CN109932448A - The content assaying method of effective component in a kind of line leaf broom top and its product - Google Patents

Abstract

The invention belongs to food quality control technology fields, specifically provide the content assaying method of effective component in line leaf broom top and its product, the assay step including following Lutonaretin: the assay of A Lutonaretin；Prepare Lutonaretin reference substance solution；The preparation of test solution；Chromatographic condition and system suitability: according to high effective liquid chromatography for measuring, using Waters Symmetry C18 (4.6mm × 250mm, 5 μm) chromatographic column or Diamonsil C18 (4.6mm × 250mm, 5 μm) 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；The content for measuring and calculating Lutonaretin in test solution, the present invention provides a kind of content assaying methods for quickly, accurately, easily measuring effective component in line leaf broom top and its product.

Description

The content assaying method of effective component in a kind of line leaf broom top and its product

Technical field

The invention belongs to food quality control technology field, relate in particular in line leaf broom top and its product effectively at The content assaying method divided.

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 effective component in a kind of line leaf broom top and line leaf goldspink flower extract and line leaf goldspink jasmine tea is established Content assaying method have great importance for the development and utilization of line leaf broom top food, health care product or drug.

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, and unstable, and measurement accuracy is low, expensive, In addition, line leaf broom top ingredient is various, complicated, impurity content is more, at present temporary not wired leaf quality control standard gorsy. The HPLC measuring method of aspalathin in doctor's tea is disclosed in " assay of aspalathin in doctor's tea " document, in the party In method, line leaf quality gorsy is determined by measuring the concentration of aspalathin, there are extraction operation complexity, separation for this method Spend the problem of low, peak type is poor, at high cost, long operational time (see Cha Shenghua etc., the assay of aspalathin, day in doctor's tea Right Study on product and exploitation, 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 it is a kind of quickly, it is accurate, conveniently, in cheap ground detection line leaf broom top effective component content assaying method.

Summary of the invention

For this purpose, that technical problem to be solved by the present invention lies in the detection method measurement results of the prior art is single, extracts It is complicated for operation, separating degree is low, peak type is poor, long operational time, the problem of can not evaluating its quality accurately, comprehensively, and then provide one Kind quickly, accurate, conveniently, different Polygonum orientale in cheap measurement line leaf broom top and line leaf goldspink flower extract and line leaf goldspink jasmine tea The detection method of glycosides, orientoside and aspalathin content, and then the comprehensively and accurately matter of control line leaf broom top and its product Amount.

In order to solve the above technical problems, of the invention provide a kind of containing for effective component in line leaf broom top and its product Quantity measuring method, the assay step including following Lutonaretin:

The assay of A Lutonaretin

Prepare Lutonaretin reference substance solution；

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, using Waters Symmetry C18 (4.6mm × 250mm, 5 μm) chromatographic column or Diamonsil C18 (4.6mm × 250mm, 5 μm) chromatographic column；It is stream with acetonitrile Dynamic 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；According to Gradient elution program carries out chromatography, and control flow rate of mobile phase is 0.8-1.2ml/min；Detection wavelength be 275~295nm and 340~360nm；

Measuring method: drawing Lutonaretin reference substance solution respectively and test solution injects liquid chromatograph, measures and counts Calculate the content of Lutonaretin in test solution.

Further, further include the assay step of following orientoside and aspalathin:

The assay of B orientoside and aspalathin

The mixed reference substance solution of Lutonaretin, orientoside reference substance solution and aspalathin；

Lutonaretin reference substance, orientoside reference substance and aspalathin reference substance are taken, addition alcoholic solution is made a series of dense The mixed reference substance solution of the reference substance containing Lutonaretin, orientoside reference substance and aspalathin reference substance spent, in the chromatography Under the conditions of, it is injected separately into liquid chromatograph, the standard curve of Lutonaretin, orientoside and aspalathin is made respectively, utilizes mark The slope of directrix curve calculates the relative correction factor of orientoside and aspalathin；

Pass through Lutonaretin, the peak value of orientoside and aspalathin and orientoside and aspalathin in test solution map Relative correction factor, pass through internal standard method calculate test solution in orientoside and aspalathin content.

Preferably, the sample to be tested is line leaf broom top, line leaf goldspink flower extract, line leaf goldspink jasmine tea, line leaf gold At least one of sparrow anther compositions or line leaf broom top pharmaceutical preparation.

Further, the line leaf broom top include fermentation molded line leaf broom top and non-fermented molded line leaf broom top in extremely Few one kind, the line leaf goldspink flower extract include that fermentation molded line leaf goldspink flower extract and non-fermented molded line leaf broom top are extracted At least one of object.

Preferably, the orientoside, aspalathin the relative correction factor of characteristic peak be respectively 1.066 and 1.168.

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 or Diamonsil 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 following gradient elution journey Sequence carries out chromatography: 0-12min, mobile phase A: Mobile phase B 12%:88%；12-25min, mobile 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 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 Control flow rate of mobile phase is 1ml/min；Detection wavelength is 288 and 350nm.

Preferably, in the preparation of the test solution, the alcoholic solution is ethanol solution or methanol solution；Described In extraction process, the amount ratio of the sample to be tested and alcoholic solution are as follows: 0.2~0.5g:20~50ml.

Further, the volumetric concentration of the alcoholic solution is 20~80%, it is preferred that 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 the online leaf broom top of any of the above-described content assaying method, line leaf broom tops to extract 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.

1. the content assaying method of effective component in line leaf broom top of the present invention and its product, efficient by controlling Liquid phase chromatogram condition, screens suitable mobile phase and gradient elution program, effectively overcome because complicated component in line leaf broom top, Dopant species and content are more and the problem of easily cause Lutonaretin principal component peak to interfere and Lutonaretin and orientoside be with point Isomers, inferior separating effect the problem of interfering with each other, not only simplify the pretreated step of sample to be tested, and improve different The separating degree of orientoside and orientoside to each other, and be effectively separated with other impurity peaks, the HPLC method of foundation is not only special Attribute 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.

2. the content assaying method of effective component, further includes orientoside in line leaf broom top of the present invention and its product With the assay step of aspalathin, by the suitable mobile phase of aforementioned screening and gradient elution program, effectively overcome because Complicated component, dopant species and content easily cause orientoside and aspalathin and other impurity peaks cannot more in line leaf broom top The problem of efficiently separating, while realizing using same flow phase system, it can disposably complete different Polygonum in line leaf goldspink jasmine tea The assay of careless glycosides, orientoside and aspalathin, can quick, the easy quality condition for knowing the product, solve existing The problem of single component can only be measured, can not control quality of medicinal material comprehensively by having the detection method of technology, passes through to realize The content of measurement effective component Lutonaretin, orientoside and aspalathin comes control line leaf broom top and its extract and line leaf gold The purpose of the quality of sparrow jasmine tea, and the content assaying method is with simple and quick, reliable and stable, precision is high, is easy to grasp Advantage, in addition, commented using Lutonaretin cheap and easy to get as one survey of internal reference object progress, it can Accurate Determining line leaf broom top. more And its content of extract and Lutonaretin, orientoside and aspalathin in line leaf goldspink jasmine tea, save the cost, while improving inspection The accuracy of survey.

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；Wherein A is the offline leaf broom top of 288nm The chromatogram of tea negative control solution, B are the chromatogram of the offline leaf broom top test solution of 288nm；C is the offline leaf of 350nm The chromatogram of goldspink jasmine tea negative control solution, D are the chromatogram of the offline leaf broom top test solution of 350nm；

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 the molded line that ferments under 288nm The chromatogram of leaf goldspink flower extract test solution, B are the molded line leaf goldspink flower extract test solution that ferments under 350nm Chromatogram；C is the chromatogram of non-fermented molded line leaf goldspink flower extract test solution under 288nm, and D is non-fermented under 350nm The chromatogram of molded line leaf goldspink flower extract test solution；

Fig. 6 is the chromatogram of embodiment 10 and the test solution in embodiment 11；Wherein A is 288nm in embodiment 10 The chromatogram of lower fermentation molded line leaf goldspink flower extract test solution, B are molded line leaf goldspink of fermenting under 350nm in embodiment 10 The chromatogram of flower extract test solution；C is non-fermented molded line leaf goldspink flower extract test sample under 288nm in embodiment 11 The chromatogram of solution, D are the chromatogram of non-fermented molded line leaf goldspink flower extract test solution under 350nm in embodiment 11；

Fig. 7 is test solution in the comparative example 1 non-fermented molded line leaf goldspink flower extract test solution at 350nm Chromatogram；

Fig. 8 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；

Fig. 9 is the chromatogram of the test solution in comparative example 3；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 4；Wherein A is that fermentation molded line leaf broom top mentions under 288nm The chromatogram of object test solution is taken, B is the chromatogram of fermentation molded line leaf goldspink flower extract test solution under 350nm；C is The chromatogram of non-fermented molded line leaf goldspink flower extract test solution under 288nm, D are non-fermented molded line leaf goldspink under 350nm The chromatogram of flower extract test solution.

Figure 11 is the chromatogram of mixed reference substance solution and test solution in comparative example 5, 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 Lutonaretin reference substance solution: it is appropriate that precision weighs Lutonaretin reference substance, and it is molten that 60vt% ethyl alcohol is added Lutonaretin reference substance solution is respectively prepared in liquid dissolution；

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；

Measurement: it is accurate respectively to draw Lutonaretin reference substance solution and each 5 μ l of test solution, liquid chromatograph is injected, The content for measuring and calculating Lutonaretin in test solution is surveyed using one and comments method more, using Lutonaretin as internal standard compound, passes through confession The peak value of Lutonaretin, orientoside and aspalathin and the relative correction factor of orientoside and A Siba in test sample solution map The relative correction factor in spit of fland calculates the content of orientoside and aspalathin in test solution by internal standard method.

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, respectively By " chromatographic condition and system suitability " measurement in 1.2 test methods.Shown in the result is shown in Figure 1, as the result is shown Under 288nm and 350nm, it is showed no and Polygonum different in test solution chromatogram in line leaf goldspink jasmine tea negative control solution chromatogram The chromatographic peak of careless glycosides, orientoside identical retention time with aspalathin shows detection of the negative controls solution to principal component peak It does not interfere with.

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；Take fermentation molded line leaf goldspink flower extract and non-fermented molded line leaf Goldspink flower extract presses under 1.2 the line leaf goldspink flower extract test sample solution recorded in " preparation of test solution " respectively Line leaf goldspink flower extract test solution is made in preparation method；Take fermentation molded line leaf broom top and non-fermented molded line leaf broom top 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 control series 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 calculating of 1.12 relative correction factors

By the contrast solution of 6 concentration made from the lower test method of item 1.5, each three parts, as reference substance solution A, control Product solution B and reference substance solution C inject different liquid chromatographs in following different times respectively, record retention time and peak face Product, and draw curve.Using Lutonaretin as internal standard, the relative correction factor fsi and aspalathin of orientoside are calculated separately Relative correction factor fsi show that the relative correction factor of orientoside is 1.068, and the relative correction factor of aspalathin is 1.165, as a result see the table below it is shown,

^*Note: 1 result measured on 1260 instrument of Agilent for reference substance solution A；2 be reference substance solution A in 10 days The result measured on Agilent1260 instrument afterwards；3 results measured on 1260 instrument of Agilent for reference substance solution B； 4 results measured on 1260 instrument of Agilent after 10 days for reference substance solution B；5 for reference substance solution B after 10 days The result measured on Waters UPLC H-Class instrument；6 be reference substance solution C on Waters UPLC H-Class instrument The result measured；7 results measured on Waters UPLC H-Class instrument after 1 day for reference substance solution C.

The calculating of 1.13 relative retention times

Under 1.2 chromatographic condition of item, take by the lower mixed reference substance solution prepared of item 1.2 and 1.2 it is lower prepare for examination Product solution injects different liquid chromatographs in different time respectively, records retention time.Using Lutonaretin as object of reference, calculate The ratio of the retention time of orientoside, aspalathin and Lutonaretin, obtain orientoside relative retention time be 1.066, Ah The relative retention time of Si Bating is 1.168.

* note: the 1 result average value measured on 1260 instrument of Agilent for reference substance solution；2 be reference substance after 2 days The result average value that solution measures on Agilent1260 instrument；3 measure on 1260 instrument of Agilent for test solution Result average value；4 be the result average value that test solution measures on 1260 instrument of Agilent after 2 days；5 be after 5 days The result average value that reference substance solution and test solution measure on 1260 instrument of Agilent；6 exist for reference substance solution The result average value measured on Waters UPLC H-Class instrument；7 be test solution in Waters UPLC H-Class The result average value measured on instrument；8 be the knot that reference substance solution measures on Waters UPLC H-Class instrument after 1 day Fruit average value；9 be the result average value that test solution measures on Waters UPLC H-Class instrument after 1 day.

The durability of 1.14 relative correction factors and relative retention time is investigated

The standard solution for taking item 1.2 to prepare investigates flow velocity (0.9mL/min~1.1mL/min), (23 DEG C of column temperature respectively ~30 DEG C), different batches chromatographic column and different instrument (1.Agilent 1260, chromatographic column Waters Symmetry C18, batch Number be 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 orientoside and aspalathin relative correction factor and relative retention time, as a result see the table below institute Show.

The result shows that the opposite school of the minor change of flow velocity and column temperature condition, different instruments to orientoside and aspalathin Positive divisor and relative retention time all have no significant effect (RSD < 5%), and the reproducibility in different batches chromatographic column is preferable.

1.15 1 surveys comments method compared with external standard method result

Line taking leaf broom top, line leaf goldspink flower extract and line leaf goldspink jasmine tea prepare mark by the test method under item 1.2 Quasi- product solution and test solution are measured by the lower chromatographic condition of item 1.2, survey by one and comment method to determine each chromatographic peak to be measured more Peak position, and calculate its content.Again with the content of each ingredient of external standard method.As a result it see the table below.The result shows that relative retention value method Can accurate positioning aspalathin chromatographic peak, and by contents obtained by 2 kinds of methods through rank sum test analysis the two difference, as a result Polygonum P value is 0.739 between two groups of data of careless glycosides, and P value is 0.075 between two groups of data of aspalathin, is all larger than 0.05, thus orientoside, Ah Si Bating external standard method measured value and one survey comment between method calculated value that there was no significant difference, show that establish one surveys and comments method (QAMS) more Method is accurately feasible.

Embodiment 1

The content assaying method of effective component, 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: taking fermentation molded line leaf goldspink flower extract 0.2g, accurately weighed, sets 25mL volumetric flask In, 60vt% ethanol solution about 20mL is added, is ultrasonically treated 30min, supersonic frequency 35Hz is let cool, molten with 60vt% ethyl alcohol Liquid is settled to scale, shakes up, and with 0.45 μm of organic membrane filtration, takes subsequent filtrate molten to get line leaf goldspink flower extract test sample Liquid；

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 2

The content assaying method of effective component, 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: negated fermentation molded line leaf goldspink flower extract 0.2g, it is accurately weighed, set 50mL volumetric flask In, 60vt% ethanol solution about 40mL is added, is ultrasonically treated 40min, supersonic frequency 40Hz is let cool, molten with 60vt% ethyl alcohol Liquid is settled to scale, shakes up, and with 0.45 μm of organic membrane filtration, takes subsequent filtrate molten to get line leaf goldspink flower extract test sample Liquid；

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 3

The content assaying method of effective component, includes the following steps: in line leaf goldspink jasmine tea described in the present embodiment

The preparation of Lutonaretin reference substance solution: it is appropriate that precision weighs Lutonaretin reference substance, and it is molten that 60vt% ethyl alcohol is added Lutonaretin reference substance solution is respectively prepared in liquid dissolution；

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 Lutonaretin reference substance solution and each 5 μ l of test solution, inject liquid chromatogram Instrument measures and calculates the content of Lutonaretin in test solution, surveys using one and comments method more, using Lutonaretin as internal standard compound, leads to Cross Lutonaretin, the peak value of orientoside and aspalathin and the relative correction factor of orientoside in test solution map and Ah The relative correction factor of Si Bating calculates the content of orientoside and aspalathin in test solution by internal standard method.

Embodiment 4

The content assaying method of effective component, includes the following steps: in line leaf broom top described in the present embodiment

The preparation of Lutonaretin reference substance solution: it is appropriate that precision weighs Lutonaretin reference substance, and it is molten that 60vt% ethyl alcohol is added Lutonaretin reference substance solution is respectively prepared in liquid dissolution；

The preparation of test solution: taking fermentation molded line leaf broom top 0.5g, accurately weighed, sets in 25mL volumetric flask, is added 60vt% ethanol solution about 20mL, is ultrasonically treated 30min, 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 Lutonaretin reference substance solution and each 5 μ l of test solution, inject liquid chromatogram Instrument measures and calculates the content of Lutonaretin in test solution, surveys using one and comments method more, using Lutonaretin as internal standard compound, leads to Cross Lutonaretin, the peak value of orientoside and aspalathin and the relative correction factor of orientoside in test solution map and Ah The relative correction factor of Si Bating calculates the content of orientoside and aspalathin in test solution by internal standard method.

Embodiment 5

The content assaying method of effective component, includes the following steps: in line leaf broom top described in the present embodiment

The preparation of Lutonaretin reference substance solution: it is appropriate that precision weighs Lutonaretin reference substance, and it is molten that 60vt% ethyl alcohol is added Lutonaretin reference substance solution is respectively prepared in liquid dissolution；

The preparation of test solution: negated fermentation molded line leaf broom top 0.5g, it is accurately weighed, it sets in 50mL volumetric flask, adds Enter 60vt% ethanol solution about 40mL, is ultrasonically treated 30min, supersonic frequency 35Hz is let cool, with 60vt% ethanol solution constant volume It to scale, shakes 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 Lutonaretin reference substance solution and each 5 μ l of test solution, inject liquid chromatogram Instrument measures and calculates the content of Lutonaretin in test solution, surveys using one and comments method more, using Lutonaretin as internal standard compound, leads to Cross Lutonaretin, the peak value of orientoside and aspalathin and the relative correction factor of orientoside in test solution map and Ah The relative correction factor of Si Bating calculates the content of orientoside and aspalathin in test solution by internal standard method.

Embodiment 6

The content assaying method of effective component 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, in test solution Lutonaretin, orientoside and aspalathin peak and adjacent unknown peak point 1.5 are all larger than from degree, good separation, however retention time and instrument runing time are longer.

Embodiment 7

The content assaying method of effective component 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, Lutonaretin, orientoside and A Siba in test solution The separating degree at Ting Feng 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 8

The content assaying method of effective component in fermentation molded line leaf goldspink flower extract described in the present embodiment, including it is as follows Step:

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 takes hybrid standard stock solution and aspalathin standard reserving solution B that alcoholic solution is added and a series of containing for concentration is made The mixed reference substance solution of Lutonaretin reference substance, orientoside reference substance and aspalathin reference substance；

The preparation of Lutonaretin reference substance solution: it is appropriate that precision weighs Lutonaretin reference substance, and it is molten that 60vt% ethyl alcohol is added Lutonaretin reference substance solution is respectively prepared in liquid dissolution；

The preparation of test solution: taking fermentation molded line leaf goldspink flower extract 0.2g, accurately weighed, sets 25mL volumetric flask In, 60vt% ethanol solution about 20mL is added, is ultrasonically treated 30min, supersonic frequency 35Hz is let cool, molten with 60vt% ethyl alcohol Liquid is settled to scale, shakes up, and with 0.45 μm of organic membrane filtration, takes subsequent filtrate molten to get line leaf goldspink flower extract test sample Liquid；

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 Lutonaretin reference substance solution and each 5 μ l of test solution, inject liquid chromatogram Instrument measures and calculates the content of Lutonaretin in test solution, and method of more commenting is surveyed using one, and precision draws a series of the mixed of concentration Close reference substance solution be injected separately into liquid chromatograph under the chromatographic condition, respectively be made Lutonaretin, orientoside and Ah The standard curve of Si Bating calculates orientoside and aspalathin using the slope of standard curve using Lutonaretin as internal standard compound Relative correction factor；By Lutonaretin, the peak value of orientoside and aspalathin and orientoside in test solution map and The relative correction factor of aspalathin calculates the content of orientoside and aspalathin in test solution by internal standard method.

Embodiment 9

The content assaying method of effective component in non-fermented molded line leaf goldspink flower extract described in the present embodiment, including such as Lower step:

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 takes hybrid standard stock solution and aspalathin standard reserving solution B that alcoholic solution is added and a series of containing for concentration is made The mixed reference substance solution of Lutonaretin reference substance, orientoside reference substance and aspalathin reference substance；

The preparation of Lutonaretin reference substance solution: it is appropriate that precision weighs Lutonaretin reference substance, and it is molten that 60vt% ethyl alcohol is added Lutonaretin reference substance solution is respectively prepared in liquid dissolution；

The preparation of test solution: negated fermentation molded line leaf goldspink flower extract 0.2g, it is accurately weighed, set 25mL volumetric flask In, 60vt% ethanol solution about 20mL is added, is ultrasonically treated 30min, supersonic frequency 35Hz is let cool, molten with 60vt% ethyl alcohol Liquid is settled to scale, shakes up, and with 0.45 μm of organic membrane filtration, takes subsequent filtrate molten to get line leaf goldspink flower extract test sample Liquid；

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 Lutonaretin reference substance solution and each 5 μ l of test solution, inject liquid chromatogram Instrument measures and calculates the content of Lutonaretin in test solution, and method of more commenting is surveyed using one, and precision draws a series of the mixed of concentration Close reference substance solution be injected separately into liquid chromatograph under the chromatographic condition, respectively be made Lutonaretin, orientoside and Ah The standard curve of Si Bating calculates orientoside and aspalathin using the slope of standard curve using Lutonaretin as internal standard compound Relative correction factor；By Lutonaretin, the peak value of orientoside and aspalathin and orientoside in test solution map and The relative correction factor of aspalathin calculates the content of orientoside and aspalathin in test solution by internal standard method.

Embodiment 10

The content assaying method of effective component 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 Waters Symmetry C18 (4.6mm × 250mm, the 5 μm) column used in example 8 is applied, as a result as shown in fig. 6, Lutonaretin Retention time is 22.445min, and the retention time of orientoside is 23.661min, and the retention time of aspalathin is 25.342min；Using Lutonaretin as reference, calculate orientoside relative retention time be 1.0542, aspalathin it is opposite Retention time is 1.1291, meets a survey and method is commented to require relative retention time error range need to be within ± 5% more.

Embodiment 11

The content assaying method of effective component in non-fermented molded line leaf goldspink flower extract described in the present embodiment, with implementation The difference of example 9 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. 6, Lutonaretin Retention time be 22.456min, the retention time of orientoside is 23.672min, and the retention time of aspalathin is 25.346min；Using Lutonaretin as reference, calculate orientoside relative retention time be 1.0542, aspalathin it is opposite Retention time is 1.1287, can satisfy a survey and method is commented to require relative retention time error range need to be within ± 5% more.

Comparative example 1

The content assaying method of effective component 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 fig. 7, 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 effective component 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 8, can be seen that under 288nm from A, point of aspalathin and adjacent unknown peak It is 1.10 from degree, 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 effective component 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, as a result as shown in figure 9, its In, from figure A it is found that under 288nm, the separating degree at aspalathin and adjacent unknown peak is 1.24, and less than 1.5, separating degree cannot Meet the requirement of chromatography.

Comparative example 4

The content assaying method of effective component in line leaf goldspink flower extract described in this comparative example, the area with embodiment 1 It is not only that chromatographic column difference, is substituted in this comparative example using Agilent Extend-C18 (4.6mm × 250mm, 5 μm) column real Waters Symmetry C18 (4.6mm × 250mm, the 5 μm) column used in example 1 is applied, the results are shown in Figure 10, Lutonaretin Retention time be 15.795min, the retention time of orientoside is 18.188min, and the retention time of aspalathin is 20.774min；Using Lutonaretin as reference, calculate orientoside relative retention time be 1.1515, aspalathin it is opposite Retention time is 1.3152, is not able to satisfy a survey and method is commented to require relative retention time error range need to be in ± 5% more.

Comparative example 5

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 effective component in a kind of line leaf broom top and its product, which is characterized in that including following different Polygonum The assay step of careless glycosides:

The assay of A Lutonaretin

Prepare Lutonaretin reference substance 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, using Waters Symmetry C18 (4.6mm × 250mm, 5 μm) chromatographic column or Diamonsil C18 (4.6mm × 250mm, 5 μm) chromatographic column；It is flowing with acetonitrile 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；According to ladder It spends elution program and carries out chromatography, control flow rate of mobile phase is 0.8-1.2ml/min；Detection wavelength be 275~295nm and 340~360nm；

Measuring method: drawing Lutonaretin reference substance solution respectively and test solution injects liquid chromatograph, measures and calculates confession The content of Lutonaretin in test sample solution.

2. content assaying method according to claim 1, which is characterized in that further include following orientoside and aspalathin Assay step:

The assay of B orientoside and aspalathin

Lutonaretin reference substance, orientoside reference substance and aspalathin reference substance are taken, alcoholic solution is added, a series of concentration is made The mixed reference substance solution of reference substance containing Lutonaretin, orientoside reference substance and aspalathin reference substance, in the chromatographic condition Under, it is injected separately into liquid chromatograph, the standard curve of Lutonaretin, orientoside and aspalathin is made respectively, utilizes standard song The slope of line calculates the relative correction factor of orientoside and aspalathin；

Pass through the phase of the peak value and orientoside and aspalathin of Lutonaretin, orientoside and aspalathin in test solution map To correction factor, the content of orientoside and aspalathin in test solution is calculated by internal standard method.

3. content assaying method according to claim 1 or 2, which is characterized in that the sample to be tested be line leaf broom top, In line leaf goldspink flower extract, line leaf goldspink jasmine tea, line leaf broom top pharmaceutical composition or line leaf broom top pharmaceutical preparation extremely Few one kind.

4. content assaying method according to claim 1 to 3, which is characterized in that the line leaf broom top includes hair At least one of ferment molded line leaf broom top and non-fermented molded line leaf broom top, the line leaf goldspink flower extract includes fermented type At least one of line leaf goldspink flower extract and non-fermented molded line leaf goldspink flower extract.

5. content assaying method according to any one of claims 1-4, which is characterized in that the orientoside, aspalathin The relative correction factor of characteristic peak be respectively 1.066 and 1.168.

6. any content assaying method in -5 according to claim 1, 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 or Diamonsil C18 (4.6mm × 250mm, 5 μm) chromatographic column；Using acetonitrile as mobile phase A, with volumetric concentration 1% acetum is Mobile phase B；Chromatography: 0-12min is carried out according to following gradient elution program, mobile phase A: flowing Phase B is 12%:88%；12-25min, mobile 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 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.

7. any content assaying method in -6 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 Measure ratio are as follows: 0.2~0.5g:20~50ml.

8. any content assaying method in -7 according to claim 1, which is characterized in that the volumetric concentration of the alcoholic solution It is 20~80%；Preferably, the volumetric concentration of the alcoholic solution is 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.