CN114942297B - Developing agent for thin layer identification method of Taohong four-ingredient soup and thin layer identification method - Google Patents
Developing agent for thin layer identification method of Taohong four-ingredient soup and thin layer identification method Download PDFInfo
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Abstract
The invention discloses a developing agent for a thin layer identification method of Taohong four-substance soup and a thin layer identification method, and belongs to the technical field of medicine detection. According to the invention, ethyl acetate, methanol and water are mixed according to the volume ratio of 20 (4-6) (2-4) to be used as developing agents, the developing agents are used for thin-layer identification of Taohong four-ingredient soup, one developing agent is used, the same sample is used for preparing a sample solution, the sample solution is spotted on the same thin-layer plate, the sample solution is developed under the same developing system, four medicinal herbs of white paeony root, peach seed, rehmannia root and safflower in Taohong four-ingredient soup can be identified simultaneously under one inspection condition, and six medicinal herbs of angelica sinensis, ligusticum wallichii, white paeony root, peach seed, rehmannia root and safflower in Taohong four-ingredient soup can be identified simultaneously under two inspection conditions; the method has clear spots, good separation degree, strong specificity, no interference on the background, no interference on negative control and good durability; the quality information of the Taohong four-ingredient soup can be reflected by one-time identification, the identification is more comprehensive, the quality control of the Taohong four-ingredient soup is facilitated, the test times are reduced, and the test efficiency is high.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a developing agent for a thin layer identification method of Taohong four-substance soup and a thin layer identification method.
Background
The Taohong Siwu decoction is from Miao Bing Jian (self-cleaning and Chai Dehua for women), is one of hundreds of classical prescriptions published by the national administration of traditional Chinese medicine for the first time, and belongs to a traditional Chinese medicine compound preparation managed according to the classical prescriptions. The Taohong Siwu decoction is prepared from six medicines including rehmannia root, chinese angelica, white peony root, szechuan lovage rhizome, peach seed and safflower, wherein the prepared rehmannia root in the Siwu decoction is monarch, gan Wenzi is greasy and is good for nourishing nutrient and blood; chinese angelica is ministerial, pungent and warm in flavor, enters blood system mainly, can tonify blood, and has middle-jiao and middle-jiao, volume 16 of Ben Cao gang mu is called "harmonizing blood"; paeonia lactiflora is adjuvant, acidic and cold in taste, and has effects of nourishing blood, astringing yin, and softening liver. Chuan Xiong is pungent and warm in nature and can promote blood circulation and promote qi circulation, dispel blood stasis and alleviate pain, and is combined with the nourishing and tonifying herbs of prepared rhizome of rehmannia, white peony root and Chinese angelica, and can be used as an adjuvant drug without stagnation. Peach kernel and safflower are added on the basis of the four-ingredient soup, and the peach kernel and safflower are mainly used for activating blood and dissolving stasis; is suitable for treating blood stasis.
The thin layer identification method is a main means for qualitative identification in the quality standard of the Chinese herbal medicine compound preparation. The following requirements are set in the "drafting description of quality standard of Chinese herbal compound preparation" of the technical requirement of research on Chinese herbal medicine: the corresponding identification project can be established according to prescription composition and research data, and each prescription medicine flavor is subjected to experimental research in principle, and is selected to be listed in the standard according to experimental conditions. At present, the quality standard (identification) of the traditional Chinese medicine compound preparation comprising Chinese pharmacopoeia is mainly set according to identification medicine taste, 1 identification item of 1 prescription medicine taste, namely 1 thin layer plate, is unfolded for 1 time, and 1 prescription medicine taste is identified. If the thin-layer chromatography identification method is adopted to realize basic coverage of the prescription medicine taste of the traditional Chinese medicine compound preparation, the required test times are often more [ reference document: the thinking about the new mode of 'whole identification' of the compound preparation of the new traditional Chinese medicine is disclosed. The Taohong four-material decoction consists of six medicines, at most, two to three medicines are simultaneously identified by a thin-layer identification method of the Taohong four-material decoction reported in the literature, test sample solutions of the thin-layer identification methods of other medicines are generally different, the thin-layer chromatography identification of each medicine taste of the Taohong four-material decoction is completed, at least 3 or more different test sample solutions are required to be prepared, and 3 different unfolding systems are required to be used for unfolding, so that time and labor are wasted. The Chinese patent document CN107478749 discloses a detection method of a medicinal preparation of Taohong Siwu decoction in 2017, 12 and 15 days, wherein 3 sets of thin-layer chromatography methods are adopted for identifying four medicinal materials in a prescription, and the first set of methods simultaneously identify peach kernels and white paeony root; the second set of method is used for identifying the angelica and the ligusticum wallichii at the same time; the third set of method identifies safflower; the preparation methods and developing agents of the sample solutions of the three sets of methods are different; the Chinese patent document CN105535237A discloses a preparation method and a quality control method of a Taohong Siwu decoction formula granule in the year of 2016, wherein the quality control method comprises infrared fingerprint spectrum, thin-layer qualitative identification and HPLC content measurement, a perfect quality standard is established, a thin-layer chromatography one-measurement-multi-evaluation technology is adopted, the quality control is carried out by combining the infrared spectrum and the chromatography, the quality of the compound granule can be effectively controlled, but only the simultaneous detection of 3 decoction pieces of white paeony root, chinese angelica and szechuan lovage rhizome in the formula can be realized, and an organic solvent is required to be firstly used for extraction and purification in the preparation of a test solution.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a developing agent for a thin layer identification method of Taohong four-ingredient soup and a thin layer identification method, wherein ethyl acetate-methanol-water is mixed according to a specific volume ratio to be used as the developing agent, the developing agent is not layered, can be used after being mixed, and is not required to be placed for a long time at low temperature and then used; the thin layer identification method for the Taohong four-material soup provided by the application has the advantages of clear spots, good separation degree, strong specificity, no interference on the background and good durability; the quality information of six medicines can be reflected by one identification test, the identification is more comprehensive, the quality control of Taohong four-ingredient soup is more facilitated, the test times of thin-layer identification are reduced, the reagent consumption is reduced, the time is saved, the test efficiency is improved, and the problems that various test sample solutions and developing agents are required to be prepared in the prior art, and the detection is troublesome are solved.
The invention provides a developing agent for a thin-layer identification method of Taohong four-material soup, which consists of ethyl acetate, methanol and water, wherein the volume ratio of the ethyl acetate to the methanol to the water is 20 (4-6) (2-4).
The invention also provides a thin layer identification method for simultaneously identifying six medicinal materials in the whole recipe of the Taohong four-ingredient decoction, which comprises the following steps:
s1, preparing a sample solution: weighing Taohong four-ingredient decoction composition, adding solvent for dilution and dissolution, extracting, mixing diethyl ether solution, volatilizing, and adding methanol for dissolution to obtain an ether layer sample solution; passing the aqueous layer solution through AB-8 macroporous adsorption resin column, eluting with ammonia water solution, discarding ammonia solution, eluting with water again, discarding water solution, eluting with ethanol continuously, collecting eluate, evaporating to dryness, and dissolving the residue with methanol to obtain aqueous layer sample solution;
s2, preparing a control medicinal material solution: precisely weighing semen Persicae, radix Paeoniae alba, radix rehmanniae and Carthami flos, respectively adding solvent, heating and reflux extracting, filtering while it is hot, passing the filtrate through AB-8 macroporous adsorbent resin column, eluting with ammonia water solution, discarding ammonia solution, eluting with water, discarding water solution, eluting with ethanol, collecting eluate, evaporating to dryness, and dissolving the residue with appropriate amount of methanol to obtain semen Persicae, radix Paeoniae alba, radix rehmanniae and Carthami flos reference medicinal material solution; adding solvents into radix Angelicae sinensis and rhizoma Ligustici Chuanxiong control materials respectively, heating and reflux extracting, filtering, extracting with diethyl ether, mixing diethyl ether solutions, volatilizing, and dissolving residues with methanol to obtain radix Angelicae sinensis and rhizoma Ligustici Chuanxiong control medicinal materials solution;
s3, preparing a reference substance solution: adding appropriate amounts of amygdalin control, paeoniflorin control, rehmannia glycoside D control, and hydroxysafflor yellow A control into methanol to obtain mixed control solution 1, adding appropriate amounts of ferulic acid control and ligustilide control into methanol to obtain mixed control solution 2;
s4, detecting by thin layer chromatography: sucking the sample solution prepared in the step S1, the control medicinal material solution prepared in the step S2 and the control medicinal material solution 1-2 prepared in the step S3, respectively putting the sample solution on a silica gel G plate, mixing ethyl acetate, methanol and water according to the volume ratio of 20 (4-6) (2-4) as developing agents, developing, airing, and inspecting under an ultraviolet lamp, wherein fluorescent spots with the same color are displayed on positions corresponding to the ferulic acid control, the ligustilide control, the angelica sinensis and the ligusticum chuanxiong hort control medicinal material chromatograms in an ether layer sample chromatogram; in the water layer sample chromatogram, fluorescence spots with the same color are displayed at the positions corresponding to the hydroxysafflor yellow A control and the safflower control medicine chromatogram;
then spraying the mixture into a color reagent, heating the mixture at 105 ℃ until spots develop, and displaying spots with the same color on the positions corresponding to the chromatograms of the amygdalin reference substance and the peach kernel reference substance in the chromatogram of the water layer sample; spots with the same color are displayed on the positions corresponding to the chromatograms of the paeoniflorin reference substance and the white peony root reference substance; spots with the same color appear on the positions corresponding to the chromatograms of the rehmannia glycoside D reference substance and the rehmannia reference medicinal material; and fluorescent spots with the same color appear at the positions corresponding to the color spectrum of the hydroxysafflor yellow A reference substance and the color spectrum of the safflower reference medicine.
Further, the ammonia solution in the steps S1 and S2 has a volume concentration of 4%.
Further, the volume concentration of the ethanol elution in the step S1 is 20%.
Further, the solvent in the steps S1 and S2 is water.
Further, the diethyl ether extraction process in the step S2 is as follows: the mixture was extracted 2 times with 50ml of diethyl ether.
Further, the filtration in the step S2 is filtration with a 0.45um microporous filter membrane.
Further, the concentration of the mixed reference substance solution 1 and the mixed reference substance solution 2 in the step S3 is 0.5mg of reference substance per 1 ml.
Further, the color-developing agent in the step S4 is phosphomolybdic acid solution obtained by dissolving 2g of phosphomolybdic acid in 20ml of water, slowly adding 30ml of sulfuric acid, and mixing uniformly.
The invention also provides a thin layer identification method for simultaneously identifying four medicinal herbs of white paeony root, peach seed, rehmannia root and safflower in the Taohong four-ingredient decoction, which comprises the following steps of:
s1, preparing a sample solution: placing a sample into a beaker, diluting or dissolving with water, passing through a macroporous adsorption resin column, washing the beaker with water, passing the washing solution through the macroporous adsorption resin column, eluting with ammonia solution, discarding ammonia solution, eluting with water again, discarding water solution, continuing eluting with 20% ethanol, collecting eluent, evaporating to dryness, and dissolving the residue with appropriate amount of methanol to obtain a sample solution;
s2, preparing a negative sample: respectively weighing a boiled peach kernel negative sample, a white peony root negative sample, a rehmannia root negative sample and a safflower negative sample with equal decoction piece amount, and respectively preparing a boiled peach kernel negative sample solution, a white peony root negative sample solution, a rehmannia root negative sample solution and a safflower negative sample solution according to the sample preparation method of the step S1;
s3, preparing a control medicinal material solution: adding water into the mountain peach kernel control medicinal material, the white peony root control medicinal material, the radix rehmanniae control medicinal material and the safflower control medicinal material respectively, heating and reflux-extracting, filtering while the mixture is hot, and preparing a pair of medicinal material solutions from the filtrate according to the method for preparing the sample solution in the step S1 by using a macroporous adsorption resin column;
s4, preparing a reference substance solution: taking appropriate amounts of amygdalin reference substance, paeoniflorin reference substance, rehmannia glycoside D reference substance and hydroxysafflor yellow A reference substance, and adding methanol to obtain reference substance solution;
s5, detecting by thin layer chromatography: sucking 15 mu l of each of the test solution, the negative test solution, the control medicinal solution and the control solution, respectively, putting the mixture of ethyl acetate, methanol and water according to the volume ratio of 20 (4-6) (2-4) on the same silica gel G thin layer plate, developing the mixture as developing agent, taking out the developing agent, immediately spraying the phosphomolybdic acid sulfuric acid solution, heating the developing agent until spots develop clearly, and developing spots with the same color on the positions corresponding to the control medicinal material and the control chromatograph in the test chromatograph.
The thin layer identification method provided by the invention can also be applied to detection of the water decoction of the basic sample of the Taohong four-material decoction, the concentrated extract of the basic sample of the Taohong four-material decoction, the freeze-dried powder of the basic sample of the Taohong four-material decoction, the preparation of the Taohong four-material decoction and the extracting solution, the concentrated solution and the spray-dried powder of the intermediate thereof.
Compared with the prior art, the invention has the beneficial effects that:
1. the developing agent provided by the invention is formed by mixing ethyl acetate, methanol and water according to the volume ratio of 20 (4-6) (2-4), the developing agent is not layered, can be used after being mixed according to a proportion, is not required to be used after being placed for a long time at low temperature, and can identify the six medicinal materials in the Taohong four-ingredient soup by only using one developing agent, so that the reagent consumption is reduced, and the operation is more convenient;
2. the method for simultaneously identifying six medicinal materials provided by the invention is that two sample solutions are prepared by using one sample, the sample solutions are spotted on the same thin layer plate, the sample solutions are unfolded under the same unfolding agent system, and the six medicinal materials of the full formula of the Taohong four-ingredient soup can be simultaneously identified under two inspection conditions; the method has clear spots, good separation degree, strong specificity, no interference on the background, no interference on negative control and good durability; the quality information of six medicines can be reflected by one identification test, the identification is more comprehensive, the integral quality characteristics of the medicines can be reflected more favorably, the test times of thin-layer identification are reduced, the reagent consumption is reduced, the time is saved, and the inspection efficiency is improved;
3. according to the preparation method of the sample solution, provided by the invention, the preparation of the sample solution can be started after the sample is diluted or dissolved by directly using water as a solvent, and the extraction and purification by using an organic solvent are not needed, so that the pretreatment method of the sample solution is simplified;
4. the preparation method of the sample solution of the thin-layer identification method of the traditional Chinese medicine compound preparation is generally complex, the extraction steps are complicated, time and labor are wasted, and the sample solutions of the thin-layer identification methods of different medicines are generally different, but the invention only needs to weigh the sample once, and two sample solutions are prepared after the sample is extracted, and the ether layer sample solution is used for identifying the angelica and the ligusticum wallichii; the water layer sample solution is used for identifying four medicinal herbs of white paeony root, peach kernel, rehmannia root and safflower, and different medicinal herbs adopt the same sample solution, so that the detection efficiency is greatly improved;
5. in the preparation of the control medicinal material solution, water is firstly used instead of an organic solvent for heating reflux extraction, and the filtrate is treated by adopting the same method as the sample solution to be tested of the Taohong four-material decoction, and because the standard sample and the preparation of the classical name Taohong four-material decoction also use water as extraction solvents and use water as solvents for sample pretreatment of the control medicinal material, the types of components contained in the control medicinal material solution and the sample solution to be tested are basically the same, the TLC spots of the control medicinal material are basically the same as the spot information of the corresponding medicinal components in the Taohong four-material decoction, and the judgment accuracy of the identification result is further improved;
6. the method for simultaneously identifying four medicines of white paeony root, peach kernel, rehmannia root and safflower in the Taohong four-ingredient decoction composition provided by the invention is characterized in that under the same thin layer system, one sample solution, one unfolding system and one color developing agent are used, and under the same inspection condition, the four medicines of white paeony root, peach kernel, rehmannia root and safflower can be simultaneously identified.
Drawings
FIG. 1 shows fluorescence spots of the thin layer chromatography plate of example 1 under 365nm light before color development, wherein the numbers 1-14 are radix Angelicae sinensis control, rhizoma Ligustici Chuanxiong control, mixed control 2, 1 diethyl ether layer, 2 diethyl ether layer, 3 diethyl ether layer, rehmanniae radix control, carthami flos control, semen Persicae control, radix Paeoniae alba control, mixed control, 1 water layer, 2 water layer and 3 water layer, respectively;
FIG. 2 shows spots under fluorescent lamps after the thin layer chromatography plate of example 1 is developed, wherein the numbers 1-14 are respectively radix Angelicae sinensis control, rhizoma Ligustici Chuanxiong control, mixed control 2, diethyl ether layer sample 1, diethyl ether layer sample 2, diethyl ether layer sample 3, rehmanniae radix control, carthami flos control, semen Persicae control, radix Paeoniae alba control, mixed control 1, water layer sample 2 and water layer sample 3;
fig. 3 is a chart of results of investigation of thin layer chromatography specificity of peach kernel and white peony root, which are obtained by burning peach kernel and four materials in peach kernel, and a chart of results of investigation of thin layer chromatography specificity of white peony root, which are obtained by taking a thin layer chromatography plate of example 2, wherein the plate 2 is a chart of results of investigation of thin layer chromatography specificity of rehmannia root and safflower;
FIG. 4 is a spot diagram under a fluorescent lamp after the thin layer chromatography plate of example 3 is developed;
FIG. 5 is a graph of temperature durability test results of the thin layer identification method of the present invention;
FIG. 6 is a graph showing the results of examining the humidity durability of the thin layer discriminating method of the present invention;
FIG. 7 is a graph of results of investigation of durability of a thin layer plate according to the thin layer discriminating method of the present invention;
FIG. 8 is a graph of results from different developer studies;
FIG. 9 is a graph of results of various visualizations;
fig. 10 is a view of the results of the examination of different sample preparation methods, from left to right: h211202 concentrate (method for preparing a sample solution of this patent), H211202 spray-dried powder (method for preparing a sample solution of this patent), concentrate of comparative example 3 (method for preparing a sample solution of patent CN 107478749), spray-dried powder of comparative example 3 (method for preparing a sample solution of patent CN 107478749).
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
The present invention will be further described in detail with reference to the following embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Instrument information used in the examples:
Control and control information in examples:
amygdalin control (China food and drug inspection institute, lot number: 110820-201608);
paeoniflorin reference (Chinese food and drug verification institute, lot number: 110736-202044);
rehmannia root glycoside D reference substance (Shanghai Shiadad Standard technical service Co., ltd., lot number: 7534);
hydroxysafflor yellow A control (China food and drug inspection institute, lot number: 111637-201810);
ferulic acid reference substance (Chinese food and drug inspection institute, batch number: 110773-201915);
ligustilide control (China food and drug inspection institute, lot number: 111737-201910);
semen Persicae reference material (Chinese food and drug inspection institute, lot number 121560-201302);
white peony root control medicine (China food and drug inspection institute, lot number: 120905-201610);
rehmannia root (radix rehmanniae) control medicinal material (China food and drug inspection institute, batch number: 121180-201506);
safflower control (China food and drug inspection institute, lot number: 120907-201713);
chinese angelica control medicine (Chinese food and medicine verification institute, batch number: 120927-202118);
ligusticum wallichii control medicine (Chinese food and drug verification institute, lot number: 120918-201813);
peach red four soup (homemade 3 batches of samples, batch number: TH210411H, TH210412H, TH 210413H);
peach red four soup reference sample lyophilized powder (homemade 3 batches, lot number: TH210415H, TH210425H, TH 210506H);
peach red four-ingredient soup concentrate (homemade 2 batches: H211201 concentrate, H211202 concentrate);
peach red four-material soup spray-dried powder (homemade 2 batches: H211201 spray-dried powder and H211202 spray-dried powder).
Example 1 thin layer identification method for simultaneously identifying six medicinal materials including white peony root, peach kernel, rehmannia root, safflower, angelica sinensis and ligusticum wallichii in Taohong four-ingredient decoction
S1, preparing a sample solution:
taking 3.5g of Taohong four-material soup particles, adding 40ml of water, heating to dissolve, cooling to obtain Taohong four-material soup solution (homemade 3 batches: batch number: TH210411H, TH210412H, TH 210413H), transferring into a separating funnel, shaking and extracting for 2 times with diethyl ether for 50ml each time, mixing diethyl ether solutions, adding 1g of anhydrous sodium sulfate, filtering, volatilizing, and dissolving residues with 2ml of methanol to obtain an ether layer sample solution; passing the water layer through AB-8 macroporous adsorbent resin column (inner diameter is 1.5cm, column height is 8cm, pre-washing with water until no alcohol smell is generated), eluting with 30ml ammonia water solution with volume concentration of 4%, discarding ammonia water solution, eluting with water of 20ml, discarding water solution, eluting with 50ml ethanol water solution with volume concentration of 20%, collecting eluate, evaporating to dryness, and dissolving residue with methanol of 2ml to obtain water layer sample solution;
s2, preparation of reference substance solution
Preparation of mixed control solution 1: taking appropriate amounts of amygdalin reference, paeoniflorin reference, rehmannia glycoside D reference and hydroxysafflor yellow A reference, preparing into mixed reference solution containing 0.5mg of each 1ml of methanol, and mixing to obtain mixed reference solution 1;
preparation of mixed control solution 2: taking ferulic acid reference substance and ligustilide reference substance, preparing mixed reference substance solution containing 0.5mg of ligustilide reference substance per 1ml with methanol, and mixing to obtain mixed reference substance solution 2;
s3, preparation of a control medicinal material solution
Preparing a peach kernel reference medicinal material solution: taking about 0.5g of peach kernel reference medicine, adding 40ml of boiling water, heating, refluxing and extracting for 1 hour, filtering while the medicine is still hot, passing the filtrate through an AB-8 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 8cm, adding water for pre-washing until no alcohol smell exists), eluting with 30ml of ammonia water solution with the volume concentration of 4%, discarding ammonia water solution, eluting with 20ml of water again, discarding water solution, eluting with 50ml of ethanol water solution with the volume concentration of 20%, collecting eluent, evaporating to dryness, adding methanol into residues to dissolve 2ml, filtering with a 0.45um microporous filter membrane, and obtaining a subsequent filtrate to obtain peach kernel reference medicine solution;
preparation of white peony root reference medicinal material solution: taking about 0.5g of white paeony root reference medicinal material, adding 40ml of water, heating, refluxing and extracting for 1 hour, filtering while the white paeony root reference medicinal material is hot, passing the filtrate through an AB-8 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 8cm, adding water for pre-washing until no alcohol smell exists), eluting by 30ml of ammonia water solution with the volume concentration of 4%, discarding ammonia water solution, eluting by 20ml of water again, discarding water solution, eluting by 50ml of ethanol water solution with the volume concentration of 20%, collecting eluent, evaporating to dryness, adding 2ml of methanol into residues for dissolving, filtering by a microporous filter membrane with 0.45um, and obtaining a white paeony root reference medicinal material solution from subsequent filtrate;
preparation of rehmannia control medicinal material solution: taking about 0.5g of rehmannia root reference medicinal material, adding 40ml of water, heating, refluxing and extracting for 1 hour, filtering while the mixture is hot, passing the filtrate through an AB-8 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 8cm, adding water for pre-washing until no alcohol smell exists), eluting with 30ml of ammonia water solution with the volume concentration of 4%, discarding ammonia water solution, eluting with 20ml of water again, discarding water solution, eluting with 50ml of ethanol water solution with the volume concentration of 20%, collecting eluent, evaporating to dryness, adding 2ml of methanol into residues for dissolution, filtering with a 0.45um microporous filter membrane, and obtaining a subsequent filtrate to obtain rehmannia root reference medicinal material solution;
preparation of safflower control medicinal material solution: taking about 0.5g of safflower reference medicinal material, adding 40ml of water, heating, refluxing and extracting for 1 hour, filtering while the safflower reference medicinal material is hot, passing the filtrate through an AB-8 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 8cm, adding water for pre-washing until no alcohol smell exists), eluting with 30ml of ammonia water solution with the volume concentration of 4%, discarding ammonia water solution, eluting with 20ml of water again, discarding water solution, eluting with 50ml of ethanol water solution with the volume concentration of 20%, collecting eluent, evaporating to dryness, adding 2ml of methanol into residues for dissolution, filtering with a 0.45um microporous filter membrane, and obtaining a subsequent filtrate to obtain safflower reference medicinal material solution;
preparing a angelica control medicinal material solution: taking about 0.5g of angelica control medicinal material, adding 40ml of water, heating, refluxing and extracting for 1 hour, filtering while the mixture is hot, transferring filtrate into a separating funnel, shaking and extracting for 2 times by using diethyl ether, 50ml each time, combining diethyl ether liquid, adding 1g of anhydrous sodium sulfate, filtering, volatilizing, dissolving residues in 2ml of methanol, filtering by using a microporous filter membrane of 0.45um, and taking subsequent filtrate to obtain a angelica control medicinal material solution;
preparing rhizoma Ligustici Chuanxiong control medicinal material solution, collecting about 0.5g of rhizoma Ligustici Chuanxiong control medicinal material, adding 40ml of water, heating and reflux-extracting for 1 hr, filtering while it is hot, transferring filtrate into separating funnel, shaking and extracting with diethyl ether for 2 times, 50ml each time, mixing diethyl ether solutions, adding anhydrous sodium sulfate 1g, filtering, volatilizing, dissolving residue with 2ml of methanol, filtering with 0.45um microporous membrane, and collecting subsequent filtrate to obtain rhizoma Ligustici Chuanxiong control medicinal material solution;
s4, detection by thin layer chromatography
According to a thin layer chromatography (four-part rule 0502 of the 2020 edition of Chinese pharmacopoeia), 20ul of each of the test sample solution and the reference medicinal material solution is sucked, 10ul of the reference sample solution is respectively spotted on the same silica gel G thin layer plate, and ethyl acetate, methanol and water are mixed according to a volume ratio of 20:5:3 mixing to obtain developing agent, developing, air drying, inspecting under ultraviolet lamp (365 nm), and displaying fluorescent spots of the same color on the positions corresponding to the chromatography of ferulic acid reference substance, ligustilide reference substance, radix Angelicae sinensis and rhizoma Ligustici Chuanxiong reference substance in the chromatography of diethyl ether layer sample; in the water layer sample chromatogram, fluorescence spots with the same color appear at the positions corresponding to the hydroxysafflor yellow A control and the safflower control chromatogram, specifically referring to figure 1;
spraying phosphomolybdic acid sulfuric acid solution (phosphomolybdic acid 2g, adding water 20ml for dissolving, slowly adding sulfuric acid 30ml, mixing well to obtain), heating at 105deg.C until the color of spots is clear, and displaying spots with the same color on the positions corresponding to the color spectrum of amygdalin reference substance and semen Persicae reference substance in the color spectrum of the water layer sample; spots with the same color are displayed on the positions corresponding to the chromatograms of the paeoniflorin reference substance and the white peony root reference substance; spots with the same color appear on the positions corresponding to the chromatograms of the rehmannia glycoside D reference substance and the rehmannia reference medicinal material; spots of the same color appear at the positions corresponding to the chromatograms of the hydroxysafflor yellow A control and the safflower control, see specifically FIG. 2.
Example 2 thin layer identification method for simultaneously identifying four medicinal herbs of Taohong Siwu decoction white peony root, peach seed, rehmannia root and safflower
S1, preparing a sample solution:
taking peach red four-soup reference sample freeze-dried powder (homemade 3 batches, batch number: TH210415H, TH210425H, TH 210506H) with the total decoction piece amount of about 5g (about 0.45g of boiled peach kernel decoction pieces, 0.675g of white peony root decoction pieces, 1.375g of rehmannia root decoction pieces, 0.5g of safflower decoction pieces), placing in a 50ml beaker, adding 10ml of water for dissolution, passing through an AB-8 type macroporous adsorption resin column (with the inner diameter of 1.5cm and the column height of 8cm, adding water for pre-washing until no alcohol smell), washing the beaker with 10ml of water, passing through the macroporous adsorption resin column together with the washing liquid, eluting with 30ml of aqueous ammonia solution with the volume concentration of 4%, discarding ammonia solution, eluting with 20ml of water again, eluting with 50ml of aqueous ethanol with the volume concentration of 20%, collecting eluent, evaporating to dryness, adding a proper amount of methanol into residues for dissolution, and taking the residues to 2ml as a sample solution;
s2, preparing a negative sample:
respectively weighing a boiled peach kernel negative sample, a white peony root negative sample, a rehmannia root negative sample and a safflower negative sample which are equivalent to the same decoction piece amount in the step S1, and respectively preparing a boiled peach kernel negative test solution, a white peony root negative test solution, a rehmannia root negative test solution and a safflower negative test solution according to the method of the step S1;
s3, preparing a control medicinal material solution:
taking 0.45g of mountain peach kernel control medicine, 0.675g of white peony root control medicine, 0.5g of radix rehmanniae control medicine and 0.5g of safflower control medicine, respectively adding 20ml of water, heating and reflux-extracting for 1 hour, filtering while the mixture is hot, and preparing a control medicine solution according to the method of the example 1;
s4, preparing a reference substance solution:
taking appropriate amounts of amygdalin reference substance, paeoniflorin reference substance, rehmannia glycoside D reference substance and hydroxysafflor yellow A reference substance, and respectively adding methanol to prepare reference substance solutions containing 0.5mg per ml;
s5, detection by thin layer chromatography
According to the thin layer chromatography (rule 0502 of four parts in 2020 edition of Chinese pharmacopoeia), the above solutions were sucked 15 μl each, and due to the too many negative samples, the solution was spotted on two identical silica gel G thin layer plates [ silica gel G plate of Merck ], ethyl acetate, methanol and water were mixed according to a volume ratio of 20:5:3 mixing as developing agent, developing at 25deg.C and 74% relative humidity, spreading at about 17cm, taking out, immediately spraying phosphomolybdic acid sulfuric acid solution (phosphomolybdic acid 2g is dissolved by adding water 20ml, and sulfuric acid 30ml is slowly added, mixing), heating at 105deg.C until the color of spots is clear, and in the sample chromatogram, spots with the same color appear at the positions corresponding to the control medicinal material and the control chromatogram, wherein spots in the sample solution of Tao-hong-Siwu decoction can correspond to those of the amygdalin control solution, and the spots also correspond to the corresponding spots of the mountain kernel control medicinal material, and no interference occurs in the peach kernel negative sample, see B, C, D line frame in figure 3; in the compound sample solution of the Taohong four-substance decoction, spots can correspond to spots of the paeoniflorin reference substance solution, two spots in the white peony root reference medicine can correspond to spots in the compound, one of the spots is paeoniflorin, and the white peony root negative sample has no interference to the two spots, and the white peony root negative sample is shown in a line box A in the figure 3; the negative samples of the radix rehmanniae and the flos Carthami are free from interference, and the three batches of standard sample lyophilized powder (TH 210415H, TH210425H, TH 210506H) of the Taohong four-substance soup show spots with the same color on the corresponding positions of the rehmannoside D, the hydroxy carthamin yellow A and the radix rehmanniae control medicinal material and the flos Carthami control medicinal material.
Example 3 thin layer identification method for simultaneously identifying four medicinal herbs of white peony root, peach seed, rehmannia root and safflower in Taohong Siwu decoction
S1, preparing a sample solution: taking peach red four-material soup concentrate (homemade 2 batches: H211201 concentrate and H211202 concentrate) with the total decoction piece amount of about 5g (about 0.45g of boiled peach kernel decoction pieces, 0.675g of white peony root decoction pieces and 1.375g of rehmannia root decoction pieces, 0.5g of safflower decoction pieces), placing the peach red four-material soup concentrate in a 50ml beaker, adding water to dilute the peach red four-material soup concentrate to 10ml, passing through an AB-8 type macroporous adsorption resin column (with the inner diameter of 1.5cm and the column height of 8cm, adding water to pre-wash until no alcohol taste), washing the beaker with 10ml of water, passing through the macroporous adsorption resin column together with the washing liquid, eluting with 30ml of aqueous ammonia solution with the volume concentration of 4%, discarding the ammonia solution, eluting with 20ml of water again, eluting with 50ml of aqueous ethanol solution with the volume concentration of 20%, collecting the eluent, evaporating the eluent, adding a proper amount of methanol to dissolve the residue to 2ml, and taking the residue as a sample solution; the preparation method comprises the steps of (1) preparing a sample solution from the sample preparation method of the embodiment 1 from the step of passing through an AB-8 type macroporous adsorption resin column, wherein the sample solution is prepared by taking peach red four-material soup spray-dried powder (homemade 2 batches: H211201 spray-dried powder and H211202 spray-dried powder) with the total decoction piece amount of about 5g (about 0.45g of boiled peach kernel decoction pieces, 0.675g of white peony root decoction pieces, 1.375g of rehmannia root decoction pieces and 0.5g of safflower decoction pieces);
s2, preparing a control medicinal material solution: taking 0.45g of mountain peach kernel control medicine, 0.675g of white peony root control medicine, 0.5g of radix rehmanniae control medicine and 0.5g of safflower control medicine, respectively adding 20ml of water, heating and reflux-extracting for 1 hour, filtering while the mixture is hot, and preparing a filtrate from a sample solution by using an AB-8 type macroporous adsorption resin column, wherein the preparation method of the control medicine solution in the embodiment 1 is the same as that of the control medicine solution;
s3, preparing a reference substance solution: taking appropriate amounts of amygdalin reference substance, paeoniflorin reference substance, rehmannia glycoside D reference substance and hydroxysafflor yellow A reference substance, and adding methanol to prepare a mixed reference substance solution containing 0.5mg per ml;
s4, detecting by thin layer chromatography: according to a thin layer chromatography (four-part rule 0502 of the 2020 edition of Chinese pharmacopoeia), the above solutions are sucked into 15 μl each and respectively spotted on the same silica gel G thin layer plate [ silica gel G plate of Merck ] with a volume ratio of 20:5:3, developing at 25deg.C with a relative humidity of 74%, spreading at about 17cm, taking out, immediately spraying phosphomolybdic acid sulfuric acid solution (phosphomolybdic acid 2g, adding water 20ml for dissolving, slowly adding sulfuric acid 30ml, mixing), heating at 105deg.C until the color of the spot becomes clear, and displaying the same color spot on the position corresponding to the control material and the control material chromatogram in the sample chromatogram, see figure 4.
Comparative example 1
3 batches of Taohong four-material soup extract, 2 batches of Taohong four-material soup concentrate and 4 batches of Taohong four-material soup spray dry powder are prepared according to the preparation method of the test sample in the embodiment 2, and then the developing agent adopts chloroform: ethyl acetate: methanol: mixing water according to the volume ratio of 15:40:22:10, then placing the mixture at the temperature of 5-10 ℃ for 12 hours, taking the lower layer solution as a developing agent, and identifying the drug taste of peach kernels according to the thin layer identification method of the embodiment 2 to obtain a thin layer chromatogram.
Comparative example 2
In the comparative example, vanillin sulfuric acid solution with the volume percentage concentration of 5% is used as a color reagent, and then two medicinal herbs of white paeony root and peach kernel are identified according to the method and the steps of the example 2, so as to obtain a thin-layer chromatogram.
Comparative example 3
The comparative example was prepared by preparing 1 lot of concentrated solution and 1 lot of spray-dried powder of the sample solution according to the sample solution preparation method in patent CN107478749a, and then carrying out thin layer identification of the sample solution on 1 lot of concentrated solution, 1 lot of spray-dried powder prepared in example 3 and 1 lot of concentrated solution and 1 lot of spray-dried powder of the sample solution prepared in the comparative example, thereby obtaining a thin layer chromatogram.
Test examples, methodological investigation
1. Investigation of specificity
The chromatograms obtained in comparative examples 1-3 are analyzed, and referring to fig. 1, 2, 3 and 4, the developing agent and the thin layer identification method for the thin layer identification method of the Taohong four-ingredient decoction provided by the invention are clear in spots, good in separation degree, strong in specificity and free from interference of negative control, and the chromatograms obtained by simultaneously identifying all six medicinal materials or four medicinal materials including white paeony root, peach seed, rehmannia root and safflower are clear in spots, good in separation degree and strong in specificity when detecting the reference sample freeze-dried powder of the Taohong four-ingredient decoction, the Taohong four-ingredient decoction preparation (such as particles) and intermediates (extract, concentrated solution and spray-dried powder) thereof.
2. Durability inspection
(1) The operation was the same as in example 2 except that the film was developed at a temperature of 4℃and a relative humidity of 74%, and referring to FIG. 5, it was found that the spot clarity, separation and specificity of the film chromatography were not affected by both low temperature (4 ℃) and room temperature (25 ℃) under the same high relative humidity (RH: 74%), indicating that the film identification method provided by the present invention was excellent in temperature durability;
(2) The operation was the same as in example 2 except that the development was performed at 25℃and 25% relative humidity, and the obtained 2 thin-layer plate chromatograms were shown in FIG. 6, and it is understood that the low relative humidity (RH: 25%) and the high relative humidity (RH: 74%) did not affect the spot clarity, specificity and separation of the thin-layer chromatograms at the same room temperature (25 ℃), indicating that the thin-layer identification method provided by the present invention has good humidity durability;
(3) The other operations were the same as in example 2 except that the silicon gel G plate of the Qingdao ocean chemical plant was used for development, and the obtained 2 thin layer plate chromatograms were shown in fig. 7, and it was found that the thin layer identification method provided by the present invention was excellent in durability, as the spots were slightly diffused when the silicon gel G plate of the Qingdao ocean chemical plant was used for development, but the judgment of the results was not affected.
3. Examination of the developing Agents
As can be seen from FIG. 8, the thin-layer chromatogram obtained in comparative example 1 is analyzed, the developing agent in comparative example 1 is prepared by mixing chloroform, ethyl acetate, methanol and water according to the volume ratio of 15:40:22:10, and then the lower solution is placed for 12 hours at the temperature of 5-10 ℃ as the developing agent, unlike the developing agent of the invention, the obtained thin-layer plate for identifying one drug of peach kernel has a wavy pattern, the difference of the spreading distance of spots can affect judgment, and the obtained thin-layer chromatogram has clear spots and strong specificity only by using the developing agent for the Taohong four-soup thin-layer identification method provided by the application and combining the thin-layer identification method provided by the application.
4. Investigation of the color developer
As can be seen from fig. 9, the thin-layer chromatogram obtained in comparative example 2 is analyzed, and when the comparative example 2 uses vanillin sulfuric acid solution with concentration of 5% as a color developer, 2 medicinal flavors of white paeony root and peach kernel are identified, and the color of spots is different or the spots are not clear enough due to uneven heating of a thin-layer plate when the thin-layer plate is heated, so that judgment is affected, and therefore, the color of the spots can be developed stably and clearly only by using the phosphomolybdic acid sulfuric acid solution color developing agent in the application and combining the thin-layer identification method in the application.
5. Investigation of sample preparation method
Analysis of the thin-layer chromatogram obtained in comparative example 3, as shown in fig. 10, the spots obtained in comparative example 3 were not clear enough according to the method for preparing the sample solution in CN107478749 a; the preparation method of the sample solution provided by the patent does not need to firstly extract the to-be-detected object by methanol ultrasonic, directly uses water as a solvent, and can start the preparation of the sample solution after diluting or dissolving the to-be-detected object, does not need to firstly extract and purify by using an organic solvent, simplifies the pretreatment method, has clearer spots and has no interference on the background.
It should be noted that, in the present specification, specific features, structures, materials, or characteristics may be arbitrarily combined, and in order to simplify the description, all possible combinations of the features in the foregoing embodiments are not described, and those skilled in the art may combine and combine the features of the different embodiments and the different embodiments described in the present specification without contradiction.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (6)
1. A thin layer identification method for simultaneously identifying six medicinal materials in the whole recipe of Taohong Siwu decoction is characterized by comprising the following steps:
s1, preparing a sample solution: weighing Taohong four-ingredient decoction composition, adding water for dilution and dissolution, extracting, mixing diethyl ether solution, volatilizing, and adding methanol for dissolution to obtain an ether layer sample solution; passing the aqueous layer solution through AB-8 macroporous adsorption resin column, eluting with ammonia water solution, discarding ammonia solution, eluting with water again, discarding water solution, eluting with ethanol continuously, collecting eluate, evaporating to dryness, and dissolving the residue with methanol to obtain aqueous layer sample solution;
s2, preparing a control medicinal material solution: precisely weighing semen Persicae, radix Paeoniae alba, radix rehmanniae and Carthami flos, respectively adding water, heating and reflux extracting, filtering while hot, passing the filtrate through AB-8 macroporous adsorbent resin column, eluting with ammonia water solution, discarding ammonia solution, eluting with water, discarding water solution, eluting with ethanol, collecting eluate, evaporating to dryness, and dissolving the residue with appropriate amount of methanol to obtain semen Persicae, radix Paeoniae alba, radix rehmanniae and Carthami flos reference medicinal material solution; adding solvents into radix Angelicae sinensis and rhizoma Ligustici Chuanxiong control materials respectively, heating and reflux extracting, filtering, extracting with diethyl ether, mixing diethyl ether solutions, volatilizing, and dissolving residues with methanol to obtain radix Angelicae sinensis and rhizoma Ligustici Chuanxiong control medicinal materials solution;
s3, preparing a reference substance solution: adding appropriate amounts of amygdalin control, paeoniflorin control, rehmannia glycoside D control, and hydroxysafflor yellow A control into methanol to obtain mixed control solution 1, adding appropriate amounts of ferulic acid control and ligustilide control into methanol to obtain mixed control solution 2;
s4, detecting by thin layer chromatography: sucking the sample solution prepared in the step S1, the control medicinal material solution prepared in the step S2 and the mixed control solution 1 and 2 prepared in the step S3, respectively putting the sample solution and the mixed control solution on a silica gel G plate, spreading the sample solution by using a spreading agent, airing the sample solution, putting the sample solution under an ultraviolet lamp for inspection, and displaying fluorescent spots with the same color on the positions corresponding to the ferulic acid control, the ligustilide control, the angelica sinensis and the ligusticum chuanxiong hort control medicinal material chromatograms in the diethyl ether layer sample chromatogram; in the water layer sample chromatogram, fluorescence spots with the same color are displayed at the positions corresponding to the hydroxysafflor yellow A control and the safflower control medicine chromatogram;
then spraying the mixture into a color reagent, heating the mixture at 105 ℃ until spots develop, and displaying spots with the same color on the positions corresponding to the chromatograms of the amygdalin reference substance and the peach kernel reference substance in the chromatogram of the water layer sample; spots with the same color are displayed on the positions corresponding to the chromatograms of the paeoniflorin reference substance and the white peony root reference substance; spots with the same color appear on the positions corresponding to the chromatograms of the rehmannia glycoside D reference substance and the rehmannia reference medicinal material; fluorescent spots with the same color are displayed on the positions corresponding to the color spectrum of the hydroxysafflor yellow A reference substance and the color spectrum of the safflower reference medicine;
the developing agent consists of ethyl acetate, methanol and water, wherein the volume ratio of the ethyl acetate to the methanol to the water is 20 (4-6) (2-4);
the color reagent in the step S4 is phosphomolybdic acid sulfuric acid solution obtained by dissolving 2g of phosphomolybdic acid in 20ml of water, slowly adding 30ml of sulfuric acid and uniformly mixing.
2. The thin layer identification method for simultaneously identifying six medicinal materials in the whole body of the Taohong four-ingredient decoction according to claim 1, wherein the volume concentration of the ammonia water solution in the steps S1 and S2 is 4%.
3. The thin layer identification method for simultaneously identifying six medicinal materials in the whole body of the Taohong four-ingredient decoction according to claim 1, wherein the volume concentration of ethanol elution in the step S1 is 20%.
4. The thin-layer identification method for simultaneously identifying six medicinal materials in the whole body of the Taohong four-ingredient decoction according to claim 1, wherein the diethyl ether extraction process in the step S2 is as follows: the mixture was extracted 2 times with 50ml of diethyl ether.
5. The thin-layer identification method for simultaneously identifying six medicinal materials in the whole body of the Taohong four-ingredient decoction according to claim 1, wherein the filtering in the step S2 is performed by using a 0.45um microporous filter membrane.
6. A thin layer identification method for simultaneously identifying four medicinal herbs of white paeony root, peach seed, rehmannia root and safflower in Taohong four-ingredient decoction is characterized by comprising the following steps of:
s1, preparing a sample solution: placing a sample into a beaker, diluting or dissolving with water, passing through a macroporous adsorption resin column, washing the beaker with water, passing the washing solution through the macroporous adsorption resin column, eluting with ammonia solution, discarding ammonia solution, eluting with water, discarding water solution, continuing eluting with ethanol, collecting eluent, evaporating to dryness, and dissolving the residue with appropriate amount of methanol to obtain a sample solution;
s2, preparing a negative sample: respectively weighing a boiled peach kernel negative sample, a white peony root negative sample, a rehmannia root negative sample and a safflower negative sample with equal decoction piece amount, and respectively preparing a boiled peach kernel negative sample solution, a white peony root negative sample solution, a rehmannia root negative sample solution and a safflower negative sample solution according to the sample preparation method of the step S1;
s3, preparing a control medicinal material solution: adding water into the mountain peach kernel control medicinal material, the white peony root control medicinal material, the radix rehmanniae control medicinal material and the safflower control medicinal material respectively, heating and reflux-extracting, filtering while the mixture is hot, and preparing a pair of medicinal material solutions from the filtrate according to the method for preparing the sample solution in the step S1 by using a macroporous adsorption resin column;
s4, preparing a reference substance solution: taking appropriate amounts of amygdalin reference substance, paeoniflorin reference substance, rehmannia glycoside D reference substance and hydroxysafflor yellow A reference substance, and adding methanol to obtain reference substance solution;
s5, detecting by thin layer chromatography: sucking 15 mu l of each of the sample solution, the negative sample solution, the control medicinal material solution and the control solution, respectively, spotting the sample solution, the negative sample solution, the control medicinal material solution and the control solution on the same silica gel G thin layer plate, adopting a developing agent, developing, taking out, immediately spraying a phosphomolybdic acid sulfuric acid solution, heating until spots develop clearly, and developing spots with the same color on the positions corresponding to the control medicinal material and the control chromatogram in the sample chromatogram;
the developing agent consists of ethyl acetate, methanol and water, wherein the volume ratio of the ethyl acetate to the methanol to the water is 20 (4-6) (2-4);
the phosphomolybdic acid sulfuric acid solution is prepared by dissolving 2g of phosphomolybdic acid in 20ml of water, slowly adding 30ml of sulfuric acid, and uniformly mixing.
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