Phytochemical study of Seriphidium khorassanicum (syn. Artemisia khorassanica) aerial parts: sesquiterpene lactones with anti-protozoal activity

Abstract Two new eudesmane-type sesquiterpene lactones, 1β,3α,8α-trihydroxy-11β,13-dihydroeudesma-4(15)-en-12,6α-olide (1) and 1β,4α,8α-trihydroxy-11β,13-dihydroeudesma-12,6α-olide (2), and an unprecedented elemane-type sesquiterpene lactone, 1β,2β,8α-trihydroxy-11β,13-dihydroelema-12,6α-olide (3) along with a known eudesmanolide artapshin (4) were isolated from Seriphidium khorassanicum. Structures were elucidated by NMR, HR-ESI-MS, and ECD spectral data analysis. The anti-protozoal activity was evaluated against Leishmania major promastigotes and amastigote-infected macrophages. They showed dose- and time-dependent activity against L. major amastigotes with IC50 values in the range of 4.9 to 25.3 μM being favourably far below their toxicity against normal murine macrophages with CC50 values ranging from 432.5 to 620.7 μM after 48 h of treatment. Compound 3 exhibited the strongest activity and the highest selectivity index (SI) with IC50 of 4.9 ± 0.6 μM and SI of 88.2 comparable with the standard drug, meglumine antimoniate (Glucantime), with IC50 and SI values of 15.5 ± 2.1 μM and 40.0, respectively. Graphical Abstract

Compound    (Rustaiyan et al. 1979;Herz 1977).Thermal conversion in the lab is true in simple cases, including elemadienes and elemadienolides.But, in the cases in which double bonds of the elemane system are further modified as in oxidatively modified systems like vernolepin from Vernonia hymenolepis (Asteraceae) or micordilin from Mikania cordifolia (Asteraceae) in which the C1 ¼ C2 and C3 ¼ C4 double bonds have undergone oxidation reactions after the Cope rearrangement; such elemanolide compounds are not artefacts and have only been naturally extracted (Kupchan et al. 1968;Herz et al. 1977;Herz 1977).There are other elemanolides reported in the Asteraceae family, including onopordopicrin in Onopordon leptolepis, hierapolitanins A and B from Centaurea hierapolitana, shonachalin D from Artemisia fragrans, and epivernadolol from Vernonia lasiopus (Rustaiyan et al. 1979;Karamenderes et al. 2007;Serkerov and Aleskerova 1987;Koul et al. 2003).
Compound 4 was elucidated as artapshin based on the NMR and mass spectral data analysis as well as on comparison with the reported spectral data (Serkerov and Aleskerova 1983) (Figure S.32,S.33).

Anti-promastigote leishmanial activity
Compounds 1-4 were tested against L. major promastigotes for 24 h and 48 h of treatment.They showed a weak dose-and time-dependent anti-proliferative activity with IC 50 >200 lM.Amphotericin B as the standard drug suppressed L. major promastigotes with IC 50 values of 7.9 ± 0.42, and 1.7 ± 0.69 lM, after 24 and 48 h, respectively (Figure 2).

Anti-amastigote leishmanial activity
Although the isolated compounds exhibited weak inhibition on the growth rate of promastigotes, treatment of amastigote-infected macrophages with the isolated compounds 1-4 demonstrated a remarkable dose-and time-dependent activity against L. major amastigotes with IC 50 values in the range of 4.9 to 25.3 lM being favourably far below their toxicity against normal murine macrophages with CC 50 values ranged from 432 to 620.7 lM after 48 h (Figure 2, Table 1).Among all, after 48 h, compound 3 exhibited the strongest activity and the highest selectivity index (SI) with IC 50 of 4.9 ± 0.6 lM and SI of 88.2 is comparable to those of the standard drug, meglumine antimonite, with IC 50 and SI values of 15.5 ± 2.1 lM and 40.0, respectively.The IC 50 values against two forms of L. major after 24 and 48 h of treatment as well as the CC 50 values against J774 murine macrophages and selectivity indices, are given in Table 1.

Plant material
Seriphidium khorassanicum (Podlech) K.Bremer & Humphries plant sample was collected from Bojnord, North Khorasan province, northeast of Iran (Latitude: 36 38'26" N; Longitude: 59 52'32" E).The aerial parts of the plant were harvested in mid-September 2016 at the early stages of the flowering season and authenticated by Valiolah Mozafarian at the Department of Botany, Research Institute of Forests and Rangelands, Tehran, Iran.A voucher specimen (SAM-4006 of the Herbarium) has been deposited at the School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.

ECD calculations
Conformers in an energy window of 2 Kcal/mol distance from global minima were optimized using the DFT/B3LYP/3-21G ÃÃ level of theory in MeOH.Theoretical calculation of ECD spectra of conformers was performed by the time-dependent density function theory (TDDFT) method at B3LYP/3-21G ÃÃ in MeOH using the SCRF (selfconsistent reaction field) method.All calculations were carried out using the Gaussian 09 software package (Ghanadian et al. 2020).

Anti-promastigote leishmanial activity
Briefly, L. major (MRHO/IR/75/ER) were cultured first in NNN medium and then in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), Penicillin (100 IU/mL), and Streptomycin (100 mg/mL) at 25 C ± 1 C to produce a mass of the parasite.Then the promastigotes were seeded at 7.5 Â 10 5 per well in two 96-well microplates and 100 lL of various concentrations (1, 10, 50, 100, and 200 mM) of isolated compounds were added to each well and incubated at 25 ± 1 C to treat the promastigotes for 24 and 48 h.The wells with promastigotes in medium with no drug were considered as the negative control and those with Amphotericin B were supposed as the positive control.For each treatment, the cell density was determined after 24 and 48 h of incubation in a hemocytometer (Improved Double Neubauer) using an optical microscope (Varshosaz et al. 2018).

Anti-amastigote leishmanial activity
In order to evaluate the effect of the isolated compounds on intracellular amastigotes, the J774 mouse macrophage cell line was first grown in culture flasks containing RPMI-1640 medium supplemented with 20% FBS, penicillin (100 IU/mL), and streptomycin (100 lg/mL) being incubated at 37 in 5% CO 2 atmosphere.Cells were passaged when they reached about 80% confluence.Subsequently, the macrophages were seeded at the density of 25 Â 10 4 cells/well into 6-well plates in which 24 Â 24 mm sterile coverslips were placed on the bottom of each well and the incubation was repeated at 37 C and 5% CO 2 overnight.
The adherent macrophages infected with methacrylic L. major promastigotes (parasite/macrophage ratio 8:1; logarithmic growth phase) were further incubated for 6 h in the same conditions for phagocytosis of promastigotes.Next, free promastigotes were removed by extensive washing with 0.01 M phosphate-buffered saline (PBS; pH 7.2).Fresh enriched RPMI was then added and the plates were re-incubated for 24 h in order to form amastigotes into the macrophages.After that, the infected macrophages were treated with various concentrations (1, 10, 50, 100, and 200 mM) of isolated compounds and meglumine antimoniate (glucantime) as the positive control, while some wells were maintained untreated as negative controls.The cells were incubated for 24 and 48 h, at 37 C in 5% CO 2, and after that, the coverslips were washed with 0.01 M PBS and then fixated with MeOH and stained using Giemsa staining protocol.The stained coverslips were examined using oil-immersion light microscopy and the number of amastigotes inside the macrophages was counted (100 macrophages per coverslip).These tests were performed in duplicate (Varshosaz et al. 2018).

Statistical analysis
Statistical analysis was performed by non-parametric one-way analysis of variance (ANOVA) using the software GraphPad Prism by Dunnett's post-hoc test.Each experiment was carried out in triplicate.P-value < 0.05 was considered a statistically significant difference.All data were expressed as means ± SD.

Conclusion
Leishmaniasis is a series of vector-borne diseases, the causative parasite of which transmits by almost ninety sandfly species threatening hundreds of millions of individuals worldwide by either visceral or cutaneous leishmaniasis types.The risk of drug resistance and serious side effects of currently available drugs in the absence of a vaccine and adequate therapy has raised an urgent need for new potential lead compounds.Cutaneous leishmaniasis is the most prevalent form of the disease for which Leishmania major is one of the protozoan parasites responsible.Compounds isolated from Seriphidium khorassanicum (1-4) significantly hinder the growth of intra-macrophage amastigotes of L. major with IC 50 and SI values being considerably comparable to those of the control drug; therefore, they may be considered as promising lead compounds with effective anti-leishmanial activity.

Disclosure statement
We declare no competing interests.
NATURAL PRODUCT RESEARCH

Compound 1
was deduced to be C 15 H 22 O 5 based on its [M þ Na] þ sodium adduct ion peak in the HR-ESI-MS at m/z 305.1357 (calc.for C 15 H 22 O 5 Na þ m/z 305.1359,D À0.66 ppm) (Figure S.10).The IR spectrum displayed characteristic bands at max 1766 and 3437 cm À1 for carbonyl and hydroxyl functions, respectively. 1H-and 13 C-NMR data showed resonances for two methyls (Figure S.1, S.2,

Table 1 .
Anti-protozoal activity of compounds 1-4 against Leishmania major and cytotoxicity on normal J774 murine macrophages.

Table S .
1) resembled compound 2 except for changes in ring A. The 1 H-1 H COSY correlations (Figure S.27) confirmed the spin system H-5-H-6-H-7(H-11-H-13)-H-8-H 2 -9.The HMBC correlations(Figure S.26) of H-2 with C-1, C-10, and C-4, along with the deshielding downfield resonance of C-2 (d C 97.62) affirmed the presence of a hemiacetal methine (C-2) connected to C-4 through an ether linkage.The combination of 1 H-1 H COSY, HSQC, and HMBC demonstrated the constitution of ring A as a six-membered heterocyclic ring containing an oxygen atom.Finally, the relative configuration of 3 was determined through the NOESY experiment.
Considering b-orientation of Me-14 and a-orientation of H-5, the NOESY correlations of H 3 -14/H-6, H-8; H-6/H-8, H-11, H 3 -15; H 3 -15/2-OH indicate that 2-OH, H-6, H-8, H-11, and H 3 -15 are all co-facially b-oriented(Figure S.28, S.29).The small coupling constant of two hydrogen nuclei, H-2a, and broad singlet H-1, supported their syn conformation and ultimately determined the a-orientation of H-1.Altogether, the structure of 3 was elucidated as a novel elemane-type sesquiterpene and was named 1b,2b,8a-trihydroxy-11b,13-dihydroelema-12,6a-olide.The biogenesis pathway of compound 3 is depicted in Figure S.31.The formation of elemanolides involves a Cope rearrangement in related germacranolides.It could occur in the laboratory at a temperature above 100 C or by an enzyme-mediated process in plants that is not yet fully recognized