CN113150048B - Cyclocarya paliurus extract and application thereof in resisting rheumatoid arthritis - Google Patents
Cyclocarya paliurus extract and application thereof in resisting rheumatoid arthritis Download PDFInfo
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Abstract
The invention relates to a new moneyThe active site extracted from the cyclocarya paliurus is a n-butanol extraction concentrated solution of the cyclocarya paliurus, the main component of the active site is an isopentenyl flavonoid compound, and the structural general formula of the isopentenyl flavonoid compound is as follows:
Description
Technical Field
The invention relates to an extract of cyclocarya paliurus, in particular to an isopentenyl flavonoid compound, 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 the human body.
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 the influx of Ca 2+ into vascular smooth muscle, directly dilates 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, proteins, flavonoids, polysaccharides, triterpenes and other compounds, wherein prenylflavonoids are widely concerned due to the diversity of structures and pharmacological activities of the prenylflavonoids. At present, the research on the components mostly focuses on reducing blood sugar and inflammation, and the research on the activity and mechanism in other aspects is less, so that the specific elucidation of other application activities and action mechanisms of the prenylflavonoids 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 an isopentenyl flavone active site and an isopentenyl flavonoid compound of cyclocarya paliurus, and a system research on an active component and a pharmacodynamic substance basis for resisting rheumatoid arthritis.
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:
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-glucose, 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-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
Preferably, the isopentenyl flavonoid compound has the following structural formula:
the invention also provides a method for extracting the isopentene 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, removing interference components such as pigments and tannins from Fr.C by adopting polyamide column chromatography, and carrying out 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, which is the n-butanol extraction concentrated solution.
The invention also claims the application of the active site in preparing the medicine for resisting rheumatoid arthritis.
Further, the specific process of separating and purifying the prenylflavonoids 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 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 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.c1.2.23-Fr.c1.2.25 by semi-preparative liquid phase (ACN-H) 2 O 21％,0.1％CH 3 COOH, v/v,220nm,3mL/min) gave compound 28(8.6mg),29 (5.9)mg) and 30(7.4 mg). Fr.c1.2.29-Fr.c1.2.30 by semi-preparative liquid phase (ACN-H) 2 O24%, v/v,220nm,3mL/min) to give compound 27(13.6mg),35(11.7mg) and 40(15.7mg), Fr. c.1.2.35-Fr. c.1.2.36 via a semi-preparative liquid phase (ACN-H) 2 O31%, v/v,220nm,3mL/min) to yield 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 semi-preparative liquid phase (ACN-H) 2 O 18％,0.1％CH 3 COOH, v/v,220nm,3mL/min) gave compound 36(10.8mg),37(4.9 mg). Fr. c1.3.15 by semi-preparative liquid phase (ACN-H) 2 O22%, v/v,220nm,3mL/min) gave compound 38(7.4mg),39(11.3 mg). Fr. c1.8 by semi-preparative liquid phase (ACN-H) 2 O29%, 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.c. 2.4.18 by semi-preparative liquid phase (ACN-H) 2 O 20％,0.1％CH 3 COOH, v/v,220nm,3mL/min) gave compound 53(33.7 mg); fr.c2.4.24-Fr.c2.4.25 by semi-preparative liquid phase (ACN-H) 2 O22%, v/v,220nm,3mL/min) to give a mixture of compound 34 and compound 51, which was purified by thin layer preparative chromatography (developing solvent: water-saturated n-butanol) to give compound 34(7.6mg), compound 51(28.5 mg); fr. c2.4.26 semi-preparative liquid phase (ACN-H) 2 O25%, v/v,220nm,3mL/min) to give compound 50(26.4mg), compound 51(76.2 mg); fr.c. 2.4.28 by semi-preparative liquid phase (ACN-H) 2 O25%, 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 by semi-preparative liquid phase (ACN-H) 2 O 28％,0.1％CH 3 COOH, v/v,220nm,3mL/min) gave compound 31(10.6 mg); fr.c. 2.6.20 by semi-preparative liquid phase (ACN-H) 2 O27%, v/v,220nm,3mL/min) to give compound 46(33.7mg),47(21.9mg),48(9.4 mg); fr.c2.6.22 Jing semi-finished productLiquid preparation phase (ACN-H) 2 O 32％,0.1％CH 3 COOH, v/v,220nm,3mL/min) gave compound 41(104.2 mg); fr.c. 2.6.25 by semi-preparative liquid phase (ACN-H) 2 O39%, v/v,220nm,3mL/min) to give compound 52(7.5 mg); fr.c. 2.6.28 by semi-preparative liquid phase (ACN-H) 2 O44%, v/v,220nm,3mL/min) gave compound 42(7.5 mg).
The structural formulas of the compounds 27-53 are:
the invention also provides application of the isopentene flavonoid compounds 27-53 in preparation of a medicament for resisting rheumatoid arthritis.
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 a 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 a new application of the n-butyl alcohol active site and the isopentenyl flavonoid compound 27-53 of the cyclocarya paliurus in resisting rheumatoid arthritis.
Drawings
FIG. 1 is the HSQC spectrum of compound 27
FIG. 2 shows HMBC spectra of compound 27
FIG. 3 is a 1H-1H COSY spectrum of compound 28
FIG. 4 is the HSQC spectrum of Compound 28
FIG. 5 is an HMBC spectrum of compound 28
FIG. 6 shows the 1H-1H COSY spectrum of compound 28
FIG. 7 is the HSQC spectrum of Compound 29
FIG. 8 is an HMBC spectrum of compound 29
FIG. 9 shows the 1H-1H COSY spectrum of compound 29
FIG. 10 is the HSQC spectrum of Compound 30
FIG. 11 is an HMBC spectrum of compound 30
FIG. 12 shows the 1H-1H COSY spectrum of compound No. 30
FIG. 13 is the HSQC spectrum of Compound 31
FIG. 14 is an HMBC spectrum of compound 31
FIG. 15 is a 1H-1H COSY spectrum of compound 31
FIG. 16 is the HSQC spectrum of Compound 32
FIG. 17 is an HMBC spectrum of compound 32
FIG. 18 shows the 1H-1H COSY spectrum of compound No. 32
FIG. 19 is the HSQC spectrum of compound 33
FIG. 20 is an HMBC spectrum of compound 33
FIG. 21 shows the 1H-1H COSY spectrum of compound 33
FIG. 22 is an HSQC spectrum of Compound 34
FIG. 23 is an HMBC spectrum of compound 34
FIG. 24 is the 1H-1H COSY spectrum of compound 34
FIG. 25 is the HSQC spectrum of Compound 35
FIG. 26 is an HMBC spectrum of compound 35
FIG. 27 is the 1H-1H COSY spectrum of compound 35
FIG. 28 is HSQC spectrum of compound 36
FIG. 29 is an HMBC spectrum of compound 36
FIG. 30 is the 1H-1H COSY spectrum of compound 36
FIG. 31 is the HSQC spectrum of compound 37
FIG. 32 is an HMBC spectrum of compound 37
FIG. 33 is the 1H-1H COSY spectrum of compound 37
FIG. 34 is an HSQC spectrum of Compound 38
FIG. 35 is an HMBC spectrum of compound 38
FIG. 36 is a 1H-1H COSY spectrum of compound 38
FIG. 37 is the HSQC spectrum of Compound 39
FIG. 38 is an HMBC spectrum of compound 39
FIG. 39 is a 1H-1H COSY spectrum of compound 39.
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. Dispersing the extract with water, extracting with n-butanol 10L for 3 times, concentrating the extractive solution at 60 deg.C for 24 hr to obtain n-butanol extractive concentrated solution.
The n-butanol fraction is flavonoid. Denoted QQL-HT.
1.2 the biological activity oriented screening of cyclocarya paliurus active sites: the degree of resisting RA is comprehensively evaluated by indexes such as swelling degree of toes, arthritis index score, synovial tissue morphology observation and the like by adopting an adjuvant arthritis rat animal model.
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.
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 through ODS-AA medium pressure column, eluting with methanol-water system (5:95-75:5), and HPLC-DAD analysis to obtain Fr.c1.2.1-Fr.c1.2.45. Fr.c1.2.23-Fr.c1.2.25 by semi-preparative liquid phase (ACN-H) 2 O 21％,0.1％CH 3 COOH, 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 by semi-preparative liquid phase (ACN-H) 2 O24%, v/v,220nm,3mL/min) to give compound 27(13.6mg),35(11.7mg) and 40(15.7mg), Fr.c. 1.2.35-Fr.c. 1.2.36 as a semi-preparative liquid phase (ACN-H) 2 O31%, v/v,220nm,3mL/min) to yield 32(13.6mg) and 33(6.4 mg). 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. c1.3.13 by semi-preparative liquid phase (ACN-H) 2 O 18％,0.1％CH 3 COOH, 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-H) 2 O22%, v/v,220nm,3mL/min) gave compound 38(7.4mg),39(11.3 mg). Fr. c1.8 by semi-preparative liquid phase (ACN-H) 2 O29%, 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 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.c. 2.4.18 by semi-preparative liquid phase (ACN-H) 2 O 20％,0.1％CH 3 COOH, v/v,220nm,3mL/min) gave compound 53(33.7 mg); fr.c2.4.24-Fr.c2.4.25 by semi-preparative liquid phase (ACN-H) 2 O22%, v/v,220nm,3mL/min) to give a mixture of compound 34 and compound 51, which was purified 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 by semi-preparative liquid phase (ACN-H) 2 O25%, v/v,220nm,3mL/min) to give compound 50(26.4mg), compound 51(76.2 mg); fr.c. 2.4.28 by semi-preparative liquid phase (ACN-H) 2 O25%, 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.c. 2.6.16 by semi-preparative liquid phase (ACN-H) 2 O 28％,0.1％CH 3 COOH, v/v,220nm,3mL/min) gave compound 31(106 mg); fr.c. 2.6.20 by semi-preparative liquid phase (ACN-H) 2 O27%, v/v,220nm,3mL/min) to give compound 46(33.7mg),47(21.9mg),48(9.4 mg); fr.c. 2.6.22 by semi-preparative liquid phase (ACN-H) 2 O 32％,0.1％CH 3 COOH, v/v,220nm,3mL/min) to give compound 41(104.2 mg); fr. c2.6.25 by semi-preparative liquid phase (ACN-H) 2 O39%, v/v,220nm,3mL/min) to give compound 52(7.5 mg); fr.c. 2.6.28 by semi-preparative liquid phase (ACN-H) 2 O44%, v/v,220nm,3mL/min) gave compound 42(7.5 mg).
1.3.2 Structure 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 spectral characterization and 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 of Compounds 27-53
The structural characterization data are:
compound 27: a yellow amorphous powder;
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)。
chemical combinationSubstance 28: a yellow amorphous powder;
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;
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;
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 a mixture of a yellow amorphous powder,
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;
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;
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;
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;
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;
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 for711.2500)。
compound 37: a yellow amorphous powder;
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;
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: a yellow amorphous powder;
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
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
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
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
a Measured in DMSO-d 6 at 400MHz； b Measured in DMSO-d 6 at 500MHz；Overlapped signals indicated by(o)
Example 2
Application of 1 QQL-HT and isopentenyl flavonoid compound in preparation of medicine for treating rheumatoid arthritis
1.1 bioactive targeting active site screening: by adopting an adjuvant arthritis rat animal model, the influence of the traditional Chinese medicine extract on the toe swelling degree of a rat and the influence on inflammatory factors in joint fluid of the rat can be used as indexes of preliminary in vivo activity screening and in vivo efficacy evaluation.
(1) The rat animal model with adjuvant arthritis is adopted to comprehensively evaluate the degree of resisting RA according to indexes such as toe swelling degree, arthritis index score, synovial tissue morphological observation and the like.
(2) Detecting the rheumatoid factor PGE in joint fluid by adopting an adjuvant arthritis rat animal model and an ELISA method 2 Levels, the extent of anti-RA was assessed in combination.
1.2 anti-RA Activity of candidate Compounds and preliminary mechanism of action
(1) For inflammatory factor PGE 2 Measuring the content; (2) screening COX-2 activity inhibition, and primarily discussing the action mechanism of the active compound.
1.3 medicine:
1.3.1 dried leaves of cyclocarya paliurus are provided by Hunan and Nature Biotechnology development Ltd.
1.3.2 Positive control: celecoxib capsules.
1.4 Experimental animals: SD rats 40, male, were purchased from Silikedada laboratory animals, Inc., Hunan.
1.5 main reagents: sodium carboxymethylcellulose (CMC-Na) (Chemicals, Inc., national drug group), bovine type II collagen solution and complete Freund's adjuvant were purchased from Sigma, USA, and COX-2ELISA kit was purchased from Solebao scientific, Inc.
1.6 Main instruments: rat ankle volume measurement (Chengdutai alliance technologies, Inc.); LABOSPECT003 model automatic biochemical analyzer (number: 001, manufactured by Hitachi, Japan);
2 method of experiment
2.1 cyclocarya paliurus medicinal material treatment
Dried cyclocarya paliurus (20Kg), reflux-extracting with distilled water under heating (300L; 2 × 2h), concentrating at 60 deg.C under reduced pressure to obtain extract,
2.2 research on the effect of cyclocarya paliurus water extract on rheumatoid arthritis resistance:
experimental SD rats with adjuvant rat model for resisting RA effect are male, the weight of the SD rats is 180-220 g, 10 rats are taken as a normal control group, the rest animals are taken as model control groups, 0.1mL of Freund's complete adjuvant is injected into the right hind foot sole part of the model control group subcutaneously, after 7d of molding, toe swelling and swelling degree is measured by adopting a toe volume meter, the SD rats are randomly divided into the model control group according to the toe swelling degree, the positive control group has different sample doses of 150mL/kg, and each 6 animals. Before daily administration, each compound is prepared into suspension with corresponding concentration by 0.5% CMC-Na, each group of rats is administrated with suspension with corresponding concentration by 10mL/kg intragastric administration for 1 time/day for 21 consecutive days, the model control group is administrated with equal volume of 0.5% CMC-Na by intragastric administration, and the normal control group is administrated with equal volume of distilled water by intragastric administration.
(1) The swelling degree of the feet of the rats in each group was measured with a foot swelling instrument after 7 days, 14 days and 21 days of the administration, and the degree of the systemic joint lesion was observed and recorded, and the arthritis index score (AI) was calculated. Systemic lesions are evaluated according to a grade 5 grading method, and the polyarthritis index is calculated according to the accumulated integral of the lesion degrees of the rest 3 limbs which are not injected with the adjuvant. 0 minute: no red swelling; 1 minute: red and swollen toe joints; and 2, dividing: swelling of the toe joints and metatarsals; and 3, dividing: paw swelling below the ankle joint; and 4, dividing: the paw including the ankle joint was swollen, and the cumulative score of each joint was the AI for each rat.
(2) After anesthetizing each group of rats, the swollen toes were cut at a position 0.5cm above the ankle joint from the site of inflammation, and cut longitudinally with 5mL of raw material
And fully washing the inside of the swollen joint by normal saline, placing the swollen toes in the washing solution for soaking for 2h, centrifuging at 3000rpm, taking the supernatant, and detecting the levels of rheumatoid factors RF, TNF-alpha, IL-1 and PGE2 in the supernatant by using an ELISA kit.
(3) Swollen toe joints were taken, formalin-fixed, paraffin-embedded, sectioned, HE-stained, histopathological examination of joints and synovium was performed, and pathological scoring was performed.
(4) The immunohistochemical ABC method is used for detecting the protein expression level of the nuclear transcription factor COX-2 in synovial tissues.
2.3 statistical methods: statistical analysis was performed using SPSS 25.0, with the level of statistical significance set at P < 0.05. The mean. + -. standard deviation (+ -s) is used for the measurement data. The method of Leven's test is used for checking the normality and the homogeneity of the variance. If the normality and the homogeneity of variance are met, carrying out statistical analysis by using One-way ANOVA (One-way ANOVA) and post Hoc LSD; if the normality and variance are not met, the Kruskal-Wallis test is used. If the Kruskal-Wallis Test is statistically significant (P <0.05), a comparative analysis is performed using Dunnett's Test (nonparametric method).
3, experimental results:
3.1. biological activity guide screening cyclocarya paliurus active site
As shown in table 5 and table 6, qqql-HT improved adjuvant rat toe volume and significantly different synovial fluid cytokine levels (P <0.05 or P <0.01) compared to the model control group, and was comparable to the positive control drug celecoxib.
3.1.1 Effect on rat toe Capacity
As shown in table 6, the toe volume of the model control rats significantly increased before, 7 days after, and 14 days after the administration of the drug (P <0.01) compared to the normal control group, indicating that the model replication was successful. After 7 days of continuous administration, the toe volume of the rats in the QQL-HT group is remarkably reduced (P <0.05 or P <0.01), and the toe swelling degree of the rats in the QQL-HT group is remarkably reduced (P <0.05 or P < 0.01); after 14 days of continuous administration, the toe swelling degree of the rats in the QQL-HT group is obviously reduced (P <0.05 or P <0.01) compared with that in the model control group, and the effect is caused by a positive control group.
TABLE 6 influence of QQL-HT on left hind paw volume of SD rats: (
n＝6)
Note: comparing with normal control group ++ P<0.01; comparison with model control group * P<0.05， ** P<0.01。
3.1.2 Effect on inflammatory factors in rat synovial fluid
As shown in Table 7, the levels of TNF- α, IL-6, PGE2, COX-2, and RF in the synovial fluid of the rats in the model control group were increased (P <0.05 or P <0.01) compared to the normal control group, indicating successful model replication; compared with the model control group, the positive control group, the QQL-HT group, the TNF-alpha, the IL-6, the PGE2 and the COX-2 level are obviously reduced (P <0.05 or P < 0.01). The QQL-HT group has a lower NF- κ B content than the model control group (P <0.05 or P < 0.01).
TABLE 7 influence of QQL-HT on inflammatory factors in synovial fluid of SD rats ((
n＝6)
Note: comparing with normal control group ++ P<0.01; comparison with model control group * P<0.05， ** P<0.01。
3.2 screening of prenylflavonoids against RA in vitro Activity
3.2.1 Induction of RAW.264.7 by LPS to produce PGE 2 Influence of (2)
As shown in Table 8, at low dose (1. mu.M), the compounds 28, 36 and 45 in the drug groups all inhibited the secretion of PGE from RAW.264.7 cells induced by LPS 2 And reducing the release of inflammatory factors, with compound 36 being the most effective. At high doses (10 μ M), compounds 32 and 42 were the best, comparable to the positive control, and had no side effects.
TABLE 8 PGE production by LPS-induced RAW.264.7 cells 2 Influence of (2)
In contrast to the model set, # p<0.05, ## p<0.01
inhibition of COX-2 Activity by active Compounds
According to the research basis of the anti-RA activity of the candidate compound, the COX-2 enzyme activity inhibitor screening kit is adopted to detect the inhibition effect of the compound on the COX-2 activity. As shown in Table 9, all candidate compounds were able to inhibit COX-2 to some extent, with Compound 36 showing a better COX-2 activity inhibition.
TABLE 9 inhibition of COX-2 activity
3. Small knot
The research separates the n-butyl alcohol active site and 27 isopentenyl flavones of cyclocarya paliurus from the active site of the cyclocarya paliurus, and performs the activity determination of the anti-rheumatoid arthritis on the n-butyl alcohol active site and the 27 isopentenyl flavones of the cyclocarya paliurus, QQL-HT can improve the volume of the toes of an adjuvant rat, and the content of the joint fluid cytokines is obviously different from that of a model control group (P is a P-QL-HT)<0.05 or P<0.01) which has the same effect with the positive control drug celecoxib. The QQL-HT group can significantly reduce the levels (P) of inflammatory factors TNF-alpha, IL-6, PGE2 and COX-2<0.05 or P<0.01). Simultaneously, preliminary mechanism research is carried out, and the prenylflavone can inhibit LPS (LPS) -induced RAW.264.7 cells from secreting PGE (PGE) 2 And reducing the release of inflammatory factors, with compound 36 being the most effective. The compound 36 is found to remarkably inhibit COX-2, shows good COX-2 inhibitor activity and has better activity than a prenylflavone compound with a similar structure disclosed in the prior art, so that the protection effect of the prenylflavone on LPS-induced RAW.264.7 cells is supposed to be to play a role in improving RA by inhibiting COX-2 protein. The research result shows that the isopentene flavonoid compound is the drug effect substance basis of the cyclocarya paliurus for resisting the rheumatoid arthritis, and provides a thought and a direction for the later deep molecular action mechanism research.
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 (3)
1. A method for extracting isopentenyl flavonoid compounds from cyclocarya paliurus is characterized by comprising the following steps:
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;
s3, analyzing, separating and purifying the n-butanol extraction concentrated solution by HPLC-DAD, TLC and UPLC-MS/MS methods;
the structural formula of the isopentene flavonoid compound is as follows:
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 2 hours, the concentration temperature is 60 ℃, and the concentration time is 2-3 days;
in S2, n-butanol is used for extraction for three times, 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;
in S3, the n-butanol extraction part is subjected to gradient elution by macroporous resin with the proportion of 0:100-0:95 of EtOH: H2O, a sample is analyzed by HPLC-DAD, and 5 parts a-e are obtained by concentration and combination;
TLC thin layer analysis of a-e part, heating and developing with 10% concentrated sulfuric acid/ethanol, wherein Fr.c thin layer plate shows yellow band;
firstly, removing pigments and tannin interference components by adopting polyamide column chromatography for Fr.c, and carrying out 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 prenyl flavonoids by a UPLC-MS/MS method in the whole separation process, and effectively separating the prenyl flavonoids by ultraviolet characteristic absorption peaks; and finally, purifying the target compound by adopting a semi-preparative high performance liquid phase.
2. The application of an active site in preparing a medicament for resisting rheumatoid arthritis is characterized in that the active site is composed of isopentenyl flavonoid compounds, and the structural formula of the isopentenyl flavonoid compounds is as follows:
3. an application of an isopentene group flavonoid compound in preparing a medicament for resisting rheumatoid arthritis is characterized in that the structural formula of the isopentene group flavonoid compound is as follows:
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