Enzyme extract derived from immature strawberries and preparation method therefor

Abstract

The present invention relates to: a method for extracting an enzyme from immature strawberry fruits; and a cosmetic composition for skin improvement, comprising an enzyme prepared by the method. By the method according to an aspect, an enzyme can be extracted from immature strawberry fruits with excellent efficiency, and a composition comprising the enzyme exhibits skin moisturizing, skin texture improvement, and dead skin cell improvement effects without exhibiting cytotoxicity, and thus can be effectively used for skin improvement.

Description

Enzyme extract derived from immature strawberries and method for preparing the same

[0001] The present invention relates to a method for extracting enzymes from immature strawberries and a cosmetic composition for skin improvement comprising the enzymes prepared thereby.

[0002] Strawberries (Fragaria xananassa) are a fruit belonging to the Rosaceae family and are cultivated worldwide. They are one of the popular fruits consumed either fresh or processed into various products. Currently, strawberries cultivated globally originate mainly from varieties resulting from the crossbreeding of two species (Fragaria chiloensis and Fragaria virginiana), and there are various varieties depending on size, color, taste, harvest time, etc.

[0003] Strawberries are generally harvested when they are fully ripe and turn red, and those that have not yet passed a certain period after flowering are referred to as unripe or immature strawberries. Immature strawberries have not yet fully developed their color, have a strong sour taste, and may have hard or crunchy flesh; therefore, except for specific varieties or cases requiring long-term transport or storage before shipment, they are difficult to harvest and utilize.

[0004] Against this backdrop, the inventors of the present invention sought to develop a method for extracting protease from immature strawberries with excellent efficiency, and confirmed that the enzyme produced according to this method of extracting enzyme from immature strawberries can be utilized as a cosmetic composition for skin improvement, thereby completing the present invention.

[0005] One aspect provides a method for extracting enzymes from immature strawberries.

[0006] Another aspect provides a cosmetic composition for skin improvement comprising an enzyme derived from immature strawberries, prepared by a method of extracting an enzyme from immature strawberries.

[0007] Another aspect provides a use of an enzyme derived from immature strawberries, produced by a method of extracting enzymes from immature strawberries, for improving skin condition.

[0008] Another aspect provides a method for improving skin condition comprising the step of administering to an individual an enzyme derived from immature strawberries, prepared by a method of extracting an effective amount of enzyme from immature strawberries.

[0009] One aspect is to provide a method for extracting enzymes from immature strawberries.

[0010] Specifically, a method for extracting enzymes from immature strawberries according to one aspect may include the step of preparing immature strawberries; the step of obtaining an extract by mixing the immature strawberries with a eutectic solvent and a surfactant composed of a mixture of one or more hydrogen bond donors (HBD) and one or more hydrogen bond acceptors (HBA); and the step of separating enzymes from the extract.

[0011] Protease can be extracted with excellent efficiency by the method of extracting enzymes from immature strawberries according to the present invention, and a composition containing said enzyme exhibits skin moisturizing, skin texture improvement, and exfoliation improvement effects without exhibiting cytotoxicity, and can be usefully utilized for skin improvement purposes.

[0012] In this specification, the term "unripe strawberry (Fragariaxananassa)" or "unripe strawberry" means a strawberry that is not fully ripe, which may mean a strawberry fruit harvested 24 to 28 days after flowering.

[0013] The above enzyme may be a protease and may be papain, ficin, or bromelin, but is not particularly limited thereto.

[0014] In the above method for extracting enzymes from immature strawberries, the hydrogen bond donor may be one or more selected from the group consisting of formic acid (FA), lactic acid, acetic acid, propionic acid, glycerol, ethylene glycol, diethylene glycol, triethylene glycol, and foramide, and more specifically, may be formic acid, but is not particularly limited thereto.

[0015] In addition, the hydrogen bonding receptor may be one or more selected from the group consisting of proline, betaine, alanine, glycine, choline chloride, sodium acetate, and nicotinamide, and more specifically, may be betaine, but is not particularly limited thereto.

[0016] The hydrogen bond donor and hydrogen bond acceptor constituting the above eutectic solvent may be mixed in a ratio of 1:2 to 1:4, and more specifically, may be mixed in a ratio of 1:2.

[0017] A method for extracting enzymes from immature strawberries according to one aspect may exhibit a synergistic effect of protein extraction due to the combination of the above-mentioned eutectic solvent and surfactant.

[0018] Specifically, the surfactant may be one or more selected from the group consisting of polyglyceryl-10 stearate, polyglyceryl-10 laurate, polyglyceryl-6 stearate, polyglyceryl-4 oleate, polyglyceryl-4 oleate, and sodium stearyl lactate, and more specifically, may be polyglyceryl-10 stearate, but is not particularly limited thereto.

[0019] In the step of obtaining an extract by mixing the above-mentioned eutectic solvent and surfactant with the above-mentioned immature strawberry fruit, conventional extraction methods in the art may be used, for example, static extraction, ultrasonic extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, filtration, or reflux extraction. Preferably, static extraction may be used, but is not particularly limited thereto.

[0020] In one aspect, the extraction may be performed at a temperature of 2 to 10°C, 2 to 8°C, 2 to 6°C, 2 to 4°C, 3 to 10°C, 3 to 8°C, 3 to 6°C, 3 to 4°C, 4 to 10°C, 4 to 8°C, or 4 to 6°C. Additionally, the extraction may be performed for 2 to 24 hours, but is not particularly limited thereto.

[0021] Another aspect provides a cosmetic composition for skin improvement comprising an extract prepared by the method of extracting enzymes from the aforementioned immature strawberry fruit, or an enzyme derived from the immature strawberry fruit isolated from the said extract. The same parts as described above apply equally to the said cosmetic composition.

[0022] In this specification, the term "extract" includes all substances obtained by extracting components of a natural product, regardless of the extraction method, extraction solvent, extraction conditions, extracted components, or the form of the extract, and also includes substances that may be obtained by processing or treating the components of the natural product by other methods after extraction. For example, the processing or treatment may include dilution, concentration, drying, purification, fractionation, filtration, fermentation, enzymatic treatment, etc. Accordingly, the extract may include an extract solution, a diluted or concentrated extract solution, a dried product obtained by drying the extract solution, a modified or purified product thereof, or a fraction obtained by fractionating the same.

[0023] In a cosmetic composition according to one aspect, the skin-improving agent may be for skin moisturization, skin texture improvement, or exfoliation improvement.

[0024] In one embodiment, it was confirmed that a serum containing an enzyme derived from immature strawberries showed a concentration-dependent skin moisturizing effect compared to a comparative example that did not contain an enzyme derived from immature strawberries, and it was confirmed that it exhibited sustained moisturizing power, with an increase in skin moisture up to 3 hours after application to the skin.

[0025] In addition, in another embodiment, it was confirmed that a serum containing enzymes derived from immature strawberries showed concentration-dependent improvement in skin texture and significantly superior exfoliation effects compared to the comparative example.

[0026] The above cosmetic composition comprises an extract prepared by a method of extracting an enzyme from immature strawberry fruit according to one aspect, or an enzyme derived from immature strawberry fruit isolated from said extract, wherein said enzyme may be a protease.

[0027] The above skin-improving cosmetic composition may be any one formulation selected from the group consisting of softening lotion, nourishing lotion, astringent lotion, skin toner, lotion, mist, essence, serum, cream, massage cream, pack, makeup base, BB cream, foundation, powder, cleansing foam, soap, cleansing cream, cleansing oil, and cleansing water, and specifically may be a powder or serum formulation, but is not particularly limited thereto.

[0028] In addition to the extract or immature and derived enzyme according to one aspect, the above cosmetic composition may further include ingredients commonly used in cosmetic compositions, functional additives, etc., such as, for example, antioxidants, stabilizers, solubilizers, surfactants, dispersants, thickeners, preservatives, vitamins, pigments, fragrances, etc., and cosmetically acceptable carriers.

[0029] Another aspect provides a use of an enzyme derived from immature strawberries, produced by a method of extracting enzymes from immature strawberries, for improving skin condition.

[0030] Another aspect provides a method for improving skin condition comprising the step of administering to an individual an enzyme derived from immature strawberries, prepared by a method of extracting an effective amount of enzyme from immature strawberries.

[0031] In the above aspects, any terms or elements mentioned that are identical to those already mentioned are as stated above.

[0032] In the above aspects, “effective amount” means an amount of a composition containing an active ingredient in the composition in a sufficient quantity to provide an improvement, therapeutic, or preventive effect to an individual to whom the composition is to be administered. The effective amount may include an “improvement effective amount,” a “preventive effective amount,” or a “therapeutic effective amount.”

[0033] In the above aspects, “individual” may mean any individual requiring improvement of skin condition. The individual may include or exclude humans, and may include mammals, and the mammals may include, for example, humans, dogs, cats, mice, rats, guinea pigs, horses, cattle, pigs, sheep, monkeys, chimpanzees, etc.

[0034] In the above aspects, “administration” means administering an effective amount of the composition directly to an individual to form it within the individual’s body. The administration may be oral or parenteral, and may include transdermal administration, subcutaneous administration, or application to the skin. Suitable dosages of the pharmaceutical composition may be prescribed differently depending on factors such as the formulation method, mode of administration, patient’s age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity.

[0035] In the present invention, enzymes can be extracted with excellent efficiency by a method for extracting enzymes from immature strawberries, and a composition containing said enzymes exhibits skin moisturizing, skin texture improvement, and exfoliation improvement effects without exhibiting cytotoxicity, and can be usefully utilized for skin improvement purposes.

[0036] Figure 1 shows the results of BCA protein quantification according to the composition and dilution ratio of the eutectic solvent used for extracting immature strawberries.

[0037] Figure 2 shows the results of a cytotoxicity evaluation following treatment with an enzyme (protease) derived from immature strawberries.

[0038] Figure 3 shows the results of the irritation relief evaluation following treatment with enzymes derived from immature strawberries.

[0039] Figure 4 shows the results of evaluating the hyaluronic acid production-promoting effect following treatment with an enzyme derived from immature strawberries.

[0040] Figure 5 shows the evaluation results of moisturizing and long-lasting moisturizing power according to treatment with a serum containing enzymes derived from immature strawberries.

[0041] Figure 6 shows the results of the skin texture improvement evaluation following treatment with a serum containing enzymes derived from immature strawberries.

[0042] Figure 7 shows the results of the skin exfoliation improvement evaluation following treatment with a serum containing enzymes derived from immature strawberries.

[0043] The present invention will be described in more detail below through the attached drawings and embodiments. However, these drawings and embodiments are for illustrative purposes only and the scope of the present invention is not limited to these embodiments.

[0044] Experimental Example 1. Confirmation of enzyme extraction content according to the composition and ratio of the eutectic solvent composition ratio

[0045] Formic acid was selected as the hydrogen bond donor (HBD) constituting the eutectic solvent, and proline, betaine, or alanine was selected as the hydrogen bond acceptor (HBA). The mixing ratio of these was adjusted to 1:2 to 1:4, and the eutectic solvent was prepared by heating at 80°C for 1 hour. The prepared eutectic solvent was diluted to 5–40%, and twice the weight of the immature strawberries was added. After extraction, the mixture was left to stand at 4°C for 2 hours, followed by crushing with a bead-shaped homogenizer. The extractable content of the protein (enzyme) in the supernatant was analyzed using a BCA assay.

[0046] Specifically, 180 µL of BCA protein reagent was added to 20 µL of each standard or sample and mixed, then left at 37°C for 30 minutes, after which the absorbance was measured at 562 nm. Subsequently, a standard curve was constructed using the protein concentrations of each standard, and the protein concentration of the sample was calculated.

[0047] As a result, it was confirmed that the enzyme extraction efficiency was best when betaine was used as the hydrogen bond acceptor (betaine approx. 11.2 μg / mL > alanine approx. 9.3 μg / mL > proline approx. 9.5 μg / mL > no addition 5.7 μg / mL), and that the extraction efficiency was high when mixed at a ratio of 1:2 compared to 1:3 or 1:4. In addition, it was confirmed that the best extraction efficiency was similarly observed at eutectic solvent dilution ratios of 5% or 10% (Fig. 1).

[0048] Experimental Example 2. Confirmation of Enzyme Extraction Content According to Eutectic Solvent and Surfactant Combinations and Concentrations

[0049] Formic acid was used as the hydrogen bond donor, and betaine, which was confirmed to have the best extraction efficiency in Experimental Example 1, was used as the hydrogen bond acceptor. The extraction efficiency of the enzyme according to the combination and concentration of different surfactants was to be determined.

[0050] Specifically, a eutectic solvent (diluted to a 5% ratio) mixed with formic acid and betaine in a 1:2 ratio was added, and 0.34% Disodium EDTA, 0.24% TRIS, 0.8% NaCl, 0.02% KCl, and each surfactant (Polyglyceryl-10 Stearate, Polyglyceryl-10 Laurate, Polyglyceryl-6 Stearate, polyglyceryl-4 oleate, polyglyceryl-4 oleate, Sodium Stearoyl Lactylate) were mixed at a concentration of 0.03–0.1%. Triton X-100 was used as a comparative example. Subsequently, the mixture was extracted at 4°C for 2 hours in the same manner as in Experimental Example 1, then crushed using a bead-shaped homogenizer, and the protein (enzyme) extraction content of the supernatant was analyzed by BCA Assay.

[0051] As a result, as shown in Table 1 below, prior to the addition of the eutectic solvent, the protein content was found to be highest in the order of Triton X-100 > Polyglyceryl-10 Stearate > Polyglyceryl-10 Laurate > Polyglyceryl-6 Stearate > polyglyceryl-4 oleate > polyglyceryl-4 oleate > Sodium Stearoyl Lactylate; however, after addition with the eutectic solvent, the protein content was found to be highest in the order of Polyglyceryl-10 Stearate > Polyglyceryl-10 Laurate > Polyglyceryl-6 Stearate > polyglyceryl-4 oleate > Sodium Stearoyl Lactylate > Triton X-100 > Sorbitan Stearate. From this, it was confirmed that there is a synergistic effect on protein extraction depending on the combination of the eutectic solvent and the surfactant according to the specific pattern.

[0052] [Table 1]

[0053]

[0054] Experimental Example 3. Measurement of extracted enzyme activity and extraction efficiency

[0055] To measure the activity of the extracted enzyme (protease), the experiment was conducted by hydrolyzing casein (0.6%) as a substrate at pH 6.0 and a temperature of 40°C for 60 minutes. The unhydrolyzed substrate was precipitated with trichloroacetic acid and removed by filtration, and the dissolved tyrosine was measured using the absorbance method at 660 nm with Folin reagent. The experiment was conducted based on the amount of enzyme that releases an equivalent of 1 μg of tyrosine in 1 minute. Disodium EDTA 0.34%, TRIS 0.24%, NaCl 0.8%, KCl 0.02%, and each surfactant (Polyglyceryl-6 Stearate, polyglyceryl-4 oleate, Sorbitan Stearate) at a concentration of 0.07% were mixed into a eutectic solvent (diluted to 5%) mixed with formic acid and betaine in a 1:2 ratio. Triton X-100 was used as a comparative example.

[0056] As a result, protease activity was confirmed to be 6.9 U / g, 5.5 U / g, 5.3 U / g, 4.1, and 3.3 U / g in the order of Polyglyceryl-6 Stearate, polyglyceryl-4 oleate, Triton X-100, Sorbitan Stearate, and no treatment, respectively. From this, it was confirmed that the enzyme (protease) extraction efficiency was best when Polyglyceryl-6 Stearate was used in combination with a eutectic solvent as a surfactant.

[0057] Experimental Example 4. Preparation of Enzyme Powder and Evaluation of Efficacy

[0058] 0.34g of Disodium EDTA, 0.24g of TRIS, 0.8g of NaCl, 0.02g of KCl, 0.07g of Polyglyceryl-10 Laurate, and a eutectic solvent (diluted to 5%) mixed in a 1:2 ratio of formic acid to betaine were added in an amount equal to twice the weight of immature strawberries, and the mixture was left to stand at room temperature for 2 hours to extract the enzyme (protease). After recovering the crude enzyme by adding Ammonium sulfate (AS), powdering was performed by adding an excipient (maltodextrin).

[0059] 4.1 Cytotoxicity Assessment

[0060] 1.5 x 10⁻⁶ HaCaT in a 96-well plate 4 Cells were seeded at cells / well and cultured under cell culture conditions. After 24 hours, the culture medium was discarded, washed with PBS, and the cells were starved using DMEM medium without supplements. The next day, test substances at specific concentrations (1.5, 3, 6, 12, 25, 50 ppm) were added and cultured for 24 hours. 100 µl of WST-1 reagent, diluted 10-fold in the medium, was added to each well and cultured for 2 hours. The absorbance was measured at 450 nm, and the results are shown in Table 2 below. It was confirmed that the highest concentration with a cell viability of over 90% was 12 ppm (Fig. 2).

[0061] [Table 2]

[0062]

[0063] 4.2 Cytoprotective Assessment (Stimulation Alleviation Assessment)

[0064] 1.5 x 10⁻⁶ HaCaT in a 96-well plate 4Cells were seeded at cells / well and cultured under cell culture conditions. After 24 hours, the culture medium was discarded, washed with PBS, and the cells were starved using DMEM medium that did not contain FBS. The next day, test substances at specific concentrations (0.75, 1.5, 3, 6, 12 ppm) were added along with a specific amount of SDS and cultured for 24 hours. 100 µl of WST-1 reagent, diluted 10-fold, was added to each well and cultured for 2 hours. The absorbance was measured at 450 nm, and the results are shown in Table 3 below. From this, it was confirmed that cell viability, which had decreased by approximately 46.2% due to SDS, increased by up to approximately 18.5% due to the immature strawberry-derived enzyme (protease) (Fig. 3).

[0065] [Table 3]

[0066]

[0067] 4.3 Evaluation of Hyaluronic Acid Production-Promoting Ability

[0068] 1.5 x 10⁻⁶ HaCaT in a 96-well plate 4 Cells were seeded at wells and cultured under cell culture conditions. After 24 hours, the culture medium was discarded, washed with PBS, and the cells were starved using DMEM medium free of FBS. The following day, test substances at specific concentrations (1.5, 3, 6, and 12 ppm) were added, and the cells were cultured for 24 hours. Absorbance was measured at 450 nm using the Hyaluronic Acid ELISA kit, and retinoic acid was used as a positive control.

[0069] The results are shown in Table 4 below. From this, it was confirmed that the enzyme (protease) derived from immature strawberries promotes the production of hyaluronic acid in a concentration-dependent manner, and showed a maximum promotion of hyaluronic acid production of approximately 27.1% (Fig. 4).

[0070] [Table 4]

[0071]

[0072] Experimental Example 5. Preparation of serum containing protease derived from immature strawberries and evaluation of efficacy

[0073] 5.1 Preparation of Serum Using Enzymes Derived from Immature Strawberries

[0074] Glycerin, butylene glycol, disodium EDTA, purified water, and xanthan gum were weighed and dispersed using an Agi Mixer at 1500 rpm for 3 minutes at a temperature of 45°C. 1,2-hexanediol and ethylhexylglycerin were added and stirred for 3 minutes, after which the mixture was cooled to 30°C. Subsequently, different amounts of protease derived from immature strawberries were added and stirred at 1500 rpm for 3 minutes to prepare Examples 1 and 2. For the Comparative Example, a serum formulation was prepared using the same method as the Examples, except that protease derived from immature strawberries was not added. The ingredient list for each serum formulation is shown in Table 5 below.

[0075] [Table 5]

[0076]

[0077] 5.2 Evaluation of Moisturizing and Long-lasting Moisturizing Power

[0078] During the preparation phase, the test site was kept clean and dry to ensure uniform measurement conditions for the subjects. Evaluation was conducted after skin stabilization for at least 30 minutes in an environment maintaining constant temperature and humidity (22±2℃, RH 40–60%). During the measurement phase, the test product was applied to a selected test site (5cm x 4cm) on the forearm using a micropipette at a dose of 2 mg / cm². 2 It was applied in an amount of [amount]. Measurements were taken a total of three times: before use, after use, and 3 hours later, and the average value was calculated using the three values. Skin moisture was measured using an Aphrodite moisture meter (Aphroditemoisturechecker, MC-1000).

[0079] As a result, it was confirmed that serum Examples 1 and 2, which were applied with enzymes derived from immature strawberries, showed a concentration-dependent skin moisturizing effect compared to the comparative example, and that they exhibited sustained moisturizing power with moisture increases of approximately 8.8% and 15.4%, respectively, up to 3 hours after application to the skin (Fig. 5).

[0080] 5.3 Skin Texture Improvement Evaluation

[0081] For 10 subjects, the serum of the above comparative example or example was applied to the face once a day, and the skin condition was photographed using Antera3D® (Miravex, Ireland) before use, after use, 3 days later, and 7 days later, and the degree of improvement in skin texture was compared and analyzed using an imaging program.

[0082] As a result, it was confirmed that the serums Examples 1 and 2, to which enzymes derived from immature strawberries were applied, showed a concentration-dependent reduction in skin roughness compared to the comparative example, thus demonstrating an excellent skin texture improvement effect (Fig. 6).

[0083] 5.4 Evaluation of Skin Exfoliation Improvement

[0084] Similar to the skin texture improvement evaluation above, the serum of the comparative example or example above was applied to the face of 10 subjects once a day, and the skin condition was photographed using Antera3D® (Miravex, Ireland) before use, after use, 3 days later, and 7 days later, and the degree of improvement in skin exfoliation was measured using an imaging program.

[0085] As a result, it was confirmed that the serums Examples 1 and 2, to which enzymes derived from immature strawberries were applied, showed a significantly superior exfoliation reduction effect compared to the comparative example, thus demonstrating an excellent skin exfoliation improvement effect (Fig. 7).

[0086] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A method for extracting enzymes from immature strawberries, The stage of preparing immature strawberries; A step of obtaining an extract by mixing an eutectic solvent composed of a mixture of one or more hydrogen bond donors (HBD) and one or more hydrogen bond acceptors (HBA), and a surfactant with the above-mentioned immature strawberry fruit; A method for extracting enzymes from immature strawberries, comprising the step of isolating enzymes from the above extract.

2. A method for extracting enzymes from immature strawberry fruit according to claim 1, wherein the immature strawberry fruit is collected 24 to 28 days after flowering.

3. A method for extracting an enzyme from immature strawberries according to claim 1, wherein the enzyme is a protease.

4. A method for extracting an enzyme from an immature strawberry, wherein, in claim 1, the hydrogen bond donor is one or more selected from the group consisting of formic acid (FA), lactic acid, acetic acid, propionic acid, glycerol, ethylene glycol, diethylene glycol, triethylene glycol, and formamide.

5. A method for extracting enzymes from immature strawberries, characterized in that, in claim 1, the hydrogen bond donor is formic acid.

6. A method for extracting an enzyme from an immature strawberry, wherein, in claim 1, the hydrogen bond receptor is one or more selected from the group consisting of proline, betaine, alanine, glycine, choline chloride, sodium acetate, and nicotinamide.

7. A method for extracting an enzyme from an immature strawberry, characterized in that, in claim 1, the hydrogen bond receptor is betaine.

8. A method for extracting enzymes from immature strawberries according to claim 1, wherein the hydrogen bond donor and the hydrogen bond acceptor are mixed in a ratio of 1:2 to 1:

4.

9. A method for extracting enzymes from immature strawberries according to claim 1, wherein the surfactant is one or more selected from the group consisting of polyglyceryl-10 stearate, polyglyceryl-10 laurate, polyglyceryl-6 stearate, polyglyceryl-4 oleate, polyglyceryl-4 oleate, and sodium stearyl lactate.

10. A cosmetic composition for improving skin, comprising an extract prepared by the method of any one of claims 1 to 9 or an enzyme isolated from said extract.

11. A cosmetic composition for improving skin, wherein, in paragraph 10, the enzyme is a protease.

12. A cosmetic composition for improving skin according to claim 10, characterized in that the skin-improving agent is for skin moisturization, skin texture improvement, or exfoliation improvement.

13. A cosmetic composition for improving skin according to claim 10, wherein the cosmetic composition for improving skin is any one formulation selected from the group consisting of softening lotion, nourishing lotion, astringent lotion, skin toner, lotion, mist, essence, serum, cream, massage cream, pack, makeup base, BB cream, foundation, powder, cleansing foam, soap, cleansing cream, cleansing oil, and cleansing water.

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