A rose polysaccharide, a preparation method thereof and application of the rose polysaccharide in skin anti-wrinkle tightening, beauty and improvement of anemia

Abstract

The present application belongs to the technical field of natural product extraction and preparation, and particularly relates to a rose waste liquid polysaccharide, a preparation method thereof and application of the rose waste liquid polysaccharide in skin anti-wrinkle and firming, beauty and skin care and improvement of anemia. Specifically, the present application takes rose waste liquid generated in the process of preparing rose essential oil as raw material, optimizes the extraction process conditions, and finally obtains the rose waste liquid polysaccharide. Research shows that the rose waste liquid polysaccharide has good effects of antioxidation, inhibition of melanin formation to achieve whitening effect, promotion of skin tissue growth, promotion of skin collagen formation (anti-wrinkle and firming) and blood replenishment (ruddy complexion and improvement of anemia), and can be widely applied in the fields of food, medicine, daily chemical products and feed, effectively improves the comprehensive utilization value of rose flowers, realizes secondary recycling of rose flower industrial waste, and therefore has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and preparation technology, specifically relating to a rose waste liquid polysaccharide, its preparation method, and its application in skin anti-wrinkle and firming, beauty and skin care, and improvement of anemia. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Rose( Rosa rugosa Thunb., a traditional Chinese medicinal and edible plant, has seen its essential oil and hydrosol processing industry expand rapidly. Statistics show that in 2022, the raw material processing volume in major rose-producing areas across China exceeded 500,000 tons, but this generated 1.5-2 million tons of high-concentration organic wastewater annually. This wastewater originates from the residue after distilling and extracting essential oils, with a soluble solids content as high as 8%-15%. Testing revealed that its main components are insufficiently recovered polysaccharides, phenolic substances, and a small amount of protein. However, current industry practices for wastewater treatment remain rudimentary. This contradiction of "high yield - low utilization" not only results in the waste of tens of thousands of tons of rose polysaccharide resources annually but also places increasingly severe environmental regulatory pressure on enterprises.

[0004] Recent studies have revealed that plant-derived polysaccharides possess multiple benefits, including immune regulation, antioxidant effects, and tissue repair promotion. For example, wolfberry polysaccharides have been developed into health products to improve anemia, while tremella polysaccharides are widely used in skincare products. However, research on the polysaccharide components in rose processing wastewater remains lacking. Studies on the bioactivity of rose polysaccharides have largely focused on their antioxidant properties, with few reports on their effects on skin collagen metabolism regulation and hematopoietic function. Therefore, developing functional products with rose wastewater polysaccharides as the main active ingredient is an effective way to improve rose utilization, increase rose added value, and extend the rose industry chain. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a rose waste sap polysaccharide, its preparation method, and its applications in skin anti-wrinkle and firming, beauty enhancement, and anemia improvement. Through experimental research, this invention has discovered that the rose waste sap polysaccharide extracted from rose essential oil processing waste sap possesses multiple beauty and skin-enhancing effects, including antioxidant properties, inhibition of melanin formation (leading to whitening), promotion of skin tissue growth, promotion of collagen formation, and improvement of anemia and complexion, thus enriching its application areas. Based on the above research results, this invention is thus completed.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: The first aspect of the invention provides the use of rose waste sap polysaccharides in all of the following (a)-(e): (a) Antioxidant or preparation of antioxidant products; (b) Whitening or preparing whitening products; (c) Promoting skin tissue growth or preparing products that promote skin tissue growth; (d) Promote the formation of collagen in the skin to achieve anti-wrinkle and firming effects, or prepare products that promote the formation of collagen in the skin to achieve anti-wrinkle and firming effects; (e) Improve anemia to achieve a rosy complexion or prepare products that improve anemia to achieve a rosy complexion.

[0007] The products include, but are not limited to, food, pharmaceuticals, daily chemical products, and animal feed.

[0008] The rose waste liquid polysaccharide in this invention is prepared by extraction from rose waste liquid; and the rose waste liquid is the waste generated during the extraction of rose essential oil.

[0009] A second aspect of the present invention provides a method for preparing rose waste liquid polysaccharide, the method comprising extracting and preparing the rose waste liquid polysaccharide from rose waste liquid.

[0010] Specifically, the preparation method includes: S1. Add ethanol to the rose waste liquid, mix and let stand, centrifuge to collect the precipitate and dry to obtain solid RCPS. S2. The solid RCPS is reconstituted, the protein is removed, and then separated and purified by column chromatography to obtain the final product.

[0011] The inventors have demonstrated through experiments that the rose waste sap polysaccharide component, especially RCPS-2, obtained by the above method has excellent antioxidant, melanin inhibition, skin tissue growth promotion, skin collagen formation promotion, and anemia improvement effects.

[0012] A third aspect of the present invention provides a product that has antioxidant properties, inhibits melanin formation, promotes skin tissue growth, promotes skin collagen formation, and achieves anti-wrinkle and firming effects as well as improving anemia, wherein the product comprises the above-mentioned rose waste sap polysaccharide.

[0013] The products include, but are not limited to, food, pharmaceuticals, daily chemical products, and animal feed.

[0014] The beneficial technical effects of one or more of the above technical solutions are as follows: The above technical solution uses rose waste liquid generated during the preparation of rose essential oil as raw material. By optimizing the extraction process conditions, rose waste liquid polysaccharide is finally obtained. Studies have found that this rose waste liquid polysaccharide has good antioxidant properties, inhibits melanin formation to achieve whitening effects, promotes skin tissue growth, promotes skin collagen formation to achieve anti-wrinkle and firming effects, and improves anemia to achieve a rosy complexion. It can be widely used in food, pharmaceuticals, daily chemical products, and feed, effectively improving the comprehensive utilization value of roses and realizing the secondary recycling of waste from the rose industry. Therefore, it has good practical application value. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The images show the separation and purification curves of (A) RCPS on the DEAE-52 cellulose ion exchange column and (B1) RCPS-1, (B2) RCPS-2 and (B3) RCPS-3 on the Sephadex G-100 dextran gel column in Example 1 of this invention.

[0017] Figure 2 The ion chromatograms of (A) standard sugar, (B1) RCPS, (B2), RCPS-1, (B3) RCPS-2 and (B4) RCPS-3 in Example 2 of the present invention are shown below; wherein, 1: Fuc; 2: Rha; 3: Ara; 4: Gal; 5: Glc; 6: Xyl; 7: Man; 8: Fru; 9: Rib; 10: Gal-UA; 11: Gul-UA; 12: Glc-UA; 13: Man-UA.

[0018] Figure 3 The HPSEC-MALLS chromatograms of A: RCPS, B: RCPS-1, C: RCPS-2, and D: RCPS-3 in Example 3 of this invention are shown.

[0019] Figure 4 The infrared spectrum of RCPS and its components in Example 4 of this invention is shown.

[0020] Figure 5 The experimental spectrum of RCPS and its component potassium iodide in Example 5 of the present invention is shown.

[0021] Figure 6 The spectrum of RCPS and its component Congo Red in Example 6 of this invention is shown.

[0022] Figure 7 The results of the in vitro antioxidant experiments of RCPS and its components in Example 7 of this invention; A: Total antioxidant capacity of RCPS and its components; B: Free radical scavenging capacity of ABTS; C: Free radical scavenging capacity of hydroxyl radicals.

[0023] Figure 8 This illustrates the inhibitory effect of RCPS-2 on melanin formation in zebrafish skin in Example 8 of the present invention. A: Schematic diagram of the inhibitory effect of RCPS-2 on melanin formation in zebrafish skin; B: Statistical chart of melanin area in zebrafish skin of each group.

[0024] Note: NC: Blank control group (fish tank water); PC: Positive control group (arbutin, 20 μg / mL); Groups I, II, and III: (RCPS-2 were 1, 10, and 20 μg / mL, respectively). Compared with NC, p<0.05, p<0.01, p<0.001.

[0025] Figure 9 In embodiment 9 (A) of the present invention dct, (B) oca2 and (C) silv Gene expression status; compared with NC, p<0.05 ， p<0.01 ， p<0.001.

[0026] Figure 10 This is an example of the promoting effect of RCPS-2 on the growth of zebrafish caudal fin skin tissue in Example 10 of the present invention. (A) Schematic diagram of RCPS-2 promoting the regeneration of damaged zebrafish caudal fin. (B) Experimental data on the promotion of zebrafish caudal fin skin tissue growth by RCPS-2; a. Regenerated caudal fin area (red dashed area); b. Regenerated caudal fin length (black dashed length). NC: Blank control (no tail amputation); T0: (tail amputation 0 h); MC: Model control (tail amputation without drug); Groups I, II, and III: (tail amputation + RCPS-2 intervention, RCPS-2 concentrations were 1, 10, and 20 μg / mL, respectively). Compared with NC, ##p<0.01; compared with MC, p<0.05, p<0.01, p<0.001.

[0027] Figure 11 This illustrates the intervention effect of RCPS-2 on the expression of collagen-related genes in Example 11 of this invention. A: col1a1a Relative mRNA level; B: col1a2 Relative mRNA levels; NC: blank control (fish tank water); Groups I, II, and III: (RCPS-2; RCPS-2 concentrations were 1, 10, and 20 μg / mL, respectively). Compared with NC, p<0.05, p<0.01, p<0.001.

[0028] Figure 12 This illustrates the anemia-improving activity of RCPS-2 in Example 12 of this invention. A: Schematic diagram of the intervention effect of RCPS-2 on zebrafish erythrocyte formation; B: Numerical values ​​of the intervention effect of RCPS-2 on zebrafish erythrocyte formation (erythrocyte IOD value). NC group (no drug added); MC group (0.1 μg / mL phenylhydrazine); Groups I, II, and III: (0.1 μg / mL phenylhydrazine + different concentrations of RCPS-2). Compared with NC, ## p<0.01; compared with MC, p<0.05, p<0.01, p<0.001. Detailed Implementation

[0029] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, 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.

[0030] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0031] In a typical specific embodiment of the present invention, rose waste sap polysaccharide is provided in all of the following (a)-(e): (a) Antioxidant or preparation of antioxidant products; (b) Whitening or preparing whitening products; (c) Promoting skin tissue growth or preparing products that promote skin tissue growth; (d) Promote the formation of collagen in the skin (anti-wrinkle and firming) or prepare products that promote the formation of collagen in the skin (anti-wrinkle and firming); (e) To replenish blood or to prepare blood-replenishing products (to improve complexion and anemia).

[0032] The products include, but are not limited to, food, pharmaceuticals, daily chemical products, and animal feed.

[0033] The rose waste liquid polysaccharide in this invention is prepared by extraction from rose waste liquid; and the rose waste liquid is the waste generated during the extraction of rose essential oil.

[0034] Through experimental research, this invention has discovered that the rose waste sap polysaccharide prepared by this invention possesses excellent total antioxidant capacity and ABTS and hydroxyl radical scavenging ability, thus exhibiting significant antioxidant effects. Simultaneously, it can inhibit pigment deposition by reducing tyrosinase activity, thereby suppressing melanin formation and achieving a whitening effect. Furthermore, it promotes skin tissue growth and collagen formation (anti-wrinkle and firming), and also has blood-tonifying effects (rosy complexion, improvement of anemia). Therefore, this invention demonstrates that the rose waste sap polysaccharide has broad market value in the development and application of food, pharmaceuticals, daily chemical products, and feed.

[0035] Furthermore, it should be noted that the rose varieties selected in this invention are not limited to Pingyin rose, Grasse rose, Damask rose, and Kushui rose.

[0036] In another specific embodiment of the present invention, a method for preparing rose waste liquid polysaccharide is provided, the method comprising extracting and preparing the rose waste liquid polysaccharide from rose waste liquid.

[0037] Specifically, the preparation method includes: S1. Add ethanol to the rose waste liquid, mix and let stand, centrifuge to collect the precipitate and dry to obtain solid RCPS. S2. The solid RCPS is reconstituted, the protein is removed, and then separated and purified by column chromatography to obtain the final product.

[0038] In step S1, The rose waste liquid is specifically the rose waste liquid generated during the extraction of rose essential oil. Further, 1-5 times (preferably 1.5 times, mass-volume ratio, g / mL) of water is added to the rose flowers, and the mixture is heated at 60-110 ℃ (preferably 100 ℃) for 1-5 h (preferably 3 h) to obtain rose cooking residue. The rose cooking residue is filtered, and the filtrate is the rose waste liquid.

[0039] The ethanol is high-concentration ethanol, and the ethanol can be 80% or higher concentration ethanol, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% ethanol. In one specific embodiment of the present invention, the ethanol is 95% ethanol.

[0040] The volume ratio of the rose waste liquid to ethanol is 1:1 to 5, such as 1:1, 1:2, 1:3, 1:4, 1:5. In one specific embodiment of the present invention, the volume ratio of the rose waste liquid to ethanol is 1:3.

[0041] The settling time is 1-36 hours, such as 3 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, and 36 hours. In one specific embodiment of the present invention, the settling time is 12 hours.

[0042] The specific centrifugation conditions are 8000-12000 r / min for 5-20 min, preferably 10000 r / min for 10 min.

[0043] The drying process can be carried out by oven drying or freeze drying. In one specific embodiment of the present invention, an oven drying process (e.g., 50-60°C) is selected.

[0044] In step S2, the specific method for resolution of the solids includes adding the solids to deionized water, wherein the mass-to-volume ratio of the solids to the deionized water is 1:10-100 (preferably 1:50, g / mL).

[0045] The method for removing proteins can be any method known in the art, such as the Sevag method, and is not specifically limited here.

[0046] The column chromatography was performed by sequential separation and purification using a DEAE-52 cellulose ion exchange column and a Sephadex G-100 dextran gel column. At this time, the rose polysaccharide was purified into three main components, which were named RCPS-1, RCPS-2 and RCPS-3, respectively.

[0047] Specifically, the method for separation and purification via column chromatography includes: Purification was performed using a DEAE-52 cellulose column, eluting sequentially with water and 0.1, 0.3, and 0.5 mol / L NaCl solutions at a flow rate of 0.5-2 mL / min (preferably 1 mL / min). For each elution concentration, 30 tubes of eluent were collected, each with a volume of 0.5-4 mL (preferably 2 mL). Tubes 6-26 (water eluent), tubes 35-50 (0.1 mol / L NaCl eluent), and tubes 65-80 (0.3 mol / L NaCl eluent) were collected. All eluents were lyophilized and then purified using a Sephadex G-100 dextran gel column, specifically eluting with water at a flow rate of 0.05-0.5 mL / min (preferably 0.1 mL / min) and a volume of 0.5-4 mL (preferably 2 mL / min) per tube. (mL), tubes 15-31, 11-41 and 11-31 were collected and freeze-dried to obtain RCPS-1, RCPS-2 and RCPS-3 respectively.

[0048] The inventors have demonstrated through experiments that the rose waste sap polysaccharide component, especially RCPS-2, obtained by the above method has excellent antioxidant, melanin inhibition, skin tissue growth promotion, skin collagen formation promotion, and anemia improvement effects.

[0049] In another specific embodiment of the present invention, a product is provided that has antioxidant properties, inhibits melanin formation, promotes skin tissue growth, promotes skin collagen formation, and / or improves anemia, the product comprising the above-mentioned rose waste sap polysaccharide.

[0050] The products include, but are not limited to, food, pharmaceuticals, daily chemical products, and animal feed.

[0051] It should be noted that the term "food" used in this invention should be interpreted broadly, and can be understood as any form that can be eaten. For example, food in this invention includes ordinary food and special food. The special food mentioned in this invention includes health food and food for special medical purposes. Ordinary food, in contrast to special food, is food suitable for everyone.

[0052] The food includes, but is not limited to, solid food and liquid food; the solid food includes, but is not limited to, baked goods, candy, and solid beverages; the liquid food includes, but is not limited to, liquid beverages.

[0053] The drug can be administered in unit doses, and the dosage form can be liquid or solid. Liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, emulsions, granules, suppositories, lyophilized powder for injection, inclusion complexes, implants, patches, and liniments.

[0054] The daily chemical products mentioned can be laundry detergents, personal hygiene cleaners, and cosmetics (including skin care products), such as toothpaste, mouthwash, laundry disinfectant, shampoo, hair lotion, hair gel, shower gel, soap, face mask, face cream, facial cleanser, etc.

[0055] The feed refers to the food for animals raised in agriculture or animal husbandry. The rose waste liquid polysaccharide of the present invention can be added as a feed additive to any type of feed, including but not limited to complete compound feed, concentrated feed and premixed feed.

[0056] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and not for limiting the scope of the invention. In the examples, rose waste liquid is obtained as follows: rose petals (Grass roses) are placed in a rose essential oil / rose water distillation apparatus, 1.5 times (g / mL) of deionized water is added, and the mixture is heated at 100 °C for 3 h to obtain rose cooking residue. The rose cooking residue is then filtered, and the filtrate is the rose waste liquid.

[0057] Example 1 Extraction and purification of polysaccharides from waste liquid Waste liquid from rose essential oil processing was mixed with 3 volumes of 95% ethanol and allowed to stand for 12 h. The mixture was centrifuged (10000 r / min, 10 min), and the precipitate was dried at 60℃ to obtain crude rose waste liquid polysaccharide (named RCPS). RCPS was dissolved in deionized water, and protein was removed using the Sevag method. Purification was performed using a DEAE-52 cellulose column (1.6 cm × 30 cm), eluting sequentially with distilled water, 0.1, 0.3, and 0.5 mol / L NaCl solutions at a flow rate of 1 mL / min. Thirty tubes were collected for each elution concentration, and the eluent (2 mL / tube) was collected using an automated fraction collector. Elution curves were plotted tube by tube using the phenol-sulfuric acid method, with the tube number as the x-axis and absorbance (A490 nm) as the y-axis.

[0058] Collect the eluents from distilled water (tubes 6 to 26), 0.1 mol / L NaCl solution (tubes 35 to 50), and 0.3 mol / L NaCl solution (tubes 65 to 80), and freeze-dry them. Dissolve 0.1 g of each polysaccharide fraction in 4 mL of deionized water, filter through a 0.45 μm filter, and purify using a Sephadex G-100 dextran gel column (1.6 cm × 50 cm). Elute with deionized water at a rate of 0.1 mL / min, collecting 2 mL from each tube. Analyze each tube using the phenol-sulfuric acid method, measuring absorbance at 490 nm and plotting elution curves. Collect tubes 15-31, 11-41, and 11-31, respectively, and freeze-dry to obtain RCPS-1, RCPS-2, and RCPS-3. Determine the polysaccharide content of each fraction using the phenol-sulfuric acid method.

[0059] Figure 1 As shown, the yields of the three components RCPS-1, RCPS-2, and RCPS-3, eluted with distilled water, 0.1 mol / L NaCl solution, and 0.3 mol / L NaCl solution, were 317.37 ± 15.87 mg / g, 289.65 ± 14.48 mg / g, and 131.74 ± 6.59 mg / g, respectively. According to the Sephadex G-100 elution curves, all three components exhibited a single peak.

[0060] Example 2 Determination of monosaccharide composition of RCPS and its components The monosaccharide composition of RCPS and its components was analyzed by ion chromatography. A clean chromatographic vial was used, and an appropriate amount of polysaccharide sample was weighed and added to 1 mL of 2M trifluoroacetic acid (TFA) solution. The sample was heated at 121 °C for 2 h. Nitrogen gas was then introduced, and the sample was dried. The sample was washed with 99.99% methanol, dried again, and this process was repeated three times. The sample was then dissolved in sterile water and transferred to a chromatographic vial for analysis. The chromatographic system used was a Thermo ICS5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA). A Dionex™ CarboPac™ PA20 (150 × 3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1 M NaOH, 0.2M NaAc), flow rate 0.5 mL / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V). Chromatographic data were processed using the software Chromeleon (version 7.3.2, USA).

[0061] The specific data is as follows: RCPS contains: arabinose (29.62%), glucose (24.65%), galactose (19.47%), rhamnose (6.94%), galacturonic acid (10.65%), mannose (3.72%), xylose (2.93%), glucuronic acid (1.2%), and fucose (0.83%).

[0062] RCPS-1 contains: glucose (74.13%), mannose (14.01%), galactose (5.78%), arabinose (3.74%), and xylose (2.34%).

[0063] RCPS-2 contains: arabinose (41.22%), glucose (22.36%), galactose (18.52%), and may contain trace amounts of: rhamnose (7.2%), galacturonic acid (6.14%), mannose (1.91%), xylose (1.43%), glucuronic acid (0.77%), and fucose (0.45%).

[0064] RCPS-3 contains: arabinose (42.61%), galactose (24.59%), rhamnose (13.47%), glucose (14.56%), fucose (1.57%), galacturonic acid (1.87%), and glucuronic acid (1.33%).

[0065] Example 3 Molecular weight determination of RCPS and its components The molecular weights of RCPS and its components were determined using HPSEC-MALLS. Chromatographic conditions: 18-angle laser light scattering high gel permeation chromatography system (GPC gel permeation chromatography unit pump, Waters 515), 18-angle laser light scattering instrument (Wyatt Dawn Heleos 11), differential refractive index detector (Optilab T-rex), and Shodex SBOHPAK-806-803 column. Mobile phase: ultrapure water (0.02% sodium azide, pH=6); column temperature: 40 ℃; flow rate: 1 mL / min; injection volume: 500 µL. The molecular weights of RCPS and its components were analyzed using ASTRA software.

[0066] like Figure 3 As shown, the Mw of RCPS is 1.22 × 10 5 The Mw of RCPS-1 is 5.72 × 10⁻⁶. 4 Da, the Mw of RCPS-2 is 5.06 × 10 4 The Mw of RCPS-3 is 2.09 × 10⁻⁶. 4 Da.

[0067] Example 4 FT-IR analysis of RCPS and its components The FT-IR spectra of the polysaccharide were obtained using Fourier transform infrared spectroscopy. The dried polysaccharide component (1 mg) was thoroughly mixed and ground with KBr powder (100 mg), pressed into transparent sheets using a tool, and then examined at 4000-400 cm⁻¹. -1 Scanning measurement within the range.

[0068] like Figure 4 As shown; at 3300-3500 cm - ¹The broad peaks in this region are usually associated with -OH stretching vibrations, indicating the presence of -OH groups in these polysaccharide molecules. 2900-3000 cm⁻¹ - The peak in region ¹ is typically associated with CH stretching vibrations. (1600-1700 cm⁻¹) - The peaks in region ¹, which are typically associated with C=O stretching vibrations, indicate the presence of carbonyl groups in these polysaccharide molecules. (1000-1300 cm⁻¹) -The peak in region ¹ is typically associated with CO stretching vibrations. (1400 cm⁻¹) - ¹The absorption peaks near the CH bond originate from the angle-shifting vibrations of the CH bond. 1200~1000 cm⁻¹ - ¹The strong absorption peaks in the region are formed by the superposition of sugar ring vibrations, COC glycosidic bond stretching vibrations, and C-OH side group vibrations, which are typical characteristics of polysaccharide skeletal structures. 1000 cm⁻¹ - The peaks below ¹ are typically associated with skeletal vibrations of polysaccharides. At 844 cm⁻¹ -1 The peaks on the left and right represent CH-coupling, indicating pyranoses linked by α-glycosidic bonds. (891 cm⁻¹) -1 The characteristic absorption peaks on the left and right indicate that the pyranose is linked by β-glycosidic bonds. Therefore, RCPS and RCPS-2 are mainly pyranose linked by α- and β-glycosidic bonds, RCPS-1 is mainly pyranose linked by α-glycosidic bonds, and RCPS-3 is mainly pyranose linked by β-glycosidic bonds.

[0069] Example 5 Determination of RCPS and its component branching structure The branched structure of polysaccharides was determined using the potassium iodide iodide assay. 2 mL of polysaccharide solution (RCPS and its components) was added to 1.2 mL of I2-KI solution (0.2 g / L I2 and 2 g / L KI), mixed well, and the UV absorption spectra of RCPS and its components in the range of 240–600 nm were measured.

[0070] I₂-KI can react with polysaccharides of different structures and exhibit different absorption peak characteristics in the UV-Vis spectral range. For example... Figure 5 As shown, the maximum absorption peaks appear at 288 nm and 350 nm, while there is no maximum absorption peak at 565 nm. This indicates that the sample has a complex chain structure with long side chains and many branches.

[0071] Example 6 Determination of the triple helical structure of RCPS and its components The triple helix structure of polysaccharides was determined using the Congo red method. Congo red solution (1 mL, 80 μmol / L) and polysaccharide sample solution (1 mL, 1 mg / mL) were mixed. Distilled water was used as a blank control instead of polysaccharide. Then, different concentrations of NaOH solution (1 mL) were added to achieve a final NaOH concentration ranging from 0 to 3.0 mol / L. After standing for 5 min, the sample was scanned at wavelengths between 480 and 520 nm with 1 nm intervals to determine the maximum absorption wavelength (λmax).

[0072] When polysaccharides possess a triple-helix structure, Congo red can intercalate into the triple-helix structure of the polysaccharide, forming a stable complex. Under alkaline conditions, the formation of this complex causes a redshift in the maximum absorption wavelength of Congo red. For example... Figure 6 As shown: In the concentration range of 0-3 mol / L NaOH solution, after RCPS and its components were mixed with Congo red reagent, the maximum absorption did not show a significant red shift as the NaOH solution concentration increased, indicating that RCPS and its components do not have a triple helix structure.

[0073] Example 7 Determination of total antioxidant capacity of RCPS and its components RCPS and its components were used to prepare stock solutions, which were then used to prepare a series of polysaccharide aqueous solutions with different concentrations (200, 400, 600, 800, 1000, 1500, 2000 mg / L). A polysaccharide sample solution (0.4 mL) was mixed with 2 mL of LP solution (4 mL, 4 mmol / L ammonium molybdate, 28 mmol / L trisodium phosphate, and 0.6 mol / L sulfuric acid), with vitamin C as the positive control. The mixture was incubated in a water bath (95°C, 90 min), and after cooling, the absorbance of the mixture was measured at a wavelength of 695 nm.

[0074] Determination of hydroxyl radical scavenging rate of RCPS and its components Take 1 mL of each grade of polysaccharide aqueous solution (200-2000 mg / L), and mix it with H2O2 solution (1 mL, 8.8 mmol / L), FeSO4 solution (1 mL, 9 mmol / L), and salicylic acid-ethanol solution (1 mL, 9 mmol / L). Incubate the mixture in a water bath (37°C, 30 min), centrifuge (6000 r / min, 10 min), and measure the absorbance (A) of the supernatant at 510 nm. Zero the sample using deionized water (A0). Vc is used as a positive control. Calculate the hydroxyl radical scavenging rate using the formula [(A0 - A) / A0] × 100%.

[0075] Determination of ABTS radical scavenging rate of RCPS and its components ABTS solution (7 mmol / L) and potassium persulfate solution (4.9 mmol / L) were mixed in equal volumes and incubated (for 20 h in the dark). The ABTS stock solution was then diluted with phosphate buffer (pH 7.4, 0.1 mol / L) to an absorbance of 0.7 ± 0.2 at 734 nm. ABTS working solution (3 mL) was mixed with polysaccharide sample solution (1 mL) and incubated in the dark for 6 min. The absorbance A was measured at 734 nm. A0 (deionized water + ABTS working solution), A1 (sample solution + phosphate buffer), and Vc was the positive control. The ABTS clearance rate was calculated using the formula [1 - (A - A1) / A0] × 100%.

[0076] Table 1. In vitro antioxidant capacity of RCPS and its components

[0077] Note: Total antioxidant activity is calculated based on absorbance at 2000 mg / L. Data labeled with the same letter in the same row showed no significant differences (P>0.05).

[0078] according to Figure 7 As shown in Table 1, the in vitro antioxidant activity of RCPS and its components is concentration-dependent. The total antioxidant activity assay showed that RCPS-2 had significantly higher total antioxidant activity than RCPS-3 and RCPS-1. The ABTS free radical scavenging assay showed that RCPS-2 exhibited the strongest activity (EC50 = 66.61 ± 3.33 mg / L), an increase of approximately 14.8% compared to RCPS (77.93 ± 3.90 mg / L), and a significant difference from RCPS-1 (670.44 ± 33.52 mg / L), demonstrating an activity advantage of more than 10 times. In the hydroxyl radical scavenging assay, RCPS-2 also performed best, with the activity intensity ranking as RCPS-2 > RCPS-3 > RCPS > RCPS-1.

[0079] Example 8 Inhibitory effect of rose polysaccharides on melanin formation in the skin Healthy fertilized embryos at 21 hpf (hours post-fertilization) were used as experimental animals to study the inhibitory effect of RCPS-2 on melanin production. The egg membrane was removed with a 1.0 mg / mL streptomycin solution, and the embryos were transferred to 6-well plates, divided into 5 groups of 15 embryos per well. The blank control group (NC) consisted of fish tank water; the positive control group (PC) consisted of fish tank water containing 20 mg / mL arbutin; and the low, medium, and high concentration RCPS-2 intervention groups (Ⅰ, Ⅱ, Ⅲ) consisted of fish tank water containing 1, 10, and 20 μg / mL RCPS-2, respectively. All embryos were incubated at 28 ℃ for 24 h. Melanin production was observed and photographed under a fluorescence microscope (Zeiss, AXIO-V16, Germany), and the melanin area was calculated using ImageJ software (1.0, USA).

[0080] Based on the melanin display diagram and area statistical bar chart of zebrafish, the melanin area in the RCPS-2 intervention groups (I, II, and III) decreased in a gradient with increasing concentration, by 14.83%, 12.05%, and 9.58%, respectively, indicating a positive correlation between the whitening effect and concentration. Group III (20 μg / mL) showed the most significant effect, with a melanin area reduction of 40.8% compared to the NC group (p<0.01), suggesting that high concentrations of RCPS-2 can significantly inhibit melanin synthesis. Groups II (10 μg / mL) and I (1 μg / mL) showed reductions of 25.5% and 8.3% compared to the NC group, respectively, with statistically significant differences. Therefore, rose polysaccharide RCPS-2 has whitening effects.

[0081] Example 9 Rose polysaccharides regulate the expression of genes related to skin melanin formation. Zebrafish embryos at 2 hours post-fertilization (hpf) were placed in six-well plates and divided into 5 groups of 35 embryos each. The RCPS-2 intervention groups (I, II, and III) were treated with different concentrations of RCPS-2 (1 μg / mL, 10 μg / mL, and 20 μg / mL), respectively. The positive control group (PC) received arbutin at 20 mg / mL in its culture water; the blank control group (NC) received no added drugs. After 72 h of culture, samples from each group were collected in EP tubes, 500 μL of lysis buffer was added, and the mixture was mechanically homogenized for 2 min in a tissue homogenizer. After centrifugation at 11000 r / min for 5 min, the supernatant was aspirated and added to a cleansing column, centrifuged at 11000 r / min for 2 min. 350 μL of the liquid was aspirated and added to an equal volume of 70% ethanol solution, quickly mixed, and then transferred to an adsorption column. The mixture was centrifuged again for 1 min at 11000 r / min. Add 700 μL of protein removal buffer, incubate at room temperature for 1 min, centrifuge at high speed for 1 min, and then rinse twice with 500 μL of washing buffer. Finally, place the adsorption column into an empty centrifuge tube (RNase-free) and add 40 μL of pure water (RNase-free water). Incubate at room temperature for 5 min, centrifuge at high speed for 1 min, and immediately place the extracted RNA on an ice box. Determine the RNA concentration using a micro spectrophotometer, strictly controlling the A260 / A280 ratio between 2.0 and 2.5.

[0082] Immediately use a C1000Touch gradient PCR instrument to reverse RNA into cDNA, strictly following the instructions of the reverse transcription kit (gDNA Purge). The concentration of cDNA in each group was measured using an ultra-micro spectrophotometer. The reverse-transcribed cDNA was diluted with double-distilled water to a concentration of 100 ng / μL, immediately aliquoted, and stored at -20°C. The reverse-transcribed cDNA from each group was used as a template for... rpl13a As the internal reference gene, the test sample and primers were added to 100 μL eight-tube EP tubes, and the gene expression changes in each group were detected in real time using a Roche real-time quantitative PCR instrument. After amplification, the expression of each group and the internal reference gene were output. rpl13a The target gene CT (Cycle Threshold) value was calculated based on the 2-ΔΔCT relative quantification method to determine the relative expression level of the gene in different groups.

[0083] PCR amplification program: initial step 95 ℃ for 30 s, followed by 40 cycles, each cycle 95 ℃ for 10 s and 58 ℃ for 30 s. qRT-PCR results were analyzed using 2-... Ct The methods were analyzed, and the gene primer sequences are shown in Table 2: Table 2 Primers used for qRT-PCR

[0084] dct It is a key enzyme controlling the melanin synthesis pathway and belongs to the tyrosinase-associated protein family (TRP-2). It catalyzes the conversion of the melanin precursor dopachrome into 5,6-dihydroxyindole-2-carboxylic acid (DHICA), affecting the type and stability of melanin.

[0085] oca2 It encodes a transmembrane transport protein (P protein) that is primarily involved in the transport of tyrosine residues and pH regulation within the melanosome. It influences melanin synthesis efficiency by maintaining the slightly acidic environment of the melanosome and ensuring the activity of tyrosinase (TYR). oca2 Loss of function (such as albinism caused by mutation) prevents tyrosinase from activating properly, hindering melanin synthesis and resulting in reduced melanin in the skin, hair, and eyes (manifested as the albinism phenotype).

[0086] silv The PMEL17 protein is a major component of the amyloid fibrils within melanosomes. These fibrils provide a scaffold structure for melanin deposition, aiding in the assembly and storage of melanin granules. During melanosome maturation, the PMEL17 protein is cleaved into fragments and self-assembles into fibrous matrix. Melanin precursors (such as dopachrome and DHICA) attach to these fibrils and gradually deposit to form mature melanin granules. silv Defects in this area can lead to abnormal melanin granule structure, affecting pigmentation in the skin and eyes.

[0087] like Figure 9 As shown, RCPS-2 pairs dct, oca2 and silv The inhibition of gene expression was concentration-dependent. Compared with the NC group, the RCPS-2 intervention groups (I, II, and III) showed significantly lower levels of inhibition. dct, oca2 and silv Relative mRNA expression levels were significantly reduced in all groups (P<0.05). High-dose RCPS-2 intervention group (III) dct, oca2 and silv The relative mRNA expression levels were 0.59, 0.41, and 0.19, respectively, representing decreases of 41%, 59%, and 81% compared to the NC group. Furthermore, the positive control group (PC) showed... dct, oca2 and silv The relative mRNA expression levels were 0.64, 0.52, and 0.25, respectively. Therefore, RCPS-2 has a good melanin-inhibiting effect, which inhibits pigment deposition by blocking melanosome acidification (reducing tyrosinase activity).

[0088] Example 10 The promoting effect of rose polysaccharides on skin tissue growth Wild-type AB zebrafish in good condition and fertilized for 72 hours post-fertilization were placed in 6-well plates, 15 fish per well. A blank control group (NC), a model group (MC), and low, medium, and high-dose RCPS-2 intervention groups were set up (Ⅰ: 1 μg / mL; Ⅱ: 10 μg / mL and Ⅲ: 20 μg / mL). The blank control group received no treatment, while the caudal fins of the model control group and RCPS-2 intervention groups were cut off using the same method. The zebrafish were incubated in the dark under the experimental conditions of each group (28℃, 48 h). Images were taken under a visible light microscope, and the caudal fin length and area were measured and recorded using ImageJ (1.0, USA) image processing software.

[0089] The zebrafish tail fin is mainly composed of a fin membrane and fin rays, containing collagen, elastin, and other components of the extracellular matrix. These components are also present in human skin; for example, collagen is one of the main structural proteins of the skin. Zebrafish tail fins have an extremely strong regenerative capacity. During regeneration, epithelial cells cover the wound and secrete signaling molecules (such as FGF and Wnt), interacting with the underlying mesenchymal cells. This mechanism is similar to the epidermal-dermal interaction in human skin wound healing. By observing the regenerative capacity of zebrafish tail fins under drug intervention through tail amputation experiments, the effects of drugs on skin tissue growth can be reflected, and the regeneration status of zebrafish tail fins can also indirectly characterize the amount of collagen produced.

[0090] Depend on Figure 10 (B) indicates that after 48 hours, the average caudal fin area of ​​the MC group was 1.17 × 10⁻⁶. 4 The area of ​​the NC group (3.17 × 10 μm², length 43.8 μm) was significantly smaller than that of the NC group (area 3.17 × 10 μm², length 43.8 μm). 4 The fin area (μm²) and length (141.41 μm) decreased by 63.09% and 69.01% respectively (p<0.01), indicating limited natural repair capacity after injury. The polysaccharide experimental group showed a concentration-dependent effect; the high-dose RCPS-2 intervention group (tail fin area 1.79×10 μm², length 141.41 μm) showed a 63.09% and 69.01% decrease respectively (p<0.01), indicating limited natural repair capacity after injury. 4 The lesion area (70.8 μm², 70.8 μm in length) showed a 52.99% (p<0.05) and 61.64% (p<0.001) increase in size compared to the MC group, respectively, significantly accelerating the closure of the damaged area. The difference was statistically significant. Therefore, RCPS-2 promotes skin tissue growth.

[0091] Example 11 Regulation of collagen-related gene expression 96 hpf zebrafish were cultured in six-well plates, 35 fish per well. Different concentrations of RCPS-2 (1 μg / mL, 10 μg / mL, and 20 μg / mL) were added to the water in the low, medium, and high dose RCPS-2 intervention groups (I, II, and III), respectively. A blank control group (NC) was also set up, with no drug added to the NC group. Zebrafish in each group were incubated for 24 h at 28℃ under the experimental conditions, and samples were collected. RNA extraction, cDNA synthesis, and qRT-PCR amplification were performed. rpl13a As a steward's gene, testing col1a2、cola1a Gene expression levels. Amplification and result processing conditions are the same as in Example 7.

[0092] Table 3 Primers used for qRT-PCR

[0093] Collagen, a structural protein in the skin, contains a large number of hydrophilic moisturizing factors, providing mechanical strength and structural support for tissues such as skin, bones, and tendons. It maintains skin firmness and elasticity, and promotes skin reconstruction and repair. Type I collagen is a trimer, mainly composed of two α1 chains and one α2 chain, which are... col1a1a and col1a2 Genetic encoding. Through detection... col1a1a or col1a2 The relative expression level of genes can indicate the effect of a sample on collagen production.

[0094] Depend on Figure 11 It can be seen that the zebrafish in the RCPS-2 intervention groups (I, II, and III) col1a1a and col1a2 Relative mRNA levels gradually increased with increasing RCPS-2 intervention dose. The high-dose RCPS-2 intervention group (III) showed a significantly higher level than the NC group (p<0.05). col1a2 and col1a1a The relative mRNA levels were 1.93 and 1.83, respectively, representing increases of 93% and 83% compared to the NC group (p<0.05). Therefore, RCPS-2 can upregulate collagen-related genes. col1a1a and col1a2 The expression of [something] promotes the formation of collagen in the skin.

[0095] Example 12 Evaluation of activity in improving anemia Healthy wild-type AB zebrafish reaching 56 hpf (hour post-fertilization) were placed in six-well plates and divided into 5 groups of 10 fish each. The blank control group (NC) was given culture water; the anemia model group (MC) was supplemented with 0.1 μg / mL phenylhydrazine; and the low, medium, and high-dose RCPS-2 intervention groups (I, II, and III) were supplemented with 0.1 μg / mL phenylhydrazine and different concentrations of RCPS-2 (1 μg / mL, 10 μg / mL, and 20 μg / mL). The zebrafish in each group were incubated for 3 dpf (day post-fertilization). Samples were treated with an anesthetic (cocaine) for 1 min, and embryos were stained with o-anisidine solution in the dark for 20 min. Images were acquired under a microscope. The optical density (IOD) of the stained areas was quantified using ImageJ (1.0, USA) software.

[0096] The hemoglobin in mature red blood cells can catalyze the oxidation of o-anisidine by hydrogen peroxide, resulting in a rust-colored (reddish) appearance. Benzene traps can destroy red blood cells, creating an anemia model. In anemic juvenile fish (with a reduced number of mature red blood cells), the average optical density value of red blood cell staining at the heart region will decrease.

[0097] Depend on Figure 12 It was found that after model induction with 0.1 μg / mL phenylhydrazine, the IOD value of the heart in the MC group of zebrafish larvae was significantly lower than that in the NC group (p<0.01). Phenylhydrazine effectively inhibited erythropoiesis, indicating that the zebrafish anemia model was successfully established. Under low, medium, and high doses of RCPS-2 intervention, the IOD value of the heart in zebrafish larvae gradually increased, showing a clear dose-response relationship. The IOD values ​​of zebrafish in groups I, II, and III were 0.77 × 10⁻⁶. 6 1.14×10 6 and 1.46×10 6 These values ​​represent increases of 51.26%, 123.71%, and 185.45% respectively compared to the MC group (IOD value). Furthermore, the IOD value for juvenile zebrafish in the NC group was 1.46 × 10⁻⁶. 6 There was no significant difference between the group treated with the high-dose RCPS-2 intervention (20 μg / mL). Therefore, rose polysaccharide (RCPS-2) showed a significant hematopoietic effect in the zebrafish anemia model, significantly restoring red blood cell levels in anemic states, demonstrating the potential of RCPS-2 as a natural hematopoietic agent.

[0098] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Applications of rose waste sap polysaccharides in the following (a)-(e): (a) To prepare antioxidant products, or, (b) To prepare skin whitening products, or, (c) Prepare products that promote skin tissue growth, or, (d) Prepare products that promote the formation of collagen in the skin, or, (e) Preparation of blood-tonifying products; The method for preparing the rose waste sap polysaccharide includes extracting and preparing the rose waste sap polysaccharide from rose waste sap; The preparation method includes: S1. Add ethanol to the rose waste liquid, mix and let stand, centrifuge to collect the precipitate and dry to obtain solid RCPS. The ethanol is ethanol with a concentration of 80% or higher; S2. The solid RCPS is reconstituted, deproteinized, and then purified by column chromatography to obtain the final product; the specific method for reconstituted the solid includes adding the solid to deionized water; The column chromatography was performed by sequential separation and purification using a DEAE-52 cellulose ion exchange column and a Sephadex G-100 dextran gel column. The separation and purification method by column chromatography includes: purification using a DEAE-52 cellulose column, eluting sequentially with distilled water, 0.1, 0.3, and 0.5 mol / L NaCl solutions at a flow rate of 0.5-2 mL / min, collecting 30 tubes of eluent for each elution concentration, with each tube containing 0.5-4 mL; collecting tubes 35 to 50 of the elution fraction eluted with 0.1 mol / L NaCl solution; lyophilizing the eluent fraction and then purifying it using a Sephadex G-100 dextran gel column, specifically eluting with water at a flow rate of 0.05-0.5 mL / min, with each tube containing 0.5-4 mL; collecting tubes 11-41; and lyophilizing to obtain RCPS-2. The RCPS-2 has a molecular weight of 5.06 × 10⁻⁶. 4 Da contains 41.22% arabinose, 22.36% glucose, and 18.52% galactose; The rose waste liquid is specifically the rose waste liquid generated during the extraction of rose essential oil. Add 1-5 times the mass-volume ratio of water (g / mL) to the rose petals and heat at 60-110 ℃ for 1-5 h to obtain rose cooking residue. Filter the rose cooking residue, and the filtrate is the rose waste liquid.

2. The application as described in claim 1, characterized in that, The product that promotes the formation of collagen in the skin is used to achieve anti-wrinkle and firming effects.

3. The application as described in claim 1, characterized in that, The blood-enriching products specifically improve complexion and reduce anemia.

4. The application as described in claim 1, characterized in that, The products include food, pharmaceuticals, daily chemical products, and animal feed.

5. The application as described in claim 1, characterized in that, In the preparation of the rose waste liquid, 1.5 times the mass-volume ratio of water (g / mL) is added to the rose petals, and the mixture is heated at 100°C for 3 hours.

6. The application as described in claim 1, characterized in that, In step S1 The volume ratio of the rose waste liquid to ethanol is 1:1~5; The settling time is 1-36 hours; The specific centrifugation conditions were 8000-12000 r / min for 5-20 min. The drying process employs either oven drying or freeze drying.

7. The application as described in claim 1, characterized in that, In step S2, the mass-to-volume ratio of the solid to the deionized water is 1:10-100, g / mL; The protein removal method is the Sevag method.

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