CN106266587B - Orchid extract and its preparation method and application - Google Patents

Abstract

The present invention is a kind of orchid extract and its preparation method and application, wherein the orchid is Phalaenopsis, and the orchid extract is obtained as prepared by the following steps: (1) extracting: the orchid and solvent being carried out co-grinding or broken wall obtains orchid slurry；Wherein the solvent is at least one of or mixtures thereof water or alcohols；(2) it is separated by solid-liquid separation: the orchid slurry of step (1) being separated by solid-liquid separation, and impurity is removed, liquid is left and obtains orchid filtrate；(3) activity divides: by the orchid filtrate of step (2) with specific molecular sieve film processing purifying, obtaining orchid extraction screening liquid；(4) it is concentrated: the orchid extraction screening liquid of step (3) being handled into concentration or vacuum with molecular sieve film or boiling mode carries out partial concentration, obtains active sludge and active liquid reactive extract liquor.Active sludge of the invention and active liquid reactive extract liquor have whitening function, resistance to oxidation and crease-resistant (anti-aging) ability.

Description

Orchid extract and its preparation method and application

Technical field

The present invention relates to chemicals fields, are especially to provide a kind of orchid extract and its preparation method and application, should Orchid extract has the effect of whitening function, anti-oxidant and crease-resistant (anti-aging).

Background technique

The color of normal skin depends on: 1, in skin various pigments content and distribution situation；2, blood in skin blood The redox ratio of Lactoferrin；3, the scattering of the thickness of skin and light in skin surface.Due to the influence of external environment The air of pollution (ultraviolet light), incorrect life style (incorrect beauty, cosmetics inferior, electronic display spoke Penetrate), the aging (immunocompetence decline, microcirculation disappearance, metabolic capability reduce) of human skin can all cause skin chromogenesis Deposition, Magnifying chromoscopy are uneven, skin tarnishes generate it is low and deep.Melanocyte (Melanocytes) is located at skin base layer It into the cell, is single celled secretory, cell is rounded, and for melanocyte by growing and the tubular protrusion of branch, these are prominent It rises and shuttles in epidermal cell.Melanin is formed in the deep layer of epidermis, and is migrated to the surface of skin, but melanocyte sheet Body can't be migrated and then, but be responsible for shifting the work of melanin by horn cell, and horn cell goes to these melanin The surface of skin, to form natural protective barrier.

Tyrosinase activity in melanocyte can be promoted under ultraviolet light by getting sun or exposing, and increase melanin production Raw, skin generates spontaneous chemical reaction and ferment via the synthesis path (Raper-Mason pathway, Fig. 2) of melanin Catalytic action and form melanin.Melanin is a kind of high molecular complex, and when skin is irradiated by ultraviolet light, melanin is thin Tyrosinase (tyrosinase) intracellular can be activated, and Tyrosine (tyrosine) in activated cell is accelerated to be oxidized to dihydroxy Phenylpropylamine acid (dihydroxyphenylalanine, abbreviation DOPA), and dihydroxy phenyl alanine (DOPA) is again by tyrasamine Sour enzymatic is DOPA quinone (dopachrome), and the GCMS computer path of melanin involves the tyrasamine acid catalysis by Tyrosinase Hydroxyl turns to L-3, and 4- dihydroxy phenyl alanine (L-3,4-dihydroxyphenylalanine, L-DOPA) and L-DOPA turn Become DOPA quinone (dopachrome)；And DOPA quinone is to be changed into melanin via oxidation reaction, it is anti-through a series of oxidation It answers, eumelanin (eumelanin) is formed by DOPA quinone.

Tyrosinase (tyrosinase) is a kind of polyphenol oxidase of copper ions, is synthesized by melanocyte, is lived Property can be because source be different and different, the active binding site of Tyrosinase and Tyrosine (tyrosine) have active at three Center copper ion, research now is related to be inhibited on the active machine of Tyrosinase turns, and there are mainly two types of modes: (1) Tyrosinase Deactivate: this reaction machine turns to be primarily due on ferment molecular structure with copper ion, and mortifier is used to be chelated using similar copper The mode of agent (copper-chelating agent) chelates copper ion or vies each other with copper ion, uses reduction copper ion Binding force, and then reduce the activity of Tyrosinase, to block melanin production mechanism, this inhibits the ingredient that most represents of type For kojic acid (kojic acid).(2) competitive to replace Tyrosinase by matter: this reaction machine switchs to mortifier and Tyrosine or DOPA Deng being vied each other by matter and Tyrosinase is reacted, cause to be reduced with what Tyrosinase acted on by matter, and reduces melanin shape At it includes hydroquinone (hydroquinone), Arbutin that this competitiveness, which replaces by the ingredient that matter type most represents, (arbutin) etc..

Whitening is one of the cosmetics function of most being paid attention to by Asia women.Traditionally, whether assessment cosmetic material Potentiality with whitening mainly have following several modes:

(1) whether Tyrosinase is suppressed: since Tyrosinase is the important ferment that melanin is formed, if this ferment Element is suppressed, then the formation of melanin is likely to reduced (Fig. 2).

(2) K-1735 of mouse is united (B16 cell line): since raw material (or ingredient) is wanted effectively to inhibit Tyrosinase, then this effect can be reached by needing to can enter cell in the premise of the ingredient mechanism of action.Therefore also there is scholar with mouse K-1735 system (B16 cell line) be used as mode, inquire into ingredient whether have inhibit melanocyte growth Or the whitening potentiality for inhibiting melanin to be formed.But B16 cell line is the tumor cell line of melanocyte, and melanin generates Amount and ingredient to the attainable inhibitory effect of B16 cell institute, it is often different from the integumentary system of Healthy People.

(3) human melanoma's cell (human melanocyte) of Initial culture (primary culture): due to being The Initial culture of the melanocyte of the mankind, therefore closest to the true Skin Cell system of human body.Would generally with it is harmless at Maximum dose (such as constituent concentration that 95% cell survival can be maintained) is divided whether may be used to melanocyte generation melanin The test screened using reaching inhibitory effect as whitening.

Three of the above mode can see the ingredient for having obvious whitening effect and few in most cases；If any such effect Fruit, then showing the ingredient really has preferable whitening potentiality.

Competent authority, various countries audit new cosmetics function raw material it is whether mutatis mutandis when, also to the security assessment result of raw material Extremely pay attention to.Cosmetics new raw material, which needs the safety evaluation that does, to be carried out in a manner of zoopery, wherein most It is important and substantially be skin irritation, skin corrosivity and hypersensitive test etc..But European Union is in July, 2013 Implement disabling animal experiment (animal ban), it is specified that forbidding peddling (Marketing ban) with the product of animal testing, so It is international to have begun a large amount of money of investment and time in substitution zoopery (Alternative testing), endeavour out Hair substitution zoopery method, that is, with the experimental method that so-called 3D mankind's bionics skin tissue and OECD are approved come into The security evaluation of row cosmetic material is tested.This research take the lead in equipment Imported From German EMH Company 3D human skin tissue carry out cosmetics The skin irritation (OECD 439) and skin corrosivity of raw material test (OECD 431).Separately also tests plus do with human clinical The test of hypersensitive, and verify the skin irritation and skin corrosivity test result of 3D skin.

Due to that need to take into account the safety and validity of functional component, current Department of Health is announced in a manner of front table column Workable whitening composition and its concentration, these ingredients include kojic acid (kojic acid), Arbutin (arbutin), ellagic acid (ellagic acid) and chamomile extract object (chamomile ET) etc., are detailed in the following table 1.From the point of view of table 1, workable whitening Ingredient is not more, and acid is mostly containing the excitement of local use.In recent years since Japanese beauty's treasured company uses Du The problem of cuckoo flower alcohol causes consumer's hickie disease, therefore find safety and whitening effective and with substantial application Cosmetic raw material, the cosmetics new raw material even simultaneously with other function characteristic other than whitening, just at many dealers research Target.

Table 1

Summary of the invention

Based on this, it is necessary to provide a kind of orchid extract and its preparation method and application.

The purpose of the present invention is to provide a kind of orchid extract, wherein the orchid is Phalaenopsis, the orchid Extract is obtained as prepared by the following steps: (1) it extracts: by the orchid and solvent with weight ratio for 0.5:1~10:1, into Row co-grinding or broken wall obtain orchid slurry；Wherein the solvent include water or alcohols (alcohols, such as: ethyl alcohol, propyl alcohol, fourth At least one of or mixtures thereof alcohol)；(2) it is separated by solid-liquid separation: step (1) the orchid slurry being separated by solid-liquid separation, by impurity Removal, leaving liquid is orchid filtrate；(3) activity divides: step (2) the orchid filtrate is handled with " molecular sieve " film (thin film manufacture process) purifying obtains orchid extraction screening liquid；(4) it is concentrated: by step (3) the orchid extraction screening liquid with " molecule Sieve " film processing concentration or other vacuum or boiling mode carry out partial concentration, obtain active sludge and active liquid State reactive extraction liquid.

The orchid is to be selected from Phalaenopsis Sogo Yukidian kind " rising sun Dong Weishi in one of the embodiments, Avoid " (Phalaenopsis Sogo Yukidian ' Shiuh-Dong Whishkey '), which is Plant variety right certificate product Kind power word the A00595th, is derived from green hill orchid Biotechnology Co., Ltd.

Orchid described in step (1) and the preferred solvents are with the side of weight ratio 5:1 in one of the embodiments, Formula is extracted (mashing).

The temperature of separation of solid and liquid described in step (2) is 0~60 DEG C in one of the embodiments,.

The temperature of affiliated separation of solid and liquid is 0~35 DEG C in step (2) in one of the embodiments,.

Centrifugal process or filtration method, the filtration method can be used to adopt when step (2) is separated by solid-liquid separation in one of the embodiments, Filtering material is that diatomite, perlite, filter paper, filter cloth and (0.1~5 μm) of filtering element (cartridge) are at least one.

In one of the embodiments, step (3) activity divide and step (4) described concentration described at film Reason adopts two sections of steps: first segment uses inorganic ceramic membrane or organic hybrid films；Second segment using nanofiltration membrane (NF, ) or reverse osmosis membrane nanofiltrationmembrane.

The inorganic ceramic membrane is pellumina, zirconium oxide film or oxidation single in one of the embodiments,.

The organic hybrid films are PS membrane, cellulose acetate film, polytetrafluoroethylene film or poly- in one of the embodiments, Propylene film.

The nanofiltration membrane or reverse osmosis membrane can be single film or laminated film in one of the embodiments, and material is Composite membrane (TFM, thin film membrane), Kynoar (PVDF), polysulfones (PS), polyether sulfone (PES) and polypropylene At least one of nitrile (PAN), wherein the pressure of second segment separation is 150-400psi, optimum pressure 200-300psi.

Step (4) the active sludge Structural Identification is with nuclear magnetic resonance (NMR) and matter in one of the embodiments, Spectrometer (MS) carries out analyzing and identifying for chemical structure.

Another object of the present invention is to provide a kind of medical composition, comprising a effective amount of above-mentioned liquid reactive extract liquor, Active sludge (compound) or combinations thereof and its pharmaceutically acceptable excipient.

The purposes for having the drug of whitening function is used to prepare another object of the present invention is to provide a kind of orchid extract, Wherein the orchid extract is above-mentioned liquid reactive extract liquor, active sludge or combinations thereof.

The orchid extract is to be used to prepare the whitening medicaments for inhibiting Tyrosinase or work in one of the embodiments, Property ingredient.

In one of the embodiments, the orchid extract be used to prepare inhibit melanin formed whitening medicaments or Active constituent.

Another object of the present invention is to provide a kind of orchid extracts to be used to prepare the medicine that treatment free radical causes disease The purposes of object, wherein the orchid extract is above-mentioned liquid reactive extract liquor, active sludge or combinations thereof.

The orchid extract is to be used to prepare the drug or activity of quickly removing free radical in one of the embodiments, Ingredient.

Another object of the present invention is to provide the purposes that a kind of orchid extract is used to prepare crease-resistant (anti-aging) drug, Wherein the orchid extract is above-mentioned liquid reactive extract liquor, active sludge or combinations thereof.

The orchid extract is to be used to prepare treatment collagen to lack diseases caused in one of the embodiments, Drug or active constituent.

The orchid extract is to be used to prepare to inhibit the crease-resistant of matrix metalloproteinase in one of the embodiments, (anti-aging) drug or active constituent.

It is used to prepare when the orchid extract in one of the embodiments, and inhibits the crease-resistant (anti-ageing of Collagenase Change) drug or active constituent.

The orchid extract is to be used to prepare to inhibit the crease-resistant (anti-ageing of elastoser in one of the embodiments, Change) drug or active constituent.

Another object of the present invention is to provide a kind of orchid extracts, wherein the orchid is Phalaenopsis, the orchid Extracts are obtained as prepared by the following steps: (1) it extracts: by the orchid and solvent with weight ratio for 0.5:1~10:1, It carries out co-grinding or broken wall obtains orchid slurry；Wherein the solvent be water or alcohols (alcohols, such as: ethyl alcohol, propyl alcohol, fourth At least one of or mixtures thereof alcohol)；(2) it is separated by solid-liquid separation: step (1) the orchid slurry being separated by solid-liquid separation, by impurity Removal, leaving liquid is orchid filtrate.

Another object of the present invention is to provide a kind of orchid extracts, wherein the orchid is Phalaenopsis, the orchid Extracts are obtained as prepared by the following steps: (1) it extracts: by the orchid and solvent with weight ratio for 0.5:1~10:1, It carries out co-grinding or broken wall obtains orchid slurry；Wherein the solvent be water or alcohols (alcohols, such as: ethyl alcohol, propyl alcohol, fourth At least one of or mixtures thereof alcohol)；(2) it is separated by solid-liquid separation: step (1) the orchid slurry being separated by solid-liquid separation, by impurity Removal, leaving liquid is orchid filtrate；(3) activity divides: step (2) the orchid filtrate is pure with the processing of " molecular sieve " film Change, obtains orchid extraction screening liquid；(4) it is concentrated: step (3) the orchid extraction screening liquid is dense with the processing of " molecular sieve " film Contracting or other vacuum or boiling mode carry out partial concentration, obtain active sludge and active liquid reactive extract liquor.

Another object of the present invention is to provide a kind of methods for preparing orchid active extract, wherein the orchid is butterfly Phalaenopsis category comprises the steps of (1) extraction: the orchid and solvent is mixed with weight ratio for 0.5:1~10:1 It crushes or broken wall obtains orchid slurry；Wherein the solvent is water or alcohols (alcohols, such as: ethyl alcohol, propyl alcohol, butanol) or it is mixed Close at least one of object；(2) it is separated by solid-liquid separation: step (1) the orchid slurry being separated by solid-liquid separation, impurity is removed, is left Liquid is orchid filtrate；(3) activity divides: step (2) the orchid filtrate being handled with " molecular sieve " film and is purified, orchid is obtained Flower extraction screening liquid；(4) it is concentrated: step (3) the orchid extraction screening liquid being handled with " molecular sieve " film and is concentrated, Huo Zheqi His vacuum or boiling mode carry out partial concentration, obtain active sludge and active liquid reactive extract liquor.

Compared with prior art, the invention has the following advantages:

Inventor is studied for many years, it is found that specific orchid (Phalaenopsis) passes through special extraction processing procedure, extraction Object has activity, resistance to oxidation and crease-resistant (anti-aging) ability for inhibiting Tyrosinase simultaneously.This extract at least covers three The similar flavonoids of kind of structure, Tyrosinase, mouse B16 melanocyte and mankind's Initial culture melanocyte, And the effect of whitening, while the analysis through biochemistry and molecular biology are all seen in human experimentation, also find it with anti-oxidant With crease-resistant (anti-aging) effect.This specific orchid can differentiate its difference with other orchids in a manner of base chiller.

Whole process is had the active extract of several functional characteristics with low temperature, the environment-friendly preparation method thereof of water phase simultaneously, For the crystallization with specific flavonoid structure or the reactive extraction liquid containing this flavonoids.The active extract has Tyrosinase Rejection ability, and it is provided simultaneously with the ability for inhibiting melanocyte to generate melanin and anti-oxidant and crease-resistant (anti-aging) Effect, cell experiment and human experimentation confirm its safety in vitro, have actual commodity application.

Detailed description of the invention

Fig. 1 is orchid extraction step process, wherein

110 take orchid flower

Orchid and solvent are carried out co-grinding or broken wall by 120

130 are separated by solid-liquid separation

140 carry out active division

150 are concentrated

160 obtain active sludge and liquid reactive extract liquor；

Fig. 2 by Tyrosinase (tyrosinase) synthesis of melanin path (Raper-Mason pathway)；

Fig. 3 is that the active sludge 1H-NMR of orchid extract analyzes map (Varian 500) (A)；And 13C-NMR It analyzes map (Varian 500) (B)；

Fig. 4 is that the mass spectrograph (mass spectrometry, MS) of the active sludge of orchid analyzes result (A)；And mark The mass spectrograph (mass spectrometry, MS) of quasi- product (Apigenin-6-ribosido-7-glucoside) analyzes result (B)；

Fig. 5 is that the mass spectrograph (mass spectrometry, MS) of the active sludge of orchid analyzes result (A)；And mark The mass spectrograph (mass spectrometry, MS) of quasi- product (Saponarin) analyzes result (B, C)；

Fig. 6 is that the mass spectrograph (mass spectrometry, MS) of the active sludge of orchid analyzes result (A)；And mark The mass spectrograph (mass spectrometry, MS) of quasi- product (Apigenin 7-glucoside) analyzes result (B)；

Fig. 7 is that orchid extracts concentrate progress vitro skin corrosion test result (A)；And orchid extract concentrate into Row vitro skin irritation test result (B)；

Fig. 8 is that orchid extracts concentrate after the ethyl alcohol of various concentration and water extraction, the content (gallic of total polyphenols Acid equivalent, GAE)；

Fig. 9 is that orchid extracts concentrate after the ethyl alcohol of various concentration and water extraction, DPPH free radical scavenging activity (A)；It is blue The free radical scavenging activity (B) of flower extraction concentrate and BHT；Orchid, Lan Ye extract the free radical scavenging ability of concentrate and ABTS (C)；

Figure 10 is orchid filtrate after the ethyl alcohol of various concentration and water extraction, is carried out the assessment of Tyrosinase inhibiting rate (A)； Concentrate (object) is extracted for the orchid of orchid different step not of the same race, with water extraction test Tyrosinase inhibiting rate (B)；For orchid Flower, Lan Ye extraction concentrate test Tyrosinase rejection ability (C)；

Figure 11 is that orchid extracts concentrate or active sludge after the ethyl alcohol of various concentration and water extraction, and melanin is thin Born of the same parents inhibit melanin experimental result；

Figure 12 A to H is the knot that different rising sun east whiskey orchid extract liquors carries out cell survival rate experiment to HMC-4 cell Fruit；

The orchid extract liquor of Figure 13 different rising sun east whiskeys inhibits the test result of melanin performance, and wherein M254 is M254 culture medium, M2 are M2 culture medium, and KA is kojic acid (Kojic acid)；

Figure 14 is that orchid extracts crease-resistant (anti-aging) ability of concentrate test.

Specific embodiment

Orchid extract of the invention and its extracting process and application are made below in conjunction with specific embodiment further detailed Explanation.

All technical and scientific term described in this specification is all the fields unless in addition being defined The meaning that can be understood jointly with usual those skilled in the art.

As used herein, term " extract " refers to by product prepared by extraction.The extract can be with The solution form that is dissolved in solvent is presented or extract can be to be free of or generally not solvent-laden concentrate or essence are presented. As further discussed below, extract is also adjustable assigned in medical composition or food.Term extract can be from specific extraction The single extract or extract that step or a series of extraction steps obtain also can be the extract obtained from independent extraction step Combination.Therefore, which is also covered by term " extract " through combined extract.

" raw material " typically refers to material of vegetable origin as used herein, one or more comprising individual entire plant or plant The combination of a component part, comprising leaf, root (including but not limited to main root, root of the tail and fibrous root), stem, skin, berry, seed and Flower, wherein the plant or component part may include it is original, through drying, through boiling, heated or otherwise through Physical Processing In favor of the material of processing, can further include it is complete, mince, dice, milling, grinding or otherwise processed with shadow Ring the size of the vegetable material and the material of entity integrity.Term " raw material " can be used for the material for indicating to be used for Additional extraction processing procedure Expect the extraction product in source.

In addition, can be Dermatologic preparation composition in the present invention, form in addition to above-mentioned orchid extract, base can be according to need It is suitable for adding for ingredient used in the skin preparations for extenal use such as general cosmetics and pharmaceuticals, such as moisturizer, antioxidant, oil Property ingredient, ultraviolet absorbing agent, interfacial agent, tackifier, alcohols, powdered ingredients, toner, water composition, water, various skins Nutritional agents etc..Also can suitably add other natrium adetates, edetate trisodium, sodium citrate, polyphosphate sodium, sodium metaphosphate, The blocking metal agent of gluconic acid etc.；Caffeine, tannic acid, Pa meters of Wella (verapamil), amine first naphthenic acid and its derivative, Radix Glycyrrhizae extract, glycyrrhizin, water hot extraction's object of firethorn fruit, various crude drugs, Tocopherol acetate ester, glycyrrhizic acid and its derivative Or the medicament of its salt etc.；Other whitenings of vitamin C, magnesium ascorbyl phosphate, ascorbic acid glucoside, arbutin, kojic acid etc. Agent；The carbohydrate etc. of glucose, fructose, mannose, sucrose, trehalose etc..

Dermatologic preparation composition of the invention be for example ointment, creams, lotion, liquid medicine, facial mask (pack), baths, The external preparation for skin dosage form such as creme, as long as can be without its dosage form of special provision for known Dermatologic preparation composition person.

Term " treatment ", " in treatment " and its class term mean to delay, improve, reduce or reverse just torments patient at present The illness or the relevant any symptom of the illness method and the method for preventing the illness or its any symptom just occurred.

Term " pharmaceutically acceptable " means that substance or composition must be compatible with other compositions of composite, and to patient It is harmless.

Term " pharmaceutically acceptable excipient ", person as used herein, it is intended that knowing well can be with orchid known to this skill person The compatible any physiology inertia of the physics and chemical characteristic of extract, pharmacologically not active substance.It is pharmaceutically acceptable Excipient include, but are not limited to polymer, resin, plasticizer, filler, lubricant, diluent, binder, disintegrating agent, molten Agent, totally one solvent, interfacial agent, preservative, sweetener, flavoring agent, the dyestuff of pharmacy grade or pigment and viscosity agent.

Term " medical component (pharmaceutical composition) " be a kind of solid or liquid compositions, Form, concentration and purity level are suitble to administering that can induce after bestowing to sufferer (such as mankind or animal sufferer) and be intended to give birth to Reason variation.Medical component is sterile and non-heat generation person (non-pyrogenic).

Term " effective quantity " as used herein refers to amount necessary to generation expectation biological respinse.Such as they's common skill in the industry Patient it is understood that the effective quantity of compound or bioactivator visually the factors such as following and change: it is expected biological terminal, quasi- Deliver bioactivator, the composition of encapsulation matrix, destination organization etc..

The present invention is to be demonstrated to illustrating with the following examples, but the present invention is not limited by following embodiments.This hair Bright orchid used, raw material, biomaterial is all commercially available is easy to obtain, following merely illustrative obtainable pipeline.

One orchid extract extracting process of embodiment

The extracting process of orchid, step is as shown in Figure 1, taking orchid is Phalaenopsis Sogo Yukidian kind " rising sun Dong Weishi Avoid " (Phalaenopsis Sogo Yukidian ' Shiuh-Dong Whishkey ', hereinafter referred to as rising sun east whiskey), the orchid Flower is that Plant variety right certificate kind weighs word the A00595th, to obtain prepared by the following steps:

(1) it extracts: with weight ratio being 0.5:1~10:1 by the orchid and solvent, preferably 5:1；Time is 3~60 Minute, preferably 10 minutes；Temperature is 0~60 DEG C, preferably 0~35 DEG C；It is carried out using the extractions such as ultrasonic, grinding mode It extracts, preferably ultrasonic, carries out co-grinding or broken wall obtains orchid slurry；The solvent is that water, alcohols or alcohols are water-soluble Liquid, wherein the alcohols is ethyl alcohol, propyl alcohol or butanol.

(2) be separated by solid-liquid separation: the orchid slurry that step (1) is extracted carries out destarch, and the destarch time is 20~60 minutes, preferably It is 30~40 minutes；0~60 DEG C of temperature, preferably 0~35 DEG C；The destarch in a manner of being centrifuged, squeeze etc., preferably it is centrifuged 1000~ 3000rpm；Most preferably it is centrifuged 1500~2000rpm.Leaving liquid is the thick extract liquor of orchid, abandons colored slag；

(3) coarse filtration: carrying out coarse filtration for the thick extract liquor of the orchid of step (2), and diatomite, pearl can be used in when coarse filtration Rock, filter paper, filter cloth or filtering element (cartridge) are preferably used using (0.1~5 μm) diatomite, perlite and filtering element (cartridge) collocation, obtain orchid filter Liquid；

(4) activity divides: by the orchid filtrate of step (3) with " molecular sieve " film processing (thin film manufacture process) purifying, film Divide two-phase seperation；First segment: film adopts inorganic ceramic membrane (pellumina, zirconium oxide film, the oxidation single etc. in 0.04~1 μm of aperture Inorganic ceramic membrane) or the organic hybrid films (organic composites such as PS membrane (PS), cellulose acetate film, polytetrafluoroethylene film, polypropylene screen Film), preferable membrane pore size is 0.04 μm.Fine particles are separated, penetrating fluid is left.Second segment: film adopts filter membrane (nanofiltrationmembrane, NF) or reverse osmosis membrane, 100~1000MW of molecular cut off, best molecular cut off 150MW.Wherein the film can be organic single or composite membrane, material be include composite membrane (TFM, thin film Membrane), at least one of Kynoar (PVDF), polysulfones (PS), polyether sulfone (PES), polyacrylonitrile (PAN)；Its In the thin film separation pressure be 150-400psi, optimum pressure 200-300psi.Extra moisture and organic matter are separated, Active sludge and active liquid reactive extract liquor are obtained, as orchid extracts concentrate；

(5) it filters: after the orchid extraction concentrate filtering of step (4), obtaining active sludge.Active sludge It washed, dry to obtain white-yellow crystal, then using DMSO as solvent, through 1H-NMR (Fig. 3 A) with pulse train (Pulse Sequence be) s2pul, 25 DEG C, it is relax.Delay 1.0 seconds, 45 DEG C of pulse (pulse), 3.000 seconds Acq. time, wide (Width) 8000.0Hz, 52repetitions, Observe H1,499.8789802MHz, Line broadening 0.1Hz, FT size 65536, divide total time 4 16 seconds；With 13C-NMR (Fig. 3 B), with pulse train (Pulse Sequence) For s2pul, 25 DEG C, relax.Delay 0.5 second, 40 DEG C of pulse (pulse), 1.042 seconds Acq. time, wide (Width) 31446.5Hz、896repetitions、Observe C13,125.6946696MHz、Decouple H1, 499.8814989MHz, Power 36dB, Line broadening 1.0Hz, FT size 131072, total time 50 divide 50 Second.After analysis, it was initially believed that being flavone compound.

The structure in the active sludge of above-mentioned orchid is analyzed by mass spectrograph (mass spectrometry, MS), discovery should The active sludge of orchid is the composition of flavonoids (flavone)；The composition is largely the flavone for connecing disaccharide, Having small part is the flavone of order sugar.

Primary structure in the composition includes at least:

i.C₂₆H₂₈O₁₄: MW:564 for the flavone for meeting disaccharide (ribosido and glucoside) is contained in composition High ingredient is measured, foreign member Sui flavine -6- riboside -7- glucoside (Apigenin-6-ribosido-7- can be estimated as Glucoside the) (active sludge and Fig. 4 (B) standard items Apigenin-6-ribosido-7- of Fig. 4 (A) orchid glucoside)；

ii.C₂₇H₃₀O₁₅: MW:594 is that content is high in composition for the flavone for meeting disaccharide (2 glucoside) Ingredient can be estimated as saponarin (Saponarin) (active sludge and (B, C) standard items Saponarin of Fig. 5 (A) orchid)；

iii.C₂₁H₂₀O₁₀: MW:432 is the flavone of order sugared (1 glucoside), is that content is less in composition Ingredient, can be estimated as ocean member Sui flavine -7- glucoside Apigenin 7-glucoside (Fig. 6 (A) orchid activity precipitating Object and (B) standard items Apigenin 7-glucoside).

Two orchid of embodiment extracts skin irritatin and the sensitivity tests of concentrate

A. orchid extracts concentrate without skin irritation and corrosivity-cell tests

Referring to skin corrosivity code test in vitro Skin Corrosion:Reconstructed Human Epidermis (RHE) Test Method (edition 4 .3,2012/11) (OECD 431), and referring to skin irritation code test In vitro Skin Irritation:Reconstructed Human Epidermis Test Method (version 3 .1, 2012/11) (OECD 439), purchased from Germany's3D epithelial cell group tested.

Vitro skin corrosivity/irritation test is carried out according to the standard method of above-mentioned OECD431 and OECD439.In vitro It take water as positive control group as negative sense control group, potassium hydroxide in corrosion test, the orchid of rising sun east whiskey extracts concentration With the PS film in 0.04 μm of aperture after the concentration of liquid (EW-DK), as step (4), and it is the TFM film of 150MW with molecular cut off, obtains Concentrate is extracted to orchid, is determined as no skin corrosivity (Fig. 7 A) through the external corrosion test of OECD 431.Stimulated in vitro examination In testing, using Du Shi phosphate buffer (Dulbecco's Phosphate-Buffered Saline) as negative sense control group, 5% SDS solution is as positive control group.The orchid extraction concentrate warp of rising sun east whiskeyStimulated in vitro test determine For no skin irritation (Fig. 7 B).

B. orchid extracts test-cell tests of the concentrate without eye irritation

The present embodiment extracts concentrate using the rising sun east whiskey orchid that concentration is 5.7% and 6.7%, thin with cervix cancer The test of born of the same parents (HeLa cell) Lai Jinhang eye irritation；The rising sun east whiskey orchid extracts concentrate system using in embodiment one The membrane separation step of step (4) with 0.04 μm of the PS film in aperture, and is obtained again with molecular cut off for the TFM film of 150MW , the extraction concentrate that examination solids level concentration is respectively 6.7wt% and 5.7wt% is finally measured with infrared moisture again, directly It is tested.

Wherein the method for eye irritation test is as follows:

The cervical cancer cell for the people that defrosting is stored in liquid nitrogen, and cultivated in the MEM culture medium containing 5%FBS Until that can be used to test required cell quantity.It is continuous with 10 times of base of MEM training containing 5%FBS by 10,000 μ g/mL It is diluted to the experiment materials of 5 concentration.When cell is sufficiently used for experiment, after collecting cell with trypsase, by cell inoculation Enter and (shown below with dish cart) in a hole in 96 hole micropore disks, is cultivated before carrying out 3 days.With the whole rising sun east whiskey orchid extraction of training keynote Concentrate (experiment materials) is added in disk, is further cultured for 48 hours after being suitble to the concentration of experiment.

Culture medium is removed from disk, after being cleaned with phosphate buffer (PBS), is replaced into the culture medium containing MTT (being indicated below with MTT) cultivates 2 hours；MTT is removed from disk, and after cleaning with PBS (-), hydrochloric isopropanol is added, it will The Formazan dye generated into the cell is extracted, and after confirmation Formazan dye is gone out by uniform extraction, it is bright to survey suction in 570nm Degree, and calculate experiment materials concentration (EC when cell survival rate is 50%₅₀).This experiment carries out 2 times, is averaged and is commented Estimate.

The rising sun east whiskey orchid that test result display density is 5.7% and 6.7% extracts concentrate without eye irritation (table 2)

2 orchid of table extracts concentrate without the irritating test of eyes

* it in the case where not having to animal to test, to obtain entirely without irritating judgement, it is necessary to which cells survival rate is Concentration (EC when 50%₅₀) it is 5,000 μ g/mL or more.EC in table 2₅₀Greater than 5,000 μ g/mL, therefore, it is determined that being nonirritant.

Table 3 is that the rising sun east whiskey orchid of various concentration extracts influence of the concentrate to cell survival rate.

3rd table

* % is the solid content (solid content) in orchid extraction concentrate, is concentration expressed in percentage by weight.

C. orchid extracts test-human test of the concentrate without skin irritatin and sensibility

The experiment is to extract concentrate by the laboratory AMA (AMA LABORATORIES, INC.) with rising sun east whiskey orchid The subject of 52 different sexes and all ages and classes is tested, subject is 9 males of the age between 18-69 years old With 43 women, including 38 high family's rope people (Caucasian), 12 Hispanics (Hispanic) and 2 Asia descendants (Asian)；Each subject receives 9 tests respectively, the test result (reference of AMA laboratory report as shown in table 4 Number are as follows: MS12.RIPT.M6496OP50.FEN.REV.2)；This as the result is shown rising sun east whiskey orchid extract liquor to skin not Have irritation and sensitization；Wherein rising sun east whiskey orchid extraction concentrate system utilizes the concentration of step (4) in embodiment one Step with 0.04 μm of the PS film in aperture, and is obtained again with molecular cut off for the TFM film of 150MW.

4 orchid of table extracts concentrate to human body without skin irritatin and sensibility

Wherein C: it indicates high family's rope people, H: indicating Hispanic, A: indicating Asia descendants；F: women, M: male；9 times tested Numerical value in reaction indicates no any reaction (no evidence of effect) if 0, and Dc indicates that test is interrupted, by Examination person does not appear tested person；N/A in score indicates to interrupt because of test, so its score can not be calculated.According to upper table, 52 by In survey person, only 2 because of individual factor interrupt experiments, remaining 50 non-testee is without generating any skin irritation (skin ) or allergic reaction (skin sensitization) irritation.The skin safe of obvious orchid extraction concentrate Property.

Three orchid of embodiment extracts concentrate Determination of Polyphenols

Experimental method: (1) influence of the sodium carbonate to gallic acid absorption spectrum is tested, takes the 1 of 0.25mL, 000 μ g/mL does not have Gallate-based (gallic acid), 20%, 2%, 0.2%, 0.02%, 0.002% and 0.0002% sodium carbonate of 0.5mL, adds The distilled water of 4.25mL after reacting 25 minutes at normal temperature, is detected in 200-800nm with light splitting luminance meter and inhales brightness.Galla turcica The final concentration of 50 μ g/mL of acid.(2) shadow that the sequence of addition Folin-Ciocalteu reagent and sodium carbonate measures gallic acid It rings to modify Folin-Ciocalteu method test method, takes the gallic acid of 0.25mL and the Folin- of same volume The distilled water of Ciocalteu reagent, 20% sodium carbonate of 0.5mL and 4mL, the ultimate density of gallic acid are 0,20,60 and 100 μ g/mL.After reacting 25 minutes at normal temperature, is detected with light splitting luminance meter in 730nm and inhale brightness.

Experiment is extracted using water or different 20,40,60,80,100% concentration ethanols, and step is as in the first embodiment, Xu Dong Whiskey is extracted to the coarse filtration liquid experimental result of one step of embodiment (3) from Fig. 8 it can be found that orchid is with different concentration ethanol Determination of Polyphenols data are shown after extraction, and water phase extraction extracts the Polyphenols of available higher amount out compared with alcohol phase, usually total The high person's resistance to oxidation of polyphenol content is stronger.

Example IV orchid extracts concentrate tool anti-oxidation function assessment

Experimental method: scavenging ability of DPPH free radical measurement, take 2ml0.2g/ml test specimen and 5mg/ml 2, 6- bis- third butyl paracresol (BHT) ethanol solution, is separately added into Fresh 2.5 × 10^-4Mα,α-diphenyl-β- The ethanol solution 0.5ml of picrylhydrazyl (DPPH), uniformly mixing was protected from light standing after 30 minutes, and UV-VIS sets scanning area Section 400nm-700nm, surveys the light absorption value of its 517nm, calculates its clearance rate, the lower expression Scavenging activity of light absorption value is stronger.More than Experiment carries out three and repeats.Scavenging ability EC₅₀The experimental data measured according to experiment one is measured, before taking clearance rate respectively Three various concentration test specimens survey its clearance rate, and according to debita spissitudo needed for different tests sample, test specimen ethyl alcohol is molten Fresh 2.5 × 10 is added in liquid 2ml^-4M concentration DPPH0.5ml, uniformly mixing was protected from light standing after 30 minutes, UV-Vis setting Section 400nm-700nm is scanned, the light absorption value of its 517nm is surveyed.The above experiment carries out three and repeats.Using concentration as y-axis, clearance rate It draws the calibration curve of test sample respectively for x-axis and calculates the EC of clearance rate 50%₅₀Concentration.The extraction concentration of rising sun east whiskey orchid With the PS film in 0.04 μm of aperture after the concentration of the concentration of liquid (EW-DK), as one step of embodiment (4), and with molecular cut off it is The TFM film of 150MW obtains orchid extraction concentrate, is to try its solid content using infrared moisture measurement, takes 2mg/ml as one The concentration of cause.

Experimental result is from Fig. 9 A it is found that Determination of Polyphenols testing result, orchid have been confirmed in the test of DPPH free radical scavenging activity It is extracted with water phase, possesses stronger free radical scavenging ability, while also Determination of Polyphenols with higher.Fig. 9 B is shown simultaneously, Orchid extraction concentrate and BHT (2,6-Di-tert-butyl-4-methylphenol) is taken to compare oxidation resistance, Ke Yifa Now compared to BHT, orchid extraction concentrate can quickly remove free radical, reach oxidation resistant effect in the short time very much. In addition, Fig. 9 C is shown, for ABTS (2,2'-azino-bis [3-ethylbenz thiazoline-6-sulphonic Acid]) free radical scavenging ability is as a result, IC₅₀It is that display may achieve free radical scavenging ability as a result, orchid extraction concentrate is compared Yu Lanye extracts the ability that concentrate has more radicals scavenging.

Five orchid extract liquor of embodiment has whitening function-inhibition Tyrosinase

A. inhibit Tyrosinase test-different concentration ethanol extraction

Ferment functional assays mode is that the shape of product dopachrome in melanin forming process is carried out using ferment Determine that ferment is functional at content, dopachrome has absorption in UV/VIS wavelength 475nm, can be with by ferment functionality inhibiting rate Observe that this compound makes the no ability for having and reducing melanin production.1. test 980 μ l of solution contains 0.8mM L- Tyrosine and 0.9M phosphate buffer (pH6.8) after evenly mixing, stand room temperature 10 minutes.2. by 440.5unit Tyrosinase and 10 μ l of test sample mix rapidly, and with UV/VIS wavelength 475nm measure 3 minutes Change of absorption.Tyrasamine Sour enzyme inhibition rate (%)={ (control group-experimental group)/control group } × 100%.

Rising sun east whiskey orchid is using 0~100% ethyl alcohol as extractant, with the orchid of embodiment one step (2) method extraction Flower filtrate, is measured using infrared moisture and tries its content, take 2mg/ml as consistent concentration.Experimental result is as shown in Figure 10 A, 6 kinds of test samples carry out inhibiting Tyrosinase functional assessment, by 4 kinds of test samples of the first and second batch as the result is shown (60%, 40%, 20% ethyl alcohol and water phase), reaches 75% or more Tyrosinase inhibiting rate.(note: 80% ethyl alcohol is because cause Tyrosinase is denaturalized when external test tube test, therefore result is disregarded).With Department of Health's core can whitening composition function compared with (this reality Test the Arbutin using 3mM), inhibition of the orchid filtrate for Tyrosinase shows good effect in low concentration, and It is tested via two batches, has reproducibility, display orchid filtrate has outstanding whitening function begetting power.

B. inhibit Tyrosinase test-different cultivars different step extract liquor (object)

The extract liquor (object) of detailed various orchids and different step is all extractant extraction using water, and difference is from step (3) Start, is measured with infrared moisture and try its solid content, take 2mg/ml as consistent concentration.As listed by the following table 5.

The acquisition pattern of the orchid active extract of the invention of table 5

Experimental result is as shown in Figure 10 B, and analysis belongs to rising sun east whiskey and beauty not of the same race, all has inhibition Tyrosine Enzyme function, preferably with the functionality of rising sun east whiskey；But different kinds of green hill clovershrubs are not belonged to more, data show that green hill is bright red The extract liquor of robe, unrestraint Tyrosinase function.So being iris, but according to different cultivars, there is different functional Weakly heterogeneous.

C. the effect of inhibiting Tyrosinase test-Hua Yuye

Rising sun east whiskey orchid and Lan Ye, after being extracted using water, until with the PS film in 0.04 μm of aperture in step (4) concentration, Orchid, Lan Ye extraction concentrate are obtained, is measured with infrared moisture and tries its solid content, taken 2mg/ml as consistent concentration, survey It tries Tyrosinase and inhibits situation.

Experimental result as illustrated in figure 10 c, IC50 show may achieve inhibit Tyrosinase as a result, orchid extraction concentration liquid phase Compared with Lan Ye extraction concentrate with more the activity for inhibiting Tyrosinase.

Embodiment six

A. orchid extract liquor tool whitening function-inhibition mouse melanoma cells generate melanin

Experimental method cultivates mouse melanoma cells (B16) in 6-well culture plate for 1., and DMEM is added and (contains 10% FBS) culture solution is placed on 37 DEG C, 5%CO₂In incubator, the test agent of various concentration is added in culture solution every other day.2. will not With being cultivated 3 days in the test agent addition culture solution of concentration, appropriate trypsin/EDTA is added with PBS cleaning cell and stands 3 points Cell is taken out and is cleaned with PBS after cell is separated with culture dish by clock, is centrifuged 5000rpm 5 minutes.3. removing after centrifugation Supernatant is added PBS and is slowly mixed together uniformly with cell precipitate, is centrifuged 5000rpm 5 minutes.4. step 3 repeats once. 5. finally removing supernatant, and the 1N NaOH that 1ml is added in cell precipitate is slowly broken up into cell, is evenly distributed on it In NaOH solution.6. centrifugation 3000rpm 10 minutes takes 1ml supernatant to survey UV/VIS wavelength 405nm and absorbs.What 7. measurement obtained Numerical value is divided by survivaling cell number, as melanin content measurement experiment result.

Fresh orchid inhibits mouse melanoma cells to generate melanin, and rising sun east whiskey orchid utilizes various concentration solvent It extracts (water, 20% ethyl alcohol, 80% ethyl alcohol, 100% ethyl alcohol), until the orchid filtrate (product item 1-4) of step (2), dense to step (4) With the PS film in 0.04 μm of aperture after contracting, and the TFM film with molecular cut off for 150MW obtains orchid extraction concentrate；Using The active sludge (product item 5) obtained, Arbutin 5mM (product item 6), control group (product item 7,1%DMSO) are separated, all with infrared ray Moisture meter tests its solid content, takes 2mg/ml as consistent concentration, as listed by the following table 6.

The different extract explanations of each test tube of table 6 addition

Product item	Remarks
		1	The extraction of 100% water
2	The extraction of 20% ethyl alcohol
		3	The extraction of 80% ethyl alcohol
4	The extraction of 100% ethyl alcohol
		5	100% water extractive crystallization sediment
6	Arbutin 5mM
		7	Control group

Experimental result is as shown in figure 11, inhibits melanin experimental result to show by mouse melanoma cells, Arbutin, orchid Flower alcohol extraction liquid, orchid water phase white crystals all have the obvious effect for inhibiting melanoma cells (B16).

B. orchid extract liquor has whitening function-inhibition mankind Initial culture (primary culture) melanocyte (HMC-4) generating melanin experimental method is with different rising sun east whiskey orchid extract liquors, in mankind's Initial culture Carry out inhibiting the test of melanin performance on the melanocyte (HMC-4) of (primary culture)；And in carrying out inhibiting black Before the test of pigment performance, cell survival rate (cell is first carried out to HMC-4 cell with different rising sun east whiskey orchid extract liquors Viability experiment), then the full test dosage (concentration) with cell survival rate greater than 95% carries out inhibiting melanin table Existing test.

Table 7 and table 8 are the difference for inhibiting rising sun east whiskey orchid extract liquor used in the test of melanin performance Dosage and extracting process, Figure 12 A-H are that each rising sun east whiskey orchid extract liquor carries out cell to HMC-4 cell in table 7 and table 8 Survival experiment as a result, Figure 13 is then the test result that different rising sun east whiskey orchid extract liquor inhibits melanin performance； Though whiskey orchid extract liquor dosage in all tested person rising suns east is greater than 95% full test with cell survival rate as shown in Figure 13 Dosage (concentration) inhibit the test of melanin performance, and the extract liquor of the right concentration all can inhibit the performance of melanin.

Table 7 inhibits the dosage and extracting process of rising sun east whiskey orchid extract liquor used in the test of melanin performance

Table 8 inhibits the dosage and extracting process of rising sun east whiskey orchid extract liquor used in the test of melanin performance

Seven orchid extract liquor of embodiment has crease-resistant (anti-aging) effect

One, Fibroblast collagenase (MMP-1)

MMP-1 (Fibroblast collagenase, Matrix Metalloproteinase-1) is the collagen point of common name Solve one kind of enzyme, that is to say, that MMP-1 is responsible for the collagen degradation of dermal tissue.The present embodiment utilizes rising sun east whiskey step (4) with the PS film in 0.04 μm of aperture after being concentrated, and it is again the TFM film of 150MW with molecular cut off, obtains orchid extraction concentration Liquid.Examination solids level concentration is measured with infrared moisture and is weight percentage 6.7% and 5.7% respectively, is directly tested.

Experimental result is as shown in figure 14, wherein crease-resistant (anti-aging) the efficacy test method and step are as follows:

1. diploid fibroblast (TIG-118, Normal the human diploid of liquid nitrogen will be stored in Fibroblasts it) thaws, and to contain the MEM culture medium (Minimum of 5%FBS (Fetal Bovine Serum, fetal calf serum) Essential Medium) enough cell number of the culture to needed for testing.

2. confirming that substance to be tested will not generate the concentration of toxicity to cell (TIG-118) before carrying out preliminary experiment Afterwards, and from 2 times of concentration dilution of highest (big) when optimum official testing, one there are be made into 3 concentration (1%, 0.5%, 0.25%) test concentrations are used as.

3. will test in above-mentioned steps 1 after required enough cells obtain with trypsin treatment, and it is inoculated in 35mm training Supporting in ware (makes its 50,000 cells of cell density/mL).

4. then the cell for being inoculated in 35mm culture dish, by broth out, is diluted after culture 24 hours with step 2 The good testing liquid containing test substances replaces, and after being cultivated for 24 hours, extracts cell mRNA and (extracts reagent set: RNeasy Mini kit, Qiagen), MMP- is analyzed with real-time quantitative polymerase chain reaction (Quantitative real-time PCR) 1 gene performance amount.

The condition of Real-time PCR analysis:

Use equipment: ABI PRISM 7900HT, Applied Biosystems Co., Ltd.

PCR temperature condition are as follows:

Stage1:42 DEG C of 5min, 95 DEG C of 10sec (1cycle)

Stage2:95 DEG C of 5sec, 60 DEG C of 34sec (40cycle)

Stage3:95 DEG C of 15sec, 60 DEG C of 1min, 95 DEG C of 15sec (1cycle)

Two, elastoser (elastase)

The curling meeting of elastomer (elastic fibers) is so that skin is formed in the reduction of skin elasticity and skin corium Wrinkle, and elastoser (elastase) inhibitor can inhibit the activity of elastoser, prevent the damage of elastic fibers of skin Wound, to facilitate the formation of mitigation wrinkle.

The present embodiment extracts concentrate using the rising sun east whiskey orchid that concentration is 5% and 1%, by it respectively with Tris- Hydrochloride buffer is diluted, total to be made into 5 kinds of concentration respectively, carries out the test (table 9) of the activity suppression of elastoser, In 5% and 1% rising sun east whiskey orchid extraction concentrate all have the maximum inhibition of extraordinary elastoser.Simultaneously simultaneously It is the IC of 5% and 1% rising sun east whiskey orchid extraction concentrate inhibition elastoser₅₀Test (table 10).The wherein rising sun Eastern whiskey orchid extraction concentrate is the concentration step for utilizing step (4) in embodiment one, with 0.04 μm of the PS film in aperture, and It is obtained again with molecular cut off for the TFM film of 150MW.

Maximum inhibition of the rising sun east whiskey orchid extraction concentrate of 9 various concentration of table to elastoser

The rising sun east whiskey orchid extraction concentrate of 10 various concentration of table inhibits the IC of elastoser₅₀

Wherein the elastase activity inhibits the method for test as follows:

The rising sun east whiskey orchid of various concentration is extracted into concentrate, enzyme liquid (elastase solution), matrix solution (N- Succiny-Ala-Ala-Ala-p-Nitroanilide it) mixes, after 37 DEG C heat 15 minutes, is surveyed in 415nm and inhale brightness, and The maximum inhibition of elastoser is calculated by following formula.

A: the suction brightness after blank solution reaction

B: the suction brightness after experimental solution reactions

A0, B0: the suction brightness instead of enzyme liquid, when with Tris- hydrochloride buffer.

Three, Collagenase (collagenase)

Collagenase can degrade the collagen in skin, in turn result in skin wrinkle and aging, and collagen egg White enzyme inhibitor can inhibit Collagenase, prevent skin wrinkle and aging.The present embodiment using concentration be 5.7% and 6.7% rising sun east whiskey orchid extracts concentrate, by it respectively with 2 times of serial dilutions of citrate buffer solution at 5 kinds of concentration, into The test (table 11) of the activity suppression of row Collagenase, wherein 5.7% and the 6.7% of 100% concentration rising sun east whiskey is blue Flower extraction concentrate has the maximum inhibition of optimal Collagenase；Wherein rising sun east whiskey orchid extraction concentrate is It with 0.04 μm of the PS film in aperture, and is again 150MW's with molecular cut off using the concentration step of step (4) in embodiment one TFM film obtains, and finally measures the extraction concentrate that examination solids level concentration is respectively 6.7% and 5.7% again with infrared moisture, Directly tested.

Maximum inhibition of the rising sun east whiskey orchid extraction concentrate of 11 various concentration of table to Collagenase

Wherein the collagenase activity inhibits the method for test as follows:

By rising sun east whiskey orchid extraction concentrate, the victory peptide solution (Pz-Pro-Leu-Gly-Pro-D- of various concentration Arg-OH it) mixes, after being heated at 37 DEG C, is added enzyme liquid (collagenase), after 37 DEG C of reactions, 0.1M is added Tris buffer (containing 20mM CaCl2, pH7.1) stops reaction, and ethyl acetate is added, is centrifugated in 3,000rpm.In 320nm surveys the suction brightness of ethyl acetate layer, calculates collagenase activity inhibiting rate (%) by following formula.

A: the suction brightness after blank solution reaction

B: the suction brightness after experimental solution reactions

A₀、B₀: suction brightness when enzyme liquid is replaced with Purified Water.

Eight orchid extract liquor of embodiment is tested in the skin-care effect of human body

The present embodiment using after rising sun east whiskey step (4) concentration with the PS film in 0.04 μm of aperture, and again to retain molecule Amount is the TFM film of 150MW, obtains orchid extraction concentrate.Measuring examination solids level concentration with infrared moisture is respectively 1.0%, Directly tested.Human skin efficacy test is carried out in platform large hospital to the testee of 31 all ages and classes, gender, and in survey Its result is analyzed in examination after two weeks.

In four kinds of spot indexs that current image test is assessed, comprising: visible speckles, ultraviolet light color spot, brown spot and Red grouper；Wherein visible speckles are the black splotch that visually can be seen under standard white light；Ultraviolet light color spot is ultraviolet using 365nm The black splotch seen under light photography (ultraviolet photography can highlight the spot part being invisible to the naked eye)；Brown spot is to use RBX The deep skin spot assessment that polarisation patented technology is simulated；Red grouper is is simulated using RBX polarisation patented technology The skin redness spot assessment arrived；And in table numerical value number represent quantity number and the feature of score value proportional representation detection project Degree (density).

There is no testee's adverse reaction and sense of discomfort after two weeks in using.

Test result, should be as a result, it has been found that using as shown in table 12 and table 13 in visible spot, brown spot and red grouper number There is statistical downward trend after two weeks, but in characteristic length (density) part then without statistical difference, and is using two The analysis result in week, which can be seen, has the effect of reducing melanin spot quantity (visible spot and deep layer spot) and reduction red grouper quantity.

12 orchid extract liquor of table measures numerical value in the skin-care effect of human body

The result statistic that 13 orchid extract liquor of table is tested in human body skin-care effect

The lower numerical value the better

Invention utilizes unique variety white iris (the i.e. Phalaenopsis Sogo Yukidian kind rising sun of green hill orchid cultivation Eastern whiskey orchid), inhibiting Tyrosinase after extracting, mouse B16 melanocyte and mankind's HMC-4 cell is inhibited to generate In the experiment such as melanin and human body, good whitening capability is shown, while also there is crease-resistant (anti-aging), ageing resistance, And after various kinds of cell experiment, testing in vitro and human experimentation, the corrosion-free irritation of safety is confirmed, also without hypersensitive. Vegetable active is studied for many years by this research team, can be in multiple systems (ferment, zooblast system, mankind Skin Cell primary System and clinical trial) verify whitening effect active constituent and mild non-stimulated ingredient it is extremely rare.Thus It can be seen that the superiority of this raw material and preparation method.

In terms of preparation method, after extract is handled via film, in addition to volume production can be achieved, and it is available isolate and purify it is dense The effect of contracting.Orchid extract liquor after concentration, can generate a large amount of sediment, and through researching and analysing after collection, which belongs to The similar chromocor compound group of three kinds of structures.Extract liquor and sediment have good whitening capability, crease-resistant (anti-aging) energy Power and resistance to oxidation.This preparation method belongs to green processing procedure, does not generate toxic organic solvent and amplification is easy, can also connect Tie the flavonoid active compound of microorganism control, the safe and healthy reactive extraction concentrate of output and then precipitable acquisition.

Attempted other orchids not belonged to, no Tyrosinase rejection ability；And compared with another orchid belonged to, though Has Tyrosinase rejection ability, but preferable with kind rejection ability of the invention.Preparation method and reagent used in this patent, Allow to be used in cosmetics.

Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Claims (23)

1. a kind of orchid extract, which is characterized in that the orchid is Phalaenopsis Sogo Yukidian kind rising sun east whiskey, The orchid extract is obtained as prepared by the following steps:

(1) it extracts: with weight ratio being 0.5:1~10:1 by the orchid and solvent, carry out co-grinding or broken wall obtains Orchid slurry, the solvent is water, alcohols or alcohol solution, wherein the alcohols is ethyl alcohol, propyl alcohol or butanol；

(2) it is separated by solid-liquid separation: the orchid slurry of step (1) extraction being separated by solid-liquid separation, leaving liquid is orchid filtrate；

(3) activity divides: the separated orchid filtrate of step (2) being handled with molecular sieve film and is purified, orchid extraction sieve is obtained Liquid separation；The molecular cut off of the molecular sieve film is 100~1000MW；

(4) it is concentrated: the orchid extraction screening liquid of step (3) purifying is handled into concentration or vacuum or boiling with molecular sieve film Mode carries out partial concentration, obtains active sludge and active liquid reactive extract liquor.

2. orchid extract according to claim 1, which is characterized in that orchid described in step (1) and solvent are with weight The mode of ratio 5:1 is extracted.

3. orchid extract according to claim 1, which is characterized in that the temperature of separation of solid and liquid described in step (2) is 0 ~60 DEG C.

4. orchid extract according to claim 3, which is characterized in that the temperature of separation of solid and liquid described in step (2) is 0 ~35 DEG C.

5. orchid extract according to claim 1, which is characterized in that the method for step (2) described separation of solid and liquid be from Heart method or filtration method, the filtering material that the filtration method uses is at least one of diatomite, perlite and filtering element (cartridge).

6. orchid extract according to claim 1, which is characterized in that step (3) activity divides and step (4) institute The molecular sieve film processing in concentration is stated using two sections of steps: first segment uses inorganic ceramic membrane or organic hybrid films；The Two sections use nanofiltration membrane or reverse osmosis membrane.

7. orchid extract according to claim 6, which is characterized in that the inorganic ceramic membrane is pellumina, oxidation Zirconium film or oxidation single film.

8. orchid extract according to claim 6, which is characterized in that the organic hybrid films are PS membrane, acetic acid fibre Tie up film, polytetrafluoroethylene film or polypropylene screen.

9. orchid extract according to claim 6, which is characterized in that the nanofiltration membrane or reverse osmosis membrane are single film Or laminated film, material are at least one of composite membrane, Kynoar, polysulfones, polyether sulfone and polyacrylonitrile.

10. orchid extract according to claim 1, which is characterized in that the structure of step (4) described active sludge is reflected It surely is analyzed and identified with nuclear magnetic resonance and mass spectrograph.

11. a kind of medical composition, comprising liquid reactive extract liquor described in claim 1, active sludge or combinations thereof and Its pharmaceutically acceptable excipient.

12. a kind of orchid extract is used to prepare the purposes of lightening compositions, wherein the orchid extract is claim 1 institute The liquid reactive extract liquor stated, active sludge or combinations thereof.

13. purposes according to claim 12, which is characterized in that the orchid extract is used to prepare inhibition Tyrosinase Whitening medicaments.

14. purposes according to claim 12, which is characterized in that the orchid extract, which is used to prepare, inhibits melanin Whitening medicaments.

15. a kind of orchid extract is used to prepare the purposes that treatment free radical causes the drug of disease, wherein the orchid extracts Object is liquid reactive extract liquor described in claim 1, active sludge or combinations thereof.

16. purposes according to claim 15, which is characterized in that the orchid extract, which is used to prepare, quickly to be removed freely The drug of base.

17. a kind of orchid extract is used to prepare crease-resistant and antiageing medicine purposes, wherein the orchid extract is right It is required that liquid reactive extract liquor, active sludge or combinations thereof described in 1.

18. purposes according to claim 17, which is characterized in that the orchid extract is used to prepare treatment collagen Lack the drug of diseases caused.

19. purposes according to claim 17, which is characterized in that the orchid extract is used to prepare inhibition matrix metal The crease-resistant or antiageing medicine of protease.

20. purposes according to claim 17, which is characterized in that the orchid extract is used to prepare inhibition collagen The crease-resistant or antiageing medicine of enzyme.

21. purposes according to claim 17, which is characterized in that the orchid extract is used to prepare inhibition elastin laminin The crease-resistant or antiageing medicine of enzyme.

22. a kind of orchid extract, which is characterized in that the orchid is Phalaenopsis Sogo Yukidian kind rising sun east whiskey, The orchid extract is obtained as prepared by the following steps:

(1) it extracts: with weight ratio being 0.5:1~10:1 by the orchid and solvent, carry out co-grinding or broken wall obtains Orchid slurry, the solvent is water, alcohols or alcohol solution, wherein the alcohols is ethyl alcohol, propyl alcohol or butanol；

(2) it is separated by solid-liquid separation: the orchid slurry of step (1) extraction being separated by solid-liquid separation, leaving liquid is orchid filtrate；

(3) activity divides: the orchid filtrate of step (2) separation being handled with molecular sieve film and is purified；The molecular sieve film Molecular cut off is 100~1000MW.

23. a kind of method for preparing orchid extract, which is characterized in that the orchid is Phalaenopsis Sogo Yukidian kind Rising sun east whiskey, comprises the steps of

(1) it extracts: with weight ratio being 0.5:1~10:1 by the orchid and solvent, carry out co-grinding or broken wall obtains Orchid slurry, the solvent is water, alcohols or alcohol solution, wherein the alcohols is ethyl alcohol, propyl alcohol or butanol；

(2) it is separated by solid-liquid separation: the orchid slurry of step (1) extraction being separated by solid-liquid separation, leaving liquid is orchid filtrate；

(3) activity divides: the orchid filtrate of step (2) separation being handled with molecular sieve film and is purified, orchid extraction screening is obtained Liquid；The molecular cut off of the molecular sieve film is 100~1000MW；

(4) it is concentrated: the orchid extraction screening liquid of step (3) purifying is handled into concentration or vacuum or boiling with molecular sieve film Mode carries out partial concentration, obtains active sludge and active liquid reactive extract liquor.