Method for preparing rice bran extract composition and its use in skincare

Abstract

To provide a preparation method of a rice bran extract composition characterized by recycling rice bran which tends to be discarded but is rich in health care ingredients, by preparing a rice bran extract composition and adding it to personal care products to improve skin care effects such as skin whiteness of a product, anti-aging, anti-inflammatory, and antiallergy effects, further by recovering reduced pressure-concentrated solvent in a preparation step to be able to reuse the same as a solvent of the present invention, and to obtain effects of resource saving and cyclic economy.SOLUTION: The present invention provides a method for preparing a rice bran extract composition and its application in skin care, the preparation method including a step of mixing rice bran with a solvent having a concentration of more than 80% to obtain a mixed solution, and extracting while stirring in an environment of 40-55°C., a step of centrifugation or filtering the mixed solution to collect a supernatant or filtrate, and a step of vacuum concentrating the supernatant or filtrate to obtain a rice bran extract, and a step of processing the rice bran extract into a rice bran extract composition.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a rice bran extract composition and use thereof in skin care, and particularly has effects such as whitening, anti-aging, anti-inflammation and anti-allergy, and can be applied to skin care. [Background Art]

[0002] Rice bran is a product of the process of milling brown rice into polished white rice, and contains 12-16% protein, 16-22% fat, 20-25% dietary fiber, as well as a large amount of trace minerals and phytochemicals such as γ-oryzanol, plant sterols, vitamin E, squalene and glucosylceramide. However, rice bran is difficult to preserve and prone to rancidity, so few organic growers inactivate or roast it for consumption or sale, and it is not popular in the market due to its rough texture and poor taste. In the past, rice bran was used as feed, and excess rice bran was directly discarded as waste, imposing a burden on the environment. It has been reported in many literatures that phytochemicals contained in rice bran are beneficial for skin care, and therefore rice bran extract is added to many products to enhance the efficacy and value of the products. However, rice bran is a common material that is easy to obtain. Without detailed extraction methods and product forms, rice bran extract is only an abstract noun, and there are doubts about the reproducibility of its efficacy and ease of use.

[0003] Regarding the extraction of rice bran, in the prior art, rice bran is treated with hot water exceeding 75°C in an amount 6 to 10 times that of the rice bran, cooled to less than 40°C, then extracted with less than 80% ethanol for at least 30 minutes, and the first supernatant (filtrate) after filtration or centrifugation is used. Alternatively, the mixture is further concentrated under reduced pressure to obtain a layered supernatant and a precipitate, and then the precipitate is treated with an aqueous alcohol (methanol, ethanol, propanol) to obtain a second supernatant and a second precipitate. However, the above process has complicated steps, and toxic solvents such as n-hexane are also used when spray-drying the first supernatant of defatted rice bran.

[0004] In conventional techniques, methanol, acetone, ethanol, etc., are mixed with water in a volume ratio of 30-70:70-30 to create an extract, with a converted solvent concentration of approximately 30%-70%, and then extracted using a ratio of 1:1-20 (weight) of rice bran to extract. The temperature is set to 50-70°C and the extraction is carried out for 1-3 hours. Various test tube biochemical tests (in vitro) are performed on the extract. In conventional techniques, rice bran is first treated with enzymes, and then extracted with a mixed solution of water and ethanol (ratio 1:1-25:1, equivalent to 50%-3.8% ethanol) at a temperature of 4-40°C for 6 hours-7 days.

[0005] From the conventional techniques described above, it has become clear that the difficulty of the rice bran extraction process varies greatly depending on whether or not degreasing of the rice bran is performed first, the type and ratio of solvents used for extraction, whether or not enzymes are used, and the extraction temperature and time. Furthermore, the methods for testing effectiveness also differ, with some being test tube tests and others cell experiments, and there are no indicator components to compare differences between lots and between different effectiveness levels.

[0006] Furthermore, many plant-derived chemicals contained in rice bran, such as oryzanol (γ-oryzanol), plant sterols, vitamin E, squalene, and glucosylceramide, have skin-improving effects, but most are oil-soluble, and if the solvent concentration is too low, oil-soluble substances are difficult to extract. Raising the temperature helps to extract oil-soluble substances, and conventional techniques involve first treating the rice bran with hot water exceeding 75°C, and then extracting it with a solvent after cooling (below 40°C). Another conventional technique raises the extraction temperature to 50-70°C, but the solvent concentration is less than 70%. Yet another conventional technique can achieve this when the solvent concentration is low (less than 50%) by using enzymes and extending the extraction time. However, these conventional techniques are complicated and time-consuming, requiring two stages of water treatment and solvent treatment, or extending the extraction time. While raising the temperature may make extraction easier, high-temperature treatment tends to destroy and oxidize oil-soluble substances. [Overview of the project] [Problems that the invention aims to solve]

[0007] Considering the above, the inventors deeply understand the shortcomings and drawbacks of the prior art, and have been diligently pursuing improvement and innovation. After many years of research, they have finally developed a method that can significantly simplify the rice bran extraction process. At the same time, the prepared rice bran extract composition can be added to personal care products to improve the healthcare effects of the products, such as skin whitening, anti-aging, anti-inflammatory, and anti-allergic properties, providing a novel and low-cost healthcare strategy for skincare. Furthermore, it can enhance the value of rice bran, create a new economy, reduce the environmental burden of agricultural waste, and practice the spirit of resource recycling and reuse.

[0008] The objective of this invention is to simplify the extraction process, increase the solvent concentration to over 80%, lower the extraction temperature (below 55°C), avoid destroying the effects and activity of rice bran, and shorten the extraction time to at least one hour. The effects of the rice bran extract are demonstrated through a series of cell tests, and its industrial applicability is realized by making the rice bran extract into an easily usable form (rice bran extract composition). [Means for solving the problem]

[0009] To achieve the above objectives, the main object of the present invention is to provide a method for preparing a rice bran extract composition, the preparation method comprising the steps of: mixing rice bran with a solvent of a concentration exceeding 80% to obtain a mixed solution and extracting it while stirring in an environment of 40 to 55°C; centrifuging or filtering the mixed solution to collect the supernatant or filtrate; concentrating the supernatant or filtrate under reduced pressure to obtain a rice bran extract; and processing the rice bran extract into a rice bran extract composition.

[0010] In one embodiment of the present invention, the rice bran is defatted or undefatted rice bran, the mixing ratio of rice bran weight to solvent volume is 1:4 to 1:8, the unit of weight is grams, and the unit of volume is milliliters.

[0011] In one embodiment of the present invention, the solvent is a solvent with an ethanol volume:water volume ratio of 100 to 80:0 to 20, or a solvent with a propanol volume:water volume ratio of 100 to 80:0 to 20.

[0012] In one embodiment of the present invention, the preparation method includes a step of decolorizing and purifying the supernatant or filtrate using activated carbon before concentrating the supernatant or filtrate under reduced pressure, and the reduced pressure concentration involves concentrating the volume of the supernatant or filtrate 18 to 22 times using a reduced pressure distillation method to obtain a rice bran extract.

[0013] In one embodiment of the present invention, the processing involves preparing a rice bran extract composition by adding an excipient, the excipient comprising water, oil, emulsifier, preservative, and cosolvent, and the rice bran extract composition is in liquid dosage form containing 20-30% rice bran extract.

[0014] In one embodiment of the present invention, the processing involves preparing a rice bran extract composition by adding plant starch, wherein the ratio of plant starch addition is 1:0.4 to 1:2 in terms of rice bran extract volume to plant starch weight, and the rice bran extract composition is spray-dried to form a powder, with the weight unit being grams and the volume unit being milliliters.

[0015] In one embodiment of the present invention, the preparation method includes adding an emulsifier, a preservative, and a cosolvent before spray drying, and the plant starch includes maltodextrin, corn starch, wheat starch, rice starch, or tapioca starch.

[0016] In one embodiment of the present invention, the ratio of rice bran extract to plant starch is adjusted by measuring the glucosylceramide concentration in the rice bran extract.

[0017] The present invention further provides applications for rice bran extract compositions in skincare, wherein the rice bran extract composition is prepared by the above preparation method, and the rice bran extract composition is free from cytotoxicity, phototoxicity, and skin irritation, and the skincare applications include skin whitening, anti-aging, anti-inflammatory, and anti-allergic effects.

[0018] In one embodiment of the present invention, the effective amount of rice bran extract is 7.8 to 1000 μg / ml. [Effects of the Invention]

[0019] In the process of the method for preparing the rice bran extract composition of the present invention, the temperature used is an important factor. This is because when rice bran extract is treated at a high temperature, for example, in a water bath at 70°C for 0.5 to 1 hour, the efficacy of the rice bran extract composition (powder and liquid) prepared by subsequent processing will be weakened.

[0020] The present invention breathes new life into rice bran that was originally treated as waste, recycles and reuses natural and environmentally friendly rice bran, prepares a rice bran extract composition while greatly simplifying the rice bran extraction process, and furthermore, the solvent concentrated under reduced pressure in the preparation process can be recovered and reused as the solvent of the present invention. That is, the present invention can achieve the effects of resource saving and circular economy. Furthermore, the prepared rice bran extract composition can be added to personal care products to improve the healthcare effects of the products such as skin whitening, anti-aging, anti-inflammation, and anti-allergy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] [Figure 1] It is a cytotoxicity test of rice bran extract according to one embodiment of the present invention. [Figure 2] It is a phototoxicity test of rice bran extract according to one embodiment of the present invention. [Figure 3] It is a skin irritation test of rice bran extract according to one embodiment of the present invention. [Figure 4] It is an in vitro inhibition test of tyrosinase activity of rice bran extract according to one embodiment of the present invention (in vitro inhibition of tyrosinase). [Figure 5] It is a melanogenesis inhibition test of rice bran extract according to one embodiment of the present invention (anti-melanogenesis in B16F10 cells). [Figure 6] It is a melanosome phagocytosis test of rice bran extract according to one embodiment of the present invention (melanosome transfer to keratinocytes). [Figure 7] It is an anti-aging test of rice bran extract according to one embodiment of the present invention. [Figure 8] It is an anti-inflammatory test of rice bran extract according to one embodiment of the present invention. [Figure 9]This is an anti-allergic test of rice bran extract according to one embodiment of the present invention. [Figure 10] This is a melanin formation inhibition test of a rice bran extract composition according to one embodiment of the present invention. [Figure 11] This is a melanosome phagocytosis test of a rice bran extract composition according to one embodiment of the present invention. [Figure 12] This shows the mass spectrometry parameters and analytical spectrum of glucosylceramide according to one embodiment of the present invention. [Figure 13] This is a test to inhibit melanin formation of a rice bran extract composition by high-temperature treatment, according to one embodiment of the present invention. [Figure 14] This is a melanosome phagocytosis test of a rice bran extract composition by high-temperature treatment, according to one embodiment of the present invention. [Modes for carrying out the invention]

[0022] [Term definition]

[0023] This specification makes extensive use of many technical and chemical terms commonly used in the field of biotechnology. The following definitions are provided to ensure a clear and consistent understanding of this specification, the scope of the patent application, and the scope assigned to these terms. Other terms not specifically defined below have meanings commonly understood by experts in the field.

[0024] The terms “encompassing” and “including” are unrestrictive open conjunctions. The preceding paragraph is for systematic reference only and should not be construed as a limitation on the subject matter of the invention.

[0025] In this specification, "%" means "weight percentage (wt%)" unless otherwise specified. Numerical ranges (e.g., A is 10% to 11%) include upper and lower limits unless otherwise specified (i.e., 10% ≤ A ≤ 11%). If the lower limit of a numerical range is not defined (e.g., B is less than 0.2%, or B is less than 0.2%), it means that the lower limit may be 0 (i.e., 0% ≤ B ≤ 0.2%). The proportional relationships of "weight percentages" for each component may also be replaced with proportional relationships of "parts by weight".

[0026] All figures disclosed herein may have a standard technical measurement error (standard deviation) of ±10%. The term “approximately” is intended to mean ±10%, ±5%, ±2.5%, or ±1% of a given value, i.e., “approximately 20%” means 20±2%, 20±1%, 20±0.5%, or 20±0.25%.

[0027] As used herein, “effective dose” means an amount of an active substance administered to a subject that is sufficient to prevent or reduce one or more disease symptoms or physiological conditions, the result of which is reduction and / or mitigation of signs, symptoms, or etiologies, or an intentional alteration of other physiological systems.

[0028] [Rice bran raw material]

[0029] The state of the rice bran used in this invention is not limited and may include, for example, untreated rice bran or treated rice bran (e.g., fermented, defatted, etc.). In one embodiment, the rice bran used in this invention is defatted or undefatted rice bran. Furthermore, unless otherwise specified, all materials used in this invention are commercially available and readily obtainable.

[0030] [Mixed extraction process]

[0031] The method for preparing the rice bran extract composition of the present invention includes the steps of mixing rice bran with a solvent of a concentration exceeding 80% to obtain a mixed solution, and extracting it while stirring in an environment of 40 to 55°C. The concentration of the solvent may be a weight percentage concentration exceeding 80% or a volume percentage concentration exceeding 80%, depending on the type of solvent, and the present invention is not limited thereto.

[0032] The mixing ratio of rice bran to solvent is not limited, but from the viewpoint of improving the extraction efficiency of rice bran, the mixing ratio of rice bran weight (in grams) to solvent volume (in milliliters) is preferably about 1:4 to 1:8, for example, about 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, or a ratio between any two of the above values. The mixing extraction process can be carried out in batches, and the mixing ratio of rice bran to solvent used in each mixing extraction process may be the same or different.

[0033] The temperature at which the rice bran and solvent are mixed must not exceed approximately 55°C, that is, it must be less than approximately 55°C. While the specific temperature is not limited, from the viewpoint of improving the extraction efficiency of the rice bran, it is preferably less than 50°C. For example, it may be approximately 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or any two of the above values, with the most preferred being approximately 40-50°C. The mixing and extraction process can be carried out in batches, and the temperature of each mixing and extraction process may be the same or different.

[0034] The stirring and extraction time after mixing the rice bran and solvent is not limited, but from the viewpoint of improving the extraction efficiency of the rice bran, the stirring and extraction time is preferably at least about 1 hour, and may be, for example, about 1.5 hours, 2 hours, 2.5 hours, 3 hours, more than 3 hours, or any two of the above values. The mixing and extraction process can be carried out in batches, and the stirring and extraction time for each mixing and extraction process may be the same or different.

[0035] The solvent concentration must exceed approximately 80%, and while the specific concentration is not limited, from the viewpoint of improving the extraction efficiency of rice bran, the solvent is preferably ethanol or propanol at a volume percentage concentration of approximately 80% to 100%, for example, ethanol or propanol at a volume percentage concentration of approximately 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or any two of the above values. The ethanol and propanol mentioned above may also include any isomers thereof, such as n-propanol and isopropanol. That is, the solvent is preferably a solvent with an ethanol volume:water volume ratio of about 100-80:0-20, or a solvent with a propanol volume:water volume ratio of about 100-80:0-20, for example, about 99:1, 98:2, 96:4, 94:6, 92:8, 90:10, 88:12, 86:14, 84:16, 82:18, or a ratio between any two of the above values. The mixed extraction process can be carried out in batches, and the type and concentration of the solvent used in each mixed extraction process may be the same or different.

[0036] This invention does not require the use of toxic solvents such as n-hexane; in other words, it prioritizes the selection of solvents that are gentle on the human body and the environment. Furthermore, this invention does not require the use of hot water for extraction; in other words, it prioritizes solvent extraction (at a temperature not exceeding 55°C).

[0037] [Mixed solution separation process]

[0038] The method for preparing the rice bran extract composition of the present invention includes the step of separating the rice bran and solvent by centrifugation or filtration of the extracted mixed solution to obtain a supernatant or filtrate.

[0039] In embodiments using centrifugal separation, the specific centrifugal speed and number of centrifuges are not limited, as long as the centrifugal separation can separate the rice bran from the solvent. Similarly, in embodiments using filtration, the equipment used for filtration (e.g., filter screen, filter element, etc.) and the pore size are not limited, as long as the rice bran can be separated from the solvent, and can be adjusted as appropriate depending on the separation conditions.

[0040] In one embodiment, the mixing extraction step and the mixed solution separation step are performed in batches, for example, by dividing the mixture into three steps to obtain a total of three supernatants or filtrates. After collecting all the supernatants or filtrates, the subsequent decolorization and purification steps (which may be omitted in some cases) and the reduced-pressure concentration step can be performed. In one embodiment, depending on the extraction and separation status, each batch can be extracted and separated individually, or the same batch can be separated (filtered) and then extracted one or two times.

[0041] [Decolorization and purification process]

[0042] In one embodiment, after the mixed solution separation step, the separated supernatant or filtrate can be decolorized and purified, followed by a subsequent vacuum concentration step. The materials used for decolorization and purification are not limited, but activated carbon is preferred from the viewpoint of improving the quality of the rice bran extract.

[0043] [Depressurized concentration process]

[0044] The method for preparing the rice bran extract composition of the present invention includes the step of obtaining a rice bran extract by concentrating the supernatant or filtrate under reduced pressure. The method of reduced pressure concentration is not limited and may include, for example, reduced pressure distillation or vacuum concentration.

[0045] In one embodiment, the solvent can be recovered in a vacuum concentration step, and the concentration of the recovered solvent is over 80%. This recovered solvent can be used as the solvent of the present invention to achieve resource conservation and circular economy effects. The specific concentration of the recovered solvent depends on the concentration of the solvent used in the mixed extraction step, but the present invention is not limited thereto.

[0046] In one embodiment, vacuum concentration involves concentrating the volume of the supernatant or filtrate by about 18 to 22 times (for example, about 19 times, 20 times, 21 times, or any two of the above values) using a vacuum distillation method to obtain a rice bran extract (concentrate) for subsequent processing, but the present invention is not limited thereto.

[0047] In one embodiment, the concentrate can be further concentrated to obtain a more viscous rice bran extract. For example, the volume of the supernatant or filtrate can be concentrated to about 23, 24, or 25 times to obtain the rice bran extract (concentrate). In one embodiment, the weight of the extracted rice bran extract is about 12-18% (for example, about 13%, 14%, 15%, 16%, 17%, or any two of the above weight percentages) compared to the total weight of the rice bran raw material, but the present invention is not limited thereto.

[0048] [Processing process]

[0049] The method for preparing the rice bran extract composition of the present invention includes a processing step of processing the rice bran extract into a rice bran extract composition as a product, and the specific method is appropriately adjusted according to the dosage form of the rice bran extract composition.

[0050] In embodiments for making the rice bran extract composition into a liquid, the processing step involves preparing the rice bran extract composition by adding an excipient to the rice bran extract, where the excipient includes water, oil, emulsifier, preservative, cosolvent, etc., and the rice bran extract composition becomes a liquid dosage form containing 20-30% of the rice bran extract (for example, 22%, 24%, 26%, 28%, or any two of the above values).

[0051] The specific types and proportions of each component, such as water, oil, emulsifier, preservative, and co-solvent, contained in the excipient of the present invention are not limited and can be appropriately adjusted depending on the state of the product (rice bran extract composition).

[0052] In an embodiment for powdering the rice bran extract composition, the processing step involves adding vegetable starch to the rice bran extract to prepare the rice bran extract composition, where the ratio of vegetable starch addition is 1:0.4 to 1:2 (for example, about 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or any two of the above values), and the rice bran extract composition is then powdered by spray drying.

[0053] In one embodiment, the specific type of plant starch is not limited, but from the viewpoint of improving the quality of the powder, the plant starch preferably includes maltodextrin, corn starch, wheat starch, rice starch, or tapioca starch.

[0054] In one embodiment, the rice bran extract (concentrate) is uniformly mixed, and before subsequent spray drying, an emulsifier, preservative, and co-solvent may be added. The specific types and content ratios of the emulsifier, preservative, co-solvent, and other components used are not limited and can be appropriately adjusted depending on the state of the product (rice bran extract composition).

[0055] In one embodiment of the present invention, the rice bran extract (concentrate) is prepared in liquid or powder form (rice bran extract composition), and the reproducibility of its effect is demonstrated by cell testing.

[0056] [Quality control]

[0057] Since rice bran is a natural product, its quality can vary even with the same process, depending on factors such as the rice's origin and the climate of the year. Therefore, indicator components are needed to identify differences between batches and adjust the spray-drying ratio of concentrated rice bran extract to plant starch.

[0058] Examples of indicator components that can be used include oryzanol, vitamin E, squalene, and glucosylceramide (GluCer). Of these, glucosylceramide is easily identifiable in trace amounts, so in this invention, it is preferable to adjust the addition ratio of rice bran extract to plant starch by measuring the glucosylceramide concentration in the rice bran extract.

[0059] [Uses in skincare]

[0060] The present invention further provides applications for rice bran extract in skincare, wherein the rice bran extract is prepared by the above preparation method, and the rice bran extract is cytotoxic, phototoxic, and skin irritant, and the skincare applications include skin whitening, anti-aging, anti-inflammatory, and anti-allergic effects.

[0061] In one embodiment, the safety of the rice bran extract of the present invention was confirmed by cell testing, and its safety included cell viability / cytotoxicity, phototoxicity, and skin irritation (in vitro skin irritation). Furthermore, the effects of the rice bran extract of the present invention were confirmed by cell testing, and its effects included skin whitening, anti-aging, anti-inflammatory, and anti-allergic effects.

[0062] In one embodiment, the effective amount of rice bran extract contained in the rice bran extract composition of the present invention is 7.8 to 1000 μg / ml, and may be, for example, 15.6 μg / ml, 31.2 μg / ml, 62.5 μg / ml, 125 μg / ml, 250 μg / ml, 500 μg / ml, or an effective amount between any two of the above values.

[0063] To illustrate the method for preparing the rice bran extract composition of the present invention, several examples and comparative examples are listed below. These are not intended to limit the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Furthermore, all materials used below are commercially available and readily obtainable. [Examples]

[0064] Experiment 1: Preparation of rice bran extract

[0065] In this experiment, 100g of rice bran was added to a solvent (a mixed solution of 400mL of ethanol and 50mL of water, i.e., 89% ethanol), extracted at 42°C for 1 hour with stirring, and the supernatant was collected by centrifugation. The extraction was repeated two more times, yielding a total of three supernatants. All the supernatants were combined to a volume of approximately 1000-1200mL, treated with activated carbon, and concentrated under reduced pressure to obtain approximately 15g of viscous extract (rice bran extract), with an extraction rate of approximately 15%.

[0066] Experiment 2: Cell viability / cytotoxicity test

[0067] In this experiment, first, 8 × 10 3 Individual mouse L929 fibroblast cells were inoculated into 96-well plates. After 24 hours of cell adhesion, the cells were treated with different concentrations of rice bran extract for 24-48 hours. The culture medium was then removed, and MTS reagent was added to allow the cells to react for 2-4 hours. Absorbance at a wavelength of 490 nm was measured using a microplate reader. The negative control group (NC) consisted of cells treated with a culture medium without rice bran extract (1% DMSO), and cell viability was confirmed. The positive control group (PC) consisted of cells treated with 5% DMSO, and cell death was confirmed. The absorbance of the negative control group was set as 100% cell viability, and a comparative analysis was performed with the experimental group. A cell viability of 80% was established as the safe concentration reference value for the sample.

[0068] Figure 1 shows the results of a cytotoxicity test of the rice bran extract. From Figure 1, it can be seen that the rice bran extract obtained by the preparation method of the present invention showed a survival rate of over 90% at 500 μg / mL, and still showed a survival rate of 70% even at a high concentration of 1000 μg / mL.

[0069] Experiment 3: Phototoxicity Test

[0070] In this experiment, we followed OECD Guideline No. 432, using 1 × 10⁻¹⁰ 4Two 96-well plates were inoculated with one mouse 3T3 cell line and cultured for 24 hours. Then, rice bran extract and CPZ (chlorpromazine, positive control) were added to each plate, with each group containing 8 concentrations. After culturing in the dark at 37°C for 1 hour, the cells from one plate were exposed to UVA (5 J / cm²). 2 The cells were exposed to UV light for 0.5 to 1 hour, and the other plate was stored in the dark. The cells were then replaced with fresh cell culture medium and cultured for 18 hours. Neutral red dye was added and allowed to react for 3 hours. The cells were washed with phosphate buffer, and the neutral red from within the cells was extracted. The absorbance at a wavelength of 540 nm was measured using a microplate reader, with higher readings indicating higher cell viability. Cell viability response curves corresponding to the concentration of the test substance were plotted, and the IC50 was calculated. The formula for photostimulatory factor (PIF) [IC50(-UV) / IC50(+UV)] ≥ 6 indicates a risk of phototoxicity.

[0071] Figure 2 shows the results of a phototoxicity test of rice bran extracts. A shift to the left in the cell survival curve after light irradiation indicates phototoxicity. From Figure 2, it can be seen that the PIF values ​​of the rice bran extracts obtained by the preparation method of the present invention range from 1.28 to 1.67 (some batches were not even able to calculate the curve). From the above results, compared to the positive control, the CPZ was greater than 31 in all cases, and the PIF values ​​of the rice bran extracts were all less than 2. In other words, the phototoxicity risk of the rice bran extracts of the present invention is extremely low.

[0072] Experiment 4: Skin irritation test (in vitro skin irritation)

[0073] In this experiment, following OECD guideline No. 439, keratinocytes were cultured in the stratum corneum of skin-mimicking reconstituted cells for testing. A test concentration (usually the highest concentration added to the product or 2-3 times that concentration) was selected and applied to the surface of the skin-mimicking tissue (apical surface) and allowed to react at room temperature for 15 minutes. The positive control group (PC) was coated with 5% SDS, and the negative control group (NC) was coated with the solvent (mineral oil) used to prepare rice bran extract. After removing the skin surface samples and washing the surface three times with phosphate buffer, the MTS reagent was added and allowed to react for 24 hours. The absorbance at a wavelength of 490 nm was measured using a microplate reader. The absorbance of the negative control group was defined as 100% viability, and the cell viability of the positive control group had to be less than 50%. If the cell viability of the tested sample exceeded 50%, it was considered that there was no skin irritation.

[0074] Figure 3 shows the results of a skin irritation test of rice bran extract. From Figure 3, it can be seen that the rice bran extract obtained by the preparation method of the present invention does not cause skin irritation even at a high concentration of 50 mg / mL.

[0075] Experiment 5: Whitening Test: In vitro tyrosinase activity inhibition test (in vitro inhibition of tyrosinase)

[0076] In this experiment, buffer solutions containing samples of different concentrations were added to 96-well plates. The positive control group (PC) contained kojic acid (final concentration 6.25 μg / ml), while the negative control group (NC) contained buffer solution without the test substance (2% DMSO). 125 U of mushroom tyrosinase was added and allowed to react at room temperature for 5 minutes. Then, the substrate L-tyrosine was added, and the change in absorbance per minute at a wavelength of 475 nm was measured for a total of 30 minutes using a microplate reader. If tyrosinase activity was not inhibited, the absorbance increased over time, and an increase or decrease in absorbance indicated inhibition of activity. To compare tyrosinase activity, the change in absorbance per unit time from 0 to 15 minutes was calculated.

[0077] Figure 4 shows the results of in vitro inhibition of tyrosinase activity by rice bran extract. From Figure 4, it can be seen that the rice bran extract obtained by the preparation method of the present invention has a positive correlation with its concentration in inhibiting tyrosinase activity.

[0078] Experiment 6: Whitening Test: Melanin Formation Inhibition Test (Anti-melanin formation in B16F10 cells)

[0079] In this experiment, first, 2 × 10 4 Each melanoma cell line (B16 / F10) was inoculated into a 6-well culture plate. After 24 hours of adhesion, the culture medium was removed, 100 ng / mL of melanocyte-stimulating hormone (α-MSH) was added and the mixture was allowed to react for 30 minutes. Then, an equal volume of cell culture medium containing twice the concentration of the extract was added. The negative control group (NC) was cell culture medium without rice bran extract (treated with α-MSH but without rice bran extract), the positive control group (PC) was 0.5 mg / mL of kojic acid (treated with α-MSH), and the cell background group (baseline) was not treated with α-MSH. After 72 hours of incubation, the culture medium was removed, washed with phosphate buffer, and 0.25% trypsin was added to detach the cells. The cells were collected by centrifugation, then washed 2-3 times with sterile phosphate buffer. 100 µl of M-PER cell lysate was added to the precipitated cell mass to disrupt the cells, followed by centrifugation. The supernatant was collected and the protein concentration was quantified. 1N sodium hydroxide solution was added to the precipitate and reacted at 80°C for 2 hours. The supernatant was then transferred to a 96-well plate, and the absorbance at a wavelength of 405 nm was measured using a microplate reader. A lower absorbance indicated less melanin. After normalizing the melanin absorbance by its own protein concentration, the values ​​of the experimental group and the negative control group were compared, and the melanin content of the negative control group was set to 100%.

[0080] Figure 5 shows the results of a melanin formation inhibition test using rice bran extract. From Figure 5, it can be seen that the rice bran extract obtained by the preparation method of the present invention showed a tendency to inhibit melanin formation at a concentration of 50 μg / mL, and the inhibitory effect at 250 μg / mL was superior to that of kojic acid at 500 μg / mL.

[0081] Experiment 7: Skin Whitening Test: Melanosome Phagocytosis Test (Melanosome Transfer to Keratinocytes)

[0082] In the two whitening tests, Experiments 5 and 6, differences were observed in the results of the extracellular (in vitro) and cellular tests. This may be because rice bran extract has multiple physiological mechanisms related to whitening. Therefore, in order to further verify the whitening effect and to examine the whitening mechanism of the rice bran extract obtained by the preparation method of the present invention from a different perspective, another cellular test was conducted.

[0083] Melanin possesses strong antioxidant properties. When keratinocytes are stimulated by ultraviolet light, they phagocytose melanosomes secreted by melanocytes to protect the genetic material, ribonucleic acid, by surrounding the cell nucleus. This normal defense mechanism is one of the causes of skin darkening. Under the stimulation of low growth factors (i.e., starvation), endocytosis by keratinocytes is promoted. Therefore, by simulating melanosomes with fluorescent beads corresponding to the size of melanosomes and observing whether the extract can inhibit melanosome phagocytosis by keratinocytes, we aim to understand the mechanism by which darkening is inhibited.

[0084] In this experiment, first, 1 × 10 4Each keratinocyte was inoculated into a 24-well plate, cultured in 10% serum (growth factor) medium to allow adhesion, then rice bran extract was added under 0-2% serum culture conditions, and the cells were co-cultured for 4-6 hours. Finally, red fluorescent microbeads (0.5 μm) with a particle size equivalent to melanosomes were added and allowed to react for 2 hours. Unphagocytosed fluorescent microbeads were removed, the cells were fixed in 4% formalin solution, and the cell nuclei were stained with DAPI. The amount of phagocytosed fluorescent microbeads in 50 cells was observed and counted using a fluorescence microscope (ex / em: 580 / 605 nm), and the average value was calculated. 0-5 beads: 0 points, 6-10 beads: 1 point, 11-15 beads: 2 points, 16-20 beads: 3 points, 21-25 beads: 4 points, 26 or more beads: 5 points. The blank control group (PC) was cultured in a 10% growth factor concentration (10% serum, sufficient nutrition, keratinocytes not phagocytosed), while the negative control group (NC) was cultured in 0-2% serum without extracts (e.g., 2% serum, starvation stimulates keratinocyte phagocytosis).

[0085] Figure 6 shows the results of a melanosome phagocytosis test using rice bran extract. From Figure 6, it can be seen that the rice bran extract obtained by the preparation method of the present invention showed an effect of inhibiting melanosome phagocytosis by keratinocytes in a positive correlation with concentration at concentrations of 16 to 40 μg / mL.

[0086] Experiment 8: Anti-aging test

[0087] In this experiment, first, 5 × 10 4Fibroblasts were inoculated into 6-well plates and cultured overnight. The culture medium was then replaced with fresh, serum-free medium, and 8 mM hydroxyurea and extract were added for 24 hours. After another 24 hours of culture with fresh, serum-free medium, the cells were washed twice with phosphate buffer, fixed with 3.7% formalin, and stained with β-galactosidase. The baseline group (background cells) were not treated with hydroxyurea, while the negative control group (NC) was treated with hydroxyurea but without rice bran extract. Hydroxyurea induces aging and leads to an increase in intracellular β-galactosidase (SA-β-galactosidase), causing cells to show a blue color after staining. A lower percentage of blue indicates that the cells possess anti-aging properties. The cells were observed and photographed using a light microscope, and the percentage of blue reaction in 100 cells across 3-5 fields was counted.

[0088] Figure 7 shows the results of an anti-aging test of rice bran extract. From Figure 7, it can be seen that the rice bran extract obtained by the preparation method of the present invention can reduce the blue color ratio of cells, shows a positive correlation with concentration, and exhibits anti-aging properties.

[0089] Experiment 9: Anti-inflammatory test

[0090] In this experiment, first, 2 × 10 4 Macrophages (RAW264.7) were inoculated into 96-well plates. After cell attachment, the original culture medium was removed and a new culture medium containing 250 ng / mL of lipopolysaccharide (LPS) and rice bran extract was added. The negative control group (NC) received a culture medium without rice bran extract (no nitroarginine was administered), while the positive control group (PC) received 1 mM nitroarginine (LNNA). LPS induces an increase in nitrite levels, while LNNA suppresses this increase. After 24 hours of cell culture, the culture medium was collected and the amount of nitrite produced by reacting with Griess reagent was measured. Nitrite can be used as an indicator of nitric oxide (NO), an intracellular inflammation regulator. Suppression of NO production indicates an anti-inflammatory effect; therefore, a lower amount of nitrite production indicates a higher anti-inflammatory effect.

[0091] Figure 8 shows the results of an anti-inflammatory test of rice bran extract. From Figure 8, it can be seen that the rice bran extract obtained by the preparation method of the present invention can clearly suppress nitrite at 500 μg / mL and has an anti-inflammatory effect.

[0092] Experiment 10: Anti-allergic test

[0093] In this experiment, first, 3 × 10 5 100 basophils (RBL-2H3) were inoculated into 24-well plates and cultured for 24 hours, after which the original culture medium was removed. A culture medium containing rice bran extract was added to the experimental group, and the cells were cultured for 4-6 hours. Then, 500 ng / mL of the drug A23187 was added and stimulated for 30 minutes, and the culture supernatant was collected to measure hexosaminidase (β-hexosaminidase) activity. The cell background group (baseline) consisted of cells that were not treated with ketotifen or A23187, the negative control group (NC) consisted of a culture medium without rice bran extract (no ketotifen was administered, but A23178 was administered to induce enzyme activity), and the positive control group (PC) consisted of cells that were administered 0.3 mM ketotifen followed by A23178 to induce enzyme activity. 30 μL of culture supernatant and 50 μL of enzyme substrate solution (p-nitrophenyl N-acetyl-D-glucosamine, concentration 1.3 mg / mL) were reacted at 37°C for 1 hour, and the reaction was stopped by adding 0.5 M sodium hydroxide solution. The absorbance at a wavelength of 405 nm was measured using a microplate reader. Lower absorbance indicates lower β-hexosaminidase activity and a higher allergy mitigation effect. A23187 induces degranulation of RBL-2H3 cells and releases many inflammatory mediators, among which β-hexosaminidase is often used as a biochemical indicator to detect whether or not an allergy is occurring.

[0094] Figure 9 shows the results of an anti-allergic test of rice bran extract. From Figure 9, it can be seen that the rice bran extract obtained by the preparation method of the present invention can clearly inhibit the activity of β-hexosaminidase at 500 μg / mL and has an anti-allergic effect.

[0095] Experiment 11: Processing Steps - Liquid Preparation

[0096] This experiment was divided into two parts: the oil phase and the aqueous phase. Oil phase: Rice bran extract, emulsifier, glycerin, and preservative were heated to approximately 50°C and mixed. Aqueous phase: Water, preservative, co-solvent, etc., were heated to approximately 50°C and mixed. Next, the oil phase was poured into the aqueous phase and mixed uniformly to prepare a rice bran extract composition (liquid) containing 20-30% rice bran extract.

[0097] Experiment 12: Processing Steps - Powder Preparation

[0098] Considering that powdered raw materials are more convenient for weighing and compounding when manufacturing skincare products, and further verifying the industrial applicability of the preparation method of the present invention, in this experiment, the extraction scale was increased 100-fold, and the rice bran extract was further powdered.

[0099] 10 kg of rice bran was added to a solvent (a mixed solution of 60 L of ethanol and 7.5 L of water) and extracted at 42°C for 1 hour while stirring. After centrifugation, the supernatant was taken and the rice bran was filtered out. A second supernatant was obtained by adding another solvent (a mixed solution of 50 L of ethanol and 6.25 L of water). The supernatants were combined and treated with activated carbon to obtain approximately 100 L. After concentrating under reduced pressure to obtain approximately 5 L of concentrated liquid (rice bran extract), the preservative Spectrastat® E and butanediol were added, and the mixture was spray-dried with 3000 g of maltodextrin to obtain a rice bran extract composition (powder).

[0100] Experiment 13: Whitening effect of rice bran extract composition

[0101] The aforementioned experiment was conducted using rice bran extract, and it was confirmed that rice bran extract has the highest whitening effect. Therefore, in this experiment, a cell test related to whitening will be performed on a rice bran extract composition (powder) in the same manner as for rice bran extract, and it will be proven that the rice bran extract composition obtained by the preparation method of the present invention also has a whitening effect.

[0102] The method of this experiment is the same as that of the melanin formation inhibition test (anti-melanin formation in B16F10 cells) and the melanosome phagocytosis (melanosome transfer to keratinocytes) test described in Experiments 6-7 above, except that the rice bran extract was replaced with a rice bran extract composition (powder).

[0103] Figures 10 and 11 show the results of a melanin formation inhibition test and a melanosome phagocytosis test of the rice bran extract composition, respectively. From Figures 10 and 11, it can be seen that the rice bran extract composition obtained by the preparation method of the present invention has a melanin inhibitory effect and an effect of inhibiting melanosome phagocytosis by keratinocytes at a concentration of 0.1%, comparable to that of 500 μg / mL of kojic acid.

[0104] Experiment 14: Indicator components of rice bran extract composition

[0105] Indicator components are used to confirm differences between extraction batches and to adjust the spray-drying ratio of the concentrate (rice bran extract) to the plant starch. In this experiment, 0.5-0.25 g of the rice bran extract composition was mixed uniformly with 10 mL of water, diluted 20-50 times with 100% methanol, filtered, and 5 μL was injected for quantitative and qualitative analysis of glucosylceramide (GluCer) using LC-MS. The column used was Agilent Poroshell 120 EC-C18 4.6*50 mm 2.7-Micron, the mobile phase was 100% methanol containing 0.5-5 mM ammonium acetate, the flow rate was 1 mL / min, and the temperature was 40°C. Galactosylceramide can be added as an internal standard if necessary.

[0106] Table 1 and Figure 12 below show the ion parameters and chromatograms for each GluCer. From Table 1 and Figure 12, it can be seen that the GluCer concentration in the rice bran extract composition obtained by the preparation method of the present invention is approximately 0.01 to 0.03%.

[0107] [Table 1]

[0108] Experiment 15: The Importance of Temperature

[0109] This experiment is used to illustrate the importance of temperature as defined by the preparation method of the present invention. 4 kg of rice bran was added to a solvent (a mixed solution of 24 L of ethanol and 3 L of water), extracted at 42°C for 1 hour with stirring, and after centrifugation, the supernatant was taken and the rice bran was filtered. A second supernatant was obtained by adding another solvent (a mixed solution of 20 L of ethanol and 2.5 L of water). The supernatants were combined and treated with activated carbon to obtain approximately 40 L. After concentrating under reduced pressure to obtain approximately 1.9 L of concentrated liquid (rice bran extract), the preservative Spectrastat® E and butanediol were added, and the mixture was spray-dried with 2500 g of maltodextrin. The GluCer concentration was measured by LC-MSMS and found to be 0.019%, which was within the normal range.

[0110] 10 kg of rice bran was mixed with a solvent (a mixture of 60 L of ethanol and 7.5 L of water) and extracted at 42°C for 1 hour with stirring. After centrifugation, the supernatant was taken and the rice bran was filtered out. A second supernatant was obtained by adding another solvent (a mixture of 50 L of ethanol and 6.25 L of water). The supernatants were combined and treated with activated carbon to obtain approximately 105 L. Approximately 5 L of concentrated solution (rice bran extract) was obtained by vacuum concentration. After adding the preservative Spectrastat® E and butanediol, approximately 2.5 L of the concentrated solution was taken and subjected to high-temperature treatment in a 70°C water bath for 1 hour. Subsequently, it was spray-dried with 3000 g of maltodextrin, and the GluCer concentration was measured by LC-MSMS, which was approximately 0.029%, indicating the high-temperature treatment group. The GluCer concentration in the rice bran extract composition is relatively high, and theoretically it contains a relatively high whitening composition, so the results of cell tests should be better.

[0111] The experimental method was the same as in Experiments 6-7, except that the rice bran extract was replaced with a normal group and a high-temperature treated group. Figures 13-14 show the results of the melanin formation inhibition test and the melanosome phagocytosis test for the rice bran extract composition (normal group and high-temperature treated group), respectively. From Figures 13-14, it can be seen that although the high-temperature treated group had a high GluCer concentration (0.029%), the results of the melanin formation inhibition test and the melanosome phagocytosis test were only half that of the normal group (0.019% GluCer), indicating that the extraction temperature greatly affects the whitening effect of the composition. Therefore, in order to ensure the whitening effect, the temperature specified by the preparation method of the present invention must be less than 55°C, preferably 40-50°C.

[0112] In summary, the present invention recycles rice bran, which is often discarded but is rich in healthcare components, to prepare a rice bran extract composition. The preparation process is simple, and the reduced-pressure concentrated solvent can be recovered and reused as the solvent for the present invention. In other words, the present invention can achieve resource conservation and circular economy effects and belongs to the realm of green technology. Furthermore, the rice bran extract composition has effects such as skin whitening, anti-aging, anti-inflammatory, and anti-allergic properties.

[0113] The embodiments described above are solely for the purpose of illustrating the technical idea and features of the present invention, and their purpose is to enable those skilled in the art to understand and implement the present invention; however, where they cannot be used to limit the scope of the invention, i.e., all equivalent changes or modifications made in accordance with the spirit disclosed herein should still be included within the scope of the invention.

Claims

1. A method for preparing a rice bran extract composition, The process involves mixing rice bran with a solvent of a concentration exceeding 80% to obtain a mixed solution, and then extracting it while stirring in an environment of 40°C to less than 50°C. The process involves centrifuging or filtering the mixed solution to collect the supernatant or filtrate, A step of obtaining rice bran extract by concentrating the supernatant liquid or filtrate under reduced pressure, The process includes a step of processing the aforementioned rice bran extract into a rice bran extract composition, The mixing ratio of the rice bran weight to the solvent volume is 1:4 to 1:8, the unit of weight is grams, and the unit of volume is milliliters. The solvent is a solvent with an ethanol volume:water volume ratio of 90-80:10-20, or a solvent with a propanol volume:water volume ratio of 90-80:10-20. A method for preparing a rice bran extract composition.

2. A method for preparing a rice bran extract composition according to claim 1, wherein the rice bran is defatted or undefatted rice bran.

3. A method for preparing a rice bran extract composition according to claim 1, comprising the step of decolorizing and purifying the supernatant or filtrate using activated carbon before concentrating it under reduced pressure, wherein the reduced pressure concentration is performed by concentrating the volume of the supernatant or filtrate 18 to 22 times using a reduced pressure distillation method to obtain the rice bran extract.

4. The method for preparing a rice bran extract composition according to claim 1, wherein the processing involves adding an excipient to prepare a rice bran extract composition, the excipient comprising water, oil, emulsifier, preservative, and cosolvent, and the rice bran extract composition is in a liquid dosage form containing 20-30% of the rice bran extract.

5. The method for preparing a rice bran extract composition according to claim 1, wherein the processing involves adding plant starch to prepare a rice bran extract composition, the ratio of the plant starch to the volume of rice bran extract: the weight of plant starch is 1:0.4 to 1:2, and the rice bran extract composition is spray-dried to form a powder, the weight of which is in grams and the volume of which is in milliliters.

6. A method for preparing a rice bran extract composition according to claim 5, comprising the step of adding an emulsifier, a preservative, and a cosolvent before the spray drying, wherein the plant starch comprises maltodextrin, corn starch, wheat starch, rice starch, or tapioca starch.

7. A method for preparing a rice bran extract composition according to claim 5, wherein the addition ratio of the rice bran extract to the plant starch is adjusted by measuring the concentration of glucosylceramide in the rice bran extract.

8. Uses of a rice bran extract composition in skincare, wherein the rice bran extract composition is prepared by the preparation method described in claim 1, the rice bran extract composition is cytotoxic, phototoxic, and skin irritant, and the skincare includes skin whitening, anti-aging, anti-inflammatory, and anti-allergic effects.

9. The use of the rice bran extract composition in skincare according to claim 8, wherein the effective amount of the rice bran extract is 7.8 to 1000 μg / ml.

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