CN100465639C - Inspection of manyprickle acanthopanax - Google Patents

Inspection of manyprickle acanthopanax Download PDF

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CN100465639C
CN100465639C CNB2006100169265A CN200610016926A CN100465639C CN 100465639 C CN100465639 C CN 100465639C CN B2006100169265 A CNB2006100169265 A CN B2006100169265A CN 200610016926 A CN200610016926 A CN 200610016926A CN 100465639 C CN100465639 C CN 100465639C
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wilsonii
herbal medicine
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CN1895312A (en
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由天艳
周晓光
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Changzhou Institute Of Energy Storage Materials & Devices
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Changchun Institute of Applied Chemistry of CAS
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Abstract

A method for testing acanthopanax bark features that the simple ultrasonic extracting in methanol is combined with the capillary electrophoresis type electrochemical analysis method, and the quality of acanthopanax bark is determined by comparing the contents of active components and the fingerprint.

Description

A kind of detection method of wilsonii
Technical field
The invention belongs to the detection method of Chinese medicine wilsonii, be specifically related to a kind of capillary electrophoresis electrochemical detection method of wilsonii.
Background technology
Wilsonii is a kind of herbal medicine that extensively is planted in Hebei, Jilin, Liaoning and North China.But the wilsonii that only originates in the Heilongjiang Province is considered to genunie medicinal materials.In Chinese Pharmacopoeia, the Chinese herbal medicine wilsonii is dry root and rhizome or the stem of Araliaceae wilsonii (Acanthopanax senticosus (Ru pr.Et Maxim.) Harms).In addition, the leaf of plant wilsonii is also pharmaceutically acceptable.The pharmaceutically active of wilsonii is similar to genseng, has the effect that improves the health.Therefore, caused researcher's extensive interest about the research of wilsonii.Isofraxidin, chlorogenic acid and rutin are the important biomolecule active components in the wilsonii.Wherein, the content of isofraxidin is the important indicator of formulating the acanthopanax senticosus pharmaceutical standards.A lot of methods have been used the composition of analyzing wilsonii, and the existing patent documentation report of the quality testing of the acanthopanax (patent No.: 01104005).But, the discriminating of the Caulis Et Caulis Acanthopanacis Senticosi in the different places of production and the different parts of wilsonii, and the distribution of these active components in different parts also do not analyzed and patent report up to now.
At present, the method that is used for the Chinese herbal medicine fingerprint analysis mainly is the high performance liquid chromatography uv detection method.Capillary Electrophoresis (capillary electrophoresis, CE) the method analysis that is used for Chinese herbal medicine is compared with it and is had many advantages: 1. capillary column easy cleaning; 2. simply 3. sample pre-treatments lacks analysis time; 4. because post is imitated height, might make same separation condition be fit to multi-component analysis in the several samples; 5. extra small sample volume and low solvent consumption; 6. clastotype is many, is suitable for all kinds of chemical composition analysis in the Chinese herbal medicine.Uv detection method has good versatility, but short kapillary light path makes that its sensitivity is lower.By contrast, (electrochemical detection ED) provides high sensitivity to electrochemical assay, and device is simple.Therefore, adopt that CE efficiently and sensitive ED fingerprint analysis Chinese herbal medicine can guarantee better that it is true, quality, safety and drug effect.
Summary of the invention
The invention provides a kind of detection method of wilsonii, be a kind of instrument cheapness, simple to operate and quick and sensitive capillary electrophoresis electrochemical analytical approach, thereby be fingerprint map analyzing and active component content to be measured combine control and estimate herbal medicine wilsonii method for quality.
Method of the present invention is divided following step:
(1) preparation of buffer solution
Directly mix NaH 2PO 4And Na 2B 4O 7Aqueous solution, and use 0.20molL -1HCl or 0.20molL -1NaOH regulates its pH value and is 5.0-9.0.
(2) preparation and the cyclic voltammetry experiment thereof of active component standard solution in the herbal medicine wilsonii
The main active of herbal medicine wilsonii is isofraxidin, chlorogenic acid and rutin, and they become concentration with dissolve with methanol respectively is 3-8mol L -1Solution.
The cyclic voltammetry experiment process of active component standard solution is:
With 33 μ m carbon fiber microdisk electrodes (CFE) at the NaH that contains isofraxidin, chlorogenic acid and rutin respectively 2PO 4And Na 2B 4O 7Its cyclic voltammetric behavior is investigated in scan round in the mixed solution.
(3) optimization of testing conditions, and under this optimal conditions, carry out the investigation of reappearance, detectability and the range of linearity
Change-detection current potential, 5.0 * 10 between 0.45-1.35V -3-8.5 * 10 -3Mol L -1With 5.0 * 10 -3-9.0 * 10 -3Mol L -1Between change NaH 2PO 4And Na 2B 4O 7Concentration, 5.0-9.0 between change to change between pH value of buffer solution, 8-35s and change separation voltage between sample injection time and 5-17.5kV and investigate influence peak current, transit time, degree of separation and separation efficiency.
(4) preparation of herbal medicine sample solution
Pulverize the herbal medicine sample with the plant comminutor that has 20-80 mesh sieve, take by weighing the herbal medicine sample of 0.3-0.8g, with the ultrasonic 15-30min of methyl alcohol water-bath, inclining afterwards supernatant, and this extraction process repeats 1-3 time again.At last, the extract decompression distillation is closely dried, uses dissolve with methanol residue and constant volume in the methyl alcohol of 3-8mL.Before the operation, all sample solutions are all with the cellulose esters membrane filtration of 0.22 μ m and be diluted to needed concentration direct injection analysis.
(5) acquisition of wilsonii CE-ED finger-print
The acquisition of wilsonii CE-ED finger-print is to carry out electrophoretic analysis under the top condition that is obtained to obtain in step (3).
(6) peak height of active component is determined the content of active component in the actual sample in contrast standard mixed solution and the actual sample;
(7) contrast the CE-ED finger-print that is obtained, differentiate the different parts of herbal medicine wilsonii and the Caulis Et Caulis Acanthopanacis Senticosi in the different places of production from several different fingerprint fragments respectively.
The advantage of herbal medicine wilsonii set forth in the present invention quality control and fingerprint discrimination method is:
CE-ED method instrument is simple: only need the kapillary of a cheapness, little dish working electrode, high direct voltage instrument (± 30kV), electrochemical analyser; It is few to consume reagent and solution: sampling volume has only several liters of receiving usually, and is very little to environmental impact; Efficient and fast: as long as CE finishes a testing process a few minutes to tens minute usually, and can obtain the theoretical cam curve up to hundreds of thousands/rice; Because it has response to electroactive component, so its selectivity is good, and detectability is low, can reach 10 usually -8Mol L -1, the range of linearity reaches 2-3 the order of magnitude.
Description of drawings
Fig. 1 is different places of production Caulis Et Caulis Acanthopanacis Senticosi (that is: a cap mountain; The b Wudalianchi; C 5 constant virtues; D Mudanjiang) CE-ED finger-print contrast; I, II, III, II ' and III ' are respectively fingerprint fragment I, II, III, II ' and III ' among the figure; Peak 1 is an isofraxidin, and peak 3 is a chlorogenic acid.
Fig. 2 is herbal medicine wilsonii different parts (that is: an a root; The b leaf; The c rhizome; The d stem) CE-ED finger-print contrast; I, II, III and III ' are respectively fingerprint fragment I, II, III and III ' among the figure; Peak 1 is an isofraxidin, and peak 2 is a rutin, and peak 3 is a chlorogenic acid.
Embodiment
Below, the invention will be further described in conjunction with the embodiments.
Embodiment 1: the detection by quantitative of different places of production Caulis Et Caulis Acanthopanacis Senticosi and fingerprint discriminatory analysis
(1) preparation of buffer solution, its concrete grammar is:
Directly mix NaH 2PO 4And Na 2B 4O 7Aqueous solution, the pH value is respectively 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0 solution 0.20mol L -1HCl or 0.20mol L -1NaOH regulates.
(2) preparation of active component standard solution and cyclic voltammetry experiment thereof, process is as follows:
The preparation of standard solution: concentration is 5.0 * 10 -3Mol L -1The solution of isofraxidin, rutin and chlorogenic acid is used dissolve with methanol respectively, and lays in 4 ℃ refrigerator, is diluted to desired concn with secondary water before using.
The cyclic voltammetry experiment of standard solution: at first with 33 μ m CFE electrodes at NaH 2PO 4And Na 2B 4O 7One week of scan round as a setting in the mixed solution.Then this electrode is being contained 1.0 * 10 respectively -4Mol L -1Isofraxidin, 1.0 * 10 -4Mol L -1Rutin and 1.0 * 10 -4Mol L -1The NaH of chlorogenic acid 2PO 4And Na 2B 4O 7In one week of scan round in the mixed solution, investigate their cyclic voltammetric behavior.
(3) optimization of testing conditions, and under this optimal conditions, carry out the investigation of reappearance, detectability and the range of linearity;
The detailed process of testing conditions optimization is:
Change-detection current potential, 5.0 * 10 between 0.45-1.35V -3-8.5 * 10 -3Mol L -1With 5.0 * 10 -3-9.0 * 10 -3Mol L -1Between change NaH 2PO 4And Na 2B 4O 7Concentration, 5.0-9.0 between change to change between pH value of buffer solution, 8-35s and change separation voltage between sample injection time and 5-17.5kV and investigate influence peak current, transit time, degree of separation and separation efficiency.
Under the separation and testing conditions of the best: the detection current potential is 1.20V; Buffer solution is 7.0 * 10 -3MolL -1NaH 2PO 4+ 7.5 * 10 -3Mol L -1Na 2B 4O 7(pH=7.0); 17cm height sample introduction 25s; Separation voltage is 15kV, reaches baseline separation and has high sensitivity at 6min inner analysis thing isofraxidin, rutin and chlorogenic acid.
Under the described top condition, tested the series of standards mixed solution in the above, concentration range is from 1.0 * 10 -7To 1.0 * 10 -4Mol L -1Isofraxidin, 2.0 * 10 -7To 1.0 * 10 -4Mol L -1Rutin and 1.5 * 10 -6To 5.0 * 10 -5Mol L -1Chlorogenic acid.Obtained crossing over the range of linearity of 1-3 the order of magnitude, related coefficient surpasses 0.99.Adopt this CE-ED method actual detected to the detectability of isofraxidin, rutin and chlorogenic acid be respectively 1.0 * 10 -7Mol L -1, 2.0 * 10 -7Mol L -1With 1.5 * 10 -6Mol L -1(signal to noise ratio (S/N ratio) is 3).
Under top condition, advance the standard biased sample continuously: 1.0 * 10 -5Mol L -1Isofraxidin, 2.0 * 10 -5Mol L -1Rutin and 2.0 * 10 -5Mol L -1Chlorogenic acid, the reappearance in a few days of investigation the method.Continuous 6 sample introductions, the RSDs of their peak current and transit time (n=6) value is respectively 1.5,0.63%, 2.4,0.69% and 1.2,0.70%.
(4) preparation of herbal medicine sample solution, detailed process is:
Pulverize the herbal medicine sample with the plant comminutor that has 40 mesh sieve, take by weighing the herbal medicine sample of 0.5g, with the ultrasonic 20min of methyl alcohol water-bath, inclining afterwards supernatant, and this extraction process repeats 2 times again.At last, the extract decompression distillation is closely dried, uses dissolve with methanol residue and constant volume in the methyl alcohol of 5mL.Before the experiment, all sample solutions are all with the cellulose esters membrane filtration of 0.22 μ m and be diluted to needed concentration direct injection analysis.
(5) acquisition of Caulis Et Caulis Acanthopanacis Senticosi CE-ED finger-print;
The acquisition of Caulis Et Caulis Acanthopanacis Senticosi CE-ED finger-print is to carry out (as shown in Figure 1) that electrophoretic analysis obtains under the top condition that is obtained in (3), can be respectively from several different fingerprint fragments the spectrogram of the different places of production of obtain Caulis Et Caulis Acanthopanacis Senticosi is carried out visual analysis, directly debates knowledge.
(6) peak height of active component is determined the content of active component in the actual sample in contrast standard mixed solution and the actual sample.Active component isofraxidin, rutin and chlorogenic acid contents are all listed in the table 1 in the Caulis Et Caulis Acanthopanacis Senticosi in the different places of production.
The assay result of active component in the Caulis Et Caulis Acanthopanacis Senticosi in the different places of production of table 1
Figure C200610016926D00081
NF: expression does not detect.
By table 1 as seen, in the Caulis Et Caulis Acanthopanacis Senticosi in all places of production, all do not detect rutin, and isofraxidin also is different with chlorogenic acid contents for the Caulis Et Caulis Acanthopanacis Senticosi in the different places of production.Similar from the content of isofraxidin in the Caulis Et Caulis Acanthopanacis Senticosi in 5 constant virtues and Mudanjiang, and be the highest.On the contrary, be minimum from the content of isofraxidin in the Caulis Et Caulis Acanthopanacis Senticosi of Wudalianchi, have only 1/10th of 5 constant virtues, and the content of isofraxidin is 3/10ths of 5 constant virtues in the Caulis Et Caulis Acanthopanacis Senticosi of cap mountain.Chlorogenic acid contents is the highest in the Caulis Et Caulis Acanthopanacis Senticosi in Mudanjiang, and chlorogenic acid contents is minimum in the Caulis Et Caulis Acanthopanacis Senticosi of Wudalianchi.
Embodiment 2: the detection by quantitative of herbal medicine wilsonii different parts and fingerprint discriminatory analysis
(1) preparation of buffer solution, its concrete grammar is:
Directly mix NaH 2PO 4And Na 2B 4O 7Aqueous solution, the pH value is respectively 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0 solution 0.20mol L -1HCl or 0.20mol L -1NaOH regulates.
(2) preparation of standard solution and cyclic voltammetry experiment thereof, process is as follows:
The preparation of standard solution: concentration is 5.0 * 10 -3Mol L -1The solution of isofraxidin, rutin and chlorogenic acid is used dissolve with methanol respectively, and lays in 4 ℃ refrigerator, is diluted to desired concn with secondary water before using.
The cyclic voltammetry experiment of standard solution: at first with 33 μ m CFE electrodes at NaH 2PO 4And Na 2B 4O 7One week of scan round as a setting in the mixed solution.Then this electrode is being contained 1.0 * 10 respectively -4Mol L -1Isofraxidin, 1.0 * 10 -4Mol L -1Rutin and 1.0 * 10 -4Mol L -1The NaH of chlorogenic acid 2PO 4And Na 2B 4O 7In one week of scan round in the mixed solution, investigate their cyclic voltammetric behavior.
(3) optimization of testing conditions, and under this optimal conditions, carry out the investigation of reappearance, detectability and the range of linearity;
The detailed process of testing conditions optimization is:
Change-detection current potential, 5.0 * 10 between 0.45-1.35V -3-8.5 * 10 -3Mol L -1With 5.0 * 10 -3-9.0 * 10 -3Mol L -1Between change NaH 2PO 4And Na 2B 4O 7Concentration, 5.0-9.0 between change to change between pH value of buffer solution, 8-35s and change separation voltage between sample injection time and 5-17.5kV and investigate influence peak current, transit time, degree of separation and separation efficiency.
Under the separation and testing conditions of the best: the detection current potential is 1.20V; Buffer solution is 7.0 * 10 -3MolL -1NaH 2PO 4+ 7.5 * 10 -3Mol L -1Na 2B 4O 7(pH=7.0); 17cm height sample introduction 25s; Separation voltage is 15kV, reaches baseline separation and has high sensitivity at 6min inner analysis thing isofraxidin, rutin and chlorogenic acid.
Under the described top condition, tested the series of standards mixed solution in the above, concentration range is from 1.0 * 10 -7To 1.0 * 10 -4Mol L -1Isofraxidin, 2.0 * 10 -7To 1.0 * 10 -4Mol L -1Rutin and 1.5 * 10 -6To 5.0 * 10 -5Mol L -1Chlorogenic acid.Obtained crossing over the range of linearity of 1-3 the order of magnitude, related coefficient surpasses 0.99.Adopt this CE-ED method actual detected to the detectability of isofraxidin, rutin and chlorogenic acid be respectively 1.0 * 10 -7Mol L -1, 2.0 * 10 -7Mol L -1With 1.5 * 10 -6Mol L -1(signal to noise ratio (S/N ratio) is 3).
Under top condition, advance the standard biased sample continuously: 1.0 * 10 -5Mol L -1Isofraxidin, 2.0 * 10 -5Mol L -1Rutin and 2.0 * 10 -5Mol L -1Chlorogenic acid, the reappearance in a few days of investigation the method.Continuous 6 sample introductions, the RSDs of their peak current and transit time (n=6) value is respectively 1.5,0.63%, 2.4,0.69% and 1.2,0.70%.
(4) preparation of herbal medicine sample solution, detailed process is:
Pulverize the herbal medicine sample with the plant comminutor that has 40 mesh sieve, take by weighing the herbal medicine sample of 0.5g, with the ultrasonic 20min of methyl alcohol water-bath, inclining afterwards supernatant, and this extraction process repeats 2 times again.At last, the extract decompression distillation is closely dried, uses dissolve with methanol residue and constant volume in the methyl alcohol of 5mL.Before the experiment, all sample solutions are all with the cellulose esters membrane filtration of 0.22 μ m and be diluted to needed concentration direct injection analysis.
(5) acquisition of wilsonii different parts CE-ED finger-print;
The acquisition of wilsonii different parts CE-ED finger-print is to carry out (as shown in Figure 2) that electrophoretic analysis obtains under the top condition that is obtained in (3), can be respectively from several different fingerprint fragments the spectrogram of acquisition wilsonii different parts is carried out visual analysis, directly debates knowledge.
(6) peak height of active component is determined the content of active component in the actual sample in contrast standard mixed solution and the actual sample, and active component isofraxidin, rutin and chlorogenic acid contents are all listed in the table 2 in the different parts of herbal medicine wilsonii.
The measurement result of active component content in the table 2 herbal medicine wilsonii different parts
NF: expression does not detect.
By table 2 as seen, for the different parts of herbal medicine wilsonii, the content of analyte also is visibly different.Only having detected rutin in the leaf of wilsonii, is comparable in the content of isofraxidin and the rhizome in the stem, and is in the leaf ten times.And be similar in the content of isofraxidin and the root in the leaf.Chlorogenic acid contents is the highest in the root, is minimum in the leaf.

Claims (1)

1. the detection method of a wilsonii is characterized in that the capillary electrophoresis electrochemical method is used for fingerprint differentiates the wilsonii in the different places of production and the different parts of wilsonii, and its step and condition are as follows:
(1) preparation of buffer solution
Directly mix NaH 2PO 4And Na 2B 4O 7Aqueous solution, and use 0.20mol L -1HCl or 0.20molL -1NaOH regulates its pH value and is 5.0-9.0;
(2) preparation and the cyclic voltammetry experiment thereof of active component standard solution in the herbal medicine wilsonii
The main active of herbal medicine wilsonii is isofraxidin, chlorogenic acid and rutin, and they become concentration with dissolve with methanol respectively is 3-8mol L -1Solution;
The cyclic voltammetry experiment process of active component standard solution is:
With 33 μ m carbon fiber microdisk electrodes (CFE) at the NaH that contains isofraxidin, chlorogenic acid and rutin respectively 2PO 4And Na 2B 4O 7Its cyclic voltammetric behavior is investigated in scan round in the mixed solution;
(3) optimization of testing conditions, and under this optimal conditions, carry out the investigation of reappearance, detectability and the range of linearity
The optimizing process of testing conditions is: change-detection current potential, 5.0 * 10 between 0.45-1.35V -3-8.5 * 10 -3Mol L -1With 5.0 * 10 -3-9.0 * 10 -3Mol L -1Between change NaH 2PO 4And Na 2B 4O 7Concentration, 5.0-9.0 between change to change between pH value of buffer solution, 8-35s and change separation voltage between sample injection time and 5-17.5kV and investigate influence peak current, transit time, degree of separation and separation efficiency; The top condition that obtains is: the detection current potential is that 1.20V, buffer solution are 7.0 * 10 -3Mol L -1NaH 2PO 4+ 7.5 * 10 -3Mol L -1Na 2B 4O 7(pH=7.0), 17cm height sample introduction 25s, separation voltage are 15kV;
(4) preparation of herbal medicine sample solution
Pulverize the herbal medicine sample with the plant comminutor that has 20-80 mesh sieve, take by weighing the herbal medicine sample of 0.3-0.8g, with the ultrasonic 15-30min of methyl alcohol water-bath, inclining afterwards supernatant, and this extraction process repeats 1-3 time again; At last, the extract decompression distillation is closely dried, and in the methyl alcohol of 3-8mL, before the operation, all herbal medicine sample solutions are all with the cellulose esters membrane filtration of 0.22 μ m and be diluted to needed concentration direct injection analysis with dissolve with methanol residue and constant volume;
(5) acquisition of wilsonii CE-ED finger-print
The acquisition of wilsonii CE-ED finger-print is to carry out electrophoretic analysis under the top condition that is obtained to obtain in step (3);
(6) peak height of active component is determined the content of active component in the wilsonii actual sample in contrast wilsonii standard mixed solution and the wilsonii actual sample;
(7) contrast the CE-ED finger-print that is obtained, differentiate the different parts of herbal medicine wilsonii and the Caulis Et Caulis Acanthopanacis Senticosi in the different places of production from several different fingerprint fragments respectively.
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CN101813626B (en) * 2009-08-26 2011-09-07 黑龙江大学 Method for simultaneously measuring arsenic and stibium in Chinese medicinal manyprickle acathopanax root
CN104407037A (en) * 2014-12-12 2015-03-11 广西科技大学 Method for separating and measuring chlorogenic acid through large-volume sample injection-reverse current flow accumulation-capillary zone electrophoresis
CN105067683A (en) * 2015-03-16 2015-11-18 查孝柱 Electrochemical fingerprint identification of traditional Chinese medicine pawpaw

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CN1369704A (en) * 2001-02-16 2002-09-18 中国科学院长春应用化学研究所 Method for testing quality of acanthopanax leaf

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Publication number Priority date Publication date Assignee Title
CN1369704A (en) * 2001-02-16 2002-09-18 中国科学院长春应用化学研究所 Method for testing quality of acanthopanax leaf

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Title
刺五加及其注射液中槲皮素的含量测定. 陈勇川等.中国药房，第12卷第3期. 2002 *

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