CN116399966B - Construction method of feature map of cotton rose leaves - Google Patents
Construction method of feature map of cotton rose leaves Download PDFInfo
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- 235000003973 Hibiscus mutabilis Nutrition 0.000 title claims abstract description 87
- 238000010276 construction Methods 0.000 title claims abstract description 8
- 241000035884 Filago Species 0.000 title 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 132
- 244000048199 Hibiscus mutabilis Species 0.000 claims abstract description 131
- 239000012488 sample solution Substances 0.000 claims abstract description 51
- 239000013558 reference substance Substances 0.000 claims abstract description 49
- 238000001228 spectrum Methods 0.000 claims abstract description 41
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 claims abstract description 40
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- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims abstract description 37
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- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 claims abstract description 37
- 235000005493 rutin Nutrition 0.000 claims abstract description 37
- 229960004555 rutoside Drugs 0.000 claims abstract description 37
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 claims abstract description 20
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- 229940074360 caffeic acid Drugs 0.000 claims abstract description 20
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 claims abstract description 20
- DVGGLGXQSFURLP-FIZCXTQCSA-N potengriffioside A Natural products O[C@@H]1[C@@H](COC(=O)C=Cc2ccc(O)cc2)O[C@@H](Oc2c(oc3cc(O)cc(O)c3c2=O)-c2ccc(O)cc2)[C@H](O)[C@H]1O DVGGLGXQSFURLP-FIZCXTQCSA-N 0.000 claims abstract description 19
- LTRRTGCXRIMDTF-UHFFFAOYSA-N tiliroside Natural products OC1C(COC(=O)C=Cc2ccc(O)cc2)OC(OC3=C(Oc4cc(O)cc(O)c4C3)c5ccc(O)c(O)c5)C(O)C1O LTRRTGCXRIMDTF-UHFFFAOYSA-N 0.000 claims abstract description 19
- DVGGLGXQSFURLP-VWMSDXGPSA-N tribuloside Chemical compound C([C@@H]1[C@H]([C@@H]([C@@H](O)[C@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=CC(O)=CC=2)=O)O1)O)O)OC(=O)\C=C\C1=CC=C(O)C=C1 DVGGLGXQSFURLP-VWMSDXGPSA-N 0.000 claims abstract description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 17
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- XUDNWQSXPROHLK-OACYRQNASA-N 2-phenyl-3-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=CC=CC=2)OC2=CC=CC=C2C1=O XUDNWQSXPROHLK-OACYRQNASA-N 0.000 claims abstract description 11
- NLZCOTZRUWYPTP-MIUGBVLSSA-N 5-hydroxy-2-(4-methoxyphenyl)-7-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one Chemical compound C1=CC(OC)=CC=C1C(OC1=C2)=CC(=O)C1=C(O)C=C2O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 NLZCOTZRUWYPTP-MIUGBVLSSA-N 0.000 claims abstract description 11
- NLZCOTZRUWYPTP-UHFFFAOYSA-N acacetin-7-O-beta-D-galactoside Natural products C1=CC(OC)=CC=C1C(OC1=C2)=CC(=O)C1=C(O)C=C2OC1C(O)C(O)C(O)C(CO)O1 NLZCOTZRUWYPTP-UHFFFAOYSA-N 0.000 claims abstract description 11
- GWOKWCRSUJQOMD-UHFFFAOYSA-N tilianin Natural products C1=CC(OC)=CC=C1C(OC1=C2)=CC(=O)C1=CC=C2OC1C(O)C(O)C(O)C(CO)O1 GWOKWCRSUJQOMD-UHFFFAOYSA-N 0.000 claims abstract description 11
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
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- 238000001514 detection method Methods 0.000 claims description 11
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 10
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- 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
- G01N2030/8813—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 biological materials
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- 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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Abstract
The invention discloses a construction method of a characteristic map of cotton rose leaves, which comprises the following steps: extracting folium Hibisci Mutabilis with methanol or ethanol to obtain sample solution; extracting caffeic acid reference substance, rutin reference substance, tiliroside reference substance and flos Wallichii flavonol glycoside I reference substance with methanol or dissolving to obtain reference substance solution; and (3) measuring the sample solution and the reference solution by adopting a liquid chromatograph to obtain a characteristic spectrum, the rutin content and the tilianin content. The characteristic map obtained by implementing the invention has rich information quantity of characteristic peaks, particularly has more characteristic peaks of flavonoid components, and can rapidly and comprehensively reflect the quality of the cotton rose hibiscus leaves by measuring the multi-index components.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a construction method of a characteristic spectrum of cotton rose leaves.
Background
The Chinese pharmacopoeia 2020 edition records that the Hibiscus sabdariffa leaf is a dry leaf of Hibiscus sabdariffa Hibiscus mutabilis L. Collected in summer and autumn, and dried. It has slightly pungent taste, and has effects of clearing heat and detoxicating, detumescence and expelling pus, cooling blood and stopping bleeding and relieving pain, and can be used for treating carbuncle, swelling and pain, wound, snake dan, scald, conjunctival congestion and swelling and pain, traumatic injury, etc. The flowers, leaves and roots of Hibiscus sabdariffa can be used as medicinal parts, and the description of the' materia medica schema: the cottonrose hibiscus leaves, which are smooth in smell, not cold or hot, slightly pungent in taste and slimy in saliva, have the effect of treating carbuncle and swelling, and are used in combination with cottonrose hibiscus leaves, root bark, flower, raw or ground or dried and ground into powder, and are mixed with honey to be spread around the swelling, the head is left in the middle, and the dry nature is changed frequently. The first person is cool and refreshing, and the pain is relieved. The already established one, pus and toxin are accumulated. The wearer is easy to astringe after pus is discharged. Not to say, it can be seen that the leaves, flowers and roots of the cottonrose hibiscus have the effect of enhancing the curative effect. According to the herb examination, the leaves of Hibiscus are called frost-resistant leaves in Shi Yi De Fang (effective prescription of medicine), hibiscus flower leaves in Pu Ji Fang (prescription of medicine), and Tie Qian san in Hunan Yao Zhi (medicinal emotion of medicine). The cotton rose leaves are mainly distributed in Hunan, hubei, guangxi, guangdong, sichuan and Guizhou provinces.
In recent years, researches on chemical components and pharmacological actions of cottonrose hibiscus are mainly focused on leaves of cottonrose hibiscus, and the cottonrose hibiscus is recorded in Chinese pharmacopoeia of 2020 edition as a new medicinal material, and is widely used for relieving pain, detoxifying, eliminating phlegm and the like. Recent pharmacological studies show that the cotton rose leaves have the activities of resisting infection, inhibiting bacteria and viruses outside antibodies, protecting chronic liver injury and the like, and are clinically applied for many years. The chemical components contained in the Hibiscus Mutabilis leaf comprise flavonoids, organic acids, coumarin, triterpenes, lignans, volatile components, etc. The main chemical components of the Hibiscus Mutabilis leaf are flavonoids, and have antioxidant, cardiovascular protecting, antitumor, anticancer, antibacterial, antiinflammatory, antiviral, and immunity enhancing effects. Therefore, in order to ensure the quality of the cotton rose leaves, the flavonoid components of the cotton rose leaves need to be researched.
The method provides a quality detection method of a standard decoction of Hibiscus Mutabilis leaves, which uses rutin as a reference substance and acetonitrile-0.1% phosphoric acid solution as a mobile phase for liquid chromatograph analysis, so as to construct a characteristic spectrum of the standard decoction of Hibiscus Mutabilis leaves, determine 7 characteristic peaks and identify 3 characteristic peaks. However, the method has poor separation degree, the response value of each characteristic peak is low, the characteristic spectrum contains less information quantity of the characteristic peak, and the quality characteristics of the cotton rose hibiscus leaves are not fully reflected.
Disclosure of Invention
The technical problem to be solved by the invention is to provide the construction method of the characteristic spectrum of the cotton rose hibiscus leaves, which has rich characteristic peak information and can comprehensively reflect the quality characteristics of the cotton rose hibiscus leaves.
In order to solve the technical problems, the invention provides a construction method of a characteristic map of cotton rose leaves, which comprises the following steps:
extracting folium Hibisci Mutabilis with extraction solvent to obtain sample solution; wherein the extraction solvent is methanol or ethanol;
extracting caffeic acid reference substance, rutin reference substance, tiliroside reference substance and flos Wallichii flavonol glycoside I reference substance with methanol or dissolving to obtain reference substance solution;
measuring the sample solution and the reference solution by adopting a liquid chromatograph to obtain a characteristic map, the content of rutin and the content of tilianin;
wherein, the liquid chromatograph uses octadecylsilane chemically bonded silica as a stationary phase, a mixed solution of acetonitrile and tetrahydrofuran as a mobile phase A, and 0.05-0.3 vol% of phosphoric acid as a mobile phase B for gradient elution, and the gradient elution curve is as follows:
0 min-12 min, mobile phase A from 0% to 2%, mobile phase B from 100% to 98%;
12-25 min, mobile phase A from 2% -9%, mobile phase B from 98% -91%;
25-40 min, mobile phase A from 9% -15%, mobile phase B from 91% -85%;
40-48 min, mobile phase A from 15% -32%, mobile phase B from 85% -68%;
48 min-54 min, mobile phase A is 32%, mobile phase B is 68%.
As an improvement of the technical scheme, in the mobile phase A, the volume ratio of acetonitrile to tetrahydrofuran is (2-5): 1, wherein the mobile phase B is 0.1vol% phosphoric acid.
As an improvement of the above technical scheme, in mobile phase a, the volume ratio of acetonitrile to tetrahydrofuran is 3:1.
as an improvement of the technical scheme, the particle size of the stationary phase is 1.5-2 μm;
the column length of a chromatographic column of the liquid chromatograph is 100-200 mm, the column diameter is 2-5 mm, the column temperature is 40-50 ℃, and the flow rate is 0.38-0.42 mL/min;
the detection wavelength of the liquid chromatograph is 278 nm-330 nm.
As an improvement of the above technical scheme, the particle size of the stationary phase is 1.8 μm;
the column length of a chromatographic column of the liquid chromatograph is 150mm, the column diameter is 2.1mm, the column temperature is 45 ℃, and the flow rate is 0.38 mL/min-0.42 mL/min;
the detection wavelength of the liquid chromatograph is 315nm.
As an improvement of the technical scheme, the extraction solvent is 50-70 vol% methanol or 50vol% ethanol, the extraction mode is heating reflux extraction or ultrasonic extraction, and the extraction time is 10-60 min.
As an improvement of the technical scheme, the preparation method of the sample solution comprises the following steps: taking 0.1g to 1g of cottonrose hibiscus leaf medicinal material or cottonrose hibiscus leaf decoction pieces, adding 20mL to 40mL of 50vol% -70 vol% methanol, weighing, heating and refluxing for 15min to 60min, cooling, filtering, taking 50vol% -70 vol% methanol to complement the lost weight, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the extract;
or (b)
The preparation method of the sample solution comprises the following steps: taking 0.1g to 1g of standard decoction of the cotton rose leaves or traditional Chinese medicine formula particles of the cotton rose leaves, adding 20mL to 40mL of 50vol% -70 vol% methanol, weighing, adopting ultrasonic treatment with the frequency of 20kHz to 50kHz for 20min to 40min, taking out, cooling, filtering, taking 50vol% -70 vol% methanol to complement the lost weight, shaking uniformly, filtering, and taking subsequent filtrate to obtain the cotton rose tea.
As an improvement of the technical scheme, the preparation method of the sample solution comprises the following steps: taking 0.5g of Hibiscus Mutabilis leaf medicinal material or Hibiscus Mutabilis leaf decoction pieces, adding 25mL of 70vol% methanol, weighing, heating and refluxing for 30min, cooling, weighing again, taking 70vol% methanol to supplement the weight of loss, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the final product;
or (b)
The preparation method of the sample solution comprises the following steps: taking 0.2g of standard decoction of Hibiscus Mutabilis leaf or traditional Chinese medicine granule of Hibiscus Mutabilis leaf, adding 25mL of 70vol% methanol, performing ultrasonic treatment at 40kHz for 30min, taking out, cooling, filtering, taking 70vol% methanol to supplement the weight of loss, shaking, filtering, and collecting subsequent filtrate.
As an improvement of the above technical solution, the preparation method of the reference solution comprises: mixing tiliroside reference substance, rutin reference substance, caffeic acid reference substance, and flos Hedyotidis Diffusae flavonol glycoside I reference substance with methanol to obtain mixed solution containing tiliroside reference substance 20 μg, rutin reference substance 40 μg, caffeic acid reference substance 50 μg, and flos Hedyotidis Diffusae flavonol glycoside I reference substance 10 μg per 1 mL.
As improvement of the technical scheme, the characteristic spectrum has 13 peaks, wherein, peak 7 is caffeic acid, peak 11 is rutin, peak 12 is tiliroside, and peak 13 is hopogonidol I;
calculating relative retention times of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 8, peak 9, peak 10 and the S1 peak with respect to each other, the relative retention times of the peaks being within ±10% of a prescribed value, wherein the prescribed value of peak 1 is 0.30, the prescribed value of peak 2 is 0.41, the prescribed value of peak 3 is 0.44, the prescribed value of peak 4 is 0.47, the prescribed value of peak 5 is 0.78, the prescribed value of peak 6 is 0.96, the prescribed value of peak 8 is 1.05, the prescribed value of peak 9 is 1.22, and the prescribed value of peak 10 is 1.49, with respect to the S1 peak;
the relative retention time of the peak 13 and the S2 peak was calculated with the peak 12 as the S2 peak, and the relative retention time of the peak 13 was within.+ -. 10% of the prescribed value, wherein the prescribed value of the peak 13 was 1.01.
The implementation of the invention has the following beneficial effects:
the invention establishes the characteristic map of the cotton rose hibiscus leaf, has rich information of characteristic peaks, particularly has more characteristic peaks of flavonoid components, can rapidly and comprehensively reflect the quality of the cotton rose hibiscus leaf by measuring multi-index components. The characteristic spectrum construction method and the multi-index content detection method are simultaneously suitable for relevant detection of the cottonrose hibiscus leaf medicinal materials, cottonrose hibiscus leaf decoction pieces, cottonrose hibiscus leaf standard decoction and cottonrose hibiscus leaf traditional Chinese medicine formula particles, and have wide range of application.
Drawings
FIG. 1 is a characteristic map of Hibiscus Mutabilis leaf in example 1 using different extraction solvents;
FIG. 2 is a characteristic map of Hibiscus Mutabilis leaf in example 1 using different extraction methods;
FIG. 3 is a characteristic map of Hibiscus Mutabilis leaf at different extraction times in example 1;
FIG. 4 is a characteristic spectrum of Hibiscus Mutabilis leaf (condition 1 to condition 6) in example 1 using different gradient elution procedures and mobile phases;
FIG. 5 is a characteristic map of Hibiscus Mutabilis leaf (condition 7 to condition 11) in example 1 using different gradient elution procedures and mobile phases;
FIG. 6 is a characteristic spectrum of Hibiscus Mutabilis leaf in example 1 using different absorption wavelengths;
FIG. 7 is a diagram of the results of specific investigation of the characteristic pattern of Hibiscus Mutabilis leaf in example 1;
FIG. 8 is a characteristic spectrum of Hibiscus Mutabilis leaf measured by different personnel using different instruments in example 1;
FIG. 9 is a graph of the results of investigation of different column temperatures of the characteristic spectrum of Hibiscus Mutabilis leaf in example 1;
FIG. 10 is a graph of the results of investigation of different flow rates of the characteristic spectrum of Hibiscus Mutabilis leaf in example 1;
FIG. 11 is a diagram of the results of different chromatographic columns of the characteristic spectrum of Hibiscus Mutabilis leaf in example 1;
FIG. 12 is a pattern diagram of the characteristics of Hibiscus Mutabilis leaf medicinal material in example 1;
FIG. 13 is a comparison characteristic map of Hibiscus Mutabilis leaf medicinal material in example 1;
FIG. 14 is a pattern diagram of the feature map of the Hibiscus Mutabilis decoction pieces in example 1;
FIG. 15 is a graph of the comparison characteristics of Hibiscus Mutabilis decoction pieces in example 1;
FIG. 16 is a pattern diagram of the standard decoction feature of Hibiscus Mutabilis leaf in example 1;
FIG. 17 is a feature map of the standard decoction of Hibiscus Mutabilis leaf in example 1;
FIG. 18 is a pattern diagram of the characteristics of the Chinese medicinal granule in Hibiscus Mutabilis in example 1;
fig. 19 is a characteristic spectrum of the cotton rose leaf traditional Chinese medicine formula granule in example 1.
Detailed Description
The present invention will be described in further detail with reference to the drawings and the detailed description, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
Example 1 method for establishing characteristic Property of Hibiscus Mutabilis leaf
1. Instrument, reagent and reagent
Instrument: thermo ultra-high performance liquid chromatography (Vanquish, simer Feishi technologies (China), waters ultra-high performance liquid chromatography (H-Class, waters, agilent 1290, agilent), waters HSS T3 chromatography column (2.1 mm. Times.150 mm,1.8 μm), one-ten-thousandle balance (ME 204E, metrel-Toli), part-per-day flat (XP 26, metrel-Toli), digital controlled ultrasonic cleaner (KQ 500D, kunshan ultrasonic apparatus, inc.), thermostatted water bath (HW 28, shanghai) ultrapure water systems (Milli-Q Direct, g).
Reagent: ethanol (limited of the scientific sciences of the ridge) and methanol (limited of the scientific sciences of the ridge) are all analytically pure; the liquid phase was HPLC chromatography grade with phosphoric acid (Miro Euro chemical reagent Co., tianjin, inc.), acetonitrile, tetrahydrofuran (Merck Co., ltd.) and the water was ultra pure water (laboratory self-made).
Reagent: rutin control (lot number: 100080-201811, content is 91.6%, chinese food and drug verification institute), caffeic acid control (lot number: 110885-201603, content is 99.7%, chinese food and drug verification institute), tiliroside control (lot number: 5369, content is 94.9%, shanghai Shidand standard technical service Co., ltd.), telescoping flavonol glycoside I control (lot number: CFS202002, content is 98%, chem Faces).
The Chinese pharmacopoeia 2020 edition one specifies that cottonrose She Jiyuan is a dried leaf of cottonrose Hibiscus mutabilis L. All the medicinal materials used in the research are identified by Chinese medical institute of Chinese traditional medicine department. Specific information is shown in Table 1.
TABLE 1 sample Source information Table
2. Measurement method
2.1 chromatographic conditions
The column was a Waters HSS T3 (2.1 mm. Times.150 mm,1.8 μm); gradient elution was performed as specified in table 2 with acetonitrile-tetrahydrofuran (3:1) mixed solution as mobile phase a and 0.1% phosphoric acid solution as mobile phase B; the flow rate was 0.40mL per minute; the column temperature is 45 ℃; the detection wavelength was 315nm. The theoretical plate number is not less than 5000 according to rutin peak. The sample loading was 1. Mu.L.
TABLE 2 gradient elution table
2.2 preparation of sample solutions
Medicinal materials and decoction pieces: taking proper amount of Hibiscus Mutabilis leaf medicinal material or decoction piece powder (sieving with a third sieve), taking about 0.5g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 70% methanol, weighing, heating and refluxing for 30min, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking, filtering, and collecting the subsequent filtrate.
Standard decoction and traditional Chinese medicine formula granule: taking a proper amount of Hibiscus Mutabilis leaf standard decoction or traditional Chinese medicine formula granule, grinding, taking about 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 25mL of 70% methanol, weighing, performing ultrasonic treatment (power 250W, frequency 40 KHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, and taking subsequent filtrate.
2.3 preparation of reference solutions
Mixing tiliroside reference substance, rutin reference substance, caffeic acid reference substance, and flos Hedyotidis Diffusae flavonol glycoside I reference substance with methanol to obtain mixed solution containing tiliroside reference substance 20 μg, rutin reference substance 40 μg, caffeic acid reference substance 50 μg, and flos Hedyotidis Diffusae flavonol glycoside I reference substance 10 μg per 1 mL.
2.4 assay
Precisely sucking 1 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
3. Determination of chromatographic conditions
3.1 examination of the preparation method of the sample solution
And (3) respectively examining the influence of the extraction solvent, the extraction mode and the extraction time on the characteristic spectrum of the cotton rose hibiscus leaf medicinal material through single factor analysis so as to determine an optimal sample pretreatment method.
(1) Investigation of the extraction solvent
Taking about 0.5g of Hibiscus Mutabilis leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, parallel-weighing 4 groups, 2 parts of each group, placing into a conical flask with a plug, respectively adding 25mL of 70% methanol, 50% methanol, 70% ethanol and 50% ethanol, weighing, performing ultrasonic treatment (power 300W, frequency 50 kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the reduced weight with corresponding solvent, shaking uniformly, filtering, and taking the subsequent filtrate for sample injection measurement according to the conditions under 2.1. The results of investigation of different extraction solvents of the characteristic spectrum of the cotton rose hibiscus leaf medicinal material are shown in table 3 and figure 1.
TABLE 3 investigation results table of different extraction solvents of characteristic patterns of Hibiscus Mutabilis leaf
By comparing the characteristic patterns of 4 extraction solvents, it can be found that the total peak area/sample amount is obviously different when different solvents are used for extraction, and the total peak area/sample amount is the largest when 70% ethanol and 70% methanol are used, but the corresponding chromatographic peak pattern is poor when 70% ethanol is used, and the peak patterns of each chromatographic peak are better when 70% methanol is used as the extraction solvent, so that 70% methanol is used as the extraction solvent of the characteristic pattern of the cotton rose hibiscus leaf medicinal material.
(2) Investigation of the extraction method
Taking about 0.5g of Hibiscus Mutabilis leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing 2 groups, 2 parts each group, placing into a conical flask with a plug, adding 25mL of 70% methanol, weighing, respectively performing ultrasonic treatment (power 300W and frequency 50 kHz) for 30 minutes, heating and refluxing for 30 minutes, taking out, cooling, supplementing the lost weight with 70% methanol, shaking, filtering, taking out the subsequent filtrate, and performing sample injection measurement according to the conditions under 2.1. The results of different extraction modes of the characteristic spectrum of the cotton rose hibiscus leaf are shown in table 4 and figure 2.
TABLE 4 investigation results table of different extraction modes of Hibiscus Mutabilis leaf feature patterns
The feature map obtained by heating reflux extraction is found to be higher in total peak area/sample amount by comparing ultrasonic treatment and reflux extraction, so that the heating reflux is selected as an extraction mode.
(3) Investigation of extraction time
Taking about 0.5g of Hibiscus Mutabilis leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, parallel-weighing 4 groups, 2 parts each group, placing into a conical flask with a plug, precisely adding 25mL of 70% methanol, respectively heating and refluxing for 15min, 30min, 45 min and 60min, taking out, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and carrying out sample injection measurement according to the conditions under 2.1. The results of investigation of different extraction time of the characteristic spectrum of the cotton rose hibiscus leaf medicinal material are shown in table 5 and figure 3.
TABLE 5 investigation results table of different extraction times of Hibiscus Mutabilis leaf feature patterns
By comparing the influence of different extraction times on the characteristic spectrum of the cotton rose hibiscus leaf medicinal material, the influence of different extraction times on the characteristic spectrum of the cotton rose hibiscus leaf medicinal material is not great, and heating reflux is selected for 30 minutes to ensure complete extraction.
3.2 gradient elution procedure, investigation of mobile phase
Taking about 0.5g of Hibiscus Mutabilis leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, preparing a sample solution according to the sample solution preparation method determined under 2.2, measuring according to the conditions of Table 6, and performing sample injection analysis under other chromatographic conditions as specified under 2.1. The results are shown in FIGS. 4 and 5.
TABLE 6 chromatographic conditions of Hibiscus Mutabilis leaf feature map
According to the investigation result, different elution gradients and different organics have obvious influence on the separation of each chromatographic peak of the cotton rose characteristic spectrum, and compared with different organic phase chromatograms of the same chromatographic condition of the condition 5 and the condition 6, the chromatographic peak separation effect is better when acetonitrile-tetrahydrofuran (3:1) is used; when the using condition 11 is optimized by the chromatographic condition, the separation degree of each chromatographic peak is better, and the chromatographic peak distribution is more uniform. Thus, it was confirmed that the gradient elution was carried out as defined in condition 11 using acetonitrile-tetrahydrofuran (3:1) mixed solution as mobile phase A and 0.1% phosphoric acid as mobile phase B.
3.3 investigation of absorption wavelength
Taking a proper amount of the standard decoction of the cotton rose hibiscus leaves, grinding, taking about 0.2g, precisely weighing, and preparing a sample solution according to the sample solution preparation method determined under the condition of 2.2. And measured according to the following chromatographic conditions:
chromatographic conditions: waters HSS T3 column (2.1 mm. Times.150 mm,1.8 μm) column; gradient elution was performed with acetonitrile-tetrahydrofuran (3:1) mixed solution as mobile phase a and 0.1% phosphoric acid solution as mobile phase B, as specified in condition 10 of table 6; the flow rate was 0.40mL per minute; the column temperature was 45℃and the measurement wavelengths were 254nm, 278nm, 315nm, 330nm and 365nm, respectively. The results are shown in FIG. 6.
By comparing the 5 detection wavelength chromatograms, the chromatographic peak information is more when 315nm is selected as the detection wavelength, so 315m is selected as the detection wavelength of the cotton rose hibiscus leaf characteristic map.
4. Methodological verification
4.1 investigation of specificity
Precisely sucking 1 μl of each of the sample solution, reference solution and blank solvent of folium Hibisci Mutabilis (MFRY 06), and measuring with liquid chromatograph under 2.1 chromatographic conditions. The results are shown in FIG. 7.
The result shows that the sample chromatogram has the same chromatographic peak at the retention time corresponding to the reference chromatogram, and the negative is not interfered, which indicates that the method has good specificity.
4.2 precision investigation
About 0.5g of the cotton rose hibiscus leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, preparing a sample solution according to the sample solution preparation method under 2.2, analyzing according to chromatographic conditions under 2.1, taking the peak corresponding to the caffeic acid reference peak as an S1 peak, calculating relative retention time of the peak 1, the peak 2, the peak 3, the peak 4, the peak 5, the peak 6, the peak 8, the peak 9, the peak 10 and the S1 peak, taking the peak corresponding to the basswood glycoside reference peak as an S2 peak, calculating relative retention time of the peak 13 and the S2 peak and relative peak area of each characteristic peak, and calculating an RSD value. The experimental results are shown in tables 7 and 8.
Table 7 Hibiscus sabdariffa She Tezheng map precision investigation result table (relative retention time)
Table 8 results table (relative peak area) of the precision investigation of Hibiscus Mutabilis She Tezheng map
The result shows that the same sample solution is continuously sampled for 6 times, the relative retention time RSD value of each characteristic peak is in the range of 0.01-0.22%, the relative peak area RSD value is in the range of 1.94-4.25%, and the RSD values are all less than 5%, which indicates that the instrument precision is good.
4.3 repeatability investigation
About 0.5g of the cotton rose hibiscus leaf medicinal material powder (sieving with a No. three sieve, batch number: MFRY 06), precisely weighing in parallel, preparing a sample solution according to the sample solution preparation method under 2.2, analyzing according to chromatographic conditions under 2.1, calculating relative retention time of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 8, peak 9, peak 10 and S1 peak by taking the peak corresponding to the caffeic acid reference peak as S1 peak, calculating relative retention time of peak 13 and S2 peak and relative peak area of each characteristic peak by taking the peak corresponding to the basswood glycoside reference peak as S2 peak, and calculating RSD value. The experimental results are shown in tables 9 and 10.
TABLE 9 results table of repeatability of feature patterns of Hibiscus Mutabilis (relative retention time)
TABLE 10 results table of repeatability of feature patterns of Hibiscus Mutabilis (relative peak area)
The result shows that the relative retention time RSD value of each characteristic peak and S peak is in the range of 0.00% -0.11%, and the relative peak area RSD value is in the range of 3.23% -4.95%, which are all less than 5.0%, thus the method has good repeatability.
4.4 stability investigation
About 0.5g of the cotton rose hibiscus leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, preparing a sample solution according to the sample solution preparation method under 2.2, carrying out sample injection analysis under 2.1 chromatographic conditions for 0,2,4,8, 10 and 24 hours respectively, taking the peak corresponding to the caffeic acid reference peak as an S1 peak, calculating relative retention time of the peak 1, the peak 2, the peak 3, the peak 4, the peak 5, the peak 6, the peak 8, the peak 9, the peak 10 and the S1 peak, taking the peak corresponding to the tilianin reference peak as an S2 peak, calculating relative retention time of the peak 13 and the S2 peak and relative peak area of each characteristic peak, and calculating an RSD value. The experimental results are shown in tables 11 and 12.
TABLE 11 results table for investigating stability of Hibiscus Mutabilis leaf feature map (relative retention time)
Table 12 results table (relative peak area) for investigating stability of characteristic spectrum of Hibiscus Mutabilis leaf
The results show that the same sample solution is analyzed in 0,2,4,8, 10 and 24 hours, the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.01-0.12 percent, the relative peak area RSD value is in the range of 2.33-4.82 percent, and the relative retention time RSD value is less than 5.0 percent, which indicates that the sample solution is relatively stable in 24 hours.
4.5 intermediate precision investigation
Taking about 0.5g of Hibiscus Mutabilis leaf medicinal powder (sieved by a third sieve, batch number: MFRY 06), precisely weighing in parallel 6 parts, preparing 6 parts of sample solution according to the sample solution preparation method under 2.2, preparing 6 parts of sample solution according to the chromatographic condition under 2.1, sampling and analyzing, taking the peak corresponding to the caffeic acid reference substance peak as S1 peak, calculating the relative retention time of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 8, peak 9, peak 10 and S1 peak, taking the peak corresponding to the tilianin reference substance peak as S2 peak, calculating the relative retention time of peak 13 and S2 peak and the relative peak area of each characteristic peak, and calculating the RSD value. The experimental results are shown in table 13, table 14 and fig. 8.
Table 13 results table (relative retention time) of intermediate precision investigation of Hibiscus Mutabilis leaf feature patterns
Table 14 results table (relative peak area) of the Hibiscus Mutabilis leaf feature map for intermediate precision investigation
The results show that different analysts operate on different instruments at different times, the same batch of samples is repeatedly measured for 6 times by the personnel 2, the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.00-1.40%, and the relative peak area RSD value is in the range of 2.65-4.89%; the RSD value of the relative retention time and the RSD value of the 6 data of the repeatability test are in the range of 0.05% -3.26%, the relative peak area of each characteristic peak and the RSD value of the 6 data of the repeatability test are in the range of 3.58% -10.38%, the relative retention time RSD value is less than 5%, the fluctuation of the RSD value of the relative peak area of the intermediate precision is large, and the RSD value of part of the characteristic peaks exceeds 5%, which indicates that the intermediate precision of different chromatographs is good, and the relative peak areas of the peaks 2-4, 6, 7 and 10 are poor, so the relative peak area limit of the characteristic peak is not specified temporarily.
4.6 durability inspection
(1) Investigation of different column temperatures
Taking about 0.5g of Hibiscus Mutabilis leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, preparing a sample solution according to the sample solution preparation method under 2.2, carrying out sample injection analysis under the chromatographic conditions under 2.1, wherein the column temperature is controlled to be 40 ℃, 45 ℃ and 50 ℃, taking the peak corresponding to the caffeic acid reference peak as an S1 peak, calculating the relative retention time of the peak 1, the peak 2, the peak 3, the peak 4, the peak 5, the peak 6, the peak 8, the peak 9, the peak 10 and the S1 peak, taking the peak corresponding to the tilianin reference peak as an S2 peak, calculating the relative retention time of the peak 13 and the S2 peak and the relative peak area of each characteristic peak, and calculating the RSD value. The experimental results are shown in table 15, table 16 and fig. 9.
Table 15 column temperature investigation result table (relative retention time) of Hibiscus sabdariffa She Tezheng map
Table 16 column temperature investigation result table (relative peak area) of Hibiscus sabdariffa She Tezheng map
The result shows that the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.04% -2.36% and is less than 5.0% under different column temperatures, which shows that the change of the column temperature has less influence on the relative retention time of each characteristic peak when the column temperature is +/-5 ℃; the relative peak area RSD value is in the range of 0.26% -4.63% and less than 5.0%, indicating that the change of the column temperature has less influence on the relative peak area of each characteristic peak when the column temperature is + -5 deg.c.
(2) Investigation of different flow rates
Taking about 0.5g of Hibiscus Mutabilis leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, preparing a sample solution according to the sample solution preparation method under 2.2, preparing a sample solution according to the chromatographic conditions under 2.1, controlling the flow rates to be 0.38mL/min, 0.40mL/min and 0.42mL/min respectively, carrying out sample injection analysis, taking the peak corresponding to the caffeic acid reference peak as an S1 peak, calculating the relative retention time of the peak 1, the peak 2, the peak 3, the peak 4, the peak 5, the peak 6, the peak 8, the peak 9, the peak 10 and the S1 peak, taking the peak corresponding to the basswood glycoside reference peak as an S2 peak, calculating the relative retention time of the peak 13 and the S2 peak and the relative peak area of each characteristic peak, and calculating the RSD value. The experimental results are shown in Table 17, table 18 and FIG. 10.
Table 17 Hibiscus sabdariffa She Tezheng map flow velocity investigation result table (relative retention time)
Table 18 Hibiscus sabdariffa She Tezheng map flow velocity investigation result table (relative peak area)
The result shows that the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.03% -1.95% and is less than 5.0% under different flow rates, and the change of the flow rate has less influence on the relative retention time of each characteristic peak when the flow rate is +/-0.02 mL/min; the relative peak area RSD value is in the range of 3.29% to 4.85% and less than 5.0%, indicating that the change in flow rate has less effect on the relative peak area of each characteristic peak when the flow rate is ±0.02 mL/min.
(3) Investigation of different chromatographic columns
Taking about 0.5g of Hibiscus Mutabilis leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, preparing a sample solution according to the sample solution preparation method under 2.2, preparing relative retention time of peak 13 and peak S2 and relative peak area of each characteristic peak according to chromatographic conditions under 2.1, wherein the adopted chromatographic columns are respectively Waters CORTECS T3 columns (with numbers of BH-317, BH-302 and BH-278), analyzing by sample injection, calculating relative retention time of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 8, peak 9, peak 10 and peak S1 by taking peak corresponding to caffeic acid reference as peak S2, calculating relative retention time of peak 13 and peak S2 and relative peak area of each characteristic peak, and calculating RSD value. The experimental results are shown in Table 19, table 20 and FIG. 11.
Table 19 Hibiscus sabdariffa She Tezheng chromatographic column investigation results table (relative retention time)
Table 20 Hibiscus sabdariffa She Tezheng chromatographic column investigation result table (relative peak area)
The results show that the relative retention time RSD value of each characteristic peak and S peak is in the range of 0.01% -0.60%, the relative peak area RSD value is in the range of 2.28% -4.87% and is less than 5%, and the durability of the Waters HSS T3 chromatographic columns (2.1 mm multiplied by 150mm,1.8 μm) of different batches is good.
(4) Durable knot
The durability investigation result shows that the characteristic spectrum method has poor durability on chromatographic columns of different brands, and adopts Waters HSS T3 chromatographic columns, and the peak type and the whole separation effect of each characteristic peak are good, so the fixed chromatographic column is suggested to be the Waters HSS T3 chromatographic column under the chromatographic condition of the characteristic spectrum. The peak corresponding to the caffeic acid reference peak is taken as an S1 peak, the peak corresponding to the linden glycoside reference peak is taken as an S2 peak, the relative retention time of each characteristic peak and the S peak is less influenced by column temperature, flow rate and chromatographic columns of different batches of the same brand, and the relative peak area of each characteristic peak is greatly different among different chromatographs, so that the relative peak area range of each characteristic peak is temporarily not specified by individual chromatographic peaks.
5. Determination of characteristic patterns
Taking the cottonrose hibiscus leaf medicinal materials and decoction pieces, preparing a sample solution according to the sample preparation method under the 2.2 item, carrying out sample injection measurement according to the chromatographic condition under the 2.1 item to obtain a cottonrose hibiscus leaf characteristic map, generating a control map according to an average method (or a median method) by using Chinese medicine chromatographic characteristic map similarity evaluation software, establishing the cottonrose hibiscus leaf characteristic map, and carrying out common peak identification on the cottonrose hibiscus leaf characteristic map, as shown in figures 12-13 and 14-15.
Taking a standard decoction of the cotton rose hibiscus leaves, preparing a sample solution according to the sample preparation method under the 2.2 item, carrying out sample injection measurement according to the chromatographic condition under the 2.1 item to obtain a cotton rose hibiscus leaf characteristic spectrum, generating a control spectrum according to an average method (or a median method) by using Chinese medicine chromatographic characteristic spectrum similarity evaluation software, establishing the cotton rose hibiscus leaf characteristic spectrum, and carrying out common peak identification on the cotton rose hibiscus leaf characteristic spectrum, as shown in figures 16-17.
Taking the Chinese medicinal herb formula particles of the cotton rose leaves, preparing a test sample solution according to the test sample preparation method under the 2.2 items, carrying out sample injection measurement according to the chromatographic conditions under the 2.1 items to obtain a cotton rose leaf characteristic spectrum, generating a control spectrum according to an average method (or a median method) by using Chinese medicinal chromatographic characteristic spectrum similarity evaluation software, establishing the cotton rose leaf characteristic spectrum, and carrying out common peak identification on the cotton rose leaf characteristic spectrum, as shown in figures 18-19.
Example 2 determination of rutin and Tiliaside index content in Hibiscus Mutabilis leaves
1. Measurement method
The same as in example 1, part 2.
2. Methodological verification
2.1 linear relationship investigation
Accurately weighing rutin control 3.451mg in a 10mL measuring flask, and adding methanol to obtain solution containing rutin 338.198 μg per 1 mL.
Accurately weighing the tiliroside reference substance 2.127mg in a 10mL measuring flask, and adding methanol to prepare a solution containing 201.852 mug of tiliroside per 1 mL.
And (3) accurately sucking the rutin control solution and the tiliroside control solution into a 20mL measuring flask respectively by 2mL, adding methanol to fix the volume to a scale, and shaking uniformly to obtain a mixed solution containing 33.820 mug of rutin and 20.185 mug of tiliroside in each 1 mL.
Respectively precisely sucking 0.5mL, 1.0mL, 2.0mL, 3.0mL and 5.0mL of the mixed reference substance solution into a 10mL measuring flask, and adding methanol to the scale to prepare mixed reference substance solutions containing rutin 1.691 mug, 3.382 mug, 6.764 mug, 10.146 mug and 16.901 mug and tilianin 1.009 mug, 2.185 mug, 4.037 mug, 6.056 mug and 10.093 mug in each 1 mL. And respectively precisely sucking 1 mu L of the 5 reference substance solutions with different concentrations and the reference substance mother solution for sample injection measurement, and recording chromatographic peak areas. The peak area is taken as an ordinate (y), and the sample injection concentration of the reference substance is taken as an abscissa (x), and the sample injection concentration is shown in Table 21.
Table 21 Linear regression equation of rutin and Tilia Miqueliana Maxim glycoside content determination method in Hibiscus Mutabilis leaves
2.2 repeatability investigation
About 0.5g of the cotton rose hibiscus leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, and preparing 6 parts in parallel, preparing a sample solution according to the sample solution preparation method under the condition of 2.2 in example 1, and measuring rutin and linden glycoside content in the sample solution according to the condition sample injection measurement under the condition of 2.1 in example 1, wherein the measurement results are shown in Table 22.
Repeated investigation result of rutin and linden glycoside content determination method in table 22 cotton rose leaves
The results show that: the same batch of samples are repeatedly measured for 6 times, the RSD value of rutin content is 0.82%, the RSD value of tiliroside content is 1.06%, and the RSD is less than 3.0%, which shows that the repeatability of the analysis method is good.
2.3 inspection of sample recovery rate
About 0.25g of the cottonrose hibiscus leaf medicinal material powder (sieving with a third sieve, batch number: MFRY 06), precisely weighing, and carrying out parallel 3 groups, wherein 3 parts of each group are added according to the proportion of the tiliroside or rutin reference substance to the sample content of 1:0.5,1:1 and 1:1.5, 9 parts of the test sample solution is prepared according to the test sample solution preparation method under the condition of 2.2 in example 1, the rutin and tiliroside content in the test sample solution are measured according to the condition sample injection measurement under the condition of 2.1 in example 1, and the measurement results are shown in Table 23.
Sample-adding recovery rate investigation result of rutin and tilianin content determination method in Table 23 cotton rose leaves
The results show that: the average sample recovery rate of the tiliroside is 97.66%, and the RSD value is 2.00%; the average rutin sample adding recovery rate is 99.62%, the RSD value is 2.65%, and the accuracy is good.
3. Content determination
Taking the cottonrose hibiscus leaf medicinal materials, decoction pieces, standard decoction and traditional Chinese medicine formula granules respectively, and testing according to a determined method. The measurement results are shown in tables 24 to 27.
Table 24 determination results of rutin and Tilia Miqueliana glycoside content in the materials of the cotton rose leaves of the batch 24
Table 25 determination results of rutin and Tilia Miqueliana glycoside content in the Hibiscus Mutabilis leaf decoction pieces of 15 batches
Table 26 determination results of rutin and Tilia Miqueliana glycoside content in the 15 batches of Hibiscus Mutabilis leaf standard decoction
Table 27 determination results of rutin and Tiliaside content in Chinese medicinal formulation granule of batch of Hibiscus Mutabilis leaves
According to the determination result, the rutin content in the cottonrose hibiscus leaf medicinal material is determined to be 0.7 mg/g-2.0 mg/g, and the tilianin content is determined to be 0.4 mg/g-1.5 mg/g; the decoction pieces are the same as the medicinal materials; the rutin content in the cotton rose leaf standard decoction is 1.5 mg/g-5.0 mg/g, and the tilianin content is 0.7 mg/g-3.0 mg/g; the rutin content in the Chinese medicinal granule of the cotton rose leaves is 1.0 mg/g-4.0 mg/g, and the tilianin content is 0.7 mg/g-3.0 mg/g.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.
Claims (9)
1. The construction method of the characteristic spectrum of the cotton rose leaves is characterized by comprising the following steps:
extracting folium Hibisci Mutabilis with extraction solvent to obtain sample solution; wherein the extraction solvent is 50-70 vol% methanol or 50vol% ethanol;
extracting caffeic acid reference substance, rutin reference substance, tiliroside reference substance and flos Wallichii flavonol glycoside I reference substance with methanol or dissolving to obtain reference substance solution;
measuring the sample solution and the reference solution by adopting a liquid chromatograph to obtain a characteristic map;
the liquid chromatograph uses a T3 column as a chromatographic column, the particle size of a stationary phase of the liquid chromatograph is 1.5-2 mu m, a mixed solution of acetonitrile and tetrahydrofuran with the volume ratio of 3:1 is used as a mobile phase A, 0.05-0.3 vol% of phosphoric acid is used as a mobile phase B for gradient elution, and a gradient elution curve is as follows:
0 min-12 min, mobile phase A from 0% to 2%, mobile phase B from 100% to 98%;
12-25 min, wherein the mobile phase A is from 2% to 9%, and the mobile phase B is from 98% to 91%;
25-40 min, wherein the mobile phase A is from 9% to 15%, and the mobile phase B is from 91% to 85%;
40-48 min, wherein the mobile phase A is 15% -32%, and the mobile phase B is 85% -68%;
48 min-54 min, 32% of mobile phase A and 68% of mobile phase B;
the detection wavelength of the liquid chromatograph is 274 nm-330 nm.
2. The method of claim 1, wherein the mobile phase B is 0.1 vol.% phosphoric acid.
3. The method for constructing a characteristic spectrum according to claim 1, wherein the column length of a chromatographic column of the liquid chromatograph is 100 mm-200 mm, the column diameter is 2 mm-5 mm, the column temperature is 40-50 ℃, and the flow rate is 0.38-0.42 mL/min.
4. The method for constructing a feature map according to claim 1 or 3, wherein the particle diameter of the stationary phase is 1.8 μm;
the column length of a chromatographic column of the liquid chromatograph is 150mm, the column diameter is 2.1mm, the column temperature is 45 ℃, and the flow rate is 0.38 mL/min-0.42 mL/min;
the detection wavelength of the liquid chromatograph is 315nm.
5. The method for constructing a characteristic spectrum according to claim 1, wherein in the step of extracting the cotton rose leaves by using an extraction solvent to obtain the sample solution, the extraction mode is heating reflux extraction or ultrasonic extraction, and the extraction time is 10-60 min.
6. The method for constructing a characteristic spectrum according to claim 1, wherein the method for preparing the sample solution comprises the steps of: taking 0.1 g-1 g of a cottonrose hibiscus leaf medicinal material or cottonrose hibiscus leaf decoction pieces, adding 20-40 mL of 50-70 vol% methanol, weighing, heating and refluxing for 15-60 min, cooling, filtering, taking 50-70 vol% methanol to complement the lost weight, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the extract;
or (b)
The preparation method of the sample solution comprises the following steps: taking 0.1g to 1g of standard decoction of the cotton rose leaves or prescription granules of the cotton rose leaves, adding 20mL to 40mL of 50vol% to 70vol% of methanol, weighing, adopting ultrasonic treatment with the frequency of 20kHz to 50kHz for 20min to 40min, taking out, cooling, filtering, taking 50vol% to 70vol% of methanol to supplement the lost weight, shaking uniformly, filtering, and taking subsequent filtrate to obtain the cotton rose tea.
7. The method for constructing a characteristic spectrum according to claim 6, wherein the method for preparing the sample solution comprises the steps of: taking 0.5g of Hibiscus Mutabilis leaf medicinal material or Hibiscus Mutabilis leaf decoction pieces, adding 25mL of 70vol% methanol, weighing, heating and refluxing for 30min, cooling, weighing again, taking 70vol% methanol to supplement the weight of loss, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the final product;
or (b)
The preparation method of the sample solution comprises the following steps: taking 0.2g of standard decoction of Hibiscus Mutabilis leaf or formulated granule of Hibiscus Mutabilis leaf, adding 25mL of 70vol% methanol, ultrasonic treating at 40kHz for 30min, taking out, cooling, filtering, taking 70vol% methanol to supplement the weight of loss, shaking, filtering, and collecting the subsequent filtrate.
8. The method for constructing a feature map according to claim 1, wherein the reference solution is prepared by: mixing tiliroside reference substance, rutin reference substance, caffeic acid reference substance, and flos Hedyotidis Diffusae flavonol glycoside I reference substance with methanol to obtain mixed solution containing tiliroside reference substance 20 μg, rutin reference substance 40 μg, caffeic acid reference substance 50 μg, and flos Hedyotidis Diffusae flavonol glycoside I reference substance 10 μg per 1 mL.
9. The method for constructing a characteristic spectrum according to claim 1, wherein the characteristic spectrum has 13 peaks, wherein peak 7 is caffeic acid, peak 11 is rutin, peak 12 is tilianin, and peak 13 is schwann flower flavonol glycoside i;
calculating relative retention times of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 8, peak 9, peak 10 and the S1 peak with respect to each other, the relative retention times of the peaks being within ±10% of a prescribed value, wherein the prescribed value of peak 1 is 0.30, the prescribed value of peak 2 is 0.41, the prescribed value of peak 3 is 0.44, the prescribed value of peak 4 is 0.47, the prescribed value of peak 5 is 0.78, the prescribed value of peak 6 is 0.96, the prescribed value of peak 8 is 1.05, the prescribed value of peak 9 is 1.22, and the prescribed value of peak 10 is 1.49, with respect to the S1 peak;
the relative retention time of the peak 13 and the S2 peak was calculated with the peak 12 as the S2 peak, and the relative retention time of the peak 13 was within.+ -. 10% of the prescribed value, wherein the prescribed value of the peak 13 was 1.01.
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| 木芙蓉叶中芦丁的含量测定方法研究;李华 等;今日药学;20100131;第20卷(第01期);34-36 * |
| 木芙蓉叶标准汤剂质量指标的建立;万丽娟 等;中国药师;20221231;第25卷(第12期);2234-2239 * |
| 木芙蓉药材HPLC指纹图谱研究;邓亚利 等;中国实验方剂学杂志;20110331;第17卷(第06期);96-99 * |
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