CN111198244A - Thin-layer chromatography detection method for nauclea officinalis extract syrup - Google Patents

Thin-layer chromatography detection method for nauclea officinalis extract syrup Download PDF

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CN111198244A
CN111198244A CN202010041368.8A CN202010041368A CN111198244A CN 111198244 A CN111198244 A CN 111198244A CN 202010041368 A CN202010041368 A CN 202010041368A CN 111198244 A CN111198244 A CN 111198244A
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methanol
detection method
thin
layer chromatography
chromatography detection
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谢晨星
张鹏
李华焕
张岭伟
唐伟茂
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Hainan Senqi Pharmaceutical Co ltd
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Hainan Senqi Pharmaceutical Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/94—Development
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/95—Detectors specially adapted therefor; Signal analysis

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Abstract

The invention relates to the technical field of thin-layer chromatography detection, in particular to a thin-layer chromatography detection method for nauclea officinalis extract syrup. The method comprises the following steps: mixing the lignum naucleae extract syrup with ethanol, heating in water bath under reflux, cooling, filtering, evaporating the filtrate to dryness, and adding methanol to obtain a sample; mixing lignum naucleae medicinal material with ethanol, heating under reflux in water bath, cooling, filtering, evaporating the filtrate, and adding methanol to obtain standard substance; respectively spotting the sample and the standard substance on the same thin-layer plate, developing in a developing agent, and detecting the color of the fluorescent spot under ultraviolet light after air drying; the developing solvent is a mixture of dichloromethane, ethyl acetate and methanol. The detection method provided by the invention realizes that the national standard or Chinese pharmacopoeia does not have a thin-layer identification method for the product by taking the vinca lactam as a target object, solves the problems of inferior-quality and counterfeit products of the product and the like, is simple and practical, consumes less time, and has strong reproducibility, specificity and durability, and the monitoring time of the product is greatly reduced.

Description

Thin-layer chromatography detection method for nauclea officinalis extract syrup
Technical Field
The invention relates to the technical field of thin-layer chromatography detection, in particular to a thin-layer chromatography detection method for nauclea officinalis extract syrup.
Background
Lignum et radix Naucleae belongs to genus Pterocarpus of Rubiaceae, and is a famous south drug, also named as lignum Santali albi, herba Urticae Cannabinae and radix Rumicis Japonici. Lignum naucleae is bitter in taste and cold in nature, has the effects of clearing heat, removing toxicity, relieving swelling and pain and the like, and is commonly used for treating cold, fever, bronchitis, pneumonia, acute tonsillitis, pharyngolaryngitis, mastitis, cholecystitis, enteritis, bacillary dysentery, urinary tract infection, ulcer of lower limbs, tinea pedis infection, burn infection, furuncle and eczema. Danmu has been used as a folk medicine all the time, and was first recorded in books of Guangzhou army, such as "handbook of common Chinese herbal medicine" and "compilation of Chinese herbal medicine", and was also recorded in the section of "Chinese pharmacopoeia" published in 1977. As early as 1977, the successful cure of leptospirosis has been reported clinically with nauclea officinalis preparation.
The lignum naucleae extract syrup is a traditional Chinese medicine preparation developed and researched from single medicinal materials of lignum naucleae, and is a yellow brown to tan viscous liquid; sweet and bitter taste, and is clinically used mainly for clearing away heat and toxic material, relieving swelling and alleviating pain. Can be used for treating acute tonsillitis, acute pharyngitis, acute conjunctivitis, and upper respiratory infection.
At present, the quality control method for the nauclea officinalis extract syrup and other preparations is detected by adopting a high performance liquid chromatography. However, the high performance liquid chromatography is complex to operate, has high requirements on instrument conditions, and is not beneficial to popularization in the environment that instruments of most basic detection departments fall behind and the quality of operators is generally low in China.
Thin layer chromatography is a chemical separation and analysis method, which is simple to operate and has the functions of qualitative identification, quality control and impurity detection, and the classical identification of chemistry is to separate or divide them into single components by using over-dissolving extraction or reagent treatment. The lignum naucleae extract syrup is subjected to thin-layer identification by taking target strictosamide as a standard, a good detection method is not available at the present stage, the national standard and Chinese pharmacopoeia do not have corresponding detection standards, and the problems of authenticity of the lignum naucleae extract syrup preparation or adulteration of inferior products are difficult to ensure.
Disclosure of Invention
In view of the above, the invention provides a thin-layer chromatography detection method for nauclea officinalis extract syrup. The method is simple and practical, has less time consumption and strong reproducibility, specificity and durability, and greatly reduces the monitoring time of the product.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a thin-layer chromatography detection method of nauclea officinalis extract syrup, which comprises the following steps:
mixing the lignum naucleae extract syrup with ethanol, heating in water bath under reflux, cooling, filtering, evaporating the filtrate to dryness, and adding methanol to obtain a sample;
mixing lignum naucleae medicinal material with ethanol, heating under reflux in water bath, cooling, filtering, evaporating the filtrate, and adding methanol to obtain standard substance;
respectively spotting the sample and the standard substance on the same thin-layer plate, developing in a developing agent, and detecting the color of the fluorescent spot under ultraviolet light after air drying; the developing solvent is a mixture of dichloromethane, ethyl acetate and methanol.
Preferably, the volume ratio of the dichloromethane, the ethyl acetate and the methanol in the developing solvent is (12-18): (8-12): 0.5-1.5).
Preferably, the volume ratio of the dichloromethane to the ethyl acetate to the methanol in the developing solvent is 15:10: 1.
Preferably, in the preparation method of the test sample, the volume ratio of the nauclea officinalis extract syrup to the ethanol to the methanol is (1-3): (18-22): 0.5-1.5).
Preferably, in the preparation method of the test sample, the volume ratio of the lignum naucleae extract syrup to the ethanol to the methanol is 2:20: 1.
Preferably, in the preparation method of the standard substance, the ratio of the medicinal material of Nauclea officinalis, ethanol and methanol is (1-3): (18-22): 0.5-1.5) in g/mL/mL.
Preferably, in the preparation method of the standard substance, the ratio of the medicinal material of the Nauclea officinalis Pierrc ex Pitard, the ethanol and the methanol is 2:20:1 in g/mL/mL.
Preferably, the time of the water bath reflux heating is 0.5-2.5 h.
Preferably, the time for heating the water bath under reflux is 1 h.
Preferably, the sample amount of the sample or the standard is 3 to 8. mu.L.
Preferably, the sample amount of the test sample or the standard sample is 5. mu.L.
Preferably, the wavelength of the ultraviolet light is 360-370 nm.
Preferably, the wavelength of the ultraviolet light is 365 nm.
The invention provides a thin-layer chromatography detection method of nauclea officinalis extract syrup. The method comprises the following steps: mixing the lignum naucleae extract syrup with ethanol, heating in water bath under reflux, cooling, filtering, evaporating the filtrate to dryness, and adding methanol to obtain a sample; mixing lignum naucleae medicinal material with ethanol, heating under reflux in water bath, cooling, filtering, evaporating the filtrate, and adding methanol to obtain standard substance; respectively spotting the sample and the standard substance on the same thin-layer plate, developing in a developing agent, and detecting the color of the fluorescent spot under ultraviolet light after air drying; the developing solvent is a mixture of dichloromethane, ethyl acetate and methanol. The invention has the technical effects that:
the dichloromethane-ethyl acetate-methanol is used as a developing agent, the developing agent can selectively transfer a target component of vinca glycinamide in the nauclea officinalis, and the transfer effect of the developing agent is better than that of other developing agents;
the invention provides a detection method of thin-layer chromatography of nauclea officinalis extract syrup, which realizes that the national standard or Chinese pharmacopoeia does not have a thin-layer identification method for the product by taking vinca galanthamine as a target object, solves the problems of inferior-quality and counterfeit products and the like of the product, is simple and practical, consumes less time, and has strong reproducibility, specificity and durability, and greatly reduces the monitoring time of the product.
Drawings
FIG. 1 shows that the specificity of the method of example 1 is good;
FIG. 2 shows that the reproducibility of the method of example 1 is good;
FIGS. 3-5 illustrate the durability of the method of example 1;
fig. 6 shows a comparison of the effects of the processes of example 1, comparative example 1 and comparative example 2.
Detailed Description
The invention discloses a thin-layer chromatography detection method of nauclea officinalis extract syrup, which can be realized by appropriately improving process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Preparing a test sample: taking 2mL of the nauclea officinalis extract syrup, adding 20mL of ethanol, heating in a water bath for 1h under reflux, cooling, filtering, steaming the filtrate in a water bath until the filtrate is nearly dry, and adding 1mL of methanol to dissolve the filtrate to obtain a test sample for later use.
Preparing a standard substance: taking 2g of the medicinal material nauclea officinalis, adding 20mL of ethanol, heating in a water bath under reflux for 1h, cooling, filtering, steaming the filtrate in a water bath until the filtrate is nearly dry, and adding 1mL of methanol to dissolve the filtrate to obtain a test sample.
The detection method comprises the following steps: respectively sucking the test solution and the reference substance, dropping on the same thin layer plate, developing with dichloromethane-ethyl acetate-methanol as developing agent, taking out, air drying, and inspecting under ultraviolet lamp.
As a result: 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.
Preferably, the ratio of dichloromethane to ethyl acetate to methanol of the developing agent is (15:10: 1).
Preferably, the sample size is 5. mu.L in the present invention.
Preferably, the present invention is inspected with an ultraviolet lamp at a wavelength of 365 nm.
The reagent or instrument used in the thin-layer chromatography detection method of the nauclea officinalis extract syrup provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
The thin-layer chromatography detection method for the nauclea officinalis extract syrup in the embodiment comprises the following steps:
1. preparing a test sample: taking 2mL of the nauclea officinalis extract syrup, adding 20mL of ethanol, heating in a water bath for 1h under reflux, cooling, filtering, steaming the filtrate in a water bath until the filtrate is nearly dry, and adding 1mL of methanol to dissolve the filtrate to obtain a test sample for later use.
2. Preparing a standard substance: taking 2g of the medicinal material nauclea officinalis, adding 20mL of ethanol, heating in a water bath under reflux for 1h, cooling, filtering, steaming the filtrate in a water bath until the filtrate is nearly dry, and adding 1mL of methanol to dissolve the filtrate to obtain a test sample.
3. The detection method comprises the following steps: respectively sucking sample solution and control sample each 5 μ L, spotting on the same thin layer plate, developing with dichloromethane-ethyl acetate-methanol (15:10:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).
Experimental example 1 methodological verification:
(1) specificity
Respectively sucking 10 μ L of the above test solution, sequentially spotting on the same thin layer plate, developing, taking out, air drying, and inspecting under ultraviolet lamp (365nm), as shown in FIG. 1. The test sample shows the same color spot on the corresponding position of the chromatogram of the reference substance as shown in figure 1, which indicates that the specificity of the method is good.
(2) Repeatability of
Taking three parts of a test sample and a reference sample of the nauclea officinalis extract syrup of the same batch number, respectively accounting for 5 mu L of the test sample and the reference sample, and dotting on the same thin-layer plate: the results are shown in FIG. 2A, FIG. 2B, FIG. 2C
(3) Durability
1) Adjustment and investigation of sample application amount:
respectively sucking sample solution 4 μ L, 5 μ L, and 6 μ L, placing on the same thin layer plate, developing with dichloromethane-ethyl acetate-methanol (15:10:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The results are shown in FIG. 3A, FIG. 3B, and FIG. 3C, respectively.
The sample presents the same color spot on the position corresponding to the chromatogram of the reference substance, and the difference between the thin-layer plates is not obvious after the sample amount is finely adjusted, which shows that the fine adjustment sample amount has no influence on the thin-layer identification of the strictosamide.
2) Adjustment of thin-layer plates of different brands:
thin-layer plate 1: silica gel GF254 thin layer plate (Yangtze river friend silica gel development Co., Ltd.) is shown in FIG. 4A;
thin-layer plate 2: silica gel GF254 thin layer plate (Qingdao ocean chemical plant division) as shown in FIG. 4B;
thin-layer plate 3: the self-made silica gel GF254 thin layer plate is shown in figure 4C.
The test product shows the same color spots on the corresponding positions of the chromatogram of the reference product, and the change of the different brand thin-layer plates has no obvious difference, which shows that the change of the different brand thin-layer plates has no influence on the thin-layer identification of the strictosamide.
3) Adjusting the developing solvent:
developing agent 1: dichloromethane-ethyl acetate-methanol (15:9:2) is shown in FIG. 5A;
developing agent 2: dichloromethane-ethyl acetate-methanol (15:10:1) is shown in FIG. 5B;
developing agent 3: dichloromethane-ethyl acetate-methanol (16:9:1) is shown in FIG. 5C.
The sample amount is adjusted, different brands of thin-layer plates are replaced, and the adjustment of the proportion of the developing agent has no influence on the thin-layer identification of the strictosamide, so that the durability of the method meets the requirement. By inspecting the thin-layer chromatography of the strictosamide, the repeatability, specificity and durability of the method meet the requirements, and the method is suitable for thin-layer identification of the strictosamide in the nauclea officinalis extract syrup.
Comparative example 1
The thin-layer chromatography detection method for the biliary tree extract syrup in the comparative example comprises the following steps:
1. preparing a test sample: taking 2mL of the nauclea officinalis extract syrup, adding 20mL of ethanol, heating in a water bath for 1h under reflux, cooling, filtering, steaming the filtrate in a water bath until the filtrate is nearly dry, and adding 1mL of methanol to dissolve the filtrate to obtain a test sample for later use.
2. Preparing a standard substance: taking 2g of the medicinal material nauclea officinalis, adding 20mL of ethanol, heating in a water bath under reflux for 1h, cooling, filtering, steaming the filtrate in a water bath until the filtrate is nearly dry, and adding 1mL of methanol to dissolve the filtrate to obtain a test sample.
3. The detection method comprises the following steps: respectively sucking sample solution and control sample each 5 μ L, dispensing on the same thin layer plate, developing with n-hexane-ethyl acetate-methanol (15:10:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).
Comparative example 2
The thin-layer chromatography detection method for the biliary tree extract syrup in the comparative example comprises the following steps:
1. preparing a test sample: taking 2mL of the nauclea officinalis extract syrup, adding 20mL of ethanol, heating in a water bath for 1h under reflux, cooling, filtering, steaming the filtrate in a water bath until the filtrate is nearly dry, and adding 1mL of methanol to dissolve the filtrate to obtain a test sample for later use.
2. Preparing a standard substance: taking 2g of the medicinal material nauclea officinalis, adding 20mL of ethanol, heating in a water bath under reflux for 1h, cooling, filtering, steaming the filtrate in a water bath until the filtrate is nearly dry, and adding 1mL of methanol to dissolve the filtrate to obtain a test sample.
3. The detection method comprises the following steps: respectively sucking sample solution and control sample each 5 μ L, dispensing on the same thin layer plate, developing with dichloromethane-ethyl acetate-formic acid (15:10:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).
Test example 2 comparison of effects:
the developing agents of example 1, comparative example 1 and comparative example 2 were used to test the same sample.
A solution of 0.5% sodium hydroxide was sprayed onto commercially available silica gel GF254 thin layer plates before use, as shown in fig. 6: example 1 the developing agent is dichloromethane-ethyl acetate-methanol (15:10:1) in the chromatogram of the test solution, and the fluorescent spots with the same color are shown on the corresponding positions of the chromatogram of the reference solution. When comparative example 1 n-hexane-ethyl acetate-methanol (15:10:1) and comparative example 2 dichloromethane-ethyl acetate-formic acid (15:10:1) were selected as developing agents, spots were not clear, and no fluorescent spots of the same color were observed.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A thin-layer chromatography detection method of nauclea officinalis extract syrup is characterized by comprising the following steps:
mixing the lignum naucleae extract syrup with ethanol, heating in water bath under reflux, cooling, filtering, evaporating the filtrate to dryness, and adding methanol to obtain a sample;
mixing lignum naucleae medicinal material with ethanol, heating under reflux in water bath, cooling, filtering, evaporating the filtrate, and adding methanol to obtain standard substance;
respectively spotting the sample and the standard substance on the same thin-layer plate, developing in a developing agent, and detecting the color of the fluorescent spot under ultraviolet light after air drying; the developing solvent is a mixture of dichloromethane, ethyl acetate and methanol.
2. The thin layer chromatography detection method of claim 1, wherein the volume ratio of dichloromethane, ethyl acetate and methanol in the developing solvent is (12-18): (8-12): (0.5-1.5).
3. The thin layer chromatography detection method of claim 1, wherein the volume ratio of dichloromethane, ethyl acetate and methanol in the developing solvent is 15:10: 1.
4. The thin layer chromatography detection method of claim 1, wherein the volume ratio of the nauclea officinalis extract syrup to the ethanol to the methanol in the sample preparation method is (1-3): (18-22): (0.5-1.5).
5. The thin layer chromatography detection method of claim 1, wherein in the sample preparation method, the volume ratio of the lignum naucleae extract syrup to the ethanol to the methanol is 2:20: 1.
6. The thin layer chromatography detection method of claim 1, wherein in the preparation method of the standard, the ratio of the Nauclea officinalis material to the ethanol to the methanol is (1-3): (18-22): (0.5-1.5) in g/mL/mL.
7. The thin layer chromatography detection method of claim 1, wherein in the preparation method of the standard, the ratio of the nauclea officinalis kuntze medicinal material, the ethanol and the methanol is 2:20:1 in g/mL/mL.
8. The thin layer chromatography detection method of claim 1, wherein the time for heating by water bath reflux is 0.5-2.5 h.
9. The thin layer chromatography detection method according to claim 1, wherein the sample amount of the test sample or the standard sample is 3 to 8 μ L.
10. The thin layer chromatography detection method of any one of claims 1 to 9, wherein the wavelength of the ultraviolet light is 360 to 370 nm.
CN202010041368.8A 2020-01-15 2020-01-15 Thin-layer chromatography detection method for nauclea officinalis extract syrup Pending CN111198244A (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN101185692A (en) * 2006-11-15 2008-05-28 江苏中康药物科技有限公司 Nauclea officinalis extract and preparation and use thereof
CN109298116A (en) * 2018-11-22 2019-02-01 海南森祺制药有限公司 A kind of detection method of nauclea officinalis medicinal extract piece

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101185692A (en) * 2006-11-15 2008-05-28 江苏中康药物科技有限公司 Nauclea officinalis extract and preparation and use thereof
CN109298116A (en) * 2018-11-22 2019-02-01 海南森祺制药有限公司 A kind of detection method of nauclea officinalis medicinal extract piece

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