Xanthomonas oryzae exopolysaccharide as well as preparation method and application thereof

Abstract

The invention discloses a xanthomonas oryzae exopolysaccharide as well as a preparation method and application thereof, and the weight-average molecular weight of the xanthomonas oryzae exopolysaccharide is 1 * 1052 * 107000. The preparation method comprises the following steps: inoculating a bacterial liquid into an NA liquid culture medium, removing small molecular impurities from the fermented bacterial liquid, freeze-drying to obtain refined xanthomonas oryzae extracellular crude polysaccharide, separating the xanthomonas oryzae extracellular polysaccharide, respectively taking 0.2 mol / L NaCl and 0.3 mol / L NaCl as eluents, and determining the content of the xanthomonas oryzae extracellular polysaccharide according to a chromatogram drawn by a phenol-sulfuric acid method. And collecting fractions with the same signal peak, concentrating, dialyzing by using a 3500 Da dialysis bag, and freeze-drying to obtain the xanthomonas oryzae exopolysaccharide. The pharmaceutical composition can resist A549 non-small cell lung cancer and PC-3 human prostate cancer cells, and the preparation method is simple.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to an exopolysaccharide of Xanthomonas oryzae, a preparation method of the exopolysaccharide, and application of the exopolysaccharide of Xanthomonas oryzae in resisting A549 non-small cell lung cancer and PC-3 human prostate cancer cells. Background Art

[0002] Cancer is a disease caused by the abnormal proliferation and spread of cells. Normally, cell growth, division, and death are controlled, but cancer cells experience a breakdown in these regulatory mechanisms, leading to uncontrolled proliferation and the formation of tumors (benign or malignant). Malignant tumors can invade surrounding tissues and spread to other sites through the bloodstream or lymphatic system. Cancer is a major impediment to increased life expectancy. The 2024 National Cancer Report from the National Cancer Center of China reveals that the top five malignant tumors are lung cancer, colorectal cancer, thyroid cancer, liver cancer, and stomach cancer. Lung cancer is the leading cause of both morbidity and mortality from malignant tumors in China. Prostate cancer is also increasing in incidence at an average annual rate of 1.4%. Currently, the main treatments for tumors include surgery, radiotherapy, chemotherapy, and hormone therapy. While these treatments have some effectiveness for early-stage tumors, they are often ineffective for patients with advanced and metastatic disease. However, in many cases, they also carry serious side effects. For example, chemotherapy drugs can damage or even kill normal cells, causing serious side effects such as hematological, gastrointestinal, and epithelial reactions. These treatments often cause significant physical and mental distress to patients, leading to the development of novel anticancer drugs with improved efficacy and minimal adverse reactions. Polysaccharides, a class of biomacromolecules, have attracted widespread research interest in recent years due to their safety, anticancer activity, and potential to enhance the efficacy of conventional chemotherapy drugs with minimal side effects.

[0003] Xanthomonas oryzae is a Gram-negative bacterium with blunt ends, short rods, and a single polar flagellum approximately 6-8 microns long. It lacks spores and bud membranes and is aerobic. It primarily parasitizes rice and is a plant pathogen that causes rice diseases. Xanthomonas oryzae synthesizes a type of macromolecular carbohydrate intracellularly and releases it onto the cell surface. This exopolysaccharide (xanthan gum) is composed of repeating pentose units and is protein-free, single-component, with a molecular weight of less than 2×10 6 Da polysaccharides have excellent emulsification stability, viscosity, acid and alkali resistance, and compatibility, and are widely used in food, medicine, environmental protection, cosmetics and other fields. However, their anti-tumor activity has not been reported. Summary of the Invention

[0004] The present invention aims to overcome the above disadvantages and provide an exopolysaccharide of Xanthomonas oryzae that is resistant to A549 non-small cell lung cancer and PC-3 human prostate cancer cells and has a simple preparation method.

[0005] Another object of the present invention is to provide a method for preparing the Xanthomonas oryzae exopolysaccharide.

[0006] Another object of the present invention is to provide the application of the Xanthomonas oryzae exopolysaccharide in resisting A549 non-small cell lung cancer and PC-3 human prostate cancer cells.

[0007] The Xanthomonas oryzae exopolysaccharide of the present invention has a weight average molecular weight of 1×10 5 -2×10 7 Ten thousand.

[0008] The above-mentioned Xanthomonas oryzae exopolysaccharide preferably has a weight average molecular weight of 1.75×10 6 -1.89×10 6 Ten thousand.

[0009] The present invention provides a method for preparing Xanthomonas oryzae exopolysaccharide, comprising the following steps:

[0010] (1) Prepare NB liquid medium: add 15.0g glucose, 7.5g peptone, 1.5g yeast powder, and 4.5g beef extract to 1.5L ultrapure water, and adjust the pH to 7.0-7.2 with 3mol / L sodium hydroxide solution; prepare NA solid medium: add 3.0g agar to 200mL NB liquid medium. Pour NB and NA medium into 250mL conical flasks, seal with sealing film, and sterilize in an autoclave at 121℃ for 20min. Pour the NA medium into a glass culture dish while it is still hot, wait for it to cool and solidify, and seal with plastic wrap for later use;

[0011] (2) Thaw the Xanthomonas oryzae stored in a -80°C refrigerator, draw 5 μL of the bacterial solution and inoculate it into NA liquid medium in a biosafety cabinet, culture it in a 28°C incubator for three days, pick a single colony containing Xanthomonas oryzae and inoculate it into 50 mL of NB liquid medium, shake and culture it in a shaker at 240 rpm at the same temperature for 24-36 h (OD600 = 1.0), draw the bacterial solution into NB medium at a ratio of bacterial solution: NB medium = 1:100 and expand the culture for 3 days;

[0012] (3) The fermented bacterial liquid was centrifuged at 9500 rpm for 5 min in a refrigerated centrifuge, and the supernatant was collected. Anhydrous ethanol 3 times the volume of the supernatant was added to the supernatant for precipitation overnight. After centrifugation at 9500 rpm for 15 min, the precipitate was collected, and the precipitate was redissolved and dialyzed with a 3500 Da dialysis bag for 48 h to remove small molecular impurities. After freeze-drying, the refined extracellular crude polysaccharide was obtained and stored in a refrigerator at -20°C.

[0013] (4) 100 mg of crude extracellular polysaccharide was dissolved in distilled water and filtered through a 0.45 μm filter membrane. The extracellular polysaccharide of Xanthomonas oryzae was separated using an anion exchange column (diethylaminoethyl cellulose) (2.6 cm × 20 cm). The eluents were 0.2 mol / L and 0.3 mol / L NaCl, respectively. According to the chromatogram drawn by the phenol-sulfuric acid method, the fractions with the same signal peak were collected, concentrated, dialyzed using a 3500 Da dialysis bag, and freeze-dried to obtain the extracellular polysaccharide of Xanthomonas oryzae.

[0014] The invention discloses an application of an extracellular polysaccharide of Xanthomonas oryzae in resisting A549 non-small cell lung cancer and PC-3 human prostate cancer cells.

[0015] Compared with existing technologies, the present invention has significant advantages. As can be seen from the above technical solutions, the Xanthomonas oryzae exopolysaccharide composition of the present invention uses readily available raw materials and is simple to prepare. Experiments have demonstrated significant anti-A549 non-small cell lung cancer and PC-3 human prostate cancer cell activity, while also being non-toxic, residue-free, and environmentally friendly.

[0016] The beneficial effects of the present invention are further illustrated by the following specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the infrared spectra of EPS-H2 and EPS-H3;

[0018] Figure 2 This is the UV full wavelength scanning spectrum of EPS-H2 and EPS-H3;

[0019] Figure 3 are the scanning electron micrographs of EPS-H2 (A) and EPS-H3 (B);

[0020] Figure 4 This is the Congo red test image of EPS-H2 and EPS-H3;

[0021] Figure 5 is the ABTS+ free radical scavenging capacity of EPS-H2 and EPS-H3;

[0022] Figure 6 It is the DPPH free radical scavenging ability of EPS-H2 and EPS-H3. DETAILED DESCRIPTION

[0023] Example 1:

[0024] A method for preparing Xanthomonas oryzae exopolysaccharide EPS-H2 comprises the following steps:

[0025] (1) Prepare NB liquid culture medium: add 15.0g glucose, 7.5g peptone, 1.5g yeast powder, and 4.5g beef extract to 1.5L ultrapure water, and adjust the pH to 7.0-7.2 with 3mol / L sodium hydroxide solution. Prepare NA solid culture medium: add 3.0g agar to 200mL NB liquid culture medium. Pour NB and NA culture media into 250mL conical flasks respectively, seal them with sealing film, and sterilize them in a high-pressure sterilizer at 121℃ for 20min. Pour the NA culture medium into a glass culture dish while it is still hot, wait for it to cool and solidify, and seal it with plastic wrap for later use;

[0026] (2) Thaw the Xanthomonas oryzae (provided by the Fine Chemicals Center of Guizhou University) stored in a -80°C refrigerator, draw an appropriate amount of bacterial liquid and inoculate it into NA liquid culture medium in a biosafety cabinet, culture it in a 28°C incubator for three days, pick a single colony containing Xanthomonas oryzae and inoculate it into 50 mL NB liquid culture medium, and culture it in a shaking incubator at 240 rpm at the same temperature for 24-36 h (OD 600 =1.0), the bacterial solution was added to NB medium at a ratio of 1:100 and cultured for 3 days;

[0027] (3) The fermented bacterial liquid was centrifuged at 9500 rpm for 5 min in a refrigerated centrifuge, and the supernatant was collected. Anhydrous ethanol 3 times the volume of the supernatant was added to the supernatant, and the supernatant was precipitated at 4°C overnight. The precipitate was collected after centrifugation at 9500 rpm for 15 min, and the precipitate was redissolved and dialyzed with a 3500 Da dialysis bag for 48 h to remove small molecular impurities. The precipitate was freeze-dried according to the following procedure: the initial temperature was set at -40°C, and the temperature was lowered to -40°C and maintained for 1 h; then the temperature was programmed to 40°C at a rate of 0.5°C / min and maintained for 8 h; the refined extracellular crude polysaccharide was obtained and stored in a -20°C refrigerator;

[0028] (4) Dissolve 100 mg of crude extracellular polysaccharide in distilled water, filter through a 0.45 μm filter membrane, and separate the extracellular polysaccharide of Xanthomonas oryzae using an anion exchange column (diethylaminoethyl cellulose) (2.6 cm × 20 cm). The eluent is 0.2 mol / L NaCl. According to the chromatogram drawn by the phenol-sulfuric acid method, the fractions with the same signal peak are collected. The solution is concentrated to 2-4 mL by vacuum distillation at 45°C, dialyzed using a 3500 Da dialysis bag, and finally the solution in the dialysis bag is concentrated again to 1 mL. The dialyzed product is freeze-dried according to the following procedure: the initial temperature is set at -40°C, the temperature is lowered to -40°C and maintained for 1 hour; then the temperature is programmed to 40°C at a rate of 0.5°C / min and maintained for 8 hours. Finally, the dried extracellular polysaccharide EPS-H2 of Xanthomonas oryzae is obtained.

[0029] Example 2:

[0030] A method for preparing Xanthomonas oryzae exopolysaccharide EPS-H3 comprises the following steps:

[0031] (1) Same as Example 1;

[0032] (2) Same as Example 1;

[0033] (3) Same as Example 1;

[0034] (4) 100 mg of crude extracellular polysaccharide was dissolved in distilled water and filtered through a 0.45 μm filter membrane. The exopolysaccharide of Xanthomonas oryzae was separated using an anion exchange column (diethylaminoethyl cellulose) (2.6 cm × 20 cm). The eluent was 0.3 mol / L NaCl. According to the chromatogram drawn by the phenol-sulfuric acid method, the fractions with the same signal peak were collected. The solution was concentrated to 2-4 mL by vacuum distillation at 45°C, dialyzed using a 3500 Da dialysis bag, and finally the solution in the dialysis bag was concentrated again to 1 mL. The dialyzed product was freeze-dried according to the following procedure: the initial temperature was set at -40°C, and the temperature was lowered to -40°C and maintained for 1 hour; then the temperature was programmed to 40°C at a rate of 0.5°C / min and maintained for 8 hours. Finally, the dried exopolysaccharide EPS-H3 of Xanthomonas oryzae was obtained.

[0035] Test Example 1: Qualitative determination of extracellular polysaccharides of Xanthomonas oryzae:

[0036] 1. Determination of molecular weight of homogeneous Xanthomonas oryzae exopolysaccharide

[0037] (1) Experimental steps: 2 mg of the extracellular polysaccharide of Xanthomonas oryzae obtained in Example 1-2 was accurately weighed, dissolved in ultrapure water to a final concentration of 2 mg / mL, and filtered through a 0.22 μm microporous filter membrane for later use.

[0038] Pullulan standards of varying molecular weights (Mw = 6.4, 9.8, 21.9, 49.4, 107, 216, 348, and 739 kDa) were prepared into 1 mg / ml solutions and filtered through a 0.22 μm microporous membrane for later use. The standards and Xanthomonas oryzae exopolysaccharide samples were then analyzed using a 7.8×300 mm SEC-500 column at 30°C, ultrapure water as the mobile phase, and a 50 μL injection volume. Dextran standards were injected sequentially from small to large molecules, and the corresponding retention times of the standards were recorded. A standard curve was then plotted using retention time as the abscissa and the logarithm of the molecular weight as the ordinate. The molecular weight range of the Xanthomonas oryzae exopolysaccharide was calculated based on the standard curve.

[0039] (2) Test results: The results are shown in Table 1. The weight average molecular weight (Mw) of EPS-H2 is 1.75×10 6Da, the number average molecular mass (Mn) is 1.56×10 6 Da, Mw / Mn value is 1.12; the weight average molecular weight (Mw) of EPS-H3 is 1.89×10 6 Da, the number average molecular mass (Mn) is 1.65×10 6 The Da, Mw / Mn value is 1.14. Mw / Mn represents the molecular weight distribution width. The closer the value is to 1, the narrower the molecular weight distribution is and the higher the uniformity of the polysaccharide. This shows that EPS-H2 and EPS-H3 are uniform polysaccharides with narrow molecular weight distribution.

[0040] Table 1 Molecular weight range of Xanthomonas oryzae exopolysaccharides

[0041]

[0042] 2. Analysis of monosaccharide composition of Xanthomonas oryzae exopolysaccharides

[0043] (1) Experimental procedures: The polysaccharide was hydrolyzed with 2 mol / L trifluoroacetic acid at 100°C for 6 h. After removing the excess trifluoroacetic acid, the polysaccharide hydrolyzate was derivatized pre-column with 200 μL of 0.5 mol / L PMP methanol solution in a 70°C water bath for 1 h. After the reaction was complete, the polysaccharide was extracted three times with chloroform to remove unreacted PMP. The upper aqueous phase was filtered through a 0.22 μm membrane and analyzed by liquid chromatography.

[0044] (2) Test results: The test results are shown in Table 2. By comparing the retention time of the monosaccharide standard reference substance, it can be determined that EPS-H2 mainly contains mannose, glucose, and glucuronic acid, with a molar ratio (%) of 1.54:2.13:1.07; EPS-H3 mainly contains mannose, glucose, and glucuronic acid, with a molar ratio (%) of 1.09:1.37:0.72.

[0045] Table 2 Monosaccharide composition of polysaccharides

[0046]

[0047] 3. Monosaccharide linkage analysis of Xanthomonas oryzae exopolysaccharides

[0048] (1) Experimental steps: Weigh about 10 mg of polysaccharide and dissolve it with an appropriate amount of distilled water, add 300 mg of CMC (1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate) to activate the carboxyl group, reduce the carboxyl group with 2 mol / L NaBH4 solution, and obtain the carboxyl group reduction sample by dialysis and freeze drying. Add 2 mL of anhydrous DMSO to the reduced sample under N2 protection and ultrasonically dissolve it. Add 50 mg of freshly ground dry NaOH powder and react at room temperature for 10 minutes. Slowly add 1.0 ml of iodomethane under N2 protection, ultrasonicate at room temperature for 30.0 minutes, and repeat three times. After the reaction is completed, add 1 ml of water to terminate the methylation reaction. Add equal volumes of chloroform and aqueous solution for extraction. Take a small amount of chloroform layer from the methylated sample and detect it by infrared spectroscopy at 3700 cm -1 -3100cm -1 , with no characteristic absorption peaks near hydroxyl groups. The fully methylated polysaccharide sample was added to 2.0 mL of 2 mol / L trifluoroacetic acid and reacted at 100°C for 3 h for acid hydrolysis. After completion, the hydrolyzed product was rotary evaporated to dryness. 2 mL of 35 mg / mL NaBD4 solution (freshly prepared) was added, and the product was reduced at room temperature for 2 h after shaking. After the reaction, 4 mol / L glacial acetic acid (acetic acid) was added dropwise to decompose the excess sodium borohydride and adjust the pH to 7.0. The reaction solution was evaporated to dryness under reduced pressure at 50°C. 3.0 mL of freshly prepared pyridine / acetic anhydride (1:1) was added and the reaction was continued at 100°C for 2.5 h. After cooling to room temperature, 1 mL of deionized water was added and the solution was allowed to stand for 10 min. The solution was then evaporated to dryness under reduced pressure at 50°C. The solution was reconstituted in 1 mL of dichloromethane and analyzed by GC / MS.

[0049] (2) Test results:

[0050] Table 3 Methylation data of EPS-H2

[0051]

[0052] Table 4 Methylation data of EPS-H3

[0053]

[0054] According to Tables 3 and 4, EPS-H2 has four types of glycosidic bonds, while EPS-H3 has five types of glycosidic bonds. Combined with infrared spectroscopy analysis, it can be determined that both EPS-H2 and EPS-H3 contain β-configuration glycosidic bonds. It is speculated that the glycosyl linkage pattern of EPS-H2 is composed of four sugar residues: 1→)-β-D-Manp, →1)-β-D-Glcp-(6→, →1)-β-D-Glcp-(2→, and →1)-β-D-Glcp-(4→); the glycosyl linkage pattern of EPS-H3 is speculated to be composed of five sugar residues: 1→)-β-D-Manp, →1)-β-D-Glcp-(2→, →1)-β-D-Manp-(4→, →1)-β-D-Glcp-(4→, and →1)-β-D-Glcp-(3,4→).

[0055] 4. Infrared characterization of uniform Xanthomonas oryzae exopolysaccharide

[0056] (1) Experimental steps: The polysaccharide sample was subjected to infrared spectroscopy analysis using the thin film method. Weigh 2 mg of freeze-dried sample and spread it into a uniform film (thickness required to be between 10-30 μm). Place it in the infrared spectrometer and use the film without EPS as a blank test to adjust the baseline. Scan in transmission mode. The Fourier transform infrared spectrometer scan range is 4000-400 cm -1 .

[0057] (2) Test results: Figure 1 It can be seen that EPS-H2 and EPS-H3 are at 3302cm -1 The strong and broad absorption peak at 2910 cm -1 The absorption peak observed at 1728 cm is the CH stretching vibration absorption peak and is a typical carbohydrate characteristic peak. -1 The peak at 1634 cm was attributed to the stretching vibration of C=O, which is a characteristic of carboxylic acid ester, indicating that EPS-H2 and EPS-H3 may be an acidic polysaccharide. -1 The absorption peak at 1412 cm-1 is attributed to the asymmetric stretching vibration of C=O, which indicates the presence of uronic acid in EPS-H2 and EPS-H3. -1 The absorption peak at 1038 cm is attributed to the stretching and bending vibration of CH. -1 The absorption peak at 900 cm indicates the presence of pyran rings in EPS-H2 and EPS-H3. -1 The characteristic absorption peak at β-glycosidic bond indicated the presence of β-glycosidic bond. FT-IR results showed that EPS-H2 and EPS-H3 were probably acidic heteropolysaccharides containing pyranose ring, β-glycosidic bond and glucuronic acid.

[0058] 5. UV Characterization of Uniform Xanthomonas oryzae Exopolysaccharide

[0059] (1) Test steps: The polysaccharide sample was prepared into a solution of a certain concentration using distilled water, and the polysaccharide was detected using a UV-visible spectrometer UH5300 with a scanning range of 200-600 nm.

[0060] (2) Test results: Figure 2 It can be seen that EPS-H2 and EPS-H3 have no absorption peaks near 260nm and 280nm, indicating that EPS-H2 and EPS-H3 contain no or very small amounts of proteins and nucleic acids.

[0061] 6. Electron microscopy analysis of uniform Xanthomonas oryzae exopolysaccharide

[0062] (1) Experimental steps: The freeze-dried polysaccharide samples were gold-sprayed, and the morphologies of EPS-H2 and EPS-H3 were characterized by scanning electron microscopy.

[0063] (2) Test results: Figure 3 The results showed that the surface morphology of EPS-H2 was mainly thin flakes, while that of EPS-H3 was mainly thin flakes, but also contained relatively thick rods, which were cross-linked. This may be related to the structural conformation of the polysaccharide.

[0064] 7. Uniform triple helical structure of Xanthomonas oryzae exopolysaccharide

[0065] (1) Experimental steps: Prepare 80 μmol / L Congo red solution and 1 mol / L NaOH solution and set aside. Add various reagents according to the table to make the molar concentrations of NaOH 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L, respectively. Mix well and let stand for 10 minutes. Zero with deionized water. Use a UV-visible spectrophotometer to perform a full wavelength scan in the range of 400-700 nm and measure the maximum absorption wavelength of the sample solution at different concentrations of NaOH solution. In another reaction system, EPS is not added, and other reagents remain unchanged. The maximum absorption wavelength of the sample solution at different concentrations of NaOH solution is measured using the same method. This is the trend of the maximum absorption wavelength of Congo red solution changing with NaOH concentration.

[0066] Table 5 Addition amount of various reagents in Congo red test

[0067]

[0068] (2) Test results:

[0069] Depend on Figure 4It can be seen that when the concentration of NaOH increases from 0 to 0.3 mol / L, the characteristic absorption peak position of the complex (λ max ) showed an obvious red shift phenomenon, indicating that the molecular conformation began to change significantly at a lower alkali concentration (0.1 mol / L). When the NaOH concentration exceeded 0.2 mol / L and 0.3 mol / L, respectively, in a strong alkaline environment, the three-dimensional helical structure of EPS-H2 and EPS-H3 was completely unwound and transformed into a disordered single-stranded conformation, resulting in the loss of its specific binding ability to Congo red. At this time, the λ max The value gradually decreased and finally approached the characteristic absorption peak of free Congo red. This series of changes confirmed that EPS-H2 and EPS-H3 had a typical triple-stranded helical structure in their natural state.

[0070] Test Example 2: In vitro anti-tumor and antioxidant activity test of homogeneous Xanthomonas oryzae exopolysaccharide

[0071] 1. In vitro antitumor activity of homogeneous Xanthomonas oryzae exopolysaccharides

[0072] (1) Experimental steps: MTT method was used to test the anti-tumor activity of polysaccharides: The MTT method was used to test the anti-tumor activity of polysaccharide samples on five cell lines, including A549 (human lung cancer cells), K562 (human myeloid leukemia cells), PC-3 (human prostate cancer cells), 5637 (human bladder cancer cells) and T47D (human ductal mammary cancer cells), and the toxicity was evaluated on HEK-293T (human embryonic kidney cells).

[0073] (2) Test results:

[0074] Table 6 Preliminary screening of antitumor activity of homogeneous Xanthomonas oryzae exopolysaccharide at 1000 μg / mL

[0075]

[0076] Table 7 Antitumor activity rescreening of EPS-H3

[0077]

[0078] As shown in Table 6, the anti-tumor activity test results of the uniform Xanthomonas oryzae exopolysaccharide at a test concentration of 1000 μg / mL. The Xanthomonas oryzae exopolysaccharide described in the table has a certain inhibitory activity against PC-3 and A549 tumor cells, among which EPS-H3 has an inhibitory activity of 55% against PC-3 at a concentration of 1000 μg / mL; and an inhibitory activity of 50% against A549. In addition, the results showed that EPS-H3 had no toxicity to HEK-293T cells at a concentration of 1000 μg / mL, and had the effect of promoting normal cell proliferation. As shown in Table 7, the IC activity of EPS-H3 on PC-3 and A549 tumor cells was tested. 50 The results showed that the IC value of EPS-H3 on A549 tumor cells 50 The value was 422.96 μg / mL, and the IC 50 The value is 411.98μg / mL.

[0079] 2. In vitro antioxidant activity of EPS-H3

[0080] (1) Determination of ABTS free radical scavenging rate

[0081] 1) Experimental steps: First, prepare ABTS stock solution, mix thoroughly and place in the dark to react for 15 hours. Then, dilute the reaction solution with 95% ethanol to an absorbance of 0.70±0.02 at a wavelength of 734 nm to obtain ABTS. + Working solution: When measuring, take 200 μL working solution and mix it with 100 μL polysaccharide solution to be tested. Incubate in the dark for 10 minutes and then measure the absorbance change at 734 nm. Two control groups were set up in the experiment: the sample control group was replaced with ABTS by an equal volume of 95% ethanol. + The blank control group used 95% ethanol instead of polysaccharide sample solution. By comparing the absorbance differences of each group, the free radical scavenging ability of the sample was calculated. The calculation formula is as follows:

[0082]

[0083] Where: A1 represents the absorbance value of the polysaccharide solution to be tested, A0 is the absorbance value of the blank control group without sample, and A2 is the absorbance value of the background control to eliminate background interference.

[0084] 2) Test results: EPS-H3 on ABTS + Free radical scavenging ability Figure 5 As shown in Figure 2, EPS-H2 and EPS-H3 scavenged ABTS in a dose-dependent manner within the concentration range of 0.005–10.0 mg / mL. + When the concentration of EPS-H2 and EPS-H3 solution reached 4.0 mg / mL, the free radicals reached equilibrium and the ABTS+ The free radical scavenging abilities of VC were 65.94% and 58.83% respectively. At this concentration, VC had a strong + The free radical scavenging activity of EPS-H2 and EPS-H3 was 99.97%. + Free radicals have a certain scavenging ability, but the scavenging ability is lower than VC.

[0085] (2) Determination of DPPH free radical scavenging rate

[0086] 1) Experimental Procedure: First, weigh 9.86 mg of DPPH reagent, dissolve it thoroughly in 100 mL of anhydrous ethanol, and store it in a dark environment at 4°C until use. Take 1 mL of the polysaccharide solution of different concentrations and mix it with an equal volume of DPPH working solution. After thorough vortexing, react for 30 minutes in the dark. After the reaction is complete, use a microplate reader to measure the absorbance of each reaction system at 517 nm. For the blank control group, replace the polysaccharide sample solution with 1 mL of anhydrous ethanol, and for the sample control group, replace the DPPH working solution with 1 mL of anhydrous ethanol. By comparing the absorbance differences between the groups, the free radical scavenging rate of the sample can be accurately calculated. The calculation formula is as follows:

[0087]

[0088] Where: A1 represents the absorbance value of the polysaccharide solution to be tested, A0 is the absorbance value of the blank control group without sample, and A2 is the absorbance value of the background control to eliminate background interference.

[0089] 2) Test results: Figure 6 As shown in the experimental results, within the EPS-H3 concentration range of 0.005–10.0 mg / mL, its DPPH radical scavenging ability exhibited a clear concentration-dependent effect. With increasing concentrations of EPS-H2 and EPS-H3, the free radical scavenging rate also increased significantly, demonstrating a good dose-effect relationship. Equilibrium was reached at 8 mg / mL for EPS-H2 and EPS-H3, with scavenging rates of 45.79% and 54.07%, respectively. At this concentration, VC exhibited a 96.67% DPPH radical scavenging activity. These results indicate that EPS-H2 and EPS-H3 also possessed some DPPH radical scavenging capacity, albeit at a lower level than VC.

[0090] The above method is only a preferred embodiment of the present invention and does not impose any formal limitation on the present invention. Without departing from any technical solution content of the present invention, any simple modification, equivalent change and modification made to the above implementation case based on the technical essence of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. An exopolysaccharide from Xanthomonas oryzae, having a weight average molecular weight of 1 × 10 5 ‒2 × 10 7 Ten thousand.

2. The Xanthomonas oryzae exopolysaccharide according to claim 1, having a weight average molecular weight of 1.75 × 10 6 ‒1.89× 10 6 Ten thousand.

3. The method for preparing an exopolysaccharide of Xanthomonas oryzae according to claim 1 or 2, comprising the following steps: (1) Prepare NB liquid medium: add 15.0 g glucose, 7.5 g peptone, 1.5 g yeast powder, and 4.5 g beef extract to 1.5 L ultrapure water, and adjust the pH to 7.0-7.2 with 3 mol / L sodium hydroxide solution; prepare NA solid medium: add 3.0 g agar to 200 mL NB liquid medium. Pour NB and NA medium into 250 mL conical flasks, seal them with sealing film, and sterilize them in an autoclave at 121 °C for 20 min. Pour the NA medium into a glass culture dish while it is still hot, wait for it to cool and solidify, and seal it with plastic wrap for later use. (2) Thaw the Xanthomonas oryzae stored in a −80°C refrigerator, aspirate 5 µL of the bacterial solution and inoculate it into NA liquid medium in a biosafety cabinet, culture it in a 28°C incubator for three days, pick a single colony containing Xanthomonas oryzae and inoculate it into 50 mL of NB liquid medium, culture it in a shaker at 240 rpm at the same temperature for 24–36 h (OD600 = 1.0), aspirate the bacterial solution and add it to NB medium at a ratio of 1:100 to expand the culture for 3 days; (3) The fermented bacterial liquid was centrifuged at 9500 rpm for 5 min in a refrigerated centrifuge, and the supernatant was collected. Anhydrous ethanol 3 times the volume of the supernatant was added to the supernatant for precipitation overnight. After centrifugation at 9500 rpm for 15 min, the precipitate was collected and redissolved. The precipitate was dialyzed with a 3500 Da dialysis bag for 48 h to remove small molecular impurities. After freeze-drying, the refined extracellular crude polysaccharide of Xanthomonas oryzae was obtained and stored in a refrigerator at −20 °C. (4) 100 mg of crude extracellular polysaccharide was dissolved in distilled water and filtered through a 0.45 μm filter membrane. The extracellular polysaccharide of Xanthomonas oryzae was separated using an anion exchange column (diethylaminoethyl cellulose) (2.6 cm × 20 cm). The eluents were 0.2 mol / L and 0.3 mol / L NaCl, respectively. According to the chromatogram drawn by the phenol-sulfuric acid method, the fractions with the same signal peak were collected, concentrated, dialyzed using a 3500 Da dialysis bag, and freeze-dried to obtain the extracellular polysaccharide of Xanthomonas oryzae.

4. Use of the Xanthomonas oryzae exopolysaccharide according to claim 1 or 2 in inhibiting A549 non-small cell lung cancer and PC-3 human prostate cancer cells.

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