Plant collection and preparation of extract
Mentha piperita (MP), Mentha longifolia (ML), and Origanum majorana (OM) were collected from the local cultivations in Taif region of Saudi Arabia during the periods of February- March, 2022. Fresh plant leaves were dried under sun for 8 days at 18oC, and in oven at 40°C until dry, individually. The dried samples were milled, and powder was stored at room temperature, respectively. 1g of sample was dissolved with 20 mL of distilled water and boiled for 10 min. Then, allowed to reach till room temperature and centrifuged at 3000×g for 10 min. The sample was filtered with a filter paper (Whattman No. 2). The concentrated extracts (50 mg/mL) were collected and stored at 4oC for further use (Fig. 1).
Qantification of total polyphenol content
Total polyphenolic contents (TPC) were qantified according to the previously published method by Salamatullah et al. [18]. In brief, 25 µL of extract was mixed with 1500 µL of water and 125 µL of folin–ciocalteau reagent was added. After 1 min incubation, 375 µL of sodium carbonate (20% w/v) have been added and mixed with 475 µL of distilled water. Finally, the total content was kept at room temperature for 30 min. The absorbance was recorded with a spectrophotometer at 760 nm. The amount of TPC have been presented as milligram of gallic acid equivalent per gram dry weight of the test sample (mg GAE/g DW).
Quantification of total flavonoid content
To qantify the total flavonoid content (TFC), the extract (250 µL) have been mixed with 1000 µL of distilled water. Then, 75 µL of sodium nitrite (5% w/v) and 75 µL of aluminum trichloride (10% w/v) were added. After incubation at room temperature for 5 min, 500 µL of sodium hydroxide (1M) and 600 µL of distilled water were added, as previously described by the method [18]. The absorbance was recorded with a spectrophotometer at 510 nm. The TFC was expressed as milligram catechin equivalent per gram dry weight of the sample (mg CE/g DW).
DPPH scavenging
The free radical scavenging capacity (DPPH) of the extracts were assayed using the method described by Salamatullah et al [18]. In dark condition, 130 µL of extract have been mixed with 2.5 mL of DPPH solution and incubated for 30 min. The absorbance was determined at 517 nm using a spectrophotometer
Reducing power
Antioxidant power (RP) was assessed throgh the ferric ion reducing capacity of the extract according to the method described by Salamatullah et al [18]. 500 µL of extract was mixed with 1250 µL of 0.2 M-phosphate buffer (pH 6.6) and 1250 µL of 2% potassium ferricyanide and incubated for 20 min at 50°C. Then, 1250 µL of 10% trichloroacetic acid was added, and the reaction mixture was spinned at 3000×g for 10 min. An aliquot 1250 µL was obtained from the supernatant, then 1250 µL water and 250 µL of ferric chloride was added. The absorbance was measured spectrophotometrically at 700 nm.
GC-MS analysis
GC-MS analysis conducted on a Perkin Elmer Clarus 600 T gas chromatography coupled with a Turbo mass spectrometer following the method of Nasr et al [19] with minor modification. 1 µL of sun dried extract have been injected into the Elite-5MS column of 30 meter, 0.25 µm film thickness, 0.25 mm internal diameters. The initial oven temperature was set for 40ºC for 2 min, further increasing to 150ºC at a rate of 5ºC for 2 min, and finaly set with 300ºC at a rate of 5 ºC for 5 min. The injector temperature have been set as 280ºC and the interface temperature set as 250ºC. Helium gas at a flow rate of 1.0 mL/min was used as the mobile phase. The electron ionization mode have been used to detect mass spectra between the scanning of 40 to 600(m/z). Finally, mass ranges of unknown hits were comapred and identified by the National Institute of Standard and Technology library (NIST) database.
Green synthesis of silver nanoparticles
30 mL of sun dried extracts (each sample have two extract concentrations: 1 and 2 mg/mL) was added to 90 mL of 2 mM silver nitrate (AgNO3) solutions in room temperature. The total reaction was carried out with the stable pH of 12 adjusted using 0.1M NaOH solution (dissolved in deionized water) with continuous stirring with magnetic stirrer (200 rpm, 90 min). The formation of Ag+ to Ag0 was confirmed by the color change from colorless to brown color of the reaction mixture. The total reaction mixture was allowe to dry by keeping them in oven at 60°C for 24 h (Fig. 2). The samples became as follows: AgNO3 2 mM, OM extract, MP extract, ML extract, OM-AgNPs (1 and 2 mg/mL), MP-AgNPs (1 and 2 mg/mL), ML-AgNPs (1 and 2 mg/mL).
Structural characterization of silver nanoparticles
To validate the formation of silver nanoparticles, the reduction of Ag+ ions was examined by a UV-visible spectrophotometer (UV-2450 double-beam, Shimadzu, Tokyo, Japan). The UV-visible spectra of all the samples were measured with a random wavelength range of 200–900 nm. To determine the functional groups, Nicolet 6700 Fourier- transform infrared (FT-IR) spectrometer (Waltham, MA, USA) was used between the wavelength range of 500–4000 cm− 1. Scanning electron microscope (SEM) (JEM-1011, JEOL Ltd., Tokyo, Japan) working at an acceleration voltage of 160 kV was used to charecterize the morphology of synthesiszed nanoparticles. The distribution of nanomaterials size was determined by using a Zeta sizer (ZEN3600 Malvern, Malvern, UK) [20].
In vitro cell culture
Chemicals
Human umbilical vein endothelial cells (HUVECs) were purchased from American Type of culture collection (ATCC, Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM), Ethylenediaminetetraacetic acid (EDTA), and trypsin were purchased from Gibco (Paisley, UK). Cell culture materials such as FBS (fetal bovine serum) was obtained from Hyclone Laboratories, United States. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], nuclear stain propidium iodide, JC-1 stain, and all other fine chemicals have been purchased from Sigma-Aldrich (St. Louis, MO, United States).
Human umbilical vein endothelial cell (HUVECs) culture
HUVECs were allowed to grown in 10% DMEM and 1% antibiotics (Penicillin/Streptomycin complex) at 37°C in a humidified 5% CO2 incubator. HUVECs were seeded in a T75 flask and cultured until 80% confluence and continued with the subculture process every 3 days.
Cytotoxicity analysis
HUVECs were seeded in 96- well plate at a number of 1 × 104 cells/well with growth medium, then allowed to adhere overnight at 37°C and 5% CO2 incubator. After confirming the 80% confluence, the growth medium replaced with fresh media containing an increasing concentration (0, 5, 10, 20, 40, 80, and 160 µg/mL) of OM, MP, and ML extract and their respective silver nanoparticles (namely OM-AgNPs, MP-AgNPs, and ML-AgNPs) were added to their respective well; control cells were cultured without treatment. After 48 h incubation, 20 µL/well of freshly prepared MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] at a concentration of 5 mg/mL was added and incubated in dark at 37°C and 5% CO2. After 4 h, the supernatant solution was removed gently, the formazan crystals were disolved using DMSO (100 µL/well). The optical density of the solution was measured at 490 nm using Spectro star nano microplate reader from BMG Labtech (Ortenberg, Germany). All experiments were performed 4 times and the average was calculated. Cells viability (%) was determined by the mean absorbance of the sample/ mean absorbance of the control × 100.
Experimental design
According to the results of cytotoxicity 20 µg/mL of OM, MP, and ML extracts and their respective nanoparticles have been selected for further cell morphology and molecular biology analysis. Two sets of HUVECs were seeded in 24 well plate and allowed to grow for 24 h to reach confluence. After confluence, one set is maintained as normal observation without H2O2; the second set of HUVECs were treated with 10 µM of H2O2 for 30 min to induce oxidative stress. After 30 min incubation, the HUVECs were treated with 20 µg/mL of OM, MP, and ML extracts and their respective nanoparticles for 48 h. At the end of experiment, normal and H2O2 induce oxidative stressed cells were compared to determine the nuclear damage using propidium iodide staining, mitochondrial oxidative capacity by JC-1 staining and antioxidant & tumor suppressor related gene expression analysis have been studied.
Analysis of nuclear damage
Changes in the cellular morphology for characteristic nuclear damage, pyknosis or apoptotic variations in OM, MP, and ML extracts and their respective silver nanoparticles treatment in normal and H2O2 induced oxidative stress were determined using light microscopy and propidium iodide (PI) staining images were analysed using fluorescence microscopy.
Quantitative real-time PCR analysis
Normal and oxidative stress induced vehicle control, OM, MP, and ML extracts and their respective nanoparticles treated HUVEC’s total RNA was isolated, then the cDNA was synthesized using Fastlane® Cell cDNA kit using qPCR instrument (Applied Biosystems). The mRNA expression levels of oxidative stress (LPO), antioxidant (SOD, GPx, and GSK-3β), and tumor suppressor (CYP1a and p53) and β-actin (house keeping gene) have been analyzed. The gene amplification values (ΔCt) were determined by the difference between Ct (treated) and Ct (control). Gene expression were plot using the expression of 2−ΔΔCt value.
Statistical analysis
The experiments were run in triplicate and the data have been presented as mean values ± SD (standard deviation). Statistical package for social sciences (SPSS-Version 29, 2000–2008; SAS Institute Inc., NC, USA) have been used to analyze the differences between and withing the groups (ANOVA) and the significant differences were analyzed using Duncan’s multiple range test at a confidence interval of 95% (p ≤ 0.05 and p ≤ 0.001).