JP7090886B2 - Hydrolyzate of watery rose apple extract and method for producing the hydrolyzate and use of the hydrolyzate - Google Patents

Description

The present invention relates to a hydrolyzate of a water extract of Syzygium samarangense and a method for producing the hydrolyzate. The present invention also relates to the use of the hydrolyzate for whitening the skin, improving the moisturizing property of the skin, improving wound healing, and reducing oxidative stress.

The skin is the largest barrier in the human body and can prevent water loss, pathogen invasion, and damage from various harmful environments. However, excessive exposure to ultraviolet (UV), ionizing radiation, drugs, and xenobiotic substances produces reactive oxygen species (ROS) and free radicals in the skin. When the amount of accumulated reactive oxygen species and free radicals exceeds the antioxidant capacity of cells and tissues, it causes oxidative stress. Specifically, reactive oxygen species and free radicals react with cellular components (DNA, proteins, lipids, etc.), which adversely affects the skin. It has been reported that reactive oxygen species and free radicals not only play an important role in melanin production, but also delay wound healing (Kim Y.J. and Yokozawa T. (2009), Biol. Pharm. Bull. , 32: 1155-1159; and Kurahashi T. and Fujii J. (2009), J. Dev. Biol., 3: 57-70).

Melanin formation is the process by which melanocyte tyrosine is converted to melanin under the catalysis of tyrosinase and a series of redox reactions. Melanin formation is usually induced by UV irradiation (particularly UV-B). When the skin is exposed to UV light, ROS and free radicals produced by keratinocytes induce the expression of tumor protein 53 (p53), thereby causing the expression of the proopiomelanocortin gene (POMC gene), α-melanocytes. Releases POMC-derived peptides such as (α-MSH) and adrenocorticotropic hormone (ACTH). In addition, the tyrosine gene in melanocytes is expressed and large amounts of melanin accumulate on the skin.

When the skin is damaged (eg, congestion, trauma, surgical injury, contusion, etc.) and a wound is created, an inflammatory reaction is triggered at the wound site. At this time, a huge amount of ROS and free radicals are released in order to enhance the antipathogen action of immune cells. However, excess ROS and free radicals cause damage to the tissue surrounding the wound. On the other hand, in the process of wound healing, fibroblasts aggregate and proliferate at the wound site to release growth factors, which promote angiogenesis, epithelialization, collagen remodeling and the like. However, excess ROS and free radicals not only inhibit angiogenesis, but also inactivate enzymes involved in signaling pathways, thus hindering wound healing.

In recent years, there has been an increasing demand for skin whitening, moisturizing, promoting wound healing and antioxidants. In particular, the use of natural ingredients that do not cause unwanted and harmful side effects on the skin has become a trend in the field of cosmetology medicine. Therefore, researchers in the health and cosmetics industries have found that traditional Chinese medicine (TCM) or plants are safe active ingredients that are effective in whitening, moisturizing, wound healing and antioxidants to meet such great demands. I am trying to find out.

Syzygium samarangense (scientific name: Syzygium samarangense, general name: apple apple, Chinese pin-in: lian wu) belongs to the family Myrtaceae and the genus Syzygium. It is a perennial evergreen tree that blooms axillary flowers with a flower crown and bears inverted cone-shaped fruits. Watery rose apple is distributed in Taiwan, Indonesia, Philippines, Malaysia, etc. Common varieties of Javaft peach fruit include dark red varieties, pale red varieties, "Black Pearl", "Black King Kong", and "Black Diamond". Listed and widely edible. In Chinese folk medicine, the fruit of Javafum peach is used for lung nourishment, antitussive, expectorant, cool blood, and promotion of convergence. Furthermore, the fruit of Watery rose apple is considered to be effective in reducing fever, diuresis, and relieving stress.

The application of the extract of Watery rose apple to the pharmaceutical field is being studied. For example, in the paper "Evaluation of Antioxidant and Free Radical Scavenging Activity of Taiwan-specific Fruits" by Pei-Wen Lo, Department of Food and Nutrition, Sujin University (Taiwan), anti-antioxidant of Javanese peach fruit and other kinds of fruits. The results of the investigation of the oxidative effect have been reported. Specifically, 14 kinds of fruits (orange (Citrus sinensis Osbeck), tankan (Citrus tankan Hayata), pomelo (Citrus grandis (L) Osbeck), evening white yuzu (Citrus grandis Osbeck), kinkan (Citrus micro carpa), Strawberry (Fragaria ananassa Duch), dragon fruit (Hylocereus undatus), Javaft peach (Syzygium samarangense), persimmon (Diospyros kaki L.), mango (Mangifera indica L.), papaya (Carica papaya L.), nuts (Zizyphus jujuba) , Star fruit (Averrhoa carambola L.), and Guava (Psidium guajava L.) fruit) are frozen and dried and then ground into powder. 10 g of each fruit powder was extracted with 100 mL of methanol for 6 hours, filtered with a filter paper, and the obtained filtrate was concentrated to dryness. Antioxidant activity was tested on the methanol extracts of the 14 fruits thus obtained. Experimental results showed that the methanol extracts of the above 14 fruits exhibited different levels of activity in different tests. Specifically, according to the test of α, α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging ability, the methanol extract of the fruit material of Javaft peach (including the peel and pulp) has the DPPH radical scavenging ability. It turned out to be the lowest. Furthermore, as a result of testing of the ability to inhibit superoxide anion production, it was revealed that the methanol extract of the fruit material of Javaft peach could not inhibit superoxide anion production, but rather induces superoxide anion production. became.

Also, in the paper "Application of Extracts from Wax Apple Flowers on Antioxidantion" by Yi-Zhen Chen of the Cosmetics Research Institute of Kanan Pharmaceutical University (Taiwan). As described, the antioxidant potency and tyrosinase inhibitory ability in the flowers of the watery rose apple have been investigated. Specifically, 50 g of the dry powder of the watery rose apple is extracted with 500 ml of 80% methanol for 1 hour. Then, it is filtered using a filter paper. The two obtained filtrates were concentrated and lyophilized to obtain a methanol extract of Javafto peach flower and a water extract of Javaft peach flower. The extracts were tested for antioxidant potency and tyrosinase inhibitory activity. Most antioxidant activity tests (ie, superoxide anion scavenging ability, lipid peroxide inhibitory activity, ferrous ion chelating ability). In the test), it was found that the antioxidant activity of the water extract was superior to that of the watery rose apple. However, these results show that the tyrosinase inhibitory ability of the watery rose apple is superior to that of the watery rose apple. It shows that it is significantly superior to the tyrosinase inhibitory ability of the substance.

Taiwan Patent No. I465258 discloses an ethanol extract of Javanese myrtaceae flower (which has skin whitening and anti-aging effects) and a method for preparing the same. The above-mentioned preparation method includes a step of extracting a dry powder of a watery rose apple with 95% ethanol, performing a filtration treatment, and concentrating the obtained filtrate. In addition, it has been verified from experiments that the ethanol extract of the Javanese peach flower acts as an antioxidant and a tyrosinase inhibitor to promote collagen production.

Chinese Patent Application Publication No. 102228207 discloses a method for processing Javamyrtaceae and the products thus obtained. In this method, Javaft peach and water are mixed, the pH is adjusted to 3.5 with hydrochloric acid, and the enzymatic hydrolysis reaction is carried out with pepsin. Subsequently, the pH of the obtained pepsin hydrolyzate is adjusted to 7.5 using NaOH, and an enzymatic hydrolysis reaction is carried out using trypsin. The trypsin hydrolyzate thus obtained is centrifuged, and the obtained supernatant is collected and filtered. The product is obtained by concentrating the filtrate thus obtained. Experiments have shown that the products treated in this way are effective in treating dry lungs and coughs, hemorrhoidal bleeding, dyspepsia, enteritis and dysentery.

Although the above literature already exists, researchers in this field are trying to find a more satisfying bioactive ingredient in the watery rose apple. In previous studies, the applicant of the present application has found that the water extract of the fruit material of Myrtaceae japonicum has some effect on inhibiting the activity of tyrosinase, improving the moisturizing property of the skin, and improving the healing of wounds (note that it should be noted). Such a discovery has not been announced). In further research, Applicants of the present application found that the hydrolyzate prepared by hydrolyzing the water extract obtained from the fruit material of Javaft peach with bromelain was surprisingly compared to the water extract itself, tyrosinase. It was found that it can more effectively inhibit the activity of Bromelain, improve the moisturizing property of the skin, and improve wound healing. Therefore, the hydrolyzate of the watery rose apple extract is expected to serve as an active ingredient for cosmetics or pharmaceutical compositions.

Taiwan Patent No. I465258 Chinese Patent Application Publication No. 102228207

Kim Y.J. and Yokozawa T. (2009), Biol. Pharm. Bull., 32: 1155-1159 Kurahashi T. and Fujii J. (2009), J. Dev. Biol., 3: 57-70 Sukejin University (Taiwan) Department of Food and Nutrition, Pei-Wen Lo, "Evaluation of Antioxidant and Free Radical Erasing Activity of Taiwan-specific Fruits" Chia Nan Univiers (Taiwan), Cosmetics Research Institute, Yi-Zhen Chen, "Application of Extracts from Wax Apple Flowers on Antioxidantion"

Therefore, in the first aspect, the present invention comprises Java extraction treatment of a fruit material of Javanese peach with water to obtain a water extract, and hydrolysis treatment of the water extract with bromeline. Provided is a method for producing a hydrolyzate of an aqueous extract of Futomomo.
In a second aspect, the present invention is produced by a method comprising extracting a fruit material of Javanese peach with water to obtain a water extract, and hydrolyzing the water extract with bromelain. Provided is a hydrolyzate of a water extract of Bromelain.
In a third aspect, the present invention provides a composition containing a hydrolyzate of the water extract of Myrtaceae.
In a fourth aspect, the invention provides a means for skin whitening. The means include administration of the above composition to a subject.
In a fifth aspect, the present invention provides a means for improving the moisturizing property of the skin. The means include administration of the above composition to a subject.
In a sixth aspect, the invention provides a means for improving wound healing. The means include administration of the above composition to a subject.
In a seventh aspect, the invention provides a means for reducing oxidative stress. The means include administration of the above composition to a subject.
Other features and advantages of the invention will become apparent in the detailed description of the following embodiments with reference to the accompanying drawings.

It is an HPLC elution profile of BHWESS (bromelain hydrolyzate of watery rose apple extract) produced in Example 1. The ferrous ion chelating ability of BHWESS produced in Example 1 and APHWESS (animal-derived protease hydrolyzate of watery rose apple extract) produced in Example 3 is shown. The BHWESS and WESS (watery rose apple extract) produced in Example 1 show an inhibitory effect on tyrosinase activity, and the symbol "###" indicates that p <0.001 as compared with the control sample. "***" indicates that p <0.001 as compared with the WESS sample. The BHWESS and WESS produced in Example 1 show an improving effect on wound healing, the symbol "##" indicates that p <0.01 as compared with the control group, and the symbol "**" indicates that the WESS group. It shows that p <0.01 by comparison. The improvement effect of BHWESS produced in Example 1 and APHWESS produced in Example 3 in wound healing was shown, and the symbol "###" indicates that p <0.001 as compared with the control group, and the symbol ". *** ”Indicates that p <0.001 as compared with the APHWESS group. The effect of improving the water content of the stratum corneum of BHWESS and WESS produced in Example 1 is shown, and the symbol “###” indicates that p <0.001 as compared with the control area, and the symbol “**”. , "***" indicate that p <0.01 and p <0.001 as compared with the WESS area, respectively. The effect of improving the water content of the stratum corneum of BHWESS produced in Example 1 and APHWESS produced in Example 3 is shown.

The following description may refer to prior art documents, but it should be understood that these references do not acknowledge that they form part of the general knowledge in the art to which the present invention belongs. ..
Further, in the following description, "including" means "including but not limited to".
Unless otherwise defined, all technical and academic terms used herein are used in the sense that they are usually understood by those with ordinary knowledge in the field to which the present invention belongs.
Further, one of ordinary skill in the art will appreciate that many methods and materials similar to or equivalent to those described herein can be used in the practice of the present invention. The present invention is not limited to the specified methods and materials.

The applicant of the present application has attempted to obtain an extract of Watery rose apple using various methods in order to develop a skin care composition that is safe for long-term use. The applicant of the present application has stated that the hydrolyzate produced by hydrolyzing the water extract of Javanese peach fruit with bromelain promotes ferrous ion chelation (that is, as an antioxidant) rather than the water extract itself. It has been found to be more effective in improving tyrosinase inhibitory activity, skin moisturizing activity, and wound healing.

Therefore, the present invention provides a hydrolyzate of a watery rose apple extract and a method for producing the same. The hydrolyzate of the water extract of Javaft peach according to the present invention is obtained by extracting the fruit material of Javaft peach with water to obtain a water extract, and hydrolyzing the water extract with bromeline. Manufactured by a method comprising.

In the present invention, the fruit material of Watery rose apple includes the pulp of Watery rose apple. In some embodiments, the fruit material of the watery rose apple further comprises the skin and seeds of the watery rose apple.

In the present invention, the fruit material of Watery rose apple can be obtained from various Myrtaceae fruits of different cultivars. Depending on the embodiment, the fruit materials of Java peach are "Black Pearl", dark red variety, pale red variety, pink variety (both South Sea species), "Black Diamond", and "Black Kongo (Black Diamond)". It is obtained from one or more Javaft peach fruits belonging to the cultivated varieties selected from "Black King Kong)". In an exemplary embodiment, the fruit material of the watery rose apple is obtained from the fruit of the watery rose apple belonging to the "Black Pearl" variety.

In the present invention, the Myrtaceae fruit for obtaining the fruit material of Watery rose apple may have a range of maturity. In some embodiments, the fruit material of the watery rose apple is obtained from the immature watery rose fruit harvested one month after flowering.

In the present invention, the fruit material of Watery rose apple extracted with water may be a fruit material of fresh Watery rose apple, or may be dried, crushed, chopped, finely pulverized, or a combination thereof. The treatment may be performed before the extraction treatment with water.

In the present invention, the extraction treatment with water can be carried out using techniques well known to those skilled in the art. For example, the extraction process with water can be carried out according to the method shown in the above-mentioned paper by Yi-Zhen Chen of the Cosmetics Research Institute of Chia Nan Univiers (Taiwan).

In the disclosure of the present invention, the weight ratio of the fruit material of Watery rose apple to water is in the range of 1: 0.5 to 1: 5. In one embodiment of the present invention, the weight ratio of the fruit material of Watery rose apple to water is 1: 1.5.

In the present invention, the extraction treatment with water can be performed at a temperature of 35 ° C to 65 ° C. In one embodiment of the present invention, the extraction treatment with water is performed at a temperature of 50 ° C.

The conditions of the water extraction treatment vary depending on several factors (for example, pretreatment of the fruit material of Watery rose apple, weight ratio of the fruit material of Watery rose apple to water, etc.) in order to obtain a desired extraction result.

In the disclosure of the present invention, the weight ratio of water extract to bromelain is in the range of 150: 1 to 600: 1. In one embodiment of the invention, the weight ratio of the water extract to bromelain is 200: 1.

In the present invention, the hydrolysis treatment with bromelain can be carried out in a temperature range of 30 ° C. to 60 ° C. for 3 hours to 6 hours. In one embodiment of the invention, the hydrolysis treatment with bromelain is performed at 45 ° C. for 4 hours.

The conditions for the hydrolysis treatment with bromelain vary depending on various factors (for example, the weight ratio of bromelain to water) in order to obtain a desired hydrolysis treatment result.

In the present invention, the step of producing the hydrolyzate of the water extract of Javaft peach may further include a step of performing a solid-liquid separation treatment after the hydrolysis treatment to remove the solid content generated by the hydrolysis treatment. The solid-liquid separation treatment can be performed using a technique well known to those skilled in the art. Specific examples of the solid-liquid separation treatment include, but are not limited to, filtration, centrifugation, decantation and the like. In one embodiment of the invention, the solid-liquid separation process comprises a centrifugation step followed by a filtration step.

The applicant of the present application has found that the hydrolyzate of the water extract of Watery rose apple according to the present invention has the biological effects described below.

First, it was confirmed by in vitro experiments that the hydrolyzate of the watery rose apple extract according to the present invention can effectively inhibit the tyrosinase activity. Therefore, the hydrolyzate of the water extract of Myrtaceae according to the present invention is expected to be effective for skin whitening.

In addition, in this specification, "whitening skin", "lightening skin color", "whitening skin", "lightening skin", "lightening melanin", "suppressing melanin production" The terms "to" and "inhibit melanin synthesis" are used interchangeably.

Second, as a result of preliminary human tests by the applicant of the present application, the hydrolyzate of the water extract of Watery rose apple according to the present invention effectively increases the water content of the stratum corneum without causing undesired side effects. It was shown that it can be done. Therefore, the hydrolyzate of the water extract of Myrtaceae according to the present invention is expected to be useful for enhancing the moisturizing property of the skin.

Third, the hydrolyzate of the water extract of Jawaft peach according to the present invention was proved by in vitro experiments to be effective in improving the closure of the wound opening mediated by fibroblasts. As described above, it is considered that the hydrolyzate of the watery rose apple extract according to the present invention can be used for improving wound healing.

Fourth, it was proved by in vitro experiments that the hydrolyzate of the water extract of Watery rose apple according to the present invention has an excellent ferrous ion chelating ability. Therefore, the hydrolyzate of the water extract of Watery rose apple according to the present invention can be used as an antioxidant for reducing oxidative stress.

Fifth, since the hydrolyzate of the water extract of Watery rose apple according to the present invention has antioxidant activity, it is expected to be effective in preventing skin aging and delaying aging.

As used herein, the term "delayed" treats, alleviates, alleviates, improves, sedates or suppresses one or more clinical signs of a disease or disorder, and the severity of the symptom or symptom. It means to slow down, stop, or reverse the progress of.

Also, as used herein, the term "skin aging" includes naturally occurring endogenous skin aging and extrinsic skin aging caused by environmental factors (eg, ultraviolet light). Intended to be. Skin aging phenomena include dilation of capillaries, thinning of the epidermis, atrophy of the skin, roughening of the skin, dryness, wrinkle formation and pigmentation (eg moles, freckles, hypopigmentation or hyperpigmentation). ), But not limited to these.

Therefore, the present invention includes the hydrolyzate of the above-mentioned water extract of Javaft peach, which is suitable for skin whitening, improvement of skin moisturization, improvement of wound healing, reduction of oxidative stress, prevention and delay of skin aging. A composition (hereinafter also simply referred to as a composition) is provided.

The present invention also provides the following means using the above composition.
First, the present invention provides a method for skin whitening, which comprises administering the above composition to a subject. An effective amount of the composition may be administered for a sufficient time until the subject's skin is significantly whitened. The above composition does not cause harmful side effects on the subject's skin even in an effective amount.

In the present invention, the dose and frequency of the composition for skin whitening vary depending on the following factors. That is, the dose and frequency of the composition vary depending on the initial state of the target area of melanin lightening in the skin, the route of administration, and the degree of whitening effect desired to be achieved. For example, the dose of the composition for topical administration according to the invention can be 1-10 mg per square cm of skin area and can be administered 1-3 times per day. In certain embodiments, the dose of the composition for oral administration can be 0.05-5 mg / kg body weight and can be administered 1-3 times daily.

Secondly, the present invention provides a means for improving the moisturizing property of the skin, which comprises administering the above composition to a subject. An effective amount of the composition may be administered for a sufficient period of time until the moisturizing properties of the subject's skin are significantly improved. The above composition does not cause harmful side effects on the skin even in an effective amount.

In the present invention, the dose and frequency of the composition for enhancing the moisturizing property of the skin are changed by the following factors. That is, the dose and frequency of the composition vary depending on the initial state of the target area for improving the moisturizing property in the skin, the administration route, and the degree of the moisturizing property improving effect to be achieved. For example, the dose of the composition for topical administration according to the invention can be 1-10 mg per square cm of skin area and can be administered 1-3 times per day. In certain embodiments, the dose of the composition for oral administration can be 0.05-5 mg / kg body weight and can be administered 1-3 times daily.

Third, the present invention provides means for improving wound healing, including administering the above composition to a subject. An effective amount of the composition may be administered for a sufficient amount of time until the wound area is significantly reduced. The composition does not cause any harmful side effects on the wound, even in effective amounts.

In the present invention, the dosage and frequency of the composition for improving wound healing will vary depending on the following factors: That is, the dose and frequency of the composition will vary depending on the extent of the wound, the route of administration, and the degree of healing effect desired to be achieved. For example, the dose of the composition for topical administration according to the invention can be 1-10 mg per square cm of skin area and can be administered 1-3 times per day. In certain embodiments, the dose of the composition for oral administration can be 0.05-5 mg / kg body weight and can be administered 1-3 times daily.

Fourth, the present invention provides means for reducing oxidative stress, including administering the above composition to a subject. An effective amount of the composition may be administered for a sufficient time until the oxidative stress is significantly reduced. The composition does not cause side effects to the subject even in an effective amount.

In the present invention, the dose and frequency of the composition for reducing oxidative stress will vary depending on the following factors: That is, the dose and frequency of the composition will vary depending on the degree of oxidative stress, the route of administration, and the degree of antioxidant effect desired to be achieved. For example, the dose of the composition for topical administration according to the invention can be 1-10 mg per square cm of skin area and can be administered 1-3 times per day. In certain embodiments, the dose of the composition for oral administration can be 0.05-5 mg / kg body weight and can be administered 1-3 times daily.

Fifth, the present invention provides means for preventing or delaying skin aging, which comprises administering the above composition to a subject. An effective amount of the composition may be administered for a sufficient time until at least one type of skin aging phenomenon is significantly improved. The composition does not cause side effects to the subject even in an effective amount.

In the present invention, the dose and frequency of the composition for preventing or delaying skin aging depends on the following factors: That is, the dose and frequency of the composition will vary depending on the degree of skin aging, the route of administration, and the degree of effect desired to be achieved. For example, the dose of the composition for topical administration according to the invention can be 1-10 mg per square cm of skin area and can be administered 1-3 times per day. In certain embodiments, the dose of the composition for oral administration can be 0.05-5 mg / kg body weight and can be administered 1-3 times daily.

Due to the above-mentioned biological activity and safety of the hydrolyzate of the water extract of Javaft peach, the hydrolyzate (skin whitening, skin moisturizing improvement, wound healing improvement, oxidative stress reduction, skin aging The composition comprising (suitable for prevention and delay) can be a pharmaceutical composition or a cosmetic composition.

The pharmaceutical composition according to the present invention can be formulated into an appropriate dosage form for oral or topical administration using techniques well known to those skilled in the art.

The pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable carrier widely used in pharmaceuticals. Pharmaceutically acceptable carriers include, for example, solvents, buffers, emulsifiers, suspending agents, degrading agents, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, preservatives, wetting agents. , Lubricants, diluents, absorption retarders, liposomes, sweeteners, flavors, colorants and the like, but not limited to these. The choice and amount of pharmaceutically acceptable carrier is within the expertise of one of ordinary skill in the art.

Examples of orally administered dosage forms include sterile powders, tablets, troches, drops, pills, capsules, powders or granules, liquids, suspending agents, emulsions, syrups, elixirs, slurries and the like. Not limited to.

Examples of topical dosage forms include emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, instillations, suspending agents, ointments, bandages, etc. Not limited to these.

In an exemplary embodiment of the invention, the pharmaceutical composition is externally applied by mixing a hydrolyzate of a watery rose apple extract with a base well known and commonly used in the art. Formed as an agent.

In the present invention, suitable bases include water, alcohol, glycol, hydrocarbons (petrolatum and white petrolatum), waxes (paraffin and yellow wax), preservatives, antioxidants, surfactants, absorption enhancers, and stables. Agents, gelling agents (eg Carbopol® 974P, microcrystalline cellulose and carboxymethyl cellulose, etc.), activators, wetting agents, deodorants, fragrances, pH adjusters, chelating agents, emulsifiers, blockages. Examples thereof include those containing one or more selected from agents, skin softeners, thickeners, solubilizers, penetration enhancers, antioxidants, colorants, propellants and the like. The choice and amount of these additives is within the expertise of one of ordinary skill in the art.

The cosmetic composition according to the present invention may further contain an adjuvant that is widely used in cosmetics manufacturing techniques and is acceptable for cosmetics. Acceptable adjuvants for cosmetics include, for example, solvents, gelling agents, activators, preservatives, antioxidants, shielding agents, chelating agents, surfactants, colorants, thickeners, fillers, fragrances, etc. Examples include those containing one or more selected from deodorants. The choice and amount of these additives is within the expertise of one of ordinary skill in the art.

The cosmetic composition according to the present invention can be in the form of a skin care or make-up product using techniques well known to those skilled in the art. Such forms include aqueous solutions, aqueous alcoholic or oily solutions, emulsions, gels, ointments, creams, masks, patches, packs, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, suspensions, etc. Includes, but is not limited to, drops, mousses, sunscreens, tonic waters, foundations, eye shadows, makeup removers, soaps and other body cleansing products.

The cosmetic composition according to the present invention can be used together with at least one kind of external preparations listed below. External agents include whitening agents (tretinoin, catechin, succinic acid, arbutine, vitamin C, etc.), moisturizers, anti-inflammatory agents, bactericides, UV absorbers, algae extracts (including aloe extracts), skin nutrition. Agents, anesthetics, anti-acne agents, anti-itch agents, painkillers, antiperspirants, anti-dermatitis agents, anti-hyperkeratolytic agents, dry skin improvers, anti-aging agents, anti-wrinkle agents, anti-lipid agents, wound healing Agents, corticosteroids, hormones. The choice and amount of these agents is within the expertise of one of ordinary skill in the art.

Hereinafter, the present invention will be further described with reference to examples. However, it should be understood that the following examples are for illustration purposes only and should not be construed as limiting the invention.

[Example 1: Production of bromelain hydrolyzate of water extract of Myrtaceae]
3 to 6 g of Javaft peach fruit material (including peel, pulp and seeds) harvested in Fangliao Township, Pingtung County, Taiwan is crushed, and the obtained crushed product and water are weighed at a weight ratio of 1: 1.5. They were mixed and heated at 50 ° C. for 1 hour. As a result, a water extract of Syzygium samarangense (hereinafter abbreviated as WESS) was obtained.

Subsequently, WESS and 1600 IU / g bromelain (manufactured by NEO-ONE BIOTECH CO., LTD., Product number: 1600GD) were mixed at a weight ratio of 200: 1 to carry out an enzymatic hydrolysis reaction. It was allowed to proceed at 45 ° C. for 4 hours. The obtained reaction solution was centrifuged, and the supernatant was recovered. This supernatant is sequentially used with filters having pore sizes of 5 μm, 1 μm, and 0.5 μm (manufactured by Tech Seed Enterprise Co., Ltd., product numbers: PureMax, SCB2000-001, SP2000-00A). And filtered. The obtained filtrate was freeze-dried to obtain a bromelain hydrolysate of water extract of Syzygium samarangense (hereinafter abbreviated as BHWESS) as a freeze-dried powder.

For comparison with BHWESS, WESS was also subjected to the above-mentioned centrifugation, filtration and freeze-drying treatment to obtain a powder form.

[Example 2: HPLC analysis of watery rose apple extract in bromelain hydrolyzate]
In order to measure the main component of BHWESS according to the present invention, the BHWESS obtained in Example 1 was dissolved in distilled deionized water to prepare a sample having a BHWESS concentration of 1 mg / mL, and HPLC ( High performance liquid chromatography) analysis was performed.

The HPLC instrument used was an HP Agilent 1100 series HPLC system with a UV-Vis detector and a C18 column (Purosphere® STAR RP-18, product number: 150359, size: 250 mm x 4.6 mm).

The applicable HPLC operating conditions are as follows. The mobile phase is acetonitrile / deionized water (100: 0, v / v) and the flow rate of the mobile phase is 0.7 mL / min. Gradient elution with the mobile phase was performed for 35 minutes as follows: acetonitrile was reduced from 100% to 90% between 0-10 minutes and 90% to 80% between 10-30 minutes. , Increased from 80% to 100% in 30 minutes and maintained at 100% from 30 to 35 minutes.

〔result〕
FIG. 1 is an HPLC elution profile of BHWESS produced in Example 1. As shown in FIG. 1, four major peaks (marked with a, b, c, d, respectively) were observed during the dwell time from 0 to 35 minutes.

[Example 3: Evaluation of antioxidant activity of watery rose apple extract in bromelain hydrolyzate]
In order to investigate whether BHWESS according to the present invention has an antioxidant effect, the ferrous ion chelating ability of BHWESS produced in Example 1 was analyzed. In addition, for comparison with BHWESS, animal protease hydrolysate of watery rose apple extract, largely according to the method described in Chinese Patent Application Publication 102228207A (with minor modifications). A water extract of Syzygium samarangense (hereinafter abbreviated as APHWESS) was produced, and the same experiment as the BHWESS according to the present invention was carried out on this. Before explaining the experimental procedure, a method for producing APHWESS is shown below.

20 g of Javaft peach fruit material (including peel, pulp and seeds) harvested in Fangliao Township, Pingtung County, Taiwan was crushed, and the obtained crushed product and water were mixed at a weight ratio of 1: 1.5. , 95 ° C. for 15 minutes. This gave a watery rose apple extract.

After cooling to 50 ° C., the pH of the watery rose apple extract was adjusted to 3.68 with 4.5% citric acid. Subsequently, a water extract of Watery rose apple and 2500 IU / g pepsin (manufactured by Sigma, product number: P7012) were mixed at a weight ratio of 50,000: 1, and the enzymatic hydrolysis reaction was allowed to proceed at 50 ° C. for 2 hours.

The pH of the obtained pepsin hydrolyzate was adjusted to 7.65 with potassium hydroxide 4 g / mL and then heated to 95 ° C. for 15 minutes. The pepsin hydrolyzate and 2000 IU / mg trypsin (manufactured by Himedia, product number: RM618) were mixed at a weight ratio of 27000: 1 and the enzyme hydrolysis reaction was allowed to proceed at 50 ° C. for 3 hours.

The pH of the tryptic hydrolyzate thus obtained was adjusted to 6.28 with 4.5% citric acid, followed by centrifugation, and the obtained supernatant was recovered. The supernatant was filtered using filters having pore sizes of 5 μm, 1 μm, and 0.5 μm, respectively. The obtained filtrate was freeze-dried to obtain APHWESS in the form of a freeze-dried powder.

[Experimental procedure]
An appropriate amount of BHWESS produced in Example 1 was dissolved in distilled deionized water to prepare a BHWESS sample having a BHWESS concentration of 0.35% (w / w). Then, 1 mL of the BHWESS sample, 3.7 mL of ethanol, and 0.1 mL of a _{2 mM FeCl 2.4H 2 O} solution were mixed for 30 seconds. The obtained mixture was uniformly mixed with 0.2 mL of 5 mM ferrozine, and then left at a constant temperature for 10 minutes in a no-light environment and at room temperature. Absorbance (OD ₅₆₂ ) at 562 nm was measured for this mixture using a Colibri spectrophotometer (manufactured by Titertek-Berthold). The above experimental procedure was also performed on 1 mL of a 95% ethanol solution as a control sample. Further, an appropriate amount of the above-mentioned APHWESS produced generally according to the method described in Chinese Patent Application Publication 102228207A is dissolved in distilled deionized water to prepare an APHWESS sample having an APHWESS concentration of 0.35% (w / w). The same experimental procedure as for the BHWESS sample was performed on this APHWESS sample to analyze the ferrous ion chelating ability of APHWESS.
The test results of the sample show that the lower the value of OD ₅₆₂ , the stronger the antioxidant effect.

The ferrous ion chelation rate (%) was calculated using the following formula (I).
A = [1- (B / C)] x 100 (I)
During the ceremony
"A" is the ferrous ion chelation rate (%).
"B" is the value of OD ₅₆₂ for each of BHWESS and APHWESS.
"C" is the value of OD ₅₆₂ of the control sample.
All experiments were repeated 3 times. Experimental data is expressed as mean ± standard deviation.

〔result〕
FIG. 2 shows the ferrous ion chelation rate of the BHWESS sample, the control sample, and the APHWESS sample. As shown in FIG. 2, the ferrous ion chelation rate of the BHWESS sample is significantly higher than that of the control sample and the APHWESS sample. Based on this result, the applicant of the present application found that the hydrolyzate of the watery rose apple according to the present invention has a sufficient antioxidant effect even when compared with the animal-derived protease hydrolyzate of the watery rose apple. I think it can be demonstrated.

[Example 4: Evaluation of melanin production inhibitory effect of watery rose apple extract in bromelain hydrolyzate]
In this example, the melanin production inhibitory effect of BHWESS according to the present invention was evaluated based on the tyrosinase activity. Further, for comparison, the same experiment as BHWESS was performed on the WESS produced in Example 1.

[Experimental procedure]
500 mL of 0.1 M Na ₂ HPO ₄ solution and 500 mL of KH ₂ PO ₄ solution were mixed, and the pH of the obtained phosphate buffer was adjusted to 6.8 with sodium hydroxide. An appropriate amount of tyrosine (manufactured by Alfa Aesar, lot number: 10174330) was added to an appropriate amount of phosphate buffer to prepare a 2 mM tyrosine solution.

Subsequently, appropriate amounts of BHWESS and WESS produced in Example 1 were added to an appropriate amount of phosphate buffer to obtain a BHWESS sample having a BHWESS concentration of 10 mg / mL and a WESS sample having a WESS concentration of 10 mg / mL. .. 21.6 μL of BHWESS and WESS samples, respectively, were mixed with 250 μL of tyrosinase (Sigma, lot number: SLBJ5647V) (115.42 U / mL in an appropriate amount of phosphate buffer) and 540 μL of tyrosine solution. The obtained mixture was left at a constant temperature of 37 ° C. for 30 minutes in a no-light environment. Then, the absorbance (OD ₄₇₅ ) at 475 nm was measured using a spectrophotometer. The above experimental procedure was also performed on a 290 μL phosphate buffer solution as a control sample. The test results of the sample show that the lower the value of OD ₄₇₅ , the stronger the tyrosinase inhibitory activity.

The tyrosinase inhibitory activity rate (%) was calculated using the following formula (II).
D = [1- (E / F)] x 100 (II)
During the ceremony
"D" is the tyrosinase inhibitory activity rate (%),
"E" is the value of OD ₄₇₅ for each of BHWESS, WESS, and the control sample.
"F" is the value of OD ₄₇₅ of the control sample.

All experiments were repeated twice. Experimental data were expressed as mean ± standard deviation and analyzed by Student's t-test to assess differences between BHWESS, WESS and control samples. Statistical significance is p <0.05.

〔result〕
FIG. 3 shows the percentage inhibition of tyrosinase activity in BHWESS samples, WESS samples and control samples. As shown in FIG. 3, the percent tyrosinase activity inhibition of the BHWESS sample is significantly higher than that of the WESS sample and the control sample. This experimental result shows that the hydrolyzate of the watery rose apple extract according to the present invention has a better tyrosinase inhibitory ability as compared with the watery rose apple extract. Therefore, the applicant of the present application considers that the hydrolyzate of the watery rose apple according to the present invention has a skin whitening effect because it can suppress the production of melanin by suppressing the tyrosinase activity.

[Example 5: Evaluation of wound healing improving effect in bromelain hydrolyzate of water extract of Myrtaceae]
In this example, the wound healing improving effect of BHWESS according to the present invention was evaluated by a wound healing cell migration assay. Further, for comparison, the WESS produced in Example 1 and the APHWESS produced in Example 3 were evaluated in the same manner as BHWESS.

[Experimental material]
(1) Human skin fibroblast cell line Detroit551
The human skin fibroit cell line Detroit 551 used in this example is the Bioresource Collection and Research Center (Higashi-ku, Shintake City, Taiwan) of the Food Industry Research and Development Institute in Taiwan. Purchased from Food Road 331).

Detroit 551 cells contain 10% fetal bovine serum (FBS) and 10% penicillin-streptomycin (GIBCO®, model number: 15140122) supplemented with minimal essential medium (MEM, Sigma, product number: M2279). The cells were cultured in Petri dishes and then in an incubator (37 ° C., 5% CO ₂ ). The medium was changed every two days, and cell passage was performed when the cultured cells became 80% to 90% confluent.

[Experimental procedure]
(A) Comparison of wound healing improving effect between BHWESS and WESS The Detroit 551 cells obtained in (1) of the experimental material were divided into three groups, a BHWESS group, a WESS group, and a control group. Each group of Detroit 551 cells was cultured at 2 × 10 ⁵ cells / well in a 24-well plate containing 500 μL of MEM (filled with 10% FBS) and then in an incubator (37 ° C., 5% CO ₂ ) for 24 hours. did.

After changing the medium to fresh, 200 μL of plastic pipette tips were used to scratch Detroit 551 cells in each well of the plate along the diameter line. This formed a 500 μm wide, cell-free wound area along the diameter line of each well. Washing was performed twice with phosphate buffer (PBS). After media exchange with fresh MEM (filled with 5% FBS), the cell cultures of the BHWESS group and the WESS group were treated with appropriate amounts of BHWESS and WESS prepared in Example 1, respectively, and the BHWESS and WESS groups were treated with appropriate amounts, respectively. The final concentrations were 0.03% (w / w) BHWESS and 0.03% (w / w) WESS, respectively. The cell cultures of the control group were not treated as described above.

Then, the cells were cultured in an incubator (37 ° C., 5% CO ₂ ) for 12 hours. At 0 hours (before treatment) and 12 hours after treatment, the wound area of each group was observed using an inverted microscope (TE300 manufactured by Nikon Corporation) with a magnification of 40 times, and photographed using a digital camera (D90 manufactured by Nikon Corporation). The area was calculated using the ImageJ software based on the obtained photographs.

The wound healing rate (%) was calculated using the following formula (III).
G = [1- (H / I)] x 100 (III)
During the ceremony
"G" is the wound healing rate (%)
"H" is the area of the wound area 12 hours after the procedure.
"I" is the area of the wound area before treatment.

All experiments were repeated twice. Experimental data were expressed as mean ± standard deviation and analyzed by Student's t-test to assess differences between the BHWESS, WESS and control groups. Statistical significance is p <0.05.
(B) Comparison of wound healing improving effect between BHWESS and APHWESS

First, a SPLScar® block (manufactured by Taiwan Bio-genesis Technologies, model number: 21902) having a width of 500 μm was placed along the diameter line of each well of the 24-well plate. Subsequently, the Detroit 551 cells obtained in (1) of the experimental material were divided into three groups, a BHWESS group, an APHWESS group, and a control group. Each group of Detroit 551 cells was cultured at 5 × 10 ⁴ cells / well in a 24-well plate containing 500 μL of MEM (filled with 10% FBS) and then in an incubator (37 ° C., 5% CO ₂ ) for 24 hours. did.

Subsequent removal of the SPLScar block formed a 500 μm wide, cell-free wound area. Washing was performed twice with phosphate buffer (PBS). After media exchange with fresh MEM (filled with 5% FBS), the cell cultures of the BHWESS group and the APHWESS group were treated with appropriate amounts of BHWESS prepared in Example 1 and APHWESS prepared in Example 3, respectively. , BHWESS group and APHWESS group were set to 0.025% (w / w) BHWESS and 0.03% (w / w) APHWESS, respectively. The cell cultures of the control group were not treated as described above.

Then, the cells were cultured in an incubator (37 ° C., 5% CO ₂ ) for 12 hours. At 0 hours (before treatment) and 12 hours after treatment, the wound area of each group was observed using an inverted microscope (IX73 manufactured by Olympus) at a magnification of 40 times, and photographed using a digital camera (DP80 manufactured by Olympus). The area was calculated using the ImageJ software based on the obtained photographs. Then, the wound healing rate (%) was calculated using the formula (III).

All experiments were repeated 3 times. Experimental data were expressed as mean ± standard deviation and analyzed by Student's t-test to assess differences between the BHWESS group, APHWESS group and control group. Statistical significance is p <0.05.

〔result〕
FIG. 4 shows the wound healing rates of Detroit 551 cells treated with BHWESS or WESS and those cells that were not treated. As shown in FIG. 4, no significant difference was observed in the wound healing rate between the WESS group and the control group. On the other hand, the wound healing rate in the BHWESS group was significantly higher than that in the control group and the WESS group.

FIG. 5 shows the wound healing rates of Detroit 551 cells treated with BHWESS or APHWESS and those cells that were not treated. As shown in FIG. 5, no significant difference was observed in the wound healing rate between the APHWESS group and the control group. However, the wound healing rate in the BHWESS group was significantly higher than that in the control group and the APHWESS group.

From the above experimental results, it was clarified that the hydrolyzate of the water extract of Jawaft peach according to the present invention is effective in improving wound closure by fibroblasts and can promote wound healing.

[Example 6: Evaluation of the effect of improving the moisturizing property of the skin on the bromelain hydrolyzate of the water extract of Myrtaceae]
In order to investigate the effect of BHWESS according to the present invention on the moisturizing property of human skin, the following experiments were conducted.

(A) Preparation of Essence In this example, BHWESS and WESS produced in Example 1 and APHWESS produced in Example 3 were used. Four kinds of essences (BHWESS essence, WESS essence, APHWESS essence, control essence) were prepared using the recipes shown in Table 1.

The process for preparing BHWESS essence, WESS essence, and APHWESS essence is as follows. That is, butylene glycol and hydroxyethyl cellulose were dissolved in deionized water at 75 ° C. with stirring, and then cooled to 40 ° C. to 45 ° C. BHWESS, WESS and APHWESS were added, respectively, and then mixed with Dermosoft OMP with stirring. Next, using sodium hydroxide, the pH of each obtained mixture was adjusted to about 6 to 7, and BHWESS essence, WESS essence, and APHWESS essence were obtained.

The process for preparing the control essence is similar to the process for preparing the BHWESS essence, except that additional deionized water was used in place of BHWESS.

(B) Comparison of skin moisturizing effect between BHWESS and WESS Yeun Diing Enterprise CO., LTD. In Pingtung County, Taiwan and Hong Hong Biotechnology Co., Ltd. in Yunlin County, Taiwan (MH) Subjects recruited from employees of Biotechnology Co., Ltd.) were screened according to the exclusion criteria shown in Table 2. As a result, 10 eligible subjects (4 males and 6 females aged 25-40 years) participated in the study below.

The subject's right arm was randomly divided into three test areas (ie, BHWESS area, WESS area and control area) each having a width of four fingers, and each area was prepared by the above-mentioned "(A) Preparation of essence". BHWESS essence, WESS essence or control essence was applied twice daily at a dose of 0.5 mL at a time. The test period for each test area was 28 days in total.

On day 0 (ie, before application of the test essence) and at designated points in time (days 7, 14, 21, 28) after the start of application of the test essence, the test area of each subject is electrostatically charged. The water content of the stratum corneum was measured using a capacitance meter (Corneometer (registered trademark) CM285, manufactured by Course & Khazaka).

The improvement rate (%) of the water content of the stratum corneum was calculated using the following formula (IV).
J = [(LK) / K] x 100 (IV)
During the ceremony
"J" is the improvement rate (%) of the water content of the stratum corneum at each designated time point (7th day, 14th day, 21st day, 28th day).
"K" is the water content of the stratum corneum on the 0th day.
"L" is the water content of the stratum corneum at each specified time point.

Water content measurements were repeated 3 times for each subject's test area. Experimental data were expressed as mean ± standard deviation and analyzed by Student's t-test to assess differences between test areas. Statistical significance is p <0.05.

In addition, a safety assessment was performed by the investigator on day 0 and at each designated time point thereafter (day 7, 14, 21, 21). On day 0, a small amount of essence was applied to the inside of the subject's upper arm for safety assessment and was investigated for adverse skin reactions (itch, irritation and erythema, etc.).

(C) Comparison of skin moisturizing effect between BHWESS and APHWESS The subjects recruited from employees of MH Biotechnology Co., Ltd. were screened according to the exclusion criteria shown in Table 2. .. As a result, 9 eligible subjects (6 males and 3 females aged 25-40 years) participated in the study below.

The application of the test essence, the measurement of the water content of the stratum corneum, and the safety evaluation were carried out generally according to the procedure described in (B) above of this example, except for the following differences. The differences are that the APHWESS essence was replaced with the WESS essence and tested, the area where the APHWESS essence was applied was the APHWESS area (that is, the WESS area was replaced with the APHWESS area), and the measurement of the water content of the stratum corneum was the test essence. It was carried out only at one designated time point (14th day) after the start of application of.

〔result〕
(1) Measurement result of the water content of the stratum corneum FIG. 6 shows the improvement rate of the water content of the stratum corneum at each of BHWESS, WESS and the control area at different time points. As shown in FIG. 6, on the 14th, 21st, and 28th days after the start of application of the test essence, the improvement rate of the water content of the stratum corneum in the BHWESS area was significantly higher than that in the WESS area and the control area. high. Furthermore, the water content of the stratum corneum in the BHWESS area increased significantly over time, but the improvement in the water content of the WESS area was very slow.

FIG. 7 shows the improvement rate of the water content of the stratum corneum on the 14th day of each of BHWESS, APHWESS and the control area. As shown in FIG. 7, the improvement rate of the water content of the stratum corneum in the BHWESS area is higher than that in the APHWESS area and the control area.

The above data suggests that the hydrolyzate of the watery rose apple extract according to the present invention can be used to effectively enhance the moisturizing property of the skin.

(2) Safety evaluation results No adverse skin reaction was observed in all subjects during the test period (data not shown).

From the above, the hydrolyzate of the water extract of Jawaft peach according to the present invention exhibits excellent antioxidant activity, suppresses melanin production, enhances the moisturizing property of the skin, and promotes wound healing. Thereby, the applicant of the present application considers that the hydrolyzate of the water extract of Myrtaceae according to the present invention can be developed into a skin care product for long-term use that does not induce harmful side effects.

All patents and references cited herein are incorporated herein by reference in their entirety. However, if a contradiction arises between the referenced content and the description relating to the invention of the present application, the description relating to the invention of the present application, including the definition, takes precedence.

Although the embodiments that are considered to be exemplary of the present invention have been described above, the present invention is not limited thereto, and all modifications and equalities are made as various forms included in the spirit and scope of the broadest interpretation. It shall include various configurations.

Claims (19)

To obtain a watery rose apple by extracting the fruit material of the watery rose apple with water,
Hydrolyzing the water extract with bromelain and
A method for producing a hydrolyzate of a watery rose apple extract, including. The method according to claim 1, wherein the weight ratio of the fruit material of the watery rose apple to water is in the range of 1: 0.5 to 1: 5. The method according to claim 1 or 2, wherein the weight ratio of the water extract to bromelain is in the range of 150: 1 to 600: 1. The method according to any one of claims 1 to 3, further comprising performing a solid-liquid separation treatment after the hydrolysis treatment to remove the solid content generated by the hydrolysis treatment. The method according to any one of claims 1 to 4, wherein the fruit material of the watery rose apple contains the pulp of the watery rose apple. The method according to claim 5, wherein the fruit material of the watery rose apple further comprises the skin of the watery rose apple and the seed of the watery rose apple. To obtain a watery rose apple by extracting the fruit material of the watery rose apple with water,
A hydrolyzate of a watery rose apple extract produced by a method comprising hydrolyzing the watery rose apple with bromelain. The hydrolyzate according to claim 7, wherein the weight ratio of the fruit material of the watery rose apple to water is in the range of 1: 0.5 to 1: 5. The hydrolyzate according to claim 7 or 8, wherein the weight ratio of the water extract to bromelain is in the range of 150: 1 to 600: 1. The hydrolyzate according to any one of claims 7 to 9, further comprising performing a solid-liquid separation treatment after the hydrolysis treatment to remove the solid content generated by the hydrolysis treatment. .. The hydrolyzate according to any one of claims 7 to 10, wherein the fruit material of the watery rose apple contains the pulp of the watery rose apple. The hydrolyzate according to claim 11, wherein the fruit material of the watery rose apple further comprises the peel of the watery rose apple and the seed of the watery rose apple. A composition comprising the hydrolyzate according to any one of claims 7 to 12. The composition according to claim 13, which is used for skin whitening. The composition according to claim 13, which is used for improving the moisturizing property of the skin. 13. The composition of claim 13, which is used to improve wound healing. The composition according to claim 13, which is used for reducing oxidative stress. The composition according to any one of claims 13 to 17, which is formed in a topically administered dosage form. The composition according to any one of claims 13 to 17, which is formed in an orally administered dosage form.

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