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DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA agrawal@naturalproduct.us

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PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain afbarre@ugr.es

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HONORARY EDITOR

PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

axuf64@dsl.pipex.com

Anti-oxidant, Anti-inflammatory and Anti-proliferative Activities of Moroccan Commercial Essential Oils

Smail Aazzaa,b, Badiaa Lyoussib, Cristina Megíasc, Isabel Cortés-Giraldoc, Javier Vioquec, A. Cristina Figueiredod and Maria G. Miguele* aUniversidade do Algarve, Faculdade de Ciências e Tecnologia, Departamento de Química e Farmácia, Campus de Gambelas 8005-139 Faro, Portugal

bLaboratory of Physiology, Pharmacology and Environmental Health, Faculty of Sciences Dhar El Mehraz, BP 1796 Atlas, University Sidi Mohamed Ben Abdallah, Fez 30 000, Morocco

cInstituto de la Grasa (C.S.I.C.), Avda. Padre García Tejero 4, 41012-Sevilla, Spain

dUniversidade de Lisboa, Faculdade de Ciências de Lisboa, Departamento de Biologia Vegetal, Instituto de Biotecnologia e Bioengenharia, Centro de Biotecnologia Vegetal, C2, Piso 1, Campo Grande, 1749-016 Lisboa, Portugal

eUniversidade do Algarve, Faculdade de Ciências e Tecnologia, Departamento de Química e Farmácia, Instituto de Biotecnologia e Bioengenharia, Centro Biotecnologia Vegetal, Campus de Gambelas 8005-139 Faro, Portugal mgmiguel@ualg.pt

Received: December 12th, 2013; Accepted: January 5th, 2014

Essential oils (EO) possess antimicrobial, anti-inflammatory, insect repellent, anti-cancer, and antioxidant properties, among others. In the present work, the antioxidant, anti-inflammatory and anti-proliferative activities of Moroccan commercial EOs (Citrus aurantium, C. limon, Cupressus sempervirens, Eucalyptus globulus, Foeniculum vulgare and Thymus vulgaris) were evaluated and compared with their main constituents. T. vulgaris EO showed the best free radicals scavenging capacity. This EO was also the most effective against lipid peroxidation along with C. limon and F. vulgare EOs. C. sempervirens EO was the most effective in scavenging NO free radicals, whereas C. limon EO showed the best chelating power. Not all of the major compounds of the EO were responsible for the whole activity of the EOs. T. vulgaris EO showed the best anti-proliferative activity against THP-1 cells in contrast to that of F. vulgare. The antioxidant and anti-inflammatory activities of the EOs were plant species dependent and not always attributable to the EOs main components. Nevertheless, the EOs anti-proliferative activities were more related to their main components, as with T. vulgaris, C. limon, E. globulus and C. sempervirens. Keywords: Chemical composition, Biological properties, Essential oils, MTT. For food and beverage consumption, the essential oil products used are those on the Generally Recognised as Safe (GRAS) list approved by the Food and Drug Administration (FDA). For medical purposes essential oils need to fulfil national and international Pharmacopoeia recommendations. The maximum quantities and uses of some essential oils, as well as their single components, are regulated by the International Fragrance Association (IFRA), the Bundesinstitut für Risikobewertung (BfR), the Research Institute for Fragrance Materials (RIFM) and the Scientific Committee Consumer’s Safety (SCCS). Physical standards of essential oils are also specified by the Association Française de Normalisation (AFNOR), as well as the International Organization for Standardization (ISO). The need for this control and standardization lies in the fact that the chemical composition varies depending on plant health, growth stage, edaphic and climate factors, harvesting time, part of plant used, and agronomic conditions, among other factors [1,2]. The use of essential oils in the pharmaceutical, agricultural and nutritional fields are due to their antimicrobial, antiviral, nematicidal, antifungal, insecticidal, antioxidant and anti-inflammatory activities [3]. In the present work, six traded essential oils belonging to the first {Citrus aurantium L., C. limon (L.) Burman f. and Eucalyptus globulus Labil.) and second (Thymus vulgaris L.) groups of global

production, along with Cupressus sempervirens L., and Foeniculum vulgare Mill. were chemically analyzed. Also, antioxidant and anti-proliferative activities of these essential oils were investigated. Essential oils are composed of many compounds and their biological activities can be attributed to the major components and/or to minor ones. Hence, in the present work, the biological activities of the essential oils were compared with those of their main components in order to ascertain if the activities of these compounds reflect the biological activities of the respective essential oils. Essential oils composition: The detailed composition of the essential oil (EO) components of Citrus aurantium (leaves), C. limon (peel), Cupressus sempervirens, Eucalyptus globulus, Foeniculum vulgare and Thymus vulgaris are listed in Table 1, in order of their elution from a DB-1 column. In a previous work, only the main components (>10%) were reported [4]. The volatile profile of some of the studied species was in general in accordance with previous studies, namely those of C. aurantium [5], C. sempervirens [6,7] and F. vulgare [8]. On the contrary, other essential oils showed some differences from those previously reported, such as those of C. limon [9], although some authors [10] also reported high percentages of limonene in some lemon taxa essential oils (81% to 96%); E. globulus [11] and T. vulgaris [12].

NPC Natural Product Communications 2014 Vol. 9 No. 4

587 - 594

588 Natural Product Communications Vol. 9 (4) 2014 Aazza et al.

The EO yields can vary considerably, as well as the exact composition of any EO will be variable depending, among other factors, on the particular plant part material used in the isolation procedure, on the cultivation conditions, cultivar, harvesting time and distillation [2,13]. This chemical variability may contribute to the existence of EO chemotypes, which may partly explain the contrasting properties of the essential oils isolated from the same species. Antioxidant activity: The antioxidant capacity of the studied commercial EOs was determined by evaluating their ability to scavenge free radicals (ABTS, hydroxyl, peroxyl and NO), lipid peroxidation (liposomes) and chelating ability (Table 2). T. vulgaris EO showed the best capacity for scavenging ABTS, hydroxyl and peroxyl radicals. Nonetheless, it was not as effective for either scavenging nitric oxide radicals or for chelating iron metal (Table 2). C. sempervirens EO was the most effective in scavenging NO free radicals, and C. limon EO showed the most effective chelating power. The antioxidant properties of T. vulgaris essential oils have been reported in previous studies using either the same methods or different ones from those used in the present work [4,14,15]. C. limon, along with F. vulgare and T. vulgaris EOs showed the highest prevention of liposome peroxidation, in contrast to that of C aurantium in which the IC50 was not possible to determine and E. globulus EO (Table 2). This EO also had a weak capability for scavenging free peroxyl radicals. C. sempervirens EO was the most effective for scavenging NO radicals and, along with F. vulgare EO, was a good chelator of metal ions (Table 2). As mentioned, EOs are complex mixtures of lipophilic and volatile compounds at different concentrations. The activity can be attributed to major EO compounds, but also minor compounds may reveal antioxidant activity. Moreover, the association between major and minor compounds may result in an antagonistic result. In the present work, the antioxidant activities of entire EOs were compared with those of their main components to determine the potential correlation between the EOs and their major constituents. The activity of T. vulgaris EO, which was one of the best antioxidants in almost all the assays used, seems to be predominantly due to thymol and carvacrol. Borneol and p-cymene had only very weak activity (Table 2), which is in accordance with previous studies [16]. The capacity of T. vulgaris EO for scavenging hydroxyl radicals was the best, whereas in the remaining assays thymol was generally the best antioxidant (Table 2). The antioxidant activities of thymol and carvacrol have already been demonstrated by Puertas-Mejía et al. [17], both alone and in combination. The authors concluded that these phenols combined in a 1:1 ratio had a synergistic effect. The antioxidant activity C. limon EO may be attributed to limonene, independently of the method used for assay. Limonene was practically the sole terpene in this EO (Table 1). The low capacity of limonene for scavenging ABTS free radicals (<50%) was also reported by Roberto et al. [18] when using DPPH as free radicals. Limonene, as well as C. limon EO, had the capacity for preventing lipid peroxidation of liposomes. Ruberto and Baratta [16] found a low capacity for preventing lipid peroxidation of limonene through the modified thiobarbituric acid reactive species and the rate of conjugated diene formation from linoleic acid. The chelating

capacity of limonene-rich essential oils detected in the present work was also reported by Gursoy et al. [19], this activity being dose-dependent. The capacity for either scavenging ABTS radicals or preventing lipid peroxidation by C. aurantium EO cannot be attributed to linalool and linalyl acetate, the dominant monoterpenes in this EO, since the activities were much lower than that of C. aurantium EO. The weak ability of linalool to prevent oxidation either by inhibiting lipid peroxidation or scavenging free radicals was also previously reported [16,20]. Hence other EO components may be responsible for the activity of C. aurantium EO and/or their synergism. All EOs showed metal chelating activity and the capacity for scavenging peroxyl radicals, although linalool and linalyl acetate had higher activities, suggesting that these monoterpenes and/or other components present in the EO showed an antagonistic effect, lowering the antioxidant capacity of the Citrus EO. Linalool, one of the main components of Hedychium coronarium and Diplazium squamigerum EOs exhibited moderate ferrous chelating activity [21], as well as a capacity for scavenging peroxyl radicals [22]. Only E. globulus EO was able to scavenge ABTS free radicals (Table 2). 1,8-Cineole had very little ability to scavenge these free radicals. Hence, the activity may be the result of several components of the EO and not only to one of the major compounds. Limonene had the best capacity for scavenging peroxyl radicals in contrast to that of 1,8-cineole (Table 2). The combination of 1,8-cineole and p-cymene or other combinations with minor components seemed to determine the activity of E. globulus EO. 1,8-Cineole was also the worst in terms of prevention of lipid peroxidation, in contrast to limonene (Table 2). E. globulus EO was better as an antioxidant than 1,8-cineole, but the worst when compared to the remaining samples (Table 2). This compound also had very little ability for preventing lipid peroxidation when egg yolk was used as the lipid model in the thiobarbituric acid reactive substances assay [16]. Neither 1,8-cineole nor p-cymene showed NO radical scavenging activity. Only E. globulus EO and limonene showed this property, although the EO activity was poorer than that of limonene. Such results indicate that an antagonistic effect may have occurred that is responsible for the lower activity of the eucalyptus EO (Table 2). In contrast to the results reported in the other assays, 1,8-cineole was the best for scavenging hydroxyl radicals. The EO presented an intermediate value between 1,8-cineole and limonene. 1,8-Cineole’s low IC50 was quite different from that reported by Singh et al. [23]. According to them, E. tereticornis EO was better for scavenging hydroxyl radicals than the pure constituents, such as 1,8-cineole. Comparing the antioxidant and chelating activities of C. sempervirens EO with its main components (Table 2), -pinene was the least active in almost all the assays, with the exception of lipid peroxidation prevention. In most cases, it was even impossible to calculate the IC50 for -pinene due to its very low antioxidant activity (TEAC, hydroxyl and nitric oxide scavenger and chelator). -Pinene was reported as having DPPH free radical scavenging ability, although lower than that of 1,8-cineole, myrcene and thymol, but better than rosemary EO, in which that monoterpene prevailed [24].

Biological properties of essential oils Natural Product Communications Vol. 9 (4) 2014 589

Table 1: Percentage compositions of the commercial essential oils isolated by hydrodistillation.

Apiaceae Cupressaceae Lamiaceae Myrtaceae Rutaceae

Components RI Foeniculum Cupressus Thymus Eucalyptus Citrus Citrus

vulgare sempervirens vulgaris globulus aurantium limon Tricyclene 921 t 0.2 α-Thujene 924 t t 0.1 t α-Pinene 930 9.0 47.3 3.4 3.5 0.1 0.3 Camphene 938 t 0.7 4.7 t Sabinene 958 0.7 0.4 t 0.1 t 0.2 1-Octen-3-ol 961 t β-Pinene 963 0.7 0.8 0.6 0.1 2.5 0.1 β-Myrcene 975 1.7 0.2 0.2 0.4 1.3 t α-Phellandrene 995 t t 1.8 δ-3-Carene 1000 17.6 α-Terpinene 1002 0.3 p-Cymene 1003 0.3 t 24.6 37.8 t 1,8-Cineole 1005 0.3 0.5 29.3 β-Phellandrene 1005 0.3 t 0.5 t t Limonene 1009 8.3 31.4 1.5 26.1 0.3 96.7 Z-β-Ocimene 1017 t t 0.3 E-β-Ocimene 1027 t 1.9 γ-Terpinene 1035 0.1 t 1.2 t t n-Octanol 1045 t Z-Linalool oxide 1045 t Fenchone 1050 9.1 2,5-Dimethyl styrene 1059 t t E-Linalool oxide 1059 t Terpinolene 1064 0.4 0.3 Linalool 1074 t 3.4 t 54.0 0.3 Isopentyl isovalerate 1084 t Z-Limonene oxide 1095 0.5 Camphor 1102 0.7 E-Pinocarveol 1106 0.4 E-Limonene oxide 1112 0.3 Borneol 1134 15.5 Terpinen-4-ol 1148 t t t 0.1 Z-Dihydrocarvone 1159 0.9 α-Terpineol 1159 t 3.5 t 8.5 0.1 Estragole (= Methyl chavicol) 1163 t Verbenone 1164 t n-Decanal 1180 0.1 E-Carveol 1189 t 0.3 Bornyl formate 1199 0.1 Anisaldehyde 1200 t Z-Carveol 1202 t t Nerol 1206 0.6 Pulegone 1210 5.4 Carvone 1210 t t t Methyl carvacrol oxide 1224 1.4 Geraniol 1236 3.1 Linalyl acetate 1245 21.1 E-Anethole 1254 69.4 Bornyl acetate 1265 0.7 Thymol 1275 11.8 Carvacrol 1286 15.4 Limonene-1,2-diol 1314 0.2 α-Terpenyl acetate 1334 0.7 t Neryl acetate 1353 1.4 Geranyl acetate 1370 3.3 α-Copaene 1375 0.1 β-Caryophyllene 1414 2.9 0.2 0.8 β-Copaene 1426 0.1 α-Humulene 1447 0.1 γ-Muurolene 1469 t Germacrene-D 1474 t Valencene 1484 0.1 Bicyclogermacrene 1487 0.1 0.4 γ-Cadinene 1500 t 0.1 Calamenene 1505 t δ-Cadinene 1505 0.1 t β-Caryophyllene oxide 1561 t Cedrol 1574 0.1 T-Cadinol (= epi-α-Cadinol) 1616 t % of Identification 99.9 99.9 99.7 99.8 99.9 99.3 Grouped components Monoterpene hydrocarbons 21.1 98.8 37.3 69.8 6.7 97.3 Oxygen-containing monoterpenes 9.4 0.7 59.3 29.7 92.0 1.8 Sesquiterpene hydrocarbons 0.3 3.1 0.3 1.2 0.1 Oxygen-containing sesquiterpenes 0.1 t Phenylpropanoids 69.4 Others t t t 0.1

RI, in lab calculated retention index relative to C9–C17 n–alkanes on a DB–1 column; t, trace (<0.05%)

590 Natural Product Communications Vol. 9 (4) 2014 Aazza et al.

Table 2: Antioxidant and anti-inflammatory activities of essential oils and their main constituents.

Plant / Standard TEAC Hydroxyl NO ORAC Chelating Liposomes Lipoxygenase IC50 (mg mL-1) IC50 (g mL-1) IC50 (mg mL-1) TE* (mol mg-1) IC50 (mg mL-1) IC50 (mg mL-1) IC50 (mg mL-1) Thymus vulgaris 0.0100.000aF 0.400.00cF 28.70.4aC 1788.0244.5cA - 0.460.01bC 0.190.00aC Carvacrol 0.0030.000b 1.100.00a 11.20.4b 24891.4244.5b - 0.040.01c 0.110.00b Thymol 0.0020.000cc 0.900.00b 9.90.4b 27156.5244.5a - 0.040.01c 0.200.00a Borneol - - - 7.2244.5d - - - p-Cymene - - - 3.7244.5d - 0.990.01a - Citrus limon 28.000.53A 4.000.00aC 19.61.0aD 57.13.9aC 0.030.00aD 0.540.02aC 0.300.01aB Limonene - 4.200.00a 18.71.0a 51.13.9a 0.030.00a 0.510.02a 0.290.01a Citrus aurantium 22.540.23B 1.600.00bE 78.014.4aA 161.92.1aB 0.060.00aA - 0.940.25bA Linalool - 4.100.00b 51.914.4b 31.52.1b 0.030.00c - - Linalyl acetate - 237.400.00a - 23.02.1c 0.040.00b - 1.370.25a Eucalyptus globulus 1.430.05E 2.900.00bD 60.422.9aB 5.90.5bD 0.050.00bB 3.660.00bA 0.160.07bC 1,8-Cineole - 0.060.00c - 1.80.5d 0.100.00a 4.200.00a - Limonene - 4.200.0001a 18.722.9b 51.10.5a 0.030.00c 0.510.00d 0.290.07a p-Cymene - - - 3.70.5c - 1.000.00c - Cupressus sempervirens 3.620.12D 0.010.00aA 6.56.7bE 26.00.6bCD 0.040.01aC 2.170.03aB 0.170.07bC -Pinene - - - 1.20.6c - 1.020.03b - Limonene - 0.0040.00b 18.76.7a 51.10.6a 0.030.01b 0.510.03c 0.290.07a Foeniculum vulgare 9.280.08C 0.010.007bB 16.81.7aD 58.930.4bC 0.040.02aC 0.470.01aC 0.040.01aD E-Anethole - 0.030.007a 14.31.7b 114.330.4a 0.010.02b 0.460.01a 0.020.01b

-: without activity or very poor activity. TE*: Trolox Equivalent. For each plant, values in the same column followed by the same upper letter case are not significant by the Tukey’s multiple range test (p<0.05). Data are the mean of at least three replicates. For each plant/standard, values in the same column followed by the same lower letter case are not significant by the Tukey’s multiple range test (p<0.05) or by the Paired Student t test (no differences at 5% significance). Data are the mean of at least three replicates. The capacity of -pinene for preventing lipid peroxidation was worse than that of limonene, as already reported [16], although using a different lipid substrate. In the present work, the lower capacity for inhibiting lipid oxidation of C. sempervirens EO suggests the influence of either other minor components or the involvement of limonene + -pinene in the EO in the antagonism processes, which were not evaluated. The iron chelating inability of -pinene agrees with data from Boulanouar et al. [25] that showed a lack of chelating activity in the -pinene-rich Juniperus phoenicea EO. The chelating ability of C. sempervirens EO may be attributed to other minor components. The weak capacity for scavenging free radicals of E-anethole and E-anethole-rich fennel EO has already been reported [8,17]. The capacity for inhibiting peroxidation of lipids, measured through the TBRAS method, was reported by Miguel et al. [8]. However, the authors found that high concentrations of E-anethole-rich fennel EO had a pro-oxidant activity. In the present study, the activity was measured using liposomes as the lipid substrate and differences between the EO and the phenylpropanoid were not observed. In contrast, E-anethole was significantly better than the respective EO for scavenging peroxyl radicals and nitric oxide, as well as metal chelating (Table 2). The capacity for iron binding of the EO from some cultivars of fennel, particularly those richest in E-anethole, was previously reported [26]. In the present study, E-anethole was a significantly better chelating agent than fennel EO. The iron binding property can be attributed to this compound, although other components of fennel EO may antagonize its action. The same may explain the lowest capacity of the essential oil for scavenging peroxyl (Table 2). Anti-inflammatory activity: The lipoxygenase assay was used as an indication of the anti-inflammatory and antioxidant activities of the EOs. Lipoxygenase catalyses the addition of molecular oxygen to fatty acids containing a Z,Z-1,4-pentadiene system originating from unsaturated fatty acid hydroperoxides. Compounds which are able to inhibit this enzyme, which is responsible for the production of these peroxides, can be considered as antioxidants. At the same time, those products are converted into others that play a key role in

inflammatory processes. F. vulgare EO had the highest activity in contrast to the lowest observed with C. aurantium EO (Table 2). Several works have shown that EOs may possess anti-inflammatory activity. This has been attributed to limonene, 1,8-cineole, -terpinene, and -pinene, among other components [27,28]. In the present work, 1,8-cineole, -pinene, p-cymene, linalool and borneol either did not show anti-inflammatory activity or it was very low, which did not allow IC50 determination (Tables 2 and 3). F. vulgare EO activity may be attributed to its major compound, E-anethole. Nevertheless, other EO components may partially antagonize this phenylpropanoid activity, since the EO showed less activity than E-anethole. -Pinene, limonene, and fenchone at >8% may have contributed to the lower activity of fennel EO when compared with that of E-anethole (Table 2). It is noteworthy that, among the pure compounds tested, this phenylpropanoid had the best anti-inflammatory activity. The EO of C. limon and limonene standard, which dominates the EO, showed similar activities, which allowed us to conclude that the activity of the whole EO may be attributed to this monoterpene. On the other hand, limonene was also a dominant component of E. globulus and C. sempervirens EOs, but never reaching percentages as elevated as those observed in C. limon EO. In both cases, the activities of the EOs were always higher than that of limonene, which may mean that other EO components are also responsible for the activity, acting by synergism with limonene. In C. aurantium EO, linalyl acetate, along with other components, contributed, by synergism, to the activity of the EO since the activity of linalyl acetate was lower than that of the respective EO (Table 2). Anti-proliferative activity: The cytotoxic activities of C. aurantium, C. limon, C. sempervirens, E. globulus, F. vulgare and T. vulgaris EOs and their main constituents were studied on the THP-1 leukemia cell line by treating these cells with increasing doses of either the EOs or their main components for 24 or 96 h.

Biological properties of essential oils Natural Product Communications Vol. 9 (4) 2014 591

B

Figure 1: Antiproliferative activity of the essential oils on THP-1 cell line with (A) 24 h exposure and (B) 96 h exposure. The mean absorbance values for the negative control (DMSO treated cells) was standardized as 100% absorbance (i.e. no growth inhibition) and results were displayed as absorbance (% of control) vs. essential oil concentration. T. vulgaris EO showed the best anti-proliferative activity (Figure 1A) after 24 h of cell treatment, in contrast to F. vulgare EO. After 96 h of cell exposure, the trend was similar (Figure 1B). All samples inhibited cell proliferation in a concentration-dependent manner. T. vulgaris EO at >200 g mL-1 practically prevented the growth of THP-1 cells after 96 h of exposure, and at 100 g mL-1 only T. vulgaris EO had significantly higher anti-proliferative capacity. At this concentration <40 % of the cells survived. T. vulgaris EO’s main components are p-cymene, borneol, carvacrol and thymol (Table 1). Comparing the anti-proliferative activities of these monoterpenes with that of the EO showed that the activity of the oil may be attributed to carvacrol and thymol (Figure 2A). p-Cymene was the least effective since more than 80% cell survival was observed at > 400 g mL-1 (Figure 2A). p-Cymene and borneol might be responsible for the lower activity of the EO compared with carvacrol and thymol (Figure 2A). Limonene also showed relatively high anti-proliferative activity (Figures 2B and 2C), being better for preventing THP-1 cell growth than C. limon and E. globulus EOs. C. limon EO is almost entirely dominated by limonene. Maybe for this reason, its cytotoxic activity (Figure 2B) was higher than that of E. globulus EO (Figure 2C), that also possesses p-cymene and 1,8-cineole in relatively high amounts (Table 1). p-Cymene and 1,8-cineole were less active than limonene and are probably responsible for the weaker activity of E. globulus EO. The anti-proliferative activities of both EOs, as well as those of the standards, were dose-dependent. Limonene alone or as an EO component has been reported to inhibit colon cancer (SW480) cell proliferation [29], MCF-7 breast tumor cells [30], and a lymphoma cell line [18], as well as acting against carcinogen-induced mammary tumors in rats [31]. Nevertheless, β-pinene, α-terpineol, γ-terpinene and trans-α-bergamotene, along with limonene in some Citrus fruit peel EOs, were critical to obtain a better anti-proliferative activity on MCF-7 and HeLa cell lines [32]. The present results demonstrated that p-cymene and 1,8-cineole may also interfere with the activity of limonene on the growth of THP-1 cells. C. sempervirens EO also had anti-proliferative activity on THP-1 cells (Figure 2D), similar to that of limonene, one of the major constituents of the EO, in contrast to that of -pinene, which only possessed weak activity. The cytotoxic activity of -3-carene, also in relative high percentage in the EO, on THP-1 cells was not evaluated. However, the similar anti-proliferative activity of the EO and limonene may reveal a weak cytotoxic activity of -3-carene and -pinene on THP-1 cells, or at least there was not an antagonistic effect between these two monoterpenes and limonene.

C. aurantium EO had a higher capacity to reduce THP-1 cell growth than the main oil components, linalool and linalyl acetate (Figure 2E). Linalyl acetate was more effective than linalool, at concentrations >400 g mL-1, although lower than the EO. O. vulgare, a linalool and linalyl acetate-rich EO, was reported as possessing considerable cytotoxicity against breast cancer MCF-7 and androgen-sensitive human prostate adenocarcinoma cell lines [33]. Linalool, a minor component of Platycladus orientalis, Prangos asperula and Cupressus sempervirens ssp. pyramidalis EOs was cytotoxic to amelanotic melanoma C32 and renal cell adenocarcinoma cells, in contrast to -pinene, the major component, which was inactive on tumor cell population growth and proliferation [34]. However, Tundis et al. [35] found that linalool was inactive against LNCaP and MCF-7 cell lines, despite its cytotoxic efficacy against tamelanotic melanoma C32 and renal cell adenocarcinoma cells. These findings demonstrate that one compound’s cytotoxic activity largely depends on the evaluated cell type. According to Prashar et al. [36], linalyl acetate present in lavender EO was more cytotoxic to human skin cells in vitro (endothelial cells and fibroblasts) than linalool, another lavender EO component. Using non-cancer cells, Prashar et al. [36] showed that the acetate group (linalyl acetate) would be responsible for higher cytotoxicity than the respective alcohol, linalool. The results obtained by Prashar et al. [36] and those obtained in the present study support the view that linalyl acetate may show anti-proliferative activity against normal cells and THP-1 leukemia cells. E-Anethole, fennel EO’s main component, showed low anti-proliferative activity, even at high concentrations (Figure 2F). This may explain the weak action of fennel EO on THP-1 cell growth. These results contrast with those of al-Harbi [37], in which E-anethole was active against Erlich tumor (EAT)-cells in Swiss albino mice paw. However, the present results agree with those reported by Firuzi et al. [38] since the E-anethole-rich Heracleum persicum EO was inactive against human cervical adenocarcinoma (HeLa), human colon adenocarcinoma (LS180) and human B lymphoma (Raji) cell lines. Therefore, the cytotoxicity of EOs and/or their components depend on the type of cancer cell lines. Conclusion: The chemical composition of the commercial EOs did not fit within the typical composition recognized by Food and Health Institutions, which reinforces the need to control the EO trade. T. vulgaris EO was the best for scavenging ABTS, hydroxyl and peroxyl radicals, whereas C. sempervirens EO was the best for scavenging NO radicals. The best chelating activity was found in C. limon EO. T. vulgaris, F. vulgare and C. limon EOs had similar capacities for preventing lipid peroxidation. Fennel EO had the best

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592 Natural Product Communications Vol. 9 (4) 2014 Aazza et al.

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Figure 2. Anti-proliferative activity of (A) T. vulgaris, (B) C. limon, (C) E. globulus, (D) C. sempervirens, (E) C. aurantium and (F) F. vulgare EOs and their main components on THP-1 cell line (96 h exposure). The mean absorbance values for the negative control (DMSO treated cells) was standardized as 100% absorbance (i.e. no growth inhibition) and results are displayed as absorbance (% of control) vs EOs concentration.

capacity for inhibiting lipoxygenase activity, thus showing the best anti-inflammatory activity, although greatly test dependent. The EOs’ antioxidant activity was also greatly dependent on several compounds and not only on their major components. T. vulgaris EO had the best anti-proliferative activity on THP-1 cells in contrast to F. vulgare EO. T. vulgaris EO’s activity may be attributed to thymol and carvacrol, whereas its weak anti-proliferative activity may be attributed to E-anethole. Limonene, present in relative high amounts in Citrus limon, E. globulus and Cupressus sempervirens EO also showed good anti-proliferative activity on THP-1 cells. Experimental Essential oils provenance and analysis: Citrus aurantium (leaves), Citrus limon (peel), Cupressus sempervirens, Eucalyptus globulus, Foeniculum vulgare and Thymus vulgaris essential oils were

provided by the Zaraphyt Company from Rabat, Morocco. The EOs were analyzed by gas chromatography, and gas chromatography coupled to mass spectrometry, as previously detailed [39]. Antioxidant activity ABTS radical cation scavenging capacity: The ABTS radical cation decolorization assay was carried out using the method described in [25]. The capability to scavenge the ABTS + was calculated using the formula: ABTS.+ scavenging activity (%) = [(A0-A1)/ A0] × 100 (%), where A0 is the absorbance of the control (without sample) and A1 is the absorbance in the presence of the sample. The sample concentration providing 50% inhibition (IC50) was obtained by plotting the inhibition percentage against EOs concentrations.

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Biological properties of essential oils Natural Product Communications Vol. 9 (4) 2014 593

Hydroxyl radical scavenging activity: OH-scavenging activity (%) was assessed as in Boulanouar et al. [25], and calculated using the equation: Inhibition = [(A0-A1)/ A0] × 100 (%) where A0 is the absorbance of the control (without sample) and A1 is the absorbance in the presence of the sample. Tests were carried out in triplicate. The sample concentration providing 50% inhibition (IC50) was obtained by plotting the inhibition percentage against EOs concentrations. Nitric oxide scavenging capacity: The nitric oxide (NO) scavenging activity was measured as in [40a]. Fifty µL of a serially diluted sample of each EO and its major components were added to 50 µL of 10 mM sodium nitroprusside in phosphate buffer saline (PBS) in a 96-well plate, which was incubated at room temperature for 90 min. Finally, an equal volume of Griess reagent was added to each well and the absorbance was read at 546 nm. Several concentrations of samples were made and the percentage inhibition calculated from the formula: [1 - (Asample - Asample blank)/( Acontrol - Acontrol blank)]x100, where (Asample - Asample blank) is the difference in the absorbance of a sample, with or without 10 mM sodium nitroprusside, and (Acontrol - Acontrol blank) is the difference in the absorbance of the PBS control, with or without 10 mM sodium nitroprusside. The sample concentration providing 50% inhibition (IC50) was obtained by plotting the inhibition percentage against EOs concentrations. Oxygen radical absorbance capacity (ORAC): The procedure was as reported by [25] for EOs. The results were expressed as micromoles Trolox equivalent (TE). Chelating metal ions: EOs and their major components ferrous ion chelating degree was evaluated as in [40b]. The chelating ability percentage was determined according to: [(A0 − A1)/A0 x 100], in which A0 is the absorbance of the control and A1 the absorbance of sample. The values of IC50 were determined as above. Inhibition of lipid peroxidation of lecithin liposomes: Lipid peroxidation (LP) was measured according to [41]. Liposomes were obtained from 0.4 g lecithin in 80 mL chloroform. This solution was dried under vacuum in a rotary evaporator (<50°C) to yield a thin, homogenous film, which was submitted to a nitrogen flux for 30 s. Liposomes were then submitted to vacuum for at least 2 h until complete dryness. The film was then dispersed in 80 mL of phosphate saline buffer 0.01 M, pH 7.0. The mixture was sonicated to obtain a homogeneous suspension of liposomes and kept at 4ºC until the assay. The percentage of LP inhibition was calculated by: I (%) = (A0-A1)/A0 x 100, where A0 was the absorbance of the control reaction (full reaction, without the test compound) and A1 was the absorbance in the presence of the inhibitor.

Anti-inflammatory activity 5-Lipoxygenase inhibitory activity: Lipoxygenase is known to catalyze the oxidation of unsaturated fatty acids containing 1-4 diene structures. The conversion of linoleic acid to 13-hydroperoxy linoleic acid was followed spectrophotometrically by the appearance of a conjugate diene at 234 nm on a UV/visible spectrophotometer, as in [42]. The concentration giving 50% inhibition (IC50) was calculated as above. Anti-proliferative activity Cell culture: THP-1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) non-essential amino acids, 100 U mL-1 penicillin, and 100 g mL-1 streptomycin. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere. Anti-proliferative activity: The growth-inhibitory effect of EOs was measured using the standard 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay adapted from Mosmann [43]. THP-1 cells were seeded in a 96 well plate at 5.103 cells well-1 and exposed to different EO concentrations (10-500 µg mL-1) for 1 and 4 days at 37°C in 5% CO2/95% air in complete medium containing serum. All test substances were dissolved in dimethyl-sulfoxide (DMSO). The solvent concentration in the incubation medium never exceeded 0.5%. Control cultures received the equivalent concentration of DMSO. After treatment, cells were incubated for 1 h in the usual culture conditions after addition of the same volume of medium containing MTT (2 mg mL-1). After this incubation, 150 µL HCl (0.1 M) in isopropanol was added to dissolve the blue formazan crystals formed by reduction of MTT. Absorbance at 570 nm using a background reference wavelength of 630 nm was measured using a dual-wavelength plate reader. The mean absorbance values for the negative control (DMSO treated cells) were standardized as 100% absorbance (i.e. no growth inhibition) and results were displayed as absorbance (% of control) vs essential oil concentration. Statistical analysis: Data were analyzed by one-way analysis of variance (ANOVA) using IBM SPSS Statistics version 20. Tukey test was used to determine the difference at the 5% significance level. Paired Student t test was used in some tests to determine differences at 5% significance. Acknowledgements - Partially funded by Fundação para a Ciência e a Tecnologia (FCT) under Pest-OE/EQB/LA0023/2011. Cristina Megias is recipient of a JAE-Doc (C.S.I.C.) contract from the "Junta para la Ampliación de Estudios" program (cofinanced by the European Social Fund). Isabel Cortés-Giraldo is recipient of a JAE-Pre (C.S.I.C.) fellowship from the "Junta para la Ampliación de Estudios" program (cofinanced by the European Social Fund)

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Natural Product Communications Vol. 9 (4) 2014 Published online (www.naturalproduct.us)

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5-(Hydroxymethyl)-2-furaldehyde Inhibits Adipogenic and Enhances Osteogenic Differentiation of Rat Bone Mesenchymal Stem Cells Xiang-ling Tan, Yan-Hong Zhang, Jian-Ping Cai, Li-Hua Zhu, Wen-Jie Ge and Xian Zhang 529

Variation of Glucosinolate Accumulation and Gene Expression of Transcription Factors at Different Stages of Chinese Cabbage Seedlings under Light and Dark Conditions Yeon Bok Kim, Jin-Hyuk Chun, Hye Ran Kim, Sun-Ju Kim, Yong Pyo Lim and Sang Un Park 533

Identification of the Hydroxamate Siderophore Ferricrocin in Cladosporium cladosporioides Nina Pourhassan, René Gagnon, Thomas Wichard and Jean-Philippe Bellenger 539 Structure Characterization and Adhesive Ability of a Polysaccharide from Tendrils of Parthenocissus heterophylla Li Zhang and Wenli Deng 541

Two Peptides, Cycloaspeptide A and Nazumamide A from a Sponge Associated Marine Actinobacterium Salinispora sp. Utpal Bose, Mark P. Hodson, P. Nicholas Shaw, John A. Fuerst and Amitha K. Hewavitharana 545

Full Assignments of the 1H, 13C and 15N Magnetic Resonance Spectra of Two Porphyrin Compounds Qi-Feng Chen, Yao-Nan Wang, Ling Wang, Xi-Xian Jian, Dong-Lin Chen, Ming Zhao and Feng-Peng Wang 547

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Korean Propolis Suppresses Angiogenesis through Inhibition of Tube Formation and Endothelial Cell Proliferation Seon-Il Park, Toshiro Ohta, Shigenori Kumazawa, Mira Jun and Mok-Ryeon Ahn 555

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Cytotoxicity and Antimicrobial Activity of the Essential Oil from Satureja montana subsp. pisidica (Lamiceae) Tatjana Kundaković, Tatjana Stanojković, Branka Kolundžija, Stevan Marković, Branka Šukilović, Marina Milenković and Branislava Lakušić 569

Chemical Composition of Essential Oils of Grindelia squarrosa and G. hirsutula Katalin Veres, Orsolya Roza, Eszter Laczkó-Zöld and Judit Hohmann 573

Seasonal Influence on the Essential Oil of Eucalyptus microcorys Flávia N. M. Oliveira, Gilmara A. C. Fortes, José R. Paula, Pedro H. Ferri and Suzana C. Santos 575

Composition of the Essential Oil of Wild Grown Caraway in Meadows of the Vienna Region (Austria) Remigius Chizzola 581

Volatile Compounds from Roots, Stems and Leaves of Angelica acutiloba growing in Taiwan Hsin-Chun Chen, Yi-Jr Tsai, Li-Yun Lin, Chin-Sheng Wu, Shan-Pao Tai, Yu-Chang Chen and Hsiu-Mei Chiang 583

Anti-oxidant, Anti-inflammatory and Anti-proliferative Activities of Moroccan Commercial Essential Oils Smail Aazza, Badiaa Lyoussi, Cristina Megías, Isabel Cortés-Giraldo, Javier Vioque, A. Cristina Figueiredo and Maria G. Miguel 587

Natural Product Communications 2014

Volume 9, Number 4

Contents

Original Paper Page

Activation of Cell-mediated Immunity by Morinda citrifolia Fruit Extract and Its Constituents Kazuya Murata, Yumi Abe, Megumi Futamura-Masuda, Akemi Uwaya, Fumiyuki Isami, and Hideaki Matsuda 445

A New Pyrrolosesquiterpene from the Terrestrial Streptomyces sp. Hd7-21 Dong-Ze Liu and Bo-Wen Liang 451

Structure-Activity Relationships of Tanshinones in Activating Nrf2. A DFT Study and Implications for Multifunctional Antioxidant Discovery You-Min Sun, Zheng-Tao Xiao and Hong-Yu Zhang 453

Chemical Modifications of Cinchona Alkaloids Lead to Enhanced Inhibition of Human Butyrylcholinesterase Daniela Karlsson, Adyary Fallarero, Pravin Shinde, Anju CP, Igor Busygin, Reko Leino, C. Gopi Mohan and Pia Vuorela 455

Alkaloids from Marine Sponges as Stimulators of Initial Stages of Development of Agricultural Plants Mikhail M. Anisimov, Elena L. Chaikina and Natalia K. Utkina 459

Crinane Alkaloids of the Amaryllidaceae with Cytotoxic Effects in Human Cervical Adenocarcinoma (HeLa) Cells Jerald J. Nair, Lucie Rárová, Miroslav Strnad, Jaume Bastida, Lee Cheesman and Johannes van Staden 461

Alkaloids from Xylariaceae sp., a Marine-derived Fungus Xu-Hua Nong, Xiao-Yong Zhang, Xin-Ya Xu, Yun-Lin Sun and Shu-Hua Qi 467

Occurrence of a Taurine Derivative in an Antarctic Glass Sponge Marianna Carbone, Laura Núñez-Pons, M. Letizia Ciavatta, Francesco Castelluccio, Conxita Avila and Margherita Gavagnin 469

Two New Thyminenol Derivatives from the Marine Sponge Haliclona sp. Bin Wang, Yaocai Lin, Yinning Chen and Riming Huang 471

Decorosides A and B, Cytotoxic Flavonoid Glycosides from the Leaves of Rhododendron decorum Mostafa E. Rateb, Hossam M. Hassan, El-Shaimaa A. Arafa, Marcel Jaspars and Rainer Ebel 473

In vitro Cultures of Bituminaria bituminosa: Pterocarpan, Furanocoumarin and Isoflavone Production and Cytotoxic Activity Evaluation Francesca D’Angiolillo, Laura Pistelli, Cecilia Noccioli, Barbara Ruffoni, Simona Piaggi, Roberto Scarpato and Luisa Pistelli 477

In Vitro Antioxidant Activity and Phenolic Content of Cedrus brevifolia Bark Elena Cretu, Juha-Pekka Salminen, Maarit Karonen, Anca Miron, Christiana Charalambous, Andreas I. Constantinou and Ana Clara Aprotosoaie 481

Evaluation of Bioactive Components and Antioxidant and Anticancer Properties of Citrus Wastes Generated During Bioethanol Production Soon Jae Im, Jae-Hoon Kim and Min Young Kim 483

A New Coumarin and Cytotoxic Activities of Constituents from Cinnamomum cassia Tran Minh Ngoc, Nguyen Xuan Nhiem, Nguyen Minh Khoi, Doan Cao Son, Tran Viet Hung and Phan Van Kiem 487

Coumarin Compounds in Coronilla scorpioides Callus Cultures Anna Piovan, Raffaella Filippini and Gabbriella Innocenti 489

A New Isocoumarin from Cajanus cajan (Fabaceae) Virginia F. Rodrigues, Rodrigo R. Oliveira and Maria Raquel G. Vega 493

Styryllactones and Acetogenins from the Fruits of Goniothalamus macrocalyx Quy Hung Trieu, Huong Doan Thi Mai, Van CuongPham, Marc Litaudon, Françoise Gueritte, Pascal Retailleau, Isabelle Schmitz-Afonso, Olinda Gimello, Van Hung Nguyen and Van Minh Chau 495

Potent Acetylcholinesterase Inhibitory Compounds from Myristica fragrans To Dao Cuong, Tran Manh Hung, Hyoung Yun Han, Hang Sik Roh, Ji-Hyeon Seok, Jong Kwon Lee, Ja Young Jeong, Jae Sue Choi, Jeong Ah Kim and Byung Sun Min 499

Clastogenic Effect of Atranorin, Evernic acid, and Usnic Acid on Human Lymphocytes Gordana S. Stojanović, Miroslava Stanković, Igor Ž. Stojanović, Ivan Palić, Vesna Milovanović and Sofija Rančić 503

MAO-A Inhibition Profiles of Some Benzophenone Glucosides from Gentiana verna subsp. pontica Duygu Kaya, Anna K. Jäger, Funda N. Yalçın and Tayfun Ersöz 505

Phytochemical Investigations of Lonchocarpus Bark Extracts from Monteverde, Costa Rica Caitlin E. Deskins, Bernhard Vogler, Noura S. Dosoky, Bhuwan K. Chhetri, William A. Haber and William N. Setzer 507

Immune Enhancing Effects of Echinacea purpurea Root Extract by Reducing Regulatory T Cell Number and Function Hyung-Ran Kim, Sei-Kwan Oh, Woosung Lim, Hyeon Kook Lee, Byung-In Moon and Ju Young Seoh 511

Isocorilagin, a Cholinesterase Inhibitor from Phyllanthus niruri Yee-Hui Koay, Alireza Basiri, Vikneswaran Murugaiyah and Kit-Lam Chan 515

Antioxidant Activity and Phenolic Content of Bergenia crassifolia, B. x ornata and B. ciliata Helena Hendrychová, Anna Vildová, Nina Kočevar-Glavač, Lenka Tůmová, Elnura Abdykerimova Kanybekovna and Jiří Tůma 519

Antioxidant Activity and Total Phenolic Contents of Three Bupleurum Taxa Hyeusoo Kim, Sea Hyun Kim and Kyeong Won Yun 523

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