In vitro antileishmanial and cytotoxicity activities of essential oils from Haplophyllum tuberculatum A. Juss leaves, stems and aerial parts

Background Plants used for traditional medicine produce diverse and complex secondary metabolites exhibiting various medicinal properties. The medicinal plant Haplophyllum tuberculatum is used by native people against malaria and parasitic infections. Methods In this study and in order to contribute for the search of new natural drugs for leishmaniasis, the essential oils of H. tuberculatum leaves, stems and aerial parts (leaves+stems) collected in two different periods, 2013 and 2015, and their components by GC/FID and GC/MS analyses were investigated. Those collected in 2013 were also re-analyzed two years later. The extracted oils were screened in vitro for anti-leishmanial activity on Leishmania mexicana mexicana (L.m.m.) promastigotes and cytotoxicity on the Chinese Hamster Ovary (CHO) cell line. Limonene (1.5 – 8%), its isomers (R- (+)-limonene and S-(-)-limonene), linalool and octanol were also tested. Results Results showed that the chemical composition varied according to the year of collection. Though major compounds remain almost the same, qualitative and quantitative variations in the composition of the EOs can be observed between the two years of collection, with some minor compounds identified only in one type of samples. Variation in the composition were also observed in the re-analyzed volatile oils, showing stability concerns. The essential oils and R-(+)-limonene showed moderate anti-leishmanial activity. Their IC50 range from 6.48 to 50.28 μg/ml. Cytotoxicity assays for theses volatile extracts, R- (+)-limonene and S- (-)-limonene on CHO cells showed relatively potent cytotoxicity with a selectivity index <10. Their CC50 range from 27.79 to 82.56 μg/ml. Conclusions The findings of the present study demonstrated that H. tuberculatum might not be considered as a natural source for production of new anti-leishmanial agents without further analyzing its eventual in vivo toxicity as well as that of major pure compounds.


Background
Leishmaniasis are parasitic diseases. More than 20 Leishmania species have about 70 natural reservoir hosts and vectors including animals, such as dogs or rodents, and human bodies, and are transmitted by more than 90 sand fly species. These diseases are considered as a serious health concern which are occurring in Africa, Asia, Southern Europe and Latin America. Their prevalence rate was estimated from 900 000 to 1.3 million new cases and from 20 000 to 30 000 deaths annually. The parasite may survive for decades in asymptomatic infected people and can also be transmitted directly from person to person. Many factors can increase leishmaniasis like malnutrition, migration, poor housing, a weak immune system, environmental and climatic changes such as deforestation and temperature variation and lack of financial resources [1][2][3]. In the absence of effective and sustainable vaccines, its control is still considered as a major public health problem [4]. The chemotherapy and the drugs based on pentavalent antimonials as sodium stibugluconate (Pentostam®) and meglumine antimoniate (Glucantime®) are the current means of treatment [5]. Therefore, the required long-term treatment, toxicity, grave side-effects, pain, cost, drug resistance associated with treatment failures point out the crucial need for new agents in the treatment of leishmaniasis [4,6].
Natural products traditionally used in folk medicine to treat several diseases are analyzed worldwide. In this regard, complex mixtures or purified compounds obtained from organic/aqueous extracts and essential oils of roots, stems, leaves, flowers, fruits and spices are various sources of diverse bioactive natural constituents [7][8][9]. The investigations showed that wild and cultivated plants exhibited various pharmacological activities such as antimicrobial, anticancer, anti-inflammatory, antiparasitic including anti-leishmanial ones [10]. Moreover, some plants essential oils and their main constituents displayed anti-leishmanial activities [6].
The main aim of the present study is to analyze the volatile oils of different H. tuberculatum parts collected during two different seasons, to evaluate their antileishmanial effects against L. mexicana mexicana promastigote forms and their cytotoxic activities against CHO cells in an in vitro model. In order to relate the oils bioactivities to pure components ones, a major compound, limonene, and two minor ones, octanol and linalool, were tested in vitro. The other identified major compounds, cis-p-menth-2-en-1-ol, trans-p-menth-2en-1-ol, cis-piperitol are not commercially available.

Plant material
Plant material from the H. tuberculatum Forssk. (A. Juss) species was collected at the end of December 2012 and May 2015 from Beni Ghzayel, Medenine, an arid region in Tunisia (33°21′17″ North 10°30′19″ East). The herbarium specimens were authenticated with their morphological and anatomical features in the Botany Department, Faculty of Pharmacy Monastir Tunisia and Botany Department of Faculty of Sciences Sfax Tunisia, according to the flora of Tunisia [21] and a voucher specimen (H.t-01.03) was deposited in the Biological Laboratory of the Faculty of Pharmacy of Monastir. The leaves, stems and roots were cut into small pieces and weighed before extraction of volatile compounds.

Extraction of essential oils
About 100 g of fresh plant parts (leaves and/or stems or roots) was subjected to a 3 h hydrodistillation with 500 ml of distilled water using a Clevenger-type apparatus (Clevenger, 1928). The leaves (L), stems (S), and leaves+stems (LS) essential oils (EO) obtained were separated from the distilled water and dried on anhydrous sodium sulphate. No oil was obtained from the roots part. The volatile extracts were stored in sealed glass vials at 4-5°C.

Analytical GC-FID and GC-MS
Three analyzes were done to identify the differential components in the three oils. Analyze 1 (Anlz1) referred to the oils extracted in 2013 and analyzed in 2014. The oils samples were re-analyzed in 2016, mentioned as Analyze 2 (Anlz2), in order to study the stability of the components. The third one (Anlz3) referred to samples extracted in 2015 and analyzed in 2016.

Identification and quantification of oils components
The identification of the constituents was based on comparison of retention times with those of reference data or pure compounds based on their LRIs with the series of nhydrocarbons. Moreover, identification of compounds also used gas chromatography-chemical ionization mass spectrometry, using methanol as the chemical ionizing gas [22][23][24][25][26][27] and computer matching against commercial (NIST 98 and ADAMS) and home-made library mass spectra (built up from pure substances and components of known oils and mass spectra literature data). The identified constituents were quantified by the normalization procedure using FID data.

Anti-leishmanial activity
The in vitro anti-leishmanial effects of essential oils from the leaves (LEO), stems (SEO) and the aerial parts of H. tuberculatum (LSEO) were investigated on Leishmania mexicana mexicana (L.m.m.) as described by [28]. The solutions were prepared in DMSO at 20 mg/ml. Parasites in the logarithmic growth phase were seeded in 96well culture plates. Essential oils and compounds were tested in eight serial threefold dilutions (0.05-100 μg/ml, 2 wells/concentration). Amphotericin B was used as positive control with an initial concentration of 1μg/ml. The plate was kept at 28°C and after 72 h of incubation, Leishmania viability was calculated by quantification of Alamar Blue fluorescence (10 μl diluted two times in PBS/well incubated 4h), using an excitation wavelength of 530 nm and emission one of 590 nm on a SpectraMax M2e (Molecular Devices) spectrophotometer. Assays were performed in triplicate to calculate IC 50 .

Cytotoxicity assay and selectivity index
A tetrazolium salt colorimetric method has been used to determine survival of Chinese Hamster Ovary (CHO) as described by Bero et al., 2009, with minor modifications [29]. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay is based on the conversion of MTT into formazan crystals by mitochondrial dehydrogenase in living cells. The solutions of essential oils were prepared in DMSO at 20 mg/ml. The essential oils and compounds were firstly diluted in medium in eight serial threefold dilutions (0.5-100 μg/ml, 3 wells/ concentration) in 96-well microtiter plate. Secondly, 20 μl of diluted solutions were transferred to 180 μl of 5 10 3 cells previously incubated during 24 h. Finally, after 72 h of incubation, the medium was replaced by a 10% MTT solution (3 mg/ml in PBS) in fresh medium and after 45 min, the medium was replaced again by 100μl of DMSO added to solubilize formed formazan crystals. Absorbance was recorded at 570 and 620 nm on a Spec-traMax M2e (Molecular Devices) spectrophotometer. Camptothecin was used as positive control at an initial concentration of 25μg/ml. Assays were performed in duplicate to calculate the CC 50 . The selectivity index (SI), expressed by the CC 50 /IC 50 ratio [30], defines the balance between cytotoxicity and anti-leishmanial activity. If the SI value is higher than 10, treatment is considered as safe for the cells (CHO) at the therapeutic concentration [31,32].

Statistical analysis
The IC 50 and CC 50 of the bioassays were calculated by the least square ordinary fit method based on a sigmoidal curve. Statistical analysis was performed using Microsoft Excel as the average ± SE for triplicates and duplicates of the anti-leishmanial and cytotoxicity assay, respectively. Data analysis was carried out by using SPSS statistical package version 16.0. Differences between the tests were analyzed by Duncan-test. P<0.05 was considered as statistically significant.

Chemical profiles of the oils
The comparison of the data obtained for the same essential oil (EO) obtained from H. tuberculatum analyzed in 2014 and re-analyzed in 2016 showed qualitative and quantitative variations. Moreover, different chemical profiles were also observed between the EO extracted in 2013 and in 2015 ( Table 1).

Anti-leishmanial activity
In this study, promastigotes of L.m.m. were incubated in the presence of various concentrations of essential oils and pure compounds. The cell viability was determined after 72 h using Alamar Blue assay. This is the first time that the anti-leishmanial activity of H. tuberculanum essential oils is shown with IC 50 range from 6.48 to >100 μg/ml and dose-dependent responses (P≤0.05: Table 2). The

Cytotoxicity and selectivity index
Cytotoxic effects of H. tuberculatum oils were determined in CHO cells using MTT assay. The obtained finding indicated significant cytotoxicity of the essential oils and the tested pure compounds on CHO cells with CC 50 range from 27.79 to >100 μg/ml ( Table 2). The selectivity index (SI) for LEO extracted in 2013 and 2015 and LSEO 2013 were 4.86, 4.15 and 4.29, respectively, showing that they were less toxic on CHO cells than Leishmania parasites. However, in all cases, they are lower than 10, the minimum value defined for the selection of an anti-parasitic hit [31,32]. Thus these oils could perhaps be used safely at therapeutic concentration in short term treatments but R-(+)-limonene (SI=1.79) cannot be considered as a drug candidate for the development of anti-leishmanial agents.

Discussion
Chemical profiles of the oils Literature review showed variation between chemical compositions, depending on the location and stages of development of H. tuberculatum. Reports from Oman [19], Iran [33] and the United Arab Emirates [34] showed a difference between the major compounds. The comparison of the chemical composition from the different parts of our H. tuberculatum with that of H. tuberculatum from Oman showed that the main compounds varied [19]. Indeed, the main compounds of H. tuberculatum from Oman were beta-phellandrene (23.3%), limonene (12.6%), (Z)-beta-ocimene (12.3%), beta-caryophyllene (11.6%), myrcene (11.3%), and alpha-phellandrene (10.9%) [19]. Another study showed that the main constituents of the EO from the aerial parts were limonene (27.3%) and αpinene (21.9%) [33]. Furthermore, the composition of the EO varied also according to the collecting season.
Conversion and degradation reactions can be involved in these changes because of the metabolic relation between terpenoid biosynthesis and the effect of different factors such as temperature or light. This was already observed i.e. for laurel and fennel [35,36]. The differences in composition observed between our results and those of the previous works might be related to the analyzed plant part (leaves, stems, leaves+stems and aerial part), the geographic origin of the populations, the ecological conditions in which they grow and also chemical instability or transformation, but may also suggest the existence of a new chemotype.

Anti-leishmanial activity
This variability in anti-leishmanial activity may be explained by the qualitative and quantitative differences in chemical compositions of each essential oils. Indeed, literature showed that the chemical composition of essential oils and so their biological activities may be affected by the growing seasons. Some factors may be considered as responsible for these variations as temperature, rainfall and humidity which can affect plant metabolism and lead to composition differences [37].

Cytotoxicity and selectivity index
Both isomers, R-(+)-limonene and S-(-)-limonene, can account for at least a part of the cytotoxic activity of the EOs. Thus, once again, the cytotoxic variabilities of the samples can be related to the studied plant part and the season of collection [37].
The cytotoxicity of R-(+)-limonene was a little higher than that described by Kpoviessi et al. (2014) but the difference (factor less than 2) may be explained by the general biological variability of cells in culture. Moreover, our study showed that 1-octanol and linalool (CC 50 >100μg/ ml) had no cytotoxicity on CHO cells. Some studies analyzed the specific cellular targets of single components on the cells. Recent research demonstrated that linalool was not cytotoxic on some cell types (CC 50 > 200 μg/ml): Vero, Macrophages, A-549, HeLa, HT-29 cells Saulo [41] but can inhibit mitochondrial complexes I and II, increase reactive oxygen species and inhibit the HepG2 cells viability (IC 50 =0.4 μM) [42].
Literature data indicate that H. tuberculatum organic extracts possess a high cytotoxicity [53,54]. This cytotoxicity may be explained by the cytotoxic lignans already identified in this species [55]. However, it cannot explain cytotoxicity of its essential oils which should not contain these lignans.
Previous studies showed that promastigotes cultures are a validated model for primary screening. Promastigotes are known to be less sensitive than amastigotes to drugs and allow a restricted selection of the most active samples before performing an intracellular amastigotes test [56]. However, as shown by the present results, the EOs have high cytotoxicity and the selectivity indices are not encouraging enough to proceed to the intracellular tests.

Conclusions
Results showed that the chemical composition varied according to the year of collection, but that stabilities issues may also modify the composition n along the time, as shown by the variation observed in the re-analyzed volatile oils.
Our results showed that the chemical composition of the essential oils of different parts (leaves, stems and leaves+stems) from H. tuberculatum varied according the period of collection. The re-analyzed volatile oils indicated that stabilities issues may also modify their composition along the time. The tested essential oils and one of their major component (R-(+)-limonene) were biologically active against L. mexicana mexicana coupled to a cytotoxic effect on CHO in vitro.
However, as limonene is only present at concentration < 10%, other compounds and/or synergistic effects remain to be analyzed to explain the observed activities. Thus, the biological activities of the identified major compounds, cis-p-menth-2-en-1-ol, trans-p-menth-2en-1-ol, cis-piperitol should be evaluated.
The results obtained in this study confirm the importance of chemical and biological investigations of essential oils but also toxicity risks. So in vitro studies are needed to assess the potential of these oils as antileishmanial agents and analyze deeper toxicity risks.
To our knowledge this is the first report of the antileishmanial and cytotoxic activity of the essential oils of H. tuberculatum.

Acknowledgments
We are grateful to Madame Maude Bourlet for her assistance to carry out the bioassays.

Funding
This work is partly supported by the Belgian Fund of Scientific Research (FNRS) T.0190.13.

Availability of data and materials
The plant materials and methods used were available upon request. All data obtained have been included into the manuscript.
Author's contributions AH collected plant material, extracted essential oils, tested the biological activities of essential oils and analyzed data; JB developed the methods; CB controlled experiments; GF identified chemical composition of essential oils; ZM identified the plant; YVH contributed to manuscript preparation and JQL supervised the study and edited the manuscript. All authors have read and approved the manuscript.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing of interests
The authors declare no commercial, financial or any other conflict of interest.