CN113049732A - Rapid thin-layer identification method for astragalus root and poria cocos granules with one detection and multiple medicines - Google Patents
Rapid thin-layer identification method for astragalus root and poria cocos granules with one detection and multiple medicines Download PDFInfo
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Abstract
The invention relates to a detection method of a traditional Chinese medicine preparation, in particular to a rapid thin-layer identification method of astragalus and poria cocos granules for detecting multiple medicines; the detection method is thin-layer chromatography and comprises the following steps: (1) preparation of a test solution: extracting radix astragali and Poria with methanol; (2) preparation of control solutions: adding methanol into control of QILINGGEN granule to obtain control solution; (3) identifying by thin-layer chromatography: and (3) sampling each solution on the same silica gel thin-layer plate, and developing and comparing by taking petroleum ether-ethyl acetate-methanol-anhydrous formic acid as a developing agent in a certain ratio. Compared with the traditional thin-layer chromatography, the method has the greatest advantages that three medicinal materials are identified on the same thin-layer plate, a color developing agent is not needed, the solvent toxicity is low, the specificity is high, and the durability is good.
Description
Technical Field
The invention relates to a detection method of a traditional Chinese medicine preparation, in particular to a rapid thin-layer identification method of astragalus-poria granules for detecting multiple medicines.
Background
Thin-layer chromatography is a common method for identifying the quality of traditional Chinese medicine products, and can rapidly separate and analyze various substances. Most of traditional Chinese medicines and compound preparations thereof have complex components, low content of index components and a plurality of types of interference substances, so that the medicinal taste identification of the traditional Chinese medicine compound preparations mostly adopts an independent thin-layer identification method.
Conventionally, a plurality of medicinal materials are in thin-layer identification of a traditional Chinese medicine compound preparation, and a preparation method, a thin-layer developing agent, developing conditions and the like of a test sample of each medicinal material are independently optimized. At present, the development of hospital preparations is a great direction of the development of traditional Chinese medicines, but most of the quality standard medium-thin layer identification methods of hospital preparations are carried out according to single medicines in a prescription, and the qualitative and quantitative detection of main medicines in the prescription is not carried out according to the guiding principle of 'monarch, minister, assistant and guide' or the selected detection components are not representative, so that the quality of the compound preparation cannot be effectively controlled. The detection method of the traditional Chinese medicine compound preparation is simple, convenient and quick, the detection efficiency is improved, the detection cost is reduced, and the method is a necessary breakthrough in the quality control of the traditional Chinese medicine.
The astragalus and poria cocos granules are clinical proved formulas of Hangzhou traditional Chinese medicine hospitals, are prepared from astragalus (monarch drug), salvia (monarch drug), rhizoma smilacis glabrae, gentiana macrophylla, coix seeds, loranthus parasiticus and edible tulip, have the functions of strengthening spleen and replenishing qi, removing blood stasis and discharging turbidity, and clearing heat and promoting diuresis, can be clinically used for treating hyperuricemia, and have the effects of reducing blood uric acid and blood fat of patients. The clinical data of the Qiling granules show good effect, but the Qiling granules have no scientific and reasonable quality standard for a while. The prescription has complex composition, and the quality of the medicinal materials seriously influences the curative effect of the compound preparation. How to quickly identify the quality of medicinal materials in the compound preparation has an important effect on improving the quality of products. Therefore, it is imperative to establish a 'one-test-multiple-drug' thin-layer identification method, i.e. the same sample processing method is used, and the same development conditions are adopted on the same thin-layer plate, so that the multiple drug flavors in the compound preparation can be quickly identified.
The Chinese invention patent CN107102093B discloses a detection method of compound Danyin ointment, wherein identification of Salvia miltiorrhiza and Gentiana macrophylla is involved, tanshinone IIA and gentiopicroside are taken as reference substances, the reference substances are spotted on the same silica gel G thin-layer plate, and benzene and ethyl acetate in a volume ratio of 20-22: 0.5-2 are taken as developing agents. The Chinese patent CN107014945B discloses a detection method of a traditional Chinese medicine preparation for treating acute infectious hepatitis, and the components of salvia miltiorrhiza and gentiana macrophylla are identified: tanshinone IIA and gentiopicrin are respectively used as reference substances, a silica gel G thin layer plate is used, benzene and ethyl acetate in a volume ratio of 19-23: 0.5-2 are used as developing agents, and identification is carried out by using a thin layer chromatography.
In the method for simultaneously identifying the salvia miltiorrhiza and the gentiana macrophylla, the developing solvent contains benzene which is a carcinogen and brings great adverse effects on the health and the environment of detection personnel.
Disclosure of Invention
The invention aims to establish a rapid thin-layer identification method for one-detection-multiple-drug in Qiling granules, which can simultaneously identify specific components such as salvianolic acid B, astilbin, gentiopicrin and the like in the Qiling granules on the same thin-layer plate by using the same test solution and the same developing agent, so that the developed spots are abundant, the time required by detection is shortened, the consumption of reagents is reduced, and the detection cost is greatly reduced.
The purpose of the invention is realized by the following technical scheme:
the detection method for detecting multiple medicines by using astragalus and poria cocos granules is characterized by being thin-layer chromatography and comprising the following steps:
(1) preparation of a test solution: extracting radix astragali and Poria granule with methanol, and collecting supernatant or filtrate;
(2) preparation of control solutions: adding methanol into control of QILINGGEN granule to obtain mixed control solution; the radix astragali and Poria granule reference substance is one or more of salvianolic acid B, astilbin and gentiopicrin;
(3) identifying by thin-layer chromatography: and (3) sucking the sample solution and the mixed reference solution to be spotted on the same silica gel thin-layer plate, developing in a developing container by taking petroleum ether-ethyl acetate-methanol-formic acid as a developing agent, taking out, inspecting and comparing.
Preferably, the volume-to-mass ratio of the methanol added in the step (1) to the astragalus and poria cocos granules is 8-12:1 mL/g.
Preferably, the extraction in step (1) is ultrasonic extraction, and the time of ultrasonic extraction is 10-40 min.
Preferably, the concentration of salvianolic acid B, astilbin and gentiopicrin in the mixed reference solution in the step (2) is 0.8-1.5 mg/mL.
Preferably, the volume ratio of the developing solvent petroleum ether-ethyl acetate-methanol-anhydrous formic acid in the step (3) is (1-3): (6-8): (0.8-1.2): (0.4-0.6);
preferably, the volume ratio of the developing solvent petroleum ether-ethyl acetate-methanol-anhydrous formic acid in the step (3) is 2:7:1: 0.5.
Preferably, the silica gel thin layer plate in the step (3) is silica gel GF254Thin layer plates, viewed under UV 254 nm.
Preferably, the expansion vessel is saturated for more than 5 minutes, preferably 5-20min, before expanding in step (3);
preferably, the taking out is carried out when the spread is more than 6cm, and the spread is preferably 7cm-9 cm.
Preferably, the sample amount of the test solution in step (3) is 2-10 μ L, and the sample amount of the mixed control solution is 1-5 μ L.
Preferably, the step of thin layer chromatography further comprises the preparation of a negative control solution comprising the steps of: extracting the negative granules of the radix salviae miltiorrhizae, the rhizoma smilacis glabrae and the gentiana macrophylla with methanol, and taking supernate or filtrate to obtain a radix salviae miltiorrhizae negative control solution, a rhizoma smilacis glabrae negative control solution and a gentiana macrophylla negative control solution respectively; preferably, the volume-to-mass ratio of the methanol to the negative granules is 8-12:1 mL/g; preferably, the extraction is ultrasonic extraction, and the time of ultrasonic extraction is 10-40 min.
Preferably, the negative control solution is spotted in an amount of 2 to 10. mu.L.
The invention also aims to provide the application of any one of the detection methods in identifying one or more of the components of salvianolic acid B, astilbin and gentiopicrin or identifying one or more of the medicinal materials of salvia miltiorrhiza, glabrous greenbrier rhizome and large-leaved gentian;
preferably, any one of the detection methods is applied to the analysis and detection of astragalus membranaceus and poria cocos particles, and the astragalus membranaceus and poria cocos particles are prepared from astragalus membranaceus (monarch drug), salvia miltiorrhiza (monarch drug), rhizoma smilacis glabrae, radix gentianae macrophyllae, semen coicis, parasitic loranthus and edible tulip.
The invention has the beneficial effects that:
(1) the method is based on the thin-layer chromatography, can simultaneously identify three index components, namely salvianolic acid B, astilbin and gentiopicrin, in the Qiling granules, is simple and quick to operate, and provides an effective basis for identifying the Qiling granules. In addition, the separation degree related in the technical scheme is good, negative interference is effectively eliminated, the inspection is clear, the specificity and the reproducibility are very good, and more comprehensive quality control of the Qiling granules can be effectively realized.
(2) The developing solvent used in the method is petroleum ether-ethyl acetate-methanol-anhydrous formic acid, and each reagent is a solvent with low toxicity, compared with toluene and chloroform used in the identification method of salvia miltiorrhiza (trichloromethane-toluene-ethyl acetate-methanol-formic acid) and glabrous greenbrier rhizome (toluene-ethyl acetate-formic acid) in 2015 edition of Chinese pharmacopoeia. And the salvia miltiorrhiza needs to be developed for the second time in the 'Chinese pharmacopoeia' of 2015 edition, and the method has the characteristic of more quickly and accurately identifying the salvianolic acid B, astilbin and gentiopicrin in the Qiling granules.
(3) In the invention, 3 medicinal flavors are identified by using a thin-layer plate, and the simultaneous identification of three traditional Chinese medicine components of salvianolic acid B, astilbin and gentiopicrin is realized for the first time, so that the solvent and labor cost are effectively saved, the identification time is shortened, only a single sample preparation method and an expansion system are needed, the identification of the three components in the Qiling granules can be realized, meanwhile, a negative granule reference substance is also free of interference, and the identification accuracy is ensured.
Drawings
FIG. 1 is a thin-layer chromatogram developed in example 1 (in the figure, numbers 1-3 are different Qiling granules, batch numbers are 21001, 21002 and 21003, number 4 is a mixed reference solution of salvianolic acid B, astilbin and gentiopicrin, number 5 is a control solution of negative granules lacking Salvia miltiorrhiza, number 6 is a control solution of negative granules lacking Smilax glabra, and number 7 is a control solution of negative granules lacking radix Gentianae Marcrophyllae);
FIG. 2 is a thin layer chromatogram for examining different sample application modes and sample application amounts of a sample solution in example 2 (from top to bottom: dot-sample application and strip-sample application; sample amounts of 1-3 are 2 μ L in the figure, and astragalus root and poria cocos particles with different lot numbers 21001, 21002 and 21003 are sequentially numbered; sample amounts of 4-6 are 4 μ L in the figure, and astragalus root and poria cocos particles with different lot numbers 21001, 21002 and 21003 are sequentially numbered; sample amounts of 7-9 are 8 μ L in the figure, and astragalus root and poria cocos particles with different lot numbers 21001, 21002 and 21003 are sequentially numbered; sample amounts of 10-12 are 10 μ L in the figure, and astragalus root and poria cocos particles with different lot numbers 21001, 21002 and 21003 are sequentially numbered 13, and a mixed reference solution of salvianolic acid B, astilbin and gentiopicrin is provided, and reference solution is reference solution lacking in Salvia miltiorrhiza negative particles, reference solution is reference solution lacking in the number 14, reference solution lacking in rhizoma smilacis glabrae negative particles is reference solution lacking in the number 15, and;
FIG. 3 is a thin-layer chromatogram for the spot size examination of each negative control solution in example 3 (from top to bottom: lack of Salvia miltiorrhiza negative chromatogram, lack of rhizoma Smilacis Glabrae negative chromatogram, lack of radix Gentianae Marcrophyllae negative chromatogram; number 1 spot sample size in each figure is 2. mu.L, number 2 spot sample size is 4. mu.L, number 3 spot sample size is 8. mu.L, number 4 spot sample size is 10. mu.L, number 5 is salvianolic acid B, astilbin, gentiopicrin mixed control solution, number 6 is 21001 batches of Qiling granule test solution);
FIG. 4 is a thin layer chromatogram for examining the amount of the mixed control solution in example 3 (in the figure, number 1 indicates the mixed control solution, the amount of the mixed control solution is 1. mu.L, number 2 indicates the mixed control solution, the amount of the mixed control solution is 3. mu.L, number 3 indicates the mixed control solution, the amount of the mixed control solution is 5. mu.L, and number 4 indicates 21001 batches of the Qiling granule test solutions);
FIG. 5 is a chromatogram examination of different thin-layer plates of example 4 (from top to bottom: Merck plate, Qingdao sea silica gel plate and Nicoti Jiangyou silica gel plate; in each figure, numbers 1-3 are different Qiling granules, lot numbers 21001, 21002, 21003, number 4 is salvianolic acid B, astilbin, gentiopicrin mixed reference solution, number 5 is salvia negative granule lacking reference solution, number 6 is glabrous greenbrier negative granule lacking reference solution, and number 7 is gentiana macrophylla negative granule lacking reference solution);
FIG. 6 is a chromatogram obtained by examining different temperatures in example 5 (from top to bottom: 10. + -. 2 ℃, 20. + -. 2 ℃, 40. + -. 2 ℃; in each figure, numbers 1-3 are different stilbene powder particles, lot numbers: 21001, 21002, 21003, number 4 is salvianolic acid B, astilbin, gentiopicrin mixed reference solution, number 5 is salvia miltiorrhiza negative particle-lacking reference solution, number 6 is glabrous greenbrier negative particle-lacking reference solution, number 7 is gentiana macrophylla negative particle-lacking reference solution);
FIG. 7 is a chromatogram obtained by examining different humidities in example 6 (from top to bottom: 20. + -. 5%, 40. + -. 5% and 80. + -. 5%; in each figure, numbers 1-3 are different stilbene powder particles, lot numbers: 21001, 21002, 21003, number 4 is salvianolic acid B, astilbin, gentiopicrin mixed control solution, number 5 is Salvia miltiorrhiza negative particle-lacking control solution, number 6 is Smilax rhizoma negative particle-lacking control solution, number 7 is gentiana macrophylla negative particle-lacking control solution);
FIG. 8 is a chromatogram developed in example 7 (in the figure, numbers 1-3 are different Qiling granules, batch numbers are 21001, 21002 and 21003, number 4 is a mixed reference solution of salvianolic acid B, astilbin and gentiopicrin, number 5 is a control solution of negative granules lacking Salvia miltiorrhiza, number 6 is a control solution of negative granules lacking Smilax glabra, and number 7 is a control solution of negative granules lacking radix Gentianae Marcrophyllae);
FIG. 9 is a chromatogram developed in example 8 (in the figure, numbers 1-3 are different Qiling granules, batch numbers are 21001, 21002 and 21003, number 4 is a mixed reference solution of salvianolic acid B, astilbin and gentiopicrin, number 5 is a control solution of negative granules lacking Salvia miltiorrhiza, number 6 is a control solution of negative granules lacking Smilax glabra, and number 7 is a control solution of negative granules lacking radix Gentianae Marcrophyllae);
FIG. 10 is a thin layer chromatogram of the comparative examples 1-3, batch No. 21001 radix astragali and Poria granule developed in respective developing solvent (different developing solvent system from left to right, respectively petroleum ether-ethyl acetate-methanol-formic acid system, petroleum ether-ethyl acetate-toluene-formic acid system, petroleum ether-ethyl acetate-chloroform-formic acid system).
FIG. 11 is a thin layer chromatogram of the developed stilbene-poria particles of 21001 of comparative example 1, example 1 and comparative example 4 in respective developing agents (different ratios of petroleum ether-ethyl acetate-methanol-formic acid systems developing from left to right, 2:7:1:1, 2:7:1:0.5, 2:7:1:0.3, respectively).
Detailed Description
The negative granules in the following examples are red sage root negative granules, glabrous greenbrier rhizome negative granules and large-leaved gentian negative granules which are prepared by respectively lacking the medicinal materials of red sage root, glabrous greenbrier rhizome and large-leaved gentian according to the preparation process of astragalus and poria granules.
Example 1
Thin layer identification of Qiling granules:
(1) preparing a test solution: taking 0.5g of Qiling granules, adding 5mL of methanol, carrying out ultrasonic treatment for 30min, and taking supernatant to obtain the traditional Chinese medicine.
(2) Preparation of a reference solution: mixing salvianolic acid B, astilbin, and gentiopicrin with methanol to obtain mixed reference solution containing salvianolic acid B and gentiopicrin each 1mg/mL and astilbin 1.5 mg/mL.
(3) Preparation of negative control solution: taking 0.5g of each negative particle, adding 5mL of methanol, performing ultrasonic treatment for 30min, and taking supernatant to obtain the product.
(4) Dropping 4 μ L of the sample solution, 4 μ L of the reference solution and 4 μ L of the negative reference solution on the same silica gel GF254Spreading petroleum ether-ethyl acetate-methanol-anhydrous formic acid (2:7:1:0.5) as developing agent on the thin layer plate, spreading with spreading cylinder filter paper, saturating for 20min, spreading with spreading distance of 7cm, taking out, air drying, and inspecting under ultraviolet lamp (254 nm).
The results show that: the test chromatogram showed the same spot at the same position as the control chromatogram, and the negative particle control solution showed no spot at the same position, and the method was well-defined (as shown in FIG. 1).
Example 2
In this embodiment, different sample application modes and sample application amounts of sample solutions are considered to further explain the technical scheme.
(1) Preparing a test solution: taking 0.5g of Qiling granules, adding 5mL of methanol, carrying out ultrasonic treatment for 30min, and taking supernatant to obtain the traditional Chinese medicine.
(2) Preparation of a reference solution: taking salvianolic acid B, astilbin, and gentiopicrin, adding methanol to obtain mixed reference solution of salvianolic acid B, gentiopicrin 1mg/mL, and astilbin 1.5 mg/mL.
(3) Negative control solution preparation: taking 0.5g of each negative particle, adding 5mL of methanol, performing ultrasonic treatment for 30min, and taking supernatant to obtain the product.
(4) Dropping 2, 4, 8 and 10 μ L of each sample solution, 4 μ L of each control solution and 4 μ L of each negative control solution on the same silica gel GF254Spreading petroleum ether-ethyl acetate-methanol-anhydrous formic acid (2:7:1:0.5) as developing agent on the thin layer plate, spreading with spreading cylinder filter paper, saturating for 20min, spreading with spreading distance of 7cm, taking out, air drying, and inspecting under ultraviolet lamp (254 nm).
The results show that: the identification purpose can be achieved by point sample application and strip sample application, and the sample application amount is between 2 muL and 10 muL, which has good separation effect (shown in figure 2).
Example 3
In this example, different sample amounts of the control solution and the negative control solution are examined to further explain the technical scheme.
(1) Preparation of a reference solution: taking salvianolic acid B, astilbin, and gentiopicrin, adding methanol to obtain mixed reference solution of salvianolic acid B, gentiopicrin 1mg/mL, and astilbin 1.5 mg/mL.
(2) Negative control solution preparation: taking 0.5g of each negative particle, adding 5mL of methanol, performing ultrasonic treatment for 10min, and taking supernatant to obtain the product.
(3) Preparing a test solution: taking 0.5g of Qiling granules, adding 5mL of methanol, performing ultrasonic treatment for 10min, and taking supernatant to obtain the traditional Chinese medicine.
(4) Pipetting 1. mu.L, 3. mu.L and 5. mu.L of each of control solutions and 2. mu.L, 4. mu.L, 8. mu.L and 10. mu.L of negative control solutions, and spotting on silica gel GF254Thin layer plateAnd (3) taking petroleum ether-ethyl acetate-methanol-anhydrous formic acid (2:7:1:0.5) as a developing agent, developing with filter paper in a developing cylinder, saturating for 5 minutes, developing with a developing distance of 7cm, taking out, drying, and inspecting under an ultraviolet lamp (254 nm).
The results show that: the negative control solution has good separation effect in the range of 2-10. mu.L, and the control solution has good separation effect in the range of 1-5. mu.L (as shown in FIGS. 3 and 4).
Example 4
In this embodiment, the durability of the technical solution is examined by using thin-layer plates of different manufacturers to further explain the technical solution.
(1) Preparing a test solution: taking 0.5g of Qiling granules, adding 5mL of methanol, carrying out ultrasonic treatment for 30min, and taking supernatant to obtain the traditional Chinese medicine.
(2) Preparation of a reference solution: taking salvianolic acid B, astilbin, and gentiopicrin, adding methanol to obtain mixed reference solution of salvianolic acid B, gentiopicrin 1mg/mL, and astilbin 1.5 mg/mL.
(3) Negative control solution preparation: taking 0.5g of each negative particle, adding 5mL of methanol, performing ultrasonic treatment for 30min, and taking supernatant to obtain the product.
(4) Sucking 4 μ L of the sample solution, 4 μ L of the reference solution, and 4 μ L of the negative reference solution, and respectively dropping onto GF of different manufacturers254Silica gel plate (Merck plate, Qingdao ocean plate, and Nicoti river-friend plate), developing with petroleum ether-ethyl acetate-methanol-anhydrous formic acid (2:7:1:0.5) as developing agent, developing with developing tank filter paper for 20min, developing with a developing distance of 7cm, taking out, air drying, and inspecting under ultraviolet lamp (254 nm).
The results show that: under selected conditions, the thin-layer plates of different manufacturers can achieve the purpose of identification, and the durability of the thin-layer plate obtained by the method is good (as shown in figure 5).
Example 5
In this embodiment, the durability of the present technical solution is examined with different temperatures to further explain the technical solution.
(1) Preparing a test solution: taking 0.5g of Qiling granules, adding 5mL of methanol, carrying out ultrasonic treatment for 30min, and taking supernatant to obtain the traditional Chinese medicine.
(2) Preparation of a reference solution: taking salvianolic acid B, astilbin, and gentiopicrin, adding methanol to obtain mixed reference solution of salvianolic acid B, gentiopicrin 1mg/mL, and astilbin 1.5 mg/mL.
(3) Negative control solution preparation: taking 0.5g of each negative particle, adding 5mL of methanol, performing ultrasonic treatment for 30min, and taking supernatant to obtain the product.
(4) Fixing relative humidity at 20%, sucking 4 μ L of the sample solution, 4 μ L of the control solution and 4 μ L of the negative control solution at different temperatures (10 deg.C, 20 deg.C and 40 deg.C), and dropping onto the same GF254On a silica gel plate, using petroleum ether-ethyl acetate-methanol-anhydrous formic acid (2:7:1:0.5) as a developing agent, developing in a developing cylinder with filter paper, saturating for 20 minutes, developing with a developing distance of 7cm, taking out, air drying, and inspecting under an ultraviolet lamp (254 nm).
The results show that: under different temperature conditions, the purpose of rapid identification can be achieved, which shows that the temperature and humidity durability of the method is good (as shown in figure 6).
Example 6
In this embodiment, the durability of the present technical solution is examined with different humidity to further explain the technical solution.
(1) Preparing a test solution: taking 0.5g of Qiling granules, adding 5mL of methanol, carrying out ultrasonic treatment for 30min, and taking supernatant to obtain the traditional Chinese medicine.
(2) Preparation of a reference solution: taking salvianolic acid B, astilbin, and gentiopicrin, adding methanol to obtain mixed reference solution of salvianolic acid B, gentiopicrin 1mg/mL, and astilbin 1.5 mg/mL.
(3) Negative control solution preparation: taking 0.5g of each negative particle, adding 5mL of methanol, performing ultrasonic treatment for 30min, and taking supernatant to obtain the product.
(4) The fixation temperature is 20 ℃, under the condition of different relative humidities (20%, 40%, 80%), sucking 4 μ L of the test solution, 4 μ L of the reference solution and 4 μ L of the negative reference solution, and spotting on the same GF254On a silica gel plate, using petroleum ether-ethyl acetate-methanol-anhydrous formic acid (2:7:1:0.5) as a developing agent, developing in a developing cylinder with filter paper, saturating for 20 minutes, developing with a developing distance of 7cm, taking out, air drying, and inspecting under an ultraviolet lamp (254 nm).
The results show that: under different humidity conditions, the purpose of rapid identification can be achieved, which shows that the temperature and humidity durability of the method is good (as shown in fig. 7).
Example 7
(1) Preparing a test solution: taking 0.5g of Qiling granules, adding 4mL of methanol, performing ultrasonic treatment for 40min, and taking supernatant to obtain the traditional Chinese medicine.
(2) Preparation of a reference solution: taking salvianolic acid B, astilbin, and gentiopicrin, adding methanol to obtain mixed reference solution of salvianolic acid B, gentiopicrin 1mg/mL, and astilbin 1.5 mg/mL.
(3) Preparation of negative control solution: taking 0.5g of each negative particle, adding 4mL of methanol, performing ultrasonic treatment for 40min, and taking supernatant to obtain the compound preparation.
(4) Dropping 4 μ L of the sample solution, 4 μ L of the reference solution and 4 μ L of the negative reference solution on the same silica gel GF254Spreading petroleum ether-ethyl acetate-methanol-anhydrous formic acid (1:8:0.8:0.6) as developing agent on the thin layer plate, spreading with spreading jar filter paper for 5 min, spreading with spreading distance of 9cm, taking out, air drying, and inspecting under ultraviolet lamp (254nm) (as shown in FIG. 8).
Example 8
(1) Preparing a test solution: taking 0.5g of Qiling granules, adding 6mL of methanol, carrying out ultrasonic treatment for 30min, and taking supernatant fluid to obtain the traditional Chinese medicine.
(2) Preparation of a reference solution: taking salvianolic acid B, astilbin, and gentiopicrin, adding methanol to obtain mixed reference solution of salvianolic acid B, gentiopicrin 1mg/mL, and astilbin 1.5 mg/mL.
(3) Preparation of negative control solution: taking 0.5g of each negative particle, adding 6mL of methanol, performing ultrasonic treatment for 30min, and taking supernatant to obtain the compound preparation.
(4) Dropping 4 μ L of the sample solution, 4 μ L of the reference solution and 4 μ L of the negative reference solution on the same silica gel GF254Spreading petroleum ether-ethyl acetate-methanol-anhydrous formic acid (3:6:1.2:0.4) as developing agent on the thin layer plate, spreading with spreading cylinder filter paper for 20min, spreading with spreading distance of 7cm, taking out, air drying, and inspecting under ultraviolet lamp (254nm) (as shown in FIG. 9).
Comparative example 1
Comparative example andexample 1 is distinguished in that the ratio of developing solvent is different, the preparation method of the sample is the same as that of example 1, 4. mu.L of sample solution is sucked and spotted on the silica gel GF254Spreading with petroleum ether-ethyl acetate-methanol-anhydrous formic acid (2:7:1:1) as developing agent in spreading cylinder, saturating with filter paper for 20min, spreading with spreading distance of 7cm, taking out, air drying, and inspecting under ultraviolet lamp (254nm) (see fig. 10).
Comparative example 2
The difference between the comparative example and example 1 is that the developing agent is different, the preparation method of the sample is the same as that of example 1, 4. mu.L of sample solution is absorbed, and the solution is spotted on the silica gel GF254Spreading with petroleum ether-ethyl acetate-toluene-anhydrous formic acid (2:7:1:1) as developing agent in spreading cylinder, saturating with filter paper for 20min, spreading with spreading distance of 7cm, taking out, air drying, and inspecting under ultraviolet lamp (254nm) (see fig. 10).
Comparative example 3
The difference between the comparative example and example 1 is that the developing agent is different, the preparation method of the sample is the same as that of example 1, 4. mu.L of sample solution is absorbed, and the solution is spotted on the silica gel GF254Spreading petroleum ether-ethyl acetate-chloroform-anhydrous formic acid (2:7:1:1) as developing agent on the thin layer plate, spreading with spreading cylinder filter paper, saturating for 20min, spreading with spreading distance of 7cm, taking out, air drying, and inspecting under ultraviolet lamp (254nm) (see fig. 10).
Comparative example 4
The difference between the comparative example and the example 1 is that the ratio of developing agent is different, the preparation method of the sample is the same as the example 1, 4 mu L of sample solution is absorbed, and the sample solution is spotted on the silica gel GF254Spreading petroleum ether-ethyl acetate-methanol-anhydrous formic acid (2:7:1:0.3) as developing agent on the thin layer plate, spreading with spreading cylinder filter paper, saturating for 20min, spreading with spreading distance of 7cm, taking out, air drying, and inspecting under ultraviolet lamp (254nm) (see fig. 11).
The results show that: the selected developing solvent is as follows: the petroleum ether-ethyl acetate-methanol-anhydrous formic acid can achieve the purpose of rapid identification under the strict mixture ratio of (1-3): (6-8): 0.8-1.2): 0.4-0.6.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. The detection method for detecting multiple medicines by using astragalus and poria cocos granules is characterized by being thin-layer chromatography and comprising the following steps:
(1) preparation of a test solution: extracting radix astragali and Poria granule with methanol, and collecting supernatant or filtrate;
(2) preparation of control solutions: adding methanol into control of QILINGGEN granule to obtain control solution; the radix astragali and Poria granule reference substance is one or more of salvianolic acid B, astilbin and gentiopicrin;
(3) identifying by thin-layer chromatography: and (3) absorbing each solution to sample on the same silica gel thin layer plate, developing in a developing container by taking petroleum ether-ethyl acetate-methanol-anhydrous formic acid as a developing agent, taking out, inspecting and comparing.
2. The detection method according to claim 1, wherein the volume-to-mass ratio of the methanol added in step (1) to the Qiling granules is 8-12:1 mL/g.
3. The detection method according to claim 1, wherein the extraction in step (1) is ultrasonic extraction, and the time of ultrasonic extraction is 10-40 min.
4. The detection method according to claim 1, wherein the concentration of salvianolic acid B, astilbin and gentiopicrin in the control solution in the step (2) is 0.8-1.5 mg/mL; preferably 1-1.5 mg/mL.
5. The detection method according to claim 1, wherein the volume ratio of the developing solvent petroleum ether-ethyl acetate-methanol-anhydrous formic acid in the step (3) is (1-3): (6-8): (0.8-1.2): (0.4-0.6), preferably 2:7:1: 0.5.
6. According to claimThe detection method according to claim 1, wherein the silica gel thin layer plate in the step (3) is silica gel GF254Thin layer plates, viewed under UV 254 nm.
7. The assay of claim 1, wherein in step (3) the expansion vessel is saturated for more than 5 minutes, preferably 5-20min, prior to expansion; preferably, the taking out is carried out when the spread is more than 6cm, and the spread is preferably 7cm-9 cm.
8. The detection method according to claim 1, wherein the sample solution in step (3) is spotted in an amount of 2 to 10. mu.L, and the mixed control solution is spotted in an amount of 1 to 5. mu.L.
9. The detection method according to claim 1, wherein the step of thin layer chromatography further comprises preparation of a negative control solution comprising the steps of: extracting the negative granules of the radix salviae miltiorrhizae, the rhizoma smilacis glabrae and the gentiana macrophylla with methanol, and taking supernate or filtrate to obtain a radix salviae miltiorrhizae negative control solution, a rhizoma smilacis glabrae negative control solution and a gentiana macrophylla negative control solution respectively; preferably, the volume-to-mass ratio of the methanol to the negative granules is 8-12:1 mL/g; preferably, the extraction is ultrasonic extraction, and the time of the ultrasonic extraction is 10-40 min; preferably, the amount of spotting of the negative control solution is 2-10. mu.L.
10. Use of the detection method according to any one of claims 1 to 9 for identifying one or more of the components salvianolic acid B, astilbin and gentiopicrin or for identifying one or more of the medicinal materials Salvia miltiorrhiza, Smilax glabra and Gentianae Macrophyllae; preferably, the detection method is applied to analysis and detection of astragalus and poria cocos particles, and the astragalus and poria cocos particles are prepared from astragalus, salvia miltiorrhiza, rhizoma smilacis glabrae, gentiana macrophylla, coix seeds, loranthus parasiticus and edible tulip.
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