Method for preparing pinocembrin from litchi kernel and application thereof

Abstract

This invention discloses a method for preparing pine resin from litchi seeds and its applications, belonging to the field of agricultural product processing. The structural formula of pine resin is shown in formula (I). This invention uses an enzyme-assisted ultrasonic extraction method to prepare and isolate pine resin from litchi seeds, with a yield of 25.3–181.3 mg / kg (purity 89–97%). Activity experiments demonstrate that this compound has a significant inhibitory effect on erastin-induced HT22 cell damage, and can effectively improve and prevent ferroptosis-related diseases, showing broad application prospects. This invention helps to increase the added value and market competitiveness of litchi seeds, and is of great significance for promoting the intensive processing and utilization of litchi and fostering the sustainable development of the industry.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product processing, specifically relating to a method for preparing pine resin from litchi seeds and its application. Background Technology

[0002] Lychee, belonging to the Sapindaceae family, is known as the "King of Fruits in Lingnan" and is one of my country's distinctive tropical and subtropical fruits. With its unique flavor, delicious taste, and rich nutrition, lychee is beloved by consumers. my country is the world's earliest country to cultivate lychee, boasting the largest cultivation area and yield globally. According to the "2023 China·Maoming Lychee Industry Development White Paper," China's lychee planting area in 2023 was estimated at approximately 7.9 million mu (about 567,000 hectares), with a yield of about 3.29 million tons. Lychee seeds, also known as lychee kernels or lychee pits, are the mature seeds of the lychee fruit. They possess the effects of soothing the liver and regulating qi, promoting qi circulation and relieving stagnation, and dispelling cold and pain. They can be used to treat hernia, testicular swelling and pain, chronic stomach pain, dysmenorrhea, and postpartum abdominal pain. Lychee seeds are rich in various active ingredients, exhibiting antioxidant, anti-inflammatory, blood sugar-lowering, and blood lipid-lowering effects. However, as a waste product from lychee processing, lychee seeds have low utilization rates, resulting in significant resource waste. Therefore, it is necessary to further explore the added value of lychee seeds and promote the comprehensive utilization of lychee.

[0003] Pisorcinol is a natural flavonoid compound. Previous studies have found that pisorcinol possesses excellent biological activities, such as antioxidant, antitumor, anti-inflammatory, lipid-lowering, and neuroprotective effects, and can be widely used in the food, pharmaceutical, and cosmetic industries, showing broad application prospects. Currently, reports on pisorcinol mainly focus on plants such as propolis, ginger family, and oregano. Some studies have also shown that litchi seeds contain small amounts of pisorcinol, but there are currently no reports on the isolation and purification of pisorcinol from litchi seeds. Previous research in this invention has revealed that the content of pisorcinol in litchi seeds is extremely low, and conventional extraction methods are insufficient to achieve high yields. Therefore, it is necessary to conduct research on the preparation process, structural identification, and activity evaluation of pisorcinol from litchi seeds, which will help improve the added value and market competitiveness of litchi seeds and promote the development of the litchi processing industry. Summary of the Invention

[0004] Therefore, the first objective of this invention is to provide a method for preparing pine resin from litchi seeds, which not only significantly improves the yield of pine resin but also yields a product with high purity.

[0005] The jugain of the present invention has the structural formula shown in formula (I):

[0006]

[0007] Formula (I).

[0008] The method for preparing pine resin from litchi seeds includes the following steps: litchi seed powder is added to 3-10 times its volume of ultrapure water, shaken evenly, and then a 0.5%-3% (v / v) dissolving enzyme solution is added. The mixture is reacted at 40-50°C for 5-15 h, followed by ultrasonic extraction with 2-4 times its volume of methanol or ethanol solution. The extract is concentrated to remove methanol or ethanol, and then extracted successively with petroleum ether and ethyl acetate. The ethyl acetate phase is concentrated to obtain an extract. The extract is subjected to C18 reversed-phase column chromatography with methanol / water as the solvent, eluting from a volume ratio of 20 / 80 to 95 / 5. The fraction eluted with methanol / water at a volume ratio of 60 / 40 is collected. This fraction is subjected to gel column chromatography with methanol as the elution solvent. The main fraction is collected, concentrated, and dried to obtain pine resin.

[0009] Preferably, the litchi seed powder is litchi seed powder obtained by grinding fresh litchi seeds with liquid nitrogen or by sun-drying or oven-drying and then pulverizing them through a 60-mesh sieve.

[0010] Preferably, the lysing enzyme solution is a plant complex hydrolase Viscozyme L (Sigma-Aldrich).

[0011] Preferably, the ultrasonic extraction is performed at 50-70°C for 5-30 minutes, with an ultrasonic frequency of 20 kHz and an ultrasonic power of 200 W.

[0012] Preferably, the sequential extraction with petroleum ether and ethyl acetate involves first extracting with petroleum ether 3 to 5 times, and then extracting with ethyl acetate 3 to 8 times.

[0013] Preferably, the flow rate for gradient elution in the C18 reversed-phase column chromatography is 2.0~5.0 mL / min, and the detection wavelength is 280 nm.

[0014] A second objective of this invention is to provide the application of the above-described method for preparing pine resin from litchi seeds in the preparation of products for the prevention and treatment of ferroptosis-related diseases.

[0015] Preferably, the ferroptosis is erastin-induced ferroptosis.

[0016] Preferably, the product includes pharmaceuticals.

[0017] Preferably, the cell is a neuronal HT22 cell.

[0018] A third objective of this invention is to provide a product for the prevention and treatment of ferroptosis-related diseases, comprising pinealin prepared according to the above-described preparation method as an active ingredient and a pharmaceutically acceptable carrier.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The method of this invention extracts pine resin from litchi seeds, significantly improving the yield and purity of pine resin. The yield is 25.3~181.3 mg / kg, and the purity is 89~97%. Activity experiments show that the pine resin has a significant inhibitory effect on erastin-induced HT22 cell damage, and can effectively improve and prevent ferroptosis-related diseases, showing broad application prospects.

[0021] 2. High-purity arborin with significant ferroptosis-inhibiting effect was isolated from litchi seeds, expanding the pathway for obtaining arborin from natural sources.

[0022] 3. A modern enzymology and ultrasound combined technology was developed to extract and separate monomeric components with good biological activity from litchi seeds, promote the deep processing and utilization of litchi seeds, turn waste into treasure, improve the comprehensive utilization of litchi, enhance the added value of the litchi industry, and promote the sustainable development of the litchi industry. Attached Figure Description

[0023] Figure 1 This is a secondary mass spectrum of gerundine (I).

[0024] Figure 2 It is of the genus 1 (I) 1 H nuclear magnetic resonance spectrum.

[0025] Figure 3 The effect of geraniol on erastin-induced HT22 cell viability is shown in the figure, where different letters indicate significant differences in the mean (p < 0.05). Detailed Implementation

[0026] To make the technical problem to be solved and the technical solution of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0027] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0028] Example 1: Isolation, purification and structural identification of galactopyrin

[0029] Fresh litchi seeds were ground with liquid nitrogen, and the powder was added to 3 times (v / m) of ultrapure water. After shaking and homogenization, 3% (v / v) Viscozyme L solution was added, and the mixture was reacted at 45℃ for 6 h. Subsequently, 4 times the volume of methanol solution (v / v) was added for ultrasonic extraction at a frequency of 20 kHz and a power of 200 W (50℃, 20 min). The extract was concentrated to remove methanol, and then extracted 5 times with petroleum ether and 7 times with ethyl acetate. The ethyl acetate phase was concentrated to obtain a paste, which was then subjected to C18 reversed-phase column chromatography with methanol / water as the solvent, eluting from a volume ratio of 20 / 80 to 95 / 5 at a flow rate of 4.0 mL / min, with each gradient eluting for 4 column volumes. The detection wavelength was 280 nm. nm, the fraction eluted with methanol / water at a volume ratio of 60 / 40 was collected. This fraction was then subjected to gel column chromatography (methanol as the elution solvent), and the major fraction was collected. The fraction was then concentrated and dried by evaporation at 40–60 °C. The substance was identified as galenacin by mass spectrometry and nuclear magnetic resonance spectroscopy. Mass spectrometry data can be found in [link to mass spectrometry data]. Figure 1 The nuclear magnetic resonance spectroscopy data can be found in Figure 2 And Table 1 (sample dissolved in deuterated dimethyl sulfoxide).

[0030] The yield of pine resin obtained by this method was 62.4–181.3 mg / kg (calculated on a dry weight basis based on litchi seeds), and the purity was 90–95%.

[0031] Table 1 shows the proton NMR spectrum of geraniol (I). 1 H-NMR data

[0032] No. <![CDATA[ 1 H-NMR(ppm)]]> 2 5.59 (dd, 1H, J=3.0, 13.0) 3 2.78 (dd, 1H, J=3.0, 12.0), 3.37 (dd, 1H, J=12.0, 17.5) 6 5.89 (d, 1H, J=2.0) 8 5.91 (d, 1H, J=2.0) 2’, 6’ 7.52 (d, 2H, J=7.0) 3’, 4’, 5’ 7.37~7.45 (m, 3H) 5-OH 12.13 (s, 1H) 7-OH 10.84 (s, 1H)

[0033] Example 2: Isolation, purification and structural identification of galactopyrin

[0034] Fresh lychee seeds were dried and then crushed using a traditional Chinese medicine pulverizer. The powder was passed through a 60-mesh sieve. After sieving, ultrapure water was added at a ratio of 8:1 (v / m), and the mixture was shaken evenly. Then, 0.5% (v / v) Viscozyme L solution was added, and the mixture was reacted at 45℃ for 15 h. Subsequently, 2:1 (v / v) methanol solution was added for ultrasonic extraction at a frequency of 20 kHz and a power of 200 W (60℃, 30 min). The extract was concentrated to remove methanol, and then extracted 3 times with petroleum ether and 8 times with ethyl acetate. The ethyl acetate phase was concentrated to obtain a paste. The paste was subjected to C18 reversed-phase column chromatography with methanol / water as the solvent, eluting at a gradient from 20 / 80 to 95 / 5 (v / v) at a flow rate of 5.0 mL / min, with 4 column volumes eluted per gradient. The detection wavelength was 280 nm. nm, collect the fraction eluted with methanol / water at a volume ratio of 60 / 40, the fraction is subjected to gel column chromatography (methanol as the elution solvent), collect the main component, evaporate and concentrate at 40~60℃ and dry, the substance is identified as geraniol by mass spectrometry and nuclear magnetic resonance spectroscopy.

[0035] The yield of pine resin obtained by this method was 25.3–106.9 mg / kg (calculated on a dry weight basis based on litchi seeds), and the purity was 89–97%.

[0036] Example 3: Isolation, purification and structural identification of galactopyrin

[0037] Fresh litchi seeds were dried and then crushed using a traditional Chinese medicine pulverizer. The powder was passed through a 60-mesh sieve. After sieving, ultrapure water was added at a ratio of 6:1 (v / m), and the mixture was shaken until homogeneous. Then, 2% (v / v) Viscozyme L solution was added, and the mixture was reacted at 45℃ for 10 h. Subsequently, 3:1 (v / v) methanol solution was added for ultrasonic extraction at a frequency of 20 kHz and a power of 200 W (70℃, 10 min). The extract was concentrated to remove methanol, and then extracted 4 times with petroleum ether and 5 times with ethyl acetate. The ethyl acetate phase was concentrated to obtain a paste. The paste was subjected to C18 reversed-phase column chromatography with methanol / water as the solvent, eluting at a gradient ratio of 20 / 80 to 95 / 5 at a flow rate of 4.0 mL / min, with 4 column volumes eluted for each gradient. The detection wavelength was 280 nm. nm, collect the fraction eluted with methanol / water at a volume ratio of 60 / 40, the fraction is subjected to gel column chromatography (methanol as the elution solvent), collect the main component, evaporate and concentrate at 40~60℃ and dry, the substance is identified as geraniol by mass spectrometry and nuclear magnetic resonance spectroscopy.

[0038] The yield of pine resin obtained by this method was 46.8–125.2 mg / kg (calculated on a dry weight basis based on litchi seeds), and the purity was 89–96%.

[0039] Example 4: Isolation, purification and structural identification of galactopyrin

[0040] Fresh litchi seeds were ground with liquid nitrogen, and the powder was mixed with 3 times (v / m) ultrapure water. After shaking, 3% (m / v) pectinase (Sigma-Aldrich) was added, and the mixture was reacted at 45℃ for 6 h. Subsequently, 4 times the volume of methanol solution (v / v) was added for ultrasonic extraction at a frequency of 20 kHz and a power of 200 W (50℃, 20 min). The extract was concentrated to remove methanol, and then extracted 5 times with petroleum ether and 7 times with ethyl acetate. The ethyl acetate phase was concentrated to obtain a paste, which was then subjected to C18 reversed-phase column chromatography with methanol / water as the solvent, eluting from a volume ratio of 20 / 80 to 95 / 5 at a flow rate of 4.0 mL / min, with 4 column volumes eluted for each gradient. The detection wavelength was 280 nm. nm, collect the fraction eluted with methanol / water at a volume ratio of 60 / 40, the fraction is subjected to gel column chromatography (methanol as the elution solvent), collect the main component, evaporate and concentrate at 40~60℃ and dry, the substance is identified as geraniol by mass spectrometry and nuclear magnetic resonance spectroscopy.

[0041] The yield of pine resin obtained by this method was 5.7–19.2 mg / kg (calculated on a dry weight basis based on litchi seeds), and the purity was 78–90%.

[0042] Example 5: Isolation, purification and structural identification of galactopyrin

[0043] Fresh litchi seeds were ground with liquid nitrogen, and the powder was extracted with 4 times its volume of methanol solution (v / v) using ultrasonic extraction at a frequency of 20 kHz and a power of 200 W (50℃, 20 min). The extract was concentrated to remove methanol, and then extracted 5 times with petroleum ether and 7 times with ethyl acetate. The ethyl acetate phase was concentrated to obtain an extract, which was then subjected to C18 reversed-phase column chromatography with methanol / water as the solvent, eluting at a gradient from 20 / 80 to 95 / 5 (v / v) at a flow rate of 4.0 mL / min, with each gradient eluting 4 column volumes. The detection wavelength was 280 nm. The fraction eluted with methanol / water at a volume ratio of 60 / 40 was collected and subjected to gel column chromatography (methanol as the elution solvent). The main component was collected, concentrated, and dried by evaporation at 40–60℃. The substance was identified as galactopyrin by mass spectrometry and nuclear magnetic resonance spectroscopy.

[0044] The yield of pine resin obtained by this method was 1.4–4.9 mg / kg (calculated on a dry weight basis based on litchi seeds), and the purity was 82–90%.

[0045] Example 6: HT22 cell culture

[0046] The HT22 cell line is a mouse hippocampal neuronal cell line. This cell line is an excellent model for in vitro studies of glutamate toxicity and has proven effective in many neurodegenerative diseases, such as Alzheimer's Disease and Parkinson's Disease. HT22 cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum, 50 units / mL penicillin, and 50 µg / mL streptomycin. Cells were cultured at 37°C in 5% CO2. Medium was changed and passaged as needed based on cell growth.

[0047] Example 7: Effect of geraniol on erastin-induced viability of HT22 cells

[0048] Erastin is a common cell death inducer. It works by directly inhibiting the activity of the cysteine / glutamate antitransporter system (Xc-), activating endoplasmic reticulum stress, thereby reducing glutathione levels, inducing lipid peroxidation, and ultimately inducing cell death. Erastin-induced cell death is called ferroptosis. Ferrroptosis is closely related to central nervous system injury.

[0049] In cell experiments, the above-mentioned geraniol (prepared in Example 1) was prepared into a 100 mM stock solution using dimethyl sulfoxide (DMSO) and stored at -20°C for later use. Cell viability was detected using the Cell Counting Kit-8 (CCK8) reagent kit. The CCK8 kit is a widely used kit for rapid and highly sensitive detection of cytotoxicity and cell proliferation based on 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt (WST-8). In the presence of electron coupling reagents, WST-8 can be reduced by some dehydrogenases in mitochondria to form water-soluble orange-yellow crystalline formazan. The greater the cytotoxicity, the lighter the color; while the more and faster the cell proliferation, the darker the color. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, which can indirectly reflect the number of viable cells. Within a certain cell number range, the absorbance value is linearly related to the cell number.

[0050] Take 100 μL of HT22 cells in logarithmic growth phase (5 × 10⁻⁶ cells). 3 Cells were seeded per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. After discarding the growth medium (DMEM high-glucose medium as described in Example 6), 100 μL of medium containing different concentrations (0.5 μM, 1.0 μM, 2.0 μM, 4 μM, 8 μM, and 10 μM) of the present invention's terpineol and 400 μM erastin (containing 0.1% DMSO) was added to the control wells. An equal volume of DMEM high-glucose medium containing 0.1% DMSO was added to the control wells. After further incubation for 12 h, the medium was discarded, and 110 μL of DMEM medium containing CKK8 solution (CKK8 solution:DMEM medium = 1:10, v / v) was added. The plates were incubated at 37°C in the dark for 1 h, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated according to Formula II. The experiment was repeated three times.

[0051] Formula II

[0052] Among them: A 对照 This indicates cells treated with only 0.1% DMSO medium; A 样品 This indicates that the cells were treated with geraniol or 0.1% DMSO and erastin.

[0053] The results are as follows Figure 3As shown in the figure. The results indicated that erastin treatment significantly induced HT22 cell death, with a cell survival rate of only 29.36% in the model group (0 μM arbutin concentration). Higher concentrations of arbutin (2–10 μM) significantly increased erastin-induced neuronal cell damage, raising the HT22 cell survival rate to 40.41%–89.42%, exhibiting a concentration gradient effect.

[0054] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A method for preparing pine resin from litchi seeds, characterized in that, Includes the following steps: Litchi seed powder was mixed with 3-10 times its volume of ultrapure water and shaken until homogeneous. Then, 0.5%-3% (v / v) of Viscozyme L, a plant complex hydrolase, was added and reacted at 40-50℃ for 5-15 h. Subsequently, 2-4 times its volume of methanol or ethanol solution was added and ultrasonically extracted at 50-70℃ for 5-30 min. The extract was concentrated to remove methanol or ethanol. It was then extracted 3-5 times with petroleum ether and 3-8 times with ethyl acetate. The ethyl acetate phase was concentrated to obtain an extract. The extract was subjected to C18 reversed-phase column chromatography with methanol / water as the solvent, eluting from a volume ratio of 20 / 80 to 95 / 5. The fraction eluted with methanol / water at a volume ratio of 60 / 40 was collected. This fraction was then subjected to gel column chromatography with methanol as the elution solvent. The main fraction was collected, concentrated, and dried to obtain pine oleanolic acid.

2. The method for preparing pine resin from litchi seeds according to claim 1, characterized in that, The litchi seed powder mentioned above is litchi seed powder made by grinding fresh litchi seeds with liquid nitrogen or by sun-drying or oven-drying and then pulverizing them through a 60-mesh sieve.

3. The method for preparing pine resin from litchi seeds according to claim 1, characterized in that, The C18 reversed-phase column chromatography was performed with a gradient elution flow rate of 2.0~5.0 mL / min and a detection wavelength of 280 nm.

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