TW201420127A - Extract of Phalaenopsis amabilis meristem, and the preparation process and uses thereof - Google Patents

Abstract

Disclosed herein are an extract of Phalaenopsis amabilis meristem and the preparation process thereof. Also disclosed is that the extract of Phalaenopsis amabilis meristem can be used in promoting skin whitening, enhancing the moisture-retaining capacity of the skin, and preventing and/or retarding skin aging.

Description

Extract of white flower phalaenopsis meristem and preparation method thereof And use

The invention relates to an extract of Phalaenopsis amabilis meristem and a preparation method thereof. The present invention also relates to the use of the extract of the genus Phalaenopsis meristem to promote skin whitening, enhance skin moisturizing ability, and prevent and/or delay skin aging.

Skin is the biggest barrier to protect humans. It has the function of resisting water loss, pathogens and various environmental damages. Exposure to large amounts of ultraviolet (UV), ionizing radiation, drugs, or xenobiotics can cause the skin to produce reactive oxygen species (ROS) and free radicals. When the amount of active oxygen species and free radicals accumulated exceeds the antioxidant capacity of the cell or tissue itself, the reactive oxygen species and free radicals react with intracellular components (including DNA, proteins, lipids, etc.) and The skin has an undesired effect. It has been reported that reactive oxygen species and free radicals play an extremely important role in melanogenesis and skin aging (Kim YJand Yokozawa T. (2009), Biol . Pharm . Bull ., 32 :1155-1159).

Melanogenesis refers to the process by which tyrosine is converted into melanin by catalysis of tyrosinase and a series of redox reactions in melanocytes, which are mainly ultraviolet rays. [Specially caused by ultraviolet-B (UV-B)] irradiation. When the skin is exposed to ultraviolet light, the reactive oxygen species and free radicals generated in keratinocytes induce the expression of tumor protein 53 (p53), which promotes the pro-brain melanocyte The gene (proopiomelanocortin gene, POMC gene) expresses and releases POMC-derived peptides (such as α-melanocyte stimulating hormone (α-MSH) and adrenal cortex The adrenocorticotropic hormone (ACTH), which in turn causes the tyrosinase gene in melanocytes to be expressed, eventually causing a large amount of melanin to accumulate on the skin.

Skin aging can be divided into the following two categories: (1) intrinsic skin aging [also known as chronological aging]: it is a natural aging process over time, mainly because The oxidative cellular metabolism produces a reactive oxygen species and the continuous accumulation of free radicals; and (2) extrinsic skin aging [also known as photoaging]: it It is premature aging caused by environmental factors (especially ultraviolet radiation). In the process of skin aging The reactive oxygen species and free radicals produced by oxidative cellular metabolism and ultraviolet radiation induce a series of matrix metalloproteinases by promoting the formation of transcription factor 1 (AP1). The performance of MMPs, in turn, promotes the degradation of collagen and other constituents of the extracellular matrix [eg, elastin and proteoglycan], which ultimately leads to skin aging. Symptoms of skin aging include: telangiectasia, epidermal thinning, skin atrophy, reduction of collagen fibers and elastic fibers, elastic tissue degeneration (elastosis), skin elasticity ( Skin elasticity), skin texture roughening, dryness, wrinkle formation, and pigmentary change [eg, lentigines, freckles, hypopigmentation) ) or hyperpigmentation].

In recent years, human demand for skin whitening, antiaging, and moisturizing has increased. In particular, the use of natural ingredients does not cause undesired adverse side effects on the skin, and thus has become the current medical beauty. Market trends. Therefore, researchers in the pharmaceutical and cosmetic industries are trying to find active ingredients that are safe and have whitening, anti-aging and moisturizing effects from traditional Chinese medicines (TCM) or plants for the vast majority of medicine. The needs of the beauty market.

White Phalaenopsis (Latin name: Phalaenopsis amabilis; English common name: Moon Orchid) orchids (Orchidaceae) genus Phalaenopsis (Phalaenopsis) of the perennial herb; short stems, are tightly coated with the sheath; leaves oval appearance , oblong or sickle-shaped oblong, leathery; flowers are white, flowers like butterflies. The production areas are distributed in Taiwan, India and the Philippines.

At present, there have been studies on the water-extracted products of Phalaenopsis chinensis and the use of alcohol-extracted products for skin whitening. For example, CN 100589790 C discloses a skin whitening or depigmenting cosmetic comprising from 0.005 to 5% of the total weight of the cosmetic, water or alcohol or water/alcohol extract of leaves, flowers and stems of Phalaenopsis, and 0.5 to 5% of the total weight of the cosmetic kiwi extract. According to the specification of this Continental Patent, an extraction temperature in the range of 10-40 ° C can be used in the preparation of water, alcohol or water/alcohol extract of the leaves, flowers and stems of Phalaenopsis sinensis, and in one yuan. An alcohol (for example, ethanol, propanol, isopropanol or a mixture thereof) or a polyol (for example, propylene glycol) is used as an extraction solvent. In this embodiment of the Continental Patent, a product of propylene glycol extracted from propylene glycol and an ethanol-extracted product is obtained, and the propylene glycol-extracted product of the white flower Phalaenopsis is further extracted with ethanol from one of the kiwis. Together, they were made into a whitening cream which was confirmed to have depigmentation effects through human trials.

In the book "Comparison of ten flowers on the Whitening effect" by Zeng Meijun of the Department of Biotechnology, Nantai University of Science and Technology, Zeng Meizhen has 6 kinds of flowers [that is, the color of the flowers is white and yellow White with yellow lip, white orchid, red white with red lip, red impatiens walleriana , white white balsam, white rose ( Rosa rugosa) The petals of var.alba Ware) and red rose ( Rosa rugosa var.rosea Rehd) were taken separately and chopped separately, followed by soaking extraction with 50 mL of 95% ethanol at 60 ° C for 24 hours. Thus, the ethanol-extracted product of the six kinds of flowers was obtained. Zeng Meizhen took the ethanol-extracted product of the six kinds of flowers for the test of tyrosinase inhibition, and the experimental results showed that the flower color was the product of the ethanol-extracted product of the white flower phalaenopsis petals of the white flower yellow heart. There was no significant difference between the aminase inhibition ability and the white flower Phalaenopsis whose color was red and white. The tyrosinase inhibitory ability of the ethanol-extracted product of red rose petals is among the white flowers of the phalaenopsis, the red balsam, the white African balsam, the white rose and the red rose. The best.

Upon investigation, the applicant unexpectedly discovered that extracts of the genus Phalaenopsis meristem [including lipid soluble extract, water soluble extract, and alcohol soluble extract] have Promotes skin whitening, enhances skin's moisturizing ability and anti-aging effect. Therefore, the extract of the genus Phalaenopsis meristem is expected to be available as a skin care product.

Summary of invention

Thus, in a first aspect, the present invention provides an extract of Phalaenopsis amabilis meristem, which is produced by a method comprising the following steps: supercritical CO ₂ (supercritical CO) ₂ ) To extract the melon tissue of the white flower, and obtain a fat-soluble extract of the butterfly orchid meristem.

In a second aspect, the present invention provides an extract of the genus Phalaenopsis meristem, which is obtained by a method comprising the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ , borrowing Thus, a fat-soluble extract of a genus Phalaenopsis meristem and a residue are obtained; and the residue is extracted with water to obtain a water-soluble extract of the genus Phalaenopsis meristem.

In a third aspect, the present invention provides an extract of the genus Phalaenopsis meristem, which is obtained by a method comprising the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ , borrowing Thus, a fat-soluble extract of the genus Phalaenopsis meristem and a residue are obtained; and the residue is extracted with ethanol to obtain an alcohol-soluble extract of the genus Phalaenopsis meristem.

In a fourth aspect, the present invention provides a method for preparing an extract of a genus Phalaenopsis meristem, comprising the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ , thereby obtaining a A fat-soluble extract of the genus Phalaenopsis meristem and a residue.

According to the present invention, the method may further comprise the steps of: extracting the residue with water to obtain a white flower of the Phalaenopsis meristem Water soluble extract.

Further, according to the present invention, the method may further comprise the steps of: extracting the residue with ethanol to obtain an alcohol-soluble extract of a butterfly flower meristem of white flowers.

In a fifth aspect, the present invention provides a composition for whitening skin comprising any one of the extracts of three kinds of white-flowered Phalaenopsis meristems as described above, or a combination thereof.

In a sixth aspect, the present invention provides a method for whitening skin comprising administering to a subject in need of promoting skin whitening a composition as described above.

In a seventh aspect, the present invention provides a composition for enhancing the moisturizing ability of the skin, which comprises any one of the extracts of three kinds of white-flowered Phalaenopsis meristems as described above, or a combination thereof.

In an eighth aspect, the present invention provides a method for enhancing the moisturizing ability of the skin, which comprises administering to a subject in need of enhancing the moisturizing ability of the skin a composition as described above.

In a ninth aspect, the present invention provides a composition for anti-skin aging comprising any one of the extracts of three species of Phalaenopsis meristems as described above, or a combination thereof.

In a tenth aspect, the present invention provides a method for anti-skin aging comprising administering to a subject in need of prevention and/or delaying skin aging a composition as described above.

The above and other objects, features, and advantages of the present invention will become apparent by reference to the appended claims Will become obvious.

Detailed description of the invention

For the purposes of this specification, it will be clearly understood that the words "comprising" means "including but not limited to" and the words "comprises" have a corresponding meaning.

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the former publication forms a common general in the art. Part of the knowledge.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

In order to obtain natural active components having a skin-care effect from plants, Applicants have attempted to extract different parts of Phalaenopsis morifolium in various different ways, and further take the obtained extracts into living bodies. In vitro test and human test found: lip soluble extract, water soluble extract and alcohol soluble extract of Phalaenopsis amabilis meristem (alcohol soluble extract) has the effect of promoting skin whitening, enhancing skin's moisturizing ability and anti-aging skin.

Accordingly, the present invention provides a fat-soluble extract of the genus Phalaenopsis meristem, which is obtained by a method comprising the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ , thereby A fat-soluble extract of the white-flowered Phalaenopsis meristem and a residue are obtained.

According to the present invention, the residue is further subjected to extraction treatment with water, whereby a water-soluble extract of the genus Phalaenopsis meristem is obtained.

Further, according to the present invention, the residue is further subjected to extraction treatment with ethanol, whereby an alcohol-soluble extract of a butterfly flower meristem of white flower is obtained.

The invention also provides a method for preparing an extract of the genus Phalaenopsis meristem, which comprises the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ , thereby obtaining a white flower phalaenopsis The fat-soluble extract of the tissue and a residue.

According to the invention, the method further comprises the step of extracting the residue with water and extracting a water-soluble extract of a butterfly flower melon tissue.

Further, according to the present invention, the method further comprises the steps of: extracting the residue with ethanol to obtain an alcohol-soluble extract of a butterfly flower meristem of white flower.

As used herein, the term "meristem" means a cell comprising cells capable of dividing and eliciting similar cells or which will differentiate to produce tissues or organs (eg, roots, stems, leaves, and flowers). Formal plant tissue.

According to the present invention, the genus Phalaenopsis meristem is selected from the group consisting of: shoot apical meristem, root apical meristem, floral meristem (floral) Meristem), inflorescence meristem, and axillary meristem. In a preferred embodiment of the invention, the white-flowered phalaenopsis meristem is a twin meristem.

As used herein, the term "supercritical CO ₂ (supercritical CO _2)" or "supercritical carbon dioxide (supercritical carbon dioxide)" is used to mean CO ₂ is maintained at a temperature equal to or greater than a threshold (i.e., 31.1 ℃ And a state of a critical pressure (ie, 1100 psi), in which CO ₂ has a viscosity similar to a gas and a density of a liquid, and thus can be used as an extraction Solvent.

The operating procedures and parameter conditions for the extraction treatment using supercritical CO ₂ fall within the scope of professional literacy and routine techniques of those skilled in the art. In this regard, reference can be made, for example, to Pan Jianliang and Yang Shiwei (2010), Mining and Metallurgy, 54:83-89, and a master's thesis by Wu Junyi of the National Institute of Chemistry, National Cheng Kung University [Name: "Study on the extraction of seal oil by supercritical carbon dioxide""Study on extraction of seal oil with supercritical fluid carbon dioxide"", and a master's thesis by Lin Shuangqiu of the National Institute of Chemistry, National Cheng Kung University [Name: "Supercritical carbon dioxide extraction of Chinese herbal medicine and its extracts to inhibit melanin production Studies on the supercritical carbon dioxide extraction of the Chinese herbal medicine and its extract for inhibition of melanin formation)].

According to the present invention, the extraction of the supercritical CO ₂ is carried out at a temperature ranging from 35 ° C to 100 ° C and a pressure ranging from 1500 psi to 7250 psi. In a preferred embodiment of the invention, the supercritical CO ₂ extraction is carried out at a temperature of 40 ° C and a pressure of 4350 psi.

According to the present invention, the water-soluble extract of the genus Phalaenopsis meristem is obtained by mixing the residue with water and performing an ultrasonic treatment.

According to the present invention, the alcohol-soluble extract of the genus Phalaenopsis meristem is obtained by mixing the residue with ethanol and performing an ultrasonic treatment.

According to the invention, the ultrasonic treatment is carried out at a temperature ranging from 10 ° C to 50 ° C and a frequency falling within the range of 20 KHz to 100 KHz. In a preferred embodiment of the invention, the ultrasonic processing is performed at a temperature of 25 ° C and a frequency of 30 KHz.

According to the present invention, a further filtering treatment may be performed after the ultrasonic treatment to remove insoluble impurities. This filtration process can be carried out using techniques well known and customary to those skilled in the art. For example, the filtration treatment can be carried out by using a cheese cloth or a filter paper or a filter membrane having a selected pore size. In a preferred embodiment of the invention, the filtering process is carried out by using a filter paper having a pore size of 6 μm.

Extract of the genus Phalaenopsis meristem in accordance with the present invention It has been confirmed by in vitro experiments that it can effectively inhibit melanin production and tyrosinase activity of skin melanoma cells, and has the clearance of α,α-diphenyl-β-bitteryl thiol (α, α -diphenyl-β-picryhydrazyl, DPPH) The ability of free radicals. Further, the extract of the genus Phalaenopsis meristem according to the present invention has been confirmed to be effective for enhancing the brightness of skin color by human experiment.

Based on the above, the extract of the genus Phalaenopsis meristem according to the present invention is expected to have an effect of promoting skin whitening, and thus can be used for the preparation of a pharmaceutical for whitening skin.

Accordingly, the present invention provides a composition for whitening skin comprising any one of the extracts of three kinds of white-flowered Phalaenopsis meristems as described above, or a combination thereof.

As used herein, the terms "whitening" and "skin color lightening", "bleaching", "whitening", "brightening", "blackening", " "Black" and "lightening the melanin" can be used interchangeably.

In a preferred embodiment of the present invention, the composition comprises a fat-soluble extract of the phalaenopsis meristem of the white flower as described above and a water-soluble extract of the genus Phalaenopsis meristem.

In another preferred embodiment of the present invention, the composition comprises an alcohol-soluble extract of the moth orchid melon tissue as described above and a water-soluble extract of the moth orchid melon tissue.

In accordance with the present invention, the composition can be made to be topically applied to the substrate by techniques well known to those skilled in the art. External preparation on the skin, including but not limited to: emulsion, gel, ointment, cream, patch, liniment , powder, aerosol, spray, lotion, serum, paste, foam, drop, suspension , soap, salve, bandage, and the like.

In accordance with the present invention, the external formulation is prepared by mixing the compositions of the present invention with a base well known to those skilled in the art.

According to the invention, the substrate may comprise one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons [such as petroleum jelly and white). White petrolatum, wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers (absorption) enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 941 (carbopol ^® 941), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)], the activity of Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents Occlusive agents), emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants Colorant (Colorants), and propellant (PROPELLANTS) and the like. The selection and quantity of these additives falls within the professionalism and routine technology of those skilled in the art.

The present invention also provides a method for whitening skin comprising administering to a subject in need of promoting skin whitening a composition as described above. Preferably, the method comprises applying an effective amount of the composition to the skin of the individual and for a sufficient period of time until the melanin of the skin is significantly faded.

As used herein, the terms "administering" and "administering" and "application" are used interchangeably.

As used herein, the term "effective amount" means that when the composition of the present invention is administered to an individual in need of promoting skin whitening, one is sufficient to provide the desired whitening effect and does not produce to the skin. Safe use of undesired adverse side effects.

As used herein, the terms "effective amount" and "effective dose", "whitening effective amount", and "whitening effective dose" are used interchangeably.

In accordance with the present invention, the effective amount and frequency of application of the composition will vary depending on the initial condition of the skin area to be lightened by melanin and the ultimate whitening effect desired. In general, when the composition according to the present invention is topically applied to the skin, the effective amount per administration is usually 1 The skin area to 6 mg/cm 2 is about 1 to 3 times a day.

Further, the extract of the genus Phalaenopsis meristem according to the present invention has been confirmed by human experiment to effectively enhance the barrier function and water content of the stratum corneum of the skin, and to reduce the loss of moisture of the skin. .

Based on the above, the extract of the genus Phalaenopsis meristem according to the present invention is expected to have the effect of enhancing the moisturizing ability of the skin, and thus can be used for the purpose of preparing a medicine for improving the moisturizing ability of the skin.

Accordingly, the present invention provides a composition for enhancing the moisturizing ability of the skin, which comprises any one of the extracts of three kinds of white-flowered Phalaenopsis meristems as described above, or a combination thereof.

In a preferred embodiment of the present invention, the composition comprises a fat-soluble extract of the phalaenopsis meristem of the white flower as described above and a water-soluble extract of the genus Phalaenopsis meristem.

In another preferred embodiment of the present invention, the composition comprises an alcohol-soluble extract of the moth orchid melon tissue as described above and a water-soluble extract of the moth orchid melon tissue.

In accordance with the present invention, the route of administration, dosage form, and substrate available for use of the composition are as described above.

The present invention also provides a method for enhancing the moisturizing ability of the skin, which comprises administering to a subject in need of enhancing the moisturizing ability of the skin a composition as described above. Preferably, the method comprises applying an effective amount of the composition to the skin of the individual and for a sufficient period of time until the skin The moisturizing ability of the skin is significantly improved.

As used herein, the term "effective amount" means that when the composition of the present invention is administered to an individual who needs to enhance the moisturizing ability of the skin, it is sufficient to provide the desired moisturizing effect and not to A safe amount of unpleasant side effects of the skin.

As used herein, the terms "effective amount" and "effective dose", "moisture-retaining effective amount", and "moisture-retaining effective dose" are used interchangeably. .

According to the present invention, the effective amount and frequency of application of the composition vary depending on the initial condition of the skin area to be moisturized and the desired moisturizing effect desired. In general, when the composition according to the present invention is topically applied to the skin, the effective amount per application is usually from 1 to 6 mg/cm 2 of skin area, about 1 to 3 times per day.

Further, the extract of the genus Phalaenopsis meristem according to the present invention has been confirmed to be effective for enhancing the elasticity of the skin through human trials.

Based on the above, the extract of the genus Phalaenopsis meristem according to the present invention is expected to have an effect of preventing and/or delaying skin aging, and thus can be used for the preparation of a medicament for anti-skin aging.

Accordingly, the present invention provides a composition for anti-skin aging comprising any one of the extracts of three species of Phalaenopsis meristems as described above, or a combination thereof.

As used herein, the term "retarding" means One or more clinical signs of treating, reducing, alleviating, ameliorating, relieving, or controlling a disease or disorder. And lowering, stopping, or reversing the progression of severity of a condition or symptom being treated.

As used herein, the term "skin aging" is intended to encompass naturally occurring intrinsic skin aging, as well as extrinsic skin aging caused by environmental factors (especially ultraviolet light exposure) (extrinsic skin). Aging). Symptoms of skin aging include, but are not limited to, telangiectasia, epidermal thinning, skin atrophy, reduction of collagen fibers and elastic fibers, and elastic tissue degeneration (elastosis). , skin elasticity deterioration, skin texture roughening, dryness, wrinkle formation, and pigmentary change [eg, lentigines, freckles, Hypigmentation or hyperpigmentation, etc.

In a preferred embodiment of the present invention, the composition comprises a fat-soluble extract of the phalaenopsis meristem of the white flower as described above and a water-soluble extract of the genus Phalaenopsis meristem.

In another preferred embodiment of the present invention, the composition comprises an alcohol-soluble extract of the moth orchid melon tissue as described above and a water-soluble extract of the moth orchid melon tissue.

In accordance with the present invention, the route of administration, dosage form, and substrate available for use of the composition are as described above.

The present invention also provides a method for anti-skin aging comprising administering to a subject in need of prevention and/or delaying skin aging a composition as described above. Preferably, the method comprises administering an effective amount of the composition to the skin of the individual and for a sufficient period of time until the symptoms of skin aging are significantly improved.

As used herein, the term "effective amount" means that when the composition of the present invention is administered to an individual in need of prevention and/or delaying skin aging, it is sufficient to provide the desired anti-skin aging. A safe dosage that does not cause undesired adverse side effects on the skin.

As used herein, the terms "effective amount" and "effective dose", "anti-aging effective amount", and "anti-aging effective dose" may be used. Used interchangeably.

In accordance with the present invention, the effective amount and frequency of administration of the composition will vary depending on factors such as the initial condition of the area of the skin that is intended to prevent and/or delay the aging and the ultimate anti-skin aging effect desired. In general, when the composition according to the present invention is topically applied to the skin, the effective amount per application is usually from 1 to 6 mg/cm 2 of skin area, about 1 to 3 times per day.

In particular, the fat-soluble extract, water-soluble extract and alcohol-soluble extract of the genus Phalaenopsis meristem according to the present invention are also expected to be used for preparing a moisturizing energy for promoting skin whitening and lifting the skin. The use of force and cosmetics to prevent and/or delay skin aging.

Accordingly, the present invention provides a cosmetic composition comprising any one of the extracts of three kinds of white-flowered Phalaenopsis meristems as described above, or a combination thereof.

Preferably, the cosmetic composition comprises a fat-soluble extract of the butterfly flower melon tissue of the white flower as described above and a water-soluble extract of the butterfly flower melon tissue of the white flower.

Preferably, the cosmetic composition comprises an alcohol-soluble extract of the butterfly flower melon tissue of the white flower as described above and a water-soluble extract of the butterfly flower melon tissue of the white flower.

According to the present invention, the cosmetic composition may further comprise a cosmetically acceptable adjuvant which is widely used in cosmetic manufacturing techniques. For example, the cosmetically acceptable adjuvant may comprise one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, Chelating agents, interfacial active agents, coloring agents, thickening agents, fillers, perfumes, and odor absorbers. The selection and quantity of these reagents falls within the professional literacy and routine skills of those skilled in the art.

In accordance with the present invention, the cosmetic composition can be made into a form suitable for skincare or makeup using techniques well known to those skilled in the art, including, but not limited to, aqueous solutions. , aqueous-alcohol solution or oily solution (oily solution), oil-in-water type, water-in-oil type or composite emulsion, gel, ointment, cream, mask, Patch, pack, liniment, powder, aerosol, spray, lotion, serum, paste, foam, dispersion, drops, mousse, sunblock, lotion ( Tonic water), foundation, eyeshadow, makeup remover products, soap, and other body cleansing products.

In a preferred embodiment of the invention, the cosmetic composition is manufactured as a water-alcohol solution.

According to the present invention, the cosmetic composition may also be used in combination with one or more external use agents selected from the following activities: whitening agents [such as tretinoin, children) Catechin, citric acid, arbutin, and vitamin C], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts ) [such as aloe extract], skin nutrients, anesthetics, anti-acne agents, antipruritic, analgesic, anti-skin Antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents ), anti-seborborheic agents, wound-healing agents , corticosteroids and hormones. The selection and quantity of these external preparations falls within the professional literacy and routine technology of those who are familiar with the technology.

Other features and effects of the present invention will be apparent from the following description of the drawings, wherein: Figure 1 shows the fat-soluble extraction of the moth orchid's axillary meristem obtained according to Example 1 of the present invention. The elution pattern obtained by HPLC was carried out, wherein the peaks a1 to a6 represent the six main components occurring during the retention period of the 0th to 60th minute; and FIG. 2 shows the preparation of the first embodiment according to the present invention. The eluted pattern obtained by HPLC was obtained from the water-soluble extract of the genus Phalaenopsis mycelium, wherein the peaks b1 to b5 represent the five main components appearing during the 0 to 60 minute retention period; The elution pattern obtained by HPLC was carried out according to the alcohol-soluble extract of the genus Phalaenopsis chinensis meristem obtained in Example 1 of the present invention, wherein the peaks c1 to c6 indicate the retention period during the 0th to 60th minute. The six main components appearing; Figure 4 shows the elution pattern obtained by HPLC based on the ethanol extract of the butterfly flower of the Phalaenopsis chinensis obtained from CN 100589790 C, wherein the peaks d1 to d4 are indicated at the 0th Up to 60 minutes Arising during the stay four main ingredients; Figure 5 shows the United States will be based on the topic was written by Michelle student Department of Biotechnology of Southern Taiwan University of ethanol extract (the same above) prepared petals of white Phalaenopsis The elution pattern obtained by HPLC was taken, wherein the peaks e1 to e2 represent the two main components appearing during the retention period of 0 to 60 minutes; FIG. 6 shows that the skin of the subject's left and right faces is ITA values measured over time after administration of the white phalaenopsis essence 1 of the present invention and the control serum, respectively; FIG. 7 shows that the skin of the left and right faces of the subject are respectively administered with the present invention. The ITA value measured with time after the white flower phalaenopsis essence 2 and the control essence; FIG. 8 shows that the skin of the left and right faces of the subject are respectively administered with the white flower phalaenopsis essence 3 of the present invention and ITA value measured over time after control of the serum; Figure 9 shows that the skin of the subject's left and right faces was administered with the white phalaenopsis essence 4 of the present invention and the control serum, respectively, over time. Measured ITA value; Figure 10 shows the ITA values measured over time by the subject's left and right face skins after application of the white phalaenopsis essence 5 of the present invention and the control serum, respectively; Figure 11 shows that the subject's left and right face skins are respectively The water content of the stratum corneum measured with time after administration of the white phalaenopsis essence 1 of the present invention and the control essence; FIG. 12 shows that the skin of the left and right faces of the subject are respectively administered with the present invention. The water content of the stratum corneum measured over time after the white flower Phalaenopsis Essence 2 and the control serum; Figure 13 shows that the skin of the left and right faces of the subject was administered separately The water content of the stratum corneum measured with time after the white butterfly moth orchid essence 3 and the control essence of the present invention; FIG. 14 shows that the skin of the left and right faces of the subject are respectively applied with the white butterfly of the present invention. The water content of the stratum corneum measured with time after the blue essence 4 and the control essence; FIG. 15 shows that the skin of the left and right faces of the subject are respectively administered with the white flower phalaenopsis essence 5 of the present invention. The water content of the stratum corneum measured after the control of the serum over time; Figure 16 shows that the skin of the left and right faces of the subject were administered with the white flower phalaenopsis essence 1 of the present invention and the control serum, respectively. The measured value of the transdermal water dispersion measured by time; FIG. 17 shows that the skin of the subject's left and right faces was measured over time after being administered with the white flower phalaenopsis essence 2 of the present invention and the control essence, respectively. The obtained skin water dispersion loss value; FIG. 18 shows that the skin of the subject's left and right faces is applied to the skin after the application of the white flower phalaenopsis essence 3 of the present invention and the control essence, respectively. Water dispersion loss value; Figure 19 shows the subject's The skin of the face and the right face was subjected to the transdermal water loss value measured with time after the application of the white phalaenopsis essence 4 of the present invention and the control essence, respectively; FIG. 20 shows the subject's left and right faces. The transdermal water loss value measured with time after the application of the white phalaenopsis essence 5 of the present invention and the control serum respectively; FIG. 21 shows that the skin of the subject's left face and right face are respectively Being applied The R2 value measured with time after the white flower phalaenopsis essence 1 of the present invention and the control essence solution; FIG. 22 shows that the skin of the subject's left and right faces are respectively applied with the white flower phalaenopsis essence of the present invention. The R2 value measured with time after the liquid 2 and the control serum; FIG. 23 shows that the skin of the left and right faces of the subject are respectively administered with the white flower phalaenopsis essence 3 of the present invention and the control essence. R2 value measured over time; Figure 24 shows that the skin of the subject's left and right faces was measured over time after administration of the white phalaenopsis essence 4 of the present invention and the control serum, respectively. The R2 value; and Figure 25 shows the R2 values measured over time by the subject's left and right face skins after application of the white phalaenopsis essence 5 of the present invention and the control serum, respectively.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example General experimental materials: 1. MS medium:

The MS medium used in the following examples had the formulations shown in Table 1 below.

2. Source and culture of cell lines:

The mouse skin melanoma cell line B16F10 (BCRC 60031) (corresponding to ATCC CRL-6475) used in the following examples is a biological resource conservation and research center purchased from the Food Industry Development Research Institute (FIRDI) in Taiwan ( Biosource Collection and Research Center, BCRC).

B16F10 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (purchased from Deyi Technology Co., Ltd.) [added with 10% fetal bovine serum (FBS), In a 10-cm petri dish of 100 U/mL penicillin and 100 U/mL streptomycin, followed by an incubator with a culture condition set to 37 ° C and 5% CO ₂ Cultivate in the middle. After that, fresh medium was replaced approximately every 2 days. When the cell density reached about 80-90% confluence, the medium was removed and the cells were washed twice with serum-free medium, followed by trypsin-EDTA (trypsin-EDTA) to make the cells Detached from the bottom of the dish. Thereafter, fresh medium is added to neutralize the activity of trypsin and the medium is repeatedly aspirated by a pipette to fully break up the cells, and then the formed cell suspension is dispensed into a new dish and cultured. The conditions were set to be cultured in an incubator of 37 ° C and 5% CO ₂ .

Example 1. Preparation of extracts of Phalaenopsis amabilis axillary meristem experimental method: A. Preparation of lip soluble soluble extracts of the genus Hymenoptera:

First, the first section of the flower stalk of the flowering stalk of the white flower phalaenopsis purchased from the Taiwan Orchid Biotechnology Park (Tainan City, Taiwan) and the stem segment of the flower bud that has been differentiated from the upper end are removed. The stem is then surface disinfected with 70% alcohol. Next, the peduncle is cut to obtain several individual There is a stem segment having a length of about 5 cm and having 1 node at a central position. Thereafter, the batter coated outside the nodes of the stem segments is removed, followed by surface disinfection of the stem segments with a 0.5% sodium hypochlorite solution (with a few drops of Tween-20) for 10 to 20 minutes. Then, it is washed 2 to 3 times with sterile water. Thereafter, the ends of the stem segments were each cut 1 cm with a scalpel, and then inserted into MS medium (the formulation for MS medium is shown in Table 1 above), and then placed at a temperature maintained at 28 ° C. The illuminance was maintained in an incubation chamber of 1,500 lux and a dark period of 16 hours light/8 hours dark. After 4 to 6 weeks of cultivation, the cultivated stem segments are harvested, and then the axillary meristem is removed from the nodes of the stem segments by scalpel in a sterile state, followed by washing with sterile water. It was dried 2 to 3 times and then dried in an oven at a temperature of 50 °C.

Thereafter, 60 grams of dried axillary meristems were weighed and placed in a supercritical fluid extraction system (purchased from Taichao Extracting and Finishing Machine Co., Ltd., model OV-SCF- In an extraction vessel of 10000), CO _{2 was} then introduced into the extraction vessel, and then extraction was carried out at 40 ° C and a pressure of 4350 psi for 1 hour. During the extraction process, CO ₂ is passed through the extraction vessel at a flow rate of 6 mL/min, and then enters a collection container under normal pressure, thereby utilizing a fat-soluble property of the yellow-green paste-like white-flowered Phalaenopsis mycelium meristem. The extract is obtained in the collection container. The obtained fat-soluble extract of the Phalaenopsis chinensis meristem was dissolved in 95% ethanol to form a stock solution having a concentration of 4 mg/mL. In addition, the residue of the genus Phalaenopsis mycelium obtained in the extraction container was subjected to the experiments of the following items B and C.

B. Preparation of water soluble extract of the white-flowered Phalaenopsis mycorrhizal meristem:

Weigh 100 g of the residue of the genus Phalaenopsis acuminata according to item A above and add it to a beaker, then add 1000 mL of pure water and mix well, then form the mixture at 25 ° C. An ultrasonic processor (purchased from Taichao Extraction and Purification Co., Ltd., model ES600N) was used and the oscillation was performed for a period of 4.5 hours at a frequency of 30 kHz, followed by a filter paper having a pore size of 6 μm. (ADVANTEC, No. 1 Qualitative Filter Papers) was filtered to obtain a filtrate. Thereafter, the filtrate was freeze-dried in a freeze dryer (FDU 2200, EYELA) for 72 hours to obtain a water-soluble extract of a lyophilized powder of Phalaenopsis chinensis. The obtained water-soluble extract of the genus Phalaenopsis mycelium was dissolved in pure water to form a stock solution having a concentration of 4 mg/mL.

C. Preparation of alcohol soluble extract of the butterfly melon meristem of white flower:

Weigh 100 grams of the residue of the genus Phalaenopsis mycelium obtained according to item A above and add it to a beaker, then add 1000 mL of 95% ethanol and mix well, then form the mixture Use an ultrasonic processor at 25 ° C and use a 30 KHz The frequency was oscillated for 4.5 hours, and then filtered through a filter paper having a pore size of 6 μm to obtain a filtrate. Thereafter, the filtrate was concentrated by a vacuum concentrator (available from Suntech Instruments Co., Ltd., model RA-3000V1-B) for 5 hours to obtain a yellow-green gelatinous white flower phalaenopsis. A tissue-soluble alcohol-soluble extract. The obtained alcohol-soluble extract of the genus Phalaenopsis mycelium was dissolved in 75% ethanol to form a stock solution having a concentration of 4 mg/mL.

Example 2. High performance liquid chromatography (HPLC) analysis of the extract of the butterfly melon meristem of white flower

In order to understand the main component distribution of the extract of the moth orchid genus meristem of the present invention, the extracts of the three species of the genus Phalaenopsis sinensis (the genus Hymenoptera) are obtained according to the above Example 1. The fat-soluble extract, the water-soluble extract, and the alcohol-soluble extract) were subjected to high performance liquid chromatography analysis.

Experimental Materials:

For comparison, the applicant prepared an ethanol extract of the butterfly melon meristem (hereinafter referred to as the ethanol extract of the conventional axillary meristem) and an ethanol extract of the petunia flower petals according to a conventional method (hereinafter Referred to as the ethanol extract of the conventional petals, they were taken for the same HPLC analysis. The ethanol extract of the conventional twin meristem is described with reference to the method described in CN 100589790 C with minor modifications. Briefly, the axillary meristem of Phalaenopsis chinensis and the 95% ethanol were uniformly mixed at a ratio of 1:10 (wt/wt), followed by 25 ° C. The extraction was carried out for 24 hours, whereby the conventional ethanol extract of the meristem meristem was obtained.

In addition, the ethanol extract of the conventional petal is referred to by the method described in the book of the student of Zeng Meijun of the Department of Biotechnology of Nantai University of Science and Technology (the same as above), and is slightly modified. Briefly, 2.5 g of the petals of the white flower Phalaenopsis were weighed and cut, and then heated and extracted with 50 mL of 95% ethanol at 60 ° C for 24 hours, thereby obtaining the ethanol extract of the conventional petals. Things.

experimental method:

The HPLC analytical instruments used in this experiment were as follows: ATIMA-Isocratic/400 high performance liquid chromatography system (CVC Technologies, Inc.) and UV-Vis detector (UVD) -100, CVC Technologies, Inc.), and various operating parameters and conditions for HPLC are shown in Table 2 below.

result:

1 to 3 respectively show that the liposoluble extract, the water-soluble extract and the alcohol-soluble extract of the genus Phalaenopsis chinensis meristem obtained according to the above Example 1 were obtained by high performance liquid chromatography. The elution profile, and Figure 4 and Figure 5 respectively show the ethanol extract of the genus Phalaenopsis mycelium produced according to CN 100589790 C and the study by Zeng Meijun of the Department of Biotechnology, Nantai University of Science and Technology. The ethanol extract of the petals of Phalaenopsis chinensis obtained from the topic (same as above) was used for the elution pattern obtained by high performance liquid chromatography.

As can be seen from Fig. 1, the fat-soluble extract of the genus Moth orchid of the present invention has six major elution peaks during the 0 to 60 minute retention period (labeled as crests a1, a2, a3, respectively). A4, a5 and a6). As can be seen from Fig. 2, the water-soluble extract of the butterfly flowering meristem of the present invention has five major elution peaks during the 0 to 60 minute retention period (labeled as peaks b1, b2, b3, respectively). B4 and b5). As can be seen from Fig. 3, the alcohol-soluble extract of the genus Phalaenopsis mycelium of the present invention exhibits six major elution peaks during the 0 to 60 minute retention period (labeled as crests c1, c2, c3, respectively). C4, c5 and c6).

In addition, as can be seen from Figure 4, the ethanol extract of the conventional twin meristem has four major elution peaks during the 0 to 60 minute retention period (labeled as peaks d1, d2, d3, and d4, respectively). . As can be seen from Figure 5, the ethanol extract of the conventional petal appeared to have two major elution peaks during the 0 to 60 minute retention period (labeled as peaks e1 and e2, respectively).

By comparing the peak positions of FIGS. 1 to 3, it is clearly found that the components of the fat-soluble extract, the water-soluble extract, and the alcohol-soluble extract of the genus Phalaenopsis mycelium obtained according to the present invention are different from each other. . In addition, it can be clearly seen by comparing the peak positions of FIG. 1 to FIG. 5 that the components of the extracts of the three species of Phalaenopsis hybrids obtained according to the present invention are significantly different from the white flowers obtained according to the conventional method. Ethanol extract of the genus Moth orchid, and the butterfly of the white flower phalaenopsis Alcohol extracts are available.

Example 3. Cell viability analysis of extracts of Phalaenopsis chinensis meristems

The water-soluble extract and the alcohol-soluble extract of the Phalaenopsis chinensis meristem obtained according to the above Example 1 were subjected to the following experiment to evaluate the cytotoxicity of the two extracts.

experimental method:

The B16F10 cells subcultured according to the second item "Source and culture of cell strain" of "General Experimental Materials" above were divided into two groups, including one control group and one experimental group. Each group of cells was cultured in a 96-well plate containing 100 μL of DMEM (10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin) in an amount of 1×10 ⁴ cells/well (96). -well plate) and culture in an incubator (37 ° C, 5% CO ₂ ) for 24 hours. Next, an appropriate amount of a stock solution of the water-soluble extract of the genus Phalaenopsis mycelium obtained according to the item B of the above Example 1 was added to the cell culture of the experimental group so that it had a final concentration of A water-soluble extract of 1.25% (w/v, g/L) of the genus Phalaenopsis. As for the cell culture of the control group, no treatment was performed. Each group of cell cultures was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, after 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra Toluene bromide {3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide} (MTT, 1 mg/mL, 100 μL) was added to each well and cultured for 3 hours. After that, the liquid in each well was removed, and then 100 μL of dimethylsulfoxide (DMSO) was added and mixed well, and then each ELISA reader (SunriseTM, TECAN) was read at a wavelength of 570 nm to read each. The absorbance of the well (OD ₅₇₀ ).

The cell viability assay for the alcohol-soluble extract of the Phalaenopsis chinensis meristem is generally carried out with reference to the procedure described above for the water-soluble extract of the Phalaenopsis mycelium meristem, with the following exceptions: A stock solution of the water-soluble extract of the Phalaenopsis mycelium meristem was replaced by a stock solution of the alcohol-soluble extract of the Phalaenopsis chinensis meristem obtained according to Item C of Example 1 above.

The cell activity percentage (%) is calculated by substituting the measured absorbance value (OD ₅₇₀ ) into the following formula (1): Formula (1): A = (B/C) × 100

Where: A = percentage of cell viability (%)

B = OD ₅₇₀ absorbance measured by each group

C = OD ₅₇₀ absorbance measured in the control group

result:

Table 3 below shows the percentage of cell viability measured by B16F10 cells treated with a water-soluble extract of 1.25% (w/v) of the Phalaenopsis sinensis meristem and an alcohol-soluble extract, respectively. As can be seen from Table 3, the percentage of cell viability of the experimental group was as high as 95% or more, and there was no significant difference compared with the control group. The results of this experiment show that the water-soluble extract and the alcohol-soluble extract of the mycelium of the genus Phalaenopsis in accordance with the present invention do not cause damage to cells, and thus are not cytotoxic.

^α : Experimental data is expressed as mean ± standard deviation (Standard Deviation, SD).

Example 4. Evaluation of the effect of extracts of the genus Hypophthalmia meristem on the inhibition of melanogenesis

In this example, the Applicant evaluates the water-soluble extract and the alcohol-soluble extract of the butterfly melon mycelium of the present invention by measuring the melanin content and the tyrosinase activity. Inhibits the utility of melanogenesis.

experimental method: A, determination of melanin content:

The determination of melanin content was commissioned by SGS Taiwan Ltd. and generally referred to the method described in Koo JH et al. (2008), Exp. Mol. Med., 40:313-319. get on. Briefly, B16F10 cells subcultured according to item 2 "Source and culture of cell lines" of "General Experimental Materials" above were divided into two groups, including one control group and one experimental group. Each group of cells was cultured in an amount of 1.5 × 10 ⁶ cells in an appropriate amount of DMEM [added with 10% FBS, penicillin, streptomycin, and 0.1 mM 3-isobutyl-1-methylxanthine (3 -isobutyl-1-methylxanthine, IBMX)] in a 10-cm petri dish and cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, allowing B16F10 cells to be induced melanin production . Next, an appropriate amount of a stock solution of the water-soluble extract of the genus Phalaenopsis mycelium obtained according to the item B of the above Example 1 was added to the cell culture of the experimental group so that it had a final concentration of A water-soluble extract of 1.25% (w/v, g/L) of the genus Phalaenopsis. As for the cell culture of the control group, no treatment was performed. Each group of cell cultures was cultured in an incubator (37 ° C, 5% CO ₂ ) for 24 hours, and then washed twice with Phosphate Buffered Saline (PBS), followed by 1 mL of pancreas. Protease-EDTA is used to detach the cells from the bottom of the dish. Thereafter, an appropriate amount of fresh medium was added to neutralize the activity of trypsin, and then the resulting mixed solution was pipetted into a centrifuge tube and centrifuged. After the supernatant was removed, an appropriate amount of lysis buffer [containing 20 mM Tris and 0.1% (v/v) Triton X-100] was added to dissolve the cells and centrifuged. Next, a portion of the supernatant was collected and the total protein concentration in the supernatant was determined by Bradford protein assay.

Further, a portion of the supernatant liquid was collected and an appropriate amount of 20% trichloroacetic acid (TCA) was added to cause a precipitate to be precipitated. After centrifugation, the supernatant was removed and washed with 10% trichloroacetic acid, then sequentially washed with an organic solvent containing ethanol and diethyl ether and diethyl ether, and then air dried. Thereafter, the obtained precipitate was sufficiently dispersed with an appropriate amount of 0.85 M KOH, and then an appropriate amount of the volume was taken out of the formed mixture, and the absorbance (OD ₄₄₀ ) was measured by an ELISA reader at a wavelength of 440 nm. .

The effect of the alcohol-soluble extract on the meristem of the Phalaenopsis sinensis on the melanin content is generally analyzed with reference to the procedure described above for the water-soluble extract of the genus Phalaenopsis mycelium, with the following differences: A stock solution of the water-soluble extract of the Phalaenopsis mycelium meristem was replaced by a stock solution of the alcohol-soluble extract of the Phalaenopsis chinensis meristem obtained according to Item C of Example 1 above.

The melanin content (%) is calculated by substituting the measured absorbance value (OD ₄₄₀ ) into the following formula (2): Formula (2): D = (E/F) × 100

Where: D = melanin content (%)

E = measured OD ₄₄₀ absorbance of each group

F = OD ₄₄₀ absorbance measured in the control group

B. Determination of tyrosinase activity:

First, 0.5 g of L-tyrosine (L-tyrosine, Sigma, T8566) was added to 100 mL of PBS and thoroughly mixed, followed by standing at room temperature for 30 minutes. Next, centrifugation was carried out at 3000 rpm for 15 minutes, and then the supernatant was aspirated to obtain a tyrosine buffer solution.

Thereafter, an appropriate amount of a stock solution of the water-soluble extract of the Phalaenopsis chinensis meristem obtained according to the item B of the above Example 1 is separately added to the secondary water (ddH2O) to obtain a phalaenopsis The water-soluble extract concentrations of the twin meristems were 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, and 2.0% (w/v, g/L) of the samples to be tested (ie, experimental groups 1 to 6). ). Next, 100 μL of each sample to be tested was taken, and then 5 μL of tyrosine (tyrosinase, Sigma, T3824) solution (7 U/μL in PBS) and 900 μL of tyrosine buffer solution were added. In addition, the control was replaced with 100 μL of tyrosine buffer solution to test the sample. After thorough mixing, the resulting mixture was placed at 37 ° C for 1 hour. Thereafter, 100 μL of each mixture of each group was taken and added to a 96-well culture dish, and then the absorbance (OD ₄₅₀ ) of each well was measured by an ELISA reader at a wavelength of 450 nm.

The effect of alcohol-soluble extracts on the tyrosinase activity of the butterfly melons in the genus Phalaenopsis is generally based on the above-described procedures described for the water-soluble extracts of the genus Phalaenopsis mycelium. The present invention is to replace the stock solution of the water-soluble extract of the genus Phalaenopsis mycelium by the stock solution of the alcohol-soluble extract of the genus Phalaenopsis mycelium obtained according to the item C of the above Example 1.

The inhibition rate (%) of tyrosinase activity is calculated by substituting the measured absorbance value (OD ₄₅₀ ) into the following formula (3): Formula (3): G = [1-(H/I) )]×100

Where: G = inhibition rate of tyrosinase activity (%)

H = absorbance of OD ₄₅₀ measured by each group

I = OD ₄₅₀ absorbance measured in the control group

result: A, determination of melanin content:

Table 4 below shows the melanin content of B16F10 cells induced by melanin after treatment with a water-soluble extract and an alcohol-soluble extract of 1.25% (w/v) of respectively. As can be seen from Table 4, compared with the control group, the melanin content of the cells in the experimental group was significantly reduced. The results of this experiment show that the water-soluble extract and the alcohol-soluble extract of the butterfly-derived meristem of the genus Phalaenopsis according to the present invention have a strong inhibitory effect on the melanin production of the IBMX-treated B16F10 cells.

^α : Experimental data is expressed as mean ± standard deviation.

B. Determination of tyrosinase activity:

Table 5 below shows the inhibition rates of tyrosinase activity measured by water-soluble extracts and alcohol-soluble extracts of different concentrations of the Phalaenopsis mycelium meristem. As can be seen from Table 5, compared with the control group, the inhibition rate of tyrosinase activity in the experimental groups 1 to 6 was improved, and the water-soluble extract or alcohol solubility of the melon tissue of the Phalaenopsis sinensis was also observed. The concentration of the extract is more pronounced and more pronounced. The results of this experiment show that: according to the invention Both the water-soluble extract and the alcohol-soluble extract of the white-flowered Phalaenopsis mycelium have a superior effect of inhibiting tyrosinase activity.

Based on the above experimental results, it can be seen that the water-soluble extract and the alcohol-soluble extract of the genus Moth orchid of the genus Phalaenopsis can effectively inhibit the melanin production and tyrosinase activity of the skin melanoma cells, thereby achieving the skin. The effect of skin whitening.

Example 5. Evaluation of Antioxidation Efficacy of Extracts from Hybrids of Phalaenopsis chinensis

In this example, the applicant removes α,α-diphenyl-β-bitteryl thiol (α,α-diphenyl- by analyzing the water-soluble extract of the genus Hydrangea chinensis meristem and the alcohol-soluble extract. The ability of β-picryhydrazyl, DPPH) to evaluate the antioxidant activity of these two extracts.

experimental method:

First, an appropriate amount of a stock solution of the water-soluble extract of the Phalaenopsis chinensis melon tissue obtained according to the item B of the above Example 1 is separately added to the methanol solution, thereby obtaining a white-flowered butterfly orchid meristem. The water-soluble extract concentrations were 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, and 2.0% (w/v, g/L) of the samples to be tested (ie, experimental groups 1 to 6). Next, 0.5 mL of each sample to be tested was taken, and then 2 mL of methanol and 0.5 mL of a methanol solution containing 0.2 mM DPPH were added. In addition, the control group was replaced with 0.5 mL of methanol to test the sample. After thorough mixing, the resulting mixture was allowed to stand at room temperature for 30 minutes, and the absorbance (OD ₅₁₇ ) was measured with a spectrophotometer at a wavelength of ₅₁₇ nm. The lower the measured absorbance value (OD ₅₁₇ ), the better the ability to remove DPPH free radicals.

The ability of the alcohol-soluble extract of the genus Hydrangea chinensis in the removal of DPPH free radicals is generally analyzed with reference to the procedure described above for the water-soluble extract of the genus Phalaenopsis mycelium, which is different. The present invention is to replace the stock solution of the water-soluble extract of the genus Phalaenopsis mycelium by the stock solution of the alcohol-soluble extract of the genus Phalaenopsis mycelium obtained according to the item C of the above Example 1.

The DPPH radical scavenging rate (%) is calculated by substituting the measured absorbance value (OD ₅₁₇ ) into the following formula (4): Formula (4): J = [1-(K) /L)]×100

Where: J = DPPH free radical scavenging rate (%)

K = OD ₅₁₇ absorbance measured for each group

L = OD ₅₁₇ absorbance measured in the control group

result:

Table 6 below shows DPPH free radical scavenging rates measured by water-soluble extracts and alcohol-soluble extracts of different concentrations of Phalaenopsis chinensis. It can be seen from Table 6 that compared with the control group, the DPPH free radical scavenging rate of the experimental group 1 to 6 is improved, and the water-soluble extract or the alcohol-soluble extract of the meristem of the Phalaenopsis sinensis The concentration is more pronounced and more obvious. The results of this experiment show that the water-soluble extract and the alcohol-soluble extract of the genus Phalaenopsis sinensis according to the present invention have a superior DPPH radical scavenging ability. The applicant infers from this that the water-soluble extract and the alcohol-soluble extract of the mycelium of the genus Phalaenopsis in accordance with the present invention can inhibit the formation of melanin by removing free radicals in the skin cells, thereby achieving skin whitening ( Skin whitening). Therefore, the Applicant believes that the water-soluble extract and the alcohol-soluble extract of the genus Phalaenopsis mycelium according to the present invention have a high potential to develop into a skin whitening agent.

Example 6. Evaluation of the skincare effect of human individual extracts of the extract of Phalaenopsis chinensis

To understand the fat-soluble extract, the water-soluble extract, the alcohol-soluble extract, the combination of the above-mentioned fat-soluble extract and the water-soluble extract, and a combination of the above-mentioned fat-soluble extract and the water-soluble extract, according to the present invention The following tests were carried out for the skin care effects of the above-mentioned combination of the water-soluble extract and the alcohol-soluble extract, respectively, for human subjects.

Experimental materials, individuals, and methods: A. Prepare an extract containing the extract of the white-flowered Phalaenopsis mycelium meristem:

a stock solution of the fat-soluble extract of the Phalaenopsis chinensis meristem obtained according to the item A of the above Example 1, and a stock solution of the water-soluble extract of the genus Phalaenopsis mycelium obtained from the item B and Alcohol-soluble extract of the subspecies of Phalaenopsis chinensis obtained from Item C The stock solutions of the extracts were separately prepared into five kinds of essences (hereinafter referred to as white flower moth orchid essences 1 to 5) which can be applied to human skin. In addition, for comparison, an essence (hereinafter referred to as a control serum) containing no extract of the genus Phalaenopsis mycelium was prepared. The composition and formulation of these six essences are shown in Table 7 below.

Preparation of Essence 1 is related to white moth orchid in accordance with the following steps carried out: First, the Carbopol ^® 941 and sodium alginate dissolved in purified water, and then added in dipropylene glycol, polyethylene glycol 400, and based on the above A stock solution of the water-soluble extract of the genus Phalaenopsis sinensis, which was obtained in the item B of Example 1, was thoroughly mixed, thereby obtaining an aqueous solution. Further, sorbitan monooleate, polyoxyethylene (20) sorbitan monostearate, oleyl alcohol, propyl paraben, and according to item A of the above Example 1 A stock solution of the fat-soluble extract of the genus Phalaenopsis sinensis is dissolved in ethanol, thereby obtaining an alcoholic solution. Next, the alcoholic solution is slowly added to the aqueous solution to carry out microemulsion, and then a potassium hydroxide solution is added to the obtained mixture and thoroughly mixed, thereby obtaining the white flower Phalaenopsis essence. Liquid 1.

In addition, about the white flower phalaenopsis essence 2 to 5 and the control essence are generally prepared according to the above steps, the difference is: in the process of preparing the alcoholic solution of the white flower phalaenopsis essence 2, according to the above A stock solution of the alcohol-soluble extract of the Phalaenopsis chinensis meristem obtained in Item C of Example 1 is used instead of the stock solution of the fat-soluble extract of the Phalaenopsis mycelium of the white flower; in the preparation of the white flower Phalaenopsis essence 3 of the aqueous solution was not added to the process of the white flower Phalaenopsis a stock solution of the water-soluble extract, and the volume lacking is made up of an equal volume of pure water in the process of formulating the aqueous solution; no white butterfly is added during the preparation of the alcoholic solution of the white flower Phalaenopsis essence 4 a stock solution of the fat-soluble extract of the orchid meristem, and the volume lacking is made up of an equal volume of pure water in the process of formulating the aqueous solution; in preparing the alcoholic solution of the white flower Phalaenopsis essence 5 In the process, a stock solution of the alcohol-soluble extract of the genus Phalaenopsis mycelium is replaced by a stock solution of the fat-soluble extract of the genus Phalaenopsis mycelium, and no white butterfly is added during the preparation of the aqueous solution. a stock solution of the water-soluble extract of the melon tissue, and the volume lacking is supplemented with an equal volume of pure water during the preparation of the aqueous solution; and during the preparation of the aqueous solution of the control serum Adding a stock solution of the water-soluble extract of the genus Phalaenopsis to the meristem, and the process of preparing the alcoholic solution of the control essence Stock solution was not added liposoluble white phalaenopsis extract axillary meristematic tissue, and the lack of volume in the process of preparation of the aqueous solution to an equal volume of purified water to make up.

B. Screening of experimental individuals:

The subjects participating in the trial were faculty members from Hong Kong University of Science and Technology who must not have any of the exclusion criteria shown in Table 8 below. In addition, all of the individuals involved in the trial have obtained their informed consent.

There were a total of 100 subjects in the trial, and they were randomly divided into 5 groups, namely the elite group 1 to 5 (n=20 per group). The facial skin of each group of subjects was administered with different white flower phalaenopsis essence according to the method described in item C below, and then evaluated according to the method described in items D to G below. The effect of blue essence on skin care. The clinical information (including gender, mean age, and age range) for each group of subjects and the type of serum applied were shown in Table 9 below.

C. Application of white flower phalaenopsis essence:

First, the left face skin of each group of subjects was used as the experimental area, and the right face skin was used as the control area, and then the experimental areas of each group of subjects were respectively administered to obtain the white flower phalaenopsis essence according to item A above. The liquid (the types of serum applied to each group are as shown in Table 9 above), and the control areas of each group of subjects were administered to control the serum. The application doses of the experimental area and the control area were as follows: 2 mg of the essence was applied every 2 square centimeters, twice a day, and the test period lasted for 28 days. At day 0 (ie before the application of the serum) and on days 7, 14, 21 and 28 after the application of the serum, according to the methods described in items D, E, F and G below The skin of the cheek bone site of the experimental zone and the control zone of each group of subjects was measured.

D, determination of colorimetry of skin:

At the 0th day (ie before the application of the serum) and at the 7th, 14th, 21st and 28th days after the application of the serum, the test area of each group of subjects and the tibia of the control area The skin was measured for skin color by using a colorimeter (Chromameter ^® CR-10, Minolta), thereby obtaining an L value and a b value, respectively.

The ITA value is calculated by substituting the measured L value and the b value into the following formula (5): Equation (5): M = {Arc TAN [(N-50) / O]} × 180 / π

Where: M = ITA value

N = L value measured at each test time

O = b value measured at each test time

The higher the ITA value measured, the brighter the color representing the skin.

E, determination of water content of stratum corneum:

At the 0th day (ie before the application of the serum) and at the 7th, 14th, 21st and 28th days after the application of the serum, the test area of each group of subjects and the tibia of the control area The skin was measured for water content in the stratum corneum by using a capacitance meter (Corneometer ^® CM 285, Courage & Khazaka).

F, determination of transepidermal water loss (TEWL):

At the 0th day (ie before the application of the serum) and at the 7th, 14th, 21st and 28th days after the application of the serum, the test area of each group of subjects and the tibia of the control area The skin was measured by the use of an evaporimeter (Tewameter ^® TM300, Courage & Khazaka) for transdermal water loss.

G, determination of skin viscoelasticity (viscoelasticity of skin):

At the 0th day (ie before the application of the serum) and at the 7th, 14th, 21st and 28th days after the application of the serum, the test area of each group of subjects and the tibia of the control area The skin was measured for skin viscoelasticity by using a skin elasticity meter (Cutometer ^® MPA580, Courage & Khazaka), thereby obtaining an R2 value. If the measured R2 value approaches 1, the better the elasticity of the skin.

result: A. Determination of skin color:

6 to 10 respectively show the ITA values measured at different time points in the experimental zone to which the white-flowered phalaenopsis extracts 1 to 5 of the present invention were applied. It can be seen from Fig. 6 to Fig. 10 that the ITA values measured in the experimental area are significantly increased compared with the control area, and will become more apparent over time. Conversely, the ITA values measured in the control area did not show significant changes throughout the experiment. The results of this experiment show that the white flower phalaenopsis essence according to the present invention can effectively whiten the skin.

B. Determination of water content in the stratum corneum:

Fig. 11 to Fig. 15 respectively show the water content of the stratum corneum measured at different time points in the experimental area to which the white flower phalaenopsis essences 1 to 5 of the present invention were applied. As can be seen from Fig. 11 to Fig. 15, the water content of the stratum corneum measured in the experimental area was significantly increased compared with the control area, and it became more obvious with time. Conversely, the water content of the stratum corneum measured in the control zone will slowly increase over time. The results of this experiment show that the white flower phalaenopsis essence according to the present invention can effectively enhance the moisture-retaining capacity of the skin.

C. Determination of the loss of transdermal water:

16 to 20 respectively show the values of the transdermal water dispersion loss measured at different time points in the experimental zone to which the white-flowered phalaenopsis extracts 1 to 5 of the present invention were applied. As can be seen from Fig. 16 to Fig. 20, compared with the control area, the measured value of the transdermal water loss in the experimental area was significantly reduced, and it became more obvious with time. Conversely, the value of the transdermal water loss measured in the control zone may slowly decrease over time. This The results of the experiments show that the white flower phalaenopsis essence according to the present invention can effectively enhance the barrier function of the stratum corneum of the skin.

D, determination of skin viscoelasticity:

21 to 25 respectively show the R2 values measured at different time points in the experimental zone to which the white-flowered phalaenopsis extracts 1 to 5 of the present invention were applied. It can be seen from Fig. 21 to Fig. 25 that the R2 values measured in the experimental area are significantly increased compared with the control area, and will become more pronounced with time. Conversely, the R2 values measured in the control zone did not show significant changes throughout the experiment. The results of this experiment show that the white flower phalaenopsis essence according to the present invention can effectively enhance skin elasticity.

According to the above experimental results, the fat-soluble extract, the water-soluble extract, the alcohol-soluble extract, a combination of the above-mentioned fat-soluble extract and the water-soluble extract of the moth orchid And a combination of the above water-soluble extract and an alcohol-soluble extract can achieve skin whitening, moisturizing and anti-aging effects. Therefore, the Applicant believes that the extracts of the three species of Phalaenopsis acuminata meristems according to the present invention (that is, the fat-soluble extracts, the water-soluble extracts, and the alcohol-soluble extracts of the white-flowered Phalaenopsis mycelium) and their The combination has the potential to develop into a skin care product.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

Although the present invention has been described with reference to the specific embodiments described above, it is obvious that many modifications can be made without departing from the scope and spirit of the invention. Change and change. It is therefore intended that the invention be limited only by the scope of the appended claims.

Claims (44)

An extract of the genus Phalaenopsis meristem, which is prepared by a method comprising the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ and obtaining a white flower phalaenopsis meristem a fat-soluble extract. An extract of the genus Phalaenopsis meristem, which is prepared by a method comprising the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ , thereby obtaining a white flower phalaenopsis a fat-soluble extract of the tissue and a residue; and extracting the residue with water to obtain a water-soluble extract of the genus Phalaenopsis meristem. An extract of the genus Phalaenopsis meristem, which is prepared by a method comprising the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ , thereby obtaining a white flower phalaenopsis a fat-soluble extract of the tissue and a residue; and extracting the residue with ethanol to obtain an alcohol-soluble extract of a butterfly flower melon tissue. The extract of the genus Phalaenopsis meristem, according to any one of claims 1 to 3, wherein in the method, the phalaenopsis meristem is selected from the group consisting of: apical meristem , root tip meristem, flower meristem, inflorescence meristem and twin meristem. An extract of the genus Phalaenopsis meristem, as claimed in claim 4, wherein In the method, the white-flowered Phalaenopsis meristem is a twin meristem. The extract of the genus Phalaenopsis meristem, according to any one of claims 1 to 3, wherein in the method, the extraction of the supercritical CO ₂ is at a temperature ranging from 35 ° C to 100 ° C and A range is carried out at a pressure ranging from 1500 psi to 7250 psi. An extract of the genus Phalaenopsis meristem of claim 2, wherein in the method, the water-soluble extract of the phalaenopsis meristem is subjected to an ultrasonic treatment by mixing the residue with water. given. An extract of the genus Phalaenopsis meristem, as claimed in claim 3, wherein in the method, the alcohol-soluble extract of the phalaenopsis meristem is subjected to an ultrasonic treatment by mixing the residue with ethanol. given. An extract of the genus Phalaenopsis meristem, as claimed in claim 7 or 8, wherein in the method, the ultrasonic treatment is carried out at a temperature ranging from 10 ° C to 50 ° C. An extract of the genus Phalaenopsis meristem, as claimed in claim 7 or 8, wherein in the method, a filtration treatment is further performed after the ultrasonic treatment. A method for preparing an extract of the genus Phalaenopsis meristem, comprising the steps of: extracting the genus Phalaenopsis meristem by supercritical CO ₂ , thereby obtaining the fat solubility of the phalaenopsis meristem of the white flower Extract and a residue. The method of claim 11, further comprising the steps of: extracting the residue with water, and obtaining a water-soluble solution of the genus Phalaenopsis meristem. Sexual extract. The method of claim 11, which further comprises the step of extracting the residue with ethanol to obtain an alcohol-soluble extract of a butterfly flower melon tissue. The method of any one of clauses 11 to 13, wherein the genus Phalaenopsis meristem is selected from the group consisting of: apical meristem, apical meristem, floral meristem, inflorescence The meristem and the twins. The method of claim 14, wherein the white-flowered Phalaenopsis meristem is a twin meristem. The method of any one of claims 11 to 13, wherein the supercritical CO ₂ extraction is carried out at a temperature ranging from 35 ° C to 100 ° C and a pressure ranging from 1500 psi to 7250 psi. . The method of claim 12, wherein the water-soluble extract of the genus Phalaenopsis meristem is obtained by mixing the residue with water and performing an ultrasonic treatment. The method of claim 13, wherein the alcohol-soluble extract of the genus Phalaenopsis meristem is obtained by mixing the residue with ethanol and performing an ultrasonic treatment. The method of claim 17 or 18, wherein the ultrasonic treatment is performed at a temperature ranging from 10 ° C to 50 ° C. The method of claim 17 or 18, wherein a filtering process is further performed after the ultrasonic processing. A composition for whitening skin comprising as claimed in claim 1 An extract of the genus Phalaenopsis meristem from any of the three. The composition of claim 21, which comprises a fat-soluble extract of the genus Phalaenopsis meristem and a water-soluble extract of the genus Phalaenopsis meristem. The composition of claim 21, which comprises the alcohol-soluble extract of the phalaenopsis mycorrhizal meristem and the water-soluble extract of the genus Phalaenopsis meristem. The composition of claim 21, which is in the form of a dosage form for topical administration. A composition for enhancing the moisturizing ability of the skin, which comprises the extract of the phalaenopsis mycelium meristem of any one of claims 1 to 3. The composition of claim 25, which comprises the fat-soluble extract of the phalaenopsis meristem meristem and the water-soluble extract of the phalaenopsis mycorrhizal meristem. The composition of claim 25, which comprises the alcohol-soluble extract of the genus Phalaenopsis meristem and the water-soluble extract of the genus Phalaenopsis meristem. The composition of claim 25, which is in the form of a dosage form for topical administration. A composition for anti-skin aging comprising an extract of the genus Phalaenopsis meristem as claimed in any one of claims 1 to 3. The composition of claim 29, which comprises a fat-soluble extract of the genus Phalaenopsis meristem and a water-soluble extract of the genus Phalaenopsis meristem. The composition of claim 29, which comprises the white flower phalaenopsis meristem A woven alcohol soluble extract and a water soluble extract of the white flower of the Phalaenopsis meristem. The composition of claim 29, which is in the form of a dosage form for topical administration. A composition comprising an extract of the genus Phalaenopsis meristem of any one of claims 1 to 3 for use in the preparation of a pharmaceutical or cosmetic for whitening skin. The use of claim 33, wherein the composition comprises a fat-soluble extract of the genus Phalaenopsis meristem and a water-soluble extract of the genus Phalaenopsis meristem. The use of claim 33, wherein the composition comprises an alcohol-soluble extract of the phalaenopsis meristem, and a water-soluble extract of the phalaenopsis mycorrhizal meristem. The use of claim 33, wherein the pharmaceutical or cosmetic is in a form for topical administration. A composition comprising an extract of a phalaenopsis mycorrhizal meristem of any one of claims 1 to 3 for use in the preparation of a pharmaceutical or cosmetic for enhancing the moisturizing ability of the skin. The use of claim 37, wherein the composition comprises a fat-soluble extract of the genus Phalaenopsis meristem and a water-soluble extract of the genus Phalaenopsis meristem. The use of claim 37, wherein the composition comprises an alcohol-soluble extract of the Phalaenopsis mycorrhizal meristem and a water-soluble extract of the Phalaenopsis mycorrhizal meristem. The use of claim 37, wherein the pharmaceutical or cosmetic product is presented Form of topical application. A composition comprising an extract of the genus Phalaenopsis meristem as defined in any one of claims 1 to 3 for use in the preparation of a medicament or cosmetic for anti-skin aging. The use of claim 41, wherein the composition comprises a fat-soluble extract of the genus Phalaenopsis meristem and a water-soluble extract of the genus Phalaenopsis meristem. The use of claim 41, wherein the composition comprises an alcohol-soluble extract of the Phalaenopsis mycorrhizal meristem and a water-soluble extract of the Phalaenopsis mycorrhizal meristem. The use of claim 41, wherein the pharmaceutical or cosmetic is in a form for topical administration.