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 specifically discloses a method for separating and purifying salidroside and a product thereof. The method for separating and purifying salidroside of the present invention does not use an organic solvent; it includes the steps of eluting the salidroside fermentation broth with an alkaline buffered salt solution after being adsorbed by a macroporous resin at pH 3 to 5 . The method does not need chromatographic separation, only simple adsorption and desorption, short cycle, no use of organic solvent in the whole process, cost saving and simple process operation. The product purity and content are all more than 99%, which is suitable for industrial production of salidroside.

Description

A method for separating and purifying salidroside and its product

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 its product.

Background technique

Rhodiola rosea is a perennial herbaceous plant, which mainly grows in areas with an altitude of 1600-4000 meters in high cold, dry, anoxic, strong ultraviolet radiation, and large temperature difference between day and night. It has strong environmental adaptability and vitality. It is recorded in "Four Medical Codes" and "Compendium of Materia Medica". Studies have proved that salidroside has various pharmacological effects such as anti-fatigue, anti-aging, immune regulation, and scavenging free radicals.

Among the ingredients of Anpu Capsules, salidroside also has a corresponding effect. Salidroside can interfere with cell metabolism, change the properties of the cell coat, inhibit tumor cell proliferation, and at the same time increase the transformation rate of T lymphocytes and phagocyte activity, enhance immunity, inhibit tumor growth, increase white blood cells, and resist microwaves radiation etc.

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

At present, the production methods of salidroside are mainly chemical synthesis and fermentation. The chemical synthesis has many steps, many by-products and is difficult to remove.

At this stage, the focus is on the purification of salidroside from Rhodiola rosea. It has been reported that the use of Sephadex gel adsorption and desorption, simulated moving bed chromatography, countercurrent extraction, macroporous resin adsorption, alcohol desorption, reversed phase C18 preparative chromatography Separation, complex enzymatic hydrolysis, mixed solvent crystallization and other extraction methods.

In Chinese patent CN103467540A, macroporous resin is used for adsorption, high-concentration alcohol is eluted, the eluent is concentrated, dissolved in absolute ethanol, and then separated by simulated moving bed chromatography and crystallized. In the process, one time of chromatography and simulated moving bed chromatography is separated, the cycle is long, and the process uses organic solvents, and the cost and safety need to be considered.

CN105085588A uses silica gel column chromatography for enrichment, microporous resin for impurity removal, reversed-phase liquid phase preparative chromatography for purification, and the use of organic solvents in the whole process is costly.

CN106117281A, CN108727441A, CN109929002A, CN107686492A, CN101392011 and other patents all adopt macroporous resin adsorption and organic solvent desorption. CN105924481A adopts the biological enzyme method, which has problems of enzyme inactivation and recovery. At the same time, continuous countercurrent extraction also uses a large amount of organic solvents. CN106279310A uses ether, and CN104892696A uses acetone, so the safety is difficult to guarantee. U.S. Patent US201816224257 discloses the principle, gene construction method and separation and purification method of salidroside produced by transgenic strains, 70% ethanol extraction, concentration, sequential extraction of n-hexane, chloroform and butanol respectively, LH20 separation, water-methanol gradient is 0-100% purified. The currently disclosed salidroside extraction methods almost all use macroporous resin organic solvent chromatography and chromatographic separation or preparative chromatography organic solvent chromatographic separation, which has cost problems and safety risks, so it is necessary to provide a new separation and purification method for salidroside Tianglycoside method.

SUMMARY OF THE INVENTION

Aiming at the problems in the prior art, the purpose of the present invention is to provide a process technology for preparing high-purity salidroside which is quick, easy and low-cost (without using organic solvents).

In order to realize this object, technical scheme of the present invention is as follows:

A method for separating and purifying salidroside, the method does not use an organic solvent; the method comprises the step of eluting the salidroside fermentation liquid with an alkaline buffered saline solution after being adsorbed by a macroporous resin at a pH of 3 to 5 step.

The salidroside fermentation liquid of the present invention is a fermentation liquid containing salidroside produced in the form of microbial fermentation. Preferably, after the macroporous resin is adsorbed, it is washed with water first, and then eluted with an alkaline solution.

In the invention, the salidroside is firstly adsorbed on the resin under specific acidic conditions, and the highly polar substances, pigments, and impurities precipitated by acid are left in the clear liquid. After the resin is washed with water, it is desorbed under specific alkaline conditions, so that the less polar impurities and some pigments are left on the resin to achieve effective purification, which greatly improves the purity of the product and removes more colors.

In the present invention, the pH value of the alkaline buffered saline solution is 8-12, preferably 9-11;

The alkaline buffer salt in the alkaline buffer saline solution is ammonium bicarbonate, sodium bicarbonate, ammonium phosphate, sodium phosphate, sodium citrate or quaternary ammonium salt, preferably sodium phosphate, such as sodium dihydrogen phosphate. It has a strong buffering capacity under the pH condition of the eluent, and the pH value is less affected by material fluctuations.

In the present invention, the concentration of the alkaline buffered saline solution is 0.25-1%;

Preferably, the elution is gradient elution with 0.5% basic buffer saline solution and 0.75% basic buffer saline solution, which can achieve ideal yield.

In the present invention, after elution, the step of treating with activated carbon at pH 2-5 and 40-80° C. is also included.

Preferably, the treatment is carried out with activated carbon at pH 2-4, 50-60°C.

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

In accordance with the characteristics of the solution treated in the above specific adsorption and desorption steps, the present invention sets the step of further removing macromolecular substances and pigments by using activated carbon under specific acidic conditions. This step can be carried out under high temperature conditions, which can effectively improve the treatment effect.

In the present invention, after activated carbon treatment, the step of crystallization by cooling is also included; the step of crystallization by cooling is specifically:

The starting temperature is 60°C, and after cooling down to 40°C at a rate of 1°C per hour, it is then lowered to 5°C at a rate of 1°C every 20 minutes.

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

In the present invention, the concentration of the salidroside fermentation liquid is 40-60 mg/ml before being adsorbed by the macroporous resin; the concentration before the activated carbon treatment is 60-100 mg/ml, preferably 60-70 mg/ml; The concentration before crystallization is 400 mg/ml-700 mg/ml, preferably 400-600 mg/ml.

Concentration can be adjusted by rotary evaporation.

In the present invention, the salidroside fermentation liquid is sequentially filtered, ultra-filtered and nano-filtered before being adsorbed by the macroporous resin; the pore diameter of the filter is 50nm; the ultra-filtration is carried out at pH 7-9, using The ultrafiltration membrane is a polyethersulfone membrane with a molecular weight of 2000Da; the nanofiltration membrane used in the nanofiltration is a polyethersulfone membrane with a molecular weight of 100Da.

Filtration can be performed using ceramic membranes.

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

Preferably, the present invention comprehensively adopts salidroside fermented liquid ceramic membrane filtration, the filtrate is subjected to ultrafiltration and nanofiltration, adsorbed and desorbed through macroporous adsorption resin, activated carbon removes impurities, concentrates, and carries out cooling crystallization and drying technology.

The method of the present invention also includes the steps of separation and drying under reduced pressure after the temperature-lowering crystallization.

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

The beneficial effects of the present invention are at least:

The invention provides a method for separating and purifying salidroside. The method does not need chromatographic separation, but simply absorbs and desorbs, has a short cycle, does not use organic solvents in the whole process, saves costs, and is easy to operate. At the same time, the quality level and yield level of the method of the invention are very high. Salidroside solids with chromatographic purity and content greater than 99% can be obtained, which is suitable for industrial production of salidroside.

Description of drawings

Fig. 1 is the chromatogram of the salidroside fermentation liquid used in the embodiment of the present invention.

Fig. 2 is the final product chromatogram of embodiment 2 of the present invention.

Fig. 3 is an appearance view of the final product of Example 2 of the present invention.

Fig. 4 is the photograph after the filtrate after activated carbon treatment is diluted to the same concentration under different pHs in Comparative Example 2 of the present invention, the test tube on the left is the result after treatment at pH 6.0, and the middle test tube is the result after treatment at pH 5.0 , the right tube is the result after treatment at pH 3.5.

Fig. 5 is the concentrated solution photo (concentration is 500mg/ml) before and after treatment with activated carbon in the comparative example 2 of the present invention at pH3.5, the left side is before treatment, and the right side is after treatment.

Detailed ways

Preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are given for the purpose of illustration only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The salidroside-rich raw material used in the present invention is derived from a fermented liquid fermented by a transgenic strain. For the principle of production of salidroside by the transgenic strain and the gene construction and fermentation method, please refer to US Patent US201816224257 (US 2019/0264221A1) (such as paragraph 131). Filtrate the fermentation broth through a 50nm ceramic membrane to obtain a ceramic membrane filtrate rich in salidroside. The following examples and comparative tests all adopt this ceramic membrane filtrate. The chromatogram is shown in Figure 1, and the specific peak detection information is shown in Table 1. The chromatographic conditions are: 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 adopted in the purity test are 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 total 1487881 143445 100.000

Example 1

The present embodiment provides a method for separating and extracting salidroside of the present invention, specifically as follows:

Adjust the salidroside ceramic membrane filtrate to pH 8.0, carry out ultrafiltration with a polyethersulfone membrane with a molecular weight of 2000Da, perform nanofiltration with a polyethersulfone membrane with a molecular weight of 100Da, and then concentrate it into a concentrated solution with a concentration of 50 mg/ml. Adjust the concentrated solution to pH 4, add HP20 resin, stir statically and adsorb at room temperature for 2 hours, sieve, wash with water, then pack the resin into a column, use 0.5% disodium hydrogen phosphate-pH10.5 solution and 0.75% disodium hydrogen phosphate-pH10.5 The solution (5 times volume each) was subjected to gradient elution, and the eluate was collected. After chromatographic testing, the purity of the eluate was 93.72%, and the yield was 92.10%. Concentrate the eluent by rotary evaporation to a concentration of 60 mg/ml, adjust to pH 3.5, add 0.3% (mass volume ratio) of activated carbon at 60 ° C to remove impurities and stir for 30 minutes, filter, and continue to concentrate the filtrate by rotary evaporation to a concentration of 500 mg/ml ml, take it out, start cooling at 60°C by 1°C per hour to 40°C, then cool down to 5°C at a rate of 1°C every 20 minutes, separate to obtain white crystals, dry under reduced pressure (-0.09MPa, 60°C, 20h), The finished product is tested, and the chromatographic purity is: 99.55%, the content: 99.22%, and the yield is 94.07%. The final overall yield was 80.44%.

Example 2

The present embodiment provides a method for separating and extracting salidroside of the present invention, specifically as follows:

Adjust the salidroside ceramic membrane filtrate to pH 8.0, carry out ultrafiltration with a polyethersulfone membrane with a molecular weight of 2000Da, perform nanofiltration with a polyethersulfone membrane with a molecular weight of 100Da, and then concentrate it into a concentrated solution with a concentration of 50 mg/ml. The concentrated solution was adjusted to pH 3.0, and HP20 resin was added to statically stir and adsorb at room temperature for 2 hours, sieved, washed with water, and then the resin was packed into a column, and 0.5% disodium hydrogen phosphate-pH10.5 solution and 0.75% disodium hydrogen phosphate- The pH 10.5 solution (each with 5 volumes) was subjected to gradient elution, and the eluate was collected and tested by chromatography. The purity of the eluate was 94.38%, and the yield was 92.52%. Concentrate the eluent by rotary evaporation to a concentration of 67 mg/ml, adjust to pH 3.5, add 0.3% (mass volume ratio) of activated carbon at 60°C and stir to remove impurities for 30 minutes, filter, and continue to concentrate the filtrate by rotary evaporation to a concentration of 500 mg/ml ml, take it out, start at 60°C and drop the temperature by 1°C per hour to 40°C, then cool down to 5°C at a rate of 1°C every 20 minutes, separate and obtain white crystals (see Figure 3), dry under reduced pressure (-0.09MPa, 60 ℃, 20h), the finished product is tested, and the chromatographic purity is: 99.91% (see Figure 2, see Table 2 for the specific detection information of each peak), the content: 100.86%, and the yield is 96.45%. The final overall yield was 82.75%.

Table 2

peak# keep time area area% 1 6.145 3106027 99.912 2 7.216 384 0.012 3 9.499 2358 0.076 total 3108769 100.000

Example 3

The present embodiment provides a method for separating and extracting salidroside of the present invention, specifically as follows:

Adjust the salidroside ceramic membrane filtrate to pH 8.0, carry out ultrafiltration with a polyethersulfone membrane with a molecular weight of 2000Da, perform nanofiltration with a polyethersulfone membrane with a molecular weight of 100Da, and then concentrate it into a concentrated solution with a concentration of 50 mg/ml. Adjust the concentrated solution to pH5, add HP20 resin and stir statically at room temperature for 2 hours, sieve, wash with water, then pack the resin into a column, and use 0.5% disodium hydrogen phosphate-pH10.5 solution and 0.75% disodium hydrogen phosphate-pH10.5 The solution (5 times volume each) was subjected to gradient elution, and the eluate was collected and tested by chromatography. The purity of the eluent was 92.03%, and the yield was 93.01%. Concentrate the eluent by rotary evaporation to a concentration of 70 mg/ml, adjust to pH 3.5, add 0.5% (mass volume ratio) of activated carbon at 60°C and stir to remove impurities for 30 minutes, filter, and continue to concentrate the filtrate by rotary evaporation to a concentration of 500 mg/ml ml, take it out, start cooling at 60°C by 1°C per hour to 40°C, then cool down to 5°C at a rate of 1°C every 20 minutes, separate to obtain white crystals, dry under reduced pressure (-0.09MPa, 60°C, 20h), The finished product is tested, and the chromatographic purity is: 99.21%, the content: 99.07%, and the yield is 93.15%. The final overall yield was 80.07%.

Comparative Example 1

This comparative example compares the effects of different eluents on the separation and extraction of salidroside. Specifically, the resin after absorbing the concentrated solution in Example 2 is evenly stirred and divided into 4 parts, respectively with 1. 0.25% disodium hydrogen phosphate-pH10.5 solution, 2. 0.5% disodium hydrogen phosphate-pH10.5 solution, 3.0 .75% disodium hydrogen phosphate-pH10.5 solution, ④1% disodium hydrogen phosphate-pH10.5 solution for desorption (the elution times are the same, both are 5 times), and the yield is calculated.

Finally, after desorption with ①0.25% disodium hydrogen phosphate-pH10.5 solution, the yield was 15.78%, and the purity was 88.75%. The yield after desorption with ②0.5% disodium hydrogen phosphate-pH10.5 solution is 39.57%, and the purity is 93.52%, and the yield after desorption with ③0.75% disodium hydrogen phosphate-pH10.5 solution is 52.78% , Purity: 94.39%, the yield after desorption with ④1% disodium hydrogen phosphate-pH10.5 solution: 89.97%, purity: 91.98%.

Comparative Example 2

This comparative example compares the effect of different pH values on the separation and extraction of salidroside during the treatment of activated carbon. Specifically, the eluate obtained in Example 2 was concentrated by rotary evaporation to a concentration of 67 mg/ml, adjusted to pH 3.5 and pH 5.0 and pH 6.0 respectively, and added 0.3% (mass volume ratio) of activated carbon at 60 ° C to remove Mix for 30 minutes, filter, calculate the yield, and dilute the filtrate to the same concentration, observe the color of the filtrate, see Figure 4. It can be seen from Figure 4 that the treatment effect is better with pH3.5.

The final yield after activated carbon treatment at pH 3.5 was 92.6%. The yield after activated carbon treatment at pH 5.0 was 89.15%, and the yield after activated carbon treatment at pH 6.0 was 83.9%.

In addition, this comparative example also compares the color of the concentrate before and after treatment with activated carbon at pH 3.5 (concentrations before and after are both 500 mg/ml). See Figure 5. It further embodies the decolorization effect after being treated with pH3.5.

Comparative Example 3

This comparative example compares the effects of different cooling crystallization methods on the separation and extraction of salidroside. Specifically, the activated carbon-treated 500mg/ml concentrated solution in Example 2 was crystallized at a rate of 3°C per hour from 60°C to 5°C, and the yield and purity were calculated. Starting at ②60°C, the temperature was lowered by 1°C to 50°C per hour, and then the temperature was lowered by 3°C per hour to 5°C for crystallization, and the yield and purity were calculated. ③The crystallization was carried out by cooling the temperature from 2°C to 40°C per hour starting at 60°C, and then cooling down to 5°C at a rate of 3°C per hour, and calculating the yield and purity. The crystallization was carried out by cooling down to 40°C by 1°C per hour starting at 60°C, and then down to 5°C at a rate of 3°C per hour, and the yield and purity were calculated.

Finally, the yield after crystallization was 93.77% and the purity was 98.15% after cooling down to 5°C at a rate of 3°C per hour starting from 60°C. Starting at ②60°C, the temperature was lowered by 1°C to 50°C per hour, and then the temperature was lowered by 3°C per hour to 5°C. The yield after crystallization was 93.65%, and the purity was 98.77%. The yield after crystallization was 94.05%, and the purity was 99.01%.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

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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