Preparation method of red rice skin extract as well as product and application of red rice skin extract
Red rice peel was extracted by leaching mixed solvent of alcohol and water and multi-step treatment, and red rice peel extract was prepared, which solved the problem of difficulty in extracting active ingredients of red rice peel, achieved its antioxidant, anti-inflammatory and anti-aging effects, and provided efficient and stable ingredients for cosmetics development.
Patent Information
- Application Number
- CN202411647300.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-18
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art is difficult to effectively extract and utilize the active ingredients in red rice peels, which limits its application prospects in the market.
The red rice peel was leaching with a mixed solvent of alcohol and water with a volume ratio of 90:10-40:60, and the red rice peel extract was prepared by pulverizing, screening, centrifugation and drying.
It has achieved efficient extraction of active ingredients in red rice peel, improved its antioxidant, anti-inflammatory and anti-aging effects, and provided the necessary conditions for the development of efficient and stable cosmetics.
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Figure CN119499155A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cosmetics, and in particular to a preparation method of a red rice peel extract, a product thereof and an application thereof. Background Art
[0002] Red rice (Oryza sativa L.) is a traditional regional rice variety that has attracted widespread attention from academia and industry due to the red pigment in its husk. As an important agricultural cultural heritage of the Yuanyang Rice Terraces in Honghe Prefecture, Yunnan Province, while creating a beautiful terraced landscape, it has also brought many health benefits to the Hani people who have lived there for generations, such as lowering blood pressure and blood lipids, and is known as the "plateau red agate". Compared with ordinary white rice, red rice is rich in a variety of functional ingredients, such as anthocyanins, dietary fiber, minerals and vitamins, showing more outstanding nutritional value and potential health benefits. As people's demand for healthy diets increases, red rice has gradually been regarded as a representative of functional grains and has become an important subject of research in agricultural breeding, food science and biotechnology.
[0003] In recent years, scientific research related to red rice has been carried out one after another, and the exploration of bioactive substances has made a lot of progress. However, due to many unfavorable factors such as low extraction rate of active substances, poor stability, and high raw material costs, its market application prospects have not been very clear. However, as a by-product of red rice processing, red rice skin has long been used as animal feed or directly discarded, and the exploration of its active substances has been rarely reported.
[0004] On this basis, the present application conducts research and development on red rice peel, creates a preparation method of red rice peel extract and its product and application, so that it can effectively extract the effective ingredients in the red rice peel, provide the necessary conditions for exploring the potential activity of its effective ingredients, and achieve the purpose of fully utilizing the antioxidant, anti-inflammatory and anti-aging activities of the red rice peel. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a preparation method of a red rice peel extract, so that the effective components of the red rice peel can be effectively extracted, and the necessary conditions are provided for exploring the potential activity of the effective components, so as to fully utilize the antioxidant, anti-inflammatory and anti-aging activities of the red rice peel.
[0006] In order to solve the above technical problems, in a first aspect, the present invention provides a method for preparing a red rice peel extract, the method comprising the following steps:
[0007] (1) after husking the red rice, the red rice husk is ground and crushed, and passed through a 40-mesh sieve to obtain a red rice husk extraction raw material;
[0008] (2) extracting the red rice husk raw material with a mixed solvent of alcohol and water in a volume ratio of 90:10-40:60, wherein the extraction temperature is 30-70° C., and the extraction time is 1-24 h. After vacuum filtering the extract, the extract is concentrated under reduced pressure to obtain a water-soluble suspension of red rice husk;
[0009] (3) centrifuging the red rice skin water-soluble suspension at a speed of 3000-8000 rpm for 10-30 min, filtering to remove insoluble precipitates, and then standing for stratification to remove the clear liquid to obtain a red rice skin water-soluble concentrated solution;
[0010] (4) drying the red rice peel water-soluble concentrate to obtain the red rice peel extract.
[0011] Further improvement, the mass of the red rice husk extracted in step (1) accounts for 10% of the mass of the whole rice after shelling; in step (2), the alcohol-water mixed solvent is a methanol-water mixed solvent or an ethanol-water mixed solvent, and the volume of the alcohol-water mixed solvent added is more than 8-10 times the volume of the red rice husk extraction raw material.
[0012] Further improvement, step (2) is specifically: using an alcohol-water mixed solvent with a volume ratio of 90:10-70:30 to extract the red rice peel extraction raw material, stirring continuously, the stirring speed is 300 rpm, the extraction temperature is 50°C, the extraction time is 2h, after vacuum filtering the extract, it is concentrated under reduced pressure at 40°C until the solution volume is reduced to 1 / 4 of the original, to obtain a red rice peel water-soluble suspension.
[0013] As a further improvement, in step (3), the centrifugal speed is 4000 rpm and the centrifugal time is 10 min.
[0014] As a further improvement, in step (4), the red rice peel water-soluble concentrate is dried by freeze drying or spray drying.
[0015] In a second aspect, the present invention provides a red rice peel extract, which is prepared by the above-mentioned preparation method of the red rice peel extract.
[0016] In a third aspect, the present invention further provides a cosmetic comprising the red rice peel extract.
[0017] The added amount of the red rice peel extract in the cosmetic is 0.1%-5%.
[0018] The cosmetics are skin care lotions, skin care emulsions or skin care essences with anti-oxidation, anti-inflammatory and anti-aging functions.
[0019] In a fourth aspect, the present invention also provides a use of the red rice peel extract in the preparation of cosmetics with antioxidant, anti-inflammatory and anti-aging effects. The red rice peel extract in cosmetics can improve the antioxidant capacity of the skin, promote skin metabolism, delay aging, and have a moisturizing effect.
[0020] After adopting such a design, the present invention has at least the following advantages:
[0021] The invention effectively extracts the active components of red rice peel, which is not only safe and reliable, but also has a high extraction rate and is simple and convenient. It can also make full use of red rice by-products, improve the use value of red rice, and effectively exert the antioxidant, anti-inflammatory and anti-aging effects of the red rice peel, thereby providing necessary conditions for developing efficient and stable cosmetic products containing red rice peel extracts and meeting the application needs in the existing cosmetics field.
[0022] The red rice peel extract is used in cosmetics, can effectively enhance the antioxidant capacity, delay skin aging, and improve the water-oil balance of the skin, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.
[0024] Figure 1 These are the results of the effect of red rice peel extract on the sensitizing activity of THP-1 cells.
[0025] Figure 2 These are the test results of the antioxidant activity of red rice peel extract.
[0026] Figure 3 , 4 , 5 are the test results of red rice peel extract inhibiting the release of NO, IL-6 and TNF-α in the LPS-induced Raw264.7 cell model.
[0027] Figure 6 These are the results of the anti-non-enzymatic glycation test of red rice peel extract.
[0028] Figure 7 , 8 These are the evaluation results of the anti-glycation activity of red rice peel extract in the MGO-induced BJ cell glycation model.
[0029] Fig. 9 This is the test result of the inhibition rate of red rice peel extract on elastase activity.
[0030] Fig.10 The staining results of β-galactosidase expression in the hydrogen peroxide-induced BJ cell anti-aging model of red rice peel extract.
[0031] Fig.11 This is the result of the antioxidant capacity of red rice peel extract in skin care lotions, emulsions and essences.
[0032] Fig.12 The results show the moisturizing effect of red rice peel extract in skin care lotion.
[0033] Fig.13 The results show the moisturizing effect of red rice peel extract in skin care lotion.
[0034] Fig.14 This is the result of the moisturizing effect of red rice peel extract in skin care essence. DETAILED DESCRIPTION
[0035] The exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art. The specific embodiments are as follows.
[0036] Example 1 Preparation of red rice peel extract
[0037] 1.1 Raw material processing
[0038] 10 kg of red rice produced in Honghe Prefecture, Yunnan Province was taken, the outer husk was removed, and the rice skin accounting for 10% of the total rice weight was ground using a rice mill and then crushed to obtain a coarse red rice skin sample, which was passed through a 40-mesh sieve to remove impurities and dust, and then dried at 60° C. to ensure that the moisture content was less than 10%, which was the raw material for red rice skin extraction.
[0039] 1.2 Solvent extraction
[0040] 500 g of red rice husk extract raw material was added to the reaction kettle, and then 10 times the volume of ethanol-water mixed solvent (volume ratio of 70:30) was added, and the extraction was carried out at 50° C. for 2 hours with a stirring speed of 300 rpm. Then, the extract was vacuum filtered and the filtrate was collected.
[0041] 1.3 Vacuum concentration
[0042] The collected filtrate is concentrated under reduced pressure at a low temperature of 40° C. to remove volatile alcohol components until the volume of the solution is reduced to 1 / 4 of the original volume, thereby obtaining a red rice peel water-soluble suspension.
[0043] 1.4 Impurity separation
[0044] The red rice skin water-soluble suspension was centrifuged at 4000 rpm for 10 min and filtered. After removing the insoluble precipitate, the suspension was allowed to stand for stratification for 30 min. The upper layer of grease was removed and the lower layer of clear liquid was taken to obtain the red rice skin water-soluble concentrated solution for standby use.
[0045] 1.5 Dry powder preparation
[0046] The red rice peel water-soluble concentrated liquid after impurities removal is spray-dried to obtain the red rice peel extract dry powder, which is stored in a sealed container for later use.
[0047] Example 2 Preparation of Whole Red Rice and Red Rice Peel Extract Sample Pretreatment and Extraction
[0048] 500 g of each red rice sample after hulling treatment in Example 1 was selected, one portion was used as the raw material for whole red rice extraction without any treatment, and the other portion was milled by a rice mill to obtain the outer layer of red rice skin accounting for 10% by weight, and then crushed and sieved through a 40-mesh sieve to remove impurities and dust as the raw material for red rice skin extraction.
[0049] Two portions of the extracted raw materials were dried at 60°C to obtain pretreated samples. Two portions of the pretreated samples of the same mass were weighed and extracted with 80% ethanol solution with a solid-liquid ratio of 1:10, respectively. After the extracts were concentrated and filtered, extracts of the two samples were obtained, namely, red rice whole rice extract and red rice skin extract. The extraction rate was calculated, and the results are shown in Table 1 below.
[0050] Example 3 Comparative Analysis of Active Components of Whole Red Rice and Red Rice Peel Extracts
[0051] 3.1 Active ingredient detection method
[0052] Take two extracts obtained in Example 2 and test their active component indicators. The test indicators include:
[0053] 1) Anthocyanin compound content: The agricultural industry standard "NY / T 2640-2014 Determination of Anthocyanins in Plant-derived Foods by High Performance Liquid Chromatography" was used to determine the contents of six anthocyanins in two samples, including delphinium pigment, cyanidin, petunia pigment, pelargonium pigment, peony pigment and malva pigment;
[0054] 2) Flavonoid content: The total flavonoid content in the two samples was determined using the method described in Article 15 of Chapter 2 “Determination of total flavonoids in health foods” in the “Technical Guidelines for Inspection and Evaluation of Physical, Chemical and Hygienic Indicators of Health Foods (2020 Edition)”;
[0055] 3) Antioxidant activity: The antioxidant capacity of two samples was tested using the experimental method described in Example 5 below.
[0056] 3.2 Experimental Results
[0057] The results are shown in Table 1 below, which shows the extraction rate and active component detection results of whole red rice and red rice peel extracts.
[0058] Table 1 Comparison of test results of whole red rice and red rice peel extract
[0059]
[0060]
[0061] 3.3 Experimental analysis
[0062] 1) As shown in Table 1, red rice skin accounts for about 9.72% of the total weight of red rice, and its solvent extraction rate is about 2.19 times that of the whole red rice.
[0063] 2) The results of flavonoid content determination showed that the total flavonoid content in red rice peel extract was 4.91 times that in whole red rice extract.
[0064] 3) The results of anthocyanin compound content determination showed that delphinium pigment, petunia pigment and peony pigment were not detected in both groups of samples, while cyanidin, geranium pigment and malva pigment were detected in both groups of samples, and the corresponding contents in red rice peel extract were 7.70 times, 9.70 times and 7.27 times that of red rice whole rice extract, respectively.
[0065] 4) The results of antioxidant activity determination showed that the half-clearance rate of antioxidant activity of red rice peel extract was 22.27 μg / mL, while that of whole red rice extract was 183.83 μg / mL, indicating that the activity difference was nearly 9 times, so red rice peel extract had higher research value.
[0066] 3.4 Experimental Conclusion
[0067] Through comparative analysis of the content of active substances and antioxidant activity in whole red rice and red rice skin, the results show that the content of anthocyanin compounds and flavonoid compounds in red rice skin extract is significantly higher than that in whole red rice extract, and the antioxidant activity of red rice skin extract is also significantly higher than that in whole red rice extract. Therefore, red rice skin, as a by-product of red rice processing, shows extremely high application potential in the fields of functional foods, health products and cosmetics.
[0068] Example 4: Evaluation of THP-1 Cell Sensitization of Red Rice Peel Extract
[0069] 4.1 Reagents and Materials
[0070] 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), β-mercaptoethanol (β-ME), DPBS, bovine serum albumin (BSA), dinitrochlorobenzene (DNCB), lactic acid (LA), 7-AAD, FITC anti-human CD86Antibody, PE anti-human CD54 Antibody, FITC Mouse IgG1κIsotype ControlAntibody, PE Mouse IgG1κIsotype Control Antibody.
[0071] 4.2 Instruments
[0072] CO2 incubator, biological safety cabinet, inverted microscope, flow cytometer.
[0073] 4.3 Cell lines
[0074] Human acute monocytic leukemia cells (THP-1) were purchased from the Cell Bank of the Chinese Academy of Sciences.
[0075] 4.4 Experimental Grouping
[0076] Blank control group: 1640 complete medium;
[0077] Solvent control group: 0.2% DMSO, prepared with 1640 complete medium;
[0078] Positive control group: 3 μg / mL DNCB, prepared in 1640 complete medium;
[0079] Negative control group: 1 mg / mL lactic acid, prepared with 1640 complete medium;
[0080] Drug administration group: the red rice peel extract obtained in Example 1 was prepared at concentrations of 100, 50, 25, and 12.5 μg / mL using 1640 complete medium.
[0081] 4.5 Experimental methods
[0082] Take THP-1 cells in good exponential growth phase, centrifuge to remove supernatant, add culture medium to resuspend, and adjust the cell density to 1.5×10 6 / mL, take the cell suspension and inoculate it in a 48-well plate, 200μL / well. According to the experimental grouping, prepare the corresponding test solution with a concentration of 2 times. Add 200μL / well of the prepared test sample to the corresponding wells, and incubate in a 37℃, 5% CO2 incubator for 24h.
[0083] After incubation, the cell suspension in each well was collected and divided into two parts, one for CD86 and CD54 staining, and the other for isotype control staining. After staining, the cells were fixed with paraformaldehyde, and the mean fluorescence intensity (MFI) of FITC, PE, and 7-AAD dyes in each group of cells was detected by flow cytometry, and the relative fluorescence intensity (RFI) of each group of test substances was calculated using the following formula (1).
[0084]
[0085] 4.6 Experimental Principles and Results
[0086] When allergens come into contact with the skin, dendritic cells differentiate and mature in the process of moving to lymphoid organs, and upregulate the expression of a series of surface molecules. By staining the cell surface molecules CD86 and CD54 with fluorescent antibody dyes and measuring them with flow cytometry, it can be determined whether the test substance is allergenic.
[0087] The experimental results are shown in Figure 1 The positive control group produced positive expression of cells (RFI CD86 >150, RFI CD54 >200) and cell survival rate>50%; negative control group produced negative expression of cells (RFI CD86 <150, RFI CD54 <200 and cell survival rate>50%; the solvent control group had negative expression of cells (RFI CD86 <150, RFI CD54 <200) and cell viability>90%, indicating that the cells passed the stability test.
[0088] At all concentrations tested, the red rice peel extract showed negative expression of CD86 and CD54 in THP-1 cells, and the cell viability was >90%, indicating that the red rice peel extract had no allergenicity.
[0089] Example 5: Evaluation of DPPH radical scavenging activity of red rice peel extract
[0090] 5.1 Reagents and Materials
[0091] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH), anhydrous ethanol, 96-well plate.
[0092] 5.2 Instruments
[0093] ELISA reader, 1 / 100,000 microbalance.
[0094] 5.3 Solution preparation
[0095] DPPH working solution: 50 μg / mL, prepared with anhydrous ethanol;
[0096] Red rice peel extract: the red rice peel extract obtained in Example 1, tested at concentrations of 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL, prepared with distilled water;
[0097] 5.4 Experimental methods
[0098] According to Table 2 below, prepared red rice peel extract solutions of different concentrations, DPPH working solution, distilled water, and anhydrous ethanol were added to a 96-well plate; the solutions in each well were mixed, reacted at room temperature in the dark for 30 minutes, and then the absorbance of the solution in each well was read using an enzyme reader at a wavelength of 517 nm.
[0099] Table 2 Ratio of components in the evaluation experiment of DPPH free radical scavenging activity of red rice peel extract
[0100]
[0101] The scavenging rate of each group of samples to be tested on DPPH free radicals was calculated according to the following formula (2):
[0102]
[0103] Among them, T, T0, C, and C0 represent the absorbance of the sample group, sample background group, DPPH group, and solvent background group at a wavelength of 517 nm, respectively.
[0104] 5.5 Experimental Principles and Results
[0105] DPPH is a stable, long-lived free radical that is dark purple when dissolved in anhydrous ethanol and has strong absorption near 517nm. When there is a free radical scavenger (reducing agent), DPPH reacts with its single electron pairing, causing the characteristic purple color of the DPPH ethanol solution to gradually disappear and turn into colorless or light yellow, resulting in a weakening of the light absorption of DPPH. In this experiment, different concentrations of red rice peel extract were incubated with DPPH solution to evaluate its antioxidant activity.
[0106] The experimental results are as follows Figure 2 As shown, red rice peel extract has a strong DPPH free radical scavenging ability, and its half-scavenging rate is about 11.3μg / mL.
[0107] Example 6: Evaluation of anti-inflammatory activity of red rice peel extract
[0108] 6.1 Reagents and Materials
[0109] DMEM high glucose medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, lipopolysaccharide (LPS), dexamethasone (DEX), Mouse TNF-α ELISA detection kit, Mouse IL-6 ELISA detection kit, NO detection kit.
[0110] 6.2 Instruments
[0111] CO2 incubator, biological safety cabinet, inverted microscope, and microplate reader.
[0112] 6.3 Cell lines
[0113] Mouse mononuclear macrophage leukemia cells (Raw264.7) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences.
[0114] 6.4 Solution preparation
[0115] LPS working solution: 25 ng / mL, prepared in DMEM medium containing 1% FBS
[0116] Dexamethasone working solution (Dex): 10 μg / mL, prepared using LPS working solution;
[0117] Red rice peel extract: The red rice peel extract obtained in Example 1 was prepared at test concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 μg / mL using LPS working solution.
[0118] 6.5 Experimental methods
[0119] Take Raw264.7 cells that are in good exponential growth phase, use a cell scraper to remove the cells, and adjust the cell density to 1.5×10 5 / mL of cell suspension. Take the above cell suspension and inoculate it in a 48-well plate, 500μL / well, and incubate it in a 37℃, 5% CO2 incubator for 16-24h. When the cell fusion rate reaches 30-50%, remove the supernatant, and add 400μL of the above test samples to each of the plates except the control group, and incubate them in a 37℃, 5% CO2 incubator for 24h. After the incubation is completed, collect the supernatant, centrifuge to remove cell debris, and detect the concentration of nitric oxide (NO), IL-6 and TNF-α in the supernatant of each group according to the steps in the kit instructions.
[0120] 6.6 Experimental Principles and Results
[0121] Lipopolysaccharide can activate cells to synthesize and release a variety of cytokines and inflammatory mediators through the cell signal transduction system. The accumulation of inflammatory factors can cause vasodilation, redness, swelling, tingling, and itching, resulting in a state of skin tingling sensitivity. In this experiment, LPS-induced Raw264.7 cells were selected to establish an inflammatory model and evaluate the anti-inflammatory activity of red rice peel extract.
[0122] The experimental results are as follows Figures 3 to 5 As shown, compared with the control group, the expression levels of NO, TNF-α and IL-6 in the Raw264.7 cells in the LPS group after LPS induction were significantly increased, indicating that the Raw264.7 cell inflammation model was successfully established. When the red rice peel extract concentration was 2.5-20 μg / mL, it could significantly inhibit the release of IL-6, NO and TNF-α of Raw264.7 cells induced by LPS. It shows that the red rice peel extract of the present invention has good anti-inflammatory activity.
[0123] Example 7: Evaluation of non-enzymatic saccharification inhibitory activity of red rice peel extract
[0124] 7.1 Reagents and Materials
[0125] Bovine serum albumin (BSA), D-anhydrous glucose, DPBS, penicillin / streptomycin (P / S).
[0126] 7.2 Instruments
[0127] ELISA reader, 1 / 100,000 microbalance.
[0128] 7.3 Solution preparation
[0129] BSA working solution: 40 μg / mL, prepared in DPBS containing 1% P / S;
[0130] Glucose working solution: 480 μg / mL, prepared in DPBS containing 1% P / S;
[0131] Red rice peel extract: The red rice peel extract obtained in Example 1 was prepared at concentrations of 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL using DPBS containing 1% P / S.
[0132] 7.4 Experimental methods
[0133] According to Table 3 below, the prepared red rice peel extract solutions of different concentrations, BSA working solution, glucose working solution and DPBS were added to the 96-well plate, sealed with a sealing film, and placed in a 37°C incubator incubated in the dark for 7 days. After the incubation, the fluorescence intensity was detected using an ELISA reader under the conditions of an excitation wave of 370 nm and an emission wave of 440 nm.
[0134] Table 3 Group ratios of red rice peel extract non-enzymatic saccharification inhibition activity evaluation experiment
[0135]
[0136]
[0137] According to the following formula (3), the AGEs inhibition rate (P) and half inhibition concentration (IC 50 ).
[0138]
[0139] Among them, T, T0, C, and C0 represent the fluorescence intensity of the sample group, sample background group, saccharification reaction group, and BSA group under the conditions of excitation wave 370nm and emission wave 440nm, respectively.
[0140] 7.5 Experimental Principles and Results
[0141] Non-enzymatic glycation (NEG) refers to the non-enzymatic condensation reaction between the carbonyl group on reducing sugars (such as glucose, fructose, etc.) and the free amino group on macromolecules such as proteins, lipids or nucleic acids. The stable covalent addition products generated by the reaction are called advanced glycation end products (AGEs). This experiment evaluates the anti-glycation ability of each test substance by measuring its ability to inhibit the production of AGEs.
[0142] The experimental results are as follows Figure 6 As shown in the results, red rice peel extract has significant non-enzymatic glycation inhibitory activity, and its half inhibitory concentration (IC 50 ) was 76.6μg / mL.
[0143] Example 8: Evaluation of anti-glycation activity of red rice peel extract
[0144] 8.1 Reagents and Materials
[0145] DMEM high glucose medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, methylglyoxal (MGO), aminoguanidine hydrochloride (AG), and fluorescent probe (H2DCFDA).
[0146] 8.2 Instruments
[0147] CO2 incubator, biological safety cabinet, and fluorescence inverted microscope.
[0148] 8.3 Cell lines
[0149] Human skin fibroblasts (BJ) were purchased from the Cell Bank of the Chinese Academy of Sciences.
[0150] 8.4 Solution preparation
[0151] Methylglyoxal (MGO) working solution: 800 μM, prepared in DMEM complete medium;
[0152] Aminoguanidine (AG) working solution: 30 μg / mL, prepared using methylglyoxal working solution;
[0153] Red rice peel extract: The red rice peel extract obtained in Example 1 was prepared at test concentrations of 0.5, 1, and 2 μg / mL using methylglyoxal working solution.
[0154] 8.5 Experimental methods
[0155] Take BJ cells that are in good exponential growth phase, use 0.25% Trypsin-EDTA to remove the cells from the culture dish, and adjust the cell concentration to 3×10 after centrifugation. 4 / mL of cell suspension. Take the above cell suspension and inoculate it in a 12-well plate, 1mL / well, and incubate it in a 37℃, 5% CO2 incubator for 16-24h. When the cell fusion rate reaches 60%, remove the supernatant, and add 1mL of the above test samples to each well except the control group, and incubate it in a 37℃, 5% CO2 incubator for 24h. After the incubation is completed, remove the supernatant and wash with DPBS, add 500μL of the prepared ROS staining working solution (10μM H2DCFDA) to each well, place it in a 37℃, 5% CO2 incubator, and incubate it in the dark for 30 minutes. After the incubation is completed, take pictures under a fluorescence microscope and use Image J to analyze the average fluorescence intensity.
[0156] 8.6 Experimental Principles and Results
[0157] Advanced Glycation End-products (AGEs) produced by glycation reactions are harmful products of uncontrolled condensation reactions in the human body, which have a very adverse effect on human health, especially skin health. Accumulated AGEs can not only directly deposit in the skin to cause pigmentation, but also further cause pigmentation and oxidative damage to the skin by interacting with the receptor for advanced glycation end products (RAGE) and free radicals. In addition, the binding of collagen and elastin to sugar molecules will cause irreversible changes or even damage to their properties and structures, causing the skin to lose elasticity, collapse in structure, and eventually form wrinkles. In this experiment, methylglyoxal was used to induce glycation in human primary skin fibroblasts (BJ). After incubation with the test samples for 24 hours, the production of reactive oxygen species (ROS) in BJ cells was detected to evaluate the anti-glycation activity of the test substances.
[0158] The experimental results are as follows Figure 7 and Figure 8 As shown, compared with the control group, the fluorescence intensity of ROS in the BJ cells in the MGO group was significantly enhanced after MGO induction, indicating that the BJ cell glycation model was successfully established; the red rice peel extract concentrations of 0.5, 1, and 2 μg / mL significantly weakened the fluorescence intensity of ROS. This shows that the red rice peel extract of the present invention has the effect of inhibiting the glycation of BJ cells induced by MGO.
[0159] Example 9: Evaluation of the Elastase Inhibitory Activity of Red Rice Peel Extract
[0160] 9.1 Reagents and Materials
[0161] Elastase, N-succinyl-Ala-Ala-Ala-pNA (NSAAAP), 1 M Tris-HCl buffer (pH=8.0), and DPBS.
[0162] 9.2 Instruments
[0163] ELISA reader, 1 / 100,000 microbalance.
[0164] 9.3 Solution preparation
[0165] NSAAAP working solution: 0.5 mg / mL, prepared in 50 mM Tris-HCL buffer;
[0166] Red rice peel extract: the red rice peel extract obtained in Example 1, tested at concentrations of 0.41, 1.23, 3.7, 11.1, 33.33, 100, and 300 μg / mL, prepared using 50 mM Tris-HCL buffer;
[0167] Elastase working solution: 5 U / mL, prepared with 50 mM Tris-HCL buffer.
[0168] 9.4 Experimental Methods
[0169] According to Table 4 below, the prepared elastase working solution, different concentrations of red rice peel extract solutions, and 50 mM Tris-HCL buffer were added to the 96-well plate, and incubated at 25°C for another 10 min, and then NSAAAP working solution or 50 mM Tris-HCL buffer was added and incubated at 25°C for another 30 min. After the incubation, the absorbance at a wavelength of 410 nm was detected using an enzyme reader.
[0170] Table 4 Group ratios of red rice peel extract elastase inhibitory activity evaluation experiment
[0171]
[0172] According to the following formula (4), the elastase activity (P) and half inhibition concentration IC of the sample to be tested were calculated: 50 :
[0173]
[0174] Wherein, T, T0, C, and C0 represent the absorbance of the sample group, sample background group, reaction group, and solvent group at 410 nm, respectively.
[0175] 9.5 Experimental Principles and Results
[0176] Inhibiting elastase activity is beneficial to improving skin aging. In this experiment, NSAAAP is used as a substrate. Elastase can hydrolyze it into Nsuccinyl-Ala-Ala-Ala and p-nitroaniline (pNA) in 50mM Tris-HCl buffer at pH 8.0. The absorbance of pNA can be measured at 410nm in a spectrophotometer. The test substance with elastase activity inhibition can reduce the degradation of elastin, thereby reducing the absorbance value. This experiment evaluates the inhibitory effect of the test substance on elastase activity based on the change in absorbance value.
[0177] The experimental results are as follows Fig. 9 As shown in Figure 2, the activity of elastase (P) decreased with the increase of the concentration of red rice peel extract, and its half inhibition concentration IC 50 The results show that the red rice peel extract of the present invention has the anti-aging effect of inhibiting elastase.
[0178] Example 10: Evaluation of the anti-aging activity of red rice peel extract
[0179] 10.1 Reagents and Materials
[0180] DMEM high-glucose culture medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, 30% H2O2 (analytical grade), vitamin E (VE), X-Gal, and cell senescence β-galactosidase staining kit.
[0181] 10.2 Instruments
[0182] CO2 incubator, biological safety cabinet, inverted microscope.
[0183] 10.3 Cell lines
[0184] Human skin fibroblasts (BJ) were purchased from the Cell Bank of the Chinese Academy of Sciences.
[0185] 10.4 Solution preparation
[0186] H2O2 working solution: 600 μM, prepared in serum-free DMEM medium;
[0187] VE working solution: 50 μg / mL, prepared with DMEM complete medium;
[0188] Red rice peel extract: The red rice peel extract obtained in Example 1 was prepared at test concentrations of 0.1, 0.3, and 1 μg / mL using DMEM complete medium.
[0189] 10.5 Experimental Methods
[0190] Take BJ cells that are in good exponential growth phase, use 0.25% Trypsin-EDTA to remove the cells from the culture dish, and adjust the cell concentration to 4×10 after centrifugation. 4 / mL of cell suspension. Take the above cell suspension and inoculate it in a 12-well plate, 1mL / well, and incubate it in a 37℃, 5% CO2 incubator for 16h. When the cell fusion rate reaches 60%, remove the supernatant, add 1mL of 600μM H2O2 working solution to each well except the blank control group, add 1mL of serum-free DMEM culture medium to each well of the blank control group, and continue to culture in the incubator for 2h. After the induction is completed, wash and add 1mL of the above test samples in turn, and continue to culture in a 37℃, 5% CO2 incubator for 72h. After incubation, use the cell senescence β-galactosidase staining kit for staining, and observe and take pictures under an inverted microscope.
[0191] 10.6 Experimental Principles and Results
[0192] H2O2 can cause oxidative stress response, leading to intracellular redox imbalance and DNA damage, thus causing cell senescence. When cells undergo senescence, the lysosome content in the cells increases significantly, leading to overexpression of β-galactosidase (β-gal), so the activity of β-gal in senescent cells increases abnormally. In this experiment, H2O2 was used to induce BJ cells to establish an oxidative aging model. After adding the test substance and incubating the cells, the ratio of senescent cells was detected by β-galactosidase staining to evaluate the anti-aging activity of the test substance in BJ cells.
[0193] The experimental results are as follows Fig.10 As shown, compared with the blank control group, after H2O2 induction, the number and staining intensity of BJ cell β-galactosidase-positive cells increased significantly, indicating that the H2O2-induced BJ cell aging model was successfully established. When the concentration of red rice peel extract in the drug administration group was 0.3 and 1 μg / mL, the number and staining intensity of β-galactosidase-positive cells were significantly reduced. This shows that the red rice peel extract of the present invention has the effect of inhibiting H2O2-induced BJ cell aging.
[0194] Example 11: Application of red rice peel extract in skin care lotion
[0195] 11.1 Formulation Design
[0196] Red rice peel extract (0.3%), butylene glycol (5%), hyaluronic acid (1%), thickener 0.5%, moisturizer 5%, flavor (appropriate amount), preservative (appropriate amount), deionized water (balance).
[0197] 11.2 Preparation method
[0198] Mix all the aqueous phase ingredients (butylene glycol, hyaluronic acid, moisturizer, thickener and deionized water) at 85°C, stir evenly until completely dissolved, cool to room temperature, add red rice peel extract, preservatives and flavors, continue to stir evenly, and finally obtain a skin care lotion with antioxidant and moisturizing effects.
[0199] 11.3 Efficacy Testing
[0200] 11.3.1 Test of Antioxidant Capacity
[0201] The DPPH free radical scavenging activity evaluation test method of Example 5 was used, and the test samples were 0.1%, 0.5%, 1%, 2%, 5%, 10%, and 20% skin care lotions. The antioxidant capacity of different test samples was tested. The experimental results are as follows: Fig.11 shown.
[0202] from Fig.11It can be seen that the skin care lotion with added red rice peel extract can significantly reduce the generation of DPPH free radicals, has strong DPPH free radical scavenging ability, and has strong antioxidant capacity.
[0203] 11.3.2 Testing the moisturizing effect on skin
[0204] 11.3.2.1 Reagents and materials
[0205] Dry facial tissue, latex finger cots.
[0206] 11.3.2.2 Instruments
[0207] Skin moisture tester Corneometer CM 825.
[0208] 11.3.2.3 Experimental methods
[0209] Ten subjects who have not used any products (cosmetics or topical drugs) for 2-3 days were selected. It was ensured that the test area did not come into contact with water for 1-3 hours before the test. The inner forearms of both hands were cleaned uniformly 1 hour before the test and wiped dry with facial tissue. Five test areas of 3cm×3cm were marked. Before the formal test, the subjects sat quietly in a standard room with a constant temperature of 20-22℃ and a constant humidity of 40-60% for 30 minutes. Drinking water was prohibited and they were required to stay relaxed. After the experiment began, a latex finger cot was used to press the test sample liquid at (2.0±0.1)mg / cm 2 Apply a certain amount of the sample solution quickly and evenly on the test area once, and test the changes in skin moisture content before and after application of the sample solution.
[0210] The specific implementation method is: before using the sample solution and 2h, 7h, and 14h after using the sample solution, use a skin moisture content tester to test the values of the five test areas in turn, and take the average to obtain the skin moisture content value of the subject.
[0211] 11.3.2.3 Experimental Results
[0212] The experimental results are as follows Fig.12 As shown. Fig.12 The results show that the skin care lotion with red rice peel extract can significantly increase the moisture content of the skin and has a significant skin moisturizing effect.
[0213] Example 12: Application of red rice peel extract in skin care lotion
[0214] 12.1 Formulation Design
[0215] Red rice peel extract (0.4%), butylene glycol (5%), hyaluronic acid (0.8%), emulsifier (3%), thickener 1%, moisturizer 5%, oil 3%, flavor (appropriate amount), preservative (appropriate amount), deionized water (balance).
[0216] 12.2 Preparation method
[0217] All water phase ingredients (butylene glycol, hyaluronic acid, moisturizer, thickener and deionized water) are mixed at 85°C and stirred evenly until they are completely dissolved, and then the oil phase ingredients (oils, emulsifiers) are gradually added and emulsified at high speed until they are uniform. After cooling to room temperature, red rice peel extract, preservatives and flavors are added and stirred evenly to obtain a skin care lotion with antioxidant and moisturizing effects.
[0218] 12.3 Efficacy Test
[0219] 12.3.1 Test of Antioxidant Capacity
[0220] The DPPH free radical scavenging activity evaluation test method of Example 5 was used, and the test samples were 0.1%, 0.5%, 1%, 2%, 5%, 10%, and 20% skin care lotions. The antioxidant capacity of different test samples was tested. The experimental results are as follows: Fig.11 shown.
[0221] from Fig.11 It can be seen that the skin care lotion with added red rice peel extract can significantly reduce the generation of DPPH free radicals, has strong DPPH free radical scavenging ability, and has strong antioxidant capacity.
[0222] 12.3.2 Testing the moisturizing effect on skin
[0223] The skin moisturizing effect test method of 11.3.2 in the above embodiment 11 was used to test the skin lotion samples. The results are shown in the attached Fig.13 .from Fig.13 The results show that the skin care lotion with red rice peel extract can significantly increase the moisture content of the skin and has a significant skin moisturizing effect.
[0224] Example 13: Application of red rice peel extract in skin care essence
[0225] Recipe Design
[0226] Red rice peel extract (0.5%), glycerin (5%), hyaluronic acid (1%), thickener 1%, moisturizer 5%, flavor (appropriate amount), preservative (appropriate amount), deionized water (balance).
[0227] 13.2 Preparation method
[0228] Mix all the aqueous phase ingredients (glycerin, hyaluronic acid, humectant, thickener and deionized water) at 85°C, stir evenly until completely dissolved, cool to room temperature, add red rice peel extract, preservatives and flavors, continue to stir evenly, and finally obtain a skin care essence with antioxidant and moisturizing effects.
[0229] 13.3 Efficacy Test
[0230] 13.3.1 Test of Antioxidant Capacity
[0231] The DPPH free radical scavenging activity evaluation test method of Example 5 was used, and the test samples were 0.1%, 0.5%, 1%, 2%, 5%, 10%, and 20% of the skin care essence. The antioxidant capacity of different test samples was tested. The experimental results are as follows: Fig.11 shown.
[0232] from Fig.11 It can be seen that the skin care essence with added red rice peel extract can significantly reduce the generation of DPPH free radicals, has strong DPPH free radical scavenging ability, and has strong antioxidant capacity.
[0233] 13.3.2 Testing the moisturizing effect on skin
[0234] The skin moisturizing effect test method of 11.3.2 in the above embodiment 11 was used to test the skin care essence samples. The results are shown in the attached Fig.14 .from Fig.14 The results show that the skin care essence with added red rice peel extract can significantly increase the moisture content of the skin and has a significant skin moisturizing effect.
[0235] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.
Claims
1. A method for preparing a red rice peel extract, characterized in that: The method comprises the following steps: (1) after husking the red rice, the red rice husk is ground and crushed, and passed through a 40-mesh sieve to obtain a red rice husk extraction raw material; (2) extracting the red rice husk raw material with a mixed solvent of alcohol and water in a volume ratio of 90:10-40:60, wherein the extraction temperature is 30-70° C., and the extraction time is 1-24 h. After vacuum filtering the extract, the extract is concentrated under reduced pressure to obtain a water-soluble suspension of red rice husk; (3) centrifuging the red rice skin water-soluble suspension at a speed of 3000-8000 rpm for 10-30 min, filtering to remove insoluble precipitates, and then standing for stratification to remove the clear liquid to obtain a red rice skin water-soluble concentrated solution; (4) drying the red rice peel water-soluble concentrate to obtain the red rice peel extract.
2. The method for preparing the red rice peel extract according to claim 1, characterized in that: The mass of the red rice husk extracted in step (1) accounts for 10% of the mass of the whole rice after shelling; the alcohol-water mixed solvent in step (2) is a methanol-water mixed solvent or an ethanol-water mixed solvent, and the volume added of the alcohol-water mixed solvent is more than 8-10 times the volume of the red rice husk extraction raw material.
3. The method for preparing the red rice peel extract according to claim 2, characterized in that: Step (2) specifically comprises: extracting the red rice husk extraction raw material with an alcohol-water mixed solvent having a volume ratio of 90:10-70:30, stirring continuously, with a stirring speed of 300 rpm, an extraction temperature of 50° C., and an extraction time of 2 h; vacuum filtering the extract, and concentrating under reduced pressure at 40° C. until the volume of the solution is reduced to 1 / 4 of the original volume, thereby obtaining a red rice husk water-soluble suspension.
4. The method for preparing the red rice peel extract according to claim 1, characterized in that: In step (3), the centrifugal speed is 4000 rpm and the centrifugal time is 10 min.
5. The method for preparing the red rice peel extract according to claim 1, characterized in that: In step (4), the red rice peel water-soluble concentrate is dried by freeze drying or spray drying.
6. A red rice peel extract, characterized in that The red rice peel extract is prepared by the preparation method of the red rice peel extract according to any one of claims 1 to 5.
7. A cosmetic, characterized in that: The invention comprises the red rice peel extract according to claim 6.
8. The cosmetic according to claim 7, characterized in that: The added amount of the red rice peel extract is 0.1%-5%.
9. The cosmetic according to claim 8, characterized in that: The cosmetics are skin care lotions, skin care emulsions or skin care essences with anti-oxidation, anti-inflammatory and anti-aging functions.
10. Use of the red rice peel extract according to claim 6 in preparing cosmetics having antioxidant, anti-inflammatory and anti-aging effects.