Polygonatum sibiricum polysaccharide extraction and purification process and application of polygonatum sibiricum polysaccharide in antioxidant health-care products

Abstract

The invention relates to the technical field of fine processing of traditional Chinese medicinal materials, in particular to a polygonatum polysaccharide extraction and purification process and application of the polygonatum polysaccharide extraction and purification process in anti-oxidation health care products, and the polygonatum polysaccharide extraction and purification process comprises the following steps: taking raw polygonatum rhizome, washing, slicing, drying, powdering, carrying out ethanol reflux extraction, carrying out suction filtration while hot, merging extracting solutions, and drying; then transferring into a round-bottom flask, boiling and extracting by taking water as an extraction solvent, combining water extraction filtrates, and concentrating under reduced pressure; under a continuous stirring condition, adding ethanol until the concentration is 80%; standing and carrying out alcohol precipitation in a refrigerator; centrifugally collecting the precipitate, adding distilled water to completely dissolve, and freeze-drying to obtain crude polygonatum sibiricum polysaccharide; dissolving the crude polysaccharide with distilled water, and adsorbing on a macroporous resin column; and eluting with distilled water, and freeze-drying to obtain pure polygonatum polysaccharide. The extraction of the crude polygonatum sibiricum polysaccharide effectively improves the extraction purity of polygonatum sibiricum polysaccharide and the content of active ingredients, and provides powerful support for efficient utilization of polygonatum sibiricum resources in the fields of medicine and health care.

Description

Technical Field

[0001] The present invention relates to the field of Chinese medicinal material processing, and in particular to a process for extracting and purifying polygonatum polysaccharide and application thereof in antioxidant health products. The polygonatum polysaccharide components are extracted efficiently by utilizing an innovative and improved extraction method, and have good antioxidant effects. The polysaccharide components are pure natural antioxidant active ingredients with excellent safety, and are of great significance for promoting the in-depth development and utilization of polygonatum resources in the big health industry. Background Art

[0002] In the field of traditional medicine, Polygonatum sibiricum is a traditional Chinese medicine with multiple health and medicinal effects, and its active ingredient, Polygonatum sibiricum polysaccharide, has attracted much attention. For a long time, many researchers have been committed to exploring the extraction process of Polygonatum sibiricum polysaccharide to meet the growing needs of the pharmaceutical and health care industries.

[0003] However, there are many problems with the existing extraction technology of Polygonatum sibiricum polysaccharide.

[0004] On the one hand, most extraction processes lack specificity and often use a general plant polysaccharide extraction process without fully considering the structural characteristics and composition characteristics of Polygonatum sibiricum itself. For example, in the selection and dosage of the extraction solvent, the control of the extraction temperature and time, they are not optimized according to the unique chemical composition of Polygonatum sibiricum, resulting in a large number of impurities in the extraction process, which are extracted together with Polygonatum sibiricum polysaccharides, making subsequent separation and purification more difficult.

[0005] On the other hand, due to the imperfection of the extraction process, the obtained polygonatum polysaccharide component is not ideal. Under conventional extraction methods, the content of effective active ingredients in polygonatum polysaccharide is low, and some polysaccharide branch structures with key pharmacological effects are destroyed or not fully released during the extraction process, resulting in poor performance of the final product in terms of pharmacological activity, bioavailability, etc., and the efficacy is not outstanding. This not only limits the in-depth application of polygonatum polysaccharide in drug research and development, but also makes it difficult to give full play to its due health care functions in the field of health care products, and it is impossible to provide consumers with high-quality and high-efficiency polygonatum polysaccharide related products. Therefore, there is an urgent need for a new and targeted polygonatum polysaccharide extraction process to solve the above problems in order to promote the efficient utilization of polygonatum resources and industrial development. Summary of the invention

[0006] The purpose of the present invention is to provide a process for extracting and purifying polygonatum polysaccharide and its application in antioxidant health products in view of the defect that the extraction process of polygonatum polysaccharide in the prior art is not perfect.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A process for extracting and purifying polygonatum polysaccharide comprises the following steps:

[0009] S1. Pretreatment: Take raw rhizomes of polygonatum, wash and slice them, dry them at 50-80°C for 10-24h, and grind them into powder using a grinder to obtain polygonatum powder;

[0010] S2, ethanol extraction: take Polygonatum sibiricum powder, add 3-6 times the amount of 90v% or more ethanol, reflux extraction at 50-60°C for 1-4 times, the first extraction time is 1.5-2h, and the subsequent extraction time is 0.5-1.2h each time, filter while hot, and combine to obtain the first extract;

[0011] The filter residue is washed 1-3 times with hot ethanol with a purity of more than 90v%, the washing solution is degreased, and then the washing solution is combined with the first extract;

[0012] Then, drying the first extract at 50-65°C;

[0013] S3, water extraction: the dried product of the first extract is placed in a round-bottom flask, and water is used as the extraction solvent, and the boiling extraction is performed 1-2 times according to the weight ratio of the feed liquid g / mL of 1:7-15, and the timing is started from the boiling of the water each time, and the extraction is performed for 2-6 hours; after the completion of the water extraction, the water extraction filtrate is combined, and the second extract is obtained by concentrating under reduced pressure at 50-65°C;

[0014] S4, alcohol precipitation and drying: adding an ethanol solution with a concentration of 90% or more to the second extract under continuous stirring until the ethanol concentration in the second extract is 80%;

[0015] Then, the mixture is placed in a refrigerator at 1-4°C for 18-30 hours to allow alcohol precipitation; the precipitate is collected by centrifugation, the precipitate is completely dissolved in distilled water, and freeze-dried to obtain a raw product of Polygonatum sibiricum crude polysaccharide;

[0016] S5, taking a resin sample, immersing it in anhydrous ethanol for activation, wet-loading it into a column, and preparing an elution column; the resin is a macroporous resin;

[0017] The raw Polygonatum sibiricum polysaccharide obtained in S4 was dissolved in distilled water to prepare a 10-50 mg / mL Polygonatum sibiricum polysaccharide solution.

[0018] The crude polysaccharide solution of polygonatum sibiricum is loaded on the column for adsorption, and then eluted with distilled water of the same volume as the adsorption column, and the eluate from the 30% to the 70% portion is collected. The collected elution product is freeze-dried to obtain pure polygonatum sibiricum polysaccharide.

[0019] In the present invention, "first extract" and "second extract" are only used to distinguish between extracts extracted by ethanol and extracts extracted by water, and "first" and "second" do not constitute substantial limitations.

[0020] The present invention adopts alcohol extraction and water precipitation and water alcohol precipitation process for purification and extraction to obtain high-purity crude polygonatum polysaccharide, and then uses macroporous resin for adsorption and purification to remove impurities such as protein, and the product has high purity. After double extraction and purification, the polygonatum polysaccharide is obtained. After in vitro antioxidant activity experiments, it is shown that the polygonatum polysaccharide is purified and the free radical scavenging rate is greatly improved by more than 110%. The corresponding polygonatum polysaccharide has higher medicinal value, which is of great significance for the development of new products in the high value-added health industry of polygonatum.

[0021] Further, in S1, pretreatment: take raw Polygonatum sibiricum rhizome, wash it and cut it into small pieces with a thickness of 2-5 mm, and dry it at a temperature range of 50° C.-70° C. for 12 h-24 h.

[0022] Further, in S1, raw rhizomes of Polygonatum sibiricum are taken, screened, and parts that are rotten, deteriorated, or have diseases and insect pests are removed, and then washed and sliced.

[0023] Further, in S2, ethanol extraction: weigh a certain amount of polygonatum powder, add 3-5 times the amount of 90%-98% ethanol, reflux extract twice at 55°C-65°C, each extraction time is 1h-2h, after the extraction is completed, filter while hot, and combine to obtain the first extract.

[0024] Further, in S2, filtering is performed while hot by using one or more combinations of filter paper filtration, filter cloth filtration or microporous membrane filtration.

[0025] Furthermore, in S2, the degreasing process can be separated and degreased using a separatory funnel.

[0026] Furthermore, in S2, the heating device during reflux extraction is a constant temperature water bath.

[0027] Further, in S3, water extraction: the dried sample is placed in a round-bottom flask, water is used as the extraction solvent, and boiling extraction is performed twice in a material-liquid weight ratio of 1:8-12, with each time starting from the boiling of the water, and the extraction is performed for 3-5 hours; after the water extraction is completed, it is filtered, the water extraction filtrate is combined, and the second extract is concentrated under reduced pressure at 55-65°C.

[0028] Further, in S4, alcohol precipitation and drying: under continuous stirring, an ethanol solution with a concentration of 95v% or more is added to the second extract until the ethanol concentration in the second extract reaches 80v%.

[0029] Further, the mixture is placed in a refrigerator at 2-4°C for 18-24 hours in S4 to allow alcohol precipitation; the precipitate is then collected by centrifugation at 3000-5000 rpm for 10-20 minutes; the precipitate is completely dissolved in distilled water, and freeze-dried to obtain raw Polygonatum sibiricum crude polysaccharide.

[0030] Further, in S4, distilled water is added to the precipitate, stirred to fully dissolve, and then placed in a freeze dryer for freeze drying to obtain raw Polygonatum sibiricum crude polysaccharide.

[0031] Preferably, the freezing temperature of the freeze dryer is -50°C to -80°C, and the drying time is 24h to 48h.

[0032] Further, in S5, preferably, the macroporous resin is one of NKA-9, D101, HPD600, HP20, AB-8, and HPD750. Preferably, NKA-9 macroporous adsorption resin is used.

[0033] Furthermore, in S5, the purity of the obtained pure polygonatum polysaccharide reaches more than 70%.

[0034] The present invention also provides a new use of the polygonatum polysaccharide prepared by the method.

[0035] An application of the polygonatum polysaccharide obtained by the above polygonatum polysaccharide extraction and purification process, especially in the preparation of health care products. Preferably, the polygonatum polysaccharide is used in the preparation of antioxidant health care products.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The method of the present invention adopts a combination of high-concentration ethanol extraction + water extraction to extract selective polysaccharide components. The obtained polygonatum polysaccharide component has higher selectivity and the corresponding polygonatum polysaccharide has better activity performance, which is of great significance for the development of downstream high-value-added applications of polygonatum polysaccharide.

[0038] 2. The method for extracting raw polygonatum polysaccharide by the method of the present invention combines the coordinated optimization of multiple factors to fully purify polygonatum polysaccharide and obtain a high-purity polygonatum polysaccharide product with low lipid and protein content. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the elution curve of each component.

[0040] Figure 2 This is the infrared absorption chromatogram of crude Polygonatum sibiricum polysaccharide before and after purification by macroporous adsorption resin.

[0041] Figure 3 The scavenging ability of Polygonatum sibiricum polysaccharide on DPPH free radicals.

[0042] Figure 4 The scavenging ability of Polygonatum sibiricum polysaccharide on ABTS+ free radicals.

[0043] Figure 5 The scavenging ability of Polygonatum sibiricum polysaccharide on hydroxyl free radicals.

[0044] Figure 6 It is the total reducing capacity of Polygonatum sibiricum polysaccharide. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is described below in conjunction with specific implementation methods. It should be understood that the corresponding description is only for the convenience of explaining the implementation methods and processes of the relevant technical solutions, and is not a limitation to the technical solution of the present invention. Any technical solution based on the same concept of the present invention should be included in the protection scope of the present invention.

[0046] 1. Experimental instruments and materials

[0047] 1.1 Instruments and reagents

[0048] MAPADA UV-visible spectrophotometer (Shanghai Mepda Instrument Co., Ltd.); electronic analytical balance (Sartorius Scientific Instrument (Beijing) Co., Ltd.); stepless power regulation ultrasonic cleaning machine (Xiaomei Ultrasonic Instrument (Kunshan) Co., Ltd.); EYELA rotary evaporator (Shanghai Ailang Instrument Co., Ltd.); electric blast drying oven (Beijing Zhongxing Weiye Century Instrument Co., Ltd.); TDZ5-WS desktop low-speed centrifuge (Changsha Xiangyi Centrifuge Instrument Co., Ltd.); freeze dryer (Beijing Yaxing Instrument Technology Development Co., Ltd.), etc.

[0049] NKA-9, D101, HPD600, HP20, AB-8, and HPD750 macroporous resins were purchased from Zhengzhou Hecheng New Material Technology Co., Ltd.; Coomassie Brilliant Blue G250 was purchased from Shanghai Maikun Chemical Co., Ltd., production batch number 20230316; glucose reference substance was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., production batch number wkq22111408; 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), L(+)-ascorbic acid (vitamin C), o-phenanthroline, phenol, concentrated sulfuric acid, anhydrous ethanol, potassium persulfate, anhydrous sodium dihydrogen phosphate, potassium ferrocyanide, and trichloroacetic acid were purchased from Chengdu Cologne Chemical Co., Ltd.; sterile PBS was purchased from Bost Biotechnology Co., Ltd.; 30% hydrogen peroxide was purchased from Shanghai Wokai Biotechnology Co., Ltd.; ferrous sulfate was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; anhydrous disodium hydrogen phosphate was purchased from Sinopharm Chemical Reagent Co., Ltd.; anhydrous ferric chloride was purchased from Shanghai McLean Biochemical Co., Ltd. All of the above reagents were analytical grade.

[0050] 1.2 Medicinal Materials

[0051] The polygonatum cyrtonema Hua medicinal material used in the experiment was identified by Associate Professor Long Fei of the Pharmacognosy Teaching and Research Section, School of Pharmacy, Chengdu University of Traditional Chinese Medicine as the dried rhizome of Polygonatum cyrtonema Hua, a plant of the Liliaceae family.

[0052] 2. Experimental Methods

[0053] 2.1 Extraction of raw Polygonatum sibiricum

[0054] Take 200g of raw rhizome of Polygonatum sibiricum, wash it with clean water, cut it into small pieces, and bake it in a 60℃ oven for 18h until the Polygonatum sibiricum is dried, grind it into powder with a grinder, weigh 200g of Polygonatum sibiricum powder, add 4 times the amount of 95% ethanol, reflux extract at 60℃ for 1.5h and 1h respectively, filter it while hot, remove fat, wash the residue with 95% hot ethanol 3 times, and dry it at 60℃. Take the dried sample and place it in a round-bottom flask, boil it in an electric heating mantle with water as the extraction solvent (solid-liquid ratio 1:10) for 4h (start timing after the water boils), extract it twice, filter it after the reaction is completed, combine the filtrate, and concentrate it under reduced pressure at 60℃ to obtain 700mL of extract.

[0055] Under continuous stirring, add 95% ethanol to the above extract to a final concentration of 80%, and place it in a 4°C refrigerator for 24 hours. Perform alcohol precipitation twice, centrifuge (4000rpm×15min), collect the precipitate, add distilled water to completely dissolve the precipitate, and place it in a freeze dryer for freeze drying to obtain raw Polygonatum sibiricum crude polysaccharide.

[0056] 2.2 Determination of the absorption wavelength of pigments

[0057] According to the principle of color complementation, the color of the solution is determined by the wavelength it absorbs. The solution of Polygonatum sibiricum polysaccharide is orange-yellow, indicating that it absorbs blue light with a wavelength of about 450nm. Therefore, 450nm was selected as the wavelength for pigment determination. The decolorization rate calculation formula is:

[0058] (A 脱色前 -A 脱色后 ) / A 脱色前 ×100%

[0059] 2.3 Determination of total protein content and polysaccharide content

[0060] The total protein content in Polygonatum cyrtonema polysaccharide was determined using the Coomassie Brilliant Blue method. The test solution was prepared according to the instructions of the kit, and the dilution ratio of the polysaccharide solution was adjusted to ensure that the protein concentration was less than 1.3 g / mL. After standing for 5 minutes, the absorbance value was measured at a wavelength of 595 nm using a UV-visible spectrophotometer. The protein removal rate was calculated as follows:

[0061] (D 脱蛋白前 -D 脱蛋白后 ) / D 脱蛋白前 ×100%

[0062] The phenol-sulfuric acid method was used to determine the sugar content in Polygonatum cyrtonema polysaccharide, and the polysaccharide retention rate was calculated as follows:

[0063] C 后 / C前 ×100%

[0064] 2.4 Resin pretreatment

[0065] Take a resin sample, soak it in anhydrous ethanol for 12 hours, wet-pack it into a column, release the soaking liquid, and continue washing with ethanol until the washing liquid is not turbid when adding water and there is no absorption peak under UV scanning. Then use a large amount of distilled water as the mobile phase to wash the resin column until the eluent has no ethanol smell, and store it in a wet state at low temperature.

[0066] 2.5 Static adsorption experiment

[0067] Take 10 mL of each of the 6 different types of macroporous resins (NKA-9, D101, HPD600, HP20, AB-8, HPD750) that have been treated and place them in a 100 mL conical flask, 6 portions of each, add 20 mL of 30 mg / mL crude Polygonatum cyrtonema polysaccharide solution, adsorb on a slow shaker at 30°C for 12 hours, and then filter. Subsequently, the resin was washed with 20 mL of deionized water, adsorbed again for 12 hours, filtered, and the filtrate was combined to 25 mL, and the absorbance at 450 nm was measured.

[0068] The comprehensive score S is calculated by the polysaccharide retention rate A (40% weight), protein removal rate B (30% weight) and decolorization rate C (30% weight), that is, S = 0.4A + 0.3B + 0.3C. SPSS25.0 was used for single-factor variance analysis to determine the resin with the best purification effect. In this process, the macroporous resin retains the polysaccharide in the eluent by adsorbing the pigment and protein in the solution to achieve purification.

[0069] 2.6 Dynamic adsorption experiment

[0070] Resins with excellent static screening performance were selected for wet column loading (column height 50cm, diameter 2cm), and each resin was operated in parallel for 6 times, and the wet volume of the resin was 50mL. About 28mg / mL of crude Polygonatum sibiricum polysaccharide solution was added to half of the resin bed volume, and the flow rate was controlled to be 1min / mL, so that the polysaccharide, pigment and protein were allowed to stand and adsorb in the column for 30 minutes to achieve saturated adsorption. Subsequently, distilled water was used for elution at a flow rate of 1min / mL until the Molish reaction of the eluate was negative, and collection was stopped. According to the scoring criteria under item 2.5, a one-way analysis of variance was performed using SPSS25.0 to determine the resin with the best effect of purifying Polygonatum sibiricum polysaccharide under dynamic conditions.

[0071] 2.7 Single factor loading concentration study

[0072] On the basis of determining the optimal resin model, the column was loaded according to item 2.6, the flow rate was controlled at 1 mL / min, and the raw Polygonatum sibiricum crude polysaccharide solutions were loaded with mass concentrations of 0.01, 0.02, 0.03, 0.04, and 0.05 g / mL, respectively. The distilled water was used for elution. The results of each condition were analyzed based on the weighted values ​​of polysaccharide retention rate, pigment removal rate, and protein removal rate, and a comprehensive score was obtained to determine the optimal loading concentration of the raw Polygonatum sibiricum polysaccharide solution under this column loading condition.

[0073] 2.8 Investigation of single factor elution flow rate

[0074] Determine the column packing conditions and sample concentration according to the above experimental results, and pack the column according to 2.6. The flow rates are controlled at 0.5, 1.0, 1.5, and 2.0 min / mL, respectively. Calculate the optimal elution flow rate using the same procedure as above.

[0075] 2.9 Investigation of single factor loading volume

[0076] Determine the column packing conditions, sample concentration, and elution flow rate according to the above experimental results, and pack the column according to 2.6. The sample volume is controlled to 5, 10, 15, and 20 mL respectively. Calculate the optimal sample volume using the same operation as above.

[0077] 2.10 Investigation of elution curve and elution volume

[0078] 2.10.1 Drawing of elution curve

[0079] Determine the column packing conditions, sample concentration, elution flow rate, and sample volume according to the above experimental results. Pack one column according to the operation in 2.6. Elute with distilled water, collect 5 mL of a portion (one tube), and measure the absorbance of each tube at 450 nm (pigment), 490 nm (polysaccharide), and 595 nm (protein) in sequence using a UV-visible spectrophotometer. Draw an elution curve with the collection volume as the horizontal axis and the absorbance of each component as the vertical axis. The purpose is to screen out a suitable elution volume.

[0080] 2.10.2 Determination of elution volume

[0081] According to the elution curve, 45 mL (from the 3rd tube to the 12th tube), 40 mL (from the 4th tube to the 12th tube), and 35 mL (from the 5th tube to the 12th tube) were collected. The absorbance of each portion was measured at 450 nm (pigment), 490 nm (polysaccharide), and 595 nm (protein) using a UV-visible spectrophotometer in sequence. The results of each condition were analyzed with the weighted values ​​of polysaccharide retention rate, pigment removal rate, and protein removal rate, and a comprehensive score was obtained to determine the optimal elution volume.

[0082] 2.11 Orthogonal Experimental Design

[0083] Through the previous single-factor experiment, the three-factor three-level experimental factors and level table as shown in Table 1 were determined, and L9 (3 4 ) orthogonal table, with sample mass concentration (A), flow rate (B), elution volume (C) as independent variables, and weighted comprehensive scores of polysaccharide retention rate, decolorization rate and protein removal rate as dependent variables, an orthogonal experiment was carried out, and finally the optimal purification process of raw Polygonatum sibiricum crude polysaccharide was obtained.

[0084] Table 1 Experimental factors and levels

[0085] level A B C Loading concentration mg / mL Elution flow rate min / mL Elution volume mL 1 30 0.5 35 2 40 1 40 3 50 1.5 45

[0086] 2.12 FT-IR spectral feature analysis

[0087] Take appropriate amount of freeze-dried samples of raw Polygonatum sibiricum crude polysaccharide before and after adsorption, press them into tablets by KBr method, and use IRAffinity-1s Fourier transform infrared spectrophotometer at 4000-400 cm -1 The infrared range is scanned and the absorption spectrum is recorded.

[0088] 2.13 In vitro antioxidant activity test

[0089] 2.13.1 DPPH free radical scavenging effect

[0090] Referring to the method of Tahidul Islam, Yao F, Kang W et al. in the article A systematic study on mycochemical profiles, antioxidant, and anti-inflammatory activities of 30 varieties of Jew's ear (Auricularia auricula-ju dae) [J]. Food Science and Human Wellness, 2022, 11 (04): 781-794., a series of concentrations (0.2-1.0 mg·mL-1) of purified and processed Polygonatum sibiricum solutions were prepared. Accurately weigh 2 mg DPPH, dissolve it in anhydrous ethanol and dilute to 100 mL. Take test tubes respectively, add 2 mL of extract and 2 mL of DPPH solution, and measure the absorbance Ai; add 2 mL of extract and 2 mL of anhydrous ethanol, and measure the absorbance Aj; add 2 mL of 50% ethanol and 2 mL of DPPH solution, and measure the absorbance Ac. After 30 minutes of light-proof reaction, measure the absorbance at a wavelength of 517 nm and calculate the clearance rate. At the same time, vitamin C solution with the same concentration was used as a positive control.

[0091] DPPH free radical scavenging rate (%) = [1-(Ai-Aj) / Ac] × 100% (1)

[0092] 2.13.2 ABTS+ free radical scavenging effect

[0093] According to the method of Cui Qiaoyu et al. (Cui Qiaoyu, Shen Wenjuan, Sun Xiaolu, et al. Optimization of the extraction process of total polyphenols from tulips and study of their antioxidant activity [J]. Chinese Patent Medicine, 2022, 44(06): 1918-1922.), ABTS+ working solution was prepared: 7mmol / LABTS and 2.45mmol / L potassium persulfate were mixed in equal volumes, allowed to stand at room temperature in the dark for 12 hours, and diluted with anhydrous ethanol to an absorbance of 0.700±0.005 at 734nm. Next, take a test tube and add 4mL ABTS solution and 1mL of different concentrations (0.2-1.0mg·mL-1) of processed Polygonatum sibiricum solution before and after purification, and measure the absorbance Ai; take another test tube and add 4mL anhydrous ethanol and 1mL of different concentrations (0.2-1.0mg·mL-1) of processed Polygonatum sibiricum solution before and after purification, and measure the absorbance Aj; and add 4mL ABTS+ solution and 1mL ultrapure water, and measure the absorbance as Ac; after reacting at room temperature and avoiding light for 6 minutes, measure the absorbance at a wavelength of 734nm, and calculate the ABTS+ clearance rate. At the same time, use the same concentration of vitamin C solution as a positive control.

[0094] ABTS+ free radical scavenging rate (%) = [1-(Ai-Aj) / Ac] × 100% (2)

[0095] 2.13.3 Hydroxyl radical scavenging effect

[0096] The determination method of Xiong Ying et al. ([9] Xiong Ying, Fan Junfu, Xue Junwen, et al. Optimization of ultrasonic-assisted extraction process and antioxidant activity of tea polyphenols [J]. Chinese Modern Applied Pharmacy, 2020, 37(02): 175-179. DOI: 10.13748 / j.cnki.issn1007-7693.2020.02.010.) was used. 5×10 -3 1.0 mL of 1 mol / L o-phenanthroline solution, 0.5 mL of 7.5×10-3 mol / L ferrous sulfate solution, 1.0 mL of PBS buffer, 1.0 mL of purified and processed Polygonatum sibiricum solution of different concentrations (0.2-1.0 mg·mL-1), and 0.5 mL of 0.1% hydrogen peroxide were added to distilled water to make up to 10 mL. After 30 minutes in a 37°C water bath, the absorbance was measured at a wavelength of 510 nm, and the clearance rate was calculated according to formula (2). At the same time, a vitamin C solution of the same concentration was used as a control.

[0097] Hydroxyl radical scavenging rate (%) = (A1-A2) / (A3-A2) × 100% (3)

[0098] Wherein, A1 is the extracting solution; A2 is water replacing the extracting solution; A3 is water replacing the extracting solution and H2O2.

[0099] 2.13.4 Determination of total reducing capacity

[0100] Referring to the method of An Min, Liu Huanming, Zhong Qiuhong et al. (Screening of marine lactic acid bacteria producing extracellular polysaccharides and optimization of polysaccharide fermentation process [J]. Journal of Guangdong Ocean University, 2023, 43(05): 106-112.), the total reducing capacity of processed Polygonatum sibiricum polysaccharide was determined by potassium ferrocyanide reduction method. After the preliminary test, 1.0 mL of processed Polygonatum sibiricum solution before and after purification with different concentrations (0.2-1.0 mg·mL-1) was taken, 1.0 mL of 0.2 mol / L phosphate buffer (pH 6.6) and 1.0 mL of 1% potassium ferrocyanide solution were added, mixed and reacted at 50°C for 20 minutes. Then, 1.0 mL of 10% trichloroacetic acid solution and 1.0 mL of 0.1% ferric chloride solution were added, mixed and allowed to stand for 10 minutes, and the absorbance at 700 nm was measured. At the same time, distilled water was used as a blank control, and the same concentration of vitamin C solution was used as a positive control.

[0101] Total reducing power (H) = A1-A0 (4)

[0102] Where A0 is the absorbance of the blank control group; A1 is the absorbance of the sample group.

[0103] 3. Experimental results

[0104] 3.1 Results of static and dynamic adsorption experiments

[0105] Table 2 Comprehensive score table of static adsorption of different resins ( n=3)

[0106]

[0107]

[0108] According to the static adsorption results (Table 2), D3520 resin exhibited the strongest adsorption capacity with an adsorption rate of (59.61±0.498)%, while HP-20 had the weakest adsorption capacity with an adsorption rate of (10.89±0.549)%.

[0109] The static desorption capacity of all resins was generally weak, which may be due to the failure to reach desorption equilibrium. NKA-9 resin had the highest desorption rate (95.14±0.42)%, while HPD-600 had the lowest desorption rate (72.14±4.90)%. In order to screen out the best resin, the weighted value (adsorption rate × 50% + desorption rate × 50%) was used for evaluation. The results showed that D101 macroporous adsorption resin had the highest score, and HPD600 had the lowest score. The scores were ranked from high to low as D101, D3520, NKA-9, HPD750, HP-20, and HPD600. Therefore, four resins, NKA-9 (77.11±0.39)%, D3520 (74.94±0.91)%, D101 (67.81±0.61)%, and HPD750 (67.80±0.83)%, were selected for further dynamic adsorption screening.

[0110] Table 3 Comprehensive score table of dynamic adsorption of different resins

[0111] Resin Model Polysaccharide retention rate (%) Protein elution rate (%) Pigment elution rate (%) Comprehensive score (%) D101 55.31±6.71 83.91±3.47 61.91±5.36 65.87±4.13 NKA-9 69.00±4.39 81.47±2.62 48.76±6.00 66.67±1.14 D3520 48.97±0.50 89.62±1.43 27.48±10.49 54.72±3.23 HPD750 51.24±6.71 78.22±2.98 37.00±6.99 56.27±3.10

[0112] According to the dynamic adsorption results of different resins in Table 3, the highest comprehensive score of purified raw Polygonatum sibiricum crude polysaccharide was NKA-9 macroporous resin (66.67±1.14)%, and the lowest was D3520 (54.72±3.23)%. Considering the comprehensive economic benefits, NKA-9 resin was selected as the best dynamic adsorption resin and used in subsequent experiments.

[0113] 3.2 Single factor investigation results

[0114] 3.2.1 Sample concentration

[0115] The effect of loading mass concentration on the purification of Polygonatum cyrtonema polysaccharide by NKA-9 resin (Table 4) shows that when the loading mass concentration is 0.04 g / mL, the comprehensive score of the purification effect reaches the maximum value, so the loading mass concentration of 0.04 g / mL is selected as the midpoint of the response surface star point design.

[0116] Table 4 Effect of sample concentration on purification of Polygonatum cyrtonema polysaccharide by NKA-9 resin

[0117]

[0118] 3.2.2 Elution flow rate

[0119] Effects of different flow rates on the purification of Polygonatum cyrtonema polysaccharide by NKA-9 resin (Table 5, Figure 1 ), it can be seen that the comprehensive score reaches the maximum value when the flow rate is 1.0 min / mL, so the flow rate of 1.0 min / mL is selected as the midpoint of the response surface star point design.

[0120] Table 5 Effect of different flow rates on the purification of Polygonatum cyrtonema polysaccharide by NKA-9 resin

[0121] Flow rate / (min / mL) Polysaccharide retention rate / % Decolorization rate / % Protein removal rate / % Overall Rating 0.5 80.65 59.38 40.19 62.13 1 84.51 60.95 43.72 65.21 1.5 87.58 48.27 43.69 62.62 2 91.63 34.04 39.81 58.81

[0122] 3.2.3 Sample loading

[0123] The effect of different sample loading amounts on the purification of Polygonatum cyrtonema polysaccharide by NKA-9 resin (Table 6). It can be seen from the data that the comprehensive score reaches the maximum value when the sample loading amount is 5 mL, so the sample loading amount of 10 mL is selected as the midpoint of the response surface star point design.

[0124] Table 6 Effect of sample loading on purification of Polygonatum cyrtonema polysaccharide by D101 resin

[0125] Sample volume / mL Polysaccharide retention rate / % Decolorization rate / % Protein removal rate / % Overall Rating 5 85.03 31.38 45.44 57.06 10 77.44 23.11 23.32 44.90 15 89.59 19.17 20.57 47.76 20 84.61 16.63 23.13 45.77

[0126] 3.2.4 Elution curve

[0127] Starting from the 4th tube, the absorbance of protein and pigment increased significantly, and the absorbance of polysaccharide increased slightly, indicating that when the elution solution reached 20mL, pigment and protein were eluted in large quantities, while polysaccharide was only eluted in small quantities. Figure 1 ), according to the elution curve, collect 45mL (from the 3rd tube to the 12th tube), 40mL (from the 4th tube to the 12th tube), and 35mL (from the 5th tube to the 12th tube). The absorbance of each portion is measured at 450nm (pigment), 490nm (polysaccharide), and 595nm (protein) in sequence using a UV-visible spectrophotometer. The results of each condition are analyzed with the weighted values ​​of polysaccharide retention rate, pigment removal rate, and protein removal rate, and a comprehensive score is obtained to determine the optimal elution volume. Figure 1 shown.

[0128] 3.2.4 Elution volume

[0129] The effect of different elution volumes on the purification of Polygonatum cyrtonema polysaccharide by NKA-9 resin (Table 7) showed that the comprehensive score was the maximum when the elution volume was 40 mL, so the elution volume of 40 mL was determined as the midpoint of the response surface star point design.

[0130] Table 7 Effect of elution volume on purification of Polygonatum cyrtonema polysaccharide by NKA-9 resin

[0131] Elution volume / (mL) Polysaccharide retention rate / % Decolorization rate / % Protein removal rate / % Overall Rating 35 63.60 52.36 67.11 61.28 40 88.04 49.78 64.43 69.48 45 93.36 43.99 49.06 65.26

[0132] 3.3 Orthogonal test results

[0133] As can be seen from the table, the order of influence of these three factors on the experimental results is A>C>B, that is, the factor that has the greatest impact on the experimental results is the sample concentration, followed by the elution volume, and finally the elution flow rate. As can be seen from the table, the two factors that have a significant effect (P < 0.05) on the purification of raw Polygonatum sibiricum crude polysaccharides are the sample concentration and the elution volume, while the elution flow rate has no significant effect on the experimental results. Finally, combining intuitive analysis and variance analysis, it was determined that the optimal process for the purification of raw Polygonatum sibiricum polysaccharides is A2B1C2, with a sample concentration of 40 mg / mL, an elution flow rate of 0.5 mL / min, and an elution volume of 40 mL. However, in order to further determine the accuracy and authenticity of the test results, further verification experiments are needed.

[0134] Table 8 Orthogonal test results and analysis

[0135]

[0136]

[0137] Table 9 Analysis of variance

[0138] Sources of variance Sum of Squares of Deviations Degrees of Freedom Mean Square F-number P-value A 172.179 2 86.090 249.222 0.004 B 0.291 2 0.146 0.421 0.703 C 100.062 2 50.031 144.835 0.007 error 0.691 2 0.345

[0139] 3.4 Verification Experiment

[0140] According to the obtained model, the optimal process conditions are predicted: using a column with a length of 50 cm and a diameter of 2 cm, a sample mass concentration of 40.00 mg / mL, a flow rate of 0.5 min / mL, an elution volume of 40.00 mL, and a sample volume of 5 mL. Under these conditions, the weighted value of extracting Polygonatum sibiricum polysaccharide can reach 69.07%. In order to eliminate the influence of experimental errors and determine the stability and accuracy of the optimal process, 2.00 g of raw Polygonatum sibiricum crude polysaccharide was accurately weighed, added to distilled water to dissolve, and fixed to a 50 mL volumetric flask. Three columns with a length of 50 cm and a diameter of 2 cm were taken, and the experiment was carried out according to the above optimal process.

[0141] The results showed that the polysaccharide retention rate was (84.94±0.95)%, the protein removal rate was (67.06±0.93)%, and the pigment removal rate was (57.22±1.09)%, which were close to the predicted optimal process results. Therefore, the optimal process for the purification of raw Polygonatum sibiricum polysaccharide was determined as follows: 50 mL of wet volume of NKA-9 macroporous adsorption resin was used for column loading (column length 50 cm, diameter 2 cm), the loading mass concentration of raw Polygonatum sibiricum polysaccharide was 40 mg / mL, the elution flow rate was 0.5 min / mL, the elution volume was 40 mL, and the loading amount was 5 mL.

[0142] 3.5 Structural changes of Polygonatum cyrtonema polysaccharide before and after adsorption by macroporous adsorption resin

[0143] like Figure 2As shown in the FT-IR spectra before and after adsorption, the -1 A strong absorption peak of hydroxyl stretching vibration appeared near the surface, indicating the existence of stretching vibration of OH or intermolecular hydrogen bonds; at 2928.2cm -1 There is a weak absorption peak near it, which is the CH stretching vibration; at 1628.9cm -1 The characteristic absorption peak near 1026.2cm is the symmetrical stretching vibration of the C=O bond of the carboxyl group, indicating the possible presence of a -COOH group; -1 The characteristic absorption peak near 930cm represents the stretching vibration of the COC glycosidic bond on the pyranose ring; -1 The characteristic absorption peak near represents the α-glycosidic bond. Comparing the changes before and after adsorption, it was found that the main characteristic signals of the polysaccharide FT-IR absorption spectrum did not change significantly. The results showed that after the raw Polygonatum sibiricum crude polysaccharide was adsorbed by NKA-9 macroporous resin, the main functional group structure did not change, and the interference of impurity absorption signals was reduced by removing impurities.

[0144] 3.6 In vitro antioxidant activity

[0145] 3.6.1 Scavenging activity against DPPH free radicals

[0146] The results of scavenging rate of raw Polygonatum sibiricum polysaccharide on DPPH free radicals before and after purification are as follows Figure 3 . As shown in the raw polygonatum polysaccharide concentration range of 1-5 mg / mL, as the concentration of polysaccharide increases, its scavenging ability for DPPH free radicals continues to increase, and the purified raw polygonatum polysaccharide has a stronger scavenging effect on DPPH free radicals than the raw polygonatum polysaccharide before purification, but both are lower than the positive control group VC group, with weaker antioxidant capacity. The results show that the raw polygonatum polysaccharide before and after purification can scavenge DPPH free radicals, and the purified raw polygonatum polysaccharide has a stronger scavenging effect on DPPH free radicals than the raw polygonatum polysaccharide before purification.

[0147] 3.6.2 Scavenging activity against ABTS+ free radicals

[0148] The results of scavenging rate of ABTS+ free radicals by processed Polygonatum sibiricum polysaccharide before and after purification are as follows Figure 4 As shown. Compared with VC, the scavenging effect of processed Polygonatum sibiricum polysaccharides before and after purification on ABTS+ was dose-dependent, among which the purified Polygonatum sibiricum polysaccharide had a stronger scavenging effect. Raw Polygonatum sibiricum had a weak scavenging effect on ABTS+ free radicals in the concentration range of 1-5 mg / mL, and its scavenging rate reached 42.50% when the concentration after purification was 5 mg / mL. The results showed that raw Polygonatum sibiricum polysaccharides before and after purification had a certain scavenging effect on ABTS+ free radicals, and the purified polysaccharide had a stronger scavenging ability than the polysaccharide before purification.

[0149] 3.6.3 Scavenging activity against hydroxyl radicals

[0150] The results of scavenging rate of raw Polygonatum sibiricum polysaccharide on hydroxyl free radicals before and after purification are as follows Figure 5 As shown in the figure, as the concentration of raw Polygonatum sibiricum increases, its scavenging activity against hydroxyl free radicals is constantly increasing. However, compared with Vc, there is still a lot of gap. The results show that the purified raw Polygonatum sibiricum polysaccharide has a stronger scavenging effect on hydroxyl free radicals than the purified one.

[0151] 3.6.4 Total reduction capacity

[0152] The total reducing capacity of raw Polygonatum cyrtonema polysaccharide before and after purification is as follows Figure 6 As shown. The stronger the reducing ability, the stronger the antioxidant property. As can be seen from the figure, with the increase of the concentration of raw Polygonatum sibiricum polysaccharide, the absorbance value continues to increase, indicating that its total reducing ability continues to increase. And the purified raw Polygonatum sibiricum polysaccharide has stronger total reducing power than the purified one. But compared with Vc, there is still a lot of gap.

[0153] 4. Conclusion

[0154] Pigment and protein are the main impurities in polysaccharide research, affecting structural analysis, macroporous resin, due to its unique composition and structure, combines adsorption and screening functions, compared to other adsorbents or gel-type resins, with higher adsorption capacity, easy elution, high strength and strong anti-pollution ability, widely used in various fields. The present invention adopts macroporous resin method to purify polygonatum polysaccharide, and experimental results show that the optimal process for the purification of raw polygonatum polysaccharide selects 50mL wet volume of NKA-9 macroporous adsorption resin to load the column (column length 50cm, diameter 2cm), the loading mass concentration of raw polygonatum polysaccharide is 40mg / mL, the elution flow rate is 0.5min / mL, the elution volume is 40mL, and the loading amount is 5mL. Polysaccharide retention rate (84.94±0.95)%, protein removal rate (67.06±0.93)%, pigment removal rate (57.22±1.09)%, and the predicted optimal process result is close. After purification, the polysaccharide content of processed polygonatum increased from 41.65% to 71.71%; after purification, the polysaccharide content of raw polygonatum increased from 47.23% to 64.22%.

[0155] In a specific embodiment of the present invention, the purified polygonatum polysaccharide product obtained by the above method can be added to food and auxiliary materials to prepare a health product.

[0156] For example, to prepare tablets:

[0157] Take 2 g of the purified polygonatum polysaccharide product, 50 g of medicinal starch, and 5 g of magnesium stearate to prepare 300 film-coated tablets.

[0158] Purified polygonatum polysaccharide is mixed with medicinal starch and magnesium stearate in equal increasing amounts, and a small amount of 60v% ethanol is added to obtain microgranules, which are then made into tablet granules, dried and granulated, and magnesium stearate is added to mix the mixture, pressed into tablets, and film-coated. The obtained film-coated tablets are the antioxidant health products of polygonatum polysaccharide.

[0159] For example, to prepare granules:

[0160] 2g of polygonatum polysaccharide product, 88g of dextrin, and 10g of sucrose are mixed evenly in equal increasing amounts, sprayed with a small amount of 60v% ethanol as an excipient, and made into granules using a granulator. According to 5g per bag, 20 bags are packed to obtain the polygonatum polysaccharide antioxidant health product granules.

Claims

1. A process for extracting and purifying polygonatum polysaccharide, characterized in that: The following steps are involved: S1. Pretreatment: Take raw rhizomes of polygonatum, wash and slice them, dry them at 50-80°C for 10-24h, and grind them into powder using a grinder to obtain polygonatum powder; S2, ethanol extraction: take Polygonatum sibiricum powder, add 3-6 times the amount of 90v% or more ethanol, reflux extraction at 50-60°C for 1-4 times, the first extraction time is 1.5-2h, the subsequent extraction time is 0.5-1.2h, filter while hot, and combine to obtain the first extract; The filter residue is washed 1-3 times with hot ethanol with a purity of more than 90v%, the washing solution is degreased, and then the washing solution is combined with the first extract; Then, drying the first extract at 50-65°C; S3, water extraction: the dried product of the first extract is placed in a round-bottom flask, and water is used as the extraction solvent, and the boiling extraction is performed 1-2 times according to the weight ratio of the feed liquid g / mL of 1:7-15, and the timing is started from the boiling of the water each time, and the extraction is performed for 2-6 hours; after the completion of the water extraction, the water extraction filtrate is combined, and the second extract is obtained by concentrating under reduced pressure at 50-65°C; S4, alcohol precipitation and drying: under continuous stirring, adding an ethanol solution with a concentration of 90% or more to the second extract until the ethanol concentration in the second extract is 80%; Then, the mixture is placed in a refrigerator at 1-4°C for 18-30 hours to allow alcohol precipitation; the precipitate is collected by centrifugation, the precipitate is completely dissolved in distilled water, and freeze-dried to obtain a raw product of Polygonatum sibiricum crude polysaccharide; S5, taking a resin sample, immersing it in anhydrous ethanol for activation, wet-loading it into a column, and preparing an elution column; the resin is a macroporous resin; The raw Polygonatum sibiricum polysaccharide obtained in S4 was dissolved in distilled water to prepare a 10-50 mg / mL Polygonatum sibiricum polysaccharide solution; The crude polysaccharide solution of polygonatum is loaded on the column for adsorption; then it is eluted with distilled water of the same volume as the adsorption column, and the eluate from the 30% to the 70% portion is collected; The collected eluted products were freeze-dried to obtain pure polygonatum polysaccharide.

2. The process for extracting and purifying polygonatum polysaccharide according to claim 1, characterized in that: In S1, pretreatment: take raw Polygonatum cyrtonema rhizome, wash and cut into small pieces with a thickness of 2-5 mm, and dry at a temperature range of 50° C.-70° C. for 12 h-24 h.

3. A process for extracting and purifying polygonatum polysaccharide according to claim 2, characterized in that: In S1, take the raw rhizomes of Polygonatum cyrtonema, screen them, remove the rotten, deteriorated or diseased and insect-infested parts, and then wash and slice them.

4. The process for extracting and purifying polygonatum polysaccharide according to claim 1, characterized in that: In S2, ethanol extraction: weigh a certain amount of Polygonatum sibiricum powder, add 3-5 times the amount of 90%-98% ethanol, reflux extract twice at 55℃-65℃, each extraction time is 1h-2h, after the extraction is completed, filter while hot, and combine to obtain the first extract.

5. The process for extracting and purifying polygonatum polysaccharide according to claim 4, characterized in that: In S2, filtering is performed while hot using one or more combinations of filter paper filtration, filter cloth filtration or microporous membrane filtration.

6. The process for extracting and purifying polygonatum polysaccharide according to claim 5, characterized in that: In S2, the heating device during reflux extraction is a constant temperature water bath.

7. The process for extracting and purifying polygonatum polysaccharide according to claim 1, characterized in that: In S3, water extraction: the dried sample is placed in a round-bottom flask, and water is used as the extraction solvent. The sample is boiled and extracted twice in a weight ratio of 1:8-12 to the liquid. The timing starts from the boiling of the water each time, and the extraction is performed for 3-5 hours. After the water extraction is completed, the water extraction filtrate is combined, and the second extract is concentrated under reduced pressure at 55-65°C.

8. The process for extracting and purifying polygonatum polysaccharide according to claim 1, characterized in that: In S4, alcohol precipitation and drying: under continuous stirring, an ethanol solution with a concentration of 95% by volume or more is added to the second extract until the ethanol concentration in the second extract reaches 80% by volume.

9. The process for extracting and purifying polygonatum polysaccharide according to claim 1, characterized in that: In S5, the purity of the obtained pure polygonatum polysaccharide reached more than 70%.

10. An application of polygonatum sibiricum polysaccharide obtained by the extraction and purification process of polygonatum sibiricum polysaccharide according to any one of claims 1 to 9, characterized in that: The application of polygonatum polysaccharide in the preparation of antioxidant health products.

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