CN108107129A - A kind of method for measuring ursolic acid total content in Verbena officinalis - Google Patents

A kind of method for measuring ursolic acid total content in Verbena officinalis Download PDF

Info

Publication number
CN108107129A
CN108107129A CN201711367980.9A CN201711367980A CN108107129A CN 108107129 A CN108107129 A CN 108107129A CN 201711367980 A CN201711367980 A CN 201711367980A CN 108107129 A CN108107129 A CN 108107129A
Authority
CN
China
Prior art keywords
verbena officinalis
ursolic acid
propylene glycol
measuring
total content
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201711367980.9A
Other languages
Chinese (zh)
Inventor
陈学松
钟水桥
梁峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuzhou Institutes for Food and Drug Control
Original Assignee
Wuzhou Institutes for Food and Drug Control
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuzhou Institutes for Food and Drug Control filed Critical Wuzhou Institutes for Food and Drug Control
Priority to CN201711367980.9A priority Critical patent/CN108107129A/en
Publication of CN108107129A publication Critical patent/CN108107129A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • G01N2030/042—Standards
    • G01N2030/047—Standards external
    • 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

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a kind of methods for measuring ursolic acid total content in Verbena officinalis, belong to field of chemical detection.It is intended to provide ursolic acid content in a kind of measure Verbena officinalis, and easy to operate, high sensitivity, the ASE HPLC methods that have good stability.This method extracts the Verbena officinalis after crushing with propylene glycol using ASE methods, collects propylene glycol extract liquor；The content of ursolic acid in Verbena officinalis is measured using HPLC methods, wherein chromatographic column used is Thermo Acclaim C30.The present invention can be used for the quality control and evaluation of Verbena officinalis.

Description

A kind of method for measuring ursolic acid total content in Verbena officinalis
Technical field
The invention belongs to field of chemical detection, especially a kind of method for measuring ursolic acid total content in Verbena officinalis.
Background technology
Verbena officinalis is the dry aerial parts of Verbenaceae.Its is cool in nature, bitter, nontoxic；With clearing heat and detoxicating, blood-breaking The effect of promoting menstruation, promoting blood circulation to remove blood stasis, inducing diuresis to remove edema.Research shows that Verbena officinalis has apparent anti-inflammatory, Robust speaker feature and anti-human chorionic cancer Effect, further research learns that the active ingredient of anticancer is ursolic acid.
Ursolic acid is isomer, and structure and properties are extremely similar, and often symbiosis is mutually deposited in natural plants, separation It measures more difficult.Version in 2005《Chinese Pharmacopoeia》(one) measures ursolic acid in Verbena officinalis using Thin-layer scanning chromatography and contains Amount, this method are influenced by factors such as lamellae, temperature, point sample, colour developings, easily lead to quantitative analysis results deviation, and can not be accurate Really determine the content of another active ingredient-ursolic acid.
The content of the invention
Against the above deficiency, the present invention is intended to provide a kind of easy to operate, high sensitivity, the measure horsewhip having good stability The method of ursolic acid total content in grass.
In order to realize above-mentioned technique effect, technical solution provided by the invention is such：Bear in a kind of measure Verbena officinalis The method of tartaric acid total content, comprises the following steps successively：
Step 1：The Verbena officinalis after crushing is extracted with propylene glycol using ASE methods, collects propylene glycol extract liquor；
Step 2：The content of ursolic acid in Verbena officinalis is measured using HPLC methods, wherein chromatographic column used is Thermo Acclaim C30。
Further, the specification of the chromatographic column described in step 2 is 3 μm, 2.1*150mm.
Further, the step 1 includes following sub-steps：
Step S1：Verbena officinalis sample comminution is crossed into No. three sieves, 0.25g is taken to be mixed with 1g diatomite；
Step S2：Step 1 gained mixture is put in the 10mlASE abstraction pools equipped with fibrous filter membrane, add diatomite to Pond mouthful is parallel；
Step S3：Extracted with 25ml propylene glycol, after with propylene glycol be settled to 25ml, obtain propylene glycol extract liquor.
Further, the extraction described in step S3, parameter are as follows：Extraction temperature is 100 DEG C, extraction time 6min, extraction It is 1 time, pressure 1500psi to take number, flush volume 60%, purge time 60s.
Further, the detection parameters of the HPLC methods described in step 2 are as follows：Column temperature is 40 DEG C；Flow velocity is 0.3mL/min； Mobile phase is -0.2% acetic acid of propylene glycol；Detection wavelength is 205nm.
Further, the volume ratio of -0.2% acetic acid of propylene glycol of the mobile phase is 90:10.
Further, instrument used in the ASE methods is the ASE350 Accelerate solvent extractions of DIONEX companies of the U.S. Instrument is -1290 high performance liquid chromatograph of Agilent used by instrument, HPLC methods.
The present invention has the following advantages compared with conventional method：
1. the present invention once investigated the solvent that Accelerate solvent extraction uses, there is paper to mention and extracted using 70% ethyl alcohol Ursolic acid relative amount is higher, therefore to propylene glycol and 70% ethyl alcohol and absolute ethyl alcohol is used to carry out solvent investigation, the results show Propylene glycol, 70% ethyl alcohol are consistent with absolute ethyl alcohol extraction ursolic acid content, and impurity peaks are essentially identical, therefore use propylene glycol conduct Extraction solvent；
2. the present invention, which has investigated extraction times, influences it, sample is taken to carry out extraction 1 time, 2 times, 3 times to it in experiment, point Analysis is as a result, the result shows that extraction once almost, therefore tests the method using extraction once；
3. the present invention, which improves temperature, reduces the viscosity of solvent, reduce the prevention that solvent enters sample matrices, add Solvent enters the diffusion of sample matrices, reduces the surface tension between solvent and sample matrices, makes the appearance of solvent dissolving determinand Amount increases；
4. due to liquid to the solvability of solute much larger than gas to the solvability of solute, therefore the boiling point of extraction liquids It is improved with pressure rise, so that solvent remains at liquid at high temperature under high pressure.
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, any limited number of time modification made in the claims in the present invention protection domain, still the present invention's In claims.
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.), high-efficient liquid phase color Spectrometer (1290)
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；
Propylene glycol (CH4O)：Chromatographically pure (phase chromatography-use)；
Ethyl alcohol (C2H5OH)：It analyzes pure.
2 methods
The preparation of 2.1 reference substance solutions：
Take ursolic acid reference substance, ursolic acid reference substance appropriate, it is accurately weighed, add propylene glycol that every 1ml 50 μ containing ursolic acid are made G, the mixed solution of ursolic acid 0.1mg to get.
2.2 the preparation of test solution
2.2.1《Chinese Pharmacopoeia》Version one prepares test sample method within 2015：
This product powder (crossing No. four sieves) about 0.5g is taken, it is accurately weighed, it puts in conical flask with cover, precision adds in absolute ethyl alcohol 25ml, weighed weight, be heated to reflux 4 it is small when, let cool, then weighed weight, the weight of less loss supplied with absolute ethyl alcohol, is shaken up, filter It crosses.Precision measures subsequent filtrate 10ml, adds 1% ammonia spirit 3ml, and mixing shakes extraction 3 times for (30~60 DEG C), often with petroleum ether Secondary 15ml discards petroleum ether liquid, ethanol is taken to be evaporated, and residue adds propylene glycol to dissolve, and is transferred in 5ml measuring bottles, adds propylene glycol extremely Scale shakes up, filtration, take subsequent filtrate to get.
2.2.2 Accelerate solvent extraction method (ASE) prepares test sample method：
Sample is crushed through pulverizer, is crossed No. three sieves, is taken 0.25g, accurately weighed, is uniformly mixed with about 1g diatomite, for use, 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 exist with Chi Kou In same horizontal line, abstraction pool upper cover is tightened, is put into rapid extracting device and is extracted.After extraction, extract liquor is shifted Into 25ml volumetric flasks, clean extraction flask with a small amount of propylene glycol and be incorporated into volumetric flask for 3 times, scale is settled to using propylene glycol, To obtain the final product.
2.3ASE extraction conditions
The extraction, parameter are as follows：Extraction temperature is 100 DEG C, extraction time 6min, and extraction times are 1 time, pressure For 1500psi, flush volume 60%, purge time 60s.
2.4 chromatographic conditions and system suitability
Using octadecylsilane chemically bonded silica as filler；With -0.2% acetum (82.5 of propylene glycol:17.5) it is stream Dynamic phase；Evaporative light scattering detector detects.Number of theoretical plate is calculated by ursolic acid peak should be not less than 5000.
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 10 μ l of test solution, inject liquid chromatograph, measure to get.
Detection parameters are as follows：Instrument：Agilent-high performance liquid chromatograph (1290)；Chromatographic column is Thermo AcclaimC30,3 μm, 2.1*150mm；Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is that volume ratio is 90:The third of 10 - 0.2% acetic acid of glycol；Detection wavelength is 205nm.
The requirement of 2.6 standard limited values
This product is calculated by dry product, and the total amount containing ursolic acid (C30H48O3) and ursolic acid (C30H48O3) must not be less than 0.30%.
2.7 calculate (external standard method)
C in formulaR--- reference substance solution concentration, unit are micro- gram per liter (mg/L)；
AX--- the peak area of test sample；
AR--- reference substance peak area.
Pay attention to：
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 leakage in sealing ring；
Abstraction pool fills elastic moderate during sample, and too loose to be easy to cause extracting solution excessive；
Check whether gas cylinder air pressure reaches 1Mpa before start；
It is cleaned out after use, abstraction pool will be dried and (got rusty easily) in time.
3 results
3.1 repetitive test
Take the sample (lot number of identical lot number：20150801) 0.5g, it is totally 6 parts, accurately weighed, it is extracted by ASE extracting methods Test solution, sample size are 3 μ L, and with above-mentioned chromatographic condition parallel test, the content for measuring ursolic acid content in sample is shown in Table 1, RSD 2.4%, experiments have shown that ASE extracting methods repeatability is good.
Table 1
3.3 sample size measurement results (3 batches)
The present invention has used two Accelerate solvent extraction instrument and official method to extract Verbena officinalis sample respectively, extracts It the results are shown in Table 2：
Table 2
The data comparison of ASE1 and ursolic acid total content in official method extraction sample the results are shown in Table 3：
Table 3
Lot number ASE1 Pharmacopeia Average value RAD%
15062801 0.368 0.316 0.342 7.60
1507103 0.414 0.355 0.385 7.67
20150801 0.400 0.343 0.372 7.67
The data comparison of ASE2 and ursolic acid total content in official method extraction sample the results are shown in Table 4：
Table 4
Lot number ASE2 Pharmacopeia Average value RAD%
15062801 0.385 0.316 0.351 9.84
1507103 0.401 0.355 0.378 6.08
20150801 0.382 0.343 0.363 5.38
Data comparison between two ASE instruments is shown in Table 5：
Table 5
4 discuss
The selection of 4.1 ASE Extraction solvents：
In experiment, once the solvent that Accelerate solvent extraction uses was investigated, has paper to mention and is extracted using 70% ethyl alcohol Ursolic acid relative amount is higher, therefore to propylene glycol and 70% ethyl alcohol and absolute ethyl alcohol is used to carry out solvent investigation, the results show Propylene glycol, 70% ethyl alcohol are consistent with absolute ethyl alcohol extraction ursolic acid content, and impurity peaks are essentially identical, therefore use propylene glycol conduct Extraction solvent.
The optimization of 4.2 ASE extraction conditions
On above-mentioned experiment basis, extraction times are mainly investigated in this experiment influences it, and sample is taken to carry out it in experiment Extraction 1 time, 2 times, 3 times, analysis is as a result, the result shows that extract once almost.
Therefore method of the experiment using extraction once.
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 modifications, equivalent substitutions and improvements etc., should be included within the scope of the present invention.

Claims (7)

  1. A kind of 1. method for measuring ursolic acid total content in Verbena officinalis, which is characterized in that comprise the following steps successively：
    Step 1：The Verbena officinalis after crushing is extracted with propylene glycol using ASE methods, collects propylene glycol extract liquor；
    Step 2：The content of ursolic acid in Verbena officinalis is measured using HPLC methods, wherein chromatographic column used is Thermo Acclaim C30。
  2. A kind of 2. method for measuring ursolic acid total content in Verbena officinalis according to claim 1, which is characterized in that step 2 The specification of the chromatographic column is 3 μm, 2.1*150mm.
  3. 3. a kind of method for measuring ursolic acid total content in Verbena officinalis according to claim 1, which is characterized in that described Step 1 includes following sub-steps：
    Step S1：Verbena officinalis sample comminution is crossed into No. three sieves, 0.25g is taken to be mixed with 1g diatomite；
    Step S2：Step 1 gained mixture is put in the 10mlASE abstraction pools equipped with fibrous filter membrane, add diatomite to and Chi Kou It is parallel；
    Step S3：Extracted with 25ml propylene glycol, after with propylene glycol be settled to 25ml, obtain propylene glycol extract liquor.
  4. A kind of 4. method for measuring ursolic acid total content in Verbena officinalis according to claim 3, which is characterized in that step S3 The extraction, parameter are as follows：Extraction temperature is 100 DEG C, extraction time 6min, and extraction times are 1 time, and pressure is 1500psi, flush volume 60%, purge time 60s.
  5. A kind of 5. method for measuring ursolic acid total content in Verbena officinalis according to claim 1, which is characterized in that step 2 The detection parameters of the HPLC methods are as follows：Column temperature is 40 DEG C；Flow velocity is 0.3mL/min；Mobile phase is -0.2% vinegar of propylene glycol Acid；Detection wavelength is 205nm.
  6. 6. a kind of method for measuring ursolic acid total content in Verbena officinalis according to claim 5, which is characterized in that described The volume ratio of -0.2% acetic acid of propylene glycol of mobile phase is 90:10.
  7. 7. a kind of method for measuring ursolic acid total content in Verbena officinalis according to claim 1, which is characterized in that described Instrument used in ASE methods is that instrument is used by the ASE350 Accelerate solvent extractions instrument of DIONEX companies of the U.S., HPLC methods - 1290 high performance liquid chromatograph of Agilent.
CN201711367980.9A 2017-12-18 2017-12-18 A kind of method for measuring ursolic acid total content in Verbena officinalis Pending CN108107129A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711367980.9A CN108107129A (en) 2017-12-18 2017-12-18 A kind of method for measuring ursolic acid total content in Verbena officinalis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711367980.9A CN108107129A (en) 2017-12-18 2017-12-18 A kind of method for measuring ursolic acid total content in Verbena officinalis

Publications (1)

Publication Number Publication Date
CN108107129A true CN108107129A (en) 2018-06-01

Family

ID=62210980

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711367980.9A Pending CN108107129A (en) 2017-12-18 2017-12-18 A kind of method for measuring ursolic acid total content in Verbena officinalis

Country Status (1)

Country Link
CN (1) CN108107129A (en)

Similar Documents

Publication Publication Date Title
CN108169356A (en) A kind of method that ASE-HPLC methods measure spinosin content in spina date seed
CN108107129A (en) A kind of method for measuring ursolic acid total content in Verbena officinalis
CN108020624A (en) A kind of method for measuring oleanolic acid total content in Verbena officinalis
CN107064347A (en) A kind of ASE HPLC methods determine Psoralen, the method for Isopsoralen content in Psoralen ester
CN107219315A (en) A kind of method that ASE HPLC methods determine oleanolic acid and ursolic acid total content in Verbena officinalis
CN108107127A (en) A kind of method of ginsenoside Re's content in measure ginseng
CN108088928A (en) A kind of method for measuring Content Determination of Indirubin in folium isatidis
CN106831914A (en) A kind of method that ASE methods extract aurantiamarin in dried orange peel
CN106885860A (en) A kind of method that ASE HPLC methods determine spinosin content in spina date seed
CN106872611A (en) A kind of method that ASE HPLC methods determine amentoflavone content in Selaginella tamariscina
CN108169353A (en) A kind of method that ASE-HPLC methods measure Determination of hesperidin in pericarpium citri reticulatae
CN108169352A (en) A kind of method that ASE-HPLC methods measure amentoflavone content in Selaginella tamariscina
CN108101949A (en) A kind of method of aurantiamarin in ASE methods extraction dried orange peel
CN106955502A (en) A kind of method of Geniposidic acid and acteoside in ASE methods extraction plantain seed
CN106885861A (en) A kind of method that ASE HPLC methods determine isoferulic acid content in rattletop
CN107014927A (en) A kind of method that ASE HPLC methods determine Determination of hesperidin in pericarpium citri reticulatae
CN108132310A (en) A kind of method of Determination of Content of Ginsenoside Rg_1 in measure ginseng
CN108169359A (en) A kind of method of castor-oil plant alkali content in measure castor bean
CN108169355A (en) A kind of method of magnelin in ASE methods extraction flower bud of lily magnolia
CN107892706A (en) A kind of method of spinosin in ASE methods extraction spina date seed
CN107064384A (en) A kind of method that ASE methods extract spinosin in spina date seed
CN107064344A (en) A kind of method that ASE HPLC methods determine ginsenoside Rg_1 and Re content in ginseng
CN106872610A (en) A kind of method that ASE HPLC methods determine castor-oil plant alkali content in castor bean
CN106918666A (en) A kind of method that ASE HPLC methods determine the curculigoside content in thizoma curculiginis
CN108088939A (en) A kind of method that ASE-HPLC methods measure magnelin content in the flower bud of lily magnolia

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20180601