Thin-layer identification method for rhizoma alismatis in dampness-removing stomach-poria decoction

Abstract

The invention provides a thin-layer identification method for rhizoma alismatis in dampness-eliminating stomach-poria decoction, and belongs to the technical field of drug detection, the method comprises the processes of preparation of a test solution, preparation of a reference medicinal material solution and thin-layer chromatography development detection, and in the preparation of the test solution, dichloromethane is adopted as an extraction solvent to extract a dampness-eliminating stomach-poria decoction sample. According to the thin-layer identification method for the rhizoma alismatis in the dampness-removing stomach-poria decoction, the quality of the rhizoma alismatis medicinal material can be conveniently, accurately and qualitatively detected, and the method is high in specificity and relatively high in reliability and stability.

Description

Technical Field

[0001] The present invention relates to a thin-layer identification method for Alisma orientale, and particularly to a thin-layer identification method for Alisma orientale in Chushi Weiling Decoction, belonging to the technical field of drug detection. Background Art

[0002] Chushi Weiling Decoction: It has the effects of clearing and drying dampness, strengthening the spleen to dry dampness, and regulating the middle-jiao and promoting diuresis. It is composed of Atractylodes lancea (parched), Magnolia officinalis (parched with ginger), Citrus reticulata Blanco, Polyporus umbellatus, Alisma orientale, Poria rubra, Atractylodes macrocephala Koidz. (fried with earth), Talc, Saposhnikovia divaricata, Gardenia jasminoides Ellis (raw, ground), Akebia quinata, Cinnamomum cassia Presl, and Glycyrrhiza uralensis Fisch. Indications: Herpes zoster (dampness-excessive type of shingles), eczema (damp skin ulcer), psoriasis (damp-cold type of pityriasis versicolor), etc.

[0003] Thin-layer chromatography (TLC) is a method in which a suitable stationary phase is coated on a glass plate, plastic, or aluminum substrate to form a uniform thin layer. After spotting and developing, the retention factor (Rf) is compared with that of the chromatogram obtained in the same way with a suitable reference substance for the identification, impurity inspection, or content determination of drugs. Thin-layer chromatography is a very important experimental technique for the rapid separation and qualitative analysis of small amounts of substances and is also used to monitor the progress of reactions.

[0004] At present, there is little research on the quality of Chushi Weiling Decoction, and there is no identification method for its constituent medicinal materials. Alisma orientale is the dried rhizome of the plant Alisma orientale (Sam.) Juzep. of the Alismataceae family. It is dug when the stems and leaves begin to wither in winter, washed, dried, and the fibrous roots and rough bark are removed. It is spherical, oval, or ovoid in shape, 2-7 cm in length and 2-6 cm in diameter. Alisma orientale is sweet, bland, and cold; it belongs to the kidney and bladder meridians and has the effects of promoting diuresis and excreting dampness, clearing heat, and reducing turbidity and lipid. It is used for oliguria, edema, diarrhea with scanty urine, phlegm-fluid dizziness, acute strangury with pain, hyperlipidemia. It is mainly produced in Fujian, Sichuan, Jiangxi provinces, and Guangxi Zhuang Autonomous Region, and is now mostly artificially cultivated. The thin-layer chromatographic analysis of Alisma orientale in Chushi Weiling Decoction helps to screen qualified medicinal materials and provides a basis for the quality control of Chushi Weiling Decoction. Summary of the Invention

[0005] In view of the above problems, the present invention provides a thin-layer identification method for Alisma orientale in Chushi Weiling Decoction to conveniently and accurately qualitatively detect the quality of Alisma orientale medicinal materials.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A thin-layer identification method for Alisma orientale in Chushi Weiling Decoction includes the preparation of a test solution, the preparation of a reference medicinal material solution, and the process of thin-layer chromatography test. In the preparation of the test solution, dichloromethane is used as the extraction solvent to extract the Chushi Weiling Decoction sample.

[0008] Furthermore, the steps of preparing the test solution are: taking a sample of Dehumidifying Weiling Decoction, dissolving it in water, filtering it, extracting the filtrate with dichloromethane to obtain a dichloromethane solution, evaporating it to dryness, and dissolving the residue with ethyl acetate to obtain the test solution.

[0009] Furthermore, the Dehumidifying Weiling Decoction sample is Dehumidifying Weiling Decoction powder.

[0010] Furthermore, the specific steps for preparing the test solution are: take 4g of Dehumidification Weiling Decoction powder, add 20ml of water to dissolve, filter, extract the filtrate twice with dichloromethane, 20ml each time, combine the dichloromethane liquid, evaporate to dryness, and dissolve the residue with 0.5ml of ethyl acetate to obtain the test solution.

[0011] Furthermore, the control medicinal material solution is prepared by taking a control medicinal material of Alisma orientalis, adding water, decocting, filtering, extracting the filtrate with dichloromethane to obtain a dichloromethane solution, evaporating to dryness, and dissolving the residue with ethyl acetate to obtain the control medicinal material solution.

[0012] Furthermore, the specific steps for preparing the control medicinal material solution are: take 2g of Alisma orientalis control medicinal material, add 50ml of water, decoct for 30min, filter, extract the filtrate with dichloromethane twice, 20ml each time, combine the dichloromethane liquid, evaporate to dryness, and dissolve the residue with 0.5ml of ethyl acetate to obtain the control medicinal material solution.

[0013] Furthermore, the thin layer chromatography test process is to perform thin layer identification according to thin layer chromatography using ethyl acetate-cyclohexane as a developing solvent.

[0014] Furthermore, the thin layer chromatography test process is to absorb 6-10 μl of the test solution and the control medicinal material solution, respectively, spot them on the same silica gel H thin layer plate, use ethyl acetate-cyclohexane as the developing agent, develop, take out, dry, spray with 5% silicotungstic acid ethanol solution, and heat at 105°C until the spots are clearly colored.

[0015] Furthermore, the volume ratio of ethyl acetate to cyclohexane in the developing solvent is 1:1.

[0016] Further, 8 μl of the test solution and 6 μl of the control medicinal material solution were aspirated.

[0017] The beneficial effects of the thin layer identification method of orientalis in the dehumidifying Weiling decoction of the present invention are:

[0018] The invention provides a thin layer identification method of oriental rhizoma in dehumidifying weiling decoction, which can be used for convenient and accurate qualitative detection of the quality of oriental rhizoma medicinal materials, and the method has strong specificity and high reliability and stability.

[0019] By optimizing the preparation method of the test sample and using dichloromethane as the extraction solvent, the spots in the chromatogram have obvious colors and good resolution. Using the method of the present invention, in the test sample chromatogram obtained, at the positions corresponding to the chromatogram of the control crude drug, spots of the same color appear, and there is no interference from the negative sample, indicating that the method is feasible. Through the durability investigation, it can be seen that the method of the present invention has good durability for different thin-layer silica gel plates, different developing temperatures and humidities, and the main spots are clear. Description of the Drawings

[0020] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0021] Figure 1 It is the thin-layer chromatography result diagram in Example 1 of the present invention;

[0022] Figure 2 It is the thin-layer chromatography result diagram of the sample application amount investigation test in Example 2 of the present invention;

[0023] Figures 3 - 5 It is the thin-layer chromatography result diagram of the durability investigation test in Example 3 of the present invention;

[0024] Figures 6 - 7 It is the thin-layer chromatography result diagram of the thin-layer identification test of different batches of dry extract powder in Example 4 of the present invention;

[0025] Figure 8 It is the thin-layer chromatography result diagram in Comparative Example 1 of the present invention;

[0026] Figure 9 The thin-layer chromatography result diagram in Comparative Example 2 of the present invention. Specific Embodiments

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0028] A thin-layer identification method for Alisma orientale in Chushi Weiling Decoction includes the following steps:

[0029] Step S1: Preparation of the test sample solution: Take the powder of Chushi Weiling Decoction, dissolve it in water, filter, extract the filtrate with dichloromethane to obtain dichloromethane solution, evaporate to dryness, and dissolve the residue in ethyl acetate to obtain the test sample solution;

[0030] Step S2: Preparation of the reference medicinal material solution: Separately take the reference medicinal material of Alisma orientale, add water, decoct, filter, extract the filtrate with dichloromethane to obtain a dichloromethane solution, evaporate to dryness, and dissolve the residue in ethyl acetate to obtain the reference medicinal material solution;

[0031] Step S3: According to the thin-layer chromatography method in General Principles 0502 of the Fourth Part of the Chinese Pharmacopoeia 2015 Edition, respectively absorb 6 - 10 μl of the test solution and the reference medicinal material solution, and spot them on the same silica gel H thin-layer plate. Use ethyl acetate - cyclohexane as the developing agent, develop, take out, air dry, spray with 5% silicotungstic acid ethanol solution, and heat at 105 °C until the spots are clearly developed.

[0032] The preparation method of the Dampness-Removing and Stomach-Regulating Decoction of the present invention is as follows:

[0033] Weigh the daily prescription amount of the cut crude drugs: stir-fried Atractylodes lancea 3.75 g, Magnolia officinalis var. biloba 3.75 g, Citrus reticulata Blanco 3.75 g, Polyporus umbellatus 3.75 g, Alisma orientale 3.75 g, Poria rubra 3.75 g, stir-fried Atractylodes macrocephala 3.75 g, Talc 3.75 g, Saposhnikovia divaricata 3.75 g, Gardenia jasminoides Ellis 3.75 g, Akebia quinata 3.75 g, Cinnamomum cassia Presl 1.13 g, Glycyrrhiza uralensis Fisch 1.13 g, Juncus effusus L. 0.14 g, place them in a casserole (specification: capacity is about 1200 mL, inner diameter at the bottom is 13.5 cm, inner diameter at the top is 18 cm, height is 8 cm), add 400 mL of water, heat with an open flame, cover and decoct, bring to a boil over high heat, and decoct over low heat for about 20 minutes (decoct until about 270 mL), filter through a 200-mesh filter cloth to obtain the Dampness-Removing and Stomach-Regulating Decoction.

[0034] The preparation method of the powder (dry extract powder) of the Dampness-Removing and Stomach-Regulating Decoction is as follows:

[0035] Take the Dampness-Removing and Stomach-Regulating Decoction, centrifuge for 2 minutes (centrifugation speed is 1000 revolutions per minute), take the supernatant and concentrate it under reduced pressure (at 60 - 70 °C, -0.09 Mpa to -0.1 Mpa) to the feeding amount: the volume ratio of the concentrated solution is about 0.9:1 (g:mL), and perform freeze-drying (pre-freezing: temperature: -20 to -45 °C, time: at least 30 minutes; drying: cold trap temperature -60 to -70 °C, vacuum degree: <100 Pa, freeze-drying time: at least 18 hours) to obtain the powder (dry extract powder) of the Dampness-Removing and Stomach-Regulating Decoction.

[0036] Example 1

[0037] A thin-layer identification method for Alisma orientale in the Dampness-Removing and Stomach-Regulating Decoction, comprising the following steps:

[0038] Take 4 g of the Chushi Weiling Decoction powder, add 20 ml of water to dissolve it, filter, extract the filtrate with dichloromethane twice, 20 ml each time. Combine the dichloromethane solutions, evaporate to dryness, dissolve the residue in 0.5 ml of ethyl acetate to obtain the test solution. Additionally, take 2 g of the prepared Alisma orientale reference crude drug, add 50 ml of water, decoct for 30 minutes, filter, and prepare the reference crude drug solution in the same manner starting from "extract with dichloromethane twice"; take 4 g of the negative sample without Alisma orientale and prepare the negative sample solution in the same way. According to the thin-layer chromatography method (General Principles 0502), absorb 10 μl of each of the above three solutions and spot them on the same silica gel H thin-layer plate. Use ethyl acetate - cyclohexane (1:1) as the developing solvent, develop, take out, air dry, spray with 5% silicotungstic acid ethanol solution, and heat at 105 °C until the spots are clearly visible. The results are as Figure 1 shown, Figure 1 the silica gel thin-layer plate used in Figure 1 is the Qingdao silica gel H plate (batch number: 201710260). When developing, the temperature T is 19.9 °C and the humidity RH is 40.0%. In Figure 1 label 1 is the solution of the Alisma orientale reference crude drug, labels 2 - 4 are the test solutions, and label 5 is the negative sample solution without Alisma orientale. It can be seen from the figure that in the chromatogram of the test solution, at the position corresponding to the chromatogram of the reference crude drug, spots of the same color appear, and there is no interference from the negative sample. Therefore, the method is determined to be feasible.

[0039] Example 2

[0040] Test on the sample application volume

[0041] On the basis of Example 1, the sample application volume of the test solution was investigated. Specifically, the same method as in Example 1 was used, and the sample application volumes were selected as 2 μl, 4 μl, 6 μl, and 8 μl respectively. The test results are as Figure 2 shown. Figure 2 The silica gel thin-layer plate used in Figure 2 is the Qingdao silica gel H plate. When developing, the temperature T is 20.0 °C and the humidity RH is 40.0%. In Figure 2 label 1 is the solution of the Alisma orientale reference crude drug with a sample application volume of 8 μl, and labels 2 - 5 are the test solutions with sample application volumes of 2 μl, 4 μl, 6 μl, and 8 μl respectively. It can be Figure 2 seen that when the sample application volume is 4 - 8 μl, spots of the same color appear at the position corresponding to the chromatogram of the reference crude drug, and the spots are clearest when the sample application volume is 6 μl. Therefore, the optimal sample application volume of the test solution is 6 μl, and that of the reference crude drug solution is 8 μl.

[0042] Example 3

[0043] Durability investigation test

[0044] On the basis of Example 1, a durability test was carried out. Using the method in Example 1, factors such as thin-layer silica gel H plates from different batches of the same manufacturer (batch number of Qingdao Ocean Chemical Factory: 20170808), different developing temperatures (19.9 °C and 4 °C), and different relative humidities (40.0% and 85%) were investigated. The specific test results are as follows Figures 3 - 5 shown, where Figure 3 The silica gel plate used was a Qingdao silica gel H plate (batch number: 20170808). When developing, the temperature T was 22.1 °C and the humidity RH was 35%. In the figure, label 1 is the solution of the control crude drug of Alisma orientale, labels 2 - 4 are the test solution, and label 5 is the negative sample solution without Alisma orientale. Figure 4 is the chromatogram under low-temperature conditions. The silica gel plate is a Qingdao silica gel H plate (batch number: 20170808). When developing, the temperature T is 4 °C and the humidity RH is 47.2%. In the figure, label 1 is the solution of the control crude drug of Alisma orientale, labels 2 - 4 are the test solution, and label 5 is the negative sample solution without Alisma orientale. Figure 5 is the chromatogram under high-humidity conditions. The silica gel plate is a Qingdao silica gel H plate (batch number: 20170808). When developing, the temperature T is 19.8 °C and the humidity RH is 85%. In the figure, label 1 is the solution of the control crude drug of Alisma orientale, labels 2 - 4 are the test solution, and label 5 is the negative sample solution without Alisma orientale. From the above Figures 3 - 5 shown, it can be seen that the present invention has good durability under the above various conditions, and the main spots are all clear.

[0045] Example 4

[0046] Thin-layer identification test of dry extract powder of different batches

[0047] On the basis of Example 1, a thin-layer identification test of dry extract powder of different batches was carried out. Using the method in Example 1, 4 g of the corresponding physical objects (dry extract powder) of different batches were weighed respectively, and the test solution was prepared according to the preparation method of the test sample in Example 1, and thin-layer identification was carried out. The results are as follows Figure 6 and Figure 7 shown. Among them, Figure 6 and Figure 7 the silica gel plates are both Qingdao silica gel H plates. When developing, the temperature T is 22.1 °C and the humidity RH is 33%. Figure 6Among them, No. 1 is the reference medicinal material of Alisma orientale, and No. 2-16 are the test samples with batch numbers: J-181108-01-1, J-181108-02-1, J-181109-03-1, J-181109-04-1, J-181110-05-1, J-181110-06-1, J-181111-07-1, J-181112-08-1, J-181112-09-1, J-181113-10-1, J-181113-11-1, J-181114-12-1, J-181114-13-1, J-181117-14-1, J-181117-15-1; Figure 7 Among them, No. 1 is the reference medicinal material of Alisma orientale, and No. 2-16 are the test samples with batch numbers: J-181108-01-2, J-181108-02-2, J-181109-03-2, J-181109-04-2, J-181110-05-2, J-181110-06-2, J-181111-07-2, J-181112-08-2, J-181112-09-2, J-181113-10-2, J-181113-11-2, J-181114-12-2, J-181114-13-2, J-181117-14-2, J-181117-15-2. As can be seen from Figure 6 and Figure 7 shown, in the test sample chromatograms of different batches of samples, at the positions corresponding to the chromatogram of the reference medicinal material, spots of the same color appear.

[0048] Comparative Example 1

[0049] A thin-layer identification method for Alisma orientale in Chushi Weiling Decoction, referring to the thin-layer identification method under Alisma orientale in the Chinese Pharmacopoeia (Volume I) 2015 Edition. Specifically: Take 4 g of the powder of this product (batch number: J-180730-01), add 20 ml of water to dissolve it, filter, extract the filtrate with ethyl acetate twice, 20 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of ethyl acetate to prepare the test sample solution. Separately take 2 g of the reference medicinal material of Alisma orientale, add 50 ml of water, decoct for 30 minutes, filter, and from "extract with ethyl acetate twice", prepare the reference medicinal material solution in the same way. Then take 4 g of the negative sample without Alisma orientale and prepare the negative sample solution in the same way. According to the test of thin-layer chromatography (General Rule 0502), absorb 10 μl of each of the above three solutions, spot them on the same silica gel H thin-layer plate respectively, use ethyl acetate-cyclohexane (1:1) as the developing agent, develop, take out, dry in the air, spray with 5% silicotungstic acid ethanol solution, and heat at 105 °C until the spots are clearly developed. As a result, the spots in the test sample chromatogram are lighter in color and have poor resolution. The results are shown in Figure 8As shown, the silica gel plate used in the figure is Qingdao silica gel H plate. The temperature during development is 25°C and the humidity is 51.7%. Label 1 is the solution of Alisma orientale control medicinal material, label 2 is the test solution, and label 3 is the negative test solution.

[0050] Comparative Example 2

[0051] A thin-layer identification method for Alisma orientale in Chushi Weiling Decoction. On the basis of Comparative Example 1: The preparation method of the test sample is changed, and the extraction solvent is changed to ethyl acetate - petroleum ether (30 - 60°C) = 1:1, and the rest is the same as Comparative Example 1. As a result, the color of the spots in the test sample chromatogram is still relatively light and the resolution is poor. The results are shown in Figure 9 As shown. The silica gel plate used in the figure is Qingdao silica gel H plate. The temperature during development is 24.2°C and the humidity is 69.4%. Label 1 is the solution of Alisma orientale control medicinal material, label 2 is the test solution, and label 3 is the negative test solution.

[0052] Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A thin layer chromatography identification method for oriental rhizoma in dehumidifying weiling decoction, comprising the preparation of a test solution, the preparation of a control medicinal material solution and a thin layer chromatography test process, characterized in that: In the preparation of the test solution, dichloromethane is used as the extraction solvent to extract the Dehumidifying Weiling Decoction sample.

2. The thin layer identification method of Rhizoma Alismatis in Dehumidifying Weiling Decoction according to claim 1, characterized in that: The steps of preparing the test solution are: taking a sample of Dehumidification Weiling Decoction, adding water to dissolve it, filtering it, extracting the filtrate with dichloromethane to obtain dichloromethane liquid, evaporating it to dryness, and dissolving the residue with ethyl acetate to obtain the test solution.

3. The thin layer identification method of Rhizoma Alismatis in Dehumidifying Weiling Decoction according to claim 1 or 2, characterized in that: The Dehumidifying Weiling Decoction sample is Dehumidifying Weiling Decoction powder.

4. The thin layer identification method of Rhizoma Alismatis in Chushi Weiling Decoction according to claim 3, characterized in that: The specific steps of preparing the test solution are: take 4g of Dehumidification Weiling Decoction powder, add 20ml of water to dissolve, filter, extract the filtrate twice with dichloromethane, 20ml each time, combine the dichloromethane liquid, evaporate to dryness, and dissolve the residue with 0.5ml of ethyl acetate to obtain the test solution.

5. The thin layer identification method of Rhizoma Alismatis in Chushi Weiling Decoction according to claim 1, characterized in that: The control medicinal material solution is prepared by taking a control medicinal material of Alisma orientalis, adding water, decocting, filtering, extracting the filtrate with dichloromethane to obtain a dichloromethane solution, evaporating to dryness, and dissolving the residue with ethyl acetate to obtain the control medicinal material solution.

6. The thin layer identification method of orientalis in Chushiweiling decoction according to claim 5, characterized in that: The specific steps of preparing the control medicinal material solution are: taking 2g of Alisma orientalis control medicinal material, adding 50ml of water, decocting for 30min, filtering, extracting the filtrate twice with dichloromethane, each time with 20ml, combining the dichloromethane liquid, evaporating to dryness, and dissolving the residue with 0.5ml of ethyl acetate to obtain the control medicinal material solution.

7. The thin layer identification method of Rhizoma Alismatis in Chushi Weiling Decoction according to claim 1, characterized in that: The thin layer chromatography test process is to perform thin layer identification according to the thin layer chromatography method using ethyl acetate-cyclohexane as a developing solvent.

8. The thin layer identification method of Rhizoma Alismatis in Chushi Weiling Decoction according to claim 7, characterized in that: The thin layer chromatography test process is to take 6-10 μl of the test solution and the control medicinal material solution, respectively, spot them on the same silica gel H thin layer plate, use ethyl acetate-cyclohexane as the developing solvent, develop, take out, dry, spray with 5% silicotungstic acid ethanol solution, and heat at 105°C until the spots are clearly colored.

9. The thin layer identification method of Rhizoma Alismatis in Chushi Weiling Decoction according to claim 8, characterized in that: The volume ratio of ethyl acetate to cyclohexane in the developing solvent is 1:

1.

10. The thin layer identification method of Rhizoma Alismatis in Chushi Weiling Decoction according to claim 8, characterized in that: Pipette 8μl of test sample solution and 6μl of control medicinal material solution.

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