CN105911194B - The detection method of Determination of Gardenoside in a kind of bee glue soft capsule deep processed product - Google Patents
The detection method of Determination of Gardenoside in a kind of bee glue soft capsule deep processed product Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The present invention relates to Quality Control Technology field, more particularly to the detection method of Determination of Gardenoside in a kind of bee glue soft capsule deep processed product.The detection method includes：Gelatine capsule is mixed with water, is ultrasonically treated, obtains gelatin solution；Gelatin solution is mixed with acetic acid zinc solution, potassium ferrocyanide solution, absolute ethyl alcohol, is staticly settled, is centrifuged, supernatant is taken, obtains test liquid；The content of Gardenoside in test liquid is detected using high-efficient liquid phase chromatogram technology.Gardenoside can be sufficiently separated by detection method provided by the invention from gelatine capsule, so as to accurately detect the content of Gardenoside in gelatine capsule；Detection method is linear, repeatability, has good stability, and precision is high, and blank is substantially noiseless to measurement result, and detection limit, quantitative limit are low, and the rate of recovery is high.
Description
Technical field
The present invention relates to Quality Control Technology field, more particularly to Gardenoside contains in a kind of bee glue soft capsule deep processed product
The detection method of amount.
Background technology
Containing abundant and unique flavonoids, terpenoid substance in propolis, have significantly to various bacteria, fungi, virus etc.
Inhibition and killing effect.Can be used for treat tinea pedis, onychomycosis, eczema, various ulcer, periodontitis, laryngitis, rhinitis, gastritis,
Disease caused by the pathogenic microorganisms such as urinary system infection contamination, flu, hemorrhoid, diarrhea.Dosage is small, quick, does not generate drug resistance,
It does not destroy beneficial to bacterium colony.From propolis it is separated go out several natural materials (PRCA) that can directly inhibit, kill cancer cell, make
Obtain propolis has prevention and treatment effect well to tumour, polyp.Flavonoid substances rich content in propolis has good
Promoting blood circulation and removing blood stasis, softening blood vessel, prevents platelet aggregation, improves the effects that microcirculation reducing blood lipid.To vascular sclerosis, infraction, blood
Bolt, hyperlipidemia etc. have prevention and treatment effect well.
Bee glue soft capsule is added in salad oil in appropriate proportions, in standard using after the processing dissolving of high-quality bee glue purifying
Soft capsule is made in the workshops GMP, its main feature is that absorb rapidly, it is in good taste convenient for taking and carrying, it is easy to preserve.Bee glue soft capsule
The capsule skin used is the former material of gelatin, and gelatin capsule material itself is transparent, and propolis content is it is easy to appear lamination, from
And influence the sensory experience of consumer.Gardenia Yellow can be added in order to improve the sensory experience of consumer, in gelatin capsule material to carry out
Color is dyed opaque, uniform faint yellow.
Gardenia Yellow, alias crocin are commonly called as Yellow Fructus Gardeniae, belong to carotenoid series, it is the xanthein in cape jasmine, point
Minor is C44H64O24, relative molecular mass 976.97.Gardenia Yellow can be used for juice-type beverage, assembled alcoholic drinks, color make-up, ice on cake
Rod, ice cream, dilated food, jelly, flour cake, candy and chestnut can etc..Gardenia Yellow main component includes carotenoids
Crocin and crocetin, Gardenoside and flavones, chlorogenic acid containing iridoid glycosides.It is provided according to GB2760-2014
The maximum additive amount of Gardenia Yellow is 0.3g/kg, because Gardenoside is the main component of Gardenia Yellow, therefore in order to control propolis flexible glue
The additive amount of Gardenia Yellow in the former material of capsule, needs the content for detecting Gardenoside in former material.
However, during atual detection, since the organic solvent for dissolving Gardenoside can not dissolve gelatin, and in addition
The content of Gardenoside is micro, so it is difficult to accurately detecting the content of Gardenoside in gelatine capsule.
Invention content
In view of this, the present invention provides a kind of detection methods of Determination of Gardenoside in bee glue soft capsule deep processed product.
The detection method can accurately detect the content of Gardenoside in gelatine capsule.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of detection methods of Determination of Gardenoside in gelatine capsule, including：
Step 1：Gelatine capsule is mixed with water, is ultrasonically treated, obtains gelatin solution；
Step 2：Gelatin solution is mixed with acetic acid zinc solution, potassium ferrocyanide solution, absolute ethyl alcohol, is staticly settled, from
The heart takes supernatant, obtains test liquid；
Step 3：The content of Gardenoside in test liquid is detected using high-efficient liquid phase chromatogram technology.
Preferably, the temperature being ultrasonically treated is 40~50 DEG C.
Preferably, the temperature of supersound process is 40 DEG C.
Preferably, the usage ratio of agents useful for same is in detection method：
In embodiment provided by the invention, the usage ratio of agents useful for same is in the detection method：
Preferably, the temperature staticly settled is 2~8 DEG C, the time is 1~2h.
Preferably, the temperature staticly settled is 4 DEG C, time 2h.
Preferably, the rotating speed of centrifugation is 5000~8000r/min, the time is 3~5min.
In embodiment provided by the invention, the rotating speed of centrifugation is 8000r/min, time 5min.
Further include pre-treatment step in embodiment provided by the invention, before step 1, pre-treatment includes：It includes content to take
The gelatine capsule of object is removed content, is cleaned using the first organic solvent and the second organic solvent, the first organic solvent is
Petroleum ether and/or ether, the second organic solvent are absolute methanol or absolute ethyl alcohol.
In embodiment provided by the invention, content is propolis.
In embodiment provided by the invention, petroleum ether is petroleum ether I.
Preferably, further including the steps that filtering between taking supernatant to obtain test liquid in step 2.
In embodiment provided by the invention, filtered using 0.45 μm of organic phase filter membrane.
Preferably, the mobile phase of high performance liquid chromatography is acetonitrile solution or methanol aqueous solution.
Preferably, the volume ratio of acetonitrile and water is 15 in acetonitrile solution:85.
Preferably, the volume ratio of methanol and water is 35 in methanol aqueous solution:65.
Preferably, the flow velocity of mobile phase is 0.8~1.0mL/min.
Preferably, the flow velocity of mobile phase is 1.0mL/min.
Preferably, the Detection wavelength of high performance liquid chromatography is 238~240nm.
Preferably, the Detection wavelength of high performance liquid chromatography is 238nm.
Preferably, the column temperature of high performance liquid chromatography is 30~40 DEG C.
Preferably, the column temperature of high performance liquid chromatography is 40 DEG C.
Preferably, the chromatographic column of high performance liquid chromatography is Phenomenex Gemini-NX chromatographic columns, Phenomenex
Synergi Hydro-RP chromatographic columns, Agilent ZORBAX SB-C18 chromatographic columns or Inertsil ODS-SP chromatographic columns.
In embodiment provided by the invention, the model C18 of chromatographic column, specification be 250 × 4.6mm, 5 μm.
The present invention provides a kind of detection methods of Determination of Gardenoside in bee glue soft capsule deep processed product.The detection method
Including：Gelatine capsule is mixed with water, is ultrasonically treated, obtains gelatin solution；By gelatin solution and acetic acid zinc solution, ferrous iron
Potassium cyanide solution, absolute ethyl alcohol mixing, staticly settle, centrifuge, take supernatant, obtain test liquid；Using high-efficient liquid phase chromatogram technology
Detect the content of Gardenoside in test liquid.The present invention at least has one of following advantage：
1, Gardenoside can be sufficiently separated by detection method provided by the invention from gelatine capsule, so as to accurately detect
The content of Gardenoside in gelatine capsule；
2, detection method is linear, repeatability, has good stability, and precision is high, blank to measurement result substantially without
Interference, detection limit, quantitative limit are low, and the rate of recovery is high, meets GB/T27404-2008《Good Laboratory controls specification》Requirement,
It proves that the Determination of Gardenoside detection method is scientific and effective, the mesh of quality control can be played to the Determination of Gardenoside of bee glue soft capsule
's.
Description of the drawings
Fig. 1~5 are respectively the chromatogram of standard serial number STD1~STD5 reference substances, show the linear of detection method
Relationship；
Fig. 6 shows the standard working curve that the concentration according to Gardenoside in standard serial number STD1~STD5 reference substances is drawn；
Fig. 7~12 are 6 obtained chromatograms of reference substance solution replication, show the precision of detection method；
Figure 13~18 are 6 obtained chromatograms of sample replication, show the repeatability of detection method；
Figure 19~24 are that reference substance solution places the chromatogram obtained after 0h, 1h, 2h, 4h, 8h, 12h at room temperature, show this
The stability of invention detection method；
Figure 25 shows the chromatogram of blank solution；
Figure 26 shows the detection limit of detection method；
Figure 27 shows the quantitative limit of detection method；
Figure 28~36 are the chromatogram of 1~9 sample of serial number, show the rate of recovery of detection method.
Specific implementation mode
The invention discloses a kind of detection method of Determination of Gardenoside in bee glue soft capsule deep processed product, art technologies
Personnel can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that all similar replacements and changing
Dynamic apparent to those skilled in the art, they are considered as being included in the present invention.The present invention method and answer
With being described by preferred embodiment, related personnel can obviously not depart from the content of present invention, in spirit and scope
Method described herein and application are modified or are suitably changed and combined, to realize and apply the technology of the present invention.
The testing principle of detection method：The content of bee glue soft capsule is washed away with petroleum ether and methanol, at ultrasound
Reason dissolves by heating capsule material with water and is centrifuged off with acetic acid zinc solution, potassium ferrocyanide solution and absolute ethyl alcohol protein precipitation
Precipitation, is detached Gardenoside with high performance liquid chromatography C18 columns, and quantified by external standard method is used in combination in detector detection.
Agents useful for same or instrument in the detection method of Determination of Gardenoside in bee glue soft capsule deep processed product provided by the invention
Device is available on the market.
With reference to embodiment, the present invention is further explained：
The detection of 1 Determination of Gardenoside of embodiment
Instrument：1260 high performance liquid chromatograph of Agilent (matches VWD or DAD detectors).
Reagent reagent：Petroleum ether I (AR)；Methanol (AR)；Absolute ethyl alcohol (AR)；Zinc acetate (AR)；Potassium ferrocyanide (AR)；
Acetonitrile (chromatographically pure)；Level-one water.
Gardenoside reference substance source：National Institute for Food and Drugs Control, lot number:110749-201115, purity:
99.7%.
Analytical procedure：
1, chromatographic condition：
Mobile phase：Acetonitrile：Water=15：85；
Flow velocity：1.0mL/min；
Wavelength：238nm；
Column temperature：40℃
Run time：25min
Chromatographic column：Phenomenex Gemini-NX, C18,250 × 4.6mm, 5 μm.
2, the preparation of storing solution is compareed：
Precision weighs Gardenoside reference substance 5.09mg, is placed in 50mL brown volumetric flasks, and flowing phased soln and constant volume is added
To scale, shake up to get storing solution.Storing solution is placed in 4 DEG C of refrigerator and stores for future use.
3, standard working curve is drawn：
Precision draws 100 μ L of Gardenoside storing solution, is placed in 25mL brown volumetric flasks, and mobile phase is added and is settled to scale,
It shakes up to get working solution, difference is accurate to draw 2 μ L of working solution, 5 μ L, 10 μ L, 20 μ L, 30 μ L, in above-mentioned chromatographic condition
Under, analysis measurement is carried out, standard working curve is drawn.
4, prepared by sample：
Precision weighs bee glue soft capsule sample 4 (about 2.7g), half-and-half cuts off capsule skin, removes content, and capsule skin is set
In 50mL centrifuge tubes, 25mL petroleum ethers I are added and shake 10 seconds, discard petroleum ether I, repetition washes capsule skin 5 times；Be added 25mL without
Water methanol shakes 10 seconds, discards absolute methanol；Capsule skin sets another clean centrifuge tube, and 10mL water, 40 DEG C of ultrasounds is added to shake frequently
It is completely dissolved to capsule skin；It lets cool, adds 0.5mL acetic acid zinc solutions (219g/L) and 0.5mL potassium ferrocyanide solutions (106g/L),
It is settled to 25mL with absolute ethyl alcohol, after shaking up, puts to 4 DEG C of refrigerator and staticly settles 2h；8000r/min centrifuges 5min, takes supernatant,
It is filtered to get test liquid through 0.45 μm of organic phase filter membrane.Precision draws 10 μ L of test liquid, under above-mentioned chromatographic condition, carries out
Analysis measures.
5, result calculates：
X=C × V/M × K
In formula：The content of Gardenoside, mg/kg in X-sample；
The quality of M-sample, g；
K-unit conversion factor (K=1)；
The extension rate of V-sample, mL；
The concentration of Gardenoside in C-sample solution, μ g/mL.
6, methodology validation：
(1) range of linearity confirms：
Test data is shown in Table 1：(chromatogram is shown in attached drawing 1~5)
1 range of linearity validation test result of table
With a concentration of abscissa, peak area is ordinate, and it is as shown in Figure 6 to draw standard working curve.
Linear test conclusion：Coefficient R is 0.99976, so measuring Gardenoside a concentration of with this method
0.0811957 μ g/mL are good linear to being presented between 1.218 μ g/mL, meet GB/T27404-2008《Good Laboratory control
Specification processed》Requirement【GB/T27404-2008 requires coefficient R >=0.99】.
(2) precision test
Test method：The reference substance solution of Gardenoside is pressed into above-mentioned chromatographic condition replication 6 times, calculates its peak area
RSD (%).
Test data is shown in Table 2：(chromatogram is shown in attached drawing 7~12)
2 Precision test result of table
Conclusion (of pressure testing)：The RSD (%) of 6 peak areas of Gardenoside replication is 2.0%, shows that this method has preferable essence
Density.
(3) repetitive test：
Test method：6 parts of samples are weighed, sample is handled by said sample preparation method, detects sample size, calculate it
RSD (%).
Test data is shown in Table 3：(chromatogram is shown in attached drawing 13~18)
3 repetitive test result of table
Conclusion (of pressure testing)：The RSD of 6 parts of sample Determination of Gardenoside is 1.7%, shows that this method has preferable repeatability, meets
GB/T27404-2008《Good Laboratory controls specification》Requirement【GB/T27404-2008 requires RSD (%)≤7.5%】.
(4) stability test
Test method：After Gardenoside reference substance solution is placed 0h, 1h, 2h, 4h, 8h, 12h at room temperature respectively, by upper
It states chromatographic condition and measures peak area, calculate its RSD (%).
Test data is shown in Table 4：(chromatogram is shown in attached drawing 19~24)
4 stability test result of table
Conclusion (of pressure testing)：After Gardenoside reference substance solution places 0h, 1h, 2h, 4h, 8h, 12h at room temperature respectively, RSD (%)
It is 2.9%, shows Gardenoside reference substance solution having good stability in 12 hours at room temperature.
(5) blank test
Test method：Sample is not weighed, blank solution is handled by said sample preparation method, is measured by above-mentioned chromatographic condition
Blank solution is compared with the appearance time of Gardenoside reference substance solution.
Test result is shown in Figure 25.
Conclusion (of pressure testing)：Blank solution, without absorption peak, shows blank to measurement result base at the appearance time of Gardenoside
This is noiseless.
(6) detection limit is tested with quantitative limit
Detection limit and quantitative limit chromatogram are shown in 26,27 respectively.
The quantitative limit and detection limit of analysis method are calculated by signal-to-noise ratio (S/N).When signal-to-noise ratio (S/N) is 3, detection is limited to
0.024359μg/mL；The detection for obtaining method is limited to 0.226mg/kg.When signal-to-noise ratio (S/N) is 10, quantitatively it is limited to
0.081196μg/mL；It obtains quantifying for method and is limited to 0.752mg/kg.
(7) recovery test：
Test method：
Mark-on：Precision weighs 9 parts of (Determination of Gardenoside of known sample of about 2.7g samples：3.518mg/kg), it is divided into 3 groups,
Every group 3 parts, sample is handled by said sample preparation method.In " adding 10mL water, 40 DEG C of ultrasounds " step, each group is accurate respectively to be added
Enter 15 μ L of storing solution, 45 μ L, 120 μ L of Gardenoside.
Test data such as table 5：(chromatogram is shown in attached drawing 28~36)
5 recovery test result of table
Measure addition=mark-on sample measured amount-sample measured amount
The rate of recovery (%)=measure addition/theoretical addition amount
Conclusion (of pressure testing)：Average recovery rate is：95.3%, relative standard deviation (RSD) is 1.6%, meets GB/T27404-
2008《Good Laboratory controls specification》Requirement【It is 90-110% that GB/T27404-2008, which requires the rate of recovery,】.
Conclusion：
By to the content assaying method of Gardenoside carry out linear, precision, repeatability, stability, blank, detection limit,
Quantitative limit, recovery test, meet GB/T27404-2008《Good Laboratory controls specification》Requirement, it was demonstrated that assay
Methodological science is effective, and the purpose of quality control can be played to the Determination of Gardenoside of bee glue soft capsule.
The detection of 2 Determination of Gardenoside of embodiment
Instrument：1260 high performance liquid chromatograph of Agilent (matches VWD or DAD detectors).
Reagent reagent：Petroleum ether I (AR)；Methanol (AR)；Absolute ethyl alcohol (AR)；Zinc acetate (AR)；Potassium ferrocyanide (AR)；
Acetonitrile (chromatographically pure)；Level-one water.
Gardenoside reference substance source：National Institute for Food and Drugs Control, lot number:110749-201115, purity:
99.7%.
Analytical procedure：
1, chromatographic condition：
Mobile phase：Acetonitrile：Water=15：85；
Flow velocity：0.9mL/min；
Wavelength：239nm；
Column temperature：35℃；
Run time：25min
Chromatographic column：PHenomenex Synergi Hydro-RP chromatographic columns.
2, the preparation of storing solution is compareed：
Precision weighs Gardenoside reference substance 5.09mg, is placed in 50mL brown volumetric flasks, and flowing phased soln and constant volume is added
To scale, shake up to get storing solution.Storing solution is placed in 4 DEG C of refrigerator and stores for future use.
3, standard working curve is drawn：
Precision draws 100 μ L of Gardenoside storing solution, is placed in 25mL brown volumetric flasks, and mobile phase is added and is settled to scale,
It shakes up to get working solution, difference is accurate to draw 2 μ L of working solution, 5 μ L, 10 μ L, 20 μ L, 30 μ L, in above-mentioned chromatographic condition
Under, analysis measurement is carried out, standard working curve is drawn.
4, prepared by sample：
Precision weighs 4 (known Determination of Gardenoside of sample:3.0mg/kg), capsule skin is half-and-half cut off, content, glue are removed
Capsule skin is placed in 50mL centrifuge tubes, and 30mL petroleum ethers I are added and shake 10 seconds, discards petroleum ether I, and repetition washes capsule skin 5 times；It is added
30mL absolute methanols shake 10 seconds, discard absolute methanol；Capsule skin sets another clean centrifuge tube, adds 8mL water, 45 DEG C of ultrasounds, no
When shake to capsule skin and be completely dissolved；It lets cool, adds 0.8mL acetic acid zinc solutions (219g/L) and 0.8mL potassium ferrocyanide solutions
(106g/L) is settled to 25mL with absolute ethyl alcohol, after shaking up, puts to 2 DEG C of refrigerator and staticly settles 1.5h；8000r/min is centrifuged
5min takes supernatant, filters that (theoretical concentration of Gardenoside is to get test liquid through 0.45 μm of organic phase filter membrane：0.3240μg/
mL).Precision draws 10 μ L of test liquid and carries out analysis measurement under above-mentioned chromatographic condition.
5, result calculates：
X=C × V/M × K
In formula：The content of Gardenoside, mg/kg in X-sample；
The quality of M-sample, g；
K-unit conversion factor (K=1)；
The extension rate of V-sample, mL；
The concentration of Gardenoside in C-sample solution, μ g/mL.
6, content detection result：
6 Determination of Gardenoside testing result of table
The detection of 3 Determination of Gardenoside of embodiment
Instrument：1260 high performance liquid chromatograph of Agilent (matches VWD or DAD detectors).
Reagent reagent：Petroleum ether I (AR)；Methanol (AR)；Absolute ethyl alcohol (AR)；Zinc acetate (AR)；Potassium ferrocyanide (AR)；
Acetonitrile (chromatographically pure)；Level-one water.
Gardenoside reference substance source：National Institute for Food and Drugs Control, lot number:110749-201115, purity:
99.7%.+
Analytical procedure：
1, chromatographic condition：
Mobile phase：Methanol：Water=35：65；
Flow velocity：0.8mL/min；
Wavelength：240nm；
Column temperature：30℃；
Run time：25min
Chromatographic column：Agilent ZORBAX SB-C18 chromatographic columns.
2, the preparation of storing solution is compareed：
Precision weighs Gardenoside reference substance 5.09mg, is placed in 50mL brown volumetric flasks, and flowing phased soln and constant volume is added
To scale, shake up to get storing solution.Storing solution is placed in 4 DEG C of refrigerator and stores for future use.
3, standard working curve is drawn：
Precision draws 100 μ L of Gardenoside storing solution, is placed in 25mL brown volumetric flasks, and mobile phase is added and is settled to scale,
It shakes up to get working solution, difference is accurate to draw 2 μ L of working solution, 5 μ L, 10 μ L, 20 μ L, 30 μ L, in above-mentioned chromatographic condition
Under, analysis measurement is carried out, standard working curve is drawn.
4, prepared by sample：
Precision weighs 4 (known Determination of Gardenoside of sample:3.8mg/kg), capsule skin is half-and-half cut off, content, glue are removed
Capsule skin is placed in 50mL centrifuge tubes, and 15mL ether is added and shakes 10 seconds, discards ether, and repetition washes capsule skin 5 times；Be added 15mL without
Water-ethanol shakes 10 seconds, discards absolute ethyl alcohol；Capsule skin sets another clean centrifuge tube, and 5mL water, 50 DEG C of ultrasounds is added to shake frequently
It is completely dissolved to capsule skin；It lets cool, adds 1mL acetic acid zinc solutions (219g/L) and 1mL potassium ferrocyanide solutions (106g/L), with nothing
Water-ethanol is settled to 25mL, after shaking up, puts to 8 DEG C of refrigerator and staticly settles 1h；8000r/min centrifuges 5min, takes supernatant, passes through
0.45 μm of organic phase filter membrane filters that (theoretical concentration of Gardenoside is to get test liquid：0.4104μg/mL).Precision, which is drawn, to be supplied
10 μ L of test solution carry out analysis measurement under above-mentioned chromatographic condition.
5, result calculates：
X=C × V/M × K
In formula：The content of Gardenoside, mg/kg in X-sample；
The quality of M-sample, g；
K-unit conversion factor (K=1)；
The extension rate of V-sample, mL；
The concentration of Gardenoside in C-sample solution, μ g/mL.
6, content detection result：
7 Determination of Gardenoside testing result of table
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (7)
1. the detection method of Determination of Gardenoside in a kind of gelatine capsule, which is characterized in that including：
Step 1：Gelatine capsule is mixed with water, is ultrasonically treated, obtains gelatin solution；
Step 2：Institute's gelatine solution is mixed with acetic acid zinc solution, potassium ferrocyanide solution, absolute ethyl alcohol, is staticly settled, from
The heart takes supernatant, obtains test liquid；
Step 3：The content of Gardenoside in the test liquid is detected using high-efficient liquid phase chromatogram technology；
The mobile phase of the high performance liquid chromatography is methanol aqueous solution, and the volume ratio of methanol and water is in the methanol aqueous solution
35:65；The flow velocity of mobile phase is 0.8~1.0mL/min；The Detection wavelength of the high performance liquid chromatography is 240nm, column temperature 30
℃；The chromatographic column of the high performance liquid chromatography is Agilent ZORBAX SB-C18 chromatographic columns；The specification of the chromatographic column is
250 × 4.6mm, 5 μm.
2. detection method according to claim 1, which is characterized in that the temperature of the supersound process is 40~50 DEG C.
3. detection method according to claim 1, which is characterized in that the usage ratio of agents useful for same in the detection method
For：
4. detection method according to claim 1, which is characterized in that the temperature staticly settled is 2~8 DEG C, the time
For 1~2h.
5. detection method according to claim 1, which is characterized in that the rotating speed of the centrifugation is 5000~8000r/min,
Time is 3~5min.
6. detection method according to claim 1, which is characterized in that further include pre-treatment step before the step 1, it is described
Pre-treatment includes：Take the gelatine capsule for including content, remove content, using the first organic solvent and the second organic solvent into
Row cleaning, first organic solvent are petroleum ether and/or ether, and second organic solvent is absolute methanol or anhydrous second
Alcohol；The content is propolis.
7. detection method according to claim 1, which is characterized in that taken described in step 2 supernatant obtain test liquid it
Between further include the steps that filtering.
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