Stock solutions of 10,000 ppm cedrol in ethanol were prepared by adding 150 mg of (+)-cedrol (≥99.0%, sum of enantiomers, Sigma-Aldrich) to 15 ml of absolute ethanol (puriss. pa, absolute, ≥99.8% (GC), Sigma-Aldrich). Dilutions were made by adding the appropriate amount of stock solution to lake water. For example, to make a 5-ppm solution of cedrol in water, 3.5 mL of the stock solution was added to 7 L of lake water; for each round 2.5 L of this was used for cage bioassays and 4.5 L for semi-field experiments with free-flying mosquitoes. The same formulation procedures were used to create 5-ppm cedrol preparations for all traps in the field.
Cedrol
Manufactured by Merck Group
Sourced in United States, Germany
Cedrol is a laboratory equipment product manufactured by Merck Group. It is a clear, colorless liquid that is used as a standard in analytical procedures and scientific research. Cedrol has a specific chemical structure and purity that makes it suitable for use as a reference material in various analytical techniques.
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Lab products found in correlation
Nerolidol, by Merck Group (2 mentions)
β caryophyllene, by Merck Group (2 mentions)
Valencene, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Picrotoxin, by Merck Group (1 mentions)
Parthenolide, by Merck Group (1 mentions)
Farnesol, by Merck Group (1 mentions)
Farnesyl acetate, by Merck Group (1 mentions)
Nootkatone, by Merck Group (1 mentions)
Soybean lecithin, by Merck Group (1 mentions)
Cromolyn sodium, by Merck Group (1 mentions)
Prednisolone, by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Fura 2 am, by Merck Group (1 mentions)
Medium 199, by HiMedia (1 mentions)
O phthalaldehyde opt, by Merck Group (1 mentions)
Triolein, by Merck Group (1 mentions)
9 protocols using cedrol
1
Cedrol Preparation for Mosquito Bioassays
2
Culturing K562 and HT29 Cell Lines
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K562 and HT29 cell lines purchased from the American Type Culture Collection (ATCC) were maintained in RPMI 1640 medium (Gibco Invitrogen, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, United States), 2 mM L -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco Invitrogen) at 37°C in humidified atmosphere of 5% CO2. Cedrol was purchased from Sigma Chemicals.
3
Sesquiterpene Compound Acquisition
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Eight sesquiterpenes including β-Caryophyllene (Cat # W225207), (+)-Cedrol (Cat # W521418-1KG-K), Farnesol (Cat # F203-100G), Farnesyl acetate (Cat # 45895-10ML-F), Nerolidol (Cat # W277207-1KG-K), (+)-Nootkatone (Cat # 74437-2.5G), Parthenolide (Cat # P0667-25MG), and Picrotoxin (Cat # P1675-5G), as shown in Figure 2 , were purchased from Sigma Aldrich, UK.
4
Nanoparticle Formulation Preparation
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Compritol® 888 ATO was supplied by Gattefosse India Pvt Ltd (Mumbai, India). Triolein, soybean lecithin, TPGS, sodium deoxycholate, compound 48/80, O-phthalaldehyde (opt), 4-nitrophenyl N-acetyl-β-d -glucosaminide, Fura 2-AM, cromolyn sodium, prednisolone, and cedrol were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Fetal bovine serum (RM10432 HiMedia), RPMI 1640, and Medium 199 were supplied by HiMedia (Mumbai, India).
5
Cedrol Identification and Purity Validation
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The identity of ID 276 was confirmed using an authentic standard: (+)-cedrol, ≥99.0% sum of enantiomers, GC, optical activity αD20 + 10.5 ± 1° (Sigma-Aldrich, Steinheim, Germany). The compound was diluted in MTBE to 0.8 mg/ml and 1 μl was injected in a CIS-injector, set to a splitless mode, held at 40°C for 0.5 minutes and then heated at a rate of 12°C/second to 260°C. All other GC-MS parameters were as for the soil infusion samples above.
6
Identification of Volatile Organic Compounds
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The synthetic standards including hexanal, α-thujene, α-pinene, camphene, o-cymene, sabinene, β-pinene, myrcene, α-terpinene, β-terpinene, terpinolene, decanal, tridecane, pentadecane, cedrol, hexadecane and the solvent dichloromethane were purchased from Sigma Aldrich (USA) whilst δ-2-carene, α-phellandrene, δ-3-carene, p-cymene, limonene, β- phellandrene, β-ocimene [mixture of (E) and (Z)], dihydrotagetone, δ-elemene, β-elemene, α-cedrene, (E)-caryophyllene, α-humulene, (E, E)-α-farnesene were purchased from Merck (France) and were used to confirm the identified compounds. The chemical purity of the synthetic standards except for sabinene (75%) and α-phellandrene (85%) ranged between 90–99%.
7
Sesquiterpenoid Extraction and Quantification
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The clean tissue culture seedlings were dried at 37 °C for 36 h to constant weight. After grinding to powder in a mortar, a four-fold volume of cyclohexane (W: V) was added, followed by extraction in the dark for 8 h. The sesquiterpenoid content was detected by gas chromatography (GC). The Agilent 7890A GC (Agilent Technologies, Santa Clara, CA) with HP-5 capillary column (30 m × 0.32 mm × 0.10 μm) and flame ionization detector (FID) were used, and samples were tested according to the following heating program: 70 °C for 1 min, 8 °C min−1 to 200 °C, 20 °C min−1 to 300 °C for 5 min. The temperature of the sample was 240 °C, the temperature of the detector was 350 °C, the carrier gas was high-purity nitrogen, the flow rate was 0.92266 mL min−1, and the injection volume was 1 μL. Twelve kinds of sesquiterpenoids were analyzed by GC. Among them, caryophyllene oxide, cedrol, nerolidol, farnesene, β-caryophyllene and valencene were purchased from Sigma-Aldrich with purity > 99%; germacrene d and zingiberene were purchased from Chengdu MUST Bio-Technology Co., Ltd with purity > 95%; β-sesquiphellandrene was purchased from Toronto Research Chemicals with purity > 95%; Atractylone, β-eudesmol and hinesol were isolated and purified by our research group with purity > 95%.
8
Radioimmunoassay of Eicosanoids in Blood
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Hexane, ethyl acetate and methanol used were of analytical grades. Radiolabelled PGE 2 ([ 3 H]-PGE 2 , 50 μCi/mmol) and TXB 2 ([ 3 H]-TXB 2 , 25 μCi/mmol) were purchased from Amersham (Buckinghamshire, UK). Unlabelled PGE 2 , unlabelled TXB 2 , anti-PGE 2 and anti-TXB 2 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Radiolabeled PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, 125 Ci/mmol) was purchased from Amersham. Unlabeled PAF and cedrol were obtained from Sigma Chemical Co. Bovine Serum Albumin (BSA) was purchased from Boehringer Mannheim Co. (Mannheim, West Germany). Other chemicals were purchased from Merck Co. (Darmstadt, Germany) and BDH Laboratory Supplies (Poole, UK). Ethylenediamine tetraacetic acid (EDTA) 2% was used as an anticoagulant. Lipopolysaccharide (LPS) 1 mg/mL was used to induce prostaglandin endoperoxide synthesis in whole blood. Phosphate buffer solutions (PBS) 0.01M, pH 7.4 was used as buffer for assays. Dextran charcoal (0.4% dextran, 2% charcoal) was used to separate the free and bound ligand. Scintillation cocktail was made up of 2,5-diphenyloxazole (PPO, 0.26%), 2,2'-p-phenylene-bis(5-phenyloxazole (POPOP, 0.006%), toluene (500 mL) and Triton X (250 mL). Radioactivity was measured by a liquid scintillation counter (LSC) (Packard Tri-Carb, models 2100TR/2300TR, Hamburg, Germany).
9
Extraction and Characterization of Calendula officinalis
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Dry Calendula officinalis flowers were purchased from a local herbalist supplier (Murcia, Spain).
The flowers were ground (particle size smaller than 500 µm) in a knife mill (Grindomix GM200 RETSCH). Extra virgin olive oil was purchased from a local supermarket.
Thymol, β-caryophillene, valencene, cedrol, β-sitosterol, tocopherol and alkane mixture (C7-C30) were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). The standard α-amyrin was from Extrasynthese (Genay, France).
Trizma, maleic acid, Amano lipase A from Aspergillus niger, pepsin, pancreatin from porcine pancreas, bile salts, phosphatidyl choline from egg yolk and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
The flowers were ground (particle size smaller than 500 µm) in a knife mill (Grindomix GM200 RETSCH). Extra virgin olive oil was purchased from a local supermarket.
Thymol, β-caryophillene, valencene, cedrol, β-sitosterol, tocopherol and alkane mixture (C7-C30) were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). The standard α-amyrin was from Extrasynthese (Genay, France).
Trizma, maleic acid, Amano lipase A from Aspergillus niger, pepsin, pancreatin from porcine pancreas, bile salts, phosphatidyl choline from egg yolk and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) were from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).
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