Homogenized pitaya polysaccharide as well as preparation method and application thereof

Abstract

The invention provides homogenized pitaya polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of polysaccharide extraction and purification. The homogenized pitaya polysaccharide is a polysaccharide of which the monosaccharide is composed of mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, the relative molecular weight is 8.08 * 10 < 4 > Da, and the corresponding molar ratio is (0.181): (0.250): (16.544): (12.762): (3.793): (0.739): (51.014): (14.716). The invention further provides a preparation method of the homogenized pitaya polysaccharide and application of the homogenized pitaya polysaccharide in preparation of lipid-lowering and sugar-lowering drugs. The homogenized pitaya polysaccharide disclosed by the invention has an obvious lipid-lowering and blood-glucose-reducing effect, is from a natural product, so that the homogenized pitaya polysaccharide has no toxic or side effect and can be used for preparing lipid-lowering and blood-glucose-reducing medicines.

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 to be 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 related to certain types of cancer. A notable feature of obesity is the abnormality of lipid metabolism in the body, which is a series of complex biochemical reactions, including the synthesis and decomposition of lipids such as fatty acids, triglycerides and cholesterol. Lipid metabolism disorders are often manifested as elevated blood lipids, and high blood lipids will further increase the risk of atherosclerosis, cirrhosis and cardiovascular and cerebrovascular diseases. At present, the clinical treatment of lipid metabolism diseases is mainly symptomatic treatment, using drugs to relieve symptoms. However, these drugs are often accompanied by certain toxic side effects, which bring 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, scientific name Hylocereus undatus, also known as red dragon fruit, green dragon fruit, fairy honey fruit, etc., belongs to the genus Hylocereus of the Cactaceae family and is a perennial climbing plant. Pitaya is a fruit widely grown in tropical and subtropical regions and is loved by people for its rich nutrients and unique flavor. Pitaya is rich in nutrients and contains a large amount of minerals, especially potassium, magnesium, calcium, iron and copper; as well as vitamins such as vitamin C, biotin, K1 and D2. In the prior art, there is a lack of utilization of pitaya extract. 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] A 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 8.08×104Da, 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 for 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) Purifying the ion-purified pitaya polysaccharide obtained in step (4) by gel column chromatography, collecting the eluate containing the polysaccharide, and dialyzing to obtain homogenized pitaya polysaccharide.

[0013] In the present invention, in step (1), during water extraction, the solid-liquid ratio of pitaya powder to water is 1:20-40; the extraction temperature is 70-90°C, and the extraction time is 2-4h; 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 35-55min; 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), a Sephadex G-100 gel column is used for purification, and the loading concentration is 10 to 30 mg / mL; the elution method is water elution, the elution time is 100 to 300 min, and the eluate is collected; the elution flow rate is 0.2 to 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 adjuvants.

[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 a raw material, prepares a single-component pitaya polysaccharide for the first time, and simultaneously characterizes the structure of the polysaccharide. The homogenized pitaya polysaccharide has a novel structure, a chemical structure, and a uniform composition. The present invention verifies the hypolipidemic activity of pitaya polysaccharide by inducing an obesity model in C57BL / 6 male mice through a high-fat diet, and finds 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 conditions of the homogenized pitaya polysaccharide of the present invention are mild, simple to operate, and environmentally friendly. The obtained polysaccharide has high purity and stable structure, and the activity of the polysaccharide is maintained to the greatest extent, which is 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 The liquid chromatograms of pitaya polysaccharide SDFP-2 (a) and monosaccharide standard (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 of the effect of SDFP-2 on the body shape (a) and body weight (b) of mice fed a high-fat diet are shown. The horizontal axis is the time of the experiment after the animals were grouped. **, P < 0.01, indicating that there is a very significant difference between the model group (HFD) and the control group (ND group); ##, P < 0.01, indicating that there is a very significant difference between the SDFP-2 administration 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 effect of SDFP-2 on TG and TC in the serum of mice fed a high-fat diet. **, P < 0.01, indicating that there is a very significant difference between the model group (HFD) and the control group (ND group); #, P < 0.05, indicating that there is a significant difference between the SDFP-2 administration group and the model group (HFD); ##, P < 0.01, indicating that there is a very significant difference between the SDFP-2 administration group and the model group (HFD).

[0031] Fig. 9 The results show the effect of SDFP-2 on HDL-C and LDL-C in the serum of mice fed a high-fat diet. **, P < 0.01, indicating that there is a very significant difference between the model group and the control group; #, P < 0.05, indicating that there is a significant difference between the SDFP-2 administration group and the model group; ##, P < 0.01, indicating that there is a very significant difference between the SDFP-2 administration group and the model group.

[0032] Fig.10 The results show the effect of SDFP-2 on NEFA in the serum of mice fed a high-fat diet. **, P < 0.01, indicating that there is a very significant difference between the model group and the control group; #, P < 0.05, indicating that there is a significant difference between the SDFP-2 administration group and the model group.

[0033] Fig.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 solution of the present invention will be clearly and completely described 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 Vietnamese white-heart dragon fruit (Hylocereus undatus, purchased from Xianlin Suguo Supermarket in Qixia District, Nanjing) with the skin removed was washed, cut into slices, freeze-dried at -50°C for 48 h, crushed using a stirring mill, passed through a 40-mesh sieve, and the sieved 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 distilled water to the pitaya powder at a solid-liquid ratio of 1:29 (g / mL), mix well, extract at 78°C for 3.5 h, repeat the extraction three times under the same extraction conditions, and combine the extracts obtained three times. The solid-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 supernatant was rotary evaporated at 55°C and 85 rpm for 45 min to remove excess water to obtain a concentrated solution.

[0051] (4) Alcohol precipitation was then performed by adding 3 times the volume of 95% (volume percentage) ethanol aqueous solution to the concentrated solution obtained in step (3), treating at 4° C. for 12 h, then pouring off the excess ethanol, centrifuging at 4° C. and 8000 rpm for 15 min, and collecting 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 the precipitate was dissolved by stirring 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 drawn, and placed in a dialysis bag (MD44mm, molecular weight cutoff 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 equilibrated with distilled water for 24 hours for use. The crude pitaya polysaccharide 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 loading volume was 10 mL, and the column was eluted with 0, 0.2, and 0.3 M NaCl aqueous solutions for 125, 175, and 150 min, respectively. The flow rate was 2 mL / min, and the column was collected by an automatic collector. 10 mL per tube was used to track and monitor the polysaccharide concentration in the eluate using the phenol-sulfuric acid method, and an elution curve was prepared with the number of tubes and absorbance as the horizontal and vertical coordinates. Figure 1As shown in (a), two components were obtained by eluting with NaCl solutions of three concentrations of 0 to 0.3 mol / L, respectively named DFP-1 (component obtained by water elution, corresponding to the tubes 10 to 18) and DFP-2 (component obtained by eluting with 0.2 M NaCl aqueous solution, corresponding to the tubes 38 to 50), wherein DFP-2 is an acidic polysaccharide. The component 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), dialyzed with running water for 48 h to remove the salt therein, and freeze-dried at -50°C for 48 h to obtain a freeze-dried powder of the component DFP-2, which was stored at -20°C for later use.

[0055] (2) Purification by Sephadex G-100: The component DFP-2 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 component DFP-2 was dissolved in distilled water to a concentration of 10 mg / mL, and loaded on 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 made with the number of tubes and absorbance as the horizontal and vertical coordinates, respectively. The elution curve is shown in the figure. Figure 1 (b), component DFP-2 was purified again to obtain a uniform component, named SDFP-2 (corresponding to tubes 5 to 15), with a purity of 94.73±0.1%. Component SDFP-2 was concentrated by rotary evaporation at 55°C and 85rpm for 45min, then placed in a dialysis bag (molecular weight cutoff of 3500Da), dialyzed with flowing water to remove small molecules such as inorganic salts, and freeze-dried at -50°C for 48h to obtain uniform pitaya polysaccharide SDFP-2, which was stored at -20°C for later 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, Japan Shodex) 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) Plotting the standard curve: Accurately weigh dextran standards of different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards), prepare a 5 mg / ml solution with 0.05 M NaCl solution (precisely prepared, filtered through a 0.22 μm filter membrane, ultrasonicated for 20 min, and stored at room temperature), and filter through a 0.22 μm filter membrane for later use. Perform linear regression with the logarithmic value of the relative molecular mass of the standard (lg 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, the supernatant was collected, filtered through a 0.22 μm microporous filter membrane, transferred to a 2 mL injection vial, injected, and 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) Dragon fruit 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, glucosamine hydrochloride, and galactosamine hydrochloride, and 10 mg of fucose in turn, dissolve in water, and use a volumetric flask to make up to 10 ml to prepare a standard stock solution. Then perform gradient dilution and make up to the volume to obtain a monosaccharide standard solution, and filter through a 0.22 μm microporous filter membrane before placing into a sample injection bottle.

[0069] (2) Accurately weigh 5 mg (±0.05 mg) of polysaccharide SDFP-2 sample, add 1 ml of 2M TFA (trifluoroacetic acid) aqueous solution, and heat at 121°C for 2 hours. Pass 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 completed, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the NaOH, add 1 mL of chloroform, vortex extract 3 times to remove excess PMP, discard the chloroform layer, take 0.3 mL and add water to make the volume to 1 mL, and then pass through a 0.22 μm filter membrane for detection. Chromatographic conditions: Chromatographic column: ZORBAX EclipseXDB-C18, mobile phase is acetonitrile: phosphate buffer (containing 12 g / L potassium dihydrogen phosphate and 2M NaOH in water, pH = 6.8), isocratic elution, the volume ratio of acetonitrile to phosphate buffer is 17:83, the flow rate is 0.8 mL / min, the column temperature is 30 ° C, the UV detector, the detection wavelength is 250 nm, and the injection volume is 10 μL.

[0070] 2.2 Determination results 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 shown in 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, and the corresponding molar ratios are: 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 sample 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 (Galactose) 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 treatments, 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, add 1 mL of water to dissolve, add 1 mL of 100 mg / mL carbodiimide solution, and react for 2 hours. Add 1 mL of 2M imidazole solution, add 1 mL of 30 mg / mL NaBD4 solution, and react for 3 hours. Add 100 μL of glacial acetic acid to terminate the reaction. Then, put it into a dialysis bag, dialyze the sample with water for 48 hours, and freeze-dry the sample after dialysis. Take 1-3 mg of freeze-dried sample, add 500 μL of DMSO to dissolve. Add 1 mg of NaOH and incubate for 30 minutes. Add 50 μL of iodomethane solution and react for 1 hour. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat water washing 3 times. Take the lower dichloromethane phase and evaporate it to dryness. Add 100 μL of 2 M TFA aqueous solution and react at 121°C for 90 minutes. Evaporate to dryness at 30°C. Add 50μL 2M ammonia solution and 50μL 1M NaBD4 solution, mix well, and react at room temperature for 2.5h. Add 20μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250μL methanol, and blow dry with nitrogen. Add 250μL acetic anhydride, vortex mix well, and react at 100℃ for 2.5h. Add 1mL water and let stand for 10min. Add 500μL dichloromethane, vortex mix well, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Take the lower dichloromethane phase and detect it using Agilent 7890A-5977B gas spectrometer.

[0077] The chromatographic system used was an Agilent gas chromatograph system (Agilent 7890A; Agilent Technologies, USA), an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), a carrier gas of high-purity helium (purity not less than 99.999%), a flow rate of 1.0 mL / min, and an injection port temperature of 260°C. The injection volume was 10 μl. The program temperature conditions were: 50°C for 1.0 min, 50°C / min to 130°C, 3°C / min to 230°C, and kept for 2 min.

[0078] The mass spectrometry system used a quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) from Aiglent, USA, equipped with an electron impact ion source (EI) and a MassHunter workstation. The electron impact ion source (EI), the injection port temperature was 230°C, the quadrupole temperature was 150°C, and the electron energy was 70 eV. The scanning mode was full scan mode (SCAN), and the mass scanning range (m / z): 30-600.

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

[0080] Methylation test results Figure 3 , as shown in Table 3. The sugar residues and their molar ratios contained in SDFP-2 were obtained based on the retention time and peak area, and the composition and connection mode of the glycosidic bonds can also be obtained 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), 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 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. Its 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 spectra ( 13 C-NMR) can be used to 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 resonance region of the anomeric proton (H-1) is between δ4.5 and 5.5 ppm. 1 The characteristic signals of H-NMR can identify certain sugar residues or groups. For example, the methyl proton signal of the 6-deoxy sugar appears in the high field area of ​​δ0.8~1.4ppm; the methyl proton signal of the acetyl group (CH3COO- or -OAc) appears in the low field area of ​​δ1.9~2.2ppm; the methyl proton signal of the methyl ester (-COOCH3 or -COOMe) is located at δ3.0~3.8ppm. The chemical shift of the anomeric carbon (C-1) signal of the polysaccharide is generally between δ90 and 110ppm, among which the α-configuration anomeric carbon signal usually appears between δ95 and 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 glycosylation shift and will move to the low field. The unsubstituted C-6 signal is near δ60ppm, and the substituted C-6 signal moves to the low field 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 ​​δ15-20ppm; the carbonyl signal of uronic acid (-COOH) and its methyl ester or acetyl group appears in the low field area of ​​δ170-180ppm; the methyl carbon signal of methyl ester is located in the δ50-60ppm area; the methyl carbon signal of acetyl group appears in the higher field area of ​​δ20-25.

[0088] like Figure 4 (a) shows that SDFP-2 1 A large number of proton resonance signals on the H-NMR spectrum are concentrated in the δ3.0-5.5ppm region, with serious signal overlap. Multiple anomeric hydrogen signals were found in the δ4.3-5.5ppm region, indicating that there are multiple sugar residues, and there is a certain overlap, which is 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)) has 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 4(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 located in the high field region of δ1.10-1.30 ppm is a 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 δ1.90-2.20 ppm region is the methyl proton signal of the O-acetyl group. 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 δ20.00 ppm. 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 δ172-176 ppm signal in the low field region of the carbon spectrum. Combined with the monosaccharide composition determination results 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 revealed 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) In the anomeric region of the HSQC hydrogen spectrum, there were strong cross peaks near the δ4.90-5.00 ppm region and the δ97-98 ppm region. In the COSY spectrum, 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. Then, the COSY spectrum was cross-contaminated to determine the chemical shift of the H-1 of the sugar residue to be δ4.93 ppm. 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 in the HSQC spectrum. The cross peaks near δ52ppm of the methyl proton carbon signal of methyl ester (-COOMe) are not found in the HSQC spectrum, indicating that there are no obvious esterified galacturonic acid residues in the sample. In the HMBC spectrum ( Figure 5 c) found the signal cross peak δ3.75 / 173.62ppm, it can be judged that δ173.62ppm belongs to the C-6 signal of the galacturonic acid residue. The chemical shifts of C-1 and C-4 shifted to the low field, indicating that the residue was substituted at the C-1 and C-4 positions of the sugar ring. Combined with the methylation results and literature reports, it is inferred that the sugar residue is →4)-α-GalpA-(1→, marked as GA 1,4 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 / 4.03ppm (H-1 / H-2). Combining the results of methylation analysis and literature reports, it can be inferred that the cross peak δ5.15 / 98.84ppm in the anomeric region of HSQC mainly belongs to the anomeric signal of the sugar residue α-Rhap. COSY and HSQC were used for deduction, and combined with the results of methylation analysis 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 of δ4.64 / 100.66 ppm was found in the anomeric region of HSQC. COSY and HSQC were used to deduce, 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) In the anomeric region of HSQC, there are strong cross-peak signals near the hydrogen spectrum δ4.30-4.45ppm and the carbon spectrum δ102.0-104.0ppm. In the COSY spectrum, it can be found that the anomeric hydrogen in this region and the hydrogen spectrum δ3.40-3.60ppm have strong cross-peak signals δ4.34 / 3.44ppm, δ4.38 / 3.44ppm, and δ4.43 / 3.58ppm. 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→, labeled 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 with 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 are strong cross peak signals near the δ4.40~4.49ppm region of the hydrogen spectrum and the δ102~103ppm region of the carbon spectrum in the HSQC anomeric region, and the cross signals of δ4.45 / 3.25ppm and δ4.44 / 3.29ppm of the anomeric hydrogen in this region are found in the COSY spectrum. Combined with the results of methylation analysis 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 was determined to be δ4.45ppm and δ4.44ppm by HSQC and COSY. Similarly, COSY and HSQC were used for deduction, and combined with the HMBC spectrum ( Figure 5 c) There are cross peaks at δ3.71 / 174.72ppm and δ3.69 / 174.72ppm, and the H-5 signals are δ3.71ppm and δ3.69ppm, and the C-6 signal is δ174.72ppm. The chemical shift assignments are shown in Table 1. The C-1 chemical shift of the anomeric signal δ4.45 / 102.86ppm is shifted to the 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 were derived using COSY and HSQC and with the help of literature reports. 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 samples also contain 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 in the HMBC long-range correlation spectrum, and the two protons at the connection sites of adjacent sugar residues are close in space, they are easy to produce strong NOE signals. The HMBC long-range correlation spectrum and NOESY spectrum can further infer the mutual connection order between each sugar residue. HMBC correlation spectrum of polysaccharide sample ( Figure 5 c) and NOESY spectra ( 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 existence 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 The C-1 (δ98.84ppm) has 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 The H-2 (δ4.03ppm) has a related signal peak (GA 1,4 H-1 / Rha 1,2 or Rha 1,2,4 H-2), indicating 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 NOESY spectrum of 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→ and →2,4)-α-L-Rhap-(1→ connected, and the connection site is 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 The H-4 (δ4.09ppm) has 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 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 spectrum t2 H-1 (δ5.11ppm) and sugar residue A 1,2 There is a related signal peak (A t2 H-1 / A 1,2C-2), indicating the presence of α-L-Araf-(1→2)-α-L-Araf-(1→;(8) HMBC spectrum of sugar residue G 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 O-6. 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 sugar residue O-3 were found on HMBC, and the anomeric hydrogen of these two sugar residues was not found to be connected to 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 spectrum t H-1 (δ4.45ppm) and sugar residue G 1,6 There is a related signal peak at C-6 (δ69.56ppm) (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 spectrum 1,4 H-1 (δ4.44ppm) and sugar residue G 1,6 There is a related signal peak at C-6 (δ69.56ppm) (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 1,4The C-1 (δ102.86ppm) has 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 existence of →4)-β-D-GlcpA-(1→6)-β-D-Galp-(1→ connection; (11) sugar residue Rha in the HMBC spectrum t H-1 (δ 4.64 ppm) 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 The H-4 (δ3.50ppm) has 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 spectrum 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 spectrum 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 spectrum 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 The H-3 (δ3.62ppm) has 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→ connected, with the connection 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→ connected, and the connection site is located at the O-3 position; (16) sugar residue A in the HMBC spectrum 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 The C-1 (δ107.33ppm) has 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 NMR information analysis, it is inferred that the pitaya polysaccharide SDFP-2 sample contains HG-type and RG-Ⅰ type pectin structures, and contains 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 Fig.11 shown.

[0097] Example 3 Effect of pitaya polysaccharide SDFP-2 on the improvement of obesity in mice induced by high-fat diet

[0098] 1 Animal grouping and experimental design

[0099] After one week of adaptive feeding, 40 6-week-old C57BL / 6 male mice were randomly divided into 4 groups. Each group had 10 mice, 5 mice per cage. Grouping: 1) ND group (control group): fed with normal diet (XTCON50J, Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.); 2) HFD group (model group): fed with high-fat diet (XTHF60, Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.); 3) LSDFP-2 group: fed with high-fat diet (XTHF60), and given oral administration of pitaya polysaccharide SDFP-2 at a dose of 75 mg / kg / d, 0.1 ml / 10 g; 4) HSDFP-2 group: fed with high-fat diet, and given oral administration of pitaya polysaccharide SDFP-2 at a dose of 150 mg / kg / d, 0.1 ml / 10 g. In addition to the above feed, the ND group and the HFD group were also given distilled water with the same volume as pitaya polysaccharides.

[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 the intervention of pitaya polysaccharide SDFP-2, the body size was significantly reduced, and the hair of mice fed with a high-fat diet became shiny.

[0103] The weight index of mice is the most direct manifestation of testing whether pitaya polysaccharide SDFP-2 can prevent mouse obesity. The weekly weight of mice is recorded during the feeding cycle, and the weight gain curve is 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 in 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), which indicates 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 8 weeks of intervention with pitaya polysaccharide, the mice in each group were fasted and deprived of water for 8-12 hours, and then oral glucose tolerance test (OGTT) was performed. After the baseline blood glucose value (0 min) was measured, the mice were given 2g / kg glucose solution by gavage, and the blood glucose values ​​of the mice were measured and recorded at 30, 60, 90 and 120 min.

[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, mice were subjected to OGTT. Figure 7 As shown, the blood glucose levels of mice in the HFD group were higher than those in other groups at all points. When glucose was given for 30 minutes, 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 given pitaya polysaccharide SDFP-2 were close to those of mice fed with normal feed, indicating that pitaya polysaccharide SDFP-2 can improve the glucose tolerance of obese mice.

[0107] 4. Determination of lipid indexes in serum

[0108] The experiment was terminated 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 instructions 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 figure, 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), close to 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 Fig. 9As shown in the results, compared with the ND group, the serum HDL-C content of mice in the HFD group was significantly decreased (P < 0.01), indicating that the metabolism of cholesterol from the liver to the liver was blocked; the serum LDL-C content in the HFD group was significantly higher than that in the ND group, indicating that the transport of cholesterol to extrahepatic tissues increased. The decrease in HDL-C content and the increase in LDL-C content may lead to hypercholesterolemia and lipid metabolism disorders. After treatment with pitaya polysaccharides, the serum HDL-C content of mice on a high-fat diet increased significantly, and the LDL-C content decreased significantly, which helps to reduce the cholesterol content in the serum and thus improve the obesity of mice.

[0111] Fat decomposition produces free fatty acids, and their excessive accumulation can cause dyslipidemia. Fig.10 As shown in the figure, compared with the ND group, the NEFA content in the serum of the mice in the HFD group 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 of obese mice induced by high-fat diet.

[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 principle of the present invention. These improvements and modifications should also be regarded as 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, the relative molecular weight is 8.08×104Da, and 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.

2. The method for preparing the homogenized pitaya polysaccharide according to claim 1, characterized in that: The steps include: (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 pitaya crude polysaccharide; (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; (5) Purifying the ion-purified pitaya polysaccharide obtained in step (4) by gel column chromatography, collecting the eluate containing the polysaccharide, and dialyzing to obtain homogenized pitaya polysaccharide.

3. The preparation method according to claim 2, characterized in that: In step (1), during water extraction, the solid-liquid ratio of pitaya powder to water is 1:20-40; the extraction temperature is 70-90°C, and the extraction time is 2-4h; 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 35-55min; 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.

4. The preparation method according to claim 3, characterized in that: 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, the elution time of each solution is 125-175 min, and the eluate obtained by eluting with 0.2 mol / L NaCl aqueous solution is collected; the flow rate of the elution is 1-4 mL / min.

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

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

7. The preparation method according to claim 6, characterized in that: A dialysis bag with a molecular weight cut-off of 2500 to 4500 Da is used in the dialysis.

8. Use of the homogenized pitaya polysaccharide according to claim 1 or the homogenized pitaya polysaccharide obtained by the preparation method according to any one of claims 2 to 7 in the preparation of lipid-lowering and blood sugar-lowering drugs.

9. A lipid-lowering and blood sugar-lowering pharmaceutical composition, characterized in that: The invention comprises the homogenized pitaya polysaccharide according to claim 1 or the homogenized pitaya polysaccharide obtained by the preparation method according to any one of claims 2 to 7 and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that The dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, drop pill, pill or powder.

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