Ferric chloride fecl3

3-hexathiophene (3HT, 98.0% purity, TCI) was the starting monomer. Ferric chloride (FeCl₃; 97% purity, Sigma Aldrich, St. Louis, MO, USA) was the oxidant used. Chloroform (CHCl₃; AR grade, RCI Labscan, Bangkok, Thailand) was the solvent. Dodecylbenzenesulfonic acid (DBSA; 98% purity, Sigma Aldrich), p-toluenesulfonic acid (PTSA; 98.5% purity, Sigma Aldrich), sodium dioctyl sulfosuccinate (AOT; 96% purity, Sigma Aldrich), and sodium dodecyl sulfate (SDS; 98% purity, Sigma Aldrich) were the surfactants. Methanol (99.8%), toluene (99.5%), and hexane (99.8%) were obtained from RCI Labscan. For the de-doping and doping processes, ammonium hydroxide (NH₄OH; 30% v/v, Panreac, Barcelona, Spain) was used to de-dope the P3HT_DBSA powder, whereas perchloric acid (HClO₄; 70% v/v, Panreac) was the dopant. All reagents were of analytical reagent grades and were used without modification.

The preliminary phytochemical screening was performed using selective derivatization reactions for the characterization of secondary metabolites present in the
n-hexane, chloroform, ethyl acetate and
n-butanol extracts obtained from inner bark, as previously reported for
T. rosea^{6 (link)}. The extracts were evaluated using normal and reverse phase thin layer chromatography (TLC). Chromatographic plates were revealed with aluminum chloride (AlCl
₃, Sigma Chemical Co, Saint Louis, MO, USA, Cat No. 563919-25G) and ferric chloride (FeCl
₃, Sigma Chemical Co, Saint Louis, MO, USA, Cat No. 157740-1KG) for detection of flavonoids, phenols and phenolic acids; potassium hydroxide (KOH, Merck, Darmstadt, Germany, Cat No. 1050331000) in analytical grade ethanol for detection of anthrones, quinones and coumarins; oleum (Sigma Chemical Co, Saint Louis, MO, USA, Cat No. 778990-500ML) for detection of sesquiterpenic lactones; and the Liebermann-Burchard reagent for detection of terpenes and steroids.

Spleens were isolated from tumor-naive female C57Bl/6N mice. After lysis of erythrocytes using the Mouse Erythrocyte Lysing Kit (R&D Systems) 2.5 × 10⁵ splenocytes per well were then seeded in a 96-well round bottom plate and stimulated with 4 µg/ml plate-bound or 1 µg/ml soluble rat anti-mouse CD3 (clone 17A2; BD Pharmingen). Ferric chloride FeCl₃ (Sigma Aldrich), ferric sulfate Fe₂(SO₄)₃ (Sigma Aldrich), ferric citrate FeC₆H₅O₇ (Sigma Aldrich), and holo-transferrin were added at concentrations of 2.5µM, 5 µM, 10 µM and 20 µM elementary iron. Splenocytes were cultured in RPMI-1640 medium (PAN Biotech) supplemented with 10% FCS (Biochrom), 2% sodium pyruvate (Sigma), 1× non-essential amino acids (Gibco), 0.01% β-mercaptoethanol (Roth), 1% penicillin/streptomycin (Lonza) and 2 mM L-glutamine (Lonza).

The chemicals used in this study, including Folin–Ciocalteu phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid, quercetin, Trolox, HPLC-grade methanol, 2,4,6-tripyridyl-s-triazine (TPTZ), glacial acetic acid, sodium acetate trihydrate, and ferric chloride (FeCl₃), were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA).

The U937 human macrophage cell line and fetal bovine serum (FBS) were purchased from the American Type Culture Collection (Masassas, VA, USA). RPMI 1640 and penicillin/streptomycin were procured from Invitrogen (Carlsbad, CA, USA). Potassium chloride (KCl) and hydrochloric acid (HCl) were obtained from BDH Chemicals Ltd. (Poole, United Kingdom). PMA (phorbol 12-myristate 13-acetate), 2,2’-diphenyl-1-picrylhydrazyl (DPPH), gallic acid, Folin-Ciocalteu reagent, sodium carbonate (Na₂CO₃), lipopolysaccharide,and quercetin were purchased from Sigma Aldrich (Germany). Aluminum chloride (AlCl₃), potassium acetate, sodium nitrate, sodium hydroxide, and 2,2-azinobis (3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) were purchased from Merck (Germany). Diammonium salt, 2,4,6-Tri (2- pyridyl)-s-triazine (TPTZ), ferric chloride (FeCl₃), and potassium persulfate were obtained from Sigma Aldrich (Poole, Dorset, UK). Chloroform was obtained from R & M Marketing (Essex, UK). Methanol and absolute alcohol were purchased from Riedel-de Haën (Seelze, Germany), and λ-carrageenan was procured from Sigma Aldrich (St. Gallen, Switzerland).