CN107064344A - A kind of method that ASE HPLC methods determine ginsenoside Rg_1 and Re content in ginseng - Google Patents

A kind of method that ASE HPLC methods determine ginsenoside Rg_1 and Re content in ginseng Download PDF

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CN107064344A
CN107064344A CN201710195525.9A CN201710195525A CN107064344A CN 107064344 A CN107064344 A CN 107064344A CN 201710195525 A CN201710195525 A CN 201710195525A CN 107064344 A CN107064344 A CN 107064344A
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ginsenoside
ase
ginseng
content
hplc methods
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廖强
韦日伟
陈学松
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Wuzhou Institutes for Food and Drug Control
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Wuzhou Institutes for Food and Drug Control
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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
    • 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
    • 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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  • Life Sciences & Earth Sciences (AREA)
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Abstract

The invention discloses a kind of method that ASE HPLC methods determine ginsenoside Rg_1 and Re content in ginseng, belong to field of chemical detection, for above-mentioned deficiency, it is intended to effectively combine ASE (Accelerate solvent extraction method), GCB (Graphon) and HPLC (high performance liquid chromatography) three, so as to improve the accuracy rate and stability of measure, the method that a kind of repeated ASE HPLC methods of raising method determine content of ginsenoside in ginseng, and this method can determine the ginsenoside Rg_1 and Re in ginseng simultaneously.This method carries out methanol extraction using ASE to the ginseng after crushing, takes 1ml extracts to be mixed with 75mg Graphons, filters, obtains methanol extraction liquid；The content of ginsenoside Rg_1 and Re in methanol extraction liquid is detected using HPLC methods.The present invention can be used for the content of control ginseng and its ginsenoside in preparation.

Description

A kind of method that ASE-HPLC methods determine ginsenoside Rg_1 and Re content in ginseng
Technical field
The invention belongs to field of chemical detection, especially a kind of ASE-HPLC methods determine ginsenoside Rg_1 and Re in ginseng The method of content.
Background technology
Ginseng is most widely used representative in China's authentic medicinal herbs, and ginsenoside is the main pharmacodynamics composition of ginseng, people Joining saponin(e has enhancing memory, improves immunity, improves cardiovascular, adjusts endocrine, anti-aging and the function such as antitumor, The assay of ginsenoside has become the focus studied both at home and abroad.
But in continuous mode, because the pigment impurity in sample is excessive, so as to cause measurement result inaccurate, therefore Set up effective removal of impurities and to determine quality control of the accurate ginsenoside detection method to ginseng and its preparation significant.
The content of the invention
For above-mentioned deficiency, it is contemplated that ASE (Accelerate solvent extraction method), GCB (Graphon) and HPLC is (high Effect liquid phase chromatogram method) three effectively combines, so as to improve the accuracy rate and stability of measure, improves repeated one of method The method that ASE-HPLC methods determine content of ginsenoside in ginseng is planted, and this method can determine the ginsenoside in ginseng simultaneously Rg1 and Re.
In order to solve the above-mentioned technical problem, the technical scheme that the present invention is provided is such：A kind of ASE-HPLC methods are determined The method of ginsenoside Rg_1 and Re content, comprises the steps successively in folium panacis japonici cum caule：
Step 1：Methanol extraction is carried out to the ginseng after crushing using ASE, takes 1ml extracts to be mixed with 75mg Graphons Close, filtering obtains methanol extraction liquid；
Step 2：The content of ginsenoside Rg_1 and Re in methanol extraction liquid is detected using HPLC methods.
Further, described step 1 includes following sub-steps：
Step S1：By sample comminution, No. three sieves are crossed, the powder 0.2g after sieving is taken；
Step S2：0.2g Ginseng Root Powder is mixed with 1g diatomite, loaded on the 5ml ASE abstraction pools for being placed with filter membrane In, plus diatomite is to mouthful parallel with pond；
Step S3：With methanol extraction, it is evaporated, plus methanol dissolving, and with methanol constant volume to 10ml；
Step S4：1ml step S3 resulting solutions are taken to be mixed with 75mgGCB, vortex oscillation 1min, centrifugation takes supernatant mistake Methanol extraction liquid is obtained after 0.22 micron membrane filter.
Further, the extraction described in step S3, parameter is as follows：Extraction temperature is 110 DEG C, and extraction time is 7min, extraction It is 3 times to take number of times, and flush volume is 80%, and purge time is 60s.
Further, the detection parameter of the HPLC methods described in step 2 is：Chromatographic column is Thermo Syncronis Dim.AQ, 1.7 μm, 50 × 2.1mm；Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is that volume ratio is equal to 18:82 second Nitrile-water；Detection wavelength：203nm.
Further, the parameter of noncentricity described in step S4 is：Speed is 10000r/min, and the time is 4min.
Further, the instrument that the extraction described in step 1 is used for DIONEX companies of the U.S. ASE350 fast solvents Abstraction instrument；HPLC methods described in step 2 detect that used instrument is the high performance liquid chromatographs of Agilent 1290.
The present invention is compared with conventional method, with advantages below：
1. the present invention is once investigated to the solvent that Accelerate solvent extraction is used, the methanol and first of n-hexane saturation were used Two kinds of solvents of alcohol are extracted, and as a result show that methanol is extracted consistent with the content of official method and impurity peaks are essentially identical, just oneself The sample size that the methanol of alkane saturation is extracted is relatively low, therefore uses the Extraction solvent consistent with pharmacopeia；
2. the present invention is once purified to the sample extracted and after constant volume, the sample 1.00mL after constant volume is taken to add respectively respectively Enter to after 2ml centrifuge tube vortexs 1min, the 10000r/min centrifugation 4min of the GCB equipped with 25mg, 50mg, 75mg, 100mg and draw Machine is determined on supernatant liquid filtering.As a result show that GCB energy Preferential adsorption pigments make the impurity in sample substantially reduce, but when pigment quilt Composition to be measured is possible to GCB absorption in sample after absorption completely, therefore selects 75mg GCB additions.
3. it is of the invention by ASE (Accelerate solvent extraction method), GCB (Graphon) and HPLC (high performance liquid chromatography) three Person effectively combines, so as to improve the accuracy rate and stability of measure, improves the repeated method of method, and this method can be same When determine ginseng in ginsenoside Rg_1 and Re.
Brief description of the drawings
Fig. 1 is that ginsenoside Rg1 carries out linear regression graph with concentration (mg)-peak area；
Fig. 2 is that ginsenoside Re carries out linear regression graph with concentration (mg)-peak area；
Fig. 3 is ginsenoside Rg_1 and Re Re reference substance collection of illustrative plates figures；
Fig. 4 is the sample drawing spectrogram that ASE methods are extracted；
Fig. 5 is the sample collection of illustrative plates that official method is extracted；
Fig. 6 is the influence figure to sample impurity using different amounts of GCB.
Embodiment
With reference to embodiment, the claim to the present invention is described in further detail, but is not constituted pair Any limitation of the present invention, the modification of any limited number of time made in the claims in the present invention protection domain, still in the present invention Claims within.
Embodiment 1
1. instrument and equipment and reagent
1.1 instrument：
Electronic analytical balance (XA205DU), ASE350 Accelerate solvent extractions instrument (DIONEX companies of the U.S.), Agilent 1290 high performance liquid chromatographs
1.2 reagents (in addition to especially indicating, this experiment agents useful for same is that analysis is pure)：
Water：Meet one-level water as defined in GB/T 6682；
Methanol (CH3OH)：Chromatographically pure (phase chromatography-use).
2 methods
The preparation of 2.1 reference substance solutions：
Take ginsenoside Rg1's reference substance, ginsenoside Re's reference substance appropriate, it is accurately weighed, plus every 1ml is respectively prepared in methanol 0.2mg containing ginsenoside Rg1, ginsenoside Re 0.2mg solution.
The preparation of 2.2 need testing solutions
2.2.1《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：
This product powder about 0.2g is taken, it is accurately weighed, put in apparatus,Soxhlet's, plus chloroform 30ml, it is heated to reflux 1 small When, chloroform liquid is discarded, the dregs of a decoction fling to chloroform, plus methanol 30ml, are heated to reflux 3 hours, extract solution low temperature is evaporated, plus Water 10ml makes dissolving, plus (30~60 DEG C) of petroleum ether extract 2 times, and each 10ml discards ether liquid, and aqueous is inhaled by D101 types macropore Attached resin column (internal diameter is 1.5cm, and column length is 15cm), is eluted with water 50ml, discards aqueous.Eluted again with 20% ethanol 50ml, 20% ethanol eluate is discarded, is eluted after with 80% ethanol 80ml, collects eluent 70ml, be evaporated, residue adds methanol to dissolve, turned Move in 10ml measuring bottles, plus methanol is to scale, shakes up, filtration, takes subsequent filtrate, produces.
2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method：
The size-reduced machine of sample is crushed, and crosses No. three sieves, and about 0.2g is accurately weighed, is well mixed with 1g diatomite, stand-by, moves Enter and add appropriate diatomite into the ASE 5ml abstraction pools for putting filter membrane well in advance, gently shaking is allowed to Chi Kou same On horizontal line, tighten and covered on abstraction pool.After extraction terminates, extract is transferred to after being evaporated in evaporating dish and is dissolved and determined with methanol Hold to 10mL volumetric flasks, then draw 1ml into the 2ml centrifuge tubes equipped with 75mgGCB, vortex oscillation 1min is centrifuged, and is filtered, filter Liquid, which is crossed after 0.22 micron membrane filter, enters HPLC measure.
2.3 ASE extraction conditions
Extraction temperature is 110 DEG C, and extraction time is 7min, and extraction times are 3 times, and flush volume is 80%, purge time For 60s.
2.4 chromatographic conditions and system suitability
Using octadecylsilane chemically bonded silica as filler；With acetonitrile-water (18:82) eluted for eluent gradient；Detection Wavelength is 203nm.Number of theoretical plate is calculated by ginsenoside Re peak should be not less than 1500.
2.5 determination method
Determined according to high performance liquid chromatography (general rule 0512)
It is accurate respectively to draw reference substance solution and each 1 μ l of need testing solution, liquid chromatograph is injected, determines, produces.
The detection parameter of HPLC methods is：Chromatographic column is Thermo Syncronis Dim.AQ, 1.7 μm, 50 × 2.1mm；Post Temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is that volume ratio is equal to 18:82 acetonitrile-water；Detection wavelength：203nm.
The requirement of 2.6 standard limited values
This product (C containing ginsenoside Rg142H72O14) and ginsenoside Re (C48H82O18) total amount must not be less than 2.25%.
2.7 calculate
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：
Cleaned out 1. should be dismantled using preceding extraction bottom of pond portion, otherwise easily cause pressure instability；
2. otherwise the filter paper of abstraction pool bottom should cause seepage in sealing ring；
3. abstraction pool fills elastic moderate during sample, too loose to be easily caused extract solution excessive；
4. check whether gas cylinder air pressure reaches 1Mpa before start；
5. using being cleaned out after terminating, abstraction pool will in time dry and (get rusty easily).
3 results
3.1 linear relationship
Take ginsenoside Rg1's reference substance, ginsenoside Re's reference substance, it is accurately weighed, plus methanol be made every 1ml containing about 0.2mg mixed solution, is produced, and then the accurate solution 0.5 μ l, 1 μ l, 1.5 μ l, 2 μ l, the 3 μ l of drawing are determined into LC respectively, And determined according to the above method, ginsenoside Rg1's linear test the results are shown in Table 1, and ginsenoside Re's linear test the results are shown in Table 2.
Linear regression is carried out with concentration (mg)-peak area, the regression equation of ginsenoside Rg1 is tried to achieve：Y=1264.2x+ 7.6763, R2=0.99979, it is in good linear relationship in the range of 0.0911~0.5466mg；Refer to Fig. 1.
Linear regression is carried out with concentration (mg)-peak area, the regression equation of ginsenoside Re is tried to achieve：Y=1148.8x- 0.96132, R2=0.99998, it is in good linear relationship in the range of 0.1013~0.6078mg；Refer to Fig. 2.
Table 1
Table 2
3.2 replica test
Take the sample (lot number of identical lot number：15071601) 0.2g, it is totally 3 parts, accurately weighed, by 2.2.2ASE extracting methods Need testing solution is extracted, sample size is 1 μ L, with above-mentioned chromatographic condition parallel test, measures ginsenoside Rg_1 and Re in sample Total content to be shown in Table 3, RSD be 0.5%, experiment shows that ASE extracting methods repeatability is good.
Table 3
3.3 precision and stability test
Take ginsenoside Rg1's reference substance solution (0.1822mg/ml), and ginsenoside Re's reference substance solution (0.2026mg/ Ml), continuous sample introduction 6 times, record peak area, and peak area RSD ginsenoside Rg1s are 1.1%, and ginsenoside Re is 0.5%, is shown Instrument precision is good, refers to Fig. 3, wherein ginsenoside Rg1 (tR：9.443) with Re (tR：10.021).
Same need testing solution is taken, is placed at room temperature, is determined in accordance with the law respectively at 0,2,8,12h.Record peak area, peak area RSD ginsenoside Rg1s are 0.6%, and ginsenoside Re is 0.9%, show that need testing solution is stable in 12h.
3.4 samples difference instrument extract result and with official method results contrast (3 batches)
Sample difference ASE instruments are extracted the results detailed in Table 4, and collection of illustrative plates refers to Fig. 4；Extracted using ASE methods with using pharmacopeia side The comparative result that method extracts result refers to table 5, and the collection of illustrative plates that official method extracts sample refers to Fig. 5.
Table 4
Sample lot number ASE1 methods measure content (%) ASE2 methods measure content (%) RAD (%)
15071601 3.45 3.27 2.7%
120303 3.20 3.12 1.3%
1407221 1.90 1.85 1.3%
Table 5
Sample lot number ASE1 methods measure content (%) Official method measures content (%) RAD (%)
15071601 3.45 3.43 0.3%
120303 3.20 3.13 1.1%
1407221 1.90 1.93 0.8%
4 discuss：
The selection of 4.1ASE Extraction solvents：
In experiment, once the solvent that Accelerate solvent extraction is used was investigated, the methanol and first of n-hexane saturation was used Two kinds of solvents of alcohol are extracted, and as a result show that methanol is extracted consistent with the content of official method and impurity peaks are essentially identical, just oneself The sample size that the methanol of alkane saturation is extracted is relatively low, therefore uses the Extraction solvent consistent with pharmacopeia.
4.2ASE extraction and cleaning
In experiment, once the sample extracted and after constant volume was purified, takes the sample 1.00mL after constant volume to add respectively respectively Enter to after 2ml centrifuge tube vortexs 1min, the 10000r/min centrifugation 4min of the GCB equipped with 25mg, 50mg, 75mg, 100mg and draw Machine is determined on supernatant liquid filtering.As a result show that GCB energy Preferential adsorption pigments make the impurity in sample substantially reduce, but when pigment quilt Composition to be measured is possible to GCB absorption in sample after absorption completely, therefore selects 75mg GCB additions.
Influence figure using different amounts of GCB to sample impurity refers to Fig. 6.
Above-described is only presently preferred embodiments of the present invention, all timess made in the range of the spirit and principles in the present invention What modifications, equivalent substitutions and improvements etc., should be included in the scope of the protection.

Claims (6)

1. a kind of method that ASE-HPLC methods determine ginsenoside Rg_1 and Re content in folium panacis japonici cum caule, it is characterised in that include successively Following step：
Step 1：Methanol extraction is carried out to the ginseng after crushing using ASE, takes 1ml extracts to be mixed with 75mg Graphons, Filtering, obtains methanol extraction liquid；
Step 2：The content of ginsenoside Rg_1 and Re in methanol extraction liquid is detected using HPLC methods.
2. the method that a kind of ASE-HPLC methods according to claim 1 determine ginsenoside Rg_1 and Re content in ginseng, its It is characterised by, described step 1 includes following sub-steps：
Step S1：By sample comminution, No. three sieves are crossed, the powder 0.2g after sieving is taken；
Step S2：0.2g Ginseng Root Powder is mixed with 1g diatomite, loaded on being placed with the 5ml ASE abstraction pools of filter membrane, plus Diatomite is extremely parallel with pond mouthful；
Step S3：With methanol extraction, it is evaporated, plus methanol dissolving, and with methanol constant volume to 10ml；
Step S4：1ml step S3 resulting solutions are taken to be mixed with 75mgGCB, vortex oscillation 1min, centrifugation takes supernatant to cross 0.22 Methanol extraction liquid is obtained after micron membrane filter.
3. the method that a kind of ASE-HPLC methods according to claim 2 determine ginsenoside Rg_1 and Re content in ginseng, its It is characterised by, the extraction described in step S3, parameter is as follows：Extraction temperature is 110 DEG C, and extraction time is 7min, and extraction times are 3 Secondary, flush volume is 80%, and purge time is 60s.
4. the method that a kind of ASE-HPLC methods according to claim 1 determine ginsenoside Rg_1 and Re content in ginseng, its It is characterised by, the detection parameter of the HPLC methods described in step 2 is：Chromatographic column is Thermo Syncronis Dim.AQ, 1.7 μm, 50×2.1mm；Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is that volume ratio is equal to 18:82 acetonitrile-water；Detect ripple It is long：203nm.
5. the method that a kind of ASE-HPLC methods according to claim 2 determine ginsenoside Rg_1 and Re content in ginseng, its It is characterised by, the parameter of noncentricity described in step S4 is：Speed is 10000r/min, and the time is 4min.
6. the method that a kind of ASE-HPLC methods according to claim 1 determine ginsenoside Rg_1 and Re content in ginseng, its Be characterised by, the instrument that the extraction described in step 1 is used for DIONEX companies of the U.S. ASE350 Accelerate solvent extraction instrument；Step HPLC methods described in rapid 2 detect that used instrument is the high performance liquid chromatographs of Agilent 1290.
CN201710195525.9A 2017-03-29 2017-03-29 A kind of method that ASE HPLC methods determine ginsenoside Rg_1 and Re content in ginseng Pending CN107064344A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105651925A (en) * 2016-04-15 2016-06-08 广西壮族自治区梧州食品药品检验所 Determination method of ginsenosides Rg1 and Re in ginseng leaves

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105651925A (en) * 2016-04-15 2016-06-08 广西壮族自治区梧州食品药品检验所 Determination method of ginsenosides Rg1 and Re in ginseng leaves

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
宋文斌等: "加速溶剂萃取-液相色谱-紫外检测法测定人参中多种人参皂甙含量", 《现代科学仪器》 *

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Application publication date: 20170818