Shifocui tea polysaccharide and its extraction method and application

Abstract

The invention discloses a Shifocui tea polysaccharide and an extraction method and application thereof. The extraction method comprises drying and crushing Shifocui tea leaves to obtain tea powder, defatting and decolorizing, adding the obtained pretreated tea powder and a composite enzyme into a phosphate buffer for enzymolysis, passivating and centrifuging the enzymolysis product, adjusting the pH of the first supernatant, adding the precipitate into water for temperature reaction to obtain a water extraction product, centrifuging the water extraction product, adjusting the pH of the second supernatant, mixing with the first supernatant for evaporation and concentration, adding anhydrous ethanol, standing and centrifuging, drying the precipitate and removing protein, standing and centrifuging, adjusting the third supernatant to neutral, dialysis, evaporation concentration and freeze drying to obtain Shifocui tea polysaccharide. The Shifocui polysaccharide prepared by the invention exhibits good free radical scavenging efficiency in vitro, exhibits a good improvement effect on the reduction of cell activity caused by free radical damage, and has a repairing effect on oxidative damage at the cellular level, and has broad application prospects in the fields of food, medicine, cosmetics, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of application of natural product polysaccharides, and relates to a Shifocui tea polysaccharide and an extraction method and application thereof, and specifically relates to a summer and autumn tea tree fresh leaf polysaccharide - Shifocui tea polysaccharide and an extraction method thereof and a new application in repairing oxidatively damaged cells. Background Art

[0002] Various reactive oxygen species (ROS) free radicals naturally existing in cells are mainly generated in the mitochondria of various tissues and cells. However, mitochondria are also one of the most sensitive organelles in cells to physiological changes. Once cells are stressed by different physical and chemical factors, mitochondrial function will be inhibited, which is manifested as mitochondrial morphological fracture, membrane potential loss and even more ROS generation. ROS (especially H2O2 and ·OH) as a small molecule can freely pass through the cytoplasmic membrane to enter and exit the cell. Therefore, the accumulation of free radicals, whether exogenously taken or endogenously induced, will cause the generation of cell pathological states, such as aging, neurological decline and cancer. Oxidative damage to cells caused by exogenous oxidants will also be reflected in the levels of various antioxidant enzymes in cells. The antioxidant activity of polysaccharides from different sources will also show an increase in antioxidant enzyme activity. Antioxidant activity is often closely related to biological functions such as anti-aging, immunomodulatory activity, hypoglycemia and anti-tumor, especially immunomodulatory activity, because oxygen free radicals are closely related to the regulation of certain physiologically active substances and inflammatory immune processes. Under pathological conditions, cell oxidative damage caused by free radicals will also act on the immune system, causing a decrease in immune function.

[0003] Among the various physiological activities of tea polysaccharides, the hypoglycemic activity of tea polysaccharides is the most well-known. In addition, the antioxidant activity of tea polysaccharides is also well-known. As a polyhydroxy compound, polysaccharides have antioxidant activity, which is a natural property of polysaccharides. Although there are many studies on the antioxidant activity of tea polysaccharides, there are always some disputes. As a complex natural macromolecule, the biological activity of tea polysaccharides is easily affected by many factors, from the cultivation environment and tea tree varieties to the sampling site, sampling season, finished tea production method, and even the extraction process. The biological activity of tea polysaccharides can be affected by many factors. Southwest Anhui, as the main production area of ​​Anhui tea, has planted many national-level fine varieties. The Shifocui (Camellia sinensis (L.) O. Kuntze cv. 'Shifocui') variety used in this project began to be bred in 1989 and was identified as a national-level fine variety in 2010. So far, this variety of tea tree resources are only used for spring tea production, but its excellent cold-resistant properties make it an abundant source of summer, autumn and winter tea. However, research on the functional components of this variety of tea tree has not been reported.

[0004] Hot water extraction is the most commonly used method. This method has the advantages of being simple, convenient, and low in cost. Its disadvantages are that it is time-consuming and has a low yield. However, plant polysaccharides, as plant cell inclusions, need to break the cell wall to be dissolved. Therefore, it is necessary to participate in auxiliary water extraction and alcohol precipitation methods, such as acid extraction, alkali extraction, and ultrasound-assisted extraction. The inventors of this application found that as a cold-resistant and excellent variety, the cellulose and pectin content of the cell wall of Shifocui is much higher than that of other tea tree varieties. Based on the high efficiency of enzymes, it is feasible to extract Shifocui polysaccharides using enzyme-assisted methods. The use of enzymes and their subsequent water extraction and alcohol precipitation methods will affect the activity of tea polysaccharides.

[0005] Therefore, based on biological activity, high-activity tea polysaccharides can be obtained by reasonable extraction methods, and systematic and in-depth pharmacological research is the future research direction, in order to provide a certain theoretical basis for the development of tea polysaccharide products and the high value of tea resources in southwestern Anhui, improve resource utilization, and help solve the problem of low economic benefits for tea farmers caused by the serious abandonment of summer and autumn tea and tea tree flowers in the tea area of ​​southwestern Anhui. In addition, based on the good application prospects of tea functional components, domestic and foreign researchers have achieved certain results in the selection and breeding of varieties with specific functional components, but there are few reports on high-quality tea polysaccharide germplasm resources. Therefore, further exploration of the development and application of tea polysaccharides also has certain guiding significance for the selection and breeding of high-polysaccharide tea varieties. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a Shifocui Tea polysaccharide having good free radical scavenging efficiency, showing a good improvement effect on the reduction of cell activity caused by free radical damage and having a repairing effect on oxidative damage at the cellular level, as well as an extraction method and application thereof.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0008] A method for extracting polysaccharides from Shifocui Tea, comprising the following steps:

[0009] (1) drying, crushing and sieving Shifocui tea leaves to obtain tea powder, soaking the tea powder in petroleum ether, stirring, and filtering to defatted the tea powder, soaking the defatted tea powder in anhydrous ethanol, stirring, and filtering to decolorize the tea powder, and drying to obtain pretreated tea powder;

[0010] (2) adding the pretreated tea powder and the composite enzyme into a phosphate buffer, wherein the composite enzyme is cellulase and macerate, and the enzymatic activity ratio of the cellulase to the macerate is 1:1-2, and performing an enzymatic hydrolysis reaction at 45°C-55°C for 2h-3h to obtain an enzymatic hydrolysis product;

[0011] (3) passivating and centrifuging the enzymatic hydrolysis product, adjusting the pH value of the obtained first supernatant to 6.9-7.2 for standby use, adding the obtained precipitate into water and heating it to 70° C.-80° C., reacting for 1 h-3 h, and obtaining a water extraction product;

[0012] (4) centrifuging the water-extracted product, adjusting the pH value of the obtained second supernatant to 6.9-7.2, then mixing it with the first supernatant, evaporating and concentrating it, adding anhydrous ethanol, standing it at 4°C-6°C for 12h-16h, and then centrifuging it again, drying the obtained precipitate, adding trichloroacetic acid solution to remove protein, and controlling the pH value to 3.5-4.2;

[0013] (5) The above-mentioned protein-removed product is placed at 4°C to 6°C for 2h to 3h, and after centrifugation, the third supernatant is obtained and the pH value is adjusted to 6.9 to 7.2, and then dialyzed, evaporated and concentrated, and freeze-dried to obtain Shifocui Tea polysaccharide.

[0014] In the above-mentioned method for extracting Shifocui tea polysaccharides, preferably, in step (2), the mass volume ratio of the pretreated tea powder to the phosphate buffer is 1g:30mL~40mL, the mass fraction of the complex enzyme in the phosphate buffer is 0.004wt%~0.006wt%, and the pH value of the phosphate buffer is 4.5~5.

[0015] In the above-mentioned method for extracting polysaccharides from Shifocui Tea, preferably, in step (4), evaporation and concentration are performed until the volume of the concentrate is 1 / 10 of the sum of the volume of the phosphate buffer used in step (2) and the volume of the water used in step (3); the protein removal process must ensure that the total volume of the final reaction system is 1 / 3 to 1 / 4 of the volume of the phosphate buffer used in step (2).

[0016] In the above-mentioned method for extracting polysaccharides from Shifocui Tea, preferably, in step (3), the passivation time is 30 min to 50 min, and the centrifugal speed is 4000 r·min -1 ~4500r·min -1 The centrifugation time is 10 min to 12 min, the water extraction time is 1 h to 2 h, and the amount of water added is calculated based on the mass volume ratio of the pretreated tea powder to water of 1 g: 10 mL to 20 mL.

[0017] In the above-mentioned method for extracting polysaccharides from Shifocui Tea, preferably, in step (4), the centrifugal speed is 4000 r·min -1 ~4500r·min -1 The centrifugal time is 10-12 minutes, the volume ratio of the evaporation concentrate to the anhydrous ethanol is 1:3-4, and the speed of the second centrifugal is 4000 r / min. -1 ~4500r·min -1The centrifugation time is 10 min to 12 min, and the mass fraction of trichloroacetic acid in the trichloroacetic acid solution is 3% to 5%.

[0018] In the above-mentioned method for extracting polysaccharides from Shifocui Tea, preferably, in step (5), the centrifugal speed is 4000 r·min -1 ~4500r·min -1 The centrifugation time is 10 min to 12 min. The dialysis refers to placing the third supernatant after the pH value is adjusted into a dialysis bag with a molecular retention capacity of 8000Da to 13000Da for dialysis. The water is replaced every 4h to 6h, and the dialysis is performed for a total of 24h to 48h. The vacuum freeze-drying pressure is 10Pa to 15Pa, the temperature is -40℃ to -35℃, and the freeze-drying time is 36h to 48h.

[0019] In the above-mentioned method for extracting polysaccharides from Shifocui tea, preferably, in step (1), the drying temperature is 60°C to 75°C, the drying time is 4h to 5h, the tea powder is turned over every 1h to 1.5h, and the ratio of the volume of tea powder to the volume of petroleum ether is 1:10 to 15.

[0020] In the above-mentioned method for extracting polysaccharides from Shifocui Tea, preferably, during the degreasing process, the soaking and stirring time is 8h to 12h, and the soaking, stirring and filtration process is repeated 2 to 4 times.

[0021] In the above-mentioned method for extracting Shifocui tea polysaccharides, preferably, during the decolorization process, the immersion and stirring time is 6 hours to 8 hours, and the immersion and stirring and filtration process is repeated 2 to 4 times until the tea powder is colorless.

[0022] In the above-mentioned method for extracting Shifocui tea polysaccharides, preferably, in step (1), the Shifocui tea leaves are Shifocui summer and autumn tea leaves.

[0023] As a general inventive concept, the present invention also provides a Shifo Cui Camellia polysaccharide extracted by the above-mentioned Shifo Cui Camellia polysaccharide extraction method, wherein the Shifo Cui Camellia polysaccharide has free radical scavenging activity.

[0024] As a general inventive concept, the present invention also provides an application of the above-mentioned Camellia japonica polysaccharide in the field of medicine or cosmetics, wherein the Camellia japonica polysaccharide is used to repair oxidative stress cell damage caused by free radicals.

[0025] In the present invention, the enzyme activity ratio of cellulase to macerate refers to the ratio of the total enzyme activity of cellulase to the total enzyme activity of macerate. The mass ratio of the two can be obtained according to the enzyme activity ratio and the unit enzyme activity of the manufacturer.

[0026] Compared with the prior art, the advantages of the present invention are:

[0027] 1. The tea polysaccharide prepared by the present invention is an extract of fresh tea leaves from Shifocui Tea, a national excellent variety recognized in 2010. It is prepared by enzyme-assisted water extraction and alcohol precipitation. Compared with the acid extraction and alkali extraction auxiliary methods in the prior art, it is simple to operate, reduces the use of acid and alkali, and is safer and more environmentally friendly. Compared with ultrasound-assisted extraction, enzyme-assisted extraction is milder and is conducive to retaining the biological activity of tea polysaccharides. The present application further optimizes the type, proportion and action time of the complex enzyme, which is conducive to accelerating the dissolution of tea polysaccharides while avoiding the decomposition of polysaccharides, and can maximize the retention of the activity of tea polysaccharides, which has good application prospects in biological research.

[0028] 2. The tea polysaccharide of the present invention has good free radical scavenging activity in vitro and has the excellent property of improving the survival rate of damaged cells in cell models damaged by free radicals from different sources.

[0029] 3 The research on the oxidative damage repair mechanism of the tea polysaccharides of the present invention shows that it is different from the traditional antioxidant damage repair method of increasing the antioxidant enzymes in cells. The repair of damaged cells by the Shifocui summer and autumn tea polysaccharides of the present application is an antioxidant damage mechanism of directly removing free radicals that is consistent with the in vitro antioxidant effect.

[0030] Biological experiments of the present invention show that:

[0031] 1. In terms of protecting cells from free radical oxidative damage, the cell survival rates in both exogenous and endogenous free radical oxidative damage models increased with the increase of WTPs concentration. -1 When the test results were compared, they were as high as 87.41% and 85.84%, which was significantly improved compared with the model group (47.67% and 48.03%).

[0032] 2. WTPs showed good antioxidant activity in vitro, but had no obvious effect on the antioxidant enzyme activity of damaged cells. Further research results on the oxidative damage repair mechanism of summer and autumn tea polysaccharides showed that tea polysaccharides can directly remove endogenous or exogenous free radicals that cause cell damage caused by endogenous or exogenous free radicals.

[0033] 3. The tea polysaccharide prepared by the present invention has high antioxidant activity. Vitamin C (Vc) was used as a positive control to investigate its antioxidant activity. Tea polysaccharide has a high scavenging effect on DPPH and ·OH free radicals. EC 50 The values ​​were 209.5 μg·mL -1 and 535.2 μg mL -1 , the maximum removal efficiency is equivalent to that of Vc.

[0034] 4. It also has a certain protective effect on the endogenous free radical damage cell model established by macrophage RAW264.7. From the perspective of cell morphology, WTPs has a certain repair effect on cell differentiation caused by oxidative damage.

[0035] The Shifocui tea polysaccharide prepared by the water extraction and alcohol precipitation method with enzyme-assisted treatment in the present invention has been shown through systematic biological studies to exhibit good free radical scavenging efficiency in vitro, an improvement effect on the reduction of cell activity caused by free radical damage, and a protective effect on oxidative damage at the cellular level. It is expected to be developed into medicines, cosmetics or related products with antioxidant effects, providing a new way to high-value utilization of the remaining comprehensive tea resources in the Shifocui tea gardens in southwestern Anhui. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The free radical scavenging ability result graph of WTPs prepared in Example 1 of the present invention, wherein: (a) is the scavenging ability result graph of WTPs for DPPH free radicals; (b) is the scavenging rate result graph of WTPs for hydroxyl free radicals.

[0037] Figure 2 The results of the effects of WTPs prepared in Example 1 of the present invention on the proliferation and growth of different cells are shown in Figure 1, wherein: (a) is the effect of different WTPs concentrations on the survival rate of HepG2 cells; (b) is the effect of different WTPs concentrations on the survival rate of RAW264.7 macrophages.

[0038] Figure 3 The diagram shows the effect of WTPs prepared in Example 1 of the present invention on the physiological state of cells, wherein: (a) is the physiological state of mitochondria in cells of the blank control group and the distribution diagram of mitochondrial signals; (b) is the physiological state of mitochondria in cells and the distribution diagram of mitochondrial signals after WTPs treatment for 24 hours, and the scale bar is 20 μm.

[0039] Figure 4 The model of the effect of exogenous hydroxyl free radicals on cells was established, and the WTPs repair effect on the viability of damaged cells was shown, where: (a) is the effect of different concentrations of exogenous hydroxyl free radicals on the survival rate of HepG-2 cells; (b) is the effect of different concentrations of WTPs on the viability of HepG-2 cells induced by hydroxyl free radicals. After statistical analysis, compared with the control group: *P<0.05, **P<0.01; compared with the model group: #P<0.05, ##P<0.01.

[0040] Figure 5The model of the effect of endogenous hydroxyl free radicals on cells was established, and the effect of WTPs on the viability of RAW264.7 macrophages induced by PMA was shown; (a) is the effect of different concentrations of PMA-induced endogenous hydroxyl free radicals on the survival rate of RAW264.7 macrophages; (b) is the effect of different concentrations of WTPs on the viability repair of RAW264.7 macrophage damage induced by endogenous hydroxyl free radicals; after statistical analysis of the data, it was found that compared with the control group: *P<0.05, **P<0.01; compared with the model group: #P<0.05, ##P<0.01.

[0041] Figure 6 The figures are the effect of WTPs on the repair of oxidative damage in HepG-2 cells, where: (a) is the confocal imaging of the hydroxyl radical content in cells of different treatment groups, and the scale is 20 μm; (b) is the comparison of the fluorescence intensity of hydroxyl radicals in cells of different treatment groups. Data statistical analysis shows that compared with the control group: *P<0.05, **P<0.01; compared with the model group: #P<0.05, ##P<0.01.

[0042] Figure 7 The figures show the effect of WTPs on the repair of PMA-induced oxidative damage in RAW264.7 macrophages, where: (a) is the confocal imaging of the hydroxyl radical content in cells of different treatment groups, and the scale bar is 20 μm; (b) is the comparison of the fluorescence intensity of hydroxyl radicals in cells of different treatment groups, and the statistical analysis of the data shows that compared with the control group: *P<0.05, **P<0.01; compared with the model group: #P<0.05, ##P<0.01. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following embodiments are all commercially available.

[0044] The human liver cancer cells HepG-2 and mouse mononuclear macrophage RAW264.7 cells involved in the present invention are both from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0045] Embodiment 1:

[0046] A method for extracting polysaccharides from Shifocui Tea, comprising the following steps:

[0047] (1) Drying Shifocui tea leaves at 70° C., crushing and sieving to obtain tea powder, soaking the tea powder in petroleum ether 10 times the volume of the tea powder, stirring for 12 hours, filtering, repeating the petroleum ether soaking and filtering process twice to defatt the tea powder, soaking the defatted tea powder in anhydrous ethanol 10 times the volume of the tea powder, stirring for 6 hours, filtering, repeating the anhydrous ethanol soaking and filtering process until the anhydrous ethanol is colorless, this embodiment is repeated twice to decolorize the tea powder, drying at 60° C. for 4 hours, turning over once every 1 hour to reduce the drying time and ensure that the contents in the tea leaves are evenly heated, and storing after drying to obtain pretreated tea powder;

[0048] (2) Accurately weigh 30 g of pretreated tea powder and place it in a clean and dry beaker, add it to a phosphate buffer of pH 4.5 according to the material-liquid ratio, that is, the mass volume ratio of tea powder to phosphate buffer is 1 g:30 mL, the phosphate buffer is prepared by NaH2PO4·2H2O and Na2HPO4·12H2O, and shake well; add a composite enzyme thereto so that the mass fraction of the composite enzyme in the phosphate buffer is 0.006 wt %, the composite enzyme is cellulase and macerate, the commodity information of cellulase is: Source Leaf Biology S10041, the unit enzyme activity is 50 U / mg, the commodity information of macerate is: Source Leaf Biology S10082, the unit enzyme activity is 3 U / mg, the enzyme activity ratio of cellulase to macerate is 1:2, and the enzymatic hydrolysis reaction is carried out at 45°C for 3 h to obtain an enzymatic hydrolysis product;

[0049] (3) The enzymatic hydrolysate was inactivated at room temperature for 30 min and then heated at 4000 r / min. -1 Centrifuge at a speed of 10 minutes, adjust the pH value of the first supernatant to neutral (6.9-7.2) for standby use, add distilled water to the precipitate according to the solid-liquid ratio, that is, the mass volume ratio is 1g:20mL, heat to 70°C, react for 2 hours, and obtain the water extraction product;

[0050] (4) The water extraction product was heated at 4000 r / min -1 Centrifuge at 1000 rpm for 10 min, discard the lower precipitate, adjust the pH value of the obtained second supernatant to neutral (6.9-7.2), then mix it with the first supernatant, and concentrate the volume to about 150 mL by rotary evaporation. Add 3 times the volume of anhydrous ethanol, let it stand at 4 ° C for 12 h, and then rotate at 4000 r / min. -1 Centrifuge for 10 minutes, discard the supernatant, dry the precipitate, add trichloroacetic acid solution to remove protein, the mass fraction of trichloroacetic acid in the trichloroacetic acid solution is 5%, and ensure that the total volume of the final reaction system is 300 mL and the pH is 3.5-4.2;

[0051] (5) The above protein removal product was placed at 4°C for 3 h and then rotated at 4000 r / min. -1The mixture was centrifuged at a speed of 10 min, the lower precipitate was discarded, the third supernatant was taken and the pH value was adjusted to neutral (6.9-7.2), and then placed in a dialysis bag with a molecular cutoff of 13000Da for dialyze, and the distilled water was replaced every 6 hours for a total of 24 hours. The mixture was concentrated by rotary evaporation at a pressure of 10Pa and freeze-dried at -35°C for 36 hours to obtain Shifocui Tea polysaccharides, recorded as WTPs.

[0052] The yield of Shifocui Tea polysaccharide extracted in this example is 1.73%.

[0053] Comparative Example 1:

[0054] A method for extracting Shifocui tea polysaccharides, the steps are basically the same as those in Example 1, the only difference is that: the complex enzyme in the enzyme reaction uses cellulase and pectinase, the enzyme activity ratio of cellulase to pectinase is 1:2, and the yield of Shifocui tea polysaccharides extracted is 0.86%, indicating that Shifocui is an excellent cold-resistant variety, and the degradation effect of the combined action of cellulase and pectinase is not enough to degrade the cell wall structure, thereby affecting the extraction yield of tea polysaccharides.

[0055] Comparative Example 2:

[0056] A method for extracting polysaccharides from Schisandra chinensis, the steps of which are basically the same as those in Example 1, with the only difference being that the reaction time in the enzyme reaction is 1.5 h, and the yield of the extracted Schisandra chinensis polysaccharides is 0.92%, indicating that the reaction time of the complex enzyme can significantly affect the extraction rate of the polysaccharide.

[0057] Comparative Example 3:

[0058] A method for extracting tea polysaccharides from Shifocui tea, the steps are basically the same as those in Example 1, the only difference is that the reaction time in the enzyme reaction is 4h, the yield of the extracted Shifocui polysaccharides is 1.68%, but the tea polysaccharides obtained by the extraction method are less than 1000μg·mL -1 The maximum scavenging rate of hydroxyl free radicals at the concentration of 1200 μg·mL –1 The maximum free radical scavenging rate under the concentration conditions can reach 94.61%, indicating that the reaction time of the complex enzyme can significantly affect the extraction rate of polysaccharides, and too long enzyme treatment time will significantly reduce the antioxidant activity of polysaccharides.

[0059] Comparative Example 4:

[0060] A method for extracting polysaccharides from schefflera tea, the steps of which are basically the same as those in Example 1, except that the enzyme activity ratio of cellulase to macerate in the enzyme reaction is 1:3, the yield of the schefflera tea polysaccharide extracted is 1.68%, which is slightly lower than that in Example 1, and the polysaccharide extracted in this comparative example has significantly reduced antioxidant activity against hydroxyl radicals, with a maximum scavenging rate of 1000 μg·mL-1 The concentration reached 80.2% under the condition, and the calculated EC 50 The value is 725.3 μg·mL -1 This indicates that the enzymatic activity of the analyte will significantly affect the antioxidant activity of tea polysaccharides. When the enzymatic activity of the analyte in the complex enzyme exceeds a certain proportion, the antioxidant activity of the extracted tea polysaccharides will be reduced.

[0061] The following characterizes the various properties and effects of Shifocui Tea polysaccharides:

[0062] 1. In vitro antioxidant activity experiment of Shifocui tea polysaccharide

[0063] The antioxidant activity of the polysaccharides extracted from Shifocui Tea in Example 1 of the present invention was investigated to explore the antioxidant activity of the polysaccharides in Shifocui Tea at 50 μg·mL -1 , 100 μg·mL -1 , 200 μg·mL -1 , 400 μg·mL -1 , 600 μg·mL -1 , 800 μg·mL -1 , 1000μg·mL -1 and 1200 μg mL -1 The free radical scavenging ability within the concentration range of Figure 1 As shown in the figure, the same concentration of vitamin C (Vc) was used as a positive control to investigate its antioxidant activity. Among them, Shifocui Tea polysaccharide had a higher scavenging effect on DPPH and ·OH free radicals. The EC 50 The values ​​were 209.5 μg·mL -1 and 535.2 μg mL -1 , the concentration of polysaccharide in Shifocui tea is 600 μg·mL -1 The maximum scavenging efficiency of DPPH is shown in Figure (a), and the concentration is 1200 μg·mL -1 The maximum scavenging efficiency for ·OH is shown in Figure (b), which is equivalent to Vc, indicating that the prepared Shifocui summer and autumn tea polysaccharide has good antioxidant activity.

[0064] 2. Effect of Shifocui Tea Polysaccharide on Cell Survival Rate

[0065] Human hepatoma cell line HepG-2 and macrophage RAW264.7 were cultured to the logarithmic phase and seeded in 96-well plates with 5×10 3 After the cells adhered to the wall, the experimental group was set to 50 μg mL -1 , 100 μg·mL -1 , 200 μg·mL -1 , 400 μg·mL -1 , 600 μg·mL-1 and 800 μg mL -1 100 μL of Shifocui Tea polysaccharide concentration gradient was added to each well. Pure culture medium was added to the control group as a control. After adding the sample, it was placed in a CO2 incubator for 24 h, and then the culture medium was removed. 100 μL MTT (0.5 mg mL -1 Serum-free medium was used for the preparation, and the cells were incubated for another 4 h. The supernatant was then removed, and 100 μL of DMSO was added to dissolve the formazan crystals. The cell culture plate was shaken in the dark for 10 minutes, and the absorbance of each group at 490 nm was detected. Six parallel experiments were set up for each group. The cell viability was measured by MTT method, and the cell viability (i.e., cell survival rate) was calculated according to the following formula: Cell viability (%) = (OD 实验组 -OD 空白孔 ) / (OD 对照组 -OD 空白孔 )*100%. The result is Figure 2 As shown, at 50 μg mL -1 ~800μg·mL -1 Within the concentration range, the survival rates of HepG-2 cells (Figure a) and RAW264.7 cells (Figure b) were maintained at about 95% after 24h intervention with Shifocui Tea polysaccharide. In addition, cell morphology studies found that the intracellular ROS-producing organelles - mitochondria are the most sensitive organelles in the cell, such as Figure 3 As shown in the figure, after the intervention of tea polysaccharides, the signal distribution of the two groups of mitochondria was analyzed, and the distribution data showed that the mitochondrial morphology remained intact in the filamentous shape, indicating that tea polysaccharides had no obvious inhibitory effect on the normal proliferation and growth of the two cells, that is, tea polysaccharides had no obvious effect on the physiological activity of cells.

[0066] 3. Establishment of exogenous free radical cell damage model and cell viability determination

[0067] Model establishment: HepG-2 cells were cultured to the logarithmic phase and seeded in 96-well plates, with 5×10 3 After the cells attached to the wall, the model of endogenous OH addition in the cells was established as follows: 1 mM EDTA-Fe 2+ , using Fenton reaction to generate ·OH, that is, the experimental group was added with H2O2 concentration gradient diluted with culture medium to 50μM, 100μM, 200μM, 400μM, 600μM and 800μM, 100μL was added to each well, and the blank control group was added with pure culture medium as a control, while retaining EDTA-Fe 2+ The treated group was used as a control. After adding the sample, the cells were placed in a CO2 incubator for 4 h, and then the culture medium was removed. The cell viability was measured using the MTT method in the second characterization. Figure 4(a) The results show that the cell survival rate is close to 50% when the H2O2 concentration is 400 μM, which can be used as a modeling concentration.

[0068] Referring to the cell culture method in the second characterization above, an optimized 400 μM concentration of H2O2 was selected to establish an exogenous ·OH model group, and a blank control group, an injury model group, a positive control group, and an experimental group were set up respectively. After the modeling was completed, the supernatant of the model group was removed and washed twice with PBS to remove the influence of residual H2O2. After that, complete culture medium was added to the blank control group and the model group, different concentration gradients of Shifocui Tea polysaccharide WTPs solution were added to the experimental group, and 200 μg / mL of Vc solution was added to the positive control group. Each group continued to culture for 24 hours, and the cell viability was measured according to the MTT method. Figure 4 As shown in (b), the cell survival rate after treatment with different concentrations of polysaccharides was significantly improved. Compared with the model group, 200 μg·mL -1 The cell survival rate of the WTPs-treated experimental group began to show significant differences (P<0.05). -1 After that, it showed a very significant difference. The experimental results showed that Shifocui Tea polysaccharide can effectively improve the survival rate of exogenously damaged cells and show a protective effect on cell oxidative damage.

[0069] 4. Establishment of endogenous free radical cell damage model and cell viability determination

[0070] Model establishment: RAW264.7 cells were cultured to the logarithmic phase and seeded in 96-well plates, with 5×10 cells per well. 3 After the cells adhered to the wall, phorbol ester (PMA) was used to establish an endogenous ·OH addition model in the cells. The steps were as follows: the experimental group was added with a PMA concentration gradient diluted with culture medium to 1μg / mL, 2μg / mL, 4μg / mL, 6μg / mL, and 8μg / mL, and 100μL was added to each well. The blank control group was added with pure culture medium as a control. After adding the sample, it was placed in a CO2 incubator for 4 hours and then the culture medium was removed. PMA will induce cell damage and cause cell dendritic differentiation. Cell viability was measured according to the MTT method. Figure 5 (a) up to 4 μg mL -1 When the cell survival rate is about 50%, nearly half of the cells are dead. Therefore, we determined that 4 μg mL -1 The cells were cultured with PMA for 4 h as the culture condition for subsequent modeling.

[0071] Referring to the cell culture method in the second characterization, the cells were cultured in a 96-well plate for 24 h and then a blank control group, injury model group, positive control group, and experimental group were set up. -1The endogenous OH model group was established using 4 μg mL -1 After culturing cells with PMA for 24 hours, the supernatant of the model group was removed and washed twice with PBS to remove the influence of residual PMA. Complete culture medium was then added to the blank control group and model group, WTPs polysaccharide solutions of different concentration gradients were added to the experimental group, and 200 μg·mL of Vc solution was added to the positive control group. -1 Each group was cultured for 24 h, and the cell viability was measured by MTT method. Figure 5 (b) Compared with the model group, 400 μg·mL -1 The cell survival rate of the WTPs-treated experimental group began to show extremely significant differences (P<0.01). The experimental results showed that tea polysaccharides WTPs can effectively improve the survival rate of cells damaged by endogenous ·OH, showing a protective effect against endogenous oxidative damage.

[0072] 5. Detection of antioxidant enzyme content in oxidatively damaged cells

[0073] A blank control group, an injury model group, and an experimental group were set up respectively, and 4 μg·mL -1 The endogenous free radical damage model was established by adding PMA at a concentration of 100 μg mL -1 , 200 μg·mL -1 , 400 μg·mL -1 , 800 μg·mL -1 WTPs polysaccharide solution was added, and the cells were collected with a cell scraper, washed twice with 4°C pre-cooled PBS, and then added with cold cell lysis solution (500μL of protein lysis solution containing 2μL of protease inhibitors and phosphatase inhibitors) to the cell pellet and placed in ice water for 20min to 30min to fully lyse the cells. Then, the content of antioxidant enzymes in the cell lysates of different groups was detected according to the instructions of the catalase detection kit and the glutathione reductase detection kit. The cell viability identification experiment showed that the oxidatively damaged cells showed damage protection after polysaccharide treatment, and the results of the intracellular antioxidant enzyme activity determination showed that after the intervention of polysaccharide concentrations from low to high, as shown in Table 1, the content of the two antioxidant enzymes in the experimental group cells showed no significant difference from the model group, indicating that Shifocui tea polysaccharides do not repair oxidative stress damage by increasing the activity of antioxidant enzymes at the cellular level.

[0074] Table 1 Effects of WTPs treatment on glutathione reductase (GSH-PX) and catalase (CAT) in RAW264.7 oxidatively damaged cells (mean ± SD)

[0075]

[0076] Compared with the control group: **P<0.01

[0077] 6. Protective mechanism of Shifocui tea polysaccharide against cellular oxidative damage

[0078] 5 × 10 4 HepG-2 cells were modeled and different treatment groups were set up according to the method in the fourth characterization. After the culture, the cells were treated with 10 μM dichlorodihydrofluorescein-acetoacetate (DCFH-DA) and directly used for laser confocal imaging analysis. In the model group, strong fluorescence signals of ROS were shown throughout the cells. The strong signal in the cytoplasm may be due to the addition of exogenous ·OH, while the stronger signal in the mitochondria may be due to the cell oxidative damage caused by exogenous free radicals, which produced more ROS. Figure 6 As shown in the figure, in the WTPs-treated group, the intracellular fluorescence signal was significantly reduced, while the fluorescence signal in the cytoplasm was significantly lower than that in the model group, indicating that the exogenous free radicals that caused cellular oxidative damage were directly removed by WTPs, so the ROS signal in the mitochondria in the experimental group was also significantly lower than that in the model group, ultimately manifesting as the repair of cellular oxidative damage activity.

[0079] 1×10 5 RAW264.7 cells were modeled and treated in different groups according to the method in the fifth characterization. After the culture, they were washed three times with PBS, treated with the RAW264.7 endogenous OH commercial dye HKOH-1r (5μM, 30min), and washed three times with PBS before being directly used for imaging. The excitation wavelength of HKOH-1r is 500nm, and the emission band is 510nm~540nm. Figure 7 As shown in the figure, compared with the blank control group, the endogenous ·OH in the PMA model group cells increased significantly and showed mitochondrial morphology, which is consistent with the result that ROS is generated in mitochondria. The cytoplasm also showed obvious ROS signals, which may be due to the diffusion of ROS generated in mitochondria into the cytoplasm, further accelerating the oxidative damage of cells. And from the cell open image, it can be seen that the morphology of RAW264.7 cells has changed from the original round and smooth to bifurcated, which also proves that the cells have oxidative stress response. In the WTPs treatment experimental group and V C In the treatment group, the fluorescence signal showed a significant decrease in both the cytoplasm and mitochondria, indicating that the oxidative stress response in the cells was repaired and the cell morphology was restored.

[0080] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for extracting polysaccharides from Shifocui Tea, It is characterized in that The following steps are involved: (1) drying, crushing and sieving Shifocui tea leaves to obtain tea powder, soaking the tea powder in petroleum ether, stirring, and filtering to defatted the tea powder, soaking the defatted tea powder in anhydrous ethanol, stirring, and filtering to decolorize the tea powder, and drying to obtain pretreated tea powder; (2) adding the pretreated tea powder and a composite enzyme into a phosphate buffer, wherein the composite enzyme is cellulase and macerate, and the enzymatic activity ratio of the cellulase to the macerate is 1:2, and performing an enzymatic hydrolysis reaction at 45° C. to 55° C. for 3 h to obtain an enzymatic hydrolysis product; (3) passivating and centrifuging the enzymatic hydrolysis product, adjusting the pH value of the obtained first supernatant to 6.9-7.2 for standby use, adding the obtained precipitate into water and heating it to 70° C.-80° C., reacting for 1 h-3 h, and obtaining a water extraction product; (4) centrifuging the water-extracted product, adjusting the pH value of the obtained second supernatant to 6.9-7.2, then mixing it with the first supernatant, evaporating and concentrating it, adding anhydrous ethanol, standing it at 4°C-6°C for 12h-16h, and then centrifuging it again, drying the obtained precipitate, adding trichloroacetic acid solution to remove protein, and controlling the pH value to 3.5-4.2; (5) placing the above protein-removed product at 4°C to 6°C for 2h to 3h, centrifuging to obtain a third supernatant, adjusting the pH value to 6.9 to 7.2, and then dialyzing, evaporating and concentrating, and freeze-drying to obtain Shifocui Tea polysaccharide; In step (2), the mass volume ratio of the pretreated tea powder to the phosphate buffer is 1 g: 30 mL to 40 mL, the mass fraction of the complex enzyme to the phosphate buffer is 0.004 wt % to 0.006 wt %, and the pH value of the phosphate buffer is 4.5 to 5; In step (3), the passivation time is 30 min to 50 min, and the centrifugal speed is 4000 r / min. -1 ~4500r·min -1 The centrifugal time is 10 min to 12 min, the water extraction time is 1 h to 2 h, and the amount of water added is calculated based on the mass volume ratio of the pretreated tea powder to water of 1 g: 10 mL to 20 mL; In step (4), evaporation and concentration are performed until the volume of the concentrate is 1 / 10 of the sum of the volume of the phosphate buffer used in step (2) and the volume of the water used in step (3); the protein removal process needs to ensure that the total volume of the final reaction system is 1 / 3 to 1 / 4 of the volume of the phosphate buffer used in step (2); In step (5), the centrifugal speed is 4000 r / min -1 ~4500r·min -1 The centrifugation time is 10 min to 12 min. The dialysis refers to placing the third supernatant after the pH value is adjusted into a dialysis bag with a molecular retention capacity of 8000Da to 13000Da for dialysis. The water is replaced every 4h to 6h, and the dialysis is performed for a total of 24h to 48h. The freeze-drying pressure is 10Pa to 15Pa, the temperature is -40℃ to -35℃, and the freeze-drying time is 36h to 48h.

2. The method for extracting polysaccharides from Shifocui Tea according to claim 1, It is characterized in that In step (4), the centrifugal speed is 4000 r / min -1 ~4500r·min -1 The centrifugal time is 10-12 minutes, the volume ratio of the evaporation concentrate to the anhydrous ethanol is 1:3-4, and the speed of the second centrifugal is 4000 r / min. -1 ~4500r·min -1 The centrifugation time is 10 min to 12 min, and the mass fraction of trichloroacetic acid in the trichloroacetic acid solution is 3% to 5%.

3. The method for extracting polysaccharides from Shifocui Tea according to claim 1, It is characterized in that In step (1), the drying temperature is 60° C. to 75° C., the drying time is 4 h to 5 h, the tea powder is turned over once every 1 h to 1.5 h, and the ratio of the volume of the tea powder to the volume of the petroleum ether is 1:10 to 15; During the degreasing process, the soaking and stirring time is 8h to 12h, and the soaking and stirring and filtration process is repeated 2 to 4 times; During the decolorization process, the soaking and stirring time is 6 hours to 8 hours, and the soaking and stirring and suction filtering process is repeated 2 to 4 times until the tea powder is colorless; And / or, in step (1), the Shifocui tea leaves are Shifocui summer and autumn tea leaves.

4. A Shifocui Tea polysaccharide extracted by the Shifocui Tea polysaccharide extraction method according to any one of claims 1 to 3.

5. The Shifocui Tea polysaccharide according to claim 4, It is characterized in that The Shifocui Tea polysaccharide has free radical scavenging activity.

6. A use of the Shifocui Tea polysaccharide as claimed in claim 5 in the preparation of medicines or cosmetics, It is characterized in that The Shifocui Tea polysaccharide is used to repair oxidative stress cell damage caused by free radicals.

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