CN110818668B - Method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems - Google Patents
Method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems Download PDFInfo
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- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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Abstract
The invention belongs to the technical field of natural products, and discloses a method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems, which comprises the following steps: preparing a total flavone extracting solution; enriching total flavonoids; separating and purifying by high-speed counter-current chromatography; separating and purifying by preparative chromatography; preparing quercetin, luteolin, apigenin, tetrahydroxyflavone and 7-methylether baicalein with the purity of more than 98%. The method can separate and purify five flavonoid glycoside compound monomers from the gynura procumbens at one time, and effectively improves the extraction efficiency, the separation purity and the yield of the flavonoid glycoside compounds in the gynura procumbens.
Description
Technical Field
The invention belongs to the technical field of natural products, relates to the technical field of extraction and separation of active ingredients of natural products, and particularly relates to a method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems.
Background
Gynura procumbens (Lour.) Merr. is a plant of Panax of Compositae, also called Panax notoginseng, Sambucus nigra, Angelica keiskei, is pungent, slightly bitter and cool in nature, is a perennial herbal and edible plant, and is widely distributed in China, Malaysia, Thailand, Indonesia, Korea, Philippine and other countries. Gynura procumbens has wide pharmacological effects, in the first half of 2009, toxicology inspection, hygiene inspection and inspection of various components are completed by relevant units such as the Chinese disease prevention and control center, the national food quality supervision and inspection center, the Chinese food fermentation industry research institute and the like, and the conclusion is that: no toxicity and no teratogenic influence; the effective components of the medicine have the effects of clearing and activating the channels and collaterals, diminishing inflammation and relieving cough, dissipating blood stasis and reducing swelling, promoting blood circulation and promoting tissue regeneration, treating traumatic injury, bronchopneumonia, phthisis and the like; can delay aging, activate immunocyte, improve organism immunity, enhance metabolism and improve dysmnesia; has the obvious effects of reducing blood pressure, blood fat and blood sugar, resisting oxidation and ulcer, preventing chronic nephropathy and inhibiting hepatitis B; has certain curative effect on preventing and treating cardiovascular and cerebrovascular diseases, diabetes and the like; it also has antiviral, antibacterial, and bone marrow cancer and shiga-like toxin cell inhibiting effects. On the other hand, gynura procumbens is a unique plant which can be used as both medicine and food and has extremely good nutritional value, so that the gynura procumbens is widely applied to the fields of food and medicine industry, daily chemical industry and the like and is a plant which can be used as both medicine and food and has extremely high potential and high economic value. However, so far, the research on the effective components and the action mechanism thereof is not deep enough.
Research shows that the gynura procumbens stem and branch mainly comprise the following active components: flavonoids, triterpenoid glycosides, sterol compounds, and phenolic acid compounds such as vanillin, syringaldehyde, p-hydroxybenzoic acid, p-hydroxycinnamic acid, protocatechuic acid, etc. In the prior art, the flavonoids extracted from gynura procumbens stems are all total flavonoids, which is shown in the following steps: fan welcome, etc., research on the extraction process of total flavonoids in gynura procumbens stems, guidance and periodical of food safety, stage 34 in 2015; zhengguo and the like, the research on the process for extracting the total flavonoids in the gynura procumbens stem by microwave assistance, Jiangsu agricultural science, 2015, 08; only a few studies have analyzed flavonoids from gynura procumbens whole plant, see: the chemical composition research (I) of gynura procumbens, Chinese herbal medicines, the 2016 period of 11, is carried out by consolidating the general information of the plant, separating and purifying by methods such as repeated silica gel column chromatography, Sephadex LH-20 gel column chromatography, medium-pressure column chromatography, semi-preparative high performance liquid chromatography and the like, and identifying the structure of a compound by NMR, MS and the like to obtain 16 compounds from a 70% ethanol extract of gynura procumbens. However, no relevant report exists for separating flavonoid compounds from gynura procumbens stems.
In the prior art, the flavonoid compounds are mostly extracted and separated by traditional methods such as column chromatography separation and recrystallization, and the methods need repeated column chromatography separation, have complex operation, limited conditions, long time consumption, large solvent consumption, large sample loss and poor effect.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a simple, efficient and rapid method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems, which can separate and purify a plurality of flavonoid glycoside compound monomers from gynura procumbens stems.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems comprises the following steps:
step 1, preparing a total flavone extracting solution: drying and crushing gynura procumbens stems as raw materials in the shade, and performing ultrasonic extraction by using ethanol with volume concentration of 60-80% as a solvent, wherein the material-liquid ratio g/mL of gynura procumbens stem powder to ethanol solution is 1: 10-1: 20, extracting for 1-3 times, extracting for 1-3 hours, performing ultrasonic power of 350-500W, filtering, combining filtrates, and performing vacuum concentration under reduced pressure until no solvent exists to obtain a total flavone extract;
step 2, enriching total flavonoids: adsorbing and enriching the total flavone extract obtained in the step 1 by using macroporous adsorption resin, eluting by using water with the volume of 8 times of the column volume, sequentially eluting by using ethanol with the volume concentration of 20%, 40%, 60%, 80% and 90% in 6 times of the column volume respectively, collecting the eluent with the volume concentration of 40%, merging, decompressing and recovering the solvent, and freeze-drying to obtain crude flavone;
and 3, high-speed counter-current chromatography separation and purification: the first two-phase solvent system is obtained by uniformly mixing ethyl acetate, n-butyl alcohol and water according to the volume ratio of 1:1: 1-1: 4:8, standing and layering, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, the stationary phase is firstly injected into a high-speed counter-current chromatograph, the mobile phase is then injected after the whole column is filled with the stationary phase, and the rotating speed of the counter-current chromatograph is adjusted to 700-900 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the crude flavone obtained in the step (2) in the two-phase solvent system, and carrying out high-speed countercurrent chromatographic separation, wherein the sample injection concentration is 10-50 mg/mL, and the sample injection volume is 20-50 mL; during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 200-350 nm, respectively collecting corresponding peak components according to chromatographic peaks, concentrating and drying under reduced pressure, and sequentially obtaining fractions I, II, III, IV and V; the fraction I contains quercetin; the fraction II contains luteolin; the fraction III contains apigenin; the fraction IV contains tetrahydroxy flavone; the fraction V contains 7-methyl ether baicalein;
and 4, separating and purifying by preparative chromatography: the second two-phase solvent system is obtained by uniformly mixing n-heptane, ethyl acetate, methanol and water according to the volume ratio of 1:1:1 to 1:4:6:8, standing and layering, wherein the upper phase is a stationary phase, and the lower phase is a mobile phase; injecting the stationary phase into a spiral tube for preparing the chromatogram, starting a host machine after the spiral tube is completely filled with the stationary phase, slowly adjusting the rotating speed of the spiral tube to 750-950 r/min, simultaneously pumping the mobile phase, when the mobile phase flows out from the tail end of the spiral tube, enabling the system to reach dynamic balance, respectively injecting the different fractions collected in the step 3 into the spiral tube through a sample injection valve, performing preparative chromatogram separation, detecting by using an ultraviolet detector with the wavelength of 200-300 nm, and respectively collecting the highest peak fractions of the different fractions in the step 3; then, the peak fractions are respectively subjected to reduced pressure concentration and freeze drying to obtain quercetin, luteolin, apigenin, tetrahydroxyflavone and 7-methylether baicalein with the purity of more than 98%.
Further, in the above step 1, the volume concentration of the ethanol solution is preferably 75%.
Further, in the step 1, the ratio g/mL of the gynura procumbens stem powder to the ethanol solution is preferably 1: 15.
Further, in the step 2, the macroporous adsorption resin is HPD-300, HPD-400, HPD-600, AB-8 or D-101 macroporous resin.
Further, in the step 2, the collected eluent with the volume concentration of 40% is the eluent containing the flavone component, and is measured by a sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method.
Furthermore, in the step 3, the volume ratio of ethyl acetate, n-butanol and water in the two-phase solvent system is 1 (2-4) to (4-8).
Further, in the step 3, when the high-speed counter-current chromatography is performed, the flow rate of the stationary phase is 10 to 20mL/min, and the flow rate of the mobile phase is 1 to 3 mL/min.
Further, in the step 3, when the high-speed countercurrent chromatography is performed, the rotation speed of the separation column is 800rpm, the flow rate of the mobile phase is 3mL/min, the wavelength of the ultraviolet detector is 255nm, the sample injection concentration is 30mg/mL, and the sample injection volume is 30 mL.
Furthermore, in the step 3, the peak emergence time of the fractions I, II, III, IV and V is 25-35 min, 40-60 min, 140-155 min, 155-165 min and 195-215 min in sequence.
Further, in the step 4, the volume ratio of n-heptane, ethyl acetate, methanol and water in the solvent system is 1 (2-4) to (3-6): (4-8).
Due to the adoption of the technical scheme, the invention has the following advantages:
the method for separating and purifying the high-purity flavonoid glycoside compounds from the gynura procumbens stems has the advantages of wide raw material sources, easiness in obtaining, simple process method, mild extraction conditions, simplicity and convenience in operation, low comprehensive cost, economy and environmental friendliness, can separate and purify five flavonoid glycoside compound monomers from the gynura procumbens stems at one time, effectively improves the extraction efficiency, the separation purity and the yield of the flavonoid glycoside compounds in the gynura procumbens stems, and is large in separation amount, high in product purity and small in sample loss.
Drawings
FIG. 1 is a chromatogram of the separation and purification of gynura procumbens stem flavonoid glycoside compounds by high-speed counter-current chromatography using a first two-phase solvent system according to the method steps of the invention in example 1;
in fig. 1, i: quercetin; II: luteolin; III: apigenin; IV: tetrahydroxy flavone; v: 7-methyl ether baicalein.
Detailed Description
The present invention will be further described in detail with reference to the following examples; however, the following examples are merely illustrative, and the present invention is not limited to these examples.
Example 1
A method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems comprises the following specific steps:
(1) 1.0kg of naturally dried gynura procumbens stems are taken, crushed and subjected to ultrasonic extraction by using ethanol with the volume concentration of 75% as a solvent, the material-liquid ratio of gynura procumbens stem powder to ethanol solution is 1g:15mL, the extraction time is 2h, the ultrasonic power is 350W, and then the filtration is carried out, and the filter residues are repeatedly treated for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain total flavone extractive solution;
(2) adsorbing and enriching the obtained total flavone extract by HPD-400 macroporous adsorption resin, eluting by using deionized water with 8 times of column volume, sequentially eluting by using ethanol with the volume concentration of 20%, 40%, 60%, 80% and 90% and 6 times of column volume, respectively, collecting the eluent with the volume concentration of 40%, merging, decompressing and recovering the solvent, freezing and drying to obtain crude flavone, and storing in a refrigerator at 4 ℃ for later use; the method for determining flavone contained in the eluent with volume concentration of 40% is sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method;
(3) the first two-phase solvent system is obtained by uniformly mixing ethyl acetate, n-butyl alcohol and water according to the volume ratio of 1:3:6, standing and layering, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonic degassing is performed, the stationary phase is firstly injected into a high-speed counter-current chromatograph at the flow rate of 10mL/min, after the whole column is filled with the stationary phase, the mobile phase is then injected at the flow rate of 3mL/min, and the rotating speed of the counter-current chromatograph is adjusted to 800 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the crude flavone obtained in the step (2) in the two-phase solvent system to prepare a separation sample, and performing high-speed countercurrent chromatographic separation on the crude flavone, wherein the concentration of the sample entering a high-speed countercurrent chromatograph is sample introduction concentration, the sample introduction concentration is 30mg/mL, and the sample introduction volume is 30 mL; during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 255nm, respectively collecting corresponding peak components according to chromatographic peaks, concentrating and drying under reduced pressure, and sequentially obtaining fractions I, II, III, IV and V according to peak emergence time of 25-35 min, 40-60 min, 140-155 min, 155-165 min and 195-215 min, wherein the fractions I, II, III, IV and V are shown in figure 1;
(4) the second two-phase solvent system is obtained by uniformly mixing n-heptane, ethyl acetate, methanol and water according to the volume ratio of 1:2:4:3, standing and layering, wherein the upper phase is a stationary phase, and the lower phase is a mobile phase; injecting the stationary phase into a spiral tube for preparing the chromatogram at the flow rate of 15mL/min, starting a host machine after the spiral tube is completely filled with the stationary phase, slowly adjusting the rotating speed of the spiral tube to 800r/min, simultaneously pumping the mobile phase at the flow rate of 4mL/min, enabling the system to reach dynamic balance when the mobile phase flows out of the tail end of the spiral tube, respectively injecting the different fractions collected in the step 3 into the spiral tube through a sample injection valve, performing preparative chromatographic separation, detecting by an ultraviolet detector with the wavelength of 255nm, and respectively collecting the highest peak fractions of the different fractions in the step 3;
(5) identifying the collected fractions by 1H-NMR and 13C-NMR respectively to obtain monomer compounds of quercetin 8mg, luteolin 35mg, apigenin 12mg, tetrahydroxyflavone 6mg and 7-methylether baicalein 43 mg; detecting each collected fraction by HPLC, calculating by a chromatographic peak area normalization method, measuring the area of each impurity peak and the total chromatographic peak area, calculating the percentage of each impurity peak area and the sum of the impurity peak areas in the total peak area, wherein the sample purity is the percentage of the total peak area without the impurity peak sum; the peak area of the impurity peak is 3 percent and the total chromatographic peak area is 1 percent, the purity of the quercetin is 98.6 percent, the purity of the luteolin is 99.1 percent g, the purity of the apigenin is 98.9 percent, the purity of the tetrahydroxyflavone is 99.2 percent and the purity of the 7-methyl ether baicalein is 98.8 percent.
Example 2
A method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems comprises the following specific steps:
(1) 1.5kg of naturally dried gynura procumbens stems are taken, crushed and subjected to ultrasonic extraction by using ethanol with the volume concentration of 70% as a solvent, the material-liquid ratio of gynura procumbens stem powder to ethanol solution is 1g to 20mL, the extraction time is 2h, the ultrasonic power is 350W, and then the filtration is carried out, and the filter residues are repeatedly treated for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain total flavone extractive solution;
(2) adsorbing and enriching the obtained total flavone extract by AB-8 macroporous adsorption resin, eluting with deionized water with 8 times of column volume, sequentially eluting with ethanol with volume concentration of 20%, 40%, 60%, 80% and 90% and 6 times of column volume respectively, collecting the eluent with volume concentration of 40%, combining, decompressing and recovering the solvent, freeze-drying to obtain crude flavone, and storing in a refrigerator at 4 ℃ for later use; the method for determining flavone contained in the eluent with volume concentration of 40% is sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method;
(3) the first two-phase solvent system is obtained by uniformly mixing ethyl acetate, n-butyl alcohol and water according to the volume ratio of 1:4:7, standing and layering, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonic degassing is carried out, the stationary phase is firstly injected into a high-speed counter-current chromatograph at the flow rate of 15mL/min, after the whole column is filled with the stationary phase, the mobile phase is then injected at the flow rate of 3mL/min, and the rotating speed of the counter-current chromatograph is adjusted to 800 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the crude flavone obtained in the step (2) in the two-phase solvent system to prepare a separation sample, and performing high-speed countercurrent chromatographic separation on the crude flavone, wherein the concentration of the sample entering a high-speed countercurrent chromatograph is sample introduction concentration, the sample introduction concentration is 30mg/mL, and the sample introduction volume is 30 mL; during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 254nm, respectively collecting corresponding peak components according to chromatographic peaks, concentrating and drying under reduced pressure, and sequentially obtaining fractions I, II, III, IV and V according to the peak emergence time of 25-35 min, 40-60 min, 140-155 min, 155-165 min and 195-215 min;
(4) the second two-phase solvent system is obtained by uniformly mixing n-heptane, ethyl acetate, methanol and water according to the volume ratio of 1:2:3:6, standing and layering, wherein the upper phase is a stationary phase, and the lower phase is a mobile phase; injecting the stationary phase into a spiral tube for preparing the chromatogram at the flow rate of 15mL/min, starting a host machine after the spiral tube is completely filled with the stationary phase, slowly adjusting the rotating speed of the spiral tube to 800r/min, simultaneously pumping the mobile phase at the flow rate of 4mL/min, when the mobile phase flows out of the tail end of the spiral tube, enabling the system to reach dynamic balance, respectively injecting the different fractions collected in the step 3 into the spiral tube through a sample injection valve, performing preparative chromatographic separation, detecting by an ultraviolet detector with the wavelength of 254nm, and respectively collecting the highest peak fractions of the different fractions in the step 3;
(5) and detecting the purity of the monomer compound by using high performance liquid chromatography in the same manner as in the step (5) in the embodiment 1.
Example 3
A method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems comprises the following specific steps:
(1) taking 2.0kg of naturally dried gynura procumbens stems, crushing, carrying out ultrasonic extraction by taking ethanol with volume concentration of 80% as a solvent, wherein the material-liquid ratio of gynura procumbens stem powder to ethanol solution is 1g to 10mL, the extraction time is 3h, the ultrasonic power is 350W, then filtering, and repeatedly treating filter residues for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain total flavone extractive solution;
(2) adsorbing and enriching the obtained total flavone extract by using D-101 macroporous adsorption resin, eluting by using deionized water with 8 times of column volume, sequentially eluting by using ethanol with the volume concentration of 20%, 40%, 60%, 80% and 90% and 6 times of column volume, respectively, collecting eluent with the volume concentration of 40%, merging, decompressing and recovering the solvent, freezing and drying to obtain crude flavone, and storing in a refrigerator at 4 ℃ for later use; the method for determining flavone contained in the eluent with volume concentration of 40% is sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method;
(3) the first two-phase solvent system is obtained by uniformly mixing ethyl acetate, n-butyl alcohol and water according to the volume ratio of 1:1:1, standing and layering, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonic degassing is carried out, the stationary phase is firstly injected into a high-speed counter-current chromatograph at the flow rate of 18mL/min, after the whole column is filled with the stationary phase, the mobile phase is then injected at the flow rate of 2mL/min, and the rotating speed of the counter-current chromatograph is adjusted to 750 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the crude flavone obtained in the step (2) in the two-phase solvent system to prepare a separation sample, and performing high-speed countercurrent chromatographic separation on the crude flavone, wherein the concentration of the sample entering a high-speed countercurrent chromatograph is sample introduction concentration, the sample introduction concentration is 30mg/mL, and the sample introduction volume is 40 mL; during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 300nm, respectively collecting corresponding peak components according to chromatographic peaks, concentrating and drying under reduced pressure, and sequentially obtaining fractions I, II, III, IV and V according to the peak emergence time of 25-35 min, 40-60 min, 140-155 min, 155-165 min and 195-215 min;
(4) the second two-phase solvent system is obtained by uniformly mixing n-heptane, ethyl acetate, methanol and water according to the volume ratio of 1:1:1:1, standing and layering, wherein the upper phase is a stationary phase, and the lower phase is a mobile phase; injecting the stationary phase into a spiral tube for preparing the chromatogram at the flow rate of 18mL/min, starting a host machine after the spiral tube is completely filled with the stationary phase, slowly adjusting the rotating speed of the spiral tube to 820r/min, simultaneously pumping the mobile phase at the flow rate of 5mL/min, when the mobile phase flows out of the tail end of the spiral tube, enabling the system to reach dynamic balance, respectively injecting the different fractions collected in the step 3 into the spiral tube through a sample injection valve, performing preparative chromatographic separation, detecting by an ultraviolet detector with the wavelength of 300nm, and respectively collecting the highest peak fractions of the different fractions in the step 3;
(5) and detecting the purity of the monomer compound by using high performance liquid chromatography in the same manner as in the step (5) in the embodiment 1.
Example 4
A method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems comprises the following specific steps:
(1) taking 1.8kg of naturally dried gynura procumbens stems, crushing, carrying out ultrasonic extraction by taking ethanol with the volume concentration of 65% as a solvent, wherein the material-liquid ratio of gynura procumbens stem powder to ethanol solution is 1g:15mL, the extraction time is 2.5h, the ultrasonic power is 400W, then filtering, and repeatedly treating filter residues for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain total flavone extractive solution;
(2) adsorbing and enriching the obtained total flavone extract by HPD-600 macroporous adsorption resin, eluting by using deionized water with 8 times of column volume, sequentially eluting by using ethanol with the volume concentration of 20%, 40%, 60%, 80% and 90% and 6 times of column volume, respectively, collecting the eluent with the volume concentration of 40%, merging, decompressing and recovering the solvent, freezing and drying to obtain crude flavone, and storing in a refrigerator at 4 ℃ for later use; the method for determining flavone contained in the eluent with volume concentration of 40% is sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method;
(3) the first two-phase solvent system is obtained by uniformly mixing ethyl acetate, n-butyl alcohol and water according to the volume ratio of 1:4:8, standing and layering, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonic degassing is performed, the stationary phase is firstly injected into a high-speed counter-current chromatograph at the flow rate of 20mL/min, after the whole column is filled with the stationary phase, the mobile phase is then injected at the flow rate of 3mL/min, and the rotating speed of the counter-current chromatograph is adjusted to 880 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the crude flavone obtained in the step (2) in the two-phase solvent system to prepare a separation sample, and performing high-speed countercurrent chromatographic separation on the crude flavone, wherein the concentration of the sample entering a high-speed countercurrent chromatograph is sample introduction concentration, the sample introduction concentration is 50mg/mL, and the sample introduction volume is 50 mL; during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 320nm, respectively collecting corresponding peak components according to chromatographic peaks, concentrating and drying under reduced pressure, and sequentially obtaining fractions I, II, III, IV and V according to the peak emergence time of 25-35 min, 40-60 min, 140-155 min, 155-165 min and 195-215 min;
(4) the second two-phase solvent system is obtained by uniformly mixing n-heptane, ethyl acetate, methanol and water according to the volume ratio of 1:4:6:8, standing and layering, wherein the upper phase is a stationary phase, and the lower phase is a mobile phase; injecting the stationary phase into a spiral tube for preparing the chromatogram at the flow rate of 20mL/min, starting a host machine after the spiral tube is completely filled with the stationary phase, slowly adjusting the rotating speed of the spiral tube to 900r/min, simultaneously pumping the mobile phase at the flow rate of 4mL/min, when the mobile phase flows out of the tail end of the spiral tube, enabling the system to reach dynamic balance, respectively injecting the different fractions collected in the step 3 into the spiral tube through a sample injection valve, performing preparative chromatographic separation, detecting by an ultraviolet detector with the wavelength of 320nm, and respectively collecting the highest peak fractions of the different fractions in the step 3;
(5) and detecting the purity of the monomer compound by using high performance liquid chromatography in the same manner as in the step (5) in the embodiment 1.
Example 5
A method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems comprises the following specific steps:
(1) taking 2.5kg of naturally dried gynura procumbens stems, crushing, carrying out ultrasonic extraction by taking ethanol with volume concentration of 80% as a solvent, wherein the material-liquid ratio of gynura procumbens stem powder to ethanol solution is 1g to 20mL, the extraction time is 3h, the ultrasonic power is 500W, filtering, and repeatedly treating filter residues for 2 times; mixing filtrates, vacuum concentrating to remove ethanol smell to obtain total flavone extractive solution;
(2) adsorbing and enriching the obtained total flavone extract by HPD-300 macroporous adsorption resin, eluting by using deionized water with 8 times of column volume, sequentially eluting by using ethanol with the volume concentration of 20%, 40%, 60%, 80% and 90% and 6 times of column volume, respectively, collecting the eluent with the volume concentration of 40%, merging, decompressing and recovering the solvent, freezing and drying to obtain crude flavone, and storing in a refrigerator at 4 ℃ for later use; the method for determining flavone contained in the eluent with volume concentration of 40% is sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method;
(3) the first two-phase solvent system is obtained by uniformly mixing ethyl acetate, n-butyl alcohol and water according to the volume ratio of 1:3:7, standing and layering, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, ultrasonic degassing is carried out, the stationary phase is firstly injected into a high-speed counter-current chromatograph at the flow rate of 20mL/min, after the whole column is filled with the stationary phase, the mobile phase is then injected at the flow rate of 3mL/min, and the rotating speed of the counter-current chromatograph is adjusted to 900 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the crude flavone obtained in the step (2) in the two-phase solvent system to prepare a separation sample, and performing high-speed countercurrent chromatographic separation on the crude flavone, wherein the concentration of the sample entering a high-speed countercurrent chromatograph is sample introduction concentration, the sample introduction concentration is 50mg/mL, and the sample introduction volume is 50 mL; during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 350nm, respectively collecting corresponding peak components according to chromatographic peaks, concentrating and drying under reduced pressure, and sequentially obtaining fractions I, II, III, IV and V according to peak emergence time of 25-35 min, 40-60 min, 140-155 min, 155-165 min and 195-215 min;
(4) the second two-phase solvent system is obtained by uniformly mixing n-heptane, ethyl acetate, methanol and water according to the volume ratio of 1:3:5:8, standing and layering, wherein the upper phase is a stationary phase, and the lower phase is a mobile phase; injecting the stationary phase into a spiral tube for preparing the chromatogram at the flow rate of 20mL/min, starting a host machine after the spiral tube is completely filled with the stationary phase, slowly adjusting the rotating speed of the spiral tube to 950r/min, simultaneously pumping the mobile phase at the flow rate of 6mL/min, when the mobile phase flows out of the tail end of the spiral tube, enabling the system to reach dynamic balance, respectively injecting the different fractions collected in the step 3 into the spiral tube through a sample injection valve, performing preparative chromatographic separation, detecting by an ultraviolet detector with the wavelength of 350nm, and respectively collecting the highest peak fractions of the different fractions in the step 3;
(5) and detecting the purity of the monomer compound by using high performance liquid chromatography in the same manner as in the step (5) in the embodiment 1.
The invention relates to a method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems, and the separated and purified flavonoid compounds have a plurality of biological activities beneficial to human health, including various pharmacological and biological activities of oxidation resistance, allergy resistance, anti-inflammatory immunity, bacteria resistance, virus resistance, tumor resistance, blood pressure reduction, blood sugar reduction, cardiovascular protection, lipid metabolism regulation, immunity improvement and the like.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems is characterized by comprising the following steps: which comprises the following steps:
step 1, preparing a total flavone extracting solution: drying and crushing gynura procumbens stems as raw materials in the shade, and performing ultrasonic extraction by using ethanol with volume concentration of 60-80% as a solvent, wherein the material-liquid ratio g/mL of gynura procumbens stem powder to ethanol solution is 1: 10-1: 20, extracting for 1-3 times, extracting for 1-3 hours, performing ultrasonic power of 350-500W, filtering, combining filtrates, and performing vacuum concentration under reduced pressure until no solvent exists to obtain a total flavone extract;
step 2, enriching total flavonoids: adsorbing and enriching the total flavone extract obtained in the step 1 by using macroporous adsorption resin, eluting by using water with the volume of 8 times of the column volume, sequentially eluting by using ethanol with the volume concentration of 20%, 40%, 60%, 80% and 90% in 6 times of the column volume respectively, collecting the eluent with the volume concentration of 40%, merging, decompressing and recovering the solvent, and freeze-drying to obtain crude flavone;
and 3, high-speed counter-current chromatography separation and purification: the first two-phase solvent system is obtained by uniformly mixing ethyl acetate, n-butyl alcohol and water according to the volume ratio of 1:1: 1-1: 4:8, standing and layering, wherein the upper phase is a stationary phase, the lower phase is a mobile phase, the stationary phase is firstly injected into a high-speed counter-current chromatograph, the mobile phase is then injected after the whole column is filled with the stationary phase, and the rotating speed of the counter-current chromatograph is adjusted to 700-900 rpm; when the two-phase solvent system reaches dynamic balance in a countercurrent column, dissolving the crude flavone obtained in the step (2) in the two-phase solvent system, and carrying out high-speed countercurrent chromatographic separation, wherein the sample injection concentration is 10-50 mg/mL, and the sample injection volume is 20-50 mL; during the high-speed counter-current chromatographic separation, detecting by using an ultraviolet detector with the wavelength of 200-350 nm, respectively collecting corresponding peak components according to chromatographic peaks, concentrating and drying under reduced pressure, and sequentially obtaining fractions I, II, III, IV and V; the fraction I contains quercetin; the fraction II contains luteolin; the fraction III contains apigenin; the fraction IV contains tetrahydroxy flavone; the fraction V contains 7-methyl ether baicalein;
and 4, separating and purifying by preparative chromatography: the second two-phase solvent system is obtained by uniformly mixing n-heptane, ethyl acetate, methanol and water according to the volume ratio of 1:1:1 to 1:4:6:8, standing and layering, wherein the upper phase is a stationary phase, and the lower phase is a mobile phase; injecting the stationary phase into a spiral tube for preparing the chromatogram, starting a host machine after the spiral tube is completely filled with the stationary phase, slowly adjusting the rotating speed of the spiral tube to 750-950 r/min, simultaneously pumping the mobile phase, when the mobile phase flows out from the tail end of the spiral tube, enabling the system to reach dynamic balance, respectively injecting the different fractions collected in the step 3 into the spiral tube through a sample injection valve, performing preparative chromatogram separation, detecting by using an ultraviolet detector with the wavelength of 200-300 nm, and respectively collecting the highest peak fractions of the different fractions in the step 3; then, the peak fractions are respectively subjected to reduced pressure concentration and freeze drying to obtain quercetin, luteolin, apigenin, tetrahydroxyflavone and 7-methylether baicalein with the purity of more than 98%.
2. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 1, wherein the method comprises the following steps: in step 1, the volume concentration of the ethanol solution is preferably 75%.
3. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 1 or 2, wherein the method comprises the following steps: in the step 1, the material-liquid ratio g/mL of the gynura procumbens stem powder to the ethanol solution is preferably 1: 15.
4. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 1, wherein the method comprises the following steps: in the step 2, the macroporous adsorption resin is HPD-300, HPD-400, HPD-600, AB-8 or D-101 macroporous resin.
5. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 1, wherein the method comprises the following steps: in the step 2, the collected eluent with the volume concentration of 40 percent is the eluent containing flavone components, and is measured by a sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method.
6. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 1, wherein the method comprises the following steps: in the step 3, the volume ratio of ethyl acetate, n-butanol and water in the two-phase solvent system is 1 (2-4) to (4-8).
7. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 1, wherein the method comprises the following steps: in the step 3, when the high-speed countercurrent chromatography separation is carried out, the flow rate of the stationary phase is 10-20 mL/min, and the flow rate of the mobile phase is 1-3 mL/min.
8. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 7, wherein the method comprises the following steps: in the step 3, when the high-speed countercurrent chromatography separation is carried out, the rotating speed of the separation column is 800rpm, the flow rate of the mobile phase is 3mL/min, the wavelength of the ultraviolet detector is 255nm, the sample injection concentration is 30mg/mL, and the sample injection volume is 30 mL.
9. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 1, wherein the method comprises the following steps: in the step 3, the peak emergence time of the fractions I, II, III, IV and V is 25-35 min, 40-60 min, 140-155 min, 155-165 min and 195-215 min in sequence.
10. The method for separating and purifying high-purity flavonoid glycoside compounds from gynura procumbens stems as claimed in claim 1, wherein the method comprises the following steps: in the step 4, the volume ratio of n-heptane, ethyl acetate, methanol and water in the solvent system is 1 (2-4) to (3-6): (4-8).
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