CN105503780A - Pestalotic acid compound with antibacterial activity and application thereof - Google Patents
Pestalotic acid compound with antibacterial activity and application thereof Download PDFInfo
- Publication number
- CN105503780A CN105503780A CN201510955816.4A CN201510955816A CN105503780A CN 105503780 A CN105503780 A CN 105503780A CN 201510955816 A CN201510955816 A CN 201510955816A CN 105503780 A CN105503780 A CN 105503780A
- Authority
- CN
- China
- Prior art keywords
- compound
- relevant
- antibacterial activity
- proton
- esi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
- C07D303/02—Compounds containing oxirane rings
- C07D303/38—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
-
- 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
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
- A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
-
- 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
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing within the same carbon skeleton a carboxylic group or a thio analogue, or a derivative thereof, and a carbon atom having only two bonds to hetero atoms with at the most one bond to halogen, e.g. keto-carboxylic acids
-
- 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
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/20—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom three- or four-membered rings
-
- 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
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
- C07C59/56—Unsaturated compounds containing hydroxy or O-metal groups containing halogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/76—Unsaturated compounds containing keto groups
- C07C59/90—Unsaturated compounds containing keto groups containing singly bound oxygen-containing groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a Pestalotic acid compound with antibacterial activity. Hyperparasitism Pestalotiopsis neglecta strains are cultured in a fermented mode, extracted and separated to obtain the compound. The Pestalotic acid compound is easy to obtain, has high antibacterial activity and can be used as an antibacterial agent for preventing and controlling plant bacterial wilt, and a new path is provided for developing antibacterial agents and preventing and controlling plant diseases.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to Pestaloticacid compound and application thereof that a class has antibacterial activity.
Background technology
Plant endogenesis epiphyte (fungalendophyte) refers to the certain phase of the life history or all stage life and health plant respectively organize qualified intraorganic fungi.They and host plant define symbiotic relationship closely by interaction, can promote the growth of plant, and provide plant to tackle the adaptive faculty of coercing, some plant endogenesis epiphytes also have pharmaceutical use.Find from plant endogenesis epiphyte and find that active compound has become the another focus of research both at home and abroad, as Chinese patent literature CN201310194836.5 discloses in a strain rough gentian endogenetic fungus (Metarrhizium) LD421 being separated and obtaining, can be used for control rough gentian leaf blight; Patent application CN201210067829.4 then discloses and be separated the endogenetic fungus fusarium solani T-7 obtained from the tissue such as Radix Ginkgo, stem, leaf, has good restraining effect to P. capsici, tomato wilt bacterium, Valsa mali etc.
The Main Means controlling crop diseases and pest crop smothering at present in agricultural production process is chemical pesticide control, it is to alleviating disease pest and weed, ensure that the good harvest of farm crop high yield plays a positive role, but due to the mutual restriction interdependence between nature biotechnology, do not advise now too relying on chemical pesticide in the process carrying out disease; Therefore, to the research of plant endogenesis epiphyte, utilizing the relation of plant endogenesis epiphyte and the symbiosis of host plant biological activity, screen the endogenetic fungus with high biological activity, obtain active result further, is a large focus of Recent study.
Summary of the invention
Object of the present invention aims to provide Pestaloticacid compound and the antibacterial activity application thereof that a class has antibacterial activity.
The Pestaloticacid compound with antibacterial activity of the present invention is after superparasitism being intended pestalotia bacteria strain fermentation culture, then the compound obtained after Extraction and isolation, and structural formula is as follows:
The preparation method with the Pestaloticacid compound of antibacterial activity of the present invention, comprises following processing step:
Step is 1.: cultivate 10-30 days under superparasitism being intended pestalotia bacteria strain substratum room temperature;
Step is 2.: by described step 1. in substratum be together cut to bulk together with the bacterium colony on it, then use mixed solvent soak extraction 3 times; 3 extracts are merged concentrated after, be extracted to color with extraction agent and no longer change, be then evaporated to dry medicinal extract with Rotary Evaporators 40-50 DEG C;
Step is 3.: by described step 2. in medicinal extract dissolution with solvents after recycle silicon glue mix sample, through normal phase column chromatography, then petroleum ether-ethyl acetate, chloroform-methanol system gradient elution is used, eventually pass TLC chromatography to detect, compound component containing identical Rf value and colour developing situation is combined, be evaporated to dry respectively, obtain 10 component Fr.1 to Fr.10;
Step is 4.: by described step 3. in compound component Fr.1 to Fr.10 to be separated or one or more combinations in elution process are separated repeatedly, obtain having the Pestaloticacid compound of antibacterial activity after wash-out with conventional respectively, described routine be separated or elution process be following listed by method:
(1) SephadexLH-20 is separated;
(2) NP7000 type-high performance liquid chromatograph is separated;
(3) with silica gel mixed sample, wash-out post is loaded as GF254 silica gel, carries out isocratic elution by sherwood oil-acetone system.
Described step 1. in the composition (improvement M-1-D substratum) of substratum: NaH
2pO
4h
2o20mg, FeCl
32.0mg, MgSO
4360mg, KCl60mg, Ca (NO
3) 22280mg, KNO
380mg, sucrose 30g, ammonium tartrate 5g, yeast extract 0.5g, MnSO
45.0mg, ZnSO
47H
2o2.5mg, H
3bO
41.4mg, KI0.7mg, distilled water 1000mL, agar 15-20g, pH nature; Described step 1. in the volume of substratum be 30L.
Described step 1. in superparasitism to intend pestalotia bacteria strain be that pestalotia bacteria strain cr014 is intended in superparasitism.
Described step 2. middle mixed solvent is ethyl acetate: methyl alcohol: acetic acid=80:15:5 (V/V/V), extraction agent is ethyl acetate.
The described silica gel for mixing sample is 100-200 order, described step 3. middle normal phase column chromatography used silica gel is 200-300 order, petroleum ether-ethyl acetate, chloroform-methanol system gradient elution PetroChina Company Limited. ether: ethyl acetate is 10:1 → 6:4, chloroform: methyl alcohol is 20:1 → 0:100.
Described step 4. in SephadexLH-20 be separated time eluent used be chloroform: the one in methyl alcohol=1:1, methyl alcohol, acetone; Described step sherwood oil when 4. middle sherwood oil-acetone system carries out isocratic elution: acetone is 8:2-7:3.
Molecular structure of compounds formula (1) of the present invention is to (10) corresponding compound 1 to 10 respectively:
Compound 1: brown oil, in conjunction with
13cNMR and DEPT composes, high resolution mass spectrum HR-ESI-MS ([M-H]
-m/z349.1658) determine that its molecular formula is C
19h
26o
6, from
13cNMR spectrum and DEPT spectrum can be found out in compound 7 containing 6 quaternary carbon signal (δ
c197.1,171.4,151.4,133.0,130.3 and 64.6), 5 methyne (δ
c143.3,136.1,127.3,66.4 and 59.2), 6 methylene radical (δ
c55.1,35.2,32.8,31.0,29.8 and 23.7) and 2 methyl (δ
c14.5 and 13.0).
According to
1h-NMR data presentation: owing to having a unimodal methyl (δ
h1.89, s) He one triplet methyl (δ
h0.94, t, J=7.0Hz), show that the two dimensional structure of compound 1 is similar to the analogue of ambuicacid.Data in being composed by COSY can find out the crucial reference point (H-3/H-4 of this compound; H-11/H-12/H-13/H-14/H-15/H-16/H-17), this compound can be derived thus there is-C-3-C-4-and-C-11 – C-12 – C-13-C-14-C-15 – C-16 – C-17-2 fragment.The structure that this compound is detailed is determined by 2D-NMR, the data presentation according on HMBC: the proton δ of 3 thiazolinyls
h6.84 (H-3) and δ
c171.4 (C-1), 133.0 (C-2), 31.0 (C-4), 64.6 (C-5) and 13.0 (C-18) are relevant, the proton δ on 18 methyl
h1.89 (H-18) and δ
c171.4 (C-1), 133.0 (C-2) and 136.1 (C-3) are relevant; Proton δ on 4 methylene radical
h3.18 and 2.74 (H-4) and δ
c133.0 (C-2), 136.1 (C-3), 64.6 (C-5) and 59.2 (C-10) are relevant; The proton δ of 6
h4.85 (H-6) and δ
c151.4 (C-7), 130.3 (C-8), 127.3 (C-11) and 31.0 (C-4) are relevant; The proton δ of 19
h4.49 and 4.28 (H-19) and δ
c197.1 (C-9), 151.4 (C-7) and 130.3 (C-4) are relevant.By in conjunction with other reference points, the two dimensional structure of this compound can be inferred.
The relative configuration of compound 1 is tested by NOESY and is determined, NOE data presentation: H-4, H-6 are relevant with H-10 determines corresponding C-5, C-6 and C-10 configuration, and from
1the coupling constant that H-NMR calculates H-11 and H-12 is 15.9Hz, and disclosing the double bond being positioned at C-11 is E, has drawn the relative configuration of compound 1 thus, and by its called after pestaloticacidA.
Compound 2: brown oil, in conjunction with
13cNMR and DEPT composes, high resolution mass spectrum HR-ESI-MS ([M-H]
-m/z349.1658; Calc.345.1338) determine that its molecular formula is C
19h
22o
6, from
13cNMR spectrum and DEPT spectrum can be found out in compound 2 containing 9 quaternary carbon signal (δ
c169.1,162.1,148.5,148.1,140.8,129.7,122.6,119.2 and 110.7), 3 methyne (δ
c195.0,138.5 and 100.4), 5 methylene radical (δ
c32.1,29.1,28.2,24.9 and 23.1) and 2 methyl (δ
c14.3 and 12.7).
The carbon modal data of table 1 compound 1-9
Position | 1 a | 2 b | 3 a | 4 a | 5 a | 6 a | 7 a | 8 a | 9 a |
1 | 171.4 | 169.1 | 171.8 | 172.0 | 171.1 | 171.2 | 171.2 | 171.2 | 172.0 |
2 | 133.0 | 129.7 | 132.0 | 129.1 | 132.8 | 132.0 | 131.9 | 131.8 | 132.7 3 --> |
3 | 136.1 | 138.5 | 137.6 | 141.3 | 135.5 | 136.3 | 136.6 | 136.6 | 135.6 |
4 | 31.0 | 24.9 | 36.9 | 28.8 | 28.0 | 28.3 | 28.75 | 28.8 | 28.7 |
5 | 64.6 | 119.2 | 79.7 | 128.6 | 62.7 | 62.3 | 61.3 | 61.3 | 61.2 |
6 | 66.4 | 148.1 | 69.9 | 146.3 | 59.7 | 61.6 | 61.1 | 61.1 | 61.0 |
7 | 151.4 | 122.6 | 156.1 | 127.9 | 193.3 | 64.5 | 65.9 | 65.9 | 65.8 |
8 | 130.3 | 110.7 | 124.5 | 112.0 | 144.6 | 157.0 | 151.7 | 151.1 | 150.8 |
9 | 197.1 | 148.5 | 193.0 | 148.3 | 134.2 | 132.0 | 131.3 | 131.9 | 131.9 |
10 | 59.2 | 140.8 | 70.0 | 121.1 | 194.8 | 190.2 | 195.8 | 196.0 | 196.1 |
11 | 127.3 | 100.4 | 130.5 | 125.4 | 122.6 | 86.8 | 122.2 | 124.9 | 123.0 |
12 | 143.3 | 162.1 | 143.0 | 133.2 | 148.0 | 129.4 | 142.0 | 136.5 | 139.9 |
13 | 35.2 | 29.1 | 34.7 | 34.6 | 35.5 | 134.2 | 73.5 | 42.6 | 34.5 |
14 | 29.8 | 28.2 | 29.7 | 30.5 | 29.6 | 32.9 | 38.0 | 71.8 | 26.4 |
15 | 32.8 | 32.1 | 32.8 | 32.9 | 32.7 | 32.3 | 28.79 | 40.2 | 39.6 |
16 | 23.7 | 23.1 | 23.7 | 23.8 | 23.7 | 23.1 | 23.7 | 19.9 | 68.4 |
17 | 14.5 | 14.3 | 14.5 | 14.6 | 14.5 | 14.3 | 14.4 | 14.5 | 23.5 |
18 | 13.0 | 12.7 | 13.2 | 12.9 | 13.0 | 12.8 | 12.8 | 12.8 | 13.0 |
19 | 55.1 | 195.0 | - | - | 58.7 | 75.1 | 60.1 | 60.3 | 60.3 |
1′ | - | - | - | - | 172.2 | - | - | - | - |
2′ | - | - | - | - | 20.7 | - | - | - | - |
anuclear magnetic data is at CD
3obtain in OD.
bnuclear magnetic data is at CD
3cOCD
3obtain
By
1h-NMR,
13cNMR and DEPT modal data can find out that compound 2 is analogues of ambuicacid, and difference is that 6 rings in ambuicacid have become phenyl ring in this compound.Data in being composed by COSY can find out the crucial reference point (H-3/H-4 of this compound; H-13/H-14/H-15/H-16/H-17), this compound can be derived thus there is-C-3-C-4-and-C-13-C-14-C-15-C-16-C-17-2 a fragment.The fine structure of this compound is determined by 2D-NMR, can find out phenyl ring is connected with 3 hydroxyls, 1 aldehyde radical and 1 isopentene group group according to the data of HMBC spectrum; The proton δ of 3 thiazolinyls
h6.96 (H-3) and δ
c169.1 (C-1), 129.7 (C-2), 24.9 (C-4), 119.2 (C-5) (w) and 12.7 (C-18) are relevant, the proton δ on 18 methyl
h2.06 (H-18) and δ
c169.1 (C-1), 129.7 (C-2) and 138.5.0 (C-3) are relevant; Proton δ on 4 methylene radical
h3.87 (H-4) and δ
c169.1 (C-1), 129.7 (C-2), 138.5 (C-3), 119.2 (C-5), 148.1 (C-6) and 140.8 (C-10) are relevant; The proton δ of 11 thiazolinyls
h6.97 (H-11) and δ
c148.1 (C-6), 122.6 (C-7), 162.1 (12) and 29.1 (C-13) are relevant; The proton δ of 13
h2.84 (H-13) and δ
c100.4 (C-11), 162.1 (C-12), 28.2 (C-14) and 32.1 (C-15) are relevant; Proton δ on 19 aldehyde radicals
h10.34 (H-19) and δ
c148.1 (C-6), 122.6 (C-7), 110.7 (C-8), 148.5 (C-9) and 140.8 (C-10) are relevant.
Finally use total degree of unsaturation of compound except the degree of unsaturation of double bond, two ketone groups and phenyl ring, also have a degree of unsaturation, implied to also have an extra ring texture on the skeleton of this compound: this ring is that C-6 and C-12 is connected to form by oxo bridge.The structure of compound 2 can be inferred, finally by its called after pestaloticacidB based on above-mentioned data.
Compound 3: brown oil, in conjunction with
13cNMR and DEPT composes, high resolution mass spectrum HR-ESI-MS ([M+Na]
+m/z379.1284) determine that its molecular formula is C
18h
25 35clO
5, and can observe in the molecular weight of compound 3 from the experimental data of the ESI-MS of compound 3 and have one for m/z381 [M+2+Na]
+isotopic peak, its abundance is another isotopic peak m/z379 [M+Na]
+1/3rd, therefore can judge in compound 3 containing a Cl replacing group.
The crucial reference point of compound 3 in HMBC and COSY spectrum and compound 1 about the same is δ except having lacked a chemical shift in compound 3
cthe carbon of 55.1, and in 6 rings many Cl replacing groups.In compound 3, the proton δ of 6
h4.43 (H-6) and δ
c156.1 (C-7), 130.5 (C-11), 124.5 (C-8), 79.7 (C-5), 70.0 (C-10) and 36.9 (C-4) are relevant, the proton δ on 18 thiazolinyls
h1.91 (H-18) and δ
c171.8 (C-1), 132.0 (C-2), 137.6 (C-3) and 36.9 (C-4) are relevant; Proton δ on 10 precedence methyl
h5.02 (H-10) and δ
c193.0 (C-9) He 79.7 (C-5) are correlated with.In order to determine the position of Cl replacing group, determine the nuclear magnetic data of compound 3 in deuterated acetone.Result shows: a hydroxyl signal (δ
h5.19) be positioned on C-6, another hydroxyl signal (δ
h4.69) be positioned on C-5, and hydroxyl proton δ
h5.19 is relevant with C-7, C-5 and C-6, hydroxyl proton δ
h4.69 relevant with C-4, C-5, C-6 and C-10.
Corresponding C-5, C-6 and C-10 configuration is determined as can be seen from NOESY experimental result: H-4 and H-6 is relevant with H-10, and from
1the coupling constant that H-NMR calculates H-11 and H-12 is 15.3Hz, and disclosing the double bond being positioned at C-11 is E, based on above-mentioned experimental result, has drawn the relative configuration of compound 3 thus, and by its called after pestaloticacidC.
Compound 4: brown oil, in conjunction with
13cNMR and DEPT composes, high resolution mass spectrum HR-EI-MS ([M]
+m/z338.1285) determine that its molecular formula is C
18h
23 35clO
4, 6 rings be in compound 9 with the difference of compound 3 of compound 4 have become phenyl ring in compound 4.
Based on the related data that following HMBC composes, can find out that 2 hydroxyls in compound 4 and 1 Cl replacing group lay respectively at C-6, on C-9 and C-10: the proton δ on 3 thiazolinyls
h6.72 (H-3) and δ
c172.0 (C-1), 128.6 (w) (C-5), 28.8 (C-4) and 12.9 (C-18) are relevant, the proton δ of 4
h3.70 (H-4) and δ
c(w) 172.0 (C-1), 146.3 (C-6), 141.3 (C-3), 129.1 (C-2), 128.6 (C-5) and 121.1 (C-10) are relevant; Proton δ on 8 aromatic rings
h6.88 (H-8) and δ
c148.3 (C-9), 146.3 (C-6), 125.4 (C-11) and 121.1 (C-10) are relevant; Proton δ on 11 thiazolinyls
h6.64 (H-11) and δ
c146.3 (C-6), 127.9 (C-7), 112.0 (C-8) and 34.6 (C-13) are relevant.
From
1the coupling constant that H-NMR calculates H-11 and H-12 is 15.6Hz, and disclosing the double bond being positioned at C-11 is E, based on above-mentioned experimental result, has drawn the relative configuration of compound 4 thus, and by its called after pestaloticacidD.
Compound 5: brown oil, in conjunction with
13cNMR and DEPT composes, high resolution mass spectrum HR-ESI-MS ([M+H]
+m/z413.2665; Calc.413.2668) determine that its molecular formula is C
21h
26o
7, according to
1h-,
13c-NMR and DEPT modal data can judge that compound 5 is analogues of ambuicacid
[9 , 13].
By comparing rear discovery with ambuicacid, the proton δ on 6 methyl
h3.82 (H-6) and δ
c193.3C-7), 134.2 (C-9) and 62.7 (C-5) are correlated with, 19 proton δ containing Oxymethylene
h4.79 and 5.08 (H-19) and δ
c193.3 (C-7), 172.1 (C-1), 144.6 (C-8) and 134.2 (C-9) are relevant, and above related description 9-OH is oxidized to ketone group, and an ethanoyl is connected with 19-OH.
Corresponding C-5 and C-10 configuration is determined as can be seen from NOESY experimental result: H-4 with H-10 is relevant, and from
1the coupling constant that H-NMR calculates H-11 and H-12 is 15.8Hz, and disclosing the double bond being positioned at C-11 is E, based on above-mentioned experimental result, has drawn the relative configuration of compound 5 thus, and by its called after pestaloticacidE.
Compound 6: brown oil, in conjunction with
13cNMR and DEPT composes, high resolution mass spectrum HR-ESI-MS ([M+H]
+m/z371.1464; Calc.371.1471) determine that its molecular formula is C
19h
24o
6, according to
1h-spectrum,
13c-spectrum and DEPT modal data can judge that compound 6 is analogues of ambuicacid.
The hydrogen modal data of table 2 compound 1-9
anuclear magnetic data is at CD
3obtain in OD.
bnuclear magnetic data is at CD
3cOCD
3obtain
By comparing rear discovery with ambuicacid, be moved to C-12/C-13 by relevant discovery double bond being positioned at C-11/C-12 of HMBC, C-11 then connected by oxo bridge and has gone up a methylene radical (C-19): a proton δ on 19 methylene radical
h4.79 (H-19) and δ
c190.2 (C-10), 157.0 (C-8), 132.0 (C-9), 129.4 (C-12) and 86.8 (C-11) are relevant, 11 proton δ containing oxygen methyne
h5.29 (H-11) and δ
c157.0 (C-8) He 134.2 (C-13) are correlated with.
Corresponding C-5, C-6 and C-10 configuration is determined as can be seen from NOESY experimental result: H-4 and H-6 is relevant with H-10, and from
1the coupling constant that H-NMR calculates H-12 and H-13 is 18.3Hz, and disclosing the double bond being positioned at C-12 is E, based on above-mentioned experimental result, has drawn the relative configuration of compound 6 thus, and by its called after pestaloticacidF.
Compound 7: brown oil, in conjunction with
13c spectrum and DEPT spectrum, high resolution mass spectrum HR-ESI-MS ([M+H]
+m/z389.1575; Calc.389.1576) determine that its molecular formula is C
19h
26o
7.
After comparing with ambuicacid, and found by HMBC, C-13 is oxidized, has connected a hydroxyl and has been positioned on C-13: the proton δ on 12 thiazolinyls
h5.92 (H-12) and δ
c131.3 (C-9), 122.2 (C-11), 73.5 (C-13) and 38.0 (C-14) are relevant, 13 proton 4.14 (H-13) and δ containing oxygen methyne
c142.0 (C-12), 122.2 (C-13), 38.0 (C-14) and 28.79 (C-15) are relevant.
Corresponding C-5, C-6 and C-10 configuration is determined as can be seen from NOESY experimental result: H-4 and H-6 is relevant with H-10, and from
1the coupling constant that H-NMR calculates H-12 and H-13 is 16.0Hz, and disclosing the double bond being positioned at C-12 is E, based on above-mentioned experimental result, has drawn the relative configuration of compound 7 thus, and by its called after pestaloticacidG.
Compound 8: brown oil, in conjunction with
13c spectrum and DEPT spectrum, high resolution mass spectrum HR-ESI-MS ([M+H]
+m/z389.1573; Calc.389.1576) determine that its molecular formula is C
19h
26o
7.
After comparing with ambuicacid, and found by HMBC, C-14 is oxidized, and a hydroxyl is positioned on C-14: the proton δ on 11 thiazolinyls
h6.23 (H-11) and δ
c196.0 (C-10), 151.1 (C-8), 136.5 (C-12), 131.9 (C-9), 71.8 (C-14) and 42.6 (C-13) are relevant; Proton δ on 13 methylene radical
h2.35 (H-13) and δ
c136.5 (C-12), 124.9 (C-11), 71.8 (C-14) and 40.2 (C-15) are relevant; 14 containing the proton δ on oxygen methyne
h3.67 (H-14) and δ
c136.5 (C-12), 40.2 (C-15) and 19.9 (C-16) are relevant.
Corresponding C-5, C-6 and C-10 configuration is determined as can be seen from NOESY experimental result: H-4 and H-6 is relevant with H-10, and from
1the coupling constant that H-NMR calculates H-11 and H-12 is 15.9Hz, and disclosing the double bond being positioned at C-11 is E, based on above-mentioned experimental result, has drawn the relative configuration of compound 8 thus, and by its called after pestaloticacidH.
Compound 9: brown oil, in conjunction with
13c spectrum and DEPT spectrum, high resolution mass spectrum HR-ESI-MS ([M+H]
+m/z389.1573; Calc.389.1576) determine that its molecular formula is C
19h
26o
7.
After comparing with ambuicacid, and found by HMBC, C-16 is oxidized, and a hydroxyl is positioned on C-16: the proton δ on 17 methyl
h1.16 (H-17) and δ
c68.4 (C-16) He 39.6 (C-15) are correlated with; 16 containing the proton δ on oxygen methyne
h3.75 (H-16) and δ
c39.6 (C-15), 26.4 (C-14) and 23.5 (C-17) are relevant.
Corresponding C-5, C-6 and C-10 configuration is determined as can be seen from NOESY experimental result: H-4 and H-6 is relevant with H-10, and from
1the coupling constant that H-NMR calculates H-11 and H-12 is 15.5Hz, and disclosing the double bond being positioned at C-11 is E, based on above-mentioned experimental result, has drawn the relative configuration of compound 9 thus, and by its called after pestaloticacidI.
The antibacterial activity application with the Pestaloticacid compound of antibacterial activity of the present invention.
The present invention has the following advantages relative to prior art:
1, pestaloticacid compound of the present invention obtains simple, has antibacterial activity, can as antibacterial agent for preventing and treating plant-bacterial-wilt.
2, pestaloticacid compound of the present invention is development antibacterial agent, and controlling plant diseases provides new approach.
3, pestaloticacid compound antibacterial activity of the present invention is high, can as antibacterial agent for preventing and treating plant-bacterial-wilt.
Accompanying drawing explanation
Fig. 1 is the structural formula of pestaloticacid compound of the present invention.
Embodiment
By the following examples the present invention is described in further detail, but the present invention is not limited to following embodiment.
Embodiment 1:
The separation preparation of pestaloticacid compound:
A) substratum and culture condition
Improvement M-1-D substratum: NaH
2pO
4h
2o20mg, FeCl
32.0mg, MgSO
4360mg, KCl60mg, Ca (NO
3) 22280mg, KNO
380mg, sucrose 30g, ammonium tartrate 5g, yeast extract 0.5g, MnSO
45.0mg, ZnSO
47H
2o2.5mg, H
3bO
41.4mg, KI0.7mg, distilled water 1000mL, agar 15-20g, pH nature.
Culture condition: bacterial strain cr014 improvement M-1-D substratum fermentation 30L, cultivates 20d under room temperature.
B) Extraction and isolation of tunning
The improvement Fries substratum of the 20d that ferments together is cut to tiny bulk together with the bacterium colony on it, by ethyl acetate: methyl alcohol: acetic acid=80:15:5 (V/V/V) mixed solvent soak extraction 3 times, 3 extracts are merged after concentrating, be extracted with ethyl acetate again to color and no longer change, be finally evaporated to dry medicinal extract (37.337g) with Rotary Evaporators 45 DEG C.
By the medicinal extract (37.337g) of gained after extraction into ethyl acetate with after appropriate dissolution with solvents, sample is mixed with silica gel (100-200 order), through normal phase column chromatography (200-300 order), with sherwood oil: ethyl acetate=(10:1 → 6:4), chloroform: methyl alcohol=(20:1 → 0:100) system gradient elution, detect through TLC chromatography, component containing identical Rf value and colour developing situation compound is combined, be evaporated to dry respectively, be divided into 10 component: Fr.1-Fr.10.
Fr.4 (8.423g) obtains 4 components (Fr.4.1-Fr.4.4) after SephadexLH-20 (chloroform: methyl alcohol=1:1) is separated; Wherein Fr.4.2 (635mg) is after SephadexLH-20 (methyl alcohol) is separated, then obtains compound 5 (1.2mg) and compound 4 (3.9mg) through NP7000 type-high performance liquid chromatograph separation; Fr.4.4 (63mg) obtains compound 2 (12.2mg) after SephadexLH-20 (chloroform: methyl alcohol=1:1) is separated.
Fr.5 (2.377g) obtains 5 components (Fr.5.1-Fr.5.5) after SephadexLH-20 (methyl alcohol) is separated; Wherein Fr.5.4 (329mg) is after SephadexLH-20 (methyl alcohol) is separated, sample mixed by recycle silicon glue (100-200 order), wash-out post is loaded as GF254 silica gel, with sherwood oil: acetone=(8:2) system carries out isocratic elution, obtains compound 3 (9.8mg).
Fr.6 (213mg) obtains 4 components (Fr.6.1-Fr.6.4) after SephadexLH-20 (chloroform: methyl alcohol=1:1) is separated; Wherein sample mixed by Fr.6.2 silica gel (100-200 order), and wash-out post is loaded as GF254 silica gel, with sherwood oil: acetone=(8:2) system carries out isocratic elution, obtains compound 10 (9.8mg); Fr.6.3 obtains Fr.6.3.1 after SephadexLH-20 (chloroform: methyl alcohol=1:1) is separated; Sample mixed by Fr.6.3.1 silica gel (100-200 order), wash-out post is loaded as GF254 silica gel, sherwood oil with containing 0.3% formic acid: after acetone=(7:2) system carries out isocratic elution, then obtain compound 1 (1.1mg) after SephadexLH-20 (methyl alcohol) purifying.
Fr.8 (386mg) obtains 3 components (Fr.8.1-Fr.8.3) after SephadexLH-20 (acetone) is separated; Wherein Fr.8.1 is separated through NP7000 type-high performance liquid chromatograph and obtains 3 subfractions (Fr.8.1.1-Fr.8.1.3); Sample mixed by Fr.8.1.1 silica gel (100-200 order), and wash-out post is loaded as GF254 silica gel, the sherwood oil with containing 0.3% formic acid: after acetone=(7:3) system carries out isocratic elution, obtain compound 9 (4.5mg); Fr.8.1.3 is separated through NP7000 type-high performance liquid chromatograph and obtains 4 subfractions (Fr.8.1.3.1-Fr.8.1.3.4); Sample mixed by Fr.8.1.3.3 silica gel (100-200 order), and wash-out post is loaded as GF254 silica gel, the sherwood oil with containing 0.3% formic acid: after acetone=(7:3) system carries out isocratic elution, obtain compound 8 (14.0mg).Sample mixed by Fr.8.1.3.4 silica gel (100-200 order), wash-out post is loaded as GF254 silica gel, sherwood oil with containing 0.3% formic acid: after acetone=(7:3) system carries out isocratic elution, obtain compound 7 (17.4mg) and compound 6 (1.0mg).
Embodiment 2:
Pestaloticacid compound activity is studied:
A) test compound and reagent
Test compound: compound 2, compound 3, compound 4, compound 5, compound 7, compound 8, compound 9;
Positive control: Cefotaxime;
For examination bacterial classification: Ralstoniasolanacearum, Salmonellatyphi, Escherichiacoli, Staphylococcusaureus.
B) Cleaning Principle and method
The MICs value of bacterium adopts the microorganism By Dilution of improvement.4 pathogen strain bacteriums cultivate 18 to 24h at 25 DEG C on nutrient agar, then collect pathogenetic bacteria thalline with aseptic transfering loop, cultivate in the centrifuge tube that the aseptic physiological saline of 10mL is housed.The concentration of bacterial suspension is be adjusted to 108CFU/mL under the standard conditions of 0.10 in 630nm optical density (OD).The bacterial suspension of this concentration dilutes 100 times before use, and ultimate density is 106CFU/mL.All tested compounds are all dissolved in DMSO, are finally made into the solution that concentration is 4 μ g/ μ L.Adopt coubling dilution when measuring active, add in 96 hole versions by the testing compound of different concns and the meat soup containing bacterial suspension, end reaction volume fixes on 200 μ L (ultimate density of DMSO is equal to or less than 5%).Using DMSO as negative control, the bacterial suspension not adding compound and DMSO as blank, to add Cefotaxime as positive control.24h is cultivated at finally 96 orifice plates being placed in 37 DEG C.After 24h, determine bacterium increment by measuring extinction (OD) value, compound concentration when bacterium increment is almost 0 is MIC value.Each process all arranges 3 repetitions.
Research determines the activity of the anti-4 kinds of pathogenetic bacterias (R.solanacearum, Salmonellatyphi, E.coli, Staphylococcusaureus) of compound 2-5 and compound 7-9.All determined compounds all have antibacterial activity, the results are shown in Table 3.Can find out that from result the ability that compound 2, compound 3, compound 7 and compound 8 suppress R.Solanacearum to grow is equally strong with positive control Cefotaxime, therefore this several compound can as antibacterial agent for preventing and treating plant-bacterial-wilt.
Table 3 compound antibacterial activity (μ g/mL)
Note: non-activity during "-" expression MIC>100 μ g/mL.
Embodiment 3:
Pestaloticacid compound physicochemical constant and spectral data:
PestaloticacidA (1): brown oil,
uV (MeOH) λ max (log ε): 292 (4.10), 206 (4.25); NMRdataseeTables1and2; ESI-MS:349 [M – H]
-; HR-ESI-MS:349.1658 (349 [M – H]
-, calc.349.1651).
PestaloticacidB (2): brown oil,
UV(MeOH)λ
max(logε):318(4.16),232(4.30),207(4.40);ESI-MS:345[M-H]
-;HR-ESI-MS:345.1338([M-H]
-,calc.345.1338)。
PestaloticacidC (3): brown oil,
uV (MeOH) λ
max(log ε) nm:285 (4.31), 204 (4.20); ESI-MS:357 [M+H]
+, 359 [M+2+H]
+, 379 [M+Na]
+, 381 [M+2+Na]
+; HR-ESI-MS:379.1284 ([M+Na]
+, calc.379.1288).
PestaloticacidD (4): brown oil,
uV (MeOH) λ
max(log ε) nm:317 (3.82), 217 (4.52); ESI-MS:337 [M-H]
-, 339 [M+2-H]
-; HR-EI-MS:338.1285 ([M]
+, calc.338.1285).
PestaloticacidE (5): brown oil,
uV (MeOH) λ
max(log ε): 310 (3.47), 208 (4.07); ESI-MS:413 [M+H]
+; HR-ESI-MS:413.2665 ([M+H]
+, calc.413.2668).
PestaloticacidF (6): brown oil,
UV(MeOH)λ
max(logε):257(3.73),203(4.26);ESI-MS:371[M+Na]
+;HR-ESI-MS:371.1464([M+Na]
+,calc.371.1471)。
PestaloticacidG (7): brown oil,
UV(MeOH)λ
max(logε):269(3.81),211(4.59);ESI-MS:389[M+Na]
+;HR-ESI-MS:389.1575([M+Na]
+,calc.389.1576)。
PestaloticacidH (8): brown oil,
UV(MeOH)λ
max(logε):274(3.60),211(4.41);ESI-MS:389[M+Na]
+;HR-ESI-MS:389.1573([M+Na]
+,calc.389.1576)。
PestaloticacidI (9): brown oil,
UV(MeOH)λ
max(logε):273(3.59),208(4.37);ESI-MS:389[M+Na]
+;HR-ESI-MS:389.1573([M+Na]
+,calc.389.1576)。
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. having the Pestaloticacid compound of antibacterial activity, is after superparasitism being intended pestalotia bacteria strain fermentation culture, then the compound obtained after Extraction and isolation.
2. have the Pestaloticacid compound of antibacterial activity as claimed in claim 1, it is characterized in that, structural formula is as follows:
3. there is the antibacterial activity application of the Pestaloticacid compound of antibacterial activity as claimed in claim 2.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510955816.4A CN105503780B (en) | 2015-12-17 | 2015-12-17 | Pestalotic acid compound and its application with antibacterial activity |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510955816.4A CN105503780B (en) | 2015-12-17 | 2015-12-17 | Pestalotic acid compound and its application with antibacterial activity |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105503780A true CN105503780A (en) | 2016-04-20 |
CN105503780B CN105503780B (en) | 2019-08-13 |
Family
ID=55712149
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510955816.4A Expired - Fee Related CN105503780B (en) | 2015-12-17 | 2015-12-17 | Pestalotic acid compound and its application with antibacterial activity |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105503780B (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106947781A (en) * | 2017-04-21 | 2017-07-14 | 云南大学 | A kind of fungi intends disk stey extract and its application |
CN108624527A (en) * | 2018-05-12 | 2018-10-09 | 湖南科技学院 | A kind of ginkgo source growth-promoting preparation of prevention ginger bacterial wilt |
CN111393492A (en) * | 2020-04-03 | 2020-07-10 | 中国科学院南海海洋研究所 | Alkyne-alkene hydroquinone glucoside, preparation method thereof and application of alkyne-alkene hydroquinone glucoside in preparation of liver X receptor agonist |
-
2015
- 2015-12-17 CN CN201510955816.4A patent/CN105503780B/en not_active Expired - Fee Related
Non-Patent Citations (4)
Title |
---|
FAN ZHANG, ET. AL.: "Isolation, antimicrobial activity, and absolute configuration of the furylidene etronic acid core of pestalotic acids A–G", 《ORG. BIOMOL. CHEM.》 * |
GANG DING, ET AL.: "Ambuic Acid and Torreyanic Acid Derivatives from the Endolichenic Fungus Pestalotiopsis sp.", 《J. NAT. PROD.》 * |
GOVERDHAN MEHTA AND KABIRUL ISLAM: "Total Synthesis of the Novel NF-kB Inhibitor (-)-Cycloepoxydon", 《ORG. LETT.》 * |
JIANGTAO GAO, ET AL: "Antimicrobial and antiprotozoal activities of secondary metabolites from the fungus Eurotium repens", 《MED CHEM RES》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106947781A (en) * | 2017-04-21 | 2017-07-14 | 云南大学 | A kind of fungi intends disk stey extract and its application |
CN106947781B (en) * | 2017-04-21 | 2019-08-23 | 云南大学 | A kind of fungi intends disk stey extract and its application |
CN108624527A (en) * | 2018-05-12 | 2018-10-09 | 湖南科技学院 | A kind of ginkgo source growth-promoting preparation of prevention ginger bacterial wilt |
CN111393492A (en) * | 2020-04-03 | 2020-07-10 | 中国科学院南海海洋研究所 | Alkyne-alkene hydroquinone glucoside, preparation method thereof and application of alkyne-alkene hydroquinone glucoside in preparation of liver X receptor agonist |
Also Published As
Publication number | Publication date |
---|---|
CN105503780B (en) | 2019-08-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhou et al. | New azaphilones and tremulane sesquiterpene from endophytic Nigrospora oryzae cocultured with Irpex lacteus | |
CN105503780A (en) | Pestalotic acid compound with antibacterial activity and application thereof | |
CN103665071B (en) | Elaiophylin derivative and the application in antimicrobial agent and resistance m tuberculosis infection thereof | |
CN105906650B (en) | Macrolide new compounds and preparation method and application | |
CN113637010B (en) | Hydrogenated xanthone derivative and preparation method and application thereof | |
CN109400444B (en) | Sesquiterpenoids for inhibiting plant pathogenic fungi and preparation method thereof | |
Shen et al. | Azaphilones from the Fungus Penicillium multicolor LZUC‐S2 and Their Antibacterial Activity | |
CN109988129A (en) | Compound and its preparing the application in antituberculotic | |
CN112961168B (en) | Macrolide new compound and preparation method and application thereof | |
Yehia | Multi-function of a new bioactive secondary metabolite derived from endophytic fungus Colletotrichum acutatum of Angelica sinensis | |
Milagre et al. | Novel agents for enzymatic and fungal hydrolysis of stevioside | |
Piska et al. | Terpenoid as antibacterial produced by endophyte Fusarium oxysporum LBKURCC41 from Dahlia variabilis tuber | |
CN110698541B (en) | Natural Rakicidins compound Rakicidin J and fermentation extraction method thereof | |
CN105837590A (en) | Compound with anti-Candida albicans activity, preparation method and application thereof | |
CN115124582B (en) | Derivatives containing 2,9-dimethyl hexadecanoic macrolide parent nucleus for resisting rhizoctonia solani, and preparation method and application thereof | |
CN106957216B (en) | Preparation method and antifungal application of antifungal compound | |
CN109575040B (en) | Compound with antibacterial activity and preparation method thereof | |
CN108002998B (en) | Naphthalene compound and preparation method and application thereof | |
CN110437314B (en) | Natural Rakicidins compound Rakicidin B1-1 and fermentation extraction method thereof | |
CN107573394A (en) | Dechlorination platform hooks mould chlorins compound and preparation method thereof and the application in antibacterials are prepared | |
CN114634518B (en) | Two macrolide new compounds, pharmaceutical composition, and preparation method and application thereof | |
CN113402385B (en) | Antibacterial compound derived from fungal metabolite, preparation method and application | |
CN110698537B (en) | Natural Rakicidins compound Rakicidin B1-2 and fermentation extraction method thereof | |
CN114853574B (en) | Sesquiterpenoids and preparation method and application thereof | |
LU503011B1 (en) | Preparation method of Trienomycin A, Trienomycin A and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190813 Termination date: 20201217 |