A method for the isolation and purification of amatoxins in poisonous mushrooms

Abstract

The present invention discloses a method for the isolation and purification of amatoxins in poisonous mushrooms. The specific steps include: S1. Firstly, separating the mushroom extract containing amatoxins by high-speed counter-current chromatography; S2. Secondly, separating the product after the first separation by preparative liquid chromatography. The present invention uses HSCCC combined with Pre-HPLC to isolate and purify amatoxins in Amanita mushrooms, collects the target substances in groups, and finally conducts purity analysis and structural confirmation, thereby establishing a method for isolating and purifying four components, namely α-AMA, β-AMA, carboxy-tricholoro-phalloidin, and carboxy-dihydroxy-phalloidin, in Amanita mushrooms. This method is simple to operate, fast and efficient, with high purity, good reproducibility, and high sample recovery rate.

Description

Technical Field

[0001] The present invention relates to the technical field of the separation of amanitin toxins, in particular to a method for separating and purifying amanitin toxins from poisonous mushrooms. Background Art

[0002] Mushroom poisoning has become one of the acute diseases with the highest fatality rate in foodborne diseases. Mushroom poisoning incidents are characterized by seasonal and wide geographical distribution. The overall fatality rate is 11.69% - 42.30%. This disease progresses rapidly, has a complex condition, and a high fatality rate, and has become one of the acute diseases with the highest fatality rate in foodborne diseases. Among them, the fatality rate of poisoning by hepatotoxic Amanita species such as Amanita fuliginea, Amanita exitialis, Amanita rimosa, Amanita pallidorosea, and Amanita pseudopallidorosea is as high as 80%. Poisoning by mushrooms containing amanitin toxins directly accounts for more than 95% of the death cases, and they are the most common and lethal mushroom species and mushroom toxins.

[0003] Amanitin toxins are cyclic peptide compounds composed of 7 - 8 amino acids. According to their structures, they can be divided into three categories: amanitin (AMA), phalloidin, and virotoxin. There is currently no evidence that virotoxin is harmful to humans. Phalloidin has a rapid onset of toxicity, and AMA has a delayed onset of toxicity. Currently, 9 types of AMA have been isolated and identified from nature. Among them, α-AMA has the highest content and toxicity and is considered the main cause of liver injury and death in patients. In addition, in the field of life sciences, α-AMA is also used as a specific inhibitor of eukaryotic RNAPⅡ and is widely used in the study of the transcription process from DNA to mRNA, which has important value.

[0004] Currently, the scientific community's understanding of poisonous mushroom species and their toxins is not complete. There is a lack of an effective method for quickly distinguishing poisonous mushrooms from edible mushrooms, and there are certain deficiencies in the understanding and detection methods of mushroom toxins. The separation and purification of substances have always been an essential means and basis for scientific research. However, the existing extraction processes of amanitin toxins mainly use traditional techniques such as solvent extraction and silica gel column chromatography. The preparation cycle is long, impurities are not easily removed, the purity is low, and the use of solid fillers will cause irreversible adsorption and sample loss of the sample. Therefore, it is of great significance to establish a purification and preparation method for quickly extracting high-purity amanitin toxins from mushrooms using new technologies. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for separating and purifying amanitin toxins from poisonous mushrooms, which can simply and quickly extract 4 types of amanitin toxins from mushrooms containing amanitin toxins and provide a technical solution for the separation and purification of high-purity amanitin.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for separating and purifying amatoxins in poisonous mushrooms, comprising the following steps:

[0008] S1. Initially separate the mushroom extract containing amatoxins by high-speed counter-current chromatography;

[0009] S2. Secondly separate the product obtained from the initial separation by preparative liquid chromatography.

[0010] In some implementable embodiments of the present application, the mushroom extract is obtained by extraction with absolute ethanol;

[0011] wherein, the solid-liquid mass ratio during extraction is 1:20 - 30;

[0012] The extraction method is ultrasonic extraction for 30 - 120 min or heating under reflux for 60 - 120 min;

[0013] In some implementable embodiments of the present application, the mushroom extract is first extracted with absolute ethanol and then obtained by extraction with petroleum ether.

[0014] In some implementable embodiments of the present application, in step S1, the mushroom extract is initially separated by high-speed counter-current chromatography, and the effluent A containing a mixture of β-amanitin and carboxytricholoperatoxin, the effluent B containing α-amanitin, and the effluent C containing carboxydihydroxyphalloidin are collected according to the chromatographic peaks or time.

[0015] In some implementable embodiments of the present application, the solvent system of the high-speed counter-current chromatography includes n-butanol, water, and an auxiliary solvent; the volume ratio of n-butanol, water, and the auxiliary solvent is 3 - 7:5:0.5 - 2.

[0016] The auxiliary solvent includes any one or more of formic acid, acetic acid, and ethyl acetate.

[0017] In some implementable embodiments of the present application, the volume ratio of n-butanol, water, and the auxiliary solvent is 4:5:1.

[0018] In some implementable embodiments of the present application, the upper phase of the solvent system in the high-speed counter-current chromatography is the stationary phase, and the lower phase is the mobile phase; the flow rate is 10 - 50 mL / min, the rotation speed is 800 - 2000 revolutions per minute, and the detection wavelengths are 254 nm and 300 nm.

[0019] In some implementable embodiments of the present application, in step S2, according to the preparative liquid chromatography conditions, the concentrated effluent A is secondly separated by preparative liquid chromatography, the effluent components are received according to the chromatographic peaks, and each effluent component is concentrated and dried respectively to obtain β-amanitin and carboxytricholoperatoxin.

[0020] As some feasible embodiments of the present application, the treatment methods for the effluents B and C are direct concentration treatment or secondary separation under the conditions of preparative liquid phase.

[0021] As some feasible embodiments of the present application, the conditions of the preparative liquid chromatography are as follows:

[0022] The mobile phase is acetonitrile - 0.1% formic acid water, and the volume ratio is 8 - 20:92 - 80.

[0023] The flow rate is 5 - 40 mL / min, and the detection wavelengths are 254 and 300 nm.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The present invention uses HSCCC combined with Pre - HPLC to separate and purify the amatoxins in Amanita mushrooms; by optimizing the solvent separation conditions, collecting the target substances in groups, and finally performing purity analysis and structure confirmation, a method for separating and purifying four components, namely α - AMA, β - AMA, phallisacin (PSC), and phallacidin (PCD), from Amanita mushrooms is established. This method is simple, rapid, highly efficient, has high purity, good reproducibility, and high sample recovery rate. Description of the Drawings

[0026] Figure 1 It is the process flow chart of the present invention;

[0027] Figure 2 It is the high - performance liquid chromatography diagram of the mushroom extract obtained by the present invention;

[0028] Figure 3 It is the HSCCC chromatogram of the separation of the mushroom extract of the present invention;

[0029] Figure 4 It is each component separated by the preparative liquid phase of the present invention;

[0030] Figure 5 It is the high - performance liquid chromatography diagram of each fraction after concentration separated by HSCCC and preparative liquid phase of the present invention. Detailed Embodiments

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope protected by the present application.

[0032] In the prior art, the extraction process of amanitins mainly relies on traditional techniques such as solvent extraction and silica gel column chromatography. This process has a long preparation cycle, impurities are difficult to remove, the purity is low, and the use of solid packing materials can lead to irreversible adsorption of samples and sample loss.

[0033] Based on this, the present invention proposes a method for separating and purifying amanitins from poisonous mushrooms, which includes the following steps:

[0034] S1. First, use high-speed counter-current chromatography to preliminarily separate the mushroom extract containing amanitins;

[0035] S2. Then, use preparative liquid chromatography to perform secondary separation on the product after the preliminary separation.

[0036] In the above solution, preparative liquid chromatography (pre-HPLC) has the advantages of high separation efficiency and accurate collection, is suitable for industrial preparation, and is widely used in the separation and preparation of high-purity natural products. However, this method has high requirements for samples, and the samples need to be well pretreated before injection to ensure the safety of the preparation column and improve the separation efficiency.

[0037] Based on this, before using preparative liquid chromatography for separation, the present invention first performs preliminary separation using high-speed counter-current chromatography (HSCCC).

[0038] High-speed counter-current chromatography is an efficient chromatographic technique that separates substances based on the different distribution coefficients of the substances to be separated in two immiscible liquid phases. It does not require solid packing materials, avoiding sample losses such as irreversible adsorption of samples, and has unique advantages such as flexible elution methods, high recovery rates, large preparation amounts, low economic costs, and good reproducibility. It has broad application prospects in the fields of biomedical research and development, natural product separation, chemical engineering, and food production, and is particularly suitable for the pretreatment of samples with complex components. Therefore, combining HSCCC technology and preparative liquid chromatography technology can become an effective method for simultaneously separating and preparing multiple high-purity monomers in the laboratory.

[0039] The present invention uses HSCCC combined with Pre-HPLC to separate and purify amanitins from Amanita mushrooms, collect the target substances in groups, and finally perform purity analysis and structure confirmation, establishing a method for separating and purifying four components, namely α-AMA, β-AMA, phallisacin (PSC), and phallacidin (PCD), from Amanita mushrooms. This method is simple, rapid, highly efficient, has high purity, good reproducibility, and high sample recovery rate.

[0040] In order to further improve the extraction purity of the extract, as some feasible embodiments of the present application, the extraction method of the extract is limited, that is, the mushroom extract is obtained by extraction with absolute ethanol.

[0041] Since the amatoxins are polypeptide components with relatively large polarity and good water solubility, the reported extraction methods mostly involve alcohol + water extraction. The solvents are difficult to recover, and tannins and sugars in the mushrooms will be extracted together, introducing impurities and causing difficulties in subsequent separation and purification, such as low sample loading amount in countercurrent chromatography. Therefore, in the present invention, absolute ethanol is used for ultrasonic extraction, which is simple and easily available, avoiding water-soluble impurities, and the solvent can be recycled and reused during industrial production.

[0042] Among them, during extraction, the solid-liquid mass ratio during extraction is limited to 1:20 - 30; the extraction method is ultrasonic extraction for 30 - 120 min or heating under reflux for 180 min; the number of extraction times is 3 - 5 times.

[0043] By limiting the above extraction conditions, the extraction rate of amatoxins can be effectively increased.

[0044] Furthermore, through single-factor investigation, the optimal extraction conditions are determined as a solid-liquid mass ratio of 1:30, ultrasonic extraction for 60 minutes, and extraction for 3 times. In this way, the extraction rate of amatoxins can reach over 95%.

[0045] In order to further increase the sample loading amount in countercurrent chromatography, as some feasible embodiments of the present application, the extract is limited, that is, the mushroom extract is first extracted with absolute ethanol and then extracted with petroleum ether. In this scheme, the product after extraction with absolute ethanol is further extracted with petroleum ether, which can not only maintain the high extraction rate of the mushroom extract but also increase the sample loading amount in countercurrent chromatography.

[0046] In order to further improve the primary separation effect, as some feasible embodiments of the present application, the effluent collected in step S1 is limited, that is, in step S1, high-speed countercurrent chromatography is used to initially separate the mushroom extract, and according to the chromatographic peaks or time, the effluent A containing a mixture of β-amanitin and carboxyphalloidin, the effluent B containing α-amanitin, and the effluent C containing carboxydehydrophalloidin are collected. Among them, the effluent A needs to be further separated in step S2, and the effluent B and the effluent C can either be directly concentrated and dried or can be further separated in step S2 to improve the purity.

[0047] In order to further improve the primary separation effect, in some feasible embodiments of the present application, the solvent system of high-speed counter-current chromatography is further defined, that is, the solvent system of the high-speed counter-current chromatography includes n-butanol, water and an auxiliary solvent; the auxiliary solvent includes any one or more of formic acid, acetic acid, and ethyl acetate. It is found through experiments that in order to effectively separate the extract in the present invention, the solvent system must contain n-butanol, water, and any one or more of formic acid, acetic acid, and ethyl acetate. Otherwise, the separation effect is extremely poor and even separation cannot be achieved (for example, when the solvent system only contains n-butanol and water, separation cannot be carried out). Among them, the volume ratio of the n-butanol, water and the auxiliary solvent is 3-7:5:0.5-2.

[0048] In order to further improve the primary separation effect, in some feasible embodiments of the present application, the dosages of the components in the solvent system of high-speed counter-current chromatography are further defined, that is, the volume ratio of the n-butanol, water and the auxiliary solvent is 4:5:1.

[0049] In order to further improve the primary separation effect, in some feasible embodiments of the present application, the relevant conditions of high-speed counter-current chromatography are further defined, that is, the upper phase of the solvent system in the high-speed counter-current chromatography is the stationary phase, and the lower phase is the mobile phase; the flow rate is 10-50 mL / min, the rotation speed is 800-2000 revolutions per minute, and the detection wavelength is 254 nm and 300 nm.

[0050] Up to now, there is no report at home and abroad on the separation and purification of amatoxins by counter-current chromatography. The reason is that the counter-current chromatography solvent system used has not been invented, and the relevant processes and technical details are unknown, so counter-current chromatography cannot be used for separation. At present, the separation and purification of amatoxins mainly focuses on the research of α-amanitin, and the obtained varieties are few; traditional methods such as solvent extraction, macroporous adsorption resin, and column chromatography are used for extraction, with many steps and high costs; and because most amatoxins are water-soluble, the use of conventional silica gel column chromatography and the like usually has poor effects, and it is difficult to avoid irreversible adsorption of samples caused by solid fillers, resulting in low purity and poor recovery of the final product.

[0051] Therefore, inventing a suitable solvent system is the key to developing a counter-current chromatography method. After systematic screening of three types of solvent systems: 1) n-hexane-ethyl acetate-methanol-water; 2) dichloromethane-methanol-water; 3) n-butanol-water with different proportion combinations, a solvent system of ethyl acetate-n-butanol-water (1:4:5) was finally selected, and the partition coefficients (K values) of α-amanitin, β-amanitin, carboxytricholide, and carboxydihydroxyphalloidin are appropriate (0-2.5); subsequently, the elution method, rotation speed, temperature and detection wavelength were further investigated, and the elution time of each component was determined by liquid chromatography, and high-purity monomers of four components were obtained by combining preparative liquid chromatography.

[0052] In order to re-separate the mixture in the effluent A, as some feasible embodiments of the present application, the effluent A is re-separated by preparative liquid chromatography. That is, in the step S2, according to the preparative liquid chromatography conditions, the concentrated effluent A is secondarily separated by preparative liquid chromatography, the effluent components are received separately according to the chromatographic peaks, and each effluent component is concentrated and dried respectively to obtain β-amanitin and carboxy-trihydroxyphalloidin.

[0053] The treatment methods for the effluents B and C are direct concentration treatment or secondary separation according to the preparative liquid conditions.

[0054] Specifically, α-amanitin is obtained after the effluent B is directly concentrated and dried, and carboxy-dihydroxyphalloidin is obtained after the effluent C is directly concentrated and dried.

[0055] In order to further improve the purity of α-amanitin and carboxy-dihydroxyphalloidin, the effluents B and C need to be re-purified by preparative liquid chromatography. That is, in the step S2, according to the preparative liquid chromatography conditions, the concentrated effluents B and C are secondarily separated by preparative liquid chromatography respectively, the effluent components are received according to the chromatographic peaks, and the effluent components are concentrated and dried. Compared with the concentration treatment, higher-purity α-amanitin and carboxy-dihydroxyphalloidin can be obtained by the secondary separation.

[0056] In order to further improve the secondary separation effect, as some feasible embodiments of the present application, the conditions of the preparative liquid chromatography are further defined, that is, the conditions of the preparative liquid chromatography are as follows:

[0057] The mobile phase is acetonitrile - 0.1% formic acid water, and the volume ratio is 8 - 20:92 - 80; the flow rate is 5 - 40 mL / min, and the detection wavelengths are 254 and 300 nm.

[0058] Preparative liquid chromatography is a common preparative separation instrument, and the elution conditions are the key to successful separation. Because the amatoxin toxins have a large polarity, especially β-amanitin will cause a shift in the liquid phase peak time with the change of pH. How to effectively elute and detect is a technical difficulty. The present invention has developed a liquid phase detection method with a mobile phase of formic acid water with pH = 4.0 and acetonitrile and a preparative liquid chromatography elution method with a mobile phase of 0.1% formic acid aqueous solution and acetonitrile to secondarily separate the fractions obtained by countercurrent chromatography. The obtained components have high purity, short separation time, and simple method.

[0059] The following further details the separation and purification method of the amatoxin toxins described in the present application in combination with specific embodiments.

[0060] It should be noted that in the liquid-phase detection methods of α-amanitin, β-amanitin, carboxytrihydroxyphalloidin, and carboxydihydroxyphalloidin, the detection conditions of high-performance liquid chromatography are as follows: chromatographic column C18 (4.6 mm × 250 mm, 5 μm), mobile phase A (formic acid water with pH = 4.0) and mobile phase B (acetonitrile) for gradient elution: 0 - 10 min, mobile phase B (5 - 15%); 10 - 25 min, B (15 - 25%); 25 - 30 min, B (25 - 90%); 30 - 35 min, B (90%); flow rate is 1.0 mL / min; injection volume is 10 μL; detection wavelengths are 254 and 300 nm.

[0061] Example 1 (for the specific process flow, see Figure 1 )

[0062] S1 Preparation of mushroom extract

[0063] Take 50 g of dried mushroom powder of Amanita, use anhydrous ethanol as the extraction solvent, with a solid-liquid ratio of 1:30, extract 3 times by ultrasonic for 60 minutes, filter, combine the extraction solutions, recover ethanol under reduced pressure, and concentrate to dryness to obtain 8 g of ethanol extract. Calculated by α-amanitin, the extraction rate reaches over 95%.

[0064] S2 Separation of mushroom ethanol extract by high-speed counter-current chromatography

[0065] Take the mushroom extract and use the high-speed counter-current chromatography conditions: use ethyl acetate - n-butanol - water (1:4:5) as the solvent system, reverse-phase elution (the upper phase is used as the stationary phase and the lower phase is used as the mobile phase), flow rate 10 mL / min, rotation speed 1000 rpm, temperature 20 °C, detection wavelength 300 nm. Dissolve it in the lower-phase solution of the solvent system to 50 mg / mL, and after the hydrodynamic equilibrium of CCC is established, load the sample. The loading volume is 40 mL; under these conditions, the retention rate of the stationary phase is between 40% and 80%. The HSCCC separation chromatogram is shown in detail in Figure 2 .

[0066] Collect the elution fractions at 35 - 57, 79 - 92, and 109 - 114 minutes (or collect according to the chromatographic peaks in the chromatogram), and record them as effluent A, effluent B, and effluent C respectively. The HSCCC chromatogram is as shown in Figure 3 shown.

[0067] Effluent A continues to step S3 for preparative liquid-phase separation; effluent B and effluent C can be directly concentrated and dried to obtain 90.4 mg of α-amanitin and 36.8 mg of carboxydihydroxyphalloidin, with a purity > 96%, or choose to perform step S3 again to make the purity > 99.5%. The results are shown in Figure 5 .

[0068] S3 Purification of compounds by preparative liquid chromatography

[0069] Take the effluent A and use preparative liquid chromatography conditions: chromatographic column C18 (20 ID × 250 mm), the mobile phase is 0.1% formic acid aqueous solution containing 12.5% by volume of acetonitrile, the flow rate is 10 mL / min, the detection wavelengths are 254 and 300 nm, and collect the elution fractions according to the chromatographic peaks (see Figure 4 ), concentrate and dry, and 48.8 mg of β-amanitin and 56.5 mg of carboxytricholide can be obtained respectively. After detection by HPLC, the purity is greater than 98% (calculated by area normalization method), see Figure 5 .

[0070] Determination of Compound S4

[0071] Take the HSCCC obtained from the above S2 and S3 and the eluent of the preparative liquid chromatography, and compare them with the standards of α-amanitin, β-amanitin, carboxytricholide and carboxydihydrotricholide. Their retention times and ultraviolet absorption spectra are consistent.

[0072] In this example, 50 g of dry fruiting body powder of Amanita exitialis in the genus Amanita was extracted once, 8 g of ethanol extract could be obtained, the single injection amount of countercurrent chromatography reached 2 g, and 90.4 mg of α-amanitin, 48.8 mg of β-amanitin, 56.5 mg of carboxytricholide and 36.8 mg of carboxydihydrotricholide could be obtained after separation by preparative liquid chromatography. The purities were 99.6%, 98.2%, 99.5% and 99.2% in turn.

[0073] Example 2

[0074] Compared with Example 1, change the countercurrent chromatography solvent system to a volume ratio of ethyl acetate, n-butanol and water of 0.5:3:5. After countercurrent chromatography separation, directly concentrate and dry the effluents B and C to obtain α-amanitin and carboxydihydrotricholide respectively, with purities of 85% and 88%. After secondary purification by preparative liquid chromatography, the purities can reach 98.6% and 98.2%.

[0075] In this example, after changing the countercurrent chromatography solvent system, the purities of α-amanitin and carboxydihydrotricholide decreased compared with Example 1, but the effects of the present invention could still be achieved.

[0076] Example 3

[0077] Compared with Example 1, on the basis of extraction with absolute ethanol, carry out petroleum ether extraction, specifically as follows:

[0078] Take 50 g of dried mushroom powder of the genus Amanita, use anhydrous ethanol as the extraction solvent, with a solid-liquid ratio of 1:30, extract 3 times by ultrasonic for 60 minutes, filter, combine the extracts, recover ethanol under reduced pressure, concentrate to a dry extract, and then extract the extract with petroleum ether (solid-liquid ratio of 1:5 - 10), discard the petroleum ether layer, dry and then perform countercurrent chromatography separation, and then perform preparative liquid chromatography separation. The results show that the separation and purification effect of the present invention can be achieved, that is, the purities of α-amanitin, β-amanitin, carboxytricholochin, and carboxydihydroxyphalloidin are generally consistent with those in Example 1, and the sample loading amount of countercurrent chromatography can also be increased by about 10% - 30%. The petroleum ether layer is the fat-soluble substances in the mushroom and can also be reserved for subsequent analysis.

[0079] Comparative Example 1

[0080] Compared with Example 1, the ultrasonic method was used to extract the mushroom powder with a solid-liquid ratio of 1:10 and an extraction time of 30 minutes. After liquid phase detection, the extraction rate for three extractions was 55%.

[0081] This shows that the solid-liquid ratio has a great influence on the extraction rate during extraction.

[0082] Comparative Example 2

[0083] Compared with Example 1, the reflux extraction method was used to extract the mushroom powder with a solid-liquid ratio of 1:30 and an extraction time of 30 minutes. After liquid phase detection, the extraction rate for two extractions was 70% - 80%.

[0084] This shows that the reflux extraction time is short, seriously affecting the extraction rate.

[0085] Comparative Example 3

[0086] Compared with Example 1, the solvent system of countercurrent chromatography was changed to a volume ratio of ethyl acetate, n-butanol, and water of 0.2:2:5. The results show that the results of the present invention cannot be achieved and the four components cannot be effectively separated.

[0087] Comparative Example 4

[0088] Compared with Example 1, after the mushroom powder was extracted, it was directly subjected to preparative liquid chromatography separation without passing through countercurrent chromatography separation. The results show that the results of the present invention cannot be achieved and the four components cannot be effectively separated.

[0089] The above are only the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and environments, and can be changed within the scope conceived herein through the above teachings or the techniques or knowledge in related fields. And any changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention shall fall within the protection scope of the appended claims of the present invention.

Claims

1. A method for separating and purifying amanitin toxins from poisonous mushrooms, characterized in that, It includes the following steps: S1. Use high-speed countercurrent chromatography to conduct primary separation on the mushroom extract containing amatoxins. The specific steps are as follows: Use high-speed countercurrent chromatography to initially separate the mushroom extract, and collect the effluent A containing a mixture of β-amanitin and carboxytricholomic acid, the effluent B containing α-amanitin, and the effluent C containing carboxydihydrotricholomic acid according to the chromatographic peaks or time; The solvent system of the high-speed countercurrent chromatography includes n-butanol, water, and an auxiliary solvent; the volume ratio of n-butanol, water, and the auxiliary solvent is 3-7:5:0.5-2; the auxiliary solvent is ethyl acetate; S2. Use preparative liquid chromatography to conduct secondary separation on the product after primary separation. The specific steps are as follows: According to the preparative liquid chromatography conditions, use preparative liquid chromatography to conduct secondary separation on the concentrated effluent A, receive the effluent components according to the chromatographic peaks respectively, concentrate and dry each effluent component respectively to obtain β-amanitin and carboxytricholomic acid; The preparative liquid chromatography conditions are as follows: The mobile phase is acetonitrile - 0.1% formic acid water, and the volume ratio is 8-20:92-80; The flow rate is 5-40 mL / min, and the detection wavelengths are 254 nm and 300 nm.

2. The method for separating and purifying amanitin toxins in poisonous mushrooms according to claim 1, characterized in that, The mushroom extract is obtained by extraction with absolute ethanol; Among them, the solid-liquid mass ratio during extraction is 1:20-30; The extraction method is ultrasonic extraction for 30-120 min or heating under reflux for 60-120 min.

3. The method for separating and purifying amanitin toxins in poisonous mushrooms according to claim 2, characterized in that, The mushroom extract is first extracted with absolute ethanol and then extracted with petroleum ether.

4. The method for separating and purifying amanitin toxins in poisonous mushrooms according to claim 1, characterized in that, The volume ratio of n-butanol, water, and the auxiliary solvent is 4:5:

1.

5. The method for separating and purifying amanitin toxins in poisonous mushrooms according to claim 1, characterized in that, In the high-speed countercurrent chromatography, the upper phase of the solvent system is the stationary phase, and the lower phase is the mobile phase; the flow rate is 10-50 mL / min, the rotation speed is 800-2000 revolutions per minute, and the detection wavelengths are 254 nm and 300 nm.

6. The method for separating and purifying amatoxins in poisonous mushrooms according to claim 1, characterized in that The treatment methods for the effluents B and C are direct concentration treatment or secondary separation according to the preparative liquid chromatography conditions.

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