A process for the preparation of tenuazonic acid

Abstract

The application discloses a method for preparing fine fusaric acid, which comprises the following steps: toxin production culture, sample extraction, preliminary purification, reverse-phase preparation, gel chromatography separation and normal-phase preparation. The method for preparing fine fusaric acid provided by the application improves material steady state and preparation efficiency, greatly reduces production cost, and has a purity of more than 99%, and can be used as a standard product for fine fusaric acid detection, prevention and control and toxicology research in food and feed.

Description

Technical Field

[0001] This invention relates to the fields of food safety and natural product separation and purification technology, specifically to a method for preparing Alternaria alterniflora ketoacid. Background Technology

[0002] Tenuazonic acid (TeA) is produced by Alternaria species (Alternaria). Alternaria spp . Alternaria toxin is a nitrogen-containing mycotoxin produced by fungi after they infect crops under suitable temperature and humidity conditions. It was first discovered by Roset in 1953 and is a brown, oily substance with the molecular formula C. 10 H 15 O3N, molecular weight: 197.23, is readily soluble in organic solvents such as acetone, chloroform, ethanol, methanol and ethyl acetate.

[0003] TeA can cause mold growth on grains, vegetables, fruits, and field crops not only during field, transportation, and storage, but it can also grow in low-temperature and humid environments, leading to the spoilage of refrigerated fruits and vegetables. TeA has the highest toxicity levels among all Alternaria toxins and can cause serious harm to human and animal health through the food chain. Studies have shown that the toxic mechanism of TeA in mammals is mainly through inhibiting the release of newly synthesized proteins from the proteosome into the plasma. It can selectively form complexes with certain trace metal ions in the body and act on the active site of a 60S transaminase in the peptide bond formation step of protein synthesis.

[0004] The European Food Safety Authority (EFSA) uses the Toxicological Concern Threshold (TTC) to assess the potential risks of TeA to humans, with a TTC value of 1.5 µg / kg body weight / day. However, there are currently no applicable limits for TeA in food and feed, and research on the metabolic distribution of TeA is limited. Its pharmacokinetic behavior after entering the animal body remains unclear, and studies on the toxicology of TeA, its migration and transformation in vivo, analytical detection, and risk assessment require a large number of reference standards.

[0005] Currently, the main method for preparing TeA is chemical synthesis, which is cumbersome, consumes large amounts of organic reagents, and is time-consuming and expensive. Separation methods include solvent partitioning, macroporous resin methods, and thin-layer chromatography, but these methods are all time-consuming and produce low purity of the target compound, making them unsuitable for large-scale preparation. Janardhannan et al. used glucose-yeast liquid medium as the toxin-producing medium and ethyl acetate as the extraction solvent to separate TeA via thin-layer chromatography, but the yield was low (5.7 mg / L), the extraction solvent was highly toxic, and the preparation steps were complex, making it unsuitable for industrial-scale preparation. Current TeA standards are expensive, averaging around 20,000 yuan / mg. However, currently reported methods do not specifically address the purity of the obtained TeA, making it impossible to determine whether they meet the requirements for standards.

[0006] Therefore, there is an urgent need to develop new methods for purifying and preparing TeA. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing TeA ketoacid from Alternaria alternata, the method specifically comprising the following steps: (1) Inoculate Alternaria alternata ATCC 36381 onto potato-dose-agar medium and activate it in the dark at 25-28℃ for 5 days; (2) Take small pieces from the mycelia of the above potato-glucose-agar medium and inoculate them into a new medium for culture. The culture conditions are: potato-glucose-agar medium or potato-glucose-water medium (initial pH of medium is 5.0-6.5), add L-isoleucine (0.5%, m / m) or L-phenylalanine (0.3%, m / m), and culture in the dark at 25-28℃ for 10 days. (3) Transfer the culture medium to an Erlenmeyer flask, add acetonitrile / water (50 / 50, v / v), sonicate for 30 min, and then vortex at 180 rpm for 30 min. Repeat this step and collect the extract. (4) Filter the extract into a rotary evaporator flask using gauze, and adjust the rotary evaporator until all the liquid has evaporated to dryness; (5) Preliminary purification: The concentrated solid was redissolved in pure water and transferred to a separatory funnel. An equal volume of n-butanol was added, and the mixture was shaken vigorously. The mixture was then allowed to stand and allow the liquid to separate into layers. The upper layer was collected. This step was repeated, and the liquid collected after extraction (n-butanol layer) was concentrated to dryness by rotary evaporation to obtain the solid substance. Or: The concentrated solid was redissolved in pure water and transferred to an Erlenmeyer flask. 1 / 100 volume of DA201 macroporous resin was added, and the mixture was shaken at 120 rpm for 10 h to adsorb TeA. Then, the adsorption was carried out using an eluent (ethanol / water, 90 / 10, v / v), and the eluent was collected. The eluent was then concentrated to dryness by rotary evaporation to obtain the solid substance. (6) Reverse-phase preparation: The solid substance was dissolved in acetonitrile / water (1 / 1, v / v) and filtered through a 0.22 μm filter membrane. A single-phase preparation was performed using a medium-pressure preparative liquid phase. The preparation conditions were: C 18 AQ-N preparative chromatographic column (20 mm × 250 mm, 5 μm); mobile phase A is 0.1% (v / v) formic acid aqueous solution, B is acetonitrile, flow rate is 15 mL / min, detection wavelength is 278 nm, injection volume is 1 mL, collect the distillate after 9-10 min, concentrate the distillate to dryness by rotary evaporation to obtain solid substance; (7) Gel chromatography separation: The solid was dissolved in methanol / dichloromethane (1 / 1, v / v), and then added to an LH-20 gel chromatography column. The mobile phase was methanol / dichloromethane (1 / 1, v / v). The pale yellow TeA segment was collected, and the collected liquid was evaporated to dryness to obtain the solid substance. (8) Normal phase preparation: The solid was dissolved in hexane / isopropanol (4 / 1, v / v) and filtered through a 0.22 μm filter membrane. Secondary normal phase preparation was performed using medium-pressure preparative liquid chromatography. An Ultimate SiO2 preparative column (21.2 mm × 250 mm, 5 μm) was used. The mobile phase A was hexane and B was isopropanol. The flow rate was 12 mL / min, the detection wavelength was 277 nm, the injection volume was 0.2 mL, and the fraction was collected after 7.0-8.0 min. Mix all the distillate liquids, dry them with nitrogen at room temperature in the dark, and then dry them in a freeze vacuum dryer to obtain a yellow oily substance, which is TeA.

[0008] The purity of the prepared TeA was determined by the area normalization method of ultra-high performance liquid chromatography (UPLC-PDA).

[0009] This invention provides a simple, rapid, inexpensive, high-purity, and large-scale method for preparing TeA. The method uses an optimized potato-glucose-agar / water medium with high toxin production, low impurities, and short culture time as the toxin-producing medium. After preliminary purification via extraction, extraction, or macroporous resin decolorization, precise separation is achieved through a three-stage orthogonal preparation process involving reverse-phase preparation, gel size exclusion, and normal-phase preparation, ultimately yielding the high-purity target compound. This process is simple to operate, highly efficient, and significantly reduces production costs. Furthermore, the purity of the final product reaches over 99%, making it suitable as a standard for the detection, control, and toxicological research of TeA.

[0010] Compared with the prior art, the present invention has the following beneficial technical effects: (1) Short fermentation cycle and high toxin yield: Alternaria alternata ATCC 36381 innovatively creates a stress toxin-producing environment by adding toxin synthesis precursor amino acids in potato-glucose-agar / water medium. After optimization of culture conditions, the yield of TeA can reach as high as 1003.1 mg / kg in just 10 days. (2) The crude extract has few impurities and high preparation efficiency: Two crude purification methods, liquid-liquid extraction and macroporous resin adsorption-desorption, were established based on the molecular polarity of TeA. Under the premise of ensuring that the structure of highly active weak acidic keto acid is not destroyed, high-efficiency impurity removal and pre-decolorization (purity >80%) are achieved, protecting the chromatographic column and greatly improving the preparation efficiency.

[0011] (3) High purity: Due to the small molecular weight of TeA (197.23), it is easily mixed with structurally similar isomers and small molecule pigments. It is difficult to completely remove similar impurities using only reversed-phase chromatography, and isomers with configuration inversion may appear during the preparation process. This invention constructs a three-stage orthogonal purification route of "reverse-phase preparation - gel size exclusion - normal-phase preparation" to obtain TeA with a purity >99%, which significantly improves the purity and preparation efficiency of the standard substance while maintaining the steady state of the standard substance. Attached Figure Description

[0012] Figure 1 The infrared spectrum of the TeA prepared in Example 1. Figure 2 The 1H NMR spectrum (A) and 1C NMR spectrum (B) of the TeA prepared in Example 1 are shown. Figure 3 The 1H NMR spectrum (A) and 1C NMR spectrum (B) of the TeA prepared in Example 2 are shown. Figure 4 The UV absorption spectrum of the prepared TeA Figure 5 For ESI + and ESI - Time-of-flight mass spectra of TeA prepared in Example 1 under the model Figure 6 The liquid chromatogram of the TeA collection solution in Example 1 Figure 7 The liquid chromatogram of the TeA collection solution in Example 2. Detailed Implementation Example 1: Toxin production culture (1) Alternaria alternata ATCC 36381 (purchased from the American Type Culture Collection (ATCC)) was inoculated on potato-dose-agar medium (potato-dose-agar powder, Beijing Luqiao Technology Co., Ltd.) and activated in the dark at 25 ℃ for 5 days; (2) Use a sterile toothpick to take a small piece from the edge of the culture medium and inoculate it into freshly prepared potato-glucose-agar medium (potato-glucose-agar powder, Beijing Luqiao Technology Co., Ltd.), add L-isoleucine (0.5%, m / m), the initial pH of the culture medium is 6.4, and the culture conditions are culture temperature 25 ℃ and culture time 10 d.

[0013] Sample extraction (3) Transfer the above potato-glucose-agar medium to a 1 L Erlenmeyer flask, add 900 mL of acetonitrile / water (50 / 50, v / v), sonicate for 30 min, and then vortex at 180 rpm / min for 2 h. Repeat this step three times.

[0014] (4) Filter the extract through four layers of gauze (100 mesh) into a rotary evaporator flask and concentrate it in a rotary evaporator at 45°C until the liquid is completely evaporated.

[0015] (5) Preliminary purification The solid, dried by rotary evaporation, is reconstituted with water (until no particles remain) and transferred to a separatory funnel. An equal volume of n-butanol is added, and the mixture is shaken vigorously about 50 times. The mixture is allowed to stand and allow the liquid to separate into layers. The supernatant (n-butanol layer) is collected. This step is repeated 3 times. The extracted liquid is then concentrated to dryness by rotary evaporation to obtain the solid substance.

[0016] (6) Reverse phase preparation The solid substance was dissolved in acetonitrile / water (1 / 1, v / v) and then filtered through a 0.22 μm filter membrane. The preparation was performed using a medium-pressure preparative liquid chromatograph (AutoP preparative liquid chromatograph, Waters Corporation, USA).

[0017] Chromatographic conditions: C 18 AQ-N preparative chromatographic column (20 mm × 250 mm, 5 μm, Zhejiang Huapu Xinchuang Technology Co., Ltd.); mobile phase A was 0.1% (v / v) formic acid aqueous solution, and mobile phase B was acetonitrile; flow rate was 15 mL / min; detection wavelength was 278 nm, and injection volume was 1 mL. Elution gradient is shown in Table 1.

[0018] Table 1 Gradient elution conditions for reverse-phase preparation

[0019] Fraction collection conditions: via C 18 AQ-N preparative chromatography column separation completely separates impurities from the target analyte TeA. The TeA fraction is collected at fixed intervals of 9-10 minutes to prevent cross-contamination between impurities and the target analyte. The fraction is then concentrated to dryness by rotary evaporation to obtain a solid substance.

[0020] The TeA collection time settings are shown in Table 2.

[0021] Table 2 TeA fraction collection time

[0022] (7) Gel chromatography separation The solid was dissolved in methanol / dichloromethane (1 / 1, v / v) and then added to an LH-20 gel chromatography column (Sephadex, Sigma-Aldrich, USA). The mobile phase was methanol / dichloromethane (1 / 1, v / v). The pale yellow TeA segment was collected, and the collected liquid was evaporated to dryness to obtain the solid substance.

[0023] (8) Normal phase preparation The solid was dissolved in hexane / isopropanol (4 / 1, v / v) and filtered through a 0.22 μm filter membrane; normal phase preparation was performed using a medium-pressure preparative liquid chromatograph (AutoP preparative liquid chromatograph, Waters Corporation, USA).

[0024] Chromatographic conditions: A preparative SiO2 column (21.2 mm × 250 mm, 5 μm, Shanghai Yuexu Technology Co., Ltd.) was used; mobile phase A was hexane, and mobile phase B was isopropanol; flow rate was 12 mL / min; detection wavelength was 277 nm, and injection volume was 0.2 mL. The elution gradient is shown in Table 3.

[0025] Table 3 Gradient elution conditions for normal phase preparation

[0026] Fraction collection conditions: TeA and its analogues were further separated using a normal-phase column prepared with Ultimate SiO2. The TeA fraction was collected at fixed intervals of 7.0–8.0 min to prevent cross-contamination between impurities and target analytes. The collection time for TeA is shown in Table 4. All fractions were turbidized, dried under nitrogen at room temperature in the dark, and then dried in a refrigerated vacuum dryer to obtain a yellow oily substance, which was stored at -20°C.

[0027] Table 4. TeA fraction collection time

[0028] Example 2: Toxin production culture (1) Alternaria alternata ATCC 36381 (purchased from the American typeculture collection (ATCC)) was inoculated on potato-dose-agar medium (potato-dose-agar powder, Beijing Luqiao Technology Co., Ltd.) and activated in the dark at 25 ℃ for 5 days; (2) Use a sterile toothpick to take a small piece from the edge of the culture medium and inoculate it into the newly prepared potato-glucose-water culture medium (potato-glucose-water powder, Beijing Luqiao Technology Co., Ltd.), add L-phenylalanine (0.3%, m / m), the initial pH of the culture medium is 5.5, and the culture conditions are a culture temperature of 25 ℃ and a culture time of 10 days.

[0029] Sample extraction (3) Transfer the above potato-glucose-water culture medium to a 1 L Erlenmeyer flask, add 900 mL of acetonitrile / water (50 / 50, v / v), sonicate for 30 min, and then vortex at 180 rpm for 2 h.

[0030] (4) Filter the extract through four layers of gauze (100 mesh) into a rotary evaporator flask and concentrate it in a rotary evaporator at 45°C until the liquid is completely evaporated.

[0031] (5) Preliminary purification The rotary-evaporated solid was reconstituted with water (until no particles remained) and transferred to a 1 L Erlenmeyer flask. 1 / 100 volume of DA201 macroporous resin (Shanghai Yuanye Biotechnology) was added, and the mixture was shaken at 120 rpm for 10 h to adsorb TeA. Then, an eluent (ethanol / water, 90 / 10, v / v) was added to remove the adsorbate, and the eluent was collected. The eluent was then concentrated to dryness by rotary evaporation to obtain the solid substance.

[0032] (6) Reverse phase preparation The solid substance was dissolved in acetonitrile / water (1 / 1, v / v) and then filtered through a 0.22 μm filter membrane. The preparation was performed using a medium-pressure preparative liquid chromatograph (AutoP preparative liquid chromatograph, Waters Corporation, USA).

[0033] Chromatographic conditions: C 18 AQ-N preparative chromatographic column (20 mm × 250 mm, 10 μm, Zhejiang Huapu Xinchuang Technology Co., Ltd.); mobile phase A was 0.1% (v / v) formic acid aqueous solution, and mobile phase B was acetonitrile; flow rate was 15 mL / min; detection wavelength was 278 nm, and injection volume was 1 mL. Elution gradient is shown in Table 5.

[0034] Table 5 Gradient elution conditions for reverse-phase preparation

[0035] Fraction collection conditions: via C 18 The AQ-N preparative chromatography column ensures complete separation of impurities and the target analyte TeA. The TeA fraction is collected at fixed intervals of 9.0–10.0 min to prevent cross-contamination between impurities and the target analyte.

[0036] The TeA collection time settings are shown in Table 6.

[0037] Table 6 TeA fraction collection time

[0038] (7) Gel chromatography separation The solid was dissolved in methanol / dichloromethane (1 / 1, v / v) and then added to an LH-20 gel chromatography column (Sephadex, Sigma-Aldrich, USA). The mobile phase was methanol / dichloromethane (1 / 1, v / v). The pale yellow TeA segment was collected, and the collected liquid was evaporated to dryness to obtain the solid substance.

[0039] (8) Normal phase preparation The solid was dissolved in hexane / isopropanol (4 / 1, v / v) and filtered through a 0.22 μm filter membrane. The solution was then prepared using a medium-pressure preparative liquid chromatograph (AutoP preparative liquid chromatograph, Waters Corporation, USA).

[0040] Chromatographic conditions: A preparative SiO2 column (21.2 mm × 250 mm, 5 μm, Shanghai Yuexu Technology Co., Ltd.) was used; mobile phase A was n-hexane, and mobile phase B was isopropanol; flow rate was 12 mL / min; detection wavelength was 277 nm, and injection volume was 0.2 mL. The elution gradient is shown in Table 7.

[0041] Table 7 Gradient elution conditions for normal phase preparation

[0042] Fraction collection conditions: TeA and its analogues were further separated using a normal-phase column prepared with Ultimate SiO2. The TeA fraction was collected at fixed intervals of 7.0–8.0 min to prevent cross-contamination between impurities and target analytes. The collection time for TeA is shown in Table 8. All fractions were mixed, dried under nitrogen at room temperature in the dark, and then dried in a refrigerated vacuum dryer to obtain a yellow oily substance, which was stored at -20°C.

[0043] Table 8 TeA fraction collection time

[0044] Example 3: Qualitative analysis and purity determination were performed on the TeA prepared in Example 1 or Example 2. 1. Infrared spectroscopy identification Infrared spectroscopy is a powerful tool for characterizing the major constituent groups of compounds. The FT-IR spectrum of the product prepared in Example 1 is shown below. Figure 1 As shown, several major absorption bands appeared in the FT-IR spectrum. The major absorption bands of the FT-IR spectrum of the product prepared in Example 2 are consistent with those of the product prepared in Example 1, located at 3241, 3072, 2965, 1660, 1455, 1225, and 813 cm⁻¹, respectively. -1 This indicates that the prepared product is TeA.

[0045] 2. Nuclear magnetic resonance identification NMR is a relatively effective method for analyzing and identifying the structure of compounds, and is often used for sample preparation identification and purity determination. The TeA solid prepared in Example 1 was dissolved in CDCl3 (99.9% purity). 1 H and 13 C NMR spectrum as follows Figure 2 As shown, the NMR spectrum of the TeA prepared in Example 2 is as follows. Figure 3 As shown, this further confirms that the prepared product is TeA. (A represents TeA) 1 H NMR spectrum, B is 13 (C NMR spectrum) 3. Ultraviolet Identification UV spectroscopy of the preparations in Examples 1 and 2 Figure 1 To, as Figure 4 As shown, the prepared product exhibits ultraviolet absorption at 219.5 nm and 277.1 nm, further confirming that the prepared product is TeA.

[0046] 4. Time-of-flight mass spectrometry identification LC-Q-TOF / MS conditions: ACQUITY BEH C 18 The chromatographic column (2.1 mm × 100 mm, 1.7 μm, Waters Corporation, USA) was used. Mobile phase A was acetonitrile, and mobile phase B consisted of 3 mmol / L ammonium acetate and 0.1% formic acid-water. Gradient elution was used: 0–2 min, 5%A–15%A; 2–8 min, 15%A–60%A; 8–13 min, 60%A–90%A; 13–16 min, 90%A–90%A; 16–16.5 min, 90%A–5%A; 16.5–18 min, 5%A–5%A. The flow rate was 0.4 mL / min, and the injection volume was 5 μL. Nebulization was performed using an electrospray ionization (ESI) source at 550 °C and a spray voltage of 3.5 kV (ESI). + ) and 5.5 kV (ESI) –The declustering potential and collision energy are 80 V and 10 V, respectively; the scanning range is 100~900 (m / z); the fragmentation voltage is 1.5 V, the scanning force is 3500 Pa; the sheath pressure is 45 Pa; and the auxiliary pressure is 15 Pa.

[0047] The obtained TeA preparations were fully scanned in both positive and negative ion modes, yielding single, high-precision chromatographic peaks in both cases. Further precursor ion scans were performed on the characteristic peaks in both positive and negative ion modes. In positive ion mode, the spectra of the compounds obtained in Examples 1 and 2 were essentially consistent, with the main molecular ion peak at 198.11 [M+H]. + The main secondary fragments of the main molecular ion peaks are 142.04, 125.02, and 153.09, further confirming that the prepared product is TeA (the spectrum of Example 1 is...). Figure 5 (A is the primary mass spectrum, and B is the secondary mass spectrum).

[0048] 5. Ultra-high performance liquid chromatograph with diode array detector (UPLC-PDA) full scan Chromatographic conditions: ACQUITY BEH C 18 Chromatographic column (2.1 mm × 100 mm, 1.7 μm, Waters Corporation, USA); mobile phase A was acetonitrile, and mobile phase B was 0.1% formic acid aqueous solution; 0–30.0 min, 5%A–100%A; 30.0–30.5 min, 100%A–90%A; 30.5–35.0 min, 90%A–90%A; 35.0–35.5 min, 90%A–40%A; 35.5–40.0 min, 5%A–5%A; flow rate 0.4 mL / min; injection volume 1 μL; UV detector; wavelength 210 nm–400 nm.

[0049] Purity analysis of the TeA preparation obtained by UPLC-PDA full scanning showed that the purity of TeA, as determined by the area normalization method, reached over 99% (the chromatogram of the preparation in Example 1 is shown below). Figure 6 The chromatogram of the product prepared in Example 2 is as follows: Figure 7 ).

[0050] In summary, the present invention provides an efficient method for preparing TeA, with a cultivation cycle of only 10 days. The optimized potato-glucose-agar / water culture medium extract contains fewer impurities, protecting the chromatographic column, improving substance stabilization and preparation efficiency, significantly reducing production costs, and achieving a purity of over 99%. It can be used as a standard for the detection, control, and toxicological research of TeA in food and feed.

Claims

1. A method for preparing Alternaria alterniflora ketoacid, characterized in that... The method includes the following steps: (1) Inoculate Alternaria alternata ATCC 36381 onto potato-dose-agar medium and activate it in the dark at 25-28℃ for 5 days; (2) Take a small piece from the mycelium of the above potato-glucose-agar medium and inoculate it into a new medium for culture. The culture conditions are: potato-glucose-agar medium or potato-glucose-water medium, initial pH of medium 5.0-6.5, add L-isoleucine or L-phenylalanine, and culture in the dark at 25-28℃ for 10 days. (3) Transfer the culture medium to an Erlenmeyer flask, add acetonitrile / water, 50 / 50, v / v, sonicate for 30 min, and then vortex at 180 rpm for 30 min. Repeat this step and collect the extract. (4) Filter the extract into a rotary evaporator flask using gauze, and adjust the rotary evaporator until all the liquid has evaporated to dryness; (5) Preliminary purification: The concentrated solid was redissolved in pure water and transferred to a separatory funnel. An equal volume of n-butanol was added, and the mixture was shaken vigorously. The mixture was then allowed to stand and allow the liquid to separate into layers. The upper layer was collected. This step was repeated. The n-butanol layer collected after extraction was concentrated to dryness by rotary evaporation to obtain the solid substance. Or: The concentrated solid was redissolved in pure water and transferred to an Erlenmeyer flask. 1 / 100 volume of DA201 macroporous resin was added, and the mixture was shaken at 120 rpm for 10 h to adsorb TeA. Then, the adsorbed solid was adsorbed using an ethanol / water ratio of 90 / 10, v / v. The adsorbed solid was collected. The adsorbed solid was concentrated to dryness by rotary evaporation to obtain the solid substance. (6) Reverse-phase preparation: The solid substance was dissolved in acetonitrile / water at a ratio of 1 / 1, v / v, and then filtered through a 0.22 μm filter membrane. The preparation was carried out in a single step using a medium-pressure preparative liquid phase under the following conditions: C 18 AQ-N preparative chromatographic column, 20 mm × 250 mm, 5 μm; mobile phase A is 0.1% formic acid aqueous solution, B is acetonitrile, flow rate is 15 mL / min, detection wavelength is 278 nm, injection volume is 1 mL, collect the distillate after 9-10 min, concentrate the distillate to dryness by rotary evaporation to obtain solid substance; (7) Gel chromatography separation: The solid was dissolved in methanol / dichloromethane, 1 / 1, v / v, and then added to an LH-20 gel chromatography column. The mobile phase was methanol / dichloromethane, 1 / 1, v / v. The pale yellow TeA segment was collected, and the collected liquid was evaporated to dryness to obtain the solid substance. (8) Normal phase preparation: The solid was dissolved in hexane / isopropanol, 4 / 1, v / v, and then filtered through a 0.22 μm filter membrane. Secondary normal phase preparation was performed using medium-pressure preparative liquid chromatography. The Ultimate SiO2 preparative column was 21.2 mm × 250 mm, 5 μm. The mobile phase A was hexane and B was isopropanol. The flow rate was 12 mL / min, the detection wavelength was 277 nm, the injection volume was 0.2 mL, and the fraction was collected after 7.0-8.0 min. Mix all the distillate liquids, dry them with nitrogen at room temperature in the dark, and then dry them in a freeze vacuum dryer to obtain a yellow oily substance, which is TeA.

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