CN106163534A - Hyaluronic acid synthesis accelerant, HAS2mRNA express accelerator, have the hyaluronic acid synthesis pharmaceuticals of facilitation, food or cosmetic preparation and have the HAS2mRNA expression pharmaceuticals of facilitation, food or cosmetic preparation - Google Patents
Hyaluronic acid synthesis accelerant, HAS2mRNA express accelerator, have the hyaluronic acid synthesis pharmaceuticals of facilitation, food or cosmetic preparation and have the HAS2mRNA expression pharmaceuticals of facilitation, food or cosmetic preparation Download PDFInfo
- Publication number
- CN106163534A CN106163534A CN201580010029.0A CN201580010029A CN106163534A CN 106163534 A CN106163534 A CN 106163534A CN 201580010029 A CN201580010029 A CN 201580010029A CN 106163534 A CN106163534 A CN 106163534A
- Authority
- CN
- China
- Prior art keywords
- fraction
- hyaluronic acid
- has2mrna
- acid synthesis
- chrysin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- 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)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- 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
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Botany (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Alternative & Traditional Medicine (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Cosmetics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
A kind of hyaluronic acid synthesis accelerant or HAS2mRNA express accelerator, its contain by aqueous organic solvent from the fraction that the seed of Semen Oroxyli is extracted including at least the 1st fraction of chrysin, other fraction obtained by purification the 1st fraction or chrysin are as effective ingredient.Additionally, the problem of the present invention is to provide the hyaluronic acid synthesis accelerant containing the present invention or HAS2mRNA to express the pharmaceuticals of accelerator, food or cosmetic preparation.It is that the useful material using fraction or composition obtained by the Semen Oroxyli that safety is high forms that the hyaluronic acid synthesis accelerant of the present invention, HAS2mRNA express accelerator, pharmaceuticals, food or cosmetic preparation.
Description
Technical field
The present invention relates to hyaluronic acid synthesis accelerant, HAS2mRNA expresses accelerator, have hyaluronic acid synthesis promotion
Pharmaceuticals, food or the cosmetic preparation of effect and there is HAS2mRNA express the pharmaceuticals of facilitation, food or cosmetic preparation.
Background technology
Hyaluronic acid (also referred to as hyaluronic acid) is macromolecule (the amino Portugal being made up of N-acetyl-glucosamine and glucuronic acid
Polysaccharide), there is high moisture holding capacity.Known hyaluronic acid be as the gelatinous material containing water extensively see in organism thin
Extracellular matrix, the viscoelasticity holding with biological tissue's (particularly skin histology), the moisturizing of biological tissue, the merit of joint portion cartilage
Energy maintenances etc. are relevant.Entered into intracellular, regulation cell by extracellular additionally, hyaluronic acid also has prevention polymer substance
Move or breed, becoming the function in the place etc. of information transmission (signal transduction).
, the amount of the hyaluronic acid in organism because of various pressure, ultraviolet, add age etc. and reduce.Known organism
In the amount of hyaluronic acid when reducing, abnormal (wrinkle, lax and dry coarse) because being dried the skin that causes will occur
Or the symptom of arthralgia etc..The immediate cause reduced as the amount of the hyaluronic acid in organism can be enumerated: due to hyaluronic acid
Enzyme (hyaluronic acid catabolic enzyme) or ultraviolet and make hyaluronic acid be decomposed, or the generation amount of the hyaluronic acid in organism subtracts
Few.
Therefore, if the amount of the hyaluronic acid in organism can be supplemented or increase, then the abnormal of skin or joint can be expected
Pain etc. symptom prevention, suppress and improve or normalization.
In the past as the composition of the synthesis facilitation with hyaluronic acid in organism, tretinoin in epidermis,
In corium, TGF-β 1 is widely known by the people.Additionally, as the material of the synthesis facilitation with hyaluronic acid in organism, it is known that
Natural product miscellaneous and fraction (fraction) (such as from the extract of mushroom (Agaricus)) thereof is had (such as to join
Illuminate the way with document 1).
On the other hand, it is known that with from Semen Oroxyli (Oroxylum indicum, sometimes referred to as Semen Oroxyli or wood butterfly
Butterfly, in detail aftermentioned.) extract be the antioxidant (referring for example to quoting document 2) that effective ingredient contains.Should say
Bright, Semen Oroxyli is the natural product of the safety the most also used as eating in original producton location.
But, for containing using as follows as hyaluronic acid synthesis accelerant still the unknown of effective ingredient: from Semen Oroxyli
Extract or its purified, the fraction extracted from the seed of Semen Oroxyli especially by aqueous organic solvent, divided by this fraction
The fraction of the regulation cut or be contained in the chrysin (chrysin) of Semen Oroxyli.
Moreover it is known that there be (particularly cell surface) in vivo to carry out the HAS2 of the enzyme of the synthesis of hyaluronic acid (thoroughly
Bright matter acid enzyme 2).HAS2 is to be expressed by HAS2mRNA and be synthesized.It is to say, in order to supplement or increase in organism
The amount of hyaluronic acid, promote that the expression of HAS2mRNA is effective.
As the situation of hyaluronic acid synthesis accelerant, for containing using as follows as the HAS2mRNA table of effective ingredient
Reach accelerator also unknown: the fraction that extracted by aqueous organic solvent or be contained in the white of Semen Oroxyli from the seed of Semen Oroxyli
Yang Su.
Prior art literature
Patent documentation
Patent documentation 1 Japanese Laid-Open 2013-035835 publication
Patent documentation 2 Japanese Laid-Open 2006-321730 publication
Summary of the invention
Technical task to be solved by this invention " uses the fraction obtained by the Semen Oroxyli that safety is high or one-tenth for providing
The hyaluronic acid synthesis accelerant divided "." use high from safety additionally, its technical task to be solved lies also in offer
Fraction or the HAS2mRNA of composition obtained by Semen Oroxyli express accelerator ".Additionally, its technical task to be solved lies also in
" the hyaluronic acid synthesis accelerant containing the present invention has the hyaluronic acid synthesis pharmaceuticals of facilitation, food or change in offer
Makeup material ".And then, its technical task to be solved lies also in offer, and " HAS2mRNA containing the present invention expresses accelerator, has
HAS2mRNA expresses the pharmaceuticals of facilitation, food or cosmetic preparation ".
The result that the inventors of the present invention study deeply, distinguished from the extract of Semen Oroxyli or its purified it
In, " fraction extracted from the seed of Semen Oroxyli by aqueous organic solvent ", " by the fraction of the regulation of this fraction fractional distillation " and " white
Yang Su " there is hyaluronic acid synthesis facilitation.Additionally, by the research for its mechanism (mechanism), also distinguished above-mentioned
Each fraction and composition have HAS2mRNA and express facilitation.
The inventors of the present invention have been reached to complete the present invention by above knowledge.The present invention is made up of following item.
(1) a kind of hyaluronic acid synthesis accelerant, it is characterised in that contain by aqueous organic solvent from the kind of Semen Oroxyli
The fraction that son is extracted includes at least the 1st fraction of chrysin as effective ingredient.
(2) a kind of hyaluronic acid synthesis accelerant, it is characterised in that containing with water and ethyl acetate to organic by aqueous
Solvent, when the 1st fraction that the seed of Semen Oroxyli is extracted carries out liquid-liquid partition, is dispensed on the fraction of described ethyl acetate side
In including at least the 2nd fraction of chrysin as effective ingredient.
(3) a kind of hyaluronic acid synthesis accelerant, it is characterised in that containing with water and ethyl acetate to organic by aqueous
The 1st fraction that solvent is extracted from the seed of Semen Oroxyli carries out liquid-liquid partition, by 90% methanol and hexane further to being divided
Fit over the 2nd fraction of described ethyl acetate side when carrying out liquid-liquid partition, be dispensed in the fraction of described 90% methanol side at least
Comprise the 3rd fraction of chrysin as effective ingredient.
(4) a kind of hyaluronic acid synthesis accelerant, it is characterised in that containing with water and ethyl acetate to organic by aqueous
The 1st fraction that solvent is extracted from the seed of Semen Oroxyli carries out liquid-liquid partition, by 90% methanol and hexane further to being divided
The 2nd fraction fitting over described ethyl acetate side carries out liquid-liquid partition, by silica gel chromatography to being dispensed on described 90% methanol
When 3rd fraction of side carries out fractional distillation, by chloroform: methanol is at least to wrap in the mixed solvent of the 9:1 fraction by the dissolution of silica gel institute
The 4th fraction containing chrysin is as effective ingredient.
(5) a kind of hyaluronic acid synthesis accelerant, it is characterised in that containing chrysin as effective ingredient.
(6) a kind of HAS2mRNA expresses accelerator, it is characterised in that contain by aqueous organic solvent from the kind of Semen Oroxyli
The fraction that son is extracted includes at least the 1st fraction of chrysin as effective ingredient.
(7) a kind of HAS2mRNA expresses accelerator, it is characterised in that containing with water and ethyl acetate to organic by aqueous
Solvent, when the 1st fraction that the seed of Semen Oroxyli is extracted carries out liquid-liquid partition, is dispensed on the fraction of described ethyl acetate side
In including at least the 2nd fraction of chrysin as effective ingredient.
(8) a kind of HAS2mRNA expresses accelerator, it is characterised in that containing with water and ethyl acetate to organic by aqueous
The 1st fraction that solvent is extracted from the seed of Semen Oroxyli carries out liquid-liquid partition, by 90% methanol and hexane further to being divided
Fit over the 2nd fraction of described ethyl acetate side when carrying out liquid-liquid partition, be dispensed in the fraction of described 90% methanol side at least
Comprise the 3rd fraction of chrysin as effective ingredient.
(9) a kind of HAS2mRNA expresses accelerator, it is characterised in that containing with water and ethyl acetate to organic by aqueous
The 1st fraction that solvent is extracted from the seed of Semen Oroxyli carries out liquid-liquid partition, by 90% methanol and hexane further to being divided
The 2nd fraction fitting over described ethyl acetate side carries out liquid-liquid partition, by silica gel chromatography to being dispensed on described 90% methanol
When 3rd fraction of side carries out fractional distillation, by chloroform: methanol is at least to wrap in the mixed solvent of the 9:1 fraction by the dissolution of silica gel institute
The 4th fraction containing chrysin is as effective ingredient.
(10) a kind of HAS2mRNA expresses accelerator, it is characterised in that containing chrysin as effective ingredient.
(11) a kind of have the hyaluronic acid synthesis pharmaceuticals of facilitation, food or cosmetic preparation, it is characterised in that contains
Hyaluronic acid synthesis accelerant according to any one of above-mentioned (1)～(5).
(12) a kind of have the HAS2mRNA expression pharmaceuticals of facilitation, food or cosmetic preparation, it is characterised in that contains
HAS2mRNA according to any one of above-mentioned (6)～(10) expresses accelerator.
According to the present invention, it is possible to from test example described later, it is possible to provide " use obtained by the Semen Oroxyli that safety is high
Fraction or the hyaluronic acid synthesis accelerant of composition ", " use fraction obtained by the Semen Oroxyli that safety is high or composition
HAS2mRNA expresses accelerator ", " hyaluronic acid synthesis accelerant containing the present invention and there is hyaluronic acid synthesis facilitation
Pharmaceuticals, food or cosmetic preparation " and " HAS2mRNA containing the present invention expresses accelerator and has HAS2mRNA expressing promoting
Enter the pharmaceuticals of effect, food or cosmetic preparation ".
Should illustrate, in this specification and each accompanying drawing, when (%) is only recited as concentration, be to represent with volume/body
The percentage concentration that long-pending (V/V) calculates.
Under chrysin (chrysin, 5,7-dihydroxy-2-phenyl-4H-chromen-4-one) in the present invention is
The compound described in formula (1) of row,
Accompanying drawing explanation
The flow chart that Fig. 1 is the 1st fraction for illustrating in embodiment 1～5～the 4th fraction and chrysin shows.
Fig. 2 is for the flow chart that sample a～e in test example 1～5 shows is described.
Detailed description of the invention
Hereinafter, based on the embodiment hyaluronic acid synthesis accelerant to the present invention, HAS2mRNA expresses accelerator, has
The hyaluronic acid synthesis pharmaceuticals of facilitation, food or cosmetic preparation and have HAS2mRNA express facilitation pharmaceuticals,
Food or cosmetic preparation illustrate.
[embodiment]
The flow chart that Fig. 1 is the 1st fraction for illustrating in embodiment 1～5～the 4th fraction and chrysin shows.Should
Illustrate, be connected as be described hereinafter with dotted line between the 4th fraction and chrysin in FIG, be in order to the chrysin in embodiment 5 not by
It is defined in the material isolated by the 4th fraction.
The hyaluronic acid synthesis accelerant of the present invention and HAS2mRNA express accelerator and contain described in above-mentioned (1)～(10)
The 1st fraction, the 2nd fraction, the 3rd fraction, the 4th fraction or chrysin as effective ingredient.
Hereinafter each above-mentioned effective ingredient is illustrated by Fig. 1 and embodiment 1～5.Additionally, above-mentioned is contained thoroughly
Bright matter acid synthesis accelerant or HAS2mRNA are expressed the pharmaceuticals of accelerator, food or cosmetic preparation and are said by embodiment 6
Bright.
[embodiment 1]
The hyaluronic acid synthesis accelerant relevant with embodiment 1 and HAS2mRNA express accelerator for containing organic by aqueous
Solvent includes at least the 1st fraction of chrysin as effective ingredient from the fraction that the seed of Semen Oroxyli is extracted.
Semen Oroxyli (Oroxylum indicum) is that Lagerstroemia indica L. section (Bignoniaceae) Semen Oroxyli belongs to (Oroxylum), the height of tree
The high wood plant of fallen leaves of 7m～12m.Original producton location is Southeast Asia, India and China, naturally grows the oblique of Yushan Hill in these regions
Face or naturally grow along streams.The florescence of Semen Oroxyli is 7～August, and seed can gather in 10～December.The seed of Semen Oroxyli
For the general circulation goods that can obtain in these regions.
Should illustrate, the fruit of Semen Oroxyli, young leaves, petal etc. are used as edible product in Southeast Asia etc..Additionally, a thousand sheets
Paper is considered have drug effect, is used in hoarseness or the treatment of aphasis, the treatment (hemostasis) of injury, stomach invigorating, cough-relieving
Deng.
The seed of Semen Oroxyli (is used for making seed fly except plumule, the thin film also outside endosperm (endosperm) with wing
It is scattered to the position in a distant place), become thin plates on the whole.
Also above-mentioned thin film can be attached, it is possible to remove above-mentioned thin film in order to obtain the seed of the Semen Oroxyli that the 1st fraction is used.At this
In description and each accompanying drawing, the seed of Semen Oroxyli comprises the most above-mentioned thin film and both above-mentioned thin film of removing.
Accelerator is expressed, such as in order to obtain the hyaluronic acid synthesis accelerant relevant with embodiment 1 and HAS2mRNA
Can use and the seed gathered by Semen Oroxyli is dried up the dried object making its natural drying.Additionally, be used as the seed of Semen Oroxyli
Without machining object, heat drying thing, lyophilization thing, frozen material etc..
When extracting, such as, the seed of Semen Oroxyli be can be used directly, it is possible to chopping uses, it is possible to do powdered making
With.
In this manual, " aqueous organic solvent " refers to the solvent with hydrophilic organic solvent as main constituent." hydrophilic
The organic solvent of property " refer to not be separated from water and the organic solvent that mixes.Hydrophilic organic solvent can illustrate lower aliphatic
Alcohol (lower aliphatic alcohol) and acetone.Lower aliphatic alcohols can illustrate the one of methanol, ethanol, propanol, butanol etc.
Unit's alcohol, or the polyhydric alcohol of 1,3 butylene glycol, propylene glycol, 1,3-PD, pentanediol, isoprene etc..
Aqueous organic solvent also can be containing organic solvents more than two kinds.
Aqueous organic solvent also can contain water.Should illustrate, when aqueous organic solvent contains water, hydrophilic organic molten
Whether agent is main constituent, will consider the water removed among constituent.Even it is to say, water is than hydrophilic organic solvent
Many situations, if the hydrophilic organic solvent in organic solvent is main constituent, then for mention in this manual " aqueous has
Machine solvent ".
When aqueous organic solvent contains water, aqueous organic solvent preferably comprise more than 30% above-mentioned hydrophilic organic molten
Agent, more preferably contains more than 50%.
Can enumerate as the concrete example of the 1st fraction and be obtained by using the extraction of aqueous organic solvent by the seed of Semen Oroxyli
The extracting solution arrived, the diluent of this extracting solution or concentrated solution, this extracting solution is dried and the dried object that obtains and theirs is the purest
Compound or purified.
Should illustrate, shown in test example as be described hereinafter, the seed of Semen Oroxyli use aqueous organic solvent to extract
Fraction includes chrysin.Therefore, as long as not carrying out as from extracting solution, concentrated solution, diluent, dried object, coarse-fine thing processed, essence
Things processed etc. remove the such process of chrysin, just meet the condition of " comprising chrysin ".It is with the 2nd fraction～the 4th fraction also
Sample.
As long as extracting the fraction dissolution of solubility of the seed that can be included in Semen Oroxyli in aqueous organic solvent, the most not by
It is particularly limited to, can carry out according to common method.When extracting without using special method or apparatus, arbitrary method can be used
Or device.
Example as the method extracted can enumerate the method at extract at room temperature, or is heated to the boiling point of aqueous organic solvent
Following temperature and the method extracted, in the method being heated to reflux lower extraction.Now, only impregnate Semen Oroxyli seed can also,
Stirring together with solvent can also.The amount of aqueous organic solvent can be such as 5～50 times amount (weight ratio) extracting raw material.Extract
Time can be such as 1～72 hour.Extracting temperature can be such as room temperature～95 DEG C.
Before the extraction carrying out utilizing aqueous organic solvent, it is possible to carry out using pentane, hexane, heptane, octane, petroleum ether
Deng the ungrease treatment of non-polar solven.
Can filter by after the fraction dissolution making solubility goes out, extraction residue is removed in the process of centrifugation etc.
Obtain extracting solution.In order to be obtained the diluent of this extracting solution, concentrated solution, dried object, thick purified, pure by obtained extracting solution
Compound etc., it is possible to be further diluted according to common method, concentrate, be dried, the process of purification etc..
Purification as the 1st fraction, it is possible to the purification for the purpose of carrying out with decolouring, deodorize etc..This purification can use such as
Various adsorption treatment or various separating treatment (such as use activated carbon, adsorbent resin, ion exchange resin, silica gel, gel filtration
The process of material).This point in the 2nd fraction described later～the 4th fraction too.
[embodiment 2]
The hyaluronic acid synthesis accelerant relevant with embodiment 2 and HAS2mRNA express accelerator and contain with water and ethyl acetate
To by aqueous organic solvent when the 1st fraction that the seed of Semen Oroxyli is extracted carries out liquid-liquid partition, be dispensed on described second
The fraction of acetoacetic ester side includes at least the 2nd fraction of chrysin as effective ingredient.
1st fraction can be by being obtained by the method described in above-mentioned embodiment 1.
As long as liquid-liquid partition just can be specially limited by the 1st fraction seperation the 2nd fraction, can carry out according to common method.
Implementing without using special method or apparatus of liquid-liquid partition, can use arbitrary method or apparatus.
[embodiment 3]
The hyaluronic acid synthesis accelerant relevant with embodiment 3 and HAS2mRNA express accelerator and contain with water and ethyl acetate
The 1st fraction extracted from the seed of Semen Oroxyli by aqueous organic solvent is carried out liquid-liquid partition, by 90% methanol Radix Chloranthi Serrati
When alkane carries out liquid-liquid partition to the 2nd fraction being dispensed on described ethyl acetate side further, it is dispensed on described 90% methanol
The fraction of side includes at least the 3rd fraction of chrysin as effective ingredient.
1st fraction can be obtained by the method described in above-mentioned embodiment 1.2nd fraction can pass through above-mentioned embodiment 2
Described method obtains.
As long as liquid-liquid partition just can be specially limited by the 2nd fraction seperation the 3rd fraction, can carry out according to common method.
Implementing without using special method or apparatus of liquid-liquid partition, can use arbitrary method or apparatus.
[embodiment 4]
The hyaluronic acid synthesis accelerant relevant with embodiment 4 and HAS2mRNA express accelerator and contain with water and ethyl acetate
The 1st fraction extracted from the seed of Semen Oroxyli by aqueous organic solvent is carried out liquid-liquid partition, by 90% methanol Radix Chloranthi Serrati
Alkane carries out liquid-liquid partition to the 2nd fraction being dispensed on described ethyl acetate side further, by silica gel chromatography to allocated
When the 3rd fraction of described 90% methanol side carries out fractional distillation, by chloroform: methanol is that the mixed solvent of 9:1 is by the dissolution of silica gel institute
Fraction in including at least the 4th fraction of chrysin as effective ingredient.
1st fraction can be obtained by the method described in above-mentioned embodiment 1.2nd fraction can pass through above-mentioned embodiment 2
Described method obtains.3rd fraction can be obtained by the method described in above-mentioned embodiment 3.
As long as silica gel chromatography just can be specially limited by the 3rd fraction seperation the 4th fraction, can enter according to common method
OK.Without using special method or apparatus when carrying out silica gel chromatography, arbitrary method or apparatus can be used.
Embodiment 5
The hyaluronic acid synthesis accelerant relevant with embodiment 5 and HAS2mRNA express accelerator and contain chrysin as effectively
Composition.
Chrysin is the following compound described in formula (1),
Shown in chrysin test example as be described hereinafter, it is contained in the 1st above-mentioned fraction～the 4th fraction.Hyaluronic acid synthesis promotees
Enter agent and HAS2mRNA to express the chrysin that accelerator used and can obtain (with reference to test example described later) from the seed of Semen Oroxyli,
Also can natural product beyond the seed of Semen Oroxyli, and the natural product containing chrysin obtains, it is possible to for commercially available product or conjunction
Finished product.
When obtaining chrysin from the seed of Semen Oroxyli, such as can be by the 4th fraction list described in embodiment 4
From.The isolated purification of chrysin can be suitable for using silica gel chromatography and gel-filtration chromatography, but the invention is not restricted to this.
As long as isolated for chrysin purification can be just not particularly limited by silica gel chromatography and gel-filtration chromatography, can depend on
Carry out according to common method.Without using special method or apparatus when carrying out silica gel chromatography and gel-filtration chromatography, can adopt
Use arbitrary method or apparatus.
The hyaluronic acid synthesis accelerant relevant with above-mentioned embodiment 1～5 and HAS2mRNA express accelerator and can pass through
Its hyaluronic acid synthesis facilitation and HAS2mRNA express the amount that facilitation is supplemented or increased hyaluronic acid in organism,
Its result, can expect the prevention of the symptom of the abnormal of skin or arthralgia etc., suppress and improve or normalization.But, with this
Inventing relevant hyaluronic acid synthesis accelerant and HAS2mRNA and express accelerator in addition to these purposes, it is right also to can be suitably used for
In playing hyaluronic acid synthesis facilitation and the HAS2mRNA expression meaningful all of purposes of facilitation.
[embodiment 6]
The pharmaceuticals relevant with embodiment 6, food or cosmetic preparation, containing embodiment institute any in above-mentioned embodiment 1～5
The hyaluronic acid synthesis accelerant recorded or HAS2mRNA express accelerator.
When the pharmaceuticals relevant with embodiment 6 are interior pharmaceuticals, medication is not particularly limited, such as can be with:
Oral administration, drop rectum with drug etc. through enteral administration；The mucosa delivery of nose administration etc.；Intravenous administration, subcutaneous administration etc.
Drug administration by injection.The dosage form of the interior pharmaceuticals of the present invention can be in the way of applicable medication, such as can be with: lozenge, powder, thin
Granule, granule, capsule, powder, pill, solid formulation containing tablet etc.；Solution, suspension, Emulsion, syrup, injection
Deng liquor；Gelatinous preparation.As the pharmaceuticals relevant with embodiment 6, it is possible to by arbitrary reality in embodiment 1～5
Execute the hyaluronic acid synthesis accelerant described in mode or HAS2mRNA expresses the sterling of accelerator, purified, thick purified etc.
It is directly administered, it is possible to be administered together with the excipient being pharmacologically allowed.Monosaccharide, disaccharidase class, many can be used as excipient
Saccharide, inorganic salts, oils and fats, distilled water etc. are as the usual spendable material of preparation.By the interior doctor relevant with embodiment 6
During medicine preparation, it is possible to use binding agent, lubricant, dispersant, suspending agent, emulsifying agent, diluent, buffer agent, antioxidation
The additive of agent, antibacterial etc..
When the pharmaceuticals relevant with embodiment 6 are external pharmaceuticals, its form is not specially limited, such as can be with:
Ointment, unguentum, paste (cataplasm), adhesive tape agent, external preparation.The external pharmaceuticals relevant with embodiment 6, except reality
Execute in mode 1～5 the hyaluronic acid synthesis accelerant described in any embodiment or HAS2mRNA expresses outside accelerator, also
Other medical components or excipient etc. can be contained as required.When manufacturing the external pharmaceuticals relevant with embodiment 6 also
The additive of binding agent, dispersant, suspending agent, emulsifying agent, diluent, buffer agent, antioxidant, antibacterial etc. can be used.
The pharmaceuticals relevant with embodiment 6 can (be medication, agent if interior pharmaceuticals according to various data
Type, hyaluronic acid synthesis accelerant or HAS2mRNA express effective dosage of accelerator itself, are administered as the effective of preparation
The data of amount etc.) set optimal hyaluronic acid synthesis accelerant or the content of HAS2mRNA expression accelerator.
The form of the food relevant with embodiment 6, such as, can be made into any embodiment in embodiment 1～5 and remembered
The hyaluronic acid synthesis accelerant carried or HAS2mRNA express the sterling of accelerator, partial purification product, synthesize containing hyaluronic acid
Accelerator or HAS2mRNA express the paste (paste) of accelerator, coordinate hyaluronic acid synthesis accelerant or HAS2mRNA to express
The normal food of accelerator.Concrete form can enumerate health beverage, fruit jelly, cookies, tea, lozenge, pill, soft capsule, hard
Capsule, powder, granula subtilis, granule, as long as the form that can serve as food offer then can use any form.Manufacture with
During the relevant food of embodiment 6, auxiliary material is used as excipient, binding agent, lubricant, dispersant, suspending agent, emulsifying
The additive of agent, diluent, buffer agent, antioxidant, antibacterial etc..
The form of the cosmetic preparation relevant with embodiment 6 such as can be made into astringent, beautifying liquid, emulsion, cream, gel,
Facial film, beauty treatment muffin, Cleansing Foam, bath shampoo etc., as long as can serve as the form that cosmetic preparation uses, then can use any form.
The cosmetic preparation relevant with embodiment 6 promotes except the hyaluronic acid described in any embodiment in embodiment 1～5 synthesizes
Agent or HAS2mRNA express outside accelerator, it is possible to contain the various gradation compositions being matched with cosmetic preparation as required.Fit in
Divide and such as can use solid oil, lard, liquid oil, low molecule wetting agent, macromolecule wetting agent, fat-soluble wetting agent, profit
Lubrication prescription (emollient), surfactant, preservative, antioxidant, pH adjusting agent, ethanol, water.
Pharmaceuticals, food and the cosmetic preparation relevant with above-mentioned embodiment 6 can pass through relevant with embodiment 1～5 saturating
Bright matter acid synthesis accelerant and HAS2mRNA express hyaluronic acid synthesis facilitation and the HAS2mRNA table that accelerator is had
Reach the amount that facilitation is supplemented or increased hyaluronic acid in organism, its result, the abnormal of skin or arthralgia can be expected
Deng the prevention of symptom, inhibitor improve or normalization.But, pharmaceuticals, food and the cosmetic preparation relevant with embodiment 6 remove
Beyond this purposes, could be used that in expressing facilitation and have for playing hyaluronic acid synthesis facilitation and HAS2mRNA
The all of purposes of meaning.
Although additionally, hyaluronic acid synthesis accelerant, HAS2mRNA described in embodiment 1～6 express accelerator, doctor
The mankind are suitable for being suitable for by medicine, food and cosmetic preparation, but as long as each action effect can be reached, also can be suitably used for beyond the mankind
Animal.
[test example]
Hereinafter, as test example, enumerate " 1. isolated 1st fraction of graduation～the 4th fraction and the test example of chrysin ", " 2. use
In the test example confirming hyaluronic acid synthesis facilitation " and " 3. for confirming that HAS2mRNA expresses the test example of facilitation "
Thus illustrate in greater detail the present invention.Should illustrate, following test example is to represent that the method for fractional distillation etc. or effect confirm
Result, the present invention is not limited by tests below example.
1. isolated 1st fraction of graduation～the 4th fraction and the test example of chrysin
Fig. 2 is sample a～e for illustrating in test example 1～5 and the flow chart that shows.Should illustrate, due in test example 6
Sample f be and sample a～the sample of e different system that therefore Fig. 2 does not records.
Illustrate the fractional distillation of the 1st fraction～the 4th fraction and chrysin is isolated below by way of test example 1～test example 6.
Should illustrate, in following test example, water uses distilled water, methanol, ethyl acetate and hexane to use mountain one chemistry work
The Japan of industry Co., Ltd. produces the methanol of one-level, ethyl acetate and hexane.Additionally, experimental apparatus uses general product (such as
100mL conical flask and 30mL conical flask, the conical flask of Iwaki Glass Co., Ltd).
[test example 1]
Test example 1 be for obtain by aqueous organic solvent from the fraction that the seed of Semen Oroxyli extracts including at least chrysin
The test example of the 1st fraction.As in figure 2 it is shown, using in test example 1 by the 1st fraction of graduation as sample a.
Use methanol as aqueous organic solvent in test example 1.
First, the seed 1kg of Semen Oroxyli (Oroxylum indicum) is prepared.The seed of Semen Oroxyli uses general circulation goods
Dried object (with the thin film person of wing).
Secondly, with shears, the seed of Semen Oroxyli is cut off and broken into pieces, be immersed in methanol and extract.It is extracted in stainless steel
Container is carried out.Extracting method, with merceration (room temperature, methanol 20L, 24 hours extraction times), adds up to and carries out 3 extractions.
Then, at room temperature carry out natural filtration, obtain extracting solution (the transparent liquid of fluorescent yellow-green).And then to this
Extracting solution concentrating under reduced pressure (40 DEG C, 60mmHg), obtains sample a (the 1st fraction) 31.2g of the solid of black.Kind with Semen Oroxyli
Solid yields on the basis of the weight (1kg) of son is 3.12%.
Should illustrate, although do not carry out whether sample a comprises the test of chrysin, but shown in test example 5 as be described hereinafter, because of
The result of the fractional distillation purification of repeat sample a has obtained chrysin, therefore specify that sample a comprises chrysin.This point is aftermentioned
Test example 2～test example 4 in sample b～sample d too.
Test example 2
Test example 2 be for obtain with water and ethyl acetate to being extracted from the seed of Semen Oroxyli by aqueous organic solvent the 1st
When fraction carries out liquid-liquid partition, it is dispensed on the fraction of ethyl acetate side and the test of the 2nd fraction including at least chrysin
Example.Using the 2nd fraction that is fractionated in test example 2 as sample b.
First, water 800mL is added to sample a (the 1st fraction) 31.2g that test example 1 obtains as suspension, to this suspension
Liquid total carries out 3 times utilizing the extraction (room temperature, ethyl acetate 100mL) of ethyl acetate.Extract and use separatory funnel to carry out.
Then reclaim ethyl acetate, carry out concentrating under reduced pressure (40 DEG C, 75mmHg), obtain the sample b (the 2nd fraction) of the solid of black
11.8g.Solid yields on the basis of the weight of the seed of Semen Oroxyli is 1.18%.
Test example 3
Test example 3 be for obtain with water and ethyl acetate to being extracted from the seed of Semen Oroxyli by aqueous organic solvent the 1st
Fraction carries out liquid-liquid partition, is carried out the 2nd fraction being distributed in ethyl acetate side further by 90% methanol and hexane
During liquid-liquid partition, it is dispensed on the fraction of 90% methanol side, and the test example of the 3rd fraction including at least chrysin.With in examination
Test divided 3rd fraction of example 3 as sample c.
First, sample b (the 2nd fraction) 11.8g being added to 90% methanol 300mL obtain in test example 2 as suspension,
This suspension is added up to and carries out 3 times utilizing the extraction (room temperature, hexane 300mL) of hexane.Extract and use separatory funnel to carry out.
Then reclaim 90% methanol, carry out concentrating under reduced pressure (40 DEG C, 75mmHg), obtain the sample c (the 3rd fraction) of the solid of black
4.0g.Solid yields on the basis of the weight of the seed of Semen Oroxyli is 0.40%.
Test example 4
Test example 4 be for obtain with water and ethyl acetate to being extracted from the seed of Semen Oroxyli by aqueous organic solvent the 1st
Fraction carries out liquid-liquid partition, is carried out the 2nd fraction being dispensed on ethyl acetate side further by 90% methanol and hexane
Liquid-liquid partition, when the 3rd fraction being dispensed on 90% methanol side being carried out fractional distillation by silica gel chromatography, passes through chloroform: methanol
For the mixed solvent of 9:1 by the fraction of silica gel dissolution, and the test example of the 4th fraction including at least chrysin.With in test example 4
By the 4th fraction of graduation as sample d.
First, the sample c obtained in test example 3 is made with a small amount of chloroform of (tens of about mL) and the mixed solvent of methanol
(the 3rd fraction) 4.0g dissolves as solution.Secondly, this solution is placed on it is filled with silica gel (with Guang Chun medicine Co., Ltd.
Wakosil C-200) post
Then, by chloroform: the mixed solvent 300mL of methanol=9:1 carries out dissolution, obtains component A-1.Then, chloroform is passed through:
The mixed solvent 300mL of methanol=4:1, chloroform: the mixed solvent 300mL of methanol=1:1, methanol 300mL carry out dissolution successively,
Obtain component A-2, component A-3, component A-4 successively.
Wherein component A-1 is carried out concentrating under reduced pressure (40 DEG C, 60mmHg), obtains coffee color～the sample d of blackish green extract shape
(the 4th fraction) 2.32g.Solid yields on the basis of the weight of the seed of Semen Oroxyli is 0.232%.
[test example 5]
Test example 5 is the test example for obtaining chrysin.Using in test example 5 by isolated chrysin as sample e.
Should illustrate, in silica gel chromatography in test example 5 and gel-filtration chromatography, take on a small quantity each fraction
The sample of (20～about 30mL), confirms point with TCL.By color or the reaction of UV lamp are confirmed a little.Appearance for point
Identical fraction, processes as a component.The amount of the solution of each fraction not necessarily, also has sight
Examine the color etc. of solution while the situation of point fraction.
First, with the mixed solvent of a small amount of chloroform and methanol make in the sample d (the 4th fraction) that test example 4 obtains by
The 2.27g of part that later half fraction obtains dissolves as solution.Secondly, this solution is placed on that to be filled with silica gel (pure with light
The Wakosil C-200 of medicine Co., Ltd.) post
Then, by chloroform: the mixed solvent 500mL of methanol=9:1 carries out dissolution, the 3fraction of first half obtain component
B-1, is obtained component B-2 by later half 2fraction.Then, carry out dissolution by methanol 300mL, obtain component B-3.
Wherein component B-1 is carried out concentrating under reduced pressure (40 DEG C, 60mmHg), obtains the fraction 1.04g from component B-1.
And then, make the fraction 1.04g from component B-1 dissolve as molten using the mixed solvent of a small amount of chloroform and methanol
Liquid.Secondly, this solution is placed on it is filled with the post of silica gel (with the Wakosil C-200 of Guang Chun medicine Co., Ltd.)
Then, carry out dissolution by chloroform 500mL, the 1fraction of first half obtain component C-1, by later half 3fraction
Obtain component C-2.Then, chloroform is passed through: the mixed solvent 300mL of methanol=19:1, methanol 300mL carry out dissolution successively, depend on
Secondary component C-3 that obtains, component C-4.Wherein component C-2 is carried out concentrating under reduced pressure (40 DEG C, 60mmHg), obtains from component C-2
Fraction 0.35g.
And then, make the fraction 0.35g from component C-2 dissolve as molten using the mixed solvent of a small amount of chloroform and methanol
Liquid.Secondly, this solution is placed on it is filled with the post of silica gel (with the Wakosil C-200 of Guang Chun medicine Co., Ltd.)
Then, carry out dissolution by chloroform 500mL, the 1fraction of first half obtain component D-1, by later half 3fraction
Obtain component D-2.Then, carry out dissolution by methanol 300mL, obtain component D-3.
Wherein component D-2 is carried out concentrating under reduced pressure (40 DEG C, 60mmHg), obtains the fraction 0.31g from component D-2.
And then, make the fraction 0.31g from component D-2 dissolve as molten using the mixed solvent of a small amount of chloroform and methanol
Liquid.Next, be placed on the gel filtration material (Sephadex of Sigma-Aldrich company being filled with chromatography by this solution
LH-20) post
Then, by chloroform: the mixed solvent 500mL of methanol=1:1 carries out dissolution, initial 2fraction obtain component
E-1, is obtained component E-2 by next 1fraction, next one 1fraction obtains component E-3.
Wherein component E-3 is carried out concentrating under reduced pressure (40 DEG C, 60mmHg), obtains the fraction 0.07g from component E-3.
And then, make the fraction 0.07g from component E-3 dissolve as molten using the mixed solvent of a small amount of chloroform and methanol
Liquid.Next, be placed on the gel filtration material (Sephadex of Sigma-Aldrich company being filled with chromatography by this solution
LH-20) post
Then, by chloroform: the mixed solvent 500mL of methanol=1:1 carries out dissolution, initial 1fraction obtain component
F-1, is obtained component F-2 by next 3fraction, next one 1fraction obtains component F-3.
Wherein component F-2 is carried out concentrating under reduced pressure (40 DEG C, 60mmHg), obtains sample e (chrysin) about 10mg.With Semen Oroxyli
Solid yields on the basis of the weight of seed is about 0.001%.
For being obtained 1H-NMR spectrum money by isolated sample e (chrysin) by nuclear magnetic resonance spectrometry like that to above-mentioned
Material.The acquirement of this data employs the JNM-ECA500 of JEOL RESONANCE Co., Ltd..
Its result viewing is to following peak (with outline value, only record main peak), with the 1H-NMR of commonly known chrysin
SPECTRAL DATA is substantially uniform.
1H-NMR(acetone-d6): δ (ppm)=6.29 (1H, d, J=1.7Hz, H-6), 6.58 (1H, d, J=
1.7Hz, H-8), 6.79 (1H, s, H-3), 7.60 (2H, m, H-3 ', H-5 '), 7.61 (1H, m, H-4 '), 8.07 (2H, dd, J=
8.1,1.7Hz, H-2 ', H-6 ')
And, the analysis utilizing high performance liquid chromatography has been carried out for sample e.This analysis employs Shimadzu Seisakusho Ltd.'s strain
The diode array detector SPD-M20A of formula commercial firm, feed unit LC-20AB, post baking oven CTO-20A and Shiseido strain formula meeting
The HPLC column CAPCELL PAK MG II 4.6mm I.D. × 250mm of society.In analysis, A liquid is phosphate buffer
(pH6.5), B liquid is 100% methanol, and linear gradient is 40 DEG C with A:B=95:5～20:80 (60 points), column temperature.Detection method makes
With diode array (190-800nm).
It is analyzed with above-mentioned condition firstly, for chrysin standard substance (purchased from Tokyo HuaCheng Industry Co., Ltd),
Confirm to produce maximum peak at 53.7 points.Secondly, sample e is analyzed with identical condition, it is thus identified that at 52.6 points
Produce maximum peak.And, it is thus identified that the spectrum of the maximum peak of chrysin standard substance and sample e is consistent.
As it has been described above, can confirm that sample e is mainly by chrysin by nuclear magnetic resonance spectrometry and high performance liquid chromatography
Constitute.
Additionally, due to sample e is the material isolated by sample a～d fractional distillation, therefore can confirm that sample a～d also comprises Cortex Populi dividianae
Element.
[test example 6]
Test example 6 be for obtain by aqueous organic solvent from the fraction that the seed of Semen Oroxyli extracts including at least chrysin
Another test example of the 1st fraction.Using the 1st fraction that is fractionated in test example 6 as sample f.
Use 50% ethanol as aqueous organic solvent in test example 6.
First, the seed 10kg of Semen Oroxyli (Oroxylum indicum) is prepared.The seed of Semen Oroxyli uses general circulation
The dried object (with the thin film of wing) of product.
Secondly, the seed of Semen Oroxyli is shredded, is immersed in 50% ethanol and extracts.Extracting method is with under being heated to reflux
Extract (80 DEG C, 50% ethanol 150L, 2 hours extraction times).
Then, at room temperature carry out natural filtration and obtain extracting solution, so to this extracting solution concentrating under reduced pressure (heat, but 60 DEG C
Below), obtain testing f (the 1st fraction).
2. for confirming the test example of hyaluronic acid synthesis facilitation
[test example 7]
Test example 7 is for confirming for the 1st fraction in the present invention, the 3rd fraction, the 4th fraction and the hyaluronic acid of chrysin
The test example of synthesis facilitation, carries out the mensuration of hyaluronic acid synthesis facilitation.
First test method is illustrated
Pass on as sowing and use containing 10% (V/V) hyclone by culture medium that (FBS, purchased from NICHIREI bioscience
Co., Ltd., CCB company) minimum must culture medium α (MEM α, purchased from GIBCO, Cat.No.11900-024).Use this cultivation
Base, (NB1RGB, grinds Biological Resource Center (Riken BioResource by human neonatal dermal's fibroblast purchased from reason
Center), Resource.No.RBRC-RCB022) it is seeded in 96 well culture plates to become 2 × 104cells/well.Confirm
Cell adhesion after 24 hours, in order to remove the serum in culture medium culture medium α must carry out twice cleaning without the minimum of FBS.
Then, by chemical examination culture medium must culture medium α add all well to containing 0.5% (V/V) hyclone is minimum, with one-tenth
It is 100 μ L/well.Then, the culture medium comprising various sample of 100 μ L/well is added (with the culture medium system chemically examined
Standby, with become final concentration times concentration) and do not comprise the culture medium (comparison with) of sample, further carry out training in 72 hours
Support.
After cultivation terminates, ELISA is used to measure the Quantikine ELISA Hyaluronan of external member culture supernatant
Immunoassay (R&D Systems company) and the Multiskan Spectrum (Thermo of extinction micro-dish analyser
Fisher Scientific Co., Ltd. (Thermo Fisher Scientific K.K.)), measure hyaluronic acid amount.And
And, measure cells survival rate by dimethyl diaminophenazine chloride method.Set with cells survival rate 80% for standard substantially, with the concentration of sample not
The scope further giving the impact of cells survival rate is evaluated.The mensuration of the hyaluronic acid amount carried out by ELISA method and
The measuring of cells survival rate carried out by dimethyl diaminophenazine chloride method is passed through conventional because of respective according to the using method measuring external member or machine
Method is carried out, and therefore omits detailed description.
As sample, use the sample f of test example 6 as the 1st fraction respectively, use the sample c of test example 3 as the 3rd
Fraction, uses the sample d of test example 4 as the 4th fraction, uses the sample e of test example 5 as chrysin.
Secondly, result of the test is illustrated
Table 1 is the table of the result (hyaluronic acid synthesis facilitation produced by each sample) representing test example 7.Additionally, at table 1
In the concentration of " concentration " each sample when being to represent cultivation, unit is μ g/mL.And, " live vol " be with comparison in transparent
Matter acid generation amount is the hyaluronic acid generation amount of the situation of 100, and unit is %.And then, " cells survival rate " is with in comparison
Cells survival rate is the cells survival rate of the situation of 100, and unit is %.
[table 1]
Hyaluronic acid synthesis facilitation produced by each sample
As shown in table 1, for the 1st fraction in the present invention, the 3rd fraction, the 4th fraction and chrysin, can confirm that the mankind
Neonatal dermal fibroblast blast cell display hyaluronic acid synthesis facilitation.And, in the sample that test example 7 uses, can be true
Recognize sample c (the 3rd fraction) and sample e (chrysin) and show the tendency of high hyaluronic acid synthesis facilitation.
Although additionally, do not carry out the test about the 2nd fraction in test example 7, but because of for other fraction and chrysin
All can confirm that hyaluronic acid synthesizes facilitation, therefore it is contemplated that be that the 2nd fraction has hyaluronic acid synthesis promotion the most certainly
Effect.
3. for confirming that HAS2mRNA expresses the test example of facilitation
Test example 8
Test example 8 is for confirming that HAS2mRNA expresses the test of facilitation for the 1st fraction in the present invention and chrysin
Example, has carried out HAS2mRNA and has expressed the mensuration of facilitation.
First test method is illustrated.
It is to use as above-mentioned test example 7 that sowing is passed on by the culture medium of culture medium and chemical examination.Use sowing is passed on
By culture medium, human neonatal dermal's fibroblast (as above-mentioned test example 7) is seeded in 12 well culture plates to become 1
×105Cells/well, after 24 hours with 1 × PBS (-) solution once cleans.Then, the culture medium of chemical examination is added
It is added on all well, to become 700 μ L/well.Then, the culture medium comprising various sample of 700 μ L/well is added (with chemical examination
Culture medium be prepared, with become final concentration times concentration) and do not comprise the culture medium (comparison with) of sample.During cultivation
Between be that 4 are divided into for same sample, 6 stages of 8,12,16,20,24 hours and carry out.After cultivation terminates, remove the upper of each hole
Clearly, with 1 × PBS (-) solution 1mL is carried out.
For the extraction of RNA, use NucleoSpinRNA (Takara Shuzo Co., Ltd. of totalRNA purification external member
(TAKARABIO INC.)) and carry out.
With 1 × PBS (-) after the cleaning of solution, by Buffer RA1 (Cell lysis buffer of totalRNA purification external member)
Mixing reagent with 2 mercapto ethanol (2-mercaptoethanol) is added to each hole.Buffer RA1 and the amount of 2 mercapto ethanol
It is with every 2 hole 350 μ L and 3.5 μ L.Reclaim mixing reagent from each hole, move to sample and manage (the sterilizing 1.5mL pipe of RNA) respectively,
Often pipe imposes eddy mixer (vortex mixer) and stirs.Purification external member purification totalRNA is used from the content of pipe,
-80 DEG C of keepings.Because the extraction of RNA is carried out by common method according to the operating process of purification external member, therefore omit specifically
Bright.
For the totalRNA extracted like this, use SmartSpec Plus spectrophotometer (Bio-Rad company), by
Absorbance (260nm) measures RNA concentration.Reverse transcription reaction uses PrimeScript RT Master Mix (Perfect Real
Time) (Takara Shuzo Co., Ltd.), is cultivated 15 minutes at 37 DEG C by hot block, cultivates 5 seconds 500ng's at 85 DEG C
TotalRNA, obtains cDNA10 μ L.Then by forward and each 1 μ L of reverse primer (10 μMs), the SYBR Premix EX of each gene
Taq II (Takara Shuzo Co., Ltd.) 12.5 μ, as the RNase Free H of balance2O is added to prepared cDNA2 μ L, and total amount is
251μL.Use 5'-GACAGGCATCTCACGAACCG-3' as the forward primer of HAS2,5'-
CAACGGGTCTGCTGGTTTAGC-3' is as reverse primer.Internal standard uses glyceraldehyde-3-phosphate
dehydrogenase(GAPDH).Thermal Cycler Dice Real Time System II (Takara Shuzo Co., Ltd.)
Carry out real-time PCR, measure the expression of HAS2mRNA.
And, test example 8 carries out the mensuration of cells survival rate also by the method as test example 7.
Sample only uses the sample f of test example 6 as the 1st fraction, uses the sample e of test example 5 as chrysin.
Secondly, result of the test is illustrated.
Table 2 is the table of the result (HAS2mRNA produced by each sample expresses facilitation) representing test example 8.Additionally, at table 2
In the concentration of " concentration " each sample when being to represent cultivation, unit is μ g/mL.And, " promotion rate " is with in comparison
HAS2mRNA expression is the multiplying power of HAS2mRNA expression when 1, and unit is again.Additionally, comprise the training of the culture medium of sample
The foster time is divided into 6 stages of 4～14 hours in above-mentioned, records the result of the incubation time maximum about promotion rate in table 2.
With parantheses signifier as incubation time after the numerical value of promotion rate." cells survival rate " is
The cells survival rate of the situation of 100, unit is %.
[table 2]
HAS2mRNA produced by each sample expresses facilitation
As shown in table 2, for the 1st fraction and chrysin in the present invention, it is thus identified that female to human neonatal dermal's fiber
Cell display HAS2mRNA expresses facilitation, therefore it is contemplated that the 2nd fraction～the 4th fraction have HAS2mRNA expression the most certainly
Facilitation.
Embodiment
The making of embodiment 1. ointment
Use the 1st fraction prepared according to the method described in above-mentioned test example 1, by following prescription with common method system
Make ointment (100g).
(oil-phase component)
(water-phase component)
(preparation method)
Oil-phase component and water-phase component it is heated separately to 80 DEG C and makes it uniform, by stirring, aqueous phase being added to oil
Phase, after emulsifying, cooling has made ointment.
The making of embodiment 2. adhesive tape agent
Use the 2nd fraction prepared according to the method described in above-mentioned test example 2, by following prescription with common method system
Make adhesive tape agent (100g).
(sticker solvent)
(active ingredient)
2nd fraction 0.01g
Ethanol 7.99g
(transdermal absorption accelerator)
Oleyl alcohol 0.80g
All amounts 100g
(preparation method)
Make sticker solvent and active ingredient uniform respectively, active ingredient and transdermal absorption accelerator be added to sticker solvent,
It is stirred at room temperature making compositions.Said composition is extended in and has carried out, on the polyester film that silicon processes, being dried cold at 120 DEG C
But, after, make adhering agent layer be transferred to polyethylene film, make adhesive tape agent.
The making of embodiment 3. astringent
Use the 3rd fraction prepared according to the method described in above-mentioned test example 3, by following prescription with common method system
Make astringent (100g).
(oil-phase component)
Polyoxyethylene (60 moles) castor oil hydrogenated 1.0g
Shark alkane 0.05g
(water-phase component)
(preparation method)
Make oil-phase component and water-phase component uniform dissolution respectively.Oil phase is heated, stirs the component being added to aqueous phase
And be prepared, this is prepared liquid is added to remaining aqueous phase, makes astringent.
The making of embodiment 4. emulsion
Use the 4th fraction prepared according to the method described in above-mentioned test example 4, by following prescription with common method system
Make emulsion (100g).
(preparation method)
Above-mentioned raw materials is stirred, mixes and make emulsion.
The making of embodiment 5. beautifying liquid
Use the chrysin prepared according to the method described in above-mentioned test example 5, by following prescription with common method system
Make beautifying liquid (100g).
(preparation method)
Above-mentioned raw materials is stirred, mix, dissolves and make beautifying liquid.
The making of embodiment 6. lozenge
Use the 1st fraction prepared according to the method described in above-mentioned test example 6, make lozenge by following prescription (every
One ingot 500mg).
(preparation method)
1st fraction (2g), dried corn starch (2g), Talcum (1.8g), calcium stearate (0.2g) are made an addition to lactose (94g) and
Mix.Then, use one-shot Ingot pressing machine (single stroke tablet press) and manufacture lozenge with common method.
The making of embodiment 7. hard capsule
Use the 1st fraction prepared according to the method described in above-mentioned test example 6, make hard capsule by following prescription
(each capsule 360mg).
(preparation method)
Lactose (220g) and corn starch (110g) are made an addition to the 1st fraction (5g) and mix, by hydroxypropyl cellulose
(25g) aqueous solution makes an addition to wherein and carries out mediating (kneading).Then, use Squeezinggranulator by common method system
Make granule.By this is particles filled to snap fit capsule (gelatin hard capsule) making hard capsule.
The making of embodiment 8. soft capsule
Use and prepare the 1st fraction according to the method described in above-mentioned test example 6, make soft capsule by following prescription
(each capsule 170mg).
1st fraction 5mg
Soybean oil 165mg
(preparation method)
1st fraction (5g) is made an addition to soybean oil (165g) and mixes.Then, by use rotational automatic forming machine according to
Common method is filled into soft capsule and makes soft capsule.
The making of embodiment 9. pill
Use the 1st fraction prepared according to the method described in above-mentioned test example 6, make pill by following prescription (every
One 100mg).
(preparation method)
With above-mentioned cooperation mixing raw material, after adding water in right amount, manufacture the kneaded material of homogenizing, the kneaded material pressure that will obtain with kneader
Prolong, after using pellet processing machine pill, be dried making pill.
The making of embodiment 10. fruit jelly
Use the 1st fraction prepared according to the method described in above-mentioned test example 6, by following kitchen with common method system
Make fruit jelly (100g).
(preparation method)
Mixing mentioned component, is heated to 90 DEG C.After the dissolving confirming gelatin, it is filled into container, cools down.By making gelatin
Solidify and make fruit jelly.
Claims (12)
1. a hyaluronic acid synthesis accelerant, it is characterised in that contain by aqueous organic solvent from the seed institute of Semen Oroxyli
The fraction extracted includes at least the 1st fraction of chrysin as effective ingredient.
2. a hyaluronic acid synthesis accelerant, it is characterised in that containing with water and ethyl acetate to passing through aqueous organic solvent
When the 1st fraction that the seed of Semen Oroxyli is extracted carries out liquid-liquid partition, it is dispensed in the fraction of described ethyl acetate side extremely
Comprise the 2nd fraction of chrysin less as effective ingredient.
3. a hyaluronic acid synthesis accelerant, it is characterised in that containing with water and ethyl acetate to passing through aqueous organic solvent
The 1st fraction extracted from the seed of Semen Oroxyli carries out liquid-liquid partition, by 90% methanol and hexane further to being dispensed on
When 2nd fraction of described ethyl acetate side carries out liquid-liquid partition, it is dispensed in the fraction of described 90% methanol side and includes at least
3rd fraction of chrysin is as effective ingredient.
4. a hyaluronic acid synthesis accelerant, it is characterised in that containing with water and ethyl acetate to passing through aqueous organic solvent
The 1st fraction extracted from the seed of Semen Oroxyli carries out liquid-liquid partition, by 90% methanol and hexane further to being dispensed on
2nd fraction of described ethyl acetate side carries out liquid-liquid partition, by silica gel chromatography to being dispensed on described 90% methanol side
When 3rd fraction carries out fractional distillation, by chloroform: methanol is that the mixed solvent of 9:1 is white by including at least in the fraction of silica gel institute dissolution
4th fraction of Yang Su is as effective ingredient.
5. a hyaluronic acid synthesis accelerant, it is characterised in that containing chrysin as effective ingredient.
6. a HAS2mRNA expresses accelerator, it is characterised in that contain by aqueous organic solvent from the seed institute of Semen Oroxyli
The fraction extracted includes at least the 1st fraction of chrysin as effective ingredient.
7. a HAS2mRNA expresses accelerator, it is characterised in that containing with water and ethyl acetate to passing through aqueous organic solvent
When the 1st fraction that the seed of Semen Oroxyli is extracted carries out liquid-liquid partition, it is dispensed in the fraction of described ethyl acetate side extremely
Comprise the 2nd fraction of chrysin less as effective ingredient.
8. a HAS2mRNA expresses accelerator, it is characterised in that containing with water and ethyl acetate to passing through aqueous organic solvent
The 1st fraction extracted from the seed of Semen Oroxyli carries out liquid-liquid partition, by 90% methanol and hexane further to being dispensed on
When 2nd fraction of described ethyl acetate side carries out liquid-liquid partition, it is dispensed in the fraction of described 90% methanol side and includes at least
3rd fraction of chrysin is as effective ingredient.
9. a HAS2mRNA expresses accelerator, it is characterised in that containing with water and ethyl acetate to passing through aqueous organic solvent
The 1st fraction extracted from the seed of Semen Oroxyli carries out liquid-liquid partition, by 90% methanol and hexane further to being dispensed on
2nd fraction of described ethyl acetate side carries out liquid-liquid partition, by silica gel chromatography to being dispensed on described 90% methanol side
When 3rd fraction carries out fractional distillation, by chloroform: methanol is that the mixed solvent of 9:1 is white by including at least in the fraction of silica gel institute dissolution
4th fraction of Yang Su is as effective ingredient.
10. a HAS2mRNA expresses accelerator, it is characterised in that containing chrysin as effective ingredient.
11. 1 kinds have the hyaluronic acid synthesis pharmaceuticals of facilitation, food or cosmetic preparation, it is characterised in that containing having the right
Require the hyaluronic acid synthesis accelerant according to any one of 1～5.
12. 1 kinds have HAS2mRNA and express the pharmaceuticals of facilitation, food or cosmetic preparation, it is characterised in that containing having the right
Require that the HAS2mRNA according to any one of 6～10 expresses accelerator.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014-039981 | 2014-02-28 | ||
| JP2014039981A JP5976025B2 (en) | 2014-02-28 | 2014-02-28 | Method for producing hyaluronic acid synthesis promoter, hyaluronic acid synthesis promoter, method for producing HAS2 mRNA expression promoter, and HAS2 mRNA expression promoter |
| PCT/JP2015/054462 WO2015129525A1 (en) | 2014-02-28 | 2015-02-18 | HYALURONIC ACID SYNTHESIS PROMOTER, HAS2 mRNA EXPRESSION PROMOTER, PHARMACEUTICAL, FOOD, OR COSMETIC HAVING HYALURONIC ACID SYNTHESIS-PROMOTING ACTION, AND PHARMACEUTICAL, FOOD, OR COSMETIC HAVING HAS2 mRNA EXPRESSION-PROMOTING ACTION |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106163534A true CN106163534A (en) | 2016-11-23 |
| CN106163534B CN106163534B (en) | 2020-05-19 |
Family
ID=54008846
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201580010029.0A Expired - Fee Related CN106163534B (en) | 2014-02-28 | 2015-02-18 | Hyaluronic acid synthesis promoter, HAS2mRNA expression promoter, and pharmaceutical, food or cosmetic containing the same |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JP5976025B2 (en) |
| CN (1) | CN106163534B (en) |
| TW (1) | TWI625133B (en) |
| WO (1) | WO2015129525A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006321730A (en) * | 2005-05-17 | 2006-11-30 | Maruzen Pharmaceut Co Ltd | Antioxidant, antiaging agent, skin cosmetics, and food or drink |
| JP2007161681A (en) * | 2005-12-16 | 2007-06-28 | Mitsui Chemicals Inc | Agent for preventing wrinkle and improving skin condition |
| US20080262080A1 (en) * | 2004-10-05 | 2008-10-23 | Kyushu University, National University Corporation | Allergy Suppressive Agent |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI382846B (en) * | 2009-10-07 | 2013-01-21 | Univ China Medical | Pharmaceutical compositions and extracts for inhibiting formation and/or activation of osteoclasts and uses of the same |
| JP6002510B2 (en) * | 2012-09-06 | 2016-10-05 | 花王株式会社 | Estrogen receptor β activator |
-
2014
- 2014-02-28 JP JP2014039981A patent/JP5976025B2/en active Active
-
2015
- 2015-02-16 TW TW104105309A patent/TWI625133B/en not_active IP Right Cessation
- 2015-02-18 WO PCT/JP2015/054462 patent/WO2015129525A1/en active Application Filing
- 2015-02-18 CN CN201580010029.0A patent/CN106163534B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080262080A1 (en) * | 2004-10-05 | 2008-10-23 | Kyushu University, National University Corporation | Allergy Suppressive Agent |
| JP2006321730A (en) * | 2005-05-17 | 2006-11-30 | Maruzen Pharmaceut Co Ltd | Antioxidant, antiaging agent, skin cosmetics, and food or drink |
| JP2007161681A (en) * | 2005-12-16 | 2007-06-28 | Mitsui Chemicals Inc | Agent for preventing wrinkle and improving skin condition |
Non-Patent Citations (3)
| Title |
|---|
| 仝会娟等: "木蝴蝶药材中黄芩素和白杨素的含量测定", 《中国中医药信息杂志》 * |
| 文景兵等: "木蝴蝶种子中黄酮类化学成分研究", 《中国药学杂志》 * |
| 杜晓燕等: "HPLC法测定中药木蝴蝶中黄芩苷和白杨素", 《西北药学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5976025B2 (en) | 2016-08-23 |
| TWI625133B (en) | 2018-06-01 |
| TW201532628A (en) | 2015-09-01 |
| CN106163534B (en) | 2020-05-19 |
| JP2015164901A (en) | 2015-09-17 |
| WO2015129525A1 (en) | 2015-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105853326A (en) | Infant lip gloss and making method thereof | |
| CN101543546A (en) | Antioxidative substance and composition and use thereof | |
| CN108697685A (en) | Containing moracin, Phellinus copper G or the root bark of white mulberry, the prevention of muscle disease and treatment is used or muscular function improvement composition | |
| TW201900151A (en) | Partial muscat grape formulation for cosmetic use | |
| JP2008540582A (en) | System and method for promoting hair growth and improving hair and scalp health | |
| CN110037981A (en) | A kind of skin care item and the preparation method and application thereof | |
| CN102335217B (en) | Promoter for generation of glutathione | |
| CN105462793A (en) | Health-care medicinal liquor and preparation method thereof | |
| KR20180050331A (en) | Honkiol and Magnolol Formulations Having Increased Stability and Improved Absorbency, and Methods of Using Them | |
| CN106163534A (en) | Hyaluronic acid synthesis accelerant, HAS2mRNA express accelerator, have the hyaluronic acid synthesis pharmaceuticals of facilitation, food or cosmetic preparation and have the HAS2mRNA expression pharmaceuticals of facilitation, food or cosmetic preparation | |
| Zennaf et al. | Ethnobotanical and Phytochemical Study of the Medicinal Plant Atriplex halimus and Its Importance in the Traditional Algerian Pharmacopoeia | |
| TWI674103B (en) | Extracts of dragon fruit flower, preparation method for the same, use, and skin complexion improved compositions thereof | |
| KR20130087928A (en) | COMPOSITION COMPRISING AND HEALTHY FOOD LUTEOLIN 5-O-β-D-GLUCOPYRANOSIDE OR THE EXTRACT OF CIRSIUM MAACKII FOR PREVENTING OR TREATING INFLAMMATORY DISEASE | |
| JP2016121123A (en) | Agent for inhibiting production of advanced glycation end products | |
| Herdiani et al. | Effect Of Roselle Petal Extract On Decreased Levels Of MDA In Rats With Type 2 Diabetes | |
| Malek et al. | investigation of Syrian cactus leaf extricates gel and shell/shell collagen | |
| JP2016013981A (en) | Tubulin polymerization activity regulator, food and agent comprising the same, and method of producing tubulin polymerization activity regulator | |
| CN110325195A (en) | The manufacturing method of Adipocyte Differentiation inhibitor, the food compositions for inhibiting Adipocyte Differentiation and Adipocyte Differentiation inhibitor | |
| KR102386953B1 (en) | Non-comedogenic cosmetic compositions for acne skin | |
| JP7455350B2 (en) | Damage inhibitors, cosmetics, and food and beverages caused by air pollutants | |
| JP2019516777A (en) | Whitening agent | |
| JP6606636B2 (en) | Betulin derivative exhibiting cell membrane stabilizing action and production method thereof | |
| Rahmadianti et al. | Antioxidant Gel from Brown Algae (Ascophyllum nodosum) and Binahong Leaves for Diabetic Wound Healing | |
| Abbas et al. | Potential Role and Mechanism of Mulberry Extract in Immune Modulation: Focus on Chemical Compositions, Mechanistic Insights, and Extraction Techniques | |
| Alshaikhi et al. | Nutritional aspects, bioactive phytochemicals and biomedical traits of Opuntia spp.: current trends and applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200519 Termination date: 20220218 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |