CN107325140B - A kind of chromone ketoside compounds isolated and purified from green bamboo mark and extracting method - Google Patents
A kind of chromone ketoside compounds isolated and purified from green bamboo mark and extracting method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 33
- OTAFHZMPRISVEM-UHFFFAOYSA-N chromone Chemical compound C1=CC=C2C(=O)C=COC2=C1 OTAFHZMPRISVEM-UHFFFAOYSA-N 0.000 title claims abstract description 18
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- 239000000287 crude extract Substances 0.000 claims description 28
- 238000005377 adsorption chromatography Methods 0.000 claims description 27
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- 238000001514 detection method Methods 0.000 claims description 25
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
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Classifications
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- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
- C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
- C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Saccharide Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of chromone ketoside compounds isolated and purified from green bamboo mark and extracting method, isolated 6 kinds of chromone ketoside compounds from green bamboo mark: 5- hydroxy-2-methyl -7-O- α-L- rhamnose chromogen ketoside；5- hydroxy-2-methyl -7-O- β-D- xylose chromogen ketoside；5- hydroxy-2-methyl -7-O- [6-OAc- β-D-Glucose base-(1-3)]-α-L- rhamnose chromogen ketoside；5- hydroxy-2-methyl -7-O- [β-D-Glucose base-(1-2)]-α-L- rhamnose chromogen ketoside；2- methylol -5,7- dihydroxy -11-O- β-D-arabinose chromogen ketoside；2- methylol -5,7- dihydroxy -11-O- β-D-arabinose chromogen ketoside；5,7- dihydroxy -2- methyl -8-C- β-D-Glucose chromogen ketoside.The extracting method, raw material sources are extensive, and preparation process is simple, and economic, safety, yield is high, and resulting 6 noval chemical compounds all have anti-inflammatory activity, and toxic side effect is low, has good prospect in medicine.
Description
Technical Field
The invention relates to the technical field of chromone ketoside compounds and preparation methods thereof, in particular to a chromone ketoside compound separated and purified from a green bamboo label and an extraction method thereof.
Background
The chromone glycoside compound is an active substance widely distributed in various families and has the effects of resisting inflammation, easing pain, resisting platelet aggregation, resisting oxidation, inhibiting histamine release and the like. The inflammatory reaction is a complex process that various mediators participate and inflammatory factors continuously fight with the body until a balance is reached, and the traditional Chinese medicine plays an anti-inflammatory role through multiple ways and multiple links. The traditional Chinese medicine has the characteristics of various chemical components, wide pharmacological activity, multiple effects, few adverse reactions and the like, and has very important development and utilization values.
The green bamboo label (Scindapsus of fiscinalis Schott) is named as Millettia odorata, Millettia speciosa, climbing tree dragon, golden bamboo label, and the whole plant of the Araceae Marsdenia tenacissima, mainly distributed in Yunnan, Guizhou and Guangxi, and is a rare ethnic medicine widely applied in local places. Recorded in Yunnan Chinese herbal medicine selection, the medicine has the effects of removing blood stasis, relieving pain, moistening lung and relieving cough, and can be used for treating traumatic injury, fracture, rheumatic numbness, bronchitis and pertussis; recorded in Guangxi medicine plant famous book, the medicine has the effects of relieving swelling and pain, and treating traumatic injury, rheumatism and carbuncle sore; the book of Guizhou medicine plant records that the medicine can remove blood stasis, promote tissue regeneration and relieve pain. The research and report on the new chromone ketoside compound separated and purified from the green bamboo label and the extraction method are not found through the literature search.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a chromone ketoside compound which is separated and purified from a green bamboo label and has anti-inflammatory activity.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a chromogen ketoside compound separated and purified from green bamboo is compound I, compound II, compound III, compound IV, compound V or compound VI; wherein,
the structural formula of the compound I is
The structural formula of the compound II is
Structure of Compound IIIIs of the formula
The structural formula of the compound IV is
The structural formula of the compound V is
The structural formula of the compound VI is
The second purpose of the invention is to provide an extraction method of the chromone ketoside compound, which comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15: 1;
(3) passing the concentrated fraction of elution sites with gradient elution volume ratio of 15:1 from step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, and 30 + -1% MeOH;
(4) passing the 30 + -1% MeOH elution site of step (3) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(5) eluting 45 + -1% MeOH from step (4) with CH3CN and H2And preparing the compound I by using a mobile phase with the volume ratio of O being 20:80 under the conditions that the flow rate is 3mL/min and the detection wavelength is 210 nm.
The invention also aims to provide an extraction method of the chromone ketoside compound, which comprises the following steps of:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 15:1 in the step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(4) passing the 45 + -11% MeOH elution site of step (3) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(5) eluting 45 + -1% MeOH from step (4) with CH3CN and H2And (3) preparing the compound II by using a mobile phase with the volume ratio of O being 18:82 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
The fourth object of the present invention is to provide a method for extracting the chromone glycoside compound, wherein the step of extracting the compound III is:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10: 1;
(3) passing the concentrated fraction of elution sites with gradient elution volume ratio of 10:1 from step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 30 + -1% MeOH, 40 + -1% MeOH, and 60 + -1% MeOH;
(4) passing the 60 +/-1% MeOH elution part of the step (3) through Sepadex LH-20 gel column chromatography, and eluting with MeOH to obtain 9 elution parts;
(5) using CH for the 6 th elution part in the step (4)3CN and H2And preparing the compound III by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
The fifth purpose of the invention is to provide an extraction method of the chromone glycoside compound, wherein the step of extracting the compound IV is as follows:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and eluting with 5 + -1% MeOH and 30 + -1% MeOH in sequence;
(4) passing the 30 +/-1% MeOH elution part in the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting 6 th elution part of MeOH in the step (4) with CH3CN and H2And (3) preparing a compound IV by using a mobile phase with the volume ratio of O being 18:82 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
The sixth purpose of the invention is to provide an extraction method of the chromone glycoside compound, wherein the step of extracting the compound V is as follows:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 30 + -1% MeOH, and 40 + -1% MeOH;
(4) passing the 40 +/-1% MeOH elution part of the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting 13 th elution part of MeOH in the step (4) with CH3CN and H2And (3) preparing the compound V by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
The seventh object of the present invention is to provide a method for extracting the above chromone glycoside compound, wherein the step of extracting the compound VI is:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH and 12 + -1% MeOH;
(4) passing the 12 +/-1% MeOH elution part in the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting the 5 th elution part of MeOH in the step (4) with CH3CN and H2And (3) preparing the compound VI by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
The eighth purpose of the invention is to provide an application of the chromone ketoside compound or the chromone ketoside compound prepared by the extraction method in preparing anti-inflammatory drugs.
The invention has the beneficial effects that:
the new chromone ketoside compound separated and purified from the green bamboo label and the preparation method thereof take the green bamboo label as a raw material, have wide sources, simple preparation process, economy, safety and high yield, and the obtained 6 new compounds all have anti-inflammatory activity, wherein the middle compound III has better anti-inflammatory activity, the compound V has moderate anti-inflammatory activity, and has low toxic and side effects and good medicinal prospect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 is a drawing showing 5-hydroxy-2-methyl-7-O-alpha-L-rhamnose chromone glycoside1H NMR；
FIG. 2 is a drawing showing 5-hydroxy-2-methyl-7-O-alpha-L-rhamnose chromone glycoside13C NMR；
FIG. 3 is a HRESI-MS cation diagram of 5-hydroxy-2-methyl-7-O- α -L-rhamnose chromone glycoside;
FIG. 4 is a drawing showing 5-hydroxy-2-methyl-7-O-beta-D-xylosylchromone glycoside1H NMR；
FIG. 5 is a drawing showing 5-hydroxy-2-methyl-7-O-beta-D-xylosylchromone glycoside13C NMR；
FIG. 6 is the HRESI-MS cation diagram of 5-hydroxy-2-methyl-7-O- β -D-xylosylchromone glycoside;
FIG. 7 shows 5-hydroxy-2-methyl-7-O- [ 6-OAc-beta-D-glucosyl- (1-3)]Method for producing alpha-L-rhamnosolone glycosides1H NMR；
FIG. 8 shows 5-hydroxy-2-methyl-7-O- [ 6-OAc-beta-D-glucosyl- (1-3)]Method for producing alpha-L-rhamnosolone glycosides13C NMR；
FIG. 9 is a HRESI-MS cation diagram of 5-hydroxy-2-methyl-7-O- [6-OAc- β -D-glucosyl- (1-3) ] - α -L-rhamnose chromone glycoside;
FIG. 10 shows 5-hydroxy-2-methyl-7-O- [ beta-D-glucosyl- (1-2)]Method for producing alpha-L-rhamnosolone glycosides1H NMR；
FIG. 11 is 5-hydroxy-2-methyl-7-O- [ beta-D-glucosyl- (1-2)]Method for producing alpha-L-rhamnosolone glycosides13C NMR；
FIG. 12 is the HRESI-MS cation diagram of 5-hydroxy-2-methyl-7-O- [ β -D-glucosyl- (1-2) ] - α -L-rhamnosoloside;
FIG. 13 is a drawing showing the synthesis of 2-hydroxymethyl-5, 7-dihydroxy-11-O-beta-D-arabinochromone glycoside1H NMR；
FIG. 14 is a drawing showing the synthesis of 2-hydroxymethyl-5, 7-dihydroxy-11-O-beta-D-arabinochromone glycoside13C NMR；
FIG. 15 is a HRESI-MS cation diagram of 2-hydroxymethyl-5, 7-dihydroxy-11-O- β -D-arabinochromone glycoside;
FIG. 16 is a drawing showing 5, 7-dihydroxy-2-methyl-8-C-. beta. -D-glucono-ketoside1H NMR；
FIG. 17 is a drawing showing 5, 7-dihydroxy-2-methyl-8-C-. beta. -D-glucono-ketoside13C NMR；
FIG. 18 is an HRESI-MS anion diagram of 5, 7-dihydroxy-2-methyl-8-C-. beta. -D-glucono-chromone glycoside.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
All the percentages in the present application are volume percentages unless otherwise specified.
The elution procedure in the application adopts the macroporous resin column as an open type, the flow rate is not fixed, and the time is not fixed.
As described in the background art, there is no record of a new chromone ketoside compound separated and purified from a green bamboo mark and an extraction method in the prior art, and in order to solve the technical problems, the application provides a chromone ketoside compound separated and purified from a green bamboo mark and an extraction method.
A typical embodiment of the application provides a chromogen ketoside compound separated and purified from a green bamboo mark, which is a compound I, a compound II, a compound III, a compound IV, a compound V or a compound VI; wherein,
the structural formula of the compound I isIs named as 5-hydroxy-2-methyl-7-O-alpha-L-rhamnose chromone glycoside;
the structural formula of the compound II isNamed as 5-hydroxy-2-methyl-7-O-beta-D-xylosylchromone glycoside;
the structural formula of the compound III isNamed as 5-hydroxy-2-methyl-7-O- [ 6-OAc-beta-D-glucosyl- (1-3)]- α -L-rhamnose chromone glycoside;
the structural formula of the compound IV isIs named as 5-hydroxy-2-methyl-7-O- [ beta-D-glucosyl- (1-2)]- α -L-rhamnose chromone glycoside;
the structural formula of the compound V isNamed as 2-hydroxymethyl-5, 7-dihydroxy-11-O-beta-D-arabinochromone glycoside;
the structural formula of the compound VI isNamed as 7-dihydroxy-2-methyl-8-C-beta-D-glucochromone glycoside.
In a second exemplary embodiment of the present invention, there is provided a method for extracting the chromone glycoside compound, wherein the method for extracting the compound i comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15: 1;
(3) passing the concentrated fraction of elution sites with gradient elution volume ratio of 15:1 from step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, and 30 + -1% MeOH;
(4) passing the 30 + -1% MeOH elution site of step (3) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(5) eluting 45 + -1% MeOH from step (4) with CH3CN and H2And preparing the compound I by using a mobile phase with the volume ratio of O being 20:80 under the conditions that the flow rate is 3mL/min and the detection wavelength is 210 nm.
In a third exemplary embodiment of the present invention, there is provided a method for extracting chromone glycoside compound, wherein the step of extracting compound ii is:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15: 1;
(3) gradient elution volume of step (2)Concentrated fractions of elution sites with a 15:1 ratio were passed through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(4) passing the 45 + -11% MeOH elution site of step (3) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(5) eluting 45 + -1% MeOH from step (4) with CH3CN and H2And (3) preparing the compound II by using a mobile phase with the volume ratio of O being 18:82 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
In a fourth exemplary embodiment of the present invention, there is provided a method for extracting a chromone glycoside compound, wherein the method for extracting a compound iii comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10: 1;
(3) passing the concentrated fraction of elution sites with gradient elution volume ratio of 10:1 from step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 30 + -1% MeOH, 40 + -1% MeOH, and 60 + -1% MeOH;
(4) passing the 60 +/-1% MeOH elution part of the step (3) through Sepadex LH-20 gel column chromatography, and eluting with MeOH to obtain 9 elution parts;
(5) using CH for the 6 th elution part in the step (4)3CN and H2And preparing the compound III by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
In a fifth exemplary embodiment of the present invention, there is provided a method for extracting the chromone glycoside compound, wherein the method for extracting the compound iv comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and eluting with 5 + -1% MeOH and 30 + -1% MeOH in sequence;
(4) passing the 30 +/-1% MeOH elution part in the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting 6 th elution part of MeOH in the step (4) with CH3CN and H2And (3) preparing a compound IV by using a mobile phase with the volume ratio of O being 18:82 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
In a sixth exemplary embodiment of the present invention, there is provided a method for extracting a chromone glycoside compound, wherein the method for extracting the compound v comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 30 + -1% MeOH, and 40 + -1% MeOH;
(4) passing the 40 +/-1% MeOH elution part of the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting 13 th elution part of MeOH in the step (4) with CH3CN and H2And (3) preparing the compound V by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
In a seventh exemplary embodiment of the present invention, there is provided a method for extracting the chromone glycoside compound, wherein the method for extracting the compound vi comprises the steps of:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH and 12 + -1% MeOH;
(4) passing the 12 +/-1% MeOH elution part in the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting the 5 th elution part of MeOH in the step (4) with CH3CN and H2And (3) preparing the compound VI by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm.
In order to better obtain the crude extract of the green bamboo mark, the crude extract of the green bamboo mark in the step (1) is preferably extracted by heating and refluxing with 95% ethanol.
Preferably, the extraction process of the crude extract of the green bamboo mark comprises the steps of taking a green bamboo mark medicinal material, crushing, heating and refluxing with 95% ethanol at a solid-to-liquid ratio of 1:3 for three times of 2h, 1h and 1h, respectively, combining filtrates, carrying out reduced pressure rotary evaporation, and carrying out freeze drying to obtain the crude extract of the green bamboo mark.
In order to disperse the crude extract of the green bamboo mark into water more uniformly, the crude extract of the green bamboo mark in the step (1) is preferably added into water for ultrasonic dispersion.
The instrument used for preparing the compounds I to VI in the step (5) is a semi-preparative high performance liquid chromatograph.
Pre-and post-concentration fractions as referred to herein refer to each gradient wash CH as the gradient elution is performed2Cl2Volume of MeOH V, concentrate to 1 fraction per 1/2V pool, i.e. 2 concentrated fractions per gradient pool, first 1/2V to pre-concentrated fraction and last 1/2V to post-concentrated fraction.
In an eighth embodiment of the present application, there is provided an application of the above-mentioned chromone glycoside compound or the chromone glycoside compound prepared by the above-mentioned extraction method in preparing an anti-inflammatory drug.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific examples and comparative examples.
Examples
The preparation method of the compounds I-VI comprises the following steps:
(1) taking 5kg of the green bamboo standard medicinal material, crushing, heating and refluxing with 95% ethanol at a solid-liquid ratio of 1:3 for three times of 2h, 1h and 1h respectively, combining the filtrates, carrying out reduced pressure rotary steaming, and freeze-drying to obtain 1kg of the green bamboo standard crude extract;
(2) adding appropriate amount of water into the crude extract, ultrasonic pulverizing, extracting with petroleum ether, ethyl acetate and n-butanol, filtering the extractive solution, and concentrating under reduced pressure to obtain petroleum ether, ethyl acetate, n-butanol and water extract;
(3) dissolving 180g of the obtained ethyl acetate part in methanol, adding 300g of 200-300-mesh silica gel for sample mixing, volatilizing the solvent, and performing volume ratio CH2Cl2Gradient elution with MeOH (100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5:1 → 1:1 → 0:1) to give elution sites, concentration, with each gradient pooled into 2 fractions before and after (5000 mL per gradient wash, pooled and concentrated into 1 fraction per 2500mL, i.e., 2 fractions per gradient pooled, the first 2500mL being the front fraction, and the second 2500mL being the back fraction).
(4) Will CH2Cl2The MeOH (15:1) preflux is passed through C18Adsorption resin chromatographyElution was performed with 5%, 12%, 30%, 45%, 60%, 80%, and 100% MeOH in this order to obtain elution sites. C is to be18The 30% MeOH portion of the column was then passed through C18And (4) performing resin adsorption chromatography, and eluting with 5%, 12%, 30%, 45%, 60%, 80% and 100% MeOH in sequence to obtain each elution part. C is to be18The 45% MeOH fraction of the column was prepared using Shimadzu LC-6AD semi-preparative HPLC, and two mobile phases AB were each set to CH3CN and H2O，CH3CN and H2The volume ratio of O is 20:80(v/v) and the flow rate is 3mL min-1The detection wavelength was 210nm, and 80. mu.L of each sample was injected to give 55mg of Compound I.
(5) Will CH2Cl2after-MeOH (15:1) the aliquot is passed through C18And (4) performing resin adsorption chromatography, and eluting with 5%, 12%, 30%, 45%, 60%, 80% and 100% MeOH in sequence to obtain each elution part. C is to be18The 45% MeOH portion of the column was then passed through C18And (4) performing resin adsorption chromatography, and eluting with 5%, 12%, 30%, 45%, 60%, 80% and 100% MeOH in sequence to obtain each elution part. C is to be1845% MeOH portion of the column with CH3CN/H2O (18:82, v/v) was prepared (Shimadzu LC-6AD semi-preparative HPLC, flow rate: 3mL min-1(ii) a Detection wavelength: 252nm) to give 110mg of compound II.
(6) Will CH2Cl2The MeOH (10:1) front stream was passed through C18And (4) performing resin chromatography, and eluting with 5%, 30%, 40%, 60%, 80% and 100% MeOH in sequence to obtain each elution part. C is to be18The 60% MeOH fraction from the column was chromatographed on a SephadexLH-20 gel column, eluting with MeOH, to give 9 elution fractions. The 6 th elution site was treated with CH3CN/H2O (28:72, v/v) was prepared (Shimadzu LC-6AD semi-preparative HPLC, flow rate: 3mL min-1(ii) a Detection wavelength: 252nm) to yield 60mg of compound III.
(7) Will CH2Cl2The MeOH (5:1) preflux is passed through C18Adsorbing with adsorbent resin chromatography sequentially at 5%, 30%, 40%, 60%, 80%, 10%Elution with 0% MeOH gave individual elution sites. C is to be18The 30% MeOH fraction from the column was chromatographed on a Sepadex LH-20 gel column, eluting with MeOH, to give 9 elution fractions. The 6 th elution site was treated with CH3CN/H2O (18:82, v/v) was prepared (Shimadzu LC-6AD semi-preparative HPLC, flow rate: 3mL min-1(ii) a Detection wavelength: 252nm) to yield 80mg of compound IV. C is to be18The 40% MeOH fraction from the column was chromatographed on a Sepadex LH-20 gel column eluting with MeOH to give 18 elution fractions. The 13 th elution site was treated with CH3CN/H2O (28:72, v/v) was prepared (Shimadzu LC-6AD semi-preparative HPLC, flow rate: 3mL min-1(ii) a Detection wavelength: 252nm) to yield 180mg of compound V.
(8) Will CH2Cl2after-MeOH (5:1) the aliquot is passed through C18And (4) performing resin chromatography, and eluting with 5%, 12%, 30%, 60% and 100% MeOH in sequence to obtain each elution part. C is to be18The 12% MeOH fraction from the column was chromatographed on a Sepadex LH-20 gel column eluting with MeOH to give 7 elution fractions. Subjecting the 5 th elution site to CH3CN/H2O (18:82, v/v) was prepared (Shimadzu LC-6AD semi-preparative HPLC, flow rate: 3mL min-1(ii) a Detection wavelength: 252nm) to yield compound VI 430 mg.
And (3) structural identification: and (3) respectively measuring MS and NMR spectrums of the separated monomer components by using an Agilent 5973N mass spectrometer and a Burker 400MHz nuclear magnetic resonance spectrometer, wherein the obtained nuclear magnetic data are shown in tables 1-2, and identifying the structures of 6 new chromone glycoside compounds I, II, III, IV, V and VI.
A compound I: the 5-hydroxy-2-methyl-7-O-alpha-L-rhamnose chromone glycoside is yellow powder, the chemical structure representation of which is shown in figure 1-3, HR-ESIMS gives a molecular ion peak M/z 339.1030[ M + H ] 339.1030]+(calcd for C16H19O8339.1035), in combination1H NMR and13the C NMR spectrum assumed that Compound I has the formula C16H18O8. IR shows hydroxyl group (3400 cm)-1)，Conjugated carbonyl (1667 cm)-1) And double bonds (1624,1581,1508 cm)-1) The absorption peak of (1).1The H NMR spectrum shows the characteristic signals of 2-methyl-5, 7-dioxy substituted chromone compounds: deltaH2.38(3H, s, Me-11), 6.26(1H, s, H-3), 6.67(1H, d, J ═ 2.4Hz, H-8), 6.43(1H, d, J ═ 2.4Hz, H-6), 12.82(s, OH-5). In addition to this, the present invention is,1the H NMR spectrum also gives the terminal hydrogen signal of 1 saccharide: deltaH4.91(1H, d, J ═ 10.0Hz, H-1') and 1 bimodal methyl signal: deltaH 1.11(3H,d,J＝6.0Hz,H-6')。13C and HSQC, HMBC spectra show that compound I has 16 carbon atoms in total, wherein 11 carbons are aglycon signals, and 6 carbons are sugar signals. Comparison of Compound I with 5,7-dihydroxy-2-methyl chromone1H and13c NMR data, which were found to be structurally similar, the main difference being that 5,7-dihydroxy-2-methyl chromone had an OH substitution at C-7, but no OH substitution at C-2 of compound I, as further demonstrated by the dehydroxylation shifts of compound I, C-6(+0.7), C-7(-2.7), C-8(+0.1) and C-10(+ 0.4). Compound I was subjected to acid hydrolysis and detected by GC to contain an L-rhamnose. The position of the sugar attachment is determined by HMBC spectroscopy: deltaH4.91(H-1' of α -L-rhamnose) and δC161.5 (C-7). In conclusion, the structure of the compound I is determined to be 5-hydroxy-2-methyl-7-O-alpha-L-rhamnose chromone glycoside.
Compound ii: the chemical structural characterization of 5-hydroxy-2-methyl-7-O-beta-D-xylosylchromone glycoside yellow powder is shown in figures 4-6, and HR-ESIMS gives a molecular ion peak M/z 325.2520[ M + H ] 325.2520]+(calcd for C15H17O8325.0879), in combination1H NMR and13the C NMR spectrum assumed that Compound II has the formula C15H16O8. IR shows a hydroxyl group (3350 cm)-1) Conjugated carbonyl group (1669 cm)-1) And double bonds (1623,1580,1509 cm)-1) The absorption peak of (1). Comparison of Compounds II and I1H NMR and13c NMR spectrum data show that the structures of the two are similar, and the main difference is the sugar chain connected to the C-7 position. Compound II was subjected to acid hydrolysis and was found to contain a D-xylose by GC. The position of the sugar attachment is determined by HMBC spectroscopy: deltaH5.06(H-1' of β -D-xylose) and δC163.1 (C-7). In conclusion, the structure of the compound II is determined to be 5-hydroxy-2-methyl-7-O-beta-D-xylosylchromone glycoside.
Compound iii: 5-hydroxy-2-methyl-7-O- [ 6-OAc-beta-D-glucosyl- (1-3)]The chemical structure of the-alpha-L-rhamnose tropone glycoside yellow powder is characterized as shown in figures 7-9, and HR-ESIMS gives a molecular ion peak M/z 543.1794[ M + H ]]+(calcd for C24H31O14543.1669), in combination1H NMR and13c NMR spectrum presumes that the formula of the compound III is C24H30O14. IR shows a hydroxyl group (3395 cm)-1) Carbonyl group (1742 cm)-1) Conjugated carbonyl group (1669 cm)-1) And double bonds (1625,1590,1505 cm)-1) The absorption peak of (1). Comparison of Compounds III with I and II1H NMR and13c NMR spectrum data show that the two structures are similar, and the main difference is that the sugar chain connected to the C-7 position is more than 1 CH in the compound III3CO-functional group [ delta ]H 1.99(3H,s,CH 3CO-),δC 21.1(CH 3CO-) and 170.6 (CH)3 CO-)]。1The H NMR spectrum gives the terminal hydrogen signals of 2 sugars: 5.55(1H, s, H-1') and 4.40(1H, d, J ═ 8.0Hz, H-1") and 1 bimodal methyl signal: deltaH1.15(3H, d, J ═ 5.6Hz, H-6'). Compound III was subjected to acid hydrolysis and detected by GC to contain 1L-rhamnose and 1D-glucose. Sugar and CH3The CO-junction location was determined by HMBC spectroscopy: deltaH 1.99(3H,s,CH 3CO-) with 64.1(C-6 '), 4.40 (H-1' of beta-D-glucose) and deltaC82.1(C-3'), 5.55(H-1' of α -L-rhamnose) and δC161.9 (C-7). In conclusion, the structure of the compound III is determined to be 5-hydroxy-2-methyl-7-O- [ 6-OAc-beta-D-glucosyl- (1-3)]- α -L-rhamnosoloside.
A compound IV: 5-hydroxy-2-methyl-7-O- [ beta-D-glucosyl- (1-2)]The chemical structure of the-alpha-L-rhamnose tropone glycoside yellow powder is characterized as shown in figures 10-12, and HR-ESIMS gives a molecular ion peak M/z 501.1561[ M + H ]]+(calcdfor C22H29O13501.1563), in combination1H NMR and13the C NMR spectrum of the compound IV presumes that the molecular formula is C22H28O13. IR shows a hydroxyl group (3409 cm)-1) Conjugated carbonyl group (1665 cm)-1) And double bonds (1626,1590,1502 cm)-1) The absorption peak of (1). Comparison of Compounds IV with I, II, III1H NMR and13c NMR spectrum data show that the structures of the two are similar, and the main difference is the sugar chain connected to the C-7 position.1The H NMR spectrum gives the terminal hydrogen signals of 2 sugars: 5.55(1H, s, H-1') and 4.43(1H, d, J ═ 8.0Hz, H-1") and 1 bimodal methyl signal: deltaH1.21(3H, d, J ═ 5.6Hz, H-6'). Compound III was subjected to acid hydrolysis and detected by GC to contain 1L-rhamnose and 1D-glucose. The position of linkage of the sugar chain is determined by HMBC spectroscopy: deltaH4.40(H-1 "of. beta. -D-glucose) and. deltaC81.2(C-2'), 5.55(H-1' of. alpha. -L-rhamnose) and. deltaC161.9 (C-7). In conclusion, the structure of the compound IV is determined to be 5-hydroxy-2-methyl-7-O- [ beta-D-glucosyl- (1-2)]- α -L-rhamnosoloside.
Compound v: the chemical structure of the 2-hydroxymethyl-5, 7-dihydroxy-11-O-beta-D-arabinopyranoside is characterized as shown in figure 13-15, and HR-ESIMS gives a molecular ion peak M/z 341.1627[ M + H ] 341.1627]+(calcd forC15H17O9341.0828), in combination1H NMR and13the molecular formula of the compound V is presumed to be C by C NMR spectrum15H16O9. IR shows hydroxyl group (3400 cm)-1) Conjugated carbonyl group (1662 cm)-1) And double bonds (1630,1589,1515 cm)-1) The absorption peak of (1).1The HNMR spectra show the characteristic signals of 2-hydroxymethyl-5, 7-dihydroxy-substituted chromonic compounds: deltaH4.73(1H, d, J ═ 15.6Hz, Ha-11), 4.59d (1H, d, J ═ 15.6Hz, Hb-11), 6.20(1H, s, H-8), 6.36(1H, s, H-6), 6.53(1H, s, H-3), 10.00(s,7-OH), 12.82(s, OH-5). In addition to this, the present invention is,1the H NMR spectrum also gives the terminal hydrogen signal of 1 saccharide: deltaH 4.47(1H,d,J＝6.8Hz,H-1')。13C and HSQC, HMBC spectra show that compound V has 15 carbon atoms in total, wherein 11 carbons are aglycone signals, and 5 carbons are sugar signals. Comparison of Compound V with 5, 7-dihydroxy-2-hydroxyymethylOf chromone1H and13c NMR data, found to be structurally similar, the main difference being that C-11 of 5,7-dihydroxy-2-hydroxymethyl chromone has an OH substitution, while C-11 of compound V has no OH substitution, which is further confirmed by the dehydroxylation shift C-11(+6.6) of compound V. Compound V was subjected to acid hydrolysis and was detected by GC to contain a D-arabinosine. The position of the sugar attachment is determined by HMBC spectroscopy: deltaH4.47(H-1' of. beta. -D-arabinosine) with. deltaC66.1 (C-11). In conclusion, the structure of the compound V is determined to be 2-hydroxymethyl-5, 7-dihydroxy-11-O-beta-D-arabinochromone glycoside.
Compound vi: 5, 7-dihydroxy-2-methyl-8-C-beta-D-glucochromone glycoside yellow powder, the chemical structure of which is characterized as shown in figure 16-18, and HR-ESIMS gives a molecular ion peak M/z 353.0851[ M-H]+(calcd for C16H17O9353.0984), in combination1H NMR and13the molecular formula of the compound V is presumed to be C by C NMR spectrum16H18O9. IR shows a hydroxyl group (3385 cm)-1) Conjugated carbonyl group (1664 cm)-1) And double bonds (1623,1593,1517 cm)-1) The absorption peak of (1).1The H NMR spectrum shows the characteristic signals of the chromone compounds of 2-methyl-5, 7-dihydroxy-8-carbon glycoside: deltaH2.35(3H, s, Me-11), 6.19(1H, s, H-3), 6.26(1H, s, H-6), 10.70(s,7-OH), 13.00(s, 5-OH). In addition to this, the present invention is,1the H NMR spectrum also gives the terminal hydrogen signal of 1 saccharide: deltaH 4.63(1H,d,J＝9.2Hz,H-1')。13C, HSQC and HMBC spectra show that the compound VI has 16 carbon atoms, wherein 11 carbons are aglycone signals, and 6 carbons are sugar signals. Comparison of Compound VI with 5,7-dihydroxy-2-methyl chromone1H and13c NMR data found to be structurally similar, the main difference being that the 5,7-dihydroxy-2-methyl chromone has no C-8 substitution, while compound VI has a carbon glycoside substitution at the C-8 position, which is further evidenced by the glycosidation shifts of compound VI, C-7(-1.5), C-8(+10.2), and C-9 (-5.0). Compound VI was subjected to acid hydrolysis and was found to contain a D-glucose by GC. The position of the sugar attachment is determined by HMBC spectroscopy: deltaH4.63(H-1' of beta-D-glucose) and deltaC105.0 (C-8). In conclusion, the structure of the compound VI is determined to be 5, 7-dihydroxy-2-methyl-8-C-beta-D-glucochromone glycoside.
TABLE 1 of Compounds I to VI1H NMR spectroscopic data (400MHz, DMSO-d)6,δppm,J,Hz)
TABLE 2 of Compounds I to VI13C NMR spectroscopic data (400MHz, DMSO-d)6,δppm)
Pharmacological experiment:
toxicity of the compound to cells: the culture medium was discarded from the above 24-well plate by the MTT method, and a freshly prepared serum-free medium containing 0.50mg/mL MTT was added to each well, followed by further culture at 37 ℃ for 30min and then the supernatant was removed. Add 50. mu.L DMSO per well to dissolve the formazan pellet. The optical density at 570nm was determined on a Perkin Elmer EnSpire type microplate reader.
Anti-inflammatory activity: taking macrophage of RAW264.7 mouse, counting, and dividing by 5 × 105Perwell was seeded in 24-well cell culture plates. Using DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin, cells were incubated at 37 ℃ with 5% CO2The wall is attached to the incubator for 12h under saturated humidity. The experiment was divided into a blank control group, an LPS group, an administration group and a DEX group, wherein only the culture medium was added to the control group, only 1. mu.g/mL LPS was added to the LPS group, 1. mu.g/mL LPS and 10. mu.M dexamethasone were added to the DEX group, 1. mu.g/mL LPS and 10. mu.M of the sample to be tested were added to the administration group, and the reaction was carried out at 37 ℃ under 5% CO2Culturing in an incubator with saturated humidity for 24 h. Taking out 60 μ L of supernatant, adding Griess reagent for 10 min, measuring OD (optical density) value with enzyme labeling instrument at 570nm, and calculating NO generation according to NO standard curveAmount of the compound (A).
TABLE 3.6 inhibition of macrophage NO release in RAW264.7 mice by Compounds
And (4) conclusion: toxicity of 6 compounds on LPS-induced RAW264.7 mouse macrophage and inhibition effect on cell NO release are determined by adopting an MTT method, and detection results are shown in Table 3, wherein 6 compounds have NO inhibition effect on proliferation of LPS-induced RAW264.7 mouse macrophage and show inhibition effects of different degrees on NO release of LPS-induced RAW264.7 mouse macrophage.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (8)
1. A method for extracting chromone glycoside compounds is characterized in that the method for extracting the compounds I comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15: 1;
(3) passing the concentrated fraction of elution sites with gradient elution volume ratio of 15:1 from step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, and 30 + -1% MeOH;
(4) passing the 30 + -1% MeOH elution site of step (3) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(5) eluting 45 + -1% MeOH from step (4) with CH3CN and H2Preparing a compound I by using a mobile phase with the volume ratio of O being 20:80 under the conditions that the flow rate is 3mL/min and the detection wavelength is 210 nm;
the structural formula of the compound I is
2. A method for extracting chromone glycoside compounds is characterized in that the method for extracting the compounds II comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 15:1 in the step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(4) passing the 45 + -11% MeOH elution site of step (3) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 12 + -1% MeOH, 30 + -1% MeOH, and 45 + -1% MeOH;
(5) eluting 45 + -1% MeOH from step (4) with CH3CN and H2Preparing a compound II by a mobile phase with the volume ratio of O being 18:82 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm;
the structural formula of the compound II is
3. A method for extracting chromone ketoside compounds is characterized in that the method for extracting the compounds III comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10: 1;
(3) passing the concentrated fraction of elution sites with gradient elution volume ratio of 10:1 from step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 30 + -1% MeOH, 40 + -1% MeOH, and 60 + -1% MeOH;
(4) passing the 60 +/-1% MeOH elution part of the step (3) through Sepadex LH-20 gel column chromatography, and eluting with MeOH to obtain 9 elution parts;
(5) using CH for the 6 th elution part in the step (4)3CN and H2Preparing a compound III by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm;
the structural formula of the compound III is
4. A method for extracting chromone glycoside compounds is characterized in that the method for extracting the compounds IV comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and eluting with 5 + -1% MeOH and 30 + -1% MeOH in sequence;
(4) passing the 30 +/-1% MeOH elution part in the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting 6 th elution part of MeOH in the step (4) with CH3CN and H2Preparing a compound IV by using a mobile phase with the volume ratio of O to O of 18:82 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm;
the structural formula of the compound IV is
5. A method for extracting a chromone ketoside compound is characterized in that the method for extracting the compound V comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH, 30 + -1% MeOH, and 40 + -1% MeOH;
(4) passing the 40 +/-1% MeOH elution part of the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting 13 th elution part of MeOH in the step (4) with CH3CN and H2Preparing a compound V by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm;
the structural formula of the compound V is
6. A method for extracting chromone glycoside compounds is characterized in that the method for extracting the compounds VI comprises the following steps:
(1) dispersing the crude extract of the green bamboo mark into water, sequentially extracting by using petroleum ether and ethyl acetate, and removing the solvent from the extracted ethyl acetate organic phase to obtain an ethyl acetate extract;
(2) dissolving the ethyl acetate extract in methanol, and performing gradient elution with CH through silica gel chromatographic column2Cl2-MeOH volume ratio of 100:1 → 50:1 → 25:1 → 15:1 → 10:1 → 5: 1;
(3) passing the concentrated fraction of the elution part with the gradient elution volume ratio of 5:1 in the step (2) through C18Performing resin adsorption chromatography, and sequentially eluting with 5 + -1% MeOH and 12 + -1% MeOH;
(4) passing the 12 +/-1% MeOH elution part in the step (3) through Sepadex LH-20 gel column chromatography, and eluting MeOH;
(5) eluting the 5 th elution part of MeOH in the step (4) with CH3CN and H2Preparing a compound VI by using a mobile phase with the volume ratio of O being 28:72 under the conditions that the flow rate is 3mL/min and the detection wavelength is 252 nm;
the structural formula of the compound VI is
7. The method according to any one of claims 1 to 6, wherein the crude extract of the Phyllostachys nigra of step (1) is extracted by heating and refluxing with 95% ethanol.
8. The extraction method according to any one of claims 1 to 6, wherein the crude extract of the green bamboo mark in the step (1) is added into water for ultrasonic dispersion.
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