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HPLC method for quantification of naphthoquinones in plant extracts

Plants produce a variety of secondary metabolites with a potential for treatment of human diseases. One such group of metabolites are naphthoquinones (NQs) which recently sparked an interest for use as novel therapeutics [1]. The aim of this study was to optimize a method for quantification of naphthoquinones (NQs), in particular 2-hydroxy-1, 4-naphthoquinone (2-HNQ) and 2-methoxy-1, 4-naphthoquinone (2-MNQ) and to determine the level of NQs in species Impatiens glandulifera Royle and Impatiens balfourii Hook.f., present in Croatia. The leaves and the flowers of I. glandulifera and I. balfourii were collected in the continental part of Croatia (Čučerje) in the summer of 2016. After air-drying, the plant material was grounded and for each plant sample two types of extractions were used: ultrasound extraction and decoction. Quantification of NQs was performed on HPLC Agilent 1100 (Santa Clara, CA, USA), which consisted of a gradient pump, an autosampler and a diode-array detector controlled by the ChemStation for LC 3D software. For the mobile phase, methanol and 2% acetic acid were used (gradient elution: 0-10 min, 75:25 ; 10-20 min, 68:32 ; 20-35 min 55:45, 35-42 min, 75:25) with a flow rate set to 1 mL/min. The injection volume was 20 μL and separation was achieved using a RP-C18 analytical column (150 mm x 4.6 mm, particle size 5 μm ; Merck, Darmstadt, Germany). Chromatograms were recorded at 280 nm. Retention time of 2-HNQ was 16.45 min and of 2-MNQ was 21.68 min. The method was validated using 2- HNQ and 2-MNQ standards prepared in the concentration range from 2 to 100 μg/mL. Both calibration curves were linear ; the correlation coefficient (R2) for 2-HNQ was 0.9841 and for 2-MNQ it was 0.9826, while the calibration curve intercepts were -0.0028 and -0.003, respectively. For both, 2-HNQ and 2-MNQ, the calculated limit of detection (LOD) was 0.1 μg/mL and the limit of quantification (LOQ) was 0.5 μg/mL while the relative standard deviation (RSD) for the intra-day precision was below 5% and for the inter-day precision it was below 10%. Validation parameters indicate that the method is linear and precise and therefore can be used for the quantification of 2-HNQ and 2-MNQ in the prepared I. glandulifera and I. balfourii plant extracts. In both the leaves and the flowers extracts of I. glandulifera and I. balfourii only 2-MNQ was detected, while the concentration of 2-HNQ was under the LOD of the method (Table 1). From the two tested extraction procedures, decoction enabled a detection of a higher 2-MNQ concentration than ultrasound extraction. Both the I. glandulifera and the I. balfourii species had a higher 2-MNQ concentration in flowers than in leaves. In general, I. balfourii had a lower 2-MNQ concentration compared to I. glandulifera. In conclusion, the developed method enabled detection of NQs in plant extracts. Additionally, the decoction extraction procedure, compared to the ultrasound extraction, enabled detection of higher levels of NQs. Due to higher 2-MNQ levels, I. glandulifera can be considered as a better source of NQs than I. balfourii.

Impatiens glandulifera ; Impatiens balfourii ; naphthoquinones ; HPLC

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