Evaluation of Antioxidant Activity, Antimicrobial and Fungicidal Effect of Organic Extracts of Cork Oak Acorns Infested by Carpophagous Insects

Y. Adjami, M.L. Ouakid, A. Saouli, H. Bensafi and N. Soltani Laboratoire de Biologie Animale Applique Dpartement de Biologie Facult des Sciences Universit de Annaba 23000 Annaba Algria E.A. Hayouni Laboratoire dEcologie et de Technologie Microbienne Institut National des Sciences Appliques et de la Technologie (INSAT) BP. 676, 1080 Tunis Tunisia

C. Everarts CNRS UMR 5548 Dveloppement Communication chimique chez les Insectes Universit de Bourgogne Facult des Sciences 6 Bd gabriel, 21000 Dijon France Keywords: acorns, antimicrobial and fungicidal effect, antioxidant activity essential oil, Quercus suber L. Abstract The Algerian cork oak forest is constituted with numerous essences (Quercus faginea, Quercus coccifera, Quercus suber). This last one establishes an essential element of the physical, climatic and especially socioeconomic balance of the populations of the rural zones and the country in general. An appeal to the regeneration would be indispensable for the rehabilitation of these forests, where from the importance of acorns, which play a major role in the regeneration of this essence. The purpose of this work is to estimate antioxidant activity, antimicrobial and fungicidal effect of the essential oil of organic extracts of the cork oak acorns attacked with the procession of insects called Carpophagous Insects. We extracted acorns of cork oak infested with insects by using organic solvents as the acetone, the chloroform, the ethanol and also extract of water. We measured at first the total lipids according to Goldsworthy's method (1972). The antioxidant activity of samples was determined by two methods, the one based on the discoloration of a solution of cation ABTSo +. The original product, ABTS is going to generate cations ABTSo + which are free radicals and the other one based on the discoloration of a solution of -carotene (-carotene bleaching method). Extracts, obtained were individually estimated against some phytopathogenes: Penicillium digitalum, Penicillium italicum, Botrytis fabae, Botrytis cinera, Ascophyta fabae, Fusarium oxysporum, as well as against pathogenic microorganisms such as Geotrichum candidum, Aspergillus, Candida galbrata, Candida albicans, and Saccharomyces cerevisiae. Extracts were so individually estimated against a sample group of 4 bacteria including: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922. Results show that all the extracts stemming from attacked acorns contain less lipids than the extracts of the healthy acorns. These same extracts present an antioxidant activity, a significant antimicrobial and fungicidal effect. This biologic activity is a function of various used solvents.
Proc. IS on MAP SIPAM 2009 Eds.: M. Neffati et al. Acta Hort. 853, ISHS 2010

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INTRODUCTION The cork oak is considered as one of the forested essences of which the area, naturally unstretchable is strictly limited to the Occidental Mediterranean pond (Boudy, 1950). The ecological and socioeconomic importance of the cork oak incites to increase efforts with the aim of the rehabilitation of cork oak forest. The problems from which suffers this forest are numerous. Decline for example, entice the attention of numerous researchers and in particular that of the phytopathologistes, during these last years. It is considered as one of the main causes of the regression of cork oak forest (Asmrfc, 1998), it drags a embrittlement of the populations that becomes homes of predilection for different antagonistic agents as the mushrooms, bacteria and especially the bugs who are of the devastating of the acorns of oaks (Bakry et al., 1999; Anderson, 1992; Fukumoto and Kajimura, 2000). An effort of regeneration imposes itself. The cork oak multiple of two way, a natural and the other artificial, reproduction or natural regeneration makes or by seedling fallen acorns) or by dismissals of stumps. The regeneration by natural seedling depends strictly on several factors: quantity, power and faculty of germination of acorns on one hand, conditions of the physical environment and anthropic action, on the other hand (Boudy, 1950; Bouchafra and Fraval, 1991). On the harvested quantities important of seeds, a big part is lost or altered, these losses are due to multiple reasons as the mushrooms Ciboria batshiana responsible for the black rot of the acorns and Myrioconium castanea (Khaladi et al., 1999), as well as the devastating of the acorns, and all attack of the insects can decrease the regularity and the abundance of the acorn product. However, insects evolving inside the acorns of the North of Algeria in El-Kala's region are a Tortricidae (Lepidopera) Cydia fagiglandana, Cydia splendana, and beetle Curculionidae Curculio sp. This last one is an important devastating of Quercuss acorns (Coutin, 1960; Bovey et al., 1975; Bellal, 2008; Adjami, 2009). This work suggests determining the effect of the attack of insects in the lipidic contents of acorns, in the antioxidant activity, in the antimicrobial activity and fungicide of extracts of acorns with various solvents. MATERIAL AND METHODS Dosage of the Total Lipids The concentration of the total lipids was estimated according to Glodsworthy method (Glodsworthy et al., 1972) by the sulfophosphovanillic using lyophilized human serum (Lyotrol N, Boehringer) as a standard. Biological Activity of the Extract of Cork Oak Acorn 1. Extraction. Fruits were ground to a fine powder with a grinder. Powders (100 g) were extracted in a Soxhelt extractor with hexane for 6 h at 65C to remove the fatty materials. Then the defatted powder was divided into four fractions. Each fraction was separately reextracted in a Soxhelt apparatus for 5 h with 250 ml of one of these solvents: absolute chloroform, absolute acetone, Chloroform, respectively. For water extraction, the fourth fraction was infused with 100 ml of freshly boiled distilled water for 10 min. The infusion was filtered through Whatman No. 1 and rapidly cooled under tap water. All obtained organic extracts were concentrated by rotary evaporation under vacuum at 45C, using a HACH UVVis spectrophotometer (Model DR/4000, Colorado, USA), to get crude extracts, whereas infusions were lyophilised. 2. Quantification of Total Antioxidant Activity ABTS Assay. ABTS radical scavenging activity of extracts was determined according to Re et al. (1999). In this test, we measured the relative capacity of antioxidants to scavenge the ABTS+ radical compared to the antioxidant potency of L (+) ascorbic acid (Vitamin C) used as a standard. The ABTS+ radical was freshly prepared by adding 5 ml of a 4.9 338

mM potassium persulfate (K2S2O8) solution to 5 ml of a 14 mM ABTS solution and kept for 16 h in the dark. Before usage, this solution was diluted to get an absorbance of 0.7000,020 at 734 nm with PBS at pH 7.4 (5 mM NaH2PO4, 5 mM Na2HPO4 and 153.84 mM of NaCl). The spectrophotometer is preliminary blanked with PBS. The final reaction mixture of standard group was made up to 1 ml with 950 l of ABTS+ solution and 50 l of Vit-C. Similarly, in the test group 1 ml reaction mixture comprised 950 l of ABTS+ solution and 50 l of the extract solution. The reaction mixture was vortexed for 10 s and its absorbance at 734 nm was recorded each minute after initial mixing. Appropriate solvent blanks were run in each assay, and all measurements were done at least 6 min. The results, determined from regression equation of the calibration curve (y = 0.0446x 0.0076, R2 = 0.987), were expressed as mg ascorbic acid equivalents per gram dry weight of raw material (mg Vit-C/g dry weight). -Carotene Bleaching Test. The antioxidant activity of fruit extracts was evaluated according to a slightly modified version of the -carotene bleaching method. -carotene (0.2 mg), 20 mg of linoleic acid and 200 mg of Tween 40 (polyoxyethylene sorbitan monopalmitate) were mixed in 0.5 ml of chloroform. Chloroform was removed at 45C under vacuum using a rotary evaporator (Heidolph, Germany). The resulting mixture was immediately diluted with 10 ml of triple-distilled water and was mixed well for 12 min. The emulsion was further made up to 50 ml with oxygenated distilled water. Aliquots (4 ml) of this emulsion were transferred into different test tubes containing 0.2 ml of test samples. BHA was used for comparative purposes. A control, containing 0.2 ml of corresponding solvent and 4 ml of the above emulsion, was prepared. The tubes were placed at 50C in a water bath. Absorbencies of all the samples at 470 nm were taken at zero time (t = 0), measurement of absorbance was continued until the colour of the carotene disappeared in the control reaction (t = 180 min) at 15 min intervals. A mixture prepared as above without -carotene served as blank. All determinations were performed in triplicate. The antioxidant activity (AA) of the extracts was evaluated in terms of bleaching of the -carotene using the following formula:
( A At ) AA = % inhibition = 100 .1 0 A0 At

where A0 and are the absorbance values measured at zero time of the incubation for test sample and control, respectively. At and At are the absorbance measured in the test sample and control, respectively, after incubation for 180 min. 3. Determination of Antibacterial Effect Microbial Strains. The extracts, obtained from both populations of Quercus, were individually tested against some phytopathogens: Penicillium digitalum, Penicillium italicum, Botrytis fabae, Botrytis cinera, Ascophyta fabae, Fusarium oxysporum, as well as against clinical, food borne and water borne fungi: Geotrichum candidum, Aspergillus niger and yeasts: Candida galbrata, Candida albicans, Saccharomyces cerevisiae. The extracts were also individually tested against a panel of 4 bacteria including: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922. Activity against Bacteria and Yeasts (Disc-Diffusion Assay). The agar disc diffusion method was employed for the determination of antimicrobial activities of the extracts as recommended by NCCLS (1997). Briefly, a suspension of the tested bacteria or yeasts (100 l of suspension containing 108 CFU/ml) was spread on nutrient agar (NA). Filter paper discs (12.7 mm in diameter) were impregnated with 100 l of 3 mg/ml extracts (300 g/disc) and placed on the inoculated plates. Negative controls were prepared using the corresponding solvent. Streptomycin B (30 g/disc) was used as positive reference standards to determine the sensitivity of one strain/isolate in each bacterial specie tested. The inoculated plates were incubated at 37C for 24 h. Antimicrobial activity was evaluated by measuring the zone of inhibition against the tested bacteria and yeasts. All tests were performed in triplicate and experiments were repeated twice. Activity Against Fungi. Antifungal activity on tested strains was obtained by aseptically 339

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adding, the extracts, to sterile media at 45C in order to obtain desired concentrations. Controls were set up with equivalent quantities of appropriate solvent. The cultures were obtained by transplanting mycelium disks (diameter 10 mm) from a single culture in the stationary phase. Then they were incubated at 301C on SDA (Sabouraud dextrose agar) on thin cellophane sheets until logarithmic growth phase was reached. Subsequently, the cultures were transferred to Petri plates with a medium containing the extracts. The diametral development was measured after 72 h of incubation. Statistical Analysis. All data were expressed as means standard errors of triplicate measurements. One-way analysis of variance (ANOVA) was carried out to test any differences between the solvents used. Statistical comparisons between variables (phenolic contents and antioxidant activity) were performed with Students t-test. Correlation between the antioxidant, antimicrobial activities and total phenolic content was carried out using the correlation and regression in the EXEL program. Differences were considered significant at p 0.05. RESULTS The Content in Lipids of the Acorns of the Cork Oak The content in lipids of the cork oak acorns varies in function that the acorn is healthy, attacked or greatly attacked. The healthy acorn presents to the level of the fine content of about 50 g/g.MS, this concentration decreases as for the acorn is attacked and that it presents a hole to 22.613.61 g/g.MS. When the acorn is attacked strongly, one records a fall of the lipids to 6.183.96 g/g.MS. It has a significant difference between these 3 averages (F=23.12, p=0 ,0001). On the other hand the content in lipids in the pericardic area of the healthy acorns is weak in relation to the fine about 16 g/g.MS and in spite of a light increase of these concentrations in the pericardic area of the acorns attacked these averages are not significant difference (F=1.06, p=0.8) (Fig. 1). Biologic Activities of the Extracts of Cork Oak Acorn 1. Antioxidant Activity. The results of the antioxidant activity of the different extracts of acorns tested represented in the Figure 2, show a strong potential in the prevention of the peroxydation of the linoleic acid, and this some either the used solvent. Indeed, in any case the extract of Q. suber is capable to prevent the bleaching of the -carotene solution. The extracts of the ethanol and the acetone are the most efficient extract in relation to the synthetic BHA, at least during the first hour of incubation. The extracts of chloroform and water are less efficient in the activity of inhibition. It seems that it is the polar solvents that assure the best antioxidant activity of the extracts of cork oak acorns. In terms of trappers of the radicals free ABTS. + bleaching the solution (Fig. 2), these are always the polar solvents that present the best activity. The acetone presents 15.661.08 mg equivalent Vitamin C/g dry weight, followed by the ethanol with 12.120.56 mg equivalent Vitamin C/g dry weight these two averages, are significant difference. Water and chloroform present different values but near. 2. Antimicrobial Activity. All tested extracts present an important activity facing all tested microorganismes. The results show that the extracts of the acorns by the different solvents used against some pathogenic bacteria as: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, ATCC 25922 are positive. It comes out again that it is the aqueous extract of Q. suber that has the strongest activity against all estimated bacteria; this activity varies between 20.4 and 23.8 mm. The chloroformic extract present also a strong activity that varies between 18.5 and 22.2 mm, these two extracts are apolar. The antimicrobial activity the excerpt to the acetone varies from 12.7 to 15.8 mm while the one to the ethanol varies from 10.4 to 15 mm, these two solvents are polar. Him ya place to note a significant interrelationship between the polyphnolic content and the antimicrobial activity (R2 = 7378%). (Table 2). 340

Concerning the antimycotic activities of the studied extracts we noted a substantial activity against all estimated mildews. Indeed P. italicum and P. digitalum was the most sensitive (of the zones of inhibition spreading between 12 mm and 16 mm). The strongest activity (16.6 mm) has been recorded when the extract of acetone has been valued against Ascophyta fabae. Once again, the extracts are active against the majority of estimated phytopathogenes. Indeed, for the chloroformic extract, we recorded 14.4 mm (against T. fabae and S. oxysporum) 13 mm (against G. candidum). As for the antioxidant activity, strong interrelationships between the antimycotic activities and the content of polypheol have been calculated (R2 = 7586%). Some similar results have occurred while valuing the extracts of cork oak acorns against yeasts as Candida albicans, Candida glabrata and Saccharomyces cerevisae (Table 2). DISCUSSION Because of their wealth in food reserve, the acorns of the various species of oak have been consumed more or less everywhere in the world by all populations of the moderate zones, this use persisted, to the modern times the acorns constituting a complementary food then currently in the difficult periods (food of scarcity) the acorns are only used for the food of livestock. The soft acorn coffee gets ready in Spain; it constitutes an excellent beverage in the cases of the digestive difficulties. According to the catalog of the main medicinal plants, aromatic of the forest development institute (IDF), the oak to numerous medicinal virtues, the acorns in mash are efficient against the diarrheas, the coffee of acorns is invigorating. The acorns of cork oak are especially rich in metabolites when they are not attacked. The lipidic content is especially very important to the level of the almond; this content decreases eight times when the acorns are attacked strongly. The pericardic area also contains big quantities of lipids that vary according to the attack. The use of the acorns of Quercus spp. in the nutrition a long history has in the region of the Mediterranean. The acorns of oak have been used traditionally in medicine; they are a rich source of hydrates of carbon, amino acids, proteins, lipids and various sterols (Cup et al., 2005). Next to the food components, they contain various biologically active compounds (tannins, gallic and ellagic acidic and different derivative of hexahydroxydiphenoyl) that possess a antioxidant activity (Cantos et al., 2003; Chiou, 1989; Lee et al., 1992; Rakic', 2000; Rakic' et al., 2004, 2006). A lot of plants compounds are biologically active, antimicrobial, allopathic, antioxidants and have some properties bio-regulating. The extracts of plant are the most estimated as antioxidant and antimicrobial (Dorman et al., 2000). The last two decades the accent to be put on the antioxidants, antimicrobial and the natural insecticides from plants. The plant reign offers a large range of polyphenolic compounds polar and apolar that are antioxidants with properties redox, allowing them to act as donors of hydrogens (Pietta, 2000). The new discoveries of their biologic activities provide the basis to the interest of use of natural antioxidants and antimicrobial, as tools of biologic control against the insect. The tannins, a group of phenolic compounds is known to be responsible for the defense of the acorns against the insects and the predatory vertebrates (Fox, 1982; Weckerly et al., 1989; Steele et al., 1993; Smallwood et al., 2001; Vander Wall, 2001). In order to test the pharmacological effect of the extracts of cork oak acorns results showed that all extracts present an important activity facing all tested microorganismes. The extracts of the acorns by the different solvents are efficient against pathogenic bacteria, mildews, the phytopathogenes and the yeasts. This activity is correlated strongly to the polyphenolic content. Literature Cited Adjami, Y. 2009. Etat sanitaire des subraies du Nord-Est Algrien. Etudes des facteurs de dprissements du chne-lige (Quercus suber L.). Essais insecticides contre les 341

insectes du gland. Mmoire de magistre Universit de Annaba. p.145. Anderson, C. 1992. The effect of weevil and fungal attacks on the germination of Quercus robur acorns. Ecol. Manage 50:247257. Asmrfc. 1998. Actes du Sminaire Mditerrane sur la rgnration des forts de Chnelige, Tabarka 22-24 Octobre 1996, Annales de lINRGREF, N0 spcial p.259. Bakry, M., Sbay, H., Abourouh, M. and Satrani, B. 1999. Ractions de diffrentes provenances de chne-lige laction pathogne de Diplodia mutila. Integrated Protection in Oak Forests, IOBC wprs Bull. 22(3):1924. Bellal, W. 2008. Inventaire de lentomophone du chne-lige dans la subraies du Nordest Algrien, Mmoire de magistre Universit de Annaba. p.80. Bouchafra, A. and Fraval, A. 1991. Prsentation du chne-lige et de la subraie. In Villemant C.et Fraval A. : La faune du chne-lige. Actes Edition, Rabat, 126. Boudy, P. 1950. Economie forestire nord africaine. Tome 2(1): Monographie et traitements des essences forestires. Larousse, Paris. p.525. Bovey, P., Linder, A. and Mller, O. 1975. Recherches sur les insectes des chtaignes au Tessin (Suisse). Schweizerischen Zeitschrift fr Forstwesen 126:781820. Cantos, E., Espin, J., Carlos-Lopez, B.C., Delahoz, L., Ordonez, J.A. and TomasBarberan, F.A. 2003. Phenolic compounds and fatty acids from acorns (Quercus spp.), the main dietary constituent of free ranged Iberian pigs. J. Agric. Food Chem. 51:62486255. Chiou, J.W. 1989. The antioxidant activity and the chemical structure of selected components acorns and their potential use as inhibitors of milk oxidation. A Dissertation of Cornell University, USA. Coutin, R. 1960. Estimation de l'importance des populations d'imagos de Balanus elephas Gyll. dans une chtaigneraie cvenole. Revue de Zoologie Agricole et Applique 59:15. Dorman, H.J.D. and Deans, S.G. 2000. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88:308316. Fukumoto, H. and Kajimura, H. 2000. Seed-insect fauna of pre-dispersal acorns and acorn seasonal fall patterns of Quercus variabilis and Q. serrata. Central Japan. Entomological Science 2: 197203. Khaladi, A., Benjama, M.L. and Stiti, B. 1999. Les glands de chne-lige et leurs agents pathogne: essai de conservation et de lute. IOBC/Bull. 22(3):2936. Glodsworthy, G.J., Mordue, W. and Guthkelch, J. 1972. Studies on insect adipokinetic hormones. Genr. Compare. Endocrinol 18:545551. Lee, M.H., Jeong, J.H. and Oh, M.J. 1992. Antioxidative activity of gallic acid in acorn extract.Hanguk Yong yang Siklyong Hakhoechi, 21:693700 mridionales. Tome 1 .CNRS, Paris. p.565. NCCLS (National Committee for Clinical laboratory Standards). 1997. Performance standards for antimicrobial disk susceptibility test. 6th ed. Approved Standard. M2A6, Wayne, PA. Pietta, P.G. 2000. Flavonoids as antioxidants. J. Nat. Prod., 63:10351042. Raki, S. 2000. Effect of oak acorn extracts an lipid oxidation kinetics. J. Agri. Sci. Edited by Agricultural Faculty Belgrade. 45:139145. Raki, S., Maleti, R., Perunovi, M. and Svrzi, G. 2004. Influence of thermal treatment on tannin content and antioxidation effect of oak acorn Quercus cerris extract. J. Agri. Sci. Edited by Agricultural Faculty Belgrade. 49:97106. Rakic, S., Povrenovic D., Tesevic, V., Simic, M. and Maletic, R. 2006. Oak acorn, polyphenols and antioxidant activity in functional food. Journal of Food Engineering. 74:416423. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorizung assay. Free Radical Bio. Med. 26(9/10):12311237. Steele, M.A., Knowles, T., Bridle, K. and Simms, E.L. 1993. Tannins and partial consumption of acorns: implication for dispersal of oaks by seed predators. Am. Midl. 342

Nat. 130:229238. Smallwood, P.D., Steele, M.A. and Faeth, S.H. 2001. The ultimate basis of the caching preferences of rodents, and the oak-dispersal syndrome: tannins, insects, and seed germination. Am. Zool. 41:840851. Vander Wall, S.B. 2001. The evolutionary ecology of nut dispersal. Bot. Rev. 67:741 17. Weckerly, F.W., Sugg, D.W. and Semlitsch, R.D. 1989. Germination success of acorns (Quercus): insect and tannins. Can. J. Forest Res. 19:811815. Tables Table 1. The effect of different solvents on the free radical scavenging capacity of Quercus suber L. fruit extracts. Results of the ABTS+ assay are expressed as mg equivalent Vitamin C/g dry weight (means standard deviation of three measurements). Solvents Water Acetone Ethanol Chloroform Quercus suber 9.430.89a 15.661.08b 12.120.56c 8.190.33d

For each solvent, values in the same lane bearing different letters are significantly different at p 0.05 (at least). For each sample, values in the same column bearing different letters are significantly different at p 0.05 (at least).

Table 2. The antimicrobial activities of extracts abtained from infested acorns of Q. suber. Water Bacteria P.aeruginosa S. aureus E. faecalis E. coli Fungi P. digitalum P. italicum B. fabae B. cinerea A. fabae F. oxysporum G. candidum A. niger Yeasts C. glabrata C. albicans S. cerevisiae 21 23.8 22.6 20.4 14.2 16 14.2 15 15 14.8 12 12.3 14.4 16 16.8 Acetone 15.8 15 12.7 13.5 12 12.6 14.6 14 16.6 15.3 13.4 10.5 16 16.4 16.2 Ethanol 15 14 12.8 10.4 12.4 14.1 12 10.6 14.7 13.6 12.8 9.6 13 11 14 Chloroform 19 19 22.2 18.5 13.9 14 13.9 13 14.4 14.4 13 12.5 15 15.6 15

Results are expressed as diameter (mm) of inhibition zones minus diameter of inhibition zones of negative control as determined by the paper disc diffusion method. NA: not active.

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Figures

Fig. 1. Variations of the lipidic contents in of the healthy and infested acorns: in the almond (A) and in the pericardic area (B) (S: healthy; 1T: 1 hole; 2T: 2 holes).

Fig. 2. Antioxidant activity of the different extracts of the acorns pierced of Quercus suber determined by the -carotene bleaching test.

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