CN105315187A - Guaiane-type sesquiterpenoids compound, and preparation method and application thereof - Google Patents
Guaiane-type sesquiterpenoids compound, and preparation method and application thereof Download PDFInfo
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- CN105315187A CN105315187A CN201410392171.3A CN201410392171A CN105315187A CN 105315187 A CN105315187 A CN 105315187A CN 201410392171 A CN201410392171 A CN 201410392171A CN 105315187 A CN105315187 A CN 105315187A
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Abstract
The invention discloses a guaiane-type sesquiterpenoids compound, and a preparation method and an application thereof. The preparation method comprises the following steps: (1) a Reduning injection finished product is subjected to HP-20 macroporous resin column chromatographic separation; gradient elution is carried out sequentially with water, 30% ethanol and 90-100% ethanol; elution liquids are respectively collected; and concentration is carried out, such that a 90-100% ethanol elution fraction is obtained; (2) the 90-100% ethanol elution fraction is sequentially subjected to silica gel column chromatographic separation, Sephadex LH-20 column chromatographic separation, ODS column chromatographic separation and semi-preparative liquid-phase HPLC separation, such that the novel compound provided by the invention is obtained. 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 art, pockwood alkane type sesquiterpene compound particularly 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 pockwood 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 pockwood alkane type sesquiterpene compound, its structure as indicated with 1:
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 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, use chloroform-methanol gradient elution, with elution 3 ~ 5BV that chloroform-methanol volume ratio is 95: 5, elutriant merges, be labeled as fraction A, fraction A is through SephadexLH-20 pillar layer separation, chloroform-methanol equal-volume, isocratic elution, after removing pigment zone, collect cut, be labeled as A-2, fraction A-2 is through ODS pillar layer separation, use methanol-water gradient elution, wherein methanol concentration is 50% ~ 70%, collect the fraction A-2-2 that methanol-water volume ratio is 3: 2, fraction A-2-2 is through partly preparing liquid phase separation, obtain the compounds of this invention.
In above-mentioned preparation method, step (2) described semi-preparative liquid chromatography, with ratio be the acetonitrile-water of 25: 75 for moving phase, determined wavelength is 208 and 220nm, flow velocity 4mL/min, and column temperature is normal temperature.In half preparation liquid phase, the retention time of the compounds of this invention is respectively 28.4min.
Contriver by physico-chemical property and the Modern spectroscopy section of learning to do (UV, IR, MS, CD,
1h-NMR,
13c-NMR with 2D-NMR) carry out Structural Identification to being separated the compound obtained, be confirmed that it is structure pockwood alkane type sesquiterpene compound as shown in Equation 1.
Another object of the present invention is to provide the application of compound shown in a kind of Fig. 1 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 Fig. 1.
Present invention also offers a kind of pharmaceutical composition for the treatment of hand foot mouth disease, the above-mentioned Fig. 1 compound 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 1
Fig. 2 is compound 1
1h-NMR composes
Fig. 3 is compound 1
13c-NMR spectrum and DEPT-135 spectrum
Fig. 4 is the structure fragment a of compound 1, b and c
Fig. 5 is the HMBC spectrum of compound 1
Fig. 6 is compound 1
1h-
1hCOSY composes
Fig. 7 is the NOESY spectrum of compound 1
Fig. 8 is the CD spectrum of compound 1
Fig. 9 is the hsqc spectrum of compound 1
Figure 10 is the structural formula of compound 1
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 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, use chloroform-methanol gradient elution, with elution 3 ~ 5BV that chloroform-methanol volume ratio is 95: 5, elutriant merges, be labeled as fraction A 7.8g, fraction A is through SephadexLH-20 pillar layer separation, chloroform-methanol equal-volume, isocratic elution, after removing pigment zone, collect cut, be labeled as A-25.4g, fraction A-2 is through ODS pillar layer separation, use methanol-water gradient elution, wherein methanol concentration is 50% ~ 70%, collect the fraction A-2-285mg that methanol-water volume ratio is 3: 2, fraction A-2-2 is through partly preparing liquid phase separation, obtain the compounds of this invention.
Half preparation liquid phase HPLC is separated, and the acetonitrile-water with 25: 75 is moving phase, and determined wavelength is 208 and 220nm, flow velocity 4mL/min, and in half preparation liquid phase, the retention time of the compounds of this invention is respectively 28.4min.Separating obtained solution is dry, obtains the compounds of this invention 14.5mg.
The Structural Identification of embodiment 2 the compounds of this invention
Compound 1 is yellow indefiniteness solid.ESI-MS (positive) provides m/z323 [M+Na]
+; ESI-MS (negative) provides m/z299 [M-H]
-, 345 [M+HCOO]
-, prompting compound molecular weight is 300.HR-ESI-Q-TOF-MS provides m/z301.1646 [M+H]
+(calculated value is 301.1651) (see Fig. 1) deterministic adduct molecule formula is C
15h
24o
6, calculating degree of unsaturation is 5.
Compound 1
1h-NMR (600MHz, inCD
3oD) high field region of (see Fig. 2) can observe 4 obvious methyl signals, and wherein 3 methyl are unimodal [δ 1.36 (3H, s), 1.38 (3H, s), 1.76 (3H, s)], show that it is directly connected with quaternary carbon; Other 1 methyl signals is d peak [δ 1.06 (3H, d, J=7.1Hz)], shows that it is directly connected with tertiary carbon.
13c-NMR (100MHz, inCD3OD) shows 15 carbon signals altogether in conjunction with DEPT-135 spectrum (see Fig. 3), comprises 4 methyl (δ 26.4,25.4,18.5,8.9), 3 methylene radical (δ 40.7,37.5,29.8), 2 methynes (δ 77.4,34.1), and 6 quaternary carbon signals [1 carbonyl (δ 209.7), 3 company oxygen SP
3hydridization quaternary carbon (δ 94.0,79.1,79.0), 1 replaces double bond [(δ 166.3,144.6)] to four.Comprehensive above NMR data and degree of unsaturation information, can infer that the compounds of this invention may be 1 sesquiterpenoids, containing 4 methoxyl groups, 2 rings, 1 ketone carbonyl and 1 double bond in structure.According to the hsqc spectrum (see Fig. 9) of compound, the hydrocarbon signal of compound is belonged to.
As shown: according to HMBC (Fig. 5) relevant peaks H-15/C-3,4,5 and H-2/C-1,3,5, release the structure fragment a (Fig. 4) of α, β-unsaturated cyclopentanone.
1h-
1in HCOSY experiment (see Fig. 6), visible H-8/H-9/H-10, H-10/CH
3the relevant peaks of-14, in conjunction with H-6/C-1, the HMBC of 4,5,7 is long-range relevant; CH
3-14/C-1, the HMBC of 10,9 and H-8/C-7,9,10 is long-range relevant, builds the structure fragment b (Fig. 4) of suberane.Two unimodal methyl CH
3-12 and CH
3-13 all to have HMBC long-range relevant to C-7 and C-11, infers in structure containing fragment c (Fig. 4).In 3 structure fragments estimated, we can find 1, and 5,7 carbon atoms are public carbon, thus to connect be the sesquiterpene skeleton of 1 guainane type, binding molecule formula C
15h
24o
6, determine in structure, also to there are 3 hydroxyls and 1 hydroperoxyl radical is δ 94.0 according to the chemical shift of 1 carbon, determine that the link position of hydroperoxyl radical is 1.To sum up, the two dimensional structure of deterministic compound is 7,8,11-trihydroxy-1-hydroperoxy-4-guaien-3-one.
The relative configuration of this compound is determined by NOESY (see Fig. 7) experiment, can see 10-H/8-H, 12-CH in NOE spectrum
3relevant, show 8-OH and 10-CH
3be in the same side (β) of molecule, C-6 (δ 2.22)/8-H, 12-CH can be observed simultaneously
3; C-6 (δ 2.76)/13-CH
3(δ 1.38), 15-CH
3(δ 1.76) is correlated with.By consulting literatures, we find that the substituting group many places of C-7 position in most guainane type sesquiterpenoid are in β key.In the CD spectrum of compound 1 (see Fig. 8), 249.2nm (Δ ε 0.99) shows positive Cotton effect, 312.4nm (Δ ε-1.95) shows a negative Cotton effect, and compare with document, the C-1 position of deterministic compound 1 has S configuration.
Comprehensive above analyzing, is (1S, 7R, 8R, 10S)-7,8,11-trihydroxy--1-hydroperoxyl radical-4-alkene-3-ketone by the Structural Identification of compound 1.All hydrocarbon signals assignment are see table 1.Through SciFinderScholar network retrieval, find that compound 1 has no bibliographical information, show that it is a new sesquiterpenoids.
Table 1 compound 1 nuclear magnetic data
(deuterated methanol,
1h-NMR600MHz,
13c-NMR150MHz)
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, laboratory passage is preserved.
1.2 cell model monkey-kidney cells system Vero, laboratory passage is preserved.Culture condition: DMEM+10% foetal calf serum, 37 DEG C, 5%CO
2.
2. principle and method
The cytotoxicity of 2.1 medicines detects
Have employed
(Invitrogen) test kit 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) 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, 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 examine under a microscope most cells become justify and have cracking, also have overt toxicity when (100 μMs), 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 (7)
1. a pockwood alkane type sesquiterpene compound, its structure as indicated with 1:
。
2. prepare a method for compound according to claim 1, it is characterized in that, comprise 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, use chloroform-methanol gradient elution, with elution 3 ~ 5BV that chloroform-methanol volume ratio is 95: 5, elutriant merges, be labeled as fraction A, fraction A is through SephadexLH-20 pillar layer separation, chloroform-methanol equal-volume, isocratic elution, after removing pigment zone, collect cut, be labeled as A-2, fraction A-2 is through ODS pillar layer separation, use methanol-water gradient elution, wherein methanol concentration is 50% ~ 70%, collect the fraction A-2-2 that methanol-water volume ratio is 3: 2, fraction A-2-2 is through partly preparing liquid phase separation, obtain the compounds of this invention.
3. preparation method according to claim 2, it is characterized in that, the described semi-preparative liquid chromatography condition of step (2) comprises: with volume ratio be the acetonitrile-water of 25: 75 for moving phase, determined wavelength is 208 and 220nm, flow velocity 4mL/min, column temperature is normal temperature.
4. preparation method as claimed in claim 2, is characterized in that, in half preparation liquid phase, the retention time of the compounds of this invention is respectively 28.4min.
5. the application of compound described in claim 1 in preparation treatment hand foot mouth disease medicine.
6. a pharmaceutical composition, is characterized in that, comprises compound described in claim 1.
7. 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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112047826A (en) * | 2019-06-05 | 2020-12-08 | 江苏康缘药业股份有限公司 | Guaiane type sesquiterpenoids and preparation method and application thereof |
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| CN101279964A (en) * | 2008-02-29 | 2008-10-08 | 中国药科大学 | Guaiane type sesquiterpenes, preparation and medical use thereof |
| CN103242275A (en) * | 2013-05-13 | 2013-08-14 | 沈阳药科大学 | Sesquiterpenoids in zedoary as well as preparation method and application of sesquiterpenoids |
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2014
- 2014-08-03 CN CN201410392171.3A patent/CN105315187A/en active Pending
Patent Citations (2)
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| CN101279964A (en) * | 2008-02-29 | 2008-10-08 | 中国药科大学 | Guaiane type sesquiterpenes, preparation and medical use thereof |
| CN103242275A (en) * | 2013-05-13 | 2013-08-14 | 沈阳药科大学 | Sesquiterpenoids in zedoary as well as preparation method and application of sesquiterpenoids |
Non-Patent Citations (5)
| Title |
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| A novel systems pharmacology model for herbal medicine injection: a case using reduning injection;Haixing Yang, et al.;《BMC Complementary and Alternative Medicine》;20141104;第14卷;1-19 * |
| HAI-BO LI, ET AL.: "Two new sesquiterpenoids from Artemisia annua", 《MAGN. RESON. CHEM.》 * |
| HAIXING YANG, ET AL.: "A novel systems pharmacology model for herbal medicine injection: a case using reduning injection", 《BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112047826A (en) * | 2019-06-05 | 2020-12-08 | 江苏康缘药业股份有限公司 | Guaiane type sesquiterpenoids and preparation method and application thereof |
| CN112047826B (en) * | 2019-06-05 | 2022-02-08 | 江苏康缘药业股份有限公司 | Guaiane type sesquiterpenoids and preparation method and application thereof |
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Application publication date: 20160210 |