Eugenol, β-escin, curcumin, berberine hydrochloride, sclareol, capsaicin, parthenolide, ellagic acid, glutathione, L -ascorbic acid were from Sigma–Aldrich (United Kingdom); osthole and pterostilbene were from Stratech (United Kingdom) and mitoquinol from Cayman Chemical Company (United Kingdom); all other NPs were components of the Puretitre natural compound library from Caithness Biotechnologies (United Kingdom). All of the above except Eugenol (70% ethanol), glutathione, and L -ascorbic acid (dH2O) were dissolved in dimethyl sulfoxide (DMSO, Sigma United Kingdom) and added to growth media from the following stock solutions prepared in those solvents: Eugenol, 500 mM; glutathione, 375 mM, L -ascorbic acid, 500 mM; osthole, 200 mM; pterostilbene, 200 mM; β-escin, 50 mM; curcumin, 50 mM; berberine hydrochloride, 200 mM; sclareol, 130 mM; capsaicin, 200 mM; parthenolide, 130 mM; ellagic acid, 33.3 mM, mitoquinol, 2.94 mM.
Sclareol
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
Sclareol is a natural compound extracted from the Salvia sclarea plant. It is a colorless, crystalline solid that has various applications in the chemical industry, particularly in the production of fragrances and pharmaceuticals.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ellagic acid, by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Curcumin, by Merck Group (1 mentions)
Capsaicin, by Merck Group (1 mentions)
Eugenol, by Merck Group (1 mentions)
Parthenolide, by Merck Group (1 mentions)
Osthole, by Merck Group (1 mentions)
Mitoquinol, by Cayman Chemical (1 mentions)
Pterostilbene, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
β escin, by Merck Group (1 mentions)
Berberine hydrochloride, by Merck Group (1 mentions)
Potato dextrose broth (pdb), by BD (1 mentions)
Topo ta cloning kit dual promoter, by Thermo Fisher Scientific (1 mentions)
Absolute ethanol, by Scharlab (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Nucleospin plasmid kit, by Macherey-Nagel (1 mentions)
Acetone, by Merck Group (1 mentions)
Squalene, by Merck Group (1 mentions)
10 protocols using sclareol
1
Natural Compounds Screening Protocol
2
Sclareol Induction in Haberlea roseoniger
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H. roseoniger suspension cultures grown in PDB (Becton Dickinson, Woodmead, South Africa) were initiated from frozen cell stocks. Saturated overnight cultures were prepared and sterile PDB growth medium (20 mL in 250 mL Erlenmeyer flasks) was inoculated to reach a starting OD600 = 0.015. Cells were grown in Erlenmeyer flasks in an orbital shaker at 160 rpm in a temperature-controlled room at 24 • C for 14 days. Cells were pre-treated with 20 mg/200 mL sclareol (0.01% m/v) (Sigma-Aldrich, Munich, Germany) for a 3 d induction period, followed by the addition of 1 g sclareol/200 mL (0.5% m/v). The experimental design is illustrated in Figure S1 with the assigned phases (I-V) based on the growth curve of H. roseoniger in batch culture. The experiments were repeated at least three times as independent biological replicates.
3
Molecular cloning and yeast manipulation
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Potassium hydroxide pellets, acetone (≥98%), ergosterol (≥98%) and squalene (≥98%) used as standards and petroleum ether (b.p. = 40–60°C) were all purchased from Sigma-Aldrich, while ethanol absolute was supplied from Scharlau.; Sclareol, kindly provided by VIORYL, SA, (−)-trans-caryophyllene (Sigma, C9653-5), were used as standard compounds; MyTaq DNA polymerase (BIO-21105, Bioline), and Accuzyme DNA polymerase (BIO-21051, Bioline) were used in PCR amplifications; NucleoSpin Plasmid Kit (REF 740588.250, Macherey-Nagel) was used for plasmid DNA purification; QIAquick Gel Extraction Kit (#28704, Qiagen) was used for gel extraction and DNA purification.
Yeast media: D (+)-Glucose monohydrate (16301, Sigma); Yeast Nitrogen Base w/o AA, carbohydrate & w/AS (Y2025, US Biologicals); Complete Minimal (CM) medium is composed of 0.13% (w/v) dropout powder (all essential amino acids) [45 ], 0.67% (w/v) yeast nitrogen base w/o AA, 2% glucose; TOPO TA Cloning Kit Dual Promoter (K4610-20, Invitrogen). All yeast transformations were done by lithium acetate transformation.
Yeast media: D (+)-Glucose monohydrate (16301, Sigma); Yeast Nitrogen Base w/o AA, carbohydrate & w/AS (Y2025, US Biologicals); Complete Minimal (CM) medium is composed of 0.13% (w/v) dropout powder (all essential amino acids) [45 ], 0.67% (w/v) yeast nitrogen base w/o AA, 2% glucose; TOPO TA Cloning Kit Dual Promoter (K4610-20, Invitrogen). All yeast transformations were done by lithium acetate transformation.
4
Antibody Procurement for Cellular Analyses
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Sclareol, bortezomib, E64 and pepstatin A were obtained from Sigma-Aldrich; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Cav1, SOD1, β-tubulin and p62 antibodies were obtained from ProteinTech Group, Inc., Chicago, IL, USA), the LC3 antibody was obtained from Sigma-Aldrich; Thermo Fisher Scientific, Inc.), the Flag antibody was obtained from Prospec-Tany TechnoGene, Ltd., (East Brunswick, NJ, USA), the HA tag antibody was obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).
5
Microdilution Assay for Antifungal Activity
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A microdilution assay was performed based on the description of a previous work [20 (link)]. Sclareol (Sigma-Aldrich, St. Louis, MO, USA) was dissolved with dimethyl sulfoxide (DMSO, Junsei, Tokyo, Japan) as a 10 mg/mL stock solution. Candida strains were cultured in culture tubes overnight and diluted to absorbances at OD600 = 0.01. Amounts totaling to 100 µL of C. albicans cells were added into each well of a 96-well plate (SPL, Gyeonggi-do, Seoul, Republic of Korea) with serial two-fold dilution of Sclareol (3.125–200 µg/mL). After incubating at 30 °C for 24 h, the minimum inhibitory concentration (MIC) of the compound was measured by the naked eye. After MIC detection, 50 µL of the cultures was spread onto a YPD agar plate and incubated for 24 h at 30 °C to obtain the minimum fungicidal concentration (MFC) value as a no-cell-growth determinant. Growth control without Sclareol treatment and sterilized medium control were performed in each test [21 (link)].
6
Phytosterol and Lovastatin Treatments on Plants
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Sclareol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Campesterol, stigmasterol, and lovastatin were purchased from Tokyo Chemical Industry (Tokyo, Japan). Sclareol, Campesterol, stigmasterol, and lovastatin were dissolved in methanol and diluted in water to various concentrations. Methanol concentrations did not exceed 0.1% (v/v) in any experiment.
The procedure of chemical treatment, including fractions obtained from fractionation, was performed according to previous reports [16 (link),18 (link)]. Briefly, plants were removed from the fertilizer, transferred to a glass Petri dish (9 cm in diameter) containing sterile distilled H2O, and preincubated for 24 h to diminish the influence of any stress wounds caused by the transfer. After discarding water with a pipette, plant roots were treated by gently adding a solution containing adequate concentrations of each fraction or compound for 48 h. The treated plants were harvested and used for chlorophyll measurements, gene expression analysis, and phytosterol quantification. Five to six plants were used for each treatment and regarded as one biological replicate.
For the lovastatin treatment, we used 100 μM lovastatin, 100 μM Sclareol, and a mixture of 100 μM lovastatin and 100 μM Sclareol in a 50:50 ratio.
The procedure of chemical treatment, including fractions obtained from fractionation, was performed according to previous reports [16 (link),18 (link)]. Briefly, plants were removed from the fertilizer, transferred to a glass Petri dish (9 cm in diameter) containing sterile distilled H2O, and preincubated for 24 h to diminish the influence of any stress wounds caused by the transfer. After discarding water with a pipette, plant roots were treated by gently adding a solution containing adequate concentrations of each fraction or compound for 48 h. The treated plants were harvested and used for chlorophyll measurements, gene expression analysis, and phytosterol quantification. Five to six plants were used for each treatment and regarded as one biological replicate.
For the lovastatin treatment, we used 100 μM lovastatin, 100 μM Sclareol, and a mixture of 100 μM lovastatin and 100 μM Sclareol in a 50:50 ratio.
7
Sclareol and Trypsin in A549 Cell Line
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Sclareol and trypsin (0.25%) were bought from Sigma Aldrich Co. (Poole, UK). A549 human lung epithelial cancer cell line was bought from the national cell bank of Iran, Pasteur Institute (Tehran, Iran). RPMI 1640 medium, fetal bovine serum (FBS), cell culture instruments were acquired from Gibco, Invitrogen (Paisley, UK), and IWAKI (Japan) respectively. The chemicals were provided by Merck Co. (Darmstadt, Germany). Normal melting point agarose (NMP) was purchased from Gibco, Invitrogen (Paisley, UK).
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Sclareol and trypsin (0.25%) were bought from Sigma Aldrich Co. (Poole, UK). A549 human lung epithelial cancer cell line was bought from the national cell bank of Iran, Pasteur Institute (Tehran, Iran). RPMI 1640 medium, fetal bovine serum (FBS), cell culture instruments were acquired from Gibco, Invitrogen (Paisley, UK), and IWAKI (Japan) respectively. The chemicals were provided by Merck Co. (Darmstadt, Germany). Normal melting point agarose (NMP) was purchased from Gibco, Invitrogen (Paisley, UK).
8
Vascular Contractility Assay Reagents
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Acetylcholine (ACh), 1H-[1,2,4] oxadiazole [4,3-a]quinoxalin-1-one (ODQ),
phenylephrine (Phe) and Sclareol were purchased from Sigma Chemical Company (St.
Louis, MO, USA). Nω-nitro-L-arginine methyl ester (L-NAME) was obtained
from Calbiochem (San Diego, CA, USA). Isoflurane from Abbott. All the salts used
for Krebs solution preparation were furnished by Vetec Química Fina Ltda.
Almost all the drugs were prepared with distilled water, except for indomethacin
(which was dissolved in ethanol) and Sclareol (solubilized in dimethyl sulfoxide
and diluted in ethanol + water).
phenylephrine (Phe) and Sclareol were purchased from Sigma Chemical Company (St.
Louis, MO, USA). Nω-nitro-L-arginine methyl ester (L-NAME) was obtained
from Calbiochem (San Diego, CA, USA). Isoflurane from Abbott. All the salts used
for Krebs solution preparation were furnished by Vetec Química Fina Ltda.
Almost all the drugs were prepared with distilled water, except for indomethacin
(which was dissolved in ethanol) and Sclareol (solubilized in dimethyl sulfoxide
and diluted in ethanol + water).
9
Molecular Profiling of Cell Signaling
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Sclareol (CAS: 515-03-7, 98%), oxytocin, sodium bicarbonate, carbachol, acetylcholine (Ach), mannitol, glucose, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, estradiol, and dimethyl sulfoxide (DMSO) were purchased from (Sigma-Aldrich, St. Louis, MO, USA). PGF2α and Bay K 8644 were purchased from (Cayman Chemical Company, Ann Arbor, MI, USA).
10
Sclareol, Sclareolide, and ACC Root Treatment
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Stock solutions (100 mM) of sclareol (Sigma) and sclareolide (Sigma) in methanol and a stock solution (100 mM) of ACC (Sigma) in water were diluted in water to various concentrations. Methanol concentrations did not exceed 0.1% in all the experiments.
Roots of plants grown in the sand system were treated by soaking the pot itself in a solution containing various concentrations of each chemical in a tray, with their roots submerged in the solution. After soaking for 24, 48, or 72 h, the treated plants were transferred to a new tray containing water and used for RKN penetration assays; or the roots were pulled out of the pots, washed with water, and used for real-time RT-PCR analysis or quantification of thioglycolic acid lignin.
For treatment of the roots of hydroponically grown plants, plants were pulled out of the fertilizer, transferred to a petri dish containing water with their roots submerged in the water, and preincubated for 48 h to diminish the influence of any wound stresses that could be caused by removal. After discarding water using a pipette, the plant roots were treated by gently adding a solution containing various concentrations of each chemical to the petri dish until the roots were submerged in the solution, incubated for adequate time periods, and used for phytohormone measurements, immune complex kinase assays, or histochemical analysis of lignin.
Roots of plants grown in the sand system were treated by soaking the pot itself in a solution containing various concentrations of each chemical in a tray, with their roots submerged in the solution. After soaking for 24, 48, or 72 h, the treated plants were transferred to a new tray containing water and used for RKN penetration assays; or the roots were pulled out of the pots, washed with water, and used for real-time RT-PCR analysis or quantification of thioglycolic acid lignin.
For treatment of the roots of hydroponically grown plants, plants were pulled out of the fertilizer, transferred to a petri dish containing water with their roots submerged in the water, and preincubated for 48 h to diminish the influence of any wound stresses that could be caused by removal. After discarding water using a pipette, the plant roots were treated by gently adding a solution containing various concentrations of each chemical to the petri dish until the roots were submerged in the solution, incubated for adequate time periods, and used for phytohormone measurements, immune complex kinase assays, or histochemical analysis of lignin.
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