CN101327224A - Identification method of propolis medicinal materials and preliminary classification method of propolis medicinal material growing district - Google Patents

Abstract

The invention relates to a propolis (as a medicinal material) distinguishing method and a preliminary classification method of propolis production areas. First test sample solution is prepared, then control solution is prepared. The measurement method is as follows: 10mul of the reference solution and 10mul of the test sample solution are respectively and accurately extracted and then injected into a high performance liquid chromatograph; the test is performed according to the high performance liquid chromatograph; with the control solution as the reference, the chromatogram is measured and recorded; then a propolis HPLC finger-print map is established. The invention has the advantages of convenient method, stability, high accuracy, good reproduction and easiness in mastering, and also has the advantages that the trueness of the propolis can be mastered from the whole characteristic appearance of the chromatogram, and the propolis distinguishing method can be used as one of the indexes for controlling the propolis quality and distinguishing the trueness of the propolis. Meanwhile the propolis from different production areas also undergoes preliminary classification.

Description

The preliminary classification method in a kind of discrimination method of propolis medicinal material and propolis medicinal material producing region

Technical field

The present invention relates to the discrimination method of propolis medicinal material and the preliminary classification method in propolis medicinal material producing region, particularly a kind of high performance liquid chromatography with medelling (HPLC) finger printing is differentiated the propolis medicinal material method.

Background technology

Propolis (propolis) is the natural gum of Apis from plant sprout bud and trunk collection, sneaks into a kind of gluey solid content with fragranced of formation such as Apis mandibular gland secretions and Cera Flava.The propolis chemical constituent is relevant with the vegetation of honeybee kind and Apis life surrounding area, known propolis chemical constituent has hundreds of, if the inherent quality of propolis and prescribed preparation thereof is described with the active component of one, two propolis, have certain one-sidedness, should be controlled its material group integral body.So, except " micro analysis ", also use certain " macroscopic analysis " method, effectively characterize Chinese medicine quality on the whole.Propolis is a kind of Flavonoid substances pure natural bee product that is rich in, and the natural gum of plant secretions such as willow is the raw material of propolis, and Apis gathers natural gum after making has formed propolis.Multiple medical health care functions such as that propolis has is antibiotic, antiviral, antioxidation, blood sugar lowering, blood fat reducing, raising immunity are a kind of health product raw materials with wide market.Domestic propolis annual production in 2003 has reached 300 tons, and the version Chinese Pharmacopoeia was recorded propolis as Chinese crude drug in 2005.But adulterate in the society, with vacation when genuine case of common occurrence, not only can't determine its place of production, even the phenomenon of propolis occur pretending to be with natural gum.And at present to the quality control of propolis, mainly be to be index (Chinese Pharmacopoeia 2005 version one one 249 pages) with chrysin, galangin equal size.Yet because propolis comes from natural gum, its composition also is similar to natural gum, therefore, existing official method and industry standard etc. all can't be distinguished natural gum and propolis, make the propolis quality control be difficult to carry out, appreciable impact the foreign exchange earning of people's quality of life and propolis.Therefore need application model fingerprint pattern technology etc. to take the lead in setting up propolis Quality Control platform, guarantee that propolis is stable and controllable for quality.

Summary of the invention

The objective of the invention is by setting up a kind of pattern finger printing application technology of distinguishing the propolis producing region, can distinguish natural gum and propolis well, and tentatively distinguish the place of production of propolis, clear and definite its range of application aspect medical and health and food chemical industry, thereby significantly improve propolis Quality Control level, guarantee its using value and economic benefit.

The discrimination method of a kind of propolis medicinal material of the present invention comprises the steps:

(1), sets up the finger printing of propolis

(a) preparation of need testing solution: get many batches of propolis medicinal material powder respectively, the accurate title, decide, and puts in the apparatus,Soxhlet's, be extracted into extracting liquid colourless with the acetone Soxhlet, extracting solution water bath method, residue dissolve with methanol, put in the volumetric flask, methanol constant volume is need testing solution to scale;

(b) preparation of reference substance solution: respectively accurate to claim decide rutin, Quercetin, chrysin, galangin reference substance an amount of, dilutes with mobile phase, makes reference substance solution;

(c) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler, and mobile phase is: A: methanol, B: sour water; Adopt the gradient elution mode; Flow velocity 1.0mL/min; Detect wavelength: 283nm or 256nm or 365nm or 360nm; Column temperature: 20-30 ℃.

(d) measure: accurately draw each 10 μ l of reference substance solution and need testing solution respectively, injecting high performance liquid chromatograph respectively, test according to high performance liquid chromatography, is object of reference with the reference substance, measure and the record chromatogram, obtain propolis HPLC finger printing (seeing accompanying drawing 1-Fig. 2).

(2), measure the finger printing (seeing accompanying drawing 3) of false propolis such as yang gum with above-mentioned identical method.

(3), described propolis finger printing has 29 total peaks, finger printing contrast with yang gum, from the difference of peak height and size ordering thereof more as can be seen: parameters such as the peak height at No. 19 total peaks and No. 21 total peaks and big or small ordering thereof reflect the difference of yang gum and propolis, therefore, No. 19 total peaks (RT=66.61min) and No. 21 total peaks (RT=78.37min) have formed characteristic peak gummy and that propolis is distinguished mutually jointly.

(4), measure the finger printing of testing sample with above-mentioned identical method.

(5), with the contrast of the finger printing of propolis sample finger printing to be measured and propolis, screening meets the propolis medicinal material of propolis standard finger-print.

In the first step of above-mentioned steps, the concrete preparation process of described propolis need testing solution is: get the about 1g of multiple propolis medicinal material powder respectively, the accurate title, decide, and puts in the apparatus,Soxhlet's, uses acetone 100ml, Soxhlet is extracted into extracting liquid colourless, the extracting solution water bath method, the residue dissolve with methanol is put in the 100ml measuring bottle, methanol constant volume is need testing solution to scale.

Described chromatographic condition is a filler for the chromatographic column adopting octadecylsilane chemically bonded silica, and mobile phase is: A: methanol, the phosphoric acid water of B:0.4%; Adopt the gradient elution mode: 0min → 10min → 20min → 30min → 35min → 50min → 60min → 120min, methanol: 35% → 35% → 45% → 45% → 50% → 50% → 55% → 55%, 0.4% phosphoric acid water: 65% → 65% → 55% → 55% → 50% → 50% → 45% → 45%; Flow velocity: 1.0mL/min; Detect wavelength: 283nm; Column temperature: 25 ℃.

Contain rutin 0.268mg, Quercetin 0.1096mg, chrysin 0.0296mg, galangin 0.0206mg among the every ml of reference substance solution described in the above-mentioned steps first step.

Propolis described in the above-mentioned steps first step is the propolis that produce in the different places of production of 26 batches of China.

Above-mentioned steps is in the 5th step:

(a) there are not significant difference in total peak retention time and propolis finger printing, there is not exclusive peak yet, and obvious No. 25 total peaks (chrysin RT=88.76min) are arranged, emphasis is differentiated No. 19 and No. 21 total peaks in the collection of illustrative plates, No. 19 the high value of total peak-to-peak does not reach the tenth-largest peak, and it is true propolis that No. 21 high values of total peak-to-peak do not reach the third-largest peak; And No. 19 high values of total peak-to-peak reach the fourth-largest peak, and the high values of while No. 21 total peak-to-peaks reach first peak person and are yang gum.

(b), total peak retention time and the propolis finger printing person that has the significant difference, belong to gummy or other the false propolis of other classes.

The relative standard deviation RSD of the relative retention time at described 29 total peaks is all less than 3%, and wherein the average relative retention time RT of rutin (No. 11 total peaks) is 29.5min, and relative standard deviation RSD is 0.23%; The average relative retention time RT of Quercetin (No. 16 total peaks) is 46.3min, and relative standard deviation RSD is 0.95%; The average relative retention time RT of chrysin (No. 25 total peaks) is 88.8min, and relative standard deviation RSD is 1.6%, and the average relative retention time RT of galangin (No. 27 total peaks) is 98.0min, and relative standard deviation RSD is 2.0%.

The preliminary classification method in a kind of propolis medicinal material producing region comprises the steps:

Adopt national Bureau of Drugs Supervision to recommend the MATLAB software that uses, propolis HPLC finger printing is analyzed comparison, the result, the similarity of 26 batches of propolis samples (hair glue) is between 61%-94%, therefore differ greatly (the seeing accompanying drawing 7) of its similarity, classify to the HPLC finger printing employing principal component analysis and the similarity mode of combining of 26 batches of propolis samples (hair glue), tentatively 26 batches of propolis samples (hair glue) are fallen into 5 types, the similarity of every class all is higher than 90% (seeing accompanying drawing 8-Figure 12).

Relatively carry out analytic induction according to table 1 to the data of table 3, the difference of propolis and gummy HPLC finger printing is embodied on this parameter of peak height.

With the peak height is the parameter analysis, and propolis is divided into 5 classes: the 1st class is that No. 6 total peaks (RT=17.62min) are the 1st big peak, and No. 28 total peaks (RT=100.02min) are the 2nd big peak; The 2nd class is that No. 20 total peaks (RT=75.84min) are the 2nd or 1 big peak, and No. 24 total peaks (RT=84.7min) are the 19th big peak, and No. 25 total peaks (chrysin RT=88.76min) do not reach the 3rd big peak simultaneously; The 3rd class is that No. 20 total peaks (RT=75.84min) are the 1st big peak, and No. 11 total peaks (RT=29.50min) do not reach the 11st big peak, and No. 23 total peaks (RT=81.11min) are the 2nd big peak; The 4th class is that No. 20 total peaks (RT=75.84min) are the 1st big peak, and No. 25 total peaks (chrysin RT=88.76min) are the 3rd big peak; The 5th class is that No. 20 total peaks (RT=75.84min) are the 1st or 2 big peaks, and No. 25 total peaks (RT=88.76min) are the 2nd, the 1st or 4 big peaks.

Annotate: 1 expression has the peak in the table 1-table 2, but is not integrated to owing to little; Table 3 hollow BAIGE represents that the peak is arranged, but is not integrated to owing to little.

The analysis result that table 4 peak height and similarity combine

Numeral 1,2,3 in the table 4 ... the expression peak is from by force to weak.1 expression highest peak, 2 expressions time strong peak,

Principle of the present invention is to be one of its main active ingredient according to the flavones ingredient in the propolis, set up the HPLC finger printing of propolis flavone constituents, derive from the natural gum difference of the bee colony collection in the different places of production, cause the difference on its stratographic whole facial feature, can set up the propolis classification by medelling.Though propolis comes from natural gum simultaneously, through chewing of Apis, behind jaw glandular secretion thing on its incorporation and the Cera Flava, also can cause the difference of both finger printing, distinguish by medelling.

The present invention has that method is easy, stable, precision is high, favorable reproducibility, the characteristics that are easy to grasp.The detection wavelength that pharmacopeia is used to measure chrysin and galangin is 256nm (Chinese Pharmacopoeia 2005 version one one 249 pages), and the detection wavelength that is used to measure alpinin and galangin in the document is 360nm (Song Aiqing etc., China's bee-keeping, 2001,52 (3): 10); The detection wavelength that is used to measure Quercetin and kaempferol is 365nm (Du Haiyan etc., Chinese herbal medicine, 2002,23 (11): 997); The detection wavelength that is used to measure chrysin be 268nm (burnt celebrating connects etc., Chinese patent medicine, 2005,27 (8): 961), but finger printing is not for measuring the accurate content of certain composition, but will fully reflect the information of chemical constituent.Compare with officinal detection wavelength with document, detect with 283nm, it is more to go out the peak, and the information of reflection is more complete, so adopt 283nm for detecting wavelength.

Description of drawings

Describe the present invention in detail below in conjunction with accompanying drawing:

Fig. 1 reference substance collection of illustrative plates.

Fig. 2 propolis HPLC finger printing

Fig. 3 pseudo-gums (natural gum) finger printing

The replica test similarity evaluation stacking chart of 5 parts of need testing solutions of the parallel preparation of the same test sample of Fig. 4

Fig. 5 precision test similarity evaluation stacking chart

Fig. 6 stability test similarity evaluation stacking chart

6 batches of propolis samples of Figure 72 (hair glue) HPLC finger printing similarity evaluation stacking chart

(1,2 are followed successively by Jiangxi sample 2, Jiangxi sample 3 to Fig. 8 first kind (Jiangxi 1 class) propolis sample similarity evaluation stacking chart among the figure.)

Fig. 9 second class (other classes) propolis sample similarity evaluation stacking chart (1 is the Yunnan sample among the figure, and 2 is Jiangxi sample 8)

(1,2,3,4,5 are followed successively by Jiangxi sample 1, Jiangxi sample 4, Jiangxi sample 5, Jiangxi sample 6, Jiangxi sample 7 to Figure 10 the 3rd class (Jiangxi 2 classes) propolis sample similarity evaluation stacking chart among the figure.)

(1 is that Shandong sample 1,2 is an Inner Mongol sample to Figure 11 the 4th class (northern class) propolis sample similarity evaluation stacking chart among the figure, and 3 is the Shanxi sample, 4 is Liaoning sample, and 5 is the Jilin sample, and 6 is Shandong sample 2,7 is the Qinghai sample, and 8 is the Hebei sample, and 9 is the northeast sample.)

(1 is that Henan sample 1,2 is that Henan sample 2,3 is the Gansu sample to Figure 12 the 5th class (Henan, Hubei Province class) propolis sample similarity evaluation stacking chart among the figure, and 4 is the Jing Zhou sample, and 5 is Jingmen sample, and 6 is the Anhui sample, and 7 is that Henan sample 3,8 is the Yichang sample.)

The specific embodiment

The preferred embodiments of the present invention are as follows:

1, instrument, reagent and material

1.1 instrument and material: German Dionex P680A type high performance liquid chromatograph (PDA-100 detector, STH585 column oven, P680HPLC pump, ASI-100 automatic sampler), the general work station of chromeleon color; 8953Dietikon type electronic analytical balance (Switzerland, precision 0.1mg); AS2060B type Ultrasound Instrument (Tianjin Ao Tesaiensi Instr Ltd.) etc.

1.2 reagent: control substance of Rutin (080-9705), Quercetin reference substance (081-9003), chrysin (111701-200501), galangin (111699-200501) all have Chinese pharmaceutical biological product to identify that institute provides, propolis (hair glue, provide by the precious circle company limited of giving birth in Guangzhou, account for row through Guangzhou bee product institute Lu and identify, be propolis).The used methanol of mobile phase is chromatograph alcohol, other reagent as: methanol, phosphoric acid etc. are analytical pure.

2, method

(1) sets up the finger printing of propolis

(a), the preparation of need testing solution: get 26 batches of propolis medicinal material-10 respectively and get the about 1g of powder with the pulverizer pulverizing after ℃ freezing a couple of days, the accurate title, decide, put in the apparatus,Soxhlet's, use acetone 100ml, Soxhlet is extracted into extracting liquid colourless, the extracting solution water bath method, the residue dissolve with methanol is put in the 100ml measuring bottle, and methanol constant volume is to scale, shake up, promptly get and be need testing solution.

(b), the preparation of reference substance solution: respectively accurate to claim to decide rutin, Quercetin, chrysin, galangin reference substance an amount of, dilute with mobile phase, make reference substance solution, contain rutin 0.268mg, Quercetin 0.1096mg, chrysin 0.0296mg, galangin 0.0206mg among the every ml of described reference substance solution.

(c), chromatographic condition is: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler, and mobile phase is: A: methanol, the phosphoric acid water of B:0.4%; Adopt the gradient elution mode: 0min → 10min → 20min → 30min → 35min → 50min → 60min → 120min, methanol: 35% → 35% → 45% → 45% → 50% → 50% → 55% → 55%, 0.4% phosphoric acid water: 65% → 65% → 55% → 55% → 50% → 50% → 45% → 45%; Flow velocity: 1.0mL/min; Detect wavelength: 283nm; Column temperature: 25 ℃.

(d) measure: accurately draw each 10 μ l of reference substance solution and need testing solution respectively, injecting high performance liquid chromatograph respectively, test according to high performance liquid chromatography, is object of reference with the reference substance, measure and the record chromatogram, obtain 26 batches of propolis HPLC finger printing.

(2), measure the finger printing of false propolis such as yang gum with above-mentioned identical method.

(3), described propolis finger printing has 29 total peaks, finger printing contrast with false propolis such as yang gum, from the difference of peak height and size ordering thereof more as can be seen: parameters such as the peak height at No. 19 total peaks and No. 21 total peaks and big or small ordering thereof reflect the difference of yang gum and propolis, therefore, No. 19 total peaks (RT=66.61min) and No. 21 total peaks (RT=78.37min) have formed characteristic peak gummy and that propolis is distinguished mutually jointly.The relative standard deviation RSD of the relative retention time at described 29 total peaks is all less than 3%, and wherein the average relative retention time RT of rutin (No. 11 total peaks) is 29.5min, and relative standard deviation RSD is 0.23%; The average relative retention time RT of Quercetin (No. 16 total peaks) is 46.3min, and relative standard deviation RSD is 0.95%; The average relative retention time RT of chrysin (No. 25 total peaks) is 88.8min, and relative standard deviation RSD is 1.6%, and the average relative retention time RT of galangin (No. 27 total peaks) is 98.0min, and relative standard deviation RSD is 2.0%.The statistical result of 26 batches of propolis medicinal material retention times sees Table 1.

(4), measure the finger printing of testing sample with above-mentioned identical method.

(5), with the contrast of the finger printing of propolis sample finger printing to be measured and propolis, screening meets the propolis medicinal material of propolis standard finger-print, concrete screening process is as follows:

(a), there are not significant difference in total peak retention time and propolis finger printing, there is not exclusive peak yet, and obvious No. 25 total peaks (chrysin RT=88.76min) are arranged, emphasis is differentiated No. 19 and No. 21 total peaks in the collection of illustrative plates, No. 19 the high value of total peak-to-peak does not reach the tenth-largest peak, and it is true propolis that No. 21 high values of total peak-to-peak do not reach the third-largest peak; And No. 19 high values of total peak-to-peak reach the fourth-largest peak, and the high values of while No. 21 total peak-to-peaks reach first peak person and are yang gum.

(b), total peak retention time and the propolis finger printing person that has the significant difference, belong to gummy or other the false propolis of other classes.

3, finger printing repeatability experiment

Get with 5 parts of a test samples (Henan propolis medicinal material), preparation method according to need testing solution, 5 parts of need testing solutions of parallel preparation, detect with method, the chromatographic fingerprinting that 5 parts of need testing solutions record is intuitively observed, the overall picture that shows finger printing does not have significant change, calculate with similarity, the chromatographic fingerprinting that records at same instrument and the similarity of its gained common pattern collection of illustrative plates are respectively 0.9894,0.9956,0.9951,0.9969,0.9977, all greater than 0.95, and the concordance of investigation retention time and peak area, rutin, the RSD of Quercetin retention time RSD and peak area is all less than 1.8% (table 5, Fig. 4).

Table 5 replica test result (n=6)

4, finger printing precision experiment

Get with a test sample (Henan propolis medicinal material) solution, continuous sample introduction 5 times, calculate with similarity, the chromatographic fingerprinting that records at same instrument and the similarity of its gained common pattern collection of illustrative plates are respectively 0.9937,0.9952,0.9973,0.9969,0.9959, satisfy similarity and be not less than 0.950, and the concordance of investigation chromatographic peak retention time and peak area, the RSD of rutin, Quercetin, chrysin, galangin retention time RSD and peak area is all less than 1.7%.(table 6, Fig. 5)

Table 6 Precision test result (n=5)

5, finger printing stability experiment

Get with a need testing solution, respectively at 0h, 2h, 4h, 10h, the 14h different time points detects, the overall picture of observing finger printing directly perceived does not have significant change, calculate with similarity, the chromatographic fingerprinting that records at same instrument and the similarity of its gained common pattern collection of illustrative plates are respectively 0.9922,0.9932,0.9886,0.9959,0.9951, satisfy similarity and be not less than 0.950, and the concordance of investigation retention time and peak area, rutin, Quercetin, chrysin, the RSD of galangin retention time RSD and peak area is all less than 1.6%, show at this moment between in the composition of need testing solution be stable (table 7, Fig. 6).

Table 7 stability test result (n=5)

More than experiment shows that this assay method is reliable and stable.

The preliminary classification method in a kind of propolis medicinal material producing region comprises the steps:

Adopt national Bureau of Drugs Supervision to recommend the MATLAB software that uses, propolis HPLC finger printing is analyzed comparison, the result, the similarity of 26 batches of propolis samples (hair glue) is between 61%-94%, therefore differ greatly (the seeing accompanying drawing 7) of its similarity, classify to the HPLC finger printing employing principal component analysis and the similarity mode of combining of 26 batches of propolis samples (hair glue), tentatively 26 batches of propolis samples (hair glue) are fallen into 5 types, the similarity of every class all is higher than 90% (seeing accompanying drawing 8-Figure 12).

Relatively carry out analytic induction according to table 1 to the data of table 3, the difference of propolis and gummy HPLC finger printing is embodied on this parameter of peak height.

With the peak height is the parameter analysis, and propolis is divided into 5 classes: the 1st class is that No. 6 total peaks (RT=17.62min) are the 1st big peak, and No. 28 total peaks (RT=100.02min) are the 2nd big peak; The 2nd class is that No. 20 total peaks (RT=75.84min) are the 2nd or 1 big peak, and No. 24 total peaks (RT=84.7min) are the 19th big peak, and No. 25 total peaks (chrysin RT=88.76min) do not reach the 3rd big peak simultaneously; The 3rd class is that No. 20 total peaks (RT=75.84min) are the 1st big peak, and No. 11 total peaks (RT=29.50min) do not reach the 11st big peak, and No. 23 total peaks (RT=81.11min) are the 2nd big peak; The 4th class is that No. 20 total peaks (RT=75.84min) are the 1st big peak, and No. 25 total peaks (chrysin RT=88.76min) are the 3rd big peak; The 5th class is that No. 20 total peaks (RT=75.84min) are the 1st or 2 big peaks, and No. 25 total peaks (RT=88.76min) are the 2nd, the 1st or 4 big peaks.

Claims (6)

1, a kind of discrimination method of propolis medicinal material comprises the steps:

(1), sets up the finger printing of propolis

(a) preparation of need testing solution: get many batches of propolis medicinal material powder respectively, the accurate title, decide, and puts in the apparatus,Soxhlet's, be extracted into extracting liquid colourless with the acetone Soxhlet, extracting solution water bath method, residue dissolve with methanol, put in the volumetric flask, methanol constant volume is need testing solution to scale;

(b) preparation of reference substance solution: respectively accurate to claim decide rutin, Quercetin, chrysin, galangin reference substance an amount of, dilutes with mobile phase, makes reference substance solution;

(c) chromatographic condition: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler, and mobile phase is: A: methanol, B: sour water; Adopt the gradient elution mode; Flow velocity 1.0mL/min; Detect wavelength: 283nm or 256nm or 365nm or 360nm; Column temperature: 20-30 ℃.

(d) measure: accurately drawing each 10 μ l of reference substance solution and need testing solution respectively, inject high performance liquid chromatograph respectively, test according to high performance liquid chromatography, is object of reference with the reference substance, measures and the record chromatogram, obtains propolis HPLC finger printing.

(2), measure the finger printing of false propolis such as yang gum with above-mentioned identical method.

(3), described propolis finger printing has 29 total peaks, finger printing contrast with false propolis such as yang gum, from the difference of peak height and size ordering thereof more as can be seen: parameters such as the peak height at No. 19 total peaks and No. 21 total peaks and big or small ordering thereof reflect the difference of yang gum and propolis, therefore, No. 19 total peaks (RT=66.61min) and No. 21 total peaks (RT=78.37min) have formed characteristic peak gummy and that propolis is distinguished mutually jointly.

(4), measure the finger printing of testing sample with above-mentioned identical method.

(5), with the contrast of the finger printing of propolis sample finger printing to be measured and propolis, screening meets the propolis medicinal material of propolis standard finger-print.

2, the discrimination method of propolis medicinal material according to claim 1, the concrete preparation process that it is characterized in that described propolis need testing solution is: get the many crowdes of about 1g of propolis medicinal material powder respectively, the accurate title, decide, and puts in the apparatus,Soxhlet's, uses acetone 100ml, Soxhlet is extracted into extracting liquid colourless, the extracting solution water bath method, the residue dissolve with methanol is put in the 100ml measuring bottle, methanol constant volume is need testing solution to scale.

3, the discrimination method of propolis medicinal material according to claim 1, it is characterized in that described chromatographic condition is: the chromatographic column adopting octadecylsilane chemically bonded silica is a filler, and mobile phase is: A: methanol, the phosphoric acid water of B:0.4%; Adopt the gradient elution mode: 0min → 10min → 20min → 30min → 35min → 50min → 60min → 120min, methanol: 35% → 35% → 45% → 45% → 50% → 50% → 55% → 55%, 0.4% phosphoric acid water: 65% → 65% → 55% → 55% → 50% → 50% → 45% → 45%; Flow velocity: 1.0mL/min; Detect wavelength: 283nm; Column temperature: 25 ℃.

4, the discrimination method of propolis medicinal material according to claim 1 and 2 is characterized in that:

(a) there are not significant difference in total peak retention time and propolis finger printing, there is not exclusive peak yet, and obvious No. 25 total peaks (chrysin RT=88.76min) are arranged, emphasis is differentiated No. 19 and No. 21 total peaks in the collection of illustrative plates, No. 19 the high value of total peak-to-peak does not reach the tenth-largest peak, and it is true propolis that No. 21 high values of total peak-to-peak do not reach the third-largest peak; And No. 19 high values of total peak-to-peak reach the fourth-largest peak, and the high values of while No. 21 total peak-to-peaks reach first peak person and are yang gum.

(b), total peak retention time and the propolis finger printing person that has the significant difference, belong to gummy or other the false propolis of other classes.

5, propolis medicinal material discrimination method according to claim 1 and 2, it is characterized in that: the relative standard deviation RSD of the relative retention time at described 29 total peaks is all less than 3%, wherein the average relative retention time RT of rutin (No. 11 total peaks) is 29.5min, and relative standard deviation RSD is 0.23%; The average relative retention time RT of Quercetin (No. 16 total peaks) is 46.3min, and relative standard deviation RSD is 0.95%; The average relative retention time RT of chrysin (No. 25 total peaks) is 88.8min, and relative standard deviation RSD is 1.6%, and the average relative retention time RT of galangin (No. 27 total peaks) is 98.0min, and relative standard deviation RSD is 2.0%.

6, the preliminary classification method in a kind of propolis medicinal material producing region comprises the steps:

Adopt national Bureau of Drugs Supervision to recommend the MATLAB software that uses, propolis HPLC finger printing is analyzed comparison, the result, the similarity of 26 batches of propolis samples (hair glue) is between 61%-94%, therefore differing greatly of its similarity, classify to the HPLC finger printing employing principal component analysis and the similarity mode of combining of 26 batches of propolis samples (hair glue), tentatively 26 batches of propolis samples (hair glue) are fallen into 5 types, the similarity of every class all is higher than 90%.

With the peak height is the parameter analysis, and propolis is divided into 5 classes: the 1st class is that No. 6 total peaks (RT=17.62min) are the 1st big peak, and No. 28 total peaks (RT=100.02min) are the 2nd big peak; The 2nd class is that No. 20 total peaks (RT=75.84min) are the 2nd or 1 big peak, and No. 24 total peaks (RT=84.7min) are the 19th big peak, and No. 25 total peaks (chrysin RT=88.76min) do not reach the 3rd big peak simultaneously; The 3rd class is that No. 20 total peaks (RT=75.84min) are the 1st big peak, and No. 11 total peaks (RT=29.50min) do not reach the 11st big peak, and No. 23 total peaks (RT=81.11min) are the 2nd big peak; The 4th class is that No. 20 total peaks (RT=75.84min) are the 1st big peak, and No. 25 total peaks (chrysin RT=88.76min) are the 3rd big peak; The 5th class is that No. 20 total peaks (RT=75.84min) are the 1st or 2 big peaks, and No. 25 total peaks (RT=88.76min) are the 2nd, the 1st or 4 big peaks.

Images