Thin-layer chromatography for identifying extract of Aspidistra elatior and extract of A. sinensis

Abstract

The application provides a kind of stone lotus seed extract, bitter stone lotus extract thin layer identification method, comprising: taking stone lotus seed extract pretreatment, to obtain test solution;Take bitter stone lotus extract pretreatment, to obtain bitter stone lotus extract solution;Take stone lotus control medicinal material, and water decoction is obtained control medicinal material solution;Test solution, bitter stone lotus extract solution and control medicinal material solution are detected by thin layer chromatography, and thin layer plate is silica gel G thin layer plate;Developing agent is n-butanol-isopropyl alcohol-water-glacial acetic acid;Under the observation of ultraviolet light, if test solution and control medicinal material solution have the same color fluorescent spot at Rf equal place, while there is no fluorescent spot at Rf=0.64-0.79, it is stone lotus seed extract;If there is fluorescent spot at Rf=0.64-0.79, it is bitter stone lotus extract.The application is used for distinguishing stone lotus seed extract and bitter stone lotus extract by whether there is fluorescent spot in the range of relative migration value in thin layer chromatogram.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and in particular to a method for identifying lotus seed extract and bitter lotus seed extract by thin layer chromatography. BACKGROUND

[0002] Lotus seed is the dry mature fruit of Nelumbo nucifera Gaertn. of the family Nymphaeaceae. It has the effects of tonifying spleen and stomach, clearing heart and relieving heat, and removing heat and toxic substances, and is used for treating dysentery, cold and hot vomiting, food retention, and turbid urine.

[0003] Bitter lotus seed is the dry mature seed of Millettia pinnata, which is a misused product of lotus seed. It has the effects of removing blood stasis and relieving pain, clearing heat, and removing dampness, and is used for treating vomiting, dysentery, turbid urine, hematuria, and traumatic injury.

[0004] Lotus seed and bitter lotus seed are similar in size, shape, color, and texture, so some pharmacies and Chinese medicine shops regard them as one thing and replace each other or confuse each other in application. Physical and chemical identification and property identification can be used to distinguish the two medicinal materials, but the extracts thereof lose their properties after extraction and cannot be used for identifying the extracts.

[0005] Therefore, a method with stronger specificity, stronger repeatability, and effective controllability is needed to provide an accurate basis for the qualitative identification of lotus seed extract. SUMMARY

[0006] Therefore, the present application aims to provide a method for identifying lotus seed extract and bitter lotus seed extract by thin layer chromatography.

[0007] The terms "comprise", "contain" and "have" are used interchangeably in this document and are intended to mean the inclusivity of the scheme, meaning that the scheme can have other elements in addition to the listed elements. It should also be understood that the use of "comprise", "contain" and "have" in this document also provides a "consisting of" scheme.

[0008] In this application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B, which can represent the following three cases: A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural.

[0009] In this application, "at least one" means one or more, and "multiple" means two or more. "At least one" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0010] It should be understood that the size of the sequence number of the following processes in various embodiments of the present application does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence, and the execution order of the processes should be determined according to its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0011] The present application provides a method for identifying lithospermi radix extract, and the lithospermi radix extract and lithospermi radix control drug material thin layer chromatogram are compared, and the same color spot is displayed. The test product and the lithospermi radix control drug material thin layer chromatogram are compared, and no fluorescent spot of lithospermi radix appears at the position of the relative migration value of 0.64-0.79, while the pseudo-product of bitter lithospermi radix extract has obvious blue fluorescent spot, which can be used as a discrimination point for distinguishing lithospermi radix extract and bitter lithospermi radix extract. The thin layer method can quickly and effectively identify the lithospermi radix extract which has lost the form of drinking pieces, and distinguish the lithospermi radix extract and the bitter lithospermi radix extract, and has the advantages of simple operation, high precision and sensitivity, good stability and good application prospect.

[0012] The present application provides a method for identifying lithospermi radix extract and bitter lithospermi radix extract, comprising:

[0013] A) pretreatment of lithospermi radix extract to obtain a test solution;

[0014] Pretreatment of bitter lithospermi radix extract to obtain a bitter lithospermi radix extract solution;

[0015] B) taking lithospermi radix control drug material, adding water to decoct, to obtain a control drug material solution;

[0016] C) thin layer chromatography detection of the test solution, the bitter lithospermi radix extract solution and the control drug material solution, and the thin layer plate is a silica gel G thin layer plate; the developing agent is n-butanol-isopropyl alcohol-water-glacial acetic acid;

[0017] D) inspection under ultraviolet light, if the test product and the control drug material solution have the same color fluorescent spot at the same Rf, and no fluorescent spot at Rf=0.64-0.79, then it is lithospermi radix extract; if there is a fluorescent spot at Rf=0.64-0.79, then it is bitter lithospermi radix extract.

[0018] The present application provides lithospermi radix extract and bitter lithospermi radix extract, which are first pretreated to obtain a test solution.

[0019] The pretreatment of the lithospermi radix extract or the bitter lithospermi radix extract according to the present application is specifically:

[0020] a) the extract of the plant of the genus Aspidistra is wetted with ammonia water, then refluxed with chloroform, filtered, evaporated, the residue is dissolved in water, and then passed through a C18 solid phase extraction column, eluted with 30% methanol, the eluate is discarded, then eluted with methanol, and the eluate is collected;

[0021] b) the eluate is recovered to dryness, the residue is dissolved in dilute ethanol, then passed through a neutral alumina column, eluted with ethanol, the eluate is collected, recovered to dryness, and the residue is dissolved in 70% ethanol by ultrasonic treatment.

[0022] The extract of the plant of the genus Aspidistra is wetted with ammonia water, then refluxed with chloroform for 20-30 min, filtered, evaporated, the residue is dissolved in water, and then passed through a C18 solid phase extraction column, eluted with 30% methanol, the eluate is discarded, then eluted with methanol, and the eluate is collected

[0023] In some embodiments of the present application, the mass / volume ratio of the extract of the plant of the genus Aspidistra, water, 30% methanol and methanol is 1 g:10 mL:10 mL:10 mL.

[0024] The eluate is recovered to dryness, the residue is dissolved in dilute ethanol, then passed through a neutral alumina column, eluted with ethanol, the eluate is collected, recovered to dryness, and the residue is dissolved in 70% ethanol by ultrasonic treatment.

[0025] In some embodiments of the present application, the volume ratio of the dilute ethanol, ethanol and 70% ethanol is 5:25:10.

[0026] In some embodiments of the present application, the ultrasonic treatment time is 20-30 min.

[0027] In some embodiments of the present application, the ultrasonic treatment time is 30 min.

[0028] The specific steps for the pretreatment of the extract of the plant of the genus Aspidistra are not limited, and are well known to those skilled in the art.

[0029] The control medicinal material of the plant of the genus Aspidistra is boiled with water to obtain a control medicinal material solution.

[0030] The mass / volume ratio of the control medicinal material of the plant of the genus Aspidistra and water is 1 g:50 mL; the boiling time is 50-70 min; and more preferably, 60 min.

[0031] The test sample solution, the extract of the plant of the genus Aspidistra and the control medicinal material solution are subjected to thin layer chromatography detection.

[0032] Preferably, the test sample solution and the control medicinal material solution are taken and spotted on the same silica gel G thin layer plate, and then developed with a developing agent, taken out, dried, and observed under ultraviolet light with a wavelength of 365 nm.

[0033] According to the application, the developing agent is n-butanol-isopropyl alcohol-water-glacial acetic acid;

[0034] In one preferred embodiment of the application, the mass ratio of the n-butanol-isopropyl alcohol-water-glacial acetic acid is 7:5:1:1.

[0035] The thin layer plate according to the application is a silica gel G thin layer plate; preferably a GF254 thin layer plate. It can be selected from Tianjin Silida, Merck, Qingdao Marine Prefabricated Silica Gel GF254 plate. The results show that the method has good durability, and can meet the identification requirements. Compared with the control drug material spectrum, the test sample spectrum and the control drug material spectrum show the same color spots at the corresponding positions.

[0036] Under the ultraviolet light, if the test sample and the control drug material solution have the same color fluorescent spots at the same Rf, and no fluorescent spots at Rf=0.64-0.79, it is a Gasteria extract;

[0037] If there are fluorescent spots at Rf=0.64-0.79, it is a Gasteria croucheri extract.

[0038] In a preferred embodiment of the application, if the test sample and the control drug material solution have the same color fluorescent spots at the same Rf, and no fluorescent spots at Rf=0.714, it is a Gasteria extract;

[0039] If there are fluorescent spots at Rf=0.714, it is a Gasteria croucheri extract.

[0040] The thin layer chromatography test sample sample amount according to the application is 1-7 μL, and the control drug material solution sample amount is 5-15 μL.

[0041] The viewing temperature according to the application is 4-25°C.

[0042] The method has good durability to different temperatures. Compared with the control drug material spectrum, the test sample spectrum and the control drug material spectrum show the same color spots at the corresponding positions.

[0043] The method has good durability to different humidity. Compared with the control drug material spectrum, the test sample spectrum and the control drug material spectrum show the same color spots at the corresponding positions.

[0044] The application provides a lithopsida extract and a lithopsida extract thin-layer identification method, which comprises the following steps: A) pretreating lithopsida extract to obtain a test sample solution; pretreating lithopsida extract to obtain a lithopsida extract solution; B) taking lithopsida control medicinal materials, adding water to decoct, and obtaining a control medicinal material solution; C) performing thin-layer chromatography detection on the test sample solution, the lithopsida extract solution and the control medicinal material solution, using a silica gel G thin-layer plate as the thin-layer plate, and using n-butanol-isopropyl alcohol-water-glacial acetic acid as the developing agent; D) observing under ultraviolet light, if the test sample solution and the control medicinal material solution have the same fluorescent spot at the same Rf value, and no fluorescent spot is present at Rf=0.64-0.79, then the lithopsida extract is obtained; if a fluorescent spot is present at Rf=0.64-0.79, then the lithopsida extract is obtained. The lithopsida extract thin-layer identification chromatography method established by the application is used for quickly, accurately and effectively identifying lithopsida extract. Meanwhile, whether a fluorescent spot is present in a certain Rf value range is observed as an identification point, and is used for distinguishing lithopsida extract and lithopsida extract. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Sample application amount investigation;

[0046] Figure 2 Specificity investigation;

[0047] Figure 3 Different thin-layer plates investigation-Tianjin Silida;

[0048] Figure 4 Different thin-layer plates investigation-Merck;

[0049] Figure 5 Different thin-layer plates investigation-Qingdao Yumin Source;

[0050] Figure 6 Different temperatures-4 DEG C;

[0051] Figure 7 Different temperatures-25 DEG C;

[0052] Figure 8 Different humidities-32%;

[0053] Figure 9 Different humidities-75%;

[0054] Figure 10 Different times of verification;

[0055] Figure 11 Rf value determination for lithopsida extract and lithopsida extract identification points;

[0056] Figure 12 Method result graph of the comparative example 1;

[0057] Figure 13 Method results chart for Comparative Example 2;

[0058] Figure 14 Method results chart for Comparative Example 3;

[0059] Figure 15 Method results chart for Comparative Example 4. DETAILED DESCRIPTION

[0060] In order to further illustrate the present application, the following provides a detailed description of a method for constructing a UPLC characteristic chromatogram of Guanchun medicinal material, decoction piece, standard decoction and formula granules according to the present application in combination with examples.

[0061] Hot plate, ultrasonic machine, thin layer imaging system: CAMAG TLC Visualizer, silica gel GF254 thin layer plate (Qingdao Marine Chemical Plant, batch number: 20180527, Tianjin Silida Technology Co., Ltd., batch number: 191016, Merck, batch number: HX87183353).

[0062] Ethanol, n-butanol, isopropyl alcohol, glacial acetic acid are all analytical pure, and water is ultrapure water (laboratory self-made).

[0063] Gymnadenia conopsea control medicinal material (Shanghai Hongyong Biological Technology Co., Ltd., batch number: 230011-202201). Gymnadenia conopsea extract (Sichuan Xinglv Pharmaceutical Technology Development Co., Ltd., batch number: SY2211001; SY2211002; SY2211003). Eulophia zollingeri extract (Sichuan Xinglv Pharmaceutical Technology Development Co., Ltd., batch number: SY2212001; SY2212002; SY2212003).

[0064] Example 1

[0065] Preparation of test sample solution

[0066] Take the product powder 1 g, add a few drops of ammonia wet, then add chloroform reflux 30 min, filter, dry, the residue dissolved in water 10 ml, through C18 (300 mg) solid phase extraction column, eluted with 30% methanol, discard the eluent, then eluted with methanol 10 ml, collect the eluent, recover the solvent to dry, the residue dissolved in dilute ethanol 5 ml through neutral alumina column, eluted with ethanol 25 ml, collect the eluent, recover the solvent to dry, the residue added 70% ethanol 10 mL ultrasonic 30 min, take the filtrate as the test solution. According to the test method of thin layer chromatography (general rule 0502), take 10 μl of the control drug, 5 μl of the test solution, respectively, and point them on the same silica gel GF254 thin layer plate. Use n-butanol-isopropyl alcohol-water-glacial acetic acid (7:5:1:1) as the developing agent, develop, take out, dry, and observe under ultraviolet light (365 nm). In the test solution chromatogram, the same color fluorescent spots appear at the corresponding positions of the control drug chromatogram.

[0067] Preparation of the control drug solution

[0068] Take 1 g of the control drug of Gynura medica, add 50 ml of water, boil for 1 h, filter, and evaporate to dryness. Then prepare the control drug solution as the test solution.

[0069] Methodological investigation of Example 2

[0070] 2.1 Investigation of the sample size

[0071] Under the above proposed experimental conditions, take 5 μl, 10 μl, 15 μl of the control drug solution of Gynura medica, and 1 μl, 3 μl, 5 μl, 7 μl of the extract solution of Gynura medica, respectively, and point them on the same silica gel G thin layer plate. Develop according to the proposed thin layer chromatography conditions, and observe. The results are shown in Table 1. Figure 1 As can be seen from the figure, when the sample size of the control drug solution is 5-15 μl and the sample size of the test solution is 1-7 μl, the fluorescent spots in the thin layer chromatogram are clear. Therefore, the sample size of the control drug solution is determined to be 5-15 μl, and the sample size of the test solution is determined to be 1-7 μl. Compared with the control drug chromatogram, the test chromatogram shows the same color spots at the corresponding positions.

[0072] Figure 1 Investigation of the sample size. Note: 1-3 are the sample sizes of the control drug of Gynura medica, which are 5 μl, 10 μl, and 15 μl, respectively. 4-7 are the sample sizes of the extract of Gynura medica, which are 1 μl, 3 μl, 5 μl, and 7 μl, respectively.

[0073] 2.2 Investigation of specificity

[0074] Prepare the control drug solution of Gynura medica, the extract solution of Gynura medica, and the negative solution according to the above test solution preparation method, and point them on the same thin layer plate. Develop according to the proposed thin layer chromatography conditions, and observe. The results are shown in Table 2. Figure 2, the results showed that the negative samples had no interference on the test samples of Aspidii Rhizoma and the control drug material, and the method had good specificity. Compared with the control drug material chromatogram, the test sample chromatogram and the control drug material chromatogram showed the same color spots at the corresponding positions.

[0075] Figure 2 Specificity investigation. Note: 1 Note: 1 is a negative solution; 2 is Aspidii Rhizoma control drug material; 3 is Aspidii Rhizoma extract.

[0076] 2.3 Investigation of durability

[0077] 2.3.1 Comparison of different thin layer plates

[0078] Select Tianjin Silida, Merck, Qingdao Haoyang pre-prepared silica gel GF254 plates, respectively, according to the proposed test method for testing, see Figures 3-5 , the results of three brands of thin layer plates can meet the identification requirements, indicating that the method has good durability. Compared with the control drug material chromatogram, the test sample chromatogram and the control drug material chromatogram showed the same color spots at the corresponding positions.

[0079] The results are as follows Figure 3 , Figure 3 Different thin layer plate investigation-Tianjin Silida. Note: 1 Aspidii Rhizoma control drug material; 2 is Aspidii Rhizoma extract. Figure 4 Different thin layer plate investigation-Merck Note: 1 Aspidii Rhizoma control drug material; 2 is Aspidii Rhizoma extract. Figure 5 Different thin layer plate investigation-Qingdao Yuminyuan, Note: 1 Aspidii Rhizoma control drug material; 2 is Aspidii Rhizoma extract.

[0080] 2.3.2 Comparison of different temperatures

[0081] After taking the sample, the thin layer plate was developed at low temperature 4℃ and room temperature 25℃, respectively. From Figures 6-7 It can be seen that the method has good durability to different temperatures. Compared with the control drug material chromatogram, the test sample chromatogram and the control drug material chromatogram showed the same color spots at the corresponding positions. Figure 6 Different temperature 4℃; Note: 1 Aspidii Rhizoma control drug material; 2 is Aspidii Rhizoma extract. Figure 7 Different temperature 25℃; Note: 1 Aspidii Rhizoma control drug material; 2 is Aspidii Rhizoma extract.

[0082] 2.3.3 Comparison of different humidity

[0083] After taking the sample, the thin layer plate was developed at 32% and 75% humidity, respectively, see Figures 8-9 . From the figure, it can be seen that the method has good durability to different humidity. Compared with the control drug material chromatogram, the test sample chromatogram and the control drug material chromatogram showed the same color spots at the corresponding positions. Figure 8Different humidity-32%, note: 1 note: 1 GYPSOPHILA PALLIDA control medicinal materials; 2 GYPSOPHILA PALLIDA extract; Figure 9 Different humidity-75%, note: 1 note: 1 GYPSOPHILA PALLIDA control medicinal materials; 2 GYPSOPHILA PALLIDA extract.

[0084] 2.3.4 Verification

[0085] 3 GYPSOPHILA PALLIDA extract thin layer identification verification, the experimental results are shown in Figure 10 , the results show that, compared with the control medicinal materials atlas, the test product atlas and the control medicinal materials atlas in the corresponding position, the same color spots appear.

[0086] Figure 10 Different batches of verification. Note: 1 note: 1 GYPSOPHILA PALLIDA control medicinal materials; 2-4 GYPSOPHILA PALLIDA extract SY2211001; SY2211002; SY2211003.

[0087] 2.4 GYPSOPHILA PALLIDA extract and GYPSOPHILA SERRATA extract identification point Rf value determination

[0088] GYPSOPHILA PALLIDA extract and GYPSOPHILA SERRATA extract were tested by the same method as in example 1, the results are shown in Figure 11 , Figure 11 GYPSOPHILA PALLIDA extract and GYPSOPHILA SERRATA extract identification point Rf value determination; note: 1 note: 1 GYPSOPHILA PALLIDA control medicinal materials; 2-5 GYPSOPHILA PALLIDA extract SY2211001; SY2211002; SY2211003; 6-8 GYPSOPHILA SERRATA extract.

[0089] The results show that at the position of 0.714, GYPSOPHILA PALLIDA has no fluorescent spot, while its adulterant GYPSOPHILA SERRATA extract has obvious blue fluorescent spot, which can be used as identification point to distinguish GYPSOPHILA PALLIDA from GYPSOPHILA SERRATA extract.

[0090] Comparative example 1

[0091] Take GYPSOPHILA PALLIDA extract 1 g, add concentrated ammonia 2 ml wet, then add 70% ethanol 10 ml, ultrasonic for 30 minutes, take the filtrate, filter to get. Another GYPSOPHILA PALLIDA control medicinal materials 1 g, add water and decoct for 30 min, filter, evaporate to dryness, add concentrated ammonia 2 ml wet, and prepare the control medicinal material solution by the same method. According to the test of thin layer chromatography (general rule 0502), take 5-15 μl of the above two kinds of solution respectively, and point them on the same silica gel GF254 thin layer plate, use toluene-acetone (7:2) as developing agent, develop, take out, dry, and observe under ultraviolet lamp (365 nm). The experimental results are shown in Figure 12 .

[0092] Figure 12The method results of Example 1 are shown in the figure. Note: 1, 3, 5 are the sample amount of Gynura japonica medicinal material 5 μl, 10 μl, 15 μl; 2, 4, 6 are the sample amount of Gynura japonica extract 5 μl, 10 μl, 15 μl.

[0093] The results show that the spots of Gynura japonica extract are consistent with the spots of Gynura japonica medicinal material by using the above thin layer identification method. However, the thin layer chromatography effect is poor, and is not suitable for the identification of Gynura japonica extract.

[0094] Example 2

[0095] Take 1 g of Gynura japonica extract, add 70% ethanol, cold soak overnight, filter, concentrate the filtrate to 1 ml, filter again as the test sample solution. Take 1 g of Gynura japonica medicinal material, add water and decoct for 30 minutes, filter, evaporate to dryness, and prepare the medicinal material solution in the same way as the test sample solution. Test by thin layer chromatography (General Rule 0502), take 5-15 μl of the test sample solution and 5-15 μl of the medicinal material solution, and spot them on the same thin layer silica gel GF254 plate. Use n-butanol-isopropyl alcohol-ammonia water-glacial acetic acid (7:5:1:1) as the developing agent, develop, take out, dry, and observe under ultraviolet light (365 nm). The experimental results are shown in the figure. Figure 13

[0096] Figure 13 The method results of Example 2 are shown in the figure. Note: 1-3 are the sample amount of Gynura japonica medicinal material 5 μl, 10 μl, 15 μl; 4-6 are the sample amount of Gynura japonica extract 5 μl, 10 μl, 15 μl.

[0097] The results show that the spots of Gynura japonica extract are consistent with the spots of Gynura japonica medicinal material by using the above thin layer identification method. However, the thin layer chromatography effect is poor, and is not suitable for the identification of Gynura japonica extract.

[0098] Example 3

[0099] Take 1 g of Gynura japonica extract, add a few drops of ammonia water to wet, then add chloroform and reflux for 30 minutes, filter, dry, dissolve the residue in 10 ml of water, pass through a C 18 (300 mg) solid phase extraction column, elute with 30% methanol 10 ml, discard the eluent, then elute with methanol 10 ml, collect the eluent, recover the solvent to dryness, dissolve the residue in 5 ml of dilute ethanol, pass through a neutral alumina column, elute with ethanol 25 ml, collect the eluent, recover the solvent to dryness, dissolve the residue in 10 mL of 70% ethanol for 30 minutes, take the filtrate as the test sample solution. Take 1 g of Gynura japonica medicinal material, add water 50 ml and decoct for 1 hour, filter, evaporate to dryness, then prepare the medicinal material solution in the same way as the test sample solution. Test by thin layer chromatography (General Rule 0502), take 10 μl of the medicinal material and 5 μl of the test sample, and spot them on the same silica gel G F254 ​Thin layer plate, with n-butanol-isopropanol-ammonia-ice acetic acid (7:5:1:1) as developing agent, development, take out, dry, placed under UV light (365 nm) to view, the experimental results are shown in Figure 14

[0100] Figure 14 The method results of the comparative example 3 are shown in the figure. Note: 1, 3, 5 are the sample amount of Pteridium aquilinum L. 5 μl, 10 μl, 15 μl; 2, 4, 6 are the sample amount of Pteridium aquilinum L. extract 5 μl, 10 μl, 15 μl.

[0101] The results show that the spots of Pteridium aquilinum L. extract are consistent with the spots of Pteridium aquilinum L. by using the above thin layer identification method. However, the thin layer chromatography effect is poor, and it is not suitable for the identification of Pteridium aquilinum L. extract.

[0102] Comparative example 4

[0103] Take 1 g of the product powder, add a few drops of ammonia to wet it, then add chloroform to reflux for 30 minutes, filter, dry, dissolve the residue in 10 ml of water, pass through a C 18 (300 mg) solid phase extraction column, elute with 30% methanol, discard the eluent, then elute with 10 ml of methanol, collect the eluent, recover the solvent to dryness, dissolve the residue in 5 ml of dilute ethanol, then pass through a neutral alumina column, elute with 25 ml of ethanol, collect the eluent, recover the solvent to dryness, ultrasonic the residue in 10 mL of 70% ethanol for 30 minutes, take the filtrate as the test solution. Take 1 g of Pteridium aquilinum L. control drug, add 50 ml of water to decoct for 1 hour, filter, evaporate to dryness, then prepare the control drug solution in the same way as the test solution. Test by thin layer chromatography (General rule 0502), take 10 μl of the control drug and 5 μl of the test solution, spot on the same silica gel G F254 Thin layer plate, with n-butanol-isopropanol-ammonia-ice acetic acid (7:5:1:1) as developing agent, development, take out, dry, placed under UV light (365 nm) to view, the experimental results are shown in

[0104] Figure 15 The method results of the comparative example 4 are shown in the figure. Note: 1-3 are the sample amount of Pteridium aquilinum L. 5 μl, 10 μl, 15 μl; 4-6 are the sample amount of Pteridium aquilinum L. extract 5 μl, 10 μl, 15 μl.

[0105] The results show that the spots of Pteridium aquilinum L. extract are consistent with the spots of Pteridium aquilinum L. by using the above thin layer identification method. However, the thin layer chromatography effect is poor, and it is not suitable for the identification of Pteridium aquilinum L. extract.

[0106] ​The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for identifying an extract of Aspidium and an extract of bitter Aspidium, comprising: A) pretreating the extract of Aspidium to obtain a test sample solution; pretreating the extract of bitter Aspidium to obtain a solution of the extract of bitter Aspidium; the pretreatment of the extract of Aspidium or the extract of bitter Aspidium specifically comprises: a) wetting the extract of Aspidium or the extract of bitter Aspidium with ammonia water, then refluxing with chloroform, filtering, evaporating, dissolving the residue in water, passing through a C18 solid phase extraction column, eluting with 30% methanol, discarding the eluate, then eluting with methanol, and collecting the eluate; b) recovering the solvent from the eluate to dryness, dissolving the residue in dilute ethanol, passing through a neutral alumina column, eluting with ethanol, collecting the eluate, recovering the solvent to dryness, and ultrasonically dissolving the residue in 70% ethanol to obtain the solution; B) boiling the control medicinal material of Aspidium in water to obtain a solution of the control medicinal material; C) detecting the test sample solution, the solution of the extract of bitter Aspidium, and the solution of the control medicinal material by thin layer chromatography, using a silica gel G thin layer plate as the thin layer plate, and using n-butanol-isopropyl alcohol-water-glacial acetic acid as the developing agent, wherein the mass ratio of n-butanol-isopropyl alcohol-water-glacial acetic acid is 7:5:1:1; D) observing under a UV light, if the test sample solution and the solution of the control medicinal material have the same color fluorescent spots at the same Rf, and no fluorescent spots at Rf = 0.64-0.79, then the solution is the extract of Aspidium; if there are fluorescent spots at Rf = 0.64-0.79, then the solution is the extract of bitter Aspidium.

2. The method of claim 1, wherein, The refluxing time in step a) is 20-30 min; the mass / volume ratio of the extract of Aspidium / extract of bitter Aspidium, water, 30% methanol, and methanol is 1 g:10 mL:10 mL:10 mL. The volume ratio of dilute ethanol, ethanol, and 70% ethanol in step b) is 5:25:10; the ultrasonic time is 20-30 min.

3. The method of claim 1, wherein, The mass / volume ratio of the control medicinal material of Aspidium and water in step B) is 1 g:50 mL; The boiling time is 50-70 min.

4. The method of claim 1, wherein, The sample spotting amount of the test sample in the thin layer chromatography is 1-7 μL, and the sample spotting amount of the solution of the control medicinal material is 5-15 μL.

5. The method of claim 1, wherein, The wavelength of the UV light in step D) is 365 nm.

6. The method of claim 1, wherein, The silica gel G thin layer plate in step D) is a GF254 thin layer plate.

7. The method of claim 6, wherein, The observation temperature in step D) is 4-25°C; and the observation humidity is 32%rh-75%rh.

8. The method of claim 1, wherein, The Rf in step D) is 0.714.

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