Isolation of Compounds in Brazilian Propolis Active against the Honey Bee Pathogen "Paenibacillis Larvae".

Propolis, a plant resin used by honeybees, is collected from specific botanical sources in different regions worldwide yielding different types with distinct color, aroma, and chemical composition. Among other biological properties, propolis has exhibited antimicrobial activity against several organisms including Paenibacillus larvae, the causative organism of the honey bee disease American Foulbrood (AFB). The longer term goals of this study are to isolate the chemical components of propolis which could be used as an alternative treatment for AFB and other diseases. Brown propolis from Mariana, Minas Gerais (Brazil) was extracted in 65% isopropanol and filtered. This propolis solution was adjusted to pH 2, 7, and 8.5 and partitioned against ethyl acetate. A spectrophotometric assay of bacterial growth was conducted to determine if any of the fractions inhibited P. larvae. It was postulated that only pH 2, organic acid fraction would likely inhibit growth. Using solid phase extraction (SPE), two aromatic acid reference compounds with different pKa’s were used to standardize the system with elution step gradients of KCl (10 mM, 15 mM, 25 mM, 35mM, 50 mM, 60 mM, 75 mM, 1 M). Acids were detected by UV280; they were shown to separate as expected by pKa. This SPE method was employed on the propolis extracts and fractions collected. SPE fractions were assayed for inhibition of P. Larvae growth using the spectrophotmetric bacterial assay. We found that the 10 mM KCl fraction from the pH 2 ethyl acetate extraction inhibited the P. Larvae. Future experiments will be directed towards analyzing active fractions with LC/MS and LC/MS/MS. [This study was supported by USDA Capacity Building/NSF HBCU-UP grants awarded to Dr. Sarwan Dhir Ph.D. Professor & Director, Center for Biotechnology, Fort Valley State University, Fort Valley, GA 31030]