CN113897406A - Method for extracting and purifying salidroside from rhodiola rosea powder - Google Patents
Method for extracting and purifying salidroside from rhodiola rosea powder Download PDFInfo
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- CN113897406A CN113897406A CN202111150297.6A CN202111150297A CN113897406A CN 113897406 A CN113897406 A CN 113897406A CN 202111150297 A CN202111150297 A CN 202111150297A CN 113897406 A CN113897406 A CN 113897406A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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- C07H1/00—Processes for the preparation of sugar derivatives
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- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
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- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
Abstract
The invention relates to a method for extracting and purifying salidroside from rhodiola powder, which comprises the following steps: (1) pretreatment: mixing the rhodiola powder with a phosphoric acid solution, carrying out heat treatment, adjusting the pH, adding a complex enzyme, and carrying out enzymolysis to obtain a rhodiola crude extract; (2) preparing yeast seed liquid; (3) fermenting with yeast to obtain radix Rhodiolae fermentation liquid; (4) separating and purifying to obtain salidroside purified solution. After enzymolysis, fermentation extraction and purification according to the method of the invention, the extraction rate of salidroside reaches 90.8-92.3%, the purity is improved from 2.8% to 50-55%, and the extraction yield and purity of salidroside are greatly improved. The method has the characteristics of low cost, simple operation, high efficiency, high extraction rate and continuous production, and has good application prospect for producing high-purity salidroside on a large scale.
Description
Technical Field
The invention belongs to the technical field of natural product extraction, and particularly relates to a method for extracting and purifying salidroside from rhodiola rosea powder.
Background
The content of the salidroside is shown in the specification,i.e. 2- (4-hydroxyphenyl) ethyl-beta-D-glucopyranoside (C)14H20O7) Tyrosol glycoside compounds isolated and identified from various natural rhodiola plants, such as rhodiola crenulata (rhodiolaccrenulata), rhodiola rosea (rhodiolarosa L), and rhodiola sachalinensis (rhodiolaca chaliensis), are one of the most effective active ingredients in rhodiola plants, and have been used as traditional Chinese medicines for the first time. Salidroside has been shown to have good physiological effects of resisting altitude stress, inflammation, oxidation, fatigue, energy, osteoporosis, aging, etc. In recent years, researchers have also found that the compound also exerts remarkable curative effects in the treatment of certain cancers and tumors, and also shows a certain therapeutic effect on diabetes. Salidroside attracts more and more attention due to its outstanding pharmacological and health-care effects, and has been widely applied to industries such as medicine, cosmetics, special medical food and the like as a functional component, and market demand is expanding day by day, so that research on extraction and purification methods of effective components in rhodiola is more important, and a salidroside extraction and purification method which is low in production cost, simple in operation and easy for large-scale industrial production is urgently needed to be developed.
At present, the prior art for extracting and purifying salidroside mainly comprises the following steps: the first is an extraction method, which utilizes the principle of similarity and intermiscibility to separate and extract salidroside according to the solubility difference of different components in a solvent. The rhodiola root and stem material is usually ground into powder and then the salidroside is extracted by water or ethanol. Extracting effective component salidroside in rhodiola rosea by respectively cooking Rongwei and the like with water and ethanol, wherein the water cooking conditions are as follows: steaming with water twice for 30min each time; the ethanol cooking conditions are as follows: steaming with 70% ethanol twice for 30min each time; the yield of the two is 0.16% and 0.14% respectively (Wangwei, Liuzhong, Xianzhilong. novel process for extracting the salidroside from the mountain [ J ]. Chinese herbal medicine, 1999,30(11):824 and 826.). The other is an organic solvent combined resin method, the scheme is that after salidroside is roughly extracted by using ethanol, ether and the like, the purity of salidroside is improved by combining the extraction effect of macroporous resin, and the yield of the invention patent CN201610688538.5 is 1.6-2.1%. In addition, patent CN201910168702.3 firstly uses ethanol reflux to extract salidroside from rhodiola rosea pretreated powder, and finally uses macroporous adsorption resin to perform adsorption purification, but does not provide extraction rate and purity value of salidroside. The leaching method and the resin purification method combined with organic reagents have the advantages of relatively poor separation and selection effects, large loss of target components, low extraction rate and low purity, difficult post-treatment process, high alcohol consumption and high production cost. Thirdly, the rhodiola rosea is crushed in the invention patents CN201810718980.7, CN201610918396.7, CN201310463903.9 and CN201510273811.3, the pretreatment of crude extract is carried out by physical ultrasonic wave or microwave-assisted ethanol, and then the crude extract is purified by alcohol precipitation or macroporous adsorption resin chromatography; in addition, the invention patent CN201710694349.3 activates rhodiola root powder by high frequency magnetic field, and then adds solvents such as ethanol, acetone, ethyl acetate, n-hexane, etc. to separate salidroside; and the latest invention patent CN202110049505.7 provides a process for extracting salidroside by means of magnetic particle materials, wherein the magnetic particles are prepared by fixing nickel or palladium complex on the surface of magnetic silica particles through tyramine, then mixing and incubating with rhodiola powder, placing in a magnetic field for attraction and separation, soaking the obtained magnetic particles in eluent, placing in a magnetic field for attraction and separation again, then acetylating the obtained sample liquid, reacting with pentaacetylglucose under the catalysis of the magnetic particles, and obtaining salidroside after the deacetylation treatment of reactants. The method has the disadvantages of complicated separation and purification process, high technical requirements on operators, high production cost (including equipment or materials and the like), and difficulty in industrial application because the method is basically in a laboratory exploration stage at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for extracting and purifying salidroside from rhodiola powder, which mainly obtains a salidroside product with high extraction rate and higher purity by using a natural yeast strain after enzymolysis and performing resin chromatography after fermentation, and has the advantages of simple purification process, energy saving and environmental protection. The extraction and purification of salidroside by a biological method is not reported.
The technical scheme of the invention is as follows:
a method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: mixing rhodiola root powder with a phosphoric acid solution, carrying out heat treatment, adjusting the pH value to 4.5-5.8, and adding a complex enzyme, wherein the complex enzyme consists of glucanase, feruloyl esterase and cellulase; carrying out enzymolysis for 9-12 h at 30-60 ℃ to obtain a rhodiola rosea crude extract;
(2) preparing yeast seed liquid: activating saccharomycete, inoculating to seed culture medium and culturing to obtain saccharomycete seed liquid;
(3) fermentation: inoculating the yeast seed liquid in the step (2) into the rhodiola rosea crude extract in the step (1), standing and fermenting for 24-72 hours at 28-40 ℃ to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) to obtain supernatant, and purifying to obtain salidroside purified liquor.
According to the invention, the rhodiola rosea powder in the step (1) is preferably rhodiola rosea powder obtained by directly crushing dry roots and stems of rhodiola rosea or rhodiola rosea extract powder obtained by extracting the rhodiola rosea with an organic solvent.
According to the invention, the concentration of the phosphoric acid solution in the step (1) is preferably 0.01 to 0.1 wt%.
According to the invention, the mass-volume ratio of the rhodiola rosea powder to the phosphoric acid solution in the step (1) is preferably 1-3: 1, and the unit is g/mL.
According to the invention, the heat treatment method in the step (1) is preferably as follows: heat treatment is carried out for 14-45 minutes at 121-160 ℃, and then cooling is carried out to 50-60 ℃.
According to the invention, the mass ratio of the glucanase, the feruloyl esterase and the cellulase in the complex enzyme in the step (1) is 1:1: 2.
According to the invention, the feeding mass ratio of the complex enzyme to the rhodiola root powder in the step (1) is (0.05-0.1): (10-50).
Preferably, the complex enzyme in the step (1) is solid powder.
Preferably, in step (2), the yeast is Saccharomyces cerevisiae, Kluyveromyces marxianus or Kluyveromyces lactis.
According to the invention, the activation in the step (2) is preferably performed by inoculating yeast into a YPD solid medium and performing static culture at 20-37 ℃.
Preferably, the seed culture medium in step (2) is a YPD liquid culture medium.
Preferably, the culture conditions in step (2) are 20-37 ℃ and 100-200 rpm, and the culture is carried out until the yeast seed liquid OD6000.6 to 0.8.
According to the invention, the inoculation amount of the yeast seed liquid in the step (3) is preferably 5-10% by mass.
According to the invention, the centrifugation condition in the step (4) is preferably 8000-12000 rpm for 10-20 min.
Preferably, in the step (4), the purification is that the supernatant is filtered, decolorized and decontaminated by a 400kDa nano membrane, purified by HP20 macroporous adsorption resin chromatography, eluted by 2% ethanol and evaporated in a rotary manner to obtain the salidroside purified solution.
According to the invention, the salidroside purified liquid in the step (4) can be dried to obtain salidroside purified powder.
In the invention, the rhodiola rosea powder raw material is dissolved by adding water, and then the purity of the rhodiola rosea powder raw material is 2.8% by HPLC-UV detection; according to the method, after dissolution of a phosphoric acid solution, enzymolysis of a complex enzyme, microbial fermentation, centrifugation, nanofiltration and chromatography and purification of HP20 macroporous adsorption resin, the extraction rate of the salidroside reaches 90.8-92.3%, the purity is improved from 2.8% to 50-55%, and the extraction yield and purity of the salidroside are greatly improved.
Has the advantages that:
1. in the invention, three enzymes of glucanase, feruloyl esterase and cellulase (mass ratio of 1:1:2) are used for the first time to cooperatively pretreat the rhodiola rosea powder raw material, so that salidroside in the raw material is dissolved out to the maximum extent, the enzyme reaction conditions are mild, the protection of the biological activity of salidroside is facilitated, and the extraction rate is ensured to be remarkably improved.
2. The rhodiola rosea glycoside purification liquid is obtained by fermenting and purifying rhodiola rosea powder by using saccharomyces cerevisiae, kluyveromyces marxianus and kluyveromyces lactis, and the extraction rate and purity of the rhodiola rosea glycoside are obviously improved. Meanwhile, rhodiola rosea powder which is currently sold on the market is generally obtained by grinding and grinding the rhodiola rosea powder or simply extracting the rhodiola rosea powder by using an organic reagent (methanol, ethanol and the like), so that the extraction rate and the purity of salidroside are lower. The method has the advantages of mild conditions, easy product separation, short production period, easy regulation and control and the like, uses less organic reagents, greatly protects the edible and medicinal values of the salidroside, and is favorable for large-scale industrial production and popularization.
3. The invention can solve the problems of low extraction efficiency, low product purity and more complex separation and purification process of rhodiola rosea powder prepared by directly crushing rhodiola rosea plants or extracting salidroside by an organic solvent method. After enzymolysis, fermentation extraction and purification according to the method of the invention, the extraction rate of salidroside reaches 90.8-92.3%, the purity is improved from 2.8% to 50-55%, and the extraction yield and purity of salidroside are greatly improved. In conclusion, the method has the characteristics of low cost, simple operation, high efficiency and continuous production, and has good application prospect for producing high-purity salidroside on a large scale.
Drawings
FIG. 1 is an HPLC chromatogram of a raw material rhodiola root powder dissolved in pure water.
FIG. 2 is an HPLC chromatogram of the purified solution of salidroside of example 1.
FIG. 3 is an HPLC chromatogram of the purified solution of salidroside of example 2.
FIG. 4 is an HPLC chromatogram of the purified solution of salidroside of example 3.
FIG. 5 is an HPLC chromatogram of the purified solution of salidroside of comparative example 1.
FIG. 6 is an HPLC chromatogram of the purified solution of salidroside of comparative example 2.
FIG. 7 is an HPLC chromatogram of the purified solution of salidroside of comparative example 3.
FIG. 8 is an HPLC chromatogram of the purified solution of salidroside of comparative example 4.
FIG. 9 is an HPLC chromatogram of the purified solution of salidroside of comparative example 5.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to examples, comparative examples and figures of the specification, which are used to illustrate the present invention, but the scope of the present invention is not limited thereto.
In the present invention, unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The rhodiola rosea powder used in the invention is rhodiola rosea extract powder (the purity is 3%) which is obtained by crude extraction through an organic solvent and is a common commercial product; the salidroside standard product, dextranase, feruloyl esterase, cellulase and HP20 macroporous adsorbent resin are all common commercial products; saccharomyces cerevisiae, Kluyveromyces marxianus, and Kluyveromyces lactis are also commercially available products, and are available from Jinan Sheng and chemical engineering Co., Ltd, Shanghai Xinyu Biotech Co., Ltd, or Fengshu (Shanghai) Biotech Co., Ltd.
YPD liquid medium: 2% of tryptone, 1% of yeast extract and 2% of glucose; 1.5 percent of agar powder is additionally added into the solid culture medium, and the mass percent is all.
Preparing a salidroside standard gradient solution: adding 0.1mg of salidroside standard into 100 μ L of ultrapure water, mixing to obtain 1mg/mL primary standard solution, and standing at-20 deg.C for use; taking 10 mu L of the primary standard substance solution, adding 90 mu L of ultrapure water to obtain a standard substance solution with the concentration of 100mg/L, and respectively diluting the standard substance solution into different concentrations step by step to obtain a standard substance gradient solution of salidroside; each gradient standard was then detected using High Performance Liquid Chromatography (HPLC). The detection conditions are that the wavelength is 280nm, the flow rate is 0.5mL/min, and the mobile phase is acetonitrile-0.1 percent formic acid waterAnd an ultraviolet detector. According to the detection result, drawing a standard curve of y-2295997 x-34651, R21, the linear relation is good at 0.01-2mg/mL, and the salidroside peak time is 10.080-13.100 min.
Detecting salidroside in raw material rhodiola root powder by adopting high performance liquid chromatography: dissolving 10mg rhodiola rosea powder in 10mL of pure water, centrifuging at 12000rpm for 20min, taking supernatant, filtering by using a 0.22 mu m filter membrane, and performing HPLC detection under the following detection conditions: wavelength of 280nm, flow rate of 0.5mL/min, mobile phase of acetonitrile-0.1% formic acid water, ultraviolet detector. The results of the detection are shown in FIG. 1.
As can be seen from the liquid phase diagram in fig. 1, the raw material rhodiola rosea powder has many peaks, and is calculated by combining the standard curve and the HPLC diagram, and the concentration of the salidroside in the raw material rhodiola rosea powder is 0.028mg/mL, the content of the salidroside is 28mg/g of the rhodiola rosea powder, and the purity of the salidroside is 2.8% calculated by using a formula of "mass of the salidroside/mass of the rhodiola rosea powder:" 100%.
Example 1
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: putting 10g of rhodiola rosea powder into a triangular flask, adding 10mL of 0.1 wt% phosphoric acid solution, carrying out constant temperature treatment at 121 ℃ for 30 minutes, standing and cooling to 60 ℃, adjusting the pH to 5.8 by using a sodium hydroxide solution, adding 100mg of complex enzyme consisting of glucanase, ferulic acid esterase and cellulase, wherein the mass ratio of the glucanase, the ferulic acid esterase and the cellulase is 1:1:2, adding pure water to a constant volume of 500mL, placing in a shaking table, and carrying out enzymolysis at 150rpm at 50 ℃ for 12 hours to prepare a rhodiola rosea crude extract;
(2) preparing yeast seed liquid: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in 30 deg.C constant temperature incubator, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in shaking table at 30 deg.C and 200rpm, performing shake-flask culture to OD6000.8, obtaining saccharomyces cerevisiae seed liquid;
(3) fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculation amount of 5 wt%, standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min, taking supernatant, carrying out suction filtration on the centrifuged supernatant, decoloring and removing impurities through a 400kDa nano-membrane, purifying by chromatography through HP20 macroporous adsorption resin, eluting with 2% ethanol, and carrying out rotary evaporation to obtain salidroside purified liquor.
Detecting the obtained salidroside purified solution by using High Performance Liquid Chromatography (HPLC), wherein the detection conditions are as follows: wavelength of 280nm, flow rate of 0.5mL/min, mobile phase of acetonitrile-0.1% formic acid water, ultraviolet detector, and detection method consistent with radix Rhodiolae standard. The results of the detection are shown in FIG. 2.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside purified liquid obtained in the embodiment, wherein the salidroside concentration is 0.50mg/mL, the mass is 254.5mg, and the extraction rate of the salidroside is 90.9%.
The salidroside purified solution obtained in the example is dried to obtain high-purity salidroside powder with the purity of 50.0%.
Example 2
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: putting 15g of rhodiola rosea powder into a triangular flask, adding 6mL of 0.01 wt% phosphoric acid solution, carrying out constant temperature treatment at 155 ℃ for 30 minutes, standing and cooling to 50 ℃, adjusting the pH to 4.5 by using sodium hydroxide solution, adding 50mg of complex enzyme consisting of glucanase, ferulic acid esterase and cellulase, wherein the mass ratio of the glucanase, the ferulic acid esterase and the cellulase is 1:1:2, adding pure water to fix the volume to 500mL, putting the mixture into a shaking table, and carrying out enzymolysis at 200rpm at 50 ℃ for 9 hours to prepare rhodiola rosea crude extract;
(2) preparing yeast seed liquid: inoculating Kluyveromyces marxianus in YPD solid culture medium, activating and culturing at 30 deg.C in constant temperature incubator, inoculating activated Kluyveromyces marxianus in 1.5L YPD liquid culture medium, placing in shaker at 25 deg.C and 200rpm, shake-flask culturing to OD6000.6, obtaining kluyveromyces marxianus seed liquid;
(3) fermentation: inoculating the kluyveromyces marxianus seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculation amount of 10 wt%, standing at 30 ℃, fermenting and culturing for 56 hours to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 8000rpm for 10min, taking supernatant, carrying out suction filtration on the centrifuged supernatant, decoloring and removing impurities through a 400kDa nano-membrane, purifying by chromatography through HP20 macroporous adsorption resin, eluting with 2% ethanol, and carrying out rotary evaporation to obtain salidroside purified liquor.
The obtained salidroside purified solution was detected by High Performance Liquid Chromatography (HPLC) under the same conditions as in example 1. The results of the detection are shown in FIG. 3.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside purified liquid obtained in the embodiment, wherein the salidroside concentration is 0.52mg/mL, the mass is 382.7mg, and the extraction rate of the salidroside is 91.1%.
The salidroside purified solution obtained in the example is dried to obtain high-purity salidroside powder with the purity of 51.9%.
Example 3
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: putting 20g of rhodiola rosea powder into a triangular flask, adding 10mL of 0.01 wt% phosphoric acid solution, carrying out constant temperature treatment at 140 ℃ for 20 minutes, standing and cooling to 55 ℃, adjusting the pH to 5.0 by using sodium hydroxide solution, adding 80mg of complex enzyme consisting of glucanase, ferulic acid esterase and cellulase, wherein the mass ratio of the glucanase, the ferulic acid esterase and the cellulase is 1:1:2, adding pure water to a constant volume of 500mL, placing in a shaking table, and carrying out enzymolysis at 120rpm at 40 ℃ for 10 hours to prepare rhodiola rosea crude extract;
(2) preparing yeast seed liquid: inoculating Kluyveromyces lactis into YPD solid culture medium, performing activation culture in 30 deg.C constant temperature incubator, inoculating activated Kluyveromyces lactis into 2L YPD liquid culture medium, placing in shaker at 37 deg.C, performing shake-flask culture at 200rpm to OD600Is 0.7, and the Kluyveromyces lactis seed liquid is obtained;
(3) fermentation: inoculating the Kluyveromyces lactis seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculation amount of 8 wt%, standing at 35 ℃, fermenting and culturing for 64h to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 10000rpm for 15min, taking supernatant, carrying out suction filtration on the centrifuged supernatant, decoloring and removing impurities through a 400kDa nano membrane, purifying by chromatography through HP20 macroporous adsorption resin, eluting with 2% ethanol, and carrying out rotary evaporation to obtain salidroside purified liquor.
The obtained salidroside purified solution was detected by High Performance Liquid Chromatography (HPLC) under the same conditions as in example 1. The results of the detection are shown in FIG. 4.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside purified liquid obtained in the embodiment, wherein the salidroside concentration is 0.54mg/mL, the mass is 516.7mg, and the extraction rate of the salidroside is 92.3%.
The salidroside purified solution obtained in this example was dried to obtain a high purity salidroside powder with a purity of 54.1%.
Comparative example 1
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: putting 10g of rhodiola rosea powder into a triangular flask, directly adding pure water to a constant volume of 500mL, putting the mixture into a shaking table, shaking uniformly at 150rpm at 50 ℃ for 12h, and preparing a rhodiola rosea crude extract;
(2) preparing yeast seed liquid: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in 30 deg.C constant temperature incubator, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in shaking table at 30 deg.C and 200rpm, performing shake-flask culture to OD6000.8, obtaining saccharomyces cerevisiae seed liquid;
(3) fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculation amount of 5 wt%, standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min, taking supernatant, carrying out suction filtration on the centrifuged supernatant, decoloring and removing impurities through a 400kDa nano-membrane, purifying by chromatography through HP20 macroporous adsorption resin, eluting with 2% ethanol, and carrying out rotary evaporation to obtain salidroside purified liquor.
The obtained salidroside purified solution was detected by High Performance Liquid Chromatography (HPLC) under the same conditions as in example 1. The results of the detection are shown in FIG. 5.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside purified liquid obtained in the comparative example, wherein the salidroside concentration is 0.29mg/mL, the mass is 174.3mg, and the extraction rate of the salidroside is 62.3%; drying to obtain salidroside powder with purity of 29.0%. The HPLC detection of the chromatogram shows that many peaks are present, and the extraction rate and purity of salidroside are reduced, which indicates that some impurity substances can not be degraded by yeast and exist in the rhodiola rosea extract after the rhodiola rosea powder which is not pretreated by dilute acid and enzymolysis is directly fermented and chromatographed, so that the purity is reduced. In addition, a part of salidroside may be lost with the solid impurities of suction filtration, resulting in a reduction in extraction yield.
Comparative example 2
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: putting 10g of rhodiola rosea powder into a triangular flask, adding 10mL of pure water, carrying out constant temperature treatment at 121 ℃ for 30 minutes, standing and cooling to 60 ℃, adjusting the pH to 5.8 by using a sodium hydroxide solution, adding 100mg of complex enzyme consisting of glucanase, ferulic acid esterase and cellulase, wherein the mass ratio of the glucanase, the ferulic acid esterase and the cellulase is 1:1:2, adding the pure water to a constant volume of 500mL, putting the mixture into a shaking table, shaking uniformly at 150rpm at 50 ℃ for 12 hours, and preparing a rhodiola rosea crude extract;
(2) preparing yeast seed liquid: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in 30 deg.C constant temperature incubator, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in shaking table at 30 deg.C and 200rpm, performing shake-flask culture to OD6000.8, obtaining saccharomyces cerevisiae seed liquid;
(3) fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculation amount of 5 wt%, standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min, taking supernatant, performing suction filtration on the centrifuged supernatant, decoloring and removing impurities through a 400kDa nano-membrane, performing chromatographic purification through HP20 macroporous adsorption resin, eluting with 2% ethanol, and performing rotary evaporation to obtain salidroside purified liquor.
The obtained salidroside purified solution was detected by High Performance Liquid Chromatography (HPLC) under the same conditions as in example 1. The results of the detection are shown in FIG. 6.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside purified liquid obtained in the comparative example, wherein the salidroside concentration is 0.36mg/mL, the mass is 196.3mg, and the extraction rate of the salidroside is 70.1%; drying to obtain salidroside powder with purity of 35.8%.
Compared with example 1, the extraction rate and the extraction purity of salidroside in the comparative example 2 are both obviously reduced, and the HPLC (high performance liquid chromatography) spectrum has more impurity peaks; compared with the comparative example 1, the HPLC chromatogram of the comparative example 2 has a little amount of peaks, and the extraction rate and purity of the obtained salidroside are improved. The results show that after the compound enzyme is added to treat the rhodiola rosea raw material powder, the influence of some cellulose impurities on fermentation, extraction and purification of target products is reduced, but the influence of some impurity substances on the extraction and purification of the salidroside is also influenced because the impurity substances cannot be utilized by saccharomycetes without the acidolysis treatment.
Comparative example 3
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: putting 10g of rhodiola rosea powder into a triangular flask, adding 10mL of 0.1 wt% phosphoric acid solution, carrying out constant temperature treatment at 121 ℃ for 30 minutes, standing and cooling to 60 ℃, adjusting the pH to 5.8 by using sodium hydroxide solution, only adding 100mg of dextranase, then fixing the volume to 500mL by using pure water, putting the mixture into a shaking table, shaking uniformly at 150rpm and 50 ℃ for 12 hours to prepare a rhodiola rosea crude extract;
(2) preparing yeast seed liquid:inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in 30 deg.C constant temperature incubator, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in shaking table at 30 deg.C and 200rpm, performing shake-flask culture to OD6000.8, obtaining saccharomyces cerevisiae seed liquid;
(3) fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculation amount of 5 wt%, standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min, taking supernatant, performing suction filtration on the centrifuged supernatant, decoloring and removing impurities through a 400kDa nano-membrane, performing chromatographic purification through HP20 macroporous adsorption resin, eluting with 2% ethanol, and performing rotary evaporation to obtain salidroside purified liquor.
The obtained salidroside purified solution was detected by High Performance Liquid Chromatography (HPLC) under the same conditions as in example 1. The results of the detection are shown in FIG. 7.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside purified liquid obtained in the comparative example, wherein the salidroside concentration is 0.41mg/mL, the mass is 225.1mg, and the extraction rate of the salidroside is 80.4%; drying to obtain salidroside powder with purity of 40.9%.
Compared with example 1, the extraction rate and the extraction purity of salidroside in the comparative example 3 are reduced, and the HPLC chromatogram has more impurity peaks; compared with the comparative example 1, the HPLC chromatogram of the comparative example has a little amount of peaks, and the extraction rate and purity of the obtained salidroside are improved. The results show that after the rhodiola rosea raw material powder is treated by acid and glucanase, the influence of impurities such as cellulose on the fermentation, extraction and purification of target products is reduced, but the enzymolysis effect of the glucanase is not as good as that of the compound enzyme only.
Comparative example 4
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: putting 10g of rhodiola rosea powder into a triangular flask, adding 10mL of 0.1 wt% phosphoric acid solution, carrying out constant temperature treatment at 121 ℃ for 30 minutes, standing and cooling to 60 ℃, adjusting the pH to 5.8, only adding 100mg of ferulic acid esterase, then using pure water to fix the volume to 500mL, putting the mixture into a shaking table, shaking uniformly at 150rpm at 50 ℃ for 12 hours, and preparing a rhodiola rosea crude extract;
(2) preparing yeast seed liquid: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in 30 deg.C constant temperature incubator, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in shaking table at 30 deg.C and 200rpm, performing shake-flask culture to OD6000.8, obtaining saccharomyces cerevisiae seed liquid;
(3) fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculation amount of 5 wt%, standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min, taking supernatant, performing suction filtration on the centrifuged supernatant, decoloring and removing impurities through a 400kDa nano-membrane, performing chromatographic purification through HP20 macroporous adsorption resin, eluting with 2% ethanol, and performing rotary evaporation to obtain salidroside purified liquor.
The obtained salidroside purified solution was detected by High Performance Liquid Chromatography (HPLC) under the same conditions as in example 1. The detection results are shown in fig. 8.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside purified liquid obtained in the comparative example, wherein the salidroside concentration is 0.45mg/mL, the mass is 228.2mg, and the extraction rate of the salidroside is 81.5%; drying to obtain salidroside powder with purity of 45.2%.
Compared with example 1, the extraction rate and the extraction purity of salidroside in the comparative example 4 are reduced, and the HPLC chromatogram has more impurity peaks; compared with the comparative example 1, the HPLC chromatogram of the comparative example has a little amount of peaks, and the extraction rate and purity of the obtained salidroside are improved. The results show that after the rhodiola rosea raw material powder is treated by acid and ferulic acid esterase, the influence of impurities such as cellulose on the fermentation, extraction and purification of target products is reduced, but the enzymolysis effect is not as good as that of the compound enzyme only by adopting ferulic acid esterase for enzymolysis.
Comparative example 5
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) pretreatment: putting 10g of rhodiola rosea powder into a triangular flask, adding 10mL of 0.1 wt% phosphoric acid solution, carrying out constant temperature treatment at 121 ℃ for 30 minutes, standing and cooling to 60 ℃, adjusting the pH to 5.8, only adding 100mg of cellulase, then adding pure water to a constant volume of 500mL, putting the mixture into a shaking table, shaking uniformly at 150rpm at 50 ℃ for 12 hours, and preparing a rhodiola rosea crude extract;
(2) preparing yeast seed liquid: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in 30 deg.C constant temperature incubator, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in shaking table at 30 deg.C and 200rpm, performing shake-flask culture to OD6000.8, obtaining saccharomyces cerevisiae seed liquid;
(3) fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculation amount of 5 wt%, standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min, taking supernatant, performing suction filtration on the centrifuged supernatant, decoloring and removing impurities through a 400kDa nano-membrane, performing chromatographic purification through HP20 macroporous adsorption resin, eluting with 2% ethanol, and performing rotary evaporation to obtain salidroside purified liquor.
The obtained salidroside purified solution was detected by High Performance Liquid Chromatography (HPLC) under the same conditions as in example 1. The detection results are shown in fig. 9.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside purified liquid obtained in the comparative example, wherein the salidroside concentration is 0.48mg/mL, the mass is 241.6mg, and the extraction rate of the salidroside is 86.3%; drying to obtain salidroside powder with purity of 47.7%.
Compared with example 1, the extraction rate and the extraction purity of salidroside in the comparative example 5 are reduced, and the HPLC chromatogram has more impurity peaks; compared with the comparative example 1, the HPLC chromatogram of the comparative example has a little amount of peaks, and the extraction rate and purity of the obtained salidroside are improved. The results show that after the rhodiola rosea raw material powder is treated by acid and cellulase, the influence of impurities such as cellulose on the target products of fermentation extraction and purification is reduced, but the enzymolysis effect of the cellulase is not as good as that of the compound enzyme only by adopting the enzymolysis of the cellulase.
The analysis results of salidroside prepared in examples 1 to 3 and comparative examples 1 to 5 are shown in Table 1.
TABLE 1 test and analysis results of examples 1 to 3 of the present invention and comparative examples 1 to 5
| Extraction rate of salidroside | Purity of purified powder of salidroside | |
| Example 1 | 90.8％ | 50％ |
| Example 2 | 91.1％ | 51.9％ |
| Example 3 | 92.3％ | 54.1％ |
| Comparative example 1 | 62.3％ | 29.0％ |
| Comparative example 2 | 70.1％ | 35.8％ |
| Comparative example 3 | 80.4％ | 40.9％ |
| Comparative example 4 | 81.5％ | 45.2％ |
| Comparative example 5 | 86.3％ | 47.7％ |
In conclusion, by adopting the method disclosed by the invention, after acidolysis, compound enzyme enzymolysis and yeast fermentation, the extraction rate and the product purity of the purified salidroside are greatly improved, the extraction rate of the salidroside reaches 90.8-92.3%, and the purity is 50-55%. In the rhodiola powder raw material, impurities such as cellulose restrict the release of salidroside, which is not beneficial to the extraction and purification of salidroside. In addition, the compound enzyme adopted by the invention is prepared by mixing glucanase, ferulic acid esterase and cellulase according to the mass ratio of 1:1:2, and the comparison result of comparative examples 3-5 and example 1 shows that compared with the single enzyme for enzymolysis, the compound enzyme has the best enzymolysis effect, and the three enzymes supplement each other in action and jointly promote the degradation of impurities such as cellulose in the rhodiola rosea powder.
Claims (10)
1. A method for extracting and purifying salidroside from rhodiola powder is characterized by comprising the following steps:
(1) pretreatment: mixing rhodiola root powder with a phosphoric acid solution, carrying out heat treatment, adjusting the pH value to 4.5-5.8, and adding a complex enzyme, wherein the complex enzyme consists of glucanase, feruloyl esterase and cellulase; carrying out enzymolysis for 9-12 h at 30-60 ℃ to obtain a rhodiola rosea crude extract;
(2) preparing yeast seed liquid: activating saccharomycete, inoculating to seed culture medium and culturing to obtain saccharomycete seed liquid;
(3) fermentation: inoculating the yeast seed liquid in the step (2) into the rhodiola rosea crude extract in the step (1), standing and fermenting for 24-72 hours at 28-40 ℃ to obtain rhodiola rosea fermentation liquid;
(4) separation and purification: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) to obtain supernatant, and purifying to obtain salidroside purified liquor.
2. The method according to claim 1, wherein the rhodiola rosea powder in step (1) is rhodiola rosea powder obtained by directly pulverizing dry roots and stems of rhodiola rosea or rhodiola rosea extract powder obtained by extracting rhodiola rosea with an organic solvent.
3. The method according to claim 1, wherein the concentration of the phosphoric acid solution in the step (1) is 0.01 to 0.1 wt%;
preferably, the mass-volume ratio of the rhodiola root powder to the phosphoric acid solution in the step (1) is 1-3: 1, and the unit is g/mL.
4. The method of claim 1, wherein the heat treatment in step (1) is performed by: heat treatment is carried out for 14-45 minutes at 121-160 ℃, and then cooling is carried out to 50-60 ℃.
5. The method of claim 1, wherein the mass ratio of the glucanase, the feruloyl esterase and the cellulase in the complex enzyme in the step (1) is 1:1: 2;
preferably, the feeding mass ratio of the complex enzyme to the rhodiola root powder in the step (1) is (0.05-0.1): (10-50).
6. The method of claim 1, wherein the complex enzyme in step (1) is in solid powder form.
7. The method of claim 1, wherein the yeast in step (2) is Saccharomyces cerevisiae, Kluyveromyces marxianus or Kluyveromyces lactis;
preferably, the activation in the step (2) is to inoculate yeast into YPD solid culture medium and perform static culture at 20-37 ℃.
8. The method of claim 1, wherein the seed medium in step (2) is a YPD liquid medium;
preferably, the culture conditions in the step (2) are 20-37 ℃ and 100-200 rpm, and the culture is carried out until the yeast seed liquid OD is reached6000.6 to 0.8.
9. The method according to claim 1, wherein the inoculation amount of the yeast seed liquid in the step (3) is 5-10% by mass;
preferably, the centrifugation in the step (4) is carried out for 10-20 min at 8000-12000 rpm.
10. The method as claimed in claim 1, wherein in the purification in step (4), the supernatant is filtered, decolorized and decontaminated by a 400kDa nanofiltration membrane, purified by HP20 macroporous adsorption resin chromatography, eluted by 2% ethanol and evaporated in a rotary manner to obtain salidroside purified solution;
preferably, the salidroside purified liquid in the step (4) is dried to obtain salidroside purified powder.
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