Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3672

Full Length Research Paper

Chemical composition, in vitro antioxidant and antiparasitic properties of the essential oils of three plants used in traditional medicine in Benin.

Didier Kpadonou
  • Didier Kpadonou
  • Laboratory of Physic and Synthesis Organic Chemistry (LaCOPS), Faculty of Sciences and Technics (FAST), University of Abomey-Calavi (UAC), BP: 4521 Cotonou, Benin.
  • Google Scholar
Salomé Kpoviessi
  • Salomé Kpoviessi
  • Laboratory of Physic and Synthesis Organic Chemistry (LaCOPS), Faculty of Sciences and Technics (FAST), University of Abomey-Calavi (UAC), BP: 4521 Cotonou, Benin.
  • Google Scholar
Joanne Bero
  • Joanne Bero
  • Laboratory of Physic and Synthesis Organic Chemistry (LaCOPS), Faculty of Sciences and Technics (FAST), University of Abomey-Calavi (UAC), BP: 4521 Cotonou, Benin.
  • Google Scholar
Pierre Agbani
  • Pierre Agbani
  • Laboratory of Applied Ecology (LEA), Faculty of Agronomic Sciences (FSA), University of Abomey-Calavi (UAC), 03 BP: 1974 Cotonou, Benin.
  • Google Scholar
Fernand Gbaguidi
  • Fernand Gbaguidi
  • Laboratory of Physic and Synthesis Organic Chemistry (LaCOPS), Faculty of Sciences and Technics (FAST), University of Abomey-Calavi (UAC), BP: 4521 Cotonou, Benin.
  • Google Scholar
Bénédicta Kpadonou-Kpoviessi
  • Bénédicta Kpadonou-Kpoviessi
  • Laboratory of Physic and Synthesis Organic Chemistry (LaCOPS), Faculty of Sciences and Technics (FAST), University of Abomey-Calavi (UAC), BP: 4521 Cotonou, Benin.
  • Google Scholar
Brice Sinsin
  • Brice Sinsin
  • Laboratory of Applied Ecology (LEA), Faculty of Agronomic Sciences (FSA), University of Abomey-Calavi (UAC), 03 BP: 1974 Cotonou, Benin.
  • Google Scholar
Michel Frédérich
  • Michel Frédérich
  • Université de Liège, Drug Research Center, Laboratoire de Pharmacognosie, Av. de l’Hôpital 1, B36, B-4000 Liège, Belgium.
  • Google Scholar
Joëlle Quetin-Leclercq
  • Joëlle Quetin-Leclercq
  • Pharmacognosy Research Group, Louvain Drug Research Institute, Université Catholique de Louvain, B1 7203 Av. E. Mounier 72, B-1200 Bruxelles, Belgium.
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  •  Received: 23 May 2019
  •  Accepted: 08 August 2019
  •  Published: 30 September 2019

 ABSTRACT

Sclerocarya birrea (Sb), Psidium guajava (Pg) and Eucalyptus camaldulensis (Ec) are widely used in traditional medicine for the treatment of many diseases, some of which were related to oxidative stress and parasitic diseases. Their essential oils (EO) were analyzed by GC/MS and FID and tested in vitro for their antioxidant activities (DPPH), their anti-trypanosomal and anti-plasmodial activities against Trypanosoma brucei brucei (Tbb) (strain 427) and Plasmodium falciparum (Pf) (strain 3D7), respectively. Cytotoxicity was evaluated in vitro against CHO and WI38 cells (MTT) to evaluate the selectivity. They were shown to possess low antioxidant but a strong anti-trypanosomal and a good antiplasmodial activity with a good selectivity, except Ec oil whose anti-plasmodial activity was less interesting. Sb oil was the most active against Tbb(IC50 = 0.46 ± 0.28 µg/ml) and Pf (5.21 ± 1.12 µg/ml). All tested oils had low or no cytotoxicity against CHO and WI38 cells. GC/MS and GC/FID analysis revealed that composition of Sb (49 compounds) was characterised by the presence as main constituents of 7-epi-α-selinene, α-muurolene and valencene; Pg (60 compounds) by β-bisabolene, ar-curcumene and β-bisabolol; Ec (43 compounds) by γ-terpinene and p-cymene. The activity of these oils seems to be the result of a synergistic action of all their constituents, including minor ones. This study shows that essential oils of Sb and Pg can be good sources of anti-trypanosomal and anti-plasmodial agents.

Key words: Essential oil, S. birrea, P. guajava, E. camaldulensis, antimalarial, antitrypanosomal, antioxidant.

 

Abbreviation: Sb: Sclerocarya birrea, Pg: Psidium guajava, Ec: Eucalyptus camaldulensis.

 INTRODUCTION

The emergence of parasites resistant to current chemotherapies highlights the importance of the search of  potential   novel  anti-parasitic  agents  which  may  be used as alternatives or adjuvants to current  anti-parasitic therapies (Cheikh-Ali et al., 2011; Nibret and Wink, 2010). Similarly   the  overproduction  of  free  radicals  in cells induces an oxidative stress inplicated in atherosclerosis, cardiovascular diseases, hypertension, ischemia/reperfusion injury, diabetes mellitus, neuro-degenerative diseases, immuno-inflammatory and malaria (Maloueki et al., 2015; Rashid et al., 2013; Valko et al., 2007; Djordjević et al., 2008; Ayoola et al., 2008). To escape these serious consequences related to oxidative stress and parasitic diseases, the use of aromatic and medicinal plants, and especially their essential oils have been the subject of several studies (Kpoviessi et al., 2014; Safaei-Ghomi et al., 2009).

Sclerocarya birrea (A. Rich.) Hochst (Anacardiaceae), Psidium guajava L. (Myrtaceae) and Eucalyptus camaldulensis Dehnh (Myrtaceae) are aromatic plants used as food for men and cattle, for firewood, wood carving and in traditional medicine for many diseases (Kabiru et al., 2013; Gouwakinnou et al, 2011; Gutiérrez et al., 2008). The stem bark aqueous extract of S. birrea has been used to treat malaria in Benin (Gouwakinnou et al, 2011). Bark aqueous and methanolic extracts were shown by Gathirwa et al. (2008) to possess in vitro anti-plasmodial and in vivo anti-malarial efficacy alone or in combination with other medicinal plant extracts. Maceration, infusion or decoction in water of different parts of P. guajava are used in several countries as febrifuge or in skin problems (Gutiérrez et al., 2008; Hermans et al., 2004; Ajaiyeoba et al., 2003). Aqueous decoctions and various extracts from leaves and flowers of P. guajava, alone or in combination with other medicinal plant extracts possess in vitro anti-plasmodial activities (Kaushik et al., 2015; Tarkang et al. 2014; Rajendran et al., 2014; Chinchilla, et al., 2012). E. camaldulensis leaves are used alone and in combination with other plants to treat malaria and typhoid fevers in some Northern parts of Nigeria and ethanolic extracts possess in vivo anti-trypanosomal activities (Kabiru et al., 2013).

Essential oils of these plants are known for antimicrobial, antifungal, antioxidant, analgesic, anti-inflammatory, anti-nociceptive, antiradical, larvicidal, and insecticidal properties (Ghalem and Mohamed, 2014; Njume et al., 2011). Furthermore, these oils are used orally (drops) or by inhalation in traditional medicine for the treatment of malaria or its symptoms or sleeping sickness (Knezevic, 2016; Rasoanaivo et al., 1992; Gelfand et al., 1985).The direct activity of these essential oils against Trypanosoma brucei and Plasmodium falciparum was not very documented except for essential oil of E. camaldulensis from Nigeria. This oil was reported to kill in 4 mins T. brucei brucei parasites at a concentration of 0.4 g/ml in vitro (Habila et al., 2010). So, it seemed  interesting  to  study  the  anti-plasmodial  and anti-trypanosomal activities of these essential oils and their components.

T. brucei is the parasite responsible for human African trypanosomiasis or sleeping sickness, an illness affecting 300,000 African people, while up to 60 million people in 36 countries are at risk of contracting the disease and 6314 cases were recorded in 2013 (WHO, 2015). This parasite is transmitted by the bite of infected Tse-tse flies of the genus Glossina. Malaria is also a disease caused by a protozoan parasite of Plasmodium specie and still remains a major public health problem in the world. According to the latest estimates, 219 million cases of this disease occurred globally in 2017 (uncertainty range 203 to 262 million) and the disease led to 435 000 deaths (WHO, 2018).

These two parasitic diseases are the cause of considerable mortality and morbidity throughout the world and parasites develop resistance to most of the drugs used (WHO, 2018). Some of these drugs need a long course parenteral administration, show toxicity and a variable efficacy between strains or species. Free radicals also cause several diseases whose treatments are very expensive for the population. There is a need to search for new anti-trypanosomal, anti-plasmodial and antioxidant lead compounds with new mechanism of action from medicinal plants (Bero et al., 2011).

The present study aims to evaluate in vitro anti-trypanosomal, anti-plasmodial and antioxidant activities, along with cytotocycity against chinese hamster ovary cells (CHO) and a human non cancer fibroblast cell line (WI38) for the determination of selectivity, of essential oils from three plants: S. birrea, P. guajava and E. camaldulensis used in traditional medicine in Benin.

 


 MATERIALS AND METHODS

Plant material

Fresh leaves of S. birrea (A. Rich.) Hochst (Anacardiaceae), P. guajava L. (Myrtaceae) and E. camaldulensis Dehnh. (Myrtaceae) were collected in March 2014, from the Botanical Garden of the Abomey-Calavi University. Voucher specimens (n°AA6384, AA6536 and AA6590/HNB respectively) were conserved at the University of Abomey-Calavi Herbarium.

Chemicals and drugs

Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F12 culture media were purchased from Life technologies corporation (Grand Island, NY 14072, USA); Dulbecco’s Phosphate Buffered Saline (DPBS 1X) from Invitrogen (Grand Island, NY 14072, USA); tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium-bromide) (MTT), DPPH (2,2-diphenyl-1- picrylhydrazyl),   (S) - (+),    ascorbic   acid,     (S)-(+)-camptothecin, suramine, chloroquine,  artemisinin, dimethyl sulfoxide (DMSO) and n-alkanes “C7-C28” were obtained from Sigma-Aldrich (Steinhein, Germany), Acros Organics (New jersey, USA), and Fluka Chemie (Buchs, Switzerland). All compounds were of analytical standard grade. Ter-Butyl methyl ether (TBME) was an analytical grade solvent purchased from Fluka Chemie, and anhydrous Na2SO4 was of analytical reagent grade from UCB (Brussels, Belgium).

Isolation of essential oils

Five hundred grams (500 g) of fresh leaves were steam distillated for 3 h in a modified Clevenger-type apparatus (Bruneton, 2009). The extraction was carried out in triplicate. The oils were preserved in a sealed vial at 4°C. The essential oil yields were calculated based on the fresh plant material (Kpoviessi et al., 2014).

Chemical analysis of essential oils

GC/MS analysis

GC/MS analysis was carried out using a TRACE GC 2000 series (Thermo-Quest, Rodano, Italy), equipped with an autosampler AS2000 Thermo-Quest. The GC system was interfaced to a Trace MS mass spectrometer (ThermoQuest) operating in the electronic impact mode at 70 eV. HP 5MS column (30 m × 0.25 mm, film thickness: 0.25 mm) was used; injection mode: splitless; injection volume: 1 µl (TBME solution); split flow: 10 ml/min; splitless time: 0.80 min; injector temperature:  260°C; oven temperature was programmed as following: 50 to 250°C at 6°C/min and held at 250°C for 5 min; the carrier gas was helium with a constant flow of 1.2 ml/min. The coupling temperature of the GC was 260°C and the temperature of the source of the electrons was 260°C. The data were recorded and analyzed with the Xcalibur 1.1 software (ThermoQuest) (Kpoviessi et al., 2014).

Identification of oil components

Individual components of the volatile oils were identified by comparison with computer matching of their retention times against those of commercial EI-MS spectra library (NIST/EPA/NIH, 1998; Adams, 2007), home-made mass spectra library made from pure substances and components of known oils (Kpoviessi et al., 2011). Mass spectrometry literature data were also used for the identification, which was confirmed by comparison of the GC retention indices (RI) on a non-polar column (determined from the retention times of a series of n-alkanes “C7 - C28” mixture) (VanDenDool and Kratz, 1963). The minimum Relative Strength Index (RSI) for MS analysis was 937. The Kovats indices (KI) calculated were in agreement with those reported by Adams (Adams, 2007). Quantification (expressed as percentages) was carried out by the normalization procedure using peak areas obtained by FID. Values are expressed as mean ± standard deviation (n = 3).

In vitro test for antioxidant activity

The DPPH method was used to evaluate the antioxidant activity of oils. In a 96-well microplate, a series of 10 successive dilutions (at 1/2) of each oil, was prepared from sample solutions at 150 μL/ml in methanol. For each concentration, three (03) tests were carried out by adding 100 μl of DPPH at 100 μg/ml in methanol at all dilutions in cascade. Thus, the DPPH was tested at a single concentration of 50 μg/ml. The plate was incubated in the dark for 20 min and the absorbance at 517 nm using a spectrophotometer. The negative control  consists  of  1 ml of  methanolic solution and 1 ml  of  DPPH solution (100 µ/ml). Positive control was the solution of Ascorbic acid (1 mg/ml) (Otohinoyi et al., 2014; Brand-Williams et al., 1995)

The antiradical activity was estimated according to the following equation:


  

The extract concentration that reduces the absorbance of DPPH by 50% (EC50) was obtained with the GraphPadPrism 4.0 software.

Parasites, cell lines and media

T. brucei brucei strain 427 (Molteno Institute in Cambridge, UK) bloodstream forms were cultured in vitro in HMI9 medium containing 10% heat-inactivated foetal bovine serum (Hirumi and Hirumi, 1994). P. falciparum chloroquine-sensitive strain 3D7 (from Prof. Grellier of Museum d’Histoire Naturelle, Paris-France) asexual erythrocytic stages were cultivated continuously in vitro according to the procedure described by Trager and Jensen (1976) at 37°C and under an atmosphere of 5% CO2, 5% O2 and 90% N2. The host cells were human red blood cells (A or O Rh+). The culture medium was RPMI 1640 (Gibco) containing 32 mM NaHCO3, 25 mM HEPES and 2.05 mM L-glutamine. The medium was supplemented with 1.76 g/L glucose (Sigma–Aldrich), 44 mg/mL hypoxanthin (Sigma–Aldrich), 100 mg/L gentamycin (Gibco) and 10% human pooled serum (A or O Rh+). Parasites were subcultured every 3 to 4 days with initial conditions of 0.5% parasitaemia and 1% haematocrit.

The macrophage-like cell line, CHO Chinese Hamster Ovary cells  (ATCC N° CCL-61, batch 4765275), were cultivated in vitro in Ham’s F12 Nutrient Mixture 21765 medium (Gibco) containing 2 mM L-glutamine supplemented with 10% heat-inactivated foetal bovine serum (Gibco) and penicillin–streptomycin (100 UI/mL to 100 mg/mL). The human non cancer fibroblast cell line, WI38 (ATCC N° CCL - 75 from LGC Standards) was cultivated in vitro in DMEM medium (Gibco) containing 4 mM L-glutamine, 1 mM sodium pyruvate supplemented with 10% heat-inactivated foetal bovine serum (Gibco) and penicillin–streptomycin (100 UI/mL to 100 mg/mL).

In vitro test for antiplasmodial activity

Parasite viability was measured using parasite lactate dehydrogenase (pLDH) activity according to the method described by Makler et al. (1993). The in vitro test was performed as described by Murebwayire et al. (2008). Chloroquine (Sigma) or artemisinin (Sigma) were used as positive controls in all experiments with an initial concentration of 100 ng/mL. First stock solutions of essential oils and pure compounds were prepared in DMSO at 20 mg/mL. The solutions were further diluted in medium to give 2 mg/mL stock solutions. The highest concentration of solvent to which the parasites were exposed was 1%, which was shown to have no measurable effect on parasite viability. Essential oils were tested in eight serial threefold dilutions (final concentration rang: 200 to 0.09 mg/mL, two wells/concentration) in 96-well microtiter plates. The parasitaemia and the haematocrit were 2 and 1%, respectively. All tests were performed in triplicate.

In vitro test for anti-trypanosomal activity

The in vitro test was performed as described by Hoet et al. (2004). Suramine (a commercial antitrypanosomal drug, MP Biomedicals, Eschwege, Germany) was used as positive control in all experiments with  an  initial  concentration  of  1 mg/mL.  First  stock solutions of essential oils and compounds were prepared in DMSO at 20 mg/mL. The solutions were further diluted in medium to give 0.2 mg/mL stock solutions. Essential oils and compounds were tested in eight serial threefold dilutions (final concentration range: 100 to 0.05 mg/mL, two wells/concentration) in 96-well microtiter plates. All tests were performed in triplicate.

Cytotoxicity assay

The cytotoxicity of the oils against CHO and WI38 cells was evaluated as described by Stevigny et al. (2002), using the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (Sigma)) colorimetric method based on the cleavage of the reagent by dehydrogenases in viable cells. Camptothecin (Sigma) was used as positive cytotoxic reference compound. Stock solutions of compounds and essential oils were prepared in DMSO at 10 mg/mL. The solutions were further diluted in medium with final concentrations of 200 to 6.25 mg/mL. The highest concentration of solvent to which the cells were exposed was 1%, which was shown to be non-toxic. Each oil was tested in six serial fourfold dilutions in 96-well microtitre plates. All experiments were made at least in duplicate.

Statistical analysis

Student’s t-test was used to test the significance of differences between sets of results for different samples, and between results for samples and controls (GraphPad Prism 4.0; GraphPad Software Inc., San Diego, USA). Statistical significance was set at P < 0.05.

 


 RESULTS AND DISCUSSION

Chemical composition of the essential oils

Yields (w/w) of oils extracted from fresh leaves of Sb, Pg and Ec (0.24, 0.78 and 1.38%, respectively) collected in the same place at the same time are given in Table 1. The yield (1.38%) of Ec leaves oil obtained in the present study confirms the work of Moudachirou et al. (1999) who reported the highest rate (1.30%) for this plant in Benin at Calavi in the period of February and March or at Kétou between April and May 1996. However, this yield was higher than that obtained in Morocco (0.84%) (Farah et al., 2002) and between the values 0.75 and 1.42% obtained from Tunisia (Haouel et al., 2010). For Pg, the yield (0.78%) was closer to that reported for this plant at Tchaada in Benin (0.82%) (Noudogbessi et al., 2013) and in Nigeria (0.75%) (Ogunwande et al., 2003) but different from the one described by Noudogbessi et al. (2013) in Missérété (0.25%) and Adjarra (0.30%) in outhern Benin. The leaves of Sb gave an oil yield (0.24%) in accordance with that indicated by Kpoviessi et al. (2011) for the same plant in the same area during the rainy season. These authors had also showed that this yield varies depending on the season (Kpoviessi et al., 2011). The difference between essential oil yields or chemical composition of the same plant could be explained by the influence of the location, season, and time of  harvest  in  the  day  or  the vegetative stage of the plant (Noudogbessi et al., 2013; Kpadonou-Kpoviessi et al., 2012; Kpoviessi et al., 2011; Moudachirou et al., 1999).

 

 

A total of 49 (Sb), 43 (Ec) and 60 (Pg) compounds, representing respectively 97.96% (Sb), 98.50% (Ec) and 96.10% (Pg) of hydrodistillate, were identified (Table 1). These oils contained more hydrocarbon compounds (60.60 to 92.55%) than oxygenated ones. Sesquiterpenes were the major terpenoids in Sb and Pg oils (95.12 and 93.81%, respectively) while Ec oil was characterized by the predominance of monoterpenes (96.95%) (Table 2).

 

 

The essential oil of Sb was characterized by the presence of 7-epi-α-selinene (37.86 ± 0.03%) of α-muurolene (25.03 ± 0.03%), and valencene (17.12 ± 0.06%) as major constituents followed by β-selinene (4.32 ± 0.01%), β-caryophyllene (3.24 ± 0.02%), epoxy-allo aromadendrene (1.54 ± 0.03%), 14-hydroxy-α-humulene (1.51 ± 0.03%) and α-copaene (1.20 ± 0.04%). The study of this oil was not very documented. Its chemical composition was close to that described by Kpoviessi et al. (2011) by GC/FID and GC/MS analysis methods.

In the Ec oil, γ-terpinene (57.24 ± 0.04%) predominated followed in decreasing order of rate by p-cymene (18.22 ± 0.02%), terpinen-4-ol (7.50 ± 0.07%), 1,8-cineole (7.49 ± 0.07%), limonene (1.82 ± 0.02%) and terpinolene (1.02 ± 0.01%). This composition was similar to that described at Calavi (Moudachirou et al., 1999) but different from those studied in Spain (Verdeguer et al., 2009), Jerusalem (Chalchat et al., 2001), Tunisia (Haouel et al., 2010), Australia, Morocco and Ivory Coast (Kanko et al., 2012), which were richer in p-cymene, spathulenol, cryptonne or 1, 8-cineole. 

No component of the Pg oil exceeded a rate of 15%. Over twenty compounds exhibit a percentage higher than 1% with β-bisabolene (14.38 ± 0.03%), ar-curcumene (12.39 ± 0.02%), β-bisabolol (11.40 ± 0.08%) and β-caryophyllene (8.04 ± 0.03%) as major compounds. These results were more similar to those obtained in the locality of Banigbe (Benin) by Noudogbessi et al. (2013) than those obtained in other parts of the country by the same authors. Furthermore, the content of 1,8-cineole (0.44 ± 0.01%) in Benin oil was lower than that in Brazil (21.40%; with GC/MS method), Taiwan (12.40%, with GC/FID and GC/MS methods) and China (18.90% with GC/MS method) ones (Chen et al., 2007; Da Silva et al., 2003). 

Anti-trypanosomal, anti-plasmodial activities and cytotoxicity

All studied oils were tested in vitro for their anti-trypanosomal and anti-plasmodial activities respectively on T. brucei brucei and P. falciparum 3D7 and their cytotoxicity  against  WI38 and CHO cells. The results are summarized in Table 3.

 

 

These oils show an interesting anti-trypanosomal activity, the most interesting being Pg (IC50 = 1.16 ± 0.16 mg/ml) and Sb (IC50 = 0.46 ± 0.28 mg/ml). According to Bero et al. (2014), Ec oil has a moderate anti-trypanosomal activity (2 ≤ IC50 ≤ 20 mg/ml). While the other oils exhibited good activities (IC50 ≤ 2 mg/ml) and could be of interest for future development (Bero et al., 2014). The activity of Sb oil was not significantly different (P value = 0.1628> 0.1) than that of suramin (IC50 = 0.11 ± 0.02 µg/ml), the standard compound used against this parasite. The selectivity index of the three tested oils (Sb = 79; Ec > 19 and Pg = 33) showed that Sb was also the most selective. In vivo  studies  should   be  performed  to assess its efficacy on sleeping sickness and determine if the essential oil from Sb already consumed by livestock and extensively used in traditional medicine, can be recommended for the treatment of this illness. It will be necessary to search for adequate formulation as LBDDS (lipid based drug delivery systems) (Mu et al., 2013) and to verify the absence of toxicity. To our knowledge, this is the first report of the activity of the essential oil of these three plants from Benin on T. brucei brucei except Habila et al. (2010) who showed that a concentration of 0.4 g/ml of Ec oil from Nigeria killed T. brucei brucei parasites in 4 min. Essential oils of plants from the same family (Myrtaceae) as Leptospermum scoparium Forst., Melaleuca  alternifoliaSyzygium   aromaticum  (L.) Merrand L. M. Perry Cheel and Kunzea ericoides (A. Rich) Joy Thomps, showed anti-trypanosomal activities, respectively with IC50 values of 16.90, 0.50, 1.90 and 13.60 µg/ml (Bero et al., 2014). It was also reported that the ethanolic extract of Pg leaf was able to produce alterations in the biochemical parameters in the kidney and liver of rats experimentally infected with T. brucei brucei (Adeyemi and Akanji, 2011).

Concerning the anti-plasmodial activity against the chloroquine-sensitive 3D7 P. falciparum strain, Sb (IC50 = 5.21 ± 1.12 mg/ml) and Pg (IC50 = 12.02 ± 2.99 mg/ml) essential oils showed moderate activity with 2 ≤ IC50 ≤ 20 mg/ml. The Ec (IC50 = 51.30  ±   4.35 mg/ml)   oil   was  less   interesting against this parasite. Sb aqueous extract in combination with three other medicinal plants was reported to exhibit high malaria parasite suppression (chemo-suppression >90%) in vivo with a doses of 100 mg/kg/d at different ratios tested interperitoneally or per os (Gathirwa et al., 2008). Recently, promising in vitro antiplasmodial activity against 3D7 (IC50 ≤ 20 μg/ml), was seen in leaves ethyl acetate extracts and methanol extracts of Pg (Kaushik et al., 2015) and synergistic activities of combination with ethanol and water macerations of Mangifera indica, Carica papaya, Cymbopogon citratus, Citrus sinensis, and Ocimum gratissimum were reported against P. falciparum 3D7 and Dd2 strains (Tarkang et al., 2014). Aqueous decoctions of Pg also showed anti-plasmodial activity against chloroquine resistant P. berghei (Rajendran et al., 2014).

With a selectivity index > 6 and 3, respectively, the essential oils of Sb and Pg can also be good candidates for bio-guided fractionation to yield a more active and less toxic fraction against P. falciparum. These results may explain, at least in part the use of these plants in the treatment of malaria in Benin (Gouwakinnou et al., 2011; Hermans et al., 2004). Moreover, these results indicate the selectivity of the activity of the studied oils on T. brucei brucei as compared to P. falciparum (SI > 10 for all studied oils). The cytotoxicity  tests  against  the   Chinese  Hamster Ovary (CHO) cells and the human non cancer fibroblast cell line (WI38) showed that all tested oils and components had a low cytotoxicity (IC50 > 31 mg/ml) (Table 3).

Correlation of activity and chemical composition of essential oils

The antitrypanosomal activity of available major compounds of these studied oils was also evaluated or obtained from literature (Table 4).

 

 

The essential oil of Sb contains active compounds as myrcene (IC50 = 2.24 mg/ml), limonene (IC50 = 4.24 mg/ml), α-pinene (IC50 = 4.09 mg/ml), while sabinene (IC50 = 17.67 mg/ml), aromadendrene (IC50 = 18.77 mg/ml), β-caryophyllene (IC50 = 13.76 mg/ml) and caryophyllene oxide (IC50 = 17.67 mg/ml) had moderate activity. Other compounds as β-pinene (IC50 = 47.37 mg/ml), p-cymene (IC50 = 76.32 mg/ml), linalool (IC50 = 39.26 mg/ml) and thymol (IC50 = 22.83 mg/ml) had low activities (Bero et al., 2014). All these compounds do not explain the interesting activity (IC50 = 0.46 mg/ml) of this essential oil. The absence of described activity for not available and major compounds as 7-epi-α-selinene (37.86 ± 0.03%), α- muurolene (25.03 ± 0.03%), and valencene (17.12 ± 0.06%) could not help to explain this activity.

The first four major constituents of the essential oil of Ec: γ-terpinene (57.24%; IC50 = 136.91 mg/ml), p-cymene (18.22%; IC50 = 76.32 mg/ml), 1,8-cineole (7.49%; IC50 = 83.02 mg/ml) and terpinen-4-ol (7.50%; IC50 = 39.51 mg/ml) have very low anti-trypanosomal activities with IC50 values > 20 mg/ml which could not explain the moderate activity (IC50 = 2.65 mg/ml) observed for this oil. Furthermore, the oil contains minor compounds showing activity close to the activity of the crude oil with IC50 values < 5 mg/ml. Indeed, myrcene, citronellal and α-pinene with a concentration lower than 1%, showed IC50 values of 2.24 mg/ml; 2.76 mg/ml and 4.09 mg/ml respectively. Limonene (1.82%) had also a low IC50 value (IC50 = 4.24 µg/ml). These components seemed to act synergistically in the oil. These results confirm those obtained by Kpoviessi et al. (2014) that described the possibility of synergy effect in the essential oil of Cymbopogon spp.

No compound in Pg oil does exceed the concentration of 15%. β-caryophyllene (8.04 ± 0.03%), the fourth compound of this oil, showed moderate activity (IC50 = 13.76 mg/ml), while caryophyllene oxide (2.20%; IC50 = 17.67 mg/ml), α-cedrene (1.01%; IC50 = 4.06 mg/ml) and limonene (0.13%; IC50 = 4.24 mg/ml) were more effective. (E)–nerolidol (2.38 %) showed an interesting activity with an IC50 value of 1.70 mg/ml (Bero et al., 2014) similar to that  (IC50=1.16 mg/ml) obtained from the oil. Furthermore, β-bisabolene (14.38 ± 0.03%), ar-curcumene (12.39 ± 0.02%) and β-bisabolol (11.40 ± 0.08%), the three first major constituents of this oil, were not available and could not be tested. Recently, bisabolol oxide derivatives from Artemisia persica ethyl acetate extracts, exhibited in vitro antimalarial activity against P. falciparum, with IC50 values ranging from 1.14 to 7.92 µg/ml (Moradi-Afrapoli et al., 2013).

Given the activity observed for pure compounds, these essential oils seem to be the result of a synergistic action of all its constituents, including minor ones.

Antioxidant activity

The antioxidant activity of the studied oils was expressed in IC50 values and recorded in Table 3. The essential oil of Sb (IC50 = 5106 μg/ml) showed the highest activity, followed by that of Ec (IC50 = 9510 μg/ml) and by that of Pg (IC50 = 19290 μg/ml). The studied oils were all active, but less than ascorbic acid (IC50 = 20 μg/ml), the reference compound used in the test (Figure 1). This activity is quite low and could be explained by the presence in these oils of some high active components as β-caryophyllene (IC50 = 3.68 µ/ml; Pujiarti et al., 2012) and some less active components as β-pinene (IC50 = 20.05± 0.03 µg/ml; Kazemi, 2015); p-cymene (IC50 = 20.05± 0.4 µg/ml; Kazemi, 2015). Safaei-Ghomi et al. (2009), showed that the antioxidant activity of the major components   tested    separately    gives    lower   results compared to the activity of the whole components of the essential oil. Furthermore, presence of allylic compounds and / or benzyls (less than 1% in the studied oils; Table 2) could contribute to this activity. Therefore, the antioxidant activity of our oils could be explained by a synergy of action between their different constituents (Safaei-Ghomi et al., 2009; Vardar-Unlu et al., 2003).

 

 

 

 

 

 


 CONCLUSION

Our study shows that the essential oils of S. birrea, E. camaldulensis and P. guajava from Benin were more active on T. brucei brucei than on Plasmodium falciparum (3D7) and very weakly antioxidants. The essential oils of S. birrea and P. guajava already used extensively in traditional medicine and consumed by livestock were the most active and could be interesting for the treatment of sleeping sickness but may also have some interest on Plasmodium. These plants contain components with low, moderate or very good activities, which appear to act synergistically in their essential oils. These oils had a low cytotoxicity against CHO and WI38 cells. This is the first report on the activities of these essential oils against T. brucei brucei, P. falciparum and their cytotoxicity.

 


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interest.

 



 REFERENCES

Adams RP (2007). Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. 4th Edition Allured Publishing Corporation, Carol Stream, USA pp. 57-332.

 

Adeyemi OS, Akanji MA (2011). Biochemical changes in the kidney and liver of rats following administration of ethanolic extract of Psidium guajava leaves. Human and Experimental Toxicology 30(9):1266-1274.
Crossref

 

Ajaiyeoba OE, Oladepo O, Fawole OI, Bolaji OM, Akinboye DO, Ogundahunsi OAT, Falade CO, Gbotosho GO, Itiola OA, Happi TC, Ebong OO, Ononiwu IM, Osowole O, Oduola OO, Ashidi AMJ, Oduola AMJ (2003). Cultural categorization of febrile illnesses in correlation with herbal remedies used for treatment in Southwestern Nigeria. Journal of Ethnopharmacology 85:179-185.
Crossref

 

Ayoola GA, Coker HAB, Adesegun SA, Adepoju-Bello AA, Obaweya K, Ezennia EC, Atangbayila TO (2008). Phytochemical screening and antioxidant activities of some selected medicinal plants used for malaria therapy in Southwestern Nigeria. Tropical Journal of Pharmaceutical Research 7(3):1019-1024.
Crossref

 

Bero J, Hannaert V, Chataigné G, Hérent M-F, Quetin-Leclercq J (2011). In vitro antitrypanosomal and antileishmanial activity of plants used in Benin in traditional medicine and bio guided fractionation of the most active extract. Journal of Ethnopharmacology 137(2):998-1002.
Crossref

 

Bero J, Kpoviessi S, Quetin-Leclercq J (2014). Anti-parasitic activity of essential oils and their active constituents againts Plasmodium, Trypanosoma and Leishmania. Novel Plant Bioresources: Applications in Food, Medicine and Cosmetics pp. 455-469.
Crossref

 

Brand-Williams W, Cuvelier ME, Brest C (1995). Use of a method to evaluate antioxidant actvity. Lebensmittel-Wissenschaft and Technologie 28:25-30.
Crossref

 

Bruneton J (2009). Pharmacognosie: Phytochimie, plantes médicinales. 2ème édition. Paris: Technique et Documentation-Lavoisier. pp. 565-595.

 

Chalchat JC, Kundakovic T, Gomnovic MS (2001). Essential oil from the leaves of Eucalyptus camaldulensis Dehnh (Myrtaceae) from Jerusalem. Journal of Essential Oil Research 13(2):105-107.
Crossref

 

Cheikh-Ali Z, Adiko M, Bouttier S, Bories C, Okpekon T, Poupon E, Champy P (2011). Composition, and antimicrobial and remarkable antiprotozoal activities of the essential oil of rhizomes of Aframomum sceptrum K. Schum. (Zingiberaceae). Chemistry and Biodiversity 8(4):658-667.
Crossref

 

Chen HC, Sheu MJ, Lin LY, Wu CM (2007). Chemical composition of the leaf essential oil of Psidium guajava L. from Taiwan. Journal of Essential Oil Research 19(4):345-347.
Crossref

 

Chinchilla M, Valerio I, Sánchez R, Mora V, Bagnarello V, Martínez L, Gonzalez A, Vanegas JC, Apestegui A (2012). In vitro antimalarial activity of extracts of some plants from a biological reserve in Costa Rica. Revista de biologia tropical 60(2):881-891.
Crossref

 

da Silva JD, Luz AIR, da Silva MHL, Andrade EHA, Zoghbi MDGB, Maia JGS (2003). Essential oils of the leaves and stems of four Psidium spp. Flavour and Fragrance Journal 18(5):240-243.
Crossref

 

Djordjević VB, Zvezdanović L, Cosić V (2008). Oxidative stress in human diseases. Srpski Arhiv Za Celokupno Lekarstvo 136(2):158-165.
Crossref

 

Farah A, Fechtall M, Chaouch A, Zrira S (2002). The essential oils of Eucalyptus camaldulensis and its natural hybrid (clone 583) from Morocco. Flavour and Fragrance Journal 17(5):395-397.
Crossref

 

Gathirwa JW, Rukunga GM, Njagi ENM, Omar SA, Mwitari PG, Guantai AN, Tolo FM, Kimani CW, Muthaura CN, Kirira PG, Ndunda TN, Amalemba G, Mungai GM, Ndiege IO (2008). The in vitro anti-plasmodial and in vivo anti-malarial efficacy of combinations of some medicinal plants used traditionally for treatment of malaria by the Meru community in Kenya. Journal of Ethnopharmacology 115(2):223-231.
Crossref

 

Gelfand M, Mavi S, Drummond RB, Ndemera B (1985). The traditional medical practitioner in Zimbabwe. Mambo press, Gweru, Zimbabwe P. 411.

 

Ghalem BR, Mohamed B (2014). Antibacterial activity of essential oil of North West Algerian Eucalyptus camaldulensis against Escherichia coli and Staphylococcus aureus. Journal of Coastal Life Medicine 2(10):799-804.

 

Gouwakinnou GN, Lykke AM, Assogbadjo AE, Sinsin B (2011). Local knowledge, pattern and diversity of use of Sclerocarya birrea. 7 Journal of Ethnobiology and Ethnomedicine 7(1):8.
Crossref

 

Gutiérrez RMP, Mitchell S, Solis RV (2008). Psidium guajava: A review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology 117(1):1-27.
Crossref

 

Habila N, Agbaji AS, Ladan Z, Bello IA, Haruna E, Dakare MA, Atolagbe TO (2010). Evaluation of in vitro activity of essential oils against Trypanosoma brucei brucei and Trypanosoma evansi. Journal of Parasitology Research Article ID 534601, 5p.
Crossref

 

Haouel S, Mediouni-Ben JJ, Khouja ML (2010). Postharvest control of the date moth Ectomyelois ceratoniae using Eucalyptus essential oil fumigation. Tunisian Journal of Plant Protection Research 5:201-212.

 

Hermans M, Akoegninou A, Van Der Maesen L, Los G (2004). Medicinal plants used to treat malaria in Southern Benin. Economic Botany 58(Supplement):239-252.
Crossref

 

Hirumi H, Hirumi K (1994). Axenic culture of African trypanosome bloodstream forms. Parasitology Today 10(2):80-84.
Crossref

 

Hoet S, Stevigny C, Herent M-F,Quetin-Leclercq J (2004). Antitrypanosomal compounds from the leaf essential oil of Strychnos spinosa. Planta Medica 72(5):480-482.
Crossref

 

Kabiru YA, Ogbadoyi EO, Okogun JI, Gbodi TA, Makun HA (2013). Anti-trypanosomal potential of Eucalyptus camaldulensis British Journal of Pharmacology and Toxicology 4(2):25-32.
Crossref

 

Kanko C, Kone S, Ramiarantsoa H, Tue Bi B, Chalchat J-C, Chalard P, Figueredo G, Ahibo-Coffy A (2012). Monoterpene hydrocarbons, major components of the dried leaves essential oils of five species of the genus Eucalyptus from Côte d'Ivoire, Natural Science 4:106-111.
Crossref

 

Kaushik NK, Bagavan A, Rahuman AA, Zahir AA, Kamaraj C, Elango G, Jayaseelan C, Kirthi AV, Santhoshkumar T, Marimuthu S, Rajakumar G, Tiwari SK, Sahal D (2015). Evaluation of antiplasmodial activity of medicinal plants from North Indian Buchpora and South Indian Eastern Ghats. Malaria Journal 14:65.
Crossref

 

Kazemi M (2015). Gas Chromatography-Mass Spectrometry analyses for detection and identification of antioxidant constituents of Achillea tenuifolia Essential Oil. International Journal of Food Properties 18(9):1936-1941.
Crossref

 

Knezevic P, Aleksic V, Simin N, Svircev E, Petrovic A, Mimica-Dukic N (2016). Antimicrobial activity of Eucalyptus camaldulensis essential oils and their interactions with conventional antimicrobial agents multi-drug resistant Acinobacter baumannii. Journal of Ethnopharmacology 178:125-136.
Crossref

 

Kpadonou-Kpoviessi BGH, Yayi-Ladekan E, Kpoviessi DSS, Gbaguidi F, Yehouenou B, Quetin-Leclercq J, Figueredo G, Moudachirou M, Accrombessi GC (2012). Chemical variation of essential oil constituents of Ocimum gratissimum L. from Benin, and impact on antimicrobial properties and toxicity against Artemia salina Leach. Chemistry and Biodiversity 9(1):139-150.
Crossref

 

Kpoviessi DSS, Gbaguidi FA, Kossouoh C, Agbani P, Yayi-Ladekan E, Sinsin B, Moudachirou M, Accrombessi GC, Quetin-Leclercq J (2011). Chemical composition and seasonal variation of essential oil of Sclerocarya birrea (A. Rich.) Hochst subsp birrea leaves from Benin. Journal of Medicinal Plant Research 5(18):4640-4646.

 

Kpoviessi S, Bero J, Agbani P, Gbaguidi F, Kpadonou-Kpoviessi B, Sinsin B, Accrombessi G, Frédérich M, Moudachirou M, Quetin-Leclercq J (2014). Chemical composition, cytotoxicity and in vitro antitrypanosomal and anti-plasmodial activity of the essential oils of four Cymbopogon species from Benin. Journal of Ethnopharmacology 151(1):652-659.
Crossref

 

Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL, Hinrichs DJ (1993). Parasite lactate-dehydrogenase as an assay for Plasmodium falciparum drug-sensitivity. American Journal of Tropical Medicine and Hygiene 48(6):739-741.
Crossref

 

Maloueki U, Kunyima KP, Mbomba ID, Dani NA, Lukuka KA, Lami NJ, Mpiana PT, Ngbolua KN, Ndimbo KSP, Mbomba NB, Musuyu Muganza CD (2015). Activités antioxydante et antiplasmodiale d'extraits de Massularia acuminata (Rubiaceae). Phytothérapie 13(6):389-395.
Crossref

 

Moradi-Afrapoli F, Ebrahimi SN, Smiesko M, Raith M, Zimmermann S, Nadjafi F, Brun R, Hamburger M (2013). Bisabololoxide derivatives from Artemisia persica, and determination of their absolute configurations. Phytochemistry 85:143-152.
Crossref

 

Moudachirou M, Gbenou JD, Chalchat JC, Chabard JL, Lartigue C (1999). Chemical composition of essential oils of Eucalyptus from Benin: E. citriodora and E. camaldulensis. Influence of location, harvest time, storage of plants and time of steam distillation. Journal of Essential Oil Research 11(1):109-118.
Crossref

 

Mu H, Holm R, Müllertz A (2013). Lipid-based formulations for oral administration of poorly water-soluble drugs. International Journal of Pharmaceutics 453(1):215-224.
Crossref

 

Murebwayire S,Frederich M, Hannaert V, Jonville MC, Duez P (2008). Antiplasmodial and antitrypanosomal activity of Triclisias acleuxii (Pierre) Diels. Phytomedicine 15(9):728-733.
Crossref

 

National Institute of Standard and Technology/environmental protection agency/national institutes of health [NIST/EPA/NIH] (1998). Mass Spectral Database, Standard Reference Database N° 1A, version 1.6. NIST/EPA/NIH, Gaithersburg, MD.

 

Nibret E, Wink M (2010). Trypanocidal and antileukaemic effects of the essential oils of Hagenia abyssinica, Leonotis ocymifolia, Moringa stenopetala, and their main individual constituents. Phytomedicine 17(12):911-920.
Crossref

 

Njume C, Afolayan AJ, Green E, Ndip RN (2011). Volatile compounds in the stem bark of Sclerocarya birrea (Anacardiaceae) possess antimicrobial activity against drug-resistant strains of Helicobacter pylori. International Journal of Antimicrobial Agents 38(4):319-324.
Crossref

 

Noudogbessi JP, Chalard P, Figueredo G, Alitonou GA, Agbangnan P, Osseni A, Avlessi F, Chalchat JC, Sohounhloue DC, (2013). Chemical compositions and physical characteristics of volatile extracts of leaves of Psidium guajava Linn and Lantana camara Linn of Benin. Research Journal of Pharmaceutical, Biological and Chemical Sciences 4(1):28-37.

 

Ogunwande IA, Olawore NO, Adeleke KA, Ekundayo O, Koenig WA (2003). Chemical composition of the leaf volatile oil of Psidium guajava L. growing in Nigeria. Flavour and Fragrance Journal 18(2):136-138.
Crossref

 

Otohinoyi DA, Ekpo O, Ibraheem O (2014). Effect of ambient temperature storage on 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a free radical for the evaluation of antioxidant activity. International Journal of Biological and Chemical Sciences 8(3):1262-1268.
Crossref

 

Pujiarti R, Ohtani Y, Ichiura H (2012). Chemical compositions, antioxidant and antifungal activities of Melaleuca leucadendron Linn. leaf oils from ndonesia. Wood Research Journal 3(1):23-29.

 

Rajendran C, Begam M, Kumar D, Baruah I, Gogoi HK, Srivastava RB, Veer V (2014). Antiplasmodial activity of certain medicinal plants against chloroquine resistant Plasmodium berghei infected white albino BALB/c mice. Journal of Parasitic Diseases 32(2):148-152.
Crossref

 

Rashid MK, Alam R, Khan S, Prakash V (2013). Oxidative stress marker and antioxidant status In Falciparum Malaria In relation to the intensity of parasitaemia. International Journal of Biological and Medical Research 4(3):3469-3471.

 

Rasoanaivo P, Petitjean A, Rakoto-Ratsimamanga A (1992). Medicinal plants used to treat malaria in Madagascar. Journal of Ethnopharmacology 37(2):117-127.
Crossref

 

Safaei-Ghomi J, Ebrahimabadi AH, Djafari-Bidgoli Z, Batooli H (2009). Analyse GC / MS et activité antioxydante in vitro d'extraits d'huile essentielle et de méthanol de Thymus caramanicus Jalas et de son constituant principal, le carvacrol. Food Chemistry 115(4):1524-1528.
Crossref

 

Stevigny C, Block S, Pauw-Gillet MC, deHoffmann E, Llabres G, Adjakidje V, Quetin-Leclercq J (2002). Cytotoxic aporphine alkaloids from Cassytha filiformis. Planta Medica 68(11):1042-1044.
Crossref

 

Tarkang PA, Franzoi KD, Lee S, Lee E, Vivarelli D, Freitas-Junior L, Liuzzi M, Nolé T, Ayong LS, Agbor GA, Okalebo FA, Guantai AN (2014). In vitro antiplasmodial activities and synergistic combinations of differential solvent extracts of the polyherbal product, Nefang. BioMed Research International, Article ID 835013, 10 p.
Crossref

 

Trager W, Jensen JB (1976). Human malaria parasites in continuous culture. Science 193:673-675.
Crossref

 

Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J (2007). Free radicals and antioxidants in normal physiological functions and human disease. International Journal of Biochemistry and Cell Biology 39(1):44-84.
Crossref

 

VanDenDool H, Kratz PD (1963). A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. Journal Chromatography A 11:463-471.
Crossref

 

Vardar-Ünlü G, Candan F, Sökmen A, Daferera D, Polissiou M, Sökmen M, Tepe B (2003). Antimicrobial and antioxidant activity of the essential oil and methanol extracts of Thymus pectinatus Fisch. et Mey. Var. pectinatus (Lamiaceae). Journal of Agricultural and Food Chemistry 51(1):63-67.
Crossref

 

Verdeguer M, Blazquez MA, Boira H (2009). Phytotoxic effects of Lantana camara, Eucalyptus camaldulensis and Eriocephalus africamis essential oils in weeds of Mediterranean summer crops. Biochemical Systematics and Ecology 37(4):362-369.
Crossref

 

World Health Organisation (WHO) (2015). World Health Statistics. 

View

 

World Health Organisation (WHO) (2018). World Health Statistics. 

View

 




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