Separation and purification method of salidroside

Abstract

The invention relates to the technical field of separation and purification, and discloses a salidroside separation and purification method which comprises the following steps: (1) filling macroporous resin serving as a filler into an organic medium-pressure-resistant chromatographic column, and mounting the organic medium-pressure-resistant chromatographic column at a chromatographic column position of a high performance liquid chromatograph; (2) dissolving the salidroside crude product, adjusting the pH value of the salidroside crude product, and taking the salidroside crude product as a loading solution; (3) loading the loading solution to an organic-resistant medium-pressure chromatographic column, adopting a high performance liquid chromatograph as a liquid conveying system, eluting the loading solution by adopting an ethanol water solution, and collecting eluent in sections; and the macroporous resin is macroporous resin LX-32. The provided separation and purification method can rapidly obtain a high-purity salidroside product, the separation and purification process is simple and convenient, the purity is high, the recovery rate is large, and the problems that a large-scale industrial extraction and separation method in the market is immature, the process is complex, the stability is low, the recovery rate is low, the number of repeated use times is low, and the extraction rate is low can be effectively solved.

Description

Technical Field

[0001] The present application relates to the technical field of separation and purification, and mainly to a method for separation and purification of salidroside. Background Art

[0002] Rhodiola rosea is a perennial herb that mainly grows in areas with an altitude of 1,600 to 4,000 meters, high cold, dry, hypoxic, strong ultraviolet radiation, and large temperature differences between day and night. It has strong environmental adaptability and vitality. Salidroside is a compound extracted from the dried roots and rhizomes or dried whole grass of the large plant Rhodiola rosea of ​​the Crassulaceae family. It has the functions of preventing tumors, enhancing immune function, delaying aging, anti-fatigue, anti-hypoxia, anti-radiation, bidirectional regulation of the central nervous system, and repairing and protecting the body. my country has been using Rhodiola rosea for a long time, and it is recorded in the "Four Medical Classics" and "Compendium of Materia Medica". Modern research has also proved that the active ingredients of salidroside have multiple pharmacological effects such as anti-fatigue, anti-aging, immune regulation, and scavenging free radicals.

[0003] At present, the development and production of salidroside mainly include natural extraction and chemical synthesis, but both methods have disadvantages, which limit the large-scale production of salidroside. Rhodiola rosea has a unique growth environment, and large-scale artificial cultivation has not yet been achieved. The natural resources that can be collected in the wild are very limited. Crassulaceae plants such as Rhodiola rosea are listed as national second-level protected plants. The natural extraction method is mainly due to the shortage of Rhodiola rosea on the raw material side, with an annual demand gap of nearly 2,000 tons. In addition, the content of salidroside in Rhodiola rosea is low, the extraction process is complex, and the cost is high. In addition, natural Rhodiola rosea also contains the toxic substance radix salidroside, which puts higher requirements on the purification process. At present, most chemical synthesis methods have the disadvantages of requiring selective protection, activation or the use of expensive metal catalysts. After synthesis, there are often small or trace amounts of other toxic chemicals. Not only is the production cost high, but it also has certain insecurity. Compared with natural extraction and chemical synthesis, biological fermentation has mild reaction conditions, simple process, low cost, high yield, and is conducive to protecting the ecological environment, so it has been widely studied.

[0004] In the existing technology of salidroside separation, before the sample was adsorbed with macroporous resin, the Chinese invention patent CN113416223A was filtered, ultrafiltered and nanofiltered three times in sequence. The macroporous resin model used is Japan Mitsubishi HP20. The starting temperature of the crystallization process is 60°C. After cooling down to 40°C at a rate of 1°C per hour, it is cooled down to 5°C at a rate of 1°C every 20 minutes. The crystallization time takes at least 30 hours. The experimental process is cumbersome, the time cycle is long, the material cost is high, and it relies on foreign materials. Chinese invention patent CN105085588A uses silica gel column chromatography for enrichment, microporous resin for impurity removal, and then refined by reverse liquid preparative chromatography. The use of organic solvents throughout the process is costly and dangerous. Chinese invention patent CN106699822A uses a dynamic countercurrent device for extraction, and the extraction rate of salidroside reaches 6.16%. The dynamic countercurrent device is complex to build, and there is still room for improvement in the extraction rate.

[0005] Since there are many species of Rhodiola rosea plants on the market, the components of different species of Rhodiola rosea vary greatly, and the content of extracted salidroside is also different. In the prior art, due to the immature extraction and separation methods, the process is complicated, the stability is not high, and the extraction rate is low, the price of salidroside on the market is relatively expensive. Therefore, it is necessary to develop a set of methods that can be simple, fast, stable and efficient in separating and purifying salidroside. Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a method for separating and purifying salidroside, aiming to solve the problems of low stability and low extraction rate of the existing extraction and separation methods for salidroside.

[0007] The technical solution of this application is as follows: The present application provides a method for separating and purifying salidroside, which comprises the following steps: (1) Filling a macroporous resin as a filler into an organic-resistant medium-pressure chromatography column, and installing the organic-resistant medium-pressure chromatography column in the chromatography column position of a high performance liquid chromatograph; (2) Dissolving the crude salidroside and adjusting the pH of the crude salidroside to prepare the sample solution; (3) loading the sample solution onto the organic-resistant medium-pressure chromatography column, using a high performance liquid chromatograph as an infusion system, sequentially eluting the sample solution with ethanol aqueous solutions of gradient concentrations, and collecting the eluate in sections; The macroporous resin is macroporous resin LX-32.

[0008] The present application provides an integrated technical method capable of separating and purifying salidroside, which can quickly obtain a high-purity salidroside product. The separation and purification process is simple, the purity is high, and the recovery rate is large. It can effectively solve the problems of immature large-scale industrial extraction and separation methods on the market, complex processes, low stability, low recovery rate, low number of reuses, and low extraction rate.

[0009] Furthermore, in step (2), the preparation step of the crude salidroside comprises: (21) adding an antioxidant to the salidroside fermentation broth, dissolving it with a solvent, and centrifuging to obtain a supernatant; (22) rotary evaporating the supernatant to obtain a concentrated solution; (23) extracting the concentrated solution with water-saturated n-butanol, and collecting the lower layer of extract; (24) evaporating the lower layer extract to dryness to obtain the crude salidroside; The volume ratio of the concentrate to water-saturated n-butanol is 1:1; In step (21), the antioxidant is one of BHT and VC, and the concentration of the antioxidant in the salidroside fermentation broth is 0.5-1 g / L.

[0010] Furthermore, after collecting the eluate in sections, the following step (4) is also included: (4) detecting the purity of salidroside in the eluate. If the purity of salidroside reaches 100%, decolorizing the eluate with activated carbon, and drying the filtrate to obtain salidroside; If the purity of salidroside is less than 100%, the eluate is used as the loading solution and steps (3) and (4) are repeated.

[0011] Furthermore, the salidroside concentration of the eluent was detected by ultra-high performance liquid chromatography, the chromatographic column of the ultra-high performance liquid chromatography was C18 (50 mm×2 mm), the mobile phase was methanol and ultrapure water, the injection volume was 5 μL, the wavelength was 280 nm, and the flow rate was 0.4 mL / min; The elution conditions of the mobile phase were as follows: first elution at a volume ratio of methanol to ultrapure water of 4:6 from 0 to 1 min, then elution at a volume ratio of methanol to ultrapure water of 4:6 from 1 to 7 min, and then elution at a volume ratio of methanol to ultrapure water of 4:6 from 7 to 15 min.

[0012] Furthermore, in step (2), after the crude salidroside is dissolved, the concentration of salidroside in the prepared solution is 8 g / L.

[0013] Furthermore, in step (2), the pH value of the loading solution is 5-8.

[0014] Furthermore, in step (4), the step of decolorizing the eluent using activated carbon comprises: The activated carbon is ultrasonically cleaned with ultrapure water until it becomes colorless, immersed in acetone and filtered, and dried for later use; The activated carbon was weighed and placed in a container, and an eluent was added. The container was sealed and placed in a shaker at 25° C. and 160 rpm for 1 hour.

[0015] Furthermore, in step (4), the activated carbon is 6-8 mesh activated carbon, and the mass volume ratio of the 6-8 mesh activated carbon to the eluent is 0.1-1:10 (g / ml).

[0016] Furthermore, in step (3), the elution rate is 3-8 ml / min; The ethanol aqueous solution with gradient concentration includes ethanol aqueous solutions with volume proportions of anhydrous ethanol of 5%, 20%, 50% and 100% respectively.

[0017] Furthermore, in step (21), the solvent is one of anhydrous ethanol or methanol, the centrifugal speed is 5000-8000 rpm / min, and the centrifugal time is 5-10 min.

[0018] Beneficial effects: The present application provides an integrated technical method for separating and purifying salidroside, which can quickly obtain high-purity salidroside products. The separation and purification process is simple, the purity is high, and the recovery rate is large. It can effectively solve the problems of immature large-scale industrial extraction and separation methods on the market, complex processes, low stability, low recovery rate, low number of reuses, and low extraction rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a schematic diagram of the process of separating and purifying salidroside in the present application.

[0020] Figure 2 This is the ultra-high performance liquid chromatography spectrum of the crude salidroside before purification by macroporous resin LX-32 in Example 1 of the present application.

[0021] Figure 3 This is the ultra-high performance liquid chromatography spectrum of crude salidroside purified by macroporous resin LX-32 in Example 1 of the present application.

[0022] Figure 4 This is a schematic diagram of the molecular ion peak of salidroside in ultra-high performance liquid time-of-flight mass spectrometry of the eluate after purification in Example 1 of the present application.

[0023] Figure 5 This is the NMR detection image of salidroside collected after purification in Example 1 of the present application.

[0024] Figure 6This is a comparison chart of the eluent of Example 5 of the present application before and after decolorization.

[0025] Figure 7 This is a graph showing the test results of salidroside concentration and recovery rate of the eluate of Example 1 and the filtrate of Examples 5-7 of the present application. DETAILED DESCRIPTION

[0026] The present application provides a method for separating and purifying salidroside. To make the purpose, technical scheme and effect of the present application clearer and more specific, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0027] like Figure 1 As shown, the present application provides a method for separating and purifying salidroside, which comprises the following steps: (1) Pretreatment of the macroporous resin: filling the macroporous resin as a filler into an organic-resistant medium-pressure chromatography column, and installing the organic-resistant medium-pressure chromatography column in the chromatographic column position of a high performance liquid chromatograph; (2) Dissolving the crude salidroside and adjusting the pH of the crude salidroside to prepare the sample solution; (3) The sample solution is loaded onto an organic resistant medium pressure chromatography column, and a high performance liquid chromatograph is used as an infusion system. The sample solution is eluted with ethanol aqueous solutions of gradient concentrations in sequence, and the eluate is collected in sections.

[0028] The purity and concentration of salidroside in the obtained eluate are relatively high, and high-purity salidroside can be obtained by drying and collecting.

[0029] The present application provides an integrated technical method capable of separating and purifying salidroside, which can quickly obtain a high-purity salidroside product. The separation and purification process is simple, the purity is high, and the recovery rate is large. It can effectively solve the problems of immature large-scale industrial extraction and separation methods on the market, complex processes, low stability, low recovery rate, low number of reuses, and low extraction rate.

[0030] Furthermore, in step (1), the macroporous resin is macroporous resin LX-32. Through the optimization of the process of separation and purification of salidroside in the present application, the applicant has conducted multiple experiments and found that the macroporous resin LX-32 used can achieve a good impurity removal effect of salidroside, and the obtained sample can reach 100% chromatographic purity.

[0031] In the present application, the separation and purification method provided is capable of dealing with most salidroside raw materials, from which salidroside can be separated and purified, such as salidroside natural products, salidroside Chinese medicine granules, salidroside cosmetics and salidroside fermentation broth.

[0032] Preferably, the crude salidroside is obtained by salidroside fermentation broth, and the salidroside fermentation broth synthesized by microorganisms is used for separation, which has the characteristics of low cost, high efficiency and easy large-scale preparation.

[0033] Furthermore, in step (2), the preparation steps of crude salidroside include: (21) adding an antioxidant to the salidroside fermentation broth, dissolving it with a solvent, and centrifuging to obtain a supernatant; (22) Rotary evaporation of the supernatant to obtain a concentrated solution; (23) extracting the concentrated solution with water-saturated n-butanol, and collecting the lower layer of extract; (24) The lower layer extract was evaporated to dryness to obtain crude salidroside; The volume ratio of the concentrate to the water-saturated n-butanol is 1:1.

[0034] More specifically, the steps of crude extraction of salidroside fermentation broth include: (21a) adding antioxidants to the salidroside fermentation broth, dissolving it with a solvent by ultrasonication for 5-10 min, and centrifuging to obtain a supernatant; (22a) rotary evaporating the supernatant at 45°C to obtain a concentrated solution; (23a) extracting the concentrate with water-saturated n-butanol, the volume ratio of the concentrate to water-saturated n-butanol was 1:1, extracting three times, and combining the lower extracts; (24a) evaporating the lower layer extract to dryness to obtain crude salidroside; In step (21), the solvent is one of anhydrous ethanol or methanol, the centrifugal speed is 5000-8000 rpm / min, and the centrifugal time is 5-10 min; in step (22), rotary evaporation is used to remove the solvent; in step (23), after combining the lower layer extract, if an emulsion layer appears, 1-10 ml of isopropanol can be added to every 200 ml of extract to break the emulsion.

[0035] Furthermore, in step (21), the antioxidant is one of BHT and VC, and the concentration of the antioxidant in the salidroside fermentation broth is 0.5-1 g / L.

[0036] In the present application, by adding a specific concentration of antioxidant to the salidroside fermentation broth, the following effects can be achieved: (1) the oxidation reaction can be inhibited, which helps to maintain the physical and chemical properties of salidroside and make the subsequent separation and purification operations more controllable; (2) the antioxidant can also inhibit the side reactions caused by oxidation, reduce the formation of by-products and impurities, and help simplify the purification steps and improve the product purity, while maintaining the stability of the fermentation broth components and avoiding affecting the adsorption effect on salidroside; (3) the stability of salidroside is increased, and the adsorption and elution behavior of salidroside in the macroporous resin LX-32 are more stable, which is conducive to reducing the loss during the separation process, and is conducive to obtaining a better separation effect and improving the recovery rate.

[0037] Specifically, the composition of the salidroside fermentation broth is complex, and if the concentration of the antioxidant is too low, it is difficult to ensure sufficient antioxidant effect. If the concentration is too high, it will affect the pH value of the salidroside fermentation broth, and it may also react with the other components to change the polarity thereof and affect the adsorption effect of salidroside. If there is too much residue in the crude product, it may also occupy the adsorption site of the macroporous resin LX-32 or change the surface properties of the macroporous resin LX-32. The present application is conducive to ensuring the separation and purification effect of the macroporous resin LX-32 on salidroside by controlling the concentration of the antioxidant within a suitable range.

[0038] Furthermore, before step (1), the method further includes the following steps: (10) Accurately weigh 0.1 mg of salidroside standard and dilute to 10 mL with methanol in a volumetric flask to obtain a salidroside standard solution; The mass concentration of salidroside was used as the horizontal coordinate, and the peak area value of ultra-high performance liquid chromatography (UPLC) was used as the vertical coordinate to obtain the detection standard curve of salidroside by ultra-high performance liquid chromatography. The chromatographic column used was C18 (50mm×2 mm), the mobile phase was methanol, ultrapure water, the injection volume was 5μL, the wavelength was 280nm, the detection time was 15min, the flow rate was 0.4 mL / min, and the regression equation of the obtained curve was y = 4896.7x - 9087.6, R² = 0.9993. The obtained standard curve shows that salidroside presents a good linear relationship within the linear range. Among them, the retention time of the chromatographic peak is 3.614min, which is 8-9min less than the retention time of 12min of the conventional HPLC liquid chromatography peak.

[0039] Specifically, by measuring the response value of a standard solution of salidroside of known concentration, an ultra-high performance liquid chromatography detection standard curve of salidroside is drawn, and a quantitative relationship between the concentration of salidroside and the detection signal is established, so that its concentration can be inferred based on the response value of the eluent.

[0040] Furthermore, in step (3), the diameter and height of the organic medium-pressure chromatography column is 4*23 cm, and the loading volume of the loading liquid is 30-80 ml.

[0041] Furthermore, in step (2), after the crude salidroside is dissolved, the concentration of salidroside in the prepared solution is 8 g / L. Specifically, the crude salidroside can be dissolved in one of anhydrous ethanol or methanol. In the present application, the crude salidroside is dissolved in anhydrous ethanol and used as the loading solution. Since the macroporous resin LX-32 is used, if the concentration of salidroside is too high, it will adhere to the macroporous resin LX-32 and will not be easy to elute. If it is too dilute, it will be difficult to be fully absorbed by the macroporous resin LX-32. Maintaining it at an appropriate concentration helps the elution proceed smoothly.

[0042] Furthermore, in step (2), the pH value of the loading solution is 5-8. In the present application, by controlling the pH value of the loading solution within a suitable range, the separation effect between salidroside and impurities can be enhanced, and by adjusting the pH, the impurities in the fermentation broth can be precipitated while salidroside remains in a dissolved state, and at the same time, salidroside can be preferentially adsorbed on the separation medium and impurities can be excluded, thereby improving the selectivity and effect of separation.

[0043] Furthermore, the elution rate is 3-8 ml / min. In the present application, by controlling the elution rate of the ethanol aqueous solution, the elution effect of the macroporous resin LX-32 under a certain column volume is better.

[0044] Furthermore, in step (3), the ethanol aqueous solution with gradient concentrations includes ethanol aqueous solutions with volume proportions of anhydrous ethanol of 5%, 20%, 50%, and 100%, respectively. Specifically, after elution, the ethanol aqueous eluate can be collected, and the purity of the eluate can be detected by ultra-high performance liquid chromatography.

[0045] Preferably, when collecting the eluate, it is preferred to collect an ethanol-water eluate in which the volume ratio of anhydrous ethanol is 20%. When performing gradient elution, the ethanol-water solution at this concentration has the best resolution effect on the salidroside adsorbed in the macroporous resin LX-32, and the collected eluate has high purity.

[0046] Specifically, in the present application, after being eluted with an ethanol-water solution with a volume ratio of 50% and 100% of anhydrous ethanol, the macroporous resin LX-32 can be reused more than 8 times, and the chromatographic purity of salidroside collected by elution with an ethanol-water solution with a volume ratio of 20% of anhydrous ethanol can reach more than 99%. Among them, the macroporous resin can be regenerated and reused, which is in line with green environmental protection benefits.

[0047] Further, the salidroside concentration of the eluent was detected by ultra-high performance liquid chromatography, wherein the chromatographic column used was: C18 (50 mm×2 mm), the mobile phase was: methanol, ultrapure water, the injection volume was: 5 μL, the wavelength was: 280 nm, and the flow rate was: 0.4 mL / min. Specifically, the elution conditions of the mobile phase were first eluted at a volume ratio of methanol to ultrapure water of 4:6 for 0-1 min, then eluted with methanol for 1-7 min, and then eluted at a volume ratio of methanol to ultrapure water of 4:6 for 7-15 min.

[0048] Furthermore, after the eluate is collected in sections, the following steps (4) are also included: (4) Detecting the purity of salidroside in the eluate. If the purity of salidroside reaches 100%, decolorizing the eluate with activated carbon. After decolorization, the filtrate is dried to obtain salidroside; If the purity of salidroside is less than 100%, the eluate is used as the loading liquid of the high performance liquid chromatography, and steps (3) and (4) are repeated until the purity reaches the standard.

[0049] The purity of the eluted eluate can also be tested by ultra-high performance liquid chromatography.

[0050] The concentration of salidroside in the filtrate can also be determined by ultra-high performance liquid chromatography response value, and the concentration of salidroside in the decolorized eluate can be determined according to the standard curve.

[0051] The separation and purification method provided in the present application can separate and purify high-purity salidroside.

[0052] Furthermore, in step (4), the step of decolorizing the eluent using activated carbon comprises: (41) The activated carbon was ultrasonically cleaned with ultrapure water until it became colorless, then soaked in acetone and filtered, and dried for later use; (42) Weigh the activated carbon into a container, add the eluent, seal the container, and place it in a shaker at 25 °C and 160 rpm for 1 h.

[0053] Furthermore, the activated carbon can be 6-8 mesh activated carbon, 10-24 mesh activated carbon or 200 mesh activated carbon. After decolorization, the decolorization of the activated carbon can be observed, and the concentration of salidroside in the eluate after decolorization by the activated carbon can be determined by ultra-high performance liquid chromatography to calculate the recovery rate.

[0054] Specifically, after decolorization by activated carbon, filtration can be performed and the filtrate can be retained, and the filtrate can be dried to obtain high-purity salidroside, wherein the drying can be freeze-drying or oven-drying.

[0055] Further, in step (4), the activated carbon is 6-8 mesh activated carbon, and the mass volume ratio of 6-8 mesh activated carbon to eluent is 0.1-1:10 (g / ml), preferably 0.4:10 (g / ml). In the present application, the activated carbon will adsorb salidroside. By using 6-8 mesh activated carbon, the concentration of salidroside can be maintained while reducing the chromaticity in the mass volume ratio range of 0.1-1:10 (g / ml). At a ratio of 0.4:10 (g / ml), the chromaticity and recovery rate are well balanced, which is conducive to the large-scale separation and purification of salidroside.

[0056] Furthermore, the drying can be performed by placing the activated carbon in an oven at 80-120° C. for heat treatment for 2 hours to activate the pores through heat treatment.

[0057] The invention is further described below by means of specific examples.

[0058] In the embodiment of the present application, the chromatographic conditions for determining the purity of salidroside by ultra-high performance liquid chromatography are as follows: the chromatographic column is C18 (50 mm×2 mm), the mobile phase is methanol, ultrapure water, the injection volume is 5 μL, the wavelength is 280 nm, and the flow rate is 0.4 mL / min. The elution conditions of the ultra-high performance liquid chromatography are shown in Table 1 for elution by the mobile phase: Table 1 Time period / min Methanol / (volume %) Ultrapure water / (volume %) 0-1 40 60 1-7 100 0 7-15 40 60

[0059] Example 1 The method for separating and purifying salidroside of Example 1 comprises the following steps: (1) Take Escherichia coli producing salidroside, ferment to prepare 50mL of fermentation broth containing salidroside, dissolve it in anhydrous ethanol by ultrasonic treatment for 7 minutes, add antioxidant VC, centrifuge at 8000rpm for 8 minutes to obtain supernatant, and the concentration of antioxidant VC in the salidroside fermentation broth is 0.8g / L.

[0060] (2) The obtained supernatant was evaporated at 45°C to remove anhydrous ethanol to obtain a concentrated solution.

[0061] (3) The concentrated solution obtained in step (2) is extracted with water-saturated n-butanol using a separatory funnel for three times, and the lower layer extracts are combined; wherein the volume ratio of the concentrated solution to the water-saturated n-butanol is 1:1.

[0062] (4) Evaporate the extract to dryness to obtain crude salidroside.

[0063] (5) Pretreatment of macroporous resin: using macroporous resin LX-32 as filler, filling it into an organic-resistant medium-pressure chromatography column by wet column packing, and installing the organic-resistant medium-pressure chromatography column in the chromatographic column position of a high performance liquid chromatograph; (6) Dissolve the crude salidroside in anhydrous ethanol as a loading solution, the concentration of salidroside in the prepared solution is 8 g / L, and adjust the pH value of the loading solution to 7; (7) The sample liquid is loaded onto an organic-resistant medium-pressure chromatography column, and a high performance liquid chromatograph is used as the infusion system. The sample liquid is eluted with ethanol-water solutions with a volume ratio of 5%, 20%, 50%, and 100% of anhydrous ethanol in sequence. The diameter of the organic-resistant medium-pressure chromatography column is 4*23 cm, the loading volume of the sample liquid is 80 mL, the flow rate is controlled at 6 ml / min, and the eluate obtained by eluting with ethanol-water solutions with a volume ratio of 20% of anhydrous ethanol is collected.

[0064] Comparative Example 1 The separation and purification steps of Comparative Example 1 are basically the same as those of Example 1, except that Comparative Example 1 does not use macroporous resin for purification, but directly dissolves the crude salidroside to a salidroside concentration of 8 g / L, and then tests the purity.

[0065] Comparative Example 2 The separation and purification steps of Comparative Example 2 are basically the same as those of Example 1, except that the macroporous resin used in Comparative Example 2 is macroporous resin LX-12, and the eluate of Comparative Example 2 is collected.

[0066] Comparative Example 3 The separation and purification steps of Comparative Example 3 are basically the same as those of Example 1, except that the macroporous resin used in Comparative Example 3 is macroporous resin LX-18, and the eluate of Comparative Example 3 is collected.

[0067] Example 1 The ultra-high performance liquid chromatography spectra of crude salidroside before and after purification are as follows Figure 2 and Figure 3 As shown, the eluate obtained after purification in Example 1 was used for ultra-high performance liquid time-of-flight mass spectrometry to determine the molecular ion peak of salidroside. The results are as follows Figure 4 As shown, the purified salidroside sample of Example 1 was subjected to nuclear magnetic resonance detection, and the nuclear magnetic resonance detection image is as shown in Figure 5 As shown, it can be found that the collected salidroside has a higher purity.

[0068] The eluates obtained by eluting with ethanol-water solution having a volume ratio of 20% of anhydrous ethanol in Example 1 and Comparative Examples 1-3 were collected, and the purity of salidroside was determined by ultra-high performance liquid chromatography, and the chromaticity was calculated. The data are shown in Table 2.

[0069] Table 2

[0070] The eluents collected in Example 1 and Comparative Examples 1-3 were measured for the purity of salidroside by ultra-high performance liquid chromatography. The purity of Example 1 reached 100%, the purity of the crude salidroside product without macroporous resin purification in Comparative Example 1 was 43.74%, the purity of Comparative Example 2 after purification by macroporous resin LX-12 reached 64.72%, and in Comparative Example 3, salidroside was adsorbed on macroporous resin LX-18, and no salidroside component was eluted, and the chromatographic purity was 0%. By comparison, it can be found that 100% purity of salidroside can be obtained by using macroporous resin LX-32.

[0071] Example 2 The separation and purification steps of Example 2 are basically the same as those of Example 1, except that in step (7), the eluate obtained by eluting with an ethanol-water solution having a volume percentage of 5% of anhydrous ethanol is collected.

[0072] Example 3 The separation and purification steps of Example 3 are basically the same as those of Example 1, except that in step (7), the eluate obtained by eluting with an ethanol-water solution having a volume ratio of 50% anhydrous ethanol is collected.

[0073] Example 4 The separation and purification steps of Example 4 are basically the same as those of Example 1, except that in step (7), the eluate obtained by eluting with an ethanol-water solution having a volume ratio of 100% anhydrous ethanol is collected.

[0074] The eluate collected in Example 2-4 was used to determine the purity of salidroside by ultra-high performance liquid chromatography, and the chromaticity was calculated. The data are shown in Table 3.

[0075] Table 3

[0076] The eluate collected in Examples 2-4 was used to measure the purity of salidroside by ultra-high performance liquid chromatography. The purity of the eluate eluted by 5% ethanol aqueous solution in Example 2 reached 2.51%, the purity of Example 3 reached 67.45%, and no salidroside component was measured in Example 4. By comparison with Example 1, it can be found that the ethanol water eluate at a concentration of 20% by volume of anhydrous ethanol has the best adsorption-analysis effect on salidroside.

[0077] Example 5 The separation and purification steps of Example 5 are basically the same as those of Example 1, except that the following steps are further included: (8) Ultrasonic cleaning of 6-8 mesh activated carbon was performed with ultrapure water until it became colorless, then the activated carbon was immersed in acetone and filtered, and dried in an oven at 100°C for 2 h for later use.

[0078] (9) Weigh 1.00 g of the treated 6-8 mesh activated carbon into a conical flask, add 10 ml of the eluate obtained by eluting with a 20% ethanol aqueous solution, seal the conical flask, and shake it in a shaker at 25°C and 160 rpm for 1 h. After decolorization, filter and obtain the filtrate of Example 5.

[0079] Example 6 The separation and purification steps of Example 6 are basically the same as those of Example 5, except that the activated carbon used is 10-24 mesh activated carbon, and the filtrate of Example 6 is obtained by filtration after decolorization.

[0080] Example 7 The separation and purification steps of Example 7 are basically the same as those of Example 5, except that the activated carbon used is 200 mesh activated carbon, and the filtrate of Example 7 is obtained by filtration after decolorization.

[0081] The effect diagram of the eluent before and after decolorization of Example 5 is as follows Figure 6 shown.

[0082] The filtrate of Example 5-7 was tested by ultra-high performance liquid chromatography to determine the concentration of salidroside. The test results of the salidroside concentration and recovery rate of the eluate of Example 1 and the filtrate of Example 5-7 are as follows: Figure 7 As shown. The filtrate of Example 5 measured the salidroside concentration to be 0.96 mg / ml, the filtrate of Example 6 measured the salidroside concentration to be 0.44 mg / ml, the filtrate of Example 7 measured the salidroside concentration to be 0.24 mg / ml, and the eluate of Example 1 without adding activated carbon measured the salidroside concentration to be 1.57 mg / ml. By comparison, it can be seen that the decolorization effect is best under 6-8 mesh activated carbon decolorization, which not only reduces the chromaticity but also maintains a certain salidroside concentration.

[0083] Examples 8-12 The separation and purification steps of Examples 8-12 are basically the same as those of Example 5, except that the weights of 6-8 mesh activated carbon used for decolorization in Examples 8-12 are 0.80 g, 0.60 g, 0.40 g, 0.20 g, and 0.10 g, respectively. The filtrate obtained by filtration after decolorization in Examples 8-12 is measured by ultra-performance liquid chromatography to determine the concentration of salidroside, and the chroma and recovery rate are calculated. The test data are shown in Table 4.

[0084] Table 4 6-8 mesh activated carbon dosage Salidroside concentration in the filtrate Chroma Recovery rate Example 5 1.00 g 0.96 mg / ml 0.2 61.12% Example 8 0.80 g 1.04 mg / ml 0.3 66% Example 9 0.60 g 1.13 mg / ml 0.4 72% Example 10 0.40 g 1.25 mg / ml 0.5 79.5% Embodiment 11 0.20 g 1.40 mg / ml 0.6 89.2% Example 12 0.10 g 1.42 mg / ml 0.7 90.3% It can be seen from the experimental data that the chromatographic purity of the separation and purification method provided in this application can reach 100%. Under the premise of maintaining high purity, decolorization is carried out by activated carbon. Among them, the recovery rate of the preferred embodiment 10 can reach 79.5%, and the chromaticity can also be reduced to 0.5, which is suitable for large-scale production.

[0085] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made according to the above description, and all these improvements and changes should fall within the scope of protection of the present application.

Claims

1. A method for separating and purifying salidroside, characterized in that: The following steps are involved: (1) Filling a macroporous resin as a filler into an organic-resistant medium-pressure chromatography column, and installing the organic-resistant medium-pressure chromatography column in the chromatography column position of a high performance liquid chromatograph; (2) Dissolving the crude salidroside and adjusting the pH of the crude salidroside to prepare the sample solution; (3) loading the sample solution onto the organic-resistant medium-pressure chromatography column, using a high performance liquid chromatograph as an infusion system, eluting the sample solution with an ethanol aqueous solution of gradient concentration, and collecting the eluate in sections; The macroporous resin is macroporous resin LX-32.

2. The method for separation and purification of salidroside according to claim 1, characterized in that: In step (2), the preparation steps of the crude salidroside include: (21) adding an antioxidant to the salidroside fermentation broth, dissolving it with a solvent, and centrifuging to obtain a supernatant; (22) rotary evaporating the supernatant to obtain a concentrated solution; (23) extracting the concentrated solution with water-saturated n-butanol, and collecting the lower layer of extract; (24) evaporating the lower layer extract to dryness to obtain the crude salidroside; The volume ratio of the concentrate to water-saturated n-butanol is 1:1; In step (21), the antioxidant is one of BHT and VC, and the concentration of the antioxidant in the salidroside fermentation broth is 0.5-1 g / L.

3. The method for separation and purification of salidroside according to claim 1, characterized in that: After the eluate is collected in sections, the following step (4) is also included: (4) detecting the purity of salidroside in the eluate. If the purity of salidroside reaches 100%, decolorizing the eluate with activated carbon, and drying the filtrate to obtain salidroside; If the purity of salidroside is less than 100%, the eluate is used as the loading solution and steps (3) and (4) are repeated.

4. The method for separation and purification of salidroside according to claim 1, characterized in that: The salidroside concentration of the eluent was detected by ultra-high performance liquid chromatography, the chromatographic column of the ultra-high performance liquid chromatography was C18 (50 mm×2 mm), the mobile phase was methanol and ultrapure water, the injection volume was 5 μL, the wavelength was 280 nm, and the flow rate was 0.4 mL / min; The elution conditions of the mobile phase were as follows: first elution at a volume ratio of methanol to ultrapure water of 4:6 from 0 to 1 min, then elution at a volume ratio of methanol to ultrapure water of 4:6 from 1 to 7 min, and then elution at a volume ratio of methanol to ultrapure water of 4:6 from 7 to 15 min.

5. The method for separation and purification of salidroside according to claim 1, characterized in that: In step (2), after the crude salidroside is dissolved, the concentration of salidroside in the prepared solution is 8 g / L.

6. The method for separation and purification of salidroside according to claim 1, characterized in that: In step (2), the pH value of the sample solution is 5-8.

7. The method for separation and purification of salidroside according to claim 3, characterized in that: In step (4), the step of decolorizing the eluent using activated carbon comprises: The activated carbon is ultrasonically cleaned with ultrapure water until it becomes colorless, immersed in acetone and filtered, and dried for later use; The activated carbon was weighed and placed in a container, and an eluent was added. The container was sealed and placed in a shaker at 25° C. and 160 rpm for 1 hour.

8. The method for separation and purification of salidroside according to claim 7, characterized in that: In step (4), the activated carbon is 6-8 mesh activated carbon, and the mass volume ratio of the 6-8 mesh activated carbon to the eluent is 0.1-1:10 (g / ml).

9. The method for separation and purification of salidroside according to claim 1, characterized in that: In step (3), the elution rate is 3-8 ml / min; The ethanol aqueous solution with gradient concentration includes ethanol aqueous solutions with volume proportions of anhydrous ethanol of 5%, 20%, 50% and 100% respectively.

10. The method for separation and purification of salidroside according to claim 2, characterized in that: In step (21), the solvent is one of anhydrous ethanol or methanol, the centrifugal speed is 5000-8000 rpm / min, and the centrifugal time is 5-10 min.

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