Use of a plant-derived glycolipid in the preparation of anti-inflammatory products

Abstract

The application discloses application of a plant-derived glycolipid in preparation of anti-inflammatory products, the inflammation is an inflammation reaction of RAW264.7 cells induced by LPS, and the plant-derived glycolipid realizes anti-inflammation by inhibiting expression of inflammatory factors. Through column chromatography combined with chromatographic analysis, the application realizes efficient extraction, accurate separation and structure identification of the plant GL, and provides important technical support for plant lipid research. The plant GL can significantly inhibit the inflammation reaction of RAW264.7 macrophages, including reducing LPS-induced cell morphological change, down-regulating mRNA expression of inflammatory factors and reducing expression level of CD86. The application provides a solid molecular level theoretical basis for application of the plant glycolipid in development of anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of active substance extraction and application, and particularly relates to application of a plant-derived glycolipid in preparation of anti-inflammatory products. BACKGROUND

[0002] Inflammatory response is an immune response triggered by the body to respond to infection or tissue damage, usually manifested as redness, fever, pain and dysfunction, etc. This process is to remove harmful substances and repair damaged tissues, so as to restore the normal physiological function of the body and rebalance the internal and external environment. However, long-term or excessive inflammatory response can disrupt this balance, leading to abnormal activation of the immune system. In particular, in the case of adverse inflammatory immune response, excessive activation of macrophages can lead to the release of a large amount of cytokines, including TNF-α, interferon-γ (IFN-γ), IL-1β, IL-6 and interleukin-17a (IL-17a), etc. Excessive release of these cytokines can disrupt the body's immune regulation, triggering a variety of chronic inflammatory diseases including sepsis, atherosclerosis and inflammatory bowel disease, etc.

[0003] LPS is the main component of the cell wall of gram-negative bacteria, which can activate macrophages through Toll-like receptor 4 (TLR4) binding, and is a classic research tool for inducing inflammatory response. In vitro, RAW264.7 cells are highly sensitive to LPS and can simulate the inflammatory response of macrophages in vivo, and are often used for research on inflammatory mechanisms and screening of anti-inflammatory drugs.

[0004] Plant glycolipids, as a natural bioactive ingredient, have multiple biological activities such as antiviral, antibacterial, antitumor and anti-inflammatory. However, so far, the research on the biological activity of plant glycolipids is still limited, and most of them are focused on the research of microbial glycolipids. The role of plant glycolipids in macrophage-mediated inflammatory response, especially its regulatory role in LPS-induced RAW264.7 cell inflammatory model, has not been systematically explored. Therefore, it is of great scientific significance to study the role of plant glycolipids in anti-inflammatory. SUMMARY

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

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

[0007] One of the purposes of the present application is to provide the application of plant-derived glycolipids in the preparation of anti-inflammatory products, which can significantly inhibit the inflammatory response of RAW264.7 macrophages, and provide a solid molecular level theoretical basis for the application of plant glycolipids in the development of anti-inflammatory drugs.

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

[0009] As a preferred scheme of the application of plant-derived glycolipids in the preparation of anti-inflammatory products, wherein: the plant-derived glycolipids achieve anti-inflammation by inhibiting the expression of inflammatory factors.

[0010] As a preferred scheme of the application of plant-derived glycolipids in the preparation of anti-inflammatory products, wherein: the inflammatory factors are IL-6, IL-12 and IL-16.

[0011] As a preferred scheme of the application of plant-derived glycolipids in the preparation of anti-inflammatory products, wherein: the plant-derived glycolipids achieve anti-inflammation by inhibiting the expression of CD86.

[0012] As a preferred scheme of the application of plant-derived glycolipids in the preparation of anti-inflammatory products, wherein: the plants include one or more of water spinach, Chinese cabbage, cabbage, mustard green, lettuce, and lettuce.

[0013] As a preferred scheme of the application of plant-derived glycolipids in the preparation of anti-inflammatory products, wherein: the preparation method of the plant-derived glycolipids comprises:

[0014] The plant material is placed in boiling water, cooled and crushed, and a chloroform-methanol solution is added and mixed thoroughly;

[0015] The mixed slurry is subjected to suction filtration, and chloroform and pure water are added to the filtrate, respectively, and the lower layer solution is recovered after standing and layering;

[0016] Anhydrous sodium sulfate is added to the lower layer solution, and it is dehydrated by standing, and the full lipid is obtained after filtration;

[0017] Silica gel is dissolved in chloroform, stirred uniformly, and then poured into a chromatography column;

[0018] The full lipid is dissolved in chloroform and added to the chromatography column for elution, and the glycolipid is collected.

[0019] As a preferred scheme of the application of the plant-derived glycolipid in the preparation of anti-inflammatory products, wherein: the inflammatory factors are IL-1beta, IL-6, IL-16, A4galt, FPR1, IL1rn, IL36a.

[0020] As a preferred scheme of the application of the plant-derived glycolipid in the preparation of anti-inflammatory products, wherein: the plant-derived glycolipid is monogalactosylglycerol or digalactosyldiacylglycerol.

[0021] As a preferred scheme of the application of the plant-derived glycolipid in the preparation of anti-inflammatory products, wherein: the preparation method of the plant-derived glycolipid comprises:

[0022] The plant material is placed in boiling water, crushed after cooling, and mixed with a trichloromethane-methanol solution;

[0023] The mixed slurry is filtered, trichloromethane and pure water are added to the filtrate, and the lower layer solution is recovered after standing and layering;

[0024] Anhydrous sodium sulfate is added to the lower layer solution, and the water is removed by standing, and the total lipid is obtained after filtration;

[0025] The silica gel is dissolved in trichloromethane, stirred uniformly, and poured into a chromatography column;

[0026] The total lipid is dissolved in trichloromethane and added to the chromatography column for elution, and the glycolipid is collected;

[0027] The silica gel is dissolved in chloroform, stirred uniformly, and poured into a chromatography column;

[0028] The glycolipid is dissolved in chloroform and added to the chromatography column for elution, and the digalactosyldiacylglycerol is collected by using different proportions of trichloromethane-acetone solution in turn.

[0029] As a preferred scheme of the application of the plant-derived glycolipid in the preparation of anti-inflammatory products, wherein: the trichloromethane-methanol solution, the volume ratio of trichloromethane to methanol solution is 1:2, and the mixture is mixed at room temperature;

[0030] The trichloromethane and pure water are added, and the final volume ratio of trichloromethane:methanol:water is 1:1:0.9;

[0031] The anhydrous sodium sulfate is added, and the mass of anhydrous sodium sulfate is one-tenth of the volume of the solvent;

[0032] The filtration, and the nitrogen blowing after filtration.

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

[0034] The application realizes efficient extraction, accurate separation and structure identification of plant GL by column chromatography combined with chromatographic analysis, and provides important technical support for plant lipid research. Research shows that plant GL can significantly inhibit the inflammatory response of RAW264.7 macrophages, including reducing LPS-induced cell morphological changes, down-regulating inflammatory factor mRNA expression, and reducing the expression level of CD86. The above research results provide a solid molecular level theoretical basis for the application of plant glycolipids in the development of anti-inflammatory drugs. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

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

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

[0038] Figure 3 The effects of different plant glycolipids on RAW264.7 cell viability; wherein (a) is cabbage, (b) is kale, (c) is agaric, (d) is small cabbage, (e) is lettuce, (f) is hollow stem, (g) is lettuce, and (h) is spinach.

[0039] Figure 4 The morphological characteristics (200x) of RAW264.7 cells under different treatment conditions; wherein (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 effects of different plant glycolipids on the secretion of IL-6 in RAW264.7 cells.

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

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

[0043] Figure 8 Effect of glycolipids in Example 1 on IL-6 secretion of RAW264.7 cells.

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

[0045] Figure 10 Thin layer chromatography qualitative results of eight plant glycolipids.

[0046] Figure 11 Thin layer chromatography results of spinach glycolipid subgroups; 1 is a chloroform elution component; 2 is a chloroform-acetone (9:1) elution component; 3 is a chloroform-acetone (7:3) elution component; 4 is a chloroform-acetone (4:6) elution component; 5 is a chloroform-methanol (2:8) elution component; and 6 is an acetone elution component.

[0047] Figure 12 qRT-PCR verification of differential genes in Example 8; (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 objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the description and examples.

[0049] In the following description, a lot of specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the concept of the present application, so the present application is not limited to the specific examples disclosed below.

[0050] Secondly, "one embodiment" or "embodiment" referred to herein means that a specific feature, structure or characteristic can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is separate or alternative to other embodiments.

[0051] Unless otherwise specified, the raw materials used in the examples are commercially available.

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

[0053] Table 1

[0054]

[0055] The cell culture method used in the embodiments of the present application:

[0056] (1) Cell culture

[0057] RAW264.7 cells were cultured with complete medium (containing 10% special fetal bovine serum + 90% high-sugar medium), and the cell culture bottle was placed in a 37°C, 5% CO2 constant temperature incubator, and the fresh medium was replaced every 24 hours. When the cells grew to the confluence state (preferably 90% cell adhesion), they were passed down after being blown by a pipette gun. In the experiment, RAW264.7 cells in the logarithmic growth phase and in good condition were selected for operation.

[0058] (2) Cell passage

[0059] The old culture medium in the T25 culture bottle was discarded, and the cells were washed twice with 3-4 mL of phosphate buffered saline (PBS). The PBS was discarded, and the culture medium was blown to the bottom of the cell adhesion to make it fall off completely. The cell suspension was transferred to a 10 mL centrifuge tube, centrifuged (1000 r / min, 5 min), and the supernatant was discarded. 2 mL of fresh complete medium was added to resuspend the cells, and the cell suspension was transferred to a new T25 cell culture bottle according to the proportion (1:2-1:4), 4-6 mL of fresh complete medium was added, and it was placed in a 37°C incubator for culture.

[0060] (3) Cell freezing

[0061] The cells in the T25 culture bottle with good growth state and density reaching 90% were blown down with a pipette gun, centrifuged at 1000 rmp for 5 minutes, the supernatant was discarded, 1 mL of freezing solution (serum: dimethyl sulfoxide = 9:1) was added to resuspend, and it was transferred to a cell freezing tube and placed in a programmed cooling box. The programmed cooling box was first placed in a -80°C refrigerator for 24 hours, and then the cell freezing tube was transferred to liquid nitrogen or a -80°C refrigerator.

[0062] Example 1

[0063] (1) Plant lipid extraction

[0064] 100 g of plant material (water spinach, lettuce, agaric, Chinese cabbage, spinach, cabbage, cabbage, lettuce) was placed in boiling water for 5 min, dried and cooled, then ground in a grinder, then transferred to a beaker, 30 mL of chloroform-methanol solution (1:2) was added, and mixed well at room temperature. The mixed slurry was poured into a suction filtration device for suction filtration, and the filtrate was transferred to a separatory funnel, 10 mL of chloroform and 18 mL of pure water were added respectively, and the lower layer was recovered after standing and separating. Add an appropriate amount of anhydrous sodium sulfate to the lower layer solution and stand overnight to dehydrate. After filtration, remove the chloroform using a rotary evaporator, dry under nitrogen and weigh to obtain the total lipid and calculate the total lipid yield. The total lipid is redissolved with a small amount of chloroform and stored in a -4 ℃ refrigerator for use.

[0065] (2) Isolation and purification of crude glycolipids

[0066] Dissolve 20 g of silica gel in 60 mL of chloroform, stir well and pour into a chromatography column. Then, dissolve 1 g of total lipid sample from step (1) in 5 mL of chloroform and add it to the chromatography column. Elute with 125 mL of chloroform, 150 mL of acetone and 125 mL of methanol in sequence, and collect the three components as neutral lipids, glycolipids and phospholipids, respectively. Remove the solvents using a rotary evaporator, dry under nitrogen and weigh to calculate the yield of each type of lipid.

[0067] Figure 1 The contents of total lipids, neutral lipids, glycolipids and phospholipids in 8 plants are shown. The content of total lipids in each plant sample varies greatly, ranging from 0.64 to 7.09 mg / g. The content of glycolipids in spinach is the highest in total lipids, reaching 66.12 %, followed by agaric, accounting for 52.31 %, while the proportions of neutral lipids and phospholipids in these plants are relatively low. This result shows that plants with darker leaves and higher chlorophyll content generally have higher total lipid and glycolipid content. The total lipid and glycolipid content of spinach, lettuce and cabbage is significantly higher than that of lettuce, Chinese cabbage and other green vegetables with relatively light color.

[0068] Example 2

[0069] This example studies the effect of different concentrations of LPS on the viability of RAW264.7 cells. The cell viability is determined by CCK8 method, and the specific method is as follows:

[0070] (1) Take RAW264.7 cells in logarithmic growth phase and inoculate them in a 96-well plate at a concentration of 5×10 4 μL / well) to prevent edge effects, and incubate in a cell culture incubator at 37 ℃ with 5 % CO2.

[0071] (2) After the cells adhered to the wall, 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 and only added culture medium; the concentration of the LPS group was 0.5, 1, 2, 4, 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 placed in the incubator for 24 h of continuous culture.

[0072] (3) Remove the old culture medium and wash the cells twice with PBS, then add 100 μL of complete culture medium (containing 10 μL of CCK-8) to each well.

[0073] (4) Place the 96-well plate in the cell incubator for 2 hours of incubation.

[0074] (5) Measure the absorbance at 450 nm with a microplate reader.

[0075] Cell viability (%) = [A (drug) - A (blank)] / [A (0 drug) - A (blank)] x 100;

[0076] Wherein, A (drug) is the absorbance of the well with cells, CCK-8 solution and drug solution;

[0077] A (blank) is the absorbance of the well with culture medium and CCK-8 solution without cells;

[0078] A (0 drug) is the absorbance of the well with cells and CCK-8 solution without drug solution.

[0079] Figure 2 The effect of different concentrations of LPS (0, 0.5, 1, 2, 4, 8 μg / mL) on the viability of RAW264.7 cells was determined using the CCK8 method. With the increase of LPS concentration, the cell viability gradually decreased, and when the LPS concentration increased to 2 μg / mL, the cell viability was significantly lower than that of the control group, and the difference was statistically significant (P <0.05). Therefore, 1 μg / mL of LPS concentration was selected as the standard concentration for inducing inflammatory response in subsequent experiments.

[0080] Example 3

[0081] This example studies the effect of different types of GL on the viability of RAW264.7 cells. The same CCK8 method as in Example 2 was used to determine cell viability. The experiment was divided into a blank control group and different plant glycolipid groups. Only the blank group was not inoculated with cells and only added culture medium; the concentration of 8 plant glycolipids (i.e. the glycolipids separated in step (2) of Example 1) was set to 100, 500, 1000, 1500, 2000, 2500, 3000, 3500 μg / mL; other steps were the same as Example 2.

[0082] Figure 3 The effects of different concentrations of GLs from different plants on the viability of RAW264.7 cells after 24 h of treatment are shown. From the results, it can be seen that GLs from different plants have a significant impact on cell survival rate. At lower concentrations (such as 50-100 μg / mL), the cell viability generally remains at a high level, however, as the concentration increases, the cell viability decreases significantly, especially at high concentrations (such as 500 μg / mL and above), the cell viability is significantly inhibited, and in some treatment groups, the cell viability is almost even reduced to 0%. For example, GLs from spinach, lettuce, water spinach, Chinese cabbage, and lettuce, when their concentrations are below 500 μg / mL, the cell viability is maintained within the normal range compared to the control group, while GLs from wood ear, cabbage, and cabbage when their concentrations exceed 300 μg / mL and 100 μg / mL respectively, have a significant inhibitory effect on the survival rate of RAW264.7 cells.

[0083] Example 4

[0084] Under normal conditions, RAW264.7 macrophages are round or oval in shape without pseudopodia. In this example, the morphological changes of cells in each group were observed under an inverted microscope.

[0085] 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 1 μg / mL concentration of LPS for 24 h; the GL treatment group was treated with 1 μg / mL concentration of LPS and then immediately added with 500 ug / ml concentration of water spinach-derived glycolipids, and treated for 24 h.

[0086] Figure 4 The morphological changes of RAW264.7 cells under different treatment conditions are shown, which reveals the effects of different experimental conditions on the state of cells from the perspective of cell morphology. Under normal (control) conditions, RAW264.7 cells mainly present as small round shape, with smooth cell edge and uniform cytoplasm distribution, without obvious pseudopodia. Figure 4 -a). However, after LPS stimulation, the cell morphology changed significantly, showing 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 are activated after LPS stimulation and polarize to M1 type macrophages, i.e. pro-inflammatory phenotype. In the GL treatment group, the morphology of cells is obviously restored, the number of cell pseudopodia is significantly reduced, and most of the cells tend to be round, with a morphology closer to the normal resting state. Figure 4- c). This phenomenon indicates that GL has the effect of inhibiting LPS-induced M1 polarization, keeping RAW264.7 cells in an unactivated or lowly activated state, thereby possibly reducing the release of inflammatory factors and alleviating inflammatory reactions.

[0087] Example 5

[0088] This example studies the effect of plant glycolipids on the secretion of IL-6, IL-12 and IL-16 by RAW264.7 cells.

[0089] Experimental method:

[0090] (1) Extraction of total cell RNA

[0091] All the following steps are operated on ice:

[0092] RAW264.7 cells with a density of 80-90% and in good growth state were inoculated in a 12-well plate, with a cell inoculation density of 2 x 10 6 / mL (2 mL / well). Subsequently, the well plate was placed in an incubator (37 °C, 5% CO2) for culture for 24 h, the old cell culture medium in the culture bottle was poured out, and the cells were washed twice with PBS. The experimental concentration of glycolipids was set to 500 μg / mL, the inflammatory concentration of LPS was 1 μg / mL, and glycolipids-free was used as a control group. Co-culture was performed in a 37 °C, 5% CO2 cell incubator for 24 h.

[0093] Before the experiment, the high-pressure sterilized experimental supplies were placed in the ultraclean table for ultraviolet irradiation for 30 minutes. Take the complete culture medium and 1x PBS buffer from the 4 °C refrigerator in advance and balance to room temperature. After ultraviolet irradiation for 30 minutes, open the ultraclean table and fan, sterilize the table with 75% alcohol cotton ball, and light the alcohol lamp. Pour out the culture solution and wash once with 1x PBS. Add 500 μL of RNA extraction solution to the culture cells growing in the 12-well plate, and place it horizontally for a while. Use a pipette gun to blow it thoroughly to completely lyse it. Transfer the lysate containing cells to a 1.5 mL centrifuge tube, and observe whether it is completely absorbed. Add 100 μL of chloroform (RNA extraction solution: chloroform = 5:1 (v / v)) and vortex for 15-30 s. After standing for 5 min, centrifuge at 10,000xg, 4 °C for 15 min. At this time, the sample is divided into three layers, a colorless aqueous phase (upper layer), an intermediate layer, and a pink organic phase (lower layer). The volume of the aqueous phase is about 50% of the RNA extraction reagent used. Transfer the colorless aqueous phase to a new centrifuge tube, add 0.5 mL of isopropanol (isopropanol: water = 1:1 (v / v)) for every 1 mL of RNA extraction reagent used, and mix well by inverting. Incubate at room temperature for 10 min. Centrifuge at 10,000xg, 4 °C for 10 min. Discard the supernatant, and the tube side and bottom form a gelatinous precipitate. Add 1 mL of 75% ethanol (DEPC-treated water preparation), and vortex vigorously (add 75% ethanol 1 mL for every 1 mL of RNA extraction reagent used). Centrifuge at 7500xg, 4 °C for 5 min. Discard the supernatant, and dry in the fume hood. Dissolve the precipitate in 20-50 μL of RNA dissolution solution. Incubate at 55-60 °C for 10 min, and store the sample at -20 °C for long-term use.

[0094] (2) Fluorescent quantitative PCR

[0095] (2-1) Reverse transcription reaction:

[0096] (a) Genomic DNA removal

[0097] Prepare the following mixture in an RNase-free centrifuge tube:

[0098] Table 2

[0099]

[0100] Gently mix the prepared reaction mixture with a pipette and centrifuge at a slow speed for 5 s. Place it in a PCR instrument and set the conditions: 42 °C incubation for 2 min.

[0101] (b) Preparation of reverse transcription reaction system

[0102] Directly add 5x HiScript Ill qRT SuperMix to the reaction tube of step (a) as shown in Table 3.

[0103] Table 3

[0104]

[0105] (c) Perform reverse transcription reaction

[0106] The prepared reaction mixture was pipetted and gently blown to mix, and centrifuged at low speed for 5 s. It was placed in a PCR instrument, and the conditions were set as follows: 37 °C incubation for 15 min, and heating at 85 °C for 5 s. After dilution 5 times with 80 μL ddH2O, the PCR tube in which the reverse transcription was completed was stored at -20 °C.

[0107] (2-2) Fluorescent qPCR reaction

[0108] Synthesis of mouse gene primer sequences: The primer sequences synthesized by Shanghai Generay Biotech Co., Ltd. are as follows: before use, the dry powder primer is dissolved and diluted with ddH2O (PH = 8.0) according to the instructions, and the forward primer is mixed with the reverse primer according to the proportion. The primer list is shown in Table 4.

[0109] Table 4

[0110]

[0111] (a) Take 0.1 mL PCR eight-tube labeled group, take the primers and qPCR kit from the -20 °C refrigerator, and dissolve on ice. Shake all reagents before use, and use after slow centrifugation. The qPCR reactions of GAPDH (glyceraldehyde-3-phosphate dehydrogenase), interleukin 6 (IL-6), interleukin 12 (IL-12), and interleukin 16 (IL-16) use Nuoyuan qPCR kit, and the reaction system is shown in Table 5, and the running program is shown in Table 6.

[0112] Table 5

[0113]

[0114] Table 6

[0115]

[0116] (b) After completing the preparation and centrifugation of the above mixture, the eight-tube is placed in the fluorescent quantitative PCR instrument, and the reaction conditions are set as shown in the following table. The eight-tube is labeled on the instrument program, and then the machine cover is automatically closed by the program, and then the running is clicked.

[0117] According to the Ct value measured by real-time fluorescent quantitative PCR, the Ct value of the target gene of the test group is subtracted from the Ct value of the internal reference gene of the test group, and the result is the △Ct value. The △Ct value of the experimental group is subtracted from the △Ct value of the control group, which is the △△Ct value.

[0118] Figure 5 The effects of glycolipids from different plant sources on the IL-6 secretion level of RAW264.7 cells were demonstrated. The results showed that the IL-6 mRNA expression level of the LPS treatment group was significantly increased, indicating that LPS could strongly induce the inflammatory response of RAW264.7 cells, making them enter the pro-inflammatory state. In the LPS-induced inflammation model, the treatment of glycolipids from different plant sources could significantly inhibit the secretion of IL-6 (P < 0.05), suggesting that these compounds might have certain anti-inflammatory effects.

[0119] However, there were differences in the inhibitory effects of glycolipids from different plant sources on IL-6 expression, among which the inhibitory effect of the agaric group was relatively weak, lower than that of other plant treatment groups. This result indicated that plant glycolipids might affect IL-6 secretion through different regulatory mechanisms, and their anti-inflammatory effects differed due to different plant sources.

[0120] Figure 6 The effects of plant glycolipids on the IL-12 mRNA expression of RAW264.7 cells were demonstrated. The results showed that the IL-12 expression level of the LPS treatment group was significantly increased (P < 0.05). In the LPS-induced inflammation model, glycolipids from different plant sources could inhibit IL-12 expression, but the inhibitory effects differed, among which, lettuce, cabbage, Chinese cabbage, and cabbage had stronger inhibitory effects on IL-12 expression, while the inhibitory effect of lettuce was relatively weak.

[0121] Figure 7 The effects of plant glycolipids on the IL-16 mRNA expression of RAW264.7 cells were demonstrated. The results showed that the IL-16 mRNA expression level of the LPS treatment group was significantly increased (P < 0.05), indicating that LPS could effectively induce the activation of RAW264.7 cells and promote the expression of pro-inflammatory factors, triggering the inflammatory response. Similar to the change trend of IL-6 and IL-12, glycolipids from different plant sources could significantly inhibit the expression of IL-16, but the inhibitory effects differed due to different plant species.

[0122] Comparative Example 1

[0123] Comparative Example 1 was based on Example 5, in which the glycolipids of the experimental group were replaced with glycolipids from black tea, and the other steps were consistent with Example 5.

[0124] Figure 8This study demonstrated the effect of glyceroglycolipids derived from black tea on IL-6 secretion levels in RAW264.7 cells. The results showed that treatment with glyceroglycolipids derived from black tea did not produce an inhibitory effect in an LPS-induced inflammation model, indicating that glyceroglycolipids derived from black tea do not possess anti-inflammatory properties.

[0125] Example 6

[0126] This study investigated the effects of plant glycerol glycolipids on M1 polarization in RAW264.7 cells.

[0127] Experimental methods:

[0128] RAW264.7 cells with a density of 80-90% and in good growth condition were seeded into 6-well plates at a seeding density of 5 × 10⁶ cells / well. 6 Cells / mL (5 mL / well). The plates were then incubated in an incubator (37 ℃, 5% CO2) for 24 h. The old cell culture medium in the culture flasks was discarded, and the cells were washed twice with PBS. The experimental concentration of glycerol glycolipids from water spinach was set at 500 μg / mL, and the inflammatory concentration of LPS was set at 1 μg / mL. The experiment was divided into four groups: ① Control group, ② LPS group, ③ LPS+GL group, and ④ GL group. Each group was repeated 5 times. (All the following steps were performed on ice.)

[0129] (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.

[0130] (2) Resuspend the cells in PBS, count them, and then dispense them into flow cytometry tubes, with each tube containing approximately 1.5 × 10⁻⁶ cells. 6 Centrifuge each sample at 300×g for 5 min and discard the supernatant.

[0131] (3) Resuspend in 100 μL PBS, then add 2 μL CD16 / 32 and incubate on ice in the dark for 10 min.

[0132] (4) Each flow cytometer tube was labeled. In addition to the experimental group, the experiment also set up an isotype control group and a single staining group. The experimental groups and antibody addition are shown in Table 7.

[0133] (5) Add fluorescently labeled antibody (CD86 2.5 μL / tube) according to the dosage in the instructions, mix well, and incubate at 4°C in the dark for 20 min;

[0134] (6) Centrifuge at 300×g for 5 min, discard the supernatant, and wash twice with PBS;

[0135] (7) After resuspending the cells with 500 μl PBS, the cells were put into flow cytometer for detection;

[0136] (8) The flow cytometry results were analyzed and visualized using FlowJo 10 software.

[0137] Table 7

[0138]

[0139] To further clarify the effect of plant glycolipids on inflammation, a M1 polarization model was constructed by stimulating RAW264.7 cells with LPS, and the level change of M1 polarization marker CD86 was analyzed by flow cytometry. The experimental results are shown in Figure 9 The results show that compared with the control group, the expression of CD86 on the surface of RAW264.7 cells in the LPS stimulation group was significantly increased (P<0.05). Compared with the LPS group, the expression of CD86 in the LPS+GL group was significantly reduced (P<0.05), but if only GL was used to treat RAW264.7 cells, there was no effect on the expression of CD86. These data show that plant glycolipids can effectively inhibit the expression of CD86 induced by LPS, suggesting that they can alleviate inflammatory response by reversing M1 polarization.

[0140] Example 7

[0141] In this example, the crude glycolipids obtained in Example 1 were further separated and analyzed as follows.

[0142] (1) Qualitative analysis of glycolipids

[0143] The qualitative analysis of glycolipids was performed by TLC method using chloroform-methanol-water as the mobile phase. After development, the color was developed at 130 °C using anthrone-sulfuric acid spray. The qualitative analysis was performed according to the Rf values of the standard samples. The mobile phase was chloroform:methanol:water=32.5:8:1, and the color developing agent was 0.1 g anthrone and 2 g thiourea in 200 mL 66% sulfuric acid.

[0144] The glycolipids separated in step (2) of Example 1 are a complex mixture. TLC technology can effectively separate and analyze the subgroups of the mixture. Figure 10 The TLC results of different plant glycolipids are shown. As can be seen from the figure, although the subgroups of glycolipids in different plants are basically the same, they all contain various components such as pigments, monogalactosyldiacylglycerol (MGDG), sterol glycoside (SG), monogalactosylmonoacylglycerol, digalactosyldiacylglycerol (DGDG), etc., but the relative content and distribution may be different between different plants. This indicates that although the plant species are different, the main components of their glycolipids are similar, but the specific lipid composition may be related to the physiological characteristics and growth environment of the plants.

[0145] (2) Isolation of glycolipid subgroups

[0146] Silica gel 50 g was dissolved in chloroform and packed into a column. 600 mg of crude glycolipids (i.e. glycolipids isolated in step (2) of Example 1) were dissolved in 5 mL of chloroform and loaded onto the column. According to the Rf values of the qualitative results of step (1), different proportions of chloroform-acetone were used to elute the column in gradient elution, and the gradient elution solvent proportions are shown in Table 8.

[0147] Table 8

[0148]

[0149] Each eluted component was dried with a nitrogen blower, and then a small amount of chloroform was added to dissolve each component again. Each component was then identified again using TLC in step (1).

[0150] Because the glycolipid components of spinach are complex and have a high content, the glycolipids from spinach were selected as the research object for column chromatography to isolate glycolipid subgroups. According to the Rf values of different glycolipid subgroups, appropriate solvents were selected for gradient elution, and each subgroup component of spinach glycolipids was successfully eluted and verified by TLC technology.

[0151] As shown in Table 9, the components separated by column chromatography showed the following characteristics: 1, 2 components were pigments, 3 components contained high-purity MGDG and a small amount of pigments, 4 components were SG, and 5, 6 components were DGDG. These results showed that MGDG and DGDG had high purity, and their separation degree was good, indicating that most plant glycolipids could achieve high purity and separation effect by column chromatography. Figure 11 (3) Quantitative analysis of glycolipid subgroups in plants

[0152] The quantitative analysis of glycolipid subgroups was performed by HPLC. The retention time of glycolipid standards was used to identify each component, and the peak area of each standard at different concentrations was used to establish a standard curve. Then, the peak area of each glycolipid subgroup in various plants was substituted into the standard curve to calculate the content.

[0153] Each sample was dissolved in chromatographic-grade chloroform to 1 mg / mL, and filtered using a 0.22 μm filter before loading.

[0154] The HPLC conditions were as follows: normal phase HPLC column (4.6 x 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 size, 10 μL; detector, ultraviolet detector, wavelength 215 nm.

[0155] ​

[0156] By using different concentrations of MGDG and DGDG standard, the standard curve of liquid chromatography was established, and the R² of MGDG and DGDG standard curve was greater than 0.98. According to the standard curve, the content of each plant glycolipid subclass was calculated as shown in Table 9.

[0157] Table 9

[0158]

[0159] Among the 8 plants in this study, the MGDG content of wood ear was the highest, reaching 2.25 and 2.10 mg / g, followed by the MGDG content of spinach (1.53 mg / g) and lettuce (1.32 mg / g). In contrast, the MGDG content of lettuce was the lowest, reaching 0.50 mg / g. The DGDG content also showed obvious differences, with the DGDG content of wood ear being 1.15 mg / g, and the DGDG content of lettuce being only 0.18 mg / g. Overall, the glycolipid content in dark green plants was generally higher than 1.00 mg / g, while the glycolipid content in light green plants was generally lower than 1.00 mg / g.

[0160] Example 8

[0161] In this example, 7 differentially expressed genes (IL-1β, IL-6, IL-12, IL-16, A4galt [α 1,4-galactosyltransferase], FPR1 [formyl peptide receptor 1], IL1rn [interleukin 1 rn], IL36 [interleukin 36a]) were screened, and the mRNA expression levels of the genes were verified in RAW264.7 cells treated with plant glycolipid monomers (MGDG and DGDG).

[0162] The experimental method was basically the same as that described in Example 5.

[0163] Synthesis of mouse gene primer sequences: The sequences of the primers synthesized by Shanghai Generay Biotech Co., Ltd. are as follows: before use, the dry powder primers are dissolved and diluted with ddH2O according to the instructions, and the forward primers are mixed with the reverse primers in proportion. The primers are shown in Table 10.

[0164] Table 10

[0165]

[0166] The results are as follows Figure 12As shown, in the LPS treatment group, the mRNA expression levels of all detected inflammatory factors (IL-6, IL-1β, IL-12, etc.) were significantly up-regulated, indicating that LPS successfully induced inflammation, and the expression levels of inflammatory factors in the GL treatment group (MGDG and DGDG) were significantly lower than those in the LPS group, indicating that plant glycolipids have significant anti-inflammatory effects.

[0167] Further analysis found that MGDG and DGDG treatment can inhibit the expression of inflammatory factors, but in the above 7 genes (IL-1β, IL-6, IL-16, A4galt, FPR1, IL1rn, IL36a), the inhibitory effect of DGDG is more obvious than that of MGDG, suggesting that DGDG may play a stronger role in inflammation regulation.

[0168] The present application adopts chloroform-methanol solvent system to extract plant total lipid, and obtains GL crude product and GL subclass through two column chromatography separation. Then, GL is qualitatively and quantitatively analyzed by combining TLC and HPLC.

[0169] Under the stimulation of 1 μg / mL LPS, the inflammation model of RAW264.7 cells was successfully constructed. The present application found that plant GLs of different sources can significantly reduce the mRNA expression levels of pro-inflammatory factors IL-1β, IL-6, IL-12 and IL-16, and effectively inhibit the expression of LPS-induced M1 type macrophage surface specific antigen CD86, thereby significantly alleviating the inflammatory state of cells. This result shows that plant GLs can inhibit the polarization of RAW264.7 cells to M1 type, thereby inhibiting the inflammatory response, and play a significant anti-inflammatory role in macrophages.

[0170] In the glycolipid subclass analysis, MGDG and DGDG treatment can inhibit the expression of inflammatory factors, but DGDG has more significant effect, and DGDG shows more significant anti-inflammatory activity than MGDG.

[0171] The present application realizes efficient extraction, precise separation and structure identification of plant GLs through column chromatography combined with chromatographic analysis, which provides important technical support for plant lipid research. Research shows that plant GLs can significantly inhibit the inflammatory response of RAW264.7 macrophages, including reducing LPS-induced cell morphological changes, down-regulating inflammatory factor mRNA expression, and reducing the expression level of CD86. Multi-omics analysis further reveals the potential molecular mechanism, suggesting that the anti-inflammatory effect of plant GLs may be closely related to phospholipid metabolism, NF-κB signaling pathway and metabolic regulation. The above research results provide a solid molecular level theoretical basis for the application of plant glycolipids in anti-inflammatory drug development.

[0172] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced, without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. Use of a plant-derived glycolipid in the preparation of an anti-inflammatory medicament, characterized in that: The inflammation is an inflammation reaction of RAW264.7 cells induced by LPS; The plant-derived glycolipid realizes anti-inflammation by inhibiting the expression of CD86 and the expression of inflammatory factors IL-6, IL-12 and IL-16; The plant is selected from one or more of the following: water spinach, Chinese cabbage, cabbage, mustard green, lettuce and lettuce; The preparation method of the plant-derived glycolipid comprises: The plant material is placed in boiling water, and after cooling, it is crushed and mixed with a trichloromethane-methanol solution; The mixed slurry is filtered, trichloromethane and pure water are added to the filtrate, and after standing and layering, the lower solution is recovered; Anhydrous sodium sulfate is added to the lower solution, and after standing and dehydration, the full lipid is obtained after filtration; Silica gel is dissolved in trichloromethane, stirred uniformly, and then poured into a chromatography column; The full lipid is dissolved in trichloromethane and added to the chromatography column for elution, and the glycolipid is collected.

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