A novel inhibitor of plant pathogenic bacteria extracted from propolis residues

A novel inhibitor of plant pathogenic bacteria extracted from propolis residues JIANGSU UNIVERSITY OF SCIENCE AND TECHNOLOGY YAN XU

1. Background 2.2 研究内容与方法 Ralstonia solanacearum Ralstonia solanacearum 2 nd Plant pathogenic bacteria in the world Ralstonia solanacearum (Smith) Bacterial wilt Distribution widest; Most harm Hardest to control; Most lost Direct economic losses $50,000,000,000 10

The prevention 2.2 研究内容与方法 of plant disease is a worldwide problem Control Traditional Biological Breeding for disease resistance More difficult, Long term A Avirulent strain Crop rotation cycles Geographic restrict, Difficult to promote Natural agent D B Biocontrol bacterium strain Chemical pesticide Toxic very much Pollute the environment C Induced resistance

1. Background 2.2 研究内容与方法 Natural source material Caffeic Acids Derivatives Caffeic acid and its ester derivatives are a kind of natural phenolic acids which are widely found in fruits, vegetables, cereals, and bee products, which have biological activities such as antioxidant, anti-inflammatory and scavenging free radicals. Name Abb. R- Caffeic acid CA -- Methyl caffeate MC CH 3 Ethyl caffeate EC CH 2 CH 3 Propyl caffeate PC CH 2 CH 2 CH3 Isopropyl caffeate IpC CH 2 CH 2 CH3 Rhodiola rosea L. Butyl caffeate BC (CH 2 ) 3 CH 3 Amyl caffeate AC (CH 2 ) 4 CH 3 Isoamyl caffeate IaC (CH 2 ) 4 CH 3 Colla Apis, Propolis Hexyl caffeate HexC (CH 2 ) 5 CH 3 Heptyl caffeate HepC (CH 2 ) 6 CH 3 Octyl caffeate OCC (CH 2 ) 7 CH 3 Caffeic acid phenethyl ester CAPE (C 6 H 6 )CH 2 CH 3

1. Background Propolis Colla Apis, Propolis 4,000 Y ago Alleviate inflammation by Greek 3,000 Y ago Formalined cadaver by Egyptians Widely application now

1. Backgound Propolis Residues Annual production: 400-450 tons in China Active ingredient: phenolic acid, flavone, terpene Purple gold: antibacterial, antiviral, antioxidant Propolis Residues: 250-300 tons per year Sforcin J M, Bankova V. Propolis: Is there a potential for the development of new drugs?[j]. Journal of Ethnopharmacology. 2011, 133(2): 253 260. Cottica S M, Sabik H, Bélanger D, et al. Use of propolis extracts as antioxidant in dairy beverages enriched with conjugated linoleic acid[j]. European Food Research and Technology. 2015, 241(4): 543 551.

2. Inhibitory activity Inhibition rate The inhibitory activity of the extract of propolis residues Chloroform extract(d) and Ethanol extract(f)>50% for 5 Races Ethanol extract(f): GIM1.74:99.73±0.13% RS-5:91.61±4.75% Fig.2.1 Bacterial grow inhibition (%) of various extracts from propolis residue against R. solanacearum strains including GMI1000, GIM1.76, GIM1.74, GIM1.71 and RS-5. Samples: CK, agricultural streptomycin, 0.30mg/mL (proposed dosage); A, B, C, D, E, F were 35% ethanol extract, methanol extract, ethanol extract, chloroform extract, ethyl acetate extract, petroleum ether extract from propolis residues, respectively, their concentrations were all 0.10 mg/ml.(%)

2. The antibacterial activities of propolis residues MIC MBC The inhibitory activity on Ralstonia solanacearum of propolis residues extracts Table 2.1 The MIC and MBC concentration (mg/ml) of R. solanacearum treated by 35% ethanol extract and ethanol extract from propolis residue Bacterial Strain 35% Ethanol Extract Ethanol Extract Race MIC MBC MIC MBC GMI1000 1 2 0.25 1 GIM1.76 1 2 0.25 0.5 GIM1.74 0.06 2 0.06 0.5 GIM1.71 0.06 1 0.03 0.25 RS-5 0.25 2 0.12 0.5 The antibacterial activity of wax propolis residues is mainly found in ethanol extract.

2. The antibacterial activities of propolis residues Swimming motility Pathogenicity Flagellum swimming Realize diffusion Pathogen transmission Fig.2.2 Swimming motility inhibition of the ethanol extract of propolis residue against R. solanacearum strains including GMI1000 (A), GIM1.76 (B), GIM1.74 (C), GIM1.71 (D) and RS-5 (E), respectively. 0.20-0.50 mg/ml ethanol extract of propolis residue against R. solanacearum 5 strains: good, RS-5 BEST

SEM Effect on the morphology of mulberry Ralstonia solanacearum treated with propolis residues extract Shapes vary in size Growth defect Rough surface Fig. 2.3 SEM images of R. solanacearum RS-5 untreated and treated with the 35% ethanol extract of propolis residues. (A) Untreated (ruler units: 2.0 μm); (B) CK-35% ethanol (ruler units: 500 nm); (C) Treated (ruler units: 2.0 μm); (D) Treated (ruler units: 1.0 μm). Growth inhibition and cell structure change

TEM Cell irregular sunken Cytoplasmic density not uniform Substance outflow, appears cavitation Permeability of cell membrane barrier Fig. 2.4 TEM images of R. solanacearum RS-5 untreated and treated with the 35% ethanol extract of propolis residues (A) Untreated; (B) 35% ethanol; (C) 0.20 mg/ml of 35% ethanol extract of propolis residues; (D) 0.20 mg/ml of 35% ethanol extract of propolis residues (ruler units: 0.2 μm).

3.Caffic acid and its ester in Propolis residues LC-MS Qualitative identification of caffeic acid derivatives in ethanol extract of propolis residues by LC-MS Fig. 2.5 LC-MS TIC spectra of the standards (A) and the ethanol extract of propolis residue (B) The peak time of CA, PC, BC, CAPE, AC is 0.34 min 14.70 min 20.06 min 33.60 min and 34.21 min

LC-MS CA, CAPE more Fig. 2.6 Mass spectra of caffic acid and its alkyl esters in the ethanol extract of propolis residues. Sample ion peaks: m/z 178.96 221.01 234.95 282.95 248.95 Standard molecular weight [M-H] - : CA-179 PC-221 BC-235 CAPE-283 AC-249

LC-MS Table 2.2 Qualitative analysis of caffic acid and its alkyl esters in the propolis residue extract No. t R /min m/z 化合物归属 Chemical compound 化合物缩写 Chemicals abbreviation 1 0.34 179 Caffeic acid CA Table 2.3 Distribution of caffic acid and its alkyl esters in the grading extracts of propolis residue Extracts of propolis residue 35% Ethanol extract CA PC BC CAPE AC + + + + + 2 14.70 221 Propyl caffeate PC 3 20.06 235 Butyl caffeate BC 4 33.60 283 Caffeic acid phenethyl ester CAPE 5 34.21 249 Amyl caffeate AC Methanol extract Ethanol extract Chloroform extract + + + + + + + + + + + - - + - Ethyl acetate extract + - + + + Petroleum ether extract + - - - - Freely soluble in polar solvents.

LC-MS/MS Quantitative analysis of main caffeic acid ingredients in propolis residues by LC-MS/MS Table 2.4 Contents of CA and CAPE from grading solvent extracts of propolis residue (mg/g) Extracts of propolis residue Batch CA Std Dev CAPE Std Dev 35% Ethanol extract Methanol extract Ethanol extract Chloroform extract Ethyl acetate extract Petroleum ether extract A 0.64 0.01 0.23 0.06 B 0.44 0.01 0.39 0.00 A 1.81 0.03 2.91 0.02 B 1.43 0.04 0.22 0.24 A 1.97 0.02 6.53 0.66 B 1.48 0.01 1.31 0.27 A 0.74 0.02 3.93 0.06 B 0.53 0.05 1.69 0.29 A 1.46 0.02 2.33 0.11 B 1.39 0.12 1.31 0.26 A 0.22 0.02 - - B - - - - CA: 1.97mg/g, CAPE: 6.53mg/g Production place of origin OR Conditions of supercritical extraction process-difference

SUMMARY Propolis residue of different polar solvent extracts had antibacterial activity on 5 Races of Ralstonia solanacearum Ethanol extract is the best. Qualitative and quantitative analysis of the Caffic acid and its ester compound Based on anion full scanning and selective reaction monitoring mode : LC-MS LC-MS/MS It provides a new way for the development of the natural source of mulberry blight.

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