Method for preparing morin from black red roses and application of morin in whitening cosmetics

Abstract

The invention provides a method for preparing morin from black red roses and application of the morin in whitening cosmetics, and belongs to the technical field of effective component extraction. The method comprises the following steps: (1) weighing black red rose sample powder, adding ethanol, carrying out water bath extraction, and centrifuging after the extraction is finished, so as to obtain supernate; (2) drying the supernate to obtain a red rose crude extract; (3) performing column elution separation, rotary evaporation and drying on the red rose crude extract to obtain a macroporous resin extract; and (4) purifying the macroporous resin extract to obtain a dark red rose purified product, namely the morin. According to the invention, it is found and proved for the first time that the rosa rugosa is excellent in tyrosinase inhibition rate and total flavone synergistic effect, the crude extract activity of the rosa rugosa is obviously superior to that of common rosa rugosa, and the unique position of the rosa rugosa as an efficient whitening raw material is determined.

Description

Technical Field

[0001] This invention belongs to the field of active ingredient extraction technology, specifically relating to a method for preparing mulberry pigment from dark red rose and its application in whitening cosmetics. Background Technology

[0002] Skin pigmentation and excessive melanin production are core issues that urgently need to be addressed in the field of skin whitening. Tyrosinase, as a key rate-limiting enzyme in melanin synthesis, has become a crucial target for developing skin whitening products, and finding safe and effective tyrosinase inhibitors has become a major research and development strategy in this field.

[0003] Currently, mainstream skin-whitening ingredients such as vitamin C and its derivatives, arbutin, and niacinamide, while widely used, still suffer from limitations such as poor stability, slow effectiveness, and potential skin irritation at high concentrations. Glycyrrhizin, a highly effective skin-whitening ingredient extracted from licorice root, is hailed as "skin-whitening gold" due to its exceptional tyrosinase inhibitory effect; however, its high price and limited availability severely restrict its industrial application. Therefore, developing a novel natural skin-whitening ingredient with comparable or even superior efficacy to glycyrrhizin, while possessing a stable source and controllable costs, has become a pressing technological bottleneck that needs to be overcome in this field.

[0004] Roses, as a traditional medicinal and edible plant, are rich in flavonoids, polyphenols, vitamins, etc. They are often added to cosmetics in the form of extracts, mainly to exert fragrance, moisturizing and basic antioxidant functions.

[0005] For example, Chinese patent CN120391663A discloses a method for preparing and applying rose extract, which utilizes microbial fermentation technology to increase the content of water-soluble vitamins in roses. This method uses *Lactobacillus plantarum*, *Saccharomyces cerevisiae*, and *Bacillus subtilis* strains, adjusting the strain ratio to achieve a viable count ratio of (1-3):(1-2):(1-2) to form a compound microbial agent, which is then added to pretreated roses for staged fermentation. This ultimately increases the content of water-soluble vitamins in the rose extract, while retaining rose polysaccharides and increasing the total antioxidant activity by approximately 35%. This rose extract is suitable for providing raw materials for functional foods, health products, and cosmetics.

[0006] However, existing research has significant limitations: First, explorations of the whitening effects of roses are mostly limited to crude extracts, lacking a systematic comparison of efficacy differences between different varieties; second, the core whitening active ingredients have not been thoroughly isolated and identified, leading to unclear product efficacy and uncontrollable quality; finally, the efficacy evaluation system is incomplete, lacking systematic verification from tyrosinase inhibition to melanin production inhibition at the cellular level, making it difficult to convincingly demonstrate its whitening potential. Current technology has not yet revealed whether specific components comparable to glycyrrhizin exist in roses, nor does it possess a technical solution for systematically screening and precisely locating core whitening substances from multiple varieties.

[0007] Therefore, there is a need to develop a method for preparing mulberry pigment from dark red roses and its application in whitening cosmetics. Summary of the Invention

[0008] In view of the shortcomings of the existing technology, the present invention provides a method for preparing mulberry pigment from dark red rose and its application in whitening cosmetics.

[0009] This invention employs an activity-tracking separation strategy, utilizing modern preparative liquid chromatography to directionally separate and purify the extract of Mo Hong rose. It successfully enriched, purified, and structurally identified the key whitening active ingredient, morin, from a complex matrix, thus clearly defining morin as the core material basis for the whitening efficacy of Mo Hong rose for the first time. Through an in vitro melanoma cell model evaluation system, it was confirmed that the comprehensive whitening efficacy of morin derived from Mo Hong rose in inhibiting tyrosinase activity and blocking melanin production is significantly superior to the positive control glycyrrhizin, providing a scientific basis for developing a highly effective and commercially viable alternative to glycyrrhizin.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A method for preparing morin from dark red roses includes the following steps: (1) Weigh the powder of dark red rose sample, add ethanol and extract in a water bath. After extraction, centrifuge to obtain the supernatant. (2) The supernatant obtained in step (1) is dried to obtain crude extract of dark red rose; (3) The crude extract of dark red rose obtained in step (2) was reconstituted with ethanol, then separated by column elution, rotary evaporation and drying to obtain macroporous resin extract; (4) Take the macroporous resin extract prepared in step (3), add ethanol to redissolve and then purify to obtain the purified dark red rose extract, which is the mulberry pigment.

[0012] The volume fraction of ethanol in step (1) above is 55-65%; preferably 60%. The ratio of sample powder to ethanol in step (1) above is 1g:10-20mL; preferably 1g:15mL.

[0013] The water bath extraction temperature described in step (1) above is 55-70℃; preferably 60℃.

[0014] The extraction time mentioned in step (1) above is 60-100 min; preferably 70 min.

[0015] The extraction is performed 1-3 times in step (1) above; preferably 2 times.

[0016] The centrifugation rate in step (1) above is 4000-6000 r / min and the time is 8-15 min; preferably, the centrifugation rate is 5000 r / min and the time is 10 min.

[0017] The drying described in step (2) above is spray drying.

[0018] The volume fraction of ethanol mentioned in step (3) above is 55-65%; preferably 60%. The column separation described in step (3) above uses AB-8 as a macroporous resin column and water-ethanol as the mobile phase for separation.

[0019] The elution described in step (3) above involves gradient elution with water, 30% ethanol, 60% ethanol, 80% ethanol and 100% ethanol, respectively, and collecting the 60% ethanol eluent.

[0020] The drying process described in step (3) above is freeze drying.

[0021] The purification process described in step (4) above consists of the following steps: PrePulite XP C18 (5 μm, 10 × 250 mm) was used as a semi-preparative chromatographic column; Gradient elution was performed using acetonitrile as mobile phase A and 0.2% phosphoric acid as mobile phase B: 0-1 min, 5% A; 1-2 min, 5% → 10% A; 2-8 min, 10% → 11% A; 8-11 min, 11% → 15% A; 11-14 min, 15% A; 14-16 min, 15% → 19% A; 16-19 min, 19% A; 19-25 min, 19% → 40% A. The flow rate is 5 mL / min; The column temperature is 35 ℃; The detection wavelength is 254 nm; The injection volume was 500 μL.

[0022] Collect the eluent with a retention time of 19-20 min, and dry the eluent with nitrogen to obtain the morin.

[0023] The present invention also provides a mulberry pigment prepared by the above method.

[0024] This invention also provides the application of the mulberry pigment prepared by the above method in the preparation of whitening cosmetics.

[0025] A cosmetic product with whitening effect, wherein the cosmetic product comprises mulberry pigment prepared by the above method and cosmetically acceptable excipients.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Innovation in raw material screening: Extracts were prepared using dark red rose as raw material. For the first time, it was discovered and confirmed that the dark red rose exhibits outstanding performance in tyrosinase inhibition rate and total flavonoid synergistic effect. Its crude extract activity is significantly better than that of common rose varieties, establishing the unique status of dark red rose as a highly effective whitening raw material.

[0027] (2) Innovative ingredient identification: Precisely targeting the core whitening factor, mulberry pigment. Using an activity-tracking separation strategy, the extract of Mo Hong Rose was directionally separated and purified by modern preparative liquid chromatography. The key whitening active ingredient was successfully enriched, purified and structurally identified from the complex matrix as morin, which is the first time that morin has been identified as the core material basis for the whitening effect of Mo Hong Rose.

[0028] (3) Innovation in efficacy verification: Achieving whitening efficacy that meets the industry's gold standard Using an in vitro melanoma cell model evaluation system, it was confirmed that the comprehensive whitening efficacy of morin derived from Mohong rose in inhibiting tyrosinase activity and blocking melanin production is significantly better than that of the positive control glycyrrhizin, providing a scientific basis for developing a glycyrrhizin alternative that is both highly effective and commercially feasible. Attached Figure Description

[0029] Figure 1 TIC chromatogram of component identification of the purified dark red rose extract prepared in Example 1; Figure 2 Secondary mass spectrum of morin, the purified morin from dark red rose prepared in Example 1; Figure 3 Comparative graph of the effects of different concentrations of purified dark red rose extract on melanin production in B16F10 cells; Note: "Indicates a significant difference compared to the control group, P < 0.05;" "" indicates a highly significant difference from the control group, P < 0.01; Figure 4 Comparative figure of the effects of different concentrations of purified dark red rose extract on tyrosinase activity in B16F10 cells; Note: "Indicates a significant difference compared to the control group, P < 0.05;" "" indicates a highly significant difference from the control group, P < 0.01. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] "Chromatography" refers to a physical separation method in which the components to be separated (i.e., chemical constituents) are distributed between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves in a definite direction. The mobile phase can be a gas ("gas chromatography", "GC") or a liquid ("liquid chromatography", "LC"). Chromatographic output data can be used in implementations of the methods described herein.

[0033] A "chromatogram" is a chromatographic representation of mass spectrometry data, where the x-axis represents time and the y-axis represents total ion intensity. This data represents mass spectrometry data available for acquisition using a liquid chromatography-mass spectrometry (LC-MS) system. Liquid chromatography separates mixtures of organic molecules, while mass spectrometry provides molecular formula assignments for individual organic molecules. Therefore, the x-axis represents the retention time on the LC column of the LC-MS system.

[0034] "Separation" refers to the process of separating a complex mixture into its component molecules or metabolites. Common exemplary laboratory separation techniques include electrophoresis and chromatography.

[0035] "Retention time" refers to the elapsed time in a chromatographic process since the sample was introduced into the separation device. The retention time of a sample component refers to the elapsed time in a chromatographic process between the time the sample is injected into the separation device and the time the sample component is partially eluted (e.g., leaves) from the separation device containing the stationary phase.

[0036] The following embodiments use the following common instrument materials: instrument: NS4205 preparative liquid chromatograph: purchased from Jiangsu Hanbang Technology Co., Ltd.; Thermo-Orbitrap-QEHF liquid chromatograph-mass spectrometer: purchased from Thermo Fisher Scientific.

[0037] Reagents: Methanol: purchased from Beijing Mairuida Technology Co., Ltd.; Ethanol: purchased from Beijing Yili Fine Chemicals Co., Ltd.; Acetonitrile: purchased from Thermo Fisher Scientific.

[0038] The dark red roses were purchased from Yunnan Yuanshangcao Company, and are produced in Yunnan.

[0039] Example 1: A method for preparing morin from dark red rose. Includes the following steps: (1) Weigh 50g of dark red rose sample powder, add 60% ethanol at a material-liquid ratio of 1g:15mL and extract in a water bath at 60℃ for 75min. Extract twice, combine the extracts, and extract at 5000 r / min for 10min to obtain the supernatant. (2) The supernatant obtained in step (1) is spray-dried to obtain crude extract of dark red rose; (3) The crude extract of dark red rose obtained in step (2) was reconstituted with 60% ethanol. Then, AB-8 was used as a macroporous resin column and water-ethanol was used as the mobile phase. Gradient elution was performed with water, 30% ethanol, 60% ethanol, 80% ethanol and 100% ethanol respectively. The 60% ethanol eluent was collected, rotary evaporated and freeze-dried to obtain macroporous resin extract. (4) The macroporous resin extract prepared in step (3) was redissolved in ethanol and then purified to obtain the purified dark red rose extract, which is the morin mentioned above. The specific purification steps are as follows: PrePulite XP C18 (5 μm, 10 × 250 mm) was used as a semi-preparative chromatographic column; Gradient elution was performed using acetonitrile as mobile phase A and 0.2% phosphoric acid as mobile phase B: 0-1 min, 5% A; 1-2 min, 5% → 10% A; 2-8 min, 10% → 11% A; 8-11 min, 11% → 15% A; 11-14 min, 15% A; 14-16 min, 15% → 19% A; 16-19 min, 19% A; 19-25 min, 19% → 40% A. The flow rate is 5 mL / min; The column temperature is 35 ℃; The detection wavelength is 254 nm; The injection volume was 500 μL.

[0040] Collect the eluent with a retention time of 19-20 min, and dry the eluent with nitrogen to obtain the morin.

[0041] Example 2: A method for preparing morin from dark red rose. Includes the following steps: (1) Weigh 50g of dark red rose sample powder, add 65% ethanol at a material-liquid ratio of 1g:10mL and extract in a water bath at 55℃ for 100min. Extract twice, combine the extracts, and extract at 4000 r / min for 15min to obtain the supernatant. (2) The supernatant obtained in step (1) is spray-dried to obtain crude extract of dark red rose; (3) The crude extract of dark red rose obtained in step (2) was reconstituted with 65% ethanol. Then, AB-8 was used as a macroporous resin column and water-ethanol was used as the mobile phase. Gradient elution was performed with water, 30% ethanol, 60% ethanol, 80% ethanol and 100% ethanol respectively. The 60% ethanol eluent was collected, rotary evaporated and freeze-dried to obtain macroporous resin extract. (4) The macroporous resin extract prepared in step (3) was redissolved in ethanol and then purified to obtain the purified dark red rose extract, which is the morin mentioned above. The specific purification steps are as follows: PrePulite XP C18 (5 μm, 10 × 250 mm) was used as a semi-preparative chromatographic column; Gradient elution was performed using acetonitrile as mobile phase A and 0.2% phosphoric acid as mobile phase B: 0-1 min, 5% A; 1-2 min, 5% → 10% A; 2-8 min, 10% → 11% A; 8-11 min, 11% → 15% A; 11-14 min, 15% A; 14-16 min, 15% → 19% A; 16-19 min, 19% A; 19-25 min, 19% → 40% A. The flow rate is 5 mL / min; The column temperature is 35 ℃; The detection wavelength is 254 nm; The injection volume was 500 μL.

[0042] Collect the eluent with a retention time of 19-20 min, and dry the eluent with nitrogen to obtain the morin.

[0043] Example 3: A method for preparing morin from dark red rose. Includes the following steps: (1) Weigh 50g of dark red rose sample powder, add 55% ethanol at a material-liquid ratio of 1g:20mL and extract in a water bath at 70℃ for 60min. Extract twice, combine the extracts, and extract at 6000 r / min for 8min to obtain the supernatant. (2) The supernatant obtained in step (1) is spray-dried to obtain crude extract of dark red rose; (3) The crude extract of dark red rose obtained in step (2) was reconstituted with 55% ethanol. Then, AB-8 was used as a macroporous resin column and water-ethanol was used as the mobile phase. Gradient elution was performed with water, 30% ethanol, 60% ethanol, 80% ethanol and 100% ethanol respectively. The 60% ethanol eluent was collected, rotary evaporated and freeze-dried to obtain macroporous resin extract. (4) The macroporous resin extract prepared in step (3) was redissolved in ethanol and then purified to obtain the purified dark red rose extract, which is the morin mentioned above. The specific purification steps are as follows: PrePulite XP C18 (5 μm, 10 × 250 mm) was used as a semi-preparative chromatographic column; Gradient elution was performed using acetonitrile as mobile phase A and 0.2% phosphoric acid as mobile phase B: 0-1 min, 5% A; 1-2 min, 5% → 10% A; 2-8 min, 10% → 11% A; 8-11 min, 11% → 15% A; 11-14 min, 15% A; 14-16 min, 15% → 19% A; 16-19 min, 19% A; 19-25 min, 19% → 40% A. The flow rate is 5 mL / min; The column temperature is 35 ℃; The detection wavelength is 254 nm; The injection volume was 500 μL.

[0044] Collect the eluent with a retention time of 19-20 min, and dry the eluent with nitrogen to obtain the morin.

[0045] Comparative Example 1: A method for preparing morin from dark red rose. The difference from Example 1 is as follows: In step (3), the gradient elution program of the macroporous resin (AB-8) is changed to: gradient elution is performed using water, 40% ethanol, 70% ethanol and 90% ethanol respectively, and the 70% ethanol eluent is collected; the remaining steps and parameters are exactly the same as in Example 1.

[0046] Comparative Example 2: A method for preparing morin from dark red rose. The difference from Example 1 is as follows: In the purification step (4), mobile phase A (acetonitrile) was replaced with methanol, while mobile phase B remained 0.2% phosphoric acid. The gradient elution program was adjusted as follows: 0-1 min, 10% A; 1-5 min, 10% → 15% A; 5-10 min, 15% → 20% A; 10-15 min, 20% → 25% A; 15-20 min, 25% → 35% A; 20-25 min, 35% → 50% A. The remaining chromatographic conditions (column, flow rate, column temperature, detection wavelength, injection volume) were the same as in Example 1, and the fraction corresponding to the target peak (morula pigment) was collected according to the chromatogram.

[0047] Comparative Example 3: A method for preparing morin from dark red rose. The difference from Example 1 is as follows: In step (4), the purification step involves replacing the 0.2% phosphoric acid in mobile phase B with a 0.1% formic acid aqueous solution, while mobile phase A remains acetonitrile. The gradient elution procedure is the same as in Example 1; the remaining chromatographic conditions are the same as in Example 1.

[0048] Effect detection: 1. Determination of flavonoid content 1.1 Plotting the Rutin Standard Curve A standard curve was plotted using rutin as a reference standard via the sodium nitrite-aluminum nitrate method. 100 mg of rutin standard was accurately weighed and placed in a 100 mL volumetric flask protected from light, and the volume was diluted to obtain the rutin standard solution. 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the rutin standard solution were transferred to 10 mL volumetric flasks, respectively. 0.4 mL of 5% NaNO₂ was added sequentially, and the mixture was shaken well and allowed to stand at room temperature for 6 min. Next, 0.4 mL of 10% Al(NO₃)₃ was added, and the mixture was shaken well and allowed to stand at room temperature for 6 min. Finally, 4 mL of 4% NaOH was added, and the mixture was allowed to stand at room temperature for 15 min. The volume was then diluted to 10 mL with distilled water. The absorbance was measured at a wavelength of 510 nm. A standard curve was plotted with rutin concentration on the x-axis and absorbance on the y-axis.

[0049] 1.2 Determination of flavonoid content in pure pre-flowering dark red rose extract The dilution factor of the macroporous resin extract obtained in step (3) of Examples 1-3 was adjusted by preliminary experiment. The sample of dark red rose extract was transferred into a 10 mL volumetric flask and the OD value was measured at 510 nm according to the operation method in step 1.1. The obtained OD value was substituted into the rutin standard curve to calculate the flavonoid content.

[0050] Formula for calculating the flavonoid content (mg / g) of dark red rose In the formula: Y -- Total flavonoid content, mg / g; C -- The standard curve equation is used to calculate the mass concentration of the sample solution, in mg / mL. D -- Dilution factor; V -- Volume of sample solution, mL; m -- the mass of the sample taken, in grams.

[0051] The test results are shown in Table 1 below.

[0052] Table 1

[0053] Note: "--" indicates that no detection was performed.

[0054] As can be seen from the test results in Table 1 above, the flavonoid content in the extracts obtained by the preparation methods of Examples 1-3 of the present invention is all above 10 mg / g, indicating that the preparation method of the present invention can better enrich the flavonoid components in the dark red rose and has a high extraction effect.

[0055] 2. Tyrosinase activity inhibition experiment The rose extract samples obtained in step (4) of Examples 1-3 and Comparative Examples 1-3 were diluted to multiple concentrations using disodium hydrogen phosphate-citrate buffer. Referring to Table 2 below, 10 mL test tubes were used to set up sample tubes (T), sample background (T0), enzyme reaction tubes (C), and solvent background (C0). Three parallel tubes were set up for each sample tube (T) at each test concentration, and three parallel tubes were also set up for each enzyme reaction tube (C). 1 mL of the same concentration of sample solution was added to each of the sample tubes (T) and sample background (T0), while 1 mL of disodium hydrogen phosphate-citrate buffer was added to each of the enzyme reaction tubes (C) and solvent background (C0). 0.5 mL of tyrosinase solution was added to each of the sample tubes (T) and enzyme reaction tubes (C). The sample background (T0) and solvent background (C0) were replaced with 0.5 mL of disodium hydrogen phosphate-citrate buffer. The sample and tyrosinase were thoroughly mixed and incubated in a 37°C water bath for 10 min. Add 2 mL of levodopa solution to each tube in sequence, control the reaction time of each tube to 5 min, and immediately transfer the reaction solution of each tube into a cuvette and measure the absorbance at 475 nm.

[0056] Table 2 Sample Addition Requirements

[0057] Calculate the tyrosinase inhibition rate:

[0058] In the formula: T—Absorbance of the sample tube, i.e., the absorbance of the solution after the sample reacts with tyrosinase; T0—Sample background absorbance; C—The average of three absorbance values ​​of the enzyme reaction tube, i.e. the absorbance values ​​of the tyrosinase and dopa reaction without the addition of sample; C0 — Solvent background absorbance.

[0059] The test results are shown in Table 3 below.

[0060] Table 3

[0061] According to the test results in Table 3 above, the rose extract prepared by the method of Examples 1-3 of this invention contains more flavonoid active ingredients, which can better inhibit tyrosinase and thus have a better whitening effect; while in Comparative Examples 1-3, changing the purification conditions will significantly destroy the flavonoid components in the rose extract or reduce the content of flavonoid components, thereby reducing tyrosinase and weakening the whitening effect.

[0062] 3. Instrumental analysis conditions for purified mass spectrometry of dark red rose petals 3.1 Sample Pretreatment After homogenizing the purified solution of the dark red rose prepared in Example 1, take 150 μL of the sample into a 1.5 mL centrifuge tube, add 150 μL of 70% methanol-water (containing mixed internal standard, 2 μg / mL), vortex for 1 min, sonicate in an ice-water bath for 60 min, centrifuge for 10 min (12000 rpm, 4℃), and take 200 μL of the supernatant into an LC-MS vial with an inner liner for analysis.

[0063] 3.2 Chromatographic conditions The chromatographic column was an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B: 0.0–3.0 min, 100% A; 3.0–18.5 min, 100%→80% A; 18.5–20.0 min, 80%→65% A; 20.0–26.0 min, 65%→60% A; 26.0–35.0 min, 60%→5% A; 35.0–38.0 min, 5% A; 38.0–38.1 min, 5%→100% A; 38.1–40.0 min, 100% A. The flow rate was 0.35 mL / min; the column temperature was 45℃; the detection wavelength was 254 nm; and the injection volume was 2 μL.

[0064] 3.3 Mass Spectrometry Conditions Ion source: HESI; sample mass spectrometry signal acquisition adopted positive and negative ion scanning modes respectively.

[0065] Data acquisition mode: DDA Scanning method: Full MS / dd-MS2 (TOP 10).

[0066] LC-MS analysis of the purified dark red rose extract was performed according to... Figure 1 and Figure 2 Analysis of the total ion spectrum (TIC) and secondary mass spectrometry revealed that the whitening active ingredient in the purified dark red rose extract is morin.

[0067] 4. Cell experiments 4.1 Determination of the safe concentration of purified dark red rose extract for melanocytes The purified sample of dark red rose prepared in Example 1 was used to treat B16F10 cells at different concentrations for 48 h. The supernatant was discarded, and 110 μL of culture medium containing 10% CCK-8 solution was added to each well. The OD value of each well was measured at 450 nm and calculated according to the following formula:

[0068] 4.2 Determination of melanin content in cells Cells were seeded at the same density into 12-well plates and divided into a normal control group, a positive control group, and different concentration sample groups (containing different concentrations of samples). The cell control group was cultured in DMEM containing 10% fetal bovine serum, while the sample groups were cultured in addition to different concentrations of samples. Cells were collected, washed twice with PBS, centrifuged, and the supernatant was discarded. 1 mL of 1 M NaOH solution containing 10% DMSO was added, and the cells were incubated at 80°C for 1 h. 200 μL of the solution was added to a 96-well plate, and the A405 nm concentration was measured. The experiment was repeated three times. The relative melanin production was calculated as follows:

[0069] 4.3 Assay of tyrosinase activity in cells Cells were seeded at the same density into 12-well plates and divided into a normal control group, a positive control group, and sample groups with different concentrations (containing different concentrations of samples). The cell control group was cultured in DMEM medium containing 10% fetal bovine serum, while the sample groups were cultured in medium and contained different concentrations of samples. Cells were collected, washed twice with PBS, centrifuged, and the supernatant was discarded. Cells were lysed with 1 ml of 1% Triton X-100 solution, rapidly incubated at -80°C for 30 min, thawed at room temperature for approximately 20 min, mixed, and incubated at 4°C for 20 min. The supernatant was collected by centrifugation, and 100 μL of the cell extract was added to a 96-well plate. 100 μL of L-levodopa solution was added, mixed, and incubated at 37°C for 1 h. OD475 was measured, and the total protein P content (mg) in each well was measured using a BCA kit. The experiment was repeated three times. The melanin production inhibition rate was calculated as follows:

[0070] The test results are shown in Table 3 below. Figure 3 , Figure 4 .

[0071] Table 3

[0072] According to Table 3 and Figure 3 The results showed that, compared with the melanin production of the normal group, different concentrations of the purified dark red rose extract inhibited melanin production, and the inhibition was concentration-dependent, with the highest inhibition rate of 41.07%. The positive control selected in the experiment (glycyrrhizin 10 μg / mL) inhibited cellular melanin production by 37.55%, indicating that the whitening effect of the purified dark red rose extract was comparable to that of glycyrrhizin.

[0073] According to Table 3 and Figure 4 The results showed that, compared with the normal group, different concentrations of the purified black rose extract had a highly significant inhibitory effect on tyrosinase activity, and the inhibition was concentration-dependent, with the highest inhibition rate being 46.62%. The positive control group (10 μg / mL of glycyrrhizin) inhibited cytotyrosinase activity by 39.76%, indicating that the whitening effect of the purified black rose extract was comparable to that of glycyrrhizin.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing morin from dark red rose, characterized in that: Includes the following steps: (1) Weigh the powder of dark red rose sample, add ethanol and extract in a water bath. After extraction, centrifuge to obtain the supernatant. (2) The supernatant obtained in step (1) is dried to obtain crude extract of dark red rose; (3) The crude extract of dark red rose obtained in step (2) was reconstituted with ethanol, then separated by column elution, rotary evaporation and drying to obtain macroporous resin extract; (4) The macroporous resin extract prepared in step (3) was redissolved in ethanol and then purified to obtain the purified dark red rose extract, which is the morin mentioned above. The column separation in step (3) is performed using AB-8 macroporous resin column and water-ethanol as mobile phase; the elution is performed by gradient elution with water, 30% ethanol, 60% ethanol, 80% ethanol and 100% ethanol respectively, and the 60% ethanol eluent is collected.

2. The method according to claim 1, characterized in that: The volume fraction of ethanol in step (1) is 55-65%; preferably 60%.

3. The method according to claim 1, characterized in that: The ratio of sample powder to ethanol in step (1) is 1g:10-20mL; preferably 1g:15mL.

4. The method according to claim 1, characterized in that: The water bath extraction temperature in step (1) is 55-70℃; the extraction time is 60-100 min; and the number of extractions is 1-3.

5. The method according to claim 1, characterized in that: The centrifugation rate in step (1) is 4000-6000 r / min and the time is 8-15 min.

6. The method according to claim 1, characterized in that: The volume fraction of ethanol mentioned in step (3) is 55-65%.

7. The method according to claim 1, characterized in that: The purification process described in step (4) consists of the following steps: PrePulite XP C18 was used as a semi-preparative chromatographic column; Gradient elution was performed using acetonitrile as mobile phase A and 0.2% phosphoric acid as mobile phase B: 0-1 min, 5% A; 1-2 min, 5% → 10% A; 2-8 min, 10% → 11% A; 8-11 min, 11% → 15% A; 11-14 min, 15% A; 14-16 min, 15% → 19% A; 16-19 min, 19% A; 19-25 min, 19% → 40% A. The flow rate is 5 mL / min; The column temperature is 35 ℃; The detection wavelength is 254 nm; The injection volume was 500 μL; Collect the eluent with a retention time of 19-20 min, and dry the eluent with nitrogen to obtain the morin.

8. The mulberry pigment prepared by the method according to any one of claims 1-7.

9. The use of mulberry pigment prepared by the method according to any one of claims 1-7 in the preparation of whitening cosmetics.

10. A cosmetic product with whitening effect, characterized in that: The cosmetic product comprises mulberry pigment prepared by the method of any one of claims 1-7 and cosmetic excipients.

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