A kind of Chlorella SDEC-18 polysaccharide and its extraction method and application

Abstract

The present invention belongs to the field of microalgae biotechnology, and specifically relates to a chlorella SDEC-18 polysaccharide and its extraction method and application. The chlorella SDEC-18 polysaccharide of the present invention is extracted from chlorella SDEC-18, and contains 4 parts of polysaccharides with different molecular weights, and its peak molecular weights are 1030Da, 2735Da, 10.187KDa and 244.933KDa respectively; the chlorella SDEC-18 polysaccharide is composed of arabinose, galactose and talose, wherein galactose is the main component; the chlorella polysaccharide can significantly remove the excess ROS produced in the cell, reduce the MDA content, and increase the T-AOC and SOD content, and has the effect of removing free radicals and reducing oxidative stress, and has excellent antioxidant effect on the body, and has a wide range of application prospects, including for the preparation of oxidative stress products, the preparation of biopharmaceutical products, functional foods and daily chemical products that delay skin aging and improve the body's immune ability, etc.

Description

Technical Field

[0001] The invention belongs to the field of microalgae biotechnology, and specifically relates to Chlorella SDEC-18 polysaccharide and an extraction method and application thereof. Background Art

[0002] The generation of oxidative stress is closely related to a variety of chronic diseases and is very detrimental to human health. When the antioxidant defense system in the body is out of balance, a large number of reactive oxygen free radicals will be produced, causing oxidative stress damage to the body, ultimately leading to aging, cell apoptosis, organ damage, and chronic metabolic diseases such as diabetes and Alzheimer's disease. Finding compounds with antioxidant effects from natural products and developing new skin care products is of great significance, which is not only beneficial to human health, but also can meet the market demand for new products.

[0003] Algae are rich in high-value bioactive substances, including polysaccharides, peptides, proteins, pigments, amino acids and vitamins. These high-value bioactive substances are beneficial to human health, have the characteristics of non-toxicity, antioxidant, anti-cancer, antibacterial, neuroprotective activity and natural ingredients, and are an important source of natural products for the cosmetics or medical industries.

[0004] The application of microalgae in cosmetics, health care and functional foods has increased year by year. It is widely used to treat skin problems, such as anti-aging, whitening and reducing pigmentation. It can also be used as a functional factor in some drug development and a major additive in food. Although some metabolites of algae have been used in some cosmetic formulas, such as moisturizers and thickeners, or as providers of trace nutrients in functional foods, their role as the main active ingredients is still insufficient. For example, astaxanthin extracted from microalgae is extremely unstable. To solve this problem, microliposome encapsulation technology is usually used to improve its stability, but it also greatly reduces the activity of astaxanthin. Polysaccharides extracted from microalgae have multiple biological activities, but their mechanism of action is not clear. Many microalgae metabolites have not yet been fully developed. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a Chlorella SDEC-18 polysaccharide and an extraction method and application thereof.

[0006] The present invention relates to Chlorella SDEC-18, which was deposited in China Center for Type Culture Collection on December 23, 2015, with a deposit number of CCTCC NO: M2015769.

[0007] The present invention provides a method for extracting Chlorella SDEC-18 polysaccharide, which specifically comprises the following steps:

[0008] S1. Hot water extraction: Weigh 2 to 5 g of lyophilized powder of Chlorella SDEC-18, add 100 mL of deionized water, and extract in a water bath at 80 to 90 ° C for 3 to 4 hours;

[0009] S2. Concentration and centrifugation: The extract was centrifuged at 6000-9000 rpm for 10-20 min to obtain a precipitate and a supernatant; the supernatants were combined and concentrated to 1 / 10 of the original volume at 50-60°C in vacuum;

[0010] S3. Alcohol precipitation treatment: add ethanol to a final concentration of 60-80% (v / v), stand at 0-4°C for 12-16 hours, centrifuge at 5000-8000 rpm for 10-30 minutes, and collect the polysaccharide precipitate;

[0011] S4. Protein removal: Dissolve the collected polysaccharide precipitate, add 1 / 3 of its volume of Sevage reagent, mix and shake, let stand for 10 to 30 minutes, centrifuge, and remove the denatured protein at the junction by taking the water layer;

[0012] S5. Decolorization treatment: The polysaccharide obtained in step S4 is passed through a macroporous resin exchange column to obtain a Chlorella SDEC-18 polysaccharide sample.

[0013] The Chlorella SDEC-18 used for extracting active substances in the present invention is grown to a stable stage.

[0014] The present invention analyzes and measures the molecular weight distribution and monosaccharide composition of the prepared Chlorella SDEC-18 polysaccharide; wherein the Chlorella SDEC-18 polysaccharide contains 4 parts of polysaccharides with different molecular weights, and the peak molecular weights are 1030Da, 2735Da, 10.187KDa and 244.933KDa respectively; the Chlorella SDEC-18 polysaccharide is composed of arabinose (12.28%), galactose (58.80%) and talose (28.92%), wherein galactose is the main component.

[0015] The chlorella polysaccharide can significantly remove excess ROS produced in cells, reduce MDA content, increase T-AOC and SOD content, has the function of removing free radicals and reducing oxidative stress, has excellent antioxidant effect on the body, and has broad application prospects.

[0016] Specifically, the SDEC-18 polysaccharide prepared by the present invention has excellent antioxidant effect and can be used as an antioxidant; specifically comprising:

[0017] The use of the Chlorella SDEC-18 polysaccharide in the preparation of oxidative stress products, including the oxidative stress products in which the Chlorella SDEC-18 polysaccharide inhibits the ROS, MDA, T-AOC and SOD contents of HepG2 cells induced by H2O2;

[0018] The Chlorella SDEC-18 polysaccharide is used in the preparation of biopharmaceutical products, functional foods and daily chemical products for delaying skin aging and improving the body's immune ability.

[0019] The present invention uses chlorella SDEC-18, and the polysaccharide extracted from the chlorella can significantly inhibit the influence of oxidative stress damage caused by H2O2-induced HepG2 cells, and has an antioxidant effect. Its antioxidant property has obvious advantages in delaying skin aging, improving the body's immune ability, etc., and can be applied to the development of biomedicine, functional food and daily chemical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a microscopic morphological photograph of Chlorella SDEC-18 provided in an embodiment of the present invention.

[0021] Figure 2 The invention provides a freeze-dried powder of Chlorella SDEC-18 polysaccharide.

[0022] Figure 3 This is a molecular weight distribution diagram of Chlorella SDEC-18 polysaccharide provided in an embodiment of the present invention.

[0023] Figure 4 This is a monosaccharide analysis diagram of Chlorella SDEC-18 polysaccharide provided in an embodiment of the present invention.

[0024] Figure 5 This is a diagram of the H2O2-induced oxidative stress model of HepG2 cells provided in an embodiment of the present invention.

[0025] Figure 6 This is an analysis chart of the survival rate of HepG2 cells induced by H2O2 and acted on Chlorella SDEC-18 polysaccharide provided in an embodiment of the present invention.

[0026] Figure 7 This is a graph showing the ROS, MDA, T-AOC, and SOD contents of HepG2 cells induced by H2O2 in response to the Chlorella SDEC-18 polysaccharide provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described below through specific embodiments in conjunction with the accompanying drawings.

[0028] The present invention provides a method for extracting Chlorella SDEC-18 polysaccharide, comprising the following steps:

[0029] S1. Hot water extraction: Weigh 2 g of lyophilized powder of Chlorella SDEC-18, add 100 mL of deionized water, and centrifuge in a water bath at 80 ° C for 4 hours, add 100 mL of deionized water to the precipitate, continue to extract for 2 hours, and mix the two extracts;

[0030] S2. Concentration and centrifugation: The extract was centrifuged at 9000 rpm for 20 min to obtain a precipitate and a supernatant; insoluble matter was removed, the supernatant was combined, and vacuum concentrated at 60°C to 1 / 10 of the original volume;

[0031] S3. Alcohol precipitation treatment: add ethanol to the polysaccharide concentrate to a final concentration of 60% (v / v), let stand at 4°C for 12 hours, centrifuge at 8000 rpm for 10 minutes, and collect the polysaccharide precipitate;

[0032] S4. Protein removal: The collected polysaccharide precipitate was redissolved, and 1 / 3 of its volume of Sevag reagent (chloroform: n-butanol = 4:1) was added, mixed and shaken, and then allowed to stand for 30 minutes, centrifuged (4000 rpm, 5 minutes), and the water layer was obtained to remove the denatured protein at the junction; this process was repeated 3 to 4 times until no white precipitate appeared at the junction of the reagent layer after centrifugation;

[0033] S5. Decolorization treatment: The polysaccharide obtained in step S4 is passed through a D280 macroporous resin exchange column to obtain a Chlorella SDEC-18 polysaccharide sample; the sample is decolorized for 1 hour, and the chlorophyll is substantially removed after detection by an ultraviolet spectrophotometer.

[0034] The present invention determines the composition of Chlorella SDEC-18 polysaccharide, including molecular weight determination analysis and monosaccharide composition analysis.

[0035] The invention provides application of Chlorella SDEC-18 polysaccharide in oxidative stress, which mainly includes that Chlorella SDEC-18 polysaccharide inhibits ROS, MDA, T-AOC and SOD contents of HepG2 cells induced by H2O2.

[0036] The present invention provides the use of Chlorella SDEC-18 polysaccharide in the preparation of antioxidant products; Chlorella SDEC-18 polysaccharide exhibits strong antioxidant capacity and is easy to prepare, and can be used in the daily chemical industry to prepare antioxidant products such as facial masks, sunscreens or freeze-dried powders, and can also be used in antioxidant oral preparations in the medical and health care industry and as a main additive for functional products in the food industry, etc., and has broad prospects.

[0037] Example 1

[0038] Collection and observation of Chlorella SDEC-18

[0039] The Chlorella SDEC-18 deposit number is CCTCC NO:M2015769. It was deposited by China Center for Type Culture Collection on December 23, 2015 and was isolated from the lake in Quancheng Park, Jinan City.

[0040] (1) Sample collection

[0041] Samples were collected from the lake in Quancheng Park, Jinan City in March 2011.

[0042] (2) Observation of Chlorella SDEC-18

[0043] Algae cells appear green under an optical microscope. They are spherical or nearly spherical. Their size varies greatly with the growth period. The cell diameter is 7.5-18.0 μm. The microstructure is as follows: Figure 1 shown.

[0044] Example 2

[0045] Extraction of polysaccharides from Chlorella SDEC-18

[0046] (1) Hot water extraction: Weigh 2 g of lyophilized powder of Chlorella SDEC-18, add 100 mL of deionized water, and extract in a water bath at 80°C for 4 hours, then centrifuge. Add 100 mL of deionized water to the precipitate and continue to extract for 2 hours. Mix the two extracts.

[0047] (2) Concentration and centrifugation: centrifuge the extract from step (1) at 9000 rpm for 20 min to obtain a precipitate and a supernatant; remove insoluble matter, combine the supernatants, and concentrate at 60° C. in a vacuum concentration chamber to 1 / 10 of the original volume;

[0048] (3) Alcohol precipitation treatment: add ethanol to the extract of step (2) to a final concentration of 60% (v / v), let stand at 4°C for 12 hours, centrifuge at 8000 rpm for 10 minutes, and collect the polysaccharide precipitate;

[0049] (4) Protein removal: add 1 / 3 of the volume of the extract from step (3) to Sevag reagent (chloroform: n-butanol = 4:1), mix and shake, let stand for 30 min, centrifuge (4000 rpm, 5 min), remove the denatured protein at the interface of the water layer; repeat 3 to 4 times until no white precipitate appears at the interface of the reagent layer after centrifugation;

[0050] (5) Decolorization treatment: the extract of step (4) is passed through a D280 macroporous resin exchange column to obtain a concentrated solution of Chlorella SDEC-18 polysaccharide; the solution is decolorized for 1 hour, and the chlorophyll is substantially removed after detection by an ultraviolet spectrophotometer;

[0051] (6) The concentrated solution in step (5) was freeze-dried in a freeze dryer at -50°C for 48 hours to obtain a freeze-dried powder of Chlorella SDEC-18. Figure 2 shown.

[0052] Example 3

[0053] (1) Molecular weight distribution analysis of Chlorella SDEC-18 polysaccharide

[0054] Using high performance gel permeation chromatography, appropriate amounts of Chlorella SDEC-18 polysaccharide and glucan standards T-1000, T-300, T-150, T-10, and T-5 were dissolved in the mobile phase to prepare a 10 mg / mL polysaccharide solution for sample loading and detection. Before detection, the solution was filtered through a 0.22 μm filter membrane.

[0055] Chromatographic conditions: Ultrahydrogel TM Linear gel chromatography column, 7.8mmid×300mm; column temperature 40℃; mobile phase 0.1mol / L sodium nitrate; flow rate 0.5mL / min; detectors are differential refractive index detector (2410) and Empower workstation, constant temperature 35℃; standard curves are drawn based on the retention time and peak area of ​​different molecular weight dextran standards, and the molecular weight of polysaccharide samples is calculated. The results are as follows Figure 3 As shown;

[0056] from Figure 3 The analysis showed that Chlorella SDEC-18 polysaccharide contained four parts of polysaccharides with different molecular weights, and their peak molecular weights were 1030Da, 2735Da, 10.187KDa and 244.933KDa respectively;

[0057] (2) Analysis of monosaccharide composition distribution of Chlorella SDEC-18 polysaccharide

[0058] The monosaccharide composition of Chlorella SDEC-18 polysaccharide was determined by gas chromatography-mass spectrometry. 10 mg of Chlorella SDEC-18 polysaccharide was accurately weighed and 1 mL of 4 mol·L -1 The polysaccharide was hydrolyzed with trifluoroacetic acid (TFA) at 110 °C for 3 h, and then dried under reduced pressure at 70 °C in a vacuum drying oven. The hydrolyzed Chlorella SDEC-18 polysaccharide was mixed with 1 mL of pyridine, and 1 mL of silanization reagent (N, O-bistrimethylsilyltrifluoroacetamide (BSTFA): trimethylchlorosilane (TMCS) = 99:1) was quickly added. After shaking until it was fully dissolved, it was reacted in a constant temperature drying oven at 70 °C for 30 min, and then the temperature was gradually lowered and the sample was tested.

[0059] Chromatographic conditions: chromatographic column: DB-17 (30m×0.32mm×0.25μm), injection port temperature: 280℃, FID detector temperature: 300℃, carrier gas: nitrogen, column pressure: 73.0kPa, column flow rate: 1.00mL·min -1, split ratio 10:1, temperature program from 50℃ for 3min, at 10℃·min -1 Raise to 280℃, keep for 5min, and inject 1μL. Figure 4 shown.

[0060] from Figure 4 It can be seen that the polysaccharide of Chlorella SDEC-18 is composed of arabinose (12.28%), galactose (58.80%) and talose (28.92%), among which galactose is the main component.

[0061] Example 4

[0062] Oxidative stress test of Chlorella SDEC-18 polysaccharide

[0063] Human liver cancer cell HepG2 cells have the characteristics of simple and convenient culture conditions, high cell survival rate, short cell growth cycle, and difficulty in differentiation. HepG2 cells can also participate in redox reactions, and the generation of free radicals such as reactive oxygen is also related to them. Therefore, HepG2 cells can be selected as an oxidative stress damage model to explore the antioxidant activity of Chlorella SDEC-18 polysaccharide.

[0064] H2O2 is a very strong exogenous reactive oxygen species that can easily penetrate cell membranes and is stable in nature. After decomposition, it produces a large number of free radicals. The free radicals are extremely active, highly oxidizing and unstable. At the same time, free radicals will attack cell membranes and mitochondrial membranes, causing oxidative stress in the body, causing harm to the skin and even the body.

[0065] (1) HepG2 culture

[0066] Place the culture dish containing HepG2 cells (DMEM: fetal bovine serum: double antibody = 6:3:1) in an incubator, and control the incubator conditions to 5% CO2, 95% air, 37°C saturated humidity. Observe the cell growth status every 24 hours and replace with fresh complete medium. Generally, after 24 hours, the cell growth saturation can reach about 60%.

[0067] (2) Establishment of H2O2 oxidative stress model

[0068] HepG2 cells in the logarithmic growth phase were inoculated in a 96-well plate at a cell density of 1×10 5 / mL. The volume of each well is 100μL, and the cells are cultured in an incubator with 5% CO2, 95% air, and saturated humidity at 37℃ for 24h. After the cells adhere to the wall, the supernatant is discarded, and then H2O2 with concentrations of 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5mM is added to the culture medium. The control group is added with serum-free culture medium. After culturing for 24h at 37℃ and 5% CO2, the supernatant is discarded. 100μL MTT solution (final concentration is 5mg / mL) is added to each well, and incubated in an incubator in the dark for 4h, the supernatant is carefully discarded, and 150μL DMSO reagent is added. Oscillate for 3min, let it stand until the MTT purple crystalline product is completely dissolved, and then use an enzyme reader to measure its absorbance value at 490nm. Six parallel experiments are set up for each group to calculate the cell viability. The results are as follows Figure 5 shown.

[0069] Cell viability (%) = (absorbance value of sample group / absorbance value of control group) × 100.

[0070] from Figure 5 The analysis showed that the cell survival rate decreased significantly after different concentrations of H2O2 acted on HepG2 cells. When the concentration of H2O2 was 1.0mM after 24h of induction, the cell survival rate was 51.3%, reaching half lethality. Therefore, the 1.0mM concentration of H2O2 for 24h was selected as the modeling condition for subsequent experiments.

[0071] (3) Effect of SDEC-18 polysaccharide from Chlorella vulgaris on the survival rate of HepG2 cells induced by H2O2

[0072] 100 μL of HepG2 cells in the logarithmic growth phase were taken and cultured in an incubator with 5% CO2, 95% air, and saturated humidity at 37°C for 24 h. The waste liquid was discarded and 1.0 mM H2O2 was added. After culturing for 24 h, the supernatant was discarded. Different concentrations of Chlorella SDEC-18 polysaccharides were added to the culture medium. The control group (no polysaccharide was added, the concentration was 0 μM) was added with serum-free culture medium. After culturing for 24 h, the supernatant was discarded. The cell survival rate of each group was then determined by the MTT method. The specific method was the same as the determination method in (2). Six parallel experiments were set up for each group. The cell survival rate was as follows: Figure 6 shown.

[0073] Cell survival rate (%) = (absorbance value of sample group / absorbance value of control group) × 100

[0074] In this experiment, the concentration range of 0-400 μg / mL of Chlorella SDEC-18 polysaccharide was selected to detect its effect on the survival rate of HepG2 cells. Figure 6The analysis showed that when the concentration of Chlorella SDEC-18 polysaccharide was 0-100 μg / mL, it had no significant effect on cell survival rate; but when the concentration was 200-400 μg / mL, it had a significant effect on cell survival rate. Therefore, low, medium and high concentrations of 25, 50 and 100 μg / mL of Chlorella SDEC-18 polysaccharide were selected for subsequent experiments.

[0075] (4) Effects of Chlorella SDEC-18 polysaccharide on ROS, MDA, T-AOC, and SOD contents in HepG2 cells induced by H2O2

[0076] Take 100 μL of HepG2 cells in the logarithmic growth phase, culture them in an incubator with 5% CO2, 95% air, and 37°C saturated humidity for 24 hours, discard the waste liquid, add 1.0 mM H2O2 except the control group (Con), discard the supernatant after culturing for 24 hours, add SDEC-18 polysaccharides of high, medium, and low dose groups to the culture medium, add 100 μg / mL ascorbic acid to the positive control group, add serum-free culture medium to the model group (Mod) and the control group (Con), discard the supernatant after culturing for 24 hours, load DCFH-DA (10 μM) in each well, react in the dark at 37°C for 40 minutes, discard the reaction solution, wash the cells twice, then digest the cells with trypsin, discard the supernatant after centrifugation, resuspend the cells in PBS, move into a 96-well ELISA plate black board, and measure the relative intensity of ROS (excitation wavelength 502 nm, emission wavelength 530 nm). Set up 6 parallel experiments for each group.

[0077] According to the method of step (4), the content of MDA, T-AOC and SOD was determined according to the instructions of the MDA, T-AOC and SOD kits. The results are as follows Figure 7 shown.

[0078] from Figure 7 Analysis showed that compared with the Con group, the ROS content of the Mod group cells was significantly increased (p<0.05), which successfully verified the establishment of the oxidative stress model; after intervention with Chlorella SDEC-18 polysaccharide, the ROS and MDA contents in the cells were significantly reduced in a dose-dependent manner, and the T-AOC and SOD were significantly increased in a dose-dependent manner. It can be proved that Chlorella SDEC-18 polysaccharide can significantly remove excess ROS produced in the cells and increase the content of T-AOC and SOD. It has a good antioxidant effect and can be used for the development of antioxidant cosmetics.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The application of Chlorella SDEC-18 polysaccharide in the preparation of cell antioxidants, characterized in that: In the application, the concentration of the Chlorella SDEC-18 polysaccharide is 25-100 μg / mL. The Chlorella SDEC-18 polysaccharide at this concentration has no significant effect on cell survival rate, but can significantly remove excess ROS generated in cells and increase T-AOC and SOD content. The Chlorella SDEC-18 is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M2015769; The extraction method of the Chlorella SDEC-18 polysaccharide is: S1. Hot water extraction: Weigh 2-5 g of lyophilized powder of Chlorella SDEC-18, add 100 mL of deionized water, and extract in a water bath at 80-90 ° C for 3-4 hours; S2. Concentration and centrifugation: centrifuge the above extract at 6000-9000 rpm for 10-20 min to obtain a precipitate and a supernatant; combine the supernatants and concentrate them at 50-60°C in a vacuum to 1 / 10 of the original volume; S3. Alcohol precipitation: add ethanol to a final concentration of 60-80% (v / v), stand at 0-4°C for 12-16 hours, centrifuge at 5000-8000 rpm for 10-30 minutes, and collect the polysaccharide precipitate; S4. Protein removal: Dissolve the collected polysaccharide precipitate, add 1 / 3 of its volume of Sevage reagent, mix and shake, let stand for 10-30 minutes, centrifuge, and remove the denatured protein at the interface by taking the water layer; S5. Decolorization treatment: The polysaccharide obtained in step S4 is passed through a macroporous resin exchange column to obtain the Chlorella SDEC-18 polysaccharide product.

2. The use of the cellular antioxidant according to claim 1 in the preparation of a product capable of improving cellular antioxidant activity, characterized in that: The products capable of improving the antioxidant capacity of cells include daily chemical products capable of improving the antioxidant capacity of cells.

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