A method for preparing total phenolic amines from bee pollen and its application in mitigating zearalenone toxicity.

Abstract

This invention discloses a total phenolic amine from bee pollen, its preparation method, and its application in alleviating zearalenone (ZEA) toxicity, belonging to the field of agricultural technology. The total phenolic amines extracted from bee pollen by this invention can reduce the bioaccumulation of ZEA and alleviate organ pathological damage; it can inhibit lipid peroxidation by activating the Nrf2 / GPX4 signaling pathway, upregulate BTB core protein, and restore the integrity of the barrier function; furthermore, TPP can regulate sex hormone balance, reverse the ZEA-induced decrease in testosterone levels and abnormal increase in estradiol, thereby comprehensively alleviating the toxic effects of ZEA.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, and in particular to a total phenolic amine in bee pollen, its preparation method, and its application in alleviating the toxicity of zearalenone in zeaxanthin. Background Technology

[0002] Bee pollen is an oval-shaped substance collected by bees from the male flowers of flowering plants, incorporating secretions from their glands, saliva, and nectar during the collection process. It contains all the nutrients necessary for plant growth, serving as bee food for the colony and an essential source of nutrition for royal jelly production and reproduction. Clinical trials have shown that bee pollen has multiple health benefits. Consuming bee pollen helps improve sleep quality, relieve nervous tension, prevent cardiovascular diseases, and regulate blood lipid levels. It can also optimize gastrointestinal function, enhance digestion and absorption, and significantly improve habitual constipation. Furthermore, it is effective in regulating the endocrine system, boosting immunity, and slowing the aging process, and helps prevent anemia, diabetes, and fatigue. Bee pollen has extremely wide applications in nutritional products, cosmetics, and medicine, and is known as a "treasure among natural foods," "edible cosmetics," "green gold, the essence of plant life," and "a natural miniature nutrient storehouse." Single-flower pollen is usually classified according to the main pollen source plant collected by bees. For example, lindenbee pollen has a variety of physiological functions, such as lowering blood lipids, inhibiting cancer cells, regulating the central nervous system, relieving fatigue, preventing prostate and cardiovascular diseases, and regulating human immunity. It can also prevent high blood sugar, effectively improve brain function to activate brain cell growth, and stimulate most areas of the brain, enabling people to concentrate better, improve work efficiency, and enhance memory.

[0003] Phenolamides, also known as polyamine derivatives, are the main phenolic components of plant reproductive organs and seeds. They play important functional roles in plant growth, development, and resistance to environmental damage as metabolic intermediates or end products. High concentrations of phenolamides are mostly closely related to the growth of their organs, especially flower formation and development. Phenolamide compounds have been detected in the anthers or pollen of almost all angiosperms. Multiple reports indicate that bee pollen contains abundant phenolamides; for example, Mongolian oak pollen contains 18 phenolamides, Mimosa pollen contains 13, and rapeseed pollen contains 13. Eighteen phenolamides have been identified in bee pollen from Colombia, Italy, and Spain, and 64 phenolamides have been identified in 20 Chinese bee pollen samples. Structurally, phenolamides are formed by one or more hydroxycinnamic acids (such as p-coumaric acid, ferulic acid, and caffeic acid) linked to polyamines (such as putrescine, cadaverine, spermidine, and spermine) via amide bonds. The types and contents of phenolic amines vary greatly among different pollen varieties. Their structural diversity depends primarily on the type of hydroxycinnamoyl substitution (coumaryl, caffeoyl, or sinapicoyl, etc.), the degree of substitution of the polyamine chain (mono-, di-, tri-, or tetra-substituted, etc.), and the length of the polyamine acceptor carbon chain (putrescine, spermidine, or spermine, etc.). However, different plant sources have different metabolic pathways, resulting in significant differences in the types of phenolic amines observed in the pollen of single-flowered bees.

[0004] Zearalenone (ZEA) is an estrogen-like toxin produced by Fusarium fungi. It is widely present in the environment, readily contaminating grain feed and posing a serious threat to animal health. Its chemical structure is stable, allowing it to enter animals and humans through feed and the food chain, causing various toxic effects. It is one of the major mycotoxins threatening livestock production and human health globally. Statistics from the Food and Agriculture Organization of the United Nations (FAO) show that approximately 30%-40% of grains worldwide are contaminated with mycotoxins to varying degrees, with ZEA contamination rates exceeding 70% in hot and humid climates. ZEA contamination is widespread and diverse. Major contaminated crops include corn, wheat, barley, oats, sorghum, rice, and legumes (such as soybeans). During food processing, ZEA remains in byproducts such as flour, bread, and beer, and due to its heat-resistant properties (requiring heating at 110°C for over 1 hour to destroy it), conventional processing methods are insufficient to completely remove it. The fibrous parts of moldy grains (such as bran and germ) have even higher ZEA concentrations after processing. Furthermore, after animals ingest feed contaminated with ZEA, its metabolites can be transferred to milk and eggs, further threatening human health.

[0005] In recent years, the widespread contamination of ZEA in feed ingredients has endangered public health and safety. ZEA not only significantly reduces the reproductive performance of boars through estrogen-like effects, manifesting as typical symptoms such as pathological atrophy of testicular tissue, increased apoptosis rate of spermatogenic cells, and decreased sperm motility, but it may also threaten human health through the food chain.

[0006] In addition to essential nutrients such as carbohydrates, proteins, lipids, vitamins, and minerals, bee pollen also contains abundant plant compounds, including phenolic acids, flavonoids, and phenolic amines. These plant compounds are the material basis for the numerous functional activities of bee pollen. Phenolic amines have many health benefits, including combating chronic prostatitis and benign prostatic hyperplasia, inhibiting tyrosinase, preventing diabetes, protecting nerves, and exhibiting significant antioxidant and anti-inflammatory activities. However, no research has yet shown that phenolic amines in bee pollen can alleviate ZEA-induced reproductive toxicity. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing total phenolic amines in bee pollen and their application in alleviating zearalenone (ZEA) toxicity, thereby addressing the problems existing in the prior art. Current academic research does not fully understand the mechanism of ZEA-induced testicular damage in pigs, and existing prevention methods cannot effectively remove ZEA from feed; therefore, there are currently no effective means to prevent this phenomenon. In view of this situation, and given that phenolic amines in bee pollen have shown significant antioxidant stress resistance in current research, this invention innovatively proposes a total phenolic amine intervention strategy in bee pollen.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] One of the technical solutions of this invention is a method for preparing total phenolic amines from bee pollen, comprising the following steps:

[0010] (1) Defatted bee pollen was extracted by ultrasonic extraction, the extract was centrifuged, and the supernatant was collected by filtration; the extracts were combined after repeated extraction; the extracts were concentrated under reduced pressure to obtain crude extract concentrate;

[0011] (2) Add distilled water to the concentrate, mix with an equal volume of ethyl acetate for extraction and dehydration, and evaporate under reduced pressure to dryness to obtain the extract;

[0012] (3) Take the extract and load it onto a 200-300 mesh silica gel column. Elute with a gradient of dichloromethane-methanol-0.5% acetic acid, with each gradient having an elution volume of 600 mL. Monitor the thin-layer chromatography. The developing solvent is chloroform-methanol-acetic acid. Simultaneously spot N-trans-p-coumaryltyramine reference standard. Collect the fraction with a ratio shift value Rf≈0.4 consistent with the reference standard. Concentrate under reduced pressure to obtain an off-white crude product.

[0013] (4) The off-white crude product was loaded onto an ODS-AQ medium-pressure column, and the loading amount was controlled to be 0.5% to 1% of the column volume. The product was eluted with a methanol-water-formic acid gradient at a flow rate of 10 mL / min. The fractions with a retention time of 8.9 min and UV spectra consistent with the reference standard were combined by analytical HPLC and freeze-dried to obtain the enriched product.

[0014] (5) The Shimadzu LC-20AP preparation system was used, with a Shiseido Prep ODS-BP column. The mobile phase A was 0.1% formic acid aqueous solution, and B was 0.1% formic acid-acetonitrile solution. The gradient program was: 0-15 min, 20%→30% B; 15-25 min, 30%→40% B; 25-30 min, 40%→90% B; the flow rate was 10 mL / min, and the detection wavelength was 310 nm. The enriched product was dissolved in 20% acetonitrile aqueous solution and then loaded onto the sample. The target peak components from 29.5 to 31.0 min were collected by peak purity analysis. The same components were combined, concentrated under reduced pressure, and freeze-dried to obtain the total phenolic amines of bee pollen.

[0015] The second technical solution of the present invention is the total phenolic amines in bee pollen prepared by the preparation method.

[0016] The third technical solution of the present invention is the application of the total phenolic amines in bee pollen in the preparation of drugs that alleviate the toxicity of zearalenone.

[0017] The fourth technical solution of the present invention is a drug for alleviating the toxicity of zearalenone, comprising the total phenolic amines in bee pollen.

[0018] Based on the above technical solution, the present invention has the following technical effects:

[0019] This invention discloses a total phenolic amine from bee pollen, its preparation method, and its application in alleviating the toxicity of zearalenone (ZEA). The total phenolic amines extracted from bee pollen by this invention can reduce the bioaccumulation of ZEA and alleviate organ pathological damage; by activating the Nrf2 / GPX4 signaling pathway, it inhibits lipid peroxidation, upregulates the BTB core protein, and restores the integrity of the barrier function; in addition, TPP can regulate the balance of sex hormones, reverse the ZEA-induced decrease in testosterone levels and abnormal increase in estradiol, thereby comprehensively alleviating the toxic effects of ZEA. Attached Figure Description

[0020] Figure 1 This is the UV-Vis spectrum of N-trans-p-coumaryltyramine. Where Abs represents absorbance.

[0021] Figure 2This is the infrared scan spectrum of N-trans-p-coumaryltyramine. Wherein, Wavenumber: wavenumber, used to represent the wavenumber of electromagnetic waves in infrared spectra, etc.; Transmittance: transmittance, reflecting the proportion of a substance's ability to transmit radiation such as light.

[0022] Figure 3 This is the mass spectrum of N-trans-p-coumaryltyramine.

[0023] Figure 4 The NMR spectrum of N-trans-p-coumaryltyramine is shown.

[0024] Figure 5 The effect of TPP on serum testosterone levels in pigs poisoned by ZEA (n=6). * indicates that compared with the CON group, 0.01 < P < 0.05, ** indicates that P < 0.01, # indicates that compared with the ZEA group, 0.01 < P < 0.05, and ## indicates that P < 0.01. The same applies below.

[0025] Figure 6 The effect of TPP on the changes in estradiol levels in pig serum induced by ZEA poisoning (n=6).

[0026] Figure 7 The effect of TPP on the changes in progesterone levels in pig serum induced by ZEA poisoning (n=6).

[0027] Figure 8 The effects of TPP on the changes in organ coefficients in pigs induced by ZEA poisoning are shown. Specifically, a) represents the effect of TPP on the liver coefficient in pigs induced by ZEA poisoning; b) represents the effect of TPP on the kidney coefficient in pigs induced by ZEA poisoning; and c) represents the effect of TPP on the testicular coefficient in pigs induced by ZEA poisoning.

[0028] Figure 9 The histological changes in porcine heart induced by TPP poisoning. Magnification: 100×.

[0029] Figure 10 The image shows the histopathological changes in the spleen of pigs induced by TPP-induced ZEA poisoning. Blue arrow: Lymph nodes. Magnification: 100×.

[0030] Figure 11 The image shows the histopathological changes in pig kidneys induced by TPP-induced ZEA poisoning. Red arrow: swelling of renal tubular epithelial cells. Magnification: 100×.

[0031] Figure 12 The image shows the histopathological changes in pig liver induced by TPP-induced ZEA poisoning. Green arrow: balloon-like swelling of hepatocytes. Magnification: 100×.

[0032] Figure 13The histopathological changes in the porcine testis induced by TPP poisoning with ZEA. Red arrow: vacuolar structure; yellow arrow: detachment of luminal cells. Magnification: 100×.

[0033] Figure 14 The image shows the histopathological changes in the porcine jejunum induced by TPP-induced ZEA poisoning. Blue arrows indicate broken intestinal villi and increased gap between the epithelial tissue and lamina propria. Magnification: 40×.

[0034] Figure 15 The effect of TPP on oxidative stress parameters in porcine testicular tissue induced by ZEA poisoning was investigated (n=6). Specifically, a) the effect of TPP on MDA content in porcine testicular tissue induced by ZEA poisoning, as detected by TBA method; b) the effect of TPP on H2O2 content in porcine testicular tissue induced by ZEA poisoning, as detected by visible spectrophotometry; and c) the effect of TPP on GSH content in porcine testicular tissue induced by ZEA poisoning, as detected by colorimetric method.

[0035] Figure 16 To detect changes in iron content in porcine testicular tissue induced by ZEA poisoning using a colorimetric method (n=6).

[0036] Figure 17 The mRNA expression levels of Keap1, Nrf2, and GPX4 in porcine testicular tissue induced by TPP poisoning (n=6) were shown. Among them, a represents the mRNA expression level of Keap1 in porcine testicular tissue; b represents the mRNA expression level of Nrf2 in porcine testicular tissue; and c represents the mRNA expression level of GPX4 in porcine testicular tissue. Detailed Implementation

[0037] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0038] This invention provides a method for preparing total phenolic amines from bee pollen, comprising the following steps:

[0039] (1) Defatted bee pollen was extracted by ultrasonic extraction, the extract was centrifuged, and the supernatant was collected by filtration; the extracts were combined after repeated extraction; the extracts were concentrated under reduced pressure to obtain crude extract concentrate;

[0040] (2) Add distilled water to the concentrate, mix with an equal volume of ethyl acetate for extraction and dehydration, and evaporate under reduced pressure to dryness to obtain the extract;

[0041] (3) Take the extract and load it onto a 200-300 mesh silica gel column. Elute with a gradient of dichloromethane-methanol-0.5% acetic acid, with each gradient having an elution volume of 600 mL. Monitor the thin-layer chromatography. The developing solvent is chloroform-methanol-acetic acid. Simultaneously spot N-trans-p-coumaryltyramine reference standard. Collect the fraction with a ratio shift value Rf≈0.4 consistent with the reference standard. Concentrate under reduced pressure to obtain an off-white crude product.

[0042] (4) The off-white crude product was loaded onto an ODS-AQ medium-pressure column, and the loading amount was controlled to be 0.5% to 1% of the column volume. The product was eluted with a methanol-water-formic acid gradient at a flow rate of 10 mL / min. The fractions with a retention time of 8.9 min and UV spectra consistent with the reference standard were combined by analytical HPLC and freeze-dried to obtain the enriched product.

[0043] (5) The Shimadzu LC-20AP preparation system was used, with a Shiseido Prep ODS-BP column. The mobile phase A was 0.1% formic acid aqueous solution, and B was 0.1% formic acid-acetonitrile solution. The gradient program was: 0-15 min, 20%→30% B; 15-25 min, 30%→40% B; 25-30 min, 40%→90% B; the flow rate was 10 mL / min, and the detection wavelength was 310 nm. The enriched product was dissolved in 20% acetonitrile aqueous solution and then loaded onto the sample. The target peak components from 29.5 to 31.0 min were collected by peak purity analysis. The same components were combined, concentrated under reduced pressure, and freeze-dried to obtain the total phenolic amines of bee pollen.

[0044] In some specific implementations, in step (1), the ultrasonic extraction conditions are as follows: using 80% ethanol with a volume fraction of 0.05% ascorbic acid as the extraction solvent, the ratio of bee pollen to extraction solvent is 1:10 (g:mL), ultrasonic extraction is performed at 25℃ and 300W for 30min; the centrifugation conditions are as follows: centrifugation at 3500r / min for 10min; the ratio of the volume of the concentrated product to the defatted bee pollen is 1:1 (mL:g).

[0045] In some specific implementations, in step (2), the volume ratio of the concentrate to distilled water is 1:2.5.

[0046] In some specific implementations, in step (3), the conditions for elution with dichloromethane-methanol-0.5% acetic acid gradient are 95:5:0.5→90:10:0.5→85:15:0.5→80:20:0.5, v / v / v; and the volume ratio of chloroform-methanol-acetic acid is 80:20:1.

[0047] In some specific implementations, in step (4), the conditions for elution with methanol-water-formic acid gradient are 40:60:0.1→45:55:0.1→50:50:0.1→55:45:0.1→60:40:0.1, v / v / v.

[0048] In some specific implementations, in step (5), the freeze-drying conditions are: pre-freezing at -80°C for 1 hour, followed by freeze-drying at 66Pa / -58°C for 24 hours.

[0049] The present invention also provides total phenolic amines in bee pollen prepared by the preparation method described above.

[0050] This invention also provides the application of the total phenolic amines from bee pollen in the preparation of drugs to alleviate the toxicity of zearalenone.

[0051] This invention also provides a drug for alleviating the toxicity of zearalenone, comprising the total phenolic amines from bee pollen.

[0052] This invention constructs a ZEA-poisoned Luchuan pig model to observe the regulatory effects of total phenolamides in bee pollen (TPP) on testicular histopathology, sex hormone levels, and oxidative damage markers. For the first time, it reveals the molecular mechanism of testicular damage induced by TPP in pigs from the perspective of ferroptosis and verifies the intervention effect of TPP through the Nrf2 / GPX4 pathway. This natural product-based detoxification strategy not only provides a new approach to overcoming reproductive barriers in pigs but also holds promise for reducing treatment costs, alleviating the economic burden on farmers, and improving economic efficiency.

[0053] Example 1

[0054] 1. Experimental materials:

[0055] 1.1 Sample Collection:

[0056] Linden bee pollen was collected in July 2024 from Yangmuqiao Village, Qinghe Town, Ji'an City, Jilin Province.

[0057] 1.2 Obtaining total phenolic amines from bee pollen:

[0058] Total phenolic amines in bee pollen refer to natural polyamine compounds, primarily phenolic amines, extracted from bee pollen. The main component is N-trans-p-coumaryltyramine. Total phenolic amines in bee pollen are prepared in the laboratory, and the main operational steps are as follows:

[0059] ① Weigh 200g of defatted bee pollen and add 80% ethanol containing 0.05% ascorbic acid at a material-to-liquid ratio of 1:10 (g:mL). Extract the pollen in a 25℃, 300W ultrasonic cleaner for 30min. Centrifuge the extract at 3500r / min for 10min, filter and collect the supernatant. Repeat the extraction of the residue under the same conditions for 20min, and combine the two extracts. Concentrate the crude extract to 200mL using a rotary evaporator (40℃, 0.08MPa vacuum) to obtain a concentrated crude extract.

[0060] ② Dilute the concentrate with distilled water at a volume ratio of 1:2.5, mix with an equal volume of ethyl acetate, shake for 10 min, let stand for 20 min to separate the layers, and collect the upper organic phase; repeat the extraction 3 times, combine the organic phases, dehydrate with anhydrous sodium sulfate, and evaporate to dryness under reduced pressure at 35℃ to obtain the extract.

[0061] ③ Take the above extract and load it onto a 200-300 mesh silica gel column (100cm×5cm). Elute with a gradient of dichloromethane-methanol-0.5% acetic acid (95:5:0.5→90:10:0.5→85:15:0.5→80:20:0.5, v / v / v), with each gradient having an elution volume of 600mL. Monitor by thin-layer chromatography (TLC). The developing solvent is chloroform-methanol-acetic acid (80:20:1, v / v / v). Simultaneously spot N-trans-p-coumaryltyramine reference standard. Collect fractions with the same Rf value (Rf≈0.4) as the reference standard and concentrate under reduced pressure to obtain a white crude product.

[0062] ④ The purified product from the silica gel column was loaded onto an ODS-AQ medium-pressure column (20–45 μm, 50 cm × 2.6 cm), with the loading volume controlled at 0.5%–1% of the column volume. Elution was performed using a methanol-water-formic acid gradient (40:60:0.1→45:55:0.1→50:50:0.1→55:45:0.1→60:40:0.1, v / v / v) at a flow rate of 10 mL / min. Fractions with a retention time of 8.9 min and UV spectra (280–320 nm) consistent with the reference standard were combined by analytical HPLC and freeze-dried to obtain the enriched product.

[0063] ⑤ A Shimadzu LC-20AP preparative system was used, with a Shiseido Prep ODS-BP column (250 mm × 20 mm, 5 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid-acetonitrile solution. The gradient program was: 0–15 min, 20% → 30% B; 15–25 min, 30% → 40% B; 25–30 min, 40% → 90% B; flow rate 10 mL / min; detection wavelength 310 nm. 5–8 mg of the enriched sample was dissolved in 20% acetonitrile aqueous solution and loaded onto the sample. Peak purity analysis was performed, and the target peak fraction from 29.5–31.0 min was collected. Identical fractions were combined, concentrated under reduced pressure at 35 °C, pre-frozen at -80 °C for 1 h, and freeze-dried at 66 Pa / -58 °C for 24 h to obtain 18.7 mg of a white purified total phenolic amine.

[0064] Using the above method, 200g of linden bee pollen yielded 18.7mg of phenolic amine compound after extraction and purification, resulting in a yield of 93.5μg / g, meaning that 93.5 micrograms of phenolic amine compound can be extracted from each gram of raw material. The obtained phenolic amine compound has high purity and few impurities.

[0065] Structural identification of 1,3N-trans-p-coumaryltyramine

[0066] (1) Ultraviolet analysis

[0067] The structure of the purified phenolic amine compound from linden bee pollen prepared above was identified. A small amount of the purified phenolic amine sample was taken and prepared with methanol (MEOH) as solvent to a concentration of 0.01 mg / ml. The phenolic amine solution sample was scanned using a TU-1810 ultraviolet spectrophotometer. Figure 1 The absorbance (Abs) at different wavelengths (190-900 nm) is shown in Table 1. The purified compound exhibits characteristic absorption peaks at 225.00 nm, 292.00 nm, and 309.00 nm, with an absorption valley at 248.00 nm. The absorption peaks near 292.00 nm and 309.00 nm can be attributed to the π→π* transition of the conjugated system formed by the coumarin double bond (C=C) and the aromatic ring in the molecule, a typical UV absorption characteristic of this type of coumarin-containing compound, suggesting the presence of a coumarin structural unit in the molecule. The absorption peak at 225.00 nm may be related to the E2 band absorption of the aromatic ring, further confirming the presence of the aromatic ring structure in the molecule. These characteristics provide important evidence for the structural characterization of this purified compound.

[0068] Table 1. UV Analysis of N-trans-p-coumaryltyramine

[0069]

[0070] (2) Infrared analysis

[0071] A small amount of purified phenolamine sample was mixed with 200 mg of potassium bromide, pressed into 1 mm thick sheets, and then analyzed using Fourier transform infrared spectroscopy. The infrared spectral analysis results are shown below. Figure 2 The results show that the spectrum is located between 500 and 3500 cm⁻¹. -1 Multiple characteristic absorption peaks are observed within the range, mainly including 3432.38, 3305.70, and 3189.36 cm⁻¹. -1 The absorption at [location] (possibly attributable to the OH / NH stretching vibration of the phenolic hydroxyl or amide group) is observed at 3024.29 and 2942.04 cm⁻¹. -1 Absorption at 1660.70 cm⁻¹ (possibly corresponding to aromatic ring and saturated CH stretching vibrations), 1660.70 cm⁻¹ -1 The strong absorption at 1622.01 and 1603.87 cm⁻¹ (possibly due to the C=O stretching vibration of the amide carbonyl group) is observed. -1 The absorption at 1535.11 cm⁻¹ (possibly related to the C=C stretching vibration of the trans double bond and the skeletal vibration of the aromatic ring) and 1513.78 cm⁻¹ -1 The absorption at [location] (further confirming the aromatic ring structure), and at 1243.27 and 1049.21 cm⁻¹ -1 Absorption at the location (possibly due to CO stretching vibration).

[0072] The results of ultraviolet analysis, along with the characteristic absorptions of the amide carbonyl group, trans double bond, and aromatic ring in the infrared spectrum, corroborate the absorption characteristics of the conjugated system in the ultraviolet spectrum. This suggests that the compound contains structural units such as p-coumaryl group (containing trans double bond and para-substituted aromatic ring), amide bond, and phenolic hydroxyl group, which are consistent with the structural characteristics of N-trans-p-coumaryltyramine, providing important evidence for its structural characterization.

[0073] (3) Mass spectrometry analysis

[0074] The purified phenolamine sample was detected using LC / MS. The mass spectrometry analysis results are shown below. Figure 3 The results show that characteristic peaks of m / z 284.1 and m / z 285.1 appear in the positive ion spectrum at a retention time of 0.802 min. Among them, m / z 284.1 can be attributed to the protonated molecular ion peak [M+H] of this compound. + The molecular weight is consistent with that of N-trans-p-coumaryltyramine. In DAD analysis by liquid chromatography, the area percentages of the main peak with a retention time of 0.708 min at wavelengths of 254 nm and 214 nm were 99.60% and 100.00%, respectively, indicating high purity of this component in the sample. The molecular weight information from mass spectrometry and the main peak purity data from liquid chromatography corroborate each other, suggesting that the structure of the main component in the sample is consistent with that of N-trans-p-coumaryltyramine, providing important evidence for the structural characterization of this compound.

[0075] (4) Nuclear magnetic resonance analysis

[0076] The NMR spectra of the purified phenolamine were acquired using a Bruker 400MHz NMR spectrometer. The results are shown in the figure. Figure 4 The results showed that the spectrum exhibited multiple characteristic signals in the 3-10 ppm range. Among them, the multiple peaks in the aromatic region (6.777-8.044 ppm, such as 6.777, 6.798, 7.002, 7.022, 7.292, 7.331, etc.) could be attributed to protons on the benzene ring in the coumaroyl and tyramine groups, reflecting the spin system characteristics of the para-substituted benzene ring; the signals in the 6.377-6.692 ppm range (such as 6.377, 6.417, 6.672, 6.6...)... 92) The protons are presumed to be from a trans double bond (-CH=CH-), consistent with the structural characteristics of a coumaroyl group; the series of peaks from 2.507 to 4.038 ppm (e.g., 2.507, 2.624, 3.314, 3.331, 3.348, 3.378, etc.) may correspond to the methylene (-N-CH2-) protons bonded to the nitrogen atom; the low-field signals at 9.200 and 9.850 ppm may be the active hydrogen protons of the phenolic hydroxyl (-OH) or amide (-NH-) groups. The above 1H NMR data are consistent with the molecular structural characteristics of N-trans-p-coumaryltyramine and can serve as important evidence for the structural characterization of this compound.

[0077] Combined with ultraviolet ( Figure 1 ), infrared ( Figure 2 Mass spectrometry information Figure 3 ) and NMR information ( Figure 4 Based on the spectral information, the phenolic amine compound extracted by this method is N-trans-p-coumaryltyramine, 284.1 m / z [M+H]. + The molecular formula is C 17 H 17 NO3 has a high purity.

[0078] 2. Animal experiments

[0079] This experiment used 24 male Luchuan miniature pigs, aged 30 days.

[0080] 2.1 Animal model establishment and grouping:

[0081] Twenty-four male Luchuan miniature pigs were housed in cages, one pig per cage, for a pre-feeding period of one week until their weight reached approximately 2.0 ± 0.28 kg before the experiment began. The 24 experimental pigs were randomly divided into four groups of six pigs each. The grouping and treatment methods are shown in Table 2.

[0082] Table 2 Animal Grouping (n=6)

[0083]

[0084] The drug was administered once daily for a total of 28 days. ZEA (98%) was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), and TPP was prepared in the laboratory and extracted, separated, and purified from bee pollen using the method described in 1.2.

[0085] 2.2 Effects of total phenolic amines from bee pollen on changes in serum testosterone levels in pigs induced by zearalenone

[0086] To assess the effects of TPP and ZEA on porcine sex hormone levels, serum sex hormone levels were measured in this study. The biochemical results for serum testosterone (T) levels are shown below. Figure 5 Experimental data showed that, compared to the CON group, the serum testosterone concentration in the ZEA group was significantly lower (P<0.05), which was consistent with the clinical manifestation of anestrus. After TPP intervention, the testosterone level in the ZEA / TPP group significantly increased compared to the ZEA group (P<0.05), suggesting that TPP may have a potential role in antagonizing the reproductive toxicity of ZEA. The results indicate that TPP has a therapeutic effect on the decrease in serum testosterone levels in pigs induced by ZEA.

[0087] 2.3 Effects of total phenolic glycosides from bee pollen on changes in serum estradiol levels in pigs induced by zearalenone

[0088] Comparative analysis of serum estradiol (E2) concentration data showed that ( Figure 6 Compared to the CON group, the serum estradiol level in the ZEA group showed a significant upward trend (P<0.05). Further analysis revealed that the estradiol concentration in the ZEA / TPP group was significantly lower than that in the ZEA group (P<0.05). This data suggests that TPP intervention can effectively antagonize the abnormal increase in serum estradiol induced by ZEA, demonstrating its regulatory role in sex hormone homeostasis.

[0089] 2.4 Effects of total phenolic amines from bee pollen on changes in serum progesterone levels in pigs induced by zearalenone.

[0090] Comparative analysis of serum progesterone (PGN) concentration data can be found in [link to relevant data]. Figure 7 Compared with the CON group, the serum PGN level in the ZEA group was statistically significantly lower (P<0.01). The PGN concentration in the ZEA / TPP group showed an upward trend compared to the ZEA group, but the difference was not statistically significant (P>0.05). This data pattern suggests that TPP may have a potential role in alleviating ZEA-induced progesterone secretion suppression.

[0091] 2.5 Changes in organ coefficients in pigs induced by zearalenone poisoning from total phenols in bee pollen

[0092] To clarify whether ZEA poisoning causes morphological changes in the liver, kidneys, and testes of pigs, and whether TPP can alleviate these organ changes, this experiment measured the body weight of pigs in the CON group, ZEA group, TPP group, and ZEA / TPP group. Liver, kidney, and testes were then collected from each of the four groups for weighing and statistical analysis. The results are as follows: Figure 8 As shown in the figure, compared with the CON group, the liver and kidney volumes in the ZEA group were significantly larger, and their organ indices showed statistically significant differences (P<0.05). The liver and kidney organ indices in the ZEA / TPP group were significantly lower than those in the ZEA group (P<0.01), indicating that ZEA can cause changes in the morphology and weight of pig liver and kidneys, and that TPP can have a certain therapeutic effect. Meanwhile, compared with the CON group, the testes in the ZEA group were smaller, lighter, and had a lower organ coefficient. After TPP treatment, the testicular coefficient increased significantly (P<0.05).

[0093] 2.6 Pathological and histological changes in porcine heart induced by zearalenone poisoning from total phenolic amines in bee pollen

[0094] like Figure 9 As shown in the microscopic observation, the heart sections of the CON group pigs showed no overall inflammatory cell infiltration, myocardial degeneration (such as vacuolation or necrosis), hemorrhage, or thrombosis, and the tissue structure of each layer was intact, consistent with the morphological characteristics under normal physiological conditions. Compared with the CON group, the cardiomyocytes of the ZEA group pigs had clear striations, neatly arranged striated muscle fibers, elongated oval nuclei centered, and a step-like structure at the junction of intercalated discs without breakage. A small number of erythrocytes were visible between the myocardial fibers.

[0095] 2.7 Pathological and histological changes in pig spleen induced by zearalenone poisoning from total phenols in bee pollen

[0096] like Figure 10 As shown, the porcine spleen tissue sections exhibited a typical lymphoid organ structure, consisting of a capsule, white pulp, red pulp, and a marginal zone. Microscopic observation revealed that the reticular fibers in the interstitium of the CON group were evenly distributed, without hemorrhage, inflammatory cell infiltration, or amyloid deposition, and the overall structure was intact, consistent with the morphological characteristics of a normal spleen. Compared with the CON group, the ZEA group showed scattered distribution of white pulp in the pig spleen, including periarterial lymphatic sheaths and lymphoid nodules with diffuse hemorrhage. The smooth muscle layer of the central arterial wall was clear, and the peripheral lymphocytes were sparser and more deeply stained than those in the CON group. The red pulp consisted of splenic cords and splenic sinuses. The splenic cords were rich in lymphocytes, macrophages, and erythrocytes, while the endothelial cells of the splenic sinuses were elongated spindle-shaped with blue-stained nuclei. The sinus cavities were open, without congestion or thrombosis. The marginal zone, located at the junction of the white and red pulp, had a lower lymphocyte density, with a small number of scattered macrophages. After TPP intervention, the above pathological changes improved.

[0097] 2.8 Histopathological changes in pig kidneys induced by zearalenone poisoning from total phenols in bee pollen

[0098] like Figure 11 As shown in the microscopic observation, in the CON group, numerous glomeruli were visible in the cortical region of the pig kidneys, with open capillary loops, clear Bowman's capsule cavities, and surrounding proximal and distal convoluted tubules. The tubular structure was intact and clearly visible. The medullary region consisted of the descending and ascending limbs of the loop of Henle and the collecting ducts, with regular lumens. The epithelial cells of the collecting ducts were tall columnar with round, neatly arranged nuclei. Interstitial vessels were evenly distributed without congestion or hemorrhage. The connective tissue was sparse, without inflammatory cell infiltration, fibrosis, or calcification, consistent with the morphological characteristics of a normal kidney. Compared with the CON group, the ZEA group showed enlarged glomeruli, swollen tubular (proximal convoluted tubule) epithelial cells that bulged into the lumen (red arrows), and narrowed tubular spaces. After TPP treatment, the swelling of the tubular epithelial cells was relieved, indicating that TPP can reverse the damage to pig kidney tissue caused by ZEA.

[0099] 2.9 Histopathological changes in pig liver caused by zearalenone poisoning from total phenols in bee pollen

[0100] like Figure 12 As shown in the microscopic observation, the liver tissue sections of the CON group pigs exhibited a typical lobular structure, with hepatocyte cords arranged radially around the central vein. The hepatocytes were polygonal, with abundant, eosinophilic cytoplasm, large, round, centrally located nuclei, and no vacuolar degeneration or necrosis. Hepatic sinusoids were clearly visible, with flattened endothelial cells and blue-stained nuclei, and the sinus cavities were patent. The portal region of the TPP group contained interlobular bile ducts, interlobular arteries, and interlobular veins, with intact structures and no inflammatory infiltration or fibrosis. The central vein wall was thin, and the surrounding hepatocytes showed no congestion or lipofuscin deposition. The interstitial connective tissue was sparse, without fibrosis or pseudolobule formation, and the overall structure conformed to the morphological characteristics of a normal liver. In contrast, the hepatocyte cord structure of the ZEA pig liver was unclear, the hepatocytes were significantly swollen, the cytoplasm was pale, and some hepatocytes swelled like balloons (green arrows). After TPP treatment, the above pathological phenomena were alleviated.

[0101] 2.10 Histopathological changes in pig testes induced by zearalenone poisoning from total phenols in bee pollen

[0102] like Figure 13As shown, microscopic observation revealed that the testicular tissue sections of the CON group exhibited a typical spermatogenic structure: the seminiferous tubules were tightly arranged, with the tubule walls composed of multiple layers of spermatogenic cells and supporting cells. The nuclei of the spermatogenic cells varied in depth, and the branched cytoplasm of the supporting cells at their base was clearly visible, with triangular or oval nuclei. In the interstitial tissue, testicular interstitial cells were clustered, with abundant eosinophilic cytoplasm, large, round, and centrally located nuclei, surrounded by capillaries and loose connective tissue, without inflammatory infiltration or fibrosis. In the testicular tissue sections of the TPP group, spermatocytes were observed close to the lumen, and mature sperm could be seen extending their tails into the lumen. The seminiferous tubules were patent, without spermatogenic arrest or accumulation of detached cells. The interstitial vessels showed no congestion or hemorrhage, and the overall structure was intact, consistent with the morphological characteristics of a normal testis. In contrast, the number of spermatogonia in the seminiferous tubules of the ZEA group was sparse, with numerous vacuolated structures (red arrows), and detached tubular cells accumulated within the seminiferous tubules (yellow arrows). After TPP treatment, the above pathological changes were alleviated.

[0103] 2.11 Histopathological changes in the jejunum of pigs induced by total phenolic amines from bee pollen poisoning with zearalenone

[0104] like Figure 14 As shown in the microscopic observation, the jejunal tissue sections of the CON group pigs exhibited a typical layered structure of the small intestine: the mucosa layer formed dense, finger-like villi, covered with a single layer of columnar epithelium, with clear striated borders visible on the free surface; the lamina propria contained numerous intestinal glands, with columnar and goblet cells as the glandular epithelium, and a small number of lymphocytes infiltrating at the base; the muscularis mucosae consisted of a thin layer of smooth muscle, arranged neatly; the submucosa was loose connective tissue containing blood vessels and nerve plexuses, without edema or fibrosis. Overall, no villi atrophy, diffuse infiltration of inflammatory cells, hemorrhage, or necrosis was observed; the structure was intact, consistent with the morphological characteristics of a normal jejunum. Compared with the CON group, the jejunal tissue sections of the ZEA group pigs showed partial villi breakage and an increased gap between the epithelial tissue and the lamina propria (blue arrows). After TPP treatment, the above pathological changes were alleviated.

[0105] 2.12 Changes in oxidative stress indicators in porcine testicular tissue induced by zearalenone poisoning from total phenols in bee pollen

[0106] To investigate whether ZEA causes oxidative stress damage to porcine testicular tissue and whether TPP can exert an antioxidant effect in testicular tissue, this experiment used an MDA kit, an H2O2 kit, and a GSH kit to detect the oxidative stress level in porcine testicular tissue. The results are as follows: Figure 15As shown, compared with the CON group, the ZEA group showed increased MDA and H2O2 content and decreased GSH content in the testicular tissue of pigs, with significant differences in all three (P < 0.05); compared with the ZEA group, the ZEA / TPP group showed significantly increased GSH content in the testicular tissue of pigs (P < 0.05), while the MDA and H2O2 content showed a decreasing trend, but the differences were not significant.

[0107] 2.13 Changes in iron ion content in porcine testicular tissue induced by zearalenone poisoning from total phenols in bee pollen

[0108] To clarify the effects of TPP and ZEA on iron ion content in porcine testicular tissue, this study used a commercially available kit to detect the total iron content in porcine testicular tissue. The results are as follows: Figure 16 As shown in the figure, iron metabolism analysis of testicular tissue revealed a significantly increased iron accumulation level in the testes of pigs in the ZEA group compared to the CON group (P<0.05). After TPP intervention, the testicular iron concentration in the ZEA / TPP group significantly decreased compared to the ZEA group (P<0.01). These results indicate that TPP can have a certain therapeutic effect on the increased total iron content in pig testicular tissue caused by ZEA poisoning.

[0109] 2.14 Changes in Nrf2 / GPX4 protein expression levels in porcine testicular tissue induced by zearalenone poisoning from total phenolamines in bee pollen

[0110] To investigate the mechanism by which TPP alleviates ZEA-induced ferroptosis in porcine testicular tissue, the mRNA expression levels of Keap1, Nrf2, and GPX4 in porcine testicular tissue were measured using real-time quantitative PCR. The results are as follows: Figure 17 As shown in the analysis compared with the CON group, the transcription level of Keap1 gene in porcine testicular tissue was significantly upregulated in the ZEA group (P < 0.05), while the mRNA expression of the antioxidant regulator Nrf2 and its downstream target gene GPX4 was significantly downregulated (P < 0.05). The ZEA / TPP group showed a significant reversal effect: the expression level of Keap1 mRNA was significantly downregulated compared with the ZEA group (P < 0.01), while the expression of Nrf2 and GPX4 genes was significantly restored (P < 0.01), suggesting that TPP may reverse ZEA-induced oxidative damage by activating the Nrf2 signaling pathway.

[0111] In summary, this invention, by establishing a ZEA poisoning model in male Luchuan pigs, revealed the direct damage of ZEA to the pig's reproductive system, liver and kidney function, and immune organs, as well as its disruptive effect on the integrity of the blood-testis barrier (BTB) and oxidative stress balance. Furthermore, by using TPP as a therapeutic agent, the mechanism by which TPP alleviates ZEA poisoning was revealed through an antioxidant pathway.

[0112] (1) Total phenolic amines in bee pollen alleviate liver and intestinal damage in pigs caused by zearalenone poisoning:

[0113] Regarding hepatobiliary function, ZEA significantly increased the liver coefficient. HE staining and microscopic observation revealed significant swelling and irregular arrangement of hepatocytes, central venous dilation and congestion, accompanied by inflammatory cell infiltration and even hepatocyte necrosis. Compared with the CON group, the liver coefficient in the ZEA group was significantly increased. Simultaneously, liver histopathology showed ballooning degeneration of hepatocytes, suggesting that ZEA damages hepatocyte membrane structure through oxidative stress and lipid peroxidation, leading to abnormal liver function. ELISA detection revealed varying degrees of ZEA accumulation in the liver, kidneys, testes, and jejunum of pigs exposed to ZEA, suggesting that this toxin may enter the bloodstream by disrupting the intestinal barrier integrity, subsequently distributing to multiple organs and causing direct toxic damage. Further analysis showed that after TPP intervention, the accumulation of ZEA in the above organs was significantly reduced. Combined with the results of intestinal epithelial barrier structure repair in the TPP group observed in intestinal histopathology, it is considered that TPP can effectively block the enterogenic absorption pathway of ZEA by repairing the intestinal epithelial barrier. ZEA reduces the activity of liver antioxidant enzymes SOD and CAT. This oxidative stress response leads to lipid peroxidation in hepatocytes, increased MDA concentration, and further weakens the liver's ability to metabolize toxins due to liver dysfunction, forming a closed-loop toxicity network in the "gut-liver" cycle.

[0114] (2) Total phenolic amines in bee pollen alleviate kidney damage in pigs caused by zearalenone poisoning:

[0115] Mouse experiments showed that ZEA, alone or in synergy with other toxins, can cause renal dysfunction. Exposure to ZEA significantly increased serum creatinine and blood urea nitrogen levels, indicating impaired glomerular filtration function. Microscopic pathology revealed increased lymphocyte infiltration and apoptosis in renal tissue, associated with endoplasmic reticulum stress and activation of apoptotic pathways. HE staining revealed glomerular enlargement and increased cell number in the ZEA-treated pig kidneys, with swollen tubular (proximal convoluted tubule) epithelial cells protruding into the lumen. Statistical analysis of organ coefficients showed a significantly increased renal coefficient in the ZEA-treated pig kidneys compared to the CON group.

[0116] (3) Total phenolic amines in bee pollen alleviate damage to the porcine blood-testis barrier caused by zearalenone poisoning:

[0117] Experimental results showed that ZEA-induced decreases in the testicular coefficient, damage to seminiferous tubule structure, and ultrapathological changes in Sertoli cell mitochondria indicated that it exacerbated reproductive toxicity through the ferroptosis pathway. The blood-testis barrier is a core structure maintaining the spermatogenic microenvironment, and its integrity depends on the expression and distribution of tight junction proteins between Sertoli cells. Transmission electron microscopy further confirmed that tight junctions between Sertoli cells were broken in the ZEA group, while the structure in the TPP treatment group tended to be normal. This result is closely related to the mechanism by which TPP enhances antioxidant capacity by activating the Nrf2 pathway.

[0118] (4) Total phenolic amines in bee pollen alleviate oxidative stress damage to porcine testicular tissue caused by zearalenone poisoning:

[0119] Following ZEA treatment, MDA levels significantly increased, indicating enhanced lipid peroxidation of the cell membrane. ZEA-induced oxidative stress is a key driver of BTB damage. This invention found that the ZEA group showed significantly increased MDA and H2O2 levels and significantly decreased GSH levels in testicular tissue, suggesting that lipid peroxidation and antioxidant system imbalance jointly exacerbated cell damage. After TPP treatment, GSH levels significantly rebounded, suggesting that it exerts a protective effect by enhancing glutathione synthesis or reducing ROS generation. This phenomenon is consistent with the experimental results of TPP upregulating SLC7A11 expression, indicating that TPP may inhibit ferroptosis by promoting cystine uptake and increasing GSH biosynthesis efficiency.

[0120] (5) Total phenolic amines in bee pollen alleviate hormonal imbalances in pigs caused by zearalenone poisoning:

[0121] ZEA disrupts sex hormone balance by mimicking estrogen effects, leading to a significant decrease in serum testosterone levels and a significant increase in estradiol levels in pigs, directly related to its estrogenic activity. ZEA's estrogen-like activity interferes with reproductive endocrine function through a dual mechanism: its metabolite α-ZOL competitively binds to the estrogen receptor ERα, interfering with the synthesis and metabolism of endogenous hormones, thereby disrupting the regulatory function of the hypothalamic-pituitary-gonadal axis. Furthermore, the estrogenic effect of α-ZOL is approximately 2-4 times stronger than that of ZEA. An in vitro experiment showed that administration of 50 μM ZEA inhibited the activity of 17β-hydroxysteroid dehydrogenase (HSD17B3) and reduced its mRNA expression level, while ZEA concentrations of 10 and 20 mg / kg reduced the protein expression of CYP17A1, a key enzyme in testosterone synthesis. In a transgenerational toxicity study, ZEA caused a decrease in STAR gene expression in rat offspring testes. TPP significantly reversed this effect. Serum testosterone levels were significantly higher in the TPP / ZEA group and significantly lower in the ZEA group than in the TPP / ZEA group. This may be related to the expression of key enzymes in TPP-regulated testosterone synthesis (such as CYP17A1).

[0122] (6) The molecular mechanism by which total phenolic amines in bee pollen inhibit ferroptosis through the Nrf2 / GPX4 pathway:

[0123] The ZEA group exhibited cristae breakage and vacuolation in mitochondria, consistent with the morphological characteristics of ferroptosis. Total iron content analysis of testicular tissue showed a significant increase in iron ion content in the ZEA group, which decreased significantly after TPP intervention. The core mechanism of ferroptosis lies in the imbalance between lipid peroxidation and the antioxidant system, with GSH depletion and GPX4 inactivation as markers. SLC7A11 / SLC3A2 mediates cysteine ​​uptake to synthesize GSH, and GPX4 utilizes GSH to reduce lipid peroxides, its inhibition directly inducing ferroptosis. Polyunsaturated fatty acid peroxidation is the core driving factor, dependent on enzymatic reactions such as lipoxygenase, manifested as increased MDA, GSH depletion, and decreased GPX4 activity.

[0124] Nrf2 is a core transcription factor for antioxidant defense, regulating the expression of key genes such as GPX4 and SLC7A11. This invention found that TPP significantly upregulated Nrf2 and GPX4 protein expression in testicular tissue and reversed the ZEA-induced decrease in SLC7A11 mRNA levels. This indicates that TPP enhances GPX4-mediated lipid peroxide scavenging by activating the Nrf2 pathway, thereby inhibiting ferroptosis. TPP exerts its core antioxidant effect by activating the Nrf2 pathway, through mechanisms including promoting Nrf2 nuclear translocation and inhibiting Keap1 binding to enhance the expression of downstream antioxidant genes, thus improving endogenous antioxidant capacity. In an oxidative damage model, TPP significantly reduced ROS levels, decreased the accumulation of lipid peroxidation product MDA, and maintained mitochondrial membrane potential stability. In summary, ZEA induces multiple damages by disrupting mitochondrial function and redox homeostasis, while TPP provides a key strategy for its toxicity prevention and control through targeted regulation of the Nrf2 / GPX4 pathway.

[0125] In summary, ZEA exposure can induce multi-system damage: physiologically, it manifests as weight loss, diarrhea, and histopathological damage to organs such as the liver, kidneys, and testes, and promotes the accumulation of ZEA in target organs; it promotes ferroptosis in testicular Sertoli cells, disrupts the structural integrity of the BTB; and it interferes with sex hormone homeostasis, manifested as a significant decrease in serum testosterone levels and an abnormal increase in estradiol.

[0126] TPP antagonizes ZEA toxicity through multiple pathways: TPP can reduce the bioaccumulation of ZEA and alleviate organ pathological damage; it can inhibit lipid peroxidation by activating the Nrf2 / GPX4 signaling pathway, upregulate the BTB core protein, and restore the integrity of the barrier function; in addition, TPP can regulate the balance of sex hormones, reverse the ZEA-induced decrease in testosterone levels and abnormal increase in estradiol, thereby comprehensively alleviating the toxic effects of ZEA.

[0127] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method of preparing total phenolic amine of bee pollen, characterized by, The main component of the total phenylamine in the bee pollen is N-trans-p-coumaroyl tyramine; The method comprises the following steps: (1) ultrasonic extraction of defatted bee pollen, centrifugation of the extract, collection of supernatant by suction filtration, repeated extraction, and combination of the extract, concentration under reduced pressure, and obtaining of a concentrated crude extract; (2) adding distilled water to the concentrated extract, mixing with an equal volume of ethyl acetate, extracting, dehydrating, and rotary evaporation under reduced pressure until dryness to obtain an extract; (3) taking the extract, loading it onto a 200-300 mesh silica gel column, gradient elution with dichloromethane-methanol-0.5% acetic acid, each gradient elution volume being 600 mL, monitoring by thin layer chromatography, developing agent being chloroform-methanol-acetic acid, synchronous sample loading of N-trans-p-coumaroyl tyramine reference substance, collection of fractions with Rf≈0.4 consistent with the reference substance, and concentration under reduced pressure to obtain a white-like crude product; the gradient elution conditions of dichloromethane-methanol-0.5% acetic acid are 95:5:0.5→90:10:0.5→85:15:0.5→80:20:0.5, v / v / v; the volume ratio of chloroform-methanol-acetic acid is 80:20:1; (4) loading the white-like crude product onto an ODS-AQ medium pressure column, controlling the loading amount to be 0.5%-1% of the column volume, gradient elution with methanol-water-formic acid at a flow rate of 10 mL / min, monitoring by analytical HPLC, combining fractions with a retention time of 8.9 min and a UV spectrum consistent with the reference substance, and freeze-drying to obtain an enriched product; the gradient elution conditions of methanol-water-formic acid are 40:60:0.1→45:55:0.1→50:50:0.1→55:45:0.1→60:40:0.1, v / v / v; (5) using a Shimadzu LC-20AP preparation system, a Shiseido Prep ODS-BP column, mobile phase A being 0.1% formic acid aqueous solution, B being 0.1% formic acid-acetonitrile solution, gradient program: 0-15 min, 20%→30% B; 15-25 min, 30%→40% B; 25-30 min, 40%→90% B; flow rate 10 mL / min, detection wavelength 310 nm; dissolving the enriched product in 20% acetonitrile aqueous solution and loading it, collecting target peak components of 29.5-31.0 min through peak purity analysis, combining the same components, concentrating under reduced pressure, and freeze-drying to obtain the total phenylamine in the bee pollen.

2. The production method according to claim 1, characterized by, In step (1), the ultrasonic extraction conditions are as follows: 80% ethanol containing 0.05% ascorbic acid is used as the extraction solvent, the solid-liquid ratio of defatted bee pollen to the extraction solvent is 1:10 (g:mL), ultrasonic extraction is carried out at 25°C and 300 W for 30 min; the centrifugation conditions are 3500 r / min for 10 min; and the volume ratio of the concentrated product to defatted bee pollen after concentration under reduced pressure is 1:1 (mL:g).

3. The preparation method according to claim 1, characterized in that, In step (2), the volume ratio of the concentrated extract to distilled water is 1:2.

5.

4. The production method according to claim 1, characterized by, In step (5), the freeze-drying condition is pre-freezing at -80℃ for 1 h and freeze-drying at 66 Pa / -58℃ for 24 h.

5. The use of the total phenolic amine in bee pollen prepared by the method of any one of claims 1-4 in the preparation of a medicine for relieving the toxicity of zearalenone.

Images