
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12849-6
10.1016/j.heliyon.2024.e36818
e36818
Research Article
Bioassay-guided isolation of dehydrocostus lactone from Echinops kebericho as a leishmanicidal drug
Abdo Bekri Melka bekrimelka2003@yahoo.com
bekri.melka@eiar.gov.et
a⁎
Asfaw Bizuayehu Tesfaye b
Choudhary M. Iqbal c
Yousuf Sammer c
Mengesha Wendawek Abebe d
Mekonnen Solomon Abate e
a Wendo Genet Natural Product Research Laboratory, Ethiopian Institute of Agricultural Research, Addis Ababa, 2003, Ethiopia
b Plant and Horticultural Science Department, Hawassa University, Hawassa, Ethiopia
c International Center for Chemical and Biological Science, University of Karachi, Karachi, 75270, Pakistan
d Department of Molecular, Cellular, and Microbial Biology, Addis Ababa University, Addis Ababa, Ethiopia
e Food Science and Nutrition Research Process, Ethiopian Institute of Agricultural Research, Addis Ababa 2003, Ethiopia
⁎ Corresponding author. bekrimelka2003@yahoo.combekri.melka@eiar.gov.et
29 8 2024
15 9 2024
29 8 2024
10 17 e3681818 6 2024
13 8 2024
22 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Several strains of Leishmania parasite are involved in the occurrence of leishmaniasis infections, which makes its prevention and treatment very challenging. Currently, all forms of leishmaniasis are being treated with chemical drugs, which have limitations and adverse effects. Discovering antileishmanial agents from natural sources can lead to novel drugs against this dreadful disease. The essential oils and nonpolar solvent extracts of the roots of E. kebericho exhibit antileishmanial activity. Thus, the isolation of the leishmanicidal compounds from the roots of E. kebericho through a bioassay-guided technique was carried out in this study. The present finding showed that the essential oil and hexane fraction of crude extract from the roots of E. kebericho possessed significant leishmanicidal activity against L. major and L. tropica. Dehydrocostus lactone (1), one of the major constituents of the essential oil and hexane fraction, was more active than the standard drug miltefosine against L. major and L. tropica promastigotes. The presence of α-methylene, γ-lactone is the responsible moiety of dehydrocostus lactone towards the leishmanicidal activity against the tested Leishmania species. The MTT assay of dehydrocostus lactone showed inactive toxicity against the human cervical carcinoma HeLa cells. In addition, dehydrocostus lactone exhibits a broad spectrum of antibiotic activities. Based on this interesting finding, dehydrocostus lactone was identified as a potential lead for treating infections caused by Leishmania.

Keywords

Dehydrocostus lactone
Echinops kebericho
Leishmaniasis
Leishmania major
Leishmania tropica
==== Body
pmc1 Introduction

Leishmaniasis infection is a major health problem in tropical regions. It is caused by more than 20 species of the protozoa genus Leishmania. Over 90 species of sandflies serve as transmitting vectors, and more than 70 animal species including humans are zoonotic hosts [1]. The involvement of several strains of Leishmania makes their diseases difficult to control and treat various forms of leishmaniasis. Worldwide, 50,000 to 90,000 and 600,000 to 1 million cases of visceral and cutaneous forms of leishmaniasis, respectively, are reported annually [1]. Cutaneous leishmaniasis is the most common form in Ethiopia, with an estimated 20,000 to 50,000 cases annually, and L. aethiopica, L. tropica, and L. major are the causative agents [2].

Affordable, safe, and short-course treatments are required for the successful treatment of leishmaniasis, as per the World Health Organization [3]. Currently, pentavalent antimonial, paromomycin, amphotericin B, liposomal amphotericin, pentamidine, and miltefosine are generally used. These drugs have numerous limitations such as drug resistance, high cost, low availability, toxicity, and painful routes of administration [[4], [5], [6]]. Therefore, it is important to search for new leishmanicidal compounds with good efficacy and safety. Medicinal plants are excellent sources of secondary metabolites with diverse biological activities [7]. Echinops kebericho Mesfin is an endemic and endangered Ethiopian medicinal plant belonging to the genus Echinops and family Asteraceae. Its roots are traditionally used for the treatment of various ailments, such as dispelling nightmares in children, constipation, headache, heart pain, stomachache, typhus, as a fumigant after childbirth, intestinal pains, lung TB, leprosy, syphilis, cough, ward off the evil eye, toothache, and vomiting [[8], [9], [10], [11], [12], [13], [14]]. Additionally, it is used as a snake and mosquito repellant [15,16]. The organic solvent extracts from the roots of Echinops kebericho have been identified as antimalarial, antimicrobial, antileishmanial, antischistosomal, antidiarrheal, and spasmolytic active agents [[17], [18], [19], [20], [21], [22]].

The essential oil, extracted from the roots of E. kebericho, possesses constituents, such as eudesm-7 (11)-en-4-ol, caryophyllene oxide, τ-cadinol, β-cubebene, β-patchoulene, longifolene, cyperene, dehydrocostus lactone, β-phellandrene, germacrene B, α-selinene, isoshyobunone, modephene, α-pinene, and β-pinene [23,24]. Moreover, modephene, lappaconitine, pyrocurzerenone, dehydrocostus lactone, dihydrodeydrocostus lactone, 3aR,6aR,9aR,9bR)-decahydro-4 hydroxy3,6,9-trimethyleneazuleno[4,5-b] furan-2(9bH)-one, β-sitosterol, stigmasterol, campesterol, β-amyrene, lupeol, and ursolic acid, together with a series of fatty acids are obtained from the solvent extract of E. kebericho roots [[25], [26], [27], [28]].

The essential oil, petroleum ether, and chloroform extracts obtained from the roots of E. kebericho possess activity against several Leishmania species [22]. The biological activities of the isolated ingredients from the roots of E. kebericho are not adequately studied. The current study aimed to evaluate the leishmanicidal activity of the crude fractions and isolated compounds against the promastigote stage of L. major and L. tropica strains. The in vitro leishmanicidal and cytotoxic activities were evaluated through a 96-well plate assay with microscope detection and 3- [4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide assay, respectively [[29], [30], [31]].

2 Materials and methods

2.1 Plant collection and processing

The roots of Echinops kebericho Mesfin were collected from the experimental field of Wendo Genet Agricultural Research Center, Ethiopia (1819 m a.s.l., 07°05′664″ N, 038°38′897″ E). All the steps, such as extraction, isolation, structure elucidation, and biological activities, were conducted at the H.E.J. Research Institute (ICCBS), Pakistan.

2.2 Preparation of plant sample

The matured (12 months old) E. kebericho roots (500 g) were harvested. The roots were washed with tap water, then chopped and dried in the shade. The dried roots were crushed with mortar and pestle. The powdered sample was stored in a plastic bag until extraction.

2.3 Essential oil extraction

The powdered roots (200 g) were distilled through hydro distillation by using a Clevenger-type apparatus for 4 h. The distillate was collected using a measuring pipette and stored in cleaned amber vials after drying with anhydrous Na2SO4. The essential oil yield was calculated according to the formula described in Equation (1). The essential oil was subjected to the antileishmanial activity test and chemical composition analysis (Fig. 1).(1) Essentialoilyield(ww(%))=Amountofdistilledoil(g)Amountoftherootsample(g)×100

Fig. 1 Diagram showing the workflow of extraction, compound isolation (blue line), and antileishmanial activity tested samples (red arrow). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1

2.4 Chemical composition analysis of essential oil

A GC-MS (Agilent model 7890 A, Agilent, Santa Clara, CA, USA) equipped with GC sampler 120 was used to determine the chemical profile of the essential oil. The instrument was conditioned with a split/splitless injector mode, MS detector (7000 Triple Quad, Agilent, Santa Clara, CA, USA), and Zebron ZB-5 capillary column (320 μm internal diameter × 30 m length × 0.25 μm film thickness, Phenomenex, USA). The injector was operated in a split ratio of 1:20 with an injection volume of 1.2 μL. The injector and detector temperature were set at 250 °C. The MSD was operated on scan mode in the m/z 40–700 range, and the interface temperature was set to 260 °C. Helium was used as a carrier gas in a controlled constant flow mode at a linear velocity of 44.64 cm/s. The oven was programmed to start at 50 °C and held for 5 min; then, the temperature was ramped at 3 °C/min to 200 °C and held for 15 min; subsequently, the temperature was ramped at 10 °C/min to 300 °C and held for 20 min. The solvent delay time was 5 min and took 100 min for the total run.

2.5 Crude extraction and fractionation

The powdered sample of E. kebericho roots (892.1 g) was macerated and divided into two 2000 mL volume round bottom flasks using a mixture of dichloromethane and methanol (1:1) solvents (1600 mL for each flask) for 48 h with periodical shaking. The extracted solution was filtered using a muslin cloth, and the residue was repeatedly extracted with 800 mL maceration solvent. All the extract solutions were collected and filtered with filter paper and finally concentrated using a rotary evaporator. The crude extract was added into a 2000 mL separatory flask containing 500 mL distilled water for further partitioning with liquid–liquid extraction using different organic solvents, such as hexane, dichloromethane, ethyl acetate, and n-butanol, starting from a low to high polarity gradient. The extraction was achieved with 1000 mL solvent in the separatory flask having crude extract in distilled water. The extraction was repeated four times with 1000 mL organic solvent. Hexane, dichloromethane, ethyl acetate, and n-butanol solvent extracts were obtained. For dichloromethane solvent, the lower layers were collected and concentrated. Finally, the remaining water extract was concentrated using a freeze drier. The extract yield was calculated according to the formula described in Equation (2).(2) Extractyield(ww(%))=Amountofextract(g)Amountoftherootsample(g)×100

2.6 Isolation of compounds

The major compounds were isolated by using column chromatography (column diameter of 4 cm, silica gel of 70–230 pore size (Merck, Darmstadt, Germany), and a 40 cm pack length). Hexane was used to prepare the slurry of silica gel for column packing. The hexane fraction (5 g) was loaded into the column. Gradient elution of a mobile phase was used starting from 200 mL hexane and continued with 1000 mL hexane–ethyl acetate (95:5); 1000 mL hexane–ethyl acetate (90:10) and 1000 mL hexane–ethyl acetate (86:14). An eluate of 100 mL volume was collected as one fraction and transferred to vials (20 mL) after being concentrated. The purity of the isolated fraction was checked with TLC through a hexane–ethyl acetate (86:14) solvent system. The spots on the TLC were detected with UV light at 254 nm and 366 nm wavelengths for UV-active compounds. The non-UV-active compounds were stained by using a vanillin–sulfuric acid reagent. Finally, the purified compounds were submitted to spectral analysis of EI-MS (JEOL JSM 600H-1, Jeol, Japan) and 1H NMR (Avance NEO, 500 MHz, Bruker, Switzerland).

2.7 Derivatization of dehydrocostus lactone

Michael addition was performed to derivatize the α-β unsaturation of dehydrocostus lactone (1) [32]. Dehydrocostus lactone (140 mg) was dissolved in hexane (HPLC grade) and mixed through a vortex shaker. The solution was boiled in a water bath for 2 min, and 140 μL of 2N potassium hydroxide in methanol (HPLC grade) was added. The solution was mixed with a vortex shaker for 30 s and centrifuged. The supernatant hexane solution was separated, and the derivatized compound was checked with TLC through a hexane–ethyl acetate (86:14) solvent system. The derivatized compound was purified using column chromatography (column diameter of 10 mm, a silica gel (flash, 20 g), (Merck, Darmstadt, Germany)). Finally, the purified derivative compound was submitted to spectral analysis of EI- MS (JEOL JSM 600H-1, Jeol, Japan) and NMR (1H, 13C, DEPT (135° and 90°), HSQC, and HMBC) (Avance NEO, 500 MHz, Bruker, Switzerland).

2.8 Antileishmanial activity test

An in vitro test with a 96-well plate assay described by Bouyahya et al. (2018) [33] was used for antileishmanial activity against Leishmania major 50155 (ATCC) and Leishmania tropica 50129 (ATCC). Leishmania promastigotes were grown in RPMI-1640 media with 10 % fetal bovine serum. The parasites at the log phase were centrifuged at 200 rpm for 10 min, and the supernatant was discarded. Fresh media was added to dilute the pallet material till the final density of 106 cells/mL. The media (100 μL) was added in all well tissue culture plates except the first column, which added 180 μL. The last two rows were used for negative (5 % DMSO in media) and positive control (Amphotericin B, Pentamidine, and Miltefosine). The test samples (1 mg) (fractioned extract, essential oil, and isolated compounds) (Fig. 1) were dissolved in 50 μL DMSO and diluted with 950 μL of RPMI-1640 media. The tested solution (20 μL) was added to the first well plate and mixed. A serial dilution of the next well plate was followed. The plates were incubated in the dark at 23 °C for 72 h. After 72 h, the antileishmanial activities of the fractioned extract, essential oil, isolated compounds, and standard drugs were assessed microscopically using an improved Neubauer chamber.

2.9 Cytotoxicity activity test

Cytotoxic activities of the extracts and isolated compound were evaluated in 96-well flat-bottomed microplates by using the standard MTT (3- [4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) colorimetric assay [34]. HeLa cells (Human Cervical Cancer) were cultured in Minimum Essential Medium Eagle, supplemented with 5 % of fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 μg/mL of streptomycin in 75 cm2 flasks, and kept in 5 % CO2 incubator at 37oC. Exponentially growing cells were harvested, counted with a hemocytometer and diluted with a particular medium. Cell culture with a concentration of 6x104 cells/mL was prepared and introduced (100 μL/well) into 96-well plates. After overnight incubation, the medium was removed and 200 μL of fresh medium was added with different concentrations of test samples (1–30 μM). After 48 h, 200 μL of MTT (0.5 mg/mL) was added to each well and incubated further for 4 h. Subsequently, 100 μL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 570 nm, using a microplate reader (Spectra Max plus, Molecular Devices, CA, USA). The cytotoxicity was expressed as concentration causing 50 % growth inhibition (IC50) for HeLa cell. The results of percent of inhibitions were processed by using Soft- Max Pro software (Molecular Device, USA). The percent of inhibition was calculated by using the formula described in Equation (3).(3) Inhibition(%)=100−((Absorbanceoftestsample–AbsorbanceofnegativecontrolAbsorbanceofpositivecontrol–Absorbanceofnegativecontrol)×100)

2.10 Statistical analysis

The significance variation of the IC50 value from leishmanicidal and cytotoxicity activities of the extracts, isolated compounds, and the standard drugs were determined by F test. The means and standard deviations were used for data analysis.

3 Results and discussion

Hydro-distillation of dried roots of E. kebericho yielded 0.16 % (w/w) of essential oil. The IC50 value of the leishmanicidal and cytotoxicity activity test showed that highly significant difference between the tested samples and the standards at p < 0.001 (Table 1). The oil showed in vitro leishmanicidal activity with an IC50 value of 38.3 μg/mL against L. major and 55.16 μg/mL against L. tropica (Table 2). Furthermore, the essential oil profile was characterized, and a total of 43 compounds were identified (Table 4). Dehydrocostus lactone (1) (24.95 %), β-guaiene (11.13 %), cis lanceol (5.92 %), Juniperol (4.98 %), (±)-cadinene (3.94 %), δ-neoclovene (3.68 %), aromadendrene oxide (3.29 %), and caryophyllene (3.05 %) were the major constituents of the essential oil.Table 1 Descriptive statistics showing the significant variation of IC50 value of crude extract, isolated compound, and the standard drugs from leishmanicidal and cytotoxicity activity test.

Table 1Activities	Tested organisms	Mean ± SE	Range	F test	CV (%)	LSD (α = 0.05)	
Leishmanicidal	L. major	21.11 ± 0.12	3.39–38.3	***	1.40	0.79	
L. tropica	23.52 ± 0.15	3.41–55.16	***	1.58	0.88	
Cytotoxicity	HeLa cell	9.37 ± 0.14	1.19–15.9	***	2.6	0.65	
SE = standard error; *** = highly significant at p < 0.001; F test = fisher test; CV = coefficient of variance; LSD = list significant difference.

Table 2 In vitro antileishmanial activity test of crude extract, essential oil, and isolated compounds from the roots of E. kebericho.

Table 2Type of Samples	Tested Samples	L. major	L. tropica	
Crude extract
IC50(μg/mL) ± SD	Hexane fraction	33.3 ± 0.7	36.6 ± 0.5	
Dichloromethane fraction	NT	NT	
Ethyl acetate fraction	>100ND	>100ND	
N-Butanol fraction	>100ND	>100ND	
Water fraction	>100ND	>100ND	
Essential oil	38.3 ± 0.8	55.16 ± 0.9	
Compounds
IC50(μM) ± SD	Dehydrocostus lactone	15.3 ± 0.03	14.2 ± 0.2	
13-Methoxy dehydrocostus lactone	>100ND	>100ND	
Amphotericin B	3.39 ± 0.03	3.41 ± 0.02	
Pentamidine	4.56 ± 0.01	4.56 ± 0.01	
Miltefosine	31.8 ± 0.2	27.2 ± 0.6	
SD = standard deviation; NT = biological activity not tested; >100 = inactive against the tested biological activity; ND = not determined the statistical analysis.

Dichloromethane–methanol maceration of the dried roots of E. kebericho yielded a crude extract of 10.38 % (92.63 g). Different yields of fractions were obtained through liquid-liquid partition, such as hexane (55.61 g), dichloromethane (8.53 g), ethyl acetate (4 g), n-butanol (11.70 g), and water fraction (12.79 g). From the in vitro assay, the hexane fraction showed an antileishmanial activity with IC50 values of 33.3 and 36.6 μg/mL against L. major and L. tropica, respectively. All other crude fractions were not active against the tested Leishmania strains. The dichloromethane fraction was not tested due to solubility issues.

The major compound from the hexane fraction was isolated using column chromatography, with an Rf value of 0.57 through elution with the solvent system of hexane–ethyl acetate (86:14). Compound 1 was a colorless crystalline solid. Its structure was identified using EI-MS, which gave a molecular mass ion peak ([M]+) at m/z 230.2, having the molecular formula C15H18O2. The 1H NMR spectra (500 MHz, CDCl3) showed peaks at δ(ppm): 6.19 (1H, d, J = 3.5 Hz, H-13a), 5.46 (1H, d, J = 3.2 Hz, H-13b), 5.24 (1H, brs, J = 2.4 Hz, J = 1.5 Hz, H-15a), 5.04 (1H, brs, H-15b), 4.87 (1H, brs, H-14a), 4.79 (1H, brs, H-14b), 3.94 (1H, t, J = 9.3 Hz, H-6), 2.93 (1H, m, H-5), 2.89–2.79 (2H, m, H-3a, H-9a), 2.59–2.41 (3H, m, H-1, H-7, H-2a), 2.22 (1H, m, H-9b), 2.13 (1H, m, H-2b), 1.97–1.79 (2H, m, H-8a, H-8b), and 1.39 (1H, m, H-3b). From the spectral analysis and comparisons with the literature [35,36], the structure of compound 1 was characterized as dehydrocostus lactone (1), a sesquiterpene lactone of the guaianolides class (Fig. 2).Fig. 2 Chemical structure of isolated compounds from the roots of E. kebericho; (1) Dehydrocostus lactone, (2) 13-Methoxy dehydrocostus lactone.

Fig. 2

Compound 2 was a colorless, amorphous solid with an Rf value of 0.43 through elution with the solvent system of hexane–ethyl acetate (86:14). The EI-MS gave a molecular mass ion peak ([M]+) at m/z 262.16, having the molecular formula C16H22O3. The 1H NMR spectrum (500 MHz, CDCl3) showed peaks at δ (ppm) 1H: 5.17 (1H, brs, J = 2.4, J = 0.8 Hz, H-15a), 5.02 (1H, brs, J = 2.3, J = 0.9 Hz, H-15b), 4.85 (1H, brs, J = 1.0 Hz, H-14a), 4.75 (1H, brs, J = 1.3 Hz, H-14b), 3.91 (1H, t, J = 9.3 Hz, H-6), 3.68 (1H, d, J = 9.8, 4.4 Hz, H-13a), 3.62 (1H, d, J = 9.8, 3.3 Hz, H-13b), 3.35 (3H, s, OCH3), 2.92–2.85 (1H m, H-5), 2.85–2.77 (1H, m, H-11), 2.57–2.31 (5H, m, H-3a, 9a, 2a, 1, 7), 2.21–2.12 (1H, m, H-9b), 2.05 (1H,m, H-2b), 1.98–1.87 (1H, m, H-8a), 1.83 (1H, m, H-8b) and 1.30 (1H, m, H-3b). The 13C NMR (201 MHz, CDCl3) δ 175.99 (=C=O, C-12), (=C = , C-4), 150.01 (=C = , C-10), 109.16 (=CH2, H-15), 111.77 (=CH2, C-14), 85.56 (O-CH, C-6), 69.11 (CH2, C-13), 59.35 (O-CH3), 51.82 (CH, C-11), 48.10 (CH, C-7), 47.04 (CH, C-5), 44.22 (CH, C-1), 37.75 (CH2, C-2), 32.65 (CH2, C-9), 32.58 (CH2, C-3), and 30.2 (CH2, C-8). The DEPT-135° 13C NMR spectrum showed a total number of six CH3 and CH carbons and seven CH2 carbons. The DEPT-90° 13C NMR spectrum showed a total of five CH carbons. The 1H - 13C correlation was confirmed by HSQC and HMBC spectra, respectively. From the spectral analysis results, compound 2 was characterized as 13-methoxy dehydrocostus lactone (2) (Fig. 2).

In vitro, a leishmanicidal test result of dehydrocostus lactone (1) showed IC50 values of 15.3 μM for L. major and 14.2 μM for L. tropica. 13-Methoxy dehydrocostus lactone (2) was found inactive against the tested Leishmania species. The standard drugs showed IC50 values of 3.39 and 3.41 μM (Amphotericin B), 4.56 μM (Pentamidine), and 31.8 and 27.2 μM (Miltefosine) against L. major and L. tropica, respectively.

The antileishmanial assay showed that the essential oil and hexane fraction of crude extract from the roots of E. kebericho have significant activity against L. major and L. tropica. Dehydrocostus lactone (1) is the most abundant constituent of the essential oil and hexane fraction of the crude extract from the roots of E. kebericho, which showed potent antileishmanial activity against L. major and L. tropica promastigotes. Similar results of the chemical composition of essential oils of the roots of E. kebericho were reported by Tariku et al. (2011) [24], which included dehydrocostus lactone (41.83 %), β-phellandrene (10.84 %), germacrene B (5.38 %), α-selinene (4.13 %), α-pinene (3.63 %), and β-pinene (3.62 %). A supportive result has been reported as the essential oils of E. kebericho showed a concentration-dependent growth inhibitory effects against promastigote forms of L. donovani (MIC = 0.0765 μL/mL) and L. aethiopica (MIC = 0.0097 μL/mL) [23].

Dehydrocostus lactone (1) is twice more active than the standard drug miltefosine against the tested Leishmania species. Amphotricin B is the most active standard drug against the tested Leishmania species. Some compounds, such as physalin B, 5β, 6β-epoxyphysalin B, and physalin H, have reported comparable antileishmanial activity with amphotericin B against L. major promastigotes with an IC50 value (3.04–3.76 μM) [37]. Similarly, Azadbakht et al. (2020) [38] reported that compounds isolated from Colchicum kurdicum have antileishmanial activities against L. major promastigotes with an IC50 value of colchicoside (0.22 μg/mL), colchicine (0.39 μg/mL), and the standard drug amphotricin B (0.12 μg/mL). Moreover, physalin B, 5β, 6β-epoxyphysalin B, and physalin (IC50 = 9.59–18.53 μM) have comparable activities with dehydrocostus lactone (1) against L. tropica promastigotes [37].

The 13-methoxy-derivative of dehydrocostus lactone (2) is not active against the tested Leishmania species. From the structure-activity relationship (SAR) of dehydrocostus lactone (1) and 13-methoxy dehydrocostus lactone (2), the presence of α-methylene, γ-lactone is responsible for leishmanicidal activity against the tested Leishmania species. Similar results of the structure–activity relationship have been reported on the antitrypanosomal effects and cytotoxicity of dehydrocostus lactone [39]. However, the 13-amino derivative of dehydrocostus lactone has shown better anticancer activity and reduced cytotoxicity than dehydrocostus lactone [40].

The in vitro cytotoxicity activity test was determined with a cut of value of 50 % inhibition. The Tested samples having inhibition value of below 50 % was considered as inactive toxicity against the human cervical carcinoma HeLa cells. The hexane fraction and essential oil showed significant toxicity against the human cervical carcinoma HeLa cells with IC50 value of 15.9 μg/mL and 11.03 μg/mL, respectively (Table 3). The reference standard doxorubicin was scored an IC50 value of 1.19 μM. Dehydrocostus lactone (1) was non-toxic to the human cervical carcinoma HeLa cells.Table 3 In vitro cytotoxicity activity test of crude extract, essential oil, and isolated compound from the roots of E. kebericho.

Table 3Tested Samples	Test Conc.	Inhibition (%)	IC50± SD	
Hexane fraction	30 μg/mL	92.91	15.90 ± 0.15 (μg/mL)	
Dichloromethane fraction	NT	NT	NT	
Ethyl acetate fraction	30 μg/mL	8.88	Inactive	
N-Butanol fraction	30 μg/mL	15.77	Inactive	
Water fraction	30 μg/mL	12.95	Inactive	
Essential oil	30 μg/mL	91.66	11.03 ± 0.54 (μg/mL)	
Dehydrocostus lactone	30 μM	46.67	Inactive	
Doxorubicin	30 μM	98.7	1.19 ± 0.04 (μM)	
Conc. = concentration.

Table 4 Chemical composition of essential oil from the roots of E. kebericho.

Table 4No.	RT (Minutes)	MW (g/mole)	MF	Name	Relative Concentration (%)	
1	18.1	152	C10H16O	(S)-Cis-Verbenol	0.21	
2	19.1	154	C10H18O	Borneol	0.18	
3	23.5	154	C10H18O	p-Menth-2-en-7-ol, trans-	0.28	
4	24.4	196	C12H20O2	Borneol, acetate	2.08	
5	26.7	204	C15H24	(±)-Cadinene	3.94	
6	27.1	204	C15H24	δ-Neoclovene	3.68	
7	27.5	204	C15H24	β-Elemene	2.67	
8	28.4	204	C15H24	α-Guaiene	2.36	
9	28.7	204	C15H24	Caryophyllene	3.05	
10	30.5	204	C15H24	α-Humulene/α-Caryophyllene	1.36	
11	30.8	204	C15H24	(−)-Alloaromadendrene	1.07	
12	31.7	204	C15H24	γ-Muurolene	0.15	
13	32.1	204	C15H24	β-Eudesmene/β-Selinene	0.11	
14	32.4	204	C15H24	α-Selinene	0.43	
15	33.5	204	C15H24	γ-Cadinene	2.19	
16	33.7	204	C15H24	δ-Cadinene	0.33	
17	34.1	204	C15H24	β-Guaiene	11.13	
18	35.6	222	C15H26O	±-Trans-Nerolidol	0.48	
19	37.7	220	C15H24O	Caryophyllene oxide	2.33	
20	37.8	220	C15H24O	Aromadendrene oxide-(2)	0.74	
21	38.2	220	C15H24O	Juniperol	4.98	
22	38.7	220	C15H24O	Diepi-α-cedrene epoxide	0.37	
23	38.9	222	C15H26O	Germacrene D-4-ol	1.84	
24	39.1	222	C15H26O	Cubenol	0.18	
25	39.8	222	C15H26O	τ-Cadinol	0.33	
26	39.9	222	C15H26O	τ-Muurolol	1.32	
27	40.1	222	C15H26O	δ-Cadinol, (−)-	0.08	
28	40.3	222	C15H26O	α-Cadinol	1.10	
29	40.5	220	C15H24O	γ-Gurjunenepoxide-(2)	0.90	
30	40.8	204	C15H24	Globulol	0.33	
31	41.0	220	C15H24O	Aromadendrene oxide-(1)	3.29	
32	42.0	232	C16H24O	9-Methoxycalamenene	1.43	
33	42.6	220	C15H24O	Cedren-13-ol, 8-	0.09	
34	42.8	220	C15H24O	Ledene oxide-(II)	0.43	
35	44.0	220	C15H24O	Lanceol, cis	5.92	
36	44.6	220	C15H24O	Santalol, cis, α-	0.80	
37	45.5	220	C15H24O	2-(4a,8-Dimethyl-1,2,3,4,4a,5,6,7-octahydro-naphthalen-2-yl)-prop-2-en-1-ol	1.95	
38	46.2	236	C15H24O2	Bicyclo[4.4.0]dec-5-ene, 1,5-dimethyl-3-hydroxy-8-(1-methylene-2-hydroxyethyl-1)-	0.39	
39	46.6	220	C15H24O	α-Copaen-11-ol	0.08	
40	47.5	236	C15H24O2	Bicyclo[4.4.0]dec-2-ene-4-ol, 2-methyl-9-(prop-1-en-3-ol-2-yl)-	0.18	
41	49.0	230	C16H22O	Cycloisolongifolene, 8,9-dehydro-9-formyl-	0.20	
42	50.5	202	C15H22	Aromadendrene, dehydro-	0.19	
43	56.9	230	C15H18O2	Dehydrocostus lactone	24.95	
RT: Retention time; MW: molecular weight; MF: molecular formula.

Although, dehydrocostus lactone (1) showed inactive toxicity, a significant cytotoxicity of hexane fraction and essential oil were observed in this study. Previous studies reported that the essential oil of E. kebericho showed no mortality of rats in acute oral doses of up to 2000 mg/kg body weight and was well tolerated in 200 mg/kg sub-acute toxicity with repeated dose exposure [23]. Additionally, the decoction of E. kebericho root showed LD50 greater than 5000 mg/kg in acute toxicity test and was well tolerated up to a dose of 600 mg/kg in sub-acute toxicity [41]. Moreover, an in vivo cytotoxicity test of dehydrocostus lactone (1) against laryngeal carcinoma showed inhibition of the growth of the Hep-2 nude mouse xenograft model with no significant signs of toxicity in the organs of nude mice [42]. The pharmacokinetics profile of dehydrocostus lactone in rat plasma after oral administration and intravenous administration have been reported as peak concentration (Cmax) (493.00 ng/mL and 5.79 μg/mL) and area under the plasma drug concentration-time curve (AUC) (7884.51 ng h/mL and1.87 μg h/mL), respectively [43,44].

Several researchers reported that dehydrocostus lactone (1) exhibits a range of biological activities such as anticancer activity against different cancer cells [[45], [46], [47], [48]], and it has antibacterial [49], antifungal [50], anti-inflammatory [51,52], anti-allergy [53], and anti-depressant properties [54]. Ancklandia and berberine tablets are clinically available as dehydrocostus lactone-containing drugs, which have been used for the treatment of digestive tract diseases with its anti-inflammatory and anti-microbial activities [47]. Overall, the broad-spectrum antibiotic activities of dehydrocostus lactone could be a potential drug lead for co-infection caused by Leishmania and other pathogens.

Dehydrocostus lactone (1) transduces different mechanism of actions for various biological activities. Dehydrocostus lactone as an anticancer agent through inhibition of the NF-κB/COX-2 signaling pathway, causing cell cycle arrest, inducing apoptosis and differentiation, promoting the aggregation of microtubule protein, inhibiting the activity of telomerase, inhibiting metastasis, and invasion, reversing multidrug resistance, restraining angiogenesis [[55], [56], [57], [58]]. Dehydrocostus lactone treats the inflammation through the inhibition of pro-inflammatory agents (IL1β, IL6, and TNFα), phosphorylation of p38 MAPK, degradation of IκBα, and the activation and nuclear translocation of NF-κB p65 [59,60]. Regarding dehydrocostus lactone (1) mechanism of action as antileishmanial activity has not yet been reported.

4 Conclusions

Dehydrocostus lactone (1) is the major constituent of essential oil and hexane fraction of the crude extract from the roots of E. kebericho. The leishmanicidal evaluation indicated that the roots of E. kebericho possess potent activity, most likely due to the presence of sesquiterpene lactones. Particularly, dehydrocostus lactone has shown more potent leishmaniacidal activity than the antileishmanial standard drug miltefosine. The present in vitro cytotoxicity activity test and previous study report showed that dehydrocostus lactone is non-toxic to normal cells and the human cervical carcinoma HeLa cells. Therefore, dehydrocostus lactone is expected to have a high value of selectivity and safety as a leishmanicidal agent. The presence of α-methylene, γ-lactone is the responsible moiety of dehydrocostus lactone towards the leishmanicidal activity against the tested Leishmania species. In addition, the broad-spectrum antibiotic activities of dehydrocostus lactone make this a potential drug lead against co-infection caused by Leishmania and other pathogens. The diverse range of antileishmanial activities of dehydrocostus lactone needs to be validated against the different Leishmania species both in vivo and in vitro tests. Further, the mechanism of action and potential side effects of dehydrocostus lactone should be tested.

CRediT authorship contribution statement

Bekri Melka Abdo: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Bizuayehu Tesfaye Asfaw: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. M. Iqbal Choudhary: Writing – review & editing, Supervision, Methodology, Funding acquisition. Sammer Yousuf: Resources. Wendawek Abebe Mengesha: Supervision. Solomon Abate Mekonnen: Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank the International Center for Chemical and Biological Science, University of Karachi, Pakistan; United Nations Educational, Scientific, and Cultural Organization; The World Academy of Science, Trieste, Italy; Ethiopian Institute of Agricultural Research.
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