Preparation and application of pseudomonas defending FD6 anti-tumor fermentation product

Abstract

The invention belongs to the field of microbial pharmacy, and particularly relates to a preparation method and an application of an anti-tumor fermentation product of a Pseudomonas protegens (Pseudomonas protegens) FD6. The PS culture medium and the PD culture medium are adopted to ferment P.protein FD6 respectively, the PS culture medium fermentation liquor crude extract can remarkably inhibit the human non-small cell lung cancer cell strain A549, the IC50 value is 8.45 + / -0.74 microgram / mL, and the application prospect in the lung cancer treatment aspect is good.

Description

Technical Field

[0001] The invention belongs to the field of microbial pharmacy, and specifically relates to the preparation and application of anti-tumor fermentation products of Pseudomonas protegens FD6. Background Art

[0002] Malignant tumors are one of the major diseases that threaten the lives and health of Chinese residents. Chemotherapy is currently the main method for treating malignant tumors, but clinical chemotherapy drugs have many side effects, such as digestive tract reactions, gastrointestinal reactions, and nervous system damage. In addition, there are also problems such as drug resistance and high prices. Therefore, the development of new anti-tumor drugs is particularly important.

[0003] Pseudomonas bacteria have strong biosynthetic potential. Antibacterial ingredients such as 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluterorin (PLT), phenazine-1-carboxylic acid (PCA), pyrrolnitrin (PRN), hydrocyanic acid (HCN), mupirocin and lipopeptides have been discovered from Pseudomonas, which have broad application prospects in the fields of medicine and pesticides. Among them, nitropyrrolidine exhibits strong inhibitory activity against various pathogenic fungi, especially skin fungi, and is used in Japan to treat superficial fungal infections of the genus Trichophyton; mupirocin is a polyketide antibiotic isolated for the first time from Pseudomonas aeruginosa NCIMB 10585. It has specific inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) and is mainly used for local treatment of bacterial skin infections.

[0004] Pseudomonas protegens FD6 is a bacterium isolated from the rhizosphere of Qingkou vegetable in Minhou, Fujian Province. Studies have found that P.protegens FD6 can also produce abundant antimicrobial active ingredients, such as 2,4-diacetylphloroglucinol, pyrrolidine, pyocyanin and hydrocyanic acid. There are metabolic pathways in microorganisms that are not expressed under conventional culture conditions. The existence of silent metabolic pathways means the existence of more abundant metabolites. Studies have found that slight changes in culture conditions can significantly affect the biosynthesis of microbial secondary metabolites. The OSMAC strategy has become a simple, rapid and effective way to improve the chemical diversity of microbial secondary metabolites. Among them, changing the fermentation medium is the most common method. Based on the OSMAC strategy, the culture medium of P.protegens FD6 was optimized. The results showed that the chemical composition of the fermentation broth of KB culture medium was the richest. The fermentation conditions were further optimized based on KB culture medium. It was found that when the nitrogen source (tryptone) was removed and the carbon source (5‰ glycerol) was added, the fermentation broth had the strongest inhibitory effect on tomato gray mold, with an inhibition rate of up to 92.37%. Summary of the invention

[0005] The present invention provides a PS medium fermentation broth crude extract of P.protegens FD6, which is characterized by being prepared by the following method: (1) inoculating a single colony of P.protegens FD6 into an LB medium and culturing it for 24 hours at 28° C. and 180 rpm to obtain a seed solution for standby use;

[0006] (2) The seed liquid obtained in step (1) is inoculated into PS culture medium at an inoculum amount of 1‰, and cultured at room temperature to 30°C for 48-72 hours to obtain a fermentation liquid.

[0007] (3) The fermentation broth obtained in step (2) was centrifuged at 4° C. and 10,000 rpm for 30 min, the supernatant was collected, extracted with ethyl acetate 2-3 times, and concentrated under reduced pressure to obtain the crude extract of the PS medium fermentation broth.

[0008] The yield of the crude extract of the PS culture medium fermentation broth is 56.1±13.7 mg / L.

[0009] The PS culture medium fermentation broth crude extract 1 The H-NMR diagram is basically as follows Figure 2 As shown below.

[0010] The LC-MS graph of the crude extract of the PS culture medium fermentation broth is basically as follows Figure 3 shown.

[0011] Another embodiment of the present invention provides a crude extract of PD medium fermentation broth of P. protegens FD6, characterized in that it is prepared by the following method:

[0012] (1) A single colony of P. protegens FD6 was inoculated into LB medium and cultured at 28°C and 180 rpm for 24 h to obtain seed solution for later use;

[0013] (2) inoculating the seed solution obtained in step (1) into PD culture medium at an inoculum size of 1‰, and culturing at room temperature to 30° C. for 48-72 hours to obtain a fermentation liquid;

[0014] (3) The fermentation broth obtained in step (2) was centrifuged at 4° C. and 10,000 rpm for 30 min, the supernatant was collected, extracted with ethyl acetate 2-3 times, and concentrated under reduced pressure to obtain the crude extract of the PD medium fermentation broth.

[0015] The yield of the crude extract of the PD culture medium fermentation broth is 73.2±36.0 mg / L.

[0016] The crude extract of the PD culture medium fermentation broth 1 The H-NMR diagram is basically as follows Figure 2 As shown above.

[0017] The LC-MS graph of the crude extract of the PD culture medium fermentation broth is basically as follows Figure 4 shown.

[0018] Another embodiment of the present invention provides a method for preparing the crude extract of the PS and / or PD culture medium fermentation broth, characterized by comprising the above steps.

[0019] Another embodiment of the present invention provides the use of the crude extract of PS and / or PD culture medium fermentation broth in the preparation of anti-tumor drugs. The tumor is preferably a human non-small cell lung cancer cell line A549.

[0020] Another embodiment of the present invention provides a composition, characterized in that the composition uses the above PS and / or PD culture medium fermentation broth crude extract as an effective ingredient. The composition may also include other anti-tumor drugs. The composition may also contain pharmaceutically acceptable salts. The dosage form of the composition is selected from a solid preparation, a liquid preparation or a semi-solid preparation.

[0021] The formula of the LB culture medium of the present invention is 5.0 g of yeast powder, 10.0 g of tryptone, 10.0 g of NaCl per liter of water, and pH=7.0-7.2; the formula of the PD culture medium is 200 g of peeled potatoes and 20 g of glucose per liter of water; the formula of the PS culture medium is 200 g of peeled potatoes and 20 g of sucrose per liter of water.

[0022] The "Pseudomonas protegens FD6" described in the present invention, referred to as "P.protegensFD6", is a bacterium isolated from the rhizosphere of Qingkou Qingcai in Minhou, Fujian Province. Its ITS sequence has been submitted to the NCBI website, and the GenBank number is CP031396. P.protegens FD6 has been disclosed in the inventor's previous research paper "Australasian Plant Pathology, 2020, 49: 307-317" and has been deposited in the College of Horticulture and Plant Protection of Yangzhou University where the applicant is located. The public can obtain P.protegens FD6 according to the method described in the inventor's above-mentioned research paper, or purchase it from the College of Horticulture and Plant Protection of Yangzhou University where the applicant is located. The applicant promises that the College of Horticulture and Plant Protection of Yangzhou University can provide P.protegens FD6 to the public 20 years from the date of application of this invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the TLC chart of the crude extract of the fermentation broth of P. protegens FD6 in PD medium and PS medium (developing solvent: chloroform-methanol v:v 10:1).

[0024] Figure 2 It is the crude extract of PD medium and PS medium fermentation broth of P.protegens FD6 1 H-NMR chart (DMSO-d6, 400 MHz).

[0025] Figure 3 This is the LC-MS graph of the crude extract of PS medium fermentation broth of P. protegens FD6.

[0026] Figure 4 This is the LC-MS graph of the crude extract of the PD medium fermentation broth of P. protegens FD6.

[0027] Figure 5 The inhibitory effect of the crude extract of PS culture medium fermentation broth of P. protegens FD6 on the proliferation of A549 cells. (A) Crude extract of PS culture medium fermentation broth; (B) positive control cisplatin. DETAILED DESCRIPTION

[0028] In order to facilitate further understanding of the present invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the invention and are not intended to limit the scope or implementation principles of the present invention. The implementation methods of the present invention are not limited to the following contents.

[0029] Example 1 Liquid fermentation of Pseudomonas protegens FD6 and chemical composition analysis of the fermentation broth 1 Instruments and materials

[0030] DD2 nuclear magnetic resonance spectrometer (400 MHz) (Agilent Technologies, USA); Triple TOF 4600 ultra-high performance liquid chromatograph-quadrupole tandem time-of-flight mass spectrometer (AB SCIEX, USA); SHP-250 constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.); SKY-200B constant temperature oscillating shaker (Shanghai Sukun Industrial Co., Ltd.), etc.

[0031] Silica gel 60F254 aluminum plate (20 cm × 20 cm), mass spectrometry methanol (Merck, Germany); Atlantis T3 chromatographic column (2.1 mm × 100 mm, 3 μm) (Waters, USA); DMSO-d6 (Cambridge Isotope Laboratories, USA); glucose, sucrose, sodium chloride, ethyl acetate, chloroform, methanol (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); yeast powder (biochemical reagent, Beijing Aoboxing Biotechnology Co., Ltd.); trypsin (biochemical reagent, Shanghai Bowei Biotechnology Co., Ltd.), etc.

[0032] 2. Strain source and strain fermentation

[0033] Pseudomonas defensus P. protegens FD6 (GenBank No.: CP031396) was a bacterium isolated from the rhizosphere of Qingkou vegetable in Minhou, Fujian Province, and is now deposited in the College of Horticulture and Plant Protection of Yangzhou University.

[0034] (1) Pick a single colony of freshly activated P. protegens FD6 and inoculate it into LB medium (5.0 g of yeast powder, 10.0 g of tryptone, 10.0 g of NaCl, 1000 mL of water, pH = 7.0-7.2), and culture it at 28°C and 180 rpm for 24 h to obtain seed solution for later use.

[0035] (2) The seed liquid was inoculated into PD medium (200 g peeled potato, 20 g glucose, 1000 mL water) and PS medium (200 g peeled potato, 20 g sucrose, 1000 mL water) at an inoculum of 1‰, and 400 mL of the medium was placed in a 1000 mL Erlenmeyer flask and cultured at 28°C and 180 rpm for 60 h. Three bottles of each medium were fermented in parallel.

[0036] (3) The fermentation broth was centrifuged at 4°C and 10,000 rpm for 30 min, the supernatant was collected, extracted with ethyl acetate three times, and concentrated under reduced pressure to obtain a crude extract of the fermentation broth. The yields of the crude extracts of the fermentation broths in the PD medium and PS medium were 73.2±36.0 and 56.1±13.7 mg / L, respectively.

[0037] Analysis of chemical components in 3P.protegens FD6 fermentation broth

[0038] (1) Thin layer chromatography analysis of crude extract of P. protegens FD6 fermentation broth

[0039] Thin layer chromatography (TLC) analysis showed that the parallel fermentation products of the two culture media had basically the same thin layer spots, and the fermentation was relatively stable; under the ultraviolet analyzer at 254nm and 365nm, the crude extract of the PD culture medium fermentation broth was f 0.86 and R f There is an obvious spot at 0.27, while the crude extract of PS medium fermentation broth is at R f 0.93, R f 0.80 and R f There is an obvious spot at 0.40. The chemical composition of the fermentation broth of P. protegens FD6 in PD medium and PS medium is significantly different ( Figure 1 ).

[0040] (2) P. protegens FD6 fermentation broth crude extract 1 H-NMR spectrum analysis

[0041] The crude extracts of PD medium and PS medium fermentation broth were sampled (about 25 mg) and dissolved in DMSO-d6. Agilent DD2 NMR spectrometer was used for determination 1 H-NMR spectra, processed by MestReNova 14.0 software 1 H-NMR spectrum. Figure 2 As shown, the PS medium fermentation product showed four characteristic hydrogen signals δ H 0.67(dd,28.0,4.0Hz),δ H 0.77~0.91(m),δ H 1.14 (d, 8.0 Hz) and δ H 1.25~2.25(m); while the fermentation products of PD medium have δ H 2.88(t,8.0Hz), 2.99~3.12(m), δ H 4.26 (d, 8.0 Hz) and δ H4.90 (d, 4.0 Hz) and the four characteristic hydrogen signals; the fermentation products of PS medium and PD medium H There are abundant hydrogen signals at 3.20-4.00, but the hydrogen signals of the fermentation products in PD medium are significantly stronger than those in PS medium. H In the range of 6.00 to 8.50, the fermentation products of both culture media have weak hydrogen signals, but the hydrogen signals of the fermentation products of PD culture medium are richer.

[0042] (3) LC-MS / MS spectral analysis of crude extract of P. protegens FD6 fermentation broth

[0043] The crude extracts of the fermentation broth of P. protegens FD6 in PD medium and PS medium were sampled (about 1 mg / mL) and measured for LC-MS / MS spectra.

[0044] UPLC conditions: Atlantis T3 column (2.1 mm × 100 mm, 3 μm), flow rate 0.3 mL / min, injection volume 3 μL. 0.1% formic acid-water as mobile phase A, methanol as mobile phase B, elution gradient: 0 min, 65% A; 7 min, 45% A; 13 min, 30% A; 17 min, 20% A; 27 min, 5% A; 30 min, 2% A.

[0045] LC-MS / MS conditions: electrospray ion source (ESI), positive ion scan mode; primary mass spectrometry scan range m / z 100-2000, secondary mass spectrometry scan range m / z 50-2000. Spray voltage: 5500 V; ion source temperature: 550 °C; curtain gas pressure: 35 psi; nebulizer gas pressure: 55 psi; auxiliary gas pressure: 55 psi; declustering voltage: 80 V; collision energy: 35 ev; ion release delay: 30 V; ion release width: 15 V.

[0046] Figure 3 and Figure 4These are the LC-MS graphs of crude extracts of PS medium and PD medium fermentation broth, where TWC is Total Wavelength Chromatogram, XWC is Extract Wavelength Chromatogram, TIC is Total Ion Chromatogram, and BPC is Base Peak Chromatogram. The TWC graph and XWC graph show that the PS medium fermentation broth has a main peak at a retention time of 9.1 min, while the chromatographic peak signal of the PD medium fermentation broth is relatively weak and has no obvious main peak. The BPC graph shows that the ion signals of the PS medium and PD medium fermentation broth are relatively rich, and there are many similar signals, but there are also obvious differences; the PS medium fermentation broth has a significant signal of m / z 1296.8377 at a retention time of 20.1 min, and there is also an obvious ion signal at 2.1 min; the signal of the PD medium fermentation broth is relatively weak, and it has multiple weak characteristic signals at a retention time of 24.9-25.9 min ( Figure 3 and Figure 4 ).

[0047] Example 2 Inhibitory effect of crude extract of Pseudomonas protegens FD6 fermentation broth on proliferation of human non-small cell lung cancer cell line A549

[0048] 1. Instruments and Materials

[0049] Bio-Tek SYNERGY2 microplate reader (BioTek Co., Ltd., USA); Thermo SeriesⅡCO2 constant temperature incubator (Thermo Fisher Scientific, USA); CX-23 inverted microscope (Olympus, Japan); MDF-U53V-80℃ ultra-low temperature refrigerator (SANYO, Japan), etc.

[0050] Human non-small cell lung cancer cell line A549 (Shanghai Institutes for Biological Sciences Cell Bank); RPMI-1640 culture medium (Gibco, USA); fetal bovine serum (Nanjing Bio-Channel); penicillin-streptomycin mixture (Israel Biological Industries); 0.25% trypsin (Shanghai Bio-Tech Biotechnology Co., Ltd.); PBS (Jiangsu KeyGen Biotechnology Co., Ltd.); mesothiocyanate (MTT) (Beijing Solebold Technology Co., Ltd.); cisplatin injection (production batch number: 181103, Jiangsu Hausen Pharmaceutical Co., Ltd.); DMSO (analytical grade, Sinopharm Chemical Reagent Co., Ltd.), etc.

[0051] 2MTT assay to determine the in vitro cytotoxic activity of fermentation products

[0052] The crude extract of P.protegens FD6 fermentation broth is dissolved in culture medium [RPMI-1640 culture medium: fetal bovine serum: penicillin-streptomycin mixture (v:v:v) 89:10:1] and diluted into a working solution of a certain concentration (a small amount of DMSO is used to assist dissolution, and the final concentration of DMSO shall not exceed 0.1%), and is prepared and used immediately.

[0053] The in vitro cytotoxic activity of the samples was determined by MTT method, and cisplatin was used as positive control. The cell suspension in logarithmic growth phase was taken and 1.0×10 4 Cells / well were inoculated in a 96-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2 for 24h. A certain concentration of sample solution or positive control drug was added to each well, and 100μL culture medium was added to the negative control group and the blank control group (5 replicate wells were set in the experiment). After 48h of culture, 10μL MTT (final concentration of 5mg / mL) was added to each well, and the culture was continued for 4h, then the culture medium was discarded, 100μL DMSO was added to each well, and the crystals were fully dissolved by shaking at 37°C for 10min. The absorbance value of each well was measured by an enzyme marker at a wavelength of 490nm. The experiment was repeated three times, and the experimental results were expressed as mean±SD; Graphpad Prism was used for one-way analysis of variance, and Tukey test was used for comparison between groups. P<0.05 was significantly different, P<0.01 was very significantly different, and P<0.001 was extremely significantly different.

[0054] like Figure 5 As shown in the results, the PS medium fermentation broth extract of strain FD6 can significantly inhibit the proliferation of A549 cells in a dose-effect relationship; at 100 μg / mL, its inhibition rate on A549 cells is as high as 89.52%±2.68%, and IC 50 The value was 8.45±0.74μg / mL, which was comparable to the positive drug cisplatin (IC 50 The crude extract of PD medium fermentation broth of strain FD6 had a weak and significant inhibitory effect on A549 cells, and the inhibition rate was only 20.11%±2.06% at 200μg / mL.

Claims

1. A crude extract of PS medium fermentation broth of P. protegens FD6, characterized in that Prepared by the following method: (1) A single colony of P. protegens FD6 was inoculated into LB medium and cultured at 28°C and 180 rpm for 24 h to obtain seed solution for later use; (2) The seed liquid obtained in step (1) is inoculated into PS culture medium at an inoculum amount of 1‰, and cultured at room temperature to 30°C for 48-72 hours to obtain a fermentation liquid. (3) The fermentation broth obtained in step (2) was centrifuged at 4° C. and 10,000 rpm for 30 min, the supernatant was collected, extracted with ethyl acetate 2-3 times, and concentrated under reduced pressure to obtain the crude extract of the PS medium fermentation broth.

2. The crude extract according to claim 1, characterized in that Its yield was 56.1±13.7mg / L.

3. The crude extract according to any one of claims 1 to 2, characterized in that That 1 The H-NMR graph is basically as shown in Figure 2 below.

4. The crude extract according to any one of claims 1 to 3, characterized in that The LC-MS graph thereof is basically shown in FIG3 .

5. A crude extract of PD culture medium fermentation broth of P. protegens FD6, characterized in that Prepared by the following method: (1) A single colony of P. protegens FD6 was inoculated into LB medium and cultured at 28°C and 180 rpm for 24 h to obtain seed solution for later use; (2) inoculating the seed solution obtained in step (1) into PD culture medium at an inoculum size of 1‰, and culturing at room temperature to 30° C. for 48-72 hours to obtain a fermentation liquid; (3) The fermentation broth obtained in step (2) was centrifuged at 4° C. and 10,000 rpm for 30 min, the supernatant was collected, extracted with ethyl acetate 2-3 times, and concentrated under reduced pressure to obtain the crude extract of the PD medium fermentation broth.

6. The method for preparing a crude extract of PS and / or PD culture medium fermentation broth according to any one of claims 1 to 5, characterized in that The method comprises the steps described in any one of claims 1 to 5.

7. Use of the crude extract of PS and / or PD culture medium fermentation broth according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that The tumor is preferably human non-small cell lung cancer cell line A549.

9. A composition characterized in that The composition uses the PS and / or PD culture medium fermentation broth crude extract as claimed in any one of claims 1 to 5 as an effective component.

10. The composition of claim 9, characterized in that The composition may further include other anti-tumor drugs; the composition may further include pharmaceutically acceptable salts; the dosage form of the composition is selected from solid preparations, liquid preparations or semi-solid preparations.

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