CN109021046B - A kind of method for simultaneously extracting quercitrin and kaempferol from stems and leaves of Luo Han Guo - Google Patents

Abstract

A method for simultaneously extracting quercetin and kaempferol from stems and leaves of Luo Han Guo, comprising the following steps: (1) drying fresh stems and leaves of Luo Han Guo, crushing, adding water, continuous countercurrent flash extraction, and centrifugal filtration; (2) Ultrafiltration, adsorption on a macroporous adsorption resin column, washing with water, discarding the washing liquid, eluting with organic solvent, collecting the eluate, washing with water, collecting the washing liquid, concentrating and drying; (3) Add to alcohol solution, heat to dissolve, and cool , let stand, filter, concentrate, and dry; (4) dissolve with mobile phase, filter, enter into preparative liquid chromatography column, eluate isocratically, collect eluate of target segment, concentrate, and dry to obtain quercetin and quercetin respectively. Kaempferol. The purity of the quercitrin product obtained by the method of the invention is ≥98.2%, the yield is ≥90.4%, the purity of the kaempferol product is ≥98.8%, and the yield is ≥91.3%; the method of the invention has simple and stable operation process, low energy consumption and low cost , which can realize continuous large-scale production.

Description

A kind of method for simultaneously extracting quercitrin and kaempferol from stems and leaves of Luo Han Guo

technical field

The invention relates to a method for extracting flavonoid glycosides, in particular to a method for simultaneously extracting quercitrin and kaempferol from stems and leaves of Luo Han Guo.

Background technique

Luo Han Guo is a perennial plant of the Cucurbitaceae family, mainly produced in Guilin, Guangxi. The whole body of Luo Han Guo is a treasure: the fresh fruit of Luo Han Guo is rich in glycosides, proteins and polysaccharides and other components, especially Mogroside as a natural sweetener, which has huge market and economic value; Starch has high nutritional value; Luo Han Guo seeds are rich in oil, and its main active ingredient is squalene, which has anti-cancer, anti-cancer and moisturizing effects; Luo Han Guo stems and leaves contain more flavonoid glycosides, mainly For quercetin and kaempferol, flavonoid glycosides have significant pharmacological and physiological activities, can dilate blood vessels, increase blood circulation, inhibit thrombosis, and have anti-inflammatory, anti-cancer, antibacterial, hypoglycemic and hypolipidemic effects. .

At present, mogrosides are extracted from the fruit of Luo Han Guo, which has been developed earlier and more mature. After the fresh fruit of Luo Han Guo is picked, its stems, leaves and rattan are usually thrown away, which not only pollutes the environment, but also causes huge waste of resources.

CN106668234A discloses a process for extracting and purifying total flavonoids of roses. The process flow is: solvent extraction→macroporous resin purification→polyamide resin purification→C18 preparation and purification. Although this process can obtain a product with a total flavonoid purity of ≥90%, the entire process is complex, consumes a lot of organic solvent, is difficult to recover, and has a low recovery rate of flavonoids.

CN102138958A discloses a process for extracting and purifying flavonoids of Luo Han Guo plants, the process flow is as follows: ethanol extraction→recovery solvent→macroporous resin impurity removal→elution→concentration and drying→ alcohol-dissolving impurity removal→concentration and drying. Although this process can obtain products with a purity of 50% or more of flavonoid glycosides, and the process is simple and stable, the purity of the purified flavonoids is not high, and although one-step alcohol dissolution is simple and effective, the recovery rate is low.

CN107698638A discloses an extraction and purification process, detection method and application of total flavonoids of wolfberry fruit. The process flow is: drying → crushing → extraction → extraction → macroporous resin purification → polyamide resin purification. Although the total flavonoids of Lycium barbarum obtained by this process are more than 40%, 80% ethanol extraction and petroleum ether extraction are used in the extraction process, which consumes a large amount of solvent, requires high production equipment, and has high cost.

CN102399252A discloses a method for preparing flavonoid glycoside monomer of Wangbuliu. The technological process is as follows: raw material extraction → macroporous resin enrichment → high-efficiency preparation liquid phase separation → macroporous resin re-enrichment → elution → concentration → drying. Although the flavonoid flavonoid monomer obtained by this process is greater than or equal to 99%, the process steps are complicated, and the macroporous resin is used twice for enrichment, which not only consumes a large amount of solvent, but also has a low recovery rate.

CN102050845A discloses a method for simultaneously preparing a chemical reference substance of verbascoside and formononetin. The process flow is: silica gel column chromatography enrichment→resin column impurity removal→reverse high performance liquid preparative chromatography refining→vacuum drying. Although this process can simultaneously prepare verbascoside and formononein with a purity of ≥98%, the method of enrichment by silica gel chromatography has a small processing capacity, a large consumption of organic solvents, and high requirements on the production environment. Large-scale production costs are high.

CN102503998A discloses a method for rapidly separating quercetin from Albizia Julibrissin flowers. The technological process is as follows: raw material extraction → macroporous resin enrichment → concentration and crystallization → high-efficiency preparation liquid phase separation → cooling and crystallization → drying. Although this process can obtain quercetin monomer, the recovery rate is low due to secondary crystallization.

CN104592327A discloses a preparation method of rutin and isoquercitrin in hops and the detection of their antiallergic and antioxidant activities. The technological process is: raw material extraction→extraction and impurity removal→macroporous resin enrichment→semi-preparation of high-efficiency liquid phase separation. Although this process can obtain rutin and isoquercitrin monomers, due to the need to sequentially use petroleum ether, ethyl acetate and n-butanol to extract and remove impurities, the process is complicated, the solvent consumption is large, and the cost is high.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the above-mentioned defects in the prior art, and provide a product with high purity, high yield, simple and stable operation process, low energy consumption and cost, and can realize continuous large-scale production. Simultaneous extraction of quercitrin and kaempferol from stems and leaves of Luo Han Guo.

The technical scheme adopted by the present invention to solve the technical problem is as follows: a method for simultaneously extracting quercetin and kaempferol from the stems and leaves of Luo Han Guo, comprising the following steps:

(1) Crushing and extraction: drying the fresh stems and leaves of Luo Han Guo, crushing, adding water, continuous countercurrent flash extraction, and centrifugal filtration to obtain an extract;

(2) Ultrafiltration and macroporous resin adsorption: the extract obtained in step (1) is subjected to ultrafiltration, adsorbed on a macroporous adsorption resin column, washed with water until the effluent is colorless, clear, and translucent, discarded the water washing liquid, and washed with an organic solvent. Take off, collect the eluent, wash with water until there is no organic solvent, collect the water wash, concentrate under reduced pressure, and vacuum dry to obtain crude flavonoid glycoside A;

(3) alcohol-dissolving impurity removal: adding the crude flavonoid glycoside A obtained in step (2) into the alcohol solution, heating and dissolving, cooling, standing, filtering, concentrating under reduced pressure, and vacuum drying to obtain crude flavonoid glycoside B;

(4) Preparative liquid chromatography separation: Dissolve the crude flavonoid glycoside B obtained in step (3) with the mobile phase, filter, enter the preparative liquid chromatography column for separation, and then perform isocratic elution with the mobile phase, and collect the target segment washes respectively. Deliquoring, concentration under reduced pressure, and vacuum drying to obtain quercitrin and kaempferol respectively.

Preferably, in step (1), in the fresh stems and leaves of Luo Han Guo, the mass content of quercetin is 0.04-0.08%, and the mass content of kaempferol is 0.5-1.0%.

Preferably, in step (1), the fresh stems and leaves of Luo Han Guo are crushed to 20-40 meshes.

Preferably, in step (1), the amount of water used is 4 to 20 times (more preferably 5 to 15 times) the mass of fresh stems and leaves of Luo Han Guo. Since the mangosteen flavonoid glycosides are easily soluble in water, especially hot water, using water as a solvent is not only low cost, but also environmentally safe.

Preferably, in step (1), the temperature of the continuous countercurrent flash extraction is 50-90°C, the number of extractions is ≥ 2 times, and the time of each extraction is 60-200s (more preferably 100-180s). Due to the special nature of easy oxidation of flavonoids, if the extraction temperature is too high or the extraction time is too long, the extraction recovery rate will decrease. Therefore, continuous countercurrent flash extraction can realize continuous and large-scale production, thereby shortening the extraction time. , while increasing the recovery rate.

Preferably, in step (1), the rotation speed of the centrifugation is 10000-80000 r/min (more preferably 40000-60000 r/min).

Preferably, in step (1), the centrifugal filtration method is first horizontal spiral centrifugal filtration, and then butterfly centrifugal filtration.

Preferably, in step (2), the ultrafiltration membrane used for ultrafiltration is a ceramic membrane, and the pore size of the ceramic membrane is 0.2-2.0 μm (more preferably 0.3-1.0 μm).

Preferably, in step (2), the flow rate of the upper column is 1.0-4.0 BV/h. Since flavonoid glycosides are highly polar substances, they are easily adsorbed by macroporous adsorption resins, so as to achieve the purpose of purification and separation.

Preferably, in step (2), the model of the macroporous adsorption resin is one or more of D-101 type, AB-8 type, DM-130 type, X-7 type or XDA-7 type, etc. .

In the method of the present invention, before the macroporous adsorption resin is used, soak it with 1-3 BV ethanol with a volume fraction of 90-99% for 20-30 hours, and then wash it with 90-99% ethanol by volume until the effluent is colorless and free of For odor, wash with water until there is no alcohol smell, then use 3~5BV sodium hydroxide solution with 4~6% mass concentration to alkali wash, then wash with water until neutral, and finally pickle with 3~5BV mass concentration 4~6% hydrochloric acid solution, Rinse with water until neutral, set aside.

Preferably, in step (2), the volume-to-mass ratio (L/kg) of the macroporous adsorption resin to the fresh stems and leaves of Luo Han Guo is 1:1-20 (more preferably 1:5-15).

Preferably, in step (2), the diameter-height ratio of the macroporous adsorption resin column is 1:2-8 (more preferably 1:4-6).

Preferably, in step (2), the flow rate of the water washing is 1.0-4.0 BV/h. The purpose of washing is to remove impurities with strong water solubility, such as monosaccharides, polysaccharides and pectin.

Preferably, in step (2), the amount of the organic solvent used is 1-4BV.

Preferably, in step (2), the flow rate of the organic solvent elution is 0.25-2.00 BV/h (more preferably 0.5-1.0 BV/h). The purpose of the organic solvent elution is to elute the flavonoid glycosides adsorbed by the resin.

Preferably, in step (2), the volume fraction of the organic solvent is 50-90%.

Preferably, in step (2), the organic solvent is food grade ethanol.

Preferably, in step (2), the temperature of the vacuum concentration is 40 to 70° C., the pressure is -0.1 to -0.07 MPa, and the concentration is concentrated to a solid content of 30 to 60%.

Preferably, in step (2), the temperature of the vacuum drying is 40-70° C., the pressure is -0.1-0.07MPa, and the time is 4-12h.

Preferably, in step (3), the mass-volume ratio (kg/L) of the crude flavonoid glycoside A to the alcohol solution is 1:8-20 (more preferably 1:9-15). Alcohol-soluble impurity removal mainly removes a class of impurities with good water solubility but poor alcohol solubility, such as proteins.

Preferably, in step (3), the heating temperature is 50-80°C.

Preferably, in step (3), the alcohol solution is food-grade anhydrous methanol or food-grade ethanol with a volume fraction of 90-99%.

Preferably, in step (3), the cooling is performed to room temperature. Plate cooling is preferably used.

Preferably, in step (3), the standing time is 2-4 hours. The purpose of standing is to make the impurity removal sufficient, the impurities are easy to be layered, and the subsequent filtration is convenient.

Preferably, in step (3), the temperature of the vacuum concentration is 40-70° C., the pressure is -0.1-0.07 MPa, and the concentration is concentrated to a solid content of 30-60%.

Preferably, in step (3), the temperature of the vacuum drying is 40-70° C., the pressure is -0.1-0.07MPa, and the time is 4-12h.

Preferably, in step (4), the mass-volume ratio (kg/L) of the crude flavonoid glycoside B to the mobile phase is 0.08-0.30:1. The effect of separation and purification is better under the stated ratio.

Preferably, in step (4), the mobile phase is an organic solvent-water solution with a volume ratio of 15-40:95-60. Under the stated ratio, the effect of separation and purification is better, and the recovery rate is higher.

Preferably, in step (4), the organic solvent is one or more of ethanol, methanol or acetonitrile. More preferably, ethanol. Using the solvent has better separation effect, is convenient for recovery, and has lower cost.

Preferably, in step (4), the filter membrane used for filtration is an organic phase filter membrane with a pore size of 0.20-0.45 μm. The main purpose of filtration is to remove a small amount of suspended impurities and prevent the preparation of liquid phase from blocking the column and affecting the separation effect.

Preferably, in step (4), the flow rate of the injection is 0.25-1.00 BV/h.

Preferably, in step (4), the prepared liquid chromatography column is an ODS column, and the filler is C-18 reverse-phase bonded silica gel. The inventors of the present invention have found that the use of the column type and packing material has more obvious separation and purification effect, higher separation degree and higher recovery rate.

Preferably, in step (4), the flow rate of the elution is 0.5-2.0 BV/h. The inventors have found that the separation effect is better at the flow rate, and the recovery rate of the target component is higher.

Preferably, in step (4), after the flow elution, according to the residence time of two independent peaks appearing on the signal display screen of the UV detector of the preparation liquid phase, the elution of the target products of quercitrin and kaempferol are collected respectively. liquid.

Preferably, in step (4), the temperature of the vacuum concentration is 40-70°C, the pressure is -0.1-0.07MPa, and the concentration is concentrated to a solid content of 30-60%.

Preferably, in step (4), the temperature of the vacuum drying is 40-70°C, the pressure is -0.1--0.07MPa, and the time is 4-12h.

In steps (3) and (4), the filtration is preferably plate-and-frame filtration or plate-and-frame filter press.

The beneficial effects of the inventive method are as follows:

(1) Both the quercitrin product and kaempferol product obtained by the method of the present invention are yellow, the purity of quercitrin in the quercitrin product is ≥98.2%, the yield is ≥90.4%, and the purity of kaempferol in the kaempferol product is ≥98.2%. 98.8%, yield ≥91.3%;

(2) Since the solvent extracted by the method of the present invention is water, compared with traditional organic solvent extraction, it is more environmentally friendly, safe and low in cost; using continuous countercurrent flash extraction, compared with traditional reflux extraction, both continuous and large-scale production can be ensured , and the extraction time is short and the recovery rate is high;

(3) The preparation liquid phase of the method of the present invention adopts an organic solvent-water mobile phase and is eluted isocratically. Compared with other organic mixed solvents, the cost is lower, the operation is simple, and the mobile phase can be recycled and reused, which is suitable for large-scale industrialization Production;

(4) The method of the present invention adopts the combination of macroporous resin adsorption purification → alcohol dissolution and impurity removal → preparation liquid chromatography separation, which not only has a simple and stable process, but also ensures the high purity of the product.

Detailed ways

The present invention will be further described below in conjunction with the examples.

The stems and leaves of Luo Han Guo used in the embodiment of the present invention were purchased from Guilin, Guangxi, and detected by HPLC, the mass content of quercetin was 0.06%, and the mass content of kaempferol was 0.73%; The 0.5 μm ceramic membranes were purchased from Jiangsu Jiu’anyuan Environmental Protection Technology Co., Ltd.; the D-101 and AB-8 macroporous adsorption resins used in the examples of the present invention were purchased from Xi’an Lanxiao Technology New Materials Co., Ltd.; The X-5 macroporous adsorption resin used in the embodiment of the present invention was purchased from Tianjin Nankai Hecheng Technology Co., Ltd.; the organic phase filter membranes with pore diameters of 0.22 μm and 0.45 μm used in the embodiment of the present invention were purchased from Shanghai Xinya Purification device factory; the ODS column and C-18 reverse-phase bonded silica gel used in the embodiment of the present invention were purchased from Jiangsu Hanbang Technology Co., Ltd.; the raw materials and chemical reagents used in the embodiment of the present invention, unless otherwise specified, were Obtained by conventional commercial means.

Reference Example 1

Before using the macroporous adsorption resin of the embodiment of the present invention, soak it with 2BV and 95% ethanol for 24 hours, then wash it with 95% ethanol until the effluent is colorless and has no peculiar smell, and then wash with water until there is no alcohol smell. Alkaline wash with 5% sodium hydroxide solution of 4BV mass concentration, then wash with water to neutrality, finally pickle with 4BV mass concentration of 5% hydrochloric acid solution, then wash with water to neutrality, standby.

Example 1

(1) Crushing and extraction: Sun-dried 10t of fresh stems and leaves of Luo Han Guo, crushed to 20 mesh, added 100t of water, at 60°C, continuously countercurrent flash extraction for 2 times, each time for 150s, and then at 60000r/min, first Centrifugal filtration with a horizontal screw centrifuge, and then centrifugal filtration with a butterfly centrifuge to obtain 95t of extract;

(2) Ultrafiltration and macroporous resin adsorption: The 95t extract obtained in step (1) was subjected to ultrafiltration with a 0.5 μm ceramic membrane, and the ultrafiltration permeate was subjected to AB-8 macroporous adsorption at a flow rate of 3BV/h. Resin column (the volume of AB-8 macroporous adsorption resin is 1000L, the ratio of diameter to height is 1:4) is adsorbed, and then washed with water at a flow rate of 2BV/h until the effluent is colorless, clear and translucent. 2BV, food grade ethanol with a volume fraction of 80%, elute at a flow rate of 0.5BV/h, collect the eluate, wash with water until there is no ethanol, collect the water wash, and at 50 ℃, under -0.1MPa, the collected eluate The liquid and the washing liquid were concentrated under reduced pressure to a solid content of 45%, and vacuum-dried at 40°C under -0.1MPa for 6h to obtain 131.29kg of crude flavonoid glycoside A;

(3) Alcohol-dissolved impurity removal: 131.29 kg of crude flavonoid glycoside A obtained in step (2) was added to 1500 L of food-grade ethanol solution with a volume fraction of 95%, heated and dissolved at 60 °C, cooled to room temperature by plate type, and stood for 2 h , plate and frame filtration, the filtrate was concentrated under reduced pressure to a solid content of 50% at 40 ° C under -0.1 MPa, and vacuum-dried at 40 ° C under -0.1 MPa for 8 hours to obtain 93.23 kg of crude flavonoid glycosides;

(4) Preparative liquid chromatography separation: Dissolve 93.23 kg of crude flavonoid glycoside B obtained in step (3) with 1100L mobile phase ethanol-water solution (volume ratio of 30:70), and use the organic phase filter plate with a pore size of 0.22 μm for the dissolved solution Frame pressure filtration, then, at a flow rate of 0.25BV/h, enter a preparative liquid chromatography column (ODS column, the filler is C-18 reversed-phase bonded silica gel) for separation, and then use mobile phase ethanol-water solution (volume ratio of 30 : 70), carry out isocratic elution at a flow rate of 1.0BV/h, and collect 10-25BV quercetin target segment and The eluate of the target segment of 30-40BV kaempferol was concentrated under reduced pressure to a solid content of 55% at 40°C and -0.1MPa, and vacuum-dried at 40°C and -0.1MPa for 6 hours to obtain quercitrin 5.58. kg and kaempferol 67.89kg.

Both the quercitrin product and the kaempferol product obtained in the embodiment of the present invention are yellow, and the HPLC detection shows that the purity of quercitrin in the obtained quercitrin product is 98.2%, the yield is 91.3%, and the purity of kaempferol is 99.1% %, the yield is 92.2%.

Example 2

(1) Crushing and extraction: Sun-dried 10t of fresh stems and leaves of Luo Han Guo, crushed to 40 mesh, added 150t of water, at 80°C, continuously countercurrent flash extraction for 3 times, 100s each time, and then at 40000r/min, first Centrifugal filtration with a horizontal screw centrifuge, and then centrifugal filtration with a butterfly centrifuge to obtain an extract of 145t;

(2) Ultrafiltration and macroporous resin adsorption: The 145t extract obtained in step (1) was subjected to ultrafiltration with a 0.3 μm ceramic membrane, and the ultrafiltration permeate was subjected to X-5 type macroporous adsorption at a flow rate of 4BV/h. Resin column (the volume of X-5 macroporous adsorption resin is 1500L, the ratio of diameter to height is 1:6) for adsorption, and then at a flow rate of 4BV/h, wash with water until the effluent is colorless, clear, and translucent. 4BV, food grade ethanol with a volume fraction of 60%, elute at a flow rate of 1.0BV/h, collect the eluate, wash with water until there is no ethanol, collect the water wash, and at 70 ℃, under -0.08MPa, the collected eluate The liquid and the washing liquid were concentrated under reduced pressure to a solid content of 50%, and vacuum-dried at 60°C under -0.08MPa for 8h to obtain 125.6kg of crude flavonoid glycoside A;

(3) Alcohol-dissolved impurity removal: Add 125.6 kg of crude flavonoid glycoside A obtained in step (2) into 1200 L of food-grade anhydrous methanol solution, heat and dissolve at 80°C, plate-type cooling to room temperature, stand for 4 hours, plate frame Filtration, the filtrate was concentrated under reduced pressure at 60°C under -0.08MPa to a solid content of 40%, and vacuum-dried at 60°C under -0.08MPa for 6 hours to obtain 91.8kg of crude flavonoid glycosides B;

(4) Preparative liquid chromatography separation: 91.8kg of crude flavonoid glycoside B obtained in step (3) was dissolved with 800L mobile phase methanol-water solution (volume ratio of 20:80), and the dissolved solution was filtered with an organic phase filter plate with a pore size of 0.45 μm Frame pressure filtration, then, at a flow rate of 0.75BV/h, enter a preparative liquid chromatography column (ODS column, the packing is C-18 reversed-phase bonded silica gel) for separation, and then use mobile phase methanol-water solution (volume ratio of 20 : 80), carry out isocratic elution at a flow rate of 2.0BV/h, and collect 10-25BV quercetin target segment and The eluate of 30-40BV kaempferol target segment was concentrated under reduced pressure to a solid content of 60% at 60°C under -0.08MPa, and vacuum-dried at 60°C under -0.08MPa for 10h to obtain quercitrin 5.5. kg and kaempferol 66.8kg.

Both the quercitrin product and the kaempferol product obtained in the embodiment of the present invention are yellow, and as detected by HPLC, the purity of quercitrin in the obtained quercitrin product is 98.6%, the yield is 90.4%, and the purity of kaempferol is 99.8% %, the yield is 91.3%.

Example 3

(1) Crushing and extraction: Sun-dried 10t of fresh stems and leaves of Luo Han Guo, crushed to 30 mesh, added 120t of water, at 70°C, continuously countercurrent flash extraction twice, 120s each time, and then at 50000r/min, first Centrifugal filtration with a horizontal screw centrifuge, and then centrifugal filtration with a butterfly centrifuge to obtain 95t of extract;

(2) Ultrafiltration and macroporous resin adsorption: The 95t extract obtained in step (1) was subjected to ultrafiltration with a 0.5 μm ceramic membrane, and the ultrafiltration permeate was subjected to D-101 macroporous adsorption at a flow rate of 2BV/h. Resin column (the volume of D-101 macroporous adsorption resin is 1200L, the ratio of diameter to height is 1:5) for adsorption, and then washed with water at a flow rate of 3BV/h until the effluent is colorless, clear and translucent. 3BV, food grade ethanol with a volume fraction of 70%, elute at a flow rate of 0.75BV/h, collect the eluate, wash with water until there is no ethanol, collect the water wash, and at 60 ° C, under -0.09MPa, the collected eluate The liquid and the washing liquid were concentrated under reduced pressure to a solid content of 48%, and vacuum-dried at 50°C under -0.09MPa for 11h to obtain 140.3kg of crude flavonoid glycoside A;

(3) Alcohol-dissolved impurity removal: 140.3 kg of crude flavonoid glycoside A obtained in step (2) was added to 1500 L of food-grade ethanol solution with a volume fraction of 92%, heated and dissolved at 70 °C, cooled to room temperature by plate type, and allowed to stand for 3 hours. , plate and frame filtration, the filtrate was concentrated under reduced pressure to a solid content of 47% at 50°C under -0.09MPa, and vacuum-dried at 50°C under -0.09MPa for 10h to obtain 89.5kg of crude flavonoid glycosides B;

(4) Preparative liquid chromatography separation: 89.5kg of crude flavonoid glycoside B obtained in step (3) was dissolved in 1000L mobile phase acetonitrile-water solution (volume ratio of 26:74), and the dissolved solution was filtered with an organic phase filter plate with a pore size of 0.22 μm. Frame pressure filtration, then, enter a preparative liquid chromatography column (ODS column, the filler is C-18 reversed-phase bonded silica gel) at a flow rate of 0.5BV/h for separation, and then use mobile phase acetonitrile-water solution (volume ratio is 26: 74), carry out isocratic elution at a flow rate of 1.5BV/h, according to the residence time of two independent peaks appearing on the signal display of the preparative liquid phase UV detector, collect the target segment of 5-15BV quercitrin and 20 The eluate of the target segment of ~30BV kaempferol was concentrated under reduced pressure to a solid content of 56% at 50°C and -0.09MPa, and vacuum-dried at 50°C and -0.09MPa for 8 hours to obtain 5.75kg of quercitrin. And kaempferol 70.5kg.

Both the quercitrin product and the kaempferol product obtained in the embodiment of the present invention are yellow, and the HPLC detection shows that the purity of quercitrin in the obtained quercitrin product is 98.4%, the yield is 94.3%, and the purity of kaempferol is 98.8% %, the yield is 95.4%.

Claims (12)

1. A method for simultaneously extracting quercetin and kaempferitrin from stem and leaves of momordica grosvenori is characterized by comprising the following steps:

(1) crushing and extracting: sun drying fresh stem and leaf of fructus Siraitiae Grosvenorii, crushing, adding water, performing continuous countercurrent flash extraction, centrifuging, and filtering to obtain extractive solution;

(2) ultrafiltration and macroporous resin adsorption: carrying out ultrafiltration on the extracting solution obtained in the step (1), adsorbing the extracting solution by using a macroporous adsorption resin column, washing with water until an effluent liquid is colorless, clear and transparent, discarding a washing liquid, eluting with an organic solvent, collecting an eluent, washing with water until no organic solvent exists, collecting the washing liquid, concentrating under reduced pressure, and drying in vacuum to obtain a crude product A of the flavonoid glycoside;

(3) alcohol dissolving and impurity removing: adding the crude flavonoid glycoside A obtained in the step (2) into an alcohol solution, heating for dissolving, cooling, standing, filtering, concentrating under reduced pressure, and drying in vacuum to obtain crude flavonoid glycoside B;

(4) preparing liquid phase chromatographic separation: dissolving the flavonoid glycoside crude product B obtained in the step (3) by using a mobile phase, filtering, carrying out separation by using a preparative liquid chromatography column, carrying out isocratic elution by using the mobile phase, respectively collecting target section eluent, carrying out reduced pressure concentration, and carrying out vacuum drying to respectively obtain quercetin glycoside and kaempferitrin;

in the step (1), crushing fresh stems and leaves of momordica grosvenori to 20-40 meshes; the amount of the water is 4-20 times of the mass of the fresh stems and leaves of the momordica grosvenori; the temperature of the continuous countercurrent flash extraction is 50-90 ℃, the extraction frequency is more than or equal to 2 times, and the extraction time is 60-200 s each time;

in the step (2), the ultrafiltration membrane used for ultrafiltration is a ceramic membrane, and the aperture of the ceramic membrane is 0.2-2.0 μm;

in the step (2), the model of the macroporous adsorption resin is one or more of D-101 type, AB-8 type, DM-130 type, X-7 type or XDA-7 type; the volume-mass ratio of the macroporous adsorption resin to fresh stems and leaves of the momordica grosvenori is 1: 1-20;

in the step (2), the flow rate of the water washing is 1.0-4.0 BV/h; the dosage of the organic solvent is 1-4 BV; the flow rate of the organic solvent elution is 0.25-2.00 BV/h; the volume fraction of the organic solvent is 50-90%; the organic solvent is food grade ethanol;

in the step (3), the mass-to-volume ratio of the crude flavonoid glycoside A to the alcoholic solution is 1: 8-20; the heating temperature is 50-80 ℃; the alcoholic solution is food-grade anhydrous methanol or food-grade ethanol with the volume fraction of 90-99%; the standing time is 2-4 h;

in the step (4), the mass-to-volume ratio of the crude flavonoid glycoside B to the mobile phase is 0.08-0.30: 1; the mobile phase is an organic solvent-water solution with a volume ratio of 15-40: 95-60; the organic solvent is one or more of ethanol, methanol or acetonitrile; the filter membrane used for filtering is an organic phase filter membrane, and the aperture is 0.20-0.45 mu m; the flow rate of the sample introduction is 0.25-1.00 BV/h; the preparative liquid chromatography column is an ODS column, and the filler is C-18 reverse phase bonded silica gel.

2. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 1, wherein: in the step (1), the rotating speed of the centrifugation is 10000-80000 r/min; the centrifugal filtration mode is horizontal spiral centrifugal filtration and then butterfly centrifugal filtration.

3. The method for simultaneously extracting quercetin and kaempferitrin from momordica grosvenori stem and leaf according to claim 1 or 2, wherein: in the step (2), the flow rate of the upper column is 1.0-4.0 BV/h; the diameter-height ratio of the macroporous adsorption resin column is 1: 2-8.

4. The method for simultaneously extracting quercetin and kaempferitrin from momordica grosvenori stem and leaf according to claim 1 or 2, wherein: in the step (2), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.

5. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 3, wherein: in the step (2), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.

6. The method for simultaneously extracting quercetin and kaempferitrin from momordica grosvenori stem and leaf according to claim 1 or 2, wherein: in the step (3), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.

7. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 3, wherein: in the step (3), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.

8. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 4, wherein: in the step (3), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.

9. The method for simultaneously extracting quercetin and kaempferitrin from momordica grosvenori stem and leaf according to claim 1 or 2, wherein: in the step (4), the flow rate of elution is 0.5-2.0 BV/h; the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.

10. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 3, wherein: in the step (4), the flow rate of elution is 0.5-2.0 BV/h; the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.

11. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 4, wherein: in the step (4), the flow rate of elution is 0.5-2.0 BV/h; the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.

12. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 6, wherein: in the step (4), the flow rate of elution is 0.5-2.0 BV/h; the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.