CN110075128A - A kind of pharmaceutical compositions for being used to enhance immune function comprising enzyme treated bee milk powder as effective component - Google Patents
A kind of pharmaceutical compositions for being used to enhance immune function comprising enzyme treated bee milk powder as effective component Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L21/00—Marmalades, jams, jellies or the like; Products from apiculture; Preparation or treatment thereof
- A23L21/20—Products from apiculture, e.g. royal jelly or pollen; Substitutes therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/64—Insects, e.g. bees, wasps or fleas
- A61K35/644—Beeswax; Propolis; Royal jelly; Honey
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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Abstract
The present invention relates to the pharmaceutical compositions for being used to enhance immune function comprising enzyme treated bee milk (Enzyme treated royal jelly, ERJ) powder as effective component.Enzyme treated bee milk (ERJ) powder of the invention does not include the albumen to cause allergic reaction, anti-oxidant and anti-inflammatory activity is outstanding, actively proliferating immune cells, the outstanding activity of induced natural killer cell, to which brilliant immune function reinforcing effect be presented, therefore the pharmaceuticals or health functional food of enhancing immune function can be effectively served as.
Description
Technical field
The present invention relates to be used as to have comprising enzyme treated bee milk (Enzyme treated royal jelly, ERJ) powder
The pharmaceutical compositions for being used to enhance immune function of effect ingredient.
Background technique
As modern gets higher the concern of Health and Living quality, such as cancer, immune deficiency, idiocrasy autoimmunity
Relevant to immune class continuing disease increases, the research and product for enhancing immune health functional food be in it is increased become
Gesture.Therefore, although domestic and international economic depression, health functional food market scale is 100,000,000 7,920 hundred million yuan in South Korea in 2013,
Increase by 5.2% than last year, the past 5 years average growth rates per annum are 11.5%, and lasting growth trend is presented.
Also, improve relevant product to immune function according to the functional red ginseng of health functional food, chlorella etc.
Occupation rate is 25%, occupies highest specific gravity, and is parsed into the consumer demand for reflecting a variety of stratum, more various
Immune function improves relevant health functional food raw material and product sustainable growth.
Royal jelly digests and assimilates the substance prepared after honey and pollen by bottleneck throat as worker bee, is used as small children in bee colony
The food of the larva of worm and queen bee.The white gels form of royal jelly tart flavour Qiang Bingcheng local fragrance, protein abundance especially remove
Comprising including dimension life except fatty acid 10-HAD (10-HAD, 10-hydroxy-2-decenoic acid)
Element and pantothenic acid etc..It was reported that the effect of as royal jelly, is effective to hyperlipidemia, artery sclerosis, aging, immune etc., in food
In public allusion quotation using royal jelly raw material and product benchmark specification by 10-HAD content as benchmark.As young worker bee
The ingredient that decomposes and synthesize in vivo is also known as bee milk after intake honey and pollen, begin to function as in the past nutritious subsidy food or
Pharmaceuticals, it is well known that in royal jelly ingredient 10-HAD to the raising of immunity, pre- anti-osteoporosis,
Beautifying skin etc. has prevention and improvement, absorbs in the whole world as health functional food.On the other hand, in South Korea
Health functional food functional raw materials in 2010 examine again in because scientific effect/Evaluation of Functional data is insufficient from health care function
It can be deleted in food functionality raw material project, to cannot function as health functional food sale.Royal jelly not only in overseas, and
And the sustainable requests of consumer relevant to the functional raw material of royal jelly are high also in South Korea, but currently without functionality
The royal jelly product authenticated, it is therefore desirable to the royal jelly product that research and development are assert by food and drug safety department.
Enzyme treated bee milk includes by handling protease to royal jelly freeze-dried powder, to obtain the step of enzymatic treatment object
Suddenly, the protease for being used in it includes endo protease, exoproteinase, exoproteinase and endo protease.Existing bee
Royal jelly includes to generate anaphylactoid various anaphylaxis substances to organism, but it is logical to be used in enzyme treated bee milk of the invention
It crosses and destroys or change the structure of anaphylaxis substance to reduce anaphylaxis, total free amino acid content increases, as in food public affairs allusion quotation
The contents of 10-HAD of index components keep constant.
As described above, it is well known that a variety of pharmacological effects relevant to royal jelly, but do not illustrate at enzyme so far
Whether reason royal jelly powder has effects that enhance immune function, and is in the state studied this.
In this regard, the present inventor executes immune function enhancing research, knot to enzyme treated bee milk powder for developing new drug object
Fruit confirms that enzyme treated bee milk powder has immune function reinforcing effect, so as to complete the present invention.
Summary of the invention
It is an object of the present invention to provide immune for enhancing as effective component comprising enzyme treated bee milk powder
The pharmaceutical compositions of function.
It is a further object of the present invention to provide exempt from as effective component for enhancing comprising enzyme treated bee milk powder
The food compositions of epidemic disease function.
Another object of the present invention is to provide exempt from as effective component for enhancing comprising enzyme treated bee milk powder
The health functional food of epidemic disease function.
A further purpose of the present invention is to provide the method for the immune function of enhancing animal, including will be at a effective amount of enzyme
The step of reason royal jelly powder is administered into the animal for needing to enhance immune function other than the mankind.
To achieve the goals above, the present invention is provided comprising enzyme treated bee milk powder as effective component for enhancing
The pharmaceutical compositions of immune function.
Also, the present invention is provided comprising enzyme treated bee milk powder as effective component for enhancing the food of immune function
Product composition.
Also, the present invention is provided comprising enzyme treated bee milk powder as effective component for enhancing the guarantor of immune function
Health-care function food.
Also, the present invention provides the method for the immune function of enhancing animal, including by a effective amount of enzyme treated bee milk powder
End is administered into the step of animal for needing to enhance immune function other than the mankind.
Enzyme treated bee milk (ERJ) powder of the invention does not include the albumen to cause allergic reaction, anti-oxidant and anti-inflammatory
Activity is outstanding, actively proliferating immune cells, the outstanding activity of induced natural killer cell, so that brilliant immune function be presented
Energy reinforcing effect, therefore the pharmaceuticals or health functional food of enhancing immune function can be effectively served as.
Detailed description of the invention
Fig. 1 is the figure for showing the anaphylactogen of removal enzyme treated bee milk powder.
Fig. 2 is the figure for showing enzyme treated bee milk powder and having an impact to Spleen cell proliferation.
Fig. 3 a, Fig. 3 b are the figure for showing enzyme treated bee milk powder and having an impact to macrophage proliferation.
Fig. 4 a is to show enzyme treated bee milk powder to the α as Antioxidant Indexes-diphenyl-β-picryl hydrazine (DPPH, α-
Diphenyl- β-picrylhydrazyl) figure that has an impact of activity.
Fig. 4 b is to show enzyme treated bee milk powder to synthesize (ROS, Reactive to the active oxygen as Antioxidant Indexes
Oxygen synthesis) figure that has an impact of activity.
Fig. 4 c be show enzyme treated bee milk powder to as Antioxidant Indexes superoxide dismutase (SOD,
Superoxide dismutase) figure that has an impact of activity.
Fig. 4 d is to show enzyme treated bee milk powder to the glutathione (GSH, Glutathione) as Antioxidant Indexes
The figure that activity has an impact.
Fig. 4 e is to show enzyme treated bee milk powder in peritoneal macrophage to nitric oxide (NO, Nitric oxide)
The figure that has an impact of expression quantity.
Fig. 5 a is to show enzyme treated bee milk powder to the tumour for generating the intracellular inflammation in participation peritoneal macrophage
The figure that necrosis factor-alpha (TNF-α, Tumor necrosis factor- α) has an impact.
Fig. 5 b is to show enzyme treated bee milk powder to do to the γ-for participating in the intracellular inflammation in peritoneal macrophage is generated
Disturb the figure that plain (IFN-γ) has an impact.
Fig. 6 is the nitric oxide shown in the peritoneal macrophage of the experimental animal of intake enzyme treated bee milk powder
(NO) figure that expression quantity has an impact.
Fig. 7 is the figure for showing enzyme treated bee milk powder and having an impact to the natural killer cell activity in splenocyte.
Fig. 8 a is the figure shown related to the measurement of animal model dietary amount.
Fig. 8 b is to determine the related figure shown to animal model weight changed measurement.
Fig. 9 a is to show enzyme treated bee milk powder to the interleukin-1 ' beta ' (IL-1 that index occurs as internal inflammation
The figure that activity β) has an impact.
Fig. 9 b is to show enzyme treated bee milk powder to the interleukin-6 (IL-6) that index occurs as internal inflammation
The figure that has an impact of activity.
Fig. 9 c is to show enzyme treated bee milk powder to the tumor necrosis factor-alpha (TNF- that index occurs as internal inflammation
The figure that activity α) has an impact.
Fig. 9 d is to show enzyme treated bee milk powder to the interleukin 10 (IL- that index occurs as internal inflammation
10) figure that activity has an impact.
Fig. 9 e is to show enzyme treated bee milk powder to the interleukin 2 (IL-2) that index occurs as internal inflammation
The figure that has an impact of activity.
Fig. 9 f is to show enzyme treated bee milk powder to the gamma interferon (IFN-γ) that index occurs as internal inflammation
The figure that activity has an impact.
Figure 10 is the figure for showing enzyme treated bee milk powder and having an impact to the Spleen cell proliferation in animal model.
Figure 11 is to show enzyme treated bee milk powder to have an impact the natural killer cell activity in animal model
Figure.
Specific embodiment
Hereinafter, referring to the examples and drawings that are described in detail later, the clear advantages of the present invention of energy, feature and realize that these are excellent
The method of point and feature.But the invention is not limited to embodiments as disclosed below, can be with mutually different variform
It embodies, the present embodiment is served only for keeping disclosure of the invention more complete, and in order to the common skill of the technical field of the invention
Art personnel completely inform scope of the invention and provide that the present invention is only defined by inventing claimed range.It is complete in specification
Wen Zhong, identical appended drawing reference indicate identical structural element.Also, and/or include each related structural element and one
A above whole combinations.Term used in the present specification is not intended to restrict the invention for illustrating embodiment.?
In this specification, as long as not specifically mentioned in sentence, then singular shape further includes plural shape.Used in the description
" including (comprises) " and/or " including (comprising) " in addition to related structural element, step, movement and/or
Element, it is not excluded that the presence or addition of more than one another structural element, step and/or movement.
Hereinafter, the reagent used in embodiment and experimental example is as the reagent that can be obtained on the market, using most quality product,
Unless otherwise stated, being directed to following reality using the reagent of Sigma-Aldrich (Sigma-Aldrich) company of being purchased from
It tests as a result, find out average and standard deviation if necessary, and detection statistics conspicuousness.
The present invention is provided comprising enzyme treated bee milk powder as effective component for enhancing the pharmacy group of immune function
Close object.
In the present specification, term " enzyme " becomes the catalyst chemically reacted in life entity, refers to that biological cell produces
Protein macromolecule organic catalyst.There are various enzymes in all biologicals such as animal, plant, and participate in life entity into the cell
Chemical reaction.
Above-mentioned enzyme treated bee milk powder may include to freeze-drying royal jelly processing protease (proteinase (Protease) or
Person's peptase (Peptidase)) it is carried out to obtain the enzymatic treatment step of enzymatic treatment object.
At this point, above-mentioned protease can be for selected from by endo protease and exoproteinase, exoproteinase and inscribe
One or more of the group of protease composition, it is preferable that can be for selected from by fungoid endo protease and exoproteinase, true
One or more of the group of bacterium property exoproteinase and bacillary endo protease composition, it is highly preferred that can for selected from by
The group of fungoid endo protease and exoproteinase, fungoid exoproteinase and bacillary Alcalase composition
One or more of.It is processed into the freeze-drying royal jelly fungoid relative to 100 parts by weight in one embodiment of this invention
Endo protease and exoproteinase, fungoid exoproteinase and bacillary Alcalase content are respectively 1 weight
Measure part.
Fungoid endo protease and exoproteinase, for example, can be aminopeptidase (aminopeptidase) and carboxylic peptide
The mixed enzyme of enzyme (carboxypeptidase) can be Probeef 2000P, Sumizyme FP-G etc. as commercially available example.
Aminopeptidase and carboxypeptidase mixed enzyme can derive from fungi, and fungi as described above can be aspergillus oryzae (Aspergillus
oryzae).As commercially available example, include the aminopeptidase from aspergillus oryzae (Aspergillus oryzae)
(aminopeptidase) and carboxypeptidase (carbo xypeptidase) mixed enzyme.
Also, fungoid exoproteinase for example, can be leucine aminopeptidase (leucyl aminope ptidase),
It can be Prozyme 2000P etc. as commercially available example.Leucine aminopeptidase can derive from fungi, and fungi as described above can be with
For aspergillus oryzae (Aspergillus oryzae).Commercially available example includes the bright ammonia from aspergillus oryzae (Aspergillus oryzae)
Acyl aminopeptidase (leucyl aminopeptidase).
Also, bacillary endo protease for example, can for Zinc metalloproteinase (metalloendopeptidase) or
Serine proteinase (serine protease).Zinc metalloproteinase can be for example bacilysin (bacillolysin), make
It can be Alphalase NP etc. for commercially available example.Also, serine proteinase (serin protease) for example can be withered grass bar
Bacterium proteinase (subtilisin) can be food-grade alkali proteinase (Foodpro alkaline protease) as commercially available example.
Zinc metalloproteinase as described above can be one kind of bacillary neutral endo protease.Also, serine proteinase can be thin
One kind of bacterium property Alcalase.
Bacterium can be bacillus amyloliquefaciens (Bacillus amyloliquefaciens) or bacillus licheniformis
(Bacillus licheniformis)。
That is, bacillary endo protease can be from bacillus amyloliquefaciens (Bacillus
) or bacillus licheniformis (Bacillus licheniformis) amyloliquefaciens.
It is highly preferred that bacillary neutrality endo protease can be from fungi bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens (example, bacilysin)).
Also, bacillary Alcalase can be from bacillus licheniformis (Bacillus
Licheniformis (example, hay bacillus proteinase)).
At this point, " deriving from " refers to including from " culture solution acquirement ".
Relative to the royal jelly of 100 parts by weight, the above-mentioned protease of 1 parts by weight to 10 parts by weight can be handled, it is highly preferred that
3 parts by weight can be handled.The only insufficient worry of non-enzymatic activity in range as described above can relative to enzyme amount when being more than
There are the worries of resolution ratio reduction.At this point, the royal jelly relative to 100 parts by weight, the protease of 3 parts by weight refers to is with weight
Benchmark, relative to royal jelly 100, the ratio of protease 3.Hereinafter, " parts by weight " can also be solved in the same manner as described above
It releases.Also, at this point, the above-mentioned protease of 3 parts by weight can be the fungoid endo protease and circumscribed egg relative to 1 parts by weight
White enzyme is mixed by the fungoid exoproteinase of 1 parts by weight and the bacillary endo protease of 1 parts by weight.That is, it is preferred that
Ground, relative to the royal jelly of 100 parts by weight, mixes 3 kinds of protease respectively with the same amount of 1 parts by weight and carries out for protease
Processing.
When being handled using protease, can be handled in the solution, relative to the royal jelly of 100 parts by weight,
Solvent can be 100 parts by weight to 1000 parts by weight.At this point, solvent can be water.As set forth above, it is possible to according to the amount of royal jelly
The ratio of different setting solvents.That is, the ratio of solvent is reduced in the royal jelly of opposite volume, in the royal jelly of relatively small amount
In the ratio of solvent can be improved.For example, in the royal jelly of opposite volume, relative to the royal jelly of 100 parts by weight, solvent can be with
For 500 parts by weight, in the royal jelly of relatively small amount, relative to the royal jelly of 100 parts by weight, solvent can be 1000 weight
Part.As described above, protease and royal jelly can be made by the solvent for adding opposite height ratio in the royal jelly of relatively small amount
Fully contact.Also, in the royal jelly of opposite volume, even if the solvent of the relatively low ratio of addition, also absolute molten
On dosage face, protease is contacted with royal jelly to sufficiently.By reducing the ratio of the solvent relative to royal jelly, can reduce
The scale of processing unit, and can also reduce energy consumption.
Also, it, can be at 40 DEG C in royal jelly powder after mixing protease when using Protease Treatment royal jelly powder
Said mixture is reacted under the conditions of to 60 DEG C of temperature, it is preferable that can 50 DEG C to 60 DEG C at a temperature of, carry out first
Secondary processing, 40 DEG C to 50 DEG C at a temperature of, carry out second and handle, 45 DEG C to 55 DEG C at a temperature of, carry out at third time
Reason, 50 DEG C to 60 DEG C at a temperature of, carry out the 4th time processing, it is highly preferred that can 55 DEG C at a temperature of carry out for the first time
Processing, 45 DEG C at a temperature of carry out second and handle, 50 DEG C at a temperature of carry out third time processing, 55 DEG C at a temperature of
Reacted under the conditions of the temperature of 4th processing, however, it is not limited to this.
Enzymatic treatment object can also become inactive disactivation enzymatic treatment object for protease.I.e., it is possible to be by excessive egg
White enzyme carries out inactive state by the method for heating etc..Disactivation can be based on heating, for instance, it is preferred that heating can be
Implement at a temperature of 70 DEG C to 90 DEG C.And, it is preferable that heating time can be 15 minutes to 30 minutes.
Heat 15 minutes to 30 minutes with 70 DEG C to 90 DEG C of temperature to realize that is, disactivation can be.It is more excellent
Selection of land when solvent is 1000 parts by weight, can be heated 15 minutes when the royal jelly relative to 100 parts by weight with 90 DEG C.Also,
When the royal jelly relative to 100 parts by weight, when solvent is 500 parts by weight, can be heated 30 minutes with 70 DEG C of temperature.Institute as above
It states, it can be different according to the ratio heating condition of the solvent to royal jelly.In the case where the ratio of solvent is big, under the high temperature conditions
Short time processing is carried out, in the case where the ratio of solvent is small, long time treatment can be carried out under the conditions of lower temperature and come pair
Enzyme carries out disactivation.Also, at relatively low temperatures when disactivation, carbonization phenomenon or thermal deformation can be effectively further prevented
Deng.
Freeze-drying step obtains the step of freeze-drying object as being freeze-dried to concentrate, using freezing
Drying machine etc. is implemented.Freeze-drying, for example, (- 40 DEG C of shelf temperature is hereinafter, trap freezing-in specified conditions using freeze drier
65 DEG C hereinafter, vacuum degree 250mTorr, 23 DEG C of drying temperature) under implement.
The present inventor confirms that there is enzyme treated bee milk powder immune function reinforcing effect specifically to pass through dodecyl
Sodium sulfonate (SDS) gel electrophoresis coloration result, it is thus identified that enzyme treated bee milk of the invention does not include the egg to cause allergic reaction
White (47kDa~55kDa), and by it is anti-oxidant (α-diphenyl-β-picryl hydrazine, active oxygen synthesis, superoxide dismutase,
Glutathione, nitric oxide) and inflammation index (tumor necrosis factor-alpha, gamma interferon) be analyzed to identify and non-enzymatic treated bee
Royal jelly (RJ) is compared, and the activity degree of enzyme treated bee milk (ERJ) is high.And further acknowledge the enzymatic treatment compared with non-enzymatic treated royal jelly
Royal jelly makes the tendency that B cell (B cell) and T cell (T cell) as immunocyte are actively proliferated from splenocyte.And
And further acknowledge the activity of natural killer cells relevant to vivo immunization reaction, it is thus identified that the lactic acid of concentration dependent in ER
Increase to the numerical value conspicuousness of salt dehydrogenase (LDH).As described above, enzyme treated bee milk immune function reinforcing effect of the invention
It is outstanding, therefore can effectively serve as being conducive to the health functional food or pharmaceuticals of the prevention of immunological diseases.
Composition of the invention can be additionally included in commonly used carrier appropriate, figuration in the preparation of pharmaceutical compositions
Agent and diluent.Also, powder, granule, tablet, capsule, suspension, cream can be melted into according to usual way preparation
The oral type dosage form of liquid, syrup, aerosol etc., external preparation, suppository and sterilizing injecting solution form come using.Preferably,
It uses as well known suitable preparation in the art in document (Remington's Pharmaceutical
Science, newest, Mack Publishing Company, Easton PA) disclosed in.As carrier, the figuration that may include
Agent and diluent have comprising lactose, glucose, sucrose, D-sorbite, mannitol, xylitol, erythrite, maltitol, shallow lake
Powder, acacia gum, alginates, gelatin, calcium phosphate, calcium silicates, cellulose, methylcellulose, microcrystalline cellulose, polyethylene pyrrole
Pyrrolidone, water, methyl hydroxybenzoate, nipasol, talcum, hard fatty acids, mineral oil etc..By above-mentioned composition
In the case where formulation, by using usually used filler, incremental agent, bonding agent, wetting agent, disintegrating agent, surface-active
It is prepared by the diluent or excipient of agent etc..As for oral administration solid pharmaceutical preparation include tablet, pill, powder,
Granula, capsule etc., this solid pharmaceutical preparation by mixing at least one excipient in above-mentioned composition come, such as mixing starch,
Calcium carbonate (calcium carbonate), sucrose, the preparation such as lactose, gelatin.And it other than simple excipient, also uses
Lubricant such as magnesium stearate, talcum etc..It include suspension, interior solution, lotion, syrup as liquid formulations for oral administration
Deng may include various excipient other than as the water of usually used diluent, atoleine, such as wetting agent, sweet taste
Agent, aromatic, antistaling agent etc..It include the aqueous solution of sterilizing, non-aqueous solvent, suspension as the preparation for non-oral administration
Agent, emulsion, freeze-dried preparation, suppository.It include suspending agent, interior liquor, emulsion, syrup as liquid formulations for oral administration
Deng, may include a variety of excipient other than as the water of commonly used diluent, atoleine, such as include wetting
Agent, sweetener, aromatic, antistaling agent etc..It include the aqueous solution to sterilize as the preparation for non-oral administration, non-aqueous molten
Agent, emulsion, freeze-dried preparation, suppository.Plant oil, such as propylene glycol can be used as non-aqueous solvent, suspending agent
(propyleneglycol), polyethylene glycol, olive oil etc.；The ester of injectable, such as ethyl oleate.It, can as the matrix of suppository
To use witepsol, polyethylene glycol, Tween61, cocoa paper, laruyl alcohol, glycerin gelatine etc..
Term " administration " as used in the present invention, which refers to, utilizes any suitable method by defined group of the invention
Object is closed to provide to individual.
The preferred dosage of pharmaceutical composition of the invention according to the state and weight of patient, disease degree, drug form, give
Medicine approach and period and difference, but can suitably be selected by those skilled in the art.In order to obtain preferred effect, enzyme of the invention
The daily dose of processing royal jelly powder can be administered with the amount of 1mg/kg to 10000mg/kg, and can be administered once a day also
It is segmented into and is administered several times.
Pharmaceutical compositions of the invention can be administered into individual through a variety of ways.It is contemplated that whole modes of administration, example
Such as, oral, rectum or vein, intramuscular, subcutaneous, uterus endosclerite or cerebrovascular inner injecting and administering can be passed through.
For the enhancing of immune function or the prevention of immunological diseases, composition of the invention can individually or with perform the operation, put
Beta ray therapy, hormone therapy, chemotherapy and using biologically conditioning agent method and use and use.
Also, the present invention is provided comprising enzyme treated bee milk powder as effective component for enhancing the food of immune function
Product composition and health functional food.
It, can in the case where enzyme treated bee milk powder of the invention is used as food compositions or health functional food
It directly adds above-mentioned enzyme treated bee milk powder or is used simultaneously with other food or food composition, it can be suitable according to usual way
Locality uses.The combined amount of effective component can be according to using purpose (prevention, health or rapeutic treatment) suitably to determine.It is logical
Often, when preparing food or beverage, relative to raw material, 15 weight percent enzyme treated bee milk powder of the invention below is added
End, it is preferable that addition 10 weight percent amount below.But by health and health as a purpose or by health adjust make
For the purpose of absorb for a long time in the case where, can be above range hereinafter, due to not had any problems in terms of safety,
Effective component can also be used with amount more than above range.
The type of above-mentioned food is not specially limited.Example as the food that can add above-mentioned substance has meat, sausage, face
Packet, chocolate, carbohydrate, fast food class, Biscuits, Piza, instant noodles, other noodles, chewing gum class, ice cream dairy products,
Various soup, beverage, tea, freshener, alcoholic beverage and compound vitamin, the health food being all contained on ordinary meaning.
For example common beverage of health beverage composition of the invention may include that various flavouring agents or natural carbohydrate etc. are made
For additional ingredient.Monosaccharide, such as glucose, fructose can be used in above-mentioned natural carbohydrate；Disaccharides, such as maltose, sucrose；
And natural sweetener, such as dextrin, cyclodextrin；Synthetic sweetener, such as saccharin, Aspartame.In general, above-mentioned natural compounds
The ratio of object is the composition about 0.01 to 10g, preferably from about 0.01 to 0.1g of the invention relative to 100ml.
In addition to that mentioned above, composition of the invention may include various nutritional agents, vitamin, electrolyte, flavouring agent, coloring
Agent, pectic acid and its salt, alginic acid and its salt, organic acid, protecting colloid thickener, pH adjusting agent, stabilizer, preservative,
Glycerol, ethyl alcohol, the carbonating agent for being used in soda etc..In addition to this, composition of the invention may include fruit juice, fruit
Juice beverage and the pulp for being used to prepare vegetable beverage.This ingredient independent or combined can use.The ratio of this additive is simultaneously
It is inessential, in general, the composition of the invention relative to 100 parts by weight selects under the range of 0.01 to 0.1 parts by weight.
Also, the present invention provides the method for the immune function of enhancing animal, including by a effective amount of enzyme treated bee milk powder
End is administered into the step of animal for needing to enhance immune function other than the mankind.
Hereinafter, being easy to understand the present invention prompt preferred embodiment, experimental example.But following embodiment, experimental example are only
It is further easy to understand and provides, the contents of the present invention are not limited to embodiment, experimental example and preparation example.
The preparation of 1. enzyme treated bee milk powder of embodiment
1kg relative to 100 parts by weight is freeze-dried royal jelly powder (10-HAD (10-hydroxy-2-
Decenoic acid) 5.0% or more；Jiangshan of Zhejiang Province apiculture Co., Ltd (ZHEJIANG JIANGSHAN BEE
ENTERPRISE CO., LTD)) be added 1000 parts by weight purified water mixing after, while mixed fungus endo protease, make
Probeef 2000P for exoproteinase, the Prozyme 2000P as fungoid exoproteinase and as bacillary
The Foodpro Alkaline proteinase of Alcalase.Each albumen is bought from (strain) Vision Biochem (South Korea)
Enzyme, and it is mixed into the above-mentioned royal jelly powder relative to 100 parts by weight, content is 1 parts by weight (total 3 parts by weight) respectively.Then
55 DEG C at a temperature of carry out first time processing, second is carried out at a temperature of 45 DEG C and is handled, 50 DEG C at a temperature of carry out the
Handle three times, 55 DEG C at a temperature of carry out the 4th time processing temperature under the conditions of reacted, 70 DEG C at a temperature of carry out
Heat treatment in 30 minutes carries out the disactivation of protease.After heat treatment, freeze drier (PVTE300KL, (strain) are utilized
Ilsinraep) be freeze-dried condition: -40 DEG C of shelf temperature is hereinafter, trap freezes -65 DEG C hereinafter, vacuum degree 250mTorr, dry
23 DEG C of temperature), to prepare the enzyme treated bee milk of powder morphology.On the other hand, confirm enzyme treated bee milk powder of the invention
Before being freeze-dried after protease mixing, pair based on whether the pro-inflammatory cytokine expression quantity there are reduced pressure process does not have
There is significant difference.Another feature is that it is different from the preparation method of our company's granted patent (KR 10-1900258 B1), it saves
Process slightly is concentrated under reduced pressure and prepares.
Experimental example 1. is true using protein electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE))
Recognize with the presence or absence of anaphylactogen (allergen)
In order to analyze in the enzyme treated bee milk powder obtained in above-described embodiment 1 with the presence or absence of causing allergic reaction
Protein measures protein concentration, and is pressed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis of 10% gel strength
Molecular weight protein isolate matter.After electrophoresis, after 1% Coomassie blue (Coomassie Blueg-250) stained gel,
Non-staining partial decolorization is measured in de-inking solution (45% methanol, 10% acetic acid), and its result is shown in Fig. 1.In order to than
Compared with if embodiment 1 is simultaneously using the non-enzymatic treated royal jelly (RJ) without enzymatic treatment.
As shown in Figure 1, observing the protein expression of 47~55kDa in non-enzymatic treated royal jelly, but in enzymatic treatment bee
It is not shown in royal jelly.By above-mentioned, it is thus identified that enzyme treated bee milk does not remove only the egg of the 47~55kDa to cause allergic reaction
White matter, and keep the albumen of macromolecular degraded.
Splenocyte and macrophage proliferation effect of the experimental example 2. based on enzyme treated bee milk powder
The Spleen cell proliferation effect of 2-1. enzyme treated bee milk powder
It, will be from just in order to analyze the Spleen cell proliferation ability of the enzyme treated bee milk powder obtained in above-described embodiment 1
The splenocyte that normal BALB/c mouse obtains is with 5 × 105Cell (cells)/hole mode is inoculated in 48 orifice plates, and handles by concentration
After sample, cultivate 24 hours and 48 hours.Then, 3- (4,5- dimethylthiazole -2) -2,5- diphenyl of 250 μ g/mL is added
Tetrazole bromide (MTT, 3- (4,5-dimethythiazol-2-yl) -2-5-diphenytetrazolium bromide) is molten
Liquid removes culture solution, and the dimethyl sulfoxide (DMSO, dimethyl sulfoxide) of 200 μ L is added after cultivating 4 hours,
It reacts at room temperature after ten minutes, absorbance is measured under the conditions of 570nm, and be shown in Table 2 its result.
As shown in Fig. 2, Spleen cell proliferation ability dramatically increases when handling enzyme treated bee milk powder of the invention.And
And after processing enzymatic treatment roral jelly powder last (ERJ), behind 24 hours and 48 hours, confirms the proliferative capacity of splenocyte, tie
Fruit is compared with 24 hours, and enzyme treated bee milk was in 48 hours presentation high proliferation abilities.In particular, at same concentrations (100 μ g/ml)
Under, when comparing enzyme treated bee milk powder and non-enzymatic treated royal jelly (RJ), the proliferative capacity of enzyme treated bee milk is than non-enzymatic place
Reason royal jelly dramatically increases.
The macrophage proliferation effect of 2-1. enzyme treated bee milk powder
In order to confirm the cultivation effect in peritoneal macrophage, by peritoneal macrophages with 5 × 104The side of cells/well
Formula is inoculated in 96 orifice plates, and after handling each sample by concentration, in addition to other processing groups processing rouge of non-enzymatic treated royal jelly is more
Sugared (LPS), and cultivate 24 hours.Then, 3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide of 250 μ g/mL is added
Bromide solution and cultivate 4 hours after, remove culture solution, be added 200 μ L dimethyl sulfoxide react at room temperature 10 minutes
Afterwards, absorbance is measured under the conditions of 570nm.
As shown in Figure 3a, 3b, it is thus identified that in individually processing enzymatic treatment roral jelly powder end and the feelings with lipopolysaccharides stimulation
Under condition, cell Proliferation is dramatically increased, and at same concentrations (100 μ g/ml), compared with non-enzymatic treated royal jelly, at enzyme
Cell Proliferation dramatically increases in reason royal jelly.
The antioxidant activity of 3. enzyme treated bee milk powder of experimental example
The α of 3-1. enzyme treated bee milk powder-diphenyl-β-picryl hydrazine (DPPH) activity increases analysis
In order to confirm α-diphenyl-β-picryl hydrazine activity of the enzyme treated bee milk powder obtained in above-described embodiment 1,
Together, sun will be used as with as the well-known ascorbic acid of typical Antioxidant Indexes (ascorbic acid) (vitamin C)
Property control group non-enzymatic treated royal jelly and the sample solution concentration of enzyme treated bee milk be aligned to 1mg/mL, mix the sample of 10 μ L
After α-diphenyl-β-picryl hydrazine solution (126 μ g/mL) of 90 μ L, place 10 minutes under normal temperature conditions, then in 517nm
Under the conditions of measure absorbance, and its result is shown in Fig. 4 a.
As shown in fig. 4 a, at same concentrations (100 μ g/ml), compared with non-enzymatic treated royal jelly, enzyme treated bee milk and
The significant increase of DPPH free radical scavenging activity in Vc.In particular, confirming the α-of enzyme treated bee milk compared with non-enzymatic treated royal jelly
Diphenyl-β-picryl hydrazine free radical scavenging ability increase about 30%.
The active oxygen of 3-2. enzyme treated bee milk powder synthesizes (ROS) activity analysis
It is in order to analyze the active oxygen synthesizing activity of the enzyme treated bee milk powder obtained in above-described embodiment 1, peritonaeum is huge
Phagocyte is with 5 × 104The mode of cells/well is inoculated in 96 orifice plates.After 48 hours, by each concentration handle sample after, in addition to
Other processing groups of carrier (Vehicle) handle lipopolysaccharides with the concentration of 1 μ g/ml, and cultivate 15 minutes.It, will be with after 15 minutes
Phosphate buffer solution (PBS): 2 ', 7 '-dichlorofluorescein diacetate esters (DCFDA, 2 ', 7 '-Dichlorofluorescin
Diacetate the diluted solution of)=5000:1 is mixed with culture medium same amount, come handle with each hole, react at room temperature
After twenty minutes, measurement fluorescence radiation is bright, and shows its result in Fig. 4 b.
As shown in Figure 4 b, compared with carrier (Vehicle), the active oxygen synthesizing activity of control group increases, with control group phase
Than substantially reducing to the active concentration dependence of active oxygen synthesis in enzyme treated bee milk.Especially, it is thus identified that in same concentrations
Under (100 μ g/ml), active oxygen synthesizing activity is substantially reduced compared with non-enzymatic treated royal jelly, in enzyme treated bee milk.
Superoxide dismutase (SOD) activity analysis of 3-3. enzyme treated bee milk powder
In order to confirm the superoxide dismutase activity of the enzyme treated bee milk powder obtained in above-described embodiment 1, lead to
It crosses and uses superoxide dismutase detection kit (Assay kit) (Michigan, USA Cayman chemistry (Cayman
Chemical, Ann Arbor, MI, USA)) it is measured.By the giant cell separated from cavum peritoneale at culture dish (100cm2)
After middle culture 24 hours, the lipopolysaccharides of sample and 1 μ g/mL is handled, and also cultivate 24 hours.Then it will be diluted by cell smashing fluid
Made of buffer as sample, in 10 μ L samples, be added 200 μ L free radical detection agent (radical d etector),
The xanthine oxidase (Xanthine oxidase) of 20 μ L, and under normal temperature conditions after placement 30 minutes, in 440-460nm item
Absorbance is measured under part, and its result is shown in Fig. 4 c.
As illustrated in fig. 4 c, compared with carrier (Vehicle), the active oxygen synthesizing activity of control group is reduced, with control group phase
Than dramatically increasing to the active concentration dependence of active oxygen synthesis in enzyme treated bee milk.Especially, it is thus identified that in same concentrations
Under (100 μ g/ml), compared with non-enzymatic treated royal jelly, active oxygen synthesizing activity increases in enzyme treated bee milk.
Glutathione (GSH) activity analysis of 3-4. enzyme treated bee milk powder
In order to analyze the glutathione activity of the enzyme treated bee milk powder obtained in above-described embodiment 1, by using
Glutathione detection kit (Assay kit) (Michigan, USA Ann Arbor city Cayma n Chemical company (Cayman
Chemical, Ann Arbor, MI, USA)) it is measured.After peritoneal macrophage culture 24 hours, handled by each concentration
Sample, and lipopolysaccharides is handled with the concentration of 1 μ g/ml to other processing groups in addition to carrier (Vehicle)
(Lipopolysaccharide) it after, also cultivates 24 hours.Then the buffer of cell smashing fluid mixes as sample dilution
Close sample, inoculation mixing the MES buffer, co-factor mixture (cofactor mixture), enzymatic mixture (enzyme of 50 μ L
Mix) and the reaction buffer (reaction buffer) of the 150 μ L of DTNB, and carry out after shading comes 30 minutes 405~
Absorbance is measured under the conditions of 414nm, and its result is shown in Fig. 4 d.
As shown in figure 4d, compared with carrier (Vehicle), the glutathione activity of control group is reduced, compared with the control group,
In enzyme treated bee milk, dramatically increase to the active concentration dependence of glutathione.Especially, it is thus identified that in same concentrations (100 μ
G/ml under), compared with non-enzymatic treated royal jelly, enzyme treated bee milk Glutathione peptide activity increases.
Nitric oxide (NO) expression analysis of 3-5. enzyme treated bee milk powder
In order to analyze the expression of nitric oxide amount of the enzyme treated bee milk powder obtained in above-described embodiment 1, by making
It is measured with giress reagent system (giress reagent system) (Promega, Madiso n, WI).Abdominal cavity is huge
Phagocyte is with 5 × 104The mode of cells/well is inoculated in 96 orifice plates, after adhering to 4 hours, sample is handled by each concentration, to removing
Other processing groups of carrier (Vehicle) handle lipopolysaccharides (Lipopolysaccharide) with the concentration of 1 μ g/ml, and train
It supports 24 hours.Then same amount of Griess reagent (griess reagent) is mixed in the culture solution of 100 μ L, in room temperature
Under the conditions of react 10 minutes, extinction is measured under the conditions of 550nm, and its result is shown in Fig. 4 e.
As shown in fig 4e, compared with carrier (Vehicle), the nitric oxide production generation of control group increases, with control group phase
Than substantially reducing to nitric oxide production generation concentration dependent in enzyme treated bee milk.Especially, it is thus identified that in same concentrations
Under (100 μ g/ml), compared with non-enzymatic treated royal jelly, nitric oxide production generation is reduced in enzyme treated bee milk.
The anti-inflammatory activity of 4. enzyme treated bee milk powder of experimental example is evaluated
4-1. tumor necrosis factor-alpha (TNF-α) activity
In order to analyze the life of the tumor necrosis factor-alpha (TNF-α) for participating in intracellular inflammation in peritoneal macrophage
At, by using murine tumor necrosis factor-α enzyme-linked immunosorbent assay kit (TNF-α ELISA kit) (Abcam,
Cambridge, MA) it is measured.After peritoneal macrophage culture 24 hours, sample is handled by each concentration, in addition to carrying
After other processing groups of body (Vehicle) handle lipopolysaccharides with the concentration of 1 μ g/ml, cultivate 24 hours.Also, into 96- orifice plate
The culture solution (sample) of 100 μ L is added, after reacting 30 minutes 2 hours at room temperature, the biotinylation of 100 μ L is added
(biotinylated) tumor necrosis factor-alpha, it is at room temperature, horseradish peroxidase-strepto- of 100 μ L is affine
Plain (HRP-streptabvidin) solution is added 1 hour, at room temperature, by the one step substrate of tetramethyl benzidine of 100 μ L
Reagent (TMB one step substr ate reagent) is added 1 hour, after placing 30 minutes at room temperature, is added
Terminate liquid (sto p solution) measures absorbance under the conditions of 450nm, and shows its result in Fig. 5 a.
As shown in Figure 5 a, compared with carrier (Vehicle), the generation of the tumor necrosis factor-alpha of control group increases, and right
It compares according to group, in enzyme treated bee milk, substantially reduces to the generation concentration dependent of tumor necrosis factor-alpha.Especially, it is thus identified that
At same concentrations (100 μ g/ml), compared with non-enzymatic treated royal jelly, the life of tumor necrosis factor-alpha in enzyme treated bee milk
At reduction.
4-2. gamma interferon (gamma interferon) activity
In order to analyze the generation of the gamma interferon (gamma interferon) for participating in intracellular inflammation in peritoneal macrophage,
By using mouseγ-interferon enzyme-linked immunosorbent assay kit (Massachusetts, United States Sai Mo scientific & technical corporation (Thermo
Scientific, MA, USA)) it is measured.After peritoneal macrophage culture 24 hours, sample is handled by each concentration, it is right
In addition to other processing groups of carrier (Vehicle) handle lipopolysaccharides with the concentration of 1 μ g/ml, and cultivate 24 hours.Also, to 96-
The culture solution (sample) of 100 μ L is added in orifice plate, after reacting 30 minutes 2 hours at room temperature, the biotin of 100 μ L is added
The gamma interferon for changing (biotinylated) detects antibody, at room temperature, by horseradish peroxidase-strepto- of 100 μ L
Avidin solution is added 1 hour, and at room temperature, the one step substrate reagent of tetramethyl benzidine of 100 μ L is added 1 hour,
After placing 30 minutes at room temperature, terminate liquid is added, measures absorbance under the conditions of 450nm, and Figure 5b shows that its knots
Fruit.
As shown in Figure 5 b, it is thus identified that compared with carrier (Vehicle), the generation of the gamma interferon of control group increases, and right
It compares according to group, in enzyme treated bee milk, substantially reduces to the generation concentration dependent of gamma interferon.
The immunocompetence of 5. enzyme treated bee milk powder of experimental example is evaluated
In order to confirm the influence of expression generation of the enzyme treated bee milk powder to immunoreactive protein, to peritoneal macrophage
In the generation of nitric oxide (NO) analyzed, and it is right by using giress reagent system (Promega, Madison, WI)
It is measured.By peritoneal macrophage with 5 × 104The mode of cells/well is inoculated in 96 orifice plates, after adhering to 4 hours, by each
A concentration handles sample, and has cultivated 24 hours.Then same amount of Griess reagent is mixed in the culture solution of 100 μ L, come
It reacts 10 minutes at room temperature, extinction is measured under the conditions of 550nm, and its result is shown in Fig. 6.
As shown in fig. 6, nitric oxide production generation increases in enzyme treated bee milk compared with carrier (Vehicle), in particular,
It confirmed at same concentrations (100 μ g/ml), compared with non-enzymatic treated royal jelly, nitric oxide production life in enzyme treated bee milk
At increase.
Natural killer cell activity (the Natural killer cell of 6. enzyme treated bee milk powder of experimental example
Activity, NK cell activity)
In order to analyze the influence of the generation of the natural killer cell activity in enzymatic treatment roral jelly powder foot couple splenocyte, cream is utilized
Hydrochlorate dehydrogenase reagent box (LDH cytotoxicity detection kit) is evaluated.It will be at sample by each concentration
It manages in splenocyte, after cultivating 24 hours, when (YAC-1) cell quantity of yeast artificial chromosome -1 is 1 × 104When cells/well,
By splenocyte: being inoculated in 96- orifice plate in a manner of yeast artificial chromosome -1=200:1, and cultivate 4 hours.Then, to the hole 96-
The culture solution of 100 μ L and the reaction mixture (reaction mixture) of 100 μ L is added in plate, and anti-in 37 DEG C of incubator
After answering 30 minutes, absorbance is measured under the conditions of 490nm, and its result is shown in FIG. 7.
As shown in fig. 7, confirming the enzyme of the enzyme treated bee milk of 50 μ g/ml and 100 μ g/ml compared with carrier (Vehicle)
Processing royal jelly cytotoxicity dramatically increases.Especially, it is thus identified that compared with non-enzymatic treated royal jelly, the enzymatic treatment bee of 100 μ g/ml
Cytotoxicity is significantly high in royal jelly.
It is fixed that 7. animal model dietary amount of experimental example and changes of weight measure
For the dietary amount and weight of measurement experiment animal, BALB/c mouse is bought from more objects scientific (Taejon, Korea)
(25g), and light and shade is adjusted with defined humidity (40~60%), defined temperature (22 ± 2 DEG C) and with 12 hour period
Experimental situation under, adapt to 1 week.With the temperature and humidity of 24 hours unit confirmation raising rooms during feeding.It is divided into enzyme treated bee milk
Processing group (50mg/kg, 150mg/kg, 300mg/kg, 500mg/kg) and non-enzymatic treated royal jelly processing group (500mg/kg).It will
Enzyme treated bee milk and non-enzymatic treated royal jelly are prepared into solid feed and use, when assuming with one day dietary amount for 4g, often
Group 10, when absorbing 1.2kg altogether within 4 weeks, calculating is incorporated in the amount of feed to prepare.The enzyme treated bee milk feed of preparation is used as certainly
By diet regimen.It is 4 weeks during free diet, the food intake (food intake) of each group of every weekly check.With regard to each group
Individual amount for, after being set as 10 by each concentration, carry out repeating test, and its result be shown in Fig. 8.
As shown in Figure 8 a, in all experimental groups, increased tendency over time is presented in diet regimen amount, this
It is judged as the natural phenomena occurred during week old is got higher.As shown in Figure 8 b, it in all experimental groups, presents with the time
The how much increased tendency of passage weight, this is judged as the natural phenomena occurred during week old is got higher.
8. cell factor of experimental example (interleukin-1 ' beta ', interleukin-6, TNF-α, IL-10, interleukin 2,
Gamma interferon) secretion capacity measurement
The activity of 8-1. interleukin-1 ' beta '
The serum of the BALB/c normal mouse model of above-mentioned experimental example 7-1 is measured using enzyme-linked immunosorbent assay kit
With the amount for the cell factor secreted from the culture solution of macrophage.Using culture solution as sample to the 96- orifice plate being ready for according to
The biotinylated interleukin-1 ' beta ' detection antibody of 100 μ L is put into 2 at room temperature by the secondary sample for being put into 100 μ L
30 minutes hours the horseradish peroxidase of 100 μ L-solution of streptavidin were added 1 hour, in room at room temperature
Under the conditions of temperature, the one step substrate reagent of tetramethyl benzidine of 100 μ L is added 45 minutes, is placed 30 minutes at room temperature
Afterwards, the terminate liquid (stop solution) of 50 μ L is added, absorbance is measured under the conditions of 450nm, and its knot is shown in Fig. 9 a
Fruit.
As illustrated in fig. 9, interleukin-1 ' beta ' inflammation index is presented in macrophage-conditioned media and serum similar inclines
To.Confirmed handle enzyme treated bee milk after, the content of the interleukin-1 ' beta ' measured in macrophage-conditioned media and
The interleukin-1 ' beta ' measured in serum is presented and is substantially reduced, at same concentrations (500mg/kg), enzyme treated bee milk
The production quantity for being substantially less than the interleukin-1 ' beta ' of non-enzymatic treated royal jelly is presented.
The activity of 8-2. interleukin-6
The serum of the BALB/c normal mouse model of above-mentioned experimental example 7-1 is measured using enzyme-linked immunosorbent assay kit
With the amount for the cell factor secreted from the culture solution of macrophage.Using culture solution as sample to the 96- orifice plate being ready for according to
The secondary sample for being put into 50 μ L at room temperature puts the Ms IL-6biotin-conjugate solution solution of 100 μ L
Enter 2 hours, at room temperature, by Streptavidin-horseradish peroxidase (streptavidin-HRP) solution of 100 μ L
Addition 30 minutes, at room temperature, by Stabilized Chromagen addition 30 minutes of 100 μ L, at room temperature
After placing 30 minutes, the terminate liquid of 100 μ L is added, absorbance is measured under the conditions of 450nm, and its result is shown in Fig. 9 b.
As shown in figure 9b, interleukin-6 inflammation index is presented in macrophage-conditioned media and serum similar inclines
To.Confirmed handle enzyme treated bee milk after, the content of the interleukin-6 measured in macrophage-conditioned media and
The interleukin-6 measured in serum is presented and is substantially reduced, at same concentrations (500mg/kg), enzyme treated bee milk
The production quantity for being substantially less than the interleukin-1 ' beta ' of non-enzymatic treated royal jelly is presented.
The activity of 8-3.TNF- α
The serum of the BALB/c normal mouse model of above-mentioned experimental example 7-1 is measured using enzyme-linked immunosorbent assay kit
With the amount for the cell factor secreted from the culture solution of macrophage.Using culture solution as sample to the 96- orifice plate being ready for according to
The biotinylated interleukin-1 ' beta ' detection antibody of 100 μ L is put into 2 at room temperature by the secondary sample for being put into 100 μ L
30 minutes hours the horseradish peroxidase of 100 μ L-solution of streptavidin were added 1 hour, in room at room temperature
Under the conditions of temperature, the one step substrate reagent of tetramethyl benzidine of 100 μ L is added 30 minutes, is placed 30 minutes at room temperature
Afterwards, the terminate liquid (stop solution) of 50 μ L is added, absorbance is measured under the conditions of 450nm, and its knot is shown in Fig. 9 c
Fruit.
As is shown in fig. 9 c, tumor necrosis factor-alpha inflammation index is presented in macrophage-conditioned media and serum similar inclines
To.In particular, processing enzyme treated bee milk after, the content of the tumor necrosis factor-alpha measured in macrophage-conditioned media and
The tumor necrosis factor-alpha measured in serum is presented and is substantially reduced, under the Serum samples of same concentrations (500mg/kg),
The production quantity for being substantially less than the interleukin-1 ' beta ' of non-enzymatic treated royal jelly is presented in enzyme treated bee milk.
The activity of 8-4.IL-10
The serum of the BALB/c normal mouse model of above-mentioned experimental example 7-1 is measured using enzyme-linked immunosorbent assay kit
With the amount for the cell factor secreted from the culture solution of macrophage.Using culture solution as sample to the 96- orifice plate being ready for according to
The analysis buffer of the secondary sample for being put into 50 μ L and 50 μ L, at room temperature, by the biotinylated interleukins-of 50 μ L
10 detection antibody are put into 3 hours, at room temperature, the Streptavidin of 100 μ L-horseradish peroxidase solution are added 1
Hour, at room temperature, the one step substrate reagent of tetramethyl benzidine of 100 μ L is added 30 minutes, is put at room temperature
After setting 30 minutes, the terminate liquid of 100 μ L is added, absorbance is measured under the conditions of 450nm, and its result is shown in Fig. 9 d.
As shown in figure 9d, interleukin 10 inflammation index is presented in macrophage-conditioned media and serum similar inclines
To.It confirmed, especially after handling enzyme treated bee milk, the interleukin 10 that is measured in macrophage-conditioned media
Content and the interleukin 10 measured in serum, which are presented, to be substantially reduced, in the serum of same concentrations (500mg/kg)
In sample, the production quantity for being substantially less than the interleukin-1 ' beta ' of non-enzymatic treated royal jelly is presented in enzyme treated bee milk.
The activity of 8-5. interleukin 2
The serum of the BALB/c normal mouse model of above-mentioned experimental example 7-1 is measured using enzyme-linked immunosorbent assay kit
With the amount for the cell factor secreted from the culture solution of macrophage.Using culture solution as sample to the 96- orifice plate being ready for according to
The analysis buffer of the secondary sample for being put into 50 μ L and 50 μ L, at room temperature, by the biotinylated interleukins-of 50 μ L
2 antibody reagents are put into 3 hours, at room temperature, it is small that the Streptavidin of 100 μ L-horseradish peroxidase solution are added 1
When, at room temperature, the one step substrate reagent of tetramethyl benzidine of 100 μ L is added 30 minutes, is placed at room temperature
After 30 minutes, the terminate liquid of 100 μ L is added, absorbance is measured under the conditions of 450nm, and its result is shown in Fig. 9 e.
As shown in figure 9e, interleukin 2 inflammation index is presented in macrophage-conditioned media and serum similar inclines
To.It confirmed, especially after handling enzyme treated bee milk, the interleukin 10 that is measured in macrophage-conditioned media
Content and the interleukin 2 measured in serum, which are presented, to be substantially reduced, and is tried in the serum of same concentrations (500mg/kg)
In sample, the production quantity for being substantially less than the interleukin-1 ' beta ' of non-enzymatic treated royal jelly is presented in enzyme treated bee milk.
The activity of 8-6. gamma interferon
The serum of the BALB/c normal mouse model of above-mentioned experimental example 7-1 is measured using enzyme-linked immunosorbent assay kit
With the amount for the cell factor secreted from the culture solution of macrophage.Using culture solution as sample to the 96- orifice plate being ready for according to
The biotinylated gamma interferon antibody reagent of 50 μ L is put into 2 hours by the secondary sample for being put into 50 μ L at room temperature,
Under room temperature, the Streptavidin of 100 μ L-horseradish peroxidase solution is added 1 hour, at room temperature, by 100
The one step substrate reagent of tetramethyl benzidine of μ L is added 30 minutes, and after placing 30 minutes at room temperature, the end of 100 μ L is added
Only liquid measures absorbance under the conditions of 450nm, and shows its result in Fig. 9 f.
As shown in figure 9f, similar tendency is presented in macrophage-conditioned media and serum in gamma interferon inflammation index.Really
It accepts, especially after handling enzyme treated bee milk, the content of the gamma interferon measured in macrophage-conditioned media and in blood
The gamma interferon measured in clear, which is presented, to be substantially reduced, in the Serum samples of same concentrations (500mg/kg), enzymatic treatment bee
The production quantity for being substantially less than the gamma interferon of non-enzymatic treated royal jelly is presented in royal jelly.
9. Spleen cell proliferation effect of experimental example
9-1. Spleen cell proliferation ability
By the Spleen cell suspensions separated from the BALB/c normal mouse model of above-mentioned EXPERIMENTAL EXAMPLE 7-1 with 5 × 106
Cells/well is inoculated in 96 orifice plates, and handling the mitogen (Mitogen) that is activated to immune cell propagation, (rouge is more
Sugar, concanavalin A (ConA)), cell survival rate (cell viability) then is measured after 44 hours, and it is shown in Figure 10
As a result.
As shown in Figure 10, it is thus identified that compared with carrier (Vehicle), mitogen whole enzymatic treatment bees before and after the processing
Royal jelly has cell Proliferation effect.It confirmed, especially at identical capacity (500mg/kg), compared with non-enzymatic treated royal jelly,
There is significant Spleen cell proliferation effect in enzyme treated bee milk.
Measurement (Natural killer cell activity, the NK cell of 10. natural killer cell activity of experimental example
activity)
Using lactate dehydrogenase kit to obtaining the spleen separated in BALB/c normal mouse model from above-mentioned experimental example 7-1
Cell is evaluated.With same concentrations inoculation (seeding) intake enzyme treated bee milk mouse splenocyte, 24 hours
Afterwards, when -1 cell quantity of yeast artificial chromosome is 1 × 104When cells/well, with splenocyte: yeast artificial chromosome -1=
The mode of 200:1 is inoculated in 96- orifice plate, and cultivates 4 hours.Then, the culture solution and 100 μ L of 100 μ L are added to 96- orifice plate
Reaction mixture, and after being reacted 30 minutes in 37 DEG C of incubator, absorbance is measured under the conditions of 490nm, and in Figure 11
Its result is shown.
As shown in figure 11, when absorbing enzyme treated bee milk, increase to lactate dehydrogenase concentration dependent, by above-mentioned
It confirmed that natural killer cells (NK) activity increases.It confirmed, especially at identical capacity (500mg/kg), with non-enzymatic treated bee
Royal jelly is compared, and natural killer cell activity increases in enzyme treated bee milk.
As described above, passing through experiment in vitro and enzyme treated bee milk using enzyme treated bee milk powder evaluation of the invention
The effect of immune function in the experimental animal of diet enhances, as a result in dodecyl sodium sulfate gel electrophoresis coloration result really
It accepts without the protein (47kDa~55kDa) to cause allergic reaction, passes through anti-oxidant (α-diphenyl-β-picryl hydrazine, activity
Oxygen synthesis, superoxide dismutase, glutathione, nitric oxide) anti-oxidant (α-diphenyl-β-picryl hydrazine, active oxygen synthesis,
Superoxide dismutase, glutathione, nitric oxide) and inflammation index (tumor necrosis factor-alpha, gamma interferon) analysis, really
It accepts compared with non-enzymatic treated royal jelly (RJ), the activity degree for being freeze-dried royal jelly (enzyme treated bee milk) is high.Also, it is also true
It accepts compared with royal jelly, enzyme treated bee milk is proliferated B cell and T cell as immunocyte actively from splenocyte
Tendency.And, it is thus identified that activation natural killer cells relevant to vivo immunization reaction, it is newborn in enzyme treated bee milk processing group
It dramatically increases to the numerical concentration dependence of hydrochlorate dehydrogenase, to confirmed that outstanding immune function reinforcing effect is presented.
Hereinafter, to the medicine for being used to enhance immune function comprising enzyme treated bee milk powder as effective component of the invention
The preparation example for learning composition and food compositions is illustrated, but is not to limit the present invention to be only specifically illustrated.
1. pharmaceutical formulations of preparation example
The preparation of 1-1. powder
Enzyme treated bee milk 20mg
Lactose 100mg
Talcum 10mg
Mentioned component is mixed, and is filled in close bag to prepare dispersing agent.
The preparation of 1-2. tablet
Enzyme treated bee milk 10mg
Cornstarch 100mg
Lactose 100mg
Magnesium stearate 2mg
After mixing mentioned component, ingot is beaten to prepare tablet according to the preparation method of common tablet.
The preparation of 1-3. capsule
Enzyme treated bee milk 10mg
Avicel cellulose 3mg
Lactose 14.8mg
Magnesium stearate 0.2mg
Mentioned component is mixed according to the preparation method of common capsule, and is filled in gelatine capsule to prepare capsule.
The preparation of 1-4. injection
Enzyme treated bee milk 10mg
Mannitol 180mg
Injection sterile purified water 2974mg
Na2HPO42H2O26mg
According to the preparation method of common injection, prepared in a manner of each ampoule (2ml) mentioned component content.
The preparation of 1-5. liquor
Enzyme treated bee milk 20mg
Isomerized sugar 10g
Mannitol 5g
Purified Water q. s
Each ingredient is added in purified water according to common liquor preparation method, makes it dissolve, suitable lemon is added
After fragrance, mentioned component is mixed, and purified water is added, be adjusted to total 100ml, be then filled into brown bottle and sterilized and prepared
Liquor.
2. food formulation of preparation example
The preparation of 2-1. health food
Enzyme treated bee milk 100mg
Appropriate vitamin mixtures
70 μ g of vitamin A acetate
Vitamin e1 .0mg
VitaminB10 .13mg
Vitamin B2 0.15mg
Vitamin B6 0.5mg
0.2 μ g of vitamin B12
Vitamin C 10mg
10 μ g of biotin
Niacin hydroxyacyl amine 1.7mg
50 μ g of folic acid
Calcium pantothenate 0.5mg
Inanimate matter mixture is appropriate
Ferrous sulfate 1.75mg
Zinc oxide 0.82mg
Magnesium carbonate 25.3mg
Potassium dihydrogen phosphate 15mg
Dipotassium hydrogen phosphate 55mg
Potassium citrate 90mg
Calcium carbonate 100mg
Magnesium chloride 24.8mg
Although the ratio of components of said vitamin and mineral mixture is formed with preferred embodiment mixing and is relatively more suitable for
The ingredient of health food, but arbitrarily change even its match ratio implemented, it can be mixed according to the preparation method of common health food
After closing mentioned component, particle is prepared, is used in the preparation of health-care food composition according to usual way.
The preparation of 2-2. health drink
Enzyme treated bee milk 100mg
Vitamin C 15g
Vitamin E (powder) 100g
Ferrous lactate 19.75g
Zinc oxide 3.5g
Niacin hydroxyacyl amine 3.5g
Vitamin A 0.2g
VitaminB10 .25g
Vitamin B2 0.3g
Water is quantitative
After mixing mentioned component according to the preparation method of common health drink, in 85 DEG C of at a temperature of agitating and heating about 1
After hour, it is attained at the 2l container of sterilizing by filtering prepared solution, and seal stored under refrigeration after sterilizing, then used
In preparing health beverage composition of the invention.
For above-mentioned ratio of components, the ingredient for being more suited to hobby beverage is formed with preferred embodiment mixing, but can
The region, national characteristic preference degree such as stratum, demand country, usage according to demand, arbitrarily change its match ratio implement also without
Harm.
Claims (8)
1. a kind of for enhancing the pharmaceutical compositions of immune function, which is characterized in that being used as comprising enzyme treated bee milk powder has
Imitate ingredient.
2. according to claim 1 for enhancing the pharmaceutical compositions of immune function, which is characterized in that above-mentioned enzyme is albumen
Enzyme.
3. according to claim 2 for enhancing the pharmaceutical compositions of immune function, which is characterized in that above-mentioned protease is
Selected from what is be made of fungoid endo protease and exoproteinase, fungoid exoproteinase and bacillary endo protease
One or more of group.
4. according to claim 3 for enhancing the pharmaceutical compositions of immune function, which is characterized in that above-mentioned bacillary interior
Cutting protease is bacillary Alcalase.
5. according to claim 1 for enhancing the pharmaceutical compositions of immune function, which is characterized in that relative to 100 weights
The royal jelly of part is measured, the above-mentioned enzyme of 1 parts by weight to 10 parts by weight is handled.
6. a kind of for enhancing the food compositions of immune function, which is characterized in that being used as comprising enzyme treated bee milk powder has
Imitate ingredient.
7. a kind of for enhancing the health functional food of immune function, which is characterized in that include the conduct of enzyme treated bee milk powder
Effective component.
8. a kind of for enhancing the method for the immune function of animal, which is characterized in that including by a effective amount of enzyme treated bee milk
Powder is administered into the step of animal for needing to enhance immune function other than the mankind.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20180010121 | 2018-01-26 | ||
| KR10-2018-0010121 | 2018-01-26 | ||
| KR10-2019-0006152 | 2019-01-17 | ||
| KR1020190006152A KR102173591B1 (en) | 2018-01-26 | 2019-01-17 | Pharmaceutical composition for enhancing immune function comprising enzyme treated royal jelly as an active ingredient |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110075128A true CN110075128A (en) | 2019-08-02 |
Family
ID=67413040
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910082362.2A Pending CN110075128A (en) | 2018-01-26 | 2019-01-28 | A kind of pharmaceutical compositions for being used to enhance immune function comprising enzyme treated bee milk powder as effective component |
Country Status (1)
| Country | Link |
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| CN (1) | CN110075128A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113647591A (en) * | 2021-08-20 | 2021-11-16 | 山东丰采健康产业有限公司 | Royal jelly zymolyte and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526444A (en) * | 2003-03-07 | 2004-09-08 | 株式会社山田养蜂场 | Composition for immunological activation |
| JP2008194031A (en) * | 2007-01-17 | 2008-08-28 | Yamada Bee Farm Corp | Stabilization of enzyme-treated royal jelly |
| CN101606706A (en) * | 2009-07-10 | 2009-12-23 | 江西汪氏蜜蜂园有限公司 | A kind of health food of compound bee product raw material |
| KR101418067B1 (en) * | 2012-12-20 | 2014-07-14 | 주식회사 삼양제넥스 | Preparation method of hypoallergenic royal jelly by enzyme treatment |
-
2019
- 2019-01-28 CN CN201910082362.2A patent/CN110075128A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1526444A (en) * | 2003-03-07 | 2004-09-08 | 株式会社山田养蜂场 | Composition for immunological activation |
| JP2008194031A (en) * | 2007-01-17 | 2008-08-28 | Yamada Bee Farm Corp | Stabilization of enzyme-treated royal jelly |
| CN101606706A (en) * | 2009-07-10 | 2009-12-23 | 江西汪氏蜜蜂园有限公司 | A kind of health food of compound bee product raw material |
| KR101418067B1 (en) * | 2012-12-20 | 2014-07-14 | 주식회사 삼양제넥스 | Preparation method of hypoallergenic royal jelly by enzyme treatment |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113647591A (en) * | 2021-08-20 | 2021-11-16 | 山东丰采健康产业有限公司 | Royal jelly zymolyte and preparation method and application thereof |
| CN113647591B (en) * | 2021-08-20 | 2023-11-10 | 山东丰采健康产业有限公司 | Royal jelly zymolyte and preparation method and application thereof |
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