Application of plant-derived glyceroglycolipid in preparation of anti-inflammatory products

Abstract

The invention discloses application of plant-derived glyceroglycolipid in preparation of an anti-inflammatory product, inflammation is RAW264.7 cell inflammatory response induced by LPS (lipopolysaccharide), and the plant-derived glyceroglycolipid can resist inflammation by inhibiting expression of inflammatory factors. By combining column chromatography with chromatographic analysis, efficient extraction, precise separation and structural identification of the plant GL are realized, and important technical support is provided for research of plant lipids. The plant GL can significantly inhibit inflammatory response of RAW264.7 macrophages, including reduction of LPS-induced cellular morphological change, down-regulation of inflammatory factor mRNA expression and reduction of CD86 expression level. The invention provides a solid molecular level theoretical basis for the application of the plant glycerol glycolipid in the development of anti-inflammatory functional foods and medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of active substance extraction and application, and specifically relates to the application of a plant-derived glyceroglycolipid in the preparation of an anti-inflammatory product. Background Art

[0002] Inflammation is an immune response triggered by the body in response to infection or tissue damage, typically manifesting as symptoms such as redness, swelling, fever, pain, and dysfunction. This process aims to eliminate harmful substances and repair damaged tissue, thereby restoring the body's normal physiological functions and rebalancing the internal and external environments. However, prolonged or excessive inflammation can disrupt this balance, leading to abnormal activation of the immune system. In particular, in adverse inflammatory immune responses, excessive activation of macrophages leads to the release of a large number of cytokines, including TNF-α, interferon-γ (IFN-γ), IL-1β, IL-6, and interleukin-17a (IL-17a). Excessive release of these cytokines can disrupt the body's immune regulation and trigger a variety of chronic inflammatory diseases, including sepsis, atherosclerosis, and inflammatory bowel disease.

[0003] LPS, a major component of the cell wall of Gram-negative bacteria, can activate macrophages through binding to Toll-like receptor 4 (TLR4), making it a classic research tool for inducing inflammatory responses. In vitro, RAW264.7 cells are frequently used for studying inflammatory mechanisms and screening anti-inflammatory drugs due to their high sensitivity to LPS and their ability to mimic the inflammatory response of macrophages in vivo.

[0004] Plant glycerol glycolipids, as natural bioactive ingredients, possess multiple biological activities, including antiviral, antibacterial, antitumor, and anti-inflammatory properties. However, to date, research on the bioactivity of plant glycerol glycolipids remains limited, with most studies focusing on microbial glycolipids. The role of plant glycerol glycolipids in macrophage-mediated inflammatory responses, particularly their regulatory effects in the LPS-induced RAW264.7 cell model of inflammation, has not been systematically explored. Therefore, studying the anti-inflammatory effects of plant glycerol glycolipids is of great scientific significance. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] One of the objectives of the present invention is to provide a plant-derived glyceroglycolipid for use in the preparation of an anti-inflammatory product, which can significantly inhibit the inflammatory response of RAW264.7 macrophages, providing a solid molecular-level theoretical basis for the application of plant glyceroglycolipids in the development of anti-inflammatory functional foods and drugs.

[0008] To solve the above technical problems, the present invention provides the following technical solution: an application of plant-derived glyceroglycolipid in the preparation of an anti-inflammatory product, wherein the inflammation is an inflammatory response of RAW264.7 cells induced by LPS.

[0009] As a preferred embodiment of the use of the plant-derived glyceroglycolipid of the present invention in the preparation of an anti-inflammatory product, the plant-derived glyceroglycolipid achieves anti-inflammation by inhibiting the expression of inflammatory factors.

[0010] As a preferred embodiment of the use of the plant-derived glyceroglycolipid of the present invention in the preparation of anti-inflammatory products, the inflammatory factors are IL-6, IL-12 and IL-16.

[0011] As a preferred embodiment of the use of the plant-derived glyceroglycolipid of the present invention in the preparation of an anti-inflammatory product, the plant-derived glyceroglycolipid achieves anti-inflammatory effect by inhibiting the expression of CD86.

[0012] As a preferred embodiment of the use of the plant-derived glyceroglycolipids of the present invention in the preparation of anti-inflammatory products, the plants include one or more of water spinach, pakchoy, cabbage, kale, lettuce, and romaine lettuce.

[0013] As a preferred embodiment of the use of the plant-derived glyceroglycolipid of the present invention in the preparation of anti-inflammatory products, the preparation method of the plant-derived glyceroglycolipid comprises:

[0014] Place the plant material in boiling water, crush it after cooling, add chloroform-methanol solution and mix thoroughly;

[0015] The mixed slurry was filtered, chloroform and pure water were added to the filtrate respectively, and the lower layer solution was recovered after standing and stratification;

[0016] Anhydrous sodium sulfate was added to the lower solution, and the solution was allowed to stand for dehydration, and then filtered to obtain the complete lipids;

[0017] Dissolve silica gel in chloroform, stir well and pour into the chromatography column;

[0018] The whole lipids were dissolved in chloroform and added to the chromatography column for elution to collect the glycolipids.

[0019] As a preferred embodiment of the use of the plant-derived glyceroglycolipid of the present invention in the preparation of anti-inflammatory products, the inflammatory factors are IL-1β, IL-6, IL-16, A4galt, FPR1, IL1rn, and IL36a.

[0020] As a preferred embodiment of the use of the plant-derived glyceroglycolipid of the present invention in the preparation of anti-inflammatory products, the plant-derived glyceroglycolipid is monogalactosylglycerol or digalactosyldiacylglycerol.

[0021] As a preferred embodiment of the use of the plant-derived glyceroglycolipid of the present invention in the preparation of anti-inflammatory products, the preparation method of the plant-derived glyceroglycolipid comprises:

[0022] Place the plant material in boiling water, crush it after cooling, add chloroform-methanol solution and mix thoroughly;

[0023] The mixed slurry was filtered, chloroform and pure water were added to the filtrate respectively, and the lower layer solution was recovered after standing and stratification;

[0024] Anhydrous sodium sulfate was added to the lower solution, and the solution was allowed to stand for dehydration, and then filtered to obtain the complete lipids;

[0025] Dissolve silica gel in chloroform, stir well and pour into the chromatography column;

[0026] The whole lipids were dissolved in chloroform and added to the chromatography column for elution to collect the glycolipids;

[0027] Dissolve silica gel in chloroform, stir well and pour into the chromatography column;

[0028] The glycolipid is dissolved in chloroform and added to a chromatography column for elution, and elution is performed using chloroform-acetone solutions in different ratios in sequence to collect digalactosyldiacylglycerol.

[0029] As a preferred embodiment of the use of the plant-derived glyceroglycolipid of the present invention in the preparation of an anti-inflammatory product, the chloroform-methanol solution has a volume ratio of chloroform to methanol solution of 1:2 and is fully mixed at room temperature;

[0030] Said adding chloroform and pure water, the final volume ratio of chloroform:methanol:water is 1:1:0.9;

[0031] The anhydrous sodium sulfate is added in an amount of one tenth of the solvent volume;

[0032] The filtration was followed by drying with nitrogen.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This study, using column chromatography combined with chromatographic analysis, achieves efficient extraction, precise separation, and structural identification of plant GLs, providing important technical support for plant lipid research. Studies have shown that plant GLs can significantly inhibit the inflammatory response of RAW264.7 macrophages, including reducing LPS-induced cell morphological changes, downregulating mRNA expression of inflammatory factors, and reducing CD86 expression. These findings provide a solid molecular-level theoretical basis for the application of plant glycerol glycolipids in the development of anti-inflammatory functional foods and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0036] Figure 1 The total lipid content and various lipid compositions of 8 kinds of plants; among them, (a) is the total lipid content in 100 g of plants; (b) is the proportion of neutral lipids in total lipids; (c) is the proportion of glycolipids in total lipids; (d) is the proportion of phospholipids in total lipids.

[0037] Figure 2 The results of the cell viability experiment of RAW264.7 cells treated with different concentrations of LPS.

[0038] Figure 3 The effects of different plant glycerol glycolipids on the viability of RAW264.7 cells; among them, (a) is cabbage, (b) is kale, (c) is mallow, (d) is Chinese cabbage, (e) is lettuce, (f) is water spinach, (g) is lettuce, and (h) is spinach.

[0039] Figure 4 The morphological characteristics of RAW264.7 cells under different treatment conditions (200×); (a) is the control group; (b) is the LPS group: 1 μg / mL; (c) is the GL group: LPS (1 μg / mL) + GL (500 μg / mL).

[0040] Figure 5 The effect of different plant glycerol glycolipids on IL-6 secretion in RAW264.7 cells.

[0041] Figure 6 The effect of different plant glycerol glycolipids on IL-12 secretion in RAW264.7 cells.

[0042] Figure 7The effect of different plant glycerol glycolipids on IL-16 secretion in RAW264.7 cells.

[0043] Figure 8 This is the effect of glyceroglycolipid in comparative example 1 on the IL-6 secretion of RAW264.7 cells.

[0044] Figure 9 The effect of plant glycerol glycolipids on the expression of surface antigen CD86 of M1 RAW264.7 cells; (a) is the flow cytometry graph, and (b) is the flow cytometry analysis result.

[0045] Figure 10 These are the qualitative results of thin layer chromatography of 8 plant glycerol glycolipids.

[0046] Figure 11 These are the results of thin-layer chromatography of spinach glyceroglycolipid subclasses; among them, 1 is the chloroform elution fraction; 2 is the chloroform-acetone (9:1) elution fraction; 3 is the chloroform-acetone (7:3) elution fraction; 4 is the chloroform-acetone (4:6) elution fraction; 5 is the chloroform-methanol (2:8) elution fraction; and 6 is the acetone elution fraction.

[0047] Figure 12 This is the qRT-PCR verification of the differentially expressed genes in Example 8; among them, (a) is IL-6; (b) is IL-16; (c) is IL-1β; (d) is FPR1; (e) is IL-36a; (f) is IL1rn; and (g) is A4galt. DETAILED DESCRIPTION

[0048] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0049] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0050] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0051] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0052] The experimental materials used in the examples of the present invention are shown in Table 1.

[0053] Table 1

[0054] The cell culture method used in the embodiment of the present invention is:

[0055] (1) Cell culture

[0056] RAW264.7 cells were cultured in complete medium (10% premium fetal bovine serum + 90% high-glucose medium) in a 37°C, 5% CO2 incubator. Fresh medium was replaced every 24 hours. When cells reached confluence (90% adherence was considered optimal), they were pipetted and passaged. For this experiment, RAW264.7 cells in the logarithmic growth phase and in good condition were selected.

[0057] (2) Cell passaging

[0058] Discard the old medium in the T25 flask and wash the cells twice with 3-4 mL of phosphate-buffered saline (PBS). Discard the PBS and use a pipette to remove any cells adhering to the bottom of the flask by pipetting. Transfer the cell suspension to a 10 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant. Resuspend the cells in 2 mL of fresh complete medium. Transfer the cell suspension to a new T25 cell culture flask at a ratio of 1:2 to 1:4, add 4-6 mL of fresh complete medium, and incubate in a 37°C incubator.

[0059] (3) Cell cryopreservation

[0060] Pipette cells from a well-grown T25 culture flask reaching 90% density. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend in 1 mL of freezing buffer (serum:dimethyl sulfoxide = 9:1). Transfer the cells to a cryovial and place them in a programmed cooling box. Place the programed cooling box in a -80°C freezer for 24 hours before transferring the cryovial to liquid nitrogen or a -80°C freezer.

[0061] Example 1

[0062] (1) Plant lipid extraction

[0063] 100 g of plant material (water spinach, lettuce, broccoli, pakchoy, spinach, kale, cabbage, and lettuce) was placed in boiling water for 5 minutes. After drying and cooling, the slurry was ground in a grinder. The slurry was then transferred to a beaker and 30 mL of a 1:2 chloroform-methanol solution was added and mixed thoroughly at room temperature. The mixed slurry was then filtered through a vacuum filtration device. The filtrate was transferred to a separatory funnel, and 10 mL of chloroform and 18 mL of pure water were added. After standing for separation, the lower layer was collected. An appropriate amount of anhydrous sodium sulfate was added to the lower layer, and the solution was allowed to stand overnight for dehydration. After filtration, the chloroform was removed using a rotary evaporator. The slurry was dried with nitrogen and weighed to obtain the total lipids, and the total lipid yield was calculated. The total lipids were reconstituted with a small amount of chloroform and stored at -4°C until use.

[0064] (2) Separation and purification of crude saccharolipids

[0065] Dissolve 20 g of silica gel in 60 mL of chloroform, stir well, and pour into the chromatography column. Subsequently, dissolve 1 g of the whole lipid sample from step (1) in 5 mL of chloroform and add it to the chromatography column. Use 125 mL of chloroform, 150 mL of acetone, and 125 mL of methanol for elution in sequence. The three collected components are neutral lipids, glycolipids, and phospholipids. Use a rotary evaporator to remove the solvent, blow dry with nitrogen, and weigh them. Calculate the yield of each lipid type.

[0066] Figure 1 The contents of total lipids, neutral lipids, glycolipids, and phospholipids in 8 plant species are shown. The total lipid content in each plant sample varies greatly, ranging from 0.64 to 7.09 mg / g. Spinach has the highest glycolipid content in total lipids, reaching 66.12%, followed by wood ear vegetable, which accounts for 52.31%. In contrast, the neutral lipid and phospholipid ratios of these plants are relatively low. This result shows that plants with darker leaf color and more chloroplasts generally have higher total lipid and glycolipid content. The total lipid and glyceroglycolipid content of the three green plants, spinach, lettuce, and kale, are significantly higher than those of relatively lighter green vegetables such as lettuce and pakchoy.

[0067] Example 2

[0068] This example studies the effect of different concentrations of LPS on the viability of RAW264.7 cells. The CCK8 assay was used to determine cell viability. The specific method is:

[0069] (1) Take 5×10 RAW264.7 cells in the logarithmic growth phase and 4 Cells were seeded in a 96-well plate at 100 μL / mL (100 μL / well). 100 μL PBS was added to the outermost wells of the plate to prevent edge effects. 100 μL was added to each well and the plate was cultured in a cell culture incubator at 37°C containing 5% CO2.

[0070] (2) After the cells adhered to the wall and grew, the old culture medium was discarded. The experiment was divided into a blank control group and an LPS group. Only the blank group was not inoculated with cells but only culture medium was added; the concentrations of the LPS group were 0.5, 1, 2, 4, and 8 μg / mL; each concentration was repeated 5 wells, and 100 μL of culture medium was added to each well of the 96-well plate, and the plate was placed in an incubator and cultured for 24 hours.

[0071] (3) Remove the old culture medium and wash the cells twice with PBS, then add 100 μL complete culture medium (containing 10 μL CCK-8) to each well.

[0072] (4) Place the 96-well plate in a cell culture incubator and incubate for 2 hours.

[0073] (5) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0074] Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100;

[0075] Wherein, A(drug added) is the absorbance of the wells with cells, CCK-8 solution, and drug solution;

[0076] A (blank) is the absorbance of the wells with culture medium and CCK-8 solution but no cells;

[0077] A(0 drug addition) is the absorbance of the well containing cells and CCK-8 solution but no drug solution.

[0078] Figure 2 The effect of different LPS concentrations (0, 0.5, 1, 2, 4, and 8 μg / mL) on RAW264.7 cell viability was measured using the CCK8 assay. Cell viability gradually decreased with increasing LPS concentration. When the LPS concentration reached 2 μg / mL, cell viability was significantly reduced compared to the control group, and the difference was statistically significant (P < 0.05). Therefore, an LPS concentration of 1 μg / mL was selected as the standard concentration for inducing inflammatory responses in subsequent experiments.

[0079] Example 3

[0080] In this example, the effects of different types of GL on the viability of RAW264.7 cells were studied. The cell viability was determined using the same CCK8 method as in Example 2. The experiment was divided into a blank control group and different plant glycerol glycolipid groups. Only the blank group was not inoculated with cells but only supplemented with culture medium. The concentrations of the eight plant glycerol glycolipids (i.e., the glycolipids isolated in step (2) of Example 1) were all set to 100, 500, 1000, 1500, 2000, 2500, 3000, and 3500 μg / mL. The other steps were the same as in Example 2.

[0081] Figure 3 The results show the effects of different GLs at different concentrations on RAW264.7 cell viability after 24 hours of treatment. The results show that GLs from different plant sources have a significant effect on cell viability. At low concentrations (e.g., 50-100 μg / mL), cell viability generally remains high. However, as the concentration increases, cell viability decreases significantly. High concentrations (e.g., 500 μg / mL and above) significantly inhibit cell viability, with some treatment groups experiencing almost zero viability. For example, GLs from spinach, lettuce, water spinach, pakchoy, and broccoli maintained normal cell viability compared to the control group at concentrations below 500 μg / mL. However, GLs from jalapenos, cabbage, and kale significantly inhibited RAW264.7 cell viability at concentrations exceeding 300 μg / mL and 100 μg / mL, respectively.

[0082] Example 4

[0083] Normally, RAW264.7 macrophages are round or oval in shape and have no pseudopodia. In this example, the morphological changes of cells in each group were observed under an inverted microscope.

[0084] The experiment was divided into blank control group, LPS group and GL treatment group. The blank group was not inoculated with cells but only added culture medium. The LPS group was treated with LPS at a concentration of 1 μg / mL for 24 hours. The GL treatment group was treated with glyceroglycolipids derived from water spinach at a concentration of 500ug / ml immediately after the addition of LPS at a concentration of 1 μg / mL for 24 hours.

[0085] Figure 4 The morphological changes of RAW264.7 cells under different treatment conditions are shown, revealing the effects of different experimental conditions on cell status from the perspective of cell morphology. Under normal (control) conditions, RAW264.7 cells mainly appear as small round cells with smooth cell edges, evenly distributed cytoplasm, and no obvious pseudopodia ( Figure 4 -a). However, after LPS stimulation, the cell morphology changed significantly, showing a long spindle shape, and a large number of protruding slender pseudopodia appeared on the cell surface ( Figure 4 -b). This change indicates that RAW264.7 cells were activated after LPS stimulation and polarized into M1 macrophages, a pro-inflammatory phenotype. In the GL-treated group, the cell morphology was significantly restored, the number of pseudopodia was significantly reduced, and most cells tended to be round, with a morphology closer to the normal resting state ( Figure 4This phenomenon suggests that GL has the effect of inhibiting LPS-induced M1 polarization, keeping RAW264.7 cells in an unactivated or low-activated state, thereby potentially reducing the release of inflammatory factors and alleviating the inflammatory response.

[0086] Example 5

[0087] This example studies the effects of plant glycerol glycolipids on the secretion of IL-6, IL-12, and IL-16 in RAW264.7 cells.

[0088] Experimental methods:

[0089] (1) Extraction of total cell RNA

[0090] All the following steps were performed on ice:

[0091] RAW264.7 cells with a density of 80-90% and good growth status were seeded in a 12-well plate at a cell seeding density of 2×10 6 The plates were then incubated in an incubator (37°C, 5% CO2) for 24 hours. The old cell culture medium was discarded and the cells were washed twice with PBS. The experimental concentration of glyceroglycolipids was set at 500 μg / mL, and the inflammatory concentration of LPS was set at 1 μg / mL. A control group containing no glyceroglycolipids was used. The cells were co-cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0092] Before the experiment, place autoclaved laboratory supplies in a clean bench under ultraviolet light for 30 minutes. Remove complete culture medium and 1× PBS buffer from a 4°C refrigerator and equilibrate to room temperature. After 30 minutes of ultraviolet light exposure, turn on the clean bench and fan. Disinfect the benchtop with 75% alcohol cotton balls and light an alcohol burner. Pour out the culture medium and rinse once with 1× PBS. Add 500 μL of RNA extraction solution to the cultured cells growing in a 12-well plate, place horizontally for a moment, and pipette thoroughly to completely lyse the cells. Transfer the cell lysate to a 1.5 mL centrifuge tube and observe for complete aspiration. Add 100 μL of chloroform (RNA extraction solution: chloroform = 5:1 (v / v)), vortex for 15-30 seconds, let stand for 5 minutes, and centrifuge at 10,000 × g at 4°C for 15 minutes. The sample will now separate into three layers: a colorless aqueous phase (upper layer), a middle layer, and a pink organic phase (lower layer). The volume of the aqueous phase is approximately 50% of the volume of the RNA extraction solution used. Transfer the colorless aqueous phase to a new centrifuge tube and add 0.5 mL of isopropanol (isopropanol: water = 1:1 (v / v)) for every 1 mL of RNA extraction solution used. Mix by inversion and incubate at room temperature for 10 minutes. Centrifuge at 10,000 × g at 4°C for 10 minutes. Remove the supernatant; a gel-like precipitate will form on the sides and bottom of the tube. Add 1 mL of 75% ethanol (prepared with DEPC-treated water) and vortex vigorously (at least 1 mL of 75% ethanol should be added for every 1 mL of RNA extraction solution used). Centrifuge at 7500 × g at 4°C for 5 minutes. Discard the supernatant, air dry in a hood, and dissolve the pellet in 20–50 μL of RNA dissolution buffer. Incubate at 55–60°C for 10 minutes. Store the sample at -20°C for long-term use.

[0093] (2) Fluorescence quantitative PCR

[0094] (2-1) Reverse transcription reaction:

[0095] (a) Genomic DNA removal

[0096] Prepare the mixture shown in Table 2 in an RNase-free centrifuge tube:

[0097] Table 2

[0098] Gently pipette the prepared reaction mixture to mix thoroughly, centrifuge slowly for 5 seconds, and place in a PCR instrument, setting the conditions to incubate at 42°C for 2 minutes.

[0099] (b) Prepare the reverse transcription reaction system

[0100] Add 5×HiScript II qRT SuperMix directly to the reaction tube in step (a), as shown in Table 3.

[0101] Table 3

[0102] (c) Perform reverse transcription reaction

[0103] Gently pipette the prepared reaction mixture to mix thoroughly and centrifuge slowly for 5 seconds. Place the mixture in a PCR instrument and set the following conditions: incubate at 37°C for 15 minutes, then heat at 85°C for 5 seconds. Add 80 μL of ddH2O to the PCR tube where reverse transcription has been completed, dilute the mixture 5-fold, and store at -20°C.

[0104] (2-2) Fluorescence qPCR reaction

[0105] Synthesis of mouse gene primer sequences: The sequences of the primers synthesized by Shanghai Bioengineering Co., Ltd. are as follows. Before use, the dry powder primers were dissolved and diluted with ddH2O (pH = 8.0) according to the instructions, and the front primer and the back primer were mixed evenly in proportion. The primer list is shown in Table 4.

[0106] Table 4

[0107] (a) Label eight 0.1 mL PCR tubes, remove primers and qPCR kits from a -20°C freezer, and thaw on ice. Vortex all reagents before use and centrifuge slowly before use. qPCR reactions for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), interleukin-6 (IL-6), interleukin-12 (IL-12), and interleukin-16 (IL-16) use the Novozymes qPCR kit. The reaction system is shown in Table 5, and the run protocol is shown in Table 6.

[0108] Table 5

[0109] Table 6

[0110] (b) After preparing the mixture and centrifuging, place the eight-tube strip into the quantitative PCR instrument and set the reaction conditions as shown in the table below. Mark the eight-tube strip on the instrument program. After the program automatically closes the lid, click Run.

[0111] According to the Ct value measured by real-time fluorescence quantitative PCR, the Ct value of the target gene in the test group was subtracted from the Ct value of the internal reference gene in the test group, and the result was the △Ct value. The △△Ct value was obtained by subtracting the △Ct value of the control group from the △Ct value of the experimental group.

[0112] Figure 5This study demonstrated the effects of glyceroglycolipids from different plant sources on IL-6 secretion in RAW264.7 cells. The results showed a significant increase in IL-6 mRNA expression in the LPS-treated group, indicating that LPS can strongly induce an inflammatory response in RAW264.7 cells, shifting them into a pro-inflammatory state. In this LPS-induced inflammation model, treatment with glyceroglycolipids from different plant sources significantly inhibited IL-6 secretion (P<0.05), suggesting that these compounds may have anti-inflammatory properties.

[0113] However, the inhibitory effects of glyceroglycolipids from different plant sources on IL-6 expression varied, with the Auricularia auricula treatment exhibiting a relatively weaker inhibitory effect than the other plant-treated groups. This result suggests that plant-derived glyceroglycolipids may influence IL-6 secretion through different regulatory mechanisms, with their anti-inflammatory effects varying depending on the plant source.

[0114] Figure 6 The study demonstrated the effects of plant-derived glyceroglycolipids on IL-12 mRNA expression in RAW264.7 cells. The results showed that IL-12 expression levels were significantly elevated in the LPS-treated group (P < 0.05). In the LPS-induced inflammation model, plant-derived glyceroglycolipids from different sources all inhibited IL-12 expression, but the inhibitory effects varied. Lettuce, kale, pakchoy, and cabbage exhibited stronger inhibitory effects on IL-12 expression, while romaine lettuce had a relatively weaker inhibitory effect.

[0115] Figure 7 The study demonstrated the effects of plant-derived glycerol glycolipids on IL-16 mRNA expression in RAW264.7 cells. Results showed a significant increase in IL-16 mRNA expression in the LPS-treated group (P<0.05), indicating that LPS effectively induces RAW264.7 cell activation and promotes the expression of proinflammatory cytokines, triggering an inflammatory response. Similar to the trends observed for IL-6 and IL-12, plant-derived glycerol glycolipids from different sources significantly inhibited IL-16 expression, although the inhibitory effect varied depending on the plant species.

[0116] Comparative Example 1

[0117] In this comparative example 1, based on Example 5, the glyceroglycolipid of the experimental group was replaced with glyceroglycolipid derived from black tea, and the other steps remained the same as those of Example 5.

[0118] Figure 8 The study demonstrated the effect of black tea-derived glyceroglycolipids on IL-6 secretion in RAW264.7 cells. The results showed that treatment with black tea-derived glyceroglycolipids had no inhibitory effect in the LPS-induced inflammation model, indicating that black tea-derived glyceroglycolipids do not have anti-inflammatory effects.

[0119] Example 6

[0120] This example studies the effect of plant glyceroglycolipids on M1 polarization of RAW264.7 cells.

[0121] Experimental methods:

[0122] RAW264.7 cells with a density of 80-90% and good growth status were seeded in 6-well plates at a cell seeding density of 5×10 6 The plates were then incubated in an incubator (37°C, 5% CO2) for 24 hours. The old cell culture medium in the culture flasks was discarded and the cells were washed twice with PBS. The experimental concentration of water spinach-derived glyceroglycolipids was set at 500 μg / mL, and the inflammatory concentration of LPS was set at 1 μg / mL. The experiment was divided into the following four groups: ①Control group, ②LPS group, ③LPS+GL group, and ④GL group. Each group was repeated five times. (All following steps were performed on ice.)

[0123] (1) Collect cells from each group, centrifuge at 300 × g for 5 min, discard the supernatant, and collect the cells. Wash the remaining old culture medium with pre-cooled PBS, centrifuge at 300 × g for 5 min, and discard the supernatant.

[0124] (2) Add PBS to resuspend the cells, count and dispense into flow cytometry tubes, with the number of cells per tube being 1.5×10 6 Centrifuge at 300 × g for 5 min and discard the supernatant.

[0125] (3) Resuspend in 100 μL PBS, then add 2 μL CD16 / 32 and incubate on ice in the dark for 10 min.

[0126] (4) Each flow cytometry tube was labeled. In addition to the experimental group, this experiment also set up an isotype control group and a single staining group. The experimental groups and antibody additions are shown in Table 7.

[0127] (5) Add fluorescently labeled antibody (CD86 2.5 μL / tube) according to the instructions, mix well, and incubate at 4°C in the dark for 20 min;

[0128] (6) Centrifuge at 300 × g for 5 min, discard the supernatant, and wash twice with PBS;

[0129] (7) Resuspend the cells in 500 μl PBS and place them in a flow cytometer for detection;

[0130] (8) Use FlowJo 10 software to analyze and visualize the flow cytometry results.

[0131] Table 7

[0132] To further clarify the effect of plant glycerol glycolipids on inflammation, LPS was used to stimulate RAW264.7 cells to construct an M1 polarization model, and the level changes of M1 polarization marker CD86 were analyzed by flow cytometry. Figure 9 As shown in the results, the expression of CD86 on the surface of RAW264.7 cells was significantly increased in the LPS-stimulated group compared with the control group (P<0.05). Compared with the LPS group, the expression of CD86 in the LPS+GL group was significantly decreased (P<0.05), but treatment of RAW264.7 cells with GL alone had no effect on CD86 expression. These data demonstrate that plant glyceroglycolipids can effectively inhibit LPS-induced CD86 expression, suggesting that they may alleviate inflammatory responses by reversing M1 polarization.

[0133] Example 7

[0134] In this example, the crude glycolipid obtained in Example 1 was further separated and analyzed as follows.

[0135] (1) Qualitative analysis of glycolipids

[0136] Glycolipids were characterized by TLC using chloroform-methanol-water as the mobile phase. After development, anthrone-sulfuric acid spray was used at 130°C for color development. Qualitative analysis was performed based on the Rf values of the standards. The mobile phase consisted of chloroform:methanol:water (32.5:8:1), and the color developer was 0.1 g of anthrone and 2 g of thiourea in 200 mL of 66% sulfuric acid.

[0137] The glycolipids separated by step (2) in Example 1 are a complex mixture, and the TLC technique can be used to effectively separate and analyze its subclasses. Figure 10 The TLC results of glycolipids from different plants are shown. As can be seen from the figure, although the glycolipid subclass composition of different plants is basically the same, including multiple components such as pigments, monogalactosyldiacylglycerol (MGDG), sterol glycosides (SG), monogalactosylmonoacylglycerol, and digalactosyldiacylglycerol (DGDG), the relative content and distribution of these components may vary between different plants. This suggests that although the main components of glycolipids are similar across different plant species, the specific lipid composition may be related to the plant's physiological characteristics and growth environment.

[0138] (2) Separation of glycolipid subclasses

[0139] 50 g of silica gel was dissolved in chloroform and loaded onto the column. 600 mg of crude glycolipid (i.e., glycolipid separated in step (2) of Example 1) was dissolved in 5 mL of chloroform and loaded onto the column. Based on the Rf value of the qualitative result in step (1), elution was performed using chloroform-acetone solutions in different ratios. The gradient elution solvent ratios are shown in Table 8.

[0140] Table 8

[0141] Each eluted component was blown dry with a nitrogen blower, and then redissolved in a small amount of chloroform. The resulting components were identified again by TLC in step (1).

[0142] Because spinach glycolipids are complex and high in content, spinach-derived glycolipids were selected for column chromatography to separate glycolipid subclasses. Based on the Rf values of the different glycolipid subclasses, appropriate solvents were selected for gradient elution, successfully separating the various subclasses of spinach glycolipids. This separation was then verified using TLC.

[0143] like Figure 11 As shown, the fractions separated by column chromatography exhibit the following characteristics: fractions 1 and 2 are pigments, fraction 3 contains highly pure MGDG and a small amount of pigment, fraction 4 is SG, and fractions 5 and 6 are DGDG. These results indicate that MGDG and DGDG are of high purity and well separated, demonstrating that most plant glycolipids can be separated and purified to a high degree using column chromatography.

[0144] (3) Quantitative analysis of glyceroglycolipid subclasses in plants

[0145] The quantification of glyceroglycolipid subclasses was performed by HPLC. The retention time of the glyceroglycolipid standard was used to calibrate each component. The peak area of each standard at different concentrations was used to establish a standard curve. The peak area of the glyceroglycolipid subclasses in various plants was then substituted into the standard curve to calculate the content.

[0146] Each sample was dissolved in chromatography-grade chloroform to 1 mg / mL and filtered through a 0.22 μm filter before loading.

[0147] The HPLC conditions were as follows: normal-phase HPLC column (4.6 × 50 mm, 5 μm Sepax HP-Silica, Dr. Maisch); mobile phase (chloroform: 95% methanol + 5% pure water = 99:1); flow rate, 1 mL / min; sample volume, 10 μL; detector, UV detector, wavelength 215 nm.

[0148] Liquid chromatography calibration curves were established using MGDG and DGDG standards at varying concentrations. The R² values for the MGDG and DGDG calibration curves were >0.98. The content of each plant glycerol glycolipid subclass was calculated based on the calibration curves, as shown in Table 9.

[0149] Table 9

[0150]

[0151] Among the eight plant species studied, MGDG content was highest in edible stalks (Hydroxysporum multiflorum) and spinach (Spinach), reaching 2.25 and 2.10 mg / g, respectively, followed by water spinach (1.53 mg / g) and lettuce (1.32 mg / g). In contrast, romaine lettuce had the lowest MGDG content, at 0.50 mg / g. DGDG content also showed significant variation, with Hydroxysporum multiflorum at 1.15 mg / g and lettuce at only 0.18 mg / g. Overall, glyceroglycolipid content in dark green plants was generally higher than 1.00 mg / g, while that in light green plants was generally lower than 1.00 mg / g.

[0152] Example 8

[0153] In this example, seven differentially expressed genes significantly associated with inflammatory pathways (IL-1β, IL-6, IL-12, IL-16, A4galt [α 1,4-galactosyltransferase], FPR1 [formyl peptide receptor 1], IL1rn [interleukin 1rn], IL36 [interleukin 36a]) were screened and their mRNA expression levels were verified in RAW264.7 cells treated with plant glycerol glycolipid monomers (MGDG and DGDG).

[0154] The experimental method is basically the same as that described in Example 5.

[0155] Synthesis of Mouse Gene Primer Sequences: The primer sequences synthesized by Shanghai Bioengineering Co., Ltd. are as follows. Before use, dissolve the dry primer powder in ddH2O according to the instructions and mix the forward and backward primers in the appropriate proportions. The primers are shown in Table 10.

[0156] Table 10

[0157]

[0158] The results are as follows Figure 12 As shown in the data, in the LPS-treated group, the mRNA expression levels of all detected inflammatory factors (IL-6, IL-1β, IL-12, etc.) were significantly upregulated, indicating that LPS successfully induced an inflammatory response, while the expression levels of inflammatory factors in the GL-treated groups (MGDG and DGDG) were significantly lower than those in the LPS group, indicating that plant glycerol glycolipids have significant anti-inflammatory effects.

[0159] Further analysis found that both MGDG and DGDG treatments could inhibit the expression of inflammatory factors, but among the above 7 genes (IL-1β, IL-6, IL-16, A4galt, FPR1, IL1rn, IL36a), the inhibitory effect of DGDG was more obvious than that of MGDG, suggesting that DGDG may play a stronger role in inflammatory regulation.

[0160] The present invention uses a chloroform-methanol solvent system to extract plant full lipids, and separates GL crude product and GL subclasses through two-step column chromatography. Subsequently, TLC and HPLC are combined to perform qualitative and quantitative analysis of GL.

[0161] A RAW264.7 cell inflammation model was successfully established under 1 μg / mL LPS stimulation. The present invention found that plant GLs from different sources significantly reduced the mRNA expression levels of the pro-inflammatory factors IL-1β, IL-6, IL-12, and IL-16, while effectively inhibiting LPS-induced expression of the M1 macrophage surface antigen CD86, thereby significantly alleviating the cellular inflammatory state. This result suggests that plant GLs can inhibit the polarization of RAW264.7 cells to the M1 type, thereby suppressing the inflammatory response and exerting a significant anti-inflammatory effect in macrophages.

[0162] In the glycolipid subclass analysis, both MGDG and DGDG treatments could inhibit the expression of inflammatory factors, but the effect of DGDG was more significant, and DGDG exhibited more significant anti-inflammatory activity than MGDG.

[0163] The present invention achieves efficient extraction, precise separation and structural identification of plant GL through column chromatography combined with chromatographic analysis, providing important technical support for plant lipid research. Studies have shown that plant GL can significantly inhibit the inflammatory response of RAW264.7 macrophages, including reducing LPS-induced cell morphological changes, downregulating the expression of inflammatory factor mRNA, and reducing the expression level of CD86. Multi-omics analysis further revealed its potential molecular mechanism, suggesting that the anti-inflammatory effect of plant GL may be closely related to phospholipid metabolism, NF-κB and other signaling pathways and metabolic regulation. The above research results provide a solid molecular-level theoretical basis for the application of plant glycerol glycolipids in the development of anti-inflammatory functional foods and drugs.

[0164] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of a plant-derived glyceroglycolipid in the preparation of an anti-inflammatory product, characterized in that: The inflammation is an inflammatory response of RAW264.7 cells induced by LPS.

2. The use of the plant-derived glyceroglycolipid according to claim 1 in the preparation of an anti-inflammatory product, characterized in that: The plant-derived glyceroglycolipid achieves anti-inflammation by inhibiting the expression of inflammatory factors.

3. The use of the plant-derived glyceroglycolipid according to claim 2 in the preparation of an anti-inflammatory product, characterized in that: The inflammatory factors are IL-6, IL-12 and IL-16.

4. The use of the plant-derived glyceroglycolipid according to claim 1 in the preparation of an anti-inflammatory product, characterized in that: The plant-derived glyceroglycolipid achieves anti-inflammation by inhibiting the expression of CD86.

5. Use of the plant-derived glyceroglycolipid according to any one of claims 1 to 4 in the preparation of an anti-inflammatory product, characterized in that: The plants include one or more of water spinach, Chinese cabbage, cabbage, kale, lettuce and lettuce.

6. Use of the plant-derived glyceroglycolipid according to claim 5 in the preparation of an anti-inflammatory product, characterized in that: The preparation method of the plant-derived glyceroglycolipid comprises: Place the plant material in boiling water, crush it after cooling, add chloroform-methanol solution and mix thoroughly; The mixed slurry was filtered, chloroform and pure water were added to the filtrate respectively, and the lower layer solution was recovered after standing and stratification; Anhydrous sodium sulfate was added to the lower solution, and the solution was allowed to stand for dehydration, and then filtered to obtain the complete lipids; Dissolve silica gel in chloroform, stir well and pour into the chromatography column; The whole lipids were dissolved in chloroform and added to the chromatography column for elution to collect the glycolipids.

7. The use of the plant-derived glyceroglycolipid according to claim 2 in the preparation of an anti-inflammatory product, characterized in that: The inflammatory factors are IL-1β, IL-6, IL-16, A4galt, FPR1, IL1rn, and IL36a.

8. The use of the plant-derived glyceroglycolipid according to claim 7 in the preparation of an anti-inflammatory product, characterized in that: The plant-derived glyceroglycolipid is monogalactosylglycerol or digalactosyldiacylglycerol.

9. The use of the plant-derived glyceroglycolipid according to claim 8 in the preparation of an anti-inflammatory product, characterized in that: The preparation method of the plant-derived glyceroglycolipid comprises: Place the plant material in boiling water, crush it after cooling, add chloroform-methanol solution and mix thoroughly; The mixed slurry was filtered, chloroform and pure water were added to the filtrate respectively, and the lower layer solution was recovered after standing and stratification; Anhydrous sodium sulfate was added to the lower solution, and the solution was allowed to stand for dehydration, filtered, and nitrogen blown to obtain the complete lipids; Dissolve silica gel in chloroform, stir well and pour into the chromatography column; The whole lipids were dissolved in chloroform and added to the chromatography column for elution to collect the glycolipids; Dissolve silica gel in chloroform, stir well and pour into the chromatography column; The glycolipid is dissolved in chloroform and added to a chromatography column for elution, and elution is performed using chloroform-acetone solutions in different ratios in sequence to collect digalactosyldiacylglycerol.

10. Use of the plant-derived glyceroglycolipid according to claim 6 or 9 in the preparation of an anti-inflammatory product, characterized in that: The chloroform-methanol solution, with a volume ratio of chloroform to methanol solution of 1:2, was fully mixed at room temperature; Said adding chloroform and pure water, the final volume ratio of chloroform:methanol:water is 1:1:0.9; The anhydrous sodium sulfate is added in an amount of one tenth of the solvent volume; The filtration was followed by drying with nitrogen.

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