Mateine and its application in skin preparations for extenal useTechnical field
The invention belongs to field of phytochemistry, and in particular to the preparation technology of a kind of mateine and its outside skinWith the application in agent or cosmetics.
Background technology
Skin refers to tissue of the body surface bag outside muscle, is the maximum organ of human body, mainly carries protection bodyBody, perspire, feel the functions such as cold and hot and pressure.Skin covers whole body, protects each tissue and organ in human body micro- from cause of diseaseThe invasion and attack of the mechanical damage such as the external substances such as biology and physics, effectively resist environmental stimuli；Skin also maintains human bodyThe loss of moisture, electrolyte and nutriment etc., effectively maintains the stabilization of human internal environment, is the natural screen for protecting human bodyBarrier.
Skin is made up of epidermis, corium and the part of hypodermis three, and difference of the different skin in structure can be to the colour of skinTool has a certain impact.Epidermis is made up of cuticula, hyaline layer, stratum granulosum, spinous layer and basalis again, and its mesocuticle is positionIn 5~20 layers of dead pinacocyte of body surface, when cuticula is blocked up or during low water content, the ability reduction of skin reflex light,So as to which the colour of skin of dimness is presented；Melanin is mainly distributed in the basalis of epidermis, and melanin is most strong to the absorbability of light,Melanin content is more, then the colour of skin presented is deeper.Corium is located at epidermis deep layer, is made up of dense connective tissue, its interior pointCloth various phoirocytes and substantial amounts of collagenous fibres, elastomer.Collagenous fibres in corium assign skin tension andToughness, its skewness or structure change cause light uniformly to be reflected so that the colour of skin is uneven and lackluster；In addition, if angioplerosis degree in corium is not enough or poor blood circulation, hemochrome cohesion or pigment content can decline,So as to cause the colour of skin dark red or pale.
Although skin texture has a certain impact to the colour of skin, the colour of skin depends primarily on the content of various pigments contained by skinAnd type.Human skin mainly contain the melanin of brown-black, red oxygenated haemoglobin, the reduced hemoglobin of blueness and4 kinds of biochromes such as carrotene of yellow, wherein carrotene are dna exogenous pigment, and mainly by diet regimen, remaining 3 kinds areEndogenous pigment, is synthesized by body itself.Research find, black race, the melanin content of yellow and white skin andThere is the difference of conspicuousness in form：Black race's melanin granule is maximum, in brown, dark brown or black；Yellow's melanin granuleIt is small compared with black race, it is bigger compared with white people, in light brown, brown or brownish black；White people's melanin granule is relatively fine, in pale red,Yellowish red color or rufous.Therefore, the content and form of melanin are the principal elements for determining the colour of skin depth in skin.
Pigment be biology in order to be produced to uvioresistant, melanin is pigment most powerful in all pigments.BlackElement is a kind of pigment of dark brown present in animal skin or hair, and a kind of special cell is melanin in organismCell is synthesized by substrate of tyrosine, and its building-up process is by tyrosinase (Tyrosinase, TYR), dopachrome changeEnzyme (Dopaehrometautomerase), 5,6- dihydroxy indole -2- carboxylic oxidases (DHICAoxidase) and the black cell of rushThe regulation and control of a variety of enzymes such as hormone (Melanocyte stimulating hormone, MSH) or hormone, wherein tyrosinase is blackPlayed a decisive role in the biosynthesis of pigment.Tyrosine is converted into DOPA by melanocyte in the presence of tyrosinase,It is changed into melanin through the biochemical process of a series of complex again.Therefore, the activity of tyrosinase is higher, i.e., acted on tyrosineThe melanin of generation will increase, and find expression on skin of face and produce various blackspots or freckle etc..Therefore, tyrosine enzyme activity is suppressedProperty be a kind of effective important channel for reducing melanin generation, domestic and foreign scholars are often using suppressing tyrosinase activity experimental studyThe medicine of the screening treatment excessive disease of cutaneous pigmentation, and some can be applied to whitening as the material of tyrosinase inhibitorIn cosmetics.
With the enhancing of people's living standard and self maintenance consciousness, the basic nursing except whitening, moisturizing etc. to skinEffect is outer, and the various skin problems that sensitiveness skin is brought also result in the extensive concern of people.Especially in recent years, climate changeAbnormal, air pollution is serious, emerge in an endless stream cosmetics species and the cosmetic composition of complexity, improve sensitive skin appearanceAnd receive the possibility of environmental stimuli, the possibility also plus according to various scytitises and allergic symptom occurred, people's skinThe consciousness of health and the cognition of sensitive skin are also being stepped up.
Skin sensitivity refers to susceptible to many factors and the subjective sensation symptom of induction skin condition, i.e. dermoreactionThe even easy allergy of high, poor resistance.Sensitive skin takes place mostly in face, also other positions such as trick, scalp.Sensitive skinBe mainly characterized by more dry, easily it is tight and rubescent, by minimal irritation, you can produce itch, furfur even burn andShouting pain etc. feels that severe patient can produce unhealthy emotion or mental symptom.Therefore, for the maintenance effect of sensitive skin, includingThe daily nursing carried out to sensitive skin and improvement, suppress skin wound repair, inflammation of releiving and allergy, to improve skin selfBarrier function, resists the stimulation of outer bound pair skin, improves skin state and has great importance.
The origin cause of formation of sensitive skin has a variety of, mainly includes barrier function defect, nerves reaction too strong or inflammatory reactionVicious circle, is divided into barrier function maintenance, neuroleptic by antiallergic activity on this basis and suppresses the type of inflammation 3.Wherein,It is to induce various inflammation due to extraneous adverse factor invasion and the loss of endotrophic material caused by skin barrier function defectWith the major reason of skin sensitivity.Therefore, strengthen the maintenance and reparation to skin barrier function, can effectively improve skin shapeState, improve skin stimulate to external world or allergy sex factor resistivity.
Hyaluronic acid (Hyaluronic acid, HA) is that content is most in extracellular matrix (ECM), proportion is maximumComposition, with very strong water binding ability and viscosity, extracellular matrix volume can be maintained, regulating cell growth factor and thinThe secretion of intracellular cytokine, influences sticking, grow, breed and breaking up for cell, thus is maintaining moisture of skin and elasticity, wound healingIt is main with serving during vascularization etc..Hyaluronidase (Hyaluronidase, HAase) is the spy of hyaluronic acidDifferent in nature lyases, it can make hyaluronic acid degraded, so as to reduce the activity of internal hyaluronic acid, suppress hyaluronidaseActivity can ensure the content and normal function of hyaluronic acid.Therefore, the experiment of hyaluronic acid enzyme level is that most typical antiallergic is livedProperty in-vitro evaluation method, using hyaluronic acid enzyme inhibition rate as the antiallergic activity of metrics evaluation material, hyaluronic acid enzyme inhibition rateBigger, then the antiallergic activity of material is stronger.
Compared with normal skin, there is barrier function damage or the characteristics of abnormality of nerve function in sensitive skin, it more difficult inThe invasion and attack of imperial environmental stimuli thing and anaphylactogen, scytitis reacts caused by easily occurring a series of stimulations or allergy, Ran HoujinOne step damages skin barrier structure and nerve endings, causes vicious circle.Therefore, inflammatory reaction is the another important of sensitive skinFactor, by suppressing the evaluation of inflammation effect to tested substance, can reflect that it contacts external substance (stimulant to sensitive skinOr anaphylactogen) when caused adverse reaction inhibitory action it is strong and weak.
Macrophage is a kind of white blood cell in tissue, belongs to immunocyte, anti-in immune response and inflammationPlay an important roll in answering.During inflammatory reaction, macrophage is under bacterium and its endotoxic stimulation, and can secrete substantial amounts of hasThe endogenous chemicals of proinflammatory effect --- inflammatory factor, such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-α) and whiteβ of cytokine 1 (lL-1 β) etc., these inflammatory factors again can further activated macrophage, the macrophage after activation can releaseAmplification quantity NO, can cause the injury and the diastole that blood vessel is excessive of cell, eventually cause serious inflammatory response and complication.NOAs a kind of inflammatory mediator, the adjustment effect of key is played in inflammation and immune response, is suppressed caused by macrophage activationNO release, can reduce inflammation to a certain extent.In addition, NO can with super oxide anion formation peroxynitrite ion andBe decomposed into supervirulent OH, once into target cell or close on cell will cause cytotoxic effect even damage groupKnit.Therefore, reducing activated macrophage, excessively release NO has certain anti-inflammatory meaning.Inflammatory mediator is discharged to macrophageInhibitory action research is the common method of the anti-delayed-type hypersensitivity effect of detectable substance confrontation.
Ilex paraguarensis (Yerba Mate tea), makees mate again, is Aquifoliaceae (Aquifoliaceae) Ilex(Ilex) the drying blade of perennial woody plant mate tea (Ilex Paraguarensis), is that the torrid zone or subtropical zone are moreYear raw evergreen shrubs.Wild mate tea originates in South American region, is distributed widely in Argentina, Chile, Peru and Brazil, meshBefore be widely cultivated in many tropic countries；As a kind of traditional drink, mate some Latin American countries as Brazil,Argentina, Uruguay, Paraguay etc. are popular, are described as Argentine state's tea.
Have and contain abundant polyphenols in document report, Ilex paraguarensis, but its polyphenol component has significantly not with common teaTogether, contain in mate in a large amount of common teas and contain seldom or do not contain chlorogenic acid, and the catechins enriched in teaDo not contained but in mate, in addition, also containing flavonoid class materials such as a small amount of Kaempferol, rutin, Quercetins.Ilex paraguarensisIn also contain the class material such as a certain amount of purine alkaloid (caffeine etc.) and saponin(e (horse black pigment used by women in ancient times to paint their eyebrows saponin(e etc.).Pharmacological research shows,Ilex paraguarensis has anti-oxidant, hypoglycemic, antibacterial, antiviral, cholagogic, aid digestion, fat-reducing, regulating lipid metabolism, resisting cardiovascularA variety of effects such as disease, antitumor, excitor nerve system.
The present inventor have passed through extensively and discovery after in-depth study：Mateine has suppression simultaneouslyMake the effect of external tyrosinase and hyaluronidase activity, the work also with suppression or the NO burst sizes for reducing macrophageWith.Therefore, Ilex paraguarensis can be added to skin preparations for extenal use as functional additive, especially in cosmetics, for skin whitening, anti-mistakeThe effects such as quick and anti-inflammatory, acts on.
The content of the invention
Present invention firstly discovers that mateine has a tyrosinase inhibitory action, hyaluronic acid enzyme inhibition andSuppress and reduce the effect of NO releases.
On the one hand, the invention provides a kind of preparation method of mateine, the preparation method is selected from：Decoction is carriedTake, extraction, ultrasonic extraction.
Some preferred embodiment in, the preparation method of mateine is extracted using decocting, and decoction was extractedJourney is further comprising the steps of：
B) filter, filtrate alcohol precipitation, centrifugation takes supernatant, obtains mateine.
Some preferred embodiment in, the preparation method of mateine uses extraction, extraction mistakeCheng Caiyong solvent is selected from：Deionized water, methanol, ethanol, acetone, propane diols, butanediol, glycerine or its combination.
Some preferred embodiment in, the preparation method of mateine uses ultrasonic extraction, ultrasonic extraction mistakeJourney is further comprising the steps of：
B) filter, filtrate alcohol precipitation, filtering obtains mateine.
On the other hand, the invention provides extract obtained mateine by the method for the invention.
Another aspect, the invention further relates to mateine answering in skin-whitening, resisting skin allergy, skin anti-inflammatoryWith.
Some preferred embodiment in, it is described the invention mainly relates to the skin-whitening application of mateineSkin-whitening acts through suppression tyrosinase and realized.
Some preferred embodiment in, the invention mainly relates to the resisting skin allergy application of mateine, instituteState resisting skin allergy and act through suppression hyaluronidase realization.
Some preferred embodiment in, it is described the invention mainly relates to the skin inflammatory applications of mateineSkin antiinflammatory action discharges NO realizations by suppressing macrophage.
Detailed description of the invention
The invention discloses a kind of preparation method of mateine.Its specific extraction process includes：Extract, decoct,Refluxing extraction, ultrasonic extraction, Microwave Extraction, Dynamic Extraction etc..
In some embodiments, Ilex paraguarensis raw material can be pre-processed before extraction.For example, pretreatment mode canWith including crushing, degreasing etc..
In general, the extraction process of Ilex paraguarensis is as follows：By Ilex paraguarensis (for example, Fujian Ya Tonglv gardens Tea Industry Co., LtdIlex paraguarensis) after pretreatment (if any), add appropriate solvent, extracted, remove slag and take extract solution, concentrate, be made oneDetermine the Ilex paraguarensis concentrate of crude drug concentration.
In some specific embodiments, mateine is prepared using solvent extraction method.It is for instance possible to useSolvent include hydrophilic solvent and its mixed solvent such as deionized water, methanol, ethanol, acetone, propane diols, butanediol, glycerineDeng.
In some specific embodiments, mateine is prepared by decocting extraction method using deionized water.After extraction is decocted, then filtering carries out alcohol precipitation with ethanol.Some preferred embodiment in, using 70% ethanolCarry out alcohol precipitation.It is then centrifuged for, takes supernatant to can obtain mateine after being concentrated under reduced pressure.
In some specific embodiments, mateine is prepared using solvent extraction method.It is for instance possible to useEthanol is extracted.Some preferred embodiment in, extracted using 70% ethanol.Then, filtered, filtrateAfter being concentrated under reduced pressure, centrifugation.Supernatant is taken afterwards, and mateine is can obtain after filtering.
In some specific embodiments, mateine is prepared using ultrasonic extraction.It is for instance possible to useDeionized water carries out ultrasound under conditions of 50 DEG C.Then, filtered, after filtrate decompression concentration.Then, alcohol is carried out with ethanolIt is heavy.Some preferred embodiment in, using 70% ethanol carry out alcohol precipitation.Then filter, after filtrate decompression concentration, filteringIt can obtain mateine.
Present invention firstly discovers that mateine has the effect for suppressing tyrosinase activity.Therefore, Ilex paraguarensis is extractedThing can be added in cosmetics as the additive with white-skinned face function, effectively suppressed the increase of melanin, played skin whiteningOr desalinate the effect such as freckle.
In some embodiments, concentration is used for 1-1000ug/ml mateine to suppress tyrosine enzyme activityProperty.Some preferred embodiment in, tyrosinase activity is suppressed using 1-100ug/ml mateine.AnotherIn some preferred embodiments, tyrosinase activity is suppressed using 50-60ug/ml mateine.
Present invention firstly discovers that mateine has the effect for suppressing hyaluronidase activity.Therefore, Ilex paraguarensis is carriedTake thing can act on antiallergic act on additive add cosmetics in, maintain cell or tissue in hyaluronic acid content andNormal function, keeps epidermal cell elasticity and normal function, moisturizing and antiallergic is played a part of to skin.
In some embodiments, concentration is used for 0.1-1000mg/ml mateine to suppress hyaluronic acidEnzymatic activity.Some preferred embodiment in, hyaluronidase is suppressed using 0.1-100mg/ml mateineActivity.Some preferred embodiment in, hyaluronidase activity is suppressed using 1-100mg/ml mateine.Some preferred embodiment in, hyaluronidase activity is suppressed using 10-100mg/ml mateine.
Present invention firstly discovers that mateine, which can reduce or suppress lipopolysaccharides stimulating expression of macrophage, discharges an oxidationNitrogen.Therefore, mateine can be added in cosmetics as the additive with antiinflammatory action, significantly suppress LPS thornsA large amount of releases of the macrophage to NO after swashing, thus suppress or the reaction that reduces inflammation generation, play a part of anti-inflammatory.
In some embodiments, concentration is used to be released for 1-1000 μ g/ml mateine to suppress nitric oxidePut.Some preferred embodiment in, nitric oxide releasing is suppressed using 10-500 μ g/ml mateine.Some preferred embodiment in, nitric oxide releasing is suppressed using 100-500 μ g/ml mateine.At someIn most preferred embodiment, nitric oxide releasing is suppressed using 400 μ g/ml mateine.
In another aspect of this invention there is provided a kind of skin preparations for extenal use, the skin preparations for extenal use is included according to the present inventionWhat method was prepared includes mateine and the acceptable excipient of cosmetic field.
The skin preparations for extenal use is typically used for the general designation concept of all the components outside skin, for example, can be cosmetic preparationComposition or pharmaceutical compositions.Can be foundation make up material, facial dressing cosmetic preparation, body in the cosmetic combinationMakeup material, hair nursing cosmetic preparation etc., can reasonable selection according to different purposes to its formulation without specifically limited.
Bu Tong also allowing containing different cosmeceutical aspects according to formulation and purpose in the cosmetic combinationMedium or matrix excipients.
It can be used for cosmetics, dermatology or the pharmaceutically acceptable excipient of Dermatologic preparation composition of the present inventionFor the form of aqueous phase, oil phase, gel, water bag wax pattern emulsion, emulsion oil-in-water or water-in-oil emulsion.Aqueous phase is a kind of or manyThe mixture of water-soluble or dispersed component is planted, it can be liquid, semi-solid or solid under room temperature (25 DEG C).Excipient bagInclude or can be the suspension in water or water-alcohol excipient, the form of dispersion liquid or solution, it can contain thickener or solidifyingJelly.The suitable product form of knowledge-chosen that those skilled in the art can be grasped based on those skilled in the art, wherein wrappingThe component contained.
Described composition can include aqueous phase, and the aqueous phase can contain water or water and at least one hydrophilic organic solventMixture, described hydrophilic organic solvent such as alcohol, the rudimentary unitary of straight or branched especially containing 2-5 carbon atomAlcohol, such as ethanol or propyl alcohol；Polyalcohol, such as propane diols, sorbierite, glycerine, panthenol or polyethylene glycol and its mixture.
When the composition of invention is emulsion form, said composition can also optionally include surfactant.
Described composition can also include film forming polymer, such as polyurethanes, polyacrylic acid homopolymer or copolymerizationThing, polyester, resin and/or silicone resin based on hydrocarbon.It can dissolve a polymer in or be scattered in the acceptable tax of cosmeticsOptionally merge in shape agent and with plasticizer.
The composition of the present invention can also include oil phase, and described oil phase contains under room temperature (25 DEG C) for the oily molten of liquidProperty or oil-dispersing property component and/or at room temperature be oily or wax-like material, such as wax, semisolid, natural gum and its mixture.ShouldOil phase can also contain organic solvent.
Generally it is liquid at room temperature, suitable oily matter includes：From the oil based on hydrocarbon of animal, such as perhydrogenatingSqualene；The triglyceride of the C4-10 aliphatic acid of vegetable oil based on hydrocarbon, such as liquid, such as enanthic acid or Trivent OCGClass, or oil, such as sunflower oil, corn oil, soybean oil, grape-kernel oil, castor oil, avocado oil, caprylic/capric triglycerideClass, jojoba oil；The straight or branched hydro carbons of mineral or synthesis source, such as liquid paraffin and its derivant, vaseline；SynthesisThe esters of esters and ethers, particularly fatty alcohol, such as isopropyl myristate, palmitic acid 2- ethylhexyls, stearic acid 2- are pungentBase dodecyl ester, isostearyl isostearate ester；Hydroxylating esters, such as lactic acid isooctadecanol ester, octyl hydroxystearate,Octyl hydroxystearate, hydroxy stearic acid octyldodecyl, the enanthic acid esters of fatty alcohol, sad esters and capric acid lipid；Polyalcohol esters, such as propylene, the heptanoate of neopentyl glycol two, the isononoate of diethylene glycol (DEG) two and pentaerythritol esterClass；Aliphatic alcohols containing C12-26, such as octyldodecanol, 2- butyl octanol, 2- hexyl decyl alcohols, 2- undecyls 15Alkanol, oleyl alcohol；Fluorocarbon oil and/or fluorosilicon oil based on part hydrocarbon, silicone oil is at room temperature liquid or semisolid volatility or non-Volatile straight chain or cyclo-methicone, such as X 2-1401 and dimethyl silicone polymer, its is optionalInclude phenyl, such as Silicone DC 556, siloxanes and its mixture.
The composition of the present invention can be further comprising any component being usually used in cosmetic field.These components includePreservative, water phase thickener (extract biopolymer, synthetic polymer) and fatty phase thickener, aromatic, hydrophily andLipophilic active agent and its mixture.
The composition of the present invention can also include other particle phase, and described particle mutually can be in cosmetic compositionThe pigment and/or pearling agent and/or filler used.
Pigment may reside in composition, and suitable inorganic pigment includes titanium oxide, zirconium oxide and cerium oxide and oxygenChange zinc, iron oxide and barba hispanica；Suitable organic pigment includes barium, strontium, calcium and aluminum lake and carbon black.
Pearling agent may reside in composition, and suitable pearling agent includes being coated with titanium oxide, iron oxide or natural faceThe mica of material.
Filler may reside in composition, and suitable filler includes talcum powder, silica, zinc stearate, cloudMother, kaolin, nylon powder, polyethylene powders, Teflon, starch, borazon, copolymer microsphere, such as silicone resinMicroballon.
The oil phase of the present composition can include one or more waxes, natural gum or its mixture.Wax is included based on hydrocarbonWax, fluorine wax and/or siloxane wax, and plant, mineral, animal and/or synthesis source can be derived from.Suitable wax includes honeybeeWax, Brazil wax, candelila wax, paraffin, microwax, ceresine；Synthetic wax includes Tissuemat E, the siloxanes containing C16-45Wax.Natural gum is generally dimethyl silicone polymer or sodium carboxymethylcellulose or extracts species, and semi-solid material is generally baseIn the compound of hydrocarbon, such as lanolin and its derivative.
The composition of the present invention can be configured to any suitable product form.This kind of product form includes, but does not limitIn aerosol spray, creme, emulsion, solid, liquid, dispersion, foam, gel, toner, mousse, ointment, pulvis,Patch, brilliantine, solution, hand press pump-type spray, club, facial mask and hygenic towelette.The composition of the present invention can be passed throughVarious methods well-known in the art are advantageously available for preparing or produced as cosmetics, dermatology or medicine local applicationProduct.
The Dermatologic preparation composition of the present invention can include one or more following ingredients：It is anti-allergic agent, antimicrobialAgent, antioxidant, chelating agent, colouring agent depigmentation, emollient, emulsifying agent, excoriation agent, film forming agent, spices, moisturizingIt is agent, insect repellent, lubricant, pharmaceutically active agents, humidizer, photostabilizer, preservative, skin conditioner, skin penetration enhancer, anti-Shine agent, stabilizer, surfactant, thickener, viscosity modifier, vitamin or its any combination.
Embodiment
Present inventors discovered unexpectedly that：Mateine can effectively suppress the activity of tyrosinase, fromAnd can effectively suppress the increase of melanin, play the effect such as skin whitening or desalination freckle；Mateine can also haveEffect ground suppresses the activity of hyaluronidase, so as to maintain the content and normal function of hyaluronic acid in cell or tissue, keeps tableChrotoplast elasticity and normal function, moisturizing and antiallergic are played a part of to skin；Mateine can significantly suppress LPSA large amount of releases of the macrophage to NO after stimulation, thus suppress or the reaction that reduces inflammation generation, play a part of anti-inflammatory.
The present invention is expanded on further with reference to specific embodiment.It should be appreciated, however, that these embodiments are only used forIt is bright the present invention and be not meant to limit the scope of the invention.The test method of unreceipted actual conditions in the following example, generallyAccording to normal condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, all percentage and number be by weightMeter.
Embodiment 1：The preparation of mateine
Weigh Ilex paraguarensis appropriate, measure deionization water boiling and extraction 2 times with 15 and 10 times respectively, filtering, filtrate decocting and concentratingTo 1 times of crude drug amount, 70% ethanol alcohol precipitation, high speed centrifugation takes supernatant to be concentrated under reduced pressure, and produces mateine, crude drug concentration：1.0g/g, solids content：20.65%.
Embodiment 2：The preparation of mateine
Weigh Ilex paraguarensis appropriate, extracted with 10 times of 70% alcohol at normal temperature of amount, filtering, filtrate decompression is concentrated into 1 times of crude drug amount,High speed centrifugation, takes supernatant, filtering, produces mateine, crude drug concentration：1.0g/g, solids content：18.51%.
Embodiment 3：The preparation of mateine
Ilex paraguarensis is crushed, sieving, and with 10 times of amount 50 DEG C of deionized water ultrasonic extractions 2 times, each 30min (expands auspicious grand circulationUltrasonic extractor, model：TGCXN-2B, power 900W, ultrasonic transformer parameter phi=10), filtering, filtrate decocting and concentrating is to about 1 timesCrude drug amount, 70% ethanol alcohol precipitation, filtering, filtrate decompression concentration, filtering produces mateine, crude drug concentration：1.0g/g,Solids content：19.48%.
Ilex paraguarensis obtained by Example 1-3 extracts concentrate as efficacy in vitro test sample, and small size is dispensed after -20DEG C preserve.
Embodiment 4：Influence to tyrosinase activity
Instrument and reagent：
1st, pH 6.8 phosphate buffer
1. 0.2M disodium hydrogen phosphates (analysis pure)：71.63g is dissolved in 1000ml distilled water and shakes up set aside for use；
2. 0.1M citric acids (analysis pure)：21.04g is dissolved in 1000ml distilled water and shakes up set aside for use；
3. the preparation of the phosphate buffers of pH 6.8：0.2M disodium hydrogen phosphate 154.5ml are taken, 0.1M citric acids are added45.5ml, mixes, is configured to 200ml phosphate buffer.
2nd, tyrosinase solution (Sigma)：The solution that concentration is 1mg/50ml is configured to the phosphate buffers of pH 6.8.
3rd, L-DOPA solution (Sigma)：The solution that concentration is 1g/100ml is configured to the phosphate buffers of pH 6.8.
4th, ultraviolet specrophotometer.
5th, cuvette：10mm.
Sample cell (T), sample controls pipe (T are set up in this experiment respectively0), positive control pipe (C) and negative control pipe (C0)。Each sample solution for adding 1.0ml corresponding concentrations, positive control pipe and negative control Guan Ze in sample cell and sample controls pipeIt is separately added into 1.0ml pH6.8 phosphate buffer；Each 0.5ml concentration that adds is 1mg/ in sample cell and positive control pipe50ml tyrosinase solution (with buffer), sample controls pipe is slow with 0.5ml pH6.8 phosphoric acid with negative control pipeFliud flushing is replaced.All reaction tubes are shaken, each reaction solution is fully mixed, respectively the 1% of addition 2.0ml in each reaction tubeL-DOPA (with buffer), is reacted 10 minutes under the conditions of 30 DEG C, and the OD value of each reaction tube is determined at 475nm(OD)。
Table 1：Influence of the different embodiment samples under various concentrations to tyrosinase activity
Note：Inhibitory activity against tyrosinase (%)=(1 ﹣ (T ﹣ T0)/(C ﹣ C0)) × 100%
In formula：T：Sample cell OD；T0：Sample controls pipe OD；C：Positive control pipe OD；C0：Negative control pipe OD.
Tyrosinase activity body outer suppressioning experiment result shows (table 1) that embodiment 1-3 sample is right under various concentrationsTyrosinase activity has a different degrees of inhibitory action, wherein the sample of embodiment 1 when activity is 57ug/mL to tyrosineThe maximum inhibition highest of enzyme, reaches 67.70%.Illustrate that there is embodiment 1-3 sample tyrosinase activities certain suppression to makeWith.
Embodiment 5：Influence to hyaluronidase activity
Instrument and reagent：
1st, 0.1M acetic acid buffer solutions (pH 3.5)：0.1N acetic acid (analysis is pure) 16ml+0.1M sodium acetates (analysis is pure)1ml。
2nd, hyaluronidase (Sigma) solution：The 0.1M acetate buffers (pH 3.5) of 6000u/ml ox hyaluronidases.
3rd, Sodium Hyaluronate (cosmetics-stage) solution：0.24g is dissolved in 100ml pH3.5 acetate buffer.
4th, 0.4N sodium hydroxides (analysis is pure) solution：0.4g sodium hydroxides are dissolved in 25ml water.
5th, 0.4M potassium borates (analysis is pure) solution：30.55g potassium borate/250ml.
6th, dimethylaminobenzaldehyde (analysis is pure) solution：1g dimethylaminobenzaldehydes/350ml acetate buffers
7th, ultraviolet specrophotometer.
8th, cuvette：10mm.
Sample cell (T), sample controls pipe (T are set up in this experiment respectively0), positive control pipe (C) and negative control pipe (C0)。In sample cell (T) and sample controls pipe (T0) in each sample solution for adding 400ul corresponding concentrations, positive control pipe (C) and the moonProperty control tube (C0) in replaced with the buffer solution of equivalent；50ul hyaluronidase solutions are added in sample cell and positive control pipe to mixEven, the acetate buffer that sample controls pipe and negative control pipe add 50ul pH3.5 is mixed, 37 DEG C of water-baths 20 minutes；Each reactionPipe is separately added into sodium hyaluronate solution 350ul, 37 DEG C of water-baths 40 minutes；Each reaction tube is separately added into 0.4N sodium hydroxides again100ul and 0.4M potassium borate 100ul, boiling water is heated 3 minutes, is cooled to room temperature；It is separately added into dimethylaminobenzaldehyde solution3ml, 37 DEG C of water-baths 20 minutes.Each reaction solution is moved into cuvette, the colorimetric estimation light at spectrophotometer 585nm wavelengthDensity value (OD).
Table 2：Influence of the different embodiment samples under various concentrations to hyaluronidase activity
Note：Hyaluronic acid enzyme inhibition rate (%)=(1 ﹣ (T ﹣ T0)/(C ﹣ C0)) × 100%
In formula：T：Sample cell OD；T0：Sample controls pipe OD；C：Positive control pipe OD；C0：Negative control pipe OD.
Hyaluronidase activity body outer suppressioning experiment result shows (table 2) that embodiment 1-3 samples are under various concentrations to saturatingThe sour enzyme of bright matter has different degrees of inhibitory action, and the wherein sample of embodiment 1 has certain to the inhibitory action of hyaluronidaseConcentration dependant sexual intercourse；Inhibitory action highest of the sample of embodiment 1 when concentration is 100mg/ml to hyaluronidase activity,Reach 69.5%.Illustrate that embodiment 1-3 samples have good inhibiting effect to hyaluronidase activity.
Embodiment 6：The influence of nitric oxide (NO) is discharged to lipopolysaccharides stimulating expression of macrophage
This experiment carries out experiment detection using the mononuclear macrophage strains (ATCC) of mouse RAW 264.7, is gone out using containing 10%The DMEM nutrient solutions (HyClone) of hyclone (FBS, HyClone) living, 100U/ml penicillin and 100 μ g/ml streptomysins,37 DEG C, 5%CO2Cultivate, pass on 1 time in incubator within every 3 days.Detect the given the test agent of various concentrations to macrophage using CCK-8 methodsThe survival rate influence of cell, as a result shows：Each given the test agent is free of toxic effects to macrophage under experimental concentration.
Raw264.7 cells are inoculated in 96 orifice plates, are put into adherent 4h in incubator, add various concentrations given the test agent, addBacteria lipopolysaccharide (LPS, Sigma) (the μ g/ml of final concentration 10), separately sets corresponding cell controls group and stimulates control group(RAW264.7 cells add above-mentioned concentration LPS), in 37 DEG C, 5%CO2Cultivated in incubator after 24h, after supernatant is centrifuged,Supernatant is taken to freeze, it is to be detected.
NO burst size is detected using Griess methods.Take 50 μM of NaNO2Titer, which half-and-half dilutes, amounts to 7 concentration, each holeIt is middle to add the Standard Applying Solution or each 50 μ l of testing sample diluted respectively, 50 μ l Griess reagent As are added into each hole(85% concentrated phosphoric acid 6ml, deionized water 10ml, anhydrous p-aminobenzene sulfonic acid 1.0g, fully dissolve and be settled to 100ml), in 37Reacted 10 minutes in DEG C incubator, 50 μ l reagents B (0.1mg/ml N-1- naphthodiamide hydrochloric acid saline solution) added into each hole,Reacted 10 minutes in 37 DEG C of incubators.96 orifice plates are gently vibrated for several times, after each hole reaction solution is mixed completely, in 540nm ripplesThe OD values in long each hole of detection, according to gained OD values, are fitted NO reaction normal curves, and calculate each testing sample group by standard curveIn NO contents.
Table 3：Different embodiment samples discharge NO influence under various concentrations to RAW264.7 cells
Discharge NO influence result to RAW264.7 cells under various concentrations from different embodiment samples, it is different realThe macrophage RAW264.7 releases NO that apply a sample stimulates LPS under various concentrations has different degrees of inhibitory action,Wherein the sample of embodiment 1 discharges NO inhibiting rate maximum when concentration is 400 μ g/ml to macrophage, reaches 82.6%.ExplanationEmbodiment 1-3 samples can effectively suppress macrophage release NO, with certain antiinflammatory action.
Embodiment 4-6's test result indicates that, mateine can effectively suppress the activity of tyrosinase so thatThe increase of melanin can effectively be suppressed, the effect such as skin whitening or desalination freckle is played；Mateine can also be effectiveGround suppresses the activity of hyaluronidase, so as to maintain the content and normal function of hyaluronic acid in cell or tissue, keeps epidermisCell elasticity and normal function, moisturizing and antiallergic are played a part of to skin；Mateine can significantly suppress LPS thornsA large amount of releases of the macrophage to NO after swashing, thus suppress or the reaction that reduces inflammation generation, play a part of anti-inflammatory.
The mateine prepared in Example 1-3, the preparation for skin preparations for extenal use.The skin preparations for extenal use is excellentCosmetic composition is elected as, such as toner, Essence, creams.The consumption of the mateine is 0.001%-20% (w/w).It is preferred that percentage by weight be 0.01%-20% (w/w).Preferred percentage by weight is 0.01%-10%(w/w).Most preferred percentage by weight is 0.1%-5% (w/w).
The following is the embodiment of the concrete application in the skin preparations for extenal use containing mateine, and its these formulationsFormula and preparation method."-" represents no added in following table.
Embodiment 7：The preparation of face cream
Embodiment 8：The preparation of emulsion
Embodiment 9：The preparation of gel
Embodiment 10：The preparation of toner
Embodiment 11：The preparation of Essence
Embodiment 12：The preparation of facial mask
Embodiment 13：The preparation of eye cream
Embodiment 14：The preparation of aerosol (cleaning bubble)
Embodiment 15：The preparation of spraying
Embodiment 16：The preparation of shower cream
Embodiment 17：The preparation of mildy wash