CN102320947B - Polyphenol compound contained in tobacco, preparation method and application thereof - Google Patents
Polyphenol compound contained in tobacco, preparation method and application thereof Download PDFInfo
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- CN102320947B CN102320947B CN2011101445545A CN201110144554A CN102320947B CN 102320947 B CN102320947 B CN 102320947B CN 2011101445545 A CN2011101445545 A CN 2011101445545A CN 201110144554 A CN201110144554 A CN 201110144554A CN 102320947 B CN102320947 B CN 102320947B
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Abstract
The invention discloses a polyphenol compound (I) contained in tobacco, a preparation method and an application thereof. Tobacco samples are crushed and are extracted through ultrasound for three times by 95 percent ethanol, the extracting solutions are combined, filtered and concentrated into extract through pressure reduction, the extract is primarily separated through silica gel column chromatography, then, the high-efficiency semi-preparative liquid chromatography is adopted for further separation, and the required novel compound is obtained. The antioxidant activity and the anti-human immunodeficiency virus (HIV)-1 activity screening are carried out on the compound, and experiment results show that the compound has higher oxidation resistance and anti-HIV-1 activity.
Description
Technical field
The invention belongs to the tobacco chemistry field, more particularly, the present invention relates to contained phenolic compound of a kind of new tobacco and preparation method thereof, simultaneously, also relate to its application in anti-oxidant and anti-HIV-1.
Background technology
Tobacco is maximum a kind of containing chemical substance in each kind of plant of being familiar with of the mankind, and through the research of decades, people identify that monomer chemical substance out just surpasses kind more than 3000 at present from tobacco, and also have many compositions not yet to identify out.Tobacco, except being mainly used in the cigarette smoking purposes, also can therefrom be extracted the multiple chemical composition that the value utilized is arranged, and therefrom finds that there is the guiding compound of value of exploiting and utilizing.Therefore, except as cigarette consumption, the comprehensive utilization of strengthening tobacco and waste thereof is significant.
Polyphenol (Polyphenol) is that a class extensively is present in the polyphenol compound in plant materials, and the content in vascular plant is only second to Mierocrystalline cellulose, hemicellulose and xylogen, extensively is present in the skin, root, leaf, fruit of plant, and content can reach 20%.Polyphenolic compound has potential promotion health effect, as: anti-oxidant, the Scavenger of ROS free radical, blocking-up lipid peroxidation process, improve the activity of human body endoenzyme, thereby play the effect of anti-mutation, anticancer; Prevent and treat the disease that hyperlipidaemia causes; Hypoglycemic; Antithrombotic; Improve the effect of the comprehensive immunological competence of human body; Although polyphenol extensively is present in plant food, its content and structure of in different plants has very big-difference.The present invention separates and has obtained a kind of anti-oxidant and new polyphenolic compound Anti-HIV-1 Active of having from tobacco, and this compound it is not yet seen relevant report.
Summary of the invention
The object of the present invention is to provide a kind of new polyphenolic compound.
Another object of the present invention is to provide a kind of preparation method of described compound.
Further aim of the present invention is to provide described compound in the application in anti-oxidant and anti-HIV-1.
Purpose of the present invention is achieved by following technical proposals.
* except as otherwise noted, the percentage ratio adopted in the present invention is mass percent.
A. the present invention isolates a kind of new polyphenolic compound from tobacco, and this compound has following structural formula
The called after of this compound: tobacco phenol C (Tobphenol C).
B. the invention provides a kind of preparation method of described compound, the method adopts following steps:
(1) tobacco sample divides and uses supersound extraction 3 times with 95% ethanol after pulverizing, and united extraction liquid concentrating under reduced pressure become medicinal extract;
(2) medicinal extract just divides with silica gel column chromatography, then adopts high performance liquid phase half preparative chromatography further to separate, and obtains this required new compound.
C. the present invention has carried out anti-oxidant activity detection and anti-HIV-1 effect test to described new compound, and compound demonstrates good anti-oxidant activity and anti-HIV-1 effect.
The invention provides a kind of new contained polyphenolic compound of tobacco---tobacco phenol C, and disclosed its good pharmaceutical use had by test, for the comprehensive utilization of tobacco provides useful new way.
The accompanying drawing explanation
The high resolution mass spectrum that Fig. 1 is compound (HRESIMS);
The proton nmr spectra that Fig. 2 is compound (
1H NMR);
The nucleus magnetic resonance charcoal spectrum that Fig. 3 is compound (
13C NMR);
The HSQC Correlated Spectroscopy that Fig. 4 is compound;
Fig. 5 is compound
1H-
1H COSY Correlated Spectroscopy;
The HMBC Correlated Spectroscopy that Fig. 6 is compound;
The ROESY Correlated Spectroscopy that Fig. 7 is compound;
The CD spectrum that Fig. 8 is compound.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1
---the preparation of compound
Tobacco sample is adopted in Yunnan Yuxi, and kind is the large gold dollar of safflower.Tobacco sample is sampled to 1.2 kg and be crushed to 30 orders, supersound extraction 3 times of the ethanol with 95%, extracting solution merges, and filters, and concentrating under reduced pressure becomes medicinal extract, obtains medicinal extract 65.8 g.Medicinal extract is with after appropriate dissolve with methanol, with the thick silica gel of 100 g (80-100 order), mixing sample, 1.5 kg silica gel (160～200 order) dress post carries out silica gel column chromatography, chloroform: acetone (1:0 → 0:1) gradient elution, the TLC monitoring merges identical part, obtain 8 part (pure chloroforms, chloroform-acetone 20:1, chloroform-acetone 9:1, chloroform-acetone 8:2, chloroform-acetone 3:2, chloroform-acetone 1:1, chloroform-acetone 1:2, pure acetone), wherein chloroform-acetone (1:1) wash-out part 6.5 g separate with prompt logical sequence 1,100 half preparative high-performance liquid chromatographics of peace, 30% the methyl alcohol of take is moving phase, Zorbax SB-C
18(21.2 * 250 mm, 5 μ m) preparative column is stationary phase, flow velocity is 20 ml/min, and it is 254 nm that UV-detector detects wavelength, each sample introduction 200 μ L, collect the chromatographic peak of 14.3 min, repeatedly cumulative rear evaporate to dryness, again use pure dissolve with methanol, then take methyl alcohol as moving phase, separate with Sephadex LH-20 gel filtration chromatography, get final product to obtain this new compound.
---the evaluation of compound
This patent compound is white powder; UV spectrum (solvent is methyl alcohol), λ
max(log
ε) λ
max(log
ε) 320 (3.12), 288 (4.02), 210 (4.67) nm; Infrared spectra (pressing potassium bromide troche) ν
max3482,2955,2870,1721,1625,1526,1475,1442,1268,956 cm
-1HRESIMS (figure-1) provides quasi-molecular ion peak m/z 415.1360 [M+Na]
+(calculated value 415.1369).In conjunction with
1H and
13C NMR spectrum provides a molecular formula C
20H
24O
8, degree of unsaturation is 9.From
1H and
13CNMR spectrum (figure-2 and scheme-3, attribution data is in Table-1) signal can find out from
1H and
13The CNMR spectrum can be seen in compound 2 phenyl ring (comprises 6 two key methine carbons, δ
C111.7,116.1,120.6,111.9,114.8,123.1); Also has in addition 1 carbonyl (δ
C198.1), the methyne (δ of 2 oxidations
C73.3,86.1), 1 methylene radical (δ
C42.1), the methylene radical (δ of 2 oxidations
C58.4,61.8), 2 methoxyl group (δ
C55.8,55.7).By H-7/H-8/H-9's
1H-
1H COSY relevant (figure-5) can infer that C-7 and C-8, C-8 and C-9 are connected, and by H-7 and C-1, C-2 and C-6, exists HMBC relevant (figure-6) can infer that C-7 is connected with phenyl ring C-1 place; By H-8 ' and H-9 ', existed
1H-
1H COSY is relevant, and H-8 ', H-9 ' exist with C-7 ' that HMBC is relevant can infer that C-7 ' is connected with C-8 ', and C-8 ' is connected with C-9 '; The relevant susceptible of proof C-7 ' of HMBC that passes through H-2 ', H-6 ' and C-7 ' locates to be connected with another phenyl ring C-1 '; Chemical displacement value (the δ of and C-4 ' relevant according to the HMBC of C-8 and H-4 '
C154.2) susceptible of proof C-8 is connected with C-4 ' by Sauerstoffatom; Have with C-3, C-3 ' respectively from two methoxyl group signals that HMBC is relevant infers that two methoxyl groups are substituted in respectively C-3 and C-3 ' position, exist the relevant susceptible of proof phenolic hydroxyl group of HMBC to be substituted in the C-4 position by fragrant hydroxyl signal with C-3, C-4, C-5; But the two dimensional structure of deterministic compound thus.Two chiral carbon (C-7 and C-8) are arranged in compound, according to C-7 position in compound
JValue is that 7.1 deducibility compounds are erythro form, the absolute configuration of compound can be determined by its ROESY (figure-7) and CD (figure-8) spectrum, at 239 nm places, positive Cotton effect is arranged according to its CD spectrum, and H-7 and H-8, H-8 and H-2, between H-8 and H-6, exist obvious ROESY relevant, the susceptible of proof compound is 7
S, 8
S-configuration. the structure of this patent compound is determined thus.
Table-1 compound
1
H and
13
C NMR data (C
5
ND
5
)
---compound with oxidation resistance is active to be detected
Anti-oxidant activity means with the size of scavenging ability of DPPH free radical; Take 50 μ g/mL as primary dcreening operation concentration, measure the activity that it removes fat free love base DPPH.Get costar 96 orifice plates, add freshly prepared DPPH ethanolic soln (6.5 * 10
5Mol/L) 190 μ L/ holes, add testing sample l0 μ L/ hole, and blank well adds l0 μ L physiological saline, fully mix, by standing 30 minutes of lucifuge under room temperature after shrouding film shrouding, measure each hole absorbance on determinator on the UV2401 spectrophotometer, measuring wavelength is 517 nm; Sample is calculated as follows fat free love base DPPH clearance rate:
DPPH clearance rate (%)=(A
Blank-A
Sample)/A
Blank* 100%
A
Blank: blank group absorbance; A
Sample: add the sample sets absorbance.
Sample detects for parallel 5 times, and calculating median elimination concentration IC50 measurement result is 2.23 μ g/L, shows that compound has good anti-oxidant activity.
Embodiment 4
---HIV (human immunodeficiency virus)-resistant activity to the compounds of this invention detects
(1) HIV-1 infectious titration: by the described method improvement of Johnson & Byington, carry out titration; Calculate viral TCID by the Reed&Muench method
50(50% Tissue Culture Infection Dose).
(2) the cytotoxicity inspection of sample to the C8166 host cell: 4 * 10
5/ ml C8166 cell suspension 100 ul mix with testing compound solution, establish three repeating holes.The control wells that does not contain compound, 37 ℃ of temperature, 5% CO are set simultaneously
2Cultivate three days, adopt MTT colorimetric determination cytotoxicity.ELx800 ELISA instrument is measured the OD value, and the mensuration wavelength is 595nm, and reference wavelength is 630 nm.Calculate CC
50Value (50% Cytotoxic Concentration), the compound concentration while 50% normal T lymphocyte series C8166 being produced to toxicity.
(3) sample is to HIV-1
IIIBInduce the inhibition test of C8166 cytopathy (CPE): by 8 * 10
5/ mL C8166 cell 50 μ L/ holes are inoculated on the 96 porocyte culture plates that contain 100 μ L/ hole doubling dilution compounds, then add the HIV-1 of 50 μ L
IIIBDilution supernatant (M.O.I. 0.0016).If three repeating holes.The normal cell control wells that does not contain compound is set simultaneously.37 ℃, 5% CO
2Cultivate three days, under inverted microscope, (100 *) count plasmodial formation.EC
50(50% Effective Concentration) is the compound concentration while suppressing Syncytium formation 50%.
(4) the provide protection test of sample to the HIV cells infected: by 8 * 10
5/ ml MT
4 Cell 50 μ l/ holes are inoculated on the 96 porocyte culture plates that contain 100 μ l/ hole doubling dilution compounds, and half hole of culture plate adds the HIV-1 of 50 μ l
IIIBDilution (M.O.I.0.006), second half hole adds 50 μ l substratum.2 repeating holes of each concentration gradient arrange control wells and the blank hole that does not contain compound, 37 simultaneously
0C, 5% CO
2Cultivate, within the 3rd day, 100 μ l fresh cultures are added in every hole, within the 5th day or the 6th day, adopt MTT colorimetric determination cell survival rate.ELx800 ELISA instrument is measured the OD value, and the mensuration wavelength is 595nm, and reference wavelength is 630 nm.Calculate compound to Normocellular toxicity with to HIV-1 with formula
IIIBThe provide protection of cells infected.
(5) calculation formula: draw dose response curve according to experimental result, by the Reed&Muench method, calculate the 50% effective concentration (EC that compound suppresses virus
50), 50% cell growth inhibiting concentration (CC
50) and the therapeutic index TI value (Therapeutic index) of Anti-HIV-1 Active be: TI=CC
50/ EC
50.
Growth of Cells survival rate (%)=experimental port OD value/control wells OD value * 100
Inhibitory rate of cell growth (%)=(1-experimental port OD value/control wells OD value) * 100
The cytopathogenic inhibiting rate of HIV-1 (%)=(1-experimental port synplasm number/control wells synplasm number) * 100
The protection ratio of cells infected (%)=(experimental port OD value-positive control hole OD value)/(negative control hole OD value-positive control hole OD value) * 100
(6) experimental result: experimental result clearly illustrates that, this compound demonstrates certain Anti-HIV-1 Active, and its therapeutic index is 128.5, and disclosed compound of the present invention has good application prospect in the medicine of preparation AIDS resisting.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (1)
1. the compound with following structural formula is in the application for the preparation of in anti-HIV-1 medicines.
The called after of this compound: tobacco phenol C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101445545A CN102320947B (en) | 2011-05-31 | 2011-05-31 | Polyphenol compound contained in tobacco, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101445545A CN102320947B (en) | 2011-05-31 | 2011-05-31 | Polyphenol compound contained in tobacco, preparation method and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN102320947A CN102320947A (en) | 2012-01-18 |
| CN102320947B true CN102320947B (en) | 2013-12-04 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102675076B (en) * | 2012-04-24 | 2014-02-26 | 云南烟草科学研究院 | Polyphenol active compound in aromatic tobacco and preparation method and application thereof |
| CN102675105A (en) * | 2012-04-24 | 2012-09-19 | 云南烟草科学研究院 | Phenolic compound in tobacco as well as preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005002052A (en) * | 2003-06-12 | 2005-01-06 | Aotsubu:Kk | Extract of cruciferae plants and its use |
| CN101973883A (en) * | 2010-09-26 | 2011-02-16 | 云南烟草科学研究院 | Phenolic compound in tobaccos and preparation method and application thereof |
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2011
- 2011-05-31 CN CN2011101445545A patent/CN102320947B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005002052A (en) * | 2003-06-12 | 2005-01-06 | Aotsubu:Kk | Extract of cruciferae plants and its use |
| CN101973883A (en) * | 2010-09-26 | 2011-02-16 | 云南烟草科学研究院 | Phenolic compound in tobaccos and preparation method and application thereof |
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| CN102320947A (en) | 2012-01-18 |
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Granted publication date: 20131204 Termination date: 20140531 |