CN104415214B - Purposes of the black garlic extract in microbial population of animal intestinal tract is adjusted - Google Patents
Purposes of the black garlic extract in microbial population of animal intestinal tract is adjusted Download PDFInfo
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- CN104415214B CN104415214B CN201310390623.XA CN201310390623A CN104415214B CN 104415214 B CN104415214 B CN 104415214B CN 201310390623 A CN201310390623 A CN 201310390623A CN 104415214 B CN104415214 B CN 104415214B
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- black garlic
- garlic extract
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- intestinal tract
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/4841—Filling excipients; Inactive ingredients
- A61K9/4866—Organic macromolecular compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention discloses purposes of the black garlic extract in microbial population of animal intestinal tract is adjusted, purposes of the black garlic extract in medicine is prepared, medicine and food and its purposes in microbial population of animal intestinal tract is adjusted, and the method for regulation microbial population of animal intestinal tract.Inventor has found, black garlic extract can significantly improve the quantity of the probiotics such as Bifidobacterium in animal intestinal tract, Ruminococcus, so as to significantly adjust the gut flora of animal, and then its immunity, the disease such as prevention or treatment obesity, diabetes, hypertension, high fat of blood or tumour are strengthened by the effect of gut flora.
Description
Technical field
The present invention relates to the new application of black garlic extract, in particular it relates to which black garlic extract is in regulation microbial population of animal intestinal tract
In purposes.More specifically, the purposes the present invention relates to black garlic extract in microbial population of animal intestinal tract is adjusted, black garlic extract are being made
Purposes in standby medicine, medicine and food and its purposes in microbial population of animal intestinal tract is adjusted, and regulation microbial population of animal intestinal tract
Method.
Background technology
Substantial amounts of symbiosis flora in normal human be present, these bacteriums are largely colonized in the enteron aisle of people, and quantity exceedes
1000000000000000, equivalent to 10 times of human body cell sum, its microbial gene quantity is about 3,000,000, about mankind's base
Because of more than 100 times of group gene dosage.The gene of such magnanimity can help microorganism to adapt to changeable environment, at the same form with
The inseparable mutualistic symbiosis relation of human body.The completion of the Human Genome Project so that the mankind are more deep to the understanding of oneself,
But the genome for understanding the mankind can not completely hold the health of the mankind, the micropopulation forever settled down in human body intestinal canal
Influence to human health is extremely far-reaching, small to environment in alimentary canal, digestive problems, greatly to fat or thin problem, hypertension, glycosuria
Disease, high fat of blood, cancer etc., the life and health of the mankind is closely bound up with them, therefore human body intestinal canal microbial genome
It is described as second set of genome of the mankind.
At present, research confirm human body a variety of diseases such as diabetes, hypertension, high fat of blood, cancer etc. all with gut flora
It is closely related.Gut flora fermentation polysaccharides metabolite mainly has short key aliphatic acid（Acetic acid, propionic acid, butyric acid etc.）, gas（Hydrogen
Gas, carbon dioxide, hydrogen sulfide and methane）And ammonia, wherein hydrogen and carbon dioxide is the main component of gas in colon.People's enteron aisle
In vitro study shows that butyric acid can promote intestinal mucosa reparation and its functional rehabilitation, and suppresses the formation of inflammatory cytokine, and
The secretion of the TNF of intestinal epithelial cell can be reduced, plays anti-inflammatory and antitumor action.In addition, separately there is research to report
In road gut flora Bacteroides and（Or）The change of Clostridia population and bacterium Uncultured bacterium clone
Nbw1009b01c1 appearance, the occurrence and development of Kazak ethnic population hypertension may be influenceed.Different gut flora spectrums
It can cause a disease, can also diseases prevention.
However, the research in terms of regulation microbial population of animal intestinal tract still needs to be goed deep at present.
The content of the invention
It is contemplated that at least solves one of technical problem present in prior art.Therefore, one object of the present invention
It is to propose a kind of means that can effectively adjust microbial population of animal intestinal tract.
Inventor, which studies, to be found, black garlic extract can significantly improve the probiotics such as Bifidobacterium in enteron aisle, Ruminococcus
Quantity, the gut flora of animal is adjusted, and then strengthen immunity, fat-reducing, hypoglycemic, drop are played by the effect of gut flora
Blood pressure, reducing blood lipid, antitumor and other effects, and without any side effect, can be long-term use of., wherein it is desired to explanation, black
The main component of garlic extract is diallyl trisulfide（DATS）And diallyl disulfide（DADS）, wherein main
Effective active composition be diallyl trisulfide（DATS）That is allicin (allicin), it is slightly soluble in water, be dissolved in ethanol,
The organic solvents such as benzene, ether, at this stage and have no the research of black garlic extract and allicin in terms of gut flora is adjusted and
Report.
Thus, according to an aspect of the present invention, the invention provides black garlic extract in microbial population of animal intestinal tract is adjusted
Purposes.According to an embodiment of the invention, black garlic extract can significantly improve the benefits such as Bifidobacterium in animal intestinal tract, Ruminococcus
The quantity of raw bacterium, so as to significantly adjust the gut flora of animal, and then exempted from by the effect of gut flora to play enhancing
Epidemic disease power, fat-reducing, hypoglycemic, lowering blood pressure and blood fat, antitumor and other effects.Thus, black garlic extract is administered to animal can be effective
Ground adjusts its gut flora, so as to strengthen its immunity, prevention or treatment obesity, diabetes, hypertension, high fat of blood or tumour etc.
Disease.
According to an embodiment of the invention, black garlic extract of the invention can be prepared by following steps：From big
Small uniform fresh black garlic, the garlic clove of peeling is cleaned in peeling, and clothing film is gone to the greatest extent, with stainless steel crusher in crushing 5min.With squeezing
Juice machine（8000rpm）Squeeze the juice 10min, then produces black garlic extract after centrifuge filters 5min with 60 mesh screen cloths.
It should be noted that " black garlic extract is in microbial population of animal intestinal tract is adjusted for expression way used in herein
Purposes " in the signified animal species of term " animal " be not particularly limited.According to an embodiment of the invention, the animal can be with
It is optimal to choose for any animal with enteron aisle organ, preferably mammal, more preferably rat, mouse, people.In addition, herein
Used in expression way " prevention or treatment " be primarily referred to as prevention or auxiliary treatment obesity, diabetes, hypertension, high fat of blood
And the disease such as tumour.
According to an embodiment of the invention, taking black garlic extract does not have any side effect, thus the administration of black garlic extract
Dosage is not particularly limited, as long as the quantity for being administered the probiotics such as Bifidobacterium in animal intestinal tract, Ruminococcus can be improved
And effectively adjust the gut flora of animal.According to the present invention some specific examples, by the black garlic extract with
0.3-1.0mg allicins/kg dosage is administered to the animal., will be described black according to other embodiments of the present invention
Garlic extract is administered with 0.5mg allicins/kg dosage to the animal.Thus, it is administered bifid bar in animal intestinal tract
The quantity increase of the probiotics such as bacterium, Ruminococcus is obvious, i.e. the significant effect of black garlic extract regulation microbial population of animal intestinal tract.
According to another aspect of the invention, the work based on above-mentioned black garlic extract in terms of microbial population of animal intestinal tract is adjusted
With present invention also offers purposes of the black garlic extract in medicine is prepared, the medicine is used to adjust microbial population of animal intestinal tract.
Said medicine can be used in adjusting microbial population of animal intestinal tract, and then can play enhancing by the effect of gut flora and exempt from
Epidemic disease power, fat-reducing, hypoglycemic, lowering blood pressure and blood fat, antitumor and other effects.Thus, according to an embodiment of the invention, the medicine
For strengthen immunity, prevention or treatment obesity, diabetes, hypertension, high fat of blood or tumour.
According to an embodiment of the invention, 0.3-1.0mg allicins/kg black garlic extract the animal is administered daily.
Thereby, it is possible to significantly improve to be administered the quantity of the probiotics such as Bifidobacterium in animal intestinal tract, Ruminococcus, the intestines of animal are adjusted
Road flora, and then effectively prevent or treat obesity, diabetes, hypertension, high fat of blood or tumour etc. by the effect of gut flora
Disease.
According to another aspect of the present invention, present invention also offers a kind of medicine.According to an embodiment of the invention, the medicine
Comprising：Black garlic extract；And pharmaceutically acceptable excipient.It is surprisingly found by the inventors that medicine of the invention can show
The quantity for improving and being administered the probiotics such as Bifidobacterium in animal intestinal tract, Ruminococcus is write, adjusts the gut flora of animal, and then
Effectively prevent or treat the diseases such as obesity, diabetes, hypertension, high fat of blood or tumour by the effect of gut flora.
According to an embodiment of the invention, the species of the excipient is not particularly limited, and is held as long as can be formed medicine
The formulation being easily administered.According to some specific examples of the present invention, the excipient is selected from adhesive, filler, painting
Membrane polymer, plasticizer, glidant, at least one of disintegrant and lubricant.
According to an embodiment of the invention, the formulation of medicine of the invention is not particularly limited, if can be convenient for
Medicine.According to some specific examples of the present invention, the medicine is in selected from capsule, pill, tablet, granule, oral liquid
Body, oral pastes, at least one form of aerosol and spray.It is described according to some currently preferred embodiments of the present invention
Medicine is in the form of capsule.Thus, it is easy to be administered.
According to an embodiment of the invention, the dosage of medicine of the invention is not particularly limited, can be with practical application
Flexibly selected according to the health status of administration object.According to some embodiments of the present invention, the dosage of the medicine is
0.3-1.0mg allicins/kg.Thus, the quantity for being administered the probiotics such as Bifidobacterium in animal intestinal tract, Ruminococcus significantly increases
Add, adjust the significant effect of microbial population of animal intestinal tract.
According to an embodiment of the invention, the medicine is used to adjust microbial population of animal intestinal tract.And then the work for passing through gut flora
With strengthen immunity, fat-reducing, hypoglycemic, lowering blood pressure and blood fat, antitumor and other effects can be played.Thus, according to the present invention's
Other embodiments, the medicine are used for strengthen immunity, prevention or treatment obesity, diabetes, hypertension, high fat of blood or swollen
Knurl.
In accordance with a further aspect of the present invention, present invention also offers a kind of food.According to an embodiment of the invention, the food
Comprising：Black garlic extract；And acceptable additive in bromatology.According to an embodiment of the invention, food energy of the invention
The quantity for being administered the probiotics such as Bifidobacterium in animal intestinal tract, Ruminococcus is enough significantly improved, effectively adjusts the enteron aisle of animal
Flora, and then effectively prevent or treat the diseases such as obesity, diabetes, hypertension, high fat of blood or tumour by the effect of gut flora
Disease.Meanwhile food of the invention also there are all raw materials to come from naturally, securely and reliably, raw material sources are easy to get extensively, preparation technology
Simply, store and transport and application is safe, low production cost, steady quality, advantage with long preservation period.
It should be noted that herein used in term " food " should broadly understood, its can be it is any can quilt
Edible form, i.e., in addition to the food form of routine, food of the invention can also be health products（Also sometimes referred to as " work(
Energy food "）, drink etc..According to some specific examples of the present invention, food of the invention is selected from biscuit, jam, candy, cake
At least one of point.
According to another aspect of the present invention, present invention also offers foregoing medicine or food, in regulation animal
Purposes in gut flora.Inventor has found, foregoing medicine or food of the invention is administered to animal, can significantly carry
Height is administered the quantity of the probiotics such as Bifidobacterium in animal intestinal tract, Ruminococcus, effectively adjusts the gut flora of animal, and then
Effectively prevent or treat the diseases such as obesity, diabetes, hypertension, high fat of blood or tumour by the effect of gut flora.
It should be noted that term " administration " used in herein should broadly understood, except the medicine of routine is given
Outside medicine, for can be understood as feeding animal or providing the food during food.
According to another aspect of the invention, present invention also offers a kind of method for adjusting microbial population of animal intestinal tract.According to this
The embodiment of invention, this method are to animal administration black garlic extract, foregoing medicine or food.According to this hair
Bright embodiment, it can be significantly improved using the method for the present invention and be administered the benefits such as Bifidobacterium in animal intestinal tract, Ruminococcus
The quantity of raw bacterium, the gut flora of animal is effectively adjusted, and then effectively prevented by the effect of gut flora or treat fat, sugar
Urinate the diseases such as disease, hypertension, high fat of blood or tumour.
In addition, according to an embodiment of the invention, black garlic extract and food of the invention comprising black garlic extract or
Medicine, strengthen effect of the effect better than the Radix Astragali of single component, FOS, chitosan oligosaccharide or yeast dextran of animal immunizing power
Effect.
It should also be noted that, new application --- the use in microbial population of animal intestinal tract is adjusted of the black garlic extract of the present invention
On the way, exactly the present inventor just has been surprisingly found that by arduous creative work and substantial amounts of experimental work.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Embodiment
Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining this hair
It is bright, and be not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to text in the art
Offer described technology or condition（Such as write with reference to J. Pehanorm Brookers etc., what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》,
The third edition, Science Press）Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, it is
Can be by the conventional products of acquisition purchased in market, such as can purchase from Illumina companies.
Black garlic extract of the present invention is commercially available, or is prepared on demand according to existing conventional techniques extraction
Arrive, do not tire out one by one state herein.The preparation method of black garlic extract is described in example 1 below, but is not to the present invention
Limitation.
Embodiment 1：The preparation of black garlic extract
Raw material：Fresh black garlic
Preparation method：From fresh black garlic of uniform size, the garlic clove of peeling is cleaned in peeling, and clothing film is gone to the greatest extent, with not
Steel crusher in crushing of becoming rusty 5min.Use juice extractor（8000rpm）Squeeze the juice 10min, then filters 5min through centrifuge with 60 mesh screen cloths
After produce black garlic extract.
Embodiment 2：The preparation of black garlic extract capsule
Raw material：Black garlic extract, granular starch
Preparation method：The parts by weight of black garlic extract 8, the parts by weight of granular starch 6 are weighed, using research of super-pine crush equipment processing powder
It is broken, black garlic extract, granular starch fine powder that grain diameter is 100 microns are obtained, homogeneous is into mixing fine powders, then by content
Material is placed in soft capsule pellet press, and soft capsule is pressed into according to the filling of existing soft capsule production technology.
Embodiment 3：The preparation of black garlic extract biscuit
Raw material：Flour, black garlic extract, sweetener, phosphatide, flavor enhancement, sodium bicarbonate, vegetable oil, milk powder, fresh hen egg, water
Preparation method：5 parts by weight black garlic extracts, 100 parts by weight flour are well mixed, add 4 parts by weight sugar, 0.5
Parts by weight phosphatide, 1.0 parts by weight refined salt, 0.2 parts by weight sodium bicarbonate, 8 parts by weight vegetable oil, 8 parts by weight milk powder, 2 parts by weight are fresh
Egg and 20 parts by weight drinking water, after above-mentioned raw materials are mixed, through adjusting powder, shaping, baking process that black garlic extract biscuit is made.
Embodiment 4：The preparation of black garlic extract jam
Raw material：Fresh fruit, black garlic extract, honey, essence
Preparation method：10 parts by weight black garlic extracts, 50 parts by weight fresh fruits, 40 parts by weight honey, 2 parts by weight are fragrant
Essence is well mixed, and black garlic extract jam is made through dry packing technique.
Embodiment 5：The preparation of black garlic extract candy
According to the molten sugar of the manufacture craft of candy, filtering, stir off, add black garlic extract when reconciling other additives, so
Mix, cool down afterwards, being molded, packaging, the candy containing black garlic extract is made.The technique in addition to black garlic extract process is added,
Other process ways and general candy（Such as sesame seed candy）Technique is identical.
Embodiment 6：The preparation of black garlic extract cake
Raw material：Wheat flour, vegetable oil, NMF, emulsifying agent, carbohydrate, black garlic extract
Preparation method：By 30 parts by weight vegetable oil, 1.0 parts by weight NMFs, 5 parts by weight Emulsifier, 30 parts by weight of saccharide,
25 parts by weight black garlic extracts mixing after be filled with gas, modulate creamy material, then mixed with 100 parts by weight wheat flours, into
Shape, baking obtain black garlic extract cake.
Embodiment 7：Rat acute toxicity test
By 6 week old specific-pathogen frees（SPF）SD rats 15 are only randomly divided into 3 groups, every group 5.By the embodiment of the present invention
The food that 2-6 is prepared is suspended in 0.5% methocel solution, respectively with 1g/kg body weight, 5g/kg body weight and 10g/
The dosage of kg body weight is once administered orally to every group of 5 SD rats, and death, clinical symptoms and the body weight for observing rat become
Change, and carry out blood test and hematochemical test, in postmortem, any exception of visual inspection thorax abdomen gastrointestinal tissue is existing
As.
Experimental result is shown：Black garlic extract product of the present invention does not cause any specific clinical symptoms, body weight
Change is dead, and blood test, hematochemical test and postmortem also do not change.Thus, due to until the agent of 10g/kg body weight
Amount will not cause any poisoning of rat to change, and can determine whether its median lethal dose（LD50）It is far longer than 10g/kg body weight, this hair
Bright described black garlic extract product is rated as safe and non-toxic material.
Embodiment 8：Black garlic extract regulation human body intestinal canal flora influences experiment
By black garlic extract product prepared by embodiment 2-6 according to the Ministry of Public Health《Health food is examined and assessment technique specification
（2003）》Regulation gut flora function human experiment experimental evaluation method evaluated.It is specific as follows：
1st, the packet of test-meal experiment：
The packet situation of test-meal experiment see the table below 1：
The test-meal experiment packet table of table 1
2nd, human experiment experimental method
Subject is randomly divided into 5 groups, every group of 10 people, group result is as follows：Group 1 is normal population, and group 2 is II types sugar
Patient is urinated, group 3 is hyperlipemic patients, and group 4 is hyperglycemic patients, and group 5 is tumor patient.Before subject's test-meal, equal nothing
Bacterium takes subject's excrement, and gut flora, the reference as background level are examined in 16S rDNA sequencings.
By the packet situation of table 1, group 1 takes black garlic extract capsule, group 2 takes black garlic extract biscuit, group 3 take it is black
Garlic extract jam, group 4 take black garlic extract candy, group 5 takes black garlic extract cake, after 4 weeks, take subject's excrement
Just, gut flora is examined in 16S rDNA sequencings, is contrasted with background level, is contrasted the increased multiple of its relative abundance.
3rd, gut flora detection method
Fecal sample carries out DNA extractions first, and PCR expands 16SrDNA V3~V5 regions, is then sequenced with 454 platforms,
Direction V5- is sequenced>V3.Each average 7795 tag of sample initial data, read long 400bp or so.Eventually for the tag of analysis
Number average out to 3235, reads long 265bp or so.
16S analyses mainly use mothur softwares（http://www.mothur.org/wiki/Mothur_manual）Bag
The analyses such as Quality Control, OTU clusters, annotation are included, and on the basis of data species taxonomy is completed, analyze the relatively rich of each species
Degree.
4th, experimental result
The result of the test of the present embodiment is as shown in table 2 below.
The sequencing data abundance contrast of each Pseudomonas of people's gut flora of table 2（Improved again compared with the background level before test-meal
Number）
Table 2 test result indicates that, test-meal the present invention black garlic extract product after, normal population, diabetes, high blood
Sugar, high fat of blood, the gut flora of tumor patient have obtained different degrees of improvement, black garlic extract product of the invention regulation
Gut flora significant effect.
Embodiment 9：The experiment of black garlic extract strengthen immunity function
According to following steps, influence of the research gained black garlic extract biscuit of the embodiment of the present invention 3 to animal immunizing power：
1st, test material
Experimental animal：BALB/C1 monthly age secondary mouses, male and female half and half, 20 ± 2g of body weight, are in a good state of health, by Wuhan
University Medical College Experimental Animal Center provides, production licence number：SCXK（Hubei Province）2008-0004.Every batch of animal is grouped at random.
The recommended dose of the black garlic extract biscuit of the gained of embodiment 3 is the daily 250mg/kgbw of mouse (body weight), point
Not using mouse recommend 0.5 times, 1 times and 2 times as low dose group (125mg/kgbw), middle dose group (250mg/kgbw),
High dose group (500mg/kgbw).
If 4 positive controls,
1 group of positive control is astragalus polyose capsule（Primary raw material：The Radix Astragali, oligoisomaltose, the double strange medicine company stocks in the Inner Mongol
Part Co., Ltd, the strong word G20040082 of state's food）,
2 groups of positive control is FOS oral liquid（Primary raw material：FOS, the limited public affairs of Zhuhai sage protozoa science and technology
Department, the strong word G20050336 of state's food）,
3 groups of positive control is chitosan oligosaccharide capsule（Primary raw material：Chitosan oligosaccharide, Xiamen Blue Bay Science and Technology Co., Ltd., the strong word of state's food
G20110158）,
4 groups of positive control is yeast dextran capsule（Primary raw material：Yeast dextran, Nanjing great Yuan beauty and health cares are limited
Company, the strong word G20110445 of state's food）.
Other main reagent reagents are：Sheep red blood cell (SRBC)（SRBC）, Hank ' s liquid, guinea pig serum, prepared Chinese ink.
The wherein compound method of Hank ' s liquid：
（1）Store liquid：
NaCl：80.00g KCl：4.00g Na2HPO4·12H2O：1.52g KH2PO4:0.60g, glucose：4.0g
0.4% phenol red liquid：50.00mL, double distilled waters, which add, is settled to 100mL, 115 DEG C of autoclaving 15min, stored frozen.
（2）Working solution：
Double distilled waters of 9 times of volumes, 115 DEG C of autoclaving 15min are added to storage liquid.Face the 5.6% of used time sterilizing
NaHCO3Solution adjusts pH value to 7.2-7.4 (solution is in orange red).
The preparation of physiological saline：9.00g NaCl are dissolved in produced in the double distilled waters of 991g 0.9% NaCl solution.
2nd, test method
Administration by gavage is taken in experiment.Once, blank control group fills equal volume distilled water to daily gavage.Each group mouse is continuously given
After giving tested material 30 days, indices are determined.
The influence of 2.1 pairs of mouse weights, thymus gland/body weight, spleen/body weight
Mouse stomach takes off neck and put to death after 30 days, weigh.Thymus gland and spleen are taken, weighs thymus gland and spleen weight, measure respectively
Thymus gland/body weight value, and spleen/body weight value.
2.2 pairs of mouse antibodies cellulations（PFC）Influence
The measure of antibody-producting cell, hemolytic plaque test, it is that a kind of vitro detection single antibody forms cell（Slurry is thin
Born of the same parents）Method.
Every mouse is immunized in intraperitoneal injection 0.2mL2% (v/v) SRBC.After immune 5 days, take mouse spleen that cell is made dense
Spend for 5 × 106Individual/mL splenocyte liquid.L% solution is made in dissolving agarose, is incubated in 48 DEG C of water-baths, dense with 2 times of equivalent
Hank ' the s liquid mixing of degree, is dispensed into small test tube, often pipe 0.5mL.0.05mL10%SRBC and 0.01mL splenocytes are added to hang
Liquid, it is rapid to mix, it is poured on slide.1h is incubated in 37 DEG C of insulating boxs, by complement（Guinea pig serum）It is added in slide groove, then
Incubate 1.5h.Count plaque.
2.3 the influence to mouse hemolytic antibody nucleus formation（The measure of serum hemolysin）
Every mouse is immunized in intraperitoneal injection 0.2mL2% (v/v) SRBC.After immune 5 days, pluck eyeball and take blood in centrifuge tube,
1h is placed, 3000rpm is centrifuged 5 minutes, collects serum.400 times of normal saline dilution of serum, sequentially add the mouse blood of dilution
Clear 1.0mL, 10% (v/v) SRBC0.5mL, complement (use physiological saline 1：10 dilutions) 1.00mL.Separately set with physiological saline generation
The control tube of the not increase serum replaced.30 minutes stopped reactions of DEG C refrigerator, 3000rpm centrifugations are set to 0 after putting 37 DEG C of water-baths 20 minutes
5min.Supernatant 1.0mL is taken, OD value is determined at spectrophotometer 540nm wavelength.
The influence of 2.4 pairs of mouse carbonic clearances
4 times of prepared Chinese ink, 0.1mL/10g body weight are diluted to every mouse tail vein injection.The timing immediately after prepared Chinese ink injection.
After prepared Chinese ink is injected l/min, take blood 0.02mL to add to 2.0mL0.1%Na within 10 minutes2CO3Solution（That is 1g/L）In, in
OD value is determined at 600nm wavelength, with Na2CO3Solution compares.Separately liver and spleen is taken to weigh.Come with phagocytic index (α)
Represent the ability of mouse carbonic clearance.
3rd, result of the test
3.1 body weight, thymus gland/body weight, the measurement result of spleen/body weight
Each tested material is to mouse weight, thymus gland/body weight ratio, and the influence of spleen/body weight ratio, measurement result such as table
3rd, shown in 4 and 5.
Influence of the 3 each tested material of table to mouse weight
As shown in Table 3, before the tested material of orally administration mouse various dose, and after 30 days, basic, normal, high 3 dosage groups
Compared with blank control group and positive controls, body weight is without significant difference (P>0.05), i.e., to mouse feed 3 it is low, in,
The sample of high dose group and 4 positive controls is on the body weight of mouse without influence.
Influence of the tested material of table 4 to mouse thymus/body weight ratio
| Group | Dosage | Number of animals (only) | Thymus gland/body weight ratio (g/g) | P values |
| Blank control group | 0 | 12 | 0.0017±0.0004 | - |
| 1 group of positive control | 1 | 12 | 0.0021±0.0003 | 0.0321 |
| 2 groups of positive control | 0.5mL | 12 | 0.0020±0.0002 | 0.0321 |
| 3 groups of positive control | 1 | 12 | 0.0021±0.0001 | 0.0352 |
| 4 groups of positive control | 1 | 12 | 0.0020±0.0006 | 0.0259 |
| Low dose group | 125mg/kg·bw | 12 | 0.0024±0.0004 | 0.0254 |
| Middle dose group | 250mg/kg·bw | 12 | 0.0025±0.0002 | 0.0215 |
| High dose group | 500mg/kg·bw | 12 | 0.0028±0.0008 | 0.0289 |
As shown in Table 4, the tested material of orally administration mouse various dose is after 30 days, compared with blank control group, each dosage
There were significant differences (P ＜ 0.05) for group thymus gland/body weight ratio, i.e., the sample of 3 dosage groups and 4 positive controls is fed to mouse
Product increased to thymus gland/body weight ratio of mouse.
Influence of the tested material of table 5 to mouse spleen/body weight ratio
| Group | Dosage | Number of animals (only) | Thymus gland/body weight ratio (g/g) | P values |
| Blank control group | 0 | 12 | 0.0041±0.0001 | - |
| 1 group of positive control | 1 | 12 | 0.0048±0.0003 | 0.0386 |
| 2 groups of positive control | 0.5mL | 12 | 0.0049±0.0005 | 0.0425 |
| 3 groups of positive control | 1 | 12 | 0.0050±0.0005 | 0.0415 |
| 4 groups of positive control | 1 | 12 | 0.0054±0.0002 | 0.0423 |
| Low dose group | 125mg/kg·bw | 12 | 0.0055±0.0004 | 0.0302 |
| Middle dose group | 250mg/kg·bw | 12 | 0.0059±0.0008 | 0.0332 |
| High dose group | 500mg/kg·bw | 12 | 0.0060±0.0003 | 0.0456 |
As shown in Table 5, the tested material of orally administration mouse various dose is after 30 days, compared with blank control group, each dosage
There were significant differences (P ＜ 0.05) for group spleen/body weight ratio, i.e., the sample of 3 dosage groups and 4 positive controls is fed to mouse
Product increased to spleen/body weight ratio of mouse.
3.2 antibody-producting cell（PFC）Measurement result
Antibody-producting cell（PFC）Testing result is as shown in table 6.
Influence of the tested material of table 6 to mouse antibodies cellulation number
| Group | Dosage | Number of animals (only) | Antibody-producting cell number | P values |
| (Ⅹ±S) | ||||
| Blank control group | 0 | 12 | 70±19 | - |
| 1 group of positive control | 1 | 12 | 87±13 | 0.0458 |
| 2 groups of positive control | 0.5mL | 12 | 85±18 | 0.3210 |
| 3 groups of positive control | 1 | 12 | 82±21 | 0.3321 |
| 4 groups of positive control | 1 | 12 | 85±73 | 0.2104 |
| Low dose group | 125mg/kg·bw | 12 | 96±18 | 0.0348 |
| Middle dose group | 250mg/kg·bw | 12 | 99±13 | 0.0401 |
| High dose group | 500mg/kg·bw | 12 | 85±17 | 0.0647 |
As shown in Table 6, the tested material of orally administration mouse various dose is after 30 days, positive right compared with blank control group
According to 1 group and low, middle dose group PFC quantity there were significant differences (P ＜ 0.05), illustrate high dose group and 3 positive controls
Group is on mouse PFC without influence, and 1 group of positive control, low dose group, middle dose group have a significant impact to mouse PFC.
The measurement result of 3.3 serum hemolysins
The measurement result of serum hemolysin is as shown in table 7.
Influence of the tested material of table 7 to mice serum hemolysin
| Group | Dosage | Number of animals (only) | HC50 | P values |
| Blank control group | 0 | 12 | 120±17 | - |
| 1 group of positive control | 1 | 12 | 135±24 | 0.1556 |
| 2 groups of positive control | 0.5mL | 12 | 137±20 | 0.2574 |
| 3 groups of positive control | 1 | 12 | 140±13 | 0.0325 |
| 4 groups of positive control | 1 | 12 | 130±15 | 0.2568 |
| Low dose group | 125mg/kg·bw | 12 | 162±21 | 0.0089 |
| Middle dose group | 250mg/kg·bw | 12 | 167±18 | 0.0075 |
| High dose group | 500mg/kg·bw | 12 | 142±23 | 0.0698 |
As shown in Table 7, the tested material of orally administration mouse various dose is after 30 days, compared with blank control group, low dosage
The HC of group, middle dose group50There are pole significant difference (P ＜ 0.01), the HC that 3 groups of positive control50There were significant differences (P ＜ 0.05),
Illustrate low dose group and middle dose group of the present invention, can pole significantly increase mice serum hemolysin HC50, it is positive
Mice serum hemolysin HC can be significantly improved by compareing 3 groups50, and 1 group of positive control, 2 groups of positive control, 4 groups of positive control, height
Dosage group is to mice serum hemolysin HC50Without influence.
3.4 mouse carbonic clearance experimental results
Mouse carbonic clearance experimental result is as shown in table 8.
Influence of the tested material of table 8 to mouse carbonic clearance
| Group | Dosage | Number of animals (only) | Phagocytic index α (Ⅹ ± S) | P values |
| Blank control group | 0 | 12 | 3.19±0.21 | - |
| 1 group of positive control | 1 | 12 | 4.17±0.39 | 0.0349 |
| 2 groups of positive control | 0.5mL | 12 | 4.19±0.41 | 0.0321 |
| 3 groups of positive control | 1 | 12 | 3.82±0.23 | 0.2100 |
| 4 groups of positive control | 1 | 12 | 3.98±0.45 | 0.5236 |
| Low dose group | 125mg/kg·bw | 12 | 4.00±0.21 | 0.0325 |
| Middle dose group | 250mg/kg·bw | 12 | 4.53±0.21 | 0.0072 |
| High dose group | 500mg/kg·bw | 12 | 4.75±0.49 | 0.0086 |
As shown in Table 8, the tested material of orally administration mouse various dose is after 30 days, compared with blank control group, the present invention
Middle dose group, high dose group there is pole significant difference (P to mouse macrophage phagocytic activity<0.01), low dose group, sun
Property control 1 group, 2 groups of positive control there is significant difference (P to mouse macrophage phagocytic activity<0.05), remaining group pair
Mouse carbonic clearance ability is without influence.This shows that black garlic extract product of the present invention has enhancing mononuclear macrophage phagocytosis
The effect of function, the non-specific immune function of mouse can be strengthened.
Result shows before and after the experiment of immunity function is improved as the black garlic extract product described in the invention described above, respectively
Mouse weight, thymus gland/body weight ratio, spleen/body weight ratio are criticized without significant difference.Detected in mouse antibodies cellulation real
Test, in the experiment of serum hemolysin determination experiment and mouse carbonic clearance, its result has significant difference.Thus, it is believed that this hair
Bright described black garlic extract product has function and the effect of strengthen immunity, and black garlic extract of the present invention is produced
Effect of the product to strengthen immunity is better than the Radix Astragali, FOS, chitosan oligosaccharide, the action effect of yeast dextran of single component.
Embodiment 10：The hypoglycemic zoopery of black garlic extract
According to the Ministry of Public Health《Health food is examined and assessment technique specification（2003）》Auxiliary hyperglycemic function animal it is real
Evaluation method is tested, black garlic extract product obtained by embodiment 3-6 is evaluated.It is specific as follows：
1st, test method：Male SD rat 40 is taken, preparing low dose of streptozotocin according to standard method causes diabetes big
Mouse model, 4 groups, every group 10 are randomly divided into according to rat blood sugar value after three days.Each group continuous gavage administration 8 days, one time a day,
Dosage is 4.5mg allicins/kg, and last time two hours after administration takes blood, determines blood glucose value.
2nd, subjects：Tested SD rats are randomly divided into 4 groups, every group 10, tested SD rat primaries diet control and
Activity is constant, and group 1 takes black garlic extract biscuit, group 2 takes black garlic extract cake, group 3 takes black garlic extract jam, group
4 take black garlic extract candy.
3rd, experimental result
The hypoglycemic result of different experiments group is as shown in table 9.
The different experiments group blood sugar decreasing effect of table 9 compares
Note：（1）The * P compared with blank group<0.01；（2）Compared with blank group△P<0.05
The animal test results of table 9 show that black garlic extract product of the present invention is to Experimental Diabetes of Mice
Hyperglycaemia is respectively provided with decline effect, shows the black garlic extract product of the present invention and have hypoglycemic function have to diabetes
Significant auxiliary therapeutic action.
Embodiment 11：The zoopery of black garlic extract reducing blood lipid
According to the Ministry of Public Health《Health food is examined and assessment technique specification（2003）》Auxiliary lipid-lowering function animal it is real
Evaluation method is tested, black garlic extract product obtained by embodiment 3-6 is evaluated.It is specific as follows：
1st, test material and method：
Wistar male rats 40,200 ± 20g of body weight, are provided by Wuhan University's Experimental Animal Center.It is high to induce rat
Blood fat disease pathological model formula（High lipid food）：1% cholesterol is added in basal feed, 10% yolk, 10% lard, is dried into
Block.
The preparation method of Hyperlipemia model rat：Wistar male rats are randomly divided into 4 groups (every group 10), i.e., it is high
Blood fat rat（Hyperlipemia model control group）, feed high lipid food（Feed 93.8%, cholesterol based on high lipid food formula
1.0%th, lard 5.0%, cholate 0.2%）, do not give tested material；
Medication：
Tested Hyperlipemia model rat is randomly divided into 4 groups, every group 10, tested Hyperlipemia model rat original is drunk
Food control and activity are constant, and group 1 takes black garlic extract biscuit, group 2 takes black garlic extract cake, group 3 takes black garlic extraction
Thing jam, group 4 take black garlic extract candy.
All animals are raised 20 days under identical environment, and dosage is 4.5mg allicins/kg.Last give respectively by
After the 4h for trying thing, hematometry serum total cholesterol (TC mmol/L, iron chloride development process), serum triacylglycerol (TG mmol/ are taken
L, acetylacetone method), HDL-C (HDL-C mmol/L, phosphotungstic acid method).
2nd, experimental result
Each group rat blood serum total TG, TC and HDL-C content are shown in Table 10.
The different experiments group of table 10 drop TC, TG, HDL-C effect compare
Note：（1）* P is represented<0.05；（2）* represents P<0.01
Table 10 test result indicates that：After test-meal 30 days, the black garlic extract product of 3-6 of embodiment of the present invention preparations is taken
The cholesterol of rat, triglyceride compared with there is obvious reduction before test-meal, HDL is compared with there is obvious liter before test-meal
Height, show that black garlic extract product of the present invention has significant auxiliary therapeutic action to hyperlipidemia.
Embodiment 12：Black garlic extract is hypoglycemic, the experiment of the human experiment of reducing blood lipid
According to the Ministry of Public Health《Health food is examined and assessment technique specification（2003）》Auxiliary hyperglycemic, hypolipemic function
Human experiment experimental evaluation method, black garlic extract product obtained by embodiment 2-6 is evaluated.It is specific as follows：
1st, human experiment and test method：
50 type II diabetes people are selected by the principle of voluntariness, the wherein people of male 35, the people of women 15, the range of age 43-75 year,
Without complication such as serious conscience kidneys, in taking before test-meal after contrast design.
Subject is randomly divided into 5 groups, every group of 10 people, subject is former to use medicine species, diet control and activity not
Become, group 1 takes black garlic extract capsule, group 2 takes black garlic extract biscuit, group 3 takes black garlic extract cake, group 4 is taken
Black garlic extract jam, group 5 take black garlic extract candy, daily 1 time, in one after each meal, continuously take 30 days, are administered
Dosage is 0.5mg allicins/kg.
The change of patient blood pressure, stool and urine, body weight is monitored, and determines the blood glucose with postprandial 2 hours on an empty stomach（With grape glycosyloxy
Chemical-enzyme method）；Determine blood fat（T-CHOL TC, triglycerides TG and HDL HDL-C）.
2nd, judgment criteria：
It is effective：Cardinal symptom disappears, and declines on an empty stomach or before postprandial 2h blood glucose relatively treatment and is no more than 30.0%.
Effectively：Cardinal symptom is obviously improved, and declines before relatively being treated with postprandial 2h blood glucose on an empty stomach and is no more than 10.0%.
It is invalid：Cardinal symptom is not improved, and 10.0% is dropped by less than before relatively being treated with postprandial 2h blood glucose on an empty stomach.
3rd, human experiment result：
After test-meal 30 days, take the patient of each black garlic extract product, compared with before test-meal, the blood pressure of patient, stool and urine,
Body weight there are no significant difference, but fasting blood-glucose, 2h-plasma glucose have obvious attenuating, and concrete outcome is shown in Table 11.
On the influence to the blood fat before and after test-meal：The patient of each black garlic extract product is taken, compared with before test-meal, examination
Compared with there is obvious reduction before test-meal, HDL is bright compared with having before test-meal for food cholesterol of patient after 30 days, triglyceride
Aobvious rise, concrete outcome are shown in Table 12.
As can be seen here, black garlic extract product of the present invention has obvious auxiliary hyperglycemic, the health care of reducing blood lipid
Function, and to the harmless effect of tested population health.
Table 11 foretastes crowd's blood glucose test results
Table 12 foretastes crowd's lipids detection result
Note：（1）* P is represented<0.05；（2）* represents P<0.01
Embodiment 13：Black garlic extract antitumor animal is tested
According to the Ministry of Public Health《Health food is examined and assessment technique specification（2003）》Adjunct antineoplastic function animal it is real
Evaluation method is tested, black garlic extract product obtained by embodiment 3-6 is evaluated.It is specific as follows：
1st, test material and method：
BALB/c mouse (4-6 week old, male, body weight 20-24g) 50, by the hepatoma cells of exponential phase
It is subcutaneous that MM45T.Li is seeded in the right armpit of BALB/c mouse, every inoculation 5 × 107Individual cell, establishes Transplanted tumor model.
Medication：
Tested rat model is randomly divided into 5 groups, every group 10, original diet control and activity are constant, and group 1 takes black garlic
Extract biscuit, group 2 take black garlic extract cake, group 3 takes black garlic extract jam, group 4 takes black garlic extract candy,
Group 5 takes physiological saline, successive administration 30 days, and dosage is 4.5mg allicins/kg.Observe mouse general feelings before and after treatment
Condition and tumor size change.
1 tumour most major diameter (a) and most minor axis (b) are measured with slide measure within every 2 days, after into knurl by formula TV=a × b2/
2 obtain knurl body approximate volumes (TV：Volume).Medicine tumor-inhibiting action is evaluated by the following method：It is dynamic in putting to death for the 2nd day for last dose
Thing weighs tumor weight, and as the following formula：
Calculate medicine tumour inhibiting rate (IR)：Tumour growth tumour inhibiting rate=(the average knurl weight-black garlic extract product group of control group is put down
Equal knurl weight) average knurl weight × 100% of/control group.Acted on inhibition rate of tumor growth IR evaluation Antitumor Activity of Drugs, curative effect is commented
Price card is accurate：IR ＜ 30 are invalid, and IR >=30 are effective.
Result of the test see the table below 13.
The effect of the different experiments group Tumor growth inhibition of table 13 compares
The result of the test of table 13 shows：Compared with control group, the rat of the black garlic extract product of the present invention, test-meal are taken
Medicine tumour inhibiting rate has obvious reduction after 30 days, shows that black garlic extract product of the present invention has significantly to antitumor
Auxiliary therapeutic action.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
Claims (4)
1. purposes of the black garlic extract in medicine is prepared, the medicine is for improving Bifidobacterium in animal intestinal tract, excrement bar of dwelling
Bacterium, Eubacterium, Ross visit the quantity of auspicious Salmonella, fecal bacteria, butyric acid vibrios, lactobacillus and galactococcus,
Wherein, the preparation method of the black garlic extract is：Clothing film black garlic 5min is removed with the fresh peeling of crusher in crushing, with squeezing
Juice machine is squeezed the juice 10min with 8000rpm rotating speed, then filters 5min through centrifuge with 60 mesh screen cloths.
2. purposes according to claim 1, it is characterised in that the dosage to the animal is 0.3-1.0mg daily
Allicin/kg.
3. purposes according to claim 1, it is characterised in that the dosage to the animal is 0.5mg garlics daily
Element/kg.
4. purposes of the black garlic extract in food is prepared, the food is for improving Bifidobacterium in animal intestinal tract, excrement bar of dwelling
Bacterium, Eubacterium, Ross visit the quantity of auspicious Salmonella, fecal bacteria, butyric acid vibrios, lactobacillus and galactococcus,
Wherein, the preparation method of the black garlic extract is：Clothing film black garlic 5min is removed with the fresh peeling of crusher in crushing, with squeezing
Juice machine is squeezed the juice 10min with 8000rpm rotating speed, then filters 5min through centrifuge with 60 mesh screen cloths.
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| 大蒜素对新生乳鼠肠道细菌移位的影响;牛志民，等;《中国生化药物杂志》;20121231;第33卷(第4期);第411页摘要，右栏第2段-第412页左栏第2段 * |
| 黑蒜的营养价值及保健作用的研究进展;雷逢超，等;《食品工业科技》;20121231;第33卷(第13期);第430页左栏第4-5段，第431页右栏第2-4段 * |
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