Polygonum multiflorum composition and application thereof in anti-aging

Abstract

The invention relates to a fleece-flower root composition, which is prepared from homologous fleece-flower root raw materials through a composite extraction process, and comprises two active components including stilbene glucoside and polysaccharide, the content of the stilbene glucoside is 40-60% (w / w), and the content of the polysaccharide is 30-60%. The composite extraction process comprises the steps of graded alcohol extraction, low-temperature concentration, ethanol washing sedimentation and further purification. The composition has the anti-aging effects of inhibiting MMP-1 and TNF-alpha and the like, and is particularly suitable for preventing and treating skin photoaging caused by ultraviolet rays.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product extraction, and particularly relates to a Polygonum multiflorum synergistic composition based on a composite extraction process, a preparation method thereof, and an application thereof in anti-aging cosmetics. Background Art

[0002] Skin aging is a complex physiological process affected by multiple factors. Among them, ultraviolet (UV) radiation is the main cause of skin photoaging, accounting for about 80% of the causes of skin aging. Ultraviolet irradiation can stimulate the generation of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) in the body, and these molecules are closely related to skin cancer and accelerated aging.

[0003] At present, anti-aging products on the market, such as skin care products and vitamin drugs, although widely used, often face problems such as unstable efficacy, high cost, and potential side effects. Therefore, developing natural, effective, and safe anti-aging products has become an important research goal in the daily chemical field.

[0004] Polygonum multiflorum, as a traditional herb, is famous for its rich bioactive components such as stilbene glycosides, anthraquinones, polymeric procyanidins, and polysaccharides, and has multiple pharmacological effects such as anti-aging, liver protection, promoting blood cell regeneration, and cardiovascular health. In recent years, the application of Polygonum multiflorum in skin anti-aging has gradually attracted attention, but there is still a lack of systematic research at present. In particular, how to effectively extract and retain the synergistic effect of its bioactive components remains a technical problem.

[0005] Traditional extraction processes of Polygonum multiflorum usually use single-solvent extraction, such as water extraction or high-concentration ethanol extraction. However, these methods have significant defects, resulting in an imbalance in the proportion of extracted active ingredients. For example, water extraction mainly extracts polysaccharides, but the recovery rate of alcohol-soluble stilbene glycosides is extremely low; when high-concentration ethanol is used to extract stilbene glycosides, the extraction rate of polysaccharides drops sharply. In addition, when physically mixing stilbene glycosides and polysaccharides, it is difficult to achieve the efficacy and safety achieved after natural extraction, probably because certain trace substances are retained during the natural extraction process or the co-solvent system is more conducive to absorption and utilization.

[0006] In view of the above problems, it is urgent to develop a new Polygonum multiflorum composition using a new extraction process, which can form a suitable ratio of stilbene glycosides and polysaccharides, thereby effectively exerting their synergistic effect, and can form a stable complex with good solubility, in order to achieve a breakthrough in the field of skin anti-aging. The realization of this technology will provide a more effective natural ingredient basis for the research and development of anti-aging products and may significantly improve the effect of anti-aging treatment. Summary of the Invention

[0007] The object of the present invention is to provide a polygonum multiflorum composition, which is prepared from polygonum multiflorum raw materials by a composite extraction process and contains the following components: a) stilbene glucoside; b) polysaccharide content.

[0008] Furthermore, the polygonum multiflorum composition of the present invention is prepared from polygonum multiflorum raw materials by a composite extraction process and contains the following components:

[0009] a) The content of stilbene glucoside is 40-60% (w / w);

[0010] b) The polysaccharide content is 30-60%;

[0011] The mass ratio of the stilbene glucoside to the polysaccharide is (0.7-2.0):1, preferably 1:1.

[0012] Furthermore, the content of the stilbene glucoside is 45-55% (w / w).

[0013] Furthermore, the polysaccharide content is 40-55%.

[0014] In some embodiments, the composite extraction process includes the following steps:

[0015] a) Subject the polygonum multiflorum raw materials to fractional ethanol extraction and collect the 30-50% ethanol extract;

[0016] b) Adjust the pH of the extract to 5.0-6.0 and perform low-temperature concentration;

[0017] c) Add ethanol to the concentrated solution, sediment, filter, and separately collect the sediment containing polysaccharides and the mother liquor containing stilbene glucoside;

[0018] d) After the sediment and the mother liquor are further separated and purified respectively, then mixed, add an appropriate amount of deionized water, heat to 40-45°C, fully dissolve, add activated carbon (dosage 1%, w / v), decolorize at 40-45°C for 30 minutes, filter, concentrate, and dry.

[0019] The object of the present invention also lies in providing a preparation method of the polygonum multiflorum composition, which is characterized by including the following steps:

[0020] a) Subject the polygonum multiflorum raw materials to fractional ethanol extraction and collect the 30-50% ethanol extract;

[0021] b) Adjust the pH of the extract to 5.0-6.0 and perform low-temperature concentration;

[0022] c) Add ethanol to the concentrated solution, sediment, filter, and separately collect the sediment containing polysaccharides and the mother liquor containing stilbene glucoside;

[0023] d) After the sediment and the mother liquor are further separated and purified respectively, they are mixed, an appropriate amount of deionized water is added, heated to 40 - 45 °C, fully dissolved, activated carbon (dosage 1%, w / v) is added, decolorized at 40 - 45 °C for 30 minutes, filtered, concentrated, and dried.

[0024] Further, in step d) of the preparation method, the sediment is subjected to secondary alcohol precipitation to further remove impurities and obtain a high-purity polysaccharide component.

[0025] Further, in step d) of the preparation method, the mother liquor is separated by a macroporous resin column to obtain a high-purity stilbene glucoside component.

[0026] Further, the polysaccharide component and the stilbene glucoside component are mixed at a mass ratio of (0.7 - 2.0):1, preferably 1:1, an appropriate amount of deionized water is added, heated to 40 - 45 °C, fully dissolved, activated carbon (dosage 1%, w / v) is added, decolorized at 40 - 45 °C for 30 minutes, filtered, concentrated, and dried.

[0027] Another object of the present invention is to provide an application of a polygonum multiflorum thunb composition in the preparation of anti-aging cosmetics.

[0028] Further, the polygonum multiflorum thunb composition has at least one of the following effects:

[0029] a) Promote an increase in the synthesis amount of collagen;

[0030] b) Inhibit the generation of MMP-1;

[0031] c) Inhibit the secretion of inflammatory factors TNF-α and IL-β by cells;

[0032] d) The antioxidant ability to scavenge DPPH free radicals.

[0033] Further, the aging is skin photoaging caused by ultraviolet rays.

[0034] The object of the present invention also lies in providing an anti-aging cosmetic, which contains the polygonum multiflorum thunb composition prepared according to the preparation method of the present invention. Description of the Drawings

[0035] Figure 1 It is a comparison chart of cell viability data of the stilbene glucoside group of polygonum multiflorum thunb, the polysaccharide group of polygonum multiflorum thunb, and the polygonum multiflorum thunb composition group 1 and the polygonum multiflorum thunb composition group 2.

[0036] Figure 2 It is a comparison chart of the data on the influence of the stilbene glucoside group of polygonum multiflorum thunb, the polysaccharide group of polygonum multiflorum thunb, and the polygonum multiflorum thunb composition group 1 and the polygonum multiflorum thunb composition group 2 on the TNF-α mRNA expression (x±s, n = 3).

[0037] Figure 3It is a data comparison chart (x±s, n = 3) of the effects of the stilbene glucoside group of Polygonum multiflorum, the polysaccharide group of Polygonum multiflorum, Polygonum multiflorum composition group 1, and Polygonum multiflorum composition group 2 on the expression of IL-1β mRNA.

[0038] Figure 4 It is a data comparison chart (mean, n = 3) of the DPPH free radical scavenging rates of the stilbene glucoside group of Polygonum multiflorum, the polysaccharide group of Polygonum multiflorum, Polygonum multiflorum composition group 1, and Polygonum multiflorum composition group 2.

[0039] Figure 5 It is a data comparison chart (x±s, n = 3) of the effects of the stilbene glucoside group of Polygonum multiflorum, the polysaccharide group of Polygonum multiflorum, Polygonum multiflorum composition group 1, and Polygonum multiflorum composition group 2 on the expression of MMP-1.

[0040] Figure 6 It is a data comparison chart (x±s, n = 3) of the effects of the stilbene glucoside group of Polygonum multiflorum, the polysaccharide group of Polygonum multiflorum, Polygonum multiflorum composition group 1, and Polygonum multiflorum composition group 2 on the total collagen content (μg / mL). Detailed implementation manners

[0041] The present invention is further described in conjunction with specific embodiments, but does not limit the scope of the present invention.

[0042] Unless otherwise specified, the methods used in the following embodiments are all conventional methods. The materials or reagents required in the following embodiments are all commercially available unless otherwise specified.

[0043] Example 1 Preparation of Polygonum multiflorum composition 1

[0044] 1. Raw material pretreatment

[0045] Take the dried tuberous roots of the homologous cultivated Polygonum multiflorum Thunb. of the Polygonaceae family, screen and remove impurities, and then crush them into 20-40 mesh particles.

[0046] 2. Gradient alcohol extraction

[0047] (1) First extraction: Take 1 kg of the treated raw materials, add 8 times the amount of 50% ethanol solution (v / v), reflux and extract at 50±5 °C for 2 hours, filter, and collect the filtrate;

[0048] (2) Second extraction: Add 6 times the amount of 30% ethanol solution to the residue, reflux and extract at 50±5 °C for 1.5 hours, filter and collect the filtrate;

[0049] (3) Combine the two filtrates, filter, and obtain about 10 L of light brown clear extract.

[0050] 3. pH adjustment and concentration

[0051] Slowly add 0.1 mol / L hydrochloric acid solution to the extract, stirring while adding, and adjust the pH to 5.0 ± 0.2. Concentrate it to a relative density of 1.12 - 1.15 (measured at 25°C) under the conditions of a vacuum degree of -0.08 MPa and a temperature of 50 ± 2°C using a rotary evaporator to obtain about 2.5 L of concentrated solution.

[0052] 4. Alcohol precipitation separation

[0053] (1) Slowly add anhydrous ethanol to the concentrated solution to make the ethanol concentration in the system reach 75%, maintain a constant temperature water bath at 40 ± 2°C, and stir at 300 rpm for 30 minutes.

[0054] (2) Let it stand for sedimentation for 12 hours, filter, and collect respectively:

[0055] The sediment containing polysaccharides (about 120 g wet weight)

[0056] The mother liquor containing stilbene glycoside (about 1.8 L)

[0057] 5. Separation and purification

[0058] (1) Treatment of polysaccharide component:

[0059] The sediment is redissolved with 10 times the amount of deionized water, activated carbon is added (dosage 1%, w / v), decolorized at 50°C for 30 minutes, and after centrifugation (4000 rpm, 15 min), the supernatant is treated with an ultrafiltration membrane with a molecular weight cut-off of 50 kDa. Collect the permeate, concentrate it to a solid content of about 20%, add anhydrous ethanol to a final concentration of 85%, let it stand at 4°C for 12 hours, centrifuge to collect the precipitate, and dry to obtain the polysaccharide component (the polysaccharide content is about 90% detected by UV method, calculated on a dry basis).

[0060] (2) Treatment of stilbene glycoside component:

[0061] The mother liquor is adsorbed by an AB-8 macroporous resin column (column volume 2 L, diameter-height ratio 1:8). First, wash with 3 BV of water to remove impurities, and then elute the target component with 4 BV of 60% ethanol at a flow rate of 2 BV / h. Collect the eluate, concentrate it, and dry it to obtain the stilbene glycoside component (the stilbene glycoside content is about 90% detected by HPLC, calculated on a dry basis).

[0062] 6. Complex preparation

[0063] Mix the polysaccharide component and the stilbene glycoside component in a ratio of 1:1.3 (w / w), add an appropriate amount of deionized water, heat to 45°C, stir until fully dissolved, add activated carbon (dosage 1%, w / v), decolorize at 45°C for 30 minutes, filter, concentrate, and dry to obtain a light yellow composite extract powder.

[0064] 7. Detection indexes:

[0065] Polysaccharide content: 41.2% (UV method, calculated on dry basis)

[0066] Stilbene glycoside content: 51.3% (HPLC method, calculated on dry basis)

[0067] Loss on drying: ≤8.0% (dried to constant weight at 105°C)

[0068] Preparation of Polygonum multiflorum Thunbergii composition 2 in Example 2

[0069] 1. Raw material pretreatment

[0070] Take the dried tuberous roots of the homologous cultivated Polygonum multiflorum Thunb. in the Polygonaceae family. After screening to remove impurities, crush them into 20 - 40 mesh particles.

[0071] 2. Gradient ethanol extraction

[0072] (1) First extraction: Take 1 kg of the treated raw material, add 8 times the amount of 50% ethanol solution (v / v), reflux and extract at 55 ± 5°C for 2 hours, filter and collect the filtrate;

[0073] (2) Second extraction: Add 6 times the amount of 30% ethanol solution to the residue, perform ultrasonic - assisted extraction (power 300 W, frequency 40 kHz) at 60 ± 5°C for 1.5 hours, filter and collect the filtrate;

[0074] (3) Combine the two filtrates, filter to obtain about 9.5 L of a light - brown clear extract.

[0075] 3. pH adjustment and concentration

[0076] Slowly add 0.1 mol / L hydrochloric acid solution to the extract while stirring, adjust the pH to 6.0 ± 0.2. Concentrate it to a relative density of 1.12 - 1.15 (measured at 25°C) under the conditions of a vacuum degree of - 0.08 MPa and a temperature of 50 ± 2°C using a rotary evaporator to obtain about 2.5 L of concentrated solution.

[0077] 4. Alcohol precipitation and separation

[0078] (1) Slowly add anhydrous ethanol to the concentrated solution to make the ethanol concentration in the system reach 75%, maintain a constant - temperature water bath at 40 ± 2°C, and stir at 300 rpm for 30 minutes

[0079] (2) Let it stand for sedimentation for 12 hours, filter, and collect respectively:

[0080] The sediment containing polysaccharides (about 150 g wet weight)

[0081] The mother liquor containing stilbene glycoside (about 2.0 L)

[0082] 5. Separation and purification

[0083] (1) Treatment of polysaccharide component:

[0084] The sediment was redissolved with 15 times the amount of deionized water, activated carbon was added (dosage 1%, w / v), and it was decolorized at 60 °C for 30 minutes. After centrifugation (4000 rpm, 15 min), the supernatant was treated with an ultrafiltration membrane with a molecular weight cut-off of 20 kDa. The permeate was collected, concentrated to a solid content of about 25%, absolute ethanol was added to a final concentration of 85%, and it was allowed to stand at 4 °C for 12 hours. The precipitate was collected by centrifugation. Absolute ethanol was added to the precipitate to a final concentration of 85%, and it was allowed to stand at 4 °C for 12 hours. The precipitate was collected by centrifugation and dried to obtain the polysaccharide component (the polysaccharide content was detected by the UV method to be about 95%, calculated on a dry basis).

[0085] (2) Treatment of stilbene glucoside component:

[0086] The mother liquor was adsorbed on a D101 macroporous resin column (column volume 2 L, diameter-height ratio 1:8). First, it was eluted with 4 BV of water to remove impurities, and then the target component was eluted with 6 BV of 60% ethanol at a flow rate of 2 BV / h. The eluate was collected, concentrated, and dried to obtain the stilbene glucoside component (the stilbene glucoside content was detected by HPLC to be about 95%, calculated on a dry basis).

[0087] 6. Preparation of the complex

[0088] The polysaccharide component and the stilbene glucoside component were mixed in a ratio of 1:1 (w / w), an appropriate amount of deionized water was added, and it was heated to 45 °C and stirred until fully dissolved. Activated carbon was added (dosage 1%, w / v), and it was decolorized at 45 °C for 30 minutes. It was filtered, concentrated, and dried to obtain a light yellow composite extract powder.

[0089] 7. Detection indexes:

[0090] Polysaccharide content: 48.8% (UV method, calculated on a dry basis)

[0091] Stilbene glucoside content: 50.1% (HPLC method, calculated on a dry basis)

[0092] Loss on drying: ≤8.0% (dried to constant weight at 105 °C)

[0093] Example 3 Preparation of Polygonum multiflorum Thunb. polysaccharide extract (polysaccharide content 98%)

[0094] The polysaccharide component (about 95% purity) obtained in Example 2 was redissolved with 5 times the amount of deionized water, heated to 45 °C, stirred for 2 hours, filtered, concentrated to 1 / 3 of the original volume, absolute ethanol was added, the ethanol concentration was adjusted to 90%, and it was stirred at room temperature for 30 minutes to precipitate the polysaccharide.

[0095] After static sedimentation for 24 hours, the precipitate was collected by centrifugation, washed again with a small amount of absolute ethanol to remove impurities, and finally the precipitate was dried to obtain a polysaccharide extract with a purity of about 98%.

[0096] Example 4 Preparation of Polygonum multiflorum Stilbene Glucoside Extract (stilbene glucoside content 98%)

[0097] The stilbene glucoside-enriched fraction (about 95% purity) obtained in Example 2 was collected, dissolved in an appropriate amount of deionized water, the concentration was adjusted to 30 g / L, heated to 45 °C and stirred until completely dissolved, allowed to stand at 4 °C for 48 hours, the precipitate was collected by centrifugation, and dried to obtain a pale yellow powder with a stilbene glucoside content of about 98% (HPLC method).

[0098] Example 5 Determination of Anti-inflammatory Efficacy

[0099] 1 Test Purpose and Principle

[0100] LPS (lipopolysaccharide, LPS) is a complex of lipid and polysaccharide, mainly extracted from the cell wall of Gram-negative bacteria, and has strong immunogenicity. LPS can stimulate skin keratinocytes to secrete pro-inflammatory cytokines (IL-1β, TNF-α, etc.). In this test, the expression levels of TNF-α and IL-1β were used as evaluation criteria to determine the anti-inflammatory ability of the product, and to compare the anti-inflammatory abilities of stilbene glucoside 98%, Polygonum multiflorum polysaccharide 98%, Polygonum multiflorum composition 1, and Polygonum multiflorum composition 2.

[0101] 2 Main Instruments, Reagents and Consumables

[0102] 2.1 Instruments

[0103] Carbon dioxide incubator

[0104] Microplate reader

[0105] 2.2 Reagents and Consumables Dimethyl sulfoxide (DMSO)

[0106] LPS

[0107] Dexamethasone (DEX)

[0108] Human keratinocytes (HaCaT)

[0109] CCK-8 kit

[0110] ELISA kit

[0111] DMEM high-glucose medium Fetal bovine serum (FBS)

[0112] 3 Test Conditions

[0113] Test substance concentration: Determine the concentration range of the test substance through cytotoxicity detection, and select the concentration with cell viability ≥ 90%. Positive control (dexamethasone DEX): The up-regulation rate of the positive control content ≥ 20%.

[0114] 4 Cytotoxicity test

[0115] This test will use a CCK-8 kit to detect the cytotoxicity of 98% stilbene glucoside (TSG), 98% polygonum multiflorum polysaccharide (PMP), polygonum multiflorum composition 1, and polygonum multiflorum composition 2. Seed HaCaT cells into a 24-well plate at a density of 7.0×10 4 cells per well and incubate stably for 24 hours. Dilute 98% stilbene glucoside, 98% polygonum multiflorum polysaccharide, polygonum multiflorum composition 1, and polygonum multiflorum composition 2 to different concentrations and use them to treat HaCaT cells for 24 hours. After aspirating the culture medium supernatant, add a mixture of phenol red-free DMEM and EZ-Cytox prepared at a ratio of 10:1 to each well, and then react for 30 minutes. In living cells, the reaction solution will change from colorless to yellow. Measure the optical density (OD value) at 450 nm using an enzyme-linked immunosorbent assay reader to determine cell viability.

[0116] 5 Pro-inflammatory cytokine inhibition assay

[0117] 5.1 RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)

[0118] This test will be carried out by enzyme-linked immunosorbent assay. To extract total RNA, use Trizol reagent to lyse the cultured cells. Subsequently, add 200 μL of chloroform and vortex thoroughly, then react at room temperature for 5 minutes. Then, centrifuge at 14000 r / min for 15 minutes at 4°C to obtain the supernatant. Mix the supernatant with isopropanol at a ratio of 1:1 and centrifuge again at 14000 r / min for 15 minutes at 4°C. Use the ReverTra Ace-α kit to react at 42°C for 20 minutes, 99°C for 5 minutes, and then at 4°C for 5 minutes to reverse transcribe RNA into cDNA. The RT-PCR reaction is performed using a StepOnePlus real-time PCR system. Use Taqman probes for each gene. The PCR reaction includes 45 cycles, each cycle including a reaction at 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute.

[0119] 5.2 Western blotting Extract total protein by using RIPA lysis buffer. Set up the following test groups:

[0120]

[0121]

[0122] After measuring the total protein content, each test group was loaded onto a 10% polyacrylamide gel and electrophoresed at 160 V for 1 hour for separation according to protein size, and then transferred to a polyvinylidene fluoride (PVDF) membrane at room temperature for 1 hour. After blocking the PVDF membrane with 10% (w / v) skim milk powder, it was washed with 1×TBST buffer (50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4) and incubated with the primary antibody (diluted 1:1000) at 4°C for 12 hours. Subsequently, it was washed 3 times with 1×TBST buffer for 10 minutes each time. Then, the membrane was incubated with the secondary antibody (diluted 1:10000) diluted with 4% skim milk powder for 1 hour. After washing 3 times again with 1×TBST buffer, Western ECL substrate was dropped onto the PVDF membrane.

[0123] 6 Repeatability

[0124] The absolute difference of no less than 3 independent determination results obtained under repeatability conditions does not exceed 20% of the arithmetic mean.

[0125] 7 Data processing

[0126] All experimental data were analyzed by t-test, using the mean and standard deviation of three measurements. If the p-value in the t-test is less than 0.05, statistical significance is considered. Compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with the groups of polygonum multiflorum thunb composition 1 and polygonum multiflorum thunb composition 2 with the same concentration of the test substance, *P < 0.05, **P < 0.01, ***P < 0.001.

[0127] 8 Result analysis

[0128] 8.1 Results of cytotoxicity test

[0129] From Figure 1 It can be seen that the 4 components have no obvious toxicity to HaCaT cells at a concentration not exceeding 100 μg / ml, and the cell survival rate > 90%. At 200 μg / ml, obvious cytotoxicity appears. Therefore, it is concluded that 98% stilbene glycoside, 98% polygonum multiflorum thunb polysaccharide, and polygonum multiflorum thunb composition 1 and polygonum multiflorum thunb composition 2 are safe for HaCaT cells at a concentration below 100 μg / ml, and this concentration range provides a reference for the following test items.

[0130] 8.2 Results of pro-inflammatory cytokine inhibition test

[0131] As Figure 2 、Figure 3 As shown, compared with the blank group, the expression of inflammatory factors TNF-α mRNA and IL-1β mRNA in the model group cells increased significantly (P < 0.001), verifying the effectiveness of the induction. Compared with the model group, the positive control group significantly reduced the expression of TNF-α mRNA and IL-1β mRNA (P < 0.001), indicating the effectiveness of the test system. Compared with the TSG group and the PMP group, after treatment with the test groups of Polygonum multiflorum composition 1 and Polygonum multiflorum composition 2, the expression of IL-1β mRNA decreased significantly (P < 0.05, P < 0.001), and a better inhibitory effect on the expression of TNF-α mRNA was also shown.

[0132] The results showed that the inhibitory effects of the four products on pro-inflammatory factor cells were in the following order: Polygonum multiflorum composition 2 > Polygonum multiflorum composition 1 > stilbene glucoside 98% (TSG) > polygonum multiflorum polysaccharide 98% (PMP). Polygonum multiflorum composition 1 and Polygonum multiflorum composition 2 could significantly reduce IL-1β and TNF-α secreted by LPS-induced HaCaT cells, and their inhibitory effects were positively correlated with the doses of Polygonum multiflorum composition 1 and Polygonum multiflorum composition 2, showing excellent anti-inflammatory effects.

[0133] Example 6 Determination of antioxidant efficacy

[0134] 1 Test purpose and principle

[0135] In this test, the antioxidant effects of stilbene glucoside 98%, polygonum multiflorum polysaccharide 98%, Polygonum multiflorum composition 1, and Polygonum multiflorum composition 2 were compared by the DPPH free radical scavenging method. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable free radical. Its anhydrous ethanol solution is dark purple and has a strong absorption peak at 517 nm in the visible light region. When the DPPH free radical combines with the electrons provided by the antioxidant test substance for electron pairing, the characteristic purple color of the DPPH free radical will disappear and turn colorless or light yellow. This color change is proportional to the number of electrons provided by the test substance. Therefore, the antioxidant ability of the test substance can be evaluated by measuring the change in absorbance after the reaction. The DPPH free radical scavenging method can quantify the scavenging ability of the test substance on DPPH free radicals, and this quantitative evaluation helps to compare the antioxidant abilities of stilbene glucoside 98%, polygonum multiflorum polysaccharide 98%, and Polygonum multiflorum composition 1 and Polygonum multiflorum composition 2.

[0136] 2 Main instruments, reagents and consumables

[0137] 2.1 Instruments

[0138] Microplate reader

[0139] Electronic balance

[0140] 2.2 Reagents and consumables

[0141] DPPH solution

[0142] Methanol

[0143] 3 Test conditions

[0144] Concentration of the test substance: Determine the concentration range of the test substance through cytotoxicity detection, and select the concentration with cell viability ≥ 90%. (See 2.8.1 for the results of cytotoxicity tests)

[0145] Positive control (BHT): The up - regulation rate of the positive control content ≥ 20%.

[0146] 4 Test procedures

[0147] Accurately weigh 6.4 g of 98% stilbene glucoside, 98% polygonum multiflorum polysaccharide, 12.81 g of polygonum multiflorum composition 1, and polygonum multiflorum composition 2 respectively. Dilute them to 25 mL with methanol and further dilute to make the final concentration 2.5 - 60 μg / ml. Add 0.5 mL of diluted solutions with different concentrations to a 96 - well microplate in sequence. Then, accurately pipette 2.5 mL of freshly prepared DPPH solution (0.6 mmol / L) into the corresponding methanol solutions of the test substances. Finally, make the total volume of the solutions in each well of the microplate 5 mL with methanol to prepare gradient - concentration solutions of the test substances. Put the covered 96 - well microplate into a microplate reader, turn on the oscillator to mix the solutions evenly, and react in the dark for 1 h at a constant temperature of 37°C. After the reaction ends, take out the plate cover and then put the plate back in. Use 5 mL of methanol as the blank control group, use BHT as the positive control, measure the absorbance of each well in the 96 - well microplate at a detection wavelength of 517 nm. Calculate the scavenging rate of DPPH free radicals according to the absorbance values, plot a curve of the free - radical scavenging rate corresponding to the concentration of the sample solution. When the inhibition rate of the sample on DPPH free radicals is defined as 50%, the concentration of the corresponding solution is the half - inhibitory concentration (IC 50 ) of DPPH free radicals. Each group of experiments is repeated three times, and the scavenging rate of DPPH free radicals is expressed as the average value ± standard deviation of the three groups of experiments.

[0148] 5 Result calculation

[0149]

[0150] Wherein:

[0151] A s is the absorbance of the methanol solution of the test substance after scavenging DPPH free radicals;

[0152] A b is the absorbance of the methanol solution of the test substance;

[0153] A D is the absorbance of the methanol solution of DPPH free radicals.

[0154] 6 Repeatability

[0155] The absolute difference of no less than 3 independent determination results obtained under repeatability conditions does not exceed 20% of the arithmetic mean.

[0156] 7 Data Analysis

[0157] From Figure 4 the DPPH free radical scavenging rate and IC 50 statistically analyzed in it, it can be concluded that the ability of the 4 products to scavenge free radicals from strong to weak is: Polygonum multiflorum Thunb. composition 2 > Polygonum multiflorum Thunb. composition 1 > Stilbene glycoside 98% > Polygonum multiflorum Thunb. polysaccharide 98%. Compared with Stilbene glycoside 98% and Polygonum multiflorum Thunb. polysaccharide 98%, Polygonum multiflorum Thunb. composition 1 and Polygonum multiflorum Thunb. composition 2 obtained by the composite extraction process have relatively excellent antioxidant capacity.

[0158] Example 7 Determination of anti-aging effect (MMP-1 inhibition test)

[0159] 1 Test purpose and principle

[0160] Ultraviolet stimulation can activate the cell surface receptor and induce the production of reactive oxygen species (ROS), initiate the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways, and then activate specific transcription factors (such as AP-1 and NF-κB) to regulate the expression of target genes such as matrix metalloproteinase (MMP) and collagen. Compared with UVA, UVB can activate MMP more quickly, and the over-activated MMP in the body will cause the excessive degradation of collagen and elastin that support the skin structure in the dermis of the skin, thus triggering aging symptoms such as skin relaxation and wrinkle formation.

[0161] In this test, a human skin fibroblast (HSF) was cultured in vitro, and a low-dose, repeated ultraviolet B (UVB) irradiation method was used to induce a skin cell aging model. On the basis of this model, 4 test substances with different concentrations (Stilbene glycoside 98%, Polygonum multiflorum Thunb. polysaccharide 98%, Polygonum multiflorum Thunb. composition 1, Polygonum multiflorum Thunb. composition 2) were used for intervention, and the changes of aging-related biological markers were observed. In order to comprehensively compare the anti-aging effects of Stilbene glycoside 98%, Polygonum multiflorum Thunb. polysaccharide 98%, Polygonum multiflorum Thunb. composition 1, and Polygonum multiflorum Thunb. composition 2, we used the determination results of the matrix metalloproteinase-1 (MMP-1) activity inhibition experiment as the main evaluation basis.

[0162] 2 Instruments and reagents

[0163] 2.1 Instruments

[0164] Microplate reader

[0165] UVB generator

[0166] Centrifuge

[0167] 2.2 Reagents and consumables

[0168] Human skin fibroblasts (HSF) with MMP-1 enzyme

[0169] High-glucose DMEM medium, fetal bovine serum (FBS)

[0170] Penicillin and streptomycin (0.99 U / mL)

[0171] 25 cm 2 Culture flask

[0172] 96-well plate, 6-well plate, 100 mm cell culture dish

[0173] PBS (Phosphate Buffered Saline)

[0174] Human MMP-1 ELISA kit

[0175] TMB chromogenic solution

[0176] 3 Experimental procedures

[0177] (I) Cell resuscitation and culture:

[0178] Resuscitate human skin fibroblasts (HSF) and inoculate them into a 25 cm 2 culture flask. Culture using high-glucose DMEM medium (containing 10% fetal bovine serum (FBS), 0.99 U / mL penicillin and streptomycin).

[0179] Place the cells in an incubator at 37°C and 5% CO 2 for incubation.

[0180] (II) Cell seeding and grouping:

[0181] Select cells in the logarithmic growth phase from the 4th to the 10th passage and seed them into 96-well plates, 6-well plates, and 100 mm cell culture dishes at the predetermined density respectively.

[0182] The experimental groups are designed as follows:

[0183]

[0184] (III) UVB irradiation treatment:

[0185] When the cells grow to 60% to 70% confluence, remove the medium and wash once with PBS. Cover the cell surface with a thin layer of PBS and start UVB irradiation. Place the light source of the UVB generator 10 cm above the culture plate, with a single irradiation duration of 20 seconds and a single dose of 10 mJ / cm 2 . After each irradiation, remove the PBS.

[0186] Replace the model control group with DMEM medium containing 1% FBS and continue culturing. Replace the UVB + test substance groups at each concentration with DMEM medium containing 1% FBS and the corresponding concentration of TSG and continue culturing. The total irradiation dose is 60 mJ / cm 2 (Irradiate twice a day for three consecutive days).

[0187] (IV) Post-irradiation treatment:

[0188] After the last UVB irradiation, remove the PBS. Replace the model control group with DMEM medium containing 10% FBS and continue culturing. Replace the UVB + test substance groups at each concentration with DMEM medium containing 10% FBS and the corresponding concentration of the test substance and continue culturing.

[0189] (V) ELISA detection of MMP-1 content:

[0190] 48 hours after the last UVB irradiation, collect the cell culture supernatants of each group. Centrifuge at 2000 - 3000 rpm for 20 minutes, take the supernatant and aliquot it. Add 100 μL of each concentration standard product to seven wells in a row of the enzyme-linked immunosorbent assay (ELISA) plate in turn, set the zero well (only add 100 μL of sample diluent) and the TMB blank color development well. After labeling the samples of each group, add them to the corresponding wells (100 μL / well). Operate strictly according to the ELISA kit instructions. After the reaction, measure the absorbance value at 450 nm with an ELISA reader. Draw a standard curve, substitute the absorbance value of the sample into the standard curve equation, and calculate the MMP-1 concentration.

[0191] 4 Result calculation

[0192] Use the 2-ΔΔCt method to calculate and process the data. The analysis is repeated three times (n = 3), and the results are expressed as the mean. One-way analysis of variance is used for comparison among multiple groups, and Tukey's test is used for pairwise comparison between groups. Compared with the induction group, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with the test substance polygonum multiflorum composition 1 group at the same concentration, *P < 0.05, **P < 0.01, ***P < 0.001.

[0193] 5 Data analysis

[0194] ByFigure 5 It can be seen that, compared with the blank group, the model group can significantly increase the concentration of MMP-1, that is, UVB irradiation can stimulate fibroblasts to secrete an increased level of MMP-1; under the condition of the same active ingredient concentration, compared with the TSG group and the PMP group, the polygonum multiflorum composition 1 group and the polygonum multiflorum composition 2 group can significantly inhibit the level of MMP-1. And the ability of the TSG group with the same active ingredient concentration to inhibit MMP-1 is slightly better than that of the PMP group. It can be inferred from this that polygonum multiflorum compositions 1 and 2 obtained by the composite extraction process can inhibit the high expression of the photoaging-related factor MMP-1 in HSF after UVB irradiation, have a photoprotective effect on HSF, and have more excellent anti-aging ability.

[0195] Example 8 Determination of anti-aging efficacy (effect on collagen content)

[0196] 1 Test purpose and principle

[0197] Fibroblasts are the main effector cells of the dermis and can produce various extracellular matrices (ECMs) such as collagen, laminin, and elastin. The water retained by fibroblasts and the ECM matrix they produce is the material basis for maintaining skin elasticity. The content of total collagen in the dermis accounts for 70% - 80% of the total dermal proteins and is the main protein component in the dermal layer of the skin and the main component for maintaining skin structure and strength. The effects of 98% stilbene glucoside, 98% polygonum multiflorum polysaccharide, polygonum multiflorum composition 1, and polygonum multiflorum composition 2 on the synthesis of total collagen in human skin fibroblasts (HSF) cultured in vitro were detected by ELISA method to evaluate and compare the anti-aging effects of several groups of products.

[0198] 2 Main instruments, reagents and consumables

[0199] 2.1 Instruments

[0200] Carbon dioxide incubator

[0201] Microplate reader

[0202] Centrifuge

[0203] UVB generator

[0204] 2.2 Reagents and consumables Human skin fibroblasts (HSF)

[0205] High-glucose DMEM medium Fetal bovine serum (FBS)

[0206] Penicillin-streptomycin double antibody (1%)

[0207] Total collagen ELISA detection kit

[0208] PBS buffer Cell lysis buffer (RIPA buffer)

[0209] Dimethyl sulfoxide (DMSO)

[0210] 3 Experimental procedures

[0211] Resuscitate HSF cells and culture them in high-glucose DMEM medium (containing 10% FBS and 1% double antibiotics) at 37 °C and 5% CO 2 condition. When the cells reach 80% - 90% confluence, passage them. Seed HSF cells in logarithmic growth phase into 6-well plates at a density of 2×10 5 cells per well.

[0212] The experimental groups are as follows: The group without TSG and stimulant is the blank group; The group with only the stimulant (UVB irradiation treatment) is the model control group; The TSG treatment groups are those adding 2 μmol / L, 10 μmol / L, and 50 μmol / L TSG respectively; The group adding the known retinoic acid (RA) is the positive control group.

[0213] The experimental design is as follows:

[0214]

[0215]

[0216] Except for the blank control group, add the corresponding concentrations of TSG or the positive control drug to the other groups and pretreat for 4 hours. Add the stimulant (UVB irradiation) to the model control group and the TSG treatment groups and continue to culture for 24 hours.

[0217] After the experiment, discard the medium and wash the cells twice with PBS. Add 200 μL of cell lysis buffer (RIPA buffer) to each well and lyse on ice for 30 minutes. Collect the lysate, centrifuge at 12000 rpm for 10 minutes, take the supernatant and aliquot it for storage at -80 °C for later use. Operate strictly according to the ELISA kit instructions. After the reaction, measure the absorbance value (OD value) at 450 nm with an enzyme-linked immunosorbent assay reader and draw a standard curve based on the OD values of the standards.

[0218] Substitute the OD value of the sample into the standard curve equation to calculate the total collagen content. Compare the total collagen contents of each group to evaluate the effects of 98% stilbene glycoside, 98% polygonum multiflorum polysaccharide, polygonum multiflorum composition 1, and polygonum multiflorum composition 2 on collagen synthesis.

[0219] 4 Repeatability

[0220] Under repeatability conditions, the absolute difference of no less than 3 independent measurement results does not exceed 20% of the arithmetic mean.

[0221] 5 Data processing

[0222] All experimental data were analyzed using Student's t-test, with the mean and standard deviation of three measurements used. Statistical significance was considered if the p-value in the Student's t-test was less than 0.05. Compared with the blank group, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with the groups of polygonum multiflorum compositions 1 and 2 with the same concentration of the test substance, *P < 0.05, **P < 0.01, ***P < 0.001.

[0223] 6 Result Analysis

[0224] It can be seen from Figure 6 that compared with the blank group, after UVB irradiation, the total collagen protein content in the model group decreased significantly (P < 0.01), indicating that the test system was effective; compared with the blank group, the total collagen content in the positive control group increased significantly (P < 0.01), indicating that the test system was effective; at the same concentration of the active ingredient, compared with the TSG group and the PMP group, the polygonum multiflorum composition 1 group and the polygonum multiflorum composition 2 group could more significantly increase the total collagen content (P < 0.01); at the same concentration of the active ingredient, the ability of the TSG group to promote the synthesis of total collagen was slightly higher than that of the PMP group. It can be concluded that the polygonum multiflorum composition 1 and the polygonum multiflorum composition 2 treatment groups can significantly promote the synthesis of total collagen in fibroblasts, showing a dose-dependent effect and having a more excellent anti-aging effect.

[0225] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. The non-essential improvements, adjustments or replacements made by those skilled in the art according to the content of this specification all fall within the scope protected by the present invention.

Claims

1. A Polygonum multiflorum composition, characterized in that It is made from Polygonum multiflorum raw materials through a composite extraction process and contains the following components: a) stilbene glycosides; b) Polysaccharide content.

2. A Polygonum multiflorum composition, characterized in that It is made from Polygonum multiflorum raw materials through a composite extraction process and contains the following components: c) a stilbene glycoside content of 40-60% (w / w); d) polysaccharide content of 30-60%; The mass ratio of the diphenylethylene glycoside to the polysaccharide is (0.7-2.0):

1.

3. The Polygonum multiflorum composition according to claim 2, characterized in that The mass ratio of the diphenylethylene glycoside to the polysaccharide is 1:

1.

4. The Polygonum multiflorum composition according to claim 2 or 3, characterized in that The content of the stilbene glycoside is 45-55% (w / w).

5. The Polygonum multiflorum composition according to claim 2 or 3, characterized in that: The polysaccharide content is 40-55%.

6. The Polygonum multiflorum composition according to any one of claims 1 to 5, characterized in that The composite extraction process comprises the following steps: a) subjecting the raw material of Polygonum multiflorum to graded alcohol extraction, and collecting 30-50% ethanol extract; b) adjusting the pH of the extract to 5.0-6.0 for low temperature concentration; c) adding ethanol to the concentrated solution, allowing the solution to settle, filtering, and collecting the polysaccharide-containing sediment and the mother liquor containing stilbene glycosides respectively; d) After the precipitate and the mother liquor are further separated and purified, they are mixed again, an appropriate amount of deionized water is added, and the mixture is heated to 40-45° C. to fully dissolve. Activated carbon (1% in amount, w / v) is added to decolorize the mixture at 40-45° C. for 30 minutes, and the mixture is filtered, concentrated, and dried.

7. A method for preparing a Polygonum multiflorum composition, characterized in that: The steps include: a) subjecting the raw material of Polygonum multiflorum to graded alcohol extraction, and collecting 30-50% ethanol extract; b) adjusting the pH of the extract to 5.0-6.0 for low temperature concentration; c) adding ethanol to the concentrated solution, allowing the solution to settle, filtering, and collecting the polysaccharide-containing sediment and the mother liquor containing stilbene glycosides respectively; d) After the precipitate and the mother liquor are further separated and purified, they are mixed again, an appropriate amount of deionized water is added, and the mixture is heated to 40-45° C. to fully dissolve. Activated carbon (1% in amount, w / v) is added to decolorize the mixture at 40-45° C. for 30 minutes, and the mixture is filtered, concentrated, and dried.

8. The method according to claim 7, characterized in that: The precipitate in step d) is subjected to secondary alcohol precipitation to further remove impurities and obtain a high-purity polysaccharide component; the mother liquor is separated by a macroporous resin column to obtain a high-purity diphenylethylene glycoside component; the polysaccharide component and the diphenylethylene glycoside component are mixed in a mass ratio of (0.7-2.0):1, an appropriate amount of deionized water is added, heated to 40-45°C, fully dissolved, activated carbon (dosage 1%, w / v) is added, decolorized at 40-45°C for 30 minutes, filtered, concentrated, and dried.

9. Use of the composition according to any one of claims 1 to 5 in the preparation of anti-aging cosmetics.

10. The use according to claim 9, wherein the aging is skin photoaging caused by ultraviolet rays.

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