Cupressus lusitanica has been reported to have phytochemical, ethno-botanical, agricultural, ornamental and industrial importance (Kamatenesi-Mugisha et al., 2013; Teke et al., 2013). In other research reports, the essential oil C. listanica has been shown to have antibacterial and antifungal activities against Bacillus cereus and Aspergillus niger, respectively (Hassanzadeh et al., 2010). On the other hand O. americanum is useful in medical practice in treatment of dysentery, leprosy, pruritus, parasitic infections helminthiasis, anorexia, dyspepsia, flatulence, vomiting, poisonous affections, fever among others (Yamada et al., 2013). The screening of L. javanica for pharmacological activity has indicated that its activities include antimalarial, antimicrobial, antioxidant, antiplasmodial, anticancer, antiamoebic, antidiabetic, and pesticidal properties (Viljoen et al., 2005; Van Wyk, 2011; Mujovo et al., 2008).

It is also a known fact that plants with medicinal properties are likely to have reproductive inhibition antifeedant, toxic, repellent properties against field and stored product insect pests (Pandey et al., 2017). Furthmore, reports on the qualitative and quantitative variations of chemical constituents among plants from same species and growing in different parts of the world may be associated to environmental conditions and genetic differences. Variations in analytical methods can also be responsible. Therefore, it is scientifically stimulating to screen essential oils from different plant species to determine whether they possess pesticidal properties. In addition, few research efforts have directed towards determining chemical constituents of aromatic plants of medicinal and pesticidal importance in the Eastern African region. Therefore, the main focus of the current study was to determine chemical constituents of essential oils of C. lusitnica, O. americanum and L. iavanica growing in Kenya.

Plant materials collection and essential oil extraction

The fresh  plant  leaves  were  collected  from  different  geographic regions in Kenya: C. lusitanica from Busia (0º25’20.02”N, 35º7’45.00”E, 1215 MASL), O. americanum from Homa Bay (0º22’03’’S, 35º16’59’’E, 2002 MASL) and L. javanica from Nakuru (0º20’23.52’’S, 35º56’34.67’’, 2250 MASL), Kenya in December, 2019. The fresh plant leaves were separately packed in paper bags and transported to the laboratory for essential oil extraction. The identification of plant species was carried out on sight based on professional expertise, literature materials and pictorial aids (Bett et al., 2016). The preserved samples of each plant species were presented to Plant Taxonomist, from Department of Biological Sciences, Egerton University for confirmation of identity. The preserved voucher samples of plant materials were then deposited at the herbarium of Egerton University. The fresh leaves of C. lusitanica, O. americanum and L. javanica weighing 500g were water extracted using a modified Clevenger-type apparatus. After 4 (h) of hydro-distillation the floating oil on top of the water was collected. The remaining water in the oil removed over anhydrous sodium sulphate (Na2SO4) and dried oil stored in the refrigerator at below 4 ºC until use (Bett et al., 2016).

GC-MS analysis and determination of essential oil constituents

The chemical constituents of essential oils from plant leaves were analysed using an HP-7890A Gas Chromatograph (GC) connected to an HP 5975 C Mass Spectrometer (MS) (Agilent, Wilmington, USA) at the International Centre of Insect Ecology and Physiology (ICIPE), Nairobi. From each sample 1mg was separately weighed and dissolved in 1 mL dichloromethane, dried using anhydrous sodium sulphate (Na2SO4) to make a stock solution (1 mg/mL). The experimental sample was obtained whose final concentration was 100 ng/µL. The samples were analyzed on an GC-MS in in full scan mode and a HP-5 MS low bleed capillary column (30 m × 0.25 mm i.d., 0.25 μm) (J and W, Folsom, CA, USA). The carrier gas was Helium (1.25 ml/min, constant flow mode) and the injection mode was split mode. The oven temperature was programmed at 35 ºC (for 5 min) to 280 ºC at 10 ºC/ min(5.5min) and then at 285ºC @50ºC/min (14.9 min) and the total run time was 50 min. The electron ionization mass spectra were acquired at 70 eV within a mass range of 38–550 Daltons (Da) during a scan time of 0.73 scans s-1 whereas the ion source was maintained at a temperature of 230 ºC. The essential oil constituents were identified by comparing mass spectra with library data (NIST05a and Adams MS HP, USA) and by comparison of the retention times with mass spectra (Adams et al., 1997).

The percentage yields (w/w) of essential oil extraction indicate that C. lusitanica leaves were richer (0.35 ± 0.2%) in essential oils as compared to O. americanum (0.27 ± 0.06%) and L. javanica (0.15 ± 0.09%). Tables 1 to 3 shows the retention time (min) identified chemical constituents and percentage concentration of oils obtained from C. lusitanica, O. americanum and L. javanica. The results of C. lusitanica, O. americanum and L. javanica essential oil extraction and GC-MS analysis showed that the oils were dominated by monoterpenes. However, each of the essential oils had different major chemical constituents. In C. lusitanica oil, 91 compounds were identified, corresponding to 99.8% of the total essential oil composition. The monoterpene hydrocarbons were 38.63%- dominated by α-pinene (13.8%), limonene (6.64%) and ortho-cymene (4.07%). On the other hand oxygenated monoterpenes were 15.77% with umbellulone (12.66%) and Terpinen-4-ol (1.37%) as major compounds.

The sesquiterpene hydrocarbons and oxygenated sesquiterpenes were 35.37 % and 6.56% respectively. The major sesquiterpene hydrocarbons and oxygenated sesquiterpenes were cadinene (7.47%) and α-cadinol (3.99%), respectively (Table 1 and Figure 1).

Table 2 and Figure 2 shows the retention time (min) identified chemical constituents and percentage concentration of oils obtained from O. americanum. In total, 72 compounds were identified, corresponding to 99.5 % of the total essential oil composition. The leaf essential oil contained 36.32, 36.19, 24.4 and 1.85% monoterpenes hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes, respectively.

The major monoterpenes hydrocarbons were1, 8-cineole (17.48%), camphor (7.55%) and myrecene (2.75%) whereas oxygenated monoterpenes were geraniol (18.72%), Eugenol (6.40%), and geranyl propanoate (3.91%). On  the  other  hand,  sesquiterpenes hydrocarbons were represented by elemicin (8.20%), b-bisabolene (3.78%) and caryophyllene (E) (2.22%) whereas oxygenated sesquiterpenes were in trace amounts. The composition of the essential oil of L. javanica is listed in Table 3 and the peaks depicted in Figure 3. As shown, a total of forty-seven (47) compounds were identified, consisting of mainly of oxygenated monoterpenes (67.82%) followed by sesquiterpene hydrocarbons (19.35%), monoterpene hydrocarbons (10.95%) and oxygenated sesquiterpenes (1.34%). It’s noted that the major monoterpene hydrocarbon was myrcene(7.04%) whereas oxygenated monotwerpenes were mainly ipsdienone (26.07%), ocimenone (14.32%), and bicyclo [3.1.1]hept-3-en-2-one, 4,6,6-trimethyl-, (1S)-(10.91%), On the other hand sesquiterpene hydrocarbons were dominated by caryophyllene(E) (5.11%) and germacrene D (4.78%). When the chemical constituents of the three essential oils were compared, the highest amounts of monoterpenes were found in L. javanica (87.77%), followed by O. americanum (72.51%) and C. lusitanica (54.40%) in decreasing  amounts, respectively. The converse was the situation with sesquiterpenes, with the highest percentage detected in C. lusitanica (41.93 %) followed by O. americanum (26.25%) and L. javanica (20.69 %), respectively. Diterpenes were found in trace amounts (2.14%) in C. lusitanica leaf essential oils.

The chemical constituents of C. lusitanica, O. americanum and L. javanica essential oils revealed in the present study indicate a variation in yield, number and concentration of chemical constituents. However, the main chemical constituents in oils of the three plant species were dominated by monoterpene hydrocarbons. It is noted that in C. lusitanica oil, the major compounds were; ??pinene??umbellulone and Limonene??The finding of the current study are also comparable to previous researches which have shown  chemical  composition  of C. lusitanica, O. americanum and L. javanica varied with countries and region. For instance Teke et al. (2013) found C. lusitanica to contain mainly germacrene D (18.5%), epi-zonarene (8.2%), cis-calamenene (8.2%), terpinen-4-ol (6.3%), linalool (6.0%) and umbellulone (6.0%). Similarly, the main components C. lusitanica oils has been found also to contain α-pinene (70.1%), δ-3-carene (45.4%), umbellulone (15.2%), β-phellandrene (10.8%)??terpinen-4-ol (9.7%) and myrcene (5.8%) (Bett et al., 2016). On the other hand, the main chemical constituents in O. americanum essential oil were Geraniol, 1, 8-cineole, elemicin and Camphor. In other studies, the main chemical constituents in O. americanum oil have been reported to include citral, linalool, geraniol, citronellol and 1, 8-cineole (Ilboudo et al., 2010; Yamada et al., 2013). In the same way, L. javanica oil was dominated by ipsdienone, ocimenone, and bicyclo [3.1.1] hept-3-en-2-one, 4, 6, 6-trimethyl-, (1S)-,  myrcene,  caryophyllene  (E)  and  germacrene  D.  

Several other authors have indicated that Lippia javanica oil is rich in caryophyllene, carvone, ipsenone, ipsdienone, limonene, linalool, myrcene, myrcenone, ocimenone, p-cymene, piperitenone, sabinene, and tagetenone (Viljoen et al., 2005; Lukwa et al., 2009; Chagonda and Chelchat, 2015; Maroyi, 2017).

However, Viljoen et al. (2005) using cluster analysis, found that L. javanica chemotypes growing in South Africa and Swaziland, were rich in myrcenone (36 to 62%), carvone- (61 to 73%), piperitenone- (32 to 48%), ipsdienonerich (42 to 61%), and linalool (>65%). The variations observed in chemical constituents and concentrations may be influenced by climatic and soil conditions in different the regions, which directly affect the metabolic processes of the plant (Chéraif et al., 2007), but also may arise from interaction of different biotic components. In addition, it has also been found that chemical constituents of essential oils could differ with species of plant, season, geographic region, and age of the plant, and the method used to extract essential oil extraction (Brooker and Kleinig, 2006, Bernard et al., 2020; Baccaria et al., 2020).

The chemical constituents found in oils in current study have been found in previous studies to have insecticidal properties against insect pests (Lee et al., 2003; Rosman et al., 2007 and Nivea et al., 2013). This supports the findings of Ilboudo et al. (2010); Olivero-Verbel et al. (2013) who reported that 1, 8-cineole, limonene and α-pinene were associated with contact toxicity of essential oils against insect pests. Similarly, Rozman et al. (2007) reported essential oils constituents containing, eugenol, 1,8-cineole, camphor and linalool to cause mortality of 85-100, 80-100 and 0-13% mortality of adult Sitophilus oryzae, Rhizopherta dominica and Tribolium castaneum, respectively.

The repellent properties of the major chemical constituents of essential oils of plant leaves for instance 1, 8-cineole, terpineol and α-pinene have also been reported in different researches (Tapondjou et al., 2005; Toloza et al., 2006; Bett et al., 2016). Furthermore, constituents of essential oils such as 1, 8-cineole, p-cymene, and γ-terpinene and α-pinene have earlier been reported to possess reproductive inhibition properties against insect  pests (Sedaghat et al., 2011; Alzogaray et al., 2011 and Gomah et al., 2015).  

For instance essential oils of Cupressus arizonica been found to have larvicidal activity against larvae of Anophhleles stephensi. The main essential oils constituents in the oil C. arizonica include limonene (14.44%), umbellulone (13.25%) and α-pinene (11%) (Sedaghat et al., 2011). Hence, the findings of this study is a proof that C. lusitanica, O. americanum and L. javanica and others possess pesticidal properties that may be exploited sustainably and cheaply for use in agriculture and related applications.

From the results of this study, it may be concluded that essential composition of C. lusitanica, O. americanum and L. javanica composition and classification were into various chemotypes varied with plant species. The essential oil constituents consisted mainly of monoterpenes that are known to have antifeedant, repellent, reproductive inhibition and insecticidal properties against  insect pests of field and stored products insect pests. The presence of potentially insecticidal bioactive compounds in C. lusitanica, O. amicanum and L. javanica provides scientifically stimulating since natural insecticides are ecologically and environmentally friendly for the management of insect pests.

The authors recommended more studies to be carried out on the bioactivity of the essential oils against various field and stored insect pests’ product. Therefore, the results of present study may lead to the formulation of new natural insecticides for management of insect pests.

The authors wish to appreciate Egerton University, through the Division of Research and Extension, for their financial and technical support towards this study. They wish also to thank the International Centre of Insect Physiology and Ecology (ICIPE) for assisting with GC-MS analysis apparatus and Mr Xavier Cheseto for providing technical support during GC-MS analysis process of essential oils.

The authors have not declared any conflicts of interests.

The authors have not declared any conflicts of interests.