CN112083097B - Thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin - Google Patents
Thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin Download PDFInfo
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Abstract
The invention discloses a thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin, belonging to the technical field of identification of traditional Chinese medicine compound preparations. In the technical scheme, three traditional Chinese medicine active ingredients including ferulic acid, calycosin glucoside and hesperidin are qualitatively identified at the same time by preparing a test solution, preparing a reference solution, detecting by thin-layer chromatography and each specific control condition, and meanwhile, the involved separation degree is good, negative interference is effectively eliminated, the inspection is clear, the method has better specificity and good reproducibility, the quality control of a compound preparation can be effectively realized, the drug production standardization is realized, and the safety, the production process stability and the controllability of the drug are better met; in addition, the solvent and labor cost are effectively saved, and the identification time is shortened by about 70%.
Description
Technical Field
The invention relates to a thin-layer identification method of a traditional Chinese medicine compound preparation, in particular to a thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin, belonging to the technical field of identification of traditional Chinese medicine compound preparations.
Background
Ferulic acid is a phenolic acid widely existing in plants, is mostly present in Chinese medicines such as angelica sinensis, ligusticum wallichii and the like, has wide pharmacological activity application, and has the effects of resisting atherosclerosis, resisting platelet aggregation and thrombus, resisting bacteria, diminishing inflammation, resisting tumors, resisting mutation, increasing immune function, enhancing the activity and the motility of sperms of a human body and the like. Calycosin glucoside belongs to isoflavone compounds, is an active monomer component extracted from dried roots of astragalus mongholicus and astragalus capsulatus of leguminous plants, is also one of the most active main components of astragalus mongholicus commonly used in China, and has obvious effects of resisting oxidation and viruses, inhibiting melanin formation, inhibiting immunity and the like. Hesperidin is widely present in plants of Leguminosae, Labiatae, dished flower, Rutaceae, and Citrus, and has pharmacological activities of resisting inflammation, resisting oxidation, resisting bacteria, resisting cancer, regulating immunity, preventing radiation, and protecting cardiovascular system.
In the existing traditional Chinese medicine composition for treating qi-blood deficiency symptoms, the traditional Chinese medicine composition is prepared from five medicines of astragalus, angelica, Chinese date, medlar and dried orange peel or extracts thereof, the astragalus and the angelica are combined to generate yin and grow, so that qi is vigorous and blood is generated, and meanwhile, the medlar, the Chinese date and the dried orange peel are supplemented, so that the whole formula has the effects of nourishing liver and kidney, tonifying spleen and regulating qi, benefiting defense and consolidating exterior, and strengthening body resistance and consolidating foundation, thereby enhancing the immune function of an organism. However, a set of standard and effective detection methods for ensuring the product quality is not established for the quality control of the traditional Chinese medicine composition, so that a set of simple and reliable quality standards is urgently needed to ensure the administration effect of the medicine.
In order to better control the quality of the combination, a method for simultaneously measuring effective components of astragalus, dried orange peel, angelica and the like is established, so that a simpler, faster and more accurate identification means is realized while the quality control method is improved, and the cost is saved.
At present, because the components of a traditional Chinese medicine compound preparation are complex, if a traditional thin-layer identification method is adopted, namely if a thin-layer identification method is established for each medicinal material or each effective component, the operation process is complicated and complicated, the time consumption is long, the efficiency is low, and the retrieval result accuracy is poor.
The study on the determination of the contents of various components in the pill for tonifying middle-jiao and replenishing qi by the one-test-multiple-evaluation method mentions that: the one-test-multiple-evaluation method utilizes the internal relation among medicinal compounds, can realize the synchronous measurement and control of one component on a plurality of components, better embodies the quality evaluation mode of the Chinese medicinal components, and accords with the overall view of the traditional Chinese medicinal theory. In addition, the method realizes the multi-group synchronous measurement of the pill for tonifying middle-jiao and qi through the hesperidin reference substance by constructing the internal relation between various components in the pill for tonifying middle-jiao and qi and the hesperidin, and provides reference for the supplement and the improvement of the quality standard control method of the pill for tonifying middle-jiao and qi.
The paper "quality standard research of compound qi-tonifying cough-relieving granules, wang jing and the like" specifically discloses: adding methanol, pre-treating to obtain mixed reference solution of calycosin glucoside and hesperidin, and identifying by thin layer chromatography.
In 2016, month 07 and day 13, a patent document with publication number CN105758985A entitled "a quality detection method for tongkang tablets" is disclosed, which includes: inspecting under 365nm ultraviolet lamp, spraying 10% ethanol solution of aluminum trichloride, heating at 105 deg.C to obtain clear spots, and identifying calycosin glucoside and hesperidin; a patent document with publication No. CN104931640A entitled "thin layer chromatography detection method of a Chinese medicinal composition for improving animal immunity" was disclosed in 2015, 9/23, and includes: detecting 1mg/mL calycosin glucoside reference solution by thin layer chromatography at detection wavelength of 254nm, and making into silica gel plate of silica gel GF254 thin layer plate; in 2012, 03/28, a patent document with publication number CN102393440A entitled "thin layer chromatography detection method of hesperidin in huoxiang zhengqi tablet" was disclosed, wherein the patent document discloses: detecting hesperidin control solution by thin layer chromatography, spraying aluminum trichloride solution, and inspecting under 365nm ultraviolet lamp.
Although the theoretical basis of simultaneously measuring multiple components in the traditional Chinese medicine by adopting liquid chromatography and the detection of the effective components (calycosin glucoside or hesperidin) in the traditional Chinese medicine are disclosed in the prior art, ferulic acid, calycosin glucoside and hesperidin cannot be effectively and simultaneously identified for different detection objects and traditional Chinese medicine compositions with more complex components.
Disclosure of Invention
The invention aims to solve the problems of the prior art and provides a thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin. In the technical scheme, three traditional Chinese medicine effective components of ferulic acid, calycosin glucoside and hesperidin are qualitatively identified simultaneously by preparing a test solution, preparing a reference solution, detecting by thin-layer chromatography and each specific control condition; the identification method is simple and rapid to operate, and provides effective basis for identification of the compound preparation.
In order to achieve the technical purpose, the following technical scheme is proposed:
a thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin comprises the following steps:
A. preparing a test solution: taking 3g of the traditional Chinese medicine compound preparation, adding 20mL of ethyl acetate, heating and refluxing for 1h, filtering, and evaporating filtrate to dryness; adding 1mL of methanol into the filter residue, and dissolving to obtain a test solution;
wherein, the drying by distillation specifically means that the solvent in the filtrate is zero;
wherein, the heating reflux adopts a conventional electronic constant temperature water bath (for example, model DK-98-11, Beijing Zhongwei apparatus Co., Ltd.);
the traditional Chinese medicine compound preparation comprises the following components in parts by weight: 25-35 parts of astragalus membranaceus or extract thereof, 5-7 parts of angelica sinensis or extract thereof, 8-12 parts of Chinese date or extract thereof, 5-10 parts of medlar or extract thereof, and 5-8 parts of dried orange peel or extract thereof, wherein the traditional Chinese medicine composition at least comprises the following effective components: calycosin glucoside is greater than or equal to 0.10 mg/g, astragaloside is greater than or equal to 0.45mg/g, ferulic acid is greater than or equal to 0.05mg/g, lycium barbarum polysaccharide is greater than or equal to 8.0mg/g, and hesperidin is greater than or equal to 5.0 mg/g;
B. preparation of control solutions: respectively taking a ferulic acid reference substance, a calycosin glucoside reference substance and an hesperidin reference substance, adding methanol, and respectively preparing a 1mg/L ferulic acid reference substance solution, a 1mg/L calycosin glucoside solution and a 1mg/L hesperidin reference substance solution for later use;
C. preparing a reference medicinal material solution: respectively taking 0.5g of angelica sinensis reference medicinal material, 1g of astragalus membranaceus reference medicinal material and 1g of dried orange peel reference medicinal material, adding 28-32mL of water, boiling for 30min, standing to normal temperature, filtering, and evaporating filtrate to dryness until the water content is zero; adding 1mL of methanol into the filter residue, and dissolving to obtain three reference medicinal material solutions;
D. and (3) detection: respectively taking 5-20 muL of a test solution, 2-15 muL of a ferulic acid reference solution, 2-15 muL of a calycosin glucoside reference solution, 5-15 muL of an hesperidin reference solution and 10 muL of each of three reference medicinal material solutions by adopting a thin-layer chromatography, respectively dropping the solutions on the same GF254 thin-layer plate, spreading the solution by using a developing agent, taking out the solution, drying the solution until the developing agent is volatilized, inspecting fluorescent spots at 254nm, spraying a 10% aluminum trichloride ethanol solution of a color developing agent, drying the color developing agent, heating the solution at 105 ℃ until the spots on the plate are clearly developed, and inspecting the spots under an ultraviolet lamp (365 nm);
D. and (4) analyzing results: in the chromatogram of the test sample under 254nm, the same spots are displayed on the corresponding positions of the ferulic acid reference solution and the calycosin glucoside reference solution in the chromatogram; and in the chromatogram of the test solution under 365nm, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram with the hesperidin reference solution.
Preferably, 10 μ L of the test solution, 5 μ L of the ferulic acid control solution, 5 μ L of the calycosin glucoside control solution and 10 μ L of the hesperidin control solution are respectively spotted on the same GF254 thin layer plate.
Preferably, the developing solvent is chloroform-methanol-acetone = 15: 3.5: 1.5.
by adopting the technical scheme, the beneficial technical effects brought are as follows:
1) the method is based on the thin-layer chromatography, can simultaneously identify three effective components of the traditional Chinese medicine, namely ferulic acid, calycosin glucoside and hesperidin, is simple and quick to operate, and provides an effective basis for identifying the compound preparation. In addition, the separation degree related in the technical scheme is good, negative interference is effectively eliminated, the inspection is clear, the specificity and the repeatability are better, the quality control of the compound preparation can be effectively realized, the drug production standardization is realized, and the safety, the production process stability and the controllability of the drug are better met;
2) in the step A, the ethyl acetate is used as a solvent, and the effective components in the traditional Chinese medicine compound preparation with complex components are greatly extracted according to the similar compatibility principle, so that a good separation effect is obtained, and the accuracy and precision of later-stage detection and judgment are facilitated. Adding methanol into the filter residue, wherein the polarity of the methanol is greater than that of the ethyl acetate, and the methanol can effectively ensure that the extracted components of the ethyl acetate are dissolved to a greater extent;
3) in the step D of the invention, a GF254 thin-layer plate is selected, a specific fluorescent agent is added into the GF254 thin-layer plate, the fluorescent agent emits green fluorescence under the wavelength of 254nm, and most of compounds with aromatic rings or conjugated systems can absorb ultraviolet under the wavelength of 254nm, so that the green fluorescence is covered to form dark spots; under the wavelength of 254nm, calycosin glucoside and ferulic acid can form obvious dark spots, and if the calycosin glucoside and ferulic acid are replaced by G plates and other plates, the effect is not good, so that the calycosin glucoside and ferulic acid cannot be distinguished well; in addition, fluorescence can be checked under 366nm by using the GF254 thin-layer plate, so that hesperidin can be better distinguished; in addition, the color developing agent is dried, so that the problem of poor color developing effect caused by the fact that the color developing agent is not dried during heating is effectively solved.
The hesperidin belongs to flavanone glycosides, a chemical reaction can be generated by spraying 10% aluminium trichloride ethanol solution as a color developing agent, the fluorescence of the hesperidin can be excited by ultraviolet rays with the wavelength of 365nm, and after aluminum ions are complexed with the color developing agent, the relatively obvious fluorescence can be displayed at 365nm, namely, the identification method is reasonable and strict in design according to the principle, and can effectively improve the accuracy of simultaneous identification of three traditional Chinese medicine components, namely ferulic acid, calycosin glucoside and hesperidin;
4) in the invention, 3 medicinal flavors are identified through a GF254 thin-layer plate under two inspection conditions, the simultaneous identification of three traditional Chinese medicine components of ferulic acid, calycosin glucoside and hesperidin is realized for the first time, the solvent and labor cost are effectively saved, the identification time is shortened by about 70 percent, the method can be used for identifying other components containing the three components by only a single sample preparation method, and meanwhile, the interference of other complex components in the traditional Chinese medicine is overcome, and the identification accuracy is ensured.
Drawings
Fig. 1 is a developed view of thin layer chromatography (one), in which No. 1: calycosin glucoside; number 2: hesperidin; number 3: ferulic acid; number 4-6: a sample;
fig. 2 is a developed view of thin layer chromatography (ii), in which No. 1: calycosin glucoside; number 2: hesperidin; number 3: ferulic acid; number 4-6: a sample;
fig. 3 is a developed view of thin layer chromatography (iii), in which No. 1: calycosin glucoside; number 2: hesperidin; number 3: ferulic acid; number 4-6: a sample;
fig. 4 is a developed view of thin layer chromatography (iv), in which No. 1: calycosin glucoside; number 2: hesperidin; number 3: ferulic acid; number 4-6: a sample;
fig. 5 is a developed view of thin layer chromatography (v), in which No. 1: calycosin glucoside; number 2: hesperidin; number 3: ferulic acid; number 4-6: a sample;
fig. 6 is a developed view (six) of thin layer chromatography, in which No. 1: calycosin glucoside; number 2: hesperidin; number 3: ferulic acid; number 4-6: a sample;
fig. 7 is a developed view (seven) of thin layer chromatography, in which No. 1: calycosin glucoside; number 2: hesperidin; number 3: ferulic acid; number 4-6: a sample;
fig. 8 is a developed view (eight) of thin layer chromatography, in which No. 1: calycosin glucoside; number 2: hesperidin; number 3: ferulic acid; number 4-6: a sample;
fig. 9 is a developed view (nine) of thin layer chromatography, in which No. 1: a sample; number 2: radix Angelicae sinensis as reference material; number 3: radix astragali as reference material; number 4: fructus Lycii as reference material; number 5: chinese date as reference medicinal material; number 1: pericarpium Citri Tangerinae control medicinal material;
fig. 10 is a developed view (ten) of thin layer chromatography, in which No. 1: a sample; number 2: radix Angelicae sinensis as reference material; number 3: radix astragali as reference material; number 4: fructus Lycii as reference material; number 5: chinese date as reference medicinal material; number 1: pericarpium Citri Tangerinae control medicinal material;
FIG. 11 is a thin layer chromatography development (254 nm) of a control sample amount test, in which sample amounts of numbers 1 to 15 are: 2. mu.L, 5. mu.L, 8. mu.L, 10. mu.L, 15. mu.L, 2. mu.L, 5. mu.L, 8. mu.L, 10. mu.L, 15. mu.L; number 1-5: ferulic acid; number 6-10: calycosin glucoside; number 11-15: hesperidin;
FIG. 12 is a development view (365 nm) of thin layer chromatography for examining the number of spots of a control sample, wherein the numbers 1 to 15 are respectively: 2. mu.L, 5. mu.L, 8. mu.L, 10. mu.L, 15. mu.L, 2. mu.L, 5. mu.L, 8. mu.L, 10. mu.L, 15. mu.L; number 1-5: ferulic acid; number 6-10: calycosin glucoside; number 11-15: hesperidin;
FIG. 13 is a thin layer chromatography development (254 nm) of the test sample application amount, wherein the sample application amounts of numbers 1 to 4 are respectively: 5. mu.L, 10. mu.L, 15. mu.L, 20. mu.L;
FIG. 14 is a development view (365 nm) of thin layer chromatography for examining the sample amount of a sample, wherein the sample amounts of numbers 1 to 4 are respectively: 5. mu.L, 10. mu.L, 15. mu.L, 20. mu.L;
FIG. 15 is a developed thin layer chromatography (Si Li Da, 254 nm) of thin layer plate investigation of different manufacturers, wherein the number 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
FIG. 16 is a development of thin layer chromatography for thin layer plate examination of different manufacturers (Silida, 365 nm), wherein the number 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 17 is a developed view of thin layer chromatography for thin layer plate investigation of different manufacturers (Qingdao ocean, 254 nm), wherein the number 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 18 is a development of thin layer chromatography for thin layer plate investigation of different manufacturers (Qingdao ocean, 365 nm), wherein, number 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 19 is a developed diagram of thin layer chromatography (254 nm, Yumin, Qingdao) of thin layer plate investigation of different manufacturers, wherein the number 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 20 is a developed diagram of thin layer chromatography of thin layer plate investigation of different manufacturers (Yumin, 365 nm), wherein the number 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 21 is a thin layer chromatogram (254 nm) developed in an environment at 25 ℃, wherein No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 22 is a thin layer chromatogram (365 nm) developed in an environment at 25 ℃, wherein No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 23 is a thin layer chromatogram (254 nm) developed in an environment at 8 ℃, in which No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 24 is a thin layer chromatogram (365 nm) developed in an environment at 8 ℃, wherein No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 25 is a thin layer chromatogram (254 nm) developed in an environment with a humidity of 32%, in which No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 26 is a thin layer chromatogram (365 nm) developed in an environment with a humidity of 32%, in which No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 27 is a thin layer chromatogram (254 nm) developed in an environment with humidity of 75%, in which No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 28 is a thin layer chromatogram (365 nm) developed in an environment with a humidity of 75%, in which No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 29 is a confirmation thin layer chromatogram (254 nm), in which No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: a sample;
fig. 30 is a validation thin layer chromatogram (365 nm), with No. 1: ferulic acid; number 2: radix Angelicae sinensis as reference material; number 3: a sample; number 4: a sample; number 5: calycosin glucoside; number 6: radix astragali as reference material; number 7: a sample; number 8: a sample; number 9: hesperidin; number 10: pericarpium Citri Tangerinae control medicinal material; number 11: a sample; number 12: and (3) sampling.
Detailed Description
In the following, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, reference is made to an apparatus comprising:
a semi-automatic thin layer sample applicator (CAMAG Lionmat-5), a thin layer imaging system (CAMAG TLC Vis μ equalizer), an ultra-pure water machine (cell type 1810A, Shanghai Mole scientific instruments Co., Ltd.), an ultrasonic cleaner (KQ 5200DB, 600W, 40 KHz; Kunshan City ultrasonic instruments Co., Ltd.), a silica gel G thin layer plate (Qingdao ocean division, Tianjin Silida science Co., Ltd., Qingdao Yu chemical plant;
in the following examples, the reagents involved included: methanol, ethyl acetate, chloroform, acetone and the like which are analytically pure;
in the following examples, reference controls include: ferulic acid (China institute for food and drug assay, batch No. 110773-201614); hesperidin (China institute for food and drug assay, lot number: 110721-201818); calycosin glucoside (China institute for testing and testing food and drug, lot number: 111920-201608);
in the following examples, reference drug materials include: chinese angelica as the reference (China institute for testing and testing food and drug, lot number 120927-201617); radix astragali (Mongolia) reference drug (China institute for food and drug testing, lot number 120974-; pericarpium Citri Tangerinae control drug (China institute for food and drug assay, lot number 120969-201510); the related Chinese herbal compound preparation is angelica sinensis, astragalus mongholicus and fructus lycii jujube paste, wherein the ingredients are angelica sinensis, astragalus mongholicus, jujube, dried orange peel and wolfberry fruit.
Example 1
A thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin comprises the following steps:
A. preparing a test solution: taking 3g of the traditional Chinese medicine compound preparation, adding 20mL of ethyl acetate, heating and refluxing for 1h, filtering, and evaporating filtrate to dryness; adding 1mL of methanol into the filter residue, and dissolving to obtain a test solution;
wherein, the drying by distillation specifically means that the solvent in the filtrate is zero;
wherein, the heating reflux adopts a conventional electronic constant temperature water bath (for example, model DK-98-11, Beijing Zhongwei apparatus Co., Ltd.);
the traditional Chinese medicine compound preparation comprises the following components in parts by weight: 25-35 parts of astragalus membranaceus or extract thereof, 5-7 parts of angelica sinensis or extract thereof, 8-12 parts of Chinese date or extract thereof, 5-10 parts of medlar or extract thereof, and 5-8 parts of dried orange peel or extract thereof, wherein the traditional Chinese medicine composition at least comprises the following effective components: calycosin glucoside is greater than or equal to 0.10 mg/g, astragaloside is greater than or equal to 0.45mg/g, ferulic acid is greater than or equal to 0.05mg/g, lycium barbarum polysaccharide is greater than or equal to 8.0mg/g, and hesperidin is greater than or equal to 5.0 mg/g;
B. preparation of control solutions: respectively taking a ferulic acid reference substance, a calycosin glucoside reference substance and an hesperidin reference substance, adding methanol, and respectively preparing a 1mg/L ferulic acid reference substance solution, a 1mg/L calycosin glucoside solution and a 1mg/L hesperidin reference substance solution for later use;
C. preparing a reference medicinal material solution: respectively taking 0.5g of angelica sinensis reference medicinal material, 1g of astragalus membranaceus reference medicinal material and 1g of dried orange peel reference medicinal material, adding 28-32mL of water, boiling for 30min, standing to normal temperature, filtering, and evaporating filtrate to dryness until the water content is zero; adding 1mL of methanol into the filter residue, and dissolving to obtain three reference medicinal material solutions;
D. and (3) detection: respectively taking 10 mu L of test solution, 5 mu L of ferulic acid reference solution, 5 mu L of calycosin glucoside reference solution, 10 mu L of hesperidin reference solution and 10 mu L of each of three reference medicinal material solutions by adopting a thin-layer chromatography, respectively dropping the solution on the same GF254 thin-layer plate, and respectively adding a developing agent chloroform-methanol-acetone = 15: 3.5: 1.5, taking out after development, airing until the developing agent is volatilized, inspecting fluorescent spots under 254nm, spraying 10% aluminum trichloride ethanol solution of a color-developing agent, airing the color-developing agent, heating at 105 ℃ until the spots on the plate are clearly developed, and inspecting under an ultraviolet lamp (365 nm);
D. and (4) analyzing results: in the chromatogram of the test sample under 254nm, the same spots are displayed on the corresponding positions of the ferulic acid reference solution and the calycosin glucoside reference solution in the chromatogram; and in the chromatogram of the test solution under 365nm, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram with the hesperidin reference solution.
Example 2
The present embodiment studies the influence of different deployment systems on the result of the present technical solution to further explain the present technical solution.
Preparing a test solution according to a formulated method by taking 3g of the angelica-astragalus-wolfberry jujube paste formula, and preparing a reference solution according to the formulated method;
the development system was one, chloroform: methanol: acetone = 15: 3.5: 1.5;
development system two, dichloromethane: methanol: acetone = 15: 3.5: 1.5;
respectively taking 5 mu L of test solution and reference solution prepared by a proposed method, respectively, spotting on the same GF254 plate, respectively developing in a first developing system and a second developing system, taking out and drying, and inspecting according to a proposed inspection method, wherein the first developing system has better effect.
Example 3
In this embodiment, the influence of specificity on the result of the technical solution is studied to further explain the technical solution.
Since the angelica, astragalus and wolfberry jujube paste is a compound preparation, the components are complex, and the situation that a plurality of medicinal materials coexist with the same component can exist, so the interference of special investigation is large, and negative control is not considered, so five control medicinal materials (angelica, astragalus, Chinese date, dried orange peel and medlar) are added with 30mL of water, heated and decocted for 30min, cooled and filtered, the filtrate is evaporated to dryness, and 20mL of ethyl acetate is added to prepare a control medicinal material solution by the same method.
Taking the reference medicinal material solution, the reference substance solution and the sample solution, and spotting on the same thin layer plate by the same method to compare the component sources. The results show that: the chromatogram of the test solution and the chromatogram of the reference material show the same color of main fluorescent spot at corresponding positions, and the method can identify three effective components (shown in FIGS. 9-10).
Example 4
In this embodiment, the influence of the sampling amounts of the ferulic acid reference solution, the hesperidin reference solution, the calycosin glucoside reference solution and the test solution on the result of the technical scheme is studied to further explain the technical scheme.
Dispensing 2 μ L, 5 μ L, 8 μ L, 10 μ L and 15 μ L of 3 kinds of control solutions, respectively spotting on the same GF254 plate, and inspecting according to the proposed inspection method, the results are shown in FIGS. 11-12; dispensing sample solutions of 5 μ L, 10 μ L, 15 μ L and 20 μ L on the same GF254 plate, and inspecting according to the proposed inspection method, with the results shown in FIGS. 13-14;
the results show that: the ferulic acid reference solution and the calycosin glucoside reference solution have clear fluorescent spots in 2-15 μ L, the hesperidin reference solution has clear fluorescent spots in 5-15 μ L, and the sample amount of the test solution is clear in 5-20 μ L; therefore, based on sample cost, experimental conditions, and the sharpness of fluorescent spots, etc., it is preferable that: the sampling amount of ferulic acid control solution is 5 μ L, the sampling amount of calycosin glucoside control solution is 5 μ L, and the sampling amount of hesperidin control solution is 10 μ L; the sample amount of the sample is 10 mu L.
Example 5
In this embodiment, the influence of the thin-layer plates of different manufacturers on the result of the technical scheme is studied to further explain the technical scheme.
Selecting silica gel GF of three different manufacturers254Thin layer plate (Tianjin Silida science and technology Co., Ltd., Qingdao ocean chemical plant division and Qingdao Yumin chemical plant division) is prepared by respectively dispensing ferulic acid reference solution with a dispensing amount of 5 μ L, calycosin glucoside reference solution with a dispensing amount of 5 μ L and hesperidin reference solution with a dispensing amount of 10 μ L, and dispensing the same silica gel GF254Developing on the thin layer plate according to the selected test method, taking out, air drying, inspecting at 254nm, spraying 10% aluminum trichloride ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight and ultraviolet lamp (365 nm). Testing the unfolding effect of different thin-layer plates, and adopting the unfolding result of the cuttable plate of Tianjin Silida technology Limited, as shown in FIGS. 15-16; the results of the development of the glass plates from the division of the Qingdao maritime plant were used, as shown in FIGS. 17-18; the result of spreading the glass plate of the Yu Min chemical plant of Qingdao is adopted, as shown in figures 19-20.
The results show that: analyzing and comparing thin-layer chromatograms developed by different thin-layer plates, and under the selected thin-layer chromatographic condition, the different thin-layer plates can achieve the identification purpose.
Example 6
In this embodiment, the influence of different temperatures and humidity on the result of the technical solution is studied to further explain the technical solution. The conventional standard requirements are combined, the thin-layer plate is placed at 8 ℃ and 25 ℃ to be respectively unfolded under the condition that the relative humidity is 60%, and comparison is carried out; and the selected thin-layer plate is placed under the conditions of relative humidity of 32% and 75% to be respectively unfolded under the condition of 25 ℃ for comparison.
Respectively changing the temperature of a thin layer developing environment under the condition that the relative humidity is 60%, and developing the thin layer plate subjected to sample application in the environment of 8 ℃ and 25 ℃; then taking out, air drying, inspecting at 254nm, spraying 10% ethanol solution of aluminum trichloride, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight and ultraviolet lamp (365 nm). Thin layer chromatograms developed at 25 deg.C and the results are shown in FIGS. 21-22; the results of thin layer chromatograms developed at 8 ℃ are shown in FIGS. 23-24.
Respectively changing the relative humidity of the thin layer development environment at 25 deg.C, developing the sample-applied thin layer plate in the environment with relative humidity of 32% and 75%, taking out, air drying, inspecting at 254nm, spraying 10% aluminum trichloride ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight and ultraviolet lamp (365 nm). The development results in an environment with 32% relative humidity, as shown in FIGS. 25-26; the results were developed in an environment with 75% relative humidity, as shown in FIGS. 27-28.
The results show that: under different temperature and humidity and selected thin-layer chromatography conditions, the aim of identification can be achieved at different temperature and humidity.
Example 7
In this embodiment, a sample of a Chinese herbal compound preparation, gui qi fructus ziziphi jujuba paste, is used for verifying to further explain the technical scheme.
The sample is verified according to a drawing method, and the result shows that: displaying the same spots on the chromatogram of the test sample at 254nm at the positions corresponding to the chromatogram of ferulic acid control and calycosin glucoside control; and displaying fluorescent spots with the same color at the corresponding positions of the chromatogram of the hesperidin control in the chromatogram of the test sample under the condition of 365 nm.
Claims (8)
1. A thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin is characterized by comprising the following steps:
A. preparing a test solution: adding ethyl acetate into the Chinese medicinal compound preparation, heating and refluxing, filtering, and evaporating filtrate; adding methanol into the filter residue, and dissolving to obtain a test solution;
B. preparation of control solutions: respectively taking ferulic acid reference substance, calycosin glucoside reference substance and hesperidin reference substance, adding methanol, and respectively preparing ferulic acid reference substance solution, calycosin glucoside reference substance solution and hesperidin reference substance solution for use;
C. preparing a reference medicinal material solution: respectively taking radix Angelicae sinensis control medicinal material, radix astragali control medicinal material and pericarpium Citri Tangerinae control medicinal material, boiling in water, standing to normal temperature, filtering, and evaporating to dryness; adding methanol into the residue, and dissolving to obtain three reference medicinal solutions;
D. and (3) detection: respectively taking 5-20 muL of a test solution, 2-15 muL of a ferulic acid reference solution, 2-15 muL of a calycosin glucoside reference solution, 5-15 muL of an hesperidin reference solution and 10 muL of each of three reference medicinal material solutions by adopting a thin layer chromatography, respectively dropping the solutions on the same GF254 thin layer plate, spreading the solutions by using a developing agent, taking out the solutions, airing the solutions until the developing agent is volatilized, inspecting fluorescent spots at 254nm, spraying a 10% aluminum trichloride ethanol solution of a color developing agent, airing the color developing agent, heating the solutions at 105 ℃ until the spots on the plate are clearly developed, and inspecting the fluorescent spots at 365 nm;
wherein the developing solvent is chloroform-methanol-acetone = 15: 3.5: 1.5;
D. and (4) analyzing results: in the chromatogram of the test sample under 254nm, the same spots are displayed on the corresponding positions of the ferulic acid reference solution and the calycosin glucoside reference solution in the chromatogram; and in the chromatogram of the test solution under 365nm, fluorescent spots with the same color are displayed at the corresponding positions of the chromatogram with the hesperidin reference solution.
2. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein said Chinese medicinal compound preparation comprises radix astragali, radix Angelicae sinensis, fructus Jujubae, fructus Lycii, pericarpium Citri Tangerinae or their extracts.
3. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin according to claim 1, wherein the compound traditional Chinese medicine preparation comprises the following components in parts by weight: 25-35 parts of astragalus membranaceus or extract thereof, 5-7 parts of angelica sinensis or extract thereof, 8-12 parts of Chinese date or extract thereof, 5-10 parts of medlar or extract thereof, and 5-8 parts of pericarpium citri reticulatae or extract thereof.
4. The thin-layer chromatography identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, 2 or 3, wherein the Chinese medicinal composition at least comprises the following effective ingredients: calycosin glucoside is greater than or equal to 0.10 mg/g, astragaloside is greater than or equal to 0.45mg/g, ferulic acid is greater than or equal to 0.05mg/g, lycium barbarum polysaccharide is greater than or equal to 8.0mg/g, and hesperidin is greater than or equal to 5.0 mg/g.
5. The thin-layer identification method for identifying ferulic acid, calycosin glucoside and hesperidin simultaneously as claimed in claim 1, wherein in step A, 3g of the compound Chinese medicinal preparation is taken, 20mL of ethyl acetate is added, heating and refluxing are carried out for 1h, filtering is carried out, and the filtrate is evaporated to dryness; and adding 1mL of methanol into the filter residue, and dissolving to obtain a test solution.
6. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein the concentrations of ferulic acid control solution, calycosin glucoside control solution and hesperidin control solution are all 1 mg/L.
7. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein in step C, 0.5g of radix Angelicae sinensis control drug, 1g of radix astragali control drug and 1g of pericarpium Citri Tangerinae control drug are respectively taken, added with 28-32mL of water, boiled for 30min, placed to normal temperature, filtered and evaporated to dryness; adding 1mL of methanol into the filter residue, and dissolving to obtain three reference medicinal material solutions.
8. The thin-layer identification method for simultaneously identifying ferulic acid, calycosin glucoside and hesperidin as claimed in claim 1, wherein in step D, 10 μ L of test solution, 5 μ L of ferulic acid control solution, 5 μ L of calycosin glucoside control solution and 10 μ L of hesperidin control solution are respectively spotted on the same GF254 thin-layer plate.
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