1. MIHAELA BERINDE 1,2, MAURIZIO RUZZI 2, ROSARIO MULEO 2, MARIA TOFANA 1*, SONIA SOCACI 1 1-Universitaty of Agricultural Science and Veterinary Medicine, Cluj-Napoca 2 - University of Tuscia, Viterbo, Italy * Corresponding author: tofanam@yahoo.com tofanam@yahoo.com Purpose: To determine the polyphenols composition of apple (fruit pulp and leaf) from two varieties: Italian Red Passion and Annurca; Preliminary evaluation of the antimicrobial activity of this apple varieties POLYPHENOLS CONTENT IN APPLE ITALIAN RED PASSION AND ANNURCA Introduction Abstract Apples of seedlings, M3, M4 and M5 from the experimental field of the University of Tuscia Italy, belonging to the Italian Red Passion group are investigated about the polyphenols content. These fruits are characterized by red coloration of the pulp. In this work we analyzed comparatively the polyphenols content of apples M3 and M4 and leaves of apples M4 and M5. As control are used Annurca apples and Annurca leaves. Materials and MethodsResults Literature The assortment of polyphenols present in apple fruit is wide, but differs between flesh and peel both in its range and in the total content (Wolfe et al., 2003). Typical apple fruit phenolic acids are hydroxy-cinnamic acids, dihydro-chalcones, flavonols, anthocyanin, flavanols and procyanidin (Tsao et al., 2003). The content changes of the main phenols in apples and their contributions to the total antioxidant activity are not very clear. Numerous epidemiological studies, published toward the end of the twentieth century, have provided evidence that increased consumption of fruit and vegetables provides protection against cardiovascular diseases, various kinds of cancer, and other chronic diseases. The risk of a heart attack has been shown to decrease by 49% for apple consumers in diet at a daily amount of >110 g compared to those at <18 g. In fruits such as apple, red coloration of the skin is a much valued trait. In the last 20 years the interest on the polyphenolic content of the fruit is continuously growing up, due to their health-related characteristics (Boyer and Liu, 2004); from this point of view, red fleshed apple could be a varietal innovation with favorable health-related characteristics and a strong appeal to consumers. The accessions were obtained from cross pollinated by a plant of red apple from the wild germplasm Lazio in the experimental field at the farm of the University of Tuscia. In this work we used only apples M3 and M4 and leaves of apples M4 and M5. As control was used Annurca apples and Annurca leaves. Extract preparation: Extract preparation: Lyophilized material Solvent extraction  Methanol/Water/Acetic acid (70:29:1)  Acetone/Water/Acetic acid (70:29:1)  Methanol/Acetone/Water/Acetic acid (60:20:19:1) Dried samples were resuspended in pure MetOH Wolfe K, Liu RH. (2003), “ Apple peels as a value-added food ingredient”. J Agric Food Chem. 2003, 51:1676–1683. Tsao Rong, Yang Raymond, Young J. Christopher and Zhu Honghui, (2003), ”Polyphenolic Profiles in Eight Apple Cultivars Using High Performance Liquid Chromatography (HPLC)”, J. Agric. Food Chem., 51: 6347-6353. Alberto María Rosa, Matías Andrés, CanavosioRinsdahl, Manca de NadraMaría Cristina,(2006), “Antimicrobial effect of polyphenols from apple skins on human bacterial pathogens”, Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.9 No.3, Special Issue, by Pontificia Universidad Católica de Valparaíso – Chile. Boyer J.; Liu, R.H,(2004), “Apple phytochemicals and their health benefits”, Nutrition Journal, 3: 5. Kondo Satoru, Tsuda Kazuhiko, Muto Norio, Ueda Jin-etsu, (2002) “Antioxidative activity of apples skin or flesh extracts associated whit fruit development on selected apple cultivars”,ScientiaHorticulturae, 96: 177-185. Mari Angela, Tedesco Idolo, Nappo Annunziata, Russo Gian Luigi, Malorni Antonio, Carbone Virginia, (2010) “Phenolic compound characterization and anti-proliferative activity of ‘‘Annurca” apple, a southern Italian cultivar”, Food Chemistry, 123: 157–164. D’AngeloStefania, Cimmino Amelia, Raimo Marianna, Salvatore Anna, Zappia Vincenzo, and GallettiPatrizia, (2007) “Effect of Reddening– Ripening on the Antioxidant Activity of Polyphenol Extracts from Cv. ‘Annurca’ Apple Fruits”,J. Agric. Food Chem, 55:9977–9985. HPLC Analysis was performed using a HPLC-DAD apparatus at 5 wavelengths (280, 307, 316, 365 and 520 nm), following the method used by Jeffrey et al. (1999) Bacterial antimicrobial susceptibility plate test: modified Kirby-Bauer Method Bacterial antimicrobial susceptibility liquid test: Broth Dilution Method Growth inhibition obtained using acetone extracts from fruit pulp of different Italian Red Passion genotypes (M3, M4) and Annurca SOLVENTSAMPLE INHIBITION ZONE DIAMETER (cm) 24h48h E. coli DH 5α M3 0.325±0.001250.300±0.0 M40.600±0.00.575±0.00125 Annurca 0.450±0.0050.450±0.0 B.subtilis PB19 M3 0.275±0.001250.250±0.0 M40.550±0.0050.525±0.00125 Annurca 0.375±0.00.350±0.00125 P.aeruginosa M3 0.225±0.00125 M4 0.400±0.0 Annurca 0.400±0.0 Control(methanol)0.0 Higher antimicrobial activity was obtained using fruit pulp extracts of M4 genotype, independently from the solvent system SOLVENTSAMPLE GROWTH INHIBITION (%) 25 µl50 µl Acetone M3none25.20±0.00172 M419.52±0.005367.87±0.0004 Annurcanone68.40 Methanol M3none25.02±0.01533 M435.00±0.015653.32±0.0013 Annurca25.65±0.0134659.18±0.00019 Mix M3none21.66±0.00274 M435.55±0.000860.25±0.0016 Annurca33.90±0.0027361.84±0.00047 Functional compounds in apple: organic acids, polyphenols, flavonoids 6 th International Conference on Polyphenols Applications June 7 & 8, 2012 – Paris, France The total polyphenols content from all this samples were extracted with 3 types of solvents: Methanol/Water/Acetic Acid:70:29:1, Acetone/Water/Acetic Acid:70:29:1 and Methanol/ Acetone/Water /Acetic Acid: 60:20:19:1. The obtained extract was evaporated, lyophilized and resuspended in methanol and then analyzed by HPLC. The HPLC analysis was performed, simultaneously, at 5 wavelengths (280, 307, 316, 365 and 520 nm). Comparative analysis of HPLC data confirmed that Italian Red Passion extracts from both fruit and leaf samples contained a larger array of compounds compared to Annurca samples. This compounds, belong to different chemical groups, such as anthocyanin, hydroxycinnamic acids, flavonols, procyanidins and dihydrochalcones. The better solvent systems for the chemical analysis of fruit pulp and leaf samples are acetone and methanol respectively. ANTIMICROBIAL ACTIVITY OF EXTRACTS FROM APPLE ITALIAN RED PASSION AND ANNURCA HPLC chromatogram of anthocyanins (A520 ) Compounds identified Graphical representation of HPLC results from methanol extracts of M4 fruit pulp