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Chemical Composition and Biological Activities of the Essential Oil of Athanasia brownii Hochr. (Asteraceae) Endemic to Madagascar by Philippe Rasoanaivo a ) b ), Richard Fortune´ Randriana a ), Filippo Maggi* c ), Marcello Nicoletti d ), Luana Quassinti c ), Massimo Bramucci c ), Giulio Lupidi c ), Dezemona Petrelli e ), Luca A. Vitali c ), Fabrizio Papa c ), and Sauro Vittori c ) a
) De´partement de Ge´nie Chimique, Ecole Supe´rieure Polytechnique, Universite´ dAntananarivo, Antananarivo, Madagascar b ) Institut Malgache de Recherches Applique´es, Antananarivo, Madagascar c ) School of Pharmacy, University of Camerino, Via Pontoni 5, I-62032 Camerino (phone: þ 39 0737 404506; fax: þ 39 0737 404508; e-mail: [email protected]) d ) Department of Environmental Biology, La Sapienza University, I-Rome e ) School of Biosciences and Biotechnology, University of Camerino, I-Camerino
The essential oil obtained from hydrodistillation of flowering aerial parts of Athanasia brownii (Asteraceae) was studied for its chemical composition by GC/FID and GC/MS, and for biological activity, namely, antioxidant, antimicrobial, and chemopreventive potential, by DPPH ( ¼ 2,2-diphenyl-1picrylhydrazyl), ABTS ( ¼ 2,2’-azinobis[3-ethylbenzothioline-6-sulfonic acid), and FRAP ( ¼ ferric reducing antioxidant power), disk diffusion test, and MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, respectively. The oil was characterized by a high content of oxygenated sesquiterpenes (71.2%), with selin-11-en-4a-ol (24.6%), caryophyllene oxide (8.7%), humulene epoxide II (5.1%), and (E)-nerolidol (4.9%) as the predominant compounds. The oil showed a moderate activity against streptococci as well as radical-scavenging potential, while the inhibitory effects against human cancer cells examined such as A375 (malignant melanoma) and HCT 116 (colon carcinoma) were significant, with IC50 values of 19.85 and 29.53 mg/ml, respectively.
Introduction. – In an inventory of aromatic plants of Madagascar, 110 species were identified as having aromatic properties, of which 52 are introduced, 50 are endemic to the island, and eight are autochthon [1]. The first investigations of aromatic plants growing in Madagascar were mainly focused on introduced species such as YlangYlang, Ravintsara, cloves, cinnamon, pepper, etc. for commercial purposes [2]. Since 1970, increasing attention has been paid to the exploration of endemic aromatic species for scientific and economic reasons. In our continuing efforts directed towards the investigation of endemic aromatic plants of Madagascar [3] [4], we studied the chemical composition and the biological activities of Athanasia brownii Hochr. (syn. Inulanthera brownii (Hochr.) Kllersj˛) [5]. The genus Inulanthera, which is found in southwestern Africa, was formerly included in Athanasia, but now it is considered as a well-established taxon on the basis of morphological and chemotaxonomic evidence [6]. This plant, endemic to Madagascar, is the only species of the genus Athanasia found in the island. It is known under the vernacular name ramijengy, and found growing wild in the Central and middle South Highlands. 2013 Verlag Helvetica Chimica Acta AG, Zrich
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From a phytochemical point of view, some secondary metabolites were previously isolated from the genus Athanasia such as furanosesquiterpenes and keto lactones [7]. Herein, we report for the first time the chemical composition of the essential oil obtained from the aerial parts of A. brownii, together with biological properties, namely antioxidant, antimicrobial, and antiproliferative activities. Results and Discussion. – 1. Composition of the Essential Oil. Here, we present the first phytochemical investigation of A. brownii essential oil the constituents of which are compiled in Table 1. A total of 73 components were identified, corresponding to 84.4% of the total composition. The essential oil was dominated by the fraction of oxygenated sesquiterpenes (71.2%), with selin-11-en-4-a-ol (24.6%), caryophyllene oxide (8.7%), humulene epoxide II (5.1%), and (E)-nerolidol (4.9%) as the major compounds (Fig.).
Figure. Chemical structures of the major volatiles of Athanasia brownii. a) Selin-11-en-4-a-ol, b) caryophyllene oxide, c) humulene epoxide II, d) (E)-nerolidol.
In this fraction, it is notable that two unknown compounds (4.5 and 2.3%, resp.) occur, whose mass-fragmentation patterns are indicated in Table 1. A minor contribution was provided by sesquiterpene hydrocarbons (11.9%), with (E)-caryophyllene (2.1%), a-humulene (2.4%), and ar-curcumene (1.5%) as the most abundant. Finally, the fraction of monoterpenes was poor (6.1%), with 1,8-cineole (1.8%) as the most abundant. The eudesmane alcohol selin-11-en-4-a-ol was first isolated from the leaves of Podocarpus dacrydioides [11]. Significant amounts of this sesquiterpene were found in the family of Asteraceae, notably in the essential oil of Artemisia asiatica Nakai (12.32%). Selin-11-en-4a-ol was established to possess a high activity against fungi, but a low activity against bacteria and especially against Gram-negative bacteria [12]. This compound was found also in soft corals, and it was effective in inhibiting the proliferation of murine leukemia P388 cell lines [13]. Caryophyllene oxide is the oxidation product of (E)-caryophyllene and is one of the oxygenated sesquiterpenes most frequently detected in essential oils. It has been approved by the FDA (US Food and Drug Administraion) as a food and cosmetic preservative, and has been included by the European Council in the list of natural and synthetic flavoring substances. Previously, it was established to be effective in the treatment of onychomycosis [14],
RF b )
1.08 1.08 1.08 1.08 1.08 1.45 1.09 1.08 1.40 1.08 1.40 1.45 1.65 0.96 1.45 1.40 1.40 1.40 1.45 1.40 1.45 1.68 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.40 1.05
Component a )
a-Pinene Camphene Sabinene b-Pinene Myrcene Isoamyl isobutyrate p-Cymene Limonene 1,8-Cineole g-Terpinene Linalool Isopentyl 2-methylbutanoate Nonanal Undecane Isopentyl isovalerate Borneol Terpinen-4-ol a-Terpineol Isobornyl formate Piperitone Bornyl acetate Eugenol a-Copaene b-Bourbonene b-Panasinsene (E )-Caryophyllene a-trans-Bergamotene Aromadendrene a-Humulene allo-Aromadendrene Geranylacetone (E )-b-Farnesene
Entry
1 2 3 4 5 6 7 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
932 945 971 974 991 1017 1026 1030 1032 1061 1100 1100 1106 1100 1108 1164 1176 1189 1234 1253 1283 1358 1370 1377 1389 1409 1432 1440 1445 1452 1455 1458
HP-5MS
RI (calc.) c )
1586 1580 1632 1658 1634 1854 1666
1297 1700 1601 1700 1239 1716 1576 2163 1484 1509
1270 1198 1211 1247 1556 1281 1393
1026 1070 1124 1112 1166
DB-Wax 939 954 975 979 990 1009 1024 1029 1031 1059 1096 1100 1100 1100 1103 1169 1177 1188 1568 1252 1288 1359 1376 1388 1382 1419 1434 1441 1454 1460 1455 1456
ADAMS 931 945 971 973 989 1017 1026 1028 1032 1062 1101 1100 1106 1100 1109 1164 1176 1189 1239 1253 1284 1358 1370 1377 1385 1410 1432 1440 1445 1452 1455 1458
NIST08
apolar phase
RI (lit.) d )
Table 1. Essential-Oil Composition of Athanasia brownii
polar phase
1587 1586 1632 1658 1635 1854 1666
1296 1700 1599 1700 1574 h ) 1717 1575 2164 1485 1510
1270 1198 1211 1247 1558 1280 1393
1027 1070 1124 1113 1167
NIST08 0.6 0.1 0.1 0.2 0.4 0.2 0.1 0.3 1.8 0.2 0.4 0.1 Tr g ) Tr 0.1 0.4 0.3 0.1 0.1 0.1 0.2 0.1 Tr Tr 0.3 2.1 0.3 0.1 2.4 0.3 1.3 0.1
% e)
Std Std RI, MS Std Std RI, MS Std Std Std Std Std RI, MS RI, MS Std RI, MS Std Std Std RI, MS Std Std Std RI, MS RI, MS RI, MS Std RI, MS RI, MS Std RI, MS RI, MS RI, MS
ID f )
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Germacrene D g-Selinene ar-Curcumene d-Selinene a-Muurolene b-Dihydroagarofuran b-Bisabolene 7-epi-a-Selinene d-Cadinene a-Agarofuran a-Calacorene cis-Cadinene ether b-Calacorene Palustrol ( E)-Nerolidol Spathulenol Caryophyllene oxide Viridiflorol Humulene epoxide II 10-epi-g-Eudesmol 1,10-Diepicubenol Muurola-4,10(14)-dien-1b-ol Caryophylla-4(12),8(13)-dien-5a-ol Caryophylla-4(12),8(13)-dien-5b-ol a-Eudesmol Selin-11-en-4-a-ol ( Z)-a-Santalol M þ 222, 69 (100), 95 (96), 109 (87), 179 (72), 41 (67), 93 (60), 137 (55), 107 (2), 55 (2), 67 (2) Helifolenol A a-Bisabolol Eudesma-4(15),7-dien-1b-ol
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
Component a )
Entry
Table 1 (cont.)
1.20 1.20 1.20
1.05 1.05 1.05 1.05 1.05 1.20 1.05 1.05 1.05 1.20 1.05 1.20 1.05 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20
RF b )
1676 1679 1685
1473 1475 1479 1483 1494 1496 1504 1507 1517 1535 1535 1548 1558 1558 1564 1568 1572 1592 1597 1608 1620 1622 1622 1627 1644 1649 1656 1663
HP-5MS
RI (calc.) c )
2299
2026 2099 2061 2308 2288 2294 2215 2226 2372 2282
1680 1767 1713 1716 1709 1721 1749 1749 1877 1901 1872 1945 1918 2049 2123 1969
DB-Wax
1675 1685 1688
1480 1492 1500 1503 1505 1522 1523 1550 1545 1553 1568 1568 1563 1578 1583 1592 1608 1623 1619 1631 1640 1640 1653 1659 1675
1485
ADAMS
1680
1644 1648 h ) 1660
1625 h )
1548 h ) 1557 1564 1569 1572 1592 1598 1609 h )
1473 1477 1479 1483 1494 1495 h ) 1504 1514 h ) 1517 1536 h ) 1528 h )
NIST08
apolar phase
RI (lit.) d ) polar phase
2212 2241 h )
2026 2094 h )
1915 2049 2124 1967
1749 1861 h ) 1906 h )
1685 1767 1717 1716 1706 h ) 1720
NIST08
0.7 Tr 0.9
0.1 1.1 1.5 0.7 0.2 0.3 0.4 1.2 0.8 0.2 0.2 0.2 0.2 0.2 4.9 0.6 8.7 0.8 5.1 1.4 2.4 1.1 1.1 1.7 1.7 24.6 0.7 4.5
% e)
RI, MS RI, MS RI, MS
RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS Std RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS Std RI, MS Std RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS MS i )
ID f )
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1.45 1.20 1.43
1.20 1.20 1.20 1.20 1.45 1.20 1.20 1.20 1.20
RF b )
1773 1783 2166
1694 1697 1700 1704 1714 1720 1740 1753 1759
HP-5MS
RI (calc.) c )
2562 2829
2542 2577 2822
2394
2375
DB-Wax
1788 1789
1714 1690 1717 1725 1756
1689
ADAMS
2201
1703 1715
NIST08
apolar phase
RI (lit.) d ) NIST08
polar phase
1.8 4.3 11.9 71.2 1.3 0.7
84.4
0.5 1.1 0.4
0.6 3.2 Tr 1.1 0.1 0.4 0.6 0.2 2.3
% e)
RI, MS RI, MS MS i )
RI, MS MS i ) RI, MS RI, MS RI, MS RI, MS RI ,MS MS i ) MS i )
ID f )
a ) Compounds are listed in order of their elution from a HP-5MS column. Their nomenclature was in accordance with Adams [8]. b ) Relative response factor ( RF ) of FID detector for the main chemical groups occurring in essential oil. c ) Linear retention index on HP-5MS and DB-Wax column. Experimentally determined using homologous series of C8 – C30 alkanes. d ) Linear retention index taken from Adams [8] and/or NIST08 [9]) for apolar columns, and from NIST08 for polar columns. e ) Percentage values are means of three determinations, with a RSD% for the main components below 5% in all cases. f ) Identification methods: Std, based on comparison with authentic compounds; MS, based on comparison with Wiley, Adams, and NIST08 MS database; RI, based on comparison of RI values with those reported in Adams and NIST08. g ) Tr, traces (mean value below 0.1%). h ) RI Values taken from The Pherobase, database of pheromones and semiochemicals [10]. i ) Tentative identification.
Total identified [%] Grouped compounds [%] Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Esters Others
( Z )-Apritone Cedr-8-en-15-ol 14-Hydroxy-a-humulene ( Z )-a-trans-Bergamotol ( E )-Nerolidyl acetate ( Z )-Nuciferol ( E )-Nuciferol 3,7,11-Trimethyldodeca-2,6,10-trienoic acid M þ 220, 93 (100), 43 (50), 133 (44), 79 (42), 91 (40), 119 (36), 41 (34), 121 (33), 120 (32), 69 (31) 8-Cedren-13-ol acetate b-Bisabolenol Geranylgeraniol
64 65 66 67 68 69 70 71 72
73 74 75
Component a )
Entry
Table 1 (cont.)
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and as potential therapeutic candidate for both the prevention and treatment of human prostate and breast cancers [15]. Humulene epoxide II is an oxidation product of ahumulene and it has been suggested to contribute a hoppy aroma in beer [16]. The tertiary alcohol (E)-nerolidol constitutes the major floral volatile in the commercially important kiwifruit species (Actinidia chinensis) as well as in strawberry, grape, snapdragon, and maize. This terpene is likely to play a role in attracting pollinators or seed dispersal agents, and it is also produced by plants in response to insect herbivory [17]. Within the Asteraceae, this compound was found in high amount in Baccharis dracunculifolia D.C. [18]. (E)-Nerolidol is approved by the FDA as a food-flavoring agent, and it currently undergoes testing as a skin penetration enhancer for the transdermal delivery of therapeutic drugs. Further, it was shown to inhibit carcinogenesis on azoxymethane-induced neoplasia in rats [19] by an uncharacterized mechanism, and it was found effective against some Leishmania species [20]. 2. Antioxidant Activity. We evaluated the antioxidant power of the essential oil from A. brownii using Trolox as positive control. Results are compiled in Table 2. Of the various chemical methods in practice to evaluate the antioxidant property, DPPH and ABTS methods are the best and commonly accepted. These methods are based on the scavenging ability of antioxidants (test molecules) towards the stable 2,2-diphenyl-1picrylhydrazyl radical and 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid, respectively. Table 2. In vitro Radical-Scavenging Activities of the Essential Oil of Athanasia brownii DPPH
Essential oil Trolox
ABTS
FRAP
TEAC a ) [mmol TE/g]
IC50 b ) [mg/ml]
TEAC a ) [mmol TE/g]
IC50 b ) [mg/ml]
TEAC a ) [mmol TE/g]
441 ( 5.2)
910 ( 34) 10.1 ( 0.2)
167 ( 3.7)
880 ( 28) 3.7 ( 0.2)
17.2 ( 0.8)
a
) TEAC, Trolox equivalent ( TE) antioxidant concentration. b ) IC50 , the concentration of compound that affords a 50% reduction in the assay.
From our data, the essential oil exhibited antioxidant activities with IC50 values of 100- and 200-fold lower than that of Trolox in DPPH and ABTS assays, respectively. The FRAP ( ¼ ferric reducing antioxidant power) assay revealed that A. brownii oil have a low capacity for iron binding that is related to its possible action as peroxidation protector. Our results showed that the essential oil has a mild ability as a free-radical inhibitor. It is very difficult to attribute the antioxidant effect to one or a few active principles, because it contains a mixture of different chemical compounds among which also minor compounds may contribute to the oils activity. 3. Antimicrobial Activity. In Table 3, the antimicrobial activities are compiled, expressed as inhibition zone diameters for all the microorganisms screened. The essential oil exhibited a modest inhibition, as evidenced by the modest inhibition zone diameters (7 to 10 mm), against the growth of Gram-positive bacteria, with a stronger activity against streptococci than S. aureus and E. faecalis. P. aeruginosa and E. coli growth was not affected by the essential oil, while an appreciable activity
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Table 3. In vitro Antimicrobial Activities of the Essential Oil from Athanasia brownii Microorganism
Inhibition zone diameter [mm] a )
Staphylococcus aureus Enterococcus faecalis Escherichia coli Pseudomonas aeruginosa Streptococcus pyogenes Streptococcus pneumoniae Candida albicans
71 61 60 60 10 1 10 1 71
a ) Values refer to the diameter of the inhibition zone SD. Sterile filter paper discs (6 mm in diameter) were placed on the surface of inoculated plates and spotted with 10 ml of a 1 : 1 dilution of each oil in DMSO.
was exhibited against the fungus C. albicans. Taking into account the high fraction of selin-11-en-4a-ol, and hypothesizing its dominant putative contribution to the overall activity of the oil, our results support those reported for Artemisia asiatica Nakai [12]. 4. Antiproliferative Activity. The essential oil of A. brownii was tested in vitro for their potential tumor cell growth-inhibitory effect on MDA-MB 231 human breast adenocarcinoma, A375 human malignant melanoma, and HCT116 human colon carcinoma cell lines, by using MTT assay. The results, collected in Table 4, show that the essential oil exhibited an inhibitory effect against the human cancer cells examined, although significantly lower than that of the anticancer drug cisplatin. The highest activity was observed on A375 cell line, with an IC50 value of 19.9 mg/ml. Lower activity was obtained on MDA-MB 231, with an IC50 value of 51.7 mg/ml, while medium was the inhibition against HCT 116 (IC50 of 29.53 mg/ml). The cytotoxic activity of the A. brownii essential oil may be attributed to specific components of the oil. A few of the compounds found in A. brownii essential oil have been tested for cytotoxic properties. Of the major compounds present in the oil, only selin-11-en-4a-ol (24.6%) appears to possess some cytotoxic activity against mouse lymphocytic leukemia P-388 [13]. There is still controversy over whether caryophyllene oxide (8.7%) is cytotoxic or not. Kubo et al. [21] and Sibanda et al. [22] reported that it exhibited a modest cytotoxic activity, while some reports stated that it Table 4. In vitro Cytotoxic Activities of Essential Oil from Athanasia brownii Cell line ( IC50 [mg/ml]) a )
A. brownii essential oil 95% C.I. Reference Cisplatin 95% C.I.
MDA-MB 231 b )
HCT116 c )
A375 d )
51.7 48.3 – 55.3
29.5 27.9 – 31.2
19.9 16.8 – 23.5
2.47 2.18 – 2.73
2.79 2.54 – 2.95
0.34 0.29 – 0.38
a ) IC50 , The concentration of compound that affords a 50% reduction in cell growth (after 72 h of incubation). C.I., Confidence Interval. b ) Human breast adenocarcinoma cell line. c ) Human colon carcinoma cell line. d ) Human malignant melanoma cell line.
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was inactive against tumor cell lines [23 – 25]. Recently, nerolidol was shown to be cytotoxic against Balb/c 3T3-A31 fibroblast [26]. However, the low concentrations of selin-11-en-4a-ol (24.6%), caryophyllene oxide (8.7%), and nerolidol (4.9%) cannot fully explain the cytotoxic activity of A. brownii essential oil, including that some other compounds, probably sesquiterpenes, are active in the essential oil. It is also possible that minor components may be involved in some type of synergism with the other active compounds [27] [28]. Conclusions. – The essential oil from the aerial parts of A. brownii endemic to Madagascar was investigated for the first time. The majority of the oil was constituted by oxygenated sesquiterpenes, among which selin-11-en-4a-ol was the most abundant compound. The antimicrobial activity and the antioxidant potential of the oil were negligible. The cytotoxic effects exhibited particularly on malignant melanoma cell line encourage further investigations of the mechanisms of action of the oil, and in vivo studies for applications as natural anticancer drug. Experimental Part Plant Material. Aerial parts of Athanasia brownii were collected in Ambatofotsy-Ambalavao (central highlands of Madagascar) at ca. 30 km from Antananarivo, in the Route Nationale 7, in August, 2012, during flowering. The plant was identified by botanists at the Department of Botany of the Parc Botanique et Zoologique de Tsimbazaza, Antananarivo, by comparaison with a authentic specimen. A voucher specimen was deposited with the Herbarium of the Institut Malgache de Recherches Applique´es (IMRA), under the accession code MAD-0917 2.2. Extraction of Essential Oil. The essential oil was obtained from freshly collected aerial parts (5 kg) of A. brownii by steam distillation for 3 h using a field alembic to yield 0.004% of a yellowish oil. The oil yield was estimated on a dry-weight basis (w/w). Before analysis, the oil was dried (Na2SO4 ), transferred into an amber glass flask, and kept at 208 before GC and biological experiments. Chemicals. For identification of volatiles the following anal. standards purchased from SigmaAldrich (I-Milan) were used: a-pinene, camphene, b-pinene, myrcene, p-cymene, limonene, 1,8-cineole, g-terpinene, linalool, n-undecane, borneol, terpinen-4-ol, a-terpineol, piperitone, bornyl acetate, eugenol, (E)-caryophyllene, a-humulene, b-bisabolene, (E)-nerolidol, caryophyllene oxide. For retention-index (RI) determination, a mixture of hydrocarbons, ranging from octane (C8 ) to triacontane (C30) (Supelco, USA), was used and run under the exper. conditions reported below. All compounds were of anal. standard grade. Anal.-grade hexane solvent was purchased from Carlo Erba (I-Milan); it was successively distilled through a Vigreux column before use. GC/FID and GC/MS Analysis. For GC separations, an Agilent 4890D instrument coupled to an ionization flame detector (FID) was used. Volatile components were separated on a HP-5 cap. column (5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d.; 0.17-mm film thickness; J and W Scientific, Folsom, CA), with the following temp. program: 5 min at 608, subsequently 48/min up to 2208, then 118/min up to 2808, held for 15 min, for a total run of 65 min. Injector and detector temps. were 2808. He was used as the carrier gas, at a flow rate of 1.4 ml/min; injection volume, 1 ml; split ratio, 1 : 34. A mixture of aliphatic hydrocarbons (C8 – C30 ; Sigma, I-Milan) in hexane was directly injected into the GC injector under the above temp. program, in order to calculate the retention index (RI) of each compound. Oil samples were diluted to 1 : 100 in hexane and injected at a volume of 1 ml. Analysis was repeated three times. Data were collected by using HP3398A GC Chemstation software (Hewlett Packard, Rev. A.01.01). The rel. amounts of essential-oil components, expressed as percentages, were obtained by FID peak-area normalization by calculating the response factor (RF) of the FID for nine different classes of volatiles occurring in the essential oil analyzed [4]. GC/MS Analysis was performed on an Agilent 6890N gas chromatograph coupled to a 5973N mass spectrometer using two different columns: a HP-5 MS (5%
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phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1-mm film thickness; J & W Scientific, Folsom), and a DB-WAX (polyethylene glycol; 30 m, 0.25 mm i.d., 0.25-mm film thickness; J & W Scientific, Folsom) cap. columns. The temp. program was the same as described above. Injector and detector temps. were 2808. He was used as the carrier gas, at a flow rate of 1 ml/min. Split ratio, 1 : 50; acquisition mass range, m/ z 29 – 400. All mass spectra were acquired in electron-impact (EI) mode with an ionization voltage of 70 eV. Oil samples were diluted to 1 : 100 in hexane, and the volume injected was 2 ml. Data were analyzed by using MSD ChemStation software (Agilent, Version G1701DA D.01.00). About half of the constituents (21 out of 73 compounds), corresponding to 23.8% of essential oil, were identified by coinjection with authentic standards. Otherwise, the peak assignment was carried out on the basis of the standard of the International Organization of the Flavor Industry (IOFI, http://www.iofi.org/) statement [29], i.e., by the interactive combination of chromatographic linear RI values that were consistent with those reported in [8 – 10] for apolar and polar stationary phases, and MS data consisting in the computer matching with the WILEY275, NIST 08, Adams, and a home-made (based on the analyses of reference oils and commercially available standards) libraries. Evaluation of Antioxidant Activity. DPPH Free-radical scavenging activity was evaluated on a microplate anal. assay according to the procedure described in [30], while radical-scavenging capacity (ABTS assay) was evaluated by a modified method originally described by Re et al. [31] for application to a 96-well microplate assay [32]. Trolox was used as the positive control. Determination of antioxidant activity by ferric reducing antioxidant power (FRAP) assay was carried out according to Mller et al. [33]. The ability of the test samples to scavenge the different radicals in the assays was compared to that of Trolox used as standard, and the activity of the essential oil was expressed as tocopherol equivalent antioxidant capacity mmol TE/gr of product. Each experiment was repeated three times. Antimicrobial Activity. Plain essential oil was tested directly against a panel of microorganisms including Staphylococcus aureus ATCC 25923 (American Type Culture Collection, Rockville, MD, USA), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Streptococcus pyogenes, Streptococcus pneumoniae, and Candida albicans ATCC 24433. Growth conditions and antimicrobial activity tests were as described in [3]. Briefly, a suspension of the tested microorganism (1 – 2 108 cells per ml in saline/106 per ml for Candida) was spread on the solid media plates using a sterile cotton swab. Sterile paper discs (6-mm in diameter) were placed on the surface of inoculated plates and spotted with 10 ml of a 1 : 1 dilution of each oil in DMSO. The plates were incubated 18 h at 35 18 (48 h for C. albicans). Streptococcus spp. were incubated in 5% CO2 atmosphere. The diameters of zone inhibition (including the 6-mm disc) were measured with a calliper. A reading of more than 6 mm indicated growth inhibition. No zone inhibition was observed using DMSO alone. Ciprofloxacin (5 mg disc) and Fluconazole (25 mg disc) were used as reference antimicrobials against bacteria and fungi, resp. Each test was repeated three times. MTT Cytotoxicity Assay. HCT116 Cells (human colon carcinoma cells) were cultured in RPMI1640 medium with 2 mm l-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; PAA Laboratories GmbH, Austria), MDA-MB 231 (human breast adenocarcinoma cells) and A375 cells (human malignant melanoma cells) were cultured in Dulbeccos Modified Eagles Medium (DMEM) with 2 mm l-glutamine, 100 IU/ml penicillin, 100 mg/ ml streptomycin, and supplemented with 10% HI-FBS. Cells were cultured in a humidified atmosphere at 378 in presence of 5% CO2 . The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay was used as a relative measure of cell viability. Cell-viability assays were carried out as described in [34]. Briefly, cells were seeded at the density of 2 104 cells/ml. After 24 h, samples were exposed to different concentrations of essential oil (1.56 – 400 mg/ml). Cells were incubated for 72 h in a humidified atmosphere of 5% CO2 at 378. At the end of incubation, each well received 10 ml of MTT (5 mg/ml in phosphate-buffered saline (PBS)), and the plates were incubated for 4 h at 378. The extent of MTT reduction was measured spectrophotometrically at 540 nm using a Titertek Multiscan microElisa (Labsystems, FI-Helsinki). Experiments were conducted in triplicate. The chemotherapy drug cisplatin was used as a reference compound. Cytotoxicity was expressed as the concentration of the essential oil inhibiting cell growth by 50% (IC50). The IC50 values were determined with GraphPad Prism 4 computer program (GraphPad Software, S. Diego, CA, USA).
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Statistical Analysis. Each experiment was repeated at least three times. The Unpaired t test was used for comparisons between two groups. Statistical significance was indicated by p-values < 0.001. Data are reported as the mean SD. The authors are grateful to Prof. Suzanne Ratsimamanga, President of the Institut Malgache de Recherches Applique´es, for her precious support during this study. We thank the local communities of Analalava for helping us to collect A. brownii.
REFERENCES [1] L. H. Rakotovao, E. Randrianjohany, in Bioge´ographie de Madagascar, Ed. R. Wilson LourenÅo, ORSTOM Publications, Paris, 1996, p. 187. [2] P. Rasoanaivo, P. de la Gorce, Herbalgram 1998, 43, 31. [3] M. Nicoletti, F. Maggi, F. Papa, S. Vittori, L. Quassinti, M. Bramucci, G. Lupidi, D. Petrelli, L. A. Vitali, E. Ralaibia, P. Rasoanaivo, Nat. Prod. Res. 2012, 26, 2291. [4] F. Maggi, R. F. Randriana, P. Rasoanaivo, M. Nicoletti, L. Quassinti, M. Bramucci, G. Lupidi, D. Petrelli, L. A. Vitali, F. Papa, S. Vittori, Chem. Biodiversity 2013, 10, 356. [5] H. Humbert, in Flore de Madagascar et des Comores, Compose´es, 189e Famille, Tome III, Publication du Muse´um National d Histoire Naturelle, Paris, 1963, p. 670. [6] M. Kllersjç, Nordic J. Bot. 1985, 5, 527. [7] C. Zdero, L. Lehmann, F. Bohlmann, Phytochemistry 1991, 30, 1161. [8] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th edn., Allured, Carol Stream, 2007. [9] NIST 08, Mass spectral library (NIST/EPA/NIH) National Institute of Standards and Technology, Gaithersburg, USA, 2008. [10] The Pherobase, Database of Pheromones and Semiochemicals, http://www.pherobase.com. [11] R. E. Corbett, R. A. J. Smith, Tetrahedron Lett. 1967, 11, 1009. [12] D. Kalemba, D. Kusewicz, K. S´wia˛der, Phytother. Res. 2002, 16, 288. [13] C. Duh, S. Wang, Y. Weng, M. Y. Chiang, C. Dai, J. Nat. Prod. 1999, 62, 1518. [14] D. Yang, L. Michel, J. Chaumont, J. Millet-Clerc, Mycopathologia 1999, 148, 79. [15] K. Park, D. Nam, H. Yun, S. Lee, H. Jang, G. Sethi, S. K. Cho, K. S. Ahn, Cancer Lett. 2011, 312, 178. [16] X. Yang, C. Lederer, M. McDaniel, M. Deinzer, J. Agric. Food Chem. 1993, 41, 1300. [17] S. A. Green, X. Chen, N. J. Nieuwenhuizen, A. J. Matich, M. Y. Wang, B. J. Bunn, Y. Yauk, R. G. Atkinson, J. Exp. Bot. 2011, 63, 1951. [18] J. P. B. De Sousa, R. F. Jorge, M. F. Leite, N. A. J. C. Furtado, J. K. Bastos, A. A. Da Silva Filho, C. L. Queiroga, P. M. De Magalhaes, A. E. E. Soares, J. Essent. Oil Res. 2009, 21, 308. [19] L. W. Wattenberg, Carcinogenesis 1991, 12, 151. [20] D. C. Arruda, F. L. DAlexandri, A. M. Katzin, S. R. B. Uliana, Antimicrob. Agents Chemother. 2005, 49, 1679. [21] I. Kubo, S. K. Chaudhuri, Y. Kubo, Y. Sanchez, T. Ogura, T. Saito, H. Ishikawa, H. Haraguchi, Planta Med. 1996, 62, 427. [22] S. Sibanda, G. Chigwada, M. Poole, E. T. Gwebu, J. A. Noletto, J. M. Schmidt, A. I. Rea, W. N. Setzer, J. Ethnopharmacol. 2004, 92, 107. [23] N. Kanada, J. M. Pezzuto, A. D. Kinghorn, N. R. Earnsworth, T. Santisuk, P. Tuchinda, J. Udchachon, V. Reutrakul, J. Nat. Prod. 1992, 55, 654. [24] J. Legault, W. Dahl, E. Debition, A. Pichette, J. C. Maldemont, Planta Med. 2003, 69, 402. [25] Y. Tambe, H. Tsujiuchi, G. Honda, Y. Ikeshiro, S. Tanaka, Planta Med. 1996, 62, 469. [26] S. A. Mendanha, S. S. Moura, J. L. Anjos, M. C. Valadares, A. Alonso, Toxicol. In Vitro 2013, 27, 323. [27] N. Lattaoui, A. Tantaoui-Elaraki, Riv. Ital. EPPOS 1994, 13, 13. [28] J. Q. Yu, J. Q. Lei, H. D. Yu, X. Cai, G. L. Zou, Phytochemistry 2004, 65, 881. [29] C. Bicchi, C. Cagliero, P. Rubiolo, Flavour Fragrance J. 2011, 26, 321. [30] R. Srinivasan, M. J. N. Chandrasekar, M. J. Nanjan, B. Suresh, J. Ethnopharmacol. 2007, 113, 284.
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[31] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free Radic. Biol. Med. 1999, 26, 1231. [32] J. R. Esparza Rivera, M. B. Stone, C. Stushnoff, E. Pilon-Smits, P. A. Kendall, J. Food Sci. 2006, 71, S270. [33] L. Mller, K. Frçhlich, V. Bçhm, Food Chem. 2011, 129, 139. [34] L. Quassinti, G. Lupidi, F. Maggi, G. Sagratini, F. Papa, S. Vittori, A. Bianco, M. Bramucci, Nat. Prod. Res. 2013, 27, 862. Received May 6, 2013
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Chemical Composition and Biological Activities of the Essential Oil of Athanasia brownii Hochr. (Asteraceae) Endemic to Madagascar by Philippe Rasoanaivo a ) b ), Richard Fortune´ Randriana a ), Filippo Maggi* c ), Marcello Nicoletti d ), Luana Quassinti c ), Massimo Bramucci c ), Giulio Lupidi c ), Dezemona Petrelli e ), Luca A. Vitali c ), Fabrizio Papa c ), and Sauro Vittori c ) a
) De´partement de Ge´nie Chimique, Ecole Supe´rieure Polytechnique, Universite´ dAntananarivo, Antananarivo, Madagascar b ) Institut Malgache de Recherches Applique´es, Antananarivo, Madagascar c ) School of Pharmacy, University of Camerino, Via Pontoni 5, I-62032 Camerino (phone: þ 39 0737 404506; fax: þ 39 0737 404508; e-mail: [email protected]) d ) Department of Environmental Biology, La Sapienza University, I-Rome e ) School of Biosciences and Biotechnology, University of Camerino, I-Camerino
The essential oil obtained from hydrodistillation of flowering aerial parts of Athanasia brownii (Asteraceae) was studied for its chemical composition by GC/FID and GC/MS, and for biological activity, namely, antioxidant, antimicrobial, and chemopreventive potential, by DPPH ( ¼ 2,2-diphenyl-1picrylhydrazyl), ABTS ( ¼ 2,2’-azinobis[3-ethylbenzothioline-6-sulfonic acid), and FRAP ( ¼ ferric reducing antioxidant power), disk diffusion test, and MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, respectively. The oil was characterized by a high content of oxygenated sesquiterpenes (71.2%), with selin-11-en-4a-ol (24.6%), caryophyllene oxide (8.7%), humulene epoxide II (5.1%), and (E)-nerolidol (4.9%) as the predominant compounds. The oil showed a moderate activity against streptococci as well as radical-scavenging potential, while the inhibitory effects against human cancer cells examined such as A375 (malignant melanoma) and HCT 116 (colon carcinoma) were significant, with IC50 values of 19.85 and 29.53 mg/ml, respectively.
Introduction. – In an inventory of aromatic plants of Madagascar, 110 species were identified as having aromatic properties, of which 52 are introduced, 50 are endemic to the island, and eight are autochthon [1]. The first investigations of aromatic plants growing in Madagascar were mainly focused on introduced species such as YlangYlang, Ravintsara, cloves, cinnamon, pepper, etc. for commercial purposes [2]. Since 1970, increasing attention has been paid to the exploration of endemic aromatic species for scientific and economic reasons. In our continuing efforts directed towards the investigation of endemic aromatic plants of Madagascar [3] [4], we studied the chemical composition and the biological activities of Athanasia brownii Hochr. (syn. Inulanthera brownii (Hochr.) Kllersj˛) [5]. The genus Inulanthera, which is found in southwestern Africa, was formerly included in Athanasia, but now it is considered as a well-established taxon on the basis of morphological and chemotaxonomic evidence [6]. This plant, endemic to Madagascar, is the only species of the genus Athanasia found in the island. It is known under the vernacular name ramijengy, and found growing wild in the Central and middle South Highlands. 2013 Verlag Helvetica Chimica Acta AG, Zrich
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From a phytochemical point of view, some secondary metabolites were previously isolated from the genus Athanasia such as furanosesquiterpenes and keto lactones [7]. Herein, we report for the first time the chemical composition of the essential oil obtained from the aerial parts of A. brownii, together with biological properties, namely antioxidant, antimicrobial, and antiproliferative activities. Results and Discussion. – 1. Composition of the Essential Oil. Here, we present the first phytochemical investigation of A. brownii essential oil the constituents of which are compiled in Table 1. A total of 73 components were identified, corresponding to 84.4% of the total composition. The essential oil was dominated by the fraction of oxygenated sesquiterpenes (71.2%), with selin-11-en-4-a-ol (24.6%), caryophyllene oxide (8.7%), humulene epoxide II (5.1%), and (E)-nerolidol (4.9%) as the major compounds (Fig.).
Figure. Chemical structures of the major volatiles of Athanasia brownii. a) Selin-11-en-4-a-ol, b) caryophyllene oxide, c) humulene epoxide II, d) (E)-nerolidol.
In this fraction, it is notable that two unknown compounds (4.5 and 2.3%, resp.) occur, whose mass-fragmentation patterns are indicated in Table 1. A minor contribution was provided by sesquiterpene hydrocarbons (11.9%), with (E)-caryophyllene (2.1%), a-humulene (2.4%), and ar-curcumene (1.5%) as the most abundant. Finally, the fraction of monoterpenes was poor (6.1%), with 1,8-cineole (1.8%) as the most abundant. The eudesmane alcohol selin-11-en-4-a-ol was first isolated from the leaves of Podocarpus dacrydioides [11]. Significant amounts of this sesquiterpene were found in the family of Asteraceae, notably in the essential oil of Artemisia asiatica Nakai (12.32%). Selin-11-en-4a-ol was established to possess a high activity against fungi, but a low activity against bacteria and especially against Gram-negative bacteria [12]. This compound was found also in soft corals, and it was effective in inhibiting the proliferation of murine leukemia P388 cell lines [13]. Caryophyllene oxide is the oxidation product of (E)-caryophyllene and is one of the oxygenated sesquiterpenes most frequently detected in essential oils. It has been approved by the FDA (US Food and Drug Administraion) as a food and cosmetic preservative, and has been included by the European Council in the list of natural and synthetic flavoring substances. Previously, it was established to be effective in the treatment of onychomycosis [14],
RF b )
1.08 1.08 1.08 1.08 1.08 1.45 1.09 1.08 1.40 1.08 1.40 1.45 1.65 0.96 1.45 1.40 1.40 1.40 1.45 1.40 1.45 1.68 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.40 1.05
Component a )
a-Pinene Camphene Sabinene b-Pinene Myrcene Isoamyl isobutyrate p-Cymene Limonene 1,8-Cineole g-Terpinene Linalool Isopentyl 2-methylbutanoate Nonanal Undecane Isopentyl isovalerate Borneol Terpinen-4-ol a-Terpineol Isobornyl formate Piperitone Bornyl acetate Eugenol a-Copaene b-Bourbonene b-Panasinsene (E )-Caryophyllene a-trans-Bergamotene Aromadendrene a-Humulene allo-Aromadendrene Geranylacetone (E )-b-Farnesene
Entry
1 2 3 4 5 6 7 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
932 945 971 974 991 1017 1026 1030 1032 1061 1100 1100 1106 1100 1108 1164 1176 1189 1234 1253 1283 1358 1370 1377 1389 1409 1432 1440 1445 1452 1455 1458
HP-5MS
RI (calc.) c )
1586 1580 1632 1658 1634 1854 1666
1297 1700 1601 1700 1239 1716 1576 2163 1484 1509
1270 1198 1211 1247 1556 1281 1393
1026 1070 1124 1112 1166
DB-Wax 939 954 975 979 990 1009 1024 1029 1031 1059 1096 1100 1100 1100 1103 1169 1177 1188 1568 1252 1288 1359 1376 1388 1382 1419 1434 1441 1454 1460 1455 1456
ADAMS 931 945 971 973 989 1017 1026 1028 1032 1062 1101 1100 1106 1100 1109 1164 1176 1189 1239 1253 1284 1358 1370 1377 1385 1410 1432 1440 1445 1452 1455 1458
NIST08
apolar phase
RI (lit.) d )
Table 1. Essential-Oil Composition of Athanasia brownii
polar phase
1587 1586 1632 1658 1635 1854 1666
1296 1700 1599 1700 1574 h ) 1717 1575 2164 1485 1510
1270 1198 1211 1247 1558 1280 1393
1027 1070 1124 1113 1167
NIST08 0.6 0.1 0.1 0.2 0.4 0.2 0.1 0.3 1.8 0.2 0.4 0.1 Tr g ) Tr 0.1 0.4 0.3 0.1 0.1 0.1 0.2 0.1 Tr Tr 0.3 2.1 0.3 0.1 2.4 0.3 1.3 0.1
% e)
Std Std RI, MS Std Std RI, MS Std Std Std Std Std RI, MS RI, MS Std RI, MS Std Std Std RI, MS Std Std Std RI, MS RI, MS RI, MS Std RI, MS RI, MS Std RI, MS RI, MS RI, MS
ID f )
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Germacrene D g-Selinene ar-Curcumene d-Selinene a-Muurolene b-Dihydroagarofuran b-Bisabolene 7-epi-a-Selinene d-Cadinene a-Agarofuran a-Calacorene cis-Cadinene ether b-Calacorene Palustrol ( E)-Nerolidol Spathulenol Caryophyllene oxide Viridiflorol Humulene epoxide II 10-epi-g-Eudesmol 1,10-Diepicubenol Muurola-4,10(14)-dien-1b-ol Caryophylla-4(12),8(13)-dien-5a-ol Caryophylla-4(12),8(13)-dien-5b-ol a-Eudesmol Selin-11-en-4-a-ol ( Z)-a-Santalol M þ 222, 69 (100), 95 (96), 109 (87), 179 (72), 41 (67), 93 (60), 137 (55), 107 (2), 55 (2), 67 (2) Helifolenol A a-Bisabolol Eudesma-4(15),7-dien-1b-ol
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
Component a )
Entry
Table 1 (cont.)
1.20 1.20 1.20
1.05 1.05 1.05 1.05 1.05 1.20 1.05 1.05 1.05 1.20 1.05 1.20 1.05 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20 1.20
RF b )
1676 1679 1685
1473 1475 1479 1483 1494 1496 1504 1507 1517 1535 1535 1548 1558 1558 1564 1568 1572 1592 1597 1608 1620 1622 1622 1627 1644 1649 1656 1663
HP-5MS
RI (calc.) c )
2299
2026 2099 2061 2308 2288 2294 2215 2226 2372 2282
1680 1767 1713 1716 1709 1721 1749 1749 1877 1901 1872 1945 1918 2049 2123 1969
DB-Wax
1675 1685 1688
1480 1492 1500 1503 1505 1522 1523 1550 1545 1553 1568 1568 1563 1578 1583 1592 1608 1623 1619 1631 1640 1640 1653 1659 1675
1485
ADAMS
1680
1644 1648 h ) 1660
1625 h )
1548 h ) 1557 1564 1569 1572 1592 1598 1609 h )
1473 1477 1479 1483 1494 1495 h ) 1504 1514 h ) 1517 1536 h ) 1528 h )
NIST08
apolar phase
RI (lit.) d ) polar phase
2212 2241 h )
2026 2094 h )
1915 2049 2124 1967
1749 1861 h ) 1906 h )
1685 1767 1717 1716 1706 h ) 1720
NIST08
0.7 Tr 0.9
0.1 1.1 1.5 0.7 0.2 0.3 0.4 1.2 0.8 0.2 0.2 0.2 0.2 0.2 4.9 0.6 8.7 0.8 5.1 1.4 2.4 1.1 1.1 1.7 1.7 24.6 0.7 4.5
% e)
RI, MS RI, MS RI, MS
RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS Std RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS Std RI, MS Std RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS MS i )
ID f )
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013) 1879
1.45 1.20 1.43
1.20 1.20 1.20 1.20 1.45 1.20 1.20 1.20 1.20
RF b )
1773 1783 2166
1694 1697 1700 1704 1714 1720 1740 1753 1759
HP-5MS
RI (calc.) c )
2562 2829
2542 2577 2822
2394
2375
DB-Wax
1788 1789
1714 1690 1717 1725 1756
1689
ADAMS
2201
1703 1715
NIST08
apolar phase
RI (lit.) d ) NIST08
polar phase
1.8 4.3 11.9 71.2 1.3 0.7
84.4
0.5 1.1 0.4
0.6 3.2 Tr 1.1 0.1 0.4 0.6 0.2 2.3
% e)
RI, MS RI, MS MS i )
RI, MS MS i ) RI, MS RI, MS RI, MS RI, MS RI ,MS MS i ) MS i )
ID f )
a ) Compounds are listed in order of their elution from a HP-5MS column. Their nomenclature was in accordance with Adams [8]. b ) Relative response factor ( RF ) of FID detector for the main chemical groups occurring in essential oil. c ) Linear retention index on HP-5MS and DB-Wax column. Experimentally determined using homologous series of C8 – C30 alkanes. d ) Linear retention index taken from Adams [8] and/or NIST08 [9]) for apolar columns, and from NIST08 for polar columns. e ) Percentage values are means of three determinations, with a RSD% for the main components below 5% in all cases. f ) Identification methods: Std, based on comparison with authentic compounds; MS, based on comparison with Wiley, Adams, and NIST08 MS database; RI, based on comparison of RI values with those reported in Adams and NIST08. g ) Tr, traces (mean value below 0.1%). h ) RI Values taken from The Pherobase, database of pheromones and semiochemicals [10]. i ) Tentative identification.
Total identified [%] Grouped compounds [%] Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Esters Others
( Z )-Apritone Cedr-8-en-15-ol 14-Hydroxy-a-humulene ( Z )-a-trans-Bergamotol ( E )-Nerolidyl acetate ( Z )-Nuciferol ( E )-Nuciferol 3,7,11-Trimethyldodeca-2,6,10-trienoic acid M þ 220, 93 (100), 43 (50), 133 (44), 79 (42), 91 (40), 119 (36), 41 (34), 121 (33), 120 (32), 69 (31) 8-Cedren-13-ol acetate b-Bisabolenol Geranylgeraniol
64 65 66 67 68 69 70 71 72
73 74 75
Component a )
Entry
Table 1 (cont.)
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and as potential therapeutic candidate for both the prevention and treatment of human prostate and breast cancers [15]. Humulene epoxide II is an oxidation product of ahumulene and it has been suggested to contribute a hoppy aroma in beer [16]. The tertiary alcohol (E)-nerolidol constitutes the major floral volatile in the commercially important kiwifruit species (Actinidia chinensis) as well as in strawberry, grape, snapdragon, and maize. This terpene is likely to play a role in attracting pollinators or seed dispersal agents, and it is also produced by plants in response to insect herbivory [17]. Within the Asteraceae, this compound was found in high amount in Baccharis dracunculifolia D.C. [18]. (E)-Nerolidol is approved by the FDA as a food-flavoring agent, and it currently undergoes testing as a skin penetration enhancer for the transdermal delivery of therapeutic drugs. Further, it was shown to inhibit carcinogenesis on azoxymethane-induced neoplasia in rats [19] by an uncharacterized mechanism, and it was found effective against some Leishmania species [20]. 2. Antioxidant Activity. We evaluated the antioxidant power of the essential oil from A. brownii using Trolox as positive control. Results are compiled in Table 2. Of the various chemical methods in practice to evaluate the antioxidant property, DPPH and ABTS methods are the best and commonly accepted. These methods are based on the scavenging ability of antioxidants (test molecules) towards the stable 2,2-diphenyl-1picrylhydrazyl radical and 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid, respectively. Table 2. In vitro Radical-Scavenging Activities of the Essential Oil of Athanasia brownii DPPH
Essential oil Trolox
ABTS
FRAP
TEAC a ) [mmol TE/g]
IC50 b ) [mg/ml]
TEAC a ) [mmol TE/g]
IC50 b ) [mg/ml]
TEAC a ) [mmol TE/g]
441 ( 5.2)
910 ( 34) 10.1 ( 0.2)
167 ( 3.7)
880 ( 28) 3.7 ( 0.2)
17.2 ( 0.8)
a
) TEAC, Trolox equivalent ( TE) antioxidant concentration. b ) IC50 , the concentration of compound that affords a 50% reduction in the assay.
From our data, the essential oil exhibited antioxidant activities with IC50 values of 100- and 200-fold lower than that of Trolox in DPPH and ABTS assays, respectively. The FRAP ( ¼ ferric reducing antioxidant power) assay revealed that A. brownii oil have a low capacity for iron binding that is related to its possible action as peroxidation protector. Our results showed that the essential oil has a mild ability as a free-radical inhibitor. It is very difficult to attribute the antioxidant effect to one or a few active principles, because it contains a mixture of different chemical compounds among which also minor compounds may contribute to the oils activity. 3. Antimicrobial Activity. In Table 3, the antimicrobial activities are compiled, expressed as inhibition zone diameters for all the microorganisms screened. The essential oil exhibited a modest inhibition, as evidenced by the modest inhibition zone diameters (7 to 10 mm), against the growth of Gram-positive bacteria, with a stronger activity against streptococci than S. aureus and E. faecalis. P. aeruginosa and E. coli growth was not affected by the essential oil, while an appreciable activity
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Table 3. In vitro Antimicrobial Activities of the Essential Oil from Athanasia brownii Microorganism
Inhibition zone diameter [mm] a )
Staphylococcus aureus Enterococcus faecalis Escherichia coli Pseudomonas aeruginosa Streptococcus pyogenes Streptococcus pneumoniae Candida albicans
71 61 60 60 10 1 10 1 71
a ) Values refer to the diameter of the inhibition zone SD. Sterile filter paper discs (6 mm in diameter) were placed on the surface of inoculated plates and spotted with 10 ml of a 1 : 1 dilution of each oil in DMSO.
was exhibited against the fungus C. albicans. Taking into account the high fraction of selin-11-en-4a-ol, and hypothesizing its dominant putative contribution to the overall activity of the oil, our results support those reported for Artemisia asiatica Nakai [12]. 4. Antiproliferative Activity. The essential oil of A. brownii was tested in vitro for their potential tumor cell growth-inhibitory effect on MDA-MB 231 human breast adenocarcinoma, A375 human malignant melanoma, and HCT116 human colon carcinoma cell lines, by using MTT assay. The results, collected in Table 4, show that the essential oil exhibited an inhibitory effect against the human cancer cells examined, although significantly lower than that of the anticancer drug cisplatin. The highest activity was observed on A375 cell line, with an IC50 value of 19.9 mg/ml. Lower activity was obtained on MDA-MB 231, with an IC50 value of 51.7 mg/ml, while medium was the inhibition against HCT 116 (IC50 of 29.53 mg/ml). The cytotoxic activity of the A. brownii essential oil may be attributed to specific components of the oil. A few of the compounds found in A. brownii essential oil have been tested for cytotoxic properties. Of the major compounds present in the oil, only selin-11-en-4a-ol (24.6%) appears to possess some cytotoxic activity against mouse lymphocytic leukemia P-388 [13]. There is still controversy over whether caryophyllene oxide (8.7%) is cytotoxic or not. Kubo et al. [21] and Sibanda et al. [22] reported that it exhibited a modest cytotoxic activity, while some reports stated that it Table 4. In vitro Cytotoxic Activities of Essential Oil from Athanasia brownii Cell line ( IC50 [mg/ml]) a )
A. brownii essential oil 95% C.I. Reference Cisplatin 95% C.I.
MDA-MB 231 b )
HCT116 c )
A375 d )
51.7 48.3 – 55.3
29.5 27.9 – 31.2
19.9 16.8 – 23.5
2.47 2.18 – 2.73
2.79 2.54 – 2.95
0.34 0.29 – 0.38
a ) IC50 , The concentration of compound that affords a 50% reduction in cell growth (after 72 h of incubation). C.I., Confidence Interval. b ) Human breast adenocarcinoma cell line. c ) Human colon carcinoma cell line. d ) Human malignant melanoma cell line.
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was inactive against tumor cell lines [23 – 25]. Recently, nerolidol was shown to be cytotoxic against Balb/c 3T3-A31 fibroblast [26]. However, the low concentrations of selin-11-en-4a-ol (24.6%), caryophyllene oxide (8.7%), and nerolidol (4.9%) cannot fully explain the cytotoxic activity of A. brownii essential oil, including that some other compounds, probably sesquiterpenes, are active in the essential oil. It is also possible that minor components may be involved in some type of synergism with the other active compounds [27] [28]. Conclusions. – The essential oil from the aerial parts of A. brownii endemic to Madagascar was investigated for the first time. The majority of the oil was constituted by oxygenated sesquiterpenes, among which selin-11-en-4a-ol was the most abundant compound. The antimicrobial activity and the antioxidant potential of the oil were negligible. The cytotoxic effects exhibited particularly on malignant melanoma cell line encourage further investigations of the mechanisms of action of the oil, and in vivo studies for applications as natural anticancer drug. Experimental Part Plant Material. Aerial parts of Athanasia brownii were collected in Ambatofotsy-Ambalavao (central highlands of Madagascar) at ca. 30 km from Antananarivo, in the Route Nationale 7, in August, 2012, during flowering. The plant was identified by botanists at the Department of Botany of the Parc Botanique et Zoologique de Tsimbazaza, Antananarivo, by comparaison with a authentic specimen. A voucher specimen was deposited with the Herbarium of the Institut Malgache de Recherches Applique´es (IMRA), under the accession code MAD-0917 2.2. Extraction of Essential Oil. The essential oil was obtained from freshly collected aerial parts (5 kg) of A. brownii by steam distillation for 3 h using a field alembic to yield 0.004% of a yellowish oil. The oil yield was estimated on a dry-weight basis (w/w). Before analysis, the oil was dried (Na2SO4 ), transferred into an amber glass flask, and kept at 208 before GC and biological experiments. Chemicals. For identification of volatiles the following anal. standards purchased from SigmaAldrich (I-Milan) were used: a-pinene, camphene, b-pinene, myrcene, p-cymene, limonene, 1,8-cineole, g-terpinene, linalool, n-undecane, borneol, terpinen-4-ol, a-terpineol, piperitone, bornyl acetate, eugenol, (E)-caryophyllene, a-humulene, b-bisabolene, (E)-nerolidol, caryophyllene oxide. For retention-index (RI) determination, a mixture of hydrocarbons, ranging from octane (C8 ) to triacontane (C30) (Supelco, USA), was used and run under the exper. conditions reported below. All compounds were of anal. standard grade. Anal.-grade hexane solvent was purchased from Carlo Erba (I-Milan); it was successively distilled through a Vigreux column before use. GC/FID and GC/MS Analysis. For GC separations, an Agilent 4890D instrument coupled to an ionization flame detector (FID) was used. Volatile components were separated on a HP-5 cap. column (5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d.; 0.17-mm film thickness; J and W Scientific, Folsom, CA), with the following temp. program: 5 min at 608, subsequently 48/min up to 2208, then 118/min up to 2808, held for 15 min, for a total run of 65 min. Injector and detector temps. were 2808. He was used as the carrier gas, at a flow rate of 1.4 ml/min; injection volume, 1 ml; split ratio, 1 : 34. A mixture of aliphatic hydrocarbons (C8 – C30 ; Sigma, I-Milan) in hexane was directly injected into the GC injector under the above temp. program, in order to calculate the retention index (RI) of each compound. Oil samples were diluted to 1 : 100 in hexane and injected at a volume of 1 ml. Analysis was repeated three times. Data were collected by using HP3398A GC Chemstation software (Hewlett Packard, Rev. A.01.01). The rel. amounts of essential-oil components, expressed as percentages, were obtained by FID peak-area normalization by calculating the response factor (RF) of the FID for nine different classes of volatiles occurring in the essential oil analyzed [4]. GC/MS Analysis was performed on an Agilent 6890N gas chromatograph coupled to a 5973N mass spectrometer using two different columns: a HP-5 MS (5%
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phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1-mm film thickness; J & W Scientific, Folsom), and a DB-WAX (polyethylene glycol; 30 m, 0.25 mm i.d., 0.25-mm film thickness; J & W Scientific, Folsom) cap. columns. The temp. program was the same as described above. Injector and detector temps. were 2808. He was used as the carrier gas, at a flow rate of 1 ml/min. Split ratio, 1 : 50; acquisition mass range, m/ z 29 – 400. All mass spectra were acquired in electron-impact (EI) mode with an ionization voltage of 70 eV. Oil samples were diluted to 1 : 100 in hexane, and the volume injected was 2 ml. Data were analyzed by using MSD ChemStation software (Agilent, Version G1701DA D.01.00). About half of the constituents (21 out of 73 compounds), corresponding to 23.8% of essential oil, were identified by coinjection with authentic standards. Otherwise, the peak assignment was carried out on the basis of the standard of the International Organization of the Flavor Industry (IOFI, http://www.iofi.org/) statement [29], i.e., by the interactive combination of chromatographic linear RI values that were consistent with those reported in [8 – 10] for apolar and polar stationary phases, and MS data consisting in the computer matching with the WILEY275, NIST 08, Adams, and a home-made (based on the analyses of reference oils and commercially available standards) libraries. Evaluation of Antioxidant Activity. DPPH Free-radical scavenging activity was evaluated on a microplate anal. assay according to the procedure described in [30], while radical-scavenging capacity (ABTS assay) was evaluated by a modified method originally described by Re et al. [31] for application to a 96-well microplate assay [32]. Trolox was used as the positive control. Determination of antioxidant activity by ferric reducing antioxidant power (FRAP) assay was carried out according to Mller et al. [33]. The ability of the test samples to scavenge the different radicals in the assays was compared to that of Trolox used as standard, and the activity of the essential oil was expressed as tocopherol equivalent antioxidant capacity mmol TE/gr of product. Each experiment was repeated three times. Antimicrobial Activity. Plain essential oil was tested directly against a panel of microorganisms including Staphylococcus aureus ATCC 25923 (American Type Culture Collection, Rockville, MD, USA), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, Streptococcus pyogenes, Streptococcus pneumoniae, and Candida albicans ATCC 24433. Growth conditions and antimicrobial activity tests were as described in [3]. Briefly, a suspension of the tested microorganism (1 – 2 108 cells per ml in saline/106 per ml for Candida) was spread on the solid media plates using a sterile cotton swab. Sterile paper discs (6-mm in diameter) were placed on the surface of inoculated plates and spotted with 10 ml of a 1 : 1 dilution of each oil in DMSO. The plates were incubated 18 h at 35 18 (48 h for C. albicans). Streptococcus spp. were incubated in 5% CO2 atmosphere. The diameters of zone inhibition (including the 6-mm disc) were measured with a calliper. A reading of more than 6 mm indicated growth inhibition. No zone inhibition was observed using DMSO alone. Ciprofloxacin (5 mg disc) and Fluconazole (25 mg disc) were used as reference antimicrobials against bacteria and fungi, resp. Each test was repeated three times. MTT Cytotoxicity Assay. HCT116 Cells (human colon carcinoma cells) were cultured in RPMI1640 medium with 2 mm l-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; PAA Laboratories GmbH, Austria), MDA-MB 231 (human breast adenocarcinoma cells) and A375 cells (human malignant melanoma cells) were cultured in Dulbeccos Modified Eagles Medium (DMEM) with 2 mm l-glutamine, 100 IU/ml penicillin, 100 mg/ ml streptomycin, and supplemented with 10% HI-FBS. Cells were cultured in a humidified atmosphere at 378 in presence of 5% CO2 . The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay was used as a relative measure of cell viability. Cell-viability assays were carried out as described in [34]. Briefly, cells were seeded at the density of 2 104 cells/ml. After 24 h, samples were exposed to different concentrations of essential oil (1.56 – 400 mg/ml). Cells were incubated for 72 h in a humidified atmosphere of 5% CO2 at 378. At the end of incubation, each well received 10 ml of MTT (5 mg/ml in phosphate-buffered saline (PBS)), and the plates were incubated for 4 h at 378. The extent of MTT reduction was measured spectrophotometrically at 540 nm using a Titertek Multiscan microElisa (Labsystems, FI-Helsinki). Experiments were conducted in triplicate. The chemotherapy drug cisplatin was used as a reference compound. Cytotoxicity was expressed as the concentration of the essential oil inhibiting cell growth by 50% (IC50). The IC50 values were determined with GraphPad Prism 4 computer program (GraphPad Software, S. Diego, CA, USA).
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Statistical Analysis. Each experiment was repeated at least three times. The Unpaired t test was used for comparisons between two groups. Statistical significance was indicated by p-values < 0.001. Data are reported as the mean SD. The authors are grateful to Prof. Suzanne Ratsimamanga, President of the Institut Malgache de Recherches Applique´es, for her precious support during this study. We thank the local communities of Analalava for helping us to collect A. brownii.
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