Chemical Characterization and Insecticidal ... - Wiley Online Library

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)

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Chemical Characterization and Insecticidal Properties of Essential Oils from Different Wild Populations of Mentha suaveolens subsp. timija (B Br i q.) Ha r l e y from Morocco by Ayoub Kasrati a ), Chaima Alaoui Jamali a ), Khalid Bekkouche a ), Robert Spooner-Hart b ), David Leach b ) c ), and Abdelaziz Abbad* a ) a

) Laboratoire de Biotechnologie, Protection et Valorisation des Ressources V¦g¦tales (URAC 35), Facult¦ des Sciences, Semlalia, Universit¦ Cadi Ayyad, Marrakech, Maroc (phone: þ 212-524-434649; fax: þ 212-524-437412; e-mail: [email protected], [email protected]) b ) School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith NSW 2751, Australia c ) Southern Cross Plant Science, Southern Cross University, Lismore NSW 2480, Australia

The present study is the first investigation of the volatile-oil variability and insecticidal properties of the endemic Moroccan mint Mentha suaveolens subsp. timija (mint timija). The yield of essential oils (EOs) obtained from different wild mint timija populations ranged from 0.20 0.02 to 1.17 0.25% (v/w). GC/MS Analysis revealed the presence of 44 oil constituents, comprising 97.3 – 99.9% of the total oil compositions. The main constituents were found to be menthone (1.2 – 62.6%), pulegone (0.8 – 26.6%), cis-piperitone epoxide (2.9 – 25.5%), piperitone (0.3 – 35.5%), trans-piperitone epoxide (8.1 – 15.7%), piperitenone (0.2 – 9.6%), piperitenone oxide (0.5 – 28.6%), (E)-caryophyllene (1.5 – 11.0%), germacrene D (1.0 – 15.7%), isomenthone (0.3 – 7.7%), and borneol (0.2 – 7.3%). Hierarchical-cluster analysis allowed the classification of the EOs of the different mint timija populations into four main groups according to the contents of their major components. This variability within the species showed to be linked to the altitude variation of the mint timija growing sites. The results of the insecticidal tests showed that all samples exhibited interesting activity against adults of Tribolium castaneum, but with different degrees. The highest toxicity was observed for the EOs belonging to Group IV, which were rich in menthone and pulegone, with LC50 and LC90 values of 19.0 – 23.4 and 54.9 – 58.0 ml/l air in the fumigation assay and LC50 and LC90 values of 0.17 – 0.18 and 0.40 – 0.52 ml/cm2 in the contact assay.

Introduction. – The genus Mentha, commonly known as mint, is one of the most important genera in the Lamiaceae family. This genus includes spontaneous and cultivated forms, comprising about 19 species and 13 natural hybrids widely distributed across Europe, Africa, Asia, Australia, and North America [1]. Several species of Mentha have been reported to possess numerous pharmacological and biological properties. In fact, the aerial parts of these species are commonly used in form of powders, infusions, and decoctions as anti-inflammatory, carminative, antiemetic, diaphoretic, antispasmodic, analgesic, stimulant, emmenagogue, and antitussive remedies [2] [3]. The essential oils (EOs) of different mint species have been reported to have excellent antimicrobial, antioxidant, and insecticidal activities [1] [4] [5]. These properties have been ascribed to the presence of numerous oxygenated monoterpenes, including menthone, menthol, pulegone, piperitone oxide, piperitenone, and carvone [1] [6 – 8]. Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich

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In Morocco, the genus Mentha is represented by ten species, two of which are endemic [9]. Among these endemic species, Mentha suaveolens subsp. timija (Briq.) Harley, commonly known as mint timija, is an aromatic perennial herbaceous plant, with a pungent minty odor, reaching up to 40 cm height. This species grows wild in semiarid and subhumid areas along rivers in plains and mountains [9]. Mint timija products have been used in the Moroccan folk medicine as a powder or infusion to treat different ailments including whooping cough, bronchitis, and ulcerative colitis, as an antispasmodic, as well as an excellent carminative [10]. As an aromatic plant, mint timija is considered an important flavoring agent, extensively used in herbal teas for its tonic and stimulant potencies. The antimicrobial, antioxidant, and insecticidal activities of its EOs have also been reported [7] [8]. The literature reports that the yield of Mentha EOs as well as their chemical constituents and biological activities are influenced by several external factors, including geographical origin, growing conditions, and harvest season [6] [11 – 15]. However, to the best of our knowledge, there are no comprehensive studies on the chemical variability and biological properties of mint timija oils with respect to geographical variation. Thus, the aims of the present study were to evaluate, for the first time, the chemical variability of the EOs from ten natural populations of this endemic Moroccan species and any consequent effect on the insecticidal properties against Tribolium castaneum. Results and Discussion. – Chemical Composition of the Essential Oils. Prospecting conducted during 2011 over the natural distribution area of M. suaveolens subsp. timija in southwestern Morocco led to the sampling of ten populations, located at different altitudes and in various geographical regions. Four populations, i.e., those from Tifnout (Population T1), Ait Lkak (Population T2), Oukaimeden (Population T4), and Iguer (Population T7), grew at high altitude ( > 1700 m.a.s.l.), two populations, the one from Tadla (Population T3) and the one from Ourika (Population T10), were located at lower altitude ( < 1000 m.a.s.l.), and four populations, i.e., those from Ait Ourir (Population T6), Ouirgane (Population T5), Imlil (Population T8), and Toufliht (Population T9), were situated at an intermediate altitude (1049 – 1565 m.a.s.l.; Table 1). Table 1. Collection Site and Geographical Coordinates of the Ten Studied Mint Timija Populations and Essential Oil Yield of Their Aerial Parts Population

Collection site

Voucher specimen

Latitude/Longitude

Altitude [m.a.s.l.]

Oil Yield [% (v/w)]

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

Tifnout Ait Lkak Tadla Oukaimeden Ouirgane Ait Ourir Iguer Imlil Toufliht Ourika

MST01 MST02 MST03 MST04 MST10 MST06 MST07 MST08 MST09 MST05

N 31823’/W 07846’ N 31814’/W 07848’ N 32820’/W 06821’ N 31811’/W 07853’ N 31810’/W 08840’ N 31827’/W 07832’ N 30852’/W 09824’ N 31819’/W 07854’ N 31828’/W 07826’ N 31821’/W 07847’

1747 1800 930 2100 1406 1049 1917 1190 1565 900

0.57 0.07 0.40 0.10 1.17 0.25 0.20 0.02 0.47 0.13 0.38 0.10 0.88 0.21 0.60 0.18 0.43 0.09 1.00 0.22


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Hydrodistillation of the aerial parts of samples of these ten mint timija populations gave pale yellowish to deep yellow oils with a yield ranging from 0.20 0.02 to 1.17 0.25% (v/w based on dry weight of the plant material; Table 1). The average EO yield (0.61%) achieved in this study is comparable with those published in our previous reports [7] [8]. The results of the GC/MS analysis of the volatile-oil constituents of the different mint timija populations (percentage content of each compound, elution order, retention index (RI), and structural subclass) are summarized in Table 2. In total, 44 compounds were identified, accounting for 97.3 – 99.9% of the total oil composition. For an easier comparison of the oils, the components were divided into four compound classes: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes. Oxygenated monoterpenes (relative contents of 35.0 – 91.8%) constituted the principal compound class for the majority of the EO samples, with the exception of the oil obtained from Population T4 (Oukaimeden), which showed a higher content of sesquiterpene hydrocarbons (47.6%). The predominant compounds were found to be menthone (1.2 – 62.6%), pulegone (0.8 – 26.6%), cis-piperitone epoxide (2.9 – 25.5%), piperitone (0.3 – 35.5%), trans-piperitone epoxide (8.1 – 15.7%), piperitenone (0.2 – 9.6%), piperitenone oxide (0.5 – 28.6%), (E)-caryophyllene (1.5 – 11.0%), germacrene D (1.0 – 15.7%), isomenthone (0.3 – 7.7%), and borneol (0.2 – 7.3%). These compounds had frequently been found in EOs of other subspecies of M. suaveolens, but at different relative contents [13] [16] [17]. Hierarchical-cluster analysis (HCA) based on nine major EO compounds allowed the differentiation between the analyzed mint timija samples. The resulting dendrogram showed that the oil samples were divided into four main groups (Fig.). The EOs of Group I were further classified into three subgroups. The first subgroup (Subgroup Ia) included oil samples T1 and T7, obtained from plants collected in the localities Tifnout and Iguer, respectively, which were rich in piperitenone oxide (28.6 and 19.2%, resp.), trans-piperitone epoxide (8.1 and 15.7%), and germacrene D (8.1 and 9.2%). The second subgroup (Subgroup Ib) was represented by Sample T5, which was collected in Ouirgane and provided oil with high levels of piperitenone oxide (27.9%), cis-piperitone epoxide (17.8%), and piperitenone (6.8%). The third subgroup (Subgroup Ic) comprised Sample T9, which was collected in Toufliht and characterized by an oil with high contents of piperitenone oxide (22.6%), pulegone (23.4%), and menthone (10.1%). In the EOs of Group II, consisting of samples T2 and T4 collected in Ait Lkak and Oukaimeden, respectively, the dominant compounds were cispiperitone epoxide (25.5 and 19.4%, resp.) and germacrene D (9.1 and 15.7%). The EOs of Group III, comprising samples T6 and T8 collected in Ait Ourir and Imlil, were characterized by high contents of piperitone (35.5 and 17.7%), piperitenone oxide (9.4 and 6.5%), and germacrene D (6.4 and 6.9%). The fourth group, represented by samples T3 and T10 collected in Tadla and Ourika, had oils with a chemical composition dominated by menthone (62.6 and 46.8%) associated with pulegone (22.8 and 26.6%) and isomenthone (3.7 and 7.7%). Based on the studied samples, Moroccan mint timija EOs showed a notable variation in the chemical composition. These differences appeared to be linked to the variation in altitude of the growing sites. For example, the EO samples obtained from plants collected in the localities with the lowest altitudes (Ourika and Tadla) were

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Table 2. Chemical Composition of the Essential Oils Isolated from the Aerial Parts of Mint Timija Collected from Ten Wild Populations ( T1 – T10) Compound name and class a ) a-Pinene Camphene Sabinene b-Pinene Myrcene Limonene cis-Ocimene ( Z )-b-Ocimene 1.8-Cineole trans-Sabinene hydrate Linalool Octenyl acetate Menthone Isomenthone Borneol cis-Isopulegone Terpinen-4-ol a-Terpineol Pulegone cis-Piperitone epoxide Piperitone trans-Piperitone epoxide Menthyl acetate Bornyl acetate Thymol Carvacrol Piperitenone Piperitenone oxide a-Copaene b-Bourbonene p-Menthane-1,2,3-triol ( Z )-Jasmone b-Elemene ( E )-Caryophyllene a-Humulene 9-epi-( E )-Caryophyllene Germacrene D Bicyclogermacrene g-Cadinene d-Cadinene Germacrene D-4-ol Spathulenol Caryophyllene oxide a-Cadinol

RI b ) 936 951 975 980 991 1030 1033 1036 1059 1068 1100 1110 1156 1166 1168 1177 1179 1192 1242 1254 1255 1256 1272 1288 1291 1302 1342 1367 1381 1390 1397 1400 1417 1425 1459 1467 1486 1502 1519 1528 1581 1534 1589 1645

Content [%] T1 c )

T2

T3

T4

T5

T6

T7

T8

T9

T10

2.5 2.7 2.6 1.9 0.6 3.6 1.0 – 0.7 1.3 1.1 1.4 1.3 tr 4.8 – – 0.4 3.4 2.9 – 8.1 – 0.7 – – 5.4 28.6 – – 2.3 0.4 3.8 5.0 0.7 0.7 8.1 1.8 0.3 0.4 0.5 0.4 0.3 0.4

1.8 1.6 8.7 1.8 0.4 3.7 1.6 – 0.6 2.4 1.1 – – – 2.5 – – – 0.8 25.5 – – – 0.4 0.4 – 4.7 7.2 1.1 0.7 2.8 – 7.3 6.2 1.0 0.8 9.1 2.3 – 1.0 1.1 0.5 0.5 0.4

1.0 0.2 0.5 1.0 0.2 0.5 – – 0.1 0.2 – 0.2 62.6 3.7 0.2 1.4 – – 22.8 – 0.3 – – – – – 0.2 – 0.2 0.2 – – – 1.5 0.2 – 1.0 0.2 – – – 0.3 0.3 0.2

1.0 0.9 5.5 1.0 – d) 2.1 1.7 0.5 – 1.3 1.3 – 1.2 – 2.0 – – – 0.9 19.4 – – – 0.6 0.5 – 4.1 3.7 0.8 1.1 4.0 – 4.8 11.0 1.9 1.3 15.7 4.9 0.8 1.2 2.0 0.8 1.0 1.0

2.4 2.3 5.3 2.3 0.5 4.3 1.3 – 0.6 2.2 1.0 0.6 0.8 0.3 3.5 – – – 1.0 17.8 – – – 0.5 0.3 – 6.8 27.9 – – 0.7 – 4.1 4.9 0.8 – 5.8 0.9 – 0.4 – – – –

0.8 0.8 1.1 0.7 – 1.1 0.5 0.6 – 3.1 1.3 tr 2.1 2.3 4.8 – 1.0 – 1.3 – 35.5 – 0.6 0.6 0.6 1.3 9.6 9.4 – 0.6 – – 5.4 5.4 0.8 – 6.9 1.9 – – 0.2 – – –

2.9 4.4 2.0 1.9 0.2 5.0 0.4 – tr 0.2 0.4 0.3 – – 7.3 – – 0.3 – – – 15.7 – 0.8 0.3 0.7 2.2 19.2 1.0 0.3 5.4 – 7.3 4.7 0.7 0.8 9.2 2.9 tr 0.7 0.5 0.6 tr 0.3

2.7 1.7 1.2 2.1 0.6 5.6 tr – 2.5 2.2 0.7 0.5 14.2 6.3 2.6 – –

1.7 0.2 1.2 1.7 0.6 3.8 0.8 – 0.3 0.1 0.5 0.3 10.1 2.9 – 1.8 – 0.3 23.4 – – 9.1 2.7 – 0.2 0.8 1.6 22.6 0.2 0.2 – – 1.7 2.2 0.4 – 3.2 0.2 0.4 0.3 0.9 0.4 0.7 0.7

1.1 1.1 0.5 1.1 tr e ) 0.9 0.2 – 0.9 0.4 0.3 – 46.8 7.7 2.3 1.6 – tr 26.6 – 0.4 – – tr – – 0.3 0.5 – – – – – 2.5 0.3 – 1.2 0.2 – – – 0.6 1.1 0.2

5.7 – 17.7 – 1.5 tr – 0.5 4.3 6.5 0.5 0.5 0.5 – 4.3 5.0 0.9 0.7 6.4 1.6 – 0.6 – – – –


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Table 2 (cont.) Compound name and class a )

RI b )

Content [%] T1 c )

T2

T3

Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes

14.9 60.1 23.9 1.0

19.5 45.5 31.7 1.5

3.4 91.8 2.9 0.7

Total identified [%]

99.9

98.2

98.8

T4

T5

T6

T7

T8

T9

T10

12.7 35.0 47.6 2.8

18.5 63.2 17.4 0.0

5.5 73.4 20.4 0.0

16.8 47.4 32.2 0.9

13.8 65.2 20.0 0.0

10.0 76.7 9.2 1.9

4.9 87.9 4.3 1.9

98.1

99.1

99.3

97.3

99.0

97.8

99.0

a ) Compounds listed in order of elution from the non-polar DB-5 column. b ) RI: Retention index determined relative to n-alkanes (C9 – C24 ) on the non-polar DB-5 column. c ) The population codes and details are given in Table 1. d ) –: Not detected. e ) tr: Traces ( < 0.1%).

Figure. Dendrogram obtained by cluster analysis of the essential oils isolated from the aerial parts of mint timija collected from ten populations (T1 – T10). The letters A – J and a – j classify in decreasing order the mean insecticidal activity of the EOs in the contact and fumigation assays, respectively. The population codes and details are given in Table 1.

highly dominated by the two oxygenated monoterpenes menthone and pulegone. It can be hypothesized that wild-growing plants in these localities might be subjected to more stressful conditions (such as water deficit and high temperatures), and that under these conditions, they prioritize the pathway of cis-isopulegone and its derivatives (pulegone and menthone). The accumulation of menthone and pulegone under harsh environmental conditions has been previously reported [7] [18]. On the other hand, in the oil samples collected in sites with higher altitude, characterized by less stressful conditions (lower temperature and more humid conditions), the piperitenone pathway was favored, generating piperitenone, piperitone, and their derivatives (oxides and

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epoxides). These findings are in agreement with what has been previously reported for some other mint species collected in high-altitude areas [17] [19] [20]. The activation of the piperitenone pathway under these conditions might be explained by the accumulation of the enzyme terpenone isomerase, responsible for the conversion of isopiperitenone to piperitenone, and eventually the production of piperitenone oxide, piperitone, and cis- or trans-piperitone epoxide [21]. Insecticidal Activity. The toxicity of the EOs extracted from the aerial parts collected from the ten mint timija populations against T. castaneum adults, which are important stored-product pest insects, was evaluated using contact and fumigation assays. The lethal concentrations (LC50 and LC90 values) obtained in the contact and fumigation assays are given in Tables 3 and 4, respectively. Lower LC values reflect a higher efficacy of the EOs. Table 3. Contact Toxicity against Tribolium castaneum ( LC50 and LC90 values) of the Essential Oils Isolated from the Aerial Parts of Mint Timija Collected from Ten Wild Populations ( T1 – T10) Population a )

LC50 [ml/cm2 ] b )

LC90 [ml/cm2 ] b )

Slope SE

c2

df

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

0.27 (0.20 – 0.34) 0.46 (0.22 – 1.34) 0.17 (0.08 – 0.26) 0.41 (0.18 – 1.10) 0.26 (0.17 – 0.39) 0.34 (0.20 – 1.02) 0.31 (0.21 – 0.56) 0.29 (0.22 – 0.80) 0.32 (0.20 – 0.93) 0.18 (0.14 – 0.23)

0.82 (0.47 – 1.31) 2.42 (2.07 – 3.81) 0.40 (0.33 – 0.83) 1.96 (1.20 – 4.02) 0.99 (0.61 – 1.41) 1.33 (1.00 – 3.01) 0.92 (0.73 – 2.23) 0.60 (0.37 – 1.07) 1.08 (0.85 – 2.71) 0.52 (0.35 – 0.61)

3.19 1.10 4.93 1.75 2.61 0.90 3.12 1.30 2.21 1.09 3.49 1.90 2.71 1.24 4.11 1.58 2.00 0.97 5.40 1.47

0.38 0.16 0.16 0.42 0.13 0.28 0.03 0.37 0.08 0.28

2 2 2 2 2 2 2 2 2 2

a ) Details concerning the populations are given in Table 1. b ) LC50 and LC90 values are given as means with 95% confidence intervals in parentheses.

Table 4. Fumigant Toxicity against Tribolium castaneum ( LC50 and LC90 values) of the Essential Oils Isolated from the Aerial Parts of Mint Timija Collected from Ten Wild Populations ( T1 – T10) Population a )

LC50 [ml/l air] b )

LC90 [ml/l air] b )

Slope SE

c2

df

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

47.3 (29.2 – 79.7) 99.7 (80.6 – 134.0) 23.4 (20.8 – 39.0) 95.5 (62.0 – 132.0) 68.5 (50.5 – 112.0) 78.6 (57.4 – 121.0) 81.4 (68.6 – 113.0) 71.8 (55.9 – 111.0) 34.3 (26.3 – 72.5) 19.0 (13.7 – 28.1)

131.0 (115.0 – 228.0) 282.0 (243.0 – 454.0) 58.0 (44.1 – 112.0) 225.0 (207.0 – 331.0) 121.0 (90.1 – 184.0) 162.0 (138.0 – 220.0) 186.0 (127.0 – 250.0) 159.0 (81.9 – 175.0) 105.0 (89.9 – 146.0) 54.9 (36.8 – 91.4)

2.29 0.99 1.83 0.65 4.55 1.19 1.74 0.34 4.33 1.28 3.29 1.15 5.61 2.24 5.01 2.30 2.04 0.93 2.78 1.05

0.13 0.06 3.10 0.44 1.12 0.26 0.24 0.27 0.69 1.33

2 2 2 2 2 2 2 2 2 2

a ) Details concerning the populations are given in Table 1. b ) LC50 and LC90 values are given as means with 95% confidence intervals in parentheses.


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It was shown that the tested EOs revealed an interesting insecticidal potency towards adults of T. castaneum, but to variable degrees. The order of the mean insecticidal activity of the mint timija EO samples in relation to their chemical composition is presented in the Figure. In both the contact and fumigation assays, the mint timija EO samples collected in Tadla (T3) and Ourika (T10), rich in pulegone and menthone and comprising Group IV, exhibited superior insecticidal activity. In the fumigation assay, the corresponding LC50 values were 23.4 and 19.0 ml/l air and the LC90 values 58.0 and 54.9 ml/l air, respectively, while the LC50 and LC90 values for the contact assay were 0.17 and 0.18 ml/cm2 as well as 0.40 and 0.52 ml/cm2, respectively. The least efficiency was observed for the oil samples collected in Ait Lkak (T2) and Oukaimeden (T4), rich in cis-piperitone epoxide and germacrene D and constituting Group II, with LC50 values of 99.7 and 95.5 ml/l air and LC90 values of 282 and 225 ml/l air in the fumigation assay and LC50 and LC90 values of 0.46 and 0.41 ml/cm2 as well as of 2.42 and 1.96 ml/cm2, respectively, in the contact assay. The oil samples belonging to Groups I and III expressed intermediate toxicity in both assays (LC50 and LC90 values ranging from 34.3 to 81.4 and from 105 to 186 ml/l air, resp., in the fumigation assay as well as from 0.26 to 0.34 and from 0.60 to 1.33 ml/cm2, resp., in the contact test). The present results confirm previous findings, which highlighted the strong toxicity of EOs from Mentha species against various insect species [22 – 25]. Generally, the insecticidal activity of plant EOs was attributable to their chemical composition and especially to their major compounds [26]. Thus, the different levels of toxicity against T. castaneum found for the studied mint timija EO samples may be attributed primarily to the variation of their different major components, particularly of the contents of menthone, pulegone, and piperitenone derivatives. In fact, the toxic effect of these oxygenated monoterpenes against storage-pest insects has been previously reported [1] [22] [23], and the comparison of the insecticidal activity between these major compounds showed that pulegone and menthone presented the highest toxicity [1]. This can explain the superior insecticidal potency of the two oils comprising Group IV, which were characterized by considerably higher contents of these monoterpene compounds, compared to the other oils. These results may also be partially explained by the likely increased phytophagous insect pressure at lower, warmer altitudes, as the two most potential insecticidal mint timija EOs were obtained from the two populations collected at altitudes below 1000 m.a.s.l. Conclusions. – The present study highlights, for the first time, differences in yield and chemical composition of mint timija EOs collected from different wild Moroccan populations. Based on the content of their major compounds, four main EO groups were characterized. This variability within the species was shown to be linked to the altitude variation of the collection sites. The EOs of the mint timija samples evaluated in this study also displayed interesting and different insecticidal activity against adults of T. castaneum. The two EOs belonging to Group IV, characterized by high contents of menthone and pulegone, exhibited the highest potency. Hence, they might have potential as botanical insecticides and could be considered for practical applications, particularly for stored-product insect pest control.

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Experimental Part Plant Material. Samples of the aerial parts of Mentha suaveolens subsp. timija (Briq.) Harley were collected from ten natural populations distributed throughout southwestern Morocco. They were harvested in 2011 at the vegetative stage, as described by Bellakhdar [10]. The plants were identified by A. A., and voucher specimens were deposited with the Laboratory of Biotechnology, Protection and Valorisation of Plant Resources, Faculty of Sciences, Semlalia, Marrakech, Morocco. Extraction of Essential Oils. The collected plant material was air-dried at r.t. (ca. 258) in the shade and subjected to hydrodistillation using a Clevenger-type apparatus for 3 h until total recovery of oil. The hydrodistillations were performed in triplicate (3 200 g), and the EOs obtained were dried (anh. Na2SO4 ), weighed, and stored at 48 until use. GC/MS Analysis. The analytical GC/MS system used was an Agilent 6890/5973 GC/MSD system (Agilent Technologies) equipped with an Agilent DB-5ms cap. column (30.0 m 0.25 mm i.d., film thickness 0.25 mm; model number 122-5532). The oven temp. was programmed rising from 60 to 2468 at 3 8/min; injector, transfer, source, and quadrupole temps. were 260, 280, 230, and 1508, resp.; carrier gas, He (high purity; constant linear velocity of 37 cm/s); injection volume, 1.0 ml (of samples of 60 ml of EO diluted with 2 ml of acetone); split ratio, 1 : 50; ionisation voltage, 70 eV; m/z range, 41 – 450 amu. Compound Identification and Quantification. The identification of the individual components was based on i) the comparison of their mass spectra with those of authentic reference compounds where possible and with those listed in the WILEY275 and NBS75K libraries and Adams terpene library [27] and ii) the comparison of their retention indices (RIs) determined on a DB5 cap. column (nonpolar, 5% phenyl polysilphenylene-siloxane) rel. to the retention times (tR ) of a series of n-alkanes (C9 – C24 ) with linear interpolation, with those of authentic compounds or literature data. For semi-quantification purposes, the normalized peak area of each compound was used without any correction factors, to establish relative contents. Insecticidal Activity. Insect Cultures. Colonies of the red flour beetle (Tribolium castaneum (Herbst, Coleoptera: Tenebrionidae)) were maintained in the laboratory without exposure to any insecticide. They were reared in glass containers (16 cm diameter 22 cm height) covered by a fine-mesh cloth for ventilation. Each container contained a mixture of wheat flour, wheat germ, and yeast extract (13 : 6 : 1 w/ w/w). The cultures were maintained in a growth chamber at 26 18, with a relative humidity (RH) of 70 – 85%, and a 16 : 8 h light : dark photoperiod. Only adults (7 – 14-d-old) were used for the test. All experimental procedures were conducted under environmental conditions identical to those of the cultures. In all bioassays, insects where considered dead when no leg or antennal movements were observed. The bioassays were designed to assess the median lethal concentration (LC50 and LC90 values, i.e., doses that kill 50 and 90% of the exposed insects, resp.). Contact-Toxicity Bioassay. The contact insecticidal activity of the EOs obtained from the ten mint timija populations against T. castaneum adults was determined by assessing the contact toxicity using filter paper discs (Whatman No. 1, 9 cm diameter). The EOs were dissolved in acetone at concentrations of 0.09, 0.16, 0.24, 0.31, 0.39, and 0.48 ml/cm2. Several preliminary tests were conducted to select the doses to be used for each EO. An aliquot of 1 ml of each soln. was uniformly dispensed on the surface of the filter paper, which was then placed in its designated glass Petri dish. Control filter papers were treated with acetone only. After 10 min, once the solvent had been evaporated, 10 unsexed adults were deposited into each dish. Each concentration and control was replicated three times, repeating each assay twice. Mortality was recorded at 24 h. Fumigant-Toxicity Bioassay. To assess the fumigant toxicity of the mint timija EOs, filter papers discs (Whatman No. 1, 2-cm diameter) were impregnated with different EO doses (1, 2, 3, 4, 5, and 6 ml). The impregnated filter papers were then immediately attached to the inside of the screw caps of 60-ml Plexiglas bottles, to give calculated fumigant concentrations of 16.66, 33.33, 50.00, 66.66, 83.33, and 100.00 ml/l air, resp. Caps were screwed tightly on the vials, each of which contained 10 unsexed adults. Each concentration and control was replicated three times, repeating each assay twice. Mortality was recorded at 24 h. Data Analysis. Probit analysis [28] was conducted for the dose-mortality data, following AbbottÏs correction for control mortality [29], to estimate lethal concentrations (LC50 and LC90) with their 95%


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confidence intervals by using SPSS 12.0 statistical software; LC values were considered significantly different when their respective 95% confidence intervals did not overlap. REFERENCES [1] P. Kumar, S. Mishra, A. Malik, S. Satya, Ind. Crops Prod. 2011, 34, 802. [2] G. Is¸can, N. Kirimer, M. Kîrkcîog˘lu, K. H. C. Baser, F. Demirci, J. Agric. Food Chem. 2002, 50, 3943. [3] A. Kamkar, A. Jebelli Javan, F. Asadi, M. Kamalinejad, Food Chem. Toxicol. 2010, 48, 1796. [4] M. Mahboubi, G. Haghi, J. Ethnopharmacol. 2008, 119, 325. [5] H. J. D. Dorman, M. Kosar, K. Kahlos, Y. Holm, R. Hiltunen, J. Agric. Food Chem. 2003, 51, 4563. [6] A. Hussain, F. Anwar, P. Nigam, M. Ashraf, A. Gilani, J. Sci. Food Agric. 2010, 90, 1827. [7] A. Kasrati, C. Alaoui Jamali, K. Bekkouche, H. Lahcen, M. Markouk, H. Wohlmuth, D. Leach, A. Abbad, Nat. Prod. Res. 2013, 27, 1119. [8] A. Kasrati, C. Alaoui Jamali, K. Bekkouche, H. Wohlmuth, D. Leach, A. Abbad, J. Food Sci. Technol. 2015, 52, 2312. [9] M. Fennane, M. Ibn Tattou, A. Ouyahya, J. El oualidi, Trav. Inst. Sci. S¦r. Bot. 2007, 2, 1. [10] J. Bellakhdar, ÐMedicinal Plants in North Africa and Basic Care, Handbook of Modern Herbal MedicineÏ, Le Fennec, Casablanca, 2006. [11] L. Rodrigues, O. Povoa, G. Teixeira, A. C. Figueiredo, M. Molda, A. Monteiro, Ind. Crops Prod. 2013, 43, 692. [12] S. Teles, J. A. Pereira, C. H. B. Santos, R. V. Menezes, R. Malheiro, A. M. Lucchese, F. Silva, Ind. Crops Prod. 2013, 46, 1. [13] S. Sutour, P. Bradesi, J. Casanova, F. Tomi, Chem. Biodiversity 2010, 7, 1002. [14] D. Segev, N. Nitzan, D. Chaimovitsh, A. Eshel, N. Dudai, Chem. Biodiversity 2012, 9, 577. [15] L. Riahi, M. Elferchichi, H. Ghazghazi, J. Jebali, S. Ziadi, C. Aouadhi, H. Chograni, Y. Zaouali, N. Zoghlami, A. Mlikia, Ind. Crops Prod. 2013, 49, 883. [16] H. Oumzil, S. Ghoulami, M. Rhajaoui, A. Ilidrissi, S. Fkih-Tetouani, M. Faid, A. Benjouad, Phytother. Res. 2002, 16, 727. [17] G. Koliopoulos, D. Pitarokili, E. Kioulos, A. Michaelakis, O. Tzakou, Parasitol. Res. 2010, 107, 327. [18] A. J. Burbott, W. D. Loomis, Plant Physiol. 1967, 42, 20. [19] S. Kokkini, V. P. Papageorgiou, Planta Med. 1988, 54, 166. [20] F. S. Sharopov, V. A. Sulaimonova, W. N. Setzer, J. Med Act. Plants 2012, 1, 76. [21] R. Croteau, F. Karp, K. C. Wagschal, D. M. Satterwhite, D. C. Hyatt, C. B. Skotland, Plant Physiol. 1991, 96, 744. [22] K. K. Aggarwal, A. K. Tripathi, A. Ahmad, V. Prajapati, N. Verma, S. Kumar, Insect Sci. Appl. 2001, 21, 229. [23] V. Pavlidou, I. Karpouhtsis, G. Franzios, A. Zambetaki, Z. Scouras, P. Mavragani-Tsipidou, J. Agric. Urban Entomol. 2004, 21, 39. [24] R. Pavela, Parasitol. Res. 2008, 102, 555. [25] P. Kumar, S. Mishra, A. Malik, S. Satya, Ind. Crops Prod. 2012, 39, 106. [26] P. Kumar, S. Mishra, A. Malik, S. Satya, Acta Tropica 2011, 122, 212. [27] R. P. Adams, ÐIdentification of Essential Oil Components by Gas Chromatography/Mass SpectrometryÏ, 4th edn., Allured Publishing Corporation, Carol Stream, IL, 2007. [28] D. J. Finney, ÐProbit AnalysisÏ, 3rd edn., Cambridge University Press, London, 1971. [29] W. S. Abbott, J. Econ. Entomol. 1925, 18, 265. Received June 27, 2014