Identification of trans-2-cis-8-Matricaria-ester from the Essential Oil of Erigeron multiradiatus and Evaluation of Its Antileishmanial Potential by in Vitro and in Silico Approaches.

The essential oil (EO) composition of the aerial parts of Erigeron multiradiatus (Lindl.ex DC.) Benth growing wild in the central Himalayan region of Uttarakhand, India, was analyzed by capillary gas chromatography with a flame ionization detector and gas chromatography-mass spectrometry. A sum of 12 constituents was identified, representing 97.81% of the oil composition. The oil was composed mainly of oxygenated monoterpenes (88.95%), sesquiterpene hydrocarbons (5.61%), oxygenated sesquiterpenes (3.05%), and monoterpene hydrocarbons (0.20%). Major constituents identified were trans-2-cis-8-matricaria-ester (77.79%), cis-lachnophyllum ester (11.04%), zingiberene (4.43%), and spathulenol (1.59%). Further, the leishmanicidal effect of EO and the purified compound trans-2-cis-8-matricaria-ester has been investigated against Leishmania donovani promastigotes and intracellular amastigotes. EO and trans-2-cis-8-matricaria-ester were safer for the hamster peritoneal macrophage and lethal to promastigotes and intracellular amastigotes at different concentrations. Further, using an in silico approach, these four compounds were tested against 10 major proteins of L. donovani associated with its virulence. Out of them, only trans-2-cis-8-matricaria-ester was found to be effective against the four target proteins, namely, l-asparaginase-1-like protein, metacaspase 2, metacaspase 1, and DNA topoisomerase II of L. donovani. The results indicate that EO contains trans-2-cis-8-matricaria-ester as a major component and showed antileishmanial activity which may facilitate discovery of new lead molecules for developing herbal medicines against visceral leishmaniasis.

Introduction

Visceral leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani, which causes a fatal systemic infection. The disease is transmitted by a female sandfly (Phlebotominae). It is endemic in 62 countries of the developing world, and with an estimated population of 200 million at risk. Every year more than 100 000 new cases of VL are reported in India, and more than 90% of these cases are reported in the state of Bihar.[1] The control of leishmaniasis is not available yet due to the lack of effective vaccines and the disease control relies on chemotherapy with pentavalent antimonials which require long-term treatment and cause serious side effects.[2] Some of the latest drugs such as miltefosine and liposomal amphotericin B are effective but are extortionate. Along with the toxicity and severe side effects, numerous relapse are also a major concern. Thus based on the present clinical scenario it is desirable to develop new antileishmanial agents from natural products which are productive, cost effective and less toxic.[2−4]

The use of traditional medical practices, their validation and discovery of natural drugs could provide a new dimension for the treatment and control of leishmaniasis.[3,5,6] Different aerial and underground parts of plants are commonly used to isolate essential oils (EOs) in folk medicine to treat various types of diseases.[7] Recently antileishmanial properties have also been explored using EOs of Cymbopogon citratus (DC) Stapf (Poaceae),[8]Chenopodium ambrosioides L. (Chenopodiaceae),[9] and Vanillosmopsis arborea (Gardner) Baker (Asteraceae).[10]

Erigeron multiradiatus (Lindl.ex DC.) Benth. &Hook.f. (Asteraceae) is a natural inhabitant of the mountainous regions of India, Nepal, China, and Afghanistan. E. multiradaitus also known by its vernacular name “meiduoluomi” by traditional healers and native people, have been extensively used to cure hepatitis, meningitis, hemiparalysis, enteritis, diarrhea, adenolymphitis, rheumatism, and polyneuritis in traditional Tibetan medicine.[11,12] Further, there are several reports on Erigeron spp. viz. Erigeron canadensis having antibacterial properties,[13]Erigeron annuus with antimicrobial activity,[14]Erigeron floribundus with antitrypanosomal[15] and antiplasmodial activity,[16] and Erigeron breviscapus widely used in Cameroonian traditional medicine, reported with analgesic, immunomodulatory, trypanocidal, antidermatophytes, anti-inflammatory, antifungal, and antiplasmodial activity,[15,17−21] Moreover the role of Erigeron spp. have also been reported to inhibit platelet aggregation, improve microcirculation, dilation of blood vessel, increase cerebral blood flow,[22−24] treat dental pain, angina, headache, female infertility, and AIDS.[17,18,25−27] More importantly, E. multiradiatus having antidiabetic and anti-inflammatory activity,[11,28] but no study has been reported on its antileishmanial potential to support their traditional folk medicinal use. E. multiradiatus collected from various regions unlocked the existence of several bioactive constituents namely matricaria ester, α-pinene, lachnophyllum ester, myrcene, β-(E)-ocimene, isoledene, α-copaene, β-cubebene, p-menthatriene, caryophyllene oxide, α-cadinol, camphene, limonene, and β-eudesmol.[14,29,30]

In this study, we have reported the isolation of EO of E. multiradiatus, identification and purification of trans-2-cis-8-matricaria-ester and further evaluation of its cytotoxicity and antileishmanial potential against L. donovani. In addition, in silico screening of the components of EOs together with the miltefosine reference antileishmanial agent was done on four vital parasitic enzymes, namely, l-asparaginase-1-like protein, metacaspase-2, metacaspase-1, and DNA topoisomerase II in order to find out its probable mode of action.

Results and Discussion

EO Composition

The EO was isolated in a yield of 0.12% (v/w) from different aerial parts of E. multiradiatus and was inspected using gas chromatography with flame ionization detector (GC-FID) and gas chromatography–mass spectrometry (GC–MS) (Figure A). A sum of 12 components, corresponding to 97.81% of the total oil has been determined. The retention index of volatile compounds (RIa and RIb) and their percentage are summarized in Table . Among these, trans-2-cis-8 matricaria ester was identified as a major compound (77.79%) (Figure B) which was further confirmed by using mass, 1H NMR and 13C NMR spectral data (Table S1 and Figures S1−S3). The EO revealed the dominant existence of oxygenated monoterpenes (88.95%) followed by sesquiterpene hydrocarbons (5.61%) and oxygenated sesquiterpenes (3.05%). The monoterpene hydrocarbons accounted only for 0.20%. The other major components are cis-lachnophyllum ester (11.04%), zingiberene (4.43%), and spathulenol (1.59%) (Figure ). In an earlier study of E. multiradiatus, trans-2-cis-8 matricaria ester (50.70%) was found as a main constituent,[14] while the present study revealed higher amounts of trans-2-cis-8 matricaria ester (77.79%). The yield of EO and chemical patterns may be also be influenced by climatic (light and temperature) and geographic conditions, genetics, and the growth stage of the collected plants.[31,32]

Figure 1

Different spectra of the E. multiradiatus EO and its pure compound. (A) Gas chromatogram of the EO from the fresh aerial parts of E. multiradiatus. (B) Gas chromatogram of the purified compound trans-2-cis-8-matricaria ester.

Table 1

Chemical Composition of EO from E. multiradiatus

S. No.	compound	RIa	RIb	% composition	method of identification
1	β-(E)-ocimene	1045	1050	0.20	c
2	presilphiperfol-7-ene	1336	1336	0.15	c
3	methyl ergenol	1400	1403	0.12	c
4	α-trans-bergamotene	1430	1434	0.92	c
5	α-humulene	1451	1454	0.11	c
6	zingiberene	1489	1493	4.43	c
7	cis-lachnophyllum ester	1527		11.04	c
8	trans-2-cis-8-matricaria-ester	1545		77.79	c,d
9	spathulenol	1578	1578	1.59	c
10	humulene epoxide II	1606	1608	0.34	c
11	cubenol	1623	1628	0.25	c
12	α-cadinol	1636	1640	0.87	c
	total identified			97.81%
	monoterpene hydrocarbons			0.20%
	oxygenated monoterpenes			88.95%
	sesquiterpene hydrocarbons			5.61%
	oxygenated sesquiterpenes			3.05%
	total unidentified			2.19%
	oil yield (% v/w)			0.12

RI calculated relative to the homologous series of n-alkanes (C8–C34) on a Rtx-5 non-polar fused silica capillary column.

RI Adams.

MS, NIST, and WILEY libraries spectra and the literature.

(1H NMR, 13C NMR, COSY, HMBC and DEPT135) NMR spectra.

Figure 2

Structure of major components of EO from E. multiradiatus used in the molecular docking study.

In Vitro Antileishmanial Activity

None of the promising therapeutic treatments are available against fatal VL. Therefore, it is an urgent need to identify the promising antileishmanial candidate. In the search of new potent antileishmania agents, we have used the in vitro and in silico approach in this study extensively.

EO and trans-2-cis-8-Matricaria Ester Inhibit the Growth of L. donovani Promastigotes

The effect of EO and trans-2-cis-8-matricaria ester was evaluated against the promastigote form of L. donovani (Dd8) by MTT assay, which significantly reduced L. donovani viabilities with IC50 values < 20 μg/mL and <56 μM, respectively. Incubation of promastigotes with 30 μg/mL of EO and 100 μM of trans-2-cis-8-matricaria ester resulted in 80.95 ± 3.00 and 91.32 ± 8.48% inhibition, respectively. While at a concentration of 20 μg/mL or 50 μM the inhibition was 57.7 ± 6.08 and 48.3 ± 4.98%, respectively in promastigotes (Table ). The IC50 value of EO against promastigotes was 18.29 ± 2.1 μg/mL and that for trans-2-cis-8-matricaria ester was 55.09 ± 6.4 μM (Table ). The number of viable promastigotes was significantly reduced (P < 0.001) in all tested concentrations (5, 10, 15, 20, 25, 30 μg/mL) of EO as well as trans-2-cis-8-matricaria ester (5, 10, 25, 50, 75, 100 μM) in comparison to the untreated control (the coefficient of the variation range was 3–11%). Miltefosine as a positive control almost completely inhibited parasite proliferation (Figure A).

Table 2

Effect of EO and trans-2-cis-8-Matricaria Ester of E. multiradiatus on L. donovani Promastigotes and Intracellular Amastigotes

	activity against promastigote		activity against intracellular amastigote
test sample/drug	doses	% inhibition (mean ± SD)	doses	% inhibition (mean ± SD)
EO (μg/mL)	05	15.4 ± 1.86	05	12.4 ± 1.47
	10	24.8 ± 2.08	10	21.8 ± 2.12
	15	40.5 ± 5.00	15	35.5 ± 3.44
	20	57.7 ± 6.1	20	52.7 ± 6.7
	25	69.3 ± 7.4	25	61.3 ± 7.2
	30	80 ± 9.8	30	75 ± 8.3
trans-2-cis-8-matricaria ester (μM)	05	6.2 ± 0.86	05	4.8 ± 0.47
	10	11.6 ± 2.08	10	9.1 ± 1.12
	25	32.2 ± 3.00	25	23.9 ± 2.44
	50	48.3 ± 5.72	50	41.5 ± 4.89
	75	61.3 ± 7.9	75	58.9 ± 6.1
	100	91.7 ± 10.67	100	85.1 ± 8.9
miltefosine (μM)	3.6	92.79 ± 1.17	3.6	82.31 ± 3.19

Table 3

Antileishmanial Activity (IC50 ± sd) and Cytotoxicity Activity (CC50 ± sd) of EO and trans-2-cis-8-Matricaria Ester and the Positive Controla

test sample/drug	IC50 ± sd (promastigotes)	IC50 ± sd (intracellular amastigotes)	CC50 ± sd (hamster peritoneal macrophage)	SI-promastigotes (MQ/pro)	SI-amastigotes (MQ/ama)
EO (μg/mL)	18.29 ± 2.1	20.19 ± 2.4	285 ± 21	15.58	14.11
trans-2-cis-8-matricaria ester (μM)	55.09 ± 6.4	61.2 ± 7.9	609 ± 71	11.05	10
miltefosine (μM)	3.7 ± 0.4	4.4 ± 1	44.5 ± 7.9	12.02	10.1

Data (mean ± standard deviation) represents results of three independent experiments.

Figure 3

(A) In vitro efficacy of different concentrations of EO, EO w/o trans-2-cis-8-matricaria ester and purified trans-2-cis-8-matricaria ester of E. multiradiatus against L. donovani promastigotes (B) intracellular amastigotes and (C) the percentage of infected macrophages. The bar diagrams show the % viability of promastigotes or the number of amastigotes per 100 macrophages. The % viability of promastigotes was determined by MTT assay after 72 h exposure with varying concentrations of test compounds. Cell viability was expressed as the proportion of absorbance values normalized to the untreated control group after subtracting the blank absorbance from the sample and control. The percentage of intracellular amastigotes per 100 macrophages was determined microscopically after incubation with different concentrations of the test compounds or the positive control miltefosine (3.6 μM) for 72 h. There were three replicates in each experiment, and the data are the mean ± SD for each concentration. Significance values indicate the difference between the untreated groups and treated groups with various concentrations of EO, trans-2-cis-8-matricaria ester, or miltefosine (***, p < 0.001; **, p < 0.01; *, p < 0.05).

Investigation of Possible Cytotoxic Effects of EO and trans-2-cis-8-Matricaria Ester on Hamster Peritoneal Macrophage

Different concentrations of EO and the purified compound trans-2-cis-8-matricaria ester exhibited no cytotoxicity on hamster peritoneal macrophages; even the higher concentrations were safe without the sign of cell deformity. The CC50 value of EO against peritoneal macrophages was 285 ± 21 μg/mL and that for trans-2-cis-8-matricaria ester was 609 ± 71 μM (Table ). Further, the safety of the therapeutic agents was evaluated by the selectivity index (SI), which was expressed by the CC50/IC50 ratio. The SI value for EO and trans-2-cis-8-matricaria ester was higher than 10 (Table ), so the treatment was considered as safe for the cells (hamster peritoneal macrophages) at different therapeutic concentrations.

EO and trans-2-cis-8-Matricaria Ester Inhibit the Growth of L. donovani Intracellular Amastigote Forms

To check the activity of EO and trans-2-cis-8-matricaria ester on intracellular amastigotes, hamster peritoneal macrophages were infected with L. donovani and allowed to grow at different concentrations of EO and trans-2-cis-8-matricaria ester. The number of amastigotes/100 macrophages was counted microscopically, and the results were expressed as a percentage inhibition as compared to the control (Figure B,C). Interestingly all the tested concentrations of EO (5, 10, 15, 20, 25, 30 μg/mL) and trans-2-cis-8-matricaria ester (5, 10, 25, 50, 75, 100 μM) were found to inhibit amastigote growth and resulted in 12.40 ± 1.47 to 75.00 ± 8.30% and 4.8 ± 0.47 to 85.10 ± 8.90% inhibition, respectively (p < 0.001), while the positive control (3.6 μM) lowered the parasite load in the infected macrophages by 82.31% (p < 0.001) compared to the untreated controls (Table ). The IC50 value of EO against intracellular amastigotes was 20.19 ± 2.4 μg/mL and for trans-2-cis-8-matricaria ester was 61.2 ± 7.9 μM (Table ). Different concentrations of the EO and trans-2-cis-8-matricaria ester were nontoxic to the hamster peritoneal macrophages and effective on intracellular amastigotes which indicated the selectivity of EO and trans-2-cis-8-matricaria ester against amastigotes as compared to mammalian cells, as analyzed by qualitative microscopic examination. Thus, the outcome signifies the selectivity of EO and its major component trans-2-cis-8-matricaria ester toward amastigotes as compared to the host cells.

As it is evident from the results, EO and its component trans-2-cis-8-matricaria ester provided a hindrance against both the promastigote and intracellular amastigote forms of L. donovani. This plant has antileishmanial potential which is almost similar to the standard drugs and possesses several medicinal properties, and because of easy oral administration, it could provide a good lead over currently available antileishmanial drugs and has to be investigated in more detail during different stages of drug development.

In Silico Study

At present, in silico practices have wide application in the development and analysis of pharmacological hypothesis. These methods are prominently involved in identification and optimization of novel molecules, and having a great understanding on a target thus reduces the time to clarify absorption, distribution, metabolism, excretion, and toxicity properties as well as physicochemical characterization.

So in this study, to identify the probable targets and to understand the probable mode of action of EO and trans-2-cis-8-matricaria ester, molecular docking was carried out.

Molecular Docking Interaction Analysis Divulges the Highest Binding Affinity of trans-2-cis-8-Matricaria Ester against the Selected Leishmania Target Proteins

The oil consisted mainly of oxygenated monoterpenes and trans-2-cis-8-matricaria-ester was a major constituent identified. Further, in order to identify the free energy accountable for complex generation and to identify the molecular interactions responsable for this target-specific inhibition, we have carried out molecular docking analysis for 4 major components of EO against 10 major proteins of the parasite. Ligprep yields stable test ligand structures by altering the ring conformations of the Lewis structures, and providing stable protonation conditions. Two indispensable terms, a 3D structure of the protein and a ligand stock are entails in molecular docking analysis. A 3D structure of l-asparaginase-1-like protein (CBZ32861.1), metacaspase-2 (ABD19718.1), metacaspase-1 (ABD19717.1), and DNA topoisomerase II (AAD34021.1) of L. donovani was generated with the help of homology modeling. The 3D structure generated by homology modeling needs to be revised as it might be deficient of necessary hydrogen and have excess water. Hence, hydrogen atoms are additionally incorporated into the structure, so that a correct ionization and precise tautomeric form is maintained. Once the protein structure is modeled, validation is carried out by Ramachandran plot generation. The Ramachandran plot for 4 proteins (metacaspase-2, metacaspase-1, l-asparaginase-1-like protein, and DNA topoisomerse II) performing best result is included in Figure . SiteMap gives knowledge about the binding sites in the receptor protein. The site score for all 10 protein binding sites for each protein should be greater than 1 for druggability. While for differentiating ligand binding and nonbinding sites, the borderline site score is 0.80. In order to predict the effect of test compounds on different leishmanial proteins, we performed molecular docking interaction analysis (Table ). The docking score corresponds to its free energy involved in “binding”. The entire attribute and genuineness of the model were found appropriate for the study. Molecular docking study revealed that trans-2-cis-8-matricaria ester was the only ligand that performed phenomenal binding affinity against four different protein molecules (Figure ). Figure A–D represents interaction of ligands that manifest various interacting amino acid residues at the ligand-binding site of a protein. In Figure A, hydrogen bonding was clearly observed between the ligand and the ASP 117 and ARG 195 residue of l-asparaginase-1-like protein. Similarly hydrogen bonding was seen between the ligand and other amino acid residues of the proteins of L. donovani (Figure B–D). The higher the hydrogen bond, the stronger will be the interaction between the ligand and receptor. Other than trans-2-cis-8-matricaria ester, no other ligand showed activity and interaction with any proteins. The binding energy score of trans-2-cis-8-matricaria ester was −4.803, −4.152, −4.083, and −3.741 kcal/mol for L. donovanil-asparaginase-1-like protein, metacaspase 2, metacaspase 1, and DNA topoisomerse II, respectively, which was significantly higher than the positive control miltefosine with a docking score of −3.835, −3.821, −3.495, and −3.158 for l-asparaginase-1-like protein, metacaspase 2, metacaspase 1, and DNA topoisomerse II, respectively. Thus the results manifest that trans-2-cis-8-matricaria ester showed the highest binding affinity for l-asparaginase-1-like protein while the binding energy of metacaspase-2, metacaspase-1, and DNA topoisomerse II were stronger than other proteins. Metacaspase is essential for chromosomal separation and survival of the parasite.[33] DNA topoisomerases are the key enzymes that facilitate high precision DNA transactions inside the parasite,[34] and l-asparginases are associated with the survival of the parasite.[35] The major EO component, trans-2-cis-8-matricaria ester represents a good inhibitory effect on the four major essential parasite proteins with nontoxic properties. So these enzymes could be used as a potential drug target against the pathogen Leishmania.

Figure 4

Homology model validation by using the Ramachandran plot. The Ramachandran plot of the l-asparaginase-1-like protein (A), metacaspase-2 (B), metacaspase-1 (C), and DNA topoisomerse II (D) modeled structure was quantitatively analyzed and the values resulted were 92.2% most favoured core regions, 6.8% allowed or favourable regions and only 1% is present generously allowed and disallowed regions.

Table 4

Molecular Docking Interaction Summary of the Studya

title	docking score (kcal/mol)	glide gscore	glide emodel
l-Asparaginase-1-like Protein
miltefosine (control)	–3.835	–3.835	–27.92
trans-2-cis-8-matricaria-ester	–4.803	–4.803	–30.71
zingiberene	Ni	Ni	Ni
cis-lachnophyllum ester	Ni	Ni	Ni
Metacaspase 2
miltefosine (control)	–3.821	–3.821	–27.149
trans-2-cis-8-matricaria-ester	–4.152	–4.152	–30.857
zingiberene	Ni	Ni	Ni
cis-lachnophyllum ester	Ni	Ni	Ni
Metacaspase 1
miltefosine (control)	–3.495	–3.495	–17.192
trans-2-cis-8-matricaria-ester	–4.083	–4.083	–26.797
zingiberene	Ni	Ni	Ni
cis-lachnophyllum ester	Ni	Ni	Ni
DNA Topoisomerse II
miltefosine (control)	–3.158	–3.158	–20.192
trans-2-cis-8-matricaria-ester	–3.741	–3.741	–23.615
zingiberene	Ni	Ni	Ni
cis-lachnophyllum ester	Ni	Ni	Ni

Ni represents no interaction.

Figure 5

Molecular docking interaction between the test ligand trans-2-cis-8-matricaria ester and various target proteins, that is, l-asparaginase-1-like protein (A), metacaspase-2 (B), metacaspase-1 (C) and DNA topoisomerase II (D).

ADME Profile of the Test Ligand

Pharmacokinetic comparative analysis of the test ligand depicts the fitness of the ligand as a drug as compared to the standard drug miltefosine (Table ). Most of the absorption parameters suggest that the solubility and permeability of the ligand be in accordance with the control’s value. Distribution parameters include renal organic cation transporters, P-glycoprotein and plasma protein binding (log PPB). Metabolism data included the substrate and inhibition analysis of various drug metabolism enzymes belonging to the CYP450 enzyme superfamily. Excretion parameters include Madin-Darby canine kidney (MDCK) cell lines on in silico levels.[36]

Table 5

ADME Profile of the Test Ligand (trans-2-cis-8-Matricaria Ester) and Control (Miltefosine)

parameter	test ligand	control
Absorption
log S	–2.15	–2.29
human oral absorption	92.65	100
human intestinal absorption	93.51	97.28
log BBB	0.420	0.629
rate of membrane permeability	31.91	85.73
Caco-2 permeability	325.29	183.69
Distribution
renal organic cation transporter	non-inhibitor	non-inhibitor
P-glycoprotein substrate	non-substrate	non-substrate
P-glycoprotein inhibitor	non-inhibitor	non-inhibitor
log PPB	2.0	0.9
Metabolism
CYP450 2C9 substrate	non-substrate	non-substrate
CYP450 2D6 substrate	non-substrate	non-substrate
CYP450 3A4 substrate	substrate	non-substrate
CYP450 1A2 inhibitor	non-inhibitor	non-inhibitor
CYP450 2C9 inhibitor	non-inhibitor	non-inhibitor
CYP450 2D6 inhibitor	non-inhibitor	non-inhibitor
CYP450 2C19 inhibitor	inhibitor	non-inhibitor
CYP450 3A4 inhibitor	non-inhibitor	non-inhibitor
CYP inhibitory promiscuity	low CYP inhibitory promiscuity	low CYP inhibitory promiscuity
Excretion
MDCK	652.82	1282.43

Conclusions

In summary, we have identified the chemical components of EO of E. multiradiatus and evaluated its antileishmanial potential against L. donovani. The results show that the EO of E. multiradiatus exhibits leishmanicidal activity in vitro against L. donovani and that this activity is related to the presence of its major compound trans-2-cis-8-matricaria ester. This major compound, trans-2-cis-8-matricaria ester, manifests more than 77% of the composition of EO, and when tested separately, it also exhibited similar or more leishmanicidal activity. Further, in silico study revealed that trans-2-cis-8-matricaria ester may be an effective inhibitor of the four pathogenic proteins of Leishmania with nontoxic properties that could be further employed for in vivo examination and may enhance the pace of herbal drug development, which may be a better option for alternate chemotherapy against VL.

Experimental Section

Plant Materials

The plant material was collected in the month of September 2017 (flowering stage) from the Chiplakedar forest (Pithoragarh District), Uttarakhand, India, at an altitude of 3000 m with geographical coordinates 29° 96′ N latitudes and 80° 43′ E longitudes. A voucher (specimen no. 116031) has been deposited at the Herbarium of Botanical Survey of India, Dehradun, India; and the Department of Chemistry, Kumaun University, Almora, India.

Extractions of the EO

Fresh aerial parts (∼4 kg) were exposed to steam distillation using a copper still distiller (Scientech, India). The distillate of fresh plant material was treated with n-hexane and dichloromethane for thorough extraction of organic components. The dichloromethane and n-hexane extracts were mixed and dried over anhydrous Na2SO4. In order to obtain residual oil, solvent distillation was performed in a rotary vacuum evaporator (Perfit-RV 1240, Buchi type, India). Further using anhydrous sodium sulphate the oil was allowed to dry, filtered and stored at 4 °C until its chemical and pharmacological test analysis.

GC-FID and GC–MS Analysis

A gas chromatographic analysis of EO was performed on a Shimadzu GC-2010 Ultra gas chromatograph, Kyoto, Japan, equipped with a flame ionization detector and an Rtx-5MS fused silica capillary column. The temperature of the injector and detector were maintained at 260 and 270 °C, respectively. Helium at a flow rate of 1.21 mL/min and 69.0 kPa inlet pressure was employed as the carrier gas. The sample (1.0 μL) was injected with 10:1 spilt ratio.

GC–MS was carried out on Shimadzu GC-MS-QP2010 Ultra, Kyoto, Japan, using identical oven temperature. The MS was used under the electron impact conditions (70 eV), ion 230 °C, mass scan mode: 2.41 scan/s, mass range: 40–650 m/z; a 5% solution of oil in hexane (1.0 μL) was injected. Individual compounds were identified by calculating retention indices (RI) using homologous C8–C34 (Supplier—Restek’s ISO 9001:2008) n-alkane series, compared with available mass spectral data (NIST 11, Wiley 8 and FFNSC 2) and finally confirmed by comparing their RI with the available literature.[37]

Isolation and Characterization of trans-2-cis-8-Matricaria Ester

The EO (2 mL) of E. multiradiatus was subjected to a silica gel column chromatograph (230–400 mesh, Merck, 20 g) with hexane/diethyl ether (99:1–85:15) as the eluent and twenty fractions were collected and screened by TLC and GC to produce the compound (80 mg, >96% purity). The compound was identified by using mass, 1H NMR and 13C NMR spectral data.[29]

Parasite and Media

Promastigotes of L. donovani (Dd8) were cultured in RPMI-1640 medium (Himedia, India) containing 10% fetal bovine serum (Cell clone, Genetix, India) along with 1% antibiotic and antimycotic solution (Cell clone, Genetix, India) at 26 °C.

Animals

Laboratory inbred female golden hamsters (Mesocricetus auratus, 45–50 g) were purchased from the Central Drug Research Institute (CDRI-CSIR), Lucknow, India and were used for experimental purposes. All animals and experiments were performed in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) for the care and use of laboratory animals and the regulations of Kumaun University, Nainital, India. The use of animals was approved by the institutional animal care and ethics committee, and the protocol number is KUDOPS/109. They were kept in a climatically controlled animal house and fed with a standard rodent food pellet (Lipton India) and water ad libitum.

In Vitro Activity

Evaluation of Inhibition of L. donovani Promastigotes Growth

To determine the efficacy of the drug log phase, L. donovani promastigotes were used.[38] Briefly, 105 parasites/well were plated on 96-well cell culture plates along with different concentrations of EO and trans-2-cis-8-matricaria ester and incubated for 72 h at 26 °C. Then 20 μL of MTT (Himedia, India) stock solution (5 mg/mL) was mixed into each well and further incubated for 4 hours then allowed to centrifuge for 10 min at 1000g; the supernatant was discarded and resuspended with 100 μL (0.5%) of DMSO (Cell clone, Genetix, India) into each well. Using a microplate reader (Bio-Rad, India) the OD was measured at 540 nm. Further percentage of inhibition was calculated by comparing the % viability with the untreated control. In order to verify the results, tests were performed in triplicate. Miltefosine was used as a standard control.

Cytotoxicity of EO and trans-2-cis-8-Matricaria Ester in Hamster Peritoneal Macrophages

The cytotoxicity of EO and trans-2-cis-8-matricaria ester was evaluated on hamster peritoneal macrophages. Two hamsters were treated with thioglycollate in the peritoneal cavity to allow inflammatory response to proceed for 4 days and then euthanized. Hamster peritoneal macrophages were resuspended at 105 cells/mL in RPMI medium plated in 16-well chamber slides (Nalge Nunc, USA) and incubated for 8 days for differentiation into macrophages in a humidified 5% CO2 air atmosphere at 37 °C. Further, the differentiated hamster peritoneal macrophages were incubated with various concentrations of EO and trans-2-cis-8-matricaria ester for 72 h. The cytotoxicity of compounds was examined using MTT assay, and cell morphology and integrity were evaluated under a microscope after Giemsa staining (Himedia, India).[39] To calculate the cytotoxic concentration (CC50), tests were conducted in triplicate. SI, which is the ratio of CC50/IC50, manifests the balance between cytotoxicity and antileishmanial activity. SI value > 10 is considered to be safe for the cells at various concentrations.

Evaluation of the Inhibitory Effect on Intracellular Amastigote Growth

Hamster peritoneal macrophages were seeded and infected with stationary-phase Leishmania promastigotes (at a ratio of 1:10 macrophages to parasites). The infected macrophages were subjected to grow for 72 h along with various concentrations of EO and trans-2-cis-8-matricaria ester. Sensitivity was checked microscopically after Giemsa staining by calculating the burden of amastigotes per 100 macrophages. Miltefosine was used as the standard positive control. Percentage of inhibition was calculated as described earlier.[40]

Target Selection

The amino acid sequences of the target proteins of L. donovani cytoplasmic l-asparaginase-1-like protein (CBZ32861.1), L. donovani metacaspase-2 (ABD19718.1), L. donovani metacaspase-1 (ABD19717.1), and L. donovani DNA topoisomerase II (AAD34021.1) were retrieved using the database of NCBI (www.ncbi.nlm.nih.gov/). For interaction analysis of the selected target proteins and ligands, Schrodinger’s software is used (Schrödinger Release: Maestro, version 10.5, Schrödinger, LLC, NY 2016-1, USA). This master software consists of more than one suite that is capable of performing visualization of the structure, binding site prediction, and receptor–ligand interaction.

Ligand Selection and Preparation

The ligands used in this study were selected from chemical components of E. multiradiatus EO (Figure ). The receptors used are significant proteins, playing a major role in the infection cycle of L. donovani, hence can be used as attractive targets for targeting leishmaniasis. For ligand preparation, Ligprep application (v3.7, Schrödinger Release NY 2016-1, USA) was used for rectifying and stabilizing the structure of the ligand.[41]

Protein Structure Generation

Structure of proteins involved in the study was either obtained from RCSB (protein databank) or generated using models. In the absence of 3D structures online, we employed the homology modeling approach using PRIME for modeling of 10 proteins. Hence, the amino acid sequences (FASTA) were retrieved from UniProt and are reported here (sequence 1). The template for sequences was generated using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the template selected were further modeled to a 3D structure by Prime (version 7.0, Schrödinger Release NY 2016-1, USA).[42]

Protein Structure Validation

Determining the accuracy of the homology modeled 3D structure requires mandatory validation. This step is called preparation of protein that primarily includes minimization of energy, removal of water, and addition of hydrogen bonds. It is facilitated by Protein Preparation Wizard (version 7.0, Schrödinger Release NY 2016-1, USA). Validation of proteins 3D resulted in a refined model structure preceded by generating the binding sites on the target for the ligand to attach. The binding site for our target proteins were predicted using SiteMap (version 3.8, Schrödinger release NY 2016-1 USA).

Molecular Docking

A docking study was carried out to fit ligands and proteins into the predicted binding site of all the protein targets selected for the study. To evaluate the interaction and binding affinity between the ligand and receptor the Glide package was used (version 7.0, Schrödinger Release NY 2016-1, USA). The size of the docking area was predefined by receptor grid generation as per the presence of the binding site in respective protein structures.[43]

ADME Studies

The pharmacokinetic profile of the test ligand was analyzed in terms of its absorption, distribution, metabolism, and excretion. This ADME analysis was carried out by Qikprop (an application from Schrodinger suite 2016).[44]

Statistical Analysis

All experiments were performed in triplicates and the results were manifested as mean ± SD. Analysis of the results (pooled data of the three experiments) were performed by one-way ANOVA followed by Dunnett’s post-test. All of the analyses were done using GraphPad Prism (version 3.03) software.