Boswellia dalzielii stem bark
Boswellia dalzielii stem bark was gathered at Dutsen Hanwa area of Zaria (latitude 11° 06′ 40.61″ N, longitude 7° 43′ 21.72″ E), Kaduna state, Northern Nigeria, in the month of October, 2017, and was authenticated at Biological Sciences Department, Ahmadu Bello University, Zaria, and a voucher specimen was deposited with voucher number 900121.
Extraction, fractionation, and phytochemical identification
The chloroform fraction of Boswellia dalzielii stem bark was prepared as earlier described [11]. Further, the chloroform fraction was subjected to silica gel (70–230 mesh) column chromatography. Briefly, silica gel was mixed with n-hexane to form a homogenous suspension/slurry and stirred using a glass-stirring rod to remove bubbles. The silica gel slurry was then poured into a glass column. The sample to load on the column was prepared by dissolving 3.7 g of the chloroform fraction in 50 ml of hexane. A preparative thin layer chromatography (TLC) had been carried out in which the chloroform fraction were separated in a mobile phase of chloroform/methanol (99:1). To the solution, 10 g of silica was added and mixed by stirring with a glass rod. The mixture was allowed to dry at room temperature. The dried silica/Boswellia dalzielii fraction mixture was layered on the column layer bed. The column was first eluted with n-hexane as the mobile phase with the polarity increasing by 5% increments of chloroform. After getting to 100% chloroform, the polarity was further increased by 5% increments of methanol (Fig. 1). Six sub-fractions were collected in glass beakers. The collected fractions were concentrated to dryness at room temperature.
Cell culture
The head and neck cancer cell line (AW8507) was generously obtained from the Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi-Mumbai, 410,210, India, and were cultured in Dulbecco’s minimum essential medium (DMEM) with 10% fetal bovine serum (FBS) and 50 μg/mL antibiotics. The cells were incubated at 37 °C in CO2 incubator in an atmosphere of humidified 5% CO2 and 95% air. The cells were maintained by sub-culturing in 25-cm3 tissue culture flasks. Cells growing in the exponential phase were used for cell death determination assay.
Determination of cell death by Annexin V-propidium iodide (AnnV-PI)
Mode of cell death induced by the chloroform fraction of Boswellia dalzielii stem bark (CLBD) was evaluated by using Annexin V-FITC Apoptosis Detection Kit (BD Biosciences). Briefly, the cells of AW8507 (3 mL, 1 × 105 cells/mL) were seeded into each well of 6-well plates and incubated for 24 h (cell attachment and recovery). After incubation (24 h), the untreated and treated (20 µg/mL) cells were harvested and washed with cold phosphate-buffered saline (PBS) solution. Subsequently, the cells were mixed with 190 µL of pre-diluted binding buffer (1 ×) containing Annexin Fluorescein 5-isothiocynate (Annexin V-FITC) (5 µL) and of propidium iodide (PI) (1 µL) (100 μg/mL) and further incubated for 15 min at 37 °C in the dark. Subsequently, 400 µL of binding buffer (1 ×) was added into each tube. The percentage of cell undergoing apoptosis and necrosis was quantified using a flow cytometer (Becton Dickinson Accuri, San Diego, CA, USA) equipped with Cell Quest software within 1 h.
HPLC–MS analysis of chloroform extract sub-fractions
The sub-fractions obtained from the chloroform extract were subjected to further analysis by high-performance liquid chromatography and mass spectrometry. For the analysis, 0.5–1.0 mg/ml of CLBD sub-fractions was dissolved in methanol. After that, it was filtered through 0.45 µm membrane filter prior to the injection into HPLC–MS system. HPLC–MS system was Agilent 1260 infinity HPLC (UV Detector wavelength, 225 ± 50 nm bandwidth), auto-sampler (injection volume 1 µl), and thermostated column (Phenomenex Kinetex XB-C18, 50 × 4.6 mm, 2.6 µm) (40∘C). The mobile phase A was 99.9% water with 0.1% acetic acid, while mobile phase B was 99.9% acetonitrile with 0.1% acetic acid. The flow rate was 2 mL/min. Agilent 6130 single quadrupole mass spectrometer (scanning in ES + / − and APCI over 70–1100 m/z) and Agilent 1290 Infinity II Evaporative Light Scattering Detector (ELSD) was used for measuring mass spectra. Agilent Mass Hunter software was used for data acquisition and processing. Compounds in the chloroform extract sample were tentatively identified by comparison of their monoisotopic/exact masses, mass fingerprints, and reference literatures.
Reverse virtual screening of Acety-11-keto-beta-boswellic acid on selected proteins of the cell cycle
A reverse virtual screening (rVS) was performed to specifically determine the binding affinities of Acety-11-keto-beta-boswellic acid (AKBA) in the selected proteins [12]. Briefly, a total of 24 proteins (Forkhead box protein M1 (FOXM1), Aurora kinase, checkpoint kinase 1 (CHK1), serine/threonine polo-like kinase 1 (PLK1), Cell Division Cycle 25 homolog C (CDC25C), Cyclin B1, p-CDK1, DNA topoisomerase 2 alpha (TOP2A), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), proto-oncogene Neu (HER2), mesenchymal epithelia transition factor (c-MET), tumor protein p53 (TP53), mouse double minute 2 homolog protein (MDM2), Janus tyrosine kinase (JAK), signal transducer and activator of transcription 3 (STAT3), mitogen activated protein kinase (MAPK), vascular endothelia growth factor receptor (VEGFR), caspase 7, caspase 3, proto-oncogene tyrosine-protein kinase Src, B-Raf proto-oncogene serine/threonine-protein kinase, epidermal growth factor receptor (EGFR), and αβ tubulin) were selected to form a biomolecular library. The individual crystal structures of the proteins were retrieved from the protein data bank (PDB). The PDB entry values are 3G738, 4BYJ9, 2C3K10, 2RKU11, 3OP312, 6GU413, 6GU413, 5GWK14, 5JHB15, 3D0E16, 3PP017, 4KNB18, 5O1C19, 5LN220, 3KRR21, 6NUQ22, 5MTX23, 4AGD24, 1I5125, 1RHQ26, 3EL727, 3C4C28, 5XDL29, and 1TUB30. The protein structures were individually prepared on the graphical user interface (GUI) of University of California at San Francisco (UCSF) chimera. The proteins were further prepared on the ADT interface where non-polar hydrogens and standard charges were added. Using coordinates from the co-crystallized ligands, target binding sites of the proteins were defined using a gridbox with size and center values that varied across the proteins. Each protein was then saved in.pdbqt formats to form the protein library against which the test compound was virtually screened. The 2-dimensional (2D) structure of the test compound, acetyl-11-keto-beta-boswellic acid, was prepared using the MarvinSketch graphical user interphase, after which it was structurally and geometrically optimized with a universal force field (UFF) using the Avogadro module [13]. Afterward, acetyl-11-keto-boswellic acid was prepared on AutoDock tools, torsions adjusted and rotatable/non-rotatable bonds were set. This was also saved in.pdbqt format. Reverse virtual screening was then performed using the AutoDock Vina software. Binding scores of acetyl-11-keto-beta-boswellic acid to each protein were then obtained. On the other hand, Docetaxel (TXL) was used as the standard molecule for comparatively measuring the binding activity of acetyl-11-keto-beta-boswellic acid (AKBA). To this effect, docetaxel was retrieved from the PubChem drug repository after which it was optimized similarly to AKBA (Fig. 2). Docetaxel (TXL) was docked singly to αβ Tubulin using AutoDock tools since docetaxel reportedly targets and bind the β domain of αβ Tubulin.
