Method for microwave-assisted extraction of glucosinolate monomer from moringa oleifera

Abstract

The invention relates to the technical field of natural product extraction, and particularly discloses a method for microwave-assisted extraction of glucosinolate monomers in moringa oleifera. The moringa seed glucosinolate monomer 4-(alpha-L-rhamnosyl) benzyl glucosinolate is obtained by taking moringa seeds as a raw material and adopting a microwave-assisted method for extraction, an extraction method for separation and a macroporous adsorption resin adsorption-desorption method for purification. According to the method, the moringa seed glucosinolate monomer can be obtained without using a high performance liquid chromatograph, a liquid chromatograph-mass spectrometer and other expensive instruments, the extraction efficiency is high, the energy consumption is low, the elution solvent is green and environment-friendly, and the operation is simple and convenient.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction, and in particular to a method for microwave-assisted extraction of glucosinolate monomers in Moringa oleifera. Background Art

[0002] Moringa is a nutritious plant that is both medicinal and edible, and all parts of the plant are edible. Moringa seeds contain about 9% of glucosinolates, namely 4-(α-L-rhamnosyl)benzyl glucosinolate, and its enzymatic hydrolysis product 4-α-L-rhamnosyl-isothiocyanate benzyl ester (GMG-ITC) has extensive and significant biological activities, such as antioxidant, anti-inflammatory, and anti-cancer. Glucosinolates can be hydrolyzed to GMG-ITC by endogenous enzymes (myrosinase) present in Moringa seeds, but this reaction does not proceed in Moringa seeds. Therefore, optimizing the extraction and purification methods of 4-(α-L-rhamnosyl)benzyl glucosinolate is of great significance for the study of these bioactive molecules.

[0003] However, glucosinolate molecules have strong hydrophilicity due to the presence of pyranose glucopyranose units, and it is this property that makes it very difficult to extract, separate and purify glucosinolates. Currently, the extraction, separation and purification steps of 4-(α-L-rhamnosyl)benzyl glucosinolate are relatively cumbersome, especially the purification step. To obtain a high-purity product, expensive instruments such as high-performance liquid chromatography and liquid chromatography-mass spectrometry are required, as well as a large amount of highly polar solvents such as acetonitrile and methanol, and the purification method is single. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned prior art, the object of the present invention is to provide a method for microwave-assisted extraction of glucosinolate monomers in Moringa. The present invention uses Moringa seeds as raw materials, adopts microwave-assisted extraction, extraction separation and macroporous adsorption resin adsorption-desorption purification to obtain Moringa seed glucosinolate monomer 4-(α-L-rhamnosyl) benzyl glucosinolate. The method of the present invention does not require the use of expensive instruments such as high performance liquid chromatographs and liquid chromatography-mass spectrometry, and can obtain Moringa seed glucosinolate monomers, with high extraction efficiency (1.59%), low energy consumption, green elution solvents and simple operation.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] A method for microwave-assisted extraction of glucosinolate monomers from Moringa oleifera comprises the following steps:

[0007] (1) Extraction of total glucosinolates from Moringa oleifera seeds: Take the powder of Moringa oleifera seeds, add ethanol solution with a volume fraction of 50% - 90% according to a liquid-solid ratio of 50:1 - 55:1 mL / g, start stirring, place it in a microwave reactor for extraction. The microwave extraction temperature is 58 - 65 °C, and the microwave extraction time is 25 - 32 min. Filter the obtained extract by suction, concentrate and dry it to obtain the crude extract of Moringa oleifera seeds;

[0008] (2) Preparation of crude glucosinolates by extraction and defatting: Dissolve the crude extract of Moringa oleifera seeds obtained in (1) in distilled water, and then extract it successively with petroleum ether, ether, and n-butanol saturated with water. Collect the n-butanol layer, concentrate and dry it to obtain the crude glucosinolates;

[0009] (3) Purification of glucosinolates: Pack the pretreated NKA-9 macroporous adsorption resin into a column. Dissolve the crude glucosinolates obtained in (2) in distilled water, load the dissolved sample solution onto the column, and pre-adsorb for 0.5 - 1 h. Then, use distilled water as the eluent for elution, control the flow rate at 1.5 BV / h. After a period of time, collect the eluate and detect it with Molish reagent until it turns colorless. Then, adjust the eluent to the desorbent, keep the flow rate unchanged, and start collecting the desorbent. The desorbent is a 90 v / v% ethanol solution, and the mass ratio of the NKA-9 macroporous adsorption resin to the volume of the desorbent is 1:2 - 1:3 g / mL. Concentrate and dry the desorbent, then dissolve it in methanol and filter it through a 0.22 μm filter membrane, and concentrate and dry it to obtain the monomer of glucosinolates from Moringa oleifera seeds. The monomer of glucosinolates from Moringa oleifera seeds is 4-(α-L-rhamnosyl)benzyl glucosinolate.

[0010] Further, in step (3), the crude glucosinolates are dissolved in distilled water according to a solid-liquid ratio of 1:1 - 1.1 mg / mL, and the mass ratio of the NKA-9 macroporous adsorption resin to the volume of the sample solution is 1:1 - 2:1 g / mL, preferably 1:1 g / mL.

[0011] Further, in step (2), the crude extract of Moringa oleifera seeds is dissolved in distilled water according to a solid-liquid ratio of 10:1 - 11:1 mg / mL.

[0012] Further, in step (2), the extraction steps include: first add an equal volume of petroleum ether for extraction three times and collect the water layer, then use an equal volume of ether for extraction three times and collect the water layer, and finally use an equal volume of n-butanol saturated with water for extraction three times and collect the n-butanol layer, concentrate and dry it to obtain the crude glucosinolates.

[0013] Furthermore, in step (3), the pretreatment operation of the resin includes: first soaking in anhydrous ethanol, taking out the resin after alcohol soaking and washing it with distilled water until there is no alcohol smell; then soaking in 4wt% HCl, taking out the resin after acid soaking and washing it with distilled water until it is neutral; and finally soaking in 4wt% NaOH, taking out the resin after alkali soaking and washing it with distilled water until it is neutral.

[0014] Furthermore, in step (1), the extraction conditions are: liquid-solid ratio of 52:1-53:1 mL / g, volume fraction of ethanol solution of 70%-90%, microwave extraction temperature of 58-60°C, and microwave extraction time of 30-31 min.

[0015] Preferably, in step (1), the extraction conditions are: liquid-to-solid ratio of 53:1 mL / g, volume fraction of ethanol solution of 70%, extraction temperature of 59° C., and extraction time of 30.5 min.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] The method of the present invention is low-cost and environmentally friendly. The present invention adopts microwave-assisted extraction, extraction separation and macroporous adsorption resin adsorption-desorption purification to obtain glucosinolate monomers, without the need to use expensive instruments such as high performance liquid chromatographs, liquid chromatography-mass spectrometers, and strong polar solvents.

[0018] The method of the present invention is easy to operate. The present invention firstly extracts the total glycosides of Moringa oleifera by microwave-assisted extraction, which has the advantages of short extraction time, high efficiency and low energy consumption; the subsequent purification step only uses polar macroporous adsorption resin and ethanol solution as a desorbent, so that high-purity Moringa seed glucosinolate monomer 4-(α-L-rhamnosyl)benzyl glucosinolate can be obtained, and the method is easy to operate.

[0019] The method of the invention can obtain moringa seed glucosinolate monomers, has high extraction efficiency (1.59%), low energy consumption, green and environmentally friendly elution solvent, small dosage and low operation requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of liquid-to-solid ratio on the yield of total glycosides of Moringa oleifera;

[0021] Figure 2 The effect of microwave temperature on the yield of total glycosides of Moringa oleifera;

[0022] Figure 3 The effect of microwave time on the yield of total glycosides of Moringa oleifera;

[0023] Figure 4 is the effect of ethanol volume fraction on the yield of total glycosides of Moringa oleifera;

[0024] Figure 5Data on the adsorption rate (A) and desorption rate (B) of crude glucosinolates by three macroporous adsorption resins, D101, AB-8, and NKA-9, in the present invention;

[0025] Figure 6 is the desorption capacity of ethanol solutions with different concentrations;

[0026] Figure 7 is the effect of the ratio of resin mass to extract volume on the adsorption rate;

[0027] Figure 8 is the effect of the ratio of resin mass to desorbent volume on the desorption rate;

[0028] Figure 9 Infrared spectrum of glucosinolates obtained in the present invention;

[0029] Figure 10 Mass spectrum of glucosinolates obtained in the present invention;

[0030] Figure 11 1H nuclear magnetic resonance spectrum of glucosinolates obtained in the present invention. Detailed implementation manners

[0031] The following is a further description of the technical solution of the present invention by the applicant in combination with specific embodiments and drawings, but the scope of protection claimed in the claims of the present invention is not limited to these embodiments.

[0032] Example 1

[0033] A method for microwave-assisted extraction of glucosinolate monomers from Moringa oleifera, comprising the following steps:

[0034] (1) Extraction of total glucosides from Moringa oleifera

[0035] The effects of ethanol volume fraction, liquid-to-solid ratio, microwave temperature, and microwave time on the yield of total glucosides from Moringa oleifera were investigated by the single-factor method. Specifically:

[0036] Accurately weigh 1 g of Moringa oleifera seed powder, put it into a 250 mL three-necked flask, add the corresponding volume of ethanol with the corresponding volume fraction according to the liquid-to-solid ratio, put in a magnetic stir bar, place it in an MCR-3 atmospheric pressure microwave chemical reactor, connect a temperature sensor and a condensation device, and carry out extraction for a certain time at a certain microwave temperature (800 W). After the extraction is completed, the total glucosides in the extracted sample are colored by the vanillin-sulfuric acid colorimetric method, the absorbance value of the sample at 548 nm is measured by ultraviolet-visible spectrophotometry, and then the concentration of total glucosides in the sample is calculated. Then, the yield of total glucosides from Moringa oleifera is calculated by combining the following formula (1):

[0037]

[0038] Where: Y: yield of total glucosides, %;

[0039] C: Total glycoside concentration, mg / mL;

[0040] V: Total volume of Moringa oleifera seed extract, mL;

[0041] m: Mass of Moringa oleifera seeds, mg.

[0042] Under the conditions of microwave temperature of 60 °C, microwave time of 20 min, and ethanol volume fraction of 70%, the effect of liquid-to-solid ratio (20:1 - 60:1 mL / g) on the yield of total glycosides from Moringa oleifera was investigated. The effect of liquid-to-solid ratio on the yield of total glycosides from Moringa oleifera is shown in Figure 1 , the yield of total glycosides from Moringa oleifera first increased with the increase of liquid-to-solid ratio and then decreased sharply. When the liquid-to-solid ratio was 50 mL / g, the yield of total glycosides reached the highest, which was 1.06%.

[0043] Under the conditions of liquid-to-solid ratio of 50 mL / g, microwave time of 20 min, and ethanol volume fraction of 70%, the effect of microwave temperature (50 - 70 °C) on the yield of total glycosides from Moringa oleifera was investigated. The effect of microwave temperature on the yield of total glycosides from Moringa oleifera is shown in Figure 2 , the yield of total glycosides from Moringa oleifera increased with the increase of temperature at 50 - 60 °C. When the temperature reached 60 °C, the yield of total glycosides was 1.06%. Continuing to increase the microwave temperature, the yield of total glycosides decreased instead.

[0044] Under the conditions of liquid-to-solid ratio of 50 mL / g, microwave temperature of 60 °C, and ethanol volume fraction of 70%, the effect of microwave time (20 - 40 min) on the yield of total glycosides from Moringa oleifera was investigated. The effect of microwave time on the yield of total glycosides from Moringa oleifera is shown in Figure 3 , the yield of total glycosides from Moringa oleifera increased with the increase of time at 20 - 30 min. When the microwave time was 30 min, the yield of total glycosides was 1.29%. Continuing to extend the microwave time, the yield of total glycosides decreased significantly.

[0045] Under the conditions of liquid-to-solid ratio of 50 mL / g, microwave temperature of 60 °C, and microwave time of 30 min, the effect of ethanol volume fraction (50% - 90%) on the yield of total glycosides from Moringa oleifera was investigated. The effect of ethanol volume fraction on the yield of total glycosides from Moringa oleifera is shown in Figure 4 , the yield of total glycosides from Moringa oleifera first increased with the increase of ethanol volume fraction. When the ethanol volume fraction was 70%, the yield of total glycosides reached the highest, which was 1.29%. Continuing to increase the ethanol volume fraction, the yield of total glycosides remained basically unchanged.

[0046] The results of single-factor experiments showed that the extraction conditions with the highest yield of total glycosides from Moringa oleifera were: ethanol volume fraction of 70%, liquid-to-solid ratio of 50 mL / g, microwave temperature of 60 °C, and microwave time of 30 min.

[0047] Since ethanol with a volume fraction of 60%-80% has little effect on the yield of total glucosides of Moringa oleifera, the liquid-solid ratio, microwave temperature, and microwave time were used as independent variables, with the optimal level of each factor as the central value and the yield of total glucosides of Moringa oleifera as the response value. A response surface experiment with three factors and three levels was designed to further optimize the extraction conditions of total glucosides of Moringa oleifera to obtain a higher yield. The factor level design and test results of the response surface are shown in Tables 1 and 2.

[0048] The optimal conditions for extracting total glucosides of Moringa oleifera obtained from the response surface results were: liquid-solid ratio of 52.70 mL / g, microwave temperature of 58.92 °C, and microwave time of 30.46 min. Under these conditions, the theoretical yield of total glucosides of Moringa oleifera was 1.310%. In the actual experiment, the liquid-solid ratio was 53 mL / g, the microwave temperature was 59 °C, and the microwave time was 30.5 min as the experimental conditions for verification, and the actual yield of total glucosides of Moringa oleifera was 1.303%.

[0049] (2) Extraction and deoiling to prepare crude glucosinolates

[0050] Accurately weigh 40 g of Moringa oleifera seed powder. After extracting total glucosides of Moringa oleifera under the experimental conditions of "ethanol volume fraction of 70%, liquid-solid ratio of 53 mL / g, microwave temperature of 59 °C, and microwave time of 30.5 min", the extract was filtered by suction. It was concentrated by evaporation at 70 °C using a rotary evaporator and dried in a vacuum drying oven at 55 °C for 60 min to obtain the crude extract of Moringa oleifera seeds, about 520 mg. Then all the obtained crude extract was dissolved in 50 mL of distilled water. First, an equal volume of petroleum ether was added for extraction three times and the aqueous layer was collected. Then, an equal volume of ether was added for extraction three times and the aqueous layer was collected. Finally, an equal volume of n-butanol saturated with water was added for extraction three times and the n-butanol layer was collected, concentrated and dried to obtain the crude glucosinolates.

[0051] (3) Screening of resins

[0052] Three macroporous adsorption resins, D101, AB-8, and NKA-9, were screened.

[0053] All three macroporous adsorption resins were first subjected to pretreatment operations. The pretreatment steps were: placing the resins in beakers respectively, first soaking them in absolute ethanol for 24 h, taking out the resins after alcohol soaking and washing them with distilled water until there was no alcohol smell; then soaking them in 4 wt% HCl for 4 h, taking out the resins after acid soaking and washing them with distilled water until they were neutral; finally soaking them in 4 wt% NaOH for 4 h, taking out the resins after alkali soaking and washing them with distilled water until they were neutral.

[0054] Adsorption experiments were carried out on the three pretreated resins. The steps of the adsorption experiment were as follows: The crude glucosinolate obtained in step (2) was dissolved in 50 mL of distilled water to obtain a sample solution. After the pretreated resins were dried by suction filtration, they were weighed. 5 g of each resin was taken and added to 10 mL of the sample solution, and the mixture was oscillated at a speed of 100 rpm in a water bath at 25 °C for 6 h. Then it was filtered, the filtrate was recovered, the volume of the adsorption solution was measured, and it was colored with vanillin-sulfuric acid. The absorbance value of the adsorption solution at 548 nm was measured by ultraviolet-visible spectrophotometry, and then the total glycoside concentration in the adsorption solution was calculated. Then, the adsorption rate (A) and adsorption capacity (Q) of the resin were calculated by combining the following formulas (2) and (3):

[0055]

[0056] Where: A: Adsorption rate;

[0057] C0: Total glycoside concentration in the sample solution before adsorption, mg / mL;

[0058] C: Total glycoside concentration in the sample solution after adsorption, mg / mL;

[0059] Q: Adsorption capacity, mg / mg;

[0060] V: Volume of the sample solution after adsorption, mL;

[0061] W: Weight of the resin, mg.

[0062] After the adsorption experiment, the desorption experiment was then carried out. The steps of the desorption experiment were as follows: 20 mL of 70 v / v% ethanol solution was added to the resin adsorbed with total glycosides as the desorbent, and the mixture was oscillated at a speed of 100 rpm in a water bath at 25 °C for 6 h. Then it was filtered, the filtrate was recovered, the volume of the desorption solution was measured, and it was colored with vanillin-sulfuric acid. The absorbance value of the desorption solution at 548 nm was measured by ultraviolet-visible spectrophotometry, and then the total glycoside concentration in the desorption solution was calculated. Then, the desorption rate (E) of the resin was calculated by combining the following formula (4):

[0063]

[0064] Where: E: Desorption rate;

[0065] C1: Total glycoside concentration in the desorption solution, mg / mL;

[0066] V1: Volume of the desorption solution, mL;

[0067] Q: Adsorption capacity, mg / mg;

[0068] W: Weight of the resin, mg.

[0069] The adsorption rate (A) and desorption rate (B) data of crude glucosinolate by three macroporous adsorption resins, D101, AB-8, and NKA-9, are shown in Figure 5 . It can be seen from Figure 5 that D101 macroporous adsorption resin (non-polar macroporous adsorption resin) has the best adsorption effect, with an adsorption rate reaching 76.46%; the adsorption rates of AB-8 macroporous adsorption resin (medium-polar macroporous adsorption resin) and NKA-9 macroporous adsorption resin (polar macroporous adsorption resin) are 61.47% and 58.25% respectively. The desorption ability of NKA-9 macroporous adsorption resin is the strongest, with a desorption rate of 62.81%; the desorption rates of AB-8 macroporous adsorption resin and D101 macroporous adsorption resin are 37.38% and 34.01% respectively. The purification effect of the resin on substances needs to comprehensively consider the adsorption-desorption ability of the resin. If the resin has good adsorption ability for substances but poor desorption ability, it is difficult to elute this substance, resulting in an unsatisfactory purification effect. The desorption effects (the ratio of desorption rate to adsorption rate) of D101, AB-8, and NKA-9 macroporous adsorption resins are 0.44, 0.61, and 1.08 respectively. Therefore, the desorption effect is introduced to evaluate the purification effect of the resin. The results show that among the three types of macroporous adsorption resins, non-polar, medium-polar, and polar macroporous adsorption resins, NKA-9 polar macroporous adsorption resin has the best desorption effect on glycoside substances and the highest recovery rate. Therefore, NKA-9 macroporous adsorption resin is selected for subsequent experiments.

[0070] (4) Performance optimization experiment of the resin

[0071] After the screening in step (3), the appropriate resin (NKA-9) is used for subsequent experiments. In order to obtain better adsorption-desorption effects and achieve the best purification effect, the factors affecting the static adsorption-desorption of the resin are studied, including the selection of desorbent concentration, the ratio of resin mass to extract volume, and the ratio of resin mass to desorbent volume. Through single-factor experiments, the optimal adsorption-desorption conditions are obtained to achieve the best adsorption-desorption effects.

[0072] Let the resin adsorb according to the method in step (3). After adsorption, the filtrate is recovered, and the resin is desorbed with ethanol solutions of 30 v / v%, 50 v / v%, 70 v / v%, and 90 v / v% respectively according to the method in step (3). After desorption, the filtrate is recovered. The volumes of the two filtrates are measured respectively, and vanillin-sulfuric acid is used for color development. The absorbance at 548 nm is measured and the total glycoside content in the solution is calculated. The desorption rate E of the resin is calculated according to formula (4). The appropriate desorbent concentration is screened out through the desorption effect. The results are as Figure 6 shown. As the concentration of the ethanol solution increases, the content of glucosinolate increases. The desorption rate of the 90% ethanol solution is the highest, with a desorption rate of 93.52%. Therefore, 90% ethanol solution is selected as the desorbent.

[0073] Accurately weigh 5 portions of 5 g of resin, and add 1 mL, 2.5 mL, 5 mL, 10 mL, and 25 mL of sample solution (i.e., extract) respectively. Let the resin perform adsorption according to the method in step (3), and recover the filtrate after adsorption. Measure the volume of the adsorption solution, color it with vanillin - sulfuric acid, measure the absorbance at 548 nm, and calculate the total glycoside content in the solution. Calculate the adsorption rate A of the resin according to formula (2). Screen out the appropriate ratio of resin mass to extract volume. The results are as Figure 7 shown. As the solid - liquid ratio increases, the resin adsorption rate gradually increases. This may be because within the resin adsorption capacity limit, the larger the extract volume, the more the total glycoside content, and the more total glycosides adsorbed by the resin, resulting in a decrease in the total glycoside concentration in the sample solution after adsorption and an increase in the adsorption rate. When the ratio of resin mass to extract volume is 1:1, the adsorption rate reaches the maximum, which is 86.49%. Subsequently, the adsorption rate decreases as the solid - liquid ratio increases. This may be due to the leakage of the resin caused by excessive extract, resulting in an increase in the total glycoside content in the sample solution after adsorption and a decrease in the adsorption rate. In order to maximize the adsorption of the sample by the resin, the ratio of resin mass to extract volume of 1:1 is selected for subsequent experiments.

[0074] Under the above - optimized conditions, add 5 mL, 10 mL, 15 mL, 20 mL, and 25 mL of desorbent to the 5 portions of resin after adsorption respectively. Let the resin perform desorption according to the method in step (3). Recover the filtrate after desorption, measure the volume of the desorption solution, color it with vanillin - sulfuric acid, measure the absorbance at 548 nm, and calculate the total glycoside content in the solution. Calculate the desorption rate E of the resin according to formula (4). Screen out the appropriate ratio of resin mass to desorbent volume. The results are as Figure 8 shown. As the liquid - solid ratio increases, the resin desorption rate increases. This is because the increase in the desorbent volume results in an increase in the total glycoside content eluted, leading to an increase in the desorption rate. When the ratio of resin mass to desorbent volume is 1:2, the desorption rate reaches the maximum, which is 96.43%. Subsequently, the desorption rate levels off as the liquid - solid ratio increases, indicating that the glucosinolates adsorbed by the resin are basically eluted, and further increasing the desorbent volume cannot increase the desorption rate. Considering the actual operation and ensuring that the glucosinolates can be completely eluted, the ratio of resin mass to desorbent volume of 1:3 is selected for subsequent experiments.

[0075] (5) Purification of Glucosinolates

[0076] The NKA-9 macroporous adsorption resin was pretreated according to the steps in step (3). After pretreatment, the resin was weighed after removing water by suction filtration, and 50 g of the resin was loaded into a column. 1 g of moringa seed powder was taken and operated according to the methods in steps (1) and (2) to obtain 46.1 mg of crude glucosinolate. All of it was added to 50 mL of distilled water. After dissolution, the obtained sample solution was all injected into the NKA-9 macroporous adsorption resin for pre-adsorption for 1 h. Distilled water was added for elution, and the flow rate was controlled at 1.5 BV / h (2 mL / min). After a period of time, the eluate was collected and detected with Molish reagent (5 g of α-naphthol dissolved in 100 mL of 95% ethanol) until it became colorless (about 45 min), then the addition of distilled water was stopped, and a desorbent was added instead. The desorbent added was 90 v / v% ethanol, and the flow rate was 1.5 BV / h (2 mL / min). The eluted desorbent was collected in bottles of 30 mL each, detected by silica gel thin-layer chromatography, developed with chloroform-methanol (1:1, v / v) as the developing agent, and then colored with 10 wt% sulfuric acid-ethanol solution. The components with the same or similar Rf values were combined, and the components in the 45 - 120 min period were taken and concentrated and dried at 80 °C. Then it was dissolved in methanol and passed through a 0.22 μm filter membrane, and concentrated and dried at 60 °C to obtain a purified product. After weighing, the mass was 15.9 mg, and the yield was 1.59%.

[0077] The purified product was characterized, and its infrared spectrum is shown in Figure 9 , and the mass spectrum is shown in Figure 10 , and the nuclear magnetic resonance hydrogen spectrum is shown in Figure 11 and Table 3. In the infrared spectrum, the broad peak at 3379.72 cm -1 corresponds to the stretching vibration of the hydroxyl group, and the peak at 2928.93 cm -1 corresponds to the stretching vibration of the methylene groups on the side chain and pyranose glucose. The peak at 1611.79 cm -1 corresponds to the stretching vibration of C=N, and the peak at 1235.30 cm -1 corresponds to the stretching vibration of C-O on the benzene ring. The peak at 1059.58 cm -1 corresponds to the stretching vibration of C-O on pyranose glucose and pyranose rhamnose. The peak at 1511.42 cm -1 is the characteristic absorption peak of the benzene ring. Combining with the peak at 798.77 cm -1 it can be seen that it is para-disubstitution on the benzene ring. From the mass spectrum, the quasi-molecular ion peak (m / z 571.09778) and the molecular ion peak (m / z 570.09473) of the target substance can be seen. Combining with the nuclear magnetic resonance hydrogen spectrum data, it can be confirmed that the purified product is the high-purity monomer of moringa seed glucosinolate, 4-(α-L-rhamnosyl)benzyl glucosinolate.

[0078] Table 1 Response surface test factor level design table for extracting total moringa glycosides

[0079]

[0080] Table 2 Response surface test results for extracting total glucosides from Moringa oleifera

[0081]

[0082] Table 3 1H NMR data of the purified product

[0083]

Claims

1. A method for microwave-assisted extraction of glucosinolate monomers from Moringa oleifera, characterized in that, It includes the following steps: (1) Extraction of total glucosinolates from Moringa oleifera seeds: Take the powder of Moringa oleifera seeds, add an ethanol solution with a volume fraction of 50% - 90% according to a liquid-solid ratio of 50:1 - 55:1 mL / g, start stirring, place it in a microwave reactor for extraction, the microwave extraction temperature is 58 - 65°C, the microwave extraction time is 25 - 32 min, filter the obtained extract by suction, concentrate and dry it to obtain the crude extract of Moringa oleifera seeds; (2) Preparation of crude glucosinolate by extraction and deoiling: Dissolve the crude extract of Moringa oleifera seeds in distilled water, and then extract it successively with petroleum ether, ether, and n-butanol saturated with water, collect the n-butanol layer, concentrate and dry it to obtain the crude glucosinolate; (3) Purification of glucosinolate: Pack the pretreated NKA-9 macroporous adsorption resin into a column, dissolve the crude glucosinolate obtained in (2) in distilled water, load the obtained sample solution, after pre-adsorbing for 0.5 - 1 h; use distilled water as the eluent for elution, control the flow rate at 1.5 BV / h, after a period of time, collect the eluate and detect it with Molish reagent until it turns colorless; then adjust the eluent to the desorbent, keep the flow rate unchanged, and start collecting the desorbent. The desorbent is a 90 v / v% ethanol solution, and the mass ratio of the NKA-9 macroporous adsorption resin to the volume of the desorbent is 1:2 - 1:3 g / mL; concentrate and dry the desorbent, then dissolve it with methanol and filter it through a 0.22 μm filter membrane, concentrate and dry it to obtain the monomer of glucosinolate from Moringa oleifera seeds.

2. The method according to claim 1, wherein In step (3), the crude glucosinolate is dissolved in distilled water according to a solid-liquid ratio of 1:1 - 1.1 mg / mL, and the mass ratio of the NKA-9 macroporous adsorption resin to the volume of the sample solution is 1:1 - 2:1 g / mL.

3. The method according to claim 1, characterized in that, In step (2), the crude extract of Moringa oleifera seeds is dissolved in distilled water according to a solid-liquid ratio of 10:1 - 11:1 mg / mL.

4. The method according to claim 1, wherein In step (2), the extraction steps include: first add an equal volume of petroleum ether for extraction three times and collect the aqueous layer, then use an equal volume of ether for extraction three times and collect the aqueous layer, and finally use an equal volume of n-butanol saturated with water for extraction three times and collect the n-butanol layer, concentrate and dry it to obtain the crude glucosinolate.

5. The method according to claim 1, wherein In step (3), the pretreatment operation of the resin includes: first soak it with anhydrous ethanol, take out the resin after alcohol immersion and wash it with distilled water until there is no alcohol smell; then soak it with 4 wt% HCl, take out the resin after acid immersion and wash it with distilled water until it is neutral; finally soak it with 4 wt% NaOH, take out the resin after alkali immersion and wash it with distilled water until it is neutral.

6. The method according to claim 1, wherein In step (1), the extraction conditions are: the liquid-solid ratio is 52:1 - 53:1 mL / g, the volume fraction of the ethanol solution is 70% - 90%, the microwave extraction temperature is 58 - 60°C, and the microwave extraction time is 30 - 31 min.

7. The method according to claim 1, wherein In step (1), the extraction conditions are: the liquid-solid ratio is 53:1 mL / g, the volume fraction of the ethanol solution is 70%, the extraction temperature is 59°C, and the extraction time is 30.5 min.

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