JP2016222620A - The active extract of phalaenopsis, preparation method and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide Phalaenopsis flower extract, and to provide pharmaceuticals and cosmetic compositions containing the same.SOLUTION: The present invention provides Orchidaceae flower extract prepared by the following steps (1)-(4), and pharmaceuticals and cosmetic compositions which contain the same and have whitening function, antioxidative activity, and anti-wrinkle (anti-aging) ability. (1) Extraction: mixing a leaching agent to Orchidaceae flowers and grinding or homogenizing it to obtain Orchidaceae flower pulp, the leaching agent being water, alcohols, or a mixture thereof. (2) Separation between a solid part and a liquid part: separating between a solid part and a liquid part of Orchidaceae flower pulp (1) and removing impurities to obtain Orchidaceae flower filtrate. (3) Activity classification: purifying the filtrate of (2) through a zeolite membrane, and obtaining an Orchidaceae flower extract screening liquid. (4) Concentration: concentrating the screening liquid of (3) through the zeolite membrane, or partially concentrating with a vacuum device or a heating method or the like to obtain an active precipitate and active liquid extract.SELECTED DRAWING: None

Description

  The present invention provides an orchid flower extract that functions as a whitening effect, antioxidant, and anti-wrinkle (anti-aging), and an extraction method thereof.

Usually, the color of the skin is determined by: 1. Content and distribution of pigment in the skin, 2. Redox ratio of hemoglobin in the blood flowing through the skin, 3. Skin thickness and light scattering on the skin surface. . Influence of external environment (ultraviolet rays, air pollution), undesirable lifestyle (inaccurate beauty, poor cosmetics, exposure to electromagnetic waves on screen), skin aging (decreased immunity, loss of microcirculation, metabolic ability All of them cause pigmentation, uneven pigment distribution, loss of luster and opacity on the skin.
Melanocytes in the basal cell layer of the skin are unicellular secretory organs that have a circular shape, and branched long tubular processes bite into epidermal cells. Melanin formed in the deep layer of the epidermis is transferred to the surface of the skin, but the melanocyte body does not move along with this, and keratinocytes function to transfer melanin. The skin surface transfer of melanin by corneocytes is a somatic protective function inherent in humans.

  When exposed to direct sunlight or exposure to ultraviolet light, tyrosinase in melanocytes is activated to promote melanin production, and the skin undergoes a series of chemical reactions and activities through the melanin synthesis pathway (Raper-Mason pathway, see FIG. 2). Melanin is formed by the action of the enzyme. Melanin is a kind of polymer complex. When the skin is irradiated with ultraviolet rays, tyrosinase in melanocytes is activated, and tyrosines in melanocytes are oxidized to oxidize dihydroxyphenylalanine (common name). DOPA is formed and metabolized by tyrosinase to dopachrome. In the melanin synthesis pathway, tyrosinase tyrosine is hydroxylated to produce L-3,4-dihydroxyphenylalanine (L-3,4-dihydroxyphenylalanine, commonly known as L-DOPA, L-dopa), and L-dopa is converted to dopachrome. ) Is involved in the function of being metabolized. Then, dopachrome is oxidized to melanin, and eumelanin is synthesized from dopachrome through this series of oxidation reactions.

Tyrosinase is a polyphenol oxidase containing copper ions, synthesized by melanocytes, and the level of activity varies depending on the source. Since there are three active centers of copper ions at the site where tyrosinase binds to tyrosine, according to today's research, the following two types of mechanisms are used to suppress tyrosinase.
(1) Inhibition of tyrosinase activity: Since this reaction mechanism has a copper ion due to the structure of the enzyme molecule, copper is used as a suppressor by utilizing a method similar to a copper-chelating agent. It is a method of blocking the mechanism of melanin production by suppressing the activity of tyrosinase by chelating ions or competing with copper ions to weaken the binding power of copper ions. Ingredients include kojic acid.
(2) Competitive inhibition with tyrosinase substrate: This reaction mechanism suppresses the formation of melanin by competing the inhibitor with a substrate such as tyrosine or dopa to promote the reaction with tyrosinase and reducing the substrate acting on tyrosinase. In this method, hydroquinone and arbutin are typical components of substrate inhibition by this competition.

Whitening is one of the functions of cosmetics and skin care products that Asian women value most. The following methods are known for evaluating the presence or absence of a whitening function in a cosmetic raw material.
(1) Presence or absence of tyrosinase suppression: Since tyrosinase is an enzyme that produces melanin, if this enzyme can be suppressed, melanin production also decreases (FIG. 2).
(2) Mouse-derived melanoma cell (B16 cell line): In order for a raw material (or component) to inhibit tyrosinase, the component must be able to penetrate into the cell as a premise of the action mechanism exerted by the component. Therefore, some scholars are using mouse-derived melanoma cells (B16 cell line) to examine the presence or absence of a whitening function that suppresses melanocyte growth and melanin synthesis. However, melanoma cells are melanocyte melanoma cells, and in terms of the amount and composition of melanogenesis, the inhibitory effect obtained on melanoma cells is often different from the effect on healthy human skin.
(3) Primary culture human melanocytes: Primary cultures of human melanocytes, most similar to human true skin cells. Usually, the melanin inhibitory effect on melanocytes is confirmed and the whitening function is evaluated at the maximum amount of harmless components (for example, the component concentration at which the cell viability is 95%).
None of the above three methods show a significant whitening effect, and if such an effect is shown, the component has a relatively excellent whitening function.

In addition to the safety assessment results, national authorities in each country place importance on the results of safety assessments when examining raw materials used as new cosmetics. Evaluation of the safety of new raw materials for cosmetics has been conducted by animal experiments in the past, and the most important and basic ones are skin irritation, skin corrosivity and skin sensitization tests.
However, since the EU has banned animal experiments since July 2013 and has banned the sale of products that have conducted animal experiments (Marketing ban), the international community has decided to conduct alternative testing for animal experiments (Alternative testing). On the other hand, a considerable amount of money and time is invested to develop a test method that can replace animal experiments. So-called 3D artificial skin and OECD-approved experimental methods are used to test the safety of cosmetic raw materials. In this study, a skin irritation test (OECD 439) and a skin corrosivity test (OECD 431) of cosmetic raw materials were carried out using 3D artificial skin made in Germany (3D human skin tissue). In addition, a skin sensitization test was performed in a human clinical test, and the results of skin irritation and skin corrosivity tests using 3D artificial skin were confirmed.

  In order to ensure both the safety and effectiveness of the ingredients, the Taiwan Health Department has made available whitenings such as kojic acid, arbutin, ellagic acid, and chamomile ET. The ingredients and their concentrations are listed (see Table 1). According to Table 1, it cannot be said that there are many whitening components that can be used, and most of the acids bring about partial irritation. Recently, there has been a problem of white spots occurring in consumers using Kanebo's Tsutsujinol, and at the same time, seeking safety and efficacy, as well as seeking ingredients for whitening cosmetics that are safe to use. The goal of many manufacturers is to find new raw materials for cosmetics that have functions and characteristics other than whitening.

Table 1 Whitening ingredients announced by Taiwan Food and Drug Administration

  The inventor of the present invention has gone through many years of research, and certain orchids (Panthera orchid) have undergone a special extract extraction process, so that the extract can suppress tyrosinase activity, antioxidant and anti-wrinkle (anti-aging) It was found to have each of the actions. This extract contains three or more types of flavonoids with similar structures, and has whitening effects on tyrosinase, mouse-derived melanoma cells, primary human melanocytes, and clinical trials. Through analysis, it has been found to have antioxidant and anti-wrinkle (anti-aging) effects. In identifying orchids, it can be distinguished from other orchids by Motohara appraisal.

  An extract having a plurality of functions obtained through a green manufacturing process in which all steps are performed by low temperature and water extraction is a crystal having a specific flavone structure or an active extract containing such a flavone. This extract has the ability to suppress tyrosinase, as well as the ability to suppress melanin production by melanocytes, and the functions of antioxidant and anti-wrinkle (anti-aging), and has proved safe in external cell experiments and clinical trials. There is a possibility of commercialization.

The object of the present invention is to provide a kind of orchid flower extract, which is of the genus Phalaenopsis and can be prepared by the following procedure.
(1) Extraction: An orchid flower is mixed with a leaching agent in a weight ratio of 0.5: 1 to 10: 1 and pulverized or broken to obtain an orchid flower pulp. This leaching agent is water, alcohols (eg: Ethanol, propanol, butanol) or a mixture thereof.
(2) Solid-liquid separation: The solid and liquid of orchid flower pulp of (1) are separated, and impurities are removed to obtain a liquid orchid flower filtrate.
(3) Activity classification: The filtrate of (2) is purified through a zeolite membrane to obtain an orchid extract screening solution.
(4) Concentration: The screening solution of (3) is concentrated using a zeolite membrane or partially concentrated by a method such as a vacuum apparatus or heating to obtain an active precipitate and an active liquid extract.

  In order to achieve the above-mentioned object, the phalaenopsis orchid is a phalaenopsis genus Sogoyukidian species "Palaenopsis Sogo Yukidian 'Shiuh-Dong Whiskey'). This orchid is exhibited by the Agricultural Committee of the Agricultural Committee of Agricultural Committee, Plant Affiliation Certificate Ai No. A00595, Aoyama Orchid Biological Technology Co., Ltd.

  In order to achieve the above object, orchid flower pulp is obtained by setting the weight ratio of the orchid flower and the leachate in step (1) to 5: 1.

  In order to achieve the object, the solid-liquid separation temperature in step (2) is 0 to 60 ° C.

  In order to achieve the object, the solid-liquid separation in step (2) is 0 to 35 ° C.

  In order to achieve the above object, the centrifugal method or the filtration method used in the solid liquid separation in step (2) is diatomaceous earth, perlite, paper filter, cloth filter, filtration core (0.1 to 5 μm) as a filtration material. Use one or more of these.

  In order to achieve the above object, the membrane filtration process in step (3) active section and step (4) concentration is performed in two stages. The first stage uses an inorganic ceramic membrane or an organic composite membrane, and the second stage uses a nanofiltration membrane or a reverse osmosis membrane. The inorganic ceramic film includes an aluminum oxide film, a zirconium oxide film, or tellurium oxide. The organic composite membrane includes a polysulfone membrane, a cellulose acetate membrane, a Teflon membrane, and a polypropylene membrane. The nanofiltration membrane or reverse osmosis membrane is a single thin film or a composite thin film, and the material of the thin film is a composite film (TFM, thin film membrane), polyvinylidene fluoride (PVDF), polysulfone (PS), polyether. One or more of sulfone (PES) and polyacrylonitrile (PAN) are included, and the film separation pressure is in the range of 150-400 psi, and preferably 200-300 psi.

  In order to achieve the object, the structure of the active precipitate in the step (4) is determined by analyzing the chemical structure using nuclear magnetic resonance (NMR) and mass spectrometry (MS).

  Another object of the present invention is to provide a pharmaceutical composition comprising an effective amount of the orchid flower extract concentrate, a precipitate (compound) or a combination thereof, and a pharmaceutically acceptable excipient.

  Another object of the present invention is to provide an orchid flower extract for use in the preparation of a drug having a whitening function. This orchid flower extract is the aforementioned orchid flower extract concentrate, a precipitate, or a combination thereof, and this orchid flower extract is a tyrosinase. This orchid flower extract is used for the preparation of a whitening drug or active ingredient that suppresses the production of melanin.

  Another object of the present invention is to provide an orchid flower extract used as a drug for treating diseases caused by radicals. The orchid flower extract is the above orchid flower extract concentrate, a precipitate, or a combination thereof. Is used in the preparation of drugs or active ingredients that quickly scavenge radicals.

  Another object of the present invention is to provide an orchid flower extract used for the preparation of an anti-wrinkle (anti-aging) drug, which orchid flower extract is the above-mentioned orchid flower extract concentrate, a precipitate or a combination thereof. The extract is used to prepare drugs or active ingredients to treat diseases caused by the lack of collagen, and this orchid flower extract is used to prepare anti-wrinkle (anti-aging) drugs or active ingredients that inhibit matrix metalloproteinases. The orchid flower extract is used for the preparation of an anti-wrinkle (anti-aging) drug or active ingredient that suppresses collagenase, and this orchid flower extract is used for the preparation of an anti-wrinkle (anti-aging) drug or active ingredient that suppresses elastase.

Another object of the present invention is to provide an orchid flower active extract, which is of the genus Phalaenopsis and can be prepared by the following procedure.
(1) Extraction: An orchid flower is mixed with a leaching agent in a weight ratio of 0.5: 1 to 10: 1 and pulverized or broken to obtain an orchid flower pulp. This leaching agent is water, alcohols (eg: Ethanol, propanol, butanol) or a mixture thereof.
(2) Solid-liquid separation: The solid and liquid of orchid flower pulp of (1) are separated, and impurities are removed to obtain a liquid orchid flower filtrate.

Another object of the present invention is to provide an orchid flower active extract, which is of the genus Phalaenopsis and can be prepared by the following procedure.
(1) Extraction: An orchid flower is mixed with a leaching agent in a weight ratio of 0.5: 1 to 10: 1 and pulverized or broken to obtain an orchid flower pulp. This leaching agent is water, alcohols (eg: Ethanol, propanol, butanol) or a mixture thereof.
(2) Solid-liquid separation: The solid and liquid of orchid flower pulp of (1) are separated, and impurities are removed to obtain a liquid orchid flower filtrate.
(3) Activity classification: The filtrate of (2) is purified through a zeolite membrane to obtain an orchid extract screening solution.
(4) Concentration: The screening solution of (3) is concentrated using a zeolite membrane or partially concentrated by a method such as a vacuum apparatus or heating to obtain an active precipitate and an active liquid extract.

Another object of the present invention is to provide a method for preparing an orchid flower active extract. This orchid flower belongs to the genus Phalaenopsis and can be prepared by the following procedure.
(1) Extraction: An orchid flower is mixed with a solvent at a weight ratio of 0.5: 1 to 10: 1 and pulverized or broken to obtain an orchid flower pulp. , Propanol, butanol), or a mixture thereof.
(2) Solid-liquid separation: The solid and liquid of orchid flower pulp of (1) are separated, and impurities are removed to obtain a liquid orchid flower filtrate.
(3) Activity classification: The filtrate of (2) is purified through a zeolite membrane to obtain an orchid extract screening solution.
(4) Concentration: The screening solution of (3) is concentrated using a zeolite membrane or partially concentrated by a method such as a vacuum apparatus or heating to obtain an active precipitate and an active liquid extract.

Orchid Extract Extraction Flow Melanin synthesis pathway by tyrosinase (Raper-Mason pathway) DPI: 5,6-dihydroxyindole DHICA: 5,6-dihydroxyindole-2-carboxylic acid (5,6-dihydroxyindole-2) -Carboxylic acid) Orchid Extract Precipitate Proton Nuclear Magnetic Resonance (1H-NMR) Analysis Diagram (Varian, Varian 500) (A) and Carbon-13 Nuclear Magnetic Resonance (13C-NMR) Analysis Diagram (Varian, Varian 500) (B) Mass spectrometry (MS) analysis result (A) of orchid flower precipitate and mass spectrometry (MS) analysis result (B) of standard product (apigenin-6-riboside-7-glucoside) Mass spectrometry (MS) analysis results (A) of orchid flower sediment and mass spectrometry (MS) analysis results (B, C) of a standard product (saponarine) Mass spectrometry (MS) analysis result (A) of orchid flower sediment and mass spectrometry (MS) analysis result (B) of standard product (apigenin 7-glucoside) Skin corrosivity test result of orchid flower extract concentrate (A) and skin irritation test result of orchid flower extract concentrate (B) Total phenol content in orchid flower extract concentrates extracted with ethanol water with different ethanol concentrations (converted to gallic acid equivalent (GAE)) Diphenylpicrylhydrazyl (DPPH) radical scavenging rate (A), orchid flower extract concentrate and dibutylhydroxytoluene (BHT) radical scavenging rate (B), orchid flower and orchid in orchid flower extract concentrate extracted with ethanol water with different ethanol concentration Leaf extract concentrate and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging ability (C) Evaluation of tyrosinase inhibition rate in orchid flower extract concentrates extracted with ethanol water with different ethanol concentrations (A), tyrosinase inhibition rate of orchid flower extract concentrates (products) extracted with different methods using different types of orchids, (B), Tyrosinase inhibitory ability of orchid and orchid leaf extract concentrate (C) Results of experiments where orchid extracts or precipitates extracted with ethanol water with different ethanol concentrations suppress melanin production by melanocytes Results of cell viability experiments on human melanocytes (HMC-4 cells) using "Asahi Toshishiki" orchid flower extract with different A to H Results of experiments on melanin suppression using different “Asahi Toshijiki” orchid extracts (M254: M254 medium, M2: M2 medium, KA: Kojic acid) Anti-wrinkle (anti-aging) ability test of orchid extract

  Unless otherwise defined, technical and scientific terms used in this document are interpreted based on recognition shared by those with ordinary skill in the domain to which the term belongs.

The term “extract” as used in this document refers to a product prepared by the action of extraction. The extract may be in the form of a liquid dissolved in the leaching agent, or may be in the form of a concentrate or essence that contains no or little leaching agent. As will be described in detail later, the extract can be incorporated into a pharmaceutical composition or food.
The term “extract” may be a single extract obtained through a specific extraction procedure or a series of extraction procedures, or a combination of extracts obtained through each independent extraction procedure. is there. Therefore, the extract combined in this way is also included in the term “extract”.

  “Raw material” as used in this document usually refers to a plant as a raw material, or a single plant as a whole, or leaves, roots (including but not limited to radishes, fine roots and whiskers), stems and skins. Including a combination of one or more plant composition parts, including berries, seeds, flowers, so that the plant or its composition parts can be processed in a pristine state, dried, heated, warmed, or otherwise Physically processed materials and materials that have affected the size and entity integrity of the plant material by its original condition, chopping, cutting, grinding, polishing, or other processing methods. The term “raw material” can also denote an extract extracted from a material used in another extraction process.

The present invention can be used as a skin external preparation composition, and in addition to the above-mentioned orchid flower extract as a composition, as a base, components used in skin external preparations such as general cosmetics or pharmaceuticals (humectant, antioxidant) Agents, oily components, ultraviolet absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, etc.) may be added as appropriate.
Moreover, you may add the following suitably. Metal blocking agents such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, glucose; caffeine, tannic acid, verapamil, tranexamic acid and its derivatives, licorice extract, glycyrrhizin , Hot water extract of pyracantha fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof such as vitamin C, sodium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid, etc. Whitening agents; sugars such as glucose, fructose, mannose, sucrose, trehalose.

  Examples of the external preparation for skin in the present invention include ointments, creams, emulsions, lotions, face masks, bath preparations, gels, and the like. Any of them may be used, and the dosage form is not particularly defined.

  “Treatment”, “in therapy”, and the like refer to a condition that alleviates, ameliorates, reduces, or improves all the symptoms caused by the condition that is bothering the patient or the condition, and the condition or current condition. Refers to the method of prevention.

  “Pharmaceutically acceptable” means that the substance or composition is compatible with the other ingredients to be formulated and is not harmful to the patient.

“Pharmaceutically acceptable excipient” is used in this document in the light of the knowledge of those skilled in the art, compatible with the physical and chemical properties of orchid extract, and physiologically. In addition, it refers to a pharmaceutically inert substance.
“Pharmaceutically acceptable excipient” means polymer, resin, plasticizer, filler, lubricant, diluent, binder, disintegrant, solvent, dissolution synergist, surfactant, preservative , Sweeteners, seasonings, pharmaceutical class dyes or glazes, and adhesives, but not limited to

  A “pharmaceutical composition” is a solid or liquid composition whose form, concentration, purity is adapted for administration to a patient (sick human or animal) and is physiological after administration. It refers to something that induces change. Usually, the pharmaceutical composition is sterile and non-pyrogenic.

  “Effective amount” refers to the amount necessary to cause a desired biological response. In light of the ordinary skill in the art, the effective amount of the composite or bioactive agent will depend on factors such as the endpoint, the bioactive agent delivery system, the material of the encapsulation, and the target tissue. .

  Although the Example of this invention is described below, it is not restricted to this. The orchids, raw materials, and biological materials used in the present invention are all commercially available, and the following examples are merely representative examples.

Example 1 Orchid Flower Extract Extraction Method The orchid flower extraction method and procedure are as shown in FIG. The orchid flower is the Phalaenopsis Sogo Yukidian 'Shiuh-Dong Whiskey'. This orchid is a plant cultivar right certificate variety character No. A00595, which is prepared as follows to obtain an extract.

  (1) Extraction: to orchids, weight ratio 0.5: 1 to 10: 1 (preferably 5: 1), time 3 to 60 minutes (preferably 10 minutes), temperature 0 to 60 ° C (0 to 35 ° C is preferred) (Preferably), a leach agent is mixed, extracted by a method such as ultrasonic, polishing, etc. (ultrasonic is desirable), and pulverized or broken to obtain orchid flower pulp. The leaching agent is water, alcohols or an aqueous solution of alcohols, and the alcohols are ethanol, propanol or butanol.

  (2) Solid-liquid separation: The solid and liquid of orchid flower pulp of (1) are separated. The separation time is 20 to 60 minutes (30 to 40 minutes is desirable), the temperature is 0 to 60 ° C. (0 to 35 ° C. is desirable), and a method such as centrifugation or squeezing is used, but the rotational speed is 1000 to 3000 rpm (1500 to 2000 rpm). Is preferable). Leave only the liquid orchid crude extract and remove the impurities.

  (3) Coarse filtration: The coarse orchid extract of (2) is coarsely filtered using diatomaceous earth, perlite, paper filter, cloth filter, and filter core to obtain a coarse filtrate. A combination of diatomaceous earth, perlite, and filtration core (0.1 to 5 μm) is desirable.

(4) Activity classification: The crude filtrate of (3) is purified through a zeolite membrane. This membrane filtration process is performed in two stages.
The first stage is an inorganic ceramic film (aluminum oxide film, zirconium oxide film, tellurium oxide, etc.) or organic composite film (polysulfone film (PS), acetic acid) having a mesh diameter of 0.04 to 1 μm (preferably 0.04 μm). Cellulose membranes, Teflon membranes, polypropylene membranes, etc.) are used to separate fine particles and obtain a permeate.
The second stage uses a nanofiltration membrane (NF) or reverse osmosis membrane with a weight average molecular weight of 100-1000 MW (preferably 150 MW). This thin film is a single thin film or a composite thin film of an organic component. The material of this thin film is a composite film (TFM, thin film membrane), polyvinylidene fluoride (PVDF), polysulfone (PS), polyethersulfone (PES). Polyacrylonitrile (PAN), etc., and the thin film separation pressure is in the range of 150-400 psi (200-300 psi is preferred). An active precipitate and a liquid active extract obtained by removing excess water and organic matter are orchid flower extract concentrates.

(5) Filtration: The orchid flower extract concentrate of (4) is filtered to obtain an active precipitate. The precipitate is washed and dried to obtain white and yellow crystals.
Dimethyl sulfoxide (DMSO) as a leaching agent, proton nuclear magnetic resonance (1H-NMR) (FIG. 3A) pulse sequence (Pulse Sequence): s2pul, 25 ° C., relaxation delay: 1.0 second, pulse 45 ° C. Acquisition time 3.000 seconds, sweep width (Width) 8000.0 Hz, integration number: 52, observation H1, 499.8787892 MHz, line width: 0.1 Hz, FT size (size): 65536, total time 4 minutes 16 seconds, and carbon 13 nuclear magnetic resonance (13C-NMR) (FIG. 3B), pulse sequence (pulse sequence): s2pul, 25 ° C., relaxation delay: 0.5 seconds, pulse 40 ° C., uptake time: 1 .042 seconds, sweep width (Width): 31446.5 Hz, number of integrations: 8 6, the observation C13, 125.6946696 MHz, decoupled H1, 499.8814989 MHz, power: 36dB, line width: 1.0 Hz, FT size: 131072, total time 50 minutes 50 seconds.
As a result of analysis, it is considered to be a flavone compound.

  When the structure of the orchid flower precipitate described above is analyzed by mass spectrometry (MS), the orchid flower precipitate is a flavone composition, which is mostly a flavone combined with two saccharides. Yes, but some are bound to a single saccharide.

The structure of the composition includes at least the following:
1. C ₂₆ H ₂₈ O ₁₄ :: MW: 564, a flavone linked to two sugars (liposide and glucoside), high content in the composition, apigenin-6-liposide-7- It is presumed to be glucoside (Apigenin-6-ribosido-7-glucoside) (FIG. 4A orchid flower precipitate and FIG. 4B standard product apigenin-6-liposide-7-glucoside).
2. C ₂₇ H ₃₀ O ₁₅ : MW: 594, a flavone linked to two sugars (two glucosides), with a high content in the composition, Saponarin (FIG. 5A orchid flower precipitate and FIG. 5B, C standard product saponarine).
3. C ₂₁ H ₂₀ O ₁₀ : MW: 432, flavone combined with one sugar (one glucoside), low content in the composition, apigenin-7-glucoside (FIG. 7A orchid flower precipitate and FIG. 7B) It is estimated that the standard product apigenin-7-glucoside).

Example 2 Skin irritation and sensitization test of orchid flower extract Tests to prove that orchid extract is not skin irritant and corrosive-cell test In vitro Skin Corrosion: Reconstructed Human Epidemis (RHE) Test Method (version 4.3, 2012/11) (OECD) 431), and the skin irritation standard test (In vitro Skin Irritation: Reconstructed Human Epidermis Test Method) (version 3.1, 2012/11) (OECD 439), see Cell Systems Model 3Cep3, German Cell Systems The test was carried out using the kit.

Skin corrosion / irritation tests were performed according to the OECD431 and OECD439 test standards described above. In the corrosive test, water was used as a negative control group, and potassium hydroxide was used as a positive control group. As in step (4), “Asahihigashi” was concentrated over a PS membrane having a mesh diameter of 0.04 μm and a composite membrane having a weight average molecular weight of 150 MW. Obtain a “weaver's disappointment” orchid extract (EW-DK). As a result of the OECD431 skin corrosion test, there was no skin corrosion (FIG. 7A).
In the skin irritation test, skin stimulation using epiCS was performed using Dulbecco's Phosphate-Buffered Saline as a negative control group and 5% sodium dodecyl sulfate (SDS) solution as a positive control group. As a result of the sex test, the “Asahi Toshishikikan” orchid extract extract had no skin irritation (FIG. 7B).

B. Test to prove that orchid flower extract has no eye irritation-cell test In this example, the concentration of 5.7% and 6.7% "Asahi Toshi disappointment" orchid flower extract concentrate, cervical cancer cells (HeLa cells) The eye irritation test was performed.
As in step (4) of Example 1, after obtaining the “Asahi Toshijiki” orchid flower extract concentrated solution over a polysulfone membrane having a mesh diameter of 0.04 μm and a composite membrane having a weight average molecular weight of 150 MW, solid content was further measured using an infrared moisture meter. Was measured, and extraction concentrates with concentrations of 6.7 wt% and 5.7 wt% were prepared and tested.

The procedure for the eye irritation test is as follows.
HeLa cells that have been stored in liquid nitrogen are thawed and cultured in minimal essential medium containing 5% fetal bovine serum (FBS) to the concentration required for the experiment. Five concentrations are prepared for the experiment by serially diluting a minimum essential medium containing 5% fetal calf serum at 10,000 μg / mL. After sufficient cells are cultured for the experiment, the cells are collected with trypsin, pipetted into each well of a 96-well tray (hereinafter referred to as tray), and cultured for 3 days. After adjusting the concentration of “Asahi Toshijiki” orchid flower extract concentrate (for experiment) to a concentration suitable for the experiment, it is placed in a tray and further cultured for 48 hours.

The medium was removed from the tray, washed with phosphate buffered saline (PBS), and then [3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyltetrazolium bromide] (3- (4,5- Di-methylthiazol-2-yl) -2,5-diphenyltetrazole bromide, yellow tetrazole (MTT) is added and cultured for 2 hours.
MTT is taken out from the tray, washed with phosphate buffered saline (PBS) (−), isopropyl alcohol containing hydrochloric acid is added, and the formazan dye produced in the cells is extracted. After confirming that the formazan dye has been extracted uniformly, the absorbance is measured at 570 nm, and the experimental concentration (EC ₅₀ ) when the cell viability reaches 50% is calculated. This experiment is carried out twice and an average value is taken for evaluation.

  As a result of the test, the concentration of 5.7% and 6.7% “Asahi Toshishikikan” orchid extract was not eye irritating (Table 2).

Table 2 Orchid extract of eye irritation test
* In order to obtain a complete non-irritant determination under conditions where animal experiments are not performed, the concentration (EC ₅₀ ) needs to be 5,000 μg / mL or more when the cell viability is 50%. Since EC ₅₀ of Table 2 exceeded 5,000 μg / mL, it was determined to be non-irritating.

Table 3 shows the effect of the concentration of the “Asahi Towei Imabari” orchid extract on cell viability.

Table 3
*% The solid content in the extract expressed as a percentage by weight.

C. A study proving that orchid extract has no skin irritation and sensitization-Clinical Trials This study was conducted by AMA Laboratories, Inc. on 52 subjects of different gender and age. An extract trial was conducted. The subjects were 9 males and 43 females aged between 18 and 69. The racial breakdown was 38 Caucasians, 12 Hispanics, and 2 Asians. there were. Each subject received 9 exposures and the results of the trial are shown in Table 4 (AMA laboratory report reference code: MS12.RIPT.M6496OP50.FEN.REV.2).
The results show that the “Asahi Toshijiki” orchid flower extract has no skin irritation and skin sensitization. This “Asahi Towei Imabari” orchid flower extract concentrate was obtained by applying a polysulfone membrane having a mesh diameter of 0.04 μm and a composite membrane having a weight average molecular weight of 150 MW, as in Step (4) of Example.

Table 4 Clinical trials demonstrating that orchid extract is not irritating to the skin and sensitizing
In the table, C: Caucasian, H: Hispanic, A: Asian, F: Female, M: Male. Of the nine exposure responses, “0” indicates no evidence of effect, and Dc indicates that the study was discontinued and the subject did not appear at the study site. The point “N / A” indicates that the point cannot be calculated due to the interruption of the trial. According to this table, only 2 people discontinued the study for personal reasons and the remaining 50 did not develop any skin irritation or sensitization reactions. The safety of this orchid flower extract concentrate to the skin is shown.

Example 3 Orchid Flower Extract Total Phenol Content Experimental Method: (1) Measurement of the effect of sodium carbonate on gallic acid absorption. Sodium carbonate with a concentration of 1,000 μg / mL gallic acid 0.25 mL, 20%, 2%, 0.2%, 0.02%, 0.002% and 0.0002% sodium carbonate. After reacting 5 mL and 4.25 mL of distilled water at room temperature for 25 minutes, the absorbance is measured with a spectrophotometer 200-800 nm. The final concentration of phagolic acid will be 50 μg / mL.
(2) The influence on the measurement of gallic acid is measured by the modified forin-thiocarte method in the order of adding the Folin-Ciocalteu reagent and sodium carbonate. Take 0.25 mL of gallic acid and the same volume of folin thiocarte reagent, 0.5 mL of 20% sodium carbonate and 4 mL of distilled water to a final concentration of 0, 20, 60 and 100 μg / mL of gallic acid. . After reacting at room temperature for 25 minutes, the absorbance is measured with a spectrophotometer at 730 nm.

  In the experiment, extraction was performed with water or aqueous ethanol solutions having different concentrations of 20, 40, 60, 80, and 100%, and the procedure was the same as in Example 1. When the crude filtrate was extracted, as shown in FIG. 8, the total phenol content showed different values depending on the concentration of the ethanol solution, and the water extraction showed a higher total phenol content than the ethanol extraction. Usually, the higher the total phenol content, the stronger the antioxidant properties.

Example 4 Antioxidant Evaluation of Orchid Extract Experimental Method: Measurement of diphenylpicrylhydrazyl (DPPH) radical scavenging ability. Prepare 2 ml of 0.2 g / ml test sample and 5 mg / ml of dibutylhydroxytoluene (BHT, 2,6-Di-tert-butyl-4-methylphenol) ethanol solution, and prepare a new one for each. After adding 0.5 ml of the prepared 2.5 × 10 ⁻⁴ M α, α-diphenyl-β-picrylhydrazyl (DPPH) ethanol solution, mixing until uniform, and standing for 30 minutes avoiding light, The UV / Vis region (UV-Vis) scan range is set to 400 nm-700 nm, the absorbance is measured at 517 nm, and the erasure rate is calculated. The lower the absorbance, the higher the erasing ability.
The experiment is duplicated. Radical scavenging ability EC ₅₀ measurement is based on the data obtained from Experiment 1 and measures the erasing rate of a plurality of test samples with different concentrations, and selects three samples with high erasing rates so that each sample has an appropriate concentration. Add 0.5 ml of 2.5 × 10 ⁻⁴ M concentration of diphenylpicrylhydrazyl (DPPH) to 2 ml of the sample ethanol solution, mix until uniform, leave for 30 minutes away from light, The absorbance is measured at 517 nm with the visible scan range set at 400 nm-700 nm.
The experiment is duplicated. A calibration curve of a test sample is drawn with the concentration on the Y axis and the erasure rate on the X axis, and the EC ₅₀ concentration with an erasure rate of 50% is calculated. The concentration of the “Asahi Towei Imabari” orchid flower extract concentrate (EW-DK) was obtained over a PS membrane having a mesh diameter of 0.04 μm and a composite membrane having a weight average molecular weight of 150 MW, as in Step (4) of the Example. The solid content is measured with an infrared moisture meter, and the concentration is unified to 2 mg / ml.

The experimental results are as follows. FIG. 9A shows the result of measuring the total phenol content by measuring the diphenylpicrylhydrazyl (DPPH) radical scavenging rate. Orchid flower shows that the water extract has stronger radical scavenging ability and the total phenol content is higher. Recognize. FIG. 9B is a comparison of the antioxidant ability of orchid flower extract concentrate and dibutylhydroxytoluene. Orchid flower extract concentrate has a much faster radical scavenging rate than dibutylhydroxytoluene and exhibits an antioxidant effect in a short time. It is shown that. FIG. 9C shows the results of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (2,2′-azino-bis [3-ethylbenzthiazine-6-sulphonic acid], ABTS) radical scavenging ability. According to the IC ₅₀ value, the orchid flower extract concentrate has higher radical scavenging ability than the orchid leaf extract concentrate.

Example 5 Whitening effect of orchid flower extract, suppression of tyrosinase Tyrosinase inhibition test-ethanol extraction at different concentrations The enzyme function measurement method employs a method that determines the functionality of the enzyme using the formation of dopachrome in the melanin production process by the enzyme, / Absorb at a visible region wavelength of 475 nm, calculate the enzyme function inhibition rate, and observe whether this compound has the ability to reduce melanin production.
1. Add 0.8 mM L-tyrosine and 0.9 M phosphate buffer solution (pH 6.8) to 980 μl of the test solution, mix until uniform, and incubate at room temperature for 10 minutes.
2.4440.5 units of tyrosinase and 10 μl of test sample are mixed rapidly and the change in absorption is measured for 3 minutes at a UV / Vis wavelength of 475 nm.
Tyrosinase inhibition rate (%) = {(control group-experimental group) / control group} × 100%

  “Asahi Toshi disappointment” Orchid uses 0-100% ethanol as an extractant and measures the solid content of Orchid filtrate extracted by the method of Step 1 of Example 1 with an infrared moisture meter to unify the concentration to 2 mg / ml To do. As a result of the experiment, as shown in FIG. 10A, when six types of test samples were prepared and inhibition of tyrosinase function was evaluated, both the first group and the second group had four types of test samples (60%, 40% %, 20% ethanol extraction and water extraction) showed a tyrosinase inhibition rate of 75% or more. (Note: 80% ethanol extraction was excluded because of the change in properties of tyrosinase in the test using a test tube.) In the functional comparison with the whitening component approved by the health department (3 mM arbutin was used in this experiment) A low concentration of orchid flower extract has a remarkable tyrosinase inhibitory effect, and the experiment divided into two groups shows reproducibility. Orchid flower extract has excellent potential in terms of whitening function. I understand.

B. Tyrosinase inhibition test-extracts (products) with different orchid varieties and procedures
Using various orchids and extracts (products) obtained by different procedures, water is used as the leaching agent. The difference is that after step (3), the solid content is measured with an infrared moisture meter, and the concentration is unified to 2 mg / ml. See Table 5.

Table 5 Method for obtaining orchid flower active extract in the present invention

  The experimental results are shown in FIG. 10B. “Asahi Toshi disappointment” and “Beauty” of the same genus and different varieties have both tyrosinase inhibitory functions, but “Asahi Toshi disappointment” is superior. The extracts of “Aoyama Daikoen”, which have different genera and varieties, have no tyrosinase inhibitory function. Therefore, it can be seen that even if the same phalaenopsis orchid is different, there is a difference in the suppression effect between different varieties.

C. Tyrosinase Inhibition Test-Difference in Effect between Flowers and Leaves After extracting water with orchids and orchids from "Asahi Toshishiki", extraction of orchids and orchids through a polysulfone membrane with a mesh diameter of 0.04 μm in step (4) After obtaining the concentrate, the solid content is measured with an infrared moisture meter, and the tyrosinase inhibition test is carried out with a concentration of 2 mg / ml.

The result of the experiment is shown in FIG. 10C. IC ₅₀ shows the effect of inhibiting tyrosinase, and it can be seen that the orchid extract concentrate has a superior ability to inhibit tyrosinase than the orchid extract extract.

Example 6 Orchid flower extract whitening function-Experimental method for inhibiting melanin production in mouse-derived melanoma cells is as follows.
1. Mouse-derived melanoma cells (B16) are cultured in 6-well trays, Dulbecco's modified Eagle's minimum essential medium (DMEM) (including 10% fetal calf serum) is added, and the mixture is incubated at 37 ° C and cultured with 5% carbon dioxide. The next day, add different concentrations of reagents to the culture medium.
2. After culturing the culture solution containing different concentrations of the reagent for 3 days, washing the cells with phosphate buffer solution, adding appropriate amount of trypsin / ethylenediaminetetraacetic acid (EDTA) for 3 minutes, separating the cells from the culture dish The fat is taken out, washed with a phosphate buffer solution, and centrifuged at 5000 rpm for 5 minutes.
3. After centrifugation, the supernatant is removed, phosphate buffer solution is added, and the mixture is slowly mixed with the cell precipitate until uniform, and then centrifuged at 5000 rpm for 5 minutes.
4). Repeat step 3.
5. The supernatant is removed and 1 ml of 1N sodium hydroxide is added to the cell pellet to disperse the cells uniformly in the sodium hydroxide solution.
6). Centrifuge at 3000 rpm for 10 minutes, take 1 ml of supernatant and absorb at ultraviolet / visible wavelength 405 nm.
7). The measured value is divided by the number of viable cells to obtain the experimental result of melanin content measurement.

  In order to test the inhibitory effect of raw orchids on mouse-derived melanoma cells, extract "Asahi Toshijiki" orchids with different concentrations of leachate (water, 20% ethanol, 80% ethanol, 100% ethanol), step (2) Active precipitate obtained by further separating orchid flower extract concentrate obtained by applying orchid flower filtrate (referred to as No. 1-4), step (4) polysulfone membrane having a mesh diameter of 0.04 μm, and composite membrane having a weight average molecular weight of 150 MW (No. 5), Arbutin 5 mM (No. 6), and a control group (No. 7, 1% dimethyl sulfoxide) were prepared. To unify. See Table 6 below.

Table 6 Breakdown of the different extracts added to each test tube

  The result of the experiment is shown in FIG. In the experiment of inhibiting melanin production by mouse-derived melanoma cells, arbutin, ethanol-extracted orchid flower extract, water-extracted orchid flower extract white crystals showed remarkable melanoma cell (B16) inhibitory effect.

  B. Whitening function of orchid flower extract-Primary culture Human melanocyte (HMC-4) suppresses the melanin production using "Asahi Toshijiki" orchid flower extract with different dose and extraction method, primary culture human A test for suppressing melanin production is performed on melanocytes. Before conducting the test for melanin suppression, an experiment was conducted to examine cell viability against human melanocyte cells using the “Asahi Toshijiki” orchid flower extract with different doses and extraction methods, and the maximum cell viability was 95%. Test melanin suppression at test dose (concentration).

  Tables 7 and 8 were subjected to melanin suppression tests using “Asahi Toshijiki” orchid flower extract with different doses and extraction methods. 12A to 12H are the results of an experiment in which the cell viability against human melanocytes was examined using each “Asahi Toshijiki” orchid flower extract in Tables 7 and 8, and FIG. 13 shows different doses and extraction methods. It is a result of the melanin suppression test by the "Asahi Towei Immoral" orchid flower extract. According to FIG. 13, all doses of “Asahi Toshijiki” orchid flower extract used in the melanin suppression test were performed at the maximum test dose (concentration) with cell viability exceeding 95%. , Showed a melanin inhibitory effect.

Table 7 Dose and Extraction Method of “Asahi Toshijiki” Orchid Extract Used for Melanin Inhibition Test

Table 8 Dose and Extraction Method of “Asahi Toshijiki” Orchid Flower Extract Used in Melanin Inhibition Test

Example 7 Anti-wrinkle (anti-aging) function of orchid flower extract 1. Matrix metalloproteinase-1 (MMP-1)
MMP-1 (matrix metalloproteinase-1, Matrix Metalloproteinase-1) is known as a kind of collagen degrading enzyme, and functions to degrade and regulate collagen in dermal tissue.
In this example, first, as in step (4) of Example 1, an “Asahi Toshijiri” orchid flower extract concentrate is obtained by applying to a polysulfone membrane having a mesh diameter of 0.04 μm and a composite membrane having a weight average molecular weight of 150 MW. Furthermore, the solid content is measured with an infrared moisture meter, and the test is advanced by unifying the concentration to 6.7% and 5.7% by weight.

The result of the experiment is shown in FIG. The method and procedure for measuring anti-wrinkle (anti-aging) function are as follows.
1. Thaw normal diploid fibroblasts (TIG-118, Normal human lipid fibroblasts) stored in liquid nitrogen, and use the minimum essential medium containing 5% fetal bovine serum to obtain the number of cells required for the experiment. Incubate until.
2. Before conducting the preliminary test, after confirming that the test substance has a concentration that is not toxic to the cells (TIG-118), dilute it twice from the maximum concentration that is optimal for the formal test, A total of three concentrations (1%, 0.5%, 0.25%) are obtained as test concentrations.
3. The cells having the number of cells required for the experiment in Step 1 are trypsinized and then pipetted onto a 35 mm culture dish (dispersed to a cell density of 50,000 cells / mL).
4. After culturing the cells pipetted on a 35 mm culture dish for 24 hours, remove the culture solution, add the test solution containing the test substance diluted in Step 2, and further culture for 24 hours, then messenger ribonucleic acid (MRNA) Cells (reagent extraction kit: RNeasy Mini kit, Qiagen) are extracted, and the gene expression level of MMP-1 is measured by real-time quantitative polymerase chain reaction (Quantitative real-time PCR).

Real-time polymerase chain reaction (PCR) analysis conditions:
Equipment used: ABI PRISM 7900HT (ABI PRISM 7900HT), Applied Biosystems Co., Ltd.
Polymerase chain reaction temperature conditions:
Stage 1: 42 ° C 5 minutes, 95 ° C 10 seconds (1 cycle)
Stage 2: 95 ° C 5 seconds, 60 ° C 34 seconds (40 cycles)
Stage 3: 95 ° C for 15 seconds, 60 ° C for 1 minute, 95 ° C for 15 seconds (1 cycle)

2. Elastase
Decreased skin elasticity and degeneration of elastic fibers in the dermis layer cause wrinkles in the skin, but elastase inhibitors that suppress elastase activity prevent skin elastic fiber damage It helps to reduce the formation of wrinkles.

In this example, 5% and 1% concentrations of “Asahi Toshijikikan” orchid flower extract concentrates were diluted with Tris-HCl buffer solution, respectively, and prepared for 5 different concentrations, respectively, and tested for elastase activity inhibition ( Table 9).
The “Asahi Tozai Imabari” orchid flower extract concentrate showed a good elastase activity inhibition rate, both 5% and 1%. Next, the elastase inhibition IC ₅₀ test of 5% and 1% “Asahi Toshishikikan” orchid flower extract concentrate is performed (Table 10). By the way, the “Asahi Tozai Imabari” orchid flower extract concentrate was obtained by applying a polysulfone membrane having a mesh diameter of 0.04 μm and a composite membrane having a weight average molecular weight of 150 MW as in Step (4) of Example.

Table 9 Inhibition rate of elastase activity of "Asahi Toshishiki" orchid flower extract concentrates with different concentrations

Table 10 Elastase Inhibition IC _{50 of} “Asahi Toshijiki” Orchid Extract Concentrates with Different Concentrations

The method for carrying out this elastase activity inhibition test is as follows.
“Asahi Toshijiki” orchid flower extract concentrates with different concentrations, enzyme solution (elastase solution), substrate solution (N-succinyl-alanine-alanine-alanine-p-nitroanilide, N-Succiny-Ala-Ala-Ala- p-Nitroanilide) is mixed, heated at 37 ° C. for 15 minutes, the absorbance is measured at 415 nm, and the elastase activity inhibition rate is calculated by the following formula.
A: Absorbance after reaction of blank solution B: Absorbance after reaction of experimental solution A0, B0: Absorbance when using alternative enzyme solution and Tris-HCl buffer solution

3. Collagenase
Collagenase breaks down collagen in the skin and forms wrinkles to promote aging, but if there is a collagenase inhibitor that suppresses collagenase, wrinkle formation and aging can be prevented. In this example, 5.7% and 6.7% “Asahi Toshishikikan” orchid flower extract concentrates were added and each of them was continuously diluted twice by adding a lemon acid buffer solution. And a collagenase activity inhibition test is performed (Table 11). Among them, 100% 5.7% and 6.7% “Asahi Toshijiki” orchid flower extract concentrate showed an excellent collagenase activity inhibition rate. By the way, the “Asahi Toshijiki” orchid flower extract concentrate was obtained by applying it to a polysulfone membrane having a mesh diameter of 0.04 μm and a composite membrane having a weight average molecular weight of 150 MW as in Step (4) of the Example. Minutes were measured and the concentration was adjusted to 6.7% and 5.7% to obtain an extract concentrate, and the test was conducted.

Table 11 Inhibition rate of collagenase activity of concentrated solutions of "Asahi Toshijiki" orchid extract with different concentrations

The method for carrying out this collagenase activity inhibition test is as follows.
Concentrated "Asahi Toshijiki" orchid flower extract concentrates with different concentrations, peptide solutions (4-phenylazobenzyloxycarbonyl-L-proline-leucine-glycine-proline-D-arginine, 4-phenazobenzoyloxycarbonyl-L-pro-leu-gly-pro -D-arg dihydrate) was mixed and heated at 37 ° C., then an enzyme solution (collagenase) was added and reacted at 37 ° C., and then 0.1 M Tris buffer solution (Tris buffer, containing 20 mM calcium chloride) The reaction is stopped by adding pH 7.1), and ethyl acetate is added, followed by centrifugation at 3,000 rpm. The absorbance of the ethyl acetate layer is measured at 320 nm, and the collagenase activity inhibition rate (%) is calculated by the following formula.
A: Absorbance after reaction with blank solution B: Absorbance after reaction with experimental solution A ₀ , B ₀ : Absorbance when purified water is used instead of enzyme solution

Example 8 Human skin protective function clinical trial of orchid flower extract In this example, as in step (4) of the example, "Asahi Toshishiki" orchid flower extraction was performed on a polysulfone membrane having a mesh diameter of 0.04 μm and a composite membrane having a weight average molecular weight of 150 MW. After obtaining the concentrate, the solid content was measured with an infrared moisture meter, and the clinical trial was conducted with a concentration of 1.0%. Thirty-one subjects with different ages and genders were tested for effects on human skin at Taidai Clinic, and the results were analyzed two weeks later.

  In this image inspection, evaluation was made based on four types of visible spots, ultraviolet color spots, brown spots, and red spots. Visible spots are black spots that are visible to the naked eye under normal light. The ultraviolet color spot is a black spot that can be seen by irradiating with 365 nm ultraviolet rays (spots that cannot be seen with the naked eye can be taken by irradiating ultraviolet rays). The brown spot is a spot evaluation of the deep skin layer analyzed by the melanin / hemoglobin index image analysis function (RBX function). Red spots are evaluations of red spots analyzed by the melanin / hemoglobin index image analysis function. The numerical values in the table represent a large quantity or a small quantity, and the score represents the feature level (density) of the inspection item.

  There was no adverse reaction or discomfort in the subjects even after 2 weeks of use. As shown in Table 12 and Table 13, the number of visible spots, brown spots, and red spots decreased as a result of the trial, but there was no statistical difference in the degree of characteristics (density). Looking at the analysis results for two weeks of use, it can be seen that the number of black pigment spots (visible spots and deep spots) has decreased, and the number of red spots has decreased.

Table 12: Measured numerical values of orchid flower extract on human skin

Table 13 Statistics of the results of measuring the effects of orchid extract on human skin

The lower the value, the more remarkable the effect.
* Statistical difference p <0.05

The present invention uses a white phalaenopsis orchid that is a unique variety cultivated by orchid Aoyama, and after extracting the extract, a good whitening function is achieved through experiments and trials of tyrosinase inhibition, mouse-derived melanoma cell (B16) and human melanocyte inhibition. It has anti-wrinkle (anti-aging) function, anti-aging function, and, after many cell experiments, in vitro tests and clinical trials, none of them are corrosive, irritating and sensitizing to the skin. Demonstrated.
Although this research team has been engaged in research on plant activity for many years, active ingredients showing whitening effects have been demonstrated and acted on in many types of strains (enzymes, animal cells, primary cultured human skin cells, and clinical trials). A mild but non-irritating ingredient is rare. In this area, the superiority of this raw material and the manufacturing process is shown.

  In the production process, after the extract is subjected to a thin film treatment, mass production is possible, and the effect of separation, purification and concentration can be obtained. The concentrated phalaenopsis extract contained a large amount of precipitates, which were collected and analyzed. As a result, the precipitates were a group of three flavone compounds having similar structures. Both the extract and the precipitate have good whitening ability, anti-wrinkle (anti-aging) ability and antioxidant ability. This manufacturing process is a green manufacturing process, does not produce toxic organic solvents, can be easily expanded, and produces a safe and sanitary active extract concentrate and flavone compounds obtained by precipitation through microbial control. can do.

  When tested in different genus Phalaenopsis, there was no tyrosinase inhibitory effect, and when compared with phalaenopsis of the same genus, all showed tyrosinase inhibitory effect, but this white flower variety had the most excellent inhibitory effect. The production process and reagent used in the present invention can be used in cosmetics and skin care products.

110 Prepare the petals of orchids 120 Mix orchids and leaching agent to crush or break the walls 130 Separate solids and liquids 140 Divide actives 150 Concentrate 160 Obtain active precipitates and liquid active extracts

Claims (25)

A kind of orchid flower extract, which belongs to the genus Phalaenopsis,
(1) Extraction: An orchid flower is mixed with a leachate in a weight ratio of 0.5: 1 to 10: 1 and pulverized or broken to obtain an orchid flower pulp. In a mixture, the alcohols are ethanol, propanol or butanol;
(2) The solid and liquid of orchid flower pulp of (1) are separated, and impurities are removed to obtain a liquid orchid flower filtrate;
(3) Activity classification: The filtrate of (2) is purified through a zeolite membrane to obtain an orchid flower extract screening solution;
(4) Concentration: The screening solution of (3) is concentrated using a zeolite membrane or partially concentrated by a method such as a vacuum device or heating to obtain an active precipitate and an active liquid extract.
Orchid flower extract prepared by the procedure.   The orchid flower extract according to claim 1, wherein the orchid orchid is a phalaenopsis genus Sogoyukidian species "Palaenopsis Sogo Yukidian 'Shiuh-Dong Whiskey').   The orchid flower extract according to claim 1, wherein the orchid flower and the leachate in step (1) are mixed and extracted at a weight ratio of 5: 1.   The orchid flower extract according to claim 1, wherein the solid-liquid separation temperature in step (2) is 0 to 60 ° C.   The orchid flower extract according to claim 4, wherein the temperature of the solid-liquid separation in step (2) is 0 to 35 ° C.   In claim 1, at least one of diatomaceous earth, perlite, paper filter, and filtration core (0.1 to 5 μm) is used as a filtration material by using a centrifugal method or a filtration method in the solid liquid separation of step (2). An orchid flower extract characterized by being used.   In claim 1, the membrane filtration process in the active section of step (3) and the concentration in step (4) is the first stage is an inorganic ceramic membrane or organic composite membrane, the second stage is a nanofiltration membrane (NF, nanofiltration). An orchid flower extract characterized by being divided into two stages, ie, membrane) or reverse osmosis membrane.   8. The orchid flower extract according to claim 7, wherein the inorganic ceramic film contains an aluminum oxide film, a zirconium oxide film, or tellurium oxide.   8. The orchid flower extract according to claim 7, wherein the organic composite film includes a polysulfone film, a cellulose acetate film, a Teflon film, and a polypropylene film.   The nanofiltration membrane or reverse osmosis membrane according to claim 7 is a single thin film or a composite thin film, and the material of the thin film is a composite film (TFM, thin film membrane), polyvinylidene fluoride (PVDF), polysulfone ( An orchid flower extract comprising at least one of PS), polyethersulfone (PES), and polyacrylonitrile (PAN).   The orchid flower extract according to claim 1, wherein the structure of the active precipitate in step (4) is determined by analyzing the chemical structure using nuclear magnetic resonance (NMR) and mass spectrometry (MS).   A pharmaceutical composition comprising an effective amount of orchid flower extract concentrate according to claim 1, a precipitate or a combination thereof, and a pharmaceutically acceptable excipient.   An orchid flower extract characterized by being used for the preparation of a composition having a whitening function in the orchid flower extract concentrate, precipitate or a combination thereof according to claim 1.   The orchid flower extract according to claim 13, which is used for the preparation of a whitening drug that suppresses tyrosinase.   The orchid flower extract according to claim 13, wherein the orchid flower extract is used for the preparation of a whitening drug for suppressing melanin production.   An orchid flower extract comprising the orchid flower extract concentrate according to claim 1, a precipitate, or a combination thereof, which is used as a drug for treating a disease caused by a radical.   The orchid flower extract according to claim 16, wherein the orchid flower extract is used for the preparation of a drug for quickly eliminating radicals.   An orchid flower extract comprising the orchid flower extract concentrate according to claim 1, a precipitate, or a combination thereof, for use in preparing anti-wrinkle and anti-aging drugs.   The orchid flower extract according to claim 18, which is used for preparing a drug for treating a disease caused by lack of collagen.   The orchid flower extract according to claim 18, which is used for the preparation of an anti-wrinkle or anti-aging drug that inhibits a matrix metalloproteinase.   The orchid flower extract according to claim 18, which is used for the preparation of an anti-wrinkle or anti-aging drug that inhibits collagenase.   The orchid flower extract according to claim 18, which is used for preparation of an anti-wrinkle or anti-aging drug that suppresses elastase. An orchid extract, which belongs to the genus Phalaenopsis,
(1) Extraction: An orchid flower is mixed with a leachate in a weight ratio of 0.5: 1 to 10: 1 and pulverized or broken to obtain an orchid flower pulp. In a mixture, the alcohols are ethanol, propanol or butanol;
(2) Solid-liquid separation: Separating the solid and liquid of orchid flower pulp of (1), removing impurities and obtaining a liquid orchid flower filtrate;
Orchid flower extract to be prepared in the procedure. An orchid extract, which belongs to the genus Phalaenopsis,
(1) Extraction: An orchid flower is mixed with a leachate in a weight ratio of 0.5: 1 to 10: 1 and pulverized or broken to obtain an orchid flower pulp. In a mixture, the alcohols are ethanol, propanol or butanol;
(2) Solid-liquid separation: Separating the solid and liquid of orchid flower pulp of (1), removing impurities and obtaining a liquid orchid flower filtrate;
(3) Activity classification: The filtrate of (2) is purified through a zeolite membrane to obtain an orchid flower extract screening solution;
(4) Concentration: The screening solution of (3) is concentrated using a zeolite membrane or partially concentrated by a method such as a vacuum device or heating to obtain an active precipitate and an active liquid extract.
Orchid flower extract to be prepared in the procedure. A method for preparing an orchid extract, which is of the genus Phalaenopsis,
(1) Extraction: An orchid flower is mixed with a leachate in a weight ratio of 0.5: 1 to 10: 1 and pulverized or broken to obtain an orchid flower pulp. In a mixture, the alcohols are ethanol, propanol or butanol;
(2) Solid-liquid separation: Separating the solid and liquid of orchid flower pulp of (1), removing impurities and obtaining a liquid orchid flower filtrate;
(3) Activity classification: The filtrate of (2) is purified through a zeolite membrane to obtain an orchid flower extract screening solution;
(4) Concentration: The screening solution of (3) is concentrated using a zeolite membrane or partially concentrated by a method such as a vacuum device or heating to obtain an active precipitate and an active liquid extract.
The orchid flower extract preparation method prepared by the procedure of.