Preparation method and application of hemp seed polysaccharide

Abstract

The present invention relates to a preparation method and application of hemp seed polysaccharide. The preparation method of hemp seed polysaccharide comprises the following steps: (1) using hemp seed meal as raw material, extracting hemp seed crude polysaccharide HSP by water extraction and alcohol precipitation; (2) removing protein from the crude hemp seed polysaccharide, and separating it by ion exchange column chromatography to obtain HSP-II; (3) separating HSP-II by gel column chromatography to obtain HSP-IIa. The hemp seed polysaccharide prepared by the present invention can be used to lower blood sugar and improve the glucose consumption capacity of insulin-resistant cells.

Description

Technical Field

[0001] The invention belongs to the technical field of hemp seed active products, and in particular relates to a preparation method and application of hemp seed polysaccharide. Background Art

[0002] Hemp seed is the dried, mature seed of the hemp plant (Cannabis sativa L.), a member of the Moraceae family. It is primarily produced in the three northeastern provinces, southern China, and northern China. It is one of the "Nine Dragon Herbs" of Heilongjiang Province and a traditional Chinese medicinal and edible plant with high medicinal and nutritional value. It is mild in nature and sweet in flavor, and enters the spleen, stomach, and large intestine meridians. It has benefits such as laxative, nourishing, and tonifying, as well as promoting urination and blood circulation. Pharmacological studies have shown that hemp seed has significant pharmacological activities, including hypoglycemic, hypolipidemic, and hypotensive, antioxidant, anti-tumor, and immunomodulatory activities. Hemp seed contains up to 30% carbohydrates (including polysaccharides). Repurposing hemp seed meal, a waste product from oil extraction, can significantly increase the added value of hemp seed. Different extraction methods produce different products, which further impacts the application of hemp seed's active products. Currently, there are no reports on the application of hemp seed polysaccharides for hypoglycemic purposes. Summary of the Invention

[0003] In view of the problems existing in the prior art, the present invention provides a preparation method of hemp seed polysaccharide and its application.

[0004] The technical solution of the present invention to solve the above technical problems is as follows:

[0005] The present invention provides a method for preparing hemp seed polysaccharide, comprising the following steps:

[0006] (1) Using hemp seed meal as raw material, crude hemp seed polysaccharide (HSP) was extracted by water extraction and alcohol precipitation;

[0007] (2) After removing protein from crude polysaccharide from hemp seed, HSP-Ⅱ was obtained by ion exchange column chromatography;

[0008] (3) HSP-Ⅱ is subjected to gel column chromatography to obtain HSP-Ⅱa.

[0009] Furthermore, in step (1), the hemp seed crude polysaccharide is extracted by water extraction and alcohol precipitation method, which comprises the following steps: mixing the defatted hemp seed meal with water, decocting, cooling, centrifuging, taking the supernatant, collecting the extract, filtering, concentrating to obtain a concentrated product; precipitating the concentrated product with an ethanol solution, centrifuging, collecting the precipitate, evaporating the ethanol, concentrating, and freeze-drying.

[0010] Furthermore, the material-liquid ratio of the defatted hemp seed meal to water is 1 g:15 mL; and the final concentration of ethanol in the ethanol solution is 80%.

[0011] Furthermore, in step (2), the protein is removed by the Sevage method, and the product is separated by chromatography using a DEAE-52 cellulose ion exchange column.

[0012] Furthermore, the protein is removed by the Sevage method, which comprises the following steps: mixing the solution of crude hemp seed polysaccharide HSP, chloroform and n-butanol, shaking and then centrifuging, taking the supernatant, dialysis, concentrating and freeze-drying.

[0013] Furthermore, the separation by DEAE-52 cellulose ion exchange column chromatography includes the following steps: the aqueous solution of hemp seed polysaccharide obtained by deproteinization by the Sevage method is separated by DEAE-52 cellulose ion exchange column chromatography, sodium chloride solution is used as eluent, the components eluted by 0.3M NaCl solution are collected, dialyzed, concentrated, and freeze-dried to obtain HSP-Ⅱ.

[0014] Furthermore, in step (3), HSP-Ⅱ is separated by Sephacryl S-300 gel column chromatography, comprising the following steps: preparing an HSP-Ⅱ aqueous solution, separating it by Sephacryl S-300 gel column chromatography, eluting it with distilled water at a flow rate of 1 mL / 2 min, collecting the elution peak, and freeze-drying to obtain HSP-Ⅱa.

[0015] The invention provides a hemp seed polysaccharide, which is prepared by the method.

[0016] The present invention provides the use of the hemp seed polysaccharide in preparing a blood sugar-lowering product.

[0017] The present invention provides the use of the hemp seed polysaccharide in improving the glucose consumption capacity of insulin-resistant cells. For example, the use of the hemp seed polysaccharide in preparing a product that improves glucose consumption capacity.

[0018] The products are not limited to food, medicine, health care products, etc.

[0019] The invention extracts, separates and purifies hemp seed meal to obtain a homogeneous polysaccharide HSP-Ⅱa. HSP-Ⅱa is an acidic polysaccharide whose primary structure is mainly composed of →4)-α-GalA-(1→) and →4)-β-Ara-(1→). The total sugar content is 57.37%, the uronic acid content is 38.87%, the weight average molecular weight (Mw) is 137.01 kDa, the average particle size is 281.57 nm, and the average Zeta potential is -30.9 mV. HSP-Ⅱa can be partially digested and degraded in simulated gastrointestinal digestive fluid in vitro and exhibits good hypoglycemic activity, thereby being used as a hypoglycemic drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the Sephacryl S-300 elution curve of hemp seed polysaccharide HSP-Ⅱa.

[0021] Figure 2 The standard curve is (a: glucose; b: galacturonic acid; c: protein).

[0022] Figure 3 This is the ultraviolet absorption spectrum of hemp seed polysaccharide.

[0023] Figure 4 This is the HPGPC test result of HSP-Ⅱa.

[0024] Figure 5 This is the relationship diagram between the molecular weight distribution of HSP-Ⅱa and dextran standards.

[0025] Figure 6 This is the ion chromatogram of a mixed sample of 16 monosaccharide standards.

[0026] Figure 7 This is the ion chromatogram of HSP-Ⅱa.

[0027] Figure 8 This is the FT-IR image of hemp seed polysaccharide HSP-Ⅱa.

[0028] Figure 9 This is the nuclear magnetic resonance spectrum of hemp seed polysaccharide HSP-Ⅱa (a: 1 H NMR; b: 13 C NMR).

[0029] Figure 10 This is the TG-DSC analysis of hemp seed polysaccharide.

[0030] Figure 11 This is the particle size distribution diagram of hemp seed polysaccharide HSP-Ⅱa.

[0031] Figure 12 Zeta potential of hemp seed polysaccharide HSP-Ⅱa.

[0032] Figure 13 This is the X-ray diffraction curve of hemp seed polysaccharide.

[0033] Figure 14 This is the Congo red test of hemp seed polysaccharide HSP-Ⅱa.

[0034] Figure 15 These are scanning electron microscope images of hemp seed polysaccharide HSP-Ⅱa, from left to right: ×100, ×1000, and ×5000.

[0035] Figure 16 Atomic force microscopy images of hemp seed polysaccharide HSP-Ⅱa (a, b: 10μm×10μm, c, d: 2μm×2μm).

[0036] Figure 17The molecular weight changes of HSP-Ⅱa during in vitro simulated gastrointestinal digestion (a: gastric juice digestion; b: intestinal juice digestion).

[0037] Figure 18 The production of free monosaccharides during HSP-Ⅱa simulated gastric juice digestion in vitro.

[0038] Figure 19 The production of free monosaccharides during in vitro simulated intestinal digestion of HSP-Ⅱa.

[0039] Figure 20 Changes in the content of total sugar and reducing sugar in HSP-Ⅱa during digestion by gastrointestinal fluid (a: total sugar; b: reducing sugar).

[0040] Figure 21 The inhibitory effect of hemp seed polysaccharide HSP-Ⅱa on α-glucosidase.

[0041] Figure 22 The inhibitory effect of hemp seed polysaccharide HSP-Ⅱa on α-amylase.

[0042] Figure 23 This is the effect of insulin on glucose consumption in HepG2 cells.

[0043] Figure 24 This is the effect of different concentrations of hemp seed polysaccharide HSP-Ⅱa on glucose consumption in IR-HepG2 cells. DETAILED DESCRIPTION

[0044] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0045] The invention uses hemp seed meal as raw material, obtains hemp seed polysaccharide by extraction, separation and purification, and characterizes its primary and secondary structures. At the same time, the in vitro simulated digestion characteristics of hemp seed polysaccharide are investigated, and its in vitro hypoglycemic activity is evaluated.

[0046] The invention adopts a water extraction and alcohol precipitation method to extract crude hemp seed polysaccharide (HSP). The total sugar content of the crude hemp seed polysaccharide HSP is (29.30±1.62)%, the uronic acid content is (9.62±2.66)%, and the protein content is (23.73±2.32)%. The HSP is subjected to a Sevage method and a dialysis method to remove impurities such as protein and small molecules. The HSP is then separated and purified by DEAE-52 anion exchange column chromatography and Sephacryl S-300 gel column chromatography to obtain high-purity hemp seed polysaccharide HSP-IIa.

[0047] The primary structure of HSP-Ⅱa was elucidated using high-performance gel permeation chromatography, ion chromatography, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The higher-order structure of HSP-Ⅱa was also characterized using thermal properties, particle size and zeta potential analysis, X-ray diffraction (XRD), Congo red analysis, field emission scanning electron microscopy (FE-SEM), and atomic force microscopy (AFM). HSP-Ⅱa, the polysaccharide component of hemp seed, is a homogeneous polysaccharide with high purity. HSP-Ⅱa has a total sugar content of (57.37±1.89)%, a uronic acid content of (38.87±2.38)%, and a weight-average molecular weight (Mw) of 137.01 kDa. It is primarily composed of monosaccharides such as rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid, with a relative molar ratio of 1.9:6.3:6.1:1.6:2.3:5.5:1.0. It is an acidic polysaccharide primarily composed of →4)-α-GalA-(1→) and →4)-β-Ara-(1→). Thermal properties testing showed that HSP-Ⅱa exhibited good thermal stability at 160°C. Particle size and potential analysis revealed an average particle size of 281.57 nm, a PDI value of 0.25, and an average zeta potential of -3 0.9mV, HSP-Ⅱa has a narrow mass distribution range and good molecular weight uniformity, and HSP-Ⅱa is stable in solution. XRD results show that HSP-Ⅱa is primarily a semi-crystalline polymer with both crystalline and amorphous phases. Congo red experiments indicate that HSP-Ⅱa possesses a relatively stable triple-helical structure. FE-SEM observations revealed that HSP-Ⅱa primarily exhibits irregular flakes with a small amount of stacked strip-like structures, with a smooth surface. AFM observations revealed that HSP-Ⅱa exhibits aggregation or entanglement of sugar chains in the liquid state. Its two-dimensional planar images primarily exhibit irregular spherical structures and partially aggregated granular structures, while three-dimensional images reveal numerous cylindrical structures of varying heights. HSP-Ⅱa has a low aggregation density, is more stable in aqueous solution, and is less prone to aggregation.

[0048] The acidic polysaccharide HSP-Ⅱa, which has a relatively small particle size and can exist relatively stably in solution, was further used in this experiment to study its in vitro simulated digestion characteristics and hypoglycemic activity.

[0049] An in vitro simulated gastrointestinal digestion model was established to investigate the digestion characteristics of HSP-Ⅱa, a polysaccharide from hemp seed, in artificial gastric and intestinal fluids, using molecular weight, free monosaccharides, total sugar content, and reducing sugar content as assays. This study provided a basis for further activity studies. The results showed that the molecular weight and total sugar content of the HSP-Ⅱa digestion products decreased with prolonged simulated gastrointestinal digestion, and that partial cleavage of glycosidic bonds resulted in the release of a small amount of free monosaccharides and an increase in reducing sugar content. This indicates that the sugar chain structure of HSP-Ⅱa undergoes partial degradation after in vitro simulated gastrointestinal digestion, suggesting that gastrointestinal digestion may have a certain impact on the structure and properties of HSP-Ⅱa.

[0050] The in vitro hypoglycemic activity of HSP-Ⅱa was verified by testing its inhibitory activity against two carbohydrate hydrolases, α-glucosidase and α-amylase. A human insulin-resistant hepatocellular carcinoma HepG2 (IR-HepG2) cell model was established, and the glucose consumption capacity of IR-HepG2 cells was assessed by measuring HSP-Ⅱa. The results of HSP-Ⅱa activity assays against α-glucosidase and α-amylase showed that HSP-Ⅱa had a certain inhibitory effect on the activities of both carbohydrate hydrolases and significantly enhanced the glucose consumption capacity of IR-HepG2 cells, preliminarily revealing that HSP-Ⅱa has significant in vitro hypoglycemic activity.

[0051] Unless otherwise specified, the experimental methods used in this invention are conventional methods in the art; the materials, reagents, methods, and instruments used are conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially or prepared by conventional methods by those skilled in the art. Unless otherwise specified, the solutions described in this invention are prepared using water as the solvent.

[0052] The defatted hemp seed meal powder is prepared by the following method: shelled hemp seeds are used as raw materials, and after sorting and cleaning, they are sent to a low-temperature press, the low-temperature pressing pressure is controlled at 2.5-4.5 MPa, the temperature is controlled at 60-70 DEG C, and after low-temperature pressing, the defatted hemp seed meal is dried at 105 DEG C to obtain the defatted hemp seed meal, and the defatted hemp seed meal powder is obtained after being crushed.

[0053] DEAE-52 was purchased from Beijing Bio-Aotoda Technology Co., Ltd.; dextran gel S-300 was purchased from Beijing Solebold Technology Co., Ltd.; BCA protein quantification kit was purchased from Beyotime Biotechnology Co., Ltd.; dextran standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; monosaccharide standards were purchased from Boruitang Biotechnology Co., Ltd.; simulated gastric fluid was purchased from Shanghai Leigen Biotechnology Co., Ltd.; artificial intestinal fluid was purchased from Shanghai Leigen Biotechnology Co., Ltd.; 1-phenyl-3-methyl-5-pyrazolone (PMP) was purchased from Xiamen Anyongbo Technology Co., Ltd.; DNS was purchased from Beijing Yaanda Biotechnology Co., Ltd.; phenol was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; PBS buffer was purchased from Beijing Bio-Aosen Biotechnology Co., Ltd.; PNPG was purchased from Beijing Bio-Aotoda Technology Co., Ltd.; HepG2 cells were purchased from Shanghai Kanglang Biotechnology Co., Ltd.; fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; serum-free 1640 culture medium was purchased from Beijing Solebold Technology Co., Ltd.; insulin was purchased from Beijing Solebold Technology Co., Ltd.; and a glucose kit was purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd.

[0054] The following is an introduction through specific embodiments.

[0055] Example 1 Preparation of Hemp Seed Polysaccharide

[0056] 1.1 Extraction of hemp seed crude polysaccharides

[0057] (1) Weigh the defatted hemp seed meal powder and add distilled water at a solid-liquid ratio of 1 g:15 mL. Place the mixture in a 90°C hot water bath and boil for 3 h. After cooling, centrifuge at 4500 rpm / min for 15 min. Collect the extract and residue separately.

[0058] (2) Extract the residue 1-2 times in the same manner as in step (1), combine the extracts, filter, and concentrate the filtrate under reduced pressure at 75° C. on a rotary evaporator to 1 / 10 of the volume before the reduced pressure concentration to obtain a concentrated product. After natural cooling, slowly pour 95% ethanol into the concentrated product (while stirring) to make the final ethanol concentration 80%. Let it settle for 12 hours, then centrifuge at 4500 r / min for 15 minutes, collect the precipitate, and freeze-dry for later use to obtain the crude hemp seed polysaccharide HSP.

[0059] 1.2 Isolation and purification of hemp seed polysaccharides

[0060] (1) Sevage method for protein removal: Dissolve hemp seed crude polysaccharide HSP in distilled water to obtain a crude polysaccharide solution with a final HSP concentration of 20 mg / mL. Add chloroform and n-butanol to the crude polysaccharide solution at a ratio of crude polysaccharide solution: chloroform: n-butanol = 25:5:1 (volume ratio). Shake thoroughly for 15-20 minutes, centrifuge at 4500 r / min for 15 minutes, remove the white protein layer, and collect the supernatant. Repeat the above method 6-7 times until the white protein layer is no longer visible. Use a 3500Da dialysis bag to dialyze with water for 24 hours. The dialyzate is concentrated under reduced pressure using a rotary evaporator and freeze-dried.

[0061] (2) DEAE-52 cellulose ion exchange column chromatography

[0062] ① Activation of DEAE-52: First, immerse DEAE-52 in deionized water. After sufficient swelling treatment, soak it in 0.5M NaOH solution for 1 hour, repeatedly wash it with deionized water until it is neutral, then soak it in 0.5M hydrochloric acid solution for 1 hour, wash it with deionized water until it is neutral, repeat the NaOH aqueous solution treatment step again, and finally degas it by vacuum pressure.

[0063] ② Column loading and balancing: Before loading the column, ensure that all pipes are unobstructed. Add a small amount of deionized water along the wall of the column to remove bubbles at the bottom. Slowly stir the activated DEAE-52 clockwise to evenly mix it. Use a glass rod to drain it into the column. After the DEAE-52 has fully settled, balance and compact it with deionized water.

[0064] 3. elution chromatography and sample determination, collection: the Fructus Cannabis polysaccharides water that Sevage method is removed protein and obtains is prepared 10mg / mL Fructus Cannabis polysaccharides solution.Fructus Cannabis polysaccharides solution is evenly added in the chromatography column along tube wall, according to the order of NaCl solution from low concentration to high concentration, carry out gradient elution with the NaCl solution (0.1mol / L, 0.2mol / L, 0.3mol / L, 0.4mol / L, 0.5mol / L) of variable concentrations successively, the volume of the NaCl solution of every kind of concentration is 2 times of chromatography column volumes, and controlled flow velocity 1mL / min collects eluent by every pipe 2mL.With phenol-sulfuric acid method (with reference to embodiment 2.1) tracking every pipe eluent of detection, survey its absorbance, take the number of tubes as abscissa, and absorbance is a ordinate, draws elution curve. The eluates obtained from elution with different concentrations of NaCl solution were collected and dialyzed against water using a dialysis bag with a molecular weight cutoff of 3000 Da. The dialyzate was concentrated under reduced pressure using a rotary evaporator and lyophilized for later use. The results showed that the fraction eluted with 0.3 M NaCl solution had a high sugar content and good peak symmetry, and was named HSP-II.

[0065] (3) Sephacryl S-300 gel column chromatography

[0066] ① Pretreatment of dextran gel S-300: First, soak S-300 repeatedly in deionized water to fully swell it, and then degas it by vacuuming for use.

[0067] ② Column loading and balancing: To remove bubbles at the bottom, add a small amount of deionized water to the chromatography column to stir the swollen S-300 evenly, then slowly pour it into the chromatography column. After the S-300 is fully settled, balance it with deionized water.

[0068] Prepare 10mg / mL HSP-Ⅱ polysaccharide solution with water, add the polysaccharide solution evenly along the wall of the tube to the Sephacryl S-300 gel column, elute the hemp seed polysaccharide HSP-Ⅱ with distilled water twice the volume of the column at a flow rate of 1mL / 2min, collect the elution peak, and freeze-dry. Draw the elution curve as shown below: Figure 1 As shown, it can be seen that the elution curves all have only a single symmetrical peak, indicating good purity. The eluate was enriched, concentrated under reduced pressure using a rotary evaporator, and freeze-dried for later use. The obtained hemp seed polysaccharide was named HSP-Ⅱa.

[0069] Example 2 Determination of the content of hemp seed polysaccharide

[0070] 2.1 Determination of total sugar content by phenol-sulfuric acid method

[0071] (1) Drawing a standard curve: Prepare glucose standard solutions with concentrations of 1.0, 0.8, 0.6, 0.4, 0.2, and 0 mg / mL with deionized water, dilute to 1 mL, add 6% phenol solution to the glucose standard solutions at a volume ratio of 1:1, and then immediately add 5 mL of concentrated sulfuric acid. Mix thoroughly, heat at 100°C for 15-20 min, cool, and measure the absorbance at 490 nm. Draw a standard curve with the concentrations of the glucose standards as the horizontal axis and the absorbance as the vertical axis.

[0072] (2) Determination of samples: Determine the absorbance of the hemp seed polysaccharide sample according to the above method, and substitute it into the standard curve regression equation to calculate the total sugar content of the hemp seed polysaccharide.

[0073] 2.2 BCA method for protein content detection

[0074] (1) Drawing a standard curve: Prepare protein standards (25 mg / mL, stored at -20°C) and BCA working solution according to the operating instructions in the BCA protein quantification kit. Then dilute the protein standards to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL to obtain protein standard solutions of different concentrations. Then, add each protein standard solution and BCA working solution at a volume ratio of 1:10. Heat in a metal bath at 60°C for 30 min. Measure the absorbance at a wavelength of 595 nm. Draw a standard curve with the concentrations of the protein standards as the horizontal axis and the absorbance value as the vertical axis.

[0075] (2) Sample determination: Determine the absorbance of the hemp seed polysaccharide sample according to the above method, substitute it into the standard curve regression equation, and calculate the protein content of the hemp seed polysaccharide.

[0076] 2.3 Determination of uronic acid content by sulfuric acid-carbazole method

[0077] (1) Preparation of reagents

[0078] The preparation method of the carbazole / anhydrous ethanol solution comprises the following steps: taking anhydrous ethanol as a solvent, weighing carbazole, and preparing a 1 mg / mL carbazole / anhydrous ethanol solution.

[0079] The sodium tetraborate / sulfuric acid solution is prepared by the following steps: weighing 0.244 g of sodium tetraborate and diluting the volume into a 50 mL volumetric flask (prepared with concentrated sulfuric acid).

[0080] (2) Drawing a standard curve: Prepare the galacturonic acid standard solution into standard solutions with concentrations of 1.0, 0.8, 0.6, 0.4, 0.2, and 0 mg / mL, dilute to 1 mL, add 6 mL of the above-prepared sodium tetraborate / sulfuric acid solution, heat in a water bath at 100°C for 5 min, take out and cool, add 1 mL of the above-prepared carbazole / anhydrous ethanol solution, mix well, heat in a water bath at 100°C for 10 min, cool, and measure its absorbance at a wavelength of 525 nm. Draw a standard curve with the concentrations of the galacturonic acid standard as the horizontal axis and the absorbance as the vertical axis.

[0081] (3) Sample determination: 1 mL of the prepared hemp seed polysaccharide sample was taken, and its absorbance was determined according to the above operation. The standard curve regression equation was used to calculate the uronic acid content in the hemp seed polysaccharide.

[0082] The chemical compositions and content determination results of hemp seed polysaccharides are shown in Table 1.

[0083] In this experiment, the phenol-sulfuric acid method was used to determine the total sugar content of each component of hemp seed polysaccharide. According to the standard curve ( Figure 2a) Obtain the regression curve equation: Y = 2.2163X + 0.0232, R 2 =0.9992. According to the test results, the total sugar content of HSP was calculated to be (29.30±1.62)%, the total sugar content of HSP-Ⅱ was (49.85±1.34)%, and the total sugar content of HSP-Ⅱa was (57.37±1.89)%.

[0084] The sulfuric acid-carbazole method was used to determine the uronic acid content of each component of hemp seed polysaccharide. According to the standard curve ( Figure 2 b) Obtain the regression curve equation: Y = 0.5181X + 0.0983, R 2 =0.9984. Calculations based on the test results showed that the uronic acid content of HSP was (9.62±2.66)%, the uronic acid content of HSP-Ⅱ was (20.86±1.16)%, and the uronic acid content of HSP-Ⅱa was (38.87±2.38)%, indicating that HSP-Ⅱa is an acidic polysaccharide.

[0085] The protein content of hemp seed polysaccharide was quantitatively detected using the BCA kit. Figure 2 c) Obtain the regression curve equation: Y = 0.5181X + 0.0983, R 2 =0.9984. Calculations based on the test results showed that HSP was (23.73±2.32)%. After protein removal and preliminary separation and purification, the protein content was significantly reduced. HSP-II still contained a small amount of protein, at (5.03±1.71)%. Furthermore, after further separation and purification, HSP-IIa contained little or almost no protein, at (3.30±1.56)%.

[0086] Table 1 Summary of chemical composition

[0087]

[0088] Example 3 Ultraviolet spectrum scanning of hemp seed polysaccharide

[0089] Weigh 4 mg of HSP-Ⅱa, a polysaccharide sample of hemp seed, and dissolve it in 4 mL of deionized water to prepare a hemp seed polysaccharide solution (i.e., HSP-Ⅱa concentration is 1 mg / mL). Use a full-band UV spectrometer to scan within the range of 190-800 nm and draw a spectrum. The experimental results show that HSP-Ⅱa contains very little or almost no protein, and has no nucleic acid and pigment ( Figure 3 ), which is consistent with the previous BCA kit protein quantification result.

[0090] Example 4 Primary Structural Analysis of Hemp Seed Polysaccharide

[0091] 4.1 Purity analysis and molecular weight determination

[0092] (1) Purity analysis

[0093] Sample processing: Hemp seed polysaccharide HSP-Ⅱa was prepared into a 1 mg / mL aqueous solution with ultrapure water, filtered using a 0.45 μm needle filter membrane, and detected and analyzed on the HPGPC.

[0094] Chromatographic conditions

[0095] Gel chromatography column: TSK Gel3000SWXL (7.8 mm×300 mm); mobile phase: 0.025 M NaH2PO4 aqueous solution (pH=6.7); flow rate: 0.8 mL / min; injection volume: 20 μL; column temperature: 35°C; detector: differential refractive index detector (RID).

[0096] (2) Determination of molecular weight: The concentration range of dextran standard is 4 kDa-5000 kDa. The determination conditions are the same as the chromatographic conditions. The molecular weight is calculated based on the retention time distribution of the determination standard and sample on the GPC workstation. The molecular weight is used as the horizontal axis, and the logarithmic relationship between mass and molecular weight (dw / dlogM) and the cumulative molecular weight ratio (Ht%) are used as the vertical axis to draw a molecular weight distribution curve of the sample and dextran, and fit the standard curve regression equation.

[0097] The analysis results are as follows Figure 4 As shown, hemp seed polysaccharide HSP-Ⅱa has a single symmetrical peak at 14-24 min, and its polydispersity coefficient PD (PD=Mw / Mn) is 3.86, indicating that the hemp seed polysaccharide HSP-Ⅱa component is relatively uniform and has good purity.

[0098] Figure 5 This is a statistical graph of the molecular weight distribution of HSP-Ⅱa and dextran standards. The horizontal axis X represents the molecular weight, and the vertical axis Y represents the logarithmic relationship between mass and molecular weight (dw / dlogM) and the cumulative molecular weight percentage (Ht%). From the dextran standard curve (i.e., the red curve in the figure), the standard curve equation is obtained: Y = 16.271282 - 0.865698X + 0.014792X 2 -0.000047X 3 The molecular weight of HSP-Ⅱa was calculated by fitting, and the weight average molecular weight (Mw) of HSP-Ⅱa was 137.01kDa; the number average molecular weight (Mn) was 35.45kDa; and the peak molecular weight (Mp) was 52.62kDa.

[0099] 4.2 Ion chromatography

[0100] (1) Preparation and calculation of standard solution: 16 monosaccharide standards were prepared to prepare standard solutions (including rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, mannuronic acid, etc.). These monosaccharide standards were mixed together to prepare a mixed standard solution, i.e., a mixed standard. In the mixed standard, the concentration of each monosaccharide standard was 1 mg / mL. According to the absolute quantitative method, the mass of different monosaccharides was determined, and the molar ratio was calculated based on the molar mass of the monosaccharides. According to the formula: C 标准品 / A 标准品 =C 样品 / A 样品计算样品中各单糖相对含量 (C: concentration of monosaccharide in standard or sample; A: frontal area of ​​monosaccharide in standard or sample).

[0101] (2) Sample preparation: Accurately weigh 5.0 mg of hemp seed polysaccharide sample and place it in an ampoule. Add 2 mL of 3 M trifluoroacetic acid solution to the ampoule and place the ampoule in a 120°C oven for hydrolysis for 3 h. After the hydrolysis is completed, transfer the solution to another 5 mL centrifuge tube and blow dry. Add 5 mL of deionized water and mix it evenly by vortexing. Pipette 0.05 mL from the mixed solution and add deionized water to make up to 1 mL for dilution. Place the diluted solution in a centrifuge and centrifuge at 12,000 rpm for 5 min. Take the supernatant after centrifugation for ion chromatography (IC) analysis.

[0102] (3) Chromatographic conditions

[0103] Chromatographic column: Dionex Carbopac™ PA20 (3×150 mm), detector: electrochemical detector.

[0104] Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaAc

[0105] Column temperature: 30℃; injection volume: 25 μL; flow rate: 0.3 mL / min; elution gradient: 0 min phase A / phase B / phase C (98.8:1.2:0, V / V), 18 min phase A / phase B / phase C (98.8:1.2:0, V / V), 20 min phase A / phase B / phase C (50:50:0, V / V), 30 min phase A / phase B / phase C (50:50:0, V / V), 30.1 min phase A / phase B / phase C (0:0:100, V / V), 46 min phase A / phase B / phase C (0:0:100, V / V), 46.1 min phase A / phase B / phase C (0:100:0, V / V), 50 min phase A / phase B / phase C (0:100:0, V / V), 50.1 min Phase A / phase B / phase C (98.8:1.2:0, V / V), 80 min Phase A / phase B / phase C (98.8:1.2:0, V / V).

[0106] Figure 6 This is the IC diagram of the mixed standard of 16 monosaccharides. The peak times are shown in Table 2. The peaks at 2.0 min and 40.5 min are sodium hydroxide and sodium acetate solvent peaks, respectively. Figure 7 The ion chromatogram of hemp seed polysaccharide HSP-Ⅱa was obtained by calculating the relative content of each monosaccharide in hemp seed polysaccharide HSP-Ⅱa according to the following formula. The specific peak time, relative molar ratio and relative content are shown in Table 3. Figure 7 As shown, compared with the mixed standard chromatogram ( Figure 6 ) comparison shows that HSP-Ⅱa is mainly composed of seven monosaccharides, including rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid, with a relative molar ratio of 1.9:6.3:6.1:1.6:2.3:5.5:1.0.

[0107] Table 2 Peak elution time of monosaccharide mixed standard

[0108]

[0109] Table 3 Monosaccharide peak time and molar ratio of HSP-Ⅱa

[0110]

[0111]

[0112] 4.3 Fourier transform infrared spectroscopy

[0113] 1 mg of hemp seed polysaccharide sample was mixed with 100 mg of KBr (dried to constant weight at 100 ° C) and ground evenly. The tablets were pressed with a tablet press. The tablets were translucent and could be put into an infrared spectrometer at 4000 cm -1 -400cm-1 Scanning is performed within the range to identify the characteristic functional groups in the polysaccharide. Figure 8 The results of FT-IR analysis of functional groups are shown in Table 4. 3416cm -1 The stretching vibration absorption peak generated by the -OH of HSP-Ⅱa is 2926cm -1 The two peaks are typical CH stretching vibration absorption peaks, both of which are typical characteristic peaks of polysaccharides. -1 It is the characteristic absorption peak of uronic acid, 1630cm -1 It is the C=O stretching vibration absorption peak in HSP-Ⅱa, which shows that HSP-Ⅱa has uronic acid group, which is consistent with the detection result of monosaccharide composition. -1 Nearby is the CH angle vibration absorption peak, 1200-1000cm -1 The absorption peaks in the range are caused by the stretching vibrations of the pyranose ring COC and COH. Figure 8 Medium 828cm -1 There is an absorption peak at , indicating that HSP-Ⅱa has an α-glycosidic bond configuration. According to the results of FT-IR, HSP-Ⅱa is an acidic polysaccharide with α-pyranose as the skeleton.

[0114] Table 4 FT-IR analysis of functional groups of HSP-Ⅱa

[0115]

[0116] 4.4 Nuclear magnetic resonance spectroscopy analysis

[0117] Weigh 25 mg of hemp seed polysaccharide and dissolve it in 0.6 mL of D2O. The solution was transferred into a nuclear magnetic resonance tube, and the structure of each component of the hemp seed polysaccharide sample was determined by nuclear magnetic resonance.

[0118] In general, the isomeric region of the α configuration appears at 5.0-5.8 ppm (anomeric proton), while the isomeric region of the β configuration appears at 4.3-5.0 ppm. The main structural information of hemp seed polysaccharide HSP-Ⅱa by one-dimensional NMR is shown in Table 5.

[0119] In HSP-Ⅱa 1 H NMR ( Figure 9 In a), most proton signal peaks are located between δ0.7-5.8 ppm, and their chemical shifts are intertwined and overlapped between the signals in the range of 3.0-4.5 ppm, making accurate analysis difficult. There are two major anomeric hydrogen signals in the range of 4.9-5.4 ppm, with chemical shifts of 5.01 and 4.99 ppm, respectively, indicating that HSP-Ⅱa has an α configuration. 13 CNMR( Figure 9The corresponding anomeric carbons in b) are at 98.94 and 107.38 ppm, respectively. Combining the monosaccharide composition with the infrared analysis results, it is speculated that HSP-Ⅱa is mainly composed of →4)-α-GalA-(1→ and →4)-β-Ara-(1→ sugar residues.

[0120] Table 5 Main structural information of hemp seed polysaccharide HSP-Ⅱa in 1D NMR

[0121]

[0122] Example 5 High-Level Structural Characterization of Hemp Seed Polysaccharide

[0123] (1) Detection of thermal characteristics

[0124] Thermogravimetric and differential scanning calorimetry (TG-DSC) was used to investigate the thermal stability and possible aggregation structure of hemp seed polysaccharide samples. Experimental conditions: sample loading of 5 mg to 8 mg; test temperature of 30°C to 800°C; carrier gas: N2; heating rate: 10°C / min.

[0125] The TG-DSC analysis curve of hemp seed polysaccharide HSP-Ⅱa is as follows Figure 10 As shown in the figure, HSP-Ⅱa experienced three weight loss stages during the thermal decomposition process.

[0126] The first weight loss: HSP-Ⅱa is between 30℃-210.45℃, and the temperature inflection point TG corresponds to an exothermic peak of DSC, which is 95.74℃, and the weight loss is 9.99%, indicating that the dried HSP-Ⅱa has good water absorption and water retention properties, and the polysaccharide can be stored stably at room temperature. This stage may be due to the evaporation of free water and bound water in the hemp seed polysaccharide sample;

[0127] The second weight loss: HSP-Ⅱa is between 210.45℃ and 552.84℃. The temperature inflection point TG corresponds to an endothermic peak and an exothermic peak of DSC. The endothermic peak is 233.94℃ and the exothermic peak is 353.84℃. The weight loss is 50.01%. The weight loss is rapid in this stage, indicating that the polysaccharide sample itself has undergone a strong thermal decomposition reaction and has been degraded. This may be due to the depolymerization of HSP-Ⅱa and the breaking of chemical bonds such as CO and CC in the sugar ring, which further decomposes the polysaccharide into components such as CO2 and H2O, which volatilize and may form polycyclic aromatic hydrocarbons and graphitic carbon structures.

[0128] The third weight loss: HSP-Ⅱa lost 3.68% of its weight between 552.84℃ and 800℃. This stage is the carbonization stage, which is mainly the transformation of the remaining residual substances. These results show that HSP-Ⅱa has good thermal stability.

[0129] (2) Particle size and potential measurement

[0130] A hemp seed polysaccharide solution with a concentration of 0.2 mg / mL was prepared, fully dissolved, filtered with a 0.45 μm needle filter membrane, and detected on a Litesizer-500 nanoparticle size and zeta potential instrument.

[0131] The particle size distribution of hemp seed polysaccharide HSP-Ⅱa is as follows Figure 11 As shown, HSP-Ⅱa is a single symmetrical peak, indicating that HSP-Ⅱa is relatively uniform in aqueous solution. The average particle size of HSP-Ⅱa is 281.57nm, and the PDI value is 0.25. The PDI value of HSP-Ⅱa is less than 1, indicating that the molecular weight distribution range of hemp seed polysaccharide HSP-Ⅱa is narrow and the molecular weight distribution uniformity is good.

[0132] The results of potential determination of HSP-Ⅱa of hemp seed polysaccharide are as follows Figure 12 As shown in the figure, the average zeta potential of HSP-Ⅱa is -30.9mV, with a relatively high absolute value, and the electrostatic repulsion between molecules is relatively large, which is closely related to its presence of uronic acid groups. It can be seen that hemp seed polysaccharide HSP-Ⅱa is relatively stable in the solution system and has a relatively low aggregation degree.

[0133] (3) X-ray diffraction

[0134] 10 mg of dried hemp seed polysaccharide sample was ground into powder using a mortar and pestle. The crystal properties of the hemp seed polysaccharide sample were analyzed using an XRD instrument. Measurement conditions: 40 kV, 40 mA, a Cu target, and a Kα radiation source with a continuous rapid scan from 10-80° 2θ.

[0135] The experimental results are as follows Figure 13 As shown in the figure, within the diffraction angle 2θ range of 10-80°, there is a broad and diffuse diffraction peak at around 22°, and the rest of the area is mostly amorphous, indicating that HSP-Ⅱa has certain crystalline characteristics, but the crystallinity is low, presenting in an amorphous state. HSP-Ⅱa has two relatively obvious diffraction peaks at 2θ = 28.35° and 40.56°, which are presumably caused by residual NaCl. The above results indicate that hemp seed polysaccharide HSP-Ⅱa has very few crystalline regions and is mainly amorphous, that is, it is a semi-crystalline polymer.

[0136] (4) Congo red test

[0137] 12.0mg hemp seed polysaccharide sample was dissolved in 12mL deionized water, and then 6mL of Congo red solution (Congo red concentration was 80μmol / L) was added. This mixture was called A solution. 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL and 0.5mL of 1M NaOH solution were taken in a test tube respectively, and the volume was made up to 1mL with A solution. After mixing, a full-band UV spectrometer was utilized to scan and record the maximum absorption wavelength (λmax) within the range of 400nm-600nm. The NaOH concentration was used as the horizontal coordinate and λmax as the vertical coordinate to draw a curve. Deionized water was used as a control and the above method was used to determine whether the polysaccharide sample had a triple helical structure.

[0138] The experimental results are shown in Figure 14 Compared to Congo red, the λmax of HSP-Ⅱa mixed with Congo red solution in aqueous and weakly alkaline solutions exhibited a significant red shift, indicating the formation of a polysaccharide-Congo red complex. Furthermore, within the NaOH concentration range of 0.1 mol / L to 0.5 mol / L, the λmax of the polysaccharide-Congo red complex exhibited a red shift, but the absorption wavelength did not change significantly, indicating a metastable state, indicating that HSP-Ⅱa possesses a relatively stable triple-helical structure.

[0139] (5) Field emission scanning electron microscopy

[0140] A small amount of dried hemp seed polysaccharide sample was attached to a sample holder covered with conductive tape and then plated with gold in an ion sputtering instrument to enhance its conductivity. Finally, observation parameters were adjusted under a scanning electron microscope until a clear, ideal field of view was achieved. Working conditions: The sample's surface morphology was observed and photographed at preselected magnifications of 100x, 1000x, and 5000x. The accelerating voltage was set to 15kV.

[0141] FE-SEM images of hemp seed polysaccharide at different magnifications Figure 15 As shown, HSP-Ⅱa primarily exhibits irregular flakes with a small amount of stacked strips, and its surface is relatively smooth and dense. The stacking gaps within the HSP-Ⅱa structure are relatively loose, a phenomenon likely related to intermolecular forces. The figure also shows that HSP-Ⅱa's surface exhibits essentially no crystalline structure, remaining mostly amorphous, consistent with the XRD results.

[0142] (6) Atomic force microscopy

[0143] A 1.0 mg / mL stock solution of HSP-Ⅱa, a polysaccharide sample from hemp seed, was dissolved in ultrapure water and magnetically stirred for 2 hours to fully dissolve. The stock solution was then diluted with ultrapure water to 20 μg / mL, 1 μg / mL, and 10 ng / mL, respectively. To reduce the presence of aggregates in the polysaccharide sample solution, each dilution was magnetically stirred at 50°C for at least 3 hours to thoroughly mix the solution. The 10 ng / mL dilution was cooled after stirring and filtered through a 0.45 μm needle filter to prepare the working solution for AFM measurements (the appropriate concentration was selected based on the sample being tested; the 10 ng / mL dilution was used as an example). 5 μL of nickel chloride solution (2 M nickel chloride concentration) was added dropwise to a mica sheet, allowed to adsorb for 30 seconds, and then rinsed with ultrapure water and dried. Subsequently, 20 μL of the hemp seed polysaccharide sample working solution was dripped onto the pretreated mica sheet. The sample was evenly distributed on the mica sheet by spin coating. To remove any unabsorbed residual sample, the sheet was rinsed multiple times with ultrapure water. Once completely dry, the hemp seed polysaccharide sample was scanned and measured using an AFM. Measurement conditions: a 100 μm scanner, a probe tip with a 10 nm radius of curvature, a tip made of Si, and a force constant of 2.8 N / m to ensure stability and accuracy during the measurement process. The tapping mode was selected for non-destructive scanning and high-resolution imaging of the sample.

[0144] Figure 16 A 2μm x 2μm two-dimensional and three-dimensional image of a 10 ng / mL aqueous solution of HSP-Ⅱa was obtained. The two-dimensional image of HSP-Ⅱa exhibited irregular spherical and partially aggregated particles, while the three-dimensional image revealed numerous cylindrical structures of varying heights. While the height of a single polysaccharide chain typically ranges from 0.1 nm to 1.0 nm, the height of HSP-Ⅱa sugar chains ranged from 1.0 nm to 10.5 nm. This suggests that sugar chain aggregation or entanglement may be caused by the intertwining of single or multiple chains of hemp seed polysaccharide through intermolecular hydrogen bonding, or by forces such as van der Waals forces. Furthermore, HSP-Ⅱa, which contains a high amount of uronic acid, exhibited electrostatic repulsion with the similarly negatively charged mica flakes, leading to more pronounced clustering of HSP-Ⅱa. These results indicate that HSP-Ⅱa is stable and not prone to aggregation, a finding that is consistent with the zeta potential measurements.

[0145] Example 6 Preliminary Study on the In Vitro Simulated Digestion Characteristics of Hemp Seed Polysaccharide

[0146] 6.1 In vitro simulated gastric fluid digestion characteristics

[0147] In this experiment, hemp seed polysaccharide HSP-Ⅱa was subjected to in vitro simulated gastric digestion using artificial gastric fluid, a method used for in vitro simulated digestion. An 8 mg / mL HSP-Ⅱa solution was prepared with deionized water. 12 mL of simulated gastric fluid and 12 mL of HSP-Ⅱa solution were mixed to create a simulated mixture. Deionized water was used as a blank control. The mixture was placed in a 37°C incubator (80 rpm) and slowly rotated to simulate gastric peristalsis. At reaction time points 0, 2, 4, and 6 h, 3 mL of the mixture was removed and immediately inactivated in a boiling water bath for 15 minutes.

[0148] 6.2 In vitro simulated intestinal digestion characteristics

[0149] In this experiment, hemp seed polysaccharide HSP-Ⅱa was subjected to in vitro simulated intestinal digestion using artificial intestinal fluid, a method used for in vitro simulated digestion experiments. The digestion product of the simulated gastric digestion (presumably 6 hours of uninactivated enzymes) and simulated intestinal fluid were added to create a simulated mixed solution. The volume ratio of the digestion product of the simulated gastric digestion (presumably 6 hours of uninactivated enzymes) to the simulated intestinal fluid was 10:3. The simulated mixed solution was placed in a 37°C constant temperature shaker (80 rpm) and slowly rotated to simulate intestinal peristalsis to perform the digestion reaction. At reaction time points 0, 2, 4, and 6 hours, 3 mL of the mixture was removed and immediately placed in a boiling water bath for inactivation.

[0150] 6.3 Determination of in vitro simulated digestion characteristics of hemp seed polysaccharides

[0151] (1) Determination of polysaccharide molecular weight

[0152] ① Polysaccharide digestion fluid treatment

[0153] 1.0 mL of digestion product at different time points after in vitro simulated gastrointestinal fluid digestion was taken, dialyzed with water for 24 h using a 1000 Da dialysis bag, and freeze-dried. The freeze-dried polysaccharide digestion solution was prepared into 1 mg / mL and passed through a 0.22 μm aqueous filter membrane, and its molecular weight was determined using HPGPC.

[0154] ②Chromatographic conditions

[0155] Gel chromatography column: TSK G4000PWXL (7.8×300 mm); detector: evaporative light scattering detector; dextran standard concentration range: 4 kDa-2000 kDa, calibration curve, calculation of the molecular weight of the polysaccharide digest.

[0156] According to the HPGPC test results, the standard curve equation is Y=-2.3038X+25.654, R 2= 0.9966, x is time (min), y is the logarithm of molecular weight, and the molecular weight of undigested HSP-Ⅱa, blank gastrointestinal fluid, and each polysaccharide digestion fluid was calculated. The molecular weight (Mw) changes of HSP-Ⅱa digestion products at different time points in simulated gastric fluid and simulated intestinal fluid are shown as follows: Figure 17 a. Figure 17 b and shown in Table 6.

[0157] Figure 17 Table 6 and Figure 5 show that after HSP-Ⅱa digestion with simulated gastric fluid, the product elution time increased with increasing digestion time. At 2 h, the Mw of the digestion product decreased from (134.03 ± 0.08) kDa to (133.43 ± 0.01) kDa. With continued digestion for 6 h, the elution time of the digestion product significantly increased, and the Mw decreased to (125.68 ± 0.18) kDa, indicating that simulated gastric fluid has a certain degradation effect on HSP-Ⅱa. Figure 17 b and Table 6 show that the peak time of HSP-Ⅱa was prolonged after digestion with simulated intestinal fluid, and the peak time of the product also increased significantly with increasing digestion reaction time. The Mw of the digestion product at 0 and 2 h was (124.69 ± 0.91) kDa and (123.42 ± 0.13) kDa, respectively, with no significant difference. After continued digestion for 6 h, the Mw decreased significantly to (113.54 ± 0.29) kDa. This further decrease in the molecular weight of the polysaccharide digestion product indicates that simulated intestinal fluid also plays a significant role in the degradation of HSP-Ⅱa.

[0158] After HSP-Ⅱa was digested in simulated gastrointestinal fluid, the molecular weight of the digestion product decreased by about 20.49kDa, indicating that the gastrointestinal tract has a certain effect on the digestion, absorption and biological activity of high-molecular-weight polysaccharide compounds.

[0159] Table 6 Changes in molecular weight of HSP-Ⅱa after in vitro simulated gastrointestinal digestion

[0160]

[0161] (2) Determination of free monosaccharides in polysaccharides

[0162] Polysaccharide Digestion Solution Treatment: 1.0 mL of digestion product was collected at different time points after in vitro simulated gastrointestinal digestion. After nitrogen purging, the product was directly derivatized without hydrolysis. The composition of free monosaccharides after digestion was determined by high-performance liquid chromatography (HPLC) after filtering through a 0.22 μm aqueous filter. Based on the monosaccharide composition of HSP-Ⅱa, 3.0 mg of each of the seven monosaccharides, Rha, GlcA, GalA, Glc, Gal, Xyl, and Ara, was accurately weighed as a monosaccharide mixed standard. This was also derivatized and analyzed by HPLC.

[0163] Derivatization method: Use 3 mL of PMP-methanol solution (prepared with methanol, the final PMP concentration is 0.5 M) and 2.5 mL of sodium hydroxide solution (sodium hydroxide concentration is 0.3 M) to derivatize at 70°C for 100 minutes, then add 2.5 mL of 0.3 M hydrochloric acid to neutralize to obtain a mixture. Take 3 times the volume of chloroform to extract 3-4 times, retain the aqueous phase, filter through a 0.22 needle filter membrane, and detect and analyze using an Agilent 1100 HPLC.

[0164] Chromatographic conditions: chromatographic column C18 column (250 mm × 4.6 mm, 5 mm); detector: UV detector; mobile phase: acetonitrile-0.02 M ammonium acetate solution (acetonitrile and 0.02 M ammonium acetate solution volume ratio 15:85), flow rate: 1 mL / min, temperature: 25°C.

[0165] According to the HPLC determination results, by comparing with mixed standards and blank gastrointestinal fluid, the free monosaccharide production of HSP-Ⅱa digestion products at different time points in simulated gastrointestinal fluid is as follows: Figure 18 、 Figure 19 shown.

[0166] Figure 18 The two-dimensional plane diagram and three-dimensional stereogram show the changes in free monosaccharides of HSP-Ⅱa in simulated gastric fluid, which are compared with mixed standards and blank artificial gastric fluid. It can be seen that during the 0-2h digestion process of simulated gastric fluid, except for the PMP solvent peak, almost no other suspected characteristic peaks appear; after 4h and 6h of continuous digestion, the three-dimensional stereogram clearly shows that in addition to the solvent peak, two weaker chromatographic peaks that are suspected to be consistent with the peak times of glucose and arabinose in the mixed standard appear at both time points, which is speculated that monosaccharides may be released during this process; and after 6h of simulated intestinal fluid digestion, Figure 19 The two-dimensional and three-dimensional plots show the changes in free monosaccharides in HSP-Ⅱa in simulated intestinal fluid, compared with mixed standards and blank artificial intestinal fluid. In addition to the PMP solvent peak at each time point, two weak peaks with elution times consistent with those of glucose and arabinose in the mixed standard were also observed, suggesting that a small amount of monosaccharide molecules were continuously released during this process. Furthermore, a strong chromatographic peak was generated at each time point during the digestion process. Its elution time coincided with that of an unknown compound in the simulated intestinal fluid, and there was no corresponding monosaccharide standard. This strong peak is suspected to be an unknown compound in the intestinal fluid.

[0167] These results indicate that HSP-Ⅱa produces free monosaccharides during simulated gastrointestinal digestion, with its response intensity gradually increasing from gastric to intestinal digestion, consistent with the molecular weight trend previously described. Furthermore, the characteristic peaks produced by HSP-Ⅱa elute at approximately 38 and 50 minutes, corresponding to the elute times of glucose and arabinose in the mixed standard. It is speculated that HSP-Ⅱa may be partially degraded during gastrointestinal digestion, resulting in the release of small amounts of glucose and arabinose.

[0168] (3) Determination of total sugar and reducing sugar content of polysaccharides

[0169] 1.0 mL of digestion products at different time points after in vitro simulated gastric juice digestion were taken respectively. Using glucose as the standard, the phenol-sulfuric acid method and dinitrosalicylic acid (DNS) colorimetric method were used to detect the changes in total sugar and reducing sugar content in the digestive fluid during the simulated digestion process.

[0170] ① Prepare DNS solution: add 0.25g NaOH and 5g potassium sodium tartrate in sequence and add appropriate amount of deionized water to completely dissolve. Add 0.25g DNS to completely dissolve in the above solution, then add 0.05g redistilled phenol and 0.0075g anhydrous sodium sulfite to completely dissolve. Finally, make up to the volume to 50mL brown volumetric bottle and store in a refrigerator at 4℃ away from light for at least one week before use. The longer the storage time, the more stable it is.

[0171] ②Dinitrosalicylic acid (DNS) colorimetric method

[0172] Prepare glucose standard solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL using deionized water. Add the prepared DNS solution to the glucose standard solution at a 1:1 volume ratio. Boil in a boiling water bath for 15 minutes, cool, and dilute with 5 times the volume of deionized water. Measure the absorbance at 540 nm and plot a standard curve. Measure the absorbance of the polysaccharide digest using the same method, and calculate the reducing sugar content of the polysaccharide digest using the regression equation of the standard curve.

[0173] Gastrointestinal digestion can break the glycosidic bonds of polysaccharides and degrade their structure, resulting in a decrease in the total sugar content of polysaccharides and the production of some reducing sugars. Therefore, changes in the total sugar and reducing sugar content can intuitively reflect the digestion characteristics of polysaccharides. Figure 20 a. Figure 20 b and Table 7.

[0174] Figure 20As shown in Table 7, the total sugar content of HSP-Ⅱa decreased from (41.76±0.78) mg / mL to (37.77±0.15) mg / mL during digestion in simulated gastric fluid for 0-6 h, showing no significant change. The total sugar content of HSP-Ⅱa digested in simulated gastric fluid decreased slightly to (35.66±0.54) mg / mL after 2 h of digestion in intestinal fluid. After 4-6 h of digestion, the total sugar content decreased to (25.88±0.39) mg / mL, demonstrating a significant difference (P<0.05). The total sugar content of HSP-Ⅱa decreased significantly during digestion in simulated gastrointestinal fluid, indicating that simulated gastrointestinal fluid has a certain degradative effect on HSP-Ⅱa.

[0175] Figure 20 b and Table 7 further show that during the simulated gastric digestion of HSP-Ⅱa polysaccharide, the reducing sugar content of HSP-Ⅱa did not change significantly from 0 to 4 h. However, it increased significantly to (0.60±0.01) mg / mL from 4 to 6 h, with a significant difference (P < 0.01), indicating that HSP-Ⅱa polysaccharide was gradually degraded by gastric juice. The HSP-Ⅱa digestion products continued to digest in simulated intestinal fluid. The reducing sugar content of HSP-Ⅱa did not change significantly from 0 to 2 h, but increased significantly to (0.98±0.02) mg / mL from 2 to 6 h, with a significant difference (P < 0.01). The reducing sugar content of HSP-Ⅱa increased significantly during the simulated gastrointestinal digestion, further demonstrating that simulated gastrointestinal fluid has a certain degrading effect on HSP-Ⅱa.

[0176] Table 7 Changes in total sugar and reducing sugar content during HSP-Ⅱa digestion in gastrointestinal fluid

[0177]

[0178] Example 7 In vitro hypoglycemic activity of hemp seed polysaccharide

[0179] 7.1 Inhibitory effect of hemp seed polysaccharide on α-glucosidase

[0180] (1) Preparation of reagents

[0181] 1U / mL α-glucosidase stock solution: Dissolve α-glucosidase in 0.1mol / L PBS buffer (pH 6.8) to a concentration of 1U / mL. Store this stock solution at -20°C.

[0182] 25 mmol / L substrate (PNPG) solution: Accurately weigh 0.3765 g of PNPG and completely dissolve it in a 50 mL volumetric flask using 0.1 mol / L PBS buffer. Dose to the mark.

[0183] 0.1 mol / L Na2CO3 solution: Accurately weigh 0.53 g of Na2CO3 powder, add deionized water and dissolve it in a 50 mL volumetric flask, dilute to the mark (50 mL), and store at 4°C until use.

[0184] Hemp seed polysaccharide solution: Accurately weigh the hemp seed polysaccharide HSP-Ⅱa sample and use deionized water to prepare solutions with concentrations of 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL, respectively, for later use.

[0185] (2) Reaction system

[0186] This experiment included a drug-response group, a blank-response group, a drug-control group, and a blank-control group, with three replicates per group. The corresponding doses were added to a 5 mL centrifuge tube according to Table 8 below, with 0.1 mol / L PBS buffer as the blank solution. First, PBS buffer, hemp seed polysaccharide solution, α-glucosidase stock solution, and distilled water were mixed and reacted at 37°C for 15 minutes before removal. PNPG solution was added and the reaction continued at 37°C for 25 minutes. Finally, Na2CO3 solution was added to terminate the reaction, and the absorbance was measured at 405 nm.

[0187] Table 8 α-glucosidase inhibition test reagent addition table

[0188]

[0189] According to the test results, the inhibition rate of hemp seed polysaccharide HSP-Ⅱa on α-glucosidase was calculated according to the following formula:

[0190] Inhibition rate (%) = (A 空 -A 样 ) / A 空 ×100%

[0191] A 空 =A 空白反应组 -A 空白对照组

[0192] A 样 =A 药物反应组 -A 药物对照组

[0193] The results are as follows Figure 21 .Depend on Figure 21It can be seen that as the concentration of HSP-Ⅱa continues to increase (0.25mg / mL-4mg / mL), its inhibitory effect on α-glucosidase becomes more and more significant, and the inhibition rate gradually increases, showing a good dose-effect relationship and concentration dependence. At 4mg / mL, the inhibitory effect is the largest, at (79.78±2.40)%, IC 50 The value is 1.25 mg / mL.

[0194] 7.2 Inhibitory effect of hemp seed polysaccharide on α-amylase

[0195] (1) Preparation of reagents

[0196] 1% starch solution: Accurately weigh 0.5 g of soluble starch, add 0.1 mol / L PBS buffer and heat to fully dissolve it. Cool to room temperature and dilute to the mark in a 50 mL volumetric flask.

[0197] 0.1U / mL α-amylase solution: Accurately weigh α-amylase powder and add 0.1mol / L PBS buffer to prepare the final concentration of α-amylase at 0.1U / mL.

[0198] Hemp seed polysaccharide solution: Accurately weigh the hemp seed polysaccharide HSP-Ⅱa sample and use deionized water to prepare solutions with concentrations of 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL, respectively, and set aside.

[0199] (2) Reaction system

[0200] This experiment also set up a drug reaction group, a blank reaction group, a blank control group, and a drug control group, with three replicates in each group. Add the dosage according to Table 9 to a 5mL centrifuge tube, and use 0.1mol / L PBS buffer as the blank solution. First, add α-amylase solution, hemp seed polysaccharide solution, distilled water, and PBS buffer, and incubate at 37℃ for 10 minutes; then add starch solution, mix well, and incubate at 37℃ for 10 minutes; finally, add the prepared DNS reagent, boil in water for 5 minutes, remove, cool, and dilute 25 times with deionized water. Mix well and add the sample to a 96-well plate, 100μL per well, and measure the absorbance at 540nm.

[0201] Table 9 α-amylase inhibition test reagent addition table

[0202]

[0203] The inhibitory effect of hemp seed polysaccharide HSP-Ⅱa on α-amylase was analyzed based on the test results. The inhibition rate is shown in the following formula:

[0204] Inhibition rate (%) = (A 空 -A样 ) / A 空 ×100%

[0205] A 空 =A 空白反应组 -A 空白对照组

[0206] A 样 =A 药物反应组 -A 药物对照组

[0207] See the results Figure 22 It can be seen that HSP-Ⅱa also has a certain inhibitory effect on α-amylase. With the gradual increase of HSP-Ⅱa polysaccharide concentration (0.25mg / mL-4mg / mL), the inhibition rate of HSP-Ⅱa on α-amylase gradually increases, with a good dose-effect relationship and an obvious concentration-dependent relationship. When the concentration is 4.0mg / mL, the inhibition rate of HSP-Ⅱa on α-amylase is the highest, which is (88.08±2.42)%, and the IC of HSP-Ⅱa on α-amylase is 50 The value is 1.93 mg / mL.

[0208] 7.3 Study on the hypoglycemic activity of hemp seed polysaccharide on IR-HepG2 cells in vitro

[0209] (1) HepG2 cell recovery and culture

[0210] After removing the cell cryopreservation tube from the liquid nitrogen tube, gently shake and thaw it in a 37°C water bath, disinfect the surface of the cryopreservation tube with 75% alcohol, and then place it in an ultra-clean workbench; open the cryopreservation tube cap, gently blow the cell suspension inside with a pipette to completely suspend it in the solution to avoid cell loss, transfer the cell suspension to a 15mL centrifuge tube, and add 5 times the volume of complete culture medium of the cell suspension; centrifuge at 1000rpm / min for 5min; aspirate the supernatant, add 1mL of complete culture medium to resuspend the cell pellet at the bottom of the centrifuge tube, and blow evenly; transfer the cell solution to a cell culture flask and add 5mL of complete culture medium; finally, place the culture flask in a 37°C, 5% CO2 incubator and let it stand until the cells adhere to the wall and grow.

[0211] After the cells were grown to the logarithmic growth phase in the incubator, the supernatant was discarded; the cells were washed with phosphate-buffered saline (PBS) solution, 2 mL of 0.25% trypsin was added, and the cells were reacted in the incubator for 3 minutes. Then, 2 volumes of complete culture medium (containing 4.5 g / L D-glucose) were quickly added to terminate the trypsin reaction. The cells were centrifuged at 1000 rpm / min for 5 minutes, and the supernatant was discarded; 1 mL of complete culture medium was added to resuspend the cells and evenly pipetted; 300-400 μL of the cell suspension was inoculated into a culture flask containing 6 mL of complete culture medium; the cells were placed in a 37°C, 5% CO2 incubator again, and after the cells attached to the wall, the cells in the logarithmic growth phase were collected for subsequent experiments.

[0212] (2) Establishment of IR-HepG2 cell model

[0213] This experiment will use an in vitro insulin induction method to establish an insulin resistance model. By adding specific concentrations of insulin to the cell culture medium, the in vivo insulin resistance environment will be simulated. Preliminary experiments will screen the optimal concentration and duration of insulin action on HepG2 cells to establish the IR model. A glucose kit will be used to measure changes in glucose consumption in each cell group to determine whether the polysaccharide has hypoglycemic activity.

[0214] ①Effect of insulin concentration on glucose consumption in HepG2 cells

[0215] HepG2 cells in the logarithmic growth phase were cultured at a rate of 1×10 5 The cells / mL were counted and inoculated into 96-well plates, 100 μL was taken from each well and cultured for 24 h.

[0216] Blank group: Take 100 μL of serum-free 1640 culture medium, plate 6 replicates per group, and incubate in an incubator for 48 h; take 10 μL of supernatant from each well and add it to a 48-well plate (each well of the 48-well plate contains 1 mL of glucose detection solution), that is, 10 μL of serum-free 1640 culture medium + 1 mL of glucose detection solution.

[0217] Experimental group: 100 μL of serum-free 1640 insulin solution was taken, and the insulin concentration in the insulin solution was 1×10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 mol / L, 6 replicate wells per group; incubate in an incubator for 48 hours; take 10 μL of supernatant from each well and add it to a 48-well plate (each well of the 48-well plate contains 1 mL of glucose detection solution); 10 μL of serum-free 1640 prepared insulin solution + 1 mL of glucose detection solution.

[0218] Calibration group: 10 μL calibration solution + 1 mL glucose detection solution.

[0219] Each group was cultured in a 37°C incubator for 10 minutes, shaken evenly at low speed on a shaker, and the absorbance was measured at 505 nm. The glucose content and glucose consumption in the culture medium were calculated and analyzed according to the following formula.

[0220] Glucose content (mmol / L) = absorbance of test group / absorbance of calibration group × concentration of calibration solution

[0221] Glucose consumption = glucose content of cells in the blank group - glucose content of cells in the experimental group

[0222] Calibration solution concentration = 5.55mmol / L

[0223] ②Effect of insulin action time on glucose consumption in HepG2 cells

[0224] After determining the optimal concentration of insulin, the cells were divided into a blank group and a test group, with 6 replicates in each group. 5 The cell suspension was inoculated into 96-well plates, 100 μL per well, and placed in an incubator to wait for the cells to adhere to the wall. The blank group was supplemented with 100 μL of serum-free 1640 culture medium, and the experimental group was supplemented with 1×10 -7 mol / L insulin solution, and cultured for 24h, 36h, 48h, 60h, and 72h respectively; the glucose content at each time was measured and the glucose consumption was calculated. The detection method was the same as above, and the results of the cell activity test were combined to determine the optimal conditions for establishing the IR model.

[0225] The results are as follows Figure 23 As shown in the figure: Under the same insulin concentration conditions, the glucose consumption in cells will increase with the increase of action time; when the insulin concentration is 1×10 -9 -1×10 -8 mol / L, glucose consumption increases over time, which indicates that low concentrations of insulin have the effect of promoting cellular glucose metabolism. -8 mol / L, the glucose consumption first showed a downward trend, and then slightly rebounded. -7 mol / L, the glucose consumption of cells at different time points dropped to the lowest point, especially when the action time was 24 hours, the glucose consumption reached the lowest value, and there was a significant difference compared with the blank group. This shows that high concentrations of insulin can induce cells to establish a significant IR model. Comprehensive considerations show that the optimal insulin concentration for establishing the IR model is 1×10 -7 mol / L, action time is 24h.

[0226] (3) Effect of hemp seed polysaccharide on glucose consumption in IR-HepG2 cells

[0227] Through preliminary experiments, it has been screened that the safe dosage concentration of hemp seed polysaccharide HSP-Ⅱa for normal HepG2 cells is less than 640μg / mL, so this experiment selected 40μg / mL, 80μg / mL, and 160μg / mL as low, medium, and high dosages for experiments.

[0228] By screening the optimal insulin concentration and time, a cell model was successfully constructed and divided into a blank group, a model group, and groups treated with HSP-Ⅱa at various doses.

[0229] Blank group: 100 μL 1640 culture medium;

[0230] Model group: 100 μL insulin solution, the insulin concentration in the insulin solution was 1×10 -7 mol / L;

[0231] Administration group: 50 μL insulin solution + 50 μL HSP-Ⅱa solution (final concentrations were 40, 80, and 160 μg / mL, respectively).

[0232] Cells in the logarithmic growth phase were made into a suspension and the cells were cultured at a rate of 1×10 5 At a cell density of 100 μL / well, cells were seeded into 96-well plates. The culture medium was grouped as described above, with six wells per group. After 24 hours of culture, the supernatant was discarded. 10 μL of supernatant from each well was assayed using a glucose kit and glucose consumption was calculated for each group. The values ​​were then compared and analyzed.

[0233] The results are as follows Figure 24 As shown in the results: compared with the cells in the blank group, the glucose consumption in the model group was extremely significantly reduced, indicating that the IR-HepG2 model has been successfully established; compared with the model group, HSP-Ⅱa at 80-160μg / mL had a significant promoting effect on the glucose consumption of cells, and the difference was extremely significant (P < 0.01); among them, the promoting effect was the best when the concentration was 320μg / mL, and the increase was 63.96% compared with the model group, showing an extremely significant difference (P < 0.01).

[0234] The above results indicate that hemp seed polysaccharide HSP-Ⅱa can significantly enhance the glucose consumption capacity of IR-HepG2 cells, improve the insulin resistance state, and thus exert hypoglycemic activity.

[0235] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing hemp seed polysaccharide, characterized in that: The following steps are involved: (1) Using hemp seed meal as raw material, the crude hemp seed polysaccharide HSP was extracted by water extraction and alcohol precipitation; the final concentration of ethanol in the alcohol precipitation was 80%; (2) After removing protein from the crude polysaccharide of hemp seed, it was separated by DEAE-52 cellulose ion exchange column chromatography, and gradient eluted with 0.1 M NaCl solution, 0.2 M NaCl solution, and 0.3 M NaCl solution in sequence. The fraction eluted with 0.3 M NaCl solution was collected, which was HSP-Ⅱ; (3) Separate HSP-Ⅱ by gel column chromatography to obtain HSP-Ⅱa.

2. The method for preparing hemp seed polysaccharide according to claim 1, wherein In step (1), the crude polysaccharide of hemp seed is extracted by water extraction and alcohol precipitation method, which comprises the following steps: mixing the defatted hemp seed meal with water, decocting, cooling, centrifuging, taking the supernatant, collecting the extract, filtering, concentrating, and obtaining a concentrated product; precipitating the concentrated product with an ethanol solution, centrifuging, collecting the precipitate, evaporating the ethanol, concentrating, and freeze-drying.

3. The method for preparing hemp seed polysaccharide according to claim 2, wherein The material-liquid ratio of defatted hemp seed meal to water is 1 g:15 mL.

4. The method for preparing hemp seed polysaccharide according to any one of claims 1 to 3, wherein In step (2), the Sevage method is used to remove protein.

5. The method for preparing hemp seed polysaccharide according to claim 4, wherein The protein is removed by the Sevage method, which comprises the following steps: mixing a solution of hemp seed crude polysaccharide HSP, chloroform and n-butanol, shaking and then centrifuging, taking the supernatant, dialyzing, concentrating and freeze-drying.

6. The method for preparing hemp seed polysaccharide according to claim 4, wherein The separation by DEAE-52 cellulose ion exchange column chromatography includes the following steps: using a DEAE-52 cellulose ion exchange column chromatography to separate the aqueous solution of hemp seed polysaccharide obtained by removing protein by the Sevage method, using a sodium chloride solution as an eluent, collecting the components eluted by a 0.3 M NaCl solution, dialyzing, concentrating, and freeze-drying to obtain HSP-Ⅱ.

7. The method for preparing hemp seed polysaccharide according to any one of claims 1 to 3, wherein In step (3), HSP-Ⅱ is separated by gel column chromatography, which includes the following steps: preparing an HSP-Ⅱ aqueous solution, separating it by Sephacryl S-300 gel column chromatography, eluting it with distilled water at a flow rate of 1 mL / 2 min, collecting the elution peak, and freeze-drying it to obtain HSP-Ⅱa.

8. A hemp seed polysaccharide, characterized in that The hemp seed polysaccharide is prepared by the method according to any one of claims 1 to 7.

9. Use of the hemp seed polysaccharide according to claim 8 in the preparation of a blood sugar-lowering product.

10. The use according to claim 9, characterized in that: The application is achieved by increasing the glucose consumption capacity of insulin-resistant cells through hemp seed polysaccharide.

Images