Quantitative analysis by HPLC/DAD of luteolin and apigenin in flavonoid-enriched bioactive fraction of Arrabidaea chica (Bonpl.) Verlot

Arrabidaea chica Verlot (Bignoniaceae), popularly known as “crajiru” (Amazonas) and “pariri” (Pará) among other names, is a climbing shrub that occurs in tropical America, more particularly in the Amazon Region. Leaves of this plant are mainly used in traditional and folk medicine as anti-inflammatory and for wound healing. This species is included in the National List of Medicinal Plants of Interest to the Public Health System (Renisus), that makes necessary the standardization of its extracts to ensure quality, efficacy and safety. Previous results of our research group demonstrated the potential leishmanicidal activity of the hydroethanolic extract (IC50 = 101.5 ± 0.064 μg/ mL) and the flavonoid-enriched fraction (IC50 =
40.42 ± 0.10 μg/ mL) of A. chica against promastigote forms of Leishmania amazonensis. This work aims to quantify two of the flavones present in the flavonoid-enriched fraction, luteolin and apigenin, in order to standardize this possible active pharmaceutical ingredient. Hydroethanolic extract was obtained by maceration from dried and powdered leaves during 5 days, partitioned
by liquid-liquid extraction and successively chromatographed in Sephadex LH-20 column, affording a flavonoid-enriched fraction and the isolated flavones luteolin and apigenin. Flavonoids were identified by mass spectrometry (MS) and nuclear magnetic resonance analysis of hydrogen (1HRMN) and carbon (13CRMN). Flavonoid-enriched fraction and flavones were analysed by high-performance liquid chromatography (HPLC), using a Shimadzu® Nexera XR chromatograph, at Environmental Toxicology Laboratory (ENSP/FIOCRUZ), equipped with CBM20A controller, DGU20A degasser, LC20AD binary pump, CTO20A oven, SILA20A automatic injector and SPDM20A detector. Luteolin (Sigma-Aldrich, Brazil) and apigenin (Phytolab, Germany) analytical standards were used in an attempt to quantify such compounds in the fractions. The quantification of flavones was carried out by an analytical method previously validated, using a gradient elution system with ultrapure water (acidified with acetic acid at pH 3.0) and acetonitrile (Merck, Brazil) as mobile phases, modified C18 column (250 mm x 4.6 i.d., 5 um particle size), oven temperature 50ºC, flow rate at 0.8 mL/ min, total analysis time 45 min. The wavelength of 254 nm was chosen to monitor and to quantify the flavonoids. Analytical curves of luteolin (tR = 29.2 min) and apigenin (tR = 33.0 min) were obtained by triplicate injections of following concentrations of standards: 1, 4, 8, 12, 16 and 20 ug/ mL, resulting in the equations ABS = 83557(concentration) – 21030 (r2 = 0.9995) for luteolin and ABS = 99355 (concentration) – 85731 (r2 = 0.9937) for apigenin. Luteolin and apigenin contents in the flavonoid-enriched fraction were calculated repectively as 38.1% and 2.1%. This work will be very useful for quality control in further studies of in vivo leishmanicidal activity of enriched
fractions of A. chica.