Application of myricetin-3-O-galactoside in preparation of food for improving urinary tract infection

Abstract

The invention belongs to the field of food, and relates to a new application of myricetin-3-O-galactoside. The invention provides an application of myricetin-3-O-galactoside in preparation of a food for improving urinary tract infection. The invention also provides a food for improving urinary tract infection, and the food comprises myricetin-3-O-galactoside. The myricetin-3-O-galactoside is proved to have the effect of inhibiting the adhesion activity of urinary tract pathogenic escherichia coli for the first time, and the myricetin-3-O-galactoside can be used as an adhesion inhibiting active agent for urinary tract infection pathogen urinary tract pathogenic escherichia coli and can be used for preparing food for effectively improving urinary tract infection.

Description

Technical Field

[0001] The present invention belongs to the field of food, and relates to a new use of myricetin-3-O-galactoside, specifically to a new food use of myricetin-3-O-galactoside. Background Art

[0002] Urinary tract infection (UTI), as one of the most common bacterial infections worldwide, is mainly caused by uropathogenic Escherichia coli (UPEC). UPEC not only causes various human diseases such as cystitis, pyelonephritis, ureteritis and prostatitis, but also brings serious economic and medical burdens. Statistics show that the female population is at the greatest risk of urinary tract infection, and almost half of women will experience at least one urinary tract infection in their lifetime. UPEC is the main cause of UTI, accounting for 80% of infections. Effective colonization in the urinary tract environment is a key step for UPEC to achieve its pathogenic potential, and this process depends on the efficient adhesion and invasion of bacteria to uroepithelial cells. Therefore, from the perspective of prevention strategies, blocking the colonization process of UPEC during UTI is regarded as a potential intervention means. Among UPEC strains, key adhesion factors and major virulence factors include type I fimbriae, P fimbriae and Curli fimbriae. When urinary tract infection occurs, these fimbriae recognize and bind to specific receptors on the surface of host cells through adhesins at their tips, thereby enhancing the adhesion and invasion ability of bacteria. Although antibiotic treatment is a conventional means for UTI, the problem is that many patients will have UPEC strains detected again in the urinary tract within weeks after treatment, and the number of multi-drug resistant strains is increasing, which highlights the importance of developing alternative treatment strategies. The present invention for the first time discovers that myricetin-3-O-galactoside can be used as an adhesion active agent to inhibit uropathogenic Escherichia coli to resist the pathogenicity of UPEC.

[0003] Myricetin-3-O-galactoside (M-gal) is a flavonoid active ingredient widely distributed in plants. In addition to having better water solubility and oral bioavailability, myricetin-3-O-galactoside also exhibits various activities, such as antioxidant activity, anti-inflammatory effect and antigenotoxic potential. At present, there is no relevant research on myricetin-3-O-galactoside in the preparation of foods for improving urinary tract infection at home and abroad. Summary of the Invention

[0004] The purpose of the present invention is to provide a new use of myricetin-3-O-galactoside, specifically the application of myricetin-3-O-galactoside in the preparation of foods for improving urinary tract infection.

[0005] The present invention also provides a food for improving urinary tract infection.

[0006] The food for improving urinary tract infection according to the present invention comprises an effective amount of myricetin-3-O-galactoside.

[0007] According to a further feature of the food for improving urinary tract infections according to the present invention, the myricetin-3-O-galactoside is extracted from the fruits or leaves of one of the following plants: cranberry, grape, dilleniid dicot genus, nightshade family, myrtle, tea tree, birch tree, water lily, Chinese bayberry.

[0008] The present invention first proves that myricetin-3-O-galactoside has the effect of inhibiting the adhesion activity of uropathogenic Escherichia coli, and it can inhibit the adhesion and invasion ability of UPEC to urothelial cells, providing a new candidate natural compound for the prevention and treatment of urinary tract infections.

[0009] The present invention verified the antibacterial effect of myricetin-3-O-galactoside and its influence on the adhesion and invasion ability of UPEC to T24 cells; and analyzed the influence of myricetin-3-O-galactoside on the expression of UPEC fimbria gene and host receptor gene by RT qPCR. The results show that myricetin-3-O-galactoside can inhibit the adhesion and invasion ability of UPEC to urothelial cells, inhibit the expression of UPEC fimbria gene, inhibit the expression of cell adhesion receptor gene, and affect the fimbria morphology of UPEC. Therefore, myricetin-3-O-galactoside can be used as an inhibitory adhesion active agent for uropathogenic Escherichia coli, a urinary tract infection pathogen, for preparing an effective food for improving urinary tract infections. Description of the Drawings

[0010] Figure 1 Showing the cytotoxicity of different concentrations of M-gal to T24 cells.

[0011] Figure 2 Showing the influence of M-gal on the growth of UPEC.

[0012] Figure 3 Showing the changing trend of the average body weight of mice.

[0013] Figure 4 Showing the changes in the organ index of mice.

[0014] Figure 5 For the analysis of the number of colonies in mice, where Figure A shows the change in the number of urine colonies, and Figures B and C are the analysis diagrams of the number of colonies in the bladder and kidney respectively.

[0015] Figure 6 For the results of pathological sections of mice, where Figure A is the pathological section of the bladder, Figure B is the score of the pathological section of the mouse bladder, Figure C is the pathological section of the kidney, and Figure D is the score of the pathological section of the mouse kidney.

[0016] Figure 7 For the influence of M-gal on the adhesion activity of UPEC - flow cytometry results.

[0017] Figure 8 Effect of M-gal on the adhesion activity of UPEC - Results detected by fluorescence microscopy.

[0018] Figure 9 Show the effect of M-gal on the invasion ability of UPEC.

[0019] Figure 10 Results of observing the change in the pili morphology of UPEC by M-gal under transmission electron microscopy.

[0020] Figure 11 Show the effect of M-gal on the gene expression of cell integrin receptors.

[0021] Figure 12 Show the effect of M-gal on the gene expression of UPEC pili.

[0022] Figure 13 Show the expression level of proteins related to pili adhesion synthesis.

[0023] Figure 14 High performance liquid chromatography (HPLC) spectrum of cranberry extract. Detailed implementation methods

[0024] Human bladder transitional cell carcinoma cells (T24) were cryopreserved in the laboratory where the applicant is located. The UPEC strain was provided by the team of the Pathogenic Microbiology Laboratory of Jinan University. Myricetin-3-O-galactoside (M-gal) was purchased from Chengdu Purigen Biotech Co., Ltd., and the purity of the drug > 97%.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0027] Example 1: Cytotoxicity of M-gal on bladder epithelial cells T24

[0028] Seed T24 cells at 1×10 4 cells per well in a 96-well plate; after the cells are completely adherent, administer the drug at final concentrations of 0, 12.5, 25, 50, 100, 200 μM of M-gal; after 24 h of drug treatment, add 10 μL of CCK-8 solution to each well (note not to generate bubbles in the wells to avoid affecting the OD reading); incubate the culture plate in an incubator for 4 h; measure the absorbance at 450 nm using a microplate reader.

[0029] Cell viability (%) = [A (加药) - A (空白) ] / [A (0加药) - A (空白) ] × 100%

[0030] The results are as Figure 1 shown. After co-incubating T24 cells with different concentrations of M-gal (0, 12.5, 25, 50, 100, 200 μM) for 4 h, M-gal had no cytotoxicity to T24 cells compared with the control group.

[0031] Example 2: Inhibitory ability of M-gal against uropathogenic Escherichia coli (UPEC)

[0032] The UPEC strain was activated for 12 h. 300 μL of LB liquid medium containing M-gal (0, 12.5, 25, 50, 100, 200 μM) was added to each well of a 96-well plate, and the bacterial suspension was inoculated, with a final concentration of 1x10 6 / mL. Three replicates were set for each concentration; the UPEC was cultured at 37 °C for 6 h, and the optical density value (OD) at a wavelength of 600 nm was automatically recorded every hour; a growth curve was plotted.

[0033] The results are as Figure 2 shown. There was no obvious change in the growth rate of UPEC, which proved that M-gal had no direct antibacterial effect at a concentration of 200 μM.

[0034] Example 3: Therapeutic effect of M-gal on mice with urinary tract infection

[0035] (1) 40 μL of the cryopreserved UPEC strain was pipetted into 5 mL of LB liquid medium and incubated overnight in a shaker at 37 °C and 180 rpm until the logarithmic growth phase. It was resuspended and washed twice with PBS, and the bacterial concentration was detected at 625 nm and diluted to 2×10 9 CFU / mL for standby.

[0036] (2) The mice removed from quarantine were raised in a SPF clean environment and pre-gavaged for 4 days. A mouse model of urinary tract infection was constructed:

[0037] a) Starting from 24 h before inoculation, the mice were deprived of water. The mice were taken out, and the lower abdomen of the mice was gently pressed to drain the residual urine until there was no urine left. The mice were anesthetized with isoflurane and placed in a biosafety cabinet after anesthesia.

[0038] b) The mice were fixed supine on a foam cardboard, and the urethra and its surrounding area of the mice were smeared with alcohol for disinfection. A 1 mL syringe was used, and the tip of the injection needle was covered with a latex catheter to prevent puncturing the urethra of the mice. The syringe pre-filled with the bacterial suspension was slowly inserted into the urethra of the mice at an angle of 30° to the plane, with a depth of about 0.6 cm; 50 μL of the bacterial liquid was slowly injected into the urethra of the mice and left for 1 - 2 min. That is, each mouse was inoculated with 1×10 8 CFU of the bacterial liquid.

[0039] c) After the inoculation, the mice were put back into the cages and given normal drinking water 6 h after water deprivation.

[0040] (3) Animal grouping: The mice were divided into five groups: blank control normal group, model group, positive control group, low-dose M-gal group, and high-dose M-gal group, with 10 mice in each group, for a total of 50 mice. All mice were given normal feed and ordinary drinking water. Among them, the control group and the model group were given saline by gavage, the positive drug administration group was given levofloxacin by gavage at a concentration of 10 mg / kg, the low-dose M-gal group was given a gavage concentration of 5 mg / mL per day, and the high-dose M-gal group was given 10 mg / mL. Each mouse was given a single gavage of 100 μL once a day for 5 consecutive days. After the last gavage on the last day, the mice were fasted for 12 h, euthanized, and dissected for tissue sampling.

[0041] (4) During the experiment, the body weight of the mice was measured and recorded every day to observe the changes in body weight. Five days after drug administration, the mice were sacrificed, and the intact bladder, kidneys, and spleen were removed. The weights of each organ were recorded, and the organ index was calculated.

[0042] (5) After establishing the mouse urinary tract infection model, urine was collected from the mice on the 1st, 3rd, and 5th days. Take 10 μL of urine, dilute it ten times with PBS, and then use an LB solid plate for plating culture to count the bacterial count in the mouse urine.

[0043] (6) After continuous drug administration for five days, the mice were sacrificed and sampled. The intact bladder and kidneys of the mice were removed, weighed, placed in a grinding tube, grinding beads were added, and 1 mL of PBS was added. The tissue was thoroughly ground and mixed using a grinder. Take 50 μL of tissue fluid and use an LB solid plate for coating culture to count the bacterial count in the bladder and kidneys.

[0044] (7) Histopathological examination of the bladder and kidneys. The bladder and kidneys after sampling were immediately fixed in 4% paraformaldehyde for 24 h, embedded in xylene, and allowed to cool naturally. The embedded wax blocks were sectioned at 5 μm and stored at 4 °C. Hematoxylin-eosin staining was performed and sealed with neutral resin for storage. Finally, scanning and photographing were performed under a digital slide scanning and application system to observe the pathological changes among the groups, and the pathological sections of the bladder and kidneys were scored according to Tables 1 and 2.

[0045] Table 1: Bladder pathology scoring standard table

[0046] Classification Scoring criteria 0 Normal bladder epithelium 1 Focal and multifocal inflammation 2 Edema and diffuse inflammation 3 Obvious inflammatory cells with necrosis and neutrophils in and on the bladder epithelium 4 Inflammatory cell infiltration into the muscle 5 Loss of surface epithelium, based on score 4

[0047] Table 2: Kidney pathology scoring standard table

[0048]

[0049] The results are as Figures 3 to 6As shown, M-gal has a significant effect on reducing the bacterial load in the urinary system (including urine, bladder, and kidney) of experimental mice, and can effectively improve the degree of pathological damage to the bladder and kidney tissues. Among them, both the low-dose and high-dose groups of M-gal showed therapeutic effects, and the therapeutic effect of the high-dose group was significantly better than that of the low-dose group.

[0050] Example 4: Inhibitory effect of M-gal on the adhesion and invasion ability of uropathogenic Escherichia coli (UPEC)

[0051] Approximately 80,000 cells were seeded in each well of a 24-well plate, and then placed in an incubator for overnight culture. The cell status was observed the next day for drug administration. The drug administration groups were blank control group, negative control group, positive control group, and M-gal (12.5, 25, 50, 100, 200 μM) groups. After drug administration, the cells were cultured for another 24 h. UPEC was cultured to the logarithmic growth phase and then centrifuged at 6000 rpm for 3 min.

[0052] (1) Adhesion experiment 1: FITC was dissolved in PBS to 50 μg / mL, added to the centrifuged bacteria and resuspended. Incubated at 37 °C for half an hour, centrifuged, washed twice with PBS, and then 2 mL of PBS was added. The bacterial concentration was measured at OD625. The bacteria were resuspended in serum-free and antibiotic-free pure DMEM high-glucose medium at a multiplicity of infection of 100:1, and the bacteria were added to the 24-well plate for inoculation. Incubated at 37 °C for two hours, washed twice with PBS, digested with 200 μL of trypsin until completely detached, pipetted with 400 μL of PBS and transferred to an EP tube, and the UPEC adhesion concentration was detected by flow cytometry.

[0053] (2) Adhesion experiment 2: The pretreatment steps were the same as described above. After incubating UPEC for two hours, the fluorescence was observed using a fluorescence microscope.

[0054] (3) Invasion experiment: UPEC cultured to the logarithmic growth phase was added to the 24-well plate at a multiplicity of infection of 100:1. After incubating at 37 °C for 2 h, 500 μL of DMEM high-glucose medium containing 100 μg / mL gentamicin was added to each well and incubated at 37 °C for 1 h to kill extracellular bacteria; the well plate was washed with 3 volumes of pre-warmed PBS to thoroughly wash away gentamicin; 200 μL of 0.25% Tritonx-100 solution was added to each well to lyse the cells completely, pipetted repeatedly, and collected into a sterile EP tube. 800 μL of sterile PBS was added to each well for washing and collected into the EP tube; after thorough vortex mixing, it was serially diluted 10 - 100 times by a 10-fold dilution method, and then 100 μL of the cell lysate was pipetted and spread on an LB agar plate, incubated at 37 °C in an inverted position, and the colonies were counted the next day. Taking the invasion rate of the blank control as 100%, the relative invasion rates of the negative control group and the M-gal group were calculated.

[0055] The results are as Figures 7 to 9As shown. Figure 2 and Figure 3 In the experiment, after incubating different concentrations of M-gal with bacteria for 2 hours, the adhesion ability of M-gal to UPEC increased first and then decreased with the increase of concentration, and it had the best anti-adhesion activity at 50μM; and it had significant invasion inhibition ability at this concentration. The above results show that M-gal can prevent UPEC from adhering to and invading T24.

[0056] Example 5: Effect of M-gal on UPEC pili morphology

[0057] Activate the frozen UPEC overnight and culture to the logarithmic phase. After resuspending, take 1mL of bacterial solution and add it to the EP tubes respectively. Prepare M-gal and A2-PAC into 50μM working solutions, add them to the corresponding EP tubes, 1mL each, and mix them thoroughly as the drug-adding group, so that the concentration of the drug-adding group is 50μM. At the same time, add 1mL of LB liquid culture medium to the EP tube of another group as the control group. Put the treated samples in a shaker and incubate at 37 and 180rpm for 3h. Take out the incubated bacterial solution, centrifuge at 3000rpm for 5min, discard the supernatant, resuspend and wash the bacteria twice with PBS, remove the residual culture medium and drug solution, and finally resuspend with 1mL PBS. Take 100μL of bacterial solution from each EP tube and move it to a new EP tube, then add 100μL of 2.5% glutaraldehyde to each tube and gently blow and mix, fix at 4 for 2h, and the fixed bacterial solution can be placed in 4 for 2-3 days. After fixation, negative staining was used to treat the sample. The fixed suspension was added to a 200-mesh copper mesh with a pipette, excess liquid was absorbed with filter paper, and then the prepared 2% phosphotungstic acid (pH 7.0) was dripped in. After staining for 40 seconds, the dye was absorbed with filter paper, and after drying, the changes in bacterial morphology were observed using a transmission electron microscope.

[0058] The results are as follows Figure 10 As shown. In Figure (A), the morphology of untreated UPEC pili is shown. In the area indicated by the arrow, a large number of short and hair-like pili can be seen evenly distributed on the surface of the bacteria. This is a typical pili feature of UPEC in its natural state. When UPEC was treated with M-gal at a concentration of 50 μM, Figure (B) showed that the pili morphology changed, the surface of the bacteria became smooth, the presence of pili was almost unobservable, and only a few residual pili were present. This change indicates that M-gal can effectively induce the loss of pili structure on the surface of UPEC. With the disappearance of the pili structure, the surface of the bacteria presents a smooth morphology, which is most likely one of the important reasons why M-gal can inhibit the adhesion and invasion of UPEC to T24 cells.

[0059] Example 6: Effect of M-gal on the expression of UPEC pili genes and T24 cell adhesion receptor genes

[0060] The cultured T24 cells were digested with trypsin, resuspended in DMEM high-glucose medium, and then the cells were counted. The counted cells were inoculated into a 12-well plate, with 1.5×10 cells per well. 5 / mL cells, incubate at 37 for 24 hours, remove the supernatant, and wash with pre-warmed PBS. The groups were divided into blank control group, negative control group, positive control group, and M-gal group. UPEC was cultured to the logarithmic phase, and the bacterial count was 1×10 8 CFU / mL, remove the supernatant after centrifugation, resuspend the bacteria in serum-free and dual-antibody-free DMEM high-glucose medium at an infection multiplicity of 100:1, and then add the bacterial solution to a 12-well plate. Continue to incubate at 37 for 2 hours. After the incubation, remove the supernatant, carefully wash twice with pre-warmed PBS, and then digest the cells in the well plate with trypsin and collect them in a 1.5mL EP tube.

[0061] Take the overnight cultured UPEC and transfer it to the second generation bacteria, and culture it to the logarithmic phase. Take a 1.5mL EP tube and add 500μL of the cultured second generation bacteria to each tube. The EP tubes are divided into 2 groups. Add 500μL of M-gal with a final concentration of 50μM after dilution to the EP tubes and mix well to form the drug-dosing group. At the same time, add 500μL of LB culture medium to the control group. Put the two groups in the incubator and incubate at 37 and 180rpm for 2 hours. Take out the cultured bacterial solution.

[0062] The total RNA of cells and bacteria was extracted using the Trizol method, and the concentration and purity of the extracted RNA were detected using a Nanodrop spectrophotometer. The extracted RNA was reverse transcribed to remove genomic DNA and synthesize cDNA. Primers were designed using Primer Premier 5, and then the experiment was performed on a real-time quantitative PCR instrument according to a 20 μL system. The results were analyzed using Quant Studio Real-Time PCR Software.

[0063] The results are as follows Figure 11 and Figure 12 As shown. The gene expression of integrin a3 and integrinβ1 was significantly different. The expression levels of adhesion genes related to type I pili, type P pili and Curli pili were also significantly reduced, among which Pap G had no reducing effect. The above results indicate that M-gal can reduce the expression of urothelial receptor adhesion genes by inhibiting the expression of host adhesion receptor genes and pili genes to improve host defense ability and reduce UPEC colonization of host cells.

[0064] Example 7: Effect of M-gal on UPEC pilus protein expression

[0065] (1) Protein extraction

[0066] The cells were cultured in 6-well plates, the culture medium was removed, and the plates were washed twice with PBS. Lysis buffer (RIPA: cocktail: PMSF = 100: 1: 1) was added to each well at a ratio of 1:9. The plates were placed on ice at an angle. After 10 minutes, the lysed cell fluid was collected in a 1.5 mL EP tube and vortexed for 15 seconds three times. The plates were placed in a centrifuge and centrifuged at 4, 12000 rpm for 10 minutes. The separated supernatant was placed in ice for later use.

[0067] (2) Protein quantification

[0068] Dilute the standard to 2, 1, 0.5, 0.25, 0.125, 0.0625, 0 μg / mL. According to the number of samples, prepare BCA working solution with A:B=50:1 and mix thoroughly. Dilute the lysed supernatant 10 times, take 20 μL and add it to a 96-well plate, with 3 replicates for each sample. Add 200 μL of BCA working solution to each well, shake well, and incubate at 37 for 20 minutes. Use an enzyme reader to measure the absorbance at a wavelength of 562 nm, and calculate the protein concentration of the stock solution based on the standard curve and the dilution factor.

[0069] (3) Sample preparation

[0070] According to the actual measured protein concentration of the sample stock solution, 3.0 μg / μL was used as the standard, and it was diluted with 1 / 5 volume of 5× protein buffer (Protein Buffer), and then supplemented with PBS. The diluted sample was vortexed and reacted at 100°C for 8 minutes, then aliquoted into 20 μL per tube and stored at -20°C for later use.

[0071] (4) Western blotting

[0072] According to the amount of sample loaded, select the appropriate plate thickness and comb type to assemble the device, and make separation gel and concentration gel. Install the prepared gel into the electrophoresis tank, pull out the comb vertically, load the sample, and run at 80V constant voltage for 30 minutes, then switch to 120V constant voltage for 60 minutes (adjust the time according to the marker, and stop when bromophenol blue reaches the bottom of the gel).

[0073] Precool the transfer solution 4. Cut the 0.22μm PVDF membrane to a suitable size, activate it with methanol for 15s until it is transparent, rinse it with distilled water and balance the transfer solution for 2min. Pry the glass plate and cut the glue. Place the black side of the transfer clip on the tray and arrange it in the order of sponge-3 layers of filter paper-glue-PVDF membrane-3 layers of filter paper-sponge, and clamp it after removing bubbles. Install the transfer clip into the tank, add ice packs and transfer solution to the scale, and transfer the membrane in an ice bath at 300mA for 90min.

[0074] After transferring the membrane, place the PVDF membrane in an incubation box and wash it with TBST for 5 min. Cover the membrane with 5% skim milk powder and incubate it in a vertical shaker at room temperature for 1.5 h. Discard the blocking solution and wash the membrane with TBST for 5 min × 3 times. Remove the PVDF membrane and cover both sides of the membrane with a self-sealing bag to keep it moist. Cut out the bands corresponding to the target molecular weight required.

[0075] Dilute the antibody with the primary antibody diluent. Add the diluted primary antibody solution to the incubation box and incubate it overnight on a vertical shaker at 4 °C. After rewarming, recover the primary antibody and wash it with TBST buffer for 10 min × 3 times. Dilute the corresponding secondary antibody with 5% skim milk powder. According to different species relationships, add the corresponding secondary antibody diluent to the corresponding grid in the incubation box and incubate it on a vertical shaker for 1 h. After incubation, recover the secondary antibody, wash it, add ECL developing luminescent solution, and place it in a dark box for development in an automatic developing exposure instrument. Finally, analyze the gray scale of the bands using Image J software.

[0076] The results are as Figure 13 shown. After incubation with M-gal and UPEC, it can significantly inhibit the expression of fimbrial adhesin proteins FimA and FimH (p < 0.001), and the inhibitory effect is most significant when M-gal is at 50 μM.

[0077] Example VIII: Food containing myricetin-3-O-galactoside (M-gal)

[0078] The present invention also provides a food containing M-gal, which may also contain other active ingredients, as well as a food-acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and food-acceptable aqueous carrier medium, and the pH value can vary according to the nature of the substances being formulated, and those skilled in the art can adjust it as needed. The formulated food can be consumed by conventional routes such as oral administration or drinking.

[0079] Example IX: Extraction and isolation of M-gal from cranberry raw materials

[0080] The cranberry raw materials are from Guangdong Anthocyanin Technology Research Co., Ltd. Take 8 g of cranberry freeze-dried powder and prepare it into a 0.2 g / mL sample solution in acidified methanol (acidified with 0.1% hydrochloric acid) with a volume fraction of 6%. The conditions set for medium-pressure preparative liquid chromatography (purchased from Suzhou Lisui Technology Co., Ltd.) are as follows: flow rate 50 mL / min, using 0.1% hydrochloric acid aqueous solution as phase A and methanol as phase B, detection wavelength 500.0 nm, monitoring wavelength 280.0 nm, collection wavelength 519.0 nm, and collection threshold 500.0 mAU. Elute according to the conditions listed in Table 3.

[0081] Table 3: Elution gradient of medium-pressure liquid chromatography for preparation

[0082]

[0083] After filtering the cranberry medium-pressure sample using a 0.22 μM filter membrane, it was detected by high-performance liquid chromatography. The liquid-phase parameters were as follows: mobile phase A: 2% formic acid aqueous solution, mobile phase C: acetonitrile solution; gradient elution: Equilibration: -15 to -10 min: 90% C, -10 to -9 min: 90% to 6% C, -9 to 0 min: 6% C; Run: 0 to 30 min: 6% to 28% C, 30 to 45 min: 28% C. Flow rate: 1 mL / min, injection volume 10 μL.

[0084] The results are as Figure 14 shown. Peak 1 is M-gal, which elutes at 19.487 min, and the extraction purity is approximately 68.15%.

[0085] Example Ten: Extraction and Isolation of M-gal from Grape Extract the M-gal from grape raw materials

[0086] Wash the selected immature grapes and dry them according to the method of Waheed Ahmad. Grind them into fine powder and store them under refrigerated conditions for other analyses. Use 80% acidified methanol and 0.1% formic acid to separate phenolic compounds. In addition, extract the sample by mixing 2 g of the sample with methanol (20 mL) and 0.1% formic acid. Shake the solution in an incubator shaker at 10 and 150 rpm. After centrifuging the solution (8000 rpm, 15 min). Separate the supernatant through a syringe filter (0.45 L) and store it at -20 for detection using LC-MS / MS.

[0087] For the extraction method, refer to Reference 1: Serreli G, I, Gil KA, Z, Pacini V, Tuberoso CIG. Phenolic Compounds, Volatiles and Antioxidant Capacity of White Myrtle Berry Liqueurs. Plant Foods Hum Nutr. 2017 Jun; 72(2): 205 - 210.

[0088] Example Eleven: Extraction and Isolation of M-gal from Green Tea Extract the M-gal from green tea raw materials

[0089] Each tea leaf sample of green tea (3.0 g) was brewed in boiling water (150 mL) for 5 minutes. It was filtered through a 0.2 μM filter paper. Flavonol glycosides and flavonols were analyzed by HPLC. The HPLC conditions were as follows: injection volume was 10 μL; 5 μm C18 column (4.6 mm × 250 mm); temperature was 25; mobile phase A, acetonitrile / formic acid / aqueous solution (20:1:646, v / v / v); mobile phase B, acetonitrile / formic acid / aqueous solution (200:1:466, v / v / v). The gradient elution was: 100% A, 23 min. 100% - 50% A, 23 - 30 min, 50% - 37.5% A, 30 - 40 min, 37.5% - 20% A, 40 - 45 min, 20% - 0% A, 45 - 48 min, 0% A, 48 - 83 min. The flow rate was 1 mL / min.

[0090] For the extraction method, reference can be made to Document 2: Xu YQ, Zhang YN, Chen JX, Wang F, Du QZ, Yin JF. Quantitative analyses of the bitterness and astringency of catechins from green tea. Food Chem. 2018 Aug 30;258:16 - 24.

[0091] Example XII: Extraction and isolation of M-gal from Betula platyphylla leaves

[0092] The Betula platyphylla leaves were dried and ground using a laboratory grinder. Under reflux, 150 g of powdered leaves were extracted with 1500 mL of methanol (70%) at 80. The extract was cooled to room temperature, filtered and evaporated to dryness in a rotary evaporator. Detection was carried out according to the HPLC conditions in Example XI.

[0093] For the extraction method, reference can be made to Document 3: Germanò MP, Cacciola F, Donato P, et al. Betula pendula Roth leaves: gastroprotective effects of an HPLC - fingerprinted methanolic extract[J]. Nat Prod Res, 2013, 27(17):1569 - 1575.

[0094] Example XIII: Extraction and isolation of M-gal from Nymphaea tetragona

[0095] Approximately 0.6 g of frozen water lily petals were pulverized in liquid nitrogen using a mortar and pestle and first extracted with 3 mL of 70% aqueous methanol solution containing 0.1% HCl. After vortexing, it was sonicated for 20 min at 20 °C and centrifuged at 12,000 rpm for 10 min. Another 2 mL and 1 mL of the extraction solution were added to the residue, and the above operations were repeated two to three times. All the extracts were filtered through a 0.22 μM reinforced nylon membrane filter and analyzed by HPLC and HPLC-MS using the above methods.

[0096] For the extraction method, reference can be made to Document 4: Zhu M, Zheng X, Shu Q, et al. Relationship between the composition of flavonoids and flower colors variation in tropical water lily (Nymphaea) cultivars [J]. PLoS One, 2012, 7(4): e34335.

[0097] Example 14: Extraction and isolation of M-gal from the raw material of Dillenia indica

[0098] After drying at 45 °C, within 72 h, the fruits of Dillenia indica were powdered and thoroughly percolated with 96% ethanol at room temperature. The solvent was removed under vacuum in a rotary evaporator at 50 °C to obtain a dark green residue. A portion of the extract was percolated through silica gel (Merck, 0.2 - 5 mm mesh) to obtain n-hexane, dichloromethane, ethyl acetate, and methanol fractions. Each fraction was subjected to column chromatography using Sephadex LH-20 with methanol as the eluent. Using ethyl acetate / acetic acid / formic acid / (60:5:5:7) as the eluent and NP-PEG as the spraying reagent, the fractions were monitored by TLC on silica gel, and the fractions with similar curves were combined. After rotary evaporation and filtration, by preparative HPLC, on an ODS column (250 × 20 mm inner diameter, 10 μm; packing material, Shimadzu, Japan), eluted with methanol / water 1:1 at a flow rate of 5.0 mL / min at room temperature, an amorphous pale yellow solid was obtained. The active ingredient was detected by referring to the method of Example 9.

[0099] For the extraction method, reference can be made to Document 5: Campos JJ, Azevedo Ade O, Filho JD, et al. Bioguided isolation of myricetin-3-O-β-galactopyranoside with antinociceptive activity from the aerial part of Davilla elliptica St.-Hil[J]. J Ethnopharmacol, 2013, 150(1): 270-274.

[0100] Example 15: Extracting M-gal from other plant extraction raw materials

[0101] Plants such as Solanaceae, Myrtaceae, and Myrica also contain M-gal. The method described in Examples 9 to 14 can be referred to for extracting M-gal from their fruits or leaves.

[0102] For the extraction method, reference can be made to Document 6: Liu M, Zhu Q, Yang Y, et al. Light influences the effect of exogenous ethylene on the phenolic composition of Cabernet Sauvignon grapes[J]. Front Plant Sci, 2024, 15: 1356257.

[0103] The above are only the preferred embodiments of the present invention, but the protection scope of the present invention is not limited thereto. It should be pointed out that for those skilled in the art and any person familiar with the technical field, without departing from the overall concept of the present invention, any equivalent replacement or change made according to the technical solution and inventive concept of the present invention, as well as several changes and improvements made, should also be regarded as within the protection scope of the present invention.

Claims

1. Use of myricetin-3-O-galactoside in the preparation of a food for improving urinary tract infection.

2. A food for improving urinary tract infections, characterized in that: It includes an effective amount of myricetin-3-O-galactoside.

3. The food for improving urinary tract infection according to claim 2, wherein: The myricetin-3-O-galactoside is extracted from the fruit or leaves of one of the following plants: cranberry, grape, dilleniid dicot family, nightshade family, myrtle, tea tree, birch tree, water lily, Chinese bayberry.

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