A new ultra-high pressure liquid chromatography method for the determination of antioxidant flavonol aglycones in six Lysimachia species

Abstract UPLC-DAD method was developed and validated for the quantitative determination of free flavonol aglycones (kaempferol, quercetin and myricetin) after acidic hydrolysis in six Lysimachia species. Quantitative analyses showed that the amounts of various flavonol aglycones were significantly different in Lysimachia vulgaris, Lysimachia nummularia, Lysimachia punctata, Lysimachia christinae, Lysimachia ciliata and Lysimachia clethroides. The L. clethroides sample was found to be the richest in kaempferol (25.77 ± 1.29 μg/mg extract) and quercetin (97.67 ± 4.61 μg/mg extract), while the L. nummularia sample contained the highest amount of myricetin (20.79 ± 1.00 μg/mg extract). The antioxidant capacity of hydrolysed extracts was evaluated using in vitro DPPH• (2,2-diphenyl-1-picrylhydrazyl) and ABTS•+ [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] decolourisation tests. The observed radical scavenging capacities of the extracts showed a relationship with the measured flavonol aglycone content and composition. The acidic treatment resulted in an increased free radical scavenging activity compared to the untreated methanol extract.

available commercially. For the reduction of the complexity of genuine flavonoid compounds, various phytoanalytical assays combined with hydrolysis have already been established in the quality control of medicinal plants (eDQM 2016).
This study was undertaken to develop an UPLC-DAD method for the quantitation of flavonol aglycones in plant extracts. To justify the applicability of the developed method for quantitative analyses of herbal extracts Lysimachia genus was chosen due to its wide variety of flavonoids.
Lysimachia species are distributed worldwide in temperate climates and are widely known both as traditional herbal remedies and commercial garden plants. Phytochemical investigations of plants from this genus revealed the presence of flavonoids (Toth et al. 2014) and triterpene saponins (Zhang et al. 2007;Podolak et al. 2013). Flavonoid composition of Lysimachia ciliata, as well as constitution and content of kaempferol, quercetin and myricetin aglycones in different Lysimachia extracts were aimed to investigate for the first time. The antioxidant activity of the extracts using in vitro DPPH • and ABTS •+ decolourisation tests were also studied.

Results and discussion
In our previous work, the presence of more than 10 flavonol components was described for three Lysimachia species native to Hungary (Toth et al. 2014). The most abundant aglycones were kaempferol, quercetin and myricetin. In view of the required effort in the separation of flavonols, quantification on the basis of a reduced number of sample components may pose a feasible alternative. Given that aglycones originating from flavonol-O-glycosides were planned to be quantified, mild hydrolysis method (25 v/v% hydrochloric acid 85 ℃ and 1 h) was considered according to the european Pharmacopoeia (eDQM 2016). Under these conditions, C-glycosidic bonds are unimpaired.
The UV chromatograms of standards and Lysimachia hydrolysed extracts are shown in Figure S1. The optimised UPLC method was validated in terms of linearity, limits of quantification (LOQ) and detection (LOD), precision and accuracy. The validation parameters are summarised in Table S1 and S2.
For the quantitation of flavonoid aglycones in Lysimachia vulgaris, Lysimachia nummularia, Lysimachia punctata, Lysimachia christinae, L. ciliata and Lysimachia clethroides extracts, hydrolysis was applied prior to the UPLC analysis. The results are summarised in Table 1. The calculated total flavonol aglycone content (sum quantity of kaempferol, quercetin and myricetin) was the highest in L. clethroides and the lowest in L. christinae samples. The L. clethroides extract was found to be the most abundant in kaempferol and quercetin, while the L. nummularia sample contained the highest amount of myricetin. The L. vulgaris sample revealed relatively high amount of quercetin, also confirmed by our previous work (Toth et al. 2014). The lower quantity of flavonol aglycones, however, according to literature data, other type of flavonoids: flavone-, flavanone-and methylated flavonoid derivatives are cumulated in the herb. Furthermore, C-glycosides are also reported in Lysimachia species (Sun et al. 2013).
To the best of our knowledge, no literature data are available regarding the flavonoid composition of L. ciliata. Presence of kaempferol-, quercetin-and myricetin aglycones in the hydrolysed extract and their quantity were described for the first time. The cultivated varieties 'Firecracker' contains similarly to L. vulgaris, mainly quercetin and kaempferol, while myricetin is present in lower amounts. In L. clethroides, significant amount of quercetin and kaempferol was measured, which is in good agreement with literature data that show presence of numerous glycosides of kaempferol and quercetin (Liu et al. 2010). The amount of myricetin was the lowest in both Lysimachia clethroides and L. christinae.
In vitro assays were employed to determine the free radical scavenging potency of the isolated extracts. Table 2 summarises the results of the antioxidant capacity of the standards and hydrolysed Lysimachia extracts. All the plant extracts were capable of scavenging the mentioned radicals in a concentration-dependent fashion. L. nummularia showed the highest antioxidant activities which can be explained by the high amount of myricetin in the sample. Besides L. nummularia, L. vulgaris and L. christinae extracts have higher scavenging activity than kaempferol. These contain quercetin and myricetin in relatively high amount. It is important to note that L. clethroides hydrolysed extract showed lower antioxidant capacity despite the high amount of flavonol aglycones. Besides flavonol-O-glycosides, C-glycosides and methoxylated flavonoids may also be accumulated (Liu et al. 2010). Thus, these currently not identified and quantified components may impair the free radical scavenging effect. The crude methanol extracts of each Lysimachia possessed varying degrees of efficacy, while the acid treatment significantly increased the radical scavenging activity owing to the increase in the number of phenolic groups (Burda & Oleszek 2001).

Plant materials
Aerial parts of L. nummularia L., Lysimachia punctata L. and L. vulgaris L. were obtained from Bükk National Park, Hungary. The samples were dried at room temperature and authenticated in the Department of Pharmacognosy, Semmelweis University, Budapest, Hungary where voucher specimens (LN/201106, LP/201106, LV/2001106), are deposited. The dried aerial

Reagents and chemicals
Methanol, chloroform, ethyl acetate, hydrochloric acid, acetic acid, of reagent grade, and hexamethylenetetramine and anhydrous Na 2 SO 4 were obtained from Molar Chemicals Kft.

Extraction and acidic hydrolyses
Soxhlet extraction was performed using a laboratory-scale apparatus. Dried and milled herb (7 g) was extracted with 250 ml of chloroform for 6 h at 60 °C, followed by methanol extraction for 6 h at 90 °C. The extracts were evaporated to dryness under reduced pressure in a rotary evaporator at 50 °C and redissolved in methanol. Hydrolysis step was performed according to the method of the european Pharmacopoeia (2016) with minor modifications. Briefly, 0.1 ml (containing approximately 0.02 g dried extract) of each redissolved residue solution was mixed with 2.0 ml 25% hydrochloric acid and 10 ml methanol. The samples were heated in water bath at 85 °C for 1 h. After the reactions of acid hydrolysis were completed, each mixture was cooled down to room temperature and was transferred to a separating funnel containing 20 ml of water, and extracted three times with 15 ml of water-saturated ethyl acetate. The combined ethyl acetate fractions were dried over Na 2 SO 4 . The hydrolysates were evaporated to dryness under reduced pressure in a rotary evaporator at 40 °C, redissolved in HPLC grade methanol and filtered through Phenex-RC 15 mm 20-μm syringe filters (Gen-Lab Ltd, Budapest, Hungary).

UPLC-DAD conditions and quantitative method validation
The samples were separated by ultra performance liquid chromatography (Waters Acquity) managed by Acquity console software using Acquity UPLC HSS C18 1.8 μm 2.1 × 100 column maintained at 40 °C. The following gradient elution programme was applied at flow rate of 0.65 ml/min. Solvent 'A' was 0.2% (v/v) acetic acid, while Solvent 'B' was acetonitrile: 0 min 22% B, 3 min 40% B, 3.5 min 100% B, 4 min 22% B. The detection wavelength was set to 254 nm and 3 μl injection volume was used. The quantity of flavonol aglycones in the hydrolysed Lysimachia extracts were determined by external standard method. For the calibration, standard solutions were prepared at concentration of 1.0, 10.0, 50.0, 100.0, 300.0 and 500.0 μg/ml using kaempferol, quercetin and myricetin authentic standards in gradient grade methanol. each standard solution was prepared in triplicates and injected once. Calibration curve was constructed by plotting peak areas against corresponding concentrations. Slope, intercept and the coefficient of determination were determined by least squares polynomial quantitation regression analysis. LOD and LOQ were determined at 3:1 and 10:1 signal to noise ratios, respectively. Quality control samples were prepared at concentration of 1, 50 and 500 μg/ml for the standard solutions. These solutions were used to determine both the intraday and the interday precision (low, mid-, and high concentrations of the standard in triplicates on the same day and on three successive days, respectively). Retention time repeatability was checked with six successive runs of the L. vulgaris, L. nummularia, L. punctata, L. christinae, L. ciliata and L. clethroides hydrolysed extracts. Blank samples (pure solvent) were analysed to check the occurrence of any impurity or co-elution.

Scavenging effect on DPPH • and on ABTS •+
Antioxidant activity of Lysimachia hydrolysed extracts and quercetin, kaempferol and myricetin authentic standards were determined using DPPH • and ABTS •+ in vitro decolourisation assay (Toth et al. 2014). About 10 mg DPPH • was dissolved in HPLC grade methanol to 0.25 g/L concentration and 10 mg ABTS was dissolved in 2.6 ml HPLC grade water and reacted with 1.72 mg potassium persulphate to produce ABTS radical cation. The DPPH • and ABTS •+ stock solutions were diluted with HPLC grade methanol and spectroscopic grade ethanol. The absorbance of the diluted free radical solutions were approximately 0.90. In their radical forms, DPPH • and ABTS •+ have absorption maxima at characteristic wavelengths, at 515 and 734 nm, respectively. At least five different volumes of the diluted samples were added to 2.5 ml DPPH • and ABTS •+ solutions resulting in different final concentrations, and the decrease of the absorbance was measured after six minutes by Hitachi U-2000 spectrophotometer at the characteristic wavelengths. Percentage of inhibition was calculated from the decrease of the absorbance. The antioxidant activity was characterised by plotting the inhibition percentage of the samples as a function of sample concentration followed by linear regression. The concentrations belonging to the 50% inhibition (IC 50 ) were determined by linear regression analyses. All analyses were run in triplicates and averaged. The results are expressed as mean values and standard deviation (SD).

Conclusion
This study presents a selective UPLC-DAD quantitation method for flavonoid aglycones of six Lysimachia species for the first time. According to our results, extracts containing higher amounts of myricetin aglycone has proved to be more effective in scavenging DPPH • and ABTS •+ radicals compared to the less hydroxylated quercetin and kaempferol. The data also suggest that the acidic treatment increased the free radical scavenging potential of the crude extracts.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the OTKA [grant number PD 109373].