CN105461545A - Cycloreversed cadinane sesquiterpene compound, and preparation method and application thereof - Google Patents
Cycloreversed cadinane sesquiterpene compound, and preparation method and application thereof Download PDFInfo
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- CN105461545A CN105461545A CN201410550172.6A CN201410550172A CN105461545A CN 105461545 A CN105461545 A CN 105461545A CN 201410550172 A CN201410550172 A CN 201410550172A CN 105461545 A CN105461545 A CN 105461545A
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Abstract
The invention discloses a cycloreversed cadinane sesquiterpene compound, and a preparation method and an application thereof. The preparation method comprises the following steps: 1, taking a finished Reduning injection product, carrying out HP-20 macroporous adsorption resin column chromatography separation, sequentially carrying out gradient elution with water, 30% ethanol and 90-100% ethanol, respectively collecting all eluates, and concentrating to obtain a 90-100% ethanol elution position; 2, sequentially carrying out silica gel column chromatography, Sephadex LH-20 column chromatography and ODS column chromatography separation on the 90-100% ethanol elution position, and carrying out semi-preparative liquid phase HPLC separation to obtain the new compound. The compound can be used for treating the hand-foot-and-mouth disease.
Description
Technical field
The present invention relates to medical art, particularly driffractive ring needle juniper alkane type sesquiterpene compound extracted from existing Chinese patent medicine preparation and its preparation method and application.
Background technology
Former Chinese medicine two kind new medicine that Reduning injection (traditional Chinese medicines accurate word Z20050217) is Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov's independent research, 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 caused by affection of exogenous wind-heat, and its effect is rapid, Be very effective.
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 finds in Reduning injection first and the new chemical composition identified, and find that this compound is new compound through Scifinderscholar retrieval.
Summary of the invention
The present invention prepares and extracts one and have bioactive needle juniper alkane type sesquiterpene compound from Reduning injection, and provides its application in preparation treatment hand foot mouth disease medicine.
Specifically, the invention provides a kind of needle juniper alkane type sesquiterpene compound, its structure is such as formula shown in I:
Present invention also offers the preparation method of above-claimed cpd, comprise the steps:
(1) Reduning injection finished product is got, be separated through HP-20 macroporous adsorbent resin chromatography post, successively with water, 30% ethanol, 90 ~ 100% ethanol gradient elutions, collect each elutriant respectively, be evaporated to without alcohol taste, obtain water elution position, 30% alcohol elution, 90 ~ 100% alcohol elutions;
(2) 90 ~ 100% alcohol elutions of step (1) are got, be separated through silica gel column chromatography, chloroform-methanol gradient elution, collect chloroform-methanol volume ratio be 9: 1 elutriant wash, the fraction B of concentrating under reduced pressure, fraction B is through SephadexLH-20 pillar layer separation, chloroform-methanol isocratic elution, cut is collected after removing pigment, be labeled as B-2, fraction B-2 is through ODS pillar layer separation, and methanol-water gradient elution collects the fraction B-2-2 that methanol-water volume ratio is 3: 2, fraction B-2-2 is separated through half preparation liquid phase HPLC, obtains compound of the present invention.
In above-mentioned preparation method, step (2) described semi-preparative liquid chromatography, with ratio be the methanol-water-formic acid of 55: 45: 0.5 for moving phase, determined wavelength is 208 and 220nm, flow velocity 4mL/min, the retention time in half preparation liquid phase is 37.4min.
Contriver by physico-chemical property and the Modern spectroscopy section of learning to do (UV, IR, MS, CD,
1h-NMR,
13c-NMR, 2D-NMR and single-crystal x ray diffraction) carry out Structural Identification to being separated the compound obtained, be confirmed that it is structure such as formula the driffractive ring needle juniper alkane type sesquiterpene 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.
Another object of the present invention is to provide a kind of pharmaceutical composition, comprises compound shown in above-mentioned formula I.
Present invention also offers a kind of pharmaceutical composition for the treatment of hand foot mouth disease, the above-mentioned formula I containing treatment significant quantity and one or more pharmaceutically acceptable carriers.
Accompanying drawing explanation
Fig. 1 is the HR-ESI-Q-TOF-MS of compound
Fig. 2 is compound
1h-NMR composes
Fig. 3 is compound
13c-NMR spectrum and DEPT-135 spectrum
Fig. 4 is compound
1h-
1hCOSY composes
Fig. 5 is the hsqc spectrum of compound
Fig. 6 is the HMBC spectrum of compound
Fig. 7 is the NOESY spectrum of compound
Fig. 8 is the single-crystal x x ray diffration pattern x of compound
Fig. 9 is structural formula of compound
Embodiment
In order to make those skilled in the art understand technical scheme of the present invention better, 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) Reduning injection finished product is got, be separated through HP-20 macroporous adsorbent resin chromatography post, successively with water, 30% ethanol, 95% ethanol gradient elution, collect each elutriant respectively, be evaporated to without alcohol taste, obtain water elution position, 30% alcohol elution, 95% alcohol elution;
(2) 90 ~ 100% alcohol elutions of step (1) are got, be separated through silica gel column chromatography, chloroform-methanol gradient elution, collect chloroform-methanol volume ratio be 9: 1 elutriant concentrating under reduced pressure obtain fraction B, fraction B is through SephadexLH-20 pillar layer separation, chloroform-methanol isocratic elution, cut is collected after removing pigment, be labeled as B-2, fraction B-2 is through ODS pillar layer separation, use methanol-water gradient elution, collect the fraction B-2-2 that methanol-water volume ratio is 3: 2, amount to 105mg, fraction B-2-2 is separated through half preparation liquid phase HPLC, be that the methanol-water-formic acid of 55: 45: 0.5 is for moving phase with ratio, determined wavelength is 208 and 220nm, flow velocity 4mL/min, retention time in half preparation liquid phase is 37.4min.Separating obtained solution is dry, obtains compound 35.7mg of the present invention.
The Structural Identification of embodiment 2 the compounds of this invention
This compound is white, needle-shaped crystals.ESI-MS (positive) provides m/z587 [2M+Na]
+; ESI-MS (negative) provides 281 [M-H]
-, prompting compound molecular weight is 282.HR-ESI-Q/TOF-MS provides m/z305.1365 [M+Na]
+(calculated value is 305.1365) (see Fig. 1), deterministic adduct molecule formula is C
15h
22o
5, calculating degree of unsaturation is 5.
This compound
1h-NMR (400MHz, inCD
3oD) (see Fig. 2) spectrum shows 20 hydrogen signals altogether, low place shows 2 alkene Hydrogen Proton signals [δ 6.19 (1H, brs), 5.68 (1H, brs)], visible 1 the unimodal methyl signals [δ 2.12 (3H, s)] of high field region, shows that it is directly connected with quaternary carbon, 1 d peak methyl signals δ 0.93 (3H, d, J=6.4Hz), show that it is directly connected with tertiary carbon.
This compound
13c-NMR (100MHz, inCD
3oD) 15 carbon signals are shown altogether, comprising 4 quaternary carbon signals (3 carbonyl δ, 211.9,178.4 and 169.73,1 SP in conjunction with DEPT-135 spectrum (see Fig. 3)
2the quaternary carbon signal (δ 144.7) of hydridization, 2 methyl carbon signals (δ 29.9,20.0), 5 mesomethylene carbon signal (δ 125.8,38.7,36.3,33.6,24.7), wherein mesomethylene carbon signal δ 125.8 is the carbon signal of a terminal double link.Comprehensive above information, infers that compound is a sesquiterpenoids with 15 carbon atom skeletons, containing 1 ring in structure, and 1 double bond, 3 carbonyls.
This compound
1h-
1in HCOSY spectrum (see Fig. 4), observe between H-1/H-6/H-7/H-8/H-9/H-10 (H-14) and H-1/H-2/H-3 and there is obvious coherent signal, in conjunction with hsqc spectrum (see Fig. 5), in pushing-out structure, there is the structure A of a saturated six-ring.
In HMBC spectrum (see Fig. 6), relevant peaks H-13/C-7, C-11, C-12; H-7/C-11, C-12, C-13; H-15/C-3, C-4 and H-6/C-5 construct structure fragment B.Binding molecule formula and molecular weight, known 15 are connected hydroxyl with on 9.Thus the two dimensional structure of compound 15 is defined as 2-carboxy-4-methyl-α-methylene-3-(3-oxobutyl)-cyclohexaneaceticacid.
The relative configuration of this compound is determined by NOESY (see Fig. 7) experiment, can see that 1-H/7-H with 6-H/10-H is relevant in NOE spectrum, show that H-6 and H-10 is in the same side (β) of molecule, the absolute configuration of compound is determined by single-crystal x ray diffraction (see Fig. 8) technology simultaneously.
The Structural Identification of compound is (1S, 6R, 7R, 10R)-6-carboxy-10-methyl-α-methylene-1-(1-oxobutyl)-cyclohexaneaceticacid by comprehensive above analysis.All hydrocarbon signals assignment are see table 1.Through SciFinderScholar network retrieval, find that compound 1 has no bibliographical information, show that it is the needle juniper alkane type sesquiterpene compound (see Fig. 9) of a new driffractive ring.
The nuclear magnetic data of table 1 compound
The drug testing of embodiment 3 the compounds of this invention In Vitro Anti hand-foot-mouth disease EV 71 virus
1. material
1.1 strain hand-foot-mouth disease EV 71 virus, Chinese Academy of Sciences's Wuhan virus preservation of going down to posterity.
1.2 cell model monkey-kidney cells system Vero, laboratory passage is preserved.
Culture condition: DMEM+10% foetal calf serum (10% foetal calf serum, Tian Hang bio tech ltd, Zhejiang, lot number: 111030; DMEM substratum, Gibco company, lot number 1016022), 37 DEG C, 5%CO
2.
2. principle and method
The cytotoxicity of 2.1 medicines detects
Have employed
(Invitrogen) test kit (Invitrogen biotech firm, product batch number DAL1100) detection of drugs is to the toxic action of cell.
Experimental principle:
be a kind of oxidation-reduction indicator, absorbancy change and fluorescent signal can be produced according to metabolic activity.
soluble in water, its oxidised form enters after cell and produces measurable fluorescence and colour-change through cyclophorase reduction, for quantitative analysis and the vitro cytotoxicity research of cytoactive and cell proliferation.This mensuration is the ability based on the cell with metabolic activity, reagent being converted to fluorescence and colorimetric indicator, and it is active that impaired and non-activity cell has lower native metabolic, 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, for subsequent use after cell attachment.With cell maintenance medium (DMEM+5% serum) (DMEM substratum, Gibco company, lot number 1216960) by medicine from 2 times of continuous 3 times of gradient dilutions of initial concentration, 6 gradients, every concentration gradient single hole detects.Dosing adds after cultivating 48h
hatch 2h, fluoroscopic examination for 37 DEG C
reduction situation, exciting light 570nm, utilizing emitted light 595nm.
Cytoactive (%)=(sample well-blank)/(cell controls-blank) × 100%
2.2 medicines are to the inhibition test of EV71 virus
After the EV71 vero cells infection of reporter gene GFP, cells infected meeting expressing green fluorescent protein, by expressing the cell number of GFP green fluorescence at fluorescence microscopy Microscopic observation, just can reflect the proliferative conditions of EV71 virus.
Method steps:
Vero cell is inoculated in 96 porocyte culture plates, for subsequent use after cell attachment.Medicine is continuous 3 times of gradient dilutions 6 gradients from 4 times of test concentrations the highest; The medicine diluted is added in hand-hole, adds vial supernatant after 4h and infect, be placed in 37 DEG C of cell culture incubators and cultivate 24h, take pictures under fluorescent microscope, fluorocyte is counted.
Experiment is established without drug control hole (not adding medicine hole after virus infection), positive drug control hole (Guanidinium hydrochloride GuHCl).
Inhibiting rate (%)=(without drug control hole-sample well)/without drug control hole × 100%
3. result
3.1 drug samples detect to the toxicity detection of cell and to EV71 inhibit activities
Drug sample dilution solvent for use, the highest concentration of ordinary dissolution, the highest test concentrations, CC
50(half cytotoxic concentration), EC
50(medium effective concentration) and SI (selectivity index) are as shown in table 2.
Table 2 experimental result
4. conclusion
The compounds of this invention shows overt toxicity, and examine under a microscope most cells become justify and have cracking, also have overt toxicity when 100 μm of ol, its SI value is 10.3, and showing 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. a driffractive ring needle juniper alkane type sesquiterpene compound, its structure is such as formula shown in I:
2. the preparation method of compound described in claim 1, is characterized in that, comprises the following steps:
(1) Reduning injection finished product is got, be separated through HP-20 macroporous adsorbent resin column chromatography, successively with water, 30% ethanol, 90 ~ 100% ethanol gradient elutions, collect each elutriant respectively, be evaporated to without alcohol taste, obtain water elution position, 30% alcohol elution, 90 ~ 100% alcohol elutions;
(2) 90 ~ 100% alcohol elutions of step (1) are got, be separated through silica gel column chromatography, chloroform-methanol gradient elution, collect chloroform-methanol volume ratio be 9: 1 elutriant concentrating under reduced pressure obtain fraction B, fraction B is through SephadexLH-20 pillar layer separation, chloroform-methanol isocratic elution, cut is collected after removing pigment, be labeled as B-2, fraction B-2 is through ODS pillar layer separation, and methanol-water gradient elution, collects the fraction B-2-2 that methanol-water volume ratio is 3: 2, fraction B-2-2 is separated through half preparation liquid phase HPLC, obtains compound of the present invention.
3. preparation method according to claim 2, it is characterized in that, step (2) described semi-preparative liquid chromatography, be that the methanol-water-formic acid of 55: 45: 0.5 is for moving phase with ratio, determined wavelength is 208 and 220nm, flow velocity 4mL/min, the retention time in half preparation liquid phase is 37.4min.
4. the application of compound described in claim 1 in preparation treatment hand foot mouth disease medicine.
5. a pharmaceutical composition, is characterized in that, comprises compound described in claim 1.
6. be used for the treatment of a pharmaceutical composition for hand foot mouth disease, it is characterized in that, compound and pharmaceutically acceptable carrier described in the claim 1 containing treatment significant quantity.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410550172.6A CN105461545A (en) | 2014-10-03 | 2014-10-03 | Cycloreversed cadinane sesquiterpene compound, and preparation method and application thereof |
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|---|---|---|---|
| CN201410550172.6A CN105461545A (en) | 2014-10-03 | 2014-10-03 | Cycloreversed cadinane sesquiterpene compound, and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732255A (en) * | 2017-04-20 | 2018-11-02 | 江苏康缘药业股份有限公司 | From the assay method of the index component content of Artemisia annua in a kind of Reduning injection |
-
2014
- 2014-10-03 CN CN201410550172.6A patent/CN105461545A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 李海波: "热毒宁注射液药效物质基础研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732255A (en) * | 2017-04-20 | 2018-11-02 | 江苏康缘药业股份有限公司 | From the assay method of the index component content of Artemisia annua in a kind of Reduning injection |
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