A method for separating mogrosides by pulse electric field ultrasonic extraction and fusion preparative chromatography technology

Abstract

The application relates to a method for separating mogroside by pulse electric field ultrasonic extraction and fusion preparative chromatography, which comprises the following steps: (1) pulse electric field ultrasonic extraction: taking momordica grosvenori, crushing, adding alkali water, continuously passing through a pulse electric field ultrasonic extraction device, coarsely filtering, centrifuging, and obtaining momordica grosvenori extract; the alkali water contains a reducing agent and a cyclodextrin inclusion compound; (2) membrane filtration; (3) enriching momordica grosvenori total glycosides by macroporous adsorption resin; (4) passing macroporous adsorption resin elution liquid through an aluminum oxide resin column, concentrating and drying the effluent; (5) gradient elution is carried out by preparative chromatography, the eluent is collected in sections, the eluent is concentrated and dried respectively, and different types of momordica grosvenori glycoside components are obtained. The application inhibits the oxidation and loss of active ingredients by the "electric crushing and ultrasonic extraction" technology, especially sianen glycoside I, and finally the obtained product has the advantages of high content and high yield.

Description

Technical Field

[0001] The present invention relates to a separation technology of natural active ingredients of plants, and in particular to a method for separating mogroside by combining pulsed electric field ultrasonic extraction with preparative chromatography technology. Background Art

[0002] Luo Han Guo (Sheng Han Guo), the fruit of a perennial vine in the Cucurbitaceae family, is native to mountainous areas such as Guilin in Guangxi, Shaoyang, Huaihua, and Yongzhou in Hunan, and Qiandongnan in Guizhou. It is a prized local specialty of my country and one of the first Chinese-approved "dual-use medicinal and edible" ingredients. The fruit boasts high nutritional value, rich in vitamin C, mogrosides, fructose, glucose, protein, pectin, and lipids. According to the "Zhongyao Zhi" (Chinese Materia Medica), Luo Han Guo is sweet and cooling in nature, and enters the lung and large intestine meridians. It moistens the lungs, relieves coughs, promotes fluid production, and quenches thirst. It is suitable for coughs caused by lung heat or dryness, whooping cough, and thirst caused by summer heat. It also has laxative effects. Modern medical research confirms that the mogrosides contained in Luo Han Guo are natural sweeteners 200-600 times sweeter than sucrose. However, they are calorie-free, making them a useful sweetener for those with diabetes, obesity, and other conditions that should avoid sugar. Mogroside is safe and non-toxic as a food. The national mandatory standard, "GB2760 Food Additives Usage Standard," stipulates that mogroside can be used in all types of food without restrictions. Due to its many excellent properties, mogroside can fully or partially replace sucrose, and therefore is widely used in the food industry. Furthermore, mogroside has broad application prospects and market potential in pharmaceuticals, cosmetics, and health foods.

[0003] Mogrosides are a class of natural active ingredients unique to Momordica grosvenori, including mogroside V, 11-oxo-mogroside V, mogroside III, mogroside IV, simenoside I, and mogroside VI. To date, researchers have discovered more than ten types of mogrosides in Momordica grosvenori. The types and contents of mogrosides usually vary depending on factors such as the variety of Momordica grosvenori, planting conditions, and degree of maturity. Generally, the most abundant and main mogroside in Momordica grosvenori is mogroside V (the content in fresh fruit is about 0.3%-0.5%; it accounts for up to 80% of the total mogrosides; its sweetness is about 400 times that of sucrose).

[0004] The sweetest glycoside in Momordica grosvenori is simenoside I, which is about 560 times sweeter than sucrose. Due to its low content and unstable nature, there is relatively little research on simenoside I in academia, and even fewer reports on its isolation and purification.

[0005] CN111018933A discloses a monk fruit extract product, its preparation method, and its use. The product is prepared from monk fruit through the following steps: crushing, water reflux extraction, concentration, centrifugation, pH adjustment, macroporous resin adsorption, ethanol analysis, MCI column adsorption, ethanol gradient elution, chromatography detection, concentration, and drying to obtain different types of monk fruit extracts. This patent claims that various extracts of simianoside I, mogroside V, and 11-O-mogroside V can be obtained, but all steps and process parameter ranges are identical. However, the patent claims that all extracts can produce high content and high yield of different mogroside monomers. This patent contains errors in its principles, and its authenticity is questionable.

[0006] CN110628861A discloses a method for increasing the overall sweetness of mogrosides by increasing the content of simenoside, comprising the following steps: 1) dissolving: dissolving mogrosides in a fermentation culture medium of the same concentration, wherein the mogroside concentration is 45-50 mg / ml, and sterilizing to obtain a stock solution; 2) screening dominant strains: 2-1) different strains are isolated from the skin of Momordica grosvenori, the soil in which Momordica grosvenori grows, and the Momordica grosvenori residue, and each strain is inoculated into a culture medium with cellulose or starch as a substrate. After culturing for 24 hours, the concentration of glucose in the substrate is detected to confirm the dominant strain that can produce exogenous glycosidases. A total of five dominant strains are screened and numbered Sc001, VSc01, CN01, CN01-A, and T-CN00, all of which are Saccharomyces cerevisiae strains; 2-2) the Saccharomyces cerevisiae strains Sc001, VSc01, CN01, CN01-A, and T-CN00 that can produce exogenous glycosidases are inoculated into the culture medium. In a culture solution with mogroside V as a substrate, ferment at room temperature for 168 hours, compare the sweetness of the fermentation solution with that of the stock solution every 24 hours, and screen out the strain corresponding to the fermentation solution with the highest sweetness as the specific sake yeast CN01; 3) Fermentation: After the specific sake yeast CN01 is rejuvenated on a potato slant medium, it is inoculated on an MS medium for expansion. After two-stage expansion, it is used as a fermentation seed liquid, and then the stock solution obtained in step 1) is inoculated with the fermentation seed liquid, and the inoculation amount is 1 / 1 of the stock solution. 0-12%, controlling the fermentation temperature to 30-32°C, and naturally fermenting for 72-80 hours to obtain a fermentation broth; 4) centrifugal filtration: centrifuging and filtering the fermentation broth to collect the supernatant; 5) chromatography: separating the supernatant through a transformed ion-type chromatographic resin at a flow rate of 2 BV / h for 2 hours, and collecting the effluent within 45 minutes to 1.2 hours; 6) concentration and freeze-drying: concentrating the effluent under vacuum and freeze-drying to obtain mogroside with enhanced sweetness. This patent converts mogroside into highly sweet simenoside through specific fermentation, but does not specifically prepare simenoside I.

[0007] CN102942611A discloses a method for preparing high-purity simenoside I, comprising the following steps: 1) crushing Momordica grosvenori into coarse powder, decocting with water, and then centrifuging to collect the supernatant; 2) adsorbing the supernatant obtained in step 1) with a macroporous resin, washing with 40% (v / v) alcohol to remove impurities, and then eluting with 60-80% (v / v) alcohol, and finally recovering ethanol from the eluate to obtain an extract; 3) dissolving the extract with 60% (v / v) alcohol, centrifuging, and applying the supernatant to an ODS column, first washing with 60% (v / v) alcohol to remove impurities, and then eluting with 70-90% (v / v) alcohol; finally, recovering ethanol from the eluate and performing HPLC detection, combining the fractions containing simenoside I to obtain a crude product of simenoside I; and 4) subjecting the crude product of simenoside I to Sephadex chromatography. LH-20 column chromatography, followed by ethanol gradient elution, is performed for HPLC detection, and fractions containing simenoside I are combined. Finally, the solvent is recovered to obtain the refined simenoside I. The patented monk fruit raw material undergoes high-temperature decoction, which causes significant simenoside oxidation losses, resulting in a low simenoside yield. Furthermore, the multiple HPLC techniques used make it inconvenient for large-scale industrial production. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the defects of the existing technology and provide a technology for separating mogrosides by pulsed electric field ultrasonic extraction combined with preparative chromatography technology, which can simultaneously obtain multiple high-value mogrosides, has high product content and high yield, has strong operability in the production process, low production cost, and is suitable for large-scale industrial production.

[0009] The technical solution adopted by the present invention to solve the technical problem is as follows: a method for separating mogrosides by pulsed electric field ultrasonic extraction combined with preparative chromatography technology, comprising the following steps:

[0010] (1) Pulsed electric field ultrasonic extraction: Take the monk fruit, crush it, add alkaline water, continuously pass it through a pulsed electric field ultrasonic extraction device, coarsely filter it, and centrifuge it to obtain the monk fruit extract; the alkaline water contains a reducing agent @ cyclodextrin inclusion complex;

[0011] (2) Membrane filtration: filtering the Momordica grosvenori extract through a ceramic membrane to obtain a ceramic membrane filtrate;

[0012] (3) Enrichment of total glycosides from Momordica grosvenori: The filtrate from the ceramic membrane was passed through a macroporous adsorption resin column until the column was completely loaded, washed with pure water, and then analyzed with an alcohol-water solution. The ethanol solution was collected for later use.

[0013] (4) Purification of total glycosides from Momordica grosvenori: the alcohol-water solution is passed through an alumina resin column, the effluent is concentrated to obtain a concentrate, which is dried to obtain a total glycosides from Momordica grosvenori extract; the alumina resin column is impregnated with acid water and washed;

[0014] (5) Preparative chromatography separation: Take a portion of the total glycosides extract of Momordica grosvenori, dissolve it in pure water, filter it through a microporous filter membrane, pass the filtrate through an MCI preparative chromatography column, perform gradient elution, and collect the eluate in sections for later use;

[0015] (6) Concentration and drying: The eluates collected in sections are concentrated and dried to obtain different types of mogroside components.

[0016] Preparative chromatography is the use of chromatographic techniques to prepare pure substances, that is, to separate and collect one or more chromatographically pure substances. The concept of "preparation" in preparative chromatography refers to obtaining sufficient quantities of a single compound to meet research and other purposes.

[0017] Preferably, in step (1), the reducing agent @ cyclodextrin inclusion complex is obtained by embedding the reducing agent in a cyclodextrin substance, the reducing agent is selected from at least one of astaxanthin and β-carotene, and the cyclodextrin substance is selected from at least one of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl ether-β-cyclodextrin; the reducing agent content in the reducing agent @ cyclodextrin inclusion complex is 15-20wt%, and the content of the reducing agent @ cyclodextrin inclusion complex in alkaline water is 1-5wt%.

[0018] The preparation of the reducing agent @ cyclodextrin inclusion complex is well known in the art. The reducing agent and cyclodextrin substances are added in a molar ratio of 1:1-3, an alcohol aqueous solution is added, and the mixture is stirred under an inert atmosphere until the embedding reaches equilibrium. The mixture is then spin-dried and freeze-dried to obtain the reducing agent @ cyclodextrin inclusion complex. The alcohol aqueous solution is an ethanol solution with a volume fraction of 70-95%, and the volume ratio of the alcohol aqueous solution to the reducing agent is 3-5L:1g.

[0019] Preferably, in step (1), the alkali is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate or sodium bicarbonate, the mass percentage concentration of the alkaline water is 0.1-2wt%, preferably 0.4-0.7wt%, and the amount of the alkaline water is 3-10 times, preferably 4-5 times, the weight of the Momordica grosvenori raw material. The purpose of using alkaline water as the extraction solvent in the present invention is, firstly, to prevent the material from becoming rancid and the degradation of mogrosides due to the growth of microorganisms during extraction and subsequent processes; secondly, under an alkaline pH environment, the conductivity and ionic strength of the solution are greatly increased, which can maximize the advantages of the pulsed electric field "electrical breaking and acoustic extraction" and quickly and efficiently leach mogrosides.

[0020] Preferably, in step (1), the electric field strength of the pulsed electric field is 0.5-10 kV / cm, the number of times is 10 to 30, the power of the ultrasound is 20-1000 W, the ultrasonic frequency is 60-150 kHz, the pulsed electric field ultrasonic extraction time is 10-100 min, and the extraction temperature is 5-30° C., preferably 10-25° C. The purpose of using pulsed electric field-assisted extraction in the present invention is to simultaneously overcome the two disadvantages of partial oxidation of mogrosides during high-temperature extraction and the low extraction rate of mogrosides during low-temperature extraction by conventional methods, thereby achieving the purpose of preventing oxidation of mogrosides at low temperatures and efficiently extracting mogrosides. Simenoside I is the mogroside with the highest sweetness multiple. The inventors have found that simenoside I can be oxidized to 11-oxy-simenoside I (with a sweetness multiple of only one-tenth that of simenoside I) under high temperature conditions. The use of low-temperature alkaline water + pulsed electric field ultrasonic assisted extraction can significantly reduce the oxidation of simenoside I, thereby improving the yield of simenoside I.

[0021] In step (1), the centrifugation method includes but is not limited to horizontal spiral centrifugation, butterfly centrifugation, tubular centrifugation, and a combination of the above multiple centrifugation methods.

[0022] Preferably, in step (2), the material of the ceramic membrane is zirconium oxide, aluminum oxide, or titanium oxide; the pore size of the ceramic membrane is 0.1-1.2 μm; and the filtration pressure of the ceramic membrane is 0.6-1.0 MPa.

[0023] Preferably, in step (3), the macroporous adsorption resin used in the macroporous adsorption resin column is a non-polar macroporous adsorption resin, a medium-polar macroporous adsorption resin, a weakly polar macroporous adsorption resin, or a combination of two or more of the above resins. For example, the macroporous adsorption resin is selected from at least one of D101, LX-T28, and AB-8, the resin volume used is 10-30 L:100 kg of fresh monk fruit, the resin column height-to-diameter ratio is 3-8:1, and the column flow rate is 0.5-5 Bv / h. The amount of pure water used for washing is 2-4 BV, and the amount of alcohol-water solution used for analysis is 2-3 BV, wherein the alcohol-water solution is a 50-70% solution of methanol and / or ethanol.

[0024] Preferably, in step (4), the types of alumina include neutral alumina, acidic alumina and basic alumina, and the amount of alumina used is 1-10wt% of the weight of the Momordica grosvenori raw material. The purpose of using alumina is that alumina can specifically adsorb impurities such as flavonoids and pigments in the ethanol analysis solution, thereby improving the purity of the total glycosides in the material and reducing the interference of impurities in the subsequent preparative chromatography separation step.

[0025] Preferably, in step (4), the alumina resin column is immersed in acidic water with a pH of 3-4 for 10-60 minutes, and then washed with water until the effluent is close to neutral (pH = 7 ± 0.5), wherein the acidic water is at least one of dilute hydrochloric acid and dilute sulfuric acid. The inventors have found that various commercially available aluminas, due to the aluminum hydroxide usually attached to their surfaces, have irreversible dead adsorption to simenoside I, resulting in the loss of simenoside I. The low content of simenoside I in Momordica grosvenori, coupled with the loss during the extraction and purification process, is the main reason for the lack of simenoside I products. The inventors have found that after the alumina resin column is immersed in acidic water, it does not affect the adsorption of other impurities, but greatly reduces the adsorption of simenoside I.

[0026] Preferably, in step (5), the filler types used in the MCI preparative chromatography include polystyrene / divinylbenzene type (PS / DVB) and acrylate type, and the particle size of the filler is 4-300 μm.

[0027] Preferably, in step (5), the gradient elution is performed using an alcohol-water solution with an increasing volume fraction of alcohol, wherein the alcohol is selected from methanol and / or ethanol, and the volume fraction of the alcohol increases from 0% (i.e., pure water) to 100% (i.e., alcohol). Using alcohol solutions with different volume fractions, mogroside is eluted sequentially according to the difference in polarity, thereby obtaining a variety of high-content mogroside monomers.

[0028] More preferably, in step (5), the gradient elution is to control the flow rate to 0.5-2BV / h, and sequentially use 3-5BV pure water, 3-5BV 10-20% alcohol aqueous solution, 3-5BV 20-30% alcohol aqueous solution, 3-5BV 30-40% alcohol aqueous solution, 3-5BV 40-60% alcohol aqueous solution, 1-2BV 60-70% alcohol aqueous solution, and 1-2BV>70% alcohol aqueous solution for elution.

[0029] The pulsed electric field will have a regular effect on the permeability of the plant tissue structure; ultrasonic vibration can generate powerful energy, causing the solvent and plant tissue to oscillate continuously, forming a cavitation effect, which helps the diffusion of solutes and accelerates the leaching of effective active ingredients in plants. When the two technologies are coupled, the pulsed electric field instantaneous wall breaking and ultrasonic enhanced mass transfer extraction (i.e.: "electric breaking and acoustic extraction" technology) can be achieved simultaneously, and the plant tissue is instantly and accurately broken to form micropores of different pore sizes, realizing the short-term and efficient extraction of active ingredients at low temperature to room temperature. Thanks to the pulsed electric field wall breaking and ultrasonic enhanced mass transfer, the active ingredients are fully and efficiently leached. At the same time, due to the lower extraction temperature and shortened extraction time, the probability of active ingredients (especially heat-sensitive, photosensitivity, and easily oxidized active ingredients) being thermally decomposed and oxidatively denatured is greatly reduced, thereby reducing the loss of active ingredients and increasing the yield.

[0030] MCI (MCI GEL) preparative chromatography is a medium-pressure preparative chromatography method using chromatographic separation gel (also known as a polymer-based, small-particle chromatographic filler). Due to its high adsorption capacity, uniform particle size, excellent mechanical strength, resistance to breakage, minimal residue, and easy pretreatment, this filler is widely used in the separation and purification of natural products, fermentation products, and small organic molecules.

[0031] The beneficial effects of the method of the present invention are as follows:

[0032] (1) With the implementation of the “electrical breaking and acoustic extraction” technology, the extraction of active ingredients no longer relies on the high temperature and long-term boiling required in traditional processes, and the energy consumption of steam, water, and electricity has been greatly reduced.

[0033] (2) Compared with C-18 bonded silica gel filler, MCI filler has the advantages of more stable chemical properties and longer service life, and is more suitable for reversed-phase chromatography separation.

[0034] (3) The method of the present invention provides a new method for separating mogroside monomers. Combined with the "electrolysis and sonic extraction" extraction technology, the active ingredients are fully extracted and the oxidation is inhibited. The final product has the advantages of high content and high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the HPLC spectrum of fraction A in Example 1.

[0036] Figure 2 This is the HPLC spectrum of fraction B in Example 1.

[0037] Figure 3 This is the HPLC spectrum of fraction C in Example 1. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the embodiments.

[0039] The Momordica grosvenori raw material used in the embodiment of the present invention was purchased from Shaoyang, Hunan, wherein the mass percentage content of mogroside V was 0.452%, the mass percentage content of 11-oxo-mogroside V was 0.053%, and the mass percentage content of simenoside I was 0.015%; the macroporous adsorption resin used in the embodiment of the present invention was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.; the preparative chromatography filler MCI GEL CHP used in the embodiment of the present invention was purchased from Mitsubishi Chemical of Japan; the raw materials or chemical reagents used in the embodiment of the present invention, unless otherwise specified, were obtained through conventional commercial channels.

[0040] In the examples of the present invention, the content of various mogrosides was detected by high performance liquid chromatography external standard method.

[0041] Preparation Example 1

[0042] 10g of astaxanthin and hydroxypropyl-β-astaxanthin were added in a 1:2 molar ratio, and 40L of 95% ethanol was added. The mixture was stirred under a nitrogen atmosphere for 24 hours to reach embedding equilibrium. The mixture was rotary evaporated to a paste, and then freeze-dried to obtain a powder, which is the astaxanthin@hydroxypropyl-β-cyclodextrin inclusion complex. Testing showed that the astaxanthin content in the astaxanthin@hydroxypropyl-β-cyclodextrin inclusion complex was 15.4wt%.

[0043] Example 1

[0044] (1) Pulsed electric field ultrasonic extraction: 100 kg of fresh Momordica grosvenori fruit was crushed, and 400 L of an alkaline aqueous solution containing 0.4 wt % NaOH and 1 wt % of the inclusion compound obtained in Preparation Example 1 was added. The fruit was continuously passed through a pulsed electric field ultrasonic extraction apparatus (electric field strength of 30 kV / cm, ultrasonic frequency of 120 kHz, power of 100 W) at room temperature, coarsely filtered, and centrifuged to obtain a Momordica grosvenori extract.

[0045] (2) Membrane filtration: The Momordica grosvenori extract was filtered through an alumina ceramic membrane with a pore size of 0.4 μm and a filtration pressure of 0.8 MPa to obtain a ceramic membrane filtrate;

[0046] (3) Enrichment of total glycosides of Momordica grosvenori: The filtrate of the ceramic membrane was passed through a macroporous adsorption resin column (wherein the macroporous adsorption resin was labeled D101, the resin dosage was 15 L, and the height-to-diameter ratio of the resin column was 4:1) at a flow rate of 1 BV / hour until the column was completed. The effluent was first washed with 3 BV of pure water until it became neutral, and then eluted with 2 BV of 70% ethanol by volume. The ethanol elution solution was collected for later use.

[0047] (4) Purification of total glycosides from Momordica grosvenori: The ethanol solution was passed through an alumina resin column (wherein the type of alumina was neutral chromatography alumina, 100-200 mesh, the dosage was 5 kg, the height-to-diameter ratio of the resin column was 5:1, the alumina resin was first immersed in dilute sulfuric acid at pH 3 for 30 min, and then washed with pure water until the effluent was neutral). The flow rate of the column was 0.5 BV / h, and the effluent from the alumina resin column was collected and concentrated to be alcohol-free to obtain a purified total glycoside concentrate, which was then dried to obtain 0.741 kg of total glycosides extract from Momordica grosvenori.

[0048] (5) Preparative chromatographic separation: 200 g of Momordica grosvenori total glycosides extract was dissolved in pure water to a solid concentration of about 10%, and then filtered through a microporous filter membrane. The filtrate was passed through an MCI preparative chromatographic column (wherein the filler model was MCI GEL CHP20SS, the filler dosage was 1 L, and the column pressure was 2 bar). The flow rate was controlled at 2 BV / h. The chromatographic column was eluted with 5 BV of pure water, 3 BV of 10% methanol by volume, 3 BV of 20% methanol by volume, 5 BV of 35% methanol by volume, 3 BV of 50% methanol by volume, 1 BV of 62% methanol by volume, 1 BV of 70% methanol by volume, and 5 BV of 100% methanol by volume. The eluents from each section were collected and set aside.

[0049] (6) Concentration and drying: The eluate eluted with 20% methanol was concentrated and dried to obtain fraction A, weighing 13.58 g; the eluate eluted with 35% methanol was concentrated and dried to obtain fraction B, weighing 115.72 g; the eluate eluted with 62% methanol was concentrated and dried to obtain fraction C, weighing 3.75 g.

[0050] Determination by high performance liquid chromatography external standard method showed that the mass percentage content of 11-oxo-mogroside V in fraction A was 97.10%, and the yield of 11-oxo-mogroside was 92.18%; the mass percentage content of mogroside V in fraction B was 97.71%, and the yield of mogroside V was 92.68%; the mass percentage content of simenoside I in fraction C was 96.23%, and the yield of simenoside I was 89.14%.

[0051] Figure 1 is the HPLC spectrum of fraction A, Figure 2 is the HPLC spectrum of fraction B, Figure 3 This is the HPLC spectrum of fraction C.

[0052] Example 2

[0053] (1) Pulsed electric field ultrasonic extraction: 100 kg of fresh Momordica grosvenori fruit was crushed, and 500 L of an alkaline aqueous solution having a mass percentage concentration of 0.6 wt% KOH and 1.5 wt% of the inclusion compound obtained in Preparation Example 1 was added. The mixture was continuously passed through a pulsed electric field ultrasonic extraction apparatus (electric field strength of 50 kV / cm, ultrasonic frequency of 60 kHz, and power of 100 W) at room temperature, coarsely filtered, and centrifuged to obtain a Momordica grosvenori extract.

[0054] (2) Membrane filtration: filtering the Momordica grosvenori extract through a ceramic membrane to obtain a ceramic membrane filtrate;

[0055] (3) Enrichment of total glycosides of Momordica grosvenori: The filtrate of the ceramic membrane was passed through a macroporous adsorption resin column (wherein the macroporous adsorption resin was labeled LX-T28, the resin dosage was 15 L, and the height-to-diameter ratio of the resin column was 4:1) at a flow rate of 1 BV / hour until the column was completed. The effluent was first washed with 3 BV of pure water until it was neutral, and then 3 BV of 65% ethanol was used for analysis. The ethanol analysis solution was collected for later use.

[0056] (4) Purification of total glycosides from Momordica grosvenori: The ethanol solution was passed through an alumina resin column (wherein the type of alumina was alkaline chromatography alumina, 100-200 mesh, the amount was 4 kg, the height-to-diameter ratio of the resin column was 4:1, the alumina resin was first immersed in dilute hydrochloric acid at pH 4 for 10 min, and then washed with pure water until the effluent was neutral), the flow rate of the column was 0.5 BV / h, the effluent from the alumina resin column was collected, concentrated to be alcohol-free, and a purified total glycoside concentrate was obtained, which was dried to obtain 0.78 kg of total glycosides extract from Momordica grosvenori;

[0057] (5) Preparative chromatographic separation: 200 g of Momordica grosvenori total glycosides extract was dissolved in pure water to a solid concentration of about 10%, and then filtered through a microporous membrane. The filtrate was passed through an MCI preparative chromatographic column (wherein the filler model was MCI GEL CHP20P, the filler dosage was 1 L, and the column pressure was 2.5 bar). The flow rate was controlled at 2 BV / h. The chromatographic column was eluted with 5 BV of pure water, 3 BV of 15% ethanol by volume, 3 BV of 25% ethanol by volume, 5 BV of 40% ethanol by volume, 3 BV of 55% ethanol by volume, 1 BV of 69% ethanol by volume, 1 BV of 80% ethanol by volume, and 5 BV of 95% ethanol by volume. The eluents from each section were collected and set aside.

[0058] (6) Concentration and drying: The eluate eluted with 25% ethanol was concentrated and dried to obtain fraction A, weighing 12.83 g; the eluate eluted with 40% ethanol was concentrated and dried to obtain fraction B, weighing 109.19 g; the eluate eluted with 69% ethanol was concentrated and dried to obtain fraction C, weighing 3.61 g.

[0059] Determination by high performance liquid chromatography external standard method showed that the content of 11-oxo-mogroside V in fraction A was 97.32 wt%, and the yield of 11-oxo-mogroside V was 91.89%; the content of mogroside V in fraction B was 98.28 wt%, and the yield of mogroside V was 92.64%; and the content of simenoside I in fraction C was 95.09 wt%, and the yield of simenoside I was 89.26%.

[0060] Comparative Example 1

[0061] Step (1) adopts traditional hot water countercurrent extraction: 100 kg of fresh Momordica grosvenori fruit is taken, crushed, countercurrent extracted with 500 L of hot water at a temperature of 95° C., coarsely filtered, and centrifuged to obtain Momordica grosvenori extract. The other steps are consistent with Example 1.

[0062] Determination by high performance liquid chromatography external standard method showed that the content of 11-oxo-mogroside V in fraction A was 97.28 wt%, and the yield of 11-oxo-mogroside V was 84.90%; the content of mogroside V in fraction B was 98.35 wt%, and the yield of mogroside V was 86.17%; the content of simenoside I in fraction C was 92.48%, and the yield of simenoside I was 53.13%.

[0063] Comparative Example 2

[0064] Other conditions were the same as those in Example 1, except that in step (1), the alkaline aqueous solution contained 0.4 wt% NaOH and 1 wt% astaxanthin, i.e., astaxanthin was used instead of the inclusion complex prepared in Preparation Example 1. Final products: Fraction A had an 11-oxo-mogroside V content of 97.14 wt% and an 11-oxo-mogroside V yield of 90.06%; Fraction B had a mogroside V content of 98.30 wt% and a mogroside V yield of 91.84%; and Fraction C had a simenoside I content of 94.32 wt% and a simenoside I yield of 73.26%.

[0065] Comparative Example 3

[0066] Other conditions were the same as those in Example 1, except that in step (1), the alkaline aqueous solution contained 0.4 wt% NaOH and 1 wt% vitamin C, i.e., vitamin C was used instead of the inclusion compound prepared in Preparation Example 1. Final products: Fraction A had a 11-oxo-mogroside V content of 97.21% by mass, and a 11-oxo-mogroside yield of 92.35%; Fraction B had a mogroside V content of 97.64% by mass, and a mogroside V yield of 92.70%; and Fraction C had a simenoside I content of 96.41% by mass, and a simenoside I yield of 78.37%.

[0067] Comparative Example 4

[0068] Other conditions were the same as those in Example 1, except that in step (4), the alumina resin column was not pretreated with acid water immersion and washing. Final products: Fraction A had a 11-oxo-mogroside V content of 97.14% by mass, and a 11-oxo-mogroside yield of 92.19%; Fraction B had a mogroside V content of 97.65% by mass, and a mogroside V yield of 92.57%; and Fraction C had a simenoside I content of 93.31% by mass, and a simenoside I yield of 75.49%.

Claims

1. A method for separating mogrosides by pulsed electric field ultrasonic extraction combined with preparative chromatography, characterized in that: The following steps are involved: (1) Pulsed electric field ultrasonic extraction: Take the monk fruit, crush it, add alkaline water, continuously pass it through the pulse electric field ultrasonic extraction equipment, coarsely filter, and centrifuge to obtain the monk fruit extract; the alkaline water contains a reducing agent @ cyclodextrin inclusion complex; the reducing agent @ cyclodextrin inclusion complex is obtained by embedding the reducing agent in a cyclodextrin substance, the reducing agent is selected from at least one of astaxanthin and β-carotene, and the cyclodextrin substance is selected from at least one of β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and sulfobutyl ether-β-cyclodextrin; the electric field strength of the pulse electric field is 0.5-10 kV / cm, the number of times is 10 to 30 times, the power of the ultrasound is 20-1000w, the ultrasonic frequency is 60-150kHz, the pulse electric field ultrasonic extraction time is 10-100min, and the extraction temperature is 5-30℃; (2) Membrane filtration: Filter the Momordica grosvenori extract through a ceramic membrane to obtain a ceramic membrane filtrate; (3) Enrichment of total glycosides from Momordica grosvenori: The filtrate from the ceramic membrane was passed through a macroporous adsorption resin column until the column was completely loaded. The filtrate was first washed with pure water and then analyzed with an alcohol-water solution. The ethanol solution was collected for later use. (4) Purification of total glycosides from Momordica grosvenori: the alcohol-water solution is passed through an alumina resin column, the effluent is concentrated to obtain a concentrate, which is dried to obtain a total glycosides from Momordica grosvenori extract; the alumina resin column is impregnated with acid water and washed; (5) Preparative chromatography separation: take a portion of the total glycosides extract of Momordica grosvenori, dissolve it in pure water, filter it through a microporous filter membrane, pass the filtrate through an MCI preparative chromatography column, perform gradient elution, collect the eluate in sections, and set aside; the filler types used in the MCI preparative chromatography are polystyrene / divinylbenzene type and acrylate type; the gradient elution is performed by eluting with an alcohol aqueous solution according to the increasing volume fraction of the alcohol, the alcohol being selected from methanol and / or ethanol, and the volume fraction of the alcohol increasing from 0% to 100%; (6) Concentration and drying: The eluates collected in sections are concentrated and dried to obtain different types of mogroside components.

2. The method according to claim 1, characterized in that In step (1), the reducing agent content in the reducing agent@cyclodextrin inclusion complex is 15-20 wt %, and the reducing agent@cyclodextrin inclusion complex in alkaline water is 1-5 wt %.

3. The method according to claim 1, characterized in that In step (1), the preparation method of the reducing agent @ cyclodextrin inclusion complex comprises the following steps: adding the reducing agent and the cyclodextrin substance in a molar ratio of 1:1-3, adding an alcohol aqueous solution, stirring under an inert atmosphere until the embedding reaches equilibrium, spinning and then freeze-drying to obtain the reducing agent @ cyclodextrin inclusion complex.

4. The method according to claim 3, characterized in that In step (1), the alcohol aqueous solution is an ethanol solution with a volume fraction of 70-95%, and the volume ratio of the alcohol aqueous solution to the reducing agent is 3-5L:1g.

5. The method according to claim 1, wherein In step (1), the alkali is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate or sodium bicarbonate, the mass percentage concentration of the alkaline water is 0.1-2wt%, and the amount of the alkaline water is 3-10 times the weight of the monk fruit raw material.

6. The method according to claim 5, characterized in that The mass percentage concentration of the alkaline water is 0.4-0.7wt%, and the amount of the alkaline water is 4-5 times the weight of the monk fruit raw material.

7. The method according to claim 1, characterized in that In step (1), the extraction temperature is 10-25°C.

8. The method according to claim 1, characterized in that In step (2), the material of the ceramic membrane is zirconium oxide, aluminum oxide, or titanium oxide; the pore size of the ceramic membrane is 0.1-1.2 μm; and the filtration pressure of the ceramic membrane is 0.6-1.0 MPa.

9. The method according to claim 1, characterized in that In step (3), the macroporous adsorption resin used in the macroporous adsorption resin column is a non-polar macroporous adsorption resin, a medium-polar macroporous adsorption resin or a weak-polar macroporous adsorption resin, or a combination of two or more of the above resins.

10. The method according to claim 9, characterized in that The macroporous adsorption resin is selected from at least one of D101, LX-T28, and AB-8, the volume ratio of the resin to the mass of the fresh monk fruit is 10-30 L:100 kg, the height-to-diameter ratio of the resin column is 3-8:1, and the column flow rate is 0.5-5 Bv / h; the pure water washing amount is 2-4 BV, and the alcohol-water solution analysis amount is 2-3 BV, and the alcohol-water solution is a 50-70% solution of methanol and / or ethanol.

11. The method according to claim 1, wherein In step (4), the type of alumina is neutral alumina, acidic alumina or basic alumina, and the amount of alumina used is 1-10 wt% of the weight of the monk fruit raw material.

12. The method according to claim 1, characterized in that In step (4), the alumina resin column is immersed in acid water with a pH of 3-4 for 10-60 minutes, and then washed with water until the effluent is close to neutral.

13. The method according to claim 12, characterized in that The acid water is at least one of dilute hydrochloric acid and dilute sulfuric acid.

14. The method according to claim 1, wherein In step (5), the particle size of the filler used in the MCI preparative chromatography is 4-300 μm.

15. The method according to claim 14, characterized in that In step (5), the gradient elution is to control the flow rate to 0.5-2BV / h, and elution is carried out in sequence with 3-5BV pure water, 3-5BV 10-20% alcohol aqueous solution by volume fraction, 3-5BV 20-30% alcohol aqueous solution, 3-5BV 30-40% alcohol aqueous solution, 3-5BV 40-60% alcohol aqueous solution, 1-2BV 60-70% alcohol aqueous solution, and 1-2BV>70% alcohol aqueous solution.

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