CN111272943B - Quality detection method of spleen-strengthening and qi-tonifying oral liquid - Google Patents
Quality detection method of spleen-strengthening and qi-tonifying oral liquid Download PDFInfo
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- 239000007788 liquid Substances 0.000 title claims abstract description 75
- 238000005728 strengthening Methods 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 claims abstract description 48
- 238000004809 thin layer chromatography Methods 0.000 claims abstract description 34
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 claims abstract description 28
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 claims abstract description 28
- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 claims abstract description 28
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 claims abstract description 28
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 claims abstract description 28
- 229940025878 hesperidin Drugs 0.000 claims abstract description 28
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 210000000952 spleen Anatomy 0.000 claims abstract description 24
- -1 calycosin glucoside Chemical class 0.000 claims abstract description 21
- ZZAJQOPSWWVMBI-UHFFFAOYSA-N Calycosin Natural products C1=C(O)C(OC)=CC=C1C1=COC2=CC(O)=CC=C2C1=O ZZAJQOPSWWVMBI-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229930182478 glucoside Natural products 0.000 claims abstract description 20
- 238000012360 testing method Methods 0.000 claims abstract description 19
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 18
- JAYVHSBYKLLDJC-DSNJPTTOSA-N (E)-2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(O)C=C(O)C=C1\C=C\C1=CC=C(O)C=C1 JAYVHSBYKLLDJC-DSNJPTTOSA-N 0.000 claims abstract description 17
- 241000132012 Atractylodes Species 0.000 claims abstract description 16
- 235000006533 astragalus Nutrition 0.000 claims abstract description 14
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 14
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- 210000004233 talus Anatomy 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 103
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 87
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 36
- 239000013558 reference substance Substances 0.000 claims description 36
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 34
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 26
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000000523 sample Substances 0.000 claims description 21
- 239000000463 material Substances 0.000 claims description 19
- 239000003208 petroleum Substances 0.000 claims description 17
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 16
- 238000001704 evaporation Methods 0.000 claims description 16
- 239000013642 negative control Substances 0.000 claims description 16
- 239000012488 sample solution Substances 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 14
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000012085 test solution Substances 0.000 claims description 12
- 239000002021 butanolic extract Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 239000009636 Huang Qi Substances 0.000 claims description 7
- 230000001502 supplementing effect Effects 0.000 claims description 7
- QMNWISYXSJWHRY-YLNUDOOFSA-N astragaloside IV Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)[C@H]4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)C[C@H]3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-YLNUDOOFSA-N 0.000 claims description 6
- QMNWISYXSJWHRY-BCBPIKMJSA-N astragaloside IV Natural products CC(C)(O)[C@@H]1CC[C@@](C)(O1)[C@H]2[C@@H](O)C[C@@]3(C)[C@@H]4C[C@H](O[C@@H]5O[C@H](CO)[C@H](O)[C@@H](O)[C@H]5O)[C@H]6C(C)(C)[C@H](CC[C@@]67C[C@@]47CC[C@]23C)O[C@@H]8OC[C@@H](O)[C@H](O)[C@H]8O QMNWISYXSJWHRY-BCBPIKMJSA-N 0.000 claims description 6
- PFKIBRPYVNVMRU-UHFFFAOYSA-N cyclosieversioside F Natural products CC(C)(O)C1COC(C)(C1)C2C(O)CC3(C)C4CC(OC5OC(CO)C(O)C(O)C5O)C6C(C)(C)C(CCC67CC47CCC23C)OC8OCC(O)C(O)C8O PFKIBRPYVNVMRU-UHFFFAOYSA-N 0.000 claims description 6
- 238000000079 presaturation Methods 0.000 claims description 6
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- YEZWWUMWIFKEQM-UHFFFAOYSA-N O.OC.ClC(Cl)Cl.CCOC(C)=O Chemical compound O.OC.ClC(Cl)Cl.CCOC(C)=O YEZWWUMWIFKEQM-UHFFFAOYSA-N 0.000 claims description 5
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 claims description 5
- 239000002024 ethyl acetate extract Substances 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 241000045403 Astragalus propinquus Species 0.000 claims description 4
- 240000002948 Ophiopogon intermedius Species 0.000 claims description 4
- 238000009835 boiling Methods 0.000 claims description 4
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 241001672694 Citrus reticulata Species 0.000 claims description 3
- NOTFZGFABLVTIG-UHFFFAOYSA-N Cyclohexylethyl acetate Chemical compound CC(=O)OCCC1CCCCC1 NOTFZGFABLVTIG-UHFFFAOYSA-N 0.000 claims description 3
- 239000012670 alkaline solution Substances 0.000 claims description 3
- 230000008020 evaporation Effects 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 3
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 claims description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
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- 238000002360 preparation method Methods 0.000 description 10
- 235000018167 Reynoutria japonica Nutrition 0.000 description 9
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- 239000012088 reference solution Substances 0.000 description 7
- 238000005070 sampling Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
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- 241000283690 Bos taurus Species 0.000 description 1
- 241000721047 Danaus plexippus Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 238000013095 identification testing Methods 0.000 description 1
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- PJANXHGTPQOBST-UHFFFAOYSA-N stilbene Chemical compound C=1C=CC=CC=1C=CC1=CC=CC=C1 PJANXHGTPQOBST-UHFFFAOYSA-N 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
- G01N30/94—Development
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Physics & Mathematics (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application discloses a quality detection method of an oral liquid for strengthening spleen and tonifying qi, which comprises the following steps of qualitatively analyzing three components of astragalus, bighead atractylodes rhizome and dried orange peel in the oral liquid by using a thin layer chromatography; and detecting the content of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the oral liquid by using a high performance liquid chromatography. The method firstly uses thin layer chromatography to carry out qualitative analysis on the oral liquid for strengthening spleen and tonifying qi, utilizes the micro, quick and simple advantages of thin layer chromatography to predict, then uses high performance liquid chromatography to carry out quantitative analysis, realizes 'one test and three evaluation', has simple and easy operation, uses high performance liquid chromatography to pass the methodological verification, has high precision, good reproducibility, good stability, high recovery rate, good durability and accurate measurement result, and establishes a foundation for establishing the quality standard of the oral liquid for strengthening spleen and tonifying qi by combining qualitative and quantitative analysis.
Description
Technical Field
The application belongs to the technical field of medicine detection, and particularly relates to a quality detection method of spleen-tonifying and qi-tonifying oral liquid.
Background
In the field of traditional Chinese medicines, main active ingredients of the spleen-strengthening and qi-tonifying oral liquid are derived from astragalus, bighead atractylodes rhizome, dried orange peel and the like, and are extracted and prepared by combining various medicinal raw materials. This results in uneven composition and potency of the spleen-invigorating and qi-tonifying oral liquid from different manufacturers.
In order to ensure the quality level of the spleen-strengthening and qi-tonifying oral liquid mainly comprising astragalus, bighead atractylodes rhizome and dried orange peel, a set of efficient and accurate detection method is urgently needed, so that the spleen-strengthening and qi-tonifying oral liquid with the optimal drug effect can be screened.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present application has been made in view of the above technical drawbacks.
Therefore, as one aspect of the application, the application overcomes the defects in the prior art and provides a quality detection method of the oral liquid for strengthening spleen and tonifying qi.
In order to solve the technical problems, the application provides the following technical scheme: a quality detection method of an oral liquid for invigorating spleen and replenishing qi comprises qualitatively analyzing three components of radix astragali, atractylodis rhizoma and pericarpium Citri Tangerinae by thin layer chromatography; and detecting the content of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the oral liquid by using a high performance liquid chromatography.
As a preferred scheme of the quality detection method of the spleen-tonifying and qi-tonifying oral liquid, the application comprises the following steps: the thin layer chromatography is used for extracting three components of astragalus, bighead atractylodes rhizome and dried orange peel in the oral liquid, wherein n-butanol, petroleum ether and ethyl acetate are used for extracting the oral liquid respectively.
As a preferred scheme of the quality detection method of the spleen-tonifying and qi-tonifying oral liquid, the application comprises the following steps: the thin-layer chromatography is used for preparing three components of astragalus, bighead atractylodes rhizome and dried orange peel in the oral liquid, wherein the used developing agents are respectively in a volume ratio of 20:10:11:5, placing the lower layer solution of ethyl acetate-chloroform-methanol-water for 30min below 10 ℃ according to the volume ratio of 7:3 in a volume ratio of 18:6:1 trichloromethane-methanol-water.
As a preferred scheme of the quality detection method of the spleen-tonifying and qi-tonifying oral liquid, the application comprises the following steps: the high performance liquid chromatography uses octadecylsilane chemically bonded silica as a filler, acetonitrile-0.1% phosphoric acid solution as a mobile phase, and the detection wavelength is 260nm; the flow rate is 1.0mL/min, the column temperature is 30 ℃, and gradient elution is carried out.
As a preferred scheme of the quality detection method of the spleen-tonifying and qi-tonifying oral liquid, the application comprises the following steps: the gradient elution is specifically that the mobile phase is 17% acetonitrile and 83% 1% phosphoric acid solution at 0-12 min; 13-40 min, the mobile phase is 37% acetonitrile and 63% 1% phosphoric acid solution; 41-55 min, the mobile phase is 17% acetonitrile and 83% 1% phosphoric acid solution.
As a preferred scheme of the quality detection method of the spleen-tonifying and qi-tonifying oral liquid, the application comprises the following steps: the theoretical plate number should be not less than 4000 as calculated by hesperidin peak.
As a preferred scheme of the quality detection method of the spleen-tonifying and qi-tonifying oral liquid, the application comprises the following steps: the oral liquid for strengthening spleen and tonifying qi is 3.0 mug or more in terms of calycosin glucoside per 1mL sample; 50.0 μg or more of 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside; and 50.0 mug or more in terms of hesperidin.
The application has the beneficial effects that:
the application firstly utilizes thin layer chromatography to respectively identify three components of astragalus, bighead atractylodes rhizome and dried orange peel in the oral liquid for strengthening spleen and supplementing qi for qualitative analysis; and then the content of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the spleen-strengthening and qi-tonifying oral liquid is detected simultaneously by utilizing a high performance liquid chromatography method, and quantitative analysis is carried out. The method firstly uses thin layer chromatography to carry out qualitative analysis on the oral liquid for strengthening spleen and tonifying qi, utilizes the micro, quick and simple advantages of thin layer chromatography to predict, then uses high performance liquid chromatography to carry out quantitative analysis, realizes 'one test and three evaluation', has simple and easy operation, uses high performance liquid chromatography to pass the methodological verification, has high precision, good reproducibility, good stability, high recovery rate, good durability and accurate measurement result, and establishes a foundation for establishing the quality standard of the oral liquid for strengthening spleen and tonifying qi by combining qualitative and quantitative analysis.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a thin-layer chromatogram of Astragalus membranaceus, wherein FIG. 1 shows Astragaloside IV as a control; 2,3,4 represent test samples; 5 represents a negative sample of astragalus membranaceus lack;
FIG. 2 is a thin-layer chromatogram of Atractylodis rhizoma, wherein 1 is a control drug of Atractylodis rhizoma; 2,3,4 represent test samples; 5 represents a negative control of white atractylodes rhizome;
FIG. 3 is a thin-layer chromatogram of pericarpium Citri Tangerinae, wherein FIG. 1 shows hesperidin control; 2 represents a tangerine peel control medicinal material; 3,4,5 represent test samples; 6 represents a negative control of the dried orange peel;
FIG. 4 is a high performance liquid chromatogram of a mixed control solution;
FIG. 5 is a high performance liquid chromatogram of a test solution;
FIG. 6 is a high performance liquid chromatogram of a negative control solution.
Detailed Description
In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
a quality detection method of the oral liquid for invigorating spleen and replenishing qi, said method is to utilize thin-layer chromatography to distinguish three kinds of components of radix astragali, rhizoma Atractylodis Macrocephalae, orange rind in the oral liquid for invigorating spleen and replenishing qi separately first, carry on the qualitative analysis; and then the content of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the spleen-strengthening and qi-tonifying oral liquid is detected simultaneously by utilizing a high performance liquid chromatography method, and quantitative analysis is carried out.
The three components of astragalus, bighead atractylodes rhizome and dried orange peel in the spleen-invigorating and qi-tonifying oral liquid are respectively identified by using a thin layer chromatography for qualitative analysis, and the method comprises the following steps:
(1) Identifying radix astragali by thin layer chromatography:
1) Taking 20mL of the product, extracting with water saturated n-butanol for 3 times by shaking, 20mL each time, combining n-butanol extract, washing with 1% sodium hydroxide test solution for 3 times, 20mL each time, discarding alkaline solution, combining n-butanol solution, washing with water saturated n-butanol to neutrality, evaporating n-butanol solution to dryness, and adding 1mL of methanol into residue to dissolve, thereby obtaining the test solution.
2) Taking 20mL of a sample prepared from astragalus membranaceus, extracting with water saturated n-butanol for 3 times under shaking, 20mL each time, combining n-butanol extract, washing with 1% sodium hydroxide test solution for 3 times, 20mL each time, discarding alkaline solution, combining n-butanol solution, washing with water saturated n-butanol to neutrality, evaporating n-butanol solution to dryness, and adding 1mL of methanol into residue to dissolve, thereby obtaining a negative control solution.
3) Adding methanol into astragaloside IV reference substance to obtain solution containing 1mg astragaloside IV per 1mL, and taking the solution as reference substance solution.
4) Respectively sucking 5 μl of the reference solution, 10 μl of the sample solution and 10 μl of the negative control solution, respectively spotting on the same silica gel G thin layer plate (10×10cm, qingdao ocean chemical plant), and spotting in the form of dots with a volume ratio of 20:10:11:5, placing the lower layer solution of ethyl acetate-chloroform-methanol-water below 10 ℃ for 30min as a developing agent, developing at the temperature: 25 ℃, relative humidity: 50%, presaturation for 15min, span: 6cm, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the spot color is clear, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the positions corresponding to those of the chromatogram of the reference sample, and the spots are negative and have no spots. The results are shown in FIG. 1.
(2) Identification of Atractylodis rhizoma by thin layer chromatography:
1) 30mL of the product is taken, 30mL of petroleum ether with the temperature of 30-60 ℃ is added, shaking extraction is carried out for 1 time, the petroleum ether layer is removed, water saturated n-butanol is added for 2 times, 30mL of each time is removed, the water layer is removed, the n-butanol extract liquid is combined, evaporation is carried out, and 1mL of methanol is added into residues to dissolve the residues to be used as a test sample solution.
2) Taking 30mL of a sample prepared from the white atractylodes rhizome lack medicinal material, adding 30mL of petroleum ether at 30-60 ℃, shaking and extracting for 1 time, discarding a petroleum ether layer, adding water saturated n-butanol and extracting for 2 times, each time 30mL of the petroleum ether layer, discarding a water layer, combining n-butanol extract liquid, evaporating to dryness, and adding 1mL of methanol into residues to dissolve the residues to serve as a negative control solution.
3) 2g of bighead atractylodes rhizome reference medicinal material is taken, 50mL of water is added, boiling is carried out for 30 minutes, filtering is carried out, the filtrate is concentrated into 30mL, 30mL of petroleum ether with the temperature of 30-60 ℃ is added, shaking extraction is carried out for 1 time, a petroleum ether layer is removed, water saturated n-butanol is added for 2 times, 30mL of each time is carried out, a water layer is removed, n-butanol extract liquid is combined, evaporation is carried out, and 1mL of methanol is added into residues to dissolve the residues to be used as reference medicinal material solution.
4) Respectively sucking 10 μl of sample solution, control medicinal material solution and negative control solution, respectively spotting on the same silica gel G thin layer plate (10×10cm, qingdao ocean chemical plant), and spotting in the form of dots with volume ratio of 7:3, developing with cyclohexane-ethyl acetate as developing agent at the temperature: 25 ℃, relative humidity: 50%, presaturation for 15min, span: 6cm, taking out, air drying, spraying 5% of 5% p-dimethylaminobenzaldehyde 10% sulfuric acid ethanol solution, heating at 105 ℃ for about 10 minutes, and immediately placing under an ultraviolet lamp (365 nm) for detection. In the chromatogram of the test sample, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the control medicinal material, and the spots are negative and have no spots. The results are shown in FIG. 2.
(3) Identifying pericarpium Citri Tangerinae by thin layer chromatography:
1) The sample solution was prepared by shaking 20mL of the sample with ethyl acetate for 3 times, 20mL each time, combining the ethyl acetate extracts, evaporating to dryness, and dissolving the residue with 1mL of methanol.
2) Taking 20mL of a sample prepared from the citrus reticulata Blume medicinal material, extracting with ethyl acetate for 3 times by shaking, 20mL each time, combining ethyl acetate extract, evaporating to dryness, and adding 1mL of methanol into the residue to dissolve the residue to obtain a negative control solution.
3) Taking 1g of pericarpium citri reticulatae reference medicinal material, adding 60mL of water, boiling for 30min, filtering, concentrating the filtrate into 20mL, shaking and extracting 3 times with ethyl acetate for 20mL each time, combining ethyl acetate extract, evaporating to dryness, and adding 1mL of methanol into residues to dissolve the residues to obtain a reference medicinal material solution.
4) Taking 1.7mg of hesperidin reference substance, adding 1mL of methanol, oscillating for 30s, standing at 4 ℃ overnight, and taking supernatant to obtain saturated solution of hesperidin reference substance as reference substance solution.
5) Respectively sucking 5 μl of control solution, test solution and negative control solution, respectively, and spotting 2 μl of control solution on the same silica gel G thin layer plate (10×10cm, qingdao ocean chemical plant) by dot sample application with volume ratio of 18:6:1, a lower solution of chloroform-methanol-water is used as a developing agent, and the developing temperature is as follows: 25 ℃, relative humidity: 50%, presaturation for 15min, span: 6cm, taking out, airing, spraying 1% of aluminum trichloride test solution, and placing under an ultraviolet lamp (365 nm) for inspection. In the chromatogram of the test sample, fluorescent spots with the same color appear at the positions corresponding to the chromatograms of the control medicinal material and the control sample, and the spots are negative and have no spots. The results are shown in FIG. 3.
The present inventors have also tested thin layer chromatography identification of prepared fleece flower root, radix codonopsis pilosulae and radix ophiopogonis when identifying oral liquid for invigorating spleen and replenishing qi by thin layer chromatography. However, (1) identification of prepared fleece flower root: the thin-layer chromatography detection results of the prepared fleece-flower root of the oral liquid for strengthening the spleen and tonifying qi in different storage time are different, and the thin-layer chromatography identification of the prepared fleece-flower root in the oral liquid for strengthening the spleen and tonifying qi is recommended to use the sample within 1 month from the production date, but the thin-layer chromatography identification of the prepared fleece-flower root is not carried out in the establishment of the quality detection standard of the oral liquid for strengthening the spleen. (2) identification of radix codonopsis: the method may be characterized in that the examination is not carried out due to the factors of extremely small content of the Codonopsis pilosula acetylenic glycoside in the test sample or improper extraction method, and the like, 2 extraction methods (respectively ethanol and petroleum ether) are replaced, and the results that the spots of the reference substances are clear but the reference medicinal materials and the test sample have no corresponding spots at the same position are all generated by various different developing agents (ethyl acetate-chloroform-methanol-water, cyclohexane-ethyl acetate and chloroform-methanol-water), so that the thin-layer chromatography identification of the Codonopsis pilosula cannot be carried out. (3) identification of dwarf lilyturf tuber: the sample solution and the negative sample solution show spots with the same color at the same position as the control medicinal material, namely negative has interference, has weak specificity and cannot be qualitatively determined. Therefore, the thin layer chromatography identification of the prepared fleece flower root, the dangshen and the dwarf lilyturf tuber is not established.
Example 2
The content of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the spleen-invigorating and qi-tonifying oral liquid is detected simultaneously by utilizing a high performance liquid chromatography method, and quantitative analysis is carried out, wherein the method comprises the following steps:
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed according to table 1 using acetonitrile-0.1% phosphoric acid solution as mobile phase; the detection wavelength is 260nm; the flow rate is 1.0mL/min; column temperature is 30 ℃; the theoretical plate number should be not less than 4000 as calculated by hesperidin peak.
TABLE 1 gradient elution table
| Time/min | Acetonitrile | 0.1% phosphoric acid solution |
| 0～12 | 17 | 83 |
| 13～25 | 37 | 63 |
| 26～40 | 32 | 68 |
| 41～42 | 17 | 83 |
| 43～55 | 13 | 87 |
(2) Preparation of a mixed control solution: taking a proper amount of calycosin glucoside reference substance, precisely weighing, adding methanol to prepare a solution containing 9.6 mug calycosin glucoside per 1mL, and filtering with a 0.45 mu m microporous filter membrane to obtain a calycosin glucoside reference substance solution; taking a proper amount of 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside reference substance, precisely weighing, adding diluted ethanol to prepare a solution containing 0.29mg of 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside per 1mL, and filtering the solution by using a microporous filter membrane with the thickness of 0.45 mu m to obtain a 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside reference substance solution; taking a proper amount of hesperidin reference substance, precisely weighing, adding methanol to prepare a solution containing 0.19mg hesperidin per 1mL, and filtering with a 0.45 μm microporous filter membrane to obtain a hesperidin reference substance solution; precisely measuring 1mL of each of the three reference substance solutions, and uniformly mixing to obtain a mixed reference substance solution.
(3) Preparation of test solution: precisely measuring 2mL of the product, placing into a 10mL measuring flask, adding methanol to the scale, shaking uniformly, and filtering with a 0.45um microporous filter membrane to obtain the product.
(4) Preparation of negative control solution: weighing other medicinal materials except radix astragali, pericarpium Citri Tangerinae and radix Polygoni Multiflori Preparata according to prescription amount, making into negative sample, precisely measuring 2mL of the negative sample, placing into 10mL measuring flask, adding methanol to scale, shaking, and filtering with 0.45 μm microporous membrane.
(5) And (3) measuring: respectively precisely sucking 10 μl of the mixed reference solution, the sample solution and the negative reference solution, and injecting into a liquid chromatograph for measurement. The results are shown in FIGS. 4-6.
Spleen-invigorating and qi-tonifying oral liquid comprises calycosin glucoside (C) per 1mL sample 22 H 22 O 10 ) Not less than 3.0 mu g The method comprises the steps of carrying out a first treatment on the surface of the With 2,3,5,4' -tetrahydroxystilbene-2-O-beta-D-glucoside (C) 20 H 22 O 9 ) Not less than 50.0. Mu.g; with hesperidin (C) 28 H 34 O 15 ) And not less than 50.0. Mu.g.
The high performance liquid chromatography also carries out methodology investigation and evaluates the accuracy of the measurement method, and the specific contents are as follows:
(1) Investigation of the linear Range:
1) Taking calycosin glucoside reference substance solution, respectively precisely sucking 10 μl, and injecting into high performance liquid chromatograph to determine peak area. The results are shown in Table 2, which shows that the linear relationship of Calycosin glucoside is good in this range.
TABLE 2 Linear relationship of Calycosin glucoside
2) 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside reference substance solutions with gradient concentrations of 0.1933, 0.4833, 0.9667, 1.4500 and 1.9333mg/mL are respectively prepared, 10 mu L of each reference substance solution with different concentrations is precisely sucked, and the reference substance solutions are injected into a high performance liquid chromatograph to determine peak areas. The results are shown in Table 3, and demonstrate that the linear relationship of 2,3,5,4' -tetrahydroxystilbene-2-O-. Beta. -D-glucoside is good in this range.
TABLE 3 stilbeneGlycoside(s)Linear relation table of (2)
3) The hesperidin reference substance solutions with gradient concentrations of 0.0967, 0.2417, 0.4833, 0.7250 and 0.9667mg/mL are respectively prepared, 10 mu L of the reference substance solutions with different concentrations are precisely sucked, and the solutions are injected into a high performance liquid chromatograph to determine peak areas. The results are shown in Table 4, which shows that hesperidin has a good linear relationship in this range.
Table 4 orange peelGlycoside(s)Linear relation table of (2)
(2) Precision test
Precisely measuring 2mL of the sample, placing in a 10mL measuring flask, adding methanol to the scale, shaking uniformly, filtering with a 0.45um microporous filter membrane to prepare a sample solution, sequentially taking 10 mu L of the same sample solution, and repeatedly sampling for 6 times, wherein the results are shown in Table 5, and the precision is good.
(3) Stability test
Taking the same sample solution, standing at room temperature, and respectively sampling and measuring at 0,2,4,8, 12 and 24 hours. The results are shown in Table 6, which demonstrate that the test solutions are stable over 24 hours.
(4) Repeatability test
Taking the same batch of samples, precisely measuring 2mL of the samples each time, placing the samples into a 10mL measuring flask, adding methanol to the scale, shaking uniformly, filtering by using a 0.45um microporous filter membrane, preparing a sample solution, preparing 6 parts, and measuring 2 times by sampling each part. The results are shown in Table 7, which demonstrate that the experimental method has good reproducibility.
(5) Sample recovery test
Precisely sucking 6 parts of samples with known contents, precisely adding reference substance solutions according to the following table, mixing, taking 2mL of the obtained mixed solution, placing into a 10mL measuring flask, adding methanol to scale, shaking, filtering with a 0.45um microporous filter membrane, preparing a sample-adding recovery solution, sampling, measuring, and calculating recovery rate. The results are shown below, indicating a better accuracy of the method.
Example 3
The content of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the spleen-invigorating and qi-tonifying oral liquid is detected simultaneously by utilizing a high performance liquid chromatography method, and quantitative analysis is carried out, wherein the method comprises the following steps:
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed according to table 1 using acetonitrile-n-butanol-0.1% phosphoric acid solution as mobile phase; the detection wavelength is 260nm; the flow rate is 1.0mL/min; column temperature is 30 ℃; the theoretical plate number should be not less than 4000 as calculated by hesperidin peak.
TABLE 1 gradient elution table
| Time/min | Acetonitrile | N-butanol | 0.1% phosphoric acid solution |
| 0～12 | 12 | 10 | 78 |
| 13～33 | 21 | 19 | 60 |
| 31～38 | 18 | 19 | 63 |
| 39～42 | 12 | 10 | 78 |
| 43～55 | 9 | 13 | 78 |
The preparation method of the rest mixed reference substance solution, the preparation method of the test substance solution and the preparation method of the negative reference solution are the same as in the example 2, respectively and precisely sucking 10 mu L of the mixed reference substance solution, the test substance solution and the negative reference solution, respectively, and injecting into a liquid chromatograph for measurement.
Precisely sucking 6 parts of samples with known contents, precisely adding reference substance solutions according to the following table, mixing, taking 2mL of the obtained mixed solution, placing into a 10mL measuring flask, adding methanol to scale, shaking, filtering with a 0.45um microporous filter membrane, preparing a sample-adding recovery solution, sampling, measuring, and calculating recovery rate. The results are shown below, indicating a better accuracy of the method.
Example 4
The content of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the spleen-invigorating and qi-tonifying oral liquid is detected simultaneously by utilizing a high performance liquid chromatography method, and quantitative analysis is carried out, wherein the method comprises the following steps:
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; gradient elution was performed according to table 1 using acetonitrile-n-butanol-0.1% phosphoric acid solution as mobile phase; the detection wavelength is 260nm; the flow rate is 1.0mL/min; column temperature is 30 ℃; the theoretical plate number should be not less than 4000 as calculated by hesperidin peak.
TABLE 1 gradient elution table
The preparation method of the rest mixed reference substance solution, the preparation method of the test substance solution and the preparation method of the negative reference solution are the same as in the example 2, respectively and precisely sucking 10 mu L of the mixed reference substance solution, the test substance solution and the negative reference solution, respectively, and injecting into a liquid chromatograph for measurement.
Precisely sucking 6 parts of samples with known contents, precisely adding reference substance solutions according to the following table, mixing, taking 2mL of the obtained mixed solution, placing into a 10mL measuring flask, adding methanol to scale, shaking, filtering with a 0.45um microporous filter membrane, preparing a sample-adding recovery solution, sampling, measuring, and calculating recovery rate. The results are shown below, indicating a better accuracy of the method.
The application firstly uses thin layer chromatography to carry out qualitative analysis on the oral liquid for strengthening spleen and supplementing qi, predicts by using the advantages of trace, quick and simple thin layer chromatography, carries out thin layer chromatography identification test on monarch drugs and ministerial drugs in the formula of the oral liquid for strengthening spleen and supplementing qi, carries out thin layer chromatography identification on astragalus root, white atractylodes rhizome and dried orange peel except that the thin layer chromatography of prepared fleece-flower root, radix codonopsis pilosulae and dwarf lilyturf tuber can not be established temporarily, and shows that the chromatography identification results show that: in the chromatogram of the sample, fluorescent spots with the same color appear on the positions corresponding to the chromatograms of the control medicinal materials and the control substances, and the spots are negative and have no spots; namely, the identification effect is good, and the thin-layer chromatography identification methods of the three components can be combined to establish the standard of qualitative analysis of the oral liquid for strengthening the spleen and tonifying qi.
The application utilizes high performance liquid chromatography to carry out quantitative analysis, and simultaneously detects the contents of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the spleen-invigorating and qi-tonifying oral liquid, thereby realizing ' one measurement and three evaluation ', detecting different components under the same wavelength, ensuring that the time of each wave peak is not overlapped, has proper size and no mutual interference, and has the advantages of simplicity, rapidness, accuracy, high efficiency and low cost. Astragaloside IV and calycosin glucoside are exclusive components of radix astragali, and are often used as a reference substance for thin layer chromatography identification, and the color development effect is obviously compared; the calycosin glucoside is used as a reference substance for measuring the content by high performance liquid chromatography, and the detection result is accurate. 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin are specific components of the prepared fleece-flower root and the dried orange peel respectively, and are used as reference substances for detecting the content of the prepared fleece-flower root and the dried orange peel in the traditional Chinese medicine preparation, so that the specificity is strong and the detection result is accurate. The high performance liquid chromatography is verified by methodology, has high precision, good repeatability, good stability, high recovery rate, good durability and accurate measurement result, and combines qualitative and quantitative analysis to lay a foundation for establishing the quality standard of the oral liquid for strengthening spleen and supplementing qi
It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.
Claims (1)
1. A quality detection method of an oral liquid for strengthening spleen and tonifying qi is characterized by comprising the following steps: comprising the steps of (a) a step of,
the components of the oral liquid for strengthening spleen and supplementing qi comprise astragalus, bighead atractylodes rhizome, dried orange peel, prepared polygonum multiflorum, dangshen and dwarf lilyturf tuber;
performing qualitative analysis on three components of radix astragali, atractylodis rhizoma and pericarpium Citri Tangerinae in the oral liquid by thin layer chromatography;
detecting the content of calycosin glucoside, 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside and hesperidin in the oral liquid by using high performance liquid chromatography;
the qualitative analysis is carried out on three components of astragalus, bighead atractylodes rhizome and dried orange peel in the oral liquid by using a thin layer chromatography, wherein the oral liquid is extracted by using n-butyl alcohol, petroleum ether and ethyl acetate respectively;
the qualitative analysis is carried out on three components of astragalus, bighead atractylodes rhizome and dried orange peel in the oral liquid by using a thin-layer chromatography, wherein the used developing agents are respectively in a volume ratio of 20:10:11:5, placing the lower layer solution of ethyl acetate-chloroform-methanol-water for 30min below 10 ℃ according to the volume ratio of 7:3 in a volume ratio of 18:6:1 trichloromethane-methanol-water lower layer solution;
the qualitative analysis includes:
(1) Identifying radix astragali by thin layer chromatography:
1) Taking 20mL of oral liquid for strengthening spleen and supplementing qi, extracting with water saturated n-butanol for 3 times by shaking, each time 20mL, combining n-butanol extract, washing with 1% sodium hydroxide test solution for 3 times, each time 20mL, discarding alkaline solution, combining n-butanol solution, washing with water saturated n-butanol to neutrality, evaporating n-butanol solution to dryness, and adding 1mL of methanol into residue to dissolve, thereby obtaining a sample solution;
2) Taking 20mL of spleen-invigorating and qi-tonifying oral liquid prepared from astragalus membranaceus, extracting with water saturated n-butanol for 3 times under shaking, 20mL each time, combining n-butanol extract, washing with 1% sodium hydroxide test solution for 3 times, 20mL each time, discarding alkali liquor, combining n-butanol solution, washing with water saturated n-butanol to neutrality, evaporating n-butanol solution to dryness, and adding 1mL of methanol into residue to dissolve, thereby obtaining a negative control solution;
3) Adding methanol into astragaloside IV reference substance to obtain solution containing 1mg astragaloside IV per 1mL, and taking the solution as reference substance solution;
4) Respectively sucking 5 mu L of the reference substance solution, 10 mu L of the test substance solution and 10 mu L of the negative control solution, respectively spotting on the same silica gel G thin layer plate, and adopting dot sample application according to the volume ratio of 20:10:11:5, placing the lower layer solution of ethyl acetate-chloroform-methanol-water below 10 ℃ for 30min as a developing agent, developing at the temperature: 25 ℃, relative humidity: 50%, presaturation for 15min, span: 6cm, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clear in color development, and inspecting under an ultraviolet lamp;
(2) Identification of Atractylodis rhizoma by thin layer chromatography:
1) Taking 30mL of spleen-invigorating and qi-tonifying oral liquid, adding 30mL of petroleum ether at 30-60 ℃, shaking and extracting for 1 time, discarding a petroleum ether layer, adding water-saturated n-butanol and extracting for 2 times, each time 30mL of petroleum ether layer, discarding a water layer, combining n-butanol extract liquid, evaporating to dryness, and adding 1mL of methanol into residues to dissolve the residues to be used as a sample solution;
2) Taking 30mL of spleen-strengthening and qi-tonifying oral liquid prepared from bighead atractylodes rhizome, adding 30mL of petroleum ether at 30-60 ℃, shaking and extracting for 1 time, discarding a petroleum ether layer, adding water saturated n-butanol and extracting for 2 times, 30mL each time, discarding a water layer, combining n-butanol extract liquid, evaporating to dryness, and adding 1mL of methanol into residues to dissolve, thereby obtaining a negative control solution;
3) 2g of bighead atractylodes rhizome reference medicinal material is taken, 50mL of water is added, boiling is carried out for 30 minutes, filtering is carried out, the filtrate is concentrated into 30mL, 30mL of petroleum ether with the temperature of 30-60 ℃ is added, shaking extraction is carried out for 1 time, a petroleum ether layer is removed, water saturated n-butanol is added for 2 times, 30mL of each time is carried out, a water layer is removed, n-butanol extract liquid is combined, evaporation is carried out, and 1mL of methanol is added into residues to dissolve the residues to be used as reference medicinal material solution;
4) Respectively sucking 10 mu L of the sample solution, the control medicinal material solution and the negative control solution, respectively spotting on the same silica gel G thin layer plate, and spotting in a dot shape according to the volume ratio of 7:3, developing with cyclohexane-ethyl acetate as developing agent at the temperature: 25 ℃, relative humidity: 50%, presaturation for 15min, span: 6cm, taking out, airing, spraying 5% of a 10% sulfuric acid ethanol solution of p-dimethylaminobenzaldehyde, heating at 105 ℃ for about 10 minutes, and immediately placing under an ultraviolet lamp for inspection;
(3) Identifying pericarpium Citri Tangerinae by thin layer chromatography:
1) Taking 20mL of oral liquid for strengthening spleen and supplementing qi, shaking and extracting with ethyl acetate for 3 times, 20mL each time, combining ethyl acetate extracting solutions, evaporating to dryness, and adding 1mL of methanol into residues to dissolve the residues to obtain a sample solution;
2) Taking 20mL of spleen-invigorating and qi-tonifying oral liquid prepared from the citrus reticulata blanco, extracting with ethyl acetate for 3 times by shaking, 20mL each time, combining ethyl acetate extracts, evaporating to dryness, and adding 1mL of methanol into residues to dissolve the residues to serve as a negative control solution;
3) Taking 1g of pericarpium citri reticulatae reference medicinal material, adding 60mL of water, boiling for 30min, filtering, concentrating the filtrate into 20mL, extracting with ethyl acetate for 3 times under shaking, 20mL each time, combining ethyl acetate extract, evaporating to dryness, and adding 1mL of methanol into the residue to dissolve the residue to obtain reference medicinal material solution;
4) Taking 1.7mg of hesperidin reference substance, adding 1mL of methanol, oscillating for 30s, standing at 4 ℃ overnight, and taking supernatant to obtain saturated solution of hesperidin reference substance as reference substance solution;
5) Respectively sucking 5 mu L of control solution, 5 mu L of test solution and 5 mu L of negative control solution, respectively spotting 2 mu L of control medicinal solution on the same silica gel G thin layer plate, and spotting in a dot shape according to the volume ratio of 18:6:1, a lower solution of chloroform-methanol-water is used as a developing agent, and the developing temperature is as follows: 25 ℃, relative humidity: 50%, presaturation for 15min, span: 6cm, taking out, airing, spraying 1% of aluminum trichloride test solution, and placing under an ultraviolet lamp for inspection;
the high performance liquid chromatography uses octadecylsilane chemically bonded silica as a filler, acetonitrile-0.1% phosphoric acid solution as a mobile phase, and the detection wavelength is 260nm; the flow rate is 1.0mL/min, the column temperature is 30 ℃, and gradient elution is carried out;
the gradient elution is specifically that the mobile phase is 17% acetonitrile and 83% 0.1% phosphoric acid solution in 0-12 min; 13-40 min, wherein the mobile phase is 37% acetonitrile and 63% 0.1% phosphoric acid solution; 41-55 min, wherein the mobile phase is 17% acetonitrile and 83% 0.1% phosphoric acid solution;
the theoretical plate number is not lower than 4000 calculated according to the hesperidin peak;
the oral liquid for strengthening spleen and tonifying qi is 3.0 mug or more in terms of calycosin glucoside per 1mL sample; 50.0 μg or more of 2,3,5,4' -tetrahydroxy stilbene-2-O-beta-D-glucoside; and 50.0 mug or more in terms of hesperidin.
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