Iridoids, Phenolic Compounds and Antioxidant Activity of Edible Honeysuckle Berries (Lonicera caerulea var. kamtschatica Sevast.)

Iridoid and polyphenol profiles of 30 different honeysuckle berry cultivars and genotypes were studied. Compounds were identified by ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC-ESI-qTOF-MS/MS) in positive and negative ion modes and quantified by HPLC-PDA. The 50 identified compounds included 15 iridoids, 6 anthocyanins, 9 flavonols, 2 flavanonols (dihydroflavonols), 5 flavones, 6 flavan-3-ols, and 7 phenolic acids. 8-epi-Loganic acid, pentosyl-loganic acid, taxifolin 7-O-dihexoside, and taxifolin 7-O-hexoside were identified in honeysuckle berries for the first time. Iridoids and anthocyanins were the major groups of bioactive compounds of honeysuckle constituents. The total content of quantified iridoids and anthocyanins was between 128.42 mg/100 g fresh weight (fw) (‘Dlinnoplodnaya’) and 372 mg/100 g fw (‘Kuvshinovidnaya’) and between 150.04 mg/100 g fw (‘Karina’) and 653.95 mg/100 g fw (‘Amur’), respectively. Among iridoids, loganic acid was the dominant compound, and it represented between 22% and 73% of the total amount of quantified iridoids in honeysuckle berry. A very strong correlation was observed between the antioxidant potential and the quantity of anthocyanins. High content of iridoids in honeysuckle berries can complement antioxidant properties of phenolic compounds.


Introduction
Edible honeysuckle berries (Lonicera caerulea L. var. kamtschatica Sevast.; Caprifoliaceae family) are gaining popularity in many European countries such as Russia, Poland, the Czech Republic, and others. The attractiveness of these fruits derives from a number of factors, including early time of ripening (in Poland before strawberries), resistance to spring frost, flavor, high contents of vitamin C and polyphenol compounds, and health-related properties. The fruits are good plant material for the food industry, for the production of juices, jams, purees, and-for the pharmaceutical industry-for the production of food supplements [1][2][3].
The health benefits of honeysuckle berries have been known and used for a long time in traditional medicine in Russia and China. Current research, both in vitro and in vivo, also supports the traditional medical use of honeysuckle berries. Recent studies indicate, among others, the antioxidant, anti-inflammatory and antibacterial properties of the extract from honeysuckle berry fruits [4][5][6]. The authors explain the health benefits of honeysuckle berries by the occurrence in the fruit of polyphenol compounds, mainly glycosides of anthocyanins. In addition to the anthocyanins, phenolic acids, flavonols, flavones and flavan-3-ols are also present [3,4,7,8]. Their contents vary in fruits, and depend on many factors, including cultivar and genotypes [9][10][11][12][13][14][15]. In addition to the polyphenols, in honeysuckle berries iridoids have also been identified [16][17][18][19]. Among the 13 iridoids we have identified epimeric pairs of loganic acid and loganin, sweroside, secologanin, secoxyloganin, and additionally pentosides of loganic acid (two isomers), pentosides of loganin (three isomers), and pentosyl-sweroside [19].
In Russia, Poland, the Czech Republic, Canada, Japan, and other countries, many honeysuckle cultivars have been selected. They differ in the content of bioactive compounds, appearance (size, shape), time of ripening, yield from the bush, growing conditions, and taste. Cultivar diversity is great, because of the search for a cultivar with large and tasty fruits. Honeysuckle berry fruits are sweet and sour, with a somewhat bitter aftertaste, and resemble blackcurrants and blueberries.
In the fruits, sugars affect the degree of sweetness, while organic acids are responsible for the sour taste. The tartness of fruit is determined by the quantity and quality of polyphenols, whereas the bitterness, among other compounds, is determined by secoiridoids [20]. In addition to the fact that polyphenol and iridoid compounds may affect the taste of the fruit, they exhibit high biological activity [21]. Many authors indicate their antioxidant and anti-inflammatory properties [5,[22][23][24][25]. Iridoids, in contrast to the polyphenols, are rarely found in fruits. Exceptions include cornelian cherry fruits [26], cranberry [27], bilberry [28], and, recently investigated by us, honeysuckle berries [17][18][19]. In this study, we determined the content of loganic acid, loganin, their three derivatives, and the quantity of iridoids in berries of honeysuckle. There are no publications on the quantitative analysis of different iridoids such as loganin 7-O-pentoside or loganic acid 7-O-pentoside in honeysuckle berries. Therefore, the aim of this study was qualitative and quantitative determination of iridoids and also of polyphenols in berries of 27 cultivars and 3 genotypes of blue honeysuckle (L. caerulea var. kamtschatica) with the usage of chromatographic methods, as well as the determination of their antioxidant activity.

Qualitative Identification of Iridoids and Phenolic Compounds
The results of qualitative identification of the compounds of honeysuckle berries are presented in Table 1, Figures S1 and S2. The compounds were identified by their UPLC retention times, elution order, spectra of the individual peaks (UV/Vis, MS), spectral data and by comparison with literature data. In our research, we determined 50 compounds from two groups: monoterpenes (iridoids) and polyphenols (anthocyanins, flavonols, flavanonols, flavones, flavan-3-ols, phenolic acids). Table 1. Characterization of compounds of honeysuckle fruits determined using their spectral characteristics in positive and negative ions in ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC-ESI-qTOF-MS/MS). Among the compounds of the first analyzed group, we identified iridoids. In our previous initial studies, we determined thirteen iridoids (loganic acid (LA), loganin (Lo), sweroside (S), their derivatives, and epimeric pairs of LA and Lo) from honeysuckle berries [17][18][19]. In this study, fifteen compounds with typical UV/Vis spectra were discovered. Among them, compounds 3 and 12 were identified in honeysuckle berries for the first time. In the former, higher abundance was observed for the ion at m/z 375.1276 [M − H] − in negative electrospray ionisation (ESI) mode than for the ion at m/z 377.1440 [M + H] + in positive ESI mode ( Figure S3). This compound (t R 2.80 min) displayed the same pseudomolecular and fragment ions as LA (compound 9; t R 3.73 min) and 7-epi-LA (compound 13; t R 4.27 min), but they differed in the retention times and abundance of the major fragment ions. In The most probable structures of fragment ions of taxifolin derivatives in positive mode are shown in Figure S4 and Table S1. Flavanonols have been detected for example in Rosa canina and R. micrantha fruits [31] but not in blue honeysuckle berries. in positive mode are shown in Figure S4 and Table S1. Flavanonols have been detected for example in Rosa canina and R. micrantha fruits [31] but not in blue honeysuckle berries.  [3]. Compound 42 was identified as luteolin 7-O-glucoside, with a pseudomolecular ion at m/z 447.0918 and a fragment ion at 285.0394 obtained after the loss of 162 Da, which has been confirmed by other authors [3,7] and compared with data for the proper standard. Compound 50 was identified as 7-O-rutinoside of diosmetin (diosmin) when compared with the standard.

Quantitative Identification of Iridoids and Phenolic Compounds
Fifty compounds from the iridoid and polyphenolic groups were identified with the UPLC-qTOF-MS/MS method (Table 1), but only major compounds were quantified using HPLC-PDA detection (Tables 2-7). Quantitative analysis of constituents of honeysuckle berry was performed for 27 cultivars and 3 genotypes.
The qualitative and quantitative compositions of iridoids were very different for all cultivars and genotypes. Five of the 14 identified iridoids, i.e., 8-epi-LA, pLo, pentosyl-sweroside (pS), 7-epi-loganin (7-epi-Lo), and secoxyloganin (secoxyLo), were present in trace amounts in the studied berries. The content of a further nine iridoids in fresh honeysuckle berries is presented in Table 2. Four of them (LA, 7-epi-loganic acid 7-O-pentoside (7-epi-LAp), loganin (Lo), and sweroside (S)) were present in all cultivars and genotypes, but there were differences in their levels: LA > Lo + S > 7-epi-LAp. Five other iridoids (7-epi-LA, loganic acid 7-O-pentoside (LAp), loganin 7-O-pentoside (Lop), 7-epi-loganin 7-O-pentoside (7-epi-Lop), and secologanin (secoLo)) were present in some cultivars only. The total quantified iridoid content in honeysuckle berries covered a wide range from 119.95 mg/100 g fresh weight (fw) ('Dlinnoplodnaya') to 276.43 mg/100 g fw ('Berry Smart Blue'). The average total content of quantified iridoids, evaluated by HPLC analysis, was 180.77 mg/100 g fw. We previously observed higher, in terms of the average value, total content of quantified iridoids in cornelian cherry fruits and honeysuckle berry [18,26]. Contents of LA and its derivatives were more than twice the content of Lo, S, and their derivatives. In honeysuckle berries, the dominant compound among iridoids was LA (mean 81.09 mg/100 g fw). The lowest amount of this compound (35.22-39.21 mg/100 g fw) was found in 'Vostorg', 'Sineglazka', and 'Chelyabinka', whereas the highest (182.28 mg/100 g fw) was found in 'Kuvshinovidnaya'. LA represented between 22% and 73% of the total content of quantified iridoids in honeysuckle berry. This range is very wide and depends strongly on the cultivar or genotype. In our previous studies, we investigated LA content in many cornelian cherry cultivars, but the percentage content of iridoid in these fruits was in a narrower range, i.e., 88%-96% [26]. The high LA content is beneficial, because this compound exhibits biological activity. Many authors have shown that LA has strong anti-inflammatory properties [33][34][35]. In studies in rabbits Sozański et al. [34,35] revealed that LA diminished diet-induced dyslipidemia and atherosclerosis, increased PPAR-α and PPAR-γ expression, and exhibited anti-inflammatory activity. In the ten cultivars of analyzed berries, there was determined the epi-isomer of LA, which constituted 11% of the total amount of quantified iridoids, on average. The highest amount of this compound (45.05 mg/100 g fw) was present in 'Viola'. epi-LA, like LA, also exhibits biological activity. According to Dinda et al. [36], epi-LA exhibited strong antibacterial activity against Escherichia coli and Staphylococcus aureus.
LAp and 7-epi-LAp were the two major derivatives of isomers of LA, but the first compound was about three times more abundant than the second. Concentrations of LAp ranged from 9.37 mg/100 g fw in "Blue Velvet" to 71.16 mg/100 g fw in 'Kamchadalka'. This compound was identified in 24 cultivars, excluding 'Bakcharskaya', 'Karina', 'Kuvshinovidnaya', 'Goluboe Vereteno', and 'Wojtek'. The average content of LAp was 31.72 mg/100 g fw. 7-epi-LAp appeared in all cultivars, and the average amount was 11.70 mg/100 g fw. 'Bakcharskaya', Atut', and 'Viola' contained the lowest amount of this compound (2.86-3.69 mg/100 g fw), while 'Morena' contained the highest amount (20.43 mg/100 g fw). To our knowledge, there are no reports on the occurrence of pentose derivatives of loganic acid in other raw materials. For all cultivars, the total content of Lo and S ranged from 1.87 mg/100 g fw ('Tomichka') to 71.71 mg/100 g fw ('Wojtek'). The average content of these iridoids found in the berries was 19.00 mg/100 g fw, which accounted for 10% of all iridoids. These iridoids are not the main components of berries, but their presence in the fruit is important for their health benefits, such as antispasmodic and antibacterial activities [36,37]. The results of Ma et al. [38] showed that Lo and its derivatives were active against diabetic nephropathy. In addition, higher concentrations of L and S may increase the bitter taste of berries. Other fruits reported to contain both Lo and S are Cornus officinalis and Cornus mas [39][40][41]. According to Du et al. [39] and Zhou et al. [40], Lo, beside morroniside, is one of the main iridoids in the fruits of C. officinalis, while S is the minor iridoid in this fruit. Both compounds are present in trace amounts in C. mas fruits [41].
Among pentose derivatives of Lo, the most important compounds are Lop and 7-epi-Lop. Lop, similarly to LAp, was present in the same 24 cultivars. It was not detected in 'Bakcharskaya', 'Karina', 'Kuvshinovidnaya', 'Goluboe Vereteno', or 'Wojtek'. Concentrations of Lp ranged from 5.85 mg/100 g fw in 'Viola' to 83.85 mg/100 g fw in 'Amphora'. The average content of this compound was 35.79 mg/100 g fw, which accounted for 20% of all iridoids. 7-epi-Lop was found in only 14 cultivars. The lowest amount of this iridoid (1.83 mg/100 g fw) was found in 'Chernichka', whereas the highest (9.11 mg/100 g fw) was found in the 'Blue Velvet' cultivar. Similarly as in the case of the derivatives of LAp, there are no reports on the prevalence of the derivatives of Lop in other raw materials.
The 24 cultivars of berries contained secoLo. Among cultivars, the concentration of this compound ranged from 0.88 mg/100 g fw in 'Leningradskii Velikan' to 13.30 mg/100 g fw in 'Klon 44'. secoLo occurs in Lonicera [42,43] and Cornus [44] species. According to Graikou et al. [37], secoLo, similarly to Lo, has antimicrobial activity but only against Gram-positive bacteria. Tables 3-7 show the contents of individual phenolic compounds of 30 honeysuckle berry cultivars and genotypes. The major polyphenolic groups detected in honeysuckle berries in this study were similar to those reported in previous studies [3]. They were anthocyanins, flavonols, flavones, flavan-3-ols, and phenolic acids. Additionally, in this work, we identified compounds from another polyphenolic group, i.e., flavanonols.

Antioxidant Activities of Cultivars and Ecotypes of Honeysuckle Berries
The antioxidant activity (1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) and ferric reducing ability of plasma (FRAP)) of the blue honeysuckle berries of 30 cultivars and genotypes is shown in Table 7. The effects of compounds present in the berries on the antioxidant activity measured by the DPPH assay ranged from 8.80 to 28.77 µmol TE/g fw, and for the FRAP assay they ranged from 22.41 to 57.52 µmol TE/g fw. 'Amur', 'Jolanta', 'Klon 40', 'Kuvshinovidnaya', and 'Nympha' were characterized by high activity measured by both DPPH (26.48-28.77 µmol TE/g fw) and FRAP (50.24-57.52 µmol TE/g fw). Low activity was observed for 'Atut' (8.80 µmol TE/g fw for DPPH and 22.86 µmol TE/g fw for FRAP) and 'Karina' (8.94 µmol TE/g fw for DPPH and 22.41 µmol TE/g fw for FRAP). These results showed that DPPH and FRAP assays for 30 cultivars and genotypes revealed the same trend. Similar results for ABTS and FRAP assays have been reported previously by other authors [11]. High activity of honeysuckle berries has been confirmed by many authors [2,8,10,13], although the level of activity depends on many factors including cultivar. The differences in antioxidant activity between honeysuckle berry cultivars and genotypes may result from different qualitative and quantitative constituents.
The correlation between the antioxidant activity of honeysuckle berry extracts and the content of anthocyanins depends on the measurement method (Table S2). Correlation coefficients were higher for the FRAP method (r = 0.94) than the DPPH method (r = 0.78). A very strong correlation was observed between the antioxidant potential (FRAP) and anthocyanins (r = 0.94), and there was a strong correlation between the antioxidant potential (DPPH) and anthocyanins (r = 0.78).
These are the first studies on the correlation of the antioxidant activity of honeysuckle extract with the amount of iridoids. The obtained results showed that the amount of iridoids weakly correlated with in vitro antioxidant activity measured by DPPH and FRAP methods. This is due to the structure of iridoids, which in their molecule do not have (or they have, but only few) phenolic OH groups, which neutralize free radicals. This is consistent with the study of Pacifico et al. [49]. The authors reported that iridoids did not exhibit a good radical scavenging capacity. They explained that the moderate radical scavenging capacity is probably due to their poor hydrogen-donating ability. Iridoids exhibit higher biological activity, especially anti-inflammatory and antibacterial activity [33][34][35]; therefore their high content in blue honeysuckle berries can complement the antioxidant effect of phenolic compounds. Thus, honeysuckle berries may have wider potential than other fruits containing only polyphenols.

Extraction of Compounds for Qualitative and Quantitative Analysis
Frozen berries of honeysuckle were homogenized and 5 g of the homogenate was extracted with 50 mL of 80% aqueous methanol (v/v) acidified with 1% HCl by ultrasonication for 20 min. The extract was centrifuged and diluted (re-distilled water with the ratio 1:1, v/v). For UPLC-qTOF-MS/MS and HPLC-PDA analysis the supernatant was filtered through a Hydrophilic PTFE 0.22 and 0.45 µm membrane (Millex Samplicity Filter, Merck, Germany) and used for routine investigation.

Identification of Iridoids and Polyphenols by UPLC-qTOF-MS/MS
The method was previously described by Wyspiańska et al. [50]. Identification of compounds was performed using the Acquity ultra-performance liquid chromatography (UPLC) system, coupled with a quadrupole-time of flight (Q-TOF) MS instrument (UPLC/Synapt Q-TOF MS, Waters Corp., Milford, MA, USA), with an electrospray ionization (ESI) source. Separation was achieved on an Acquity BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm; Waters). The mobile phase was a mixture of 2.0% aq. formic acid v/v (A) and acetonitrile (B). The gradient program was as follows: initial conditions-1% B in A, 12 min-25% B in A, 12.5 min-100% B, 13.5 min-1% B in A. The flow rate was 0.45 mL/min and the injection volume was 5 µL. The column was operated at 30 • C. UV-vis absorption spectra were recorded on-line during UPLC analysis, and the spectral measurements were made in the wavelength range of 200-600 nm, in steps of 2 nm. The major operating parameters for the Q-TOF MS were set as follows: capillary voltage 2.0 kV, cone voltage 40 V, cone gas flow of 11 L/h, collision energy 28-30 eV, source temperature 100 • C, desolvation temperature 250 • C, collision gas, argon; desolvation gas (nitrogen) flow rate, 600 L/h; data acquisition range, m/z 100-2000 Da; ionization mode, negative and positive. The data were collected with Mass-LynxTM V 4.1 software (Waters Corp., Milford, MA, USA). The runs were monitored at the following wavelengths: iridoids at 245 nm, phenolic acids and their derivatives at 320 nm, flavan-3-ols, flavonols, flavanonols, flavones and flavanones at 280 and 360 nm, anthocyanins at 520 nm.
Loganic acid and its derivatives were expressed as mg of loganic acid equivalents (LAE) per 100 g fresh weight (fw), loganin, sweroside and their derivatives as loganin equivalents (LoE) per 100 g fw, anthocyanins as cyanidin 3-O-glucoside equivalents (CygE) per 100 g fw, derivatives of quercetin and taxifolin as quercetin 3-O-glucoside equivalents (QgE) per 100 g fw, luteolin -O-dihexoside-hexoside as luteolin 7-O-glucoside equivalents (LgE) per 100 g fw, caffeoylquinic acids as mg of 5-O-caffeoylquinic (chlorogenic) acid equivalents (ChAE) per 100 g fw. Solutions of standards (1 mg/ml) were dissolved in 1 mL of methanol. The appropriate amounts of stock solutions were diluted with 50% aqueous methanol (v/v) acidified with 1% HCl in order to obtain standard solutions. Analytical characteristics for determination of phenolic compounds and iridoids are shown in Table S3.

Antioxidant Capacity
The total antioxidant potential of samples was determined using a ferric reducing antioxidant power ability of plasma (FRAP) assay by Benzie and Strain [52] as a measure of antioxidant power. The FRAP reagent was prepared by mixing acetate buffer (300 µM, pH 3.6), a solution of 10 µM TPTZ in 40 µM HCl, and 20 µM FeCl 3 at 10:1:1 (v/v/v). The DPPH radical scavenging activity of samples was determined according to the method of Yen and Chen [53]. DPPH (100 µM) was dissolved in pure ethanol (96%). All determinations were performed in triplicate using a UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). The absorbance was measured after 10 min at 593 nm for FRAP and at 517 nm for DPPH. For all analyses, a standard curve was prepared using different concentrations of Trolox. Calibration curves, in the range 0.01-5.00 µmol Trolox L −1 , were used for the quantification of the three methods of antioxidant activity, showing good linearity (r 2 ≥ 0.998). The results were corrected for dilution and expressed in µmol Trolox equivalent (TE) per 100 g fw.

Statistical Analyses
Results were presented as the mean ± standard deviation of three technical replications. All statistical analyses were performed with Statistica version 12.0 (StatSoft, Tulsa, OK, USA). One-way analysis of variance (ANOVA) by Duncan's test was used to compare the mean values. Differences were considered to be significant at α = 0.05.

Conclusions
The reported research clearly shows that honeysuckle berries are an excellent source not only of polyphenols (mainly anthocyanins), but also of iridoids, which are rarely present in other fruits. The content of both iridoids and polyphenols and antioxidant activity measured by DPPH and FRAP methods depends largely on the cultivar of berries. All 27 cultivars and 3 genotypes of blue honeysuckle berries had similar anthocyanin, flavonol, flavanonol, flavone, flavan-3-ol, and phenolic acid profiles, but did not have similar iridoid profiles. Two iridoids, two flavanones, and three flavones were identified in honeysuckle berries for the first time. The content of iridoids, besides anthocyanins, may be one of the most important parameters for appraising the characterization of blue honeysuckle fruits with respect to their nutraceutical value and potential use for different purposes.