CN109503684B

CN109503684B - Iridoid glycoside compound and preparation method and application thereof

Info

Publication number: CN109503684B
Application number: CN201811562833.1A
Authority: CN
Inventors: 孟鹤; 于洋; 梅玉丹; 黄绮韵; 姚新生; 戴毅
Original assignee: GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
Current assignee: GUANGZHOU XIANGXUE PHARMACEUTICAL CO Ltd
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2020-10-09
Anticipated expiration: 2038-12-20
Also published as: CN109503684A

Abstract

The invention relates to iridoid glycoside compounds and a preparation method and application thereof. The structural formula of the iridoid glycoside compound is shown as formula I, formula II and formula III. The compounds can effectively inhibit IL-6 and can be used for treating inflammation.

Description

Iridoid glycoside compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to an iridoid glycoside compound and a preparation method and application thereof.

Background

The defensive response of living tissue with a vascular system to injury factors is called inflammation. Inflammation is a very common and important basic pathological process, and trauma infection on the body surface and most common diseases and frequently encountered diseases of various organs belong to inflammatory diseases, such as furuncle, carbuncle, pneumonia, hepatitis, nephritis and the like.

IL-6 (interleukin-6) is one of inflammation mediators, is mainly produced by mononuclear macrophages, Th2 cells, vascular endothelial cells and fibroblasts, and participates in inflammatory reaction by stimulating and activating B cell proliferation to secrete antibody, stimulating T cell proliferation and CTL activation, and stimulating liver cells to synthesize acute phase protein.

IL-6 inhibitors (IL-6i) inhibit the immune inflammatory cascade by targeting IL-6 and act as therapeutics in chronic inflammatory diseases.

Tolizumab is an antibody drug and is the only IL-6 inhibitor approved by the FDA for the treatment of arthritis. However, the tobramycin serving as an antibody drug has the defects of high price, inconvenient storage and use and the like. The development of new compounds with better IL-6 inhibition still has important application value. Effective components are searched from natural resources, and a new type of structural compound with development prospect is found to be used as a lead compound, so that the method is an effective way for new drug research.

Disclosure of Invention

Based on the above, it is necessary to provide iridoid glycoside compounds, which can inhibit the production of IL-6 and exhibit potential anti-inflammatory activity, and a preparation method and applications thereof.

An iridoid glycoside compound having a structure represented by formula I, formula II or formula III, or a pharmaceutically acceptable salt, tautomer, stereoisomer, solvate, prodrug molecule, metabolite, or mixture thereof:

the preparation method of the iridoid glycoside compound comprises the following steps:

heating and refluxing flos Lonicerae with ethanol water solution to obtain extractive solution, and removing ethanol to obtain extract;

gradient eluting the extract with ethanol water solution through macroporous resin, collecting the elution part of ethanol water solution with ethanol volume fraction of 48-52%, concentrating under reduced pressure, and freeze-drying to obtain freeze-dried powder;

gradient eluting the freeze-dried powder by using chloroform and methanol aqueous solution through normal phase silica gel column chromatography, and collecting fractions of elution parts with the volume ratio of chloroform, methanol and water being (89-91): 9-11): 0 to obtain primary fractions;

separating the primary fraction by using an ODS (ODS) column chromatography, performing gradient elution by using a methanol aqueous solution, collecting fractions of elution positions with a volume ratio of methanol to water of (48-52): (48-52), recording as a secondary first fraction, performing gradient elution by using a methanol aqueous solution after the secondary first fraction is subjected to gel chromatography, collecting fractions of elution positions with a volume ratio of methanol to water of (48-52): (48-52), and obtaining a tertiary fraction, wherein the tertiary fraction is subjected to preparative high performance liquid chromatography, a mobile phase is an aqueous solution of acetonitrile and formic acid, a volume ratio of acetonitrile to formic acid to water of acetonitrile, formic acid and water of (24-26): 0.1 (74-76), a flow rate of 3.5-4.5 ml/min, and a retention time of 15-16 min to obtain the compound of the formula I, or a retention time of 16.5-17.5 min to obtain the compound of the formula II; or

And (3) separating the primary fraction by using an ODS (ODS) column chromatography, carrying out gradient elution by using a methanol water solution, collecting a fraction of an elution part with a volume ratio of methanol to water of (67-73): (29-31), recording as a secondary second fraction, separating the secondary second fraction by using a preparative high performance liquid chromatography, wherein a mobile phase is an aqueous solution of acetonitrile and formic acid, the volume ratio of acetonitrile to formic acid to water in the aqueous solution of acetonitrile and formic acid is (29-31): 0.1, (68-72), the flow rate is 3.5-4.5 ml/min, and the retention time is 16-17 min, so as to obtain the compound shown in the formula III.

In one embodiment, when the third fraction is separated by preparative high performance liquid chromatography, the volume ratio of acetonitrile, formic acid and water in the mobile phase is 25:0.1:75, the flow rate is 4ml/min, and the retention time is 15.3min, so that the compound of the formula I is obtained.

In one embodiment, when the third fraction is subjected to high performance liquid chromatography, the volume ratio of acetonitrile, formic acid and water in the mobile phase is 25:0.1:75, the flow rate is 4ml/min, and the retention time is 17.0min, so that the compound of the formula II is obtained.

In one embodiment, when the second fraction is subjected to preparative high performance liquid chromatography, the volume ratio of acetonitrile, formic acid and water in the mobile phase is 30:0.1:70, the flow rate is 4ml/min, and the retention time is 16.3min, so as to obtain the compound shown in the formula III.

In one embodiment, when the freeze-dried powder is subjected to gradient elution by normal phase silica gel column chromatography and aqueous solution of chloroform and methanol, the gradient of the volume ratio of chloroform to methanol to water in the aqueous solution of chloroform and methanol is 95:5:0, 90:10:1, 80:20:2, 70:30:5 and 60:40: 8.

In one embodiment, the extract is subjected to gradient elution by using ethanol water solution through macroporous resin, and the elution part of the ethanol water solution with the volume fraction of 50% is collected.

The invention also provides the application of the iridoid glycoside compound or the pharmaceutically acceptable salt, tautomer, stereoisomer, solvate, prodrug molecule, metabolite or mixture thereof in preparing anti-inflammatory drugs.

The invention also provides the application of the iridoid glycoside compound or the pharmaceutically acceptable salt, tautomer, stereoisomer, solvate, prodrug molecule, metabolite or mixture thereof in preparing IL-6 inhibitors.

A medicament for treating inflammation or an IL-6 inhibitor comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises the iridoid glycoside compound or pharmaceutically acceptable salt, tautomer, stereoisomer, solvate, prodrug molecule, metabolite or mixture thereof.

The invention firstly extracts and prepares 3 new iridoid glycoside compounds from lonicera confusa, and confirms the chemical structure and physicochemical properties of the 3 iridoid glycoside compounds by modern spectral means such as HR-ESI-MS, NMR, IR, UV and the like. And the anti-inflammatory activity of the 3 new iridoid glycoside compounds is verified through cell experiments, the compounds show excellent anti-inflammatory action and can be used as lead compounds for developing novel anti-inflammatory drugs.

The preparation method of the iridoid glycoside compound has the advantages that the technological parameters are easy to control, simple and rapid, and the preparation of the iridoid glycoside compound is easier.

Drawings

FIG. 1 is a scheme for the separation of compounds I, II and III;

FIG. 2 is a HR-ESI-MS spectrum of compound I;

FIG. 3 is a UV spectrum of compound I;

FIG. 4 is an IR spectrum of Compound I;

FIG. 5 shows the preparation of compound I¹H-NMR spectrum;

FIG. 6 shows the preparation of compound I¹³A C-NMR spectrum;

FIG. 7 is a DEPT-135 spectrum of Compound I;

FIG. 8 is a drawing of Compound I¹H-¹H COSY spectrogram;

FIG. 9 is an HSQC spectrum of Compound I;

FIG. 10 is an HMBC spectrum of compound I;

FIG. 11 is a NOESY spectrum of Compound I;

FIG. 12 is an X-ray spectrum of Compound I (CDCC No. 1877498; Cambridge Crystal morphology data Centre);

FIG. 13 is a CD spectrum of compounds I and II;

FIG. 14 is a HR-ESI-MS spectrum of Compound II;

FIG. 15 is a UV spectrum of Compound II;

FIG. 16 is an IR spectrum of Compound II;

FIG. 17 is a drawing of Compound II¹H-NMR spectrum;

FIG. 18 is a drawing of Compound II¹³A C-NMR spectrum;

FIG. 19 is a DEPT-135 spectrum of Compound II;

FIG. 20 is a drawing of Compound II¹H-¹H COSY spectrogram;

FIG. 21 is an HSQC spectrum of compound II;

FIG. 22 is an HMBC spectrum of compound II;

FIG. 23 is a NOESY spectrum of compound II;

FIG. 24 is a HR-ESI-MS spectrum of Compound III;

FIG. 25 is a UV spectrum of Compound III;

FIG. 26 is an IR spectrum of Compound III;

FIG. 27 is a drawing of Compound III¹H-NMR spectrum;

FIG. 28 is a drawing of Compound III¹³A C-NMR spectrum;

FIG. 29 is a DEPT-135 spectrum of Compound III;

FIG. 30 is a drawing of Compound III¹H-¹H COSY spectrogram;

FIG. 31 is an HSQC spectrum of compound III;

FIG. 32 is an HMBC spectrum of compound III;

FIG. 33 is a NOESY spectrum of Compound III;

FIG. 34 is a graph of IL-6 inhibitory activity of compounds I, II and III;

FIG. 35 is IC of Compound III₅₀And (6) calculating the graph.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The invention relates to iridoid glycoside compounds with a structure shown in formula I, formula II or formula III, or pharmaceutically acceptable salts, tautomers, stereoisomers, solvates, prodrug molecules, metabolites or mixtures thereof:

the mixture is any two mixtures or the mixture of more than two mixtures of pharmaceutically acceptable salts, tautomers, stereoisomers, solvates, prodrug molecules and metabolites of the compounds shown as the formulas I, II and III.

The invention also relates to a preparation method of the iridoid glycoside compound, which comprises the following steps:

heating and refluxing flos lonicerae medicinal material with 70% ethanol water solution to obtain extracting solution, concentrating the extracting solution under reduced pressure to remove ethanol to obtain extract, gradient-eluting the extract with ethanol water solution (0:100, 30:70, 50:50, 95:5, v: v) through macroporous resin, collecting eluting parts of ethanol water solution with ethanol volume fraction of 48-52%, preferably collecting eluting parts of ethanol water solution with ethanol volume fraction of 48-52%, concentrating under reduced pressure and freeze drying to obtain freeze-dried powder.

Subjecting the freeze-dried powder to normal-phase silica gel column chromatography, performing gradient elution with chloroform, methanol and water (gradient of volume ratio of chloroform to methanol to water is 95:5:0, 90:10:1, 80:20:2, 70:30:5, 60:40:8), collecting fractions of elution parts with volume ratio of chloroform to methanol to water being (89-91): 9-11): 0, preferably collecting fractions of elution parts with volume ratio of chloroform to methanol to water being 90:10:0, and obtaining a primary fraction.

Separating the first fraction by ODS column chromatography, gradient eluting with methanol water solution (30:70, 50:50, 70:30, 100:0, v: v), collecting fraction of eluting part with methanol and water volume ratio of (48-52) and fraction of eluting part with methanol and water volume ratio of (67-73) (29-31), preferably collecting fraction of eluting part with methanol and water volume ratio of 50:50 and fraction of eluting part with 70:30, and respectively obtaining second first fraction and second fraction.

And (3) carrying out gradient elution on the second first fraction by using a methanol water solution, and collecting fractions of elution parts with the volume ratio of methanol to water being (48-52) to (48-52). Preferably, fractions of elution positions with the volume ratio of methanol to water being 50:50 are collected to obtain a third fraction, the third fraction is separated by preparative high performance liquid chromatography, the mobile phase is an aqueous solution of acetonitrile and formic acid, the volume ratio of acetonitrile to formic acid to water in the aqueous solution of acetonitrile and formic acid is (24-26): 0.1: (74-76), and the flow rate is 3.5-4.5 ml/min. Preferably, the volume ratio of acetonitrile to formic acid to water in the aqueous solution of acetonitrile and formic acid is 25:0.1:75, and the flow rate is 4 ml/min. The retention time is 15-16 min to obtain the compound shown in the formula I, and the retention time is 16.5-17.5 min to obtain the compound shown in the formula II. Preferably, the retention time is 15.3min to obtain the compound of formula I, and the retention time is 17min to obtain the compound of formula II.

And (3) separating the second fraction by preparative high performance liquid chromatography, wherein the mobile phase is acetonitrile and formic acid water solution, the volume ratio of acetonitrile to formic acid to water in the acetonitrile and formic acid water solution is (29-31): 0.1, (68-72), the flow rate is 3.5-4.5 ml/min, and the retention time is 16-17 min to obtain the compound shown in the formula III. Preferably, the compound of the formula III is obtained by using acetonitrile and formic acid in an aqueous solution of acetonitrile and formic acid, wherein the volume ratio of acetonitrile to formic acid to water is 30:0.1:70, the flow rate is 4ml/min, and the retention time is 16.3 min.

The invention also relates to application of the iridoid glycoside compound or pharmaceutically acceptable salt, tautomer, stereoisomer, solvate, prodrug molecule, metabolite or mixture thereof in preparing anti-inflammatory drugs.

The invention also relates to the application of the iridoid glycoside compound or pharmaceutically acceptable salt, tautomer, stereoisomer, solvate, prodrug molecule, metabolite or mixture thereof in preparing IL-6 inhibitors.

A pharmaceutical composition for treating inflammation comprises active ingredient and pharmaceutically acceptable carrier, wherein the active ingredient comprises the iridoid glycoside compound or pharmaceutically acceptable salt, tautomer, stereoisomer, solvate, prodrug molecule, metabolite or mixture thereof.

The invention prepares 3 new iridoid glycoside compounds from lonicera confusa for the first time, and confirms the chemical structure and the physicochemical property of the 3 iridoid glycoside compounds by modern spectroscopic means such as NMR, IR and the like. And the anti-inflammatory activity of the 3 new iridoid glycoside compounds is verified through cell experiments, the compounds show excellent anti-inflammatory action and can be used as lead compounds for developing novel anti-inflammatory drugs.

The present invention will be described in further detail with reference to specific examples.

1. The instrument comprises the following steps:

NMR experiment with Bruker Avance 2600 full-digital superconducting NMR spectrometer, ultraviolet spectrum, Japan spectral JASCOV-550 ultraviolet-Visible spectrometer, infrared spectrum, Japan spectral JASCO FT/IR-480plus infrared spectrometer, KBr tablet, ESI-MS FINIGAN LCQ Advantage MAX mass spectrometer, HR-ESI-MS and UPLC-Q-TOF-MS, Waters Synapt G2Q-TOF-MS, analytical high performance liquid chromatography (Waters), Waters 2695+ DAD 2998, preparative high performance liquid chromatography (Waters), Waters 1515+2489UV/Visible, chromatographic column, C18column, Phenomex Gemini,5 μm,4.6 × 250mm, C18column (Phenomex G250 mm)emini,5 μm,10 × 250mm), C18column (Waters, Aquity, BEH,1.7 μm,3.0 × 150mm), multifunctional microplate reader-Synergy HT (Bio-Tek Instruments Inc., Burleigh, USA), Thermo Scientific BB15 model CO₂Cell incubator, us thermoelectricity; nikon TS100 type inverted microscope; clean bench, AIRTECH, model: a10051560; ZW-A type micro oscillator, Changzhou Guohua instruments, Inc.; the Therom VarioSkan Flash multifunctional reading instrument; a liquid transfer device: therom company; a centrifuge: hunan instrument, model: l530.

2. Materials and reagents:

the wild honeysuckle flower medicinal materials: shandong, the origin, the supplier, Kun Yuan medicine, Inc., Bozhou; diaion HP 20: mitsubishi Chemical company; chromatography silica gel (100-: qingdao ocean chemical company; silica gel thin layer precast slab: the institute of chemical industry of cigarette tai city; sephadex LH-20: amersham Biosciences; RP-18 column chromatography packing (12nm, S-50 um): YMC; methanol (chromatographic grade): BCR; acetonitrile (chromatographic grade): merck corporation; a deuterated reagent: CIL; the other chemical reagents for chromatographic column chromatography are analytically pure and purchased from Guangzhou chemical reagent factory; DMEM/HIGH GLUCOSE, Gibco, Cat: C11995500BT, Lot: 8118035; FBS: lonsera, Cat: S711-001S, Lot: NR 06611; LPS: nanjing Dazhi Biotechnology Ltd; DMSO, alatin, cat #: d103277, lot number: d1712055; 24-well plate, JET corporation, Cat. number TCP-010-; 0.25% Trypsin-EDTA, Gibco, Lot: 1697785, respectively; mouse IL-6ELISA kit, Invitrogen, Cat: BMS603-2, Lot: 167147019.

3. The experimental process comprises the following steps:

heating and refluxing 35kg of flos lonicerae by 70% ethanol water, and concentrating the extract under reduced pressure to remove ethanol to obtain extract (LM). Gradient eluting the extract with ethanol water through macroporous resin, collecting 50% ethanol water solution eluting part (LM-3), concentrating under reduced pressure, and freeze drying to obtain lyophilized powder.

Subjecting the lyophilized powder to normal phase silica gel column chromatography, and performing gradient elution with chloroform, methanol and water (95:5:0, 90:10:0, 90:10:1, 80:20:2, 70:30:5, 60:40:8) to obtain fractions containing compounds I, II and III (first fraction, 3E, 90:10:0 elution fraction). Subjecting fraction 3E to ODS column chromatographyThe spectral separation was eluted with a methanol-water gradient (30:70, 50:50, 70:30, 100:0) to give a fraction containing compound I and compound II (second first fraction, E6, 50:50 elution site) and a fraction containing compound III (second fraction, E10, 50:50 elution site). Subjecting fraction E6 to gel column chromatography, isocratically eluting with methanol water (50:50) to obtain fraction containing compounds I and II (third fraction, E6D), subjecting fraction E6D to preparative high performance liquid Chromatography (CH)₃CN:H₂HCOOH 25:75:0.1, v: v: v, flow rate: 4ml/min) to give Compound I (7.7mg, retention time t)_R15.30min) and compound ii (4.7mg, retention time t)_R17.0 min). Fraction E10 was subjected to preparative high performance liquid Chromatography (CH)₃CN:H₂HCOOH ═ 30:70:0.1, v: v: v, flow rate: 4ml/min) to yield Compound III (4.5mg, retention time t)_R16.30 min). The scheme for the isolation of compounds I, II and III is shown in FIG. 1.

4. Structure identification of iridoid glycoside compounds

A compound I: white amorphous powder.

18.7(c 0.6,CH₃OH),UV(CH₃OH)λ_max(log):204(3.9),232(3.9)，IR(KBr)ν_max:3366,2880,2357,1700,1414,1070cm^-1. HR-ESI-MS gave M/z477.1747[ M + Na [ ]]⁺(calculated value 477.1737), the molecular formula of the compound was determined to be C₂₂H₃₀O₁₀The unsaturation was calculated to be 8.¹H-NMR(600MHz,in CD₃OD) spectrum, the low field region showing the proton signal of the olefin at the 3-position of the typical iridoid glycoside compound_H7.48(1H,d,J＝1.2Hz,H-3)]And a proton signal of the hemiacetal in the 1-position [ alpha ]_H5.21(1H,d,J＝7.1Hz,H-1)]. In addition, the low field region also has an aldehyde group hydrogen signal_H9.38(1H,s,H-1′)]An alkene hydrogen proton signal_H6.93(1H,d,J＝2.8Hz,H-3′)]And a sugar end group protonSignal [ 2 ]_H4.72(1H,d,J＝7.9Hz,H-1″)]. Process for preparing compounds I¹³C-NMR(150MHz,in CD₃OD) profile showed 22 carbon signals in total, including: 1 ester carbonyl group (_C170.9), 2 olefin quaternary carbon signals (C:)_C145.1,112.2), 1 aldehyde group: (_C196.4), 2 sp²Hybridized methine(s) ((_C158.1,153.5), 11 sp3 hybridized methines (_C100.3,98.6,78.4,78.0,74.8,71.6,46.1,38.4,36.1,35.7,35.2), 4 methylene groups ((iii)_C62.8,38.9,32.1,28.7) and 1 methyl group(s) ((iii)_C11.8)。

Acid hydrolysis and glycosyl derivatization experiments of the compound I indicate that the compound I contains D-glucose in the molecule, the coupling constant (J is 7.9Hz) of terminal protons of the bound sugar is determined, and the structure of the compound I contains β -D-glucose.¹H-¹The H COSY spectrum shows a group of spin coupling systems H-1/H-9(H-8)/H-5/H₂-6/H-7/H-8/H₂-10/H-4′(H-3′)/H₂-5′/H₃6 ', in combination with the HMBC-related signals (H-3/C-1,4,5,11, H-1/C-3,5,8,9 and H-1 '/C-7, 2 ', 3 '), the aglycone structure of Compound I was deduced, the planar structure of Compound I was determined by determining β -D-glucosyl linkage at the C-1 position further based on the remote-related peaks (H-1 '/C-1, H-1/C-1 ') of HMBC, the hydrocarbon signals were assigned to Table 1, thereafter, the absolute configuration of Compound I (1S/5S/7S/8S/9R/4 ' S) was determined based on the single crystal data (CDCC No. 1877498), thereby identifying Compound I as lonimacranalde A. HR-ESI-MS, UV, IR, E-E,¹H-NMR、¹³C-NMR、DEPT-135、¹H-¹the maps of HCOSY, HSQC, HMBC, NOESY and X-ray are shown in the attached figures 2-12.

Compound ii: white amorphous powder.

-9.7(c 0.6,CH₃OH),UV(CH₃OH)λ_max(log):206(4.1),230(3.9)，IR(KBr)ν_max:3389,2891,2345,1678,1621,1274,1070cm^-1. HR-ESI-MS gave M/z477.1738[ M + Na [ ]]⁺(calculated value 477.1737), the molecular formula of the compound was determined to be C₂₂H₃₀O₁₀The unsaturation was calculated to be 8. The compounds II and I have the same molecular weight and molecular formula, and¹H-NMR and¹³the compound II has no single crystal data, and the configuration of the compound II is determined according to biogenic pathway, NOESY diagram, analysis of coupling constant and CD diagram, wherein the 1-position glycosidic bond configuration of iridoid is β type, H-1 is α type, and H-5 and H-9 are combined in cis form and are β type.

The NOESY correlation (H-6/H-1) of compound II confirms the presumption that H-5, H-9 are β -cis-combinations and the other NOESY correlation (H-1/H-7 and H-1/H-8) suggests that H-7, H-8 and H-1 are on the same side and H-7/H-8 is α -cis-combinations in compound II, thus determining the relative configuration of C-1,5,7,8,9 (1 α/5 β/7 α/8 α/9 β) of compound II as in compound I, and further the coupling constants (J) of H-10a, H-4' and H-8_10a,4′＝11.0Hz,J_10a,811.0Hz) suggests that H-10a/H-4 ', H-10a/H-8 are all aa coupled, i.e. H-4 ' and H-8 are in the same orientation, in the α configuration, and that compounds i and ii are epimers of C-4 ' as a result of the same cotton effect found by comparison in the CD patterns of compounds i and ii (figure 13)¹H-NMR、¹³C-NMR、DEPT-135、¹H-¹The H COSY, HSQC, HMBC and NOESY maps are shown in the attached figures 14-23.

Compound iii: light yellow gum.

-150.4(c 0.3,CH₃OH),UV(CH₃OH)λ_max(log):204(3.9),234(4.2)，IR(KBr)ν_max:3400,2926,2357,1675,1627,1272,1070cm^-1. HR-ESI-MS gave M/z479.1895[ M + Na [ ]]⁺(calculated value 479.1893), the molecular formula of the compound was determined to be C₂₂H₃₂O₁₀The unsaturation was calculated to be 7. Of compounds III¹H-NMR(600MHz,in CD₃OD) spectrum, the low field region being visible as the proton signal at position 3 of the typical cycloalkene ether terpenoid compound_H7.52(1H,d,J＝1.1Hz，H-3)]And a proton signal of the hemiacetal in the 1-position [ alpha ]_H5.55(1H,d,J＝5.6Hz,H-1)]And a single substituted terminal double bond proton signal [ alpha ]_H5.77(1H,ddd,J＝17.1,10.4,8.9Hz,H-8),5.27(2H,m,H-10)]. In addition, there is an aldehyde group hydrogen [ alpha ]_H9.33(1H,s,H-1′)]An olefinic hydrogen proton [ alpha ], [ alpha ]_H6.63(1H,t,J＝7.0Hz,H-7)]And a sugar end group proton [ alpha ], [ alpha_H4.69(1H,d,J＝7.9Hz,H-1″)]The presence of β -D-glucosyl group in the structure of compound III was confirmed by the coupling constant of the terminal proton of the binding saccharide, and by the acid hydrolysis and glycosyl derivatization experiments of compound III.¹H-¹The H COSY spectrum shows that the aglycone structure of the compound III is determined in the presence of three groups of spin coupling systems (H-1/H-9/H-8/H-10, H-9/H-5/H-6/H-7, H-3 '/H-4 '/H-5 '/H-6 ') and related peaks (H-3/C-1,4,5,11, H-1/C-3,5,8, H-1 '/C-2 ', 3 ', 7) combining HSQC and HMBC.

Thereafter, HMBC-related peaks (H-1 '/C-1 and H-1/C-1 ') suggested β -D-glucosyl group to be linked to C-1, thereby defining the planar structure of compound III, the hydrocarbon signal was assigned to Table 1. the configuration of compound III was determined similarly to that of I and II, and in NOESY spectrum, distinct related peaks H-6/H-8 and H-1/H-6 were found, suggesting that H-5/H-9 is cis-fused, β configuration, corresponding to biogenic pathway, and related peaks H-1 '/H-7, suggesting that the double bond at C-7 is E formula, thereby identifying compound III as lonimacraralde C¹H-NMR、¹³C-NMR、DEPT-135、¹H-¹The H COSY, HSQC, HMBC and NOESY maps are shown in the attached figures 24-33 in detail.

TABLE 1 Compounds I,II, III Hydrocarbon Nuclear magnetic data Attribution (in CD)₃OD)

Attribution of authentication data:

secoisolaricoside hexanal acetal A (lonimacranalde A, I)

White amorphous powder;

-18.7(c 0.6,CH₃OH)；ESI-MS:m/z 477[M+Na]⁺；HR-ESI-MS:m/z477.1747[M+Na]⁺(calcd for C₂₂H₃₀O₁₀Na,477.1737)；UV(CH₃OH)λ_max(log):204(3.9),232(3.9)，IR(KBr)ν_max:3366,2880,2357,1700,1414,1070cm^-1；¹H NMR(CD₃OD,600MHz) and¹³CNMR(CD₃OD,150MHz) is shown in table 1.

Secoisolaricoside hexanal acetal B (lonimacranalde B, II)

White amorphous powder;

-9.7(c 0.6,CH₃OH)；ESI-MS:m/z 477[M+Na]⁺；HR-ESI-MS:m/z477.1738[M+Na]⁺(calcd for C₂₂H₃₀O₁₀Na,477.1737)；UV(CH₃OH)λ_max(log):206(4.1),230(3.9)；IR(KBr)ν_max:3389,2891,2345,1678,1621,1274,1070cm^-1；¹H NMR(CD₃OD,600MHz) and¹³C NMR(CD₃OD,150MHz) is shown in table 1.

Secoisolaricoside hexanal acetal C (lonimacranalde C, III)

A light yellow gum;

-150.4(c 0.3,CH₃OH)；ESI-MS:m/z 455[M-H]^-；HR-ESI-MS:m/z479.1895[M+Na]⁺(calcd for C₂₂H₃₂O₁₀Na,479.1893)；UV(CH₃OH)λ_max(log):204(3.9),234(4.2)；IR(KBr)ν_max:3400,2926,2357,1675,1627,1272,1070cm^-1；¹H NMR(CD₃OD,600MHz) and¹³C NMR(CD₃OD,150MHz) is shown in table 1.

Inflammatory factor IL-6 inhibitory Activity assay methods:

(1) the experimental process comprises the following steps:

compounds I, II, III were prepared in DMSO to give stock solutions of desired concentration (100, 50, 25, 12.5, 6.25mM), 1.2. mu.L was added to 1199. mu.L of serum-free DMEM, mixed well and added to 495. mu.L per well in 24-well plates.

The cells were digested with 0.25% trypsin (containing 0.02% EDTA), and the cell density was adjusted to 1X 105 cells/ml in 10% FBS-containing DMEM medium, and the cells were uniformly seeded into 24-well plates at 400. mu.l per well. After plating, the plates were placed in an incubator for 24 hours. After 24 hours of culture, the 24-well plate is taken out, supernatant is aspirated, and a drug-containing medium prepared by serum-free DMEM medium is added: solvent control: 495 mul serum-free DMEM medium containing one thousandth of DMSO was added to each well; model group: 495 mul serum-free DMEM medium containing one thousandth of DMSO was added to each well; ③ administration sample group: 495. mu.l of medium containing samples at the corresponding concentrations were added to each well.

After the medicine is added, putting CO into the 24-hole plate₂The cell culture box was incubated for 1 hour. After 1 hour, 5. mu.l of LPS (final concentration: 1. mu.g/ml) of 100. mu.g/ml was added to each well except for the solvent control group, 5. mu.l of serum-free DMEM medium was added to each well of the solvent control group, and after the addition of the solvent, the 24-well plate was placed in CO₂The cell incubator was continued for 18 hours. Cell culture media were collected after 18 hours and diluted 5-fold with serum-free DMEM and assayed for IL-6 content according to the Elisa kit instructions.

(2) The experimental results are as follows:

the results of the experiments show that, as can be seen from FIG. 34, compounds I, II and III all inhibit at a concentration of 100. mu.MInhibiting IL-6 production, wherein Compound III inhibits the most strongly, IC₅₀6.33 μ M (fig. 35). The compounds I, II and III have potential anti-inflammatory activity and can be used as lead compounds of anti-inflammatory drugs.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Iridoid glycoside compounds having a structure represented by formula I, formula II or formula III:

2. a method for preparing iridoid glycoside compounds according to claim 1, comprising the steps of:

3. The method of claim 2, wherein the volume ratio of acetonitrile, formic acid and water in the mobile phase is 25:0.1:75, the flow rate is 4ml/min, and the retention time is 15.3min when the third fraction is subjected to preparative high performance liquid chromatography to obtain the compound of formula I.

4. The method of claim 2, wherein the volume ratio of acetonitrile, formic acid and water in the mobile phase is 25:0.1:75, the flow rate is 4ml/min, and the retention time is 17.0min when the third fraction is separated by HPLC to obtain the compound of formula II.

5. The method of claim 2, wherein the second fraction is separated by preparative high performance liquid chromatography, wherein the volume ratio of acetonitrile, formic acid and water in the mobile phase is 30:0.1:70, the flow rate is 4ml/min, and the retention time is 16.3min, thereby obtaining the compound of formula III.

6. The method for producing iridoid glycoside compounds according to any one of claims 2 to 5, wherein when the lyophilized powder is subjected to normal phase silica gel column chromatography and gradient elution with an aqueous solution of chloroform and methanol, the gradient of the volume ratio of chloroform to methanol to water in the aqueous solution of chloroform and methanol is 95:5:0, 90:10:1, 80:20:2, 70:30:5, or 60:40: 8.

7. The method according to any one of claims 2 to 5, wherein the extract is subjected to gradient elution with an aqueous ethanol solution through a macroporous resin, and the elution portion of the aqueous ethanol solution containing 50% by volume of ethanol is collected.

8. The use of iridoid glycosides compounds or pharmaceutically acceptable salts or mixtures thereof according to claim 1 in the preparation of anti-inflammatory agents.

9. The use of iridoid glycosides according to claim 1 or pharmaceutically acceptable salts or mixtures thereof in the preparation of IL-6 inhibitors.

10. A medicament or IL-6 inhibitor for the treatment of inflammation, comprising an active ingredient comprising the iridoid glycoside compound of claim 1 or a pharmaceutically acceptable salt or mixture thereof, and a pharmaceutically acceptable carrier.