CN108132310A - A kind of method of Determination of Content of Ginsenoside Rg_1 in measure ginseng - Google Patents

Abstract

The invention discloses a kind of methods for measuring Determination of Content of Ginsenoside Rg_1 in ginseng, belong to field of chemical detection, against the above 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 of measure and stability, the method that a kind of ASE HPLC methods of the repeatability of method measure content of ginsenoside in ginseng is improved.This method carries out ethyl alcohol extraction using ASE to the ginseng after crushing, and 1ml extract liquors is taken to be mixed with 100mg Graphons, filters, obtains alcohol extraction liquid；The content of ginsenoside Rg1 in alcohol 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 of Determination of Content of Ginsenoside Rg_1 in measure ginseng

Technical field

The invention belongs to field of chemical detection, especially a kind of method for measuring Determination of Content of Ginsenoside Rg_1 in ginseng.

Background technology

Ginseng is most widely used representative in China's authentic medicinal herbs, and ginsenoside is the main pharmacodynamics ingredient of ginseng, people Joining saponin(e has enhancing memory, improves immunity, improves cardiovascular, adjusts endocrine, slows down aging and the functions such as antitumor, The assay of ginsenoside has become the hot spot studied both at home and abroad.

But in continuous mode, since the pigment impurity in sample is excessive, so as to cause measurement result inaccurate, therefore It establishes effectively to clean and measure accurate ginsenoside detection method and be of great significance to the quality control of ginseng and its preparation.

Invention content

Against the above deficiency, the present invention is directed to 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 of measure and stability, improve the one of the repeatability of method The method that kind ASE-HPLC methods measure content of ginsenoside in ginseng.

In order to solve the above technical problem, the present invention provides technical solution be such：People in a kind of measure folium panacis japonici cum caule Join the method for saponin(e Rg1 contents, include the following steps successively：

Step 1：Ethyl alcohol extraction is carried out to the ginseng after crushing using ASE, takes 1ml extract liquors and 100mg Graphons Mixing, filtering, obtains alcohol extraction liquid；

Step 2：The content of ginsenoside Rg1 in alcohol extraction liquid is detected using HPLC methods.

Further, the step 1 includes following sub-steps：

Step S1：By sample comminution, No. three sieves are crossed, take the powder 0.2g after sieving；

Step S2：The Ginseng Root Powder of 0.2g is mixed with 1g diatomite, loaded on the 5ml ASE abstraction pools for being placed with filter membrane In, add diatomite extremely parallel with pond mouthful；

Step S3：It is extracted, is evaporated with ethyl alcohol, ethyl alcohol is added to dissolve, and 10ml is settled to ethyl alcohol；

Step S4：1ml step S3 acquired solutions is taken to be mixed with 100mgGCB, vortex oscillation 1min, centrifugation takes supernatant mistake Alcohol extraction liquid is obtained after 0.22 micron membrane filter.

Further, the extraction described in step S3, parameter are as follows：Extraction temperature is 110 DEG C, extraction time 7min, extraction It is 3 times to take number, flush volume 80%, purge time 60s.

Further, the detection parameters of the HPLC methods described in step 2 are：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 equal to 22 for volume ratio:78 second Nitrile-water；Detection wavelength：203nm.

Further, the parameter of noncentricity described in step S4 is：Speed is 10000r/min, time 4min.

Further, ASE350 fast solvent of the instrument for DIONEX companies of the U.S. used by the extraction described in step 1 Abstraction instrument；Instrument is 1290 high performance liquid chromatographs of Agilent used by HPLC methods detection described in step 2.

The present invention has the following advantages compared with conventional method：

1. the present invention once investigated the solvent that Accelerate solvent extraction uses, the ethyl alcohol and second of n-hexane saturation were used Two kinds of solvents of alcohol extract, as a result show ethyl alcohol extract and impurity peaks consistent with the content of official method it is essentially identical, just oneself The sample size of the ethyl alcohol extraction of alkane saturation is relatively low, therefore uses the Extraction solvent consistent with pharmacopeia；

2. the present invention once purifies the sample after extraction and constant volume, the sample 1.00mL after constant volume is taken to add respectively respectively It is inhaled after entering to 2ml centrifuge tube vortexs 1min, the 10000r/min centrifugation 4min of the GCB equipped with 25mg, 50mg, 100mg, 100mg Machine on supernatant liquid filtering is taken to measure.The result shows that GCB energy Preferential adsorption pigments are substantially reduced the impurity in sample, but work as pigment GCB absorption is possible to by ingredient to be measured in sample after absorption completely, therefore selects the GCB additive amounts of 100mg.

It is 3. of the invention by ASE (Accelerate solvent extraction method), GCB (Graphon) and HPLC (high performance liquid chromatography) three Person effectively combines, and so as to improve the accuracy rate of measure and stability, improves the repeated method of method.

Description of the drawings

Fig. 1 carries out linear regression graph for ginsenoside Rg1 with concentration (mg)-peak area.

Specific embodiment

With reference to embodiment, the claim of the present invention is described in further detail, but do not formed pair Any restrictions 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 level-one water as defined in GB/T 6682；

Ethyl alcohol (CH₃OH)：Chromatographically pure (phase chromatography-use).

2 methods

The preparation of 2.1 reference substance solutions：

Take ginsenoside Rg1's reference substance reference substance appropriate, it is accurately weighed, add ethyl alcohol that every 1ml is respectively prepared containing ginsenoside The solution of Rg10.2mg.

The preparation of 2.2 test 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, it puts in Soxhlet extractor, adds chloroform 30ml, it is small to be heated to reflux 1 When, chloroform liquid is discarded, the dregs of a decoction fling to chloroform, add ethyl alcohol 30ml, are heated to reflux 3 hours, extracting solution low temperature is evaporated, and is added Water 10ml makes dissolving, and (30~60 DEG C) of petroleum ether is added to extract 2 times, each 10ml, discards ether liquid, and aqueous is inhaled by D101 types macropore Attached resin column (internal diameter 1.5cm, column length 15cm) is eluted with water 50ml, discards aqueous.It is eluted again with 20% ethyl alcohol 50ml, 20% ethanol eluate is discarded, is eluted after with 80% ethyl alcohol 80ml, is collected eluent 70ml, be evaporated, residue adds ethyl alcohol to dissolve, and turns Move in 10ml measuring bottles, ethyl alcohol added to shake up to scale, filter, take subsequent filtrate to get.

2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method：

Sample is crushed through pulverizer, crosses No. three sieves, and about 0.2g is accurately weighed, is uniformly mixed with 1g diatomite, for use, is moved 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, abstraction pool upper cover is tightened.After extraction, extract liquor is transferred to after being evaporated in evaporating dish and is dissolved and determined with ethyl alcohol Hold to 10mL volumetric flasks, then draw in 1ml to the 2ml centrifuge tubes equipped with 100mgGCB, vortex oscillation 1min, centrifuge, filter, filter Liquid enters HPLC measure after crossing 0.22 micron membrane filter.

2.3ASE extraction conditions

Extraction temperature is 110 DEG C, extraction time 7min, and extraction times are 3 times, flush volume 80%, purge time For 60s.

2.4 chromatographic conditions and system suitability

Using octadecylsilane chemically bonded silica as filler；With acetonitrile-water (18:82) it is eluted for eluent gradient；Detection Wavelength is 203nm.Number of theoretical plate is calculated by ginsenoside Rg1 peak should be not less than 1500.

2.5 measuring method

It is measured according to high performance liquid chromatography (general rule 0512)

It is accurate respectively to draw reference substance solution and each 1 μ l of test solution, inject liquid chromatograph, measure to get.

The detection parameters of HPLC methods are：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 equal to 22 for volume ratio:78 acetonitrile-water；Detection wavelength：203nm.

The requirement of 2.6 standard limited values

This product (C containing ginsenoside Rg1₄₂H₇₂O₁₄) total amount must not be less than 2.25%.

2.7 it calculates

C in formula_R--- reference substance solution concentration, unit are micro- gram per liter (mg/L)；

A_X--- the peak area of test sample；

A_R--- reference substance peak area.

Pay attention to：

It is cleaned out 1. should be dismantled using preceding extraction bottom of pond portion, otherwise easily causes pressure instability；

Otherwise 2. the filter paper of abstraction pool bottom should cause leakage in sealing ring；

3. abstraction pool fills elastic moderate during sample, too loose to be easy to cause extracting solution excessive；

4. check whether gas cylinder air pressure reaches 1Mpa before booting；

5. being cleaned out after use, abstraction pool will be dried and (be got rusty easily) in time.

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 all be included in the protection scope of the present invention.

Claims (6)

1. A kind of 1. method for measuring Determination of Content of Ginsenoside Rg_1 in ginseng, which is characterized in that include the following steps successively：

   Step 1：Ethyl alcohol extraction is carried out to the ginseng after crushing using ASE, 1ml extract liquors is taken to be mixed with 100mg Graphons, Filtering, obtains alcohol extraction liquid；

   Step 2：The content of ginsenoside Rg1 in alcohol extraction liquid is detected using HPLC methods.
2. 2. a kind of method for measuring Determination of Content of Ginsenoside Rg_1 in ginseng according to claim 1, which is characterized in that described Step 1 include following sub-steps：

   Step S1：By sample comminution, No. three sieves are crossed, take the powder 0.2g after sieving；

   Step S2：The Ginseng Root Powder of 0.2g with 1g diatomite is mixed, loaded on being placed in the 5ml ASE abstraction pools of filter membrane, is added Diatomite is extremely parallel with pond mouthful；

   Step S3：It is extracted, is evaporated with ethyl alcohol, ethyl alcohol is added to dissolve, and 10ml is settled to ethyl alcohol；

   Step S4：1ml step S3 acquired solutions is taken to be mixed with 100mgGCB, vortex oscillation 1min, centrifugation takes supernatant to cross 0.22 Alcohol extraction liquid is obtained after micron membrane filter.
3. A kind of 3. method for measuring Determination of Content of Ginsenoside Rg_1 in ginseng according to claim 2, which is characterized in that step Extraction described in S3, parameter are as follows：Extraction temperature is 110 DEG C, extraction time 7min, and extraction times are 3 times, and flush volume is 80%, purge time 60s.
4. A kind of 4. method for measuring Determination of Content of Ginsenoside Rg_1 in ginseng according to claim 1, which is characterized in that step 2 The detection parameters of the HPLC methods are：Chromatographic column is Thermo Syncronis Dim.AQ, 1.7 μm, 50 × 2.1mm；Column temperature It is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is equal to 22 for volume ratio:78 acetonitrile-water；Detection wavelength：203nm.
5. A kind of 5. method for measuring Determination of Content of Ginsenoside Rg_1 in ginseng according to claim 2, which is characterized in that step Parameter of noncentricity described in S4 is：Speed is 10000r/min, time 4min.
6. A kind of 6. method for measuring Determination of Content of Ginsenoside Rg_1 in ginseng according to claim 1, which is characterized in that step 1 Instrument is the ASE350 Accelerate solvent extraction instrument of DIONEX companies of the U.S. used by the extraction；HPLC described in step 2 Instrument is 1290 high performance liquid chromatographs of Agilent used by method detects.