CN114814069B - Thin-layer identification method for 8 medicinal herbs in 9 medicinal herbs in whole formulation of cassia twig, chinese herbaceous peony and anemarrhena decoction - Google Patents
Thin-layer identification method for 8 medicinal herbs in 9 medicinal herbs in whole formulation of cassia twig, chinese herbaceous peony and anemarrhena decoction Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine quality control, and in particular relates to a thin-layer identification method for 8 medicinal herbs in 9 medicinal herbs in the whole prescription of cassia twig, chinese herbaceous peony and anemarrhena decoction, which comprises the following steps: preparing decoction, lyophilized powder or granule of ramulus Cinnamomi, radix Paeoniae and rhizoma anemarrhenae decoction into ether layer test solution and water layer test solution; preparing a mixed reference substance solution 1; preparing a water layer reference medicinal material solution; taking a water layer sample solution, a water layer reference medicinal material solution and a mixed reference substance solution 1, developing according to a thin layer chromatography condition, and identifying spots of each component; preparing a mixed reference substance solution 2; preparing an ether layer control medicinal material solution; taking an ether layer sample solution, an ether layer control medicinal material solution and a mixed control solution 2, developing according to a thin layer chromatography condition, and identifying spots of each component. The thin layer identification method can identify 8 medicinal herbs in 9 medicinal herbs in the whole prescription of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, has strong specificity of thin layer identification, is simple and quick to operate, has high detection efficiency, and reduces detection cost.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine quality control, and particularly relates to a thin-layer identification method for 8 medicinal herbs in 9 medicinal herbs in the whole prescription of cassia twig, chinese herbaceous peony and anemarrhena decoction.
Background
The cassia twig, chinese herbaceous peony and anemarrhena decoction belongs to one of 100 classical names in the ancient classical name catalog (first batch) made by the national drug administration together with the national drug administration. The decoction mainly comprises Zhi Gui Pao Zhi mu Tang, which is derived from the book of Zhang Zhongjing (the key of jin Kui Yao) of the famous family of Donghan, and is mainly used for treating pain in limbs, shortness of breath, edema of the feet, dizziness, shortness of breath, wen Wenyu vomiting. ", has the effects of dispelling pathogenic wind and removing dampness, activating yang and releasing arthralgia, nourishing yin and clearing heat, and is an excellent prescription for treating rheumatoid arthritis. The prescription is prepared from nine medicines of cassia twig, aconite (processed), chinese herbaceous peony, ephedra, ginger, bighead atractylodes rhizome, rhizoma anemarrhenae, ledebouriella root and licorice through conventional water decoction. The prescription comprises the following components: ramulus Cinnamomi, radix Paeoniae, glycyrrhrizae radix, herba Ephedrae, rhizoma Zingiberis recens, atractylodis rhizoma, rhizoma anemarrhenae, radix Saposhnikoviae, radix Aconiti lateralis Preparata, and rhizoma Atractylodis Macrocephalae, decocting with seven liters of water, and Wen Fuqi, and taking three times daily. The prescription is prepared by dissolving ramulus Cinnamomi decoction, adding ramulus Cinnamomi and rhizoma Zingiberis recens for dispelling water vapor, lowering adverse flow, removing jujube fullness, adding herba Ephedrae and radix Saposhnikoviae for dispelling pathogenic wind, adding rhizoma anemarrhenae for promoting diuresis and eliminating limb edema, and removing dampness and relieving arthralgia with Atractylodis rhizoma and radix Aconiti lateralis Preparata for strengthening body constitution, warming channel, dispelling cold and relieving pain. The whole recipe treats the rheumatic arthralgia, limb swelling and qi-flowing and vomiting. The decoction is prepared for damp, and mainly comprises ramulus Cinnamomi, radix Paeoniae alba, glycyrrhrizae radix, herba Ephedrae, atractylodis rhizoma, rhizoma anemarrhenae, radix Saposhnikoviae, radix Aconiti lateralis Preparata, and rhizoma Zingiberis recens. Firstly, the cassia twig, the white paeony root, the liquorice and the ginger are the components of the cassia twig decoction, which is used for relieving the moisture in the muscle, and the components of the ephedra and the cassia twig are equivalent to the components of the ephedra decoction, which is used for removing the moisture on the body surface, especially the ephedra has the effect of opening lung qi, and the sweating is an important way for discharging the moisture, so the medicines are very practical. Secondly, the components comprise largehead atractylodes rhizome, rhizoma anemarrhenae and divaricate saposhnikovia root, and the medicines have the effect of promoting urination, so that the pathogenic factors of water dampness are a reliable way for removing sweating, and simultaneously, the effect of promoting urination is also an effective way. In order to remove the dampness, the drugs such as cassia twig, aconite root, ginger and the like are required to be powered, namely, the drugs actively promote the internal force so as to remove the dampness from the body.
Analysis of thin layer identification method carried in a formulation of Chinese pharmacopoeia of the edition 2020 reveals that thin layer identification is basically a variety, N identification is established, and N kinds of test solution, N thin layer plates are prepared, and N times of thin layer plates are unfolded to identify the traditional identification mode of N medicinal materials. For example, chinese patent application CN103901154a discloses a thin layer identification method of a traditional Chinese medicine preparation for treating diabetes, which comprises thin layer identification of radix Ophiopogonis, thin layer identification of fructus Schisandrae chinensis and thin layer identification of radix Glycyrrhizae, wherein when the thin layer identification of radix Ophiopogonis, fructus Schisandrae chinensis and radix Glycyrrhizae is performed, 3 sample solutions are prepared conventionally, 3 thin layer plates are used, and 3 identification modes are developed. In order to eliminate interference, the pretreatment procedure of the sample is complex and complicated, a large amount of organic reagents are needed for repeated purification treatment, and the method is laborious, time-consuming, reagent-consuming, environment-friendly, health-damaging and long in detection period. The detection speed severely restricts the modern production speed of the traditional Chinese medicine.
In conclusion, the simple and rapid detection method for searching the traditional Chinese medicine compound preparation, improving the detection efficiency and reducing the detection cost is a difficulty that the quality control of the traditional Chinese medicine must break through.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a thin-layer identification method for 8 medicinal herbs in 9 medicinal herbs in the whole formula of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction. The thin layer identification method provided by the invention can identify 8 medicinal herbs in 9 medicinal herbs in the whole prescription of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, has strong specificity in thin layer identification, is simple and convenient to operate and high in detection efficiency, reduces the detection cost, provides a reference basis for quality control of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, and has great significance.
The technical scheme of the invention is as follows:
the thin layer identification method of 8 medicinal herbs in 9 medicinal herbs in the whole prescription of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction comprises the following steps:
s1, preparation of a sample solution: taking decoction, lyophilized powder or granule of ramulus Cinnamomi, radix Paeoniae and rhizoma anemarrhenae decoction, and respectively preparing into ether layer test solution and water layer test solution;
s2, preparation of a mixed reference substance solution 1: taking a reference substance to prepare a mixed reference substance solution 1;
s3, preparing a water layer control medicinal material solution: extracting Glycyrrhrizae radix control medicinal material, radix Paeoniae alba control medicinal material, herba Ephedrae control medicinal material, radix Saposhnikoviae control medicinal material, and rhizoma anemarrhenae control medicinal material with water respectively, filtering, purifying the filtrate with macroporous adsorbent resin column, eluting with ammonia solution, discarding ammonia solution, eluting with water again, discarding water solution, eluting with ethanol solution continuously, collecting eluate, evaporating to dryness, and dissolving the residue with methanol to obtain aqueous layer control medicinal material solution, namely Glycyrrhrizae radix control medicinal material solution, radix Paeoniae alba control medicinal material solution, herba Ephedrae control medicinal material solution, radix Saposhnikoviae control medicinal material solution, and rhizoma anemarrhenae control medicinal material solution;
s4 thin layer chromatography analysis 1: taking the water layer sample solution obtained in the step S1, the licorice control medicinal material solution obtained in the step S3, the white paeony root control medicinal material solution, the rhizoma anemarrhenae control medicinal material solution, the ephedra control medicinal material solution and the divaricate saposhnikovia root control medicinal material solution, and the mixed control material solution 1 obtained in the step S2, developing according to the thin-layer chromatography condition, and identifying spots of each component;
S5, preparation of a mixed reference substance solution 2: taking a reference substance to prepare a mixed reference substance solution 2;
s6, preparation of an ether layer control medicinal material solution: extracting Atractylodis rhizoma control medicinal material and ramulus Cinnamomi control medicinal material with water respectively, filtering, transferring filtrate into separating funnel, shaking with diethyl ether for 2 times, mixing diethyl ether solutions, volatilizing, and dissolving residue with methanol to obtain ether layer control medicinal material solution, i.e. Atractylodis rhizoma control medicinal material solution and ramulus Cinnamomi control medicinal material solution;
s7 thin layer chromatography analysis 2: and (3) taking the ether layer sample solution obtained in the step (S1), the bighead atractylodes rhizome reference medicinal material solution obtained in the step (S6), the cassia twig reference medicinal material solution and the mixed reference material solution 2 obtained in the step (S5), developing according to the thin-layer chromatography condition, and identifying spots of each component.
Further, in the step S1, decoction, freeze-dried powder or granules of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, which is equivalent to 5-10g of total decoction pieces, are taken, 20-60mL of water is added, heating is carried out to dissolve, cooling is carried out, the mixture is transferred into a separating funnel, diethyl ether is used for shaking and extracting for 2-3 times, 30-80mL of diethyl ether is used each time, diethyl ether solutions are combined and volatilized, and 1-3mL of methanol is added into residues to dissolve the residues, so that an ether layer sample solution is obtained; taking decoction, freeze-dried powder or particles of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, which is equivalent to 5-10g of total decoction piece, adding 20-60mL of water, heating to dissolve, cooling, passing through a macroporous adsorption resin column, eluting with 25-35mL of ammonia solution, discarding ammonia solution, eluting with 15-25mL of water, discarding water solution, continuing eluting with 30-80mL of ethanol water solution with the volume fraction of 10-60%, collecting eluent, evaporating to dryness, and adding 1-3mL of methanol into residues to dissolve, thus obtaining a water layer sample solution.
Further, in the step S2, a glycyrrhizin reference substance, a apigenin reference substance, a paeoniflorin reference substance and a 5-O-methyl-Weisi amiloride reference substance are taken, and methanol is used for preparing a mixed reference substance solution 1 with the content of 0.3-0.8 mg/mL.
Further, in the step S3, 0.3-0.7g of licorice reference medicine, 0.3-0.7g of white peony root reference medicine, 0.3-0.7g of ephedra reference medicine, 0.8-1.2g of radix sileris reference medicine and 0.8-1.2g of rhizoma anemarrhenae reference medicine are respectively added with 20-60mL of water, heated and reflux extracted for 0.5-1.5 hours, filtered while hot, filtered through a macroporous adsorption resin column, eluted with 25-35mL of ammonia solution, ammonia solution is removed, 15-25mL of water is removed, water solution is removed, 30-80mL of ethanol water solution with the volume fraction of 10-60% is continuously used for eluting, eluent is collected, evaporated to dryness, 1-3mL of methanol is added into residues to dissolve, and then aqueous layer reference medicine solutions, namely licorice reference medicine solution, white peony root reference medicine solution, ephedra reference medicine solution, radix sileris reference medicine solution and rhizoma anemarrhenae reference medicine solution are obtained.
Further, in the step S4, the thin layer chromatography conditions are: adopting a silica gel G plate as a thin layer plate, wherein the sample application amount is 10-40 mu L, the developing agent is ethyl acetate-methanol-water, developing, airing, and before color development, placing under a 365nm ultraviolet lamp for inspection, and displaying fluorescent spots with the same color at the positions corresponding to the ephedra control medicine chromatograph in the sample chromatograph; spraying phosphomolybdic acid sulfuric acid solution, and heating at 105 ℃ until the spots develop clearly; inspecting under fluorescent lamp, and displaying spots of the same color on the corresponding positions of the chromatogram of the test sample with the apioside glycyrrhizin control, paeoniflorin control, glycyrrhizin control, glycyrrhrizae radix control medicinal material, radix Paeoniae alba control medicinal material, and rhizoma anemarrhenae control medicinal material; inspecting under 365nm ultraviolet lamp, and making the sample chromatogram show spots with the same color at the positions corresponding to the 5-O-methylvisamiloride and radix Saposhnikoviae control medicine chromatogram.
Further, the volume ratio of the developing agent ethyl acetate to the methanol to the water is 15-20:2-7:1.
further, the volume ratio of the developing agent ethyl acetate to methanol to water is 20:2:1.
further, in the step S5, a 6-gingerol reference substance, a atractylenolide III reference substance and an atractylenolide II reference substance are taken, and a mixed reference substance solution 2 with the content of 0.3-0.8mg/mL is prepared by using methanol.
Further, in the step S6, 0.5-1.0g of bighead atractylodes rhizome reference medicine and 0.5-1.0g of cassia twig reference medicine are taken, 20-60mL of water is respectively added, heating reflux extraction is carried out for 0.5-1.5 hours, filtering is carried out while the mixture is still hot, filtrate is transferred to a separating funnel, shaking extraction is carried out for 2-3 times by diethyl ether, 30-80mL of diethyl ether solution is used each time, diethyl ether solution is combined and volatilized, and 1-3mL of methanol is added into residues to dissolve the residues, thus obtaining the ether layer reference medicine solution.
Further, in the step S7, the thin layer chromatography conditions are: adopting a silica gel GF254 plate as a thin layer plate, wherein the sample application amount of a sample solution of an ether layer, a sample solution of a bighead atractylodes rhizome reference medicinal material and a sample application amount of a cassia twig reference medicinal material solution are 20-40 mu L, the sample application amount of a mixed reference substance solution 2 is 10-20 mu L, developing a developing agent is cyclohexane-dichloromethane-ethyl ester-formic acid, developing, airing, and placing under a 365nm ultraviolet lamp for inspection, wherein spots with the same color appear in the positions corresponding to the sample chromatogram of the cassia twig reference medicinal material; spraying 5% vanillin sulfuric acid solution, baking at 105deg.C until spots develop, inspecting under fluorescent lamp, and displaying spots of the same color on the position corresponding to the color spectrum of 6-gingerol control sample in the color spectrum of the sample; inspecting under 365nm ultraviolet lamp, and displaying spots of the same color on the corresponding positions of the target sample chromatogram with the target atractylenolide II control, the target atractylenolide III control and the target atractylenolide III control.
Further, the volume ratio of the developing agent cyclohexane-dichloromethane-ethyl ester-formic acid is 3-4:1-2:1-2:0.1-0.2.
Further, the volume ratio of the developing agent cyclohexane-dichloromethane-ethyl ester-formic acid is 4:1:1:0.1.
the extraction method of the sample solution is based on the principle of similar compatibility, and comprises the steps of extracting small polar components in Chinese herbal medicines with diethyl ether to obtain an ether layer sample solution, adsorbing with macroporous resin, removing large polar components due to no retention, retaining small polar components due to strong adsorption, and eluting medium polar components with a proper solvent to obtain a water layer sample solution. Finally, different chemical components can be identified through multi-dimensional inspection conditions such as before and after color development, ultraviolet, sunlight and the like.
Compared with the prior art, the invention has the following advantages:
(1) According to the invention, ginger, cassia twig and bighead atractylodes rhizome can be identified simultaneously by preparing 1 sample solution for the first time.
(2) According to the invention, only 1 sample solution is prepared for the first time, and the sample solution is prepared by adopting a method of passing an aqueous solution through a column, so that the radix sileris, the ephedra, the rhizoma anemarrhenae, the radix paeoniae alba and the liquorice can be identified simultaneously.
(3) In the invention, when identifying liquorice, the liquorice is identified by adopting the liquiritin, the apioside and the liquiritin reference substance and the reference medicinal material for the first time.
(4) When the invention is used for identifying the bighead atractylodes rhizome, the bighead atractylodes rhizome lactone II, the bighead atractylodes rhizome lactone III reference substance and the reference medicinal material are adopted for the first time to identify the bighead atractylodes rhizome.
(5) The thin layer identification method provided by the invention can identify 8 medicinal herbs in 9 medicinal herbs in the whole prescription of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, has strong specificity in thin layer identification, is simple and convenient to operate and high in detection efficiency, reduces the detection cost, provides a reference basis for quality control of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, and has great significance.
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FIG. 1 is a thin layer chromatogram (identifying herba Ephedrae) of a sample solution of ramulus Cinnamomi, radix Paeoniae, and rhizoma anemarrhenae Shang Shuiceng under 365nm ultraviolet lamp before color development, wherein a is main spot of herba Ephedrae control medicine; 1. mixing reference solution 1 (5-O-methylvisamiloride, apioside, paeoniflorin, and glycyrrhizin); 2. radix Saposhnikoviae is used as reference medicine; 3. herba Ephedrae control material; 4. rhizoma anemarrhenae is used as reference medicine; 5. white peony root is used as a reference medicine; 6. licorice root is used as a reference medicine; 7. sample solution 1 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 8. sample solution 2 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 9. sample solution 3 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 10. wind-proof negative; 11. negative herba Ephedrae; 12. rhizoma anemarrhenae is negative; 13. white peony root is negative; 14. licorice was negative.
FIG. 2 is a thin layer chromatogram of the sample solution of ramulus Cinnamomi, radix Paeoniae, and rhizoma anemarrhenae Shang Shuiceng (identifying rhizoma anemarrhenae, radix Paeoniae alba, and Glycyrrhrizae radix) inspected under fluorescent lamp after color development, wherein b and glycyrrhizin; c. paeoniflorin; d. apigenin; e. major spots of rhizoma anemarrhenae contrast medicinal materials; 1. mixing reference solution 1 (5-O-methylvisamiloride, apioside, paeoniflorin, and glycyrrhizin); 2. radix Saposhnikoviae is used as reference medicine; 3. herba Ephedrae control material; 4. rhizoma anemarrhenae is used as reference medicine; 5. white peony root is used as a reference medicine; 6. licorice root is used as a reference medicine; 7. sample solution 1 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 8. sample solution 2 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 9. sample solution 3 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 10. wind-proof negative; 11. negative herba Ephedrae; 12. rhizoma anemarrhenae is negative; 13. white peony root is negative; 14. licorice was negative.
FIG. 3 is a thin layer chromatogram (identifying Fangfeng) of a test solution of ramulus Cinnamomi, radix Paeoniae, and rhizoma anemarrhenae Shang Shuiceng under 365nm ultraviolet lamp after color development, wherein f, 5-O-methyl-Weisi amiloride; 1. mixing reference solution 1 (5-O-methylvisamiloride, apioside, paeoniflorin, and glycyrrhizin); 2. radix Saposhnikoviae is used as reference medicine; 3. herba Ephedrae control material; 4. rhizoma anemarrhenae is used as reference medicine; 5. white peony root is used as a reference medicine; 6. licorice root is used as a reference medicine; 7. sample solution 1 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 8. sample solution 2 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 9. sample solution 3 of the sample solution of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae Shang Shuiceng; 10. wind-proof negative; 11. negative herba Ephedrae; 12. rhizoma anemarrhenae is negative; 13. white peony root is negative; 14. licorice was negative.
FIG. 4 is a thin layer chromatogram (identifying ramulus Cinnamomi) of the ether layer of the decoction of ramulus Cinnamomi, radix Paeoniae and rhizoma anemarrhenae examined at 365nm before color development, wherein g and ramulus Cinnamomi are the main spots of the control medicinal materials; 1. mixing reference solution 2 (6-gingerol, atractylenolide III and atractylenolide II); 2. ramulus Cinnamomi control; 3. rhizoma Atractylodis Macrocephalae is used as reference material; 4. sample solution 1 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae soup ether layer; 5. sample solution 2 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae decoction ether layer; 6. sample solution 3 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae decoction ether layer; 7. ramulus Cinnamomi is negative; 8. negative Atractylodis rhizoma; 9. ginger is negative.
FIG. 5 shows a thin layer chromatogram (identified as rhizoma Zingiberis recens) of the test sample solution of ramulus Cinnamomi, radix Paeoniae, rhizoma anemarrhenae, and rhizoma anemarrhenae under fluorescent lamp after color development, wherein h and 6-gingerol; 1. mixing reference solution 2 (6-gingerol, atractylenolide III and atractylenolide II); 2. ramulus Cinnamomi control; 3. rhizoma Atractylodis Macrocephalae is used as reference material; 4. sample solution 1 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae soup ether layer; 5. sample solution 2 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae decoction ether layer; 6. sample solution 3 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae decoction ether layer; 7. ramulus Cinnamomi is negative; 8. negative Atractylodis rhizoma; 9. ginger is negative.
FIG. 6 is a thin layer chromatogram (identified by Atractylodis rhizoma) of 365nm ultraviolet lamp after color development of test solution of ramulus Cinnamomi, radix Paeoniae and rhizoma anemarrhenae decoction ether layer, wherein i, and atractylenolide II; j. atractylodes macrocephala lactone III; 1. mixing reference solution 2 (6-gingerol, atractylenolide III and atractylenolide II); 2. ramulus Cinnamomi control; 3. rhizoma Atractylodis Macrocephalae is used as reference material; 4. sample solution 1 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae soup ether layer; 5. sample solution 2 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae decoction ether layer; 6. sample solution 3 of the test sample solution of the cassia twig, chinese herbaceous peony, rhizoma anemarrhenae decoction ether layer; 7. ramulus Cinnamomi is negative; 8. negative Atractylodis rhizoma; 9. ginger is negative.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention without departing from the basic idea of the invention.
Instrument information used in the examples:
electronic balance, mertrer-tolidol, model: XPR105DR, AL104-2; electrothermal blowing drying box: DHG-9245A, shanghai-Hengsu scientific instruments Co., ltd; electric heating plate: shanghai Libang West instruments technologies Co., ltd., DB-XAB; automatic sample application instrument: swiss kama company; automatic imager: swiss kama company.
Table 1 shows the information of the reference substances and the reference medicinal materials in the examples of the present invention.
Table 1: reference substance and reference medicinal material information in the embodiment of the invention
Example 1 preparation of decoction of ramulus Cinnamomi, radix Paeoniae and rhizoma anemarrhenae
Ramulus Cinnamomi, radix Paeoniae (radix Paeoniae alba in the invention), glycyrrhrizae radix, herba Ephedrae, rhizoma Zingiberis recens, atractylodis rhizoma, rhizoma anemarrhenae, radix Saposhnikoviae, and radix Aconiti lateralis Preparata. The nine ingredients are boiled with seven liters of water, and two liters of water are boiled to obtain the Chinese herbal medicine.
Example 2 preparation of Guizhi Shaoyao Zhi Tang granule
Ramulus Cinnamomi, radix Paeoniae (radix Paeoniae alba in the invention), glycyrrhrizae radix, herba Ephedrae, rhizoma Zingiberis recens, atractylodis rhizoma, rhizoma anemarrhenae, radix Saposhnikoviae, and radix Aconiti lateralis Preparata. The nine ingredients are boiled with seven liters of water, two liters of water are boiled, the filtrate is concentrated, and the mixture is dried and made into particles.
Example 3 preparation of lyophilized powder of Guizhi Shaoyao Zhi Tang
Ramulus Cinnamomi, radix Paeoniae (radix Paeoniae alba in the invention), glycyrrhrizae radix, herba Ephedrae, rhizoma Zingiberis recens, atractylodis rhizoma, rhizoma anemarrhenae, radix Saposhnikoviae, and radix Aconiti lateralis Preparata. The nine ingredients are boiled with seven liters of water, taken out by two liters, put into penicillin bottles and freeze-dried.
Example 4, a thin layer identification method of 8 herbs in 9 herbs in the whole recipe of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, the thin layer identification method of 8 herbs in 9 herbs in the whole recipe of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction comprises the following steps:
s1, preparation of a sample solution: taking decoction of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction prepared in the embodiment 1, which is equivalent to 5g of total decoction piece, adding 50mL of water, heating to dissolve, cooling, transferring to a separating funnel, shaking and extracting for 2 times with diethyl ether, 50mL each time, combining diethyl ether solutions, volatilizing, and adding 2mL of methanol into residues to dissolve to obtain an ether layer sample solution; taking decoction of ramulus Cinnamomi, radix Paeoniae and rhizoma anemarrhenae decoction, which is equivalent to 5g of total decoction piece, adding 50mL of water, heating to dissolve, cooling, passing through macroporous adsorption resin column (the inner diameter of the macroporous adsorption resin column is 1.5cm, the height of the macroporous adsorption resin column is 8cm, adding water to pre-wash until no alcohol smell), eluting with 30mL of ammonia solution (4-100, namely, taking 4mL of concentrated ammonia solution, adding water to 100 mL), discarding ammonia solution, eluting with 20mL of water again, discarding water solution, continuing eluting with 50mL of ethanol water solution with volume fraction of 20%, collecting eluent, evaporating to dryness, and dissolving residues with 2mL of methanol to obtain water layer sample solution;
S2, preparation of a mixed reference substance solution 1: preparing a mixed reference substance solution 1 with 0.5mg/mL of glycyrrhizin, apigenin, paeoniflorin and 5-O-methylvitamin-A-D by using methanol;
s3, preparing a water layer control medicinal material solution: taking 0.5g of licorice reference medicine, 0.5g of white peony root reference medicine, 0.5g of ephedra reference medicine, 1g of divaricate saposhnikovia root reference medicine and 1g of rhizoma anemarrhenae reference medicine, respectively adding 50mL of water, heating and reflux-extracting for 1 hour, filtering while hot, passing the filtrate through a macroporous adsorption resin column (the inner diameter of the macroporous adsorption resin column is 1.5cm, the height of the column is 8cm, adding water for pre-washing until no alcohol smell exists), eluting with 30mL of ammonia solution (4-100, namely, taking 4mL of concentrated ammonia solution, adding water to 100 mL), discarding ammonia solution, eluting with 20mL of water again, discarding water solution, continuing eluting with 50mL of ethanol water solution with the volume fraction of 20%, collecting eluent, evaporating to dryness, and adding 2mL of methanol into residues for dissolving to obtain aqueous layer reference medicine solutions, namely licorice reference medicine solution, white peony root reference medicine solution, ephedra reference medicine solution, divaricate saposhnikovia root reference medicine solution and rhizoma anemarrhenae reference medicine solution;
S4 thin layer chromatography analysis 1: according to a thin layer chromatography (four parts (general rule 0502) test in 2020 edition of Chinese pharmacopoeia), sucking the water layer sample solution obtained in the step S1, the licorice control medicinal material solution obtained in the step S3, the white peony root control medicinal material solution, the rhizoma anemarrhenae control medicinal material solution, the ephedra control medicinal material solution and the divaricate saposhnikovia root control medicinal material solution, and the mixed control material solution 1 obtained in the step S2 respectively by 20 mu L, respectively, putting the mixed control material solution on the same silica gel G thin layer plate, developing with a solution with the volume ratio of ethyl acetate to methanol to water being 15:3:1 as a developing agent, airing, and before developing, placing under a 365nm ultraviolet lamp for inspection, and in the chromatogram of the sample, putting spots with the same color on the position corresponding to the chromatogram of the ephedra control medicinal material; spraying phosphomolybdic acid sulfuric acid solution (phosphomolybdic acid 2G, adding water 20mL to dissolve, slowly adding sulfuric acid 30mL, mixing well to obtain), heating at 105deg.C until the color of spots is clear, inspecting under fluorescent lamp, wherein spots with the same color appear on the positions corresponding to the chromatograms of the apigenin control, paeoniflorin control, glycyrrhizin control, licorice control, white peony root control and rhizoma anemarrhenae control are detected in the chromatogram of the sample, inspecting under 365nm ultraviolet lamp, and displaying spots with the same color on the positions corresponding to the chromatograms of 5-O-methylvisamiloride and radix Saposhnikoviae control in the chromatogram of the sample;
S5, preparation of a mixed reference substance solution 2: taking 6-gingerol reference substance, atractylenolide III reference substance and atractylenolide II reference substance, and preparing a mixed reference substance solution 2 with 6-gingerol, atractylenolide III and atractylenolide II content of 0.5mg/mL by using methanol;
s6, preparation of an ether layer control medicinal material solution: taking 1g of bighead atractylodes rhizome reference medicinal material and 1g of cassia twig reference medicinal material, respectively adding 50mL 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, volatilizing, and adding 2mL of methanol into residues to dissolve the mixture, thereby obtaining an ether layer reference medicinal material solution, namely a bighead atractylodes rhizome reference medicinal material solution and a cassia twig reference medicinal material solution;
s7 thin layer chromatography analysis 2: according to a thin layer chromatography (the general rule 0502 of the fourth edition of 2015 of Chinese pharmacopoeia), 20 mu L of the ether layer test solution obtained in the step S1, 20 mu L of the bighead atractylodes rhizome control medicinal material solution obtained in the step S6 and 20 mu L of the cassia twig control medicinal material solution are sucked, the mixed control solution obtained in the step S5 is respectively spotted on the same silica gel GF254 thin layer plate, and the volume ratio of cyclohexane, methylene dichloride, ethyl ester and formic acid is 3:2:2:0.2 as developing agent, developing, air drying, and inspecting under 365nm ultraviolet lamp to show spots of the same color on the corresponding position of the chromatogram of the sample and the chromatogram of the ramulus Cinnamomi control; spraying 5% vanillin sulfuric acid solution, baking at 105deg.C until spots develop, inspecting under fluorescent lamp, and displaying spots of the same color on the position corresponding to the color spectrum of 6-gingerol control sample in the color spectrum of the sample; inspecting under 365nm ultraviolet lamp, and displaying spots of the same color on the corresponding positions of the target sample chromatogram with the target atractylenolide II control, the target atractylenolide III control and the target atractylenolide III control.
Example 5 thin-layer identification method of 8 herbs in 9 herbs in full formula of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction
The thin-layer identification method of 8 medicinal herbs in 9 medicinal herbs in the whole formula of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction comprises the following steps of:
s1, preparation of a sample solution: taking particles of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction prepared in the embodiment 2, which is equivalent to 10g of total decoction piece, adding 60mL of water, heating to dissolve, cooling, transferring to a separating funnel, shaking and extracting for 3 times with diethyl ether, 50mL each time, combining diethyl ether solutions, volatilizing, and adding 3mL of methanol into residues to dissolve to obtain an ether layer sample solution; taking particles of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, which is equivalent to 10g of total decoction piece, adding 60mL of water, heating to dissolve, cooling, passing through a macroporous adsorption resin column (the inner diameter of the macroporous adsorption resin column is 1.5cm, the height of the macroporous adsorption resin column is 8cm, adding water to pre-wash until no alcohol smell), eluting with 35mL of ammonia solution (4-100, namely, taking 4mL of concentrated ammonia solution, adding water to 100 mL), discarding ammonia solution, eluting with 25mL of water again, discarding water solution, continuing eluting with 50mL of ethanol water solution with volume fraction of 60%, collecting eluent, evaporating to dryness, and dissolving residues with 3mL of methanol to obtain a water layer sample solution;
s2, preparation of a mixed reference substance solution 1: preparing a mixed reference substance solution 1 with 0.8mg/mL of glycyrrhizin, apigenin, paeoniflorin and 5-O-methylvitamin-A-D-A by using methanol;
S3, preparing a water layer control medicinal material solution: taking 0.7g of liquorice control medicinal material, 0.7g of white peony root control medicinal material, 0.7g of ephedra control medicinal material, 1.2g of divaricate saposhnikovia root control medicinal material and 1.2g of rhizoma anemarrhenae control medicinal material, respectively adding 45mL of water, heating and reflux extracting for 1.5 hours, filtering while the mixture is hot, passing the filtrate through a macroporous adsorption resin column (the inner diameter of the macroporous adsorption resin column is 1.5cm, the height of the column is 8cm, adding water for pre-washing until no alcohol smell), eluting with 35mL of ammonia solution (4-100, namely, taking 4mL of concentrated ammonia solution, adding water to 100 mL), discarding ammonia solution, eluting with 25mL of water again, eluting with 50mL of ethanol water solution with the volume fraction of 45%, collecting eluent, evaporating to dryness, adding 3mL of methanol into residues to dissolve, thus obtaining water layer control medicinal material solutions, namely liquorice control medicinal material solution, white peony root control medicinal material solution, ephedra control medicinal material solution, divaricate saposhnikovia root control medicinal material solution;
s4 thin layer chromatography analysis 1: according to a thin layer chromatography (four general rules of 2015 of Chinese pharmacopoeia 0502), sucking the water layer test solution obtained in the step S1, the licorice control medicinal material solution, white peony root control medicinal material solution, rhizoma anemarrhenae control medicinal material solution, ephedra control medicinal material solution and radix sileris control medicinal material solution obtained in the step S3, and the mixed control material solution 1 obtained in the step S2 respectively by 10 mu L, respectively putting the mixed control material solutions on the same silica gel G thin layer plate, wherein the volume ratio of ethyl acetate to methanol to water is 20:2:1 as developing agent, developing, air drying, inspecting under 365nm ultraviolet lamp, and displaying spots with the same color on the position corresponding to herba Ephedrae control medicine chromatogram in the sample chromatogram; spraying phosphomolybdic acid sulfuric acid solution (phosphomolybdic acid 2g, adding water 20mL to dissolve, slowly adding sulfuric acid 30mL, mixing well to obtain), heating at 105deg.C until the color of spots is clear; inspecting under fluorescent lamp, and displaying spots of the same color on the corresponding positions of the chromatogram of the test sample with the apioside glycyrrhizin control, paeoniflorin control, glycyrrhizin control, glycyrrhrizae radix control medicinal material, radix Paeoniae alba control medicinal material, and rhizoma anemarrhenae control medicinal material; inspecting under 365nm ultraviolet lamp, and displaying spots with the same color on the positions corresponding to the 5-O-methylvisamiloride and radix Saposhnikoviae control medicine chromatogram in the sample chromatogram;
S5, preparation of a mixed reference substance solution 2: taking 6-gingerol reference substance, atractylenolide III reference substance and atractylenolide II reference substance, and preparing a mixed reference substance solution 2 with 6-gingerol, atractylenolide III and atractylenolide II content of 0.8mg/mL by using methanol;
s6, preparation of an ether layer control medicinal material solution: taking 0.8g of bighead atractylodes rhizome reference medicinal material and 0.8g of cassia twig reference medicinal material, respectively adding 45mL of water, heating and refluxing for extraction for 1.5 hours, filtering while the bighead atractylodes rhizome reference medicinal material and the cassia twig reference medicinal material are hot, transferring filtrate into a separating funnel, shaking and extracting for 3 times by using diethyl ether for 50mL each time, combining diethyl ether liquid, volatilizing, and adding 3mL of methanol into residues to dissolve the residues to obtain an ether layer reference medicinal material solution, namely the bighead atractylodes rhizome reference medicinal material solution and the cassia twig reference medicinal material solution;
s7 thin layer chromatography analysis 2: according to a thin layer chromatography (the general rule 0502 of the fourth edition of 2015 of Chinese pharmacopoeia), the ether layer test solution obtained in the step S1, the bighead atractylodes rhizome control medicinal material solution obtained in the step S6 and the cassia twig control medicinal material solution are sucked to 40 mu L respectively, and the mixed control solution obtained in the step S5 is respectively dropped on the same silica gel GF254 thin layer plate, wherein the volume ratio of cyclohexane, methylene dichloride, ethyl ester and formic acid is 4:1:1: the solution of 0.1 is used as developing agent, developed and dried, and is inspected under 365nm ultraviolet lamp, and spots with the same color appear on the position corresponding to the chromatogram of the cassia twig control medicine in the chromatogram of the sample; spraying 5% vanillin sulfuric acid solution, baking at 105deg.C until spots develop, inspecting under fluorescent lamp, and displaying spots of the same color on the position corresponding to the color spectrum of 6-gingerol control sample in the color spectrum of the sample; inspecting under 365nm ultraviolet lamp, and displaying spots of the same color on the corresponding positions of the target sample chromatogram with the target atractylenolide II control, the target atractylenolide III control and the target atractylenolide III control.
Example 6 thin-layer identification method of 8 herbs in 9 herbs in full formula of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction
The thin-layer identification method of 8 medicinal herbs in 9 medicinal herbs in the whole formula of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction comprises the following steps of:
s1, preparation of a sample solution: taking freeze-dried powder of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction prepared in the embodiment 3, which is equivalent to 10g of total decoction piece, adding 50mL of water, heating to dissolve, cooling, transferring to a separating funnel, shaking and extracting for 2 times with diethyl ether, 50mL each time, combining diethyl ether solutions, volatilizing, and adding 2mL of methanol into residues to dissolve to obtain an ether layer sample solution; taking freeze-dried powder of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, which is equivalent to 10g of total decoction piece, adding 50mL of water, heating to dissolve, cooling, passing through a macroporous adsorption resin column (the inner diameter of the macroporous adsorption resin column is 1.5cm, the height of the macroporous adsorption resin column is 8cm, adding water to pre-wash until no alcohol smell), eluting with 30mL of ammonia solution (4-100, namely, taking 4mL of concentrated ammonia solution, adding water to 100 mL), discarding ammonia solution, eluting with 20mL of water again, discarding water solution, continuing eluting with 50mL of ethanol water solution with the volume fraction of 40%, collecting eluent, evaporating to dryness, and dissolving residues with 2mL of methanol to obtain a water layer sample solution;
s2, preparation of a mixed reference substance solution 1: preparing a mixed reference substance solution 1 with 0.5mg/mL of glycyrrhizin, apigenin, paeoniflorin and 5-O-methylvitamin-A-D by using methanol;
S3, preparing a water layer control medicinal material solution: taking 0.5g of licorice reference medicine, 0.5g of white peony root reference medicine, 0.5g of ephedra reference medicine, 1.0g of divaricate saposhnikovia root reference medicine and 1.0g of rhizoma anemarrhenae reference medicine, respectively adding 40mL of water, heating and refluxing for extraction for 1 hour, filtering while the mixture is hot, passing the filtrate through a macroporous adsorption resin column (the inner diameter of the macroporous adsorption resin column is 1.5cm, the height of the column is 8cm, adding water for pre-washing until no alcohol smell), eluting with 30mL of ammonia solution (4-100, namely, taking 4mL of concentrated ammonia solution, adding water to 100 mL), discarding ammonia solution, eluting with 20mL of water again, discarding water solution, continuing eluting with 50mL of ethanol water solution with the volume fraction of 40%, collecting eluent, evaporating to dryness, adding 2mL of methanol into residues for dissolution, and obtaining aqueous layer reference medicine solutions, namely licorice reference medicine solution, white peony root reference medicine solution, ephedra reference medicine solution, divaricate saposhnikovia root reference medicine solution and rhizoma anemarrhenae reference medicine solution;
s4 thin layer chromatography analysis 1: according to a thin layer chromatography (four parts (general rule 0502) test in 2020 edition of Chinese pharmacopoeia), sucking the water layer sample solution obtained in the step S1, the licorice control medicinal material solution obtained in the step S3, the white peony root control medicinal material solution, the rhizoma anemarrhenae control medicinal material solution, the ephedra control medicinal material solution and the divaricate saposhnikovia root control medicinal material solution, and the mixed control material solution 1 obtained in the step S2 respectively by 10 mu L, respectively, putting the mixed control material solution on the same silica gel G thin layer plate, developing with a solution with the volume ratio of ethyl acetate to methanol to water being 20:2:1 as a developing agent, airing, and before developing, placing under a 365nm ultraviolet lamp for inspection, and in the chromatogram of the sample, putting spots with the same color on the position corresponding to the chromatogram of the ephedra control medicinal material; spraying phosphomolybdic acid sulfuric acid solution (phosphomolybdic acid 2G, adding water 20mL to dissolve, slowly adding sulfuric acid 30mL, mixing well to obtain), heating at 105deg.C until the color of spots is clear, inspecting under fluorescent lamp, wherein spots with the same color appear on the positions corresponding to the chromatograms of the apigenin control, paeoniflorin control, glycyrrhizin control, licorice control, white peony root control and rhizoma anemarrhenae control are detected in the chromatogram of the sample, inspecting under 365nm ultraviolet lamp, and displaying spots with the same color on the positions corresponding to the chromatograms of 5-O-methylvisamiloride and radix Saposhnikoviae control in the chromatogram of the sample;
S5, preparation of a mixed reference substance solution 2: taking 6-gingerol reference substance, atractylenolide III reference substance and atractylenolide II reference substance, and preparing a mixed reference substance solution 2 with 6-gingerol, atractylenolide III and atractylenolide II content of 0.5mg/mL by using methanol;
s6, preparation of an ether layer control medicinal material solution: taking 0.5g of bighead atractylodes rhizome reference medicinal material and 0.5g of cassia twig reference medicinal material, respectively adding 40mL of water, heating, refluxing and extracting for 1 hour, filtering while the hot, transferring filtrate into a separating funnel, shaking and extracting for 2 times with diethyl ether for 50mL each time, combining diethyl ether liquid, volatilizing, and adding 2mL of methanol into residues to dissolve the residues to obtain an ether layer reference medicinal material solution, namely a bighead atractylodes rhizome reference medicinal material solution and a cassia twig reference medicinal material solution;
s7 thin layer chromatography analysis 2: according to a thin layer chromatography (four-part rule 0502 of the 2020 edition of Chinese pharmacopoeia), sucking 40 mu L of each of the ether layer test solution, the white atractylodes rhizome reference medicinal material solution and the cassia twig reference medicinal material solution obtained in the step S1, and respectively applying 2 mu L of the mixed reference substance solution obtained in the step S5 to the same silica gel GF254 thin layer plate, wherein the volume ratio of cyclohexane, dichloromethane, ethyl ester and formic acid is 4:1:1: the solution of 0.1 is used as developing agent, developed and dried, and is inspected under 365nm ultraviolet lamp, and spots with the same color appear on the position corresponding to the chromatogram of the cassia twig control medicine in the chromatogram of the sample; spraying 5% vanillin sulfuric acid solution, baking at 105deg.C until spots develop, inspecting under fluorescent lamp, and displaying spots of the same color on the position corresponding to the color spectrum of 6-gingerol control sample in the color spectrum of the sample; inspecting under 365nm ultraviolet lamp, and displaying spots of the same color on the corresponding positions of the target sample chromatogram with the target atractylenolide II control, the target atractylenolide III control and the target atractylenolide III control.
Test example one, thin layer authentication test
Three batches of freeze-dried powder samples (numbered 1, 2 and 3) of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction were prepared according to the method of example 3, and test solution (ether layer test solution, aqueous layer test solution), mixed reference solution 1, aqueous layer reference medicinal material solution (licorice reference medicinal material solution, white peony reference medicinal material solution, ephedra reference medicinal material solution, divaricate saposhnikovia root reference medicinal material solution and rhizoma anemarrhenae reference medicinal material solution), mixed reference solution 2 and ether layer reference medicinal material solution (bighead atractylodes rhizome reference medicinal material solution and cassia twig reference medicinal material solution) were prepared according to the method of example 6. Preparing a divaricate saposhnikovia root negative sample solution, a ephedra negative sample solution, a rhizoma anemarrhenae negative sample solution, a white peony root negative sample solution, a liquorice negative sample solution, a cassia twig negative sample solution, a bighead atractylodes rhizome negative sample solution and a ginger negative sample solution.
According to a thin layer chromatography (four-part rule 0502 of the 2020 edition of Chinese pharmacopoeia), a water layer test sample 1, a water layer test sample 2, a water layer test sample 3, a licorice control medicinal material solution, a white paeony root control medicinal material solution, a rhizoma anemarrhenae control medicinal material solution, an ephedra control medicinal material solution and a radix saposhnikoviae control medicinal material solution, a mixed control sample solution 1, a radix saposhnikoviae negative sample solution, an ephedra negative sample solution, a rhizoma anemarrhenae negative sample solution, a white paeony root negative sample solution and a licorice negative sample solution are respectively 10 mu L, and are respectively spotted on the same silica gel G thin layer plate, wherein the volume ratio of ethyl acetate to methanol to water is 20:2:1 is used as developing agent, and is spread, dried and subjected to inspection under 365nm ultraviolet lamp before color development, and spots with the same color appear on the positions corresponding to the chromatogram of the ephedra control medicine in the chromatogram of the sample. Spraying phosphomolybdic acid sulfuric acid solution (phosphomolybdic acid 2g, adding water 20mL to dissolve, slowly adding sulfuric acid 30mL, mixing well to obtain), heating at 105deg.C until the color of spots is clear; inspecting under fluorescent lamp, and displaying spots of the same color on the corresponding positions of the chromatogram of the test sample with the apioside glycyrrhizin control, paeoniflorin control, glycyrrhizin control, glycyrrhrizae radix control medicinal material, radix Paeoniae alba control medicinal material, and rhizoma anemarrhenae control medicinal material; inspecting under 365nm ultraviolet lamp, and making the sample chromatogram show spots with the same color at the positions corresponding to the 5-O-methylvisamiloride and radix Saposhnikoviae control medicine chromatogram.
According to a thin layer chromatography (four-part rule 0502 of the 2020 edition of Chinese pharmacopoeia), sucking an ether layer test sample 1, an ether layer test sample 2, an ether layer test sample 3, a white atractylodes rhizome control medicinal material solution, a cassia twig negative sample solution, a white atractylodes rhizome negative sample solution and a ginger negative sample solution which are 40 mu L respectively, mixing 2 mu L of the control solution and 2 mu L of the mixed control solution, respectively, putting the mixed control solution and the mixed control solution on the same silica gel GF254 thin layer plate, wherein the volume ratio of cyclohexane, methylene dichloride, ethyl ester and formic acid is 4:1:1: the solution of 0.1 is used as developing agent, developed and dried, and is inspected under 365nm ultraviolet lamp, and spots with the same color appear on the position corresponding to the chromatogram of the cassia twig control medicine in the chromatogram of the sample; spraying 5% vanillin sulfuric acid solution, baking at 105deg.C until spots develop, inspecting under fluorescent lamp, and displaying spots of the same color on the position corresponding to the color spectrum of 6-gingerol control sample in the color spectrum of the sample; inspecting under 365nm ultraviolet lamp, and displaying spots of the same color on the corresponding positions of the target sample chromatogram with the target atractylenolide II control, the target atractylenolide III control and the target atractylenolide III control.
The authentication results are shown in fig. 1-6. As can be seen from fig. 1: (1) In the water layer test sample solution samples of 3 batches of samples of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae soup (strips 7, 8 and 9), main spots (main spots of a herba ephedra control medicinal material) with the same color as that of the herba ephedrae control medicinal material of strip 3 are shown, and a strip 11 is a herba ephedra negative sample, does not show the corresponding main spots, and shows that the sample has specificity. Herba Ephedrae can be identified.
As can be seen from fig. 2: (1) In the water layer test sample solution samples of 3 batches of samples of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae soup (strips 7, 8 and 9), spots with the same color as that of the liquorice control medicinal materials in the b glycyrrhizin, d apioside and the liquorice control medicinal materials in the strip 6 are mixed with the strip 1, and the strip 14 is a liquorice negative sample, does not show corresponding spots, so that the specificity is shown. Can identify liquorice. (2) In the water layer test sample solution samples of 3 batches of samples of the cassia twig-paeonia lactiflora-anemarrhena decoction (strips 7, 8 and 9), spots with the same color are shown in c paeoniflorin and strip 5 paeonia lactiflora control medicines in the mixed reference substance solution 1 of the strip 1, and the strip 13 is a white peony negative sample and does not show corresponding spots, so that the special sample is shown. White peony root can be identified; (3) In the water layer test sample solution samples of 3 batches of samples of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction (strips 7, 8 and 9), main spots (main spots of e rhizoma anemarrhenae contrast medicinal materials) with the same color as that of the strip 4 rhizoma anemarrhenae contrast medicinal materials are shown, and a strip 12 is a rhizoma anemarrhenae negative sample, does not show the corresponding main spots, and shows that the sample has specificity. Can identify rhizoma anemarrhenae.
As can be seen from fig. 3: (1) In the water layer test sample solution samples of 3 batches of samples of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae soup (strips 7, 8 and 9), f (5-O-methyl Weisi ami glycoside) in the mixed reference substance solution 1 and the divaricate saposhnikovia root reference medicinal material in the strip 2 show the same color main spots, and the strip 10 is a divaricate saposhnikovia root negative sample, does not show corresponding spots, and shows that the divaricate saposhnikovia root reference medicinal material has specificity. Can identify the wind prevention.
As can be seen from fig. 4: in ether layer test solution samples of 3 batches of samples of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae soup (strips 4, 5 and 6), main spots (g main spots of the cassia twig control medicinal material) with the same color as that of the cassia twig control medicinal material of strip 2 are shown, and strip 7 is a cassia twig negative sample, does not show corresponding main spots, and has specificity. Ramulus Cinnamomi can be identified.
As can be seen from fig. 5: in ether layer test sample solution samples of 3 batches of samples of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae soup (strips 4, 5 and 6), main spots with the same color are displayed in h (6-gingerol) in a mixed reference substance solution 2 with a strip 1, a strip 9 is Jiang Yinxing samples, and the corresponding main spots are not displayed, so that the special effect is shown. Ginger can be identified.
As can be seen from fig. 6: in the ether layer test sample solution samples of 3 batches of samples of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae soup (strips 4, 5 and 6), main spots with the same color appear in i (atractylenolide II), j (atractylenolide III) and strip 3 atractylenolide control medicinal materials in the mixed reference substance solution 2 of the strip 1, and the strip 8 is an atractylenolide negative sample without corresponding main spots, which indicates that the special medicine has specificity. The bighead atractylodes rhizome can be identified.
The results show that the thin-layer identification method can identify 8 medicinal herbs in 9 medicinal herbs in the whole prescription of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, has strong specificity in thin-layer identification, is simple and convenient and quick to operate, has high detection efficiency, reduces detection cost, provides reference basis for quality control of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, and has great significance.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (3)
1. The thin-layer identification method of 8 medicinal herbs in 9 medicinal herbs in the whole prescription of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction is characterized by comprising the following steps of:
s1, preparation of a sample solution: taking decoction, lyophilized powder or granule of ramulus Cinnamomi, radix Paeoniae and rhizoma anemarrhenae decoction, and respectively preparing into ether layer test solution and water layer test solution;
s2, preparation of a mixed reference substance solution 1: taking a reference substance to prepare a mixed reference substance solution 1;
s3, preparing a water layer control medicinal material solution: extracting Glycyrrhrizae radix control medicinal material, radix Paeoniae alba control medicinal material, herba Ephedrae control medicinal material, radix Saposhnikoviae control medicinal material, and rhizoma anemarrhenae control medicinal material with water respectively, filtering, purifying the filtrate with macroporous adsorbent resin column, eluting with ammonia solution, discarding ammonia solution, eluting with water again, discarding water solution, eluting with ethanol solution continuously, collecting eluate, evaporating to dryness, and dissolving the residue with methanol to obtain aqueous layer control medicinal material solution, namely Glycyrrhrizae radix control medicinal material solution, radix Paeoniae alba control medicinal material solution, herba Ephedrae control medicinal material solution, radix Saposhnikoviae control medicinal material solution, and rhizoma anemarrhenae control medicinal material solution;
S4 thin layer chromatography analysis 1: taking the water layer sample solution obtained in the step S1, the licorice control medicinal material solution obtained in the step S3, the white paeony root control medicinal material solution, the rhizoma anemarrhenae control medicinal material solution, the ephedra control medicinal material solution and the divaricate saposhnikovia root control medicinal material solution, and the mixed control material solution 1 obtained in the step S2, developing according to the thin-layer chromatography condition, and identifying spots of each component;
s5, preparation of a mixed reference substance solution 2: taking a reference substance to prepare a mixed reference substance solution 2;
s6, preparation of an ether layer control medicinal material solution: extracting Atractylodis rhizoma control medicinal material and ramulus Cinnamomi control medicinal material with water respectively, filtering, transferring filtrate into separating funnel, shaking with diethyl ether for 2 times, mixing diethyl ether solutions, volatilizing, and dissolving residue with methanol to obtain ether layer control medicinal material solution, i.e. Atractylodis rhizoma control medicinal material solution and ramulus Cinnamomi control medicinal material solution;
s7 thin layer chromatography analysis 2: taking the ether layer test solution obtained in the step S1, the bighead atractylodes rhizome control medicinal material solution obtained in the step S6, the cassia twig control medicinal material solution and the mixed control solution 2 obtained in the step S5, developing according to the thin-layer chromatography condition, and identifying spots of each component;
in the step S1, decoction, freeze-dried powder or granules of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, which is equivalent to 5-10g of total decoction pieces, are taken, 20-60mL of water is added, heating is carried out to dissolve, cooling is carried out, the mixture is transferred into a separating funnel, shaking and extracting are carried out for 2-3 times by using diethyl ether for 30-80mL each time, diethyl ether liquid is combined, volatilizing is carried out, and 1-3mL of methanol is added into residues to dissolve, thus obtaining an ether layer sample solution; taking decoction, freeze-dried powder or particles of cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction, which is equivalent to 5-10g of total decoction piece, adding 20-60mL of water, heating to dissolve, cooling, passing through a macroporous adsorption resin column, eluting with 25-35mL of ammonia solution, discarding ammonia solution, eluting with 15-25mL of water, discarding water solution, continuing eluting with 30-80mL of ethanol water solution with the volume fraction of 10-60%, collecting eluent, evaporating to dryness, and adding 1-3mL of methanol into residues to dissolve to obtain a water layer sample solution;
In the step S2, preparing a mixed reference substance solution 1 with 0.3-0.8mg/mL of methanol from a glycyrrhizin reference substance, a apigenin reference substance, a paeoniflorin reference substance and a 5-O-methylvisamiloride reference substance;
in the step S4, the thin layer chromatography conditions are as follows: adopting a silica gel G plate as a thin layer plate, wherein the sample application amount is 10-40 mu L, the developing agent is ethyl acetate-methanol-water, developing, airing, and before color development, placing under a 365nm ultraviolet lamp for inspection, and displaying fluorescent spots with the same color at the positions corresponding to the ephedra control medicine chromatograph in the sample chromatograph; spraying phosphomolybdic acid sulfuric acid solution, and heating at 105 ℃ until the spots develop clearly; inspecting under fluorescent lamp, and displaying spots of the same color on the corresponding positions of the chromatogram of the test sample with the apioside glycyrrhizin control, paeoniflorin control, glycyrrhizin control, glycyrrhrizae radix control medicinal material, radix Paeoniae alba control medicinal material, and rhizoma anemarrhenae control medicinal material; inspecting under 365nm ultraviolet lamp, and displaying spots with the same color on the positions corresponding to the 5-O-methylvisamiloride and radix Saposhnikoviae control medicine chromatogram in the sample chromatogram;
the volume ratio of the developing agent ethyl acetate to the methanol to the water is 15-20:2-7:1, a step of;
In the step S5, a 6-gingerol reference substance, a atractylenolide III reference substance and an atractylenolide II reference substance are taken, and methanol is used for preparing a mixed reference substance solution 2 with the content of 0.3-0.8 mg/mL;
in the step S7, the thin layer chromatography conditions are as follows: adopting a silica gel GF254 plate as a thin layer plate, wherein the sample application amount of a sample solution of an ether layer, a sample solution of a bighead atractylodes rhizome reference medicinal material and a sample application amount of a cassia twig reference medicinal material solution are 20-40 mu L, the sample application amount of a mixed reference substance solution 2 is 10-20 mu L, developing a developing agent is cyclohexane-dichloromethane-ethyl acetate-formic acid, developing, airing, and placing under a 365nm ultraviolet lamp for inspection, wherein spots with the same color appear in the positions corresponding to the sample chromatogram of the cassia twig reference medicinal material; spraying 5% vanillin sulfuric acid solution, baking at 105deg.C until spots develop, inspecting under fluorescent lamp, and displaying spots of the same color on the position corresponding to the color spectrum of 6-gingerol control sample in the color spectrum of the sample; inspecting under 365nm ultraviolet lamp, and displaying spots of the same color on the corresponding positions of the chromatogram of the sample to be tested, namely the atractylenolide II reference substance, the atractylenolide III reference substance and the atractylenolide reference substance;
the volume ratio of the developing agent cyclohexane-dichloromethane-ethyl acetate-formic acid is 3-4:1-2:1-2:0.1-0.2.
2. The thin-layer identification method of 8 medicinal herbs in the whole formula 9 of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction according to claim 1, wherein in the step S3, 0.3-0.7g of liquorice reference medicinal material, 0.3-0.7g of white paeony root reference medicinal material, 0.3-0.7g of ephedra reference medicinal material, 0.8-1.2g of divaricate saposhnikovia root reference medicinal material and 0.8-1.2g of rhizoma anemarrhenae reference medicinal material are respectively added with 20-60mL of water, the mixture is heated and reflux-extracted for 0.5-1.5 hours, the mixture is filtered while being hot, the filtrate is filtered through a macroporous adsorption resin column, 25-35mL of ammonia solution is used for eluting, ammonia solution is discarded, 15-25mL of water solution is used for eluting, 30-80mL of ethanol aqueous solution with the volume fraction of 10-60% is continuously used for eluting, eluent is collected, evaporated, and residues are added with 1-3mL of methanol for dissolving, so that aqueous layer reference medicinal material solutions, namely liquorice reference medicinal material solution, white paeony root reference medicinal material solution, ephedra reference medicinal material solution, divaricate and rhizoma anemarrhenae reference medicinal material solution are obtained.
3. The thin-layer identification method of 8 medicinal herbs in 9 medicinal herbs in the whole formula of the cassia twig, chinese herbaceous peony and rhizoma anemarrhenae decoction according to claim 1 is characterized in that in the step S6, 0.5-1.0g of bighead atractylodes rhizome reference medicinal materials and 0.5-1.0g of cassia twig reference medicinal materials are respectively added with 20-60mL of water, the mixture is heated and refluxed for 0.5-1.5 hours, the mixture is filtered while the mixture is hot, the filtrate is transferred to a separating funnel, the mixture is extracted for 2-3 times by shaking with diethyl ether, 30-80mL of diethyl ether solution is combined, the mixture is volatilized, and 1-3mL of methanol is added into residues to dissolve the residues, so that an ether layer reference medicinal material solution is obtained.
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