A homogenized pitaya polysaccharide and its preparation method and application

Abstract

The present invention provides a homogenized pitaya polysaccharide, a preparation method and application thereof, and belongs to the technical field of polysaccharide extraction and purification. The homogenized pitaya polysaccharide is a polysaccharide whose monosaccharide composition is mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, with a relative molecular weight of 8.08×104 Da and a corresponding molar ratio of 0.181:0.250:16.544:12.762:3.793:0.739:51.014:14.716. The present invention also provides a preparation method of the homogenized pitaya polysaccharide and its application in the preparation of lipid-lowering and blood sugar-lowering drugs. The homogenized pitaya polysaccharide of the present invention has obvious lipid-lowering and blood sugar-lowering effects. Since it comes from a natural product, it has no toxic side effects and can be used to prepare lipid-lowering and blood sugar-lowering drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction and purification, and particularly relates to a homogenized pitaya polysaccharide and a preparation method and application thereof. Background Art

[0002] Obesity is a complex metabolic disease that is considered the root cause of many chronic health problems. Studies have shown that obesity not only increases the risk of diabetes, coronary heart disease, atherosclerosis, and hypertension, but is also closely associated with certain types of cancer. A prominent feature of obesity is abnormal lipid metabolism in the body, which is a series of complex biochemical reactions involving the synthesis and breakdown of lipids such as fatty acids, triglycerides, and cholesterol. Lipid metabolism disorders are often manifested as elevated blood lipids, and excessively high blood lipids further increase the risk of atherosclerosis, cirrhosis, and cardiovascular and cerebrovascular diseases. Currently, clinical treatment for lipid metabolism diseases mainly relies on symptomatic treatment, using medication to relieve symptoms. However, these drugs are often accompanied by certain toxic side effects, causing considerable pain to patients. Therefore, it is particularly important to find active ingredients from natural products with less toxic side effects for the treatment of lipid metabolism diseases.

[0003] Pitaya (Hylocereus undatus), also known as red dragon fruit, green dragon fruit, and fairy honey fruit, belongs to the genus Hylocereus in the Cactaceae family and is a perennial climbing plant. Pitaya is a fruit widely cultivated in tropical and subtropical regions, beloved for its rich nutritional profile and unique flavor. Pitaya is highly nutritious, containing significant amounts of minerals, particularly potassium, magnesium, calcium, iron, and copper; as well as vitamins such as vitamin C, biotin, K1, and D2. However, existing technologies for the utilization of pitaya extracts are lacking. Summary of the Invention

[0004] The purpose of the present invention is to provide a homogenized pitaya polysaccharide, which has obvious lipid-lowering and blood sugar-lowering effects and has no toxic side effects because it is derived from a natural product.

[0005] The purpose of the present invention is usually achieved by the following technical solutions:

[0006] The invention discloses a homogenized pitaya polysaccharide, which is a polysaccharide whose monosaccharide composition is mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, has a relative molecular weight of 8.08×104Da, and a molar ratio of mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose of 0.181:0.250:16.544:12.762:3.793:0.739:51.014:14.716.

[0007] The present invention also provides a method for preparing the homogenized pitaya polysaccharide, comprising the following steps:

[0008] (1) extracting pitaya powder with water, centrifuging and collecting the supernatant;

[0009] (2) concentrating the supernatant obtained in step (1) to obtain a concentrated solution; mixing the concentrated solution with an ethanol aqueous solution to perform alcohol precipitation, and collecting the precipitate by centrifugation;

[0010] (3) mixing the precipitate obtained in step (2) with water to obtain a polysaccharide solution, adding Sevage reagent, centrifuging, taking the supernatant for dialysis treatment, and drying to obtain pitaya crude polysaccharide;

[0011] (4) subjecting the crude pitaya polysaccharide obtained in step (3) to anion exchange column chromatography, collecting the eluate containing the polysaccharide, and dialyzing to obtain ion-purified pitaya polysaccharide;

[0012] (5) The ion-purified pitaya polysaccharide obtained in step (4) is subjected to gel column chromatography purification, the eluate containing the polysaccharide is collected, and dialyzed to obtain homogenized pitaya polysaccharide.

[0013] In the present invention, in step (1), during water extraction, the material-liquid ratio of pitaya powder to water is 1:20-40; the extraction temperature is 70-90° C., and the extraction time is 2-4 hours; the number of water extractions is 1-5 times; in step (2), the method for concentrating the supernatant is rotary evaporation, the temperature is 45-65° C., and the concentration time is 35-55 minutes; the volume ratio of the concentrated solution to the ethanol aqueous solution is 1:1-8, and the concentration of the ethanol is 85-100%; in step (3), the volume ratio of the polysaccharide solution to the Sevage reagent is 1-5:1, and the Sevage reagent is a mixed solvent of n-butanol and chloroform in a volume ratio of 1:4.

[0014] In the present invention, in step (4), the filler of the anion exchange column is DEAE Sepharose FastFlow, and the loading concentration is 10 to 30 mg / mL; the elution method is to perform gradient elution with water, 0.2 mol / L NaCl aqueous solution and 0.3 mol / L NaCl aqueous solution in sequence, the elution time of each solution is 125 to 175 min, and the eluate obtained by eluting with 0.2 mol / L NaCl aqueous solution is collected; the flow rate of the elution is 1 to 4 mL / min.

[0015] In the present invention, in step (5), Sephadex G-100 gel column is used for purification, and the loading concentration is 10-30 mg / mL; the elution method is water elution, the elution time is 100-300 min, and the eluate is collected; the elution flow rate is 0.2-1.5 mL / min.

[0016] In the present invention, the centrifugal conditions include: 7000-9000 rpm, 5-25 min, 1-6°C.

[0017] In the present invention, a dialysis bag with a molecular weight cut-off of 2500 to 4500 Da is used in the dialysis.

[0018] The present invention also provides the use of the homogenized pitaya polysaccharide in the preparation of lipid-lowering and blood sugar-lowering drugs.

[0019] The present invention also provides a lipid-lowering and blood sugar-lowering pharmaceutical composition, comprising the homogenized pitaya polysaccharide and pharmaceutically acceptable excipients.

[0020] In the present invention, the dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, pellet, pill or powder.

[0021] The effective dosage of the homogenized pitaya polysaccharide of the present invention is 50 to 350 mg / kg, preferably 70 to 200 mg / kg.

[0022] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses pitaya as raw material to prepare a single-component pitaya polysaccharide for the first time, and at the same time, the structure of the polysaccharide is characterized. The homogenized pitaya polysaccharide has a novel structure, a chemical structure, and a uniform composition. The present invention established an obesity model by inducing a high-fat diet in C57BL / 6 male mice to verify the lipid-lowering activity of pitaya polysaccharide, and found that pitaya polysaccharide has obvious lipid-lowering and blood sugar-lowering effects, and has no toxic side effects on normal organisms, providing theoretical and technical support for the development of drugs for metabolic diseases such as obesity, hyperlipidemia, and intestinal health. The preparation process of the homogenized pitaya polysaccharide of the present invention has mild conditions, simple operation, and is environmentally friendly. The obtained polysaccharide has high purity and stable structure, and the activity of the polysaccharide is maintained to the greatest extent, making it suitable for industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Elution purification curves of pitaya polysaccharide using DEAE Sepharose Fast Flow anion exchange chromatography column (a) and Sephadex G-100 gel column.

[0024] Figure 2 Liquid chromatograms of pitaya polysaccharide SDFP-2 (a) and monosaccharide standards (b).

[0025] Figure 3 GC-MS chromatogram of SDFP-2 after methylation.

[0026] Figure 4 SDFP-2 1 H-NMR ( Figure 4 a)13 C-NMR ( Figure 4 b) DEPT-135( Figure 4 c) Spectrum.

[0027] Figure 5 HSQC of SDFP-2( Figure 5 a) COSY Figure 5 b) HMBC( Figure 5 c) NOESY Figure 5 d) Spectrum.

[0028] Figure 6 The results show the effects of SDFP-2 on body size (a) and weight (b) in mice fed a high-fat diet. The horizontal axis represents the time of experimental study after the animals were grouped. **, P < 0.01, indicates a highly significant difference between the model group (HFD) and the control group (ND group); ##, P < 0.01, indicates a highly significant difference between the SDFP-2-treated group and the model group (HFD).

[0029] Figure 7 The results of the effect of SDFP-2 on oral glucose tolerance in mice fed a high-fat diet are shown. The horizontal axis represents different sampling time points after oral glucose administration, and the vertical axis represents blood glucose levels.

[0030] Figure 8 The results show the effects of SDFP-2 on serum TG and TC levels in mice fed a high-fat diet. **, P < 0.01, indicates a highly significant difference between the model group (HFD) and the control group (ND group); #, P < 0.05, indicates a significant difference between the SDFP-2-treated group and the model group (HFD); ##, P < 0.01, indicates a highly significant difference between the SDFP-2-treated group and the model group (HFD).

[0031] Figure 9 The results show the effects of SDFP-2 on serum HDL-C and LDL-C levels in mice fed a high-fat diet. **, P < 0.01, indicates a highly significant difference between the model group and the control group; #, P < 0.05, indicates a significant difference between the SDFP-2-treated group and the model group; ##, P < 0.01, indicates a highly significant difference between the SDFP-2-treated group and the model group.

[0032] Figure 10 The results show the effect of SDFP-2 on serum NEFA levels in mice fed a high-fat diet. **, P < 0.01, indicates a highly significant difference between the model group and the control group; #, P < 0.05, indicates a significant difference between the SDFP-2-treated group and the model group.

[0033] Figure 11 Structural elements of pitaya polysaccharide SDFP-2.

[0034] Side chain: ...→4)-β-D-galactose-(1→4)-β-D-galactose-(1→…;

[0035] or…→5)-α-L-arabinose-(1→5)-α-L-arabinose-(1→…;

[0036] or…→3)-β-D-galactose-(1→3)-β-D-galactose-(1→…;

[0037] or α-L-arabinose-(1→2)-α-L-arabinose-(1→….

[0038] R1:β-D-glucuronic acid-(1→6)-β-D-galactose-(1→…;

[0039] or α-L-rhamnose-(1→4)-β-D-glucuronic acid-(1→6)-β-D-galactose-(1→…;

[0040] or α-L-arabinose-(1→6)-β-D-galactose-(1→…;

[0041] or β-D-galactose-(1→6)-β-D-galactose-(1→.

[0042] R2:α-L-arabinose-(1→,β-D-galactose-(1→,→3)-α-D-galactose-(1→;

[0043] Or β-D-glucuronic acid-(1→. DETAILED DESCRIPTION

[0044] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0045] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention.

[0046] Example 1 Preparation of homogenized pitaya polysaccharide

[0047] 1. Preparation of pitaya crude polysaccharide by water extraction and alcohol precipitation

[0048] (1) The skin of Vietnamese white-heart dragon fruit (Hylocereus undatus, purchased from Xianlin Suguo Supermarket in Qixia District, Nanjing) was removed and washed, cut into slices, freeze-dried at -50°C for 48 h, crushed using a mixing grinder, passed through a 40-mesh sieve, and the sieve-free portion was taken to obtain dragon fruit powder, which was sealed in a ziplock bag and stored in a dry environment for later use.

[0049] (2) Add pitaya powder to distilled water at a material-liquid ratio of 1:29 (g / mL), mix well, and extract at 78°C for 3.5 h. Repeat the extraction three times under the same extraction conditions, and combine the three extracts. The material-liquid ratio of 1:29 (g / mL) means that 29 mL of distilled water is added to every 1 g of pitaya powder.

[0050] (3) The extract was centrifuged at 4°C and 8000 rpm for 15 min, the supernatant was collected, and the excess water was removed by rotary evaporation at 55°C and 85 rpm for 45 min to obtain a concentrated solution.

[0051] (4) Alcohol precipitation was then performed. Three times the volume of 95% (volume percentage) ethanol aqueous solution was added to the concentrated solution obtained in step (3), and the solution was treated at 4°C for 12 hours. Then, the excess ethanol was poured off, and the solution was centrifuged at 4°C and 8000 rpm for 15 minutes to collect the precipitate.

[0052] (5) The precipitate obtained in step (4) was added to distilled water at a solid-liquid ratio of 1:15 (g / mL), and stirred to dissolve the precipitate to obtain a polysaccharide solution. Sevage reagent (n-butanol: chloroform mixed at a ratio of 1:4 (V:V)) was mixed with the polysaccharide solution at a volume ratio of 1:3 and vibrated for 20 minutes, and then centrifuged at 4°C and 8000 rpm for 15 minutes. The supernatant was collected and placed in a dialysis bag (MD44mm, molecular weight cut-off 3500Da), dialyzed with running water for 48 hours to remove small molecules, and then freeze-dried at -50°C for 48 hours to obtain pitaya crude polysaccharide.

[0053] 2. Separation and purification of pitaya crude polysaccharides

[0054] (1) DEAE Sepharose Fast Flow anion exchange chromatography column: The DEAE Sepharose Fast Flow (purchased from Cytiva, model 17070901) ion exchange filler was repeatedly rinsed with distilled water until there was no ethanol smell, and then wet-packed to obtain an anion exchange chromatography column, which was then balanced with distilled water for 24 hours for later use. The crude polysaccharide of pitaya prepared in the title 1 of this embodiment was dissolved in distilled water to 30 mg / mL, and separated using an anion exchange chromatography column equipped with DEAE Sepharose Fast Flow. The sample volume was 10 mL, and the column was eluted with NaCl aqueous solutions of 0, 0.2, and 0.3 M for 125, 175, and 150 min, respectively, at a flow rate of 2 mL / min. The column was collected by an automatic collector, 10 mL per tube, and the polysaccharide concentration in the eluate was tracked and monitored by the phenol-sulfuric acid method. The elution curve was prepared with the number of tubes and the absorbance value as the horizontal and vertical coordinates. Figure 1As shown in (a), two fractions were obtained by elution with NaCl solutions of 0 to 0.3 mol / L, respectively, named DFP-1 (fraction obtained by water elution, corresponding to tubes 10 to 18) and DFP-2 (fraction obtained by elution with 0.2 M NaCl aqueous solution, corresponding to tubes 38 to 50). DFP-2 is an acidic polysaccharide. Fraction DFP-2 was rotary evaporated at 55°C and 85 rpm for 45 min, then placed in a dialysis bag (MD44 mm, molecular weight cutoff 3500 Da) and dialyzed against running water for 48 h to remove salts. The resulting lyophilized powder was then freeze-dried at -50°C for 48 h and stored at -20°C until further use.

[0055] (2) Purification by Sephadex G-100: The DFP-2 component obtained by separation by anion exchange chromatography column was purified by Sephadex G-100 gel column. The specific method is as follows: the freeze-dried powder of the component DFP-2 was dissolved in distilled water to a concentration of 10 mg / mL, and loaded onto the top of the Sephadex G-100 gel column with a loading volume of 10 mL. Distilled water was used as the eluent, the flow rate was 1 mL / min, the elution time was 200 min, and 5 mL was collected in each tube by the automatic collector. The polysaccharide content was detected by the phenol-sulfuric acid method, and a gradient elution curve was prepared with the number of tubes and absorbance as the horizontal and vertical coordinates, respectively. The elution curve is shown in FIG. Figure 1 (b) After further purification, fraction DFP-2 was still obtained as a homogeneous fraction, designated SDFP-2 (corresponding to tubes 5 to 15), with a purity of 94.73 ± 0.1%. Fraction SDFP-2 was concentrated by rotary evaporation at 55°C and 85 rpm for 45 minutes. It was then placed in a dialysis bag (molecular weight cut-off of 3500 Da) and dialyzed with running water to remove small molecules such as inorganic salts. The resulting fraction was then freeze-dried at -50°C for 48 hours to obtain homogenized pitaya polysaccharide SDFP-2, which was then stored at -20°C until further use.

[0056] Example 2 Structural Characterization of Homogenized Pitaya Polysaccharide

[0057] 1 Structural analysis of pitaya polysaccharide SDFP-2

[0058] 1.1 Molecular weight determination: Molecular weight determination of polysaccharides using HPGPC

[0059] (1) Chromatographic method: Instrument: Waters high performance liquid chromatograph; three GFC chromatographic columns OHpak SB-803HQ, Ohpak SB-804HQ, and Ohpak SB-805HQ (8×300 mm, Shodex, Japan) were connected in series; column temperature: 40°C; mobile phase: 0.05 M NaCl solution; flow rate: 0.65 mL / min, injection volume: 30 μL; detector: Waters differential detector.

[0060] (2) Standard curve preparation: Accurately weigh dextran standards of different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards) and prepare them into 5 mg / ml solutions with 0.05 M NaCl solution (precisely prepared, filtered through a 0.22 μm filter, sonicated for 20 min, and stored at room temperature). Filter through a 0.22 μm filter and set aside. Linear regression was performed using the logarithm of the relative molecular mass of the standard (1 g Mw) as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa to obtain the standard curve.

[0061] (3) A 5 mg / mL SDFP-2 solution was precisely prepared using 0.05 M NaCl solution as solvent, centrifuged at 8000 rpm / min for 10 min, and the supernatant was collected and filtered through a 0.22 μm microporous filter membrane. The supernatant was transferred to a 2 mL injection vial and injected. The molecular weight of the polysaccharide was calculated based on the standard curve.

[0062] 1.2 Molecular weight of pitaya polysaccharide SDFP-2

[0063] The results obtained through testing and calculation are shown in Table 1. The weight average molecular weight (Mw) of pitaya polysaccharide SDFP-2 is 8.08×10 4 Da, number average molecular weight (Mn) is 5.2×10 4 Da, peak molecular weight (Mp) is 6.24×10 4 Da.

[0064] Table 1 Molecular weight of SDFP-2

[0065] Mn(Da) Mw(Da) Mp(Da) Pitaya polysaccharide SDFP-2 52012 80804 62402

[0066] 2 Monosaccharide composition determination

[0067] 2.1 Monosaccharide determination was performed using liquid chromatography (ThermoU3000).

[0068] (1) Determination of standard products: Weigh 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and galactosamine hydrochloride, and 10 mg of fucose, dissolve in water, and dilute to 10 ml in a volumetric flask to prepare a standard stock solution. Perform a gradient dilution and dilute to volume to obtain a monosaccharide standard solution. Pass the solution through a 0.22 μm microporous filter and place in a sample injection vial.

[0069] (2) Accurately weigh 5 mg (±0.05 mg) of polysaccharide SDFP-2 sample, add 1 ml of 2 M TFA (trifluoroacetic acid) aqueous solution, and heat at 121°C for 2 hours. Flow nitrogen and blow dry. Add 3 mL of methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add 5 mL of sterile water to dissolve into polysaccharide hydrolyzate. Take 0.2 mL of monosaccharide standard solution and polysaccharide hydrolyzate respectively and place them in a centrifuge tube. Add 0.2 mL of 0.5 mol / L NaOH aqueous solution and 0.5 mL of 0.5 mol / L PMP (propylene glycol methyl ether propionate) methanol solution. Vortex mix and react in a 70°C water bath for 1 hour. After the reaction is complete, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the NaOH. Add 1 mL of chloroform and vortex extract 3 times to remove excess PMP. After discarding the chloroform layer, take 0.3 mL and add water to make the volume 1 mL. Pass through a 0.22 μm filter membrane and then detect. Chromatographic conditions: chromatographic column: ZORBAX EclipseXDB-C18, mobile phase: acetonitrile: phosphate buffer (containing 12 g / L potassium dihydrogen phosphate and 2 M NaOH in water, pH = 6.8), isocratic elution, the volume ratio of acetonitrile to phosphate buffer was 17:83, the flow rate was 0.8 mL / min, the column temperature was 30 ° C, the detection wavelength was 250 nm, and the injection volume was 10 μL.

[0070] 2.2 Determination of monosaccharide composition of pitaya polysaccharide SDFP-2

[0071] Figure 2 (b) is the liquid chromatogram of 11 monosaccharide standards. It is observed that the 11 monosaccharide standards are effectively separated. The monosaccharides represented by each peak in the mixed standard can be determined according to the retention time. The sequences and retention times of the 11 monosaccharides are shown in Table 2. The monosaccharide composition of SDFP-2 is as follows Figure 2 (a) Compared with the retention time of monosaccharide standards, the monosaccharide composition of SDFP-2 is: mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, with the corresponding molar ratios of 0.181:0.250:16.544:12.762:3.793:0.739:51.014:14.716, among which galactose accounts for the largest proportion.

[0072] Table 2 11 monosaccharide standards

[0073] Standard name Peak time Slope Goodness of fit 1 Man (mannose) 17.908 97.23 0.999 2 GlcN (glucosamine) 22.045 91.42 0.999 3 Rha (rhamnose) 25.188 33.10 0.995 4 GlcUA (glucuronic acid) 26.113 65.52 0.998 5 GalUA (galacturonic acid) 29.732 67.14 0.997 6 GalN (galactosamine) 34.335 84.24 0.999 7 Glc (glucose) 36.822 79.6 0.999 8 Gal (galactose) 41.758 94.36 0.999 9 Xyl (xylose) 44.458 64.02 0.995 10 Ara (arabinose) 46.092 87.59 0.995 11 Fuc (Fucose) 54.152 77.47 0.982

[0074] 3 Methylation analysis

[0075] After conventional methylation, acid hydrolysis and acetylation treatment, the connection mode of glycosidic bonds of pitaya polysaccharide SDFP-2 was analyzed by GC-MS.

[0076] 3.1 Detection Method: Weigh 10 mg of pitaya polysaccharide SDFP-2 sample, dissolve in 1 mL of water, add 1 mL of 100 mg / mL carbodiimide solution, and react for 2 h. Add 1 mL of 2 M imidazole solution and 1 mL of 30 mg / mL NaBD4 solution, and react for 3 h. Terminate the reaction by adding 100 μL of glacial acetic acid. Then, place the sample in a dialysis bag and dialyze against water for 48 h. After dialysis, freeze-dry the sample. Dissolve 1–3 mg of the lyophilized sample in 500 μL of DMSO. Add 1 mg of NaOH and incubate for 30 min. Add 50 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times. Aspirate the lower dichloromethane phase and evaporate to dryness. Add 100 μL of 2 M TFA aqueous solution and react at 121°C for 90 min. Evaporate to dryness at 30°C. Add 50 μL of 2M ammonia and 50 μL of 1M NaBD4 solution, mix well, and react at room temperature for 2.5 hours. Terminate the reaction by adding 20 μL of acetic acid, blow dry under nitrogen, wash twice with 250 μL of methanol, and blow dry under nitrogen. Add 250 μL of acetic anhydride, vortex to mix, and react at 100°C for 2.5 hours. Add 1 mL of water and let stand for 10 minutes. Add 500 μL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times. Remove the dichloromethane phase and analyze using an Agilent 7890A-5977B gas chromatography-mass spectrometer.

[0077] The chromatographic system used was an Agilent gas chromatograph (Agilent 7890A; Agilent Technologies, USA) with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA). The carrier gas was high-purity helium (≥99.999%) at a flow rate of 1.0 mL / min. The injection port temperature was 260°C. The injection volume was 10 μl. The temperature program was as follows: 50°C for 1.0 min, then 50°C / min to 130°C, then 3°C / min to 230°C, where it was held for 2 min.

[0078] The mass spectrometer system used was an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source was operated at an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The full scan mode (SCAN) was used, with a mass scan range (m / z) of 30–600.

[0079] 3.2 Methylation detection results of pitaya polysaccharide SDFP-2

[0080] Methylation test results such as Figure 3 The sugar residues and their molar ratios of SDFP-2 were determined based on retention time and peak area. The composition and connection mode of glycosidic bonds were also determined from the fragment ion distribution. The glycosidic bond connection modes of SDFP-2 mainly include t-Ara(f), t-Rha(p), 1,2-Ara(p), 1,5-Ara(f), 1,2-Rha(p), t-Glc(p), t-GlcA(p), t-Gal(p), t-GalA(p), 1,2,4-Rha(p), 1,4-Gal(p), 1,4-GalA(p), 1,4-Glc(p), 1,4-GlcA(p), 1,3-Gal(p), 1,6-Glc(p), 1,6-Gal(p), 1,3,4-GalA(p), 1,4,6-GalA(p), and 1,3,6-Gal(p).

[0081] Table 3 Analysis of results of methylated sugar alcohol acetyl ester (PMAA) of SDFP-2

[0082]

[0083]

[0084] Note: RT in the table is retention time, and MW is weight average molecular weight.

[0085] 4 Nuclear Magnetic Resonance

[0086] The freeze-dried SDFP-2 polysaccharide sample was dissolved in 0.5 mL of D2O and the one-dimensional NMR spectra were measured using a 600 MHz Bruker NMR spectrometer. 1 H-NMR, 13 C-NMR, DEPT-135 and 2D NMR COSY, HSQC, HMBC, NOESY. Calibration: HDO hydrogen δH = δ4.70ppm, TMS carbon δC = δ0.00ppm. The spectrum is as follows Figure 4 and Figure 5As shown, all H and C chemical shift information of each major sugar residue is obtained to infer the connection order between each sugar residue.

[0087] One-dimensional H NMR spectroscopy ( 1 H-NMR) and carbon spectroscopy ( 13 C-NMR) can further identify the glycosidic bond configuration of polysaccharide samples. Most of the hydrogen spectrum signals of polysaccharides are in the range of δ3.0 to 5.5 ppm, and usually the range of δ4.5 to 5.5 ppm is the resonance region of the anomeric proton (H-1). 1 The characteristic signals of H-NMR can identify certain sugar residues or groups. For example, the methyl proton signal of 6-deoxy sugar appears in the high field area of ​​δ0.8~1.4ppm; the methyl proton signal of acetyl group (CH3COO- or -OAc) appears in the low field area of ​​δ1.9~2.2ppm; the methyl proton signal of methyl ester (-COOCH3 or -COOMe) is located at δ3.0~3.8ppm. The chemical shift of the anomeric carbon (C-1) signal of polysaccharide is generally between δ90~110ppm, among which the α-configuration anomeric carbon signal usually appears between δ95~103ppm, and the β-configuration anomeric carbon signal usually appears above δ101ppm. The signal concentration area of ​​C-2~C-5 is in the δ65~85ppm region. The chemical shift of the carbon at the substitution position will show glycosidation shift and will move to the low field. The unsubstituted C-6 signal is near δ60ppm, while the substituted C-6 signal moves to the low field to near δ69ppm. According to 13 The characteristic signals of C-NMR can identify certain sugar residues or groups. For example, the methyl carbon signal of 6-deoxy sugar appears in the high field area of ​​δ15-20 ppm; the carbonyl signal of uronic acid (-COOH) and its methyl ester or acetyl group appears in the low field area of ​​δ170-180 ppm; the methyl carbon signal of methyl ester is located in the δ50-60 ppm region; and the methyl carbon signal of acetyl group appears in the higher field area of ​​δ20-25.

[0088] like Figure 4 As shown in (a), SDFP-2 1 On the H-NMR spectrum, a large number of proton resonance signals are concentrated in the δ3.0-5.5ppm region, with serious signal overlap. In the anomeric region, δ4.3-5.5ppm, multiple anomeric hydrogen signals are found, indicating that there are multiple sugar residues, and there is some overlap, making it difficult to distinguish. Other hydrogen signals are concentrated in the δ4.3-3.0ppm region, with serious signal overlap, making it difficult to attribute. 1 Compared with the H-NMR spectrum of SDFP-2 13 C-NMR spectrum ( Figure 4 (b)) There are fewer spectral lines, and there are multiple anomeric carbons in the δ90~110ppm anomeric carbon region, indicating that there are multiple sugar residues in the polysaccharide sample. Figure 4In (c) (DEPT-135 spectrum), an obvious inverted peak signal can be seen in the δ60-70 ppm region, indicating that the sugar residue contains a -CH2- group.

[0089] from 1 H-NMR and 13 In the C-NMR spectrum, some characteristic signal peaks were found: (1) The resonance signal in the high field region of δ1.10-1.30 ppm is the typical chemical shift of the methyl proton H-6 of the rhamnose (Rha) residue. The special deoxygenated structure of the methyl carbon C-6 of rhamnose is near δ16.50 ppm. Figure 5 a) A cross peak of δ1.16 / 16.50 ppm was found, indicating the presence of rhamnose residues, which is consistent with the monosaccharide composition results; (2) 1 In the H-NMR spectrum, the δ1.90~2.20ppm region is the methyl proton signal of the O-acetyl group, and there are multiple signal peaks, indicating that the substitution position of the acetyl group occurs at different positions of the sugar residue in the sugar chain. In the carbon spectrum, there is no obvious methyl signal in the acetyl group near δ20.00ppm, and the peak signal intensity is very small. Based on this, it can be inferred that there is a low degree of acetylation in this polysaccharide sample; (3) There is a δ172~176ppm signal in the low field region of the carbon spectrum. Combined with the results of the monosaccharide composition determination and literature reports, this is the C-6 signal of glucuronic acid and galacturonic acid.

[0090] Table 4 Sugar residues in polysaccharide samples 1 H and 13 Chemical shift assignment of C

[0091]

[0092]

[0093] In the monosaccharide composition test and methylation analysis results, the monosaccharide composition is mainly composed of GalA, Gal, Ara, Rha and GlcA. The methylation analysis results mainly contain 1,4-GalpA, 1,4-Galp, 1,3,6-Galp, 1,6-Galp, 1,3-Galp, t-Galp, t-Araf, 1,5-Araf, 1,2-Araf, 1,2-Rhap, 1,2,4-Rhap, t-Rhap, 1,4-GlcpA, t-GlcpA sugar residues. 1 H-NMR, 13 C-NMR ( Figure 4 )、HSQC( Figure 5 a) and COSY( Figure 5b) The NMR spectrum showed that there were multiple anomeric signals. The polysaccharide samples had multiple anomeric signal cross peaks in the HSQC and COSY spectra, which were significant and could be used for structural analysis: (1) There was a strong cross peak signal near the δ4.90-5.00 ppm hydrogen spectrum and the δ97-98 ppm carbon spectrum in the HSQC anomeric region. The COSY spectrum showed that the anomeric hydrogen in this region had a cross peak of δ4.93 / 3.81 ppm (H-1 / H-2). Combined with the methylation analysis results and literature reports, it can be inferred that the anomeric signal belongs to the sugar residue of α-GalpA. First, the H-1 chemical shift of the sugar residue was determined to be δ4.93 ppm by HSQC and COSY, and then the COSY spectrum was cross-contaminated. The cross peak can be deduced from H-2, H-3, and H-4 signals. The chemical shifts of H-2, H-3, and H-4 of the sugar residue are assigned to δ3.81ppm, δ4.03ppm, and δ4.34ppm, respectively. The chemical shifts of C-2 to C-4 on the sugar ring are assigned to δ67.71ppm, δ69.78ppm, and δ77.46ppm, respectively, through the HSQC correlation spectrum. The cross peaks of H-5 and C-5 at δ4.75 / 70.21ppm are found on the HSQC spectrum. The cross peak near δ52ppm of the methyl proton carbon signal of methyl ester (-COOMe) is not found on the HSQC spectrum, indicating that there is no obvious esterified galacturonic acid residue in the sample. In the HMBC spectrum ( Figure 5 c) A signal cross peak of δ3.75 / 173.62 ppm was found, indicating that δ173.62 ppm belongs to the C-6 signal of the galacturonic acid residue. The chemical shifts of C-1 and C-4 shifted to the downfield, indicating that the residue was substituted at the C-1 and C-4 positions of the sugar ring. Combining the methylation results and literature reports, it was inferred that the sugar residue was →4)-α-GalpA-(1→, labeled GA 1,4 , and the chemical shift assignments are shown in Table 4; (2) There is a strong cross peak signal near the hydrogen spectrum δ5.15ppm and carbon spectrum δ98.84ppm in the anomeric region of HSQC, and in the COSY spectrum ( Figure 5 b) A strong cross signal of anomeric hydrogen in this region was found at δ5.15 / 4.03 ppm (H-1 / H-2). Combining the methylation analysis results and literature reports, it can be inferred that the cross peak δ5.15 / 98.84 ppm in the anomeric region of HSQC mainly belongs to the anomeric signal of the sugar residue α-Rhap. COSY and HSQC were used to deduce, combined with the methylation analysis results and literature reports, the chemical shifts of the two sugar residues →2)-α-L-Rhap-(1→ and →2,4)-α-L-Rhap-(1→) were obtained, marked as Rha 1,2 and Rha 1,2,4A strong cross peak at δ4.64 / 100.66 ppm was found in the anomeric region of HSQC. COSY and HSQC were used to deduce the sugar residue, and combined with literature reports, it was inferred that the sugar residue was α-L-Rhap-(1→, labeled as Rha t The chemical shifts of the three rhamnose residues are assigned as shown in Table 1; (3) There are strong cross-peak signals near the hydrogen spectrum δ4.30~4.45ppm and the carbon spectrum δ102.0~104.0ppm in the anomeric region of HSQC. In the COSY spectrum, strong cross-peak signals δ4.34 / 3.44ppm, δ4.38 / 3.44ppm and δ4.43 / 3.58ppm can be found near the hydrogen spectrum δ3.40~3.60ppm in this region. Combined with the methylation analysis results and literature reports, it can be inferred that the main signal belongs to the anomeric signal of the sugar residue β-Galp. According to HSQC, the anomeric signals of the three β-Galp sugar residues are δ4.34 / 103.47ppm, δ4.38 / 103.54ppm and δ4.43 / 102.86ppm. With the help of COSY and HSQC, combined with literature reports, the chemical shifts of the three sugar residues are assigned as shown in Table 1. The chemical shifts of the anomeric signal δ4.34 / 103.47 ppm of the sugar residue C-1 and C-6 shifted to the downfield, indicating that the residue was substituted at the C-1 and C-6 positions of the sugar ring. It was inferred that the sugar residue was →6)-β-D-Galp-(1→, marked as G 1,6 The sugar residue at the anomeric signal δ4.38 / 103.54 ppm was inferred to belong to β-D-Galp-(1→, marked as G t The chemical shifts of the anomeric signal δ4.43 / 102.86 ppm of the sugar residue C-1, C-3 and C-6 shifted to the downfield, indicating that the residue was substituted at the C-1, C-3 and C-6 positions of the sugar ring. It was inferred that the sugar residue was →3,6)-β-D-Galp-(1→, marked as G 1,3,6 According to a similar method, the sugar residue at the anomeric signal δ4.59 / 103.93 ppm on the HSQC spectrum was inferred to belong to →3)-β-D-Galp-(1→, marked as G 1,3 The sugar residue at the anomeric signal δ4.55 / 104.32 ppm was inferred to belong to →4)-β-D-Galp-(1→, marked as G 1,4(4) There is a strong cross peak signal near the hydrogen spectrum δ4.40~4.49ppm and carbon spectrum δ102~103ppm in the anomeric region of HSQC, and the cross signals of anomeric hydrogen in this region are found in the COSY spectrum at δ4.45 / 3.25ppm and δ4.44 / 3.29ppm. Combined with the methylation analysis results and literature reports, it can be inferred that the main signal belongs to the anomeric sugar residue of β-GlcpA. The H-1 chemical shift of the sugar residue is determined to be δ4.45ppm and δ4.44ppm by HSQC and COSY. Similarly, COSY and HSQC are used for deduction, and combined with the HMBC spectrum ( Figure 5 c) There are cross peaks at δ3.71 / 174.72 ppm and δ3.69 / 174.72 ppm, and the H-5 signals are δ3.71 ppm and δ3.69 ppm, respectively, and the C-6 signal is δ174.72 ppm. The chemical shift assignments are shown in Table 1. The C-1 chemical shift of the sugar residue of the anomeric signal δ4.45 / 102.86 ppm shifts downfield, and the sugar residue is inferred to be β-D-GlcpA-(1→, labeled as GlcA t The chemical shifts of the sugar residue C-1 and C-4 at the anomeric signal δ4.44 / 102.86 ppm shifted to the downfield, indicating that the sugar residue was substituted at the C-1 and C-4 positions of the sugar ring. Combined with the literature reports, the sugar residue was inferred to be →4)-β-D-GlcpA-(1→, labeled as GlcA 1,4 , and the chemical shift assignments are shown in Table 1; (5) There are multiple cross-peak signals near the hydrogen spectrum δ4.96~5.31ppm and carbon spectrum δ107~110ppm in the anomeric region of HSQC. Combined with the methylation analysis results and literature reports, it can be inferred that the main signal belongs to the anomeric sugar residue of α-L-Araf. The chemical shifts of arabinose residues are derived by COSY and HSQC and with the help of literature data. The assignments are shown in Table 1. α-L-Araf-(1→ is marked as A t1 and A t2 ,→2)-α-L-Araf-(1→labeled as A 1,2 , is →5)-α-L-Araf-(1→ labeled as A 1,5 .

[0094] According to the results of methylation analysis, the polysaccharide sample also contains 1,3,4-GalpA, 1,3,4-GlcpA and other connection modes, the content is very small, the signal in NMR is very weak, and it is impossible to identify them. 1 H and 13 C is attributed.

[0095] Through the coupling signals of the anomeric hydrogen and the carbon on each sugar residue, or the coupling signals of the anomeric carbon and the hydrogen on each sugar residue on the HMBC long-range correlation spectrum, and the two protons on the connection sites of adjacent sugar residues are likely to produce strong NOE signals due to their close spatial positions, the mutual connection order between each sugar residue can be further inferred by using the HMBC long-range correlation spectrum and NOESY spectrum. HMBC correlation spectrum of polysaccharide sample ( Figure 5 c) and NOESY spectrum ( Figure 5 d) As shown in the figure, the following coupling signals can be found: (1) Sugar residue GA in NOESY spectrum 1,4 H-1 (δ4.93ppm) and sugar residue GA 1,4 There is a cross peak (GA 1,4 H-1 / GA 1,4 H-4), indicating the presence of →4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→ connection, suggesting the presence of HG type pectin structure in the sample; (2) sugar residue Rha in the HMBC spectrum 1,2 and Rha 1,2,4 H-1 (δ5.15ppm) and sugar residue GA 1,4 There is a related signal peak (Rha 1,2 H-1 / GA 1,4 C-4 or Rha 1,2,4 H-1 / GA 1,4 C-4), GA 1,4 H-4 (δ4.34ppm) and sugar residue Rha 1,2 or Rha 1,2,4 There is a related signal peak (GA 1,4 H-4 / Rha 1,2 or Rha 1,2,4 C-1), sugar residue GA in NOESY spectrum 1,4 H-1 (δ4.93ppm) and sugar residue Rha 1,2 or Rha 1,2,4 There is a related signal peak (GA 1,4 H-1 / Rha 1,2 or Rha 1,2,4 H-2) showed the existence of →2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→) linkage, and the presence of a branched chain at the O-4 position of the rhamnose residue, suggesting the presence of RG-Ⅰ type pectin structure in the sample; (3) The sugar residue G 1,3H-1 (δ4.59ppm) and sugar residue Rha 1,2,4 There is a cross peak (G 1,3 H-1 / Rha 1,2,4 H-4), indicating the existence of →3)-β-D-Galp-(1→ linked to →2,4)-α-L-Rhap-(1→, with the linking site located at the O-4 position; (4) the sugar residue G in the HMBC map 1,4 H-1 (δ4.55ppm) and sugar residue G 1,4 There is a related signal peak (G 1,4 H-1 / G 1,4 C-4), sugar residue G in NOESY spectrum 1,4 H-1 (δ4.55ppm) and sugar residue G 1,4 There is a related signal peak (G 1,4 H-1 / G 1,4 H-4), indicating the presence of →4)-β-D-Galp-(1→4)-β-D-Galp-(1→;(5) sugar residue A in the HMBC spectrum 1,5 H-1 (δ4.99ppm) and sugar residue A 1,5 There is a related signal peak (A 1,5 H-1 / A 1,5 C-5), sugar residue A in NOESY spectrum 1,5 H-1 (δ4.99ppm) and sugar residue A 1,5 There are related signal peaks (A 1,5 H-1 / A 1,5 H-5), indicating the presence of →5)-α-L-Araf-(1→5)-α-L-Araf-(1→;(6) sugar residue A in the NOESY spectrum 1,2 H-1 (δ5.28ppm) and sugar residue Rha 1,2,4 There is a cross peak (A 1,2 H-1 / Rha 1,2,4 H-4), indicating the existence of →2)-α-L-Araf-(1→ and →2,4)-α-L-Rhap-(1→ connected, and the connection site is located at the O-4 position; (7) sugar residue A in the HMBC map t2 H-1 (δ 5.11 ppm) and sugar residue A 1,2 There is a related signal peak (A t2 H-1 / A 1,2C-2), indicating the presence of sugar residue G in the HMBC spectrum of α-L-Araf-(1→2)-α-L-Araf-(1→;(8) 1,6 H-1 (δ4.34ppm) and sugar residue G 1,3,6 There is a related signal peak (G 1,6 H-1 / G 1,3,6 C-6), sugar residue G in NOESY spectrum 1,6 H-1 (δ4.34ppm) and sugar residue G 1,3,6 There are related signal peaks (G 1,6 H-1 / G 1,3,6 H-6), indicating the existence of →6)-β-D-Galp-(1→ and →3,6)-β-D-Galp-(1→ connected, and the connection site is located at the O-6 position. Since the most abundant sugar residues in the polysaccharide methylation analysis are →6)-β-D-Galp-(1→ and →3,6)-β-D-Galp-(1→, no connection signals between these two sugar residues and the O-3 position of the sugar residue were found on HMBC, and the anomeric hydrogen of these two sugar residues was not found to be connected with other sugar residues on HMBC, indicating that the polysaccharide sample contains →6)-β-D-Galp-(1→6)-β-D-Galp-(1→ connection, and there is a branch at the O-3 position of the 1,6-β-galactose residue; (9) The sugar residue GlcA in the HMBC map t H-1 (δ4.45ppm) and sugar residue G 1,6 There is a related signal peak (GlcA t H-1 / G 1,6 C-6), sugar residue GlcA in NOESY spectrum t H-1 (δ4.45ppm) and sugar residue G 1,6 There are related signal peaks (GlcA t H-1 / G 1,6 H-6), indicating the presence of β-D-GlcpA-(1→6)-β-D-Galp-(1→ connection; (10) sugar residue GlcA in the HMBC map 1,4 H-1 (δ4.44ppm) and sugar residue G 1,6 There is a related signal peak (GlcA 1, 4H-1 / G 1,6 C-6), sugar residue G 1,6 H-6 (δ3.82ppm and δ3.93ppm) and sugar residue GlcA 1,4There is a related signal peak (G 1,6 H-6 / GlcA 1,4 C-1), sugar residue GlcA in NOESY spectrum 1,4 H-1 (δ4.44ppm) and sugar residue G 1,6 There are related signal peaks (GlcA 1,4 H-1 / G 1,6 H-6), indicating the presence of →4)-β-D-GlcpA-(1→6)-β-D-Galp-(1→ connection; (11) sugar residue Rha in the HMBC map t H-1 (δ4.64ppm) and sugar residue GlcA 1,4 There is a related signal peak (Rha t H-1 / GlcA 1,4 C-4), sugar residue GlcA 1,4 H-4 (δ3.50ppm) and sugar residue Rha t There is a related signal peak (GlcA 1,4 H-4 / Rha t C-1), sugar residue Rha in NOESY spectrum t H-1 (δ4.64ppm) and GlcA 1,4 There is a related signal peak (Rha t H-1 / GlcA 1,4 ), indicating the presence of α-L-Rhap-(1→4)-β-D-GlcpA-(1→ connection; (12) sugar residue A in the HMBC map t1 H-1 (δ5.15ppm) and sugar residue G 1,6 There is a related signal peak (A t1 H-1 / G 1,6 C-6), indicating the presence of α-L-Araf-(1→6)-β-D-Galp-(1→6) linkage; (13) sugar residue G in the HMBC map t H-1 (δ4.38ppm) and sugar residue G 1,6 There is a related signal peak (G t H-1 / G 1,6 C-6), indicating the presence of β-D-Galp-(1→6)-β-D-Galp-(1→ connection; (14) sugar residue A in the HMBC map t1 H-1 (δ5.15ppm) and sugar residue G 1,3,6There is a related signal peak (A t1 H-1 / G 1,3,6 C-3), sugar residue A in NOESY spectrum t1 H-1 (δ5.15ppm) and sugar residue G 1,3,6 There is a related signal peak (A t1 H-1 / G 1,3,6 H-3), indicating the presence of α-L-Araf-(1→ and →3,6)-β-D-Galp-(1→ linked, with the linking site located at the O-3 position; (15) Sugar residue A in the HMBC map 1,5 H-1 (δ4.99ppm) and sugar residue G 1,3,6 There is a related signal peak (A 1,5 H-1 / G 1,3,6 C-3), indicating the existence of →5)-α-L-Araf-(1→ and →3,6)-β-D-Galp-(1→ linked, with the linking site located at the O-3 position; (16) sugar residue A in the HMBC map 1,5 H-1 (δ4.99ppm) and sugar residue G 1,3 There is a related signal peak (A 1,5 H-1 / G 1,3 C-3), sugar residue G 1,3 H-3 (δ3.77ppm) and sugar residue A 1,5 There is a related signal peak (G 1,3 H-1 / A 1,5 C-1), indicating the presence of a →5)-α-L-Araf-(1→3)-β-D-Galp-(1→ linker.

[0096] Based on the monosaccharide composition of the polysaccharide samples, the results of polysaccharide methylation analysis, and the one-dimensional and two-dimensional nuclear magnetic resonance information analysis, it was inferred that the pitaya polysaccharide SDFP-2 sample contained HG-type and RG-Ⅰ type pectin structures, and contained an arabinogalactan structure with "→6)-β-D-Galp-(1→6)-β-D-Galp-(1→" as the main chain and a branched chain at the O-3 position of the 1,6-β-galactose residue. Its possible structural elements include Figure 11 shown.

[0097] Example 3: Improvement of Pitaya Polysaccharide SDFP-2 on Obesity Induced by High-Fat Diet in Mice

[0098] 1 Animal grouping and experimental design

[0099] Forty six-week-old C57BL / 6 male mice were randomly divided into four groups after one week of adaptive feeding. Each group consisted of 10 mice, five per cage. Groups were divided into the following groups: 1) ND group (control group): fed a normal diet (XTCON50J, Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.); 2) HFD group (model group): fed a high-fat diet (XTHF60, Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.); 3) LSDFP-2 group: fed a high-fat diet (XTHF60) and simultaneously administered pitaya polysaccharide SDFP-2 (75 mg / kg / day, 0.1 ml / 10 g) by gavage; 4) HSDFP-2 group: fed a high-fat diet and simultaneously administered pitaya polysaccharide SDFP-2 (150 mg / kg / day, 0.1 ml / 10 g) by gavage. In addition to the above feed, the ND and HFD groups were also given distilled water with the same volume as that of pitaya polysaccharide.

[0100] 2. Weight recording and results

[0101] Each mouse was weighed once a week and recorded according to the number. Diet measurement method: the total food intake of each cage of mice in a week was measured and divided by the number of mice in each cage and the number of days to obtain the average daily food intake of each mouse.

[0102] The most representative body shape of mice in each group was selected for photographic comparison, such as Figure 6 As shown in (a), visual observation showed that after 10 weeks of high-fat diet, the body size was significantly larger than that of the normal diet group. After intervention with pitaya polysaccharide SDFP-2, the body size was significantly reduced, and the hair of the mice fed with a high-fat diet became shiny.

[0103] The weight index of mice is the most direct indicator to test whether dragon fruit polysaccharide SDFP-2 can prevent mouse obesity. The weekly weight of mice was recorded during the feeding period, and the weight gain curve was drawn. Figure 6 As shown in (b), during the 10-week high-fat diet, the weight difference between the ND group and the HFD group gradually became significant, and at the fifth week, the weight of the mice in the HFD group was 20% higher than that of the mice in the ND group, indicating that the mouse obesity model was established. After oral administration of pitaya polysaccharide SDFP-2, the weight of the mice decreased significantly compared with the HFD group (the average weight of the mice in the ND group was 28.23±0.95g, the average weight of the mice in the HFD group was 37.3±2.48g, the average weight of the mice in the LSDFP-2 group was 33.4±1.04g; the average weight of the mice in the HSDFP-2 group was 31.17±0.15g), indicating that the intervention of pitaya polysaccharide SDFP-2 can effectively improve the weight gain of mice induced by high-fat diet.

[0104] 3 Oral glucose tolerance test and results

[0105] After eight weeks of pitaya polysaccharide treatment, mice in each group were deprived of food and water for 8-12 hours before undergoing an oral glucose tolerance test (OGTT). After measuring baseline blood glucose levels (0 min), mice were gavaged with 2g / kg glucose solution, and blood glucose levels were measured and recorded at 30, 60, 90, and 120 minutes.

[0106] Oral glucose tolerance test (OGTT) is often used to evaluate the body's ability to regulate blood sugar. After 8 weeks of intervention with pitaya polysaccharides, OGTT was performed on mice. Figure 7 As shown in the figure, the blood glucose levels of mice in the HFD group were higher than those in the other groups at all points. 30 minutes after glucose administration, the blood glucose levels of mice in each group were at their highest. After 30 minutes, they gradually decreased. The blood glucose levels of mice on a high-fat diet that were given pitaya polysaccharide SDFP-2 were close to those of mice fed a normal diet, indicating that pitaya polysaccharide SDFP-2 can improve the glucose tolerance of obese mice.

[0107] 4. Determination of serum lipid markers

[0108] The experiment was ended after 10 weeks of intervention with pitaya polysaccharide. After fasting for 12 hours but not depriving water, blood was collected from the eye sockets, and the blood samples were centrifuged (10,000 rpm, 15 min). The upper serum was aspirated and the levels of TC, TG, LDL-C, HDL-C and NEFA in the serum of each mouse in each group were detected according to the operating procedures of the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and free fatty acid (NEFA) kit (Nanjing Jiancheng Bioengineering Institute).

[0109] like Figure 8 As shown in the results, compared with the ND group, the TC and TG levels in the HFD group were significantly increased, while after the intervention of pitaya polysaccharide, the TC and TG levels in the serum of the high-fat diet mice in the HSDFP-2 group were significantly reduced (P < 0.05), approaching that of the ND group, indicating that pitaya polysaccharide SDFP-2 can effectively improve the TC and TG abnormalities caused by a high-fat diet.

[0110] Depend on Figure 9As shown in the results, compared with the ND group, the serum HDL-C level of mice in the HFD group was significantly decreased (P < 0.01), indicating that the metabolism of cholesterol from extrahepatic to intrahepatic tissues was blocked; the serum LDL-C level in the HFD group was significantly higher than that in the ND group, indicating that the transport of cholesterol to extrahepatic tissues was increased. The decrease in HDL-C level and the increase in LDL-C level may lead to hypercholesterolemia and lipid metabolism disorders. After treatment with pitaya polysaccharides, the serum HDL-C level of mice on a high-fat diet increased significantly, while the LDL-C level decreased significantly, which helps to reduce serum cholesterol levels and thus improve obesity in mice.

[0111] Fat decomposition produces free fatty acids, and their excessive accumulation can cause dyslipidemia. Figure 10 As shown in the results, compared with the ND group, the NEFA content in the serum of the HFD group mice was increased, and the NEFA content in the serum of the obese mice after high-dose SDFP-2 intervention was significantly decreased (P < 0.01), indicating that pitaya polysaccharides can reduce the fatty acid content in high-fat diet-induced obese mice.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A homogenized pitaya polysaccharide, characterized in that The monosaccharide composition is mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, with a relative molecular weight of 8.08×10 4 Da, the molar ratio of mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose is 0.181: 0.250: 16.544: 12.762: 3.793: 0.739: 51.014: 14.716; The homogenized pitaya polysaccharide is prepared by a method comprising the following steps: (1) extracting pitaya powder with water, centrifuging, and collecting the supernatant; (2) concentrating the supernatant obtained in step (1) to obtain a concentrated solution; mixing the concentrated solution with an ethanol aqueous solution for alcohol precipitation, and collecting the precipitate by centrifugation; (3) mixing the precipitate obtained in step (2) with water to obtain a polysaccharide solution, adding Sevage reagent, centrifuging, taking the supernatant for dialysis treatment, and drying to obtain crude pitaya polysaccharide; (4) subjecting the crude pitaya polysaccharide obtained in step (3) to anion exchange column chromatography separation, collecting the eluate containing the polysaccharide, dialyzing, and obtaining ion-purified pitaya polysaccharide; (5) subjecting the ion-purified pitaya polysaccharide obtained in step (4) to gel column chromatography purification, collecting the eluate containing the polysaccharide, and dialyzing to obtain homogenized pitaya polysaccharide.

2. The homogenized pitaya polysaccharide according to claim 1, wherein In step (1), during water extraction, the material-liquid ratio of pitaya powder to water is 1:20-40; the extraction temperature is 70-90°C, and the extraction time is 2-4 h; the number of water extractions is 1-5 times; in step (2), the method for concentrating the supernatant is rotary evaporation, the temperature is 45-65°C, and the concentration time is 35-55 min; the volume ratio of the concentrated solution to the ethanol aqueous solution is 1:1-8, and the concentration of the ethanol is 85-100%; in step (3), the volume ratio of the polysaccharide solution to the Sevage reagent is 1-5:1, and the Sevage reagent is a mixed solvent of n-butanol and chloroform with a volume ratio of 1:

4.

3. The homogenized pitaya polysaccharide according to claim 2, wherein In step (4), the filler of the anion exchange column is DEAE Sepharose Fast Flow, and the loading concentration is 10~30 mg / mL; the elution method is to perform gradient elution with water, 0.2 mol / L NaCl aqueous solution and 0.3 mol / L NaCl aqueous solution in sequence, and the elution time of each solution is 125~175min, and the eluate obtained by elution with 0.2 mol / L NaCl aqueous solution is collected; the flow rate of the elution is 1~4 mL / min.

4. The homogenized pitaya polysaccharide according to claim 3, wherein In step (5), a Sephadex G-100 gel column is used for purification, and the loading concentration is 10-30 mg / mL; the elution method is water elution, the elution time is 100-300 min, and the eluate is collected; the elution flow rate is 0.2-1.5 mL / min.

5. The homogenized pitaya polysaccharide according to claim 4, characterized in that The centrifugal conditions include: 7000-9000 rpm, 5-25 min, 1-6°C.

6. The homogenized pitaya polysaccharide according to claim 5, wherein A dialysis bag with a molecular weight cut-off of 2500-4500 Da is used in the dialysis.

7. Use of the homogenized pitaya polysaccharide according to any one of claims 1 to 6 in the preparation of lipid-lowering and blood sugar-lowering drugs.

8. A pharmaceutical composition for lowering blood lipids and blood sugars, characterized in that: The invention comprises the homogenized pitaya polysaccharide according to any one of claims 1 to 6 and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, wherein The dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, dripping pill, pill or powder.

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