Preliminary investigation of antimicrobial effects of pomegranate (Punica granatum L.) leathery exocarp extract against some serious phytopathogens

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1 Preliminry investigtion of ntimicroil effects of pomegrnte (Punic grntum L.) lethery exocrp extrct ginst some serious phytopthogens Elshfie H.S. 1,*, Skr S.H. 2, Mng S.M. 1, Frisullo S. 3, Cmele I. 1 1 School of Agriculturl, Forestry, Food nd Environmentl Sciences, University of Bsilict, Vile dell Ateneo Lucno 10, Potenz, Itly; 2 Deprtment of Chemistry, Fculty of Science, Zgzig University, Zgzig, Egypt; 3 Deprtment of Agriculturl, Food nd Environmentl Sciences, University of Foggi, Vi Npoli 25, Foggi, Itly. 1

2 INTRODUCTION Pomegrnte (Punic grntum L.) fruits re importnt source of vitmins nd minerls nd widely used in dietry product industry. There re severl iologicl properties of pomegrnte extrct, mong them, ntiinflmmtion, nticncer nd ntioxidnt [1]. The ntimicroil properties of lethery exocrp extrct hs een investigted in the present reserch. Antifungl ctivity hs een crried out ginst 4 phytopthogenic fungi: Fusrium oxysporum Schltdl., Phytophthor cinnmomi Rnds, Penicillium digittum (Pers.) Scc. nd Botrytis cinere Pers. Wheres, the nticteril ctivity hs een crried out ginst 2 grm negtive cteri, Escherichi coli Migul nd Xnthomons cmpestris (Pmmel) Dowson nd 2 grm positive, Bcillus megterium de Bry nd Clvicter michignensis Smith. Chemicl chrcteriztion of the studied pomegrnte extrct hs een crried out using GC-MS. 2

3 Mterils nd Methods Extrction procedure. The lethery exocrp, previously sterilized, ws homogenized y liquid nitrogen (A) or distilled wter in plstic-porous g (B). The ove queous extrcts hve een suspended t concentrtion 200 mg/ml, shcked (100 rpm/24h), centrifuged (10,000 rpm/10 min), filtered nd conserved t -20 C (Figure 1). In vitro nticteril ctivity. The ctericidl effect hs een crried out ginst the previously mentioned cteri following the disc diffusion method of Bhuni et l. [2] in King B (KB) medi. Twenty µl of ech suspension ws inoculted t concentrtion 100 nd 50% nd the pltes were incuted t 22 C for 48 h. Tetrcycline nd Knmycin were used s controls s specified in the interntionl stndrds. The dimeter of inhiition zone ws mesured (mm). Fig. 1. Extrction procedures. 3

4 Mterils nd Methods In vitro ntifungl ctivity. The ove suspensions were incorported into potto dextrose gr (PDA) medi t concentrtion of 10 mg/ml. Then, 0.5 cm of fungl disk ws inoculted in ech Petri dish nd incuted t 22 C for 96 h. The fungitoxicity ginst the ove mentioned pthogenic fungi ws mesured s the percentge of growth inhiition (PGI) following Zygdlo et l. [3] method: PGI (%) = 100 x (GC-GT)/GC; where: GC represents the verge dimeter of fungl mycelium in PDA (control); GT represents the verge dimeter of fungl mycelium on the treted PDA dish. Azoxystroin nd Cycloheximide were used s controls s specified in the interntionl stndrds. GC-MS nlysis. The GC-MS nlysis hs een performed using Solid Phse Micro Extrction method (SPME) y Supelco U, mounted on Supelco support (Fig. 2) [4]. The principls ioctive sustnces hve een identified sed on their mss spectr y comprison with spectr of uthentic compounds ville in NIST 11 nd Wiley 275 lirries [5]. Fig. 2. GC-MS 4

5 G r o w t h i n h i i t i o n ( % ) Results nd Discussion 1. Anticteril ctivity 120 E. coli X. cmpestris C. michignensis B. megterium Results of nticteril ctivity showed tht extrct (B) t 100% hs the highest significnt ctivity ginst E. coli similr to c c c c knmycin nd followed y extrct (A) t 100% nd extrct (B) t 50%. Wheres the lowest significnt ctivity ws oserved in cse 0 100% 50% 100% 50% Knmycin Tetrcycline Extrct A Extrct B Cont (-ve) of tetrcycline nd 50% of extrct (A) (Fig. 3, 4). Fig. 3. Anticteril ssy. It is of gret interest to underline lso tht the two extrcts (A,B) hve showed the sme significnt ctivity of the two tested ntiiotics ginst X. cmpestris nd C. michignensis. Regrding B. megterium, the highest ctivity ws oserved in cse of two tested ntiiotics (Fig. 3, 4). A: E. coli B: B. megterium Fig. 4. Anticteril ctivity Vs. E. coli (A) nd B. megterium (B). 5

6 G r o w t h i n h i i t i o n ( % ) Results nd Discussion 2- Antifungl ctivity Results of ntifungl ctivity hve demonstrted tht the highest significnt ctivity ginst P. cinnmomi nd P. digittum ws oserved in cse of extrct (B) nd cycloheximide 0,1 µl/ml compred to ll other tretments (Fig. 5, 6). Regrding F. oxysporum nd B. cinere, the highest ctivity ws oserved in cse of the two synthetic fungicides (Fig. 5, 6). The ntimicroil ctivity of pomegrnte extrct my relted in generlly to the polyscchrides content in the lethery exocrp in gree with Miguel et l. [6]. Antifungl ctivity could e linked to the high content of phenol in peels [7]. Results of GC MS nlysis re eing processed for F. oxysporum P. cinnmomi P. digittum B. cinere 10 mg/ml 10 mg/ml Azoxystroin 1 µl/ml Cycloheximide 0.1 µl/ml Extrct A Extrct B Cont (-ve) Fig. 5. Antifungl ssy. otining the detiled structure of the principl sustnces present in the pomegrnte extrct. A: F. Oxysporum B: P. cinnmomi C: P. digittum Fig. 6. Antifungl ctivity Vs. F. Oxysporum (A), P. Cinnmomi (B) nd P. digittum (C). 6

7 Conclusions The outcomes of this study suggested tht pomegrnte lethery exocrp extrct cn e used effectively s possile nturl lterntive for controlling severl phypthogens. In ddition, the reveling of chemicl structure of the principl constituents of the pomegrnte lethery exocrp extrcts is eing processed. Further scientific reserch seems necessry in order to improve the knowledge nd investigte the mechnism(s) of ction. References 1. Akhtr S, Ismil T, Frternle D, Sestili P (2015). Food Chem. 174, Bhuni AK, Johnson MC, Ry B (1988). J. Appl. Bcteriol. 65, Zygdlo JA, Guzmn CA, Grosso NR (1994). J. Essent. Oil Res. 6, Elshfie HS, Rcioppi R, Bufo SA, Cmele I (2017). Sudi J. Biol Sci. 24, Wiley Registry of Mss Spectrl Dt, with NIST Spectrl Dt CD Rom, 7 th edn. (1998) Wiley, New York. 6. Miguel MG, Neves MA, Antunes MD (2010). J. Med. Plnts Res. 4, Al-Zoreky NS (2009). Int. J. Food Microiol. 134:

8 Thnk You Get in Touch Hzem Slheldin Elshfie Address: University of Bsilict, Potenz 85100, Itly. E-mil: Phone numer: