Oleanolic acid

Mtb H37Rv was initially isolated from a patient with pulmonary tuberculosis in 1905 by Albert Calmette and Camille Guérin at the Pasteur Institute in Paris, France. The strain was maintained and distributed by American Type Culture Collection (ATCC 27294) centre. For our study, a clinical sample from extensively drug-resistant Mycobacterium tuberculosis h37rv was obtained from the district Tuberculosis laboratory (Guna, Gujarat, India). It has a rpoB gene mutation (gene accession number: JN037845) and has resisted the first, second, and third lines of TB drugs in primary screening. In addition, we also obtained a sample for drug-sensitive tuberculosis from the same laboratory. The culture was then inoculated into the Middlebrook medium (Himedia) supplied with 0.05% Tween 80 and albumin-dextrose-catalase. The culture was maintained by transferring in the fresh medium every 30 days. Madasiatic acid, Asiaticoside A(ASA), β-sitosterol, Cystargamide B, Myristic acid, Palmitic acid, Kalmeghin, Oleanolic acid, Longicyclene, Oleanane, and Amyrin were purchased from Sigma-Aldrich.

The extraction and isolation solvents were purchased from Junsei Chemical Co., Ltd., Tokyo, Japan and Fisher Scientific company, Hampton, NH, USA. Chemicals for antioxidant assays were acquired from Fujifilm Wako Pure Chemical Corporation, Osaka, Japan. Elastase from porcine pancreas Type IV, tyrosinase from mushroom lyophilized powder, N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), L-tyrosine, oleanolic acid, vanillin, myricetin, and all buffer components were acquired from Sigma-Aldrich, St. Louis, MO, USA.
Pure momilactones A, B, and tricin were isolated previously from rice husk by our laboratory [12 (link)]. Briefly, the ethyl acetate (EtOAc) extract of 7 kg Koshihikari husks was separated by open column chromatography over silica gel with the mobile phase as combinations of hexane and EtOAc. Momilactones A and B were isolated from the eluant hexane:EtOAc (8:2) by a repeated column chromatography while tricin (a yellow powder) was purified from the last fractions of the eluant hexane: EtOAc (7:3). The identification of such pure compounds is described in detail in the following parts.

A stock 5 mg mL^-1 solution of oleanolic acid (Sigma, St. Louis, MO, USA) was freshly prepared in dimethyl sulfoxide (DMSO; Sigma). The solution was then diluted in brain heart infusion (BHI) broth (Difco, Becton, Dickinson and Company, Sparks, MD, USA) and tryptic soy broth (TSB; Difco, Becton, Dickinson and Company, Sparks, MD, USA) to 1,024 μg mL^-1 for use in the experiments.

This was performed according to a previous method with some modifications [61 (link)]. Briefly, 100 μL of each bacterial inoculum (2.5 × 10⁵ CFU/mL) was added to the wells of a 24-flat well polystyrene plate, containing TSB (1.8 mL). Heather and Manuka honeys were tested at 0.25 mg/mL. Wells containing TSB only were used as the sterile controls. Oleanolic acid (Sigma-Aldrich, Gillingham, UK), a plant-derived triterpenoid which inhibits the formation of biofilms in Gram-positive and Gram-negative bacteria, was used as a positive control [52 (link),53 (link),54 (link),55 (link)]. Following inoculation, the plates were incubated without shaking at 37 °C for 24, 48, 72, and 96 h to allow the formation of a biofilm at the bottom of the wells as well as to determine the time point at which the maximum formation of biofilm occurred. After this period, the supernatants were removed, and the biofilms were washed (×3) with sterile distilled water. The biofilms were then stained with 1% crystal violet for 5 min, and further washed (×3) with tap water. A de-staining step was performed using a 7:3 (v/v) mixture of ethanol and acetone and the OD of the suspension was measured at 550 nm. The percentage of biofilm inhibition was calculated as: (OD_control − OD_sample) × 100/OD_control.