CN105954383A - Determination method for oleanolic acid and hederagenin in clematis root - Google Patents
Determination method for oleanolic acid and hederagenin in clematis root Download PDFInfo
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- CN105954383A CN105954383A CN201610254599.0A CN201610254599A CN105954383A CN 105954383 A CN105954383 A CN 105954383A CN 201610254599 A CN201610254599 A CN 201610254599A CN 105954383 A CN105954383 A CN 105954383A
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- extraction
- radix clematidis
- oleanolic acid
- helexin
- assay method
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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
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention discloses a determination method for oleanolic acid and hederagenin in clematis root. The method comprises the following steps: step 1, crushing the clematis root and then extracting the crushed clematis root by using an ASE extraction method so as to obtain extract; step 2, subjecting the extract obtained in the previous step to centrifugation so as to obtain supernatant; and step 3, analyzing the supernatant by using liquid chromatography. The determination method for oleanolic acid and hederagenin in clematis root is simple to operate and high in detection precision and can simultaneously detect the contents of oleanolic acid and hederagenin in the clematis root.
Description
Technical field
The present invention relates to test in laboratory field, oleanolic acid and the survey of helexin in a kind of Radix Clematidis
Determine method.
Background technology
Pharmacopeia in 2010 have recorded the method for helexin in Radix Clematidis of extracting: takes this product powder (crossing No. four sieves) about
4g, accurately weighed, to put in apparatus,Soxhlet's, add ethyl acetate appropriate, be heated to reflux 3 hours, discard acetic acid ethyl fluid, medicinal residues are waved
Dry solvent, is transferred in conical flask together with filtration paper cylinder, accurate addition Diluted Alcohol 50ml, and weighed weight is heated to reflux 1 hour, puts
Cold, more weighed weight, to supply the weight of less loss with Diluted Alcohol, shake up, filter, precision measures subsequent filtrate 25ml, puts and steams in water-bath
Dry, residue adds 2mol/L hydrochloric acid solution 30ml makes dissolving, is heated to reflux 2 hours.Cool down immediately, move in separatory funnel, use water
10ml washing container by several times, washing liquid is incorporated in separatory funnel.Add ethyl acetate shaking to extract 3 times, each 15ml, merge acetic acid second
Ester liquid, less than 70 DEG C are concentrated near dry, add methanol and dissolve, are transferred in 10ml measuring bottle, add methanol to scale, shake up, to obtain final product.
It by the method for hplc determination is: with octadecylsilane chemically bonded silica as filler；With acetonitrile-water
(90:10) for flowing phase；Detection wavelength is 205nm.Number of theoretical plate presses oleanolic acid peak and the calculating of helexin peak all should
It is not less than 3000.
In order to solve to operate excessively complicated problem in official method, we have obtained one by great many of experiments and have passed through
ASE rapid extraction-ASE separates and tests the oleanolic acid in Radix Clematidis, specifically, is divided into remove impurity and carries in its extraction process
Taking step, in removal step, solvent is ethyl acetate, and the solvent in extraction step is Diluted Alcohol, and it can extraordinary mensuration prestige
Oleanolic acid in Radix Clematidis, its precision measured with official method is consistent, but finds in actual applications, uses this ASE fast
Speed extraction when measuring helexin simultaneously, and it is on the low side that it measures concentration, although helexin and oleanolic acid
Chemistry skeleton symbol is C30H48O3But, during extracting, in addition it is also necessary to adjust Extraction solvent further, Chang Chun could be measured simultaneously
Rattan sapogenin and oleanolic acid.
Summary of the invention
It is an object of the invention to provide oleanolic acid and the assay method of helexin, the method in a kind of Radix Clematidis
Simple to operate, accuracy of detection is high, it is possible to oleanolic acid and the content of helexin in the effective Radix Clematidis of mensuration simultaneously.
The technical scheme that the present invention provides is: oleanolic acid and the assay method of helexin in a kind of Radix Clematidis,
Comprise the following steps:
Step 1: use ASE extraction that extract is obtained by extraction after being pulverized by Radix Clematidis；
Wherein, described step 1 particularly as follows:
S11: just Radix Clematidis mixes with appropriate quartz sand after pulverizing；
S12: add the mixture in S11 in being equipped with diatomaceous abstraction pool, be subsequently adding quartz sand and make abstraction pool
Interior solid reaches the Chi Kou of abstraction pool, and the lid covering abstraction pool extracts；Collect extract；
Two steps it are divided into carry out during extraction, i.e. defatting step and extraction step；
Controlling parameter during described defatting step is: extractant is ethyl acetate and methyl acetate；Extraction temperature 110
℃；Static extracting time 3min；Flush volume 100%；Quiet cycle number of times 1 time；Ethyl acetate and the volume ratio of methyl acetate
For 1:1；
Controlling parameter during described extraction step is: extractant is the mixed solution of ethanol, methanol, water；Extraction temperature
120℃；Static extracting time 5min；Flush volume 100%；Quiet cycle number of times 2 times；Ethanol, methanol, water mixed solution in
Water including 30wt% ethanol, 15wt% methanol and surplus mixes；
Step 2: after extract centrifugation, obtain supernatant；
Step 3: use liquid chromatography to be analyzed supernatant；
Wherein, the analysis condition of liquid chromatography is:
A) instrument: double ternary liquid phase chromatographs
B) chromatographic column specification: carbon 30 chromatographic column 2.1*150mm 3um
C) column temperature: 20 DEG C
D) flow velocity: 0.5mL/min
E) flowing phase: acetonitrile-water, volume ratio 62:38
F) detection wavelength: 205nm.
In above-mentioned Radix Clematidis in the assay method of oleanolic acid and helexin, the volume of described abstraction pool
For 10ml.
In above-mentioned Radix Clematidis in the assay method of oleanolic acid and helexin, described defatting step and carrying
The operation pressure taken in step is 1500psi.
In above-mentioned Radix Clematidis in the assay method of oleanolic acid and helexin, described defatting step and carrying
Taking the purge time in step is 60s.
In above-mentioned Radix Clematidis in the assay method of oleanolic acid and helexin, step 2 will be particularly as follows: will extract
Liquid is put and is evaporated in water-bath, and residue adds 2mol/L hydrochloric acid solution 30ml makes dissolving, is heated to reflux 2 hours；Cool down immediately, move into separatory
In funnel, with water 10ml washing container by several times, washing liquid is incorporated in separatory funnel；Add ethyl acetate shaking extraction 3 times, every time
15ml, combined ethyl acetate liquid, less than 70 DEG C are concentrated near dry, add methanol and dissolve, be transferred in 10ml measuring bottle, add methanol to carving
Degree, shakes up, and under 15000r/min, centrifugal 3min, takes supernatant.
In above-mentioned Radix Clematidis in the assay method of oleanolic acid and helexin, described instrument is Thermo
U3000UHPLC chromatograph of liquid.
In above-mentioned Radix Clematidis in the assay method of oleanolic acid and helexin, described chromatographic column is
Thermo Acclaim C30。
In above-mentioned Radix Clematidis in the assay method of oleanolic acid and helexin, described ASE extraction institute
The instrument used is ASE350 Accelerate solvent extraction instrument.
The present invention is after using technique scheme, and it has the beneficial effect that
The detection method of the present invention is simple to operate, and precision is consistent with the precision of official method, reproducible, it is possible to effective
Measure oleanolic acid and the content of helexin in Radix Clematidis simultaneously.
Detailed description of the invention
Below in conjunction with detailed description of the invention, technical scheme is described in further detail, but do not constitute right
Any restriction of the present invention.
Embodiment 1:
Oleanolic acid and the assay method of helexin in a kind of Radix Clematidis, comprise the following steps:
Step 1: use ASE extraction that extract is obtained by extraction after being pulverized by Radix Clematidis；
Wherein, described step 1 particularly as follows:
S11: just 1g Radix Clematidis mixes with 2g quartz sand after pulverizing；
S12: add the mixture 3g in S11 in being equipped with the diatomaceous abstraction pool of 1g, be subsequently adding quartz sand and make extraction
Taking the solid in pond and reach the Chi Kou of abstraction pool, the lid covering abstraction pool extracts；Collect extract；
Two steps it are divided into carry out during extraction, i.e. defatting step and extraction step；The solution that defatting step obtains is given up, and extracts step
Suddenly the solution obtained is as extract；
Controlling parameter during described defatting step is: extractant is ethyl acetate and methyl acetate；Extraction temperature 110
℃；Static extracting time 3min；Flush volume 100%；Quiet cycle number of times 1 time；Operation pressure 1500psi；Purge time
60s；The volume ratio of ethyl acetate and methyl acetate is 1:1；
Controlling parameter during described extraction step is: extractant is the mixed solution of ethanol, methanol, water；Extraction temperature
120℃；Static extracting time 5min；Flush volume 100%；Quiet cycle number of times 2 times；Operation pressure 1500psi；Purge time
60s；Ethanol, methanol, the mixed solution of water include that the water of 30wt% ethanol, 15wt% methanol and surplus mixes；
Step 2: put by extract in water-bath and be evaporated, residue adds 2mol/L hydrochloric acid solution 30ml makes dissolving, is heated to reflux 2 little
Time；Cooling down immediately, move in separatory funnel, with water 10ml washing container by several times, washing liquid is incorporated in separatory funnel；Add ethyl acetate
Shaking is extracted 3 times, each 15ml, combined ethyl acetate liquid, and less than 70 DEG C are concentrated near dry, add methanol and dissolve, be transferred to 10ml
In measuring bottle, adding methanol to scale, shake up, under 15000r/min, centrifugal 3min, takes supernatant；It should be noted that this enforcement
Volume ratio under conditions of the parameter of related to volume ratio is 20 DEG C in example.
Step 3: use liquid chromatography to be analyzed supernatant；
Wherein, the analysis condition of liquid chromatography is:
A) instrument: the double ternary liquid phase chromatograph of Thermo U3000UHPLC
B) chromatographic column specification: Thermo Acclaim C30 2.1*150mm 3um, i.e. carbon 30 chromatographic column, diameter 2.1mm,
Length 150mm, particle diameter 3um；
C) column temperature: 20 DEG C
D) flow velocity: 0.5mL/min
E) flowing phase: acetonitrile-water, volume ratio 62:38
F) detection wavelength: 205nm.
Comparative example 1
The size-reduced machine of Radix Clematidis sample is pulverized, excessively No. three sieves, and about 1g is accurately weighed, mixs homogeneously with appropriate amount of quartz sand, treats
With, filter membrane is put well in advance10ml abstraction pool is initially charged 1g kieselguhr, the rear sample adding mix homogeneously, then
Adding appropriate amount of quartz sand, shaking is allowed at 2mm, tighten abstraction pool upper cover with Chi Kou under same level line gently.Extraction (includes
Remove impurity and extract 2 steps, be shown in Table the ASE conditions method of 1) terminate after, put and be evaporated in water-bath, residue adds 2mol/L hydrochloric acid solution 30ml
Make dissolving, be heated to reflux 2 hours.Cooling down immediately, move in separatory funnel, with water 10ml washing container by several times, washing liquid is incorporated to point
In liquid funnel.Adding ethyl acetate shaking to extract 3 times, each 15ml, combined ethyl acetate liquid, less than 70 DEG C are concentrated near dry, add
Methanol dissolves, and is transferred in 10ml measuring bottle, adds methanol to scale, shakes up, and under 15000r/min, centrifugal 3min, takes supernatant,
Enter LC to measure and get final product.
Table 1
The repeatability checking of the extracting process of 1.1 embodiments 1:
Take sample (lot number: 150410 (the Z)) 0.5g of identical lot number, totally 6 parts, accurately weighed, extract by ASE extracting method
Need testing solution, sample size is 1 μ L, with above-mentioned chromatographic condition parallel test, record in sample the oleanolic acid of Radix Clematidis and
Helexin content is shown in Table 2, and RSD is 2% and 1.68%, and test shows that ASE extracting method repeatability is good, the results detailed in
Table 2；
Table 2
The degree of accuracy test of the extracting process of 1.2 embodiments 1
Use ASE extraction and detection method, the extraction of comparative example 1 and detection method and the extraction of pharmacopeia of embodiment 1
Extracting Radix Clematidis with detection method and detect, Radix Clematidis is 3 batches, and lot number is respectively 150410 (Y), 1510124,
150410(Z)；Testing result such as table 3 below；
Table 3
Above-described be only presently preferred embodiments of the present invention, all made in the range of the spirit and principles in the present invention appoint
What amendment, equivalent and improvement etc., should be included within the scope of the present invention.
Claims (8)
1. oleanolic acid and the assay method of helexin in a Radix Clematidis, it is characterised in that comprise the following steps:
Step 1: use ASE extraction that extract is obtained by extraction after being pulverized by Radix Clematidis；
Wherein, described step 1 particularly as follows:
S11: just Radix Clematidis mixes with appropriate quartz sand after pulverizing；
S12: add the mixture in S11 in being equipped with diatomaceous abstraction pool, is subsequently adding in quartz sand makes abstraction pool
Solid reaches the Chi Kou of abstraction pool, and the lid covering abstraction pool extracts；Collect extract；
Two steps it are divided into carry out during extraction, i.e. defatting step and extraction step；
Controlling parameter during described defatting step is: extractant is ethyl acetate and methyl acetate；Extraction temperature 110 DEG C；Quiet
State extraction time 3min；Flush volume 100%；Quiet cycle number of times 1 time；The volume ratio of ethyl acetate and methyl acetate is 1:1；
Controlling parameter during described extraction step is: extractant is the mixed solution of ethanol, methanol, water；Extraction temperature 120
℃；Static extracting time 5min；Flush volume 100%；Quiet cycle number of times 2 times；Ethanol, methanol, water mixed solution in wrap
The water including 30wt% ethanol, 15wt% methanol and surplus mixes；
Step 2: after extract centrifugation, obtain supernatant；
Step 3: use liquid chromatography to be analyzed supernatant；
Wherein, the analysis condition of liquid chromatography is:
A) instrument: double ternary liquid phase chromatographs
B) chromatographic column specification: carbon 30 chromatographic column 2.1*150mm 3um
C) column temperature: 20 DEG C
D) flow velocity: 0.5mL/min
E) flowing phase: acetonitrile-water, volume ratio 62:38
F) detection wavelength: 205nm.
Oleanolic acid and the assay method of helexin in Radix Clematidis the most according to claim 1, it is characterised in that
The volume of described abstraction pool is 10ml.
Oleanolic acid and the assay method of helexin in Radix Clematidis the most according to claim 1, it is characterised in that
Operation pressure in described defatting step and extraction step is 1500psi.
Oleanolic acid and the assay method of helexin in Radix Clematidis the most according to claim 1, it is characterised in that
Purge time in described defatting step and extraction step is 60s.
Oleanolic acid and the assay method of helexin in Radix Clematidis the most according to claim 1, it is characterised in that
Step 2 is particularly as follows: put extract and be evaporated in water-bath, and residue adds 2mol/L hydrochloric acid solution 30ml makes dissolving, is heated to reflux 2 little
Time；Cooling down immediately, move in separatory funnel, with water 10ml washing container by several times, washing liquid is incorporated in separatory funnel；Add ethyl acetate
Shaking is extracted 3 times, each 15ml, combined ethyl acetate liquid, and less than 70 DEG C are concentrated near dry, add methanol and dissolve, be transferred to 10ml
In measuring bottle, adding methanol to scale, shake up, under 15000r/min, centrifugal 3min, takes supernatant.
Oleanolic acid and the assay method of helexin in Radix Clematidis the most according to claim 1, it is characterised in that
Described instrument is Thermo U3000UHPLC chromatograph of liquid.
Oleanolic acid and the assay method of helexin in Radix Clematidis the most according to claim 6, it is characterised in that
Described chromatographic column is Thermo Acclaim C30.
Oleanolic acid and the assay method of helexin in Radix Clematidis the most according to claim 1, it is characterised in that
The instrument that described ASE extraction is used is ASE350 Accelerate solvent extraction instrument.
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| CN201610254599.0A CN105954383A (en) | 2016-04-22 | 2016-04-22 | Determination method for oleanolic acid and hederagenin in clematis root |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106905405A (en) * | 2017-03-10 | 2017-06-30 | 成都大学 | A kind of method that aglycon is extracted from the root of Chinese clematis and application |
| CN106924991A (en) * | 2017-04-17 | 2017-07-07 | 广西壮族自治区梧州食品药品检验所 | A kind of method of recinine in ASE methods extraction castor bean |
| CN107014923A (en) * | 2017-04-20 | 2017-08-04 | 广西壮族自治区梧州食品药品检验所 | A kind of method that ASE HPLC methods determine content of oleanolic acid in the root of Chinese clematis |
| CN110579547A (en) * | 2019-09-27 | 2019-12-17 | 淮阴工学院 | Detection method of radix clematidis medicinal material HPLC fingerprint |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101216462A (en) * | 2007-12-29 | 2008-07-09 | 广州中医药大学科技产业园有限公司 | Clematis chinensis medicinal materials effective ingredient content determination method |
| CN103558303A (en) * | 2013-10-25 | 2014-02-05 | 贵州信邦制药股份有限公司 | Method for measuring content of oleanolic acid in glossy privet fruit and astragalus membranaceus healthy energy-strengthening preparation |
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2016
- 2016-04-22 CN CN201610254599.0A patent/CN105954383A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101216462A (en) * | 2007-12-29 | 2008-07-09 | 广州中医药大学科技产业园有限公司 | Clematis chinensis medicinal materials effective ingredient content determination method |
| CN103558303A (en) * | 2013-10-25 | 2014-02-05 | 贵州信邦制药股份有限公司 | Method for measuring content of oleanolic acid in glossy privet fruit and astragalus membranaceus healthy energy-strengthening preparation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106905405A (en) * | 2017-03-10 | 2017-06-30 | 成都大学 | A kind of method that aglycon is extracted from the root of Chinese clematis and application |
| CN106924991A (en) * | 2017-04-17 | 2017-07-07 | 广西壮族自治区梧州食品药品检验所 | A kind of method of recinine in ASE methods extraction castor bean |
| CN107014923A (en) * | 2017-04-20 | 2017-08-04 | 广西壮族自治区梧州食品药品检验所 | A kind of method that ASE HPLC methods determine content of oleanolic acid in the root of Chinese clematis |
| CN110579547A (en) * | 2019-09-27 | 2019-12-17 | 淮阴工学院 | Detection method of radix clematidis medicinal material HPLC fingerprint |
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