CN108929296A - A kind of anti-inflammatory phenylpropanoids and its extracting method and application - Google Patents
A kind of anti-inflammatory phenylpropanoids and its extracting method and application Download PDFInfo
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Abstract
The invention discloses a kind of anti-inflammatory phenylpropanoids and its extracting method and applications.The novel phenylpropanoids that the present invention is extracted from olive fruits, it is identified by physicochemical constant and Modern spectroscopy, its physicochemical property and chemical structure are specified, to provide Technical Reference to the multiple exploitation of olive fruits and high-efficiency comprehensive utilization existing resource from now on；Separation and purification method of the invention is simple, efficient and mild；The component of phenylpropanoids can be fully saved, and its structure is clear, it is quality controllable.Simultaneously, pharmacodynamics test shows that 2 kinds of novel phenylpropanoids of the present invention have preferable extracorporeal anti-inflammatory activity, there is significant anti-inflammatory activity to BV2 cell, show that phenylpropanoids, its tautomer and its pharmaceutically acceptable salt of the present invention have as the research potential for preparing novel anti-inflammatory medicine or drug for treating Alzheimer's disease, there is long-range prospect on further exploitation anti-inflammatory agent or drug for treating Alzheimer's disease.
Description
Technical field
Field of medicaments of the present invention more particularly to a kind of anti-inflammatory phenylpropanoids and its extracting method and application.
Background technique
A kind of physiology or pathological reaction of the inflammation as body reply external irritant, and be to cause being total to for a variety of major diseases
Same access participates in human infection, tumour, cardiovascular and cerebrovascular diseases, senile dementia and neurodegenerative disease, allergic disease, essence
The occurrence and development process of many major diseases such as refreshing disease.Clinically, anti-inflammatory drug is be only second to anti-infectives second largest
Class drug.
However, existing chemical synthesis anti-inflammatory drug has apparent adverse reaction, Chinese herbal medicine is because its is resourceful, curative effect
Definitely, the advantages that Small side effects, many biological active constituents from natural medicines show different degrees of anti-inflammatory on inflammation model
Effect.People increasingly pay attention to finding from natural products, develop anti-inflammatory drug.Natural drug provides chemical diversity abundant
Property, the antiinflammatory active compound of a variety of different types of structure, which has separated to identify, to be come, and carries out the structure effect of system on this basis
Relationship research finds new anti-inflammatory lead compound, and develops the anti-inflammatory drug of a new generation.
Olive (Canarium album L.) is Burseraceae (Buseraceae) Canarium (Canarium) plant, is one
Kind aiphyllium, it is originating in China.Chinese olive variety source is abundant, is most with Fujian.Olive is to write for eating raw or processing
The subtropical zone specialty fruit tree of name, fruits nutrition is abundant, medical value and health-care efficacy with higher.It is situated between according to " Chinese Plants will "
It continues, it is medicinal to control laryngitis, hemoptysis, polydipsia, enteritis, diarrhea.It is recorded in " Chinese Pharmacopoeia " (2015 editions), have " clearing heat and detoxicating, relieving sore-throat,
Promote the production of body fluid " the effect of, it can be used for treatments such as " abscess of throat, cough phlegm are glutinous, dysphoria and thirsty, fish and crab poisoning ".
Modern pharmacology experimental study shows: olive has anti-oxidant, anti-saccharification, anti-inflammatory, antiviral isoreactivity.It is previously reported
Primarily focus on the antioxidant activity of olive, and report rare for the research of anti-inflammatory activity.Benzene of the present invention to olive fruits
C prime constituents have carried out system separation, obtain two novel phenylpropanoids, chemical structure and anti-inflammatory activity are not
See relevant report.
Summary of the invention
The purpose of the present invention is to provide a kind of anti-inflammatory phenylpropanoids and its extracting method and applications.
The technical solution used in the present invention is:
Phenylpropanoids and its tautomer and its pharmaceutically acceptable salt, the knot of the phenylpropanoids
Structure formula is
Preferably, above-mentioned phenylpropanoids pharmaceutically acceptable salt be its sodium, potassium, calcium, magnesium, iron, ferrous iron, lead,
Barium, copper, ammonium or zinc salt.
The present invention also provides above-mentioned phenylpropanoids pharmaceutically acceptable salts the preparation method comprises the following steps: by Phenylpropanoid Glycosides class
Compound dissolves in a solvent with corresponding basic salt, and it is pharmaceutically acceptable that phenylpropanoids are settled out from solution
Salt.
Preferably, above-mentioned phenylpropanoids pharmaceutically acceptable salt the preparation method comprises the following steps: by Phenylpropanoid Glycosides of the present invention
Any one in class compound mixes in a solvent with basic salt, stirring and dissolving, stands, isolates sediment to get pharmacy
Upper acceptable salt.
Preferably, above-mentioned basic salt is selected from basic lead acetate, alkali formula calcium acetate, alkali formula magnesium acetate, basic ferric acetate, alkali formula
Ferrous acetate, zinc acetate basic, barium hydroxide, sodium hydroxide, any one in potassium hydroxide.
Preferably, above-mentioned solvent is selected from least one of water, ethyl alcohol, methanol, butanol, amylalcohol.
Preferably, mixing time is 0.1~30min, and whipping temp is 40~80 DEG C.
It is highly preferred that mixing time is 8~12min, whipping temp is 45~60 DEG C.
Preferably, it is placed in 0.1~60min of standing in 2~6 DEG C.
It is highly preferred that being placed in 20~40min of standing in 3~5 DEG C.
Preferably, the molar ratio of compound 1 and basic salt is 1:1~6, the molar ratio of compound 2 and basic salt be 1:1~
2。
Another object of the present invention is to provide the extracting methods of above-mentioned phenylpropanoids, include the following steps:
1) olive fruits are crushed into dry, alcohol extracting, obtains extracting solution；
2) extracting solution is successively extracted with low polar solvent, middle polar solvent and highly polar solvent, obtains middle polar layer；
3) middle polar layer being subjected to silica gel column chromatography, the mixed liquor of polarity and highly polar solvent carries out gradient elution in,
Taking middle polarity and highly polar liquor capacity than the elution fraction for 48~52:1 is live part Y；
4) silica gel column chromatography is carried out to live part Y, the mixed liquor of polarity and highly polar solvent carries out gradient elution in
Afterwards, it is analyzed by reversed phase thin-layer, to wherein middle polarity and highly polar solvent volume than the elution fraction progress for 3.8~4.2:1
ODS column chromatography；It is analyzed after lower alcohol or its aqueous solution gradient elution, then through silica gel and reversed phase thin-layer, to wherein 28~
32%v/v aqueous lower alcoholic solutions elution fraction carries out half preparation HPLC and obtains compound 1；
Alternatively, carrying out silica gel column chromatography, the mixed liquor progress gradient elution of middle polarity and highly polar solvent to live part Y
Afterwards, it is analyzed by reversed phase thin-layer, centering polarity and highly polar liquor capacity are than carrying out ODS column for the elution fraction of 7.8~8.2:1
Chromatography is analyzed after lower alcohol or its aqueous solution gradient elution, then through silica gel and reversed phase thin-layer, to wherein 28~32%v/v
Aqueous lower alcoholic solutions elution fraction carries out half preparation HPLC and obtains compound 2；
Above-mentioned low polar solvent is selected from hexamethylene, petroleum ether, hexane, isooctane, trimethylpentane, pentamethylene, heptane etc.
At least one of hydrocarbon solvent；
Polar solvent is selected from ethyl acetate, chloroform, methylene chloride, ether, methyl formate, nitromethane, acetic acid among the above
At least one of butyl ester, isopropyl ether；
Above-mentioned highly polar solvent be selected from n-butanol, methanol, the tert-butyl alcohol, propyl alcohol, isopropanol, ethyl alcohol, acetone, tetrahydrofuran,
At least one of pyridine；
Above-mentioned lower alcohol is the alkylol of C1~C6；
The structural formula of above compound 1 are as follows:
The structural formula of above compound 2 are as follows:
Preferably, it is 40 μm~70 μm that olive fruits, which are crushed to partial size, in step 1).
Preferably, in step 1), alcohol extracting is carried out using the alcoholic solution of 60~90%v/v.
It is highly preferred that step 1) carries out alcohol extracting using the alcoholic solution of 70%v/v.
Preferably, alcohol used in step 1) alcohol extracting is ethyl alcohol, and in the case where non-special declaration, of the invention is specific
The alcohol of concentration refers to the concentration in its aqueous solution.
Preferably, step 1) alcohol extracting 1 time or more.
Preferably, step 1) alcohol extracting 1~5 time.
It is highly preferred that step 1) alcohol extracting 4 times.
Preferably, the volume of alcoholic solution and the weight ratio of olive fruits are (3~5) L:1Kg in step 1).
It is highly preferred that the volume of alcoholic solution and the weight ratio of olive fruits are 4.5L:1Kg in step 1).
Preferably, above-mentioned lower alcohol is selected from least one of methanol, ethyl alcohol, propyl alcohol.
It is highly preferred that above-mentioned lower alcohol is selected from methanol.
Preferably, above-mentioned low polar solvent is selected from hexamethylene；Middle polar solvent is selected from chloroform；Highly polar solvent is selected from first
Alcohol, n-butanol, acetone.
Preferably, in step 3) gradient elution sequence are as follows: middle polar solvent, 48~52:1,18~22:1,8~12:1,
5~7:1,2.5~3.5:1；Wherein, the ratio refers to middle polar solvent: the volume ratio of highly polar solvent.
It is highly preferred that the middle polar solvent of step 3) is selected from chloroform, highly polar solvent is selected from methanol.
Preferably, in step 4), the mixed liquor of middle polarity and highly polar solvent carries out the sequence of gradient elution are as follows: 100%
Polar solvent in v/v~0%v/v.
It preferably, is the low of 10%~100%v/v with lower alcohol or the de- sequence of its aqueous solution gradient elution in step 4)
Grade alcohol solution.
Preferably, the middle polar solvent of step 4) is selected from chloroform, and highly polar solvent is selected from acetone, and lower alcohol is selected from methanol.
The present invention also provides above-mentioned phenylpropanoids and its tautomer, pharmaceutically acceptable salt are anti-in preparation
It controls anti-inflammatory drugs or/and drug for treating Alzheimer's disease or/and inhibits the application in albumen iNOS, COX-2 expression drug.
Preferably, the inflammation is neuroinflamation, pneumonia, hepatitis, mazoitis, gastritis, bursal synovitis, thrombosis-obstructive vessel
Any one inflammation scorching, in myocarditis.
The present invention also provides the drug of a kind of anti-inflammatory or treatment Alzheimer's disease, active constituent includes above-mentioned institute
The phenylpropanoids stated or its tautomer or its pharmaceutically acceptable salt.
Preferably, said medicine further includes pharmaceutically acceptable carrier, diluent, excipient, stabilizer, antioxidant.
Preferably, carrier be selected from starch, chitosan, alginic acid, agar, fibrin, collagen, polyphosphoric acid esters,
At least one of polyurethanes, polyacids anhydride, liposome, polyethylene glycol, mannose, galactolipin, povidone.
Preferably, diluent is selected from least one of microcrystalline cellulose, lactose, mannitol, starch, saccharin.
Preferably, excipient is selected from least one of mannose, glycine, lactose, sodium chloride, glucose.
Preferably, stabilizer is selected from albumin, collagen, cyclodextrin and its derivative, polyethylene glycol, tween, sapn, dextrorotation
At least one of glucosides, mannitol.
Preferably, antioxidant is selected from VC, VE, benzoic acid, citric acid and its salt, sorbic acid, sodium sulfite, bisulfite
At least one of sodium, sodium pyrosulfite, sodium thiosulfate.
Preferably, above-mentioned anti-inflammatory or drug for treating Alzheimer's disease be selected from oral agents, injection, powder, granule,
Capsule, pill, tablet, suppository, film, aerosol, spray, powder spray, sustained release and controlled release agent, targeting preparation, in pulvis
Any one dosage form.
The beneficial effects of the present invention are:
1, the present invention extracts 2 kinds of novel phenylpropanoids from olive fruits, passes through physicochemical constant and modern times
Wave Spectrum is identified, specifies its physicochemical property and chemical structure, for from now on to the multiple exploitation of olive fruits and efficiently it is comprehensive
It closes and provides Technical Reference using existing resource.
2, the separation and purification method of the application is simple, efficient, and mild, can fully save phenylpropanoids
Component, and its structure is clear, it is quality controllable.
3, pharmacodynamics test shows: 2 kinds of novel phenylpropanoids provided by the invention have preferable external anti-
Scorching activity has significant anti-inflammatory activity to BV2 cell, shows phenylpropanoids, its tautomer and its medicine of the present invention
Learning acceptable salt has as the research potential for preparing novel anti-inflammatory medicine or drug for treating Alzheimer's disease, is further opening
Sending out has long-range prospect on anti-inflammatory agent or drug for treating Alzheimer's disease.
Detailed description of the invention
Fig. 1 is compound 11H-NMR spectrum；
Fig. 2 is compound 113C-NMR spectrogram；
Fig. 3 is the HR-ESI-MS spectrogram of compound 1；
Fig. 4 is the HMBC spectrogram of compound 1；
Fig. 5 is compound 11H-1H COSY spectrogram；
Fig. 6 is the CD spectrogram of compound 1；
Fig. 7 is compound 21H-NMR spectrum；
Fig. 8 is compound 213C-NMR spectrogram；
Fig. 9 is the HR-ESI-MS spectrogram of compound 2；
Figure 10 is the HMBC spectrogram of compound 2；
Figure 11 is the CD spectrogram of compound 2；
Figure 12: (A) is protein expression figure of the compound to LPS BV2 the cell iNOS and COX-2 induced of various concentration；
(B) quantify figure for the expressing quantity to (A).
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The extraction of 1 phenylpropanoids of embodiment
(1) dry olive fruits 20.0kg is taken, refluxing extraction four times at 65 DEG C with 50 liter of 70% ethyl alcohol merge all mention
Taking liquid and being concentrated under reduced pressure into extracting liquid volume is 12L；
(2) extracting solution in step (1) is successively extracted with isometric hexamethylene, chloroform, ethyl acetate, n-butanol
It takes, obtains 197.37g chloroform layer (middle polar layer)；
(3) chloroform layer (164.39g) is subjected to silica gel column chromatography, carries out gradient elution (CHCl using chloroform-methanol3、
50:1,20:1,10:1,6:1,3:1, methanol；V/v), taking 50:1 chloroform-methanol eluent is live part Y；
(4) by live part Y through silica gel column chromatography, with chloroform-acetone mixture gradient elution (50:1~2:1；V/v),
It is divided into 13 parts, after analyzing by reversed phase thin-layer, (volume ratio of chloroform and acetone soln is the elution of 4:1 by the 11st part
Part) mesolow ODS column chromatography is carried out, after carrying out gradient elution with methanol-water (10%~100%v/v), pass through reverse phase and silicon
After glue thin-layer chromatographic analysis, half preparation HPLC is carried out to wherein the 5th fraction (30%v/v methanol-water elution fraction) again, obtained
Compound 1.
Alternatively, Y is divided into 13 parts through silica gel column chromatography with chloroform-acetone gradient elution (50:1~2:1, v/v),
It is analyzed by reversed phase thin-layer, to low in its 8th part (elution fraction that the volume ratio of chloroform and acetone soln is 10:1) progress
Press ODS column chromatography, after carrying out gradient elution with methanol aqueous solution (10%~100%), analyze by silica gel and reversed phase thin-layer, it is right
Wherein the 7th fraction (30% methanol-water elution fraction), then half preparation HPLC is carried out, obtain compound 2.
The extraction of 2 phenylpropanoids of embodiment
(1) dry olive fruits 20.0kg is taken, refluxing extraction four times at 65 DEG C with 50 liter of 90% ethyl alcohol merge all mention
Taking liquid and being concentrated under reduced pressure into extracting liquid volume is 12L；
(2) extracting solution in step (1) is successively extracted with isometric hexane, chloroform, ether, methanol, is obtained
197.37g chloroform layer (middle polar layer)；
(3) chloroform layer (164.39g) is subjected to silica gel column chromatography, carries out gradient elution using methylene chloride-n-butanol
(100:0,50:1,20:1,10:1,6:1,3:1,0:100；V/v), taking 50:1 methylene chloride-n-butanol eluent is effective portion
Divide Y；
(4) by live part Y through silica gel column chromatography, with dichloromethane-acetone mixed liquor gradient elution (50:1~2:1；v/
V), it is divided into 13 parts, after analyzing by reversed phase thin-layer, (volume ratio of chloroform and acetone soln is washing for 4:1 by the 11st part
De- part) carry out mesolow ODS column chromatography, after carrying out gradient elution with ethanol water (10%~100%v/v), by reverse phase with
After silica gel thin-layer chromatography spectrum analysis, half preparation HPLC is carried out to wherein the 5th fraction (30%v/v ethanol water elution fraction) again, is obtained
Obtain compound 1.
Alternatively, Y is divided into 13 portions through silica gel column chromatography with ether-isopropanol gradient elution (50:1~2:1, v/v)
Point, it is analyzed by reversed phase thin-layer, to low in its 8th part (elution fraction that ether-isopropanol volume ratio is 10:1) progress
Press ODS column chromatography, after carrying out gradient elution with aqueous propanol solution (10%~100%), analyze by silica gel and reversed phase thin-layer, it is right
Wherein the 7th fraction (30% propyl alcohol water elution part), then half preparation HPLC is carried out, obtain compound 2.
The preparation method of 3 phenylpropanoids pharmaceutically acceptable salt of embodiment
Phenylpropanoids 1 or 2 of the present invention are mixed in alcohol solvent with basic salt, stirring and dissolving 0.1~
30min, whipping temp are 40~80 DEG C；0.1~60min is stood in 2~6 DEG C isolates sediment to get can pharmaceutically connect
The salt received.Wherein, the molar ratio of compound 1 and basic salt is 1:1~6, and the molar ratio of compound 2 and basic salt is 1:1~2.
It is sub- that above-mentioned basic salt is selected from basic lead acetate, alkali formula calcium acetate, alkali formula magnesium acetate, basic ferric acetate, alkali formula acetic acid
Iron, zinc acetate basic, barium hydroxide, sodium hydroxide, any one in potassium hydroxide.
The compound 1 and compound 2 that extract in above-described embodiment are identified below.
1, the identification of compound 1:
The compound 1 isolated and purified is Yellow amorphous powder, and it shows lilac to 10% sulfuric acid-ethyl alcohol, right
The aobvious red of ferric trichloride, prompts to contain phenol structure unit in the compound.Supposition compound 1 is phenylpropanoids.
Further compound 1 is carried out for above-mentioned supposition1H-NMR、13C-NMR、HR-ESI-MS、HMBC、1H-1H COSY
And CD spectrum analysis determines, the result is shown in Figure 1~6:
By Fig. 1's1Known to H-NMR map: having 7 proton signals in fragrant area, wherein δH7.59 (1H, d, J=
15.7Hz, 7 '-H), the 6.59 (- H of 1H, dd, J=15.7,8.0Hz, 8 '), judged according to coupling constant, thus it is speculated that be connected with phenyl ring
Trans- alkene it is strong on hydrogen signal, remaining 5 are fragrant hydrogen signal.In low field area, δHThe 9.59 (- H of 1H, s, 9 '), prompt is one
A aldehyde radical hydrogen signal.Judged according to Fig. 5, which is connected with double bond, forms a C3Structure fragment.In high field region, δH5.56
(1H, d, J=6.6Hz, 7-H), δH3.51 (1H, m, 8-H), δH3.69 (2H, m, 9-H) supposition is a saturation type C3Structure piece
Hydrogen signal in section, and according to J7,8=6.6Hz judges that H-7 and H-8 is Soviet Union's formula conformation in the compound.Specific data are shown in Table 1
In 1aColumn；
By Fig. 2's13Known to C-NMR map: the compound shares 19 carbon signals, low field area δC194.0 be a carbonyl
Carbon signal, in conjunction with1H-NMR map is herein aldehyde radical carbon signal.Fragrant area shows 14 carbon signals, and the compound is prompted to contain
Two benzene ring units.High field region δC55.7 be a methoxyl group carbon signal, δC87.8,62.8,52.8 being Phenylpropanoid Glycosides class C3Structure
The feature carbon signal of unit, in conjunction with1H-NMR map, containing there are two C in the compound6-C3Structure fragment, initial guess this be one
A lignanoids.Specific data are shown in Table 1 in 1aColumn；
The HR-ESI-MS map of Fig. 3 shows quasi-molecular ion peak m/z 365.0992 [M+Na]+(Calcd for
C19H18O6Na, 365.1098), prompting its molecular weight is 365, in conjunction with1H-NMR and13C-NMR can determine that its molecular formula is
C19H18O6；
From the HMBC map of Fig. 4: from H-7 ' (δH7.59) it sets out, with C-2 ' (δC 116.9)、C-6’(δC
116.8)、C-8’(δC 125.7)、C-9’(δC194.0) there is correlation；Again from H-6 ' (δH7.21) it sets out, with C-8 (δC
52.8)、C-4’(δC150.2) related.So far, it can speculate that the compound parent nucleus is benzofuran type neolignans.
Fig. 5's1H-1In H COSY map, H-7 (δH 5.56)、H-8(δH 3.51)、H-9(δH3.69) related, H-7 '
(δH 7.59)、H-8’(δH 6.59)、H-9’(δH9.59) related, know two C3Hydrogen signal on linear fragment.
In the CD map of Fig. 6, between 250-270nm, positive Cotton effect is shown.In conjunction with document, thus it is speculated that compound 1 it is absolute
It is configured as 7S, 8R.
The above analysis can determine 1 structural formula of compound are as follows:
2, the identification of compound 2:
The compound 2 isolated and purified is yellowish-brown pasty masses, and it shows bottle green to 10% sulfuric acid-ethyl alcohol, right
The aobvious red of ferric trichloride, prompts to contain phenol structure unit in the compound.Supposition compound 2 is phenylpropanoids.
Further compound 2 is carried out for above-mentioned supposition1H-NMR、13C-NMR, HR-ESI-MS, HMBC and CD spectrum analysis
It determines, as a result sees Fig. 7~11:
By Fig. 7's1Known to H-NMR map: in high field region δHThere is a unimodal proton signal at 9.81 (1H, s), thus it is speculated that be
Aldehyde radical hydrogen signal.In fragrant area, δH7.07 (1H, d, J=1.7Hz, 2-H), 6.77 (1H, d, J=8.3Hz, 5-H) and 6.87
(1H, dd, J=8.3,1.7Hz, 6-H) and δHThe 7.36 (- H of 1H, d, J=1.9Hz, 2 '), 7.19 (1H, d, J=8.3Hz, 5 '-
It H) and the 7.42 (- H of 1H, dd, J=8.3,1.7Hz, 6 '), is two groups of typical ABX system nature subsignals.High field region, δH 4.70
(1H, dd, J=10.1,5.4Hz, 7-H), 4.44 (1H, d, J=5.4Hz, 8-H) speculate the two methine hydrogen letter being connected to
Number.And according to J7,8=5.4Hz speculates that the H-7 and H-8 of the compound are erythro form conformation.δH 3.85(3H,s)、3.82(3H,s)
And 3.21 (3H, s) be three methoxyl group hydrogen signals.Specific data are shown in Table 2 in 1aColumn；
By Fig. 8's13Known to C-NMR map: the compound shares 19 carbon signals, low field area δC191.2 be an aldehyde radical
Carbon signal.Fragrant area shows 12 carbon signals, in conjunction with1H-NMR map determines that the compound contains the phenyl ring there are two ABX system
Unit.High field region, δC83.9,83.3 and 61.8, it may be possible to a C3Carbon signal on linear fragment.So far, thus it is speculated that the chemical combination
Object is a Phenylpropanoid Glycosides class.Specific data are shown in Table 2 in 1aColumn；
The HR-ESI-MS map of Fig. 9 shows quasi-molecular ion peak m/z 385.1265 [M+Na]+(calcd for C19 H22
O7Na, 385.1359) molecular weight of prompt compound 2 is 385, binding compounds 21H-NMR and13C-NMR can determine it
Molecular formula is C19H22O7；
From the HMBC map of Figure 10: from H-7 (δH4.70) it sets out, with C-1 (δC 131.3)、C-2(δC 112.3)、
C-6(δC 122.0)、C-8(δCAnd C-9 (δ 83.9)C61.8) there is correlation, thus speculate C3Linear fragment is connected with C-1.From
C-7’(δC191.2) it sets out, with H-2 ' (δH 7.36)、H-6’(δH7.42) there is correlation, thus it is speculated that aldehyde radical is connected with C-1 '.From H-
8(δH4.44) it sets out, with C-4 ' (δC154.9) there is correlation, thus it is speculated that compound 2 is a 8-O-4 ' type Phenylpropanoid Glycosides class.
In the CD map of Figure 11, in, between 250-280nm, show positive Cotton effect.In conjunction with document, thus it is speculated that compound 2
Absolute configuration is 7R, 8S.
The above analysis can determine 2 structural formula of compound are as follows:
The corresponding information of the NMR spectra of 1 compound 1 and 2 of table
Note:aNMR spectra obtains on Avance III-600NMR spectrometer；
" -- " indicates without the data.
The above results explanation, separation and purification method of the invention are simple, efficient and mild；Phenylpropanoid Glycosides can fully be saved
The component of class compound, and its structure is clear, it is quality controllable.
The anti-inflammatory activity of 4 phenylpropanoids of embodiment
External inflammatory model is established using the BV2 (mouse microglia) that LPS (lipopolysaccharides) induces, using MTT and
Influence of the compounds of this invention to the BV2 inflammatory factor NO after lipopolysaccharide-induced, anti-inflammatory agent are investigated in Griess experiment
Minocycline (minocycline) is used as positive control.
1, MTT is tested
Take 96 orifice plates, interior kind of BV2 cell culture, if sample sets, control group, blank group.Every hole is added to be measured in sample sets
Sample and cell suspension；The every hole of control group, which is not loaded, only adds cell suspension；The every hole of blank group is not loaded culture solution.Culture is for 24 hours
Afterwards, every group of 3 holes add MTT respectively, continue to cultivate, and after taking-up plus SDS terminates reaction.Cell proliferation inhibition rate=(negative control group
OD value average value-sample sets OD value average value) ÷ (negative control group OD value average value-blank control group OD value average value) ×
100%, and the half-inhibitory concentration (IC for being tested sample is calculated with CalcuSyn software50)。
2, Griess is tested
Take 96 orifice plates, interior kind of BV2 cell culture, if sample sets, model group, control group.After culture for 24 hours, each Kong Pei is drawn
50 μ L of nutrient solution, is added 50 μ L Griess A reagents and 50 μ L Griess B reagents mix, and microplate reader measures OD at 546nm
Value calculates the inhibiting rate generated to NO, NO inhibiting rate=(model control group OD value average value-sample sets OD value average value) ÷
(model control group OD value average value-negative control group OD value average value) × 100%, and calculated and be tested with CalcuSyn software
Half-inhibitory concentration (the IC of sample50)。
Above-mentioned test result see the table below 2, as shown in Table 2: the compound of the present invention 1,2 all has certain anti-inflammatory activity,
And 1 effect of compound is substantially better than anti-inflammatory agent minocycline.
MTT the and Griess experimental result of table 2 the compounds of this invention 1,2 and positive drug
3, Western Blot is tested
By in exponential phase of growth cell kind in 96 orifice plates, be separately added into various concentration compound 1 (2.5,
10.0、20.0μmol·L-1), (10.0,20.0, the 40.0 μm of olL of compound 2-1), after stimulating, extracted in corresponding time point
Each group total protein, the electrophoresis in 10% polyacrylamide gel return again on NC film, close, and are incubated for, using ECL kit
Develop and is imaged.Every group of experiment is repeated 3 times.
Experimental result is shown in Figure 12, and wherein " * " in figure indicates that conspicuousness of the LPS relative to CTL, " # " indicate Phenylpropanoid Glycosides class
Conspicuousness of the compound relative to actin (actin),#P < 0.05,##P < 0.01,###P < 0.001；*P < 0.05,***p
< 0.01,***P < 0.001).
As shown in Figure 12: when concentration is respectively 2.5 and 10.0 μm of olL-1When, compound 1,2 can significantly lower LPS and lure
INOS and the COX-2 protein expression for the BV2 cell led illustrate that the compounds of this invention 1,2 can be by inhibiting iNOS's and COX-2
Expression, to lower the biosynthesis of NO, finally shows anti-inflammatory effect.
The above results explanation, the phenylpropanoids that the present invention extracts can inhibit the generation of inflammatory factor NO, and inhibit
The expression of iNOS, COX-2 albumen, and its effect is substantially better than anti-inflammatory drug minocycline, illustrate the phenylpropanoids, its
Tautomer and its pharmaceutically acceptable salt have as the considerable advantage for preparing novel anti-inflammatory drug.
Inflammation is present in the pathogenetic pathologic process of a variety of diseases, and people are when studying primary dementia disease, also known as A Er
Zi Haimo disease (AD) finds that inflammation plays very important effect in the pathologic process of its morbidity.AD is a kind of high with the age
Degree correlation, the nervous centralis degenerative disease based on progressive cognitive disorder and memory injury.Show as memory, judgement
The forfeiture of the general intelligences such as power, abstract thinking, but eyesight, locomitivity etc. are unaffected.The disease will undergo two kinds of death, first
It is spiritual dead, followed by the human body is dead, brings heavy burden to sufferers themselves, family and society.With prolonging for human longevity
Long and social senilization's problem to become increasingly conspicuous, the quantity and constant of AD patient increases.AD is related with aging, but with it is normal
Aging has essential distinction again.Patient's postmortem shows shrinking of brain tissues, especially hippocampus and Basal forebrain portion neuron loss.Most
The big Pathologic changes of the two of feature are extracellular beta-amyloid protein deposition and neurofibrillary tangles.Studies have shown that β-starch
Sample albumen plays the inflammatory reaction of AD very important effect: it activates microglia (BV2) to generate inflammatory factor, this
A little inflammatory factors cause its apoptosis to the various cell damages including neuron.And these inflammatory factors can draw
The further activation for playing BV2, leads to vicious circle.Test proves: the phenylpropanoids that the application extracts have BV2 cell
There is significant anti-inflammatory activity, so phenylpropanoids of the present invention, its tautomer and its pharmaceutically acceptable salt have
As the realistic meaning for preparing novel therapeutic Alzheimer medicine.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. phenylpropanoids and its tautomer, pharmaceutically acceptable salt, the structural formula of the phenylpropanoids are
2. phenylpropanoids according to claim 1 and its tautomer, pharmaceutically acceptable salt, feature exist
In the pharmaceutically acceptable salt is its sodium, potassium, calcium, magnesium, iron, ferrous iron, lead, barium, copper, ammonium or zinc salt.
3. a kind of method for extracting phenylpropanoids, which comprises the following steps:
1) olive fruits are crushed into dry, alcohol extracting, obtains extracting solution；
2) extracting solution is successively extracted with low polar solvent, middle polar solvent and highly polar solvent, obtains middle polar layer；
3) middle polar layer is subjected to silica gel column chromatography, in the mixed liquor of polarity and highly polar solvent carries out gradient elution, takes
Polarity and highly polar liquor capacity are live part Y than the elution fraction for 48~52:1；
4) silica gel column chromatography is carried out to live part Y, after the mixed liquor of polarity and highly polar solvent carries out gradient elution in, led to
Reversed phase thin-layer analysis is crossed, to wherein middle polarity and highly polar solvent volume than carrying out ODS column for the elution fraction of 3.8~4.2:1
Chromatography；It is analyzed after lower alcohol or its aqueous solution gradient elution, then through silica gel and reversed phase thin-layer, to wherein 28~32%v/v
Aqueous lower alcoholic solutions elution fraction carries out half preparation HPLC and obtains compound 1；
Alternatively, to live part Y progress silica gel column chromatography, after the mixed liquor progress gradient elution of middle polarity and highly polar solvent,
It is analyzed by reversed phase thin-layer, centering polarity and highly polar liquor capacity are than carrying out ODS column layer for the elution fraction of 7.8~8.2:1
Analysis is analyzed after lower alcohol or its aqueous solution gradient elution, then through silica gel and reversed phase thin-layer, low to wherein 28~32%v/v
Grade alcohol solution elution fraction carries out half preparation HPLC and obtains compound 2；
Above-mentioned low polar solvent in hexamethylene, petroleum ether, hexane, isooctane, trimethylpentane, pentamethylene, heptane extremely
Few one kind；
Among the above polar solvent be selected from ethyl acetate, chloroform, methylene chloride, ether, methyl formate, nitromethane, butyl acetate,
At least one of isopropyl ether；
Above-mentioned highly polar solvent is selected from n-butanol, methanol, the tert-butyl alcohol, propyl alcohol, isopropanol, ethyl alcohol, acetone, tetrahydrofuran, pyridine
At least one of；
Above-mentioned lower alcohol is the alkylol of C1~C6；
The structural formula of above compound 1 are as follows:
The structural formula of above compound 2 are as follows:
4. according to the method described in claim 3, it is characterized in that, being carried out in step 1) using the alcoholic solution of 60~90%v/v
Alcohol extracting.
5. according to the method described in claim 3, it is characterized in that, in step 3) gradient elution sequence are as follows: middle polar solvent,
48~52:1,18~22:1,8~12:1,5~7:1,2.5~3.5:1；Wherein, the ratio refers to middle polar solvent: high
The volume ratio of polar solvent.
6. according to the method described in claim 3, it is characterized in that, in step 4), the mixed liquor of middle polarity and highly polar solvent
Carry out the sequence of gradient elution are as follows: polar solvent in 100%v/v~0%v/v.
7. according to the method described in claim 3, it is characterized in that, in step 4), with lower alcohol or its aqueous solution gradient elution
The aqueous lower alcoholic solutions that de- sequence is 10%~100%v/v.
8. prepared by any one of claim 1~2 phenylpropanoids and its tautomer, pharmaceutically acceptable salt
It prevents and treats inflammation or/and treats the application in Alzheimer's disease or/and the drug for inhibiting albumen iNOS, COX-2 to express.
9. application according to claim 8, which is characterized in that the inflammation be neuroinflamation, pneumonia, hepatitis, mazoitis,
Gastritis, bursal synovitis, Buerger's disease, any one inflammation in myocarditis.
10. a kind of anti-inflammatory or the drug for treating Alzheimer's disease, active constituent includes Phenylpropanoid Glycosides described in claim 1
Class compound or its tautomer or its pharmaceutically acceptable salt.
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| CN110698453A (en) * | 2019-10-09 | 2020-01-17 | 广东药科大学 | Pseudorufop-gracilis phenylpropanoids compound and application thereof |
| CN113024575A (en) * | 2021-03-23 | 2021-06-25 | 上海市奉贤区中心医院 | Flavan-3-alcohol phenylpropanoid heterozygote, and preparation method and application thereof |
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| HE, SHAN等: "Nitric oxide inhibitory constituents from the barks of Cinnamomum cassia", 《FITOTERAPIA》 * |
| SAKAKIBARA等: "Lignans and Brauns" lignins from softwoods", 《HOLZFORSCHUNG 》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110698453A (en) * | 2019-10-09 | 2020-01-17 | 广东药科大学 | Pseudorufop-gracilis phenylpropanoids compound and application thereof |
| CN113024575A (en) * | 2021-03-23 | 2021-06-25 | 上海市奉贤区中心医院 | Flavan-3-alcohol phenylpropanoid heterozygote, and preparation method and application thereof |
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