CN113416223B - Method for separating and purifying salidroside and product thereof - Google Patents

Abstract

The invention relates to the technical field of organic matter separation and extraction, and particularly discloses a method for separating and purifying salidroside and a product thereof. The method for separating and purifying salidroside does not adopt an organic solvent; comprises the steps of adsorbing salidroside fermentation liquor by macroporous resin at pH of 3-5, and eluting by alkaline buffer salt solution. The method does not use chromatographic separation, only uses simple adsorption and desorption, has short period, does not use an organic solvent in the whole process, saves the cost and has simple and convenient process operation. The purity and the content of the product are both more than 99 percent, and the method is suitable for industrial production of salidroside.

Description

Method for separating and purifying salidroside and product thereof

Technical Field

The invention relates to the technical field of organic matter separation and extraction, in particular to a method for separating and purifying salidroside and a product thereof.

Background

The rhodiola rosea is a perennial herb, mainly grows in alpine, dry, anoxic, strong ultraviolet irradiation and large day-night temperature difference areas with the altitude of 1600-4000 meters, and has extremely strong environmental adaptability and vitality. The four medical classics and the compendium of materia medica are recorded, and researches prove that salidroside has various pharmacological effects of resisting fatigue, resisting aging, regulating immunity, eliminating free radicals and the like.

In the components of the Anpu capsule, salidroside also has corresponding effect. Salidroside can interfere cell metabolism, change properties of cell coat, inhibit proliferation of tumor cell, increase T lymphocyte transformation rate and phagocyte activity, enhance immunity, inhibit tumor growth, increase leukocyte, and resist microwave radiation.

Therefore, salidroside (Salidroside) has good application prospect as a medicine or a functional food. The structural formula is as follows:

at present, the production method of salidroside mainly comprises a chemical synthesis method and a fermentation method, wherein the chemical synthesis method has multiple steps, multiple byproducts and difficult removal.

The key point at the present stage is to purify salidroside from rhodiola rosea, and extraction methods such as adsorption and desorption by using sephadex, simulated moving bed chromatography, countercurrent extraction, macroporous resin adsorption, alcohol desorption, reversed phase C18 preparative chromatographic separation, composite enzymolysis, mixed solvent crystallization and the like are reported.

In the Chinese patent CN103467540A, macroporous resin adsorption, high-concentration alcohol elution, eluent concentration, absolute ethyl alcohol dissolution, simulated moving bed chromatographic separation and crystallization are adopted. The process is separated by chromatography and simulated moving bed chromatography for 1 time, the period is long, the process uses organic solvent, and the cost and the safety need to be considered.

CN105085588A is enriched by silica gel column chromatography, purified by microporous resin and refined by reversed phase liquid phase preparative chromatography, and the whole process uses organic solvent with high cost.

CN106117281A, CN108727441A, CN109929002A, CN107686492A, CN101392011 and the like all adopt macroporous resin adsorption and organic solvent desorption. CN105924481A adopts a biological enzyme method, the problems of enzyme inactivation and recovery exist, meanwhile, a large amount of organic solvent is also used in continuous countercurrent extraction, diethyl ether is adopted in CN106279310A, acetone is adopted in CN104892696A, and the safety is difficult to guarantee. The united states patent US201816224257 discloses the principle of producing salidroside by transgenic strains, a gene construction method and a separation and purification method, wherein the salidroside is extracted by 70% ethanol, concentrated, sequentially extracted by n-hexane, chloroform and butanol respectively, separated by LH20, and purified by water-methanol gradient of 0-100%. The currently disclosed salidroside extraction method almost adopts macroporous resin organic solvent chromatography and chromatographic separation or preparative chromatography organic solvent chromatographic separation, so that the cost problem and the safety risk exist, and a new method for separating and purifying salidroside is needed to be provided.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a process technology for preparing high-purity salidroside, which is rapid, simple and convenient and has low cost (no organic solvent is used).

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for separating and purifying salidroside, which does not adopt organic solvent; comprises the steps of adsorbing salidroside fermentation liquor by macroporous resin at the pH value of 3-5, and eluting by alkaline buffer salt solution.

The salidroside fermentation broth is a fermentation broth containing salidroside and produced in a microbial fermentation mode. Preferably, the macroporous resin is absorbed and then washed by water, and then eluted by alkaline solution.

The method firstly adsorbs salidroside on resin under specific acidic conditions, and leaves substances with strong polarity, pigments and impurities of acidic precipitates in clear liquid. After the resin is washed clean by water, desorption is carried out under a specific alkaline condition, so that impurities with weak polarity and part of pigments are left on the resin, effective purification is realized, the product purity is greatly improved, and more colors are removed.

In the invention, the pH value of the alkaline buffer salt solution is 8-12, preferably 9-11;

the alkaline buffer salt in the alkaline buffer salt solution is ammonium bicarbonate, sodium bicarbonate, ammonium phosphate, sodium citrate or quaternary ammonium salt, and preferably sodium phosphate salt, such as sodium dihydrogen phosphate. The buffer capacity of the eluent is strong under the pH condition of the eluent, and the pH value is slightly influenced by material fluctuation.

In the invention, the concentration of the alkaline buffer salt solution is 0.25-1%;

preferably, the elution is performed in a gradient of 0.5% basic buffered salt solution and 0.75% basic buffered salt solution, and the desired yield can be achieved.

In the invention, after elution, the method also comprises the step of treating the mixture by using activated carbon at the temperature of 40-80 ℃ and the pH value of 2-5.

Preferably, the treatment is carried out with activated carbon at a pH of 2 to 4 and a temperature of 50 to 60 ℃.

The adding amount of the active carbon is 0.1-0.5% (w/v), preferably 0.3-0.5% (w/v).

The invention sets a step of further removing macromolecular substances and pigments by adopting activated carbon under a specific acidic condition by matching the characteristics of the solution treated by the specific adsorption and desorption steps. The step can be carried out under the condition of high temperature, and the treatment effect can be effectively improved.

In the invention, after the activated carbon treatment, the method also comprises the step of cooling and crystallizing; the step of cooling and crystallizing specifically comprises the following steps:

the starting temperature is 60 ℃, the temperature is reduced to 40 ℃ at the rate of reducing the temperature by 1 ℃ per hour, and then the temperature is reduced to 5 ℃ at the rate of reducing the temperature by 1 ℃ per 20 minutes.

The specific direct water phase cooling crystallization mode of the invention can not only ensure the effective precipitation of the product, but also improve the purity of the product.

In the invention, the concentration of the salidroside fermentation liquor before being absorbed by macroporous resin is 40-60 mg/ml; the concentration before the active carbon treatment is 60-100 mg/ml, preferably 60-70 mg/ml; the concentration before temperature reduction crystallization is 400mg/ml to 700mg/ml, preferably 400mg/ml to 600mg/ml.

The concentration can be adjusted by rotary evaporation.

In the invention, the salidroside fermentation liquor is sequentially filtered, ultrafiltered and nanofiltered before being absorbed by macroporous resin; the pore diameter of the filtration is 50nm; the ultrafiltration is carried out at the pH value of 7-9, and the adopted ultrafiltration membrane is a polyether sulfone membrane with the molecular weight of 2000 Da; the nanofiltration membrane adopted by nanofiltration is a polyether sulfone membrane with the molecular weight of 100 Da.

The filtration may be carried out using a ceramic membrane.

In the invention, the macroporous resin is Mitsubishi HP20 resin.

Preferably, the method comprehensively adopts a ceramic membrane filtration of the fermentation liquor of salidroside, filtrate is subjected to ultrafiltration and nanofiltration, then is adsorbed and desorbed by macroporous adsorption resin, impurities are removed by activated carbon, and the processes of concentration, cooling, crystallization and drying are carried out.

The method also comprises the steps of separation and decompression drying after temperature reduction and crystallization.

The invention also provides a salidroside product prepared according to the method. The product is white powder with purity of 99% and content of 99%.

The invention has the beneficial effects that:

the invention provides a method for separating and purifying salidroside, which does not use chromatographic separation, only uses simple adsorption and desorption, has short period, does not use organic solvent in the whole process, saves the cost and has simple and convenient process operation. Meanwhile, the quality level and yield of the method are high. Can obtain salidroside solid with chromatographic purity and content both more than 99 percent, and is suitable for industrial production of salidroside.

Drawings

FIG. 1 is a chromatogram of a fermentation broth of salidroside used in an example of the present invention.

FIG. 2 is a chromatogram of the final product of example 2 of the present invention.

Fig. 3 is an appearance diagram of a final product of example 2 of the present invention.

FIG. 4 is a photograph showing the results of the activated carbon-treated filtrates of comparative example 2 of the present invention diluted to the same concentration at different pH values, the results of the treatment at pH6.0 in the left tube, the results of the treatment at pH5.0 in the middle tube, and the results of the treatment at pH3.5 in the right tube.

FIG. 5 is photographs (each having a concentration of 500 mg/ml) of a concentrate of comparative example 2 of the present invention before and after treatment with activated carbon at pH3.5, wherein the left side is before treatment and the right side is after treatment.

Detailed Description

The preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The raw material rich in salidroside used in the invention is derived from fermentation liquor produced by fermentation of a genetically modified constructed strain. The principle of the transgenic strain for producing salidroside and the gene construction and fermentation method are disclosed in US patent US201816224257 (US 2019/0264221A 1) (see paragraph 131). Filtering the fermentation liquid with 50nm ceramic membrane to obtain ceramic membrane filtrate rich in salidroside. The following examples and comparative experiments all used this ceramic membrane filtrate. The chromatogram is shown in FIG. 1, and the specific detection information of each peak is shown in Table 1. The chromatographic conditions are as follows: mobile phase methanol-water (15: 85); wavelength: 223nm; solvent: methanol; column: sdb c-18; flow rate: 1ml/min; concentration: 1mg/ml. The following chromatographic conditions used for the purity test were the same.

TABLE 1

Peak#	Ret.Time	Area	Height	Area％
					1	3.827	5250	556	0.353
2	5.127	1175549	121300	79.008
					3	5.868	14994	1480	1.008
4	6.234	59338	5320	3.988
					5	7.889	156288	11320	10.504
6	8.910	4934	218	0.332
					7	9.803	39346	2252	2.644
8	11.954	8501	378	0.571
					9	13.341	5673	241	0.381
10	19.266	4978	168	0.335
					11	35.995	13028	211	0.876
In total		1487881	143445	100.000

Example 1

The present embodiment provides a method for separating and extracting salidroside, which specifically comprises the following steps:

adjusting the pH of the salidroside ceramic membrane filtrate to 8.0, performing ultrafiltration by using a polyether sulfone membrane with the molecular weight of 2000Da, performing nanofiltration by using a polyether sulfone membrane with the molecular weight of 100Da, concentrating to obtain a concentrated solution with the concentration of 50mg/ml, adjusting the pH of the concentrated solution to 4, adding HP20 resin, statically stirring at room temperature, adsorbing for 2 hours, sieving, washing with water, filling the resin into a column, performing gradient elution by using 0.5% disodium hydrogen phosphate-pH 10.5 solution and 0.75% disodium hydrogen phosphate-pH 10.5 solution (each 5 times volume), collecting eluent, and performing chromatographic test to obtain the following eluent with the purity: 93.72% and the yield 92.10%. Concentrating the eluent by rotary evaporation to a concentration of 60mg/ml, adjusting to pH3.5, adding 0.3% (mass to volume ratio) of active carbon at 60 ℃, stirring for removing impurities for 30 minutes, filtering, continuously concentrating the filtrate by rotary evaporation to a concentration of 500mg/ml, taking out, cooling 1 ℃ to 40 ℃ per hour at 60 ℃, cooling 1 ℃ to 5 ℃ per 20 minutes, separating to obtain white crystals, drying under reduced pressure (-0.09MPa, 60 ℃,20 hours), detecting the finished product with the chromatographic purity: 99.55%, content: 99.22% yield 94.07%. The final overall yield was 80.44%.

Example 2

The present embodiment provides a method for separating and extracting salidroside, which specifically comprises the following steps:

adjusting the pH of the salidroside ceramic membrane filtrate to 8.0, performing ultrafiltration by using a polyether sulfone membrane with the molecular weight of 2000Da, performing nanofiltration by using a polyether sulfone membrane with the molecular weight of 100Da, concentrating to obtain a concentrated solution with the concentration of 50mg/ml, adjusting the pH of the concentrated solution to 3.0, adding HP20 resin, statically stirring at room temperature, adsorbing for 2 hours, sieving, washing with water, filling the resin into a column, performing gradient elution by using 0.5% disodium hydrogen phosphate-pH 10.5 solution and 0.75% disodium hydrogen phosphate-pH 10.5 solution (each 5 times of volume), collecting eluent, and performing chromatographic test to obtain the following eluent with the purity: 94.38%, yield 92.52%. Concentrating the eluate by rotary evaporation to a concentration of 67mg/ml, adjusting to pH3.5, adding 0.3% (mass/volume ratio) of activated carbon at 60 deg.C, stirring for 30 min to remove impurities, filtering, concentrating the filtrate by rotary evaporation to a concentration of 500mg/ml, taking out, cooling to 1 deg.C to 40 deg.C per hour at 60 deg.C, cooling to 5 deg.C at a rate of 1 deg.C per 20 min, separating to obtain white crystals (see FIG. 3), drying under reduced pressure (-0.09MPa, 60 deg.C, 20 h), and detecting the final product with chromatographic purity: 99.91% (see fig. 2, and the specific detection information of each peak is shown in table 2), content: 100.86% yield 96.45%. The final overall yield was 82.75%.

TABLE 2

Peak #	Retention time	Area of	Area%
				1	6.145	3106027	99.912
2	7.216	384	0.012
				3	9.499	2358	0.076
Total of		3108769	100.000

Example 3

The present embodiment provides a method for separating and extracting salidroside, which specifically comprises the following steps:

adjusting the pH of the salidroside ceramic membrane filtrate to 8.0, performing ultrafiltration by using a polyether sulfone membrane with the molecular weight of 2000Da, performing nanofiltration by using a polyether sulfone membrane with the molecular weight of 100Da, concentrating the filtrate into a concentrated solution with the concentration of 50mg/ml, adjusting the pH of the concentrated solution to 5, adding HP20 resin, statically stirring at room temperature, adsorbing for 2 hours, sieving, washing with water, filling the resin into a column, performing gradient elution by using 0.5% disodium hydrogen phosphate-pH 10.5 solution and 0.75% disodium hydrogen phosphate-pH 10.5 solution (each 5 times volume), collecting eluent, and performing chromatographic test to obtain the purity of the eluent: 92.03%, yield 93.01%. Concentrating the eluate by rotary evaporation to a concentration of 70mg/ml, adjusting to pH3.5, adding 0.5% (mass to volume) of activated carbon at 60 ℃, stirring to remove impurities for 30 minutes, filtering, continuously concentrating the filtrate by rotary evaporation to a concentration of 500mg/ml, taking out, cooling to 1-40 ℃ per hour at 60 ℃, cooling to 5 ℃ at a rate of 1 ℃ per 20 minutes, separating to obtain white crystals, drying under reduced pressure (-0.09MPa, 60 ℃,20 hours), and detecting the chromatographic purity of the finished product: 99.21%, content: 99.07% yield 93.15%. The final overall yield was 80.07%.

Comparative example 1

The comparative example compares the influence of different eluents on the separation and extraction effect of salidroside. Specifically, the resin having adsorbed the concentrated solution of example 2 was uniformly stirred and divided into 4 portions, and the solution was desorbed by (1) 0.25% disodium hydrogenphosphate-ph 10.5 solution, (2) 0.5% disodium hydrogenphosphate-ph 10.5 solution, (3) 0.75% disodium hydrogenphosphate-ph 10.5 solution, and (4)1% disodium hydrogenphosphate-ph 10.5 solution (same elution times, 5 times in each case), respectively, to calculate the yield.

Finally, the yield and purity of the (1) solution after desorption from 0.25% disodium hydrogenphosphate-pH10.5 were 15.78% and 88.75%, respectively. The yield after desorption of the (2) 0.5% disodium hydrogenphosphate-pH10.5 solution was 39.57%, the purity was 93.52%, the yield after desorption of the (3) 0.75% disodium hydrogenphosphate-pH10.5 solution was 52.78%, the purity: 94.39% and the yield after desorption of a solution (4)1% disodium hydrogen phosphate-ph 10.5: 89.97%, purity: 91.98 percent.

Comparative example 2

This comparative example compares the effect of different pH values on the separation and extraction effect of salidroside when activated carbon treatment was performed. Specifically, the eluate obtained in example 2 was concentrated by rotary evaporation to a concentration of 67mg/ml, adjusted to PH3.5 and PH5.0 and PH6.0, respectively, and 0.3% (mass to volume) of activated carbon was added at 60 ℃ to remove impurities by stirring for 30 minutes, filtered, the yield was calculated, and the filtrate was diluted to the same concentration and the color of the filtrate was observed, see fig. 4. As can be seen from FIG. 4, the treatment effect was better at pH 3.5.

The final yield after activated carbon treatment at pH3.5 was 92.6%. The yield after activated carbon treatment at pH5.0 was 89.15% and the yield after activated carbon treatment at pH6.0 was 83.9%.

In addition, this comparative example also compares the color of the concentrate before and after treatment with activated carbon at pH3.5 (the concentration was 500mg/ml before and after). See fig. 5. Further, the decolorization effect after treatment at pH3.5 was exhibited.

Comparative example 3

The comparative example compares the influence of different cooling crystallization modes on the separation and extraction effect of salidroside. Specifically, the 500mg/ml concentrated solution obtained in example 2 after being treated with activated carbon was crystallized by cooling to 5 ℃ at a rate of 3 ℃ per hour from 60 ℃ to calculate the yield and purity. And (2) cooling the mixture to 1-50 ℃ per hour at the temperature of 60 ℃, cooling the mixture to 5 ℃ per hour at the rate of 3 ℃, crystallizing, and calculating the yield and the purity. Crystallization is carried out in a way that the temperature of 60 ℃ is reduced to 2-40 ℃ per hour, and then the temperature is reduced to 5 ℃ per hour at the rate of reducing the temperature to 3 ℃, and the yield and the purity are calculated. Crystallization was carried out by cooling to 1 ℃ to 40 ℃ per hour starting at 60 ℃ (4), and then to 5 ℃ at a rate of 3 ℃ per hour, and the yield and purity were calculated.

Finally, the yield and the purity of the crystal are 93.77% and 98.15% after crystallization in a way that the temperature is reduced to 5 ℃ at the rate of reducing the temperature to 3 ℃ per hour from 60 ℃. The yield after crystallization is 93.65% and the purity is 98.77% in such a way that the temperature is reduced from (2) to 60 ℃ per hour by 1 ℃ to 50 ℃ and then reduced to 5 ℃ at the rate of 3 ℃ per hour. The yield after crystallization was 94.05% and the purity was 99.01% in such a manner that (3) the temperature was decreased from 60 ℃ to 2 ℃ per hour and then to 5 ℃ per hour at a rate of 3 ℃.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims (5)

1. A method for separating and purifying salidroside is characterized in that the method does not adopt organic solvent; comprises the steps of adsorbing salidroside fermentation liquor by macroporous resin at the pH of 3-5, and eluting by alkaline buffer salt solution;

filtering, ultrafiltering and nanofiltering the salidroside fermentation liquor in sequence before the salidroside fermentation liquor is adsorbed by macroporous resin; the pore diameter of the filtration is 50nm; the ultrafiltration is carried out at the pH value of 7-9, and the adopted ultrafiltration membrane is a polyether sulfone membrane with the molecular weight of 2000 Da; the nanofiltration membrane adopted by nanofiltration is a polyether sulfone membrane with the molecular weight of 100 Da;

the macroporous resin is Mitsubishi HP20 resin;

the elution is gradient elution by 0.5% of alkaline buffer salt solution and 0.75% of alkaline buffer salt solution, the pH value of the alkaline buffer salt solution is 9-11, and the alkaline buffer salt in the alkaline buffer salt solution is sodium phosphate;

after elution, the method also comprises the step of treating the mixture by using active carbon at the temperature of 50-60 ℃ and the pH of 2-4;

after the activated carbon treatment, the method also comprises the step of cooling crystallization; the step of cooling and crystallizing specifically comprises the following steps: the starting temperature is 60 ℃, the temperature is reduced to 40 ℃ at the rate of reducing the temperature by 1 ℃ per hour, and then the temperature is reduced to 5 ℃ at the rate of reducing the temperature by 1 ℃ per 20 minutes.

2. The method of claim 1, wherein the activated carbon is added in an amount of 0.1 to 0.5% (w/v).

3. The method of claim 2, wherein the activated carbon is added in an amount of 0.3 to 0.5% (w/v).

4. The method according to any one of claims 1 to 3, wherein the concentration of the salidroside fermentation broth before adsorption on macroporous resin is 40 to 60mg/ml; the concentration before the active carbon treatment is 60-100 mg/ml; the concentration before temperature reduction and crystallization is 400 mg/ml-700 mg/ml.

5. The method according to claim 4, wherein the concentration of the salidroside fermentation broth before activated carbon treatment is 60-70 mg/ml; the concentration before temperature reduction and crystallization is 400-600 mg/ml.

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