Niflumic acid

Celecoxib, niflumic acid, parecoxib, L-kynurenine (sulfate salt), dimethyl sulfoxide (DMSO), sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, sodium phosphate monobasic, sodium phosphate dibasic, glucose, distilled water, Trizma base, acetic acid, pyridoxal 5′-phosphate, 2-mercaptoethanol, pyruvate, and glutamine were obtained from Sigma-Aldrich. High-performance liquid chromatography (HPLC) reagents were purchased from J.T. Baker Chemicals and from Sigma-Aldrich.

R(+)-NAF (>99.5% ee) and S(-)-NAF (>99.5% ee) (Figure 1) were synthesized according to previous methods (Shivani et al., 2007 (link)). The internal standard (IS) Z10 (CN201110148322.7) was obtained from our laboratory. HPLC-grade methanol, acetonitrile, and acetic acid were obtained from Merck (Darmstadt, Germany). Uridine diphosphate glucuronic acid (UDPGA), alamethicin, Tris, magnesium chloride, methylum-belliferone (MU), 4-methylumbelliferone (4-MU), 4-methylum-belliferyl-β-D-glucuronide hydrate (4-MU-G), propofol, propofol glucuronide, zidovudine, zidovudine glucuronide, fluconazole, and niflumic acid, trifluoperazine dihydrochloride (TFP), and TFP-glucuronide (TFP-G) were purchased from Sigma–Aldrich (St. Louis, MO, United States). Recombinant human UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) and the commercially available microsomes from human liver (HLMs), intestine (HIMs), and kidney (HKMs), as well as those from rat liver (RLMs), intestine (RIMs), and kidney (RKMs), were purchased from BD Gentest (Woburn, MA, United States). Protein contents of the microsomes were used according to the manufacturers’ instructions. Ultrapure water was used in all experiments (Millipore, Billerica, MA, United States).

BK powder (100 mg) was suspended in 4 mL of methanol/purified water (75 : 25, v/v). The suspension was shaken and sonicated for 5 min each. After centrifugation at 1700 g for 5 min at room temperature, the supernatant was collected as the first extract solution. Then, 4 mL of methanol/purified water (50 : 50, v/v) was added to the residue, and the second extraction solution obtained by the procedure described above was mixed with the first extract solution. The mixed extract solution undiluted or diluted 10- or 100-fold with methanol was injected into the LC-MS/MS system after pooling with a respective internal standard solution, niflumic acid (Sigma-Aldrich), or vincamine (Tokyo Chemical Industry Co.). Two systems were used for analysis of the constituents: system 1, an API4000 triple quadrupole mass spectrometer (AB SCIEX, Tokyo, Japan) equipped with an Agilent 1100 system (Agilent Technologies, Inc., Tokyo, Japan); system 2, a TripleQuad6500 (AB SCIEX) equipped with an Agilent 1290 system (Agilent Technologies, Inc.). Analytical conditions are summarized in the Supplementary Tables S1 and S2 (available online at http://dx.doi.org/10.1155/2015/853846).

DP was purchased from the National Institute for the Control of Pharmaceutical and Biological Products in China, and its purity was determined to be approximately 98% by HPLC measurement. DP was dissolved in dimethylsulfoxide (DMSO) to make a 100 mmol/L stock solution and diluted prior to experiments. Niflumic acid (NFA) was purchased from Sigma-Aldrich, and T16A_inh-A01 was obtained from Alan Verkman (University of California, San Francisco, CA, USA)

(−)-[Ring-2,5,6-³H]noradrenaline was obtained from PerkinElmer (Vienna, Austria); amphotericin B, gramicidin D, oxotremorine M, staurosoporine, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole (Gö6976), 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (Gö 6983), bisindolylmaleimide I (GF 109203 X), phorbol-12-myristate 13-acetate, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE 991), disodium-4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS), niflumic acid and pertussis toxin from Sigma; 6-(1,1-dimethylethyl)-2-[(2-furanylcarbonyl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (CaCCinh-A01) and 2-[(5-ethyl-1,6-dihydro-4-methyl-6-oxo-2-pyrimidinyl)thio]-N-[4-(4-methoxyphenyl)-2-thiazolyl]acetamide (T16Ainh-A01) from Tocris (Bristol, UK); and tetrodotoxin was from Latoxan (Rosans, France). Water-insoluble drugs were first dissolved in DMSO and then diluted into buffer to yield final DMSO concentrations of up to 0.3 %, which were also included in control solutions.

The extract powders of shimotsuto (lot no. 361142800) and its constituent crude drugs (Angelicae Acutilobae Radix [lot no. 2131002010], Paeoniae Radix [lot no. 2131001010], Rehmanniae Radix [lot no. 2151011010], and Cnidii Rhizoma [lot no. 2131004010]) were supplied by Tsumura & Co. (Tokyo, Japan). These extract powders were produced by spray-drying of a hot water extract mixture of the constituent crude drugs, and their qualities were standardized and guaranteed by measuring several characteristic marker ingredients on the basis of good manufacturing practices, as defined by the Japanese Ministry of Health, Labour and Welfare. Supplementary Figure 1 shows the 3D HPLC profile of each extract powder provided by Tsumura & Co.
The standard substances of ingredients contained in shimotsuto were obtained as follows: catalpol, paeoniflorin, albiflorin, ferulic acid, ligustilide, senkyunolide A, and butylphthalide from Tsumura & Co. and bergapten from Chem Faces (Hubei, China), 8-debenzolypaeoniflorin from BioBioPha (Yunnan, China). In addition, niflumic acid, imperatorin, and swertiamarin were obtained from Sigma-Aldrich (St. Louis, MO, USA), Tsumura & Co., and Fujifilm Wako Pure Chemical Industries (Osaka, Japan), respectively, as internal standards for quantitative (targeted) analysis. All other chemicals were purchased from commercial sources.