CN101744877B - Selfheal quality control method - Google Patents
Selfheal quality control method Download PDFInfo
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- CN101744877B CN101744877B CN2010100195730A CN201010019573A CN101744877B CN 101744877 B CN101744877 B CN 101744877B CN 2010100195730 A CN2010100195730 A CN 2010100195730A CN 201010019573 A CN201010019573 A CN 201010019573A CN 101744877 B CN101744877 B CN 101744877B
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Abstract
The invention discloses a selfheal quality control method which is a high performance liquid chromatography and a thin layer chromatography the marker components of which contain compound salviaflaside. The high performance liquid chromatography comprises the following steps: preparation of comparison products: carbinol is used to dissolve the salviaflaside compound so as to prepare the comparison product solution of the salviaflaside compound; preparation of test product solution : selfheal medicinal material powder is taken to be precisely weighed, added with carbinol, and filtered by a microfiltration membrane to be used as the test product solution; and injection to a liquid chromatograph, determination and mobile phase: elution by acetonitrile acetic acid liquid. The thin layer chromatography comprises the following steps: rosmarinic acid of the comparison product solution and salviaflaside solution and the test product solution are respectively absorbed to be sampled on the same thin board, chloroform:carbinol:formic acid or chloroform: water are taken as developers, developing, taking and drying are carried out, vanillin concentrated sulphuric acid is used for developing, and heating is carried out until spots are clear. The method is simple and effective, and thereby preventing illegal producers from using roots, stems, leaves and other non-medical parts to substitute the cluster parts of the selfheal to be used for the medical purpose.
Description
Technical field
The invention belongs to the field of Chinese medicines, specifically relate to the selfheal method of quality control.
Background technology
Selfheal is the dry fruit ear of labiate selfheal Prunella vulgaris L..Have and relieve inflammation or internal heat, make eye bright, dissipating bind, the effect of detumescence is used for red eye, swell pain, order pearl nyctalgia, it is dizzy to have a headache, scrofula, goiter and tumor, acute mastitis swells and ache; Thyromegaly, scrofula, the proliferation of mammary gland, hypertension.
Ursolic acid, oleanolic acid, Rosmarinic acid, caffeic acid all are principal ingredients of selfheal, at different parts such as the root of selfheal, stem, leaf, fruit ears certain content are arranged all.
" medicinal part of 2005 editions regulation selfheals of Chinese pharmacopoeia is a fruit ear, and the quality control index of employing is a ursolic acid.Because ursolic acid does not exist only in fruit ear, also be present in positions such as root, stem, leaf simultaneously, illegal manufacturer is to this phenomenon, and root, stem and the leaf site of employing selfheal serve as the fruit ear position and are used as medicine, and cause the quality of prunella spike finished product to can not get guaranteeing.[content assaying method of caffeic acid and Rosmarinic acid research in the selfheal different parts, Zhang Lanzhen etc., Beijing University of Chinese Medicine's journal, 30 (5), 343-345] adopts caffeic acid, Rosmarinic acid as quality control index; These compositions are present in the different parts such as the root, stem, leaf, fruit ear of selfheal equally, can not be as " medicinal part--the characteristic chemical constituent of fruit ear of the selfheal of Chinese pharmacopoeia regulation.
Compound salviaflaside is water-soluble extremely strong, is light yellow amorphous powder, and ferric trichloride-potassium ferricyanide and bromophenol blue colour developing are all positive, and vanillin shows pink, is caffeoyl depside glycosides compound, and its main pharmacologically active does not appear in the newspapers.[Zhao, Lin Min; He, Wen Yi; Liang, Xiao Tian; Li; Lian Niang.Salviaflaside and salviaflaside methyl ester two new depsidic glycosides from Salvia flava.Chinese Chemical Letters (1996), 7 (5), 449-452.] the employing alcohol extract; It is water-soluble to concentrate the back; Water extracts then, and the way of precipitation with alcohol is removed macromolecular compound such as proteinpolysaccharide, after the solution concentration with CHC13, EtOAc and three parts of n-BuOH.The positive liquor-saturated part of fourth contains phenolic acid, should use the SiO2 dry chromatography, and adopt chloroform: methyl alcohol: formic acid=75: 25: 2 wash-outs, Sephadex-LH20 obtains compound salviaflaside repeatedly.The structural formula of compound salviaflaside is following:
Summary of the invention
The method of quality control that the purpose of this invention is to provide a kind of selfheal.Prevent that position such as root, stem, leaf with selfheal from serving as the fruit ear position and being used as medicine.
The technical scheme that realizes above-mentioned purpose is following:
A kind of selfheal method of quality control may further comprise the steps: detect in the selfheal medicinal material whether contain compound salviaflaside according to high performance liquid chromatography and/or thin-layered chromatography.
Salviaflaside is different from the characteristic chemical constituent of root, stem, leaf as the selfheal fruit ear, can be used as the index property composition of selfheal quality control, is used as medicine in case illegal people serves as the selfheal fruit ear with other positions of selfheal.
Preferably, may further comprise the steps according to high performance liquid chromatography: the preparation of (1) reference substance solution: precision takes by weighing the salviaflaside compound, uses dissolve with methanol, is configured to salviaflaside compound reference substance solution;
(2) preparation of need testing solution: get the selfheal medicinal powder, the accurate title, decide, and adds methyl alcohol, weighs, and ultrasonic 15～60min takes out, and leaves standstill, and cools, and benefit is heavy, and miillpore filter filters, as need testing solution
(3) accurate respectively reference substance solution and the need testing solution drawn injects liquid chromatograph, measures chromatographic condition: flow velocity: 0.5～1.5mL.min
-1Detect wavelength: 310～330nm; Column temperature: 25～40 ℃;
Moving phase: acetonitrile-acetic acid water elution.
Preferably, described moving phase is acetonitrile: (volume ratio is 10～20 to 1.0% acetic acid water: 90～80).
Except separately with the quality control index of salviaflaside as selfheal; Can also increase other index property compositions that contain in the selfheal fruit ear; Like Rosmarinic acid, ursolic acid, oleanolic acid, caffeic acid, together as the method for selfheal quality control; Can selfheal fruit ear and other positions of selfheal be differentiated so that formed, again can integrating representation selfheal quality method of quality control just.
Preferably; With salviaflaside and Rosmarinic acid jointly as the index property composition of selfheal quality control; The preparation of the reference substance solution in the described step (1) increases the preparation of Rosmarinic acid reference substance solution; Be specially: precision takes by weighing the salviaflaside compound, uses dissolve with methanol, is configured to salviaflaside compound reference substance solution; Precision takes by weighing the Rosmarinic acid reference substance, uses dissolve with methanol, is configured to the Rosmarinic acid reference substance solution; Moving phase described in the step (3) is: acetonitrile-1.0% acetic acid water system gradient elution, preferably, the gradient elution step is: 0～30min, acetonitrile are by (10～20) %～(20～30) %, and 1.0% acetic acid water is by (90～80) %～(80～70) %.
The finger-print that can also salviaflaside and Rosmarinic acid be used for the selfheal quality control.Moving phase is: acetonitrile-1.0% acetic acid water system gradient elution; The gradient elution step is: 0～10min, acetonitrile are that 5%, 1.0% acetic acid water is 95%; 10～20min, acetonitrile are by 5～8.6%, and 1.0% acetic acid water is by 95～91.5%; 20～45min, acetonitrile are by 8.6～17.6%, and 1.0% acetic acid water is by 91.5～82.4%; 45～70min, acetonitrile are by 17.6～25.1%, and 1.0% acetic acid water is by 82.4～74.9%; 70～80min, acetonitrile are by 25.1～32.1%, and 1.0% acetic acid water is by 74.9～67.9%; 80～90min, acetonitrile are by 32.1～37.1%, and 1.0% acetic acid water is by 67.9～62.9%; In the finger-print that is obtained by above method 8 characteristic peaks are arranged, the retention time of each characteristic peak is respectively: 8.0～8.5min, 17～17.5min, 26～27min, 33～34min, 46～46.5min, 46.6～47.5min, 49.5～50.5min, 58～59min; Wherein retention time is that the chromatographic peak of 49.5～50.5min, 58～59min is respectively salviaflaside and Rosmarinic acid.
In above step, preferably, need testing solution is prepared as in the described step (2): get the selfheal medicinal powder, accurate title is fixed, adds 10 times of amount methyl alcohol, weighs, and ultrasonic 30min takes out, and leaves standstill, and cools, and mends to weigh, and miillpore filter filters, as need testing solution;
Preferably, chromatographic condition is in the described step (3): sampling volume: 10 μ L, flow velocity: 1.1mL.min
-1Detect wavelength: 319nm; Column temperature: 30 ℃; Moving phase: acetonitrile-1.0% acetic acid water system gradient elution.
Preferably, the concentration of preparation salviaflaside compound reference substance solution is 0.005～0.05 μ g/ μ L, and Rosmarinic acid reference substance concentration is 0.03～1 μ g/ μ L.
The content that the employing high performance liquid chromatography records compound salviaflaside in the selfheal medicinal material is more than 0.1 ‰.
Said selfheal method of quality control also comprises the selfheal medicinal material is carried out the thin-layered chromatography discrimination method, mainly may further comprise the steps:
(1) preparation of reference substance solution: get reference substance Rosmarinic acid and compound salviaflaside respectively, add dissolve with methanol, be mixed with Rosmarinic acid and salviaflaside reference substance solution;
(2) preparation of need testing solution: get the selfheal medicinal powder, the accurate title, decide, and adds methyl alcohol, weighs, and ultrasonic 15～60min takes out, and leaves standstill, and cools, and benefit is heavy, filters, as need testing solution;
(3) point sample, expansion, colour developing: draw reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution point sample respectively on same thin layer plate; With chloroform: methyl alcohol: formic acid or chloroform: methyl alcohol: water is developping agent; Launch, take out, dry; With the colour developing of the vanillic aldehyde concentrated sulphuric acid, be heated to clear spot.
Preferably, being prepared as of described need testing solution: get the selfheal medicinal powder, accurate title is fixed, adds 10 times of amount methyl alcohol, weighs, and ultrasonic 30min takes out, and leaves standstill, and cools, and mends to weigh, and filters, as need testing solution;
Preferably, described thin layer plate is that sodium carboxymethyl cellulose is the silica gel g thin-layer plate of binder; Developer is the 1% vanillic aldehyde concentrated sulphuric acid, and developping agent is a chloroform: methyl alcohol: formic acid=7: 3: 0.5 or chloroform: methyl alcohol: water=6: 4: 1.
Preferably, the concentration of Rosmarinic acid and compound salviaflaside reference substance solution is respectively 0.1～1.0mg/mL and 0.01～0.1mg/mL.
In described method, dissolving reference substance and test sample prepare used methyl alcohol can change the ethanol equal solvent with the dissolve with methanol similar performance into.
The inventor draws through long-term experimental analysis; The salviaflaside compound only is present in the selfheal fruit ear, is the characteristic chemical constituent of selfheal fruit ear, in the art; From the selfheal medicinal material, isolate the salviaflaside compound first; Thus, through further technology exploration, make the method for quality control of selfheal of the present invention; This method is more succinct effectively, is used as medicine thereby avoid the illegal producer to use the dis-medicinal parts such as root, stem, leaf of selfheal to serve as the fruit ear position.
Description of drawings
Fig. 1 is the HPLC figure of selfheal root, stem, leaf and fruit ear constituent analysis, and wherein peak 1 is salviaflaside.
Fig. 2 is to be the selfheal quality control HPLC1 figure of index components with salviaflaside, and wherein peak 1 is salviaflaside.
Fig. 3 is to be the selfheal method of quality control HPLC figure of index components jointly with salviaflaside and Rosmarinic acid, and wherein peak 1 is salviaflaside, and peak 2 is Rosmarinic acids.
Fig. 4 is salviaflaside and the Rosmarinic acid HPLC figure in the selfheal fruit ear of the different places of production, and wherein peak 1 is salviaflaside, and peak 2 is Rosmarinic acids.
Fig. 5 is the finger-print of selfheal medicinal material.
Fig. 6 is the contrast collection of illustrative plates of selfheal medicinal material, and this collection of illustrative plates is to be the basis at the chromatographic fingerprinting with selfheal standard medicinal material, the collection of illustrative plates that obtains through chromatographic fingerprints of Chinese materia medica similarity evaluation system handles.
Fig. 7 is the thin-layer chromatogram of selfheal root, stem, leaf and fruit ear.
Fig. 8 is the thin-layer chromatogram of different places of production selfheal fruit ear.
Embodiment
The present invention is through adopting HPLC-DAD; LC-MSn carries out systematic comparison research to the chemical constitution at selfheal fruit ear, leaf, stem, four positions of root; Seek out the characteristic compound in the selfheal fruit ear,, adopt various modern chromatographic separation technologies according to the possible structure and the character of this compound; This compound is separated preparation; Obtain the monomer component of this compound, through spectroscopic techniques such as ultraviolet, infrared, mass spectrum, nuclear-magnetisms, the physicochemical property of binding compounds is identified the architecture formula (I) of this compound.[Zhao, Lin Min; He, Wen Yi; Liang, Xiao Tian; Li; Lian Niang.Salviaflaside and salviaflaside methylester two new depsidic glycosides from Salvia flava.Chinese Chemical Letters (1996); 7 (5), 449-452.] show this structural formula compound called after salviaflaside.This structural formula (I) compound exists only in the selfheal fruit ear at each position of selfheal, is not present in other positions such as root, stem, leaf of selfheal.This compound can separate from the selfheal fruit ear through following steps and obtains:
(1) extract: get the selfheal medicinal material, measure 50～70% alcohol reflux 1～3 time with 4～8 times, each 1～2h leaches soup after the cooling, be recycled to dried;
(2) separate: will reclaim the samples with water dissolving of doing, and use petroleum ether extraction, and discard extract; Water section continues to use chloroform extraction, discards extract again; Then water section is concentrated into dried; 60～200 order silica gel mixed samples, 5～15), chloroform 1～2), chloroform last silicagel column is respectively with polarity solution chloroform, chloroform from low to high: methyl alcohol (10～6:: methyl alcohol: water (60～70: 30～40:: methyl alcohol: water (50～70: 35～45: 5～15) towards post; Obtain Part1; Part2, Part3, Part4 part;
(3) purifying: the sample of Part4 part is dissolved with methanol-water, last ODS post, use methyl alcohol: (30～60: 70～40) wash-out obtains the pure article of compound of structural formula (I) to water.
Preferred separating step is following:
(1) extract: get the selfheal medicinal material, with 5 times of amount 60% alcohol reflux 2 times, 1h leaches soup after the cooling at every turn, is recycled to dried;
(2) separate: will reclaim the samples with water dissolving of doing, and use petroleum ether extraction, and discard extract; Water section continues to use chloroform extraction, discards extract again; Then water section is concentrated into dried, 60～100 order silica gel mixed samples, last silicagel column; Use chloroform, chloroform respectively: methyl alcohol (9: 1), chloroform: methyl alcohol: water (65: 35: 10), chloroform: methyl alcohol: water (60: 40: 10) is towards post; Obtain Part1, Part2, Part3, Part4 part;
(3) purifying: the sample of Part4 part is dissolved with methanol-water, last ODS post, use methyl alcohol: water (50: 50) wash-out obtains the pure article of compound of structural formula (I) salviaflaside.
Structural formula (I) salviaflaside compounds content is greater than 50%, especially greater than 95% in the pure article of the compound of above separation method gained.
Below in conjunction with accompanying drawing and embodiment this aspect is elaborated.
The constituent analysis of embodiment 1 selfheal different parts
1, test sample preparation: divide the powder 1g at another name selfheal fruit ear, leaf, stem and four positions of root, accurate title is fixed, adds methyl alcohol 10mL, and ultrasonic 30min (power 250W, frequency 40kHz) takes out, and leaves standstill, and cools, and 0.22 μ m miillpore filter filters, and supplies the HPLC analysis.
2, chromatographic condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm * 250mm, 5 μ m); Flow velocity: 1.0mL.min
-1Detect wavelength: 290nm; Column temperature: 30 ℃; Sampling volume: 10 μ L; Moving phase: acetonitrile-water (containing 1.0v/v% acetic acid) system's gradient elution is seen table 1.
Table 1 flow phase system gradient
3, result: Fig. 1 result shows that chromatographic peak (I) compound salviaflaside exists only in the selfheal fruit ear, is not present in root, stem, the leaf site, and be the characteristic chemical constituent of selfheal medicinal material (fruit ear).
The separation example 1 of characterization compound (I) salviaflaside in embodiment 2 selfheals
1, extract: the selfheal medicinal material, with 5 times of amount 60% alcohol reflux 2 times, 1h leaches soup after the cooling at every turn, is recycled to dried;
2, separate: will reclaim dried samples with water and pinch moltenly, and use petroleum ether extraction, and discard extract; Water section continues to use chloroform extraction, discards extract again.Then water section is concentrated into dried, 60～100 order silica gel mixed samples, last silicagel column; Use chloroform, chloroform respectively: methyl alcohol (9: 1), chloroform: methyl alcohol: water (65: 35: 10), chloroform: methyl alcohol: water (60: 40: 10) is towards post; Obtain Part1, Part2, Part3, Part4 part; 3, purifying: the sample of Part4 part is dissolved with methanol-water, last ODS post, use methyl alcohol: water (50: 50) wash-out, obtain the pure article of structural formula (I) salviaflaside compound, contain structural formula (I) salviaflaside 98% in these pure article.
The separation example 21 of characterization compound (I) salviaflaside in embodiment 3 selfheals, extract: get the selfheal medicinal material, with 5 times of amount 60% alcohol reflux 2 times, 1h leaches soup after the cooling at every turn, is recycled to dried;
2, separate: will reclaim dried samples with water and pinch moltenly, and use petroleum ether extraction, and discard extract; Water section continues to use chloroform extraction, discards extract again.Then water section is concentrated into dried; 60～200 order silica gel mixed samples, last silicagel column, respectively with polarity solution chloroform, chloroform from low to high: methyl alcohol (9: 1), chloroform: methyl alcohol: water (65: 35: 10), chloroform: methyl alcohol: water (60: 40: 10) is towards post; Obtain Part1; Part2, Part3, Part4 part;
3, purifying: the sample of Part4 part is dissolved with methanol-water, last ODS post, use methyl alcohol: water (60: 40) wash-out, obtain the pure article of compound of structural formula (I) salviaflaside, contain structural formula (I) salviaflaside 80% in these pure article.
1, reference substance solution preparation: the salviaflaside that precision takes by weighing embodiment 2 moderate purities 98%, use dissolve with methanol, being configured to concentration is the salviaflaside reference substance solution of 0.03 μ g/ μ L.
2, need testing solution preparation: get selfheal fruit ear powder 1g, precision is claimed fixed, adds methyl alcohol 10mL, weighs, and ultrasonic 30min takes out, and leaves standstill, and cools, and mends to weigh, and 0.22 μ m miillpore filter filters, and HPLC analyzes.
3, accurate respectively reference substance solution and the need testing solution drawn injects liquid chromatograph, measures chromatographic condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm * 250mm, 5 μ m); Flow velocity: 1.0mL.min
-1Detect wavelength: 319nm; Column temperature: 30 ℃; Sampling volume: 10 μ L; Moving phase: acetonitrile-1.0% acetic acid water (volume ratio 15: 85).
4, the drafting of typical curve: accurate respectively absorption strength is salviaflaside reference substance solution 1,2,5,10,15, the 20 μ L of 0.03 μ g/ μ L, the peak area that record is measured by above-mentioned chromatographic condition.With the peak area is ordinate (Y), and sample size (μ g) is horizontal ordinate (X), gets regression equation, Y=9E+06X+21395, r=0.9999, range of linearity 0.033664-0.67328 μ g.
5, precision test: according to " method of Chinese pharmacopoeia appendix record, the accurate absorption of difference supplies same need testing solution 10 μ L, continuous sample introduction 6 times, by the peak area of above-mentioned chromatographic condition mensuration salviaflaside, its RSD is 0.16%.
6, stability test: according to " method of Chinese pharmacopoeia appendix record, the accurate absorption supplies same need testing solution 10 μ L, and respectively 2,4,6,8,10,12, the 24h sample introduction is pressed the peak area that above-mentioned chromatographic condition is measured salviaflaside, and its RSD is 0.18%.
7, replica test: according to " method of Chinese pharmacopoeia appendix record is got 6 parts on selfheal fruit ear, and accurate the title decides, and the preparation method prepares need testing solution according to test sample, presses the peak area that above-mentioned chromatographic condition is measured salviaflaside, and its RSD is 0.72%.
8, recovery test: according to " method of Chinese pharmacopoeia appendix record is got the about 0.5g of medicinal powder of 6 parts of known content, and accurate the title decides; The accurate respectively salviaflaside reference substance solution that adds 0.1mg; Preparation method according to need testing solution processes the application of sample need testing solution, measures content, calculate recovery rate by above-mentioned chromatographic condition; Average recovery rate is 98.18% as a result, and RSD is 0.25%.
1, reference substance solution preparation: the salviaflaside that precision takes by weighing embodiment 2 moderate purities 98%, use dissolve with methanol, being configured to concentration is the salviaflaside reference substance solution of 0.005 μ g/ μ L.
2, need testing solution preparation: get selfheal fruit ear powder 1g, precision is claimed fixed, adds methyl alcohol 10mL, weighs, and ultrasonic 15min takes out, and leaves standstill, and cools, and mends to weigh, and 0.22 μ m miillpore filter filters, and HPLC analyzes.
3, chromatographic condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm * 250mm, 5 μ m); Flow velocity: 1.5mL.min
-1Detect wavelength: 319nm; Column temperature: 40 ℃; Sampling volume: 10 μ L; Moving phase: acetonitrile-1.0% acetic acid water (volume ratio 10: 90).
Other steps are with embodiment 4.
1, reference substance solution preparation: the salviaflaside that precision takes by weighing embodiment 2 moderate purities 98%, use dissolve with methanol, being configured to concentration is the salviaflaside reference substance solution of 0.3 μ g/ μ L.
2, need testing solution preparation: get selfheal fruit ear powder 1g, precision is claimed fixed, adds methyl alcohol 10mL, weighs, and ultrasonic 60min takes out, and leaves standstill, and cools, and mends to weigh, and 0.22 μ m miillpore filter filters, and HPLC analyzes.
3, chromatographic condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm * 250mm, 5 μ m); Flow velocity: 0.5mL.min
-1Detect wavelength: 310nm; Column temperature: 25 ℃; Sampling volume: 10 μ L; Moving phase: acetonitrile-1.0% acetic acid water (volume ratio 20: 80).
Other steps are with embodiment 4.
The method of employing embodiment 4,5,6 is measured the content of salviaflaside in the selfheal of the national different places of production, and the result sees table 2.
The content of salviaflaside in the selfheal of the different places of production of table 2
Fig. 2 is to be the selfheal quality control HPLC figure of index components with salviaflaside, and wherein peak 1 is salviaflaside.The result of table 2 show the content of salviaflaside in selfheal all ten thousand/more than, and the content assaying method of setting up simple, be prone to repetition, can be used as one of selfheal method of quality control.
1, reference substance solution preparation: the salviaflaside that precision takes by weighing embodiment 2 moderate purities 98%, use dissolve with methanol, being configured to concentration is 0.3 μ g/ μ L, dilution is 0.03 μ g/ μ L and 0.005 μ g/ μ L again; It is an amount of that precision takes by weighing Rosmarinic acid, and being configured to concentration is 0.47 μ g/ μ L, and dilution is 0.047 μ g/ μ L again.
2, need testing solution preparation: get the selfheal fruit ear powder 1g in the different places of production, precision is claimed fixed, adds methyl alcohol 10mL, weighs, and ultrasonic 30min takes out, and leaves standstill, and cools, and mends to weigh, and 0.22 μ m miillpore filter filters, and HPLC analyzes.
3, chromatographic condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm * 250mm, 5 μ m); Flow velocity: 1.1mL.min
-1Detect wavelength: 319nm; Column temperature: 30 ℃; Sampling volume: 10 μ L; Moving phase: acetonitrile-1.0% acetic acid water system gradient elution, gradient is 0～30min, and acetonitrile is by 10～20%, and 1.0% acetic acid water is by 90～80%.
4, the drafting of typical curve
Salviaflaside: accurate respectively absorption strength is reference substance solution 2,5,10,15, the 20 μ L of 0.3 μ g/ μ L; Concentration is reference substance solution 1,2,5,10,15, the 20 μ L of 0.03 μ g/ μ L; Concentration is reference substance solution 1,2,3,5,10,15, the 20 μ L of 0.005 μ g/ μ L, the peak area that record is measured by above-mentioned chromatographic condition.
Rosmarinic acid: accurate respectively absorption strength is reference substance solution 1,2,3,5,10,15, the 20 μ L of 0.47 μ g/ μ L and reference substance solution 1,2,3,5,10,15, the 20 μ L that concentration is 0.047 μ g/ μ L, the peak area that record is measured by above-mentioned chromatographic condition.
With the peak area is ordinate (Y), and sample size (μ g) is horizontal ordinate (X), gets regression equation, the regression equation Y=9E+06X-32842 of result: salviaflaside, related coefficient 0.9999, range of linearity 0.0067-4.2 μ g; The regression equation Y=3E+06X-33026 of Rosmarinic acid, related coefficient 0.9999, range of linearity 0.047-9.4 μ g.
5, precision test: according to " the method for Chinese pharmacopoeia appendix record; Accurate respectively the absorption supplies same need testing solution 10 μ L; Continuous sample introduction 6 times, by the peak area of above-mentioned chromatographic condition mensuration salviaflaside and Rosmarinic acid, the RSD of the two is respectively: 0.26% and 0.42%.
6, stability test: according to " method of Chinese pharmacopoeia appendix record, the accurate absorption supplies same need testing solution 10 μ L, respectively 2; 4,6,8; 10,12, the 24h sample introduction; By the peak area of above-mentioned chromatographic condition mensuration salviaflaside and Rosmarinic acid, the RSD of the two is respectively: 0.21% and 0.33%.
7, replica test: according to " the method for Chinese pharmacopoeia appendix record; Get 6 parts of same duplicate samples; The accurate title, decide; The preparation method prepares need testing solution according to test sample, and by the peak area of above-mentioned chromatographic condition mensuration salviaflaside and Rosmarinic acid, the RSD of the two is respectively: 1.86% and 1.82%.
8, recovery test: according to " method of Chinese pharmacopoeia appendix record is got 6 parts of about 0.5g of selfheal fruit ear powder, and accurate the title decides; Accurate respectively salviaflaside and the 0.47mg Rosmarinic acid reference substance solution that adds 0.1mg; Process the application of sample need testing solution, measure content, calculate recovery rate by above-mentioned chromatographic condition; The average recovery rate of salviaflaside and Rosmarinic acid is 99.99% and 100.2% as a result, and RSD is respectively 1.13 and 1.64.
Method of quality control in embodiment 8 selfheals
1, reference substance solution preparation: the salviaflaside that precision takes by weighing embodiment 2 moderate purities 98%, use dissolve with methanol, being configured to concentration is 0.3 μ g/ μ L, dilution is 0.03 μ g/ μ L and 0.005 μ g/ μ L again; It is an amount of that precision takes by weighing Rosmarinic acid, and being configured to concentration is 0.3 μ g/ μ L, and dilution is 0.03 μ g/ μ L again.
2, need testing solution preparation: get the selfheal fruit ear powder 1g in the different places of production, precision is claimed fixed, adds methyl alcohol 20mL, weighs, and ultrasonic 15min takes out, and leaves standstill, and cools, and mends to weigh, and 0.22 μ m miillpore filter filters, and HPLC analyzes.
3, chromatographic condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm * 250mm, 5 μ m); Flow velocity: 1.5mL.min
-1Detect wavelength: 325nm; Column temperature: 30 ℃; Sampling volume: 10 μ L; Moving phase: acetonitrile-1.0% acetic acid water system gradient elution, gradient is 0～30min, and acetonitrile is by 15～25%, and 1.0% acetic acid water is by 85～75%.
Other steps are with embodiment 7.
In embodiment 9 selfheals in the method for quality control
1, reference substance solution preparation: the salviaflaside that precision takes by weighing embodiment 2 moderate purities 98%, use dissolve with methanol, being configured to concentration is 0.3 μ g/ μ L, dilution is 0.03 μ g/ μ L and 0.005 μ g/ μ L again; It is an amount of that precision takes by weighing Rosmarinic acid, and being configured to concentration is 1 μ g/ μ L, and dilution is 0.1 μ g/ μ L again.
2, need testing solution preparation: get the selfheal fruit ear powder 1g in the different places of production, precision is claimed fixed, adds methyl alcohol 10mL, weighs, and ultrasonic 45min takes out, and leaves standstill, and cools, and mends to weigh, and 0.22 μ m miillpore filter filters, and HPLC analyzes.
3, chromatographic condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm * 250mm, 5 μ m); Flow velocity: 1.5mL.min
-1Detect wavelength: 325nm; Column temperature: 30 ℃; Sampling volume: 10 μ L; Moving phase: acetonitrile-1.0% acetic acid water system gradient elution, gradient is 0～30min, and acetonitrile is by 20～30%, and 1.0% acetic acid water is by 80～70%.
Other steps are with embodiment 7.
Adopt the method for embodiment 7,8,9 to measure the content of salviaflaside and Rosmarinic acid in the selfheal of the different places of production, the result sees table 3.
The content of compound and Rosmarinic acid in the selfheal of the different places of production of table 3
Fig. 3 is to be the selfheal method of quality control HPLC figure of index components jointly with salviaflaside and Rosmarinic acid, and peak 1 is salviaflaside, and peak 2 is Rosmarinic acids.Fig. 4 is salviaflaside and the Rosmarinic acid HPLC figure in the selfheal fruit ear of the different places of production, and wherein peak 1 is salviaflaside, and peak 2 is Rosmarinic acids.The content that the result of table 3 shows salviaflaside wherein all ten thousand/more than, the content of Rosmarinic acid is almost more than one of percentage.The two is the principal ingredient in the selfheal fruit ear, can form the selfheal method of quality control jointly.
Method of quality control in embodiment 10 selfheals
1, reference substance solution preparation: reference substance solution among the embodiment 7.
2, need testing solution preparation: get the about 1g of selfheal fruit ear powder, precision is claimed fixed, adds methyl alcohol 10mL, weighs, and ultrasonic 30min takes out, and leaves standstill, and cools, and mends to weigh, and 0.22 μ m miillpore filter filters, and HPLC analyzes
3, chromatographic condition: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm * 250mm, 5 μ m); Flow velocity: 1.0mL.min
-1Detect wavelength: 290nm; Column temperature: 30 ℃; Sampling volume: 10 μ L; Moving phase: acetonitrile-1.0% acetic acid water system gradient elution is seen table 4.
Table 4 flow phase system gradient
4, precision: prepare the test sample portion according to the test sample preparation method; According to above chromatographic condition; Continuous sample introduction 6 times is according to " method of Chinese pharmacopoeia appendix record, the precision of calculating retention time and peak area; The result shows that the precision of this method meets the requirements, and its RSD value is less than 5%.The result sees table 5, table 6.
The precision of table 5 method (retention time)
The precision of table 6 method (peak area)
5, reappearance: 6 parts of medicinal material need testing solutions of parallel preparation, carry out HPLC then and analyze, according to " the method for Chinese pharmacopoeia appendix record; With the principal ingredient peak area is standard; Investigation method reappearance, the result sees table 7, the reappearance that shows method is in error range.
The reappearance of table 7 method
6, stability: after being prepared into the medicinal material need testing solution according to the test sample preparation method, at room temperature place different time, carry out HPLC and analyze; Calculated by peak area with principal ingredient; Investigate stability of sample, the result sees table 8, shows that sample is stable at least in 48h.
The stability of table 8 method
7, the finger-print of selfheal medicinal material: according to above-mentioned chromatographic condition, the selfheal medicinal material in 18 places of production is processed the analysis of need testing solution sample introduction, obtain the finger-print of selfheal medicinal material.Have 8 chromatographic peaks in the finger-print of Fig. 5 as total peak.The chromatographic retention of these 8 chromatographic peaks is seen table 9, and the relative retention time at total peak is seen table 10, and the relative reservation area at total peak is seen table 11, and wherein No. 7 peaks are that 7, No. 8 peaks of salviaflaside chromatographic peak are Rosmarinic acid.
The retention time at the total peak of table 9 selfheal standard medicinal material
The relative retention time at the total peak of table 10 selfheal standard medicinal material
The relative reservation area at the total peak of table 11 selfheal standard medicinal material
9, with the processing of similarity software to the selfheal chromatographic fingerprinting: the chromatographic fingerprinting with 18 selfheal standard medicinal materials is the basis; The chromatographic fingerprints of Chinese materia medica similarity evaluation system that can issue through pharmacopeia; 8 main peaks are check point in the selection chromatogram; Set up the contrast collection of illustrative plates of selfheal medicinal material, see Fig. 6.The retention time of each characteristic peak is respectively: 8.0～8.5min, 17～17.5min, 26～27min, 33～34min, 46～46.5min, 46.6～47.5min, 49.5～50.5min, 58～59min; Wherein retention time is that No. 7 chromatographic peaks of 49.5～50.5min, No. 8 chromatographic peaks of 58～59min are respectively salviaflaside, Rosmarinic acid.
10, result
Adopt the method for HPLC to set up the chromatographic fingerprinting of selfheal; Presentation of results: the similarity between the selfheal in the different places of production is very big; Difference is less, and all contains No. 7 (characteristic peak in the selfheal fruit ear) and No. 8 (topmost chromatographic peak in the selfheal) chromatographic peaks.Wherein No. 8 peaks are Rosmarinic acid, are topmost compositions in the selfheal, also are the maximum chromatographic peaks of peak area in the dactylogram, thereby are the peaks that influences the quality of medicinal material most critical; Secondly No. 7 peaks are salviaflaside, are time big chromatographic peaks in the selfheal, also are the characteristic chromatographic peaks in the selfheal fruit ear, thereby also are the peaks that influences the quality of medicinal material most critical.The two is two peaks of decision selfheal quality of medicinal material most critical.The chromatographic process of setting up above is reasonable, can reflect the quality of selfheal medicinal material, can be used as the method for quality control of selfheal medicinal material.
Method of quality control in embodiment 11 selfheals
1, the preparation of reference substance solution: get reference substance Rosmarinic acid and salviaflaside respectively, add dissolve with methanol, be configured to concentration and be respectively 0.3074mg/mL and 0.03367mg/mL.
2, test sample preparation: get selfheal fruit ear, leaf, stem and the about 1g of root powder respectively, the accurate title, decide, and adds methyl alcohol 10mL, and ultrasonic 30min takes out, and leaves standstill, and cools, and filters.
Get the about 1g of selfheal fruit ear sample powder in the different places of production respectively, the accurate title, decide, and adds methyl alcohol 10mL, and ultrasonic 30min takes out, and leaves standstill, and cools, and filters.
3, point sample, expansion, colour developing: draw respectively reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution 15 μ L point samples in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose; With chloroform: methyl alcohol: formic acid (volume ratio 7: 3: 0.5) is developping agent; Take out when being expanded to the about 0.5cm in thin layer plate top; Dry, the colour developing of the 1% vanillic aldehyde concentrated sulphuric acid, 105 ℃ are heated to clear spot.
4, result: in the thin-layer chromatogram of Fig. 7 selfheal root, stem, leaf and fruit ear, 1-reference substance Rosmarinic acid; 2-reference substance salviaflaside; 3-selfheal fruit ear; 4-selfheal leaf; 5-selfheal stem; 6-selfheal root.The result shows to have only the selfheal fruit ear to have the spot of salviaflaside; Selfheal leaf, stem and root sample only show the spot of same color with the reference substance Rosmarinic acid on same position, and do not have corresponding spot with reference substance salviaflaside.In the thin-layer chromatogram of the different places of production of Fig. 8 selfheal fruit ear, 1-reference substance Rosmarinic acid; 2-reference substance salviaflaside; The selfheal fruit ear in the different places of production of 3-13; The result shows that different places of production selfheal is on the relevant position of reference substance chromatogram, shows the spot of same color.The presentation of results Rosmarinic acid is present among any position of selfheal, and salviaflaside only exists among the fruit ear of selfheal, belongs to the characteristic component in the selfheal fruit ear, and the TLC method of setting up above can be used as the method for quality control of selfheal.
Method of quality control in embodiment 12 selfheals
1, the preparation of reference substance solution: with embodiment 11.
2, test sample preparation: with embodiment 11.
3, point sample, expansion, colour developing: draw respectively reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution 15 μ L point samples in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose; With chloroform: methyl alcohol: formic acid (volume ratio 8: 2: 0.5) is developping agent; Take out when being expanded to the about 0.5cm in thin layer plate top; Dry, the colour developing of the 1% vanillic aldehyde concentrated sulphuric acid, 105 ℃ are heated to clear spot.
Method of quality control in embodiment 13 selfheals
1, the preparation of reference substance solution: with embodiment 11.
2, test sample preparation: with embodiment 11.
3, point sample, expansion, colour developing: draw respectively reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution 15 μ L point samples in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose; With chloroform: methyl alcohol: formic acid (volume ratio 9: 1: 0.5) is developping agent; Take out when being expanded to the about 0.5cm in thin layer plate top; Dry, the colour developing of the 1% vanillic aldehyde concentrated sulphuric acid, 105 ℃ are heated to clear spot.
Method of quality control in embodiment 14 selfheals
1, the preparation of reference substance solution: with embodiment 11.
2, test sample preparation: with embodiment 11.
3, point sample, expansion, colour developing: draw respectively reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution 15 μ L point samples in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose; With chloroform: methyl alcohol: formic acid (volume ratio 4: 5: 1) is developping agent; Take out when being expanded to the about 0.5cm in thin layer plate top; Dry, the colour developing of the 1% vanillic aldehyde concentrated sulphuric acid, 105 ℃ are heated to clear spot.
Method of quality control in embodiment 15 selfheals
1, the preparation of reference substance solution: with embodiment 11.
2, test sample preparation: with embodiment 11.
3, point sample, expansion, colour developing: draw respectively reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution 15 μ L point samples in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose; With chloroform: methyl alcohol: water (volume ratio 6: 4: 1) is developping agent; Take out when being expanded to the about 0.5cm in thin layer plate top; Dry, the colour developing of the 1% vanillic aldehyde concentrated sulphuric acid, 105 ℃ are heated to clear spot.
Method of quality control in embodiment 16 selfheals
1, the preparation of reference substance solution: with embodiment 11.
2, test sample preparation: with embodiment 11.
3, point sample, expansion, colour developing: draw respectively reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution 15 μ L point samples in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose; With chloroform: methyl alcohol: water (volume ratio 65: 35: 10) is developping agent; Take out when being expanded to the about 0.5cm in thin layer plate top; Dry, the colour developing of the 1% vanillic aldehyde concentrated sulphuric acid, 105 ℃ are heated to clear spot.
Method of quality control in embodiment 17 selfheals
1, the preparation of reference substance solution: with embodiment 11.
2, test sample preparation: with embodiment 11.
3, point sample, expansion, colour developing: draw respectively reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution 15 μ L point samples in same be on the silica gel g thin-layer plate of binder with the sodium carboxymethyl cellulose; With chloroform: methyl alcohol: water (volume ratio 7: 3: 1) is developping agent; Take out when being expanded to the about 0.5cm in thin layer plate top; Dry, the colour developing of the 1% vanillic aldehyde concentrated sulphuric acid, 105 ℃ are heated to clear spot.
Claims (6)
1. the detection method of a selfheal is characterized in that, detects in the selfheal medicinal material whether contain compound salviaflaside according to high performance liquid chromatography and/or thin-layered chromatography, and said salviaflaside structural formula is following:
Described high performance liquid chromatography mainly may further comprise the steps:
(1) reference substance solution preparation: precision takes by weighing the salviaflaside compound, uses dissolve with methanol, is mixed with salviaflaside compound reference substance solution;
(2) preparation of need testing solution: get the selfheal medicinal powder, the accurate title, decide, and adds methyl alcohol, weighs, and ultrasonic 15～60min takes out, and leaves standstill, and cools, and benefit is heavy, and miillpore filter filters, as need testing solution;
(3) accurate respectively reference substance solution and the need testing solution drawn injects liquid chromatograph, measures chromatographic condition: chromatographic column: C18 post; Flow velocity: 0.5～1.5mL.min
-1Detect wavelength: 290～330nm;
Column temperature: 25～40 ℃; Moving phase: acetonitrile-1.0% acetic acid water=10～20: 90～80;
Described thin-layered chromatography mainly may further comprise the steps:
(1) preparation of reference substance solution: get reference substance Rosmarinic acid and compound salviaflaside respectively, add dissolve with methanol, be mixed with Rosmarinic acid and compound salviaflaside reference substance solution;
(2) preparation of need testing solution: get the selfheal medicinal powder, the accurate title, decide, and adds methyl alcohol, weighs, and ultrasonic 15～60min takes out, and leaves standstill, and cools, and benefit is heavy, filters, as need testing solution;
(3) point sample, expansion, colour developing: draw reference substance solution Rosmarinic acid, salviaflaside solution, need testing solution point sample respectively on same silica gel g thin-layer plate; With chloroform: methyl alcohol: formic acid=7: 3: 0.5 or 8: 2: 0.5 or 9: 1: 0.5 or 4: 5: 1 or chloroform: methyl alcohol: water=6: 4: 1 or 6.5: 3.5: 1.0 or 7: 3: 1 is developping agent; Launch, take out, dry; With the colour developing of the vanillic aldehyde concentrated sulphuric acid, be heated to clear spot.
2. the detection method of a selfheal is characterized in that, detects in the selfheal medicinal material whether contain compound salviaflaside according to high performance liquid chromatography, and said salviaflaside structural formula is following:
Described high performance liquid chromatography mainly may further comprise the steps:
(1) reference substance solution preparation: precision takes by weighing the salviaflaside compound, uses dissolve with methanol, is mixed with salviaflaside compound reference substance solution;
Also be provided with the Rosmarinic acid reference substance: precision takes by weighing the Rosmarinic acid reference substance, uses dissolve with methanol, is configured to the Rosmarinic acid reference substance solution;
(2) preparation of need testing solution: get the selfheal medicinal powder, the accurate title, decide, and adds methyl alcohol, weighs, and ultrasonic 15～60min takes out, and leaves standstill, and cools, and benefit is heavy, and miillpore filter filters, as need testing solution;
(3) accurate respectively reference substance solution and the need testing solution drawn injects liquid chromatograph, measures chromatographic condition: chromatographic column: C18 post; Flow velocity: 0.5～1.5mL.min
-1Detect wavelength: 290～330nm; Column temperature: 25～40 ℃; Moving phase is: 0～30min, acetonitrile are by 10%-20%～20%-30%, and 1.0% acetic acid water is by 90%-80%～80%-70% gradient elution.
3. the detection method of selfheal according to claim 2 is characterized in that, detects in the selfheal medicinal material whether contain compound salviaflaside according to high performance liquid chromatography, and said salviaflaside structural formula is following:
Described high performance liquid chromatography mainly may further comprise the steps:
(1) reference substance solution preparation: precision takes by weighing the salviaflaside compound, uses dissolve with methanol, is mixed with salviaflaside compound reference substance solution;
Also be provided with the Rosmarinic acid reference substance: precision takes by weighing the Rosmarinic acid reference substance, uses dissolve with methanol, is configured to the Rosmarinic acid reference substance solution;
(2) preparation of need testing solution: get the selfheal medicinal powder, the accurate title, decide, and adds methyl alcohol, weighs, and ultrasonic 15～60min takes out, and leaves standstill, and cools, and benefit is heavy, and miillpore filter filters, as need testing solution;
(3) accurate respectively reference substance solution and the need testing solution drawn injects liquid chromatograph, measures chromatographic condition: chromatographic column: C18 post; Flow velocity: 0.5～1.5mL.min
-1Detect wavelength: 290～330nm; Column temperature: 25～40 ℃; Moving phase is: 0～10min, acetonitrile are that 5%, 1.0% acetic acid water is 95%; 10～20min, acetonitrile are by 5～8.6%, and 1.0% acetic acid water is by 95～91.5%; 20～45min, acetonitrile are by 8.6～17.6%, and 1.0% acetic acid water is by 91.5～82.4%; 45～70min, acetonitrile are by 17.6～25.1%, and 1.0% acetic acid water is by 82.4～74.9%; 70～80min, acetonitrile are by 25.1～32.1%, and 1.0% acetic acid water is by 74.9～67.9%; 80～90min, acetonitrile are by 32.1～37.1%, and 1.0% acetic acid water carries out gradient elution by 67.9～62.9%, obtain the finger-print that selfheal detects.
4. the detection method of selfheal according to claim 1 is characterized in that, being prepared as of need testing solution in the said high performance liquid chromatography: get the selfheal medicinal powder, accurate claim fixed; Add 10 times of amount methyl alcohol, weigh ultrasonic 30min; Take out, leave standstill, cool; Benefit is heavy, and miillpore filter filters, as need testing solution; Said chromatographic condition is: sampling volume: 10 μ L, flow velocity: 1.1mL.min
-1Detect wavelength: 319nm; Column temperature: 30 ℃.
5. according to the detection method of each described selfheal of claim 1-4, it is characterized in that the concentration of preparation salviaflaside compound reference substance solution is 0.005～0.3 μ g/ μ L in the said high performance liquid chromatography; The content of salviaflaside is more than 0.1 ‰ in the selfheal medicinal material.
6. the detection method of selfheal according to claim 1 is characterized in that, being prepared as of need testing solution in the described thin-layered chromatography: get the selfheal medicinal powder, accurate claim fixed; Add 10 times of amount methyl alcohol, weigh ultrasonic 30min; Take out, leave standstill, cool; Benefit is heavy, filters, as need testing solution; Described thin layer plate is that sodium carboxymethyl cellulose is the silica gel g thin-layer plate of binder; Developer is the 1% vanillic aldehyde concentrated sulphuric acid, and developping agent is a chloroform: methyl alcohol: formic acid=7: 3: 0.5 or chloroform: methyl alcohol: water=6: 4: 1; The concentration of said Rosmarinic acid and compound salviaflaside reference substance solution is respectively 0.1～1.0mg/mL and 0.01～0.1mg/mL.
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| CN108548884A (en) * | 2018-05-25 | 2018-09-18 | 南昌大学 | The method that HPLC methods detect Rosmarinic acid, carnosic acid and ursolic acid in rosemary alcohol extract |
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