CN104880528A - Construction method for finger print of Shuanghuanglian oral liquid - Google Patents
Construction method for finger print of Shuanghuanglian oral liquid Download PDFInfo
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Abstract
The invention provides a construction method for finger print of Shuanghuanglian oral liquid. The Shuanghuanglian oral liquid is prepared from lonicera japonica, radix scutellariae, fructus forsythiae, saccharose and essence. The construction method comprises the steps of preparing a test sample solution, detecting active ingredients of the Shuanghuanglian oral liquid by adopting a high-performance liquid chromatography, and detecting whether the Shuanghuanglian oral liquid contains Chinese teasel root saponin B or not; chromatographic conditions are as follows: a chromatographic column: Waters Acquitv UPLC BEH RP18; a mobile phase: acetonitrile-0.4 percent glacial acetic acid solution; gradient elution: 0 to 10 minutes, 15 percent A-85 percent B; 2 to 4 minutes, 25 percent A-75 percent B; 4 to 8 minutes, 37 percent A-63 percent B; 8 to 10 minutes, 56 percent A-44 percent B, and 10 to 11 minutes, 93 percent A-7 percent B; column temperature: 35 DEG C; detection wavelength: 300nm. The construction method is used for constructing the finger print of the Shuanghuanglian oral liquid and meanwhile can be used for detecting whether the Shuanghuanglian oral liquid contains a Chinese teasel root saponin B ingredient or not, so that a basis is provided for establishing a quality monitoring system for the Shuanghuanglian oral liquid.
Description
Technical field
The present invention relates to shuanghuanglian mixture, shuanghuanglian koufuye Multiple components to measure.More particularly, the present invention relates to the method detecting plurality of active ingredients in shuanghuanglian mixture, shuanghuanglian koufuye simultaneously and judge whether to exist Honeysuckle flower.
Background technology
Shuanghuanglian mixture, shuanghuanglian koufuye is formed through Hydrolysis kinetics by honeysuckle, the root of large-flowered skullcap and the capsule of weeping forsythia, there is relieving the exterior syndrome with drugs pungent in flavor and cool in property, heat-clearing toxin-expelling functions, be applicable to the bacterium such as the infection of the upper respiratory tract, tonsillitis, pharyngitis, viral pneumonia that anemopyretic cold causes and disease of viral infection has certain curative effect.Shuanghuanglian mixture, shuanghuanglian koufuye has good curative effect to anemopyretic cold.But because the price of honeysuckle is more and more higher, present market has occurred using Honeysuckle flower to replace honeysuckle to prepare the phenomenon of shuanghuanglian mixture, shuanghuanglian koufuye.Because honeysuckle and Honeysuckle flower all have a great difference from source and the aspect such as proterties or its chemical composition, if use Honeysuckle flower to replace honeysuckle to prepare shuanghuanglian mixture, shuanghuanglian koufuye, great impact can be produced on the curative effect of shuanghuanglian mixture, shuanghuanglian koufuye.
Honeysuckle is dry flower or the first flower opened of band of caprifoliaceae plant honeysuckle.Honeysuckle flower is dry flower or the first flower opened of band of caprifoliaceae plant largeflower-like honeysuckle flower, lonicera hypoglauca miq or Lonicera confusa.Although both containing chlorogenic acid, other effective constituents such as rutin, galuteolin, Quercetin three chemical compositions is distributed in honeysuckle, and more containing largeflower-like honeysuckle flower saponin(e second and asperosaponin second in Honeysuckle flower.
The existing quality determining method about Shuang Huanglian liquid preparation, be all only according to detecting with chlorogenic acid, the finger-print of standard is not all had as appraisal basis for other compositions such as rutin, galuteolin, Quercetin, meanwhile, whether corresponding detection method is not had yet containing largeflower-like honeysuckle flower saponin(e second and asperosaponin second.
Summary of the invention
As the result of various extensive and careful research and experiment, the present inventor has been found that, containing specific chemical composition rutin, galuteolin, Quercetin in honeysuckle, and containing asperosaponin second and largeflower-like honeysuckle flower saponin(e second special component in Honeysuckle flower.Based on this discovery, complete the present invention.
An object of the present invention is to solve at least the problems referred to above and/or defect, and the advantage will illustrated at least is below provided.
A further object of the invention is by detection method of the present invention, build the finger-print of shuanghuanglian mixture, shuanghuanglian koufuye, can detecting whether containing asperosaponin second composition in shuanghuanglian mixture, shuanghuanglian koufuye simultaneously, providing basis for setting up shuanghuanglian mixture, shuanghuanglian koufuye quality supervision system.
A further object of the invention is by detection method of the present invention, improves the HPLC liquid preparation efficiency to be measured of shuanghuanglian mixture, shuanghuanglian koufuye, to obtain better Detection results.
In order to realize, according to these objects of the present invention and other advantage, providing a kind of construction method of shuanghuanglian mixture, shuanghuanglian koufuye finger-print.Wherein said shuanghuanglian mixture, shuanghuanglian koufuye is made up of honeysuckle, the root of large-flowered skullcap, the capsule of weeping forsythia, sucrose and essence, and described construction method comprises the following steps:
Step one, prepare need testing solution, get shuanghuanglian mixture, shuanghuanglian koufuye 1.0mL, accurately weighed, add 25mL50% methanol aqueous solution, after 45 DEG C of water-bath 5-10min, carry out centrifugal immediately, get supernatant; Described supernatant is placed in 50mL round-bottomed flask, refluxing extraction 2h, weighs after cooling, supply weightlessness with 50% methanol solution, hyperfiltration; Obtain need testing solution; Because shuanghuanglian mixture, shuanghuanglian koufuye with the addition of the auxiliary element such as sucrose and essence in preparation process, sucrose and essence to be avoided in process prepared by need testing solution to remain in need testing solution, affect follow-up stratographic analysis result.So in advance by test sample water-bath at 45 DEG C, due at high operating temperatures, sucrose extremely difficulty is dissolved in methyl alcohol, the need testing solution obtained by follow-up refluxing extraction is purer to lay the foundation for later stage high performance liquid chromatography detects.
Step 2, employing high performance liquid chromatography detect the active component of described shuanghuanglian mixture, shuanghuanglian koufuye, and detect whether contain asperosaponin second; Wherein chromatographic condition is:
Chromatographic column: Waters Acquity UPLC BEH RP
18; Mobile phase: acetonitrile-0.4% glacial acetic acid water; Gradient elution: 0-10min, 15%A-85%B; 2-4min, 25%A-75%B; 4-8min, 37%A-63%B; 8-10min, 56%A-44%B; 10-11min, 93%A-7%B; Column temperature: 35 DEG C; Determined wavelength: 300nm.Theoretical cam curve calculates by chlorogenic acid peak and is not less than 1000, is greater than 2.0 with the degree of separation of adjacent peak.
Wherein in an embodiment, the active component of described shuanghuanglian mixture, shuanghuanglian koufuye comprises: chlorogenic acid, forsythin, scutelloside and galuteolin.Wherein wooden slippers grass is sweet is the characteristic component being different from Honeysuckle flower in honeysuckle, so except detecting these three kinds of active components of chlorogenic acid in Shuanghuanglian oral solution, forsythin and scutelloside, in order to ensure the accuracy of its quality testing system, the present invention select wooden slippers grass sweet conduct be detected as/mono-.
Wherein in an embodiment, described chromatographic condition also comprises: ELSD detector parameters: drift tube temperature is 80 DEG C, and flow rate of carrier gas is 2.5ml/min.
Wherein in an embodiment, described hyperfiltration specifically refers to: use 0.45 μm of filtering with microporous membrane.
Wherein in an embodiment, described construction method also comprises the preparation of reference substance solution, specifically comprise: precision takes chlorogenic acid, forsythin, scutelloside, reseda and asperosaponin second reference substance: 0.889mg, 0.886mg, 0.903mg, 0.920mg and 0.976mg and, with acetonitrile-0.4% phosphate aqueous solution for solvent, 2mL is settled to, as storing solution in brown volumetric flask.The reference substance solution of other different quality concentration is diluted by storing solution and is obtained.
Wherein in an embodiment, in shuanghuanglian mixture, shuanghuanglian koufuye, honeysuckle adopts Honeysuckle flower to replace, and its preparation method is as follows: the dry crude drug getting Honeysuckle flower, the capsule of weeping forsythia and the root of large-flowered skullcap, is ground into fine powder and crosses No. 4 sieves; Boiling 1h, filter evaporate to dryness, residue adds the ultrasonic 20min of 70% methyl alcohol, 0.45 μm of filtering with microporous membrane.Containing chlorogenic acid 1.18mg/mL, scutelloside 7.2mg/mL, forsythin 0.04mg/mL in wherein said test sample, wooden slippers grass sweet 0.013mg/mL, asperosaponin second 3.51mg/mL.
The present invention at least comprises following beneficial effect: the present invention passes through in shuanghuanglian mixture, shuanghuanglian koufuye testing process Multiple components Simultaneously test such as the chlorogenic acid of shuanghuanglian mixture, shuanghuanglian koufuye, forsythin, scutelloside, resedas, use existing equipment, under same testing conditions, finger-print and the disposable detection of various active composition are completed, simultaneously, asperosaponin the second grade composition can also be detected under such specific conditions, for the identification of the quality of shuanghuanglian mixture, shuanghuanglian koufuye.Adopt simple buffer salt solution directly to dissolve reference substance and need testing solution, successfully solve the precipitation problem in asperosaponin second reference substance high concentration situation, also solve some composition attenuation problem that need testing solution long-term room-temperature is placed; Prepare need testing solution under being used in uniform temperature, solve the impact that in shuanghuanglian mixture, shuanghuanglian koufuye, auxiliary material detects active component in need testing solution.
Part is embodied by explanation below by other advantage of the present invention, target and feature, part also will by research and practice of the present invention by those skilled in the art is understood.
Accompanying drawing explanation
Fig. 1 shows the high-efficient liquid phase chromatogram of shuanghuanglian mixture, shuanghuanglian koufuye described in one of them embodiment of the present invention;
Fig. 2 shows the high-efficient liquid phase chromatogram of shuanghuanglian mixture, shuanghuanglian koufuye described in one of them embodiment of the present invention.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to instructions word to make those skilled in the art.
Should be appreciated that used hereinly such as " to have ", other element one or more do not allotted in " comprising " and " comprising " term or the existence of its combination or interpolation.
< example 1>
The construction method of shuanghuanglian mixture, shuanghuanglian koufuye finger-print of the present invention, the instrument of employing and material:
Waters Acquity UPLC H-Class system, comprises quaternary pump, automatic sampler, ELSD detecting device etc.100000/balance (Mei Tele company); EL204 ten thousand/balance (Shanghai exact instrument company limited); KQ-250DE ultrasonic cleaner (ultrasonic instrument company limited of city of Kunshan).
Methyl alcohol and acetonitrile (chromatographically pure, Fisher company), ethanol, phosphoric acid (analyzing pure, Beijing Chemical Plant), distilled water.Reference substance chlorogenic acid (NO:110753-201313) and galuteolin (NO:111720-201107) are purchased from National Institute for Food and Drugs Control.Honeysuckle flower material is through being accredited as the dry flower of caprifoliaceae plant largeflower-like honeysuckle flower Lonicera macranthoides Hand.-Mazz..
The construction method of shuanghuanglian mixture, shuanghuanglian koufuye finger-print of the present invention.Wherein said shuanghuanglian mixture, shuanghuanglian koufuye is made up of honeysuckle, the root of large-flowered skullcap, the capsule of weeping forsythia, sucrose and essence, and described construction method comprises the following steps:
Step one, prepare need testing solution, get commercially available shuanghuanglian mixture, shuanghuanglian koufuye 1.0mL, accurately weighed, add 25mL50% methanol aqueous solution, after 45 DEG C of water-bath 5-10min, carry out centrifugal immediately, wherein centrifugal temperature such as controls between 30 DEG C-40 DEG C; Get supernatant afterwards; Described supernatant is placed in 50mL round-bottomed flask, refluxing extraction 2h, weighs after cooling, supply weightlessness with 50% methanol solution, use 0.45 μm of filtering with microporous membrane.Obtain need testing solution;
The preparation of reference substance solution, specifically comprise: precision takes chlorogenic acid, forsythin, scutelloside, reseda and asperosaponin second reference substance: 0.889mg, 0.886mg, 0.903mg, 0.920mg and 0.976mg, with acetonitrile-0.4% phosphate aqueous solution for solvent, 2mL is settled to, as storing solution in brown volumetric flask.The reference substance solution of other different quality concentration is diluted by storing solution and is obtained.
Step 2, employing high performance liquid chromatography detect the active component of described shuanghuanglian mixture, shuanghuanglian koufuye, and detect whether contain asperosaponin second; Wherein chromatographic condition is:
Chromatographic column: Waters Acquity UPLC BEH RP
18; Mobile phase: acetonitrile-0.4% glacial acetic acid water; Gradient elution: 0-10min, 15%A-85%B; 2-4min, 25%A-75%B; 4-8min, 37%A-63%B; 8-10min, 56%A-44%B; 10-11min, 93%A-7%B; Column temperature: 35 DEG C; Determined wavelength: 300nm.Theoretical cam curve calculates by chlorogenic acid peak and is not less than 1000, is greater than 2.0 with the degree of separation of adjacent peak., described chromatographic condition also comprises: ELSD detector parameters: drift tube temperature is 80 DEG C, and flow rate of carrier gas is 2.5ml/min.
As shown in Figure 1, the active component of described shuanghuanglian mixture, shuanghuanglian koufuye comprises: chlorogenic acid 1, forsythin 3, scutelloside 4 and wooden slippers grass sweet 2, the sweet 0.002mg/mL of its Content of Chlorogenic Acid 1.1mg/mL, forsythin 0.5mg/mL, scutelloside 8.2mg/mL and wooden slippers grass.
Embodiment 2
The preparation of need testing solution, gets the dry crude drug of Honeysuckle flower, the capsule of weeping forsythia and the root of large-flowered skullcap, is ground into fine powder and crosses No. 4 sieves; Boiling 1h, filter evaporate to dryness, residue adds the ultrasonic 20min of 70% methyl alcohol, 0.45 μm of filtering with microporous membrane.Containing chlorogenic acid 1.18mg/mL, scutelloside 7.2mg/mL, forsythin 0.04mg/mL in wherein said test sample, wooden slippers grass sweet 0.013mg/mL, asperosaponin second 3.51mg/mL.
The preparation of reference substance solution, specifically comprise: precision takes chlorogenic acid, forsythin, scutelloside, reseda and asperosaponin second reference substance: 0.889mg, 0.886mg, 0.903mg, 0.920mg and 0.976mg, with acetonitrile-0.4% phosphate aqueous solution for solvent, 2mL is settled to, as storing solution in brown volumetric flask.The reference substance solution of other different quality concentration is diluted by storing solution and is obtained.
Step 2, employing high performance liquid chromatography detect the active component of described shuanghuanglian mixture, shuanghuanglian koufuye, and detect whether contain asperosaponin second; Obtain Fig. 2, wherein active component is: chlorogenic acid 1, forsythin 3, scutelloside 4, galuteolin 2 and asperosaponin second 5 wherein chromatographic condition are:
Chromatographic column: Waters Acquity UPLC BEH RP
18; Mobile phase: acetonitrile-0.4% glacial acetic acid water; Gradient elution: 0-10min, 15%A-85%B; 2-4min, 25%A-75%B; 4-8min, 37%A-63%B; 8-10min, 56%A-44%B; 10-11min, 93%A-7%B; Column temperature: 35 DEG C; Determined wavelength: 300nm.Theoretical cam curve calculates by chlorogenic acid peak and is not less than 1000, is greater than 2.0 with the degree of separation of adjacent peak., described chromatographic condition also comprises: ELSD detector parameters: drift tube temperature is 80 DEG C, and flow rate of carrier gas is 2.5ml/min.
The foundation of typical curve
A, the investigation of linear relationship, detectability (LOD) and quantitative limit (LOQ)
Accurate absorption reference substance storing solution respectively, become the reference substance solution of a series of variable concentrations with mobile phase stepwise dilution, sample introduction 3 μ L, measures (n=3) respectively.Chlorogenic acid, forsythin, scutelloside, galuteolin and asperosaponin second are with concentration X for horizontal ordinate, and average peak area Y is that ordinate carries out linear regression, investigate linear relationship.As shown in Table 1, each regression equation is good linear relationship.
The typical curve of table 1 chlorogenic acid, forsythin, scutelloside, galuteolin and asperosaponin second, related coefficient, range of linearity measurement result
B, precision, repeatability and stability test
The same need testing solution 3 μ L of accurate absorption, continuous sample introduction 6 times under above-mentioned chromatographic condition, the RSD value recording chlorogenic acid, forsythin, scutelloside, galuteolin and asperosaponin second is respectively: 0.85%, 0.81%, 0.92%, 1.04%, 0.79%, result shows that the precision of instrument is good.Get same need testing solution 0.25g, totally 6 parts, accurately weighed, measure under above-mentioned chromatographic condition, the RSD value recording chlorogenic acid, forsythin, scutelloside, galuteolin and asperosaponin second is respectively 1.17%, 0.89%, 1.13%, 1.28%, 0.99%.Result shows that this method repeatability is good.Get same need testing solution, ambient temperatare is put, respectively at 0,2,4,8,16,24h sample introduction 3 μ L, measures by above-mentioned chromatographic condition, record peak area, the RSD value of chlorogenic acid, forsythin, scutelloside, galuteolin and asperosaponin second peak area is respectively 1.64%, 1.23%, 1.65%, 1.72%, 1.59%, result shows that need testing solution is stable in 24h.
C, recovery test
Adopt application of sample absorption method.Get the shuanghuanglian mixture, shuanghuanglian koufuye need testing solution 9 parts of known content, every part of about 0.125g, accurately weighed, be divided into 3 groups, by low (80%), in (100%), high (120%) concentration respectively precision adds a certain amount of chlorogenic acid, forsythin, scutelloside, galuteolin and asperosaponin second reference substance solution, prepare according to the preparation method of test sample, measure at above-mentioned chromatographic condition, calculate the recovery.Result shows, and the recovery reaches more than 97% entirely.Result confirms that the method set up has higher accuracy for the content of Simultaneously test Chlorogenic Acid of Flos Lonicerae and galuteolin.
Although embodiment of the present invention are open as above, it is not restricted to listed in instructions and embodiment utilization.It can be applied to various applicable the field of the invention completely.For those skilled in the art, can easily realize other amendment.Therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (5)
1. a construction method for shuanghuanglian mixture, shuanghuanglian koufuye finger-print, wherein said shuanghuanglian mixture, shuanghuanglian koufuye is made up of honeysuckle, the root of large-flowered skullcap, the capsule of weeping forsythia, sucrose and essence, and described construction method comprises the following steps:
Step one, prepare need testing solution, get shuanghuanglian mixture, shuanghuanglian koufuye 1.0mL, accurately weighed, add 25mL50% methanol aqueous solution, after 45 DEG C of water-bath 5-10min, carry out centrifugal immediately, get supernatant; Described supernatant is placed in 50mL round-bottomed flask, refluxing extraction 2h, weighs after cooling, supply weightlessness with 50% methanol solution, hyperfiltration; Obtain need testing solution;
Step 2, employing high performance liquid chromatography detect the active component of described shuanghuanglian mixture, shuanghuanglian koufuye, and detect whether contain asperosaponin second; Wherein chromatographic condition is:
Chromatographic column: Waters Acquity UPLC BEH RP
18; Mobile phase: acetonitrile-0.4% glacial acetic acid water; Gradient elution: O-10min, 15%A-85%B; 2-4min, 25%A-75%B; 4-8min, 37%A-63%B; 8-10min, 56%A-44%B; 10-11min, 93%A-7%B; Column temperature: 35 DEG C; Determined wavelength: 300nm.
2. the construction method of shuanghuanglian mixture, shuanghuanglian koufuye finger-print as claimed in claim 1, the active component of wherein said shuanghuanglian mixture, shuanghuanglian koufuye comprises: chlorogenic acid, forsythin, scutelloside and galuteolin.
3. the construction method of shuanghuanglian mixture, shuanghuanglian koufuye finger-print as claimed in claim 1, wherein said chromatographic condition also comprises: ELSD detector parameters: drift tube temperature is 80 DEG C, and flow rate of carrier gas is 2.5ml/min.
4. the construction method of shuanghuanglian mixture, shuanghuanglian koufuye finger-print as claimed in claim 1, wherein said hyperfiltration specifically refers to: use 0.45 μm of filtering with microporous membrane.
5. the construction method of shuanghuanglian mixture, shuanghuanglian koufuye finger-print as claimed in claim 1, wherein said construction method also comprises the preparation of reference substance solution, specifically comprise: precision takes chlorogenic acid, forsythin, scutelloside, reseda and asperosaponin second reference substance: 0.889mg, 0.886mg, 0.903mg, 0.920mg and 0.976mg and, with acetonitrile-0.4% phosphate aqueous solution for solvent, 2mL is settled to, as storing solution in brown volumetric flask.The reference substance solution of other different quality concentration is diluted by storing solution and is obtained.
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| CN111307988A (en) * | 2020-03-24 | 2020-06-19 | 苏州大学附属儿童医院 | Quality control method of cicada oral liquid |
| CN112858549A (en) * | 2019-11-27 | 2021-05-28 | 中国科学院大连化学物理研究所 | Quantitative fingerprint quality monitoring method for Shuanghuanglian oral liquid |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106290646A (en) * | 2016-08-29 | 2017-01-04 | 河北国金药业有限责任公司 | The detection method of Compound Jinyinhua Granules |
| CN106290646B (en) * | 2016-08-29 | 2019-06-28 | 河北国金药业有限责任公司 | The detection method of Compound Jinyinhua Granules |
| CN107091887A (en) * | 2017-03-29 | 2017-08-25 | 哈尔滨珍宝制药有限公司 | The detection method and method of quality control of a kind of characteristic spectrum of honeysuckle dispensing granule |
| CN107632075A (en) * | 2017-08-08 | 2018-01-26 | 武汉双龙药业有限公司 | Golden three kinds of component contents of bavin KANGGAN JIAONANG while assay method and its HPLC fingerprint map construction methods |
| CN108760903A (en) * | 2018-03-30 | 2018-11-06 | 山东省中医药研究院 | A kind of swap buffers oral preparation finger print measuring method |
| CN108760903B (en) * | 2018-03-30 | 2020-11-17 | 山东中医药大学 | Fingerprint spectrum determination method for Shuanghuanglian oral preparation |
| CN110133138A (en) * | 2019-05-22 | 2019-08-16 | 贵州天楼生物发展有限公司 | The detection method of stauntonvine Content of Chlorogenic Acid |
| CN112858549A (en) * | 2019-11-27 | 2021-05-28 | 中国科学院大连化学物理研究所 | Quantitative fingerprint quality monitoring method for Shuanghuanglian oral liquid |
| CN111307988A (en) * | 2020-03-24 | 2020-06-19 | 苏州大学附属儿童医院 | Quality control method of cicada oral liquid |
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Application publication date: 20150902 |