Source of C. myrrha resin
The resin was purchased from Boswellness (Colchester, VT) with product number 10018 and lot number CM000105. The herb was certified to be organic and free of pesticides, preservatives, and alterations as the resin was wildcrafted by experts in the Federal Republic of Somalia. The resins were stored in a dark and cool cabinet until processed. The remaining resin sample is kept in our department for future reference.
Source of HepG2 cells
Human Hepatocellular Carcinoma (HepG2) cells were kindly gifted by Dr. Ahmed Al-Qahtani, (King Faisal Specialist Hospital and Research Center; Riyadh, Saudi Arabia), originally purchased from the American Type Culture Collection (ATCC, Manassas, VA). The HepG2 cell line is derived from a fifteen-year-old Caucasian adolescent male patient diagnosed with hepatocellular carcinoma.
Chemicals and reagents
Rifampicin (USP grade, ≥97 percent) was obtained from UFC Biotechnology Fine Chemicals (Amherst, NY). Dulbecco's Modified Eagle Medium (DMEM) plus GlutaMax-1 (4.5g/l D-Glucose, 25 mM HEPES, Pyruvate), fetal bovine serum (FBS), TrypLE™ Express, and Dulbecco's phosphate-buffered saline (PBS) were provided from Gibco® (Waltham, MA). NP40 cell lysis buffer was purchased Thermo Fisher Invitrogen (Carlsbad, CA). Methanol (chromatography grade, ≥99 percent) was obtained from Honeywell Riedel-de Haen (Seelze, Germany). Purified carbon dioxide (CO2) gas was provided by Saudi Industrial Gas (Dammam, Saudi Arabia). Ultrapure water was produced using a Millipore (Billerica, MA) system with a resistivity reading of 18.2 MΩ·cm at 25°C.
Preparation of C. myrrha aqueous extracts
The extracts were prepared as 100 percent water by sonication and 100 percent water by boiling. Prior extraction, resin of C. myrrha was fine powered using an electric grinder. For the sonicated extraction, approximately 500 mg of the fine powder were mixed with 10 ml of ultra-pure water and sonicated for 30 min at high-power mode using a Sonics (Newton, CT) Vibra-Cell Ultrasonic Liquid Processor Model GEX-130 probe-sonicator. For the boiling water, approximately 500 mg of resin were mixed with 10 ml of ultra-pure water and boiled until half of the volume was evaporated. The extracts were filtered using a Sartorius stedim biotech (Göttingen, Germany) quantitive ashless paper filter and dried in an incubator set at 50˚C for 3 to 4 days. The remaining dried pellets were weighted and reconstituted with 1 ml of ultra-pure water by vortex until completely dissolved. The reconstituted extracts were stored at cool temperature in dark until used.
Fingerprinting of C. myrrha aqueous extracts by HPLC-UVD
The chromatograms of C. myrrha aqueous extracts were performed using an Agilent (Santa Clara, CA) 1260 infinity high-performance liquid chromatography (HPLC) equipped with an ultra-violet detector (UVD). The major components of C. myrrha were separated using a Phenomenex (Torrance, CA) C18 Kinetex column (250 × 4.6 mm, 5 µm). The multi-wave UVD was set at wavelengths ranging from 225 to 375 nm at 25 nm segments without reference. The gradient programming of methanol and ultra-pure water was as follows: separation-phase (5 to 100 percent methanol, 0 to 35 min), washing-phase (100 percent methanol, 35 to 40 min), and equilibrium-phase (100 to 5 percent methanol, 40 to 45 min). The injection volume was 10 µl at ambient temperature and the total run time was 45 min.
Determination of endotoxin level in C. myrrha aqueous extracts
The levels of endotoxins in the C. myrrha aqueous extracts were determined using the Thermo Scientific™ Pierce™ Chromogenic Endotoxin Quant kit (catalog number, A39553) as per the manufacturer’s instructions.
Culture and treatment of HepG2 cells
The HepG2 cells were cultured in complete DMEM Glutamax medium containing 10 percent (v/v) heat-inactivated FBS, and 1 percent (v/v) of penicillin G (100 IU/ml) and of streptomycin (100 mg/ml) and were incubated at 37˚C in 5 percent CO2 and 95 percent relative humidity. The media was changed 2 to 3 days and sub-cultured when the cell population density reached 70 to 80 percent confluency. Cells were seeded at an appropriate density according to each experimental design. For the cell viability experiment, concentrations ranging from 0.05 to 2000 µg dry extract weight per ml were used for each boiled and sonicated extract. For gene expression assessment, the HepG2 cells were treated with three different concentrations of 1, 15, and 30 µg/ml for each boiled and sonicated aqueous C. myrrha extracts. Different rifampicin concentrations ranging between 0.001 and 50 µM inducing CYP isoenzyme gene expression were used as positive controls.
Cytotoxicity of C. myrrha extracts on HepG2 cells
The percentage of cell viability was determined using Promega CellTiter-Glo® assay (Madison, WI) according to the manufacturer’s instructions. Briefly, the HepG2 cells were seeded at a density of 4×103 cells per well. The untreated and treated cells were incubated for 48 h under cell culture conditions previously described. After incubation, the plate was left at room temperature for 30 min and 100 µl of Cell Titer-Glo® reagent were added for each well and mixed on a shaker for 2min. The plate was left at room temperature for 10 min to settle and the luminescence of each well was read by the Perkin Elmer (Waltham, MA) EnVision Multilabel Reader.
Extraction of mRNA from HepG2 cells
The untreated and treated HepG2 cells were washed with PBS and harvested using TrypLE™ Express solution. The cells were transferred to a 1.5 ml tube and centrifuged at 15,000 rpm for 5 min. The cell pellet was stored at -20°C. The whole RNA was extracted from the treated and untreated cell pellets using the Qiagen (Hilden, Germany) RNeasy Plus Mini kit (catalog number, 74106) as per the manufacturer’s instructions.
Extraction of proteins from HepG2 cells
The HepG2 cells were washed with PBS and 80 µl of NP40 lysis buffer were added to the cell pellet and incubated on ice for 30 min with repeated vortexing at 10 min intervals. The cell lysate was transferred to 1.5 ml tube and centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant containing the whole cell lysate rich in proteins was transferred to fresh 1.5 tube and kept at -20°C. The amount of proteins was measured using the Molecular Probes® Life Technologies (Eugene, OR) Qubit® Protein Assay kit (catalog number, Q33211) as per the manufacturer’s instructions.
RT-qPCR for CYP Gene Expression assessment
Complementary DNAs (cDNAs) at 50.0 ng per well were first produced from the whole RNA extracts via reverse transcription using Thermo Fisher Scientific Applied Biosystems™ High Capacity cDNA Reverse Transcription kit. The quantitative real-time PCR was performed using a Thermo Fisher Scientific SYBR Green PCR Master Mix kit and performed on Applied Biosystems 7900 real-time PCR system. For each analysis, a negative control was prepared using all reagents except the cDNA template. The housekeeping gene (β-actin) was used as an internal control and cytochrome enzymes primers (Table 1) were at 10.0 pmol/µl concentration.
Western blot for CYP protein expression analysis
Protein samples (120 µg per well) were separated on 11 percent SDS–polyacrylamide gels and electro-blotted onto a Thermo Fisher polyvinylidene difluoride (PVDF) transfer membrane 0.45 mm (catalog number, 88518). Primary antibodies (Invitrogen) against mouse monoclonal anti-CYP3A4 antibody (catalog number, MA5-17064), rabbit polyclonal anti-CYP2C19 antibody (catalog number, PA5-13669), mouse monoclonal anti-CYP2C9 antibody (catalog number, MA5-25748) were used as probes to stain the PVDF membranes. Secondary LI-COR (Lincolin, NE) IRDye® goat anti mouse (catalog number, 926-32210) and goat anti- rabbit antibodies (catalog number, 926-32211) were used for detection and measurement of targeted CYP expression. In all gels, a LI-COR Chameleon Due Pre-Stained Protein Ladder (catalog number, 928-60000) was included. The housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control and was detected by Abcam (Cambridge, UK) mouse monoclonal anti-GAPDH antibody (catalog number, ab9485).
Data Analysis
The cytotoxicity values were estimated based on the plotting of common log (log10) values of dry extract weight of C. myrrha versus percentage inhibition of untreated control using the log-inhibition variable-slop (four-parameter) model in Prism GraphPad (San Diego, CA) software version 5.04. For q-PCR, the gene expression values were normalized to β-actin and considered to be positive for induction or suppression if exceeding the two-fold relative to untreated control (i.e. 0.5 or 2.0-fold cutoff) (USFDA, 2020). The results are expressed as mean ± standard deviation from a minimum of three independent cell culture experiments. For Western blot analysis, the protein expression level detected in treated cells was related to the basal protein expression level detected in untreated cells using ImageJ software (http://rsbweb.nih.gov/ij/index.html).