CN108299368B - Flavonoid compound and preparation method and application thereof - Google Patents
Flavonoid compound and preparation method and application thereof Download PDFInfo
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
- C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
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Abstract
The invention discloses a flavonoid compound, the chemical structure of which is determined by nuclear magnetic resonance hydrogen spectrum, carbon spectrum, high resolution mass spectrum and chemical method, and the chemical name of the flavonoid compound is 3- (4-hydroxyphenyl) -6, 8-dimethoxy-7- (3-methylbut-2-en-1-yl) benzopyran-4-one. Meanwhile, a corresponding preparation method is also established, namely: extracting the rattan of derris elliptica with ethanol, detecting the ethanol extract by thin-layer chromatography, performing silica gel column chromatography, performing gradient elution by using petroleum ether-ethyl acetate eluent system, and performing systematic separation and recrystallization to obtain the derris elliptica extract. DPPH free radical scavenging active IC50Close to the positive control drug vitamin C and scavenging activity IC for superoxide radical50The value is less than that of a positive control ascorbic acid, and the antioxidant activity in vitro is excellent. Therefore, the flavonoid compound has great development potential in the aspects of medicinal or non-medicinal antioxidants.
Description
Technical Field
The invention belongs to the technical field of flavonoid compounds, and particularly relates to a flavonoid compound and a preparation method and application thereof, in particular to a brand-new flavonoid derivative compound extracted and separated from derris elliptica rattan and application thereof in antioxidation.
Background
The Derris elliptica is dry rattan and rhizome of Derris elliptica (Derris eriocarpa How) of leguminous plants, is named as caulis spatholobi, rattan licorice, soil licorice and the like, belongs to climbing shrubs, is mainly distributed in Guangxi and Yunnan provinces of China, and is abundant in Derris elliptica resources and large in sales. The roots and stems of derris elliptica can be used as a medicine, the stems are brownish yellow, the surfaces of the stems are uneven, the cut surfaces of the derris elliptica show faint yellow fibers, the derris elliptica have the needle-shaped holes and are closely arranged, the texture is hard and the derris elliptica are not easy to break, the derris elliptica is commonly used in folk recipes of Guangxi Zhuang nationality, the derris elliptica has the effects of promoting urination and removing dampness, and relieving cough and reducing phlegm, and can be used for treating various diseases such as cystitis, urethritis, nephritis and the like.
The oxidative free radicals are intermediate metabolites of various biochemical and physiological reactions in the life activities of organisms, and play an important role in the life activities of the organisms. However, an excess of free radicals has a wide range of pathological effects, which can adversely affect the life activities of the body. Many studies have shown that human aging, cellular canceration and inflammatory responses, senile dementia, bradykinesia, cardiovascular and cerebrovascular diseases, etc. are directly or indirectly related to excessive free radicals in vivo. The body can fight against the excess oxidative free radicals in the body by supplementing a suitable amount of exogenous antioxidants. The natural product can remove excessive oxygen free radicals in vivo and has beneficial effect on organisms, so that the antioxidant active substance in the natural product meets the requirement, and the natural product also becomes a research hotspot in the fields of oxidation resistance and removal of oxygen free radicals of organisms.
The derris elliptica rattans are rich in various flavonoid compounds and are one of the main active ingredients. So far, the research on the derris elliptica rattan has resource investigation, the identification of medicinal material properties, thin-layer chromatography and ultraviolet fingerprint, the research on the pharmacological and pathological aspects of anti-inflammation, analgesia, cough relieving, phlegm eliminating and the like, the research on the process, the biological activity, the chemical components and the like is relatively less, and the research on the extraction process of flavonoid compounds and the research on the action of antioxidant free radicals is not reported.
Disclosure of Invention
The invention aims to solve the technical problem of providing a flavonoid compound and a preparation method and application thereof.
In order to solve the technical problems, the invention adopts the following technical scheme:
the flavonoid compound has a chemical structural formula as follows:
the chemical name is: 3- (4-hydroxyphenyl) -6, 8-dimethoxy-7- (3-methylbut-2-en-1-yl) chromen-4-one (3- (4-hydroxyphenyl) -6, 8-dimethoxy-7- (3-methyllbut-2-en-1-y 1) -4H-chromen-4-one).
The preparation method of the flavonoid compound is to extract and separate the flavonoid compound from the rattan of derris elliptica.
The preparation method of the flavonoid compound comprises the steps of extracting the stems of derris elliptica with ethanol, detecting the ethanol extract by using a thin-layer chromatography, performing silica gel column chromatography, performing gradient elution by using a petroleum ether-ethyl acetate eluent system, and further performing systematic separation and recrystallization to obtain the flavonoid compound.
The preparation method of the flavonoid compound comprises the following steps:
drying the stems of derris elliptica in the shade, crushing, sieving, weighing crushed derris elliptica stem powder, adding 10 times of 95% ethanol solution, carrying out reflux extraction for 2h in a water bath at 80 ℃, carrying out reflux extraction for three times, filtering, combining filtrate, concentrating the filtrate under reduced pressure, and drying to obtain an extract;
repeatedly extracting the extract obtained in the step (1) with petroleum ether for multiple times, combining the filtrates, concentrating under reduced pressure, and drying to obtain a petroleum ether layer extract;
separating the petroleum ether layer extract obtained in the step (2) by a reverse silica gel chromatographic column, performing gradient elution by using petroleum ether-ethyl acetate systems with different proportions, detecting by using a thin-layer chromatography, combining fractions, and performing reduced pressure concentration on the fractions by using a rotary evaporator; the eluent for gradient elution is petroleum ether-ethyl acetate mixed liquor with the volume ratio of 100:0, 45:1, 9:1 and 8: 1;
<4> performing column chromatography separation on the characteristic fractions of the flavonoid compounds shown in the step <3>, repeatedly performing gradient elution by using petroleum ether-ethyl acetate systems with different proportions, collecting and detecting combined fractions by thin-layer chromatography; mixing and concentrating the petroleum ether-ethyl acetate mixed solution eluent with the volume ratio of 8:1 to obtain a solid, finally using a preparative thin-layer chromatography, developing the obtained solid on a preparation plate, detecting by the thin-layer chromatography, and eluting a main chromatographic silica gel strip by ethyl acetate to obtain the product; the eluent for gradient elution is petroleum ether-ethyl acetate mixed liquor with the volume ratio of 9:1 and 8: 1.
The flavonoid compound is applied to the preparation of medicinal antioxidants.
The flavonoid compounds are used as non-medicinal antioxidant.
In order to discover the medicinal value of derris elliptica, the inventor conducts intensive research, extracts and separates a flavonoid compound (compound 1) from the rattan of derris elliptica, determines the chemical structure of the flavonoid compound through nuclear magnetic resonance hydrogen spectrum, carbon spectrum, high-resolution mass spectrum and chemical method, and has the chemical name of 3- (4-hydroxyphenyl) -6, 8-dimethoxy-7- (3-methylbut-2-en-1-yl) benzopyran-4-one. Examining the database of the scifrinder, and determining that the compound 1 is a novel flavonoid compound, belongs to a natural active ingredient and is easily soluble in organic solvents such as alcohol. Meanwhile, the inventor also establishes a corresponding preparation method, namely: extracting the rattan of derris elliptica with ethanol, detecting the ethanol extract by thin-layer chromatography, performing silica gel column chromatography, performing gradient elution by using petroleum ether-ethyl acetate eluent system, and performing systematic separation and recrystallization to obtain the derris elliptica extract. Antioxidant activity experiments prove that the compound 1 and DPPH free radical scavenging activity IC thereofNamely, it isClose to the positive control drug vitamin C and scavenging activity IC for superoxide radicalNamely, it isThe value is less than that of a positive control ascorbic acid, and the antioxidant activity in vitro is excellent. Therefore, the flavonoid compound has great development potential in the aspects of medicinal or non-medicinal antioxidants.
Drawings
Fig. 1 is an ultraviolet UV spectrum of flavonoid compound 1 of the present invention.
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of flavonoid compound 1 of the present invention: (1H-NMR)。
FIG. 3 is the nuclear magnetic resonance carbon spectrum of flavonoid compound 1 of the present invention: (13C-NMR)。
FIG. 4 shows a High Resolution Mass Spectrum (HRMS) of flavonoid 1 of the present invention.
Detailed Description
Preparation of Compound 1
(1) Drying and crushing rattan of derris elliptica, sieving, extracting derris elliptica powder in a 1L round-bottom flask, adding 95% ethanol with the volume being 10 times that of the derris elliptica powder, performing reflux extraction for 2 hours under the heating of a water bath at 80 ℃, extracting for three times, filtering, combining filtrate, repeating the operations, wherein the total amount of the derris elliptica powder is 5Kg, and the extracted filtrate is subjected to reduced pressure concentration and drying to obtain 0.5Kg of crude extract:
(2) repeatedly extracting the extract with petroleum ether for multiple times, mixing filtrates, concentrating under reduced pressure, and oven drying to obtain 60.5g of petroleum ether extract;
(3) separating the petroleum ether layer extract by reverse silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate system, eluting with petroleum ether-ethyl acetate mixed solution with volume ratio (V/V) of 100:0, 45:1, 9:1, 8:1, monitoring by Thin Layer Chromatography (TLC), ultraviolet 254nm, 365nm and iodine vapor, collecting eluate, mixing the same eluate fractions to obtain 7 fractions, which are represented as: Fr.1-Fr.7; fr.2 shows three points on the thin-layer plate, the fluorescence under 254nm and 365nm ultraviolet lamps is strong, the fluorescence under 254nm shows bluish purple, the second point shows yellow green under 365nm, the iodine vapor color signal is strong, the concentration is carried out by a rotary evaporator under reduced pressure, and the separation and purification are carried out in the next step;
(4) fr.2 shows the existence of flavonoid compounds by using hydrochloric acid-magnesium powder for color development, so that Fr.2 fraction is selected for reverse column chromatography separation, petroleum ether-ethyl acetate is used for gradient elution, petroleum ether-ethyl acetate mixed liquor with eluent volume ratio (V/V) of 9:1 and 8:1 is used for monitoring through thin-layer chromatography TLC, the same fractions are collected and combined, vacuum reduced pressure concentration is carried out, finally a thin-layer chromatography plate is prepared, the obtained solid is spread on the preparation plate, the thin-layer chromatography detection is carried out, and a main chromatographic silica gel strip is eluted by ethyl acetate, so that the compound 1 can be obtained.
II, determination of Structure of Compound 1
1. Structural determination
Physicochemical properties and spectral data: and (3) reacting hydrochloric acid with magnesium powder, sucking a small amount of the elution concentrated solution into a test tube, adding a small amount of magnesium powder, dropwise adding a few drops of concentrated hydrochloric acid, and displaying mauve color in 1-2 min, wherein the result shows that the flavone structure exists.
The compound is white needle crystal, has maximum absorption peak of 240nm and 260nm (figure 1) in ultraviolet full wavelength scanning, and hydrogen spectrum of nuclear magnetic resonance1H-NMR(600MHz，CDCl3) Data (fig. 2): δ 7.80(s, 1H), δ 6.65(s, 1H), δ 7.36(d, J ═ 8.2Hz, 2H), δ 6.85(J ═ 8.2Hz, 2H), δ 5.15(t, J ═ 7.0Hz, 1H), δ 3.40(d, J ═ 7.0Hz, 2H), δ 1.78(s, 3H), and δ 1.66(s, 3H), δ 3.91(s, 3H), δ 3.85(s, 3H).
Nuclear magnetic resonance carbon spectrum of compound13C-NMR(150MHz，CDCl3) Data (fig. 3): δ 175.53, δ 131.83, δ 0122.4, δ 1162.26, δ 2157.88, δ 158.04, δ 156.04, δ 150.57, δ 62.35, δ 55.95, δ 22.41, δ 25.77, δ 17.87.
High resolution mass spectrum of compound (fig. 4): HR-ESI-MS (M/z 367.1556[ M + H ]]+)。
2. Structural derivation
(1) The molecular formula is as follows: c22H22O5。
(2) Chemical structural formula:
thirdly, determination of antioxidant Activity of Compound 1
1. Experimental medicine
1.1 medicine
1.2 reagents
DPPH, ascorbic acid (Vc), Tris-HCl buffer, pyrogallol.
2. Method of producing a composite material
2.1 elimination of DPPH free radical assay
0.03943g of DPPH (MW 394.3) was added to a volumetric flask to prepare a 10mg/mL solution of DPPH in methanol, and the solution was diluted to a concentration of 1 mg/mL. 2mL of methanol extracts of different concentrations were added to a prepared 1mg/mL solution of DPPH in methanol. Shaking and mixing uniformly, standing at room temperature for 30min, and measuring the absorbance value at 517 nm. Calculated clearance SA (%) ═ 1- (Ai-Aj)/A0 X 100%, wherein:
A0-absorbance of DPPH solution without added extract;
ai-absorbance of DPPH solution after adding the extracting solution;
aj-absorbance of the mixed methanol and extract;
A02mL of DPPH solution +2mL of methanol
Ai 2mL DPPH solution +2mL sample solution
Aj 2mL sample solution +2mL methanol
2.2 superoxide anion (O)2-) Determination of clearance
Measuring by adopting a pyrogallol oxidation method, taking 4.5mL of 50mmol/L Tris-HCl buffer solution (pH 8.2), placing in a water bath at 25 ℃, preserving the heat for 20min, then respectively adding 1mL of sample solution and 0.4mL of 30mmol/L pyrogallol solution, uniformly mixing, reacting in the water bath at 25 ℃ for 5min, adding 1mL of 8mmol HCl to stop the reaction, and measuring the absorbance (Ax) at 325 nm. Calculation of superoxide anion O according to2-Clearance rate:
in the formula: ao-blank control solution absorbance; ax-absorbance of sample solution.
3. Results
TABLE 1 scavenging of DPPH free radicals and superoxide anions by Compound 1
Superoxide anion radical O2-The clearance rate experiment result shows that: compound 1 of the present invention exhibits half the effective Inhibition (IC) of superoxide anion radical scavenging50) 346.19 +/-0.10, and the Vc ascorbate used as a positive control is 578.51 +/-0.12, which shows that the compound has strong effect of eliminating superoxide anion free radicals; the effect of scavenging DPPH free radicals is inferior to that of the control positive drug.
By combining the analysis, the compound 1 has good antioxidant activity, belongs to pure natural plant active ingredients, and can be used as a novel medicinal or non-medicinal antioxidant and the like.
Claims (1)
1. A preparation method of a flavonoid compound is characterized by comprising the following steps: extracting the rattan of derris elliptica with ethanol, detecting the ethanol extract by thin-layer chromatography, performing silica gel column chromatography, performing gradient elution by using a petroleum ether-ethyl acetate eluent system, and performing systematic separation and recrystallization to obtain the derris elliptica extract; the method specifically comprises the following steps:
drying the stems of derris elliptica in the shade, crushing, sieving, adding a 95% ethanol solution with the volume being 10 times that of the stems, carrying out reflux extraction for 2 hours in a water bath at the temperature of 80 ℃, carrying out reflux extraction for three times, filtering, combining filtrate, concentrating the filtrate under reduced pressure, and drying to obtain an extract;
repeatedly extracting the extract obtained in the step (1) with petroleum ether for multiple times, combining the filtrates, concentrating under reduced pressure, and drying to obtain a petroleum ether layer extract;
separating the petroleum ether layer extract obtained in the step (2) by a reverse silica gel chromatographic column, performing gradient elution by using petroleum ether-ethyl acetate systems with different proportions, detecting by using a thin-layer chromatography, combining fractions, and performing reduced pressure concentration on the fractions by using a rotary evaporator; the eluent for gradient elution is petroleum ether-ethyl acetate mixed liquor with the volume ratio of 100:0, 45:1, 9:1 and 8: 1;
<4> performing column chromatography separation on the characteristic fractions of the flavonoid compounds shown in the step <3>, repeatedly performing gradient elution by using petroleum ether-ethyl acetate systems with different proportions, collecting and detecting combined fractions by thin-layer chromatography; mixing and concentrating the petroleum ether-ethyl acetate mixed solution eluent with the volume ratio of 8:1 to obtain a solid, finally using a preparative thin-layer chromatography, developing the obtained solid on a preparation plate, detecting by the thin-layer chromatography, and eluting a main chromatographic silica gel strip by ethyl acetate to obtain the product; the eluent for gradient elution is petroleum ether-ethyl acetate mixed liquor with the volume ratio of 9:1 and 8: 1;
the chemical structural formula of the flavonoid compound is as follows:
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Non-Patent Citations (3)
| Title |
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| Flavonoids and other bioactive constituents from Ficus thonningii Blume (Moraceae);Yannick S.F. Fongang et al.;《Phytochemistry Letters》;20141215;第11卷;139-145 * |
| Isolation and characterization of a new bioactive isoflavone from Derris eriocarpa;Li-Fang Yang et al.;《Journal of Asian Natural Products Research》;20150814;第17卷(第10期);1002-1009 * |
| 毛果鱼藤黄酮类化合物提取工艺及抑菌作用的研究;杨立芳等;《中成药》;20141130;第36卷(第11期);2413-2417 * |
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