Chemical composition and antimicrobial activities of essential oil extracted from Tithonia diversifolia ( Asteraceae ) flower

The upsurge in the prevalence of side effects of many synthetic antimicrobial agents and incidence of multidrug resistant bacteria has spurred scientists on the search for plant based antimicrobial of therapeutic potentials. The study extracted essential oils from the flowers of Tithonia diversifolia (Hemsl) A. Gray (Mexican sunflower) and assayed it for antimicrobial activities. The oils were extracted (hydro-distillation), characterized (GC-MS) and tested for antibacterial and antifungal activities. α-Pinene (34.42%), βCaryophyllene (22.34%), β-Pinene (11.14%), Germacrene-D (11.13%) and 1, 8-Cineole (8.76%) were the major constituents of the forty-five compounds characterized. The characterized compounds were general monoterpenes (44.44%), sesquiterpenes (26.67%), including alcohols and aldehydes which accounted for 28.89%. The extract concentrations of 8-120 mg/ml in Potato Dextrose Agar (PDA) medium effectively inhibited the tested fungi in vitro. At 5 mg/ml, only Bacillus megaterium and Bacillus cereus were inhibited of all gram-positive bacteria while Streptococcus pyrogens was unaffected. All gram-negative bacteria were inhibited. Growth inhibition of the gram-positive and gram-negative species increases with increased concentration of the essential oil. At 40 mg/ml, all the tested bacteria species were inhibited and the growth inhibition for the species followed the order; E. coli> Proteus mirabi> Bacillus megaterium> Klebsiella pneumonia> Bacillus cereus> Streptococcuspyrogens. The oil concentration of 72 mg/ml totally inhibited the growth of all three fungal species Cochliobolus lunatus, Fusarium solani, Fusarium lateritum. The secondary metabolites in the oil of T. diversifolia proved effective against the tested bacteria and fungi species. The finding of this study unlocks the potentials of this essential oil of Mexican sunflower for bio-pesticide production.


INTRODUCTION
Essential oils are natural volatile substances obtained from a variety of plants.Commercially, essential oils have many uses such as pharmaceuticals, flavour in many food products, odorants in fragrances and as insecticides. 1he antibacterial, antifungal and insecticidal activities of essential oils from plants have also been documented. 2,3It is difficult to pinpoint the exact period when essential oils became widely used, as plants and their oils have been sourced for their therapeutic properties over many centuries. 4Arabs were the first to develop techniques for obtaining essential oils from the naturally occurring organic materials. 5Essential oils are generally derived from one or more plant parts, including flowers, leaves, stems, wood, roots, seeds, fruits, rhizomes and gums or oleoresin exudations. 5,6,7,8Most essential oils consist of hydrocarbons, esters, terpenes, lactones, phenols, aldehydes, acids, alcohols, ketones, and esters.The part of the plant and structures containing the essential oil vary widely among plant families.The information about the oil secretory structures in plants can serve taxonomic tool (chemotaxonomy) for the associated plant families.According to Handa, 9 different plants have specialized secretory structures including glandular hairs (Lamiaceae, Verbenaceace and Geraniaceae), modified parenchyma cells (Piperaceae), resin canals (conifers), oil tubes or vittae (Apiaceae), lysigenous cavities (Rutaceae), schizogenous passages (Myrtaceae, Poaceae, Asteraceae) or gum canals (Cistacae, Burseraceae).However, not much is known about these oil secretory structures as to carefully categorize them.Although from a practical standpoint, these oils can be categorized into two main groups -superficial and subcutaneous.Based on notion that only Lamiaceae, Verbenaceace and Geraniaceae produce the former, all other plants are considered to produce the later. 9Tithonia diversifolia (Hemsl) A. Gray is a member of the family Asteraceae, native to Mexico and Central America. 10According to Akobundu and Agyakwa, 11 this plant was probably introduced into West Africa as an ornamental plant and has become naturalized in many tropical countries.In Nigeria, T. diversifolia occurs along roadsides, farmlands and lawns.It is prominent along roads in the guinea and derived savanna especially river courses (Lordbanjou, 1991; Chukwuka,  2003). 12,13It is encountered in many states of Nigeria including Oyo, Ondo, Lagos and Osun where it is found in association with a vast array of weeds (Chukwuka et  al., 2007). 14T. diversifolia or Mexican sunflower is a prolific shrub that is known for its medicinal importance.Extraction of essentials oils from this plant has been documented by researchers around the world.Moronkola 15 et al. reported the presence of Tagitinin A, a sesquiterpene.Srividya 16 et al. successfully extracted Tagitinin -A, C and E from the plant.Yemele 16 et al. reported that leaf extract of T. diversifolia consisted of Thithoniaquinone A and tithoniamide B, psoralen, and Iquebrachitol.Thithoniaquinone-A and psoralen were strongly fungicidal and antibacterial.Volatile oils found in the leaves of the plant composed of αpinene, βcaryophyllene, Germacrene D, β -pinene, 1, 8 -Cineole, Mycrene, ϒ -3 -Carene, α -Phelladrene, Bicyclogermacrene and β -Bisabolene, 15 .There are several compounds in T. diversifolia that are of great economic and medicinal values.Some of these compounds are antioxidants, antibacterial (e.g. against Staphylococcus aureus) and pests control.They can also be used as a part of medicine. 18yedokun 19 et al. assessed the pesticidal efficacy of T. diversifolia, Phyllanthus amarus and Acassia albida leaf extracts against harvester termite Macrotermes bellicosus (Isoptera: Termitidae), an emerging pest of cocoa, and discovered that aqueous and ethanolic extracts of T. diversifolia resulted in 44-88% and 36-68% mortality rates respectively in the termites.Extract and compounds isolated from the plant have demonstrated anti-malarial, anti-inflammatory, analgesic, anti-HIV-1 and-amoebic potential. 20,15Lifongo 21 et al. likewise documented the use of T. diversifolia leaf extracts in the treatment of malaria and fevers, especially in Nigeria.Information on the bactericidal and fungicidal properties of the essential oil from flower of this plant is scanty, hence this research.This study seeks to characterize the essential oils present in the flowers of T. diversifolia -an invasive weed in South-west, Nigeria, and determine the bactericidal and fungicidal potentials of the oils onsome multiple antibiotic and antifungal resistance bacteria and leafspot fungi.The study is expected to provide information on chemical composition of essential oils present in the flower of Tithonia diversifolia and their potential as biocides (bactericide and fungicide), an alternate and environmental-friendly control against bacteria and fungi as opposed to widely used synthetic biocides.

Collection of plant materials
Fresh flowers of Tithonia diversifolia Hemsl.(A.Gray) (Figure 1) were collected within the campus of University of Ibadan and confirmed at the University of Ibadan Herbarium, Department of Botany and Microbiology, University of Ibadan, Nigeria.

Extraction procedure
About 960 g of fresh flowers of T. diversifolia (Figure 2) were subjected to hydro-distillation for 3 hours using a Clevenger-type apparatus.The distillate was collected in a glass vial and stored at -4 °C prior to characterization (Figure 3).The distillate was characterized using a HP gas chromatography (model 6890) powered with HP Chem Station Rev. A 09.01 [1206] software equipped with flame ionization detector (FID) and HP-5MS capillary column (30 m x 0.25 mm, film thickness 0.25 µm).The Inlet temperature was kept at 150 °C.Column oven temperature was programmed from 40 °C to 220 °C at the rate of 5 °C/min and final temperature was held for 2 minutes.Hydrogen was used as the carrier gas with a flow rate of 1.0 ml/min.Sample unit of 1.0 µL was injected using the slit mode at a split ratio of 20:1.The chemical composition of the extract was determined from the relative percentage of the total peak area.

Antifungal activity test
The fungi used in this study were previously isolated and identified from leafspot disease of sweet potato.The fungi included Cochliobolus lunatus, Fusarium lateritium and Fusarium solani.They were maintained on Potato Dextrose Agar (PDA) slant at 4°C in the laboratory until needed.The isolates were revived twice on PDA before use.Inocula were prepared from 18 hrs old fresh culture by transferring 4 to 5 distinct colonies of each test bacterium into a5 ml sterile physiological saline with the aid of a sterile wire loop.The inocula were standardized to 0.5 Mcfarland's turbidity standard.Discs (8mm each) were prepared from Whatman filter paper No. 1 using perforating machine.The discs were wrapped in foil paper and sterilized in hot air oven at 150 °C for 1 hr.The essential oil was diluted with Tween 80 to obtain 40, 20, 10, 5, and 2.5 mg/ml and the discs were loaded by immersing in different concentration of oil.The sterile discs were impregnated with different concentrations of the test agents.The standardized inoculum (10 6 cfu/ml) was spread inoculated onto sterile Mueller-Hinton Agar (MHA) plates.The test agents containing discs were carefully and firmly placed on the surface of the inoculated MHA plates with the aid of a sterile forceps.The plates were incubated at 37 o C for 24 hrs.Diameter of zones of inhibition was measured using a transparent calibrated ruler and compared with the zone of inhibition of the control (gentamicin 100μg/ml).The minimum inhibitory concentrations (MIC) were determined.The antibacterial activity of the oils was conducted in triplicates and zones of inhibition (mm) were expressed as the mean of three measurements.
For each set of the experiment the essential oil were dissolved in dimethyl sulphoxide (DMSO) in the ratio of 1 g of extract to 10 ml of DMSO (1:10) to give the 100mg/ml concentration. 22Different concentrations (8, 16, 24, 32 and 120-mg/ml) were prepared from each of the extracts.One milliliter of each level of concentration was aseptically incorporated into 20ml of cool molten PDA in sterile test tube.Three test tubes were used for each extract concentration and the fourth test tube without the extract served as control.Each medium was thoroughly homogenized by gentle agitation before dispensing into sterile Petri dishes.The plates were allowed to set on the laboratory bench for 3 hours.This was done by inoculating the centre of the Petri plates with a mycelia disc (4mm) obtained from the colony edge of a 7-day old culture of the test fungi.Three replicates of both the control and PDA-extract plates per isolate were incubated at room temperature (28±2 ℃ ) and radial growth was measured with a metric ruler daily for seven days.The inhibition activity of the test agents on the radial growth (IR) was determined (Ni Putu and Suprata, 2012); Where: IR = inhibitory activity to the radial growth dc = average increase in mycelia growth in control plates dt = average increase in mycelia growth in treated plates Data obtained were subjected to analysis of variance (ANOVA) using statistical package for social science SPSS version 23.0 and means were separated according to Duncan's Multiple Range Test (DMRT) at 5% probability level.
The essential oils in T. diversifolia flowers were grouped based on the number of carbon atoms.They are grouped into monoterpenes, oxygenated monoterpenes, sesquiterpenes and oxygenated sesquiterpenes (Table 2).The essential oils that could not be grouped into any of the four aromatic groups are called 'Others'.Monoterpenes and oxygenated moneterpenes formed the principal components of the identified essential oils from flowers of T. diversifolia accounting for 44.44%, while sesquiterpenes and others accounted for 26.67% and 28.89% respectively.
The results of this study are in consonance with those of other studies in which several compounds, 23,24,25,26 mostly sesquiterpenes, diterpenes, monoterpenes and alicyclic compounds have been isolated from the leaves, stem and flowers of T. diversifolia.However, monoterpenes were higher than sesquiterpenes and other compounds in the flower of T. diversifolia in this study, which is in line with that of Moronkola et al.. 15 The presence of α-Pinene, cis-Ocimene, and limonene in this study also confirms those of Gbolade et al., 27 who reported the presence of α -Pinene (28.6%), cis-beta-Ocimene (43.7%), and limonene (12.0%) as the main constituents of T. diversifolia leaves.The high concentration of α-Pinene (34.42%) in this study is in line with the result of Menut 28 et al.where α-Pinene accounted for 50.8-61.0%of the 21 compounds identified.The variation in the chemical profile of the essential oils of this study with those of previous studies may be due to environmental influences, including climatic, seasonal, and geographical, as well as genetic variability. 29The total percentage of terpenes (71.10%) obtained in this study confirm the report of Zule et al. 30 that terpenes are the main constituents of essential oils synthesized in various parts of plant tissue.The most abundant quantities may be found in blossoms, leaves and cones.Other less important ingredients of essential oils are organic substances like aldehydes, esters and alcohols.Essential oils are used as raw materials in chemical, pharmaceutical, cosmetic and food producing industries. 30

Antibacterial activity of essential oil from T. diversifolia
The sensitivity pattern of the bacteria to the essential oils from T. diversifoliais presented in Tables 3. The essential oils inhibited three of the Gram-positive bacteria with Bacillus megaterium showing the highest susceptibility.Also, 2.5 mg/ml of the oil was able to inhibit Bacillus megaterium up to 6 mm while the other Gram positive bacteria (Bacillus cereus and Streptococcus pyrogens) were not inhibited at the concentrations tested.Out of the gram-negative bacteria tested, only the growth of E. coli was inhibited (17 mm) at 2.5 mg/ml.Of all the bacteria species tested at 5 mg/ml, only the growth of Bacillus megaterium and Bacillus cereus were inhibited as gram-positive bacteria while Streptococcus pyrogens was not inhibited.All the gram-negative bacteria were inhibited at this concentration.Growth inhibition for both gram-positive and gram-negative bacteria species tested were found to increase with increasing concentration of the essential oil.At 40mg/ml, all the tested bacteria species were inhibited and the growth inhibition for the species follow this order; E. coli> Proteus mirabilis > Bacillus megaterium> Klebsiella pneumonia> Bacillus cereus> Streptococcus pyrogens.
The susceptibility obtained indicates that the essential oils of T. diversifolia are active against the test isolates bacteria tested and does not indiscriminate between Gram positive and Gram negative and hence have therapeutic potentials for the ailment caused by the organisms.Obafemi 31 reported similar inhibitory effect for germacranolide type sesquiterpene lactone, obtained from leaf extract of T. diversifolia leaf extract, on Gram positive and negative bacteria.Studies by Bachir and Benali 32 (2012) on essential oils from the leaves of Eucalyptus globulus confirmed similar inhibition against Escherichia coli and Staphylococcus aureus.

Antifungal activity of essential oil of T. diversifolia
The inhibitory effects of T. diversifolia and their percentage inhibition on the test fungi are presented in Tables 4 and 5.The result showed that the extracts significantly (P<0.05)differed in their potential to inhibit the mycelia growth of the fungal pathogens.Inhibitory activity of the extract was dependent on the concentration of the oil and type of species tested.The result for the minimum inhibitory concentration (MIC) where fungal growth was observed on the essential oil is shown in Table 5.
All the three fungal pathogens recorded no inhibition at 0mg/ml while the first inhibition was observed at 8mg/ml in the following order Cochliobolus lunatus> Fusarium solani> Fusarium lateritum.Cochliobolus lunatus exhibited the highest inhibition (100%) at 56 mg/ml while Fusarium lateritum and Fusarium solani showed inhibition of 88.37% and 81.40% respectively.At 72 mg/ml, all the three fungal pathogen exhibited 100% inhibition which showed that at the concentration of the oil was effective against all three fungal pathogen.
The results of the antifungal activity assay showed that the plant extracts had inhibitory effects on the growth of the three leaf spot fungi.These results revealed that antifungal activity of the extracts was enhanced by increasing concentration (inhibition was concentration dependent).This is in agreement with the report of Ilondu 33 , Chiejina and Ukeh 34 that increase in antifungal activity was observed with corresponding increase in concentration of plant extracts.The antifungal activities of essential oil from T. diversifolia also corroborates the report by Obafemi 31  Extracts from various Asteraceae including T. diversifolia, were effective inhibitors of the mycelia growth of F. oxysporium and Trichophyton mentagrophytes. 36Although plant extracts with antifungal potential in in-vitro tests are not always effective under field conditions, 37 several works showed the effectiveness of plant extracts in controlling plant diseases in the field.Enikuomehin 38 reported that Cercospora leaf spot disease of sesame (Sesanum indicum L.) was controlled with plant extracts including extract of Tithonia diversifolia.Field evaluation of leaf extracts of some Asteraceae especially T. diversifolia were effective in the control of leaf spot disease of sweet potatoes in Abraka. 38

CONCLUSIONS
This study revealed an array of essential oils from T. diversifolia which has great pesticidal (bactericidal and fungicidal) potentials.The information from this study is useful towards developing plant based bactericides and fungicides that are ecofriendly for the management of foliar diseases of crops and the formulations of commercial botanicals.There is the need for continuous in-vivo testing to evaluate the efficacy of the characterized components in controlling incidence of bacterial and leafspot diseases in agricultural crops.Effective collaborations with plant pathologists, microbiologists, pharmacologists and chemists are crucial to see the complete development of interesting lead components into exploitable products.

Table 1 :
Concentration of constituents of the essential oil in T.

Table 2 :
Hydrocarbon characterization of the essential oils Tithonia diversifolia flower S/No.Hydrocarbons

Table 3 :
Antibacterial activity of essential oil of flower of Tithonia diversifolia at varying concentrations

Table 4 :
Percentage inhibition of three leaf spot fungi 7 days after inoculation Concentration of essential oil(mg/ml) Fungus et al. and Ragasa 35 et al. who reported antifungal activity of the leaf extracts.