Sulphanilic acid

A modified Griess reaction was used to detect nitrite and nitrate [36 (link),37 (link)]. The NO levels in samples were indirectly measured after first converting nitrates to nitrites with a nitrate reductase treatment (Aspergillus species NAD [P] H, Sigma, UK) and NADPH β-nicotinamide adenine dinucleotide phosphate (Sigma Diagnostics, St. Louis, USA). Griess reagent [5% phosphoric acid, 1% sulphanilic acid and 0.1% N-(1-naphthyl-1)-ethylendiaminedihydrochloride, all from Sigma, UK, dissolved in 100 mL deionized water] was added and proteins were subsequently precipitated by trichloroacetic acid (BDH, England). The tube contents were mixed and centrifuged (Eppendorf centrifuge 5415 C, Germany); two samples of each supernatant were transferred to a flat-bottomed microplate and their absorbances were read at 520 nm using a microplate reader (SpectraMax, Molecular Devices Inc). NO values were calculated from standard calibration plots.

For determination of total volatile fatty acids (VFA) content and ratio of VFAs and alkalinity (VFA/TAC), samples were centrifuged for 15 min and 4,696 × g at 4 °C. 10 mL of the liquid phase were then titrated with 0.05 M sulfuric acid (Carl Roth GmbH, Karlsruhe, Germany) to pH values of 5.00, 4.40, 4.30 and 4.00. VFA as well as VFA/TAC were measured in triplicates and calculated as described elsewhere⁴⁶ . Composition of VFA was analyzed by atres Analytik (München, Germany) using an in-house gaschromatographic approach. Contents of following VFAs were measured: acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, hexanoic acid and heptanoic acid.
For analyzing elemental composition (C, N, S) samples were dried at 50 °C for 48 h. Afterwards samples were grinded using mortar and pestle. 10 mg of each sample were oxidized in the combustion tube of the elemental analyzer Vario EL II (Elementar Analysensysteme GmbH, Langenselbold, Germany) at 1,150 °C according to manufacturer’s instructions. Sulphanilic acid (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was used as standard. All analyses were performed in triplicate measurements.

Nitric oxide (NO) was generated from sodium nitroprusside (SNP) (Sigma-Aldrich, Sydney, Australia) and was measured by the Griess Ilosvay reagent [25 ], using 0.1% w/v naphthylethylene-diamine-dihydrochloride (Sigma-Aldrich, Sydney, Australia) instead of 5% 1-naphthylamine. Plant extract (0.5 mL) was added to a mixture containing SNP (2 mL) and phosphate buffer saline (PBS) (0.5 mL, pH 7.4). The reaction mixture was incubated for 2.5 hrs at 25°C. Following incubation, 0.5 mL of the reaction mixture was added to 1 mL sulphanilic acid (0.33% in 20% glacial acetic acid) (Sigma-Aldrich, Sydney, Australia) and allowed to stand for 5 minutes. naphthylethylene-diamine-dihydrochloride (1 mL of 0.1% w/v) was then added to the mixture. The resulting solution was vortexed and allowed to stand for further 30 minutes. The absorbance of the chromophores formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylethylene-diamine-dichloride was read at 546 nm. Percentage inhibition was calculated as follows: $\begin{array}{c}\%\hspace{0.17em}\hspace{0.17em}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left[\frac{\left(A_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}-A_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}\right)}{A_{\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}}}\right]\ast \mathrm{100}.\end{array}$

A modified Griess reaction was used to detect nitrite and nitrate ([38 (link)], modified by [39 (link)]). The NO levels in samples were indirectly measured after first converting nitrates to nitrites with a nitrate reductase treatment (Aspergillus species NAD [P] H, Sigma, UK) and NADPH β-nicotinamide adenine dinucleotide phosphate (Sigma Diagnostics, St. Louis, USA). Griess reagent [5% phosphoric acid, 1% sulphanilic acid, and 0.1% N-(1-naphthyl-1)-ethylendiamine dihydrochloride, all from Sigma, UK, dissolved in 100 mL deionized water] was added and proteins were subsequently precipitated by trichloroacetic acid (BDH, England). The tube contents were mixed and centrifuged (Eppendorf centrifuge 5415 C, Germany); two samples of each supernatant were transferred to a flat-bottomed microplate and their absorbencies were read at 520 nm using a microplate reader (SpectraMax, Molecular Devices Inc). NO values were calculated from standard calibration plots [39 (link)].

Trichloroacetic acid (TCA), 5, 5′-dithio-bis-2-nitrobenzoic acid (DTNB), thiobarbituric acid (TBA), reduced glutathione (GSH), and sulphanilic acid were purchased from Sigma-Aldrich (USA). Kits of cholesterol, HDL-cholesterol, and triglyceride were purchased from Biosystems (Spain), glucose was purchased from Spinreact (Spain), ELISA kit of insulin and IFN-γ were purchased from DRG (USA) and Komabiotech (Korea), respectively, and IL-6 and IL-12 were purchased from Sunredbio (Shanghai). All other chemicals and reagents were of analytical grade.

Thioacetamide, betanin, N-acetylcysteine, GSH/GSSG kit, sodium chloride, procaine, heparin, paraFormaldehyde, sodium hydroxide, gallic acid, Folin–Ciocalteu reagent, catechin, ascorbic acid, dithiothreitol (DTT), DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate), Trolox^TM ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), sodium acetate, TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), ABTS^•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), potassium persulfate, AAPH (2,2′-azobis (2-methylpropionamidine) dihydrochloride), sodium nitroprusside, sulphanilic acid, N-1-naphthyl ethylenediamine were all obtained from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile (nanograde) was from Mallinckrodt. Formaldehyde, absolute methanol and glacial acetic acid were from J.T. Baker (Madrid, Spain). Hydrochloric acid and ferric chloride were from Fermont (Monterrey, México) and sodium pentobarbital was from Pisabental, Pisa Agropecuaria, México.