PROPOLIS AS A SOURCE OF NEW ANTI-PAENIBACILLUS LARVAE SUBSTANCES K. Bilikova 1, M. Popova 2, B, Trusheva 2, V. Bankova 2
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1 PRPLIS AS A SURCE F NEW ANTI-PAENIBACILLUS LARVAE SUBSTANCES K. Bilikova 1, M. Popova 2, B, Trusheva 2, V. Bankova 2 1 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia 2 Institute of rganic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
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4 Could propolis be a source of novel treatments for bees?
5 Paenibacillus larvae a pathogen of honeybees, causing the widespread fatal disease of bee larvae "American foulbrood" (AFB)
6 Total EtH extract Petrol ether fraction EtAc fraction Active against P. larvae MRE PRMISSING ACTIVITY Active against P. larvae
7 Petrol ether fraction Individual propolis constituents ( mg each) Structures confirmed by spectral methods, purity proved by chromatography
8 Pure propolis compounds Flavonoid aglycones H H Ac H H Pinocembrin Pinobanksin-3--acetate H Me H H Chrysin Tectochrysin
9 Pure propolis compounds C C H Me H Me Dimethylallyl ferulate Benzyl ferulate CH 9-oxo-10(E)-12(Z)-octadecadienoic acid
10 Mixture of caffeic acid esters Dimethyallyl caffeate 2: Isopentenyl caffeate 1: Phenylethyl caffeate (CAPE) 1: Benzyl caffeate 1
11 Tests for anti-paenibacillus larvae activity
12 Tests for anti-paenibacillus larvae activity P. larvae subsp. larvae (ERIC I.) swe159/97 swe194/09 P. larvae subsp. pulvifaciens (ERIC II.) swe223/00 swe26/02 Bacterial strains from Swedish collection of microorganism provided by Dr. Forsgren, SLU, Upsala. Classification of P.larvae strains according to Genersch E., Forsgren E., et all, (2006) IUMS, 56,
13 Tests for anti-paenibacillus larvae activity growth inhibition test performed in 96 well microtiter plate Bacterial culture of P.larvae strains in exponential phase of the growth at density of 10 5 CFU/ml was used for experiment serial dilution of propolis compounds in growth medium (MYPGP) from 1 mg/ml (~10-3 M) to 7.81 µg/ml (~10-5 M) MIC determination after 24, 36 and 48 hours of incubation at 37 C, at 200 rpm, absorbance measured at 595 nm data evaluated by Gen5 Microplate Software
14 H Me H Chrysin H Tectochrysin
15 Tests for anti-paenibacillus larvae activity growth inhibition test performed in 96 well microtiter plate Bacterial culture of P.larvae strains in exponential phase of the growth at density of 10 5 CFU/ml was used for experiment serial dilution of propolis compounds in growth medium (MYPGP) from 1 mg/ml (~10-3 M) to 7.81 µg/ml (~10-5 M) MIC determination after 24, 36 and 48 hours of incubation at 37 C, at 200 rpm, absorbance measured at 595 nm data evaluated by Gen5 Microplate Software
16 Minimal inhibitory concentration of propolis compounds for P.larvae strains after 24h of incubation MIC [µg/ml] / 24 H Sample No. compound M W P.larvae ERIC I. (swe 159/97) (swe 194/09) P.larvae ERIC II. (swe 26/02) (swe 223/00) B-1 benzyl ferulate ,00 250,00 500,00 500,00 B-2 pentenyl ferulate ,00 250,00 500,00 500,00 B-3 B-4 caffeate mixture average oxo-10E, 12Zoctadecanoic acid ,50 500,00 125,00 62,50 62, B-5 pinocembrin ,50 62,50 125,00 125,00 B-8 pinobanksin-3-acetate ,00 31,25 125,00 125,00
17 Minimal inhibitory molar concentration of propolis compounds for P.larvae strains after 24 hours of incubation Sam ple No. compound M W P.larvae ERIC I. swe 159/97 MIC [mol.l -1 ] / 24 H swe 194/09 P.larvae ERIC II. swe 26/02 swe 223/00 B-1 benzyl ferulate 284 1,9 x 10-3 M 9,7 x 10-4 M 1,9 x 10-3 M 1,9 x 10-3 M B-2 pentenyl ferulate 276 9,5 x 10-4 M 9,5 x 10-4 M 1,9 x 10-3 M 1,9 x 10-3 M B-3 B-4 caffeate mixture average 270 2,3 x 10-4 M 4,6 x 10-4 M 2,3 x 10-4 M 2,3 x 10-4 M 9-oxo-10E, 12Zoctadecanoic acid 294 2,0 x 10-3 M B-5 pinocembrin 256 2,2 x 10-4 M 2,2 x 10-4 M 4,4 x 10-4 M 4,4 x 10-4 M B-8 pinobanksin-3-acetate 314 5,4 x 10-4 M 1,3 x 10-4 M 5,4 x 10-4 M 5,4 x 10-4 M
18 Growth inhibition of P. larvae (swe 159/97 and swe 26/02) in presence of caffeate mixture (concentration in µg/ml) subsp. larvae subsp. pulvifaciens
19 Growth inhibition of P. larvae (swe 159/97 and swe 26/02) in presence of pinocembrin (concentration in µg/ml) subsp. larvae subsp. pulvifaciens
20 Growth inhibition of P. larvae (swe 159/97 and swe 26/02) in presence of pinobanksin-3-actetate (concentration in µg/ml) subsp. larvae subsp. pulvifaciens
21 Conclusions MIC for most of tested propolis compounds is close to the MIC of antibiotics used in prevention of honeybee colony against AFB. HWEVER, use of antibiotics in beekeeping is banned in EU Propolis constituents have a very important advantage: They are of plant origin and are naturally present in the hive.
22 Thank you!
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