CN101871919A - Method for distinguishing bee glue and poplar glue - Google Patents
Method for distinguishing bee glue and poplar glue Download PDFInfo
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- CN101871919A CN101871919A CN 201010180675 CN201010180675A CN101871919A CN 101871919 A CN101871919 A CN 101871919A CN 201010180675 CN201010180675 CN 201010180675 CN 201010180675 A CN201010180675 A CN 201010180675A CN 101871919 A CN101871919 A CN 101871919A
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- salicin
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Abstract
The invention provides a method for distinguishing bee glue and poplar glue. In the method, HPLC (High Performance Liquid Chromatography) is used for measuring salicin to judge the authenticity of the bee glue so as to control the quality of the bee glue, and the measurement of the salicin by using the HPLC has the advantages of high sensitivity, strong repeatability, and the like. The method used for judging the authenticity of the bee glue has the advantages of high accuracy, high sensitivity, short time and low cost. The method proposes the standard product i.e. the salicin for the first time, thus even if an operation is carried out in the presence of different conditions, the bee glue and the poplar glue. can be accurately and qualitatively distinguished according to the peak time and the ultraviolet absorption of the salicin. The method has good separation effect and high sensitivity and strong feasibility and can be used for distinguishing the bee glue in which more than 10 percent of the poplar glue doped.
Description
Technical field
The present invention relates in a kind of yang gum peculiar and phenolic glycoside class material-salicin assay method of not having in the propolis is used to identify pure propolis or the propolis that is mixed with yang gum.
Background technology
Propolis is the resin secreted of honeybee herborization leaf bud or bark crack punishment and sneaks into mandibular gland secretion and fragrant solids that beeswax forms, mainly by beeswax, resin and volatile matter are formed, wherein: beeswax is by the honeybee secretion, and resin and volatile matter with biological action then derive from glue source plant.Glue source plant mainly contains willow, birch, elm, pine tree, Oak Tree, chestnut and eucalyptus etc.Based on propolis and the plant component comparative study of glue source, the main source that generally believes area, temperate zone propolis now is that Populus and its hybridization belong to.
Propolis has biologic activity widely because of it, as anticancer, anti-oxidant, anti-inflammatory, antibiotic and antifungal activity, being subjected to people pays close attention to widely, obtained using widely in fields such as health cares, yet the production of propolis is subjected to the honeybee kind, adopts the influence of glue mode and glue source situation, output is relatively low, the situation that is in always that supply falls short of demand.Extremely similar on composition, color, smell to willow type propolis extract because of the gemma populi extract, the phenomenon of mixing yang gum (gemma populi extract) in recent years in propolis takes place repeatedly.Existing propolis quality control is primarily aimed at flavonoids and polyphenol two big constituents, and this two constituents gemma populi extract and willow type propolis exist simultaneously, and this makes differentiation propolis and yang gum become quite because of difficulty.Therefore, setting up the analytical approach that a kind of active zone divides propolis and yang gum is the key of control propolis quality.
The characteristic chemical constituent of poplar is a phenolic glycoside, with salicin, salicortin, and 2 '-benzoic acid salicin, Tremulacin content is than horn of plenty.Although The Chemical Constituents is a lot of in the propolis, the report that has any phenolic glycoside in the propolis is not arranged up to now as yet, and it is more to contain the report of derivants such as salicylic acid and acetylsalicylic acid in the propolis.Because the phenolic glycoside of finding in the poplar is the beta-glucosidase form, so we infer that honeybee transmits in the process of propolis with the phenolic glycoside hydrolysis in gathering propolis and honeycomb.
Salicin is the basic structural unit of phenolic glycoside, is one of main degradation products of more senior salicylate analog derivative, at the skin of all poplars of studying, and leaf, flower all has distribution in the bud.Salicin is equal stable existence in methyl alcohol, acidity, alkalescence and neutral medium, and the salicin standard items are easy to get low price.Therefore, we are reference with the salicin, set up high performance liquid chromatography (HPLC) method of distinguishing propolis and yang gum.
Summary of the invention
The object of the invention provides the discrimination method of a kind of propolis and yang gum, is to measure salicin with HPLC to judge the true and false of propolis, thus control propolis quality.Measure salicin with HPLC and have highly sensitively, advantage such as repeatability is strong has the accuracy height, the advantage that sensitivity is strong, the time is short and cost is low with this method judgement true and false of propolis.
The objective of the invention is to realize by following scheme:
(1) with dissolve with methanol salicin standard items, membrane filtration is analyzed with HPLC, obtains the chromatogram of salicin standard items;
(2) with pure propolis dissolve with methanol, make pure propolis solution after the filtration, membrane filtration is analyzed with HPLC, obtains the chromatogram of pure propolis;
(3) with the yang gum dissolve with methanol, make yang gum solution after the filtration, membrane filtration is analyzed with HPLC, obtains the chromatogram of yang gum;
(4) the pure propolis (W/W) that will mix 5%, 10%, 20%, 30%, 40% yang gum is used dissolve with methanol respectively, makes pure propolis solution after the filtration, and membrane filtration is analyzed with HPLC, obtains corresponding chromatogram;
(5) with propolis chromatogram, yang gum chromatogram and mix the propolis chromatogram of different proportion yang gum and salicin standard items chromatogram is relatively determined the true and false of propolis.
The condition of liquid chromatograph of the present invention is: flow velocity is 0.5～1.0mL/min, and the detection wavelength is 210～230nm, 260～275nm, sample size is 2～20 μ L, chromatographic column is Sepax HP-C18 (4.6 * 150mm, 5 μ m) or suitable chromatographic column, and column temperature is 25～35 ℃.
Moving phase of the present invention is acetonitrile: 0.5%H
3PO
4=5: 95.
Contain salicin (S peak) and A peak, unknown peak in the yang gum sample of the present invention, and do not contain this two peaks in the propolis sample.By with the salicin standard control, if detect salicin or A peak in the HPLC spectrogram, then be indicated as yang gum, if the relevant position does not go out the peak, then be propolis.
The present invention through picking up from different regions the propolis sample and the yang gum sample that market is buied verify that repeatedly prove this method good separating effect, highly sensitive, feasibility is strong, therefore, this method can qualitative identification propolis and yang gum.The present invention all can differentiate the yang gum that mixes in the propolis more than 10%.
At present, about the discrimination method of propolis and yang gum mainly is to use HPLC to carry out fingerprint and differentiate, but be subjected to condition restriction such as instrument, experiment condition, the reappearance of finger-print is very poor under different condition, brings difficulty to discriminating, and method is difficult to popularize.The present invention's product (salicin) that set up definite criterion first even operate under different condition, according to the appearance time and the uv absorption of salicin, can accurately be differentiated.
Description of drawings
Fig. 1 is example 1 salicin standard items, propolis, the pure propolis that mixes the different proportion yang gum and the chromatogram of yang gum.
Fig. 2 is the chromatogram of example 2 salicin standard items, propolis and yang gum sample.
Fig. 3 is the chromatogram of example 3 salicin standard items, propolis and yang gum sample.
Fig. 4 is the chromatogram of example 4 salicin standard items, propolis and yang gum sample.
Specific embodiments
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1
Accurately take by weighing the 1.0mg salicin, arrive 100ml with methanol constant volume.Cross 0.45 μ m filter membrane, analyze with HPLC.Adopt the chromatographic column of Sepax HP-C 18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 1.0mL/min, detects wavelength 213nm, 30 ℃ of column temperatures, sample size 5 μ L.Referring to Fig. 1 a.
Get pure propolis 1g, dissolve with methanol filters and obtains pure propolis solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 1.0mL/min, detects wavelength 213nm, 30 ℃ of column temperatures, sample size 5 μ L.Referring to Fig. 1 b.
Get the pure propolis 1g that mixes 5%, 10%, 20%, 30%, 40% yang gum respectively, dissolve with methanol filters and obtains corresponding solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 1.0mL/min, detects wavelength 213nm, 30 ℃ of column temperatures, sample size 5 μ L.Referring to Fig. 1 c, 1d, 1e, 1f, 1g is the pure propolis liquid chromatogram that mixes 5%, 10%, 20%, 30%, 40% yang gum.
Get yang gum 1g, dissolve with methanol filters and obtains yang gum solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 1.0mL/min, detects wavelength 213nm, 30 ℃ of column temperatures, sample size 5 μ L.Referring to Fig. 1 h.
Fig. 1 a: salicin standard items; B: pure propolis; C: the propolis that mixes 5% yang gum; D: the propolis that mixes 10% yang gum; E: the propolis that mixes 20% yang gum; F: the propolis that mixes 30% yang gum; G: the propolis that mixes 40% yang gum; H: yang gum.Represent the peak: S is a salicin; A is unknown peak.
Accurately take by weighing the 1.0mg salicin, arrive 100ml with methanol constant volume.Cross 0.45 μ m filter membrane, analyze with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 0.8mL/min, detects wavelength 220nm, 33 ℃ of column temperatures, sample size 2 μ L.Referring to Fig. 2.
Get pure propolis 1g respectively, dissolve with methanol filters and obtains pure propolis solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 0.8mL/min, detects wavelength 220nm, 33 ℃ of column temperatures, sample size 2 μ L.Referring to Fig. 2.
Get yang gum 1g, dissolve with methanol filters and obtains yang gum solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 0.8mL/min, detects wavelength 220nm, 33 ℃ of column temperatures, sample size 2 μ L.Referring to Fig. 2.A among the figure: salicin standard items; B: pure propolis; C: yang gum.Represent the peak: S is a salicin; A is unknown peak.
Embodiment 3
Accurately take by weighing the 1.0mg salicin, arrive 100ml with methanol constant volume.Cross 0.45 μ m filter membrane, analyze with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 0.5mL/min, detects wavelength 269nm, 35 ℃ of column temperatures, sample size 10 μ L.Referring to Fig. 3.
Get pure propolis 1g, dissolve with methanol filters and obtains pure propolis solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 0.5mL/min, detects wavelength 269nm, 35 ℃ of column temperatures, sample size 10 μ L.Referring to Fig. 3.
Get yang gum 1g, dissolve with methanol filters and obtains yang gum solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 0.5mL/min, detects wavelength 269nm, 35 ℃ of column temperatures, sample size 10 μ L.Referring to Fig. 3.A among the figure: salicin standard items; B: pure propolis; C: yang gum.Represent the peak: S is a salicin; A is unknown peak.
Accurately take by weighing the 1.0mg salicin, arrive 10ml with methanol constant volume.Cross 0.45 μ m filter membrane, analyze with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 1mL/min, detects wavelength 265nm, 25 ℃ of column temperatures, sample size 20 μ L.Referring to Fig. 4.
Get pure propolis 1g, dissolve with methanol filters and obtains pure propolis solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 1mL/min, detects wavelength 265nm, 25 ℃ of column temperatures, sample size 20 μ L.Referring to Fig. 4.
Get yang gum 1g, dissolve with methanol filters and obtains yang gum solution, to 50ml, crosses 0.45 μ m filter membrane with methanol constant volume, analyzes with HPLC.Adopt the chromatographic column of Sepax HP-C18 (4.6 * 150mm, 5 μ m), moving phase is acetonitrile: 0.5%H
3PO
4=5: 95, flow velocity is adjusted to 1mL/min, detects wavelength 265nm, 25 ℃ of column temperatures, sample size 20 μ L.Referring to Fig. 4.A among the figure: salicin standard items; B: pure propolis; C: yang gum.Represent the peak: S is a salicin; A is unknown peak.
According to said method, measured the salicin standard items respectively, propolis mixes the pure propolis of different proportion yang gum and the liquid chromatography collection of illustrative plates of yang gum, and wherein salicin and A peak all do not detect in the propolis sample, and peak area is bigger in yang gum, content is higher.The present invention can detect the existence at A peak in mixing the pure propolis of 10% yang gum, detect salicin and A peak simultaneously and exist in the pure propolis sample that mixes above yang gum 20% (containing 20%).Therefore, this method is applicable to the discriminating that is mixed with 10% above yang gum in the propolis sample.
Claims (2)
1. the discrimination method of propolis and yang gum is to measure salicin with HPLC, realizes by following scheme:
(1) with dissolve with methanol salicin standard items, membrane filtration is analyzed with HPLC, obtains the chromatogram of salicin standard items;
(2) with pure propolis dissolve with methanol, make pure propolis solution after the filtration, membrane filtration is analyzed with HPLC, obtains the chromatogram of pure propolis;
(3) with the yang gum dissolve with methanol, make yang gum solution after the filtration, membrane filtration is analyzed with HPLC, obtains the chromatogram of yang gum;
(4) the pure propolis (W/W) that will mix 5%, 10%, 20%, 30%, 40% yang gum is used dissolve with methanol respectively, makes pure propolis solution after the filtration, and membrane filtration is analyzed with HPLC, obtains corresponding chromatogram;
(5) with propolis chromatogram, yang gum chromatogram and mix the propolis chromatogram of different proportion yang gum and salicin standard items chromatogram is relatively determined the true and false of propolis.
2. the discrimination method of a kind of propolis according to claim 1 and yang gum, phase is characterised in that, the liquid chromatograph condition is: flow velocity is 0.5～1.0mL/min, the detection wavelength is 210～230nm, 260～275nm, sample size are 2～20 μ L, and chromatographic column is Sepax HP-C18, column temperature is 25～35 ℃, and moving phase is acetonitrile: 0.5%H
3PO
4=5: 95.
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| CN 201010180675 CN101871919B (en) | 2010-05-21 | 2010-05-21 | Method for distinguishing bee glue and poplar glue |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103709013A (en) * | 2013-12-24 | 2014-04-09 | 浙江大学 | Separate purification method of unique ingredients in alamo gum and application thereof |
| CN108459038A (en) * | 2017-12-13 | 2018-08-28 | 江苏中谱检测有限公司 | The nuclear-magnetism finger print method that the willow type propolis true and false quickly differentiates |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101620208A (en) * | 2009-08-06 | 2010-01-06 | 浙江大学 | Identification method for propolis and poplar gum |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101620208A (en) * | 2009-08-06 | 2010-01-06 | 浙江大学 | Identification method for propolis and poplar gum |
Non-Patent Citations (2)
| Title |
|---|
| 《世界科学技术-中医药现代化》 20081231 南垚等 蜂胶、杨树叶及杨树芽中水杨苷和芦丁含量比较 第59－61页 1－2 第10卷, 第6期 2 * |
| 《蜜蜂杂志》 20051231 周萍等 蜂胶HPLC指纹图谱真伪鉴别初探 第5－6页 1－2 , 第8期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103709013A (en) * | 2013-12-24 | 2014-04-09 | 浙江大学 | Separate purification method of unique ingredients in alamo gum and application thereof |
| CN108459038A (en) * | 2017-12-13 | 2018-08-28 | 江苏中谱检测有限公司 | The nuclear-magnetism finger print method that the willow type propolis true and false quickly differentiates |
| CN108459038B (en) * | 2017-12-13 | 2020-03-27 | 江苏中谱检测有限公司 | Nuclear magnetic fingerprint spectrum method for quickly identifying authenticity of poplar type propolis |
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