A new steroid glycoside from Begonia sp.: cytotoxic activity and docking studies

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Natural Product Research

Formerly Natural Product Letters

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A new steroid glycoside from Begonia sp.: cytotoxic activity and docking studies

Muhammad Sulaiman Zubair, Walied M. Alarif, Mohamed A. Ghandourah &

Syariful Anam

To cite this article: Muhammad Sulaiman Zubair, Walied M. Alarif, Mohamed A. Ghandourah &

Syariful Anam (2019): A new steroid glycoside from Begonia�sp.: cytotoxic activity and docking studies, Natural Product Research, DOI: 10.1080/14786419.2019.1669026

To link to this article: https://doi.org/10.1080/14786419.2019.1669026

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A new steroid glycoside from Begonia sp.: cytotoxic activity and docking studies

Muhammad Sulaiman Zubair^a, Walied M. Alarif^b, Mohamed A. Ghandourah^band Syariful Anam^a

aFaculty of Science, Department of Pharmacy, Tadulako University, Palu, Indonesia;^bFaculty of Marine Science, Department of Marine Chemistry, King Abdul Aziz University, Jeddah, Saudi Arabia

ABSTRACT

Chemical investigation on the ethyl acetate extract of the aerial parts of Begonia sp. afforded a new steroid glycoside, 9(11)a,16(17)a-dioxirane-20,25-dihydroxy-b-sitosterol-3-O-b-glucopyranoside (1) along with a known steroidal glycoside, b-sitosterol-3-O-b-D-glucopyranoside (2). The Chemical structures were elucidated by 1D and 2D NMR and mass spectroscopic analysis.

Cytotoxicity against four different cancer cell lines (HeLa, T47D, WiDr and Vero) was assessed. Compound1was more potent and selective against breast cancer cell line (T47D) than other cell lines with an IC50 value of 0.16mg/mL. Further docking study of 1 exhibited the preference of molecule to bind in the epidermal growth factor receptor tyrosine kinase (EGFR-TK) binding pockets with docking scores of 97.8800 (PLANTS) and 3.56 kcal/mol (AutoDock 4.2.6).

ARTICLE HISTORY Received 8 July 2019 Accepted 4 September 2019 KEYWORDS

Begonia sp.; steroid glycoside; spectroscopy;

molecular docking;

breast cancer

CONTACTMuhammad Sulaiman Zubair [email protected]; [email protected] Supplemental data for this article can be accessed athttps://doi.org/10.1080/14786419.2019.1669026.

ß2019 Informa UK Limited, trading as Taylor & Francis Group

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1. Introduction

The Begonia (Begoniaceae) is one of the largest genera of the flowering plants that has leaves and flowers of beautiful shapes and various colours (Hartutiningsih et al.

2011). Previous phytochemical studies on 10 Begoniaspecies revealed the presence of flavonoids, triterpenoids, steroids, glycosides and alkaloids (Zubair et al. 2016).

Reported biological activities of these plants include antiviral and cytotoxic activities (Doskotch and Hufford 1970; Frei et al. 1998; Wu et al. 2004), antihyperglycaemic (Pandikumar et al. 2009), antibacterial (Ramesh et al. 2002; Solomon and Johnson 2012), and antioxidant properties (Velusamy and Veerabahu 2012).

Benalu Batu (Begonia sp.), has been used by Wana tribe in Morowali, Central Sulawesi, Indonesia, to treat several diseases, including cancer. Our previous study established the anticancer activity of the methanol extract of this plant against breast and cervical cancer cell lines (T47D and HeLa cells) (Anam et al. 2014). Therefore, the aim of the present study was to further investigate the phytochemicals responsible for its anticancer property.

A preliminary docking study to early identify the potential anticancer compounds by using database of secondary metabolites reported from the genus ofBegonia was performed. The docking result prompted us for the isolation of glycosides from the Begoniaplant extract (Zubair et al. 2016). Previously, 2-O-b-glucopyranosyl-cucurbitacin D was reported and it showed potent inhibitory activity against colon cancer cells, HCT-116 (Zubair et al. Forthcoming). Further investigation on the ethyl acetate fraction led to the isolation of a new steroid glycoside, 9(11)a-16(17)a-dioxirane, 20,25-dihydroxy-b-sitosterol-3-O-b-glucopyranoside (1) (Figure 1) and the known glycoside- b-sitosterol-3-O-b-D-glucopyranoside (2). Cytotoxic activity of the isolated new compound was assessed against four different cell lines. Molecular docking study of the new compound is also discussed.

2. Results and discussion

Compound 1 was isolated as an amorphous white powder. A reddish spot appeared on TLC plate after spraying with p-anisaldehyde-sulphuric acid suggesting the compound being a terpenoid/steroid. The Liebermann-Burchard test gave a green colour supporting the steroidal nature of the compound. Moreover, the analysis of HRESI-MS

OH

Glc O

OH

O

1 1

3 5 6

9 8 19

10 11 12

13

14 15 16 17 18 20 22

21

23 24 25 26 27 28 29

O

Glc 2

Figure 1. Structure of steroid glycosides 1 and 2.

2 M. S. ZUBAIR ET AL.

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atm/z637.3840 [MþH]^þsupporting the molecular structure of glycosidal steroid with the molecular formula of C35H56O10.

The ¹³C NMR spectrum (Supplementary Table S1) of compound1 exhibited 35 signals of carbon atoms which were categorised by DEPT into 6 methyls, 10 methylenes, 12 methines and 7 quaternary carbons. Three of the eight degrees of unsaturation as calculated from its molecular formula were attributed to one carbon–carbon double bond (dC 139.9 and 120.1 ppm) assigned for D⁵ sterol and two oxirane rings represented by signals resonating atdC51.3, 57.9, 69.2 and 76.4 ppm. The presence of glucopyranosyl moiety was supported by the resonance of the anomeric carbon at dC

103.3, together with four oxymethines atdC79.4, 77.2, 76.2, 73.7 ppm and one methy- lene carbon at dC 62.1 ppm. Thus, the molecule was established to be pentacyclic in which one was for a glucose unit. Additional two oxygenated quaternary carbons could be deduced from two signals atdC69.9 and 76.4 ppm.

The ¹H NMR (Supplementary Table S1) revealed the skeleton of a stigmasterol par- tial structure, proven by the presence of six methyl signals characteristic for steroids resonating at dH 0.67 and 1.06 ppm assigned for two methyls at C-18 and C-19, one triplet for a methyl atdH0.89 ppm for C-29 and three downfield signals corresponding to methyl protons resonating at dH 1.15, 1.28 and 1.29 ppm, as assignable to methyl groups attached to oxygenated carbons. A broad singlet at dH 5.76 was assigned for H-6. Two broad singlets at dH 3.08 and 3.59 ppm were characteristic for protons of two oxirane rings. The shape and position of the signal observed at dH 4.67was characteristic for 3-hydroxylated steroids (John Goad and Akihisa1997) suggesting the glucose unit was linked to the C-3. The monosaccharide moiety (glucose) was confirmed from the anomeric proton signal at dH 4.37 (dd, 1.2, 3.0), together with five signals of oxygenated carbons in the range 3.42–3.76 ppm. From the ¹H-¹H COSY correlation, a spin system between H-C3 and H2-C2, H2-C4 and proton anomeric H-C1⁰ was observed, along with the presence of cross-peaks between H2-C2 and H2-C1. Long- range C-H correlation (HMBC) observed between Me-19 (dH 1.15, br s) and C-2 (29.7), C-4 (34.3), C-10 (33.7) and C-8 (34.0) established the closing of ring A, connected by glycosidic linkage at C-3 (73.7) position. The position of first oxirane ring at C-9 and C- 11 was proven by the ¹H-¹H COSY correlation between H-C11 and H2-C12, also supported by the long-range C-H correlation (HMBC) between Me-18 (dH 0.67 ppm, s) and C-13 (48.5), C-12 (47.3), C-11 (57.9) and C-16 (51.3), suggesting the position of the second oxirane ring at C-16 and C-17 as well. The connection between steroid skeleton and side chain was observed by HMBC long-range correlation between Me-21 (dH 1.06 ppm, s) and C-18 (dC18.8).The consecutive protons were observed from H2-C- 22, H2-C-23, H-C-24, H2-C-28 and H3-C-29. Long-range C-H correlation (HMBC) was observed between Me-21 and C-23 (18.8), between Me-29 and C-28 (22.7), C-23 (18.8), and C-22 (34.7), between Me-26/Me-27 and C-24 (51.2), C-23 (18.8), C-22 (34.7), C-28 (22.7), C-29 (14.1) (Supplementary Figure S2). The position of six hydroxyl groups (four for the sugar and the remaining for steroid side chain attached to C-20 and C-25), was determined by examining the chemical shift from¹H and ¹³C spectral data and supported by ¹H-¹H COSY and ¹H-¹³C HMBC spectral data. The relatively large coupling constant value of H-11 (J¼6.0 Hz) implied axial–equatorial orientation (a-position) of H-11, hence the C-9-C-11 oxirane ring occupies b-position (Sanap et al. 2010).

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Meanwhile, the second oxirane ring occupies b-position owing to the biogenetic rule of these steroids (John Goad and Akihisa 1997). Based on above data and database searching from science finder indicated compound 1 was a new steroid and it was assigned as 9(11)a,16(17)b-dioxirane-20,25-dihydroxy-b-sitosterol-3-O-b-glucopyranoside (Figure 1). Compound2was identified by comparison with the published data for b-sitosterol-3-O-b-D-glucopyranoside (Mizushina et al.2006; Khatun et al.2012).

Compounds1 and2were tested for cytotoxic activity towards four different cancer cell lines (T47D, HeLa, WiDr and Vero) (Supplementary Table S2). Compound1 showed higher cytotoxicity only against breast cancer cells (T47D) than compound 2 with the IC50 value of 0.16mg/mL. It also possessed high selectivity as this compound did not show toxicity to Vero cell line. Based on our previous study where methanol extract of Begonia sp.had high inhibition on breast cancer cells proliferation (T47D cell lines), we could conclude that compound1 might be the bioactive compound that was responsible for this specific and selective activity against breast cancer cells.

Further molecular docking study was performed for compound 1 on EGFR-TK protein targets. Compound 1 was found to have better interaction on EGFR-TK receptors than native ligand erlotinib. The binding mode of compound 1 on EGFR-TK revealed the formation of hydrogen bonds with the residues Lys 721 and Asp 831 with the docking energy score of 97.880, which was higher than native ligand erlotinib (Supplementary Table S3). The skeleton of steroid was found to insert on the hydrophobic pocket on EGFR-TK affording a hydrophobic interaction and the presence of sugar moiety increase the docking energy score in which the hydroxyl group of sugar can form hydrogen form with Lys 721, which was located in the phosphate-binding region along the sugar pocket. Hydrogen bonding was also observed between hydroxyl group of sugar with Asp 831 which is located in helix aC (Supplementary Figure S3). It was clearly shown that the docking energy score of compound 1 had lower than native ligand erlotinib affording the high affinity and selectivity only on EGFR-TK receptor that responsible for breast cancer cell lines. This might be because of the presence of two extra oxirane rings and two extra hydroxyl groups attached to side chain of steroid skeleton prompting for further investigation for thein vitromech- anism of compound1as a selective and specific agent for breast cancer.

3. Experimental 3.1. General

TLC aluminium sheets 2020 cm silica gel 60 F254 was used. Silica gel 60 (Merck) for vacuum liquid column chromatography (230–400 mesh) was used. Pre-coated TLC glass plates SIL G-25 UV254, 0.25 mm silica gel and Sephadex LH-20 (Sigma, St. Louis, MO, USA) were used for isolation and purification of the compounds. Spots on TLC were visualised by using spraying reagent of methanol-sulphuric acid andp-anisaldehyde-sulphuric acid for terpenoid/steroid detection. Nuclear magnetic resonance (NMR) was recorded for 1D and 2D on Avance^III Bruker WM 600 MHz for ¹H and 150 MHz for¹³C. Chemical shifts are givend(ppm) relative to TMS as internal standard and deuterated chloroform was used as a solvent.

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The aerial parts of Begonia sp., growing in the mountain, were collected from Morowali, Central Sulawesi, on April 2014. The plant species were identified at Biodiversity Unit, Tadulako University, Central Sulawesi, Indonesia and deposited as a dried specimen (BSP 00020414) at Phytochemistry Laboratory, Department of Pharmacy, Tadulako University.

3.2. Extraction and isolation

The aerial parts were shade-dried. The dried sample (250 g) was extracted with ethanol 96% (30.8 L, 24 h for each batch) at room temperature. The solvent was removed in vacuountil reached a residue (15 g) referred to as crude ethanol extract. The ethanol extract (10 g) was suspended in water and successively partitioned withn-hexane and ethyl acetate to obtain n-hexane and ethyl acetate soluble fraction. The ethyl acetate fraction (3 g) was chromatographed on silica gel (60–120 mesh) and the packed column was eluted by employing gradient system of solvent of n-hexane 100%, n-hexane/ethyl acetate mixture, followed by ethyl acetate/methanol mixture and methanol 100%. A total of 40 fractions of 50 mL each were collected. Similar fractions were collected together according to TLC pattern in eight fractions (F1–F8). The fraction 3 (eluted by ethyl acetate:methanol (4:1, 60 mg)), which was found to contain steroidal skeleton, was continued to further isolation using successive preparative TLC with chloroform:methanol (1:9) as a mobile phase. The first band withRf¼0.92 (purple colour with sulphuric acid-methanol) was taken to give compound2as amorphous white powder (23 mg). The fraction 4 (eluted by ethyl acetate:methanol (4:1, 55 mg) was also continued to further isolation using successive preparative TLC preparative with chloroform:methanol (2:8). The first band withRf ¼ 0.36(purple colour with sulphuric acid- methanol) was taken to give compound1as amorphous white powder (10 mg).

9(11)a,16(17)a-dioxirane-20,25-dihydroxy-b-sitosterol-3-O-b-glucopyranoside (1).

Amorphous white powder (10 mg, 0.04% of dry weight); HRESI-MS m/z 637.3840 [MþH]^þ (calculated 637.3873 for C35H56O10); H NMR (CDCl3, 600 MHz): d¼1.34 (2 H, m, H-1), 1.26 (2 H, m, H-2), 4.67–4.70 (1 H, m, H-3), 2.83–2.84 (1 H, dd,J¼1.2, 7.8 Hz, H- 4), 5.76 (1 H, m, H-6), 1.26 (2 H, m, H-7), 2.32 (1 H, m, H-8), 3.08–3.10 (1 H, t,J¼6 Hz, H- 11), 2.56–2.57 (1 H, d, J¼10.2 Hz, H-12a), 3.29–3.30 (1 H, d, J¼10.2 Hz, H-12b), 2.00–2.03 (1 H, dd,J¼4.8, 13.2, H-14), 1.22 (2 H, m, H-15), 3.60 (1 H, m, H-16), 0.67 (3 H, s, H-18), 1.15 (3 H, s, H-19), 1.06 (3 H, s, H-21), 1.34 (2 H, m, H-22), 1.26 (2 H, m, H-23), 1.57 (1 H, m, H-24), 1.28 (3 H, s, H-26), 1.29 (3 H, s, H-27), 1.22 (2 H, m, H-28), 0.89 (3 H, t, J¼5.4 Hz, H-29), 4.37 (1 H, dd, J¼1.2, 3.0 Hz, H-1⁰), 3.58 (1 H, m, H-2⁰), 3.45 (1 H, m, H-3⁰), 3.42 (1 H, m, H-4⁰), 3.60 (1 H, m, H-5⁰), 3.76 (2 H, m, H-6⁰); ¹³C NMR (CDCl3, 150 MHz): d¼23.9 (CH2, C-1), 29.7 (CH2, C-2), 73.7 (CH, C-3), 34.3 (CH2, C-4), 139.9 (C, C-5), 120.1 (CH, C-6), 31.9 (CH2, C-7), 34.0 (CH, C-8), 69.2 (C, C-9), 33.7 (C, C-10), 57.9 (CH, C-11), 47.3 (CH2, C-12), 48.5 (C, C-13), 42.8 (CH, C-14), 29.4 (CH2, C-15), 51.3 (CH, C-16), 76.4 (C, C-17), 18.8 (CH3, C-18), 19.9 (CH3, C-19), 69.9 (C, C-20), 18.1 (CH3, C-21), 34.7 (CH2, C-22), 18.8 (CH2, C-23), 51.2 (CH, C-24), 76.4 (C, C-25), 21.3 (CH3, C-26), 21.2 (CH3, C-27), 22.7 (CH2, C-28), 14.1 (CH3, C-29), 103.3 (CH, C-1⁰), 76.2 (CH, C-2⁰), 79.4 (CH, C-3⁰), 76.4 (CH, C-4⁰), 77.2 (CH, C-5⁰), 62.1 (CH2, C-6⁰).

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3.3. Cell culture

Human ductal breast epithelial tumour cell line (T47D), human cervical cancer cell line (HeLa), colon adenocarcinoma cell line (WiDr) and green African monkey renal epithelial cell (Vero) were obtained from Laboratory of Parasitology, Faculty of Medicine, Gadjah Mada University. Cells were maintained in RPMI-1640 medium supplemented with 100mg/mL streptomycin, 100 units/mL penicillin and 10% foetal bovine serum (FBS) in 5% CO2atmosphere at 37C.

3.4. Cytotoxicity test

Cytotoxicity test was performed on HeLa, T47D, WiDr and Vero cancer cell lines by MTT method. The stock samples of all compounds were diluted with RPMI-1640 medium to desired concentrations (1000 mg/mL). The final concentration of dimethyl sulphoxide (DMSO) in each sample did not exceed 1% v/v. The cancer cells were batch cultured for 10 days, then seeded in 96 well plates of 1010³cells/well in fresh com- plete growth medium in 96-well microtitre plastic plates at 37C for 24 h under 5%

CO₂ using a water-jacketed carbon dioxide incubator (Shedon.TC2323.Cornelius, OR, USA). The medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentrations of (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.7812, 0.3906, 0.1953 mg/mL). Cells were suspended in RPMI-1640 medium, complemented with 5% FBS and kanamycin (100mg/mL) in 96-well flat-bottom microplates at 37C under 5% CO2. After 24 h of incubation, cells were added with 10mL/well of MTT (5 mg/mL) and incubated 4 h in incubator at 37C in 5% CO2-humidified atmosphere. The reaction was stopped by 100mL SDS (10% in HCl 0.01 N). The plate was then shaken in shaker for 10 min and incubated overnight in the shaded room. The absorbance of each well was read at 550 nm wavelength in ELISA Reader (Biorad), using wells without cells as blanks. All experiments were performed in triplicate. The data are represented as mean ± SD. The effect of compounds on the growth inhibition of cancer cells was expressed as the % cytoviability, using the following formula: % cytoviability¼absorbance of treated cells/

absorbance of control cells100%.

3.5. Molecular docking

Molecular docking protocols were performed by using software combination of SPORES, Open babel and PLANTS1.2 as described by Yuniarti et al. (2011). The pdb file was split to the protein file and co-crystallised ligand file using thesplitpdbmodule in SPORES. The protein was then recognised, protonated and stored as protein.mol2, while the co-crystallised ligand was also recognised, protonated and stored as ligand_AQ4999_0.mol2. The co-crystallised ligand subsequently underwent conformational search to find the most stable conformer from 10 seeds, followed by 1000 steps energy minimisation using obconformer module in Open Babel (O’Boyle et al.

2011). The optimised structure of co-crystallised ligand was subjected to SPORES before submitted to the docking simulations using PLANTS1.2 (Korb et al. 2009; ten Brink and Exner 2009). The binding site in the docking configuration file was defined

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as 5 Å from the coordinates of the location where the co-crystallised ligand was located in the co-crystallised crystal structure. The bind module of PLANTS1.2 was used to automatically identify the binding site. The RMSD (root mean square devi- ation) value between the docked pose and the crystal structure pose was calculated usingrms_cur module in PyMol (Seeliger and de Groot2010). This procedure was performed iteratively 10 times. The lowest docking energy score with the RMSD < 2 Å was chosen as selected molecular docking protocol. To confirm the docking results, an additional docking simulation by using AutoDock 4.2.6 was performed by targeting the whole protein (Morris et al. 2009). Preparation for receptor and ligand were performed by using AutoDockTools (ADT) software packages. Gasteiger charges were added, grid boxes was set to 54, 60 and 44 points spaced 1.0 Å, Lamarckian Genetic Algorithm (LGA) parameters were used as follow: population size of 50, elitism of 1, mutation rate of 0.02, crossover rate of 0.80, local search rate of 0.06, 250,000 energy evaluations, and 100 search runs. The final docked conformations were clustered using a cluster tolerance of 2.0 Å RMSD. The ligand pose with the lowest predicted free binding energy was used for subsequent analysis.

The chemical structure of compound 1 was built by MarvinSketch (Chemaxon, Dordrecht, Netherlands) (Bennett et al.2009). The structure was protonated at pH 7.4 and saved as mrv format file. After that, the file was subjected to conformational search to gain 10 different conformers and then saved as mol.2 format file.

Obconformermodule in open babel was then used to continue the ligand preparation by re-conformational search to find the most stable conformer from 10 seeds and successively minimise the energy of each structure by 1000 steps. The minimised structure was then subjected to the docking molecular simulation by using PLANTS1.2 and AutoDock 4.2.6 software. The docking score obtained was then compared to the docking score of the co-crystallised ligand erlotinib.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

The authors would like to greatly acknowledge the Ministry of Research, Technology and Higher Education, Republic of Indonesia for supporting this study through INSINAS 2015 [grant No. RD- 2015-0106].

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