CN114982763A - New use of geldanamycin and its analogue - Google Patents
New use of geldanamycin and its analogue Download PDFInfo
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- CN114982763A CN114982763A CN202210373144.6A CN202210373144A CN114982763A CN 114982763 A CN114982763 A CN 114982763A CN 202210373144 A CN202210373144 A CN 202210373144A CN 114982763 A CN114982763 A CN 114982763A
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- JRZJKWGQFNTSRN-UHFFFAOYSA-N Geldanamycin Natural products C1C(C)CC(OC)C(O)C(C)C=C(C)C(OC(N)=O)C(OC)CCC=C(C)C(=O)NC2=CC(=O)C(OC)=C1C2=O JRZJKWGQFNTSRN-UHFFFAOYSA-N 0.000 title claims abstract description 38
- QTQAWLPCGQOSGP-GBTDJJJQSA-N geldanamycin Chemical compound N1C(=O)\C(C)=C/C=C\[C@@H](OC)[C@H](OC(N)=O)\C(C)=C/[C@@H](C)[C@@H](O)[C@H](OC)C[C@@H](C)CC2=C(OC)C(=O)C=C1C2=O QTQAWLPCGQOSGP-GBTDJJJQSA-N 0.000 title claims abstract description 37
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/46—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom rings with more than six members
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D225/00—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom
- C07D225/04—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
- C07D225/06—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems condensed with one six-membered ring
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Agronomy & Crop Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
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Abstract
The invention discloses a new application of geldanamycin and analogues thereof, which has broad-spectrum antibacterial effect on various plant pathogenic bacteria or plant diseases, wherein one or more of compound 1, compound 2 and compound 3 in the geldanamycin and the analogues thereof have antibacterial activity on various crop pathogenic fungi and pathogenic bacteria, and can be used as biological pesticides for preventing and treating plant diseases caused by pathogenic bacteria such as anthracnose fungi, corynebacterium fungi, fusarium fungi, phomopsis fungi, xanthomonas bacteria and the like, including but not limited to banana fusarium oxysporum, banana anthracnose bacteria, pepper corynebacterium sporum, pepper phytophthora capsici, pepper anthracnose bacteria, papaya brown pedicellus rot bacteria, papaya ulcer bacteria, cowpea gray spot bacteria, mango anthracnose bacteria, rubber anthracnose bacteria, wheat gibberellic disease, Rice blast, cucumber root rot, rice white leaf blight and mango bacterial angular leaf spot.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a new application of geldanamycin and analogues thereof, in particular to an application of geldanamycin and analogues thereof in preparing a medicine with a broad-spectrum antibacterial effect on various plant pathogenic bacteria or plant diseases.
Background
Chemical pesticides can achieve good effects on controlling plant diseases in a short time, but adverse effects of chemical pesticides are increasingly manifested over time. In addition, the concept of 'green ecology' is gradually advocated, and the research and the production of pollution-free biological pesticides become urgent. The pesticide antibiotic is one of the branches of the biological pesticide, and has the characteristics of no residue, easy decomposition, environmental friendliness and the like. However, at present, the commercialized agricultural antibiotics are few, and the agricultural production requirement cannot be met. However, part of the antibiotic control objects are single, and the application range is limited to a certain extent. Especially in tropical regions, many tropical crop disease control still mainly uses chemical pesticides, and agricultural antibiotics are applied less or have no obvious effect. Therefore, the discovery of agricultural antibiotics with control effect on crops, especially tropical crop diseases, has important production significance.
Disclosure of Invention
The invention aims to provide new application of geldanamycin and analogues thereof.
The above object of the present invention is achieved by the following technical solutions: the novel application of the geldanamycin and the analogue thereof in preparing the medicine with broad-spectrum antibacterial effect on various plant pathogenic bacteria or plant diseases is disclosed, wherein the geldanamycin and the analogue thereof are one or more of a compound 1, a compound 2 and a compound 3 with the structures shown as follows:
preferably, the plant is a variety of commercial crops.
Preferably, the plant is cucumber, pepper, papaya, cowpea, mango, banana, rice, wheat or rubber tree.
Preferably, the plant pathogenic bacteria are one or more of anthrax fungi, clavulan fungi, fusarium fungi, phytophthora fungi, trichomonad fungi, pyricularia, phomopsis fungi and xanthomonas bacteria.
Preferably, the plant diseases comprise one or more of banana wilt pathogen, banana colletotrichum, pepper corynebacterium spore leaf spot pathogen, pepper phytophthora capsici, papaya colletotrichum, papaya brown rot pathogen, papaya ulcer pathogen, cowpea grey spot pathogen, mango colletotrichum, rubber colletotrichum, wheat scab pathogen, rice blast pathogen, cucumber root rot pathogen, rice white leaf blight pathogen and mango bacterial angular leaf spot pathogen.
Preferably, the geldanamycin and the analogue thereof are prepared by the following method:
(1) selecting a fermentation strain: the fermentation strain is Streptomyces (Streptomyces sp.) HNBCa1, which is preserved in Guangdong province microorganism strain collection center at 9/3 of 2021, with the preservation registration number being GDMCC NO. 61901;
(2) preparing a seed solution: culturing the streptomycete in the step (1) by adopting a seed culture medium to obtain a seed solution;
(3) fermentation: inoculating the seed liquid in the step (2) into a fermentation culture medium for fermentation treatment to obtain fermentation liquid;
(4) extraction of fermentation products: filtering the fermentation liquor obtained in the step (3), extracting bacteria liquid by using ethyl acetate to obtain extract liquor, extracting bacteria by using methanol-dichloromethane mixed liquor to obtain extract liquor, combining the extract liquor and the extract liquor, and concentrating under reduced pressure to obtain a crude extract containing geldanamycin and analogues thereof;
(5) isolation of geldanamycin and its analogs: separating the crude extract with normal phase silica gel chromatographic column, reverse phase silica gel chromatographic column, dextran gel chromatographic column and semi-preparative high performance liquid chromatography to obtain geldanamycin and its analogues.
Preferably, the seed culture medium in step (2) contains: 15-20 g of tryptone, 2-4 g of soybean papain hydrolysate, 4-6 g of sodium chloride, 2-3 g of dipotassium phosphate, 2-3 g of glucose and 1L of distilled water, wherein the pH value of the seed culture medium is 7.3 +/-0.2.
Preferably, when the seed culture medium is adopted for culture in the step (2), the shake culture is carried out for 3-5 days at the temperature of 28-30 ℃ and the rpm of 150-170.
Preferably, the fermentation medium in step (3) contains: 15-25 g of soluble starch, 4-6 g of peptone, 2-4 g of malt extract, 2-4 g of yeast extract, 8-12 g of glucose, 0.5-1.5 g of calcium carbonate and 1L of distilled water, wherein the pH value of the fermentation medium is 7.0.
Preferably, in the step (4), the volume of the ethyl acetate is equal to that of the bacterial liquid, and the volume ratio of the methanol to the dichloromethane in the methanol-dichloromethane mixed solution is 1: 1.
preferably, when the crude extract is passed through a normal phase silica gel chromatographic column in the step (5), the crude extract is purified by a column chromatography method using a column chromatography method with a volume ratio of 100: 0. 100, and (2) a step of: 1. 100: 2. 100, and (2) a step of: 4. 100, and (2) a step of: 8. 100, and (2) a step of: 16. 100, and (2) a step of: 32. 100, and (2) a step of: 64. 0: gradient eluting with 100 portions of chloroform-methanol to obtain 9 polarity sections Fr.A-Fr.I; wherein, the compound 1 and the compound 2 are obtained by respectively purifying Fr.D and Fr.F through a reverse phase silica gel chromatographic column, a sephadex chromatographic column and a semi-preparative high performance liquid chromatograph, and the compound 3 is obtained by purifying Fr.G through a normal phase silica gel chromatographic column, a reverse phase silica gel chromatographic column, a sephadex chromatographic column and a semi-preparative high performance liquid chromatograph.
Preferably, the reverse-phase silica gel chromatographic column in the step (5) is an ODS C18 reverse-phase silica gel chromatographic column, and the Sephadex chromatographic column is a Sephadex LH-20 Sephadex chromatographic column.
Compared with the prior art, the invention has the following advantages:
(1) the compound 1, the compound 2 and the compound 3 in the geldanamycin and the analogue thereof are natural products derived from microorganisms, are easy to degrade and are environment-friendly;
(2) the production raw materials of the compound 1, the compound 2 and the compound 3 in the geldanamycin and the analogue thereof provided by the invention are easy to obtain, the process is relatively simple, the cost is low, and the geldanamycin and the analogue thereof are suitable for being used as pesticides;
(3) biological activity test experiments show that one or more of a compound 1, a compound 2 and a compound 3 in geldanamycin and analogues thereof have antibacterial activity on various crop pathogenic fungi and pathogenic bacteria, and can be used as a biological pesticide for preventing plant diseases caused by pathogenic bacteria such as anthracnose fungus, clavispora fungus, fusarium fungus, phytophthora fungus, diplodia fungus, pyricularia, phomopsis fungus and xanthomonas bacterium, including but not limited to banana fusarium oxysporum, banana colletotrichum, capsicum clavatum, capsicum phytophthora capsici, papaya colletotrichum, papaya brown stalk rot, papaya ulcer bacteria, cowpea gray spot bacteria, mango colletotrichum, rubber colletotrichum, wheat mildew, rice blast bacteria, cucumber root rot bacteria, etc, Crop diseases caused by rice bacterial blight and mango bacterial angular leaf spot.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a chemical structure of compounds 1-3 isolated and identified in example 1;
FIG. 2 is a graph showing the antibacterial effects of the compounds 1 to 3 of example 2 on part of pathogenic fungi, wherein the middle of a culture medium plate is perforated and then a compound solution is added, and both ends are inoculated with the pathogenic fungi, wherein: 1A-1D are control groups (pathogenic fungi were inoculated only at both ends of the medium plate); 2A-2D are Compound 1 (geldanamycin) -treated groups; 3A-3D are Compound 1+ Compound 2 (geldanamycin +17-O-demethyl-geldanamycin) treatment groups; 4A-4D are compound 3 (rebetastatin) treated groups; 5A-5D are chlorothalonil treatment groups; 6A-6D are carbendazim treatment groups; 7A-7D are myclobutanil treatment groups; wherein, line A is corynespora capsici leaf spot pathogen; the row B is phytophthora capsici; row C is papaya brown pedicellus melo; line D is Cucumis sativus.
Detailed Description
Example 1 fermentation of the Strain HNBCa1 and fermentation product extraction
(1) The fermentation strain is Streptomyces (Streptomyces sp.) HNBCa1, which is deposited at the Guangdong province microorganism culture collection center at 9.3.2021, and the deposition registration number is GDMCC NO.61901 (strain patent application number: 202111339491.9, application date: 11.12.2021);
(2) preparing a seed solution: the seed culture medium comprises tryptone 17.0g, soybean papain hydrolysate 3.0g, sodium chloride 5.0g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, distilled water 1L, and pH 7.3 + -0.2. Activating the Streptomyces sp HNBCa1 strain in the step (1) by a commercial ISP2 culture medium solid plate, inoculating 10 circular bacterium blocks with the diameter of 7mm into a seed culture medium, and performing shaking culture for 5 days at the temperature of 28 ℃ and the speed of 160rpm to obtain a seed solution;
(3) fermentation: the formula of the fermentation medium comprises 20.0g of soluble starch, 5.0g of peptone, 3.0g of malt extract, 3.0g of yeast extract, 10.0g of glucose, 1.0g of calcium carbonate and 1L of distilled water, the pH value is 7.0, the seed liquid in the step (2) is inoculated into the fermentation medium according to the inoculation amount (volume percentage content) of 10 percent, and the mixture is subjected to shaking fermentation for 10 days at the temperature of 28 ℃ and the rpm of 160;
(4) extraction of fermentation products: filtering the obtained 10L of fermentation liquor warp cloth, extracting the bacterial liquid with ethyl acetate in equal volume to obtain an extract, and mixing the bacterial with methanol in a volume ratio of 1: extracting with dichloromethane to obtain extractive solution, mixing the extractive solution and the extractive solution, and concentrating under reduced pressure with rotary evaporator to obtain crude extract containing geldanamycin and its analogues.
EXAMPLE 2 isolation of geldanamycin-like Compounds
And (2) carrying out silica gel (particle diameter is 45-75 mu m) chromatography on the crude extract obtained in the example 1, and carrying out column chromatography on the crude extract by using a solvent with a volume ratio of 100: 0. 100, and (2) a step of: 1. 100, and (2) a step of: 2. 100, and (2) a step of: 4. 100, and (2) a step of: 8. 100, and (2) a step of: 16. 100, and (2) a step of: 32. 100, and (2) a step of: 64. 0: gradient elution with 100 chloroform-methanol to obtain 9 polar segments (Fr.A-Fr.I), subjecting Fr.D and Fr.F to reverse phase silica gel chromatography column, Sephadex LH-20 gel column chromatography and semi-preparative high performance liquid chromatography (Waters 1525 type, the same below) to obtain compound 1(230.5mg) and compound 2(10.1mg), subjecting Fr.G to normal phase silica gel chromatography column, reverse phase silica gel chromatography column, Sephadex LH-20 gel column chromatography and semi-preparative high performance liquid chromatography to obtain compound 3(33.8mg), wherein the structural formulas of compound 1, compound 2 and compound 3 are shown in FIG. 1.
Example 3 structural identification of geldanamycin-like compounds
The chemical structure of the monomer compound separated in example 2 was determined by nuclear magnetic resonance spectroscopy (NMR) and high resolution mass spectrometry (HRESIMS), and the measured data were consistent with the data reported in the literature for the compound, and the specific data are as follows. The nuclear magnetic spectrum testing instrument is a Bruker AVIII-500MHz nuclear magnetic resonance spectrometer, and the high-resolution mass spectrum testing instrument is an Agilent 6210TOF LC-MS mass spectrometer.
Compound 1: orange powder with peak mass-to-charge ratio of M/z 559.2773[ M-H ] as measured by negative source HRESIMS]-, formula C 29 H 40 N 2 O 9 . It is composed of 1 H-NMR data (CDCl) 3 ,500MHz)δ H Respectively as follows: 8.72(1H, br s, NH-1),7.29(1H, s, H-19),6.94(1H, d, J ═ 12.1Hz, H-3),6.58(1H, t, J ═ 11.7,10.8Hz, H-4),5.90(1H, q, J ═ 11.1,9.9Hz, H-5),5.82(1H, d, J ═ 9.7Hz, H-9),5.19(1H, d, J ═ 4.9Hz, H-7),4.32(1H, d, J ═ 9.3Hz, H-6),4.13(1H, s, OCH) 3 -17),3.54(1H,d,J＝8.5Hz,H-10),3.37(3H,s,OCH 3 -12),3.31(3H,s,OCH 3 -12),3.30(3H,s,OCH 3 -6),2.79(1H,q,H-12),2.46(2H,m,H-15),2.03(3H,s,CH 3 -2),1.80(3H,s,CH 3 -8),1.65(H,br s,H-14),1.01(3H,d,J＝6.5Hz,CH 3 -10),0.97(3H,d,J＝7.0Hz,CH 3 -14)； 13 C-NMR data (CDCl) 3 -d 6 ,125MHz)δ C Respectively as follows: 185.0(C-21),184.1(C-18),168.3(C-1),157.0(C-17),156.0 (OCONH) 2 -7),138.1(C-20),136.4(C-5),134.8(C-2),133.2(C-8),133.1(C-9),127.6(C-3),127.2(C-16),126.3(C-4),111.7(C-19),81.7(C-7),81.3(C-6),81.0(C-12),72.7(C-11),61.7(OCH 3 -17),57.3(OCH 3 -6),56.7(OCH 3 -12),34.7(C-13),32.8(C-15),32.2(C-10),27.9(C-14),23.0(CH 3 -14),12.9(CH 3 -10),12.5(CH 3 -8),12.3(CH 3 -2). The above data of hydrogen spectrum and carbon spectrum are combined, and are identified as geldanamycin (geldanamycin) consistent with the literature report.
Compound 2: yellow powder with peak mass-to-charge ratio of M/z 545.2516[ M-H ] as measured by negative source HRESIMS] - Molecular formula is C 28 H 38 N 2 O 9 . It is composed of 1 H-NMR data (CDCl) 3 ,500MHz)δ H Respectively as follows: 8.96(1H, s, NH-1),7.42(1H, s, H-19),6.97(1H, d, J ═ 11.8Hz, H-3),6.58(1H, t, J ═ 11.8Hz, H-4),5.90(1H, t, J ═ 11.8Hz, H-5),5.81(1H, d, J ═ 9.5Hz, H-9),5.18(1H, s, H-7),4.32(1H, d, J ═ 9.3Hz, H-6),3.53(1H, m, H-11),3.51(1H, m, H-5),3.36(3H, s, OCH) 3 -6),3.31(3H,s,OCH 3 -12),2.79(1H,m,H-10),2.45(1H,m,H-15),2.03(3H,s,OCH 3 -8),1.80(3H,s,OCH 3 -2),1.76(1H,br s,H-13,H-14),1.00(3H,d,J＝6.3Hz,CH 3 -10),0.97(3H,d,J＝7.0Hz,CH 3 -14)。 13 C-NMR data (CDCl) 3 -d 6 ,125MHz)δ C Respectively as follows: 184.3(C-21),183.1(C-18),168.2(C-1),156 (OCONH) 2 -7),153.1(C-17),140.5(C-20),134.6(C-2),133.3(C-8),133.1(C-9),127.6(C-3),126.2(C-4),117.4(C-16),108.2(C-19),81.8(C-7),81.4(C-6),81.0(C-12),72.7(C-11),57.4(OCH 3 -8),56.7(OCH 3 -6),34.3(C-13),32.6(C-15),32.2(C-10),28.0(C-14),23.2(CH 3 -10),12.8(CH 3 -14),12.5(CH 3 -8),12.3(CH 3 -2). The above hydrogen spectrum and carbon spectrum data are combined, and the compound is identified as 17-O-demethyl-geldanamycin consistent with the literature report.
Compound 3: white powder with peak mass-to-charge ratio of M/z 547.3031[ M-H ] of excimer ions measured by negative source HRESIMS] - Molecular formula of C 29 H 44 N 2 O 8 . It is composed of 1 H-NMR data (CDCl) 3 ,500MHz)δ H Respectively as follows: 9.21(1H, s, NH-1),6.87(1H, br s,19-H),6.45(2H, br s, NH) 2 -24),6.30(1H,s,H-21),5.86(1H,br t,H-3),5.30(1H,d,J＝9.8Hz,H-9),4.86(1H,d,J＝7.4Hz,H-7),4.29(1H,d,J＝5.2Hz,OH-11),3.63(1H,s,OCH 3 -17),3.32(1H,s,OCH 3 -6),3.21(1H,s,OCH 3 -12),3.02(1H,m,J＝8.3,3.7Hz,H-12),2.56(1H,dd,J＝13.4,6.3Hz,H-15),2.36(1H,m,H-15),2.20(1H,m,J＝13.4,6.6Hz,H-4),2.10(1H,m,H-4),1.74(1H,m,J＝13.3,6.8Hz,H-14),1.68(3H,s,CH 3 -2),1.54(1H,m,H-13),1.44(3H,s,CH 3 -8),1.35(1H,m,H-5),1.26(1H,m,H-5),1.16(1H,m,H-13),0.90(3H,d,J＝6.6Hz,CH 3 -10),0.80(3H,d,J＝6.6Hz,CH 3 -14)。 13 C-NMR data (CD) 3 Cl 3 -d 6 ,125MHz)δ C Respectively as follows: 156.66 (OCONH) 2 -7),150.36(C-18),143.00(C-17),135.06(C-3),134.03(C-20),133.89(C-16),132.73(C-2),130.23(C-8),107.68(C-19),81.62(C-12),81.18(C-7),80.04(C-6),60.30(OCH 3 -17),58.76(OCH 3 -6),56.96(OCH 3 -12),36.32(C-15),34.20(C-13),31.65(C-14),30.27(C-5),24.07(C-4),20.27(C-28),13.55(C-22),12.20 (C-25). The data of the hydrogen spectrum and the carbon spectrum are combined and are consistent with the literature report, so the reblastatin is identified.
Example 4 determination of the Activity of geldanamycin and analogues thereof against crop pathogenic fungi
4.1 test pathogenic fungi: colletotrichum capsici sp, Corynespora capsici, Colletotrichum capsici, Phytophthora capsici capsicii, Corynespora cinerea sp, Colletotrichum mangiferum, Colletotrichum gloeosporioides, Lasiopodiphora papyrifera, Phytophthora chaeta, Phytopsis carota-papayaeefrata & Cif, Colletotrichum chaenomelini, Colletotrichum gloeosporioides, Colletotrichum banana, Colletotrichum graminis, Fusarium oxysporum sp.4, Fusarium oxysporum. sp.cumbense, Foc4, Pyricularia oryzae, Pyricularia graminis, Fusarium graminearum, rubber, and cucumber as an indicator of the bacterial growth of the species of cucumber.
4.2 test method: the compound isolated in example 2 and commercially available positive drugs (chlorothalonil, myclobutanil and carbendazim) were dissolved in dimethyl sulfoxide to prepare a drug solution with a concentration of 10mg/mL, 10mL of potato dextrose agar medium (dry powder medium sold by Qingdao high-tech industrial garden Haibo Biotechnology Co., Ltd.) was poured into a sterile petri dish with a diameter of 6cm, a hole (diameter of 5mm) was drilled at the center of the plate after solidification, 10. mu.L of the drug solution was added to the hole, and pathogenic fungus blocks with a diameter of 5mm were placed on both sides of the plate. The control group was inoculated with only pathogenic bacteria, chlorothalonil (75% effective content, powder sold by Rimin chemical Co., Ltd.), myclobutanil (40% effective content, powder sold by Shanghai Yueji Biotech Co., Ltd.), carbendazim (98% effective content, powder sold by Shanghai Michelin Biotech Co., Ltd.) as positive controls, and each treatment was repeated 3 times. And measuring the bacteriostasis diameter after the contrast group grows full, and calculating the bacteriostasis rate.
4.3 results against pathogenic fungi are shown in Table 1. The geldanamycin monomeric compounds 1 and 3 and the compositions of the compounds 1 and 2 have wide antibacterial spectrum, and can be used for treating banana vascular wilt, banana anthracnose, pepper corynebacterium sp.capsici, pepper phytophthora capsici, papaya anthracnose, papaya brown stem rot, papaya ulcer, cowpea gray spot and mango anthracnose, rubber colletotrichum gloeosporioides, wheat scab, rice blast and cucumber root rot all have antibacterial activity, especially the antibacterial activity on pathogenic fungi such as colletotrichum capsici, corynespora capsici, phytophthora capsici, canker chaenomeles, rice blast and cucumber root rot is higher than that of positive drugs, and the antibacterial effects of the compound 1, the compound 1+ the compound 2 (the volume ratio is 1: 1) and the compound 3 on part of pathogenic fungi are shown in figure 2. Although the antifungal activity of the geldanamycin monomer compounds 1 and 3 and the combination of compounds 1 and 2 found in the example is weaker than that of a part of positive drugs, the most important significance of the discovery is that the geldanamycin and the analogue thereof are found to have the activity of resisting plant pathogenic fungi for the first time, and the geldanamycin and the analogue thereof can be subjected to chemical structure modification in the future so as to obtain a compound with stronger activity.
TABLE 1 geldanamycin and its derivatives having anti-phytopathogenic fungi effect
Note: different lower case letters after the same row of data indicate significant differences at the P <0.05 level.
In early experiments it was found that compound 2 alone was relatively weak in general activity, but in further experiments it was found that compound 2 at the same concentration was mixed with compound 1 in a 1:1 volume ratio and tested for activity, and that it was more active against a number of pathogens than compound 1 alone.
Example 5 determination of the anti-pathogenic bacterial Activity of geldanamycin and analogs thereof
5.1 test pathogenic bacteria: rice bacterial blight (Xanthomonas oryzae pv. oryzae) and mango angular leaf spot (xanthomas campestris pv. mangiferae).
5.2 test method: the compound obtained by separation in example 2 and kanamycin purchased in the market are respectively dissolved in dimethyl sulfoxide by a Minimum Inhibitory Concentration (MIC) determination method to prepare a liquid medicine with the Concentration of 1.3mg/mL, a 96-well plate is selected, 160 mu L of culture medium and 40 mu L of samples with different concentrations are injected into the first hole of each row, the samples are sequentially diluted by a double dilution method, and finally 100 mu L of pathogen liquid is added, and the light absorption value OD600 of the bacterial liquid is 0.6-0.8. And (5) placing the mixture into a constant temperature culture chamber at 28 ℃ for 24-48 h, and observing.
5.3 results against pathogenic bacteria are shown in Table 2. The geldanamycin monomer compounds 1 and 3 and the compounds 1 and 2 have obvious antibacterial activity on mango bacterium angular leaf spot strain L3 and rice bacterial leaf blight, and the activity of the compounds is stronger than that of positive drugs. The activity tested when compound 1 and compound 2 were mixed at a 1:1 volume ratio was greater than the antimicrobial activity of compound 1 alone, indicating that compound 1 and compound 2 had a synergistic effect on antimicrobial activity.
TABLE 2 anti-pathogenic bacterial effects of geldanamycin compounds
The foregoing is a preferred embodiment of the present invention, and it should be understood that the optimization, modification, combination, substitution, etc. of the present invention based on the principle of the present invention should be considered within the scope of the present invention.
Claims (9)
1. The new application of geldanamycin and its analogue is characterized by that: the geldanamycin and the analogue thereof are applied to the preparation of medicines with broad-spectrum antibacterial effect on various plant pathogenic bacteria or plant diseases, wherein the geldanamycin and the analogue thereof are one or more of a compound 1, a compound 2 and a compound 3 with the structures shown as follows:
2. use according to claim 1, characterized in that: the plant is cucumber, pepper, pawpaw, cowpea, mango, banana, rice, wheat or rubber tree.
3. Use according to claim 1, characterized in that: the plant pathogenic bacteria are one or more of anthrax fungus, clavulan fungus, fusarium fungus, phytophthora fungus, trichoderma fungus, pyricularia fungus, phomopsis fungus and xanthomonas bacterium.
4. Use according to claim 1, characterized in that: the plant diseases comprise one or more of banana wilt pathogen, banana colletotrichum, pepper corynebacterium spore leaf spot pathogen, pepper phytophthora capsici, papaya colletotrichum, papaya brown stalk rot pathogen, papaya ulcer pathogen, cowpea gray spot pathogen, mango colletotrichum, rubber colletotrichum, wheat scab pathogen, rice blast pathogen, cucumber root rot pathogen, rice white leaf blight pathogen and mango bacterial angular leaf spot pathogen.
5. The use according to claim 1, characterized in that the geldanamycin or analogue thereof is prepared by:
(1) selecting a fermentation strain: the fermentation strain is Streptomyces (Streptomyces sp.) HNBCa1, which is preserved in Guangdong province microorganism strain collection center at 9/3 of 2021, with the preservation registration number being GDMCC NO. 61901;
(2) preparing a seed solution: culturing the streptomycete in the step (1) by adopting a seed culture medium to obtain a seed solution;
(3) fermentation: inoculating the seed liquid in the step (2) into a fermentation culture medium for fermentation treatment to obtain fermentation liquid;
(4) extraction of fermentation products: filtering the fermentation liquor obtained in the step (3), extracting bacteria liquid by using ethyl acetate to obtain extract liquor, extracting bacteria by using methanol-dichloromethane mixed liquor to obtain extract liquor, combining the extract liquor and the extract liquor, and concentrating under reduced pressure to obtain a crude extract containing geldanamycin and analogues thereof;
(5) isolation of geldanamycin and its analogs: separating the crude extract with normal phase silica gel chromatographic column, reverse phase silica gel chromatographic column, dextran gel chromatographic column and semi-preparative high performance liquid chromatography to obtain geldanamycin and its analogues.
6. Use according to claim 5, characterized in that: the seed culture medium in the step (2) contains: 15-20 g of tryptone, 2-4 g of soybean papain hydrolysate, 4-6 g of sodium chloride, 2-3 g of dipotassium hydrogen phosphate, 2-3 g of glucose and 1L of distilled water, wherein the pH value of the seed culture medium is 7.3 +/-0.2; when the seed culture medium is adopted for culture, the shaking culture is carried out for 3-5 days under the conditions of 28-30 ℃ and 150-170 rpm.
7. Use according to claim 5, characterized in that: the fermentation medium in step (3) contains: 15-25 g of soluble starch, 4-6 g of peptone, 2-4 g of malt extract, 2-4 g of yeast extract, 8-12 g of glucose, 0.5-1.5 g of calcium carbonate and 1L of distilled water, wherein the pH value of the fermentation medium is 7.0; inoculating the seed solution into a fermentation culture medium according to the inoculation amount (volume percentage content) of 5-15%, and performing shaking fermentation for 9-13 days at the temperature of 28-30 ℃ and the speed of 150-170 rpm.
8. Use according to claim 5, characterized in that: in the step (4), the volume of the ethyl acetate is equal to that of the bacterial liquid, and the volume ratio of the methanol to the dichloromethane in the methanol-dichloromethane mixed liquid is 1: 1.
9. use according to claim 5, characterized in that: and (5) when the crude extract passes through a normal phase silica gel chromatographic column in the step (5), the volume ratio of the crude extract to the normal phase silica gel chromatographic column is 100: 0. 100, and (2) a step of: 1. 100, and (2) a step of: 2. 100, and (2) a step of: 4. 100, and (2) a step of: 8. 100, and (2) a step of: 16. 100, and (2) a step of: 32. 100, and (2) a step of: 64. 0: gradient elution with 100 chloroform-methanol to obtain 9 polar sections Fr.A-Fr.I; wherein Fr.D and Fr.F are respectively subjected to reversed phase silica gel chromatographic column, sephadex chromatographic column and semi-preparative high performance liquid chromatograph to obtain compound 1 and compound 2, and Fr.G is subjected to normal phase silica gel chromatographic column, reversed phase silica gel chromatographic column, sephadex chromatographic column and semi-preparative high performance liquid chromatograph to obtain compound 3.
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| CN116064244A (en) * | 2022-09-20 | 2023-05-05 | 中国热带农业科学院热带生物技术研究所 | Marine aspergillus ITBBc1, and separated terphenyl compound and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116064244A (en) * | 2022-09-20 | 2023-05-05 | 中国热带农业科学院热带生物技术研究所 | Marine aspergillus ITBBc1, and separated terphenyl compound and application thereof |
| CN116064244B (en) * | 2022-09-20 | 2024-02-20 | 中国热带农业科学院热带生物技术研究所 | Marine aspergillus ITBBc1, and separated terphenyl compound and application thereof |
Also Published As
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| CN118020778A (en) | 2024-05-14 |
| CN118020779A (en) | 2024-05-14 |
| CN114982763B (en) | 2024-04-26 |
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