Atractylodes chinensis fibrous root polysaccharide and application thereof

Abstract

The invention belongs to the technical field of plant polysaccharide, and discloses rhizoma atractylodis chinensis fibrous root polysaccharide and application thereof. The specific structure of the atractylodes lancea fibrous root polysaccharide with the molecular weight of 3.7 kD is defined and used for researching the corresponding functional properties of the atractylodes lancea fibrous root polysaccharide, the application of the polysaccharide with the specific structure in the aspect of skin anti-aging activity is provided, and the atractylodes lancea fibrous root polysaccharide with the molecular weight of 3.7 kD can be used for preparing the anti-aging activity of the skin by promoting the proliferation effect of keratinocytes HaCAT. The activity of beta-galactosidase in keratinocytes HaCAT and the generation of ROS active oxygen are inhibited, so that the anti-aging activity of the product is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant polysaccharides, and particularly relates to a polysaccharide from the fibrous roots of Atractylodes lancea and an application thereof. Background Art

[0002] The main cause of skin aging is the combined effects of the imbalance of the body's antioxidant system, collagen damage, and long-term overexposure to sunlight, which affect cell activity, reduce collagen and elastin synthesis, and increase collagen degradation. Aging skin shows fine wrinkles, dryness, thinning, and increased temperature sensitivity. With the improvement of people's living standards, consumers have a higher pursuit of anti-aging products. Skin protection is carried out through various measures such as oral "health products" and external "skin care products", especially beauty skin care products are accepted by many consumers. At present, anti-aging skin care products in the skin care market are mainly creams, which are often added with ingredients such as A-alcohol, peptides, recombinant collagen, and phosphodiesterin. They are highly irritating, poorly stable, slow to take effect, expensive, and have certain preparation technology requirements. Therefore, the development of anti-aging skin care products is still very challenging.

[0003] Atractylodes chinensis (DC.) Koidz. is a plant of the genus Atractylodes in the Asteraceae family. Its rhizomes are mainly composed of polyphenols, flavonoids and polysaccharides, which have multiple biological activities such as anti-inflammatory, antioxidant, antibacterial and anti-tumor, and have extremely high medicinal value. In recent years, there have been many applications of Atractylodes chinensis polysaccharides, but no reports have been seen on their application in anti-oxidation and anti-aging. Compared with the rhizomes of Atractylodes chinensis, its accompanying fibrous roots are non-medicinal parts and are often discarded during the production process. The fibrous roots of Atractylodes chinensis are homologous to the rhizomes and may also contain similar active ingredients. Preliminary studies have found that the fibrous roots of Atractylodes chinensis contain a large amount of polysaccharides, and the fibrous root polysaccharides of Atractylodes chinensis have low toxicity and good water solubility, which has the advantage of being easy to develop. Studies have reported that small molecular weight plant polysaccharides have the characteristics of good permeability, high bioavailability and few side effects. They are easier to penetrate the skin barrier, penetrate into the bottom layer of the skin to exert anti-aging effects, and are easier to be absorbed and utilized by skin cells. They can be used as a safe and effective anti-aging ingredient suitable for people of all skin types. Meanwhile, there is no report on the structure and anti-aging related activity of small molecular weight polysaccharide components in the fibrous roots of Atractylodes lancea. The present invention aims to clarify the structure of small molecular weight polysaccharides in the fibrous roots of Atractylodes lancea and develop them into anti-aging skin care products, which helps to improve the comprehensive utilization value of Atractylodes lancea. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a low molecular weight polysaccharide from the fibrous roots of Atractylodes lancea and its application in anti-aging skin care products.

[0005] In order to achieve the above-mentioned object, the first object of the present invention is to provide a polysaccharide from the fibrous roots of Atractylodes lancea, using the following technical scheme:

[0006] A polysaccharide from the fibrous roots of Atractylodes lancea is an inulin-type arabinofrucan, wherein the main chain structure is →1-β-D-Fruf-2→1-β-D-Fruf-2→, the side chain structure is α-L-Araf-(1→5)-α-L-Araf-(1→, and the side chain is connected to the main chain via O-6 of →1,6-β-D-Fruf-2→; and the molecular weight of the polysaccharide from the fibrous roots of Atractylodes lancea is 3.7 kD.

[0007] It is worth noting that the monosaccharide components of the Atractylodes lancea fibrous root polysaccharide disclosed in the present invention are mainly fructose and arabinose, and only contain a small amount of galactose and glucose.

[0008] The second object of the present invention is to provide the use of the Atractylodes lancea fibrous root polysaccharide as described above.

[0009] Application of Atractylodes macrocephala fibrous root polysaccharide in the preparation of anti-aging skin care products.

[0010] Furthermore, the Atractylodes lancea fibrous root polysaccharide is used in the preparation of a product that significantly promotes the proliferation of keratinocytes HaCAT.

[0011] Furthermore, the Atractylodes lancea fibrous root polysaccharide is used in the preparation of a product for inhibiting the activity of β-galactosidase in keratinocytes HaCAT.

[0012] Furthermore, the Atractylodes lancea fibrous root polysaccharide is used in the preparation of a product for inhibiting the generation of ROS reactive oxygen species in keratinocytes HaCAT.

[0013] Furthermore, the Atractylodes lancea root polysaccharide is used in the preparation of a product for inhibiting the generation of ROS reactive oxygen species in AB type zebrafish.

[0014] Furthermore, the Atractylodes macrocephala fibrous root polysaccharide is used in the preparation of skin anti-aging cream products.

[0015] Furthermore, the skin anti-aging cream product comprises, by mass:

[0016] 1,3-Propanediol 5-8wt%, ethylenediaminetetraacetic acid 0.2-0.6wt%, squalane 4-5wt%, cetearyl alcohol 1.0-1.5wt%, polydimethylsiloxane 1-1.2wt%, isopropyl palmitate 2-2.5wt%, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer 0.1-0.3wt%, 1,2-pentanediol 0.5-0.7wt%, phenoxyethanol 0.05-0.2wt%, Atractylodes macrocephala root polysaccharide 1-5wt% and the balance water.

[0017] Compared with the prior art, the present invention clarifies the specific structure of the polysaccharide with a molecular weight of 3.7kD in the fibrous roots of Atractylodes lancea for studying its corresponding functional properties, and provides the application of the polysaccharide with a specific structure in the anti-aging activity of the skin, which can enhance the anti-aging activity of the product by promoting the proliferation of keratinocytes HaCAT and inhibiting the β-galactosidase activity and ROS reactive oxygen generation in keratinocytes HaCAT. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 This is the molecular weight result of the purified Atractylodes lancea fibrous root polysaccharide disclosed in Example 2 of the present invention.

[0020] Figure 2 This is the monosaccharide composition result of the Atractylodes lancea fibrous root polysaccharide disclosed in Example 3 of the present invention.

[0021] Figure 3 is the nuclear magnetic resonance spectrum of the Atractylodes macrocephala root polysaccharide disclosed in Example 5 of the present invention, wherein A to F are respectively 1 H. 13 C, Dept135, COSY, HSQC and HMBC; G is a schematic diagram of the structure of polysaccharides from the fibrous roots of Atractylodes lancea.

[0022] Figure 4 These are the experimental results of promoting HaCaT cell proliferation by Atractylodes lancea root polysaccharide disclosed in Example 6 of the present invention, wherein A is the effect of Atractylodes lancea root polysaccharide on HaCaT cell viability; B is the Edu fluorescence staining positive cell rate of HaCaT cells under the action of Atractylodes lancea root polysaccharide; C is the Edu fluorescence staining image of HaCaT cells under the action of Atractylodes lancea root polysaccharide.

[0023] Figure 5 This is the result of measuring β-galactosidase activity in HaCAT cells disclosed in Example 7 of the present invention.

[0024] Figure 6 This is the result of measuring ROS content in HaCAT cells disclosed in Example 8 of the present invention.

[0025] Figure 7 This is the result of measuring ROS content in zebrafish disclosed in Example 9 of the present invention. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0028] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0029] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0030] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0031] The invention discloses a polysaccharide from the fibrous roots of Atractylodes lancea and its application, belonging to the technical field of plant polysaccharides. The invention clarifies the specific structure of the polysaccharide with a molecular weight of 3.7 kD from the fibrous roots of Atractylodes lancea to study its corresponding functional properties, and provides the application of the polysaccharide with a specific structure in the anti-aging activity of the skin, which can enhance the anti-aging activity of the product by promoting the proliferation of keratinocytes HaCAT and inhibiting the activity of β-galactosidase and the generation of ROS reactive oxygen species in keratinocytes HaCAT.

[0032] In order to better understand the present invention, the present invention is further specifically described through the following examples, but it should not be understood as limiting the present invention. Some non-essential improvements and adjustments made by technicians in this field according to the above invention content are also considered to fall within the protection scope of the present invention.

[0033] The experimental materials involved in the embodiments of the present invention include:

[0034] (1) Atractylodes macrocephala fibrous roots were provided by Huahao Testing, Science and Technology Park, Jiamusi University;

[0035] (2) Reactive oxygen species (ROS) and β-galactosidase kits were provided by Biotime Biotech Co., Ltd.;

[0036] (3) Wild AB zebrafish were provided by the National Zebrafish Resource Center (Wuhan, China);

[0037] (4) HaCaT cell line was provided by Boster Biotechnology Co., Ltd. (Wuhan, China);

[0038] (5) Levodopa was provided by Shanghai Yuanye Biotechnology Co., Ltd.

[0039] Example 1: Preparation of Atractylodes macrocephala fibrous root polysaccharide

[0040] Extraction, separation and purification of crude polysaccharides from the fibrous roots of Atractylodes lancea:

[0041] Step A, crush the fibrous roots of Atractylodes lancea, sieve to obtain powder, defat with petroleum ether, dry, then soak with 10 times the amount of deionized water for 12 hours, extract by water extraction, and concentrate the extract. Add 4 times the volume of anhydrous ethanol to the concentrate for precipitation overnight, centrifuge at 4000 rpm for 10 minutes to obtain a precipitate. Deproteinize by Sevage method, dialyze the solution, and then freeze-dry to obtain crude polysaccharide.

[0042] Step B, the crude polysaccharide aqueous solution of Atractylodes macrocephala fibrous root is loaded on a DE-52 chromatographic column for chromatographic analysis, and polysaccharides with different ionic strengths are obtained by elution. The eluted fractions (15 mL / tube) are collected by an automatic collector, and then the content of polysaccharides in each tube fraction is determined by phenol-sulfuric acid phenol method, and the microplate reader is used for detection at 490 nm to draw a scatter plot. The eluents within the range of different eluents are collected, concentrated, dialyzed with a 3500Da dialysis bag, and freeze-dried.

[0043] Step C, the polysaccharide with the water part as the eluent is purified by a fully automatic gel purification system (BRT-GS), and the eluate is collected by online detection in combination with a differential detector (SHIMADZU). The collected liquid is dialyzed, concentrated by a rotary evaporator, and freeze-dried to obtain the gel-separated and purified Atractylodes lancea fibrous root polysaccharide.

[0044] Example 2: Determination of molecular weight of polysaccharide from Atractylodes macrocephala fibrous roots

[0045] The relative average molecular weight and purity were determined by high performance gel permeation chromatography (HPGPC) (LC-10A, Shimadzu, Japan) equipped with a refractive index detector (RID) (RI-10A, Shimadzu, Japan) and a 105-103-101 series gel column (8.0 mm×300 mm).

[0046] In step A, eight dextran standards with different molecular weights (P5, P10, P20, P50, P100, P200, P400, P800) (SHOWA DENKOa, Japan) were first used for chromatographic analysis.

[0047] Step B, chromatographic conditions: 0.05M NaCL as mobile phase, flow rate of 0.7mL / min, temperature of 40°C, injection volume of 25μL, and monitoring by differential refractive index (dRI). A calibration curve was established at a concentration of 5mg / mL to obtain a molecular weight calculation formula.

[0048] Step C, similarly, after the purified polysaccharide solution (5 mg / mL) is treated, 25 μL of the sample is placed on HPGPC and analyzed using the same chromatographic conditions as the standard to obtain the chromatogram and retention time. The retention time is substituted into the molecular weight calculation formula to obtain the molecular weight result.

[0049] like Figure 1 As shown, the chromatogram results show that when the retention time is 41.365min, the chromatographic peak presents a single and symmetrical spike form, Mw (weight average molecular weight) and Mn (number average molecular weight) are both 3.7kDa, the ratio of polydispersity index Mw / Mn is approximately equal to 1, the purity is 94.56%, and the distribution is concentrated, which verifies its uniformity.

[0050] Example 3: Analysis of Monosaccharide Composition of Atractylodes macrocephala fibrous Root Polysaccharide

[0051] Step A: Dissolve 5 mg of polysaccharide in 2 mL of 3 M TFA and hydrolyze at 80 °C for 2 h.

[0052] Step B: The hydrolyzed sample was blown dry with nitrogen at 60°C, and the blown-dried material was completely mixed and dissolved in 5 mL of deionized water by vortexing. The mixed material was centrifuged at 12000 rpm for 5 minutes, and 50 μL of the supernatant was aspirated and added to 950 μL of deionized water to obtain the sample to be tested.

[0053] Step C: Then, the sample was analyzed using HPAEC-PAD equipped with Dionex CarbopacTM PA20 (150 mm×3.0 mm), the mobile phase eluent consisted of A: H2O; B: 15 mM NaOH; C: 15 mM NaOH and 100 mM NaAc, the flow rate was 0.3 mL / min, and the sample volume was 25 μL.

[0054] Step D: A standard monosaccharide mixture comprising mannose, rhamnose, galacturonic acid, galactose, glucose, glucuronic acid, arabinose, xylose, fucose, glucosamine hydrochloride, N-acetyl-D-glucosamine, D-fructose, D-ribose, galactosamine hydrochloride, L-guluronic acid and D-mannuronic acid is treated using the same derivatization and analytical methods as the sample to be tested.

[0055] Step E: comparing and identifying the monosaccharide composition and molar ratio of the Atractylodes lancea fibrous root polysaccharide based on the retention time of the chromatographic peaks of the monosaccharide mixture and the sample polysaccharide.

[0056] like Figure 2 As shown, the monosaccharide composition is mainly fructose and arabinose, with only a small amount of galactose and glucose.

[0057] Example 4: Methylation analysis of Atractylodes macrocephala fibrous root polysaccharides

[0058] Step A, weigh a sample of Atractylodes macrocephala root polysaccharide (2-3 mg) and place it in a glass reaction bottle, add 1 mL of anhydrous DMSO, quickly add methylation reagent A solution, seal, dissolve under ultrasound, then add methylation reagent B solution, and react in a magnetic stirring water bath at 30°C for 60 minutes.

[0059] Step B, finally, 2 mL of ultrapure water was added to the above mixture to terminate the methylation reaction, and the mixture was dialyzed in a 1000 Da dialysis bag for 24 h and then freeze-dried.

[0060] Step C, accurately weigh 2 mg of the dialyzed sample and 200 mg of potassium bromide, press them into tablets, and use potassium bromide powder to press the blank control into tablets. Place them in Fourier transform infrared spectrometer FT-IR650 (Tianjin Gangdong Technology Development Co., Ltd.) for scanning and recording, and do subsequent experiments after confirming that the methylation is complete.

[0061] Step D: Take the methylated polysaccharide, add 1 mL of 2 M trifluoroacetic acid (TFA) and hydrolyze for 90 min, and evaporate to dryness on a rotary evaporator.

[0062] Step E: Add 2 mL of double distilled water and 60 mg of sodium borohydride to the residue for reduction for 8 hours, add glacial acetic acid to neutralize, perform rotary evaporation, and dry in an oven at 101°C.

[0063] Step F, then add 1 mL of acetic anhydride for acetylation at 100° C. for 1 h, and cool.

[0064] Step G, then add 10 mL of pure water to terminate the reaction.

[0065] Step H, dissolve the acetylated product with 3 mL CH2Cl2 and transfer to a separatory funnel. Add a small amount of distilled water and shake thoroughly, then remove the upper aqueous solution. Repeat this process 4 times. Dry the CH2Cl2 layer with an appropriate amount of anhydrous sodium sulfate, concentrate to 1 mL, and place in a liquid phase vial. The acetylated product sample was analyzed using a Thermo scientific, 1300-7000 gas chromatograph-mass spectrometer.

[0066] Test GC-MS conditions: HP-INNOWAX chromatographic column 30m*0.32mm*0.25um; program temperature conditions: starting temperature 140℃, heating to 230℃ at 1℃ / min; injection port temperature 250℃, detector temperature 250℃, carrier gas helium, flow rate 1mL / min.

[0067] As shown in Table 1, the results of methylation analysis showed that the refined polysaccharide from the fibrous roots of Atractylodes lancea contained 9 types of glycosidic bonds.

[0068] Table 1 Results of methylation analysis of polysaccharides in the fibrous roots of Atractylodes lancea

[0069]

[0070] Example 5: Nuclear Magnetic Resonance Spectroscopic Analysis of Polysaccharides from Atractylodes macrocephala fibrous Roots

[0071] Step A, weigh 50 mg of the refined polysaccharide sample from the fibrous roots of Atractylodes lancea, dissolve it in 0.5 mL of D2O, and detect it under an AVANCE600 NMR spectrometer (600 MHz, Bruker, Germany).

[0072] Step B, NMR spectroscopy includes 1 H, 13 C,Dept 135,HSQC,HMBC, 1 H / 1 H COSY.

[0073] In step C, data were analyzed using MestReNova software (Santiago de Compostela, Spain).

[0074] The results are as follows Figure 3 As shown, it can be seen that the Atractylodes macrocephala root polysaccharide ( Figure 3 , G) has a main chain of →1-β-D-Fruf-2→1-β-D-Fruf-2→, and the side chain is α-L-Araf-(1→5)-α-L-Araf-(1→ connected to the main chain through O-6 of →1,6-β-D-Fruf-2→.

[0075] Example 6: Effects of Atractylodes macrocephala fibrous root polysaccharides on HaCAT cell proliferation and cell viability

[0076] Cell proliferation and cell viability are key factors in anti-aging. Maintaining and improving cell proliferation ability and maintaining cell viability are essential for delaying the aging process.

[0077] (1) HaCAT cell proliferation test

[0078] Step A: HaCaT cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% antibiotics (penicillin, streptomycin) and placed in a humidified incubator at 37°C and 5% CO2.

[0079] Step B: Take cells in logarithmic growth phase for experiment, and use 3*10 3 HaCaT cells were seeded into 96-well cell culture plates at a density of 10 cells / well.

[0080] Step C: After the cells adhered to the wall, different doses of polysaccharide solution (200, 400, 800 μg / mL) and 1 mM H2O2 were added. At the same time, a blank control group containing only culture medium was also set up. The cells were cultured for 24 and 48 hours respectively.

[0081] Step D: Add 20 μL of MTT solution to each well and continue incubating in the incubator for 4 hours.

[0082] Step E: Then add 200 μL DMSO to each well for dissolution and shake for 10 minutes.

[0083] Step F: Measure the absorbance of each well at a wavelength of 490 nm using a microplate reader.

[0084] (2) Cell viability test

[0085] Step A: HaCaT cells were incubated with EdU reagent for 2 hours.

[0086] Step B: Fix with paraformaldehyde (4%) at room temperature for 30 minutes.

[0087] Step C: Then permeabilize with Triton X-100 (0.4%) for 10 minutes.

[0088] Step D: Stain with 1× Apollo reagent (15 μL) at room temperature for 30 minutes.

[0089] Step E: Then stain with DAPI solution at room temperature for 10 minutes.

[0090] Step F: Images were observed using a Zeiss AxioImager Z1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

[0091] Step G: Finally, the number of Edu-positive cells was analyzed using ImageJ software.

[0092] like Figure 4 As shown, with the gradual increase of the concentration of Atractylodes lancea fibrous root polysaccharide, it promoted the proliferation of HaCAT and the gradual increase of EDU-positive cells in a dose-dependent manner, proving that it has a significant effect in promoting cell proliferation.

[0093] Example 7: Determination of β-galactosidase activity in HaCAT cells

[0094] The increase of cellular β-galactosidase activity is an important sign of cell aging, and regulating the activity of this enzyme may have potential significance in anti-aging.

[0095] Step A, HaCaT cells were cultured at 3*10 3 Cells were seeded at a density of 10 cells / well in 6-well plates.

[0096] Step B, treated with different doses of polysaccharide solution (200, 400 and 800 μg / mL) and 1 mM H2O2 for 48 hours.

[0097] Step C, remove the culture medium and wash the cells with PBS.

[0098] Step D: Add 1 mL of β-galactosidase staining fixative and fix at room temperature for 15 minutes.

[0099] Step E: remove the cell fixative and wash the cells with PBS.

[0100] Step F: Add 1 mL of staining working solution to each well and incubate at 37°C for 12 h.

[0101] Step G: Observe and count under an optical microscope; cells stained dark blue are senescence-positive cells.

[0102] like Figure 5 As shown, Atractylodes macrocephala root polysaccharide inhibited H2O2-induced β-galactosidase activity in HaCaT cells in a dose-dependent manner.

[0103] Example 8: Determination of ROS content in HaCAT cells

[0104] Excessive ROS in the body can cause cumulative damage to cells, which is one of the mechanisms of aging advocated by the free radical theory. ROS react with a variety of biological macromolecules (such as DNA, proteins and lipids) in cells, causing functional changes and damage to these macromolecules.

[0105] Step A, HaCaT cells were cultured at 1*10 5 The cells were seeded at a density of 10 cells / well into 6-well cell culture plates.

[0106] Step B, treated with different doses of polysaccharide solution (200, 400 and 800 μg / mL) and 1 mM H2O2 for 48 hours.

[0107] Step C: After 48 hours, HaCaT cells were collected and placed in a 10 μM DCFH-DA solution diluted in serum-free medium and incubated at 37° C. for 20 minutes.

[0108] In step D, the cells were washed three times with serum-free medium to fully remove DCFH-DA that did not enter the cells.

[0109] In step E, the fluorescence signal of ROS was detected using a fluorescence microscope, and the fluorescence intensity was quantified using image-J software.

[0110] like Figure 6 As shown, Atractylodes macrocephala root polysaccharide inhibited the ROS content in HaCaT cells induced by H2O2 in a dose-dependent manner.

[0111] Example 9: Determination of ROS content in zebrafish

[0112] Excessive ROS in the body can cause cumulative damage to cells, which is one of the mechanisms of aging advocated by the free radical theory. ROS react with a variety of biological macromolecules (such as DNA, proteins and lipids) in cells, causing functional changes and damage to these macromolecules.

[0113] In step A, zebrafish embryos were seeded into 6-well cell culture plates at a density of 30 per well.

[0114] Step B, treated with different doses of polysaccharide solution (200, 400 and 800 μg / mL) and 1 mM H2O2 for 96 hours.

[0115] Step C: 96 hours later, the cultured zebrafish were placed in a diluted 10 μM DCFH-DA solution and incubated at 37° C. for 20 minutes.

[0116] Step D: Wash three times with deionized water to fully remove DCFH-DA that has not entered the cells.

[0117] In step E, the fluorescence signal of ROS was detected using a fluorescence microscope, and the fluorescence intensity was quantified using image-J software.

[0118] like Figure 7 As shown, Atractylodes macrocephala root polysaccharides inhibited the ROS content in zebrafish induced by H2O2 in a dose-dependent manner.

[0119] Example 10: Skin anti-aging cream product and its effect experiment

[0120] The composition of the skin anti-aging cream product is shown in Table 2.

[0121] Table 2 Cream product ingredients

[0122]

[0123]

[0124] Human evaluation experiment:

[0125] Step A: Using the VISIA tester, select 20 subjects aged 35-65 years old, all of whom have dark yellow skin, wrinkles, decreased collagen fiber elasticity, dark brown spots, etc. The subjects are healthy, have no history of skin diseases and allergies, have non-sensitive skin, and can use skin care products according to regulations.

[0126] Step B: The subjects applied the cream prepared in Example 9 to their faces in the morning and evening respectively, once a day, for continuous use, and compared the skin conditions before and after 28 days.

[0127] Step C: Calculate the wrinkle reduction rate and skin elasticity improvement rate of each group of subjects

[0128] Table 3 shows the human evaluation results of skin anti-aging cream products, indicating that the cream products containing polysaccharide components with a molecular weight of 3.7kD from the fibrous roots of Atractylodes lancea have obvious anti-aging effects.

[0129] Table 3 Human evaluation experimental results

[0130]

[0131] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polysaccharide from the fibrous roots of Atractylodes lancea, characterized in that The Atractylodes lancea fibrous root polysaccharide is an inulin-type arabinofrucan, whose main chain structure is →1-β-D-Fruf-2→1-β-D-Fruf-2→, and the side chain structure is α-L-Araf-(1→5)-α-L-Araf-(1→, and is connected to the main chain through O-6 of →1,6-β-D-Fruf-2→; and the molecular weight of the Atractylodes lancea fibrous root polysaccharide is 3.7kD.

2. Use of the Atractylodes macrocephala root polysaccharide as claimed in claim 1 in the preparation of anti-aging skin care products.

3. The use according to claim 2, characterized in that: The application of the Atractylodes macrocephala fibrous root polysaccharide in the preparation of a product that promotes the proliferation of keratinocytes HaCAT.

4. The use according to claim 2, characterized in that: The application of the Atractylodes macrocephala fibrous root polysaccharide in the preparation of a product for inhibiting the activity of β-galactosidase in keratinocytes HaCAT.

5. The use according to claim 2, characterized in that: The application of the Atractylodes lancea fibrous root polysaccharide in the preparation of a product for inhibiting the generation of ROS active oxygen in keratinocyte HaCAT.

6. The use according to claim 2, characterized in that: The application of the Atractylodes lancea fibrous root polysaccharide in preparing a product for inhibiting the generation of ROS active oxygen in AB type zebrafish.

7. The use according to claim 2, characterized in that: The application of the Atractylodes macrocephala fibrous root polysaccharide in the preparation of skin anti-aging cream products.

8. The use according to claim 7, characterized in that: The anti-aging skin cream product comprises, by mass, 5-8wt% of 1,3-propylene glycol, 0.2-0.6wt% of ethylenediaminetetraacetic acid, 4-5wt% of squalane, 1.0-1.5wt% of cetearyl alcohol, 1-1.2wt% of polydimethylsiloxane, 2-2.5wt% of isopropyl palmitate, 0.1-0.3wt% of hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, 0.5-0.7wt% of 1,2-pentanediol, 0.05-0.2wt% of phenoxyethanol, 1-5wt% of Atractylodes macrocephala root polysaccharide and the remainder of water.

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