Two new eudesmane-type sesquiterpene derivatives from Lecokia cretica (Lam.) DC

Abstract Two new sesquiterpene glucosides, 1α,6β,9β-trihydroxy-eudesm-4(15)-en-1,6-O-β-diglucopyranoside (1) and 1α,6β,9β-trihydroxy-eudesm-3-en-1,6-O-β-diglucopyranoside (2) were obtained along with the 1α,6β,9β-trihydroxy-5,10-bis-epi-eudesm-3-en-6-O-β-D-glucopyranoside (3), chlorogenic acid (4), luteolin 7-O-rutinoside (5) and luteolin 7-O- glucoside (6) from the whole plant parts of Lecokia cretica. Their structures were determined on the basis of 1 D, 2 D NMR and HRMS analyses. The in vitro cytotoxic activity of compounds 1-3 against human lung cancer cells (A549) and normal human lung cells (BEAS-2B) was determined using the MTT colorimetric assay. All the tested eudesmane derivatives were found to be inactive. Graphical Abstract


Introduction
Lecokia DC. is a monotypic genus of Apiaceae family, represented by a single species: Lecokia cretica (Lam.)DC. worldwide.It is native to Cyprus, Syria, the southern parts of T€ urkiye, and northern Iran (Stevens 1972;Menemen 2012).'Ayı baldıranı' and 'es ¸ek baldıranı' are the local Turkish names of L. cretica (Baytop 1994;Tuzlacı 2006).The seeds are crushed and used internally for antihypertensive purposes in Turkish folk medicine (Demirci and € Ozhatay 2012).The phytochemical and pharmacological evaluation of the genus is limited due to its monotypic character.Aras et al. (2019) investigated the antidiabetic, antioxidant, and anti-Alzheimer potential of L. cretica together with characterizing of some phenolic compounds via HPLC/MS/MS.
The objective of the present study is to isolate and characterize the constituents of sesquiterpene derivatives from whole parts of L. cretica, and evaluate their cytotoxic activities with regard to the previous data (Cheng et al. 2013;Nie et al. 2014;Zhao et al. 2014;Zhang et al. 2015;Yu et al. 2016;Ebada et al. 2019) on the cytotoxic activities of similar eudesmane-type sesquiterpene compounds.
Compound 1 was obtained as colorless gum, with an molecular formula of C 27 H 46 O 13 determined as its sodium adduct peak as a positive charge at m/z 601.2817 by HRESIMS corresponding to the molecular formula C 27 H 46 O 13 Na þ (calcd.for C 27 H 46 O 13 Na þ 601.2830).
In the 13 C NMR spectrum 27 carbon signals were observed, consisting of three methyls, six methylenes, sixteen methines, and two quaternary carbons.In the 1 H NMR spectrum of 1, two anomeric proton signals were found at d H 4.46 (1H, d, J ¼ 7.9 Hz) and 4.36 (1H, d, J ¼ 7.6 Hz), while in the 13 C NMR spectrum two anomeric The relative configuration of aglycone of 1 was further determined according to the NOESY.The absences of any NOESY correlation between H-5 and H 3 -14 supported a trans ring A/B junction.In the NOESY spectrum, the cross peaks between H 3 -14 and H-7, H-7 and H-6, H 3 -14 and H-9 indicated that H 3 -14, H-6, H-7 and H-9 appeared at the same side.In addition, the presence of crossed peaks between H-5 and H-1 indicates that they are on the same side.Besides these crossover peaks between H-7 and H-6, H 3 -12 and H-6 indicated that the isopropyl group at C-7 is b -oriented.The b configuration of the glucose was incited by the large coupling constant of the anomeric protons d H 4.46 (1H, d, J ¼ 7.9 Hz) and 4.36 (1H, d, J ¼ 7.6 Hz).Based on the above evidence, compound 1 could be identified as 1a,6b,9b- trihydroxy-eudesm-4(15)-en-1,6-O-b-diglucopyranoside. 1 D ( 1 H, 13 C), 2 D (COSY, HSQC, HMBC) NMR and HRMS spectra of compounds 1 were given in Figures S1-S7 (See supplementary material).
Compound 2 was obtained as colorless gum.A sodium adduct peak as a positive charge at corresponding to the molecular formula C 27 H 46 O 13 Na þ (calcd.for C 27 H 46 O 13 Na þ 601.2830).
The isolated two new and one known eudesmane-type sesquiterpene derivatives (1-3) were investigated for the cytotoxic activity against A549 cancer cells as well as BEAS-2B cell line.Inhibition effects of the compounds on cell viability were given in Table 1.A549 cells treated with compound 3 showed a statistical decline (82.7%) in cell viability at the highest dose (p < 0.0001).The IC 50 values of doxorubicin, positive control, were determined as 1.27 ± 0.23 mM, and 0.93 ± 0.12 mM on A549, and BEAS-2B, respectively.Some eudesmane-type sesquiterpenes were reported to have cytotoxic activity against different cancer cell lines (Cheng et al. 2013;Nie et al. 2014;Zhao et al. 2014;Zhang et al. 2015;Yu et al. 2016;Ebada et al. 2019).However, several studies showed that eudesmane-type sesquiterpene derivatives from different plants possess no cytotoxic effects on human cancer cell lines (Xu et al. 2010;Wang et al. 2013;Li et al. 2017).Similarly, in the current study, the eudesmane sesquiterpenes isolated from L. cretica did not exhibit significant cytotoxic activity.

General
The stationary phase for the open column chromatography was Kieselgel 60 (Merck, 0.063-0.200mm).Sephadex LH-20 was used for the general permeation chromatography (General Electric Healthcare).The flash chromatography was performed using LiChroprep C18 (Merck, 40-63 mm) and pre-packaged Flash cartridges (Sepacore, Silica 12 g, 25 g).TLC analysis was performed using Merck aluminum silica gel (60 F254) sheets that were either examined under a UV lamp or sprayed with 30% H 2 SO 4 in EtOH and then heated on a hot plate.The 1 H and 13 C NMR spectra were acquired at 25 C using TMS as the internal standard in deuterium reagent on a Bruker AM 400 MHz.Chemical shifts are expressed in parts per million.The abbreviations s (singlet), d (doublet), t (triplet), and m (multiplet) are used to denote peak patterns.The purified chemicals' mass spectra were acquired using a mass spectrometer detector (Thermo ORBITRAP Q-EXACTIVE mass spectrometry).

Plant material
Lecokia cretica (Lam.)DC. was collected from Kuruca ( Gezik)Village of Bing€ ol, T€ urkiye in April 2020.The plant was identified by Prof. Dr. Rıdvan Polat from Bing€ ol University, Faculty of Agriculture, Department of Landscape Architecture.Plant material was collected from oak fields at an altitude of 1500-1800 m.The voucher specimen was deposited in the Faculty of Agriculture with the number RP-1823.

Preparation of extracts
The air-dried whole plant parts of L. cretica (312.8 g) were powdered and macerated with methanol at room temperature for 24 h.This extraction process was repeated with the residue five times (5 Â 5 L).Following filtration, the combined methanol extracts were evaporated to dryness under pressure at 40 C. Then the MeOH extract The experimental groups were compared with the control group ( ÃÃÃÃ : p<0.0001);SEM: standard error of the mean.

Cytotoxic activity investigation
3.5.1.Cell lines and cell culture Adenocarcinoma human alveolar basal epithelial cell line (A549, ATCC), and human bronchial epithelial cell line (BEAS-2B, ATCC) were used for cytotoxicity activity investigation.A549 and BEAS-2B cells were kindly provided by Dr. Engin Ulukaya.The details from cell culture conditions were given in our previous paper (Emerce et al. 2019).

MTT assay
The cytotoxic activity of compounds 1-3 was investigated by MTT assay.Tested cells reaching sufficient confluent levels were counted by trypan blue assay.Cell suspensions in phenol red-free medium at 6 Â 103 cells/well were seeded into 96-well tissue culture plates.After 24 h, cells were treated with five increasing concentrations of the compounds (1-100 lM) dissolved in water.Doxorubicin was used as a positive control and applied at 10, 3, 1, and 0.3 mM concentrations.The plates were incubated with extracts for 48 h.After the incubation period, the supernatant was removed and fresh medium and MTT solution (1 g/L) were added to each well.The cells were incubated for 3 h at 37 C.The resulting formazan crystals were dissolved in dimethyl sulfoxide and finally, the absorbance was read at 570 nm using a spectrophotometer (SpectraMax i3x; Molecular Devices, San Jose, CA, USA).Each experiment was done in triplicates (at least n ¼ 6).The reading absorbance values were converted to the percentage of the control.The percent of cell viability in the control was assumed 100.

Statistical analysis
Data were analyzed using GraphPad Software Prism 8.0 (San Diego, CA, USA; demo version).The significance of differences in means between the cytotoxic effects of each compound at different concentrations on cells was determined by one-way analysis of variance (ANOVA).Nonlinear regression analysis (dose-response) was used to determine the IC 50 of doxorubicin.A value of p 0.05 was considered statistically significant.

Figure 1 .
Figure 1.Structures of the compounds isolated from Lecokia cretica.