiment of the present invention 1 preparation are used 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 cleaning grade ICR male mice that Jilin University Bethune medical college animal experimental center provides, production licence number: SCXK-(is lucky) 2007-0003.Feedstuff is provided by this laboratory animal technology Co., Ltd of Changchun hundred million, and production licence number: SCXK-(is lucky) 2010-0001.This laboratory animal environmental facility quality certification, lucky moving establishes word 10-1005, and laboratory animal occupancy permit number: SYXK-(is lucky) 2010-0011.Select healthy male mice 48, body weight 18g-22g, be divided into 4 groups, often organize 12, as immune one group, carry out liver/weight ratio pH-value determination pH, half hemolysis value (HC
50) mensuration and antibody-producting cell detect; Select healthy male mice 48, body weight 18g-22g, be divided into 4 groups, often organize 12, as immune two groups, carry out carbonic clearance experiment; Select healthy male mice 48, body weight 18g-22g, be divided into 4 groups, often organize 12, as immune three groups, carry out mouse lymphocyte transformation experiment and the NK cytoactive detection of ConA induction; Select healthy male mice 48, body weight 18g-22g, be divided into 4 groups, often organize 12, as immune four groups, carry out delayed allergy test; Select healthy male mice 48, body weight 18g-22g, be divided into 4 groups, often organize 12, as immune five groups, carry out Turnover of Mouse Peritoneal Macrophages and engulf chicken red blood cell experiment.
1.3 dosage choice and preparation
Composition capsule 1.8g/ day, that is: 1.8g/60kg BW, be equivalent to 0.03g/kg BW, arranges given low with 1,10 and 30 of intake of being grown up times, namely 0.03,0.30,0.90g/kg BW, each dosage group is with distilled water diluting.Make negative control group with pure water, each group mouse stomach amount is 0.2mL/10g BW, and continuous gavage surveyed every immune indexes after 30 days.
Sample thief 4.50g adds pure water and is high dose group tested material to 100mL mixing standardize solution; Sample thief 1.5g adds pure water and is middle dosage group tested material to 100mL mixing standardize solution; Sample thief 0.15g adds pure water and is low dose group tested material to 100mL mixing standardize solution.Each group of tested material Fresh, 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., sterile surgical instrument, 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: after mouse weights, dislocation is put to death, and gets spleen and thymus, removes most fascia, blot internal organs and show blood stains, weigh with filter paper, calculates spleen/body weight ratio and thymus/body weight ratio.
Result:
(1) tested material is on the impact of immune one group of Mouse Weight
Embodiment 1 prepare health product C 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, through statistical procedures, 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 namely tested material has no significant effect Mouse Weight, weightening finish.
Health product A, B, D and E prepared by embodiment 1 on the impact of immune one group of Mouse Weight and table 1 without significant difference.
(2) tested material is on the impact of immune two groups of Mouse Weights
Embodiment 1 prepare health product C 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 compares with negative control group
From table 2, through statistical procedures, 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 namely tested material has no significant effect Mouse Weight, weightening finish.
Health product A, B, D and E prepared by embodiment 1 on the impact of immune two groups of Mouse Weights and table 2 without significant difference.
(3) tested material is on the impact of immune three groups of Mouse Weights
Embodiment 1 prepare health product C 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 compares with negative control group
From table 3, through statistical procedures, per os gives tested material C30 days, each dosage group Mouse Weight, between weightening finish and negative control group comparing difference without significance (P > 0.05), namely tested material to Mouse Weight, weightening finish have no significant effect.
Health product A, B, D and E prepared by embodiment 1 on the impact of immune three groups of Mouse Weights and table 3 without significant difference.
(4) tested material is on the impact of mice organs/body weight ratio
Embodiment 1 prepare health product C on the impact of mice organs/body weight ratio in table 4.
Table 4. tested material is on the impact of mice organs/body weight ratio
Spleen/body weight ratio P
1value: each experimental group compares with negative control group;
Thymus/body weight ratio P
2value: each experimental group compares with negative control group;
From table 4, per os gives the tested material C30 days of mice various dose, each dosage group spleen/body weight ratio and thymus/between body weight ratio and negative control group, comparing difference is without significance (P > 0.05), namely tested material has no significant effect mice organs/body weight ratio.
Health product A, B, D and E prepared by embodiment 1 on the impact of mice organs/body weight ratio and table 4 without significant difference.
1.5.2 delayed allergy (the sufficient sole of the foot thickens method DTH)
Assay method: get Sanguis caprae seu ovis, brine, 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, 20 μ L/, after injection, 24h measures left back sufficient sole of the foot portion thickness three times, calculating mean value.
Result: tested material is on the impact of mouse cell immunologic function
Health product C prepared by embodiment 1 on the impact of Mouse Weight and delayed allergy (DTH) in table 5.
Table 5. tested material is on the impact of Mouse Weight and delayed allergy (DTH)
P
1value: each experimental group compares with negative control group.
P
2value: each experimental group compares with negative control
*p < 0.05
From table 5, through statistical procedures, per os gives tested material C30 days, each dosage group Mouse Weight, between weightening finish and negative control group comparing difference without significance (P > 0.05); Between each dosage group and negative control group, comparing difference all has significance (P < 0.05), and namely the tested material of each dosage all can strengthen the delayed allergy of mice.
Embodiment 1 health product A, B, D and E of preparing on the impact of Mouse Weight and delayed allergy (DTH) and table 5 without significant difference.
The mouse lymphocyte transformation experiment (mtt assay) of 1.5.3ConA inducing
Asepticly get spleen, ground by spleen in Hank ' s liquid and make single cell suspension, Hank ' s liquid washes 2 times, each centrifugal 10min(1000r/min).By the complete culture solution of cell suspension in 1mL, the blue dyeing counting viable count of platform phenol (should more than 95%), adjustment cell concentration to 3 × 10
6individual/mL.Add in 24 well culture plates by every part of cell suspension point holes, 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 DEG C of CO
272h is cultivated in incubator.Cultivation terminates 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 terminates, every hole adds 1mL acid isopropyl alcohol, and piping and druming mixing, makes purple crystal dissolve completely, determine OD afterwards
570nm.
Result:
Embodiment 1 prepare health product C on the impact of the mouse lymphocyte transformation experiment that ConA induces in table 6.
Table 6. tested material is on the impact of the mouse lymphocyte transformation experiment that ConA induces
P value: each experimental group compares with negative control group
From table 6, per os gives the tested material C30 days of mice various dose, through statistical procedures, between the lymphocytic multiplication capacity of high dose group and negative control group, comparing difference has significance (P < 0.05), and namely tested material can strengthen mouse lymphocyte increment, conversion capability.
Health product A, B, D and E prepared by embodiment 1 on the impact of the mouse lymphocyte transformation experiment that ConA induces and table 6 without significant difference.
1.5.4 antibody-producting cell detects (Jerne improves slide method)
Sanguis caprae seu ovis is placed with in the sterilizing conical flask of bead, towards a direction shake, with defiber, 4 DEG C of Refrigerator stores (2 weeks can be preserved) for subsequent use.Hematocrit SRBC is made into 2%(V/V with normal saline) cell suspension, Mus lumbar injection 0.2mL/ is only.The mice dislocation of SRBC immunity after 4-5 days is put to death, get spleen, put into Hank ' s liquid, ground by spleen and make cell suspension, filtered through gauze, Hank ' s liquid washes 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 DEG C of water bath heat preservation, mixes with Hank ' the s liquid of equivalent pH7.2-7.42 times of concentration, subpackage small test tube, often pipe 0.5mL, in pipe, add 10%(V/V again, with SA liquid preparation) hematocrit SRBC 50 μ L, splenocyte suspension 20 μ L, be poured into after mixing be brushed with agarose thin layer slide on, do parallel plate, after agar solidification, slide level is buckled and is placed on horse, CO
2incubation 1.5h in incubator, the complement (1:8) of rear SA buffer dilution joins in slide frame groove, after continuing incubation 1.5h, counting hemolysis plaque number.
Result: tested material is on the impact of humoral immunization
Embodiment 1 prepare health product C on the impact of mouse antibodies cellulation number in table 7.
Table 7. tested material is on the impact of mouse antibodies cellulation number
P value: each experimental group compares with negative control group
From table 7, per os gives the tested material C30 days of mice various dose, through statistical procedures, high dose group antibody-producting cell number and negative control comparing difference have significance (P < 0.05), and namely tested material can promote that the antibody-producting cell number of mice rises in value.
Health product A, B, D and E prepared by embodiment 1 on the impact of mouse antibodies cellulation number and table 7 without significant difference.
1.5.5 half hemolysis value (HC
50) mensuration
Getting Sanguis caprae seu ovis, brine 3 times, at every turn centrifugal (2000r/min) 10min, lumbar injection 2%(V/V, normal saline) 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, collect serum.With SA buffer by serum-dilution (400 times), the serum 1mL after dilution puts in vitro, adds 10%(V/V successively) SRBC0.5mL, complement 1mL(with SA buffer press 1:8 dilution).Separately establish the control tube of not increase serum (replacing with SA buffer).15-30min is incubated, ice bath cessation reaction in 37 DEG C of waters bath with thermostatic control.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, after placing 10min, make blank to contrast, measure each pipe OD respectively
540nm, the amount of hemolysin is with half hemolysis value (HC
50) represent, calculate:
HC
50optical density value × extension rate during=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 compares with negative control group
From table 8, per os gives the tested material C30 days of mice various dose, through statistical procedures, between high dose group half hemolysis value and negative control group, comparing difference has significance (P < 0.05), and namely tested material C can improve mice serum hemolysin level.
Health product A, B, D and E prepared by embodiment 1 are to mice half hemolysis value (HC
50) impact and table 8 without significant difference.
1.5.6 mice carbonic clearance is tested
The india ink that mouse tail vein injection 1:4 doubly dilutes, timing immediately, to inject after prepared Chinese ink 2,10min, gets blood 20 μ L respectively, and be added to 2mL0.1%Na at once 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:
k=(lgOD1-lgOD2)/(t2-t1)
Result:
Embodiment 1 prepare health product C on the impact of mouse monokaryon-macrophage carbonic clearance function in table 9.
Table 9. tested material is on the impact of mouse monokaryon-macrophage carbonic clearance function
P value: each experimental group compares with negative control group
*p < 0.05.
From table 9, per os gives the tested material C30 days of mice various dose, through statistical procedures, each dosage group and negative control group comparing difference all have significance (P < 0.05), and namely each dosage group tested material C all can improve mouse monokaryon-macrophage carbonic clearance ability.
Health product A, B, D and E prepared by embodiment 1 are to mice half hemolysis value (HC
50) impact and table 9 without significant difference.
1.5.7 Turnover of Mouse Peritoneal Macrophages engulfs chicken red blood cell experiment
Mouse peritoneal injection 20%(V/V 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 DEG C of incubator incubation 20min, rinsing in normal saline, to remove non-paster cell.Dry, methanol is fixed, 4%(V/V) dyeing of Giemsa-phosphate buffer, distilled water rinsing is dried.Count under oil mirror, every sheet counts 100 macrophages, calculates phagocytic rate and phagocytic index:
Phagocytic rate %=engulfs macrophage number × 100 of the macrophage number/counting of chicken red blood cell
Phagocytic index=by engulf chicken red blood cell sum/counting macrophage number
Result:
Health product C prepared by embodiment 1 on the impact of the ability of Mouse Weight and macrophage phagocytic chicken red blood cell in table 10.
Table 10. tested material is on the impact of the ability of Mouse Weight and macrophage phagocytic chicken red blood cell
P
1value: each experimental group compares with negative control group
Phagocytic rate (%) P
2value: each experimental group compares with negative control group
Phagocytic index P
3value: each experimental group compares with negative control group
From table 10, through statistical procedures, 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 namely tested material has the ability that promotion mouse macrophage engulfs chicken red blood cell.
Embodiment 1 health product A, B, D and E of preparing on the impact of the ability of Mouse Weight and macrophage phagocytic chicken red blood cell and table 10 without significant difference.
1.5.8NK cytoactive detection (lactate dehydrogenase L DH algoscopy)
24h Secondary Culture target cell YAC-1 before experiment, washes 3 times with Hank ' s liquid before application, containing RPMI1640 complete culture solution adjustment cell concentration to 4 × 10 of 10% calf serum
5individual/mL.Test mice dislocation is put to death, and gets spleen, makes splenocyte suspension, Hank ' s liquid washes 3 times, the centrifugal 10min of each 1500r/min, 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%), adjustment cell concentration to 2 × 10
7individual/mL, makes effect target than being 50:1.Get 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 a culture fluid 100 μ L, and the maximum release aperture of target cell adds target cell and each 100 μ L of 1%NP40; Above-mentionedly everyly all establish three parallel holes, 37 DEG C, 5%CO
2cultivate 4h in incubator, 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 matrix liquid 100 μ L, reaction 10min, and then every hole adds the HCl solution 30 μ L cessation reaction of 1mol, measures OD
490nm, calculate NK active:
NK cytoactive %=(reacting hole OD – Spontaneous release hole OD)/(maximum release aperture OD-Spontaneous release hole OD) × 100%
The preparation of LDH matrix 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 (pH8.2) in the Tris-HCl buffer of 0.2mol/L.
Result:
Embodiment 1 prepare health product C on the impact of NK cells in mice activity in table 11.
Table 11. tested material is on the impact of NK cells in mice activity
P value: each experimental group compares with negative control group
From table 11, per os gives the tested material C30 days of mice various dose, through statistical procedures, each dosage NK cytoactive and negative control group comparing difference are without significance (P > 0.05), and namely tested material has no significant effect NK cells in mice activity.
Health product A, B, D and E prepared by embodiment 1 on the impact of NK cells in mice activity and table 11 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 carbonic clearance ability of mouse monokaryon-macrophage, can promote the lymphocyte proliferation ability of mice; Can promote that the antibody-producting cell of mice rises in value; The serum hemolysin of mice can be improved; The carbonic clearance ability of mice list and macrophage can be improved; Promote macrophage phagocytosis of mice; On Mouse Weight growth, 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.
The functional evaluation of health product for protecting the liver of embodiment 3, the embodiment of the present invention 1 preparation
1 materials and methods
1.1 samples: health product A, B, C, D and E that above-described embodiment 1 prepares, people's plan dosage is 2.7g/60kgBW day.
1.2 laboratory animals: SPF level male mice in kunming, body weight 18 ~ 22 grams, is 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, temperature 20 ~ 25 DEG C, 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) provides by Li Anlongbohua (Tianjin) medical chemistry company limited, malonaldehyde (MDA) test kit, reduced glutathion (GSH) test kit, histone mensuration test kit (biuret method) build up Bioengineering Research Institute by Nanjing provides; Triglyceride (TG) reagent is provided by Beijing Jiuqiang Biotechnology Co., Ltd..
1.6 experimental techniques: adopt alcoholic liver injury model, experiment mice is divided high, medium and low three dosage groups, a blank group and a model control group, three dosage group gavages every day give tested material, and dosage is respectively 5,10,20 times that 0.15g/kgBW, 0.3g/kgBW, 0.6g/kgBW(are equivalent to people's plan dosage).Take tested material 1.5g, 3g, 6g respectively, each adding distil water is to 200ml, and gavage amount 0.2ml/10gBW, blank group and model control group give equivalent distilled water.Every day 1 time, carry out 30 days continuously.50% ethanol (with distilled water diluting) 0.14ml/10gBW is given by model control group and three dosage groups gavage at the end of experiment.After fasting 16h, put to death all animals and get liver, carry out every biochemical indicator detection and histopathological examination.
1.7 Testing index
1.7.1 liver organization biochemical indicator detects
Get liver 0.3g and add normal saline 6ml, fully 5% liver homogenate is made in grinding.The method that in liver homogenate, the mensuration of lipid peroxide catabolite malonaldehyde (MDA), reduced glutathion (GSH), triglyceride (TG) and tissue protein content all adopts test kit to provide measures.
1.7.2 pathology of hepar inspection
1.7.2.1 pathological observation material: do in the middle part of animal leftlobe of liver to draw materials in cross section, the dyeing of frozen section, oil red, Microscopic observation fat drop in distribution liver, scope and area.
1.7.2.2 pathological observation method: every routine animal liver tissue 40 times of object lens record 70 visuals field continuously, each visual field root
How many according to positive cell (hepatocyte containing fat drips) and distribute scopes, marks by 0,1,2,3,4 point, the fat stains of the meansigma methods of 70 visual field obatained scores as this routine hepatic tissue is marked.
1.7.2.3 pathological diagnosis standard: pathologic examination using hepatocyte fat stains as observation index, and quantizes according to pathological change degree " 0 ", " 1 ", " 2 ", " 3 ", " 4 ", carries out the evaluation of hepatic injury degree.Hepatocyte fat stains is divided into Pyatyi:
1.8 experimental data statistics: experimental data statistics adopts the process 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, carries out variance analysis, as P value is less than 0.05, then compares between two by LSD method; If heterogeneity of variance, then carry out data conversion, still uneven, use rank test instead, as P value is less than 0.05, then use Dunnett ' s T3 (Wilcoxon) method to compare between two.Blank group and model control group then adopt T to check.
2 results
2.1 tested materials are on the impact of Mouse Weight:
Embodiment 1 prepare health product C on the impact of Mouse Weight in Table 2-1.
Table 2-1. tested material is on the impact (g, means standard deviation) of Mouse Weight
From table 2-1, with the tested material C gavage mice 30 days of various dose, each treated animal growth, normally movable.Each treated animal weightening finish is compared with model control group, no significant difference (P>0.05).
Health product A, B, D and E of embodiment 1 preparation are on the impact of Mouse Weight and show 2-1 without significant difference.
2.2 tested materials are on the impact of MDA, GSH, TG content:
Embodiment 1 prepare health product C on the impact of MDA, GSH, TG content in Table 2-2.
Table 2-2. tested material is on the impact (means standard deviation) of MDA, GSH, TG content
*compare with model control group, difference has statistical significance (P<0.05)
From table 2-2, model control group compares with blank group, and MDA, TG are 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 30 days of various dose, in middle and high dosage group liver homogenate, MDA concentration and TG concentration are all lower than model control group, and difference all has statistical significance (P<0.05).
Health product A, B, D and E of embodiment 1 preparation are 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 the results are shown in Table 2-3 to mouse liver Steatosis score.
Table 2-3. hepatic steatosis appraisal result (means standard deviation)
*compare with model control group, difference has statistical significance (P<0.05)
From table 2-3, the scoring of model control group steatosis oil red O stain is 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 30 days of various dose, the steatosis oil red O stain scoring of middle and high dosage group is all lower than model control group, and difference has statistical significance (P<0.05).
Health product A, B, D and E of embodiment 1 preparation are to mouse liver Steatosis score result and show 2-3 without significant difference.
3 brief summaries
With the tested material gavage mice 30 days of 0.23g/kgBW, 0.45g/kgBW, 0.90g/kgBW dosage, each treated animal vegetative activity is good, each treated animal increases weight compared with model control group, no significant difference (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 score of middle and high dosage group is lower than model control group, and difference has statistical significance (P<0.05).According to the criterion of " health food inspection and assessment technical specification " (version in 2003) auxiliary protection chemical liver injury function result, health product prepared by prompting the embodiment of the present application 1 have animal alcoholic liver injury auxiliary protection function function.
Claims (6)
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: curcumin 45 parts-60 parts, Ganoderma extract 60 parts-120 parts, Ganoderma spore powder with cellular wall broken 255 parts-500 parts and yeast rich in selenium 6 parts-13 parts.
2. the health product having enhancing immunity and protect the liver function according to claim 1, is characterized in that: described health product also comprise pharmaceutically acceptable adjuvant or complementary composition.
3. the health product having enhancing immunity and protect the liver function according to claim 2, 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.
4. the health product having enhancing immunity and protect the liver function according to claim 2, is characterized in that: described pharmaceutically acceptable adjuvant or complementary composition are microcrystalline Cellulose and magnesium stearate.
5. the health product having enhancing immunity and protect the liver function according to claim 4, is characterized in that: the raw material of weight portion described in described health product is curcumin 45 parts-60 parts, Ganoderma extract 60 parts-120 parts, Ganoderma spore powder with cellular wall broken 255 parts-500 parts, yeast rich in selenium 6 parts-13 parts; Described adjuvant or complementary composition and weight portion thereof are: microcrystalline Cellulose 81 parts-153 parts and magnesium stearate 3 parts-6 parts.
6. in claim 1-5, the application in enhancing immunity and hepatic prepared by arbitrary described health product having enhancing immunity and protect the liver function.
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| CN105267692A (en) * | 2015-11-09 | 2016-01-27 | 安徽千方生物科技有限公司 | Dendrobium candidum preparation for enhancing cell repairing and preparing method of dendrobium candidum preparation |
| CN105341917A (en) * | 2015-11-17 | 2016-02-24 | 安徽千方生物科技有限公司 | Cell nutrition enhancing health product preparation and preparation method thereof |
| CN106072559A (en) * | 2016-06-29 | 2016-11-09 | 傅志勇 | A kind of health product for protecting the liver and its preparation method and application |
| CN107692218A (en) * | 2017-09-14 | 2018-02-16 | 柳州衍生科技有限公司 | A kind of health care broken wall Ultramicro-powder with liver-protecting function and preparation method thereof |
| CN108576819A (en) * | 2018-04-24 | 2018-09-28 | 北京中和鸿业医药科技有限公司 | A kind of liver-protecting combination, health food, preparation method and applications |
| CN109363169A (en) * | 2018-10-25 | 2019-02-22 | 丽睿客信息科技（北京）有限公司 | A kind of health-care food composition improved the immunity of the human body and its application |
| CN109172795A (en) * | 2018-10-31 | 2019-01-11 | 北华大学 | Glossy ganoderma composition and its preparation method and application |
| CN109349640A (en) * | 2018-12-10 | 2019-02-19 | 金木集团有限公司 | A kind of health-caring capsule improving chemical damage |
| CN111066994A (en) * | 2020-01-02 | 2020-04-28 | 广东轻工职业技术学院 | Functional beverage for dispelling effects of alcohol and protecting liver and preparation method thereof |
| CN113713075A (en) * | 2021-09-16 | 2021-11-30 | 江苏盛世康禾生物技术有限公司 | Chinese medicinal preparation with immunity enhancing function and preparation method thereof |
| CN113908224A (en) * | 2021-11-18 | 2022-01-11 | 长春守道生物科技有限公司 | A Chinese medicinal composition with immunoregulatory function, and its preparation method |
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