Application of Berninamycin C in inhibition of microorganisms and anti-inflammation
By isolating and identifying Berninamycin C from Streptocytica Deep Olive, the problem of the lack of effective inhibition of A549 cell inflammation and Gram-positive bacteria in the prior art was solved, and good antibacterial and anti-inflammatory effects were achieved, providing new drug choices.
Patent Information
- Application Number
- CN202510959449.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-08-22
AI Technical Summary
There is a lack of effective drugs in the prior art for inhibiting the inflammatory response of A549 cells and inhibiting microorganisms, especially Gram-positive bacteria, and Berninamycin C has few studies on anti-tumor and anti-inflammatory activities.
Berninamycin C was isolated from a strain of Streptocytica olive from the soil of the Tibetan Plateau, and its application in inhibiting microorganisms and anti-inflammatory, specifically used to inhibit inflammation of Gram-positive bacteria and tumor cells. It was determined by fermentation product isolation, liquid chromatography and mass spectrometry to have good antibacterial and anti-inflammatory effects at a concentration of 3.125 μM-25 μM.
Berninamycin C showed significant antibacterial and anti-inflammatory activities against Gram-positive bacteria and a variety of tumor cells, which can effectively inhibit the secretion of IL-6, NO and TNFα, and provide new antibacterial and anti-inflammatory drug options.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of anti-tumor technology, and in particular to the application of Berninamycin C in inhibiting microorganisms and anti-inflammation. Background Art
[0002] The Berninamycins family belongs to the class of thiopeptides, which are ribosomally synthesized and post-translationally modified peptides and have an oxazolyl-thiazolyl-pyridine core. Streptomyces bernensis In a study published in the journal Nature Communications, berninamycin A was found to be the primary synthetic product, followed by the subsequent reporting of other berninamycin homologues. Research on the activity of berninamycin compounds has primarily focused on antibacterial activity, with berninamycin A and berninamycin B exhibiting strong activity against Gram-positive bacteria. Furthermore, patents have shown that berninamycin A can act as a growth promoter. The thiazole antibiotic thiostrepton has been reported to act as a proteasome inhibitor in mammalian tumor cells, while berninamycin lacks proteasome inhibitory activity. Consequently, the anti-tumor and anti-inflammatory activities of berninamycin compounds have been less well-researched.
[0003] A549 cells are a type of non-small cell lung cancer (NSCLC) cell line. NSCLC is the most common type of lung cancer, accounting for over 80% of lung cancer cases. Currently, there are no specific drugs that target the inflammatory response of A549 cells and thereby inhibit the progression of inflammation. In light of this, the present invention was developed. Summary of the Invention
[0004] The purpose of the present invention is to provide the use of Berninamycin C in inhibiting microorganisms and anti-inflammation to solve the above technical problems.
[0005] The present invention is achieved in that: In a first aspect, the present invention provides a use of Berninamycin C in inhibiting microorganisms, wherein the use is not for the purpose of treating a disease, and the microorganisms are Gram-positive bacteria.
[0006] In a second aspect, the present invention provides a use of Berninamycin C in the preparation of an anti-inflammatory drug, wherein the inflammation is selected from tumor cell inflammation, immune cell inflammation or non-immune cell inflammation.
[0007] The present invention has the following beneficial effects: This study isolated Berninamycin C from the fermentation product of Streptomyces oleiferus, a strain native to the Qinghai-Tibet Plateau. The bioactivity of Berninamycin C was studied, demonstrating its excellent antimicrobial and anti-inflammatory properties. This study provides a new option for antibacterial and anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0009] Figure 1 This is the liquid chromatogram during compound preparation; Figure 2 This is the result of liquid chromatography purity analysis of the collected 15-min effluent; Figure 3 is the mass spectrum of Berninamycin C; Figure 4 Berninamycin C NMR 1 H spectrum detection result diagram; Figure 5 Berninamycin C NMR 13 C spectrum detection result diagram; Figure 6 The figure shows the IL-6 concentration test results after A549 cells were treated with Berninamycin C (Note: NT represents the blank treatment group without IL-1β induction); Figure 7 The figure shows the results of nitric oxide concentration detection in RAW264.7 cells after treatment with Berninamycin C (Note: NT represents the blank treatment group without LPS induction); Figure 8 The figure shows the results of IL-6 concentration detection in RAW264.7 cells after treatment with berninamycin C (Note: NT represents the blank treatment group without LPS induction); Figure 9 The figure shows the results of TNFα concentration detection after RAW264.7 cells were treated with berninamycin C (Note: NT represents the blank treatment group without LPS induction). DETAILED DESCRIPTION
[0010] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.
[0011] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.
[0012] Berninamycin C is a cyclic thiopeptide with the following structural formula:
[0013] Berninamycin C can be directly purchased commercially or isolated from microbial fermentation products.
[0014] In a first aspect, the present invention provides a use of Berninamycin C in inhibiting microorganisms, wherein the use is not for the purpose of treating a disease, and the microorganisms are Gram-positive bacteria.
[0015] The above uses include but are not limited to antibacterial uses in food and cosmetics.
[0016] In a preferred embodiment of the present invention, the Gram-positive bacteria are selected from Bacillus subtilis ( Bacillus subtilis ), Staphylococcus aureus ( Staphylococcus aureus ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Micrococcus lysodeikticus ( Micrococcus lysodeikticus ) and Micrococcus luteus ( Micrococcus luteus ) at least one of the following.
[0017] In a preferred embodiment of the present invention, the use for inhibiting microorganisms includes use in the preparation of an antibacterial agent, wherein the final concentration of Berninamycin C in the antibacterial agent is 3.125 μM to 25 μM. At this concentration, it exhibits good antibacterial activity. In other embodiments, the concentration may be higher than 25 μM.
[0018] In a second aspect, the present invention provides a use of Berninamycin C in the preparation of an anti-inflammatory drug, wherein the inflammation is selected from tumor cell inflammation, immune cell inflammation or non-immune cell inflammation.
[0019] The inventors found that Berninamycin C has anti-inflammatory properties and has good anti-inflammatory effects at lower treatment concentrations (12.5 μM-25 μM).
[0020] Tumor cell inflammation is selected from IL-1β-induced tumor cell inflammation, and the tumor cells are selected from non-small cell lung cancer, breast cancer, melanoma, colon cancer, renal cancer, lymphoma, mast cell tumor, liver cancer, pituitary tumor, myeloma, brain neuroma, testicular interstitial cell tumor, gastric cancer, prostate cancer or pancreatic cancer; immune cells are selected from macrophages, neutrophils, mast cells, dendritic cells, natural killer cells, Th1 cells, Th17 cells, cytotoxic T cells (CTL) or B cells; non-immune cells are selected from endothelial cells, fibroblasts, epithelial cells, astrocytes, hepatocytes or adipocytes.
[0021] In a preferred embodiment of the present invention, the final concentration of Berninamycin C in the drug is 3.125 μM-25 μM.
[0022] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.
[0023] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is selected from one or more of the following: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, flavoring agents, sweeteners, pigments and liposomes.
[0024] In a preferred embodiment of the present invention, the drug is an oral drug or an injection.
[0025] The features and performance of the present invention are further described in detail below with reference to the embodiments.
[0026] Example 1 The strain PS-15 of Streptomyces oliveri was isolated from soil of the Qinghai-Tibet Plateau and deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number: GDMCC No. 65939. The taxonomic name is: Streptomyces atroolivaceus The deposit date is February 26, 2025, and the name of the submitted biological material is: Streptomyces atroolivaceusPS-15 was identified as viable. Fermentation of the strain, separation of the fermentation products, and analysis of the extracts revealed that the strain's primary metabolite was berninamycin C.
[0027] The isolation and identification methods of Berninamycin C are as follows: 1. Strain fermentation, extraction and column chromatography steps: (1) The PS-15 strain preserved above was inoculated into TSB medium and cultured with shaking until the liquid became turbid and no large mycelial clumps were found, which could be used as seed liquid.
[0028] (2) Take 200 μL of seeds and spread them on ISP2 solid medium. After the surface is dried, place it in a 28°C incubator for about 10 days. Ferment a total of 30 L of ISP2 solid medium.
[0029] (3) The fermented culture medium was ground in a meat grinder, and the ground sludge was placed in a 2 L glass bottle. 1.5 L of ethyl acetate was added and fully dissolved. An ultrasonic cleaner was used to remove bubbles for 2 min to promote dissolution, and extraction was performed three times.
[0030] (4) Filter the extract and use a rotary evaporator to dry it to obtain a crude extract.
[0031] (5) The crude extract was mixed with a small amount of 200-300 mesh normal phase silica gel (weight ratio = 1:0.8), dissolved in a small amount of ethyl acetate, and then evaporated to dryness using a rotary evaporator.
[0032] (6) Pour in about 400 g of silica gel powder, dissolve it in ethyl acetate, stir it, and pour it into the column to allow the silica gel to settle (the chromatographic medium in the silica gel column is silica gel and ethyl acetate; the eluent is ethyl acetate and methanol).
[0033] (7) Add the evaporated powder sample to the upper layer of silica gel. After flowing for about 10 seconds, add a small amount of dry silica gel to the upper layer of the sample. Add ethyl acetate to dissolve the upper layer of silica gel and plug it with a small amount of cotton.
[0034] (8) Continue to pour in ethyl acetate to rinse the sample.
[0035] (9) When the colored layer is seen flowing to the middle of the column, start collecting fractions. When the fractions collected reach 1 L, start mixing methanol (5%, 10%, 20%, 40%) into the mobile phase. Rinse the last bottle with 100% methanol, and collect 150 mL in each bottle.
[0036] (10) The collected product was evaporated to dryness using a rotary evaporator and transferred to a vial for compound preparation.
[0037] 2. Preparation of compounds: Liquid chromatography is performed on the isolated extract of step 1 to obtain a high-purity compound: Liquid chromatography conditions for compound preparation were as follows: column temperature: 23°C, preparative column: 150 × 10 mm, Gemini 5 μm NX-C18 (Phenomenex). Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile (0.1% formic acid), gradient from 35% to 60% mobile phase B over 20 min, flow rate: 2.5 mL / min.
[0038] Preliminary experiments revealed that sharp peaks appeared at 12.5 and 15 min, respectively, and the target compound had high absorption peaks at 250 nm and 280 nm through full wavelength scanning.
[0039] Therefore, the program was set to collect the product at 12.5 and 15 min, and the signal at 250 nm and 280 nm was detected under the UV detector. The liquid chromatogram results at 250 nm were referenced to Figure 1 shown.
[0040] The product collected at 15 minutes after effluent was loaded onto the liquid chromatography column again, and the signal at 250 nm was detected under the UV detector to determine the purity of the separated product. The signal at 250 nm was referenced to Figure 2 As shown in the figure, a sharp peak appeared when the effluent was collected for 8 minutes. HPLC analysis showed that the main product of this strain was highly abundant.
[0041] The product of the collected effluent at 15 minutes was dried, and the yield of the main metabolite per liter of fermentation medium after fermentation in ISP2 medium was about 10 mg (for subsequent mass spectrometry and nuclear magnetic resonance detection).
[0042] 3. The product with the sharp peak at 15 min in the above-mentioned liquid chromatography separation (compound preparation stage) was subjected to high performance liquid chromatography-mass spectrometry and nuclear magnetic resonance identification. The HPLC analysis conditions were as follows: Column temperature: 23°C. Preparative column: 100 × 2.1 mm, Gemini 1.7 μm NX-C18 (Phenomenex). Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The elution gradient was: initial 20% B, increasing linearly to 100% B over 12 minutes, returning to 20% B at 14 minutes. The flow rate was 0.3 mL / min, and the injection volume was 5 μL.
[0043] Mass spectrometry detection conditions are as follows: Column temperature: 23°C. Preparative column: 100 × 2.1 mm, Gemini 1.7 μm NX-C18 (Phenomenex). Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. The elution gradient was: initial 20% B, increasing linearly to 100% B over 12 minutes, and returning to 20% B at 14 minutes. The flow rate was 0.3 mL / min, and the injection volume was 5 μL. Detection was in positive ion mode.
[0044] The mass spectrometry results of the sharp peak at 15 min separated by liquid chromatography refer to Figure 3 As shown, nuclear magnetic 1 H spectroscopy and NMR 13 C spectrum refers to Figure 4-Figure 5 The results show that the product with a sharp peak at 15 minutes of liquid chromatography separation is Berninamycin C. That is, the liquid chromatography peak time of Berninamycin C during the preparation in step 2 is 15 minutes.
[0045] Example 2 This example measures the minimum inhibitory concentration of Berninamycin C: (1) The bacteria tested were Gram-positive bacteria, Bacillus subtilis ( Bacillus subtilis ), Staphylococcus aureus ( Staphylococcus aureus ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Micrococcus lysodeikticus ( Micrococcus lysodeikticus ), Micrococcus luteus ( Micrococcus luteus ).
[0046] Streak each of the above strains from -80°C freezer onto LB solid medium and culture overnight. Pick a single colony and inoculate it onto fresh LB solid medium and culture until a single colony emerges. Pick a single colony from the second streak and inoculate it onto 5 mL of LB liquid medium and shake until turbid. Dip a needle into the bacterial solution and inoculate it onto MHA medium. Culture until a large single colony emerges and store at 4°C.
[0047] (2) Under a sterile environment, Berninamycin C was diluted to a concentration gradient of 80 μM-2.5 μM, with Kana as a positive control. The Berninamycin C in this example and the following examples was obtained by isolating the extract of the fermentation product. In other embodiments, it can also be directly purchased from the market.
[0048] (3) Wash the colonies on the MHA medium with 1 mL of NaPB (sterile) and dilute to OD 600 =0.006.
[0049] (4) Incubation: Add 50 μL of different concentrations of Berninamycin C compound and 50 μL of diluted bacterial solution to a 96-well plate. Repeat three times for each concentration, mix gently, and incubate at 28°C for 24 h. Observe the growth of bacteria in the wells with the naked eye and record the results.
[0050] The results are shown in Table 1. Berninamycin C showed low minimum inhibitory concentrations against the five Gram-positive bacteria tested. The minimum inhibitory concentration refers to the range from the highest compound concentration with bacterial growth (turbidity) to the lowest compound concentration without bacterial growth (clear) in the experimental group, which is the MIC.
[0051] Table 1. Minimum inhibitory concentration of Berninamycin C against Gram-positive bacteria (μM)
[0052] Example 3 In this example, an anti-inflammatory experiment of Berninamycin C on A549 cells was conducted.
[0053] 1. The steps of anti-inflammatory experiment are as follows: 1. A549 cells were seeded at a density of 15,000 cells / well in a 96-well plate. 100 μL of F12K medium containing 10% FBS was added and cultured overnight.
[0054] 2. After 48 h of starvation treatment, discard the blank culture medium.
[0055] 3. Add 200 μL of F12K culture medium containing different concentrations of Berninamycin C, and methanol as a solvent control.
[0056] 4. After treating the cells with the compounds for 30 min, 1 ng / mL of the pro-inflammatory cytokine IL-1β was added.
[0057] Incubate at 37°C for 48 h.
[0058] 6. Transfer the culture supernatant to a 1.5 mL EP tube and centrifuge at 1000 rpm for 20 min at 4°C. Collect the supernatant for IL-6 ELISA testing. (If testing cannot be performed on the same day, the sample can be stored in a -20°C refrigerator.)
[0059] 2. IL-6 ELISA detection steps: 1. Coating Antibody: Dissolve the capture antibody in coating bufffer at a ratio of 1:250. Add 50 μL to a 96-well ELISA plate and wrap the plate with tin foil. Coating should proceed overnight at 4°C.
[0060] 2. Washing: Remove the overnight coated 96-well plate, shake off the liquid, and tap to remove any remaining liquid. Add 300 μL of wash buffer to each well, let it sit for 1 minute, shake off the liquid, and tap to remove any remaining liquid. Repeat this step three times.
[0061] 3. Blocking: Add 300 μL of assay diluent solution to each well and block at room temperature for 1 h.
[0062] 4. Wash: Wash the plate three times with wash buffer.
[0063] 5. Combine primary antibody (add samples and standards): Thaw the standards and samples 15 min in advance, add 50 μL to each well, and incubate at room temperature for 2 h.
[0064] Standard dilution: Dissolve 4.5 μL of IL-6 standard in 595 μL of assay diluent. Perform serial two-fold dilutions to the seventh concentration.
[0065] Sample dilution: Dilute the sample according to actual conditions.
[0066] 6. Wash: Wash the plate three times with wash buffer (same steps as step 3).
[0067] 7. Bind the secondary antibody: Dilute the detection Ab into the assay diluent solution at a ratio of 1:250 and add an equal volume of enzyme reagent (HRP) to prepare a worker detector (use within 15 minutes). Add 50 μL of the worker detector to each well. Incubate at room temperature, protected from light, for 1 hour.
[0068] 8. Wash: Wash the plate three times with wash buffer (same steps as step 3).
[0069] 9. Color development: Place the substrate solution (TMB) at room temperature 30-60 minutes in advance. 15 minutes before use, mix Solution A and Solution B in a 1:1 ratio. Add 50 μL of the substrate solution to each well and incubate at room temperature for 10 minutes in the dark.
[0070] 10. Stop the reaction: Add 25 μL of stop solution to each well.
[0071] 11. Detection: Detect the absorbance at 450 nm and 570 nm within 30 minutes.
[0072] 12. Calculate the secretion of IL-6.
[0073] Figure 6 The results showed that Berninamycin C treatment could significantly reduce the secretion of IL-6 in A549 cells induced by IL-1β and inhibit the development of inflammation.
[0074] Experimental Example 4 Berninamycin C anti-inflammatory experimental steps for RAW264.7 cells (macrophages): 1. RAW264.7 cells were seeded at a density of 10,000 cells / well in a 96-well plate. 100 μL of DMEM medium containing 10% FBS was added and cultured for 48 h.
[0075] 2. After 48 h of culture, discard the blank culture medium.
[0076] 3. Add 200 μL of DMEM (with 5% FBS) culture medium containing different concentrations of Berninamycin C, and methanol as a solvent control.
[0077] 4. Add 1 μg / mL of the pro-inflammatory cytokine LPS 1 h after treating the cells with the compound.
[0078] Incubate at 37°C for 48 h.
[0079] 6. Transfer the culture supernatant to a 1.5 mL EP tube and centrifuge at 1000 rpm for 20 min at 4°C. Collect the supernatant for NO content detection (Solebo S0021 Nitric Oxide Detection Kit), IL-6 secretion detection (Xinbosheng EMC004, Mouse IL-6 ELISA Kit), and TNFα secretion detection (Xinbosheng EMC102, Mouse TNFα ELISA Kit).
[0080] Nitric oxide detection steps: 1. Take out Griess Reagent I (hereinafter referred to as "Reagent I") and Griess Reagent II (hereinafter referred to as "Reagent II") and return them to room temperature.
[0081] 2. Dilute the standard to concentrations of 3.125, 6.25, 12.5, 25, 50, or 100 µM in complete culture medium (DMEM + 10% FBS).
[0082] 3. Add standards and samples to a 96-well plate at a volume of 50 µL per well.
[0083] 4. Add 50 µL / well of room temperature Reagent I to each well.
[0084] 5. Add room temperature Reagent II to each well at 50 µL / well.
[0085] 6. Measure the absorbance at 540 nm using a microplate reader.
[0086] 7. Calculate the concentration of nitric oxide in the sample based on the standard curve.
[0087] The IL-6 and TNFα detection steps are the same as those in the kit.
[0088] Figure 7 、 Figure 8 and Figure 9 The results showed that Berninamycin C treatment could significantly reduce the secretion of IL-6, NO, and TNFα in RAW264.7 cells induced by LPS, which means that Berninamycin C plays a role in inhibiting tumor cell inflammation.
[0089] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.
Claims
1. Use of Berninamycin C in inhibiting microorganisms, wherein the use is not for the purpose of treating a disease, characterized in that: The microorganism is a Gram-positive bacterium.
2. The use according to claim 1, characterized in that The Gram-positive bacteria are selected from Bacillus subtilis ( Bacillus subtilis ), Staphylococcus aureus ( Staphylococcus aureus ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Micrococcus lysodeikticus ( Micrococcus lysodeikticus ) and Micrococcus luteus ( Micrococcu luteus ) at least one of the following.
3. The use according to claim 1, characterized in that The use in inhibiting microorganisms includes use in preparing an antibacterial agent, wherein the final concentration of the Berninamycin C in the antibacterial agent is 3.125 μM-25 μM.
4. Use of Berninamycin C in the preparation of an anti-inflammatory drug, characterized in that: The inflammation is selected from tumor cell inflammation, immune cell inflammation or non-immune cell inflammation.
5. The use according to claim 4, characterized in that The tumor cell inflammation is selected from IL-1β-induced tumor cell inflammation, and the tumor cells are selected from non-small cell lung cancer, breast cancer, melanoma, colon cancer, renal cancer, lymphoma, mast cell tumor, liver cancer, pituitary tumor, myeloma, brain neuroma, testicular interstitial cell tumor, gastric cancer, prostate cancer or pancreatic cancer; the immune cells are selected from macrophages, neutrophils, mast cells, dendritic cells, natural killer cells, Th1 cells, Th17 cells, cytotoxic T cells (CTL) or B cells; the non-immune cells are selected from endothelial cells, fibroblasts, epithelial cells, astrocytes, hepatocytes or adipocytes.
6. The use according to claim 4, characterized in that The final concentration of the Berninamycin C in the drug is 3.125 μM-25 μM.
7. The use according to claim 4, characterized in that The drug further includes a pharmaceutically acceptable carrier.
8. The use according to claim 7, characterized in that The medicine is an oral medicine or an injection.