CN112083090A - Thin-layer identification method for traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae - Google Patents
Thin-layer identification method for traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae Download PDFInfo
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Abstract
The invention discloses a thin-layer identification method of a traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae, which comprises the steps of firstly, preparing a test solution containing the traditional Chinese medicine preparation containing the concretio silicea bambusae or artificial concretio silicea bambusae and a reference medicinal material solution containing the concretio silicea bambusae; and analyzing the prepared test solution and the prepared reference solution according to thin-layer chromatography, wherein fluorescent spots with the same color are displayed in the chromatogram of the test solution at the corresponding positions of the chromatogram of the reference solution. The invention provides a rapid and effective quality detection method for the traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae, adopts methanol solvent, ultrasonic extraction and heating reflux for treatment, and performs analysis by thin-layer chromatography, and has the characteristics of simple operation, good reproducibility, high sensitivity, specificity and good adaptability.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a thin-layer identification method of a traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae.
Background
The quality control of the traditional Chinese medicine preparation is always an important component for ensuring the clinical curative effect of the traditional Chinese medicine, and the traditional Chinese medicine preparation such as decoction, tablets, injection and the like lose the morphological characteristics of the original decoction pieces, so the identification of the traditional Chinese medicine is extremely important in the quality control engineering of the traditional Chinese medicine preparation.
Concretio silicea Bambusae seu Schizostachyi is dried block of secretion in stem of Gramineae plant such as Bambusa vulgaris (Bambusa terrilis McClure) or Carcinia sinica (Schizostachyum chinensis Rendle). Collected in autumn and winter, mainly distributed in Yunnan, Guangdong, Guangxi, etc., has the effects of clearing heat, resolving phlegm, clearing heart fire and arresting convulsion, and is mainly used for treating fever unconsciousness, apoplexy with phlegm stagnation, infantile phlegm-heat convulsion, night cry, etc. Because of the yield limitation of natural concretio silicea bambusae, artificial concretio silicea bambusae are mostly adopted on the market at present, are seemingly natural in shape, have white color, heavy weight, hard texture and poor water absorption, and are irregular fine fragments, milky white, gray white or gray blue. The artificial concretio silicea bambusae contains silicic acid, potassium and calcium, has sweet and cold nature and taste, has the effects of clearing heat, eliminating phlegm, dispelling wind-heat and arresting epilepsy, and is mainly used for treating symptoms such as aphasia from apoplexy, fever coma and delirium, infantile acute convulsion, dysphoria, cough caused by phlegm-heat and the like.
It is known that artificial concretio silicea bambusae account for a larger proportion of the traditional Chinese medicine market, but the artificial concretio silicea bambusae have the same effect regardless of the artificial concretio silicea bambusae or the natural concretio silicea bambusae, and therefore, a method for identifying whether the traditional Chinese medicine preparation contains the artificial concretio silicea bambusae or the artificial concretio silicea bambusae is needed.
For the identification of concretio silicea bambusae in the traditional Chinese medicine preparation, a thin-layer identification method is recorded in the quality standard of concretio silicea bambusae in 2015 edition of Chinese pharmacopoeia: taking 1g of the product powder, placing in a 20ml gas phase headspace sample injection bottle or other pressure-resistant container, adding 10ml of 6mol/L hydrochloric acid solution, capping and sealing, placing in a water bath for heating for 2 hours, taking out, cooling, centrifuging, taking supernatant, evaporating to dryness, adding 2ml of diluted ethanol into residues to dissolve, and using as a sample solution. 1g of concretio silicea Bambusae seu Schizostachyi control medicinal material is prepared into control medicinal material solution by the same method. Then, taking leucine control and alanine control, and adding diluted ethanol to obtain solutions containing 0.5mg of leucine and alanine in each 1ml, respectively, as control solutions. Performing thin layer chromatography (general rule 0502) test, sucking the above four solutions 2 μ 1 respectively, dropping on the same silica gel G thin layer plate, spreading with n-butanol-glacial acetic acid-water (19: 5: 5) as developing agent, taking out, air drying, spraying ninhydrin test solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution. However, practice proves that the thin layer identification method is complex in operation and poor in reproducibility.
2008 edition processing standard of traditional Chinese medicine decoction pieces in Shanghai city is mentioned in the identification of artificial concretio silicea Bambusae seu Schizostachyi: the product is silicate gel, contains small amount of metal ions such as sodium, potassium, calcium, aluminum, and plaster, and is adsorbed with fresh succus Bambusae, when identifying, a little crystal of sodium ammonium phosphate is dipped with platinum ear, and melted into transparent small ball on flame, and the product is dipped while hot, and silicon dioxide floats on the surface of the ball before melting to form opaque small ball with net-shaped result.
At present, a thin-layer identification method for traditional Chinese medicine preparations containing concretio silicea bambusae or artificial concretio silicea bambusae is not described.
Disclosure of Invention
The invention provides a rapid and effective quality detection method for the traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae, and provides a thin-layer identification method for the traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae.
The invention is realized by the following technical scheme: a thin layer identification method of traditional Chinese medicine preparation containing concretio silicea Bambusae seu Schizostachyi or artificial concretio silicea Bambusae seu Schizostachyi comprises the following steps:
(1) preparing a test solution containing concretio silicea Bambusae seu Schizostachyi or artificial concretio silicea Bambusae seu Schizostachyi Chinese medicinal preparation, and a control solution containing concretio silicea Bambusae seu Schizostachyi;
(2) and (2) analyzing the test solution and the reference solution prepared in the step (1) according to a thin-layer chromatography, wherein fluorescent spots with the same color are displayed in the chromatogram of the test solution at the corresponding positions of the chromatogram of the reference solution.
In the step (1), 2.0-5.0 g of a traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae is taken according to the proportion of the concretio silicea bambusae or the artificial concretio silicea bambusae, dissolved by 20-50 ml of 70-80% methanol, subjected to ultrasonic treatment for 15-30 min, filtered, heated and refluxed for 10-15 min, evaporated to dryness, and dissolved by adding 1-2 ml of 40-55% methanol into residues to prepare a sample solution.
In the step (1), 0.5-1.0 g of a concretio silicea bambusae control medicinal material is taken, dissolved by 20-50 ml of 70-80% methanol, subjected to ultrasonic treatment for 15-30 min, filtered, heated and refluxed for 10-15 min, evaporated to dryness, and residues are dissolved by 1-2 ml of 40-55% methanol to prepare a control medicinal material solution.
In the step (2), the sample solution and the reference medicinal material solution are respectively absorbed by 10-25 mu 1, respectively, and are respectively spotted on the same silica gel G thin-layer plate, water-saturated n-butyl alcohol-glacial acetic acid with the ratio of 3.5-4.5: 1 as a developing agent, and the developing agent is developed, taken out, dried and inspected under an ultraviolet lamp of 365 nm.
In the step (1), the Chinese medicinal preparation is selected from decoction, paste, granules or tablets.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention provides a thin-layer identification method of a traditional Chinese medicine preparation containing concretio silicea bambusae, which is simpler and more easy to operate and has strong reproducibility compared with a thin-layer identification method of concretio silicea bambusae in Zhongtian pharmacopoeia of 2015 edition.
(2) The thin-layer identification method can also be used for detecting the traditional Chinese medicine preparation containing the artificial concretio silicea bambusae, makes up the defect that the traditional Chinese medicine preparation containing the artificial concretio silicea bambusae has no thin-layer identification method in the prior art, and has the characteristics of simple operation, good reproducibility, stability, reliability and good specificity.
Drawings
FIG. 1 is a thin-layer chromatogram for identification of artificial concretio silicea Bambusae seu Schizostachyi formula granule.
FIG. 2 is a thin layer chromatogram of different spot sizes.
FIG. 3 is a thin layer chromatogram of a non-negative control.
FIG. 4 shows T: 4 ℃ and RH: thin layer chromatogram under 75% conditions.
FIG. 5 is T: 25 ℃ and RH: thin layer chromatogram under 75% conditions.
FIG. 6 is T: 25 ℃ and RH: thin layer chromatogram under 32% conditions.
FIG. 7 is T: 25 ℃ and RH: thin layer chromatogram under 75% conditions.
FIG. 8 is a chromatogram of a selected Tianjin Silida silica gel plate.
FIG. 9 is a thin layer chromatogram of a selected Qingdao ocean silica gel plate.
FIG. 10 is a chromatogram of a silica gel plate with a Merck thin layer plate.
Detailed Description
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is to be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention claimed, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Because the existing thin-layer identification method of the concretio silicea bambusae has the defects of complicated operation and poor reproducibility and the vacancy of the artificial concretio silicea bambusae in the thin-layer identification method, the invention provides the thin-layer identification method capable of identifying the traditional Chinese medicine preparation containing the concretio silicea bambusae or the artificial concretio silicea bambusae, and provides a quality control basis for the traditional Chinese medicine preparation containing the concretio silicea bambusae or the artificial concretio silicea bambusae.
The following examples are given to illustrate specific embodiments of the present invention, but it is to be understood that the scope of the present invention is not limited to the following examples, and the percentages herein are by mass concentration.
The following examples relate to the following instruments and reagents and experimental materials:
the instrument comprises the following steps: a semi-automatic thin layer sample applicator CAMAG Lionmat-5; thin layer imaging system CAMAG TLC Visualizer; water-making machine cell type 1810A (shanghai morel scientific instruments ltd); ultrasonic cleaner model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan). Silica gel G plate Tianjin Silida science and technology, Qingdao ocean chemical, Merck.
Reagent: the chemical reagents such as n-butyl alcohol, glacial acetic acid and the like are analytical pure.
Experimental materials: artificial concretio silicea bambusae formula granules (Sichuan new green pharmaceutical science and technology development ltd. 1703079) and concretio silicea bambusae control medicinal materials (batch number: 121718-201801).
Example 1:
the embodiment relates to a thin-layer identification method for identifying traditional Chinese medicine formula granules containing concretio silicea bambusae.
The method comprises the following specific steps:
preparing a test solution: taking the traditional Chinese medicine formula granules containing the concretio silicea bambusae, putting the traditional Chinese medicine formula granules into a conical flask according to the amount of the concretio silicea bambusae contained in the preparation of 5.0g, adding 20ml of 70% methanol, carrying out ultrasonic treatment (the frequency is 20KHz, the temperature is 40 ℃) for 30min, filtering, heating and refluxing the filtrate for 10min, evaporating to dryness, and dissolving residues in 1ml of 40% methanol to prepare a sample solution.
Preparing a reference medicinal material solution: taking 1.0g of concretio silicea Bambusae seu Schizostachyi as control material, placing in a conical flask, adding 70% methanol 20ml, performing ultrasonic treatment (frequency 20KHz, temperature 40 deg.C) for 30min, filtering, heating and refluxing the filtrate for 10min, evaporating to dryness, and dissolving the residue with 40% methanol 1ml to obtain control material solution.
Preparation of negative control solution: a negative control solution was prepared in the same manner as negative control solution (maltodextrin) 5.0g of the negative control of concretio silicea Bambusae seu Schizostachyi.
Performing thin layer chromatography (general rule 0502) test, respectively sucking sample solution and control solution 10 μ 1, respectively dropping on the same silica gel G thin layer plate, developing with water saturated n-butanol-glacial acetic acid 4.5: 1 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp 365 nm. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.
Example 2:
the embodiment relates to a thin-layer identification method for identifying particles containing an artificial concretio silicea bambusae formula.
The method comprises the following specific steps:
preparing a test solution: taking the formula particles containing the artificial concretio silicea bambusae, putting the formula particles into a conical flask according to the amount of 2.5g of the artificial concretio silicea bambusae contained in the preparation, adding 50ml of 80% methanol, carrying out ultrasonic treatment (the frequency is 40KHz, the temperature is 25 ℃) for 15min, filtering, heating and refluxing the filtrate for 15min, evaporating to dryness, and dissolving residues in 2ml of 50% methanol to prepare a sample solution.
Preparing a reference medicinal material solution: taking 0.5g of concretio silicea Bambusae seu Schizostachyi control medicinal material, placing in a conical flask, adding 50ml of 80% methanol, performing ultrasonic treatment (frequency 40KHz, temperature 25 deg.C) for 15min, filtering, heating and refluxing the filtrate for 15min, evaporating to dryness, and dissolving the residue with 2ml of 50% methanol to obtain control medicinal material solution.
Preparation of negative control solution: 2.5g of negative control (maltodextrin) lacking artificial concretio silicea Bambusae seu Schizostachyi is prepared into negative control solution by the same method.
Performing thin layer chromatography (general rule 0502) test, respectively sucking the sample solution and the reference solution 15 μ 1, respectively, dropping on the same silica gel G thin layer plate, developing with water saturated n-butanol-glacial acetic acid 4: 1 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp 365 nm. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution. Referring to fig. 1, the chromatogram shows that the method has clear spots and high sensitivity.
Example 3:
the embodiment relates to a thin-layer identification method for identifying traditional Chinese medicine paste containing concretio silicea bambusae.
The method comprises the following specific steps:
preparing a test solution: taking the traditional Chinese medicine ointment containing the concretio silicea bambusae, putting the traditional Chinese medicine ointment into a conical flask according to the amount of 2.0g of the concretio silicea bambusae contained in the preparation, adding 40ml of 75% methanol, carrying out ultrasonic treatment (the frequency is 30KHz, the temperature is 25 ℃) for 20min, filtering, heating and refluxing the filtrate for 10min, evaporating to dryness, and dissolving residues in 2ml of 50% methanol to prepare a sample solution.
Preparing a reference medicinal material solution: taking 0.5g of concretio silicea Bambusae seu Schizostachyi control medicinal material, placing in a conical flask, adding 40ml of 75% methanol, performing ultrasonic treatment (frequency 30KHz, temperature 25 deg.C) for 20min, filtering, heating and refluxing the filtrate for 10min, evaporating to dryness, and dissolving the residue with 2ml of 50% methanol to obtain control medicinal material solution.
Preparation of negative control solution: negative control solution was prepared in the same manner as negative control solution (maltodextrin) 2.0g of the negative control of concretio silicea Bambusae seu Schizostachyi.
Performing thin layer chromatography (general rule 0502) test, respectively sucking sample solution and control solution 25 μ 1, respectively dropping on the same silica gel G thin layer plate, developing with water saturated n-butanol-glacial acetic acid 3.5: 1 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp 365 nm. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.
Example 4:
the embodiment relates to a thin-layer identification method for identifying traditional Chinese medicine decoction containing artificial concretio silicea bambusae.
The method comprises the following specific steps:
preparing a test solution: taking a traditional Chinese medicine decoction containing the artificial concretio silicea bambusae, putting the traditional Chinese medicine decoction into a conical flask according to the amount of 4.0g of the artificial concretio silicea bambusae contained in the preparation, adding 35ml of 80% methanol, carrying out ultrasonic treatment (the frequency is 40KHz, the temperature is 25 ℃) for 20min, filtering, heating and refluxing the filtrate for 12min, evaporating to dryness, and dissolving residues in 2ml of 45% methanol to prepare a sample solution.
Preparing a reference medicinal material solution: taking 0.8g of concretio silicea Bambusae seu Schizostachyi control medicinal material, placing in a conical flask, adding 80% methanol 35ml, performing ultrasonic treatment (frequency 40KHz, temperature 25 deg.C) for 20min, filtering, heating and refluxing the filtrate for 12min, evaporating to dryness, and dissolving the residue with 45% methanol 2ml to obtain control medicinal material solution.
Preparation of negative control solution: a negative control solution was prepared in the same manner as negative control solution (maltodextrin) 4.0g of the negative control of concretio silicea Bambusae seu Schizostachyi.
Performing thin layer chromatography (general rule 0502) test, respectively sucking the sample solution and the reference solution 20 μ 1, respectively, dropping on the same silica gel G thin layer plate, developing with water saturated n-butanol-glacial acetic acid 4: 1 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp 365 nm. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.
Example 5: examination of sample application amount
On the basis of example 2, artificial concretio silicea bambusae reference medicinal material solution and test sample solution are prepared, and different sample amounts are respectively spotted, and the result is shown in fig. 2.
In fig. 2, the sampling amounts from left to right are: 5 mu 1, 10 mu 1, 20 mu 1 and 25 mu 1 of reference medicine solution and 5 mu 1, 10 mu 1, 20 mu 1 and 25 mu 1 of test solution. The results shown in FIG. 2 show that the optimal effect can be achieved when the sample amount of the reference drug solution is 10-25 μ l and the sample amount of the sample solution is 10-25 μ l. Therefore, 10-25 μ l of the reference solution and the sample solution are selected respectively.
Example 6: specialization inspection
On the basis of example 2, artificial concretio silicea bambusae control solution, test solution and negative control solution lacking artificial concretio silicea bambusae formula particles were prepared, and the sample application amount was 15 μ l. The results are shown in FIG. 3, which shows that the negative control is not interfered, and the specificity of the method is good.
Example 7: durability examination
(I) comparison of different temperatures
On the basis of example 2, a test solution and a reference solution are prepared and spotted, and the spotted thin-layer plates are taken and developed under the temperature environments of 4 ℃ and 25 ℃ respectively, and the results show that the method has good adaptability to different temperature environments, and the results are shown in fig. 4 and 5.
(II) examination of different humidities
On the basis of example 2, a test solution and a control solution are prepared and spotted, and the spotted thin-layer plates are taken and developed under the humidity environment of 32% and 75% respectively. The results show that the method has good adaptability to different humidities, and the results are shown in fig. 6 and 7.
(III) comparison of silica gel plates from different manufacturers
The Tianjin Silida, Qingdao ocean and Merck thin-layer plate were selected and tested according to the test method set forth in example 2. The results show that the method has good adaptability to silica gel plates of different manufacturers, and the results are shown in fig. 8, 9 and 10.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.
Claims (5)
1. A thin-layer identification method of a traditional Chinese medicine preparation containing concretio silicea Bambusae seu Schizostachyi or artificial concretio silicea Bambusae seu Schizostachyi is characterized in that: the method comprises the following steps:
(1) preparing a test solution containing concretio silicea Bambusae seu Schizostachyi or artificial concretio silicea Bambusae seu Schizostachyi Chinese medicinal preparation, and a control solution containing concretio silicea Bambusae seu Schizostachyi;
(2) and (2) analyzing the test solution and the reference solution prepared in the step (1) according to a thin-layer chromatography, wherein fluorescent spots with the same color are displayed in the chromatogram of the test solution at the corresponding positions of the chromatogram of the reference solution.
2. The thin-layer identification method of a traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae as claimed in claim 1, wherein: in the step (1), 2.0-5.0 g of a traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae is taken according to the proportion of the concretio silicea bambusae or the artificial concretio silicea bambusae, dissolved by 20-50 ml of 70-80% methanol, subjected to ultrasonic treatment for 15-30 min, filtered, heated and refluxed for 10-15 min, evaporated to dryness, and dissolved by adding 1-2 ml of 40-55% methanol into residues to prepare a sample solution.
3. The thin-layer identification method of a traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae as claimed in claim 1, wherein: in the step (1), 0.5-1.0 g of a concretio silicea bambusae control medicinal material is taken, dissolved by 20-50 ml of 70-80% methanol, subjected to ultrasonic treatment for 15-30 min, filtered, heated and refluxed for 10-15 min, evaporated to dryness, and residues are dissolved by 1-2 ml of 40-55% methanol to prepare a control medicinal material solution.
4. The thin-layer identification method of a traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae as claimed in claim 1, wherein: in the step (2), the sample solution and the reference medicinal material solution are respectively absorbed by 10-25 mu 1, respectively, and are respectively spotted on the same silica gel G thin-layer plate, water-saturated n-butyl alcohol-glacial acetic acid with the ratio of 3.5-4.5: 1 as a developing agent, and the developing agent is developed, taken out, dried and inspected under an ultraviolet lamp of 365 nm.
5. The thin-layer identification method of a traditional Chinese medicine preparation containing concretio silicea bambusae or artificial concretio silicea bambusae as claimed in claim 1, wherein: in the step (1), the Chinese medicinal preparation is selected from decoction, paste, granules or tablets.
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