Acetic acid

Six organic acids (tartaric acid, malic acid, lactic acid, acetic acid, citric acid, and succinic acid) were analyzed with an HPLC (1260 series, Agilent, Santa Clara, CA, USA) equipped with a YMC-Triart C18 (4.6 × 250 mm, 5 μm) column. The mobile phase was 25 mM KH₂PO₄ (pH 2.5) and the temperature was set to 30 °C. A 210 nm diode array detector (DAD) and a 1.0 mL/min flow rate were used. The injection volume was 20 μL. Vinegar samples were diluted 10-fold with 0.4% HCl and then filtered with 0.22 μm polyvinylidene fluoride (PVDF) membrane. Standards for six organic acids were also dissolved in 0.4% HCl and used. Concentrations of standards were set to 5, 10, 25, 50, 75, 100, 150, and 200 μg/mL. Correlation values were all over 0.9999. Standards used included L (+)-tartaric acid, 99+% (purity 100%, Thermo Fisher Scientific, Waltham, MA, USA), DL-malic acid (purity 99.2%, TCI, Tokyo, Japan), DL-lactic acid (purity 90.6%, TCI, Tokyo, Japan), acetic acid (purity 99.9%, TCI, Tokyo, Japan), citric acid (purity 99.7%, TCI, Tokyo, Japan), and succinic acid (purity 98.9%, TCI, Tokyo, Japan). The organic acid analysis was performed based on the method in the previous report, with some modifications [26 (link),27 (link)].

J. gendarussa leaves were collected from Pacet, Purwodadi, Surabaya, Gempol, Makassar, and Cibodas between September 2012 to January 2013 (Table 1). Samples were properly authenticated by Department of Pharmacognosy and Phytochemistry, Airlangga University, Surabaya. Mature, dark green leaves of five different plants were collected from each location in triplicate; the leaves were air-dried and powdered. Moisture contents (MC) of the samples were 9.6 ± 1.7%, n = 105 (by using Moisture Analyzer HB43-S, Mettler Toledo). The maximum permitted level of MC of the herbal medicine was 12%, w/w [31 ].
Soil collection was performed by using composite sampling. Fifteen sub-samples were collected randomly 6–8 inches from the surface [32 ].
Methanol, 2-propanol, and formic acid were analytical reagents from Merck (Darmstadt, Germany). Purified water was from Sigma-Aldrich (St. Louis, MO, USA), acetic acid from J.T. Baker (Phillipsburg, NJ, USA), and NaOH from Agilent (Agilent solution for HPCE). All samples were filtered through a 0.2 µm Agilent Econofilter PVDF 13m.

crystal violet staining analysis was performed to evaluate the adherent biofilms on the specimens. The infected specimens cultured with E. faecalis for 7 days at 37 °C were subjected to the above experimental groups. Following treatment, the root canal dentin was stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, followed by 30% acetic acid (Fisher Scientific, Fair Lawn, NJ, USA) for 10 min. Then, the acetic acid was dispensed to a sterile 96-well microtiter plate and the optical density was determined at 595 nm (μQuant, Biotek Instrument, Winooski, VT, USA).