Journal of Applied Botany and Food Quality 87, 108 - 116 (2014), DOI:10.5073/JABFQ.2014.087.017 1Department of Life Sciences and Biotechnology (SVeB), University of Ferrara, Ferrara, Italy 2Promociòn de la investigatiòn, Universidad Estatal Amazònica, Puyo, Pastaza, Ecuador 3Centro de Investigatiòn y Valoración de la Biodiversidad “CIVABI”, Universidad Politecnica Salesiana, Wilson, Quito, Ecuador 4Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Biological and chemo-diverse characterization of Amazonian (Ecuador) Citrus petitgrains A. Guerrini1*, D. Rossi1, A. Grandini1, L. Scalvenzi2, P.F. Noriega Rivera3, E. Andreotti4, M. Tacchini1, A. Spagnoletti1, I. Poppi1, S. Maietti1, G. Sacchetti1 (Received November 20, 2013) * Corresponding author Summary Six Amazonian petitgrain samples from C. nobilis Lour., C. auran- tium L., C. limon L. and mixture of Citrus spp. (Rutaceae), named CN, CA, CL1, CL2, C1 and C2, were chemically characterized by GC-MS and 13C NMR and evaluated for antioxidant acitivity (DPPH and b-carotene bleaching tests), for antimicrobial properties (disk diffusion method) and for antifungal capacity (agar vapour assay). CN, C1, C2 samples evidenced the most interesting results: CN (g- terpinene/linalool chemotype: 14.3 % / 41.6 %, with a considerable amount of thymol: 9.0 %), and C1 (linalool, 18.3 %; sabinene, 11.6 %; thymol, 5.5 %), showed relevant antioxidant activity with both DPPH (IC50 = 3.52 and 5.48 mg/ml, respectively) and b-carotene (IC50 = 0.387 and 0.491 mg/ml, respectively). Antibacterial proper- ties of CN and C1 against P. mirabilis (MIC = 0.61 mg/ml for both) and B. subtilis (MIC = 0.61 and 0.44 mg/ml, respectively) were most probably due to thymol. C2 (geranial: 34.7 %, neral: 33.1 %) evidenced a valuable bioactivity against C. albicans (MIC = 0.44 mg/ml). The 50 % growth inhibition (IC50) of the dermatophytes T. mentagrophytes and N. cajetani was reached with amounts of C1, C2 and CN less than 4 ml/plate. Bioactivity of Amazonian Citrus spp. CN, C1 and C2 essential oils suggests their potential use as food preservatives or additives in cosmeceuticals as preventive against dermatophytic fungal infections. Introduction Herbs and spices have been employed since ancient times as flavour- ing and preservatives agents for food, but only in the last decade scientific research has focused its interest on essential oils and ex- tracts as natural sources of antimicrobial and antioxidant compounds, as safer alternative additives for food preservations (Burt, 2004; ShaaBan et al., 2012). While Citrus essential oils are usually cold-extracted with mechani- cal systems (as stated in European Pharmacopoeia, VII ed.), the so- called petitgrain oils are obtained by distillation of Citrus leaves, buds and small branches from Citrus spp. adult plants (Dugo et al., 2010). The composition of Citrus leaves essential oils are not as well de- fined as the correspondent peel oils (Dugo et al., 2010) and stu- dies concerning these aspects have been often reviewed (Lota et al., 2001a; Lota et al., 2001b; Lota et al., 2002; Dugo et al., 2010). Due to its pleasant and characteristic fragrance, bitter orange (sour orange, bigarade) petitgrain is the most important and appreciated among leaf essential oils: it is widely used in perfumery for prepa- ration of eau de Colognes, lotions and soaps because of its good resistance to alkaline medium. Sour orange plants are cultivated mainly in the Mediterranean countries (France, Italy, Spain) and in Paraguay (Lota et al., 2001a; Dugo et al., 2010). The best known and most employed species is Citrus aurantium L. Other Citrus spp. petitgrains (lemon, mandarin, etc.) are afforded at small quantities. Lemons (Citrus limon in particular) are cultivated in Mediterranean countries, southern California and Argentina while mandarins (other several Citrus spp. and hybrids) are cultivated in Mediterranean countries, Japan, Brazil, Argentina, United States (known as tange- rines) and Australia (Lota et al., 2000; Dugo et al., 2010). Although there is extensive literature on Citrus spp. petitgrain com- position, very few papers concern biological properties or report correlations among phytochemical data, chemodiversity and bio- logical activities (Dugo et al., 2010; ShaaBan et al., 2012). In fact it is known that the wide chemodiversity which characterizes this kind of phytocomplexes could be due to several variables, affect- ing the chemical profile, as geographical origin, time of collection and cultivars (guerrini et al., 2011). These variables can determine chemical diversities in essential oils obtained from the same species which necessarily could reflect different bioactivities and possible functional uses. In light of these premises, in Ecuadorian traditional ethnomedi- cine Citrus spp. leaves are used to treat stomachaches (rioS et al., 2007). The present paper represents the first report about Citrus spp. petit- grain essential oils from plants grown at the margin of the Amazonian forest (Ecuador). The aim is to evaluate possible, distinctive bio- activity properties of chemotypes driven due to this peculiar geo- graphical origin. In fact, it is known that the Amazonian biodiverse hot-spot is characterized by an biotic and abiotic set of conditions which can force the secondary plant metabolism to peculiar and unique profiles with corresponding interesting bioactivities (ryDer WiLke et al., 2010; roSSi et al., 2013). Moreover, the ethnobotanical use of Citrus spp. leaves is sometimes performed without particular attention to the single species since the shape is similar and they are collected without distinction. This fact contributes to have prepara- tions with mixed-species leaves (mainly as flavouring and anxiolytic agents) (hanazaki et al., 2000). In the present work, the study of es- sential oils obtained from mixture of Citrus leaves would mime this ethnobotanical evidence. Therefore, different Amazonian Citrus spp. petitgrains from single and mixed species leaves were evaluated for their chemical composition through GC, GC-MS and 13C NMR in order to point out the possible chemodiversity aspects related to their geographical origin and to check for functional properties (antimi- crobial, antifungal, antioxidant) to valorize their possible applicative uses in food and/or health fields. Material and methods Chemicals All the solvents employed for chemical analyses and bioassays were chromatographic grade. Solvents and pure compounds were all pur- chased from Sigma-Aldrich Italy (Milano, Italy). Thymus vulgaris essential oil, thymol-chemotype employed as reference phytocom- plex (Sacchetti et al., 2005), was purchased from Extrasynthese (Genay, France). Microbial culture media were obtained from Oxoid Italia (Garbagnate, Italy). Amazonian Citrus petitgrains 109 Plant material C. nobilis (named CN), C. aurantium (named CA), C. limon (named CL1 and CL2) fresh leaves and fresh leaves mixture of genus Citrus spp. (named C1 and C2) were purchased by Fundacion Chankuap (Quito, Ecuador), non-governmentive organization which has as main target the valorization of Amazonian sources recovering plant material to directly obtain commercial products from natives, with the cooperation of our research about Ecuadorian Amazonian bio-about Ecuadorian Amazonian bio- diversity. For what concerns the essential oil mixtures, no informa- tion have been given by Fundacion Chankuap regarding the different Citrus species employed and their quantitative ratio. Leaves were collected in September 2010 from wild adult plants growing in three different locations on the outskirts of the Wasak’entsa reserve in eastern Ecuador (77°.15” W/2°.35” S) and positively identified by Fundacion Chankuap (Quito, Ecuador). Dried specimens were de- posited at the Department of Biology and Evolution, University of Ferrara, Code C1, C2, CA1, CN1, CL1, CL2. Essential oils isolation Essential oils were in situ extracted for 8 hours through steam distil-8 hours through steam distil- lation of C. limon, C. aurantium, C. nobilis, mixture of Citrus spp. fresh leaves (approximately 10 kg) using a mobile essential oil dis-(approximately 10 kg) using a mobile essential oil dis-10 kg) using a mobile essential oil dis- tiller (Essential Oil Company, Portland, OR, USA) set up follow-Essential Oil Company, Portland, OR, USA) set up follow- ing the parameters reported in literature (horWitz, 2003). Essential oil yields have been achieved through three different distillations of fresh plant material belonging to Citrus spp. The petitgrains were dried over anhydrous sodium sulfate and stored in airtight glass vials with Teflon-sealed caps at -18.0±0.5 °C in the absence of light until analysis. Gas Chromatography Essential oil samples were analyzed and the relative peak areas for individual constituents averaged. The relative percentages were de- termined using a ThermoQuest GC-Trace gas-chromatograph equip- ped with a FID detector and a Varian FactorFour VF-5ms poly-5 % phenyl-95 %-dimethyl-siloxane bonded phase column (i.d., 0.25 mm; length, 30 m; film thickness, 0.15 μm). Operating conditions were as follows: injector temperature 300 °C; FID temperature 300 °C, carrier (Helium) flow rate 1 ml/min and split ratio 1:50. Oven tem- perature was initially 55 °C and then raised to 100 °C at a rate of 1 °C/min, then raised to 250 °C at a rate of 5 °C/min and finally held at that temperature for 15 min. One μl of each sample dissolved in CH2Cl2 was injected. The percentage composition of the oils was computed by the normalization method from the GC peak areas, without using correction factors. Gas Chromatography-Mass Spectrometry Essential oil constituents were then analyzed by a Varian GC-3800 gas chromatograph equipped with a Varian MS-4000 mass spectro- meter using electron impact and hooked to NIST library. The conditions were the same reported for GC analysis and the same column was used. The MS conditions were as follows: ionization voltage, 70 eV; emission current, 10 μAmp; scan rate, 1 scan/s; mass range, 29-400 Da; trap temperature, 150 °C, transfer line tempera- ture, 300 °C. The constituents of the volatile oils were identified by comparing their relative retention time, KI and the MS fragmenta- tion pattern with those of other essential oils of known composition, with pure compounds and by matching the MS fragmentation pat- terns and retention indices with the above mentioned mass spectra libraries and with those in the literature (aDamS, 2007). In order to determine the Kovats index of the components, a C8-C22 n-alkanes (Sigma-Aldrich) was added to the essential oil before injecting in the GC–MS equipment and analyzed under the same conditions as above. NMR spectroscopy 13C NMR spectra were recorded at 100.58 MHz and at temperature of 303 K with a Varian Gemini-400 spectrometer. The essential oils were dissolved in CDCl3 (70 mg/0.8 mL) into a 5 mm NMR and solvent signal was used for spectral calibration (central line of tri- plet at 77.0 ppm). Chemical shifts (ppm) and peak attribution were based on comparisons of the resonances in 13C NMR spectrum of the essential oil with those of pure standards and mixture of these (a-pinene, sabinene, b-pinene, D-limonene, g-terpinene, linalool, citronellal, 4-terpinenol, citral, thymol) present in our spectral li- brary (guerrini et al., 2006) or according with those of literature (kuBeczka, 2002), SDBS (Saito et al., 2009). Biological activities Antioxidant, antifungal and antimicrobial activities were performed comparing all the data with those obtained with appropriate pure synthetic compounds and/or commercial Thymus vulgaris essential oil, in order to have positive control references with single com- pounds or comparable phytocomplexes reputed for their functional bioactivities. The use of a phytocomplex known for its chemical and biological properties (e.g. thyme essential oil) as a positive reference results particularly indicative of the real functional efficacy of a test- ed extract (maietti et al., 2013). Data reported for each assay are the average of three determinations of three independent experiments. Antifungal and antimicrobial strains According to previously described methodology (guerrini et al., 2006; maietti et al., 2013), Citrus petitgrain antifungal and anti- microbial activities were performed with agar vapor method and standard disk diffusion technique respectively. For antibacterial assays, Gram-positive (Enterococcus foecalis ATCC 29212, Staphylococcus aureus ATCC 29213 and Bacillus subtilis ATCC 7003) and Gram-negative (Escherichia coli ATCC 4350, Proteus mirabilis ATCC 29852 and Klebsiella oxytoca ATCC 29516) bacterial strains were employed. Antifungal activity was as- sessed on yeast Candida albicans ATCC 48274, on phytopathogen strains (Botrytis cinerea Micheli ATCC 48339, Pythium ultimum Trow, kindly supplied by Prof. G. D’Ercole (Institute of Vegetal Pathology, University of Bologna, Italy), Magnaporthe grisea ATCC 64413) and dermatophyte strains (Trichophyton mentagrophytes var. mentagrophytes (Robin) Blanchard CBS (Centraal Bureau Voor Schimmelcultures, Baarn, the Netherlands) 160.66, Nannizzia cajetani Ajello IHME (Institute of Hygiene and Epidemiology- Mycology (IHME) Brussels, Belgium) 3441 and Trichophyton ru- brum (Castellani) Sabouraud IHME 4321). Antimicrobial activity: disks diffusion method Mother cultures of each bacteria were set up 24 h before the as- says in order to reach the stationary phase of growth. The tests were assessed by inoculating from the mother cultures Petri dishes with proper sterile media with the aim of obtaining the microorganisms concentration 106 CFU/ml. For bacteria, aliquots of dimethyl sul- foxide (DMSO) were added to the essential oils in order to obtain a 0.01-50.0 mg/ml concentration range and then deposited on sterile paper disk (6 mm diameter, Difco). Bioactivity against the yeast Candida albicans was also processed. Mother cultures were set up inoculating 100 ml YEPD liquid me- dium (Yeast Extract and Potato Dextrose) in 250 sterile flasks and 110 A. Guerrini, D. Rossi, A. Grandini, L. Scalvenzi, P.F. Noriega Rivera, E. Andreotti, M. Tacchini, A. Spagnoletti, I. Poppi, S. Maietti, G. Sacchetti incubated in the dark at 30 °C in order to assess growth curves. From each mother cultures at the stationary phase of growth, broth dilu- tions were made to obtain the strain concentration of 105 CFU/ml to inoculate Petri dishes with agarized YEPD for bioassays. Then, 10 μl of DMSO-essential oil sample solutions were prepared in or- der to have an assay range 0.01-50.0 mg/ml, and then deposited on sterile paper disk (6 mm diameter, Difco). The Petri dishes were suc- cessively incubated at 30 °C in the dark and checked for evaluat- ing the growth inhibition after 48 h. both for bacteria and Candida streams, the lowest concentration of each essential oil showing a clear zone of inhibition was taken as the MIC (Minimum Inhibitory Concentration). Negative controls were set up with 10 μl of DMSO in the test solution, while positive ones were assessed with T. vul- garis essential oil. Antifungal activity: agar vapour assay Biological activity of Citrus petitgrains against three phytopatho- genic and three dermatophytic fungi was performed by using the agar vapour method (maietti et al., 2013). They were grown in Petri plates (90 mm) supplemented with 15 ml/plate of potato dextrose agar, inoculated with 6 mm plugs from stationary-phase cultures. The plates were then incubated for 24 h at 26 ± 1 °C. Successively, sterilized filter paper discs (diameter 9.0 mm) were absorbed with different volumes of Citrus petitgrains samples ranging from 0.20 to 25.00 ml, and placed inside the upper lid of each plate. Plates were kept in an inverted position, tightly sealed with parafilm, and incu- bated for 7 days at 26 ± 1 °C. Blanks served as a negative control. Commercial T. vulgaris essential oil was prepared as described above for petigrain samples, with volumes ranging from 0.20 to 25.00 ml, and considered as a positive control reference phytocomplex. Three replicates were made for each treatment. After 7 days the results were determined as the inhibition of radial growth and expressed as the amount of essential oil that led to 50 % inhibition of growth in each fungal strain (IC50). Antioxidant activities Radical scavenging and antioxidant properties of essential oils were performed through different assays, namely the DPPH assay and the b-carotene bleaching test according to previously described methods. This approach permits the antioxidant effectiveness of an essential oil to be more carefully defined, as it is almost impossible to express the antioxidant activity as an absolute value that is universally recog- nizable, besides being expressed by only one type of assay (maietti et al., 2013). T. vulgaris essential oil was used as positive controls. Essential oils antioxidant activity was considered as the IC50, calcu- lated from inhibition curves obtained by plotting the % inhibition against oil concentration. All the data collected for each assay are the average of three determinations for three independent experiments. Statistical analysis Relative standard deviations and statistical significance (Student’s t test; P < 0.05), one-way ANOVA and LSD post hoc Fisher’s honest significant difference test, were given, where appropriate, for all data collected. All computations were made using the statistical software STATISTICA 6.0 (StatSoft Italia srl). Results Chemical fingerprinting Steam distillation of Citrus spp. leaves provided petitgrains with yield from 0.20 ± 0.02 g/100g for CN to 0.29 ± 0.03 g/100g for CL1 and density that covered a range of 0.82-0.92 g/ml (Tab. 1). The Ecuadorian CA petitgrain exhibited as major components sa- binene (38.3 %), trans-E-ocimene (6.7 %), linalool (8.8 %): other minor components were 3-carene (8.9 %), D-limonene (7.9 %), b- myrcene (3.4 %), 4-terpinenol (2.5 %), a-pinene (1.9 %), geranial (1.9 %), b-pinene (1.8 %). CL1 essential oil evidenced an high abun- CL1 essential oil evidenced an high abun- dance of limonene (52.7 %) and linalool (15.1 %) and as minor com- pounds citronellal (3.1 %), sabinene (2.7 %) and carvone (2.6 %), instead in CL2 petitgrain predominated sabinene (36.1 %) followed by limonene (24.1 %), linalool (4.7 %), 4-terpinenol (3.9 %), g-ter- pinene (3.9 %), citronellal (3.6 %), trans-b-ocimene (3.2 %), a- terpinene (2.8 %), b-myrcene (2.6 %). Citrus nobilis petitgrain evi-evi- denced high abundance of linalool (41.6 %) and appreciable contents of γ-terpinene (14.3 %) and thymol (9.0 %), followed by trans-E- ocimene (10.9 %), p-cymene (4.1 %), a-pinene (3.6 %), b-pinene (3.1 %) limonene (2.8 %) as minor compounds. Finally, linalool (18.3 %), sabinene (11.6 %), limonene (11.1 %), γ-terpinene (10.6 %), thymol (5.5 %), b-pinene (4.9 %), trans-E-ocimene (4.8 %) and p-cymene (3.4 %) were the most characteristic compounds for C1 petitgrain samples; C2 petitgrain, instead, evidenced geranial (34.7 %) and neral (33.1 %) followed by b-myrcene (5.4 %), lina- lool (4.7 %), and geraniol (3.1 %) as the most abudant chemicals (Tab. 1). To contribute to define a metabolomic fingerprinting of Citrus spp. essential oils, 13C-Nuclear Magnetic Resonance (NMR) of the most abundant chemical standard compared with whole essential oil spec- trum was performed confirming the evidences emerged by GC-MS (supplementary materials, Tab. 5 and Fig. 1). Mono-dimensional 13C spectrum revealed typical and numerous diagnostic signals for characterizing the chemical makeup of carbons and, therefore, of the functional groups typical of the examined molecules. Antioxidant activities The essential oils examined evidenced interesting antioxidant pro- perties with sligthly different among the samples (Tab. 2). In parti- cular, CN petitgrain exhibited a good radical antioxidant activity both with DPPH test (IC50 = 3.52 ± 0.25 mg/ml) and b-carotene bleaching assay (IC50 = 0.387 ± 0.021 mg/ml). These results are particularly rel- evant if compared to that obtained with commercial Thymus vulgaris essential oil (IC50 = 1.24 ± 0.10 mg/ml), taken as reference phytocom- plex (Sacchetti et al., 2005). C1 petitgrain, that showed a relative abundance of g-terpinene (10.6 %) and thymol (5.5 %), probably to relate to the interesting DPPH activity (IC50 = 5.48 ± 0.45 mg/ ml), displayed instead a lower activity in b-carotene bleaching test (IC50 = 0.491 ± 0.041 mg/ml). Thymol has been tested as pure com- pound in DPPH and b-carotene bleaching tests suggesting that the antioxidant capacity displayed by essential oils could be mainly due to the presence and the abundance of this substance. Antimicrobial activity Evaluation of antibacterial activity (Tab. 3), expressed as MIC (Minimun Inhibitory Concentration), revealed that CN petitgrain was the most active among the Citrus spp. phytocomplexes against Gram-negative as well as Gram-positive bacteria. The most interest- ing results were against P. mirabilis for CN, C1 and CA, against B. subtilis for CN and C1 and finally against E. coli for CN and CA since MICs were comparable. The antibacterial properties of CN petitgrain were relevant also against S. aureus and E. foecalis (0.78 ± 0.08 and 0.95 ± 0.09 mg/ml) if compared to the positive control T. vulgaris. No remarkable inhibition activity was observed against K. oxytoca. C2 petitgrain was instead particularly active against the yeast C. al- bicans, with a MIC of 0.44 ± 0.05 mg/ml. Amazonian Citrus petitgrains 111 Tab. 1: Chemical composition of Citrus petitgrains Compound KIa RAb CN CA CL1 CL2 C1 C2 α-Thujene 930 1.8 0.5 0.2 1.0 1.1 0.1 α-Pinene 939 3.6 1.9 0.7 2.7 2.7 0.2 Sabinene 977 0.4 38.3 2.7 36.1 11.6 0.6 β-Pinene 979 3.1 1.8 0.9 3.4 4.9 0.2 6-Methyl-5-hepten-2-one 986 - - 0.3 0.1 - 1.9 β-Myrcene 991 0.7 3.4 0.3 2.6 1.1 5.4 α-Phellandrene 1003 0.1 0.6 - 0.2 - - p-Mentha-1(7),8-diene 1004 - - - - 0.6 - 3-Carene 1009 - 8.9 - - - - α -Terpinene 1017 0.4 1.0 0.3 2.8 0.4 - p-Cymene 1025 4.1 0.5 1.9 0.3 3.4 0.5 D-Limonene 1029 2.8 7.9 52.7 24.1 11.1 0.7 1,8-Cineole 1031 - - - 0.2 0.6 - cis-Z-Ocimene 1031 0.8 0.2 - 0.8 0.4 0.4 trans-E-Ocimene 1037 10.9 6.7 - 3.2 4.8 0.6 γ-Terpinene 1051 14.3 1.6 0.3 3.9 10.6 0.6 cis-Sabinene hydrate 1060 0.1 0.3 0.4 0.8 0.1 - trans-Linalool oxide 1073 0.1 - 1.5 - - - cis-Linalool oxide 1087 - - 1.6 - - - Isoterpinolene 1088 - 0.3 - - - - Terpinolene 1089 1.6 1.7 0.5 0.8 0.9 0.2 p-Cymenene 1091 0.9 - - - 0.4 0.2 Linalool 1097 41.6 8.8 15.1 4.7 18.3 4.7 1,3,8-p-Menthatriene 1110 0.3 - - - - 0.3 cis-p-Ment-2-en-1-ol 1122 - 0.1 0.7 - - - cis-Limonene oxide 1138 - - 0.5 - - - trans-p-Ment-2-en-1-ol 1141 - - 0.8 - - 0.3 trans-Limonene oxide 1142 - - - - 0.1 0.5 Isopulegol 1150 - - 0.2 0.1 0.8 - Citronellal 1153 - 1.4 3.1 3.6 0.8 0.3 cis-Linalyl oxide 1174 - - 0.2 - - - trans-Linalyl oxide 1176 - - 0.1 - - - 4-Terpinenol 1177 0.2 2.5 0.7 3.9 0.6 0.2 α-Terpineol 1189 0.2 0.6 0.5 0.3 0.4 0.7 cis-Dihydrocarvone 1193 - - 0.6 - - - trans- Dihydrocarvone 1201 - - 0.3 - - - trans-Carveol 1217 - - 1.4 - - - Citronellol 1226 - 0.3 0.5 0.7 - 0.3 cis-Carveol 1229 - - 0.6 - - - Nerol 1230 - 0.3 - 0.7 - - Neral 1238 - 1.6 0.1 0.2 33.1 Carvone 1243 - - 2.6 - - - Geraniol 1253 - 0.1 - - - 3.1 Geranial 1267 - 1.9 - 0.1 0.3 34.7 Perillaldehyde 1272 - - 0.4 - - 1.0 112 A. Guerrini, D. Rossi, A. Grandini, L. Scalvenzi, P.F. Noriega Rivera, E. Andreotti, M. Tacchini, A. Spagnoletti, I. Poppi, S. Maietti, G. Sacchetti Compound KIa RAb CN CA CL1 CL2 C1 C2 Citronellyl formate 1274 - - 0.9 - - - 2-Undecanone 1294 - - - - - 1.3 Geranyl formate 1298 - - 0.5 - - 0.4 Carvacrol 1299 - - - - - - δ-Elemene 1338 - - - 0.1 - - Citronellyl acetate 1353 - 0.3 0.9 0.2 0.1 - Neryl acetate 1362 - 0.5 0.8 0.2 0.1 - Geranyl acetate 1381 - 0.4 - - - 0.4 β-Elemene 1391 - 1.4 - - 0.1 - Methyl methylantranilate 1406 0.3 0.2 - - 13.1 3.1 trans-β-Caryophyllene 1419 0.9 0.8 - 0.6 1.3 0.2 cis-Carvyl propanoate 1422 - - 0.6 - - - γ-Elemene 1433 - - - - - - trans-α-Bergamotene 1435 - - - - - 0.2 α-Humulene 1455 0.1 0.4 - - 0.2 - β-(E)-Farnesene 1457 - 0.2 - - - - 2-Tridecanone 1470 - - - - - 0.5 Bicyclogermacrene 1500 0.2 0.2 - 0.3 0.4 - Germacrene A 1509 - 0.7 - - 0.2 - Germacrene B 1561 - - - 0.1 - - Spathulenol 1578 - - - - 0.1 - Caryophyllene oxide 1583 - - 0.5 - - - 1-Methoxy-9(E)-octadecen 1651 - - - - 0.7 - β-Sinensal 1700 - 0.6 - - - - α-Sinensal 1757 0.3 0.1 - - 0.2 - TOTAL IDENTIFIED 99.8 99.0 96.7 98.7 98.8 98.9 Extraction yield (g/100g) 0.20±0.02 0.23±0.01 0.29±0.03 0.29±0.01 0.23±0.01 0.24±0.03 a Arithmetic indices calculated on a Varian VF-5ms column b Relative peak area calculated by GC-FID. The major components (bold letters) of samples were identified by 13C NMR Tab. 2: Antioxidant activity of Citrus petitgrains performed by DPPH, β-carotene bleaching assays and compared to commercial Thymus vulgaris essential oil and pure compound thymol. IC50± SD (mg/ml) Essential oils DPPH β-carotene bleaching CN 3.52 ± 0.25 0.387 ± 0.021 CA 7.12 ± 0.50 0.432 ± 0.037 CL1 9.90 ± 0.71 0.986 ± 0.088 CL2 7.45 ± 0.61 0.521 ± 0.038 C1 5.48 ± 0.45 0.491 ± 0.041 C2 8.41 ± 0.71 0.788 ± 0.066 Thymol 0.60 ± 0.05 0.09 ± 0.011 Thymus vulgaris 1.24 ± 0.10 0.164 ± 0.013 Antifungal activity The most interesting results concerning antifungal activities (Tab. 4) were exhibited by C2 petitgrain, particularly against dermatophytes species (T. mentagrophytes, N. cajetani), that showed IC50 less than 0.20 ml/plate, comparable to positive control T. vulgaris essential oil: however, the concentrations corresponding to the 100 % growth in- hibition was better for C2. This essential oil was the most active also against phytopathogens, but less active than the reference standard T. vulgaris. C1 and CN petitgrains also showed good activity against all tested fungi reaching values of 50 % inhibition at concentration comprised from 2 to 8 ml/plate. The most sensitive fungal strain, however, ap- pears to be T. rubrum. The study of activity of citral (mixture of neral/geranial) standard, the most abundant component in C2, against phytopathogens and dermatophytes, confirmed the best activities against T. mentagro- phytes, N. cajetani, T. rubrum: in particular, T. mentagrophytes was the most sensitive fungus since it evidenced 50 % inhibition at con- centration less than 0.20 ml/plate. Amazonian Citrus petitgrains 113 Tab. 3: Antimicrobial activity of Citrus petitgrains compared to commercial Thymus vulgaris essential oil, thymol and citral. MIC (mg/mL) ± SD Essential oils Gram-positive bacteria Gram-negative bacteria Yeast S. aureus B. subtilis E. foecalis K. oxytoca E. coli P. mirabilis C. albicans ATCC29213 ATCC7003 ATCC29212 ATCC29516 ATCC4350 ATCC29852 ATCC48274 CN 0.78 ± 0.08 0.61 ± 0.05 0.95 ± 0.09 2.17 ± 0.22 0.61 ± 0.06 0.61 ± 0.05 1.74 ± 0.19 CA 8.46 ± 0.64 1.52 ± 0.16 1.06 ± 0.11 3.38 ± 0.35 0.68 ± 0.07 0.68 ± 0.07 3.38 ± 0.34 CL1 4.06 ± 0.41 3.79 ± 0.37 1.99 ± 0.19 8.12 ± 0.61 6.32 ± 0.62 4.06 ± 0.41 0.88 ± 0.09 CL2 3.50 ± 0.31 1.73 ± 0.17 3.89 ± 0.38 6.05 ± 0.55 3.89 ± 0.38 3.89 ± 0.37 3.46 ± 0.33 C1 1.10 ± 0.12 0.44 ± 0.04 1.93 ± 0.18 4.38 ± 0.42 2.19 ± 0.22 0.61 ± 0.06 1.75 ± 0.18 C2 7.94 ± 0.59 1.94 ± 0.18 3.97 ± 0.37 6.17 ± 0.41 1.76 ± 0.17 3.97 ± 0.39 0.44 ± 0.08 thymol 0.31 ± 0.06 0.28 ± 0.03 0.52 ± 0.05 1.10 ± 0.21 0.29 ± 0.03 0.31 ± 0.02 0.90 ± 0.07 citral 0.50 ± 0.09 0.30 ± 0.07 0.58 ± 0.06 0.20 ± 0.03 0.25 ± 0.05 0.70 ± 0.07 0.42 ± 0.04 T. vulgaris 0.11 ± 0.01 0.11 ± 0.01 0.11 ± 0.01 0.40 ± 0.04 0.06 ± 0.01 0.12 ± 0.01 0.06 ± 0.01 Tab. 4: Antifungal activity of Citrus spp. petitgrains compared to commercial T. vulgaris essential oil, citral and thymol. Samples Phytopathogens Dermatophytes P. ultimum M. grisea B. cinerea N. cajetani T. mentagrophytes T. rubrum var. mentagrophytes CN 4.0 ± 0.2 6.3 ± 0.4 3.4 ± 0.3 3.7 ± 0.2 3.6 ± 0.2 2.7 ± 0.2 CA 12.7 ± 0.9 16.1 ± 1.1 15.3 ± 1.2 9.3 ± 0.9 >25 14.2 ± 1.2 CL1 8.3 ± 0.7 >25 20.7 ± 1.5 >25 19.9 ± 1.8 12.0 ± 1.1 CL2 10.0 ± 1.0 >25 18.6 ± 1.5 17.6 ± 1.9 18.3 ± 1.7 16.7 ± 1.4 C1 3.2 ± 0.4 7.9 ± 0.5 6.2 ± 0.4 6.7 ± 0.7 4.8 ± 0.3 2.0 ± 0.2 C2 2.2 ± 0.2 2.4 ± 0.3 1.9 ± 0.2 <0.20a <0.20b 1.9 ± 0.1 T. vulgaris 0.40 ± 0.10 0.38 ± 0.08 0.23 ± 0.04 <0.20c <0.20a <0.20c Citral 1.2 ± 0.2 1.3 ± 0.1 1.4 ± 0.1 0.44 ± 0.02 <0.20b 0.88 ± 0.05 Thymol 1.1 ± 0.1 2.2 ± 0.3 1.6 ± 0.2 1.1 ± 0.2 1.2 ± 0.4 0.8 ± 0.2 All the values are expressed as IC50 (ml/plate) ± standard deviation a100% growth inhibition at concentration of 2.0 ml/plate b100% growth inhibition at concentration of 1.0 ml/plate c100% growth inhibition at concentration of 5.0 ml/plate Discussion The purpose of the current study was to compare the chemical com- position of Amazonian Citrus spp. leaves essential oils with those reported in literature to determine possible different chemotypes and biological activities with the final aim to valorize their commercial use. The distillaton yields were average values among those reported for CA (BLanco tiraDo et al., 1995; Lota et al., 2001a) and CN (Lota et al., 2001b); instead for CL petitgrains were lower than those re-, 2001b); instead for CL petitgrains were lower than those re- ported for other lemon species (Vekiari et al., 2002). The Ecuadorian CA petitgrain was comparable to an atypical sa- atypical sa- binene/trans-E-ocimene chemotype, as previously reported (Lota et al., 2001b). CL1 petitgrain exhibited an atypical composition with high abundance of limonene (52.7 %) and linalool (15.1 %), as re- ported for the Meyer cultivar (C. meyeri) (Lota et al., 2002), CL2 petitgrain could be defined as limonene (24.1 %)/sabinene (36.1 %)/ linalool (4.7%) chemotype, standing out the most common lemon chemotype characterized by limonene (17.8-33.5 %), a-pinene (10.5-25.1 %), geranial (8.6-22.6 %), neral (5.9-16.1 %) (Lota et al., 2002). C. nobilis petitgrains (CN) evidenced a γ-terpinene/lina- lool chemotype, because of high abundance of linalool (41.6 %) and appreciable contents of γ-terpinene (14.3 %) and thymol (9.0 %). A similar chemotype was pointed out in a systematic research on petitgrains derived from 58 Corsical mandarin cultivars from dif- ferent species and 41 cultivars belonging to C. reticulata Blanco (Lota et al., 2000; Lota et al., 2001b). Mandarin leaves essential oil composition from plants of different geographical origins, Floridian C. tangerine Hort. ex Tan and Israelian C. reticulata, evidenced high content of linalool, thymol and γ-terpinene (attaWay et al., 1967; FLeiSher and FLeiSher, 1990; FLeiSher and FLeiSher, 1991), while Colombian C. reticulata petitgrain was characterized by an high abundance of linalool (52.66 %), but less content of γ-terpinene (1.95 %) (BLanco tiraDo et al., 1995). The chemical composition of Citrus spp. leaves essential oils (C1 and C2) does not allow to deduce any consideration about the spe- cies employed and their abundance, but it is an important starting point in making suggestions about the comparison between the bio- 114 A. Guerrini, D. Rossi, A. Grandini, L. Scalvenzi, P.F. Noriega Rivera, E. Andreotti, M. Tacchini, A. Spagnoletti, I. Poppi, S. Maietti, G. Sacchetti activities of the mixtures and the other essential oils. However, the studies concerning plants growing in Amazonia are particularly interesting since the Amazonian basin is one of the most important biodiversity hotspots where the ecological conditions and high density and diversity of species per unit area drive the plant sec- ondary metabolism to biosynthetic pathways which are particularly rich in different chemical structures (ryDer WiLkie et al., 2010; roSSi et al., 2013). This aspect could explain the slight differences in chemical composition detected for the essential oils, with particular reference to those belonging to C. limon (CL) samples. The confirmation of the gas chromatographic results by NMR ex- periments suggests this spectroscopic technique as suitable for the identification, quality control, or fraud detection of essential oils providing their good and fast discrimination. Moreover, these kinds of evidences reinforce the role of non-chromatographic approach as potential tool to discriminate chemotypes, cultivar and hybrids as already suggested elsewhere (Lota et al., 2001b; guerrini et al., 2006; guerrini et al., 2011). All these chemical profiles obtained through GC-MS and confirmed by NMR spectroscopy, evidence that Amazonian biodiversity does not induce strong chemodiversity among Citrus spp. petitgrains examined, if compared to what related literature reports, even if interesting differences regarding minor compounds were found. The examined essential oils evidenced that CN petigrain revealed the highest antioxidant activity, if compared to results obtained with commercial T. vulgaris essential oil, taken as reference phyto- complex (Sacchetti et al., 2005), C1 petitgrain, with relative abundance of g-terpinene (10.6 %) and thymol (5.5 %), showed also interesting data. The antioxidant capacity displayed by essential oils could be mainly due to the presence and the abundance of thymol, as experimental results evidenced. However, with particular reference to CN sample, the relevant abundance of g-terpinene (14.3 %) could be also suggested as responsible of this biological property (choi et al., 2001), together with the presence of thymol (9.0%) (ruBerto and Baratta, 2000), as well as methyl-N-methylanthranilate (13.1 %) (eL-ghoraB et al., 2003). Tab. 5: Chemical shifts (13C) of compounds in Citrus spp. petitgrains Compound Chemical shift 13C α-Pinene 144.5/116.0/47.0/40.7/38.0/31.4/31.2/26.3/23.0/20.9 Sabinene 154.5/101.5/37.6/32.6/30.1/29.0/27.9/19.8/19.7/16.0 β-Pinene 152.2/105.9/51.6/40.7/40.5/27.0/26.1/23.5/21.8 β-Myrcene 146.2/139.0/131.9/124.3/115.7/113.1/31.6/26.7/25.6/17.6 3-Carene 131.4/119.4/28.3/23.7/20.8/18.4/16.8/16.6/13.2 p-Cymene 145.9/135.1/129.0/126.2/33.7/24.1/20.9 D-Limonene 150.3/133.8/120.6/108.3/41.0/30.8/30.6/27.9/235/20.8 trans-E-Ocimene 1415/133.7/122.1/110.6/ 27.3/25.7/17.7/11.6 γ-Terpinene 140.6/131.2/118.9/116.0/34.5/31.6/27.5/23.0/21.3 Linalool 145.0/132.0/124.3/111.7/73.5/42.0/27.9/25.7/22.9/17.7 Citronellal 202.7/131.8/124.0/51.0/36.9/27.8/25.7/25.4/19.9 4-Terpinenol 133.8/118.4/71.7/36.8/34.6/30.8/27.0/23.3/16.8 Neral 190.9/163.9/133.7/128.6/122.2/32.6/27.0/25.6/25.1/17.7 Geraniol 139.5/131.4/124.1/123.6/59.4/39.4/26.3/25.7/17.6/16.2 Geranial 191.4/163.9/132.9/127.4/122.5/40.6/25.7/25.6/17.7/17.6 Thymol 152.5/136.6/131.8/126.2/121.5/116.0/26.7/22.7/20.9 Methyl 169.0/151.2/134.7/131.8/115.4/110.6/51.5/29.1 methylantranilate Fig. 1: 13C spectra of Citrus spp. petitgrains CN petitgrain was the most effective against all the bacteria strains: MIC values of CA and C1 samples were instead lower and compara- ble. The amounts of thymol in CN (9.0 %) and C1 (5.5 %) petitgrains could be one of the possible reasons for the antibacterial activity (Burt, 2004). C2 petitgrain was instead particularly active against the yeast C. albicans, with a MIC of 0.44 ± 0.05 mg/ml probably due to the high abundance of neral (33.1 %) and geranial (34.7 %), pre- viously described as anti-Candida spp. agents (SiLVa et al., 2008) and confirmed by our results. Trying to relate antimicrobial activity with chemical data, thymol has been assayed as pure compound, but no remarkable results were obtained. However, it should be stressed that higher antibacterial capacity of thyme essential oil than that of thymol could be due to a sinergic interaction involving more chemi- cals, thymol included. This suggestion plays certainly a role in the activities displayed by petitgrains. The most interesting results concerning antifungal activities (Tab. 4) were exhibited by C2 petitgrain due to the high abundance of citral, as confirmed by experimental data. The good activities of C1 and CN petitgrains could be explained with the relative abundance of thy- mol, tested by us as pure compound and previously described as anti- fungal agent in vitro and in vivo against dermatomycoses (SokoVic et al., 2008). Finally, the particular interesting bioactivity of the es- sential oil mixtures confirmed the amazonian ethnobotany which Amazonian Citrus petitgrains 115 often does not discriminate Citrus species in using leaves for tradi- tional preparations, emphasizing synergic expression of different ex- tracts/chemical compounds to have better biological performances. Conclusions This first report about Amazonian Citrus spp. petitgrains evidenced their chemical characterization by GC and GC-MS and remarked the use of NMR as useful tool to characterize and discriminate chemo- type for identification, quality control and fraud detection of essen- tial oils (Lota et al., 2001b; guerrini et al., 2006; guerrini et al., 2011). However, no remarkable difference emerged with other Citrus spp. petitgrains from other geographical regions, even if interesting differences regarding minor compounds were found. In particular Amazonian CN petitgrain, γ-terpinene/linalool chemotype on the basis of chemical composition defined by GC/MS and NMR, and C1 petitgrain revealed both interesting in vitro antibacterial and radical scavenging activities. Result highlights that these two essential oils could be potentially employed as food preservatives or functional constituents in food supplements and/or health herbal products. 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SokoVic, m., gLamocLiJa, J., ciric, a., kataranoVSki, D., marin, P.D., VukoJeVic, J., Brkic, D., 2008: Antifungal activity of the essential oil of Thymus vulgaris L. and thymol on experimentally induced dermato- 116 A. Guerrini, D. Rossi, A. Grandini, L. Scalvenzi, P.F. Noriega Rivera, E. Andreotti, M. Tacchini, A. Spagnoletti, I. Poppi, S. Maietti, G. Sacchetti mycoses. Drug Dev. Ind. Pharm. 34, 1388-93. Vekiari, S.a., ProtoPaPaDakiS, e.e., PaPaDoPouLou, P., PaPanicoLaou, D., Panou, c., VamVakiaS, m., 2002: Composition and seasonal varia- tion of the essential oil from leaves and peel of a Cretan lemon variety. J. Agric. Food Chem. 50, 147-153. Address of the corresponding author: Department of Life Sciences and Biotechnology (SVeB), University of Ferrara, c.so Ercole I d’Este 32, I-44121 Ferrara, Italy. E-mail: grrlsn@unife.it