Preparation of aqueous and methanol extracts was carried out by mixing 100 g of Salvadora persica powder with 1 L of distilled water (for aqueous extract) and 95% methanol (Sigma-Aldrich, St. Louis, USA), with methanol extract for 24 h. The mixture was then filtered using Whatman number 1 filter paper, and the filtrate was then evaporated in vacuum evaporator at 60°C (for aqueous) and 40°C (for methanol). The extracts were stored in sterile bottles and kept frozen at −20°C until further use [24 (link)]. Before testing, the miswak extracts were freshly reconstituted in methanol (for methanolic extract) and water (for aqueous extract) at a final concentration of 400 mg/mL which was used to further prepare serial dilutions (400–50 mg/mL).
Methanol
Manufactured by Cytiva
Sourced in United States, United Kingdom
Methanol is a colorless, volatile, flammable, and poisonous liquid chemical compound. It is commonly used as a solvent, fuel, and chemical feedstock in industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Cytiva (2 mentions)
Acetonitrile, by Cytiva (2 mentions)
Ethanol, by Cytiva (2 mentions)
Methanol, by Merck Group (2 mentions)
Axio imager, by Zeiss (2 mentions)
Axioplan microscope, by Zeiss (2 mentions)
Whatman no 1 paper, by Cytiva (1 mentions)
Whatman filter paper number 1, by Cytiva (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Formic acid, by Avantor (1 mentions)
Acetone, by Cytiva (1 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (1 mentions)
Whatman anodisc inorganic filter membrane, by Cytiva (1 mentions)
Nile red, by Merck Group (1 mentions)
Axiocam mrc5, by Zeiss (1 mentions)
Hesperidin, by Merck Group (1 mentions)
Bay 11 7082, by Bio-Techne (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
P38 mapk, by Santa Cruz Biotechnology (1 mentions)
N acetylcysteine, by Bio-Techne (1 mentions)
8 protocols using methanol
1
Extraction and Preparation of Salvadora persica Extracts
2
Extraction and Fractionation of Tabebuia leucostaphylum
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The fresh leaves (with stems) were washed, dried, and pulverized into powder at room temperature using an electric grinder. The fine powder (585 g) was soaked in 2.5 L of methanol (Merck, Darmstadt, Germany) for 15 days, with regular shaking and stirring. The resulting extract was poured through a cotton plug and further filtered through Whatman No.1 paper, before evaporation on a rotary evaporator at 45 °C to afford the crude methanol extract of T. leucostaphylum (MTL: 95.78 g). Same extraction process was repeated three times and all obtained crude extracts were combined before further extraction with solvents. Following the customized protocol of kupchan partitioning [42 (link)], the crude methanol extract (10 g) was further partitioned to yield the following fractions; n-hexane (NTL), dichloromethane (DTL), and n-butanol (BTL) fractions.
3
Extraction of Plant Compounds
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The plant was cut into sizable pieces and air dried away from sun light until dry. The dry plant material was ground into powder using model 4-E grinding mill (Thomas Scientific). 500 g of dry ground plant material was soaked in 3.0 L of dichloromethane: methanol (1:1) (Sigma Aldrich) and extracted several times until complete extraction. The residue was then extracted with 100% methanol and the two extracts were filtered using Whatman's NO.1 filter paper and concentrated separately under vacuum using an mrc rotary evaporator (ROVA-2L).
4
Antioxidant and Phytochemical Characterization
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Tara gum, pectin esterified from citrus fruits with a degree of methoxylation of 55–70%, gallic acid, (+)-hydrated catechin, L-ascorbic acid, hydrogen peroxide (H2O2, 30% v/v), Folin–Ciocalteu reagent, carbonate sodium (Na2CO3) radical 2,2′-diphenyl -1-picrylyhydrazyl (DPPH), radical 2,2′-azino-bis (3-ethylbenzothiazolin-6-sulfonic) (ABTS), potassium persulfate (K2S2O8), ammonium molybdate tetrahydrate ((NH2)2MoO4), sodium molybdate (Na2MoO4), sodium nitrite (NaNO2), sodium phosphate (Na3PO4), aluminum chloride (AlCl3), hydrochloric acid (HCl), sodium hydroxide (NaOH), absolute ethanol, methanol, Whatman No. 3 filter paper, dialysis membrane (MWCO: 12,000–14,000 Da), were purchased from Sigma Aldrich (Sigma Chemical Co., St. Louis, MO, USA). HPLC grade water, formic acid and acetonitrile were purchased from VWR (Chromasolv, VWR International Srl, Milano, Italy).
5
Cytokine Profiling and Antimicrobial Assays
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Dulbecco’s modified media, dimethyl-ethyl-sulphonyl-oxide, HANKS balanced salt solution, histopaque, ethanol, phosphate buffered saline, paraformaldehyde, acetone, dithiothreitol, iodoacetamide, trypsin from porcine pancreas of proteomics grade, formic acid, acetonitrile, HPLC-grade water, methanol and Whatman Mini-UniPrep syringeless filter devices (pore size 0.45 µm) were purchased from Sigma Aldrich (Poole, U.K.). TNF-alpha, interleukin-6 (IL-6) enzyme linked immunosorbent assays (ELISA’s) were purchased from Research and Development Systems (Abingdon, U.K.). Mueller hinton agar was purchased from Oxoid, UK. Braag’s Apple Cider Vinegar and apple cider vinegar tablets (500 mg, Troo healthcare) were purchased from commercial sources.
6
Fluorescent Microscopy Analysis of Water-Dispersible Microparticles
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For the FM analysis, small amounts of wMPs (50 μl) at 1 mg/ml were mounted onto glass slides and observed with a Zeiss Axioplan microscope equipped with an Axiocam MRc5 digital camera. Furthermore, in order to better visualize wMPs, particles were also stained with the dye Nile Red (Merck KGaA, Darmstadt, Germany). Nile Red (NR) stock solution was prepared at 1 g L−1 in acetone (Merck KGaD) and vacuum filtered using pore size 0.1 μm Whatman® Anodisc inorganic filter membrane with a diameter of 25 mm. The staining was carried out by adding 100 μl of NR stock to a solution of 5 ml methanol (Merck KGaD) and 5 ml of milliQ water to give a final of 10 μg ml−1 NR in the suspension of wMPs. An exposure time of 30 min was used (Maes et al., 2017 (link)). The suspension was then vacuum filtered (Whatman® 25 mm Anodisc inorganic filter membrane, pore size 0.1 μm), rinsed with 30 ml methanol and left to dry. The stained wMPs were then analysed by fluorescent microscope (Zeiss, Axio Imager) under green (filter 488009‐9901‐000, excitation/emission 460/525 nm) and red (filter 89060‐9901‐000, excitation/emission 565/630 nm) light.
7
Investigating Cellular Responses to Polyphenols
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Bisphenol A (BPA), methanol (purity ≥99.9%), ethanol (purity ≥99.9%), acetonitrile (purity ≥99.9%), formic acid (FA, purity ≥97.5%), Whatman® filter paper, skim milk powder, chlorogenic acid (purity ≥95.0%), caffeic acid (purity ≥99.0%), p-coumaric acid (purity ≥98.0%), naringin (purity ≥95.0%), hesperidin (purity ≥97.0%), and kaempferol (purity ≥97.0%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). β-actin, Bcl-2, Bax, cleaved caspase-3, ASC, caspase-1, NF-κBp65, IκBα, JNK, p38 MAPK, and ERK antibodies were obtained from Santa Cruz Biotechnology (Paso Robles, CA, USA). HRP goat anti-mouse/rabbit antibodies were obtained from Abcam (Cambridge, MA, USA). Bay 11-7082, PD98059, and N-acetylcysteine (NAC) were purchased from Tocris (Minneapolis, MN, USA).
8
Determination of Chlorophenols by GC-MS
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All reagents were of analytical grade. Pyridine, sodium hydroxide, and concentrated hydrochloric acid were purchased from Reanal (Budapest, Hungary). Hexane, methanol, ethyl acetate, hexamethyldisilazane (HMDS), trifluoroacetic acid (TFA), and reference chlorophenols (CPs 3,2,2,6 from Whatman (Maidstone, UK). Ultrasonic extractions were performed on the Bandelin Sonorex (RK 52 H) apparatus (Bandelin electronic, Berlin, Germany).
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