Extraction method of Fritillaria polysaccharide

Abstract

The invention discloses a method for extracting Fritillaria polysaccharide, including: step one, preparing Fritillaria powder; step two, mixing Fritillaria powder with petroleum ether, reflux degreasing in a water bath after microwave heating, and obtaining defatted Fritillaria powder; step three, adding distilled water and cellulase solution to defatted Fritillaria powder, extracting under 45-55 DEG C conditions, and obtaining Fritillaria polysaccharide crude extract; step four, heating Fritillaria polysaccharide crude extract to kill enzyme, and adding chloroform-n-butanol solution, fully oscillating, centrifuging, taking supernatant A, collecting precipitation and adding distilled water to stir, centrifuging again, taking supernatant B and merging with supernatant A, concentrating under reduced pressure, and obtaining Fritillaria polysaccharide concentrate; step five, alcohol precipitation and drying to obtain Fritillaria crude polysaccharide; step six, adding distilled water to Fritillaria crude polysaccharide to redissolve, adding pretreated macroporous adsorption resin vibration to purify, extracting the adsorbed solution after concentration and drying to obtain Fritillaria essence polysaccharide. The inventive method has a high extraction rate of Fritillaria polysaccharide and good purification effect.

Description

Technical Field

[0001] The invention relates to the technical field of plant polysaccharide extraction, and in particular to a method for extracting fritillaria polysaccharide. Background Art

[0002] As one of the hot topics in recent years, polysaccharides are involved in the regulation of various life phenomena of cells and have important health functions, including the maintenance of redox balance, chelation with heavy metals, regulation of the immune regulation system, inhibition of genotoxicity, etc., all of which depend on the structural characteristics of polysaccharides, and polysaccharides are widely distributed in nature. A large number of studies have shown that plant polysaccharides have various biological effects such as anti-aging, and are almost non-toxic to the body, so the research on their biological effects has attracted more and more attention. Therefore, screening polysaccharides with antioxidant and free radical scavenging activities from Chinese herbal medicines is of great significance to the food and pharmaceutical industries. Fritillaria belongs to the perennial herbaceous plant of the Liliaceae family. It is a plant with both medicinal and edible properties. The Fritillaria polysaccharides it contains not only have biological activities such as anti-tumor, anti-aging, and antioxidant, but also have no toxic side effects, and the drug quality can be easily controlled by chemical means. Based on this, Fritillaria polysaccharides have opened up new research ideas and directions for the development of new drugs, green food additives, functional health products, etc., so it is of great significance to design a Fritillaria polysaccharide extraction method. Summary of the invention

[0003] The invention provides a method for extracting fritillaria polysaccharide. The method can obtain fritillaria polysaccharide with high extraction rate and high purity through processes such as microwave heating defatting, cellulase extraction, chloroform-n-butanol solution and macroporous adsorption resin impurity removal.

[0004] In order to achieve these purposes and other advantages according to the present invention, a method for extracting Fritillaria polysaccharide is provided, comprising the following steps:

[0005] Step 1: Take the freshly collected Fritillaria, wash it with clean water, freeze-dry it, and grind it into ultrafine powder to obtain Fritillaria powder;

[0006] Step 2, mix the Fritillaria powder and petroleum ether in a mass volume ratio of 1: (6-8) (i.e., 1 g of Fritillaria powder is contained in every 6-8 mL of petroleum ether) and stir evenly, place in a 60-70° C. water bath for reflux degreasing for 30-40 min after microwave heating for 8-12 min, centrifuge, collect the residue, repeat the degreasing three times, add ethanol solution after the petroleum ether is evaporated, magnetically stir, centrifuge, collect the residue, evaporate to obtain defatted Fritillaria powder;

[0007] Step 3: add 10 to 20 times the weight of distilled water to the defatted Fritillaria powder, stir evenly, then add 2 to 3 times the volume of the solution of cellulase solution, extract at 45 to 55° C. for 35 to 45 minutes, and obtain a Fritillaria polysaccharide crude extract;

[0008] Step 4: Heat the crude extract of Fritillaria polysaccharide at 90-100° C. for 10-15 min to inactivate the enzyme, add 1 / 5 times the volume of chloroform-n-butanol solution, fully shake for at least 30 min, centrifuge for 15-20 min, take supernatant A, collect the precipitate and add distilled water at 45-55° C., stir for 5-10 min, centrifuge for 15-20 min, take supernatant B and combine it with supernatant A, and concentrate under reduced pressure to obtain Fritillaria polysaccharide concentrate;

[0009] Step 5, adding 3 to 5 times the volume of ethanol solution to the Fritillaria polysaccharide concentrate, stirring while adding, standing at 5 to 8° C. for 24 to 40 hours, centrifuging for 15 to 20 minutes, collecting the precipitate, and vacuum drying to obtain Fritillaria crude polysaccharide;

[0010] Step 6: Add 400 to 500 times the weight of distilled water to the Fritillaria crude polysaccharide to re-dissolve it, then add the pretreated macroporous adsorption resin, shake on a shaker at room temperature for 24 to 30 hours, extract the adsorbed solution, concentrate and dry it to obtain Fritillaria essence polysaccharide.

[0011] Preferably, in the method for extracting Fritillaria polysaccharide, in step 2, the Fritillaria powder is defatted and then an ethanol solution of not less than 5 times its volume is added.

[0012] Preferably, in the method for extracting Fritillaria polysaccharide, in step 3, the mass concentration of the cellulase solution is 4-6%, that is, 4-6 g of cellulase is contained in every 100 liters of solution.

[0013] Preferably, in the method for extracting Fritillaria polysaccharide, in step 4, the chloroform-n-butanol solution is a mixture of chloroform:n-butanol in a volume ratio of (4-6):1.

[0014] Preferably, in the method for extracting Fritillaria polysaccharide, the ethanol solution in step 2 and step 5 is an ethanol solution with a volume fraction of 80-90%.

[0015] Preferably, in the method for extracting Fritillaria polysaccharide, the pretreatment method of the macroporous adsorption resin is as follows: take the macroporous adsorption resin, fully wash it with distilled water to remove surface impurities, then add anhydrous ethanol to soak for 20-30 hours to make the resin fully swell, then discard the ethanol, wash it with distilled water until it is tasteless, then add 3-4% weak acid solution to soak for 2-3 hours, wash it with distilled water until it is neutral, then add 4-5% baking soda glycerol aqueous solution to soak for 2-3 hours, wash it with distilled water until it is neutral, finally add distilled water to soak, and store it at 4-5°C for use. The 3-4% weak acid solution contains 3-4g of weak acid per 100g of water; the 4-5% baking soda glycerol aqueous solution contains 4-5g of baking soda per 100g of glycerol aqueous solution.

[0016] Preferably, in the method for extracting Fritillaria polysaccharide, the weak acid is one of citric acid, acetic acid and lactic acid.

[0017] Preferably, in the method for extracting Fritillaria polysaccharide, the volume ratio of glycerol to water in the glycerol aqueous solution is 1:(5-8).

[0018] Preferably, in the method for extracting Fritillaria polysaccharide, in step six, every 1g of untreated macroporous adsorption resin is subjected to adsorption treatment of 3-4mL of the reconstituted solution.

[0019] Preferably, in the method for extracting Fritillaria polysaccharide, the macroporous absorption resin is AB-8 macroporous absorption resin or D-101 macroporous absorption resin.

[0020] The present invention at least has the following beneficial effects: in the method of the present invention, microwave heating is performed during the degreasing process of petroleum ether, which promotes the rupture of plant cell walls so that various substances in the cells can quickly move into the petroleum ether, thereby improving the degreasing rate, and after degreasing, small molecule alcohol components are removed by ethanol solution, thereby further improving the purity of polysaccharide extraction; cellulase is added during the polysaccharide extraction process, thereby accelerating the decomposition of plant tissues and the extraction of polysaccharides; cellulase and most of the extracted protein substances are removed from the Fritillaria polysaccharide crude extract by chloroform-n-butanol, thereby reducing the adsorption task of macroporous adsorption resin on proteins in the subsequent purification process, while more other impurities are adsorbed, thereby improving the purification efficiency. The invention discloses a method for preparing the method of the present invention, wherein the cellulase precipitate is redissolved with distilled water, and the Fritillaria polysaccharide mixed in the precipitate is extracted, thereby avoiding the waste of the extracted polysaccharide; by pretreating the macroporous resin, not only the impurities in the macroporous resin are removed, and the impurities are avoided from being substituted into the Fritillaria polysaccharide, but also the glycerol acts on the macroporous adsorption resin under the condition of a weak alkaline solution of baking soda, so that the adsorption rate of the macroporous adsorption resin to the polysaccharide is reduced, thereby more Fritillaria polysaccharide is retained in the final Fritillaria essence polysaccharide, and the extraction rate of Fritillaria essence polysaccharide is improved; the method of the invention has a high extraction rate of Fritillaria polysaccharide and a good purification effect, and the purity of the obtained Fritillaria essence polysaccharide is above 98%, and is suitable for the extraction of Fritillaria polysaccharide from various types of Fritillaria.

[0021] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can implement the invention with reference to the description.

[0023] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0024] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials, if not otherwise specified, can be obtained from commercial sources. In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "set" should be understood in a broad sense, for example, they can be fixedly connected, set, or detachably connected, set, or connected and set in one piece. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood in specific circumstances. The orientation or positional relationship indicated by the terms "transverse", "longitudinal", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc. is based on the orientation or positional relationship shown, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation of the present invention.

[0025] Embodiment 1:

[0026] Step 1: Take the freshly collected Fritillaria thunbergii, wash it with clean water, freeze-dry it, and ultrafine grind it to a particle size of 30 μm to obtain Fritillaria thunbergii powder;

[0027] Step 2: Mix the Fritillaria powder and petroleum ether in a mass volume ratio of 1:6 and stir evenly, heat in a microwave for 8 minutes, place in a 60°C water bath for reflux degreasing for 30 minutes, centrifuge, collect the residue, repeat the degreasing three times, evaporate the petroleum ether, add 5 times the volume of 80% ethanol solution, stir magnetically at 1000r / min for 30 minutes, centrifuge for 10 minutes, collect the residue, evaporate to obtain defatted Fritillaria powder;

[0028] Step 3: add 10 times the weight of distilled water to the defatted Fritillaria powder, stir evenly, then add 2 times the volume of the solution with a mass concentration of 4% cellulase solution, extract at 45°C for 35 minutes, and stir slowly for 10 seconds every 5 minutes during the extraction process to obtain a Fritillaria polysaccharide crude extract;

[0029] Step 4: Heat the crude extract of Fritillaria polysaccharide at 90°C for 10 minutes to inactivate the enzyme, add 1 / 5 times the volume of chloroform-n-butanol solution, fully shake for 30 minutes, centrifuge for 15 minutes, take supernatant A, collect the precipitate and add distilled water, stir for 5 minutes at 45°C, centrifuge for 15 minutes, take supernatant B and combine it with supernatant A, and concentrate under reduced pressure to obtain Fritillaria polysaccharide concentrate;

[0030] The chloroform-n-butanol solution is a mixture of chloroform and n-butanol in a volume ratio of 4:1;

[0031] Step 5, adding 3 times the volume of 80% ethanol solution to the Fritillaria polysaccharide concentrate, stirring while adding, standing at 5°C for 24 hours, centrifuging for 15 minutes, collecting the precipitate, and vacuum drying to obtain Fritillaria crude polysaccharide;

[0032] Step 6: Add 400 times the weight of distilled water to the crude Fritillaria polysaccharide for re-dissolution, then add the pre-treated AB-8 macroporous adsorption resin, shake on a shaker at room temperature for 24 hours, extract the adsorbed solution, concentrate and dry to obtain Fritillaria polysaccharide;

[0033] Among them, every 1g of untreated macroporous adsorption resin corresponds to adsorption treatment of 3mL of re-dissolved solution;

[0034] The pretreatment method of the macroporous adsorption resin is as follows: take the macroporous adsorption resin, wash it thoroughly with distilled water to remove surface impurities, then add anhydrous ethanol to soak for 20 hours to make the resin fully swell, then discard the ethanol, wash it with distilled water until it is odorless, then add 3% citric acid solution to soak for 2 hours, wash it with distilled water until it is neutral, then add 4% baking soda glycerol aqueous solution to soak for 2 hours, wash it with distilled water until it is neutral, finally add distilled water to soak, and store it at 4°C for use; the volume ratio of glycerol to water in the glycerol aqueous solution is 1:5.

[0035] In this embodiment, all centrifugal speeds are 4000 r / min.

[0036] Example 2

[0037] Step 1: Take the freshly collected Fritillaria cirrhosae, wash it with water, freeze-dry it, and ultrafine grind it to a particle size of 60 μm to obtain Fritillaria cirrhosae powder;

[0038] Step 2: Mix the Fritillaria powder and petroleum ether in a mass volume ratio of 1:7 and stir evenly, heat in a microwave for 10 minutes, place in a 65°C water bath for reflux degreasing for 35 minutes, centrifuge, collect the residue, repeat the degreasing three times, evaporate the petroleum ether, add 6 times the volume of 85% ethanol solution, stir magnetically at 1000r / min for 30 minutes, centrifuge for 10 minutes, collect the residue, evaporate to obtain defatted Fritillaria powder;

[0039] Step 3: add 15 times the weight of distilled water to the defatted Fritillaria powder, stir evenly, then add 2.5 times the volume of the solution with a mass concentration of 5% cellulase solution, extract at 50° C. for 40 minutes, and stir slowly for 10 seconds every 5 minutes during the extraction process to obtain a Fritillaria polysaccharide crude extract;

[0040] Step 4: Heat the crude extract of Fritillaria polysaccharide at 95°C for 13 minutes to inactivate the enzyme, add 1 / 5 times the volume of chloroform-n-butanol solution, fully shake for 40 minutes, centrifuge for 18 minutes, take supernatant A, collect the precipitate and add distilled water at 50°C, stir for 8 minutes, centrifuge for 18 minutes, take supernatant B and combine it with supernatant A, and concentrate under reduced pressure to obtain Fritillaria polysaccharide concentrate;

[0041] The chloroform-n-butanol solution is a mixture of chloroform and n-butanol in a volume ratio of 5:1;

[0042] Step 5, adding 4 times the volume of 85% ethanol solution to the Fritillaria polysaccharide concentrate, stirring while adding, standing at 6°C for 32 hours, centrifuging for 18 minutes, collecting the precipitate, and vacuum drying to obtain Fritillaria crude polysaccharide;

[0043] Step 6: Add 450 times the weight of distilled water to the crude Fritillaria polysaccharide to re-dissolve it, then add the pre-treated AB-8 macroporous adsorption resin, shake on a shaker at room temperature for 27 hours, extract the adsorbed solution, concentrate and dry it to obtain Fritillaria polysaccharide;

[0044] Among them, every 1g of untreated macroporous adsorption resin corresponds to adsorption treatment of 4mL of re-dissolved solution;

[0045] The pretreatment method of the macroporous adsorption resin is as follows: take the macroporous adsorption resin, wash it thoroughly with distilled water to remove surface impurities, then add anhydrous ethanol to soak for 25 hours to make the resin fully swell, then discard the ethanol, wash it with distilled water until it is odorless, then add 4% acetic acid solution to soak for 2.5 hours, wash it with distilled water until it is neutral, then add 4% baking soda glycerol aqueous solution to soak for 2.5 hours, wash it with distilled water until it is neutral, finally add distilled water to soak, and store it at 4°C for use; the volume ratio of glycerol to water in the glycerol aqueous solution is 1:7.

[0046] In this embodiment, all centrifugal speeds are 4000 r / min.

[0047] Example 3

[0048] Step 1: Take the freshly collected Fritillaria cirrhosae, wash it with clean water, freeze-dry it, and ultrafine grind it to a particle size of 100 μm to obtain Fritillaria cirrhosae powder;

[0049] Step 2: Mix the Fritillaria powder and petroleum ether in a mass volume ratio of 1:8 and stir evenly, heat in a microwave for 12 minutes, place in a 70°C water bath for reflux degreasing for 40 minutes, centrifuge, collect the residue, repeat the degreasing three times, add 8 times the volume of 90% ethanol solution after evaporating the petroleum ether, stir magnetically at 1000r / min for 30 minutes, centrifuge for 10 minutes, collect the residue, evaporate to obtain defatted Fritillaria powder;

[0050] Step 3: add 20 times the weight of distilled water to the defatted Fritillaria powder, stir evenly, then add 3 times the volume of the solution with a mass concentration of 6% cellulase solution, extract at 55°C for 45 minutes, and stir slowly for 10 seconds every 5 minutes during the extraction process to obtain a Fritillaria polysaccharide crude extract;

[0051] Step 4: Heat the crude extract of Fritillaria polysaccharide at 100° C. for 15 min to inactivate the enzyme, add 1 / 5 times the volume of chloroform-n-butanol solution, fully shake for 50 min, centrifuge for 20 min, take supernatant A, collect the precipitate and add distilled water, stir for 10 min at 55° C., centrifuge for 20 min, take supernatant B and combine it with supernatant A, and concentrate under reduced pressure to obtain Fritillaria polysaccharide concentrate;

[0052] The chloroform-n-butanol solution is a mixture of chloroform and n-butanol in a volume ratio of 6:1;

[0053] Step 5, adding 5 times the volume of 90% ethanol solution to the Fritillaria polysaccharide concentrate, stirring while adding, standing at 8°C for 40 hours, centrifuging for 20 minutes, collecting the precipitate, and vacuum drying to obtain Fritillaria crude polysaccharide;

[0054] Step 6: Add 500 times the weight of distilled water to the crude Fritillaria polysaccharide for re-dissolution, then add the pretreated D-101 macroporous adsorption resin, shake on a shaker at room temperature for 30 hours, extract the adsorbed solution, concentrate and dry to obtain Fritillaria polysaccharide;

[0055] Among them, every 1g of untreated macroporous adsorption resin corresponds to adsorption treatment of 4mL of re-dissolved solution;

[0056] The pretreatment method of the macroporous adsorption resin is as follows: take the macroporous adsorption resin, wash it thoroughly with distilled water to remove surface impurities, then soak it in anhydrous ethanol for 30 hours to make the resin fully swell, then discard the ethanol, wash it with distilled water until it is odorless, then soak it in 4% lactic acid solution for 3 hours, wash it with distilled water until it is neutral, then soak it in 5% baking soda glycerol aqueous solution for 3 hours, wash it with distilled water until it is neutral, finally soak it in distilled water, and store it at 5°C for later use; the volume ratio of glycerol to water in the glycerol aqueous solution is 1:8.

[0057] In this embodiment, all centrifugal speeds are 4000 r / min.

[0058] Comparative Example 1

[0059] Same as Example 2, except that in step 2, microwave heating is not performed.

[0060] Comparative Example 2

[0061] Same as Example 2, except that in step 2, no ethanol solution is used after defatting.

[0062] Comparative Example 3

[0063] Same as Example 2, except that in step 3, no cellulase solution was added.

[0064] Comparative Example 4

[0065] Same as Example 2, except that in step 4, the chloroform-n-butanol solution is replaced with distilled water.

[0066] Comparative Example 5

[0067] The same as Example 2, except that: in step 4, no distilled water is added, that is, the supernatant A is concentrated under reduced pressure to obtain the Fritillaria polysaccharide concentrate.

[0068] Comparative Example 6

[0069] Same as Example 2, except that in step 6, the macroporous adsorption resin is not pretreated.

[0070] Comparative Example 7

[0071] Same as Example 2, except that: no glycerol is used in the macroporous adsorption resin pretreatment method.

[0072] Comparative Example 8

[0073] Same as Example 2, except that: in the pretreatment method of the macroporous adsorption resin, the weak acid solution, baking soda solution, and glycerol aqueous solution are used for soaking treatment separately.

[0074] Comparative Example 9

[0075] Same as Example 2, except that in the macroporous adsorption resin pretreatment method, the weak acid solution contains glycerol and baking soda, and the glycerol aqueous solution is a baking soda aqueous solution.

[0076] Comparative Example 10

[0077] The Fritillaria polysaccharide is extracted using the traditional water extraction and alcohol precipitation method: the collected Fritillaria cirrhosa is washed, dried and crushed, and then 60 to 100 times the volume of distilled water is added, stirred evenly, extracted at 45 to 55°C for 24 to 30 hours, centrifuged, the supernatant is taken, and the Fritillaria polysaccharide is obtained by reduced pressure concentration and drying.

[0078] According to the formula: Polysaccharide extraction rate (%) = Fritillaria polysaccharide mass / Fritillaria sample mass × 100%, the polysaccharide extraction rates in the above embodiments were measured and calculated, and recorded in Table 1; the Fritillaria polysaccharides in the above embodiments were re-dissolved in distilled water, and the Fritillaria polysaccharide content was detected by the sulfuric acid-phenol method, and the result, i.e., the purity of Fritillaria polysaccharide, was measured, and the result was recorded in Table 1.

[0079] Table 1

[0080] Polysaccharide extraction rate % Polysaccharide purity% Example 1 12.61 98.6 Example 2 45.67 98.3 Example 3 20.25 98.1 Comparative Example 1 52.21 83.1 Comparative Example 2 47.37 95.3 Comparative Example 3 35.76 98.3 Comparative Example 4 57.61 73.6 Comparative Example 5 43.82 98.2 Comparative Example 6 56.23 74.9 Comparative Example 7 35.86 95.8 Comparative Example 8 35.83 95.7 Comparative Example 9 35.87 95.8 Comparative Example 10 10.76 65.3

[0081] According to the test results in Table 1, the polysaccharides extracted from Fritillaria by the method of the present invention (Example 1, Example 2, Example 3) are not only greatly improved in extraction rate but also higher in purity, both of which are above 98% compared with the traditional water extraction and alcohol precipitation method for extracting Fritillaria polysaccharides (Comparative Example 10); the polysaccharide extraction rate in Comparative Example 1 is increased compared with Example 2, while the purity is reduced, because microwave energy promotes cell wall fragmentation, transfers various substances in the cell to the solvent, and helps to degrease and remove impurities with petroleum ether. In Comparative Example 1, microwave heating is not performed, which reduces the degreasing rate of petroleum ether, leaving some lipid impurities in the final Fritillaria polysaccharide; the polysaccharide extraction rate in Comparative Example 2 is slightly increased, while the purity is slightly reduced. , because the ethanol in step 2 of Example 2 is used to remove small molecule alcohol content to improve the purity of polysaccharides, while there is no such operation in Comparative Example 2, so that some small molecule alcohol content remains in the final Fritillaria polysaccharide; the polysaccharide extraction rate in Comparative Example 3 drops sharply compared with Example 2, while the purity remains unchanged, because cellulase can accelerate the decomposition of plant tissues and promote the rapid release and extraction of Fritillaria polysaccharides. The absence of cellulase in Comparative Example 3 leads to a decrease in the amount of Fritillaria polysaccharide extracted in the same time, resulting in a decrease in the extraction rate; the polysaccharide extraction rate in Comparative Example 4 increases compared with Example 2, but the purity is significantly reduced, because the purpose of the chloroform-n-butanol solution is to remove cellulase and most of the extracted protein substances to reduce the large The macroporous adsorption resin is used to adsorb other impurities for the purpose of adsorbing proteins, but in Comparative Example 4, no chloroform-n-butanol solution is added, and the macroporous adsorption resin cannot adsorb and remove a large amount of cellulase and protein, resulting in part of the cellulase remaining in the final Fritillaria polysaccharide, increasing its amount but reducing its purity; the polysaccharide extraction rate in Comparative Example 5 is slightly lower than that in Example 2, but the purity remains unchanged, because the supernatant B in step 4 contains polysaccharides mixed in the precipitate that are dissolved in distilled water, and Comparative Example 5 does not use distilled water to extract this part of the polysaccharide, but makes this part of the polysaccharide mixed in the precipitate and removed together, so the polysaccharide extraction rate is slightly lower, but the purity remains basically unchanged; the polysaccharide extraction rate in Comparative Example 6 is improved compared with that in Comparative Example 2 , but the purity is reduced, because the macroporous adsorption resin is not pretreated to remove impurities, so that some impurities in the macroporous adsorption resin are mixed into the final Fritillaria thunbergii polysaccharide; the polysaccharide extraction rate in Comparative Example 7, Comparative Example 8, and Comparative Example 9 drops sharply compared to Comparative Example 2, and the purity decreases slightly but still remains at a high level, because although the macroporous adsorption resin has an adsorption effect on non-polysaccharide impurities, it also has an adsorption effect on polysaccharides, and through the detection data of Comparative Example 7, Comparative Example 8, Comparative Example 9, and Example 2, it can be found that glycerol will only act on the macroporous adsorption resin under the condition of a weakly alkaline solution of baking soda, thereby reducing the adsorption rate of the macroporous adsorption resin on polysaccharides, thereby retaining the polysaccharides in the final Fritillaria thunbergii polysaccharide.

[0082] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0083] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the embodiments shown and described herein.

Claims

1. A method for extracting Fritillaria polysaccharide, characterized in that: The following steps are involved: Step 1: Take the freshly collected Fritillaria cirrhosae, wash it with clean water, freeze-dry it, and ultrafinely grind it to obtain Fritillaria cirrhosae powder; Step 2: Mix the Fritillaria powder and petroleum ether in a mass volume ratio of 1: (6-8) and stir evenly, heat in a microwave for 8-12 minutes, place in a 60-70°C water bath for reflux degreasing for 30-40 minutes, centrifuge, collect the residue, repeat the degreasing three times, evaporate the petroleum ether, add ethanol solution, stir magnetically, centrifuge, collect the residue, evaporate to obtain defatted Fritillaria powder; Step 3: add 10-20 times the weight of distilled water to the defatted Fritillaria powder, stir evenly, then add 2-3 times the volume of the solution of cellulase solution, extract at 45-55° C. for 35-45 minutes, and obtain a Fritillaria polysaccharide crude extract; Step 4: Heat the crude extract of Fritillaria polysaccharide at 90-100°C for 10-15 minutes to inactivate the enzyme, add 1 / 5 times the volume of chloroform-n-butanol solution, shake thoroughly for at least 30 minutes, centrifuge for 15-20 minutes, take supernatant A, collect the precipitate and add distilled water at 45-55°C, stir for 5-10 minutes, centrifuge for 15-20 minutes, take supernatant B and combine it with supernatant A, and concentrate under reduced pressure to obtain Fritillaria polysaccharide concentrate; Step 5, adding 3 to 5 times the volume of ethanol solution to the Fritillaria polysaccharide concentrate, stirring while adding, standing at 5 to 8°C for 24 to 40 hours, centrifuging for 15 to 20 minutes, collecting the precipitate, and vacuum drying to obtain Fritillaria crude polysaccharide; Step 6: Add 400-500 times the weight of distilled water to the crude Fritillaria polysaccharide to re-dissolve it, then add the pre-treated macroporous adsorption resin, shake on a shaker at room temperature for 24-30 hours, extract the adsorbed solution, concentrate and dry it to obtain Fritillaria polysaccharide; Among them, the pretreatment method of the macroporous adsorption resin is: take the macroporous adsorption resin, wash it with distilled water to remove surface impurities, then add anhydrous ethanol to soak for 20~30 hours to make the resin fully swell, then discard the ethanol, wash it with distilled water until it is odorless, add 3~4% weak acid solution to soak for 2~3 hours, wash it with distilled water to neutral, then add 4~5% baking soda glycerol aqueous solution to soak for 2~3 hours, wash it with distilled water to neutral, finally add distilled water to soak, and store it at 4~5℃ for use.

2. The method for extracting Fritillaria polysaccharide according to claim 1, characterized in that: In step 2, the Fritillaria powder is defatted and then an ethanol solution of not less than 5 times its volume is added.

3. The method for extracting Fritillaria polysaccharide according to claim 1, characterized in that: In step 3, the mass concentration of the cellulase solution is 4-6%.

4. The method for extracting Fritillaria polysaccharide according to claim 1, characterized in that: In step 4, the chloroform-n-butanol solution is a mixture of chloroform and n-butanol in a volume ratio of (4-6):

1.

5. The method for extracting Fritillaria polysaccharide according to claim 1, characterized in that: The ethanol solutions in step 2 and step 5 are both ethanol solutions with a volume fraction of 80-90%.

6. The method for extracting Fritillaria polysaccharide according to claim 1, characterized in that: The weak acid is one of citric acid, acetic acid and lactic acid.

7. The method for extracting Fritillaria polysaccharide according to claim 1, characterized in that: The volume ratio of glycerol to water in the glycerol aqueous solution is 1:(5-8).

8. The method for extracting Fritillaria polysaccharide according to claim 1, characterized in that: In step six, every 1 g of untreated macroporous adsorption resin is subjected to adsorption treatment with 3 to 4 mL of the reconstituted solution.

9. The method for extracting Fritillaria polysaccharide according to claim 1, characterized in that: The macroporous absorption resin is AB-8 type macroporous absorption resin or D-101 type macroporous absorption resin.