First determination of anticancer, cytotoxic, and in silico ADME evaluation of secondary metabolites of endemic Astragalus leucothrix Freyn & Bornm

Isolation and characterization of anticancer activity guided secondary metabolites of endemic Astragalus leucothrix Freyn& Bornm were aimed. Aerial parts of the plant were extracted by maceration method in the solvent system methanol-chloroform (1 : 1) at room temperature. The obtained crude extract was dissolved in purified water. Then, the extract was partitioned with n-hexane, chloroform, ethyl acetate, and n-butanol, respectively. Anticancer activity tests of all the fractions were performed against HeLa and C6 cancer cells. The chloroform fraction that has highest anticancer activity was subjected to chromatographic methods such as column chromatography and thin layer chromatography. Pentyl tetratetracontanoate (1), alfalone (2), 3,6,8-tribromoquinoline (3), and 3,6,8-tribromochromenium (4) molecules were detected from this plant for the first time. The structure determinations of the isolated molecules were elucidated by methods such as 1D and 2D NMR, HPLC - TOF / MS, and GC - MS analysis. Finally, anticancer and cytotoxic activity tests of the compounds were performed. Literature review showed that 3,6,8-tribromochromenium is a new compound. IC50 values of compound 1–2 and compound 3–4 mix were determined to be 4.50 ± 0.10, 2.81 ± 0.00, 4.33 ± 0.00 μM against C6 cell, respectively. The drug likeness properties of 1–4 were obtained by SwissADME. According to Lipinski’s rule of five; 2–4 could be a new potential anticancer agent.


Plant material
A. leucothrix Freyn & Bornm was collected from Yapraklı District (Çankırı, Turkey) in June 2016.The identification of the plant samples was confirmed by botanist Melda DÖLARSLAN.The plants were stored in the Herbarium of Biology Department, Ankara University, Turkey (Herbarium number: ANK 60526).

Determination of anticancer assays
The extract, fractions, and pure compounds were investigated for their anticancer activities against human cervical adenocarcinoma (HeLa) and rat glioma cells (C6) by using BrdU ELISA assays.The tested samples and 5-FU were dissolved in dimethyl sulfoxide (DMSO).Then the stock solution was diluted with Dulbecco's Modified Eagle Medium (DMEM).DMSO concentration is below 0.1% in stock solutions.The anticancer activity tests and cell culture study were performed according to the literature [27,28].The results are means ± SD of six values.

Determination of cytotoxic assay
The cytotoxic activities of the test substances were determined according to the manufacturer's procedure using LDH cell cytotoxicity assay and cytotoxicity % was calculated [29].

Physicochemical and pharmacokinetic properties for computational methods
The SwissADME website was written in HTML, PHP5, and JavaScript, whereas the backend of computation was mainly coded in Python 2.7.The use of additional libraries or software for specific tasks is mentioned in the corresponding paragraph.
The molecule inputted through the sketcher Marvin JS (version 16.4.18,2016, www.chemaxon.com)are converted into SMILES by JChem Web Services (version 14.9.29, 2013, www.chemaxon.com)installed on one of our servers.This on-thefly conversion allows seamless paste of SMILES in the input list.The user has the possibility to edit this list as a standard text, e.g., to modify SMILES or add a name to the molecule. 1Upon calculation submission by clicking the "Run" button, the SMILES of each molecule is canonicalised by OpenBabel (version 2.3.0,2012, http://openbabel.org)and processed individually [30].Drug-likenesses and molecular property predictions of compounds are determined by the programme at http://www.molsoft.com/mprop/mprop.cgi.

Statistical analysis and determination of IC 50 values
Statistical analyses were used to evaluate anticancer and cytotoxic activity results by one-way ANOVA test.The results are means ± SD of six values.Differences between groups were determined by ANOVA method (p < 0.01).The IC 50 values were determined using ED50 plus v1.0.

Isolation and characterization
Isolation procedure was started with CHCl 3 fraction which showed the highest anticancer activity.Three compounds were isolated and identified by spectroscopic methods from the CHCl 3 fraction.After the TLC analysis, combined fractions 66-81 (274.6 mg) gave the compound 1 (pentyl tetratetracontanoate), which was determined by using 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy (Figures S1-S3).When the proton ( 1 H) NMR spectrum (600 MHz, CDCl 3 ) was examined, two triplet peaks at 0.89 were observed to belong to the terminal methyl proton.The proton of H-1' was at δ 4.06 and the H-2 proton was resonated as a triplet at δ 2.28.The proton of H-2' was signalled at δ 1.62 and the proton of H-3 at δ 1.61.Forty-six CH 2 groups were determined by utilizing the 1 H NMR spectrum integration values (Figure 1).
When the heteronuclear multiple-bond correlation (HMBC) NMR spectrum of compound 1 was examined, it was determined that the H-8 proton (shown pink) interacts with C-9 and C-7 (carbonyl carbon) carbons.The H-5 proton (shown in red) was shown to correlate with the C-3, C-4, and C-7 carbons (Figure S1).When the carbon ( 13 C) NMR spectrum and distortionless enhancement by polarization transfer (DEPT; 150 MHz, CDCl 3 ) was examined, the presence of the carbonyl group was determined in δ 173.94 (Figure S2).C-5 carbon was found to be resonance in δ 64.16.The methyl carbons were observed at δ 14.00 (Figures S2 and S3).The compound 1 was isolated from A. leucothrix and Astragalus ssp.for the first time.
HPLC/TOF-MS analysis of compound 2 gave [M] + peak at 297.0981 (C 17 H 14 O 5 ) (Figures S4 and S5).The singlet peak observed at δ 7.91 in the 1 H NMR spectrum of compound 2 is characteristic for the H-2 proton in the isoflavone skeleton [39].The singlet peak in the 1 H NMR spectrum (Table S1) that were observed in δ 3.83 and 4.01 indicates the methoxy protons linked to the C-4' and C-7 carbons, respectively, in HMBC spectrum.Protons in the A2X2 system (δ 6.97, dd, 2H, J = 2.0, 8.0 Hz; δ 7.50, dd, 2H, J = 2.0, 8.0 Hz) are observed to interact with the C-4' carbon-linked methoxy protons.In the 1 H NMR spectrum peak at δ 6.27 (1H, s) belongs to the -OH peak due to the D 2 O exchange (Figure S8).Peaks at δ 7. 65  6.97 (1H) assigned to the H-5 and H-8 protons, respectively (Figure S6).When the 13 C NMR spectrum of 2 is examined, a signal for the carbonyl group is observed at δ 175.65 and two methoxy peaks at δ 55.46 and 56.66 are observed (Figure S9).When the DEPT of 2 is examined, two CH and five CH signals are observed (Figure S10).
In 1 H NMR spectrum (600 MHz, CDCl 3 , Figure S15, Table 1) of compound 3-4 mix, H-2 proton peak at δ 9.00 (2H, s), H-4 proton at δ 8.24 (2H, s), H-6 proton at δ 7.88 (2H, s), and H-8 proton at δ 8.15 (2H, s) were determined [42].In 13 C NMR spectrum and HPLC-TOF/MS analysis of compound 3-4 mix, compound 3 was observed as [C 9 H 4 Br 3 N] + and [M] + peak m/z at 367.8055 (Figure S20).At the same time, compound 4 was detected as [C 9 H 4 Br 3 O] + , [M] + peak m/z at 365.8074 (Figure S20).The nitrogen rule in mass spectrometry states that (organic) molecules containing no or an even number of nitrogen atoms will have even masses, and molecules containing an odd number of nitrogen atoms will have odd masses [52].Thus, the fact that the molecular ion peak in the mass spectrum is a single number supports the presence of nitrogen atom in the structure.In addition, when the mass spectrum was examined, three bromine atoms were observed in both molecules (Figure S20)[53]. 13C and DEPT NMR spectra of compound 3 (150 MHz, CDCl 3 ) gave nine signals at δ 152.47, 119.24, 136.64, 121.22,128.76, 126.03, 136.31, 142.36, and 130.72, which were assigned to carbons C-2, C-3, C-4, C-5, C-6, C-7, C-8, C-9, and C-10, respectively [42].In the HMBC spectrum (600 MHz, CDCl 3 ) in Figure 3, the H-2 proton (shown in red) was found to interact with the C-3, C-4, and C-9 carbons.The H-4 proton (shown in green) correlates with the C-2, C-3, C-5, and C-10 carbons.The H-8 proton (shown in pink) interacts with C-6, C-7, C-9, and C-10 carbons (Figures 3 and S21).When the HSQC spectrum (600 MHz, CDCl 3 ) in Figure S19 was examined, the proton in δ 9.00 ppm with carbon at δ152.47 ppm, the proton in δ 8.24 ppm with carbon in δ136.64 ppm, the proton in δ 8.15 ppm with the carbon in δ136.31ppm, and the proton in δ 7.88 ppm with the carbon in the carbon δ128.66 ppm were seen to be correlated.The interactions overlap with compound 3. Bromine ranks 44th among the elements found in the earth's crust.There are many organobromine compounds synthesized by living organisms or formed as a result of natural abiotic processes [54].Because of their similar physical and chemical properties, bromides are commonly found in the environment together with sodium chloride in smaller amounts.Br has been shown to be a new and important trace element for humans and animals [55].Although various plant species can accumulate high concentrations of Br, to our knowledge, their role in plants has not been established [56].In marine plants (for example, Bonnemaisonia hamifera, Laurencia species), marine animals (for example, sponges, bryozoans, corals), mammals (for example, cat and rat), abiogenic sources, plants (for example, rapeseed, mustard, cabbage, Chinese cabbage, broccoli, pak-choi, alyssum, wild mustard, turnip, radish), fungi and lichen, bacteria (for example, Bacillus subtilis, Chromobacterium species), and insects are naturally found organobromine compounds [57].However, the compound 3 and 4 were isolated from A. leucothrix and Astragalus ssp.for the first time.

Anticancer activity
The anticancer activities of MeOH: CHCl 3 extract, n-hexane, CHCl 3 , EtOAc, n-Butanol and water fractions and 5-FU that were used as a standard against C6 and HeLa cell were investigated (Figure 4).As a result of the tests performed, an increase in the activity of all extracts due to dose increase was observed.Activity at 100 µg/mL concentration against HeLa cell (Figure 4): CHCl 3 fraction > 5-FU > n-hexane fraction > EtOAc fraction > MeOH: CHCl 3 extract > n-BuOH fraction > water fraction.When cancer activity results were examined in both cells, the most active fraction was found to be chloroform fraction.The highest activity against C6 cells was observed in n-hexane and chloroform fractions (Figure 4).Activity at 100 µg/mL concentration against C6 cell: n-hexane fraction > CHCl 3 fraction > MeOH: CHCl 3 extract > 5-FU > EtOAc fraction > water fraction > n-BuOH fraction.
In cytotoxicity studies on Astragalus chrysochlorus extracts, the highest effect was observed in chloroform extract.This extract was followed by ethyl acetate, ethanol, n-hexane, and aqueous ethanol extracts [58].Similarly, in our study, the highest effect was observed in chloroform extract.This extract was followed by n-hexane extract.We performed GC-MS analyses of n-hexane and chloroform extracts of A. leucothrix (Table 2).In the n-hexane extract, palmitic acid, linolenic acid, and linoleic acid were main components.Palmitic acid, linolenic acid, and behenic acid were major compounds in chloroform extracts.PUFA fatty acids are used in chemotherapy.They also increase the effectiveness of chemotherapeutic drugs and may reduce chemotherapy or cancer side effects.Linolenic acid in the chloroform extract inhibited various cancer cells such as GOTO, SK-N-DZ, DU145, A-549, PC-3, 36B10 cells [59].Also, palmitic acid has anticancer activity against human leukemic cell line (MOLT-4), colon 26 murine tumour cells, and human breast cancer (MCF-7).It is also known that the crude extracts are more effective than their pure compounds for pharmacologically.This effect is thought to be due to the synergistic effect of many molecules in the extracts [60].
The anticancer activities of the isolated compound 1-2, compound 3-4 mix, and 5-FU used as standard were examined against the HeLa as a result of the tests (Figure 5), an increase was observed in all molecules (except compound 3-4 mix) due to dose increase.Among the isolated molecules, the highest activity against the HeLa cell was observed in the compound 2.
Activity at a concentration of 100 µM is as follows: 5-FU > compound 2 > compound 1 > compound 3-4 mix.As a result of anticancer activity tests of compound 1-2, compound 3-4 mix, and 5-FU against C6 cells (Figure 5), an increase was observed in all molecules due to dose increase.The highest activity against the C6 cell between the isolated molecules and the 5-FU was observed in the compound 2. Cell selective activity against C6 cells was observed in all isolated molecules.The activity at 100 µM concentration is compound 2 > 5-FU > compound 3-4 mix > compound 1.The anticancer activities of the isolated compound 1-2, compound 3-4 mix, and 5-FU used as standard were examined against the HeLa and C6 cells.The IC 50 values of these compounds are given in Table 2.As in this study, the isolated compounds and extracts from Astragalus species such as Astragalus tribuloides [60], Astragalus hamosus [60][61][62], Astragalus membranaceus [25,[63][64], Astragalus ovinus [65], Astragalus vogelii [66], Astragalus complanatus [67] have anticancer activity.

Cytotoxic activity
C6 cells were used to determine cytotoxic activity.100 µg/mL concentration, which is the highest dose used in the anticancer activity tests, was also studied during the experiment.5-FU was used as a positive control.Test results were given in Table 1.The cytotoxicity values of the samples are relatively small (except compound 2) compared to 5-FU.Especially compound 3-4 mix is less toxic than 5-FU.

Drug likeness properties
The number of hydrogen bond acceptors (n-ON) and donors (n-OHNH) are within the Lipinski's rules, n-ON < 10 and n-OHNH < 5.The calculated log P must be smaller than 5.In our study, the log P values of compound 2-4 were smaller than 5.The molecular weight of the compounds is in the range of 298.29 g/mol and 719.30g/mol, respectively.The bloodbrain barrier (BBB) score: 6-High, 0-Low [68].The BBB score of compound 1-4 ranges from 3.23 to 4.28.Compound 1-4 can cross the BBB.Synthetic accessibility score of the compounds are from 1 (very easy) to 10 (very difficult).Synthetic accessibility of all the compounds is in the range of 1.79 and 6.77.Topological polar surface area (TPSA) must be <70 Å 2 .TPSA values of all the compounds were smaller than 70 Å 2 (Tables 3 and 4).
The solubility (log S) scale value ranges between -10 (insoluble), -6 (poorly soluble), -4 (soluble), -2 (very soluble), and 0 (highly soluble).The solubility values of compound 1-4 were -16.56, -3.77, -5.36, and 5.10, which respectively correspond to insoluble, soluble, moderately soluble, and moderately soluble.The more negative the skin permeation (log Kp) the less the skin-permeant the molecule.For example, Diclofenac is a good topic antiinflammatory with a predicted log Kp of -4.96 (cm/s), while Ouabain has little chance to cross skin with a predicted log Kp of -10.94 (cm/s).The log Kp values of compound 1-4 were 6.62, -6.15, -5.51, and -5.83 cm/s, respectively.The Kp values showed that compound 2-4  was good in skin permeability (Tables 3 and 4).According to Lipinski's rule of five, compound 2-4 could be a new potential anticancer agent according to calculated data (Tables 3 and 4) [69].The pink area represents the optimal range for each property (lipophilicity: LOGP between −0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å 2 , solubility: log S not higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds.In this example, compound 2-4 are predicted orally bioavailable, because of being flexible, polar, and small size.Bioavailability radar of compound 1-4 is demonstrated in Figure 6.

Figure 4 .
Figure 4.The anticancer activity of extracts against C6 and HeLa cell (* tests repeated three times and twice).

Figure 5 .
Figure 5.The anticancer activity of the compounds against C6 and HeLa cell (* tests repeated three times and twice).

Table 2 .
GC-MS analysis results of the n-hexane and chloroform extracts of A. leucothrix.

Table 4 .
SMILES, Lipinski's rule of five and drug likeness of compound 1-4 predicted using molsoft programme.

Table S1 .
1H,13C NMR, and DEPT data of compound 2 Four compound were isolated firstly from Astragalus leucothrix.2. Two tribromo compound was identified for the first time as a natural product.3.According to Lipinski's rule of five; 2 -4 could be a new potential anticancer agent.