CN110123865A - Composition comprising man and wife's grass extract - Google Patents
Composition comprising man and wife's grass extract Download PDFInfo
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- CN110123865A CN110123865A CN201910374103.7A CN201910374103A CN110123865A CN 110123865 A CN110123865 A CN 110123865A CN 201910374103 A CN201910374103 A CN 201910374103A CN 110123865 A CN110123865 A CN 110123865A
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- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
The present invention provides a kind of composition comprising man and wife's grass extract and its application, belongs to man and wife grass extract field field, includes man and wife's grass extract and the sesquiterpene lactone as shown in formula (1):The acquisition methods of man and wife's grass extract in the composition include: to obtain the first extracting solution with solvent cold soak man and wife's grass meal end；With another solvent, ultrasonic wave extraction obtains the second extracting solution at room temperature after the concentration of first extracting solution；By macroporous absorbent resin DiaionHP-20 column, gradient elution and the eluent for collecting 40~60% elution positions after the concentration of second extracting solution, it is concentrated into constant weight to obtain the final product.Man and wife's grass extract and sesquiterpene lactone in composition can play synergistic function to reach the expression and activity, the purpose for reducing the degradation of type Ⅳ collagen albumen in collagen that efficiently inhibit MMP2 albumen, maintain and increase the content of collagen in skin, promote skin health degree, alleviate scytitis, reduces skin disease incidence probability.
Description
Technical field
The invention belongs to man and wife's grass extract fields, specifically provide a kind of composition and its system comprising man and wife's grass extract
Standby and application.
Background technique
Man and wife's grass is also known as sensitive plant, is pulse family (Fabaceae) Mimosa (Mimosaceae) plant sensitive plant
The herb of (Mimosa pudica), be suffruticose herbaceous plant, also known as know it is shy grass, it is coy grass, drink grass, coffee senna, fearness it is ugly
Grass, thorn sensitive plant etc..Man and wife's grass has the effect of reducing fever and causing diuresis, preventing phlegm from forming and stopping coughing, calmness of calming the nerves, cooling blood and hemostasis.Clinic is chiefly used in
Oxyhepatitis, neurasthenia, insomnia, pulmonary tuberculosis hemoptysis, blood urine, traumatic injury, the diseases such as shingles zoster.Mainization in man and wife's grass
It studies and is divided into the ingredients such as flavonoid glycoside, phenols, organic acid, phytosterol, in the more active constituent of sensitive plant, especially with flavones
The content of class is higher and active protrusion.The flavonoid glycosides constituent structure such as orientoside, Lutonaretin contained therein is more stable, unsuitable
Be degraded destruction, the activity with anti-oxidant, blood pressure lowering, increase coronary flow, reducing blood lipid, antibacterial anti-inflammatory etc..
Matrix metalloproteinase (matrix metalloproteinases, MMPs) is a kind of important zinc ion hydrolysis
Enzyme, major function are degradation extracellular matrixs, adjust cell adhesion, can act on extracellular components or other protein ingredients and
Start potential biological function, directly or indirectly participates in embryonic development, corneal restoration, bone uptake, tissue remodeling, vascularization
And the pathologic processes such as invasion transfer of the normal physiological processes such as wound healing and inflammation, tumour.It has at least been sent out in the mankind so far
As many as existing kind, constitute MMPs superfamily.There is special substrate to be classified as five classes: (1) clostridiopetidase A according to each member of MMPs,
Its substrate specificity is mainly interstitial collagen；(2) gelatin class, substrate specificity are mainly IV type glue and gelatin；(3) matrix colloidal sol,
Proteoglycan and glycoprotein in degradable III, IV, V, Collagen Type VI and matrix, and certain MMP can be activated；(4) model glue, energy
It degrades several ECM ingredients, some can activate other MMP；(5) other classes, more complex, some substrates are unknown.Tissue metal albumen
Enzyme inhibitor (tissue inhibitor of metalloproteinases, TIMPs) is both its natural inhibitors swollen
It plays an important role in tumor generating process.MMP2 is the important member of MMPs family, is positioned at human chromosomal 16q21, by
13 exons and 12 intrones are formed, it can not only hydrolyze the main component IV in denatured collagen and extracellular basilar memebrane
Collagen type, MMP2 expression and function adjusting betide transcription, secretion, the activation of prozymogen, the combination of cell surface with
And with multiple and different level such as the interaction of inhibitor from tumour or host cell.Increasing of the MMP2 in tumour cell
It grows, play an important role in angiogenesis, infiltration metastasis, other than basilar memebrane Collagen Type VI of degrading, can also decompose a variety of important
Bioactive molecule binding protein keeps bioactive molecule free and plays a role.
With age and ultraviolet light long-term irradiation, the collagen in skin can be lost and moisture content of skin is caused to subtract
It is few, and the drying regime of skin can further result in skin elasticity variation, wrinkle generates and pore is coarse.Therefore, try to supplement
Skin collagen, the synthesis for promoting collagen and the decomposition for reducing collagen, which have become, to be slowed down skin aging, maintains skin
The research center of gravity of skin health.Due to the barrier action of skin epidermis, the skin care products containing collagen is directly applied to skin
Skin surface can not effectively supplement the collagen of deep skin.On the other hand, if collagen is absorbed with oral way, due to big
The collagen of molecule is broken down into the amino acid of small molecule in alimentary canal, and partes corporis humani position may not necessarily after obtaining those amino acid
Collagen needed for synthesis, therefore oral collagen is limited with the effect for promoting skin collagen synthesis.In view of this, grinding
Study carefully develop it is a kind of can effectively inhibit collagen decompose novel constituent with reach reduce collagen be lost, avoid skin
The target of aging is necessary.
Summary of the invention
In consideration of it, the purpose of the present invention is to provide a kind of composition comprising man and wife's grass extract, in the composition
Man and wife's grass extract and sesquiterpene lactone can play synergistic function, can reach efficiently inhibit MMP2 albumen expression and work
Property, reduce collagen in type Ⅳ collagen albumen degradation purpose, maintain and increase the content of collagen in skin, promoted
Skin health degree alleviates scytitis, reduces skin disease incidence probability, and the composition can be prepared into a variety of dosage forms such as medicine
Agent, cosmetics, beverage etc. greatly expand its application field.
[1] a kind of composition comprising man and wife's grass extract, the composition is comprising man and wife's grass extract and such as formula (1) institute
Show sesquiterpene lactone:
Wherein, the content of man and wife's grass general flavone reaches 85% or more in man and wife's grass extract；
The composition can be effectively suppressed the performance of matrix metalloproteinase 2 (MMP2) gene and/or effectively inhibit glue
Former protein breakdown.
In the preferred embodiment of the present invention, the acquisition methods of man and wife's grass extract in the composition include:
The first extracting solution is obtained with solvent cold soak man and wife's grass meal end；
With another solvent, ultrasonic wave extraction obtains the second extracting solution at room temperature after the concentration of first extracting solution；
By macroporous absorbent resin Diaion HP-20 column after the concentration of second extracting solution, with gradient elution and collect
The eluent at 40~60% elution positions, is concentrated into constant weight to obtain the final product.
In the preferred embodiment of the present invention, a solvent be water, alcohols or alcohol-water mixture, preferably 75~
85wt% ethanol solution.
In the preferred embodiment of the present invention, man and wife's grass meal end is that will dry man and wife's grass to smash it through 140~200
Obtained by mesh.
In the preferred embodiment of the present invention, the liquid-solid ratio at a solvent and man and wife's grass meal end is 6~18:1, preferably
For 12~15:1.
In the preferred embodiment of the present invention, the temperature of the cold soaking is 1~4 DEG C, preferably 1~2 DEG C.
In the preferred embodiment of the present invention, the cold soaking time is 5~45min, preferably 10~25min.
In the preferred embodiment of the present invention, first extracting solution is concentrated into density and is at least 1.05g/mL when progress
Ultrasonic wave extraction.
In the preferred embodiment of the present invention, another solvent is the mixture of water, alcohols, nitrile, is preferably contained
There is 80~90wt% ethanol solution of 1.2~1.8wt% acetonitrile.A small amount of acetonitrile is added in extractant ethanol solution to be facilitated
The content of effective component in man and wife's grass extract is improved, above-mentioned mixed solvent can strengthen the dissolution of man and wife's grass general flavone, and can make
The content of man and wife's grass general flavone reaches 85% or more in final product man and wife's grass extract, and higher purity is also beneficial to its life
The efficient performance of effect of science, can be widely applied to the separation and Extraction of man and wife's grass total flavonoids substance.
In the preferred embodiment of the present invention, the weight ratio of another solvent and the first extracting solution concentrate be 5~
20:1, preferably 12~15:1.
In the preferred embodiment of the present invention, ultrasonic frequency is 45~80KHz when the ultrasonic wave, and energy density is
0.5~0.7W/cm2, preferably ultrasonic frequency is 65~75KHz, and energy density is 0.6~0.65W/cm2。
In the preferred embodiment of the present invention, the ultrasonic wave extraction time is 5~60min, preferably 30~45min.
In the preferred embodiment of the present invention, second extracting solution is concentrated into when density is at least 1.1g/mL and passes through
The elution of macroporous absorbent resin Diaion HP-20 column.
In the preferred embodiment of the present invention, the eluent is acetonitrile solution.
It is successively extracted using different solvents using organic cold soaking and high-frequency ultrasonic extracts two kinds of extractive techniques side in combination
Method is greatly improved extraction efficiency, and a small amount of acetonitrile is added in extractant ethanol solution and helps to improve man and wife's grass extract
The content of middle effective component, above-mentioned mixed solvent can strengthen the dissolution of man and wife's grass general flavone, man and wife in gained man and wife's grass extract
The content of careless general flavone can be widely applied to the separation and Extraction of man and wife's grass total flavonoids substance up to 85% or more, higher
Purity is also beneficial to the efficient performance of its physiology effect, and the present invention is extracted with man and wife's grass of two kinds of extracted acquisitions of extractive technique
Object can reach through the gene expression for inhibiting Skin Cell mother cell matrix metalloproteinase MMP2 reduces extracellular glue
The purpose of the degradation of type Ⅳ collagen albumen in former albumen, maintains and increases the content of collagen in skin, helps to promote skin
Skin elasticity and compact degree reduce skin lines and wrinkle, postpone skin aging, maintain skin gloss jade-like stone profit, alleviate scytitis,
Skin disease incidence probability is reduced, skin health degree is promoted.
In the preferred embodiment of the present invention, the sesquiterpene lactone as shown in formula (1) in composition of the present invention
Acquisition methods include:
1) with a solvent cold soak Centipeda minima powder, then with 15~20 times of amount solvent diacolations to the nearly nothing of percolate
Color merges percolate and is concentrated into no alcohol taste；
2) with another solvent extraction 1) concentrate, combining extraction liquid is concentrated into paste；
3) the paste concentrate of step 2) carries out silicagel column (100~200 mesh) chromatography, is washed with methanol-chloroform-acetate gradient
De- liquid carries out gradient elution, and every 100mL eluent collects 1 part, and TLC chromatography checks, the similar intermediate fraction of spot merges；
4) fraction obtained by step 3) carries out repeatedly silicagel column (200~300 mesh) chromatography, preparation efficient liquid phase and Sephadex
LH-20 purifies to obtain the sesquiterpene lactone as shown in formula (1).
In the preferred embodiment of the present invention, a solvent is the ethanol solution containing organic acid, is preferably contained
96~98% ethanol solutions of 0.12~0.15% acetic acid.
In the preferred embodiment of the present invention, the Centipeda minima powder is that will dry Centipeda minima to smash it through 140
Obtained by~200 meshes.
In the preferred embodiment of the present invention, the liquid-solid ratio of a solvent and Centipeda minima powder is 6~10:1, excellent
It is selected as 8~10:1.
In the preferred embodiment of the present invention, the cold soaking temperature is 1~4 DEG C, preferably 1~2 DEG C.
In the preferred embodiment of the present invention, the cold soaking time is 5~45min, preferably 10~25min.
In the preferred embodiment of the present invention, another solvent is ethyl acetate.
In the preferred embodiment of the present invention, the methanol-chloroform-acetate gradient eluent gradient is that volume ratio is
1:0:0,50:1:1,20:1:1,10:1:1,0:1:1,0:0:1；Contain in the gradient eluent of the methanol-chloroform-acetic acid
0.1~0.15 ‰ ethyl acetate.
In the preferred embodiment of the present invention, the eluent of silicagel column repeatedly (200~300 mesh) chromatography is above-mentioned
Methanol-chloroform-acetate gradient eluent.
In the preferred embodiment of the present invention, the efficient liquid phase is eluted with methanol-water.
In the preferred embodiment of the present invention, the Sephadex LH-20 is eluted with methanol.
It successively mentioned, extracted using cold soaking, column chromatography, repeatedly silica gel column chromatography, preparation efficient liquid phase and Sephadex LH-
Sesquiterpene lactone is prepared in 20 purifying, and the purity of gained final product is higher, selects the ethanol solution cold soaking containing acetic acid, contains vinegar
The methanol-chloroform of acetoacetic ester-acetate gradient eluent carries out silicagel column elution and is greatly improved target in Centipeda minima powder
The precipitation probability of sesquiterpene lactone, and the yield of final goal sesquiterpene lactone can be made not less than 20mg/Kg, it is remarkably improved it
Physiological role and synergistic effect to man and wife's grass extract.The target sesquiterpene lactone as shown in formula (1)1H,13C-NMR number
According to as shown in table 1, the sesquiterpene lactone has matrix metalloproteinase 2 (MMP2) inhibitory activity and collagen decomposes and inhibits
Activity, bioactive sites are concentrated mainly on its C-8 Alpha-Methyl-gamma lactone ring, and this special lactone structure can
With in conjunction with the MMP2 protein-specific in cell, and then the expression of adjustment signal access middle and lower reaches albumen, therefore can be further
Main component type Ⅳ collagen albumen effectively in reduction MMP2 hydrolysis denatured collagen and extracellular basilar memebrane, maintains and increases skin
The content of middle collagen maintains skin health degree, and can prevent scytitis and aging.
Sesquiterpene lactone shown in 1 formula of table (1)1H,13C-NMR data (CDCl3, δ: ppm, J:Hz, 400Hz)
| Site | 1H | 13C |
| 1 | 3.12m | 54.34 |
| 2 | 7.63dd | 161.89 |
| 3 | 6.05dd | 130.01 |
| 4 | - | 209.48 |
| 5 | - | 64.95 |
| 6 | 5.51brs | 71.95 |
| 7 | 2.93m | 48.71 |
| 8 | 4.76m | 79.52 |
| 9 | 2.47m | 42.01 |
| 10 | 2.20m | 25.90 |
| 11 | 3.10m | 40.51 |
| 12 | - | 179.21 |
| 13 | 1.55d | 10.94 |
| 14 | 1.25d | 19.75 |
| 15 | 1.07s | 15.70 |
| 16 | - | 166.62 |
| 17 | - | 128.21 |
| 18 | 6.08brm | 138.56 |
| 19 | 1.92d | 20.45 |
| 20 | 1.72brs | 17.65 |
In the preferred embodiment of the present invention, described includes man and wife's grass extract and the sesquiterpene lactone as shown in formula (1)
Composition in, the weight ratio of man and wife's grass extract and sesquiterpene lactone is 0.05~1000:1, preferably 1~100:1,
More preferably 42~45:1.
In the present invention, with the composition that man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) are prepared, have different
The MMP2 rejection ability and collagen of Chang Youyi decomposes inhibitory activity, man and wife's grass extract and the sequiterpene as shown in formula (1)
Lactone plays synergistic function, and sesquiterpene lactone can increase cutaneous permeability, Fu Qicao in conjunction with MMP2 protein-specific
Extract further suppresses the expression and activity of MMP2 albumen, is finally reached the degradation for reducing type Ⅳ collagen albumen in collagen
Purpose, maintain and increase the content of collagen in skin, help to promote skin elasticity and compact degree, reduce skin lines
With wrinkle, skin elasticity is improved, postpones skin aging, skin gloss jade-like stone profit is maintained, alleviates scytitis, reduces skin disease morbidity
Probability promotes skin health degree.
[2] item [1] described composition is used to prepare the use for inhibiting the preparation of matrix metalloproteinase 2 (MMP2) gene performance
On the way.
In the preferred embodiment of the present invention, the preparation includes but is not limited to drug.
In the preferred embodiment of the present invention, the preparation by the compound in the composition polymorph, vertical
Body isomers, pro-drug, solvate or pharmaceutical salt are prepared as active component with pharmaceutically acceptable carrier.
[3] item [1] described composition is used to prepare the purposes for the preparation for inhibiting collagen to decompose.
In the preferred embodiment of the present invention, the collagen is type Ⅳ collagen albumen.
In the preferred embodiment of the present invention, the preparation includes but is not limited to drug.
In the preferred embodiment of the present invention, the preparation by the compound in the composition polymorph, vertical
Body isomers, pro-drug, solvate or pharmaceutical salt are prepared as active component with pharmaceutically acceptable carrier.
[4] prevention of disease relevant to matrix metalloproteinase 2 (MMP2) activity and/or treatment pharmacy preparation,
Contain item [1] described composition in the pharmacy preparation.
In the preferred embodiment of the present invention, the pharmacy preparation by the compound in the composition polymorphic
Object, stereoisomer, pro-drug, solvate or pharmaceutical salt are as active component, with pharmaceutically acceptable carrier
Preparation.
[5] ameliorative way of skin condition, including including item [1] described composition in the application of the required position of required individual
Dosage form.
The invention has the benefit that
1) it is extracted afterwards using different solvents using organic cold soaking and high-frequency ultrasonic extracts two kinds of extractive techniques side in combination
Method is greatly improved extraction efficiency, and the content of man and wife's grass general flavone, can be extensive up to 85% or more in gained man and wife's grass extract
Applied to the separation and Extraction of man and wife's grass total flavonoids substance, higher purity is also beneficial to the efficient hair of its physiology effect
It waves, the present invention can be through base in inhibition Skin Cell mother cell with man and wife's grass extract of two kinds of extracted acquisitions of extractive technique
The gene expression of matter metalloproteinases MMP2 and achieve the purpose that the degradation for reducing type Ⅳ collagen albumen in extracellular collagen；
2) it successively mentioned, extracted using cold soaking, column chromatography, repeatedly silica gel column chromatography, preparation efficient liquid phase and Sephadex
Sesquiterpene lactone is prepared in LH-20 purifying, and the purity of gained final product is higher, selects the ethanol solution cold soaking containing acetic acid, contains
There is methanol-chloroform-acetate gradient eluent of ethyl acetate to carry out silicagel column elution to be greatly improved in Centipeda minima powder
The precipitation probability of target sesquiterpene lactone, and the yield of final goal sesquiterpene lactone can be made not less than 20mg/Kg, it can significantly mention
Its high physiological role；
3) composition being prepared with man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) has exceptional
MMP2 rejection ability and collagen decompose inhibitory activity, man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) hair
Synergistic function is waved, sesquiterpene lactone can increase cutaneous permeability, man and wife's grass extract in conjunction with MMP2 protein-specific
The expression and activity for further suppressing MMP2 albumen are finally reached the mesh for reducing the degradation of type Ⅳ collagen albumen in collagen
, the content of collagen in skin is maintained and increased, helps to promote skin elasticity and compact degree, reduces skin lines and wrinkle
Line improves skin elasticity, postpones skin aging, maintains skin gloss jade-like stone profit, alleviates scytitis, and it is several to reduce skin disease morbidity
Rate promotes skin health degree.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of sesquiterpene lactone in the composition comprising man and wife's grass extract of the invention；
Fig. 2 is the human skin fibroblast of the warp of the invention compositions-treated comprising man and wife's grass extract
Collagen fractional yield schematic diagram；
Fig. 3 is the warp compositions-treated comprising man and wife's grass extract of included man and wife's grass extract of the invention
The relative performance of MMP2 gene measures schematic diagram in human skin fibroblast.
Specific embodiment
Unless otherwise defined, technical and scientific term used herein has ordinary skill of the art
The identical meaning that personnel are generally understood.The present invention uses method described herein and material；But as is generally known in the art
Other suitable methods and material can also be used.Material, method and example described herein are merely illustrative,
It is not intended for limiting.All publications, patent application case, Patent Case, Provisional Application, data base entries and herein
Other bibliography referred to etc., entirety is incorporated herein as reference.It include being defined as with this specification if there is conflict
It is quasi-.
By following detailed description, attached drawing and claim, other features and advantages of the present invention be will become apparent.
For word " such as " (" for example ") and " such as " (" such as ") and its grammatical equivalents, phrase " and
It is not limited to " (" and without limitation ") should be understood to follow common usage, unless expressly stated otherwise,.Such as this
Used in text, words and phrases " about " (" about ") refer in view of the variation due to experimental error.Unless expressly stated otherwise, this paper institute
All measurements stated, clearly use regardless of whether having, and will be understood to be corrected by words and phrases " about " word.Such as this paper institute
With singular " one ", "one" and "the" (" a, " " an, " and " the ") all include plural usage, unless the context otherwise
Clear stipulaties.
Pharmaceutically acceptable material, composition or medium are referred to for term " pharmaceutically acceptable carrier ", such as
Liquid or solid filler, diluent, excipient, solvent or encapsulating material.From the compatibility of other ingredients with preparation and not right
For in the harmful meaning of patient, each carrier must be " acceptable ".It can be used as the material of pharmaceutically acceptable carrier
Some examples of material include: (1) sugar, such as lactose, dextrose and saccharose；(2) starch, such as cornstarch, potato starch
With substituted or unsubstituted beta-cyclodextrin；(3) cellulose and its derivates, such as sodium carboxymethylcellulose, ethyl cellulose
And cellulose acetate；(4) bassora gum of powdered；(5) malt；(6) gelatin；(7) talcum；(8) excipient, for example, cupu oil and
Suppository wax；(9) oily, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil；(10) glycol, than
Such as propylene glycol；(11) polyalcohol, such as glycerol, sorbierite, mannitol and polyethylene glycol；(12) ester, such as ethyl oleate and the moon
Ethyl cinnamate；(13) agar；(14) buffer, such as magnesium hydroxide and aluminium hydroxide；(15) alginic acid；(16) without pyrogen
Water；(17) isotonic saline solution；(18) Ringer's solution；(19) ethyl alcohol；(20) phosphate buffer；And (21) are adopted in pharmaceutical preparation
Other avirulent compatible materials.In some embodiments, drug of the invention is pyrogen-free, that is, is being applied
When to required individual, significant temperature is not induced to increase.
In some embodiments, every kind of pharmaceutically acceptable carrier should be compatible with other constituents, such as with
Composition provided in the present invention forms preparation and/or pharmacy preparation and/or dosage form, does not have to biological vital tissue or organ
There are excessive toxicity, stimulation, allergic reaction, immunogenicity or other problems or complication, and has more reasonable Benefit Risk ratio.
In some embodiments, preparation and/or pharmacy preparation and/or dosage form may include pharmaceutically acceptable auxiliary
Material, to simulate physiological condition, for example pH is adjusted and buffer, toxicity modifiers etc., such as sodium acetate, sodium chloride, potassium chloride, chlorine
Change calcium, sodium lactate etc..
Terms " formulation ", " pharmacy preparation ", " dosage form " can be regarded as usually made in fields of the invention
Standby any dosage form, including such as solid dosage forms (such as tablet, capsule, powder, granule etc.) and liquid dosage form (such as aqueous solution,
Emulsion, elixir, syrup etc.), further include but is not limited to solution, suspension, emulsion, paste, gel, creams, lotion, dissipates
Powder, soap, the detergent containing surfactant, oil, powder foundation, emulsion foundation cream, wax powder and spray etc., but do not limit to
In this, more specifically, skin softening toner, nutrition toner, nourishing cream, massage cream, essence, eye cream, face cleaning can be prepared into
Frost, facial cleanser, makeup removing, facial mask, spraying and loose powder dosage form.
For term administering " can be regarded as preparation and/or pharmacy preparation and/or dosage form of the invention can be with oral
Or non-oral mode be applied to needed for individual required position, it is described applied in a manner of oral in the way of include but unlimited
In: it swallows, buccal, chewing, drip the preparation and/or pharmacy preparation and/or dosage form of the invention that takes and can take in；It is described with non-mouth
The mode that the mode of clothes is applied includes but is not limited to: coating, application, spraying, sucking, implantation or following injection systems: vein
The mode of interior injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, local administration and percutaneous dosing etc., is preferably coated with, applies, sprays
Painting mode is applied.
The method of application appropriate of preparation and/or pharmacy preparation and/or dosage form of the invention can be according to such as formulation side
Method, administration mode, the age of individual, weight, gender, morbid state, diet, administration time, administration method, drainage rate and reaction spirit
The factor of sensitivity etc. is in a variety of forms by prescription, in some embodiments, applies preparation and/or pharmacy preparation and/or agent
Type includes applying the composition provided in the present invention containing about 0.001mg~1g (ratio such as from about 0.001mg to about 1g, 0.01mg being extremely
About 1g, about 0.1mg are to about 1g, and about 1mg to about 1g, about 5mg to about 1g, about 10mg to about 1g, about 20mg to about 1g, about 30mg are extremely
About 1g, about 40mg are to about 1g, about 50mg to about 1g, about 80mg to about 1g, about 100mg to about 1g, about 150mg to about 1g, about
200mg to about 1g, about 300mg are to about 1g, and about 400mg to about 1g, about 500mg to about 1g, about 600mg to about 1g, about 700mg is extremely
About 1g, about 800mg are to about 1g, and about 900mg to about 1g, about 0.001mg to about 0.5g, about 0.01mg to about 0.5g, about 0.1mg is extremely
About 0.5g, about 1mg are to about 0.5g, and about 5mg to about 0.5g, about 10mg to about 0.5g, about 20mg to about 0.5g, about 30mg is to about
0.5g, about 40mg are to about 0.5g, and about 50mg to about 0.5g, about 80mg to about 0.5g, about 100mg to about 0.5g, about 150mg is to about
0.5g, about 200mg are to about 0.5g, about 300mg to about 0.5g, about 400mg to about 0.5g, about 0.001mg to about 50mg, about
0.01mg to about 50mg, about 0.1mg are to about 50mg, about 1mg to about 50mg, about 5mg to about 50mg, about 10mg to about 50mg, about
20mg to about 50mg, about 30mg are to about 50mg, about 40mg to about 50mg, about 0.001mg to about 5mg, about 0.01mg to about 5mg,
About 0.1mg to about 5mg, about 1mg are to about 5mg, and about 0.001mg to about 0.5mg, about 0.01mg to about 0.5mg, about 0.1mg is to about
0.5mg, about 0.001mg are to about 0.05mg, about 0.01mg to about 0.05mg).Reasonable dosage is about 0.1mg for each person every day to about
0.05g。
Test mode:
A test material: from the minimum minimal medium of Eagle ' s (the MEM training of Gibco purchase balanced salt solution Han Earle ' s
Support base), fetal calf serum (FBS), nonessential amino acid, sodium bicarbonate, Sodium Pyruvate and phosphate buffered saline solution (PBS solution).
B test cell culture: human skin fibroblast CCD-966SK (BCRC60153) is in 37 DEG C, 5%CO2Item
The MEM culture of addition 10%FBS, 0.1mM nonessential amino acid, 1.5g/L sodium bicarbonate, 1mM Sodium Pyruvate is incubated under part
Base, hereinafter referred to as cell culture medium.
C tests collagen content: analyzing set group (Sircol Soluble Collagen using soluble collagen
Assay Kit, Bicolor) collagen secreted by measurement cell content, specific steps are summarized as follows:
C1 takes 1000 μ L cell culture mediums to mix with 200 μ L collagen isolation/concentration reagents, and culture is overturn at 4 DEG C
After a period of time, mixed liquor 10min is centrifuged with the revolving speed of 12000rpm, removes supernatant；
C2 is centrifuged the stain by remainder precipitating and 1000 μ L Sircol stain mixing 30min, then with the revolving speed of 12000rpm
Mixed liquor 10min is to form collagen deposit object；
C3 supernatant to be removed first cleans the collagen deposit object with 750 μ L acidic salt solutions and removes surplus liquid,
250 μ L alkaline reagents are added to dissolve the collagen deposit object, reuse ferment immunity analysis instrument (ELISA reader,
BioTek the collagen solution) is measured in the light absorption value of wavelength 555nm；
C4 compares the standard curve of water soluble collagen standard items, can calculate to obtain collagen in cell culture medium to be measured
Protein content statisticallys analyze to obtain the final product.
D test cdna expression quantity: the present invention with quantitative poly chain reaction (Q-PCR) measurement cell in collagen
The gene performance amount of related ferment is decomposed, specific steps are summarized as follows:
D1 is according to manufacturer's operation instruction, certainly using ribonucleic acid extraction set group (RNA Extraction Kit, Geneaid)
After cell isolates RNA, with reverse transcriptase at 37 DEG C III Reverse Transcriptase
It (Invitrogen) is complementary DNA (cDNA) (cDNA) by RNA reverse transcription；
D2 utilizes Q-PCR set group (KAPA CYBR FAST qPCR Kit (2X), KAPA Biosystems), is directed to MMP2
Positive the introduction 5 '-GATACCCCTTTGACGGTAAGGA-3 ' (SEQ ID NO.1) of gene and reversed introduction 5 '-
CCTTCTCCCAAGGTCCATAGC-3 ' (SEQ ID NO.2) and Polymerase Chain Reaction instrument carry out PCR to the cDNA, to take
It obtains gene performance amount data, statisticallys analyze to obtain the final product.
The present invention is described in detail with particular preferred embodiment and comparative example below.
Embodiment 1:
The present embodiment provides a kind of composition comprising man and wife's grass extract, the composition include man and wife's grass extract and
The sesquiterpene lactone as shown in formula (1).
The acquisition methods of man and wife's grass extract in the composition include:
1 DEG C, under the conditions of 12:1 solid-to-liquid ratio, obtain the with man and wife's grass meal end that 82wt% ethanol solution cold soaking crosses 160 meshes
One extracting solution, cold soaking time are 15min；At room temperature, it is 1.05g/mL that the first extracting solution, which is concentrated into density, with containing for 12 times of weight
There is the 80wt% ethanol solution ultrasonic wave extraction of 1.5wt% acetonitrile to obtain the second extracting solution, ultrasonic frequency is 50KHz, energy
Density is 0.55W/cm2, extract 30min；Second extracting solution, which is concentrated into after density is 1.1g/mL, passes through macroporous absorbent resin
Diaion HP-20 column, with ethyl acetate gradient elution and collect 40-60% elution position eluent, after concentration i.e.
?.
It is successively extracted using different solvents using organic cold soaking and high-frequency ultrasonic extracts two kinds of extractive techniques side in combination
Method is greatly improved extraction efficiency, and the content of man and wife's grass general flavone, can be extensive up to 85.8wt% in gained man and wife's grass extract
Applied to the separation and Extraction of man and wife's grass total flavonoids substance, higher purity is also beneficial to the efficient hair of its physiology effect
It waves, the present invention can be through base in inhibition Skin Cell mother cell with man and wife's grass extract of two kinds of extracted acquisitions of extractive technique
The gene expression of matter metalloproteinases MMP2 and achieve the purpose that the degradation for reducing type Ⅳ collagen albumen in extracellular collagen,
The content for maintaining and increasing collagen in skin helps to promote skin elasticity and compact degree, reduces skin lines and wrinkle,
Skin aging is postponed, skin gloss jade-like stone profit is maintained, alleviates scytitis, reduce skin disease incidence probability, promote skin health journey
Degree.
The acquisition methods of the sesquiterpene lactone as shown in formula (1) in the composition include:
1) 1 DEG C, under the conditions of liquid-solid ratio 8:1, with the 96% ethanol solution cold soaking containing 0.12% acetic acid by 140 meshes
Centipeda minima powder, it is closely colourless to percolate with 15 times of above-mentioned solvent diacolations of amount after cold soaking 10min, merge percolate and is simultaneously concentrated into
Without alcohol taste；
2) with ethyl acetate extraction step 1) concentrate, combining extraction liquid is concentrated into paste；
3) the paste concentrate of step 2) carries out silicagel column (100~200 mesh) chromatography, is washed with methanol-chloroform-acetate gradient
De- liquid (v:v:v 1:0:0,50:1:1,20:1:1,10:1:1,0:1:1,0:0:1) carries out gradient elution, the methanol-chlorine
Ethyl acetate in imitative-acetate gradient eluent containing 0.1wt ‰, every 100mL eluent collect 1 part, and TLC chromatography checks,
The similar intermediate fraction of spot merges；
4) (eluent is above-mentioned methanol-chloroform-to silicagel column (200~300 mesh) chromatography to the progress of fraction obtained by step 3) repeatedly
Acetate gradient eluent), preparation efficient liquid phase (eluent is methanol-water) and Sephadex LH-20 (eluent is methanol) it is pure
Change to obtain the sesquiterpene lactone as shown in formula (1).
It successively mentioned, extracted using cold soaking, column chromatography, repeatedly silica gel column chromatography, preparation efficient liquid phase and Sephadex LH-
Sesquiterpene lactone is prepared in 20 purifying, and the purity of gained final product is higher, selects the ethanol solution cold soaking containing acetic acid, contains vinegar
The methanol-chloroform of acetoacetic ester-acetate gradient eluent carries out silicagel column elution and is greatly improved target in Centipeda minima powder
The precipitation probability of sesquiterpene lactone, and the yield of final goal sesquiterpene lactone can be made up to 21.5mg/Kg, it is remarkably improved its life
Neo-Confucianism effect.The target sesquiterpene lactone as shown in formula (1)1H,13C-NMR data are as shown in table 1, and the sesquiterpene lactone has
Matrix metalloproteinase 2 (MMP2) inhibitory activity and collagen decompose inhibitory activity, and bioactive sites are concentrated mainly on
On its C-8 Alpha-Methyl-gamma lactone ring, this special lactone structure can be with the MMP2 protein-specific knot in cell
It closes, and then the expression of adjustment signal access middle and lower reaches albumen, therefore further can effectively weaken MMP2 hydrolysis denatured collagen and thin
Main component type Ⅳ collagen albumen in extracellular basilar memebrane maintains and increases the content of collagen in skin, maintains skin strong
Kang Du, and scytitis and aging can be prevented.
In composition comprising man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) described in the present embodiment, the husband
The weight ratio of wife's grass extract and sesquiterpene lactone is 42:1.
In the present embodiment, with the composition that man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) are prepared, have
The MMP2 rejection ability and collagen of exceptional decompose inhibitory activity, man and wife's grass extract and the sesquialter as shown in formula (1)
Terpene lactones plays synergistic function, and sesquiterpene lactone can increase cutaneous permeability, man and wife in conjunction with MMP2 protein-specific
Careless extract further suppresses the expression and activity of MMP2 albumen, is finally reached the drop for reducing type Ⅳ collagen albumen in collagen
The purpose of solution maintains and increases the content of collagen in skin, helps to promote skin elasticity and compact degree, it is thin to reduce skin
Line and wrinkle improve skin elasticity, postpone skin aging, maintain skin gloss jade-like stone profit, alleviate scytitis, reduce skin disease hair
Sick probability promotes skin health degree.
The present embodiment also provides the above-mentioned composition comprising man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) and is used for
Preparation inhibits the purposes of the preparation of matrix metalloproteinase 2 (MMP2) gene performance.
The present embodiment also provides the above-mentioned composition comprising man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) and is used for
Preparation inhibits the purposes of the preparation of type Ⅳ collagen protein breakdown.
The present embodiment also provides the prevention of disease relevant to matrix metalloproteinase 2 (MMP2) activity and/or treatment is used
Pharmacy preparation contains the combination comprising man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) in the pharmacy preparation
Object.
The present embodiment also provides the ameliorative way of skin condition, above-mentioned including containing in the application of the required position of required individual
The dosage form of composition comprising man and wife's grass extract and the sesquiterpene lactone as shown in formula (1).
Embodiment 2:
The present embodiment provides the following termss:
[1], a kind of composition comprising man and wife's grass extract, the composition include man and wife's grass extract and such as embodiment
Sesquiterpene lactone shown in Chinese style (1).
In the preferred embodiment, the acquisition methods of man and wife's grass extract in the composition include:
The first extracting solution is obtained with solvent cold soak man and wife's grass meal end；
With another solvent, ultrasonic wave extraction obtains the second extracting solution at room temperature after the concentration of first extracting solution；
By macroporous absorbent resin Diaion HP-20 column after the concentration of second extracting solution, with gradient elution and collect
40-60% elutes the eluent at position, is concentrated into constant weight to obtain the final product.
In the preferred embodiment,
One solvent is 82wt% ethanol solution；
Man and wife's grass meal end is that will dry man and wife's grass to smash it through obtained by 180 meshes；
The liquid-solid ratio at one solvent and man and wife's grass meal end is 14:1；
2 DEG C of the temperature of the cold soaking；
The cold soaking time is 20min；
First extracting solution is concentrated into when density is 1.08g/mL and carries out ultrasonic wave extraction；
Another solvent is the 85wt% ethanol solution containing 1.6wt% acetonitrile；
The weight ratio of another solvent and the first extracting solution concentrate is 15:1；
Ultrasonic frequency is 75KHz when the ultrasonic wave, and energy density is 0.65W/cm2；
The ultrasonic wave extraction time is 40min；
Second extracting solution is concentrated into when density is 1.15g/mL and is washed by macroporous absorbent resin Diaion HP-20 column
It is de-；
The eluent is acetonitrile solution.
It is successively extracted using different solvents using organic cold soaking and high-frequency ultrasonic extracts two kinds of extractive techniques side in combination
Method is greatly improved extraction efficiency, and the content of man and wife's grass general flavone can answer extensively up to 86.5% in gained man and wife's grass extract
For the separation and Extraction of man and wife's grass total flavonoids substance, higher purity is also beneficial to the efficient performance of its physiology effect,
The present invention can be through inhibition Skin Cell mother cell mesostroma with man and wife's grass extract of two kinds of extracted acquisitions of extractive technique
The gene expression of metalloproteinases MMP2 and achieve the purpose that the degradation for reducing type Ⅳ collagen albumen in extracellular collagen, dimension
The content for holding and increasing collagen in skin helps to promote skin elasticity and compact degree, reduces skin lines and wrinkle, push away
Slow skin aging maintains skin gloss jade-like stone profit, alleviates scytitis, reduce skin disease incidence probability, promotes skin health degree.
In the preferred embodiment, the acquisition methods of the sesquiterpene lactone as shown in formula (1) in the composition include:
1) then closely colourless to percolate with 18 times of amount solvent diacolations with a solvent cold soak Centipeda minima powder, it closes
And percolate and it is concentrated into no alcohol taste；
2) with another solvent extraction 1) concentrate, combining extraction liquid is concentrated into paste；
3) the paste concentrate of step 2) carries out silicagel column (100~200 mesh) chromatography, is washed with methanol-chloroform-acetate gradient
De- liquid carries out gradient elution, and every 100mL eluent collects 1 part, and TLC chromatography checks, the similar intermediate fraction of spot merges；
4) fraction obtained by step 3) carries out repeatedly silicagel column (200~300 mesh) chromatography, preparation efficient liquid phase and Sephadex
LH-20 purifies to obtain the sesquiterpene lactone as shown in formula (1).
In the preferred embodiment,
One solvent is the 96.5wt% ethanol solution containing 0.15wt% acetic acid；
The Centipeda minima powder is that will dry Centipeda minima to smash it through obtained by 180 meshes；
The liquid-solid ratio of one solvent and Centipeda minima powder is 9:1；
The cold soaking temperature is 2 DEG C；
The cold soaking time is 25min；
Another solvent is ethyl acetate；
The methanol-chloroform-acetate gradient eluent gradient is that volume ratio is 1:0:0,50:1:1,20:1:1,10:1:
1,0:1:1,0:0:1；Ethyl acetate containing 0.12wt ‰ in the gradient eluent of the methanol-chloroform-acetic acid；
The eluent of silicagel column repeatedly (200~300 mesh) chromatography is aforementioned methanol-chloroform-acetate gradient eluent；
The efficient liquid phase is eluted with methanol-water；
The Sephadex LH-20 is eluted with methanol.
It successively mentioned, extracted using cold soaking, column chromatography, repeatedly silica gel column chromatography, preparation efficient liquid phase and Sephadex LH-
Sesquiterpene lactone is prepared in 20 purifying, and the purity of gained final product is higher, selects the ethanol solution cold soaking containing acetic acid, contains vinegar
The methanol-chloroform of acetoacetic ester-acetate gradient eluent carries out silicagel column elution and is greatly improved target in Centipeda minima powder
The precipitation probability of sesquiterpene lactone, and the yield of final goal sesquiterpene lactone can be made to reach 22.6mg/Kg, it is remarkably improved it
Physiological role.The sesquiterpene lactone has matrix metalloproteinase 2 (MMP2) inhibitory activity and collagen decomposes and inhibits
Activity, bioactive sites are concentrated mainly on its C-8 Alpha-Methyl-gamma lactone ring, and this special lactone structure can
With in conjunction with the MMP2 protein-specific in cell, and then the expression of adjustment signal access middle and lower reaches albumen, therefore can be further
Main component type Ⅳ collagen albumen effectively in reduction MMP2 hydrolysis denatured collagen and extracellular basilar memebrane, maintains and increases skin
The content of middle collagen maintains skin health degree, and can prevent scytitis and aging.
In the preferred embodiment, the composition comprising man and wife's grass extract and the sesquiterpene lactone as shown in formula (1)
In, the weight ratio of man and wife's grass extract and sesquiterpene lactone is 44:1.
In the present invention, with the composition that man and wife's grass extract and the sesquiterpene lactone as shown in formula (1) are prepared, have different
The MMP2 rejection ability and collagen of Chang Youyi decomposes inhibitory activity, man and wife's grass extract and the sequiterpene as shown in formula (1)
Lactone plays synergistic function, and sesquiterpene lactone can increase cutaneous permeability, Fu Qicao in conjunction with MMP2 protein-specific
Extract further suppresses the expression and activity of MMP2 albumen, is finally reached the degradation for reducing type Ⅳ collagen albumen in collagen
Purpose, maintain and increase the content of collagen in skin, help to promote skin elasticity and compact degree, reduce skin lines
With wrinkle, skin elasticity is improved, postpones skin aging, skin gloss jade-like stone profit is maintained, alleviates scytitis, reduces skin disease morbidity
Probability promotes skin health degree.
[2], item [1] described composition is used to prepare the preparation for inhibiting the performance of matrix metalloproteinase 2 (MMP2) gene
Purposes.
[3], item [1] described composition is used to prepare the purposes for the preparation for inhibiting collagen to decompose.
In the preferred embodiment, the collagen is type Ⅳ collagen albumen.
[4], the prevention of disease relevant to matrix metalloproteinase 2 (MMP2) activity and/or treatment pharmacy preparation,
Contain item [1] described composition in the pharmacy preparation.
[5], the ameliorative way of skin condition, including including item [1] described combination in the application of the required position of required individual
The dosage form of object.
Comparative Example V 3:
Comparative Example V 3 is substantially the same manner as Example 1, institute the difference is that, in the composition in Comparative Example V 3
It only include man and wife's grass extract described in embodiment 1.
Comparative Example V 4:
Comparative Example V 4 is substantially the same manner as Example 2, institute the difference is that, in the composition in Comparative Example V 4
It only include man and wife's grass extract as described in example 2.
Comparative Example V 5:
Comparative Example V 5 is substantially the same manner as Example 1, institute the difference is that, Comparative Example V 5 provide it is a kind of only
The sesquiterpene lactone as shown in formula (1) comprising extracting from Centipeda minima described in embodiment 1.
Comparative Example V 6:
Comparative Example V 6 is substantially the same manner as Example 2, institute the difference is that, Comparative Example V 6 provide it is a kind of only
The sesquiterpene lactone as shown in formula (1) comprising extracting from Centipeda minima described in embodiment 2.
Comparative Example V 7:
Comparative Example V 7 is substantially the same manner as Example 2, and institute is the difference is that obtain described group in Comparative Example V 7
When closing man and wife's grass extract of object, another solvent is 85wt% ethanol solution, is free of acetonitrile.
Comparative Example V 8:
Comparative Example V 8 is substantially the same manner as Example 2, and institute is the difference is that obtain described group in Comparative Example V 8
When closing the sesquiterpene lactone as shown in formula (1) in object, a solvent is 96.5wt% ethanol solution and is free of acetic acid.
Comparative Example V 9:
Comparative Example V 9 is substantially the same manner as Example 2, and institute is the difference is that obtain described group in Comparative Example V 9
When closing the sesquiterpene lactone as shown in formula (1) in object, the methanol-chloroform-acetate gradient eluent gradient is that volume ratio is
1:0:0,50:1:1,20:1:1,10:1:1,0:1:1,0:0:1 and be free of ethyl acetate.
Comparative Example V 10:
Comparative Example V 10 is substantially the same manner as Example 2, and institute is the difference is that in Comparative Example V 10, described in acquisition
When the sesquiterpene lactone as shown in formula (1) in composition, a solvent is 96.5wt% ethanol solution and is free of acetic acid；It is described
Methanol-chloroform-acetate gradient eluent gradient is that volume ratio is 1:0:0,50:1:1,20:1:1,10:1:1,0:1:1,0:0:
1 and be free of ethyl acetate.
Test analysis:
I, the husband in the acquisition composition in contrast test embodiment 1, embodiment 2 and Comparative Example V 7-V10
When wife's grass extract the content of man and wife's grass general flavone and when obtaining the sesquiterpene lactone as shown in formula (1) in the composition most
The yield of whole target sesquiterpene lactone, statistical result are as shown in table 2.As shown in Table 2, in the preferred embodiment of the method for the present invention 1
In preferred embodiment 2, when obtaining man and wife's grass extract, it is total yellow that the mixed solution containing acetonitrile, ethyl alcohol can strengthen man and wife's grass
The dissolution of ketone, and the content of man and wife's grass general flavone in final product man and wife's grass extract can be made to reach 85% or more, further
Conducive to the efficient performance of its physiology effect；Similarly, first when obtaining target product sesquiterpene lactone from Centipeda minima powder
It extracts first with the ethanol solution containing acetic acid, is secondly eluted using the methanol-chloroform containing ethyl acetate-acetate gradient in advance
Liquid carries out gradient elution, is greatly improved the precipitation probability of target sesquiterpene lactone in Centipeda minima powder, and can make final
The yield of target sesquiterpene lactone is not less than 20mg/Kg, is remarkably improved its physiological role and to man and wife's grass extract
Synergistic effect.
The content of general flavone and the yield of sesquiterpene lactone statistics in 2 man and wife's grass extract of table
| Embodiment | The content % of general flavone in man and wife's grass extract | The yield mg/Kg of sesquiterpene lactone |
| 1 | 85.8 | 21.5 |
| 2 | 86.5 | 22.6 |
| V7 | 55.9 | 22.6 |
| V8 | 86.5 | 18.5 |
| V9 | 86.5 | 14.0 |
| V10 | 86.5 | 8.5 |
It II, is composition of the inspection present invention comprising man and wife's grass extract to the shadow of the collagen production of Skin Cell
It rings, now with collagen content analysis of experiments human skin fibroblast CCD-966SK its collagen after the processing
The variation of quantum of output.Firstly, by human skin fibroblast according to 3 × 104The hole cells/ is inoculated in containing 500 μ L cell culture
Each hole of 24 porose discs of base after cultivating for 24 hours at 37 DEG C, removes cell culture medium and cleans cell with PBS solution.Secondly, will
500 μ L, the cell culture medium (being free of FBS) containing composition described in each embodiment of 1mg/mL are added to each hole cell, separately only with 500
Cell culture medium processing cell of the μ L without FBS is with as a control group.After aforementioned two classes group of cells cultivates 48h at 37 DEG C,
Cell culture medium is collected from each hole, measures wherein collagen content, and counts as shown in Figure 2.As shown in Figure 2, of the invention
The composition in preferred embodiment has important influence to the collagen production of Skin Cell, particularly preferably implements
Composition in example 2 is 16 times or more of control group to the fractional yield of collagen, particularly it can further be seen that relative to list
It solely applies man and wife's grass extract (Comparative Example V 3, V4) and is administered alone sesquiterpene lactone (Comparative Example V 5, V6), apply
Composition (preferred embodiment 1,2) containing said two devices is bigger to the yield promotion of collagen, shows the collaboration of the two
Synergistic effect can greatly increase the content of collagen in skin, help to promote skin elasticity and compact degree, reduce skin
Microgroove and wrinkle improve skin elasticity, postpone skin aging, maintain skin gloss jade-like stone profit, alleviate scytitis, reduce skin disease
Incidence probability promotes skin health degree.
It III, is composition of the inspection present invention comprising man and wife's grass extract to human skin fibroblast mesostroma gold
The influence of relative performance's amount of Proteases 2 (MMP2) gene, measures human skin fibroblast CCD-966SK with Q-PCR
The performance amount of its collagen catabolic enzyme related gene after the compositions-treated of (BCRC 60153) included man and wife's grass extract
Variation.Firstly, by human skin fibroblast according to 2 × 105A hole cells/ is inoculated in 24 holes containing 2mL cell culture medium
Each hole of disk, is placed at 37 DEG C and cultivates.Secondly, containing the cell culture medium processing of the composition with 0.5mg/mL or 1mg/mL
Cell for 24 hours, is regathered through processing cell to carry out Q-PCR, separately to handle through the cell culture medium without man and wife's grass water extract
As a control group, the relative performance of measurement each group MMP2 gene measures human skin fibroblast, and counts as shown in Figure 3.From
It can be seen firstly that inhibiting effect effect for MMP2 gene, is either administered alone the (comparison of man and wife's grass extract in Fig. 3
Embodiment V3, V4), it is still administered alone sesquiterpene lactone (Comparative Example V 5, V6), and group of the application containing said two devices
It closes object (preferred embodiment 1,2 and 7~V10 of Comparative Example V), shows dose dependent；Simultaneously it is also seen that combination is applied
The inhibiting effect of MMP2 gene is significantly improved with man and wife's grass extract and sesquiterpene lactone, it can be by it in preferred embodiment 2
Relative performance's amount is forced down to no more than 0.2, it is seen that the composition comprising man and wife's grass extract of the invention is inhibiting MMP2 albumen
Expression and activity aspect have excellent function and effect, not only can achieve reduce collagen in type Ⅳ collagen albumen drop
The purpose for solving, maintaining and increase the content of collagen in skin additionally aids alleviation scytitis, and it is several to reduce skin disease morbidity
Rate.
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Sequence table
<110>Jiaxing City Jue Tuo Science and Technology Ltd.
<120>comprising the composition of man and wife's grass extract
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gatacccctt tgacggtaag ga 22
<210> 2
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccttctccca aggtccatag c 21
Claims (10)
1. a kind of composition comprising man and wife's grass extract, it is characterised in that comprising man and wife's grass extract and as shown in formula (1) again
Sesquiterpene lactone:
The content of man and wife's grass general flavone reaches 85% or more in man and wife's grass extract；
The composition can be effectively suppressed the performance of matrix metalloproteinase 2 (MMP2) gene and/or effectively inhibit collagen egg
White decomposition.
2. composition according to claim 1, it is characterised in that: the acquisition side of man and wife's grass extract in the composition
Method includes:
The first extracting solution is obtained with solvent cold soak man and wife's grass meal end；
With another solvent, ultrasonic wave extraction obtains the second extracting solution at room temperature after the concentration of first extracting solution；
By macroporous absorbent resin Diaion HP-20 column after the concentration of second extracting solution, with gradient elution and collect 40~
The eluent at 60% elution position, is concentrated into constant weight to obtain the final product.
3. composition according to claim 2, it is characterised in that: a solvent is water, alcohols or alcohol-water mixture.
4. composition according to claim 2 or 3, it is characterised in that: another solvent be water, alcohols, nitrile it is mixed
Close object.
5. composition according to claim 1, it is characterised in that: in the sequiterpene as shown in formula (1) in the composition
The acquisition methods of ester include:
1) then closely colourless to percolate with 15~20 times of amount solvent diacolations with a solvent cold soak Centipeda minima powder, it closes
And percolate and it is concentrated into no alcohol taste；
2) with another solvent extraction 1) concentrate, combining extraction liquid is concentrated into paste；
3) the paste concentrate of step 2) carries out silicagel column (100~200 mesh) chromatography, with methanol-chloroform-acetate gradient eluent
Gradient elution is carried out, every 100mL eluent collects 1 part, and TLC chromatography checks, the similar intermediate fraction of spot merges；
4) fraction obtained by step 3) carries out repeatedly silicagel column (200~300 mesh) chromatography, preparation efficient liquid phase and Sephadex LH-
20 purify to obtain the sesquiterpene lactone as shown in formula (1).
6. composition according to claim 5, it is characterised in that: contain in the gradient eluent of the methanol-chloroform-acetic acid
There is 0.1~0.15 ‰ ethyl acetate.
7. any one of claim 1~6 composition, which is used to prepare, inhibits the performance of matrix metalloproteinase 2 (MMP2) gene
The purposes of preparation.
8. any one of claim 1~6 composition is used to prepare the purposes for the preparation for inhibiting collagen to decompose, feature
It is that the collagen is type Ⅳ collagen albumen.
9. prevention and/or the treatment pharmacy preparation of disease relevant to matrix metalloproteinase 2 (MMP2) activity, feature
It is in the pharmacy preparation containing any one of claim 1~6 composition.
10. the ameliorative way of skin condition, it is characterised in that including including claim 1 in the application of the required position of required individual
The dosage form of any one of~6 compositions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374103.7A CN110123865A (en) | 2019-05-07 | 2019-05-07 | Composition comprising man and wife's grass extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910374103.7A CN110123865A (en) | 2019-05-07 | 2019-05-07 | Composition comprising man and wife's grass extract |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110123865A true CN110123865A (en) | 2019-08-16 |
Family
ID=67576457
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910374103.7A Withdrawn CN110123865A (en) | 2019-05-07 | 2019-05-07 | Composition comprising man and wife's grass extract |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110123865A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101732383A (en) * | 2009-12-11 | 2010-06-16 | 暨南大学 | Total sesquiterpene lactone extract of centipeda minima, preparation method and application thereof |
| CN108743583A (en) * | 2018-09-04 | 2018-11-06 | 暨南大学 | Application of the sesquiterpene lactone chemical combination object in preparing the drug with antivirus action in Centipeda minima |
| CN109276588A (en) * | 2017-07-21 | 2019-01-29 | 大江生医股份有限公司 | Application of wife extract in preparing composition for inhibiting MMP2 gene expression and collagen degradation |
-
2019
- 2019-05-07 CN CN201910374103.7A patent/CN110123865A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101732383A (en) * | 2009-12-11 | 2010-06-16 | 暨南大学 | Total sesquiterpene lactone extract of centipeda minima, preparation method and application thereof |
| CN109276588A (en) * | 2017-07-21 | 2019-01-29 | 大江生医股份有限公司 | Application of wife extract in preparing composition for inhibiting MMP2 gene expression and collagen degradation |
| CN108743583A (en) * | 2018-09-04 | 2018-11-06 | 暨南大学 | Application of the sesquiterpene lactone chemical combination object in preparing the drug with antivirus action in Centipeda minima |
Non-Patent Citations (2)
| Title |
|---|
| 孔令义: "《复杂天然产物波谱解析》", 30 September 2012, 中国医药科技出版社 * |
| 林雨珊等: ""鹅不食草中倍半萜内酯成分及其抗肿瘤活性的研究"", 《中南药学》 * |
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