CN116735776A - Method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds and application of method - Google Patents
Method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds and application of method Download PDFInfo
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- CN116735776A CN116735776A CN202211173103.9A CN202211173103A CN116735776A CN 116735776 A CN116735776 A CN 116735776A CN 202211173103 A CN202211173103 A CN 202211173103A CN 116735776 A CN116735776 A CN 116735776A
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- China
- Prior art keywords
- solution
- rosmarinic acid
- isosorbide
- selfheal
- methanol
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- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 title claims abstract description 277
- ZZAFFYPNLYCDEP-HNNXBMFYSA-N Rosmarinsaeure Natural products OC(=O)[C@H](Cc1cccc(O)c1O)OC(=O)C=Cc2ccc(O)c(O)c2 ZZAFFYPNLYCDEP-HNNXBMFYSA-N 0.000 title claims abstract description 139
- DOUMFZQKYFQNTF-MRXNPFEDSA-N rosemarinic acid Natural products C([C@H](C(=O)O)OC(=O)C=CC=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-MRXNPFEDSA-N 0.000 title claims abstract description 139
- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 title claims abstract description 139
- 235000008113 selfheal Nutrition 0.000 title claims abstract description 95
- KLDXJTOLSGUMSJ-JGWLITMVSA-N Isosorbide Chemical compound O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 KLDXJTOLSGUMSJ-JGWLITMVSA-N 0.000 title claims abstract description 92
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds and application thereof. The detection method comprises the steps of simultaneously carrying out qualitative identification on isosorbide and rosmarinic acid in selfheal seeds by using a thin-layer chromatography; the method comprises the steps of directly extracting isosorbide and rosmarinic acid by a specific polar solvent extraction method, separating the isosorbide and rosmarinic acid from fat-soluble components, comparing the mixture of the isosorbide and the rosmarinic acid with a reference substance, and simultaneously measuring the content of the isosorbide and the rosmarinic acid in the selfheal seeds by a high performance liquid chromatography. The detection method has the advantages of high accuracy and precision, good stability and repeatability and the like, provides a method for detecting the quality of isosRosmarinic acid glycoside and rosmarinic acid in the development and utilization of selfheal seeds, and has stronger specificity and good repeatability.
Description
Technical Field
The invention relates to a quality analysis method of medicines, health products, foods or cosmetics, in particular to a method for simultaneously detecting isosurceolus rosmarins and rosmarinic acid in selfheal seeds and application thereof.
Background
Spica Prunellae is a dry fruit ear of Prunella vulgaris Prunella vulgaris L belonging to Labiatae (Lamia), and is named as "Shennong Ben Cao Jing" after summer, which is a perennial herb with homology of medicine and food, and has been recently carried in China pharmacopoeia of 2015 edition for thousands of years. Prunella vulgaris has bitter and pungent taste, cold property, and liver and gallbladder channel property, and has effects of clearing liver fire, improving eyesight, resolving hard mass, and relieving swelling, and can be used for treating conjunctival congestion, swelling and pain, photophobia, lacrimation, conjunctival congestion, night pain, dizziness, scrofula, goiter, breast cancer, hypertension, lymphoid tuberculosis, infiltrative pulmonary tuberculosis, simple goiter, parotitis, acute icteric infectious hepatitis, etc. Modern researches have shown that selfheal contains various chemical components: comprises triterpene, sterol, flavone, organic acid, coumarin and other compounds, and has pharmacological effects of resisting tumor, inflammation, bacteria, virus, immunity regulation, blood pressure, blood sugar and blood lipid. The selfheal seeds are dry mature seeds of selfheal Prunella vulgaris L of Labiatae (Lamiaceae), are reproductive seeds of selfheal, have genetic active substances and functions of selfheal, but have not been valued in edible, health-care or pharmaceutical application value, and have not seen related reports of simultaneous detection of isosorbide and rosmarinic acid, in particular, a quality control method for simultaneous detection of isosorbide and rosmarinic acid in selfheal seeds is more blank.
Isostearic acid glycoside (structural formula is shown in formula I) is also known as rosmarinic acid glycoside, english name: salviafaside, rosmarinic acid-3-O-glucoside, molecular formula: c (C) 24 H 26 O 13 Molecular weight 522.4; is white crystalline powder, can be dissolved in organic solvents such as methanol, ethanol, DMSO and the like, and pharmacological research shows that the isosRosmarinic acid glycoside has the effects of resisting bacteria, viruses, oxidization and inflammation, immunosuppression and thrombus. The isosorbide is formed by combining rosmarinic acid (structural formula is shown as formula II) and a molecule of glucose, and has the same or similar pharmacological action as that of rosmarinic acid. The pharmacological studies of isoserine are relatively few, while those of rosmarinic acid are relatively many. Modern pharmacological studies have shown that: rosmarinic acid has antioxidant, antibacterial, antiinflammatory, antitumor, antidepressant, anxiolytic, kidney disease improving, and liver protecting effects. The action and action mechanism are as follows: the method comprises the following steps: rosmarinic acid has strong effects of resisting oxidation and scavenging free radicals in the body, and the effect is related to the structure of the ortho-diphenol hydroxyl group; antibacterial and anti-inflammatory effects of the following materials: rosmarinic acid has obvious inhibition effect on staphylococcus aureus, micrococcus luteus, escherichia coli, bacillus subtilis and other bacteria, and has certain inhibition effect on psoriasis, dermatitis, airway inflammation, acute lung injury and other various inflammations; anti-tumor effect: rosmarinic acid has obvious inhibition effect on various tumors such as colorectal cancer, breast cancer, cervical cancer, leukemia, lung cancer, liver cancer and the like, and the anti-tumor mechanism of rosmarinic acid can be related to improving the immune function of the organism and effectively inhibiting the proliferation and invasion of tumor cells and inducing apoptosis; antidepressant and anxiolytic effects of the fourth generation: the anti-depression effect of rosmarinic acid is found by in vitro experiments, the rosmarinic acid can promote proliferation of astrocytes of newborn rats, the in vivo experiments show that the rosmarinic acid can improve depression-like behaviors of rats with chronic unpredictable stress depression models, and low dosage of rosmarinic acid can play an anti-anxiety role; protecting liver: rosmarinic acid can inhibit proliferation and differentiation of hepatic stellate cell HSCs, and can resist carbon tetrachloride-induced anti-fibrosis by inhibiting expression of TGF-beta 1 and CTGF; the kidney disease improving effect: rosmarinic acid has effects of improving kidney-related diseases, mainly by reducing uric acid production, inhibiting glomerulonephritis and delaying chronic renal insufficiency.
The invention discovers that the selfheal seeds are rich in isosorbide and rosmarinic acid components for the first time, and establishes a detection method for simultaneously detecting the isosorbide and the rosmarinic acid in the selfheal seeds for the first time, and the detection method comprises the step of simultaneously carrying out qualitative identification on the isosorbide and the rosmarinic acid in the selfheal seeds by utilizing a thin-layer chromatography; separating liposoluble component from isosorbide and rosmarinic acid polar component by polar solvent extraction method, comparing with isosorbide and rosmarinic acid mixed reference substance, and measuring content of isosorbide and rosmarinic acid in Prunellae Spica seed by high performance liquid chromatography. Provides a method and a basis for developing and utilizing the selfheal seeds and the products thereof.
Disclosure of Invention
The primary aim of the invention is to solve the problems that the quality control method of the selfheal seeds is blank, the selfheal seeds are rich in the isosorbide and rosmarinic acid components for the first time, and a detection method for simultaneously detecting the isosorbide and the rosmarinic acid in the selfheal seeds is established for the first time, so that the method for simultaneously detecting the isosorbide and the rosmarinic acid in the selfheal seeds is provided with high accuracy and precision and good stability and repeatability.
Another object of the present invention is to provide an application of the method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds.
The aim of the invention is achieved by the following technical scheme: a method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds comprises the following steps: qualitative identification of isosorbide and rosmarinic acid in Prunella vulgaris seed by thin layer chromatography; the method comprises the steps of directly extracting isosorbide and rosmarinic acid by a specific polar solvent extraction method, separating the isosorbide and rosmarinic acid from fat-soluble components, comparing the mixture of the isosorbide and the rosmarinic acid with a reference substance, and simultaneously measuring the content of the isosorbide and the rosmarinic acid in the selfheal seeds by a high performance liquid chromatography.
The thin layer chromatography detection method comprises the following specific steps:
(1) Preparation of test solution:
preparation of test sample solution of selfheal seeds:
A. adding Spica Prunellae seed powder into methanol water solution, ultrasonic extracting, filtering to obtain filtrate, and removing solvent to obtain residue;
B. dissolving the residue with methanol, and filtering to obtain test sample solution of Prunella spike seed powder;
(2) Preparation of a control solution: dissolving isosorbide and rosmarinic acid reference substance with methanol to obtain mixed solution of isosorbide and rosmarinic acid as reference substance solution;
(3) Thin Layer Chromatography (TLC) detection: absorbing the sample solution and the reference solution to be respectively spotted on the same high-efficiency silica gel GF 254 Spreading with spreading agent on the thin layer plate, taking out, and air drying;
(4) Observation of silica gel GF 254 Thin layer plate: respectively placing an ultraviolet lamp 254nm and an ultraviolet lamp 365nm for detection; and observing whether spots with the same color exist at the positions corresponding to the chromatogram of the reference substance in the chromatogram of the sample to identify whether the selfheal seeds contain isosurcumin and rosmarinic acid components.
The selfheal powder in the step (1) is preferably obtained by the following steps: pulverizing Prunellae Spica seed, breaking wall, and sieving with at least 40 mesh sieve to obtain Prunellae Spica powder.
The screen is preferably a 40-50 mesh screen.
The content of methanol in the aqueous solution of methanol in the step (1) A is preferably 40-60% by volume; more preferably 50% by volume.
The dosage of the methanol aqueous solution in the step (1) A is preferably calculated according to the proportion of 1.0-5.0 g of selfheal seed powder to 50-250 mL of methanol aqueous solution; more preferably, the ratio of the selfheal seeds to 100mL of methanol aqueous solution is calculated according to 2.0g of selfheal seeds.
The condition of ultrasonic extraction in the step (1) A is preferably that the ultrasonic extraction is performed for 20-80 minutes at the power of 50-200W and the frequency of 40-60 kHz; more preferably, the extraction is carried out for 30 to 60 minutes at a power of 80 to 150W and a frequency of 45 to 50 kHz; most preferably at a power of 150W and a frequency of 45kHz for 45 minutes.
The filtration in the step (1) A can be performed by filter paper or filter membrane.
The filter membrane is preferably a 0.45 μm filter membrane.
The solvent removal in step (1) A is preferably carried out by evaporation in a water bath.
The dosage of the methanol in the step (1) B is preferably calculated according to the proportion of 1-5 mL of methanol in 1.0-5.0 g of selfheal seed powder; more preferably, the ratio of the selfheal seeds to the methanol aqueous solution is 2.0 g.
The filtration described in step (1) B is preferably carried out using a 0.45 μm filter.
The concentration of isosrosmarinic acid in the reference solution in the step (2) is preferably 0.9-1.1 mg/mL, more preferably 0.97-1 mg/mL; the concentration of rosmarinic acid is preferably 0.9 to 1.1mg/mL, more preferably 0.94 to 1mg/mL.
The dosage of the sample solution in the step (3) is 2-10 mu L; more preferably 5. Mu.L.
The dosage of the reference substance solution in the step (3) is 2-10 mu L; more preferably 5. Mu.L.
The developing agent in the step (3) is preferably toluene-ethyl acetate-isopropanol-formic acid solution; more preferably toluene-ethyl acetate-isopropanol-formic acid solution obtained by mixing the components according to the volume ratio of 4.4-4.6:2.4-2.6:2.4-2.6:0.4-0.6; most preferred is toluene-ethyl acetate-isopropanol-formic acid solution mixed in a volume ratio of 4.5:2.5:2.5:0.5.
The specific polar solvent extraction method is that the isosorbide and the rosmarinic acid are directly extracted by a methanol aqueous solution with the volume percentage of 45-55%, and the isosorbide and the rosmarinic acid can be effectively extracted and separated from fat-soluble components.
The aqueous methanol solution is preferably 50% by volume of aqueous methanol solution.
The high performance liquid chromatography detection method comprises the following specific steps:
1) Chromatographic conditions: chromatographic condition and system applicability test uses octadecylsilane chemically bonded silica as filler, acetonitrile-methanol-0.2% (v/v) acetic acid aqueous solution as mobile phase, and detection wavelength is 320nm; the number of theoretical plates is not lower than 8000 calculated according to the peaks of isosorbide and rosmarinic acid;
2) Preparation of a control solution: precisely weighing isosorbide and rosmarinic acid reference substance, dissolving with methanol water solution, and fixing volume to obtain reference substance solution of isosorbide and rosmarinic acid;
3) Preparation of test solution: collecting Spica Prunellae seed powder, precisely weighing, placing in conical bottle with plug, precisely adding methanol water solution, precisely weighing, ultrasonic treating, cooling, weighing again, supplementing the weight with methanol water solution, shaking, filtering, and collecting filtrate;
4) And (3) measuring: precisely sucking the reference solution and the sample solution respectively, injecting into a liquid chromatograph, and measuring.
The mobile phase in the step 1) is preferably acetonitrile, methanol and 0.2% (v/v) acetic acid aqueous solution which are prepared according to the volume ratio of 14-16:11-13:72-74; more preferably acetonitrile, methanol and 0.2% (v/v) acetic acid in a volume ratio of 15:12:73.
The chromatographic column in the chromatography described in step 1) is preferably a Ultimate XB-C18 chromatographic column.
The column temperature in the chromatography in the step 1) is 24-27 ℃; more preferably 25 ℃.
The flow rate of the mobile phase in step 1) is preferably 0.8-1.2mL/min; more preferably 1mL/min.
The aqueous methanol solution in the step 2) is preferably an aqueous methanol solution with the concentration of 45-55% by volume; more preferably an aqueous methanol solution having a concentration of 50% by volume.
Each lmL of the control solution in the step 2) contains 0.05-0.06mg of isosRosmarinic acid glycoside and 0.05-0.06mg of rosmarinic acid.
The selfheal powder described in step 3) is preferably obtained by the following steps: pulverizing Prunellae Spica seed, breaking wall, and sieving with at least 40 mesh sieve to obtain Prunellae Spica powder.
The aqueous methanol solution in the step 3) is preferably an aqueous methanol solution with the concentration of 45-55% by volume; more preferably an aqueous methanol solution having a concentration of 50% by volume.
The dosage of the methanol aqueous solution in the step 3) is preferably calculated according to the proportion of 50-150mL of the methanol aqueous solution in each 0.5-1.5g of selfheal seed powder; more preferably, the ratio of the selfheal seeds to the methanol aqueous solution is 100mL per 1.0g of selfheal seeds.
The conditions of the ultrasound described in step 3) are preferably power 500-700W, frequency 30-50kHz for 40-80 minutes; more preferably at a power of 600W and a frequency of 40kHz for 30-60 minutes.
The volume of the control solution sucked in step 4) and the volume of the sample solution sucked in are preferably in a volume ratio of 1:1 proportion.
The volume of the control solution aspirated is preferably 10. Mu.L.
The pipetting volume of the sample solution is preferably 10. Mu.L.
The Prunella Spica seed contains isosRosmarinic acid glycoside (C) 24 H 26 O 13 ) Not less than 0.35%; contains rosmarinic acid (C) 18 H 16 0 8 ) Not less than 0.45%.
The method for simultaneously detecting the isosorbide and the rosmarinic acid in the selfheal seeds is applied to the selfheal seeds and the product development thereof.
Spica Prunellae is a common medicinal taste with homology of medicine and food carried in pharmacopoeia of China, the wild and planted resources are quite rich, but thousands of tons of Spica Prunellae seeds are not collected each year because the Spica Prunellae seeds are not developed and utilized, and are scattered in the wild of barren lands, so that the resource waste is caused. Compared with the prior art, the invention has the following advantages and effects:
1. the invention provides a method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds for the first time, which provides a method and a basis for developing and utilizing the selfheal seeds and the selfheal seed oil, and reduces the resource waste of the selfheal seeds;
2. the invention discovers that the selfheal seeds are rich in isosorbide and rosmarinic acid for the first time, and provides a basis for health care and medicine of the selfheal seeds;
3. the method for simultaneously detecting the isosorbide and the rosmarinic acid in the selfheal seeds has the characteristics of high accuracy and precision, good stability and repeatability and the like.
Drawings
FIG. 1 is a photograph showing the results of TLC identification of isosorbide and rosmarinic acid in Prunella vulgaris seeds at 254nm with ultraviolet light; wherein 1, 2, 3, 4, 6, 7 are test samples of selfheal seeds, and 5 is a comparison product of isoserine and rosmarinic acid.
FIG. 2 is a photograph showing the result of TLC identification of isosorbide and rosmarinic acid in Prunella vulgaris seeds at 365nm of ultraviolet light; wherein 1, 2, 3, 4, 6, 7 are test samples of selfheal seeds, and 5 is a comparison product of isoserine and rosmarinic acid.
FIG. 3 is an HPLC chromatogram of isostearic acid glycoside and rosmarinic acid control.
Fig. 4 is an HPLC chromatogram of isostearic acid glycoside and rosmarinic acid in selfheal seeds.
Fig. 5 is an HPLC chromatogram of a blank extraction solvent in a selfheal seed.
Fig. 6 is a graph of a linear fit of isosorbide to a rosmarinic acid standard solution.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1 Simultaneous Thin Layer Chromatography (TLC) detection of isosorbide and rosmarinic acid in Prunella vulgaris seeds
(1) Preparation of test solution: taking 6 parts of selfheal seeds, respectively crushing and breaking the wall, sieving with a 40-mesh sieve, accurately weighing 2.0g of each selfheal seed powder, respectively adding 100mL of aqueous solution containing 50% of methanol, extracting with ultrasound (power 150W and frequency 45 kHz) for 45min, filtering, placing the filtrate in an evaporation dish, heating in a water bath and evaporating to dryness, adding 2mL of methanol into residues to dissolve, and filtering with 0.45 mu m to obtain solutions of No. 1, no. 2, no. 3, no. 4, no. 6 and No. 7 of the selfheal seed powder to be tested respectively for later use.
(2) Preparation of a control solution: 1.94mg of isosorbide control and 1.88mg of rosmarinic acid control are weighed, put into a 2mL measuring flask, added with 2mL of methanol to dissolve, and a mixed control solution containing 0.97mg of isosorbide and 0.94mg of rosmarinic acid per lmL is prepared as a control solution, and No. 5 is used for standby.
(3) Thin Layer Chromatography (TLC) detection:
according to the thin layer chromatography (general rule 0502) test in the 2015 edition of the pharmacopoeia of the people's republic of China, the two solutions are absorbed by 5 mu L of each solution and respectively spotted on the same high-efficiency silica gel GF 254 Spreading with toluene-ethyl acetate-isopropanol-formic acid (4.5:2.5:2.5:0.5) as developing agent, taking out, and air drying; respectively placing an ultraviolet lamp (254 nm) and an ultraviolet lamp (365 nm) for detection;
and (3) detecting under an ultraviolet lamp (254 nm): in the chromatogram of the sample, the same fluorescence quenching spots are displayed at the positions corresponding to the positions of the iso-rosmarinic acid glycoside (lower) and the rosmarinic acid (upper) of the reference substance; see fig. 1.
And (3) under an ultraviolet lamp (365 nm) for detection: in the chromatogram of the sample, fluorescence spots with the same color are displayed at the positions corresponding to the positions of the iso-rosmarinic acid glycoside (lower) and the rosmarinic acid (upper) of the reference substance; see fig. 2.
(4) Thin Layer Chromatography (TLC) detection results and conclusions:
the results from the detection under the ultraviolet lamp (254 nm) and the detection under the ultraviolet lamp (365 nm) prove that: 6 selfheal seed samples all contain isosorbide and rosmarinic acid components; and the iso-rosmarinic acid glycoside in the chromatogram of 6 parts of the sample is basically the same as the rosmarinic acid component chromatogram.
Example 2 method and methodological validation for simultaneous determination of isosorbide and rosmarinic acid content in Prunella vulgaris seeds
Principle 1 and reagent
1.1 principle
The method comprises the steps of extracting the polar components of the isosorbide and the rosmarinic acid by using a polar solvent extraction method, separating the polar components of the isosorbide and the rosmarinic acid from the fat-soluble components, and measuring the content of the isosorbide and the rosmarinic acid components simultaneously by using a high performance liquid chromatography.
1.2 reagents
Acetic acid analytically pure, methanol chromatographic pure, acetonitrile chromatographic pure, isosRosmarinic acid glycoside reference substance (batch number DSTDY055001, purity: HPLC not less than 98%) Sichuan Dule Meidian medicine/Desmot biological extract; with rosmarinic acid reference (lot number 111871-202007, content: 98.1%) Chinese food and drug assay institute. Spica Prunellae seed sample, jiangxi Xinkang ecological agriculture development Co., ltd.
1.3 instruments
BY-500A high-speed universal pulverizer, yongkang city Sufeng industrial and trade Co., ltd; sieimer's U3000 series high performance liquid chromatograph (U ultraviolet detector), sieimer, germany; BT125D model one ten thousandth electronic analytical balance, cerdolis corporation, germany; SB25-12TD type ultrasonic cleaner for new sesame, nm cleaning equipment Co., ltd; an ultraviolet lamp; a water bath kettle; a common glass instrument; and (5) drying the box.
2 methods and results
2.1 preparation of control solution
Precisely weighing 5.58mg of isostearic acid glycoside reference substance and 5.81mg of rosmarinic acid reference substance, placing into a 100mL volumetric flask, dissolving with 50% (v/v) methanol, and fixing volume to obtain a mixed reference substance solution of isostearic acid glycoside and rosmarinic acid.
2.2 preparation of sample solution
Preparation of test sample solution of selfheal seeds: crushing selfheal seeds, sieving with a 40-mesh sieve, weighing about 1.0g of selfheal seed powder, precisely weighing, placing into a conical flask with a plug, precisely adding 100mL of aqueous solution containing 50% (v/v) methanol, precisely weighing, performing ultrasonic treatment (power 600W, frequency 40 kHz) for 60 minutes, cooling, weighing again, supplementing the lost weight with the aqueous solution containing 50% (v/v) methanol, shaking uniformly, filtering with 0.45 mu m, and taking subsequent filtrate to obtain the selfheal seed sample solution.
2.3 chromatographic conditions
Ultimate XB-C18 chromatographic column (4.6 mm. Times.250 mm,5 μm) was used, the column temperature was 25deg.C, the detection wavelength was 320nm, the flow rate was 1mL/min, and the sample injection amount was: the reference substance solution and the test sample solution are 10 mu L, and the mobile phase is acetonitrile-methanol-0.2% acetic acid aqueous solution (15:12:73); the chromatograms are shown in FIGS. 3-5.
As can be seen from fig. 3-5, in the HPLC chromatogram of the isosorbide and rosmarinic acid control, the absorption peak of the isosorbide control appears at about 11 minutes of retention time, and the absorption peak of the rosmarinic acid control appears at about 20 minutes of retention time; in the HPLC chromatogram of the selfheal seed, the absorption peak of the isosorbide control substance is also shown in about 11 minutes of retention time, and the absorption peak of the rosmarinic acid control substance is also shown in about 20 minutes of retention time; and the peak shape is one to one of the peak shape of the iso-rosmarinic acid glycoside reference substance and the peak shape of the rosmarinic acid reference substance in the reference substance, the separation degree is good, and the column plate numbers of the iso-rosmarinic acid glycoside and the rosmarinic acid are about 13000. The absorption peaks of the isosorbide and the rosmarinic acid are not seen in an HPLC chromatogram of the blank extraction solvent; prompting that the conditions are mature and available by HPLC (high performance liquid chromatography) of isosrosmarinic acid and rosmarinic acid in the selfheal seeds.
2.4 linear relationship investigation
Taking a reference substance solution under the item "2.1", filtering by a microporous filter membrane with the size of 0.45 mu m, taking 4, 8, 12, 16 and 20 mu L samples according to the chromatographic condition under the item "2.3", taking the sample injection quantity (mug) as an abscissa and the peak area as an ordinate, and drawing a standard curve, wherein the results are shown in Table 1 and FIG. 6; obtaining a regression equation: isosRosmarinic acid glycoside y= 30.711X-1.4282, r 2 = 0.9996; rosmarinic acid y= 31.958X-1.4117, r 2 ＝0.9995。
Table 1 experimental data on the linear relationship between isosorbide and rosmarinic acid mixture (n=5)
Isostearic acid glycoside and rosmarinic acid y= 30.711X-1.4282, r 2 = 0.9996; rosmarinic acid y= 31.958X-1.4117, r 2 ＝0.9995。
The above results indicate that: the isosorbide and rosmarinic acid have good linear relation with peak area within the range of 0.2232-1.1160 mug; rosmarinic acid has good linear relation with peak area in the range of 0.2324-1.1620 mug.
2.5 precision test: precisely sucking 10 mu L of the mixed reference substance solution of isosorbide and rosmarinic acid under the item "2.1", continuously injecting sample for 6 times according to the chromatographic condition under the item "2.3", measuring the peak area, calculating the average value of the peak area values of the isosorbide to be 15.713, the average value of the peak area values of the isosorbide to be 1.48%, the average value of the peak area values of the rosmarinic acid to be 17.158, the RSD to be 1.27%, and the RSD to be 2%, wherein the result is shown in Table 2; the result shows that the instrument precision is good.
Table 2 precision test data (n=6)
2.6 stability test: taking a selfheal seed test sample solution under the item of 2.2, respectively injecting 10 mu L of the sample solution under the chromatographic conditions under the item of 2.3 at 0 hour, 1 hour, 4 hours, 8 hours, 12 hours and 24 hours, measuring the peak area values of isosorbide and rosmarinic acid, and calculating the average value of the peak area values of the isosorbide in the selfheal seed test sample to be 14.646, the average value of the peak area values of the rosmarinic acid to be 1.62, the average value of the peak area values of the rosmarinic acid to be 17.456 and the RSD to be 1.64 percent; RSD of peak area of isosorbide and rosmarinic acid in the sample is less than 2%, and the result is shown in table 3.
Table 3 stability test data (n=6)
The test results show that the sample test solution has good stability within 24 hours.
2.7 reproducibility test: taking the same sample of Spica Prunellae seed (sample 20220314), and preparing 6 sample solutions of Spica seed according to the preparation method of the sample solution of Spica seed under item 2.2; respectively carrying out sample injection measurement according to the chromatographic conditions under the item of 2.3, and calculating the contents of isosorbide and rosmarinic acid; the average value of the isosrosmarinic acid content in the selfheal seed sample is 0.5228%, the RSD is 1.63%, the average value of the rosmarinic acid content is 0.5900%, and the RSD is 1.44%; RSD <2% of the isosorbide and rosmarinic acid content in the sample was measured. The results are shown in Table 4.
Table 4 reproducibility test data (n=6)
The test results show that the reproducibility of the measurement method is good.
2.8 recovery test: 6 parts of selfheal seeds (20220314) with the same batch number and known content of the isosorbide and the rosmarinic acid are precisely weighed, 0.5g of each part is precisely added with a proper amount of reference substance solution with the content of 100 percent of the isosorbide and the rosmarinic acid in the sample, and the sample solution is prepared according to the method of '2.2'; and then the content and recovery rate of the isosorbide and the rosmarinic acid are calculated according to the chromatographic condition sample introduction measurement under the item of 2.3, and as a result, the average recovery rate of the isosorbide in the selfheal seed sample and the rosmarinic acid content simultaneous measurement method is 99.67%, the RSD is 1.59%, the average recovery rate of the rosmarinic acid is 100.13%, and the RSD is 1.86%. The results are shown in Table 5.
Table 5 test of recovery of isosorbide and rosmarinic acid sample (n=6)
The test results show that: the recovery rate of isosrosmarin in the selfheal seeds is 98.17-101.76%, the recovery rate of rosmarinic acid is 97.94-103.14%, and the sample is well recovered by sample addition.
Example 3 simultaneous determination of the content of isosorbide and rosmarinic acid in three batches of Prunella Spica seeds
The content of isosorbide and rosmarinic acid in 3 different batches of selfheal seed samples were measured simultaneously, respectively, by the method of content measurement in example 3, and the results are shown in table 6.
Table 6 simultaneous determination of isosrosmarinic acid glycoside and rosmarinic acid content in selfheal seeds (n=3)
The content of isosrosmarinic acid in three batches of selfheal seeds is measured to be 0.4364% -0.5228%, and the content of rosmarinic acid is measured to be 0.5426% -0.6983%;
discussion: the research result shows that the content of the isosorbide and the rosmarinic acid in the selfheal seeds is measured at the same time, the linear relationship is good in the linear range, and the precision, the stability, the reproducibility and the recovery rate tests meet the requirements. The research is to find out that the selfheal seeds contain high content of isosorbide and rosmarinic acid components for the first time, and the determination method is also a detection method for simultaneously detecting the isosorbide and the rosmarinic acid in the selfheal seeds for the first time, and provides a method and a basis for quality control of the selfheal seeds; since isostearic acid glycoside is a water-soluble component, it is hardly dissolved in the selfheal seed oil, so that the selfheal seed oil contains almost no isostearic acid glycoside component. Rosmarinic acid is partially dissolved in the selfheal seed oil and can be detected in the selfheal seed oil.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds is characterized by comprising the following steps: qualitative identification of isosorbide and rosmarinic acid in Prunella vulgaris seed by thin layer chromatography; the method comprises the steps of directly extracting isosorbide and rosmarinic acid by a specific polar solvent extraction method, separating the isosorbide and rosmarinic acid from fat-soluble components, comparing the mixture of the isosorbide and the rosmarinic acid with a reference substance, and simultaneously measuring the content of the isosorbide and the rosmarinic acid in the selfheal seeds by a high performance liquid chromatography.
2. The method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to claim 1, wherein:
the thin layer chromatography detection method comprises the following specific steps:
(1) Preparation of test solution:
preparation of test sample solution of selfheal seeds:
A. adding Spica Prunellae seed powder into methanol water solution, ultrasonic extracting, filtering to obtain filtrate, and removing solvent to obtain residue;
B. dissolving the residue with methanol, and filtering to obtain test sample solution of Prunella spike seed powder;
(2) Preparation of a control solution: dissolving isosorbide and rosmarinic acid reference substance with methanol to obtain mixed solution of isosorbide and rosmarinic acid as reference substance solution;
(3) Thin layer chromatography detection: absorbing the sample solution and the reference solution to be respectively spotted on the same high-efficiency silica gel GF 254 Spreading with spreading agent on the thin layer plate, taking out, and air drying;
(4) Observation of silica gel GF 254 Thin layer plate: respectively placing an ultraviolet lamp 254nm and an ultraviolet lamp 365nm for detection; and observing whether spots with the same color exist at the positions corresponding to the chromatogram of the reference substance in the chromatogram of the sample to identify whether the selfheal seeds contain isosurcumin and rosmarinic acid components.
3. The method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to claim 2, wherein:
the selfheal powder in the step (1) is obtained by the following steps: pulverizing Prunellae Spica seed, breaking wall, and sieving with at least 40 mesh sieve to obtain Prunellae Spica powder;
the condition of ultrasonic extraction in the step (1) is that the power is 50-200W, and the frequency is 40-60 kHz, and the ultrasonic extraction is carried out for 20-80 minutes;
the content of methanol in the aqueous solution of methanol in the step (1) is 40-60% by volume;
the dosage of the methanol aqueous solution in the step (1) A is calculated according to the proportion of 1.0-5.0 g of selfheal seed powder to 50-250 mL of methanol aqueous solution;
the dosage of the methanol in the step (1) B is calculated according to the proportion of 1-5 mL of methanol of 1.0-5.0 g of selfheal seed powder;
the developing agent in the step (3) is toluene-ethyl acetate-isopropanol-formic acid solution.
4. The method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to claim 3, wherein:
the condition of ultrasonic extraction in the step (1) A is that the power is 80-150W, the frequency is 45-50 kHz, and the ultrasonic extraction is carried out for 30-60 minutes
The content of methanol in the aqueous methanol solution in the step (1) A is 50 percent by volume;
the dosage of the methanol in the step (1) B is calculated according to the proportion of 2.0g of selfheal seed powder to 2mL of methanol aqueous solution;
the developing agent in the step (3) is toluene-ethyl acetate-isopropanol-formic acid solution obtained by mixing according to the volume ratio of 4.4-4.6:2.4-2.6:2.4-2.6:0.4-0.6.
5. The method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to claim 2, wherein:
the solvent is removed in the step (1) A by water bath heating evaporation;
the filtration in step (1) B is filtration using a 0.45 μm filter membrane;
the concentration of isosRosmarin in the reference substance solution in the step (2) is 0.9-1.1 mg/mL, and the concentration of rosmarinic acid is 0.9-1.1 mg/mL;
the dosage of the sample solution in the step (3) is 2-10 mu L;
the dosage of the reference substance solution in the step (3) is 2-10 mu L.
6. The method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to claim 1, wherein:
the specific polar solvent extraction method is to directly extract isosurchin and rosmarinic acid by using 45-55% methanol aqueous solution by volume percent.
7. The method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to claim 1, wherein:
the high performance liquid chromatography detection method comprises the following specific steps:
1) Chromatographic conditions: chromatographic condition and system applicability test uses octadecylsilane chemically bonded silica as filler, acetonitrile-methanol-0.2% (v/v) acetic acid aqueous solution as mobile phase, and detection wavelength is 320nm; the number of theoretical plates is not lower than 8000 calculated according to the peak of isosRosmarinic acid;
2) Preparation of a control solution: precisely weighing isosorbide and rosmarinic acid reference substance, dissolving with methanol water solution, and fixing volume to obtain reference substance solution of isosorbide and rosmarinic acid;
3) Preparation of test solution: collecting Spica Prunellae seed powder, precisely weighing, placing in conical bottle with plug, precisely adding methanol water solution, precisely weighing, ultrasonic treating, cooling, weighing again, supplementing the weight with methanol water solution, shaking, filtering, and collecting filtrate;
4) And (3) measuring: precisely sucking the reference solution and the sample solution respectively, injecting into a liquid chromatograph, and measuring.
8. The method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to claim 7, wherein:
the mobile phase in the step 1) is obtained by proportioning acetonitrile, methanol and 0.2% (v/v) acetic acid aqueous solution according to the volume ratio of 14-16:11-13:72-74;
the chromatographic column in the chromatograph in the step 1) is a Ultimate XB-C18 chromatographic column;
the column temperature in the chromatography in the step 1) is 24-27 ℃;
the flow rate of the mobile phase in the step 1) is 0.8-1.2mL/min;
the aqueous methanol solution in the step 2) is an aqueous methanol solution with the concentration of 45-55% by volume;
each lmL of the control solution in the step 2) contains 0.05-0.06mg of isosRosmarinic acid glycoside and 0.05-0.06mg of rosmarinic acid;
the selfheal powder in the step 3) is obtained by the following steps: pulverizing Prunellae Spica seed, breaking wall, and sieving with at least 40 mesh sieve to obtain Prunellae Spica powder;
the aqueous methanol solution in the step 3) is an aqueous methanol solution with the concentration of 45-55% by volume;
the dosage of the methanol aqueous solution in the step 3) is calculated according to the proportion of 50-150mL of the methanol aqueous solution in each 0.5-1.5g of selfheal seed powder;
the condition of the ultrasonic wave in the step 3) is that the power is 500-700W, and the frequency is 30-50kHz for 40-80 minutes.
9. The method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to claim 8, wherein:
the mobile phase in the step 1) is obtained by mixing acetonitrile, methanol and 0.2% (v/v) acetic acid aqueous solution according to the volume ratio of 15:12:73;
the column temperature in the chromatography described in step 1) is 25 ℃;
the flow rate of the mobile phase in the step 1) is 1mL/min;
the aqueous methanol solution in the step 2) is an aqueous methanol solution with the concentration of 50 percent by volume;
the aqueous methanol solution in the step 3) is an aqueous methanol solution with the concentration of 50 percent by volume;
the condition of the ultrasonic wave in the step 3) is that the ultrasonic wave is processed for 30-60 minutes at the power of 600W and the frequency of 40 kHz.
10. Use of the method for simultaneously detecting isosorbide and rosmarinic acid in selfheal seeds according to any one of claims 1-9 in selfheal seeds and product development thereof.
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