Development and Validation of an HPLC Method for the Analysis of Flowers of Wild-Growing Primula veris from Epirus, Greece

Abstract

An HPLC-PDA method was developed for the determination of the flavonoids in the flowers of Primula veris from Epirus, Greece. The aim was to investigate the chemical content of the over-harvested P. veris populations of Epirus and to develop and optimize an extraction protocol to allow fast, exhaustive, and repeatable extraction. Qualitative analysis revealed that the P. veris flowers from Epirus were particularly rich in flavonoids, especially flavonol triglycosides including derivatives of quercetin, isorhamnetin, and kaempferol. A phytochemical investigation of a 70% hydromethanolic extract from the flowers afforded a new flavonoid, namely, isorhamnetin-3-Ο-β-glucopyranosyl-(1 → 2)-β-glucopyranosyl-(1 → 6)-β-glucopyranoside, which is also the main constituent of the flower extracts. Its structure elucidation was carried out by means of 1D and 2D NMR and mass spectrometry analyses. The HPLC-PDA method was developed and validated according to the International Council for Harmonisation guidelines. Since the main flavonol glycoside of the plant is not commercially available, rutin was used as a secondary standard and the response correction factor was determined. Finally, the overall method was validated for precision (% relative standard deviation ranging between 1.58 and 4.85) and accuracy at three concentration levels. The recovery ranged between 93.5 and 102.1% with relative standard deviation values < 5%, within the acceptable limits. The developed assay is fast and simple and will allow for the quality control of the herbal drug.

Comparative Phytochemical Analyses of Flowers from Primula veris subsp. veris Growing Wild and from Ex Situ Cultivation in Greece

“…The di-glucopyranosides of quercetin and isorhamnetin, the metabolite quercetin-3-O-dirhamnosyl-hexoside, as well as the tri-glucosides of quercetin, isorhamnetin and kaempferol have been found previously in an ethanolic extract of the flowers of P. veris growing wild in Greece (Epirus region) [ 21 ].…”

“…), as well as polymethoxylated flavonoids acting as chemotaxonomic markers for the genus Primula (8-methoxy-flavone; 3′,4′methylenedioxy-5′-methoxyflavone, etc.). Several triterpenic saponins are the dominating constituents in the roots of the species, contributing to the expectorant activity of the herb [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ].…”

Exploring the Chemical Content of Primula veris L. subsp. veris Wild-Growing Populations along a Climate Gradient: An HPLC-PDA-MS Quality Assessment of Flowers, Leaves and Roots for Sustainable Exploitation

“…The plant samples (Table 1) after harvesting were air-dried in the dark, at ambient temperature, were divided into different plant parts, namely the flowers, leaves, and roots, and they were subsequently ground to obtain homogenous powdered samples. For the qualitative/quantitative analyzes of the flowers and leaves, the analytical and validated protocol developed by Chintiroglou et al [23] was followed with some modifications, i.e., approximately 200 mg of the dried and powdered P. veris flowers were ultrasonicated at room temperature (Transsonic 660/H, Elma Hans Schmidbauer, operating at 35 kH, Singen, Germany) with 70% (v/v) aqueous methanol for 10 min four times (4 × 10 mL in total). The samples were filtered through a paper filter and the filtrates were combined and adjusted to 100.0 mL using 70% (v/v) aqueous methanol.…”

“…In Greece, for example, where P. veris subsp. veris is limited in the cooler higher-altitude zones, extensive illegal harvesting events have been reported to occur, which may further compromise the longterm survival of its wild-growing populations [21][22][23]. Therefore, both in situ (natural reserves, Natura 2000 sites, Important Plant Areas) and ex situ (botanic gardens, ex situ field collections, and seed banks) conservation strategies need to be employed for its protection.…”

“…In the framework of this project, flowers of P. veris subsp. veris belonging to three populations of Epirus were initially studied for the first time regarding their chemical content, and an analytical protocol has been established for the qualitative and quantitative analysis of its flowers [23]. The encouraging results prompted us to further investigate different populations of P. veris subsp.…”