CN104098630A - Iridoid glycoside compound, and preparation method and application thereof - Google Patents
Iridoid glycoside compound, and preparation method and application thereof Download PDFInfo
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- CN104098630A CN104098630A CN201310144795.9A CN201310144795A CN104098630A CN 104098630 A CN104098630 A CN 104098630A CN 201310144795 A CN201310144795 A CN 201310144795A CN 104098630 A CN104098630 A CN 104098630A
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Abstract
The invention discloses an iridoid glycoside compound and a preparation method and application thereof. The preparation method comprises the following steps: (1) respectively extracting a finished Reduning injection product with ethyl acetate and n-butanol so as to obtain n-butanol extract; (2) subjecting the n-butanol extract to HP-20 macroporous adsorption resin column chromatographic separation and ethanol-water gradient elution so as to obtain a 25-35% ethanol elution part; and (3) successively subjecting the 25-35% ethanol elution part to silica gel column chromatographic separation, ODS column chromatographic separation, HW-40 column chromatographic separation and preparative liquid phase HPLC separation so as to obtain the compound. The compound provided by the invention can be used for treating hand-foot-and-mouth disease.
Description
Technical field
The present invention relates to medical technical field, particularly a kind of iridoid glycoside compound extracting and its preparation method and application from existing Chinese patent medicine preparation.
Background technology
Reduning injection (the accurate word Z20050217 of traditional Chinese medicines) is former Chinese medicine two kind new medicines of Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov's independent research, and its prescription is sweet wormwood, Japanese Honeysuckle, cape jasmine, and auxiliary material is Polysorbate 80.Reduning injection is widely used in the clinical treatment of flu, influenza, cough, upper respiratory tract infection due to affection of exogenous wind-heat, and its effect is rapid, effect is remarkable.
Researchist has found new chemical composition in Reduning injection, and finds this compound equal stable existence in the Reduning injection of each batch.Known through By consulting literatures, this is the new chemical composition of finding in Reduning injection first and identifying, and retrieval finds that this compound is new compound through Scifinder scholar.
Summary of the invention
The present invention prepares and extracts one and have bioactive iridoid glycoside compounds from Reduning injection, and its application in preparation treatment hand foot mouth disease medicine is provided.
Specifically, the invention provides a kind of iridoid glycoside compounds, its structure is suc as formula shown in I:
The present invention also provides the preparation method of above-claimed cpd, comprises the steps:
(1) get Reduning injection finished product, extract respectively by isopyknic ethyl acetate and propyl carbinol, concentrating under reduced pressure obtains acetic acid ethyl ester extract, n-butyl alcohol extract and water layer;
(2) step (1) gained n-butyl alcohol extract is separated through HP-20 macroporous adsorbent resin column chromatography, alcohol-water gradient elution, obtains water elution position, 25～35% alcohol elutions, 95% alcohol elution;
(3) step (2) gained 25～35% alcohol elutions are separated through silica gel column chromatography, use chloroform-methanol gradient elution, collect the cut E that chloroform-methanol ratio is 9: 1, cut E separates through ODS column chromatography, use methanol-water gradient elution, collect the cut E-2 that methanol-water ratio is 1: 1, cut E-2 separates through HW-40 column chromatography, use methanol-water gradient elution, collect the cut E-2-2 that methanol-water ratio is 4: 6, cut E-2-2 separates through preparation liquid phase HPLC, obtains the compounds of this invention.
In above-mentioned preparation method, the described preparative liquid chromatography of step (3), taking ratio as the methanol-water of 4.3: 6.7 is as moving phase, detects wavelength and is 254 and 325nm, flow velocity 8mL/min, and the retention time in preparation liquid phase is 18.8min.
Contriver by physico-chemical property and the Modern spectroscopy section of learning to do (UV, IR, MS,
1h-NMR, 13C-NMR and 2D-NMR) carry out Structural Identification to separating the compound obtaining, be confirmed that it is structure suc as formula the iridoid glycoside compound shown in I.
Another object of the present invention is to provide the application of compound shown in a kind of formula I in preparation treatment hand foot mouth disease medicine.Contriver finds that the compounds of this invention has certain restraining effect to hand-foot-mouth disease EV 71 virus.
A further object of the present invention is to provide a kind of pharmaceutical composition, comprises compound shown in above-mentioned formula I.
The present invention also provides a kind of pharmaceutical composition for the treatment of hand foot mouth disease, contains above-mentioned formula I compound and one or more pharmaceutically acceptable carriers for the treatment of significant quantity.
Brief description of the drawings
Fig. 1 is the HR-ESI-Q-TOF-MS of compound
Fig. 2 is compound
1h-NMR spectrum
Fig. 3 is compound
13c-NMR spectrum
Fig. 4 is compound
1h-
1the local spectrum of amplifying of H COSY
Fig. 5 is the local spectrum of amplifying of the HMBC of compound
Fig. 6 is the local spectrum of amplifying of the HMBC of compound
Fig. 7 is the hsqc spectrum of compound
Fig. 8 is main HMBC (→) and the COSY of compound
relevant information;
Embodiment
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
The preparation of embodiment 1 the compounds of this invention
(1) get Reduning injection finished product 700g, extract respectively three times with isopyknic ethyl acetate and propyl carbinol, concentrating under reduced pressure obtains acetic acid ethyl ester extract, n-butyl alcohol extract and water layer;
(2) n-butyl alcohol extract of getting step (1) is through the separation of HP-20 macroporous adsorbent resin chromatography, and ethanol water gradient elution, obtains water elution position, 30% alcohol elution, 95% alcohol elution;
(3) step (2) gained 30% alcohol elution is separated through silica gel column chromatography, use chloroform-methanol gradient elution, collect the cut E 7.7g that chloroform-methanol ratio is 9: 1, cut E separates through ODS column chromatography, use methanol-water gradient elution, collect the cut E-2 3.2g that methanol-water ratio is 1: 1, cut E-2 separates through HW-40 column chromatography, use methanol-water gradient elution, collect the cut E-2-2 400mg that methanol-water ratio is 4: 6, cut E-2-2 separates through preparation liquid phase HPLC, taking ratio as the methanol-water of 4.3: 6.7 is as moving phase, detecting wavelength is 254 and 325nm, flow velocity 8mL/min, retention time in preparation liquid phase is 18.8min, separating obtained solution is dry, obtain the compounds of this invention 21.4mg.
The Structural Identification of embodiment 2 the compounds of this invention
Yellow unformed powder, Molisch reacting positive.ESI-MS (positive) provides m/z747[M+Na]
+, prompting compound molecular weight is 724.HR-ESI-Q-TOF-MS provides m/z747.2105[M+Na]
+(calculated value is 747.2112), (seeing Fig. 1) deterministic adduct molecule formula is C
33h
40o
18, calculating degree of unsaturation is 14.
This compound
1h-NMR (400MHz, in CD
3oD) (see Fig. 2) and
13c-NMR (100MHz, in CD
3oD) collection of illustrative plates of (seeing Fig. 3) has shown the feature hydrogen carbon signal of 1 coffee acyl, 1 glucosyl group and genipin aglycon.?
1h-
1in H COSY (seeing Fig. 4) spectrum, H-2 "/H-3 "/H-4 "/H-5 "/H-6 " between there is obvious coherent signal, in conjunction with hsqc spectrum, release skeleton fragment A:C
2 "-C
3 "-C
4 "-C
5 "-C
6 ".In HMBC spectrum (seeing Fig. 5), relevant peaks H-3 "/C-1 ", H-2 "/C-7 " and H-6 "/C-7 ", built the quininic acid structural unit of this compound.
Comprehensive above analysis, finally composes the connection site of each structure fragment of this compound to determine by HMBC.In HMBC spectrum (seeing Fig. 6), the distant relation peak of H-1 (δ 5.07)/C-1 ' (δ 100.5), H-1 ' (δ 4.70)/C-1 (δ 98.2), prompting glucosyl group is replaced in the C-1 position of genipin structural unit; " (δ 174.7) has distant relation to H-10 (δ 4.79)/C-7, and prompting quinoline acyl group is connected in the C-10 position of genipin structural unit; The distant relation peak of H-5 " (δ 5.28)/C-1 " ' (δ 168.2), prompting quinoline acyl group is connected in the C-1 " ' position of coffee acyl structural unit.To sum up, be jasmigeniposide A by this compound identification.
Comprehensive HSQC (seeing Fig. 7) and HMBC spectrum information (seeing Fig. 8), whole carbon signals and hydrogen signal to this compound have carried out belonging to (table 1).Through SciFinder Scholar network retrieval, do not find relevant report, show that this compound is a new iridoid glycoside compounds.
The nuclear magnetic data of table 1 compound (deuterated methanol,
1h-NMR 400MHz,
13c-NMR100MHz)
Embodiment 3 the compounds of this invention In Vitro Anti hand-foot-mouth disease EV 71 virus drug testings
1. material
1.1 strain hand-foot-mouth disease EV 71 virus, the laboratory preservation of going down to posterity.
1.2 cell model monkey-kidney cells are Vero, the laboratory preservation of going down to posterity.Culture condition: DMEM+10% foetal calf serum, 37 DEG C, 5%CO
2.
2. principle and method
The cytotoxicity of 2.1 medicines detects
Adopt
(Invitrogen) toxic action of test kit detection of drugs to cell.
Experimental principle:
be a kind of oxidation-reduction indicator, can produce absorbancy according to metabolic activity and change and fluorescent signal.
soluble in water, its oxidised form produces measurable fluorescence and colour-change through cyclophorase reduction after entering cell, for quantitative analysis and the vitro cytotoxicity research of cytoactive and cell proliferation.This mensuration is the ability that the cell based on having metabolic activity converts reagent to fluorescence and colorimetric indicator, and impaired and non-activity cell has lower natural metabolic activity, and corresponding signal is lower.Therefore fluorescent signal power, can reflect the height of cytoactive.
Method steps: Vero cell is inoculated in 96 porocyte culture plates, for subsequent use after cell attachment.With cell maintenance medium (DMEM+5% serum) by medicine from 6 gradients of 2 times of continuous 3 times of gradient dilutions of initial concentration, every concentration gradient single hole detects.Dosing is cultivated after 48h, adds
hatch 2h, fluoroscopic examination for 37 DEG C
reduction situation, exciting light 570nm, utilizing emitted light 595nm.
Cytoactive (%)=(sample well-blank)/(cell contrast-blank) × 100%
The inhibition test of 2.2 medicines to EV71 virus
Containing after the EV71 vero cells infection of reporter gene GFP, cells infected meeting expressing green fluorescent protein, by express the cell number of GFP green fluorescence at fluorescence microscopy Microscopic observation, just can reflect the propagation situation of EV71 virus.
Method steps:
Vero cell is inoculated in 96 porocyte culture plates, for subsequent use after cell attachment.Medicine is 6 gradients of continuous 3 times of gradient dilutions from 4 times of test concentrations the highest; The medicine having diluted is added in hand-hole, after 4h, add viral supernatant liquor to infect, be placed in 37 DEG C of cell culture incubators and cultivate 24h, under fluorescent microscope, take pictures, fluorocyte is counted.
Experiment is established without medicine control wells (not adding medicine hole after virus infection), positive drug control wells (Guanidinium hydrochloride GuHCl).
Inhibiting rate (%)=(without medicine control wells-sample well)/without medicine control wells × 100%
3. result
3.1 drug samples detect the toxicity of cell and EV71 are suppressed to active and detect
Drug sample dilution solvent for use, the highest concentration of ordinary dissolution, the highest test concentrations, CC
50, EC
50and SI (selectivity index) is as shown in table 2.
Table 2 experimental result
4. conclusion
The compounds of this invention shows overt toxicity, and examines under a microscope most cells change and justify and have cracking, when (100 μ M), also has overt toxicity, and its SI value is 10.4, shows that it has certain restraining effect to hand-foot-mouth disease EV 71 virus.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. an iridoid glycoside compounds, its structure is suc as formula shown in I:
2. the preparation method of compound described in claim 1, is characterized in that, comprises the following steps:
(1) get Reduning injection finished product, extract respectively by isopyknic ethyl acetate and propyl carbinol, concentrating under reduced pressure obtains acetic acid ethyl ester extract, n-butyl alcohol extract and water layer;
(2) step (1) gained n-butyl alcohol extract is separated through HP-20 macroporous adsorbent resin column chromatography, alcohol-water gradient elution, obtains water elution position, 25～35% alcohol elutions, 95% alcohol elution;
(3) step (2) gained 25～35% alcohol elutions are separated through silica gel column chromatography, use chloroform-methanol gradient elution, collect the cut E that chloroform-methanol ratio is 9: 1, cut E separates through ODS column chromatography, use methanol-water gradient elution, collect the cut E-2 that methanol-water ratio is 1: 1, cut E-2 separates through HW-40 column chromatography, use methanol-water gradient elution, collect the cut E-2-2 that methanol-water ratio is 4: 6, cut E-2-2 separates through preparation liquid phase HPLC, obtains the compounds of this invention.
3. preparation method according to claim 2, is characterized in that, the described preparative liquid chromatography of step (3), taking ratio as the methanol-water of 4.3: 6.7 is as moving phase, detecting wavelength is 254 and 325nm, flow velocity 8mL/min, and the retention time in preparation liquid phase is 18.8min.
4. the application of compound in preparation treatment hand foot mouth disease medicine described in claim 1.
5. a pharmaceutical composition, is characterized in that, comprises compound described in claim 1.
6. be used for the treatment of a hand foot mouth disease pharmaceutical composition, it is characterized in that, contain compound and pharmaceutically acceptable carrier described in the claim 1 for the treatment of significant quantity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105503979A (en) * | 2015-11-23 | 2016-04-20 | 江苏康缘药业股份有限公司 | Compound and preparation method and application thereof |
| CN105753915A (en) * | 2016-03-03 | 2016-07-13 | 桂林医学院 | Caffeoyl phenylethanoid glycoside compounds as well as preparation method thereof and application thereof in anti-viral hepatitis B drug |
| CN108373488A (en) * | 2018-04-11 | 2018-08-07 | 浙江工业大学 | Catalpol 6-caffeic acid ester derivative and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0505572A1 (en) * | 1990-10-09 | 1992-09-30 | TSUMURA & CO. | Iridoide derivative and its use as medicine |
| CN1706859A (en) * | 2005-05-24 | 2005-12-14 | 中国人民解放军第二军医大学 | Process of preparing high-purity jasminodin with Cape jasmine fruit |
| US20070275006A1 (en) * | 2006-03-08 | 2007-11-29 | Council Of Scientific And Industrial Research | Iridoid glycoside composition |
| CN101704857A (en) * | 2009-09-25 | 2010-05-12 | 中国中医科学院中药研究所 | Process for separating and purifying special component specnuezhenide of glossy privet fruit |
-
2013
- 2013-04-11 CN CN201310144795.9A patent/CN104098630A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0505572A1 (en) * | 1990-10-09 | 1992-09-30 | TSUMURA & CO. | Iridoide derivative and its use as medicine |
| CN1706859A (en) * | 2005-05-24 | 2005-12-14 | 中国人民解放军第二军医大学 | Process of preparing high-purity jasminodin with Cape jasmine fruit |
| US20070275006A1 (en) * | 2006-03-08 | 2007-11-29 | Council Of Scientific And Industrial Research | Iridoid glycoside composition |
| CN101704857A (en) * | 2009-09-25 | 2010-05-12 | 中国中医科学院中药研究所 | Process for separating and purifying special component specnuezhenide of glossy privet fruit |
Non-Patent Citations (1)
| Title |
|---|
| HAI-BO LI，等: "Iridoid and bis-iridoid glucosides from the fruit of Gardenia jasminoides", 《FITOTERAPIA》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105503979A (en) * | 2015-11-23 | 2016-04-20 | 江苏康缘药业股份有限公司 | Compound and preparation method and application thereof |
| CN105503979B (en) * | 2015-11-23 | 2018-05-18 | 江苏康缘药业股份有限公司 | A kind of compound and its preparation method and application |
| CN105753915A (en) * | 2016-03-03 | 2016-07-13 | 桂林医学院 | Caffeoyl phenylethanoid glycoside compounds as well as preparation method thereof and application thereof in anti-viral hepatitis B drug |
| CN108373488A (en) * | 2018-04-11 | 2018-08-07 | 浙江工业大学 | Catalpol 6-caffeic acid ester derivative and preparation method and application thereof |
| CN108373488B (en) * | 2018-04-11 | 2020-10-09 | 浙江工业大学 | Catalpol 6-caffeic acid ester derivative and preparation method and application thereof |
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