Trypanosoma brucei Inhibition by Essential Oils from Medicinal and Aromatic Plants Traditionally Used in Cameroon (Azadirachta indica, Aframomum melegueta, Aframomum daniellii, Clausena anisata, Dichrostachys cinerea and Echinops giganteus)

Essential oils are complex mixtures of volatile components produced by the plant secondary metabolism and consist mainly of monoterpenes and sesquiterpenes and, to a minor extent, of aromatic and aliphatic compounds. They are exploited in several fields such as perfumery, food, pharmaceutics, and cosmetics. Essential oils have long-standing uses in the treatment of infectious diseases and parasitosis in humans and animals. In this regard, their therapeutic potential against human African trypanosomiasis (HAT) has not been fully explored. In the present work, we have selected six medicinal and aromatic plants (Azadirachta indica, Aframomum melegueta, Aframomum daniellii, Clausena anisata, Dichrostachys cinerea, and Echinops giganteus) traditionally used in Cameroon to treat several disorders, including infections and parasitic diseases, and evaluated the activity of their essential oils against Trypanosma brucei TC221. Their selectivity was also determined with Balb/3T3 (mouse embryonic fibroblast cell line) cells as a reference. The results showed that the essential oils from A. indica, A. daniellii, and E. giganteus were the most active ones, with half maximal inhibitory concentration (IC50) values of 15.21, 7.65, and 10.50 µg/mL, respectively. These essential oils were characterized by different chemical compounds such as sesquiterpene hydrocarbons, monoterpene hydrocarbons, and oxygenated sesquiterpenes. Some of their main components were assayed as well on T. brucei TC221, and their effects were linked to those of essential oils.

A. indica, also known as 'neem tree', is considered by many people living in Africa as a miraculous plant for a wide range of uses in ethnopharmacology such as anthelmintic, antimalarial, anti-inflammatory purposes and for healing skin diseases. Most of these properties were then confirmed by scientific reports [16][17][18]. All parts of the plant can be used for medicinal purposes, including the seed oil extracted by mechanical pressure [19,20]. Notably, the ethanolic extract obtained from neem stem bark exhibited activity against T. b. brucei [21], whereas the leaf essential oil has been barely investigated to date.
A. melegueta, also known as 'alligator pepper' or 'grain of paradise', is a perennial plant native to western Africa, and its seeds are used as a spice in food due to their aromatic flavor and pungent taste or as ingredients of ethnomedical preparations for the treatment of snakebites, stomachaches, and diarrhea [22]. Antimicrobial, anti-inflammatory, anticancer, and antioxidant properties have been reported for alligator pepper [23]. Concerning seed volatile constituents, they showed repellent activity against adults of the maize weevil Sitophilus zeamais [24].
A. daniellii, also known as 'African cardamom', is an herbaceous plant traditionally used in Africa as a spice due to the pungent taste of its seeds, whereas, for medicinal purposes, the plant is employed as a laxative and for curing parasitic and other microbial infections [25,26]. The anti-inflammatory effect of its seed essential oil and the preservative properties in stored grains have also been reported [27,28].
C. anisata is an evergreen tropical tree up to 10 m tall with leaves containing secretory glands and emitting a strong smell [29]. In Africa, it is considered highly effective against insects and has also been used in the treatment of malaria [30].
D. cinerea is a tree growing in tropical areas in countries such as Cameroon, Kenya, South Africa, and Tanzania, where a decoction of its leaves and roots is used against venereal disease, eye inflammations, skin diseases, and snake bites. The root is used for chest complaints and the twigs for gonorrhoea and syphilis. The essential oil was toxic to mosquito vectors of bancroftian filariasis [26].
E. giganteus is a perennial herb widely used in African traditional medicine for the treatment of various ailments [31,32]. In previous studies, the root methanolic extract showed significant antibacterial [33], antifungal [34], and antioxidant effects [35]. The cytotoxicity of the crude methanol extract from the roots has also been demonstrated [36,37].
Overall, these Cameroonian plants are also traditionally used to control populations of arthropod pests [38][39][40]. In this research, we shed light on the growth inhibitory potential of the essential oils obtained from the leaves of A. indica, A. daniellii, and C. anisata; the seeds of A. melegueta and D. cinerea; and the roots of E. giganteus against T. brucei TC221. Selected pure constituents from the above mentioned essential oils were also evaluated.

Plant Material
Leaves of A. indica were collected during the dry season (January 2016) from a tree in the city of Guidiguis (north of Cameroon), about 70 km from Maroua. The leaves were air-dried in the shade for one week and kept in papers. Fruits (pods) of A. melegueta were collected in a forest near Foumbam (western Cameroon) in December 2015. Once harvested, the seeds were removed from their pods and dried at room temperature over a period of three weeks in the absence of sunlight. At the end of the drying process, the seeds were placed into paper bags before hydrodistillation. Leaves of C. anisata were collected in the village of Baffou, Menoua Division, Western Cameroon. Leaves of A. danielli and fruits of D. cinerea and roots of E. giganteus were collected from Bamougoum and Bafoussam's market (Cameroon, Western Region), respectively. The pericarp of D. cinerea was removed and the seeds used; the roots of E. giganteus were washed with water and sliced into small pieces. These plant parts were dried at room temperature for one week. The botanical identification of the five species was performed by a taxonomist at the Cameroon National Herbarium (Yaoundé, Cameroon), and the voucher specimens were archived with the following codes: 4447 SRFK (A. indica), 43117 HNC (A. melegueta), 43130 HNC (A. danielli), 44242/HNC (C. anisata), 42920 HNC (D. cinerea), and 23647 SRF (E. giganteus). The botanical names were also checked against The Plant List database (www.theplantlist.org).

Isolation of Essential Oil
The dry leaves of A. indica, A. daniellii, and C. anisata; the seeds of A. melegueta and D. cinerea; and the roots of E. giganteus were cut into small pieces and subjected to hydrodistillation using a Clevenger-type apparatus until no more oil was obtained. The essential oils obtained were dried using Na 2 SO 4 and stored at −20 • C in vials sealed with teflon caps and protected from light before use. The oil yields were calculated on a dry weight basis (%, w/w).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Essential Oils
The chemical constituents of the Cameroonian essential oils were analyzed on an Agilent 6890 N gas chromatograph coupled to a 5973 N mass spectrometer (Santa Clara, CA, USA) and equipped with a HP-5 MS capillary column (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 µm film thickness; J & W Scientific, Folsom, CA, USA). For the separation of the volatile constituents, the following temperature program was used: 5 min at 60 • C then 4 • C/min up to 220 • C, then 11 • C/min up to 280 • C held for 15 min. The injector and detector temperatures were: 280 • C; carrier gas: Helium; flow rate: 1 mL/min; split ratio: 1:50; acquisition mass range: 29-400 m/z; and mode: electron-impact (EI, 70 eV). The essential oil was diluted 1:100 in n-hexane, and then 2 µL of the solution was injected into the GC-MS system. For the identification of essential oil components, co-injection with the authentic standards available in our laboratory (purchased from Sigma-Aldrich) was performed, together with a comparison of the retention indices and the mass spectra of those occurring in the ADAMS, NIST 08, and FFNSC2 libraries [41][42][43]. The percentage values of the volatile components were the means of three chromatographic analyses and were determined from the peak areas without the use of correction factors.
The essential oils or pure compounds identified from these oils were dissolved in dimethyl sulfoxide (DMSO) and serially diluted with growth medium in white 96-well microtiter plates. 20,000 bloodstream forms of T. brucei or Balb/3T3 cells were added to each well in a final volume of 200 µL. In the case of mammalian cells, we also tested 2000 cells/well with similar results. To avoid any damage to the cells, the concentration of DMSO in the solution was never higher than 1% (no cell growth inhibition was observed with this concentration of DMSO). Cell viability was verified by a drug-free control for each compound.
The plates were incubated for 48 h in the CO 2 incubator; then 20 µL of 0.5 mM resazurine (Sigma Aldrich) was added to each well, and the plates were incubated for an additional 24 h before the fluorescence was measured with an Infinite M200 microplate reader (Tecan group, Ltd., Männedorf, Switzerland) equipped with 540 and 590 nm excitation and emission filters. The half maximal inhibitory concentration (IC 50 ) values were calculated on a log inhibitor versus the response curves by non-linear regression using the GraphPad prism 5.04 software (GraphPad Software, Inc., La Jolla, CA, USA).

Inhibition of Trypanosoma brucei Proliferation
Essential oils are complex mixtures of volatile compounds with multitarget actions, the antitrypanosomal effects of which are largely unknown and barely explored. On this basis, we decided to test the in vitro inhibitory effects of a pool of essential oils taken from medicinal and aromatic plants growing in Cameroon. Some of them are known for their traditional uses in the treatment of infectious diseases and malaria [53,54] and, in the case of the Neem tree, also against T. b. brucei [21].
Based on the chemical analysis performed, they exhibited different chemical profiles characterized by diverse functionalized groups such as monoterpene hydrocarbons (African cardamom), oxygenated monoterpenes (A. melegueta and D. cinerea), sesquiterpene hydrocarbons (A. indica), sesquiterpene hydrocarbons and oxygenated sesquiterpenes (E. giganteus), and phenylpropanoids (C. anisata). In this context, the main aim of our work was to identify the chemical scaffolds of possible natural lead compounds against trypanosomiasis.
Testing the essential oils obtained from Cameroonian plants, we obtained various degrees of inhibition on T. brucei proliferation, varying from not active (A. melegueta, C. anisata, and D. cinerea), to moderately active (A. danielli, E. giganteus and A. indica). Notably, the IC 50 values on T. brucei were 7.65, 10.50, and 15.21 µg/mL for the essential oils from A. danielli, E. giganteus, and A. indica, respectively (Table 2). Furthermore, the most active oils were also evaluated for the growth inhibitory effects on Balb/3T3 cells as a reference. No effect on mammalian cells was observed with concentrations as high as 100 µg/mL, showing a noteworthy selectivity against T. brucei in comparison to mammalian cells, with selectivity indexes above 6.57 in all cases.
The inhibitory effects on T. brucei exhibited by the three essential oils highlights three classes of active compounds, i.e., monoterpene hydrocarbons (for A. danielli) and sesquiterpene hydrocarbons (for A. indica and E. giganteus) (Figure 1). E giganteus also contains high amounts of oxygenated compounds.
The toxicity of monoterpene hydrocarbons against T. brucei can be attributed to the high hydrophobicity of this class of compounds, which are able to easily cross the cell membrane, causing the destabilization of phospholipid bilayers and the alteration of their permeability, leading to cell damage and death [44,55]. Among these compounds, the most abundant was sabinene (43.9%) (Figure 1) which showed an IC 50 value against T. brucei of 5.96 µg/mL, which is close to that of the African cardamom essential oil (7.65 µg/mL) ( Table 2). Another component of this oil with detectable antitrypanosomal activity was β-pinene, showing an IC 50 value of 11.4 µg/mL. The antitrypanosomal activity of sabinene was already reported in a previous study, although its mechanism of action on the protozoal cell has not been elucidated [56]. The inhibitory effects on T. brucei exhibited by the three essential oils highlights three classes of active compounds, i.e., monoterpene hydrocarbons (for A. danielli) and sesquiterpene hydrocarbons (for A. indica and E. giganteus) (Figure 1). E giganteus also contains high amounts of oxygenated compounds. The toxicity of monoterpene hydrocarbons against T. brucei can be attributed to the high hydrophobicity of this class of compounds, which are able to easily cross the cell membrane, causing the destabilization of phospholipid bilayers and the alteration of their permeability, leading to cell damage and death [44,55]. Among these compounds, the most abundant was sabinene (43.9%) ( Figure 1) which showed an IC50 value against T. brucei of 5.96 μg/mL, which is close to that of the African cardamom essential oil (7.65 μg/mL) ( Table 2). Another component of this oil with detectable antitrypanosomal activity was β-pinene, showing an IC50 value of 11.4 μg/mL. The antitrypanosomal activity of sabinene was already reported in a previous study, although its mechanism of action on the protozoal cell has not been elucidated [56].
E. giganteus as well as A. daniellii essential oils contained the bicyclic sesquiterpene (E)caryophyllene (Figure 1), which exhibited good inhibitory properties on T. brucei (IC50 value of 8.25 μg/mL). However, the content of this component is only 6.3% in E. giganteus and can therefore not explain the good activity of this essential oil. It is rather tricyclic sesquiterpenes that are the major constituent. This is the first report documenting the antitrypanosomal activity of the tricyclic sesquiterpenes-containing E. giganteus essential oil. The latter was a rich source of compounds such as silphiperfol-6-ene, presilphiperfolan-8-ol, presilphiperfol-7-ene, and cameroonan-7-α-l with an unusual skeleton (Figure 1) [26]. To date, these compounds have not been biologically investigated. Previously, the tricyclic sesquiterpene ledol was suggested to be responsible for the trypanocidal properties of Hagenia abyssinica (Bruce ex Steud.) J.F.Gmel. essential oil [13].
Among the other pure compounds, 1,8-cineole and terpinen-4-ol were inactive against T. brucei (IC50 > 100 μg/mL) ( Table 2), and this also explained the lack of activity of the A. melegueta and D. cinerea essential oils, which are dominated by these compounds.
Finally, we demonstrated for the first time that the leaf essential oil from the neem tree, which showed an IC50 value of 15.21 μg/mL, can be a source of sesquiterpenes such as germacrene B and γelemene. Since this oil is completely dominated by sesquiterpenes (97.4%), it can be assumed that they are responsible for its antitrypanosomal activity. Further studies are needed to elucidate their mode of action and the possibility of them acting as lead compounds for the discovery of E. giganteus as well as A. daniellii essential oils contained the bicyclic sesquiterpene (E)-caryophyllene (Figure 1), which exhibited good inhibitory properties on T. brucei (IC 50 value of 8.25 µg/mL). However, the content of this component is only 6.3% in E. giganteus and can therefore not explain the good activity of this essential oil. It is rather tricyclic sesquiterpenes that are the major constituent. This is the first report documenting the antitrypanosomal activity of the tricyclic sesquiterpenes-containing E. giganteus essential oil. The latter was a rich source of compounds such as silphiperfol-6-ene, presilphiperfolan-8-ol, presilphiperfol-7-ene, and cameroonan-7-α-l with an unusual skeleton (Figure 1) [26]. To date, these compounds have not been biologically investigated. Previously, the tricyclic sesquiterpene ledol was suggested to be responsible for the trypanocidal properties of Hagenia abyssinica (Bruce ex Steud.) J.F.Gmel. essential oil [13].
Among the other pure compounds, 1,8-cineole and terpinen-4-ol were inactive against T. brucei (IC 50 > 100 µg/mL) ( Table 2), and this also explained the lack of activity of the A. melegueta and D. cinerea essential oils, which are dominated by these compounds.
Finally, we demonstrated for the first time that the leaf essential oil from the neem tree, which showed an IC 50 value of 15.21 µg/mL, can be a source of sesquiterpenes such as germacrene B and γ-elemene. Since this oil is completely dominated by sesquiterpenes (97.4%), it can be assumed that they are responsible for its antitrypanosomal activity. Further studies are needed to elucidate their mode of action and the possibility of them acting as lead compounds for the discovery of antitrypanosomal drugs.

Conclusions
In conclusion, our biological investigation into the essential oils distilled from medicinal and aromatic plants growing in Cameroon identified some terpenoids as possible lead compounds of natural antitrypanosomal drugs. Further research is encouraged to disclose their mechanisms of action and in vivo efficacy.