Developing a Validated HPLC Method for the Phytochemical Analysis of Antihyperuricemic Phenylethanoid Glycosides and Flavonoids in Lippia nodiflora

Abstract

The phenylethanoid glycosides and flavonoids of Lippia nodiflora were reported as xanthine oxidase inhibitors that possessed antihyperuricemic effects in chemically-induced hyperuricemic rats. The present study developed a validated high performance liquid chromatography method for the simultaneous phytochemical analysis of five isolated antihyperuricemic constituents in L. nodiflora, comprising two phenylethanoid glycosides, arenarioside (1) and verbascoside (2), and three flavonoids, 6-hydroxyluteolin (3), 6-hydroxyluteolin-7-O-glycoside (4) and nodifloretin (5). These compounds were analysed at 340 nm, using gradient elution of 0.1 % aqueous acetic acid and acetonitrile. The limits of detection were 78.1 ng/mL for 3 and 39.1 ng/mL for the other compounds at a signal-to-noise ratio of 3. The limits of quantification were 312.5 ng/mL for 3 and 156.3 ng/mL for the rest at a signal-to-noise ratio of 12. The intra-day and inter-day accuracy for the five bioactive constituents were between 98.60 % and 100.97 % while their precision values were below 2.11 %. The method was then successfully applied for the quantification of 1-5 in the methanolic extract of L. nodiflora whole plant and its fractions (F1-F4), the methanolic plant part extracts (leaves, stems, roots, and flowers), and water extract of the L. nodiflora whole plant at room temperature.

Development, Validation, and Application of HPLC Method for Quantification of Antihyperuricemic Compounds from Lippia nodiflora in Rat Plasma

“…The method was validated through intraday and inter-day analysis of precision and accuracy according to the International Conference on Harmonisation guidelines [33]. The intraday accuracy and precision were determined for each compound by evaluating six replicate measurements of each concentration on a single day, while the inter-day accuracy and precision were determined by assessing measurements of each concentration over 6 consecutive days [24]. The recovery of the direct extraction method using methanol was calculated by comparing the peak area of the compounds after extraction with those of the compounds dissolved in the methanol at a similar concentration [24].…”

“…4), were isolated from the bioactive fraction of L. nodiflora following the protocol described previously [19] and were used as marker compounds for the method validation and quantification of rat plasma samples. The purity of the compounds was determined using an Agilent 1120 Compact LC system equipped with a variable wavelength photometric detector and Agilent EZ-Chrom Elite Compact Software (Agilent Technologies) [24]. The purities were found to be 97.71, 98.19, 96.25, 97.30, and 97.36 % for 1, 2, 3, 4, and 5, respectively.…”

“…The gradient elution was programmed as follows: 0-25 min, 15 % B; 25-30 min, 20 % B; 30-35 min, 36 % B; 35-38 min, 15 % B. This was followed by a 12-min equilibration period prior to the injection of each sample [24].…”

“…The LOD was defined as the lowest concentration that the analytical system can reliably differentiate from the background level and was calculated based on a signal-to-noise ratio of 3:1. The LOQ was defined as the lowest quantifiable concentration on the calibration curve and was calculated based on a signal-to-noise ratio of 12:1 [24,31,32].…”

“…The only study found was on the pharmacokinetic of acteoside, also known as verbascoside, from Plantago asiatica or Cistanche deserticola using liquid chromatography-mass spectrometry [20], liquid chromatographytandem mass spectrometry [21,22], or liquid chromatography with amperometric detection [23]. Previously, we developed, validated, and applied an HPLC method for the phytochemical analysis of the antihyperuricemic constituents 1-5 in the plant samples [24]. In the present study, we aimed at the development of a simple and reliable HPLC method with a UV detection method for the simultaneous determination and quantification of the antihyperuricemic constituents 1-5 in rat plasma and to apply the method in the pharmacokinetic study of those bioactive compounds in rats after oral and intravenous administration.…”