Preparation method and application of fructus cannabis polysaccharide

Abstract

The invention relates to a preparation method and application of fructus cannabis polysaccharide. The preparation method of the fructus cannabis polysaccharide comprises the following steps: (1) taking fructus cannabis meal as a raw material, and extracting crude fructus cannabis polysaccharide HSP by adopting a water extraction and alcohol precipitation method; (2) deproteinizing the crude polysaccharide of the fructus cannabis, and performing chromatographic separation by adopting an ion exchange column to obtain HSP-II; and (3) carrying out gel column chromatography separation on the HSP-II to obtain the HSP-IIa. The fructus cannabis polysaccharide prepared by the invention can be used for reducing blood sugar and improving the glucose consumption capability 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 of hemp seed polysaccharide and application thereof. Background Art

[0002] Hemp seed is the dried mature seed of Cannabis sativa L., a plant of the Moraceae family. It is mainly produced in the three northeastern provinces, South China, North China and other places. It is one of the "Long Jiuwei" authentic medicinal materials in Heilongjiang Province. It is a traditional medicinal and edible plant in my country with extremely high medicinal and nutritional value. It is flat in nature and sweet in taste. It enters the spleen, stomach, and large intestine meridians. It has the effects of moistening the intestines and relieving constipation, nourishing and replenishing deficiency, and promoting blood circulation. Pharmacological studies have shown that hemp seed has good pharmacological activities such as lowering blood sugar, lowering blood lipids, lowering blood pressure, anti-oxidation, anti-tumor and immunomodulatory. Carbohydrates in hemp seed can reach 30% (including polysaccharides). Hemp seed meal is a waste product of hemp seed oil extraction. Reusing its resources can greatly increase the added value of hemp seed. Different extraction methods produce different products, which will further affect the application of hemp seed active products. At present, there is no application of hemp seed polysaccharides in lowering blood sugar. 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 application thereof.

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

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

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

[0007] (2) After removing protein from crude polysaccharide of hemp seed, the polysaccharide is separated by ion exchange column chromatography to obtain HSP-Ⅱ;

[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, 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 by using an ethanol solution, centrifuging, collecting the precipitate, evaporating the ethanol, concentrating, and freeze-drying.

[0010] Furthermore, the material-liquid ratio of 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 Sevage method is used to remove proteins, and DEAE-52 cellulose ion exchange column chromatography is used for separation.

[0012] Furthermore, the Sevage method is used to remove protein, which includes the following steps: mixing the solution of hemp seed crude polysaccharide HSP, chloroform and n-butanol, centrifuging after shaking, 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 by Sephacryl S-300 gel column chromatography, eluting 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 above-mentioned hemp seed polysaccharide in improving the glucose consumption capacity of insulin-resistant cells. For example, the use of the above-mentioned hemp seed polysaccharide in preparing a product that improves the glucose consumption capacity.

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

[0019] The invention extracts, separates and purifies the hemp seed meal to obtain a uniform polysaccharide HSP-Ⅱa. The HSP-Ⅱa is an acidic polysaccharide with a primary structure 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. The HSP-Ⅱa can be partially digested and degraded in an in vitro simulated gastrointestinal digestive fluid, exhibits good hypoglycemic activity, and can be 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 is the standard curve (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 standard.

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

[0029] Fig.10 TG-DSC analysis of hemp seed polysaccharide.

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

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

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

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

[0034] Fig.15 This is the scanning electron microscope image of hemp seed polysaccharide HSP-Ⅱa, from left to right are ×100, ×1000, and ×5000.

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

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

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

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

[0039] Fig. 20 The changes of total sugar and reducing sugar contents in HSP-Ⅱa during digestion by gastrointestinal fluid (a: total sugar; b: reducing sugar).

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

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

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

[0043] Fig.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 in conjunction with 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, simultaneously investigates the in vitro simulated digestion characteristics of hemp seed polysaccharide, and evaluates its in vitro hypoglycemic activity.

[0046] The invention adopts a water extraction and alcohol precipitation method to extract hemp seed polysaccharide (HSP), wherein the total sugar content of the 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, and then a DEAE-52 anion exchange column chromatography method and a Sephacryl S-300 gel column chromatography method are used for separation and purification to obtain high-purity hemp seed polysaccharide HSP-Ⅱa.

[0047] The primary structure of HSP-Ⅱa was analyzed by high performance gel permeation chromatography, ion chromatography, infrared spectroscopy, and nuclear magnetic resonance spectroscopy; the advanced structure of HSP-Ⅱa was characterized by thermal property detection, particle size and Zeta potential analysis, X-ray diffraction (XRD), Congo red experiment, field emission scanning electron microscope (FE-SEM), atomic force microscope (AFM), etc. HSP-Ⅱa, the hemp seed polysaccharide component, is a homogeneous polysaccharide with good purity. The total sugar content of HSP-Ⅱa is (57.37±1.89)%, the uronic acid content is (38.87±2.38)%, and the weight average molecular weight (Mw) is 137.01kDa. It is mainly composed of monosaccharides such as rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid. The relative molar ratio is 1.9:6.3:6.1:1.6:2.3:5.5:1.0. It is an acidic polysaccharide mainly composed of →4)-α-GalA-(1→ and →4)-β-Ara-(1→. Thermal property detection shows that HSP-Ⅱa has good thermal stability at 160℃. The results of particle size and potential analysis show that the average particle size of HSP-Ⅱa is 281.57nm, the PDI value is 0.25, and the average Zeta potential is -3 0.9mV, the mass distribution range of HSP-Ⅱa is narrow, the molecular mass distribution is uniform, and HSP-Ⅱa can exist stably in the solution system. The results of XRD show that HSP-Ⅱa is mainly a semi-crystalline polymer with both crystalline and amorphous forms. Congo red experiment shows that HSP-Ⅱa has a relatively stable triple helical structure. FE-SEM observation found that HSP-Ⅱa mainly presents irregular flakes with a small amount of strip-like structure stacking, and the surface is smooth. AFM observation found that HSP-Ⅱa has aggregation or entanglement of sugar chains in liquid state. Its two-dimensional plane image mainly presents irregular spherical and partially aggregated particle structures. The three-dimensional stereoscopic image shows that there are many high and low cylindrical structures. The aggregation density of HSP-Ⅱa is low, and it is more stable in aqueous solution and not easy to aggregate.

[0048] The acidic polysaccharide HSP-Ⅱa, which has a relatively small particle size and can exist relatively stably in solution, was further used as a research object for the in vitro simulated digestion characteristics and hypoglycemic activity of this experiment.

[0049] An in vitro simulated gastrointestinal digestion model was established, and the molecular weight, free monosaccharides, total sugar content and reducing sugar content of the digestion products of hemp seed polysaccharide HSP-Ⅱa were used as detection indicators to investigate the digestion characteristics of HSP-Ⅱa in artificial gastric juice and intestinal juice, providing a reference for its further activity research. The results showed that the molecular weight and total sugar content of the HSP-Ⅱa digestion products decreased with the extension of the simulated gastrointestinal digestion time, and the partial cleavage of glycosidic bonds led to the release of a small amount of free monosaccharides and an increase in reducing sugar content. This shows that the sugar chain structure of hemp seed polysaccharide HSP-Ⅱa will be partially degraded after in vitro simulated gastrointestinal digestion, suggesting that gastrointestinal digestion may have a certain effect on the structure and properties of HSP-Ⅱa.

[0050] The in vitro hypoglycemic activity of HSP-Ⅱa was verified by testing the inhibitory activity of HSP-Ⅱa on two carbohydrate hydrolases, α-glucosidase and α-amylase, establishing an insulin resistance-HepG2 (IR-HepG2) cell model, and detecting the glucose consumption ability of HSP-Ⅱa on IR-HepG2 cells. The results of the activity detection of HSP-Ⅱa on α-glucosidase and α-amylase showed that HSP-Ⅱa had a certain inhibitory effect on the activities of these two carbohydrate hydrolases, and HSP-Ⅱa could significantly improve the glucose consumption ability of IR-HepG2 cells, preliminarily revealing that HSP-Ⅱa has significant in vitro hypoglycemic activity.

[0051] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods in the art; the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, and can be obtained through commercial channels or prepared by conventional methods by those skilled in the art. The solutions involved in the present invention, unless otherwise specified, 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 are sent to a low-temperature press after sorting and cleaning, and the low-temperature pressing pressure is controlled at 2.5-4.5 MPa and the temperature is controlled at 60-70° C. After low-temperature pressing, the defatted hemp seed meal is dried at 105° 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-Toda Technology Co., Ltd.; dextran gel S-300 was purchased from Beijing Solebow Technology Co., Ltd.; BCA protein quantification kit was purchased from Beyotime Biotechnology Co., Ltd.; dextran standard was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; monosaccharide standard was purchased from Boruitang Biotechnology Co., Ltd.; simulated gastric fluid was purchased from Shanghai Regen Biotechnology Co., Ltd.; artificial intestinal fluid was purchased from Shanghai Regen 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-Ausin Biotechnology Co., Ltd.; PNPG was purchased from Beijing Bio-Toda 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 Solebow Technology Co., Ltd.; insulin was purchased from Beijing Solebow Technology Co., Ltd.; and 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 crude polysaccharides from hemp seeds

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

[0058] (2) Extract the residue part 1-2 times in the same way as step (1), combine the extracts, filter, and use a rotary evaporator to reduce pressure and concentrate the filtrate to 1 / 10 of the volume before the reduced pressure concentration at 75°C 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 stand for 12 hours, then centrifuge at 4500r / min for 15 minutes, collect the precipitate, and freeze-dry it for use, so as to obtain the hemp seed crude 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 in a ratio of crude polysaccharide solution: chloroform: n-butanol = 25:5:1 (volume ratio), fully shake for 15 min-20 min, centrifuge at 4500 r / min for 15 min, remove the white protein layer, and take the supernatant. Repeat the above method 6-7 times until the white protein layer is no longer visible, dialyze with water using a 3500Da dialysis bag for 24 h, and concentrate the dialysate under reduced pressure using a rotary evaporator and freeze-dry.

[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, wash it repeatedly with deionized water until it is neutral, and 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, and finally degas it by vacuum pressure for use.

[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 the bubbles at the bottom, slowly stir the activated DEAE-52 clockwise, and pour it into the column with a glass rod. After the DEAE-52 is 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 deproteinized and obtained 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 NaCl solution (0.1mol / L, 0.2mol / L, 0.3mol / L, 0.4mol / L, 0.5mol / L) of different concentrations successively, the volume of the NaCl solution of every kind of concentration is 2 times of chromatography column volumes, control flow velocity 1mL / min, collect eluent by every pipe 2mL.Track and detect every pipe eluent with phenol-sulfuric acid method (with reference to embodiment 2.1), measure its absorbance, take tube number as abscissa, absorbance as ordinate, draw elution curve. The eluates obtained by eluting with NaCl solutions of different concentrations were collected respectively, and the eluates were dialyzed with water using a dialysis bag with a molecular weight cutoff of 3000Da. The dialysate was concentrated under reduced pressure using a rotary evaporator and lyophilized for later use. The results showed that the component eluted with 0.3M NaCl solution had a higher sugar content and better 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 make it fully swell, and then degas it by vacuuming for later 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, slowly pour it into the chromatography column, and balance it with deionized water after the S-300 is fully settled.

[0068] Prepare 10mg / mL HSP-Ⅱ polysaccharide solution with water, add the polysaccharide solution evenly along the tube wall to the Sephacryl S-300 gel column, elute 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 in Figure 1 As shown, it can be seen that the elution curves all have only one single symmetrical peak, and the purity is good. The eluate is enriched, concentrated under reduced pressure using a rotary evaporator, and freeze-dried for later use. The obtained hemp seed polysaccharide is named HSP-Ⅱa.

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

[0070] 2.1 Phenol-sulfuric acid method to measure total sugar content

[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 to detect protein content

[0074] (1) Drawing a standard curve: Prepare a protein standard (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 standard 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, and draw a standard curve with the concentration of the protein standard 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: 0.244 g of sodium tetraborate is weighed and fixed to volume in a 50 mL volumetric flask (prepared with concentrated sulfuric acid).

[0080] (2) Drawing a standard curve: The galacturonic acid standard solution was prepared into standard solutions with concentrations of 1.0, 0.8, 0.6, 0.4, 0.2, and 0 mg / mL, and the volume was fixed to 1 mL. Then, 6 mL of the sodium tetraborate / sulfuric acid solution prepared above was added, and the solution was heated in a water bath at 100°C for 5 min. After cooling, 1 mL of the carbazole / anhydrous ethanol solution prepared above was added, and the solution was mixed and heated in a water bath at 100°C for 10 min. After cooling, the absorbance value was measured at a wavelength of 525 nm, and a standard curve was drawn with the concentrations of the galacturonic acid standard as the horizontal axis and the absorbance value as the vertical axis.

[0081] (3) Sample determination: Take 1 mL of the prepared hemp seed polysaccharide sample, determine its absorbance according to the above operation, and substitute it into the standard curve regression equation 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 (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. Figure 2 b) Obtain the regression curve equation: Y = 0.5181X + 0.0983, R 2 =0.9984. According to the test results, 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. According to the test results, it was calculated that: HSP (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, which was (5.03±1.71)%; and HSP-Ⅱa after further separation and purification contained a small amount of protein or almost no protein, which was (3.30±1.56)%.

[0086] Table 1 Chemical composition summary

[0087]

[0088] Example 3 Ultraviolet Spectrum Scanning of Hemp Seed Polysaccharide

[0089] Weigh 4 mg of hemp seed polysaccharide sample HSP-Ⅱa accurately, dissolve it in 4 mL of deionized water to prepare hemp seed polysaccharide solution (i.e., HSP-Ⅱa concentration is 1 mg / mL), use a full-band UV spectrometer to scan in 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 (7.8mm×300mm); mobile phase: 0.025M NaH 2 PO 4 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 of the determination standard and the sample at 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 ratio of molecular weight (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 14-24min, 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 The molecular weight distribution statistics of HSP-Ⅱa and dextran standard, the horizontal axis X represents the molecular weight, 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 137.01 kDa; the number average molecular weight (Mn) was 35.45 kDa; and the peak molecular weight (Mp) was 52.62 kDa.

[0099] 4.2 Ion chromatography

[0100] (1) Preparation and calculation method of standard solution: 16 monosaccharide standards are prepared to prepare standard solutions (including rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, mannuronic acid, etc.). These monosaccharide standards are mixed together to prepare a mixed standard solution, i.e., a mixed standard. In the mixed standard, the concentration of each monosaccharide standard is 1 mg / mL. According to the absolute quantitative method, the mass of different monosaccharides is measured, and the molar ratio is calculated according to the molar mass of the monosaccharide, 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 monosaccharides in standards or samples; A: frontal area of ​​monosaccharides in standards or samples).

[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 an oven at 120°C 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 CarbopacTM PA20 (3×150 mm), detector: electrochemical detector.

[0104] Mobile phase: A:H 2 O; B: 15mM NaOH; C: 15mM NaOH&100mM NaAc

[0105] Column temperature: 30℃; injection volume: 25μL; flow rate: 0.3mL / min; elution gradient: 0min A phase / B phase / C phase (98.8:1.2:0, V / V), 18min A phase / B phase / C phase (98.8:1.2:0, V / V), 20min A phase / B phase / C phase (50:50:0, V / V), 30min A phase / B phase / C phase (50:50:0, V / V), 30.1min A phase / B phase / C phase (0:0:100, V / V), 46minA phase / B phase / C phase (0:0:100, V / V), 46.1min A phase / B phase / C phase (0:100:0, V / V), 50min A phase / B phase / C phase (0:100:0, V / V), 50.1min 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 16 kinds of monosaccharide mixed standards. The peak times are shown in Table 2. The peaks at 2.0min and 40.5min are the sodium hydroxide and sodium acetate solvent peaks, respectively. Figure 7 The ion chromatogram of hemp seed polysaccharide HSP-Ⅱa is shown in Table 3. The relative content of each monosaccharide in hemp seed polysaccharide HSP-Ⅱa is calculated 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 time of monosaccharide mixed standard

[0108]

[0109] Table 3 Peak time and molar ratio of monosaccharide peaks of HSP-Ⅱa

[0110]

[0111]

[0112] 4.3 Fourier transform infrared spectroscopy

[0113] Mix 1 mg of hemp seed polysaccharide sample with 100 mg of KBr (dried to constant weight at 100 °C) and grind them evenly. Use a tablet press to compress the sample into a semi-transparent state and then put it into an infrared spectrometer at 4000 cm -1 -400cm-1 Scanning in the range of 100 nm to identify the characteristic functional groups in polysaccharides. Figure 8 The results of FT-IR analysis of functional groups are shown in Table 4. 3416cm -1 It is the stretching vibration absorption peak generated by -OH of HSP-Ⅱa, 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 angle-variable vibration absorption peak of CH, 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, hemp seed polysaccharide 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 D 2 O, and transferred to a nuclear magnetic resonance tube, and the structure of each component of 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 the hemp seed polysaccharide HSP-Ⅱa by one-dimensional NMR is shown in Table 5.

[0119] In HSP-Ⅱa 1 H NMR ( Fig. 9 In a), most of the proton signal peaks are located between δ0.7-5.8ppm, and the chemical shifts of the protons are intertwined and overlapped between the signals at 3.0-4.5pmm, making it difficult to accurately analyze. There are two main anomeric hydrogen signals within the chemical shift of 4.9-5.4pmm, with chemical shifts of 5.01 and 4.99ppm, respectively, indicating that HSP-Ⅱa has an α configuration. 13 CNMR( Fig. 9b) 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] Thermo gravimetric and differential scanning calorimetry (TG-DSC) was used to study the thermal stability and possible aggregation structure of hemp seed polysaccharide samples. Experimental conditions: sample loading amount 5mg-8mg; test temperature: 30℃-800℃; carrier gas: N 2 ; Heating rate: 10℃ / min.

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

[0126] The first weight loss: HSP-Ⅱa is between 30℃-210.45℃, 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, 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℃-552.84℃, and 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 in this stage is rapid, 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 rupture of chemical bonds such as CO and CC in the sugar ring, which further decomposes the polysaccharide into CO 2 and H 2 O and other components volatilize, and may form polycyclic aromatic hydrocarbons and graphite 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 mostly the transformation of the remaining residual substances. The above 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] Particle size distribution of hemp seed polysaccharide HSP-Ⅱa Fig.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 Fig.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 uronic acid group. 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 with a mortar, and the crystal characteristics of the hemp seed polysaccharide sample were detected and analyzed using an XRD instrument. Determination conditions: Determination conditions: voltage 40 kV, current 40 mA, using Cu target as target material, Kα line as radiation source for scanning, 10-80°2θ for continuous rapid scanning.

[0135] The experimental results are as follows Fig.13 As shown, in the diffraction angle 2θ range of 10-80°, there is a wide and diffuse diffraction peak at about 22°, and the other areas are mostly amorphous areas, indicating that HSP-Ⅱa has certain crystalline characteristics, but the crystallinity is low, and it is presented in an amorphous state. HSP-Ⅱa has more obvious diffraction peaks at 2θ=28.35° and 40.56°, which is speculated to be caused by NaCl residue. The above results show that hemp seed polysaccharide HSP-Ⅱa has very few crystalline areas, mainly amorphous areas, that is, semi-crystalline polymers.

[0136] (4) Congo red test

[0137] Dissolve 12.0mg of hemp seed polysaccharide sample in 12mL of deionized water, and then add 6mL of Congo red solution (Congo red concentration is 80μmol / L). This mixed solution is called A solution. Take 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL and 0.5mL of 1M NaOH solution in a test tube, add A solution to 1mL, mix well, and use a full-band ultraviolet spectrometer to scan and record the maximum absorption wavelength (λmax) in the range of 400nm-600nm. Draw a curve with NaOH concentration as the horizontal coordinate and λmax as the vertical coordinate. Determine whether the polysaccharide sample has a triple helix structure by the above method with deionized water as the control.

[0138] The experimental results are shown in Fig.14 Compared with Congo red, the λmax of the mixed solution of HSP-Ⅱa and Congo red solution in aqueous solution and weak alkaline solution was significantly red-shifted, indicating that a polysaccharide-Congo red complex was produced. In addition, when the NaOH concentration was in the range of 0.1mol / L-0.5mol / L, the λmax of the polysaccharide-Congo red complex was red-shifted, but the absorption wavelength did not change significantly, and it was in a metastable state, indicating that HSP-Ⅱa had a relatively stable triple helical structure.

[0139] (5) Field emission scanning electron microscopy

[0140] Take a small amount of dried hemp seed polysaccharide sample, stick the sample on a sample stand with conductive tape, and place it in an ion sputtering instrument for gold plating to enhance its conductivity; finally, adjust the observation parameters under a scanning electron microscope until a clear and ideal observation field is obtained. Working conditions: The observation magnification is pre-selected at 100 times, 1000 times, and 5000 times, observe the sample morphology, take photos and record, and the acceleration voltage is set to 15kV.

[0141] FE-SEM images of hemp seed polysaccharide at different magnifications Fig.15 As shown in the figure. HSP-Ⅱa mainly presents irregular flakes with a small amount of strip-like structure stacking, and the surface is relatively smooth and dense. The stacking gaps in the HSP-Ⅱa structure are relatively loose, which may be related to the intermolecular force. It can also be seen from the figure that there is basically no crystal structure on the surface of HSP-Ⅱa, and it mostly exists in an amorphous state, which is consistent with the XRD test results.

[0142] (6) Atomic force microscopy

[0143] The hemp seed polysaccharide sample HSP-Ⅱa was dissolved in ultrapure water to prepare a 1.0 mg / mL sample mother solution, and magnetically stirred for 2 hours to fully dissolve it. The mother solution of the hemp seed polysaccharide sample was then diluted to 20μg / mL, 1μg / mL and 10ng / mL in sequence with ultrapure water. In order to reduce the presence of aggregates in the polysaccharide sample solution, each concentration of dilution was magnetically stirred at a temperature of 50°C for more than 3 hours to fully mix the solution. After the 10ng / mL dilution was stirred, it was cooled and filtered with a 0.45μm needle filter membrane, which was the working solution for AFM measurement (the appropriate concentration was selected according to the sample detection situation, and the 10ng / mL dilution was used as an example here). 5μL of nickel chloride solution (nickel chloride concentration was 2M) was added to the mica sheet, and after it was adsorbed for 30s, it was washed with ultrapure water and dried. Subsequently, 20 μL of the hemp seed polysaccharide sample working solution was added to the pretreated mica sheet, and the sample was evenly distributed on the mica sheet by spin coating. To remove the unabsorbed residual sample, it was rinsed with ultrapure water for several times. After it was completely dried, the hemp seed polysaccharide sample was scanned and measured using AFM. Measurement conditions: 100 pm scanner, probe tip curvature radius of 10 nm, tip material of Si, force constant of 2.8 N / m to ensure stability and accuracy during the measurement process, and tapping mode was selected as the operation mode to achieve non-destructive scanning and high-resolution imaging of the sample.

[0144] Fig.16 The two-dimensional plane and three-dimensional stereoscopic images of 10 ng / mL HSP-Ⅱa aqueous solution in 2μm×2μm. The two-dimensional plane image of HSP-Ⅱa presents irregular spherical and partially aggregated particle structures, and the three-dimensional stereoscopic image shows that there are many cylindrical structures of different heights. Usually, the height of a single polysaccharide molecule chain is 0.1nm-1.0nm, while the height of the HSP-Ⅱa sugar chain is 1.0nm-10.5nm. There is aggregation or entanglement of sugar chains. It is speculated that it may be formed by the interweaving of single or multiple chains of hemp seed polysaccharide molecules through intermolecular hydrogen bonds, or it may be caused by forces such as van der Waals forces. In addition, HSP-Ⅱa containing a large amount of uronic acid produces electrostatic repulsion with mica sheets with the same negative charge, resulting in more obvious clustering of HSP-Ⅱa. The above results show that HSP-Ⅱa is stable and not easy to aggregate, which is basically consistent with the results of Zeta potential measurement.

[0145] Example 6 Preliminary study on in vitro simulated digestion characteristics of hemp seed polysaccharide

[0146] 6.1 In vitro simulated gastric digestion characteristics

[0147] In this experiment, artificial gastric juice used for in vitro simulated digestion was used to simulate in vitro digestion of hemp seed polysaccharide HSP-Ⅱa. Deionized water was used to prepare a solution of 8 mg / mL HSP-Ⅱa, and 12 mL of simulated gastric juice and 12 mL of HSP-Ⅱa solution were mixed to make a simulated mixed solution, with deionized water as a blank control. Placed in a 37°C constant temperature shaker (80r / min), slowly rotated to simulate gastric peristalsis for digestion reaction, 3 mL was taken out from the mixed solution at reaction time points 0, 2, 4 and 6h, and immediately placed in a boiling water bath for inactivation for 15min.

[0148] 6.2 In vitro simulated intestinal digestion characteristics

[0149] In this experiment, artificial intestinal fluid used for in vitro simulated digestion experiments was used to perform in vitro simulated intestinal digestion reaction on hemp seed polysaccharide HSP-Ⅱa. The digestion products of the above-mentioned simulated gastric juice digestion for 6 hours without inactivating the enzyme and simulated intestinal fluid were added to prepare a simulated mixed solution. In the simulated mixed solution, the volume ratio of the digestion products of the simulated gastric juice digestion for 6 hours without inactivating the enzyme and the simulated intestinal fluid was 10:3. The simulated mixed solution was placed in a constant temperature shaker (80r / min) at 37°C, and slowly rotated to simulate intestinal peristalsis for digestion reaction. 3mL was taken out from the mixed solution at reaction time points 0, 2, 4 and 6h, and immediately placed in a boiling water bath for inactivation.

[0150] 6.3 Determination of in vitro simulated digestion characteristic indexes 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 respectively, dialyzed with water for 24 h in a 1000Da dialysis bag, and freeze-dried. The freeze-dried polysaccharide digestion solution was configured to 1 mg / mL, and its molecular weight was determined by HPGPC after passing through a 0.22 μm aqueous filter membrane.

[0154] ②Chromatographic conditions

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

[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) of the digestion products of HSP-Ⅱa at different time points in simulated gastric fluid and simulated intestinal fluid is shown in the following figure: Fig.17 a. Fig.17 b and Table 6.

[0157] Fig.17 a and Table 6 show that after HSP-Ⅱa was digested with simulated gastric juice, the peak time of the product was prolonged with the increase of digestion reaction time. At 2h, the Mw of the digestion product decreased from (134.03±0.08) kDa to (133.43±0.01) kDa. When digestion was continued for 6h, the peak time of the digestion product was significantly prolonged, and the Mw decreased to (125.68±0.18) kDa, indicating that simulated gastric juice had a certain degradation effect on HSP-Ⅱa. Fig.17 b and Table 6 show that after HSP-Ⅱa was digested with simulated intestinal fluid, the peak time of HSP-Ⅱa was prolonged, and the peak time of the product was also significantly prolonged with the increase of 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 in change. After continuous digestion for 6 h, its Mw was significantly reduced to (113.54±0.29) kDa. The further reduction of the molecular weight of the polysaccharide digestion product indicates that simulated intestinal fluid also plays an important role in the degradation of HSP-Ⅱa.

[0158] After HSP-Ⅱa was digested by simulated gastrointestinal fluid, the molecular weight of the digestion product was reduced 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 fluid digestion

[0160]

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

[0162] Polysaccharide digestion solution treatment: Take 1.0 mL of digestion products at different time points after in vitro simulated gastrointestinal digestion, blow dry with nitrogen blower, and directly derivatize without hydrolysis. After passing through a 0.22 μm aqueous filter, use high-performance liquid chromatography (HPLC) to determine the composition of free monosaccharides after digestion. According to the monosaccharide composition of HSP-Ⅱa: 3.0 mg of each of the 7 monosaccharides Rha, GlcA, GalA, Glc, Gal, Xyl and Ara were accurately weighed as monosaccharide mixed standards, and also derivatized and determined 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 and obtain a mixture. Take 3 times the volume of the mixture with chloroform and extract 3-4 times. Retain the aqueous phase, filter through a 0.22 syringe filter membrane, and use Agilent 1100 HPLC for detection and analysis.

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

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

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

[0167] The above results show that HSP-Ⅱa produces free monosaccharides during the simulated gastrointestinal digestion process, and its response intensity gradually increases from gastric juice to intestinal juice digestion, which is consistent with the previous molecular weight change trend. And the characteristic peaks produced by it are all around 38min and 50min, which are consistent with the peak times of glucose and arabinose in the mixed standard. It is speculated that during the gastrointestinal juice digestion process, hemp seed polysaccharide HSP-Ⅱa may be partially degraded, resulting in a small amount of glucose and arabinose monosaccharides being released and freed.

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

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

[0170] ①Prepare DNS solution: add 0.25g NaOH, 5g potassium sodium tartrate and 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, add 0.0075g anhydrous sodium sulfite to completely dissolve, finally make up to 50mL brown volume bottle, store in a 4℃ refrigerator 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 with deionized water, add the prepared DNS solution to the glucose standard solution at a volume ratio of 1:1, boil in a water bath for 15 minutes, cool to obtain a mixed solution, add 5 times the volume of deionized water to dilute the mixed solution, and finally measure the absorbance at 540 nm to draw a standard curve. The absorbance of the polysaccharide digestion solution was detected by the same method, and the reducing sugar content of the polysaccharide digestion solution was calculated according to the regression equation of the standard curve.

[0173] Digestion in the gastrointestinal tract 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, the changes in the total sugar and reducing sugar content can intuitively reflect the digestion characteristics of polysaccharides. Fig. 20 a. Fig. 20 b and Table 7.

[0174] Fig. 20a and Table 7 show that during the digestion of HSP-Ⅱa in simulated gastric juice for 0-6h, the total sugar content of the digestion product decreased from (41.76±0.78) mg / mL to (37.77±0.15) mg / mL, and the content did not change significantly. The total sugar content of the HSP-Ⅱa simulated gastric juice digestion product continued to be digested in the intestinal juice for 2h, and it did not change much, decreasing to (35.66±0.54) mg / mL. After digestion for 4-6h, the total sugar content decreased to (25.88±0.39) mg / mL, and the content change was significantly different (P<0.05). During the digestion process of simulated gastrointestinal juice, the total sugar content of HSP-Ⅱa decreased significantly, indicating that the simulated artificial gastrointestinal juice had a certain degradation effect on HSP-Ⅱa.

[0175] Fig. 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 at 0-4h; at 4-6h, the reducing sugar content of HSP-Ⅱa increased significantly to (0.60±0.01) mg / mL, with a significant difference (P<0.01), indicating that HSP-Ⅱa polysaccharide is gradually being degraded by gastric juice. The HSP-Ⅱa simulated gastric digestion product continued to digest in the simulated intestinal fluid. The reducing sugar content of HSP-Ⅱa did not change significantly at 0-2h of digestion; at 2-6h, the reducing sugar content increased significantly to (0.98±0.02) mg / mL, with a significant difference (P<0.01). During the simulated gastrointestinal digestion, the reducing sugar content of HSP-Ⅱa increased significantly, and the results once again showed that the simulated gastrointestinal fluid had a certain degradation 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 with a pH of 6.8 to a concentration of 1U / mL, and store the stock solution at -20°C.

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

[0183] 0.1mol / L Na 2 CO 3 Solution: Accurately weigh 0.53 g of Na 2 CO 3 Powder, add deionized water and dissolve in a 50mL volumetric flask, dilute to the mark (50mL), and store at 4℃ for later 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 use.

[0185] (2) Reaction system

[0186] This experiment set up drug reaction group, blank reaction group, drug control group and blank control group, with 3 replicates in each group. According to the corresponding dose in Table 8 below, add to 5mL centrifuge tube, and use 0.1mol / L PBS buffer as blank solution. First, mix PBS buffer, hemp seed polysaccharide solution, α-glucosidase mother solution and distilled water, react at 37℃ for 15min and then take out; add PNPG solution and continue to react at 37℃ for 25min; finally add Na 2 CO 3 The solution was used 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 Fig.21 .Depend on Fig.21 It can be seen that with the continuous increase of HSP-Ⅱa concentration (0.25mg / mL-4mg / mL), its inhibitory effect on α-glucosidase became more and more significant, and the inhibition rate gradually increased, showing a good dose-effect relationship and concentration dependence. At 4mg / mL, the inhibitory effect was the largest, which was (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 a 50 mL volumetric flask to the mark.

[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, for use.

[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 3 replicates in each group. Add the doses in a 5mL centrifuge tube according to Table 9, 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 bathe at 37℃ for 10min; then add starch solution, mix well, and bathe at 37℃ for 10min; finally, add the prepared DNS reagent, take it out after boiling water bath for 5min, cool it, and dilute it 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] According to the test results, the inhibitory effect of hemp seed polysaccharide HSP-Ⅱa on α-amylase was analyzed. 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] Results Fig. 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 1.93mg / 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 taking out the cell cryopreservation tube from the liquid nitrogen tube, put it in a 37℃ water bath and shake it gently to thaw. Use 75% alcohol to disinfect the surface of the cryopreservation tube, and then put it in the clean bench; open the cryopreservation tube cover, use a pipette to gently blow the cell suspension inside to make it completely suspended 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 a rate of 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 it evenly; transfer the cell solution to a cell culture bottle, and add 5mL of complete culture medium; finally, place the culture bottle in a 37℃, 5% CO 2 Place the cells in an incubator and wait for them to adhere to the wall and grow.

[0211] After the cells grow to the logarithmic growth phase in the incubator, the supernatant is discarded; the cells are washed with phosphate buffered saline (PBS) solution, 2 mL of 0.25% trypsin is added, and the cells react in the incubator for 3 minutes, and then 2 volumes of complete culture medium (containing 4.5 g / L D-glucose) are quickly added to terminate the trypsin reaction, and the cells are centrifuged at 1000 rpm / min for 5 minutes, and the supernatant is discarded; 1 mL of complete culture medium is added to resuspend the cells, and the cells are evenly blown; 300-400 μL of the cell suspension is inoculated into a culture bottle containing 6 mL of complete culture medium; the cells are placed in a 37°C, 5% CO incubator again. 2 In the incubator, after waiting for the cells to adhere to the wall, cells in the logarithmic growth phase were taken for subsequent experiments.

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

[0213] This experiment will use the insulin induction method in vitro to establish an insulin resistance model. By adding a specific concentration of insulin to the cell culture medium, the insulin resistance environment in vivo will be simulated. The optimal concentration and optimal action time of insulin on HepG2 cells will be screened through preliminary experiments to establish the IR model, and the changes in glucose consumption in each group of cells will be measured using a glucose kit 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 1×10 5 The cells / mL were counted and inoculated into a 96-well plate, 100 μL was taken from each well and cultured for 24 h.

[0216] Blank group: Take 100 μL of serum-free 1640 culture medium, place 6 replicate wells in each group in the incubator and incubate 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), that is, 10 μL of serum-free 1640 culture medium + 1 mL of glucose detection solution.

[0217] Experimental group: 100 μL of insulin solution prepared with serum-free 1640 was taken. The insulin concentration in the insulin solution was 1×10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 mol / L, 6 replicate wells in each 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 min, shaken evenly at a 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 compared 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 test 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 insulin concentration, the cells were divided into a blank group and a test group, each with 6 replicate wells. 5 The cells were inoculated into a 96-well plate with 100 μL of cell suspension per well and placed in an incubator to wait for the cells to adhere to the wall. The blank group was added with 100 μL of serum-free 1640 culture medium, and the experimental group was added 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 optimal conditions for establishing the IR model were determined based on the cell activity test results.

[0225] The results are as follows Fig.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 24h, the glucose consumption reached the lowest value, which was significantly different from the blank group. This shows that high concentrations of insulin can induce cells to establish an obvious 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] Preliminary experiments have shown 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, respectively.

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

[0229] Blank group: 100 μL 1640 medium;

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

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

[0232] The cells in the logarithmic growth phase were made into a suspension and the culture medium was 1×10 5 / well cell density, 100 μL per well was inoculated in the wells of 96-well plate, the culture medium in the wells was grouped as above, 6 wells per group, and after 24h of culture, the supernatant was discarded. 10 μL of supernatant was taken from each well and tested with a glucose kit to calculate the glucose consumption of each group, and the relevant values ​​were calculated for comparative analysis.

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

[0234] The above results show that hemp seed polysaccharide HSP-Ⅱa can significantly improve the glucose consumption ability 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 principle of the present invention should be included in the protection scope 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 hemp seed crude polysaccharide HSP was extracted by water extraction and alcohol precipitation method; (2) After removing protein from crude polysaccharide of hemp seed, the polysaccharide is separated by ion exchange column chromatography to obtain 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, characterized in that: In step (1), a water extraction and alcohol precipitation method is adopted to extract crude polysaccharides from hemp seeds, which comprises the following steps: mixing defatted hemp seed meal with water, decocting, cooling, centrifuging, taking supernatant, collecting extract, filtering, concentrating, and obtaining concentrated product; precipitating the concentrated product with ethanol solution, centrifuging, collecting precipitate, evaporating ethanol, concentrating, and freeze-drying.

3. The method for preparing hemp seed polysaccharide according to claim 2, characterized in that: The material-liquid ratio of defatted hemp seed meal to water is 1 g:15 mL; the final concentration of ethanol in the ethanol solution is 80%.

4. The method for preparing hemp seed polysaccharide according to any one of claims 1 to 3, characterized in that: In step (2), the Sevage method is used to remove proteins, and DEAE-52 cellulose ion exchange column chromatography is used for separation.

5. The method for preparing hemp seed polysaccharide according to claim 4, characterized in that: The Sevage method is used to remove protein, which includes the following steps: mixing a solution of hemp seed crude polysaccharide HSP, chloroform and n-butanol, centrifuging after shaking, taking the supernatant, dialysis, concentrating and freeze-drying.

6. The method for preparing hemp seed polysaccharide according to claim 4, characterized in that: The separation by DEAE-52 cellulose ion exchange column chromatography includes the following steps: using DEAE-52 cellulose ion exchange column chromatography to separate the aqueous solution of hemp seed polysaccharide obtained by deproteinization by the Sevage method, using sodium chloride solution as eluent, collecting the components eluted by 0.3M 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, characterized in that: In step (3), HSP-Ⅱ is separated by gel column chromatography, which includes the following steps: preparing HSP-Ⅱ aqueous solution, separating by Sephacryl S-300 gel column chromatography, eluting with distilled water at a flow rate of 1 mL / 2 min, collecting the elution peak, and freeze-drying 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. Use of the hemp seed polysaccharide according to claim 8 in improving the glucose consumption capacity of insulin-resistant cells.

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