CN106959346A - A kind of method that ASE HPLC methods determine Geniposidic acid and acteoside content in plantain seed - Google Patents
A kind of method that ASE HPLC methods determine Geniposidic acid and acteoside content in plantain seed Download PDFInfo
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- CN106959346A CN106959346A CN201710247876.XA CN201710247876A CN106959346A CN 106959346 A CN106959346 A CN 106959346A CN 201710247876 A CN201710247876 A CN 201710247876A CN 106959346 A CN106959346 A CN 106959346A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a kind of method that ASE HPLC methods determine Geniposidic acid and acteoside content in plantain seed, this method extraction time is shorter, accuracy of detection is high, belongs to field of chemical detection.This method extracts Plantago Seed using ASE methods, and collects methanol extraction liquid；The content of the Geniposidic acid and acteoside in methanol extraction liquid is determined using HPLC methods.The present invention can substitute official method and Geniposidic acid in plantain seed and acteoside are extracted, and carry out assay.
Description
Technical field
The invention belongs to field of chemical detection, especially a kind of ASE-HPLC methods determine Geniposidic acid and hair in plantain seed
The method that stamen spends Glycosides Contents.
Background technology
《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：The preparation of need testing solution takes this product powder (to cross two
Number sieve) about 1g, it is accurately weighed, put in conical flask with cover, precision adds 60% methanol 50ml, and weighed weight is heated to reflux 2 small
When, let cool, then weighed weight, the weight of less loss is supplied with 60% methanol, is shaken up, is filtered, is taken subsequent filtrate, produce.
This method return time is long, and the professional standards to technical staff require higher.
The content of the invention
For above-mentioned deficiency, the present invention is intended to provide a kind of ASE-HPLC methods determine Geniposidic acid and hair stamen in plantain seed
The method of flower Glycosides Contents, this method extraction time is shorter, accuracy of detection is high.
In order to realize above-mentioned technique effect, the technical scheme that the present invention is provided is such：A kind of ASE-HPLC methods are determined
The method of Geniposidic acid and acteoside content, comprises the steps successively in plantain seed：
Step 1：Plantago Seed is extracted using ASE methods, and collects methanol extraction liquid；
Step 2：The content of the Geniposidic acid and acteoside in methanol extraction liquid is determined using HPLC methods.
Preferably, described step 1 specifically includes following sub-steps：
Step S1：By plantain seed sample comminution, No. two sieves are crossed, take 1g to be mixed with 1g diatomite；
Step S2：By the mixture obtained by step S1 loaded on being placed with the ASE abstraction pools of fibrous filter membrane, plus diatomite to
Pond mouthful is parallel；
Step S3：With methanol extraction, and extract is settled to 50ml with methanol.
Preferably, the extraction conditions described in step S3 is：Extraction temperature is 80 DEG C, and extraction time is 8min；Cycle-index
For 3 times；Flush volume is 60%；Purge time is 60s.
Preferably, the volume of the ASE abstraction pools described in step S2 is 10ml.
Preferably, the detection parameter of the HPLC methods described in step 2 is：Chromatographic column is Thermo Syncronis Dim.AQ；
Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is methanol-acetic acid-water；Detection wavelength is 283nm.
Preferably, the specification of described chromatographic column is 1.7 μm, 50 × 2.1mm.
Preferably, described mobile phase is that volume ratio is equal to 35:4:65 methanol-acetic acid-water.
The present invention is compared with conventional method, and tool has the advantage that：
The present invention is once investigated to the solvent that Accelerate solvent extraction is used, and is had paper to mention and is extracted shaddock using 60% methanol
Skin glycosides relative amount is higher, and impurity is less, therefore to having carried out solvent investigation using methanol and 60% methanol, as a result show methanol with
60% methanol extracts plantain seed content unanimously, and impurity peaks are essentially identical, therefore uses the Extraction solvent consistent with pharmacopeia.
The present invention is investigated to extraction temperature, and extraction time will also be investigated, on above-mentioned experiment basis, I
Use 3 factor 3 horizontal quadrature experimental program L9 (33) experiment arrangement, investigated 3 factors to accelerated solvent extraction plantain seed
In Geniposidic acid, the influence of acteoside process.This experiment determines extraction temperature, static extracting time, cycle-index
And flush volume is investigation factor, it is determined that extracting from the optimal fast solvent of the Geniposidic acid in plantain seed, acteoside
Taking technique, so that it is determined that optimal extraction parameters.
Brief description of the drawings
Fig. 1 is the chromatogram of Geniposidic acid and acteoside reference substance；
Fig. 2 is the chromatogram of the need testing solution obtained by ASE methods are extracted；
Fig. 3 is the chromatogram of the need testing solution obtained by official method is extracted.
Embodiment
The claim of the present invention is described in further detail with reference to embodiment, but not constituted to this
Any limitation of invention, still any limited number of time modification made in the claims in the present invention protection domain, the power in the present invention
In the claimed scope of profit.
Embodiment 1
1st, instrument and equipment and reagent
1.1 instruments：
Electronic analytical balance (XA205DU), ASE350 Accelerate solvent extractions instrument (DIONEX companies of the U.S.), Thermo
U3000UHPLC liquid chromatographs
1.2 reagents：
Water：Meet one-level water as defined in GB/T 6682；
Methanol (CH4O)：Chromatographically pure；
Acetic acid (CH3COOH)：Analysis is pure.
2nd, method
The preparation of 2.1 reference substance solutions：
The preparation of reference substance solution takes Geniposidic acid reference substance, acteoside reference substance appropriate, accurately weighed, puts palm fibre
In colo(u)r specification bottle, plus every lml respectively mixed solutions containing 0.1mg are made in 60% methanol, produce.
The preparation of 2.2 need testing solutions
《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：The preparation of need testing solution takes this product powder (to cross two
Number sieve) about 1g, it is accurately weighed, put in conical flask with cover, precision adds 60% methanol 50ml, and weighed weight is heated to reflux 2 small
When, let cool, then weighed weight, the weight of less loss is supplied with 60% methanol, is shaken up, is filtered, is taken subsequent filtrate, produce.
Accelerate solvent extraction method (ASE) prepares test sample method：The size-reduced machine of sample is crushed, and is crossed No. two sieves, is taken 1g, accurate
It is weighed, it is well mixed with about 1g diatomite, it is stand-by, it is moved into the ASE 10ml abstraction pools for putting fibrous filter membrane well in advance and adds
Appropriate diatomite, gently shaking is allowed to Chi Kou in the same horizontal line, tighten and cover on abstraction pool, be put into rapid extracting device
Row extraction.After extraction terminates, extract is transferred in 50ml volumetric flasks, extraction flask is cleaned with a small amount of methanol and is incorporated into appearance 3 times
In measuring bottle, using methanol constant volume to scale, produce.
2.3ASE extraction conditions
Extraction temperature is that 80 DEG C of extraction times are 8min；Cycle-index is 3 times；Flush volume is 60%；Purge time is
60s。
2.4 chromatographic conditions and system suitability
A) instrument：The double ternary liquid phases (U-3000) of Thermo；
B) chromatographic column：Thermo Syncronis Dim. (mm) AQ, 1.7 μm, 50 × 2.1mm；
C) column temperature：40℃；
D) flow velocity：0.3mL/min；
E) mobile phase：Methanol-acetic acid-water (35:4:65)；
F) Detection wavelength：283nm；
Number of theoretical plate is calculated by Geniposidic acid peak should be not less than 3000.
2.5 determination methods
Determined according to high performance liquid chromatography (general rule 0512)；
It is accurate respectively to draw reference substance solution and each 10 μ l of need testing solution, liquid chromatograph is injected, determines, produces.
The requirement of 2.6 standard limited values
This product is calculated by dry product, containing Geniposidic acid (C16H22O10) 0.50%, acteoside must not be less than
(C29H36O15) 0.40% must not be less than.
2.7 calculate (external standard method)
C in formulaR--- reference substance solution concentration, unit is micro- gram per liter (mg/L)；
AX--- the peak area of test sample；
AR--- reference substance peak area.
Note：
It should be dismantled and cleaned out using preceding extraction bottom of pond portion, otherwise easily cause pressure instability；
Otherwise the filter paper of abstraction pool bottom should cause seepage in sealing ring；
Abstraction pool fills elastic moderate during sample, and too loose to be easily caused extract solution excessive；
Check whether gas cylinder air pressure reaches 1Mpa before start；
Using being cleaned out after terminating, abstraction pool will in time dry and (get rusty easily).
3rd, result
3.1 reappearance tests
Take the sample (lot number of identical lot number：20151001) 0.5g, it is totally 4 parts, accurately weighed, by above-mentioned ASE extracting methods
Need testing solution is extracted, sample size is 1 μ L, with above-mentioned chromatographic condition parallel laboratory test, measures Geniposidic acid and hair in sample
It is 1.2% that the content of stamen flower glucosides, which is shown in Table 1, RSD, and experiment shows that ASE extracting methods repeatability is good.
The Geniposidic acid of table 1ASE methods extraction and the content of acteoside
| Repeatability 1 | Repeatability 2 | Repeatability 3 | Repeatability 4 | RSD% | |
| As a result % | 2.082 | 2.064 | 2.074 | 2.027 | 1.2 |
3.2 precision and stability test
Geniposidic acid reference substance, acteoside reference substance mixed solution are taken, continuous sample introduction 6 times records peak area,
Peak area RSD is 0.5%, shows that instrument precision is good.
Same need testing solution is taken, is placed at room temperature, is determined in accordance with the law respectively at 0,2,4,8,10h.As a result Astragaloside IV peak
The RSD of area is 0.2%, shows that need testing solution is stable in 12h.
3.3 samples difference instrument extract result and with official method results contrast (3 batches)
The sample of table 2 difference instrument extract result and with official method results contrast
| ASE1 | ASE2 | Pharmacopeia | |
| 150523 | 1.574 | 1.54 | 1.971 |
| 151001 | 1.698 | 1.581 | 1.902 |
| 1511024 | 0.964 | 1.095 | 1.713 |
| 20151001 | 2.073 | 1.626 | 2.139 |
4th, discuss：
The selection of 4.1ASE Extraction solvents
The present invention is once investigated to the solvent that Accelerate solvent extraction is used, and is had paper to mention and is extracted shaddock using 60% methanol
Skin glycosides relative amount is higher, and impurity is less, therefore to having carried out solvent investigation using methanol and 60% methanol, as a result show methanol with
60% methanol extracts plantain seed content unanimously, and impurity peaks are essentially identical, therefore uses the Extraction solvent consistent with pharmacopeia.
The optimization of 4.2ASE extraction conditions
Extraction temperature is investigated, and extraction time will also be investigated, on above-mentioned experiment basis, we use 3
Factor 3 horizontal quadrature experimental program L9 (33) experiment arrangement, has investigated 3 factors to the capital Buddhist nun in accelerated solvent extraction plantain seed
The influence of flat thuja acid, acteoside process.This experiment determines extraction temperature, static extracting time, cycle-index and flushing
Volume is investigation factor, refers to table 3.It is determined that extracting from the optimal fast solvent of the Geniposidic acid in plantain seed, acteoside
Taking technique, the results are shown in Table 4-6.
The factor level of table 3
The orthogonal experiment range analysis result of the Geniposidic acid of table 4
The orthogonal experiment range analysis result of the acteoside of table 5
| Factor | Temperature | Time | Number of times | Experimental result | |
| Experiment 1 | 1 | 1 | 1 | 1 | 0.821 |
| Experiment 2 | 1 | 2 | 2 | 2 | 0.885 |
| Experiment 3 | 1 | 3 | 3 | 3 | 0.89 |
| Experiment 4 | 2 | 1 | 2 | 3 | 0.835 |
| Experiment 5 | 2 | 2 | 3 | 1 | 0.858 |
| Experiment 6 | 2 | 3 | 1 | 2 | 0.834 |
| Experiment 7 | 3 | 1 | 3 | 2 | 0.81 |
| Experiment 8 | 3 | 2 | 1 | 3 | 0.809 |
| Experiment 9 | 3 | 3 | 2 | 1 | 0.827 |
| Average 1 | 0.865 | 0.822 | 0.821 | 0.835 | |
| Average 2 | 0.842 | 0.851 | 0.849 | 0.843 | |
| Average 3 | 0.815 | 0.850 | 0.853 | 0.845 | |
| Extreme difference | 0.050 | 0.029 | 0.032 | 0.010 |
The orthogonal experiment range analysis result of the Geniposidic acid of table 6 and acteoside total amount
Extraction time influences maximum to the content of Geniposidic acid and acteoside it can be seen from range analysis, its
Secondary is Extracting temperature and extraction time.Final to determine that optimum extraction process is combined as 80 DEG C of Extracting temperature, extraction time 8min is carried
Take number of times 3 times.
Above-described is only presently preferred embodiments of the present invention, all timess done in the range of the spirit and principles in the present invention
What modification, equivalent and improvement etc., should be included within the scope of the present invention.
Claims (7)
1. a kind of method that ASE-HPLC methods determine Geniposidic acid and acteoside content in plantain seed, it is characterised in that
Comprise the steps successively：
Step 1：Plantago Seed is extracted using ASE methods, and collects methanol extraction liquid；
Step 2：The content of the Geniposidic acid and acteoside in methanol extraction liquid is determined using HPLC methods.
2. a kind of ASE-HPLC methods according to claim 1 determine Geniposidic acid and acteoside content in plantain seed
Method, it is characterised in that described step 1 specifically includes following sub-steps：
Step S1：By plantain seed sample comminution, No. two sieves are crossed, take 1g to be mixed with 1g diatomite；
Step S2：By the mixture obtained by step S1 loaded on being placed with the ASE abstraction pools of fibrous filter membrane, plus diatomite to and Chi Kou
It is parallel；
Step S3：With methanol extraction, and extract is settled to 50ml with methanol.
3. a kind of ASE-HPLC methods according to claim 2 determine Geniposidic acid and acteoside content in plantain seed
Method, it is characterised in that the extraction conditions described in step S3 is：Extraction temperature is 80 DEG C, and extraction time is 8min；Circulation time
Number is 3 times；Flush volume is 60%；Purge time is 60s.
4. a kind of ASE-HPLC methods according to claim 2 determine Geniposidic acid and acteoside content in plantain seed
Method, it is characterised in that the volume of the ASE abstraction pools described in step S2 be 10ml.
5. a kind of ASE-HPLC methods according to claim 1 determine Geniposidic acid and acteoside content in plantain seed
Method, it is characterised in that the detection parameter of the HPLC methods described in step 2 is：Chromatographic column is Thermo Syncronis
Dim.AQ；Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is methanol-acetic acid-water；Detection wavelength is 283nm.
6. a kind of ASE-HPLC methods according to claim 5 determine Geniposidic acid and acteoside content in plantain seed
Method, it is characterised in that the specification of described chromatographic column be 1.7 μm, 50 × 2.1mm.
7. a kind of ASE-HPLC methods according to claim 5 determine Geniposidic acid and acteoside content in plantain seed
Method, it is characterised in that described mobile phase be volume ratio be equal to 35:4:65 methanol-acetic acid-water.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104530147A (en) * | 2014-11-27 | 2015-04-22 | 普正药业股份有限公司 | Method for preparing verbascoside standard product from callicarpa nudiflora leaf |
| CN105954377A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Extraction method for amentoflavone in Selaginella tamariscina |
| CN106065024A (en) * | 2015-10-30 | 2016-11-02 | 浙江工业大学 | A kind of method extracting separation verbascoside from Chinese medicine Radix Rehmanniae |
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- 2017-04-17 CN CN201710247876.XA patent/CN106959346A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104530147A (en) * | 2014-11-27 | 2015-04-22 | 普正药业股份有限公司 | Method for preparing verbascoside standard product from callicarpa nudiflora leaf |
| CN106065024A (en) * | 2015-10-30 | 2016-11-02 | 浙江工业大学 | A kind of method extracting separation verbascoside from Chinese medicine Radix Rehmanniae |
| CN105954377A (en) * | 2016-04-22 | 2016-09-21 | 广西壮族自治区梧州食品药品检验所 | Extraction method for amentoflavone in Selaginella tamariscina |
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| 周忻 等: "正交实验法考察车前子水提物质量标准及不同来源品种质量比较", 《辽宁中医药大学学报》 * |
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Application publication date: 20170718 |