Application of ginseng-derived fermented polysaccharide in preparation of medicine for treating colitis-related colon cancer

Abstract

The invention relates to the technical field of biology, in particular to application of ginseng-sourced fermented polysaccharide in preparation of a medicine for treating colitis-related colon cancer, the ginseng-sourced fermented polysaccharide is ginseng-sourced probiotic fermented polysaccharide GPS-F. The probiotic fermented polysaccharide GPS-F is prepared by fermenting a ginseng polysaccharide extract with a Lacnospiraceae bacterium L.B. Strain, and the ginseng polysaccharide extract is prepared by fermenting the ginseng polysaccharide extract with the Lacnospiraceae bacterium L.B. Strain. The obtained ginseng fermented polysaccharide has good biological activity of resisting colitis-related colon cancer, mainly inhibits formation of neutrophil extracellular trapping nets and recovers intestinal barriers, so that tumor formation is inhibited, and the purpose of preventing and treating colitis-related colon cancer is achieved. The compound has a good application prospect in preparation of medicines for preventing and treating the ulcerative colitis-related colon cancer, has important significance in treatment of the ulcerative colitis-related colon cancer, and can be further used for development of health-care products and related medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and relates to the application of ginseng-derived fermented polysaccharide in the preparation of drugs for treating colitis-associated colon cancer. Specifically, it relates to a probiotic fermented polysaccharide GPS-F derived from ginseng prepared by fermenting ginseng polysaccharide extract with Lachnospiraceae bacterium and its application in preventing and treating colitis-associated colon cancer. Background Art

[0002] Colitis-Associated Colon Cancer is a type of colon cancer developed from inflammatory bowel disease with unclear etiology, caused by long-term persistent and recurrent chronic inflammation. Its pathogenesis is related to inflammatory responses, immune microenvironment, and intestinal flora disorders.

[0003] There is a complex symbiotic relationship between the intestinal flora and the host, which plays a key role in maintaining intestinal homeostasis, regulating immunity and metabolism. Probiotics (such as lactic acid bacteria, bifidobacteria, etc.), as representatives of beneficial intestinal flora, exhibit significant anti-inflammatory and anti-tumor potential. Prebiotics (such as dietary fiber), as the "nutritional substrate" of probiotics, can become an important strategy for CRC prevention and treatment by selectively promoting the proliferation of beneficial bacteria, enhancing intestinal barrier function, and regulating host metabolism. Reasonable dietary fiber supplementation is beneficial to intestinal health and can promote the growth of probiotics.

[0004] Ginseng is the dried root or rhizome of Panax ginseng C.A.Mey. of the Araliaceae family, known as the "king of all herbs" and is the most commonly used anti-tumor traditional Chinese medicine for strengthening the healthy qi and tonifying deficiency in clinical practice. Ginseng polysaccharides (GPS) are the main water-soluble components of ginseng and have significant anti-tumor potential. Early studies found that GPS can inhibit tumors and enhance immunity, and is commonly used in adjuvant chemotherapy clinically; it can also reshape the intestinal flora, enhance the efficacy of immunotherapy, or increase the abundance of probiotics as a prebiotic. However, existing technologies mostly focus on directly extracted GPS and ignore the influence of intestinal flora on its metabolism and pharmacodynamic products. It is urgent to deeply analyze its action mode from the perspective of flora-host interaction to expand the application of traditional Chinese medicine polysaccharides in anti-tumor drug development. Summary of the Invention

[0005] The object of the present invention is to overcome the defects existing in the prior art and provide a probiotic fermented polysaccharide GPS-F derived from ginseng prepared by fermenting ginseng polysaccharide extract with Lachnospiraceae bacterium and its application in drugs for treating colitis-associated colon cancer.

[0006] Application of a ginseng-derived fermented polysaccharide in the preparation of a drug for treating colitis-related colon cancer. The ginseng-derived fermented polysaccharide is the probiotic-fermented polysaccharide GPS-F from ginseng. The probiotic-fermented polysaccharide GPS-F from ginseng is prepared by fermenting the ginseng polysaccharide extract GPS with the bacterium Lachnospiraceae bacterium. The bacterium Lachnospiraceae bacterium is from the German Collection of Microorganisms and Cell Cultures DSMZ, with the deposit number DMS24404.

[0007] Application of the ginseng-derived fermented polysaccharide in the preparation of a drug for treating colitis-related colon cancer. The preparation steps of the probiotic-fermented polysaccharide GPS-F from ginseng are as follows:

[0008] (1) Preparation of the ginseng polysaccharide extract GPS;

[0009] (2) Activation and cultivation of the bacterium Lachnospiraceae bacterium;

[0010] (3) Adding the ginseng polysaccharide extract GPS to the bacterial solution of the bacterium Lachnospiraceae bacterium for fermentation, centrifuging, taking the supernatant, performing water extraction and alcohol precipitation, and freeze-drying under reduced pressure; thus obtaining the crude polysaccharide after fermentation.

[0011] (4) Subjecting the crude polysaccharide obtained in step (3) to ion exchange column chromatography, eluting, and collecting the eluate; then purifying through a gel chromatography column to obtain the probiotic-fermented polysaccharide GPS-F from ginseng.

[0012] Application of the ginseng-derived fermented polysaccharide in the preparation of a drug for treating colitis-related colon cancer. The preparation steps of the ginseng polysaccharide extract described in step (1) are as follows:

[0013] (1) Taking ginseng cut pieces, pulverizing them, performing water reflux extraction, and filtering by suction; adding water to the filter residue and continuing reflux extraction, filtering by suction, combining the filtrates, concentrating, adding ethanol, and allowing to stand and precipitate overnight.

[0014] (2) Separating the precipitate and dissolving it in water.

[0015] (3) Mixing chloroform and n-butanol in a volume ratio of 5:1 to form a Sevage reagent, adding the Sevage reagent to the precipitate, shaking well, vortexing, centrifuging, allowing to stand and separating into three layers, taking the top layer liquid, and repeating the above process 5 - 10 times until no protein layer precipitates, and freeze-drying under reduced pressure to obtain the above-mentioned ginseng polysaccharide extract GPS.

[0016] The application of the ginseng-derived fermented polysaccharide in the preparation of drugs for treating colitis-associated colon cancer. The activation and culture method of the Lachnospiraceae bacterium in the preparation step (2) is as follows: Dip the first-generation bacterial solution of the Lachnospiraceae bacterium in the preservation tube, perform streak plating on a plate, and then incubate it statically overnight at 37°C in an anaerobic workstation; Pick a single colony on the plate and inoculate it into a liquid medium for activation for 24 hours to obtain the second-generation bacterial solution. Take the second-generation bacterial solution and transfer it into the liquid medium. Absorb the bacterial solution every 2 hours, measure its absorbance value at OD600, plot the 24-hour bacterial growth curve, determine the logarithmic growth phase and the stationary phase of the bacteria. Finally, the concentration of the Lachnospiraceae bacterium in the liquid medium is 1.5x108 CFU / ml.

[0017] The application of the ginseng-derived fermented polysaccharide in the preparation of drugs for treating colitis-associated colon cancer. The concentration of the ginseng polysaccharide extract GPS in the liquid medium containing the Lachnospiraceae bacterium in the preparation step (3) is 5-10 mg / ml.

[0018] The application of the ginseng-derived fermented polysaccharide in the preparation of drugs for treating colitis-associated colon cancer. Each liter of the liquid medium consists of the following raw materials: 10 g of tryptone, 17.5 g of beef heart infusion powder, 5 g of sodium chloride, 2.5 g of sodium dihydrogen phosphate, and the balance is distilled water; The pH value is 7.2-7.6.

[0019] The application of the ginseng-derived fermented polysaccharide in the preparation of drugs for treating colitis-associated colon cancer. Colitis-associated colon cancer is Colitis-Associated Colon Cancer.

[0020] The present invention ferments the ginseng-derived probiotic polysaccharide GPS-F obtained above, and performs molecular weight, monosaccharide composition, scanning electron microscopy characterization, methylation, nuclear magnetic resonance, and structural analysis of sugar chain units on it. The present invention first provides the application of the ginseng-derived probiotic polysaccharide GPS-F in the treatment of colitis-associated colon cancer, opening up a new use for ginseng polysaccharide. The present invention confirms through a large number of in vivo and in vitro experiments that the ginseng-derived probiotic polysaccharide GPS-F reduces neutrophil recruitment, inhibits the formation of neutrophil extracellular traps, and restores the intestinal barrier, thereby inhibiting tumor formation and achieving the purpose of preventing and treating colitis-associated colon cancer. It has good application prospects in the preparation of drugs for preventing and treating ulcerative colitis or colon cancer, and is of great significance for the treatment of colitis-associated colon cancer. Brief Description of the Drawings

[0021] Figure 1The gel chromatography spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0022] Figure 2 The Fourier transform infrared spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0023] Figure 3 The monosaccharide composition spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0024] Figure 4 The molecular weight spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0025] Figure 5 The scanning electron micrograph of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0026] Figure 6 The total ion chromatogram of methylation of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0027] Figure 7 The 1H NMR spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0028] Figure 8 The 13C NMR spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0029] Figure 9 The 1H-1H COSY spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0030] Figure 10 The HMBC spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown

[0031] Figure 11 The HSQC spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0032] Figure 12 The NOESY spectrum of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown

[0033] Figure 13 The sugar chain unit structure of the probiotic-fermented polysaccharide GPS-F derived from ginseng is shown.

[0034] Figure 14 The probiotic-fermented polysaccharide GPS-F derived from ginseng inhibits early inflammation related to colitis-associated colon cancer and reduces serum inflammatory factors IL-6 and TNF-α.

[0035] Figure 15As shown, the probiotic-fermented polysaccharide GPS-F derived from ginseng protects the intestinal immune barrier in the early inflammatory stage of colitis-associated colon cancer.

[0036] Figure 16 As shown, the probiotic-fermented polysaccharide GPS-F derived from ginseng protects against colitis-associated colon cancer.

[0037] Figure 17 As shown, the probiotic-fermented polysaccharide GPS-F derived from ginseng inhibits the generation of neutrophil extracellular traps in the intestine. Detailed implementation manners

[0038] The present invention will be further described below in conjunction with specific implementation manners. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, but these equivalent forms also fall within the scope defined by the appended claims of this application.

[0039] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope protected by the present invention.

[0040] Example 1: Preparation of probiotic-fermented polysaccharide GPS-F derived from ginseng

[0041] (1) Take 500 g of ginseng slices, powder them and place them in a round-bottom flask. After refluxing and boiling with 5 L of pure water for 2 h, filter with a vacuum pump; add another 4 L of pure water to the residue and continue refluxing and extracting for 2 h, then filter. Repeat this process once; combine the filtrates, concentrate under reduced pressure to a volume of 500 ml, add 2 L of absolute ethanol, and let it stand for precipitation overnight.

[0042] (2) Separate the precipitate and dissolve it in water.

[0043] (3) Take chloroform and n-butanol mixed in a volume ratio of 5:1 to form Sevage reagent. Add Sevage reagent to the precipitate, shake well for 3 min, vortex, centrifuge at 3000 rpm for 15 min, centrifuge, and let it stand to be divided into three layers. Take the top layer liquid, repeat the above process 8 times until no protein layer precipitates, and freeze-dry under reduced pressure to obtain 36.5 g of ginseng polysaccharide extract GPS, with a yield of 7.31%.

[0044] (4) The activation and culture method of Lachnospiraceae bacterium is as follows: Dip the first-generation bacterial liquid of Lachnospiraceae bacterium in the preservation tube, perform streak plating on a plate, and then incubate it statically overnight at 37°C in an anaerobic workstation; Pick a single colony on the plate and inoculate it into a liquid medium to activate for 24 hours to obtain the second-generation bacterial liquid. Each liter of the liquid medium consists of the following raw materials: 10 g of tryptone, 17.5 g of beef heart infusion powder, 5 g of sodium chloride, 2.5 g of sodium dihydrogen phosphate, and the balance is distilled water; The pH value is 7.4. Take 1 ml of the second-generation bacterial liquid and add it to 9 ml of the liquid medium (the same as above) for subculture. Every 2 hours, pipette 200 μl of the bacterial liquid and measure its absorbance value at OD600 to plot the 24-hour bacterial growth curve to determine the logarithmic growth phase and the stationary phase of the bacteria.

[0045] (5) Add pre-fermentation ginseng polysaccharide GPS (concentration 5 - 10 mg / ml) to the L.B. bacterial liquid with a concentration of 1.5x10 8 CFU / ml, ferment in an anaerobic chamber at 37°C for 12 hours, centrifuge at 12000 rpm for 15 minutes, take the supernatant, perform water extraction and alcohol precipitation, and freeze-dry under reduced pressure; Thus, the crude polysaccharide after fermentation is obtained.

[0046] (6) Subject the crude polysaccharide obtained in step (5) to ion exchange column chromatography, elute, collect the eluate, and after concentration, dialysis, and freeze-drying, obtain a white powder; Then, purify it through a gel chromatography column to obtain the probiotic-fermented polysaccharide GPS-F derived from ginseng;

[0047] The specific steps are as follows:

[0048] Ion exchange column chromatography: Take 1 mg / ml of the crude polysaccharide after fermentation in the above step (5), select an ion exchange chromatography column filled with DEAE-52 cellulose, and elute successively with water, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mol / L sodium chloride solutions. The eluted fractions are concentrated, dialyzed, and freeze-dried to obtain a white powder.

[0049] Gel column purification: Take about 1 g of the polysaccharide sample purified by ions and add 20 ml of pure water. Centrifuge at 10000 g for 10 minutes, take the supernatant and pass it through a gel chromatography column for separation and purification at a flow rate of 1 ml / min; Use pure water to elute 1.5 times the column volume, collect one tube every 10 ml, and collect all the eluates.

[0050] The total sugar content of the eluate in each collection tube was determined by the sulfuric acid-phenol method. The specific operation was as follows: Take 100 μl of the diluted polysaccharide supernatant, add 600 μl of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v), the same below), mix well and react in the dark for 10 min, and measure the absorbance at 490 nm. And draw the gel purification elution curve.

[0051] (7) The product obtained in step (6) was subjected to the sulfuric acid-phenol method to determine the total sugar content of the eluate in each collection tube;

[0052] (8) Identify the main elution peak (by default, select the main peak, as Figure 1 seen), and merge the same elution peak;

[0053] (9) Rotary evaporate to 1 / 5 of the original volume;

[0054] (10) Dialyze with a 3 kDa dialysis bag for 24 - 48 h to remove small molecule components.

[0055] (11) Freeze-dry, and use the sulfuric acid-phenol method to identify the purity of the polysaccharide after gel purification, and 17.5 g of the probiotic-fermented polysaccharide GPS-F derived from ginseng is obtained.

[0056] Example 2: Infrared detection and analysis of the probiotic-fermented polysaccharide GPS-F derived from ginseng

[0057] The purified polysaccharide GPS-F prepared by the method of Example 1 above was subjected to infrared detection and analysis by the potassium bromide tablet pressing method. First, take 5 g of potassium bromide and dry it in an oven at 105 °C for 4 hours, and store it in a desiccator for later use. Take 200 mg of potassium bromide powder in an agate mortar and grind it under an infrared lamp. After grinding until it is completely dry and does not form lumps, add an appropriate amount of GPS-F polysaccharide sample and continue to grind to make the sample evenly mixed in potassium bromide. During the grinding process, ensure that the moisture in the GPS-F polysaccharide is completely removed and does not form lumps. Wipe the tablet pressing mold with anhydrous ethanol. After it is completely dry, add an appropriate amount of the mixed polysaccharide sample to the mold, spread it evenly, and press it into a transparent thin film with a tablet press, and put it into a Fourier transform infrared spectrometer for infrared spectrum scanning, and set the scanning range to 4000 cm -1 ~400 cm -1 .

[0058] As Figure 2 the infrared scanning spectrum of GPS-F is shown in the figure. Among them, there is a broad and blunt strong absorption peak at about 3300 cm -1 , which is caused by the stretching vibration of -OH in the polysaccharide structure. The absorption peaks that appear at about 2900 cm -1 are caused by the stretching vibration of C-H in the polysaccharide structure. These are all typical absorption peaks of sugar molecules.

[0059] Example 3: Detection of Monosaccharide Composition of Probiotic Fermented Polysaccharide GPS-F from Ginseng

[0060] For a clean chromatographic vial, weigh the ginseng probiotic fermented polysaccharide GPS-F prepared by the method of Example 1 above, add 1 ml of 2M TFA acid solution, and heat at 121 °C for 2 hours. Pass nitrogen gas and blow dry. Add 99.99% methanol for washing, and then blow dry. Repeat the methanol washing 2 - 3 times. Add sterile water to dissolve, transfer to a chromatographic vial for testing. Take an appropriate amount of the supernatant, rotary evaporate or blow dry with nitrogen gas. Add 1 ml of 2M TFA solution, and heat at 121 °C for 2 hours. Pass nitrogen gas and blow dry. Add 99.99% methanol for washing, and then blow dry. Repeat the methanol washing 2 - 3 times. Add sterile water to dissolve, transfer to a chromatographic vial for testing.

[0061] The chromatographic system used is a Thermo ICS 5000+ ion chromatographic system (ICS 5000+, Thermo Fisher Scientific, USA), and an electrochemical detector is used to analyze and detect the monosaccharide components. Using Dionex TM CarboPac TM PA20 (150 * 3.0 mm, 10 μm) liquid chromatographic column; the injection volume is 5 μl. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5 ml / min; the column temperature is 30 °C; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V).

[0062] As Figure 3 It was detected that GPS-F is composed of four monosaccharides: rhamnose (Rha), arabinose (Ara), galactose (Gal), and glucose (Glc). The monosaccharide concentration ratio is 0.4394:2.9619:5.1779:50.6018.

[0063] Example 4: Detection of Polysaccharide Molecular Weight of Probiotic Fermented Polysaccharide GPS-F from Ginseng

[0064] The ginseng probiotic fermented polysaccharide GPS-F prepared by the method of Example 1 above was dissolved in 0.1 M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) at a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm before being detected on the machine. The chromatographic system used was a gel chromatography - differential - multi - angle laser light scattering system. The liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T - rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOSⅡ (Wyatt technology, CA, USA). The specific chromatographic column and elution conditions were as follows: The gel exclusion chromatographic columns Ohpak SB - 805HQ (300×8 mm) and Ohpak SB - 803HQ (300×8 mm) were connected in series. The column temperature was 45 °C, the injection volume was 100 μL, the mobile phase A was (0.02% NaN3, 0.1 M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was: isocratic for 75 min.

[0065] As Figure 4 It was detected that the weight - average molecular weight of GPS - F was 358.238 kDa, and the number - average molecular weight was 337.065. It was about one - tenth of the weight of the polysaccharide before fermentation. The red line represents the multi - angle laser light scattering signal (i.e., LS, unit is V), and the scattering light intensity is proportional to the molecular size and molecular weight of the substance; the blue line represents the differential signal (i.e., RI, unit is RIU), and the response value depends on the change in the refractive index of the eluent after the column, and is related to the type, concentration, and molecular weight of the substance; the black line is the molecular weight fitted by the two signals, that is, the molar mass (g / mol).

[0066] Example 5: Scanning electron microscopy was used to detect the physical structure of the surface of the polysaccharide of the probiotic fermented polysaccharide GPS - F derived from ginseng

[0067] The polysaccharide GPS - F prepared by the method of Example 1 above was sieved through a 100 - mesh sieve. A small amount was taken and placed on a conductive carbon tape, and after gold spraying treatment, it was scanned and photographed using an electron microscope at a magnification of 500 - 10000 times.

[0068] Technical parameters:

[0069] Resolution: 1.0 nm @ 15 kV; 1.9 nm @ 1 kV; 0.8 nm @ 30 kV (STEM mode); Accelerating voltage: 0.02–30 kV; Beam current: 12 pA to 100 nA; Magnification: 500–10,000; Electron gun: Thermal field emission Schottky electron gun, beam current stability better than 0.2% / h; Detector: High-efficiency in-lens secondary electron detector (in-lens Duo), Everhart-Thornley secondary electron detector; Sample chamber: 330 mm (ф) × 270 mm (h); Image processing: Storage resolution up to 32768 × 24576 pixels; Multiple integration and averaging modes.

[0070] Such as Figure 5 Electron microscopy detection found that the surface structure of GPS-F was circular, mainly fluffy and porous on the surface, similar to the polysaccharide characterization of immunomodulatory and pathogenic microorganism inhibitory activities reported in many literatures.

[0071] Example 6: Polysaccharide structure analysis of ginseng-derived probiotic fermented polysaccharide GPS-F

[0072] Polysaccharide methylation

[0073] (1) Take a small amount (2–3 mg) of the ginseng probiotic fermented polysaccharide GPS-F sample prepared by the method of Example 1 above, and dissolve it in 500 μl of DMSO.

[0074] (2) Add 1 mg of NaOH and incubate for 30 min.

[0075] (3) Add 50 μl of iodomethane solution and react for 1 h.

[0076] (4) Add 1 ml of water and 2 ml of dichloromethane, vortex and mix well, centrifuge, and discard the aqueous phase. Repeat washing with water 3 times.

[0077] (5) Pipette the lower dichloromethane phase and dry it with nitrogen.

[0078] (6) Add 100 μl of 2 M TFA and react at 121 °C for 90 min.

[0079] (7) Evaporate to dryness at 0 °C.

[0080] (8) Add 50 μl of 2 M ammonia water and 50 μl of 1 M NaBD4, mix well, and react at room temperature for 2.5 h.

[0081] (9) Add 20 μl of acetic acid to terminate the reaction, dry it with nitrogen, wash it twice with 250 μl of methanol, and dry it with nitrogen.

[0082] (10) Add 250 μl of acetic anhydride, vortex and mix well, and react at 100 °C for 2.5 h.

[0083] (11) Add 1 ml of water and let stand for 10 min.

[0084] (12) Add 500 μl of dichloromethane, vortex to mix well, centrifuge, discard the aqueous phase, and repeat the water washing 3 times.

[0085] (13) Take the lower dichloromethane phase and detect it by GC-MS.

[0086] Chromatographic parameters: The chromatographic system used is an Agilent gas chromatograph system (Agilent 6890A; Agilent Technologies, USA), and the chromatographic column is TG-200 (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The injection volume is 1 μl, the split ratio is 10:1, the carrier gas is high-purity helium, and the flow rate is 1.5 ml / min; the initial temperature of the column oven is 150 °C and is maintained for 2.0 min, programmed to increase the temperature to 210 °C at a rate of 2 °C / min and maintained for 3 min, then programmed to increase the temperature to 240 °C at a rate of 2 °C / min and maintained for 5 min. The total ion chromatogram is as Figure 6 shown.

[0087] Polysaccharide NMR scanning

[0088] (1) Take an appropriate amount of purified polysaccharide and fully dissolve it in D2O to prepare a polysaccharide solution with a concentration of 40 mg / mL or more.

[0089] (2) Transfer the dissolved solution to a NMR tube, with an added volume of 0.5 mL.

[0090] (3) Place the NMR tube in a nuclear magnetic resonance spectrometer to scan the one-dimensional 1H spectrum, 13C spectrum, and two-dimensional COSY, HSQC, HMBC, and NOESY spectra.

[0091] Use a Bruker (Germany) 600 MHz nuclear magnetic resonance spectrometer to perform quantitative analysis on the target substance, and the scanning temperature is 25 °C. The liquid probe is a QXI 1H / 31P / 13C / 15N 5 mm four-resonance reverse detection probe (Z-gradient, ATM Acc), and the technical parameters are: signal-to-noise ratio (1H): 888; resolution (Hz): 0.32 (rotating) BBFO 1H-19F, 31P-15N, 1H decoupling / observe multi-nuclear forward detection probe (Z-gradient, ATM) technical parameters: signal-to-noise ratio (1H): 798; resolution (Hz): 0.26 (rotating); signal-to-noise ratio (13C): 328; resolution (Hz): 0.1.

[0092] Process of polysaccharide structure analysis

[0093] As Figure 7As shown, the hydrogen spectrum signals of the sample are mainly concentrated between δ 3.0 - 5.5 ppm. Multiple coupling signal peaks are identified in the anomeric signal region of δ 4.3 - 5.4 ppm, indicating that this sample contains multiple sugar residues. The chemical shifts of the corresponding anomeric hydrogens are δ 4.54, 4.86, 5.05, 5.25, 5.3, etc. The non-anomeric hydrogen signals are mainly concentrated in the region of δ 3.1 - 4.2 ppm. Due to severe overlap of individual signals, the chemical shifts of H2 - H6 of each sugar residue need to be assigned by combining COSY and HSQC spectra respectively. Among them, the strong signal peak near δ 4.71 ppm is the solvent peak.

[0094] As Figure 8 shown, multiple signal peaks are identified in the anomeric carbon region of the sample. Combining the cross peaks in the anomeric region of the 13C NMR spectrum and HSQC spectrum ( Figure 11 ), the anomeric signals present in this sample are determined to be: δ 5.3 / 99.57, 4.86 / 98.55, 5.25 / 99.67, 4.54 / 104.31, 5.05 / 107.11 ppm, and are denoted as sugar residues A, B, C, D, E respectively. Combining the information of the sample's bonding structure (methylation), anomeric signals, and comprehensive literature reports, it is thus speculated that sugar residue A is →4)-α-D-Glcp-(1→, sugar residue B is α-D-Glcp-(1→, sugar residue C is →4,6)-α-D-Glcp-(1→, sugar residue D is →4)-β-D-Galp-(1→, and sugar residue E is α-L-Araf-(1→.

[0095] The process of assigning the NMR signals of the main sugar residues is as follows:

[0096] Sugar residue A: The anomeric signal δ 5.3 / 99.57 ppm (H1 / C1) indicates that residue A may be an α-configuration glucose residue. As Figure 9 shown, in the COSY spectrum, according to the cross peak δ 5.3 / 3.53 ppm, H2 (3.53 ppm) of residue A is determined. According to the cross peak δ 3.53 / 3.86 ppm, H3 (3.86 ppm) of residue A is determined. According to the cross peak δ 3.86 / 3.56 ppm, H4 (3.56 ppm) of residue A is determined. According to the cross peak δ 3.56 / 3.73 ppm, H5 (3.73 ppm) of residue A is determined. According to the cross peak δ 3.73 / 3.75 ppm, H6 (3.75 ppm) of residue A is determined. Thus, the chemical shifts of the hydrogens on the sugar ring can be completely assigned. Then, as Figure 11As shown in the figure, the chemical shifts of C on the sugar ring were assigned by HSQC signals. The chemical shift of C1 of residue A was δ99.57 ppm, the chemical shift of C2 of residue A was δ71.45 ppm, the chemical shift of C3 of residue A was δ73.28 ppm, the chemical shift of C4 of residue A was δ76.57 ppm, the chemical shift of C5 of residue A was δ71.09 ppm, and the chemical shift of C6 of residue A was δ60.32 ppm. Among them, the chemical shifts of C1 and C4 shifted to the low field, indicating that substitution occurred at the O-1 and O-4 positions of the sugar ring of this residue. Combining the results of methylation analysis and literature reports, it was inferred that sugar residue A might be →4)-α-D-Glcp-(1→.

[0097] Combining the results of similar methylation analysis and literature reports, it was inferred that sugar residue B might be α-D-Glcp-(1→.

[0098] Combining the results of similar methylation analysis and literature reports, it was inferred that sugar residue C might be →4,6)-α-D-Glcp-(1→.

[0099] Combining the results of similar methylation analysis and literature reports, it was inferred that sugar residue D might be →4)-β-D-Galp-(1→.

[0100] Combining the results of similar methylation analysis and literature reports, it was inferred that sugar residue E might be α-L-Araf-(1→.

[0101] As Figure 10 and Figure 12 shown, the structure and linkage mode in this polysaccharide were analyzed by combining HMBC and NOESY spectra. According to the HMBC spectrum, there were cross-peaks of H1 of sugar residue A and C4 of sugar residue A at δ5.3 / 76.57 ppm, cross-peaks of H1 of sugar residue A and C4 of sugar residue C at δ5.3 / 76.63 ppm, cross-peaks of C1 of sugar residue A and H4 of sugar residue A at δ99.57 / 3.56 ppm, and cross-peaks of C1 of sugar residue A and H4 of sugar residue C at δ99.57 / 3.57 ppm. According to the NOESY spectrum, there were cross-peaks of H1 of sugar residue A and H4 of sugar residue A at δ5.3 / 3.56 ppm, cross-peaks of H1 of sugar residue A and H4 of sugar residue C at δ5.3 / 3.57 ppm, cross-peaks of H1 of sugar residue B and H6 of sugar residue C at δ4.86 / 3.74 ppm, and cross-peaks of H1 of sugar residue C and H4 of sugar residue A at δ5.25 / 3.56 ppm. Since the contents of sugar residues D and E were low and the signals were weak in the HMBC and NOESY spectra, they were not reflected in the sugar chain linkage.

[0102] Therefore, based on the comprehensive analysis of one-dimensional nuclear magnetic resonance (NMR), two-dimensional NMR information, and methylation results, it is inferred that the polysaccharide is mainly composed of →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→ connected to form the main chain, and the branches are mainly composed of α-D-Glcp-(1→ connected to the O-6 position of the sugar residue →4,6)-α-D-Glcp-(1→, etc. The sugar chain structure is as Figure 13 shown.

[0103] Example 7: Inhibitory effect of ginseng probiotic fermented polysaccharide GPS-F on early inflammation of colitis-related colon cancer

[0104] Experimental animals: Five-week-old male C57BL / 6J mice, provided by Jiangsu Jicui Yakang Biotechnology Co., Ltd.; mouse production license number: SCXK (Su) 2018-0008, quantity: 50.

[0105] The mice were grouped by the random grouping method, and a control group (Control), a model group (AOM+DSS), a low-dose GPS-F group (25 mg / kg) prepared by the method of Example 1, a medium-dose GPS-F group (50 mg / kg) prepared by the method of Example 1, and a high-dose GPS-F group (100 mg / kg) prepared by the method of Example 1 were set up. Ten mice were placed in each cage and raised separately. All the above mice were raised in a SPF-level environment, the indoor temperature was 24±2 °C, the normal day and night alternation was simulated in the animal room, the drinking water and food in the mouse basket were ensured to be normal and sufficient, and the bedding was kept dry. All animal experiments followed the regulations of the Animal Ethics Committee of Nanjing University of Chinese Medicine and the international animal welfare and health standards;

[0106] After one week of mouse adaptation, each group of mice was intraperitoneally injected with azoxymethane (only once). After one week of rest, the normal drinking water was replaced with 2% DSS water for 7 days, and after a two-week interval, they were again given free 2% DSS for 7 days, and this cycle was repeated. The mice in the administration group were given 200 μl of GPS-F at 25, 50, and 100 mg / kg by gavage every three days. On the 56th day, the mice were sacrificed to observe the effect of GPS-F on the early inflammation of colitis-related colon cancer.

[0107] The mice were dissected, and the colorectum of the mice was cut from the cecum to the rectum near the anus; the colon length was recorded and statistically analyzed. Mouse serum was collected and the levels of inflammatory factors IL-6 and TNF-α in the mouse serum were measured using an ELISA kit.

[0108] As Figure 14 shown, GPS-F administration can significantly reduce the inflammatory response in the early inflammatory stage of colitis-related colon cancer, including reducing the reduction of colon length and decreasing the levels of inflammatory factors IL-6 and TNF-α in the serum.

[0109] Example 8: Protection of Intestinal Barrier Integrity in the Early Stage of Colitis-Associated Colon Cancer by Ginseng Probiotic Fermented Polysaccharide GPS-F

[0110] Take the colon tissues of the mice in Example 7 to extract proteins and embed the tissues into sections, and detect the expression of intestinal barrier-related proteins in the intestines of the mice by western blotting and immunofluorescence staining. Detect the expression of tight junction proteins ZO-1 (Proteintech, 21773-AP), E-cadherin (Affinity, AF0131), and Claudin-1 (Affinity, DF6919).

[0111] As Figure 15 shown, administration of GPS-F prepared by the method of Example 1 can significantly reduce the expression of tight junction proteins (ZO-1, Claudin-1, E-cadherin) in intestinal tissues at the early inflammatory stage of colitis-associated colon cancer, and immunofluorescence also shows consistent results, indicating that GPS-F can protect the integrity of the intestinal barrier.

[0112] Example 9: Inhibition of Colitis-Associated Colon Cancer by Ginseng Probiotic Fermented Polysaccharide GPS-F

[0113] Experimental animals: Five-week-old male C57BL / 6J mice, provided by Jiangsu Jicui Yakang Biotechnology Co., Ltd.; Mouse production license number: SCXK (Su) 2018-0008, quantity: 20 mice.

[0114] The mice were grouped by the random grouping method, and a model group (AOM+DSS) and a GPS-F group (50 mg / kg) prepared by the method of Example 1 were set up. Ten mice were placed in each cage and raised separately. The above mice were all raised in an SPF-level environment, the indoor temperature was 24±2°C, the normal day and night alternation was simulated in the animal room, the drinking water and food in the mouse basket were ensured to be normal and sufficient, and the bedding was kept dry. All animal experiments followed the regulations of the Animal Ethics Committee of Nanjing University of Chinese Medicine and international animal welfare and health standards;

[0115] After one week of adaptation of the mice, each group of mice was intraperitoneally injected with azoxymethane (only once). After resting for one week, the normal drinking water was replaced with 2% DSS water for 7 days. After a two-week interval, the mice were again given free 2% DSS for 7 days. After repeating this cycle once, the mice rested for one week. Starting from the 56th day, the mice in the administration group were intragastrically administered 200 μl of GPS-F prepared by the method of Example 1 at 50 mg / kg once every three days until the 98th day. During this period, the model group received the corresponding volume of water;

[0116] On the 98th day, the mice were anesthetized and the disease conditions in the intestines of the mice were observed through a small animal colonoscope.

[0117] Dissect the mice, cut the colorectal of the mice from the cecum to the rectum near the anus, remove the intestinal contents, and record the number of tumors and tumor burden;

[0118] As Figure 16 shown, the colonoscopy results show that GPS-F prepared by the method of Example 1 can effectively inhibit the tumor proliferation of colon cancer mice. GPS-F can effectively inhibit the tumor proliferation of colon cancer mice, mainly manifested as the number of tumors in the mouse colon and the mouse tumor burden.

[0119] Example 10: Inhibiting the production of NETs in colitis-associated colon cancer by ginseng probiotic fermented polysaccharide GPS-F

[0120] Take the embedded sections of the colon tissues of the mice in Example 9, and detect the expression of neutrophil extracellular trap markers neutrophil elastase (NE) and citrullinated histone (CitH3) in the mouse intestine by immunofluorescence staining.

[0121] As Figure 17 shown, GPS-F prepared by the method of Example 1 can significantly inhibit the formation of neutrophil extracellular traps in the colon tissues of colon cancer mice, mainly manifested as the significantly reduced expression levels of NE and CitH3 compared with the model group. It is proved that the ginseng polysaccharide fermentation product GPS-F fermented by Lachnospiraceae can inhibit tumor growth by reducing the formation of neutrophil extracellular traps, thereby alleviating colon cancer.

[0122] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A use of ginseng-derived fermented polysaccharide in the preparation and treatment of colitis-related colon cancer, characterized in that: The ginseng-derived fermented polysaccharide is ginseng-derived probiotic fermented polysaccharide GPS-F, which is prepared by fermenting ginseng polysaccharide extract GPS by Lachnospiraceae bacterium, and the Lachnospiraceae bacterium is from the German National Culture Collection DSMZ, with the collection number of DMS24404.

2. The use of ginseng-derived fermented polysaccharide in the preparation of a method for treating colitis-related colon cancer as claimed in claim 1, characterized in that: The preparation steps of the ginseng-derived probiotic fermented polysaccharide GPS-F are as follows: (1) Preparation of ginseng polysaccharide extract GPS; (2) Activation and cultivation of Lachnospiraceae bacteria; (3) adding ginseng polysaccharide extract GPS to the bacterial liquid of Lachnospiraceae bacterium for fermentation, centrifuging, taking the supernatant, extracting with water and precipitating with alcohol, and freeze-drying under reduced pressure to obtain crude polysaccharide after fermentation; (4) The crude polysaccharide obtained in step (3) is eluted by ion exchange column chromatography, and the eluate is collected; and then purified by gel chromatography column to obtain ginseng-derived probiotic fermentation polysaccharide GPS-F.

3. The use of ginseng-derived fermented polysaccharide in the preparation of a treatment for colitis-related colon cancer as claimed in claim 2, characterized in that: The ginseng polysaccharide extract in the preparation step (1) is prepared by the following steps: (1) taking ginseng slices, grinding them into powder, extracting them with water by reflux, and filtering; adding water to the residue and continuing reflux extraction, filtering, combining the filtrates, concentrating, adding ethanol, and standing to precipitate overnight; (2) separating the precipitate and dissolving it in water; (3) Chloroform and n-butanol are mixed in a volume ratio of 5:1 to form Sevage reagent, Sevage reagent is added to the precipitate, the mixture is shaken thoroughly, vortexed, centrifuged, and allowed to stand to separate into three layers, the top layer is taken, and the above process is repeated 5-10 times until no protein layer is precipitated, and the mixture is freeze-dried under reduced pressure to obtain the ginseng polysaccharide extract GPS.

4. The use of ginseng-derived fermented polysaccharide in the preparation of a treatment for colitis-related colon cancer as claimed in claim 2, characterized in that: The activation and culture method of Lachnospiraceae bacterium in the preparation step (2) is as follows: dip the first generation bacterial solution of Lachnospiraceae bacterium in the storage tube, streak the plate and then culture it overnight at 37°C in an anaerobic workstation; pick a single colony on the plate and inoculate it in a liquid culture medium for activation for 24 hours to obtain a second generation bacterial solution, take the second generation bacterial solution and add it to the liquid culture medium for passage, aspirate the bacterial solution every 2 hours, detect its absorbance value at OD600, draw a 24-hour bacterial growth curve, determine the bacterial logarithmic growth phase and the plateau phase, and finally the concentration of Lachnospiraceae bacterium in the liquid culture medium is 1.5×10 8 CFU / ml.

5. The use of ginseng-derived fermented polysaccharide in the preparation of a method for treating colitis-related colon cancer as claimed in claim 2, characterized in that: In the preparation step (3), the concentration of the ginseng polysaccharide extract GPS in the liquid culture medium containing Lachnospiraceae bacteria is 5-10 mg / ml.

6. The use of ginseng-derived fermented polysaccharide in the preparation of a method for treating colitis-related colon cancer as claimed in claim 4, characterized in that: The liquid culture medium is composed of the following raw materials per liter: 10g of tryptone, 17.5g of beef heart extract powder, 5g of sodium chloride, 2.5g of sodium dihydrogen phosphate, and the balance is distilled water; the pH value is 7.2-7.

6.

7. The use of ginseng-derived fermented polysaccharide in the preparation of a preparation for treating colitis-related colon cancer according to claim 1, characterized in that: Colitis-Associated Colon Cancer.

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