Method for extracting and purifying glucosinolate GRH from fleshy roots of radishes

Abstract

The invention discloses a method for extracting and purifying glucosinolate GRH from fleshy roots of radishes, which comprises the following steps: (1) freeze-drying the fleshy roots of the radishes, grinding the fleshy roots of the radishes into powder, and adding an alcohol extract for high-temperature extraction; and (2) adjusting the pH value of the GRH crude extract obtained in the step (1), and carrying out enrichment and purification by using macroporous anion resin to obtain high-purity GRH. The yield of glucosinolate in fleshy roots of radishes is 20.31 mg / g under the optimal optimized condition, the purity of purified GRH is 83%, and the recovery rate is 84%. The optimized purification process is simple and easy to implement and easy to industrialize, the macroporous resin is low in price and easy to obtain, the whole process is low in price, small in investment and high in recovery rate, high-purity GRH can be obtained, and a new way is opened up for the extraction preparation process of GRH in fleshy roots of radishes.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product extraction, and particularly relates to a method for extracting and purifying glucosinolate GRH from radish fleshy roots. Background Art

[0002] Radish belongs to the cruciferous crop Raphanus sativus, and contains a class of natural active substances - glucosinolates, also known as glucosinolates or mustard oil glycosides, which are hydrophilic secondary metabolites rich in nitrogen and sulfur. After hydrolysis by plant myrosinase or animal intestinal microorganisms, glucosinolates will produce a series of bioactive compounds, among which isothiocyanates have an anti-biological stress effect on plants, and can reduce the risk of human diseases and cancer, playing a certain health care effect. So far, more than 200 glucosinolates have been identified, of which more than 130 are in cruciferous plants. Currently, 15 glucosinolate compounds have been identified in radish, among which 4-methylthio-3-butenyl glucosinolate (GRH) is the most important glucosinolate in radish fleshy roots and buds, accounting for more than 90% of the total glucosinolate content. Existing studies have shown that the degradation product Raphasatin of GRH has direct antioxidant ability against hydrogen peroxide and free radical compounds, can regulate energy metabolism, has potential therapeutic effects on chronic diseases such as diabetes, and is also considered to have a cancer prevention effect.

[0003] Currently, there are various methods for extracting and purifying glucosinolates from plants. The methods for extracting glucosinolates mainly include leaching and ultrasonic extraction, and usually water, methanol, ethanol-water mixture or methanol-water mixture are used as extraction solvents. Different scholars have optimized the glucosinolate extraction process in cruciferous vegetables. For example, the optimal extraction process for broccoli dry glucosinolates is 90% ethanol as the extractant, the solid-liquid ratio is 1:11, the extraction time is 1 h, and extraction is repeated 4 times, obtaining a glucosinolate purity of 15.22 mg·g-1; the process for glucosinolates in rapeseed meal was optimized through response surface experiments: 78% ethanol solution, solid-liquid ratio of 1:8, extraction temperature of 63.5 °C, extraction time of 60 min, and the glucosinolate yield was 18.72 mg / g; when studying the total glucosinolate extraction process in broccoli flower heads, the broccoli flower heads were first inactivated by adding 100 °C distilled water and then heat-treated at 80 °C for 15 min, then dried by blowing at 70 °C, and finally the highest total glucosinolate content was measured under the optimal extraction conditions of ethanol concentration of 90%, solid-liquid ratio of 1:9, extraction temperature of 60 °C, and extraction time of 30 min to be 414.98 μmol·g -1 . However, some studies believe that radish glucosinolates (especially GRH) are easily degraded by endogenous enzyme (myrosinase) at high temperatures, which is not conducive to the extraction of glucosinolates. Therefore, there is still controversy about the temperature during the extraction of radish glucosinolates.

[0004] Methods for purifying glucosinolates include column chromatography techniques, liquid chromatography purification techniques, membrane separation methods, etc. Although high-purity natural products can be obtained by liquid chromatography techniques, the low yield limits their large-scale application. Although semi-preparative chromatography techniques can separate glucosinolates with different configurations, due to the extensive use of volatile organic solvents such as acetonitrile, there is a risk of solvent residues in the production of food-grade products. Currently, the field of phytochemical purification is gradually moving towards greening, and purification strategies centered on macroporous resin adsorption materials are gradually replacing high-energy-consuming chromatography techniques. Macroporous resin adsorption is convenient, has low operating costs, consumes less solvent, has no chemical residues in the product, and has a high product recovery rate. However, the purity reported so far is relatively low. For example, the recovery rate of 4-methylsulfinylbutyl glucosinolate (RAA) extracted from Indian mustard seeds after purification by D261 strong basic anion exchange chromatography is 79.82%, but the purity is only 58.37%. The purification differences of different substances in different resins are significant. Currently, there are few reports on the purification process of glucosinolates in radish fleshy roots, and the proposed extraction conditions have not been optimized and verified. In existing literature: In the exploration of the process for extracting glucosinolates from white radishes, 120 mL of water was used as the extraction solution, and extraction was carried out at 70 °C for 2 h. The maximum extraction amount was 5.38 μmol / g, and the extraction efficiency was relatively low. Summary of the Invention

[0005] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a method for extracting and purifying glucosinolate GRH from radish fleshy roots, effectively solving the problem of the lack of an efficient extraction process for glucosinolate GRH in radish fleshy roots, and realizing a method for preparing glucosinolate GRH that is safe, convenient, and has a high yield and high purity.

[0006] Technical Solution: The present invention provides a method for extracting and purifying glucosinolate GRH from radish fleshy roots, comprising the following steps:

[0007] (1) Freeze-dry and grind radish fleshy roots into powder, add an alcohol extraction solution for high-temperature extraction;

[0008] (2) Adjust the pH of the crude GRH extraction solution obtained in step (1), and use macroporous anion resin for enrichment and purification to obtain high-purity GRH.

[0009] Among them, in step (1), the alcohol extraction solution is a methanol or ethanol solution with a volume fraction of 70-100%, and the ratio of radish freeze-dried powder to the extraction solution is 1:4-15 g / mL.

[0010] Among them, in step (1), the alcohol extraction solution is a methanol or ethanol solution with a volume fraction of 80-90%, and the ratio of radish freeze-dried powder to the extraction solution is 1:7-10 g / mL.

[0011] Among them, the high-temperature extraction in step (1) is carried out by leaching in a water bath at 70°C - 80°C for 60 - 70 minutes, and then extracting by shaking at room temperature at a speed of 200 - 250 rpm / min for 10 - 15 min.

[0012] Among them, the pH of the GRH crude extract in step (2) is adjusted to 6 - 7.

[0013] Among them, the macroporous anion resin in step (2) is any one of HPD722, D315, D201, D261, and D290.

[0014] Among them, the desorbing solution of the macroporous anion resin in step (2) is any one of methanol, CaCl2, KNO3, and Na2CO3 - NaHCO3.

[0015] Among them, the macroporous anion resin in step (2) is D261 or D290, and the desorbing solution is KNO3.

[0016] Among them, in step (2), the pH of the GRH crude extract is adjusted, loaded into the macroporous anion resin, the impurities are removed by washing with water first, and then eluted with the desorbing solution. After the eluate is concentrated and dissolved in absolute ethanol, the insoluble matter is filtered off after reaching the equilibrium state (that is, the concentrated eluate is no longer soluble in ethanol), and the concentrated filtrate is the enriched glucoraphanin GRH.

[0017] Preferably, the present invention uses leaching combined with shaking and column chromatography technology to purify the radish-specific glucosinolate GRH. The preferred process flow and parameters for extracting and purifying glucosinolate GRH from radish fleshy roots are as follows:

[0018] (1) The radish fleshy roots are sliced, freeze-dried, and ground into powder, and the alcohol extraction method is used for shaking crude extraction: 80% ethanol is used, the solid-liquid ratio is 1:10, leaching is carried out in a water bath at 70°C - 80°C for 60 - 70 minutes, and then extracting by shaking on a shaker at a speed of 200 rpm / min for 15 min to obtain the crude extract of glucosinolate GRH in the radish fleshy roots.

[0019] (2) Use macromolecular adsorption resin to enrich and purify the crude glucosinolate extract: Adjust the pH of the GRH crude extract to 6.4, use D261 macroporous anion resin, and load the sample at a speed of 42 mL / h. Then use 0.5 M KNO3 as the eluent to elute the GRH adsorbed on the resin, the elution flow rate is 35 mL / h, and the amount of eluent used is 7 times that of the loaded crude extract. After the eluate is concentrated and dissolved in absolute ethanol, the insoluble matter is filtered off after reaching the equilibrium state, and the concentrated filtrate is the enriched glucoraphanin GRH.

[0020] The yield of glucoraphanin GRH extracted and purified by the above process can reach 20.31 mg / g (48.59 μmol / g), the purity of GRH can reach 83%, and the recovery rate is 84%.

[0021] In the extraction process of the present invention, the polarity of 80% ethanol highly matches the β-D-glucose group of glucosinolate, enhancing the solvent penetration rate, and making glucosinolate more easily dissolved. The quaternary ammonium group of D261 resin adsorbs the negatively charged group of glucosinolate through electrostatic interaction, and the benzene ring hydrophobic region fixes the side chain of glucosinolate, achieving highly selective adsorption. Currently, the existing extraction techniques for glucosinolate mostly rely on organic solvents (such as methanol), and the removal of proteins therein mostly uses lead acetate precipitation method for proteins. The separation and purification of glucosinolate mostly use preparative HPLC, with toxic reagents, complex instruments, and high prices. The present invention first proposes a green combination of ethanol-D261 resin and inorganic salt desorption, solving the problems of toxicity and cost of organic solvents. At the same time, based on the complexation reaction of glucosinolate with palladium chloride (detected at 540 nm), a real-time feedback system for rapid color development of glucosinolate is established, shortening the detection time from 30 minutes for each sample by HPLC to 2 hours for batch detection, and combining with orthogonal experiments to dynamically adjust extraction parameters, greatly improving the efficiency.

[0022] The present invention first combines a low-cost color development method during the dynamic purification process of macroporous resin, quickly determining the dosage of the eluent while reducing costs. Currently, there is very little research on the purification of glucosinolate in radish, and the preparative HPLC method is used. The present invention first enriches and purifies the main glucosinolate GRH in radish by applying D261 macroporous resin in an efficient extraction method, with remarkable effects.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0024] The present invention provides a purification process for glucoraphanin GRH in radish fleshy roots. Under the optimized best conditions, the yield of glucosinolate in radish fleshy roots is 20.31 mg / g, the purity of purified GRH is 83%, and the recovery rate is 84%. The optimized purification process of the present invention is simple and easy to implement, easy to industrialize, the macroporous resin is cheap and easily available, the whole process is inexpensive, has low investment and high recovery rate, can obtain GRH with high purity, and opens up a new way for the extraction and preparation process of GRH in radish fleshy roots. The present invention solves the problems of using non-food-grade reagents and low purity in the current extraction process of radish GRH, and has important reference significance for the comprehensive utilization of functional components in radish. Description of the Drawings

[0025] Figure 1 It is a bar chart of the extraction amount of glucosinolate under different extraction reagents;

[0026] Figure 2 It is a bar chart of the extraction amount of glucosinolate under different ethanol concentrations;

[0027] Figure 3 Bar chart of glucosinolate extraction amounts at different extraction times;

[0028] Figure 4 Bar chart of glucosinolate extraction amounts at different extraction temperatures;

[0029] Figure 5 Bar chart of glucosinolate extraction amounts at different solid-liquid ratios;

[0030] Figure 6 Standard curve of GRH;

[0031] Figure 7 Bar chart of GRH adsorption rates on different resins;

[0032] Figure 8 Elution effect diagrams of different desorbing agents on D261 macroporous resin;

[0033] Figure 9 Elution effect diagrams of different desorbing agents on D290 macroporous resin;

[0034] Figure 10 Diagram for determining the volume of eluate by palladium colorimetric method;

[0035] Figure 11 GSLs extracts before and after purification by D261 resin;

[0036] Figure 12 UPLC diagrams of GRH extracts before and after purification by D261 resin. Specific embodiments

[0037] The following combines the accompanying drawings and examples to further describe in detail the specific embodiments of the present invention. The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The experimental materials used in the following examples can all be obtained from conventional biochemical reagent manufacturers unless otherwise specified.

[0038] Instruments: The freeze dryer is purchased from Qingdao Yonghe Chuangxin Electronic Technology Co., Ltd.; the multi-sample tissue grinder is purchased from Shanghai Jingxin Industrial Development Co., Ltd.; the digital display constant temperature water bath is purchased from Guohua Electric Appliance Co., Ltd.; the integrated vacuum concentrator is purchased from Beijing Jiem Technology Co., Ltd.

[0039] Materials: Commercially available white radish

[0040] Reagents: Anhydrous ethanol, methanol, and acetonitrile of chromatographic pure grade; KNO3, Na2CO3, NaHCO3, CaCl2, and ultrapure water; The standard 4-methylthio-3-butenyl glucosinolate potassium salt (i.e., the potassium salt form of GRH, CAS 245550-64-5) was purchased from Tokyo Chemical Industry Co., Ltd. (Shanghai); Palladium chloride was purchased from Shanghai Macklin Biochemical Co., Ltd.; Sodium carboxymethyl cellulose was purchased from Sinopharm Chemical Reagent Co., Ltd.; Anion exchange macroporous resins HPD722, D201, D261, D290, and macroporous adsorption resin D315 were purchased from Beijing Huideyi Technology Co., Ltd.

[0041] Example 1

[0042] 1. Extraction method of glucosinolates from radish fleshy roots

[0043] Cut white radishes into pieces, freeze-dry them in a freeze dryer, and then grind them into powder with a grinding machine. Weigh 0.1 g of radish fleshy root powder, add the extraction solution at a ratio of 1:10 g / mL (radish powder: 80% ethanol), vortex for 1 min to mix evenly, extract at 80 °C for 70 min, then shake at 200 r / min on a shaker at room temperature for 15 min, and finally centrifuge at 12,000 rpm for 15 min. Take the supernatant and use the conventional palladium ion colorimetric method to determine the glucosinolate content.

[0044] 2. Purification method of glucosinolates from radish fleshy roots

[0045] Subsequently, adjust the pH of the crude glucosinolate extract (i.e., the supernatant) in step (1) to 6.4. Take 30 mL and use D261 macroporous anion resin for enrichment and purification. Load the sample at a speed of 42 mL / h. During the loading process, use an ear bulb to remove the air bubbles in the column. Use 0.5 M KNO3 as the eluent to elute the GRH adsorbed on the resin. The elution flow rate is 35 mL / h, and the eluent consumption is 215 mL; After the eluate is concentrated, dissolve it in anhydrous ethanol. After reaching the equilibrium state, filter to remove the insoluble matter, and concentrate the filtrate to obtain the enriched GRH.

[0046] Example 2

[0047] Optimization of glucosinolate extraction process

[0048] (1) Selection of extraction solvent

[0049] Based on the process of Example 1, with other conditions unchanged, the effects of equal amounts of different extraction solvents (water, anhydrous methanol, anhydrous ethanol, anhydrous acetonitrile, anhydrous ethyl acetate) on the extraction amount of glucosinolates from radish fleshy roots were investigated respectively.

[0050] From Figure 1It can be seen that under the conditions of the same extraction temperature, extraction time, and solid-liquid ratio, there are differences in the extraction effects of different solvents on glucosinolates in radish fleshy roots. Among them, the extraction amount of acetonitrile is only 1.64 mg / g, which is the worst among the five solvents; while the extraction amount of ethanol reaches 7.73 mg / g, which is the best among the five solvents. In addition, ethanol is easily available and inexpensive, and it is also safe and reliable without toxic side effects. Considering comprehensively the extraction effect and solvent properties, ethanol is selected as the best extraction solvent for glucosinolates in radish fleshy roots in this experiment.

[0051] (2) Selection of reagent concentration

[0052] Based on the process of Example 1, under the optimized conditions obtained (ethanol as the extraction solvent) and with other conditions unchanged, the effects of different solvent concentrations (50%, 70%, 80%, 90%, 100% ethanol) on the extraction amount of glucosinolates in radish fleshy roots were studied respectively.

[0053] Figure 2 It shows that the effect of ethanol concentration on the extraction effect of glucosinolates is relatively obvious. When the ethanol concentration is in the range of 50%-80%, the extraction amount of glucosinolates in white radish fleshy roots increases with the increase of ethanol concentration; while when the concentration is in the range of 80%-90%, the extraction amount of glucosinolates decreases with the increase of ethanol concentration. The extraction amount of glucosinolates reaches the maximum value of 8.13 mg / g when the ethanol concentration is 80%. This can be explained by the principle of like dissolves like. The extraction effects of extraction solvents with different polarities on substances with different polarities are different. Therefore, an ethanol concentration similar to the polarity of glucosinolates needs to be selected to achieve the best extraction effect. To sum up, selecting an ethanol concentration of 80% can obtain the best extraction effect of glucosinolates in radish fleshy roots.

[0054] (3) Selection of extraction time

[0055] Based on the process of Example 1, under the optimized conditions obtained in Example 2(1)(2) (80% ethanol as the extractant) and with other conditions unchanged, the effects of different extraction times (5 min, 15 min, 25 min, 30 min, 40 min, 50 min, 60 min, 70 min) on the extraction amount of glucosinolates in radish fleshy roots were studied respectively.

[0056] Figure 3 It shows the effects of different extraction times on the extraction amount of glucosinolates in radish fleshy roots under the same other conditions. The results show that with the extension of the extraction time, the extraction amount of glucosinolates continuously increases. When the extraction time reaches 60 min, the extraction amount of glucosinolates reaches the highest value of 9.53 mg / g. However, continuing to extend the extraction time will lead to the degradation of glucosinolates or the dissolution of impurities. Therefore, 60 min can be considered as the best extraction time for glucosinolates in radish fleshy roots.

[0057] (4) Selection of extraction temperature

[0058] Based on the process of Example 1, under the optimized conditions (80% ethanol, extraction for 60 min) obtained in Example 2 (1)(2)(3), with other conditions unchanged, the effects of different extraction temperatures (-20°C, 4°C, 20°C, 50°C, 70°C, 80°C) on the extraction amount of glucosinolates in radish fleshy roots were studied.

[0059] As Figure 4 shown, the extraction temperature had a significant effect on the extraction effect of glucosinolates in radish fleshy roots. As the temperature increased, the extraction amount of glucosinolates gradually increased and reached the highest value at 70°C. However, continuing to increase the temperature would lead to a decrease in the extraction amount of glucosinolates. This is because the extraction process of glucosinolates is a diffusion process, and increasing the temperature can accelerate diffusion, thereby increasing the dissolution rate of glucosinolates. But if the temperature exceeds a certain range, it may trigger the degradation reaction of some glucosinolates, resulting in a reduction in the final extraction amount.

[0060] (5) Selection of solid-liquid ratio

[0061] Based on the process of Example 1, under the optimized conditions (80% ethanol, water bath temperature 70°C, extraction for 60 min) obtained in Example 2 (1)(2)(3)(4), with other conditions unchanged, the effects of different solid-liquid ratios (1:4, 1:7, 1:10, 1:13, 1:15) on the extraction amount of glucosinolates in radish fleshy roots were studied.

[0062] As Figure 5 shown, the different solid-liquid ratios had a relatively small effect on the extraction amount of glucosinolates. In the range of 1:4 to 1:10, as the extraction liquid ratio increased, the extraction amount of glucosinolates in radish fleshy roots gradually increased and reached the highest at a ratio of 1:10. However, further increasing the extraction liquid ratio would result in a decrease in the extraction amount of glucosinolates. Therefore, 1:10 was determined as the optimal extraction solid-liquid ratio.

[0063] (6) Orthogonal experiment

[0064] According to the results obtained from the single-factor experiments, an L9(3 3 ) orthogonal experiment design was used to optimize the key factors affecting the extraction content of glucosinolates in radish fleshy roots. The experimental factors included ethanol concentration, extraction time, and extraction temperature, and the levels of each factor are shown in Table 1. Through the analysis of the orthogonal experiment results, the optimal process conditions of this solvent extraction method were determined. The specific factors and levels investigated are shown in Table 1.

[0065] Table 1 Orthogonal level table of solvent extraction

[0066]

[0067] Through the analysis of the results of single-factor experiments, an orthogonal experiment was carried out on ethanol concentration, extraction temperature and extraction time, and the results were analyzed. As can be seen from Table 2, the order of the influence of these three factors on the extraction effect is: A > B > C, that is, the ethanol concentration is the main factor, followed by the extraction temperature, and the influence of the extraction time is the smallest. Through comprehensive analysis, the best optimization scheme for extracting glucosinolates from radish fleshy roots is A2B3C3, that is, the best ethanol concentration is 80%, the extraction temperature is 80 °C, and the extraction time is 70 min.

[0068] Table 2 Results of orthogonal experiment for extracting glucosinolates from white radish fleshy roots

[0069]

[0070]

[0071] To investigate the stability of the optimized extraction process by orthogonal experiment, 3 groups of parallel verification experiments were carried out under the optimal process conditions (Table 3), and the maximum extraction amount of glucosinolates from white radish fleshy roots was 20.31 mg / g (average value), which was higher than the maximum extraction amount of glucosinolates in the 9 groups of experiments in the orthogonal experiment. The water bath plus oscillation extraction of the present invention can greatly improve the yield of glucosinolates in radish fleshy roots and improve the utilization rate of radish fleshy roots.

[0072] Table 3 Verification experiment of the best process

[0073]

[0074] Example 3

[0075] Screening of static enrichment conditions for GRH

[0076] Based on the best extraction process of glucosinolates from radish fleshy roots optimized in Example 2 (water bath temperature 80 °C, extraction time 70 min, ethanol concentration 80%, solid-liquid ratio 1:10), that is, according to the method of Example 1, GRH in white radish was enriched and purified.

[0077] (1) Resin pretreatment

[0078] Five different types of anion exchange macroporous resins (HPD722, D201, D261, D290 and D315) were used in the experiment of the present invention. First, the resin was soaked in 95% ethanol overnight to make it fully swollen. Next, the swollen resin was transferred to a chromatographic column and repeatedly washed with distilled water until the residual ethanol in the resin was removed. Finally, the treated resin was prepared for subsequent experiments.

[0079] (2) Static adsorption to screen resins

[0080] Take 5 pre-treated resin samples, 3 portions for each type, 1 g for each portion, place them in 50 mL test tubes respectively, add 10 mL of crude radish extract, shake in a shaker at 200 r / min for 12 h at room temperature, and then filter. Determine the concentration of GRH in each adsorbed filtrate by UPLC-QTOF-MS. The test conditions are as follows: the column specification is a reverse chromatographic column (Shim-pack VP-ODS) with a size of 2.0 mm×150 nm and 5 μm i.d. Mobile phase A is ultrapure water containing 0.1% formic acid, and mobile phase B is acetonitrile (chromatographic grade) containing 0.1% formic acid; mobile phase gradient: 0 min–5% B; 20 min–95% B; 22 min–95% B; 28 min–5% B; flow rate is 0.3 ml / min; injection volume: 2 μl, column temperature: 35 °C (left), 20 °C (right); carrier gas temperature is 350 °C; dry gas flow rate is 10 l / min; capillary voltage is 3.5 kV; fragmentation voltage is 135 V; skimmer voltage is 65 V.

[0081] The standard curve of GRH is as Figure 6 shown. Calculate the saturated adsorption capacity and adsorption rate of each resin type using the following formula.

[0082]

[0083] In the formula: Q is the saturated adsorption capacity (μg / mL); C0 is the GRH concentration before adsorption (μg / mL); C1 is the GRH concentration in the filtrate after adsorption (μg / mL); V0 is the volume of the added radish extract (mL); M is the resin mass (g).

[0084] Use the crude glucosinolate extract from radish fleshy roots to investigate the adsorption and desorption capabilities of 5 different types of macroporous resins for GRH. The results are as Figure 7 shown. It can be seen from the experimental results that the adsorption rate of HPD722 type resin is the lowest, only 10.96%, and then in turn are D315 < D201 < D261 < D290. Select the two resins D261 and D290 with the best adsorption effect for the next experiment.

[0085] (3) Determination of static desorption performance

[0086] The present invention aims to screen desorbing agents for two types of anion exchange resins, D261 and D290. Twelve pretreated resin samples, each weighing 1 g, were prepared. 10 mL of the crude GRH extract was added to each resin sample and shaken at room temperature for 12 h, followed by filtration. Subsequently, 10 mL of pure water was added to each resin sample for washing, and then filtered again. Then, 10 mL each of 80% methanol, 0.5 M CaCl2, 0.5 M KNO3, and Na2CO3-NaHCO3 buffer (pH 9) was used as the desorbing solution and shaken at room temperature for 12 h for desorption. Finally, the concentration of GRH in each desorbing solution was determined by UPLC-QTOF-MS. The desorbing performance of each desorbing agent was calculated using the following formula.

[0087]

[0088]

[0089] In the formula: C2 is the concentration of GRH in the filtrate after desorption (μg / mL); V2 is the volume of the eluent (mL). Q is the saturation adsorption capacity (μg / mL); V0 is the volume of the radish extract added (mL); M is the mass of the resin (g).

[0090] The elution effects of different eluents on D261 and D290 macroporous resins are shown in Figure 8 and Figure 9 As shown. After the adsorption of GRH by D261 resin in the present invention and elution with 0.5 M KNO3, the recovery rate of GRH can reach over 40%, greatly improving the yield of GRH in the purification process.

[0091] Example 4

[0092] Dynamic enrichment and purification of GRH

[0093] Based on the extraction process and enrichment conditions of glucosinolates in radish fleshy roots optimized in Example 2 and Example 3, 30 mL of the glucosinolate extract with a pH of 6.4 (the supernatant in Example 1) was slowly added to a glass chromatography column filled with 50 g of D261 resin, and the loading solution flow rate was 42 mL / h. Impurities were removed by washing with water first, and then eluted with 0.5 M KNO3 at an elution flow rate of 35 mL / h. One tube was collected for every 15 mL of eluent, and the amount of eluent was determined by palladium colorimetry. After detection, the eluents containing glucosinolates were concentrated, and the filtrate was concentrated to near dryness. Then it was dissolved in absolute ethanol, and after reaching equilibrium, the insoluble matter was filtered off, and the filtrate was concentrated to obtain the purified GRH concentrate. 0.1 g of each of the above two samples (unpurified GRH crude extract and purified GRH) was weighed, dissolved in methanol, filtered through a 0.22 μm filter membrane, and then analyzed by machine.

[0094] The eluate collected by the person in charge was detected by the palladium colorimetric method, and it was found that GRH was basically eluted completely in the 15th tube. Thus, the volume of the eluate was determined to be 225 mL. The color development of the blank control and the 14 tubes of eluate containing GRH is as follows Figure 10 .

[0095] From Figure 11 it can be seen that the color of the crude extract solution of GRH before purification is relatively deep, while the color of the GRH solution after purification is relatively light. The UPLC-QTOF-MS chromatogram is shown in Figure 12 . After enrichment and purification by D261 macroporous resin, the content of GRH is 2.1 times that before enrichment. The weight of the treated GRH concentrate was weighed as 2.52 g, the purity was 83%, and the recovery rate was 84%.

[0096] To sum up, the present invention uses extraction combined with oscillating column chromatography technology to purify the specific glucosinolate GRH of radish, which can fully extract and purify the glucosinolate GRH in the fleshy roots of radish. In the present invention, the water bath temperature is 80 °C, the water bath time is 70 min, the ethanol concentration is 80%, and the solid-liquid ratio is 1:10. Through verification experiments, the yield of glucosinolate in the fleshy roots of radish is 20.31 mg / g under the optimized optimal conditions. Subsequently, D261 macroporous anion resin and 0.5 M KNO3 desorbing solution are used to enrich and purify GRH. The purity of the purified GRH is 83%, and the recovery rate is 84%. A GRH with a relatively high purity is obtained, and the method is safe and reliable and can be widely applied, opening up a new way for the extraction and preparation process of GRH in the fleshy roots of radish.

Claims

1. A method for extracting and purifying glucosinolate GRH from radish fleshy roots, characterized in that, It includes the following steps: (1) Freeze-dry the radish fleshy root and grind it into powder, add an alcohol extraction solution and perform high-temperature extraction; (2) Adjust the pH of the GRH crude extract obtained in step (1), use macroporous anion resin for enrichment and purification to obtain high-purity GRH.

2. The method for extracting and purifying glucosinolate from radish fleshy root according to claim 1, characterized in that, In step (1), the alcohol extraction solution is a methanol or ethanol solution with a volume fraction of 70-100%, and the ratio of the freeze-dried radish powder to the extraction solution is preferably 1:4-15 g / mL.

3. The method for extracting and purifying glucosinolate GRH from radish fleshy roots according to claim 1, characterized in that, In step (1), the alcohol extraction solution is a methanol or ethanol solution with a volume fraction of 80-90%, and the ratio of the freeze-dried radish powder to the extraction solution is 1:7-10 g / mL.

4. The method for extracting and purifying glucosinolate GRH from radish fleshy roots according to claim 1, characterized in that, In step (1), the high-temperature extraction is to extract at 70°C-80°C in a water bath for 60-70 minutes, and then oscillate and extract at room temperature at a speed of 200-250 rpm / min for 10-15 minutes.

5. The method for extracting and purifying glucosinolate GRH from radish fleshy roots according to claim 1, characterized in that, In step (2), the pH of the GRH crude extract is adjusted to 6-7.

6. The method for extracting and purifying glucosinolate GRH from radish fleshy roots according to claim 1, characterized in that, In step (2), the macroporous anion resin is any one of HPD722, D315, D201, D261, D290.

7. The method for extracting and purifying glucosinolate GRH from radish fleshy roots according to claim 1, characterized in that, In step (2), the desorbing solution of the macroporous anion resin is any one of methanol, CaCl2, KNO3, and Na2CO3-NaHCO3.

8. The method for extracting and purifying glucosinolate GRH from radish fleshy roots according to claim 1, characterized in that In step (2), the macroporous anion resin is D261 or D290, and the desorbing solution is KNO3.

9. The method for extracting and purifying glucosinolate GRH from radish fleshy roots according to claim 1, characterized in that, In step (2), adjust the pH of the GRH crude extract, load it into the macroporous anion resin, first wash away impurities with water, then elute with the desorbing solution, concentrate the eluate, dissolve it in absolute ethanol, filter to remove insoluble substances after reaching an equilibrium state, and concentrate the filtrate to obtain the enriched glucoraphanin GRH.

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