Lipoxygenase

Carbopol and Glycerine were purchased from Sigma, Germany. Lipoxygenase (1.13.11.12, type I-B, Soybean), linoleic acid, and LPS (lipopolysaccharide from Escherichia coli 0111:B4) were purchased from Sigma (St. Louis, MO, USA). The RAW 264.7 murine macrophage cell line and L929 healthy mouse fibroblast were purchased from ATCC (Gaithersburg, MD, USA) and DMEM was purchased from Gibco (New York, NY, USA). All the other chemicals and solvents were of analytical or HPLC grade.

Analytical grade organic solvents (such as ethanol, methanol, etc.) were purchased from Daejung Chemicals & Metals Co., Ltd., Korea. Acetylcholinesterase (AChE) Electric eel (CAS 9000-81-1), acetylthiocholine iodide (ATCI), butyrylcholinesterase (BChE) equine serum lyophilized (CAS 9001-08-5), butyrylcholine iodide (BTCI), dithiobis nitrobenzoic acid (DTNB), galanthamine, 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), trolox, daidzein, genistein, glycitein, (+)-catechin, rutin, quercetin, gallic acid, soyasaponin I, Folin–Ciocalteau’s reagent, cyanidine 3-o-glucoside chloride, Tris-HCl, lipoxygenase, aspirin, perchloric acid, linoleic acid, casein and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) were obtained from Sigma.

Bovine serum albumin, trypsin, Tris-HCl, perchloric acid, casein, lipoxygenase, linoleic acid, lutein, rutin, β-carotene, and methanol, were obtained from Sigma Aldrich, St. Louis, MO, USA, through Analytical Instrument Pvt Ltd., Colombo, Sri Lanka. All other chemicals used were of analytical grade.

1,1-Diphenyl-2-picrylhydrazyl (DPPH), NADH (nicotinamide adenine dinucleotide), lipoxygenase, linoleic acid, xanthine (99%), xanthine oxidase (25 units), BHT (butylated hydroxytoluene), PMS (phenazinemethosulfate), glutathione and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA) and silica gel TLC 60 F254 from Merck (Darmstadt, Germany).

The lipoxygenase (LOX) inhibitory effect of n-hexane, chloroform soluble extract, and its isolated compounds was determined by using published methods [25 (link)]. The lipoxygenase inhibitory effect was done by using different concentrations of chloroform fraction and its isolated compound. LOX inhibitory potential was identified by a slight modification of the published procedure spectroscopic procedure established by lipoxygenase (EC.1.13.11.12) Type 1 (soybean) and pure linoleic acid obtained from Sigma. All chemicals and reagents used in this screen test were of analytical grade and obtain from Sigma. In this screening test, 160 ml 0.1 mm (PH = 7), concentration buffer solution, and 10 μL of extract/compound solution were combined, and then the standard solution was combined. The reaction mixture was incubated for 5 min at 258 °C. After incubation 10 ml of linoleic acid was mixed with substrate solution the absorption changes with the development of (9Z,11E)-13S-hydropeoxyoctadeca-9,11-dienoate. After 10 min control and sample (compound/extract) were dissolved in methanol. The positive control used in this assay is baicalein and tenidap sodium. The results were measured and IC₅₀ was calculated with the help of the EZ-fit enzyme kinetic program according to the published method [25 (link)].

Gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), methanol (HPLC grade), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), sodium acetate solution, potassium chloride solution, α-glucosidase from Saccharomyces cerevisiae (≥10 units/mg protein), lipoxygenase from Glycine max (soybean) type I-B, 50,000 units/mg protein, α-amylase from porcine pancreas (type I-A, 700–1400 units/mg protein), pancreatin lipase (111.5 units/mg protein), linoleic acid (≥99%), p-nitrophenyl-α-d-glucopyranoside (≥97.5%), p-nitrophenyl palmitate (98%), starch solution, Triton X-100, Arabic gum, dinitrosalicylic acid (DNS), orlistat ≥ 98%, quercetin ≥ 95%, and acarbose ≥ 98% were acquired from Sigma Aldrich (Steinheim am Albuch, Germany). All chemicals and reagents utilized in the experiments were of analytical grade.

The anti-inflammatory activities of Z. leprieurii essential oils were determined by the method previously described by Nikhila [30 (link)]. In brief, the reaction mixture containing essential oils in various concentrations (100, 75, 50 and 25 µg/mL of methanol) (in triplicate for each concentration), lipoxygenase (Sigma, Darmstadt, Germany) and 35 µL (0.1 mg/mL) of a 0.2 M borate buffer solution (pH = 9.0) was incubated for 15 min at 25 °C. The reaction was then initiated by the addition of 35 µL of a substrate solution (linoleic acid 250 µM), and the absorbance was measured at 234 nm. Quercetin (Sigma, Darmstadt, Germany) was used as a standard inhibitor at the same concentration as the essential oils. The inhibition percentage of lipoxygenase activity was calculated as follows:
where Abs blank is the Absorbance (Abs) of the reaction media without the essential oil, and Absorbance sample is the Abs of the reaction media with the essential oil minus the Abs value of the diluted essential oil (to compensate for absorbance due to the essential oils themselves).