Camellia seed cake polysaccharide with immunological enhancement and antioxidant activity as well as preparation method and application of camellia seed cake polysaccharide

Abstract

The invention belongs to the technical field of extraction and preparation of natural products, and discloses camellia seed cake polysaccharide with immunological enhancement and antioxidant activity as well as a preparation method and application of the camellia seed cake polysaccharide. The method comprises the following steps: extracting camellia seed cake polysaccharide by adopting an ultrasonic-assisted method and a water extraction and alcohol precipitation method, determining the content of crude polysaccharide by adopting a phenol-sulfuric acid method, desalting, removing impurities, purifying and grading the camellia seed cake crude polysaccharide by adopting a DEAE-Sepharose FF ion exchange column and a Chromdex 75PG gel filter column to obtain camellia seed cake pure polysaccharide, and determining the molecular weight of the polysaccharide by adopting an exclusion chromatography method. And finally, determining the structure of the compound through chemical methods such as high performance liquid chromatography, full methylation combined gas chromatography-mass spectrometry, infrared spectroscopy, nuclear magnetic resonance and the like, a chromatographic method and a spectrum method. In-vitro experiments and in-vivo experiments prove that the camellia seed cake polysaccharide has immunological enhancement and antioxidant activity at the same time, and can be widely applied to the fields of medicines, daily chemical products, feeds and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction and preparation, and particularly relates to a camellia seed cake polysaccharide with immune enhancement and antioxidant activities, and a preparation method and application thereof. Background Art

[0002] Camellia oleifera Abel belongs to the Theaceae family and the genus Camellia. It is an evergreen shrub or small tree. Camellia oleifera has a wide range of medicinal values, and its leaves, seeds, seed coats and roots can all be used for medicinal purposes. In addition to pressing camellia oil, its matrix byproduct camellia seed cake extract has a wide range of uses, such as making cleaning agents and adhesives. Studies have shown that camellia seeds have the effects of inhibiting coagulation, resisting radiation, and preventing ultraviolet rays. Studies have shown that polysaccharides extracted from camellia seed cakes have rich biological activity and can be used to make medicines or health foods.

[0003] Polysaccharides are natural high molecular weight carbohydrate compounds composed of 10 or more monosaccharide molecules connected by aldose or (and) ketose through glycosidic bonds. 10 O5) n It is widely found in animals, plants and microorganisms. Modern medical research has shown that polysaccharides have multiple biological activities such as antioxidant, anti-tumor, antiviral and immunomodulatory. As a research hotspot in the field of phytochemicals, plant polysaccharides have been reported to have many excellent physical and chemical properties and biological activities. However, the inventors found that there are few studies on camellia seed cake polysaccharides, especially on the structure and mechanism of action of camellia seed cake polysaccharides. Therefore, it is necessary to conduct further in-depth research on camellia seed cake polysaccharides, so as to provide a basis for the high added value development and utilization of camellia seed cake, a by-product of camellia seed matrix. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies in the above-mentioned prior art, the primary purpose of the present invention is to provide a camellia seed cake polysaccharide with immune enhancement and antioxidant activities.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned camellia seed cake polysaccharide with immune enhancement and antioxidant activity; the method adopts a mode combining ultrasound-assisted method with water extraction and alcohol precipitation method to extract camellia seed cake crude polysaccharide (COP) from camellia seed cake (tea meal). t), the phenol-sulfuric acid method was used to determine the crude polysaccharide content, and the crude polysaccharide from camellia oleifera cake was desalted, impurity-removed, purified and graded by DEAE-Sepharose FF ion exchange column and Chromdex 75PG gel filtration column to obtain pure camellia oleifera cake polysaccharide COP. The molecular weight of the polysaccharide was measured by size exclusion chromatography, and finally the structure of camellia oleifera cake polysaccharide (COP) was determined by chemical methods, chromatographic methods and spectral methods such as high performance liquid chromatography (HPLC), permethylation combined with gas chromatography-mass spectrometry (GC-MS), infrared spectroscopy (IR) and nuclear magnetic resonance (NMR).

[0006] Another object of the present invention is to provide an application of the above-mentioned camellia oleifera seed cake polysaccharide (COP); in vitro experiments and in vivo tests prove that the above-mentioned camellia oleifera seed cake polysaccharide has both immunoenhancing effects and antioxidant activities.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A camellia oil seed cake polysaccharide with immune enhancement and antioxidant activity, wherein the monosaccharide molecular composition chain of the polysaccharide is shown in the following formula (I):

[0009]

[0010] The weight average molecular weight of the camellia seed cake polysaccharide is 8772Da;

[0011] The camellia oleifera seed cake polysaccharide consists of mannose, glucose, galactose, xylose and arabinose, wherein the molar ratio of each monosaccharide is 10.583:53.413:19.710:2.717:13.577.

[0012] The structural analysis of the camellia seed cake polysaccharide showed that →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-β-D-Galp-(1→) were the main residues, with →4)-β-D-Galp(1→ and →4)-α-D-Glcp-(1→ as the main skeleton, and the branching site was located at No. 6.

[0013] The above-mentioned method for preparing the camellia seed cake polysaccharide having immune enhancement and antioxidant activity is prepared according to the following steps:

[0014] S1, extracting the pretreated camellia seed cake powder with alcohol to remove fat-soluble small molecules and pigments to obtain medicinal residues;

[0015] S2, drying the residue obtained in step S1, and then extracting it by adding water and heating with the assistance of ultrasound, filtering, collecting the filtrate, concentrating, and cooling to obtain a concentrated solution;

[0016] S3, subjecting the concentrated solution obtained in step S2 to alcohol precipitation, and collecting the precipitate;

[0017] S4, removing protein from the precipitate obtained in step S3, and freeze-drying to obtain crude polysaccharide from camellia seed cake;

[0018] S5, using the phenol-sulfuric acid method to determine the polysaccharide content of the crude polysaccharide of the oil-tea camellia seed cake obtained in step S4; using a pretreated DEAE-Sepharose FF ion exchange column to desalt and semi-purify the crude polysaccharide of the oil-tea camellia seed cake, and eluting with distilled water, 0.2M NaCl solution, 0.5M NaCl solution and 1.0M NaCl solution in sequence, collecting the distilled water elution part rich in polysaccharide content, dialyzing, drying and freeze-drying to obtain the oil-tea camellia seed cake polysaccharide powder;

[0019] S6. The camellia oleifera cake polysaccharide powder obtained in step S5 is classified using a Chromdex 75PG gel filtration column, concentrated, freeze-dried, and collected to obtain a single peak component, which is the camellia oleifera cake polysaccharide with immune enhancement and antioxidant activity.

[0020] The pretreatment in step S1 refers to pulverizing the camellia seed cake; the alcohol extraction is to use 95% by volume ethanol for 60° C. vacuum reflux treatment for 3 times, each time for 4 hours.

[0021] The drying in step S2 is a drying treatment; the concentration is to concentrate the filtrate to a density of 1g / mL; the specific conditions of the ultrasonic-assisted water-addition and heating extraction include: adding water according to a solid-liquid ratio of 25g:1mL, ultrasonic time for 20 minutes under 300W ultrasonic power conditions, and then heating the reaction at a temperature of 60°C for 6 hours.

[0022] The alcohol precipitation treatment in step S3 is specifically to add 95% ethanol by volume to the concentrated solution, stir, precipitate polysaccharides with alcohol, and collect the precipitate after standing for 24 hours; the protein removal in step S4 is carried out by the Sevage method, specifically adding deionized water to the precipitate obtained in step S3 for dilution, and then mixing it with Sevage solution (the mass ratio of chloroform to n-butanol is 4:1) in a conical flask at a volume ratio of 4:1, shaking in a shaker for 30 minutes, centrifuging at 4000r / min for 10 minutes, collecting the upper liquid to remove the protein, and repeating the above protein removal steps 3 times; further, reducing pressure and concentrating in a fume hood to remove residual organic reagents.

[0023] In step S4, the freeze-drying is to pre-cool the camellia oleifera seed cake polysaccharide obtained after removing the protein in a -80°C refrigerator for 4 hours, and then freeze-dry it in a vacuum freeze dryer for 72 hours; the crude camellia oleifera seed cake polysaccharide obtained after freeze-drying is stored in a -20°C refrigerator.

[0024] The phenol-sulfuric acid method for determining the polysaccharide content in step S5 is specifically carried out in the following steps: 0.05 g of anhydrous D-glucose is dissolved in deionized water and fixed to volume in a 500 mL volumetric flask to obtain a 1 mg / mL solution A; 5 mL of solution A is taken and fixed to volume in a 50 mL volumetric flask to obtain a 0.1 mg / mL solution B; 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of solution B are taken into 6 test tubes, respectively, and deionized water is added to make up to 2 mL; a fresh phenol solution with a mass percentage concentration of 5% is prepared under light-proof conditions, and 1 mL of phenol is added to the above 6 test tubes respectively. The solution and 5 mL of concentrated sulfuric acid are mixed evenly, cooled to room temperature, and the absorbance is detected at 490 nm by an ultraviolet spectrophotometer to draw a glucose standard curve; 0.025 g of the crude polysaccharide sample of the camellia seed cake obtained in step S4 is accurately weighed and dissolved in 5 mL of deionized water to obtain a solution C with a concentration of 5 mg / mL; 0.25 mL of the solution C is taken and fixed to a volume in a 250 mL volumetric flask to obtain a 0.05 mg / mL test solution D, and after adding phenol and concentrated sulfuric acid with a mass percentage concentration of 5%, the absorbance is measured, and the absorbance value of the sample is substituted into the glucose standard curve to obtain the crude polysaccharide content of the camellia seed cake.

[0025] The pretreatment of DEAE-Sepharose FF in step S5 is specifically carried out according to the following steps: the DEAE-Sepharose FF gel suspension in the Buchner funnel is connected to a vacuum pump to be drained, and the filler is repeatedly washed with pure water until there is no alcohol taste, and the obtained precipitated gel is poured into a beaker, and distilled water of 1 / 2 to 1 times the volume of the precipitated gel is added and stirred, and then poured into a φ3.0×50cm XK chromatography column, wet-packed, and the peristaltic pump is started at a flow rate of 250cm / h, and ultrapure water is used to rinse and balance the gel surface;

[0026] The dialysis in step S5 is performed in a 3.5KDa dialysis bag; the drying and freeze-drying are performed under vacuum conditions.

[0027] The specific operation of using the Chromdex 75PG gel filtration column for classification in step S6 is as follows: a Chromdex 75PG column of size φ2.6×100cm is loaded, balanced with ultrapure water, the flow rate is 30cm / h, and the sample loading amount is 1% of the column volume; when all the camellia oleifera cake polysaccharide powder samples are loaded into the gel filtration column, ultrapure water is used for elution, 4.5mL / tube, and the flow rate is controlled to be 1.8mL / min; each tube of solution collected is detected online with a differential detector, and the eluent in the elution peak area with good peak symmetry and uniform peak height is collected, preferably the eluent in tubes 5-50 is collected, which is the camellia oleifera cake polysaccharide with immune enhancement and antioxidant activity.

[0028] The camellia seed cake polysaccharide prepared by the above preparation method is subjected to structural identification, and the identification method comprises:

[0029] S1. Using size exclusion chromatography to measure the molecular weight of the purified camellia oleifera seed cake polysaccharide powder;

[0030] S2. Determine the monosaccharide composition and the molar ratio of each monosaccharide of camellia oleifera cake polysaccharide by high performance liquid chromatography;

[0031] S3. Infrared spectroscopy, methylation analysis and nuclear magnetic resonance analysis were used to determine the possible repeating unit structure of camellia seed cake polysaccharide.

[0032] The above-mentioned camellia seed cake polysaccharide is used in the preparation of a product having both immune enhancement and antioxidant activities, wherein the product is food, medicine, daily chemical product or feed.

[0033] The food can be understood as any edible form. For example, the food in the present invention includes ordinary food and special food. The special food in the present invention includes health food and special medical formula food; and ordinary food is relative to special food and is a food suitable for everyone.

[0034] The drug can be administered in a unit dosage form, and the dosage form can be a liquid dosage form or a solid dosage form. The liquid dosage form can be a true solution, a colloid, a microparticle dosage form, an emulsion dosage form, or a suspension dosage form. Other dosage forms include tablets, capsules, dripping pills, aerosols, pills, powders, solutions, emulsions, granules, suppositories, freeze-dried powder injections, inclusion compounds, landfills, patches, and liniments. The drug can be for human or animal use (veterinary use).

[0035] The daily chemical products can be laundry detergents, personal hygiene cleansers and cosmetics, such as toothpaste, mouthwash, laundry disinfectant, shampoo, hair lotion, hair spray, shower gel, soap, facial mask, facial cream, facial cleanser, etc.

[0036] The feed refers to food for animals raised in agriculture or animal husbandry. The camellia oleifera seed cake polysaccharide of the present invention can be added to any variety of feed as a feed additive, including but not limited to complete feed, concentrated feed and premixed feed.

[0037] Compared with the prior art, the present invention has the following advantages and effects:

[0038] (1) The present invention uses camellia oleifera cake as a raw material, and obtains camellia oleifera cake crude polysaccharide by combining ultrasound-assisted method with water extraction and alcohol precipitation method, with a polysaccharide extraction rate of 5.7%. The polysaccharide is further purified to obtain single-purity camellia oleifera cake polysaccharide COP. Experiments in vitro (COP and spleen lymphocyte immune cells, dendritic cells co-action experiment) and in vivo (BALB / c mice) and COP antioxidant activity detection (ABTS free radical scavenging ability detection experiment, DPPH free radical scavenging ability detection experiment) prove that the camellia oleifera cake polysaccharide obtained by the above technical scheme has good immunoenhancing effect and certain antioxidant activity, so it can be widely used in the fields of medicine, daily chemical products and feed, and has good economic and social benefits for the development of camellia oleifera cake polysaccharide antioxidant and immunomodulatory activity products.

[0039] (2) The inventors have proved through experiments that the camellia seed cake polysaccharide COP can significantly promote the immune activity of mouse spleen lymphocytes after the in vitro interaction with lymphocytes in ICR mice. The proliferation of lymphocytes stimulated by COP was significantly increased; CD4 + / CD8 + The proportion of COP in the mouse spleen lymphocyte supernatant was significantly increased; the expression of FN-γ, IL-6, IL-2 and TNF-α secreted by the mouse spleen lymphocyte supernatant was upregulated to varying degrees. The results of the interaction between COP and primary dendritic cells extracted from the bone marrow of BALB / c mice showed that COP could significantly promote the immune activity of primary bone marrow-derived dendritic cells in mice and significantly upregulated MHCⅡ + 、CD80 + 、CD86 + COP can enhance the expression of IFN-γ, IL-6, IL-2 and TNF-α secreted by cell supernatant, and promote the maturation of dendritic cells. The results of in vivo activity experiments in BALB / c mice showed that COP can enhance the immune function of BALB / c mice. COP can increase the spleen index of mice, promote the activation of serum IL-2, IL-10, and IL-12p70, increase the secretion levels of IgA and IgG, and upregulate CD4 + and CD8 +The percentage of T lymphocytes, slight to moderate proliferation of spleen lymphocytes, obvious proliferation of white pulp germinal center and marginal zone lymphocytes, and comprehensive evaluation of COP-M and COP-L have the best immunoregulatory effect. The results of the antioxidant activity test of COP show that with the increase of COP concentration, the free radical scavenging rate of ABTS also increases continuously. When the COP concentration is between 50μg / mL-250μg / mL, COP has a scavenging effect on ABTS free radicals. With the increase of COP concentration, the free radical scavenging rate of DPPH shows a trend of first increasing and then decreasing. At 150μg / mL, the scavenging rate reaches a maximum value of 20.43%. When the concentration of COP is 50μg / mL-250μg / mL, it can play a certain scavenging role on DPPH. In summary, the camellia seed polysaccharide component prepared by the method of the present invention has better immunoregulatory activity and certain antioxidant effect, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the D-glucose standard curve used for the polysaccharide content determination of the present invention.

[0041] Figure 2 The crude polysaccharide COP of the camellia oleifera seed cake of the present invention t Elution profile on DEAE-Sepharose FF column chromatography.

[0042] Figure 3 This is the elution curve of the pure COP-E1 of the present invention on the Chromdex 75PG chromatography column.

[0043] Figure 4 These are the molecular weight calibration curve results of the present invention, A is the 1gMp-RT calibration curve, B is the 1gMw-RT calibration curve, and C is the 1gMn-RT calibration curve.

[0044] Figure 5 It is the HPGPC chromatogram of the camellia seed cake polysaccharide COP of the present invention.

[0045] Figure 6 This is the monosaccharide composition analysis of the camellia seed cake polysaccharide COP of the present invention, wherein A is the liquid chromatogram of the standard product and B is the chromatogram of the sample.

[0046] Figure 7 This is a diagram showing the infrared spectrum analysis result of the camellia seed cake polysaccharide COP of the present invention.

[0047] Figure 8 This is the total ion current diagram of the methylation analysis of the camellia seed cake polysaccharide COP of the present invention.

[0048] Fig. 9The nuclear magnetic analysis diagram of the camellia seed cake polysaccharide COP of the present invention, one-dimensional nuclear magnetic resonance spectrum: A is 1H-NMR, B is 13C-NMR; two-dimensional nuclear magnetic resonance spectrum: C is DEPT 135, D is HSQC, E is COSY, F is HMBC, and G is NOESY.

[0049] Fig.10 The effects of the camellia oleifera cake polysaccharide COP on the body weight, spleen index and thymus index of mice, A: comparison of the status of mice on the 14th day and the 28th day after oral administration; B: the effect of COP on the body weight of mice, n=10; C: the effect of COP on the spleen index and thymus index of mice, n=10.

[0050] Fig.11 The effect of the camellia oleifera seed cake polysaccharide COP of the present invention on the expression of serum immunoglobulin in mice, n=5, A: IgA; B: IgG.

[0051] Fig.12 The effect of the camellia seed cake polysaccharide COP of the present invention on the secretion of serum cytokines in mice, A: IL-2; B: IL-10; C: IL-12p70; D: TNF-α.

[0052] Fig.13 Figure 2 shows the effect of the camellia seed cake polysaccharide COP on the proliferation of mouse spleen lymphocytes. A shows the effect of COP alone, B shows the effect of COP in combination with LPS, and C shows the effect of COP in combination with PHA.

[0053] Fig.14 The COP of the present invention is for the CD4 + 、CD8 + Effect of T subsets, A: CD4 + T cell percentage; B: CD8 + T cell percentage; C: CD4 + / CD8 + ratio.

[0054] Fig.15 These are the tissue sections of various organs of mice under the action of the camellia seed cake polysaccharide COP of the present invention, A: histopathological changes of mouse spleen and thymus, n=5 (HE staining); B: histological changes of mouse lung, liver, kidney and heart, n=5 (HE staining).

[0055] Fig.16 In order to detect the antioxidant activity of the camellia seed cake polysaccharide COP of the present invention, the ABTS method was used to evaluate the camellia seed cake polysaccharide.

[0056] Fig.17In order to detect the antioxidant activity of the camellia seed cake polysaccharide COP of the present invention, the DPPH method was used to evaluate the camellia seed cake polysaccharide. DETAILED DESCRIPTION

[0057] The present invention is further described below with reference to specific embodiments, but they should not be construed as limiting the present invention.

[0058] Example 1

[0059] A method for preparing camellia oleifera seed cake polysaccharide (COP) with immune enhancement and antioxidant activity, specifically according to the following operating steps:

[0060] S1. Crush the camellia seed cake, weigh 300 g of the obtained camellia seed cake powder, and use 95% by volume ethanol to perform 60° C. reflux pressure treatment for 3 times, each time for 4 hours, to remove fat-soluble small molecules and pigments, to obtain medicinal residues;

[0061] S2, drying the drug residue obtained in step S1, adding water at a material-liquid ratio of 25g:1mL, ultrasonicating for 20 minutes at 300W ultrasonic power, and then heating at 60°C for 6 hours, collecting the filtrate after filtering, concentrating the filtrate to 300mL with a density of 1g / mL, and cooling to obtain a concentrated solution;

[0062] S3, adding 1.5 L of 95% by volume ethanol to the concentrated solution obtained in step S2, stirring, precipitating polysaccharides with alcohol, standing for 24 hours, and then centrifuging at 4000 r / min for 10 min to collect the precipitate;

[0063] S4, adding deionized water to the precipitate obtained in step S3 for dilution, and then mixing with Sevage solution (the mass ratio of chloroform to n-butanol is 4:1) in a conical flask at a volume ratio of 4:1, shaking in a shaker for 30 minutes, centrifuging at 4000r / min for 10 minutes, collecting the upper liquid to remove the protein, and repeating the above protein removal step 3 times; further, decompressing and concentrating in a fume hood to remove residual organic reagents; the sample obtained after protein removal was placed in a -80°C refrigerator for precooling for 4 hours, and then placed in a vacuum freeze dryer for freeze drying for 72 hours, and the freeze-dried camellia seed cake crude polysaccharide (COP t , 17.11 g) was stored in a refrigerator at -20 °C for subsequent purification;

[0064] S5. The polysaccharide content of the crude polysaccharide of the oil-tea seed cake obtained in step S4 is determined by the phenol-sulfuric acid method. The sugar content of the crude polysaccharide of the oil-tea seed cake is 30.1% as measured by the phenol-sulfuric acid method. The standard concentration of glucose is used as the horizontal axis and the absorbance detected at 490 nm is used as the vertical axis. The standard curve of anhydrous D-glucose is determined by the phenol-sulfuric acid method. Figure 1As shown, the calculated linear regression equation is Y = 0.5783x + 0.0645, R 2 =0.9992, indicating that the linear relationship is qualified and can be used to detect the sugar content in the sample. The absorbance value of the crude polysaccharide of camellia seed cake at 490nm is 0.409. According to the glucose standard curve, the sugar content of the crude polysaccharide of camellia seed cake is 30.1%;

[0065] S6. Desalting and semi-purifying the crude polysaccharide of the camellia seed cake obtained in step S4 using a pretreated DEAE-Sepharose FF ion exchange column, eluting with distilled water, 0.2M NaCl solution, 0.5M NaCl solution and 1.0M NaCl solution in sequence, and determining the polysaccharide content of each tube of eluate by anthrone-sulfuric acid method. The elution curve obtained is as follows: Figure 2 As shown, SCZ-E1 is the dH2O elution fraction, SCZ-E2 is the 0.2 M NaCl elution fraction, SCZ-E3 is the 0.5 M NaCl elution fraction, and SCZ-E4 is the 1.0 M NaCl elution fraction. Figure 2 It can be seen that the polysaccharide components mainly exist in SCZ-E1; the eluate was dialyzed in a 3.5KDa dialysis bag and freeze-dried under vacuum conditions to obtain four components: COP-E1 (dH2O elution), COP-E2 (0.2MNaCl elution), COP-E3 (0.5M NaCl elution) and COP-E4 (1.0M NaCl elution). COP-E1 with rich polysaccharide content was selected as the camellia seed cake polysaccharide powder for subsequent experiments;

[0066] S6, the polysaccharide-rich camellia seed cake polysaccharide powder COP-E1 obtained in step S5 is classified using a Chromdex 75PG gel filtration column, concentrated, freeze-dried and collected; and the camellia seed cake polysaccharide COP is obtained by gel filtration column chromatography separation and purification. Figure 3 The component COP-E1 that passed through the Chromdex 75PG dextran gel filtration column has a single elution peak curve, and the absorption peak shape between 200-250min is good, the purity is high and single, indicating that COP has been purified.

[0067] In general, 17.11 g of crude polysaccharide from 300 g of camellia seed cake powder was extracted, the extraction rate was 5.7%, and the polysaccharide content was 30.1%. The crude polysaccharide from camellia seed was chromatographed by DEAE-sepharose FF ion exchange column, desalted, impurity-removed, and semi-purified to obtain four components: COP-E1 (eluted with dH2O), COP-E2 (eluted with 0.2M NaCl), COP-E3 (eluted with 0.5M NaCl), and COP-E4 (eluted with 1.0M NaCl). Because the polysaccharide content in the eluate was detected and found to be mainly present in the dH2O eluate, that is, the polysaccharide was finally mainly concentrated in the COP-E1 part, we selected COP-E1 for Chromdex 75PG gel filtration column chromatography, purification, and classification to obtain a single elution peak, named COP, and the obtained camellia seed cake polysaccharide COP was used in Examples 2 and 3.

[0068] Example 2: Structural identification of camellia oleifera seed cake polysaccharide COP.

[0069] The structure of the camellia seed cake polysaccharide COP obtained in Example 1 was identified by the following steps:

[0070] Step 1: Use exclusion chromatography to determine the molecular weight of camellia seed cake polysaccharide COP.

[0071] Make a molecular weight calibration curve: accurately measure dextran standards of different molecular weights (1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards), add 0.05M NaCl solution respectively, and obtain 5mg / mL standard. After filtering with a 0.22μm filter membrane, measure it with a high-performance gel permeation chromatography tandem column by the HPGPC method, and draw a calibration curve with the retention time of the corresponding chromatographic peak as the horizontal coordinate and the logarithmic value of the relative molecular mass of the standard as the vertical coordinate. With the retention time T of the corresponding chromatographic peak as the horizontal coordinate and the logarithmic value of the relative molecular mass of the standard as the vertical coordinate, draw the LogMp-T (peak molecular weight), LogMw-T (weight average molecular weight), and LogMn-T (number average molecular weight) calibration curves as follows:

[0072] The equation of the LogMp-T calibration curve is as follows: Figure 4 As shown in A: Y = -0.1646T + 10.5843, R2 = 0.9931;

[0073] The equation of the LogMw-T calibration curve is as follows: Figure 4 As shown in B: Y = -0.1727T + 10.9803, R2 = 0.9933;

[0074] The equation of the LogMn-T calibration curve is as follows: Figure 4 As shown in C: Y = -0.1642T + 10.4903, R2 = 0.9948.

[0075] Prepare sample solution: accurately weigh the camellia oleifera cake polysaccharide COP sample obtained in Example 1, add salt solution to obtain a 5 mg / mL test solution, filter through a 0.22 μm filter membrane, and transfer to a 2 mL injection bottle.

[0076] HPGPC detection method: high performance liquid chromatography equipped with a differential detector was used for detection using polymer-based water-soluble SEC (GFC) columns OHpak SB-803HQ, Ohpak SB-804HQ, and Ohpak SB-805HQ (8×300mm) in series, with 0.05M NaCl solution as the mobile phase, a flow rate of 0.65mL / min, and an injection volume of 30μL.

[0077] HPGPC method to determine the molecular weight of COP: the elution chromatographic peak of the sample COP is as follows: Figure 5 As shown in Table 3, the peak time is 40.748 min, of which the mobile phase salt peak is around 48.5 min. Substituting the retention time T of the COP polysaccharide sample into the standard regression equation, the average molecular weight of COP can be obtained, as shown in Table 3.

[0078] Step 2: Determine the monosaccharide composition of camellia oleifera cake polysaccharide. Accurately weigh 5 mg of the camellia oleifera cake polysaccharide COP sample obtained in Example 1, add 1 mL of 2M TFA acid solution, and heat at 121°C for 2 h. After blowing dry with nitrogen, add 3 mL of methanol, blow dry, and repeat the washing 2-3 times. Add 5 mL of sterile water and put it into a chromatographic bottle. Take 0.2 mL of monosaccharide standard solution, add 0.2 mL of 0.5 mol / L sodium hydroxide solution, add 0.5 mL of 0.5 mol / L PMP methanol solution, vortex mix, bathe at 70°C for 1 h, add 0.2 mL of 0.5 mol / L hydrochloric acid, add 1 mL of chloroform and vortex extract 3 times to remove excess PMP, discard the chloroform layer, take 0.3 mL and add water to make up to 1 mL. The preparation of monosaccharide standards is shown in Table 1. The monosaccharide composition of COP was determined by high performance liquid chromatography (HPLC) (the specific operation method of high performance liquid chromatography is: using the Thermo U3000 liquid chromatography system, the chromatographic column ZORBAX Eclipse XDB-C18, the mobile phase is acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate, 2M NaOH to adjust pH = 6.8) isocratic elution, the volume ratio of acetonitrile to phosphate buffer is 17:83, the flow rate is 0.8 mL / min, the wavelength is 250 nm, and the injection volume is 10 μl). The liquid chromatogram of the standard is shown in the figure below. Figure 6 As shown in A, the sample chromatogram is as follows Figure 6As shown in B. Comparative analysis shows that COP is composed of mannose, glucose, galactose, xylose, and arabinose, and the molar ratio (%) is 10.583:53.413:19.710:2.717:13.577.

[0079] Table 1 Monosaccharide standard mixed standard gradient concentration information

[0080]

[0081] Step 3: Infrared spectrum determination of camellia oleifera cake polysaccharide. Accurately weigh 1-2 mg of the camellia oleifera cake polysaccharide COP sample obtained in Example 1, add 200 mg of KBr powder, grind evenly, press into tablets, and use Fourier transform infrared spectrometer to scan the FT-IR spectrum of the sample in the range of 400-4000 cm-1. OP infrared spectrum analysis is as follows: Figure 7 As shown, the sample has characteristic absorption peaks of polysaccharides.

[0082] Step 4: Methylation analysis of camellia seed cake polysaccharides. This experiment uses gas chromatography-mass spectrometry GC / MS to analyze the bond structure of polysaccharides (first derivatize the camellia seed cake polysaccharide sample, using an Agilent gas chromatography system, with a carrier gas of high-purity helium (purity not less than 99.999%), a flow rate of 1.0 mL / min, and an injection port temperature of 260°C. The injection volume is 1 μL, split injection, split ratio 10:1, and solvent delay 2.2 min. Program temperature rise: 50°C for 1 min, 50°C / min to 130°C, 3°C / min to 230°C, and hold for 2 min). According to the relative retention time of the chromatographic peak and the mass spectrum ( Figure 8 ), compared the literature data and the ion fragment mass spectra of the database of the Center for Complex Carbohydrate Research at the University of Georgia, the sugar residue type corresponding to each chromatographic peak was obtained, and the content was calculated using the corresponding sugar residue type chromatographic peak area, where the molecular weight is MW; relative molar ratio (%) = relative molar amount / the sum of relative molar amounts of each component. As shown in the table, the sugar residue connection sequence of COP is mainly 4-Glc (p), and it is composed of multiple terminals such as t-Ara (f), 4,6-Glc (p), 4-Gal (p), t-Gal (f), t-Gal (p) and 3,5-Ara (f). It proves that COP has many branches and a complex secondary structure.

[0083] Table 2 Sugar residue connection patterns of COP

[0084]

[0085] Step 5: Structural analysis of the purified camellia oleifera seed cake polysaccharide by nuclear magnetic resonance. The camellia oleifera seed cake polysaccharide COP obtained in Example 1 was dissolved in 0.5 mL of D2O, and TMSP was used as an internal standard. The one-dimensional nuclear magnetic resonance was detected by a 600 MHz Bruker nuclear magnetic resonance spectrometer. 13 C-NMR, 1 H-NMR and 2D NMR 1 H- 1 H COSY, HMBC, HSQC, NOESY, TOCSY; calibration: HDO hydrogen δH = δ4.70ppm, TMSP methyl carbon δC = δ-1.80ppm. The results are as follows Fig. 9 As shown, analysis shows that the repeating unit structure of camellia seed cake polysaccharide COP is as follows:

[0086]

[0087] In general, the molecular weight of purified COP was 8772 Da (Table), and it was composed of mannose, glucose, galactose, xylose, and arabinose with a molar ratio (%) of 10.583:53.413:19.710:2.717:13.577. COP had →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-β-D-Galp-(1→) as the main residues, with →4)-β-D-Galp(1→ and →4)-α-D-Glcp-(1→ as the main chain, and the branching site was located at 6.

[0088] Table 3 Camellia oleifera seed cake polysaccharide COP molecular weight results

[0089]

[0090] Example 3: Effect of the camellia oleifera seed cake polysaccharide (COP) obtained in Example 1 on the immune activity of mice.

[0091] Experimental methods:

[0092] Experimental groups: 40 healthy BALB / c mice were randomly divided into 4 groups (n=10), COP high-dose group (COP-H, 1200 mg / kg), COP medium-dose group (COP-M, 600 mg / kg), COP low-dose group (COP-L, 300 mg / kg), and blank control group (normal saline).

[0093] All mice were gavaged with 0.1 mL / 10 g of the drug at 2 pm every day. The control group was gavaged with an equal amount of normal saline and weighed. Subsequent experiments were performed after 28 days of continuous administration.

[0094] Determination of body weight and organ index of mice: On the 28th day of the experiment, 12 hours after the last administration, the mice were weighed, and the spleen and thymus were removed and the organs were weighed immediately. The immune organ index was calculated as follows: organ index (mg / g) = organ mass (Mg) / D (G).

[0095] Determination of the effect of the camellia seed cake polysaccharide COP obtained in Example 1 on mouse serum immunoglobulin: After blood was collected from the eyeballs of each group of mice, it was placed at room temperature for 30 minutes, centrifuged at 3000 r / min for 10 minutes, and the supernatant was taken for use. The IgA and IgG contents were detected according to the instructions of the ELISA kit.

[0096] Determination of the effect of COP on mouse serum cytokines: IL-2, IL-10, IL-12p70, and TNF-α levels were detected according to the instructions of the ELISA kit.

[0097] Determination of the effect of COP on mouse spleen cell proliferation: Four mice were randomly selected from each group. After routine sacrifice, they were disinfected with 75% alcohol for 5 minutes, taken out, and placed in a clean bench. The complete mouse spleen was separated by aseptic operation, washed with PBS, and the fat, blood and other tissues were peeled off and cut into small pieces. Rinse with PBS, grind the spleen pieces into a 200-mesh cell sieve, add PBS in small amounts and multiple times to rinse and filter until the spleen becomes a white and transparent tissue to obtain a single cell suspension. The single cell suspension collected from each group was transferred into a 15mL centrifuge tube, PBS was adjusted to the same volume in each tube, centrifuged, the supernatant was discarded, 2mL of RBC lysis solution was added, blown and mixed, reacted for 3min, 2mL of cell culture medium was added to terminate the reaction, centrifuged, and the supernatant was discarded. Resuspend in PBS, wash twice, and obtain primary spleen lymphocytes. Add RPMI 1640 culture medium containing 10% fetal bovine serum, 100mmol / L penicillin and streptomycin, transfer a portion of the cells (200μL) to a 96-well plate, and adjust the cell concentration to 5.0×10 6 / mL, make 80μL / well, add LPS (final concentration of 5μg / mL) for synergistic stimulation, set up LPS control, cell control and blank control wells; culture in a 37.5℃, 5% CO2 incubator for 48h, and record the absorbance at 450nm with an enzyme reader.

[0098] Determination of the effect of COP on CD4 lymphocytes in mouse spleen + 、CD8 + Effect of T subsets: A portion of the spleen lymphocytes extracted in the previous step was transferred to a 24-well plate at a concentration of 2.0 × 10 6 / mL, 1mL cell suspension per well, culture for 48h, collect cells, wash twice with PBS, add corresponding anti-CD3-FITC, anti-CD4-PE and anti-CD8-APC antibodies, react in the dark for 30min, centrifuge and discard supernatant, add 1mL PBS to resuspend, centrifuge at 4000r / min for 5min, discard supernatant, add 1mL PBS to resuspend, finally transfer to flow cytometer injection tube, and finally use flow cytometer to detect the effect of COP on mouse spleen CD4 + 、CD8 + Effects of T lymphocyte subsets.

[0099] Pathological examination: After the isolated thymus and spleen tissues were fixed, embedded, sectioned, and stained, the morphological changes of the liver tissues were observed under a microscope.

[0100] Result analysis:

[0101] After 28 days of intragastric administration, the mice in the medium-dose and low-dose COP groups were in good shape, larger in size, in good spirits, lively and active, and stronger than 14 days after intragastric administration. The mice in the high-dose COP group were slightly depressed, with rough and messy fur, fecal contamination in the anus, a tendency to diarrhea, secretions around the eyes, and dull eyes. Compared with the normal group, they were slightly shorter and less active, with wounds, suppuration, and nodules on their skin, which was suspected to be caused by fighting ( Fig.10 A). The weight growth trend of the medium-dose and low-dose COP groups was better than that of the blank control group, and the difference in the low-dose group was significant (P<0.05), indicating that the low-dose drug effect may be relatively better. Compared with the blank control group, the body weight of the high-dose COP group decreased, and the difference was significant (P<0.05), indicating that the high-dose COP group may be toxic. The medium-dose and low-dose COP groups promoted the growth, development and maturity of mice ( Fig.10 B). After counting the spleen index, it was found that compared with the blank control group, the spleen index of the COP medium-dose group was significantly higher than that of the control group (P<0.05), while the index difference between the high-dose group and the low-dose group was not significant, and the value was similar to that of the blank control group. After counting the thymus index, it was found that there was no statistically significant difference between the COP high-dose, medium-dose, and low-dose groups and the blank control group, and the indexes were all within the normal range ( Fig.10 COP can significantly increase the secretion of specific immunoglobulins IgA and IgG ( Fig.11 ). COP can promote the secretion of IL-2 cytokine, and its effect is optimal at low doses ( Fig.12 COP can promote the secretion of IL-10, and the effect is optimal at a medium dose (Figure 2 Fig.12 COP can promote the secretion of IL-12p70 in mouse serum, and the effect is optimal at low doses ( Fig.12C). There was no statistically significant difference in the effects of COP on the secretion of TNF-α in mouse serum between the three dose groups and the blank control group, indicating that COP cannot promote the secretion of TNF-α ( Fig.12 COP can significantly promote the proliferation of mouse spleen lymphocytes ( Fig.13 A). COP synergistically promotes the proliferation of spleen lymphocytes with a stronger effect ( Fig.13 B). COP and PHA have a stronger effect in promoting the proliferation of spleen lymphocytes ( Fig.13 C). CD4 in the medium-dose group and the low-dose group after COP + The percentage of T lymphocytes was significantly higher than that of the blank control group (P<0.001), and the COP results of the high-dose group were not significant ( Fig.14 A). COP can increase CD4 + 、CD8 + The percentage of T lymphocytes and CD4 + / CD8 + The ratio of Fig.14 B and C). Histopathological changes of spleen and thymus such as Fig.15 As shown in A. The spleens of mice in each group showed changes. Compared with the control group, microscopic examination of the spleens of mice in the medium-dose and low-dose groups under the action of COP showed slight to moderate lymphocyte hyperplasia, with obvious hyperplasia of white pulp germinal centers and marginal zone lymphocytes, and other mainly marginal zone lymphocyte hyperplasia. There were scattered lymph nodules, and slight to mild lymphocyte hyperplasia was observed in the germinal centers of the lymph nodules. There was no pathological change such as lymphopenia, lymph nodule atrophy, and congestion in the red pulp. Microscopic examination of the high-dose group after the action of COP showed slight to mild decrease in lymphocytes, mainly marginal zone lymphocyte decrease. No pathological changes were observed in the thymus of mice in the high-, medium-, and low-dose groups. The results showed that compared with the blank control group, COP can promote the activation of splenic lymphocytes and enhance the immune function of the spleen. After the action of camellia oleifera polysaccharides in the dose group and the low-dose group on mice, the histological changes of various organs are as follows Fig.15 As shown in B. It can be seen that the heart, liver, lung, and kidney structures of all groups were normal, and no pathological changes were observed.

[0102] In general, COP can promote the activation of splenic lymphocytes, enhance the immune activity of the spleen, and effectively promote the cellular immunity and humoral immunity of BALB / c mice. Comprehensive evaluation showed that the COP medium-dose group (COP-M) and COP low-dose group (COP-L) had the best immune activity enhancement.

[0103] Example 4: Antioxidant activity test of camellia oleifera cake polysaccharide (COP) obtained in Example 1

[0104] Experimental methods:

[0105] (I) ABTS free radical scavenging ability test

[0106] The free radical scavenging ability of ABTS was detected using the Solarbio kit.

[0107] 1. Add reagents to the EP tube according to the following table:

[0108]

[0109] 2. Mix thoroughly and let stand at room temperature away from light for 6 minutes.

[0110] 3. In a 96-well plate, add 200 μL to each well.

[0111] 4. Measure the absorbance at 405 nm and record them as A blank, A measurement, A control, and A positive control respectively.

[0112] 5. ABTS free radical scavenging rate calculation:

[0113] ABTS + Free radical scavenging rate % = (A-A0) / A0*100, the measured value is recorded as A, and the sample without antioxidant is recorded as A0.

[0114] (2) DPPH free radical scavenging ability test

[0115] The DPPH free radical scavenging ability was detected using the Solarbio kit.

[0116] 1. Add reagents to the EP tube according to the following table:

[0117]

[0118] 2. Vortex to mix and let stand at room temperature away from light for 30 min.

[0119] 3. In a 96-well plate, add 200 μL to each well.

[0120] 4. Measure the absorbance at 515 nm and record it as A blank, A determination, A control, and A positive control respectively.

[0121] 5. DPPH free radical scavenging rate calculation:

[0122] DPPH clearance rate % = [1-(A i -A j ) / A0]×100%; where A i is the absorbance of each sample group; A j is the absorbance of the blank group (sample solution + anhydrous ethanol); A0 is the absorbance of the control group (deionized water + DPPH alcohol solution). ).

[0123] Result analysis:

[0124] Effect of COP on ABTS free radical scavenging Fig.16 As shown. With the increase of COP concentration, the ABTS free radical scavenging rate also increased, indicating that COP has a dose-dependent effect on the scavenging of ABTS free radicals. Compared with the VC positive control group, the clearance rates of all other drug concentration groups were significantly lower than VC. The results showed that when the COP concentration was between 50μg / mL-250μg / mL, it had a scavenging effect on ABTS free radicals. The effect of COP on the scavenging of DPPH free radicals is shown in Fig.17 As shown. With the increase of COP concentration, the free radical scavenging rate of DPPH showed a trend of first increasing and then decreasing. At 150μg / mL, the scavenging rate reached a maximum of 20.43%. Compared with the VC positive control group, the scavenging rates of each drug concentration group were significantly lower than that of VC. The results showed that when the concentration of COP was 50μg / mL-250μg / mL, it could play a certain role in scavenging DPPH.

[0125] In general, COP has a certain scavenging effect on ABTS free radicals and DPPH free radicals, indicating that COP has antioxidant activity and has a certain antioxidant effect.

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A camellia oleifera seed cake polysaccharide having immune enhancement and antioxidant activities, characterized in that: The monosaccharide molecular composition chain of the polysaccharide is shown in the following formula (I): The weight average molecular weight of the camellia seed cake polysaccharide is 8772Da; The camellia oleifera seed cake polysaccharide consists of mannose, glucose, galactose, xylose and arabinose, wherein the molar ratio of each monosaccharide is 10.583:53.413:19.710:2.717:13.

577.

2. The camellia seed cake polysaccharide having immune enhancement and antioxidant activity according to claim 1, characterized in that: The structural analysis of the camellia seed cake polysaccharide showed that →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, →4)-β-D-Galp-(1→) were the main residues, with →4)-β-D-Galp(1→ and →4)-α-D-Glcp-(1→ as the main skeleton, and the branching site was located at No.

6.

3. The method for preparing the camellia seed cake polysaccharide having immune enhancement and antioxidant activity according to claim 1, characterized in that Prepared according to the following steps: S1, extracting the pretreated camellia seed cake powder with alcohol to remove fat-soluble small molecules and pigments to obtain medicinal residues; S2, drying the residue obtained in step S1, and then extracting it by adding water and heating with the assistance of ultrasound, filtering, collecting the filtrate, concentrating, and cooling to obtain a concentrated solution; S3, subjecting the concentrated solution obtained in step S2 to alcohol precipitation, and collecting the precipitate; S4, removing protein from the precipitate obtained in step S3, and freeze-drying to obtain crude polysaccharide from camellia seed cake; S5, using the phenol-sulfuric acid method to determine the polysaccharide content of the crude polysaccharide of the oil-tea camellia seed cake obtained in step S4; using a pretreated DEAE-Sepharose FF ion exchange column to desalt and semi-purify the crude polysaccharide of the oil-tea camellia seed cake, and eluting with distilled water, 0.2M NaCl solution, 0.5M NaCl solution and 1.0M NaCl solution in sequence, collecting the distilled water elution part rich in polysaccharide content, dialyzing, drying and freeze-drying to obtain the oil-tea camellia seed cake polysaccharide powder; S6. The camellia oleifera cake polysaccharide powder obtained in step S5 is classified using a Chromdex 75PG gel filtration column, concentrated, freeze-dried, and collected to obtain a single peak component, which is the camellia oleifera cake polysaccharide with immune enhancement and antioxidant activity.

4. The preparation method according to claim 3, characterized in that: The pretreatment in step S1 refers to pulverizing the camellia seed cake; the alcohol extraction is to use 95% by volume ethanol to perform a reduced pressure reflux treatment at 60° C. for 3 times, each time for 4 hours; The drying in step S2 is a drying treatment; the concentration is to concentrate the filtrate to a density of 1g / mL; the specific conditions of the ultrasonic-assisted water-addition and heating extraction include: adding water according to a solid-liquid ratio of 25g:1mL, ultrasonic time for 20 minutes under 300W ultrasonic power conditions, and then heating the reaction at a temperature of 60°C for 6 hours.

5. The preparation method according to claim 3, characterized in that: The alcohol precipitation treatment in step S3 is specifically to add 95% ethanol by volume to the concentrated solution, stir, precipitate polysaccharides with alcohol, and collect the precipitate after standing for 24 hours; the protein removal in step S4 is carried out by the Sevage method, specifically adding deionized water to the precipitate obtained in step S3 for dilution, and then mixing it with the Sevage solution in a conical flask at a volume ratio of 4:1, shaking in a shaker for 30 minutes, centrifuging at 4000r / min for 10 minutes, collecting the upper liquid to remove the protein, and repeating the above protein removal steps 3 times.

6. The preparation method according to claim 3, characterized in that: In step S4, the freeze-drying is to pre-cool the camellia oleifera seed cake polysaccharide obtained after removing the protein in a -80°C refrigerator for 4 hours, and then freeze-dry it in a vacuum freeze dryer for 72 hours; the crude camellia oleifera seed cake polysaccharide obtained after freeze-drying is stored in a -20°C refrigerator.

7. The preparation method according to claim 3, characterized in that: The DEAE-Sepharose FF pretreatment in step S5 is specifically carried out as follows: the DEAE-Sepharose FF gel suspension in the Buchner funnel is connected to a vacuum pump to be drained, and the DEAE-Sepharose FF gel suspension is repeatedly washed with pure water until the filler has no alcohol taste, and the resulting precipitated gel is poured into a beaker, and distilled water of 1 / 2 to 1 times the volume of the precipitated gel is added and stirred, and then poured into The XK chromatography column was wet packed, and the peristaltic pump was started at a flow rate of 250 cm / h. Ultrapure water was used to rinse and balance the gel surface. The dialysis in step S5 is performed in a 3.5KDa dialysis bag; the drying and freeze-drying are performed under vacuum conditions.

8. The preparation method according to claim 3, characterized in that: The specific operation of using the Chromdex 75PG gel filtration column for classification in step S6 is: The column was packed with Chromdex 75PG and balanced with ultrapure water at a flow rate of 30 cm / h and a sample loading amount of 1% of the column volume. After all the camellia oleifera cake polysaccharide powder samples were loaded into the gel filtration column, ultrapure water was used for elution at a rate of 4.5 mL / tube and a control flow rate of 1.8 mL / min. Each tube of solution collected was detected online with a differential detector, and the eluent in the elution peak area with good peak symmetry and uniform peak height was collected.

9. The preparation method according to claim 8, characterized in that: The eluent in the elution peak region with good peak symmetry and uniform peak height is collected from the eluent in tubes 5 to 50.

10. The use of the camellia seed cake polysaccharide according to claim 1 in preparing a product having both immune enhancement and antioxidant activities, characterized in that: The product is food, medicine, daily chemical product or feed.

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