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

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.

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.

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.

Gas chromatography (GC) analyses were carried out with an HP-5890 Series II instrument equipped with HP-WAX and HP-5 capillary columns (30 m×0.25 mm, 0.25 μm film thickness), working with the following temperature program: 60 °C for 10 min, ramp of 5 °C/min up to 220 °C; injector and detector temperatures 250 °C; carrier gas was helium (2 mL/min); detector dual FID; split ratio 1:30; injection of 0.5 μL (10 % hexane solution). Gas chromatography-electron impact mass spectroscopy analyses were performed with a Varian CP-3800 gas chromatograph, equipped with a HP-5 capillary column (30 m×0.25 mm; coating thickness 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions were as follows: injector and transfer line temperatures 220 and 240 °C, respectively; oven temperature programmed from 60 to 240 °C at 3 °C/min; carrier gas helium at 1 ml/min; injection of 0.2 μL (10 % hexane solution); split ratio 1:30 [22‐27].

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 n-hydrocarbons. Moreover, identification of compounds also used gas chromatography-chemical ionization mass spectrometry, using methanol as the chemical ionizing gas [22‐27] 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.

Octanol (for synthesis, Darmstadt, Germany, Merck), linalool (Buchs, Switzerland, Fluka), R- (+)-Limonen and S- (-)-Limonen (96%, Steinheim, Germany, Sigma-Aldrich) were provided by commercial suppliers and dissolved in dimethyl sulfoxide (DMSO) (Darmstadt, Germany, Merck). MTT, amphotericin B and campthotecin came from Sigma-Aldrich (Steinheim, Germany) and Alamar Blue were provided by Thermo Fisher Scientific (Aalst, Belgium).

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 96-well 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 IC₅₀.

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 5 10³ 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 SpectraMax 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 CC₅₀. The selectivity index (SI), expressed by the CC₅₀/IC₅₀ 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].

The IC₅₀ and CC₅₀ 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.

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).

The main components present in the nine EO samples are α-pinene, limonene, cis-p-menth-2-en-1-ol, trans-p-menth-2-en-1-ol, cis-piperitol, 1-octyl acetate, piperitone, isobornyl acetate with variation in their area percentages. Trans-piperitol is also a major compound in most oils but it was not detected in both LSEO analyzed in 2016. The minor compounds found in all EOs are 2-undecanone, neryl acetate, 1-octyl 2-methylbutanoate, 1-octyl isovalerate. The amounts of myrcene and camphene vary between 0.8 and 3.3 % and 0.6 and 3.2 % respectively.

By comparing the EO analyzed in 2014 and re-analyzed in 2016, new compounds were detected in that of 2016 as tricyclene, octanal, pentyl isovalerate, 1-nonanol, 1-dodecene, (Z)-3-hexenyl isovalerate, 2-nonyl acetate, hexyl isovalerate, 1-decanol, geranyl acetate, 1-decyl acetate, 2-phenylisovalerate, kessane, tetradecanal, α-cadinol, γ-dodecalactone, cis-14-nor-muurol-5-en-4-one but as minor compounds. However, α-phellandrene, α-terpinene, 3-methyl-3-hexen-1-yl butanoate, (Z)-3-tridecene, 2-undecanol, neo-iso-isopulegol acetate, 3-oxo-p-menth-1-en-7-al, 1-decanol acetate disappeared in the Anlz2, but these compounds were minor in Anlz1 (Table 1).

The non-terpene derivatives increased in all oils. They were ranging from 13.1% to 23.4%, from 15.9% to 22.8 and from 23.4% to 27.2% for LEO, SEO and LSEO respectively. The oxygenated monoterpenes increased from 60% to 64.8%; from 46.7% to 51.5% for LEO and LSEO respectively and decreased from 61.5% to 56.7% for SEO, while the monoterpene hydrocarbons decreased in the EOs of all parts, from 19.0% to 6.3%; from 16.1 to 10.2% and from 20.7 to 17.2% for LEO, SEO and LSEO respectively.

On the other hand, new compounds were detected in the Anlz3 compared to Anlz1 as ethyl isovalerate, α-thujene, (E)-β-ocimene, 2-methylbutyl 2-methylbutanoate, ethyl benzoate, 1-decanol, 1-decyl acetate, germacrene D, α-zingiberene, tetradecanal, α-cadinol but some of them were present in Anlz 2. Moreover, some compounds were not detected in the Anlz3 as α-terpinene, 1-octanol, 2-nonanone, pinocarvone, 3-methyl-3-hexen-1-yl butanoate, cumin aldehyde, (Z)-3-tridecene, 2-undecanol, neo-iso-isopulegol acetate, myrtenyl acetate, 3-oxo-p-menth-1-en-7-al, 1-decanol acetate. When comparing Anlz 1 and Anlz 3, monoterpene hydrocarbons decreased from 19.0% to 8.8% for LEO; increased from 16.1 to 23.2% and from 20.7 to 33.8% for SEO and LSEO, respectively. The oxygenated monoterpenes increased from 60.3% to 65.6% for LEO, decreased from 61.5% to 54.3% and from 46.7% to 46.0% for SEO and LSEO respectively. The non-terpene derivatives increased from 13.1% to 21.4% from 15.9% to18.6% for LES and SEO respectively and decreased from 23.4% to 19.0% in LSEO.

Dissimilarity was noted in the Anlz2 and Anlz3. Eight compounds were detected in the Anlz3 but not in Anlz2: ethyl isovalerate, α-phellandrene, α-thujene, (E)-β-ocimene, 2-methylbutyl 2-methylbutanoate, ethyl benzoate, germacrene D, α-zingiberene. However, octanal, α-terpinene, 1-octanol, 2-nonanone, pinocarvone, 1-nonanol, 1-dodecene, 3-methyl-3-hexen-1-yl butanoate, (Z)-3-hexenyl isovalerate, hexyl isovalerate, cumin aldehyde, (Z)-3-tridecene, 2-undecanol, neo-iso-isopulegol acetate, myrtenyl acetate, 3-oxo-p-menth-1-en-7-al, geranyl acetate, 1-decanol acetate, 2-phenylethyl isovalerate, kessane, γ-dodecalactone, cis-14-nor-muurol-5-en-4-one were not identified in the Anlz3. The monoterpene hydrocarbons increased in the EO of all parts, from 6.3% to 8.8%; from 10.2 to 23.2% and from 17.2 to 33.8% for LEO, SEO and LSEO respectively. Moreover, the oxygenated monoterpenes increased from 64.8% to 65.6% for LEO, decreased from 56.7% to 54.3% and from 51.4% to 46.0% for SEO and LSEO respectively. However, the non-terpene derivatives decreased in all the oils. They were ranging from 23.4% to 21.4%, from 22.8% to 18.6 and from 27.2%to 19.0% for for LEO, SEO and LSEO respectively. In general, H. tuberculatum EO were characterized by high percentages of oxygenated monoterpenes followed by hydrocarbon monoterpenes and non-terpene derivatives.

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 IC₅₀ range from 6.48 to >100 μg/ml and dose-dependent responses (P≤0.05: Table 2).

The LEO 2013 and SEO 2013 had a similar effect while LSEO 2013 was the most active on L.m.m. (IC₅₀ values: 16.69, 16.00 and 6.48 μg/ml, respectively). The LEO, SEO and LSEO, samples collected and extracted in 2015, showed IC₅₀ values of 19.44, 50.28 and 61.79 μg/ml, respectively with a significant decrease in activity for SEO and LSEO 2015. Moreover, there is no significant difference between the IC₅₀ of LEO 2013, SEO 2013 and that of LEO 2015 (p < 0.05).

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 CC₅₀ range from 27.79 to >100 μg/ml (Table 2).

R-(+)-limonene (CC₅₀ = 29.65 μg/ml) and LSEO 2013 (CC₅₀ = 27.79 μg/ml) had the highest cytotoxic effect on cells compared to the other samples. LEO 2013, LEO 2015, SEO 2015, LSEO 2015 and S-(-)-limonene exhibited a similar low cytotoxic effect (P > 0.05).

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.

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 α-pinene (21.9%) [33]. Furthermore, the composition of the EO varied also according to the collecting season.

The analysis of the aerial parts of H. tuberculatum collected in May 1997 and 2001 from the United Arab Emirates showed that the major compounds were α-phellandrene (10.7–32.9%), β-caryophyllene (6.3–12.8%), β-pinene (7.6–8.0%), limonene (4.0–9.6%) and δ-3-carene (5.5–6.0%). However, the oil distilled from plants collected in April (1998) had as major components linalool (15.0%), linalyl acetate (10.6%), β-caryophyllene (9.7%) and α-terpineol (6.7%) [34].

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.

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].

Among the pure compounds, R-(+)-limonene was the most active one (IC₅₀ = 16.59 μg/ml) compared to inactive S-(-)-limonene, linalool and 1-octanol (IC₅₀ >100μg/ml). It can explain a part of the anti-leishmanial activity but other active compounds still remain to be identified. Kpadonou Kpoviessi et al., 2014 also, showed that R-(+)-limonene had an antitrypanosomal effect with IC₅₀=4.24 ±2.27 μg/mL on Trypanosoma brucei brucei bloodstream forms [38]. As well, linalool was shown to have a strong inhibitory activity on L. amazonensis promastigotes and amastigotes with LD₅₀ of 15.5 ng/ml and 4.3 ng/ml, respectively [39].

Some of the compounds present in our volatile oils were already tested for their anti-parasitic activities. Thus, the IC₅₀ of alpha-pinene (55.3μg/mL; 4.1μg/mL), sabinene (126.6μg/mL; 17.7μg/mL), β-pinene (200.1μg/mL; 54.8μg/mL), α-phellandrene (32.8μg/mL; 9.2μg/mL), 4-terpineol (335.9μg/mL; 0.02μg/mL) were reported against promastigotes of L. major and the bloodstream forms of T. brucei (TC 221), respectively [40].

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 (CC₅₀>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 (CC₅₀ > 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 (IC₅₀=0.4 μM) [42].

Apoptosis induction was observed for linalool on MCF7 WT cells (IC₅₀=0.62–0.79 μM) [43] and on HL-60 (IC₅₀=49.53–127.14 μM) [44], limonene on K562 (IC₅₀=ND) [45], HT-29 5 (IC₅₀ >200 μg/mL) [46], alpha-pinene on U937 (IC₅₀=ND) [47], pancreatic, mammary, and prostatic tumors (IC₅₀=ND) [48].

No cytotoxicity was observed for 1-octanol. Published data on the cytotoxicity of other H. tuberculatum components showed a great activity variability according to used cell types. Alpha-pinene was cytotoxic on K562 (IC₅₀=117.3μM) [49], HL-60 (IC₅₀=2.5 μg/mL) [40], A-549 (IC₅₀=183.2 μg/mL), HeLa (IC₅₀=172.7 μg/mL), but not on Vero, macrophages and HT-29 [41]. Sabinene (IC₅₀=23.6 μg/mL) and α-phellandrene (IC₅₀= 26.9μg/mL) were cytotoxic on HL-60, respectively [40]. The cytotoxicity of β-pinene was studied on CHO and WI38 (IC₅₀>50 μg/mL), HL-60 (IC₅₀=29.6μg/mL) [40] and K562 (IC₅₀=157.4μM) [49]. Myrcene showed an activity on HepG2 (IC₅₀= 9.23μg/ml), B16-F12 (IC₅₀=12.27μg/ml) [50], but not on Vera, macrophages, A 549, HT 29, CHO, PBMC and WI38 cells [41, 51]. p-Cymene exhibited a cytotoxic effect on B16-F12 (IC₅₀=20.06 μg/mL) but not on CHO, WI38 [51], HepG2 and on PBMC [50]. 4-Terpineol demonstrated an effect on HL-60 (IC₅₀=20.5μg/mL) [40] and on AE17 (IC₅₀=0.02%; 0.01%), B16 (IC₅₀= 0.05%; 0.04%), HF32 (IC₅₀=0.1%; 0.1%) after 24 and 48h, respectively [52].

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.