CN104897809A - Method for measuring content of index components in traditional Chinese medicine, palmleaf raspberry fruit - Google Patents
Method for measuring content of index components in traditional Chinese medicine, palmleaf raspberry fruit Download PDFInfo
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Abstract
The embodiment of the invention discloses a method for measuring the content of index components in a traditional Chinese medicine, palmleaf raspberry fruit. The method comprises the following steps: (1) preparing a test sample solution; (2) preparing a mixed contrast solution; (3) performing ultra high performance liquid chromatography detection; (4) constructing standard curves of N index components; and (5) determining the content of each of the N index components in a test sample. The method for measuring the content of the index components in the traditional Chinese medicine, palmleaf raspberry fruit, is accurate and efficient and can be used for controlling the quality of the traditional Chinese medicine, palmleaf raspberry fruit; furthermore, the method can be used for simultaneously detecting the various index components in the traditional Chinese medicine, palmleaf raspberry fruit; compared with an existing method for respectively detecting different index components, the method disclosed by the invention has the advantages that the total detection time is shortened, and the reagent cost is reduced; meanwhile, the detection result is accurate, the precision is high, the repetitiveness is high, the stability is high, and the specificity is excellent.
Description
Technical field
The present invention relates to traditional Chinese medicine quality control field, particularly the assay method of Chinese medicine raspberry middle finger index composition content.
Background technology
Chinese medicine raspberry is the dry fruit of rose family rubus rubus chingii Hu (Rubus chingii Hu).Gather when early summer fruit is greenish-yellow by green change, removing stalk, leaf, put in boiling water and slightly scald or slightly steam, take out, dry, obtains Chinese medicine raspberry.Chinese medicine raspberry has supplementing the kidney to control the nocturnal contracting urine, effect of nourishing the liver to improve visual acuity, and clinical to be used for the treatment of emission and spermatorrhea, enuresis frequent micturition, impotence and premature ejaculation, order secretly dim-sighted etc.Modern pharmacological research shows that raspberry has anti-oxidant, anti-inflammatory, antithrombotic, anticancer isoreactivity, and its chemical composition mainly contains flavonoids, terpene, steroid, phenolic acid class etc.
" Chinese Pharmacopoeia " version one in 2010 has included Chinese medicine raspberry proterties and powder discriminating, does not include TLC distinguish and assay item.But, be only difficult to scientific evaluation by the discriminating of proterties and powder and control the quality of Chinese medicine raspberry.Only have the content further measuring index components in Chinese medicine raspberry, fundamentally could ensure the drug effect of Chinese medicine raspberry.
Summary of the invention
In order to evaluate and control the quality of Chinese medicine raspberry better, the embodiment of the invention discloses the assay method of Chinese medicine raspberry middle finger index composition content.Technical scheme is as follows:
The assay method of Chinese medicine raspberry middle finger index composition content, comprises the following steps:
(1) need testing solution is prepared
Be that the Chinese medicine raspberry of M mixes with V volume 0 ~ 100% methyl alcohol by quality, obtain the two mixed liquor, determining the weight M of mixed liquor
1after, extraction operation is carried out to it, then with 0 ~ 100% methyl alcohol, the weight of described mixed liquor is complemented to M
1, get supernatant, obtain need testing solution;
(2) preparation mixing reference substance solution
Take the reference substance of N kind index components in Chinese medicine raspberry respectively, dissolve with 0 ~ 100% methyl alcohol respectively, constant volume, obtains the reference substance solution of each index components with concentration known;
Measure the reference substance solution of each index components more respectively, mixing, by 0 ~ 100% methanol constant volume, obtain mixing reference substance storing solution, in described mixing reference substance storing solution, each index components has concentration known;
Reference substance mixing storing solution 0 ~ 100% methyl alcohol stepwise dilution is become a series of mixing reference substance solution respectively with different concentration known;
(3) Ultra Performance Liquid Chromatography detects
Chromatographic condition comprises: Stationary liquid: the chromatographic column adopting octadecylsilane chemically bonded silica; Mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is (3 ~ 70): (97 ~ 30); Flow velocity is 0.2 ~ 1.0mL/min, and column temperature is 30 ~ 50 DEG C, PDA detecting device, and determined wavelength is 253 ~ 344nm;
Under described chromatographic condition, by V
1the need testing solution of volume and the mixing reference substance solution of each concentration inject Ultra Performance Liquid Chromatography instrument respectively, obtain the Ultra Performance Liquid Chromatography figure of need testing solution and the Ultra Performance Liquid Chromatography figure of each concentration mixing reference substance solution;
(4) N kind index components typical curve is separately set up
With the chromatographic peak peak area of each index components in the Ultra Performance Liquid Chromatography figure of each mixing reference substance solution for ordinate or horizontal ordinate, to mix the concentration of each index components in reference substance solution for horizontal ordinate or ordinate, set up the typical curve of each index components respectively;
(5) content that N kind index components is respective in test sample is determined
According to N kind index components chromatographic peak peak area separately in need testing solution Ultra Performance Liquid Chromatography figure, and the typical curve of each index components set up, calculate N kind index components concentration C separately respectively
dense, and calculate N kind index components content separately in test sample according to the following formula respectively:
Content=C
dense* V/M.
In the preferred embodiment of the present invention, ultrasonic extraction is extracted as described in.
In the preferred embodiment of the present invention, the weight of described mixed liquor is being complemented to M
1after, centrifugal, then get supernatant.
In the preferred embodiment of the present invention, described 0 ~ 100% methyl alcohol is 70 ~ 85% methyl alcohol.
In the preferred embodiment of the present invention, described flow velocity is 0.4mL/min.
In the preferred embodiment of the present invention, described mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is 18:82.
In one of the present invention more preferably embodiment, described index components is ellagic acid, and namely during N=1, described chromatographic condition comprises: Stationary liquid: the chromatographic column adopting octadecylsilane chemically bonded silica; Mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is 18:82; Flow velocity 0.4mL/min; Column temperature 40 DEG C; PDA detecting device; Determined wavelength is 253nm.
In one of the present invention more preferably embodiment, described index components is ellagic acid and Kaempferol-O-rutinoside, and namely during N=2, described chromatographic condition comprises: Stationary liquid: the chromatographic column adopting octadecylsilane chemically bonded silica; Mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is 18:82; Flow velocity 0.4mL/min; Column temperature 40 DEG C; PDA detecting device; Determined wavelength is 253nm and 265nm.
In one of the present invention more preferably embodiment, described index components is adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside, tiliroside, namely during N=8, described chromatographic condition comprises: Stationary liquid: the chromatographic column adopting octadecylsilane chemically bonded silica; Mobile phase: acetonitrile: 0.1% formic acid ratio is 0 ~ 2min, 3:97 ~ 5:95; 2 ~ 8min, 5:95 ~ 11.5:88.5; 8 ~ 13.5min, 11.5:88.5 ~ 15.5:84.5; 13.5 ~ 15min, 15.5:84.5 ~ 18:82; 15 ~ 18min, 18:82 ~ 20:80; 18 ~ 23min, 20:80 ~ 38:62; 23 ~ 25min, 38:62 ~ 70:30; Column temperature is 30 DEG C; PDA detecting device; Determined wavelength is 256nm, 265nm, 271nm and 344nm.
The assay method of the Chinese medicine raspberry middle finger index composition content that the present invention builds, precise and high efficiency, can be used for the quality control to Chinese medicine raspberry, and the method can detect many indexes composition in Chinese medicine raspberry simultaneously, relative to the method detected respectively for different index components, shorten and detect T.T., save reagent cost.And testing result is accurate, and precision is high, reproducible, and stability is high, and specificity is strong.
Certainly, arbitrary product of the present invention is implemented or method might not need to reach above-described all advantages simultaneously.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Figure 1A is ellagic acid reference substance solution Ultra Performance Liquid Chromatography figure under 253nm wavelength;
Figure 1B is the chromatogram of Chinese medicine raspberry (Zhejiang) need testing solution under 253nm wavelength;
Fig. 2 A is ellagic acid and Kaempferol-O-rutinoside mixing reference substance solution chromatogram under 253nm wavelength;
Fig. 2 B is ellagic acid and Kaempferol-O-rutinoside mixing reference substance solution chromatogram under 265nm wavelength;
Fig. 2 C is Chinese medicine raspberry (Zhejiang) need testing solution chromatogram under 253nm wavelength;
Fig. 2 D is Chinese medicine raspberry (Zhejiang) need testing solution chromatogram under 265nm wavelength;
Fig. 3 A is the lower 8 kinds of mixing reference substance solution chromatograms of 256nm wavelength;
Fig. 3 B is the lower 8 kinds of mixing reference substance solution chromatograms of 265nm wavelength;
Fig. 3 C is the lower 8 kinds of mixing reference substance solution chromatograms of 271nm wavelength;
Fig. 3 D is the lower 8 kinds of mixing reference substance solution chromatograms of 344nm wavelength;
Fig. 3 E is the chromatogram of Chinese medicine raspberry (Zhejiang) need testing solution under 256nm wavelength;
Fig. 3 F is the chromatogram of Chinese medicine raspberry (Zhejiang) need testing solution under 265nm wavelength;
Fig. 3 G is the chromatogram of Chinese medicine raspberry (Zhejiang) need testing solution under 271nm wavelength;
Fig. 3 H is the chromatogram of Chinese medicine raspberry (Zhejiang) need testing solution under 344nm wavelength.
Embodiment
" Chinese Pharmacopoeia " version one in 2010 has included Chinese medicine raspberry proterties and powder discriminating, does not include TLC distinguish and assay item.In current known Chinese medicine raspberry composition, the content of adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside is very important to the evaluation Chinese medicine raspberry property of medicine.
The present invention establishes a kind of assay method of Chinese medicine raspberry middle finger index composition content.The method according to the needs of those skilled in the art, using one or more in Chinese medicine raspberry composition as index components, can be carried out assay, is not specifically limited this inventor to the kind of index components.
In the inventive solutions, prepare after Chinese medicine raspberry being mixed with 0 ~ 100% methyl alcohol in the step of need testing solution, weigh, after extraction, then weigh, supply weightlessness with the methyl alcohol of same volume mark, get supernatant again, above-mentioned steps can reach two objects: fully extract and constant volume.This constant volume method uses the method for Extraction solvent constant volume again relative to exhausting supernatant, can eliminate artificially supernatant all can not be exhausted produced error in experimental implementation process, and the result of acquisition is closer to theoretical actual value.This constant volume method is the improvement to traditional constant volume method.After the method constant volume, get part supernatant, the demand of subsequent experimental can be met, simplify the experimental procedure of constant volume.Those skilled in the art can expect that 0 ~ 100% methyl alcohol can realize the object index components in Chinese medicine raspberry extracted, but in order to improve extraction efficiency, preferably 70 ~ 85% methyl alcohol are as Extraction solvent, and more preferably volume fraction is that 70% methyl alcohol extracts; In order to ensure when getting supernatant, can not the dregs of a decoction be got, affecting follow-up testing result, after preferably centrifugal, get supernatant.
Technical scheme of the present invention, in preparation reference substance solution process, first dissolves constant volume respectively by index components reference substance, makes the reference substance storing solution with concentration known; The reference substance storing solution that accurate absorption is appropriate respectively again, mixing, by 0 ~ 100% methanol constant volume, obtains the mixing reference substance solution of each index components with concentration known; Finally, stepwise dilution becomes a series of mixing reference substance solution respectively with different concentration known.The object simultaneously detecting several reference substance solution can be realized like this, save the tedious steps for each reference substance difference sample introduction, save time.In the art, dilution 5-8 concentration gradient usually, process is when Criterion curve like this, and linear relationship is good, correctly can evaluate the concentration of index components in need testing solution.
The chromatographic condition of technical solution of the present invention: Stationary liquid: the specification of octadecylsilane chemically bonded silica (C18) chromatographic column is 3.0mm × 100mm, 1.7 μm; Mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, when analysis one or two kind of index components, can isocratic elution be set, namely A phase and B Phase Proportion constant, when analyze more kinds of index components time, in order to various index components is fully separated, guarantee that the composition of each chromatographic peak is single, can be set as gradient elution, namely A phase and B Phase Proportion change along with setting elution time.As in preferred version of the present invention, when the index components of Chinese medicine raspberry detected be ellagic acid or ellagic acid and Kaempferol-O-rutinoside time, mobile phase: acetonitrile: 0.1% formic acid is 18:82; When detection Chinese medicine raspberry 8 kinds of index components (adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside, tiliroside, described reference substance is adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside) time, mobile phase: acetonitrile: 0.1% formic acid ratio is 0 ~ 2min, 3:97 ~ 5:95; 2 ~ 8min, 5:95 ~ 11.5:88.5; 8 ~ 13.5min, 11.5:88.5 ~ 15.5:84.5; 13.5 ~ 15min, 15.5:84.5 ~ 18:82; 15 ~ 18min, 18:82 ~ 20:80; 18 ~ 23min, 20:80 ~ 38:62; 23 ~ 25min, 38:62 ~ 70:30.Flow velocity can be 0.2 ~ 1.0mL/min, preferred 0.4mL/min, column temperature 30 ~ 50 DEG C.The present invention is directed to the chromatographic test strip part of index components setting in Chinese medicine raspberry, the chromatographic peak peak shape of the chromatogram display index components of acquisition is sharp-pointed, does not trail, the composition in test sample thoroughly can be separated, detect data true and accurate.The PDA detecting device that the present invention selects, i.e. photodiode array detector, can detect material at multiple wavelengths, testing result is reliable and stable simultaneously.
0 ~ 100% methyl alcohol described in the present invention refers to the methanol aqueous solution that volume fraction is 0 ~ 100%, and as 70% methyl alcohol refers to that volume fraction is the methanol aqueous solution of 70%, 85% methyl alcohol refers to that volume fraction is the methanol aqueous solution of 85%; Similarly, described 0.1% formic acid to be volume fraction be 0.1 aqueous formic acid.
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Instrument and reagent
Ultra Performance Liquid Chromatography instrument (Waters ACQUITY UPLC
tMsystem, comprises column oven, online vacuum degassing machine, high pressure binary gradient pump, automatic sampler, PDA detecting device, Waters, US); 100000/balance (METTLER TOLEDO XP6, METTLER company of Switzerland); Ten thousand/balance (METTLER TOLEDO AL204; METTLER company of Switzerland); Ultrasonic cleaner (HS6150D, Tianjin permanent AudioCodes skill Development Co., Ltd); Water purification machine (Millipore company of the U.S.).
Methyl alcohol is chromatographically pure (Tianjin Concord Technology Co., Ltd.); Acetonitrile is chromatographically pure (Sigma-Aldrich); Water is self-control ultrapure water.
The reference substance of adenosine, gallic acid is purchased from National Institute for Food and Drugs Control; The reference substance of Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside, tiliroside is self-control.Self-control reference substance preparation process: Chinese medicine raspberry 95% ethanol and 60% ethanol distinguish heating and refluxing extraction 2 times, each 2h, and merge extract, recovered under reduced pressure obtains total medicinal extract.Add appropriate distilled water by total medicinal extract suspendible, use sherwood oil, methylene chloride, ethyl acetate and extracting n-butyl alcohol successively.Acetic acid ethyl ester extract is through silica gel column chromatography, gel column chromatography, preparative liquid chromatography are separated and obtain tiliroside, ellagic acid and progallin A repeatedly.N-butyl alcohol extract is separated through D101 macroporous resin column, respectively with water, 30%, 50%, 70%, the ethanol of 95% carries out gradient elution, normal butyl alcohol 30% ethanol elution part is separated through silica gel column chromatography and obtains Brervifolincaboxylic acid; Normal butyl alcohol 50% ethanol elution part is separated obtains Kaempferol-O-rutinoside and Kampferol-3-O-glucoside through silica gel column chromatography, preparative liquid chromatography.The reference substance more than prepared through high-efficient liquid phase color spectral test purity, and confirms its structure by nuclear magnetic resonance spectrum and Mass Spectrometer Method.
Chinese medicine raspberry (Zhejiang, the place of production), Chinese medicine raspberry (Shandong, the place of production), Chinese medicine raspberry (Henan, the place of production), Chinese medicine raspberry (Anhui, the place of production), Chinese medicine raspberry (Beijing, the place of production), Chinese medicine raspberry (Hubei, the place of production), Chinese medicine raspberry (Hebei, the place of production).
Term " accurately weighed " mentioned in the present invention: accurately weighed mean take weight should accurately to get the per mille of weight." accurate absorption ": mean that the accuracy measuring volume should meet the precise requirements to this measurement instrument in national standard.
In the art, the relative standard deviation (RSD) of the experimental data of assay is within 5%, and illustrative experiment reliable results, can react the content of something truly.
In the art, average recovery is in 95 ~ 105% scopes, and abnormal occurrence does not appear in illustrative experiment link, and the method set up meets assay requirement, and the experimental result obtained is reliable.
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1
(1) preparation of need testing solution
Respectively following process is done to the Chinese medicine raspberry in 7 places of production:
Pulverized by Chinese medicine raspberry, cross 40 mesh sieves, accurately weighed 0.5g, puts in conical flask, and precision adds 70% methyl alcohol 50mL, close plug, weigh 44.1g, room temperature ultrasonic process 30min, then weigh, supply the weight of loss with 70% methanol aqueous solution, shake up, centrifugal 14000 turns of 10min, get supernatant 1mL.
Each process repetition 3 times.
(2) preparation of reference substance solution
Accurately weighed ellagic acid reference substance 7.75mg, adds 100% methyl alcohol and dissolves and be settled to 25mL, be mixed with 0.31mgmL
-1reference substance storing solution.
Accurate absorption ellagic acid reference substance storing solution 1.5mL, and by 100% methanol constant volume to 5mL, be mixed with 93.00 μ gmL
-1reference substance solution, be that 100% methanol solution dilutes gradually by 1mL volume fraction, the concentration of dilution is respectively 46.50 μ gmL
-1, 23.25 μ gmL
-1, 11.63 μ gmL
-1, 5.81 μ gmL
-1, 2.91 μ gmL
-1, 1.45 μ gmL
-1.
(3) Ultra Performance Liquid Chromatography detects
Chromatographic condition: solid phase: ACQUITY BEH C18 (3.0mm × 100mm, 1.7 μm) chromatographic column; Mobile phase: A phase: acetonitrile and B phase: the ratio of 0.1% formic acid is 18:82; Flow velocity: 0.4mLmin
-1; Column temperature 40 DEG C; PDA detecting device, determined wavelength is 253nm.
The accurate ellagic acid reference substance solution 3 μ L drawing need testing solution and variable concentrations respectively, injects Ultra Performance Liquid Chromatography instrument (UPLC) and detects.The chromatogram of ellagic acid reference substance solution as shown in Figure 1A.The chromatogram of need testing solution is for the chromatogram (Figure 1B) of the Chinese medicine raspberry need testing solution in the place of production, Zhejiang.
Can find out under 253nm determined wavelength from Figure 1A, the chromatographic peak that 2.70min occurs is the chromatographic peak of ellagic acid, and namely the retention time of ellagic acid is 2.70min.Correspondingly, in fig. ib, namely the chromatographic peak that 2.70min occurs is the chromatographic peak of ellagic acid in the Chinese medicine raspberry need testing solution in the place of production, Zhejiang, and realizes good separation with other compositions.
(4) typical curve of ellagic acid is set up
With the concentration of ellagic acid in reference substance solution for horizontal ordinate, with the chromatographic peak peak area of ellagic acid in the Ultra Performance Liquid Chromatography figure of reference substance solution for ordinate, set up the typical curve of ellagic acid, with signal to noise ratio (S/N ratio) (S/N) 10 for quantitative limit, the results are shown in Table 1.As can be known from Table 1, ellagic acid is at 1.45 ~ 93 μ gmL
-1in concentration range, typical curve linear relationship is good.
The equation of linear regression of table 1 ellagic acid, related coefficient, the range of linearity, quantitative limit
(5) content of ellagic acid in test sample is determined
According to the chromatographic peak peak area of ellagic acid in need testing solution Ultra Performance Liquid Chromatography figure, and the typical curve of the ellagic acid set up, calculate the concentration of ellagic acid, and calculate the content of ellagic acid in test sample according to the following formula:
Content=C
dense* V/M.
Obtain the content of index components ellagic acid in the Chinese medicine raspberry in 7 places of production according to this formula, the results are shown in Table 2.
Index components ellagic acid assay result (Mean ± SD, n=3) in table 2 Chinese medicine raspberry
With the place of production be below Zhejiang Chinese medicine raspberry for example carry out precision, stability, repeatability and average recovery test.
(6) precision test
Need testing solution is prepared by the step (1) of embodiment 1, by embodiment 1 step (3) chromatographic condition, continuous sample introduction 6 times, each 3 μ L, detect the content of ellagic acid in need testing solution, evaluate the withinday precision of ellagic acid, the results are shown in Table 3.Prepare need testing solution by embodiment 1 step (1), by embodiment 1 step (3) chromatographic condition, continuous three days sample introductions, 6 times/day, 3 μ L/ time, measure, evaluate the day to day precision of ellagic acid, the results are shown in Table 4.Known from table 3 and table 4, the RSD of the ellagic acid content in a few days obtained with day to day precision test is respectively 0.33%, 2.31%.Result shows that ellagic acid precision is good, meets the requirement of assay.
The withinday precision test findings (mg/g, n=6) of table 3 ellagic acid
The day to day precision test findings (mg/g, n=3) of table 4 ellagic acid
(7) stability test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 0.5g, need testing solution is prepared by the method for embodiment 1 step (1), by the chromatographic condition of embodiment 1 step (3), respectively at 0,2,4,8,10 and 12h sample introduction 3 μ L, measure, investigate the stability of ellagic acid 12h in need testing solution, the results are shown in Table 5.As known from Table 5, the relative standard deviation (RSD) of ellagic acid content is 2.24%.Result shows that ellagic acid in need testing solution is basicly stable in 12h at ambient temperature.
Table 5 ellagic acid stability test result (mg/g, n=6)
(8) replica test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 0.5g, and by method parallel processing 6 parts of need testing solutions of embodiment 1 step (1), by the chromatographic condition sample introduction 3 μ L respectively of embodiment 1 step (3), measured, the results are shown in Table 6.As known from Table 6, the RSD of ellagic acid content is 0.49%.Result shows that detection method of the present invention repeatability is good.
Table 6 ellagic acid replica test result (mg/g, n=6)
(9) average recovery test
Accurately weighedly pulverize 40 object Chinese medicine raspberry 0.25g, accurately add ellagic acid reference substance storing solution (the ellagic acid reference substance Stock concentrations 0.31mgmL suitable with ellagic acid amount in test sample respectively
-1, mark-on volume 1mL), by method parallel processing 6 parts of test samples of embodiment 1 step (1), by the chromatographic condition sample introduction 3 μ L respectively of embodiment 1 step (3), measure, calculate average recovery, the results are shown in Table 7.
The average recovery test findings (n=6) of index components ellagic acid in table 7 Chinese medicine raspberry
Embodiment 2
(1) preparation of need testing solution
With embodiment 1 step (1).
(2) preparation of reference substance solution
Accurately weighed ellagic acid reference substance and Kaempferol-O-rutinoside reference substance 7.75mg, 8.18mg respectively, adds 100% methyl alcohol respectively and dissolves and be settled to 25mL, 10mL respectively, be mixed with 0.31mgmL respectively
-1ellagic acid reference substance storing solution and 0.82mgmL
-1kaempferol-O-rutinoside reference substance storing solution.
Accurate absorption ellagic acid reference substance storing solution and Kaempferol-O-rutinoside reference substance storing solution 1.5mL, 0.2mL mixing respectively, and by 100% methanol constant volume to 5mL, be mixed with 93.00 μ gmL
-1ellagic acid, 32.72 μ gmL
-1kaempferol-O-rutinoside mixing reference substance solution, be 100% methanol solution stepwise dilution by 1mL volume fraction respectively, ellagic acid concentration is respectively 46.50,23.25,11.63,5.81,2.91,1.45 μ gmL
-1, Kaempferol-O-rutinoside concentration is respectively 16.36,8.18,4.09,2.05,1.02,0.51 μ gmL
-1.
(3) Ultra Performance Liquid Chromatography detects
Chromatographic condition: Stationary liquid: ACQUITY BEH C18 (3.0mm × 100mm, 1.7 μm) chromatographic column; Mobile phase: A phase: acetonitrile and B phase: the ratio of 0.1% formic acid is 18:82; Flow velocity: 0.4mLmin
-1; Column temperature 40 DEG C; PDA detecting device, determined wavelength is 253nm and 265nm.
Accurate ellagic acid and the Kaempferol-O-rutinoside mixing reference substance solution 3 μ L drawing need testing solution and variable concentrations, injects Ultra Performance Liquid Chromatography instrument (UPLC) and detects respectively.The chromatogram of ellagic acid and Kaempferol-O-rutinoside reference substance solution respectively as shown in Figure 2 A and 2 B.The chromatogram of need testing solution is for the chromatogram of the Chinese medicine raspberry need testing solution in the place of production, Zhejiang (Fig. 2 C and Fig. 2 D).
As can be seen from Fig. 2 A, under 253nm wavelength, the retention time of ellagic acid is 2.70min.Correspondingly, in Fig. 2 C, be exactly the chromatographic peak of ellagic acid in the Chinese medicine raspberry need testing solution in the place of production, Zhejiang at the chromatographic peak at 2.70min place, and realize good separation with other compositions.As can be seen from Fig. 2 B, under 265nm wavelength, the retention time of Kaempferol-O-rutinoside is 3.85min.Correspondingly, in Fig. 2 D, be exactly the chromatographic peak of Kaempferol-O-rutinoside in the Chinese medicine raspberry need testing solution in the place of production, Zhejiang at the chromatographic peak at 3.85min place, and realize good separation with other compositions.
(4) typical curve of ellagic acid, Kaempferol-O-rutinose is set up respectively
To mix the concentration of ellagic acid reference substance in reference substance solution for horizontal ordinate, with the chromatographic peak peak area of ellagic acid in the Ultra Performance Liquid Chromatography figure mixing reference substance solution for ordinate, set up the typical curve of ellagic acid.
To mix the concentration of Kaempferol-O-rutinoside reference substance in reference substance solution for horizontal ordinate, with the chromatographic peak peak area of Kaempferol-O-rutinoside in the Ultra Performance Liquid Chromatography figure mixing reference substance solution for ordinate, set up the typical curve of Kaempferol-O-rutinoside.
The two all with signal to noise ratio (S/N ratio) (S/N) 10 for quantitative limit, the results are shown in Table 8.As can be known from Table 8, two indices composition is good in respective concentration range internal linear relation.
Equation of linear regression, related coefficient, the range of linearity, the quantitative limit of table 8 ellagic acid and Kaempferol-O-rutinoside
(5) ellagic acid and the content respective in test sample of Kaempferol-O-rutinoside is determined
Respectively according to the chromatographic peak peak area of ellagic acid and Kaempferol-O-rutinoside in need testing solution Ultra Performance Liquid Chromatography figure, and the typical curve of the ellagic acid set up and Kaempferol-O-rutinoside, calculate the concentration of ellagic acid and Kaempferol-O-rutinoside respectively, and calculate the content of ellagic acid and Kaempferol-O-rutinoside in test sample according to the following formula:
Content=C
dense* V/M.
Obtain the content of index components ellagic acid and Kaempferol-O-rutinoside in the Chinese medicine raspberry in 7 places of production according to this formula, the results are shown in Table 9.
Index components ellagic acid, Kaempferol-O-rutinoside assay result (Mean ± SD, n=3) in table 9 Chinese medicine raspberry
With the place of production be below Zhejiang Chinese medicine raspberry for example carry out precision, stability, repeatability and average recovery test.
(6) precision test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 0.5g, need testing solution is prepared by the method for embodiment 1 step (1), by embodiment 2 step (3) chromatographic condition, continuous sample introduction 6 times, each 3 μ L, detect the content of ellagic acid, Kaempferol-O-rutinoside in need testing solution, evaluate the withinday precision of ellagic acid, Kaempferol-O-rutinoside, the results are shown in Table 10.Need testing solution is prepared by embodiment 1 step (1), by embodiment 1 step (3) chromatographic condition, continuous three days sample introductions, 6 times/day, 3 μ L/ time, measure, evaluate the day to day precision of ellagic acid, Kaempferol-O-rutinoside, the results are shown in Table 11.From table 10 and 11, the ellagic acid that withinday precision test obtains, the RSD of Kaempferol-O-rutinoside content are respectively 0.33% and 1.08%, and the ellagic acid that day to day precision test obtains, the RSD of Kaempferol-O-rutinoside content are respectively 2.31% and 0.89%.Result shows that ellagic acid, Kaempferol-O-rutinoside precision are good, meets the requirement of assay.
The withinday precision test findings (mg/g, n=6) of table 10 ellagic acid and Kaempferol-O-rutinoside
The day to day precision test findings (mg/g, n=3) of table 11 ellagic acid and Kaempferol-O-rutinoside
(7) stability test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 0.5g, need testing solution is prepared by the method for embodiment 1 step (1), by the chromatographic condition of embodiment 2 step (3), respectively at 0,2,4,8,10 and 12h sample introduction 3 μ L, measure, investigate the stability of ellagic acid, Kaempferol-O-rutinoside 12h in need testing solution, the results are shown in Table 12.As known from Table 12, the RSD of ellagic acid, Kaempferol-O-rutinoside content is respectively 2.24%, 1.90%.Result shows that ellagic acid in need testing solution and Kaempferol-O-rutinoside are basicly stable in 12h at ambient temperature.
Table 12 ellagic acid and Kaempferol-O-rutinoside stability test result (mg/g, n=6)
(8) replica test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 0.5g, and by method parallel processing 6 parts of need testing solutions of embodiment 1 step (1), by the chromatographic condition sample introduction 3 μ L respectively of embodiment 2 step (3), measured, the results are shown in Table 13.As known from Table 13, the RSD of ellagic acid, Kaempferol-O-rutinoside content is respectively 0.49%, 0.42%.Result shows that detection method of the present invention repeatability is good.
Table 13 ellagic acid and Kaempferol-O-rutinoside replica test result (mg/g, n=6)
(9) average recovery test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 0.25g, accurately added respectively and ellagic acid in test sample, ellagic acid, Kaempferol-O-rutinoside reference substance storing solution (ellagic acid reference substance Stock concentrations 0.31mgmL that Kaempferol-O-rutinoside amount is suitable
-1, mark-on volume 1mL; Kaempferol-O-rutinoside reference substance Stock concentrations 0.82mgmL
-1, mark-on volume 120 μ L), by method parallel processing 6 parts of test samples of embodiment 1 step (1), by the chromatographic condition sample introduction 3 μ L respectively of embodiment 2 step (3), measure, calculate average recovery, the results are shown in Table 14.
The average recovery test findings (n=6) of index components ellagic acid and Kaempferol-O-rutinoside in table 14 Chinese medicine raspberry
Embodiment 3
(1) preparation of need testing solution
Respectively following process is done to the Chinese medicine raspberry in 7 places of production:
Pulverized by Chinese medicine raspberry, cross 40 mesh sieves, accurately weighed 1.0g, puts in conical flask, and precision adds 70% methyl alcohol 50mL, close plug, weigh 44.6g, and room temperature ultrasonic process 30min, weighs, supply the weight of loss with 70% methanol aqueous solution, shake up, centrifugal 14000 turns of 10min, get supernatant 1mL.
Each process repetition 3 times.
(2) preparation of reference substance solution
Accurately weighed adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside reference substance 5.80mg, 6.90mg, 7.43mg, 6.89mg, 6.33mg, 7.65mg and 5.89mg respectively, add 100% methyl alcohol (containing a small amount of DMSO) respectively dissolve and be settled to 5mL respectively, accurately weighed ellagic acid reference substance 2.91mg, add 100% methyl alcohol dissolve and be settled to 10mL, be mixed with 1.16mgmL respectively
-1adenosine, 1.38mgmL
-1gallic acid, 1.49mgmL
-1brervifolincaboxylic acid, 1.38mgmL
-1progallin A, 0.29mgmL
-1ellagic acid, 1.27mgmL
-1kaempferol-O-rutinoside, 1.53mgmL
-1kampferol-3-O-glucoside, 1.18mgmL
-1the reference substance storing solution of tiliroside.
Accurate adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside reference substance storing solution 200 μ L, 150 μ L, 200 μ L, 400 μ L, 4000 μ L, 500 μ L, 150 μ L and the 300 μ L of drawing mix respectively, and by 85% methanol constant volume to 10mL, be mixed with 23.20 μ gmL
-1adenosine, 20.70 μ gmL
-1gallic acid, 29.72 μ gmL
-1brervifolincaboxylic acid, 55.12 μ gmL
-1progallin A, 116.40 μ gmL
-1ellagic acid, 63.30 μ gmL
-1kaempferol-O-rutinoside, 22.95 μ gmL
-1kampferol-3-O-glucoside, 35.34 μ gmL
-1tiliroside mixing reference substance solution, and be 100% methanol solution stepwise dilution by 1mL volume fraction, adenosine concentration is respectively 11.60,5.80,2.90,1.45,0.73,0.36 μ gmL
-1, gallic acid concentration is respectively 10.35,5.18,2.59,1.29,0.65,0.32 μ gmL
-1, brevifolin acid concentration is respectively 14.86,7.43,3.72,1.86,0.93,0.46 μ gmL
-1, progallin A is respectively 27.56,13.78,6.89,3.45,1.72,0.86 μ gmL
-1, ellagic acid concentration is respectively 58.20,29.10,14.55,7.28,3.64,1.82 μ gmL
-1, Kaempferol-O-rutinoside concentration is respectively 31.65,15.83,7.91,3.96,1.98,0.99 μ gmL
-1, Kampferol-3-O-glucoside concentration is respectively 11.48,5.74,2.87,1.43,0.72,0.36 μ gmL
-1, tiliroside concentration is respectively 17.67,8.84,4.42,2.21,1.10,0.55 μ gmL
-1.
(3) Ultra Performance Liquid Chromatography detects
Chromatographic condition: Stationary liquid: ACQUITY BEH C18 (3.0mm × 100mm, 1.7 μm) chromatographic column; Mobile phase: A phase: acetonitrile and B phase: the ratio of 0.1% formic acid is acetonitrile: 0.1% formic acid ratio is 0 ~ 2min, 3:97 ~ 5:95; 2 ~ 8min, 5:95 ~ 11.5:88.5; 8 ~ 13.5min, 11.5:88.5 ~ 15.5:84.5; 13.5 ~ 15min, 15.5:84.5 ~ 18:82; 15 ~ 18min, 18:82 ~ 20:80; 18 ~ 23min, 20:80 ~ 38:62; 23 ~ 25min, 38:62 ~ 70:30; Flow velocity: 0.4mLmin
-1; Column temperature 30 DEG C; PDA detecting device, determined wavelength is 256nm, 265nm, 271nm, 344nm.
The accurate mixing reference substance solution 3 μ L drawing need testing solution and variable concentrations respectively, injects Ultra Performance Liquid Chromatography instrument (UPLC) and detects.Adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside, the chromatogram of 8 kinds of reference substance solution is respectively as shown in Fig. 3 A ~ 3D.The chromatogram of need testing solution is for the chromatogram (Fig. 3 E ~ 3H) of the Chinese medicine raspberry need testing solution in the place of production, Zhejiang.
As can be seen from Fig. 3 A, under 256nm wavelength, the retention time of adenosine is 2.16min, and the retention time of ellagic acid is 14.39min.Correspondingly, in Fig. 3 E, be respectively the chromatographic peak of adenosine and ellagic acid in the Chinese medicine raspberry need testing solution in the place of production, Zhejiang at the chromatographic peak at 2.16min and 14.39min place, and realize good separation with other compositions.As can be seen from Fig. 3 B, under 265nm wavelength, the retention time of tiliroside is 22.12min.Correspondingly, in Fig. 3 F, be exactly the chromatographic peak of tiliroside in the Chinese medicine raspberry need testing solution in the place of production, Zhejiang at the chromatographic peak at 22.12min place, and realize good separation with other compositions.As can be seen from Fig. 3 C, under 271nm wavelength, the retention time of gallic acid is 2.67min, and the retention time of progallin A is 12.44min.Correspondingly, in Fig. 3 G, be respectively the chromatographic peak of gallic acid and progallin A in the Chinese medicine raspberry need testing solution in the place of production, Zhejiang at the chromatographic peak at 2.67min and 12.44min place, and realize good separation with other compositions.As can be seen from Fig. 3 D, under 344nm wavelength, the retention time of Brervifolincaboxylic acid is 8.54min, and the retention time of Kaempferol-O-rutinoside is 17.16min, and the retention time of Kampferol-3-O-glucoside is 17.77min.Correspondingly, in Fig. 3 H, be respectively the chromatographic peak of Brervifolincaboxylic acid, Kaempferol-O-rutinoside and Kampferol-3-O-glucoside in the Chinese medicine raspberry need testing solution in the place of production, Zhejiang at the chromatographic peak at 8.54min, 17.16min and 17.77min place, and realize good separation with other compositions.
(4) typical curve of adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside is set up respectively.
Respectively to mix adenosine in reference substance, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, the concentration of Kampferol-3-O-glucoside and tiliroside is horizontal ordinate, respectively with adenosine in the Ultra Performance Liquid Chromatography figure mixing reference substance solution, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, the chromatographic peak peak area of Kampferol-3-O-glucoside and tiliroside is ordinate, set up adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside typical curve separately.All with signal to noise ratio (S/N ratio) (S/N) 10 for quantitative limit, the results are shown in Table 15.As can be known from Table 15, adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside are good in respective concentration range internal linear relation.
Equation of linear regression, related coefficient, the range of linearity, the quantitative limit of 8 kinds of reference substances in table 15 Chinese medicine raspberry
(5) content that adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside are respective in test sample is determined
Respectively according to the chromatographic peak peak area of adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside in need testing solution Ultra Performance Liquid Chromatography figure, and corresponding typical curve with it, calculate the concentration of adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside respectively, and calculate the content of 8 kinds of index components in test sample according to the following formula:
Content=C
dense* V/M.
Obtain the content of 8 kinds of index components in the Chinese medicine raspberry in 7 places of production according to this formula, the results are shown in Table 16.
8 kinds of index components assay results (Mean ± SD, n=3) in table 16 Chinese medicine raspberry
With the place of production be below Zhejiang Chinese medicine raspberry for example carry out precision, stability, repeatability and average recovery test.
(6) precision test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 1.0g, need testing solution is prepared by the method for embodiment 1 step (1), by embodiment 3 step (3) chromatographic condition, continuous sample introduction 6 times, each 3 μ L, detect the content of 8 kinds of index components in need testing solution, evaluate the withinday precision of 8 kinds of index components respectively, the results are shown in Table 17.Prepare need testing solution by embodiment 3 step (1), by embodiment 3 step (3) chromatographic condition, continuous three days sample introductions, 6 times/day, 3 μ L/ time, measure, evaluate the day to day precision of 8 kinds of index components, the results are shown in Table 18.From table 17 and 18, the adenosine that withinday precision test obtains, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, the RSD of Kampferol-3-O-glucoside and tiliroside content is respectively 0.68%, 1.74%, 1.53%, 2.45%, 1.43%, 0.58%, 0.58%, 0.90%, the adenosine that day to day precision test obtains, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, the RSD of Kampferol-3-O-glucoside and tiliroside content is respectively 0.97%, 1.49%, 1.98%, 2.78%, 1.61%, 0.68%, 1.46%, 0.94%.Show that the precision of adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside is good, meet the requirement of assay.
The withinday precision test findings (mg/g, n=6) of table 17 8 kinds of index components
The day to day precision test findings (mg/g, n=3) of table 18 8 kinds of index components
(7) stability test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 1.0g, need testing solution is prepared by the method for embodiment 1 step (1), by the chromatographic condition of embodiment 3 step (3), respectively at 0,2,4,8,10 and 12h sample introduction 3 μ L, measure, investigate the stability of 8 kinds of index components in need testing solution, the results are shown in Table 19.As known from Table 19, the RSD of adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside content is respectively 1.00%, 0.96%, 1.01%, 3.00%, 0.58%, 0.89%, 1.65%, 0.68%.Result shows that adenosine in need testing solution, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside are basicly stable in 12h at ambient temperature.
The stability test result (mg/g, n=6) of table 19 8 kinds of index components
(8) replica test
Accurately weighedly pulverized 40 object Chinese medicine raspberry 0.5g, by method parallel processing 6 parts of need testing solutions of embodiment 1 step (1), by the chromatographic condition sample introduction 3 μ L respectively of embodiment 3 step (3), measure, the results are shown in Table 20, the RSD of adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside content is respectively 1.75%, 1.69%, 1.04%, 2.60%, 1.68%, 0.96%, 1.67%, 3.65%.Result shows that detection method of the present invention repeatability is good.
The reperformance test result (mg/g, n=6) of table 20 8 kinds of index components
(9) average recovery test
Accurately weighedly pulverize 40 object Chinese medicine raspberry 0.5g, accurately add each reference substance storing solution suitable with tiliroside amount with adenosine in test sample, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside or reference substance solution (adenosine reference substance solution concentration 116 μ gmL respectively
-1, mark-on volume 500 μ L; Gallic acid reference substance solution concentration 138 μ gmL
-1, mark-on volume 320 μ L; Brervifolincaboxylic acid reference substance Stock concentrations 1486 μ gmL
-1, mark-on volume 60 μ L; Progallin A reference substance Stock concentrations 1378 μ gmL
-1, mark-on volume 120 μ L; Ellagic acid reference substance solution concentration 327 μ gmL
-1, mark-on volume 2000 μ L; Kaempferol-O-rutinoside reference substance Stock concentrations 1266 μ gmL
-1, mark-on volume 150 μ L; Kampferol-3-O-glucoside reference substance solution concentration 153 μ gmL
-1, mark-on volume 420 μ L; Tiliroside reference substance Stock concentrations 1178 μ gmL
-1, mark-on volume 80 μ L), by method parallel processing 6 parts of test samples of embodiment 1 step (1), by the chromatographic condition sample introduction 3 μ L respectively of embodiment 3 step (3), measure, calculate average recovery, the results are shown in Table 21.
The average recovery test findings (n=6) of 8 kinds of index components in table 21 Chinese medicine raspberry
It should be noted that, as can be seen from embodiment 1,2 and 3, preparing in need testing solution step, difference is only that the quality of accurately weighed Chinese medicine raspberry powder is different, and embodiment 1 and 2 is 0.5g, and embodiment 3 is 1.0g.No matter be 0.5g or 1.0g Chinese medicine raspberry powder, with the process of 50mL 70% methyl alcohol, the addition of 70% methyl alcohol is all excessive, can reach the object fully extracted.That is embodiment 1,2 is the same with 3 for the preparation method of need testing solution.Based on this, the precision test of each embodiment, stability test, replica test are also the same with the preparation method about need testing solution in average recovery test.
It should be noted that, when preparing reference substance solution, embodiment 1 and 2 uses 70% Methanol for reference substance solution, and embodiment 3 uses 85% Methanol for reference substance solution.Due in the present invention, when carrying out Ultra Performance Liquid Chromatography detection, sample size is only 3 μ L, to whole detection system---and Ultra Performance Liquid Chromatography instrument-PDA detecting device and chromatographic condition do not produce fluctuation, namely do not have an impact to the testing result of various index components.
It should be noted that, in Fig. 3, by the retention time of 8 kinds of index components in Chinese medicine raspberry, 1 represents adenosine, 2 represents gallic acid, 3 and represents Brervifolincaboxylic acid, 4 and represent progallin A, 5 and represent ellagic acid, 6 and represent Kaempferol-O-rutinoside, 7 and represent Kampferol-3-O-glucoside, 8 and represent tiliroside, wherein as shown in Figure 3A: adenosine (1) and ellagic acid (5) reference substance determined wavelength are 256nm; As shown in Figure 3 B: tiliroside (8) reference substance determined wavelength is 265nm; As shown in Figure 3 C: gallic acid (2) and progallin A (4) reference substance determined wavelength are 271; As shown in Figure 3 D: Brervifolincaboxylic acid (3), Kaempferol-O-rutinoside (6), Kampferol-3-O-glucoside (7) reference substance determined wavelength are 344nm.Fig. 3 E is corresponding with Fig. 3 A, and detect the chromatographic peak of adenosine and ellagic acid in need testing solution, Fig. 3 F is corresponding with Fig. 3 B, detects the chromatographic peak of tiliroside in need testing solution; Fig. 3 G is corresponding with Fig. 3 C, detects the chromatographic peak of gallic acid and progallin A in need testing solution; Fig. 3 H is corresponding with Fig. 3 D, detects the chromatographic peak of Brervifolincaboxylic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside in need testing solution.
It should be noted that, the maximum absorption wavelength scope of ellagic acid is at 253 ~ 256nm, and the determined wavelength setting ellagic acid in embodiment 1 and embodiment 2 is 253nm.In embodiment 3, the maximum absorption wavelength of adenosine is 256nm, and the maximum absorption wavelength difference of the two is little, in order to reduce the number of wavelengths of setting, the determined wavelength of ellagic acid being adjusted to 256nm, almost not affecting the testing result of ellagic acid.This is that those skilled in the art can adjust according to actual conditions and experience, does not affect the reliability of testing result.
It should be noted that, in embodiment 3, when preparing reference substance mixed solution, adding a small amount of dimethyl sulfoxide (DMSO) (DMSO) is to make the reference substance of 8 kinds of index components fully be dissolved in up hill and dale in 85% methyl alcohol, does not have an impact to various index components content detection result.
It should be noted that, though the present invention detects many indexes composition simultaneously, the Ultra Performance Liquid Chromatography instrument used is published picture respectively according to the determined wavelength of setting.For embodiment 2, set 2 determined wavelength, be respectively 253nm, 265nm, then, under obtaining 253nm wavelength, reference substance and each figure of test sample, under 265nm wavelength, reference substance and each figure of test sample, amount to 4 figure.
According to the technical scheme of embodiment 3; those skilled in the art are without the need to creative work; one or more in adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside can be detected according to this technical scheme simultaneously; that is; detect the technical scheme of index components in Chinese medicine raspberry according to the present invention, the index components in any one or more Chinese medicine raspberry is detected and all belongs to protection scope of the present invention.
As can be seen from the above results, the present invention utilizes Ultra Performance Liquid Chromatography instrument to measure the quantitative analysis method of index components in constructed Chinese medicine raspberry, adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside can be detected at most, 8 kinds of index components simultaneously.The method, relative to the method detected respectively for different index components, shortens and detects T.T., saves real cost.And testing result is accurate, and precision is high, reproducible, and stability is high, and specificity is strong.
The foregoing is only preferred embodiment of the present invention, be not intended to limit protection scope of the present invention.All any amendments done within the spirit and principles in the present invention, equivalent replacement, improvement etc., be all included in protection scope of the present invention.
Claims (9)
1. the assay method of Chinese medicine raspberry middle finger index composition content, comprises the following steps:
(1) need testing solution is prepared
Be that the Chinese medicine raspberry of M mixes with V volume 0 ~ 100% methyl alcohol by quality, obtain the two mixed liquor, determining the weight M of mixed liquor
1after, extraction operation is carried out to it, then with 0 ~ 100% methyl alcohol, the weight of described mixed liquor is complemented to M
1, get supernatant, obtain need testing solution;
(2) preparation mixing reference substance solution
Take the reference substance of N kind index components in Chinese medicine raspberry respectively, dissolve with 0 ~ 100% methyl alcohol respectively, constant volume, obtains the reference substance solution of each index components with concentration known;
Measure the reference substance solution of each index components more respectively, mixing, by 0 ~ 100% methanol constant volume, obtain mixing reference substance storing solution, in described mixing reference substance storing solution, each index components has concentration known;
Reference substance mixing storing solution 0 ~ 100% methyl alcohol stepwise dilution is become a series of mixing reference substance solution respectively with different concentration known;
(3) Ultra Performance Liquid Chromatography detects
Chromatographic condition comprises: Stationary liquid: the chromatographic column adopting octadecylsilane chemically bonded silica; Mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is (3 ~ 70): (97 ~ 30); Flow velocity is 0.2 ~ 1.0mL/min, and column temperature is 30 ~ 50 DEG C, PDA detecting device, and determined wavelength is 253 ~ 344nm;
Under described chromatographic condition, by V
1the need testing solution of volume and the mixing reference substance solution of each concentration inject Ultra Performance Liquid Chromatography instrument respectively, obtain the Ultra Performance Liquid Chromatography figure of need testing solution and the Ultra Performance Liquid Chromatography figure of each concentration mixing reference substance solution;
(4) N kind index components typical curve is separately set up
With the chromatographic peak peak area of each index components in the Ultra Performance Liquid Chromatography figure of each mixing reference substance solution for ordinate or horizontal ordinate, to mix the concentration of each index components in reference substance solution for horizontal ordinate or ordinate, set up the typical curve of each index components respectively;
(5) content that N kind index components is respective in test sample is determined
According to N kind index components chromatographic peak peak area separately in need testing solution Ultra Performance Liquid Chromatography figure, and the typical curve of each index components set up, calculate N kind index components concentration C separately respectively
dense, and calculate N kind index components content separately in test sample according to the following formula respectively:
Content=C
dense* V/M.
2. the assay method of Chinese medicine raspberry middle finger index composition content as claimed in claim 1, is characterized in that, described in be extracted as ultrasonic extraction.
3. the assay method of Chinese medicine raspberry middle finger index composition content as claimed in claim 1, is characterized in that, the weight of described mixed liquor is being complemented to M
1after, centrifugal, then get supernatant.
4. the assay method of Chinese medicine raspberry middle finger index composition content as claimed in claim 1, it is characterized in that, described 0 ~ 100% methyl alcohol is 70 ~ 85% methyl alcohol.
5. the assay method of Chinese medicine raspberry middle finger index composition content as claimed in claim 1, it is characterized in that, described flow velocity is 0.4mL/min.
6. the assay method of Chinese medicine raspberry middle finger index composition content as claimed in claim 1, it is characterized in that, described mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is 18:82.
7. the assay method of the Chinese medicine raspberry middle finger index composition content as described in any one of claim 1,2,3,4 or 5, it is characterized in that, described index components is ellagic acid, and namely during N=1, described chromatographic condition comprises: Stationary liquid: the chromatographic column adopting octadecylsilane chemically bonded silica; Mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is 18:82; Flow velocity 0.4mL/min; Column temperature 40 DEG C; PDA detecting device; Determined wavelength is 253nm.
8. the assay method of the Chinese medicine raspberry middle finger index composition content as described in any one of claim 1,2,3,4 or 5, it is characterized in that, described index components is ellagic acid and Kaempferol-O-rutinoside, namely during N=2, described chromatographic condition comprises: Stationary liquid: the chromatographic column adopting octadecylsilane chemically bonded silica; Mobile phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is 18:82; Flow velocity 0.4mL/min; Column temperature 40 DEG C; PDA detecting device; Determined wavelength is 253nm and 265nm.
9. the assay method of the Chinese medicine raspberry middle finger index composition content as described in any one of claim 1,2,3,4 or 5, it is characterized in that, described index components is adenosine, gallic acid, Brervifolincaboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside, tiliroside, namely during N=8, described chromatographic condition comprises: Stationary liquid: the chromatographic column adopting octadecylsilane chemically bonded silica; Mobile phase: acetonitrile: 0.1% formic acid ratio is 0 ~ 2min, 3:97 ~ 5:95; 2 ~ 8min, 5:95 ~ 11.5:88.5; 8 ~ 13.5min, 11.5:88.5 ~ 15.5:84.5; 13.5 ~ 15min, 15.5:84.5 ~ 18:82; 15 ~ 18min, 18:82 ~ 20:80; 18 ~ 23min, 20:80 ~ 38:62; 23 ~ 25min, 38:62 ~ 70:30; Column temperature is 30 DEG C; PDA detecting device; Determined wavelength is 256nm, 265nm, 271nm and 344nm.
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| CN110674972A (en) * | 2019-08-26 | 2020-01-10 | 浙江省农业科学院 | Method for predicting content of raspberry kaempferol-3-O-rutinoside and ellagic acid |
| CN112129851A (en) * | 2020-08-23 | 2020-12-25 | 浙江省农业科学院 | Establishment method of raspberry polyphenol fingerprint spectrum |
| CN112913480A (en) * | 2021-01-26 | 2021-06-08 | 浙江锦珩健康科技有限责任公司 | Method for determining picking period of medicinal raspberry |
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| CN109633002A (en) * | 2018-12-26 | 2019-04-16 | 成都普思生物科技股份有限公司 | A kind of method of Kaempferol-O- rutinoside content in measurement safflower |
| CN110674972A (en) * | 2019-08-26 | 2020-01-10 | 浙江省农业科学院 | Method for predicting content of raspberry kaempferol-3-O-rutinoside and ellagic acid |
| CN112129851A (en) * | 2020-08-23 | 2020-12-25 | 浙江省农业科学院 | Establishment method of raspberry polyphenol fingerprint spectrum |
| CN112129851B (en) * | 2020-08-23 | 2022-09-09 | 浙江省农业科学院 | Establishment method of raspberry polyphenol fingerprint spectrum |
| CN112913480A (en) * | 2021-01-26 | 2021-06-08 | 浙江锦珩健康科技有限责任公司 | Method for determining picking period of medicinal raspberry |
| CN112913480B (en) * | 2021-01-26 | 2022-04-22 | 浙江锦珩健康科技有限责任公司 | Method for determining picking period of medicinal raspberry |
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| CN104897809B (en) | 2016-12-07 |
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