A galectin-3 inhibitor and its preparation method and application

Abstract

The present invention belongs to the field of biopharmaceutical technology and specifically relates to a galectin-3 inhibitor, its preparation method, and application. The galectin-3 inhibitor provided by the present invention has a minimum inhibitory concentration of 156 μg / mL for galectin-3. Furthermore, using BxPC-3 cells as a model, the present invention verified that the galectin-3 inhibitor provided by the present invention inhibits BxPC-3 cell proliferation and migration and promotes apoptosis through cell proliferation, apoptosis, and cell scratch assays.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to a galectin-3 inhibitor, a preparation method and an application thereof. Background Art

[0002] Pancreatic cancer is a highly malignant solid tumor, conventionally treated with surgery, radiotherapy, and chemotherapy. However, since most patients are no longer suitable for surgery at the time of diagnosis, and radiotherapy and chemotherapy are prone to adverse reactions, molecularly targeted drugs, which offer more precise effects and fewer adverse reactions than conventional treatments, have become a hot topic in cancer research and development. Galectin-3 (Gal-3), a β-galactoside-binding protein, is widely distributed in cells and participates in various physiological processes, including cell proliferation, apoptosis, and immune responses. It is implicated in tumor progression and metastasis, making it a popular target for anti-pancreatic cancer drugs. Summary of the Invention

[0003] To address the above-mentioned problems, the present invention aims to provide a galectin-3 inhibitor, its preparation method, and its application. The galectin-3 inhibitor provided by the present invention has a strong inhibitory effect on galectin-3 and is of great significance for the treatment of galectin-3-related diseases.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a galectin 3 inhibitor, which is composed of 5 galactose (G) and 4 ether galactose (A). The main chain of the inhibitor is composed of 4 As and 3 Gs connected alternately by α1,4- and β1,3-linking, and the connection mode is AGAGAGA. The A No. 3 and No. 5 on the main chain are connected to the G on the side chain by 2,6-linking. The structural formula of the inhibitor is as shown in Formula I

[0006]

[0007] The present invention also provides use of the above inhibitor in the preparation of a drug for inhibiting galectin-3.

[0008] The present invention also provides the use of the above inhibitor in the preparation of anti-tumor drugs.

[0009] The present invention also provides the use of the above inhibitor in preparing a drug for inhibiting tumor cell proliferation.

[0010] The present invention also provides the use of the above inhibitor in preparing a drug for inhibiting tumor cell migration.

[0011] The present invention also provides the use of the above inhibitor in preparing a drug for promoting tumor cell apoptosis.

[0012] Preferably, the type of tumor is pancreatic cancer.

[0013] Preferably, the working concentration of the inhibitor is 1.0 to 1.5 mmol / L.

[0014] The present invention also provides a method for preparing the above inhibitor, comprising the following steps:

[0015] Extraction: mixing Gracilaria lemaneiformis and distilled water, wherein the mass volume ratio of Gracilaria lemaneiformis to distilled water is 1 g:40-60 mL; after mixing, extracting is performed using an ultrasonic-microwave synergistic extractor, wherein the extraction conditions are ultrasonic wave 50W and microwave 500W, the extraction time is 25-35 minutes, and the extraction temperature is 75-85° C.; after the extraction is completed, filtering is performed, taking the extract, and fully mixing the extract with anhydrous ethanol for precipitation, wherein the volume ratio of the extract to anhydrous ethanol is 1:2.5-3.5, the precipitation temperature is 4° C.-10° C., and the precipitation time is 10-15 hours; after precipitation, centrifuging is performed, wherein the centrifugal speed is 4000-6000 r / min, and the centrifugal time is 10-15 minutes; after centrifugation, taking the precipitate, and freeze-drying the precipitate to obtain a crude extract;

[0016] Acid hydrolysis: dissolve the crude extract in distilled water, the mass volume ratio of the crude extract to distilled water is 1g:80-120mL; after dissolution, add sulfuric acid to the solution for acid hydrolysis, the concentration of the sulfuric acid is 1.9-2.1mol / L, the volume mass ratio of the sulfuric acid to the crude extract is 0.45-0.55mL:1g, the temperature of the acid hydrolysis is 75℃-85℃, the speed of the acid hydrolysis is 100-200r / min, and the time of the acid hydrolysis is 200-240min; after the acid hydrolysis is completed, cool the acid hydrolysis reaction to room temperature with cold water, add barium carbonate powder to neutralize to neutrality, and centrifuge, the speed of the centrifuge is 4 000~6000r / min, the centrifugation time is 10~15min; after centrifugation, taking the supernatant, performing rotary evaporation concentration, the concentration ratio is 11~9:1; after concentration, adding anhydrous ethanol to the concentrate for precipitation, the volume ratio of the concentrate to anhydrous ethanol is 1:4~5, the precipitation temperature is 4℃~10℃, and the precipitation time is 10~15h; after precipitation, centrifugation is performed, the centrifugation speed is 4000~6000r / min, and the centrifugation time is 5~10min; after centrifugation, taking the supernatant, performing secondary rotary evaporation concentration, the concentration ratio is 5~6:1, and obtaining a secondary concentrated solution;

[0017] Separation and purification: The secondary concentrate was separated and purified using an XK16 / 100 chromatography column packed with Bio-Gel P-2 column material. The column had a column height of 79 cm and a volume of 158.8 mL. The mobile phase was distilled water at a flow rate of 0.2 mL / min. During the collection process, the relationship between the separation voltage and time was monitored using a differential refractive index detector. The eluate was collected for 14.2 to 14.5 min and freeze-dried to obtain the galectin-3 inhibitor.

[0018] Beneficial effects:

[0019] The present invention provides a galectin-3 inhibitor, comprising five galactose (G) groups and four ether galactose (A) groups. The main chain of the inhibitor comprises four As and three Gs linked alternately in α1,4- and β1,3-linkages, forming an AGAGAGA connection. As on the main chain, groups 3 and 5 are linked to Gs on the side chains in a 2,6-linkage pattern. A chicken blood erythrocyte agglutination assay revealed that the minimum inhibitory concentration of the galectin-3 inhibitor against galectin-3 was 156 μg / mL. Cell proliferation assays using BxPC-3 cells as a model demonstrated that within a certain concentration range, the higher the concentration of the galectin-3 inhibitor and the longer the duration, the greater the inhibitory effect on BxPC-3 cell proliferation. Cell apoptosis assays demonstrated that the galectin-3 inhibitor was capable of inducing apoptosis in BxPC-3 cells. Cell scratch assays also demonstrated that the galectin-3 inhibitor had a certain effect on inhibiting cell migration and repair, and that within a certain range, the greater the concentration, the greater the inhibitory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The chromatogram shows the detection of the acid-hydrolyzed and concentrated oligosaccharide mixture of Gracilaria lemaneiformis.

[0021] Figure 2 Overlapping chromatograms of the acid-hydrolyzed mixture of agar powder and the oligosaccharide mixture of Gracilaria lemaneiformis.

[0022] Figure 3 The chromatogram of concentrated Gracilaria lemaneiformis is overlapped with the chromatogram of odd-numbered standards.

[0023] Figure 4 This is the differential detection diagram of the oligosaccharide mixture hydrolyzed from Gracilaria lemaneiformis.

[0024] Figure 5 These are the results of the thin layer chromatography (TIC) analysis of oligosaccharides.

[0025] Figure 6 This is the fluorescence-assisted carbohydrate electrophoresis experiment (FACE).

[0026] Figure 7This is a chicken blood red blood cell anticoagulation screening test.

[0027] Figure 8 It is a test for the inhibition of chicken blood red blood cells.

[0028] Figure 9 The chromatograms are overlapped with the mixed standard and sample.

[0029] Figure 10 Infrared spectra of purified DP7, DP9 and purchased DP9 oligosaccharides.

[0030] Figure 11 The fructose standard curve.

[0031] Figure 12 This is the primary mass spectrum of ESI-MS-MS.

[0032] Figure 13 This is the NMR analysis diagram.

[0033] Figure 14 Figure 2 is the structural diagram of oligosaccharide DP9.

[0034] Figure 15 This is the cell morphology diagram after 24 hours of drug addition.

[0035] Figure 16 The cell morphology is shown 48 hours after drug addition.

[0036] Figure 17 DP9 effect on cell proliferation assay

[0037] Figure 18 This is the cell morphology after DP9 drug stimulation for 24 hours.

[0038] Figure 19 Hoechst 33258 staining and inverted fluorescence microscopy observation after DP9 drug stimulation for 24 hours

[0039] Figure 20 The cells were treated with different concentrations of DP9.

[0040] Figure 21 The relationship between the degree of cell healing and treatment time under different concentrations of DP9. DETAILED DESCRIPTION

[0041] The present invention provides a galectin 3 inhibitor, which is composed of 5 galactose (G) and 4 ether galactose (A). The main chain of the inhibitor is composed of 4 As and 3 Gs connected alternately by α1,4- and β1,3-linking, and the connection mode is AGAGAGA. The A No. 3 and No. 5 on the main chain are connected to the G on the side chain by 2,6-linking. The structural formula of the inhibitor is as shown in Formula I

[0042]

[0043] The quality and biological activity of the galectin-3 inhibitor provided by the present invention are tested by thin-layer chromatography, fluorescence-assisted sugar electrophoresis, and chicken blood red blood cell anti-coagulation test. The minimum inhibitory concentration of the galectin-3 inhibitor of the present invention on galectin-3 is 156 μg / mL, and the inhibitory effect is good.

[0044] The present invention also provides use of the above inhibitor in the preparation of a drug for inhibiting galectin-3.

[0045] The present invention also provides the use of the above inhibitor in the preparation of anti-tumor drugs.

[0046] The present invention also provides the use of the above inhibitor in preparing a drug for inhibiting tumor cell proliferation.

[0047] The present invention also provides the use of the above inhibitor in preparing a drug for inhibiting tumor cell migration.

[0048] The present invention also provides the use of the above inhibitor in preparing a drug for promoting tumor cell apoptosis.

[0049] Preferably, the type of tumor is pancreatic cancer.

[0050] Preferably, the working concentration of the inhibitor is 1.0-1.5 mmol / L; more preferably 1.424 mmol / L.

[0051] The present invention also provides a method for preparing the above inhibitor, comprising the following steps:

[0052] Extraction: Mix the agaricus and distilled water, the mass volume ratio of the agaricus and distilled water is 1g:40-60mLmL, more preferably 1g:50mL; after mixing, extract with an ultrasonic-microwave synergistic extractor, the extraction conditions are ultrasonic 50W and microwave 500W, the extraction time is 25-35min, more preferably 30min, the extraction temperature is 75℃-85℃, more preferably 80℃; after the extraction is completed, filter, take the extract, mix the extract with anhydrous ethanol and precipitate The volume ratio of the extract to anhydrous ethanol is 1:2.5 to 3.5, more preferably 1:3, the precipitation temperature is 4°C to 10°C, more preferably 4°C, and the precipitation time is 10 to 15 hours, more preferably 12 hours; after precipitation, centrifugation is performed, the centrifugal speed is 4000 to 6000 r / min, more preferably 4000 r / min, and the centrifugation time is 10 to 15 minutes, more preferably 10 minutes; after centrifugation, the precipitate is taken and the precipitate is freeze-dried to obtain a crude extract;

[0053] Acid hydrolysis: dissolving the crude extract in distilled water, the mass volume ratio of the crude extract to distilled water is 1g:80-120mL, more preferably 1g:100mL; after dissolution, adding sulfuric acid to the dissolving solution for acid hydrolysis, the concentration of the sulfuric acid is 1.9-2.1mol / L, more preferably 2mol / L, the volume mass ratio of the sulfuric acid to the crude extract is 0.45-0.55mL:1g, more preferably 0.5mL:1g; the temperature of the acid hydrolysis is 75-85°C, more preferably 80°C; the speed of the acid hydrolysis is 100-200r / min, more preferably 150r / min; the time of the acid hydrolysis is 200-240min, more preferably 240min; after the acid hydrolysis is completed, the acid hydrolysis reactant is cooled to room temperature with cold water, barium carbonate powder is added to neutralize it to neutrality, and centrifuged, the speed of the centrifugation is 4000-6000r / mi n, more preferably 4000r / min; the centrifugation time is 10-15min, more preferably 10min; after centrifugation, taking the supernatant, performing rotary evaporation concentration, the concentration ratio is 11-9:1, more preferably 10:1; after concentration, adding anhydrous ethanol to the concentrate for precipitation, the volume ratio of the concentrate to anhydrous ethanol is 1:4-5, more preferably 1:4, the precipitation temperature is 4-10°C, more preferably 4°C; the precipitation time is 10-15h, more preferably 12h; after precipitation, centrifugation is performed, the centrifugation speed is 4000-6000r / min, more preferably 4000r / min; the centrifugation time is 5-10min, more preferably 5min; after centrifugation, taking the supernatant, performing secondary rotary evaporation concentration, the concentration ratio is 5-6:1, more preferably 5:1, to obtain a secondary concentrated solution;

[0054] Separation and purification: The secondary concentrate was separated and purified using an XK16 / 100 chromatography column packed with Bio-Gel P-2 column material. The column had a column height of 79 cm and a volume of 158.8 mL. The mobile phase was distilled water at a flow rate of 0.2 mL / min. During the collection process, the relationship between the separation voltage and time was monitored using a differential refractive index detector. The eluate was collected for 14.2 to 14.5 min and freeze-dried to obtain the galectin-3 inhibitor.

[0055] To further illustrate the present invention, a galectin-3 inhibitor provided by the present invention, its preparation method and application are described in detail below with reference to the accompanying drawings and examples. However, these figures should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] Preparation of galectin-3 inhibitor of the present invention

[0058] Take 10g of Gracilaria lemaneiformis, add 500mL of distilled water, and extract using an ultrasonic-microwave synergistic extractor for 30 minutes at 50W ultrasonic power and 500W microwave power. After extraction, filter the extract through gauze. Using alcohol precipitation, thoroughly mix the Gracilaria lemaneiformis extract with 3x anhydrous ethanol and refrigerate overnight at 4°C to produce a precipitate. Centrifuge (4000 rpm, 10 minutes), and freeze-dry the precipitate to obtain a crude extract.

[0059] Take 1g of crude extract, add 100mL of distilled water and heat to completely dissolve it. Add 0.5mL of 2mol / L sulfuric acid and stir in an 80℃ water bath for hydrolysis for 240min. After the reaction is completed, cool the reactants to room temperature with cold water and neutralize them with barium carbonate powder. Centrifuge (4000r / min, 10min). Concentrate the supernatant by rotary evaporation at 70℃ to 10mL. Add 4 times the amount of anhydrous ethanol to produce a precipitate. Centrifuge (4000r / min, 5min). Concentrate the supernatant by rotary evaporation at 70℃ to obtain a secondary concentrate. The acid hydrolysis conditions of agar powder are the same.

[0060] Separation was performed on a chromatography column (XK16 / 100, 79 cm height, 158.8 mL) packed with Bio-Gel P-2 column material. 2 mL of the concentrated Gracilaria oligosaccharide mixture was loaded onto the top of the column. Distilled water was used as the mobile phase at a flow rate of 0.2 mL / min. 3 mL of sample was collected in a tube using a collector, and each tube was collected for 15 minutes. During the collection process, a differential refractive index detector was used to monitor the relationship between separation voltage and time.

[0061] A column (Shodex Asahipak NH2P-50 4e) was installed on a high-speed liquid chromatograph, and the gradient elution was set as 0-7 min, 67%-50% B; 7-20 min, 50% B (A was double-distilled water, B was acetonitrile, and the flow rate was 0.5 mL / min). The oligosaccharides collected in each tube were detected with an injection volume of 20 μL. The test results are shown in FIG. Figure 1 As the polarity of the mobile phase decreases, peaks with higher polarity elute first, while peaks with lower polarity elute later. Peaks with retention times of 11.854, 12.886, 13.656, 14.212, and 14.589 min represent oligosaccharides from Gracilaria lemaneiformis with increasing degrees of polymerization, respectively.

[0062] Related studies have shown that agar oligosaccharides can be obtained by acid hydrolysis of agar powder. Figure 2 It is a chromatogram of the overlapping mixture of oligosaccharides of Gracilaria lemaneiformis and agar powder. The overlapping parts of the two chromatographic curves are agar oligosaccharides DP3 (agarotrinose), DP5 (agapentaose), DP7 (agarehenose), DP9 (agarenonaose), and DP11 (agareundeose).

[0063] Figure 3 The chromatogram of concentrated Gracilaria lemaneiformis is overlapped with the chromatogram of odd-numbered standards. Among them, S-DP3 is agarotriose standard; S-DP5 is agaropentaose standard; S-DP7 is agarheptaose standard. Figure 3 It can be seen that the curve of the Agaricus lemaneiformis oligosaccharide mixture in the detection chromatogram has a high degree of overlap with the curve of the odd-numbered standard samples. Therefore, it can be concluded that the Agaricus lemaneiformis oligosaccharide mixture is composed of monosaccharides such as galactose and oligosaccharides with polymerization degrees of 3, 5, 7, 9, and 11.

[0064] Figure 4 This image shows the results of the differential refractive index detector (DRD) during the separation of oligosaccharides from Gracilaria lemaneiformis. Since the separation column separates by molecular size, with larger molecules exiting first and smaller molecules exiting later, the first peak to elute is associated with a higher molecular weight, while the last peak is associated with a lower molecular weight. HPLC-ELSD analysis was also performed on each collected tube to determine the relationship between the number of collected tubes, peak elution time, and oligosaccharide type.

[0065] The separated Asparagus lemaneiformis oligosaccharides were detected by HPLC-ELSD. The oligosaccharides in tubes 39, 40, and 41 completely overlapped with S-DP3, indicating that the oligosaccharides in tubes 39, 40, and 41 were DP3. Therefore, the oligosaccharides in tubes 39, 40, and 41 can be mixed and collected to collect DP3 monomers with higher purity.

[0066] The oligosaccharides in tubes 36, 37, and 38 completely overlap with S-DP5, indicating that the oligosaccharides in tubes 36, 37, and 38 are DP5. Therefore, the oligosaccharides in tubes 36, 37, and 38 can be mixed and collected to collect DP5 monomers with higher purity.

[0067] The oligosaccharides in tubes 33 and 34 completely overlap with S-DP7, indicating that the oligosaccharides in tubes 33 and 34 are DP7. Therefore, the oligosaccharides in tubes 33 and 34 can be mixed and collected to collect DP7 monomers with higher purity.

[0068] The oligosaccharides in tubes 27 and 28 completely overlap with the agar powder acid hydrolysis mixture DP9, which indicates that the oligosaccharides in tubes 27 and 28 are DP9. Therefore, the oligosaccharides in tubes 27 and 28 can be mixed and collected to collect DP9 monomer with higher purity.

[0069] The oligosaccharides in tube 25 completely overlap with the agar powder acid hydrolysis mixture DP11, which means that the oligosaccharides in tube 25 are DP11. Therefore, the oligosaccharides in tube 25 can be mixed and collected to collect DP11 monomers, but the content is very small.

[0070] Table 1 Yields of isolated and freeze-dried oligosaccharides 3, 5, 7, 9, and 11.

[0071]

[0072] Example 2 Quality and biological activity tests of isolated and purified oligosaccharides

[0073] 1 Thin layer chromatography (TLC) analysis of oligosaccharides

[0074] Take 10μL of sample and spot it on a silica gel plate (10*20cm), spread it with a spreading agent for about 4.5 hours, blow it dry naturally in a fume hood, soak it in a color developer for 1S in a plate, develop it at 85 degrees for 15 minutes, observe the results and take pictures. Figure 5 .like Figure 5 As shown, DP3 and the odd-numbered standard sample S-DP3 are essentially on the same horizontal line, DP5 and the odd-numbered standard sample S-DP5 are essentially on the same horizontal line, DP7 and the odd-numbered standard sample S-DP7 are essentially on the same horizontal line, and DP9 is between the even-numbered standards DP8 and DP10. Meanwhile, DP3, DP5, DP7, and DP9 are misaligned with the even-numbered standards. This indicates that the oligosaccharide separation effect is good, and oligosaccharides with different aggregation degrees are separated from each other with little crossover. Therefore, it is preliminarily inferred that the quality test results of the isolated Gracilaria oligosaccharides are good.

[0075] 2 Fluorescence-assisted carbohydrate electrophoresis (FACE)

[0076] Glue making

[0077] Mother solution: 14.55 g acrylamide, 0.45 g N-N' mono-bisacrylamide, 15% SDS stock solution, 50 mL double distilled water

[0078] pH = 6.8 buffer: Dissolve 6.06g of Tris in 40mL of double-distilled water, adjust the pH to 6.8 with HCl, and add double-distilled water to 50mL

[0079] pH = 8.8 buffer: Dissolve 9.08g of Tris in 40mL of double-distilled water, adjust the pH to 6.8 with HCl, and add double-distilled water to 50mL

[0080] Separation gel configuration: mother liquor 8 mL, pH = 8.8 buffer 2 mL, AP 40 μL, TEMED 4 μL;

[0081] Concentrating gel configuration: mother liquor 0.5 mL, pH = 6.8 buffer 0.5 mL, double distilled water 1.5 mL, AP 50 μL,

[0082] TEMED 5 μL.

[0083] Derivatization of oligosaccharide samples

[0084] The collected Gracilaria oligosaccharide solution was concentrated and dried with nitrogen. 50 μL of 0.1 mol / L ANTS (aminonaphthalene trisulfonic acid) in HAC-H2O and 50 μL of 1 mol / L NaCNBH3 in DMSO were added to the dried sample; the mixture was mixed and incubated at 37°C for 16 h; the sample was concentrated and dried with nitrogen at 35°C; the sample was resuspended in 300 μL of 6 mol / L urea and stored in a refrigerator at -20°C until use.

[0085] Sample addition: Add 10 μL of sample to the well without bubbles.

[0086] Electrophoresis: The electrophoresis conditions are 300 V, 150 mA, 30 W, and the electrophoresis time is about 1 h 40 min.

[0087] After electrophoresis, observe under 365nm UV light and take pictures. The results are as follows: Figure 6 .like Figure 6 As shown, the separation of ANTS derivatives from Gracilaria lemaneiformis oligosaccharides of varying degrees of polymerization is evident. DP3 is aligned with the odd-numbered standard sample S-DP3, DP5 is aligned with the odd-numbered standard sample S-DP5, and DP7 is aligned with the odd-numbered standard sample S-DP7. DP3, DP5, DP7, DP9, and DP11 are distinct from one another, with the brightest band representing the target product and minimal impurities. This further demonstrates the effectiveness of the quality control of the separated oligosaccharides.

[0088] 3 Chicken blood red blood cell anticoagulation test

[0089] 1) Preparation of oligosaccharide mother liquor

[0090] Take 50 mg of oligosaccharides with different polymerization degrees and dissolve them in 10 mL of distilled water to prepare 5000 μg / mL oligosaccharide mother solution for use.

[0091] 2) Selection of the optimal concentration of Galectin-3 for agglutination of chicken red blood cells

[0092] Place a V-bottom 96-well plate on ice, add 15 μL of PBS (pH 7.4) to each well, then add 10 μL of various concentrations of Galectin-3 protein. Let the plate rest for 30 minutes, maintaining a low temperature and placed horizontally to prevent protein denaturation and inactivation. After 30 minutes, add 25 μL of 4% chicken red blood cells to each well. After waiting for 90 minutes, observe the red blood cell agglutination results and select the minimum Galectin-3 protein concentration that allows complete agglutination of the chicken red blood cells. The minimum protein concentration for complete agglutination of the chicken red blood cells was determined to be 250 μg / mL.

[0093] Add 25 μL of PBS to the first column of wells and 20 μL of PBS to the other wells. The negative control (NC) is in the first column, and the positive control (PC) is in the second column. Add 20 μL of oligosaccharide stock solution to wells in columns 8 and 9, respectively. Take 20 μL of the solution from well 8 and add it to well 7 and mix thoroughly. Take 20 μL of the solution from well 7 and add it to well 6 and mix thoroughly. Repeat this process for the third column of wells. Discard 20 μL of the solution from each well.

[0094] Add 5 μL of galectin-3 solution (500 μg / mL) to each well in columns 2 to 9 and let it react for 30 minutes. Finally, add 25 μL of chicken red blood cells to each well and let it react for 90 minutes. Observe the experimental results after 90 minutes. Figure 7 and Figure 8 .

[0095] Figure 7 This is the result of the early large-scale screening experiment. Figure 7 It can be seen that the inhibitory ability of odd-numbered oligosaccharides increases with the increase of polymerization degree; even-numbered oligosaccharides have no inhibitory effect in the sugar concentration designed in the experiment.

[0096] In the early large-scale screening experiments, it was found that DP3 and DP5 had weak anticoagulant ability, while DP7, DP9, and DP11 had certain anticoagulant ability. Therefore, DP7, DP9, and DP11 were selected as the key experimental objects. The results are shown in Figure 2. Figure 8 .from Figure 8 From the results, it can be seen that the minimum inhibitory concentration of DP7 is 625μg / mL, the minimum inhibitory concentration of DP9 is 156μg / mL, and the minimum inhibitory concentration of DP11 is 1250μg / mL. The concentrations of DP11 and S-DP7 are 625μg / mL, which show certain inhibitory ability, but not complete inhibition. The positive drug LacNac (N-acetyl-D-lactosamine) also shows certain inhibitory ability at a concentration of 156μg / mL, but not complete inhibition. The inhibition experiment shows that DP9 has the strongest inhibitory ability, that is, the strongest binding ability with Galectin-3. Therefore, the minimum inhibitory concentrations are ranked from small to large as DP9, DP7, DP11, DP5, and DP3, and the ability to bind to Galectin-3 is ranked from strong to weak as DP9, DP7, DP11, DP5, and DP3. Therefore, DP9 was selected as the subsequent research object, that is, the Galectin-3 inhibitor of this application.

[0097] Example 3 Structural Identification of Gracilaria Lemaneiformis Oligosaccharide DP9

[0098] 1) Monosaccharide composition analysis (PMP-HPLC)

[0099] Solution preparation

[0100] 0.6M NaOH: Take 0.24g and add water to make up to 10mL

[0101] 0.5M PMP methanol solution: Take 0.87g PMP and dilute to 10mL with methanol

[0102] 0.1M phosphate buffer, pH 6.7 (filtered and ultrasonically degassed): Add 8g NaCl, 0.2g KCl, 7.1g Na2HPO4, and 6.8g KH2PO4 to 900mL with water. Adjust the pH to 6.7 with HCl / NaOH, and finally make up to 1000mL. Man, Rha, GluA, GalA, Glu, Gal, Ara, and Fuc standards: Dissolve 10mg each of Man, Rha, GluA, GalA, Glu, Gal, Ara, and Fuc in 1mL of 50% ethanol and refrigerate at 4°C until ready to use. Take 100μL of each and add 200μL of water to prepare the standard mixture.

[0103] Sample processing

[0104] Take 10mg of sample and mix it with 2mL 2mol (15%) trifluoroacetic acid, put it in an oil bath at 120℃ for 2h, take the supernatant after centrifugation and blow it dry with nitrogen, add 500μL methanol and blow it twice. After blowing dry, add 0.5mL water to dissolve it, take 200μL sample, add 200μL 0.6M NaOH and 400μL 0.5MPMP methanol solution, put it in a water bath at 70℃ for 100min, wash it three times with dichloromethane (extract and wash away PMP, PMP peak will appear within 5min, the smaller the better), add 1mL dichloromethane each time, cover it, shake it, open it to release the air and shake it vigorously, centrifuge it, wash out dichloromethane (lower layer), add 0.5mL water to dilute it, and filter the water layer with an organic nylon membrane water system polyethersulfone membrane (0.45um) for reference treatment

[0105] Standards: Take 200 μL of the standard mixture, blow dry, add 200 μL of water to reconstitute, add 200 μL of 0.6 M NaOH and 400 μL of 0.5 M PMP methanol solution, incubate at 70°C in a water bath for 100 min, and repeat the above steps.

[0106] Blank control: 200 μL of water was added with 200 μL of 0.6 M NaOH and 400 μL of 0.5 M PMP methanol solution, and the mixture was incubated at 70°C in a water bath for 100 min. The same operation was repeated as above.

[0107] HPLC experiments

[0108] Experimental conditions: Column: VisionHT C18 HL5μ, column temperature 30℃, sample volume 20μL, flow rate 1mL / min, A-0.1M phosphate buffer Ph6.7, B-acetonitrile, conditions are 82% A, 18% B isocratic elution, UV wavelength 245nm.

[0109] Test results are shown in Figure 9 The test results indicate that the monosaccharide composition is primarily galactose, with the remaining components including rhamnose, glucose, galactose, and possibly fucose. Since the amounts of rhamnose, glucose, and fucose are extremely low, they are likely impurities. Therefore, it can be inferred that the monosaccharide component is galactose.

[0110] 2) Determination results of 3,6-galactose

[0111] FTIR of separated and purified DP7 and DP9

[0112] Take a small amount of freeze-dried DP7, DP9 and purchased DP9 standard sample, press a small amount into KBr pellet, and measure the infrared spectrum with a scanning range of 400-4000 cm -1 The results are shown in Figure 10 .like Figure 10 As shown, 3416.52cm -1 The extremely strong and broad peaks appearing on the left and right are the stretching vibrations of -OH, indicating the presence of intramolecular and intermolecular hydrogen bonds; 1074.56 cm -1 is a glycosidic bond; 931.81 cm -1 There is a signal of 3,6-endogalactose at 1243 cm -1 or 847cm -1 There are no absorption bands on the left and right, indicating that there are no hydrolysis products of sulfate groups. From this, it can be inferred that DP9 is composed of galactose and ether galactose.

[0113] Determination of 3,6-galactose in purified oligosaccharides by resorcinol method

[0114] Refer to the method of Li et al. for determining 3,6-galactose using fructose and make appropriate improvements

[25] .

[0115] Prepare 100 mL of a 1.5 mg / mL resorcinol solution and store in a refrigerator at 4°C. Prepare 100 mL of a 0.04% 1,1-acetal solution in a brown reagent bottle and store in a refrigerator at 4°C. Before colorimetry, thoroughly mix 9 mL of the resorcinol solution, 1 mL of the 1,1-acetal solution, and 100 mL of 12 mol / L concentrated hydrochloric acid solution.

[0116] Prepare 100 mL of 25 μg / mL fructose dilution for use. Take 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of fructose dilution respectively into 10 mL stoppered test tubes, make up to 1 mL with distilled water, and take another 1 mL of distilled water as a blank. Cover the stopper and place in an ice-water bath for 5 min. Take 5 mL of the newly prepared resorcinol reagent respectively into each test tube, shake in an ice-water bath, place in an 80°C constant temperature water bath for 15 min, take out and place in ice water for 1.5 min, and compare color at a wavelength of 554 nm.

[0117] The sample was prepared into a 25 μg / mL aqueous solution and measured according to the operating procedures for preparing the standard curve. Each sample was measured three times.

[0118] See the results Figure 11 and Table 2.

[0119] Table 2 Determination results of 3,6-galactose in purified oligosaccharides by resorcinol method

[0120]

[0121] Therefore, the results of the determination of 3,6-galactose in the purified oligosaccharide by the resorcinol method showed that the mass percentage of 3,6-galactose was 23.70%.

[0122] 3) Electrospray ionization mass spectrometry (ESI-MS) determination of separated products

[0123] Experimental conditions:

[0124] Mobile phase: A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile Flow rate: 0.3 mL / min

[0125] Sample injection volume: 5 μL

[0126] Column temperature: 30°C

[0127] Isocratic elution: 50% A: 50% B

[0128] Liquid chromatography-mass spectrometry: positive ion mode, M / Z scan range 200-2200 amu

[0129] Yang's experiments showed that the structure of DP3 is GAG

[26] , it is inferred that the structure of DP9 may be GAGAGAGAG, and the calculated [DP9+Na] + The molecular weight is:

[0130] [DP9+Na] + =5G+4A+Na +-8H20=5*180.063385+4*162.052826+22.989771-8*18.010565=1427.43347

[0131] Compared with ESI-MS-MS primary mass spectrum ( Figure 12 ) results were consistent with those of the control group, from which it can be inferred that DP9 is composed of 5 galactoses and 4 ether galactoses.

[0132] 4) Methylation analysis of polysaccharides

[0133] Methylation reaction

[0134] Weigh 5 mg of dried sample and dissolve it in 0.5 mL of DMSO solution. Stir magnetically until the sample is fully dissolved. Add 0.5 mL of NaOH-DMSO suspension to the dissolved sample. Cover the sample with tin foil and place it on an ice box in the dark. Place the ice box on a magnetic stirrer. In a fume hood in the dark, add 1 mL of iodomethane to the sample and stir magnetically for 30 minutes. Then add 2 mL of distilled water to terminate the reaction. Transfer the sample to a dialysis bag and dialyze it with running water for 24 hours, then dialyze it with distilled water for 24 hours. Then place it in a small glass bottle and blow it with a nitrogen blower at 45°C until the liquid is concentrated to 4-5 mL.

[0135] extraction

[0136] Transfer the concentrate to a glass bottle, add an equal volume of dichloromethane, stir magnetically for 30 minutes, let it stand for 5 minutes, remove the lower layer of liquid and place it in another dry glass bottle. Add an equal volume of dichloromethane to the upper layer and stir magnetically for 20 minutes. Pipette the lower layer and combine it with the previous lower layer solution. Extract the upper layer again and stir for 10 minutes. After combining the lower layer solutions, add an equal volume of distilled water, stir magnetically for 10 minutes, let it stand for 5 minutes, discard the upper aqueous layer (to extract water-soluble substances such as DMSO), repeat the back extraction three times, and then place the glass bottle under a nitrogen blower and air dry.

[0137] Infrared detection: Add 1.2mL of dichloromethane to the dried sample and dissolve it thoroughly in a small, well-sealed glass bottle. Then place it under a nitrogen blower and blow it dry. After the sample is dried, it is best to freeze-dry it to obtain a completely dry sample. Take a small amount of sample and grind it thoroughly with KBr. Then press it into a pellet and perform infrared spectroscopy detection. If the IR spectrum is at 3400cm -1 There is no absorption peak at 1000cm -1 There are high absorption peaks around, proving that the polysaccharide is completely methylated.

[0138] Acid hydrolysis

[0139] Add 1 mL of 2 M trifluoroacetic acid to the dried sample, place the sealed reaction glass bottle in a metal bath at 120°C for 3 hours, and after the reaction, place the glass bottle under a nitrogen blower for air drying. During this period, add anhydrous ethanol to the system several times to promote its volatilization until the pH of the system is neutral.

[0140] reduction

[0141] Add 1 mL of 30 mg / mL NaBH₄ solution to the dried sample. Without completely tightening the glass cap, reduce the sample at room temperature under magnetic stirring for 6 hours. Then, slowly add 100 μL of 50% glacial acetic acid to the reaction system to terminate the reaction. Next, add approximately 1 / 2 volume of strong acid cation exchange resin and magnetically stir until the exchange is complete. Aspirate the liquid with a syringe and filter it through a 0.22 μm filter into another dry glass vial. Rinse the remaining resin in the glass vial with 3 mL of anhydrous methanol, then filter and transfer it to the previous dry vial. Air dry, repeatedly adding anhydrous methanol until neutral. Evaporate the boric acid.

[0142] Acetylation

[0143] To the dried sample, 0.5 mL each of acetic anhydride and anhydrous pyridine were added. N2 was introduced to remove moisture from the air, and the sample was placed in a metal bath at 100°C for 2 h. After the reaction, 3 mL of distilled water was added to terminate the reaction. 3 mL of dichloromethane was then added, and the sample was magnetically stirred for 20 min. The sample was allowed to stand for 5 min, and the upper layer was discarded. The sample was stripped twice with 3 mL of distilled water, the upper layer was removed, and the water was completely aspirated. The sample was then air-dried. The dried sample was re-dissolved in 1 mL of chromatographically grade dichloromethane, filtered through a 0.22 μm filter, and analyzed by gas chromatography-mass spectrometry (GC-MS). The methylation results are shown in Table 3.

[0144] Table 3 Methylation analysis results

[0145]

[0146] Analysis of the methylation results reveals that terminal (T), 1,3-, and 1,4-linkages account for a significant proportion, with 1,2,6- and 1,2,4-linkages accounting for similar proportions. Furthermore, since 1,3-linkages account for 17.77%, 1,4-linkages for 14.40%, and 1,2,4-linkages for 4.44%, and 17.77% is approximately equal to the sum of 14.40% and 4.44%, it can be inferred that 1,3-linkages, 1,4-linkages, and 1,2,4-linkages are roughly equal, and that 1,3- and 1,4-linkages are the main chain linkages. The largest proportion of T indicates the presence of branches in addition to the ends of the sugar chain, with 2-linkages being the attachment sites of the branches. Furthermore, ESI-MS indicates that DP9 is composed of five galactoses and four ether galactoses, and Yang's experiments demonstrate that the galactoses and ether galactoses are adjacently linked. The 1,2,4-linkage indicates that some monosaccharides are linked at carbon positions 1, 2, and 4. So we infer that the structure of DP9 is Figure 14 .

[0147] 5) Nuclear magnetic resonance analysis

[0148] Experimental conditions: Solvent selection: heavy water; concentration: 20 mg of DP9 dissolved in 0.5 ml of heavy water, concentration of 40 mg / ml; temperature: 25°C, accumulation time: 12 h. Test results are shown in Figure 13 .Depend on Figure 13 The analysis of the NMR results showed that the possible glycosidic bond connection modes are α1,4-linkage, β1,3-linkage, and 2,6-linkage, which correspond to the analysis of the methylation results. Figure 14 It may be the structure of DP9.

[0149] Example 4 Cell experiment of Gracilaria lemaneiformis oligosaccharide DP9

[0150] 1) Cell proliferation

[0151] Cell counting

[0152] Add 50 μL of BxPC-3 cell suspension to an equal volume of trypan blue stain, mix gently, and stain for 3 minutes. Clean the hemocytometer and coverslip with anhydrous ethanol. Place the coverslip on the counting slide. Using a pipette, quickly drip 10 μL of cell stain from the edge of the counting slide, filling the gap between the slide and the coverslip. Place the counting slide under a low-power microscope (10x10) to observe and count. Based on the principle that live cells cannot be stained, while dead cells can be stained blue, the number of live and dead cells can be determined.

[0153] The calculation formula is: Experimental steps

[0154] Take 2.6x10 5100 μL of cells with the same density were cultured in a 96-well plate with ordinary culture medium for 24 h (at 37°C, 5% CO2). The medium was replaced with 100 μL of culture medium containing different drug (DP9) concentrations (0.05%, 0.1% and 0.2%) and cultured for 24 h. The blank group was still 100 μL of ordinary culture medium with 10 μL CCK-8 solution added, incubated for 3 h, and the absorbance was measured at 450 nm.

[0155] Vitality Calculation

[0156]

[0157] A(drug added): absorbance of 100 μL cells, 10 μL CCK-8 solution, and drug solution. A(blank): absorbance of 100 μL culture medium, 10 μL CCK-8 solution, and no cells. A(0 drug added): absorbance of 100 μL cells, 10 μL CCK-8 solution, and no drug solution. Cell viability: percentage of live cells, representing the strength of cell proliferation or cytotoxicity.

[0158] Test results are shown in Figures 15-17 .

[0159] like Figure 15 As shown, when BxPC-3 cells were treated with DP9 at concentrations of 0, 0.05%, 0.1%, and 0.2% for 24 hours, the number of adherent cells decreased significantly with increasing concentrations, indicating that DP9 could effectively inhibit the proliferation of BxPC-3 cells within 24 hours.

[0160] like Figure 16 As shown, when BxPC-3 cells were treated with DP9 at concentrations of 0, 0.05%, 0.1%, and 0.2% for 48 hours, the number of adherent cells decreased significantly with increasing concentrations, indicating that DP9 could effectively inhibit the proliferation of BxPC-3 cells within 48 hours.

[0161] like Figure 17 As shown, at 12 hours, the cell viability of the drug at a concentration of 0.05% was 95.17%, the cell viability of the drug at a concentration of 0.1% was 77.04%, and the cell viability of the drug at a concentration of 0.2% was 31.58%. At 24 hours, the cell viability of the drug at a concentration of 0.05% was 69.22%, the cell viability of the drug at a concentration of 0.1% was 50.1%, and the cell viability of the drug at a concentration of 0.2% was 8.82%. The experimental results showed that cell viability decreased with increasing DP9 concentration and increasing exposure time, indicating that within a certain concentration range, the higher the DP9 concentration and the longer the exposure time, the better the inhibitory effect on the proliferation of BxPC-3 cells.

[0162] 2) Apoptosis

[0163] Cell attachment

[0164] Count the BxPC-3 cells in the logarithmic growth phase and adjust the cell concentration to 1x10 6 1 mL was aspirated and inoculated into each well of a six-well plate, and each well was cultured with a total volume of 2 mL of culture medium.

[0165] Cell apoptosis experiment

[0166] After 12 hours of culture, BxPC-3 cells had basically grown attached to the wall. All the original culture medium was aspirated, and the cells were washed with PBS 2-3 times. The medium was changed. The control group continued to be cultured with 2 mL of medium without drug. The experimental group was cultured with 2 mL of medium with medium concentration (0.1% drug concentration) and high concentration (0.2%) drug groups. Each gradient was repeated twice.

[0167] After 24 hours of incubation in the incubator, perform apoptosis staining. Aspirate the culture medium, wash twice with PBS, and add an appropriate amount of Hoechst 33258 staining solution (1 mL per well of a six-well plate). Incubate the six-well plate in the incubator for 30 minutes. After 30 minutes, aspirate the staining solution, wash two to three times with PBS, and then examine the cells using an inverted fluorescence microscope.

[0168] Test results are shown in Figure 18 and Figure 19 .

[0169] like Figure 18 As shown, when BxPC-3 cells were treated with DP9 at concentrations of 0, 0.1%, and 0.2% for 24 hours, the number of cells with densely stained nuclei increased significantly with increasing concentrations, indicating that DP9 can cause BxPC-3 cells to apoptosis within 24 hours of treatment, and the higher the concentration, the more obvious the effect of DP9 in promoting BxPC-3 cells to apoptosis.

[0170] like Figure 19 As shown, the results of 0 drug addition and 0 drug addition were compared. The number of blue dense fluorescent particles in BxPC-3 cells stimulated with DP9 was significantly greater than that of 0 drug addition. The results indicate that DP9 can induce apoptosis in BxPC-3 cells.

[0171] 3) Cell scratching

[0172] Take the well-growing, logarithmically growing BxPC-3 cells for the experiment and dilute the cell concentration to 6x10 5 BxPC-3 cells were seeded at a concentration of 6 cells / mL in a 12-well plate and placed in a cell culture incubator (37°C, 5% CO2) for 24 hours.

[0173] When the cells reached 80% to 90% confluency, two vertical scratches were gently made in the wells using a 200 μL pipette. 500 μL of PBS was added to each well to wash the remaining cells in the scratches. The wells were washed twice. After absorbing the PBS, medium containing DP9 at 0, 712 μM, and 1424 μM was added to the wells to serve as the control and experimental groups, respectively. At least three replicate wells were set up for each group. The cells under different treatments were photographed and observed at 0, 12, and 24 hours after drug addition.

[0174] See 20-21 for test results.

[0175] like Figure 20 As shown in the cell scratch assay, at 0 h, the central scratch area was the same for 0, 712 μM, and 1424 μM. At 12 h, the central scratch area decreased compared to 0 h, with 0 having the smallest area, followed by 712 μM, and 1424 μM having the largest area. At 24 h, the central scratch area decreased even more compared to 0 h, with 0 having the smallest area, followed by 712 μM, and 1424 μM having the largest area. The area of ​​the central scratch area decreased even more for 0, 712 μM, and 1424 μM, with 0 having the smallest area, making it difficult to discern with the naked eye. The area of ​​the central scratch area for 712 μM, followed by 1424 μM, remained the largest. These results suggest that DP9 has a certain effect on inhibiting cell migration and repair.

[0176] like Figure 21 As shown, this is an ImageJ area analysis statistical chart of the cell scratch experiment. Taking the area of ​​the central scratch area at 0h as 1, the repair area of ​​cells with 0 drug addition at 12h is 54.6%, the repair area of ​​cells with 712μM drug addition is 32.3%, and the repair area of ​​cells with 1424μM drug addition is 8.3%; at 24h, the repair area of ​​cells with 0 drug addition is 91.7%, the repair area of ​​cells with 712μM drug addition is 71.4%, and the repair area of ​​cells with 1424μM drug addition is 49%. The experimental results show that the drug DP9 has a certain effect on inhibiting cell migration and repair, and within a certain range, the higher the concentration, the better the inhibitory effect.

[0177] Although the above embodiments describe the present invention in detail, they are only some embodiments of the present invention, rather than all embodiments.

Claims

1. A galectin-3 inhibitor, characterized in that The inhibitor is composed of 5 galactose (G) and 4 ether galactose (A). The main chain of the inhibitor is composed of 4 As and 3 Gs connected alternately in α1, 4- and β1, 3-links in the form of AGAGAGA. A No. 3 and No. 5 A on the main chain are connected to a G on the side chain in a 2, 6-linked manner. The structural formula of the inhibitor is as shown in Formula I Formula (I).

2. Use of the inhibitor according to claim 1 in the preparation of a drug for inhibiting galectin-3.

3. Use of the inhibitor according to claim 1 in the preparation of anti-tumor drugs.

4. Use of the inhibitor according to claim 1 in the preparation of a drug for inhibiting tumor cell proliferation.

5. Use of the inhibitor according to claim 1 in the preparation of a drug for inhibiting tumor cell migration.

6. Use of the inhibitor according to claim 1 in the preparation of a drug for promoting apoptosis of tumor cells.

7. The use according to any one of claims 3 to 6, characterized in that: The type of tumor is pancreatic cancer.

8. The use according to any one of claims 2 to 6, characterized in that: The working concentration of the inhibitor is 1.0-1.5 mmol / L.

9. The method for preparing the inhibitor according to claim 1, characterized in that: The steps include: Extraction: Mix the serrata and distilled water in a mass-to-volume ratio of 1 g: 40-60 mL; after mixing, extract using an ultrasonic-microwave synergistic extractor, the extraction conditions are 50W ultrasonic wave and 500W microwave, the extraction time is 25-35 minutes, and the extraction temperature is 75°C-85°C; after the extraction is completed, filter, take the extract, and thoroughly mix the extract with anhydrous ethanol for precipitation, the volume ratio of the extract to anhydrous ethanol is 1:2.5-3.5, the precipitation temperature is 4°C-10°C, and the precipitation time is 10-15 hours; after precipitation, centrifuge at a speed of 4000-6000 r / min and a centrifugation time of 10-15 minutes; after centrifugation, take the precipitate and freeze-dry it to obtain a crude extract; Acid hydrolysis: The crude extract was dissolved in distilled water, the mass volume ratio of the crude extract to distilled water was 1g: 80~120mL; after dissolution, sulfuric acid was added to the solution for acid hydrolysis, the concentration of the sulfuric acid was 1.9~2.1 mol / L, the volume mass ratio of the sulfuric acid to the crude extract was 0.45~0.55 mL:1g, the temperature of the acid hydrolysis was 75℃~85℃, the speed of the acid hydrolysis was 100~200 r / min, and the time of the acid hydrolysis was 200~240 min; after the acid hydrolysis was completed, the acid hydrolysis reaction was cooled to room temperature with cold water, barium carbonate powder was added to neutralize it to neutrality, and centrifuged at a speed of 4000~6000 r / min, the centrifugation time is 10-15 minutes; after centrifugation, taking the supernatant and performing rotary evaporation concentration at a concentration ratio of 11-9:1; after concentration, adding anhydrous ethanol to the concentrate for precipitation, the volume ratio of the concentrate to anhydrous ethanol is 1:4-5, the precipitation temperature is 4-10°C, and the precipitation time is 10-15 hours; after precipitation, centrifugation is performed at a speed of 4000-6000 r / min and the centrifugation time is 5-10 minutes; after centrifugation, taking the supernatant and performing secondary rotary evaporation concentration at a concentration ratio of 5-6:1 to obtain a secondary concentrated solution; Separation and purification: The secondary concentrate was separated and purified using an XK16 / 100 chromatography column packed with Bio-Gel P-2 column material. The column height was 79 cm, the volume of the column was 158.8 mL, and the mobile phase was distilled water at a flow rate of 0.2 mL / min. During the collection process, the relationship between the separation voltage and time was monitored using a differential refractive index detector. The eluate from 14.2 to 14.5 min was collected and freeze-dried to obtain the galectin-3 inhibitor.

Images