CN103254259A - Iridoid glycoside compound as well as preparation method and application thereof - Google Patents
Iridoid glycoside compound as well as preparation method and application thereof Download PDFInfo
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- CN103254259A CN103254259A CN2013101450877A CN201310145087A CN103254259A CN 103254259 A CN103254259 A CN 103254259A CN 2013101450877 A CN2013101450877 A CN 2013101450877A CN 201310145087 A CN201310145087 A CN 201310145087A CN 103254259 A CN103254259 A CN 103254259A
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Abstract
The invention discloses an iridoid glycoside compound as well as a preparation method and an application thereof. The preparation method of the iridoid glycoside compound comprises the following steps of: (1), taking a reduning injection finished product for HP-20 macroporous adsorption resin column chromatography separation, carrying out gradient elution by water, 30% alcohol and 90%-100% alcohol in sequence, collecting and concentrating eluant to obtain a 90%-100% alcohol elution part; and (2), carrying out silica gel column chromatography, ODS (Ozone Depleting Substances) column chromatography separation, semi-preparation liquid phase HPLC (High Performance Liquid Chromatography) separation in sequence to obtain the iridoid glycoside compound. The iridoid glycoside compound disclosed by the invention can be used for treating hand-foot-and-mouth diseases.
Description
Technical field
The present invention relates to medical technical field, particularly a kind of iridoid glycoside compound that from existing Chinese patent medicine preparation, extracts and its preparation method and application.
Background technology
The malicious injection for curing of heat (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.The malicious injection for curing of heat is widely used in the clinical treatment of flu, influenza, cough, upper respiratory tract infection due to the affection of exogenous wind-heat, and its effect is rapid, effect is remarkable.
The researchist has found new chemical ingredients in the malicious injection for curing of heat, and finds this compound equal stable existence in the hot malicious injection for curing of each batch.Through consulting document as can be known, this is the new chemical ingredients of finding and identifying in the malicious injection for curing of heat first, and retrieval finds that this compound is new compound through Scifinder scholar.
Summary of the invention
The present invention prepares from the malicious injection for curing of heat and extracts a kind of iridoid glycoside compounds of biologically active, 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 the I:
The present invention also provides the preparation method of above-claimed cpd, comprises the steps:
(1) heat-obtaining poison injection for curing finished product, separate through HP-20 macroporous adsorbent resin chromatography post, successively with water, 30% ethanol, 90～100% ethanol gradient elutions, collect each elutriant respectively, being evaporated to does not have the alcohol flavor, obtains water elution position, 30% ethanol elution position, 90～100% ethanol elution positions;
(2) get 90～100% ethanol elution positions of step (1), separate through silica gel column chromatography, use the chloroform-methanol gradient elution, collection chloroform-methanol ratio is 95: 5 cut C, cut C separates through the ODS column chromatography, uses the methanol-water gradient elution, and collection methanol-water ratio is 1: 1 cut C-2, cut C-2 separates through partly preparing liquid phase HPLC, obtains The compounds of this invention.
Among the above-mentioned preparation method, described half preparative liquid chromatography of step (2) is that 25: 75 acetonitrile-water is moving phase with ratio, detects wavelength and be 254 and 220nm, and flow velocity 4mL/min is 16.1min in the retention time that partly prepares on the liquid phase.
The contriver by physico-chemical property and modern Wave Spectrum means (UV, IR, MS,
1H-NMR,
13C-NMR and 2D-NMR) carried out the structure evaluation to separating the compound that obtains, be confirmed that it is structure suc as formula the iridoid glycoside compound shown in the 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.The contriver finds that The compounds of this invention has certain restraining effect to hand foot mouth disease EV71 virus.
A further object of the present invention is to provide a kind of pharmaceutical composition, comprises compound shown in the 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.
Description of drawings
Fig. 1 is the HR-ESI-Q-TOF-MS of compound
Fig. 2 is compound
1The H-NMR spectrum
Fig. 3 is compound
13The C-NMR spectrum
Fig. 4 is structure fragment A and B
Fig. 5 is the local spectrum of amplifying of the HMBC of compound
Fig. 6 is the hsqc spectrum of compound
Fig. 7 is main HMBC (→) and COSY (-) relevant information of compound
Embodiment
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is described in further detail below in conjunction with specific embodiment.
The preparation of embodiment 1 The compounds of this invention
(1) heat-obtaining poison injection for curing finished product, separate through HP-20 macroporous adsorbent resin chromatography post, with water, 30% ethanol, 95% ethanol gradient elution, collect each elutriant respectively successively, being evaporated to does not have the alcohol flavor, obtains water elution position, 30% ethanol elution position, 95% ethanol elution position;
(2) get 95% ethanol elution position of step (1), separate through silica gel column chromatography, use the chloroform-methanol gradient elution, collection chloroform-methanol ratio is 95: 5 cut C7.2g, cut C separates through the ODS column chromatography, use the methanol-water gradient elution, collection methanol-water ratio is 1: 1 cut C-2195mg, cut C-2 separates through partly preparing liquid phase HPLC, is that 25: 75 acetonitrile-water is moving phase with ratio, detects wavelength and be 254 and 220nm, flow velocity 4mL/min, be 16.1min in the retention time that partly prepares on the liquid phase, separating obtained solution drying obtains The compounds of this invention 9.3mg.
The structure of embodiment 2 The compounds of this invention is identified
This compound is white unformed powder.ESI-MS (positive) provides m/z619[M+Na]
+ESI-MS (negative) provides m/z641[M+HCOO
-]
-, 1191[M-H]
-, the prompting compound molecular weight is 596.The HR-ESI-Q-TOF-MS (see figure 1) provides m/z619.2007[M+Na]
+(calculated value is 619.2003) determines that the compound molecule formula is C
28H
34O
14, calculating degree of unsaturation is 11.
This compound
1H-NMR (600MHz, in CD
3OD) spectrum has shown two groups of hydrogen signals, go up proton signal [δ 7.51 (1H comprising 3 of 2 typical iridoids, d, J=1.0Hz, H-3) and δ 7.52 (1H, d, J=0.9Hz, H-3 ")], and 3 hemiacetal proton signals [δ 5.14 (1H, d; J=7.8Hz; H-1), 4.71 (1H, d; J=7.8Hz; H-1 ") and4.70 (1H, d, J=7.8Hz, H-1 ')], by comparing with separating the compound geniposide that obtains and the collection of illustrative plates of compound genipin, can find two groups of hydrogen signals and these two compounds closely similar (Fig. 2) of this compound, infer that further this compound is the iridoid of a dimerization.
This compound
13C-NMR (150MHz, in CD
3OD) the spectrum (see figure 3) has shown two groups of carbon signals equally, by comparing with the carbon spectrum of compound geniposide and compound genipin, find basically identical, visible 2 carbonyl carbon signals [δ 169.5 (C-11) wherein, 169.6 (C-11 ")]; 4 quaternary carbon signals [δ 145.2 (C-8 "), 140.9 (C-8), 112.4 (C-4 "); 112.2 (C-4)]; 5 mesomethylene carbon signals [δ 69.3 (C-10), 62.8 (C-6 '), 61.8 (C-10 "), 39.9 (C-6), 39.8 (C-6 ")], 2 methoxyl group carbon signals [δ 51.74,51.71] with and 15 tertiary carbon signals.
By
1H-
1H COSY and HMBC spectrum can the certification structure Segment A and the existence of structure fragment B (see figure 4), and being connected of Segment A and fragment B can be composed (see figure 5) by HMBC and verified, on the HMBC spectrum, as seen (δ 103.0) are relevant for H-1 " (δ 4.71) are relevant with C-10 (δ 69.3); H-10 (δ 4.62; 4.32) and C-1 simultaneously ", and according to C-10 (δ 69.3) chemical shift obviously to low field displacement, the mode of connection of certification structure Segment A and structure fragment B is C-1 " → C10.To sum up analyzing, is the iridoid glycoside of dimerization with this compound identification, called after japonicaside B
Comprehensive HSQC (Fig. 6) and HMBC spectrum information (Fig. 7) have carried out belonging to (table 1) to whole carbon signals and the hydrogen signal of this compound.Through SciFinder Scholar network retrieval, do not find relevant report, show that this compound is a new dimerization iridoid glycoside compound, called after japonicaside B.
The nuclear magnetic data of table 1 compound (deuterated methanol,
1H-NMR600MHz,
13C-NMR150MHz)
The external anti-hand-foot-and-mouth-disease EV71 of embodiment 3 The compounds of this invention virus drugs detects
1. material
The preservation 1.1 strain hand foot mouth disease EV71 virus, laboratory go down to posterity.
1.2 the cell model monkey-kidney cells is Vero, the laboratory preservation of going down to posterity.Culture condition: DMEM+10% foetal calf serum, 37 ℃, 5%CO
2
2. principle and method
2.1 the cytotoxicity of medicine detects
Adopted
(Invitrogen) the test kit detection of drugs is to the toxic action of 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 enters cell and produces measurable fluorescence and colour-change by the cyclophorase reduction, is used for quantitative analysis and the vitro cytotoxicity research of cytoactive and cell proliferation.This mensuration is based on cell with metabolic activity and converts reagent the ability of fluorescence and colorimetric indicator to, 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: the Vero cell inoculation is in 96 porocyte culture plates, and is standby behind the cell attachment.With cell maintenance medium (DMEM+5% serum) with medicine from 6 gradients of 2 times of continuous 3 times of gradient dilutions of initial concentration, every concentration gradient single hole detects.After 48h is cultivated in dosing, add
Hatch 2h, fluoroscopic examination for 37 ℃
The reduction situation, exciting light 570nm, emission light 595nm.
Cytoactive (%)=(sample well-blank)/(cell contrast-blank) * 100%
2.2 medicine is to the inhibition test of EV71 virus
After containing the EV71 vero cells infection of reporter gene GFP, cells infected meeting expressing green fluorescent protein by observe the cell number of expressing the GFP green fluorescence under fluorescent microscope, just can reflect the propagation situation of EV71 virus.
Method steps:
The Vero cell inoculation is in 96 porocyte culture plates, and is standby behind the cell attachment.Medicine is from 6 gradients of 4 times of continuous 3 times of gradient dilutions of the highest test concentrations; The medicine that dilution is good adds in the hand-hole, adds viral supernatant liquor behind the 4h and infects, and places 37 ℃ of cell culture incubators to cultivate 24h, takes pictures under fluorescent microscope, and fluorocyte is counted.
No medicine control wells (not adding the medicine hole behind the virus infection), positive drug control wells (Guanidinium hydrochloride GuHCl) are established in experiment.
Inhibiting rate (%)=(no medicine control wells-sample well)/no medicine control wells * 100%
3. result
3.1 drug sample detects and detects the EV71 inhibition is active the toxicity of cell
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 of cell rounding and cracking is arranged, and when (100 μ M) overt toxicity is arranged also, and its SI value is 10.3, shows that it has certain restraining effect to hand foot mouth disease EV71 virus.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle 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. iridoid glycoside compounds, its structure is suc as formula shown in the I:
2. the preparation method of the described compound of claim 1 is characterized in that, may further comprise the steps:
(1) heat-obtaining poison injection for curing finished product, separate through the HP-20 macroporous adsorbent resin column chromatography, successively with water, 30% ethanol, 90～100% ethanol gradient elutions, collect each elutriant respectively, being evaporated to does not have the alcohol flavor, obtains water elution position, 30% ethanol elution position, 90～100% ethanol elution positions;
(2) get 90～100% ethanol elution positions of step (1), separate through silica gel column chromatography, use the chloroform-methanol gradient elution, collection chloroform-methanol ratio is 95: 5 cut C, cut C separates through the ODS column chromatography, uses the methanol-water gradient elution, and collection methanol-water ratio is 1: 1 cut C-2, cut C-2 separates through partly preparing liquid phase HPLC, obtains The compounds of this invention.
3. preparation method according to claim 2 is characterized in that, described half preparative liquid chromatography of step (2), be that 25: 75 acetonitrile-water is moving phase with ratio, detect wavelength and be 254 and 220nm, flow velocity 4mL/min is 16.1min in the retention time that partly prepares on the liquid phase.
4. the application of the described compound of claim 1 in preparation treatment hand foot mouth disease medicine.
5. a pharmaceutical composition is characterized in that, comprises the described compound of claim 1.
6. a pharmaceutical composition that is used for the treatment of hand foot mouth disease is characterized in that, contains the described compound of the claim 1 for the treatment of significant quantity and pharmaceutically acceptable carrier.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104072551A (en) * | 2014-05-02 | 2014-10-01 | 江苏康缘药业股份有限公司 | Dipolyiridoid glycoside compound and preparation method and application thereof |
| CN105315147A (en) * | 2014-08-03 | 2016-02-10 | 江苏康缘药业股份有限公司 | Guaiane-type sesquiterpenoids compound, and preparation method and application thereof |
| 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 |
| CN107163095A (en) * | 2017-06-07 | 2017-09-15 | 上海中药标准化研究中心 | Urnu gentian herb glycosides A extracting method and its purposes for preparing cough suppressing medicine |
| CN107513085A (en) * | 2016-06-16 | 2017-12-26 | 江苏康缘药业股份有限公司 | A kind of compound and its preparation method and application |
| CN109053831A (en) * | 2018-09-04 | 2018-12-21 | 山东中医药大学附属医院 | New compound and application in a kind of oldenlandia diffusa |
| CN109503684A (en) * | 2018-12-20 | 2019-03-22 | 广州市香雪制药股份有限公司 | Iridoid glycosides compound and its preparation method and application |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104072551A (en) * | 2014-05-02 | 2014-10-01 | 江苏康缘药业股份有限公司 | Dipolyiridoid glycoside compound and preparation method and application thereof |
| CN104072551B (en) * | 2014-05-02 | 2016-08-17 | 江苏康缘药业股份有限公司 | A kind of dimerization iridoid glycoside compound and its preparation method and application |
| CN105315147A (en) * | 2014-08-03 | 2016-02-10 | 江苏康缘药业股份有限公司 | Guaiane-type sesquiterpenoids compound, and preparation method and application thereof |
| 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 |
| CN107513085A (en) * | 2016-06-16 | 2017-12-26 | 江苏康缘药业股份有限公司 | A kind of compound and its preparation method and application |
| CN107513085B (en) * | 2016-06-16 | 2020-06-30 | 江苏康缘药业股份有限公司 | Compound and preparation method and application thereof |
| CN107163095A (en) * | 2017-06-07 | 2017-09-15 | 上海中药标准化研究中心 | Urnu gentian herb glycosides A extracting method and its purposes for preparing cough suppressing medicine |
| CN109053831A (en) * | 2018-09-04 | 2018-12-21 | 山东中医药大学附属医院 | New compound and application in a kind of oldenlandia diffusa |
| CN109503684A (en) * | 2018-12-20 | 2019-03-22 | 广州市香雪制药股份有限公司 | Iridoid glycosides compound and its preparation method and application |
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Application publication date: 20130821 |