CN113698504A - Extraction method and application of tremella polysaccharide - Google Patents
Extraction method and application of tremella polysaccharide Download PDFInfo
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Abstract
The invention relates to the technical field of traditional Chinese medicines, and particularly relates to an extraction method and application of tremella polysaccharide. The method comprises the following steps: (1) soaking Tremella in solvent; adding water, pulping, centrifuging, and collecting supernatant 1; (2) adding inorganic salt into the supernatant 1, stirring, centrifuging, and taking a supernatant 2; (3) adding the supernatant 2 into an ethanol-acetone aqueous solution, and standing; sieving with 200-300 mesh sieve to obtain polysaccharide precipitate; (4) redissolving the polysaccharide precipitate, heating to remove the organic solvent, and freeze-drying in vacuum to obtain the polysaccharide. The method has the advantages of high extraction rate, high purity and short extraction time of the tremella polysaccharide, and is beneficial to industrial mass production.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, and particularly relates to an extraction method and application of tremella polysaccharide.
Background
Tremella, also known as Tremella, is a traditional Chinese tonic health product, and has effects of nourishing yin, moistening lung, invigorating qi and regulating blood. The tremella polysaccharide is heteropolysaccharide separated and purified from tremella sporophore and submerged tremella cell fermenting spore, and many researches show that most of tremella has pharmacological activity related to tremella polysaccharide, and has multiple physiological functions of enhancing immunity, resisting aging, resisting tumor, reducing blood sugar, reducing blood fat, resisting radiation, etc. At present, the application in cosmetics and foods is more, the application in health care products and medicines is less, and the medicinal value of tremella cannot be fully exerted. The reason for this is that the extraction rate of tremella polysaccharide is not high, the extraction purity is still limited, the extraction time is long, the energy consumption is large, and the tremella polysaccharide is difficult to be industrially popularized.
Chinese invention patent CN107586349B discloses a method for extracting tremella polysaccharide from neutral buffer solution, which comprises the following steps: under the heating condition, leaching the tremella powder by using a neutral buffer solution to obtain a leaching solution: carrying out solid-liquid separation on the leaching solution to obtain a liquid component, wherein the liquid component contains tremella polysaccharide; and carrying out alcohol precipitation on the liquid components to obtain a precipitate which is the tremella polysaccharide. The extraction rate of the tremella polysaccharide is 29.69%, and the prepared polysaccharide contains protein and other impurities and has low purity.
Chinese patent CN112029006B discloses a tremella polysaccharide and a preparation method thereof, which comprises (1) leaching tremella powder by using a neutral buffer solution to obtain a leaching solution; (2) adding pectin cellulose complex enzyme into the leaching solution for enzymolysis; (3) filtering after enzymolysis, taking filtrate, and performing centrifugal standing to obtain supernatant; (4) and carrying out alcohol precipitation and centrifugation on the supernatant to obtain a precipitate which is the tremella polysaccharide. The method has high extraction rate of tremella polysaccharide, but the enzymolysis method has high cost and the purity needs to be further improved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the extraction method of the tremella polysaccharide, which has the advantages of high extraction rate, high purity of the tremella polysaccharide, short extraction time, low energy consumption and contribution to industrial production.
The purpose of the invention is realized by the following technical scheme:
a method for extracting tremella polysaccharide comprises the following steps:
(1) soaking Tremella in solvent; adding water, pulping, centrifuging, and collecting supernatant 1;
(2) adding inorganic salt into the supernatant 1, stirring, centrifuging, and taking a supernatant 2;
(3) adding the supernatant 2 into an ethanol-acetone aqueous solution, and standing; sieving with 200-300 mesh sieve to obtain polysaccharide precipitate;
(4) redissolving the polysaccharide precipitate, heating to remove the organic solvent, and freeze-drying in vacuum to obtain the polysaccharide.
Preferably, the mass-to-volume ratio of the tremella to the solvent in step (1) is 1: 1-3g/mL, wherein the soaking time is 20-40 min; the solvent is a mixture of sorbitol, polyethylene glycol, lecithin and water, and the mass ratio of the sorbitol to the polyethylene glycol to the lecithin to the water is 0.01-0.05: 0.05-0.08: 0.1-0.3: 1.
preferably, the mass-to-volume ratio of the tremella fuciformis to the water in the step (1) is 1:25-45g/mL, the beating is stopped for 3s after 5s of beating by a refiner, and the cumulative beating time is 0.5-1 min.
Preferably, the rotation speed of the centrifugation in the step (1) is 7000 9000r/min, and the time of the centrifugation is 20-40 min.
Preferably, the inorganic salt in the step (2) is a mixture of ammonium sulfate and sodium chloride, the ammonium sulfate is added into the supernatant 1 to reach the saturation degree of 55-60%, and the sodium chloride is added into the supernatant 1 to reach the saturation degree of 40-45%.
Preferably, the rotation speed of the centrifugation in the step (2) is 7000 9000r/min, and the time of the centrifugation is 5-15 min.
Preferably, the volume ratio of ethanol, acetone and water in the ethanol-acetone aqueous solution in the step (3) is 60-75: 5-15: 10-35; the standing time is 5-10 h.
Preferably, the sieving in step (3) is 300 mesh sieving.
Preferably, the redissolution in step (4) is to add water to the polysaccharide precipitate, and the mass-to-volume ratio of the polysaccharide precipitate to the water is 1: 67-90 g/mL; the organic solvent removal is to remove ethanol and acetone by rotary evaporation, and the water temperature is 55-65 ℃ under the rotary evaporation condition, and the rotating speed is 150-170 rpm.
The invention also aims to provide application of the extraction method in extracting tremella polysaccharide.
Compared with the prior art, the invention has the beneficial effects that:
(1) during pulping, tremella is very viscous in pulp, a considerable amount of water needs to be added, and a large amount of ethanol-acetone solvent needs to be consumed for polysaccharide precipitation in the later period, so that waste is caused to the reality. According to the invention, the tremella polysaccharide is soaked in the mixed solution of sorbitol, polyethylene glycol, lecithin and water in a specific ratio before pulping, so that the problem of high viscosity during pulping can be obviously solved, the tremella structure is damaged, the fluidity of the pulp is more sufficient, the tremella polysaccharide is more completely pulped, and the extraction rate of the tremella polysaccharide is improved;
(2) according to the invention, the inorganic salt is added into the white fungus supernatant 1, the affinity of the inorganic salt and water is stronger, so that the protein loses a water film, the specific inorganic salt is adopted for compounding, the synergistic removal of the protein is more efficient and complete, and the purity of the finally obtained white fungus polysaccharide is higher;
(3) in the alcohol precipitation step, the polysaccharide is efficiently separated and precipitated by adopting a specific ethanol-acetone aqueous solution, so that the purity of the tremella polysaccharide is further improved;
(4) the invention effectively removes the organic solvent residue by re-dissolving and rotary steaming, and can improve the quality of the tremella polysaccharide by a certain vacuum freeze drying process.
(5) The tremella polysaccharide prepared by the method has high content and high purity, and has good scavenging effect on superoxide anion free radicals.
Detailed Description
The present invention will be further described with reference to the following examples. All the raw materials are common commercial products.
Example 1
The extraction method of the tremella polysaccharide is characterized by comprising the following steps:
(1) weighing white mesophyll of fresh tremella, and soaking in 3 times of solvent for 30 min; adding 35 times volume of distilled water, pulping with a homogenizer for 5s and stopping for 3s, and cumulatively pulping for 1 min; centrifuging at 8000r/min for 30min to obtain supernatant 1;
wherein the solvent is a mixed dispersion solution of sorbitol, polyethylene glycol, lecithin and water, and the mass ratio of the sorbitol to the polyethylene glycol 600 to the lecithin to the water is 0.03: 0.06: 0.2: 1; lecithin was purchased from western Anjin pharmaceutical excipients, Inc.;
(2) adding ammonium sulfate into the supernatant 1 until the saturation degree is 55%, adding sodium chloride into the supernatant 1 until the saturation degree is 40%, stirring for 10min, centrifuging for 10min at the rotation speed of 8000r/min, and taking the supernatant 2;
(3) adding the supernatant 2 into an ethanol-acetone aqueous solution, and standing for 5 hours; sieving with 200 mesh sieve to obtain floccule on the sieve, i.e. polysaccharide precipitate; the volume ratio of ethanol to acetone to water in the ethanol-acetone aqueous solution is 70: 10: 20;
(4) adding water into the polysaccharide precipitate for redissolving (the mass volume ratio of the polysaccharide to the water is 1 g: 80mL), performing rotary evaporation to remove ethanol and acetone, performing rotary evaporation under the conditions of water temperature of 55 ℃ and rotation speed of 150rpm, and performing vacuum freeze drying to obtain the polysaccharide.
Example 2
The extraction method of the tremella polysaccharide is characterized by comprising the following steps:
(1) weighing white mesophyll of fresh tremella, and soaking in 1 time of solvent for 20 min; adding 25 times volume of distilled water, pulping with a homogenizer for 5s and stopping for 3s, and cumulatively pulping for 1 min; centrifuging at 7000r/min for 20min, and collecting supernatant 1; lecithin was purchased from hippophae ligustrum field food additives ltd;
wherein the solvent is a mixed dispersion solution of sorbitol, polyethylene glycol, lecithin and water, and the mass ratio of the sorbitol to the polyethylene glycol 400 to the lecithin to the water is 0.01: 0.05: 0.1: 1.
(2) adding ammonium sulfate into the supernatant 1 until the saturation degree is 60%, adding sodium chloride into the supernatant 1 until the saturation degree is 40%, stirring for 10min, centrifuging for 5min at a rotation speed of 9000r/min, and taking the supernatant 2;
(3) adding the supernatant 2 into an ethanol-acetone aqueous solution, and standing for 5 hours; sieving with 300 mesh sieve to obtain floccule and polysaccharide precipitate; the volume ratio of ethanol to acetone to water in the ethanol-acetone aqueous solution is 60: 5: 35;
(4) adding water into the polysaccharide precipitate for redissolving (the mass volume ratio of the polysaccharide to the water is 1 g: 67mL), performing rotary evaporation to remove ethanol and acetone, performing rotary evaporation under the conditions that the water temperature is 65 ℃, the rotating speed is 150rpm, and performing vacuum freeze drying to obtain the polysaccharide.
Example 3
The extraction method of the tremella polysaccharide is characterized by comprising the following steps:
(1) weighing white mesophyll of fresh tremella, and soaking in 3 times of solvent for 40 min; adding 45 times volume of distilled water, pulping with a homogenizer for 5s and stopping for 3s, and cumulatively pulping for 0.5 min; centrifuging at 7000r/min for 40min, and collecting supernatant 1;
wherein the solvent is a mixed dispersion solution of sorbitol, polyethylene glycol, lecithin and water, and the mass ratio of the sorbitol to the polyethylene glycol 600 to the lecithin to the water is 0.05: 0.08: 0.3: 1; lecithin was purchased from western Anjin pharmaceutical excipients, Inc.;
(2) adding ammonium sulfate into the supernatant 1 until the saturation degree is 55%, adding sodium chloride into the supernatant 1 until the saturation degree is 45%, stirring for 10min, centrifuging for 15min at the rotation speed of 7000r/min, and taking the supernatant 2;
(3) adding the supernatant 2 into an ethanol-acetone aqueous solution, and standing for 5 hours; sieving with 200 mesh sieve to obtain floccule and polysaccharide precipitate; the volume ratio of ethanol to acetone to water in the ethanol-acetone aqueous solution is 75: 15: 10;
(4) adding water into the polysaccharide precipitate for redissolving (the mass volume ratio of the polysaccharide to the water is 1 g: 90mL), performing rotary evaporation to remove ethanol and acetone, performing rotary evaporation under the conditions that the water temperature is 60 ℃, the rotating speed is 150rpm, and performing vacuum freeze drying to obtain the polysaccharide.
Comparative example 1
This comparative example differs from example 1 in that the soaking solvent in step (1) is a mixture of lecithin and water at 0.29: 1. The rest of the process was identical to example 1.
Comparative example 2
This comparative example differs from example 1 in that only ammonium sulfate was added to the supernatant 1 to a saturated solution, and no sodium chloride was added, and the rest was identical to example 1.
Comparative example 3
This comparative example differs from example 1 in that only 80% ethanol was used for precipitation in step (3), and the rest remained the same as example 1.
Comparative example 4
This comparative example differs from example 1 in that the solvent in step (1) is water only, and the rest is identical to example 1.
Test example 1 determination of Tremella polysaccharides
And (3) determining the content of the polysaccharide by adopting a phenol-sulfuric acid method. The extraction rate is the mass percentage of tremella polysaccharide prepared by freeze drying and obtained by the extraction method to tremella crude drug, the extraction purity is the mass percentage of tremella polysaccharide prepared by freeze drying and obtained by the extraction method to polysaccharide content measured by adopting a phenol-sulfuric acid method, and the result is shown in table 1.
TABLE 1 extraction rate and purity of Tremella polysaccharides
Test example 2 Tremella polysaccharide scavenging effect on superoxide anion radical
2.1 test materials
Tris-HCL buffer solution, pyrogallol and HCL; preparing tremella polysaccharide solution of 1mg/mL in each example and comparative example;
2.2 Experimental methods
Taking 8 10mL brown volumetric flasks numbered as pyrogallol group, example 1 group, example 2 group, example 3 group, comparative example 1 group, comparative example 2 group, comparative example 3 group and comparative example 4 group, respectively adding 4.5mL Tris-HCL buffer solution with pH9.0 and 50mmol/L, respectively adding 0.80mL pyrogallol hydrochloric acid solution with 3mmol/L to each of the other groups except the blank group, respectively adding 3mL tremella polysaccharide solution prepared by the corresponding group to each of the other groups except the blank group and the pyrogallol group, adding distilled water to fix the volume to 10mL, fully shaking, reacting for 9min at room temperature, respectively measuring the absorbance value at 325nm, and calculating the oxygen radical clearance rate R as follows:
r ═ a (each example group or comparative example group-a pyrogallol group)/a pyrogallol group × 100%;
the results of scavenging superoxide anion radicals for groups 1-3 and comparative examples 1-4 are shown in Table 2 below.
TABLE 2 scavenging ratio of superoxide anion free radical by each group of tremella polysaccharide solution
| Each group of | Clearance rate% |
| Example 1 | 92.5 |
| Example 2 | 92.4 |
| Example 3 | 92.2 |
| Comparative example 1 | 85.6 |
| Comparative example 2 | 87.2 |
| Comparative example 3 | 84.9 |
| Comparative example 4 | 79.3 |
And (4) analyzing results: the tremella polysaccharide extracts obtained by the groups 1-3 have high clearance rate on superoxide anion free radicals, and the clearance rate can reach more than 92% when the concentration is 0.3 mg/mL.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. The extraction method of the tremella polysaccharide is characterized by comprising the following steps:
(1) soaking Tremella in solvent; adding water, pulping, centrifuging, and collecting supernatant 1;
(2) adding inorganic salt into the supernatant 1, stirring, centrifuging, and taking a supernatant 2;
(3) adding an ethanol-acetone aqueous solution into the supernatant 2, and standing; sieving with 200-300 mesh sieve to obtain polysaccharide precipitate;
(4) redissolving the polysaccharide precipitate, heating to remove the organic solvent, and freeze-drying in vacuum to obtain the polysaccharide.
2. The extraction method according to claim 1, wherein the mass-to-volume ratio of the tremella to the solvent in step (1) is 1: 1-3g/mL, wherein the soaking time is 20-40 min; the solvent is a mixture of sorbitol, polyethylene glycol, lecithin and water, and the mass ratio of the sorbitol to the polyethylene glycol to the lecithin to the water is 0.01-0.05: 0.05-0.08: 0.1-0.3: 1.
3. the extraction method according to claim 1, wherein the mass-to-volume ratio of the tremella fuciformis to the water in the step (1) is 1:25-45g/mL, the beating is stopped for 3s after 5s of beating by a homogenizer, and the cumulative beating time is 0.5-1 min.
4. The extraction method as claimed in claim 1, wherein the rotation speed of the centrifugation in step (1) is 7000-9000r/min, and the time of the centrifugation is 20-40 min.
5. The extraction method according to claim 1, wherein the inorganic salt in the step (2) is a mixture of ammonium sulfate and sodium chloride, the ammonium sulfate is added to the supernatant 1 to a saturation degree of 55-60%, and the sodium chloride is added to the supernatant 1 to a saturation degree of 40-45%.
6. The extraction method as claimed in claim 1, wherein the rotation speed of the centrifugation in step (2) is 7000-9000r/min, and the time of the centrifugation is 5-15 min.
7. The extraction method according to claim 1, wherein the volume ratio of ethanol, acetone and water in the ethanol-acetone aqueous solution in the step (3) is 60-75: 5-15: 10-35; the standing time is 5-10 h.
8. The extraction method according to claim 1, wherein the sieving in step (3) is 300 mesh sieving.
9. The extraction method according to claim 1, wherein the redissolution in step (4) is that water is added into polysaccharide precipitate, and the mass-to-volume ratio of the polysaccharide precipitate to the water is 1: 67-90 g/mL; the organic solvent removal is to remove ethanol and acetone by rotary evaporation, and the water temperature is 55-65 ℃ under the rotary evaporation condition, and the rotating speed is 150-170 rpm.
10. Use of the extraction method of any one of claims 1-9 for extracting tremella polysaccharides.
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| CN115594775A (en) * | 2022-11-07 | 2023-01-13 | 福建医颜科生物科技有限公司（Cn） | Preparation method and application of tremella polysaccharide |
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