Use of plantain fruit extract in stimulating macrophages to transform into M2 type macrophages

Abstract

The application discloses application of plantain fruit extract in stimulating macrophages to transform into M2 type macrophages and belongs to the technical field of biological medicine. The application is verified by in-vitro experiments, the plantain fruit extract has a clear activation effect on LPS-induced macrophages, can effectively improve the activity of macrophages in an inflammatory state, can significantly induce the M2 type polarization of macrophages to anti-inflammation, and realizes the targeted regulation of the polarization of macrophages.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of banana fruit extract in stimulating macrophages to transform into M2 type macrophages. Background Technology

[0002] As core effector cells of the innate immune system, macrophages exhibit highly plastic functional phenotypes, polarizing into two classic subpopulations based on microenvironmental signals: the pro-inflammatory M1 type and the anti-inflammatory and repair-oriented M2 type. M1 macrophages are primarily activated by pro-inflammatory signals such as lipopolysaccharide and interferon-γ, highly expressing co-stimulatory molecules like CD80 and CD86, and secreting large amounts of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β, dominating the body's acute inflammatory responses against infection and tumors. M2 macrophages, on the other hand, differentiate under the induction of IL-4 and IL-13, highly expressing markers such as CD206 and CD163, and primarily secreting anti-inflammatory factors such as IL-10 and transforming growth factor-β (TGF-β). Their core functions are to suppress excessive inflammation, mediate tissue damage repair, and regulate immune homeostasis. Imbalance in macrophage M1 / M2 polarization is a core element in many pathological processes, including chronic inflammation, autoimmune diseases, fibrosis, and impaired wound healing. Therefore, developing safe and efficient natural active substances to precisely induce macrophage polarization toward the M2 type has become an important research direction in the field of immune regulation and tissue repair.

[0003] Plantain, a perennial herbaceous plant belonging to the genus Musa in the family Musaceae, has fruits rich in various bioactive components such as polysaccharides, flavonoids, polyphenols, terpenes, and amino acids. Modern pharmacological studies have confirmed its significant antioxidant, immunomodulatory, and tissue repair activities. Existing research indicates that plantain fruit extract can regulate lymphocyte subset differentiation, enhance NK cell function, and improve immune phenotype imbalance. However, the specific regulatory effect of plantain fruit extract on macrophage polarization, especially its induction of macrophage transformation to the M2 type, has not yet been systematically elucidated. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of banana fruit extract in stimulating macrophages to transform into M2 macrophages. Banana fruit extract increases LPS-induced macrophage activity and the gene expression levels of M2 macrophage-specific markers Arg1 and CD206, clarifying the induction effect of banana fruit extract on macrophage polarization to M2 type.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of banana fruit extract in the preparation of products that stimulate macrophages to polarize into M2 type macrophages.

[0006] Preferably, the macrophages include macrophage lines RAW264.7, THP-1, U937, bone marrow-induced macrophages BMDM, peripheral blood-derived mononuclear macrophages PBMC, tumor-associated macrophages, or primary macrophages derived from mammalian tissues.

[0007] Preferably, the stimulation of macrophages to polarize into M2 macrophages manifests as increased expression of the Arg1 and CD206 genes.

[0008] Preferably, the macrophages are LPS-induced macrophages.

[0009] Preferably, the banana fruit extract activates macrophages in an inflammatory state, improving cell survival rate.

[0010] Preferably, the preparation of the banana fruit extract includes the following steps: crushing the roots, flowers and fruits of the banana tree to obtain the extract; heating the extract and soaking it in salt water, then breaking the cell walls at low temperature to obtain a crude extract; centrifuging and allowing it to stand to obtain the upper extract; and fermenting it to obtain the banana fruit extract.

[0011] Preferably, the weight ratio of tree roots, flowers, and fruits is (5-7):(1-3):(1-3); the volume percentage of the brine is 20-30%; the crude extract is dark brown, has a pH of 12-14, and a potential difference of -230 to -250 mV; and the fermentation time is 85-95 days.

[0012] This invention provides a method for stimulating macrophages to polarize into M2 type macrophages, comprising the following steps: adding banana fruit extract to the macrophage culture system.

[0013] Preferably, the concentration of the banana fruit extract is 0.5-1%.

[0014] Preferably, the processing time for the banana fruit extract is 4-6 hours.

[0015] The beneficial effects of this invention are: This invention is the first to verify through in vitro experiments that banana fruit extract has a clear activating effect on LPS-induced macrophages, can effectively enhance the activity of macrophages under inflammatory conditions, and can significantly induce macrophages to polarize to the anti-inflammatory M2 type, thus achieving targeted regulation of macrophage polarization. Attached Figure Description

[0016] Figure 1 This is a flow cytometry analysis of the blank control group.

[0017] Figure 2 This is a flow cytometry analysis diagram of the model group.

[0018] Figure 3 This is a flow cytometry analysis of the high-dose group.

[0019] Figure 4 This is a flow cytometry analysis of the low-dose group.

[0020] Figure 5 This is a flow cytometry analysis of the positive drug group.

[0021] Figure 6 The image shows the results of RT-qPCR detection of Arg1 gene expression in each group.

[0022] Figure 7 The image shows the results of RT-qPCR detection of CD206 gene expression in each group. Detailed Implementation

[0023] This invention provides the application of banana fruit extract in the preparation of products that stimulate macrophage polarization into M2-type macrophages. The macrophages described in this invention include macrophage lines RAW264.7, THP-1, U937, bone marrow-induced macrophages (BMDM), peripheral blood-derived monocyte-macrophages (PBMCs), tumor-associated macrophages, or primary macrophages derived from mammalian tissues, preferably RAW264.7, and more preferably LPS-induced RAW264.7. Unless otherwise specified, the banana fruit extract of this invention can be obtained through commercially available channels known in the art. The banana fruit extract of this invention is preferably prepared according to a portion of the extraction process described in Example 1 of Patent TW1584814B, which is the preparation of banana extract.

[0024] In this invention, stimulating macrophages to polarize into M2-type macrophages manifests as increased expression of the Arg1 and CD206 genes. The banana fruit extract of this invention can also activate macrophages in an inflammatory state, improving cell survival. The effective concentration of the banana fruit extract of this invention is 0.5-1%, and setting the banana fruit extract to this concentration has a mild effect on macrophages without significant cell damage.

[0025] In this invention, the preferred preparation of banana fruit extract includes the following steps: pulverizing the roots, flowers, and fruits of a banana tree to obtain the extract; heating the extract and soaking it in salt water, followed by low-temperature cell wall disruption to obtain a crude extract; centrifuging and allowing it to stand to collect the supernatant extract; and fermenting the extract to obtain the banana fruit extract. The banana tree used in this invention is a mature banana tree. The weight ratio of the roots, flowers, and fruits in this invention is (5-7):(1-3):(1-3), preferably 6:2:2; the volume percentage of the salt water is 20-30%, preferably 25%; the crude extract is dark brown, has a pH of 12-14, and a potential difference of -230 to -250 mV, preferably dark brown, has a pH of 13, and a potential difference of -240 mV; the fermentation time is 85-95 days, preferably 90 days. The banana fruit extract obtained by the above preparation method of this invention can enhance the activity of LPS-induced macrophages and can polarize LPS-induced macrophages into M2 type macrophages.

[0026] In this invention, the product is preferably a pharmaceutical product, which includes oral preparations, injections, and topical preparations; the oral preparations include tablets, capsules, granules, pills, powders, oral liquids, and syrups; the injections include injection solutions and lyophilized powder injections.

[0027] This invention also provides a method for stimulating macrophages to polarize into M2 type macrophages, comprising the following steps: adding banana fruit extract to a macrophage culture system. The concentration of the banana fruit extract used in this invention is 0.5-1%, and the treatment time is 4-6 hours.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Unless otherwise specified, the following embodiments are all conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1 The preparation steps for banana fruit extract are as follows: 1. Harvest mature banana trees that are 5-7m tall, taking roots, flowers, and fruit in a weight ratio of 6:2:2, with a moisture content controlled below 30%. Wash the stems, leaves, and peel with water to remove pesticide residues, then crush them into 2-3cm fragments using a crusher to obtain the extract.

[0032] 2. The extract to be extracted is heated with a far-infrared stone layer (wavelength 9μm) and soaked in a 25% brine solution for 9 hours; the volume ratio of the extract to the brine solution is 70:30.

[0033] 3. The soaked material was treated with a low-temperature cell disruption method (-40℃ for 6 hours) to extract a dark brown, alkaline (pH 13) crude extract with a potential difference of -240mV.

[0034] 4. Centrifuge the crude extract at 3000 rpm for 1 min to separate the fibers from the liquid. Filter the liquid portion through a 100-mesh filter and let it stand in a container for 24 h. Take 80-90% of the liquid from the top half of the container and discard the sediment at the bottom to obtain the crude extract.

[0035] 5. Pour the crude extract into a fermentation tank lined with a layer of far-infrared stones (spectral wavelength 9μm) and ferment for 90 days to obtain banana fruit extract.

[0036] Example 2 1. Macrophage RAW264.7 culture Culture conditions: The culture medium used was high-glucose DMEM (containing L-glutamine and sodium pyruvate) supplemented with 10% heat-inactivated FBS and 1% double antibiotics (100U / mL penicillin and 100μg / mL streptomycin). The culture environment was 37℃ and 5% CO2.

[0037] Subculturing: Discard the old culture medium, wash once with PBS; add 2-3 mL of preheated complete culture medium, gently scrape / pipette detached cells; centrifuge at 1000 rpm for 5 min, discard the supernatant; resuspend in fresh culture medium, divide into flasks at a 1:3 ratio, and change the medium after 24 h. Cryopreservation and thawing: The cryopreservation solution is prepared with 90% complete culture medium and 10% DMSO, and the cell density is adjusted to 1×10⁻⁶ cells / mL. 6 -5×10 6 Cells / mL, prepare cell suspensions, cool them down and store them in liquid nitrogen; thaw the frozen cell suspensions rapidly in a 3°C water bath (<1 min), dilute and centrifuge, resuspend and inoculate.

[0038] 2. CCK-8 Preliminary Experiment: Seven concentration gradients of banana fruit extract were set up: 0%, 1%, 2%, 4%, 6%, 8%, and 10%. The banana fruit extract prepared in Example 1 was diluted with water to obtain these concentration gradients. RAW264.7 macrophages were seeded in 96-well plates (5 × 10⁻⁶). 3 After treating each well with 100 μL of banana fruit extract at different concentrations, 10 µL of CCK-8 reagent was added to each well. The culture plate was then incubated at 37°C and 5% CO2 for 2 hours. The absorbance at 450 nm was read using a microplate reader, and the cell viability was calculated to screen the safe and effective concentration range of banana fruit extract.

[0039] The results are shown in Table 1. The active ingredients of the banana fruit extract showed no significant toxicity to RAW264.7 macrophages within the 0-1% concentration range. At a 1% concentration, cell viability remained at 91.16%, indicating that the extract had a mild effect on macrophages within this concentration range and caused no significant cell damage. Based on this, 0.5% was determined as the low dose and 1% as the high dose, serving as the core dosage concentrations for subsequent experiments.

[0040] Table 1. Preliminary Experiment Results of CCK-8

[0041] 3. CCK-8 activation verification experiment Five experimental groups were set up: control group: cells were kept in a basic growth state without any treatment; model group: cells were treated with 100 ng / mL LPS for 24 h to induce an M1 macrophage inflammation model; low-dose group: cells were pretreated with 100 μL / well of 0.5% banana fruit extract for 5 h, followed by treatment with 100 ng / mL LPS for 24 h; high-dose group: cells were pretreated with 100 μL / well of 1% banana fruit extract for 5 h, followed by treatment with 100 ng / mL LPS for 24 h; positive control group: cells were pretreated with interleukin-4 (IL-4) for 5 h, followed by treatment with 100 ng / mL LPS for 24 h.

[0042] Cells in each group were seeded and cultured in the same manner as in the CCK-8 pre-experiment. The absorbance was detected by the CCK-8 method and the cell viability was calculated to verify the activation effect of banana fruit extract on macrophages under inflammatory conditions.

[0043] The results are shown in Table 2. Compared with the model group, the low-dose group, the high-dose group, and the positive control group all significantly activated macrophages in the inflammatory state, improved cell survival rate, and showed good cell activation effect. Among them, the activation effect of banana fruit extract showed obvious dose dependence. The activation effect of the high-dose group was better than that of the low-dose group, and only slightly weaker than that of the positive control drug IL-4.

[0044] Table 2 CCK-8 Activation Verification Results

[0045] 4. Flow cytometry-EDU detection of cell viability Log-phase RAW264.7 macrophages were harvested at a density of 1 × 10⁶ cells per well. 6Cells were seeded into 6-well plates. After overnight culture, each group was treated according to step 3 of this embodiment. After treatment, 2×EdU working solution (20 μM) was prepared. An equal volume of the preheated 37°C 2×EdU working solution (20 μM) was added to the 6-well plates to bring the final EdU concentration in the 6-well plates to 1×. Cells were incubated for another 2 hours. After EdU labeling of cells, the culture medium was removed, and adherent cells were washed twice with PBS. Cells were collected, centrifuged at 1000g for 4 min, and then gently resuspended in PBS and counted. 50,000-100,000 resuspended cells were centrifuged at 1000g for 5 min, and the supernatant was discarded. Add 1 ml of fixative (4% paraformaldehyde) and fix at room temperature for 15 min. Remove the fixative and wash each well three times with 1 ml of washing buffer for 3-5 min each time. Remove the washing buffer and incubate each well with 1 ml of permeabilization buffer (PBS containing 0.3% Triton X-100) at room temperature for 10-15 min. Remove the permeabilization buffer and wash each well 1-2 times with 1 ml of washing buffer for 3-5 min each time. Remove the washing buffer from the previous step and add 0.5 ml of Click reaction solution to each well (dissolve one tube of Click Additive in 1.3 ml of deionized water, mix until completely dissolved, aliquot, and store at -20°C). Gently shake the culture plate to ensure the reaction mixture evenly covers the sample. Incubate at room temperature in the dark for 30 min. Aspirate the Click reaction solution and wash three times with washing buffer for 5 min each time. Analyze the cells after staining.

[0046] The results are as follows Figure 1-5 The results showed that macrophages in the control group maintained normal high activity levels, while in the model group, after LPS induction, the Mean X value decreased significantly, and macrophage activity was significantly inhibited. Pretreatment with banana fruit extract significantly increased cell fluorescence intensity in both low- and high-dose groups compared to the model group, effectively enhancing macrophage activity, with the high-dose group showing the best effect. Compared to the positive control group, the effect of extract treatment was weaker, and different concentrations of extract treatment showed a drug dose-dependent effect.

[0047] 5. RT-qPCR detection of M2 biomarker expression Log-phase RAW264.7 macrophages were harvested at a density of 1 × 10⁶ cells per well. 6 Cells were seeded into 6-well plates. After overnight culture, each group was treated according to step 3 of this embodiment. Cells were collected at 1×10⁶ wells. 6 -1×10 7 Add 1 ml of TRNzol reagent to the cells.

[0048] RNA extraction: After adding TRNzol, react the sample at room temperature for 10 min. Add 200 µL of chloroform, shake vigorously for 15 s (do not use a vortex mixer), and incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 15 min at 4°C. After centrifugation, the solution will separate into three layers: a phenol-form layer, a middle white protein layer, and a clear aqueous layer. RNA is present in the aqueous layer. Transfer 400 µL of the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol (pre-cooled at 4°C), and incubate at -20°C for 15 min. Centrifuge at 12000 rpm for 10 min at 4°C. Discard the supernatant and add 1 mL of 75% ethanol (pre-cooled at 4°C) to wash the RNA precipitate. Gently invert and wash thoroughly. Centrifuge at 12000 rpm for 5 min at 4°C. Discard the supernatant and repeat the step once. Discard the supernatant, carefully aspirate any remaining liquid with a pipette, and allow to air dry. Add 50 µL of DEPC water and gently pipette to dissolve completely. RNA purity and concentration determination: The OD value and concentration of extracted RNA were determined using NanoDrop 2000. RNA integrity determination: RNA integrity was assessed using 1.5% agarose gel electrophoresis. RNA samples were stored at -80°C for extended periods.

[0049] In vitro reverse transcription: Add 4 µL 5× Reaction Buffer, 1 µL reverse transcription primer, 5 µg RNA, 1 µL L Ribolock RNase inhibitor (20 U / µL), 1 µL Revertaid M-MuLV reverse transcriptase (200 U / µL), 2 µL 10 mM dNTP Mix, and RNase / DNase-free ddH2O to a 20 µL volume. Transfer this mixture to a PCR tube, mix gently on ice, and centrifuge. Incubate at 42 °C for 60 min. Gently mix the template and primer mixture, briefly centrifuge, incubate at 65 °C for 5 min, cool on ice, centrifuge again, and place on ice once more. Terminate the reaction by heating at 70 °C for 5 min. Dilute the resulting cDNA and use it directly in the PCR reaction.

[0050] PCR amplification: Diluted cDNA (approximately 100 ng / µl) was used as the PCR template. The PCR reaction mixture was as follows: 100 ng cDNA, 0.5 µL primer F (10 µM), 0.5 µL primer R (10 µM), 10 µL SYBR mix, 0.4 µL ROX Reference Dye (50×), and RNase / DNase-free ddH2O added to a final volume of 20 µL. The PCR reaction program was as follows: 94℃ pre-denaturation for 30 s; 94℃ for 5 s, 60℃ for 30 s, 40 cycles; 95℃ for 15 s, 60℃ for 1 min, with a temperature increase of 0.3℃ every 15 s; 95℃ for 15 s.

[0051] Data analysis: The Ct values ​​of each PCR reaction were read using ABI 7500 software. The Ct value of the target gene was subtracted from the Ct value of the internal reference gene to obtain ΔCt; the mean Δt value of the experimental group was subtracted from the mean Δt value of the blank group to obtain ΔΔCt. (Using 2...) -ΔΔC The t-test was used to calculate the change in target gene expression in the experimental group relative to the control group. The results are as follows: Figure 6-7 .

[0052] Figure 6 The results showed that, compared with the control group, the Arg1 gene expression level in macrophages of the model group was significantly reduced; compared with the model group, the Arg1 gene expression level in the low-dose group, high-dose group, and positive drug group was significantly upregulated, and the upregulation effect in the high-dose group was better than that in the low-dose group, and the differences between the groups were statistically significant. p<0.0001). Figure 7 The results showed that, compared with the control group, the expression level of CD206 gene in macrophages in the model group was significantly decreased; compared with the model group, the expression level of CD206 gene in the low-dose group, high-dose group, and positive drug group was significantly increased, and the high-dose group was more effective than the low-dose group. The differences between the groups were statistically significant. p<0.0001).

[0053] The above results indicate that the active ingredients in banana fruit extract can significantly promote the gene expression of macrophage M2-specific markers Arg1 and CD206, effectively induce macrophage polarization towards the anti-inflammatory M2 type, and this effect is dose-dependent.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of banana fruit extract in the preparation of products that stimulate macrophage polarization into M2 type macrophages.

2. The application as described in claim 1, characterized in that, The macrophages include macrophage lines RAW264.7, THP-1, U937, bone marrow-induced macrophages BMDM, peripheral blood-derived mononuclear macrophages PBMC, tumor-associated macrophages, or primary macrophages derived from mammalian tissues.

3. The application as described in claim 1, characterized in that, The stimulation of macrophages to polarize into M2 macrophages manifests as increased expression of the Arg1 and CD206 genes.

4. The application as described in any one of claims 1-3, characterized in that, The macrophages mentioned are LPS-induced macrophages.

5. The application as described in claim 1, characterized in that, The banana fruit extract activates macrophages in an inflammatory state and improves cell survival rate.

6. The application as described in claim 1, characterized in that, The preparation of the banana fruit extract includes the following steps: crushing the roots, flowers and fruits of the banana tree to obtain the extract; heating the extract and soaking it in salt water, then breaking the cell walls at low temperature to obtain a crude extract; centrifuging and allowing it to stand to obtain the upper extract; and fermenting it to obtain the banana fruit extract.

7. The application as described in claim 6, characterized in that, The weight ratio of tree roots, flowers, and fruits is (5-7):(1-3):(1-3); the volume percentage of the brine is 20-30%; the characteristics of the crude extract are dark brown, pH 12-14, and potential difference of -230~-250mV; the fermentation time is 85-95 days.

8. A method for stimulating macrophages to polarize into M2 type macrophages, characterized in that, The process includes the following steps: adding banana fruit extract to the macrophage culture system.

9. The method as described in claim 8, characterized in that, The concentration of the banana fruit extract was 0.5-1%.

10. The method as described in claim 9, characterized in that, The processing time for the banana fruit extract is 4-6 hours.

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