阅读说明：本技术 五种含异戊烯基黄酮的制备方法及应用 (Preparation method and application of five prenyl-containing flavones ) 是由 高坤 王柳笛 李亚 高文颖 吴月婷 王婧 于 2020-07-28 设计创作，主要内容包括：本发明属于化工技术领域,具体涉及五种含异戊烯基黄酮化合物的制备方法及应用；本发明化合物分离自甘肃省靖远县永新乡的乌拉尔甘草(Glycyrrhiza uralensis Fisch.)干燥根和根茎,通过浸提、萃取及柱色谱制备分离得到。该五种化合物对LAG3/MHC Ⅱ或LAG3/FGL1结合具有显著抑制作用,可为研发新型抗肿瘤药物提供候选分子,为开发利用乌拉尔甘草(Glycyrrhiza uralensis Fisch.)提供了科学依据。(The invention belongs to the technical field of chemical industry, and particularly relates to a preparation method and application of five isopentenyl flavone compounds; the compound is separated from dried roots and rhizomes of Glycyrrhiza uralensis (Glycyrrhiza uralensis Fisch.) in Yongxinxiang, Jingyuan, Gansu province, and is prepared and separated by leaching, extraction and column chromatography. The five compounds have obvious inhibition effect on the combination of LAG3/MHC II or LAG3/FGL1, can provide candidate molecules for developing novel antitumor drugs, and provides scientific basis for developing and utilizing Glycyrrhiza uralensis Fisch.)

1. A compound containing isopentenyl flavone is prepared from the raw materials of (A) isopentenyl flavone,

the structural formula of the compound 5 is characterized.

2. The isoalkenylflavone-containing compound of claim 1, wherein the isoalkenylflavone-containing compound is isolated from dried root and rhizome of Glycyrrhiza uralensis Fisch.

3. The prenylflavonoid-containing compound of claim 1, wherein the preparation method comprises the steps of:

s1, extracting dried roots and rhizomes of Glycyrrhiza uralensis Fisch with methanol, concentrating the extract, dispersing the concentrated extract in a water phase, extracting with petroleum ether, ethyl acetate and n-butanol respectively, and concentrating the ethyl acetate extract to obtain a crude extract;

s2, subjecting the crude extract to chromatographic separation with macroporous resin, and sequentially eluting with ethanol-water (0: 100,30:70,50:50,80:20,100:0 (v/v)) gradient to obtain 5 fractions (fr.a-E), wherein fr.a is ethanol-water (0: 100), fr.b is ethanol-water (30: 70), fr.c is ethanol-water (50: 50), fr.d is ethanol-water (80: 20), and fr.e is ethanol-water (100: 0), and collecting the elution fractions with ethanol-water (80: 20) (fr.d) gradient;

s3 and fr.d are separated by silica gel column chromatography, and the separation is performed by gradient elution sequentially using petroleum ether-acetone (100: 0,20:1,10:1,5:1,2:1,1:1,0:1(v/v) to obtain 7 fractions (fr.da-DG), wherein fr.da is petroleum ether-acetone (100: 0), fr.db is petroleum ether-acetone (20: 1), fr.dc is petroleum ether-acetone (10: 1), fr.dd is petroleum ether-acetone (5: 1), fr.de is petroleum ether-acetone (2: 1), fr.df is petroleum ether-acetone (1: 1), fr.dg is petroleum ether-acetone (0: 1), and collection gradient is petroleum ether-acetone (10: 1) (fr.dc) and 5:1 (dd);

separating by silica gel column chromatography, sequentially eluting with petroleum ether-ethyl acetate (10: 1,5:1,2:1,1:1 (v/v)) in gradient, and detecting by TLC to obtain 4 fractions (Fr. DCA-DCD);

reverse phase silica gel RP-C for S5 Fr₁₈Separating by column chromatography, sequentially eluting with methanol-water-30: 70 to 100:0(v/v) gradient to obtain 8 fractions (fr.dcba-DCBH), wherein fr.dcba is methanol-water-30: 70, fr.dcbb is methanol-water-40: 60, fr.dcbc is methanol-water-50: 50, fr.dcbd is methanol-water-60: 40, fr.dcbe is methanol-water-70: 30, fr.dcbf is methanol-water-80: 20, fr.dcbg is methanol-water-90: 10, fr.dcbh is methanol-water-100: 0, and eluting fractions with methanol-water-80: 20(fr.dcbf) gradient are collected;

reverse phase silica gel RP-C for S6, Fr₁₈Separation was performed by column chromatography using a gradient of methanol-water ═ 65:35 to 80:20(v/v) in order, and combined by TLC detection to give 4 fractions (Fr. dcbfa-DCBFD), Fr.DCBFA is methanol-water 65:35fr.dcbfb is methanol-water 70:30, fr.dcbfc is methanol-water 75:25, fr.dcbfd is methanol-water 80: 20;

DCBFB is separated by Sephadex LH-20 column chromatography of dextran gel to obtain compound licoflavanone C (1);

purifying by semi-preparative liquid chromatography (S8, Fr. DCC) to obtain compounds of Kanzanol H (2) and Kanzanol P (3), wherein the components have retention times of 42min and 52min and ultraviolet absorption characteristic wavelengths of 282nm and 285nm respectively;

s9, fr.dd are separated by MCI column chromatography and combined by TLC detection to give 2 fractions (fr.dda and fr.ddb);

s10, fr.ddb, combined by TLC detection to give 12 fractions (fr.ddba and fr.ddbl) which are methanol insoluble white particulate compounds, glycoyrol (4);

separating S11 and fr.ddbl by silica gel column chromatography, gradient eluting sequentially with petroleum ether-ethyl acetate (10: 1,5:1,2:1,1:1(v/v), detecting by TLC, and combining to obtain 6 fractions (fr.ddbla-DDBLF);

ddble was separated on Sephadex LH-20 column chromatography using Sephadex S to give compound 5, named lichesoflavan F.

4. A class of prenylflavonoid-containing compounds according to claim 3, characterized in that: the method for preparing the ethyl acetate layer extract by the S1 comprises the following steps: pulverizing dried root and rhizome of Glycyrrhiza uralensis Fisch, leaching in 95% methanol at room temperature for 3 times (7 days each time), concentrating the leaching solution, dispersing in water phase, extracting with petroleum ether, ethyl acetate and n-butanol respectively, and concentrating the ethyl acetate extract to obtain crude extract.

5. A class of prenylflavonoid-containing compounds according to claim 3, characterized in that: and the S4Fr.DC is subjected to thin layer chromatography analysis by using petroleum ether and ethyl acetate as developing solvent, wherein the Rf value is 0.3-0.6, and the color of sulfuric acid-ethanol is black brown.

And the S5 Fr.DCB is subjected to thin layer chromatography by using petroleum ether and acetone as a developing solvent, wherein the Rf value is 0.5-0.7, and the color of sulfuric acid-ethanol is light brown.

And the S6 Fr and DCBF perform thin-layer chromatography analysis by using petroleum ether and acetone as a developing agent, wherein the Rf value is 0.4-0.5, and the sulfuric acid-ethanol develops purple black.

And the S7 Fr.DCBFB is subjected to thin-layer chromatography analysis by using petroleum ether and acetone as a developing agent, wherein the ratio of Rf is 0.3-0.4, and the color of sulfuric acid-ethanol is purple red.

Dcc with petroleum ether and acetone 3:1 as developing solvent, Rf value of 0.4-0.6, and color development of sulfuric acid-ethanol in light gray.

And the S9 Fr.DD takes petroleum ether and ethyl acetate as developing solvent to carry out thin-layer chromatography analysis, the Rf value is 0-0.5, and the sulfuric acid-ethanol is brown gray in color.

And the S10 Fr.DDB adopts petroleum ether and ethyl acetate as developing agents to carry out thin-layer chromatography analysis, the Rf value is 0.2-0.5, and the sulfuric acid-ethanol is brown black in color.

And the S11 Fr.DDBL adopts petroleum ether and ethyl acetate as developing agents to carry out thin-layer chromatography analysis, the Rf value is 0-0.5, and the sulfuric acid-ethanol develops yellow.

And the S12 Fr.DDBLE adopts petroleum ether and ethyl acetate as developing agents to perform thin-layer chromatography analysis, the Rf value is 0.3-0.5, and the sulfuric acid-ethanol develops yellow color.

6. A class of prenylflavonoid-containing compounds according to claim 3, characterized in that: the semi-preparative liquid chromatography in the S8 is performed by a Waters 1525 liquid chromatograph, a mobile phase is methanol-water with a volume ratio of 75:25, a flow rate is 2mL/min, a detector is a Waters 2998 photodiode array detector, a detection wavelength is 200-400nm, and a chromatographic column is a Waters Sunfire C₁₈Semi-preparative columns, size 10 × 250 mm.

7. A process for producing an antitumor agent, characterized by using the compound described in claim 1 as a starting material.

8. The process for preparing an antitumor agent as claimed in claim 7, wherein the process comprises administering an effective amount of the compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier to the antitumor agent.

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a preparation method and application of five prenyl-containing flavones.

Background

Plants are widely distributed and a wide variety of natural resources, and many have medicinal values. The natural compounds from plants have rich structures and diverse activities, and the research on the compounds contained in medicinal plants has certain guidance on finding potential medicine molecules. Glycyrrhiza uralensis Fisch is a traditional medicinal plant in China, is widely applied to the fields of food, cosmetics and the like, is rich in various components such as flavone and triterpene, and has various activities such as antivirus, anti-inflammatory, antioxidation and the like. Although there have been many studies on the components of glycyrrhiza uralensis, the activity of secondary metabolites has yet to be supplemented, and thus, it is desired to obtain a compound having excellent activity from this source.

Cancer is a common malignant tumor in clinic and has become a non-negligible killer for human health. In recent years, with the increase of environmental pollution and the increase of bad living habits, the incidence rate of human cancers is gradually increased, and the survival rate is still low. Therefore, the development of novel cancer therapeutic drugs is urgently needed.

Disclosure of Invention

One of the objectives of the present invention is to provide five kinds of prenyl-containing flavonoids, namely, the compounds of the present invention, namely, licoflavanone C (1), kanzonol H (2), kanzonol P (3), glycopyrrolate (4) and licoisoflavone F (5), wherein compound 5 is a novel compound which is reported for the first time and has the structural formula shown in formula (I):

another object of the present invention is to provide a process for preparing five prenylflavonoid-containing licosoflavone C (1), kanzonol H (2), kanzonol P (3), glycocoll (4) and licosoflavan F (5), characterized in that: the compounds licoflavanone C (1), kanzonol H (2), kanzonol P (3), glycyrrhol (4) and licosoflavan F (5) are prepared and separated from the extract of dried root and rhizome of Glycyrrhiza uralensis (Glycyrrhiza uralensis Fisch.), and comprise the following steps:

s1, extracting dried roots and rhizomes of Glycyrrhiza uralensis Fisch with methanol, concentrating the extract, dispersing the concentrated extract in a water phase, extracting with petroleum ether, ethyl acetate and n-butanol respectively, and concentrating the ethyl acetate extract to obtain a crude extract;

s2, subjecting the crude extract to chromatographic separation with macroporous resin, and eluting with ethanol-water ratio of 0:100,30:70,50:50,80:20,100:0(v/v) in sequence to obtain 5 fractions (fr.a-E), wherein fr.a is ethanol-water ratio of 0:100, fr.b is ethanol-water ratio of 30:70, fr.c is ethanol-water ratio of 50:50, fr.d is ethanol-water ratio of 80:20, fr.e is ethanol-water ═ 100:0, collecting the elution fraction with a gradient of ethanol-water 80:20 (fr.d);

s3 and fr.d are separated by silica gel column chromatography, and the separation is performed by gradient elution sequentially using petroleum ether-acetone (100: 0,20:1,10:1,5:1,2:1,1:1,0:1(v/v) to obtain 7 fractions (fr.da-DG), wherein fr.da is petroleum ether-acetone (100: 0), fr.db is petroleum ether-acetone (20: 1), fr.dc is petroleum ether-acetone (10: 1), fr.dd is petroleum ether-acetone (5: 1), fr.de is petroleum ether-acetone (2: 1), fr.df is petroleum ether-acetone (1: 1), fr.dg is petroleum ether-acetone (0: 1), and collection gradient is petroleum ether-acetone (10: 1) (fr.dc) and 5:1 (dd);

separating by silica gel column chromatography, sequentially eluting with petroleum ether-ethyl acetate (10: 1,5:1,2:1,1:1 (v/v)) in gradient, and detecting by TLC to obtain 4 fractions (Fr. DCA-DCD);

separating by reversed-phase silica gel RP-C18 column chromatography using S5 and fr.dcb in sequence with methanol-water-30: 70 to 100:0(v/v) gradient elution to obtain 8 fractions (fr.dcba-DCBH), wherein fr.dcba is methanol-water-30: 70, fr.dcbb is methanol-water-40: 60, fr.dcbc is methanol-water-50: 50, fr.dcbd is methanol-water-60: 40, fr.dcbe is methanol-water-70: 30, fr.dcbf is methanol-water-80: 20, fr.dcbg is methanol-water-90: 10, fr.dcbh is methanol-water-100: 0, collecting the elution fraction with methanol-water-80: 20 (fr.dcbf);

separating S6 and fr.dcbf by reversed phase silica RP-C18 column chromatography, eluting sequentially with methanol-water gradient of 65:35 to 80:20(v/v), detecting by TLC, and combining to obtain 4 fractions (fr.dcbfa-DCBFD);

DCBFB is separated by Sephadex LH-20 column chromatography of dextran gel to obtain compound licoflavanone C (1);

purifying by semi-preparative liquid chromatography (S8, Fr. DCC) to obtain compounds of Kanzanol H (2) and Kanzanol P (3), wherein the components have retention times of 42min and 52min and ultraviolet absorption characteristic wavelengths of 282nm and 285nm respectively;

s9, fr.dd are separated by MCI column chromatography and combined by TLC detection to give 2 fractions (fr.dda and fr.ddb);

s10, fr.ddb, combined by TLC detection to give 12 fractions (fr.ddba and fr.ddbl) which are methanol insoluble white particulate compounds, glycoyrol (4);

separating S11 and fr.ddbl by silica gel column chromatography, gradient eluting sequentially with petroleum ether-ethyl acetate (10: 1,5:1,2:1,1:1(v/v), detecting by TLC, and combining to obtain 6 fractions (fr.ddbla-DDBLF);

DDBLE was separated by Sephadex LH-20 column chromatography using S12 to obtain the compound, lichesoflavan F (5).

The method for preparing the ethyl acetate layer extract by the S1 comprises the following steps: pulverizing dried root and rhizome of Glycyrrhiza uralensis Fisch, soaking in 95% methanol at room temperature for 3 times, each for 7 days, concentrating the extractive solution, dispersing in water phase, extracting with petroleum ether, ethyl acetate and n-butanol for three times, mixing ethyl acetate extracts, and concentrating to obtain crude extract.

The S4Fr.DC adopts petroleum ether and ethyl acetate as developing solvent to carry out thin-layer chromatography analysis, the Rf value is 0.3-0.6, and the color development of sulfuric acid-ethanol is black brown.

And the S5 Fr.DCB is subjected to thin layer chromatography by using petroleum ether and acetone as a developing solvent, wherein the Rf value is 0.5-0.7, and the color of sulfuric acid-ethanol is light brown.

And the S6 Fr and DCBF perform thin-layer chromatography analysis by using petroleum ether and acetone as a developing agent, wherein the Rf value is 0.4-0.5, and the sulfuric acid-ethanol develops purple black.

And the S7 Fr.DCBFB is subjected to thin-layer chromatography analysis by using petroleum ether and acetone as a developing agent, wherein the ratio of Rf is 0.3-0.4, and the color of sulfuric acid-ethanol is purple red.

Dcc with petroleum ether and acetone 3:1 as developing solvent, Rf value of 0.4-0.6, and color development of sulfuric acid-ethanol in light gray.

And the S9 Fr.DD takes petroleum ether and ethyl acetate as developing solvent to carry out thin-layer chromatography analysis, the Rf value is 0-0.5, and the sulfuric acid-ethanol is brown gray in color.

And the S10 Fr.DDB adopts petroleum ether and ethyl acetate as developing agents to carry out thin-layer chromatography analysis, the Rf value is 0.2-0.5, and the sulfuric acid-ethanol is brown black in color.

And the S11 Fr.DDBL adopts petroleum ether and ethyl acetate as developing agents to carry out thin-layer chromatography analysis, the Rf value is 0-0.5, and the sulfuric acid-ethanol develops yellow.

And the S11 Fr.DDBLE adopts petroleum ether and ethyl acetate as developing agents to perform thin-layer chromatography analysis, the Rf value is 0.3-0.5, and the sulfuric acid-ethanol develops yellow color.

The liquid chromatograph of the semi-preparative liquid chromatogram in the S8 is Waters 1525, the mobile phase is methanol-water with the volume ratio of 75:25, the flow rate is 2mL/min, the detector is a Waters 2998 photodiode array detector, the detection wavelength is 200-400nm, the chromatographic column is a Waters Sunfire C18 semi-preparative column with the specification of 10 multiplied by 250 mm.

The invention also provides application of the compounds licoflavianone C (1), kanzonol H (2), kanzonol P (3), glyconol (4) and licoflavianone F (5), or pharmaceutically acceptable salts thereof in preparing antitumor drugs.

The fourth purpose of the invention is to provide five antitumor drugs, which are characterized in that: comprising as active ingredients effective amounts of the compounds licoflavanone C (1), kanzonol H (2), kanzonol P (3), glycorol (4) and licoflavanol F (5) according to claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The invention has the beneficial effects that: five compounds, licoflavdanone C (1), kanzonol H (2), kanzonol P (3), glycyrol (4) and licoflavdanone F (5), were extracted and isolated from the leachate of dried roots and rhizomes of Glycyrrhiza uralensis Fisch. The five compounds have obvious inhibition effect on the combination of LAG3/MHC II or LAG3/FGL1, can provide candidate molecules for developing novel antitumor drugs, and provides scientific basis for developing and utilizing Glycyrrhiza uralensis Fisch.

Drawings

FIG. 1 shows the structural formulae of the compounds licoflavdanone C (1), kanzonol H (2), kanzonol P (3), glycyrrhol (4) and licosoflavan F (5).

FIG. 2 is a drawing of Compound 5¹H NMR spectrum.

FIG. 3 is a drawing of Compound 5¹³C NMR spectrum.

Figure 4 is the DEPT 135 spectrum of compound 5.

FIG. 5 is a drawing of Compound 5¹H-¹H COSY spectra.

FIG. 6 is the HSQC spectrum of Compound 5.

Fig. 7 is an HMBC spectrum of compound 5.

Figure 8 is a graph of compound 1 binding to LAG3 protein.

Figure 9 is a graph of compound 2 binding to LAG3 protein.

Figure 10 is a graph of compound 3 binding to LAG3 protein.

Figure 11 is a graph of compound 4 binding to LAG3 protein.

Figure 12 is a graph of compound 5 binding to LAG3 protein.

Detailed Description