CN105439859B - A kind of new monoterpenes compound and its preparation method and application - Google Patents
A kind of new monoterpenes compound and its preparation method and application Download PDFInfo
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- CN105439859B CN105439859B CN201510875223.7A CN201510875223A CN105439859B CN 105439859 B CN105439859 B CN 105439859B CN 201510875223 A CN201510875223 A CN 201510875223A CN 105439859 B CN105439859 B CN 105439859B
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- 0 CC(OC[C@](*(C1)(C2=S)C(C=C3*)=O)I123O)=O Chemical compound CC(OC[C@](*(C1)(C2=S)C(C=C3*)=O)I123O)=O 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
- C07C69/12—Acetic acid esters
- C07C69/16—Acetic acid esters of dihydroxylic compounds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C07—ORGANIC CHEMISTRY
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- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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Abstract
The invention discloses a kind of monoterpenes compound, structural formula isThe molecular formula of the compound is C12H18O4, degree of unsaturation 4, ultraviolet spectra shows the ultraviolet absorption maximum of the compound in 203nm.Invention further relates to the extraction separation method of the compound, is extracted using ethyl acetate, and extract liquor is separated, is purified, and obtains monomeric compound of the present invention.The active function of the Compounds Against fungi provides lead compound to develop antimycotic drug, is of great significance to the new application of exploitation natural products and autonomic drug.
Description
Technical field
The invention belongs to organic compound technical fields, and in particular to a kind of new monoterpenes compound and preparation method thereof
And application.
Background technology
Tibet Paeonia ludlowii (Paeonia ludlowii) be Ranunculaceae (Ranunculacea), Paeonia
(Paeonia), Section Moutan (Sect.Moutan DC.) plant.Only be grown in the Milin in China Tibet, Linzhi, Bomi, Chayu,
In the open river valley in the Yarlung Zangbo River of the 2500~3500m of height above sea level in the counties such as Longzi and hillside border shrubbery, quantity is few, and in by
Year reduces trend, belongs to the distinctive endangered protection plant in Tibet.It is recorded according to local local chronicle, Tibetan are before the centuries
Tibet Paeonia ludlowii root skin treatment gynecological disease, cardiovascular and cerebrovascular disease, dermatophytosis etc. are just used.
At present, there is correlative study to the medicinal effects of Paeonia ludlowii root skin, but due in Paeonia ludlowii root skin
Chemical composition is varied, yet there are no relevant report for the research of its active ingredient, applicant utilizes normal phase silicagel column color
The technologies such as spectrum, reverse phase (MCI-Gel) column chromatography, gel (Sephadex LH-20) column chromatography, high performance liquid chromatography are yellow to great Hua
The ethyl acetate extraction components of moutan root bark are separated, purified, and obtain monomeric compound 23, by ultraviolet spectra, infrared
Spectrum, mass spectrum, nuclear magnetic resoance spectrum simultaneously determine its structure with reference to document report, including monoterpenes compound 7, monoterpene glycosides
Compound 3, phenolic compound 8, phenol glycosides compound 3, alkaloid compound 1, triterpene compound 1,
Middle noval chemical compound 4, it is known that compound 19, it is isolated from the Paeonia ludlowii of Tibet for the first time.
The content of the invention
The present invention provides a kind of noval chemical compound of monoterpenes and its system by the further investigation to Paeonia ludlowii root skin
Preparation Method.
The present invention is realized especially by following technical scheme：
A kind of monoterpenes compound, structural formula is shown in formula I：
Compound described in Formulas I is colorless oil, is composed ([M+Na] by ESI-MS+M/z 249) and HR-ESI-MS spectrums ([M+
Na]+m/z 249.1101,calcd for C12H18O4Na, 249.1097) the molecular formula C of the compound, is determined12H18O4, no
Saturation degree is 4.Infrared spectrum (KBr tablettings) gives hydroxyl (3435cm-1) and carbonyl (1720cm-1) signal.Ultraviolet spectra is shown
Show the ultraviolet absorption maximum of the compound in 203nm.
The preparation method of the compound of formula I, specifically includes following steps：
1) after dry Tibet Paeonia ludlowii root skin is taken to crush, 95% ethyl alcohol is added in, room temperature extracts for 24 hours, repeats to filter,
50 DEG C are concentrated under reduced pressure, and using ethyl acetate into extraction, extract liquor under the conditions of 50 DEG C are concentrated under reduced pressure, obtains dry powder A；
2) by dry powder A and polyamide in mass ratio 1:4~5 ratio mixing, utilizes the methanol-water that volumetric concentration is 40%
Solution affords component B；
3) component B 120mL methanol is dissolved, silica gel mixed sample treats that solvent volatilizees completely, carries out silica gel column chromatography, uses stone
The mixed liquor that oily ether/isopropanol volume ratio is 10 ︰ 1 elutes, and obtains component C；
4) component C is repeated into silica gel column chromatography, is eluted with petroleum ether and isopropanol volume ratio for the mixed liquor of 9 ︰ 1,
Obtain component D；
5) component D is continued through into gel column chromatography, methanol obtains Formulas I monomeric compound as eluent.
The mass volume ratio of root skin and 95% ethyl alcohol is 1 in preparation method step (1) of the present invention:10.
Dry powder A and polyamide preferably in mass ratio 1 in preparation method step (2) of the present invention:4.2 ratio mixing.
The present invention also provides application of the compound of formula I as antifungal drug.
The drug can be processed into any pharmaceutically acceptable dosage form on demand, wherein more preferably dosage form is piece
Agent, capsule, granule, syrup, freeze drying powder injection or parenteral solution.It is prepared can be public by common those skilled in the art institute
Prepared by the processing method known, i.e., after active material compound of formula I is mixed with liquid flux or solid carrier, add filling
Agent, adhesive, wetting agent, disintegrant, sorbefacient, solvent, surfactant, flavouring agent, preservative, lubricant, sweet taste
One or more of agent or pigment.
Drug of the present invention can be used through dilution or directly before use by user.
Beneficial effects of the present invention are：The present invention extracts from the Paeonia ludlowii root skin of Tibet and isolates a kind of new chemical combination
Object, the extraction is easy, quick with separation method, efficient.The compound has antimycotic active function simultaneously, compared to Tibet
The pharmacological action of Paeonia ludlowii root skin more precisely, solves complex chemical composition in the Paeonia ludlowii root skin of Tibet, and quality is difficult to
The problem of control.
Specific embodiment
With reference to embodiment, the present invention is described further, as described below, is only the preferable implementation to the present invention
Example, not limits the present invention, any person skilled in the art is possibly also with the disclosure above
Technology contents be changed to the equivalent embodiment changed on an equal basis.It is every without departing from the present invention program content, according to the present invention
Any simple modification for being made to following embodiment of technical spirit or equivalent variations, all fall in protection scope of the present invention.
Embodiment 1
Dry Tibet Paeonia ludlowii root skin is taken, 10kg is accurately weighed after crushing, according to root skin and solvent 1:10(W/V)
Ratio add in 95% ethyl alcohol, room temperature is extracted for 24 hours, filtered, and in triplicate, 50 DEG C are concentrated under reduced pressure, and then use total extract
Ethyl acetate extracts, and extract liquor is concentrated under reduced pressure under the conditions of 50 DEG C, obtains the component dry powder A of 356g.
Dry powder A is weighed into 356g and mixes sample with 1.5kg polyamide, carries out medium pressure liquid chromatography, reverse phase column material is
MCI-Gel affords 57g component B using the methanol aqueous solution that volumetric concentration is 40%, is dissolved with 120mL methanol, silicon
Glue mixes sample, treats that solvent volatilizees completely, carries out silica gel column chromatography, is eluted with the mixed liquor that petroleum ether/isopropanol volume ratio is 10 ︰ 1,
Component C is obtained, component C is repeated into silica gel column chromatography, is eluted with petroleum ether and isopropanol volume ratio for the mixed liquor of 9 ︰ 1,
Component D is obtained, component D is continued through into gel column chromatography, methanol obtains Formulas I monomeric compound 7mg as eluent.
2 monomeric compound Structural Identification of embodiment
1) wave spectrum is tested
Isolated compound, liquid chromatogram progress purity detecting, if without UV absorption, analyzes liquid phase color by analysis
Spectrum can not detect, and carry out TLC detections.And corresponding deuterated solvent is chosen according to monomeric compound polarity and is dissolved, it is examined using NMR
It surveys, tetramethylsilane (TMS) is used as internal standard.Noval chemical compound needs KBr tablettings to carry out function contained in IR authenticating compound structures
Group's species；Meanwhile appropriate compound is taken, UV detections are carried out with the dissolving of corresponding polar solvent, determine that there is purple in compound structure
Functional group's species of outer absorption.Compound molecular weight is speculated according to ESI-MS, compound is determined according further to HR-ESI-MS
Molecular weight, molecular formula and degree of unsaturation.
2) sugared configuration is definite
With reference to sour water solution thin layer chromatography, suitable compound is weighed, methanol dissolving is configured to the solution of 1mg/mL, uses
Capillary point sample on silica gel plate, and being invested in the closed developing tank equipped with the concentrated sulfuric acid, 60 DEG C of water-bath 30min, after take
Go out with hair-dryer be blown to solvent wave to the greatest extent, on silica gel plate blank space point D-Glucose, D- celerys sugar, D- xylose standard product, by chloroform/
Methanol (7:3, v/v) mixed solution water saturation takes the chloroform/methanol solution after 9mL water saturations, adds the ice of 1mL
Acetic acid is unfolded after mixing as solvent, is sprayed with the butanol solution of phthalic acid aniline, in 105 DEG C of heating
10min develops the color.
The configuration of aniline-phthalic acid color developing agent：It accurately weighs aniline 0.93g and phthalic acid 1.66g is dissolved in
The water saturated n-butanols of 100mL.
3) result and analysis
Colorless oil.It is composed ([M+Na] by ESI-MS+M/z 249) and HR-ESI-MS spectrums ([M+Na]+m/z
249.1101,calcd for C12H18O4Na, 249.1097) the molecular formula C of the compound, is determined12H18O4, degree of unsaturation
For 4.IR spectrums (KBr tablettings) give hydroxyl (3435cm-1) and carbonyl (1720cm-1) signal.Ultraviolet spectra shows the compound
Ultraviolet absorption maximum is in 203nm.
1H-NMR spectrum in, give 3 methyl hydrogen signal δ H 1.23 (s, 3H, H-8), δ H 1.15 (d, J=6.6Hz,
3H, H-10) and 2.05 (s, 3H, H-12)；3 methylene hydrogen signal δ H 1.87 (dd, J=9.0,18.6Hz, 1H, H-3a), δ H
2.24 (dd, J=9.1,18.6Hz, 1H, H-3b), δ H 1.16 (dd, J=3.6,4.8, Hz, 1H, H-7a), δ H 1.49 (dd, J
=4.8,9.6Hz, 1H, H-7b) and 4.05 (d, J=11.4Hz, 1H, H-9a), 4.29 (d, J=11.4Hz, 1H, H-9b)；2
Methine hydrogen signal δ H 2.51-2.84 (m, 1H, H-2) and δ H 1.94 (dd, J=3.0,9.6Hz, 1H, H-5).In 13C-NMR
In spectrum, 12 carbon signals are given altogether, compose to obtain with reference to DEPT spectrums and 1H-NMR：1 ketone carbonyl, 1 ester carbonyl group, 2 sp3 are miscellaneous
The quaternary carbon of change, the methine of 2 sp3 hydridization, the methylene and 3 methyl of 3 sp3 hydridization.The prompting of 1H-1H COSY spectrograms has
The presence of 2 segments：a：-CH2CH-；b：-CH2CHCH3-.
Analysis HMBC is composed and is combined its hydrogen spectrum, carbon modal data and compoundCompare, have identical
Carbon skeleton, difference lies in hydroxyls on C-9 to be acetylation.It is composed by HMBC, δ H 4.05 (d, J=11.4Hz, 1H, H-9a),
δ H 4.29 (d, J=11.4Hz, 1H, H-9b) and δ C 172.71 (s, C-11), δ H 2.05 (s, 3H, H-12) and δ C 172.71
(s, C-11) is apparent related, shows that hydroxyl is acetylation on C-9.Therefore,
Finally determine that the compound structure isNuclear magnetic data is shown in Table 1.
The hydrogen spectrum and carbon modal data (MeOD) of table 3-2 compounds Frp34
3 antifungal activity of embodiment measures
Microsporum gypseum is a kind of filamentous fungi that can cause shallow-layer dermatophytosis, can result in ringworm of the body, jock itch,
Onychomycosis, favus of the scalp etc. are clinical common superficial part pathomycetes.Effect of the medicinal plant in terms of skin disease is treated in recent years receives
Concern, it is one important to seek and treat dermopathic drug using the active ingredient design novel non-toxic in natural medicinal plant
Research direction.Minimal inhibitory concentration (Minimum inhibitory concentration, MIC) is to weigh antibacterials to live
Property size important indicator, using test tube medicine base method, not only can easily interpretation MIC value, but also easy to be economic, easily grasp
Make.
What this laboratory reference Clinical Laboratory Standard association (CLSI) was recommended《The micro broth dilution method of filamentous fungi
Drug sensitivity test scheme》(M38-A2) activity of isolated monomeric compound is studied.
1. test monomeric compound
The isolated compound of formula I of embodiment 1.
2. test strain
Microsporum gypseum bacterial strain is purchased from Chinese Academy of Medical Sciences's Nanjing Hospital of Skin Diseases.
3. test apparatus
4. test reagent
5. the preparation of culture medium
Aseptically, fresh modified form 1640 (containing L-Glutamine not carbonate containing) culture medium 1000mL is taken,
Medium's PH Value is adjusted to 7.0 by the 3- for being 0.165mol/L with concentration (N- morpholinyls)-propane sulfonic acid buffer solution, then with 0.22 μm
Filtering with microporous membrane degerming and dispense, 4 DEG C save backup.
6. the preparation of bacteria suspension
Aseptically, eugonic Microsporum gypseum test tube slant is taken to add in sterile saline, with connecing
Kind ring gently scrapes the Filamentous culture of media surface, launches in the mortar for done aseptic process and grinds, uses hemocytometer
Number plate counts, and the concentration of bacteria suspension is adjusted to 0.5 × 10 with 1640 fluid nutrient mediums of improvement3-2×103CFU/mL is spare.
7. test method
The preparation of drug sensitive plate
Accurately weigh the monomeric compound of certain mass and griseofulvin tablet powder respectively, add in certain volume DMSO and
Content is less than 1%, is configured to the storing solution of 0.8mg/mL, and storing solution then is pressed 1 with 1640 fluid nutrient mediums after improvement：25
It is diluted to 2 times of final concentrations, i.e. 32 μ g/mL.The liquid for the 32g/mL that 200 μ L have prepared is taken to add in 96 orifice plates respectively with pipettor
The 1st hole per a line；2-11 holes are separately added into 1640 fluid nutrient mediums after 100 μ L improvement；It is taken with pipettor from the 1st hole
100 μ L liquids inject the 2nd hole, are diluted to the 10th hole, and the 100 μ L liquid rejects that will be taken from the 10th hole for 2 times successively, finally obtain
1-10 holes liquor strength is respectively 32 μ g/mL, 16 μ g/mL, 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, and 0.25
μ g/mL, 0.125 μ g/mL, 0.0625 μ g/mL, ten gradient concentrations；Then 1-11 holes are separately added into 100 μ L bacteria suspensions, the 11st hole
As growth control, the 12nd hole adds in 1640 fluid nutrient medium of improvement of 200 μ L as blank control；Each sample sets three weights
Multiple, each sample sets growth control and blank control.
Antibacterial result judgement
The drug sensitive plate for being inoculated with bacteria suspension is placed in 28 (± 1) DEG C constant incubators and is cultivated, is taken out after 7 days, singulation
The drug concentration that object inhibits growth to the MIC value of the strain for 100% is closed, that is, visually observes clarification or has no growth.
8. result
In order to find monomer resistant to Microsporum gypseum in the Paeonia ludlowii root skin ethyl acetate layer of Tibet
Compound, we, which utilize to have carried out from the isolated new monomeric compound of ethyl acetate layer component, resists Microsporum gypseum
Property experiment, result of the test measure learns that the isolated compound of formula I of embodiment 1 is 4 μ to the MIC value of Microsporum gypseum
G/mL compared with 0.5 μ g/mL of reference substance griseofulvin MIC, illustrates that the compound has Microsporum gypseum certain suppression
It makes and uses.
Claims (4)
1. a kind of new monoterpenes compound, it is characterised in that：The structural formula of compound is shown in formula I：
2. the preparation method of compound described in claim 1, it is characterised in that comprise the following steps：
1) after dry Tibet Paeonia ludlowii root skin is taken to crush, 95% ethyl alcohol is added in, room temperature extracts for 24 hours, repeats to filter, 50 DEG C
It is concentrated under reduced pressure, using ethyl acetate into extraction, extract liquor under the conditions of 50 DEG C is concentrated under reduced pressure, obtains dry powder A；
2) by dry powder A and polyamide in mass ratio 1:4~5 ratio mixing, carries out medium pressure liquid chromatography, reverse phase column material
For MCI-Gel, component B is afforded using the methanol aqueous solution that volumetric concentration is 40%；
3) component B 120mL methanol is dissolved, silica gel mixed sample treats that solvent volatilizees completely, carries out silica gel column chromatography, with petroleum ether/
The mixed liquor that isopropanol volume ratio is 10 ︰ 1 elutes, and obtains component C；
4) component C is repeated into silica gel column chromatography, is eluted, obtained for the mixed liquor of 9 ︰ 1 with petroleum ether and isopropanol volume ratio
Component D；
5) component D is continued through into gel column chromatography, methanol obtains Formulas I monomeric compound as eluent.
3. preparation method according to claim 2, it is characterised in that：Dry powder A and polyamide in mass ratio 1 in step (2):
4.2 ratio mixing.
4. application of the compound of formula I described in claim 1 as antifungal drug.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948376A (en) * | 2010-09-19 | 2011-01-19 | 兰州佛慈制药股份有限公司 | Rapid extraction and separation method of paeonolum |
| CN103274919A (en) * | 2013-05-14 | 2013-09-04 | 亳州千草药业有限公司 | Extraction method of peony overground part |
| CN103524322A (en) * | 2013-10-24 | 2014-01-22 | 洛阳祥和牡丹科技有限公司 | Method for extracting paeonol from paeonia suffruticosa roots |
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- 2015-12-02 CN CN201510875223.7A patent/CN105439859B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948376A (en) * | 2010-09-19 | 2011-01-19 | 兰州佛慈制药股份有限公司 | Rapid extraction and separation method of paeonolum |
| CN103274919A (en) * | 2013-05-14 | 2013-09-04 | 亳州千草药业有限公司 | Extraction method of peony overground part |
| CN103524322A (en) * | 2013-10-24 | 2014-01-22 | 洛阳祥和牡丹科技有限公司 | Method for extracting paeonol from paeonia suffruticosa roots |
Non-Patent Citations (1)
| Title |
|---|
| Two New Monoterpenes from Tithonia diversifolia and Their Anti-Hyperglycemic Activity;Xia Li,et al.;《Rec. Nat. Prod.》;20130805;351-354 * |
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