CN104799286A - Composition for improving immunity and preparation method of composition - Google Patents
Composition for improving immunity and preparation method of composition Download PDFInfo
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- CN104799286A CN104799286A CN201510219674.5A CN201510219674A CN104799286A CN 104799286 A CN104799286 A CN 104799286A CN 201510219674 A CN201510219674 A CN 201510219674A CN 104799286 A CN104799286 A CN 104799286A
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Abstract
The invention discloses a composition for improving the immunity. The composition comprises the following components in parts by weight: 200-300 parts of natto freeze-dried powder, 10-40 parts of a ginkgo biloba extract and 40-80 parts of sea-buckthorn powder. The invention further discloses a method for preparing a plant capsule by using the composition. As the natto freeze-dried powder, the ginkgo biloba extract and the sea-buckthorn powder are taken as main raw materials, and algal polysaccharides are taken as a plant capsule material for preparing the capsule, due to the assistant functions of the three components, the purposes of stabilizing in-vivo environments of human bodies, prompting immunity of human body fluid and cells, and removing excessive free radicals in human bodies can be achieved, and the immunity function can be effectively improved.
Description
Technical field
The invention belongs to traditional Chinese medicine health care product technical field, particularly relating to a kind of composition and method of making the same for improving immunity.
Background technology
Immunity is the defense mechanism of human body self, make human bioequivalence and eliminate any foreign matter (virus, bacterium etc.) of external intrusion, process is old and feeble, damage, death, the function of mutant cell and virus infected cell in the own cells of sex change and identification and handling body.When the nutrient (the micro-lamp of protein, vitamin, amino acid, part) that human body is taken in is not enough, immunity will decline, and then increases the probability of organism infection disease, as bacterium, virus infections, and cancer etc.; But immune system exceedes normal level also can cause damage to human body, as caused allergic reaction etc.
In recent years, along with the raising of people's living standard, the change of socio-ecological environment, cultural custom and life style, dietary structure, and the quickening of rhythm of life, industrialization simultaneously, urbanization, aging progress faster, cause hypoimmunity crowd to show a rising trend at society, cause various diseases, serious harm people's is healthy.Such as, the immune organ of the elderly is aging, causes multiple infirmities of age; Chronic or patients after surgery, prolonged administration of drugs and disease consumption cause immunity degradation; Ubiquitous healthy population existence feels run-down, feels exhausted, the symptom that resistance declines, and severe patient there will be the symptoms such as apocleisis, headache, dizziness, insomnia; Common chronic disease, mainly contain cardiovascular and cerebrovascular disease, diabetes, malignant tumour, chronic respiratory disease etc., chronic diseases in China number of the infected rises fast, existing patient diagnosed 2.6 hundred million people, be great public health problem, the death that chronic disease causes has accounted for 85% of the total death of China.Along with the health education that the tissue such as national departments concerned, media promotes, our people more and more payes attention to the maintenance to own health, appreciate gradually and lower for the resistancely can cause various diseases, serious harm people are healthy, greatly increase the understanding of this type of health food and demand, market potential is huge.Simultaneously, various sudden communicable disease is multiple in recent years, and as bird flu etc., simultaneously also to endanger human body healthy for the day by day serious problem of environmental pollution such as haze weather, in the face of these factors cause patient groups's quantity significantly to increase, but be difficult to find effective method at short notice.Suffers from the patient of chronic disease, because long-term prescription and disease consumption cause immunity degradation.In this case, people have to fall back on raising autoimmunity, are that self immune system plays the ability of resisting poisoning intrusion to greatest extent.How improving immunity is also the problem that healthcare industry and medical industry are being pursued always.
An immune word in the traditional Chinese medical science, first appeared in " the immune class side " of the Ming Dynasty, it means " release plague ", i.e. the ancient traditional Chinese medical science said " healthy tendency ".The traditional Chinese medical science is thought, healthy tendency virtual loss is the pathogenetic inherent basis of disease, therefore, pays much attention to the important function of human righteousness in disease generating process.Healthy tendency fills Sheng, and premunition is strong, and pathogenic perverse trend is difficult to invasion and attack, and disease also just has no way of occurring." the Plain Questions writings left behind by the deceased acupuncture manipulation opinion " is said: " in healthy tendency deposits, heresy can not be done.Otherwise ", when human righteousness is not enough, or when healthy tendency is relatively weak, defend outer hypofunction, often anti-heresy is unable, then perverse trend may be taken advantage of a weak point, and causes body imbalance of yin and yang, zang-fu differentiation dysfunction, so that diseases induced.The immunologic homeostasis that healthy tendency can maintain the coordination of function, this function of the popular fluent of qi and blood then balances with the maintenance body inherence in immunologic function matches.Once create some pathological products in body, the healthy tendency of human body can be risen vigorously and resist it again.If positive unsaturated vapor, and these pathological products are difficult to eliminate rapidly, and perverse trend pent-up will inevitably be caused darker, healthy tendency therefore consumption wound simultaneously, very then crisis life.As the generation of long-pending card, usually because disorder of emotion, intemperance of taking food, or sense is outer evil, so that depression and stagnation of QI, hematogenous blockage, extravasated blood internal resistance, knot forms block, gradually large, eventually to not controlling." Plain Questions passages through which vital energy circulates another matter " is said: " at that time, brave person's gas is capable then, timid person then and disease also." the health products majority of existing raising immunity rests on the simple mixing of one or more nutrients, lacks the effect of theory of traditional Chinese medical science support and " strengthening the body resistance to consolidate the constitution ".
Summary of the invention
Goal of the invention: for solving problems of the prior art, the invention provides a kind of composition and method of making the same for improving immunity, reasonably composite by natto, ginkgo leaf and sea-buckthorn are carried out, make the product after three's compatibility effectively can strengthen immunity of organisms.
Technical scheme: for realizing above-mentioned technical purpose, the present invention proposes a kind of composition for improving immunity, and described composition comprises the component of following parts by weight: freeze dry powder of fermented soybean 200 ~ 300 parts, ginkgo biloba p.e 10 ~ 40 parts and seabuckthorn fruit powder 40 ~ 80 parts.Preferably, described composition comprises the component of following parts by weight: freeze dry powder of fermented soybean 250 parts, ginkgo biloba p.e 30 parts and seabuckthorn fruit powder 60 parts.
Wherein, the Flavone content of described ginkgo biloba p.e is 24 ~ 30wt%, and lactone content is 6 ~ 8wt%.
Above-mentioned freeze dry powder of fermented soybean, ginkgo biloba p.e and seabuckthorn fruit powder can purchase available product on face, ordinary city, preferably, described ginkgo biloba p.e is the ginkgo biloba p.e that careless vegetable products Co., Ltd of Huizhou City hundred produces, wherein, in described ginkgo biloba p.e, Flavone content is 24.3wt%, and lactone content is 6.1wt%.
Described composition can be prepared into various acceptable formulation, and as capsule, tablet, granule etc., preferably, the formulation of described composition is capsule.
When above-mentioned composition is prepared to capsule, described capsule is made up of the component of following parts by weight: freeze dry powder of fermented soybean 200 ~ 300 parts, ginkgo biloba p.e 10 ~ 40 parts, seabuckthorn fruit powder 40 ~ 80 parts, starch 30 ~ 60 parts, dolomol 2 ~ 4 parts.
The preparation method of above-mentioned capsule is as follows:
(1) raw material sieves: the learn from else's experience freeze dry powder of fermented soybean, ginkgo biloba p.e, seabuckthorn fruit powder and the starch that are up to the standards, for subsequent use after sieving respectively;
(2) mix: take freeze dry powder of fermented soybean, ginkgo biloba p.e, seabuckthorn fruit powder, starch and the dolomol after the sieving of formula ratio, the ginkgo biloba p.e of formula ratio and seabuckthorn fruit powder are put in mixer and mix 10 ~ 20min, obtain material A; The freeze dry powder of fermented soybean of formula ratio is dropped in mixer with the starch of formula ratio and mixes 10 ~ 20min, obtain material B; The dolomol of mixed material A and material B and formula ratio is put into mixing 15 ~ 20min in mixer, obtains homogeneous powder;
(3) preparation: utilize plant hollow capsule conventionally to make capsule the homogeneous powder that step (2) obtains.
Preferably, in above-mentioned steps, in step (1), 60 ~ 100 mesh sieves crossed by all raw materials.
Conventional plant hollow capsule is as hydroxypropyl methylcellulose (HPMC) Capsules, algal polysaccharides Capsules, pulullan Capsules, konjaku glucomannan Capsules, modified starch Capsules etc.Preferably, algal polysaccharides Capsules is selected to prepare capsule.
By adopting plant hollow capsule to prepare capsule, have the following advantages relative to traditional gelatin hollow capsule tool: plant capsule can expand applicable crowd's scope, such as vegetarian and have special religiously can relievedly to use per capita; Plant capsule water content is low, and capsule nature is stablized, and plant capsule cross-linking reaction, Maillard reaction etc. can not occur.Polysaccharide, flavonoids etc. that this raising immunity composition has high level are easy to the material with conventional capsules generation Maillard reaction, use plant capsule can reduce active component loss, weaken the impact of capsule on active function, and security is good, without the danger that animal epidemic disease source is infected, without high carcinogen as the safety issues such as pollution of chromium, medicament residue, heavy-metal residual not easily occur.
Present invention further proposes above-mentioned composition for the preparation of the application improved in the medicine of immunity, health food or functional food, after above-mentioned composition is aided with suitable auxiliary material and carrier, be prepared into medicine, health food or the functional foods such as capsule, tablet, granule.
Composition in the composition of above-mentioned raising immunity is described as follows:
Freeze dry powder of fermented soybean: natto is a kind of traditional healthy food, the edible history of 2000 is had in Japan, be called as long-lived food, scientific research confirms, containing the multiple bioactive ingredients such as multivitamin, mineral matter, amino acid, protein kinase, isoflavones, soft phosphatide, compound sugar, saponin(e, lignin, bafillus natto in natto, there is the multiple effect such as hypotensive, reducing blood lipid, anti-oxidant, thrombus dissolving, enhancing immunity of organisms.Freeze dry powder of fermented soybean is formed through fermenting bacillus natto by soybean, is a kind of traditional healthy food.Natto not only has abundant nutritive value, and has plurality of medical health care.In recent years, through Japan physician, physiologist research learn, the protein of soybean has indissolubility, and after making natto, become solvable and produce amino acid, and in raw material, non-existent various ferment can produce due to Bacillus natto and association bacterium, helps gastrointestinal disturbances to absorb.The composition of natto is: the % of moisture 61.8%, crude protein 19.26%, crude fat 8.17%, carbohydrate 6.09, crude fibre 2.2%, ash content 1.86%, and as vegetable food, crude protein, fat are the abundantest.Natto system high protein nourishing food, the alcohol contained in natto element, can to get rid of in body acidifying type lipid in part cholesterol, decomposer, make abnormal blood pressure recover normal after edible.Research in recent years shows, isoflavones and soyasaponin have the function strengthening immunity of organisms, can synthesize by Promote immunity cell, improves the phagocytic activity of macrophage, thus strengthens nospecific immunity and the specific immune function of body.Vitamin abundant in natto and mineral matter participate in the indispensable auxiliary enzymes of organism metabolism, has the effect that enhancing antibody is formed; Small protein in natto and polysaccharide can provide nutrition foundation for synthesis immunocyte, and induction produces the immune system that human interferon directly acts on human body.Containing abundant natto lignin in natto, lignin is a kind of string of efficiency natural, can in large intestine rapid adsorption carcinogen, got rid of external, and the physiological activator that formed during the fermentation of natto can supplement the deficiency of Human Physiology active material all sidedly, the effect that the atrophic diseases, glomerulonephritis, skin disease, tumour, hepatitis, virus infections etc. that cause declining because of immunity have significantly prevention and improve, therefore can say that natto is the reliable natural cover for defense in human immunity defence line.Now, natto has become convenience, the healthy food liked various countries, often edible can play improve the health, prophylactic effect.
Ginkgo biloba p.e: ginkgo leaf is the dry leaf of Relict Plant Ginkgo Biloba the most ancient in the world, as the history in medicinal existing more than 500 years.Ginkgo biloba p.e is the extract of dry leaf through being processed into of Ginkgoaceae plant Ginkgo biloba Ginkgo biloba L..According to the notice that the Ministry of Public Health manages about further specification healthy food material, ginkgo leaf belongs to the raw material that can be used for health food.Namely ginkgo biloba p.e take ginkgo leaf as raw material, obtains through extracting and developing drying.From the sixties in 20th century, the chemical composition, efficacy effect etc. of domestic and international researcher to ginkgo leaf have carried out large quantity research, confirm that ginkgo leaf and extract thereof have effects such as improving cardiovascular and cerebrovascular circulation, reduction blood fat, raising immunity of organisms, anti-inflammation, delay senility.In recent years, the develop immunitypty effect of ginkgo biloba p.e is in widespread attention, is confirmed both at home and abroad by a large amount of scientific research: ginkgo biloba p.e can Promote immunity allelotaxis, the phagocytic function of mononuclear macrophage had to facilitation and have obvious humidification to NK; The generation of energy enhancing antibody, strengthens the humoral immune function of body; Increase T cell quantity, regulate the balance of t lymphocyte subset group, have facilitation to cellular immunity; In addition can also by regulating cytokine levels, strengthening the immunologic function of hematid immunity function and mucosa dysregulation body.
Sea-buckthorn: be the Mongols, Tibetan's conventional crude drugs.For the dry mature fruit of Elaeangnaceae plant sea-buckthorn Hippophae rhamnoidesL..Sea-buckthorn is one of raw material of integration of drinking and medicinal herbs; medicinal, edible with a long history; sea-buckthorn is comprehensive nutrient source; also have multivitamin, several amino acids, various trace elements and various bioactivators; can comprehensive, multisystem, multiple organ, too many levels, the multi-level health regulating protection human body, be described as the patron saint of human health " 21 century " by academia.Sea-buckthorn, is eutrophy the source plant, particularly vitamin and amino acid whose high-load that in fruits and vegetables, rare egg collection white matter, unrighted acid, vitamin are integrated, has determined the star status of sea-buckthorn; Fructus hippophae also has the bioactivator of special health-care effect to human body containing Hippophate flavone, superoxide dismutase and omega 6 polyunsaturated fatty acid etc.China is the country utilizing sea-buckthorn maximum in the world, just has many records in ancient documents, as treated PUD D, pulmonary abscess etc. with regard to fructus hippophae on the books in " moon king medicine is examined ".Containing soluble sugar 8% ~ 15% in sea-buckthorn, containing acid 10% ~ 12%, mainly contain malic acid, citric acid, tartaric acid etc.Vitamin about 14 kinds, and content is quite abundant, wherein the highest with ascorbic content, research shows that in every 100g fructus hippophae, ascorbic content is at 100 ~ 800mg.Modern study shows, total seabuckthorn flavone has the effect increasing murine interleukin lysozyme, phagocytic function, total complement content; Can generate by enhancing antibody, antibody level of serum is raised; Increase T lymphocyte in blood ratio and the special rosette cell (SRFC) of spleen; the lymphocyte activation simultaneously making Con A activate strengthens; promote lymphocyte transformation during low concentration, and obviously the anti-sheep red blood cell (SRBC) Hemolysin formation of protection caused by cyclophosphamide reduces.Seabuckthorn fruit powder can increase the phagocytic function of mouse macrophage, makes phagocytic percentage apparently higher than control group (P < 0.01); Can promote humoral immunity, sample HD50 value improves 35% than control group; Can lymphocyte transformation be promoted, and reduce serum cholesterol content.Health beverages-Yi Liqi (main component is Hippophae (rhamnoides) original juice and lactic acid bacteria culture solution) is used to the continuous 20d of mouse, 1 times, 10 times of people's use amount, 30 multiple dose groups obviously can strengthen mouse delayed allergy and increase and periphery blood T lymphocyte ANAE positive rate, and can improve macrophage phagocytosis of mice and spleen antibody-producting cell number.
Natto, ginkgo leaf and sea-buckthorn are all health treasures.Natto is rich in abundant protein, multivitamin, the demand of human body positive nitrogen equilibrium can be kept, sufficient nutrition can be provided to the physical Health restoration of patients with chronic diseases and the elderly, being the solid good medicine just holding up this in theory of traditional Chinese medical science, is also one of fruit liking in people's daily life eating; Ginkgo leaf is the traditional Chinese medicine of China, have promoting blood circulation and removing blood stasis, remove obstruction in channels to relieve pain, astringe the lung and to relieving asthma, change effect of turbid lipopenicillinase, the effect having a surname's lung to invigorate blood circulation, have the saying of " qi and blood punching and, ten thousand diseases are not given birth to " in the traditional Chinese medical science, the effect of righting can be helped; Sea-buckthorn is one of medicinal material of medicine-food two-purpose, nutritious, taste is sour-sweet, is loved by the people, in traditional Chinese medical theory system, sea-buckthorn has effect of reinforcing spleen to promote digestion, cough-relieving apophlegmatic, be good reinforcing spleen to promote digestion medicine, spleen is periphery immune system in immune system, is the position that lymphocyte is settled down, insufficiency of the spleen, outer heresy invasion can be caused, thus cause body obstinate disease or often fall ill.In addition, the compositions such as the vitamin C in sea-buckthorn are easily oxidized, and containing oxidation-resistant active ingredient in ginkgo leaf, combination reduces scattering and disappearing of nutritional labeling, natto extract has bacteriostasis, can keep stability and the microbial safety of product to a certain extent, natto extract also has the effect of regulating intestinal canal flora, useful to absorption of nutritional labeling etc.The traditional Chinese medical science has specific view to immunity, namely strengthens the body resistance to consolidate the constitution as principle, and kidney tonifying, a surname lung, invigorating the spleen are method, thus qi and blood fills, and heresy can not be invaded, i.e. our said Immune-enhancing effect.But the health food of three's compatibility does not occur in the market at present, by the further investigation to theory of traditional Chinese medical science, reasonable combination is filled a prescription, the product after three's compatibility can be made effectively can to strengthen immunity of organisms, be better than the health products that on market, simple supplementary single component or various nutrients mix, exploitation becomes the outstanding health food really having traditional Chinese medical theory and support.
Beneficial effect: the present invention is by adopting freeze dry powder of fermented soybean, ginkgo biloba p.e, seabuckthorn fruit powder to be primary raw material, and utilize plant hollow capsule to be prepared into capsule, the effect of three kinds of compositions is complemented each other, to reach the object of stable human internal environment, promote human body fluid and cellular immunity, excessive free radicals in purged body, thus effective develop immunitypty function.
Detailed description of the invention
Illustrate below by way of specific description of embodiments of the present invention but do not limit the present invention.Wherein, the ginkgo biloba p.e that following each embodiment is selected buys from Huizhou City hundred careless vegetable products Co., Ltd, and Flavone content is 24.3wt%, and lactone content range is at 6.1wt%.
Embodiment 1
For improving a composition for immunity, it comprises the component of following parts by weight: freeze dry powder of fermented soybean 240 parts, ginkgo biloba p.e 40 parts, seabuckthorn fruit powder 60 parts, starch 56 parts, dolomol 4 parts.Utilize above-mentioned formula to prepare the capsule containing said composition, its preparation method is as follows:
A () supplementary material prepares: get each supplementary material meeting quality criteria requirements for subsequent use;
B () is sieved: freeze dry powder of fermented soybean, ginkgo biloba p.e, seabuckthorn fruit powder, starch are crossed 60 mesh sieves respectively;
C () weighs: the freeze dry powder of fermented soybean of the formula ratio be up to the standards of learning from else's experience, ginkgo biloba p.e, seabuckthorn fruit powder, starch and dolomol;
D () mixes: material ginkgo biloba p.e and seabuckthorn fruit powder are dropped into and mix 10min in HL-800 type mixer and obtain material A; Freeze dry powder of fermented soybean and starch drop into and mix 10min in HL-800 type mixer and obtain material B, then mixed material A, material B and dolomol are dropped in HL-800 type mixer and mix 15min, obtain homogeneous mixture;
(e) filling: get the plant hollow capsule (algal polysaccharides Capsules) be up to the standards, plant capsulae vacuus is added NJP-2000B type capsule filling machine test-run a machine, the material be up to the standards is added after machine works well, adjusting agent gauge rod, start formally filling after making capsules weight reach 0.38 ~ 0.42g, in production process, note observing capsule appearance, every the weight of 15min test capsule;
F () polishing: filling complete YPJ-818 type polishing machine carries out polishing to capsule, after the powder of removing capsule surface, puts into the bucket being lined with clean dry polybag;
(g) inner packing: with BS15/500B type frequency conversion amplitude modulation double end counting grain machine, capsule is loaded solid medicinal plastic bottle and (meet the quality standard of YBB 00122002 oral stable medicinal polythene bottle with high density, and carry out leak check;
H () external packing: bottle posts label, prints date of manufacture and shelf-life, load corrugated case (meeting the quality standard of GB/T 6543);
(i) inspection, warehouse-in: finished product is through being up to the standards, putting in storage.
Wherein, all in GMP workshop, 100,000 grades of clean areas carry out (a) ~ (i) item step.After testing, in the active component of said composition after extracting, Total Ginkgo Flavone-Glycoides and total seabuckthorn flavone content account for 1.7wt%.
Embodiment 2
For improving a composition for immunity, it comprises the component of following parts by weight: freeze dry powder of fermented soybean 260 parts, ginkgo biloba p.e 28 parts, seabuckthorn fruit powder 80 parts, starch 30 parts, dolomol 2 parts.Utilize above-mentioned formula to prepare the capsule containing said composition, its preparation method is as embodiment 1.After testing, in the active component of said composition after extracting, Total Ginkgo Flavone-Glycoides and total seabuckthorn flavone content account for 1.67wt%.
Embodiment 3
For improving a composition for immunity, it comprises the component of following parts by weight: freeze dry powder of fermented soybean 250 parts, ginkgo biloba p.e 30 parts, seabuckthorn fruit powder 60 parts, starch 56 parts, dolomol 4 parts.Utilize above-mentioned formula to prepare the capsule containing said composition, its preparation method is as embodiment 1.After testing, in the active component of said composition after extracting, Total Ginkgo Flavone-Glycoides and total seabuckthorn flavone content account for 1.47wt%.
Embodiment 4
For improving a composition for immunity, it comprises the component of following parts by weight: freeze dry powder of fermented soybean 242 parts, ginkgo biloba p.e 35 parts, seabuckthorn fruit powder 70 parts, starch 50 parts, dolomol 3 parts.Utilize above-mentioned formula to prepare the capsule containing said composition, its preparation method is as embodiment 1.After testing, in the active component of said composition after extracting, Total Ginkgo Flavone-Glycoides and total seabuckthorn flavone content account for 1.71wt%.
Embodiment 5
A prescription for develop immunitypty, is characterized in that comprising freeze dry powder of fermented soybean 250 parts, ginkgo biloba p.e 35 parts, seabuckthorn fruit powder 60 parts, starch 51 parts, dolomol 4 parts in mass parts.Utilize above-mentioned formula to prepare the capsule containing said composition, its preparation method is as embodiment 1.After testing, in the active component of said composition after extracting, Total Ginkgo Flavone-Glycoides and total seabuckthorn flavone content account for 1.59wt%.
The toxicological experiment of embodiment 6 composition.
1, laboratory sample and test item:
1.1 samples: composition capsule prepared by embodiment 3, by Zhong Sheng bio tech ltd, Nanjing consignment, human body RD is 1600mg/ people/sky, i.e. 26.7mg/kg b.wt.
1.2 experimental animals: healthy ICR mouse, Nanjing Medical University's Experimental Animal Center provides, cleaning grade, the quality certification number: No. 2008-0004, SCXK (Soviet Union); 4 week age healthy SD rat, the Shanghai Si Laike animal used as test environmental facility quality certification number: No. 2012-0037, SYXK (Soviet Union).
1.3 acute toxicity test in mice: select body weight 18.1 ~ 20.5gICR mouse 20, male and female half and half.Tested material dosage is 15000mg/kg b.wt, and gavage gives animal used as test, observation period 14d.
1.4 salmonella typhimurium tests: test strain is through identified gene type satisfactory TA97, TA98, TA100 and TA102, and each bacterial strain incubated overnight liquid bacterial concentration is all 10
9individual/ml or more.Sterilized water takes 500mg sample adding distil water to 10ml, then does 5 times of dilutions continuously with sterile purified water and be made into required each concentration.Establishing 5 dosage groups, 1 group of solvents, 1 blank group namely from beaming back change group altogether, separately establishing 4 positive controls (Dexon, NaN
3, 2-AF and 1,8-dihydroxy anthraquinone) adopt dull and stereotyped infiltration method, test twice altogether, each each dosage establishes Duplicate Samples 3.
1.5 mouse marrow cell micro nuclear tests: select the healthy ICR mouse 50 of body weight 25.0 ~ 29.5g, male and female half and half, mouse point male and female are divided into 5 groups at random by body weight, often organize 10.3 tested group of dosage is respectively 2500,5000,10000mg/kg b.wt, positive controls (endoxan 40mg/kg b.wt) and solvent control group (pure water).Each group all adopts 30h twice administration by gavage, and each gavage capacity is 20ml/kg, and get femur bone marrow and be suspended from direct smear in calf serum, fixing, dyeing, microscopy polychromatic erythrocyte 1000/mouse, counting is containing the cell number of micronucleus.Observe 200 polychromatic erythrocytes to count simultaneously and seen just incarnadine cell, the ratio of both calculating.
1.6 mouse testis chromosomal aberration tests: select body weight 25.4-29.4g male ICR mouse 25, be divided into 5 groups at random, often organize 5, in 3 experimental group tested material dosage be respectively 2500,5000,10000mg/kgb.wt, separately establish solvent control group and endoxan (CP) positive controls (40mg/kg b.wt).Each dosage group and solvent control group gavage give tested material, continuous 5d, positive controls lumbar injection, continuous 5 days.Each treated animal is 6h lumbar injection colchicine 4mg/kg before execution all, tests and within the 14th day, kills mouse and get testis, pull open convoluted seminiferous tubule successively, hypotonic, fixing, softening, film-making, dyeing, microscopy.
1.7 30 days feeding trials:
1.7.1 test method: 4 week age SD rat 80, male and female half and half, point sex is divided into each group by body weight, often organize 20.In 3 experimental group tested material dosage be respectively 668,1335,2670mg/kg b.wt./d (being equivalent to 25,50,100 times of RD), if basal feed control group.Take respectively 147,294 and 587g admix sterilizing basis mouse feed, with about every day, the food ration of 100g/kg b.wt. gives hello the feed that rat freely eats aforementioned proportion mixing.
1.7.2 observation index: grow, food utilization, hemoglobin, blood platelet, red blood cell, total white blood cells and neutrophil leucocyte, lymphocyte, monocyte, eosinophil, basophilic granulocyte classification, liver,kidney,spleen, testis body ratio, ALT, aspartate amino transferase, total protein, albumin, blood sugar, urea nitrogen, creatinine, T-CHOL, triglycerides, gross anatomy and liver,kidney,spleen, stomach, small intestine, testis, ovary histopathologic examination.
2, result
2.1 acute toxicity tests: have no obvious poisoning manifestations after mouse stomach, without animal dead in the observation period.At the end of observation period, gross anatomy shows no obvious abnormalities.Natto silver sour jujube capsule has no significant effect the weight of animals growth, and per os has no obvious poisoning manifestations after giving animal, without animal dead in the observation period.Male and female its mouse oral MTD value is all greater than 15000mg/kg b.wt., and according to acute oral toxicity grade scale, this sample belongs to nontoxic level.
2.2 salmonella typhimurium result of the tests:
Table 1Ames result of the test
This sample does not all detect obvious MA to standard testing bacterium TA97, TA98, TA100 and TA102.
2.3 mouse marrow cell micro nuclear test results:
Table 2 composition capsule is to mouse marrow cell micro nuclear test result
Result shows, under study dosage, does not detect this sample and causes micronucleus effect to male and female PCEMNR.
2.4 mouse testis chromosomal aberration tests
Table 3 composition capsule is to mouse testis chromosomal aberration test result
A: fracture, part, transposition, microbody etc.
Result shows, under study dosage, does not detect this sample to the chromosomal MA of male mice testis first spermatocyte.
2.5 30 days feeding trials
Mix in basal feed with the composition capsule of 835mg/kg dosage (being equivalent to 50 times of RD) and feed rat 30d, as test group, result shows: test group animal activity, growth are normally; Body weight compares there was no significant difference (p > 0.05) with control group; Male high dose group liver absolute weight and liver/body raise than all, difference tool pole conspicuousness (p < 0.01) compared with control group, but blood biochemistry index and histopathologic examination are showed no obvious abnormalities; The hematological indices detected value of each dosage group of male and female, biochemical indicator detected value are all in normal fluctuation range; High dose group male and female rat histopathologic examination does not show the pathological change obviously relevant with composition capsule.
Under this experiment condition, this sample 30 days feeding trial high dose group female rats actual absorption dosage is respectively 3142mg/kg, is equivalent to 117 times of RD.
In sum, the testing result of composition capsule prepared of the present invention is as follows:
Acute toxicity test: its mouse oral acute toxicity (MTD) of composition capsule to two kinds of sexes prepared by the embodiment of the present invention 3 is all greater than 15000mg/kg.BW, according to " health food inspection and assessment technical specification " (version in 2003) acute toxicity grading criteria, this sample belongs to nontoxic level.
Salmonella reversion test: result is negative; Mouse marrow cell micro nuclear test: result is negative; Mouse testis chromosomal aberration test: result is negative.
Embodiment 7 composition develop immunitypty function Report on Animal.
Select composition capsule prepared by embodiment 3, evaluate the effect that it strengthens animal immunizing power function, set up 3 groups of reference examples simultaneously, be contrast 1, contrast 2 and contrast 3, wherein, the difference of the capsule that the composition capsule contrasting 1 is prepared with embodiment 3 is only to contrast 1 not containing freeze dry powder of fermented soybean, the difference of capsule that the composition capsule of contrast 2 prepare with embodiment 3 is only to contrast 2 not composition containing ginkgo leaf extracts, and the difference of the capsule that the composition capsule contrasting 3 is prepared with embodiment 3 is only that contrasting 3 does not contain seabuckthorn fruit powder.
1, materials and methods
1.1 samples: composition capsule prepared by embodiment 3, the composition capsule contrasting 1, contrast 2 and contrast 3, respectively as tested group, hard shell capsules content is sundown powder.
1.2 animals used as test: 18 ~ 22g Healthy female cleaning grade ICR mouse (the animal card number: No. 2012-0002, SCXK (Shanghai)) selecting Shanghai Slac Experimental Animal Co., Ltd. to provide.
1.3 dosage choice: adult's recommended intake every day is 1600mg, be equivalent to 26.67mg/kg BW/d, experiment establishes 10 times of human body recommended amounts, namely every day, 267mg/kg BW was as test group, contrast 1, contrast 2, contrast 3 dosage consistent with test group (10 times of human body recommended amounts), solvent blank control group, with isometric(al) pure water gavage (being designated as blank group), surveys every immune indexes after continuous gavage 30d.Outside all animal process diets, according to above-mentioned dosage feeding composition capsule or blank pure water respectively.
1.4 experimental techniques:
1.4.1 dinitrofluorobenzene inducing mouse Tardive allergy (DTH)
After continuous gavage 25d, mouse web portion unhairing, 1% dinitrofluorobenzene (DNFB) acetone sesame oil solution 50ul smears sensitization.5d after sensitization, mouse right ear uniform application 1%DNFB acetone sesame oil solution 20ul attacks, and left ear is smeared acetone sesame oil solution and compared, after attacking, 24h puts to death mouse, cut left and right auricular concha, the auricle in same position cut-off footpath 8mm is weighed, and the difference of left and right auricle weight is as swelling.Get thymus gland and the spleen of mouse, using the spleen of every 10g mouse heavy (mg) and chest gland weight (mg) as spleen/body weight ratio and thymus gland/body weight ratio simultaneously.
After continuous gavage 30d, asepticly get spleen, (cell concentration is 3 × 10 to make individual cells suspension
6individual ml).Add in 24 well culture plates by a cell suspension point holes, every hole 1ml, a hole adds 75 μ l ConA liquid, and 5%CO in contrast, is put in another hole
2, 37 DEG C of CO
272h is cultivated in incubator.Cultivation terminates front 4h, and every hole sucks supernatant 0.7ml, adds 0.7ml not containing the RPMI1640 nutrient solution of calf serum, adds MTT solution (5mg/m1) 50 μ l/ hole simultaneously, continues to cultivate.After cultivation terminates, every hole adds 1ml acid isopropyl ketone, and piping and druming mixing, makes purple crystal dissolve completely.Then be dispensed in 96 well culture plates, each hole packing 3 hole (200 μ l/ hole), as Duplicate Samples, measures OD value with ELIASA with 570nm wavelength.Deduct with the OD value adding ConA hole the OD value not adding ConA hole and represent lymphocytic multiplication capacity.
1.4.2 antibody-producting cell (PFC) detects (agarose plate method)
After continuous gavage 27d, every mouse lumbar injection 2% (v/v) hematocrit SRBC 0.2ml sensitization.After immunity 4d, mouse cervical dislocation is put to death, take out spleen, make single cell suspension, wake up with a start solution plaque assays.Counting hemolysis plaque number, with plaque number/10
6splenocyte represents antibody-producting cell number.
1.4.3 serum hemolysin measures: HD50 value (HC
50) determination method
After continuous gavage 27d, every mouse lumbar injection 2% (v/v) hematocrit SRBC 0.2ml sensitization.After immunity 4d, mouse plucks eyeball blood sampling, gets serum and surveys hemolytic reaction, separately sets the control tube of not increase serum (replacing with SA buffer solution) as colorimetric blank, measures each pipe OD value respectively in 540nm.Be calculated as follows the sample HC of each mouse
50:
1.4.4 mouse carbonic clearance is tested
The india ink 0.1ml that after continuous gavage 30d, mouse every 10g body weight tail vein injection 1: 7 (v/v) is diluted, timing immediately after injection, and respectively at 2,10min gets blood 20 μ l from angular vein clump, be added in 2ml 0.1% sodium carbonate, survey OD
600nm.And get liver, spleen is weighed, by formula a=K
1/3× body weight/(liver weight+spleen weight) asks mouse phagocytic index a value (K=(lgOD
1-lgOD
2)/(t
2-t
1).
1.4.5 Turnover of Mouse Peritoneal Macrophages engulfs chicken red blood cell experiment (dripping sheet method)
After continuous gavage 27d, every mouse lumbar injection 2% (v/v) hematocrit SRBC 0.2ml, activates mouse macrophage.After 5d, cervical dislocation puts to death mouse, and lumbar injection only adds the Hanks liquid 4mL/ of calf serum, and 1% chicken red blood cell drawing abdominal cavity washing lotion and equivalent mixes.Draw 1mL mixed liquor, add in the agar circle of slide, in placement incubator, hatch 20min for 37 DEG C.With physiological saline, non-attached cell is washed out after hatching end, in methanol solution, fix 1min, Giemsa liquid dyeing 15min.By 40 × microscopic counting phagocytic rate and phagocytic index, (phagocytic rate is in every 100 macrophages, engulfs the percentage shared by macrophage of chicken red blood cell; Phagocytic index is each mouse of average each macrophage phagocytic chicken red blood cell).And judge the phagocytic activity of macrophage accordingly.
1.4.6NK cytoactive detection
After continuous gavage 30d, asepticly get spleen, (cell concentration is 2 × 10 to make individual cells suspension
7individual/mL), as effector cell.Get target cell (YAC-1 cell) and each 100 μ l of effector cell (effect target is than 50: 1), add in U-shaped 96 well culture plates; Target cell Spontaneous release hole adds target cell and each 100 μ l of nutrient solution, and the maximum release aperture of target cell adds target cell and each 100 μ l of 2.5%Triton; Above-mentionedly everyly all establish three repeating holes, in 37 DEG C, 5%CO
24h is cultivated in incubator, then by 96 well culture plates with the centrifugal 5min of 1500r/min, every hole is drawn in 96 well culture plates at the bottom of supernatant 100 μ l horizontalization, add LDH matrix liquid 100 μ l simultaneously, reaction 10min, every hole adds the HCl 30 μ l of 1mol/L, measures OD value (OD) at ELIASA 490nm place.Be calculated as follows NK cytoactive:
2, experimental result
2.1 samples are on the impact of the weight of animals before and after test
From table 4, table 5 and table 6, before and after experiment tested material test group Mouse Weight and increase weight respectively with blank group, compared with control group without statistical significant difference (p > 0.05).
The original body mass of mouse respectively organized by table 4
Tested group of test group to be organized and control group mice original body mass obtains P value through ANOVA and is all greater than 0.05 with blank.
The body weight of each group in end mouse eventually tested by table 5
Tested material test group organize and control group mice opisthosoma at end is heavy obtains P value through ANOVA and be all greater than 0.05 with blank.
The impact that table 6 sample increases Mouse Weight
Tested material test group to be organized and control group mice body weight increases and obtains P value through ANOVA and be all greater than 0.05 with blank.
2.2 samples are on the impact of internal organs/body weight ratio
From table 7, tested material test group mouse thymus/body ratio, spleen/body ratio is compared without statistical significant difference (P > 0.05) with control group with blank group.
Table 7 composition capsule is to mice organs (mg)/body weight (10g) ratio
impact
Tested material test group to be organized and control group mice spleen/body weight ratio and thymus gland/body weight ratio obtain P value through ANOVA and is all greater than 0.05 with blank.
2.3 cell immune function experiment: the mouse Tardive allergy (DTH) that dinitrofluorobenzene (DNFB) is induced
From table 8, tested material test group mice auricle swelling degree all higher than blank group, contrast 1, contrast 2 and contrast 3 groups, variance analysis and compare display between two, test group mice ear degree has remarkable significant difference (p < 0.01) compared with solvent control group, and this experimental result is positive.
Table 8 composition capsule is on the impact of Mice Auricle Tardive allergy
The mouse spleen lymphocyte transformation experiment (mtt assay) that 2.4 cell immune function experiment: ConA induces
From table 9, tested material test group optical density difference is all higher than solvent control group, result Analysis of variance and comparative statistics process between two, the optical density difference of tested material test group mouse is compared all without statistical significant difference (P > 0.05) with blank group with control group, this experimental result feminine gender.
The impact that table 9 composition capsule transforms mouse spleen lymphocyte
2.5 humoral immune function experiments: antibody-producting cell (PFC) detects (agarose plate method)
From table 10, result Analysis of variance and comparative statistics process between two, the hemolysis plaque number of tested material test group mouse is compared with control group with blank group, has statistical significant difference (P < 0.05), this experimental result positive.
Table 10 composition capsule is on the impact of mice spleen antibody-producting cell amount
2.6 humoral immune function experiments: serum hemolysin is tested
From table 11, result Analysis of variance and comparative statistics process between two, tested material test group mice serum HD50 value (HC
50) compare with control group with blank group, all without statistical significant difference (P > 0.05), this experimental result feminine gender.
Table 11 composition capsule is on the impact of serum hemolysin
2.7 monocytes/macrophages functional experiments: mouse carbonic clearance is tested.
From table 12, tested material each dosage group mouse phagocytic index a all higher than blank group, contrast 1, contrast 2 and contrast 3, variance analysis and compare display between two, the phagocytic index a of test group mouse has statistical significant difference (P < 0.01 compared with solvent control group, P < 0.05), this experimental result positive.
Table 12 composition capsule is on the impact of mouse carbonic clearance
2.8 monocytes/macrophages functional experiments: Turnover of Mouse Peritoneal Macrophages engulfs chicken red blood cell experiment.
From table 13, result Analysis of variance and comparative statistics process between two, monocytes/macrophages phagocytic percentage and the phagocytic index of tested material each dosage group mouse are compared with control group with blank group, all without statistical significant difference (P > 0.05), this experimental result feminine gender.
Table 13 composition capsule is on the impact of Turnover of Mouse Peritoneal Macrophages phagocytic activity
2.9NK cytoactive detection
From table 14, the NK cytoactive of tested material each dosage group mouse is all higher than solvent blank combination contrast 1, the value contrasting 2 and contrast 3, variance analysis and compare display between two, test group NK cells in mice activity has compared statistical significant difference (P < 0.01) with blank group with control group, this experimental result positive.
Table 14 composition capsule is on the impact of NK cells in mice activity
3, brief summary:
Per os gives the composition capsule 30d of mouse various dose, on Mouse Weight growth, thymus gland/body ratio, spleen/body ratio all without impact.In cell immune function experiment, the mouse spleen lymphocyte transformation experiment of ConA induction is negative.The experiment of dinitrofluorobenzene inducing mouse Tardive allergy is for positive.In humoral immune function experiment, antibody-producting cell test experience is positive, and serum hemolysin determination experiment is negative.Monocytes/macrophages phagocytic function experiment small mouse peritoneal macrophage engulfs chicken red blood cell experiment for negative, and mouse carbon particle clearance test is positive.The experiment of NK cytoactive detection is for positive.
In sum, according to health food develop immunitypty functional evaluation standard, composition capsule prepared by the present invention has the function of develop immunitypty, and when preparing composition capsule according to proportioning of the present invention, when freeze dry powder of fermented soybean, ginkgo biloba p.e and seabuckthorn fruit powder are all deposited, effect is optimum, by the reasonable compatibility of three, the object realizing reaching stable human internal environment can be contributed to, promote human body fluid and cellular immunity, excessive free radicals in purged body, thus effective develop immunitypty function.
Claims (8)
1. for improving a composition for immunity, it is characterized in that, described composition comprises the component of following parts by weight: freeze dry powder of fermented soybean 200 ~ 300 parts, ginkgo biloba p.e 10 ~ 40 parts and seabuckthorn fruit powder 40 ~ 80 parts.
2. the composition for improving immunity according to claim 1, is characterized in that, the Flavone content of described ginkgo biloba p.e is 24 ~ 30wt%, and lactone content is 6 ~ 8wt%.
3. the composition for improving immunity according to claim 1, is characterized in that, the formulation of described composition is capsule.
4. the composition for improving immunity according to claim 3, it is characterized in that, described capsule comprises the component of following weight fraction: freeze dry powder of fermented soybean 200 ~ 300 parts, ginkgo biloba p.e 10 ~ 40 parts, seabuckthorn fruit powder 40 ~ 80 parts, starch 30 ~ 60 parts, dolomol 2 ~ 4 parts.
5. the composition for improving immunity according to claim 4, is characterized in that, described capsule is prepared as follows:
(1) raw material sieves: the learn from else's experience freeze dry powder of fermented soybean, ginkgo biloba p.e, seabuckthorn fruit powder and the starch that are up to the standards, for subsequent use after sieving respectively;
(2) mix: take freeze dry powder of fermented soybean, ginkgo biloba p.e, seabuckthorn fruit powder, starch and the dolomol after the sieving of formula ratio, the ginkgo biloba p.e of formula ratio and seabuckthorn fruit powder are put in mixer and mix 10 ~ 20min, obtain material A; The freeze dry powder of fermented soybean of formula ratio is dropped in mixer with the starch of formula ratio and mixes 10 ~ 20min, obtain material B; The dolomol of mixed material A and material B and formula ratio is put into mixing 15 ~ 20min in mixer, obtains homogeneous powder;
(3) preparation: utilize plant hollow capsule conventionally to make capsule the homogeneous powder that step (2) obtains.
6. the composition for improving immunity according to claim 5, is characterized in that, in step (1), 60 ~ 100 mesh sieves crossed by all raw materials.
7. the composition for improving immunity according to claim 5, it is characterized in that, in step (3), described plant hollow capsule is any one in hydroxypropyl methylcellulose Capsules, algal polysaccharides Capsules, pulullan Capsules, konjaku glucomannan Capsules and modified starch Capsules.
8. composition according to claim 1 is for the preparation of the application improved in the medicine of immunity, health food or functional food.
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