CN109959735A - The content assaying method of three kinds of ingredients in line leaf broom top, line leaf goldspink flower extract and line leaf goldspink jasmine tea - Google Patents
The content assaying method of three kinds of ingredients in line leaf broom top, line leaf goldspink flower extract and line leaf goldspink jasmine tea Download PDFInfo
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Abstract
The invention belongs to food quality control technology fields, specifically provide the content assaying method of line leaf broom top, line leaf goldspink flower extract and line leaf goldspink jasmine tea, include the following steps: the preparation of mixed reference substance solution, the preparation of test solution, chromatographic condition and system suitability, according to high effective liquid chromatography for measuring, using octadecylsilane chemically bonded silica column as chromatographic column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 0.8-1.5% or using the formic acid of volumetric concentration 0.1-0.5% as Mobile phase B；Chromatography is carried out according to gradient elution program, control flow rate of mobile phase is 0.8-1.2ml/min；Detection wavelength is 275~295nm and 340~360nm；Measuring method: drawing mixed reference substance solution respectively and test solution injects liquid chromatograph, the content for measuring and calculating Lutonaretin in test solution, orientoside and aspalathin, the present invention provides a kind of content assaying methods for quickly, accurately, easily measuring line leaf broom top, line leaf goldspink flower extract and line leaf goldspink jasmine tea.
Description
Technical field
The invention belongs to food quality control technology fields, relate in particular to line leaf broom top, line leaf broom top is extracted
The content assaying method of three kinds of ingredients in object and line leaf goldspink jasmine tea.
Background technique
Line leaf broom top is the leguminous plant (Classification system: Aspalathus Linearis (Brum.f.) in South Africa
It R.Dahlgren), is the new raw-food material of national health and family planning committee member bulletin.Line leaf goldspink flower extract is line leaf gold
The water extract of sparrow flower, line leaf goldspink jasmine tea is the common drink containing wired leaf goldspink flower extract.Line leaf broom top is containing abundant
Anti-oxidant flavonoids adjusts function of human body to human free radical is removed, and prevents and control a variety of diseases to play significant curative effect, line
Decaffeinated and oxalic acid in leaf broom top, there is the effect of calm central nervous system, is suitble to lose with allergy, headache, sleep
Often, the people of the symptoms such as insomnia, nervous, slight melancholia and neural high-pressure takes；There are also the effects for alleviating skin allergy
Fruit can directly apply on infected skin, itch to skin, eczema, diaper rash, frustrating sore etc., skin allergy symptom can all have alleviation
With the effect of mitigation.Therefore establish a kind of line leaf content assaying method gorsy for line leaf broom top food, health care product or
The development and utilization of drug have great importance.
It is now recognized that flavone compound is line leaf main active ingredient gorsy, flavone compound is mainly Polygonum orientale
Glycosides, Lutonaretin, aspalathin etc..Since orientoside and Lutonaretin are isomer, more difficulty is efficiently separated, and
Aspalathin content in fermentation molded line leaf broom top is lower, is not easy to extract, in addition, line leaf broom top ingredient is various, complicated, it is miscellaneous
Matter content is more, at present temporary not wired leaf quality control standard gorsy." assay of aspalathin in doctor's tea " document
In disclose the HPLC measuring method of aspalathin in doctor's tea, in the method, only by measure aspalathin concentration come
Determine line leaf quality gorsy, measurement result is single, complete detection can not be carried out to line leaf broom top, and this method exists
(see Cha Shenghua etc., aspalathin contains the problem of extraction operation is complicated, separating degree is low, peak type is poor, long operational time in doctor's tea
It is fixed to measure, research and development of natural products, 2009,21:414-415).
Therefore, for control line leaf broom top and line leaf goldspink flower extract and the edible safety of line leaf goldspink jasmine tea,
It is necessary to establish one kind quickly, accurately, easily can while contain to what plurality of active ingredients in line leaf broom top was detected
Quantity measuring method.
Summary of the invention
For this purpose, the purpose of the present invention is to provide it is a kind of quickly, it is accurate, facilitate measurement line leaf broom top and line leaf broom top
The detection method of Lutonaretin, orientoside and aspalathin content in extract and line leaf goldspink jasmine tea, and then comprehensively and accurately
The quality of control line leaf broom top and line leaf goldspink flower extract and line leaf goldspink jasmine tea.
In order to solve the above technical problems, of the invention provide a kind of line leaf broom top, line leaf goldspink flower extract and line
The content assaying method of three kinds of ingredients, includes the following steps: in leaf goldspink jasmine tea
The preparation of mixed reference substance solution: Lutonaretin reference substance, orientoside reference substance and aspalathin reference substance are taken, is added
Enter alcoholic solution and mixed reference substance solution is made；
The preparation of test solution: taking sample to be tested, and alcoholic solution, ultrasonic extraction or circumfluence distillation is added, lets cool, and is added
Alcoholic solution dilutes constant volume, shakes up, and filters, takes subsequent filtrate to get test solution；
Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, with octadecylsilane bonded silica
Rubber column gel column is chromatographic column；Using acetonitrile as mobile phase A, with the acetum of volumetric concentration 0.8-1.5% or with volumetric concentration 0.1-
0.5% formic acid is Mobile phase B；Chromatography is carried out according to gradient elution program, control flow rate of mobile phase is 0.8-1.2ml/
min；Detection wavelength is 275~295nm and 340~360nm；
Measuring method: drawing mixed reference substance solution respectively and test solution injects liquid chromatograph, measures and calculates confession
The content of Lutonaretin, orientoside and aspalathin in test sample solution.
Further, the sample to be tested is line leaf broom top, line leaf goldspink flower extract, line leaf goldspink jasmine tea, line leaf
At least one of broom top pharmaceutical composition or line leaf broom top pharmaceutical preparation.
Preferably, the line leaf broom top include fermentation molded line leaf broom top and non-fermented molded line leaf broom top at least
One kind, the line leaf goldspink flower extract include fermentation molded line leaf goldspink flower extract and non-fermented molded line leaf goldspink flower extract
At least one of.
Further, the method for the chromatographic condition and system suitability specifically: according to high performance liquid chromatography,
Using Waters Symmetry C18 (4.6mm × 250mm, 5 μm) chromatographic column, Diamonsil C18 (4.6mm × 250mm, 5 μ
M) chromatographic column or Agilent Extend-C18 (4.6mm × 250mm, 5 μm) chromatographic column；It is dense with volume using acetonitrile as mobile phase A
The acetum of degree 1% is Mobile phase B；Chromatography: 0-12min is carried out according to following gradient elution program, mobile phase A: stream
Dynamic phase B is 12%:88%；12-25min, mobile phase A: Mobile phase B is 12%:88% → 20%:80%；25-28min, flowing
Phase A: Mobile phase B 20%:80%；28-28.01min, mobile phase A: Mobile phase B is 20%:80% → 100%:0；28.01-
38min, mobile phase A: Mobile phase B 100%:0；38-38.01min, mobile phase A: Mobile phase B is 100%:0 → 12%:
88%；38.01-50min mobile phase A: Mobile phase B 12%:88%；And controlling flow rate of mobile phase is 1ml/min；Detect wave
A length of 288 and 350nm.
Preferably, in the measuring method, a series of mixed reference substance solution of concentration is constructed, and is injected separately into liquid phase color
Spectrometer, is made the standard curve of Lutonaretin, orientoside and aspalathin respectively, and test solution injects liquid chromatograph, root
According to the standard curve, the content of Lutonaretin, orientoside and aspalathin in test solution is calculated.
Further, in the preparation of the test solution, the alcoholic solution is ethanol solution or methanol solution；Institute
It states in extraction process, the amount ratio of the sample to be tested and alcoholic solution are as follows: 0.2~0.5g:20~50ml.
Preferably, the volumetric concentration of the alcoholic solution is 20~80%.
Further, the volumetric concentration of the alcoholic solution is 60~80%.
Preferably, in the preparation of the test solution, the time of ultrasonic extraction is 30~40min, and supersonic frequency is
35~40kHz.
The present invention also provides a kind of any of the above-described online leaf broom top of content assaying method, line leaf broom tops to mention
Take object, line leaf goldspink jasmine tea, line leaf broom top pharmaceutical composition or the broom top pharmaceutical preparation quality testing of line leaf and control field
Application.
The content of three kinds of ingredients in line leaf broom top of the present invention, line leaf goldspink flower extract or line leaf goldspink jasmine tea
Measuring method, by control high-efficient liquid phase chromatogram condition, screen suitable mobile phase and gradient elution program, effectively overcome because
Complicated component, dopant species and content easily cause more in line leaf broom top, line leaf goldspink flower extract or line leaf goldspink jasmine tea
The problem of Lutonaretin, orientoside and aspalathin principal component peak interfere and Lutonaretin and orientoside are isomer,
Inferior separating effect the problem of interfering with each other, not only simplifies the pretreated step of sample to be tested, and improve Lutonaretin and
The separating degree of orientoside to each other, and be effectively separated with other impurity peaks, not only specificity is strong for the HPLC method of foundation, line
Sexual intercourse, precision, the rate of recovery are all satisfied Pharmaceutical Analysis requirement, and extracting method is simple, runing time is short, realizes simultaneously
Using same flow phase system, containing for Lutonaretin in line leaf goldspink jasmine tea, orientoside and aspalathin can be disposably completed
It is fixed to measure, can quick, the easy quality condition for knowing the product, the detection method for solving the prior art can only measure
Single component, the problem of quality of medicinal material can not be controlled comprehensively, and present invention determine that HPLC content assaying method it is not only exclusive
Property is strong, and linear relationship, precision, the rate of recovery are all satisfied Pharmaceutical Analysis requirement, and extracting method is simple, runing time is short, from
And it realizes and come control line leaf broom top and its is mentioned by the content of measurement effective component Lutonaretin, orientoside and aspalathin
The purpose of the quality of object and line leaf goldspink jasmine tea is taken, and the content assaying method has simple and quick, reliable and stable, precision
Advantage that is high, being easy to grasp.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is the chromatogram of specificity test during the method for the present invention is investigated；
Fig. 2 is mixed reference substance solution and line leaf goldspink jasmine tea in system suitability during the method for the present invention is investigated
Chromatogram；Wherein A is the chromatogram that contrast solution is mixed under 288nm, and B is the chromatogram that contrast solution is mixed under 350nm, and C is
The chromatogram of the offline leaf goldspink jasmine tea test solution of 288nm, D are the chromatography of the offline leaf goldspink jasmine tea test solution of 350nm
Figure；
Fig. 3 is ferment in system suitability during the method for the present invention is investigated molded line leaf broom top and non-fermented molded line leaf
Chromatogram gorsy；Wherein A is the chromatogram of fermentation molded line leaf broom top test solution under 288nm, and B issues for 350nm
The chromatogram of ferment molded line leaf broom top test solution, C are the chromatography of non-fermented molded line leaf broom top test solution under 288nm
Figure, D are the chromatogram of non-fermented molded line leaf broom top test solution under 350nm；
Fig. 4 is ferment in system suitability during the method for the present invention is investigated molded line leaf goldspink flower extract and non-fermented
The chromatogram of molded line leaf goldspink flower extract；Wherein A is the color of fermentation molded line leaf goldspink flower extract test solution under 288nm
Spectrogram, B are the chromatogram of fermentation molded line leaf goldspink flower extract test solution under 350nm, and C is non-fermented molded line under 288nm
The chromatogram of leaf goldspink flower extract test solution, D are non-fermented molded line leaf goldspink flower extract test solution under 350nm
Chromatogram；
Fig. 5 is the chromatogram of the present embodiment 6 and the test solution in embodiment 7；Wherein A is under 288nm in embodiment 6
The chromatogram of fermentation molded line leaf goldspink flower extract test solution, B are molded line leaf broom top of fermenting under 350nm in embodiment 6
The chromatogram of extract test solution；C is that non-fermented molded line leaf goldspink flower extract test sample is molten under 288nm in embodiment 7
The chromatogram of liquid, D are the chromatogram of non-fermented molded line leaf goldspink flower extract test solution under 350nm in embodiment 7；
Fig. 6 is the chromatogram of the test solution in embodiment 8 and 10；Wherein A is fermented type under 288nm in embodiment 8
The chromatogram of line leaf goldspink flower extract test solution, B are the molded line leaf goldspink flower extract that ferments under 350nm in embodiment 8
The chromatogram of test solution；C is the color of non-fermented molded line leaf goldspink flower extract test solution under 288nm in embodiment 10
Spectrogram, D are the chromatogram of non-fermented molded line leaf goldspink flower extract test solution under 350nm in embodiment 10；
Fig. 7 is the chromatogram of the test solution in embodiment 9 and 11；Wherein A is fermented type under 288nm in embodiment 9
The chromatogram of line leaf goldspink flower extract test solution, B are the molded line leaf goldspink flower extract that ferments under 350nm in embodiment 9
The chromatogram of test solution；C is the color of non-fermented molded line leaf goldspink flower extract test solution under 288nm in embodiment 11
Spectrogram, D are the chromatogram of non-fermented molded line leaf goldspink flower extract test solution under 350nm in embodiment 11；
Fig. 8 is chromatogram of the non-fermented molded line leaf goldspink flower extract test solution at 350nm in comparative example 1；
Fig. 9 is the chromatogram of the test solution in comparative example 2；Wherein A is that non-fermented molded line leaf broom top mentions under 288nm
The chromatogram of object test solution is taken, B is the chromatogram of non-fermented molded line leaf goldspink flower extract test solution under 350nm；
Figure 10 is the chromatogram of the test solution in comparative example 3；Wherein A is non-fermented molded line leaf broom top under 288nm
The chromatogram of extract test solution, B are the chromatography of non-fermented molded line leaf goldspink flower extract test solution under 350nm
Figure.
Figure 11 is the chromatogram of mixed reference substance solution and test solution in comparative example 4, and wherein A is to mix under 280nm
The chromatogram of reference substance solution is closed, B is the chromatogram of mixed reference substance solution under 350nm；C is fermentation molded line leaf gold under 280nm
The chromatogram of sparrow flower extract test solution, D are the chromatography of fermentation molded line leaf goldspink flower extract test solution under 350nm
Figure.
Specific embodiment
1 methodological study
1.1 main experimental instruments and reagent
Instrument: 1260 liquid chromatograph of Agilent configures diode array detector；
Reagent: acetonitrile, methanol, acetic acid, formic acid are chromatographically pure；
Line leaf broom top: it is purchased from ZhangZhou great Min Food Co., Ltd, lot number: TLE6124847 (fermented type),
TLE6124848 (non-fermented type)；
Line leaf goldspink flower extract: it is purchased from Rooibos Limited, lot number: 20151022 (fermented types), 20150203
(non-fermented type).
Line leaf goldspink jasmine tea: it is provided by perfect (Guangdong) daily necessities Co., Ltd, lot number: 20180531-1,20180531-
2 and 20180531-3.
Negative control sample without line leaf goldspink flower extract: being provided by perfect (Guangdong) daily necessities Co., Ltd, batch
Number: 20180530.
1.2 test method
The preparation of mixed reference substance solution: by shown in table 1, precision weigh Lutonaretin reference substance, orientoside reference substance and
Aspalathin reference substance is appropriate, and addition ascorbic 60vt% ethanol solution dissolution containing 0.2wt% is respectively prepared every 1ml and contains
476.016 μ g Lutonaretin reference substances and hybrid standard stock solution containing 444.074 μ g orientoside reference substances and contain 182.662
The aspalathin standard reserving solution B of μ g aspalathin reference substance, it is then accurate respectively to draw two groups of each 1ml of standard reserving solution, it sets
It is mixed in 10ml measuring bottle, the ascorbic 60vt% ethanol solution dilution containing 0.2wt% is then added and is settled to scale, shakes up,
Up to mixed reference substance solution；
The preparation of test solution: line taking leaf goldspink jasmine tea 0.3g, it is accurately weighed, it sets in 25mL volumetric flask, is added
60vt% ethanol solution 20mL is ultrasonically treated 30min, and supersonic frequency 35Hz is let cool, and is settled to quarter with 60vt% ethanol solution
Degree, shakes up, with 0.45 μm of organic membrane filtration, takes subsequent filtrate to get line leaf goldspink jasmine tea test solution；Or
Line taking leaf goldspink flower extract 0.2g, it is accurately weighed, it sets in 25mL volumetric flask, 60vt% ethanol solution is added about
20mL, is ultrasonically treated 30min, and supersonic frequency 35Hz lets cool, is settled to scale with 60vt% ethanol solution, shakes up, with 0.45
μm organic membrane filtration, takes subsequent filtrate to get line leaf goldspink flower extract test solution；Or
Line taking leaf broom top 0.5g, it is accurately weighed, it sets in 25mL volumetric flask, 60vt% ethanol solution 20mL, ultrasound is added
30min is handled, supersonic frequency 40Hz lets cool, is settled to scale with 60vt% ethanol solution, shakes up, and has machine filter with 0.45 μm
Film filtering, takes subsequent filtrate to get line leaf broom top test solution；
Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, with Waters Symmetry C18
(4.6mm × 250mm, 5 μm) column is chromatographic column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 1% as mobile phase
B；Chromatography is carried out according to gradient elution program；
The gradient elution program specifically: 0-12min, mobile phase A: Mobile phase B 12%:88%；12-25min, stream
Dynamic phase A: Mobile phase B is 12%:88% → 20%:80%；25-28min, mobile phase A: Mobile phase B 20%:80%；28-
28.01min, mobile phase A: Mobile phase B is 20%:80% → 100%:0；28.01-38min mobile phase A: Mobile phase B is
100%:0；38-38.01min, mobile phase A: Mobile phase B is 100%:0 → 12%:88%；38.01-50min mobile phase A:
Mobile phase B is 12%:88%；Control flow rate of mobile phase is 1ml/min；Detection wavelength is 288nm and 350nm；5 μ l of sample volume；
Column temperature: 25 DEG C；
Measuring method: it is accurate respectively to draw mixed reference substance solution and each 5 μ l of test solution, liquid chromatograph is injected, is surveyed
It is fixed, calculate to get.
1 standard reserving solution of table matches tabulation
The test of 1.3 specificities
The line leaf goldspink jasmine tea negative sample without line leaf goldspink flower extract is taken, according to above-mentioned 1.2 lower test methods
Line leaf goldspink jasmine tea yin is made in the preparation method for the line leaf goldspink jasmine tea test solution recorded in " preparation of test solution "
Property contrast solution, line taking leaf goldspink jasmine tea press 1.2 under test method made from line leaf goldspink jasmine tea test solution, then
It is molten by " chromatographic condition and system suitability " measurement line leaf goldspink jasmine tea negative control in 1.2 test methods respectively
Liquid and line leaf goldspink jasmine tea test solution.Shown in the result is shown in Figure 1, wherein A is that the offline leaf goldspink jasmine tea negative control of 288nm is molten
The chromatogram of liquid, B are the chromatogram of the offline leaf goldspink jasmine tea test solution of 288nm；C is the offline leaf goldspink jasmine tea yin of 350nm
Property contrast solution chromatogram, D be the offline leaf goldspink jasmine tea test solution of 350nm chromatogram, at 288nm and 350nm,
Be showed no in line leaf goldspink jasmine tea negative control solution chromatogram with Lutonaretin in test solution chromatogram, orientoside and Ah
The chromatographic peak of the identical retention time of Si Bating shows that negative controls solution does not interfere with the detection at principal component peak.
1.4 system suitability
It is molten that line taking leaf goldspink jasmine tea presses the line leaf goldspink jasmine tea test sample recorded in " preparation of test solution " under 1.2
Line leaf goldspink jasmine tea test solution is made in the preparation method of liquid；Line taking leaf goldspink flower extract (fermented type, lot number:
20151022) and line leaf goldspink flower extract (non-fermented type, lot number: 20150203) respectively by under 1.2 " test solution
It is molten that line leaf goldspink flower extract test sample is made in the preparation method for the line leaf goldspink flower extract test sample solution recorded in preparation "
Liquid；Line taking leaf broom top (fermented type, lot number: TLE6124847) and line leaf broom top (non-fermented type, lot number: TLE6124848)
Line leaf is made by the preparation method for the line leaf broom top test solution recorded in " preparation of test solution " under 1.2 respectively
Broom top test solution；Take Lutonaretin reference substance, orientoside reference substance and aspalathin reference substance respectively by under 1.2
Mixed reference substance solution is made in test method, measures by the chromatographic condition in 1.2 with system suitability；As a result such as Fig. 2-
Shown in 4, at 350nm, the retention time of Lutonaretin is about 20.7min, and the retention time of orientoside is about 22.1min,
Under 288nm, the retention time of aspalathin is about 24.2min, and theoretical cam curve is all larger than 50000, different Polygonum in test solution
The separating degree at careless glycosides, orientoside and aspalathin peak and adjacent unknown peak is all larger than 1.5, and system suitability is good.
1.5 linear relationships are investigated
The hybrid standard stock solution and A Siba by Lutonaretin and orientoside obtained under 1.2 are pipetted by 2 precision of table
Spit of fland standard reserving solution B is appropriate, and it is dense that addition ascorbic 60vt% ethanol solution dilution containing 0.2wt% is configured to a series of reality
The contrast solution of degree, the contrast solution of each concentration 5 μ l of each accurate measurement, injects liquid chromatograph, by the chromatography under 1.2
Condition and system suitability measure and record chromatogram, carry out linear regression to peak area with concentration (μ g/ml).
2 series standard solution of table is with tabulation
Conclusion: the linear equation of Lutonaretin is y=14.86237x+5.36195 (R2=0.99992), and Lutonaretin exists
Linear relationship is good in 4.7602~142.8048 μ g/mL concentration ranges；The linear equation of orientoside is y=13.79783x+
5.29306 (R2=0.99992), orientoside linear relationship in 4.4407~133.2223 μ g/mL concentration ranges are good；A Si
The linear equation of Ba Ting is y=12.34226x+0.844934 (R2=0.99997), and aspalathin is in 1.8266~54.7985 μ
Linear relationship is good in g/mL concentration range.
1.6 precision test
Precision is measured by mixed reference substance solution obtained under 1.2, by the chromatographic condition under 1.2, continuous sample introduction 6
Secondary, the peak area of measurement Lutonaretin, orientoside and aspalathin calculates the RSD value of peak area.It the results are shown in Table shown in 3, different Polygonum
The RSD of careless glycosides, the retention time of orientoside and aspalathin and peak area is respectively less than 2%, shows that instrument precision is good.
3 Precision test result of table
1.7 repetitive test
Line taking leaf goldspink jasmine tea 0.3g, line leaf goldspink flower extract 0.2g and each 6 parts of line leaf broom top 0.5g respectively, essence
It is close weighed, it is molten by method preparation line leaf goldspink jasmine tea test solution, the line leaf goldspink flower extract test sample under 1.2 respectively
Liquid and line leaf broom top test solution are measured according to the chromatographic condition under 1.2.Lutonaretin, orientoside and aspalathin
Content in chromatographic data processing software external standard method calculate, the results are shown in Table 4.As shown in Table 4,6 parts of Duplicate Samples test results
RSD less than 2%, show that this law has preferable repeatability.
4 repetitive test result table of table
1.8 accuracy and recovery test
Line taking leaf goldspink flower extract 0.1g, line leaf broom top 0.25g and each 9 parts of 0.15g of line leaf goldspink jasmine tea respectively,
It is accurately weighed, according to the ratio between component content to be measured in basic, normal, high concentrations control product additional amount and test sample 0.8:1,1:1,
1.2:1, by addition Lutonaretin standard solution, Polygonum orientale shown in table 5-1,5-2,5-3,6-1,6-2,6-3,7-1,7-2 and 7-3
Glycosides standard solution and aspalathin standard solution distinguish preparation line leaf goldspink flower extract test sample by 1.2 lower methods
Solution, line leaf broom top test solution and line leaf goldspink jasmine tea test solution are measured by the chromatographic condition under 1.2.Knot
Fruit is shown in Table shown in 5-1,5-2,5-3,6-1,6-2,6-3,7-1,7-2 and 7-3, and the rate of recovery shows between 95%-105%
This law has preferable accuracy.
The recovery test result of Lutonaretin in table 5-1 line leaf goldspink jasmine tea
The recovery test result of orientoside in table 5-2 line leaf goldspink jasmine tea
The recovery test result of aspalathin in table 5-3 line leaf goldspink jasmine tea
The recovery test result of Lutonaretin in table 6-1 line leaf goldspink flower extract
The recovery test result of orientoside in table 6-2 line leaf goldspink flower extract
The recovery test result of aspalathin in table 6-3 line leaf goldspink flower extract
The recovery test result of Lutonaretin in table 7-1 line leaf broom top
The recovery test result of orientoside in table 7-2 line leaf broom top
The recovery test result of aspalathin in table 7-3 line leaf broom top
1.9 stability test
Take same mixing reference substance test solution and test solution, by 1.2 lower chromatographic conditions, respectively at 0,8,16, for 24 hours into
Sample measurement.It the results are shown in Table 8.The result shows that the RSD of the peak area of Lutonaretin, orientoside and aspalathin peak is respectively less than 2%, table
Bright test solution is interior for 24 hours basicly stable.
8 stability test result of table
1.10 serviceability test
The test solution prepared under 1.2 is taken, investigates flow velocity (0.9mL/min and 1.1mL/min), column temperature (23 respectively
DEG C and 30 DEG C), different batches chromatographic column and different instrument (1.Agilent 1260, chromatographic column Waters Symmetry C18,
Lot number is 0309372961；2.Waters UPLC H-Class, chromatographic column Waters Symmetry C18, lot number are
0314380961；3.Waters UPLC H-Class, chromatographic column Waters Symmetry C18, lot number 0313380991)
Etc. conditions influence of the variation to chromatographic isolation and assay result, the results are shown in Table 9.
The result shows that the minor change of flow velocity and column temperature condition has no significant effect chromatographic peak separating effect, ingredient to be measured
The error of content is respectively less than 5%, and the reproducibility on different batches chromatographic column, different instruments is preferable, shows that this method is durable
Property is preferable.
9 durability of table investigates result
1.11 quantitative limits and detection limit are investigated
Take the Lutonaretin that concentration is 30.7192 μ g/mL and the orientoside hybrid standard that concentration is 29.0567 μ g/mL molten
Liquid dilutes step by step, measures according to the chromatographic condition under 1.2.The concentration of Lutonaretin is calculated by work station chromatographic software
Its signal-to-noise ratio is 13.5 when its signal-to-noise ratio is 3.6 when for 4.9ng/mL, concentration is 24.6ng/mL；The concentration of orientoside is
Its signal-to-noise ratio is 12.0 when its signal-to-noise ratio is 3.5 when 4.6ng/mL, concentration is 23.2ng/mL.That is the detection of Lutonaretin is limited to
4.9ng/mL is quantitatively limited to 24.6ng/mL；The detection of orientoside is limited to 4.6ng/mL, is quantitatively limited to 23.2ng/mL.
Taking concentration is the aspalathin standard solution of 6.893 μ g/mL, is diluted step by step, is surveyed according to the chromatographic condition under 1.2
It is fixed.The signal-to-noise ratio calculated when aspalathin concentration is 34.5ng/mL by work station chromatographic software is 2.7, and concentration is
Signal-to-noise ratio when 137.9ng/mL is 10.7.That is the detection of aspalathin is limited to 34.5ng/mL, is quantitatively limited to 137.9ng/mL.
The fermentation molded line leaf goldspink flower extract of embodiment 1
The content assaying method of three kinds of ingredients, includes the following steps: in line leaf goldspink flower extract described in the present embodiment
The preparation of mixed reference substance solution: precision weighs Lutonaretin reference substance, orientoside reference substance and aspalathin pair
It is appropriate according to product, the dissolution of 60vt% ethanol solution is added, every 1ml is respectively prepared containing 476.016 μ g Lutonaretin reference substances and contains
The hybrid standard stock solution of 444.074 μ g orientoside reference substances and aspalathin containing 182.662 μ g aspalathin reference substances
Standard reserving solution B, it is then accurate to draw two groups of each 1ml of standard reserving solution, it is placed in 10ml measuring bottle and mixes, 60vt% is then added
Ethanol solution dilution is settled to scale, shakes up to get mixed reference substance solution；
The preparation of test solution: (fermented type, lot number: 20151022), precision claims line taking leaf goldspink flower extract 0.2g
It is fixed, it sets in 25mL volumetric flask, 60vt% ethanol solution about 20mL is added, be ultrasonically treated 30min, supersonic frequency 35Hz is let cool,
It is settled to scale with 60vt% ethanol solution, is shaken up, with 0.45 μm of organic membrane filtration, takes subsequent filtrate to get line leaf broom top
Extract test solution；
Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, with Waters Symmetry C18
(4.6mm × 250mm, 5 μm) column is chromatographic column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 1% as mobile phase
B；Chromatography is carried out according to gradient elution program；
The gradient elution program specifically: 0-12min, mobile phase A: Mobile phase B 12%:88%；12-25min, stream
Dynamic phase A: Mobile phase B is 12%:88% → 20%:80%；25-28min, mobile phase A: Mobile phase B 20%:80%；28-
28.01min, mobile phase A: Mobile phase B is 20%:80% → 100%:0；28.01-38min mobile phase A: Mobile phase B is
100%:0；38-38.01min, mobile phase A: Mobile phase B is 100%:0 → 12%:88%；38.01-50min mobile phase A:
Mobile phase B is 12%:88%；Control flow rate of mobile phase is 1ml/min；Detection wavelength is 288nm and 350nm；5 μ l of sample volume；
Column temperature: 25 DEG C；
Measuring method: it is accurate respectively to draw mixed reference substance solution and each 5 μ l of test solution, liquid chromatograph is injected, is surveyed
It is fixed, calculate to get.
2 non-fermented molded line leaf goldspink flower extract of embodiment
The content assaying method of three kinds of ingredients, includes the following steps: in line leaf goldspink flower extract described in the present embodiment
The preparation of mixed reference substance solution: precision weighs Lutonaretin reference substance, orientoside reference substance and aspalathin pair
It is appropriate according to product, the ascorbic 60vt% ethanol solution dissolution containing 0.2wt% is added, every 1ml is respectively prepared containing the 476.016 different Polygonums of μ g
Careless glycosides reference substance and hybrid standard stock solution containing 444.074 μ g orientoside reference substances and contain 182.662 μ g aspalathins pair
It is then accurate to draw two groups of each 1ml of standard reserving solution according to the aspalathin standard reserving solution B of product, it is placed in 10ml measuring bottle and mixes,
The ascorbic 60vt% ethanol solution dilution containing 0.2wt% is then added and is settled to scale, shakes up molten to get mixing reference substance
Liquid；
The preparation of test solution: line taking leaf goldspink flower extract 0.2g (non-fermented type, lot number: 20150203), accurate
It is weighed, it sets in 50mL volumetric flask, 60vt% ethanol solution about 40mL is added, be ultrasonically treated 40min, supersonic frequency 40Hz is put
It is cold, it is settled to scale with 60vt% ethanol solution, is shaken up, with 0.45 μm of organic membrane filtration, takes subsequent filtrate to get line leaf goldspink
Flower extract test solution；
Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, with Waters Symmetry C18
(4.6mm × 250mm, 5 μm) column is chromatographic column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 1% as mobile phase
B；Chromatography is carried out according to gradient elution program；
The gradient elution program specifically: 0-12min, mobile phase A: Mobile phase B 12%:88%；12-25min, stream
Dynamic phase A: Mobile phase B is 12%:88% → 20%:80%；25-28min, mobile phase A: Mobile phase B 20%:80%；28-
28.01min, mobile phase A: Mobile phase B is 20%:80% → 100%:0；28.01-38min mobile phase A: Mobile phase B is
100%:0；38-38.01min, mobile phase A: Mobile phase B is 100%:0 → 12%:88%；38.01-50min mobile phase A:
Mobile phase B is 12%:88%；Control flow rate of mobile phase is 1ml/min；Detection wavelength is 288nm and 350nm；5 μ l of sample volume；
Column temperature: 25 DEG C；
Measuring method: it is accurate respectively to draw mixed reference substance solution and each 5 μ l of test solution, liquid chromatograph is injected, is surveyed
It is fixed, calculate to get.
3 line leaf goldspink jasmine tea of embodiment
The content assaying method of three kinds of ingredients, includes the following steps: in line leaf goldspink jasmine tea described in the present embodiment
The preparation of mixed reference substance solution: precision weighs Lutonaretin reference substance, orientoside reference substance and aspalathin pair
It is appropriate according to product, the ascorbic 60vt% ethanol solution dissolution containing 0.2wt% is added, every 1ml is respectively prepared containing the 476.016 different Polygonums of μ g
Careless glycosides reference substance and hybrid standard stock solution containing 444.074 μ g orientoside reference substances and contain 182.662 μ g aspalathins pair
It is then accurate to draw two groups of each 1ml of standard reserving solution according to the aspalathin standard reserving solution B of product, it is placed in 10ml measuring bottle and mixes,
The ascorbic 60vt% ethanol solution dilution containing 0.2wt% is then added and is settled to scale, shakes up molten to get mixing reference substance
Liquid；
The preparation of test solution: line taking leaf goldspink jasmine tea 0.3g, it is accurately weighed, it sets in 20mL volumetric flask, is added
60vt% ethanol solution about 15mL, is ultrasonically treated 35min, and supersonic frequency 35Hz lets cool, is settled to 60vt% ethanol solution
Scale shakes up, and with 0.45 μm of organic membrane filtration, takes subsequent filtrate to get line leaf goldspink flower extract test solution；
Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, with Waters Symmetry C18
(4.6mm × 250mm, 5 μm) column is chromatographic column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 1% as mobile phase
B；Chromatography is carried out according to gradient elution program；
The gradient elution program specifically: 0-12min, mobile phase A: Mobile phase B 12%:88%；12-25min, stream
Dynamic phase A: Mobile phase B is 12%:88% → 20%:80%；25-28min, mobile phase A: Mobile phase B 20%:80%；28-
28.01min, mobile phase A: Mobile phase B is 20%:80% → 100%:0；28.01-38min mobile phase A: Mobile phase B is
100%:0；38-38.01min, mobile phase A: Mobile phase B is 100%:0 → 12%:88%；38.01-50min mobile phase A:
Mobile phase B is 12%:88%；Control flow rate of mobile phase is 1ml/min；Detection wavelength is 288nm and 350nm；5 μ l of sample volume；
Column temperature: 25 DEG C；
Measuring method: it is accurate respectively to draw mixed reference substance solution and each 5 μ l of test solution, liquid chromatograph is injected, is surveyed
It is fixed, calculate to get.
The fermentation molded line leaf broom top of embodiment 4
The content assaying method of three kinds of ingredients, includes the following steps: in line leaf broom top described in the present embodiment
The preparation of mixed reference substance solution: precision weighs Lutonaretin reference substance, orientoside reference substance and aspalathin pair
It is appropriate according to product, the ascorbic 60vt% ethanol solution dissolution containing 0.2wt% is added, every 1ml is respectively prepared containing the 476.016 different Polygonums of μ g
Careless glycosides reference substance and hybrid standard stock solution containing 444.074 μ g orientoside reference substances and contain 182.662 μ g aspalathins pair
It is then accurate to draw two groups of each 1ml of standard reserving solution according to the aspalathin standard reserving solution B of product, it is placed in 10ml measuring bottle and mixes,
The ascorbic 60vt% ethanol solution dilution containing 0.2wt% is then added and is settled to scale, shakes up molten to get mixing reference substance
Liquid；
The preparation of test solution: line taking leaf broom top 0.5g (fermented type, lot number: TLE6124847), it is accurately weighed, it sets
In 25mL volumetric flask, 60vt% ethanol solution about 20mL is added, is ultrasonically treated 30min, supersonic frequency 35Hz is let cool, and is used
60vt% ethanol solution is settled to scale, shakes up, and with 0.45 μm of organic membrane filtration, subsequent filtrate is taken to mention to get line leaf broom top
Take object test solution；
Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, with Waters Symmetry C18
(4.6mm × 250mm, 5 μm) column is chromatographic column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 1% as mobile phase
B；Chromatography is carried out according to gradient elution program；
The gradient elution program specifically: 0-12min, mobile phase A: Mobile phase B 12%:88%；12-25min, stream
Dynamic phase A: Mobile phase B is 12%:88% → 20%:80%；25-28min, mobile phase A: Mobile phase B 20%:80%；28-
28.01min, mobile phase A: Mobile phase B is 20%:80% → 100%:0；28.01-38min mobile phase A: Mobile phase B is
100%:0；38-38.01min, mobile phase A: Mobile phase B is 100%:0 → 12%:88%；38.01-50min mobile phase A:
Mobile phase B is 12%:88%；Control flow rate of mobile phase is 1ml/min；Detection wavelength is 288nm and 350nm；5 μ l of sample volume；
Column temperature: 25 DEG C；
Measuring method: it is accurate respectively to draw mixed reference substance solution and each 5 μ l of test solution, liquid chromatograph is injected, is surveyed
It is fixed, calculate to get.
5 non-fermented molded line leaf broom top of embodiment
The content assaying method of three kinds of ingredients, includes the following steps: in line leaf broom top described in the present embodiment
The preparation of mixed reference substance solution: precision weighs Lutonaretin reference substance, careless glycosides reference substance and aspalathin control
Appropriate product are added the ascorbic 60vt% ethanol solution dissolution containing 0.2wt% and every 1ml are respectively prepared containing the 476.016 different Polygonum orientales of μ g
It glycosides reference substance and hybrid standard stock solution containing 444.074 μ g orientoside reference substances and is compareed containing 182.662 μ g aspalathins
The aspalathin standard reserving solution B of product, it is then accurate to draw two groups of each 1ml of standard reserving solution, it is placed in 10ml measuring bottle and mixes, with
The ascorbic 60vt% ethanol solution dilution containing 0.2wt% is added afterwards and is settled to scale, shakes up to get mixed reference substance solution；
The preparation of test solution: line taking leaf broom top 0.5g (non-fermented type, lot number: TLE6124848), it is accurately weighed,
It sets in 50mL volumetric flask, 60vt% ethanol solution about 40mL is added, be ultrasonically treated 30min, supersonic frequency 35Hz is let cool, and is used
60vt% ethanol solution is settled to scale, shakes up, and with 0.45 μm of organic membrane filtration, subsequent filtrate is taken to mention to get line leaf broom top
Take object test solution；
Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, with Waters Symmetry C18
(4.6mm × 250mm, 5 μm) column is chromatographic column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 1% as mobile phase
B；Chromatography is carried out according to gradient elution program；
The gradient elution program specifically: 0-12min, mobile phase A: Mobile phase B 12%:88%；12-25min, stream
Dynamic phase A: Mobile phase B is 12%:88% → 20%:80%；25-28min, mobile phase A: Mobile phase B 20%:80%；28-
28.01min, mobile phase A: Mobile phase B is 20%:80% → 100%:0；28.01-38min mobile phase A: Mobile phase B is
100%:0；38-38.01min, mobile phase A: Mobile phase B is 100%:0 → 12%:88%；38.01-50min mobile phase A:
Mobile phase B is 12%:88%；Control flow rate of mobile phase is 1ml/min；Detection wavelength is 288nm and 350nm；5 μ l of sample volume；
Column temperature: 25 DEG C；
Measuring method: it is accurate respectively to draw mixed reference substance solution and each 5 μ l of test solution, liquid chromatograph is injected, is surveyed
It is fixed, calculate to get.
Embodiment 6
The content assaying method of three kinds of ingredients in line leaf goldspink flower extract described in the present embodiment, the area with embodiment 1
It is not only that, chromatographic condition is different from system suitability, and the chromatographic condition and system suitability of the present embodiment are specific
Are as follows: according to high effective liquid chromatography for measuring, with Waters Symmetry C18 (4.6mm × 250mm, 5 μm) column for chromatographic column；
Using acetonitrile as mobile phase A, using 0.2% formic acid solution of volumetric concentration as Mobile phase B；Chromatography point is carried out according to gradient elution program
Analysis；
The gradient elution program specifically: 0-30min, mobile phase A: Mobile phase B is 10%:90% → 14%:86%；
30-35min, mobile phase A: Mobile phase B is 14%:86% → 100%:0%；35-45min, mobile phase A: Mobile phase B is
100%:0%；Control flow rate of mobile phase is 1ml/min；Detection wavelength is 288nm and 350nm；5 μ l of sample volume；Column temperature: 25 DEG C；
As a result as shown in figure 5, fermentation molded line leaf goldspink flower extract test solution in Lutonaretin, orientoside and A Si
The separating degree at Ba Tingfeng and adjacent unknown peak is all larger than 1.5, good separation, however retention time and instrument runing time compared with
It is long.
Embodiment 7
The content assaying method of three kinds of ingredients in line leaf goldspink flower extract described in the present embodiment, the area with embodiment 2
It is not only that, in chromatographic condition and system suitability, the present embodiment replaces implementing using 0.2% formic acid solution of volumetric concentration
1% acetum of volumetric concentration in example 2, as a result as shown in figure 5, different Polygonum orientale in non-thread leaf goldspink flower extract test solution
The separating degree at glycosides, orientoside and aspalathin peak and adjacent unknown peak is all larger than 1.5, good separation, however retention time
It is longer with instrument runing time.
Embodiment 8
The content assaying method of three kinds of ingredients in fermentation molded line leaf goldspink flower extract described in the present embodiment, with embodiment
1 difference is only that chromatographic column difference, uses Agilent Extend-C18 (4.6mm × 250mm, 5 μm) column in the present embodiment
Waters Symmetry C18 (4.6mm × 250mm, the 5 μm) column used in alternate embodiment 1, as a result as shown in fig. 6,
Under 350nm, the separating degree at orientoside and Lutonaretin and adjacent unknown peak is greater than under 1.5,288nm, aspalathin and it is adjacent not
Know that the separating degree at peak is greater than 1.5, good separation.
Embodiment 9
The content assaying method of three kinds of ingredients in fermentation molded line leaf goldspink flower extract described in the present embodiment, with embodiment
1 difference is only that chromatographic column difference, is substituted in the present embodiment using Diamonsil C18 (4.6mm × 250mm, 5 μm) column real
Apply Waters Symmetry C18 (4.6mm × 250mm, the 5 μm) column used in example 1, as a result as shown in fig. 7, under 350nm, Polygonum
The separating degree of careless glycosides and Lutonaretin and adjacent unknown peak is greater than under 1.5,288nm, the separation of aspalathin and adjacent unknown peak
Degree is greater than 1.5, good separation.
Embodiment 10
The content assaying method of three kinds of ingredients in non-fermented molded line leaf goldspink flower extract described in the present embodiment, with implementation
The difference of example 2 is only that chromatographic column difference, uses Agilent Extend-C18 (4.6mm × 250mm, 5 μm) in the present embodiment
Waters Symmetry C18 (4.6mm × 250mm, the 5 μm) column used in column alternate embodiment 1, as a result as shown in fig. 6,
Under 350nm, the separating degree at orientoside and Lutonaretin and adjacent unknown peak is greater than under 1.5,288nm, aspalathin and it is adjacent not
Know that the separating degree at peak is greater than 1.5, good separation.
Embodiment 11
The content assaying method of three kinds of ingredients in non-fermented molded line leaf goldspink flower extract described in the present embodiment, with implementation
The difference of example 2 is only that chromatographic column difference, is substituted in the present embodiment using Diamonsil C18 (4.6mm × 250mm, 5 μm) column
Waters Symmetry C18 (4.6mm × 250mm, 5 μm) column used in Example 1, as a result as shown in fig. 7, under 350nm,
The separating degree of orientoside and Lutonaretin and adjacent unknown peak is greater than under 1.5,288nm, point of aspalathin and adjacent unknown peak
It is greater than 1.5 from degree, good separation.
Comparative example 1
The content assaying method of three kinds of ingredients in line leaf goldspink flower extract described in this comparative example, the area with embodiment 1
It is not only that chromatographic condition difference, the chromatographic condition used in this comparative example are as follows:
According to high effective liquid chromatography for measuring, with Waters Symmetry C18 (4.6mm × 250mm, 5 μm) column for color
Compose column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 0.1% as Mobile phase B；It is carried out according to gradient elution program
Chromatography；
The gradient elution program specifically: 0-60min, mobile phase A: Mobile phase B is 5%:95% → 100%:0%,
Control flow rate of mobile phase is 1ml/min；Detection wavelength is 350nm, as a result as shown in figure 8, Lutonaretin chromatographic peak, orientoside color
Spectral peak cannot be efficiently separated with aspalathin chromatographic peak with adjacent interference peak, inferior separating effect.
Comparative example 2
The content assaying method of three kinds of ingredients in line leaf goldspink flower extract described in this comparative example, the area with embodiment 2
It is not only that chromatographic condition difference, the chromatographic condition used in this comparative example are as follows:
According to high effective liquid chromatography for measuring, with Waters Symmetry C18 (4.6mm × 250mm, 5 μm) column for color
Compose column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 0.1% as Mobile phase B；It is carried out according to gradient elution program
Chromatography；
The gradient elution program used in this comparative example specifically: 0-30min, mobile phase A: Mobile phase B 10%:90%
→ 14%:86%；30-50min, mobile phase A: Mobile phase B 14%:86%, control flow rate of mobile phase are 1ml/min；Detection
Wavelength is 288 and 350nm, as a result as shown in figure 9, from A it is found that under 288nm, the separating degree of aspalathin and adjacent unknown peak
It is 1.10, for separating degree less than 1.5, separating degree is not able to satisfy the requirement of chromatography.
Comparative example 3
The content assaying method of three kinds of ingredients in line leaf goldspink flower extract described in this comparative example, the area with embodiment 2
It is not only that chromatographic column difference, uses Agilent Eclipse XDB-C18 (4.6mm × 250mm, 5 μm) column in this comparative example
Waters Symmetry C18 (4.6mm × 250mm, the 5 μm) column used in alternate embodiment 1, the results are shown in Figure 10, from A
In it is found that at 288nm, the separating degree at aspalathin and adjacent unknown peak is 1.24, and less than 1.5, separating degree is not able to satisfy color
The requirement of spectrum analysis.
Comparative example 4
This comparative example is only that chromatographic condition is different referring to the document quoted in background technique, from the difference of embodiment 1, this
Comparative example uses SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), using the formic acid solution of volumetric concentration 0.25% as mobile phase A,
Using methanol as Mobile phase B；Chromatography is carried out according to gradient elution program；
The gradient elution program specifically: 0-5min, mobile phase A: Mobile phase B 80%:20%；5-25min, flowing
Phase A: Mobile phase B is 80%:20% → 70%:30%；25-40min, mobile phase A: Mobile phase B is 70%:30% → 65%:
35%；40-50min, mobile phase A: Mobile phase B is 65%:35% → 60%:40%；50-70min, mobile phase A: Mobile phase B
For 60%:40% → 80%:20%；70-80min, mobile phase A: Mobile phase B 80%:20%；Controlling flow rate of mobile phase is
0.4ml/min；Detection wavelength is 280nm and 350nm；10 μ l of sample volume；Column temperature: 38 DEG C.Shown in the result is shown in Figure 11, by scheming A and B
It is found that mixed reference substance solution only shows 2 chromatographic peaks under above-mentioned chromatographic condition, the wherein feature of orientoside and aspalathin
Peak is completely coincident, and can not be efficiently separated, by figure C and D it is found that the chromatography of fermentation molded line leaf goldspink flower extract test solution
Figure, each chromatographic peak separating degree is poor, and respectively less than 1.5, it therefore, can not Accurate Determining line leaf broom top under the chromatographic condition
The content of middle orientoside, Lutonaretin and aspalathin.
The investigation of 1 Extraction solvent concentration of experimental example
It is respectively 20% ethanol solution, 40% ethanol solution, 50% ethanol solution, 60% that volumetric concentration has been investigated in this experiment
Ethanol solution, 70% ethanol solution, 80% ethanol solution, 95% ethanol solution, totally 7 kinds of Extraction solvent concentration.
Fermentation molded line leaf goldspink flower extract 0.2g, non-fermented molded line leaf goldspink flower extract 0.2g, fermented type are taken respectively
Line leaf broom top 0.5g, non-fermented molded line leaf broom top 0.5g and each 7 parts of 0.3g of line leaf goldspink jasmine tea, it is accurately weighed, it sets
In 25mL volumetric flask, it is separately added into above-mentioned solution about 20mL, 30min is ultrasonically treated, lets cool, be settled to scale with corresponding solution,
It shakes up, with 0.45 μm of organic membrane filtration, takes subsequent filtrate, be measured, the results are shown in Table by the chromatographic condition recorded under 1.2
Shown in 10-1,10-2,11-1,11-2 and 12-1.The results show that using the ethanol solution of volumetric concentration 20%~80% to extract
When solvent extraction, ferment molded line leaf goldspink flower extract, non-fermented molded line leaf goldspink flower extract and line leaf goldspink jasmine tea contain
Amount relative standard deviation is respectively less than 3%, and when ethanol solution concentration is 60% and 80%, different Polygonum in the line leaf broom top measured
The content highest of careless glycosides, orientoside and aspalathin, it is therefore preferable that the ethanol solution of volumetric concentration 60% is Extraction solvent.
Extraction result table of the ethanol solution of table 10-1 various concentration to fermentation molded line leaf goldspink flower extract
Extraction result table of the ethanol solution of table 10-2 various concentration to non-fermented molded line leaf goldspink flower extract
The ethanol solution of table 11-1 various concentration is to fermentation molded line leaf extraction result table gorsy
The ethanol solution of table 11-2 various concentration is to non-fermented molded line leaf extraction result table gorsy
Extraction result table of the ethanol solution of table 12-1 various concentration to line leaf goldspink jasmine tea
The investigation of 2 Extraction solvent type of experimental example
Water, 60% ethanol solution of volumetric concentration, 60% methanol solution of volumetric concentration, 3 kinds of extraction reagents pair have been investigated in this experiment
Ferment molded line leaf goldspink flower extract, non-fermented molded line leaf goldspink flower extract, ferment molded line leaf broom top, non-fermented molded line leaf
The extraction effect of broom top and line leaf goldspink jasmine tea.
Fermentation molded line leaf goldspink flower extract 0.2g, non-fermented molded line leaf goldspink flower extract 0.2g, fermented type are taken respectively
Line leaf broom top 0.5g, non-fermented molded line leaf broom top 0.5g and each 3 parts of 0.3g of line leaf goldspink jasmine tea, it is accurately weighed, it sets
In 25mL volumetric flask, it is separately added into above-mentioned solution about 20mL, 30min is ultrasonically treated, lets cool, be settled to scale with corresponding solution,
It shakes up, with 0.45 μm of organic membrane filtration, takes subsequent filtrate, be measured, the results are shown in Table by the chromatographic condition recorded under 1.2
Shown in 13-1,13-2,14-1,14-2 and 15-1.The results show that when adopting water as Extraction solvent extraction, than using volumetric concentration
60% ethanol solution or the methanol solution of volumetric concentration 60% are that the resulting content of Extraction solvent extraction is low, the result phase of three
5% is greater than to standard deviation, when using the ethanol solution of volumetric concentration 60% as Extraction solvent extraction, molded line leaf broom top of fermenting
Extract, non-fermented molded line leaf goldspink flower extract, fermentation molded line leaf broom top, non-fermented molded line leaf broom top and line leaf gold
The more other Extraction solvents of the content of sparrow jasmine tea are high, it is therefore preferable that the ethanol solution of volumetric concentration 60% or volumetric concentration 60%
Methanol solution be Extraction solvent.
Extraction result table of the table 13-1 different solvents to fermentation molded line leaf goldspink flower extract
Extraction result table of the table 13-2 different solvents to non-fermented molded line leaf goldspink flower extract
Table 14-1 different solvents are to fermentation molded line leaf extraction result table gorsy
Table 14-2 different solvents are to non-fermented molded line leaf extraction result table gorsy
Extraction result table of the table 15-1 different solvents to line leaf goldspink jasmine tea
The investigation of 3 extraction time of experimental example
This experiment has investigated two kinds of extraction times of 30min and 60min to fermentation molded line leaf goldspink flower extract, non-fermented
The extraction of molded line leaf goldspink flower extract, ferment molded line leaf broom top, non-fermented molded line leaf broom top and line leaf goldspink jasmine tea
Effect.
Fermentation molded line leaf goldspink flower extract 0.2g, non-fermented molded line leaf goldspink flower extract 0.2g, fermented type are taken respectively
Line leaf broom top 0.5g, non-fermented molded line leaf broom top 0.5g and each 3 parts of 0.3g of line leaf goldspink jasmine tea, it is accurately weighed, it sets
In 25mL volumetric flask, it is separately added into above-mentioned solution about 20mL, the above-mentioned time is ultrasonically treated, lets cool, is settled to quarter with corresponding solution
Degree, shakes up, with 0.45 μm of organic membrane filtration, takes subsequent filtrate, be measured by the chromatographic condition recorded under 1.2, as a result see
Shown in table 16-1,16-2,17-1,17-2 and 18-1.The results show that when ultrasonic treatment (37kHz, 500w) 30min is extracted, fermentation
Molded line leaf goldspink flower extract, non-fermented molded line leaf goldspink flower extract, fermentation molded line leaf broom top, non-fermented molded line leaf goldspink
Colored and line leaf goldspink jasmine tea content results highest, it is therefore preferable that extraction time is 30min.
Extraction result table of the table 16-1 different extraction times to fermentation molded line leaf goldspink flower extract
Extraction result table of the table 16-2 different extraction times to non-fermented molded line leaf goldspink flower extract
Table 17-1 different extraction times are to fermentation molded line leaf extraction result table gorsy
Table 17-2 different extraction times are to non-fermented molded line leaf extraction result table gorsy
Extraction result table of the table 18-1 different extraction times to line leaf goldspink jasmine tea
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. the content assaying method of three kinds of ingredients in a kind of line leaf broom top, line leaf goldspink flower extract and line leaf goldspink jasmine tea,
It is characterized by comprising the following steps:
The preparation of mixed reference substance solution: taking Lutonaretin reference substance, orientoside reference substance and aspalathin reference substance, and alcohol is added
Mixed reference substance solution is made in solution；
The preparation of test solution: taking sample to be tested, and alcoholic solution, ultrasonic extraction or circumfluence distillation is added, lets cool, and it is molten that alcohol is added
Liquid dilutes constant volume, shakes up, and filters, takes subsequent filtrate to get test solution；
Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, with octadecylsilane chemically bonded silica column
For chromatographic column；Using acetonitrile as mobile phase A, with the acetum of volumetric concentration 0.8-1.5% or with volumetric concentration 0.1-0.5%'s
Formic acid is Mobile phase B；Chromatography is carried out according to gradient elution program, control flow rate of mobile phase is 0.8-1.2ml/min；Detection
Wavelength is 275~295nm and 340~360nm；
Measuring method: drawing mixed reference substance solution respectively and test solution injects liquid chromatograph, measures and calculates test sample
The content of Lutonaretin, orientoside and aspalathin in solution.
2. content assaying method according to claim 1, which is characterized in that the sample to be tested is line leaf broom top, line
In leaf goldspink flower extract, line leaf goldspink jasmine tea, line leaf broom top pharmaceutical composition or line leaf broom top pharmaceutical preparation at least
It is a kind of.
3. content assaying method according to claim 1 or 2, which is characterized in that the line leaf broom top includes fermented type
At least one of line leaf broom top and non-fermented molded line leaf broom top, the line leaf goldspink flower extract include fermentation molded line leaf
At least one of goldspink flower extract and non-fermented molded line leaf goldspink flower extract.
4. content assaying method according to claim 1 to 3, which is characterized in that the chromatographic condition and system are suitable
The method tested with property specifically: according to high performance liquid chromatography, using Waters Symmetry C18 (4.6mm × 250mm,
5 μm) chromatographic column, Diamonsil C18 (4.6mm × 250mm, 5 μm) chromatographic column or Agilent Extend-C18 (4.6mm ×
250mm, 5 μm) chromatographic column；Using acetonitrile as mobile phase A, using the acetum of volumetric concentration 1% as Mobile phase B；According to such as descending stair
It spends elution program and carries out chromatography: 0-12min, mobile phase A: Mobile phase B 12%:88%；12-25min, mobile phase A: stream
Dynamic phase B is 12%:88% → 20%:80%；25-28min, mobile phase A: Mobile phase B 20%:80%；28-28.01min,
Mobile phase A: Mobile phase B is 20%:80% → 100%:0；28.01-38min mobile phase A: Mobile phase B 100%:0；38-
38.01min, mobile phase A: Mobile phase B is 100%:0 → 12%:88%；38.01-50min mobile phase A: Mobile phase B is
12%:88%；And controlling flow rate of mobile phase is 1ml/min；Detection wavelength is 288 and 350nm.
5. content assaying method according to any one of claims 1-4, which is characterized in that in the measuring method, building
A series of mixed reference substance solution of concentration, and it is injected separately into liquid chromatograph, Lutonaretin, orientoside and A Si are made respectively
The standard curve of Ba Ting, test solution inject liquid chromatograph, according to the standard curve, calculate different Polygonum in test solution
The content of careless glycosides, orientoside and aspalathin.
6. any content assaying method in -5 according to claim 1, which is characterized in that in the system of the test solution
In standby, the alcoholic solution is ethanol solution or methanol solution；In the extraction process, the use of the sample to be tested and alcoholic solution
Amount is than being 0.2~0.5g:20~50ml.
7. any content assaying method in -6 according to claim 1, which is characterized in that the volumetric concentration of the alcoholic solution
It is 20~80%.
8. content assaying method according to claim 7, which is characterized in that the volumetric concentration of the alcoholic solution be 60~
80%.
9. any content assaying method in -8 according to claim 1, which is characterized in that in the system of the test solution
In standby, the time of ultrasonic extraction is 30~40min, and supersonic frequency is 35~40kHz.
10. a kind of -9 any online leaf broom tops of content assaying method, line leaf broom top according to claim 1 are extracted
Object, line leaf goldspink jasmine tea, line leaf broom top pharmaceutical composition or the broom top pharmaceutical preparation quality testing of line leaf and control field
Using.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111407791A (en) * | 2020-03-16 | 2020-07-14 | 鄂州职业大学 | External traditional Chinese medicine care solution for promoting wound healing |
| CN115104730A (en) * | 2022-06-28 | 2022-09-27 | 北京姿美堂生物技术股份有限公司 | Composition for improving sleep, sparrow leucomelas-asparagus sleep improvement liquid and tea powder preparation method and application |
| CN115192668A (en) * | 2022-09-15 | 2022-10-18 | 江中药业股份有限公司 | Lucid ganoderma compound extract and preparation method and application thereof |
| CN116235948A (en) * | 2021-12-07 | 2023-06-09 | 仙乐健康科技股份有限公司 | Anti-saccharification composition and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1967197A1 (en) * | 2007-03-09 | 2008-09-10 | Cognis IP Management GmbH | Use of preparations, purifications and extracts of aloe |
| CN101703599A (en) * | 2009-11-18 | 2010-05-12 | 杜守颖 | Method for detecting cape jasmine fruit extract |
| CN102219786A (en) * | 2011-06-07 | 2011-10-19 | 南京泽朗农业发展有限公司 | Preparation method of sparteine |
| EP2291172B1 (en) * | 2008-04-28 | 2017-10-25 | Aveda Corporation | Oxidative hair dye compositions, methods and products |
| CN107998220A (en) * | 2016-10-31 | 2018-05-08 | 亚宝药业集团股份有限公司 | It is a kind of to be used to reduce food intake, delay hunger, the composition of control body weight |
| CN108918734A (en) * | 2018-07-31 | 2018-11-30 | 四川新绿色药业科技发展有限公司 | A kind of efficient liquid phase method detecting folium isatidis medicinal material, medicine materical crude slice, standard decoction, granule characteristic spectrum |
-
2019
- 2019-03-29 CN CN201910253369.6A patent/CN109959735A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1967197A1 (en) * | 2007-03-09 | 2008-09-10 | Cognis IP Management GmbH | Use of preparations, purifications and extracts of aloe |
| EP2291172B1 (en) * | 2008-04-28 | 2017-10-25 | Aveda Corporation | Oxidative hair dye compositions, methods and products |
| CN101703599A (en) * | 2009-11-18 | 2010-05-12 | 杜守颖 | Method for detecting cape jasmine fruit extract |
| CN102219786A (en) * | 2011-06-07 | 2011-10-19 | 南京泽朗农业发展有限公司 | Preparation method of sparteine |
| CN107998220A (en) * | 2016-10-31 | 2018-05-08 | 亚宝药业集团股份有限公司 | It is a kind of to be used to reduce food intake, delay hunger, the composition of control body weight |
| CN108918734A (en) * | 2018-07-31 | 2018-11-30 | 四川新绿色药业科技发展有限公司 | A kind of efficient liquid phase method detecting folium isatidis medicinal material, medicine materical crude slice, standard decoction, granule characteristic spectrum |
Non-Patent Citations (4)
| Title |
|---|
| E.JOUBERT等: "Major production areas of rooibos (Aspalathus linearis) deliver herbal tea of similar phenolic and phenylpropenoic acid glucoside content", 《SOUTH AFRICAN JOURNAL OF BOTANY》 * |
| ELIZABETH J等: "Effect of Heat on Aspalathin, Iso-orientin, and Orientin Contents and Color of Fermented Rooibos (Aspalathus linearis) Iced Tea", 《J. AGRIC. FOOD CHEM》 * |
| 武钰等: "金雀花中3种黄酮成分的测定及其指纹图谱的建立", 《中成药》 * |
| 迪里木拉提*毛里明等: "金雀花中黄酮苷类组分鉴定及2种成分测定", 《中成药》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111407791A (en) * | 2020-03-16 | 2020-07-14 | 鄂州职业大学 | External traditional Chinese medicine care solution for promoting wound healing |
| CN116235948A (en) * | 2021-12-07 | 2023-06-09 | 仙乐健康科技股份有限公司 | Anti-saccharification composition and application thereof |
| CN115104730A (en) * | 2022-06-28 | 2022-09-27 | 北京姿美堂生物技术股份有限公司 | Composition for improving sleep, sparrow leucomelas-asparagus sleep improvement liquid and tea powder preparation method and application |
| CN115192668A (en) * | 2022-09-15 | 2022-10-18 | 江中药业股份有限公司 | Lucid ganoderma compound extract and preparation method and application thereof |
| CN115192668B (en) * | 2022-09-15 | 2022-12-13 | 江中药业股份有限公司 | Lucid ganoderma compound extract and preparation method and application thereof |
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