Method for extracting total bufadienolide in bufonis marini medicinal materials and application

Abstract

The application discloses a method for extracting bufadienolides in bufonis medicinals and application thereof, and belongs to the technical field of effective component extraction of traditional Chinese medicines. The method uses a deep eutectic solvent (DES) as an extracting agent, and solves the problems of low purity of bufadienolides, poor extraction efficiency and complicated process in traditional water extraction or alcohol extraction methods by optimizing the solvent composition, material ratio and extraction process. The method has high extraction efficiency, can significantly improve the total content of bufalin, resibufogenin and cinobufagin in the extract, and is simple in process, environment-friendly and easy for large-scale industrial production, thereby providing a new approach for efficient utilization of bufonis medicinals.

Description

Technical Field

[0001] This application belongs to the field of extraction technology of effective components of traditional Chinese medicine, and specifically relates to a method and application for extracting total bufotalin lactones from toad-derived medicinal materials. Background Technology

[0002] Toad-derived medicinal materials include toad skin and toad venom. Toad skin is the dried skin of the Chinese giant toad or the black-rimmed toad (Bufo gargarizans), animals belonging to the Bufonidae family. Also known as toad skin or flea skin, it is obtained by capturing the toad in summer and autumn, peeling off the entire skin, and drying it. As a traditional Chinese medicine, toad skin was first recorded in the *Benjing Fengyuan*. It is pungent, cool, and slightly toxic, possessing the effects of clearing heat and detoxifying, promoting diuresis and relieving bloating. Clinically, it is often used to treat diseases such as tumors, carbuncles, and chronic bronchitis. Modern medical research shows that the total bufotalene lactones in toad skin (mainly including bufotalin, bufotalin ligand, and bufotalin) are its core effective anti-tumor components, showing significant inhibitory effects on malignant tumors such as colon cancer, liver cancer, and pancreatic cancer, and can also reduce the toxic side effects of radiotherapy and chemotherapy.

[0003] Currently, the extraction methods for bufotalene total lactones from toad skin mainly fall into two categories: one is direct water extraction followed by alcohol precipitation, which results in extremely low purity of active ingredients and a large number of impurities in the extract; the other is ethanol extraction followed by purification with macroporous adsorption resin, which can increase the alkaloid content, but the content of key components (such as bufotalin and bufotalin) in the total bufotalene lactones is still less than 0.1%. Existing patented technologies (such as patent applications with publication numbers CN101209264A, CN101176739A, and CN108836982A) can increase the content of active ingredients, but the highest total content of bufotalin, bufotalin, and bufotalin is still low, and the process is cumbersome (such as multiple column chromatography), making it difficult to meet the needs of large-scale industrial production. Summary of the Invention

[0004] To address the aforementioned issues, in a first aspect, this application proposes a method for extracting total bufotalene lactones from toad-derived medicinal materials. The method uses a eutectic solvent as the extractant to extract the toad-derived medicinal materials, followed by extraction and drying to obtain a total bufotalene lactone extract.

[0005] The eutectic solvent is formed by dissolving hydrogen bond acceptors and hydrogen bond donors in water.

[0006] Furthermore, the toad-derived medicinal materials include toad skin and / or toad venom.

[0007] Furthermore, the method includes the following steps:

[0008] S1: After cutting the toad-like medicinal material into small pieces, add a eutectic solvent for extraction. After filtering the extract, the toad skin residue and filtrate are obtained. The filtrate is then extracted with an extraction solvent to obtain an extraction solvent layer and a eutectic solvent layer.

[0009] S2: Mix the toad skin filter residue with the eutectic solvent layer and extract repeatedly. Repeat the operation of S1 and collect the extraction solvent layer.

[0010] S3: Combine the extraction solvent layers from each extraction process, dry them, and then recover the extraction solvent under reduced pressure to obtain the total bufos sterene lactone extract.

[0011] Furthermore, the hydrogen bond acceptor is selected from one or more of choline hydrochloride, betaine hydrochloride, tetramethylammonium chloride, and tetraethylammonium bromide;

[0012] The hydrogen bond donor is selected from one or more of urea, imidazole, malic acid, citric acid, acetamide, 1,3-dimethylurea, glycerol, propylene glycol, magnesium chloride, calcium chloride, and barium chloride.

[0013] Furthermore, in the eutectic solvent, the molar ratio of hydrogen bond acceptor, hydrogen bond donor and water is 1:(1~20):(0~100);

[0014] The mass ratio of the toad-like medicinal material to the eutectic solvent is 1g:5~50g.

[0015] Furthermore, the extraction method is heating extraction or ultrasound-assisted extraction, and the number of extractions is 2-4 times;

[0016] The extraction temperature is 60~120℃, and the process is continuously stirred, with each stirring session lasting 2~3 hours; the preferred extraction temperature is 100~120℃.

[0017] Furthermore, the extraction solvent is selected from one of ethyl acetate, dichloromethane, chloroform, and n-butanol.

[0018] Furthermore, anhydrous sodium sulfate is used for drying during the drying process, and the drying time is 2-24 hours.

[0019] Secondly, this application proposes a bufotenol total lactone extract obtained by the method described above.

[0020] Thirdly, this application proposes the application of the method for extracting total bufotalene lactones from toad medicinal materials in the extraction of bufotalin, bufotalin and / or bufotalin esters from toad medicinal materials.

[0021] Compared with the prior art, this application has the following advantages:

[0022] This application employs a eutectic solvent (DES) to extract the active ingredients from toad-derived medicinal materials. Compared to traditional organic solvents, DES can efficiently dissolve the active ingredients of traditional Chinese medicine at lower temperatures while protecting the active ingredients from degradation. By selecting the type of eutectic solvent and optimizing extraction parameters, this application obtains a total bufotalene lactone extract. The content of bufotalin, bufotalin ligand, and bufotalin in the extract can be controlled by varying the composition of the DES and the extraction temperature. Extraction can be performed according to the specific needs of the target substances. Compared to traditional organic solvent extraction methods, eutectic solvent extraction not only improves extraction efficiency but is also more environmentally friendly.

[0023] This application utilizes the highly efficient dissolving properties of DES and a multi-stage extraction process to further improve the extraction rate of active ingredients, while shortening the extraction time to 5-8 hours (compared to over 9 hours for traditional alcohol extraction). Furthermore, DES has low toxicity and is recyclable and reusable, and the extraction solvent can be recovered under reduced pressure, reducing organic solvent emissions and meeting production requirements. The entire process eliminates the need for complex column chromatography steps; purification can be completed simply through extraction, drying, and purification, requiring minimal equipment and facilitating large-scale industrial production.

[0024] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The high-performance liquid chromatogram of a single bufotalin standard in an embodiment of this application is shown.

[0027] Figure 2 The high-performance liquid chromatogram of a single bufotoxin ligand standard in an embodiment of this application is shown.

[0028] Figure 3 The high-performance liquid chromatogram of a single bufotoxin-based standard in an embodiment of this application is shown.

[0029] Figure 4 The high-performance liquid chromatogram of the mixed standard of bufotoxin, bufotoxin ligand, and bufotoxin ligand in the embodiments of this application is shown.

[0030] Figure 5 The high-performance liquid chromatogram of the bufotalene total lactone extract from Example 1 of this application is shown;

[0031] Figure 6 The high-performance liquid chromatogram of the bufotalene total lactone extract from Example 2 of this application is shown;

[0032] Figure 7 The high-performance liquid chromatogram of the bufotenol total lactone extract from Example 3 of this application is shown;

[0033] Figure 8 The high-performance liquid chromatogram of the bufotenol total lactone extract from Example 4 of this application is shown;

[0034] Figure 9 The high-performance liquid chromatogram of the bufotenol total lactone extract from Example 5 of this application is shown;

[0035] Figure 10 The high-performance liquid chromatogram of the bufotenol total lactone extract from Example 6 of this application is shown;

[0036] Figure 11 The high-performance liquid chromatogram of the bufotalene total lactone extract from Example 7 of this application is shown;

[0037] Figure 12 The high-performance liquid chromatogram of the bufos sterene total lactone extract from Example 8 of this application is shown. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] This application provides a method for extracting total bufotalene lactones from toad-derived medicinal materials. The extraction method is as follows:

[0040] S1: After cutting the toad-like medicinal material into small pieces, add a eutectic solvent for extraction. After filtering the extract, the toad skin residue and filtrate are obtained. The filtrate is then extracted with an extraction solvent to obtain an extraction solvent layer and a eutectic solvent layer.

[0041] S2: Mix the toad skin filter residue with the eutectic solvent layer and extract repeatedly. Repeat the operation of S1 and collect the extraction solvent layer.

[0042] S3: Combine the extraction solvent layers from each extraction process, dry them, and then recover the extraction solvent under reduced pressure to obtain the total bufos sterene lactone extract.

[0043] Specifically, in step S1, the ratio of toad-derived medicinal materials to DES is 1g: 5-50g; the toad-derived medicinal materials include toad skin and / or toad venom.

[0044] The eutectic solvent is formed by dissolving hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) in water in a certain proportion. In the embodiments of this application, HBA is selected from one or more of choline hydrochloride, betaine hydrochloride, tetramethylammonium chloride, and tetraethylammonium bromide; HBD is selected from urea, imidazole, malic acid, citric acid, acetamide, 1,3-dimethylurea, glycerol, and propylene glycol; or selected from metal chlorides or hydrates such as magnesium chloride, calcium chloride, and barium chloride (dissolved by heating).

[0045] The molar ratio of HBA:HBD:H2O is 1:1~20:0-100;

[0046] The extraction method in step S1 is heating extraction or ultrasound-assisted extraction, with an extraction temperature of 60-120℃;

[0047] The extraction solvents are selected from ethyl acetate, dichloromethane, chloroform, and n-butanol.

[0048] The above method is illustrated below using toad skin as an example, but this application is also applicable to the extraction of bufotalin total lactones from toad venom and other toad-derived medicinal materials.

[0049] Example 1

[0050] S1: Cut 50g of dried toad skin into small pieces, add eutectic solvent (choline hydrochloride 13.9g, urea 12g, water 250g), stir for 3 hours at 60℃, filter to obtain toad skin residue and filtrate, extract the filtrate twice with ethyl acetate to obtain ethyl acetate layer (i.e., extraction solvent layer) and eutectic solvent layer.

[0051] S2: The toad skin filter residue and the eutectic solvent layer were stirred at 60°C for 2 hours, and then filtered to obtain toad skin filter residue and filtrate. The filtrate was extracted twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer.

[0052] S3: Repeat S2 to obtain the ethyl acetate layer for later use;

[0053] S4: Combine the above ethyl acetate layer and dry with anhydrous sodium sulfate for 2 hours, then recover the solvent under reduced pressure to obtain the total lactone extract of bufos sterene.

[0054] The contents of bufotoxin, bufotoxin ligand, and bufotoxin ligand in the total lactone extract of bufosterene were determined by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows:

[0055] Mobile phase A: 0.5% potassium dihydrogen phosphate solution (weigh 5.001 g of potassium dihydrogen phosphate into 1000 mL of water, and adjust the pH to 3.2 with phosphoric acid); Mobile phase B: pure acetonitrile.

[0056] Elution gradient: Mobile phase A and pure acetonitrile were eluted isocratically at a volume ratio of 50:50 for 30 min.

[0057] Column temperature: 40℃.

[0058] Detection wavelength: 296nm.

[0059] The single standards of bufotoxin, bufotoxin ligand, and bufotoxin ligand were determined according to the above chromatographic conditions, and the chromatograms are shown below. Figure 1 , Figure 2 and Figure 3 As shown in the figure, the peak times of bufotoxin, bufotoxin ligand, and bufotoxin ligand are 8.640, 11.623, and 12.133 min, respectively. When these three standards were mixed and injected at concentrations of 9.36, 8.23, and 9.46 μg / mL, the resulting chromatograms are shown in the figure. Figure 4 As shown.

[0060] The total lactone extract of bufotalene was dissolved in methanol and transferred to a 10 mL volumetric flask. 1 mL was accurately measured, diluted 200-fold with the mobile phase, filtered, and then injected in 20 μL. Chromatographic analysis was performed as described above, and the resulting chromatogram is shown below. Figure 5 As shown, chromatographic peaks appeared at retention times of 7.710, 10.463, and 11.150 min, with relative peak areas of 41.01%, 26.31%, and 32.68%, respectively. The content of bufotoxin, bufotoxin glycoside, and bufotoxin ester in the total bufotoxin lactone extract was determined to be 6.46 mg, 3.14 mg, and 4.46 mg, respectively, using the internal standard method.

[0061] Example 2

[0062] S1: Cut 50g of dried toad skin into small pieces, add eutectic solvent (choline hydrochloride 13.9g, magnesium chloride hexahydrate 41g, water 250g), stir for 3 hours at 60℃, filter to obtain toad skin residue and filtrate, extract the filtrate twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer.

[0063] S2: The toad skin filter residue and the eutectic solvent layer were stirred at 60°C for 2 hours, and then filtered to obtain toad skin filter residue and filtrate. The filtrate was extracted twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer.

[0064] S3: Repeat S2 to obtain the ethyl acetate layer for later use;

[0065] S4: Combine the above ethyl acetate layer and anhydrous sodium sulfate, dry for 2 hours, and recover the solvent under reduced pressure to obtain the total lactone extract of bufos sterene. Dissolve in methanol and transfer to a 50 mL volumetric flask. Accurately measure 1 mL, dilute 80 times with the mobile phase, filter, and inject 20 μL.

[0066] The contents of bufotalin, bufotalin ligand, and bufotalin in the total lactone extract of bufosterene were detected according to the chromatographic detection conditions in Example 1. The results are as follows: Figure 6 As shown, chromatographic peaks appeared at retention times of 7.707, 10.470, and 11.153 min, with relative peak areas of 44.57%, 23.59%, and 31.83%, respectively. The content of bufotoxin in the total lactone extract of bufosterene was determined to be 2.28 mg, the content of bufotoxin toxicant was 1.16 mg, and the content of bufotoxin ester was 1.76 mg using the internal standard method.

[0067] Example 3

[0068] S1: Cut 50g of dried toad skin into small pieces, add eutectic solvent (choline hydrochloride 13.9g, glycerol 18g, water 250g), stir for 3 hours at 60℃, filter to obtain toad skin residue and filtrate, extract the filtrate twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer.

[0069] S2: The toad skin filter residue and the eutectic solvent layer were stirred at 60°C for 2 hours, and then filtered to obtain toad skin filter residue and filtrate. The filtrate was extracted twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer.

[0070] S3: Repeat S2 to obtain the ethyl acetate layer for later use;

[0071] S4: Combine the above ethyl acetate layer and anhydrous sodium sulfate, dry for 2 hours, and recover the solvent under reduced pressure to obtain the total lactone extract of bufossone. Dissolve in methanol and transfer to a 50 mL volumetric flask. Accurately measure 1 mL, dilute 100 times with the mobile phase, filter, and inject 20 μL.

[0072] The contents of bufotalin, bufotalin ligand, and bufotalin in the total lactone extract of bufosterene were detected according to the chromatographic detection conditions in Example 1. The results are as follows: Figure 7 As shown, chromatographic peaks appeared at retention times of 7.667, 10.440, and 11.153 min, with relative peak areas of 51.07%, 18.98%, and 29.95%, respectively. The content of bufotoxin in the total lactone extract of bufotoxin was determined to be 2.85 mg, the content of bufotoxin serotonin was 0.8 mg, and the content of bufotoxin ester was 1.45 mg using the internal standard method.

[0073] Example 4

[0074] The dried toad skin was extracted according to the method in Example 3, except that 18g of glycerol was replaced with 22g of calcium chloride. The extracted bufotalene total lactone extract was dissolved in methanol and transferred to a 100mL volumetric flask. 1mL was accurately measured, diluted 100-fold with the mobile phase, filtered, and then injected in 20μL.

[0075] The contents of bufotalin, bufotalin ligand, and bufotalin in the total lactone extract of bufosterene were detected according to the chromatographic detection conditions in Example 1. The results are as follows: Figure 8 As shown, chromatographic peaks appeared at retention times of 7.693, 10.487, and 11.170 min, with relative peak areas of 54.22%, 17.17%, and 28.61%, respectively. The content of bufotoxin in the total lactone extract of bufotoxin was determined to be 3.3 mg, the content of bufotoxin serotonin was 0.8 mg, and the content of bufotoxin ester was 1.5 mg using the internal standard method.

[0076] Example 5

[0077] The dried toad skin was extracted according to the method in Example 3, except that 18g of glycerol was replaced with 41g of barium chloride. The extracted bufotenol total lactone extract was dissolved in methanol and transferred to a 100mL volumetric flask. 1mL was accurately measured, diluted 80 times with the mobile phase, filtered, and then injected into a 20μL sample.

[0078] The contents of bufotalin, bufotalin ligand, and bufotalin in the total lactone extract of bufosterene were detected according to the chromatographic detection conditions in Example 1. The results are as follows: Figure 9 As shown, chromatographic peaks appeared at retention times of 7.693, 10.480, and 11.143 min, with relative peak areas of 46.65%, 22.39%, and 30.97%, respectively. The content of bufotoxin, bufotoxin glycoside, and bufotoxin ester in the total bufotoxin lactone extract was determined to be 1.84 mg by the internal standard method.

[0079] Example 6

[0080] S1: Cut 50g of dried toad skin into small pieces, add eutectic solvent (choline hydrochloride 13.9g, urea 12g, water 250g), stir for 3 hours at 120℃, filter to obtain toad skin residue and filtrate, extract the filtrate twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer after ethyl acetate extraction.

[0081] S2: The toad skin filter residue and the eutectic solvent layer were stirred at 60°C for 2 hours, and then filtered to obtain toad skin filter residue and filtrate. The filtrate was extracted twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer.

[0082] S3: Repeat S2 to obtain the ethyl acetate layer for later use;

[0083] S4: Combine the above ethyl acetate layer and anhydrous sodium sulfate, dry for 2 hours, and recover the solvent under reduced pressure to obtain the total lactone extract of bufos sterene. Dissolve in methanol and transfer to a 20 mL volumetric flask. Accurately measure 1 mL, dilute 40 times with the mobile phase, filter, and inject 20 μL.

[0084] The contents of bufotalin, bufotalin ligand, and bufotalin in the total lactone extract of bufosterene were detected according to the chromatographic detection conditions in Example 1. The results are as follows: Figure 10 As shown, chromatographic peaks appeared at retention times of 7.853, 10.457, and 10.967 min, with relative peak areas of 85.31%, 2.07%, and 12.62%, respectively. The content of bufotoxin in the total lactone extract of bufotoxin was determined to be 15.8 mg, the content of bufotoxin toxicant was 0.28 mg, and the content of bufotoxin ester was 2.03 mg using the internal standard method.

[0085] Example 7

[0086] S1: Cut 50g of dried toad skin into small pieces, add eutectic solvent (28g of choline hydrochloride, 80g of magnesium chloride hexahydrate, and 600g of water), stir for 3 hours at 100℃, filter to obtain toad skin residue and filtrate, extract the filtrate twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer after ethyl acetate extraction.

[0087] S2: The eutectic solvent layer after extraction of toad skin residue and ethyl acetate is stirred at 60°C for 2 hours, and the ethyl acetate extraction layer is reserved.

[0088] S3: Repeat S2 to obtain the ethyl acetate layer for later use;

[0089] S4: Combine the above ethyl acetate layer and anhydrous sodium sulfate, dry for 2 hours, and recover the solvent under reduced pressure to obtain the total lactone extract of bufos sterene. Dissolve in methanol and transfer to a 100 mL volumetric flask. Accurately measure 1 mL, dilute 20 times with the mobile phase, filter, and inject 20 μL.

[0090] The contents of bufotalin, bufotalin ligand, and bufotalin in the total lactone extract of bufosterene were detected according to the chromatographic detection conditions in Example 1. The results are as follows: Figure 11As shown, chromatographic peaks appeared at retention times of 7.860, 10.473, and 10.973 min, with relative peak areas of 66.03%, 2.59%, and 31.38%, respectively. The content of bufotoxin in the total lactone extract of bufotoxin was determined to be 13.08 mg, the content of bufotoxin serotonin was 0.38 mg, and the content of bufotoxin ester was 5.4 mg using the internal standard method.

[0091] Examples 1, 6, and 7 show that increasing the extraction temperature significantly increases the bufotoxin content. This is likely because the bufotoxin ligand and the bufotoxin group contain epoxy groups, with the bufotoxin group containing an acetyl group, which degrades during extraction. Epoxyalkanes are a class of compounds containing an epoxy ring. The ring-opening reaction of epoxyalkanes generally refers to the breaking of the epoxy ring, and the reagents involved in this reaction typically perform nucleophilic attacks on the carbon atoms of the epoxy ring. Under the action of the nucleophile, epoxyalkanes undergo ring-breaking reactions, generating carbon-carbon double bonds or carbon-oxygen single bonds. The rules governing the ring-opening reactions of epoxyalkanes are determined by many factors.

[0092] Nucleophiles and reaction conditions are the main factors influencing the ring-opening reactions of epoxides. The nucleophilic nature of the nucleophile is key to the ring-opening reaction of epoxides. Generally, stronger nucleophiles react more readily with epoxides to form open products. Furthermore, reaction conditions also affect the ring-opening reaction of epoxides. Higher temperatures help accelerate the reaction rate but also easily induce side reactions, leading to the formation of other unwanted products. The structural characteristics of epoxides also affect the rules governing their ring-opening reactions. The substituents on epoxides and their positions affect the nucleotropic nature of the epoxide, thus affecting its reactivity with nucleophiles. Among epoxides with the same type and position of substituents, molecules containing larger and smaller rings are more likely to undergo ring-opening reactions. This is because molecules containing larger rings have greater ring strain, accelerating ring breaking.

[0093]

[0094]

[0095] As the extraction temperature increases, the bufalin group undergoes the aforementioned transformation, leading to a reduction in the bufalin group; the bufalin ligand is easily ring-opened and hydrolyzed to generate bufalin.

[0096] Example 8

[0097] S1: Cut 50g of dried toad skin into small pieces, add eutectic solvent (choline hydrochloride 13.9g, urea 12g, water 250g), stir for 3 hours at 110℃, filter to obtain toad skin residue and filtrate, extract the filtrate twice with n-butanol to obtain n-butanol layer and eutectic solvent layer after n-butanol extraction.

[0098] S2: The toad skin filter residue and the eutectic solvent layer after n-butanol extraction were stirred at 60°C for 2 hours, and then filtered to obtain toad skin filter residue and filtrate. The filtrate was extracted twice with n-butanol to obtain n-butanol layer and eutectic solvent layer after n-butanol extraction.

[0099] S3: Repeat S2 to obtain the n-butanol layer for later use;

[0100] S4: Combine the above n-butanol layers and dry with anhydrous sodium sulfate for 2 hours. Recover the solvent under reduced pressure to obtain the total lactone extract of bufos sterene. Dissolve the extract in methanol and transfer it to a 100 mL volumetric flask. Accurately measure 1 mL of the extract, dilute it 20 times with the mobile phase, filter it, and inject 20 μL.

[0101] The contents of bufotalin, bufotalin ligand, and bufotalin in the total lactone extract of bufosterene were detected according to the chromatographic detection conditions in Example 1. The results are as follows: Figure 12 As shown, chromatographic peaks appeared at retention times of 8.653, 11.653, and 12.163 min, with relative peak areas of 66.77%, 6.76%, and 26.47%, respectively. The content of bufotoxin in the total lactone extract of bufotoxin was determined to be 14.0 mg, the content of bufotoxin toxicant was 0.79 mg, and the content of bufotoxin ester was 3.55 mg using the internal standard method.

[0102] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for extracting total bufotalin lactones from toad-derived medicinal materials, characterized in that, The method includes the following steps: S1: Cut 50g of dried toad skin into small pieces, add a low-melting-point solvent, which is composed of 13.9g of choline hydrochloride, 12g of urea and 250g of water, stir at 120℃ for 3 hours, filter to obtain toad skin residue and filtrate, extract the filtrate twice with ethyl acetate to obtain ethyl acetate layer and low-melting-point solvent layer after ethyl acetate extraction. S2: The toad skin filter residue and the eutectic solvent layer were stirred at 60°C for 2 hours, and then filtered to obtain toad skin filter residue and filtrate. The filtrate was extracted twice with ethyl acetate to obtain ethyl acetate layer and eutectic solvent layer. S3: Repeat S2 to obtain the ethyl acetate layer for later use; S4: Combine the above ethyl acetate layer and dry with anhydrous sodium sulfate for 2 hours, then recover the solvent under reduced pressure to obtain the total lactone extract of bufos sterene.

2. A method for extracting total bufotalin lactones from toad-derived medicinal materials, characterized in that, The method includes the following steps: S1: Cut 50g of dried toad skin into small pieces, add a low-melting-point solvent, which consists of 28g of choline hydrochloride, 80g of magnesium chloride hexahydrate, and 600g of water. Stir at 100°C for 3 hours, filter to obtain toad skin residue and filtrate. Extract the filtrate twice with ethyl acetate to obtain an ethyl acetate layer and a low-melting-point solvent layer after ethyl acetate extraction. S2: The eutectic solvent layer after extraction of toad skin residue and ethyl acetate is stirred at 60°C for 2 hours, and the ethyl acetate extraction layer is reserved. S3: Repeat S2 to obtain the ethyl acetate layer for later use; S4: Combine the above ethyl acetate layer and dry with anhydrous sodium sulfate for 2 hours, then recover the solvent under reduced pressure to obtain the total lactone extract of bufos sterene.

3. A method for extracting total bufotalene lactones from toad-derived medicinal materials, characterized in that, The method includes the following steps: S1: Cut 50g of dried toad skin into small pieces, add a low-melting-point solvent, which is composed of 13.9g of choline hydrochloride, 12g of urea and 250g of water, stir at 110℃ for 3 hours, filter to obtain toad skin residue and filtrate, extract the filtrate twice with n-butanol to obtain n-butanol layer and low-melting-point solvent layer after n-butanol extraction; S2: The toad skin filter residue and the eutectic solvent layer after n-butanol extraction were stirred at 60°C for 2 hours, and then filtered to obtain toad skin filter residue and filtrate. The filtrate was extracted twice with n-butanol to obtain n-butanol layer and eutectic solvent layer after n-butanol extraction. S3: Repeat S2 to obtain the n-butanol layer for later use; S4: Combine the above n-butanol layers and dry with anhydrous sodium sulfate for 2 hours, then recover the solvent under reduced pressure to obtain the total lactone extract of bufos sterene.

4. A bufotalene total lactone extract, characterized in that, It is extracted using the method described in any one of claims 1-3.

5. The method for extracting total bufotalin lactones from toad-derived medicinal materials according to any one of claims 1-3 is used in the extraction of bufotalin from toad-derived medicinal materials.

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