Evaluation of the Chemical Composition of Selected Varieties of L. caerulea var. kamtschatica and L. caerulea var. emphyllocalyx

Lonicera caerulea fruits are a rich source of vitamins, organic acids, and phenolic compounds, which are characterised by their health-promoting properties. The content of bioactive compounds in this fruit may vary depending on the cultivar and the harvest date. The fruits of the L. caerulea var. kamtschatica cultivars ‘Duet’ and ‘Aurora’ and the L. caerulea var. emphyllocalyx cultivars ‘Lori’, ‘Colin’ and ‘Willa’ were used in this study. L. emphyllocalyx fruit, especially the cultivar ‘Willa’, was characterised as having a higher acidity by an average of 29.96% compared to L. kamtschatica. The average ascorbic acid content of the L. kamtschatica fruit was 53.5 mg·100 g−1 f.w., while L. emphyllocalyx fruit had an average content that was 14.14% lower. The antioxidant activity (determined by DPPH, FRAP, and ABTS) varied according to the cultivar and the species of fruit analysed. The total polyphenol content differed significantly depending on the cultivar analysed; fruits of the L. emphyllocalyx cultivar ‘Willa’ were characterised by the lowest content of total polyphenols—416.94 mg GAE·100 g−1 f.w.—while the highest content of total polyphenols—747.85 GAE·100 g−1 f.w.—was found in the fruits of the L. emphyllocalyx cultivar ‘Lori’. Lonicera caerulea fruits contained 26 different phenolic compounds in their compositions, of which the highest content was characterised by cyanidin 3-O-glucoside (average: 347.37 mg·100 g−1). On the basis of this study, it appears that both L. kamtschatica fruits and L. emphyllocalyx fruits, especially of the cultivars ‘Lori’ and ‘Willa’, can be used in food processing.


Introduction
The blue honeysuckle (Lonicera caerulea L.) belongs to the genus Lonicera (Caprifoliaceae), which contains more than 200 species, native to the cold lands of the Far East and central Asia. Most of them are ornamental plants; only about 17 are edible fruit-producing species [1]. Lonicera caerulea has numerous varieties, several of which are widely cultivated, originating from Russia (L. caerulea var. edulis, L. caerulea var. kamtschatica, L. caerulea var. altaica, L. caerulea var. boczkarnikovae) and the island of Hokkaido in Japan (L. caerulea var. emphyllocalyx). They are long-living (25-30 years) shrubs that can reach 0.8-3.0 m height. They need an outside pollinator to bear fruits one year after planting. After three years, approx. 500 g of fruit can be obtained from one plant. Berries are dark blue or dark purple in colour, with a size of 1.5-3.0 cm, and a mostly cylindrical shape with a wax coating. They are plants that are very resistant to frost, able to withstand temperatures up to −46 • C [2]. Lonicera caerulea var. kamtschatica is a variety of honeysuckle, commonly known as Kamchatka berry, which is one of the most popular fruits in Poland, the Czech Republic, Canada, and Russia. Several varieties native to the species have been selected, which differ, among

pH and Acidity of Fruits
The organic acid content of the fruit decreases with successive stages of ripening. The organic acids contained in the fruit are perishable and, under the influence of various factors, e.g., temperature, can change in terms of concentration in the plant material [16,17]. L. caerulea fruit are rich in organic acids (e.g., malic acid, citric acid, quinic acid and fumaric acid; [17]). The contents of individual organic acids significantly influence the taste qualities of ripe fruit and the acceptability of the consumer. The average pH values of the L. kamtschatica and the L. emphyllocalyx fruits were similar: 3.07-3.32 and 3.13-3.52, respectively ( Figure 1). acid; [17]). The contents of individual organic acids significantly influence the taste qualities of ripe fruit and the acceptability of the consumer. The average pH values of the L. kamtschatica and the L. emphyllocalyx fruits were similar: 3.07-3.32 and 3.13-3.52, respectively ( Figure 1). These results are comparable to those obtained by other authors. In a study by Gerbrand et al. [18], the fruit pH of Lonicera caerulea ranged from 2.42 to 3.10; in an experiment of Auzanneau et al. [19], the fruit pH of the same species ranged from 2.70 to 3.30, depending on the growing year and harvest date. Compared to Miyashita et al. [20] and Oszmiański et al. [21], we found a lower average pH value in L. kamtschatica and L. emphyllocalyx berries: 2.60 and 2.65, respectively. The pH value is diversified compared to other species grown in Poland, e.g., saskatoon berry (4.12-5.03) [22], red currant (3.20 and 3.27) [23], sea buckthorn (3.02-3.19) [24], highbush blueberry fruit (2.76-3.33) [25], raspberry (3.72) and mulberry (5.17) [26]. In general, both L. kamtschatica and L. emphyllocalyx are rich in organic acids that give a taste resembling blueberries, with a distinct hint of sourness.

Contents of Ascorbic Acid and Antioxidant Activity in L. kamtschatica and L. emphyllocalyx Fruit
Ascorbic acid is a powerful antioxidant that is required for the activation of many enzymes. This compound is essential for the functioning of the human body, influencing These results are comparable to those obtained by other authors. In a study by Gerbrand et al. [18], the fruit pH of Lonicera caerulea ranged from 2.42 to 3.10; in an experiment of Auzanneau et al. [19], the fruit pH of the same species ranged from 2.70 to 3.30, depending on the growing year and harvest date. Compared to Miyashita et al. [20] and Oszmiański et al. [21], we found a lower average pH value in L. kamtschatica and L. emphyllocalyx berries: 2.60 and 2.65, respectively. The pH value is diversified compared to other species grown in Poland, e.g., saskatoon berry (4.12-5.03) [22], red currant (3.20 and 3.27) [23], sea buckthorn (3.02-3.19) [24], highbush blueberry fruit (2.76-3.33) [25], raspberry (3.72) and mulberry (5.17) [26]. In general, both L. kamtschatica and L. emphyllocalyx are rich in organic acids that give a taste resembling blueberries, with a distinct hint of sourness.

Contents of Ascorbic Acid and Antioxidant Activity in L. kamtschatica and L. emphyllocalyx Fruit
Ascorbic acid is a powerful antioxidant that is required for the activation of many enzymes. This compound is essential for the functioning of the human body, influencing the immune and circulatory systems, accelerating wound healing, slowing skin ageing, and regulating collagen production [30]. The average ascorbic acid content of the L. kamtschatica fruit was 53.5 mg·100 g −1 f.w., while the L. emphyllocalyx fruit had an average ascorbic acid content of 45.93 mg·100 g −1 f.w. (Table 1). The content ascorbic acid of L. caerulea fruit differs from that reported by Celli et al. [10]; we obtained results rang-Molecules 2023, 28, 2525 4 of 12 ing from 30.5 to 186.6 mg·100 g −1 f.w. According to Jurnikova et al. [31], the content of ascorbic acid in Lonicera caerulea, higher than 70 mg·100 g −1 f.w., is associated with a lower accumulation of phenolics. The ascorbic acid content of L. kamtschatica and L. emphyllocalyx fruit was at a similar level to popular berries grown in Poland, e.g., strawberries (average: 50.1 mg·100 g −1 ), raspberries (average: 30.6 mg·100 g −1 f.w.), blueberries (average: 60.1 mg·100 g −1 f.w.) [32,33], plum blackthorn (21.94 mg·100 g −1 f.w.), blackberry (33.85 mg·100 g −1 f.w.) [33]; red currant (31.2 mg·100 g −1 -44.1 mg·100 g −1 f.w.) [23], sea buckthorn (4.0-9.1 mg·100 g −1 f.w.) [24]. According to Senica et al. [14], spreads and smoothies made out of L. caerulea berries increase the concentration of ascorbic acid by 100%. Heating plant material before crushing may influence the stability of ascorbic acid, preserving its contents. This can be used in the food industry in the production of L. caerulea products [14]. Lonicera caerulea fruit extract containing anthocyanins is a highly effective antioxidant agent, effectively reducing reactive oxygen species (ROS), which are produced by immune cells as a result of inflammation. Persistent inflammation can lead to cell damage and chronic disease. The fruit extract further reduces lipid peroxidation, which affects cellular damage under oxidative stress conditions, which can contribute to the reduction in diseases related to oxidative stress [34]. The antioxidant activity of L. kamtschatica and L. emphyllocalyx fruit, determined by the DPPH method, was at the level of 68.68-89.62% inhibition, of which the L. kamtschatica cultivar 'Duet' had the highest free radical scavenging activity (on average 13.4-30.4% more than the other cultivars analysed). These results are comparable to those obtained by Kula et al. [2] and Khattab et al. [35], in which the antiradical values of Lonicera caerluea were, respectively, 85% and 78.70%. The antioxidant activity of L. caerulea fruits is significantly higher compared to other species, e.g., sea buckthorn (74%), bilberry (37-91%), and garden rhubarb (48-98%) [36]. The iron reduction capacity (FRAP method) of L. kamtschatica range from 30.52 to 37.67 µM Fe 2+ ·g −1 f.w. and L. emphyllocalyx range from 30.52 to 37.67 µM Fe 2+ ·g −1 f.w. Results comparable to this study were obtained by Rupasinghe et al. [7], with an antioxidant value (FRAP) ranging from 27.96 to 46.90 µM Fe 2+ ·g −1 . According to studies, L. caerulea had more antioxidative properties (FRAP) compared to other species grown in Poland, e.g., strawberries (8.00 µM TE·g −1 f.w), blackberries (15.03 µM TE·g −1 f.w.), highbush blueberry (16.24 µM TE·g −1 f.w.), elderberry (29.56 mM Fe 2+ ·g −1 f.w.), blackthorn (14.74 mM Fe 2+ ·g −1 f.w.) and wild strawberry (10.99 Fe 2+ ·g −1 f.w.) [7,33]. Lonicera caerulea fruits are characterised by a higher antioxidant capacity. The antioxidant activity of the fruits of L. kamtschatica and L. emphyllocalyx, determined by the ABTS method, ranged from 1.91 to 2.21 mM TE·100 g −1 f.w. As reported by Rop et al. [37], the ABTS antiradical activity of ABTS of Lonicera caerulea was 0. (0.14 mM TE·100 g −1 f.w.) and raspberry (0.08 mM TE·100 g −1 f.w.) [26]. The high level of antioxidant capacity in L. kamtschatica and L. emphyllocalyx makes them very valuable in terms of bioactivity. Research on the antioxidant activity of extracts with L. caerulea revealed that the fruits of this plant are characterised by stronger antioxidant properties from other berries commonly regarded as effective antioxidants. Considering the relationship between modern-day diseases and long-term oxygen stress, strong antioxidant properties may indicate the potential importance of this fruit, not only in prophylaxis, but also in the treatment of many diseases [2], thus making L. caerulea products more valuable for food industry.
The analysis of phenolic compounds using the UPLC-PDA-MS/MS method allowed for the determination of the differences between the contents of individual groups of polyphenolic compounds contained in the fruit of the analysed cultivars of L. kamtschatica and L. emphyllocalyx ( Table 2). Berries of the L. kamtschatica cultivars 'Duet' and 'Aurora', and L. emphyllocalyx cultivars 'Lori', 'Willa' and 'Colin' were characterised by different contents of individual polyphenolic compounds. The total content of phenolic compounds depends, among other things, on the cultivar, the degree of fruit maturity, and also on the harvest date. The identification of compounds was carried out based on retention time (Rt), MS and MS/MS with available publications [41][42][43][44][45]. The extract prepared from the fruit contained 26 different compounds in its composition, including 14 anthocyanins, 8 flavonoids, 2 phenolic acids and 1 flavan-3-ol ( Table 2).
The highest anthocyanin content in the analysed fruits was found in the berries of L. kamtschatica 'Duet', at 456.3 mg·100 g −1 ( Table 2). Among the phenolic compounds found in the fruits of L. kamtschatica (509.29-597.29 mg GAE·100 g −1 ) and L. emphyllocalyx (416.94-747.85 mg GAE·100 g −1 ), anthocyanins represented, on average, 94% of all polyphenols, and the main representative was cyanidin 3-O-glucoside-C3G (the compound represented, on average, 82.2% of the total anthocyanin content detected in the fruits). Among the varieties analysed, the L. kamtschatica fruits of the 'Duet' variety contained the highest concentration of C3G in their composition-382.18 mg·100 g −1 ( Table 2). The results obtained in this study are comparable to those obtained by Rupasinghe et al. [9]; the C3G content of Lonicera caerulea fruit ranged from 68 to 649 mg·100 g −1 . C3G content was comparable to that reported by Khattab et al. [35], and the C3G content in the fruit of the cultivars 'Tundra', 'Berry Blue' and 'Indigo gem' reached 79-88% of the total anthocyanin content. The C3G content of the Lonicera caerulea fruit was significantly higher compared to the strawberry fruit (3.7 mg·100 g −1 ), blueberry (3.0 mg·100 g −1 ), and the cranberry (0.7 mg·100 g −1 ) [9]. C3G is the most prevalent anthocyanin in edible fruits and has been shown to have anti-inflammatory, antioxidant, chemotherapeutic, and epige-netic effects [9]. Fruits with a larger diameter and harvested at the optimal harvest time will have a higher anthocyanin content due to the larger skin area [15]. In the study of Senica et al. [15], spreads and smoothies made out of L. caerulea had higher concentrations of C3G (6.48, 5.00 mg·100 g −1 ) in comparison to fresh fruit. The fruits of this species play important roles in a wide range of physiological processes, e.g., protective effect against UV radiation for skin, protection against pathogenic strains, etc.. The total flavonoid contents ranged from 11.11 mg·100 g −1 in the L. emphyllocalyx cv. cultivar 'Colin' to 24.4 mg·100 g −1 in the case of the L. kamtschatica cv. cultivar 'Duet' ( Table 2). In the L. kamtschatica and L. emphyllocalyx fruits analysed, the average content of flavonoids accounted for 3% of all phenolic compounds and the main representative was quercetin 3-O-rutinoside, which represented an average of 41.6% of all flavonoids ( Table 2). Quercetin 3-O-rutinoside content in our study was significantly higher compared to the results of Oszmiański et al. [21]; the L. kamtschatica fruits of the cultivar 'Wojtek' were characterised by a quercetin 3-O-rutinoside content of 0.21 mg·100 g −1 f.w. Quercetin 3-Orutinoside shows a protective effect on the liver or blood vessels and has anti-inflammatory and antidiabetic properties [46].
Two phenolic acids, chlorogenic acid and neochlorogenic acid, were determined in the composition of the L. kamtschatica and L. emphyllocalyx fruits. The fruits of L. kamtschatica cultivars were characterised by a higher content of phenolic acids than those of L. emphyllocalyx cultivars. Of the L. kamtschatica and L. emphyllocalyx cultivars analysed, the L. kamtschatica cultivar 'Duet' was characterised by the highest contents of phenolic acids, on average 14.62 mg·100 g −1 , which was 51% higher than in the fruit of the L. emphyllocalyx cultivar 'Colin' ( Table 2). Chlorogenic acid, with an average of 9.77 mg·100 g −1 , was present in the analysed fruits at a significantly higher concentration than neochlorogenic acid ( Table 2). The chlorogenic acid contained in the fruits analysed constituted 91.8% of all phenolic acids, which is comparable to the results of Kithama et al. [47], who determined that, in the cultivars 'Aurora', 'Evie', 'Larissa' and 'Rebecca', the content of chlorogenic acid made up 95% of all phenolic acids contained in L. kamtschatica fruits.

Sugar Content of L. kamtschatica and L. emphyllocalyx Fruit
Of the L. kamtschatica berry cultivars analysed, the 'Duet' cultivar had the highest total sugar content, with an average of 4968 mg·100 g −1 , and glucose was the dominant sugar in the fruit of all the cultivars (Table 3). A significant difference in the contents of total sugars was observed between the fruits of the L. kamtschatica and the L. emphyllocalyx cultivars. The L. kamtschatica fruits of the cultivar 'Aurora' were characterised by the highest glucose content, on average 2963 mg·100 g −1 , while the significantly lower glucose content between the analysed cultivars was characteristic of L. emphyllocalyx fruits of the cultivar 'Willa' (Table 3). However, this cultivar was characterised by a high fructose content (comparable to that of the L. kamtschatica cultivar 'Duet') and significantly higher compared to the other cultivars ( Table 3). The sucrose content of the fruits analysed ranged from 1325 mg·100 g −1 (L. emphyllocalyx cultivar 'Willa') to 2750 mg·100 g −1 (L. kamtschatica cultivar 'Aurora'; Table 3). The sugar content results are comparable to those obtained by Senica et al. [49], who obtained a total sugar content ranging from 1557.37 to 2585.45 mg· 100 g −1 f.w. The results obtained by Gołba et al. [11] were significantly lower compared to those of this study, the total sugar content of the Lonicera caerulea fruit ranged from 1500 mg·100 g −1 to 2585 mg·100 g −1 . The average fructose content of Lonicera caerulea reported by Sharma et al. [17] was comparable with this study and ranged from 1047.53 to 1363.67 mg·100 g −1 f.w., but the content of glucose was much smaller and ranged from 750.89 to 1129.35 mg·100 g −1 f.w. A range of 3.72-126.12 mg·100 g −1 f.w. of sucrose has also been reported by Cheng et al. [50]; these values are comparable to those obtained Molecules 2023, 28, 2525 8 of 12 in the experiment. As reported by Wojdyło et al. [51], Sorbitol was previously found in Polish L. kamtschatica cultivars, with a concentration ranging from 0.1 to 0.4 mg·100 g −1 f.w. The sugar content of L. kamtschatica and L. emphyllocalyx fruit depends on environmental conditions, light intensity, fruit maturity and species, among other factors [16]. Data are expressed as mean values (n = 3) ± SD; SD-standard deviation. Mean values within rows with different letters are significantly different (p < 0.05).

Material
Fruits of the L. caerulea var. kamtschatica cultivars 'Duet' and 'Aurora' were obtained from a nursery crop located in Tyczyn (49 •  The average monthly temperatures in the period from March to June in Tyczyn were, respectively, 3.3, 7.0, 14.8, and 19.8 • C, and in Grabownica Starzeńska were, respectively, 2.2, 6.0, 13.7, and 18.5 • C. The average monthly rainfall values in the period from March to June in Tyczyn were, respectively, 20 mm, 60 mm, 50 mm, and 20 mm, and in Grabownica Starzeńska the average monthly rainfall in the period from March to June was 50 mm. The fruits of the analysed cultivars were harvested by hand at the stage of their harvest maturity (first decade of June), 1000 g each. Immediately after harvest, the fruits were subjected to chemical analysis.

Determination of pH and Acidity
The total acidity (as citric acid) and the pH of the L. kamtschatica and L. emphyllocalyx fruit were determined through the potentiometric titration of the sample for analysis with a standard 0.1 M NaOH solution at pH = 8.1 using a TitroLine 5000 (SI Analytcs, Weilheim, Germany) according to the method given in PN-EN 12147:2000 [52]. The results are expressed as g of citric acid per 100 g of fruit. The analyses were performed in triplicate.

Determination of the Contents of Bioactive Compounds in Fruit and Determination of Their Antioxidant Activity
Vitamin C (ascorbic acid) was determined according to PN-A-04019:1998 [53]. Total polyphenol content (mg GAE·100 g −1 f.w.) was determined using the Folin-Ciocalteu method according to the methodology described by Bakowska-Barczak et al. [8]. The identification of the polyphenolic profile in L. kamtschatica and L. emphyllocalyx fruit was determined according to the methodology reported by Gorzelany et al. [24].
The ability of the fruit to reduce iron ions (FRAP method) was determined according to the methodology given by Rupasinghe et al. [26], and the results are given in µM Fe 2+ ·g −1 f.w. The antioxidant activity of the fruit was determined using DPPH methods according to the methodology given by Jurčaga et al. [54], and the result is expressed as % inhibition of DPPH radicals, and through the ABTS method according to the methodology given by Gawroński et al. [1], and the results are expressed in mM TE·100 g −1 f.w. All analyses were performed in triplicate.

Determination of Sugars in L. kamtschatica and L. emphyllocalyx Fruit
The sugar content was measured using the HPLC method with refractive index detection. The chromatographic equipment SYKAM (Sykam GmbH, Eresing, Germany), consisting of sample injector S5250, pump system S1125, column oven S4120 and RI detector S3590, was used. Separation was carried out using a Cosmosil Sugar-D column 250 × 4.6 mm ( Nacalai, Kyoto, Japan). The separation was achieved with a mobile phase of 70% of acetonitrile in water in isocratic mode. The flow rate was 0.5 mL/min at column temperature set at 30 • C. The volume of injected sample was 20 µL and 15 min was needed to complete the analysis. Samples before injection were centrifuged at 5000 rpm for 10 min using Centrifuge 5430 (Eppendorf, Hamburg, Germany) and diluted with mobile phase 1:4 (v/v). All determinations were performed in triplicate.

Statistical Analysis
Using Statistica 13.3. software (TIBCO Software Inc., Tulsa, OK, USA), a statistical analysis of the results obtained was performed that included the analysis of variance (ANOVA) and NIR significance test at a significance level of α = 0.05.

Conclusions
Based on this study, differences in fruit composition were found in both individual species and the L. caerulea cultivars. The fruits of the L. kamtschatica cultivar 'Aurora' contained the highest amount of ascorbic acid in their composition, approximately 22% more compared to 'Lori', which had the highest content of this compound among L. emphyllocalyx cultivars. The highest total polyphenol content was found in the L. emphyllocalyx cultivar 'Lori', while the predominant polyphenolic compound was cyanidin 3-O-glucoside. On the basis of the results obtained, it can be concluded that the L. emphyllocalyx cultivars 'Lori' and 'Willa' can find applications in various food industries. Despite the diversity of chemical composition, the fruits have high antioxidant properties compared to the well-known varieties 'Duet' and 'Aurora'. The variety 'Lori' has the highest content of phenolic compounds among the tested varieties. 'Willa' has the highest concentration of C3G comparable to the widely known varieties of L. kamtschatica and the highest sugar content among L. emphyllocalyx. The use of Lonicera as an ingredient of functional foods, natural colourant or source of natural antioxidant seems to be promising. Compared to other types of berries grown in Poland, e.g., raspberries and red currant, L. kamtschatica and L. emphyllocalyx can be a good source of bioactive substances and sugar, making them good in food industry and processing, including juices, wines, spreads and dried fruits.