Method for identifying chaenomeles speciosa and chaenomeles speciosa as traditional Chinese medicinal materials
By using ultrasonic extraction and thin-layer chromatography, smooth-skinned papaya and wrinkled-skinned papaya can be distinguished. 3-O-acetylursolic acid is used as the identification indicator, which solves the problem of distinguishing between smooth-skinned and wrinkled papaya and achieves rapid and accurate identification.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-28
- Publication Date
- 2026-03-10
AI Technical Summary
Existing technologies are insufficient to effectively distinguish between smooth-skinned and wrinkled-skinned papayas, especially after the medicinal materials are powdered or made into traditional Chinese medicines. Furthermore, there is a phenomenon of mixing them in the market, and traditional methods of identification based on physical characteristics are highly subjective.
Ultrasonic extraction and thin-layer chromatography were used to identify the compound 3-O-acetylursolic acid. The specific developing solvent and colorimetric method of thin-layer chromatography were used to distinguish between smooth-skinned and wrinkled-skinned papaya. The structure of the compound was confirmed by nuclear magnetic resonance and mass spectrometry.
It enables rapid and accurate identification of smooth-skinned and wrinkled-skinned papayas. The operation is simple and involves few steps, has wide applicability, and the compounds to be identified are readily available.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a method for distinguishing two kinds of traditional Chinese medicinal materials, Chaenomeles speciosa Chaenomeles sinensis (Thouin Koehne) and Chaenomeles lagenaria Chaenomeles speciose (Sweet) Nakai. The method can distinguish the two kinds of traditional Chinese medicinal materials, and has the characteristics of convenience, ease and speed. BACKGROUND
[0002] The prior art shows that Chaenomeles speciosa Chaenomeles sinensis (Thouin Koehne) and Chaenomeles lagenaria Chaenomeles speciose (Sweet) Nakai have high similarity. Chaenomeles speciosa was once recorded in the Chinese Pharmacopoeia (1977 edition) as one of the origins of traditional Chinese medicine Chaenomeles speciosa, but was not recorded in subsequent editions of the Chinese Pharmacopoeia, and was only included in local traditional Chinese medicinal material standards in some regions of China such as Hunan, Sichuan and Shandong as a traditional medicinal material. However, the traditional Chinese medicine Chaenomeles speciosa currently recorded in the Chinese Pharmacopoeia (2020 edition) is Chaenomeles lagenaria. Although the dried fruits can be preliminarily judged by whether the skin is wrinkled or not, the traditional identification method is highly subjective. Moreover, it is difficult to distinguish them once the medicinal materials are ground into powder or made into Chinese patent medicines, and there are also cases of mixing the two in the market. In addition, there are many similarities between the two in terms of medicinal value and functional indications, which is one of the reasons for confusion. Therefore, it is of great significance to distinguish the two kinds of medicinal materials.
[0003] Based on the current situation and basis of the prior art, the present application provides a method for distinguishing two kinds of traditional Chinese medicinal materials, Chaenomeles speciosa SUMMARY
[0004] The purpose of the present application is to provide a method for distinguishing two kinds of traditional Chinese medicinal materials, Chaenomeles speciosa Chaenomeles sinensis (Thouin Koehne) and Chaenomeles lagenaria Chaenomeles speciose (Sweet) Nakai based on the current situation and basis of the prior art. The method can distinguish the two kinds of traditional Chinese medicinal materials by ultrasonic extraction and thin layer chromatography. The method has the characteristics of convenience, ease and speed.
[0005] In the method, first, the compound that plays a major role in identification is found by the method of phytochemical extraction and separation, and the compound is determined to be 3- O - acetyl ursolic acid by nuclear magnetic resonance and mass spectrometry. The extract sample solution is developed by thin layer chromatography using optimized conditions, and 3- O- Acetyl ursolic acid reference substance as a control, by observing and comparing whether the TLC chart of Chaenomeles speciosa and Chaenomeles lagenaria contains 3- O - The spot of acetyl ursolic acid realizes the distinction of the two, Specifically, Firstly, since the compounds that can play a role in identification in Chaenomeles speciosa and Chaenomeles lagenaria are unknown, it is necessary to find the compounds that can distinguish the two. The specific steps include: (1) Pulverization: pulverize the Chaenomeles speciosa and Chaenomeles lagenaria decoction pieces into 100-200 mesh powders; (2) Preparation of sample solution: take 1.0 g of Chaenomeles speciosa and Chaenomeles lagenaria powders in (1), add 10 mL of chloroform, ultrasonic extraction (40 kHz) for 30 minutes, then filter, and the residue obtained by distillation under reduced pressure is dissolved in 2 mL of a mixture of methanol-chloroform (1:3) to obtain a sample solution for thin layer chromatography analysis; (3) Thin layer chromatography: according to the identification method of "Papaya" in the Chinese Pharmacopoeia (2020 edition), develop the Chaenomeles lagenaria and Chaenomeles speciosa samples at the same time. The specific method is as follows: take 1-2 μL of the above samples, respectively, and point them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-acetone-formic acid (6:0.5:1:0.1) as the developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and observe under daylight and ultraviolet light (365 nm), respectively. By observing and comparing the thin layer chromatography information between Chaenomeles speciosa and Chaenomeles lagenaria, it can be found that the Chaenomeles lagenaria extract sample has a color spot at R f =0.38, which is different from the Chaenomeles speciosa sample, which is purple red under daylight, while the Chaenomeles speciosa does not have a corresponding color spot at the corresponding position; (4) Preparation of identification points: Based on the results obtained in step (3) above, the wrinkled papaya sample 4 was separated and prepared. 10 g of sample was added to 50 mL of chloroform, and ultrasonically extracted (40 kHz) for 30 minutes. After filtration, the residual solid obtained by vacuum distillation of the filtrate was dissolved in 5 mL of a methanol-chloroform (1:3) mixed solution. The same enrichment method was used 3 times, and the obtained samples were combined. Thin-layer chromatography was performed using a developing solvent of cyclohexane-ethyl acetate-acetone-formic acid = 9:1:2:0.1. Silica gel corresponding to the purple-red spots was scraped off. After multiple enrichment, the compounds were recovered by flash column chromatography. The obtained compounds were still a mixture. A second thin-layer chromatography (TLC) was performed using a toluene-ethyl acetate-acetic acid ratio of 5:1:0.1 as the developing solvent. Silica gel corresponding to the purple-red spots was scraped off, and the enriched compounds were obtained as identification points. After multiple enrichments, the compounds were recovered by flash column chromatography elution, ultimately yielding relatively pure compounds. Their structures were identified using various spectroscopic methods. Molecular weight was determined by high-performance liquid chromatography-high-resolution mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR) was used for analysis. 1 H NMR and 13 The structure was identified by C10 NMR. The results were obtained by high-resolution mass spectrometry (ESI). m / z 499.3777 [M+H] + 521.3597 [M+Na] + This indicates that its molecular formula is C. 32 H 50 O4; Nuclear magnetic resonance data: 13 C NMR (151 MHz, CDCl3) δ 182.90, 171.19, 138.10, 125.89, 81.09, 55.44, 48.09, 47.64, 47.61, 42.09, 39.65, 39.18, 38.98, 38.42, 37.84, 37.05, 36.86, 33.00, 30.75, 29.85, 28.23, 24.24, 23.72, 23.44, 21.46, 21.33, 18.32, 17.21, 17.17, 16.86, 15.69, 14.27; 1 H NMR (600 MHz, CDCl3) δ 5.24 (t, J = 3.7 Hz, 1H), 4.50 (m, 1H), 2.18 (dd, J= 11.4, 1.7 Hz, 1H), 2.04 (s, 3H), 1.30 (s, 3H), 1.07, 0.95, 0.94, 0.86,0.84, 0.78 (s, 3H×6). The above data are consistent with compound 3- O - acetyl ursolic acid data, so the identification compound of Chaenomeles speciosa and Chaenomeles lagenaria is 3- O - acetyl ursolic acid, as Figure 2 shown.
[0006] More specifically, Based on the above results, the method is further improved, and the method for identifying traditional Chinese medicinal materials Chaenomeles speciosa and Chaenomeles lagenaria is obtained, and the steps thereof include: (1) crushing: crushing the traditional Chinese medicinal materials or decoction pieces of Chaenomeles speciosa and Chaenomeles lagenaria into 100-200 mesh powders for extraction; (2) preparation of sample solution and control solution: taking 1.0 g of the powders of Chaenomeles speciosa and Chaenomeles lagenaria in (1), adding 10 mL of chloroform, and ultrasonically extracting (40 kHz) for 30 minutes, then filtering, the residue obtained by distillation under reduced pressure is dissolved in 2 mL of a methanol-chloroform (1:3) mixed solution to obtain a sample solution for thin layer chromatography analysis. The control solution is prepared by taking an appropriate amount of 3- O - acetyl ursolic acid control, and dissolving it in methanol to obtain a 0.5 mg / mL methanol solution; (3) thin layer chromatography: taking 5 μL of each of the above samples, and spotting them on the same silica gel G thin layer plate, using cyclohexane-ethyl acetate-acetone-formic acid (9:1:2:0.1) as the developing agent, developing, taking out, air-drying, spraying with 10% sulfuric acid ethanol solution, and heating at 105°C until the spots are clearly colored, and observing under daylight and ultraviolet light (365 nm), respectively; (4) comparison of results: in the thin layer chromatograms of Chaenomeles speciosa and Chaenomeles lagenaria, if the same R O - acetyl ursolic acid as the control appears, the sample is Chaenomeles lagenaria, otherwise it is Chaenomeles speciosa. f
[0007] The present application has the following advantages: (1) It can effectively solve the problem of difficulty in identifying the easily confused products of Chaenomeles speciosa and Chaenomeles lagenaria.
[0008] (2) The method adopted by the present application has few steps, simple operation, and high accuracy. The difference between the two can be found by observing the thin layer chromatogram, and the easily confused samples can be quickly identified.
[0009] (3) The identification compounds used are readily available reference standards on the market, making them easy to obtain and widely applicable. Attached Figure Description
[0010] Figure 1 The results were obtained using the TLC identification method for "Papaya" in Part I of the 2020 edition of the Chinese Pharmacopoeia. Q represents oleanolic acid reference solution, X represents ursolic acid reference solution, sample 0 is the reference material sample of papaya, samples 1-4 and 9 are wrinkled papaya samples, and samples 5-8 and 10-13 are smooth papaya samples.
[0011] Figure 2 It is identification point 3- O The structure of acetylursolic acid compounds.
[0012] Figure 3 This is a thin-layer chromatogram of a papaya sample with cyclohexane-ethyl acetate-acetone-formic acid as the developing solvent: 9:1:2:0.1, where Q represents oleanolic acid reference standard, X represents ursolic acid reference standard, and X-Ac represents 3- O - Acetylursolic acid reference standard; Sample 0 is the papaya reference medicinal material sample.
[0013] Figure 4 This is a thin-layer chromatogram of a papaya sample with toluene-ethyl acetate-acetic acid as the developing solvent: 5: 1: 0.1, where Q represents oleanolic acid reference standard, X represents ursolic acid reference standard, and X-Ac represents 3- O - Acetylursolic acid reference standard; Sample 0 is the papaya reference medicinal material sample. Detailed Implementation
[0014] The reagents used in this invention are all conventional reagents, all of which can be purchased from reagent manufacturers, wherein 3- O - Acetylursolic acid is a reference compound with high purity.
[0015] Example 1: Identification of smooth-skinned and wrinkled-skinned papaya samples Ten batches each of wrinkled-skinned and smooth-skinned papaya medicinal materials were collected, and their information is shown in Table 1. These 20 samples were ground into powder of 100-200 mesh. 1.0 g of each sample was added to 10 mL of chloroform, and the mixture was ultrasonically extracted (40 kHz) for 30 minutes, followed by filtration. The filtrate was then distilled under reduced pressure to obtain the residual solid. 2 mL of a methanol-chloroform (1:3) mixture was added to dissolve the solid, yielding the sample solution for thin-layer chromatography analysis. The papaya reference material was prepared using the same method. Ursolic acid, oleanolic acid, and 3- O Acetylursolic acid reference standard was prepared into a 1 mg / mL methanol solution.
[0016]
[0017] Table 1. Sample information of smooth-skinned papaya and wrinkled-skinned papaya
[0018] Take 2 μL of the above sample solution and each reference solution, and spot them separately on the same silica gel G thin-layer plate. Develop with cyclohexane-ethyl acetate-acetone-formic acid (9:1:2:0.1) as developing solvent 1 and toluene-ethyl acetate-acetic acid (5:1:0.1) as developing solvent 2. After development, remove the plate and allow it to stand and dry. Spray with 10% sulfuric acid ethanol solution and heat at 105℃ until the spots are clearly visible. Observe under sunlight and ultraviolet light (365 nm) respectively. The results are as follows. Figure 3 and Figure 4 As shown, Q represents oleanolic acid reference standard, X represents ursolic acid reference standard, and X-Ac represents 3- O - Acetylursolic acid reference standard; Sample 0 is the papaya reference medicinal material sample.
[0019] The results showed that, Figure 3 and Figure 4 As shown, only the left sample in each image contains 3- O -Acetylursolic acid compounds were observed in all samples from wrinkled papaya; no 3-acetylursolic acid compounds were observed in the samples on the right. O - Acetylursolic acid spots were observed in these samples, all of which were smooth-skinned papaya samples. Therefore, the results indicate that this method can rapidly and effectively identify and distinguish between smooth-skinned and wrinkled-skinned papaya samples.
Claims
1. A method for identifying Chaenomeles speciosa and Chaenomeles lagenicaulia, characterized in that, The method comprises the following steps: (1) crushing: crushing the traditional Chinese medicinal materials Chaenomeles speciosa and Chaenomeles lagenaria into powder for extraction; (2) preparing sample solution and control solution: taking the Chaenomeles speciosa and Chaenomeles lagenaria powder in (1) and adding chloroform, extracting by ultrasonic and then filtering to obtain filtrate, and then distilling the residual solid under reduced pressure to dissolve in a methanol-chloroform mixed solution to obtain a sample solution for thin layer chromatography analysis; (3) thin layer chromatography analysis: taking the above sample and spotting on the same silica gel G thin layer plate, using cyclohexane-ethyl acetate-acetone-formic acid as developing agent 1, or toluene-ethyl acetate-acetic acid as developing agent 2, developing, taking out, drying, spraying with 10% sulfuric acid ethanol solution, heating until the spots are clear, and then observing under daylight and ultraviolet light respectively; Preparation of the reference solution: take 3- O - acetyl ursolic acid reference, made into a methanol solution; 2. The method according to claim 1, wherein, (4) Result comparison: In the thin layer chromatogram of Chaenomeles speciosa and C. splendida, the same color spots as the control 3- O - Same R of acetyl ursolic acid f If the same color spots as the control appear at the position, the sample is C. splendida, otherwise it is C. speciosa. in step (1), the Chaenomeles speciosa and Chaenomeles lagenaria decoction pieces are crushed into 100-200 mesh powder for extraction; in step (3), 5 μL of the above sample is taken and spotted on the same silica gel G thin layer plate, using cyclohexane-ethyl acetate-acetone-formic acid (9: 1: 2: 0.1) as developing agent 1, or toluene-ethyl acetate-acetic acid = 5: 1: 0.1 as developing agent 2, developing, taking out, drying, spraying with 10% sulfuric acid ethanol solution, heating at 105°C until the spots are clear, and then observing under daylight and ultraviolet light (365 nm) respectively; In the step (2): take 1.0 g of Malus prunifolia (Wild.) Borkh. and Malus coronaria (L.) Moench powder in (1), add 10 mL of chloroform, ultrasonic extraction (40 kHz) for 30 minutes, then filter, and the obtained residue solid is dissolved in 2 mL of methanol-chloroform (1:3) mixed solution to obtain a sample solution for thin layer chromatography analysis; in the preparation of the control solution: take 3- O - acetyl ursolic acid control, prepare a 0.5 mg / mL methanol solution; in the thin layer chromatography analysis, the thin layer developing method is used, and the developing agent type and ratio are: cyclohexane-ethyl acetate-acetone-formic acid (9: 1: 2: 0.1); toluene-ethyl acetate-acetic acid = 5: 1: 0.
1. (4) Result comparison: In the thin layer chromatogram of Chaenomeles speciosa and C. splendida, the same Rf value of the sample and the reference substance 3- O - same Rf value of acetyl ursolic acid f If the same color spot as the reference substance appears at the position, the sample is C. splendida, otherwise it is C. speciosa.
3. The method for identifying Chaenmiles lagenicaulis and Chaenmiles macrolepis according to claim 1, characterized in that, 4. The method for identifying Chaenomelis Fructus and Malus prudum according to claim 1, characterized in that, 3- O Acetyl ursolic acid was used as the key index compound to identify the medicinal materials of Chaenomeles speciosa and C. angustidentata.