Yunnan red rose extract as well as preparation method and application thereof

Abstract

The invention discloses a Yunnan red rose extract as well as a preparation method and application thereof. The extract is rich in various active ingredients such as total polyphenols, total flavonoids, polysaccharides, proanthocyanidins and total anthocyanins. The preparation method comprises the following steps: preparing Yunnan red rose petals into freeze-dried powder, efficiently enriching active ingredients under the conditions of specific ethanol concentration, solid-liquid ratio, ultrasonic power, temperature and time by adopting an ultrasonic-assisted extraction technology, and repeatedly extracting, filtering and concentrating to obtain the high-purity Yunnan red rose extract. The Yunnan red rose extract prepared by the invention has multiple effects of resisting oxidation, whitening, blocking sun, resisting aging, resisting inflammation, relieving, controlling oil, removing acnes and the like, can be applied to preparation of cosmetics such as skin care products, sunscreen products, acne removing products and the like, and meets the requirements of green beauty markets.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and in particular relates to a Yunnan red rose extract, a preparation method and an application thereof. Background Art

[0002] Rose has been a hot ingredient in the skin care field since ancient times, and it is also a precious medicinal material. The flower buds have the effects of relieving depression, invigorating the spleen, promoting menstruation and activating blood circulation. The flower roots have the effects of relieving qi and activating blood circulation, and have antibacterial and bactericidal effects. Rose essential oil can calm the mind and promote sleep, improve skin texture, and promote metabolism. Various rose extracts are also often used in the skin care industry as antioxidants, anti-aging agents, whitening agents, etc.

[0003] The Yunnan Red Rose (Rosa rugosa 'Dian hong') is a unique highland medicinal and edible rose resource from Yunnan Province, rich in nutritional and medicinal value. Its petals contain a large amount of nutrients such as vitamin C, amino acids, and trace elements, as well as a variety of active ingredients such as proanthocyanidins, polyphenols, flavonoids, and polysaccharides. Traditional medicine believes that the Yunnan Red Rose has multiple benefits, including antioxidant, antibacterial, anti-inflammatory, and beauty-enhancing properties. However, research and utilization of the Yunnan Red Rose are limited, with insufficient systematic research data to support its use. Furthermore, its mechanism of beauty and skincare activity has not been clearly elucidated, limiting its further application in health products, pharmaceuticals, and treatments for skin diseases with anti-aging, anti-inflammatory, and whitening properties.

[0004] Currently, the main methods used to extract active ingredients from plant samples include traditional methods such as solvent extraction and steam distillation, as well as modern emerging extraction technologies such as ultrasonic-assisted extraction, supercritical fluid extraction, microwave-assisted extraction, pulsed electric field extraction, subcritical water extraction, colloid mill extraction, and enzymatic hydrolysis. Among them, ultrasonic-assisted extraction is widely used in industrial production due to its advantages such as simple operation, high efficiency, and low cost. Other extraction methods have disadvantages such as long processing time, the need to use a large amount of organic solvents, and secondary pollution. In addition, extraction solvents mainly include ethanol, methanol, petroleum ether, ether, ethyl acetate, supercritical fluids, and various enzymes. However, different extraction solvents and different solvent concentrations have a significant impact on the extraction efficiency and composition of plant active ingredients. In addition, some extraction solvents may cause the destruction or inactivation of some active ingredients, making it difficult to effectively maintain their natural structure and function. In addition, the composition of the active ingredients in Yunnan red rose is complex, and some ingredients may interfere with each other, affecting its overall efficacy.

[0005] Therefore, how to optimize the extraction process of Yunnan red rose, effectively separate and purify its active ingredients while maintaining its natural activity, and further explore its beauty and skin care mechanism has become a technical problem that needs to be solved urgently. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention aims to provide a Yunnan red rose extract, a preparation method thereof, and its application. The method uses a 50% by volume ethanol-water solution to extract Yunnan red roses, maximizing the release of active ingredients related to skin care, yielding an optimal skin care extract component. The extract can also exert beauty and skin care effects through mechanisms related to antibacterial, anti-inflammatory, antioxidant, and whitening pathways.

[0007] The first aspect of the present invention provides a Dianhong rose extract, which contains total phenols, total flavonoids, polysaccharides, proanthocyanidins and total anthocyanins, with the remainder being water, ash and other inevitable plant components;

[0008] Based on the dry weight of the extract, the weight content of each component in the Yunnan red rose extract is 20% to 22% of total phenols, 13% to 18% of total flavonoids, 29% to 45% of polysaccharides, 4% to 5% of proanthocyanidins, and 0.05% to 0.06% of total anthocyanins.

[0009] Furthermore, the total phenols include one or more of the following group: gallic acid, protocatechuic acid, p-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, epicatechin, epigallocatechin gallate, p-hydroxycinnamic acid, sinapinic acid, epicatechin gallate, tannic acid, myricetin and resveratrol.

[0010] The second aspect of the present invention provides a method for preparing the Yunnan red rose extract according to the first aspect of the present invention, the method comprising the following steps:

[0011] Step 1): collecting Dianhong rose petals, freezing them, vacuum freeze-drying them, crushing and sieving them to prepare Dianhong rose freeze-dried powder;

[0012] Step 2): immersing the freeze-dried powder of Yunnan red rose prepared in step 1) in a 30% to 80% V / V ethanol aqueous solution, and performing ultrasonic-assisted extraction under the conditions of an ultrasonic power of 200 to 300 W, an extraction time of 60 to 120 min, and an extraction temperature of 40 to 60° C.;

[0013] Step 3): repeat step 2) 1 to 3 times, combine all the extracts, filter to obtain a filtrate, and concentrate the filtrate by rotary evaporation to remove the solvent to obtain the Yunnan red rose extract.

[0014] Furthermore, the volume fraction of the ethanol aqueous solution is 50% V / V.

[0015] Furthermore, the material-liquid ratio of the Dianhong rose freeze-dried powder to the ethanol aqueous solution is 1:20 (W:V).

[0016] The third aspect of the present invention provides the use of the Dianhong Rose extract described in the first aspect of the present invention or the Dianhong Rose extract prepared by the method described in the second aspect of the present invention in any of the following aspects:

[0017] 1) preparing antioxidant products;

[0018] 2) preparing whitening products;

[0019] 3) preparing sunscreen products;

[0020] 4) preparing anti-aging products;

[0021] 5) preparing anti-inflammatory and soothing products;

[0022] 6) preparing an oil-control and acne-removing product;

[0023] Preferably, the product is a skin care product or a cosmetic.

[0024] Furthermore, the antioxidant activity refers to the enhancement of the scavenging ability of DPPH free radicals and ABTS cation free radicals;

[0025] The whitening refers to inhibiting tyrosinase activity and inhibiting the synthesis and accumulation of melanin;

[0026] Said sun protection refers to the absorption of ultraviolet radiation;

[0027] The anti-aging method refers to inhibiting the activity of elastase;

[0028] The anti-inflammatory and soothing effects refer to reducing the release of NO, lowering the level of ROS, and inhibiting the expression of inflammatory factors, including TNF-α, IL-6, and IL-1β;

[0029] The oil control and acne treatment refers to the inhibition of Staphylococcus aureus and Propionibacterium acnes.

[0030] A fourth aspect of the present invention provides a cosmetic comprising the Yunnan red rose extract described in the first aspect of the present invention.

[0031] Furthermore, the cosmetics also include a common cosmetic carrier, which includes one or more of a solvent, a moisturizer, an emulsifier, a thickener, a preservative and a fragrance.

[0032] Advantages and beneficial effects of the present invention:

[0033] The Yunnan red rose extract provided by the present invention has clear ingredients and is rich in five core active ingredients: polyphenols, flavonoids, polysaccharides, proanthocyanidins and total anthocyanins. The content of each component has reached a significant level through process optimization, and the biological activity is significantly improved through the synergistic effect of multiple targets, breaking through the limitation of a single ingredient.

[0034] The present invention provides a method for preparing Yunnan red rose extract using vacuum freeze-drying combined with ultrasound-assisted extraction. By precisely controlling the ethanol concentration, material-liquid ratio, ultrasound parameters, and extraction times, the method achieves highly selective enrichment of the target component. This method is simple, easy to operate, effectively preserves the activity of heat-sensitive substances, and is suitable for large-scale industrial production.

[0035] The Yunnan Red Rose extract provided by the present invention has multiple beauty and skin care benefits, including antioxidant, whitening, anti-aging, sun protection, anti-inflammatory and soothing, oil control and acne treatment, through the synergistic effect of its components. It is more competitive than single-effect products. Moreover, its raw materials are naturally derived and highly safe, which meets consumers' pursuit of natural skin care. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The effect of different ethanol concentrations on the yield of Dianhong rose extract, different lowercase letters indicate significant differences between the groups (p < 0.05);

[0037] Figure 2 The effects of different ethanol concentrations on the total phenolic content (A) and total flavonoid content (B) in Dianhong rose extracts. Different lowercase letters indicate significant differences among the groups (p<0.05).

[0038] Figure 3 The effect of different ethanol concentrations on the polysaccharide content in Dianhong Rose extracts. Different lowercase letters indicate significant differences between the groups (p<0.05).

[0039] Figure 4 The contents of proanthocyanidins (A) and total anthocyanins (B) in Dianhong rose extracts obtained by extraction with different ethanol concentrations. Different lowercase letters indicate significant differences among the groups (p<0.05).

[0040] Figure 5 HPLC chromatograms of Yunnan red rose extracts obtained by extraction with different ethanol concentrations;

[0041] Figure 6 DPPH free radical scavenging rate and IC of Yunnan red rose extracts obtained by extraction with different ethanol concentrations 50 Values, VC and BHT were used as positive controls, VC refers to vitamin C, BHT refers to butylated hydroxytoluene, and different lowercase letters indicate significant differences between the groups (p < 0.05);

[0042] Figure 7 ABTS cation radical scavenging rate and IC of Yunnan red rose extracts obtained by extraction with different ethanol concentrations 50Values, VC and BHT are positive controls, VC refers to vitamin C, BHT refers to butylated hydroxytoluene, different lowercase English letters indicate significant differences between groups (p < 0.05);

[0043] Figure 8 is the total antioxidant capacity of Yunnan red rose extracts obtained by extraction with different ethanol concentrations. VC and BHT are positive controls. VC refers to vitamin C, and BHT refers to butylated hydroxytoluene. Different lowercase letters indicate significant differences between the groups (p<0.05).

[0044] Figure 9 The antibacterial effects (A) and inhibition zone sizes (B) of Yunnan red rose extracts obtained from different ethanol concentrations on S. aureus and P. acnes, respectively. S. aureus refers to Staphylococcus aureus, and P. acnes refers to Propionibacterium acnes.

[0045] Figure 10 The UV absorption effects of Yunnan red rose extracts obtained by extraction with different ethanol concentrations in the UVC (200-275nm), UVB (275-320nm) and UVA (320-400nm) regions;

[0046] Figure 11 Tyrosinase inhibition rate and IC of Yunnan red rose extracts obtained by extraction with different ethanol concentrations 50 Value, kojicacid is the positive control group, different lowercase English letters indicate significant differences between groups (p < 0.05);

[0047] Figure 12 The elastase inhibition rate and IC of Yunnan red rose extracts obtained by extraction with different ethanol concentrations are shown in Table 2. 50 Value, EGCG was the positive control group, different lowercase English letters indicate significant differences between groups (p < 0.05);

[0048] Figure 13 The effects of different treatment groups on B16-F10 cell morphology, A is the blank group; B is the model group, added with 1% α-MSH; C is the positive control kojic acid group, treated with 100 μg / mL KA; D, E, and F are sample groups, treated with 15, 30, and 60 μg / mL DHE50, respectively;

[0049] Figure 14 Relative tyrosinase activity of B16-F10 cells under different treatment groups, Con refers to blank group, Mod refers to model group, KA refers to positive control kojic acid group, compared with the model group, *p<0.05, **p<0.01; compared with the blank control group, #p<0.05, ##p<0.01;

[0050] Figure 15Relative melanin content of B16-F10 cells under different treatment groups, Con refers to blank group, Mod refers to model group, KA refers to positive control kojic acid group, compared with the model group, *p<0.05, **p<0.01; compared with the blank control group, #p<0.05, ##p<0.01;

[0051] Figure 16 Effects of DHE50 on the NO content in HaCaT cells induced by LPS, compared with the model group, *p<0.05, **p<0.01; compared with the blank control group, #p<0.05, ##p<0.01;

[0052] Figure 17 Effects of DHE50 on ROS levels in HaCaT cells induced by LPS, A: control group (Con); B: model group (Mod); C: positive control group, treated with 50 μg / mL DXMS; D, E, F: low (50 μg / mL), medium (100 μg / mL), and high (200 μg / mL) doses of DHE50, respectively; G: ROS levels assessed by Image J; compared with the model group, *p<0.05, **p<0.01; compared with the blank control group, #p<0.05, ##p<0.01;

[0053] Figure 18 is the effect of DHE50 on inflammatory cytokines. A and B are TNF-α, IL-6, and IL-1β, respectively. Compared with the model group, *p<0.05, **p<0.01; compared with the blank control group, #p<0.05, ##p<0.01.

[0054] Figure 19 The effects of DHE50 on the levels of iNOS and COX-2, A, B are the levels of iNOS and COX-2, respectively; compared with the model group, *p<0.05, **p<0.01; compared with the blank control group, #p<0.05, ##p<0.01. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The materials and instruments used in the examples are all commercially available. Experimental methods in the examples where specific conditions are not specified are generally performed under conventional conditions or according to the conditions recommended by the manufacturer.

[0056] The main experimental materials, reagents and instruments used in the present invention are shown in Tables 1 and 2.

[0057] Table 1 Main materials and reagents

[0058]

[0059]

[0060] Table 2 Experimental instruments

[0061] name Manufacturer BSA224S electronic balance Ohaus Instruments (Changzhou) Co., Ltd. Model 5804R Multifunctional Desktop High-Speed Refrigerated Centrifuge Eppendorf, Germany FD-304 box-type vacuum freeze dryer Jinan Junde Instrument Co., Ltd. SB25-12DTDS ultrasonic cleaner Ningbo Xinyi Ultrasonic Equipment Co., Ltd. MD SpectraMax Plus 384 Microplate Reader BIOTEK, USA Vacuum rotary evaporator RV3V AIKA Instrument Equipment Co., Ltd. RE-52 Series Rotary Evaporator Shanghai Yarong Biochemical Instrument Factory SH2-D(Ⅲ) circulating water vacuum pump Bangxi Instrument Technology Co., Ltd. Escala LC-16 High Performance Liquid Chromatograph Shimadzu Nexera X2 LC-30AD Ultrahigh Pressure Liquid Chromatograph Shimadzu Q Exactive Plus mass spectrometer Thermo Scientific Multifunctional microplate reader Shanghai Sangon Bioengineering Co., Ltd. Inverted microscope Guangzhou Mingmei Optoelectronics Technology Co., Ltd. Inverted fluorescence microscope Nikon Precision Machinery (Shanghai) Co., Ltd. Inverted biological microscope Nikon Precision Machinery (Shanghai) Co., Ltd. Blood cell counting chamber Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.

[0062] Example 1 Preparation of Yunnan Red Rose Extract and Determination of Active Ingredients

[0063] 1. Preparation of Yunnan Red Rose Extract

[0064] The Dianhong rose of the present invention is picked from the Chaimen Manor rose planting base in Dazhuang Township, Honghe Prefecture, Yunnan Province. Fresh Dianhong rose petals are collected in the blooming period, removed of impurities, and immediately transported to a laboratory at low temperature. They are frozen at -80°C, then vacuum freeze-dried, crushed, and passed through a 60-mesh sieve to prepare Dianhong rose freeze-dried powder, which is stored at 4°C for future use. 10 g of Dianhong rose freeze-dried powder is accurately weighed, and ethanol solutions with volume fractions of 0%, 30%, 50%, 80%, and 100% are added respectively at a material-liquid ratio of 1:20 (W:V). Ultrasonic extraction is performed at 50°C and 240W for 60 minutes. The extraction is repeated twice, and the extracts are combined and vacuum filtered. The filtrate is vacuum-rotated at 100 r / min and 50°C to obtain a dry extract (DE). The yield is calculated according to the formula: extract yield (%) = extract mass / sample mass × 100. The extract is sealed and refrigerated at 4°C for future use.

[0065] The results are as follows Figure 1 As shown in the data, with the increase of ethanol concentration, the extract yield showed a trend of first increasing and then decreasing. The yields when the ethanol concentration was 0% to 80% were significantly higher than that when the ethanol concentration was 100%, and the highest yield was obtained when 50% ethanol was used as the extraction solvent, reaching 54.23%. The extract yields when the ethanol concentrations were 0%, 30%, 80% and 100% were 49.04%, 51.95%, 51.51% and 38.38%, respectively.

[0066] 2. Determination of active ingredient content in Yunnan red rose extract

[0067] (1) Determination of total phenolic and flavonoid contents

[0068] Determination of total phenolic content (TPC): 50 μL of Dianhong Rose extract sample solution diluted to an appropriate concentration was placed on a 96-well ELISA plate, and 125 μL of 10% forlin solution (V / V) and 100 μL of Na2CO3 solution (7.5% W / V) were added in sequence. After mixing, the mixture was protected from light and reacted for 30 minutes. The absorbance was measured at a wavelength of 765 nm. Methanol was used instead of Na2CO3 solution for a blank test. Under the same conditions, the absorbance of gallic acid standard solutions with mass concentrations of 10, 20, 40, 60, 80, and 100 μg / mL was measured. A gallic acid standard curve was drawn with gallic acid mass concentration as the horizontal axis and absorbance as the vertical axis, y=0.0139x+0.0297(R 2 =0.9977), and the total phenol content in the samples was calculated according to the gallic acid standard curve, and the results were expressed as mg GAE / g DE.

[0069] Determination of total flavonoid content (TFC): 40 μL of Dianhong rose extract sample solution diluted to an appropriate concentration was placed on a 96-well ELISA plate, 20 μL of NaNO2 solution (5%, W / V) was added and mixed for 6 minutes, 20 μL of Al(NO3)3 solution (10%, W / V) was added and mixed for 6 minutes, and finally 140 μL of NaOH solution (4%, W / V) was added. After reacting at room temperature for 15 minutes, the absorbance was measured at a wavelength of 510 nm. A blank test was performed using methanol solution instead of the reaction reagent. Under the same conditions, the absorbance values of rutin standard solutions with mass concentrations of 100, 200, 400, 600, 800, and 1000 μg / mL were measured. A rutin standard curve was drawn with rutin mass concentration as the horizontal axis and absorbance as the vertical axis, y=0.0011x+0.0026(R 2 =0.9971), and the flavonoid content in the samples was calculated according to the rutin standard curve, and the results were expressed as mg RT / g DE.

[0070] TPC in Dianhong rose petals extracted with different ethanol concentrations ( Figure 2 A) and TFC( Figure 2 B) Content results are as follows Figure 2 As shown in the data, with the increase of extraction ethanol concentration, TPC and TFC showed a trend of first increasing and then decreasing. The total polyphenol contents of 0%, 30%, 50%, 80% and 100% ethanol extracts of Yunnan red rose were 149.73, 202.85, 217.74, 206.81 and 196.74 mg GAE / g DE, respectively; the total flavonoid contents were 125.94, 139.76, 177.38, 165.26 and 127.77 mg RT / gDE, respectively.

[0071] (2) Determination of polysaccharide content

[0072] The polysaccharide content was determined by the phenol-sulfuric acid method: 50 μL of phenol solution (5%, W / V) was reacted with 50 μL of appropriately diluted sample, followed by the slow addition of 250 μL of concentrated sulfuric acid, and the mixture was incubated at 100°C for 10 min. The absorbance was then measured at 490 nm. Under the same conditions, the absorbance of glucose standard solutions with mass concentrations of 20, 40, 60, 80, and 100 μg / mL was measured, and a standard curve was drawn with glucose mass concentration as the horizontal axis and absorbance as the vertical axis, y = 0.0082x - 0.0156 (R 2 =0.9975), the polysaccharide content in the sample was calculated according to the glucose standard curve, and the result was expressed in mg / g DE.

[0073] The results are as follows Figure 3 As shown, the polysaccharide content decreased with the increase of ethanol concentration, and the total polyphenol contents of 0%, 30%, 50%, 80% and 100% ethanol extracts of Yunnan red rose were 572.39, 449.19, 326.90, 299.38 and 86.57 mg / gDE, respectively.

[0074] (3) Determination of procyanidins and total anthocyanin content

[0075] Determination of proanthocyanidin content: Take 3 mL of vanillin solution (5%, W / V) and mix it with 0.5 mL of appropriately diluted sample. Then, add 1.5 mL of HCl. After incubation in the dark for 15 minutes, measure the absorbance at 500 nm. Under the same conditions, measure the absorbance of proanthocyanidin standard solutions with mass concentrations of 20, 40, 60, 80, and 100 μg / mL. Draw a standard curve with proanthocyanidin mass concentration as the horizontal axis and absorbance as the vertical axis, y=1.3645x-0.0145 (R 2 =0.9996), the proanthocyanidin content in the sample was calculated according to the standard curve, and the result was expressed in mg / g DE.

[0076] Total anthocyanin content was determined using the pH differential method. Dissolve 10 mg of lyophilized sample in 10 mL of distilled water (adjusted to pH 2.5 with 1% hydrochloric acid). In a 96-well microplate, mix 30 μL of the extract with 270 μL of KI buffer (pH 1.0) and 270 μL of CH₃COONa buffer (pH 4.5). After incubation in the dark for 60 minutes, measure absorbance at 520 nm and 700 nm. Distilled water was used as a control. Total anthocyanin content was calculated according to the following formula.

[0077] ΔA=(a 520 -A 700 )pH1.0 -(A 520 -A 700 )pH 4.5

[0078]

[0079] Where: Mw, molecular molar mass of cyanidin-3-glucoside, 449.2 g / mol; DF, dilution factor; V, final volume, mL; ε, molar absorptivity of cyanidin-3-glucoside, 26900 L / (mol·cm); m, sample weight, g; L, optical path length of the cuvette, cm.

[0080] like Figure 4 As shown in Figure A, the proanthocyanidin content in Dianhong rose extracts at different ethanol concentrations initially increased and then decreased with increasing ethanol volume fraction. This trend is consistent with the variation in total phenolics, suggesting that the variation in total phenolics may be related to proanthocyanidin content. In the five experimental groups (0%, 30%, 50%, 80%, and 100% ethanol extracts), the proanthocyanidin contents were 35.20, 44.29, 48.02, 43.99, and 43.65 mg / g DE, respectively. The proanthocyanidin content in the 50% ethanol extract (48.02 mg / g DE) was the highest, significantly higher than that in the other treatment groups (p < 0.05). The proanthocyanidin content was lowest (35.20 mg / g DE) when extracted with water (0%) (p < 0.05). No significant differences were observed among the other treatment groups. This may be due to the phenolic hydroxyl groups in proanthocyanidins, which make them less soluble in highly polar liquids.

[0081] like Figure 4 As shown in Figure B, in the five extracts of Yunnan red rose at different concentrations, the total anthocyanin contents were 58.02, 56.17, 57.17, 56.17, and 57.80 mg / 100 g, respectively. The water extract (0%) contained a higher content of anthocyanins, and the anthocyanin content was the lowest when extracted with 100% ethanol, which was significantly lower than that of the other four groups (p < 0.05).

[0082] (4) HPLC determination of chemical composition

[0083] Sample Preparation: Filter the Dianhong rose extract through a 0.22 μm nylon membrane and collect in a 2 mL brown screw-in vial for later use. Fifteen marker compounds, including gallic acid, protocatechuic acid, and p-hydroxybenzoic acid, were also used as standards.

[0084] Chromatographic conditions: HPLC analysis was performed on an Escala LC-16 system (Shimadzu, Japan), and the elution conditions were modified and set according to the literature report [Li Jin, Dai Manting, Mou Yaping, et al. Study on the chemical composition, in vitro antioxidant and skin care activities of Yunnan peony stamen extract [J]. Food and Fermentation Industries, 2024, 50(17): 186-197.]. The chromatographic column was Silgreen C18 (4.6 mm × 250 mm, 5 μm), the column temperature was 35°C, the detection wavelength was 200–600 nm, the injection volume was 20 μL, and the flow rate was 0.8 mL / min. The mobile phase consisted of (A) acetonitrile and (B) 0.1% formic acid in water. The gradient elution conditions were as follows: 0–10 min, 5% A; 0–15 min, 10% A; 15–40 min, 17% A; 40–60 min, 20% A; 60–68 min, 25% A; 68–75 min, 27% A; 75–77 min, 34% A; 77–88 min, 100% A; 88–93 min, 5% A. Peaks were qualitatively and quantitatively analyzed by comparing the retention times and peak areas of 15 standards, including gallic acid and protocatechuic acid, with the standard curve.

[0085] The spectrum of each chromatographic peak can be obtained by scanning the wavelength of 200-600nm on the ultraviolet diode matrix detector. Since most polyphenols and flavonoids have large absorption at wavelengths around 280nm and 360nm, the experiment selected 280nm and 360nm as the detection wavelengths. High performance liquid chromatography (HPLC) was used to qualitatively and quantitatively analyze the chemical components of the extracts of Yunnan red rose petals with different ethanol concentrations. By calculating the relationship between the peak area and the mass concentration of the sample, the regression equation of the standard was obtained, as shown in Table 3. The chromatograms of the extracts of Yunnan red rose with different ethanol concentrations are shown in Table 3. Figure 5 The quantitative analysis results of each classification substance are shown in Table 4.

[0086] Table 3 Peaking time and standard curve of standard compounds of Dianhong rose active ingredients

[0087]

[0088] Table 4 HPLC analysis results of extracts of Dianhong rose at different ethanol concentrations (unit: μg / mg)

[0089]

[0090] Note: Different lowercase letters indicate significant differences between groups (p<0.05)

[0091] like Figure 5As shown in the figure, the chromatograms of the extracts of Yunnan red rose petals with different ethanol concentrations at 280 nm showed significant similarity. There was no obvious difference in the chromatographic peaks between them, but there was a certain difference in the extraction rate of the common peaks. A total of 15 polyphenolic compounds were identified, including gallic acid (peak 1), protocatechuic acid (peak 2), p-hydroxybenzoic acid (peak 3), 2,5-dihydroxybenzoic acid (peak 4), chlorogenic acid (peak 5), vanillic acid (peak 6), caffeic acid (peak 7), epicatechin (peak 8), epigallocatechin gallate (peak 9), p-hydroxycinnamic acid (peak 10), sinapinic acid (peak 11), epicatechin gallate (peak 12), tannic acid (peak 13), myricetin (peak 14) and resveratrol (peak 15).

[0092] Quantitative analysis (Table 4) revealed that extraction of Yunnan red roses with 50%, 80%, and 100% ethanol effectively promoted the release of polyphenolic compounds, such as p-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, epicatechin, epigallocatechin gallate, p-hydroxycinnamic acid, sinapinic acid, and tannic acid. The release of 2,5-dihydroxybenzoic acid, epigallocatechin gallate, and p-hydroxycinnamic acid initially increased and then decreased with increasing ethanol concentration, reaching peak values of 15.21 μg / mg, 0.97 μg / mg, and 1.40 μg / mg, respectively, when extracted with 50% ethanol. These three polyphenolic compounds can neutralize free radicals to prevent oxidative damage and inhibit tyrosinase activity, thereby reducing melanin production. These results indicate that the use of 50% ethanol effectively increased the extraction yield of Yunnan red rose polyphenolic compounds and also promoted the production of active ingredients with antioxidant, antibacterial, and whitening properties.

[0093] Example 2 Determination of the Antioxidant Capacity of Yunnan Red Rose Extract

[0094] 1. DPPH free radical scavenging ability determination

[0095] Take 100 μL of the Yunnan Red Rose extract sample solution prepared in Example 1 diluted to an appropriate concentration and mix it with 100 μL of DPPH solution on a 96-well ELISA plate. After reacting in the dark at room temperature for 30 minutes, the absorbance (As) is measured at 517 nm. A 70% volume fraction methanol solution is used instead of the sample solution as a blank control (Ac). The absorbance value of the reaction is Ab when a 70% volume fraction methanol solution is used instead of the DPPH solution. VC and BHT are used as positive controls. The DPPH free radical scavenging rate of the sample is calculated according to the formula: Free radical scavenging rate (%) = (1-(As-Ab) / Ac) × 100. The result is expressed as IC 50 The values are expressed as (μg / mL).

[0096] like Figure 6 As shown in Figure 2, the DPPH free radical scavenging rate of different concentrations of ethanol extracts of Yunnan Red Rose increased in a sample concentration-dependent manner ( Figure 6 A) Combined with IC 50 The value analysis found that ( Figure 6 B), IC of the sample 50 The values ranged from 9.61 to 18.29 μg / mL, among which the IC 50 The lowest value was 9.61μg / mL, which was significantly lower than the other four extraction groups and higher than the positive control VC and BHT. This shows that the Yunnan Red Rose extracted with 80% ethanol has a better DPPH free radical scavenging ability. The 80% ethanol extraction can better retain the antioxidant properties of the Yunnan Red Rose, followed by 50%, 30%, 0%, and 100%. In addition, as the ethanol volume fraction increases, the sample IC 50 The values showed a trend of first decreasing and then increasing, indicating that the sample's scavenging ability first increased and then decreased with decreasing solvent polarity. This suggests that the sample's DPPH free radical scavenging ability may be related to the active ingredients extracted under different polarity conditions. This trend is similar to the changing trends of total phenolic and total flavonoid content, suggesting that the total phenolic and total flavonoid content of Yunnan red rose extracts affects DPPH free radical scavenging ability. Overall, all five concentrations of extracts have DPPH free radical scavenging ability, with the scavenging ability showing the best performance when the ethanol volume fraction is 80%.

[0097] 2. Determination of ABTS cationic free radical scavenging ability

[0098] Take 50 μL of the Yunnan Red Rose extract sample solution prepared in Example 1 diluted to an appropriate concentration and place it in a 96-well ELISA plate. Add 200 μL of ABTS working solution and mix thoroughly. After reacting at room temperature for 5 minutes, measure the absorbance (As) at a wavelength of 734 nm. Use a 70% volume fraction methanol solution to replace the sample solution as a blank control (Ac). Use a 70% volume fraction methanol solution to replace the ABTS solution for the reaction and the absorbance is Ab. Use VC and BHT as positive controls. Calculate the ABTS cation radical scavenging rate of the sample according to the formula: Free radical scavenging rate (%) = (1-(As-Ab) / Ac) × 100. The result is expressed as IC 50 The values are expressed as (μg / mL).

[0099] like Figure 7 As shown in Figure 2, the trend of ABTS cation radical scavenging rate of Yunnan Red Rose extract after extraction with different concentrations of ethanol was consistent with that of DPPH radical scavenging rate, increasing in a sample concentration-dependent manner ( Figure 7 A) Combined with IC 50 Value Analysis ( Figure 7 B) It was found that the IC of the sample 50 The values ranged from 5.25 to 25.77 μg / mL, among which the IC 50The value was the lowest (5.25μg / mL), which was lower than the other four extraction groups, but higher than the VC and BHT positive controls. This showed that the 80% ethanol extraction of Yunnan Red Rose had the best ABTS cation free radical scavenging ability and could better preserve the antioxidant properties of Yunnan Red Rose. The next best values were 50%, 100%, 30%, and 0%. As the ethanol volume fraction increased, the IC values of the sample's ABTS cation free radical scavenging ability increased. 50 The values showed a trend of first decreasing and then increasing, which was similar to the changing trend of total phenols and total flavonoids contents.

[0100] 3. Determination of total antioxidant capacity

[0101] The FRAP method was used to determine the antioxidant capacity of Yunnan Red Rose extract. The FRAP method (Ferric reducing antioxidant potential assay) can detect the total antioxidant capacity of various body fluids, cell or tissue lysates, and plant extracts. Its principle is to reduce the amount of Fe under acidic conditions. 3+ -pyridine triazine (Fe 3+ -TPTZ) can be reduced by reducing substances in the sample to produce blue Fe 2+ -TPTZ, the compound has a maximum absorption at 593nm. The antioxidant activity of the sample can be calculated based on the absorbance value. The specific operation is as follows:

[0102] Accurately pipette 50 μL of the Yunnan Red Rose extract sample solution prepared in Example 1 diluted to an appropriate concentration into an ELISA plate, add 250 μL of FRAP working solution, shake well, react at 37°C for 10 minutes, and measure the absorbance at 593 nm. VC and BHT are used as positive controls. Under the same conditions, the absorbance of 10, 20, 40, 60, 80, and 100 μg / mL FeSO4 solutions is measured. A standard curve of FeSO4 solution is drawn with the mass concentration (X) of the FeSO4 solution as the horizontal axis and the absorbance value (Y) as the vertical axis: y = 0.0097x - 0.0016 (R 2 =0.9998), and the iron reducing ability of the sample was calculated according to the FeSO4 standard curve, and the result was expressed as mgFeSO4 / g DE.

[0103] FRAP utilizes Fe 2+ The blue-purple complex formed with TPTZ has strong absorption at 593nm, which is used to evaluate the antioxidant capacity of the sample. The larger the FRAP value, the better the antioxidant performance. Figure 8As shown, the FRAP values of the total antioxidant capacity of the samples ranged from 829.28 to 1926.24 mg FeSO4 / g DE, with 50% at 1926.24 mg FeSO4 / g DE, significantly higher than 0% (1418.30 mg FeSO4 / g DE), 80% (1740.10 mg FeSO4 / g DE), and 100% (829.28 mg FeSO4 / g DE), and lower than the positive controls VC (4018.43 mg FeSO4 / g DE) and BHT (2717.01 mg FeSO4 / g DE). Overall, the order of total antioxidant capacity from strong to weak was VC > BHT > 50% > 30% > 80% > 0% > 100%. The results indicate that the antioxidant capacity of Dianhong rose extracts initially increases and then decreases with decreasing polarity of the extraction solvent. Extraction with solutions of different polarity produces different amounts of active substances, potentially altering their structure.

[0104] Example 3 Determination of antibacterial properties of Yunnan red rose extract

[0105] The skin surface is home to a large number of microbial communities, which play a vital role in maintaining skin health. Factors such as ultraviolet radiation, environmental pollution, mechanical damage, and poor eating habits can disrupt the microbial balance of the skin. In severe cases, this disruption may lead to the development of skin diseases such as atopic dermatitis, psoriasis, and acne. These diseases may damage the skin barrier, increase susceptibility to other chronic diseases, and have a negative impact on the patient's physical and mental health. Staphylococcus aureus (S.aureus) and Propionibacterium acnes (P.acnes) play an important role in acne-related skin inflammatory reactions, so they were selected as test strains in the present invention, and the antibacterial activity of rose petal extract was evaluated by the filter paper method.

[0106] Take the different ethanol concentration extracts of Yunnan Red Rose prepared in Example 1, add sterile water to prepare a 100 mg / mL solution for later use. Staphylococcus aureus and Propionibacterium acnes were inoculated into LB and RCM liquid culture media respectively, and cultured at 37°C for 24 hours. The bacterial concentration was adjusted to 10 6 ~10 7CFU / mL. The diameter of the inhibition zone of Dianhong Rose extract was determined using the filter paper method. Under sterile conditions, sterile filter paper discs were soaked in Dianhong Rose extract solutions of varying ethanol concentrations for 45 minutes. Ampicillin at a concentration of 5 mg / mL was used as a positive control, and sterile water was used as a negative control. 100 μL of the bacterial solution was evenly spread on a solid culture medium plate. The drug-soaked filter paper discs were evenly placed on the plate. After inverted incubation at 37°C for 24 hours, the diameter of the inhibition zone was measured.

[0107] The results are as follows Figure 9 As shown, the five extracts showed good inhibitory effects on the two pathogens, with the diameters of the inhibition zones exceeding 10 mm ( Figure 9 B), 30%, 50%, and 80% ethanol extracts demonstrated more pronounced antibacterial activity, with inhibition zones ranging from 15 to 20 mm in diameter. Among these extracts, the 50% ethanol extract exhibited the strongest inhibitory effect, with an inhibition zone diameter of approximately 20 mm against both pathogens. These results suggest that Yunnan red rose petal extract, particularly that prepared with 50% ethanol, can be incorporated as an active ingredient in skincare and dermatological treatment products.

[0108] Example 4 Ultraviolet absorption capacity determination

[0109] The extracts of Yunnan red rose with different ethanol concentrations prepared in Example 1 were re-dissolved with ethanol of corresponding concentrations to a concentration of 0.5 mg / mL. The absorption spectra of the samples at 200-400 nm were measured using ethanol of different concentrations as blank solutions.

[0110] The UV absorption effect of Yunnan red rose petal extract in UVC (200-275nm), UVB (275-320nm) and UVA (320-400nm) regions is as follows: Figure 10 As shown. The results showed that the 50% ethanol extract exhibited a significant absorption effect for UVC radiation, while the water extract (0%) showed the most significant absorption effect for UVA and UVB radiation, followed by the 50% ethanol extract. UVC radiation poses a significant risk to human skin, potentially causing burns in the short term and increasing the risk of skin cancer with long-term or intense exposure. UVB radiation can reach the epidermis and cause severe damage to the skin, which may manifest as redness, inflammation, and other symptoms. On the other hand, UVA radiation can penetrate the dermis and cause melanin deposition, which darkens the skin. Therefore, the 50% ethanol extract of Yunnan red rose petals exhibits broad-spectrum UV absorption properties, making it a valuable functional additive in sunscreen formulations. Its ability to absorb UV radiation at different wavelengths suggests its potential protective effect against photodamage, pigmentation, and skin cancer.

[0111] Example 5 Determination of the Whitening Efficacy of Dianhong Rose Extract

[0112] 1. Determination of tyrosinase and elastase inhibition ability

[0113] Tyrosinase inhibitory activity assay: 200 μL of the Yunnan Red Rose extract sample solution prepared in Example 1 was mixed with 50 μL of tyrosinase (250 U / mL) in a 96-well plate, and then 50 μL of substrate (5 mM levodopa solution) was added. After slight shaking, the plate was placed in a 37°C environment and protected from light for 15 minutes. Finally, the absorbance was measured at a wavelength of 475 nm (A3). Kojic acid was used as a positive control, and PBS buffer (0.1 mol / L phosphate buffer solution with a pH of 6.47) was used instead of the sample as a blank control (A1). PBS was used instead of the sample and tyrosinase as the background absorbance of the blank control (A2), and PBS was used instead of tyrosinase as the background absorbance of the sample (A4). The tyrosinase inhibition rate was calculated according to the formula: Tyrosinase inhibition rate (%) = (1-(A3-A4) / (A1-A2)) × 100, and the results were expressed as IC 50 The values are expressed as (μg / mL).

[0114] Elastase inhibitory activity assay: First, 50 μL of the appropriate concentration of the Yunnan Red Rose extract sample solution prepared in Example 1 was mixed evenly with 25 μL of elastase (400 U / mL). Then, 25 μL of substrate was added. After gentle shaking, the 96-well plate was placed at 25°C in the dark for 30 minutes. After the reaction, the sample absorbance was measured at 410 nm (A3). Epigallocatechin gallate (EGCG) was used as a positive control. Tris buffer (10 mM pH 8 tris(hydroxymethyl)aminomethane) buffer was used instead of the sample as a blank control (A1). The background absorbance of the blank control (A2) was obtained by replacing the sample and elastase with Tris buffer. The background absorbance of the sample (A4) was obtained by replacing elastase with Tris buffer. The elastase inhibition rate was calculated according to the formula: Elastase inhibition rate (%) = (1-(A3-A4) / (A1-A2)) × 100. The results were expressed as IC 50 The values are expressed as (μg / mL).

[0115] like Figure 11 As shown in A, the tyrosinase inhibitory activity of Yunnan Red Rose extract showed a concentration-dependent enhancement, with an inhibition rate close to 80%, which is comparable to the positive control kojic acid. 50 Data analysis found that ( Figure 11 B), IC of 50% ethanol extract 50The lowest value was only 0.15 μg / mL, which was significantly lower than the other four extraction groups, but higher than the positive control. Compared with previous studies, the tyrosinase inhibitory activity of 50% ethanol extract of Yunnan red rose was significantly better than that of Damask rose (IC 50 :1351.20μg / mL), Bishop rose (IC 50 :1006.21μg / mL), Autumn rose (IC 50 :1579.22μg / mL) and Lafont rose (IC 50 :1756.71) etc. This shows that the 50% ethanol extract of Dianhong rose has significant potential in whitening skin care formulas.

[0116] like Figure 12 As shown in the figure, the elastase inhibition rate of the five Yunnan red rose extracts increased in a concentration-dependent manner, which was consistent with the results of the tyrosinase inhibition ability test. The Yunnan red rose 50% ethanol extract showed the strongest inhibition ability, with an IC 50 The value was 11.80 μg / mL, and the inhibitory effect was significantly better than that of the positive control EGCG (IC 50 The 50% ethanol extract contained a high level of quercetin (value of 16.86 μg / mL) compared to the other four extract groups. This may be related to the high content of classified substances in the 50% ethanol extract. Studies have found that quercetin can form a stable complex with elastase through multiple interactions, thereby inhibiting elastase activity and preventing skin aging. Elastase is crucial for skin elasticity. Overexpression of elastase in human skin can lead to excessive hydrolysis of elastin, resulting in the formation of wrinkles and the gradual loss of skin elasticity, which in turn promotes the aging process. Therefore, it can be inferred that Yunnan Red Rose can maintain skin elasticity and has anti-aging and anti-wrinkle potential.

[0117] 2. Effect of Yunnan Red Rose Extract on Melanin Synthesis in B16-F10 Cells

[0118] (1) Sample preparation and B16-F10 cell culture

[0119] The subsequent experiments were conducted using the Yunnan Red Rose extract (abbreviated as DHE50) extracted with the 50% by volume ethanol-water solution as the solvent in Example 1.

[0120] The mouse melanoma cells B16-F10 (STCC20013P-1) used in the present invention were purchased from Wuhan Saiweier Biotechnology Co., Ltd. Under sterile conditions, B16-F10 melanoma cells were inoculated in complete medium (15% FBS + 1% P / S + RPMI 1640) and cultured in a 37°C, 5% CO2 cell culture incubator. When the cell density reached 80% to 90%, the cells were passaged, the cell culture medium was discarded, the cells were rinsed twice with PBS phosphate buffer, 1 mL of 0.25% trypsin was added, and the cells were placed in a CO2 incubator for enzymatic hydrolysis for 3 minutes. The cells were then observed under a microscope. When the cells shrank and rounded and flowed with the enzymatic hydrolysis solution, the enzymatic hydrolysis was completed. 3 mL of complete medium was immediately added to terminate the enzymatic hydrolysis. The cells were centrifuged at 1000 r / min for 5 minutes, the supernatant was discarded, and 1 mL of complete medium was added to resuspend them. The cells were passaged at a ratio of 1:2 and the cells in the logarithmic growth phase were used for the experiment. After passage, 10 μL of cell suspension was taken into a hemocytometer and counted under an inverted biological microscope. The cell density (cells / mL) was calculated according to the formula: total number of cells in 5 squares of the hemocytometer / 5×25×10 4 After passage and counting, cells were enzymatically dissociated and centrifuged at 1000 rpm for 5 minutes. The cell pellet was resuspended in 1 mL of cell freezing solution (90% FBS + 10% DMSO), transferred to a cell cryovial, and then transferred to a programmed cooling box. First, place it at -20°C for 4-6 hours, then transfer it to a -80°C ultra-low temperature freezer overnight, and the next day, transfer it to a liquid nitrogen tank for long-term storage.

[0121] (2) Construction of α-MSH-induced melanin expression model

[0122] Melanin expression model establishment: Cells in the logarithmic growth phase were divided into 5×10 5 Cells were inoculated into 6-well cell culture plates with 2 mL per well. After culturing for 24 h until the cells were 80% full, the culture medium was discarded, the cells were rinsed twice with PBS, 2 mL of complete culture medium containing 1% α-MSH was added, and the cells were cultured in the incubator for another 24 h. At the end of the culture, the cell pellet was collected to determine the relative content of melanin. Compared with the control group (without α-MSH), the relative content of melanin was significantly increased, indicating that the model was successfully constructed.

[0123] B16-F10 cells were cultured in synthetic culture medium (15% FBS+1% P / S+RPMI 1640 basal culture medium) at 37°C and 5% CO2. Cells in the logarithmic phase were divided into experimental groups for experiments.

[0124] Experimental groups: blank group, model group (1% α-MSH), drug group (1% α-MSH induction + low (15 μg / mL), medium (30 μg / mL), high (60 μg / mL) concentrations of DHE50) and positive control kojic acid group (1% α-MSH induction + kojic acid 100 μg / mL).

[0125] (3) Cell morphology observation

[0126] After counting, dilute to 1 × 10 5 Cells were seeded at 100 μg / well in a 6-well plate and cultured for 24 h. The culture medium was discarded and the cells were rinsed three times with PBS. According to the experimental grouping described in "(2) Construction of α-MSH-induced melanin expression model", DHE50 containing different concentrations (15, 30, and 60 μg / mL) was added and co-cultured with the positive control medium for 48 h. The morphological changes induced by DHE50 in B16-F10 cells were observed using an inverted microscope.

[0127] like Figure 13 As shown in Figure 2, DHE50 had no significant effect on the morphology of B16-F10 cells. Figure 13 D, 13E and 13F) and the control group ( Figure 13 The cells in the α-MSH-induced melanin model (11B) were regularly and evenly distributed in the visual field. They were similar in size, oval or short spindle-shaped, with a clear periphery, a strong three-dimensional sense, and a bright appearance. The cells had good viability. In the α-MSH-induced melanin model (11B), compared with the control group, some B16-F10 cells had a slight change in shape, slightly wrinkled, and no obvious spindle shape. At the same time, through color comparison, it was found that the model group was darker in color, significantly darker than the control group, the low-, medium-, and high-dose drug groups, and the positive control group. The positive control group had the lightest color. This phenomenon was mainly due to the gradual production of melanin in B16-F10 cells after α-MSH induction, while the drug group and the positive control group showed improvement, which preliminarily indicated that the treatment was effective.

[0128] (4) Detection of relative activity of intracellular tyrosinase

[0129] B16-F10 cells in the logarithmic growth phase were taken, and the B16-F10 cell suspension diluted to an appropriate concentration after counting was evenly spread in a 6-well plate, 2 mL per well. After normal culture for 24 h, 2 mL of culture medium containing different mass concentrations of DHE50 was added according to the experimental grouping in the section "(2) Construction of α-MSH-induced melanin expression model". Three replicate wells were set for each group, and cultured under the same culture conditions for 48 h. After the culture was completed, the culture medium was discarded and the cells were rinsed twice with pre-cooled PBS. 300 μL of PBS solution containing 1% Triton X-100 was added to each well to lyse the cells for 10 min, placed at -80°C for 60 min, and then dissolved at room temperature. This step was repeated 3 times, and the cells were centrifuged at 12000 r / min for 20 min. 40 μL of the supernatant was mixed evenly with 100 μL L-DOPA (10 mmol / L), incubated at 37°C for 1 h, and the absorbance was measured at 475 nm. The relative tyrosinase activity of the cells was calculated according to the formula: relative tyrosinase activity (%) = A intervention group / A control group × 100.

[0130] Tyrosinase is a key enzyme in melanin synthesis. It mainly affects the production of melanin by catalyzing the conversion of tyrosine into dopa, which is then oxidized to dopaquinone, thereby affecting the skin color of the human body. Therefore, the whitening effect can be evaluated by measuring the activity of tyrosinase. Figure 14 As shown in the results, compared with the control group, the α-MSH-induced melanin model significantly increased the relative tyrosinase activity (89.08%) (p < 0.01), which in turn promoted the production of a large amount of melanin. When different concentrations of DHE50 were added to the intervention group, the relative tyrosinase activity gradually decreased with increasing drug concentration, reaching 63.98% at a drug concentration of 60 μg / mL. These results indicate that DHE50 can effectively inhibit tyrosinase activity and has a good whitening and skin care effect.

[0131] (5) Detection of relative content of intracellular melanin

[0132] The NaOH method was used to lyse B16-F10 cells and determine the intracellular melanin content. The B16-F10 cell suspension diluted to an appropriate concentration after counting was evenly plated in a 6-well plate, 2 mL per well. After normal culture for 24 hours, 2 mL of culture medium containing different mass concentrations of DHE50 was added according to the experimental grouping in the section "(2) Constructing an α-MSH-induced melanin expression model". Three replicate wells were set for each group and cultured under the same culture conditions for 48 hours. After the culture was completed, the culture medium was discarded, the cells were rinsed twice with pre-cooled PBS, 200 μL of trypsin was added for digestion, the cell fluid was collected, centrifuged, the supernatant was discarded, and 200 μL of prepared 1 mol / L NaOH solution (containing 10% DMSO) was added to each well. The mixture was pipetted and mixed. The cells were heated in an 80°C water bath for 60 minutes until the cell clusters were completely lysed and the melanin particles were completely dissolved. The absorbance was measured at a wavelength of 405 nm. The relative cellular melanin content was calculated according to the formula: Melanin content (%) = A intervention group / A control group × 100.

[0133] like Figure 15 As shown, the relative content of melanin in the α-MSH-induced model group was significantly higher than that in the control group (p<0.01); with the addition of DHE50, the melanin content decreased with the increase of Yunnan red rose extract. Combined with the observation of B16-F10 cell pellets under different treatment groups, it was found that the intervention of low-, medium-, and high-dose sample groups and the positive control group did significantly improve the accumulation of melanin. Among them, after treatment with medium and high doses of DHE50, the relative content of melanin in B16-F10 cells decreased by less than 60%. This result is consistent with the trend of changes in the relative activity of tyrosinase. There is a close connection between the relative activity of tyrosinase and melanin accumulation. Therefore, the DHE50 of the present invention can regulate melanin accumulation by inhibiting the activity of tyrosinase, thereby exerting the effect of whitening and regulating skin color.

[0134] In summary, 15-60 μg / mL of DHE50 can significantly inhibit the relative activity of tyrosinase in the α-MSH-induced B16-F10 cell melanin expression model, thereby reducing the release and accumulation of melanin, indicating that DHE50 has good whitening and skin care efficacy potential.

[0135] Example 6 Determination of the anti-inflammatory efficacy of Yunnan red rose extract

[0136] (1) Sample preparation and HaCaT cell culture

[0137] The subsequent experiments were conducted using the Yunnan Red Rose extract (abbreviated as DHE50) extracted with the 50% by volume ethanol-water solution as the solvent in Example 1.

[0138] The human immortalized keratinocytes HaCaT (STCC11801P-1) used in the present invention were purchased from Wuhan Saiweier Biotechnology Co., Ltd. A cryopreserved tube of HaCaT cells was removed from a liquid nitrogen tank, rapidly thawed in a 37°C water bath, surface disinfected with 75% ethanol, and centrifuged (1000 rpm for 5 minutes). The supernatant was discarded, and 1 mL of complete culture medium (10% FBS + 1% P / S + DMEM) was added to the cell pellet. The pellet was gently pipetted to resuspend and then inoculated into a T25 cell culture flask containing a 5 mL complete culture dish. The cells were then cultured in a cell culture incubator (containing 5% CO2, 37°C) for 24 hours until the cells were fully attached.

[0139] When the cell density reaches 80% to 90%, the cells are passaged, the cell culture medium is discarded, the cells are rinsed twice with PBS phosphate buffer, 1 mL of 0.25% trypsin is added, and the cells are placed in a CO2 incubator for enzymatic hydrolysis for 3 minutes, and then observed under a microscope. When the cells shrink and become round and flow with the enzymatic hydrolysis solution, the enzymatic hydrolysis is completed. 3 mL of complete culture medium is immediately added to terminate the enzymatic hydrolysis, centrifuged at 1000 r / min for 5 minutes, the supernatant is discarded, 1 mL of complete culture medium is added to resuspend, and the cells are passaged at a ratio of 1:2. Cells in the logarithmic growth phase are used for experiments. After passage, 10 μL of cell suspension is taken into a hemocytometer and counted under an inverted biological microscope. The formula is: cell density (cells / mL) = total number of cells in 5 squares of the hemocytometer / 5×25×10 4 After passage and counting, cells were enzymatically dissociated and centrifuged at 1000 rpm for 5 minutes. The cell pellet was resuspended in 1 mL of cell freezing solution (90% FBS + 10% DMSO), transferred to a cell cryovial, and then transferred to a programmed cooling box. First, place it at -20°C for 4-6 hours, then transfer it to a -80°C ultra-low temperature freezer overnight, and the next day, transfer it to a liquid nitrogen tank for long-term storage.

[0140] (2) Construction of LPS-induced HaCaT inflammatory cell model

[0141] HaCaT cell inflammation construction: cells in the logarithmic growth phase were divided into 5×10 5 Cells were inoculated into 6-well cell culture plates with 2 mL per well. After culturing for 24 h until the cells were 80% confluent, the culture medium was discarded, the cells were rinsed twice with PBS, and 2 mL of complete culture medium containing a final concentration of 20 μg / mL LPS was added. The cells were cultured in an incubator for another 24 h. At the end of the culture, the culture supernatant was taken to determine the NO content. Compared with the control group (without LPS), the NO content was significantly increased, indicating that the inflammatory model was successfully established.

[0142] Cells in the logarithmic growth phase were plated at 5×10 5Cells were inoculated into 6-well cell culture plates, 2 mL per well, and 3 wells were replicated for each group. After culturing for 24 h, the cells were rinsed with PBS and cultured according to the experimental groups.

[0143] The experimental groups were: control group Con (no treatment), model group Mod (treated with 20 μg / mL LPS), sample group DHE50 (20 μg / mL LPS + low (50 μg / mL), medium (100 μg / mL), and high (200 μg / mL) concentrations of Yunnan red rose extract), and positive control group (treated with 20 μg / mL LPS + 50 μg / mL dexamethasone).

[0144] (3) NO level measurement

[0145] Cells in good condition and in the logarithmic phase were prepared at a concentration of 5×10 5 Take 2 mL of the 100 μg / mL cell suspension and add it to a 6-well enzyme-labeled plate to culture for 24 hours. Remove the supernatant, wash with PBS, and treat the cells according to the experimental groups. Culture them in an incubator for 24 hours. Centrifuge at 4°C and 2500r / min for 20 minutes after the end of the culture, and collect the supernatant. Take 50 μL of the supernatant, add 50 μL GriessI and 50 μL GriessII, mix thoroughly, and measure the absorbance at 540 nm. Under the same conditions, measure the absorbance when the mass concentration of NaNO2 is 10, 20, 40, 60, 80, and 100 μg / mL, and draw a standard curve y=0.0056x-0.0038(R 2 =0.9999), the NO concentration was calculated according to the standard curve, and the results were expressed as the concentration of NO (μg / mL).

[0146] NO is an endogenously synthesized gaseous signaling molecule that can diffuse rapidly through the cell membrane. It reacts rapidly with other free radicals to generate reactive oxygen species signaling molecules. Elevated NO levels are associated with impaired skin barrier function and contribute to the development of acne by promoting inflammatory responses. Reducing LPS-stimulated NO production is one of the important means to address inflammation caused by immune cell activation. By stimulating the HaCaT cell model with LPS, the NO content in the cell supernatant after treatment with different concentrations of DHE50 was as follows: Figure 16Compared with the control group (15.33 μg / mL), the model group significantly increased NO content (18.40 μg / mL) (p < 0.01), indicating that the modeling was successful; after treatment with low, medium, and high concentrations of DHE50, the NO content in the supernatant of HaCaT cells induced by LPS was significantly reduced compared with the model group (p < 0.01), and the NO content produced after high-dose concentration treatment (15.86 μg / mL) was close to that of 50 μg / mL positive control dexamethasone (15.77 μg / mL). Compared with the model group, after high-dose concentration treatment, NO content secretion decreased by 13.78%. The results show that low, medium, and high concentrations of DHE50 can relieve skin inflammation by reducing NO release.

[0147] (4) Determination of intracellular ROS levels

[0148] The cells were treated and cultured according to the experimental groups. After the culture, the culture medium was discarded and the cells were rinsed three times with pre-cooled PBS. The ROS level of the cells was then detected according to the instructions of the reactive oxygen species (ROS) kit. The cells were fluorescently photographed and the ROS level was evaluated using Image J image processing software.

[0149] ROS are highly oxidative, capable of oxidizing cell membrane lipids, proteins, and DNA, leading to cell damage or apoptosis, metabolic dysfunction (such as mitochondrial damage), and accelerated aging (skin, wrinkles, and organ degeneration). They also activate signaling pathways such as NF-κB and MAPK, promoting the expression of inflammatory mediators and further triggering and exacerbating inflammatory responses. Inflammation also increases ROS production. Therefore, ROS levels are a key indicator of both normal cellular physiological functions and oxidative damage caused by environmental factors.

[0150] In order to explore the effect of DHE50 on LPS-induced ROS levels, HaCaT cells were stained with DCFH-DA probe and observed under an inverted fluorescence microscope, and ROS fluorescence ratio was analyzed using Image J software. The results are shown in Figure 2. Figure 17 Compared with the control group (1.19%), the LPS-induced model group produced a large area of green fluorescence, with the fluorescence accounting for as high as 31.23%, which was significantly different from the control group (p < 0.01). This indicates that LPS induction increases the ROS level in HaCaT cells and causes inflammation. However, after LPS induction, treatment with 50, 100, and 200 μg / mL DHE50 significantly reduced ROS levels in a concentration-dependent manner, with the fluorescence accounting for 28.29%, 19.37%, and 11.28%, respectively (p < 0.05). These results indicate that DHE50 can significantly reduce ROS levels in LPS-induced cells and has the potential to alleviate and improve HaCaT inflammation.

[0151] (5) Determination of TNF-α, IL-6, IL-1β, iNOS, and COX-2 levels

[0152] The cells were treated and cultured according to the experimental groups. After the culture, the cell supernatant was collected and centrifuged at 2500 r / min and 4°C for 20 min. The supernatant was collected and the secretion of TNF-α, IL-6, IL-1β, iNOS and COX-2 in the supernatant was detected by enzyme-linked immunosorbent assay kits. The standard curves were drawn according to the kits, and the formula y = 26.948x 2 +56.968x–6.3039(R 2 =0.9992), y=28.959x 2 +90.086x–6.2318(R 2 =0.9998), y=12.108x 2 +36.086x-1.5894(R 2 =0.9998), y=6.9506x 2 –4.7069x+0.7118(R 2 =0.9937), y=4.9991x 2 +25.382x–5.9827(R 2 =0.9967), and the content of each indicator was calculated according to the standard curve. The results were expressed as the concentration of TNF-α (pg / mL), IL-6 (pg / mL), IL-1β (pg / mL), iNOS (μmol / L), and COX-2 (pg / mL).

[0153] Inflammatory factors are key mediators of inflammatory responses, including proinflammatory cytokines TNF-α, IL-6, and IL-1β. They can initiate and amplify inflammatory responses, attract immune cells to sites of injury or infection, mediate vasodilation and pain responses, and excessive release of inflammatory factors may cause skin diseases such as psoriasis, eczema, acne, and skin aging. The effects of different doses of DHE50 on the content of inflammatory factors in LPS-induced HaCaT cells are shown in Figure 2. Figure 18 Compared with the control group, the HaCaT model group stimulated by 20 μg / mL LPS significantly promoted the release of TNFα, IL-1β, and IL-6 (p<0.01). Treatment with 50, 100, and 200 μg / mL DHE50 significantly reduced the expression of TNF-α in the supernatant of HaCaT cells induced by LPS, with the release amounts reduced by 16.92%, 29.24%, and 34.20%, respectively, compared with the model group. The positive control dexamethasone at 50 μg / mL reduced the release of TNF-α by 50.93%.

[0154] Figure 18 B shows the effect of DHE50 on the content of IL-6, an inflammatory factor in HaCaT cells. The results showed that compared with the model group, treatment with 50, 100, and 200 μg / mL of DHE50 significantly reduced the amount of IL-6 released in the supernatant of HaCaT cells induced by LPS (p<0.01), while there was no significant difference compared with the control group. DHE50 had a significant inhibitory effect on the expression of IL-6 inflammatory factors induced by LPS. Among them, treatment with 200 μg / mL of DHE50 reduced the secretion of IL-6 by 17.48% relative to the model group, approaching the positive control drug group (25.50%).

[0155] The effect of DHE50 on the content of inflammatory factor IL-1β in HaCaT cells is consistent with that of TNF-α and IL-6. Figure 18 As shown in Figure C, compared with the control group, the LPS-induced model group significantly promoted the expression of IL-1β, with a content of 40.05 pg / mL. As the concentration of added DHE50 increased, the release of IL-1β in the supernatant of inflammatory cells gradually decreased. When the administration concentration was 200 μg / mL, the release of IL-1β was 31.22 pg / mL, which was 22.06% lower than that of the model group and equivalent to the positive control group (26.30% lower than that of the model group).

[0156] Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are important enzymes involved in the inflammatory response process. They are induced to express under inflammatory factors or immune stimulation, thereby participating in the synthesis of NO. COX-2 has a low expression level in normal tissues, but is rapidly induced to express during inflammation or cell damage. The prostaglandins produced will promote vasodilation, resulting in pain and fever. Based on this, in order to further explore the repair effect of DHE50 on LPS-induced HaCaT cell inflammation, the enzyme-linked immunosorbent assay kit was used to measure and analyze the levels of iNOS and COX-2. The results are as follows: Figure 19 As shown. Compared with the control group, the levels of iNOS and COX-2 in the model group were extremely significantly increased (p < 0.01), increasing by 0.23 times and 1.67 times, respectively. Compared with the model group, after intervention with different concentrations of DHE50, the levels of iNOS and COX-2 were significantly decreased (p < 0.01). Among them, the iNOS produced by intervention with 200 μg / mL Yunnan Red Rose extract (6.51 μmol / L) was significantly lower than that in the positive control group (7.10 μmol / L). This indicates that DHE50 can effectively inhibit the production of iNOS and COX-2 in LPS-induced HaCaT cells, thereby inhibiting the production of NO and ultimately alleviating skin inflammatory reactions.

[0157] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A Yunnan red rose extract, characterized in that The Yunnan red rose extract contains total phenols, total flavonoids, polysaccharides, proanthocyanidins and total anthocyanins, with the remainder being water, ash and other inevitable plant components; Based on the dry weight of the extract, the weight content of each component in the Yunnan red rose extract is 20% to 22% of total phenols, 13% to 18% of total flavonoids, 29% to 45% of polysaccharides, 4% to 5% of proanthocyanidins, and 0.05% to 0.06% of total anthocyanins.

2. The Yunnan red rose extract according to claim 1, characterized in that The total phenols include one or more of the following group: gallic acid, protocatechuic acid, p-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, epicatechin, epigallocatechin gallate, p-hydroxycinnamic acid, sinapinic acid, epicatechin gallate, tannic acid, myricetin and resveratrol.

3. The method for preparing the Yunnan red rose extract according to claim 1, characterized in that: The method comprises the following steps: Step 1): collecting Dianhong rose petals, freezing them, vacuum freeze-drying them, crushing and sieving them to prepare Dianhong rose freeze-dried powder; Step 2): immersing the freeze-dried powder of Yunnan red rose prepared in step 1) in a 30% to 80% v / v ethanol aqueous solution, and performing ultrasonic-assisted extraction under the conditions of an ultrasonic power of 200 to 300 W, an extraction time of 60 to 120 min, and an extraction temperature of 40 to 60° C.; Step 3): repeat step 2) 1 to 3 times, combine all the extracts, filter to obtain a filtrate, and concentrate the filtrate by rotary evaporation to remove the solvent to obtain the Yunnan red rose extract.

4. The preparation method according to claim 3, characterized in that The volume fraction of the ethanol aqueous solution is 50% V / V.

5. The preparation method according to claim 3, characterized in that The material-liquid ratio of the Yunnan red rose freeze-dried powder to the ethanol aqueous solution is 1:20 (W:V).

6. Use of the Dianhong Rose extract according to claim 1 or the Dianhong Rose extract prepared by the method according to claim 3 in any one of the following 1) to 6): 1) preparing antioxidant products; 2) preparing whitening products; 3) preparing sunscreen products; 4) preparing anti-aging products; 5) preparing anti-inflammatory and soothing products; 6) preparing an oil-control and acne-removing product; Preferably, the product is a skin care product or a cosmetic.

7. The use according to claim 6, characterized in that The antioxidant activity is to enhance the scavenging ability of DPPH free radicals and ABTS cation free radicals; The whitening refers to inhibiting tyrosinase activity and inhibiting the synthesis and accumulation of melanin; Said sun protection refers to the absorption of ultraviolet radiation; The anti-aging method refers to inhibiting the activity of elastase; The anti-inflammatory and soothing effects refer to reducing the release of NO, lowering the level of ROS, and inhibiting the expression of inflammatory factors, including TNF-α, IL-6, and IL-1β; The oil control and acne treatment refers to the inhibition of Staphylococcus aureus and Propionibacterium acnes.

8. A cosmetic, characterized in that: The cosmetic comprises the Yunnan red rose extract according to claim 1.

9. The cosmetic according to claim 8, characterized in that The cosmetics further include a common cosmetic carrier, which includes one or more of a solvent, a moisturizer, an emulsifier, a thickener, a preservative, and a fragrance.

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