Steroids 141 (2019) 30–35
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Cytotoxicity and molecular docking studies on phytosterols isolated from Polygonum hydropiper L
T
Muhammad Ayaza, , Abdul Sadiqa,b, Abdul Wadoodc, Muhammad Junaida, Farhat Ullaha, Nadir Zaman Khand ⁎
a
Department of Pharmacy, University of Malakand, Chakdara, 18000 Dir (L), KPK, Pakistan Department of Life Sciences & Chemistry, Faculty of Health, Jacobs University Bremen, Germany c Department of Biochemistry, UCS, Shankar Abdul Wali Khan University, Mardan, Mardan 23200, Pakistan d Department of Biotechnology, University of Malakand, Chakdara 18000, Dir (L), KPK, Pakistan b
ARTICLE INFO
ABSTRACT
Keywords: Polygonum hydropiper NIH/3T3 HeLa and MCF-7 cells Cytotoxicity Tyrosine kinase Molecular docking
Based on our previous studies on cytotoxic potentials of Polygonum hydropiper L, two steroidal compounds betasitosterol and stigmasterol were isolated from the most active fraction and were subjected to cell lines cytotoxicity. Isolated compounds were tested against HeLa, MCF-7 and NIH/3T3 cell lines following MTT assay. Furthermore, the compounds were also docked against tyrosine kinase enzyme to predict the binding mode of phytosterols in the active sites of the enzyme. Beta-sitosterol exhibited considerable cytotoxicity against NIH/ 3T3, HeLa and MCF-7 cell with 67.05 ± 2.08, 79.63 ± 2.34 and 71.50 ± 1.57% lethality respectively at 1 mg/ml concentration. Median inhibitory concentrations calculated from dose response curve against NIH/3T3, HeLa and MCF-7 cells were 440, 170 and 200 µg/ml respectively. Stigmasterol was more effective against MCF-7 and NIH/3T3 cells by killing 87.50 and 81.45% cancerous cells respectively at 1 mg/ml concentration. Stigmasterol showed 77.25% cyctotoxicity against HeLA cells at 1 mg/ml concentration in MTT assay. The IC50 values for HeLA, MCF-7 and NIH/3T3 cells were 170, 60 and 140 µg/ml respectively. In docking studies, the docking score for beta-sitosterol and stigmasterol were −7.266 and −4.89 respectively. The binding energies for beta-sitosterol and stigmasterol were −41.21 and −41.04 respectively. Such lower binding energies indicate that the compounds fit into the active site more strongly. Binding affinities for both compounds were −7.76 and −7.68 respectively. Both phytosterols possess significant anticancer potentials and can be effective in the prevention and treatment of several malignancies.
1. Introduction Phytosterols are naturally occurring cell membrane components of plants and are structurally similar to cholesterol [1]. Most common phytosterols include beta-sitosterol, stigmasterol and campesterol which are abundant in dietary supplements [2]. As human body cannot prepare phytosterols, their need is fulfilled via the use of sterols rich diets like fruits, nuts, corn oil, wheat germ oil, sesame seeds, and soybean oils [3]. Phytosterols have been reported to possess significant lipid lowering properties and thus are potential alternatives to conventional drugs. Several clinical studies concluded that these compounds considerably lower plasma LDL cholesterol without any side effects [4–6]. Dietary pytosterols can offer protection and can reduce the risk of cancer by twenty percent [7]. Experimental evidences and epidemiologic data imply that phytosterols may be beneficial in the
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prevention of several types of cancers like prostate cancer, colon cancer and breast cancer [8–10]. Beta-sitosterol is natural micronutrient considered as safe and associated with several potential benefits as suggested by European Food Safety Authority (EFSA) and Joint FAO/WHO Expert Committee on Food Additives (JECFA) [11]. It possess significant anticancer, free radicals scavenging, analgesic, immunomodulatory, anti-inflammatory and anti-diabetic properties [12]. Several preparations of beta-sitosterol are available in the market which are claimed to possess beneficial effects in many diseases, yet the scientific evidence behind these claims are not clear [10,13]. Stigmasterol another popular phytosterol is known to possess anti-inflammatory, anti-osteoarthritic, anti-radicals, anti-mutagenic and anti-diabetic potentials [4,14]. Tyrosine kinases play vital role as mediators of the signaling cascade and are linked to the process of growth, cellular differentiation,
Corresponding author. E-mail addresses:
[email protected] (M. Ayaz),
[email protected] (A. Wadood),
[email protected] (N. Zaman Khan).
https://doi.org/10.1016/j.steroids.2018.11.005 Received 28 June 2018; Received in revised form 13 September 2018; Accepted 9 November 2018 Available online 13 November 2018 0039-128X/ © 2018 Elsevier Inc. All rights reserved.
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metabolism and programmed cell death (apoptosis) in response to indigenous or foreign stimuli [15]. Tyrosine kinases are implicated in the pathophysiology of various types of cancers and inhibitors of these enzymes are considered as useful targets in cancer therapy [16]. In normal cells the activity of these kinases are firmly regulated but they may transform their function due to mutation, autocrine paracrine stimulation and over expression which results in malignancy [17]. Oncogene stimulation can be hampered via the use of specific tyrosine kinase inhibitors especially from natural products [18]. Beta-sitosterol and stigmasterol were isolated from P. hydropiper L, which possess anticancer potentials and several species are reported to possess cytotoxic activities. We previously reported the anticancer potentials of P. hydropiper [19], Rumex hastatus [20] crude extracts and isolated several bioactive compounds. Based on their previously known beneficial cytotoxic potentials, both phytosterols were tested against HeLa, MCF-7 and NIH/3T3 cell lines and docked the compounds against tyrosine kinase enzyme.
2.5. Structure confirmation All the purified compounds were subjected to the rotary evaporator for the removal of possible presence of solvent. Initially, we used 1H NMR to get an idea regarding the structure and compared the spectra with the reported literature. The 13C NMR was used to get idea about the carbon skeleton of the compounds. The data was supplemented by using mass spectrometry to confirm the structures. 3. MTT cells viability assays 3.1. NIH/3T3 cell line assay Mouse embryonic fibroblast cells (NIH/3T3) were cultured in 10% FBS enriched DMEM medium following manufacturer specifications. To prevent microbial growth, antibiotics 50 units/ml penicillin and 50 units/ml streptomycin were added to the media at 37 °C using a humidified carbon incubator (5% CO2). Cytotoxicity of our test samples were evaluated following MTT assay [27,28]. Fibroblast cells were seeded into 96-well plates with cell density of 8.0 × 103 cells/well in 200 μl medium with subsequent incubation for 24 hrs. Afterwards, the culture medium was replaced with 200 μl medium containing serial dilutions (0.0325–1 mg/ml) of samples. Incubated cells without the addition of test samples acted as negative control, whereas, doxorubicin was used as standard control. Later, 20 μl of MTT solution (5 mg/ml) in PBS was added to each well, followed by incubation for 4 h. Medium having un-reacted dye was removed carefully and the absorbance was measured at a wavelength of 570 nm. cells viability and % lethality were calculated using formula;
2. Experimental 2.1. Plant collection, processing and compounds isolation P. hydropiper, a traditional folklore against cancer was collected from Talash Valley, Khyber Pakhtunkhwa (KP), Pakistan in July 2013 and was authenticated by Dr. Gul Rahim at Arid agriculture University, Rawalpindi, Pakistan. Plant sample was deposited at herbarium, University of Malakand Chakdara (Dir), Pakistan with voucher no (H.UOM.BG.107) as reported in our previous manuscripts [21,22]. Aerial parts of the plant were shade dried, powdered and extracted with increasing polarity of solvents including methanol, n-hexane, chloroform, ethyl acetate, butanal and water as reported in detail previously [23,24].
Cellviability% =
Meanofabsorbancevalueoftreatmentgroup × 100 Meanofabsorbancevalueofcontrol
3.2. HeLa cell line assay
2.2. Isolation of compounds
Cytotoxic activity of test phytosterols against HeLa cells following previously reported MTT colorimetric assay [29]. In brief, HeLa cells were cultured in MEME medium in 75 cm2 flasks, provided with 5% of FBS, streptomycin (100 µg/ml) and penicillin (100 IU/ml). Cells culture was incubated at 37 °C using 5% CO2 incubator. Cells were counted via haemocytometer and were further diluted to cells density of 6x104 cells/ml using same media. Subsequent to overnight incubation, fresh media added with increasing concentrations of test samples were added. After 48 h of incubation, MTT solution (200 µl) was added to each well and further incubated for4 hrs. Later on, DMO (100 µl) was added to each well and the extent of reduced fomazan in cell culture were analyzed via measuring absorbance at 570 nm using micro plate reader (Spectra Max plus, Molecular Devices, CA, USA). Percent inhibition was determined using formula;
The solvent fractions, especially chloroform and ethyl acetate were selected for the isolation of pure compounds. Normally, most of the organic molecules (with polar functional groups) can be likely dissolved in chloroform or ethyl acetate. Moreover, in overall activities both the chloroform and ethyl acetate were active fractions, therefore, we used both of these fractions for isolation of the compounds [25,26]. 2.3. TLC analysis Using precoated silica gel TLC plates to select a suitable solvent system which can separate maximum compounds in the selected fractions. We examined different solvent systems, but a combination of nhexane and ethyl acetate was the best to separate the components in the given fractions. Initially, we observed the TLC in n-hexane and ethyl acetate (95:5). We increased the polarity of solvent system gradually by increasing a 5% of the ethyl acetate portion.
Inhibition% 100 (MeanofO. Doftestcompounds MeanofO. Dofnegativecontrol) = × 100 MeanofO. DofPositivecontrol meanofO. Dofnegatvecontrol)
Results (% inhibition) were processed by Soft-Max Pro software (Molecular Device, USA)
2.4. Gravity column chromatography Initially, we packed a large column and load the sample absorbed on silica gel surface. Starting from non polar solvent (n-hexane 100%) we slowly precede the chromatography. We increase the polarity of solvent slowly and gradually by adding 1% ratio of the ethyl acetate. All the obtained fractions were examined for the possibility of separated components by TLC analysis. At a stage when no more possible separated compounds were observed by increasing the polarity of solvent system to 50% of ethyl acetate, the column was stopped. The fully separated components were concentrated and stored in closed vials. The semi purified compounds were subjected to small scale chromatography for further purification.
3.3. Viability assay for MCF-7 cell line MCF-7 cells from Manassas, VA, USA were seeded using 96-well micro-plate reader during the night and subsequently incubated in CO2 incubator at 37 °C. Cells density was adjusted to 0.8 × 105 cells/mL as discussed in previous section. Moreover, MCF-7 cells were treated with FLS (3–180 µM) for 24, 48, and 72 hrs and subsequently 5 mg/ml MTT was added [29]. MCF-7 cells were incubated and absorbance was measured at wavelength of 570 nm (BioTek Instruments, Winooski, VT, USA). Finally the percent cell viability was calculated using formula; 31
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%cellviability =
AbsorbanceofSample × 100 Absorbanceofcontrol
3.4. Molecular docking study To study the binding mode of ligands in the active site of tyrosine kinase, the molecular docking was carried out. The three dimensional crystal structure of tyrosine kinase (pdb ID: 1 M17) [30] was downloaded from protein databank. The 3D structure was then subjected to protonation and energy minimization using default parameters of MOE. Three dimensional structures of both compounds were built by using Molecular Builder Module program implemented in MOE and saved as a (.mdb) file for molecular docking. Subsequently, the energy of both compounds were minimized up to 0.05 Gradient using MMFF94s force field implemented in MOE. Both compounds were docked into the active site of protein using the Triangular Matching docking method (default) and 10 different conformations for each compound were generated. To obtain minimum energy structures the ligands were allowed to be flexible during docking. At the end of docking, the predicted ligand–protein complexes were analyzed for molecular interactions and their 3D images were taken by using LigPlot implemented in MOE.
Fig. 2. Results of cytotoxicty of beta-sitosterol against NIH/3T3 following MTT assay. Results represent mean ± SEM of three independent experimental assays. Values significantly different ***P < 0.001 in comparison to standard drug Doxorubicin.
weight of 412 by ESI-MS (Figure S5, Supplementary file) and the details of 1H NMR is shown in Figure S4 (assignments are shown in Supplementary file) using a 400 MHz instrument with chloroform-D as the solvent. In the current study, beta-sitosterol exhibited considerable cytotoxicity against NIH/3T3, HeLa and MCF-7 cell with 67.05 ± 2.08, 79.63 ± 2.34 and 71.50 ± 1.57% lethality respectively at 1 mg/ml concentration. Median inhibitory concentrations calculated from dose response curve against NIH/3T3, HeLa and MCF-7 cells were 440, 170 and 200 µg/ml respectively (Figs. 2–4). To discuss the interaction detail of ligands and protein, the docked conformations of the compounds were studied. It was observed that the hydroxyl group of beta-sitosterol compound shows hydrogen bonding with the carbonyl oxygen of Asp776 active site residue. Whereas the stigmasterol also shows single hydrogen bonding. The oxygen atom of hydroxyl group of this compound involve in hydrogen bonding with the hydroxyl group of Thr766 residue. As the hydroxyl groups of both compounds are involve in hydrogen bonding, so the compounds may be show activity due to the presence of this OH group. The 3D binding mode of these compounds are given in Fig. 5 and their energies and interaction details are shown in Table 1. Previous studies regarding anti-cancer potentials of beta-sitosterol reported significant cytotoxic potentials against HT-29 (colon cancer) [31,32], LNCaP (prostate cancer) [33], MDA-MB-231 (breast cancer) [33], HL60 (Caucasian promyelocytic leukemia) [34], U937 (human leukemic cells), COLO320 (human colorectal cancer cells) [35], MCA102 (fibrosarcoma cells) [36] and SiHa (uterine cervix cancer cells) [37]. Results of various studies indicate that beta-sitosterol hamper the growth of various cells linked to the activation of the sphingomyelin
3.5. Statistical analysis Results were presented as mean ± SEM of three independent experimental assays. one way ANOVA followed by Bonferroni's Multiple Comparison Test was applied to the data used for the comparison of positive control with the test groups at 95% confidence interval. Values significantly different *P < 0.05, **p < 0.01 and ***P < 0.001 in comparison to standard drug doxorubicin. 4. Results and discussion The beta-sitosterol was isolated from both the chloroform and ethyl acetate fractions in a total amount of 2.5 g. The compound appeared as white powder with 0.56 Rf value in n-hexane and ethyl acetate at a ratio of 4:1. The compound appeared with molecular weight of 414 by ESI-MS (Figure S3, Supplementary file) and the details of 1H NMR are shown in Supplementary file and Figure S1 using a 400 MHz instrument with chloroform-D as the solvent. The 13C NMR signals appeared at 12.00, 13.91, 18.73, 18.78, 19.89, 22.02, 24.67, 25.05, 27.96, 28.36, 31.11, 31.77, 32.25, 33.85, 35.47, 36.48, 37.77, 38.99, 43.25, 45.20, 49.27, 56.96, 57.75, 63.11, 63.81, 64.36, 64.88, 65.98, 66.81, 71.26, 82.39, 101.50, 121.33, 141.29, 144.89 and 148.01 as shown in Figure S2, Supplementary file. The beta-sitosterol was isolated from both the chloroform and ethyl acetate fractions in a total amount of 275 mg (Fig. 1). The compound appeared as white powder with 0.44 Rf value in n-hexane and ethyl acetate at a ratio of 4:1. The compound appeared with molecular
Fig. 1. Chemical structures of beta-sitosterol and stigmasterol.
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concentrations, modulate antioxidant enzymes and arrest cellular growth at G2/M phase, with no significant cytotoxicity against noncancerous cells [36,39,42]. In the current study, stigmasterol was observed more effective against MCF-7 and NIH/3T3 cells by killing 87.50 and 81.45% cancerous cells respectively at 1 mg/ml concentration. A dose dependent lethality was observed against all cells as shown in Fig. 6. Stigmasterol showed 77.25% cyctotoxicity against HeLA cells at 1 mg/ml concentration in MTT assay. The IC50 values for HeLA, MCF-7 and NIH/3T3 cells were 170, 60 and 140 µg/ml respectively. Standard drug doxirubicin showed 89.40, 92.00 and 88.53% cytotoxicity against NIH/3T3, HeLa and MCF-7 cell respectively. The LD50 against these cells were 15, 7 and 11 µg/ml respectively. Several investigators reported the anticancer potentials of stigmasterol. Stigmasterol isolated from Cacalia tangutica exhibited significant cytotoxicity against Spodoptera litura cells [43]. In another study, Gomez MA et al., reported that stigmasterol isolated from Achillea ageratum showed considerable cytotoxicity against McCoy and Hep-2 cells as compared to standard drug [44]. Carthami flos enriched with stigmasterol has been reported to markedly inhibit tumor growth in carcinogenesis models [45]. Stigmasterol rich extracts of Edgeworthia gardneri and Couepia polyandra possess lyase inhibitory activity of DNA polymerase β and also potentiate the cytotoxic potentials of bleomycin in A549 cell lines [46].
Fig. 3. Results of beta-sitosterol cytotoxicity against HeLa Cells in MTT assay.
5. Approval from ethical committee The study protocol was approved by Departmental Research Ethics Committee (DREC), Department of Pharmacy, University of Malakand via reference no. DREC/20160502/01. 6. Consent for publication Not applicable in this section. Fig. 4. Results of beta-sitosterol cytotoxicity against MCF-7 Cells using MTT assay.
7. Availability of supporting data The data presented in this manuscript belong to research work (Thesis) of Dr. Muhammad Ayaz and has been deposited to the repository of Higher Education Commission (HEC) of Pakistan (www.hec. gov.pk). The data is not published anywhere yet. However, the materials are available to the researchers upon request. Supplementary data is available as Figure S1,S2,S3,S4 and S5.
cycle [32,38], arrest cell cycle [39] and initiation of apoptotic cell death [40]. Beta-sitosterol is also reported to uphold apoptosis via activation of capases, first apoptosis signals [41], phosphorylation of p38 mitogen-activated protein kinase (MAPK) and signal-regulating kinase (ERK) [40]. It also inhibit malignant cells propagation at low
Fig. 5. 3D binding mode of the compounds (a) Beta-sitosterol and (b) Stigmasterol in the binding pocket of tyrosine kinase. The compounds are represented in gray color, whereas the binding site residues have green color.
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Table 1 The docking score, energies and predicted interactions of the ligands in the active site of tyrosine kinase. S. No
Compounds
Docking score
Binding Energy
Binding affinity
Predicted interactions Ligand Receptor Interaction Distance
1 2
Beta-sitosterol Stigmasterol
−7.266 −4.89
−41.21 −41.04
−7.76 −7.68
O 79 OD1 ASP776 H-donor 2.0 O 77 OG1 THR766 H-acceptor 1.8
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Fig. 6. Stigmasterol cytotoxicity against HeLa, MCF-7 and NIH/3T3 cells.
8. Competing interests The authors declare that they have no competing interests Funding This research has received no specific grant from any funding agency in the public, commercial, or not for-profit sectors. 10. Authors' contributions MA, AS, NZ conceived the project, carried out experimental work, data collection, evaluation, literature search and manuscript preparation. MJ and FU supervised research work, helped in study design and drafted the final version of the manuscript. AW performed molecular docking studies. All authors read and approved the final manuscript for publication. Acknowledgements We cordially acknowledge Department of Pharmacy, University of Malakand, Khyber Pakhtunkhwa (KP), Pakistan, for providing laboratory facilities to conduct this research. AS is thankful to the Higher Education Commission of Pakistan for financial support under project No 22-1/HEC/R&D/PPCR/2018. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.steroids.2018.11.005. References [1] R.E. Ostlund Jr, Phytosterols in human nutrition, Annu. Rev. Nutr. 22 (1) (2002) 533–549. [2] W. Ling, P. Jones, Dietary phytosterols: a review of metabolism, benefits and side effects, Life Sci. 57 (3) (1995) 195–206.
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