Extraction and preparation process and quality control and discrimination technology of acanthopanax senticosus polysaccharide

Abstract

The invention discloses an extraction process and a quality control method of acanthopanax senticosus polysaccharide and application of the acanthopanax senticosus polysaccharide. The acanthopanax senticosus polysaccharide is prepared from acanthopanax senticosus roots, stems, leaves and fruits through a water extraction and alcohol precipitation extraction method. The polysaccharides of the roots, the stems, the leaves and the fruits of the acanthopanax senticosus all contain monosaccharides which comprise 5.0% to 50% of D-mannose, 0.7% to 6.0% of L-rhamnose, 4.0% to 33% of D-glucuronic acid, 6.0% to 69% of D-glucose, 13% to 40% of D-galactose and 4.0% to 18% of D-arabinose. The quality control method ensures the quality of the acanthopanax senticosus. A standard for controlling the acanthopanax senticosus polysaccharide is established by analyzing chemical components of the acanthopanax senticosus polysaccharide, content limitation of core components of the acanthopanax senticosus polysaccharide is added to the standard, and the polysaccharide prepared from the acanthopanax senticosus selected according to the range is used for judging polysaccharides of the acanthopanax senticosus and other plants and different parts of the acanthopanax senticosus. The method is good in reproducibility, accurate and reliable, and the acanthopanax senticosus polysaccharide and medicinal materials of different parts of acanthopanax senticosus can be rapidly and accurately identified through infrared characteristics and monosaccharide composition.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and content determination of effective components of traditional Chinese medicine, and particularly relates to a method for extraction and monosaccharide composition determination of Acanthopanax senticosus polysaccharide and application thereof. Background Art

[0002] The dried root, stem, leaf and fruit of Eleutherococcus senticosus (Rupr. & Maxim.), a plant of the Araliaceae family. It is a Qi-tonifying drug. It is pungent, slightly bitter and warm in nature and enters the spleen, kidney and heart meridians. The main active ingredients of Acanthopanax senticosus include Acanthopanax glycosides, flavonoids and polysaccharides. Acanthopanax polysaccharides, as one of the main chemical components, have multiple pharmacological activities, such as lowering blood sugar, anti-tumor, immunomodulation, anti-radiation, enhancing human microcirculation, improving physical fatigue and other pharmacological activities.

[0003] Polysaccharides are widely used in medicine. However, their structure is complex and difficult to extract and separate. At present, the research on Acanthopanax senticosus polysaccharides mainly focuses on physiological activity research, while there are few reports on the separation and purification process, structural characteristics and monosaccharide content determination of Acanthopanax senticosus polysaccharides. Therefore, their chemical structure and efficacy have not been combined, and it is impossible to deeply study the unique effects of Acanthopanax senticosus polysaccharides on various pharmacological effects, which seriously limits the development and application of Acanthopanax senticosus.

[0004] There are few studies on the extraction process of Acanthopanax senticosus polysaccharides, and there is no unified and accurate method for the extraction process and monosaccharide composition determination of Acanthopanax senticosus polysaccharides. Summary of the invention

[0005] The present invention aims to provide a preparation process of Acanthopanax senticosus polysaccharide, a method for determining the monosaccharide composition and a quality control method, and to distinguish Acanthopanax senticosus polysaccharide from other plant polysaccharides and different parts of Acanthopanax senticosus.

[0006] The present invention is achieved through the following technical solutions

[0007] The Acanthopanax senticosus polysaccharide separated and purified by the present invention has a total sugar content of 8.0-74%, a uronic acid content of 4.0-23%, a monosaccharide composition of 5.0-50% D-mannose, 0.7-6.0% L-rhamnose, 4.0-33% D-glucuronic acid, 6.0-69% D-glucose, 13-40% D-galactose and 4.0-18% D-arabinose.

[0008] The invention provides a method for preparing and controlling the quality of Acanthopanax senticosus polysaccharide, which comprises the steps of extracting Acanthopanax senticosus polysaccharide and determining the monosaccharide composition.

[0009] Furthermore, the preparation method comprises the steps of obtaining crude polysaccharide by degreasing, water extraction and alcohol precipitation.

[0010] Furthermore, the preparation method also includes the step of purifying the crude polysaccharide (such as removing protein with a macroporous resin).

[0011] In one embodiment of the present invention, the preparation method comprises the following steps:

[0012] (1) pre-treating Acanthopanax senticosus to obtain defatted Acanthopanax senticosus powder;

[0013] (2) The defatted Acanthopanax senticosus powder is extracted with hot water, and the obtained Acanthopanax senticosus extract is successively subjected to the steps of removing small molecules of protein impurities, concentrating, and precipitating with alcohol to obtain Acanthopanax senticosus polysaccharide.

[0014] In step (1), the liquid-to-solid ratio of Acanthopanax senticosus powder to anhydrous ethanol is 1:5-1:10; in one embodiment of the present invention, the liquid-to-solid ratio is 1:6

[0015] In step (1), the number of extractions is 1-5 times (e.g., 1, 2, 3, 4, 5 times); in one embodiment of the present invention, the number of extractions is 3 times.

[0016] In step (1), each extraction time is 1-4 hours (such as 1, 2, 3, 4 hours); in one embodiment of the present invention, the extraction time is 2 hours.

[0017] In one embodiment of the present invention, step (1) comprises: taking Acanthopanax senticosus powder, adding anhydrous ethanol, extracting at boiling point temperature, collecting precipitate, and obtaining defatted Acanthopanax senticosus powder.

[0018] Furthermore, in step (2), the solid-liquid ratio of defatted Acanthopanax senticosus powder to water is 1:20-35 (such as 1:20, 1:25, 1:30, 1:35); in one embodiment of the present invention, the solid-liquid ratio is 1:20.

[0019] In step (2), the extraction time is 2-4 h (e.g., 2 h, 2.5 h, 3 h, 3.5 h, 4 h; in one embodiment of the present invention, the extraction time is 3 h.

[0020] In step (2), the number of extractions is 1-5 times (such as 1, 2, 3, 4, 5 times); in one embodiment of the present invention, the number of extractions is 3 times.

[0021] Furthermore, in the protein removal step, a macroporous resin method is used to remove the protein, and in the impurity small molecule removal step, a membrane separation method is used.

[0022] Furthermore, in the alcohol precipitation step, the alcohol is anhydrous ethanol, and the alcohol content reaches 85%.

[0023] In one embodiment of the present invention, step (2) comprises: extracting the defatted Acanthopanax powder obtained in step (1) in hot water, collecting the supernatant, removing the protein and small impurity molecules therein to obtain Acanthopanax polysaccharide solution, concentrating, adding anhydrous ethanol to an alcohol content of 85%, collecting the precipitate at 4°C overnight, and freeze-drying to obtain Acanthopanax polysaccharide.

[0024] The invention also provides a method for determining the composition of Acanthopanax senticosus polysaccharide.

[0025] Furthermore, the monosaccharide composition determination method comprises the steps of complete acid hydrolysis of the protein-free Acanthopanax senticosus polysaccharide and sample preparation.

[0026] The monosaccharide composition determination method also includes HPLC determination conditions, and the monosaccharide composition determination method steps are as follows:

[0027] (1) Acid hydrolysis of Acanthopanax senticosus polysaccharides

[0028] (2) Determination of monosaccharide composition of Acanthopanax senticosus polysaccharides by HPLC

[0029] Step (1) Acid hydrolysis conditions: 3 mol / L trifluoroacetic acid, the ratio of polysaccharide to trifluoroacetic acid is 0.01 mg / mL

[0030] In step (1), the acid hydrolysis time is 100 min-180 min (e.g., 100 min, 120 min, 140 min, 160 min, 180 min). In one embodiment of the present invention, the reaction time is 120 min.

[0031] In step (2), the liquid phase conditions for determining the monosaccharide composition of Acanthopanax senticosus by high performance liquid chromatography are as follows: the high performance liquid chromatography uses an ultraviolet detector, the detection conditions are a column temperature of 30° C., a detection wavelength of 245 nm, a liquid flow rate of 0.7 ml / min, and an isocratic elution using a 0.08 mol / L phosphate aqueous solution and acetonitrile in a volume ratio of 82:18;

[0032] The inventors of the present invention separated and purified Acanthopanax senticosus polysaccharide from Acanthopanax senticosus and analyzed and identified the total sugar content, uronic acid content, monosaccharide composition, infrared spectrum, chemical structure and the like.

[0033] The present invention measures polysaccharides of roots, stems, leaves and fruits of Acanthopanax senticosus from 15 production areas by a quality control method, summarizes the characteristics of Acanthopanax senticosus polysaccharides and the characteristics of different parts of Acanthopanax senticosus according to the obtained data, and distinguishes Acanthopanax senticosus polysaccharides from other plant polysaccharides and different parts of Acanthopanax senticosus according to the characteristics. In the quality control, various indicators of polysaccharides in various parts of Acanthopanax senticosus should meet the specified standards.

[0034] Preferably, the root of Acanthopanax senticosus has the following characteristics: the yield after extraction, separation and purification shall not be less than 1.00%, the total sugar content shall not be less than 35.0%, the total uronic acid content shall not be less than 6.0%, the D-mannose content in the monosaccharide composition of the polysaccharide shall not be less than 5.00%, the L-rhamnose content shall not be less than 0.900%, the D-glucuronic acid content shall not be less than 4.00%, the D-glucose content shall not be less than 33.0%, the D-galactose content shall not be less than 13.0%, and the D-arabinose content shall not be less than 4.00%.

[0035] Acanthopanax senticosus stem: The yield after extraction, separation and purification shall not be less than 1.00%, the total sugar content shall not be less than 10.0%, the total uronic acid content shall not be less than 6.0%, the D-mannose content in the polysaccharide monosaccharide composition shall not be less than 7.00%, the L-rhamnose content shall not be less than 0.700%, the D-glucuronic acid content shall not be less than 13.0%, the D-glucose content shall not be less than 7.00%, the D-galactose content shall not be less than 21.0%, and the D-arabinose content shall not be less than 7.00% Acanthopanax senticosus leaves: The yield after extraction, separation and purification shall not be less than 5.00%, the total sugar content shall not be less than 8.00%, the total uronic acid content shall not be less than 4.00%, the D-mannose content in the polysaccharide monosaccharide composition shall not be less than 21.0%, the L-rhamnose content shall not be less than 1.60%, the D-glucuronic acid content shall not be less than 14.0%, the D-glucose content shall not be less than 8.00%, the D-galactose content shall not be less than 23.0%, and the D-arabinose content shall not be less than 10.0%.

[0036] Acanthopanax fruit: The yield after extraction, separation and purification shall not be less than 8.00%, the total sugar content shall not be less than 25.00%, the total uronic acid content shall not be less than 7.00%, the D-mannose content in the polysaccharide monosaccharide composition shall not be less than 31.0%, the L-rhamnose content shall not be less than 1.40%, the D-glucuronic acid content shall not be less than 6.00%, the D-glucose content shall not be less than 6.00%, the D-galactose content shall not be less than 27.0%, and the D-arabinose content shall not be less than 4.00%. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0038] Figure 1 The attached figure is a liquid phase diagram of monosaccharide standard and monosaccharide composition analysis of Acanthopanax senticosus root polysaccharide in Experiment 4.

[0039] Figure 2 The attached figure is a liquid phase diagram of monosaccharide standard and monosaccharide composition analysis of Acanthopanax senticosus stem polysaccharide in Experiment 4.

[0040] Figure 3 The attached figure is a liquid phase diagram of monosaccharide standard and monosaccharide composition analysis of Acanthopanax senticosus leaf polysaccharide in Experiment 4.

[0041] Figure 4 The attached figure is a liquid phase diagram of monosaccharide standard and monosaccharide composition analysis of Acanthopanax senticosus fruit polysaccharide in Experiment 4.

[0042] Figure 5 The attached figure is the infrared spectrum of Acanthopanax senticosus root polysaccharide in Experiment 5.

[0043] Figure 6 The attached figure is the infrared spectrum of Acanthopanax senticosus stem polysaccharide in Experiment 5.

[0044] Figure 7 The attached figure is the infrared spectrum of Acanthopanax senticosus leaf polysaccharide in Experiment 5.

[0045] Figure 8 The attached figure is the infrared spectrum of Acanthopanax senticosus fruit polysaccharide in Experiment 5. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below in conjunction with the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0047] Example 1 Extraction, separation and purification of Acanthopanax senticosus polysaccharide

[0048] 1.1 Take an appropriate amount of dried Acanthopanax senticosus medicinal material, grind it with a grinder to obtain Acanthopanax senticosus coarse powder, heat the Acanthopanax senticosus coarse powder with 6 times the amount of anhydrous ethanol to reflux and defat it for 120 minutes, and dry it to obtain defatted Acanthopanax senticosus powder.

[0049] 1.2 Extraction of Acanthopanax senticosus polysaccharides The defatted Acanthopanax senticosus medicinal materials were extracted at a temperature of 100°C, a liquid-to-solid ratio of 1:20, and an extraction time of 3 hours. The extraction was repeated 3 times, and the filtrates were combined to obtain an Acanthopanax senticosus polysaccharide solution.

[0050] 1.3 Use hollow fiber membrane to remove impurity small molecules A method for separating and purifying Acanthopanax polysaccharide using a hollow fiber membrane: After a certain pretreatment, the Acanthopanax polysaccharide solution is ultrafiltered using a hollow cellulose membrane with a molecular weight of 5 KDa to obtain Acanthopanax polysaccharides with a molecular weight less than 5 KDa and greater than 5 KDa; the portion greater than 5 KDa is selected and frozen to obtain the Acanthopanax polysaccharide solution.

[0051] 1.4 Macroporous resin method for protein removal The type of macroporous resin was selected as HP20, and the volume of Acanthopanax senticosus polysaccharide and the macroporous resin HP20 were wet loaded in a certain ratio to pretreat the macroporous resin; first soak it in water for 18-24 hours, wash it with water until the water was clear, pour off the water and soak it in 4-5% NaOH solution for 24 hours, wash it with water until it was neutral, soak it in 4-5% HCl solution for 18-24 hours, wash it with water until it was neutral, load it into the column, and load the sample. Take an appropriate amount of Acanthopanax senticosus polysaccharide extract, treat it in a certain way, add it to the resin chromatography column, adsorb it for a certain period of time, use water as the eluent, start elution, control the flow rate at 8-10ml / min, follow up and detect it by phenol-sulfuric acid method, when the sugar-free component flows out, use 0.5-2mol / L NaCl solution of different concentrations to elute, and use the next gradient NaCl solution after the sugar-free component flows out, concentrate the eluted material to a small volume, monitor the density of the concentrated solution with a density meter, and obtain a polysaccharide concentrate with a density of 1.10-1.15 kg / L. Add anhydrous ethanol to the concentrated solution at room temperature and stir it continuously (so that the alcohol concentration is 85%), and let it stand at 4°C for 24 hours to precipitate the polysaccharide. Wash the precipitate with anhydrous ethanol and acetone respectively, add an appropriate amount of water to dissolve it, and freeze-dry it to obtain the Acanthopanax senticosus crude polysaccharide.

[0052] Example 2 Determination of polysaccharide content of Acanthopanax senticosus polysaccharide

[0053] 2.1 Determination of polysaccharide content D-glucose standard solution: accurately weigh 100 mg of D-glucose standard dried to constant weight at 40°C, dissolve it in double distilled water and dilute it to a 100 ml volumetric flask, accurately measure 5 ml of the mother solution and dilute it to 50 ml to prepare a D-glucose standard solution with a concentration of 0.1 mg / ml Drawing of standard curve: accurately measure 0, 0.2, 0.4, 0.6, 0.8, 1ml of 0.1mg / ml D-glucose standard solution, place in 10ml stoppered test tubes, add double distilled water to 2ml, add 1ml of 6% phenol reagent, shake well, quickly add 5ml of concentrated sulfuric acid, shake well immediately, place for 5min, heat in boiling water bath for 15min, take out and cool to room temperature, draw 2.0ml of distilled water, operate as blank control, and measure absorbance at 490nm. Draw standard curve with concentration of D-glucose standard solution (μg / mL) as horizontal axis and absorbance as vertical axis, and calculate regression equation.

[0054] Sample determination: Prepare a sample solution with a concentration of 1 mg / ml, take 2 ml of the sample solution, perform the above operation, and perform the determination three times in parallel. Calculate the polysaccharide content based on the standard curve regression equation.

[0055] 2.2 Test results: The standard regression equation is: y = 3.75x - 0.0342 R² = 0.994. The content of Acanthopanax senticosus polysaccharide is ≥ 35.0%.

[0056] Example 3 Determination of total uronic acid content of Acanthopanax senticosus polysaccharide

[0057] 3.1 Determination of total uronic acid content of polysaccharides Take an appropriate amount of D-galacturonic acid reference substance, weigh it accurately, and add water to make it contain 0.5 mg per 1 mL.

[0058] Preparation of standard curve: Precisely measure 0, 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of the reference solution, respectively, and place them in 10mL stoppered test tubes, add water to 1.0mL, and obtain the standard solutions containing 0, 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, and 0.5mg / mL. Then add 0.5mL of boric acid sulfuric acid solution, mix well, take out after boiling water bath for 20 minutes, and cool to room temperature. Add 0.2mL of 0.1% carbazole ethanol solution, mix well, and place at room temperature for 30 minutes. Measure the absorbance at a wavelength of 530 nm. Draw a standard curve with D-galacturonic acid concentration (mg / mL) as the horizontal axis and absorbance (OD) as the vertical axis, and calculate the regression equation.

[0059] Sample determination: Prepare a sample solution with a concentration of 1 mg / ml, take 2 ml of the sample solution, perform the above operation, and perform the determination three times in parallel. Calculate the total uronic acid content of the polysaccharide according to the regression equation of the standard curve.

[0060] 3.2 Test results: y = 0.573x + 0.0698 R² = 0.994 The total uronic acid content of Acanthopanax senticosus polysaccharide ≥ 6.00%

[0061] 3.3 Example 4 Structural Identification of Acanthopanax Senticosus Polysaccharide

[0062] 4.1 Analysis of monosaccharide composition Monosaccharide standards (D-glucose, D-mannose, D-glucuronic acid, L-rhamnose, D-galactose, D-galacturonic acid, D-arabinose) and 10.00 mg of Acanthopanax senticosus polysaccharide after complete acid hydrolysis (trifluoroacetic acid method) were mixed with NaH 2 PO 4 High performance liquid chromatography (HPLC) analysis was carried out with 2% paraformaldehyde and acetonitrile (82:18) as the mobile phase at a flow rate of 0.7 ml / min.

[0063] 4.2 Infrared spectroscopy analysis Acanthopanax senticosus polysaccharide (5 mg) was ground with a certain mass of dry KBr in a mortar and pressed into tablets using a tablet press. The obtained tablets were subjected to Fourier transform to obtain a sample spectrum in the range of 4000-400 cm -1 , resolution 4cm -1 ,Before collecting samples, it is necessary to perform background collection operation (KBr), and the obtained sample image shall be automatically baseline calibrated and automatically smoothed.

[0064] 4.3 Test results

[0065] 4.3.1 Analysis of monosaccharide composition of Acanthopanax senticosus polysaccharides After the Acanthopanax senticosus polysaccharide was hydrolyzed into monosaccharides, the monosaccharide composition was analyzed by high performance liquid chromatography. Figure 1 Its main monosaccharide components are D-glucose, D-mannose, L-rhamnose, D-glucuronic acid, D-galactose, and D-arabinose is a minor monosaccharide component.

[0066] Table 1 Determination results of monosaccharides in Acanthopanax senticosus roots from 15 different origins Sample No. Man (%) Gha (%) Glc (%) Glc (%) Gal (%) Ara (%) CWJG-1 8.09 0.95 8.27 56.1 18.1 8.47 CWJG-2 5.59 0.99 7.52 63.1 14.3 8.47 CWJG-3 6.64 1.93 11.7 47.5 23.3 8.90 CWJG-4 5.31 1.15 4.04 67.5 16.0 6.03 CWJG-5 6.82 1.64 5.58 53.5 23.5 8.99 CWJG-6 7.72 1.18 4.45 68.7 13.5 4.47 CWJG-7 6.64 1.53 12.2 40.2 25.5 13.9 CWJG-8 9.46 1.63 7.30 49.5 22.3 9.81 CWJG-9 6.79 1.64 4.00 60.1 21.3 6.16 CWJG-10 9.79 1.99 15.0 33.0 28.3 11.9 CWJG-11 12.0 1.41 5.53 55.9 19.9 5.19 CWJG-12 17.50 1.85 6.42 39.7 26.2 8.41 CWJG-13 11.0 1.78 6.47 48.4 24.7 7.76 CWJG-14 9.66 2.29 10.1 38.0 31.0 8.94 CWJG-15 16.2 1.35 6.47 45.0 22.4 8.64 Table 2 Determination results of monosaccharides in Acanthopanax senticosus stems from 15 different origins Sample No. Man (%) Rha (%) Glc (%) Glc (%) Gal (%) Ara (%) CWJJ-1 12.7 3.99 27.5 14.1 28.8 12.8 CWJJ-2 8.38 2.81 28.7 17.2 27.0 15.9 CWJJ-3 15.6 3.51 32.5 12.4 21.4 14.5 CWJJ-4 11.2 4.03 30.6 15.4 25.2 13.5 CWJJ-5 11.5 0.72 31.1 15.4 32.1 9.21 CWJJ-6 15.6 3.95 24.2 7.62 34.4 14.2 CWJJ-7 7.62 4.28 26.4 25.4 28.8 7.50 CWJJ-8 16.1 2.48 26.2 10.0 34.1 11.1 CWJJ-9 10.7 2.99 20.9 29.0 25.6 10.7 CWJJ-10 18.4 2.23 19.9 8.38 34.5 16.6 CWJJ-11 14.3 3.12 17.1 10.6 38.9 16.0 CWJJ-12 12.3 3.65 19.6 14.3 34.9 15.1 CWJJ-13 18.3 3.95 13.8 15.8 37.3 10.9 CWJJ-14 8.59 5.16 25.2 20.7 31.3 9.03 CWJJ-15 12.5 3.21 19.2 14.3 36.7 14.1 Table 3 Determination results of monosaccharides in Acanthopanax senticosus leaves from 15 different origins Sample No. Man (%) Rha (%) Glc (%) Glc (%) Gal (%) Ara (%) CWJY-1 22.1 2.27 17.4 8.95 35.5 13.8 CWJY-2 23.8 1.92 18.3 9.23 33.2 13.5 CWJY-3 27.8 2.15 15.7 9.04 33.4 12.0 CWJY-4 29.0 2.13 14.6 9.41 31.1 13.8 CWJY-5 28.2 2.17 15.2 8.69 31.5 14.3 CWJY-6 24.9 2.01 18.3 8.98 31.8 13.9 CWJY-7 27.4 2.57 18.0 8.74 29.0 14.3 CWJY-8 22.1 2.38 17.4 8.86 35.3 14.0 CWJY-9 23.1 2.07 20.3 9.27 29.4 15.9 CWJY-10 23.4 1.90 19.3 17.3 27.7 10.4 CWJY-11 26.6 1.63 17.6 15.0 23.1 16.1 CWJY-12 27.3 2.49 17.4 8.21 29.8 14.8 CWJY-13 40.1 1.90 12.5 8.62 25.7 11.2 CWJY-14 30.7 2.30 14.5 9.93 29.8 12.8 CWJY-15 21.7 1.99 18.5 17.2 27.7 12.9 Table 4 Determination results of monosaccharides in Acanthopanax senticosus fruit from 15 different origins Sample No. Man (%) Rha (%) Glc (%) Glc (%) Gal (%) Ara (%) CWJG-1 47.5 1.65 9.72 7.11 27.0 7.00 CWJG-2 40.6 1.90 8.17 6.34 37.2 5.76 CWJG-3 36.9 2.26 9.51 7.75 33.6 9.94 CWJG-4 39.0 1.93 8.33 6.36 35.8 8.60 CWJG-5 40.1 2.09 6.06 6.43 39.4 5.86 CWJG-6 42.3 1.80 6.82 7.33 34.8 6.92 CWJG-7 40.7 1.78 8.81 7.57 33.8 7.34 CWJG-8 39.9 2.02 7.89 10.2 32.8 7.20 CWJG-9 35.2 1.98 7.88 6.27 34.1 14.6 CWJG-10 39.8 1.57 10.1 6.54 29.4 12.7 CWJG-11 42.5 1.66 7.53 6.68 36.9 4.7 CWJG-12 33.0 1.60 6.66 26.5 27.0 5.25 CWJG-13 39.1 1.78 7.67 6.07 34.8 10.5 CWJG-14 38.3 1.87 6.36 12.4 29.3 11.8 CWJG-15 31.6 1.78 8.15 8.16 32.9 17.4

[0067] 4.3.2 Infrared spectroscopy analysis results of Acanthopanax senticosus polysaccharides The Fourier infrared spectrum of Acanthopanax senticosus polysaccharide is shown in Figure 2 As shown, at 3409.92 cm −1 It shows a broad tensile strength characteristic peak, which is due to the stretching vibration of OH. 2931.28 cm −1 The weak peak at 1741.31 cm −1 and 1628.99 cm −1 The two stretching peaks at indicate the presence of C=O and COO − . 1741.31 cm −1 and 1628.99 cm −1 、1416.61 cm −1 Typical absorption near 1400–1200 cm indicates the presence of uronic acid. −1The peak at 1158.33 cm −1 , 1075.00 cm −1 and 1022.31 cm −1 The peaks in the range indicate the presence of COH side groups and COC glycosidic band vibrations. −1 and 703.96 cm −1 The absorption at 850.00 cm-1 indicated that the sample contained a pyranose ring skeleton pattern, in which the root and fruit of Acanthopanax senticosus contained pyranose rings. −1 The presence of a characteristic peak near the stem and leaf of Acanthopanax senticosus indicates the presence of an α-glycosidic bond. −1 There is a characteristic absorption peak nearby, indicating the presence of a β-glycosidic bond.

[0068] Implementation case 5. Acanthopanax senticosus quality control method The quality control method of Acanthopanax senticosus includes determining the polysaccharide content by phenol-sulfuric acid method, determining the uronic acid content by carbazole-sulfuric acid method, and determining the monosaccharide composition of Acanthopanax senticosus polysaccharide by high performance liquid chromatography.

[0069] Table 5 Quality control method and quality control results of Acanthopanax senticosus polysaccharide Acanthopanax senticosus part Total sugar content Uric acid content Infrared signature Monosaccharide composition root 36.6-72.0% 6.29-9.74% <![CDATA[850 cm −1 ]]> Man 5.31-17.46% Rha0.950-2.29%GlcA 4.04-15.0% Glc 33.0-68.7%Gal 13.5-31.0% Ara4.47-13.9% stem 13.2-37.1% 6.26-14.3% <![CDATA[890 cm −1 ]]> Man 7.62-18.4% Rha0.72-5.16%GlcA 13.0-32.5% Glc 7.62-29.0%Gal 21.4-38.9% Ara7.50-16.0% leaf 8.20-23.9% 4.45-10.6% <![CDATA[890 cm −1 ]]> Man 21.7-40.1% Rha1.63-2.57%GlcA 14.5-20.3% Glc 8.21-17.3%Gal 23.1-35.5% Ara0.4-16.1% fruit 25.5-74.0% 5.44-10.0% <![CDATA[850 cm −1 ]]> Man 31.6-47.5% Rha1.47-2.26%GlcA 6.06-10.1% Glc 6.07-26.5%Gal 27.0-39.4% Ara4.70-17.4%

[0070] The embodiments described above are merely descriptions of preferred embodiments of the present invention, and are not intended to limit the concept and scope of the present invention. Each embodiment focuses on the differences from other embodiments, and the same or similar parts between the embodiments can be referred to each other. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features disclosed herein.

Claims

1. Acanthopanax senticosus polysaccharides from different parts are obtained by the preparation method, characterized in that: The polysaccharides from different parts of Acanthopanax senticosus are composed of D-mannose, L-rhamnose, D-glucuronic acid, D-glucose, D-galactose and D-arabinose.

2. The method for preparing the polysaccharide according to claim 1, wherein the polysaccharide is extracted from the root, stem, leaf and fruit of Acanthopanax senticosus, wherein the Acanthopanax senticosus polysaccharide comprises at least D-mannose, L-rhamnose, D-glucuronic acid, D-glucose, D-galactose and D-arabinose. The total sugar content is 8.0-74%, the uronic acid content is 4.0-23%, the monosaccharide composition contains D-mannose 5.0-50%, L-rhamnose 0.7-6.0%, D-glucuronic acid 4.0-33%, D-glucose 6.0-69%, D-galactose 13-40% and D-arabinose 4.0-18%.

3. Acanthopanax senticosus root polysaccharide: the total sugar content shall not be less than 35.0%, the total uronic acid content shall not be less than 6.0%, the D-mannose content in the monosaccharide composition of the polysaccharide shall not be less than 5.00%, the L-rhamnose content shall not be less than 0.900%, the D-glucuronic acid content shall not be less than 4.00%, the D-glucose content shall not be less than 33.0%, the D-galactose content shall not be less than 13.0%, and the D-arabinose content shall not be less than 4.00%.

4. Acanthopanax senticosus stem polysaccharides: total sugar content shall not be less than 10.0%, total uronic acid content shall not be less than 6.0%, D-mannose content in the polysaccharide monosaccharide composition shall not be less than 7.00%, L-rhamnose content shall not be less than 0.700%, D-glucuronic acid content shall not be less than 13.0%, D-glucose content shall not be less than 7.00%, D-galactose content shall not be less than 21.0%, and D-arabinose content shall not be less than 7.00% Acanthopanax senticosus leaf polysaccharide: total sugar content shall not be less than 8.00%, total uronic acid content shall not be less than 4.00%, D-mannose content in the polysaccharide monosaccharide composition shall not be less than 21.0%, L-rhamnose content shall not be less than 1.60%, D-glucuronic acid content shall not be less than 14.0%, D-glucose content shall not be less than 8.00%, D-galactose content shall not be less than 23.0%, and D-arabinose content shall not be less than 10.0%.

5. Acanthopanax fruit polysaccharide: the total sugar content shall not be less than 25.00%, the total uronic acid content shall not be less than 7.00%, the D-mannose content in the polysaccharide monosaccharide composition shall not be less than 31.0%, the L-rhamnose content shall not be less than 1.40%, the D-glucuronic acid content shall not be less than 6.00%, the D-glucose content shall not be less than 6.00%, the D-galactose content shall not be less than 27.0%, and the D-arabinose content shall not be less than 4.00%.

6. The method for preparing Acanthopanax senticosus polysaccharide according to claim 2, characterized in that The following steps are involved: The first step is the extraction of Acanthopanax polysaccharides: the dried roots of Acanthopanax are cut into small discs, crushed, sieved, degreased with ethanol, filtered, and dried to obtain solid medicinal powder. The solid medicinal powder is extracted multiple times with water, filtered, and the filtrates are combined to obtain a crude Acanthopanax polysaccharide solution.

7. The second step is to remove proteins and impurity small molecules from the Acanthopanax senticosus crude polysaccharide: the Acanthopanax senticosus crude polysaccharide solution obtained in the first step is subjected to membrane separation and macroporous resin methods to remove proteins and impurity small molecules, the eluate is collected, concentrated by rotary evaporation, and the Acanthopanax senticosus polysaccharide is obtained by alcohol precipitation.

8. A method for detecting the monosaccharide composition of Acanthopanax senticosus polysaccharides, characterized in that: The method is used to prepare a completely acid-hydrolyzed sample, and the method uses high performance liquid chromatography to determine the monosaccharide composition of Acanthopanax senticosus polysaccharide. The monosaccharide ratio of Acanthopanax senticosus polysaccharide is calculated by making a standard curve. The high performance liquid chromatography method uses an ultraviolet detector, and the detection conditions are: column temperature 30°C, detection wavelength 245nm, and liquid flow rate 0.7ml / min.

9. The method for determining the monosaccharide composition and content of Acanthopanax senticosus polysaccharide as claimed in claim 4, characterized in that: The detection conditions are as follows: column temperature of 30°C, detection wavelength of 245nm, and liquid flow rate of 0.7ml / min.

10. The method for determining the monosaccharide composition and monosaccharide content of Acanthopanax senticosus according to any one of claims 4 to 5, characterized in that: The high performance liquid chromatography method uses a 0.08 mol / L phosphate aqueous solution and acetonitrile in a volume ratio of 82:18 for isocratic elution.

11. The method for detecting the monosaccharide composition and monosaccharide content of Acanthopanax senticosus polysaccharide according to any one of claims 4 to 5 is characterized in that: The chromatographic column used in the high performance liquid chromatography method is a C18 liquid chromatography column; and / or, before the high performance liquid chromatography method is used to determine the monosaccharide composition and content, The method comprises the following steps: subjecting the Acanthopanax senticosus polysaccharide to acid hydrolysis.

12. A method for determining the monosaccharide composition and content of Acanthopanax senticosus polysaccharide in Acanthopanax senticosus, characterized in that: Use The method for detecting the monosaccharide composition and content of Acanthopanax senticosus according to any one of claims 4 to 8.

13. Use of a detection method in quality control of Acanthopanax senticosus, characterized in that: The method for detecting the composition and content of Acanthopanax senticosus monosaccharides as described in any one of claims 4 to 8 is adopted.

14. A method for distinguishing the difference between Acanthopanax senticosus plant polysaccharides and other plant polysaccharides based on the data obtained from the quality control method.

15. The quality control method according to claim 10, comprising the steps of determining the total sugar content by the phenol-sulfuric acid method, determining the total uronic acid content by the carbazole-sulfuric acid method, determining the infrared characteristics by Fourier transform infrared spectroscopy, and determining the monosaccharide composition and content by high performance liquid infrared spectroscopy.

16. The quality control method of claim 10, wherein the total sugar content, uronic acid content, infrared characteristics, and monosaccharide composition and content of Acanthopanax senticosus polysaccharide are determined.

17. Furthermore, the results of the polysaccharide content of Acanthopanax senticosus (root, stem, leaf and fruit) determined by the Acanthopanax senticosus quality control method were obtained.

18. The difference between the Acanthopanax senticosus plant polysaccharide obtained in claim 10 and other plant polysaccharides is that Acanthopanax senticosus polysaccharides at least include D-mannose, L-rhamnose, D-glucuronic acid, D-glucose, D-galactose, and D-arabinose.

19. The difference between the polysaccharides from different parts of Acanthopanax senticosus obtained in claim 10: First, compare the infrared characteristic spectra. −1 If there is a characteristic peak near 890, it is the Acanthopanax root or fruit polysaccharide. If there is a characteristic peak near 890, it is the Acanthopanax stem or leaf.

20. The use of the chemical characteristics of Acanthopanax senticosus polysaccharide according to any one of claims 1 to 15 in (1) and (2) (1) Distinction and identification of Acanthopanax senticosus polysaccharides and other plant polysaccharides; (2) Identification and quality control of the medicinal parts of Acanthopanax senticosus.

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