SUPPLEMENTARY MATERIAL Achillea fragrantissima (Forssk.) Sch.Bip. methanolic extract exerts potent antimicrobial activity and causes cancer cell death via induction of caspase-dependent apoptosis and S-phase arrest Mohammed Khaled Bin Breaka*, Kareem Mahmoud Younesa,b, Salem Elkahouic,d, Rahamat Unissae, Suliman Ayad Alfahidata, Khalid Salem Alshawia and Amr S. Abouzieda,f* a Department of Pharmaceutical Chemistry, College of Pharmacy, University of Hail, Hail, Saudi Arabia; bDepartment of Analytical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt; cDepartment of Biology, College of Science, University of Hail, Hail, Saudi Arabia; dLaboratory of Bioactive Substances, Center of Biotechnology of Borj Cedria (CBBC), BP 901, Hammam Lif 2050, Tunisia; e Department of Pharmaceutics, College of Pharmacy, University of Hail, Hail, Saudi Arabia; fNational Organization for Drug Control & Research, Cairo, Egypt *corresponding authors. E-mail addresses: m.binbreak@uoh.edu.sa (M.K. Break), as.ibrahim@uoh.edu.sa (A.S. Abouzied) Achillea fragrantissima (Forssk.) Sch.Bip. methanolic extract exerts potent antimicrobial activity and causes cancer cell death via induction of caspase-dependent apoptosis and S-phase arrest This study was done to evaluate the anticancer potential of Achillea fragrantissima (Forssk.) Sch.Bip. leaves methanolic extract in detail for the first time, in addition to investigating its antimicrobial activity. The antimicrobial assay revealed that the extract exerted high activity against P. vulgaris (MIC = 156.25 µg/ml) and C. albicans (MIC = 625 µg/ml), while moderate activity was observed against other microbes. The extract was also screened against HepG2, A549, HCT116 and MCF7 cancer cells and was found to be active across all cells with highest selectivity and cytotoxic activity being observed for A549 cells (IC50 = 1.21 µg/ml). Further mechanistic studies on A549 cells showed that the extract resulted in S-phase arrest and induced apoptosis via activation of caspase-3, p53 and Bax, in addition to downregulation of Bcl-2. HR-LCMS analysis indicated the presence of 3-hydroxycoumarin, quercetin 3,3'-dimethyl ether and skullcapflavone II which might be responsible for the extract’s bioactivity. Keywords: Achillea fragrantissima (Forssk.) Sch.Bip.; antimicrobial; anticancer; apoptosis; mechanism; LCMS; Compositae 3. Experimental 3.1. Chemicals and Reagents Cancer cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Methanol used for extraction was obtained from Honeywell (Germany), while DSMO, MTT and trypan blue dye were purchased from Sigma (St. Louis, Mo., USA). Fetal Bovine serum, RPMI-1640, HEPES buffer solution, Lglutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from Lonza (Belgium). PRO-PREP protein extraction kit was purchased from iNtRON Biotechnology (Korea). Anti-Bcl-2, anti-Bax and anti-cleaved caspase-3 were purchased from Cell Signaling Technology (MA, USA), while anti-p53 was obtained from Abcam and anti-β-actin was obtained from Sigma (St. Louis, Mo., USA). 3.2. Collection and extraction of plant material Achillea fragrantissima was obtained from Hail, Saudi Arabia (27°21'10.5"N 41°31'52.3"E) in April at its pre-flowering growth stage. The plant material was identified and authenticated by Dr. Ibrahim al-Muhaysen, Shaqra University, and a voucher specimen (No. UOHCOP001) has been deposited at the College of Pharmacy, University of Hail. The leaves were collected from the plant and ground using a mortar and pestle to give a powder of about 700 g. The plant material was macerated in methanol for 3 days and the resulting extract was then filtered and finally concentrated to yield the crude extract. 3.3 Antimicrobial assays 3.3.1 Target microbes Aspergillus fumigatus (RCMB 002008), Candida albicans (RCMB 005003 (1) ATCC 10231), Staphylococcus aureus (RCMB010010), Bacillus subtilis (RCMB 015 (1) NRRL B-543), Escherichia coli (RCMB 010052 ATCC 25955) and Proteus vulgaris (RCMB 004 (1) ATCC 13315). 3.3.2 Qualitative antimicrobial assay (Agar well diffusion method) Qualitative antimicrobial assay was performed using the agar well diffusion technique. In brief, a volume of the microbial inoculum was spread over the whole agar plate surface, then a hole of 6 mm was made using a sterile cork borer and 100 µl of the extract was added to the hole. The extract was added at a concentration of 10 mg/ml. The positive controls ketoconazole and gentamycin were used at a concentration of 100 µg/ml and 4 µg/ml, respectively. The resulting inhibition zone diameter was later measured. 3.3.3 Quantitative antimicrobial assay (Broth microdilution method) Quantitative antimicrobial assay was performed via the broth microdilution method. In brief, two-fold serial dilutions of the extract in a liquid growth medium was performed. The dilutions were made in a microtitre plate. A volume of the microbial suspension was then added to each well of the plate, and finally the wells containing the extracts and microbes were incubated. Minimum inhibitory concentration (MIC) was finally determined visually in accordance with the CLSI protocol. 3.4 In vitro anticancer evaluation 3.4.1 Cell culture Cancer cells were cultured in RPMI-1640 media supplemented by 10% FBS and 50 µg/ml gentamycin. Cells were maintained at 37ºC in a humidified atmosphere with 5% CO2 and were subcultured 2-3 times a week. 3.4.2 Cell viability assay Cancer cells were seeded on a 96-well plate at 5×104 cells per well and then incubated for 24 h. After incubation, the extract was added to the cells at 10 different concentrations followed by incubation for 72 h. Some cells were left untreated to act as controls. MTT solution was then added and resulting absorbance was read via a microplate reader (SunRise, TECAN, Inc, USA) at 590 nm. IC50 calculations were performed via Graphpad Prism software (San Diego, CA. USA). 3.4.3 Cell-cycle analysis Cell-cycle analysis was performed using the CycleTEST PLUS DNA Reagent Kit (Becton Dickinson Immunocytometry Systems, San Jose, CA). A549 cells were treated with the extract at IC50 concentration for 72 h and were then stained with propidium iodide stain following the procedure provided by the kit. Untreated A549 cells were also exposed to the same procedure. The cells were then analysed using the flow cytometer BD FACS Calibur (BD Biosciences, San Jose, CA), while cell-cycle distribution was calculated using CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA). 3.4.4 Annexin V-FITC assay Briefly, A549 cells were treated with extract at IC50 concentration for 72 h, and the cells were then harvested and rinsed twice in PBS (20 min each) followed by binding buffer. Cells were later re-suspended in 100 µl of kit binding buffer with the addition of 1 µl of FITC-Annexin V (Becton Dickinson BD Pharmingen, Heidelberg, Germany) followed by 40 min incubation at 4 °C. This was followed by washing the cells and re-suspending them in 150 µl of binding buffer with the addition of 1 µl of DAPI (1 µg/ml in PBS) (Invitrogen, Life Technologies, Darmstadt, Germany). Untreated A549 cells were also exposed to the same procedure. Finally, the cells were analysed using the flow cytometer BD FACS Calibur (BD Biosciences, San Jose, CA). 3.4.5 Western blot assay In brief, A549 cells were treated with the extract at IC50 concentration for 72 h. The cells were later harvested and the cell lysate was obtained. 25 µg of protein lysate was mixed with SDS‐loading buffer, denatured by boiling at 95°C, cooled on ice, vortexed for 30 seconds and finally loaded into SDS‐polyacrylamide gel. The protein lysate was then separated and transferred onto polyvinylidene fluoride membrane (Bio‐Rad), which was blocked with 5% dry milk in Tris‐buffered saline/Tween‐20 (TBST). The membranes was then incubated with the corresponding primary antibody against cleaved caspase‐3 (1:500 dilution), p53 (1:750 dilution), Bax (1:1500 dilution), Bcl-2 (1:1000 dilution) and β‐actin (1:2000 dilution) overnight at 4 °C. The blots were then washed with TBST and incubated with matched horseradish peroxidase (HRP)‐linked secondary antibodies (Dako, Denmark), followed by washing with TBST. This was followed by membrane incubation with chemiluminescence Western Lightning ECL (Perkin Elmer, Waltham, MA) and the bands were finally visualised. 3.5 HR-LCMS analysis The phytochemical constituents of Achillea fragrantissima methanolic extract were analysed using a UHPLC-PDA-Detector 323 Mass Spectrophotometer (HR-LCMS 1290 Infinity UHPLC System, 1260 Infinity Nano HPLC with Chipcube, 6550 iFunnel QTOFs) (Agilent Technologies, Santa Clara, USA). The liquid chromatographic system consisted of an HiP sampler, binary gradient solvent pump, column compartment, and Quadrupole Time of Flight Mass Spectrometer with a dual Agilent Jet Stream Electrospray ion source. 5 μl of the extract was injected, and this was separated by an SBC18 column (2.1 × 50 mm, 1.8 μm particle size). 1% formic acid in water (solvent A) and acetonitrile (solvent B) were used as solvents with a flow rate of 0.35 ml/min, while MS detection was performed via an MS Q-TOF. The acquisition time was 30 minutes and data were collected using both the positive and negative ionisation modes. The Compounds were identified based on their mass spectra and fragmentation patterns. Tools such as Compound Discoverer 2.1, ChemSpider, and PubChem were mainly used for identifying the extract’s phytochemical constituents. Table S1. Antimicrobial activity of Achillea fragrantissima methanolic extract Zone of inhibition diameter (mm)a Gram-positive Gram-negative bacteria bacteria Fungi S. aureus B. subtilis E. coli P. vulgaris A. fumigatus C. albicans Extract 11.3±0.75 12.4±0.8 13.3±1.29 24.1±1.75 11.6±0.72 14.7±1.54 Gentamycin 25.1±1.63 27.3±1.5 29.7±1.9 26.4±1.2 -- -- Ketoconazole -- -- -- -- 19.2±1.2 20.8±1.4 a Zone of inhibition diameters equal to 14 mm and above were considered to indicate significant antimicrobial activity, 9 mm–13 mm were considered to indicate moderate activity, and less than 9 mm were considered to indicate weak and insignificant activity. Zone of inhibition diameters were reported as mean (Zone of inhibition diameter±SD) of three experiments. Table S2. Minimum inhibitory concentration (MIC) of Achillea fragrantissima methanolic extract against selected bacteria and fungi MIC (µg/ml) Gram-positive Gram-negative bacteria bacteria Fungi S. aureus B. subtilis E. coli P. vulgaris A. fumigatus C. albicans Extract 2500 1250 625 156.25 1250 625 Gentamycin 9.7 4.8 4.8 4.8 -- -- Ketoconazole -- -- -- -- 156.25 312.5 Table S3. IC50 values of Achillea fragrantissima methanolic extract against HepG2, A549, HCT116 and MCF7 cancer cells IC50 (µg/ml)a a HepG2 A549 HCT116 MCF7 Extract 1.92±0.42 1.21±0.34 3.4±0.65 4.83±0.95 Vinblastine sulfate 0.93±0.21 7.12±0.38 1.48±0.38 2.10±0.46 IC50 values are reported as the mean (IC50±SD) of three replicates of two independent experiments. Table S4. IC50 values of Achillea fragrantissima methanolic extract against MRC5 normal cells IC50 (µg/ml)a Extract a Selectivity Index (SI)b MRC5 HepG2 A549 HCT116 MCF7 7.64±0.86 4.0 6.3 2.2 1.6 IC50 values are reported as the mean (IC50±SD) of three replicates of two independent experiments. b SI = (IC50 of MRC5)/(IC50 of cancer cell). Table S5. Chemical compounds identified in Achillea fragrantissima methanolic extract using HR-LCMS Compound Molecular ion MS/MS fragment ion peak (m/z) peaks (m/z) 1.111 156.1010 [M+H]+ 70.0651, 158.1172, 263.1246 3-Hydroxycoumarin 4.011 163.0379 [M+H]+ 59.0486, 117.0324, 145.0276 epi-Tulipinolide diepoxide 4.482 323.1474 [M+H]+ 155.0434, 171.1147, 281.0565 Batatasin III 5.681 245.1163 [M+H]+ 79.0530, 171.0772, 199.1115 Tetraneurin A 6.241 323.1470 [M+H]+ 145.0998, 171.1170, 203.1049 Jasmolone glucoside 7.708 365.1573 [M+Na]+ 117.0695, 199.1108, 227.1063 Allamandin 8.946 331.0793 [M+Na]+ 271.0529, 298.0462, 316.0553 Quercetin 3,3'-dimethyl 9.103 329.0677 [M-H]- 191.0566, 299.0202, 314.0436 9.325 361.0900 [M+Na]+ 118.0831, 285.038,328.0550 3-O-Acetylpadmatin 9.423 359.0785 [M-H]- 286.0124, 329.0316, 345.0574 Picrotoxinin 9.917 315.0845 [M+Na]+ 254.0552, 282.0504, 300.0609 Santin 10.117 345.0950 [M+H]+ 118.0850, 284.0668, 330.0702 Skullcapflavone II 10.529 375.1023 [M+H]+ 317.0638, 342.0716, 359.0733 Neopellitorine A 10.681 230.1529 [M+H]+ 57.0695, 91.0534, 167.1291 Dehydroisochalciporone 11.610 242.1539 [M+H]+ 69.0695, 112.0750, 128.0616 (-) - Sedamine 11.917 242.1527 [M+Na]+ 69.0690, 112.0753, 128.0618 (-) - Myrtenyl 12.140 237.1834 [M+H]+ 83.0724, 128.0612, 177.1131 2-Amino-2- Rt (min) Norbornanecarboxylic acid ether Hydrojuglone glucoside isovalerate Sarmentine 12.547 222.1841 [M+H]+ 57.0691, 93.0697, 167.1281 Homodihydrocapsaicin 13.255 344.2202 [M+H]+ 128.0613, 157.0636, 234.1816 Dihydrotetrabenazine 13.531 342.2046 [M+Na]+ 84.0807, 128.0615, 155.0589 Diphenylpyraline 13.811 282.1841 [M+H]+ 69.0692, 112.0757, 179.1294 Palmitoleamide 15.018 276.2308 [M+Na]+ 57.0692, 152.1037, 167.1264 Cycrimine 15.686 288.2311 [M+H]+ 69.0690, 112.0750, 179.1304 Khayanthone 18.134 593.2727 [M+Na]+ 461.2256, 505.2215, 533.2523 Euphornin 19.626 607.2881 [M+Na]+ 369.4218, 461.2283, 547.2676 Figure S1. Representative cell-cycle histograms showing the effect of Achillea fragrantissima methanolic extract on cell-cycle progression in A549 cells after 72 h of treatment at IC50 concentration. Figure S2. Representative apoptosis quadrant plots illustrating the apoptotic effects of Achillea fragrantissima methanolic extract on A549 cells. The cells were treated with the extract at IC50 concentration for 72 h. Figure S3. Western blot analysis of cleaved caspase-3, p53, Bax and Bcl-2 in A549 cells. Cells were treated with Achillea fragrantissima methanolic extract for 72 h at IC50 concentration. β-actin was used as an internal control. HR-LCMS Chromatograms of Achillea fragrantissima methanolic extract Figure S4. HR-LCMS chromatogram of Achillea fragrantissima methanolic extract in the positive mode Figure S5. HR-LCMS chromatogram of Achillea fragrantissima methanolic extract in the negative mode HRMS Spectra of the identified compounds from Achillea fragrantissima methanolic extract 1. 2-Amino-2-Norbornanecarboxylic acid Figure S6. HRMS and MS/MS spectra of 2-Amino-2-Norbornanecarboxylic acid identified in Achillea fragrantissima methanolic extract 2. 3-Hydroxycumarin Figure S7. HRMS and MS/MS spectra of 3-hydroxycoumarin identified in Achillea fragrantissima methanolic extract 3. epi-Tulipinolide diepoxide Figure S8. HRMS and MS/MS spectra of epi-tulipinolide diepoxide identified in Achillea fragrantissima methanolic extract 4. Batatasin III Figure S9. HRMS and MS/MS spectra of batatasin III identified in Achillea fragrantissima methanolic extract 5. Tetraneurin A Figure S10. HRMS and MS/MS spectra of tetraneurin A identified in Achillea fragrantissima methanolic extract 6. Jasmolone glucoside Figure S11. HRMS and MS/MS spectra of jasmolone glucoside identified in Achillea fragrantissima methanolic extract 7. Allamandin Figure S12. HRMS and MS/MS spectra of allamandin identified in Achillea fragrantissima methanolic extract 8. Quercetin 3,3’-dimethyl ether Figure S13. HRMS and MS/MS spectra of quercetin 3,3’-dimethyl ether identified in Achillea fragrantissima methanolic extract 9. Hydrojuglone glucoside Figure S14. HRMS and MS/MS spectra of hydrojuglone glucoside identified in Achillea fragrantissima methanolic extract 10. 3-O-Acetylpadmatin Figure S15. HRMS and MS/MS spectra of 3-O-Acetylpadmatin identified in Achillea fragrantissima methanolic extract 11. Picrotoxinin Figure S16. HRMS and MS/MS spectra of picrotoxinin identified in Achillea fragrantissima methanolic extract 12. Santin Figure S17. HRMS and MS/MS spectra of santin identified in Achillea fragrantissima methanolic extract 13. Skullcapflavone II Figure S18. HRMS and MS/MS spectra of skullcapflavone II identified in Achillea fragrantissima methanolic extract 14. Neopellitorine A Figure S19. HRMS and MS/MS spectra of Neopellitorine A identified in Achillea fragrantissima methanolic extract 15. Dehydroisochalciporone Figure S20. HRMS and MS/MS spectra of Dehydroisochalciporone identified in Achillea fragrantissima methanolic extract 16. (-) - Sedamine Figure S21. HRMS and MS/MS spectra of (-) - Sedamine identified in Achillea fragrantissima methanolic extract 17. (-) - Myrtenyl isovalerate Figure S22. HRMS and MS/MS spectra of (-) - Myrtenyl isovalerate identified in Achillea fragrantissima methanolic extract 18. Sarmentine Figure S23. HRMS and MS/MS spectra of Sarmentine identified in Achillea fragrantissima methanolic extract 19. Homodihydrocapsaicin Figure S24. HRMS and MS/MS spectra of homodihydrocapsaicin identified in Achillea fragrantissima methanolic extract 20. Dihydrotetrabenazine Figure S25. HRMS and MS/MS spectra of dihydrotetrabenazine identified in Achillea fragrantissima methanolic extract 21. Diphenylpyraline Figure S26. HRMS and MS/MS spectra of diphenylpyraline identified in Achillea fragrantissima methanolic extract 22. Palmitoleamide Figure S27. HRMS and MS/MS spectra of palmitoleamide identified in Achillea fragrantissima methanolic extract 23. Cycrimine Figure S28. HRMS and MS/MS spectra of cycrimine identified in Achillea fragrantissima methanolic extract 24. Khayanthone Figure S29. HRMS and MS/MS spectra of khayanthone identified in Achillea fragrantissima methanolic extract 25. Euphornin Figure S30. HRMS and MS/MS spectra of euphornin identified in Achillea fragrantissima methanolic extract