molecules Article
Chemical Composition and in Vitro Antimicrobial, Cytotoxic, and Central Nervous System Activities of the Essential Oils of Citrus medica L. cv. ‘Liscia’ and C. medica cv. ‘Rugosa’ Cultivated in Southern Italy Luigi Aliberti 1 , Lucia Caputo 1 , Vincenzo De Feo 1, *, Laura De Martino 1 , Filomena Nazzaro 2 and Lucéia Fátima Souza 1,3 1
2 3
*
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano (Salerno), Italy;
[email protected] (L.A.);
[email protected] (L.C.);
[email protected] (L.D.M.);
[email protected] (L.F.S.) Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche (ISA-CNR), Via Roma 64, 83100 Avellino, Italy;
[email protected] Post-Doctoral by National Counsel of Technological and Scientific Development (CNPq/Brazil), 70000-000 Brasília, Brazil Correspondence:
[email protected]; Tel.: +39-089-969-751; Fax: +39-089-969-602
Academic Editor: Luca Forti Received: 19 July 2016; Accepted: 12 September 2016; Published: 18 September 2016
Abstract: Citrus medica cv. ‘liscia’ and C. medica cv. ‘rugosa’ are two taxa of citron, belonging to the biodiversity of South Italy, in particular of Amalfi Coast, in the Campania region. The chemical composition of the essential oils (EOs) from fruit peels of both C. medica cultivars was studied by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In all, 100 compounds were identified, 82 for C. medica cv. ‘liscia’, accounting for 91.4% of the total oil, and 88 for C. medica cv. ‘rugosa’, accounting for 92.0% of the total oil. Monoterpene hydrocarbons are the main constituents in both oils of C. medica cv. ‘liscia’ (79.1%) and C. medica cv. ‘rugosa’ (80.2%). In both oils, limonene (67.2%–62.8%) and camphene (8.5%–10.9%) are the main constituents. The antimicrobial activity of the EOs was assayed against some bacterial strains: Bacillus cereus (DSM 4313), Bacillus cereus (DSM 4384), Staphylococcus aureus (DSM 25693), Pseudomonas aeruginosa (ATCC 50071), and Escherichia coli (DSM 8579). Low concentrations of C. medica cv. ‘rugosa’ EO showed an inhibitory effect on P. aeruginosa and higher concentrations inhibited more B. cereus (4384) and E. coli than S. aureus. The cytotoxicity of the EO was evaluated against SH-SY5Y cell line. The influence of the EO on the expression of adenylate cyclase 1 (ADCY1) was also studied. The antimicrobial activity registered confirm their traditional uses as food preserving agents and led us to hypothesize the possible use of these oils as antimicrobials. The alterations in ADCY1 expression suggested a role for limonene in effects on the central nervous system. Keywords: Citrus medica; essential oil; antimicrobial activity; SH-SY5Y cells; adenylate cyclase
1. Introduction Citron, native to Southeast Asia, was imported to the Mediterranean around 300 B.C. Probably, it arrived in Italy through the Hebrews who introduced the cultivation of citron on the Calabrian coasts, Amalfi Coast, and Garda Lake [1,2]. Two local cultivars of Citrus medica L. are grown on the Amalfi Coast: C. medica cv. ‘liscia’, known by the vernacular name of ‘cedro’, and C. medica cv. ‘rugosa’, known as ‘ponsino’. These two cultivars contributed to the agricultural biodiversity of this area, as well as other Citrus species. However, their
Molecules 2016, 21, 1244; doi:10.3390/molecules21091244
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diffusion is decreasing, due to the technical difficulties for their cultivation and to the competition of lemon cultivations. The taxonomy of Citrus species is complex. In fact, recent genetic analyses have shown that only three species belong to the genus Citrus: C. maxima (Burm.) Merr., C. medica L., and C. reticulata Blanco [3]. Moreover, the Citrus species are able to crossbreed, producing fruits with a wide range of morphological and organoleptic characteristics. Today, the fruits of both cultivars are used locally only for fresh alimentary consumption. In past times, both citrons have also been employed in traditional medicine as an anti-infective, an anti-inflammatory, and to treat digestive disorders. These traditional uses agree with the folkloric uses of citron. In fact, fruits and leaves are used in different countries in the treatment of allergic inflammation, for treating colds, as a decongestant, an expectorant, and a carminative or, in the case of pathologies of the intestinal tract and rectum, as well as a stomachic, an antispasmodic, a diuretic and a digestive [3,4]. The citron essential oils (EOs) are used for flavoring, for perfuming, in fruit beverages, in soft drinks, in cosmetics, and in household products [4]. Different studies reported evidence that Citrus consumption is associated with a reduced cancer incidence [5]. Menichini and coworkers [6] reported the chemical profile and the photo-induced cytotoxic activity of Citrus bergamia Risso and Poit. and Citrus medica cv. ‘Diamante’. Both oils exhibited a selective inhibition of the A375 tumoral cell line. Russo and coworkers [7] studied the cytotoxic effect of the Bergamot EO on SH-SY5Y neuroblastoma cells and its components, limonene and linalyl acetate, were able to induce cell death. Moreover, some EOs and their components are known for their activity on the central nervous system, interacting with N-methyl-D-aspartate (NMDA) receptor complex [8,9], and showing an inhibitory effect of the acetylcholine release and on the channel open time in the mouse neuromuscular junction [10]. The aims of this paper were to study the chemical composition of the EOs obtained from the peel of the fruits of the two cultivars grown in Amalfi Coast, to evaluate their potential antimicrobial activity, their cytotoxicity and the possible effects on central nervous system. 2. Results 2.1. Essential Oil Yield and Composition Hydrodistillation of the peel from fruits of C. medica cv. ‘liscia’ and C. medica cv. ‘rugosa’ gave yellow EOs characterized by a typical citrusy and floral odor, with yields of 0.9% and 0.75%, respectively. Table 1 shows the chemical composition of the two oils; the compounds are listed according to their elution order on an HP-5 MS capillary column. All 100 compounds were identified, 82 for C. medica cv. ‘liscia’, accounting for 91.4% of the total oil, and 88 for C. medica cv. ‘rugosa’ accounting for 92.0% of the total oil. Monoterpene hydrocarbons are the main constituents in both oils, 79.1% for cv. ‘liscia’ and 80.2% for cv. ‘rugosa’. In both oils, limonene (67.2%–62.8%), camphene (8.5%–10.9%), and β-pinene (1.4%–1.7%) were other main components. In the oil from C. medica cv. ‘liscia’ other components in a lesser amount are geranyl acetate (0.9%), and α-trans-bergamotene (0.5%); in the oil from cv. ‘rugosa’ geraniol (0.7%), geranial (0.7%), neral (0.5%), isopulegol (0.7%), and α-bisabolol (0.5%) are present. Table 1. Chemical composition of the essential oils (EOs) isolated from the peels of C. medica cv. ‘liscia’ (CL) and C. medica cv. ‘rugosa’ (CR) grown in Amalfi Coast. No.
Compound
LRI a
LRI b
CL
CR
Identification c
1 2 3 4 5 6 7
α-Thujene α-Pinene α-Fenchene Camphene β-Pinene α-Phellandrene δ-2-Carene
915 921 934 964 980 991 1004
930 939 952 954 979 1002 1002
0.8 0.1 8.5 1.4 0.5 0.1
0.1 1.2 0.1 10.9 1.7 0.6 0.3
1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2
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Table 1. Cont. No.
Compound
LRI a
LRI b
CL
CR
Identification c
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70
p-Cymene Limonene (Z)-β-Ocimene (E)-β-Ocimene γ-Terpinene Linalool oxide furanoid trans-Linalool oxide Terpinolene Linalool α-Pinene oxide 1,3,8-p-Menthatriene Perillene trans-Thujone Dehydro sabina ketone allo-Ocimene cis-p-Mentha-2,8-dien-1-ol cis-Limonene oxide trans-Limonene oxide Isopulegol neo allo-Ocimene Citronellal neo iso-Isopulegol Isoborneol α-Terpineol Hexyl butanoate Dihydrocarveol Methyl chavicol trans-4-Caranone Decenal 2-Decanol cis-4-Caranone endo-Fenchyl acetate Thymol methyl-ether Neral Geraniol Geranial n-Decanol trans-Carvone oxide Thymol p-Cymene-7-ol Undecen-10-en-1-al n-Nonanyl acetate Citronellic acid δ-Elemene α-Terpinyl acetate Citronellyl acetate Eugenol Neryl acetate α-Ylangene α-Copaene Geranyl acetate β-Patchoulene Methyl eugenol Italicene Sesquithujiene Longifolene β-Duprezianene γ-Elemene α-trans-Bergamotene α-Guaiene Aromadendrene (Z)-β-Farnesene (E)-β-Farnesene
1012 1022 1028 1038 1047 1064
1024 1029 1037 1050 1059 1072 1086 1088 1096 1099 1110 1103 1114 1120 1132 1137 1136 1142 1149 1144 1153 1171 1160 1188 1192 1193 1196 1196 1196 1199 1200 1220 1235 1238 1252 1267 1269 1276 1290 1290 1299 1312 1313 1338 1349 1352 1359 1361 1375 1376 1381 1381 1403 1405 1405 1407 1422 1436 1434 1439 1441 1442 1456
67.2 Tr 0.1 0.3 0.3 0.1 0.1 0.3 Tr Tr Tr 0.1 Tr Tr Tr Tr 0.8 0.7 Tr 0.3 0.3 0.1 0.9 0.1 0.9 0.1 0.3 0.1 0.1 0.1 Tr 0.4 Tr 0.1 0.7 Tr 0.9 0.1 0.1 0.1 0.1 0.5 0.1 0.1 0.5 Tr 0.1 0.1 Tr
1.0 62.8 0.1 0.3 0.7 Tr 0.3 1.3 0.1 Tr Tr 0.1 0.1 0.1 Tr 0.5 Tr 0.1 Tr 0.2 0.7 Tr 0.6 Tr Tr Tr 0.1 Tr 0.1 0.3 0.4 Tr 0.5 0.7 0.7 0.1 0.4 Tr Tr Tr Tr 0.2 0.1 0.1 Tr 0.6 Tr Tr 0,5 0.1 0.1 Tr Tr 0.6 0.1 0.1 0.4 Tr 0.1 0.1 Tr
1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2, 3 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 1, 2 1, 2, 3 1, 2 1, 2 1, 2, 3 1, 2 1, 2, 3 1, 2 1, 2, 3 1, 2, 3 1, 2, 3 1, 2
1077 1091 1095 1100 1103 1106 1111 1119 1126 1133 1140 1144 1152 1155 1167 1163 1180 1183 1185 1190 1195 1198 1202 1209 1219 1223 1231 1246 1261 1263 1276 1283 1292 1296 1301 1314 1326 1339 1343 1348 1354 1364 1368 1373 1380 1396 1399 1403 1407 1417 1422 1424 1432 1441 1445 1449
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Table 1. Cont. No.
Compound
LRI a
LRI b
CL
CR
Identification c
71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
cis-Cadin-1(6),4-diene 9-epi-(E)-Caryophyllene β-Acoradiene γ-Gurjenene α-Amorphene Aristolochene β-Selinene α-Selinene α-Cuprenene δ-Amorphene δ-Cadinene (Z)-Nerolidol γ-Cuprenene (E)-Nerolidol Caryophyllene oxide Globulol β-Oplopenone Guaiol 1-epi-Cubenol Eremoligenol α-Muurolol epi-α-Muurolol Pogostol Cedranol α-Bisabolol Eudesm-7(11)-en-4-ol Z-α-trans-Bergamotol Nootkatol (2Z,6E)-Farnesol Oplopanone Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Non terpenes Total
1457 1469 1473 1478 1482 1486 1490 1496 1502 1511 1523 1526 1530 1552 1572 1580 1597 1599 1618 1629 1631 1644 1647 1658 1674 1682 1688 1703 1711 1717
1463 1466 1470 1477 1484 1488 1490 1498 1505 1512 1523 1532 1533 1563 1583 1590 1607 1600 1628 1631 1646 1642 1653 1673 1685 1700 1690 1715 1723 1740
Tr Tr Tr 0.1 Tr 0.1 1.0 0.1 0.1 Tr Tr 0.3 Tr Tr 0.1 Tr Tr Tr 0.1 0.3 0.1 0.1 Tr 0.1 0.3 Tr Tr 79.1 4.8 4.2 2.5 0.8 91.4
Tr 0.1 Tr Tr Tr 0.1 0.6 Tr 0.1 Tr Tr 0.1 Tr Tr Tr Tr 0.1 Tr 0,1 0.5 80.2 6.9 3.2 1.6 0.1 92.0
1, 2 1, 2, 3 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2, 3 1, 2 1, 2 1, 2 1, 2 1, 2
a
Linear retention index on a HP-5MS column; b Linear retention index on a HP Innowax column; c Identification method: 1 = linear retention index; 2 = identification based on the comparison of mass spectra; 3 = Co-Gas chromatography retention time identical to authentic compounds; -: not detected; Tr = trace (10 μL 0.1 µL 0.8 µL 1 µL >10 µL 0.1 μL 0.8 μL 1 μL >10 μL 0.1 µL 0.8 µL 1 µL >10 µL
2.3. Cytotoxicity of Limonene, C. medica cv. ‘liscia’ and C. medica cv. ‘rugosa’ Essential Oils 2.3. Cytotoxicity of Limonene, C. medica cv. ‘liscia’ and C. medica cv. ‘rugosa’ Essential Oils
Limonene, C. medica cv. ‘liscia’, and C. medica cv. ‘rugosa’ EOs were evaluated for their ability to Limonene, C. medica cv. ‘liscia’, and C. medica cv. ‘rugosa’ EOs were evaluated for their ability inhibit the growth of human neuroblastoma The EOs EOsand andlimonene limonene revealed to inhibit the growth of human neuroblastomacell cellline line(SH-SY5Y). (SH-SY5Y). The revealed different cytotoxic activities. Limonene and C. medica cv. ‘rugosa’ EOs showed an IC 50 > 2000 μg/mL, different cytotoxic activities. Limonene and C. medica cv. ‘rugosa’ EOs showed an IC50 > 2000 µg/mL, instead C.C. medica IC50 50 of 718.2 μg/mL. instead medicacv. cv.‘rugosa’ ‘rugosa’EO EOshowed showed an an IC of 718.2 µg/mL. Treatment of SH-SY5Y neuroblastoma cells with 800 800µg/mL μg/mL of oflimonene limonenefor for2424h hresulted resulted Treatment of SH-SY5Y neuroblastoma cells with in in a a low cytotoxic activity. with 800 800µg/mL μg/mL of ofC. C.medica medicacv. cv.‘liscia’ ‘liscia’EOs EOs resulted low cytotoxic activity.However, However, treatment treatment with resulted in in a a stronger cytotoxicity than C. medica cv. ‘rugosa’ EO with 38% cell death (Figure 3). stronger cytotoxicity than C. medica cv. ‘rugosa’ EO with 38% cell death (Figure 3).
Figure Percentageofofcell cellviability viability after after 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl bromide Figure 3. 3. Percentage 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium tetrazolium bromide (MTT) assay.Cells Cellswere weretreated treatedwith with different different concentrations (A); C. C. medica (MTT) assay. concentrations(50–800 (50–800µg/mL) μg/mL)ofoflimonene limonene (A); medica cv. ‘liscia’ (B) and C. medica cv. ‘rugosa’ EOs, for 24 h and solvent (DMSO, 0.1%) alone. Data are thethe cv. ‘liscia’ (B) and C. medica cv. ‘rugosa’ EOs, for 24 h and solvent (DMSO, 0.1%) alone. Data are mean ± SD of three experiments * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. DMSO. mean ± SD of three experiments * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. DMSO
2.4. ADCY1: Western Blotting Analysis We investigated the effects of limonene, C. medica cv. ‘liscia’ and C. medica cv. ‘rugosa’ EOs in SH-SY5Y human neuroblastoma cells. Representative Western blots and quantitative densitometric analysis for adenylate cyclase 1 (ADCY1) protein expression in SH-SY5Y following exposure to different concentrations of limonene and EOs are shown in Figure 4. Treatments of SH-SY5Y neuroblastoma cells with 800 and 50 µg/mL of limonene for 24 h significantly influenced ADCY1 expression in different way: high concentrations appear to increase ADCY1 expression, instead low concentrations reduced the ADCY1 expression (Figure 4A). However, treatments with 400, 200, 100, 50 µg/mL of C. medica cv. ‘liscia’ and C. medica cv. ‘rugosa’ EOs appear to influence significantly ADCY1 expression with an over expression and a down expression of ADCY1, respectively (Figure 4B,C).
concentrations of limonene and EOs are shown in Figure 4. Treatments of SH-SY5Y neuroblastoma cells with 800 and 50 μg/mL of limonene for 24 h significantly influenced ADCY1 expression in different way: high concentrations appear to increase ADCY1 expression, instead low concentrations reduced the ADCY1 expression (Figure 4A). However, treatments with 400, 200, 100, 50 μg/mL of C. medica cv. ‘liscia’ and C. medica cv. ‘rugosa’ EOs appear to influence significantly ADCY1 expression Molecules 2016, 21, 1244 7 of 14 with an over expression and a down expression of ADCY1, respectively (Figure 4B,C).
Figure 4. 4. Relative Relative expression expression levels levels of of the the ADCY1 ADCY1 in in SH-SY5Y SH-SY5Y treated treated with with limonene limonene (A); (A); C. C. medica medica Figure cv. ‘liscia’ ‘liscia’ (B); (B); and and C. C. medica cv. cv. ‘rugosa ‘rugosa (C) (C) EOs. Each panel shows a representative Western Western blot blot and and cv. densitometric analysis of bands bands in in the the control control and and treated treated groups. groups. Values Valuesare arethe themean mean±± SD in each each densitometric group (n (n == 3). * p < 0.05, followed group 0.05, ** ** pp
