L 7420

The HPLC system (Hitachi, Japan) consisted of a pump (L-7000), an autosampler (L-7200), an L-7420 UV-vis detector, and a D-7000 interface module. All the test samples were analyzed on a Lichro-CART 250-4 Purospher STAR RP-18e (250 × 4.6 mm i.d., 5 μm) column, and the temperature was kept constant at room temperature. The HPLC system mobile phase was composed of methanol and water (9 : 1), the flow rate was set at 1 mL/min, and the wavelength of detection was 282 nm. The artocarpin content in the methanol extract and fractions of A. communis was determined by comparing the retention time with the artocarpin standard.

Dendrimers and their conjugates were analyzed by acidic gel electrophoresis, as previously described by Sharma and colleagues [27 (link)], and isoelectric focusing by Upadhaya and colleagues [28 (link)], and reversed phase HPLC. IEF was performed using high resolution pH 3–10 ampholyte solution (Fluka) with 10 mM phosphoric acid and 20 mM NaOH. HPLC was performed on a Hitachi system with an autosampler (L-7200), pump (L-7100), UV-visible detector (L-7420), and an interface (D-7000) with a 20-µL loop. The C18 column used was a Varian Microsorb-MV (250 mm × 4.6 mm, 300 Å) attached to a MetaGuard 4.6 mm Microsorb 300 Å 5 µ C18 guard column (Agilent Technologies, Santa Clara, CA, USA). Mobile phase A was 0.1 wt % aqueous TFA (Trifluoroacetic acid), and mobile phase B was 0.085 wt % TFA in acetonitrile. A linear gradient elution was employed, starting with 5% B and ending 90% B in 30 min, at a flow rate of 1 mL/min. Samples were prepared in HPLC grade water and filtered through a 0.45 µm syringe filter.

The levels of fecal SCFAs in mice were analyzed as described by Torii et al. [21 (link)], with some modifications. Briefly, after feeding the mice with appropriate diets for 10 weeks, their fecal samples were collected. SCFAs in feces were extracted using 70% ethanol solution. The extracted solution was mixed with 2-ethylbutyric acid (109959, Sigma-Aldrich Co., St. Louis, MO, USA) as an internal standard. Next, a reaction-assistive agent was added, and the mixture was reacted at 60 °C for 20 min. The reaction was terminated with potassium hydroxide (30,603, Sigma-Aldrich) solution; the reaction mixture was incubated at 60 °C for 20 min and then extracted with a phosphoric acid solution (B0992, Katayama Chemical Industries Co., Ltd., Osaka, Japan) and ether. The upper ether layer was collected and mixed with water for further extraction. Finally, an ether layer was obtained and air-dried. Methanol was added to dissolve fatty acid hydrazide present in the sample. The final product was analyzed using high-performance liquid chromatography (HPLC). HPLC was performed using a LaChrom L-7100 HPLC system (Hitachi Ltd., Tokyo, Japan) equipped with a NUCLEODUR C18 HTec column (MACHEREY-NAGEL, Düren, Germany) and a UV-VIS detector (L-7420, Hitachi).

High performance liquid chromatography (HPLC) analysis was performed by using L-7100, L-7200 and L-7420, Hitachi HPLC system (Hitachi, Tokyo, Japan) equipped with a reverse phase-C18 column (250 × 4.6 mm, 5 µm, MightysilKanto Chemical, Tokyo, Japan). The sample load was 10 µL. Methanol at concentrations of 20% and 100%, both in 0.3% formic acid, were used as solvents A and B, respectively. The gradient program was as follows: 0% B at the initial stage and maintained for 10 min; 0%–10% B for 10–25 min; 10%–30% B for 25–60 min; 30% B for 60–75 min; 30%–0% B for 75–76 min; and 0% B for 76–85 min. Measurement of bioactive compounds was done by using a UV-VIS detector at 280 nm. Quantification of the compounds was calculated by using standard catechins, gallic acid, and catechol as internal standards [28 (link)].

A reverse-phase high-performance liquid chromatography (RP-HPLC) system with a Merck-Hitachi separation module (Hitachi, Japan) coupled with a UV-visible detector (L-7420) was used for the quantitative assay of diazepam. Isocratic elution was performed on C18-MZ-Analitical Perfectsil Target, 250 × 4.6 mm column (MZ-Analytical, Germany) at a 1.0 mL/min flow rate and column temperature of 25°C. The mobile phase was a combination of water and acetonitrile (50:50). The run time was 16 min, and the retention time of diazepam was around 14 min. Drug standards were prepared in the mobile phase and detected at a wavelength of 254 nm (26 (link)).

Each C. carandas extract was analyzed for ursolic acid content using Hitachi HPLC series L (Tokyo, Japan) with a UV detector (Hitachi L-7420, Tokyo, Japan) set at 210 nm. A stationary phase was reverse phase column, Luna 5.0 μ C18 (2) 100A (250 × 2.0 mm, 5 μm) (Phenomenex, Torrance, CA, USA). Mixture of HPLC-grade ethanol and DI water (90:10) was used as an isocratic mobile phase with a flow rate of 0.5 mL/min. All samples were filtrated through 0.2 μm nylon filter (GNWP04700, Millipore, Darmstadt, Germany) prior to the analyzation. The injection volume of each sample was 20 μL. Ursolic acid in a concentration range of 1–500 μg/mL was used to construct a linear regression equation. The ursolic acid content of each C. carandas extracts was calculated using the following equation:
where y was area under the curve (AUC) of ursolic acid peak which was detected around 9.3 min and x was a concentration of ursolic acid (g/mg extract). Every experiment was carried out in triplicate.