CN117586327A - Dianthus superbus flavonoid glycoside compound and directional separation preparation method and application thereof - Google Patents
Dianthus superbus flavonoid glycoside compound and directional separation preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 78
- 244000183914 Dianthus superbus Species 0.000 title claims abstract description 70
- 235000013535 Dianthus superbus Nutrition 0.000 title claims abstract description 70
- 238000000926 separation method Methods 0.000 title claims abstract description 51
- -1 flavonoid glycoside compound Chemical class 0.000 title claims abstract description 41
- 229930182486 flavonoid glycoside Natural products 0.000 title claims abstract description 23
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- 229920005989 resin Polymers 0.000 claims abstract description 28
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- 238000000034 method Methods 0.000 claims abstract description 18
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
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- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 10
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- IFBHRQDFSNCLOZ-RMPHRYRLSA-N 4-nitrophenyl beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-RMPHRYRLSA-N 0.000 description 3
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- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 description 1
- 201000000736 Amenorrhea Diseases 0.000 description 1
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- XUFXOAAUWZOOIT-UHFFFAOYSA-N acarviostatin I01 Natural products OC1C(O)C(NC2C(C(O)C(O)C(CO)=C2)O)C(C)OC1OC(C(C1O)O)C(CO)OC1OC1C(CO)OC(O)C(O)C1O XUFXOAAUWZOOIT-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
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- 230000035619 diuresis Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/13—Crystalline forms, e.g. polymorphs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a fringed pink flavonoid glycoside compound, a directional separation preparation method and application thereof. The dianthus superbus flavonoid glycoside compound is discovered by first extraction and is applied to preparing the medicine for treating or improving diabetes. The preparation process comprises the following steps: extracting, performing medium-pressure chromatography coarse separation on microporous resin, performing reverse-phase medium-pressure chromatography enrichment, preparing a reverse-phase preparation column, performing reverse-phase preparation liquid chromatography purification on Fr411, and performing hydrophilic preparation liquid chromatography purification on Fr 412. The flavone C-glycoside compound obtained by separation can effectively inhibit the activity of alpha-glucosidase, and has low cost and product purity of more than 95%; the technical means adopted by the invention can carry out large-scale production, and is easy to realize large-scale production; reverse phase preparative liquid chromatography or hydrophilic preparative liquid chromatography used in separation and purification is a rapid isocratic method.
Description
Technical Field
The invention relates to the technical field of natural medicinal chemistry, relates to a fringed pink flavonoid glycoside compound, a directional separation preparation method and application thereof, in particular to a flavonoid glycoside compound which comprises a novel compound, a directional separation preparation method thereof and a novel application thereof in inhibiting alpha-glucosidase activity, and is used for preparing a medicament for treating or improving diabetes.
Technical Field
Diabetes is one of the fastest growing global health emergencies in the 21 st century, and of hundreds of millions of diabetics worldwide, 85% -90% are Type II diabetes (Type 2Diabetes Melltus,T2DM). T2DM is characterized by hyperglycemia, and ingestion of excessive high-carbon foods by the human body causes postprandial blood glucose elevation, thereby developing T2DM. However, hyperglycemia after meals can lead to not only diabetes and other complications, but also increased mortality. Therefore, controlling postprandial blood glucose is a major way to improve diabetes and can reduce mortality in diabetics. Currently, methods for controlling postprandial hyperglycemia are mainly by inhibiting the activity of alpha-glucosidase and alpha-amylase, thereby reducing their conversion of sugars to absorbable monosaccharides. Thus, the search and development of natural active substances having low toxicity and capable of effectively inhibiting the activity of α -glucosidase will have a positive effect on the treatment or amelioration of diabetes.
Dianthi (Dianthus superbus L.) is a perennial herb of the genus Caryophyllus of the family Caryophyllaceae, described in Chinese pharmacopoeia 2015 edition, and has effects of inducing diuresis for treating strangurtia, promoting blood circulation and dredging channels. It can be used for treating stranguria due to heat, stranguria with blood, stranguria with stone, difficulty in urination, and amenorrhea and blood stasis. The herba Dianthi contains various chemical components such as saponins, cyclic peptides, flavonoids, polyphenols, alkaloids, etc. Modern pharmacological research shows that it has several functions of resisting bacteria, protecting kidney, resisting early pregnancy, resisting tumor, inhibiting immunity, protecting nerve, etc. However, the flavone C-glycoside compounds are not separated, purified and identified from the fringed pink, and the literature report of inhibiting the activity of alpha-glucosidase and blood sugar is available. Therefore, there is a need to establish a method for preparing flavonoid glycoside compounds from fringed pink in a large scale with simple process, and to determine the alpha-glucosidase inhibitory activity.
Disclosure of Invention
Based on the above problems, the invention aims to provide a fringed pink flavonoid carbon glycoside compound, a directional separation preparation method and application thereof, in particular to a fringed pink flavonoid carbon glycoside compound with alpha-glucosidase inhibiting activity, a directional separation preparation method and application of the fringed pink flavonoid carbon glycoside compound in preparing a blood sugar inhibitor, and application of the fringed pink flavonoid carbon glycoside compound in preparing a medicament for treating or improving diabetes.
The fringed pink flavonoid glycoside compound is discovered by first extraction, and has the structural formula shown as follows:
the application of the dianthus superbus flavonoid glycoside compounds in preparing the alpha-glucosidase inhibitor has the structural formula shown as follows:
the dianthus superbus flavone glycoside compound is used in preparing medicine for inhibiting blood sugar, medicine for treating or improving diabetes, etc.
The method for preparing the dianthus superbus flavonoid glycoside compound by directional separation comprises the following steps:
step 1, extracting: drying the whole herb of the fringed pink in the shade, and roughly crushing the whole herb of the fringed pink according to the feed liquid ratio of 1g: extracting 5-100 mL of ethanol for 2-4 times at room temperature for 2-4 hours each time, filtering, and combining the filtrates to obtain filtrate A, wherein the filtrate A comprises the following components in percentage by weight: amount of fringed pink herb = 1: 5-15, mixing and drying under reduced pressure to obtain a sample of the fringed pink extract;
step 2, pressure chromatography coarse separation of microporous resin: separating herba Dianthi extract sample by medium pressure chromatography with microporous resin, detecting with ultraviolet detector with detection wavelength of 210nm, collecting 6 chromatographic peak fractions, drying under reduced pressure, and labeling as herba Dianthi components Fr1, fr2, fr3, fr4, fr5 and Fr6;
step 3, reverse phase medium pressure chromatography enrichment: the target component Fr4 is mixed with silica gel to obtain a sample, the sample is separated by a reverse phase medium pressure chromatographic column filled with a silica gel matrix material, is detected by an ultraviolet detector with the detection wavelength of 210nm, and is collected to prepare a 1 st chromatographic peak fraction in a chromatogram, and the fraction is dried under reduced pressure to obtain a component Fr41 containing the target component;
step 4, preparing a reverse phase preparation column: dissolving a component Fr41 containing a target component by using a methanol-water solution with the volume fraction of 50-100%, preparing a sample with the concentration of 20.0-50.0 mg/mL, filtering by using a microporous filter membrane with the concentration of 0.45 mu m to obtain a filtrate B, preparing the filtrate B by using a reverse phase preparation column, detecting by using an ultraviolet detector with the detection wavelength of 210nm, collecting two chromatographic peak fractions in a chromatographic chart prepared by the reverse phase of the filtrate B, and drying the chromatographic peak fractions under reduced pressure to obtain components Fr411 and Fr412 containing the target component;
step 5, reverse phase preparative liquid chromatography purification of fr 411: dissolving the component Fr411 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate C, purifying the filtrate C by reverse phase high pressure liquid chromatography, detecting by an ultraviolet detector with a detection wavelength of 210nm, collecting a chromatographic peak fraction Fr4111 in a chromatographic chart prepared by reverse phase high pressure of the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain 2' -O-rhamnosyllotonin with a purity of more than 95%;
step 6, hydrophilic preparation liquid chromatography purification of fr 412: dissolving the component Fr412 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate D, purifying the filtrate D by hydrophilic high pressure liquid chromatography, detecting by an ultraviolet detector with a detection wavelength of 210nm, collecting corresponding chromatographic peak fractions Fr4121, fr4122 and Fr4123 in the hydrophilic high pressure preparation chromatogram of the filtrate D, and drying the chromatographic peak fractions Fr4121, fr4122 and Fr4123 under reduced pressure to obtain Luteolin 6-C-glucoside-7-O-glucoside, luteolin-C- (6 '-O-beta-D-glucoside) -glucoside and 6' -O-rheasyluloarea with a purity of more than 95%;
further, in the steps 1, 2, 3, 4, 5 and 6, the conditions of drying under reduced pressure are as follows: the vacuum degree is 50-250 mbar, and the temperature is 40-60 ℃;
further, in the step 2, the separation working parameters of the medium pressure chromatography coarse fraction of the microporous resin are as follows: the chromatographic column has the length of 460mm and the diameter of 49mm, the stationary phase of the microporous resin column is HP20SS, the mobile phase A is water, the mobile phase B is methanol, the mobile phase C is methylene dichloride, the chromatographic conditions are 0-20 min,0% B, 20-240 min, 0-100% B, 240-300 min,100% B, 300-420 min, 0-100% C, the sample injection amount is 10-80 g, and the flow rate is 40-60 mL/min;
further, in the step 3, the working parameters of the reverse phase medium pressure chromatography enrichment are as follows: the chromatographic column has a length of 500mm and a diameter of 50mm, the reversed phase preparation column stationary phase is 50 mu m of spherial C18, the mobile phase A is water, the mobile phase B is methanol, the chromatographic conditions are 0-120 min,20% -65% B, the sample injection amount is 10-30 g, and the flow rate is 40-60 mL/min;
further, in the step 4, working parameters of the reverse phase preparation column are as follows: the size of the chromatographic column is 250 multiplied by 20mm, the stationary phase of the reversed phase preparation column is 7 mu m reversed phase column 7-X10, and the mobile phase is 6% methanol-water solution;
further, in the step 5, the working parameters of reversed-phase preparative liquid chromatography purification refer to a chromatographic column with a length of 250mm and a diameter of 20mm, a reversed-phase preparative column stationary phase of 5 μm reversed-phase column SunFire C18, and a mobile phase of 20% methanol-water solution;
further, in the step 6, the working parameters of the purification of the hydrophilic preparative liquid chromatograph are as follows: the chromatographic column size was 250X 20mm, the hydrophilic preparative column stationary phase was 5 μm zwitterionic column ClickXION, and the mobile phase was 90% acetonitrile-5% trifluoroacetic acid in water.
Compared with the prior art, the invention has the following advantages:
(1) The invention has low cost and high product purity. The extraction solvent used in the purification of microporous resin medium-pressure chromatography coarse separation, reverse-phase medium-pressure chromatography enrichment and reverse-phase hydrophilic chromatography can be recycled; the chromatographic separation materials (microporous resin, reversed phase preparation liquid chromatography and hydrophilic preparation liquid chromatography separation materials) can be reused; the recycled solvent and the recycled separation material ensure that the average cost is low in the separation process, and the purity of the product can be ensured to be more than 95% by two-step medium-pressure separation (medium-pressure chromatography coarse separation and reverse-phase medium-pressure chromatography enrichment of microporous resin) and high-pressure liquid chromatography purification.
(2) The preparation method can realize the requirement of large-scale production. The raw materials are not high in requirement, low in cost and easy to obtain, and the fringed pink herb Chinese medicinal materials which are generally planted or sold in the market can be prepared in batches easily; ethanol is extracted at room temperature, and the operation is easy; the separation adopts microporous resin column coarse separation, and the microporous resin separation material can be arranged in a medium-pressure column chromatography system, so that the large-scale separation is easy to realize; the reversed phase preparation liquid chromatography or the hydrophilic preparation liquid chromatography used in separation and purification is a rapid isocratic method and is very suitable for large-scale production.
(3) The flavonoid carbon glycoside compound in the fringed pink is a pure natural, plant-derived, nontoxic and non-irritating substance, and can remarkably inhibit the activity of alpha-glucosidase. The invention provides an application of a dianthus superbus flavone C-glycoside compound in preparing a medicament for treating or improving diabetes mellitus, and an application in preparing a medicament for inhibiting alpha-glucosidase.
Drawings
FIG. 1 is a microporous resin separation chromatogram of a sample of the present invention;
FIG. 2 is a reverse phase medium pressure chromatography enrichment chromatogram of the microporous resin separation component Fr4 of the present invention;
FIG. 3 is a diagram showing the reverse phase preparative liquid chromatography separation of the target component Fr41 of fringed pink of the present invention;
FIG. 4 is a diagram showing the reversed phase preparative liquid chromatography separation of the target component Fr411 of fringed pink according to the present invention;
FIG. 5 is a diagram showing the separation of the target frid 412 of fringed pink by liquid chromatography in a hydrophilic preparation according to the present invention;
FIG. 6 shows purity verification chromatograms of flavonoid carboglycosides Fr4111 (2 "-O-rhamnosyllabin), fr4121 (Luteolin 6-C-glucoside-7-O-glucoside), fr4122 (Luteolin 6-C- (6" -O-. Beta. -D-glucoside) -glucoside) and Fr4123 (6' "-O-rhamnosyllabin) of the present invention;
FIG. 7 is a high resolution mass spectrum of 2 "-O-rhamnosylphonarin isolated in accordance with the present invention;
FIG. 8 is a diagram of 2 "-O-rhamnosyllotonarin isolated according to the invention 1 HNMR nuclear magnetic resonance;
FIG. 9 is a diagram of 2"-O-rhamnosyllutonarin isolated in accordance with the present invention 13 C NMR nuclear magnetic pattern;
FIG. 10 is a graph of 2 "-O-rhamnosylphonarin infrared spectrum isolated according to the present invention;
FIG. 11 is a graph of the UV spectrum of 2 "-O-rhamnosylphonarin isolated according to the invention;
FIG. 12 is a chart showing the low resolution mass spectrum of Luteolin 6-C-glucoside-7-O-glucoside isolated according to the present invention;
FIG. 13 shows the isolated Luteolin 6-C-glucoside-7-O-glucoside of the present invention 1 H NMR nuclear magnetic pattern;
FIG. 14 shows the isolated Luteolin 6-C-glucoside-7-O-glucoside of the present invention 13 C NMR nuclear magnetic pattern;
FIG. 15 is an infrared spectrum of Luteolin 6-C-glucoside-7-O-glucoside isolated according to the present invention;
FIG. 16 is a chart of the ultraviolet spectrum of the isolated Luteolin 6-C-glucoside-7-O-glucoside of the present invention;
FIG. 17 is a chart showing the low resolution mass spectrum of Luteolin6-C- (6' -O-beta-D-glucoside) -glucoside isolated in accordance with the present invention;
FIG. 18 shows the isolated Luteolin6-C- (6' -O-beta-D-glucide) -glucide of the present invention 1 HNMR nuclear magnetic resonance;
FIG. 19 shows the isolated Luteolin6-C- (6' -O-beta-D-glucide) -glucide of the present invention 13 CNMR nuclear magnetic resonance;
FIG. 20 is an infrared spectrum of Luteolin6-C- (6' -O-beta-D-glucoside) -glucoside isolated according to the present invention;
FIG. 21 is a chart of the ultraviolet spectrum of Luteolin6-C- (6' -O-beta-D-glucoside) -glucoside isolated according to the present invention;
FIG. 22 is a low resolution mass spectrum of 6' -O-rhamnosylphonarin isolated in accordance with the present invention;
FIG. 23 shows a divider of the present inventionIsolated 6' -O-rhamnosyllutonarin 1 HNMR nuclear magnetic resonance;
FIG. 24 is a photograph of 6' -O-rhamnosyllabin isolated according to the present invention 13 C NMR nuclear magnetic pattern;
FIG. 25 is a chart of an infrared spectrum of 6' -O-rhamnosylphonarin isolated in accordance with the present invention;
FIG. 26 is a graph of UV spectrum of isolated 6' -O-rhamnosylphonarin according to the present invention;
FIG. 27 shows the structural diagrams of the flavonoid glycosides Fr4111 (2 "-O-rhamnosyllonin), fr4121 (Luteolin 6-C-glucoside-7-O-glucoside), fr4122 (Luteolin 6-C- (6" -O-. Beta. -D-glucoside) -glucoside) and Fr4123 (6' "-O-rhamnosyllonin) isolated according to the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method for preparing dianthus superbus flavone C-glycoside compounds by directional separation comprises the following steps:
step 1, extracting: taking 4.0kg of a sample of the dianthus superbus herb after being dried in the shade, crushing, adding ethanol with the mass of 10 times of that of the sample, extracting for 4 times at room temperature for 2 hours each time, filtering, and combining filtrate, namely filtrate A, wherein the filtrate A comprises the following components in percentage by weight: mixing the samples according to the ratio of the weight of the fringed pink herb material to the weight of 1:15, and drying under reduced pressure to obtain 827g of a sample of the fringed pink ethanol extract, wherein the condition of drying under reduced pressure is that the vacuum degree is 50mbar and the temperature is 40 ℃;
step 2, pressure chromatography coarse separation of microporous resin: the sample of the fringed pink ethanol extract is separated by a medium-pressure chromatographic column filled with a microporous resin material, detected by an ultraviolet detector with the detection wavelength of 210nm, and collected to obtain 6 main chromatographic peak fractions, and the chromatographic peak fractions are marked as fringed pink components Fr1 after drying under reduced pressure: 74g, fr2:78g, fr3:66g, fr4:155g, fr5:26g and Fr6:15g (shown in FIG. 1);
wherein the condition of decompression drying is that the vacuum degree is 50mbar and the temperature is 40 ℃; the working parameters of the micro-porous resin medium-pressure chromatographic rough separation are as follows: the chromatographic column has the length of 460mm and the diameter of 49mm, the stationary phase of the microporous resin column is HP20SS, the mobile phase A is water, the mobile phase B is methanol, the mobile phase C is dichloromethane, the chromatographic conditions are 0-20 min,0% B, 20-240 min, 0-100% B, 240-300 min,100% B, 300-420 min, 0-100% C, the sample injection amount is 80g, and the flow rate is 60mL/min;
step 3, reverse phase medium pressure chromatography enrichment: the target component Fr4 is mixed with silica gel to obtain a sample, the sample is separated by a reverse phase medium pressure chromatographic column filled with a silica gel matrix material, the sample is detected by an ultraviolet detector with the detection wavelength of 210nm, a 1 st main chromatographic peak fraction Fr41 (shown in figure 2) in a prepared chromatogram is collected, and the fraction is dried under reduced pressure to obtain the component Fr41 containing the target component: 20.5g;
wherein the condition of decompression drying is that the vacuum degree is 50mbar and the temperature is 40 ℃; the separation working parameters of the reverse phase medium pressure chromatography enrichment are as follows: the chromatographic column has a length of 500mm and a diameter of 50mm, the reversed phase preparation column stationary phase is 50 μm of spherial C18, the mobile phase A is water, the mobile phase B is methanol, the chromatographic conditions are 0-120 min,20% -65% B, the sample injection amount is 30g, and the flow rate is 60mL/min;
step 4, preparing a reverse phase preparation column: dissolving the component Fr41 containing the target component with 50% methanol-water solution, preparing 50.0mg/mL sample concentration, filtering with 0.45 μm microporous filter membrane to obtain filtrate B, preparing filtrate B by reversed phase preparation column, detecting by ultraviolet detector with detection wavelength of 210nm, collecting two main chromatographic peak fractions in reversed phase preparation chromatogram of filtrate B, and drying under reduced pressure to obtain components Fr411 and Fr412 (shown in figure 3) containing the target component;
wherein the condition of decompression drying is that the vacuum degree is 50mbar and the temperature is 40 ℃; the working parameters of the preparation of the reverse phase preparation column are as follows: the size of the chromatographic column is 250 multiplied by 20mm, the stationary phase of the reversed phase preparation column is 7 mu m reversed phase column 7-X10, the mobile phase is 6% methanol-water solution, the sample injection volume is 4mL, and the flow rate is 19mL/min;
step 5, reverse phase preparative liquid chromatography purification of fr 411: dissolving the component Fr411 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate C, purifying the filtrate C by reverse phase high pressure liquid chromatography, detecting by an ultraviolet detector with a detection wavelength of 210nm, collecting a chromatographic peak fraction Fr4111 (shown in figure 4) in a chromatographic chart prepared by reverse phase high pressure of the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain 2' -O-rhamsynellonarin with a purity of more than 95%;
wherein the condition of decompression drying is that the vacuum degree is 50mbar and the temperature is 40 ℃; the working parameters of the reverse phase preparation liquid chromatography purification of Fr411 are as follows: the chromatographic column has a length of 250mm and a diameter of 20mm, the reversed phase preparation column stationary phase is 5 mu m reversed phase column SunFire C18, the mobile phase is 20% methanol-water solution, the sample injection volume is 4mL, and the flow rate is 19mL/min;
step 6, hydrophilic preparation liquid chromatography purification of fr 412: dissolving the component Fr412 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate D, purifying the filtrate D by hydrophilic high pressure liquid chromatography, detecting with an ultraviolet detector with a detection wavelength of 210nm, collecting corresponding chromatographic peak fractions Fr4121, fr4122 and Fr4123 (shown in figure 5) in the hydrophilic high pressure preparation chromatogram of the filtrate D, and drying the chromatographic peak fractions Fr4121, fr4122 and Fr4123 under reduced pressure to obtain Luteolin 6-C-glucoside-7-O-glucoside 765.4mg, luteolin6-C- (6 '-O-beta-D-glucoside) -glucoside 8.9mg and 6' -O-rhamnosyringone 204.7mg respectively;
wherein the condition of decompression drying is that the vacuum degree is 50mbar and the temperature is 40 ℃; the working parameters of the hydrophilic preparation liquid chromatography purification of Fr412 are as follows: the chromatographic column size is 250 multiplied by 20mm, the hydrophilic preparation column stationary phase is 5 mu m zwitterionic column ClickXION, the mobile phase is 90% acetonitrile-5% trifluoroacetic acid aqueous solution, the sample injection volume is 4mL, and the flow rate is 19mL/min;
wherein, the obtained Fr4111, fr4121, fr4122 and Fr4123 are respectively 2 ' -O-rhamnosyllutonarin, luteolin 6-C-glucoside-7-O-glucoside, luteolin-C- (6 ' -O-beta-D-glucoside) -glucoside and 6 ' -O-rhamnosyllotonarin samples with purity of more than 95%, and the purity verification chromatogram (shown in figure 6);
the structural characterization and structure of flavonoid carbon glycoside compounds 2 ' -O-rhamnosyllutonarin, luteolin 6-C-glucoside-7-O-glucoside, luteolin-C- (6 ' -O-beta-D-glucoside) -glucoside and 6 ' -O-rhamnosyllabin obtained from fringed pink are shown in the accompanying figures 7-27.
Example 2
A method for preparing dianthus superbus flavone C-glycoside compounds by directional separation comprises the following steps:
step 1, extracting: taking 10kg of a sample of the dianthus superbus herb after being dried in the shade, crushing, adding ethanol with the mass of 10 times of that of the sample, extracting for 3 times at room temperature for 3 hours each time, filtering, and combining filtrate, namely filtrate A, wherein the filtrate A comprises the following components in percentage by weight: mixing the sample with the amount of the fringed pink herb material=1:8, and drying under reduced pressure to obtain 2.66kg of fringed pink herb ethanol extract mixed sample, wherein the condition of drying under reduced pressure is that the vacuum degree is 250mbar and the temperature is 60 ℃;
step 2, pressure chromatography coarse separation of microporous resin: the fringed pink ethanol extract is mixed with a sample, the sample is separated by a medium-pressure chromatographic column filled with a microporous resin material, is detected by an ultraviolet detector with the detection wavelength of 210nm, is collected to obtain 6 main chromatographic peak fractions, and is marked as fringed pink component Fr1 after being dried under reduced pressure: 184g, fr2:193g, fr3:161g, fr4:392g, fr5:7.1g and Fr6:4.0g;
wherein the condition of decompression drying is that the vacuum degree is 250mbar and the temperature is 60 ℃; the working parameters of the micro-porous resin medium-pressure chromatographic rough separation are as follows: the chromatographic column has the length of 460mm and the diameter of 49mm, the stationary phase of the microporous resin column is HP20SS, the mobile phase A is water, the mobile phase B is methanol, the mobile phase C is dichloromethane, the chromatographic conditions are 0-20 min,0% B, 20-240 min, 0-100% B, 240-300 min,100% B, 300-420 min, 0-100% C, the sample injection amount is 40g, and the flow rate is 50mL/min;
step 3, reverse phase medium pressure chromatography enrichment: the target component Fr4 is mixed with silica gel to obtain a sample, the sample is separated by a reverse phase medium pressure chromatographic column filled with a silica gel matrix material, the sample is detected by an ultraviolet detector with the detection wavelength of 210nm, the 1 st main chromatographic peak fraction Fr41 in a prepared chromatogram is collected, and the fraction is dried under reduced pressure to obtain the component Fr41 containing the target component: 50.6g;
wherein the condition of decompression drying is that the vacuum degree is 250mbar and the temperature is 60 ℃; the separation working parameters of the reverse phase medium pressure chromatography enrichment are as follows: the chromatographic column has a length of 500mm and a diameter of 50mm, the reversed phase preparation column stationary phase is 50 μm of spherial C18, the mobile phase A is water, the mobile phase B is methanol, the chromatographic conditions are 0-120 min,20% -65% B, the sample injection amount is 15g, and the flow rate is 45mL/min;
step 4, preparing a reverse phase preparation column: dissolving the component Fr41 containing the target component by using a methanol-water solution with the volume fraction of 80%, preparing a sample with the concentration of 40.0mg/mL, filtering by using a microporous filter membrane with the concentration of 0.45 mu m to obtain filtrate B, preparing the filtrate B by using a reverse phase preparation column, detecting by using an ultraviolet detector with the detection wavelength of 210nm, collecting two main chromatographic peak fractions in a chromatographic chart prepared by the reverse phase of the filtrate B, and drying the chromatographic peak fractions under reduced pressure to obtain the components Fr411 and Fr412 containing the target component;
wherein the condition of decompression drying is that the vacuum degree is 250mbar and the temperature is 60 ℃; the working parameters of the preparation of the reverse phase preparation column are as follows: the size of the chromatographic column is 250 multiplied by 20mm, the stationary phase of the reversed phase preparation column is 7 mu m reversed phase column 7-X10, the mobile phase is 6% methanol-water solution, the sample injection volume is 4mL, and the flow rate is 19mL/min;
step 5, reverse phase preparative liquid chromatography purification of fr 411: dissolving the component Fr411 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate C, purifying the filtrate C by reverse phase high pressure liquid chromatography, detecting by an ultraviolet detector with a detection wavelength of 210nm, collecting a chromatographic peak fraction Fr4111 in a chromatographic chart prepared by reverse phase high pressure of the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain 2' -O-rhamnosylphonin with a purity of more than 95% of 7.6g;
wherein the condition of decompression drying is that the vacuum degree is 250mbar and the temperature is 60 ℃; the working parameters of the reverse phase preparation liquid chromatography purification of Fr411 are as follows: the chromatographic column has a length of 250mm and a diameter of 20mm, the reversed phase preparation column stationary phase is 5 mu m reversed phase column SunFire C18, the mobile phase is 20% methanol-water solution, the sample injection volume is 4mL, and the flow rate is 19mL/min;
step 6, hydrophilic preparation liquid chromatography purification of fr 412: dissolving the component Fr412 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate D, purifying the filtrate D by hydrophilic high pressure liquid chromatography, detecting by an ultraviolet detector with a detection wavelength of 210nm, collecting corresponding chromatographic peak fractions Fr4121, fr4122 and Fr4123 in the hydrophilic high pressure preparation chromatogram of the filtrate D, and drying the chromatographic peak fractions Fr4121, fr4122 and Fr4123 under reduced pressure to obtain Luteolin 6-C-glucoside-7-O-glucoside 1.9g, luteolin6-C- (6 '-O-beta-D-glucoside) -glucoside 22.2mg and 6' -O-rhamnosynuterside 507.5mg with a purity of more than 95% respectively;
wherein the condition of decompression drying is that the vacuum degree is 250mbar and the temperature is 60 ℃; the working parameters of the hydrophilic preparation liquid chromatography purification of Fr412 are as follows: the chromatographic column size is 250 multiplied by 20mm, the hydrophilic preparation column stationary phase is 5 mu m zwitterionic column ClickXION, the mobile phase is 90% acetonitrile-5% trifluoroacetic acid aqueous solution, the sample injection volume is 4mL, and the flow rate is 19mL/min;
example 3
A method for preparing dianthus superbus flavone C-glycoside compounds by directional separation comprises the following steps:
step 1, extracting: taking 1kg of a sample of the dianthus superbus herb after being dried in the shade, crushing, adding ethanol with the mass of 10 times of that of the sample, extracting for 2 times at room temperature for 4 hours each time, filtering, and combining filtrate, namely filtrate A, wherein the filtrate A comprises the following components in percentage by weight: mixing the samples according to the ratio of the weight of the fringed pink herb to the weight of 1:5, and drying under reduced pressure to obtain 338g of fringed pink ethanol extract mixed sample, wherein the condition of drying under reduced pressure is that the vacuum degree is 150mbar and the temperature is 50 ℃;
step 2, pressure chromatography coarse separation of microporous resin: the fringed pink ethanol extract is mixed with a sample, the sample is separated by a medium-pressure chromatographic column filled with a microporous resin material, is detected by an ultraviolet detector with the detection wavelength of 210nm, is collected to obtain 6 main chromatographic peak fractions, and is marked as fringed pink component Fr1 after being dried under reduced pressure: 18.6g, fr2:19.6g, fr3:16.4g, fr4:38.7g, fr5:6.5g and Fr6:3.7g;
wherein the condition of decompression drying is that the vacuum degree is 150mbar and the temperature is 50 ℃; the working parameters of the micro-porous resin medium-pressure chromatographic rough separation are as follows: the chromatographic column has the length of 460mm and the diameter of 49mm, the stationary phase of the microporous resin column is HP20SS, the mobile phase A is water, the mobile phase B is methanol, the mobile phase C is dichloromethane, the chromatographic conditions are 0-20 min,0% B, 20-240 min, 0-100% B, 240-300 min,100% B, 300-420 min, 0-100% C, the sample injection amount is 10g, and the flow rate is 40mL/min;
step 3, reverse phase medium pressure chromatography enrichment: the target component Fr4 is mixed with silica gel to obtain a sample, the sample is separated by a reverse phase medium pressure chromatographic column filled with a silica gel matrix material, the sample is detected by an ultraviolet detector with the detection wavelength of 210nm, the 1 st main chromatographic peak fraction Fr41 in a prepared chromatogram is collected, and the fraction is dried under reduced pressure to obtain the component Fr41 containing the target component: 5.0g;
wherein the condition of decompression drying is that the vacuum degree is 150mbar and the temperature is 50 ℃; the separation working parameters of the reverse phase medium pressure chromatography enrichment are as follows: the chromatographic column has a length of 500mm and a diameter of 50mm, the reversed phase preparation column stationary phase is 50 μm of spherial C18, the mobile phase A is water, the mobile phase B is methanol, the chromatographic conditions are 0-120 min,20% -65% B, the sample injection amount is 10g, and the flow rate is 40mL/min;
step 4, preparing a reverse phase preparation column: dissolving the component Fr41 containing the target component by using a methanol solution with the volume fraction of 100%, preparing a sample with the concentration of 20.0mg/mL, filtering by using a microporous filter membrane with the concentration of 0.45 mu m to obtain filtrate B, preparing the filtrate B by using a reverse phase preparation column, detecting by using an ultraviolet detector with the detection wavelength of 210nm, collecting two main chromatographic peak fractions in a chromatographic chart prepared by the reverse phase of the filtrate B, and drying the chromatographic peak fractions under reduced pressure to obtain the components Fr411 and Fr412 containing the target component;
wherein the condition of decompression drying is that the vacuum degree is 150mbar and the temperature is 50 ℃; the working parameters of the preparation of the reverse phase preparation column are as follows: the size of the chromatographic column is 250 multiplied by 20mm, the stationary phase of the reversed phase preparation column is 7 mu m reversed phase column 7-X10, the mobile phase is 6% methanol-water solution, the sample injection volume is 4mL, and the flow rate is 19mL/min;
step 5, reverse phase preparative liquid chromatography purification of fr 411: dissolving the component Fr411 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate C, purifying the filtrate C by reverse phase high pressure liquid chromatography, detecting by an ultraviolet detector with a detection wavelength of 210nm, collecting a chromatographic peak fraction Fr4111 in a chromatographic chart prepared by reverse phase high pressure of the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain 2' -O-rhamnosyllonylarin 751.8mg with a purity of more than 95%;
wherein the condition of decompression drying is that the vacuum degree is 150mbar and the temperature is 50 ℃; the working parameters of the reverse phase preparation liquid chromatography purification of Fr411 are as follows: the chromatographic column has a length of 250mm and a diameter of 20mm, the reversed phase preparation column stationary phase is 5 mu m reversed phase column SunFire C18, the mobile phase is 20% methanol-water solution, the sample injection volume is 4mL, and the flow rate is 19mL/min;
step 6, hydrophilic preparation liquid chromatography purification of fr 412: dissolving the component Fr412 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate D, purifying the filtrate D by hydrophilic high pressure liquid chromatography, detecting by an ultraviolet detector with a detection wavelength of 210nm, collecting corresponding chromatographic peak fractions Fr4121, fr4122 and Fr4123 in the hydrophilic high pressure preparation chromatogram of the filtrate D, and drying the chromatographic peak fractions Fr4121, fr4122 and Fr4123 under reduced pressure to obtain Luteolin 6-C-glucoside-7-O-glucoside 186.7mg, luteolin6-C- (6 '-O-beta-D-glucoside) -glucoside 2.2mg and 6' -O-rhamnosynuterside 49.9mg with a purity of more than 95%;
wherein the condition of decompression drying is that the vacuum degree is 150mbar and the temperature is 50 ℃; the working parameters of the hydrophilic preparation liquid chromatography purification of Fr412 are as follows: the chromatographic column size is 250X 20mm, the hydrophilic preparation column stationary phase is 5 μm zwitterionic column ClickXION, the mobile phase is 90% acetonitrile-5% trifluoroacetic acid aqueous solution, the sample injection volume is 4mL, and the flow rate is 19mL/min.
Pharmacological test Dianthus superbus flavonoid carbon glycoside compound alpha-glucosidase inhibition activity screening
The experiment was divided into a blank group (without adding the fringed pink flavonoid glycoside and the alpha-glucosidase solution), a control group (without adding the fringed pink flavonoid glycoside), a fringed pink flavonoid glycoside measurement group and a fringed pink blank group (without adding the alpha-glucosidase solution) by using 4-Nitrophenyl-beta-D-glucopyranoside (PNPG) as a substrate, and 3 compound holes are arranged in each group.
50 μL of 0.25U/mL of alpha-glucosidase solution and 50 μL of sample solution (the concentration of the fringed pink monomer compound sample solution is 1, 10, 50, 100, 500 and 1000 μM) are added into a 96-well plate, acarbose concentration is 1, 5, 20, 50, 100 and 500 μM, the mixture is uniformly mixed for 10min at 37 ℃ in a shaking way, 50 μL of 1mmol/L PNPG solution is added, the mixture is uniformly mixed for 20min at 37 ℃ in a shaking way, and then 50 μL of 0.2mol/L sodium carbonate solution is added to terminate the reaction, and the absorbance is measured in an enzyme marker at 405 nm. The inhibition ratio of the alpha-glucosidase activity is calculated according to the formula, and the experiment is repeated for 3 times. The half maximal inhibitory concentration (IC 50) on α -glucosidase activity was calculated using SPSS (results are shown in table 1).
A c : control group, A b : blank group, A q : dianthum flavone C-glycoside group A qb : dianthi blank group
TABLE 1 screening results for alpha-glucosidase inhibitory Activity of Dianthum flavonoid C-glycosides
The test results show that Fr4111 (2 ' -O-rhamnosyllabin), fr4121 (Luteolin 6-C-glucoside-7-O-glucoside), fr4122 (Luteolin 6-C- (6 ' -O-beta-D-glucoside) -glucoside) and Fr4123 (6 ' -O-rhamnosyllabin) have obvious alpha-glucosidase activity, and the IC50 value of the fringed pink flavonoid glycoside compounds is below 1mM, so that the fringed pink flavonoid glycoside compounds can be used for preparing alpha-glucosidase inhibitors. Moreover, since the method of controlling postprandial hyperglycemia is mainly by inhibiting the activities of α -glucosidase and α -amylase, it reduces the conversion of sugar into absorbable monosaccharides. Therefore, the flavonoid carbon glycoside compound can be applied to preparing medicines for treating or improving diabetes and plays a positive role in the process of treating diabetes.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A method for preparing fringed pink flavonoid-glycoside compounds by directional separation is characterized by comprising the following steps:
step 1, extracting: drying the whole herb of the fringed pink in the shade, and roughly crushing the whole herb of the fringed pink according to the feed liquid ratio of 1g: extracting 5-100 mL of ethanol for 2-4 times at room temperature for 2-4 hours each time, filtering, and combining the filtrates to obtain filtrate A, wherein the filtrate A comprises the following components in percentage by weight: amount of fringed pink herb = 1: 5-15, mixing and drying under reduced pressure to obtain a sample of the fringed pink extract;
step 2, pressure chromatography coarse separation of microporous resin: separating herba Dianthi extract sample by medium pressure chromatography with microporous resin, detecting with ultraviolet detector with detection wavelength of 210nm, collecting 6 chromatographic peak fractions, drying under reduced pressure, and labeling as herba Dianthi components Fr1, fr2, fr3, fr4, fr5 and Fr6;
step 3, reverse phase medium pressure chromatography enrichment: the target component Fr4 is mixed with silica gel to obtain a sample, the sample is separated by a reverse phase medium pressure chromatographic column filled with a silica gel matrix material, is detected by an ultraviolet detector with the detection wavelength of 210nm, and is collected to prepare a 1 st chromatographic peak fraction in a chromatogram, and the fraction is dried under reduced pressure to obtain a component Fr41 containing the target component;
step 4, preparing a reverse phase preparation column: dissolving a component Fr41 containing a target component by using a methanol-water solution with the volume fraction of 50-100%, preparing a sample with the concentration of 20.0-50.0 mg/mL, filtering by using a microporous filter membrane with the concentration of 0.45 mu m to obtain a filtrate B, preparing the filtrate B by using a reverse phase preparation column, detecting by using an ultraviolet detector with the detection wavelength of 210nm, collecting two chromatographic peak fractions in a chromatographic chart prepared by the reverse phase of the filtrate B, and drying the chromatographic peak fractions under reduced pressure to obtain components Fr411 and Fr412 containing the target component;
step 5, reverse phase preparative liquid chromatography purification of fr 411: dissolving the component Fr411 containing the target component with 100% methanol solution, preparing a sample with a concentration of 25.0mg/mL, filtering with a 0.45 μm microporous filter membrane to obtain filtrate C, purifying the filtrate C by reverse phase high pressure liquid chromatography, detecting by an ultraviolet detector with a detection wavelength of 210nm, collecting a chromatographic peak fraction Fr4111 in a chromatographic chart prepared by reverse phase high pressure of the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain 2' -O-rhamnosyllotonin with a purity of more than 95%;
step 6, hydrophilic preparation liquid chromatography purification of fr 412: the Fr412 component containing the target component is dissolved by using a methanol solution with the volume fraction of 100%, the concentration of a prepared sample is 25.0mg/mL, a microporous filter membrane with the concentration of 0.45 mu m is used for filtering to obtain filtrate D, the filtrate D is purified by a hydrophilic high-pressure liquid chromatography, an ultraviolet detector with the detection wavelength of 210nm is used for detecting, corresponding chromatographic peak fractions Fr4121, fr4122 and Fr4123 in the hydrophilic high-pressure preparation chromatogram of the filtrate D are collected, and the chromatographic peak fractions Fr4121, fr4122 and Fr4123 are dried under reduced pressure to obtain the Luteolin 6-C-glucoside-7-O-glucoside, luteolin-C- (6 '-O-beta-D-glucoside) -glucoside and 6' -O-rheasylurenin with the purity of more than 95 percent respectively.
2. The method for directional separation and preparation of the dianthus superbus flavone C-glycoside compound according to claim 1, which is characterized in that: in the steps 1, 2, 3, 4, 5 and 6, the conditions of decompression drying are as follows: the vacuum degree is 50-250 mbar, and the temperature is 40-60 ℃.
3. The method for directional separation and preparation of the dianthus superbus flavone C-glycoside compound according to claim 1, which is characterized in that: in the step 2, the separation working parameters of the medium-pressure chromatographic coarse separation of the microporous resin are as follows: the chromatographic column has the length of 460mm and the diameter of 49mm, the stationary phase of the microporous resin column is HP20SS, the mobile phase A is water, the mobile phase B is methanol, the mobile phase C is dichloromethane, the chromatographic conditions are 0-20 min,0% B, 20-240 min, 0-100% B, 240-300 min,100% B, 300-420 min, 0-100% C, the sample injection amount is 10-80 g, and the flow rate is 40-60 mL/min.
4. The method for directional separation and preparation of the dianthus superbus flavone C-glycoside compound according to claim 1, which is characterized in that: in the step 3, the working parameters of the reverse phase medium pressure chromatographic enrichment are as follows: the chromatographic column has a length of 500mm and a diameter of 50mm, the reversed phase preparation column stationary phase is 50 μm of Sphere C18, the mobile phase A is water, the mobile phase B is methanol, the chromatographic conditions are 0-120 min,20% -65% B, the sample injection amount is 10-30 g, and the flow rate is 40-60 mL/min.
5. The method for directional separation and preparation of the dianthus superbus flavone C-glycoside compound according to claim 1, which is characterized in that: in the step 4, working parameters of the preparation of the reversed phase preparation column are as follows: the column size was 250X 20mm, the reverse phase preparation column stationary phase was 7 μm reverse phase column 7-X10, and the mobile phase was 6% methanol-water solution.
6. The method for directional separation and preparation of the dianthus superbus flavone C-glycoside compound according to claim 1, which is characterized in that: in the step 5, the working parameters of reversed-phase preparation liquid chromatography purification refer to a chromatographic column with a column length of 250mm and a diameter of 20mm, a reversed-phase preparation column stationary phase of 5 μm reversed-phase column SunFire C18, and a mobile phase of 20% methanol-water solution.
7. The method for directional separation and preparation of the dianthus superbus flavone C-glycoside compound according to claim 1, which is characterized in that: in the step 6, the working parameters of the hydrophilic preparation liquid chromatography purification are as follows: the chromatographic column size was 250X 20mm, the hydrophilic preparative column stationary phase was 5 μm zwitterionic column ClickXION, and the mobile phase was 90% acetonitrile-5% trifluoroacetic acid in water.
8. The fringed pink flavonoid glycoside compound is characterized in that the structural formula of the fringed pink flavonoid glycoside compound is shown as follows:
9. the application of the dianthus superbus flavonoid glycoside compounds in preparing the hyperglycemia inhibiting medicine is characterized in that the structural formula of the dianthus superbus flavonoid glycoside compounds is shown as follows:
10. the application of the dianthus superbus flavonoid glycoside compounds in preparing the alpha-glucosidase inhibitor is characterized in that the structural formula of the dianthus superbus flavonoid glycoside compounds is shown as follows:
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