A method for extracting Tibetan Codonopsis pilosula polysaccharide

Abstract

The present application relates to the technical field of the preparation of polysaccharides, and specifically discloses a method for extracting Tibetan Codonopsis polysaccharides, including: S1, crude extraction; S2, decolorization; S3, separation and purification, and the extraction agent used in step S1 includes choline chloride, and also includes at least one of oxalic acid, levulinic acid and glucose. The product of the present application can be used in the fields of food, medicine and cosmetics, and has the effects of strengthening the spleen and moistening the lungs, replenishing blood and fluids, anti-tumor, lowering blood sugar and anti-oxidation; in addition, the preparation method of the present application effectively increases the yield of Tibetan Codonopsis polysaccharides, promotes the rational use of Tibetan Codonopsis resources and optimizes the quality of Tibetan Codonopsis polysaccharides. The extraction rate of Tibetan Codonopsis polysaccharides can reach more than 27.5%. When the addition amount is 16 mg / mL, the DPPH free radical scavenging rate can reach more than 71%, and the hydroxyl free radical scavenging rate can reach more than 81.5%.

Description

Technical Field

[0001] The present application relates to the technical field of preparation of polysaccharides, and in particular to a method for extracting Codonopsis pilosula polysaccharides. Background Art

[0002] Tibetan Codonopsis is a famous classic perennial plant with both medicinal and edible properties. It is rich in nutrients. Modern pharmacology shows that Tibetan Codonopsis is rich in polysaccharides, phenylpropanoids, alkaloids, polyacetylene alcohols and phenolic acids, and has the effects of strengthening the spleen and moistening the lungs and replenishing blood and fluids. In addition, many studies have shown that the main active ingredient extracted from Tibetan Codonopsis, polysaccharides, can affect the growth, invasion and migration of tumor cells, and have an effective anti-proliferation effect on tumor cells. It can also relieve oxidative stress, improve lipid metabolism, increase glycolytic enzyme activity and reduce liver transaminase activity. Therefore, Tibetan Codonopsis polysaccharides also have very significant anti-tumor and hypoglycemic effects, and are well-deserved new plant-based drugs. In addition, Tibetan Codonopsis polysaccharides can also scavenge free radicals, inhibit oxidase activity and increase antioxidant enzyme activity, so they also have very efficient antioxidant capacity and can be used in the field of cosmetics.

[0003] Therefore, it has become the general trend to extract polysaccharides from Tibetan Codonopsis and make them into more efficient concentrated products. The existing methods for extracting Tibetan Codonopsis polysaccharides are usually ultrasonic extraction, microwave extraction and high-pressure extraction. Among them, the high-pressure extraction method is too complicated to operate and has high requirements for equipment. The extraction efficiency of microwave extraction and ultrasonic extraction is basically the same, but ultrasonic extraction has been more widely used due to its lower polysaccharide degradation rate. At present, the extraction rate of Tibetan Codonopsis polysaccharides can reach about 20-25%, but how to further increase the yield of Tibetan Codonopsis polysaccharides, promote the rational use of Tibetan Codonopsis resources and optimize the quality of Tibetan Codonopsis polysaccharides is still a very difficult problem. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a method for extracting Codonopsis pilosula polysaccharides.

[0005] In a first aspect, the present application provides a method for extracting Codonopsis pilosula polysaccharides, comprising the following steps: S1, crude extraction; S2, decolorization; S3, separation and purification, wherein the extraction agent used in step S1 comprises choline chloride and at least one of oxalic acid, levulinic acid and glucose.

[0006] Preferably, the extraction agent used in step S1 includes choline chloride, levulinic acid and oxalic acid.

[0007] Preferably, the molar ratio of choline chloride, glucose and oxalic acid is 2:(3-4):(1.5-2).

[0008] Preferably, the molar ratio of choline chloride, glucose and oxalic acid is 2:3.5:2.

[0009] By adopting the above technical scheme, the present application uses choline chloride and at least one of oxalic acid, levulinic acid and glucose as an extraction agent, and the extraction agent has good biocompatibility, strong extraction ability and can effectively dissolve the polysaccharides in Tibetan Codonopsis, and the crude polysaccharides extracted by the extraction agent compounded by the above substances have higher biological activity and functional characteristics, and also have a good extraction rate. After testing, the extraction rate of Tibetan Codonopsis polysaccharides can reach more than 27.5%, and when the addition amount is 16 mg / mL, the DPPH free radical scavenging rate can reach more than 71%, and the hydroxyl free radical scavenging rate can reach more than 81.5%, which effectively improves the yield of Tibetan Codonopsis polysaccharides, promotes the rational use of Tibetan Codonopsis resources and optimizes the quality of Tibetan Codonopsis polysaccharides. In addition, the present application further controls the extraction agent to be composed of choline chloride, glucose and oxalic acid in a certain molar ratio. After experimental detection, the crude polysaccharide extraction rate of step S1 can reach 35.17%, the DPPH free radical scavenging rate can reach 74.89%, and the hydroxyl free radical scavenging rate can reach 87.37%.

[0010] Specifically, the operation of step S1 is: crush Tibetan Codonopsis pilosula, and disperse it together with the extraction agent in water, heat and extract for a period of time and then centrifuge, collect the liquid and precipitate separately, extract the precipitate for the second time and collect the liquid again, and combine it with the first obtained liquid, add ethanol, refrigerate and let it stand overnight, then collect the precipitate to obtain crude polysaccharide.

[0011] Preferably, the specific operation of step S2 is: first pretreating the macroporous resin to obtain a pretreated resin column, then mixing the crude polysaccharide obtained in step S1 with water to obtain a crude polysaccharide dispersion with a crude polysaccharide concentration of 4.5-6.0 mg / L, loading the crude polysaccharide dispersion into the resin column at a flow rate of 1-3BV / h, adsorbing, filtering, eluting, and filtering twice to obtain a decolorized extract dispersion.

[0012] Specifically, the detailed steps of step S2 are: soaking the macroporous resin in ethanol, fully swelling it and then wet-packing it to ensure that the volume is 3-7BV, then repeatedly washing it with ethanol until the effluent is mixed with water and no white turbidity appears, then washing with water until there is no ethanol smell, adding HCl to soak it, washing it with water until it is neutral, and then soaking it with NaOH solution, washing it with water until it is neutral, to obtain a pretreated resin column, and then mixing the crude polysaccharide obtained in step S1 with water to obtain a crude polysaccharide dispersion with a crude polysaccharide concentration of 4.5-6.0 mg / L, and loading the crude polysaccharide dispersion onto the resin column at a flow rate of 1-3BV / h, adsorbing, filtering, eluting with an ethanol solution (2BV / h), filtering twice, collecting the liquid, and obtaining a decolorized extract dispersion.

[0013] Optionally, the macroporous resin is any one of D101 macroporous resin, HPD-300 macroporous resin, X-5 macroporous resin, AB-8 macroporous resin, DM 130 macroporous resin, NKA-9 macroporous resin and HPD-600 macroporous resin.

[0014] Preferably, the macroporous resin is AB-8 macroporous resin.

[0015] Preferably, the crude polysaccharide concentration of the crude polysaccharide dispersion is 5.0 mg / L, and the flow rate during sample loading is 2 BV / h.

[0016] Preferably, the concentration of the ethanol solution used in the elution is 40-80wt%.

[0017] Preferably, the concentration of the ethanol solution used in the elution is 50 wt %.

[0018] The crude polysaccharide of Tibetan Codonopsis pilosula polysaccharide often contains impurities such as pigments. By adopting the above technical solution, this application uses macroporous resin adsorption separation, comprehensive mechanical screening and chemical adsorption, and also uses AB-8 macroporous resin with strong spatial adsorption force and high selectivity for weak polar compounds for adsorption, and strictly controls the concentration of crude polysaccharide dispersion and the sample flow rate, effectively removes the pigment in the crude polysaccharide, effectively improves the polysaccharide decolorization rate, and optimizes the product quality of Tibetan Codonopsis pilosula polysaccharide. In addition, this application further controls the crude polysaccharide concentration of the crude polysaccharide dispersion, the flow rate during sample loading, and the concentration of the ethanol solution used during elution, further improving the polysaccharide decolorization rate and polysaccharide recovery rate, and optimizing the product quality of Tibetan Codonopsis pilosula polysaccharide.

[0019] Preferably, step S3 adopts a separation and purification method using cellulose column chromatography.

[0020] Specifically, the operation of step S3 is: ultrafiltration and concentration of the decolorized extract dispersion obtained in step S2 using an ultrafiltration membrane, removal of protein using the Sevag method, and freeze-drying for later use; dissolving the protein-free extract in water, loading it into a cellulose chromatography column, eluting it with a NaCl solution, collecting all the eluates, and freeze-drying to obtain Tibetan Codonopsis pilosula polysaccharide.

[0021] By adopting the above technical solution, the present application first ultrafilters the decolorized extract dispersion obtained in step S2 to intercept the macromolecular Tibetan Codonopsis polysaccharide and other impurities, and then uses a cellulose chromatography column to separate and purify the extract after protein removal to remove most of the impurities in the system as much as possible, thereby obtaining high-quality Tibetan Codonopsis polysaccharide. And compared with other separation and purification methods, such as gel column chromatography, cellulose column chromatography has higher separation and purification accuracy, which can further improve the product quality of Tibetan Codonopsis polysaccharide.

[0022] In a second aspect, the present application provides a Tibetan Codonopsis polysaccharide obtained by the above extraction method.

[0023] In summary, this application has the following beneficial technical effects:

[0024] 1. In the extraction method of the present application, the extraction agent prepared by compounding different components has good biocompatibility, strong extraction ability and the ability to effectively dissolve the polysaccharides in Tibetan Codonopsis pilosula, and the crude polysaccharides extracted by the extraction agent prepared by compounding the above substances have higher biological activity and functional characteristics, and also have a good extraction rate. According to the test, the crude polysaccharide extraction rate of step S1 can reach more than 55%, which effectively improves the yield of Tibetan Codonopsis pilosula polysaccharides, promotes the rational utilization of Tibetan Codonopsis pilosula resources and optimizes the quality of Tibetan Codonopsis pilosula polysaccharides;

[0025] 2. The present application strictly controls the volume of the resin column, the concentration of the crude polysaccharide dispersion and the sample loading flow rate during decolorization, thereby effectively removing the pigment in the crude polysaccharide and improving the product quality of the finally obtained Tibetan Codonopsis polysaccharide;

[0026] 3. The cellulose column chromatography of the present application removes most of the impurities in the system as much as possible. Compared with other separation and purification methods, such as gel column chromatography, cellulose column chromatography has higher separation and purification accuracy, which can further improve the product quality of Tibetan Codonopsis pilosula polysaccharide. DETAILED DESCRIPTION

[0027] Material Source

[0028] Unless otherwise specified, the raw materials used in this application are all commercially available products, specifically:

[0029] Tibetan Codonopsis was purchased from Medog Plateau Collection Business Co., Ltd.;

[0030] Choline chloride, CAS No. 67-48-1;

[0031] Oxalic acid, CAS No. 144-62-7;

[0032] Levulinic acid, CAS No. 123-76-2;

[0033] Glucose, CAS No. 50-99-7;

[0034] D101 macroporous resin, HPD-300 macroporous resin, AB-8 macroporous resin, DM 130 macroporous resin, and HPD-600 macroporous resin were purchased from the Chemical Plant of Nankai University, Tianjin;

[0035] X-5 macroporous resin and NKA-9 macroporous resin were purchased from Shaanxi Lanshen Special Resin Co., Ltd.;

[0036] Gel chromatography column, the main material is Sepharose CL-6B gel, purchased from Shanghai Yuanye Biotechnology Co., Ltd., the filling volume in the specific embodiments of this application is 1.5 cm×90 cm;

[0037] The cellulose chromatography column, whose main material is DEAE-52 cellulose, was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The filling volume in the specific embodiments of the present application is 5.5 cm×20 cm.

[0038] The present application is further described in detail below with reference to embodiments and comparative examples.

[0039] Example 1.1

[0040] A method for extracting codonopsis pilosula polysaccharide comprises the following steps:

[0041] S1. Crude extraction: 0.1 kg of Tibetan Codonopsis pilosula was crushed and dispersed in 2 kg of water together with an extraction agent (200 g of choline chloride and 200 g of oxalic acid), heated to 80°C, subjected to ultrasonic extraction at 100 W ultrasonic power for 60 min, and then centrifuged at 6000 r / min for 20 min, and the liquid and precipitate were collected separately. After the precipitate was extracted twice, the liquid was collected again and combined with the first obtained liquid, and ethanol was added until the ethanol concentration in the system reached 85 wt %. After being refrigerated and left to stand overnight, the precipitate was collected to obtain crude polysaccharide;

[0042] S2, decolorization: soak the D101 macroporous resin in 90wt% ethanol for 24h, wet-pack the column after sufficient swelling, ensure that the volume is 2BV, and then repeatedly rinse with ethanol until the effluent is mixed with water (the volume ratio of the effluent to water is 1:5) and no white turbidity appears, then wash with water until there is no ethanol smell, add 3% HCl and soak for 3h, wash with water until neutral, soak with 3% NaOH solution for 3h, wash with water until neutral, and obtain a pretreated resin column, then mix the crude polysaccharide obtained in step S1 with water to obtain a crude polysaccharide dispersion with a crude polysaccharide concentration of 4.5mg / L, load the crude polysaccharide dispersion onto the resin column at a flow rate of 3BV / h, adsorb for 24h, filter, elute with 90wt% ethanol solution at a flow rate of 2BV / h, filter twice, collect the liquid, and obtain a decolorized extract dispersion;

[0043] S3, separation and purification: dilute / concentrate the decolorized extract dispersion obtained in step S2 to 0.01 mg / mL, filter through a 45 um filter membrane, load it into a gel chromatography column, elute it with three times the column volume of distilled water at a flow rate of 5 mL / min, collect all the eluates, and freeze-dry to obtain Tibetan Codonopsis pilosula polysaccharide.

[0044] Example 1.2

[0045] A method for extracting codonopsis pilosula polysaccharide comprises the following steps:

[0046] S1. Crude extraction: 0.1 kg of Tibetan Codonopsis pilosula was crushed and dispersed in 2 kg of water together with an extraction agent (200 g of choline chloride and 200 g of levulinic acid), heated to 80°C, subjected to ultrasonic extraction at 100 W ultrasonic power for 60 min, and then centrifuged at 6000 r / min for 20 min, and the liquid and precipitate were collected separately. After the precipitate was extracted twice, the liquid was collected again and combined with the first obtained liquid, and ethanol was added until the ethanol concentration in the system reached 85 wt %. After standing overnight in the refrigerator, the precipitate was collected to obtain crude polysaccharide;

[0047] S2, decolorization: soak the HPD-300 macroporous resin in 90wt% ethanol for 24h, wet-pack the column after sufficient swelling, ensure that the volume is 2BV, and then repeatedly rinse with ethanol until the effluent is mixed with water (the volume ratio of the effluent to water is 1:5) and no white turbidity appears, then wash with water until there is no ethanol smell, add 3% HCl and soak for 3h, wash with water until neutral, then soak with 3% NaOH solution for 3h, wash with water until neutral, obtain a pretreated resin column, then mix the crude polysaccharide obtained in step S1 with water to obtain a crude polysaccharide dispersion with a crude polysaccharide concentration of 6.0mg / L, load the crude polysaccharide dispersion onto the resin column at a flow rate of 1BV / h, adsorb for 24h, filter, elute with 90wt% ethanol solution at a flow rate of 2BV / h, filter twice, collect the liquid, and obtain a decolorized extract dispersion;

[0048] S3, separation and purification: dilute / concentrate the decolorized extract dispersion obtained in step S2 to 0.01 mg / mL, filter through a 45 um filter membrane, load it into a gel chromatography column, elute it with three times the column volume of distilled water at a flow rate of 5 mL / min, collect all the eluates, and freeze-dry to obtain Tibetan Codonopsis pilosula polysaccharide.

[0049] Example 1.3

[0050] A method for extracting codonopsis pilosula polysaccharide comprises the following steps:

[0051] S1. Crude extraction: 0.1 kg of Tibetan Codonopsis pilosula was crushed and dispersed in 2 kg of water together with an extraction agent (200 g of choline chloride and 200 g of glucose), heated to 80°C, ultrasonically extracted at 100 W of ultrasonic power for 60 min, and then centrifuged at 6000 r / min for 20 min, and the liquid and precipitate were collected separately. After the precipitate was extracted twice, the liquid was collected again and combined with the first obtained liquid, and ethanol was added until the ethanol concentration in the system reached 85 wt%. After standing overnight in the refrigerator, the precipitate was collected to obtain crude polysaccharide;

[0052] S2, decolorization: soak X-5 macroporous resin in 90wt% ethanol for 24h, wet-pack it into a column after sufficient swelling, ensure that the volume is 2BV, and then rinse it repeatedly with ethanol until the effluent is mixed with water (the volume ratio of the effluent to water is 1:5) and no white turbidity appears, then wash it with water until there is no ethanol smell, add 3% HCl and soak it for 3h, wash it with water until it is neutral, soak it with 3% NaOH solution for 3h, wash it with water until it is neutral, and obtain a pretreated resin column, then mix the crude polysaccharide obtained in step S1 with water to obtain a crude polysaccharide dispersion with a crude polysaccharide concentration of 5.5mg / L, load the crude polysaccharide dispersion into the resin column at a flow rate of 1.5BV / h, adsorb for 24h, filter, elute with 90wt% ethanol solution at a flow rate of 2BV / h, filter twice, collect the liquid, and obtain a decolorized extract dispersion;

[0053] S3, separation and purification: dilute / concentrate the decolorized extract dispersion obtained in step S2 to 0.01 mg / mL, filter through a 45 um filter membrane, load it into a gel chromatography column, elute it with three times the column volume of distilled water at a flow rate of 5 mL / min, collect all the eluates, and freeze-dry to obtain Tibetan Codonopsis pilosula polysaccharide.

[0054] Examples 2.1-2.8

[0055] A method for extracting polysaccharides from Codonopsis pilosula is different from that in Example 1.1 in that the extraction agent added in step S1 is different, as shown in Table 1.1, and the rest is the same as that in Example 1.1.

[0056] Table 1.1 Specific dosage and components of the extraction agent in Example 2

[0057] Group Step S1: extracting specific dosage and components of the agent Example 2.1 Choline chloride 200g, oxalic acid 100g, glucose 100g Example 2.2 Choline chloride 200g, levulinic acid 100g, glucose 100g Example 2.3 Choline chloride 200g, levulinic acid 100g, oxalic acid 100g Example 2.4 Choline chloride 200g, oxalic acid 67g, glucose 66g, levulinic acid 67g Example 2.5 Choline chloride 138.1g, levulinic acid 172.7g, oxalic acid 89.2g Example 2.6 Choline chloride 126.3g, levulinic acid 210.5g, oxalic acid 63.2g Example 2.7 Choline chloride 129.0g, levulinic acid 187.8g, oxalic acid 83.2g Example 2.8 Choline chloride 120g, levulinic acid 144g, oxalic acid 136g

[0058] Examples 3.1-3.4

[0059] A method for extracting Tibetan Codonopsis pilosula polysaccharide is different from Example 1.1 in that in step S2, D101 macroporous resin is replaced by AB-8 macroporous resin, DM 130 macroporous resin, NKA-9 macroporous resin and HPD-600 macroporous resin, respectively, and the rest is the same as Example 1.1.

[0060] Examples 4.1-4.18

[0061] A method for extracting polysaccharides from Tibetan Codonopsis pilosula is different from that in Example 1.1 in that, in step S2, the crude polysaccharide concentration of the crude polysaccharide dispersion is different, and the flow rate during loading is different, as shown in Table 1.2, and the rest is the same as Example 1.1.

[0062] Table 1.2 Crude polysaccharide concentration and sample loading flow rate in Example 4

[0063]

[0064]

[0065] Examples 5.1-5.6

[0066] A method for extracting Tibetan Codonopsis pilosula polysaccharide is different from Example 1.1 in that in step S2, the concentrations of the ethanol solution used for elution are 80wt%, 70wt%, 60wt%, 50wt%, 40wt% and 30wt%, respectively, and the rest are the same as Example 1.1.

[0067] Example 6

[0068] A method for extracting Tibetan Codonopsis pilosula polysaccharide, which is different from Example 1.1 in that step S3 adopts a separation and purification method using cellulose column chromatography, specifically:

[0069] The decolorized extract dispersion obtained in step S2 is concentrated / diluted to 2 g / L, and the decolorized extract dispersion obtained in step S2 is ultrafiltered for 35 minutes at a pressure of 0.3 MPa using a small ultrafiltration machine with a rolled membrane and an ultrafiltration membrane to obtain a cutoff liquid, which is concentrated, and then the protein is removed by a Sevag method, and freeze-dried for later use; the extract after protein removal is dissolved in water at a volume ratio of 1:50, loaded on a cellulose chromatography column, and eluted at a flow rate of 7 mL / min with a 0.04 mol / L NaCl solution of three times the column volume, and all the eluates are collected, freeze-dried, and Tibetan Codonopsis pilosula polysaccharide is obtained.

[0070] Comparative Examples 1-4

[0071] The difference from Example 1.1 is that in step S1, 400 g of the extraction agent is replaced by 100 g of hydrochloric acid, 100 g of sodium hydroxide, 1000 g of ethanol and 400 g of choline chloride respectively, and the rest is the same as Example 1.1.

[0072] Performance Testing

[0073] Referring to the paper "Liu Qin, Song Shen, Yin Zhenxiong, et al. Study on the preparation process, chemical structure and antioxidant activity of melon seed polysaccharide sulfate [J / OL]. Food Industry Science and Technology, 2014, 35(14): 107-113.", the extraction rate and antioxidant activity of the Tibetan Codonopsis polysaccharide obtained in the examples and comparative examples were determined, and the results are recorded in Table 2. The specific detection indicators of antioxidant activity are the scavenging rate of DPPH free radicals and the scavenging rate of hydroxyl free radicals, which are determined according to the concentration of Tibetan Codonopsis polysaccharide of 16 mg / mL.

[0074] Table 2 Extraction rate and free radical scavenging rate of Tibetan Codonopsis polysaccharide

[0075]

[0076] Data Analysis:

[0077] As can be seen from Table 2, the extraction rate of Tibetan Codonopsis polysaccharides in Examples 1.1-1.3 can reach 28.95-29.21%, the DPPH free radical scavenging rate can reach 72.11-72.20%, and the hydroxyl free radical scavenging rate can reach 84.09-84.20%, proving that the present application uses choline chloride compounded with at least one of oxalic acid, levulinic acid and glucose as an extraction agent, and the extraction agent has good biocompatibility, strong extraction ability and can effectively dissolve the polysaccharides in Tibetan Codonopsis, and the crude polysaccharide extracted by the extraction agent compounded with the above substances has higher biological activity and functional characteristics, and also has a good extraction rate;

[0078] Among Examples 2.1-2.8, all data of Example 2.7 are the highest values. The difference from other Examples lies in the different components and ratios of the extraction agent. The extraction rate of Tibetan Codonopsis polysaccharide can reach 35.17%, the DPPH free radical scavenging rate can reach 74.89%, and the hydroxyl free radical scavenging rate can reach 87.37%, which proves that the present application further controls the extraction agent to be composed of choline chloride, glucose and oxalic acid in a certain molar ratio, which can further increase the yield of Tibetan Codonopsis polysaccharide, promote the rational utilization of Tibetan Codonopsis resources and optimize the quality of Tibetan Codonopsis polysaccharide;

[0079] In Examples 3.1-3.4, all data of Example 3.1 are the highest values. The difference from other examples lies in the different types of macroporous resins. The extraction rate of Tibetan Codonopsis polysaccharide can reach 31.26%, the DPPH free radical scavenging rate can reach 76.39%, and the hydroxyl free radical scavenging rate can reach 89.14%, which proves that the AB-8 macroporous resin of the present application has strong spatial adsorption and high selectivity for weakly polar compounds, effectively removes the pigment in the crude polysaccharide, effectively improves the decolorization rate of the polysaccharide, and optimizes the product quality of Tibetan Codonopsis polysaccharide;

[0080] The crude polysaccharide concentrations and sample loading flow rates of each embodiment in Examples 4.1-4.18 are different and randomly arranged. Among the limited combinations listed in this application, the data of Example 4.8 are all the highest values, and its Tibetan Codonopsis polysaccharide extraction rate can reach 32.37%, DPPH free radical scavenging rate can reach 74.98%, and hydroxyl free radical scavenging rate can reach 87.48%. It is proved that the present application effectively removes the pigment in the crude polysaccharide by strictly controlling the concentration of the crude polysaccharide dispersion and the sample loading flow rate, effectively improves the polysaccharide decolorization rate, and optimizes the product quality of Tibetan Codonopsis polysaccharide;

[0081] In Examples 5.1-5.6, all data in Example 5.4 are the highest values. The difference from other examples is that the concentration of the ethanol solution used for elution in step S2 is different. The extraction rate of Tibetan Codonopsis polysaccharide can reach 32.01%, the DPPH free radical scavenging rate can reach 74.85%, and the hydroxyl free radical scavenging rate can reach 87.32%. It is proved that the present application further improves the polysaccharide decolorization rate and polysaccharide recovery rate by strictly controlling the concentration of the ethanol solution used for elution, and optimizes the product quality of Tibetan Codonopsis polysaccharide;

[0082] The data of Example 6 are all higher than those of Example 1.1, which proves that the separation and purification method of cellulose column chromatography of the present application removes most of the impurities in the system as much as possible, thereby obtaining high-quality Tibetan Codonopsis polysaccharides, and compared with other separation and purification methods, such as gel column chromatography, cellulose column chromatography has higher separation and purification accuracy, which can further improve the product quality of Tibetan Codonopsis polysaccharides;

[0083] The data of Comparative Examples 1-4 are all lower than those of Example 1.1. The difference from Example 1.1 lies in the different extraction agents, and in Comparative Example 4, only choline chloride is used as the extraction agent, and its extraction ability and the quality of the obtained Tibetan Codonopsis polysaccharide are worse than those of Examples 1-3. This further confirms that the present application uses choline chloride in combination with at least one of oxalic acid, levulinic acid and glucose as an extraction agent, and there is a good synergistic effect between the substances. The extraction agent has good biocompatibility, strong extraction ability, and can effectively dissolve the polysaccharides in Tibetan Codonopsis, and the crude polysaccharide extracted by the extraction agent formed by the compounding of the above substances has higher biological activity and functional characteristics, and also has a good extraction rate.

[0084] The embodiments of this specific implementation method are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore, all equivalent changes made based on the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for extracting codonopsis pilosula polysaccharide, comprising the following steps: S1, crude extraction; S2, decolorization; S3, separation and purification, characterized in that: the extraction agent used in the step S1 includes choline chloride, levulinic acid and oxalic acid in a molar ratio of 2:3.5:2; the specific operation of the step S2 is: first, the macroporous resin is pretreated to obtain a pretreated resin column, and then the crude polysaccharide obtained in the step S1 is mixed with water to obtain a crude polysaccharide dispersion with a crude polysaccharide concentration of 4.5-6.0 mg / L, and the crude polysaccharide dispersion is loaded into the resin column at a flow rate of 1-3BV / h, adsorbed, filtered, eluted, and filtered twice to obtain a decolorized extract dispersion, and the macroporous resin is AB-8 macroporous resin.

2. The method for extracting Codonopsis pilosula polysaccharide according to claim 1, characterized in that: The crude polysaccharide concentration of the crude polysaccharide dispersion is 5.0 mg / L, and the flow rate during sample loading is 2 BV / h.

3. The method for extracting Codonopsis pilosula polysaccharide according to claim 1, characterized in that: The concentration of the ethanol solution used in the elution is 40-80wt%.

4. The method for extracting Codonopsis pilosula polysaccharide according to claim 3, characterized in that: The concentration of the ethanol solution used during the elution is 50wt%.

5. The method for extracting Codonopsis pilosula polysaccharide according to claim 1, characterized in that: The step S3 adopts a separation and purification method using cellulose column chromatography.

6. A Tibetan Codonopsis pilosula polysaccharide obtained by the extraction method according to any one of claims 1 to 5.

Images