CN113354539B - Morinda officinalis iridoid compound with anti-inflammatory activity and preparation method and application thereof - Google Patents
Morinda officinalis iridoid compound with anti-inflammatory activity and preparation method and application thereof Download PDFInfo
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- CN113354539B CN113354539B CN202110535687.9A CN202110535687A CN113354539B CN 113354539 B CN113354539 B CN 113354539B CN 202110535687 A CN202110535687 A CN 202110535687A CN 113354539 B CN113354539 B CN 113354539B
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Abstract
The invention discloses a morinda officinalis iridoid compound with anti-inflammatory activity and a preparation method and application thereof. Specifically provides iridoid compounds with anti-inflammatory activity, and the structural formulas of the iridoid compounds are respectively shown as a formula I and a formula II. The invention extracts and prepares two new iridoid compounds from the overground part of morinda officinalis for the first time, confirms the chemical structures and physicochemical properties of the two new iridoid compounds through modern spectral means such as HR-ESIMS, NMR, IR, UV and the like, verifies the pharmacological activity of the two new iridoid compounds through cell experiments, shows excellent anti-inflammatory action, has obvious inhibition effect on NO generation, can effectively reduce the expression of proinflammatory factors iNOS, COX-2, TNF-alpha, IL-1 beta and IL-6, can reduce inflammatory reaction caused by excessive expression of the inflammatory factors, and can be used as a lead compound for developing novel anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a morinda officinalis iridoid compound with anti-inflammatory activity, and a preparation method and application thereof.
Background
Morinda officinalis is the dried root of Morinda officinalis (Morinda officinalis How) belonging to Morinda genus of Rubiaceae family, has effects of invigorating kidney yang, strengthening tendons and bones, dispelling pathogenic wind and removing dampness, and is commonly used for treating leg weakness, infertility due to cold womb, sexual impotence, and spermatorrhea. The effective components of Morinda officinalis are rich in species, mainly including oligosaccharides, polysaccharides, iridoids, anthraquinones, etc. Modern pharmacological research shows that morinda officinalis has extensive biological activities of resisting depression, resisting oxidation, resisting osteoporosis, resisting inflammation, resisting aging, improving body immunity and the like. Meanwhile, morinda officinalis is also an important health food, and has high medicinal value, the reputation of northern Korean ginseng and southern morinda officinalis is true, and the four-great-south traditional Chinese medicines are called as fructus alpiniae oxyphyllae, fructus amomi and betel nut.
In recent years, due to the decrease of the yield of morinda officinalis, and the good effect of morinda officinalis in health care, the demand of morinda officinalis is gradually increased, and the supply of morinda officinalis in the market is short. When the morinda officinalis is harvested, the underground roots are usually dug, and a large amount of overground parts are completely discarded without reasonable utilization, so that resource waste and environmental pollution are caused to a certain extent. The reports of the aboveground parts of morinda officinalis are few at home and abroad, and intensive research is not carried out, but in recent years, research shows that a novel compound with better biological activity is separated from the aboveground parts of other plants in morinda, so that research on the chemical components and the biological activity of the aboveground parts of morinda officinalis is promoted.
The inflammatory response is typically characterized by an excessive accumulation of Nitric Oxide (NO), the production of which is regulated by nitric oxide synthase. The main chemical components in morinda officinalis are iridoid and its glycosides, the content of iridoid glycoside compounds in root can be up to about 2.0%, and good activities of resisting inflammation, relieving pain, resisting osteoporosis, etc. are represented by monotropein. Iridoid glycosides such as monoterpene glycoside and deacetyl asperulosidic acid isolated from dried root of Morinda citrifolia, such as Zhang jia Hua, have anti-inflammatory and anti-rheumatoid arthritis effects (research on the anti-rheumatoid arthritis effect of Morinda citrifolia iridoid glycosides [ D. However, the previous studies of the subject group indicate that the content of the monotropein in the leaves of morinda officinalis is higher than that in the roots of morinda officinalis, so based on the accumulation of literature research and research of the subject group, in order to make full use of plant resources, the studies have studied the chemical components and activity screening of the above-ground parts of morinda officinalis, and a foundation is expected to be laid for the development and utilization of the above-ground parts of morinda officinalis.
Disclosure of Invention
The invention aims to overcome the defects of research and development of overground part resources of morinda officinalis in the prior art, provides an iridoid compound which is obtained from the overground part of morinda officinalis and has a good anti-inflammatory effect, a preparation method of the iridoid compound, and research on anti-inflammatory activity and medical application of the iridoid compound.
The invention aims to provide iridoid compounds with the structure shown in formula I or pharmaceutically acceptable salts, tautomers and stereoisomers thereof.
The second purpose of the invention is to provide an iridoid glycoside compound with a structure shown in a formula II or pharmaceutically acceptable salt, tautomer and stereoisomer thereof.
The third purpose of the invention is to provide the application of the iridoid compound with the structure shown as the formula I or the pharmaceutically acceptable salt, tautomer and stereoisomer thereof in preparing anti-inflammatory drugs.
The fourth purpose of the invention is to provide the application of the iridoid compound shown in the structural formula I or the pharmaceutically acceptable salt, tautomer and stereoisomer thereof in preparing iNOS, and/or IL-6, and/or IL-1 beta, and/or TNF-alpha and/or COX-2 inhibitors.
The fifth purpose of the invention is to provide the application of the iridoid glycoside compound with the structure shown in the formula II or the pharmaceutically acceptable salt, tautomer and stereoisomer thereof in preparing anti-inflammatory drugs.
The sixth purpose of the invention is to provide the application of iridoid glycoside compounds with the structure shown in the formula II or pharmaceutically acceptable salts, tautomers and stereoisomers thereof in preparing iNOS, and/or IL-6, and/or IL-1 beta, and/or TNF-alpha, and/or COX-2 inhibitors.
It is a seventh object of the present invention to provide a medicament having anti-inflammatory activity.
The eighth purpose of the invention is to provide a preparation method of the iridoid compound with the structure shown in the formula I and the iridoid glycoside compound shown in the formula II.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides an iridoid compound with a structure shown as a formula I or pharmaceutically acceptable salts, tautomers and stereoisomers thereof:
iridoid glycoside compounds with the structure shown in formula II or pharmaceutically acceptable salts, tautomers and stereoisomers thereof:
the compound I with the structural formula shown as the formula I is named as offibinaloside E; the compound II with the structural formula shown as the formula II is named as offibinaloside F.
The following applications are also within the scope of the present invention:
application of iridoid compounds with a structure shown as a formula I or pharmaceutically acceptable salts, tautomers and stereoisomers thereof in preparing anti-inflammatory drugs.
Application of iridoid compounds with a structure shown as a formula I or pharmaceutically acceptable salts, tautomers and stereoisomers thereof in preparation of inhibitors of iNOS, and/or IL-6, and/or IL-1 beta, and/or TNF-alpha, and/or COX-2.
Application of iridoid glycoside compounds with structure shown in formula II or pharmaceutically acceptable salts, tautomers and stereoisomers thereof in preparing anti-inflammatory drugs.
Application of iridoid glycoside compounds with a structure shown in formula II or pharmaceutically acceptable salts, tautomers and stereoisomers thereof in preparation of iNOS, and/or IL-6, and/or IL-1 beta, and/or TNF-alpha, and/or COX-2 inhibitors.
The invention also claims a medicament with anti-inflammatory activity, which comprises one or more of an iridoid compound with a structure shown in the formula I, or a pharmaceutically acceptable salt of the iridoid compound with the structure shown in the formula I, or a tautomer of the iridoid compound with the structure shown in the formula I, or a stereoisomer of the iridoid compound with the structure shown in the formula I, or an iridoid glycoside compound with the structure shown in the formula I, or a pharmaceutically acceptable salt of the iridoid glycoside compound with the structure shown in the formula II, or a tautomer of the iridoid glycoside compound with the structure shown in the formula II, or a stereoisomer of the iridoid glycoside compound with the structure shown in the formula II.
The preparation method of the iridoid compound with the structure shown in the formula I and the iridoid glycoside compound shown in the formula II comprises the following steps:
s1, taking the overground part of morinda officinalis, performing reflux extraction for 3-5 times by using an ethanol water solution with the volume fraction of 70-95%, combining to obtain an extracting solution, and removing ethanol in the extracting solution to obtain a total extract;
s2, sequentially extracting the total extract with petroleum ether, ethyl acetate and n-butyl alcohol, retaining an n-butyl alcohol extraction phase, and removing a solvent to obtain an n-butyl alcohol extract;
s3, performing gradient elution on the n-butanol extract by using dichloromethane-methanol through silica gel column chromatography, collecting gradient eluents of each gradient, detecting through thin-layer chromatography, combining similar elution fractions, concentrating, and collecting to obtain 6 fractions A-F;
s4, separating and purifying the second fraction B obtained in the step S3 through silica gel column chromatography and semi-preparative HPLC to obtain a compound I with a structure shown in a formula I; and (4) separating the fourth fraction D obtained in the step (S3) by silica gel column chromatography and purifying by sephadex chromatography to obtain a compound II with a structure shown in a formula II.
Preferably, the aerial part of Morinda citrifolia is the stem and/or leaf of Morinda citrifolia.
Preferably, the concentration of the ethanol aqueous solution in the step S1 is 85-95%.
More preferably, the concentration of the ethanol aqueous solution in the step S1 is 95%.
Preferably, the specific process of the extraction in step S2 is: suspending the extract with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, retaining n-butanol extract phase, and concentrating under reduced pressure to obtain n-butanol extract.
Preferably, the specific separation and purification process of step S4 is as follows: performing gradient elution on the second fraction B obtained in the step S3 by using petroleum ether-ethyl acetate through silica gel column chromatography, collecting eluent, detecting through thin-layer chromatography, combining similar elution fractions, concentrating, and collecting 4 subflow fractions B1-B4; performing gradient elution on the second sub-fraction B2 by using petroleum ether-ethyl acetate through silica gel column chromatography, collecting eluent, detecting by using thin layer chromatography, combining similar elution fractions and concentrating to obtain 5 sub-fractions B2-1-B2-5, and separating the first sub-fraction B2-1 by using semi-preparative HPLC to obtain a compound I with the structure shown in the formula I;
performing gradient elution on the fourth fraction D obtained in the step S3 by using ethyl acetate-methanol through silica gel column chromatography, collecting eluent, detecting through thin layer chromatography, combining similar elution fractions, concentrating, and collecting 3 subfluids D1-D3; performing gradient elution on the second sub-fraction D2 by using methanol-water through ODS column chromatography, collecting eluent, detecting through thin-layer chromatography, combining similar elution fractions, concentrating, and collecting to obtain 6 sub-fractions D2-1-D2-6; and performing gradient elution on the sixth sub-fraction D2-6 by using ethyl acetate-methanol through silica gel column chromatography, collecting eluent, detecting through thin-layer chromatography, combining similar elution fractions, concentrating, using methanol as a mobile phase, and purifying through sephadex chromatography to obtain a compound II with a structure shown in a formula II.
Preferably, the second fraction B is eluted with a gradient of petroleum ether-ethyl acetate, the volume ratio of petroleum ether to ethyl acetate varying in a gradient of 10:0, 20:1, 10:1, 5: 1; performing gradient elution on the subfraction B2 by using petroleum ether-ethyl acetate, wherein the gradient change of the volume ratio of the petroleum ether to the ethyl acetate is 10:0, 50:1 and 10: 1; the fourth fraction D is subjected to gradient elution by ethyl acetate-methanol, and the gradient change of the volume ratio of ethyl acetate to methanol is 20:0, 20:1, 10:1, 5:1 and 3: 1; performing gradient elution on the subfraction D2 by using methanol-water, wherein the gradient change of the volume ratio of methanol to water is 1:9, 2:8, 3:7, 6:4, 8:2, 9:1 and 10: 0; subfraction D2-6 was eluted with ethyl acetate-methanol gradient with the volume ratio of ethyl acetate to methanol varying in gradient from 10:0, 50:1, 20:1, 10:1, 5: 1.
Preferably, the subfraction B2-1 is separated by semi-preparative HPLC, the mobile phase is 40% methanol aqueous solution by volume concentration, the detection wavelength is 220nm, the flow rate is 2mL/min, and the sample amount is 60 μ L.
More preferably, the sub-fraction B2-1 is subjected to semi-preparative HPLC separation by using YMC ODS-C18 chromatographic column, the mobile phase is 40% methanol aqueous solution, the detection wavelength is 220nm, the flow rate is 2mL/min, the sample injection amount is 60 μ L, and the collection retention time t is tRAnd (4) accumulating for multiple times, and evaporating to dryness to obtain a pure product of the compound I with the structure shown in the formula I.
Preferably, the silica gel chromatographic column is 100-400 meshes.
Preferably, the filler of the gel column in the purification by the Sephadex chromatography is Sephadex LH-20.
Compared with the prior art, the invention has the following beneficial effects:
the invention extracts and prepares two new iridoid compounds from the overground part of morinda officinalis for the first time, confirms the chemical structures and physicochemical properties of the two new iridoid compounds through modern spectral means such as HR-ESIMS, NMR, IR, UV and the like, verifies the pharmacological activity of the two new iridoid compounds through cell experiments, shows excellent anti-inflammatory action, has obvious inhibition effect on NO generation, can effectively reduce the expression of proinflammatory factors iNOS, COX-2, TNF-alpha, IL-1 beta and IL-6, can reduce inflammatory reaction caused by excessive expression of the inflammatory factors, and can be used as a lead compound for developing novel anti-inflammatory drugs.
The iridoid compound is derived from stems and leaves of morinda officinalis and can be developed into health-care food or medicine, the experimental steps of the preparation method are easy to control, simple and quick, the preparation of the iridoid compound is easier, the utilization of medicinal parts and medicinal resources of morinda officinalis is expanded, and the problem of environmental pollution caused by resource waste is solved.
Drawings
FIG. 1 shows the preparation of compound I1H-NMR(400MHz,CDCl3) And (4) mapping.
FIG. 2 shows the preparation of compound I13C-NMR(100MHz,CDCl3) And (4) mapping.
FIG. 3 shows the reaction of compound I in CDCl3HSQC spectra in solvent.
FIG. 4 shows the reaction of compound I in CDCl3HMBC spectrum in solvent.
FIG. 5 shows the reaction of compound I in CDCl3NOESY pattern in solvent.
FIG. 6 is an IR spectrum of compound I.
FIG. 7 shows the preparation of compound II1H-NMR(400MHz,CD3OD) profile.
FIG. 8 is a drawing of Compound II13C-NMR(100MHz,CD3OD) profile.
FIG. 9 shows the CD activity of compound II3HSQC spectra in OD solvent.
FIG. 10 shows the CD activity of compound II3HMBC mapping in OD solvent.
FIG. 11 shows the CD activity of compound II3NOESY pattern in OD solvent.
FIG. 12 is an IR spectrum of a compound II.
FIG. 13 shows the effect of compounds I (FIG. A) and II (FIG. B) on LPS-induced release of NO from RAW264.7 cells.
FIG. 14 is a graph showing the effect of compounds I (FIGS. A to E) and II (FIGS. F to J) on the expression of inflammatory factors at the mRNA level.
FIG. 15 is a graph showing the effect of compounds I (FIG. A, B) and II (FIG. C, D) on the secretion of the inflammatory factors TNF-. alpha.and IL-6.
FIG. 16 is a graph showing the effect of compounds I (FIG. A) and II (FIG. B) on the expression of inflammatory proteins iNOS and COX-2.
FIG. 17 is a molecular docking simulation of docking of compounds I (ofloxacin E) and II (ofloxacin F) with iNOS and COX-2 protein; the figure A is a molecular docking simulation diagram of a compound I and COX-2 protein, and the figure B is a molecular docking simulation diagram of a compound II and iNOS protein.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified.
Bruker-Avance III 400 NMR spectrometer (Bruker, Germany); AB Sciex Triple TOF 5600+ type high resolution mass spectrometer (AB Sciex, USA); LC-20AT type semi-preparative high performance liquid phase (Shimadzu, Japan); model N-1100 rotary evaporator (Shanghai Alang Co.); SHZ-DIII type circulating water multi-purpose vacuum pump (Shanghai Yangrong Biochemical company); sartorius electronic analytical balance (precision 0.1mg, Sartorius, switzerland); CQ-200 ultrasonic cleaner (Power 250W, frequency 80kHz, Shanghai Sound Acoustic electro-technology Corp.); sephadex LH-20 sepharose (GE Healthcare, Switzerland); analytical column YMC ODS-A (250 mm. times.4.6 mm, 5. mu.M, Japan YMC Co.); semi-preparative column YMC ODS-A (250 mm. times.10 mm, 5. mu.M, Japan YMC Co.); column chromatography silica gel of 100-200 meshes and column chromatography silica gel of 300-400 meshes, silica gel plates GF254 (Qingdao ocean chemical plant) of 200 x 200mm and 0.20-0.25 mm; the conventional reagents are analytically pure (Tianjin Fuyu fine chemical industry).
Example 1 extraction and isolation of iridoid glycosides from aerial parts of Morinda officinalis
The stem and leaf of the overground part of the Morinda officinalis are collected in the GAP planting base of Deqing county of Zhaoqing, Guangdong province in 2019 in 7-8 months, and are identified as the stem and leaf of the overground part of Morinda officinalis of Morinda of Rubiaceae by a Dingping researcher of Guangzhou Chinese medicinal university, and the voucher specimen (20190731001) is stored in a Chinese medicinal resource research and development room of Guangzhou Chinese medicinal university.
First, experiment method
1. Extraction and isolation of compounds
Air drying radix Morindae officinalis, cutting 10.0kg, extracting with 95% ethanol for three times (10L × 3) each for seven days, and reflux-extracting the residue with 95% ethanol to obtain effective components. The filtrates were combined and recovered under reduced pressure and filtered until no alcohol smell was observed, yielding about 410.1g of an extract. Suspending the extract with 1L of water, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol for three times to obtain 178.3g of petroleum ether extract, 66.7g of ethyl acetate extract, 65.9g of n-butanol extract and 156.9g of water phase extract.
Silica gel column chromatography: taking an n-butyl alcohol extract (65.9g), firstly filling the n-butyl alcohol extract into a column by using a 200-300-mesh silica gel dry method with the weight being 10 times of that of the extract, then carrying out dry sampling on 100-200-mesh silica gel with the weight being 2 times of that of the extract, carrying out gradient elution by using dichloromethane-methanol (the volume ratio gradient is 10:0, 20:1, 10:1, 5:1 and 3:1), carrying out gradient elution with 2 column volumes for each gradient, collecting gradient eluent of each gradient, carrying out thin-layer chromatography detection, replacing the next gradient elution after each gradient elution is no point on a TLC point plate, combining similar fractions, concentrating, and collecting 6 fractions (A-F) in total.
And (3) packing the fraction B (857.0mg) into a 200-300-mesh silica gel column, performing gradient elution by using petroleum ether-ethyl acetate (the volume ratio is gradient 10:0, 20:1, 10:1 and 5:1), performing gradient elution for 2 column volumes each, detecting by using thin layer chromatography, merging similar fractions, concentrating, and collecting 4 sub-fractions (B1-B4) in total. Sub-streamAnd (3) loading the fraction B2(205.0mg) into a 200-300-mesh silica gel column, performing gradient elution by using petroleum ether-ethyl acetate (the volume ratio is 10:0, 50:1 and 10:1), performing thin-layer chromatography detection on 2 column volumes of each gradient elution, combining similar fractions, concentrating, and collecting 5 sub-fractions (B2-1-B2-5) in total. Sub-fraction B2-1(36.0mg) was subjected to semi-preparative HPLC separation (10 mm. times.250 mm, 5 μm YMC ODS-C18 column with 40% aqueous methanol as mobile phase, detection wavelength 220nm, flow rate 2mL/min, sample size 60 μ L) to give Compound I (3.0mg, t:, M)R＝6.0min)。
Mixing the fraction D (998.0mg) with 200-300-mesh silica gel, loading into a column, performing gradient elution by using ethyl acetate-methanol (the volume ratio gradient is 20:0, 20:1, 10:1, 5:1 and 3:1), performing gradient elution by 2 column volumes each, detecting by using thin layer chromatography, combining similar fractions, concentrating, and collecting 3 sub-fractions (D1-D3). And eluting the sub-fraction D2(340.0mg) by ODS column chromatography in methanol-water (volume ratio gradient of 1:9, 2:8, 3:7, 6:4, 8:2, 9:1 and 10:0), detecting by thin layer chromatography, merging similar fractions, concentrating, and collecting 6 sub-fractions D2-1-D2-6. Performing silica gel column chromatography on the sub-fraction D2-6, performing gradient elution with ethyl acetate-methanol system (volume ratio gradient of 10:0, 50:1, 20:1, 10:1 and 5:1), performing thin layer chromatography, combining the same parts, concentrating, performing Sephadex LH-20 purification by Sephadex chromatography to obtain compound II (71.0 mg).
2. Identification of glucose configuration of compound II
The acid hydrolysis method comprises the following steps: 2.0mg of the compound II was weighed out precisely, 1.0mL of 1mol/L hydrochloric acid was added thereto, and the mixture was refluxed at 100 ℃ for 2 hours, and after the reflux was completed, 3.0mL of pure water was added thereto for dilution, and extraction was carried out with ethyl acetate (3X 5 mL). After the aqueous layer was dried under vacuum, the residue was dissolved in pyridine (0.5mL) containing L-cysteine methyl ester hydrochloride (2.0mg) and heated at 60 ℃ for 2 h. mu.L of o-toluyl isothiocyanate was added and the mixture was heated at 60 ℃ for 2 hours, the reaction-completed mixture was diluted to an appropriate concentration, and the reaction mixture was analyzed at a wavelength of 250nm using HITACHI Primaide high performance liquid chromatography with an ultraviolet detector. The sample was chromatographed using A YMC-Pack-ODS-A (250X 4.6mm, 5 μm) column at A column temperature of 35 ℃ in A methanol-water system (25: 75) at A flow rate of 0.8 mL/min. The retention time of the reaction mixture was compared to the retention times of D-glucose (13.1min) and L-glucose (14.4 min). The type and absolute configuration of the sugar can be confirmed if the sugar derivative of the compound exhibits a retention time very similar to that of the sugar standard.
Example 2 structural identification of iridoid Compounds
1. Structural identification of Compound I
The compound I is a colorless oily substance,
ion peak M/z [ M + Na ] given by HRESIMS mass spectrum data]+279.1451(calcd for C13H20O5Na, 279.1203) binding13The molecular formula of the C NMR data can be judged to be C13H20O5The unsaturation degree is 4.1H NMR showed 1 aldehyde group in the low field regionH9.73(1H, s, H-8); 1 pair of double bonds deltaH 5.99(1H,dd,J＝2.4,5.6Hz,H-3)，δH5.80(1H, dd, J ═ 2.4,5.6Hz, H-2); 1 butoxy group deltaH4.13(1H, m, H-10a),4.04(1H, m, H-10b),1.62(2H, m, H-11),1.37(2H, m, H-12) and 0.92(3H, t, J ═ 7.2Hz, H-13). From13C NMR spectrum, DEPT spectrum and HSQC spectrum showed 13 carbon signals including 1 aldehyde group deltaC200.4 (C-8); 1 ester carbonyl deltaC173.5 (C-9); 1 pair of double bonds deltaC133.4(C-2), 137.2 (C-3); 1 quaternary carbon with oxygen deltaC84.9 (C-1); 2 continuous oxygen methylene signal deltaC68.2(C-6), 65.1 (C-10); 2 methylene deltaC50.1(C-5) and 40.8 (C-4); 3 methine signals deltaC46.4(C-7), 30.4(C-11), 19.1(C-12) and 1 methyl signal deltaC13.7 (C-13). The cyclopentenol structure comprises a segment substituted by aldehyde at C-4 position, by oxymethylene and hydroxyl at C-1 position, and by butoxy at C-5 position, wherein the segment can be selected from1H-1H-2 is related to H-3, H-3 is related to H-4, H-4 is related to H-5 and H-7, and H-7 is related to H-8 in the H COSY spectrum. In addition, the inventive method is characterized in thatIn HMBC, H-2 is associated with C-1/C-6, H-3 is associated with C-4/C-7, H-8 is associated with C-4/C-7, H-5 is associated with C-1/C-4/C-6/C-9, H-6 is associated with C-1/C-2/C-5, and H-10 is associated with C-9/C-11/C-12, the cyclopentenol structure described above is also confirmed.
Further by analysis1H–1The relative configuration of compound i is judged by the H COSY and NOESY data. The large coupling constant J of H-5(d, J ═ 8.0Hz) indicates that H-5 is in the β configuration, see fig. 1. In NOESY, H 26 is related to H-4 and H-5, indicating H2-6, H-4 and H-5 are coplanar. In summary, compound I was named offibinaloside E. Of the above compound I1HNMR、13C-NMR, HSQC, HMBC, NOESY and IR spectra are shown in the attached figures 1-6.
Structure form of compound I of FIG. 11H-1H COSY, HMBC and NOSEY are related
In conclusion, the compound I is a colorless oil,
HRESIMS m/z[M+Na]+279.1451(calcd for C13H20O5na, 279.1203); (MeOH) λ max: 220 nm; ir (kbr) ν max 3421, 2921, 1717, 1463, 1395, 1351, 1181, 1033cm "1; 1H and 13C NMR are shown in Table 1, and the compound I (ofloxicalinside E) has a structural formula shown in a formula I and is an iridoid compound.
TABLE 1 Compound I (CDCl)3) Is1H spectrum and13c spectrum data
2. Structural identification of Compound II
The compound II is a yellow oily substance,
ion peak M/z [ M + Na ] given by HRESIMS mass spectrum data]+469.1660(calcd for C20H30O11Na, 469.1680) binding13The molecular formula of the molecular formula is C which can be judged by C NMR and DEPT data20H30O11The unsaturation degree was 6. After acid hydrolysis, chiral derivatization and thin-layer detection, the compound II is analyzed by HPLC to determine that the hydrolyzed monosaccharide is D-glucose, and the peak-off time is 13.5 min. Coupling constant delta of beta configuration of glucose through anomeric carbonH4.75(1H, d, J ═ 8.0Hz, H-1') was determined.1Hydrogen proton signal delta at position 3 in H NMRH7.66(1H, d, J ═ 1.2Hz, H-3) indicated that the compound had a typical 4-substituted iridoid enol ether structure.1H NMR and13c NMR spectrum shows that the C-1 position of the compound II has a monosaccharide structure which is almost the same as that of the deacetyl asperulosidic acid methyl ester of another iridoid compound separated by the invention, the difference is that the side chain groups are different, the component is not separated from morinda officinalis in the prior art at present, and the anti-inflammatory activity of the component is relatively poor. From14.18,1.69,1.45,0.99ppm in H NMR and13the signals at 65.2,32.0,20.4,14.3ppm in C NMR are distinguished by the substitution of the butoxy group at C-11 of compound II, whereas the methoxy group at C-11 of methyl deacetylasperulate is present.
The stereo configuration of the compound II is mainly determined by comparing chemical shift, change of coupling constant and NOESY spectrogram with the literature. Most iridoid glycoside compounds, C-1 glucose, H-5 and H-9 are in beta configuration, and the chemical shifts of C-1 and C-1' carbon spectra in alpha configuration are averagely shifted to 5-6ppm toward low field. When H-5 and H-9 are in the trans conformation, their coupling constants are generally in the range of 12-13Hz, since the coupling constant of H-9 is deltaH2.59(1H, t, J ═ 8.0Hz, H-9) so H-5 and H-9 are in the cis conformation. Because of J1,98.8Hz and deltaC108.6 > 99ppm, thereforeThe C-6-OH is in alpha configuration, and the structure is shown in figure 2. Therefore, compound II is named offibinaloside F. Of the above compounds II1HNMR、13C-NMR, HSQC, HMBC, NOESY and IR spectra are shown in the attached figures 7-12.
The structure of the compound II in FIG. 2 is key1H-1H COSY, HMBC and NOSEY are related
In conclusion, the new compound II is a yellow oil,
HRESIMS m/z[M+Na]+469.1660(calcd for C20H30O11Na，469.1680)；UV(MeOH)λmax：298nm；IR(KBr)νmax 3361，2922，2854，1736，1458，1378，1260，1185，1074，830，701cm-1；1h and13c NMR is shown in Table 2, and the structural formula of compound II (ofloxicalinoside F) is shown in formula II, and the compound is an iridoid glycoside compound.
TABLE 2 Compound II (CD)3OD) of1H spectrum and13c spectrum data
EXAMPLE 3 anti-inflammatory Activity test of iridoids
In this example, experiments were conducted to investigate the in vitro anti-inflammatory activity of compounds I (ofloxacin E) and II (ofloxacin F) on LPS-induced RAW264.7 cells.
First, experimental material and instrument
Clean benches (Suzhou Antai air technologies, Inc.); constant temperature CO2Cell culture chambers (Heraeus, Germany); an electric heating constant temperature water bath (Shanghai Boxun industries, Ltd.); inverted microscope (LEICA, germany); a multifunctional microplate reader, a common PCR amplification instrument, a real-time fluorescence quantitative PCR instrument, and an SDS-PAGE vertical electrophoresis instrument (BIO-RAD, USA); fluorescence imaging system, Trizol lysate (tiangen biochemical technology); a micro spectrophotometer (impelen, germany); 4 ℃ refrigerated centrifuge (Hettich, Germany); a metal bath heater (Hangzhou Osheng instrument); PBS phosphate buffer solution, nitric oxide kit, BCA protein quantitative kit, SDS cell lysate, primary anti-diluent, PMSF protease inhibitor, 5 × loading buffer protein loading buffer solution, pre-staining protein Marker and SDS-PAGE gel preparation kit (Byunnan biotechnology); mouse TNF-. alpha.EL ISA kit, Mouse IL-6ELISA kit, developer luminescence solution (lot 4AG092012F) (Tetraselaginella arborvitae); cell Counting Kit-8 (batch GK100013) (GLP Bio Inc. in USA); LPS O55: B5 (batch No. 039M4004V) (Sigma-Aldrich, USA); DMSO (lot No. Y181205), dexamethasone (lot No. M7084) (american MP corporation); SYBR GreenPro Taq HS premixed qPCR kit II (batch No. AG11702), Evo M-MLV reverse transcription reagent premixed solution (batch No. AG11706) (Hunan Aikery organism); goat anti-Rabbit secondary antibody, Rabbit anti-mouse secondary antibody, iNOS antibody (Rabbit 1:1000), COX-2 antibody (Rabbit 1:1000), GAPDH (Rabbit IgG 11: 6000) (Cell Signaling Technology, USA).
Second, Experimental methods
1. Detection of cytotoxic and anti-inflammatory Activity
RAW264.7 cells in logarithmic growth phase were seeded in 96-well cell culture plates, and 100. mu.L of medium was added to each well to give a cell density of 1X 105And (4) culturing the cells per mL in an incubator for 24 h. The experiment is divided into a blank group, a control group and an additive group. Adding DMEM medium into blank group, adding DMEM medium containing certain cell number into control group, adding compound I and compound II diluted with DMEM medium to different concentrations into administration groupII incubate 24h, repeat 3 wells per group. After 24h, the 96-well plate was taken out of the incubator, the supernatant was discarded, complete medium containing 10% CCK-8 was added to each well, and after incubation at 37 ℃ for 1h, the absorbance (a) value was measured at a wavelength of 450nm and the cell survival rate was calculated, and the experiment was repeated 3 times in total.
RAW264.7 cells in logarithmic growth phase were seeded in 96-well cell culture plates, and 100. mu.L of medium was added to each well to give a cell density of 2X 105One per mL. Placing in an incubator for 24 h. The experimental groups are a control group, a model group, a positive medicine group, an additive medicine group and a deacetyl asperuloside methyl ester group. The drug components are respectively added with compounds I and II (0, 1.25, 2.5, 5, 10, 20, 40, 80 mu M) with different concentrations for pretreatment for 1h, the methyl deacetyl asperuloside components are respectively added with 2.5, 5, 10, 20, 40, 80 mu M methyl deacetyl asperuloside for pretreatment for 1h, dexamethasone (10 mu M) is used as a positive control, LPS (100ng/mL) is added to stimulate cells for incubation for 18h, and each group is repeated for 3 holes. After 18h, each group sucks 50 mu L of supernatant to a 96-hole cell culture plate, then 50 mu L of Griess reagent I, Griess reagent II is sequentially added, the mixture is gently oscillated for several times to be fully mixed, the absorbance A is measured at the wavelength of 540nm by using an enzyme-labeling instrument, the NO inhibition rate is calculated according to a standard curve, and GraphPad Prism 8 software is used for obtaining a fitting curve to respectively obtain IC (integrated circuit) of the compounds I and II for inhibiting NO release50The value is obtained.
2. RT-qPCR detection of expression of inflammatory mediators
Collecting RAW264.7 cells in logarithmic growth phase, and adjusting cell density to 2 × 105one/mL, then 1mL of cell suspension per well in 12-well cell culture plates, at 37 ℃ with 5% CO2The incubation was carried out overnight in an incubator under the conditions. The experimental groups are a control group, an LPS group, a positive medicine group and an additive medicine group. The administration group is added with compounds I and II (12.5, 25 and 50 mu M) with different concentrations respectively, the positive medicine group is added with 10 mu M dexamethasone, the pretreatment is carried out for 1h, then LPS (100ng/mL) is added to stimulate cells for co-incubation, and RNA of the cells is extracted after 6 h. Detecting the concentration and purity of the extracted RNA by using a Nano Photo Meter P300 ultramicro spectrophotometer, measuring the RNA purity of the extracted sample by using an A260/A280 ratio, and considering that the ratio is 1.8-2.0For better purity, the corresponding RNA concentration was recorded.
According to the measured RNA concentration, a 10-microliter reaction system is prepared, the final RNA amount of each hole is 1000ng, DEPC water is added firstly, then RNA is added, finally reverse transcriptase is added into the cover of the PCR tube, the reaction system is uniformly mixed by vortex, and the operation is carried out on ice by a palm centrifuge for several seconds. And carrying out reverse transcription on the uniformly mixed reaction liquid. Putting a special 96-well PCR plate on ice, adding an upstream primer, a downstream primer, cDNA and a fluorescent quantitative enzyme into each well, diluting the cDNA obtained by reverse transcription with DEPC water to a proper concentration, adding the diluted cDNA into the 96-well PCR plate, adding a primer and enzyme mixed system prepared according to the proportion in the table 3 into the special 96-well PCR plate, and taking glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference. And (3) placing the prepared 96-well plate in a real-time fluorescent quantitative PCR instrument, and setting an amplification program.
TABLE 3 RNA inverse transcription proportioning table
3. ELISA for detecting content of inflammatory factors
Collecting RAW264.7 cells in logarithmic growth phase, adjusting cell density to 5 × 105one/mL, then 2mL of cell suspension per well in a 6-well cell culture plate. The cells were incubated overnight in an incubator. The experimental groups are a control group, an LPS group, a positive medicine group and an additive medicine group. Adding different concentrations of compounds I and II (12.5, 25 and 50 mu M) into an administration group, adding 10 mu M dexamethasone into a positive drug group, pretreating for 1h, adding LPS (100ng/mL) to stimulate cells for co-incubation, collecting cell supernatant after 18h, transferring the cell supernatant into an EP tube, marking, centrifuging the cell supernatant at 4 ℃ for 10min by a centrifugal force of 1000 Xg to remove particles and polymers, sucking the supernatant into a new EP tube, and subpackaging. Each sample was set with 3 biological replicate wells.
4. Expression of inflammation-related protein detected by Western blot
(1) Preparation of protein samples
After culturing cells of a control group, an LPS group, a positive drug group and an additive group for 18h, collecting the cells, adding 2mL of precooled PBS along the side corner of the dish wall, taking the cells off from ice after 2min, removing the PBS by suction, adding 100 mu L of protein lysate (RIPA: phosphatase inhibitor: protease inhibitor: 50: 1: 1), fully cracking the cells on ice for 30min, gently scraping the cells by using a cell scraper, transferring the cells to an EP tube, placing the EP tube on ice, centrifuging the cells at the rotating speed of 12000rpm at the temperature of 4 ℃ for 15min, and sucking supernatant and transferring the supernatant to a new EP tube. And (4) measuring the protein concentration after extracting the total protein, and calculating the concentration of the sample through a standard curve. Taking the sample with the minimum protein concentration in the same group as a standard, diluting the protein of each group of samples to the same concentration by PBS, adding a corresponding amount of 5 xSDS loading buffer solution, uniformly mixing by vortex, centrifuging, heating in a metal bath at 100 ℃ for 10min, and storing at-20 ℃.
(2) Western blot
Preparing 8% separation gel, adding 75% ethanol to seal the liquid surface, pouring 75% ethanol and sucking with filter paper after gel is solidified, adding prepared 5% concentrated gel, inserting into a sample comb, standing at room temperature to solidify, and preparing for sample electrophoresis. After electrophoresis, the PVDF membrane with the size of 8.5 multiplied by 5.5cm is cut, soaked by methanol, activated on a shaking table for 3min, and then transferred. And (5) after the membrane is transferred, taking out the PVDF membrane, soaking the PVDF membrane in 5% of sealing liquid, and sealing the PVDF membrane on a shaking table for 1 hour. The blocking solution was discarded, and 1 XTSST buffer was added to the solution for washing at room temperature on a shaker, and the corresponding primary antibody was added as necessary, and incubated overnight at 4 ℃. Washing, incubating for 1h at room temperature on a shaking table, exposing and developing, and analyzing data.
5. Computer molecular simulation docking
The structures of compounds I and II were drawn using ChemDraw Ultra 8.0 software, energy minimization using MM2, followed by modeling of the three-dimensional structures of the compounds using Chem3D Ultra 8.0 software and preservation. Downloading related Protein structures from a Protein Data Bank (PDB) library (http:// www.rcsb.org/PDB/index. html), analyzing and preprocessing target proteins by adopting SYBLY 2.0 software, extracting ligands in the target proteins as docking reference templates, dewatering, hydrogenating, charging and the like, and flexibly docking small molecules with related targets by running the SYBLY 2.0 software. And comprehensively scoring the molecular docking condition by using a Total-Score function, selecting the conformation with the highest Score as a docking conformation, and running PyMOL and SYBLY 2.0 programs for visualization. The conformation and position of the ligand are optimized to have the best binding mode with the receptor, and the optimal binding conformation is determined.
Third, experimental results
1. Effect of Compounds on cellular toxicity and anti-inflammatory Activity
The survival of RAW 2647 cells after 24h when 50. mu.M of compounds I and II, respectively, was shown in Table 4, which indicates that neither compounds I nor II had a toxic effect on cells at 50. mu.M.
The results of the inhibition of NO release at different concentrations are shown in FIG. 13. As can be seen from the figure, the release amount of NO in normal RAW 2647 cells is lower and is 0.8 muM, and after LPS induction, the content of NO in cell supernatant is remarkably increased and is 6.3 muM, which indicates that the model building is successful; when the different concentrations of compounds I and II intervene, the content of NO in the cell supernatant of the administration group is reduced, and the compounds I and II inhibit the IC of NO release5026.70 and 31.85 μ M (see Table 4), respectively, exhibit good anti-inflammatory effects, but the methyl deacetylasperulate, which has a structure similar to that of compound II, inhibits the IC of NO release50Above 50 μ M, the anti-inflammatory effect is relatively poor.
Compounds I and II inhibited LPS-induced NO production in a concentration-dependent manner without affecting cell viability of RAW264.7 macrophages, wherein the NO content of the 50 μ M group of compounds I and II was 1.8 μ M and 2.2 μ M, respectively, indicating that both compounds I and II significantly inhibited cellular NO release.
TABLE 4 cytotoxic and anti-inflammatory Activity of Compounds I and II on RAW 2647
2. Effect of Compounds on expression of inflammatory mediators mRNA in cells
The results are shown in FIG. 14, where iNOS, COX-2, TNF-alpha, IL-1 beta and IL-6 were expressed in cells at mRNA level under LPS stimulation, and both compounds were concentration-dependently downregulated in the expression levels of these 5 genes after treatment with compounds I and II. The experimental results show that the compounds can inhibit the expression of iNOS, COS-2, TNF-alpha, IL-1 beta and IL-6 in RAW264.7 macrophage under the induction of LPS in a concentration-dependent mode, and the compounds I and II can be used as effective regulators to relieve inflammatory diseases caused by excessive accumulation of proinflammatory cytokines.
3. Effect of Compounds on secretion of inflammatory factors IL-6, TNF-alpha in cells
The results are shown in figure 15, after the compound I and the compound II pretreat the cells for 1h at the concentration of 12.5, 25 and 50 mu M and stimulate the cells for 18h by LPS, the concentration of inflammatory factors TNF-alpha and IL-6 released by macrophages in the model group is obviously higher than that of the control group, and the construction of the inflammatory cell model is proved to be successful. After the compound treatment, the compounds I and II can reduce the expression level of two proteins of TNF-alpha and IL-6 in a concentration-dependent manner, which shows that the compounds have good anti-inflammatory activity.
4. Effect of Compounds on iNOS, COX-2 protein expression in cells
The results are shown in figure 16, the concentrations of inflammatory factors iNOS and COX-2 released by macrophages in the model group are obviously higher than those of the control group, and the construction of the inflammatory cell model composition work is illustrated; after the cells are respectively pretreated by the compounds I and II at the concentrations of 12.5, 25 and 50 mu M for 1 hour and are stimulated by LPS for 18 hours, the compounds I and II can respectively inhibit the up-regulation of iNOS and COX-2 proteins in a concentration-dependent manner, which indicates that the compounds can have good anti-inflammatory activity.
5. Molecular docking of Compounds with iNOS, COX-2, IRAK-4 proteins
The results of the simulated docking are shown in fig. 17 and table 5, and show that compound i has a strong interaction with COX-2 protein, compound ii has a strong interaction with iNOS protein, and the results of the molecular docking are consistent with the experimental results. In addition, compound I forms four hydrogen bonds with the four amino acids GLN-203, HIS-207, TRP-387, and ALA-202 in the COX-2 receptor binding site (FIG. 17A); compound II forms three hydrogen bonds with the TRP-372, GLU-377, GLY-202 amino acids in the iNOS receptor binding site (FIG. 17B). The compound I forms hydrogen bonds with carbonyl groups or peptide bonds in COX-2 receptors through C-1 hydroxyl, C-6 hydroxyl and C-9 carbonyl and C-8 carbonyl respectively; the compound II forms hydrogen bonds with carboxyl and peptide bonds in iNOS acceptors through C-10, C-4 'and C-6' hydroxyl respectively.
TABLE 5 binding sites of Compounds I and II with receptor proteins
In conclusion, the iridoid compounds I and II separated from the overground part of morinda officinalis can inhibit the generation of NO, can inhibit the expression of inflammatory factors iNOS, COX-2, TNF-alpha, IL-6 and IL-1 beta in a dose-dependent manner, and have good anti-inflammatory activity; the possible mechanism by which compounds i and ii exert anti-inflammatory effects is: affinity interaction between compound I and COX-2 protein and affinity interaction between compound II and iNOS protein.
The invention enriches the chemical components of morinda officinalis plants, provides a thought for the discovery of lead compounds with anti-inflammatory activity, and also provides a certain reference basis for the development and utilization of the above-ground morinda officinalis resources.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and that those skilled in the art can make other variations or modifications on the basis of the above description and idea, and that all embodiments are neither necessary nor exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (6)
1. An iridoid compound with a structure shown as a formula I or a pharmaceutically acceptable salt thereof:
I。
2. use of an iridoid compound of the structure of formula I according to claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of an anti-inflammatory agent.
3. Use of an iridoid compound of the structure of formula I in claim 1 or a pharmaceutically acceptable salt thereof for the preparation of an inhibitor of iNOS, and/or IL-6, and/or IL-1 β, and/or TNF- α, and/or COX-2.
4. A medicine with anti-inflammatory activity is characterized by comprising one or two of iridoid compounds with structures shown in formula I or pharmaceutically acceptable salts of iridoid compounds with structures shown in formula I.
5. The method for preparing the iridoid compound shown in the structural formula I and the iridoid glycoside compound shown in the structural formula II in the claim 1 is characterized by comprising the following steps:
s1, taking the overground part of morinda officinalis, performing reflux extraction for 3-5 times by using an ethanol water solution with the volume fraction of 70-95%, combining to obtain an extracting solution, and removing ethanol in the extracting solution to obtain a total extract;
s2, sequentially extracting the total extract with petroleum ether, ethyl acetate and n-butyl alcohol, retaining an n-butyl alcohol extraction phase, and removing a solvent to obtain an n-butyl alcohol extract;
s3, performing gradient elution on the n-butanol extract by using dichloromethane-methanol through silica gel column chromatography, collecting gradient eluents of each gradient, detecting through thin-layer chromatography, combining similar elution fractions, concentrating, and collecting to obtain 6 fractions A-F;
s4, separating and purifying the second fraction B obtained in the step S3 through silica gel column chromatography and semi-preparative HPLC to obtain a compound I shown in the structural formula I in the claim 1; separating the fourth fraction D obtained in step S3 by silica gel column chromatography and purifying by Sephadex chromatography to obtain compound II with structure shown in formula II;
II。
6. the method of claim 5, wherein the step S3 is performed by gradient elution with dichloromethane-methanol, and the gradient change of the volume ratio of dichloromethane to methanol is 10:0, 20:1, 10:1, 5:1, or 3: 1.
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| CN101156913A (en) * | 2007-11-01 | 2008-04-09 | 周兰兰 | Application of fevervine iridoid glycosides and its preparation |
| CN101812098B (en) * | 2009-12-13 | 2012-08-08 | 成都普思生物科技有限公司 | Method for extracting monotropein monomers from medicinal indianmulberry root |
| KR101787006B1 (en) * | 2016-12-08 | 2017-10-18 | 전북대학교산학협력단 | A composition comprising iridoid derivatives for treating and preventing Male Infertility |
| CN108503678A (en) * | 2017-02-24 | 2018-09-07 | 深圳曜曦生命科技有限公司 | A kind of iridoid and its preparation method and application |
| CN109568419B (en) * | 2017-09-29 | 2021-09-17 | 中国人民解放军第二军医大学 | Morinda officinalis total iridoid glycoside and preparation method and application thereof |
| CN111548380A (en) * | 2020-06-10 | 2020-08-18 | 劲牌有限公司 | Preparation method of monotropein in morinda officinalis |
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