CN113150049B - Cyclocarya paliurus extract and application thereof in resisting gout and reducing uric acid - Google Patents

Cyclocarya paliurus extract and application thereof in resisting gout and reducing uric acid Download PDF

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CN113150049B
CN113150049B CN202110424564.8A CN202110424564A CN113150049B CN 113150049 B CN113150049 B CN 113150049B CN 202110424564 A CN202110424564 A CN 202110424564A CN 113150049 B CN113150049 B CN 113150049B
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徐康平
姜德建
吴建平
朱晖
李桂花
魏希凡
陈祖辉
王钰艳
刘学武
曾宏亮
成飞
桂瑞
何小爱
欧赛玉
刘翊芊
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Hunan Helian Biotechnology Development Co ltd
Hunan Qingya Health Service Co ltd
Central South University
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Hunan Qingya Health Service Co ltd
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Abstract

The invention relates to an isopentene-based flavonoid compound extracted from cyclocarya paliurus, and particularly discloses an extraction method and application of the isopentene-based flavonoid compound, wherein the structure of the isopentene-based flavonoid compound of cyclocarya paliurus is shown as a formula I:

Description

Cyclocarya paliurus extract and application thereof in resisting gout and reducing uric acid
Technical Field
The invention relates to an extract of cyclocarya paliurus, in particular to an n-butanol active site and isopentenyl flavonoid compound of cyclocarya paliurus, and an extraction method and application thereof.
Background
Cyclocarya paliurus (Bata1) Iljinsk is a plant of cyclocarya paliurus of Juglandaceae (Juglauiaceae), is a unique single species plant in China, and is widely distributed in Anhui, Jiangsu, Zhejiang and other places. Since the 80 s in the 20 th century, experts and scholars at home and abroad mainly carry out a great deal of research on the aspects of resource cultivation, chemical components, biological activity, product development and research and the like of cyclocarya paliurus. The results show that the cyclocarya paliurus has various physiological activities and pharmacological functions beneficial to human bodies.
The isopentenyl flavone is a compound with isopentenyl group connected to the flavone mother nucleus, shows more outstanding biological activity compared with the flavonoid compound, mainly comprises the aspects of anti-inflammation, immunoregulation, cardiovascular protection, metabolic disease improvement, osteoporosis improvement, stem cell differentiation promotion, neuroprotection, anti-tumor, anti-aging, reproductive action and the like, and has wide application prospect. Icariin, a representative component of the isopentenyl flavonoids, can play a role in reducing blood pressure mainly through a mechanism of blocking beta receptors and central blood pressure reduction by sodium channel blocking. Herba Epimedii inhibits Ca 2+ influx of vascular smooth muscle, directly expands vascular smooth muscle and reduces vascular resistance. Icariin is also found to be capable of remarkably improving swelling degree of joints damaged by gouty arthritis, reducing gait scores and improving synovial tissue damage, has the effect equivalent to that of colchicine at a dose of 80mg/kg-1, and is found to remarkably reduce the leukemia number in joint effusion of an icariin treatment group, wherein IL-1 beta, IL-6, TNF-alpha and PGE2, and the icariin is related to the anti-inflammatory effect of the icariin. The epimedium total flavonoids can also reduce blood pressure by selectively blocking beta 1 receptors and reducing the content of plasma endothelin and directly expanding blood vessels.
Cyclocarya paliurus leaves contain various medicinal chemical components including carotene, protein, flavonoid, polysaccharide, triterpenes and other compounds, wherein prenylflavonoid is widely concerned due to the diversity of the structure and the pharmacological activity of the prenylflavonoid. At present, the pharmacological activity of the cyclocarya paliurus isopentenyl flavone is mostly focused on the research of blood sugar reduction and inflammation, and the research on the activity and mechanism in other aspects is less, so that the specific explanation on other application activity and action mechanism of the cyclocarya paliurus isopentenyl flavone has great significance for wide clinical application and innovative drug development.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for extracting n-butyl alcohol active site and isopentenyl flavonoid compound of cyclocarya paliurus and improve gout active ingredients and drug effect substance basis for systematic research.
In order to solve the technical problems, the technical scheme of the invention is as follows:
an isopentene group flavonoid compound has the following structural general formula:
Figure BDA0003029325720000011
wherein R is 7 Selected from the group consisting of L-rhamnose- (2 → 1) -L-rhamnose, D-xylose- (2 → 1) -L-rhamnose, D-glucose- (4 → 1) -L-rhamnose, D-glucose- (2 → 1) -L-rhamnose, D-deoxyfuranose- (2 → 1) -L-rhamnose, D-cinchose- (2 → 1) -L-rhamnose, D-glucose- (3 → 1) -L-rhamnose, L-rhamnose;
R 8 selected from hydrogen, D-glucose, L-rhamnose- (2 → 1) -D-glucoseD-xylose- (2 → 1) -L-rhamnose, D-glucose- (4 → 1) -L-rhamnose, D-glucose- (2 → 1) -L-rhamnose, D-deoxyfuranose- (2 → 1) -L-rhamnose, D-gallinarum- (2 → 1) -L-rhamnose, D-glucose- (3 → 1) -L-rhamnose, L-rhamnose;
R 9 selected from the group consisting of hydroxy, methoxy, ethoxy, propoxy;
R 10 is selected from
Figure BDA0003029325720000021
Preferably, the isopentenyl flavonoid compound has the following structural formula:
Figure BDA0003029325720000031
the invention also provides a method for extracting the isopentenyl flavonoid compound from cyclocarya paliurus, which comprises the following steps of:
s1, heating and refluxing the cyclocarya paliurus leaves by using ethanol/water, and concentrating to obtain an extract;
s2, dispersing the extract with water, extracting with n-butanol, and concentrating the extract to obtain n-butanol extraction concentrate;
and S3, analyzing, separating and purifying the n-butanol extraction concentrated solution by adopting HPLC-DAD, TLC and UPLC-MS/MS methods.
Preferably, in S1, the cyclocarya paliurus leaves are dried and pulverized.
Preferably, in S1, the concentration of ethanol is 70%, the ratio of the mass of the cyclocarya paliurus leaves to the ethanol is 1:10, the heating temperature is 120 ℃, the heating time is 2h, the concentration temperature is 60 ℃, and the concentration time is 2-3 days.
Preferably, in S2, the extraction is carried out three times by using n-butanol, the ratio of the mass of the cyclocarya paliurus leaves to the volume of the n-butanol used each time is 1:10, the concentration temperature is 65 ℃, and the concentration time is 72-96 hours.
Preferably, in S3, n-butanol extraction parts are subjected to gradient elution by macroporous resin EtOH: H2O (0:100-0:95), HPLC-DAD is used for analyzing samples, and 5 parts (a-e) are obtained by concentration and combination;
TLC thin layer analysis of a-e sites, 10% concentrated sulfuric acid/ethanol heat color development, Fr.C thin layer plate showed yellow band.
Firstly, performing polyamide column chromatography on Fr.C to remove interference components such as pigments and tannins, and performing HPLC-DAD analysis and combination to obtain 6 parts Fr.C1-C6, wherein Fr.C1 is mainly absorbed by flavonoids, and the Fr.C1 part is preliminarily determined to be a flavonoid enrichment part; then, carrying out HW-40C and reverse ODS column chromatography, tracking the isopentenyl flavone by using a UPLC-MS/MS method in the whole separation process, and effectively separating the isopentenyl flavone compounds by using ultraviolet characteristic absorption peaks; and finally, purifying the target compound by adopting a semi-preparative high performance liquid phase.
The invention also claims an active site, wherein the active site is the n-butanol extraction concentrated solution, and the main component of the active site is the isopentenyl flavone.
The invention also claims the application of the active site in the preparation of medicines for improving gout and reducing uric acid.
Further, the specific process for separating and purifying the isopentenyl flavone comprises the following steps:
the invention adopts HPLC-DAD, TLC and UPLC-MS/MS methods to track, analyze, separate and purify the n-butanol extraction concentrated solution. Subjecting 445g n-butanol fraction to macroporous resin chromatography column, isocratic eluting with ethanol-water system (0:100,30:70,50:50,70:30,95:5), and subjecting cyclocarya paliurus n-butanol fraction to five primary fractions (Fr.a-e) according to HPLC-DAD analysis result. Fr.b (106g) was passed through a polyamide column, eluted with an ethanol-water system (0:100-95:5) and divided into six fractions (Fr.b1-b6) according to HPLC-DAD analysis.
Fr.c (107g) was subjected to polyamide column chromatography, eluted with an ethanol-water system (0:100-95:5) to give six fractions (Fr.c1-C6) according to HPLC-DAD analysis, Fr.c1(22.9g) was subjected to HW-40C column chromatography, eluted with a methanol-water system (0:100-75:5), and subjected to HPLC-DAD analysis to give Fr.c1.1-Fr.c1.12 after combining the same fractions. Fr.c1.2 was subjected to ODS-AA medium pressure column, eluted with a methanol-water system (5:95-75:5), and analyzed by HPLC-DAD to give Fr.c1.2.1-Fr.c 1.2.45. Fr.c. 1.2.23-Fr.c. 1.2.25 by semipreparative liquid phase (ACN-H2O 21%, 0.1% CH3COOH, v/v,220nm,3mL/min) gave compound 28(8.6mg),29(5.9mg) and 30(7.4 mg). Fr.c. 1.2.29-Fr.c. 1.2.30 via semipreparative liquid phase (ACN-H2O 24%, v/v,220nm,3mL/min) gave compound 27(13.6mg),35(11.7mg) and 40(15.7mg). Fr.c. 1.2.35-Fr.c. 1.2.36 via semipreparative liquid phase (ACN-H2O 31%, v/v,220nm,3mL/min) isolated as 32(13.6mg) and 33(6.4 mg). Fr.c1.3 through ODS-AA medium pressure column, methanol-water system (5:95-75:5) elution, HPLC-DAD analysis, Fr.c1.3.1-Fr.c1.3.28. Fr.c. 1.3.13 by semipreparative liquid phase (ACN-H2O 18%, 0.1% CH3COOH, v/v,220nm,3mL/min) gave compound 36(10.8mg),37(4.9 mg). Fr. c1.3.15 semi-preparative liquid phase (ACN-H2O 22%, v/v,220nm,3mL/min) gave compound 38(7.4mg),39(11.3 mg). Fr.c. 1.8 by semi-preparative liquid phase (ACN-H2O 29%, v/v,220nm,3mL/min) gave compound 43(750.6mg),44(107.5mg),45(59.3 mg).
Fr.c2(20.5g) was eluted through HW-40C column chromatography using methanol-water system (0:100-75:5) and analyzed by HPLC-DAD, and the same fractions were combined to give fr.c2.1-fr.c 2.10. Fr.c2.4 through ODS-AA medium pressure column, methanol-water system (5:95-75:5) elution, HPLC-DAD analysis, Fr.c2.4.1-Fr.c2.4.40. Fr. c2.4.18 by semipreparative liquid phase (ACN-H2O 20%, 0.1% CH3COOH, v/v,220nm,3mL/min) to give compound 53(33.7 mg); fr.c. 2.4.24-Fr.c. 2.4.25 by semipreparative liquid chromatography (ACN-H2O 22%, v/v,220nm,3mL/min) to give a mixture of compound 34 and compound 51, which was separated by thin layer preparative chromatography (developing solvent: water saturated n-butanol) to give compound 34(7.6mg), compound 51(28.5 mg); fr.c. 2.4.26 via semipreparative liquid phase (ACN-H2O 25%, v/v,220nm,3mL/min) gave compound 50(26.4mg), compound 51(76.2 mg); fr.c. 2.4.28 semi-preparative liquid phase (ACN-H2O 25%, v/v,220nm,3mL/min) gave compound 49(13.1 mg). Fr.c2.6 is eluted through ODS-AA medium pressure column with methanol-water system (15:85-75:5), analyzed by HPLC-DAD, and the same fractions are combined to obtain Fr.c2.6.1-Fr.c 2.6.35. Fr.c2.6.16 via a semi-preparative liquid phase (ACN-H2O 28%, 0.1% CH3COOH, v/v,220nm,3mL/min) to give compound 31(10.6 mg); fr.c2.6.20 by semipreparative liquid phase (ACN-H2O 27%, v/v,220nm,3mL/min) gave compound 46(33.7mg),47(21.9mg),48(9.4 mg); fr.c2.6.22 via a semi-preparative liquid phase (ACN-H2O 32%, 0.1% CH3COOH, v/v,220nm,3mL/min) to give compound 41(104.2 mg); fr.c2.6.25 by semi-preparative liquid phase (ACN-H2O 39%, v/v,220nm,3mL/min) to give compound 52(7.5 mg); fr.c. 2.6.28 semi-preparative liquid phase (ACN-H2O 44%, v/v,220nm,3mL/min) gave compound 42(7.5 mg).
The structural formula of the compounds 27-53 is:
Figure BDA0003029325720000061
Figure BDA0003029325720000071
the invention also provides application of the isopentene flavonoid compounds 27-53 in preparation of medicines for improving gout and reducing uric acid.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a brand-new method for extracting isopentenyl flavonoid compounds 27-53 from cyclocarya paliurus, and the method is simple and repeatable.
2. The invention provides new application of the n-butyl alcohol active site and the isopentenyl flavonoid compound 27-53 of the cyclocarya paliurus, researches the pharmaceutical mechanism of the n-butyl alcohol active site and the isopentenyl flavonoid compound 27-53 of the cyclocarya paliurus, and provides new application prospects of reducing uric acid and resisting gout for the n-butyl alcohol active site and the isopentenyl flavonoid compound 27-53 of the cyclocarya paliurus.
Drawings
FIG. 1 is the HSQC spectrum of Compound 27
FIG. 2 is an HMBC spectrum of compound 27
FIG. 3 is a drawing of Compound 28 1 H- 1 H COSY spectrum
FIG. 4 is the HSQC spectrum of Compound 28
FIG. 5 is an HMBC spectrum of compound 28
FIG. 6 shows preparation of Compound 28 1 H- 1 H COSY spectrum
FIG. 7 is the HSQC spectrum of compound 29
FIG. 8 is an HMBC spectrum of compound 29
FIG. 9 shows Compound 29 1 H- 1 H COSY spectrum
FIG. 10 is the HSQC spectrum of Compound 30
FIG. 11 is an HMBC spectrum of compound 30
FIG. 12 shows preparation of Compound 30 1 H- 1 H COSY spectrum
FIG. 13 is the HSQC spectrum of Compound 31
FIG. 14 shows HMBC spectra of compound 31
FIG. 15 is a drawing of Compound 31 1 H- 1 H COSY spectrum
FIG. 16 is the HSQC spectrum of Compound 32
FIG. 17 is an HMBC spectrum of compound 32
FIG. 18 is a drawing of Compound 32 1 H- 1 H COSY spectrum
FIG. 19 is the HSQC spectrum of compound 33
FIG. 20 is an HMBC spectrum of compound 33
FIG. 21 is a drawing of Compound 33 1 H- 1 H COSY spectrum
FIG. 22 is the HSQC spectrum of compound 34
FIG. 23 is an HMBC spectrum of compound 34
FIG. 24 is a drawing of Compound 34 1 H- 1 H COSY spectrum
FIG. 25 is the HSQC spectrum of Compound 35
FIG. 26 is an HMBC spectrum of compound 35
FIG. 27 is a photograph of Compound 35 1 H- 1 H COSY spectrum
FIG. 28 is HSQC spectrum of compound 36
FIG. 29 is an HMBC spectrum of compound 36
FIG. 30 is a drawing of Compound 36 1 H- 1 H COSY spectrum
FIG. 31 is the HSQC spectrum of compound 37
FIG. 32 is an HMBC spectrum of compound 37
FIG. 33 is of compound 37 1 H- 1 H COSY spectrum
FIG. 34 is the HSQC spectrum of compound 38
FIG. 35 is an HMBC spectrum of compound 38
FIG. 36 is a photograph of Compound 38 1 H- 1 H COSY spectrum
FIG. 37 is the HSQC spectrum of Compound 39
FIG. 38 is an HMBC spectrum of compound 39
FIG. 39 is of Compound 39 1 H- 1 H COSY spectrum
FIG. 40 is a graph showing the effect of QQL-HT on the synovial membrane of joints in a rat gouty arthritis model.
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
1.1 cyclocarya paliurus medicinal material treatment: dried leaves (10Kg) of cyclocarya paliurus, after being crushed, are heated and refluxed at 120 ℃ by 70 percent ethanol for extraction (100L; 2 x 2h), and are concentrated at 60 ℃ to obtain extractum, and the concentration time is 2 days. And further dispersing the extract with water, sequentially extracting with dichloromethane, ethyl acetate and n-butanol for 10L each time for 3 times, and concentrating the extract at 60 deg.C for 24 hr to obtain concentrated extractive solutions of different polar parts.
The n-butanol fraction is mainly flavonoid, and is labeled as QQL-HT.
1.2 the biological activity is guided to screen the cyclocarya paliurus active site: an acute gout model is established by inducing rats with sodium urate to track and screen active sites of the n-butyl alcohol active sites of the cyclocarya paliurus for improving gout effects:
1.3 active site ingredient Studies
1.3.1 extraction, separation and purification of isopentenyl flavone: the invention adopts HPLC-DAD, TLC and UPLC-MS/MS methods to track, analyze, separate and purify the n-butanol extraction concentrated solution. Subjecting 445g of n-butanol fraction to macroporous resin chromatography column, isocratic eluting with ethanol-water system (0:100,30:70,50:50,70:30,95:5), and subjecting cyclocarya paliurus n-butanol fraction to five primary fractions (Fr.a-e) according to HPLC-DAD analysis result. Fr.b (106g) was passed through a polyamide column, eluted with an ethanol-water system (0:100-95:5) and divided into six fractions (Fr.b1-b6) according to HPLC-DAD analysis.
Subjecting Fr.c (107g) to polyamide chromatography column, eluting with ethanol-water system (0:100-95:5), separating into six fractions (Fr.c1-C6) according to HPLC-DAD analysis result, subjecting Fr.c1(22.9g) to HW-40C chromatography, eluting with methanol-water system (0:100-75:5), subjecting to HPLC-DAD analysis, and mixing the same fractions to obtain Fr.c1.1-Fr.c 1.12. Fr.c1.2 through ODS-AA medium pressure column, methanol-water system (5:95-75:5) elution, HPLC-DAD analysis, Fr.c1.2.1-Fr.c1.2.45. Fr.c. 1.2.23-Fr.c. 1.2.25 by semipreparative liquid phase (ACN-H2O 21%, 0.1% CH3COOH, v/v,220nm,3mL/min) gave compound 28(8.6mg),29(5.9mg) and 30(7.4 mg). Fr.c1.2.29-fr.c1.2.30 compound 27(13.6mg),35(11.7mg) and 40(15.7mg) were obtained by semipreparative liquid phase (ACN-H2O 24%, v/v,220nm,3mL/min), fr.c1.2.35-fr.c1.2.36 32(13.6mg) and 33(6.4mg) were isolated by semipreparative liquid phase (ACN-H2O 31%, v/v,220nm,3 mL/min). Fr.c1.3 was subjected to ODS-AA medium pressure column, eluted with a methanol-water system (5:95-75:5), and analyzed by HPLC-DAD to give Fr.c1.3.1-Fr.c 1.3.28. Fr.c. 1.3.13 by semipreparative liquid phase (ACN-H2O 18%, 0.1% CH3COOH, v/v,220nm,3mL/min) gave compound 36(10.8mg),37(4.9 mg). Fr.c. 1.3.15 by semi-preparative liquid phase (ACN-H2O 22%, v/v,220nm,3mL/min) gave compound 38(7.4mg),39(11.3 mg). Fr.c1.8 semi-preparative liquid phase (ACN-H2O 29%, v/v,220nm,3mL/min) gave compound 43(750.6mg),44(107.5mg),45(59.3 mg).
Fr.c2(20.5g) was eluted through HW-40C column chromatography using methanol-water system (0:100-75:5), analyzed by HPLC-DAD, and the same fractions were combined to give Fr.c2.1-Fr.c 2.10. Fr.c2.4 was eluted through ODS-AA medium pressure column using methanol-water system (5:95-75:5) and analyzed by HPLC-DAD to obtain Fr.c2.4.1-Fr.c 2.4.40. Fr. c2.4.18 by semipreparative liquid phase (ACN-H2O 20%, 0.1% CH3COOH, v/v,220nm,3mL/min) to give compound 53(33.7 mg); fr.c. 2.4.24-Fr.c. 2.4.25 by semipreparative liquid chromatography (ACN-H2O 22%, v/v,220nm,3mL/min) to give a mixture of compound 34 and compound 51, which was separated by thin layer preparative chromatography (developing solvent: water saturated n-butanol) to give compound 34(7.6mg), compound 51(28.5 mg); fr.c. 2.4.26 via semipreparative liquid phase (ACN-H2O 25%, v/v,220nm,3mL/min) gave compound 50(26.4mg), compound 51(76.2 mg); fr.c. 2.4.28 semi-preparative liquid phase (ACN-H2O 25%, v/v,220nm,3mL/min) gave compound 49(13.1 mg). Fr.c. 2.6 through ODS-AA medium pressure column, eluting with methanol-water system (15:85-75:5), analyzing by HPLC-DAD, and combining the same fractions to obtain Fr.c. 2.6.1-Fr.c. 2.6.35. Fr.c2.6.16 via a semi-preparative liquid phase (ACN-H2O 28%, 0.1% CH3COOH, v/v,220nm,3mL/min) to give compound 31(10.6 mg); fr.c2.6.20 by semipreparative liquid phase (ACN-H2O 27%, v/v,220nm,3mL/min) gave compound 46(33.7mg),47(21.9mg),48(9.4 mg); fr.c2.6.22 via a semi-preparative liquid phase (ACN-H2O 32%, 0.1% CH3COOH, v/v,220nm,3mL/min) to give compound 41(104.2 mg); fr.c2.6.25 by semi-preparative liquid phase (ACN-H2O 39%, v/v,220nm,3mL/min) to give compound 52(7.5 mg); fr.c. 2.6.28 semi-preparative liquid phase (ACN-H2O 44%, v/v,220nm,3mL/min) gave compound 42(7.5 mg).
1.3.2 structural confirmation: modern spectral techniques such as UV, IR, NMR, MS, CD, ORD, ECD, single crystal X-rays and the like are used for confirming the planar structure and the spatial configuration of each compound, and the system attribution of the spectral characterization and the spectral data is carried out.
The type of absorption of the compound and its presence or absence of the conjugated segment was first confirmed by uv absorption. The planar structure of the compound was then determined by 1D/2D NMR and its molecular weight was confirmed by mass spectrometry. The absolute configuration of the compound was confirmed by NOESY, ROESY, CD, ECD, etc.
1.4 chemical Structure
27 prenylflavonoids (27-53) were isolated, as shown in Table 1.
TABLE 1 molecular information for Compounds 27-53
Figure BDA0003029325720000101
Figure BDA0003029325720000111
The structural characterization data are:
compound 27: a yellow amorphous powder;
Figure BDA0003029325720000112
UV(MeOH)λ max (logε)nm:204(3.88),270(1.42),320(0.77),and 349(0.71)nm。HRESIMS,m/z:841.3125[M+H] + (calculated for 841.3130)。
compound 28: a yellow amorphous powder;
Figure BDA0003029325720000113
UV(MeOH)λ max (logε)nm:204(3.87),270(1.39),320(0.76)and 349(0.70)nm。HRESIMS,m/z 827.2974[M+H] + (calculated for 827.2974)。
compound 29: a yellow amorphous powder;
Figure BDA0003029325720000114
UV(MeOH)λ max (logε)nm:204(4.00),271(1.66),316(0.91),and 350(0.83)nm。HRESIMS,m/z 857.3074[M+H] + (calculated for 857.3079)。
compound 30: a yellow amorphous powder;
Figure BDA0003029325720000115
UV(MeOH)λ max (logε)nm:204(4.15),271(2.44),316(1.37),and 350(1.12)nm。HRESIMS,m/z 857.3074[M+H] + (calculated for 857.3079)。
compound 31: a yellow amorphous powder, which is obtained by grinding,
Figure BDA0003029325720000116
UV(MeOH)λ max (logε)nm:204(3.95),270(1.52),320(0.83),and 349(0.76)nm。HRESIMS,m/z:807.2701[M+H] + (calculated for 807.2712)。
compound 32: a yellow amorphous powder;
Figure BDA0003029325720000117
UV(MeOH)λ max (logε)nm:204(4.10),271(2.30),316(1.29),and 350(1.05)nm。HRESIMS,m/z:823.3011[M+H] + (calculated for 823.3025)。
compound 33: a yellow amorphous powder;
Figure BDA0003029325720000118
UV(MeOH)λ max (logε)nm:204(3.95),270(1.52),321(0.65),and 349(0.76)nm。HRESIMS,m/z:955.3442[M+H] + (calculated for 955.3447)。
compound 34: a yellow amorphous powder;
Figure BDA0003029325720000119
UV(MeOH)λ max (logε)nm:203(3.86),270(1.20),321(0.65),and 349(0.60)nm。HRESIMS,m/z:825.2827[M+H] + (calculated for 825.2817)。
compound 35: a yellow amorphous powder;
Figure BDA00030293257200001110
UV(MeOH)λ max (logε)nm:204(4.10),271(2.30),316(1.29),and 350(1.05)nm。HRESIMS,m/z:711.2500[M+H] + (calculated for 711.2500)。
compound 36: a yellow amorphous powder;
Figure BDA0003029325720000122
UV(MeOH)λmax(logε)nm:200(3.70),271(1.05),316(0.59),and 350(0.48)nm。HRESIMS,m/z:711.2505[M+H] + (calculated for 711.2500)。
compound 37: a yellow amorphous powder;
Figure BDA0003029325720000123
UV(MeOH)λ max (logε)nm:199(3.75),271(1.13),316(0.63),and 350(0.52)nm。HRESIMS,m/z 857.3083[M+H] + (calcd for C 39 H 53 O 21 ,857.3079)。
compound 38: a yellow amorphous powder;
Figure BDA0003029325720000124
UV(MeOH)λ max (logε)nm:199(3.75),271(1.13),316(0.63),and 350(0.52)nm。HRESIMS,m/z:825.2822[M+H] + (calcd for C 38 H 49 O 20 ,825.2817)。
compound 39: yellow colourAn amorphous powder;
Figure BDA0003029325720000125
UV(MeOH)λmax(logε)nm:199(3.75),271(1.13),316(0.63),and 350(0.52)nm。HRESIMS,m/z:839.2982[M+H] + (calcd for 839.2974)。
TABLE 2 preparation of compounds 27 to 30 1 H NMR and 13 c NMR Signal assignment
Figure BDA0003029325720000121
Figure BDA0003029325720000131
a Measured in DMSO-d 6 at 400MHz; b Measured in DMSO-d 6 at 500MHz;Overlapped signals indicated by(o)
TABLE 3 preparation of compounds 31 to 34 1 H NMR and 13 c NMR Signal assignment
Figure BDA0003029325720000132
Figure BDA0003029325720000141
Figure BDA0003029325720000151
a Measured in DMSO-d 6 at 400MHz; b Measured in DMSO-d 6 at 500MHz;Overlapped signals indicated by(o)
TABLE 4 of Compounds 35 to 37 1 H NMR and 13 c NMR Signal assignment
Figure BDA0003029325720000152
Figure BDA0003029325720000161
a Measured in DMSO-d 6 at 400MHz;Overlapped signals indicated by(o).
TABLE 5 of Compounds 38 to 39 1 H NMR and 13 c NMR Signal assignment
Figure BDA0003029325720000162
Figure BDA0003029325720000171
Figure BDA0003029325720000181
a Measured in DMSO-d 6 at 400MHz; b Measured in DMSO-d 6 at 500MHz;Overlapped signals indicated by(o)
The detailed characterization icons are shown in fig. 1-39.
Example 2
2.1 anti-gouty arthritis action of n-butanol active site and isopentenyl flavonoid of cyclocarya paliurus
2.1.1 Experimental methods:
research on the effect of QQL-HT on gouty arthritis resistance: selecting 40 quarantine certified SD rats, and randomly dividing males into 4 groups according to body weight, wherein the 4 groups are respectively a normal control group, a model control group, a cyclocarya paliurus n-butanol active site group and a gout capsule group, and each group comprises 10 animals. The corresponding liquid medicine is given to each group by intragastric administration, and the equal volume of pure water is given to the normal control group and the model control group by intragastric administration. The administration is performed 1 time per day for 8 days. Except for a normal control group, the other groups are molded 30min after administration, SD rats are inhaled and anesthetized with isoflurane (inducing 1-4 percent and maintaining 0.25-2 percent), then the peripheries of knee joints of hind legs at two sides are shaved, the skin is disinfected by medical alcohol, the knee joints are slightly bent, the needles are inserted through the side faces of the joints, 0.5mL of sodium urate solution (25mg/mL) is injected into the knee joint cavities of the rats through supraclavicular ligaments by using a No. 6 syringe needle, and the acute gouty arthritis model is copied.
Anti-inflammatory action study of candidate compounds: taking rat macrophage-like cell line (RAW.264.7) cells at 1 × 10 6 The cells were inoculated in 96-well plates at a concentration of one mL and incubated at 37 ℃ for 12 hours. Then, the test sample (1, 3, 10, 30. mu.M) was added to the cell culture solution and cultured for 1 hour, LPS (1. mu.g/mL) was added thereto, and the cells were cultured together at 37 ℃ for 24 hours. 50. mu.L of the cell culture supernatant was placed in a new 96-well plate, and 50. mu.L of LGriess I solution and Griess II solution were added to each well in succession. After standing at room temperature for 10min, the absorbance (A) of the reaction product was measured at 540nm using a microplate reader. Meanwhile, the MTT method is used for measuring the cytotoxicity of each group of medicines.
2.1.2 Experimental results:
(1) influence on swelling degree of knee joint
The circumferences of the right knee joints of the rats in each group were measured by a wire-laying method at 1h, 2h, 4h, 6h and 12h before and after the model creation, and the swelling index, which is (the circumference of the knee joint after the model creation-the circumference of the knee joint before the model creation)/the circumference of the knee joint before the model creation, was 100%, was calculated.
As shown in table 6, the percent of joint swelling in the model control rats was significantly increased (P <0.01) compared to the normal control group, suggesting successful replication of the gouty arthritis model; compared with a model control group, the joint swelling rate of rats in the QQQL-HT group after 1, 2, 4, 6 and 12h of model building is obviously reduced (P <0.05 or P <0.01), which indicates that QQQL-HT can obviously inhibit the swelling of rat gouty arthritis model caused by sodium urate; the QQQL-HT has more durable effect on inhibiting gouty arthritis swelling and better effect than gout treating capsules.
TABLE 6 influence of QQL-HT on joint swelling in rat gouty arthritis model with sodium urate (S) ((S))
Figure BDA0003029325720000183
n=10)
Figure BDA0003029325720000182
Figure BDA0003029325720000191
Note: comparing with normal control group + P<0.01; comparison with model control group * P<0.05, ** P<0.01
(2) Effects on arthritic factors
As shown in Table 7, compared with the normal control group, the contents of inflammatory factors IL-1 beta and TNF-alpha in the joint cavity washing liquid of the rats in the model control group are obviously increased (P < 0.01); compared with a model control group, IL-1 beta and TNF-alpha in the joint cavity flushing fluid of the rat in the QQL-HT group are both obviously reduced (P is less than 0.01), which indicates that QQL-HT can obviously inhibit the secretion of inflammatory factors of the rat gouty arthritis model caused by sodium urate, and meanwhile, the inhibition effect of QQL-HT on the secretion of IL-1 beta and TNF-alpha is obviously stronger than that of gout determining capsules.
TABLE 7 influence of QQL-HT on articular cavity inflammatory factor in rat gouty arthritis model induced by sodium urate ((
Figure BDA0003029325720000192
n=10)
Figure BDA0003029325720000193
Note: comparing with normal control group + P<0.01; comparison with model control group * P<0.05
(3) Histopathological examination of the synovial membrane of the knee joint
24 hours after model building, the isoflurane (inducing 1-4 percent and maintaining 0.25-2 percent) of rats in each group is inhaled for anesthesia and then the abdominal aorta is exsanguinated and euthanased, joint synovial tissues are taken and placed in 10 percent neutral formalin solution for fixation, paraffin embedding and HE staining are carried out, the pathological examination of the joint synovial tissues is carried out, and the pathological changes of the knee joint are divided into 4 grades according to the pathological changes, wherein the 1 grade is not abnormal, and the 2-4 grades are respectively divided into light, medium and severe pathological changes according to the synovial tissue hyperplasia, interstitial edema and bleeding, and the infiltration degree of inflammatory cells and fibroblasts.
As shown in table 8 and fig. 40, compared with the normal control group, the level of joint lesions of the rats in the model control group is significantly increased (P <0.01), the types of lesions are mainly synovial tissue hyperplasia of 2-3 levels, and interstitial edema, vascular congestion, inflammatory cells and fibroblast infiltration are accompanied, thus indicating that the gouty arthritis model is successfully replicated; compared with a model control group, the joint pathological changes of the rats in the QQL-HT group are obviously reduced, the number of animals with pathological changes in each degree is obviously reduced (P is less than 0.01), the QQL-HT is prompted to obviously improve the joint pathological changes of the rat gouty arthritis model caused by sodium urate, and the QQL-HT is stronger than a gouuding capsule.
TABLE 8 influence of QQL-HT on pathological changes in joints in rat gouty arthritis model induced by sodium urate ((
Figure BDA0003029325720000194
n=10)
Figure BDA0003029325720000195
Figure BDA0003029325720000201
Note: comparing with normal control group + P<0.01; comparison with model control group * P<0.05。
(4) Evaluation of anti-inflammatory Activity of prenyl flavonoid Compound
NO is closely related to gouty arthritis, and the increase of the in vivo NO level can affect pathological processes from a plurality of ways, such as the initiation of nuclear factor NF-kB, the induction of proinflammatory cytokines TNF-alpha, IL-1 and the like, and finally the stimulation of synovial cell proliferation to cause irreversible damage to cartilage. Primary screening for anti-inflammatory activity was performed using LPS-induced mouse mononuclear macrophages (raw.264.7) as a model and indomethacin (Indo) as a positive control. In the experiment, a Griess reagent is adopted to detect NO in the culture supernatant of RAW264.7 cells; and detecting the cell viability by using an MTT method, investigating the influence of the compound on NO generated by an LPS-induced RAW.264.7 cell model, and taking the NO content in the supernate as a detection index. The results are shown in Table 9.
TABLE 9 Effect on LPS-induced RAW.264.7 cell viability
Figure BDA0003029325720000202
Figure BDA0003029325720000203
Figure BDA0003029325720000211
In contrast to the model set, # p<0.05, ## p<0.01
2.2 the uric acid reducing effect of the n-butanol active site and the isopentenyl flavonoid compound of cyclocarya paliurus
2.2.1 methods of experiment:
selecting 40 male ICR mice qualified for quarantine, randomly dividing the mice into 4 groups according to weight, namely a normal control group, a model control group, a gout capsule group and a QQL-HT group, wherein each group comprises 10 animals. The corresponding concentration liquid medicine is given to each group of animals through oral gavage according to 20mL/kg, and the same volume of pure water is given to the normal control group and the model control group through gavage for 1 time every day for 7 consecutive days. A single 20mL/kg intraperitoneal injection of OAPS 300mg/kg replicates hyperuricemia model 1h before administration on day 7, and a normal control group and a blank administration group are given equal volume of 0.9% sodium chloride injection. The drug is administrated 1h after the model building, and blood is collected for measuring blood uric acid 1h after the drug administration (fasting is not forbidden one day before blood collection).
2.2.2 results of the experiment
As shown in table 10, compared with the normal control group, the serum UA of the mice in the model control group is significantly increased (P <0.01), indicating that the hyperuricemia model is successfully replicated; compared with a model control group, UA (P <0.01) of mice in the QQL-HT group is remarkably reduced after the mice are modeled, which indicates that QQL-HT can remarkably reduce the uric acid level of a hyperuricemia model caused by potassium thiocyanate, and the drug effect of QQL-HT is remarkably stronger than that of a gout determining capsule.
TABLE 10 influence of QQL-HT on potassium oxycyanate-induced mouse hyperuricemia model: (
Figure BDA0003029325720000212
n=10)
Figure BDA0003029325720000213
Note: comparing with normal control group + P<0.01; comparison with model control group * P<0.05
2.3 QQL-HT and monomeric Compounds inhibit xanthine oxidase Activity
2.3.1 methods of experiment:
xanthine Oxidase (XOD), which is a key enzyme in purine catabolism in vivo, plays an important role in maintaining uric acid balance in blood in vivo, and is a target for uric acid lowering therapy. Xanthine oxidase produces superoxide ions and uric acid by catalyzing xanthine metabolism, and therefore experiments were conducted to evaluate the inhibitory effect of compounds on xanthine oxidase by measuring the levels of superoxide ions and uric acid produced. Initial screening for uric acid lowering activity can be performed by using Allopurinol (Allopurinol) as a positive control. NBT chromogenic reaction was used to evaluate the effect of drugs on xanthine oxidase activity by measuring the content of superoxide ion, xanthine (50. mu.M), xanthine oxidase (0.1U/ml), NBT (50. mu.M) and candidate compound at each concentration or positive control allopurinol (1. mu.g/ml) were added to the reaction system, and phosphate buffer (50mM, pH 7.5) was added to the reaction system to a final volume of 100. mu.L. The reaction was started by adding xanthine oxidase, allowed to react at room temperature for 15min, and after completion of the reaction, the absorbance value was measured at 560 nm. Measurement of uric acid content the effects of the drugs on xanthine oxidase activity were evaluated by adding xanthine (50. mu.M), xanthine oxidase (0.1U/ml) and each concentration of the candidate compound or positive control allopurinol (1. mu.g/ml) to the reaction system, and adding phosphate buffer (50mM, pH 7.5) to the reaction system to a final volume of 100. mu.L. The reaction starts with the addition of xanthine oxidase, the reaction is carried out for 15min at room temperature, the absorbance value is measured at 295nm after the reaction is finished, and the inhibition rate of the xanthine oxidase is calculated.
TABLE 11 inhibition of xanthine oxidase activity in vitro
Figure BDA0003029325720000221
The data show that the QQL-HT group and isopentenyl flavonoid compounds have stronger effects of inhibiting gouty arthritis and reducing uric acid, and the effect is superior to that of a positive medicament gout determining capsule, and the QQL-HT group and isopentenyl flavonoid compounds can be used as a novel medicament. Experiments show that the effects of inhibiting gouty arthritis and reducing uric acid of the isopentene flavonoid compound are obviously superior to those of similar isopentene flavonoid compounds disclosed in the prior art, and the effects are unexpected.
The above examples are set forth so that this disclosure will be understood in all instances to be considered illustrative and not restrictive, and that various modifications and equivalent arrangements may be devised by those skilled in the art after reading this disclosure and are intended to be included within the scope of the appended claims.

Claims (2)

1. An isopentene-based flavonoid compound is characterized by having a structural formula as follows:
Figure FDA0003626947320000011
2. the use of prenylflavonoids according to claim 1 in the preparation of a medicament for the amelioration of gout.
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