CN109053667A - A kind of separation method of isoflavone derivative and application - Google Patents
A kind of separation method of isoflavone derivative and application Download PDFInfo
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- CN109053667A CN109053667A CN201810871722.2A CN201810871722A CN109053667A CN 109053667 A CN109053667 A CN 109053667A CN 201810871722 A CN201810871722 A CN 201810871722A CN 109053667 A CN109053667 A CN 109053667A
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- isoflavones
- hydroxyl
- dimethoxy
- added
- ethyl acetate
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- 230000005670 electromagnetic radiation Effects 0.000 description 1
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- 201000003914 endometrial carcinoma Diseases 0.000 description 1
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- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 1
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- 238000000684 flow cytometry Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
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- 229950006238 nadide Drugs 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003546 nucleic acid damage Effects 0.000 description 1
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- 150000004880 oxines Chemical class 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
- C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
-
- 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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
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- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
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Abstract
The invention belongs to field of medicaments, it is related to separation method and the application of a kind of isoflavone derivative.The invention discloses a kind of isoflavones in the application for preparing oxidative stress and inducing an illness in drug.The chemical formula of the isoflavones are as follows:Isoflavones can raise the II phase detoxification enzyme NQO1 and antioxidase γ GCS protein level of Nrf2 and its regulation, and activation mechanism is to inhibit Nrf2 protein degradation to realize by increasing Nrf2 protein stability；The pulmonary branches tracheal epithelium Beas-2B cellular damage model induced using arsenic, has rated the cytoprotection of isoflavones, the results showed that the compound can increase endogenous cellular antioxidant agent GSH level, inhibit arsenic induction Beas-2B cellular damage and apoptosis.
Description
Technical field
The invention belongs to field of medicaments, it is related to a kind of isoflavones and induces an illness medicine in preparation prevention or treatment oxidative stress
Application in object, health care product or cosmetics.
Background technique
The fast development of population expansion and industrial technology, causes problem of environmental pollution to increasingly sharpen.Multiple pollutant, such as carbon
Sulphur nitrogen oxides (CO, SO2、NO2Deng), heavy metal (lead, chromium, arsenic etc.), particulate matter, electromagnetic radiation, chemical organic reagent etc., no
It is open close to cross the approach such as air, water source, food and skin contact, human tissue organ is invaded, oxidative stress is caused.What body generated
A large amount of lipid peroxidation products and excessive endogeneous activity oxygen, active nitrogen, act on cell, directly or indirectly cause cell
The large biological molecules such as internal protein, lipid, nucleic acid damage mutation and function inhibitio, and then cancer is induced, neurodegenerative disease,
A series of serious diseases such as cardiovascular disease, diabetes, skin disease and chronic obstructive pulmonary disease.Therefore, enhancing body is anti-oxidant
Ability removes vivo oxidation activated product, inhibits cell oxidative damage, for the related disease that prevention and treatment is caused by oxidative stress
It is of great significance.
Nrf2 (Nuclear factor-erythroid 2-related factor 2) signal path is regulation human body oxygen
Change stress critical path, be chemopreventive agent (drug) effect good target.Nuclear factor Nrf2 is to protect cells against oxygen
The important transcription factor for changing damage, when body is in oxidative stress status, Nrf2 is just activated and indexing enters nucleus, passes through
It is combined with Antioxidation reaction original part (ARE), to encode the II phase detoxication enzyme such as GCL, UGT, NQO1, γ GCS, CAT, SOD and endogenous
Property anti-oxidant protease, to remove internal harmful poisonous substance, it is horizontal to reduce oxidation activity product, restores vivo oxidation also Yuanping City
Weighing apparatus.But body internal oxidition stress reaction is while activating Nrf2 access, the harmful substances such as a large amount of ROS of generation, free radical, also
Histoorgan is caused seriously to damage, and then induces a variety of diseases and occurs.
Therefore, when body is exposed to the environmental contaminants of electrophilicity foreign matter, chemical carcinogen equivalent damage body, Wo Menke
To utilize exogenous Nrf2 agonist, to raise the expression of protective protein enzyme in tissue in advance, activity in tissue is reduced in time
The level of oxygen and poisonous substance, enhance body self-defense ability, and then to Chronic Obstructive Pulmonary Disease, skin disease, chronic kidney disease, exhale
The occurrence and development for inhaling road inflammation, diabetes, neurodegenerative disease and tumor disease generate prevention effect.Activate Nrf2 signal logical
Road can raise II phase and detoxify enzyme level, and clearing the pollution off middle carcinogenic substance or reduces its toxicity, inhibit the DNA of carcinogenic substance induction
Damage, gene mutation, thus the generation of pre- preventing tumor.Nrf2 expression is raised, glutathione levels can be increased, is enhanced
Activities of antioxidant enzymes, active oxygen in scavenger-cell inhibit cytolipin peroxidating, reduce Chronic Obstructive Pulmonary Disease, skin disease,
The disease incidence of the diseases such as chronic kidney disease, respiratory inflammation, diabetes, neurodegenerative disease.
In recent years, artificial synthesized antioxidant is mostly phenolic compound, such as butylated hydroxy anisole (BHA), 2,6-, bis- uncle
Butyl paracresol (BHT).But due to its toxicity and carcinogenesis, BHA, BHT are limited by legislative bodies and are used.Therefore, from day
The safety with preventive and therapeutic action, natural are found in right product, is the important channel of anti-oxidation medicine research and development.
Isoflavones is a kind of natural estrin structure analog, and wherein isoflavones, has been demonstrated with estrogen-like work
With, it can be with estrogen competitive binding estrogen receptor in women body, adjusting human endocrine keeps human female inner estrogen water
Average weighing apparatus delays senescence to reach beautifying face and moistering lotion, reduces menopausal syndrome symptom, and prevention estrogen is lost caused
Coronary heart disease, atherosclerosis and bone loss disorders and other effects.Furthermore it is replaced compared to the estrogen of direct complementing estrogen
Therapy, isoflavones have lower toxic side effect, can evade climacteric women suffer from breast cancer, the risk of carcinoma of endometrium.
Hydroxyl -4 isoflavones 5-, 7 '-dimethoxy isoflavones (5-Hydroxy-4,7 '-dimethoxy-
Isoflavone), in contrast with isoflavones, benzopyran-4-one 3 substituent groups be not to hydroxyl substituted benzene,
But to methoxy substitution benzene, this structure not only strengthens the fat-soluble of drug, more conducively absorption of human body, also enhances benzo
The electrophilicity of pyrans -4- ketone 2 becomes strongly active Nrf2 agonist.But there is not document to 5- hydroxyl-at present
4,7 '-dimethoxy isoflavones progress Activity determinations.
Summary of the invention
In order to solve prior art deficiency, the present invention provides a kind of isoflavones and induces in preparation prevention or treatment oxidative stress
Application in disease medicament, health care product or cosmetics can prevent and treat Chronic Obstructive Pulmonary Disease, skin disease, chronic renal
Disease, respiratory inflammation, diabetes, neurodegenerative disease and tumor disease.
It is an object of the present invention to provide a kind of hydroxyl -4 5-, the separation methods of 7 '-dimethoxy isoflavones, by screw oil expeller
Axis grass puts into heating and refluxing extraction in ethanol solution, and concentration filtrate obtains medicinal extract, and into medicinal extract plus water is suspended after dissolution, sequentially adds
Petroleum ether and ethyl acetate extraction, by ethyl acetate portion by obtaining colourless crystallization, i.e. 5- after silica gel column chromatography gradient elution
4,7 '-dimethoxy isoflavones of hydroxyl-.
Hydroxyl -4 5-, the chemical formula of 7 '-dimethoxy isoflavones are as follows:
Preferably, specific step is as follows for the separation method:
(1) red clover is put into heating and refluxing extraction in ethanol solution, is concentrated to get ethanol extract after merging filtrate；
(2) suitable quantity of water dissolution is added in the ethanol extract obtained to step (1), adds acetic acid after petroleum ether extraction is added
Ethyl ester extraction, obtains Ethyl acetate fraction；
(3) silica gel stirring is added to the Ethyl acetate fraction, is then separated through silica gel column chromatography gradient elution, institute
The system for stating silica gel column chromatography is methylene chloride-methanol system, and nine parts are obtained after elution, are denoted as according to appearance time
Fr.1-Fr.9；
(4) Fr4 obtained in step (3) is further separated through silica gel column chromatography gradient elution, the silica gel column chromatography
System be methylene chloride-methanol system, obtain colourless crystallization, i.e. hydroxyl -4 5-, the different Huang of 7 '-dimethoxys after gradient elution
Ketone.
Preferably, the concrete operations of step (1) are as follows: red clover is put into 75% ethanol solution of 8 times of amounts and heated
It flows back three times, each 1.5h.
It is further preferred that red clover 2kg, using 8 times of 75% ethyl alcohol of amount as solvent, heating and refluxing extraction 3 times, every time
1.5 hours, merging filtrate is filtered, ethanol extract 200g is concentrated to give.
Preferably, the concrete operations of step (2) are as follows: it is dissolved in water into ethanol extract, after petroleum ether extraction is added three times,
Petroleum ether part is discarded, adds ethyl acetate extraction three times, combining extraction liquid obtains ethyl acetate extract.
It is further preferred that obtaining ethyl acetate extract 45g after extraction.
Preferably, gradient elution is carried out using silica gel column chromatography in step (3), system used is methylene chloride-methanol, ladder
Spend elution program are as follows: methylene chloride-methanol system bulk ratio is 100:0,95:5,90:10,85:15,80:20,75:25,70:
30,60:40,50:50,0:100 obtain nine parts, are denoted as Fr.1-Fr.9 after elution.
Preferably, it is eluted in step (4) using gradient elution program, specific procedure are as follows: methylene chloride-methanol system
Volume ratio is 93:7,88:12,80:20, and the fraction under 88:12 volume ratio has crystallization to be precipitated, and is hydroxyl -4 5-, 7 '-dimethoxies
Base isoflavones.
This hair second purpose with a kind of hydroxyl -4 5- are provided, 7 '-dimethoxy isoflavones are in preparation prevention or treatment oxygen
Change the application in the drug of stress induction disease.
The inventor of the present application found through research that hydroxyl -4 isoflavones 5-, 7 '-dimethoxy isoflavones can be raised
Nrf2 and its II phase detoxification enzyme NQO1 and antioxidase γ GCS protein level of regulation, activation mechanism are by increasing Nrf2
Protein stability inhibits Nrf2 protein degradation to realize；The pulmonary branches tracheal epithelium Beas-2B cellular damage model induced using arsenic, is commented
The valence cytoprotection of isoflavones, the results showed that it is horizontal that the compound can increase endogenous cellular antioxidant agent GSH, suppression
Arsenic induction Beas-2B cellular damage processed and apoptosis.
Preferably, above-mentioned oxidative stress induces an illness as Chronic Obstructive Pulmonary Disease, respiratory inflammation, cutaneous lesions, sugar
Urine disease, neurodegenerative disease or tumor disease.
Preferably, in above-mentioned application, hydroxyl -4 5-, the treatment concentration of 7 '-dimethoxy isoflavones is 6.25~12.5 μ
M, it is further preferred that being 6.25 μM.
Preferably, in above-mentioned application, hydroxyl -4 5-, 7 '-dimethoxy isoflavones are capsule, tablet, powder, gel
Agent, granule, injection, oral solution, vina, pill, mixture or tincture.
The three of the object of the invention are to provide a kind of hydroxyl -4 5-, the preparation method of 7 '-dimethoxy isoflavone tablets, general
Hydroxyl -4 isoflavones 5- are sieved after 7 '-dimethoxy isoflavones, starch and dextrin mixing, add sodium carboxymethylcellulose system
Grain, tabletting is after magnesium stearate mixing is then added up to tablet；Preferably, wherein hydroxyl -4 isoflavones 5-, 7 '-dimethoxys are different
Flavones, starch and dextrin mass ratio are 1-2:2-3:2-3.
The four of the object of the invention are to provide a kind of hydroxyl -4 5-, the preparation method of 7 '-dimethoxy isoflavone gel agent,
Carbomer and polysorbate are added to the water and are mixed, isoflavones is added and is uniformly mixed up to gelling agent；Preferably, the gelling agent
Hydroxyl -4 middle isoflavones 5-, the mass ratio of 7 '-dimethoxy isoflavones, carbomer and polysorbate are 2-3:3-4:1-2.
The five of the object of the invention are to provide a kind of cosmetics with anti-oxidation efficacy, and composition is hydroxyl -4 5-, and 7 '-two
Methoxy isoflavone and general cosmetic material.
Preferably, above-mentioned cosmetics can be facial mask, face cream, cold cream, surfactant, skin care powder or skin-care gel.
Beneficial effects of the present invention
1. the present invention provides one kind to extract hydroxyl -4 5- from red clover, the method for 7 '-dimethoxy isoflavones, and
Demonstrate its antioxidant activity.In the prior art about hydroxyl -4 5-, the research of 7 '-dimethoxy isoflavones separation methods compared with
It is few, it is even more blank about its active research, the present invention provides one kind to be directed to hydroxyl -4 5-, 7 '-dimethoxy isoflavones
Highly effective extraction method, it was demonstrated that its antioxidant activity is to realize that the research for related target drug provides by raising Nrf2
Foundation.
2. the present invention demonstrates hydroxyl -4 5-, the antioxidant activity of 7 '-dimethoxy isoflavones, the inoxidizability with it is a variety of
Sickness insurance closes, such as Chronic Obstructive Pulmonary Disease, respiratory inflammation, cutaneous lesions, diabetes, neurodegenerative disease or tumour disease
Disease etc. is each provided with new Research Thinking for the treatment of the above disease, has important medical significance.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation are not constituted an undue limitation on the present application for explaining the application.
The histogram of the induced activity test of Fig. 1 NQO1；
Fig. 1 shows hydroxyl -4 isoflavones 5-, and 7 '-dimethoxy isoflavones can induce hepa 1c1c7 cell II phase to detoxify
The expression of enzyme NQO1 simultaneously enhances its activity, and the isoflavone concentration unit in figure is μM that 2.0 μM of sulforaphen are positive control.
Hydroxyl -4 Fig. 2 different time isoflavones 5-, 7 '-dimethoxy isoflavones are to Nrf2 protein level immunoblotting assay
Figure；
Fig. 2 shows that isoflavones is able to extend Nrf2 protein half-life, and isoflavone concentration is 6.25 μM in figure.
Hydroxyl -4 Fig. 3 various concentration isoflavones 5-, the immunoblotting assay figure of 7 '-dimethoxy isoflavones；
Fig. 3 shows that isoflavones can raise Nrf2 and anti-oxidant γ GCS downstream and II phase and detoxify zymoprotein NQO1 albumen
Level, the isoflavone concentration unit in figure are μM.
Hydroxyl -4 Fig. 4 isoflavones 5-, 7 '-dimethoxy isoflavones are to cellular glutathione content histogram；
Wherein, Fig. 4 A is influence test of the various concentration isoflavones to glutathione levels, and Fig. 4 B is 6.25 μM
Influence test after isoflavones and cytosis different time, to its glutathione.Show that isoflavones being capable of concentration-time depended
Increase human bronchial epithelial Beas-2B endogenous cellular antioxidant agent glutathione level, 2.5 μM of sulforaphen are sun in figure
Property control, isoflavone concentration unit be μM；Chronomere is h.
Hydroxyl -4 Fig. 5 isoflavones 5-, the fluorogram that 7 '-dimethoxy isoflavones influence endogenous cellular active oxygen；
Fig. 5 shows that isoflavones is able to suppress the generation of excessive endogeneous activity oxygen, and isoflavone concentration is 6.25 μM in figure.
Hydroxyl -4 detection isoflavones 5- of Fig. 6 cell toxicity test, 7 '-dimethoxy isoflavones influence cell survival amount
Histogram；
Fig. 6 shows that isoflavones is able to suppress the cytotoxicity of arsenic induction, and Fig. 6 A is that various concentration isoflavones lures 10 μM of arsenic
The protective effect of guided cell toxicity, Fig. 6 B are protective effect of 6.25 μM of isoflavones to various concentration arsenic inducing cytotoxic.
Fig. 7 fluidic cell tune dies figure；
Fig. 7 shows that isoflavones is able to suppress the Apoptosis of arsenic induction, and isoflavone concentration is 6.25 μM, and the concentration of arsenic is 10
μM。
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As background technique is introduced, the prior art is insufficient to isoflavones Activity determination, in order to solve technology as above
Problem, present applicant proposes isoflavones in the application for preparing oxidative stress and inducing an illness in drug.
In a kind of exemplary embodiment of the invention, a kind of hydroxyl -4 5-, the separation side of 7 '-dimethoxy isoflavones are provided
Red clover is put into heating and refluxing extraction in ethanol solution by method, and concentration filtrate obtains medicinal extract, and into medicinal extract plus water is suspended and dissolves
Afterwards, petroleum ether and ethyl acetate extraction are sequentially added, by ethyl acetate portion by obtaining nothing after silica gel column chromatography gradient elution
Color crystallization, i.e. hydroxyl -4 5-, 7 '-dimethoxy isoflavones.
Hydroxyl -4 5-, the chemical formula of 7 '-dimethoxy isoflavones are as follows:
In preferred embodiment, specific step is as follows for the separation method:
(1) red clover is put into heating and refluxing extraction in ethanol solution, is concentrated to get ethanol extract after merging filtrate；
(2) suitable quantity of water dissolution is added in the ethanol extract obtained to step (1), adds acetic acid after petroleum ether extraction is added
Ethyl ester extraction, obtains Ethyl acetate fraction；
(3) silica gel stirring is added to above-mentioned Ethyl acetate fraction, is then separated through silica gel column chromatography gradient elution, silicon
The system of plastic column chromatography is methylene chloride-methanol system, and nine parts are obtained after elution, are denoted as Fr.1- according to appearance time
Fr.9；
(4) Fr4 obtained in step (3) is further separated through silica gel column chromatography gradient elution, the body of silica gel column chromatography
System is methylene chloride-methanol system, obtains colourless crystallization, i.e. hydroxyl -4 5-, 7 '-dimethoxy isoflavones after gradient elution.
In preferred embodiment, the concrete operations of step (1) are as follows: red clover is put into 75% ethyl alcohol of 8 times of amounts
It is heated to reflux in solution three times, each 1.5h.
In specific embodiment, red clover 2kg, using 8 times of 75% ethyl alcohol of amount as solvent, heating and refluxing extraction 3 times, every time
1.5 hours, merging filtrate is filtered, ethanol extract 200g is concentrated to give.
In preferred embodiment, the concrete operations of step (2) are as follows: be dissolved in water into ethanol extract, petroleum ether is added
After extraction three times, petroleum ether part is discarded, adds ethyl acetate extraction three times, combining extraction liquid obtains ethyl acetate extract.
Ethyl acetate extract 45g is obtained in specific embodiment, after extraction.
Preferably, gradient elution is carried out using silica gel column chromatography in step (3), system used is methylene chloride-methanol, ladder
Spend elution program are as follows: methylene chloride-methanol system bulk ratio is 100:0,95:5,90:10,85:15,80:20,75:25,70:
30,60:40,50:50,0:100 obtain nine parts, are denoted as Fr.1-Fr.9 after elution.
In preferred embodiment, eluted in step (4) using gradient elution program, specific procedure are as follows: dichloromethane
Alkane-methanol system volume ratio is 93:7,88:12,80:20, and the fraction under 88:12 volume ratio has crystallization to be precipitated, and is 5- hydroxyl-
4,7 '-dimethoxy isoflavones.
In another exemplary embodiment of the invention, a kind of hydroxyl -4 5- are provided, prepared by 7 '-dimethoxy isoflavones
The application in drug that prevention or treatment oxidative stress induce an illness.
In preferred embodiment, above-mentioned oxidative stress induces an illness as Chronic Obstructive Pulmonary Disease, respiratory inflammation, skin
Skin lesion, diabetes, neurodegenerative disease or tumor disease.
In preferred embodiment, in above-mentioned application, the treatment concentration of hydroxyl -4 5-, 7 '-dimethoxy isoflavones is
6.25~12.5 μM, be 6.25 μM in specific embodiment.
In preferred embodiment, in above-mentioned application, hydroxyl -4 5-, 7 '-dimethoxy isoflavones be capsule, tablet,
Powder, gelling agent, granule, injection, oral solution, vina, pill, mixture or tincture.
In another exemplary embodiment of the invention, a kind of hydroxyl -4 5- are provided, 7 '-dimethoxy isoflavone tablets
Hydroxyl -4 isoflavones 5- are sieved after 7 '-dimethoxy isoflavones, starch and dextrin mixing, add carboxymethyl by preparation method
Sodium cellulosate granulation, tabletting is after magnesium stearate mixing is then added up to tablet；In preferred embodiment, wherein isoflavones 5-
4,7 '-dimethoxy isoflavones of hydroxyl-, starch and dextrin mass ratio are 1-2:2-3:2-3.
In another exemplary embodiment of the invention, a kind of hydroxyl -4 5-, 7 '-dimethoxy isoflavone gel agent are provided
Preparation method, carbomer and polysorbate are added and are mixed into water, isoflavones is added and is uniformly mixed up to gelling agent；It is excellent
In the embodiment of choosing, hydroxyl -4 isoflavones 5- in the gelling agent, 7 '-dimethoxy isoflavones, carbomer and polysorbate
Mass ratio is 2-3:3-4:1-2.
In another exemplary embodiment of the invention, a kind of cosmetics with anti-oxidation efficacy, composition 5- are provided
4,7 '-dimethoxy isoflavones of hydroxyl-and general cosmetic material.
In preferred embodiment, above-mentioned cosmetics can be facial mask, face cream, cold cream, surfactant, skin care powder or shield
Skin gel.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Embodiment 1: the separation of isoflavones and structural identification
Red clover 2kg, using 8 times of 75% ethyl alcohol of amount as solvent, heating and refluxing extraction 3 times,.1.5 hours every time, filtering was closed
And filtrate, it is concentrated to give ethanol extract 200g；Add water to be suspended to dissolve, is successively extracted using petroleum ether and ethyl acetate, extract 3 respectively
It is secondary, obtain 45 grams of ethyl acetate extract.By ethyl acetate extract, silica gel is added to mix sample, silica gel column chromatography, using methylene chloride-in right amount
Methanol system gradient elution, 9 parts of merging are denoted as Fr.1-Fr.9 according to appearance time.Fr4 use silica gel column chromatography, two
Chloromethanes-methanol 95:5 has colourless crystallization precipitation, is denoted as Fr4A to 80:20 gradient elution.It is true that structure has been carried out using NMR method
Card, 1H-NMR modal data are as follows:
1H-NMR (CDCl3) 12.90 (1H, s, 5-OH), 7.89 (1H, s, H-2), 4.49 (2H, d, J=8.2Hz, H-2 ',
6 '), 7.01 (2H, d, J=8.7Hz, H-3 ', 5 '), 6.43 (1H, d, J=2.1Hz, H-6), 6.42 (1H, d, J=2.2Hz, H-
8),3.89(3H,s,7-OCH3)。
It is parsed, confirming its structure is hydroxyl -4 5-, 7 '-dimethoxy isoflavones.
Embodiment 2: hydroxyl -4 isoflavones 5-, the NQO1 induced activity evaluation of 7 '-dimethoxy isoflavones
(1) culture of murine hepatocarcinoma cell hepa 1c1c cell line
Murine hepatocarcinoma cell hepa 1c1c cell line is purchased from American Type Culture collection warehousing (ATCC), using containing 10%
The MEM culture medium of fetal calf serum (FBS) is placed in 37 DEG C, cultivates in 5%CO2 incubator.
(2) NQO1 induced activity is tested
Hepa 1c1c cell inoculation is added to the isoflavones (embodiment 1 of various concentration after on 96 orifice plates, cell is adherent
Confirmation), it handles 24 hours, using 0.8% digitonin solution lytic cell, detection liquid (tri- hydroxyl first of 1.0mL 0.5M is added
Base aminomethane-hydrochloric acid (Tris-HCl), 15mg bovine serum albumin(BSA), 6mg MTT, 150 μ L Tween-20s, 150 μ L 150mM
D-Glucose -6- phosphoric acid, 15 μ L 7.5mM flavin adenine dinucleotide (FAD)s, 27 μ L 50mM nicotinamide adenine dinucleotide phosphorus
Acid, 20 μ L 50mM menadiones), it places 3 minutes, measures luminous intensity in 630nm.
As a result: as shown in Figure 1, hydroxyl -4 isoflavones 5-, 7 '-dimethoxy isoflavones can activate hepa 1c1c7 cell
Middle NQO1 activity, maximum induced activity are 4 times (50 μM), and positive control medicine sulforaphen (2.0 μM) is blank control group
2.9 again.The above results show that isoflavones can activate II phase detoxification enzyme, have protective effect to human body cell.
Embodiment 3: hydroxyl -4 isoflavones 5-, 7 '-dimethoxy isoflavones increase Nrf2 protein stability
(1) culture of people's normal epidermis Beas-2B cell
People's normal lung bronchiolar epithelium Beas-2B cell is purchased from American Type Culture collection warehousing (ATCC), using 1640
Culture medium, and 10% fetal calf serum (FBS) is added thereto, 5% glutamine is placed in 37 DEG C, cultivates in 5%CO2 incubator.
(2) western blot analysis detects Nrf2 protein half-life
By Beas-2B cell inoculation in diameter 35mm culture dish, after culture reaches 70%-80% to density, it is added different
Then cycloheximide is added in flavones 8h, albumen was collected in timing at 0,10,20,30,40 minute respectively, carries out Western blotting point
Analysis detection, is quantified using Image J software.
As a result: as shown in Fig. 2, blank group Beas-2B cell Nrf2 protein half-life is 14.7 minutes, being handled through isoflavones
After 8h, Nrf2 protein half-life extends to 27.6 minutes, shows that isoflavones is able to extend Nrf2 protein half-life.
Embodiment 4: hydroxyl -4 isoflavones 5-, 7 '-dimethoxy isoflavones can raise Nrf2, γ GCS and NQO1 albumen
It is horizontal
Method: western blot analysis (Western blot) detects the variation of protein level in cell
By Beas-2B cell inoculation in diameter 35mm culture dish, after culture reaches 70%-80% to density, it is added not
Untested compound with concentration handles 16h, and PBS is washed 2 times, and cell pyrolysis liquid (50 μ g/ml Aprotinins, 0.5mM benzyl is added
Sulfuryl fluoride, 1mM sodium vanadate, 10mM sodium fluoride, 10mM β-phosphoglycerol), it collects albumen and egg is measured using Bradford method
White concentration.Sample protein (100 μ g) loading is respectively taken, SDS-PAGE protein isolate component is simultaneously turned protein band using electrotransfer method
It moves on cellulose nitrate film.Film through TBS prepare 5% skimmed milk power solution room temperature closing 1h after, respectively with it is each to be measured
4 DEG C of protein antibodies overnight incubations.After being separately added into the secondary antibody incubation 1h of horseradish peroxidase after TBS is washed, with increasing
Strong type ECL chemiluminescence carries out analysis of protein.
As a result: as shown in figure 3, cell is after isoflavones handles 16h, Nrf2 and downstream anti-oxidant and II phase detoxication enzyme egg
White NQO1 and γ GCS protein level is presented dose dependent and increases, and Keap1 protein level is unchanged, it was demonstrated that the compound can
Nrf2 signal path is activated on protein level.
Embodiment 5: hydroxyl -4 isoflavones 5-, 7 '-dimethoxy isoflavones can increase endogenous cellular antioxidant agent paddy
The sweet peptide level of Guang
Method: the measurement of glutathion inside cell content
By Beas-2B cell inoculation in diameter 35mm culture dish, after culture reaches 70%-80% to density, with for the moment
Between the isoflavones (confirmation of embodiment 1) of various concentration is added handles 24 hours or different time the (implementation of 6.25 μM of isoflavones be added
Example 1 is confirmed) processing cell, PBS washing 2 times, is added 0.5mL 50mM sodium phosphate and 1mM edta buffer liquid receives cell, ultrasound
1min, 10000g are centrifuged 15 minutes, take supernatant, are operated according to glutathione assay kit specification, and 412nm measures extinction
It spends and calculates glutathione content.
As a result: as shown in figure 4, isoflavones can dramatically increase glutathione levels, proper energy is gone back in enhancing into the cell
Power.And when for 24 hours, it is induced and reaches peak value.
Embodiment 6: isoflavones can reduce excessive endogeneous activity oxygen in the human bronchial epithelial Beas-2B cell of arsenic induction
Level
By Beas-2B cell inoculation in diameter 35mm culture dish, after culture reaches 30%-40% to density, it is added different
Flavones handles 8h, then acts on 10h altogether with 5 μM of As, discards and (10 μM) of DCFH-DA incubation 30min is added after culture solution, and PBS is washed
It 3 times, using fluorescence microscope and takes pictures.
As a result: as shown in figure 5, isoflavones can significantly reduce the level of the excessive endogeneous activity oxygen of arsenic induction, inhibiting
Cellular oxidation stress.
Embodiment 7: hydroxyl -4 isoflavones 5-, the human bronchial epithelial Beas-2B that 7 '-dimethoxy isoflavones induce arsenic
The protective effect of cellular damage
Method: mtt assay measures hydroxyl -4 isoflavones 5-, the guarantor for the cytotoxicity that 7 '-dimethoxy isoflavones induce arsenic
Shield effect
By Beas-2B cell inoculation on 96 orifice plates, after cell is adherent, using concentration isoflavones pretreatment cell 8 to be measured
Hour, the arsenic and concentration isoflavones to be measured (confirmation of embodiment 1) that various concentration is added are handled 24 hours, or addition MTT 3 small
Shi Hou, 590nm measurement absorbance simultaneously calculate cells survival rate.
As a result: as shown in Figure 6A, using isoflavones pretreatment cell 8 hours, the thin of 10 μM of arsenic inductions can be significantly inhibited
Cellular toxicity increases cells survival rate, wherein 6.25 μM of concentration isoflavones activity are best.As shown in Figure 6B, using 6.25 μM of concentration
Isoflavones can significantly inhibit the cytotoxicity that 5,10,20 μM of arsenic induces, increase cells survival rate as protection drug.Knot
Fruit proves that isoflavones is able to suppress the toxicity of carcinogenic substance arsenic induction.
Embodiment 8: hydroxyl -4 isoflavones 5-, 7 '-dimethoxy isoflavones are able to suppress the natural death of cerebral cells of arsenic induction
Method: the protective effect for the Apoptosis that Flow Cytometry measurement isoflavones induces arsenic
By Beas-2B cell inoculation in 35mm culture dish, 5 μM of arsenic are added after adherent or 6.25 μM of isoflavones handle 12h, inhale
Take drug containing culture solution to 15ml centrifuge tube, PBS is cleaned 1 time, with serum-free trypsin digestion cell, by culture solution, PBS cleaning solution and
Pancreatin digestive juice is collectively incorporated into 15ml centrifuge tube, and PBS is washed 1 time afterwards for centrifugation (3000rmp, 2min), collects cell and 100ul is added
1X Binding Buffer makes its suspension, and 5 μ l PI, 5 μ l Annexin V-FITC are added, and is protected from light in 2-8 DEG C and is incubated for 15min,
After 300ul 1X Binding Buffer suspension cell is added, Apoptosis is detected using streaming technology.
As a result: as shown in fig. 7, the cell that can significantly inhibit arsenic induction withered using arsenic or isoflavones coprocessing 12 hours
It dies.
Embodiment 9: the preparation of tablet
Starch 1.5g, dextrin 1.5g is added in isoflavones 1g, and sieving, addition sodium carboxymethylcellulose is appropriate, granulation.It is added
Magnesium Stearate proper quantity, mix, tabletting to get.
Embodiment 10: the preparation of gelling agent
3 grams of carbomer, 1g polysorbate, 100mL water are uniformly mixed, and isoflavones 2g is added, mixes well, and dispense, i.e.,
?.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (10)
1. red clover is put into ethanol solution and is heated back by a kind of hydroxyl -4 5-, the separation method of 7 '-dimethoxy isoflavones
Stream extracts, and concentration filtrate obtains medicinal extract；Into medicinal extract plus water is suspended after dissolution, sequentially adds petroleum ether and ethyl acetate extraction, will
Ethyl acetate portion obtains colourless crystallization, i.e. hydroxyl -4 5-, the different Huang of 7 '-dimethoxys after passing through silica gel column chromatography gradient elution
Ketone；Preferably, the specific steps are as follows:
(1) red clover is put into heating and refluxing extraction in ethanol solution, is concentrated to get ethanol extract after merging filtrate；
(2) suitable quantity of water dissolution is added in the ethanol extract obtained to step (1), obtains medicinal extract liquid, after petroleum ether extraction is added, then
Ethyl acetate extraction is added into the medicinal extract liquid, obtains Ethyl acetate fraction；
(3) silica gel stirring is added to the Ethyl acetate fraction, is then separated through silica gel column chromatography gradient elution, the silicon
The system of plastic column chromatography is methylene chloride-methanol system, and nine parts are obtained after elution, are denoted as Fr.1-Fr.9；
(4) Fr4 obtained in step (3) is separated through silica gel column chromatography gradient elution, the system of the silica gel column chromatography is two
Chloromethanes-methanol system obtains colourless crystallization, i.e. hydroxyl -4 5-, 7 '-dimethoxy isoflavones after gradient elution.
2. separation method as described in claim 1, which is characterized in that red clover described in step (1) puts into 8 times of amounts 75%
Ethanol solution in heating and refluxing extraction 3 times, each 1.5h.
3. separation method as described in claim 1, which is characterized in that after the petroleum ether extraction is added in step (2) three times,
Petroleum ether part is discarded, adds the ethyl acetate extraction three times, merging obtains the ethyl acetate extract.
4. separation method as described in claim 1, which is characterized in that the journey of silica gel column chromatography gradient elution described in step (3)
Sequence are as follows: methylene chloride-methanol system bulk ratio be 100:0,95:5,90:10,85:15,80:20,75:25,70:30,60:40,
50:50,0:100 obtain nine parts, are denoted as Fr.1-Fr.9 after elution.
5. separation method as described in claim 1, which is characterized in that methylene chloride-methanol system gradient described in step (4)
The program of elution are as follows: 93:7,88:12,80:20, the fraction under 88:12 volume ratio have crystallization be precipitated, be hydroxyl -4 5-, 7 ' -
Dimethoxy isoflavones.
Hydroxyl -4 6.5-, 7 '-dimethoxy isoflavones answering in the drug that preparation prevention or treatment oxidative stress induce an illness
With；Preferably, the oxidative stress induce an illness for Chronic Obstructive Pulmonary Disease, respiratory inflammation, cutaneous lesions, diabetes,
Neurodegenerative disease or tumor disease.
7. the use as claimed in claim 7, hydroxyl -4 5-, the treatment concentrations of 7 '-dimethoxy isoflavones is 6.25~
12.5 μM, it is preferred that be 6.25 μM；Hydroxyl -4 5-, 7 '-dimethoxy isoflavones are capsule, tablet, powder, gel
Agent, granule, injection, oral solution, vina, pill, mixture or tincture.
8. a kind of hydroxyl -4 5-, the preparation method of 7 '-dimethoxy isoflavone tablets, by hydroxyl -4 isoflavones 5-, 7 '-dimethoxies
It is sieved after base isoflavones, starch and dextrin mixing, adds sodium carboxymethylcellulose granulation, after magnesium stearate mixing is then added
Tabletting is up to tablet；Preferably, hydroxyl -4 the isoflavones 5-, 7 '-dimethoxy isoflavones, starch and dextrin mass ratio are 1-
2:2-3:2-3。
9. water is added in carbomer and polysorbate by a kind of hydroxyl -4 5-, the preparation method of 7 '-dimethoxy isoflavone gel agent
In be mixed, add isoflavones and be uniformly mixed up to gelling agent；Preferably, hydroxyl -4 the isoflavones 5-, 7 '-dimethoxys are different
The mass ratio of flavones, carbomer and polysorbate is 2-3:3-4:1-2.
10. a kind of cosmetics with anti-oxidation efficacy, ingredient is hydroxyl -4 5-, 7 '-dimethoxy isoflavones and general makeup
Product raw material, it is preferred that the cosmetics are facial mask, face cream, cold cream, surfactant, skin care powder or skin-care gel.
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