GC-MS analysis of pesticidal essential oils from four Kenyan plants

1 Zoology Department, Jomo Kenyatta University of Agriculture and Technology (JKUAT) P.O Box 62000-00200 Nairobi, Kenya. 2 Chemistry department, Jomo Kenyatta University of Agriculture and Technology (JKUAT) P.O Box 62000-00200 Nairobi, Kenya. 3 Center for Biotechnology Research and Development (CBRD), Kenya Medical Institute(KEMRI), P.O Box, 5484000200, Nairobi Kenya. 4 Statistics and Actuarial Science Department, Jomo Kenyatta University of Agriculture and Technology (JKUAT) P.O Box 62000-00200 Nairobi, Kenya.


INTRODUCTION
Essential oils are complex mixtures of volatile organic compounds produced as secondary metabolites in plants.Steam distillation of aromatic plants yields essential oils, long used as fragrances and favoring in the perfume and food industries, respectively (Bakkali et al., 2008).More recently they have become popular as agents for aromatherapy.Essential oils are characterized by a strong odor and have a generally lower density than water.Among higher plants, there are 17,500 aromatic plant species (Bruneton, 1999) and approximately 3,000 essential oils are known out of which 300 are commercially important for cosmetics, perfume, and pharmaceuticals industries apart from pesticidal potential (Chang and Cheng, 2002;Bakkali et al., 2008).Several plant families, for example, Myrtaceae, Lauraceae, Rutaceae, Lamiaceae, Asteraceae, Apiaceae, Cupressaceae, Poaceae, Zingiberaceae, and Piperaceae, have been examined for anti-insect activities.To defend themselves against herbivores and pathogens, plants naturally release a variety of volatiles including various alcohols, terpenes, and aromatic compounds.These volatiles can deter insects or other herbivores from feeding, have direct toxic effects, or involve in recruiting predators and parasitoids in response to feeding damage.They may also be used by the plants to attract pollinators, protect plants from disease, or help in interplant communication (Pichersky and Gershenzon, 2002).Since the middle-ages, essential oils have been widely used for bactericidal, virucidal, fungicidal, parasiticidal, insecticidal, medicinal, and cosmetic applications, especially in the pharmaceutical, sanitary, and cosmetic applications.Aromatic plants produce many compounds that are insect repellents or act to alter insect feeding behavior, growth and development, ecdysis (moulting), and behavior during mating and oviposition.
The composition of these oils can vary dramatically, even within species according to the part of the plant from which the oil is extracted (leaf tissue, fruits, stem, etc.), the phonological state of the plant, the season, the climate, the soil type, and other factors.For example, rosemary oil collected from plants in two areas of Italy were demonstrated to vary widely in the concentrations of two major constituents; 1,8-cineole (7 to 55%) and apinene (11 to 30%) (Flamini et al., 2002).Such variations are common and have also been described for the oils derived from Ocimum basilicum (Pascual-Villalobos and Ballesta-Acosta, 2003) and Myrtus communis (Flamini et al., 2004).In this study, we investigated the phytochemical properties of essential oils from Tagetes minuta, Fuerstia africana, T vogelii, and S. ukambensis from Machakos county Kenya.The understanding of the chemical composition of the essential oils was essential in determining their use in arthropod control, antiseptic and food industries among others.

Plant materials and extraction of oils
Plant material (Leaves) of T. minuta, F. africana, T. vogelii, and S. ukambensis were collected in April 2010 from the farms and fields in Machakos, Kenya.The fresh leaves were sliced into smaller pieces.The essential oil was isolated from the plant materials by steam distillation using Clevenger apparatus (Guenter, 1949).The condensed oils were collected in n-hexane solvent (Aldrich HPLC grade) and the solution was filtered using Whattmann grade 1 filter papers containing anhydrous sodium sulphate in a funnel to remove any remaining traces of water.Hexane was then removed by distillation at 60ºC by the use of 'Contes' Short Path distillation apparatus.When condensation stopped, the oil was collected and weighed into small amber colored vials.

GC-MS analysis
The GC-MS analysis was performed on a 7890A gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) linked to a 5975 C mass selective detector (Agilent Technologies, Inc., Santa Clara, CA, USA) by using the following conditions: inlet temperature of 270°C, transfer line temperature of 280˚C, and column oven temperature programmed from 35 to 285°C with the initial temperature maintained for 5 min then 10 C/min to 280°C *Corresponding author.E-mail: glaonya@yahoo.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License held at this temperature for 10.5 min and finally 50°C/min to 285°C and held at this temperature for 29.9 min.The GC was fitted with a HP-5 MS low bleed capillary column (30 m × 0.25 mm i.d., 0.25 μm) (J&W, Folsom, CA, USA).Helium at a flow rate of 1.25 ml/min served as the carrier gas.The mass selective detector was maintained at ion source temperature of 230°C and a quadruple temperature of 180°C.Electron impact (EI) mass spectra were obtained at the acceleration energy of 70 eV.A 1.0 μl aliquot of extract was injected in the split/splitless mode using an auto sampler 7683 (Agilent Technologies, Inc., Beijing, China).Fragment ions were analyzed over 40-550 m/z mass range in the full scan mode.The filament delay time was set at 3.3 min.Due to unavailability of most of the authentic standards, relative quantification was achieved through the use of two authentic compounds [1, 8-cineole and β-caryophyllene (Sigma, St. Louis, MO, USA) whose calibration curves were used to quantify monoterpenes and sesquiterpenes respectively].GC-MS in full scan mode was used to detect the terpenes in the distillate.Serial dilutions of authentic standards of 1, 8-cineole and β-caryophyllene (1-100 pg/µl) were also analyzed by GC-MS in full scan mode to generate linear calibration curves (peak area vs. concentration) with the following equations; 1, 8-cineole [y = 0.7694x + 3.6807 (R 2 =0.9991)], and β-caryophyllene [y =0.5999x + 2.3004 (R 2 =0.9327)] which served as the basis for the external quantification of the terepenes identified.
GC-MS analysis of the essential oils revealed compounds toxic to pests and parasites.Linalool for instance toxic to eggs and larvae of insects (Liu et al., 2011) was found in F. africana and T. vogelii.Humelene, a strong repellent against R. appendiculatus was found in all the four study plants.Terpeneol a repellant of both R. appendiculatus and I. ricinis was a constituent compound of essential oils from T. manuta and T. vogelii.α-Pinene was found in all test plants except S. ukambensis and has repellent properties against arthropod pests (Tapondjou et al., 2005).
Essential oils interfere with basic metabolic, biochemical, physiological, and behavioral functions of Arthropods.They inhale, ingest or absorb essential oils.The rapid action against some pests is indicative of a neurotoxic mode of action, and there is evidence for interference with the neuromodulator octopamine (Enan, 2005) or GABA-gated chloride channels (Priestley et al., 2003;Khater 2011).
Essential oils have been found to be useful to man in various ways.Some essential oils have larvicidal effects and the capacity to delay development and suppress emergence of adult insects of medical and veterinary importance (Khater and Shalaby, 2008;Koul et al., 2008;Khater, 2011).Thyme oil and monoterpenoids including thymol, anethole, eugenol, and citronellal combinations have been patented for pesticidal activity against cockroaches and the green peach aphid.Similarly, citronellal, cotronellol, citronellyl or a mixture of these have been patented as pest treatment composition against the human louse (Ping, 2007).Nutmeg oil has been determined to significantly impact both the maize weed, Sitophilus zeamais and the red-flour beetle, Tribolium castaneum and demonstrates both repellent and fumigant properties.Essential oils of Cinnamomum camphora, C. cassia, and C. zeylanicum repel mosquitoes (Kim et al., 2003).Oils of soybean, lemongrass, cinnamon, and the compounds 3,8-pmenthane-diol (from lemon eucalyptus), citronellal (from lemongrass), and 2-phenethylpropionate (from groundnut), are effective against mosquitoes (Fradin and Day, 2002).Lemon eucalyptus is a potent repellent.Its oil, comprising 85% citronellal, is used by cosmetic industries due to its fresh smell.Flea and tick control products for companion animals based on d limonene, a constituent of citrus peel oil, or oils of peppermint, cinnamon, clove, thyme, and lemongrass, have been introduced recently (Isman, 2010).
d-Limonene is heavily used for controlling structural pests as termite in California, and other plant oils like clove and peppermint.(Liu et al., 2011).
As can be concluded from the data presented on plant volatile, each species seems to have its own unique chemical composition with little similarity.In summary,  the results of this study, further strengthens the view that T. vogelii, T. minuta, F. africana and S. ukambensis are potential sources of anti-arthropods agents especially insects and ticks and to some extent validates the traditional use of the plants for insect pest control by the farmers in livestock keeping areas in Kenya (Table 5).

Figure 2 .
Figure 2. GC-MS chromatogram of essential oils obtained from T. minuta

Figure 3 .
Figure 3. GC-MS Chromatogram for essential oils obtained from T. vogelii

Table 1 .
Compounds obtained from GC-MS analysis of essential oils from Fuerstia africana (Labiatae).
a Compounds are listed in order of elution from an SE-52 column.b RT, Retention time.

Table 2 .
Compounds obtained from GC-MS analysis of T. minuta.
a RT b (min) Percentage maximum a Compounds are listed in order of elution from an SE-52 column.

Table 3 .
Compounds obtained from GC-MS analysis of essential oils from Tephrosia vogelii.
a Compounds are listed in order of elution from an SE-52 column.b RT, Retention time.

Table 4 .
Compounds obtained from GC-MS analysis of essential oils obtained from Sphaeranthus ukambensis.
a Compounds are listed in order of elution from an SE-52 column.b RT, Retention time.

Table 5 .
Compounds common in all the four plant species.