CN115926014A - Extraction method of fritillaria polysaccharide - Google Patents
Extraction method of fritillaria polysaccharide Download PDFInfo
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- CN115926014A CN115926014A CN202310052491.3A CN202310052491A CN115926014A CN 115926014 A CN115926014 A CN 115926014A CN 202310052491 A CN202310052491 A CN 202310052491A CN 115926014 A CN115926014 A CN 115926014A
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- fritillaria
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 135
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 135
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 135
- 241000605372 Fritillaria Species 0.000 title claims abstract description 117
- 238000000605 extraction Methods 0.000 title claims abstract description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 75
- 239000011347 resin Substances 0.000 claims abstract description 72
- 229920005989 resin Polymers 0.000 claims abstract description 72
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 67
- 239000012153 distilled water Substances 0.000 claims abstract description 50
- 238000001179 sorption measurement Methods 0.000 claims abstract description 39
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- 239000000843 powder Substances 0.000 claims abstract description 31
- 238000003756 stirring Methods 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 24
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- 230000002829 reductive effect Effects 0.000 claims abstract description 19
- 241000935235 Fritillaria meleagris Species 0.000 claims abstract description 18
- 239000003208 petroleum Substances 0.000 claims abstract description 18
- 238000005238 degreasing Methods 0.000 claims abstract description 17
- OAABHEHWRQAHEJ-UHFFFAOYSA-N butan-1-ol;chloroform Chemical compound ClC(Cl)Cl.CCCCO OAABHEHWRQAHEJ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000000284 extract Substances 0.000 claims abstract description 15
- 238000010438 heat treatment Methods 0.000 claims abstract description 15
- 239000002244 precipitate Substances 0.000 claims abstract description 15
- 238000002156 mixing Methods 0.000 claims abstract description 13
- 238000001035 drying Methods 0.000 claims abstract description 10
- 108090000790 Enzymes Proteins 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 6
- 238000010992 reflux Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 70
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 47
- 235000019441 ethanol Nutrition 0.000 claims description 28
- 239000007864 aqueous solution Substances 0.000 claims description 15
- 239000012535 impurity Substances 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- 108010059892 Cellulase Proteins 0.000 claims description 14
- 229940106157 cellulase Drugs 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 11
- 238000004140 cleaning Methods 0.000 claims description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 10
- 230000007935 neutral effect Effects 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 8
- 238000002203 pretreatment Methods 0.000 claims description 8
- CYFHBEJDHKXKGH-UHFFFAOYSA-M sodium;hydrogen carbonate;propane-1,2,3-triol Chemical compound [Na+].OC([O-])=O.OCC(O)CO CYFHBEJDHKXKGH-UHFFFAOYSA-M 0.000 claims description 7
- 238000010521 absorption reaction Methods 0.000 claims description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 6
- 229940088598 enzyme Drugs 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 230000009965 odorless effect Effects 0.000 claims description 5
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 239000003463 adsorbent Substances 0.000 claims description 3
- 239000004310 lactic acid Substances 0.000 claims description 3
- 235000014655 lactic acid Nutrition 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 6
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- 241000196324 Embryophyta Species 0.000 description 9
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 6
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- 230000008569 process Effects 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
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- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
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- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 238000003809 water extraction Methods 0.000 description 2
- 241001547127 Fritillaria cirrhosa Species 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
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- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a method for extracting fritillaria polysaccharide, which comprises the following steps: step one, preparing fritillary powder; step two, mixing the fritillaria powder with petroleum ether, heating by microwave, and carrying out reflux degreasing in a water bath to obtain degreased fritillaria powder; step three, adding distilled water and cellulose solution into degreased fritillaria powder, and extracting at the temperature of 45-55 ℃ to obtain fritillaria polysaccharide crude extract; heating the crude fritillaria polysaccharide extract to inactivate enzyme, adding a chloroform-n-butanol solution, fully oscillating, centrifuging, taking supernatant A, collecting precipitate, adding distilled water, stirring, centrifuging again, taking supernatant B, mixing with supernatant A, and concentrating under reduced pressure to obtain fritillaria polysaccharide concentrate; step five, carrying out alcohol precipitation and drying to obtain crude fritillaria polysaccharide; and step six, adding distilled water into the crude fritillaria polysaccharide for redissolving, adding the pretreated macroporous adsorption resin for oscillation and purification, extracting the adsorbed solution, concentrating and drying to obtain the refined fritillaria polysaccharide. The method has high extraction rate and good purification effect on fritillaria polysaccharide.
Description
Technical Field
The invention relates to the technical field of polysaccharide extraction from plants, in particular to a method for extracting fritillaria polysaccharide.
Background
Polysaccharide, one of the hot spots of recent research, is involved in the regulation of various vital phenomena of cells, has important health functions including maintenance of redox balance, chelation with heavy metals, regulation of immune regulatory system, inhibition of genotoxicity, etc., all depend on the structural characteristics of polysaccharide, and is widely distributed in nature. A large number of researches show that the plant polysaccharide has various biological effects such as aging resistance and the like, and almost has no toxicity to organisms, so the research on the biological effects of the plant polysaccharide draws more and more attention of people, and therefore, the polysaccharide substance with the activities of resisting oxidation and removing free radicals is screened from Chinese herbal medicines, and the plant polysaccharide has important significance on food and pharmaceutical industries. The fritillaria belongs to a perennial herb plant of the liliaceae, is a medicine-food homologous plant, contains fritillaria polysaccharide which has the biological activities of resisting tumor, resisting aging, resisting oxidation and the like, has no toxic or side effect, and can easily control the medicine quality by applying a chemical means.
Disclosure of Invention
The invention provides a method for extracting fritillaria polysaccharide, which can obtain fritillaria refined polysaccharide with high extraction rate and high purity by processes of microwave heating degreasing, cellulase extraction, chloroform-n-butanol solution and macroporous adsorption resin impurity removal and the like.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for extracting fritillary polysaccharide, comprising the steps of:
step one, taking the fritillaria which is just collected, cleaning with clear water, freeze-drying, and carrying out superfine grinding to obtain fritillaria powder;
step two, mixing fritillaria powder and petroleum ether according to a mass-volume ratio of 1: (6-8) (namely, each 6-8 mL of petroleum ether contains 1g of fritillary powder) are mixed and stirred uniformly, the mixture is placed in a water bath with the temperature of 60-70 ℃ for reflux degreasing for 30-40 min after being heated for 8-12 min by microwave, the mixture is centrifuged, residues are collected, degreasing is repeated for three times, ethanol solution is added after the petroleum ether is volatilized, the mixture is magnetically stirred and centrifuged, the residues are collected and volatilized, and the defatted fritillary powder is obtained;
step three, adding distilled water of which the weight is 10 to 20 times that of the defatted fritillaria powder into the defatted fritillaria powder, uniformly stirring the mixture, adding cellulase solution of which the volume is 2 to 3 times that of the solution into the mixture, and extracting the mixture for 35 to 45min at the temperature of between 45 and 55 ℃ to obtain crude fritillaria polysaccharide extract;
step four, heating the crude fritillaria polysaccharide extract at 90-100 ℃ for 10-15 min to inactivate enzyme, adding a 1/5 volume of chloroform-n-butanol solution, fully oscillating for at least 30min, centrifuging for 15-20 min, taking supernatant A, collecting precipitate, adding distilled water at 45-55 ℃, stirring for 5-10 min, centrifuging for 15-20 min, taking supernatant B, combining with supernatant A, and concentrating under reduced pressure to obtain fritillaria polysaccharide concentrated solution;
step five, adding 3-5 times volume of ethanol solution into the fritillaria polysaccharide concentrated solution, stirring while adding, standing for 24-40 h at 5-8 ℃, centrifuging for 15-20 min, collecting precipitate, and vacuum drying to obtain crude fritillaria polysaccharide;
and step six, adding distilled water with the weight of 400-500 times of the weight of the crude fritillaria polysaccharide into the crude fritillaria polysaccharide for redissolving, then adding the pretreated macroporous adsorption resin, oscillating for 24-30 hours at room temperature in a shaking table, extracting the adsorbed solution, concentrating and drying to obtain the refined fritillaria polysaccharide.
Preferably, in the extraction method of fritillaria polysaccharide, in the second step, ethanol solution with volume not less than 5 times of the fritillaria powder is added after the fritillaria powder is degreased.
Preferably, in the method for extracting fritillaria polysaccharide, in step three, the mass concentration of the cellulase solution is 4-6%. Namely, every 100 liters of the solution contains 4 to 6g of cellulase.
Preferably, in the extraction method of fritillaria polysaccharide, in the fourth step, the chloroform-n-butanol solution is prepared by mixing the following components in a volume ratio of (4-6): 1 chloroform: a mixture of n-butanol.
Preferably, in the extraction method of fritillaria polysaccharide, the ethanol solution in the second step and the ethanol solution in the fifth step are both 80-90% by volume.
Preferably, in the extraction method of fritillaria polysaccharide, the pretreatment method of macroporous adsorption resin comprises the following steps: taking macroporous adsorption resin, fully cleaning the macroporous adsorption resin with distilled water to remove surface impurities, adding absolute ethyl alcohol to soak for 20-30 hours to fully swell the resin, then removing the ethyl alcohol, washing the resin to be odorless with distilled water, adding 3-4% weak acid solution to soak for 2-3 hours, washing the resin to be neutral with the distilled water, adding 4-5% sodium bicarbonate glycerol aqueous solution to soak for 2-3 hours, washing the resin to be neutral with the distilled water, finally adding the distilled water to soak, and storing the resin at 4-5 ℃ for later use. 3-4% weak acid solution containing 3-4 g weak acid per 100g water; the 4-5% sodium bicarbonate glycerol aqueous solution contains 4-5 g sodium bicarbonate per 100g glycerol aqueous solution.
Preferably, in the extraction method of fritillaria polysaccharide, the weak acid is one of citric acid, acetic acid and lactic acid.
Preferably, in the extraction method of fritillaria polysaccharide, the volume ratio of glycerol to water in the glycerol aqueous solution is 1 (5-8).
Preferably, in the extraction method of fritillaria polysaccharide, in the sixth step, every 1g of untreated macroporous adsorption resin is correspondingly adsorbed with 3-4 mL of redissolution.
Preferably, in the extraction method of fritillaria polysaccharide, the macroporous absorption resin is AB-8 type macroporous absorption resin or D-101 type macroporous absorption resin.
The invention at least comprises the following beneficial effects: according to the method, microwave heating is performed in the petroleum ether degreasing process, so that the rupture of plant cell walls is promoted, various substances in cells are rapidly moved into the petroleum ether, the degreasing rate is improved, and the micromolecule alcohol content is removed through an ethanol solution after degreasing, so that the polysaccharide extraction purity is further improved; cellulase is added in the process of extracting the polysaccharide, so that the decomposition of plant tissues and the extraction of the polysaccharide are accelerated; cellulose and most of extracted protein substances are removed from the fritillaria polysaccharide crude extract by chloroform-n-butanol, so that the task of adsorbing proteins by macroporous adsorption resin in the subsequent purification process is reduced, more other impurities are adsorbed, and the purification effect is improved; the cellulose precipitate is re-dissolved by distilled water, and fritillaria polysaccharide doped in the precipitate is extracted, so that the waste of the extracted polysaccharide is avoided; by pretreating the macroporous resin, impurities in the macroporous resin are removed, the impurities are prevented from being substituted into fritillaria polysaccharide, and the glycerol acts on the macroporous adsorption resin under the condition of weak alkaline solution of sodium bicarbonate, so that the adsorption rate of the macroporous adsorption resin on the polysaccharide is reduced, more fritillaria polysaccharide is retained in the last fritillaria polysaccharide, and the extraction rate of the fritillaria polysaccharide is improved; the method has high extraction rate and good purification effect on fritillaria polysaccharide, and the purity of the obtained fritillaria refined polysaccharide is over 98 percent, so that the method is suitable for extracting fritillaria polysaccharide from various fritillaria.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified. In the description of the present invention, it should be noted that unless otherwise explicitly stated or limited, the terms "mounted," "connected," and "disposed" are to be construed broadly and can, for example, be fixedly connected, disposed, detachably connected, disposed, or integrally connected and disposed. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art. The terms "transverse," "longitudinal," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in an orientation or positional relationship indicated to be based on the orientation or positional relationship shown for ease of description and simplicity of description only, and do not indicate or imply that the device or element so referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the invention.
Example 1:
step one, taking just collected thunberg fritillary bulb, cleaning with clear water, freeze-drying, and micronizing to obtain fritillary bulb powder with the particle size of 30 microns;
step two, mixing fritillaria powder and petroleum ether according to the mass volume ratio of 1:6 mixing and stirring uniformly, heating for 8min by microwave, placing in a water bath at 60 ℃ for reflux degreasing for 30min, centrifuging, collecting residues, repeatedly degreasing for three times, volatilizing petroleum ether, adding 80% ethanol solution with the volume 5 times of that of the petroleum ether, magnetically stirring for 30min at 1000r/min, centrifuging for 10min, collecting residues, and volatilizing to obtain defatted fritillaria powder;
step three, adding distilled water of which the weight is 10 times that of the defatted fritillaria powder, uniformly stirring, adding cellulase solution of which the volume is 2 times that of the solution and the mass concentration is 4%, extracting for 35min at the temperature of 45 ℃, and slowly stirring for 10s every 5min in the extraction process to obtain crude fritillaria polysaccharide extract;
step four, heating the crude fritillaria polysaccharide extract at 90 ℃ for 10min to inactivate enzyme, adding 1/5 volume times of chloroform-n-butyl alcohol solution, fully oscillating for 30min, centrifuging for 15min, taking supernatant A, collecting precipitate, adding distilled water at 45 ℃, stirring for 5min, centrifuging for 15min, taking supernatant B, combining with supernatant A, and concentrating under reduced pressure to obtain fritillaria polysaccharide concentrated solution;
wherein the chloroform-n-butanol solution is prepared from the following components in a volume ratio of 4:1 chloroform: a mixture of n-butanol;
step five, adding 80% ethanol solution with volume 3 times of that of the fritillaria polysaccharide concentrated solution while stirring, standing for 24h at 5 ℃, centrifuging for 15min, collecting precipitate, and vacuum drying to obtain crude fritillaria polysaccharide;
step six, adding distilled water with the weight of 400 times of the crude fritillaria polysaccharide into the crude fritillaria polysaccharide for redissolving, then adding pretreated AB-8 type macroporous adsorption resin, oscillating for 24 hours in a shaking table at room temperature, extracting the adsorbed solution, concentrating and drying to obtain fritillaria refined polysaccharide;
wherein, every 1g of the untreated macroporous absorbent resin correspondingly adsorbs 3mL of redissolution;
the pretreatment method of the macroporous adsorption resin comprises the following steps: taking macroporous adsorption resin, fully cleaning with distilled water to remove surface impurities, adding absolute ethyl alcohol for soaking for 20 hours to fully swell the resin, then removing the ethyl alcohol, washing with distilled water until the resin is odorless, adding 3% citric acid solution for soaking for 2 hours, washing with distilled water until the resin is neutral, adding 4% sodium bicarbonate glycerol aqueous solution for soaking for 2 hours, washing with distilled water until the resin is neutral, finally adding distilled water for soaking, and preserving at 4 ℃ for later use; the volume ratio of glycerol to water in the glycerol aqueous solution is 1.
In the present embodiment, all the centrifugal rotation speeds are 4000r/min.
Example 2
Step one, taking the unibract fritillary bulb which is just collected, cleaning with clear water, freeze-drying, and micronizing to obtain unibract fritillary bulb powder with the particle size of 60 mu m;
step two, mixing fritillaria powder and petroleum ether according to the mass volume ratio of 1:7 mixing and uniformly stirring, heating by microwave for 10min, placing in a water bath at 65 ℃ for reflux degreasing for 35min, centrifuging, collecting residues, repeatedly degreasing for three times, volatilizing petroleum ether, adding 85% ethanol solution with 6 times of volume, magnetically stirring for 30min at 1000r/min, centrifuging for 10min, collecting residues, and volatilizing to obtain defatted fritillaria powder;
step three, adding distilled water 15 times the weight of the defatted fritillaria powder, uniformly stirring, adding cellulase solution 2.5 times the volume of the solution and having mass concentration of 5%, extracting for 40min at 50 ℃, and slowly stirring for 10s every 5min in the extraction process to obtain fritillaria polysaccharide crude extract;
heating the crude fritillaria polysaccharide extract at 95 ℃ for 13min to inactivate enzyme, adding a 1/5 volume of chloroform-n-butanol solution, fully oscillating for 40min, centrifuging for 18min, taking supernatant A, collecting precipitate, adding distilled water, stirring at 50 ℃ for 8min, centrifuging for 18min, taking supernatant B, combining with supernatant A, and concentrating under reduced pressure to obtain fritillaria polysaccharide concentrate;
wherein the chloroform-n-butanol solution is prepared from 5:1 chloroform: a mixture of n-butanol;
step five, adding 85% ethanol solution with 4 times volume of the fritillaria polysaccharide concentrated solution into the fritillaria polysaccharide concentrated solution, stirring while adding, standing for 32h at 6 ℃, centrifuging for 18min, collecting precipitate, and vacuum drying to obtain crude fritillaria polysaccharide;
step six, adding 450 times of distilled water into the crude fritillaria polysaccharide for redissolving, then adding pretreated AB-8 type macroporous adsorption resin, oscillating for 27 hours at room temperature by a shaking table, extracting the adsorbed solution, concentrating and drying to obtain refined fritillaria polysaccharide;
wherein, every 1g of the untreated macroporous adsorption resin correspondingly adsorbs 4mL of redissolution;
the pretreatment method of the macroporous adsorption resin comprises the following steps: taking macroporous adsorption resin, fully cleaning with distilled water to remove surface impurities, adding absolute ethyl alcohol for soaking for 25h to fully swell the resin, then removing the ethyl alcohol, washing with distilled water until the resin is odorless, adding 4% acetic acid solution for soaking for 2.5h, washing with distilled water until the resin is neutral, adding 4% sodium bicarbonate glycerol aqueous solution for soaking for 2.5h, washing with distilled water until the resin is neutral, finally adding distilled water for soaking, and storing at 4 ℃ for later use; the volume ratio of glycerol to water in the glycerol aqueous solution is 1.
In the present embodiment, all the centrifugal rotation speeds are 4000r/min.
Example 3
Step one, taking freshly collected fritillaria pallidiflora, cleaning with clear water, freeze-drying, and micronizing to obtain fritillaria pallidiflora powder with the particle size of 100 microns;
step two, mixing fritillaria powder and petroleum ether according to a mass-volume ratio of 1:8, mixing and uniformly stirring, heating by microwave for 12min, then placing in a 70 ℃ water bath for reflux degreasing for 40min, centrifuging, collecting residues, repeatedly degreasing for three times, volatilizing petroleum ether, adding 90% ethanol solution with the volume 8 times of that of the petroleum ether, magnetically stirring for 30min at 1000r/min, centrifuging for 10min, collecting residues, and volatilizing to obtain defatted fritillaria powder;
step three, adding 20 times of distilled water into the degreased fritillaria powder, uniformly stirring, adding 3 times of cellulase solution with the mass concentration of 6%, extracting for 45min at 55 ℃, and slowly stirring for 10s every 5min in the extraction process to obtain crude fritillaria polysaccharide extract;
step four, heating the crude fritillaria polysaccharide extract at 100 ℃ for 15min to inactivate enzyme, adding a 1/5 volume times of chloroform-n-butanol solution, fully oscillating for 50min, centrifuging for 20min, taking supernatant A, collecting precipitate, adding distilled water, stirring for 10min at 55 ℃, centrifuging for 20min, taking supernatant B, combining with supernatant A, and concentrating under reduced pressure to obtain fritillaria polysaccharide concentrated solution;
wherein the chloroform-n-butanol solution is prepared from the following components in a volume ratio of 6:1 chloroform: a mixture of n-butanol;
step five, adding 90 percent ethanol solution with the volume 5 times of that of the fritillaria polysaccharide concentrated solution while stirring, standing for 40 hours at the temperature of 8 ℃, centrifuging for 20min, collecting precipitate, and drying in vacuum to obtain crude fritillaria polysaccharide;
step six, adding 500 times of distilled water to the crude fritillaria polysaccharide for redissolving, then adding the pretreated D-101 type macroporous adsorption resin, oscillating for 30 hours at room temperature by a shaking table, extracting the adsorbed solution, concentrating and drying to obtain refined fritillaria polysaccharide;
wherein, every 1g of the untreated macroporous adsorption resin correspondingly adsorbs 4mL of redissolution;
the pretreatment method of the macroporous adsorption resin comprises the following steps: taking macroporous adsorption resin, fully cleaning with distilled water to remove surface impurities, adding absolute ethyl alcohol for soaking for 30 hours to fully swell the resin, then removing the ethyl alcohol, washing with distilled water until the resin is odorless, adding a 4% lactic acid solution for soaking for 3 hours, washing with distilled water until the resin is neutral, adding a 5% sodium bicarbonate glycerol aqueous solution for soaking for 3 hours, washing with distilled water until the resin is neutral, finally adding distilled water for soaking, and preserving at 5 ℃ for later use; the volume ratio of glycerol to water in the glycerol aqueous solution is 1.
In the present embodiment, all the centrifugal rotation speeds are 4000r/min.
Comparative example 1
The difference from example 2 is: in the second step, microwave heating is not carried out.
Comparative example 2
The difference from example 2 is: in the second step, the degreased product is not treated by an ethanol solution.
Comparative example 3
The difference from example 2 is: in step three, no cellulase solution is added.
Comparative example 4
The difference from example 2 is: in the fourth step, the chloroform-n-butanol solution was replaced with distilled water.
Comparative example 5
The difference from example 2 is: in the fourth step, no distilled water is added, namely the supernatant A is decompressed and concentrated to obtain fritillaria polysaccharide concentrated solution.
Comparative example 6
The difference from example 2 is: in the sixth step, the macroporous adsorption resin is not pretreated.
Comparative example 7
The difference from example 2 is: the macroporous adsorption resin pretreatment method does not contain glycerin.
Comparative example 8
The difference from example 2 is: in the macroporous adsorption resin pretreatment method, weak acid solution, sodium bicarbonate solution and glycerol aqueous solution are independently used for soaking treatment.
Comparative example 9
The difference from example 2 is: in the macroporous adsorption resin pretreatment method, the weak acid solution contains glycerol, and the sodium bicarbonate glycerol aqueous solution is sodium bicarbonate aqueous solution.
Comparative example 10
Extracting fritillaria polysaccharide by using a traditional water extraction and alcohol precipitation mode: cleaning, drying and crushing the collected fritillaria cirrhosa, adding 60-100 times of distilled water by volume, uniformly stirring, extracting for 24-30 h at 45-55 ℃, centrifuging, taking supernatant, concentrating under reduced pressure and drying to obtain fritillaria polysaccharide.
According to the formula: polysaccharide extraction% = fritillary extract polysaccharide mass/fritillary sample mass × 100% the polysaccharide extraction in each example above was measured and calculated and recorded to table 1; the fritillary refined polysaccharide in each example is redissolved by adding distilled water, the content of fritillary polysaccharide is detected by a sulfuric acid-phenol method, the purity of fritillary refined polysaccharide is obtained, and the result is recorded in table 1.
TABLE 1
| Polysaccharide extraction ratio% | Purity of polysaccharide% | |
| 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 |
According to the test results of table 1, the polysaccharide extracted from fritillaria by the method of the present invention (example 1, example 2, example 3) has a greatly improved extraction rate and a higher purity (more than 98%) compared to the polysaccharide extracted from fritillaria by the traditional water extraction and alcohol precipitation method (comparative example 10); compared with the polysaccharide extracted in the embodiment 2, the polysaccharide extracted in the comparative example 1 has increased extraction rate and reduced purity, because microwave can promote cell wall breaking, so that various substances in the cells are transferred into the solvent, which is beneficial to degreasing and impurity removal of petroleum ether, and the microwave heating is not carried out in the comparative example 1, so that the degreasing rate of the petroleum ether is reduced, and partial lipid impurities are left in the final fritillaria refined polysaccharide; the extraction rate of the polysaccharide is slightly increased and the purity is slightly reduced in comparative example 2 compared with that in example 2, because the ethanol in the second step of example 2 is used for removing the small molecular alcohol content to improve the purity of the polysaccharide, while in comparative example 2, the operation is not carried out, so that part of the small molecular alcohol content is left in the final fritillaria extract polysaccharide; compared with the polysaccharide obtained in the example 2, the extraction rate of the polysaccharide is suddenly reduced, the purity is unchanged, the cellulose can accelerate the decomposition of plant tissues and promote the rapid release and extraction of the fritillaria polysaccharide, and the cellulose is not added in the comparative example 3, so that the extraction amount of the fritillaria polysaccharide is reduced in the same time, and the extraction rate is reduced; comparative example 4 shows an increase in the extraction yield of polysaccharides and a significant decrease in purity compared to example 2, because the chloroform-n-butanol solution is intended to remove cellulase and most of the extracted proteinaceous material, so as to alleviate the task of adsorption of proteins by the macroporous adsorbent resin and remove other impurities, but in comparative example 4, no chloroform-n-butanol solution is added, and the macroporous adsorbent resin cannot adsorb a large amount of cellulase and proteins, resulting in a portion of cellulase remaining in the final fritillaria extract polysaccharide, increasing its amount, but decreasing its purity; comparative example 5 has a slightly lower extraction rate of polysaccharide and unchanged purity compared with example 2, because the supernatant B in step four contains the polysaccharide re-dissolved in distilled water and mixed in the precipitate, and comparative example 5 does not extract the part of polysaccharide with distilled water, but the part of polysaccharide mixed in the precipitate is removed together, so that the extraction rate of polysaccharide is slightly lower, but the purity is basically unchanged; compared with the polysaccharide in the comparative example 2, the polysaccharide extraction rate is improved, but the purity is reduced in the comparative example 6, because the macroporous adsorption resin is not pretreated to remove impurities, part of impurities in the macroporous adsorption resin are mixed into the final fritillaria refined polysaccharide; compared with the polysaccharide extraction rate suddenly reduced in the comparative examples 7, 8 and 9, the purity of the polysaccharide is slightly reduced but still kept higher in comparison with the polysaccharide extraction rate in the comparative example 2, because the macroporous adsorption resin has adsorption effect on the polysaccharide although adsorbing non-polysaccharide impurities, and the detection data of the comparative examples 7, 8, 9 and 2 also show that glycerol only acts on the macroporous adsorption resin under the condition of weak alkaline solution of sodium bicarbonate, so that the adsorption rate of the macroporous adsorption resin on the polysaccharide is reduced, and the polysaccharide is retained in the final fritillaria extract polysaccharide.
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (10)
1. The extraction method of fritillaria polysaccharide is characterized by comprising the following steps:
step one, taking the collected fritillaria, cleaning with clear water, freeze-drying, and carrying out superfine grinding to obtain fritillaria powder;
step two, mixing fritillaria powder and petroleum ether according to a mass-volume ratio of 1: (6-8) mixing and uniformly stirring, heating by microwave for 8-12 min, then placing in a water bath at 60-70 ℃ for reflux degreasing for 30-40 min, centrifuging, collecting residues, repeatedly degreasing for three times, adding an ethanol solution after petroleum ether is volatilized to dryness, magnetically stirring, centrifuging, collecting residues, and volatilizing to dryness to obtain defatted fritillaria powder;
step three, adding distilled water of which the weight is 10 to 20 times that of the defatted fritillaria powder into the defatted fritillaria powder, uniformly stirring the mixture, adding cellulase solution of which the volume is 2 to 3 times that of the solution into the mixture, and extracting the mixture for 35 to 45min at the temperature of between 45 and 55 ℃ to obtain crude fritillaria polysaccharide extract;
step four, heating the crude fritillaria polysaccharide extract at 90-100 ℃ for 10-15 min to inactivate enzyme, adding a 1/5 volume of chloroform-n-butanol solution, fully oscillating for at least 30min, centrifuging for 15-20 min, taking supernatant A, collecting precipitate, adding distilled water at 45-55 ℃, stirring for 5-10 min, centrifuging for 15-20 min, taking supernatant B, combining with supernatant A, and concentrating under reduced pressure to obtain fritillaria polysaccharide concentrated solution;
step five, adding 3-5 times volume of ethanol solution into the fritillaria polysaccharide concentrated solution, stirring while adding, standing for 24-40 h at 5-8 ℃, centrifuging for 15-20 min, collecting precipitate, and vacuum drying to obtain crude fritillaria polysaccharide;
and step six, adding distilled water with the weight of 400-500 times of the crude fritillaria polysaccharide into the crude fritillaria polysaccharide for redissolving, then adding the pretreated macroporous adsorption resin, oscillating for 24-30 h in a shaking table at room temperature, extracting the adsorbed solution, concentrating and drying to obtain the fritillaria refined polysaccharide.
2. The method for extracting fritillary polysaccharide according to claim 1, wherein in the second step, the fritillary powder is degreased and then is added with an ethanol solution with a volume not less than 5 times of the volume of the fritillary powder.
3. The method for extracting fritillaria polysaccharide as claimed in claim 1, wherein in step three, the mass concentration of the cellulase solution is 4-6%.
4. The method for extracting fritillaria polysaccharide as claimed in claim 1, wherein in the fourth step, the chloroform-n-butanol solution is prepared by mixing the following components in a volume ratio of (4-6): 1 chloroform: a mixture of n-butanol.
5. The method for extracting fritillaria polysaccharide as claimed in claim 1, wherein the ethanol solution in step two and step five is 80-90% by volume.
6. The extraction method of fritillaria polysaccharide as claimed in claim 1, wherein the pretreatment method of macroporous adsorption resin is as follows: taking macroporous adsorption resin, fully cleaning the macroporous adsorption resin with distilled water to remove surface impurities, adding absolute ethyl alcohol to soak the resin for 20 to 30 hours to fully swell the resin, then removing the ethyl alcohol, washing the resin to be odorless with distilled water, adding 3 to 4 percent weak acid solution to soak the resin for 2 to 3 hours, washing the resin to be neutral with the distilled water, adding 4 to 5 percent sodium bicarbonate glycerol aqueous solution to soak the resin for 2 to 3 hours, washing the resin to be neutral with the distilled water, finally adding the distilled water to soak the resin, and storing the resin at 4 to 5 ℃ for later use.
7. The method for extracting fritillary polysaccharide according to claim 6, wherein the weak acid is one of citric acid, acetic acid and lactic acid.
8. The method for extracting fritillaria polysaccharide as claimed in claim 6, wherein the volume ratio of glycerol to water in the glycerol aqueous solution is 1 (5-8).
9. The method for extracting fritillaria polysaccharide as claimed in claim 1, wherein in the sixth step, every 1g of the untreated macroporous adsorbent resin is treated with 3-4 mL of the redissolved solution by adsorption.
10. The method for extracting fritillaria polysaccharide as claimed in claim 1, wherein the macroporous absorption resin is AB-8 type macroporous absorption resin or D-101 type macroporous absorption resin.
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| CN108047343A (en) * | 2017-12-06 | 2018-05-18 | 中国科学院新疆理化技术研究所 | The preparation method and applications of Siberian fritillary bulb total starches |
| CN108530551A (en) * | 2018-04-25 | 2018-09-14 | 河北化工医药职业技术学院 | The preparation of fritillaria polysaccharide and application in preparation of anti-tumor drugs |
| CN111040040A (en) * | 2019-11-11 | 2020-04-21 | 佳木斯大学 | Preparation method and application of fritillaria ussuriensis polysaccharide zinc complex |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108047343A (en) * | 2017-12-06 | 2018-05-18 | 中国科学院新疆理化技术研究所 | The preparation method and applications of Siberian fritillary bulb total starches |
| CN108530551A (en) * | 2018-04-25 | 2018-09-14 | 河北化工医药职业技术学院 | The preparation of fritillaria polysaccharide and application in preparation of anti-tumor drugs |
| CN111040040A (en) * | 2019-11-11 | 2020-04-21 | 佳木斯大学 | Preparation method and application of fritillaria ussuriensis polysaccharide zinc complex |
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