CN103698420A - Resina draconis detecting method - Google Patents

Resina draconis detecting method Download PDF

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Publication number
CN103698420A
CN103698420A CN201310646198.6A CN201310646198A CN103698420A CN 103698420 A CN103698420 A CN 103698420A CN 201310646198 A CN201310646198 A CN 201310646198A CN 103698420 A CN103698420 A CN 103698420A
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China
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solution
mobile phase
phenyl
finger
print
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萧伟
王振中
秦建平
李家春
陈宝来
文红梅
吴建雄
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Jiangsu Kanion Pharmaceutical Co Ltd
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Jiangsu Kanion Pharmaceutical Co Ltd
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Abstract

The invention provides a resina draconis detecting method. The resina draconis detecting method comprises the following steps: (1) mixing a resina draconis sample to be detected and an alcohol component to obtain a solution to be detected; (2) carrying out a liquid chromatography on the solution to be detected to obtain a fingerprint of the solution to be detected, wherein an elution program of the liquid chromatography is gradient elution; and (3) obtaining contents of included components in the resina draconis sample to be detected according to the fingerprint and a pre-set standard curve of the solution to be detected and the quality of the resina draconis sample to be detected. The resina draconis detecting method adopts the gradient elution for carrying out the liquid chromatography to separate active components in the resina draconis. Furthermore, a chromatography peak form and a resolution of the active components in the obtained fingerprint are good and are good for calculation of a peak area of the chromatography peak, so as to obtain the contents of the components in the resina draconis sample to be detected. According to the method provided by the invention, the active components in the resina draconis are quantitatively determined, so as to more accurately reflect the quality of the resina draconis.

Description

The detection method that a kind of dragon's blood exhausts
Technical field
The present invention relates to Analysis of Chinese Traditional Medicine technical field, relate in particular to the detection method that a kind of dragon's blood exhausts.
Background technology
Dragon's blood exhausts the resin obtaining through extraction containing tallow wood material for Liliaceae swordleaf dragon tree, in China, mainly originates in Guangxi, Yunnan.According to < < Compendium of Materia Medica > >, record, it is warm in nature, flat that dragon's blood exhausts, taste is sweet, salty, nontoxic, enter blood system, return lung, spleen, kidney three warps, there is promoting blood circulation and removing blood stasis, swelling and pain relieving, astringing to arrest bleeding, the remarkable efficacy such as softening and resolving hard mass, regenerating tissue to heal wond.Modern medicine study confirms, dragon's blood exhausts and has the microcirculation of the body of improvement, adjusts body metabolism, improves the effects such as body's immunity.Therefore Related Component during dragon's blood exhausts has important effect to its performance drug effect, detects the focus that method that dragon's blood exhausts middle Related Component becomes people's research.
Prior art disclose a kind of dragon's blood medicinal material fingerprint atlas detection method (Song Xueying. the comparative studies [J] of import dragon's blood medicinal material and domestic Dragon Blood finger-print. Chinese Journal of Modern Applied Pharmacy magazine, 2007,4.), its detection method is specially: by dragon's blood medicinal material porphyrize, get about 0.5g, adding mass percentage concentration is 80% methanol solution, after ultrasonic extraction 30min, filters, and gets subsequent filtrate as need testing solution; Get 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one reference substance, lourerin B reference substance is appropriate, accurately weighed, adds methyl alcohol to make every 1mL approximately containing the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one reference substance of 0.2mg and the solution of lourerin B reference substance, by its product solution in contrast; Employing high performance liquid chromatography detects, and testing conditions is: mobile phase is that mass percentage concentration is 0.1% trifluoroacetic acid (TAF) and absolute methanol, carries out gradient elution; Testing result is that the principal ingredient in domestic Dragon Blood is 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one and lourerin B.
High performance liquid chromatography (HPLC) fingerprint atlas detection method that prior art also discloses a kind of dragon's blood capsule is (high beautiful, Jiang Qian, open quick. the HPLC finger-print research [J] of dragon's blood capsule. Chinese herbal medicine magazine, 2008,39:6.), its detection method is specially: adopt high performance liquid chromatography to detect, testing conditions is: Eclipse XDB-C18 chromatographic column, Phenomenex-C18 protection, and column temperature is 40 ℃; Take acetonitrile and water carries out gradient elution as mobile phase, and the volumetric flow rate of mobile phase is 110mL/min, and sampling volume is 10L/L; Detection wavelength is 275nm.Get lourerin B reference substance appropriate, accurately weighed, add methyl alcohol and dissolve, make the lourerin B reference substance solution of 12uL/mL; Get 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one and B reference substance is appropriate, accurately weighed, add the reference substance solution that methyl alcohol is made 26uL/mL 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one and 18uL/mL lourerin B; Get dragon's blood and exhaust the about 0.1g of sample, accurately weighed, be placed in the measuring bottle of 25mL, add methyl alcohol 20mL and dissolved, then add methyl alcohol and be diluted to 25mL, shake up, filter, obtain need testing solution; Testing result is that the principal ingredient during dragon's blood exhausts is 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one and lourerin B.
Related Component during the detection method that disclosed these dragon's bloods of prior art exhaust can only exhaust dragon's blood detects qualitatively, it is not carried out to quantitative detection, so the detection method that prior art provides can not fully reflect the quality that dragon's blood exhausts.
Summary of the invention
In view of this, the detection method that the object of the present invention is to provide a kind of dragon's blood to exhaust, Related Component during detection method provided by the invention can exhaust dragon's blood carries out qualitative detection and can quantitatively detect it again, and the method can comprehensively reflect the quality that dragon's blood exhausts.
The invention provides the detection method that a kind of dragon's blood exhausts, comprise the following steps:
(1), dragon's blood exhausted to testing sample mix with alcohol compound, obtain solution to be measured;
(2), described solution to be measured is carried out to liquid chromatographic detection, obtain the finger-print of described solution to be measured, mobile phase in described liquid chromatographic detection comprises mobile phase A and Mobile phase B, described mobile phase A is acetonitrile, described Mobile phase B is water or trifluoroacetic acid, and the percent by volume sum of described mobile phase A and Mobile phase B is 100%;
The elution program of described liquid chromatographic detection is gradient elution, and described gradient elution is:
In 0～12min, the percent by volume of described mobile phase A rises to 17%～19% from 12%～14%, and the percent by volume of described Mobile phase B drops to 81%～83% from 86%～88%;
In 12min～22min, the percent by volume of described mobile phase A rises to 27%～29% from 17%～19%, and the percent by volume of described Mobile phase B drops to 71%～73% from 81%～83%;
In 22min～35min, the percent by volume of described mobile phase A rises to 39%～41% from 27%～29%, and the percent by volume of described Mobile phase B drops to 59%～61% from 71%～73%;
In 35min～45min, the percent by volume of described mobile phase A remains on 39%～41%, and the percent by volume of described Mobile phase B remains on 59%～61%;
In 46min～55min, the percent by volume of described mobile phase A rises to 89%～91% from 39%～41%, and the percent by volume of described Mobile phase B drops to 9%～11% from 59%～61%;
(3), according to the finger-print of described solution to be measured, preassigned curve and dragon's blood, exhaust the quality of testing sample, obtain the content that dragon's blood exhausts ingredient in testing sample, described typical curve is the mass concentration of the dragon's blood reference substance solution that exhausts testing sample ingredient and the linearity curve between corresponding peak area.
Preferably, described alcohol compound is that C atomicity is 1～5 alcohol compound.
Preferably, described alcohol compound is ethanol or methyl alcohol.
Preferably, described alcohol compound is that mass percentage concentration is the aqueous solution of 70%～80% ethanol.
Preferably, described gradient elution is specially:
In 0～12min, the percent by volume of described mobile phase A rises to 18% from 13%, and the percent by volume of described Mobile phase B drops to 82% from 87%;
In 12min～22min, the percent by volume of described mobile phase A rises to 28% from 18%, and the percent by volume of described Mobile phase B drops to 72% from 82%;
In 22min～35min, the percent by volume of described mobile phase A rises to 40% from 28%, and the percent by volume of described Mobile phase B drops to 60% from 72%;
In 35min～45min, the percent by volume of described mobile phase A remains on 40%, and the percent by volume of described Mobile phase B remains on 60%;
In 46min～55min, the percent by volume of described mobile phase A rises to 90% from 40%, and the percent by volume of described Mobile phase B drops to 10% from 60%.
Preferably, the flow velocity of described mobile phase is 1.5mLmin -1～2.0mLmin -1.
Preferably, the detecting device of described liquid chromatographic detection is UV-detector;
Detection wavelength is 260nm～300nm.
Preferably, the chromatographic column column temperature in described liquid chromatographic detection is 30 ℃～45 ℃.
Preferably, described step (1) is:
Dragon's blood is exhausted to testing sample and be dissolved in alcohol compound, ultrasonic extraction, obtains solution to be measured.
Preferably, the time of described ultrasonic extraction is 15 minutes～60 minutes.
The invention provides the detection method that a kind of dragon's blood exhausts, comprise the following steps: (1), dragon's blood is exhausted to testing sample mix with alcohol compound, obtains solution to be measured; (2), described solution to be measured is carried out to liquid chromatographic detection, obtain the finger-print of described solution to be measured, mobile phase in described liquid chromatographic detection comprises mobile phase A and Mobile phase B, described mobile phase A is acetonitrile, described Mobile phase B is water or trifluoroacetic acid, and the percent by volume sum of described mobile phase A and Mobile phase B is 100%; The elution program of described liquid chromatographic detection is gradient elution, described gradient elution is: in 0～12min, the percent by volume of described mobile phase A rises to 17%～19% from 12%～14%, and the percent by volume of described Mobile phase B drops to 81%～83% from 86%～88%; In 12min～22min, the percent by volume of described mobile phase A rises to 27%～29% from 17%～19%, and the percent by volume of described Mobile phase B drops to 71%～73% from 81%～83%; In 22min～35min, the percent by volume of described mobile phase A rises to 39%～41% from 27%～29%, and the percent by volume of described Mobile phase B drops to 59%～61% from 71%～73%; In 35min～45min, the percent by volume of described mobile phase A remains on 39%～41%, and the percent by volume of described Mobile phase B remains on 59%～61%; In 46min～55min, the percent by volume of described mobile phase A rises to 89%～91% from 39%～41%, and the percent by volume of described Mobile phase B drops to 9%～11% from 59%～61%; (3), according to the finger-print of described solution to be measured, preassigned curve and dragon's blood, exhaust the quality of testing sample, obtain the content that dragon's blood exhausts ingredient in testing sample, described typical curve is the mass concentration of the dragon's blood reference substance solution that exhausts testing sample ingredient and the linearity curve between corresponding peak area.The present invention adopts this gradient elution to carry out liquid chromatographic detection, isolated the active component during dragon's blood exhausts, in the finger-print obtaining, the peak shape of the chromatographic peak of each active component is better, and between each chromatographic peak, there is good degree of separation, be conducive to the peak area at computer chromatography peak, thereby obtain the content that dragon's blood exhausts each composition in testing sample.The quantitative measurement of each active component during method provided by the invention has realized dragon's blood is exhausted, thus the quality that dragon's blood exhausts can be reflected more really.
Experimental result shows, the detection method that dragon's blood provided by the invention exhausts has good repeatability, stability and precision, can Accurate Determining dragon's blood exhausts the content of Related Component in testing sample.
Accompanying drawing explanation
Fig. 1 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 1;
Fig. 2 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 2;
Fig. 3 is the finger-print of the lourerin B that obtains of the embodiment of the present invention 3;
Fig. 4 is the finger-print of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that obtains of the embodiment of the present invention 4;
Fig. 5 is the finger-print of the Pterostilbene that obtains of the embodiment of the present invention 5;
Fig. 6 be the embodiment of the present invention 6 obtain 7, the finger-print of 4 '-dihydroxyflavone;
Fig. 7 is the finger-print of the resveratrol that obtains of the embodiment of the present invention 7;
Fig. 8 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 10;
Fig. 9 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 11;
Figure 10 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 14;
Figure 11 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 15;
Figure 12 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 16;
Figure 13 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 17;
Figure 14 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 34;
Figure 15 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 35;
Figure 16 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 36;
Figure 17 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 37;
Figure 18 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 38;
Figure 19 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 39;
Figure 20 is the ultraviolet absorption curve of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that obtains of the embodiment of the present invention 40;
Figure 21 is the ultraviolet absorption curve of the lourerin B that obtains of the embodiment of the present invention 40;
Figure 22 is the ultraviolet absorption curve of the resveratrol that obtains of the embodiment of the present invention 40;
Figure 23 be the embodiment of the present invention 40 obtain 7, the ultraviolet absorption curve of 4 '-dihydroxyflavone;
Figure 24 is the ultraviolet absorption curve of the Pterostilbene that obtains of the embodiment of the present invention 40;
Figure 25 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 41;
Figure 26 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 42;
Figure 27 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 43;
Figure 28 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 44;
Figure 29 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 45;
Figure 30 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 46;
Figure 31 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 47;
Figure 32 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 48;
Figure 33 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 49;
Figure 34 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 50;
Figure 35 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 51;
Figure 36 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 52;
Figure 37 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 53;
Figure 38 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 54;
Figure 39 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 55;
Figure 40 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 56;
Figure 41 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 57;
Figure 42 is the typical curve of the resveratrol that obtains of the embodiment of the present invention 60;
Figure 43 be the embodiment of the present invention 60 obtain 7, the typical curve of 4 '-dihydroxyflavone;
Figure 44 is the typical curve of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that obtains of the embodiment of the present invention 60;
Figure 45 is the typical curve of the lourerin B that obtains of the embodiment of the present invention 60;
Figure 46 is the typical curve of the Pterostilbene that obtains of the embodiment of the present invention 60;
Figure 47 is the reference fingerprint of the solution to be measured that obtains of the embodiment of the present invention 68;
Figure 48 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 70;
Figure 49 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 71;
Figure 50 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 72.
Embodiment
The invention provides the detection method that a kind of dragon's blood exhausts, comprise the following steps:
(1), dragon's blood exhausted to testing sample mix with alcohol compound, obtain solution to be measured;
(2), described solution to be measured is carried out to liquid chromatographic detection, obtain the finger-print of described solution to be measured, mobile phase in described liquid chromatographic detection comprises mobile phase A and Mobile phase B, described mobile phase A is acetonitrile, described Mobile phase B is water or trifluoroacetic acid, and the percent by volume sum of described mobile phase A and Mobile phase B is 100%; The elution program of described liquid chromatographic detection is gradient elution, and described gradient elution is:
In 0～12min, the percent by volume of described mobile phase A rises to 17%～19% from 12%～14%, and the percent by volume of described Mobile phase B drops to 81%～83% from 86%～88%;
In 12min～22min, the percent by volume of described mobile phase A rises to 27%～29% from 17%～19%, and the percent by volume of described Mobile phase B drops to 71%～73% from 81%～83%;
In 22min～35min, the percent by volume of described mobile phase A rises to 39%～41% from 27%～29%, and the percent by volume of described Mobile phase B drops to 59%～61% from 71%～73%;
In 35min～45min, the percent by volume of described mobile phase A remains on 39%～41%, and the percent by volume of described Mobile phase B remains on 59%～61%;
In 46min～55min, the percent by volume of described mobile phase A rises to 89%～91% from 39%～41%, and the percent by volume of described Mobile phase B drops to 9%～11% from 59%～61%;
(3), according to the finger-print of described solution to be measured, preassigned curve and dragon's blood, exhaust the quality of testing sample, obtain the content that dragon's blood exhausts ingredient in testing sample, described typical curve is the mass concentration of the dragon's blood reference substance solution that exhausts testing sample ingredient and the linearity curve between corresponding peak area.
The detection method that dragon's blood provided by the invention exhausts adopts liquid chromatography to detect, isolated the active component during dragon's blood exhausts, in the finger-print obtaining, the peak shape of the chromatographic peak of each active component is better, and between each chromatographic peak, there is good degree of separation, be conducive to the peak area at computer chromatography peak, thereby obtain the content that dragon's blood exhausts each composition in testing sample.The quantitative measurement of each active component during method provided by the invention has realized dragon's blood is exhausted, thus the quality that dragon's blood exhausts can be reflected more really.
The present invention exhausts testing sample by dragon's blood and mixes with alcohol compound, obtains solution to be measured; Preferably dragon's blood is exhausted after testing sample mixes with alcohol compound and carry out composition extraction, after Separation of Solid and Liquid, obtain solution to be measured.The present invention does not have special restriction to the method for described Separation of Solid and Liquid, adopts the technical scheme of Separation of Solid and Liquid well known to those skilled in the art, and in the present invention, the method for described Separation of Solid and Liquid is preferably filtration.The present invention does not have special restriction to the method for described filtration, adopts the technical scheme of filtration well known to those skilled in the art; In the present invention, the method for described filtration is preferably first carries out Filter paper filtering, then adopts filter membrane to filter the filtrate obtaining; Described filter paper is preferably quantitative filter paper; The aperture of described filter membrane is preferably 0.20 μ m～0.24 μ m, and more preferably 0.21 μ m～0.23 μ m, most preferably is 0.22 μ m.
The form that the present invention exhausts testing sample to described dragon's blood does not have special restriction, can directly dragon's blood be exhausted to testing sample and mix with alcohol compound, yet dragon's blood can be exhausted and be prepared into Powdered testing sample.In the present invention, it is 1～5 alcohol compound that described alcohol compound is preferably C atomicity, and more preferably methyl alcohol or ethanol, most preferably be ethanol, is the most preferably mass percentage concentration and is the aqueous solution of 70%～80% ethanol; It is pure that the purity of described alcohol compound is preferably analysis.The source that the present invention exhausts testing sample and alcohol compound to described dragon's blood does not have special restriction, as bought and obtain by market, concrete, the present invention can adopt the Resina Draconis-drug that originates from Yunnan as testing sample, through Nanjing University of Traditional Chinese Medicine, identifies that described Resina Draconis-drug is the resin obtaining containing the extraction of tallow wood material of liliaceous plant swordleaf dragon tree; The methyl alcohol or the ethanol that adopt Nanjing chemical reagents corporation to provide.
In the present invention, the mass ratio that described dragon's blood exhausts testing sample and alcohol compound is preferably 1:(40～130), 1:(60～110 more preferably), most preferably be 1:(70～100) and, be the most preferably 1:(80～90).The present invention, in order accurately to obtain the solution to be measured of above-mentioned mass ratio, preferably prepares solution to be measured by the following method:
Accurately weighed dragon's blood exhausts after testing sample, precision adds alcohol compound wherein, to after the weighed weight of the mixed solution obtaining, carry out composition extraction, weighed weight again after solution after composition is extracted is cooling, by above-mentioned alcohol compound complementary component, extract the weight of less loss, by supplying solution after weight, mix rear employing filter paper and filter, then adopt filter membrane again to filter the filtrate obtaining, obtain solution to be measured.In the present invention, the weighing precision that described accurately weighed dragon's blood exhausts testing sample is 0.001g, and it is 1% that described precision adds the precision of alcohol compound; Described cooling temperature is preferably 5 ℃～40 ℃, more preferably 10 ℃～35 ℃, most preferably is 10 ℃～30 ℃.
The present invention to described mixing, weighed weight, filtration, membrane filtration, accurately weighed, precision adds and cooling method does not have special restriction, adopt mixing well known to those skilled in the art, weighed weight, filtration, membrane filtration, accurately weighed, precision adds and cooling technical scheme, concrete, the present invention can adopt the method shaking up that above-mentioned solution is mixed; Adopt the electronic analytical balance of Mettler AE240 model or the electronic analytical balance of BP211D model carries out weighed weight to above-mentioned mixed solution and accurately weighed dragon's blood exhausts testing sample; Adopt the solution after the cooling method of nature is extracted mentioned component cooling.
In the present invention, the method that described composition extracts is preferably ultrasound wave and extracts or refluxing extraction; The ultrasonic power that described ultrasound wave extracts is preferably 240W～260W, and more preferably 245W～255W, most preferably is 250W; The ultrasonic frequency that described ultrasound wave extracts is preferably 30KHz～50KHz, and more preferably 35KHz～45KHz, most preferably is 40KHz.The temperature of described refluxing extraction is preferably 80 ℃～98 ℃, more preferably 85 ℃～95 ℃, most preferably is 90 ℃; The time of described refluxing extraction is preferably 40min～80min, and more preferably 50min～70min, most preferably is 60min.The present invention more preferably adopts ultrasound wave to carry out composition extraction; The present invention extracts to described ultrasound wave the instrument adopting does not have special restriction, as the KQ-250DB type numerical control ultrasonic cleaner that can adopt Kunshan Ultrasonic Instruments Co., Ltd. to provide.In the present invention, the time that described composition extracts is preferably 15min～60min, and more preferably 20min～40min, most preferably is 25min～35min.
Obtain after solution to be measured, the present invention carries out liquid chromatographic detection by solution to be measured, obtains the finger-print of described solution to be measured.In the present invention, the mobile phase in described liquid chromatographic detection comprises mobile phase A and Mobile phase B, and described mobile phase A is acetonitrile, and described Mobile phase B is water or trifluoroacetic acid, and the percent by volume sum of described mobile phase A and Mobile phase B is 100%.In the present invention, described Mobile phase B is preferably water, more preferably ultrapure water; The purity of described acetonitrile and trifluoroacetic acid is preferably chromatographically pure.The present invention does not have special restriction to the source of described acetonitrile, trifluoroacetic acid and ultrapure water, as bought and be obtained by market, concrete, acetonitrile and trifluoroacetic acid that the present invention can adopt U.S. world company to provide.
In the present invention, the elution program of described liquid chromatographic detection is gradient elution, and described gradient elution is specially:
In 0～12min, the percent by volume of described mobile phase A rises to 17%～19% from 12%～14%, and the percent by volume of described Mobile phase B drops to 81%～83% from 86%～88%; Preferably, the percent by volume of described mobile phase A rises to 18% from 13%, and the percent by volume of described Mobile phase B drops to 82% from 87%;
In 12min～22min, the percent by volume of described mobile phase A rises to 27%～29% from 17%～19%, and the percent by volume of described Mobile phase B drops to 71%～73% from 81%～83%; Preferably, the percent by volume of described mobile phase A rises to 28% from 18%, and the percent by volume of described Mobile phase B drops to 72% from 82%;
In 22min～35min, the percent by volume of described mobile phase A rises to 39%～41% from 27%～29%, and the percent by volume of described Mobile phase B drops to 59%～61% from 71%～73%; Preferably, the percent by volume of described mobile phase A rises to 40% from 28%, and the percent by volume of described Mobile phase B drops to 60% from 72%;
In 35min～45min, the percent by volume of described mobile phase A remains on 39%～41%, and the percent by volume of described Mobile phase B remains on 59%～61%; Preferably, the percent by volume of described mobile phase A remains on 40%, and the percent by volume of described Mobile phase B remains on 60%;
In 46min～55min, the percent by volume of described mobile phase A rises to 89%～91% from 39%～41%, and the percent by volume of described Mobile phase B drops to 9%～11% from 59%～61%; Preferably, the percent by volume of described mobile phase A rises to 90% from 40%, and the percent by volume of described Mobile phase B drops to 10% from 60%;
The mode that the present invention changes the percent by volume of described mobile phase does not have special restriction, adopt the percent by volume variation pattern of mobile phase in gradient elution well known to those skilled in the art, as adopted the mode of linear change, concrete, in the present invention, the percent by volume of the mobile phase A in described gradient elution is linear rising preferably, and the percent by volume of described Mobile phase B is linear decline preferably.In the present invention, the flow velocity of described mobile phase is preferably 1.5mLmin -1～2.0mLmin -1, 1.6mLmin more preferably -1～1.8mLmin -1, most preferably be 1.7mLmin -1.
In the present invention, the chromatographic column in described liquid chromatographic detection is preferably Waters Symmetry @the chromatographic column of the chromatographic column of the chromatographic column of C18 model, Agilent Zorbax Eclipse Plus C18 model or Phenomenex Kinetex 2.6u C18 100A model, the more preferably chromatographic column of Phenomenex Kinetex 2.6u C18 100A model; In the present invention, described chromatographic column filling agent is preferably octadecylsilane chemically bonded silica.
In the present invention, the chromatographic column column temperature of described liquid chromatographic detection is preferably 30 ℃～45 ℃, more preferably 35 ℃～42 ℃, most preferably is 40 ℃; The detecting device of described liquid chromatographic detection is preferably UV-detector, and the wavelength of described detection is preferably 260nm～300nm, and more preferably 270nm～290nm, most preferably is 280nm.
The instrument that the present invention uses described liquid chromatographic detection does not have special restriction, can adopt the ultrahigh pressure liquid phase chromatograph of Agilent 1290 models or the liquid chromatograph of Agilent 1200SL model, adopts DAD UV-detector.
Obtain the finger-print of described solution to be measured, the present invention preferably, according to the finger-print of the finger-print of described solution to be measured and predetermined reference substance solution, exhausts testing sample to dragon's blood and carries out qualitative detection.In the present invention, the finger-print of described predetermined reference substance solution preferably obtains according to following steps:
Preparation reference substance solution;
Reference substance solution is carried out to liquid chromatographic detection, obtain the finger-print of reference substance solution.
In the present invention, described reference substance preferably includes resveratrol, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.The present invention is preferably by resveratrol, 7, and 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B, Pterostilbene mix with alcohol compound, obtain reference substance solution; More preferably accurately weighed resveratrol, 7, is mixed after 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, then adds alcohol compound, obtains reference substance solution.In the present invention, in described reference substance solution, the mass concentration of resveratrol is preferably 40 μ g/mL～60 μ g/mL, and more preferably 45 μ g/mL～55 μ g/mL, most preferably are 50 μ g/mL; In described reference substance solution 7, the mass concentration of 4 '-dihydroxyflavone is preferably 30 μ g/mL～50 μ g/mL, and more preferably 35 μ g/mL～45 μ g/mL, most preferably are 40 μ g/mL; In described reference substance solution, the mass concentration of 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one is preferably 20 μ g/mL～40 μ g/mL, and more preferably 25 μ g/mL～35 μ g/mL, most preferably are 30 μ g/mL; In described reference substance solution, the mass concentration of lourerin B is preferably 40 μ g/mL～60 μ g/mL, and more preferably 45 μ g/mL～55 μ g/mL, most preferably are 50 μ g/mL; In described reference substance solution, the mass concentration of Pterostilbene is preferably 130 μ g/mL～150 μ g/mL, and more preferably 135 μ g/mL～145 μ g/mL, most preferably are 140 μ g/mL.The present invention does not have special restriction to described method accurately weighed and that mix, adopt the accurately weighed technical scheme with mixing well known to those skilled in the art, by resveratrol, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B, Pterostilbene weigh accurately, mix with alcohol compound.
In the present invention, described resveratrol, 7, the purity of 4 '-dihydroxyflavone and Pterostilbene is preferably greater than 99wt%; Described alcohol compound is consistent with alcohol compound described in technique scheme, does not repeat them here.The present invention is to described resveratrol, 7, the source of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B, Pterostilbene and alcohol compound does not have special restriction, can buy and obtain by market, concrete, the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one for assay and lourerin B that the present invention can adopt resveratrol that Shaanxi Yong Jian pharmaceutical Co. Ltd provides, Pterostilbene, Nat'l Pharmaceutical & Biological Products Control Institute that Hangzhou Guang Lin biological medicine Science and Technology Ltd. provides to provide; National Institute for Food and Drugs Control provide 7,4 '-dihydroxyflavone; The methyl alcohol that Nanjing chemical reagents corporation provides or ethanol.
Obtain after reference substance solution, the present invention by the reference substance solution obtaining preferably according to dragon's blood exhausted to the method that sample solution to be measured carries out liquid chromatographic detection carry out liquid chromatographic detection described in technique scheme, the finger-print that obtains reference substance solution, does not repeat them here;
Obtain after the finger-print of reference substance solution, the finger-print that the present invention exhausts sample solution to be measured by dragon's blood described in the finger-print of reference substance solution and technique scheme is compared, dragon's blood is exhausted to testing sample and carry out qualitative determination, point out out dragon's blood and exhaust the chromatographic peak in sample solution finger-print to be measured.The Related Component that method provided by the invention can exhaust in sample dragon's blood carries out quantitative detection, and the method for described quantitative detection is specially:
The finger-print of the solution to be measured obtaining according to predetermined typical curve, technique scheme and described dragon's blood exhaust the quality of testing sample, obtain the content that dragon's blood exhausts composition in testing sample; In the present invention, described typical curve is the mass concentration of the dragon's blood reference substance solution that exhausts testing sample ingredient and the linearity curve between corresponding peak area, preferably obtains in accordance with the following methods:
The reference substance that described dragon's blood is exhausted to testing sample ingredient is prepared into the reference substance solution of series concentration;
The reference substance solution of described series concentration is carried out to liquid chromatographic detection, obtain finger-print;
Mass concentration according to reference substance in the reference substance solution of the peak area of each chromatographic peak in described finger-print and correspondence, obtains typical curve.
In the present invention, described reference substance preferably includes resveratrol, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; The present invention is preferably by resveratrol, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B, Pterostilbene mix with alcohol compound, obtain the mother liquor of reference substance solution, by described mother liquor with above-mentioned alcohol compound by doubly dilution, obtain the reference substance solution of series concentration;
The present invention does not have special restriction to the method for described mixing and dilution, adopts the technical scheme of preparation reference substance solution well known to those skilled in the art; The present invention is to resveratrol, 7 in described mother liquor, the multiple of the concentration of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene and the dilution of described mother liquor does not have special restriction, can determine resveratrol, 7 in the solution after mother liquor and dilution thereof, the mass concentration value of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; In the present invention, described resveratrol, 7, resveratrol, 7 described in 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B, Pterostilbene and alcohol compound and technique scheme, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B, Pterostilbene are consistent with alcohol compound, do not repeat them here.
Prepare after the reference substance solution of series concentration, the present invention carries out liquid chromatographic detection to the reference substance solution of each concentration respectively, obtains finger-print; In the present invention, the method for described liquid chromatographic detection, with described in technique scheme, dragon's blood to be exhausted to the liquid chromatography detecting method of sample solution to be measured consistent, does not repeat them here;
Obtain after the finger-print of series concentration reference substance solution, the present invention calculates the peak area of each chromatographic peak in described finger-print.The present invention preferably be take lourerin B as object of reference in the process of computer chromatography peak area, inventor studies discovery, during exhausting, dragon's blood mainly contains flavones ingredient and organic acid composition, the retention time of lourerin B in finger-print is moderate, better separated, reference substance is easy to get, and therefore selects lourerin B as object of reference.The present invention does not have special restriction to the method for described computer chromatography peak area, adopt the technical scheme of computer chromatography peak area well known to those skilled in the art, as the present invention can adopt Agilent ChemStation software, calculate the peak area of each chromatographic peak in above-mentioned reference substance solution finger-print.
Obtain after the peak area of each chromatographic peak in described reference substance solution finger-print, the present invention, according to the mass concentration of the reference substance solution of the peak area of described chromatographic peak and correspondence thereof, obtains the typical curve that dragon's blood exhausts testing sample ingredient.The present invention is preferably using the peak area value of chromatographic peak as ordinate, and the mass concentration value of corresponding reference substance in reference substance solution, as horizontal ordinate, carried out linear fit, obtains typical curve.
The present invention is preferably with resveratrol, 7, and 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are reference substance, obtain respectively resveratrol, 7, the typical curve of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.In the present invention, the regression equation of described resveratrol typical curve is Y 1=16.906X 1-51.701, Y wherein 1for the chromatographic peak area of resveratrol in finger-print, X 1for the mass concentration of resveratrol in solution to be measured, coefficient R 1be 0.9999; Described 7, the regression equation of 4 '-dihydroxyflavone typical curve is Y 2=15.722X 2+ 0.4522, Y wherein 2for in finger-print 7, the chromatographic peak area of 4 '-dihydroxyflavone, X 2for in solution to be measured 7, the mass concentration of 4 '-dihydroxyflavone, coefficient R 2be 1; The regression equation of described 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one typical curve is Y 3=10.217X 3-1.162, Y wherein 3for the chromatographic peak area of 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one in finger-print, X 3for the mass concentration of 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one in solution to be measured, coefficient R 3be 1; The regression equation of described lourerin B typical curve is Y 4=8.1063X 4-2.4522, Y wherein 4for the chromatographic peak area of lourerin B in finger-print, X 4for the mass concentration of lourerin B in solution to be measured, coefficient R 4be 1; The regression equation of described Pterostilbene typical curve is Y 5=14.5754X 5-153.6806, Y wherein 5for the chromatographic peak area of Pterostilbene in finger-print, X 5for the mass concentration of Pterostilbene in solution to be measured, coefficient R 5be 0.9998.
Obtain after typical curve, the finger-print of the solution to be measured that the present invention obtains according to technique scheme, described typical curve and dragon's blood exhaust the quality of testing sample, obtain the content that described dragon's blood exhausts ingredient in testing sample.The present invention is preferably according to the method for computer chromatography peak-to-peak area described in technique scheme, calculate the peak area of each chromatographic peak in solution finger-print to be measured, according to the peak area of each chromatographic peak obtaining and corresponding typical curve, obtain the mass concentration of each component in solution to be measured; According to the mass concentration of each component in the volume of described solution to be measured and solution to be measured, obtain the quality of each component in described solution to be measured; According to the quality of each component in described solution to be measured and quality that dragon's blood exhausts testing sample, obtain the content that dragon's blood exhausts contained component in testing sample.The present invention preferably measures and obtains dragon's blood and exhaust resveratrol in testing sample, 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
The detection method that dragon's blood provided by the invention exhausts, adopt liquid chromatography to detect, isolated the active component during dragon's blood exhausts, and in the finger-print obtaining, the peak shape of the chromatographic peak of each active component is better, and between each chromatographic peak, there is good degree of separation, be conducive to the peak area at computer chromatography peak, thereby obtain the content that dragon's blood exhausts each composition in testing sample.The quantitative measurement of each active component during method provided by the invention has realized dragon's blood is exhausted, thus the quality that dragon's blood exhausts can be reflected more really.
The detection method that the present invention exhausts the dragon's blood providing has been carried out methodological investigation, mainly comprises: the study on the stability of solution to be measured and reference substance solution, precision are investigated and the repeatability of detection method is investigated; Average recovery and quantitative limit and the detectability of reference substance in carrying out investigation process, have been tested.The present invention does not have special restriction to described stability, precision, the investigation method of repeatability and the detection method of average recovery and quantitative limit and detectability, adopts the technical scheme of investigation stability well known to those skilled in the art, precision, repeatability and the method that detects average recovery and quantitative limit and detectability.
The study on the stability presentation of results of the present invention's solution to be measured, solution to be measured prepared by method provided by the invention had good stability in 24 hours; The study on the stability presentation of results of reference substance solution of the present invention, reference substance solution prepared by method provided by the invention had good stability in 24 hours; The precision of the present invention's solution to be measured is investigated presentation of results, and solution to be measured prepared by method provided by the invention has good precision; The precision of reference substance solution of the present invention is investigated presentation of results, and reference substance solution prepared by method provided by the invention has good precision; The repeatability of detection method of the present invention is investigated presentation of results, and the detection method that dragon's blood provided by the invention exhausts has good repeatability; The average recovery testing result explanation of reference substance of the present invention, detection method energy Accurate Determining dragon's blood provided by the invention exhausts the content of middle Related Component; The quantitative limit of reference substance of the present invention and detectability testing result be, being quantitatively limited to 4.85ng, detecting and be limited to 1.21ng of resveratrol; 7, being quantitatively limited to 2.14ng, detecting and to be limited to 1.07ng of 4 '-dihydroxyflavone; Quantitatively being limited to 7.19ng, detecting and to be limited to 0.90ng of 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one; Quantitatively being limited to 6.30ng, detecting and to be limited to 1.58ng of lourerin B; Quantitatively being limited to 7.41ng, detecting and to be limited to 1.80ng of Pterostilbene, this explanation, the mensuration that method provided by the invention exhausts the contained component of testing sample to dragon's blood has higher sensitivity.
In order further to understand the present invention, detection method dragon's blood provided by the invention being exhausted below in conjunction with embodiment is described in detail, but they can not be interpreted as to limiting the scope of the present invention.
In following embodiment, the instrument that liquid chromatographic detection adopts is: Agilent 1290 ultrahigh pressure liquid phase chromatographs, DAD UV-detector;
Ultrasound wave extracts the instrument adopting: KQ-250DB type numerical control ultrasonic cleaner;
The instrument that weighed weight adopts is: Mettler AE240 electronic analytical balance and BP211D electronic analytical balance;
Dragon's blood used exhausts and originates from Yunnan, through being accredited as the resin obtaining containing the extraction of tallow wood material of liliaceous plant swordleaf dragon tree; The batch number of 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one used is that the batch number of 111660-200402, lourerin B is 111558-200303, and 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one and lourerin B are the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one for assay and the lourerin B that Nat'l Pharmaceutical & Biological Products Control Institute provides; The batch number of Pterostilbene used is 20110723, buys in Hangzhou Guang Lin biological medicine Science and Technology Ltd.; Resveratrol used is bought in Shaanxi Yong Jian pharmaceutical Co. Ltd; Acetonitrile and trifluoroacetic acid used are chromatographically pure, buy the world company in the U.S.; It is pure that methyl alcohol used and ethanol are analysis; 7,4 '-dihydroxyflavone is bought in National Institute for Food and Drugs Control.
Embodiment 1
Accurate weighing dragon's blood exhausts powdered sample 0.5g, be placed in tool plug conical flask, to precision in tool plug conical flask, add absolute methanol 50mL, adopting the ultrasound wave of 250W, 40KHz to carry out composition the mixed solution obtaining extracts 30 minutes, solution after composition is extracted is cooled to 10 ℃, by cooled solution filter, get the filter membrane that subsequent filtrate is crossed 0.22 μ m, obtain solution to be measured;
The above-mentioned solution to be measured preparing is carried out to liquid chromatographic detection, and the condition of detection is specially:
Take octadecylsilane chemically bonded silica as chromatographic column filling agent;
Chromatographic column model is Phenomenex Kinetex 2.6u C18 100A, and column's length is 100mm, and internal diameter is 4.6mm, and particle diameter is 2.6 μ m;
Chromatographic column column temperature is 40 ℃;
Take acetonitrile as mobile phase A, take ultrapure water as Mobile phase B, the percent by volume sum of described mobile phase A and Mobile phase B is 100%, carries out gradient elution:
In 0～12min, the percent by volume of described mobile phase A rises to 18% from 13% linearity, and the percent by volume of described Mobile phase B drops to 82% from 87% linearity;
In 12min～22min, the percent by volume of described mobile phase A rises to 28% from 18% linearity, and the percent by volume of described Mobile phase B drops to 72% from 82% linearity;
In 22min～35min, the percent by volume of described mobile phase A rises to 40% from 28% linearity, and the percent by volume of described Mobile phase B drops to 60% from 72% linearity;
In 35min～45min, the percent by volume of described mobile phase A remains on 40%, and the percent by volume of described Mobile phase B remains on 60%;
In 46min～55min, the percent by volume of described mobile phase A rises to 90% from 40% linearity, and the percent by volume of described Mobile phase B drops to 10% from 60% linearity;
The flow velocity of described mobile phase A and Mobile phase B is 1.7mLmin -1;
Detection wavelength is 280nm.
The solution to be measured of the embodiment of the present invention 1 preparation is carried out to finger-print that liquid chromatographic detection obtains as shown in Figure 1, Fig. 1 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 1, the content of principal ingredient in the solution to be measured obtaining according to the finger-print calculating embodiment 1 obtaining, in described solution to be measured, the content of principal ingredient represents with the peak area of unit mass, computing method are for to be multiplied by the extension rate of solution to be measured divided by the sampling amount of solution to be measured by the peak area of principal ingredient in finger-print, result of calculation is as shown in table 1, the content of principal ingredient in the solution to be measured that table 1 obtains for the embodiment of the present invention 1 and embodiment 2.
Embodiment 2
Adopt the technical scheme of embodiment 1 to detect the finger-print that obtains solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in the solution to be measured that embodiment 2 obtains; Different, the ultrasonic extraction in the method alternative embodiment 1 of the present embodiment employing refluxing extraction, the time of refluxing extraction is 60 minutes.As shown in Figure 2 and Table 1, Fig. 2 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 2 to testing result.
The content of principal ingredient in the solution to be measured that table 1 embodiment of the present invention 1 and embodiment 2 obtain
Figure BDA0000429626030000161
Embodiment 3
After accurately weighed lourerin B, adding wherein mass percentage concentration is the aqueous solution of 70% ethanol, makes every milliliter containing the reference substance solution of lourerin B 50 μ g.
The above-mentioned reference substance solution preparing is carried out to liquid chromatographic detection according to the testing conditions described in embodiment 1, detect the finger-print of the reference substance solution obtain as shown in Figure 3, Fig. 3 is the finger-print of the lourerin B that obtains of the embodiment of the present invention 3.
Embodiment 4
After accurately weighed 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, adding wherein mass percentage concentration is the aqueous solution of 70% ethanol, makes every milliliter containing the reference substance solution of 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 30 μ g.
The above-mentioned reference substance solution preparing is carried out to liquid chromatographic detection according to the testing conditions described in embodiment 1, detect the finger-print of the reference substance solution obtain as shown in Figure 4, Fig. 4 is the finger-print of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that obtains of the embodiment of the present invention 4.
Embodiment 5
After accurately weighed Pterostilbene, adding wherein mass percentage concentration is the aqueous solution of 70% ethanol, makes every milliliter containing the reference substance solution of Pterostilbene 140 μ g.
The above-mentioned reference substance solution preparing is carried out to liquid chromatographic detection according to the testing conditions described in embodiment 1, detect the finger-print of the reference substance solution obtain as shown in Figure 5, Fig. 5 is the finger-print of the Pterostilbene that obtains of the embodiment of the present invention 5.
Embodiment 6
Accurately weighed 7, after 4 '-dihydroxyflavone, adding wherein mass percentage concentration is the aqueous solution of 70% ethanol, makes every milliliter containing 7, the reference substance solution of 4 '-dihydroxyflavone, 40 μ g.
The above-mentioned reference substance solution preparing is carried out to liquid chromatographic detection according to the testing conditions described in embodiment 1, the finger-print of the reference substance solution that detection obtains as shown in Figure 6, Fig. 6 be the embodiment of the present invention 6 obtain 7, the finger-print of 4 '-dihydroxyflavone.
Embodiment 7
After accurately weighed resveratrol, adding wherein mass percentage concentration is the aqueous solution of 70% ethanol, makes every milliliter containing the reference substance solution of resveratrol 50 μ g.
The above-mentioned reference substance solution preparing is carried out to liquid chromatographic detection according to the testing conditions described in embodiment 1, detect the finger-print of the reference substance solution obtain as shown in Figure 7, Fig. 7 is the finger-print of the resveratrol that obtains of the embodiment of the present invention 7.
Embodiment 8
Adopt the technical scheme of embodiment 1 to detect the finger-print that obtains solution to be measured, the finger-print of the reference substance that the finger-print obtaining and embodiment 3～embodiment 7 are obtained is compared, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene content; Testing result is as shown in table 2, in the solution to be measured that table 2 obtains for the embodiment of the present invention 8 and embodiment 97,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene content.
Embodiment 9
Adopt the technical scheme of embodiment 2 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene content; Testing result is as shown in table 2.
In the solution to be measured that table 2 embodiment of the present invention 8 and embodiment 9 obtain 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene content
Figure BDA0000429626030000181
By table 1 and table 2, can be found out, the no significant difference that the solution to be measured that adopts the extraction of ultrasound wave composition and backflow composition to extract preparation records, but it is easier that employing ultrasound wave carries out the operation of composition extraction, so the present invention preferably adopts ultrasound wave to carry out composition extraction preparation solution to be measured.
Embodiment 10
Adopt the technical scheme of embodiment 1 to detect the finger-print that obtains solution to be measured, and according to the finger-print obtaining calculate principal ingredient in solution to be measured content; Testing result is as shown in Fig. 8 and table 3, and Fig. 8 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 10, in the solution to be measured that table 3 obtains for the embodiment of the present invention 10 and embodiment 11 principal ingredient content.
Embodiment 11
Adopt the technical scheme of embodiment 1 to detect the finger-print that obtains solution to be measured, and according to chromatographic peak in the finger-print calculated fingerprint collection of illustrative plates obtaining; Different, the present embodiment adopts the absolute methanol in absolute ethyl alcohol alternative embodiment 1.Testing result is as shown in Fig. 9 and table 3, and Fig. 9 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 11.
In the solution to be measured that table 3 embodiment of the present invention 10 and embodiment 11 obtain principal ingredient content
Figure BDA0000429626030000182
Embodiment 12
Adopt the technical scheme of embodiment 10 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 4, the content of 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene in the solution to be measured that table 4 obtains for the embodiment of the present invention 12 and embodiment 13.
Embodiment 13
Adopt the technical scheme of embodiment 11 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 4.
In the solution to be measured that table 4 embodiment of the present invention 12 and embodiment 13 obtain 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
Figure BDA0000429626030000191
By table 3 and table 4, can be found out, ethanol is as extracting the methyl alcohol that is better than that solution to be measured that solvent prepares records, so the present invention preferably adopts ethanol to prepare solution to be measured as extracting solvent.
Embodiment 14
Adopt the technical scheme of embodiment 11 to detect the finger-print that obtains solution to be measured, and according to chromatographic peak in the finger-print calculated fingerprint collection of illustrative plates obtaining; Testing result is as shown in Figure 10 and table 5, and Figure 10 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 14, and table 5 obtains for the embodiment of the present invention 14～embodiment 17.
Embodiment 15
Adopt the technical scheme detection of embodiment 11 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Different, the absolute ethyl alcohol in the aqueous solution alternative embodiment 11 of the ethanol that the present embodiment employing mass percentage concentration is 80%.Testing result is as shown in Figure 11 and table 5, and Figure 11 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 15.
Embodiment 16
Adopt the technical scheme detection of embodiment 11 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Different, the absolute ethyl alcohol in the aqueous solution alternative embodiment 11 of the ethanol that the present embodiment employing mass percentage concentration is 70%.Testing result is as shown in Figure 12 and table 5, and Figure 12 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 16.
Embodiment 17
Adopt the technical scheme detection of embodiment 11 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Different, the absolute ethyl alcohol in the aqueous solution alternative embodiment 11 of the ethanol that the present embodiment employing mass percentage concentration is 50%.Testing result is as shown in Figure 13 and table 5, and Figure 13 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 17.
The content of principal ingredient in the solution to be measured that table 5 embodiment of the present invention 14～embodiment 17 obtains
Embodiment 18
Adopt the technical scheme of embodiment 14 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according in the method calculated fingerprint collection of illustrative plates described in embodiment 17,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 6, and table 6 obtains for the embodiment of the present invention 18～embodiment 21.
Embodiment 19
Adopt the technical scheme of embodiment 15 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 6.
Embodiment 20
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 6.
Embodiment 21
Adopt the technical scheme of embodiment 17 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 6.
In the solution to be measured that table 6 embodiment of the present invention 18～embodiment 21 obtains 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
Figure BDA0000429626030000211
By table 5 and table 6, can be found out, adopt mass percentage concentration be 70% and 80% ethanol aqueous solution as extract that solution to be measured prepared by solvent records better, therefore, preferably to adopt mass percentage concentration be that the aqueous solution of 70%～80% ethanol is prepared solution to be measured as extracting solvent in the present invention.
Embodiment 22
Adopt the technical scheme detection of embodiment 16 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Shown in testing result table 7, the content of principal ingredient in the solution to be measured that table 7 obtains for the embodiment of the present invention 22～embodiment 24.
Embodiment 23
Adopt the technical scheme detection of embodiment 16 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Different, the aqueous solution of the ethanol that the mass percentage concentration of the 50mL in the aqueous solution alternative embodiment 16 of the ethanol that the mass percentage concentration of the present embodiment employing 25mL is 70% is 70%.Testing result is as shown in table 7.
Embodiment 24
Adopt the technical scheme detection of embodiment 16 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Different, the aqueous solution of the ethanol that the mass percentage concentration of the 50mL in the aqueous solution alternative embodiment 16 of the ethanol that the mass percentage concentration of the present embodiment employing 75mL is 70% is 70%.Shown in testing result table 7.
The content of principal ingredient in the solution to be measured that table 7 embodiment of the present invention 22～embodiment 24 obtains
Figure BDA0000429626030000221
Embodiment 25
Adopt the technical scheme of embodiment 22 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according in the method calculated fingerprint collection of illustrative plates described in embodiment 17,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 8, and table 8 obtains for the embodiment of the present invention 25～embodiment 27.
Embodiment 26
Adopt the technical scheme of embodiment 23 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 8.
Embodiment 27
Adopt the technical scheme of embodiment 24 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 8.
In the solution to be measured that table 8 embodiment of the present invention 25～embodiment 27 obtains 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
Figure BDA0000429626030000231
By table 7 and table 8, can be found out, it is better that the solution to be measured that the consumption of extraction solvent prepares while being 50mL and 75mL records, so the present invention preferably adopts the extraction solvent of 50mL to prepare solution to be measured.
Embodiment 28
Adopt the technical scheme detection of embodiment 16 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Testing result is as shown in table 9, the content of principal ingredient in the solution to be measured that table 9 obtains for the embodiment of the present invention 28～embodiment 30.
Embodiment 29
Adopt the technical scheme detection of embodiment 16 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Different, the composition of 30 minutes that the present embodiment adopts the composition of 15 minutes to extract in alternative embodiment 16 extracts.Testing result is as shown in table 9.
Embodiment 30
Adopt the technical scheme detection of embodiment 16 to obtain the finger-print of solution to be measured, and according to the finger-print obtaining, calculate the content of principal ingredient in solution to be measured; Different, the composition of 30 minutes that the present embodiment adopts the composition of 60 minutes to extract in alternative embodiment 16 extracts.Shown in testing result table 9.
The content of principal ingredient in the solution to be measured that table 9 embodiment of the present invention 28～embodiment 30 obtains
Figure BDA0000429626030000232
Embodiment 31
Adopt the technical scheme of embodiment 28 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 10, the content of 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene in the solution to be measured that table 10 obtains for the embodiment of the present invention 31～embodiment 33.
Embodiment 32
Adopt the technical scheme of embodiment 29 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 10.
Embodiment 33
Adopt the technical scheme of embodiment 30 to detect the finger-print that obtains solution to be measured, by the finger-print obtaining according to the method described in embodiment 8, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, according to the method described in embodiment 1, calculate in solution to be measured 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene; Testing result is as shown in table 10.
In the solution to be measured that table 10 embodiment of the present invention 31～embodiment 33 obtains 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
Figure BDA0000429626030000241
By table 9 and table 10, can be found out, adopting composition extraction time is that the effect that records of the solution to be measured for preparing for 30 minutes and 60 minutes is suitable, so the present invention preferably carries out the composition extraction of 30 minutes in preparation solution process to be measured.
Embodiment 34
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured, as shown in figure 14, Figure 14 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 34 to testing result.
Embodiment 35
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured; Different, the present embodiment adopts Waters Symmetry @c18(4.6 * 250mm, 5 μ m) Phenomenex Kinetex 2.6u C18 100A(4.6 * 100mm in the chromatographic column alternative embodiment 16 of model, the 2.6 μ m) chromatographic column of model.As shown in figure 15, Figure 15 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 35 to testing result.
Embodiment 36
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured; Different is, the present embodiment adopts Agilent Zorbax Eclipse Plus C18(2.1 * 100mm, 1.8 μ m) Phenomenex Kinetex 2.6u C18 100A(4.6 * 100mm in the chromatographic column alternative embodiment 16 of model, the 2.6 μ m) chromatographic column of model.As shown in figure 16, Figure 16 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 36 to testing result.
Embodiment 37
Adopt the technical scheme of embodiment 34 to detect the finger-print that obtains solution to be measured, as shown in figure 17, Figure 17 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 37 to testing result.
Embodiment 38
Adopt the technical scheme of embodiment 35 to detect the finger-print that obtains solution to be measured, as shown in figure 18, Figure 18 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 38 to testing result.
Embodiment 39
Adopt the technical scheme of embodiment 36 to detect the finger-print that obtains solution to be measured, as shown in figure 19, Figure 19 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 39 to testing result.
By Figure 14～Figure 19, can be found out, adopt Phenomenex Kinetex 2.6u C18 100A(4.6 * 100mm, 2.6 μ m) it is better that the chromatographic column of model is carried out the separation of each chromatographic peak in solution finger-print to be measured that liquid chromatographic detection obtains, the present invention adopts the chromatographic column of 2 the type to carry out repeated experiment, experimental result shows that repeatability better, therefore the present invention preferably adopts Phenomenex Kinetex 2.6u C18 100A(4.6 * 100mm, 2.6 μ m) chromatographic column of model exhausts and carries out liquid chromatographic detection dragon's blood.
Embodiment 40
Accurately weighed resveratrol, 7, after 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene by its mixing, to adding mass percentage concentration in the potpourri obtaining, it is the aqueous solution of 70% ethanol, make every milliliter containing resveratrol 50 μ g, 7, the reference substance solution of 4 '-dihydroxyflavone, 40 μ g, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 30 μ g, lourerin B 50 μ g and Pterostilbene 140 μ g.
The above-mentioned reference substance solution preparing is adopted in the scope that the testing conditions of liquid chromatography in implementing regulations 1 is 190nm～400nm by DAD UV-detector at wavelength, under the frequency of 80Hz, detect, testing result is as shown in Figure 20～Figure 24, Figure 20 is the ultraviolet absorption curve of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that obtains of the embodiment of the present invention 40, Figure 21 is the ultraviolet absorption curve of the lourerin B that obtains of the embodiment of the present invention 40, Figure 22 is the ultraviolet absorption curve of the resveratrol that obtains of the embodiment of the present invention 40, Figure 23 be the embodiment of the present invention 40 obtain 7, the ultraviolet absorption curve of 4 '-dihydroxyflavone, Figure 24 is the ultraviolet absorption curve of the Pterostilbene that obtains of the embodiment of the present invention 40.
By Figure 20～Figure 24, can be found out, the maximum absorption wavelength of 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one is 278nm, and the maximum absorption wavelength of lourerin B is 276nm, the maximum absorption wavelength of resveratrol is 306nm, 7, the maximum absorption wavelength of 4 '-dihydroxyflavone is 332nm, and the maximum absorption wavelength of Pterostilbene is 308nm.
Embodiment 41
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured, as shown in figure 25, Figure 25 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 41 to testing result.
Embodiment 42
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured; Different, the detection wavelength of 280nm in the detection wavelength alternative embodiment 16 of the present embodiment employing 275nm.As shown in figure 26, Figure 26 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 42 to testing result.
Embodiment 43
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured; Different, the detection wavelength of 280nm in the detection wavelength alternative embodiment 16 of the present embodiment employing 300nm.As shown in figure 27, Figure 27 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 43 to testing result.
Embodiment 44
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured; Different, the detection wavelength of 280nm in the detection wavelength alternative embodiment 16 of the present embodiment employing 325nm.As shown in figure 28, Figure 28 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 44 to testing result.
Embodiment 45
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured; Different, the present embodiment adopts the testing conditions of liquid chromatography in implementing regulations 1, the detection wavelength of 280nm under the frequency of 80Hz, in the detection wavelength alternative embodiment 16 of 190nm～400nm.As shown in figure 29, Figure 29 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 45 to testing result.
By Figure 25～Figure 29, can be found out, adopt the detection ripple of 280nm to carry out the finger-print that liquid chromatographic detection obtains, can more comprehensively reflect that dragon's blood exhausts the chemical composition in testing sample, and in the finger-print obtaining, each chromatographic peak is better separated, baseline is steady, detects effect better.Therefore the detection wavelength that the present invention preferably exhausts 280nm as liquid chromatographic detection dragon's blood.
Embodiment 46
Adopt the technical scheme of embodiment 16 to detect the finger-print that obtains solution to be measured, as shown in figure 30, Figure 30 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 46 to testing result.The present invention has tested resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, result is as shown in table 11, table 11 is the resveratrol, 7 that the embodiment of the present invention 46 and embodiment 47 obtain, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
Embodiment 47
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, different, the ultrapure water in the aqueous solution alternative embodiment 46 of the trifluoroacetic acid that the present embodiment employing mass percentage concentration is 0.05%.As shown in figure 31, Figure 31 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 47 to testing result.The present invention has tested resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, and result is as shown in table 11.
The resveratrol, 7 that table 11 embodiment of the present invention 46 and embodiment 47 obtain, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
Figure BDA0000429626030000271
By Figure 30, Figure 31 and table 11, can be found out, acetonitrile-water system and acetonitrile-trifluoroacetic acid system are carried out the separation case of each chromatographic peak in the finger-print of the solution to be measured that liquid chromatographic detection obtains and peak shape all without significant difference as mobile phase, so the present invention's mobile phase of preferably adopting acetonitrile-water system to exhaust as liquid chromatographic detection dragon's blood.
Embodiment 48
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, shown in figure 32, Figure 32 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 48 to testing result.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, result is as shown in table 12, the resveratrol, 7 that table 12 obtains for the embodiment of the present invention 48～embodiment 51, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
Embodiment 49
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, different, the present embodiment adopts 40 ℃ of chromatographic column column temperatures in 30 ℃ of chromatographic column column temperature alternative embodiments 46.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.Testing result is as shown in Figure 33 and table 12, and Figure 33 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 49.
Embodiment 50
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, different, the present embodiment adopts 40 ℃ of chromatographic column column temperatures in 35 ℃ of chromatographic column column temperature alternative embodiments 46.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.Testing result is as shown in Figure 34 and table 12, and Figure 34 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 50.
Embodiment 51
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, different, the present embodiment adopts 40 ℃ of chromatographic column column temperatures in 45 ℃ of chromatographic column column temperature alternative embodiments 46.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.Testing result is as shown in Figure 35 and table 12, and Figure 35 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 51.
The resveratrol, 7 that table 12 embodiment of the present invention 48～embodiment 51 obtains, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
? Column temperature 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one Lourerin B 7,4 '-dihydroxyflavone Resveratrol Pterostilbene
Degree of separation Embodiment 48 1.48 2.73 - 2.61 1.87
? Embodiment 49 1.48 1.48 - 2.35 1.79
? Embodiment 50 1.42 2.50 4.46 1.93 1.89
? Embodiment 51 1.47 1.39 - 2.64 1.76
Tailing factor Embodiment 48 0.90 0.92 0.85 0.88 0.92
? Embodiment 49 0.90 0.97 0.85 0.88 0.93
? Embodiment 50 0.91 0.99 0.84 0.90 0.91
? Embodiment 51 0.90 0.97 0.84 0.91 0.92
Theoretical cam curve Embodiment 48 282402 292961 35678 50316 317067
? Embodiment 49 322192 335793 42155 73338 362953
? Embodiment 50 279251 321489 36058 51435 326835
? Embodiment 51 241128 291833 34355 46830 291514
By Figure 32～Figure 35 and table 12, can be found out, when chromatographic column column temperature is 40 ℃, carry out in the finger-print of the solution to be measured that liquid chromatographic detection obtains each chromatographic peak better separated.Therefore the present invention preferably adopts the chromatographic column column temperature of 40 ℃ to exhaust and carry out liquid chromatographic detection dragon's blood.
Embodiment 52
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, as shown in figure 36, Figure 36 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 52 to testing result.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, result is as shown in table 13, the resveratrol, 7 that table 13 obtains for the embodiment of the present invention 52～embodiment 56, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
Embodiment 53
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, different, the present embodiment adopts 1.5mLmin -1flow rate of mobile phase alternative embodiment 46 in 1.7mLmin -1flow rate of mobile phase.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.Testing result is as shown in Figure 37 and table 13, and Figure 37 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 53.
Embodiment 54
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, different, the present embodiment adopts 1.6mLmin -1flow rate of mobile phase alternative embodiment 46 in 1.7mLmin -1flow rate of mobile phase.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.Testing result is as shown in Figure 38 and table 13, and Figure 38 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 54.
Embodiment 55
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, different, the present embodiment adopts 1.8mLmin -1flow rate of mobile phase alternative embodiment 46 in 1.7mLmin -1flow rate of mobile phase.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.Testing result is as shown in Figure 39 and table 13, and Figure 39 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 55.
Embodiment 56
Adopt the technical scheme of embodiment 46 to detect the finger-print that obtains solution to be measured, different, the present embodiment adopts 2.0mLmin -1flow rate of mobile phase alternative embodiment 46 in 1.7mLmin -1flow rate of mobile phase.The present invention has calculated resveratrol in the present embodiment, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.Testing result is as shown in Figure 40 and table 13, and Figure 40 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 56.
The resveratrol, 7 that table 13 embodiment of the present invention 52～embodiment 56 obtains, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
Figure BDA0000429626030000301
From Figure 36～Figure 40 and table 13, flow rate of mobile phase is 1.7mLmin -1in the finger-print of the solution to be measured that Shi Jinhang liquid chromatographic detection obtains, the degree of separation of chromatographic peak and peak shape are better, so the present invention detects dragon's blood, and to exhaust preferred employing flow velocity be 1.7mLmin -1mobile phase carry out gradient elution.
Embodiment 57
Accurate weighing dragon's blood exhausts powdered sample 0.5g, be placed in tool plug conical flask, to the accurate mass percentage concentration that adds in tool plug conical flask, be the aqueous solution 50mL of 70% ethanol, adopting the ultrasound wave of 250W, 40KHz to carry out composition the mixed solution obtaining extracts 30 minutes, solution after composition is extracted is cooled to 10 ℃, by cooled solution filter, get subsequent filtrate and cross 0.22 μ m filter membrane, obtain solution to be measured;
The above-mentioned solution to be measured preparing is carried out to liquid chromatographic detection, and the condition of detection is specially:
Take octadecylsilane chemically bonded silica as chromatographic column filling agent;
Chromatographic column model is Phenomenex Kinetex 2.6u C18 100A, and column's length is 100mm, and internal diameter is 4.6mm, and particle diameter is 2.6 μ m;
Chromatographic column column temperature is 40 ℃;
Take acetonitrile as mobile phase A, take ultrapure water as Mobile phase B, the percent by volume sum of described mobile phase A and Mobile phase B is 100%, carries out gradient elution:
In 0～12min, the percent by volume of described mobile phase A rises to 18% from 13% linearity, and the percent by volume of described Mobile phase B drops to 82% from 87% linearity;
In 12min～22min, the percent by volume of described mobile phase A rises to 28% from 18% linearity, and the percent by volume of described Mobile phase B drops to 72% from 82% linearity;
In 22min～35min, the percent by volume of described mobile phase A rises to 40% from 28% linearity, and the percent by volume of described Mobile phase B drops to 60% from 72% linearity;
In 35min～45min, the percent by volume of described mobile phase A remains on 40%, and the percent by volume of described Mobile phase B remains on 60%;
In 46min～55min, the percent by volume of described mobile phase A rises to 90% from 40% linearity, and the percent by volume of described Mobile phase B drops to 10% from 60% linearity;
The flow velocity of described mobile phase A and Mobile phase B is 1.7mLmin -1;
Detection wavelength is 280nm.
The solution to be measured of the embodiment of the present invention 57 preparation is carried out to finger-print that liquid chromatographic detection obtains as shown in figure 41, Figure 41 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 57, the present invention has measured resveratrol, 7 according to the finger-print obtaining, the system flexibility of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene, measurement result is as shown in table 14, the resveratrol, 7 that table 14 obtains for the embodiment of the present invention 57, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
The resveratrol that table 14 embodiment of the present invention 57 obtains, 7, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
? Degree of separation Tailing factor Theoretical cam curve
Resveratrol 5.13 0.95 29468
7,4 '-dihydroxyflavone 6.69 0.96 45063
3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 1.51 1.01 307734
Lourerin B 2.73 0.96 270794
Pterostilbene 1.64 1.02 288493
By Figure 41 and table 14, can be found out, adopt the technical scheme of embodiment 57 to carry out liquid chromatographic detection, the resveratrol obtaining, 7, the system flexibility result of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene is better, so the present invention preferably exhausts and detects dragon's blood according to the detection method described in embodiment 57; And for the accuracy of detection method, the present invention's regulation is carried out dragon's blood and is exhausted number of theoretical plate while detecting and calculate and should be not less than 100000 by lourerin B peak.
Embodiment 58
The finger-print of the reference substance that the finger-print of the solution to be measured that embodiment 57 is obtained and embodiment 3～embodiment 7 obtain is compared, point out out resveratrol in finger-print, 7, the chromatographic peak of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
From Figure 41 and Fig. 3～Fig. 7, in the finger-print of the solution to be measured that embodiment 57 obtains, No. 1 peak is resveratrol, and No. 2 peak is 7,4 '-dihydroxyflavone, and No. 11 peak is 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, and No. 12 peaks are lourerin B, and No. 15 peaks are Pterostilbene.
Embodiment 59
Accurately weighed resveratrol, 7, after 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene by its mixing, to adding mass percentage concentration in the potpourri obtaining, it is the aqueous solution of 70% ethanol, make every milliliter containing resveratrol 496.8 μ g, 7,6 parts of reference substance solution of 4 '-dihydroxyflavone, 438 μ g, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 368 μ g, lourerin B 645.2 μ g and Pterostilbene 1517.2 μ g, the aqueous solution of the ethanol that wherein 5 parts of reference substance solution are 70% by mass percentage concentration is diluted respectively to 2 times～6 times, obtain 5 parts of reference substance solution after dilution;
Reference substance solution after the above-mentioned reference substance solution preparing and dilution is carried out respectively to liquid chromatographic detection according to the testing conditions described in embodiment 57, calculate resveratrol in the finger-print that detects 6 parts of reference substance solution that obtain, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the chromatographic peak area of lourerin B and Pterostilbene, result of calculation is as shown in table 15～table 19, the mass concentration of resveratrol that table 15 obtains for the embodiment of the present invention 59 and the corresponding relation of peak area, table 16 for the embodiment of the present invention 59, obtain 7, the mass concentration of 4 '-dihydroxyflavone and the corresponding relation of peak area, the mass concentration of 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that table 17 obtains for the embodiment of the present invention 59 and the corresponding relation of peak area, the mass concentration of lourerin B that table 18 obtains for the embodiment of the present invention 59 and the corresponding relation of peak area, the mass concentration of Pterostilbene that table 19 obtains for the embodiment of the present invention 59 and the corresponding relation of peak area.
The mass concentration of the resveratrol that table 15 embodiment of the present invention 59 obtains and the corresponding relation of peak area
6 parts of reference substance solution 1 2 3 4 5 6
Mass concentration (μ g/mL) 7.7625 15.525 31.5 62.1 124.2 248.4
Peak area 106.90 223.58 466.57 976.73 2014.92 4168.97
Table 16 embodiment of the present invention 59 obtain 7, the mass concentration of 4 '-dihydroxyflavone and the corresponding relation of peak area
6 parts of reference substance solution 1 2 3 4 5 6
Mass concentration (μ g/mL) 6.8438 13.6875 27.375 54.75 109.5 219
Peak area 106.13 215.65 431.55 870.09 1711.24 3446.74
The mass concentration of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that table 17 embodiment of the present invention 59 obtains and the corresponding relation of peak area
6 parts of reference substance solution 1 2 3 4 5 6
Mass concentration (μ g/mL) 5.75 11.5 23 46 92 184
Peak area 58.01 116.96 233.14 468.70 938.30 1879.19
The mass concentration of the lourerin B that table 18 embodiment of the present invention 59 obtains and the corresponding relation of peak area
6 parts of reference substance solution 1 2 3 4 5 6
Mass concentration (μ g/mL) 10.0813 21.1625 40.325 80.65 161.3 322.6
Peak area 80.60 161.64 323.07 650.64 1304.71 2613.09
The mass concentration of the Pterostilbene that table 19 embodiment of the present invention 59 obtains and the corresponding relation of peak area
6 parts of reference substance solution 1 2 3 4 5 6
Mass concentration (μ g/mL) 23.7063 47.4125 94.825 189.65 379.3 758.6
Peak area 289.72 587.31 1205.97 2512.01 5282.86 10937.70
Embodiment 60
Data in the table 15～table 19 obtaining according to embodiment 59, the peak area value of reference substance in table of take is ordinate, mass concentration value is horizontal ordinate, carry out linear fit, obtain typical curve, typical curve is as shown in Figure 42～Figure 46, Figure 42 is the typical curve of the resveratrol that obtains of the embodiment of the present invention 60, Figure 43 be the embodiment of the present invention 60 obtain 7, the typical curve of 4 '-dihydroxyflavone, Figure 44 is the typical curve of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that obtains of the embodiment of the present invention 60, Figure 45 is the typical curve of the lourerin B that obtains of the embodiment of the present invention 60, Figure 46 is the typical curve of the Pterostilbene that obtains of the embodiment of the present invention 60.
According to the typical curve of Figure 42～Figure 46, draw the range of linearity, regression equation and the related coefficient of typical curve, result is shown in table 20, the range of linearity, regression equation and the related coefficient of the typical curve that table 20 obtains for the embodiment of the present invention 60.
The range of linearity of the typical curve that table 20 embodiment of the present invention 60 obtains, regression equation and related coefficient
Figure BDA0000429626030000341
Embodiment 61
Method according to described in embodiment 57, prepares solution to be measured;
By the above-mentioned solution to be measured preparing according to the testing conditions described in embodiment 57 respectively at 0h, 3h, 6h, 10h, 16h, 20h and 24h carry out liquid chromatographic detection 7 times, the finger-print that detection is obtained be take lourerin B peak as object of reference peak, be designated as S peak, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print calculating, described main peaks is that peak area accounts for more than 5% chromatographic peak of total peak area, the chromatographic peak obtaining according to embodiment 58 is pointed out resveratrol in the finger-print that result calculates, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, result of calculation is as shown in table 21～table 23, the relative retention time at each total peak and object of reference peak in the finger-print of the solution to be measured that table 21 obtains for the embodiment of the present invention 61, the relative peak area at main peaks and object of reference peak in the finger-print of the solution to be measured that table 22 obtains for the embodiment of the present invention 61, resveratrol in the finger-print of the solution to be measured that table 23 obtains for the embodiment of the present invention 61, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding.
The relative retention time at each total peak and object of reference peak in the finger-print of the solution to be measured that table 21 embodiment of the present invention 61 obtains
Figure BDA0000429626030000342
Figure BDA0000429626030000351
The relative peak area at main peaks and object of reference peak in the finger-print of the solution to be measured that table 22 embodiment of the present invention 61 obtains
Figure BDA0000429626030000352
Resveratrol, 7 in the finger-print of the solution to be measured that table 23 embodiment of the present invention 61 obtains, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding
Figure BDA0000429626030000353
By table 21～table 23, can be found out, different sample injection times detect in the finger-print obtaining, and the relative retention time at each total peak and object of reference peak is basically identical, its relative standard deviation (RSD) < 1%; The relative peak area at main peaks and object of reference peak is basically identical, its RSD < 3%; Different sample injection times detect resveratrol, 7 in the finger-print obtaining, and the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding is basically identical, its RSD < 2%.The present invention has calculated the similarity of the finger-print that finger-print that 0h sample detection obtains and other times sample detection obtain, and result of calculation is that similarity is greater than 0.99.
The experimental result of embodiment 61 shows, solution to be measured prepared by method provided by the invention had good stability in 24 hours.
Embodiment 62
Accurately weighed resveratrol, 7, after 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene by its mixing, to adding mass percentage concentration in the potpourri obtaining, it is the aqueous solution of 70% ethanol, make every milliliter containing resveratrol 60.3 μ g, 7, the reference substance solution of 4 '-dihydroxyflavone, 51.4 μ g, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 44.1 μ g, lourerin B 77.9 μ g and Pterostilbene 189 μ g;
The above-mentioned reference substance solution preparing is carried out to liquid chromatographic detection 7 times according to the method described in embodiment 61, according to the method described in embodiment 61, calculate and detect resveratrol, 7 in the finger-print obtaining, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding, result of calculation is shown in table 24, resveratrol, 7 in the finger-print of the reference substance solution that table 24 obtains for the embodiment of the present invention 62, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding.
Resveratrol, 7 in the finger-print of the reference substance solution that table 24 embodiment of the present invention 62 obtains, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding
Figure BDA0000429626030000361
As can be seen from Table 24, different sample injection times detect resveratrol, 7 in the finger-print obtaining, and the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding is basically identical, its RSD < 2%.
The experimental result of embodiment 62 shows, reference substance solution prepared by method provided by the invention had good stability in 24 hours.
Embodiment 63
Method according to described in embodiment 57, prepares solution to be measured;
The above-mentioned solution to be measured preparing is carried out to liquid chromatographic detection 6 times continuously according to the testing conditions described in embodiment 57, the finger-print that detection is obtained be take lourerin B peak as object of reference peak, be designated as S peak, according to the method described in embodiment 61, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print calculating, and resveratrol in the finger-print calculating, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, result of calculation is as shown in table 25～table 27, the relative retention time at each total peak and object of reference peak in the finger-print of the solution to be measured that table 25 obtains for the embodiment of the present invention 63, the relative peak area at main peaks and object of reference peak in the finger-print of the solution to be measured that table 26 obtains for the embodiment of the present invention 63, resveratrol in the finger-print of the solution to be measured that table 27 obtains for the embodiment of the present invention 63, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding.
The relative retention time at each total peak and object of reference peak in the finger-print of the solution to be measured that table 25 embodiment of the present invention 63 obtains
Figure BDA0000429626030000371
The relative peak area at main peaks and object of reference peak in the finger-print of the solution to be measured that table 26 embodiment of the present invention 63 obtains
Figure BDA0000429626030000372
Figure BDA0000429626030000381
Resveratrol, 7 in the finger-print of the solution to be measured that table 27 embodiment of the present invention 63 obtains, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding
Figure BDA0000429626030000382
By table 25～table 27, can be found out, in the finger-print that 6 liquid chromatographic detection obtain, the relative retention time at each total peak and object of reference peak is basically identical, its RSD < 1%; The relative peak area at main peaks and object of reference peak is basically identical, its RSD < 2%; Resveratrol, 7 in the finger-print that 6 liquid chromatographic detection obtain, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding is basically identical, its RSD < 2%.The solution to be measured that the present invention has calculated rear 5 sample introductions detects the similarity of the finger-print that the solution detection to be measured of the finger-print that obtains and the 1st sample introduction obtains, and result of calculation is that similarity is greater than 0.99.
The experimental result of embodiment 63 shows, solution to be measured prepared by method provided by the invention has good precision.
Embodiment 64
According to the method described in embodiment 62, prepare reference substance solution;
The above-mentioned reference substance solution preparing is carried out to liquid chromatographic detection 6 times according to the method described in embodiment 63, according to the method described in embodiment 61, calculate and detect resveratrol, 7 in the finger-print obtaining, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding, result of calculation is shown in table 28, resveratrol, 7 in the finger-print of the reference substance solution that table 28 obtains for the embodiment of the present invention 64, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding.
Resveratrol, 7 in the finger-print of the reference substance solution that table 28 embodiment of the present invention 64 obtains, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding
As can be seen from Table 28, resveratrol, 7 in the finger-print that 6 liquid chromatographic detection reference substance solution obtain, the peak area of the chromatographic peak that 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene are corresponding is basically identical, its RSD < 1%.
The experimental result of embodiment 64 shows, reference substance solution prepared by method provided by the invention has good precision.
Embodiment 65
According to the method described in embodiment 57, prepare 6 parts of solution to be measured;
Above-mentioned 6 parts of solution to be measured that prepare are carried out respectively to liquid chromatographic detection according to the testing conditions described in embodiment 57, the finger-print that detection is obtained be take lourerin B peak as object of reference peak, be designated as S peak, according to the method described in embodiment 61, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print calculating, and resveratrol in the finger-print calculating, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, brings the peak area value calculating in the regression equation of table 20, according to regression equation calculation, obtains resveratrol in 6 parts of solution to be measured, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the mass concentration of lourerin B and Pterostilbene, according to the volume of above-mentioned 6 parts of solution to be measured, obtains resveratrol in above-mentioned 6 parts of solution to be measured, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quality of lourerin B and Pterostilbene, exhausts the quality of sample according to dragon's blood, calculate dragon's blood and exhaust resveratrol in sample, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene content.Result of calculation is as shown in table 29～table 31, the relative retention time at each total peak and object of reference peak in the finger-print of the solution to be measured that table 29 obtains for the embodiment of the present invention 65, the relative peak area at main peaks and object of reference peak in the finger-print of the solution to be measured that table 30 obtains for the embodiment of the present invention 65, the dragon's blood that table 31 obtains for the embodiment of the present invention 65 exhausts resveratrol in sample, 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
The relative retention time at each total peak and object of reference peak in the finger-print of the solution to be measured that table 29 embodiment of the present invention 65 obtains
Figure BDA0000429626030000401
The relative peak area at main peaks and object of reference peak in the finger-print of the solution to be measured that table 30 embodiment of the present invention 65 obtains
The dragon's blood that table 31 embodiment of the present invention 65 obtains exhausts resveratrol in sample, 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
Figure BDA0000429626030000403
Figure BDA0000429626030000411
By table 29～table 31, can be found out, 6 parts of solution to be measured carries out in finger-print that liquid chromatographic detection obtains, and the relative retention time at each total peak and object of reference peak is basically identical, its RSD < 1%; The relative peak area at main peaks and object of reference peak is basically identical, its RSD < 3%; 6 parts of dragon's bloods exhaust resveratrol in sample, 7, and the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene is basically identical, its RSD < 3%.The present invention has calculated the similarity that rear 5 parts of solution to be measured detect the finger-print that the finger-print that obtains and the 1st part of solution detection to be measured obtain, and result is that similarity is greater than 0.99.
The experimental result of embodiment 65 shows, the detection method that dragon's blood provided by the invention exhausts has good repeatability.
Embodiment 66
According to the method described in embodiment 62, prepare every milliliter containing resveratrol 496.8 μ g, 7, the reference substance solution of 4 '-dihydroxyflavone, 438 μ g, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 368 μ g, lourerin B 645.2 μ g and Pterostilbene 1517.2 μ g;
Accurately weighed dragon's blood exhausts 6 parts of powdered samples, every part of heavily about 0.25g, it is placed in respectively to six tool plug conical flasks, to accurate in each tool plug conical flask, add the above-mentioned reference substance solution 3mL preparing, the accurate mass percentage concentration that adds is the aqueous solution 47mL of 70% ethanol again, 6 parts of mixed solutions that obtain are adopted respectively to 250W, the ultrasound wave of 40KHz carries out composition and extracts 30 minutes, 6 parts of solution after composition is extracted are cooled to ℃, by cooled solution filter, get subsequent filtrate and cross 0.22 μ m filter membrane, 6 parts of solution after filtering are carried out respectively to liquid chromatographic detection according to the testing conditions described in embodiment 57, according to the method described in embodiment 65, calculate and detect resveratrol in the finger-print obtaining, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, the peak area value calculating is brought in the regression equation of table 20, according to regression equation calculation, obtain resveratrol in above-mentioned 6 parts of solution, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the mass concentration of lourerin B and Pterostilbene, according to the volume of above-mentioned 6 parts of solution, calculate resveratrol in 6 parts of solution again, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quality of lourerin B and Pterostilbene, this quality and above-mentioned 6 parts of known dragon's bloods exhaust resveratrol in sample, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the difference of the content of lourerin B and Pterostilbene is the amount of recording of reference substance, the ratio of the addition of the amount of recording of reference substance and above-mentioned known reference substance is the average recovery of reference substance.
The average recovery result of calculation of reference substance is as shown in table 32～table 36, the average recovery of the resveratrol that table 32 obtains for the embodiment of the present invention 66, table 33 for the embodiment of the present invention 66, obtain 7, the average recovery of 4 '-dihydroxyflavone, the average recovery of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that table 34 obtains for the embodiment of the present invention 66, the average recovery of the lourerin B that table 35 obtains for the embodiment of the present invention 66, the average recovery of the Pterostilbene that table 36 obtains for the embodiment of the present invention 66.
The average recovery of the resveratrol that table 32 embodiment of the present invention 66 obtains
Figure BDA0000429626030000421
Table 33 embodiment of the present invention 66 obtain 7, the average recovery of 4 '-dihydroxyflavone
Figure BDA0000429626030000422
The average recovery of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one that table 34 embodiment of the present invention 66 obtains
Figure BDA0000429626030000423
The average recovery of the lourerin B that table 35 embodiment of the present invention 66 obtains
Figure BDA0000429626030000424
Figure BDA0000429626030000431
The average recovery of the Pterostilbene that table 36 embodiment of the present invention 66 obtains
The experimental result of embodiment 66 shows, the detection method energy Accurate Determining dragon's blood that dragon's blood provided by the invention exhausts exhausts resveratrol in sample, 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
Embodiment 67
According to the method described in embodiment 62, prepare containing resveratrol 7.7625 μ g/mL, 7,4 '-dihydroxyflavone, 6.8438 μ g/mL, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 5.75 μ g/mL, the reference substance solution of lourerin B 10.0813 μ g/mL and Pterostilbene 23.7063 μ g/mL, the ethanol water that is 70% by mass percentage concentration by described reference substance solution dilutes respectively 4 times, 8 times, 16 times, 32 times and 64 times, obtains the reference substance solution of variable concentrations;
The reference substance solution of the above-mentioned variable concentrations preparing is carried out respectively to liquid chromatographic detection according to the testing conditions described in embodiment 57, according to the method described in embodiment 65, calculate and detect resveratrol in the finger-print obtaining, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, brings the peak area value calculating in the regression equation of table 20, obtains resveratrol in the solution of above-mentioned variable concentrations according to regression equation calculation, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the mass concentration of lourerin B and Pterostilbene, obtains resveratrol in the solution of above-mentioned variable concentrations according to the volume of above-mentioned variable concentrations solution, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quality of lourerin B and Pterostilbene, and then obtain resveratrol, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quantitative limit of lourerin B and Pterostilbene and detectability, result is shown in table 37, the resveratrol that table 37 obtains for the embodiment of the present invention 67, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quantitative limit of lourerin B and Pterostilbene and detectability.
The resveratrol that table 37 embodiment of the present invention 67 obtains, 7, the quantitative limit of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene and detectability
? Quantitative limit (ng) Detectability (ng)
Resveratrol 4.85（S/N=18.1） 1.21（S/N=3.9）
7,4 '-dihydroxyflavone 2.14（S/N=10.3） 1.07（S/N=4.3）
3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 7.19（S/N=16.8） 0.90（S/N=3.3）
Lourerin B 6.30（S/N=11.8） 1.58（S/N=3.3）
Pterostilbene 7.41（S/N=20.2） 1.80（S/N=4.1）
As shown in Table 37, the mensuration that method provided by the invention exhausts the contained component of testing sample to dragon's blood has higher sensitivity.
Embodiment 68
Collect ten batches of dragon's bloods and exhaust sample, according to the method described in embodiment 57, be prepared into ten parts of solution to be measured, and ten parts of solution to be measured that prepare are carried out respectively to liquid chromatographic detection according to the testing conditions described in embodiment 57, the finger-print that detection is obtained be take lourerin B peak as object of reference peak, be designated as S peak, according to the method described in embodiment 61, calculate and detect each total peak and the relative retention time at object of reference peak and the relative peak area at main peaks and object of reference peak in the finger-print obtaining, result of calculation is as shown in table 38 and table 39, the relative retention time at each total peak and object of reference peak in the finger-print of the solution to be measured that table 38 obtains for the embodiment of the present invention 68, the relative peak area at main peaks and object of reference peak in the finger-print of the solution to be measured that table 39 obtains for the embodiment of the present invention 68.The present invention is usingd the finger-print of these ten batches of solution to be measured with similarity software and is that basis obtains " common pattern " of its finger-print as the reference fingerprint of solution to be measured, as shown in figure 47, Figure 47 is the reference fingerprint of the solution to be measured that obtains of the embodiment of the present invention 68.The present invention has calculated the similarity that above-mentioned ten batches of solution to be measured carry out the finger-print that liquid chromatographic detection obtains, and result of calculation is shown in table 40, the similarity of the finger-print of the solution to be measured that table 40 obtains for the embodiment of the present invention 68.
The relative retention time at each total peak and object of reference peak in the finger-print of the solution to be measured that table 38 embodiment of the present invention 68 obtains
Figure BDA0000429626030000441
Figure BDA0000429626030000451
The relative peak area at main peaks and object of reference peak in the finger-print of the solution to be measured that table 39 embodiment of the present invention 68 obtains
Figure BDA0000429626030000452
The similarity of the finger-print of the solution to be measured that table 40 embodiment of the present invention 68 obtains
Ten batches of dragon's bloods exhaust the batch number of sample Similarity
1102012 0.979
1101010 0.981
1103040 0.953
1105009 0.986
20090602 0.991
110701 0.981
110702 0.981
120127 0.973
120301 0.962
120315 0.951
According to table 38 and table 39, can determine, dragon's blood exhausts in the finger-print of sample solution to be measured each total peak and the relative retention time at object of reference peak and the relative peak area at main peaks and object of reference peak in the limits of table 41, the limits of the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print of the solution to be measured that table 41 obtains for the embodiment of the present invention 68;
As can be seen from Table 40, the ten batches of dragon's bloods exhaust the similarity that sample solution to be measured carries out the finger-print that liquid chromatographic detection obtains and are greater than 0.90.
The limits of the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print of the solution to be measured that table 41 embodiment of the present invention 68 obtains
Figure BDA0000429626030000461
Embodiment 69
According to the method described in embodiment 65, according to Figure 47, calculating in embodiment 68 ten batches of dragon's bloods exhausts sample solution to be measured and carries out resveratrol in finger-print that liquid chromatographic detection obtains, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, the peak area value calculating is brought in the regression equation of table 20, according to regression equation calculation, obtain resveratrol in above-mentioned ten batches of solution to be measured, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the mass concentration of lourerin B and Pterostilbene, according to the volume of above-mentioned ten batches of solution to be measured, obtain resveratrol in above-mentioned ten batches of solution to be measured, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quality of lourerin B and Pterostilbene, according to above-mentioned ten batches of dragon's bloods, exhaust again the quality of sample, calculate above-mentioned ten batches of dragon's bloods and exhaust resveratrol in sample, 7, 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the content of lourerin B and Pterostilbene.Result of calculation is shown in table 42, and the dragon's blood that table 42 obtains for the embodiment of the present invention 69 exhausts resveratrol in sample, 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene.
The dragon's blood that table 42 embodiment of the present invention 69 obtains exhausts resveratrol in sample, 7, the content of 4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, lourerin B and Pterostilbene
Figure BDA0000429626030000462
Figure BDA0000429626030000471
As shown in Table 42, adopting detection method that dragon's blood provided by the invention exhausts to exhaust sample to dragon's blood detects, testing result is, dragon's blood exhausts sample and calculates by dry product, containing 7 of the resveratrol of 0.4wt%～1.0wt%, 0.3wt%～0.8wt%, the Pterostilbene of the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one of 4 '-dihydroxyflavone, 0.3wt%～0.8wt%, the lourerin B of 0.4wt%～1.0wt% and 0.9wt%～2.5wt%.
Embodiment 70
Accurately weighed dragon's blood exhausts powdered sample 0.5g, be placed in tool plug conical flask, to the accurate mass percentage concentration that adds in tool plug conical flask, be the aqueous solution 75mL of 70% ethanol, by after the weighed weight of the solution obtaining, adopt 250W, the ultrasound wave of 40kHz carries out composition to it and extracts 30min, solution after composition is extracted is cooled to 10～30 ℃, weighed weight again, the aqueous solution complementary component of the ethanol that is 70% by mass percentage concentration is extracted the weight of less loss, the solution of supplying after weight is shaken up, then by its filtration, get subsequent filtrate and cross 0.22 μ m filter membrane, obtain solution to be measured.
The above-mentioned solution to be measured preparing is carried out to liquid chromatographic detection, and testing conditions is:
Take octadecylsilane chemically bonded silica as chromatographic column filling agent;
Chromatographic column model is Phenomenex Kinetex 2.6u C18 100A, and column's length is 100mm, and internal diameter is 4.6mm, and particle diameter is 2.6 μ m;
Chromatographic column column temperature is 30 ℃;
Take acetonitrile as mobile phase A, take ultrapure water as Mobile phase B, the percent by volume sum of described mobile phase A and Mobile phase B is 100%, carries out gradient elution:
In 0～12min, the percent by volume of described mobile phase A rises to 17% from 12% linearity, and the percent by volume of described Mobile phase B drops to 83% from 88% linearity;
In 12min～22min, the percent by volume of described mobile phase A rises to 27% from 17% linearity, and the percent by volume of described Mobile phase B drops to 73% from 83% linearity;
In 22min～35min, the percent by volume of described mobile phase A rises to 39% from 27% linearity, and the percent by volume of described Mobile phase B drops to 61% from 73% linearity;
In 35min～45min, the percent by volume of described mobile phase A remains on 39%, and the percent by volume of described Mobile phase B remains on 61%;
In 46min～55min, the percent by volume of described mobile phase A rises to 89% from 39% linearity, and the percent by volume of described Mobile phase B drops to 11% from 61% linearity;
The flow velocity of described mobile phase A and Mobile phase B is 1.5mLmin -1;
Detection wavelength is 260nm.
The solution to be measured of the embodiment of the present invention 70 preparation is carried out to finger-print that liquid chromatographic detection obtains as shown in figure 48, Figure 48 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 70, the finger-print obtaining be take to lourerin B peak as object of reference peak, be designated as S peak, according to the method described in embodiment 61, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print calculating, result of calculation is shown in table 43, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print of the solution to be measured that table 43 obtains for the embodiment of the present invention 70.
The relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print of the solution to be measured that table 43 embodiment of the present invention 70 obtains
Figure BDA0000429626030000481
According to the method described in embodiment 65, according to the finger-print in Figure 48, resveratrol in calculated fingerprint collection of illustrative plates, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, brings the peak area value calculating in the regression equation of table 20, according to regression equation calculation, obtains resveratrol in above-mentioned solution to be measured, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the mass concentration of lourerin B and Pterostilbene, then obtain resveratrol in above-mentioned solution to be measured according to the volume of above-mentioned solution to be measured, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quality of lourerin B and Pterostilbene, the quality that exhausts sample according to above-mentioned dragon's blood obtains the dragon's blood that embodiment 70 detects and exhausts resveratrol in sample, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the content of lourerin B and Pterostilbene, testing result is that dragon's blood exhausts sample and calculates by dry product, containing the resveratrol of 0.4wt%, 7 of 0.3wt%, 4 '-dihydroxyflavone, the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one of 0.3wt%, the lourerin B of 0.4wt% and the Pterostilbene of 0.9wt%.
Embodiment 71
Accurately weighed dragon's blood exhausts powdered sample 0.5g, be placed in tool plug conical flask, to the accurate mass percentage concentration that adds in tool plug conical flask, be the aqueous solution 60mL of 80% ethanol, by after the weighed weight of the solution obtaining, adopt 250W, the ultrasound wave of 40kHz carries out composition to it and extracts 30min, solution after composition is extracted is cooled to 10 ℃, weighed weight again, the aqueous solution complementary component of the ethanol that is 80% by mass percentage concentration is extracted the weight of less loss, the solution of supplying after weight is shaken up, then by its filtration, get subsequent filtrate and cross 0.22 μ m filter membrane, obtain solution to be measured.
The above-mentioned solution to be measured preparing is carried out to liquid chromatographic detection, and testing conditions is:
Take octadecylsilane chemically bonded silica as chromatographic column filling agent;
Chromatographic column model is Phenomenex Kinetex 2.6u C18 100A, and column's length is 100mm, and internal diameter is 4.6mm, and particle diameter is 2.6 μ m;
Chromatographic column column temperature is 45 ℃;
Take acetonitrile as mobile phase A, take ultrapure water as Mobile phase B, the percent by volume sum of described mobile phase A and Mobile phase B is 100%, carries out gradient elution:
In 0～12min, the percent by volume of described mobile phase A rises to 19% from 14% linearity, and the percent by volume of described Mobile phase B drops to 81% from 86% linearity;
In 12min～22min, the percent by volume of described mobile phase A rises to 29% from 19% linearity, and the percent by volume of described Mobile phase B drops to 71% from 81% linearity;
In 22min～35min, the percent by volume of described mobile phase A rises to 41% from 29% linearity, and the percent by volume of described Mobile phase B drops to 59% from 71% linearity;
In 35min～45min, the percent by volume of described mobile phase A remains on 41%, and the percent by volume of described Mobile phase B remains on 59%;
In 46min～55min, the percent by volume of described mobile phase A rises to 91% from 41% linearity, and the percent by volume of described Mobile phase B drops to 9% from 59% linearity;
The flow velocity of described mobile phase A and Mobile phase B is 2.0mLmin -1;
Detection wavelength is 300nm.
The solution to be measured of the embodiment of the present invention 71 preparation is carried out to finger-print that liquid chromatographic detection obtains as shown in figure 49, Figure 49 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 71, the finger-print obtaining be take to lourerin B peak as object of reference peak, be designated as S peak, according to the method described in embodiment 61, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print calculating, result of calculation is shown in table 44, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print of the solution to be measured that table 44 obtains for the embodiment of the present invention 71.
The relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print of the solution to be measured that table 44 embodiment of the present invention 71 obtains
According to the method described in embodiment 65, according to the finger-print in Figure 49, resveratrol in calculated fingerprint collection of illustrative plates, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, brings the peak area value calculating in the regression equation of table 20, according to regression equation calculation, obtains resveratrol in above-mentioned solution to be measured, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the mass concentration of lourerin B and Pterostilbene, then obtain resveratrol in above-mentioned solution to be measured according to the volume of above-mentioned solution to be measured, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quality of lourerin B and Pterostilbene, the quality that exhausts sample according to above-mentioned dragon's blood obtains the dragon's blood that embodiment 71 detects and exhausts resveratrol in sample, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the content of lourerin B and Pterostilbene, testing result is that dragon's blood exhausts sample and calculates by dry product, containing the resveratrol of 1.0wt%, 7 of 0.8wt%, 4 '-dihydroxyflavone, the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one of 0.8wt%, the lourerin B of 1.0wt% and the Pterostilbene of 2.5wt%.
Embodiment 72
Accurately weighed dragon's blood exhausts powdered sample 0.5g, be placed in tool plug conical flask, to the accurate mass percentage concentration that adds in tool plug conical flask, be the aqueous solution 60mL of 75% ethanol, by after the weighed weight of the solution obtaining, adopt 250W, the ultrasound wave of 40kHz carries out composition to it and extracts 30min, solution after composition is extracted is cooled to 15 ℃, weighed weight again, the aqueous solution complementary component of the ethanol that is 75% by mass percentage concentration is extracted the weight of less loss, the solution of supplying after weight is shaken up, then by its filtration, get subsequent filtrate and cross 0.22 μ m filter membrane, obtain solution to be measured.
The above-mentioned solution to be measured preparing is carried out to liquid chromatographic detection, and testing conditions is:
Take octadecylsilane chemically bonded silica as chromatographic column filling agent;
Chromatographic column model is Phenomenex Kinetex 2.6u C18 100A, and column's length is 100mm, and internal diameter is 4.6mm, and particle diameter is 2.6 μ m;
Chromatographic column column temperature is 40 ℃;
Take acetonitrile as mobile phase A, take ultrapure water as Mobile phase B, the percent by volume sum of described mobile phase A and Mobile phase B is 100%, carries out gradient elution:
In 0～12min, the percent by volume of described mobile phase A rises to 18% from 13% linearity, and the percent by volume of described Mobile phase B drops to 82% from 87% linearity;
In 12min～22min, the percent by volume of described mobile phase A rises to 28% from 18% linearity, and the percent by volume of described Mobile phase B drops to 72% from 82% linearity;
In 22min～35min, the percent by volume of described mobile phase A rises to 40% from 28% linearity, and the percent by volume of described Mobile phase B drops to 60% from 72% linearity;
In 35min～45min, the percent by volume of described mobile phase A remains on 40%, and the percent by volume of described Mobile phase B remains on 60%;
In 46min～55min, the percent by volume of described mobile phase A rises to 90% from 40% linearity, and the percent by volume of described Mobile phase B drops to 10% from 60% linearity;
The flow velocity of described mobile phase A and Mobile phase B is 1.7mLmin -1;
Detection wavelength is 280nm.
The solution to be measured of the embodiment of the present invention 72 preparation is carried out to finger-print that liquid chromatographic detection obtains as shown in figure 50, Figure 50 is the finger-print of the solution to be measured that obtains of the embodiment of the present invention 72, the finger-print obtaining be take to lourerin B peak as object of reference peak, be designated as S peak, according to the method described in embodiment 61, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print calculating, result of calculation is shown in table 45, the relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print of the solution to be measured that table 45 obtains for the embodiment of the present invention 72.
The relative peak area at the relative retention time at each total peak and object of reference peak and main peaks and object of reference peak in the finger-print of the solution to be measured that table 45 embodiment of the present invention 72 obtains
Figure BDA0000429626030000521
According to the method described in embodiment 65, according to the finger-print in Figure 50, resveratrol in calculated fingerprint collection of illustrative plates, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the peak area of the chromatographic peak that lourerin B and Pterostilbene are corresponding, brings the peak area value calculating in the regression equation of table 20, according to regression equation calculation, obtains resveratrol in above-mentioned solution to be measured, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the mass concentration of lourerin B and Pterostilbene, then obtain resveratrol in above-mentioned solution to be measured according to the volume of above-mentioned solution to be measured, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the quality of lourerin B and Pterostilbene, the quality that exhausts sample according to above-mentioned dragon's blood obtains the dragon's blood that embodiment 72 detects and exhausts resveratrol in sample, 7,4 '-dihydroxyflavone, 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one, the content of lourerin B and Pterostilbene, testing result is that dragon's blood exhausts sample and calculates by dry product, containing the resveratrol of 0.7wt%, 7 of 0.5wt%, 4 '-dihydroxyflavone, the 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one of 0.6wt%, the lourerin B of 0.68wt% and the Pterostilbene of 1.7wt%.
The explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof.It should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.Above-mentioned explanation to the disclosed embodiments, makes the special technician in this area can realize or use the present invention, to the multiple modification of these embodiment, will be apparent concerning professional and technical personnel in the field.General Principle as defined herein can, in the situation that not departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention will can not be restricted to these embodiment shown in this article, but will meet the widest scope consistent with principle disclosed herein and novel features.

Claims (10)

1. the detection method that dragon's blood exhausts, comprises the following steps:
(1), dragon's blood exhausted to testing sample mix with alcohol compound, obtain solution to be measured;
(2), described solution to be measured is carried out to liquid chromatographic detection, obtain the finger-print of described solution to be measured, mobile phase in described liquid chromatographic detection comprises mobile phase A and Mobile phase B, described mobile phase A is acetonitrile, described Mobile phase B is water or trifluoroacetic acid, and the percent by volume sum of described mobile phase A and Mobile phase B is 100%;
The elution program of described liquid chromatographic detection is gradient elution, and described gradient elution is:
In 0～12min, the percent by volume of described mobile phase A rises to 17%～19% from 12%～14%, and the percent by volume of described Mobile phase B drops to 81%～83% from 86%～88%;
In 12min～22min, the percent by volume of described mobile phase A rises to 27%～29% from 17%～19%, and the percent by volume of described Mobile phase B drops to 71%～73% from 81%～83%;
In 22min～35min, the percent by volume of described mobile phase A rises to 39%～41% from 27%～29%, and the percent by volume of described Mobile phase B drops to 59%～61% from 71%～73%;
In 35min～45min, the percent by volume of described mobile phase A remains on 39%～41%, and the percent by volume of described Mobile phase B remains on 59%～61%;
In 46min～55min, the percent by volume of described mobile phase A rises to 89%～91% from 39%～41%, and the percent by volume of described Mobile phase B drops to 9%～11% from 59%～61%;
(3), according to the finger-print of described solution to be measured, preassigned curve and dragon's blood, exhaust the quality of testing sample, obtain the content that dragon's blood exhausts ingredient in testing sample, described typical curve is the mass concentration of the dragon's blood reference substance solution that exhausts testing sample ingredient and the linearity curve between corresponding peak area.
2. method according to claim 1, is characterized in that, described alcohol compound is that C atomicity is 1～5 alcohol compound.
3. method according to claim 2, is characterized in that, described alcohol compound is ethanol or methyl alcohol.
4. method according to claim 3, is characterized in that, described alcohol compound is that mass percentage concentration is the aqueous solution of 70%～80% ethanol.
5. method according to claim 1, is characterized in that, described gradient elution is specially:
In 0～12min, the percent by volume of described mobile phase A rises to 18% from 13%, and the percent by volume of described Mobile phase B drops to 82% from 87%;
In 12min～22min, the percent by volume of described mobile phase A rises to 28% from 18%, and the percent by volume of described Mobile phase B drops to 72% from 82%;
In 22min～35min, the percent by volume of described mobile phase A rises to 40% from 28%, and the percent by volume of described Mobile phase B drops to 60% from 72%;
In 35min～45min, the percent by volume of described mobile phase A remains on 40%, and the percent by volume of described Mobile phase B remains on 60%;
In 46min～55min, the percent by volume of described mobile phase A rises to 90% from 40%, and the percent by volume of described Mobile phase B drops to 10% from 60%.
6. method according to claim 1, is characterized in that, the flow velocity of described mobile phase is 1.5mLmin -1～2.0mLmin -1.
7. method according to claim 1, is characterized in that, the detecting device of described liquid chromatographic detection is UV-detector;
Detection wavelength is 260nm～300nm.
8. method according to claim 1, is characterized in that, the chromatographic column column temperature in described liquid chromatographic detection is 30 ℃～45 ℃.
9. method according to claim 1, is characterized in that, described step (1) is:
Dragon's blood is exhausted to testing sample and be dissolved in alcohol compound, ultrasonic extraction, obtains solution to be measured.
10. method according to claim 9, is characterized in that, the time of described ultrasonic extraction is 15 minutes～60 minutes.
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