CN108152405A - A kind of method of a variety of ginsenosides in measure ginseng - Google Patents

A kind of method of a variety of ginsenosides in measure ginseng Download PDF

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Publication number
CN108152405A
CN108152405A CN201711368003.0A CN201711368003A CN108152405A CN 108152405 A CN108152405 A CN 108152405A CN 201711368003 A CN201711368003 A CN 201711368003A CN 108152405 A CN108152405 A CN 108152405A
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extraction
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ginseng
acetonitrile
ginsenosides
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陈学松
钟水桥
梁峰
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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

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Steroid Compounds (AREA)

Abstract

The invention discloses a kind of methods for measuring a variety of ginsenosides in ginseng, belong to field of chemical detection, it is desirable to provide a kind of to measure the method that a variety of ginsenoside monomers, the range of linearity are wide, method is accurate, favorable reproducibility, fast and simple, reliable and stable ASE HPLC methods measure ginsenoside in ginseng simultaneously.The samples of Ginseng that the present invention crushed using ASE extraction process；Wherein, ASE extracting process includes first in Soxhlet extractor using chloroform extraction, then being extracted with ethyl alcohol in Accelerate solvent extraction instrument, collecting alcohol extraction liquid；The content of the ginsenoside in alcohol extraction liquid is detected using HPLC methods.The present invention is used for the content detection of ginseng and its a variety of ginsenosides in preparation.

Description

A kind of method of a variety of ginsenosides in measure ginseng
Technical field
The present invention relates to the detection fields of ginsenoside in ginseng, especially a kind of to measure a variety of ginsenosides in ginseng Method.
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.
With the development of society, the requirement for drug quality control is also higher and higher, but since Chemical Constituents of Panax Ginseng is answered It is miscellaneous, ginsenoside wide variety, it has been determined that and detach ginsenoside compound monomer there are more than 50 to plant, according to aglycon not Together, ginsenoside is divided into protopanoxadiol saponins, protopanaxatriol saponins and oleanolic acid saponins.
Therefore establish simultaneously, a variety of, quick and easy, reliable ginsenoside detection method is to the matter of ginseng and its preparation Amount control is of great significance.
Invention content
Against the above deficiency, the present invention is intended to provide one kind can measure simultaneously a variety of ginsenoside monomers, the range of linearity it is wide, The method that method is accurate, favorable reproducibility, fast and simple, reliable and stable ASE-HPLC methods measure ginsenoside in ginseng.
In order to solve the above technical problem, the present invention provides technical solution be such：It is a variety of in a kind of measure ginseng The method of ginsenoside, includes the following steps successively：
Step 1：The samples of Ginseng that crushed using ASE extraction process；Wherein, ASE extracting process is included first in rope It using chloroform extraction in family name's extractor, is then extracted in Accelerate solvent extraction instrument with ethyl alcohol, collects alcohol extraction liquid；
Step 2：The content of the ginsenoside in alcohol extraction liquid is detected using HPLC methods.
Further, the step 1 includes following sub-steps：
Step S1：By sample comminution, No. four sieves are crossed, take the powder 1g after sieving；
Step S2：Loaded in Soxhlet extractor, chloroform is added in, is heated to reflux 4 hours, chloroform extraction liquid house It abandons；
Step S3：1g diatomite and the dregs of a decoction are mixed, immigration is placed in the ASE 10ml abstraction pools of fibrous filter membrane, adds diatom Soil is extremely parallel with pond mouthful；
Step S4：It is put into rapid extracting device and is extracted, extractant is ethyl alcohol；
Step S5：Extract liquor is evaporated, residue adds ethyl alcohol to dissolve, and ethyl alcohol is added to be settled to 10ml, and 1ml is taken to add to and is equipped with In the 2ml centrifuge tubes of 100mgPSA, centrifugation is filtered to get alcohol extraction liquid.
Further, in the step S4, extraction parameters are：Extraction temperature is 110 DEG C, extraction time 10min, extraction It is 1 time to take number, flush volume 60%, purge time 60s.
Further, the ASE extracting process uses the ASE350 Accelerate solvent extraction instrument of DIONEX companies of the U.S..
Further, in the step 2, the detection parameters of HPLC methods are：Chromatographic column：Thermo Syncronis Dim.AQ；Column temperature：50℃；Flow velocity：0.3mL/min；Mobile phase：Acetonitrile-water；Detection wavelength：203nm.
Further, the mobile phase：The volume ratio of acetonitrile-water is 18~32：68~82；
0th minute, the volume ratio of acetonitrile-water was 18 in mobile phase：82；
10th minute, the volume ratio of acetonitrile-water was 20 in mobile phase：80；
18th minute, the volume ratio of acetonitrile-water was 30 in mobile phase：70；
24th minute, the volume ratio of acetonitrile-water was 32 in mobile phase：68；
25-28 minutes, the volume ratio of acetonitrile-water was 18 in mobile phase：82.
Further, the specification of the chromatographic column is 1.7 μm, 50 × 2.1mm；The HPLC detection methods are used Thermo U3000UHPLC liquid chromatographs.
The present invention has the following advantages compared with conventional method：
1. the present invention provides a kind of 3 kinds of ginsenoside monomer HPLC methods of measure simultaneously, the 3 kinds of ginsenoside lists measured Body is Rg1, Re, Rb1, improves efficiency；
2. the present invention only makees mobile phase with acetonitrile and water, solve phosphate and chromatographic column is lost big, cleaning systems trouble The problems such as, it is convenient that mobile phase is prepared, easy operation；
It is that 3. the present invention is measured the result shows that, 3 kinds of ginsenoside monomers are in the range of 0.5~3.0 μ l in good Linear relationship, the range of linearity are wide, and method is accurate, favorable reproducibility, fast and simple, reliable and stable.
4. the present invention can preferably analyze the content of ginsenoside monomer, the quality control for ginseng and its preparation provides reason By foundation.
Description of the drawings
Fig. 1 is the linear regression graph that ginsenoside Rg1 is carried out with concentration (mg)-peak area；
Fig. 2 is the linear regression graph that ginsenoside Re is carried out with concentration (mg)-peak area；
Fig. 3 is the linear regression graph that ginsenoside Rb1 is carried out 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.
1. instrument and equipment and reagent
1.1 instrument：
Electronic analytical balance (XA205DU), ASE350 Accelerate solvent extractions instrument (DIONEX companies of the U.S.), Thermo U3000UHPLC liquid chromatograies.
2.2 reagent：Reagent and solution (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.
Acetonitrile (C2H3N)：Chromatographically pure (phase chromatography-use).
2. method
The preparation of 2.1 reference substance solutions：
Take ginsenoside Rg1's reference substance, ginsenoside Re's reference substance and ginsenoside Rb1's reference substance appropriate, it is accurately weighed, Add ethyl alcohol that every 1ml respectively mixed solutions containing 0.2mg are made, shake up to get.
The preparation of 2.2 test solutions
2.2.1《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：
This product powder (crossing No. four sieves) about 1g is taken, it is accurately weighed, it puts in Soxhlet extractor, chloroform is added to be heated to reflux 3 Hour, chloroform liquid is discarded, the dregs of a decoction volatilize solvent, are moved into 100ml conical flasks together with filtration paper cylinder, precision plus the positive fourth of water saturation Alcohol 50ml, close plug are stood overnight, and are ultrasonically treated (power 250W, frequency 50KHz) 30 minutes, and filtration discards primary filtrate, accurate Subsequent filtrate 25ml is measured, puts in evaporating dish and is evaporated, residue adds ethyl alcohol to dissolve and is transferred in 5ml measuring bottles, and ethyl alcohol is added to be diluted to quarter Degree, shake up, filter, take subsequent filtrate to get.
2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method：
Step S1：By sample comminution, No. four sieves are crossed, take the powder 1g after sieving.
Step S2：Loaded in Soxhlet extractor, chloroform is added in, is heated to reflux 4 hours, chloroform extraction liquid house It abandons.
Step S3：1g diatomite and the dregs of a decoction are mixed, immigration is placed in the ASE 10ml abstraction pools of fibrous filter membrane, adds diatom Soil is extremely parallel with pond mouthful.
Step S4：It is put into rapid extracting device and is extracted, extractant is ethyl alcohol；
Extraction parameters are：Extraction temperature is 110 DEG C, extraction time 10min, and extraction times are 1 time, and flush volume is 60%, purge time 60s；
Using the ASE350 Accelerate solvent extraction instrument of DIONEX companies of the U.S..
Step S5：Extract liquor is evaporated, residue adds ethyl alcohol to dissolve, and ethyl alcohol is added to be settled to 10ml, and 1ml is taken to add to and is equipped with In the 2ml centrifuge tubes of 100mgPSA, centrifugation is filtered to get alcohol extraction liquid；
Step S6：The content of the ginsenoside in alcohol extraction liquid is detected using HPLC methods；
Detection parameters are：Chromatographic column：Thermo Syncronis Dim.AQ, 1.7 μm, 50 × 2.1mm, with octadecyl Silane group silica gel is filler；Column temperature：50℃；Flow velocity：0.3mL/min；Detection wavelength：203nm；Mobile phase：Volume ratio is 18~32：68~82 acetonitrile-water takes gradient elution as follows：
0th minute, the volume ratio of acetonitrile-water was 18 in mobile phase：82；
10th minute, the volume ratio of acetonitrile-water was 20 in mobile phase：80；
18th minute, the volume ratio of acetonitrile-water was 30 in mobile phase：70；
24th minute, the volume ratio of acetonitrile-water was 32 in mobile phase：68；
25-28 minutes, the volume ratio of acetonitrile-water was 18 in mobile phase：82.
HPLC detection methods use Thermo U3000UHPLC liquid chromatographs.
Number of theoretical plate is calculated by ginsenoside Rg1 peak should be not less than 6000.
2.3 measuring methods are 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 requirement of 2.4 standard limited values
This product is calculated by dry product, containing ginsenoside Rg1 (C42H72O14) and ginsenoside Re (C48H82O18) total amount not It obtains less than 0.03%, ginsenoside Rb1 (C54H92O23) 0.20% must not be less than.
2.5 calculate (external standard method)
C in formulaR- reference substance solution concentration, unit are micro- gram per liter (mg/L)；
AXThe peak area of-test sample；
AR- 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.
3 results
3.1 linear relationship
Ginsenoside Rg1's reference substance, ginsenoside Re's reference substance and ginsenoside Rb1's reference substance are taken, it is accurately weighed, add second Solution of every 1ml containing about 0.2mg is made to get then accurate respectively to draw 0.5 μ l of the solution, 1.0 μ l, 1.5 μ l, 2.0 μ in alcohol L, 3.0 μ l enter LC measure, and are measured according to the above method.
The linear test of ginsenoside Rg1 the results are shown in Table 1, carries out linear regression with concentration (mg)-peak area, refers to Fig. 1；
The linear test of ginsenoside Re the results are shown in Table 1, carries out linear regression with concentration (mg)-peak area, refers to Fig. 1；
The linear test of ginsenoside Rb1 the results are shown in Table 1, carries out linear regression with concentration (mg)-peak area, refers to Fig. 1；
It is in good linear relationship.
Table 1
Table 2
Concentration (mg) First needle peak area Second needle peak area Average peak area
0.1013 89.8636 91.1991 90.5314
0.2026 185.5257 181.4935 183.5096
0.3039 282.7353 281.9074 282.3214
0.4052 369.5435 378.2917 373.9176
0.6078 574.6941 568.6457 571.6699
Table 3
Concentration (mg) First needle peak area Second needle peak area Average peak area
0.0868 49.0303 48.6714 48.8509
0.1735 96.8173 96.5181 96.6677
0.2603 148.6210 146.6620 147.6415
0.3470 200.9043 204.7011 202.8027
0.5205 320.7283 317.2293 318.9788
3.2 precision test
Take ginsenoside Rg1's (concentration：0.1822mg/ml), ginsenoside Re's (concentration：0.2026mg/ml) and ginseng soap Glycosides Rb1 (concentration：0.1735mg/ml) reference substance mixed solution, continuous sample introduction 6 times record peak area, ginsenoside Rg1, ginseng Saponin(e Re and ginsenoside Rb1's peak area RSD is respectively 0.5%, 1.1%, and 0.5% shows that instrument precision is good.3.3 it repeats Property experiment
The sample 1g of identical lot number is taken, it is totally 3 parts, accurately weighed, test solution is extracted by 4.2.2 ASE extracting methods, Sample size is 1 μ L, and with above-mentioned chromatographic condition parallel test, the content for measuring ginsenoside in sample is shown in Table 4, Rg1, Re and The RSD of Rb1 is respectively 1.3%, 0.9% and 2.7%, experiments have shown that ASE extracting methods repeatability is good.
Table 4
Rg1 contents (%) Re contents (%) Rb1 contents (%)
Repeatability 1 0.294 0.238 0.482
Repeatability 2 0.295 0.239 0.485
Repeatability 3 0.288 0.235 0.506
Average content (%) 0.292 0.237 0.491
RSD (%) 1.3 0.9 2.7
3.4 sample difference instruments extract result and with official method results contrast (1 batch)
Sample the results are shown in Table 5 using what different instruments extracted, using the sample size that ASE methods are extracted with using official method The results contrast of the sample size of extraction is shown in Table 6.
Table 5
Table 6
4 discuss：
The selection of 4.1ASE Extraction solvents：
Once the Extraction solvents such as the ethyl alcohol to ethyl alcohol, n-hexane saturation, 50% ethyl alcohol are investigated in experiment, and as a result display is adopted It is minimum with the ethyl alcohol extraction sample impurity of n-hexane saturation, but content of ginsenoside is relatively low, and 50% ethyl alcohol and ethyl alcohol is used to carry Take content of ginsenoside and the content using official method extraction more consistent, but 50% ethyl alcohol extraction rear impurity peak ratio uses ethyl alcohol It is much higher, therefore ethyl alcohol easy to operate, at low cost is used as Extraction solvent.
The selection of 4.2ASE purifying steps：
Once purification condition is investigated in experiment, is included in abstraction pool bottom and adds in suitable neutral alumina and silica gel As cleanser, as a result, it has been found that adding the impurity that extracts of cleanser in abstraction pool and being not added with no significant difference, in experiment Also investigate after sample has been extracted, adding suitable C18, PSA, GCB using extracting solution is purified again upper machine, as a result, it has been found that PSA and C18 purifications on the content of sample substantially without influence and GCB can have saponin(e absorption, and PSA clean-up effects are best, therefore adopt It is purified with PSA.
The optimization of 4.3ASE extraction conditions
We use 3 factor 3 horizontal quadrature experimental program L9 (33) experiment arrangement, have investigated extraction temperature, static extracting Time, cycle-index, influence (be shown in Table 7) of 3 factors to ginsenoside in accelerated solvent extraction ginseng.It determines to carry from ginseng The best Accelerate solvent extraction technique of ginsenoside is taken, the results are shown in Table 8.
Table 7
Table 8
Extracting temperature influences the content of ginsenoside maximum it can be seen from range analysis, secondly during static extracting Between and extraction time.Finally determining optimum extraction process is combined as 120 DEG C, extraction time 8min of Extracting temperature, extraction time 3 It is secondary.
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 a variety of ginsenosides in ginseng, which is characterized in that include the following steps successively：
    Step 1：The samples of Ginseng that crushed using ASE extraction process；Wherein, ASE extracting process includes first carrying in Soxhlet It takes in device using chloroform extraction, is then extracted in Accelerate solvent extraction instrument with ethyl alcohol, collect alcohol extraction liquid；
    Step 2：The content of the ginsenoside in alcohol extraction liquid is detected using HPLC methods.
  2. A kind of 2. method for measuring a variety of ginsenosides in ginseng according to claim 1, which is characterized in that the step Rapid 1 includes following sub-steps：
    Step S1：By sample comminution, No. four sieves are crossed, take the powder 1g after sieving；
    Step S2：Loaded in Soxhlet extractor, chloroform is added in, is heated to reflux 4 hours, chloroform extraction liquid is given up；
    Step S3：1g diatomite and the dregs of a decoction are mixed, immigration is placed in the ASE 10ml abstraction pools of fibrous filter membrane, adds diatomite extremely It is parallel with pond mouthful；
    Step S4：It is put into rapid extracting device and is extracted, extractant is ethyl alcohol；
    Step S5：Extract liquor is evaporated, residue adds ethyl alcohol to dissolve, and ethyl alcohol is added to be settled to 10ml, and 1ml is taken to add to and is equipped with In the 2ml centrifuge tubes of 100mgPSA, centrifugation is filtered to get alcohol extraction liquid.
  3. A kind of 3. method for measuring a variety of ginsenosides in ginseng according to claim 2, which is characterized in that the step In rapid S4, extraction parameters are：Extraction temperature is 110 DEG C, extraction time 10min, and extraction times are 1 time, and flush volume is 60%, purge time 60s.
  4. 4. a kind of method for measuring a variety of ginsenosides in ginseng according to claim 1 or 2, which is characterized in that described Step 2 in, the detection parameters of HPLC methods are：Chromatographic column：Thermo Syncronis Dim.AQ；Column temperature：50℃；Flow velocity： 0.3mL/min；Mobile phase：Acetonitrile-water；Detection wavelength：203nm.
  5. A kind of 5. method for measuring a variety of ginsenosides in ginseng according to claim 4, which is characterized in that the stream Dynamic phase：The volume ratio of acetonitrile-water is 18~32：68~82；
    0th minute, the volume ratio of acetonitrile-water was 18 in mobile phase：82；
    10th minute, the volume ratio of acetonitrile-water was 20 in mobile phase：80；
    18th minute, the volume ratio of acetonitrile-water was 30 in mobile phase：70；
    24th minute, the volume ratio of acetonitrile-water was 32 in mobile phase：68；
    25-28 minutes, the volume ratio of acetonitrile-water was 18 in mobile phase：82.
  6. A kind of 6. method for measuring a variety of ginsenosides in ginseng according to claim 4, which is characterized in that the color The specification for composing column is 1.7 μm, 50 × 2.1mm；The HPLC detection methods use Thermo U3000 UHPLC liquid chromatograies Instrument.
CN201711368003.0A 2017-12-18 2017-12-18 A kind of method of a variety of ginsenosides in measure ginseng Pending CN108152405A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109212088A (en) * 2018-10-22 2019-01-15 嘉兴迈维代谢生物科技有限公司 A method of 23 kinds of ginsenosides in sample are quickly detected based on UPLC- QTRAP technology simultaneously
CN113008640A (en) * 2021-02-26 2021-06-22 九州通医药集团股份有限公司 Pretreatment method for rapidly detecting ginsenoside Re based on microfluidic immune chip
CN116124954A (en) * 2023-02-27 2023-05-16 上海市质量监督检验技术研究院 Rapid detection method for ginsenoside Rg1, re and Rb1 content in ginseng

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109212088A (en) * 2018-10-22 2019-01-15 嘉兴迈维代谢生物科技有限公司 A method of 23 kinds of ginsenosides in sample are quickly detected based on UPLC- QTRAP technology simultaneously
CN113008640A (en) * 2021-02-26 2021-06-22 九州通医药集团股份有限公司 Pretreatment method for rapidly detecting ginsenoside Re based on microfluidic immune chip
CN116124954A (en) * 2023-02-27 2023-05-16 上海市质量监督检验技术研究院 Rapid detection method for ginsenoside Rg1, re and Rb1 content in ginseng
CN116124954B (en) * 2023-02-27 2024-06-07 上海市质量监督检验技术研究院 Rapid detection method for ginsenoside Rg1, re and Rb1 content in ginseng

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Application publication date: 20180612