Small-molecular dendrobium polysaccharide, preparation method and application in resisting skin photoaging

Abstract

The invention relates to the technical field of separation and purification of plant active ingredients, in particular to micromolecular dendrobium polysaccharide, a preparation method and application in skin photoaging resistance. According to the method, acid extraction and hydroxyl free radical extraction are creatively combined, firstly, cell walls of dendrobium officinale are primarily degraded by organic acid, then hydroxyl free radicals in H2O2 are activated through ultraviolet radiation to break glucosidic bonds, and polysaccharide is further degraded, so that the molecular weight of the polysaccharide is effectively reduced, and the preparation efficiency and the product quality are remarkably improved; the molecular weight of the prepared dendrobium officinale polysaccharide is 140-180KDa, HaCaT cell experiments prove that the dendrobium officinale polysaccharide has the effect of protecting cells, can remarkably reduce cell damage caused by UVB radiation, and has the effect of resisting skin photoaging when being added into skin cosmetics.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification of plant active ingredients, and in particular to small-molecule dendrobium polysaccharide, a preparation method and application in resisting skin photoaging. Background Art

[0002] Dendrobium candidum is the dried stem of the herbaceous plant of the genus Dendrobium in the orchid family, also known as Dendrobium officinale. Dendrobium candidum has the effects of benefiting the stomach and promoting fluid production, nourishing yin and clearing away heat. Dendrobium candidum contains a variety of important active ingredients, including polysaccharides, alkaloids, amino acids and flavonoids, among which polysaccharides are the main components of Dendrobium candidum.

[0003] The physiological activity of Dendrobium candidum is closely related to the content of polysaccharides, but due to its high viscosity and poor solubility, Dendrobium candidum polysaccharides have not been used in the field of functional foods. In order to obtain polysaccharides with biological activity from Dendrobium candidum more efficiently and conveniently, many scholars at home and abroad have studied and optimized its extraction, separation, purification and analysis methods. The extraction methods of crude polysaccharides from Dendrobium candidum include hot water extraction, ultrasonic-assisted extraction, microwave-assisted extraction, enzyme-assisted extraction, steam-assisted flash extraction, alkali extraction, mechanochemical-assisted extraction and enzyme-assisted extraction. Hot water extraction is a traditional method for extracting polysaccharides, which has the advantages of low cost, simple operation and low implementation requirements, but has problems such as low extraction efficiency and large molecular weight of extracted polysaccharides. Ultrasonic-assisted extraction and microwave-assisted extraction can improve the extraction efficiency of polysaccharides and reduce environmental pollution, but they have high requirements for professional equipment and are difficult to use in industrialization. At the same time, microwaves will damage the structure of polysaccharides, thus changing the activity of polysaccharides. In addition, the molecular weight of polysaccharides degraded by this method is relatively large, which is not conducive to the biological activity of polysaccharides. The enzymatic method has mild conditions and strong specificity, but it is costly and easy to introduce proteins, affecting product quality. Degradation by simple mechanical and physical methods can easily make any similar chemical bond on the molecular chain have an equal chance of breaking, which can easily cause some cross-linking and branching reactions.

[0004] Therefore, it is of great research significance to optimize the extraction method of Dendrobium officinale polysaccharides and efficiently produce low molecular weight Dendrobium officinale polysaccharides. Summary of the invention

[0005] In order to overcome the above technical defects, the present invention provides a small molecule dendrobium polysaccharide, a preparation method and an application in anti-skin photoaging. A small molecule dendrobium polysaccharide with better biological activity is extracted and separated from Dendrobium officinale, and its biological activity is studied. It is found that it has the effect of protecting cells and can significantly reduce cell damage caused by UVB radiation.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] On the one hand, a preparation method of small molecule dendrobium polysaccharide is provided, including the following steps:

[0008] S1. Take Dendrobium officinale, after pulverization and degreasing, add an organic acid solution for extraction, concentrate the filtrate to obtain a concentrated solution;

[0009] S2. Add ethanol to the concentrated solution for precipitation, centrifuge to collect the precipitate, and freeze-dry to obtain a crude extract of dendrobium polysaccharide;

[0010] S3. Dissolve the crude extract of dendrobium polysaccharide in water, add H2O2, and carry out a degradation reaction under UV assistance to obtain low molecular weight dendrobium polysaccharide with a molecular weight of 140 - 180KDa.

[0011] Further, in S3, after the degradation reaction, add MnO2 to the solution to remove the residual H2O2, centrifuge to collect the leaching solution, dialyze and concentrate, and freeze-dry to obtain low molecular weight dendrobium polysaccharide with a molecular weight of 140 - 180KDa.

[0012] Further, in S1, the pH of the organic acid solution is 2;

[0013] Further, in S1, the mass-volume ratio of Dendrobium officinale to the organic acid is 1g:(30 - 70)mL;

[0014] In some specific embodiments, the mass-volume ratio of Dendrobium officinale to the organic acid can be selected as 1g:30mL, 1g:40mL, 1g:50mL, 1g:60mL, 1g:70mL;

[0015] Further, in S1, the organic acid solution is selected from one or more of citric acid solution, hydrochloric acid solution, and acetic acid solution. In a further preferred embodiment, the organic acid solution is citric acid solution;

[0016] Further, in S1, the extraction temperature is 90 - 110°C;

[0017] In some specific embodiments, the extraction temperature can be selected as 90°C, 95°C, 100°C, 105°C, 110°C;

[0018] Further, in S1, the extraction time is 1 - 3h;

[0019] In some specific embodiments, the extraction time can be selected as 1h, 1.5h, 2h, 2.5h, 3h;

[0020] Further, in S1, the degreasing conditions are: add petroleum ether to the Dendrobium officinale powder, degrease at room temperature for 1 - 3h, filter, evaporate the solvent to dryness, and obtain the medicinal residue for standby;

[0021] Further, S2 specifically includes adding concentrated solution to 3 - 6 times the volume of absolute ethanol, and standing still at 1 - 5 °C for 10 - 20 h; taking out the reaction solution, centrifuging, discarding the supernatant, and collecting the precipitate; adding appropriate deionized water to the precipitate, removing residual ethanol, followed by dialysis concentration and freeze - drying to obtain the crude extract of Dendrobium officinale polysaccharide;

[0022] In some specific embodiments, in S2, the volume ratio of the concentrated solution to absolute ethanol can be selected as 1:3, 1:4, 1:5, 1:6;

[0023] Further, in S3, the final concentration of H2O2 is 80 - 120 mmol / L;

[0024] In some specific embodiments, the final concentration of H2O2 can be selected as 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L;

[0025] Further, in S3, the UV irradiation power is 800 - 1000 μW / cm 2 ;

[0026] In some specific embodiments, the UV irradiation power is 800 μW / cm 2 、850 μW / cm 2 、900 μW / cm 2 、950 μW / cm 2 、1000 μW / cm 2 ;

[0027] Further, in S1, before dialysis concentration, adding Sevag reagent to the leaching solution and stirring to remove proteins;

[0028] Further, in S3, before dialysis concentration, adding Sevag reagent to the leaching solution and stirring to remove proteins;

[0029] In the second aspect, the present invention provides a small - molecule Dendrobium officinale polysaccharide, which is characterized by being prepared by the above - mentioned method.

[0030] In the third aspect, there is provided an application of a small - molecule Dendrobium officinale polysaccharide in the preparation of anti - skin photo - aging cosmetics.

[0031] Further, the mechanism of action of the cosmetics is manifested as significantly reducing the level of intracellular reactive oxygen species caused by ultraviolet irradiation and inhibiting the expression of matrix metalloproteinase - 1 (MMP - 1).

[0032] Further, the cosmetics take the small - molecule Dendrobium officinale polysaccharide as the active ingredient and are made into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

[0033] It is well understood by those skilled in the art that the pharmaceutically acceptable carriers are generally recognized for this purpose and serve as inactive ingredients of the medicaments.

[0034] The adjuvants include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.

[0035] Among them, the diluent can be one or more of mannitol, sucrose, lactose, sorbitol, xylitol, polyethylene glycol, propylene glycol, vegetable oil, and mineral oil; the disintegrant can be one or more of croscarmellose sodium, colloidal silicon dioxide, and citric acid; the binder can be one or more of starch paste, ethanol, water, and povidone alcohol solution; the preservative can be one or more of ethyl p-hydroxybenzoate, propyl hydroxybenzoate, sorbic acid, potassium sorbate, calcium propionate, sodium dehydroacetate, sodium diacetate, and sodium lactate; the antioxidant can be one or more of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, dibutylhydroxytoluene, glycine, inositol, ascorbic acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, and sodium metabisulfite; the flavoring agent can be one or more of aspartame, sucrose, xylitol, stevioside, sodium cyclamate, sorbitol, cocoa, pure vanilla, vanillin, ethyl vanillin, chocolate, malt, and mint; the suspending agent can be one or more of xanthan gum, polyvinylpyrrolidone, sodium alginate, aluminum stearate, and hydrogenated vegetable oil; the emulsifier can be one or more of alkyl sulfates, soaps, dodecylbenzenesulfonates, lactate esters, sulfosuccinates, monoglyceride sulfonates, phosphate esters, siloxanes, and taurates.

[0036] Among them, the cosmetic exists in the form of cream, lotion, gel, or aqueous solution.

[0037] The cosmetic of the present invention contains 0.1 - 90% by weight of the active ingredient.

[0038] The pharmaceutical composition can be prepared according to methods known in the art. For this purpose, if necessary, the active ingredient can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to form a suitable administration form or dosage form for human use.

[0039] In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners, or other materials can also be added to the pharmaceutical preparation.

[0040] Compared with the prior art, the present invention has the following effects:

[0041] (1) The present invention innovatively combines acid extraction and hydroxyl radical extraction. First, organic acids are used to preliminarily degrade the cell wall of Dendrobium officinale, and then hydroxyl radicals in H2O2 are activated by ultraviolet irradiation to break glycosidic bonds for further degradation of polysaccharides, thereby effectively reducing the molecular weight of polysaccharides and significantly improving the preparation efficiency and product quality;

[0042] (2) The molecular weight of the Dendrobium officinale polysaccharide prepared by the present invention is 140 - 180KDa. Verified by HaCaT cell experiments, it has the effect of protecting cells and can significantly reduce cell damage caused by UVB radiation. Therefore, Dendrobium officinale polysaccharide has important application value in the preparation of anti-skin photoaging products. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the process flow chart of the present invention;

[0044] Figure 2 is the effect of different solid-liquid ratio treatments on the extraction rate of Dendrobium polysaccharide;

[0045] Figure 3 is the effect of different UV irradiation power treatments on the molecular weight of Dendrobium polysaccharide;

[0046] Figure 4 is the infrared spectrogram of the small molecule Dendrobium polysaccharide of the present invention;

[0047] Figure 5 is the effect of different Dendrobium polysaccharide concentrations on cell viability;

[0048] Figure 6 is the effect of the small molecule Dendrobium polysaccharide of the present invention on ultraviolet irradiation damage;

[0049] Figure 7 is the effect of the small molecule Dendrobium polysaccharide of the present invention on the ROS content. DETAILED DESCRIPTION OF THE INVENTION

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. For the experimental methods without specific conditions indicated in the following examples, they are generally carried out according to conventional conditions or the conditions recommended by the manufacturer. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0051] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0052] As Figure 1 shown, the present invention aims to provide a preparation method of small molecule Dendrobium officinale polysaccharide. By precisely optimizing the usage amount of organic acid and the UV irradiation power, and using HaCaT cells treated with UV (200 μj / cm 2 ) as an experimental model, the optimal conditions are screened to prepare small molecule Dendrobium officinale polysaccharide with the best protective effect.

[0053] Based on the present invention, the small molecule Dendrobium officinale polysaccharide is prepared by the following steps: Take Dendrobium officinale and crush it. Add petroleum ether to the Dendrobium officinale powder and degrease it at room temperature for 1 - 3 h. Filter, and after evaporating the solvent, add an organic acid solution for extraction. Concentrate the filtrate to obtain a concentrated solution; Add absolute ethanol to the concentrated solution and let it stand at 1 - 5 °C for 10 - 20 h. Take out the reaction solution, centrifuge, discard the supernatant, and collect the precipitate; Add an appropriate amount of deionized water to the precipitate, remove the residual ethanol, then dialyze and concentrate, and freeze-dry to obtain a crude extract of Dendrobium officinale polysaccharide; After dissolving the crude extract of Dendrobium officinale polysaccharide in water, add H2O2 and carry out a degradation reaction under UV assistance. After the degradation reaction, add MnO2 to the solution to remove the residual H2O2. Centrifuge to collect the leaching solution. Add Sevag reagent to the leaching solution, stir to remove proteins, then dialyze and concentrate, and freeze-dry to obtain low molecular weight Dendrobium officinale polysaccharide with a molecular weight of 140 - 180 KDa.

[0054] Based on the present invention, the pH of the organic acid solution is 2;

[0055] Based on the present invention, the organic acid solution is selected from one or more of citric acid solution, hydrochloric acid solution, and acetic acid solution;

[0056] Based on the present invention, the mass-to-volume ratio of Dendrobium officinale to the organic acid is 1 g:(30 - 70) mL; during the actual operation process, the mass-to-volume ratio of Dendrobium officinale to the organic acid can be selected as 1 g:30 mL, 1 g:40 mL, 1 g:50 mL, 1 g:60 mL, 1 g:70 mL;

[0057] Based on the present invention, the extraction temperature is 90 - 110 °C; during the actual operation process, the extraction temperature can be selected as 90 °C, 95 °C, 100 °C, 105 °C, 110 °C;

[0058] Based on the present invention, the extraction time is 1 - 3 h; during the actual operation process, the extraction time can be selected as 1 h, 1.5 h, 2 h, 2.5 h, 3 h;

[0059] Based on the present invention, the volume ratio of the concentrated solution to absolute ethanol is 1:(3 - 6); during the actual operation process, the volume ratio of the concentrated solution to absolute ethanol can be selected as 1:3, 1:4, 1:5, 1:6;

[0060] Based on the present invention, the final concentration of H2O2 is 80 - 120 mmol / L; during the actual operation process, the final concentration of H2O2 can be selected as 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L;

[0061] Based on the present invention, the UV irradiation power is 800 - 1000 μW / cm 2 ; during the actual operation process, the UV irradiation power is 800 μW / cm 2 、850 μW / cm 2 、900 μW / cm 2 、950 μW / cm 2 、1000 μW / cm 2 ;

[0062] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0063] Example 1 Preparation of Crude Polysaccharide Extract

[0064] Take 20 g of defatted Dendrobium officinale powder, add it to a citric acid solution with a pH of 2, stir and extract for 2 h under boiling water bath conditions. After the extract is naturally cooled, centrifuge at a speed of 4000 rpm for 10 minutes, collect the supernatant, and then adjust the supernatant to neutral (pH = 7) using 4 M potassium hydroxide; Rotate and evaporate the neutral supernatant at 60 °C until it is concentrated to 1 / 4 of the original volume; Subsequently, add 4 times the volume of absolute ethanol and let it stand at 4 °C for 12 h; Take out the reaction solution, centrifuge, discard the supernatant, collect the precipitate, add an appropriate amount of deionized water to the precipitate, remove the residual ethanol, add Sevag reagent to the extract, stir to remove proteins; After removing the proteins, place the polysaccharide solution in a 3000 Da dialysis bag and dialyze at 4 °C for 48 h, and then perform freeze-drying to obtain the crude polysaccharide extract.

[0065] Example 1.1

[0066] Adopt the method of Example 1, wherein the mass-to-volume ratio of Dendrobium officinale to the citric acid solution is 1 g: 30 mL.

[0067] Example 1.2

[0068] Adopt the method of Example 1, wherein the mass-to-volume ratio of Dendrobium officinale to the citric acid solution is 1 g: 40 mL.

[0069] Example 1.3

[0070] Adopt the method of Example 1, wherein the mass-to-volume ratio of Dendrobium officinale to the citric acid solution is 1 g: 50 mL.

[0071] Example 1.4

[0072] Adopt the method of Example 1, wherein the mass-to-volume ratio of Dendrobium officinale to the citric acid solution is 1 g: 60 mL.

[0073] Example 1.5

[0074] Adopt the method of Example 1, wherein the mass-to-volume ratio of Dendrobium officinale to the citric acid solution is 1 g: 70 mL.

[0075] Determine the content of the crude polysaccharide extracts prepared in Examples 1.1 - 1.5 above. Figure 2It can be seen that the citric acid-to-liquid ratio has a certain effect on the extraction rate. When the ratio increases from 1:30 to 1:50, the extraction rate increases significantly, reaching a maximum value at a ratio of 1:50. The extraction rate decreases slightly at a ratio of 1:70. This shows that when the ratio is 1:50, citric acid can contact the substrate more fully, thereby improving the extraction efficiency of the target substance. However, too high a ratio may lead to excessive citric acid, which will cause excessive degradation of the polysaccharide component and reduce the extraction rate. The extraction rate of the crude polysaccharide extract is the highest when the citric acid-to-liquid ratio is 1:50.

[0076] Example 2

[0077] The crude polysaccharide obtained in Example 1.3 was dissolved in deionized water, and H2O2 was added until the final concentration of H2O2 was 100 mmol / L. The product was irradiated with UV light for 2 h. After degradation, 5% MnO2 by mass of H2O2 was added immediately. The product was stirred on a magnetic stirrer for 12 h to remove residual H2O2. The solution was centrifuged, the supernatant was concentrated, and ethanol was precipitated. The product was deproteinized 3 times using the Sevag method. The organic solvent and ethanol were removed by stirring in a water bath at 50°C. The product was then placed in a 3000Da dialysis bag, dialyzed at 4°C for 48 h, and freeze-dried to obtain Dendrobium officinale polysaccharide.

[0078] Example 2.1

[0079] The method of Example 1 is adopted, wherein the UV irradiation power is 800 μW / cm 2 .

[0080] Example 2.2

[0081] The method of Example 1 is adopted, wherein the UV irradiation power is 850 μW / cm 2 .

[0082] Example 2.3

[0083] The method of Example 1 is adopted, wherein the UV irradiation power is 900 μW / cm 2 .

[0084] Example 2.4

[0085] The method of Example 1 is adopted, wherein the UV irradiation power is 950 μW / cm 2 .

[0086] Example 2.5

[0087] The method of Example 1 is adopted, wherein the UV irradiation power is 1000 μW / cm 2 .

[0088] The molecular weight of the small molecule Dendrobium polysaccharides prepared in the above Examples 2.1-2.5 was measured.

[0089] Test method: The weight-average molecular weight (Mw) of polysaccharides from Dendrobium officinale was determined on a high performance gel permeation chromatography (HPGPC) instrument according to the following method. Briefly, a polysaccharide sample (5.0 mg) was dissolved in deionized water (1 ml), then passed through a 0.22 μm filter and applied to a gel chromatography column. The column was maintained at 25 °C, eluted with 0.1 M NaNO3 solution in PBS buffer at a flow rate of 1 ml / min, and a calibration curve was obtained using maltotriose with different molecular weights. The retention time was substituted into the calibration curve equation to calculate the molecular weight of the polysaccharide sample from Dendrobium officinale.

[0090] It is known from Figure 3 that ultraviolet light activates hydrogen peroxide to generate hydroxyl radicals, and the difference in the content of free radicals under different ultraviolet power conditions significantly affects the degradation effect and molecular weight distribution of polysaccharides. When the ultraviolet power increased from 800 μW / cm 2 to 1000 μW / cm 2 , the molecular weight of the polysaccharide gradually decreased, indicating that stronger ultraviolet power would activate H2O2 to generate more hydroxyl radicals to degrade the polysaccharide. Especially at 950 and 1000 μW / cm 2 , the degradation efficiency reached the best.

[0091] Comparative Example 1

[0092] Hot water extraction method

[0093] Comparative Example 2

[0094] Hydroxyl radical extraction method

[0095] Comparative Example 3

[0096] Alkali method combined with hydroxyl radical extraction method.

[0097] The molecular weights of the small molecule polysaccharides from Dendrobium officinale prepared in the above Comparative Examples 1-3 were measured, and the results are shown in Table 1.

[0098] Table 1

[0099] Example 2.4 Comparative Example 1 Comparative Example 2 Comparative Example 3 Molecular weight (KDa) 160.31±0.05 <![CDATA[806.33±51.78 *** > <![CDATA[296±56.02 *** > <![CDATA[206.67±27.54 *** >

[0100] Note: *** indicates P < 0.001 compared with Example 2.4

[0101] As can be seen from the above table, as shown in Table 1, the molecular weight of the polysaccharides extracted from Dendrobium officinale by the acid method combined with the hydroxyl radical method is 160.31KDa. Compared with the traditional hot water extraction method, as well as the polysaccharides extracted by the alkali method combined with the hydroxyl radical extraction method and the hydroxyl radical method, the molecular weight has decreased significantly. The biological activity of polysaccharides usually changes with the change of molecular weight. If the molecular weight of polysaccharides is too high, it is not conducive to penetrating multiple cell membrane barriers to play a pharmacological role in organisms. Therefore, a smaller molecular weight can make polysaccharides have higher activity value.

[0102] The small molecule Dendrobium officinale polysaccharides prepared by the present invention were detected by an infrared spectrometer, and the spectral diagram was obtained as Figure 4 shown. At the same time, the Dendrobium officinale polysaccharides prepared in Comparative Example 1 were detected and compared. As can be seen from Figure 4 this, the peak near 3450 cm -1 of the small molecule Dendrobium officinale polysaccharides prepared by the present invention is caused by the stretching vibration of O-H; the strong band near 2930 cm -1 is related to the stretching vibration of C-H in the sugar ring; the peak near 1750 cm -1 is the stretching vibration of C=O of the uronic acid group. There is no absorption peak at AE here, proving that it contains almost no uronic acid, which corresponds to the results in the chemical composition analysis; the weak peak near 1650 cm -1 belongs to the bending vibration of O-H; around 1400 cm -1 is caused by the vibration of C-H bonds. The results of infrared spectroscopy analysis show that compared with the polysaccharides extracted by the traditional hot water extraction method, the characteristic functional groups and backbone structures of the polysaccharides extracted by the acid method combined with the hydroxyl radical are not damaged.

[0103] Example 3 examines the protective effect of Dendrobium officinale polysaccharides on ultraviolet irradiation damage

[0104] The cell lines used in the experiment included HaCaT (Human skin keratinocyte), which was derived from the ATCC strain bank. HaCaT was cultured in a sterile DMEM high-glucose medium containing 10% FBS.

[0105] After trypsinizing HaCaT cells, centrifuging them, adjusting the cell concentration to 4×10^5 cells / mL, inoculating them into a 96-well plate at 100 μL per well, and culturing them adherently for 24 hours. After the culture, discard the medium, wash the cells once with PBS, then add Dendrobium officinale solutions at different concentrations (100 μL / well, diluted with CM medium), set up a blank control group, and incubate at 37°C for 24 hours. After incubation, aspirate and discard the medium, wash the cells once with PBS, then add methylene blue staining solution (50 μL / well), and incubate at 37°C for 1 hour. After incubation, thoroughly wash the 96-well plate under running water, dry it, and finally add 100 μL of EB solution to each well, shake it on an oscillator for 15 minutes, and measure the absorbance (OD value) of each well at a wavelength of 590 nm using a microplate reader. Calculate the cell survival rate based on the OD value.

[0106] After the cells grew adherently for 24 hours, discard the medium, wash the cells with PBS, and add 2 mL of PBS for irradiation. After UV irradiation treatment, add Dendrobium officinale polysaccharides at different concentrations and determine their effects on MMP-1 (matrix metalloproteinase-1).

[0107] It can be seen from Figure 5 that the effects of polysaccharide solutions at different concentrations on cell survival rate are relatively small. As the concentration gradually increases from 0 μg / mL to 800 μg / mL, the cell survival rate remains at a relatively high level (both higher than 80%), and no significant cytotoxic effect appears.

[0108] It can be seen from Figure 6 that compared with the sodium hyaluronate group (HA), Dendrobium officinale polysaccharide solution can inhibit the expression of MMP-1 in UVB-induced HaCaT cells, and this inhibitory effect is concentration-dependent. Specifically, as the polysaccharide concentration increases, the content of MMP-1 gradually decreases. Especially at a high concentration of 400 μg / mL, it is significantly lower than the sodium hyaluronate group (HA). This indicates that Dendrobium officinale polysaccharide has a good effect of inhibiting the expression of MMP-1 in the pathway, can effectively inhibit the degradation of collagen, and thus play a role in protecting the skin and delaying photoaging.

[0109] Example 4 examines the antioxidant effect of Dendrobium officinale polysaccharide

[0110] HaCaT cells in the logarithmic growth phase were cultured adherently in an environment of 37 °C and 5% CO2. The polysaccharide solution of Dendrobium officinale was diluted into different concentrations (100 μg / mL, 200 μg / mL, 400 μg / mL), and quercetin was used as a positive control (HA). After the cells were cultured adherently for 24 hours, the polysaccharide solution of Dendrobium officinale was added, and incubation was continued for 12 hours. After aspirating the original culture medium, serum-free medium containing DCFH-DA (20 μM) was added to each well (except for the blank group). Using a multifunctional microplate reader, the fluorescence intensity was detected in real time at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The detection period was 120 minutes, and the data were recorded every 5 minutes. According to the fluorescence values of the Dendrobium officinale polysaccharide treatment group and the blank control group, the relative fluorescence intensity was calculated, and the time point of the maximum fluorescence value was determined, so as to calculate the relative expression level of reactive oxygen species (ROS) during the reaction process.

[0111] It can be seen from Figure 7 that compared with the blank control group, the ROS level in the cells of the HA treatment group increased significantly, indicating that the scavenging effect of HA on intracellular reactive oxygen species after UVB irradiation treatment was limited. After adding the polysaccharide of Dendrobium officinale, the intracellular ROS level showed a significant downward trend, and was negatively correlated with the polysaccharide concentration: when the polysaccharide concentration was 100 μg / mL, the ROS level was still relatively high, about 200% of the control group. As the polysaccharide concentration increased to 200 μg / mL, the ROS level decreased significantly, about 150% of the control group. At a high concentration (400 μg / mL), the ROS level further decreased to about 120% of the control group, approaching the blank control level.

[0112] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Those skilled in the art can make various similar representations under the inspiration of the present invention without departing from the purpose and claims of the present invention. Such transformations all fall within the protection scope of the present invention.

Claims

1. A preparation method of small molecule dendrobium polysaccharide, characterized in that, It includes the following steps: S1. Take Dendrobium officinale, after pulverization and degreasing, add it to an organic acid solution for extraction, concentrate the filtrate to obtain a concentrated solution; S2. Add ethanol to the concentrated solution for precipitation, centrifuge to collect the precipitate, and freeze-dry to obtain a crude extract of Dendrobium officinale polysaccharide; S3. Dissolve the crude extract of Dendrobium officinale polysaccharide in water, add H2O2, and carry out a degradation reaction under UV assistance to obtain a low-molecular-weight Dendrobium officinale polysaccharide with a molecular weight of 140-180KDa.

2. The preparation method according to claim 1, wherein In S1, the pH of the organic acid solution is 2; And / or, the mass-volume ratio of Dendrobium officinale to the organic acid solution is 1g:(30-70)mL; And / or, in S1, the extraction temperature is 90-110°C; And / or, in S1, the extraction time is 1-3h.

3. The preparation method according to claim 1, wherein In S1, the degreasing conditions are: add petroleum ether to the Dendrobium officinale powder, degrease at room temperature for 1-3h, filter, evaporate the solvent to dryness to obtain the medicinal residue for standby.

4. The preparation method according to claim 1, wherein In S1, the organic acid solution is selected from one or more of citric acid solution, hydrochloric acid solution, and acetic acid solution.

5. The preparation method according to claim 1, wherein S2 is specifically as follows: add 3-6 volumes of absolute ethanol to the concentrated solution, and let it stand at 1-5°C for 10-20h; take out the reaction solution, centrifuge, discard the supernatant, and collect the precipitate; add an appropriate amount of deionized water to the precipitate, remove the residual ethanol, then dialyze and concentrate, and freeze-dry to obtain a crude extract of Dendrobium officinale polysaccharide.

6. The preparation method according to claim 1, characterized in that, In S3, the final concentration of H2O2 is 80-120mmol / L; and / or, the UV irradiation power is 800 - 1000 μW / cm 2 .

7. The preparation method according to claim 1, wherein In S3, after the degradation reaction, add MnO2 to the solution to remove the residual H2O2, centrifuge to collect the leaching solution, dialyze and concentrate, and freeze-dry to obtain a low-molecular-weight Dendrobium officinale polysaccharide with a molecular weight of 140-180KDa.

8. A small molecule dendrobium polysaccharide, characterized in that, It is prepared by the method according to any one of claims 1-5.

9. The application of a small-molecule Dendrobium officinale polysaccharide as claimed in claim 8 in the preparation of anti-skin photoaging cosmetics.

10. The application according to claim 9, characterized in that, The action mechanism of the cosmetics is manifested as significantly reducing the level of intracellular reactive oxygen species caused by ultraviolet irradiation and inhibiting the expression of MMP-1.

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