Streptomyces sp. rerS4 and metabolites and applications thereof

Abstract

The present application belongs to the technical field of Streptomyces new species, and particularly relates to a Streptomyces new species RerS4 and metabolites and applications thereof, which is isolated from the rhizosphere soil of Radix Rehmanniae infected with wheel pattern disease; the classification and naming of the Streptomyces new species RerS4 is Streptomyces antiinflammaticus , the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation date is July 4, 2025, the preservation number is CCTCC M 20251535, and the 16S rRNA gene sequence of the Streptomyces new species RerS4 is shown in SEQ ID No. 1; the present application provides a Streptomyces new species with anti-inflammatory activity, a fermentation liquor, screening of anti-inflammatory active substances in the fermentation liquor, and support for subsequent drug research and development.

Description

Technical Field

[0001] This invention belongs to the field of new species of Streptomyces, and particularly relates to a new species of Streptomyces RerS4, its metabolites, and its applications. Background Technology

[0002] Actinomycetes are a class of aerobic prokaryotes that grow in filamentous form and reproduce by spores, and are an important source of natural pharmaceutical products. Rhizosphere actinomycetes from medicinal plants possess excellent antibacterial and anti-inflammatory effects, providing the possibility of identifying antibacterial active strains from these rhizosphere actinomycetes and isolating natural drugs with antibacterial activity from their secondary metabolites. Inflammation is a common pathological change in many respiratory diseases, such as chronic obstructive pulmonary disease, pneumonia, and asthma; inhibiting the inflammatory response can effectively alleviate the progression of these diseases. Existing therapeutic drugs have certain limitations in inhibiting inflammation. The RerS4 strain of *Rehmannia glutinosa* rhizosphere actinomycete, belonging to the *Streptomyces* family, is a Gram-positive bacterium whose metabolites have broad biological activities, including antibacterial, anti-inflammatory, and antitumor effects. However, the anti-inflammatory basis of its metabolites is still unclear. This invention primarily addresses this issue. Summary of the Invention

[0003] The purpose of this invention is to provide a new species of Streptomyces RerS4, its metabolites, and its applications.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A novel Streptomyces species, RerS4, was isolated from the rhizosphere soil of Rehmannia glutinosa infected with ring spot disease. The taxonomic name of this novel Streptomyces species, RerS4, is Streptomyces antiinflammaticus. The depositary institution is the China Center for Type Culture Collection (CCTCC), the deposit date is July 4, 2025, and the accession number is CCTCC NO: M 20251535. The 16S rRNA gene sequence of this novel Streptomyces species, RerS4, is shown in SEQ ID No. 1.

[0006] Another objective of this invention is to provide a seed culture solution, which is obtained by inoculating RerS4 spores into GY liquid culture medium and culturing them at a constant temperature of 28°C and a shaking incubator at 200 rpm for 3 days.

[0007] Furthermore, the GY liquid culture medium comprises: 1% yeast extract, 1% glucose, 0.05% K2HPO4·3H2O, and 0.05% MgSO4·7H2O, sterilized at 121°C for 15 minutes under pH 7.2-7.4 conditions.

[0008] Another objective of this invention is to provide a method for preparing metabolites. The above-mentioned seed culture is inoculated into H9 fermentation medium at a 6% inoculation rate and cultured at 28°C with a shaking incubator at 200 rpm for 10 days. The supernatant and bacterial precipitate are then collected. The supernatant is adsorbed onto a macroporous adsorption resin column and eluted with 3-4 column volumes of 100% ethanol, and the eluent is collected. The bacterial precipitate is soaked in 100% ethanol overnight, filtered, and the ethanol extract is collected. The eluent and ethanol extract are concentrated into pastes for later use. The paste is mixed with silica gel at a 1:1 ratio and then dry-loaded for silica gel column chromatography. The mixture is then purified with petroleum ether. Dry matter F1 was obtained by rotary evaporation with an eluent of ethyl acetate at a volume ratio of 2:1; dry matter F2 was obtained by rotary evaporation with an eluent of petroleum ether / ethyl acetate at a volume ratio of 1:1; dry matter F1 and F2 were separated by gel column chromatography using Sephadex LH-20. When the eluent was 40% methanol / water, the eluent containing the main peak was dried to obtain crude product F1-1; when the eluent was 60% methanol / water, the eluent containing the main peak was dried to obtain crude product F2-1; crude products F1-1 and F2-1 were prepared into compound I and compound II by medium-high pressure liquid chromatography.

[0009] Another object of the present invention is to provide a compound prepared by the above-described method for preparing metabolites, wherein compound I is a white powder with the molecular formula C9H12H2O. 18 O6, structural formula is Compound II is a pale yellow powder with the molecular formula C. 15 H 20 O4, structural formula is

[0010]

[0011] Another object of the present invention is to provide an application of compounds I and II in the preparation of an anti-inflammatory drug for treating diseases caused by inflammation of A549 and THP-1 cells.

[0012] The advantages of this invention are as follows: This invention provides a new species of Streptomyces antiinflammaticus with anti-inflammatory activity and its fermentation broth, and screens out anti-inflammatory active substances in the fermentation broth; for the first time, two natural products, EI-1 and ZBH, are isolated from the fermentation broth of Streptomyces antiinflammaticus. These two compounds can significantly inhibit the secretion of inflammatory factors by alveolar epithelial cells and macrophages, which can provide support for the development of drugs for respiratory diseases with inflammatory responses as the main pathological changes. Attached Figure Description

[0013] Figure 1The study investigated the effects of eight different concentrations of fermentation broth on the levels of inflammatory factors IL-6 and TNF-α in A549 alveolar epithelial cells.

[0014] Figure 2 The study investigated the effects of different concentrations of eight fermentation broths on the levels of the inflammatory factors IL-6 and TNF-α in THP-1 cells of monocytes / macrophages.

[0015] Figure 3 This is the high-resolution mass spectra of compound I of the present invention.

[0016] Figure 4 This is the 1H NMR spectrum of compound I of the present invention.

[0017] Figure 5 This is the 13C NMR spectrum of compound I of the present invention.

[0018] Figure 6 This is the COSY spectrum of the compound I1H-1H of this invention.

[0019] Figure 7 This is the HSQC spectrum of compound I of the present invention.

[0020] Figure 8 This is the HMBC spectrum of compound I of the present invention.

[0021] Figure 9 This is the high-resolution mass spectra of compound II of the present invention.

[0022] Figure 10 This is the 1H NMR spectrum of compound II of the present invention.

[0023] Figure 11 This is the 13C NMR spectrum of compound II of the present invention.

[0024] Figure 12 This is the 1H-1H COSY spectrum of compound II of the present invention.

[0025] Figure 13 This is the HSQC spectrum of compound II of the present invention.

[0026] Figure 14 This is the HMBC spectrum of compound II of the present invention.

[0027] Figure 15 Effects of different concentrations of the novel compound IE1-2 on the expression of inflammatory cytokines mRNA in A549 and THP-1 cells.

[0028] Figure 16 Effects of different concentrations of the novel compound IIZBH on the mRNA expression of inflammatory factors in A549 and THP-1 cells.

[0029] Figure 17 This is a graph showing the effect of compound I (E1-2) on the expression of NF-κB / GSK3β pathway proteins in A549 and THP-1 cells;

[0030] Figure 18 Compound II (ZBH) is responsible for the expression of NF-κB / GSK3β pathway proteins in A549 and THP-1 cells. Detailed Implementation

[0031] A novel Streptomyces species, RerS4, was isolated from the rhizosphere soil of Rehmannia glutinosa infected with ring spot disease. The taxonomic name of this novel Streptomyces species, RerS4, is Streptomyces antiinflammaticus. The depositary institution is the China Center for Type Culture Collection (CCTCC), the deposit date is July 4, 2025, and the accession number is CCTCC M20251535. The 16S rRNA gene sequence of this novel Streptomyces species, RerS4, is shown in SEQ ID No. 1.

[0032] Example 1: Isolation and Identification of Streptomyces antiinflammaticus RerS4

[0033] 1. Isolation of strains

[0034] Streptomyces antiinflammaticus RerS4 (CCTCC NO:) was isolated from the rhizosphere soil of Rehmannia glutinosa infected with ring spot disease. Rehmannia glutinosa plants from Wenxian County, Henan Province, were dug up with soil attached, ensuring intact root tissue. Excess soil was shaken off from the rhizosphere. The rhizosphere samples were placed in sterile bags and transported to the laboratory of Henan University of Traditional Chinese Medicine in an ice box. The plant rhizosphere tissue was placed in centrifuge tubes, 5 mL of PBS buffer was added, and the mixture was sonicated for 15 min. The precipitate obtained after high-speed centrifugation was the rhizosphere soil. 5 g of the Rhizosphere soil was placed in an Erlenmeyer flask containing 45 mL of sterile water (soil concentration: 10⁻¹), and incubated at 28℃ and 250 rpm with shaking for 30 min. The soil solution after shaking incubation was then diluted again to a concentration of 10. -2 10 -3 10 -4 10 -5 10 -6 Take 10g of diluted soil solution. -4 10 -5 10 -6200 μL of each of the culture was spread onto CPA medium (formulation: 0.25% cellulose, 0.2% sodium pyruvate, 0.1% proline, 0.025% KNO3, 0.02% MgSO4·7H2O, 0.02% K2HPO4·3H2O, 0.05% CaCl2·2H2O, 0.001% FeSO4·7H2O, 2% agar, pH 7.2-7.4) and incubated at 28°C for 2-3 weeks. The cultured strain was then streaked onto fresh solid ISP3 medium (formulation: 2% oats, 0.02% KNO3, 0.05% K2HPO4·3H2O, 0.02% MgSO4·7H2O, 2% agar) until pure cultures were obtained. After streaking the strain on ISP3 slant, the spores were preserved in 20% glycerol and stored at -80°C.

[0035] 2. Identification of strains

[0036] 2.1 Morphology of the strain

[0037] Strain RerS4 produces white spores on various culture media. The aerial hyphae are gray and spiral-shaped, and the spores are smooth and cylindrical. The color of the vegetative hyphae on different culture media ranges from white to gray, while the mycelium in the substrate ranges from yellowish-white to grayish-white.

[0038] 2.2 Genomic determination and analysis of the strain

[0039] The genome of strain RerS4 was extracted, and its 16S rRNA sequence (GenBank: OP420539) was determined. The sequence was then compared with the EzBioCloud database (https: / / www.ezbiocloud.net / identify). The results showed that strain RerS4 is identical to Streptomyces racemochromogenes NRRLB-5430. T and Streptomycespolychromogenes NBRC 13072 TThe similarity was 99.72%. The whole genome of strain RerS4 contained one circular chromosome and one circular plasmid (GenBank: CP097322 and CP097323). ANI data analysis of strain RerS4 with its most similar strain (https: / / jspecies.ribohost.com / jspeciesws / ) and dDDH (https: / / ggdc.dsmz.de / ggdc.php) showed results below the thresholds for identifying new species (95% and 70%, respectively), as shown in Table 1. This indicates that strain RerS4 is a new species of Streptomyces, named *Streptomyces antiinflammaticus*.

[0040] Table 1. ANI data analysis and dDDH of strain RerS4 and its most similar strain.

[0041]

[0042] Example 2: Screening of fermentation medium for anti-inflammatory compounds produced by strain RerS4

[0043] 1. Preparation of seed liquid

[0044] 1.1 Preparation of Seed Liquid

[0045] RerS4 spores were inoculated into GY liquid medium (formulation: GY liquid medium: yeast extract 1%, glucose 1%, K2HPO4·3H2O 0.05%, MgSO4·7H2O 0.05%, pH 7.2-7.4; sterilized at 121℃ for 15 minutes to obtain GY medium) and cultured at 28℃ in a shaking incubator at 200 rpm for 3 days to obtain seed liquid.

[0046] 1.2 Preparation of Fermentation Broth

[0047] Eight different fermentation media were prepared to produce fermentation broths for different metabolites. The formulations of the eight media are as follows:

[0048] ①GYMⅠ medium: glucose 0.4%, yeast extract 0.4%, malt extract 1%, CaCO3 0.2%, pH 7.2-7.4.

[0049] ②GYMⅡ medium: glucose 0.4%, malt extract 1.0%, yeast extract 0.4%, soluble starch 4.0%, CaCO3 0.2%, trace element solution 1 mL / L, pH 7.2-7.4. Trace elements: MnCl2·4H2O 0.1%, FeSO4·7H2O 0.1%, ZnSO4·7H2O 0.1%.

[0050] ③H9 medium: soluble starch 2.0%, cottonseed meal 1.0%, yeast extract 0.5%, maltodextrin 2.0%, malt extract 0.5%, MgSO4·7H2O 0.2%, NaCl 0.2%, CaCO3 0.2%, pH 7.0-7.2.

[0051] ④HZ-2 medium: yeast extract 0.4%, FeSO4·7H2O 0.1%, malt extract 0.2%, glucose 1%, soluble starch 4%, CaCO3 0.2%, MnCl2·4H2O 0.1%, pH 7.2-7.4.

[0052] ⑤ Soybean meal culture medium: 1% skim milk powder, 2% soybean meal, 8% sucrose, 0.1% K2HPO4·3H2O, 0.05% FeSO4·7H2O, pH 7.2-7.4.

[0053] ⑥MB medium: glucose 1%, soluble starch 0.5%, yeast extract 0.2%, NaCl 0.4%, tryptone 0.2%, K2HPO4·3H2O 0.05%, MgSO4·7H2O 0.05%, CaCO3 0.2%, pH 7.2-7.4.

[0054] ⑦MD medium: 1% soluble starch, 1% glucose, 1% glycerol, 0.5% yeast extract, 0.5% peptone, 0.3% CaCO3, pH 7.2-7.4.

[0055] ⑧ISP2 medium: yeast extract 0.4%, malt extract 1%, glucose 0.4%, pH 7.2-7.4.

[0056] After sterilizing the above culture medium at 120℃ for 15 minutes, the seed liquid was inoculated into the fermentation medium at an inoculation rate of 6%, and the fermentation broth was obtained after being cultured at a constant temperature of 28℃ and a shaking incubator at 200 rpm for 10 days.

[0057] 1.3 Fermentation Broth Treatment

[0058] The fermentation broth was centrifuged at 12000 rpm for 10 min. The supernatant was extracted with an equal volume of ethyl acetate. The extract was concentrated to obtain a crude extract, which was then fully dissolved in DMSO for later use. The precipitate was extracted with methanol and concentrated under reduced pressure to obtain a crude extract, which was then dissolved in DMSO for later use.

[0059] Evaluation of the anti-inflammatory activity of 1.48 fermentation broths

[0060] Human alveolar type II epithelial cells (A549) were induced with 20 ng / mL tumor necrosis factor (TNF-α), and human monocyte / macrophage THP-1 cells were induced with 1 μg / mL lipopolysaccharide (LPS) to establish cellular inflammation models. After 24 h of intervention with eight fermentation broths for TNF-α-induced A549 cells and LPS-induced THP-1 cells, the cell culture supernatant was collected. The levels of inflammatory factors IL-6 and TNF-α in the cell supernatant were detected by ELISA. The results are shown below. Figure 1 , Figure 2 . Figure 1 High, medium, and low concentrations of GYM1, H9, HZ-2, MB, and ISP2 fermentation broths, as well as 12.5% ​​concentrations of GYM2, soybean meal, and MD fermentation broth, significantly inhibited IL-6 secretion; 12.5% ​​concentrations of GYM1, H9, HZ-2, soybean meal, and MB significantly inhibited TNF-α secretion; indicating that H9, GYM1, and HZ-2 fermentation broths had a better inhibitory effect on the inflammatory response of A549 cells. Figure 2 Eight fermentation broths at medium to low concentrations all inhibited IL-6 secretion to varying degrees; high, medium, and low concentrations of GYM1, GYM2, MB, and MD fermentation broths inhibited TNF-α secretion; 12.5% ​​H9 and soybean meal fermentation broths inhibited TNF-α secretion; and 50% HZ-2 fermentation broth inhibited TNF-α secretion. This indicates that, except for ISP2 fermentation broth, the other seven fermentation broths significantly inhibited the inflammatory response of THP-1 cells. Based on the inhibitory effects of the eight fermentation broths on inflammatory factor secretion, H9 medium was selected for subsequent isolation experiments.

[0061] Example 3: Preparation of two novel anti-inflammatory compounds

[0062] 1. Extraction of fermentation broth

[0063] After fermenting strain RerS4 in 40–50 L of H9 medium for 10 days, the supernatant and cell precipitate were collected. The supernatant was adsorbed onto a macroporous adsorption resin column and eluted with 3–4 column volumes of 100% ethanol, and the eluent was collected. The cell precipitate was soaked in 100% ethanol overnight, and the ethanol extract was collected by filtration. The eluent and ethanol extract were concentrated into pastes for later use.

[0064] 2. Extraction, separation, and purification of the compounds of this invention

[0065] 2.1 Isolation and purification of compound I

[0066] The total extract was mixed with silica gel at a 1:1 ratio and then dry-loaded for silica gel column chromatography. The eluent was a 2:1 (v / v) mixture of petroleum ether and ethyl acetate. After rotary evaporation, the dry product F1 was obtained. F1 was further separated by Sephadex LH-20 gel column chromatography with methanol as the eluent. The eluent was collected and analyzed by HPLC using a C18 column, 40% methanol / water as the eluent, a flow rate of 0.75 mL / min, and a wavelength of 254 nm. The eluent containing the main peak (retention time 7.49 min) was collected and dried to obtain crude product F1-1. Compound I was then prepared by medium-high pressure preparative liquid chromatography (1 mL / min, 80% methanol / water, v / v). The characterization data are as follows:

[0067] Compound I: White powder, as determined by HR-ESI-MS [MH] + m / z: 223.0616(C9H 18 O6, calculated molecular weight: 222.11034) Figure 3 It is speculated that the molecular formula of this compound is C9H. 18 O6. 1 The 1H NMR (500MHz, CD3OD) spectrum showed 9 proton signals: 2 olefin proton signals: δ H 7.22 (1H, q, J = 1.3 Hz) and 7.39 (1H, d, J = 1.5 Hz); 5 oxygen-bonded proton signals: δ H 3.55 (1H, t, J = 3.4Hz), δ H 3.80 (1H, t, J = 3.1Hz), δ H 3.66 (1H, dd, J=3.4, 5.3Hz), δ H 3.66 (1H, dd, J = 12.3, 5.3 Hz) and δ H 4.61 (1H, d, J = 3.69 Hz); 1 methyl proton signal: δ H 1.85 (3H, d, J = 1.1 Hz) and 1 methoxy proton signal: δ H 3.40 (1H, s), such as Figure 4 . 13 The C NMR (125MHz, CD3OD) spectrum showed 9 carbon signals ( Figure 5 ), combined with the HSQC spectrum, it can be seen that there are 2 sps 2 Hybridized carbon: δ C 153.1 and 139.13; 1 sp 3 Hybridized hydroxymethyl group: δ C 55.98; 1 sp 3 Hybridized methyl group: δC 12.10; 5 sps 3 Hybridized hydroxymethylene: δ C 103.43, 72.47, 68.10, 64.79, and 70.64, such as Figure 6 . 1 H- 1 H COSY spectrum ( Figure 4 The data shows a correlation between H-1 / H-2 / H-3 / H-4, indicating that C-1, C-2, C-3, and C4 are connected; a correlation between H-7 / H-8 indicates that C-7 and C-8 are connected. Combined with the HMBC spectrum (…), Figure 2 H-7(δ) H 7.22) is associated with C-6, H-5 (δ) H 3.66) is associated with C-4, H-3 (δ) H 3.80) is related to C-5, hydrogen H-1' (δ) H 3.40) is associated with C-1, H-4 and H5 (δ) H 3.66) and C6, such as Figure 7 Thus, the planar structure of the compound was determined, and a SciFinder search revealed that compound I was a novel compound that had not been previously reported.

[0068] 2.2 Isolation and purification of compound II

[0069] The total extract was mixed with silica gel at a 1:1 ratio and then dry-loaded for silica gel column chromatography. The eluent was a 1:1 (v / v) mixture of petroleum ether and ethyl acetate. After rotary evaporation, the dry product F2 was obtained. F2 was further separated by Sephadex LH-20 gel column chromatography with methanol as the eluent. The eluent was collected and analyzed by HPLC using a C18 column, 60% methanol / water as the eluent, a flow rate of 0.75 mL / min, and a wavelength of 254 nm. The eluent containing the main peak (retention time 10.52 min) was collected and dried to obtain crude product F2-1. Compound II was prepared by medium-high pressure preparative liquid chromatography (3 mL / min, 90% acetonitrile / water, v / v). The characterization data are as follows:

[0070] Compound 2: Pale yellow powder, [M-Na] as determined by HR-ESI-MS. + m / z:287.1648(C 15 H 20 O4, calculated molecular weight: 264.13616) Figure 9 It is speculated that the molecular formula of this compound is C. 15 H 20 O4. 1The 1H NMR (500MHz, CDCl3) spectrum showed 11 proton signals: two 1,4-disubstituted benzene ring proton signals: δ H 7.71 (1H, dd, J = 5.7, 3.3 Hz) and 7.53 (1H, dd, J = 5.7, 3.3 Hz); one hydroxymethylene proton signal: δ H 4.31 (2H, t, J = 6.7 Hz); 3 methylene proton signals δ H 1.72 (2H, m), δ H 1.44(2H, m) and δ H 1.15 (2H, d, J = 6.5 Hz); 2 methyl proton signals: δ H 0.86 (3H, d, J = 6.6 Hz) and δ H 0.96 (3H, t, J = 7.4 Hz); 1 methylene proton signal: δ H 1.50 (1H, m), such as Figure 10 . 13 The C NMR (125MHz, CDCl3) spectrum showed 15 carbon signals ( Figure 11 ), including 1 carbonyl carbon δ C 198.05 and 1 carboxyl carbon δ C 178.30; Combining the HSQC spectrum, it can be seen that there are 6 sps 2 All hybridized carbons are carbons on the benzene ring: δ C 132.45, 128.99, 131.06, 168.87, 131.06 and 128.99; 1 sp 3 Hybridized hydroxymethylene: δ C 65.73; 2 sps 3 Hybridized methyl group: δ C 13.88 and 22.82; 3 sps 3 Hybridized methylene: δ C 30.73, 19.34 and 39.21; 1 sp 3 Hybridized methine δ C 28.12, such as Figure 6 . 1 H- 1 H COSY spectrum ( Figure 12 The data shows that H-2 / H-3 is correlated, H-5 / H-6 is correlated, H-3" / H-2" / H-3"a is correlated, and H-1' / H-2' / H-3' / H-4' is correlated. Figure 13 Combined with the HMBC spectrum, H-1'(δ) H 4.31) is associated with C-4, H-3 and H5 (δ) H7.53) is associated with C-4, H-2, and H6 (δ) H 7.71) and H3' are related to C-1, hydrogen H-3"" H 1.51) Related to C-1”, such as Figure 14 Thus, the planar structure of the compound was determined, and a SciFinder search revealed that compound II was a novel compound that had not been previously reported.

[0071] The assignments of compounds I and II C and H are shown in Table 2.

[0072] Table 2. Assignment of compounds 1 and IIC and H.

[0073]

[0074] Example 4: Evaluation of the anti-inflammatory activity of two novel anti-inflammatory compounds

[0075] 1. Effects of novel compound I (E1-2) on the mRNA expression of inflammatory factors in A549 and THP-1 cells

[0076] Human alveolar epithelial cells (A549) were induced with 20 ng / mL TNF-α, and human monocyte / macrophage (THP-1) cells were induced with 1 μg / mL LPS. After 24 h of intervention with different concentrations (60, 40, and 20 μM) of E1-2, TNF-α-induced A549 cells and LPS-induced THP-1 cells, cells were collected, total RNA was extracted, and the mRNA expression of inflammatory factors IL-1β, TNF-α, IFN-γ, and IL-6 was detected by qPCR. The results showed that 60 μM E1-2 significantly inhibited the mRNA expression of IL-6, TNF-α, and IL-1β in A549 and THP-1 cells; 40 μM E1-2 significantly inhibited the mRNA expression of IL-6, IL-1β, IFN-γ, and TNF-α in A549 cells; and 40 μM E1-2 significantly inhibited the mRNA expression of IFN-γ in A549 cells. (See attached results). Figure 15 .

[0077] The anti-inflammatory effect of E1-2 may be related to the presence of more hydroxyl groups in its structure. Studies have found that compounds rich in hydroxyl groups can regulate the NF-κB, GSK3β and MAPK pathways and inhibit the secretion of inflammatory factors such as TNF-α.

[0078] 2. Effects of novel compound II (ZBH) on the mRNA expression of inflammatory factors in A549 and THP-1 cells

[0079] A549 cells were induced with 20 ng / mL TNF-α, and THP-1 cells were induced with 1 μg / mL LPS. After intervention with different concentrations (200, 150, and 100 μM) of ZBH for 24 h, cells were collected, total RNA was extracted, and the mRNA expression of inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-10 was detected by qPCR. The results showed that 200 μM ZBH significantly reduced the mRNA levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in A549 and THP-1 cells, and increased the mRNA level of IL-10. Expression; 150 μM ZBH significantly reduced IL-1β, IL-6, and TNF-α expression in THP-1 cells, and increased IL-10 mRNA expression in A549 and THP-1 cells; 200 μM and 150 μM ZBH significantly reduced IL-1β, IL-6, and TNF-α expression in THP-1 cells, and increased IL-10 mRNA expression; 50 μM ZBH significantly reduced IL-6 expression in THP-1 cells and increased IL-10 mRNA expression. Results are shown below. Figure 16 The anti-inflammatory effect of ZBH may be related to the presence of a carboxyl group in its structure. Studies have found that compounds containing carboxyl groups can reduce inflammatory responses by inhibiting the activation of the NF-κB pathway, suppressing the secretion of inflammatory factors TNF-α and IL-6.

[0080] 3. Effects of novel compound I (E1-2) on the expression of NF-κB / GSK3β pathway proteins in A549 and THP-1 cells

[0081] A549 cells were induced with 20 ng / mL TNF-α, and THP-1 cells were induced with 1 μg / mL LPS. After 24 h of intervention with different concentrations (60, 40, and 20 μM) of E1-2, A549 and THP-1 cells were collected, total cellular protein was extracted, and protein denaturation was performed. Western blotting was used to detect the expression of NF-κB / GSK3β pathway markers p-P65, p65, p-GSK3β, and GSK3β. The results showed that 60, 40, and 20 μM E1-2 significantly inhibited the phosphorylation of P65 at Ser536 and GSK3β at Tyr216 in A549 cells, while 40 μM E1-2 significantly reduced the phosphorylation of P65 at Ser536 and GSK3β at Tyr216 in THP-1 cells, thereby inhibiting the activation of the NF-κB / GSK3β pathway. This indicates that E1-2 can inhibit the secretion of downstream inflammatory factors by regulating the NF-κB / GSK3β pathway, thereby suppressing the inflammatory response of alveolar epithelial cells and macrophages. (See...) Figure 17 .

[0082] 4. Effects of novel compound II (ZBH) on the expression of NF-κB / GSK3β pathway proteins in A549 and THP-1 cells

[0083] A549 cells were induced with 20 ng / mL TNF-α, and THP-1 cells were induced with 1 μg / mL LPS. After 24 h of intervention with different concentrations (200, 150, and 100 μM) of ZBH, cells were collected, total cellular protein was extracted, and protein denaturation was performed. Western blotting was used to detect the expression of NF-κB / GSK3β pathway markers p-P65, p65, p-GSK3β, and GSK3β. The results showed that 150 and 100 μM ZBH significantly inhibited the phosphorylation of P65 at Ser536 and GSK3β at Tyr216 in A549 cells. 200, 150, and 100 μM ZBH significantly inhibited the phosphorylation of P65 at Ser536 in THP-1 cells, but had no significant inhibitory effect on the phosphorylation of GSK3β at Tyr216. This indicates that ZBH can regulate the NF-κB / GSK3β pathway and inhibit the inflammatory response of alveolar epithelial cells. (See...) Figure 18 .

Claims

1. A compound, characterized in that: This compound was obtained from strain with accession number CCTCC No: M20251535 ( Streptomyces antiinflammaticus RerS4 was obtained through fermentation and extraction; compound I was a white powder with the molecular formula C9H. 18 O6, structural formula is Compound II is a pale yellow powder with the molecular formula C. 15 H 20 O4, structural formula is .

2. The application of the compound as described in claim 1, characterized in that: Compounds I and II are used to prepare anti-inflammatory drugs, which are drugs for treating respiratory diseases.

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