Salvia ceratophylla L. from South of Jordan: new insights on chemical composition and biological activities

In Jordan, Salvia ceratophylla L. is traditionally used in the treatment of cancer, microbial infections, and urinary disorders. This study aimed: (1) to chemically characterize S. ceratophylla essential oil (EO) from South Jordan, by gas chromatography (GC) and gas chromatography-mass spectrometry (GC–MS); and (2) to evaluate in vitro the cytotoxic, anti-inflammatory, and antiprotozoal activities of the EO, it’s predominant components, and the hexane (A), ethyl acetate (B), methanol (C) and crude-methanol extracts (D). The analysis revealed that the EO has 71 compounds, with linalool (54.8%) as main constituent. Only the hexane extract (A) showed some cytotoxic activity against SK-MEL, KB, BT-549, SK-OV-3, LLC-PK1 and VERO cells lines with IC50 between 60 and > 100 µg/mL. The EO inhibited NO production (IC50 90 µg/mL) and NF-κB activity (IC50 38 µg/mL). The extracts A, B, and D inhibited NO production and NF- κB activity with IC50 between 32 and 150 µg/mL. Linalool considerably inhibited NO production (IC50 18 µg/mL). The extracts tested did not exhibit antileishmanial activity. Regarding antitrypanosomal activity, the EO exhibited significant results with IC50 2.65 µg/mL. In conclusion, Jordan S. ceratophylla EO represents a rich source of linalool and bears a promising therapeutic potential for further antitrypanosomal drug development.


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S. ceratophylla is used in traditional medicine of the Middle East against various ailments, e.g. inflammation, fungal, and nociceptive diseases [4][5][6]. In Middle Eastern countries such as Jordan, Lebanon, and Syria, the herbalists prescribe Salvia species as boiled tea to relieve abdominal pain, headaches, stomachaches, and to treat cancer, microbial infections, asthma, coughs and other pulmonary and urinary disorders [7]. It is also recommended in folk medicine to disinfect homes after sickness [8]. Several Salvia species and their essential oils are used in various traditional medicines to treat malaria [9,10] and leishmaniasis [11].
Despite the composition of S. ceratophylla essential oil (EO) grown in various habitats of the Near East countries has been analyzed and a wide variation in their composition has been reported [3,[12][13][14], there is only one report on the composition of S. ceratophylla EO originated from the middle of Jordan [15].
Few reports showed the biological activities of S. ceratophylla extracts from various locations in the Middle East. Some S. ceratophylla extracts revealed cytotoxic activity against some types of cancer cell lines [16,17].
The antioxidant activity of S. ceratophylla originated from Turkey and Iran has been assessed by different methods. Gürsoy et al. [3] showed the antioxidant activity of essential oil of S. palaestina and S. ceratophylla from Turkey. The S. ceratophylla chloroform extract inhibited the activity of butyrylcholine esterase [18]. Among 9 Salvia species from Iran, the methanolic extract of S. glutinosa and S. ceratophylla revealed the strongest antioxidant activity [17]. The anti-inflammatory activity of the EO from Salvia species is mediated by the inhibition of nitric oxide (NO) production and nuclear kappa B (NF-κB) activation [10]. Linalool and linalyl acetate, the major volatile constituents of the EO of several aromatic plants exhibit in vivo strong anti-inflammatory activity against Carrageenin-induced edema [19].
Only a few studies reported the biological activities of S. ceratophylla from Jordan. However, the anti-inflammatory activity of hexane, ethyl acetate and methanol extracts obtained from the aerial parts of Jordanian Salvia species have been evaluated by inhibition of croton oil-induced mouse ear edema [6]. Kasabri et al. [5] studied the inhibitory activity of the aqueous extracts of S. ceratophylla and other Salvia species grown in Jordan against pancreatic triacylglycerol lipase, α-amylase and α-glucosidase. S. ceratophylla exhibited cytotoxic efficacy against CaCo2 cells with an IC 50 value of 9.2 ± 0.5 μg/mL.
However, there is limited information about the chemical composition and biological activities of medicinal plants from Jordan flora. No studies have been carried out yet on S. ceratophylla EO from Dana Biosphere Reserve in southern Jordan and to the best of our knowledge, there are no previous reports on the antiprotozoal activity of this plant. Thus, the aim of this work was to evaluate for the first time the chemical composition of S. ceratophylla EO grown in Dana Biosphere Reserve (Southern Jordan), as well as, to assess the cytotoxic, antileishmanial, and antimalarial activities of its essential oil and other extracts.

Isolation of Essential Oil
To isolate EO, the crushed aerial parts of S. ceratophylla were hydro distillated during 3 h with a Clevenger-type apparatus, according to the European Pharmacopoeia [28]. The oil obtained was dried over anhydrous sodium sulphate and kept at 4 ºC until analyzed and tested.

Extraction of Non-polar and Polar Extracts
The hexane, ethyl acetate, and methanol extracts from the crude areal parts of S. ceratophylla were obtain as follows: powdered plant material (600 g) was soaked with hexane followed by ethyl acetate, and then methanol for 48 h for each solvent in ratio of 500 mL per 100 g. The process of extraction for each solvent was repeat several times to ensure complete extraction of the compounds. The obtained extracts for each solvent were combine together and subject to rotary evaporation at 40 °C. The evaporation led to dry hexane (A), ethyl acetate (B), and methanol (C) extracts with yields of 4.21%, 1.77% and 6.93%, respectively.

Extraction of Crude Methanol Extract
A sample of 50 g of powder plant materials was soaked in 250 mL of methanol for 48 h. The process of extraction was repeat several times to ensure complete extraction of the compounds. The obtained methanol extracts were filter combined and subject to rotary evaporation at 40 °C. The obtained crude methanol dry extract (D) yielded 18.96%. All extracts (A-D) were freeze-dry and stored at 4 °C for further analyses.

Chemicals
GC-grade n-hexane (> 99%) and the reference standards were purchased from Sigma-Aldrich or isolated in the National Center for Natural Products Research, University of Mississippi. A mixture of n-alkanes (C 8 H 18 -C 23 H 48 ) was use in the determination of retention indices and were purchased from PolyScience Corp.

Gas Chromatography-Mass Spectrometry (GC-MS)
Gas chromatography analysis was performed in an Agilent 7890B GC equipped with a 5975C quadruple mass spectrometer and a 7693-auto sampler. Separation was achieve using an Agilent J&W HP-1 (100% dimethyl-polysiloxane) fused silica capillary column (60 m × 0.25 mm I.D. × 0.25 µM film thickness). Helium was the carrier gas at a constant flow rate of 1.5 mL/min. Oven temperature program: 50 °C hold for 2 min, after 50-170 °C at 2 °C/ min, 170-250 °C at 6 °C/min (hold 10 min). The injector temperature was 250 °C, the injection volume was 0.5 µL and a split ratio of 50:1. The EO was diluted in n-hexane (0.5% v/v) prior to GC/MS analysis.
Mass spectra were record at 70 eV at a scan mode from m/z 40 to 500. The transfer line and the ion source temperatures were 260 °C and 230 °C, respectively. Data acquisition was made with Agilent MSD Chemstation (F.01.03.2357).

Identification of Individual Components
Compound identification was carried out through comparison of the spectra with the database (Wiley and NIST) using a probability-based matching algorithm. Further identification was based on the relative retention indices compared with reference standards and literature data [29].

Cytotoxic activity
All cell lines used in this study were purchase from American Type Culture Collection (ATCC, Rockville, MD). The cytotoxic activity of the EO, extracts isolated from S. ceratophylla and pure compounds was determined in vitro against various human cancer cells: skin malignant melanoma (SK-MEL), human KB carcinoma (KB), breast ductal carcinoma (BT-549), ovarian carcinoma (SK-OV-3), and in two non-cancerous mammalian kidney cell lines (LLC-PK1 and VERO).
Cells were seed at a density of 25,000 cells/well and incubated during 24 h. The tested EO, extracts and pure compounds were added separately at studied concentrations and cells were re-incubated during 48 h. After incubation time, the viability of cells was assessed by using Neutral Red Dye as described [30]. DMSO was use as negative control and doxorubicin as positive control.

Inhibition of Cellular Oxidative Stress Assay
The cellular antioxidant activity of S. ceratophylla EO and other extracts was measured in human HepG2 hepatoma cells This assay determines the ability of test samples to prevent intracellular generation of peroxyl radicals in response to 2,2-azobis (2-amidinopropane) dihydrochloride (ABAP, Sigma-Aldrich, St Louis, MO, USA).
HepG2 cells were culture in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin and 50 µg/mL streptomycin. Cells were seed in 96-well plates (60,000 cells/well) and incubated for 24 h. The tested samples were diluted in serum free medium containing 25 µM 2 ′ ,7′, dichoroflouresceine diacetate (DCFH-DA, Invitrogen, Carlsbad, CA, USA). The cells were wash with PBS and treated with test samples during 1 h. The medium containing tested samples was removed, ABAP (600 µM) was added and the plate was immediately placed on a Spectra Max plate reader for kinetic reading every 5 min during 1 h (37 ºC, emission at 538 nm and excitation at 485 nm). Quercetin (Sigma-Aldrich) was use as positive control. To calculate the percentage of decrease in oxidative stress, the Area Under the Curve (AUC) of fluorescence versus time was used.

Nitric Oxide (NO) Measurement
The NO inhibition by EO, extracts of S. ceratophylla and pure compounds, was assess by the following methodology [31]. Briefly, mouse macrophage cells (RAW 264.7) were culture in phenol red free RPMI medium with 10% bovine calf serum and 100 U/mL penicillin and 100 µg/mL streptomycin.
Cells were seed in 96-well plates (50,000 cells/well) and incubated during 24 h. The tested samples were dilute in serum free medium, incubated during 30 min, then lipopolysaccharide (LPS, 5 µg/mL) was added and cells were incubated for further 24 h. The level of nitrite in the cell supernatant was measure by using Griess reagent. The inhibition of nitrite production by the tested samples was calculate in comparison to the vehicle control. Parthenolide (Sigma-Aldrich) was use as positive control. IC 50 values were obtain from dose curves.

Assay for Inhibition of NF-κB Activity
The inhibition activity of tested samples against NF-κB was evaluate as described [31]. The SW 1353 human chondrosarcoma cell line was culture in a mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient (DMEM/F12) supplemented with 10% FBS, 100 U/Ml penicillin and 100 µg/mL streptomycin. Cultured cells were washed in antibiotic and FBS-free DMEM/F12 medium and then re-suspended in FBS-free DMEM/F12 medium containing 2.5% FBS. NF-κB luciferase plasmid construct was added to the cell suspension in a concentration of 50 µg/ mL and incubated for 5 min at 25ºC. The cells were transfected by electroporation at 160 V and one 70 ms pulse using BTX disposable cuvettes (model 640 in a BTX Electro Square Porator T 820-BTX I, San Diego, CA, USA), Transfected cells were plate in 96-well plates at a density of 1.25 × 10 5 cells per well in 200 µL of DMEM/F12 containing 10% FBS and antibiotics. After incubation for 24 h, the cells were treated with test samples for 30 min and then induced with PMA (70 ng/mL) for 8 h. The inhibition of NF-κB activity by EO, other extracts, and pure compounds was calculated in comparison to vehicle control. The IC 50 values were obtain from the dose curves. Parthenolide was use as positive control.

Sample Preparation
EO and extracts (A-D) at a stock concentration of 20 mg/mL were dilute to 4 mg/mL with DMSO. The obtained solutions (4 mg/mL) were four-fold diluted with incomplete RPMI medium to 1 mg/mL. Pure compounds at a stock concentration of 2 mg/mL in DMSO were diluted four-fold with incomplete RPMI medium to 0.5 mg/mL.

In Vitro Antileishmanial and Antitrypanosomal Assays
The extracts and pure compounds (highest concentrations 20 µg/mL for extracts and 10 µg/mL for pure compounds) were subject to different tests, namely Leishmania donovani promastigote assay, L. donovani axenic amastigote assay, L. donovani THP1 macrophage amastigote assay, Trypanosoma brucei trypamastigotes assay and THP1 toxicity assay. Alamar Blue assay was perform as previously described by other authors [32,33]. These assays have been adapted to 384-well micro-plate format. A 3-4 days old culture of Leishmania donovani promastigotes harvested in the exponential growth phase was diluted with RPMI medium to 1 × 10 6 cells/mL for promastigote assays. A 3-4 days old culture of Leishmania donovani axenic amastigotes was diluted with RPMI medium to 2 × 10 6 cells/mL for axenic amastigote assays. A 2 days old culture of Trypanosoma brucei harvested in the exponential phase was diluted with Iscove's Modified Dulbecco's Medium (IMDM) to 5 × 10 3 cells/mL for the antitrypanosomal assay.
Samples were added to the L. donovani promastigotes, L. donovani axenic amastigotes or T. brucei trypamastigotes cultures. The extracts were initially test at single concentrations of 20 mg/mL for primary screening. The active extracts and pure compounds were test at three or six concentrations ranging from 10 to 0.0032 µg/mL. The plates were incubated at 26 °C for 72 h (37 °C for axenic amastigotes and T. brucei trypamastigotes) and growth of cultured parasites was determined by the Alamar Blue assay [32 33].
The extracts and single compounds were also test against L. donovani intracellular amastigotes in THP1 cells employing a recently developed parasite-rescue and transformation assay [15].

Essential Oil Composition
In this study, the yield (0.34 ± 0.001%) of S. ceratophylla EO was higher than in other studies performed in the middle of Jordan, where the yield was 0.1% [15]. It is also lower than those grown in Turkey with a yield of 0.4% [13] and 0.8% [3] and in Iran (0.6%, [12]). Usually, the yield variation of S. ceratophylla EO is relate to the influence of environmental factors linked to predominant climatic and geographical differences in various growth areas [34,35]. The EO was analyzed by GC and GC/MS, the identified components and their relative percentages are listed in Table 1. Seventy-one components were identified, representing 93.5% of the total constituents ( Table 1). The major compounds of the EO were linalool (54.8%), germacrene-D (6.4%), bicyclogermacrene (5%) and the bicyclic diterpene alcoholsclareol (4.3%). As far as we know, there is no previous report on the S. ceratophylla EO composition originated from Dana Biosphere Reserve (Southern Jordan). However, there are some works reporting the chemical composition of S. ceratophylla originated from middle of Jordan [15], Turkey [3,13,14] and Iran [12].
The EO of the dried flowers of S. ceratophylla grown in the middle of Jordan was dominate by oxygenated monoterpenes (58.28%) (Al Jaber, 2016). S. ceratophylla oil grown in Elazığ, Turkey is characterized by a high amount of germacrene D (27.4%) [13].
Our results revealed that S. ceratophylla EO, originated from southern Jordan showed significant differences compared with the oils obtained from samples grown in other locations and was distinguished by its high concentration of linalool. Our results also showed that this EO is distinct by containing the bicyclic diterpene alcohol sclareol, while this compound was absent in S. ceratophylla from Middle Jordan [35], Turkey [3,13,14] and Iran [12]. This variation might be ascribed to the influence of various factors such as seasonal variation and some environmental conditions such as climate, heavy metals pollution and type of soil, in which the plants were grown [35,36]. Furthermore, harvesting time, temperature and drying period of plants, may also influence the yield of the EO and their phytochemical composition, because these factors are known to increase the concentration of some aromatic oil components, especially linalool [37]. Kumar et al. [38] reported higher contents of linalool in S. sclarea under high daily light exposure, while the highest content of germacrene D and sclareol required 50% and 75% of shade, respectively. Moreover, the EO of other Salvia species grown in Jordan were dominate by linalool as major component. Linalool was the major component in the oils of fresh and dry aerial parts of S. dominica (31.4% and 18.3%, respectively) [39]. The main compound of EO extracted from fresh S. verbenaca was mainly linalool [40].

Cytotoxic Activity Against Cancer Cells
The cytotoxic activity of S. ceratophylla EO and other extracts against cancer and non-cancer cell lines is summarize in Table 2. To the best of our knowledge, the cytotoxic activity of S. ceratophylla EO is reporting here for the first time. The EO (up to a concentration of 100 µg/mL), linalool and other tested pure compounds (up to a concentration of 10 µg/mL) did not exhibit cytotoxic activities against all cell lines tested. In agreement with our results, other reports showed that linalool did not exhibit cytotoxic activity up to a concentration of 200 μg/mL against several cancer cell lines [41]. Nevertheless, linalool had significant cytotoxic activity against various cancer cell lines, such as BCC-1/KMC, AGS, RTCC-1/KMC, U2OS, HeLa, H520, H661, OSCC-1/ Compounds listed in order to their elution on the HP-1 column RRI Relative retention indices calculated against C 8 -C 23 n-alkanes on the apolar HP-1 column % calculated from GC/MS data KMC, J82 [42]. Furthermore, it enhanced the cytotoxic activity of doxorubicin against doxorubicin-resistant MCF-7 breast cancer cells [43].
However, our results are in agreement with other previous reports. Abu-Dahab et al. [16] showed that 70% ethanol extract of S. ceratophylla originated from Middle Jordan, exhibited low cytotoxic activity against several cancer cell lines. Furthermore, compared with other Salvia species from Jordan, the cytotoxic activity of S. ceratophylla 70% ethanol extract tested by using the sulforhodamine B colorimetric assay was comparable or even higher against the obesityrelated HCT116 and Caco2 colorectal carcinoma cell lines. The cytotoxic activity of S. ceratophylla against other obesity-related colorectal cell lines including HT29, SW620, and PDL was low, but higher than other Salvia species tested [5,17] demonstrated that among various species of Salvia, S. ceratophylla methanol extract exhibited the strongest activity against human amelanotic melanoma (C32, CRL1585), and renal cell adenocarcinoma (ACHN, CRL1611) with IC 50 values of 20.8 and 27.2 μg/mL, respectively.
However, the variation in the cytotoxic activity of Salvia species may be due to the variation in their chemical composition, which can be influence by various factors such as chemotype and environment [4]. It was also reported that the cytotoxic activity of S.officinalis was affect by solvents used to obtain various extracts with various yields and quality of secondary metabolites [44].

Inhibition of Oxidative Stress and Anti-Inflammatory Activity
Nitric oxide (NO) is a free radical and its production at high levels was associated with inflammation and cancer. The metabolic pathway, which is known as the L-arginine: NO pathway is the main source for NO production in mammalian cells by a group of enzymes known as the nitric oxide synthases (NOS). Inducible NOS (iNOS; NOS2; or type II NOS) is the enzyme responsible for the involvement of NO in inflammatory processes. Accordingly, the inhibition of NO production has been considered as a potential anti-inflammatory and cancer chemo-preventive strategy [45]. On the other hand, the activation of NF-κB represents a mechanistic connection between chronic inflammation and tumorigenesis [46]. The Specificity Protein 1 (Sp1) is a transcription factor, which binds GC/GT-rich promoter elements through three C2H2-type zinc fingers that are present in their C-terminal domains [47]. Sp1 plays a major role in regulating the expression of genes involved in cell differentiation, cell cycle and apoptosis [48]. As compared with normal tissues, the activity and / or expression levels of Sp 1 were increased in some tumors such as carcinomas of breast [49], thyroid [50], and pancreas [51]. Sp1 plays an important regulatory role in controlling pathways of tumor development and progression. Thus, it may be an attractive therapeutic target for anticancer natural agents.
The EO of several Salvia species were being report to inhibit iNOS [52]. Therefore, we also evaluated, for the first time, the in vitro inhibiting intracellular oxidative stress activity of the EO and polar and nonpolar extracts S. ceratophylla from southern Jordan.
With exception of the EO, which did not decrease the oxidative stress, our results revealed that all other extracts exhibited antioxidant activity by inhibiting intracellular oxidative stress ( Table 3). The hexane (A) and ethyl acetate (B) extracts were the most active in decreasing oxidative stress. At the concentration 1000 µg/mL the decrease by the extracts was 50 and 47%, respectively, while at the concentration 500 µg/mL the decrease by the extracts was 56 and 25%, respectively. Among all tested pure compounds, only caryophyllene showed weak activity in decreasing oxidative stress by 35 and 31% at concentrations of 500 and 250 µg/ mL, respectively.
The EO showed the lowest inhibition in NO production with an IC 50 90 µg/mL and the highest inhibition activity against NF-κB with an IC 50  Linalool was the only pure compound showing high and significant NO inhibition with an IC 50 value of 18 µg/mL. Linalool, caryophyllene and α-terpineol inhibited NF-κB activity with IC 50 values of 60, 45 and 100 µg/mL, respectively. Caryophyllene was the only pure compound, which inhibited Sp-1 with an IC 50 value of 48 µg/mL.
The antioxidant activity of S. ceratophylla EO measured by different methods was moderate and slightly lower than the activity of S. palaestina essential oil [3]. Moreover, S. officinalis EO from Jordan showed anti-inflammatory effects and significantly inhibited NO production stimulated by LPS in macrophages, without affecting cell viability, in concentrations up to 0.64 μL/mL [53].
Nevertheless, the anti-inflammatory activity assessed by edema reduction was observed for S. ceratophylla ethylacetate extract with an ID 50 98 μg/cm 2 compared with indomethacin, the hexane and methanol extract showed very low activity with an ID 50 of > 300 μg/cm 2 [6]. Moreover, using the FRAP method, S. ceratophylla exhibited significant antioxidant activity 290.7 μmol FeSO 4 eq/g of dried extract [17].
However, the previously obtained results showed that linalool significantly inhibited carrageenin-induced hind paw edema (55%, p = 0.03) at a dose of 25 mg/kg [54]. Ma et al. [55] reported that the effect of linalool on cigarette smoke induced acute lung inflammation and showed that it suppressed the activation of NF-κB in a dose-dependent manner. Hassan et al. [46] showed that α-terpineol inhibited the growth of cell lung carcinoma through significant inhibition of the NF-κB pathway.
However, in agreement with Gürsoy et al. [3] it is important to mention that the anti-oxidative activity of EOs or extracts were link with the polarity of their chemical constituents. EOs with non-polar secondary metabolites such as terpenoids, were minimally or not active. The anti-inflammatory and radical scavenging activity of S. ceratophylla may be ascribe to the high total phenolic contents, thus contributing to their electron transfer/hydrogen donating capability [3,17,56].

Antileishmanial and Antitrypanosomal Activities
The present study provided for the first time, data on the activity of S. ceratophylla EO and other extracts (from Jordan) against T. brucei and L. donovani in promastigote and amastigote stages. Furthermore, this study demonstrated the antiprotozoal activity of pure compounds (linalool, α-terpineol and β-caryophyllene). Interestingly, the results of human acute monocytic leukemia cells toxicity (THP1) showed that the S. ceratophylla EO, Ethyl acetate extract (B) and α-terpineol did not exhibit cytotoxic effects on mammalian cells at the concentrations tested.
Other authors stated that extracts obtained from other Salvia species led to similar results than those of S. ceratophylla extracts, such as the activity of S. officinalis methanol extract from leaves against promastigote and amastigote forms of L. major [57]. Moreover, the liquid form of S. officinalis extract distributed in the German market did not show remarkable antileishmanial activity against L. donovani [21].
Morales et al. [25] studied the antileishmanial activity of various EO components such as linalool, carvacrol, geraniol and terpinen-4-ol against L. infantum promastigotes. Accordingly, among the compounds tested, carvacrol exhibited the highest activity at the lowest concentration of 6.25 µg/mL followed by linalool, which exhibited dosedependent antileishmanial activity. At a concentration of 37.5 µg/mL the inhibitory activity of linalool was similar to the control drug (Pentamidine). Moreover, linalool, the main EO component of Croton cajucara reduced the viability of L. amazonensis promastigotes at very low concentrations (MIC, 85.0 pg/ml) and presented no cytotoxic effects against mammalian cells [24]. Ślusarczyk et al. [63] showed that among 880 plant and fungal extracts, the dichloromethane extract of S. miltiorrhiza was the only extract that inhibited 97% of T. brucei rhodesiense growth at the lowest concentration tested (0.81 μg/mL). Accordingly, the antitrypanosomal activity of S. miltiorrhiza dichloromethane extract was correlate with the presence of tanshinone-type diterpenoids, which also exhibited antitrypanosomal activity with IC 50 values of 0.5 µM to over 30 µM against T. brucei rhodesiense [58].
Both, minor compound (nerolidol) and major compound (linalool) of Strychnos spinosa EO demonstrated high and selective activity towards Trypanosoma (SI = 35.7 and > 40, respectively, and IC 50 = 1.7 and 2.5 µg/mL, respectively). α-Terpineol was slightly active against T. brucei brucei blood stream forms [59]. However, these authors also reported that the discrepancy in the results might be due to different used methods and/or due to differences in stereoisomeric forms of the compounds tested.

Conclusion
The present study revealed that S. ceratophylla EO from Jordan was characterize by a high amount of linalool. Additionally, the EO exhibited significant antitrypanosomal activity against T. brucei. Moreover, the inhibitory activity of S. ceratophylla EO against T. brucei was stronger than that of the control (α-difluoromethylornithine).
This study highlights the potential of this EO as a good candidate for the treatment of the hard to cure neglected trypanosomiasis infections, also it is demonstrating that S. ceratophylla bears promising therapeutic potential for further drug development. Importantly, this work also clearly validated some of the traditional medicinal uses of Salvia species.