CN104098631A - 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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- CN104098631A CN104098631A CN201310145252.9A CN201310145252A CN104098631A CN 104098631 A CN104098631 A CN 104098631A CN 201310145252 A CN201310145252 A CN 201310145252A CN 104098631 A CN104098631 A CN 104098631A
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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) extracting a Reduning injection with n-butanol so as to obtain n-butanol extract; (2) subjecting the n-butanol extract to HP-20 macroporous adsorption resin column 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 to extract and a kind ofly has bioactive iridoid glycosides glycoside compound 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:
Above-claimed cpd called after of the present invention: 6 " O-is trans-asafoetide acyl group genipin O-gentibioside (6 "-O-trans-feruloylgenipin gentiobioside).
The present invention also provides the preparation method of above-claimed cpd, comprises the steps:
(1) get Reduning injection finished product, by isopyknic ethyl acetate and propyl carbinol, extract respectively, 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 is separated through ODS column chromatography, use methanol-water gradient elution, collect the cut E-2 that methanol-water ratio is 5: 5, cut E-2 is separated 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 is separated through preparation liquid phase HPLC, obtains the compounds of this invention.
In above-mentioned preparation method, the described preparative liquid chromatography of step (3), the methanol-water that the ratio of take is 4.3: 6.7 is moving phase, detects wavelength and be 254 and 325nm, flow velocity 8mL/min, the retention time in preparation liquid phase is 42.5min.
Contriver by physico-chemical property and the Modern spectroscopy section of learning to do (UV, IR, MS,
1h-NMR,
13c-NMR and 2D-NMR) the separated compound obtaining has been carried out to Structural Identification, 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.
Accompanying drawing explanation
Fig. 1 is the compounds of this invention
1h-NMR spectrogram;
Fig. 2 is the compounds of this invention
13c-NMR;
Fig. 3 is the local spectrogram that amplifies of the HMBC of the compounds of this invention;
Fig. 4 is the compounds of this invention
1h-
1h COSY spectrogram partial enlarged drawing spectrum;
Fig. 5 is the local spectrogram that amplifies of the HMBC of the compounds of this invention;
Fig. 6 is the local spectrogram that amplifies of the HMBC of the compounds of this invention;
Fig. 7 is the hsqc spectrum figure of the compounds of this invention;
Fig. 8 be the compounds of this invention main HMBC with
1h-
1h COSY 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, with isopyknic ethyl acetate and propyl carbinol, extract respectively three times, 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, alcohol-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 that chloroform-methanol ratio is 9: 1, cut E is separated through ODS column chromatography, use methanol-water gradient elution, collect the cut E-2 that methanol-water ratio is 5: 5, cut E-2 is separated 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-2-2400mg, cut E-2-2 is separated through preparation liquid phase HPLC, the methanol-water that the ratio of take is 4.3: 6.7 is moving phase, detect wavelength and be 254 and 325nm, flow velocity 8mL/min, the retention time of preparation liquid phase is 42.5min, separating obtained solution is dry, obtain the compounds of this invention 13.4mg.
The Structural Identification of embodiment 2 the compounds of this invention
Yellow unformed powder, Molish reacting positive.ESI-MS (positive) provides m/z749[M+Na]+; ESI-MS (negative) provides m/z725[M-H]-, prompting compound molecular weight is 726.HR-ESI-Q-TOF-MS provides m/z749.2369[M+Na]
+(calculated value is 749.2374), deterministic adduct molecule formula is C
33h
42o
18, calculating degree of unsaturation is 13.
The compounds of this invention
1h-NMR (600MHz, in CD
3oD) collection of illustrative plates (seeing Fig. 1), low place shows one group of trans alkene hydrogen signal [δ 7.63 (1H, d, J=15.9Hz, H-3 " '), 6.40 (1H, d, J=15.9Hz; H-2 " ')] and the fragrant proton signal of coupling mutually [δ 7.20 (1H, brs, H-9 " '), δ 7.07 (1H; brd, J=7.2Hz, H-5 " '), 6.80 (1H, d, J=7.8Hz, H-6 " ')] there are 1,3,4 trisubstituted benzene rings in prompting structure.High field region δ 3.88 (3H, s, 8 " '-OCH
3) locate to show a methoxyl group signal, in conjunction with
13c-NMR (150MHz, in CD
3oD) signal of (seeing Fig. 2): 127.6 (C-4 " '), δ 124.3 (C-5 " '), 116.5 (C-6 " '), 150.8 (C-7 " '), 149.4 (C-8 " '), 6 carbon signals on 111.7 (C-9 " ') phenyl ring.
In HMBC spectrum (seeing Fig. 3), can see alkene hydrogen proton signal H-3 " ' (δ 7.63) and C-4 " ' (δ 127.6), C-5 " ' (δ 124.3), 9 " ' (δ 111.7) and C-1 " ' (δ 169.6); H-2 " ' (δ 6.43) and C-1 " ' (δ 169.6) and C-4 " ' (δ 127.6) distant relation, the structural unit that contains an asafoetide acyl group in prompting molecule.Sugar anomeric proton signal [δ 4.71 (1H, d, J=7.8Hz, H-1 ') and 4.40 (1H, d, J=7.8Hz, H-1 ")], the configuration of two glucosyl residues of prompting is β type.In HMBC spectrum, relevant peaks H-6 ' (δ 4.11,3.76)/C-1 " (δ 105.0), H-1 " (δ 4.40)/C-6 ' (δ 70.2), pointing out 2 glucosyl groups is 1 → 6 connection, forms a gentiobiose base.
Remove 2 glucose saccharide residues, 1 C
6-C
3asafoetide acyl group fragment; in structure, also remain 10 carbon signals, comprise 1 methoxyl group (δ 51.7), 1 methylene radical (δ 61.5); (δ 153.4 for 5 methynes; 129.0,98.8,46.9; 36.8); and 3 quaternary carbon signals (wherein 1 ester carbonyl group carbon signal) (δ 169.6,144.8,112.3).Binding molecule formula and degree of unsaturation, the compounds of this invention is iridoid glycoside constituents.
?
1h-
1in H COSY spectrum (seeing Fig. 4), between H-1/H-9/H-5/H-6/H-7, there is obvious coherent signal, in conjunction with hsqc spectrum, release skeleton fragment A:C
1-C
9-C
5-C
6-C
7.
In HMBC spectrum (seeing Fig. 5), relevant peaks H-3/C-1,4,5,11 and H-7/C-5,9,10, built the aglycon of this compound: genipin (structure fragment B).Comprehensive above analysis, the compounds of this invention is for replacing the iridoid bioside that has asafoetide acyl group.The connection site of each structure fragment can be composed and be determined by HMBC.
HMBC amplifies in spectrum (seeing Fig. 6), the distant relation peak of H-1 (δ 5.13)/C-1 ' (δ 100.6) and H-1 ' (δ 4.71)/C-1 (δ 98.8), and prompting gentiobiose base is replaced in the C-1 position of aglycon; ' (δ 169.1) have distant relation to glycosyl H-6 " (δ 4.53,4.30) and carbonyl C-1 ", and prompting mustard acyl is connected to the C-6 " position of gentiobiose base.
Comprehensive above analysis is 6 by this compound identification " O-is trans-asafoetide acyl group genipin O-gentibioside.
Comprehensive HSQC (Fig. 7) and HMBC spectrum information (Fig. 8), carried out belonging to (table 1) to whole carbon signals of compound 2 and hydrogen signal.Through SciFinder Scholar network retrieval, do not find relevant report, show that the compounds of this invention is a new iridoid glycoside compounds.
The nuclear magnetic data of table 1 compound (deuterated methanol,
1h-NMR600MHz,
13c-NMR150MHz)
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 ℃, 5%CO
2.
2. principle and method
The cytotoxicity of 2.1 medicines detects
Adopted
(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 enters cell and produces measurable fluorescence and colour-change by cyclophorase reduction, for quantitative analysis and the vitro cytotoxicity research of cytoactive and cell proliferation.This mensuration is that the cell based on having metabolic activity converts reagent to the ability of fluorescence and colorimetric indicator, and impaired and non-activity cell has lower natural metabolic activity, and corresponding signal is lower.Therefore fluorescent signal is strong and weak, can reflect the height of cytoactive.
Method steps: Vero cell is inoculated in 96 porocyte culture plates, standby 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 ℃
reduction situation, exciting light 57Onm, 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, standby 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 ℃ 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 10.7, 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, by isopyknic ethyl acetate and propyl carbinol, extract respectively, 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 is separated through ODS column chromatography, use methanol-water gradient elution, collect the cut E-2 that methanol-water ratio is 5: 5, cut E-2 is separated 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 is separated 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), the methanol-water that the ratio of take is 4.3: 6.7 is moving phase, detect wavelength and be 254 and 325nm, flow velocity 8mL/min, the retention time in preparation liquid phase is 42.5min.
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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|---|---|---|---|
| CN201310145252.9A CN104098631A (en) | 2013-04-10 | 2013-04-10 | Iridoid glycoside compound, and preparation method and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1637009A (en) * | 2003-12-22 | 2005-07-13 | 罗何生 | Extraction process of total rehmannioside |
| 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 |
| CN1706860A (en) * | 2005-05-24 | 2005-12-14 | 中国人民解放军第二军医大学 | Process of preparing total iridoid glycoside with cape jasmine fruit |
| 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
- 2013-04-10 CN CN201310145252.9A patent/CN104098631A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1637009A (en) * | 2003-12-22 | 2005-07-13 | 罗何生 | Extraction process of total rehmannioside |
| 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 |
| CN1706860A (en) * | 2005-05-24 | 2005-12-14 | 中国人民解放军第二军医大学 | Process of preparing total iridoid glycoside with cape jasmine fruit |
| 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》 * |
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