CN101108841B - Compound and use thereof - Google Patents
Compound and use thereof Download PDFInfo
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- CN101108841B CN101108841B CN2007101112150A CN200710111215A CN101108841B CN 101108841 B CN101108841 B CN 101108841B CN 2007101112150 A CN2007101112150 A CN 2007101112150A CN 200710111215 A CN200710111215 A CN 200710111215A CN 101108841 B CN101108841 B CN 101108841B
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- propolis
- yang gum
- yang
- compound
- gum
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- 0 C=*c1cc(O)cc(OC2(CC3=CC=CC=I3)O)c1C2=O Chemical compound C=*c1cc(O)cc(OC2(CC3=CC=CC=I3)O)c1C2=O 0.000 description 1
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Abstract
The invention relates to a compound ''false glairin A'' separated from the yang gum, which is ''4-methoxy -2, 6-dihydroxyl-2-benzyl-[2, 3-dihydro-benzofuran-3-ketone]'', the molecular formula is C16H14O5, and the chemical structural formula refers to the drawing and the separation method and the application of the incorporation of the yang gum in the measured propolis.
Description
Technical field
The present invention relates to a kind of compound of from yang gum, separating " the plain A of pseudo-gums ", i.e. " 4-methoxyl group-2,6-dihydroxyl-2-benzyl-[2,3-dihydro-cumarone-3-ketone] ", its molecular formula is C
16H
14O
5, chemical structural formula is:
, and the application of separation method and its yang gum incorporation in measuring propolis.
Background technology
Propolis (Propolis) is the resin that apis mellifera (Apis mellifera) is gathered from the bud bud of glue source plant, a kind of colloidal solid that mixes with himself secretory product such as beeswax etc.The propolis ingredient is very complicated, and nearly kind surplus in the of 300 except that beeswax, also contains tens of kinds of Flavonoid substances such as Galangin, pinocembrin and Quercetin and various terpenes, organic acid, amino acid and mineral element etc.Because the synergy of these compositions, make propolis have antibiotic preferably, antiviral, anti-oxidant, enhancing immunity and auxiliary hyperglycemic, reducing blood-fat, medical health care function such as antitumor.Propolis has better assisting therapy effect to cardiovascular and cerebrovascular disease, cancer, diabetes, can improve the immunologic function of human body, suppresses growth of tumour cell.Simultaneously, propolis also has anti-inflammatory, eases pain and impels the ability of cell regeneration, to the illness that multiple infected by microbes causes, has better curative effect.
At present, multiple products such as propolis oral liquid, bee glue capsule, bee glue soft capsule, propolis aerosol, propolis soap, propolis washing lotion have been developed both at home and abroad.
Since the good market effect of propolis, some illegal retailer's manufacturing and marketing the fake.Modal manufacturing and marketing the fake method on the market boils with the gemma populi heating exactly, and yang gum is made in press filtration.Early stage yang gum outward appearance is harder, and smell is close with propolis, differentiates with outward appearance than being easier to; Develop so far, yang gum often is entrained in sale mutually with the propolis of orthodox school, the people is also arranged to the further purification of yang gum.Because the chemical ingredients of yang gum and propolis is closely similar, mixing yang gum in propolis becomes a kind of common propolis and mingles means.And the effective ways that are used to differentiate true and false of propolis at present seldom, two standards of propolis: a standard code that is the Ministry of Commerce issues is the propolis of SB/T10096-92, and another is that the standard code that the national Ministry of Agriculture issues is the pollution-free food propolis of NY/5136-2002.All can't differentiate propolis and yang gum with these two standards, because projects such as the glue content in the standard, total flavones, oxidation index, yang gum can reach requirement basically.CN1719247A discloses a kind of method of utilizing liquid-phase fingerprint to differentiate true and false of propolis, but really can't measure the incorporation of yang gum in the propolis accurately.Therefore all there are not to set up a kind of effective ways that are used for measuring propolis yang gum incorporation both at home and abroad, its main difficulty is need find a kind ofly not exist in natural propolis, and in yang gum the significant compound of general stable existence as detected object.
Summary of the invention
The present invention relates to a kind of compound of from yang gum, separating " the plain A of pseudo-gums ", i.e. " 4-methoxyl group-2,6-dihydroxyl-2-benzyl-[2,3-dihydro-cumarone-3-ketone] ", its molecular formula is C
16H
14O
5, chemical structural formula is:
This compound was not seen bibliographical information before the present invention, be to separate the new compound that obtains first by the contriver from yang gum called after " the plain A of pseudo-gums ".This compound has following physical chemical characteristics:
1) outward appearance: white plates crystallization (recrystallisation solvent: methyl alcohol); Fusing point is greater than 210 ℃ (decomposition).
2) solubleness: be soluble in methyl alcohol, acetone, be slightly soluble in water, chloroform.
3) mass-spectrometric data: EI-MS m/z (%): 286 (3), 268 (2), 258 (1), 225 (1), 195 (57), 167 (100), 152 (8), 124 (7), 91 (27).Wherein molecular ion peak M+ is m/z (%) 286 (3).
4) ultraviolet spectrum data: UV
MeOHNm (log ε): 219.2 (4.08), 294.4 (4.22), 324.8 (4.13).
5) ir data: IR (v, cm
-1): 3402,3085,3028,2981,1678,1601,1506,
1495,1474,1454,1441,1383,1356,1325,1302,1204,1165,1120,1076,984,835 and 696.
6) proton nmr spectra and carbon spectrum, and hydrocarbon long-range relevant spectrum data, as following table:
The plain A's of table 1 pseudo-gums
13CNMR (125MHz),
1HNMR (500MHz) and HMBC data (solvent DMSO-d
6, the ppm of unit)
| C/H | δC | δH(Jin?Hz) | HMBC(observed) |
| 2 | 105.4 | --- | 2-OH，7’-2H |
| 3 | 192.3 | --- | 2-OH，7’-2H |
| 4 | 159.1 | --- | 5-H，4-OCH3 |
| 5 | 90.7 | 5.87b | 6-OH |
| 6 | 168.7 | --- | 6-OH，5-H，7-H |
| 7 | 92.6 | 5.86b | 6-OH |
| 8 | 172.0 | --- | 7-H |
| 9 | 101.3 | --- | 5-H，7-H |
| 1’ | 134.2 | --- | 7’-2H |
| 2’ | 130.3 | 7.11-7.18m | 7’-2H |
| 3’ | 127.6 | 7.11-7.18m | |
| 4’ | 126.4 | 7.11-7.18m | |
| 5’ | 127.6 | 7.11-7.18m | |
| 6’ | 130.3 | 7.11-7.18m | 7’-2H |
| 7’ | 41.3 | 2.98d(14)，3.04d(14) | 2-OH，2’-H，6’-H |
| 4-OCH 3 | 55.5 | 3.71s | |
| 2-OH | - | 7.57s |
| 6-OH | - | 10.82s |
On the other hand, the present invention relates to separation and the purification process of the plain A of a kind of pseudo-gums, it is characterized in that this method may further comprise the steps:
A. be that raw material separates with the yang gum;
B. use ethanol preliminary purification yang gum;
C. use polyamide column chromatography to separate;
D. use silica gel column chromatography to separate;
E. use the sephadex lh-20 purifying;
F. recrystallizing methanol.
(1) separate raw materials of the plain A of pseudo-gums is a yang gum.Willow (Poplar) is the common name of Salicaceae (Salicaceae) Populus (Populus) plant defoliation arbor.Yang gum is that the tree bud with willow boils the resulting natural gum of press filtration, is to be very easy to the common false propolis bought in the market.The sample of the employed yang gum of this case is existing in the freezer of two applying units.
(2) high performance liquid phase (HPLC) color atlas with yang gum and propolis compares, in the collection of illustrative plates of yang gum and propolis sample, and corresponding to retention time 12.5min place, many chromatographic peaks in the yang gum collection of illustrative plates.The chromatogram working conditions: BWAIC LC98II system, the P98II pump, the UV98I UV-detector, Kromasil5 micron C18 post, 4.6x250mm, methyl alcohol: water: phosphoric acid (50:50:0.25v/v/v), detect wavelength 296nm, flow velocity 1.0mL/min.See Fig. 1-2:
(3) separation of the plain A of pseudo-gums and structure are identified.To 500 gram yang gum ethanol preliminary purifications, remove mechanical impurity, reclaim ethanol; Primary extract carries out polyamide column chromatography (80-100 order, 2 kilograms, water/methyl alcohol gradient elution), and each 1L collects elutriant, analyzes the part of following the tracks of the compd A place with HPLC, merges, and reclaims solvent; The part that contains compd A that obtains is separated with silica gel column chromatography (100-120 order, 1 kilogram, chloroform/methanol gradient elution), analyzes the part of following the tracks of the compd A place with HPLC, merges, and reclaims solvent; Obtain the about 100mg of compd A; With dextrane gel (LH-20,200 grams, methanol-eluted fractions) purifying, analyze the part of following the tracks of the compd A place with HPLC, merge and reclaim solvent; Recrystallizing methanol obtains the about 50mg of pure compd A.According to the spectroscopic analysis such as UV spectrum, infrared spectra, mass spectrum and nucleus magnetic resonance of compd A, the structure of compound obtains to identify, and is as above shown in Figure 1.Literature search confirms that this compound is new compound without report, the plain A of called after pseudo-gums.
In addition, the invention still further relates to the application of the plain A of pseudo-gums yang gum incorporation in measuring propolis.The present invention has set up enforceable concrete technological method, makes that above-mentioned technical problem is solved fully.The main points of technological method are to use chemico-analytic method, the method for especially first-selected high-efficient liquid phase analysis.At first measure the average content of the plain A of pseudo-gums in yang gum, measure its content in mingling propolis then, just can calculate the incorporation of yang gum in the propolis.
At last, the invention still further relates to a kind of method of measuring yang gum incorporation in the propolis, it is characterized in that passing through chemical analysis, preferred high-efficient liquid phase analysis, measure the average content of the plain A of pseudo-gums in yang gum respectively, with its content in mingling propolis, just can calculate the incorporation of yang gum in the propolis.
The present invention is by detailed finger printing comparative study, and the plain A of pseudo-gums has been identified in discovery and separation and purification finally.Studies confirm that the plain A of pseudo-gums does not exist in natural propolis, and in yang gum general stable existence, content is about 1.25%.Therefore the plain A of pseudo-gums can be used as significant compound, by measuring its content in mingling propolis, just can calculate the incorporation of yang gum.
Description of drawings
Fig. 1 is a propolis HPLC collection of illustrative plates.
Fig. 2 is the HPLC collection of illustrative plates of yang gum.
Fig. 3 is reference substance (A), yang gum-1# (B), propolis (YD004, Shunyi, Beijing) HPLC color atlas (C)
Embodiment
The following examples are mainly introduced a kind of the most frequently used technology implementation scheme, make one of skill in the art understand practical application of the present invention better.It is multiple that but concrete enforceable scheme can have, and is not subjected to the qualification of present embodiment.
Embodiment: the HPLC method is measured propolis, yang gum and is mingled the content of the plain A of pseudo-gums in the propolis
1) materials and methods
1.1 material
1.1.1 the plain A reference substance of pseudo-gums: obtain through column chromatography separation, purifying, recrystallization with yang gum.
1.1.2 sample: 10 parts of propolis sample sources see Table 2; 2 parts of yang gums, 4 parts are mingled propolis and are detected (Beijing) center by Ministry of Agriculture's bee product and provide.
1.2 key instrument and reagent
1.2.1 instrument.High performance liquid chromatograph (BWAIC LC98II system, P98II pump, UV98I UV-detector); Kromasil C
18(4.6mm * 250mm5 μ m); Ultrasonic washing instrument (KQ-100B).
1.2.2 reagent.Methyl alcohol, chromatographically pure (Deneb friend); Analytical pure (Beijing Chemical Plant); Water (WAHAHA pure water)
1.3 chromatographic condition moving phase is methyl alcohol: water: phosphoric acid (50:50:0.25v/v/v); Flow velocity 1mL/min detects wavelength 296nm, the external standard method quantitative analysis.
1.4 the preparation of solution
1.4.1 reference substance solution.Accurately take by weighing the plain A reference substance of pseudo-gums 1.08mg, place the 10mL volumetric flask, use dissolve with methanol, shake up, be mixed with that to contain pseudo-gums element A concentration be the reference substance solution of 108 μ g/mL.
1.4.2 sample solution.Accurately take by weighing propolis sample 0.1g, place the 10mL volumetric flask, add an amount of methyl alcohol supersound extraction 40min, use methanol constant volume, precision is measured 1mL and is placed the 10mL volumetric flask after shaking up, and constant volume shakes up.Get 1mL 0.45nm filtering with microporous membrane, get sample solution.
2) result and analysis
2.1 linear relationship experiment precision is measured 125 μ L, 100 μ L, 75 μ L, 50 μ L, 25 μ L, 2 μ L are settled to 1mL respectively, and is numbered 1,2,3,4,5,6 is to be measured.By 1.3 chromatographic conditions, get parallel 3 the sample introduction analyses of reference substance 10 μ L, retention time is at 12.5min.Getting its area average obtains: the equation of area average S and sample size X: S=1991937X-923.782R
2=0.9999, the sample size that shows the plain A of reference substance pseudo-gums is in 0.00216 μ g-0.135 μ g scope the time, and linear relationship is good.
2.2 the plain A reference substance solution of pseudo-gums 10 μ L sample introductions are accurately drawn in the precision test, repeat 6 times, recording compd A peak area relative standard deviation (RSD) is 0.79% (n=6), and illustration method and instrument precision are good.
2.3 the test of method circulation ratio is got with 5 parts in a collection of propolis sample, prepares sample solution by " 1.4 " method, draws 10 μ L sample introduction analyses, the peak area RSD that records the plain A of pseudo-gums is 1.16% (n=5), and favorable reproducibility is described.
2.4 the 10 μ L sample introduction analyses of the plain A reference substance solution of pseudo-gums are accurately drawn in stability test, then respectively at 0,2,4, repeat 1 time during 8h, the RSD that records the peak area of the plain A of pseudo-gums is 0.99%, shows that sample solution is stable in 8h.
2.5 average recovery test accurately takes by weighing five parts of the sample solutions of known content, every part 100 μ L joins in the 1mL volumetric bottle; Get again five parts of reference substance solution (concentration: 10.8 μ g/mL), every part 100 μ L adds respectively, and is accurate fixed molten to 1mL in above-mentioned each bottle, measure by selected chromatographic condition, calculate recovery rate, the result is 103.6%, RSD is 1.37%.
2.6 sample determination is got different yang gums, is mingled glue, the propolis sample of different areas, by " 1.4 " method preparation sample solution, accurately draws 10 μ L sample introduction analyses, and parallel 3 times, the peak area of the plain A of pseudo-gums in the working sample, external standard method is calculated content.The results are shown in Table 2.
The plain A content of table 2 propolis pseudo-gums not of the same race
3) measurement result discussion
Adopt the RP-HPLC method, with methyl alcohol: water: phosphoric acid (50:50:0.25v/v/v) be moving phase, the plain A content of pseudo-gums in the external standard method propolis, and method is easy, quick, reliable, can be used as the quantitative analysis method of pseudo-gums element A content in the propolis.The assay result shows that the plain A substances content of the pseudo-gums in propolis and the yang gum is widely different.The average content of the plain A of pseudo-gums is greatly about about 1.25% in the yang gum; The content of the plain A of pseudo-gums not in detection limit, just can not detect in the pure propolis of certified products.To the said determination result, with the following the 4th) listed formula calculates in the bar, can judge to mingle propolis-1#, mingle propolis-2#, mingle propolis-3# and mingle and be impregnated in 54.6%, 38.5%, 29.6% and 10.8% yang gum among propolis-4# respectively.
4) mingle the calculation formula of yang gum incorporation (weight percent) in the propolis
X＝(Z/Y)*100％
Wherein: X=mingles the yang gum incorporation (weight percent) in the propolis
The plain A average content (weight percent) of pseudo-gums in the Y=yang gum
Z=mingles the plain A content (weight percent) of pseudo-gums in the propolis
Claims (5)
1. compound 4-methoxyl group-2 of from yang gum, separating, 6-dihydroxyl-2-benzyl-[2,3-dihydro-cumarone-3-ketone], its molecular formula is C
16H
14O
5, chemical structural formula is:
2. a separation and purification prepares the method for compound according to claim 1, it is characterized in that this method may further comprise the steps:
A. be that raw material separates with the yang gum;
B. use ethanol preliminary purification yang gum;
C. use polyamide column chromatography to separate;
D. use silica gel column chromatography to separate;
E. use the sephadex lh-20 purifying;
F. recrystallizing methanol.
3. whether compound has been impregnated in the application of yang gum and definite yang gum incorporation in identifying the sample propolis according to claim 1, it is characterized in that mingling the content of this compound in the propolis by mensuration, and compare with the average content of this compound in the yang gum, calculate the incorporation of determining to mingle yang gum in the propolis.
4. method of measuring yang gum incorporation in the propolis, it is characterized in that passing through chemical analysis, measure average content and its content during mingling propolis of compound as claimed in claim 1 in yang gum respectively, thereby calculate the incorporation of yang gum in the propolis.
5. the method for yang gum incorporation in the mensuration propolis as claimed in claim 4 is characterized in that chemical analysis wherein is a high-efficient liquid phase analysis.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101112150A CN101108841B (en) | 2007-06-18 | 2007-06-18 | Compound and use thereof |
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|---|---|---|---|
| CN2007101112150A CN101108841B (en) | 2007-06-18 | 2007-06-18 | Compound and use thereof |
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|---|---|
| CN101108841A CN101108841A (en) | 2008-01-23 |
| CN101108841B true CN101108841B (en) | 2011-05-11 |
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| CN2007101112150A Expired - Fee Related CN101108841B (en) | 2007-06-18 | 2007-06-18 | Compound and use thereof |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103360352B (en) * | 2013-08-05 | 2015-07-08 | 中国医学科学院药用植物研究所 | Total synthesis method for 4-methoxy-2,6-dihydroxy-2-benzyl-3(2H)-benzofuranone |
| CN107941800B (en) * | 2017-12-29 | 2020-05-12 | 上海微谱化工技术服务有限公司 | Qualitative and quantitative analysis method for false propolis in health food |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1719247A (en) * | 2005-07-29 | 2006-01-11 | 浙江蜂之语蜂业集团有限公司 | Method of identifying true and false of propolis using liquid phase finger print atlas |
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2007
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1719247A (en) * | 2005-07-29 | 2006-01-11 | 浙江蜂之语蜂业集团有限公司 | Method of identifying true and false of propolis using liquid phase finger print atlas |
Non-Patent Citations (4)
| Title |
|---|
| 余兰平, 盛文胜.气相色谱- 质谱法分析蜂胶和杨树胶的化学成分.蜜蜂杂志 6.2006,(6),3-5. |
| 余兰平, 盛文胜.气相色谱- 质谱法分析蜂胶和杨树胶的化学成分.蜜蜂杂志 6.2006,(6),3-5. * |
| 周萍 等.蜂胶HPLC 指纹图谱真伪鉴别初探.蜜蜂杂志 8.2005,(8),5-6. |
| 周萍 等.蜂胶HPLC 指纹图谱真伪鉴别初探.蜜蜂杂志 8.2005,(8),5-6. * |
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