Phenolics and Carotenoid Contents in the Leaves of Different Organic and Conventional Raspberry (Rubus idaeus L.) Cultivars and Their In Vitro Activity

Raspberry leaves are a source of carotenoids and polyphenols, including ellagic acid and salicylic acid. The results of scientific research suggest that they have potential pro-health properties that contribute to human health. The aim of this study was to determine the polyphenolic and carotenoid profiles in the leaves of selected raspberry cultivars and their in vitro activity. The second aim was to determine the impact of organic and conventional farm management on the polyphenol, carotenoid, and chlorophyll contents in different raspberry cultivars: ‘Polana’, ‘Polka’, ‘Tulameen’, ‘Laszka’ and ‘Glen Ample’. Compared with conventional raspberry leaves, organic raspberry leaves were characterized by a significantly higher content of dry matter, total polyphenols, total phenolic acids, chlorogenic acid, caffeic acid, salicylic acid and quercetin-3-O-rutinoside; moreover, the organic leaves were characterized by higher antioxidant activity. Among examined cultivars, ‘Polka’ c. was characterized by the highest antioxidant status. However, raspberry leaves from conventional farms contained more total carotenoids, violaxanthin, alpha-carotene, beta-carotene, total chlorophyll and individual forms of chlorophylls: a and b.


Introduction
Raspberry (Rubus ideaus) is recognized by consumers as a tasty and healthy fruit. Recent research indicates that the leaves of berry plants, such as strawberries, raspberries, blueberries and blackcurrants, are a potential source of bioactive compounds with strong, pro-health, anticancer and anti-inflammatory properties [1][2][3][4]. Berry leaves are by-products of berry plant cultivation. Their traditional therapeutic use against several diseases, such as the common cold, inflammation, diabetes, and ocular inflammation, has been almost forgotten [5]. Raspberry leaves contain high amounts of polyphenols and can serve as a potential source of natural antioxidants for medicinal and commercial uses. Raspberry leaves contain phenolic acids, such as chlorogenic, gallic, ferulic, caffeic acids, as well as flavonoids, including quercetin and kaempferol-3-O-glucosiden [6]. However, two chemical compounds deserve special attention: ellagic and salicylic acids [7,8]. These compounds show strong biological effects in vitro that have been connected to pharmacological and nutritional effects [9]. They are mainly related to the prevention of cardiovascular diseases [10]. Many current medicines are derived from plants, including aspirin, which is a synthetic derivative of salicylic acid [11,12]. Plants produce salicylic acid as a response to biotic (pest and diseases) stresses [13,14].
Many studies have shown that organic cultivation methods increase the amount of bioactive compounds in fruits [15][16][17], mostly due to the effect of plant self-protection against pest and diseases. The latest research with raspberry fruit indicates that organic fruit contained significantly more

Plant Material Preparation
The leaves for chemical analysis were harvested early in the morning from each production farm and immediately transported to the laboratory. Each sample was divided into two parts. The first part was used for dry matter evaluation, and the second part was freeze-dried using a Labconco (2.5)

Plant Material Preparation
The leaves for chemical analysis were harvested early in the morning from each production farm and immediately transported to the laboratory. Each sample was divided into two parts. The first part was used for dry matter evaluation, and the second part was freeze-dried using a Labconco (2.5) freeze-dryer (Warsaw, Poland, −40 • C, pressure 0.100 mBa). After freeze-drying, the plant material was ground in a laboratory mill (A-11). The ground samples were then stored at −80 • C.

Dry Matter Content
The dry matter content of the raspberry leaves was measured before freeze-drying. The dry matter content was determined using the weight method. Empty glass beakers were weighed, filled with fresh leaves and weighed again. The samples were dried at 105 • C for 72 h in an FP-25W Farma Play (Tczew, Poland) dryer. After 3 days, the samples were cooled to 21 • C and weighed. The dry matter content was calculated for the leaf samples based on their mass differences and given in units of 100 g −1 FW (fresh weight) [21].

Phenolic Acid and Flavonol Separation and Identification
Polyphenols were measured by an HPLC (High Performance Liquid Chromatography) method that was described previously in detail by Hallmann et al. [22]. A total of 100 mg of freeze-dried, powdered plant material was mixed with 5 mL of 80% methanol and shaken on a Micro-Shaker 326 M (Marki, Poland). Next, all samples were extracted in an ultrasonic bath (10 min, 30 • C, 5500 Hz). After 10 min of extraction, the leaf samples were centrifuged (10 min, 3780× g, 5 • C). The supernatant was collected in a clean plastic tube and centrifuged again (5 min; 31,180× g, 0 • C). A total of 900 µL of supernatant was transferred to an HPLC vial and analysed. For polyphenol compound separation and identification, a Synergi Fusion-RP 80i Phenomenex column (250 × 4.60 mm) was used. The analysis was carried out with the use of Shimadzu equipment (USA Manufacturing Inc, Lebanon, IN, USA: two pumps LC-20AD, controller CBM-20A, column oven SIL-20AC, spectrometer UV/Vis SPD-20 AV). The phenolic compounds were separated under gradient conditions with a flow rate of 1 mL min −1 . Two gradient phases were used: 10% (V:V) acetonitrile and ultrapure water (phase A) and 55% (V:V) acetonitrile and ultrapure water (phase B). The phases were acidified by orthophosphoric acid (pH 3.0). The total time of the analysis was 38 min. The phase-time programme was as follows: 1.00-22.99 min, 95% phase A and 5% phase B; 23.00-27.99 min, 50% phase A and 50% phase B; 28.00-28.99 min, 80% phase A and 20% phase B; and 29.00-38.00 min, 95% phase A and 5% phase B. The wavelengths were 250 nm for flavonols and 370 nm for phenolic acids. The phenolic compounds were identified by using 99.9% pure standards (Sigma-Aldrich, Szelągowska, Poland) and the analysis times for the standards (Figures 2 and 3).

Carotenoid and Chlorophyll Separation and Identification
Carotenoids and chlorophylls were measured by an HPLC method, as described by Hallmann et al. [22]. Sample preparation included extraction of 100 mg of freeze-dried sample with 100% acetone using an ultrasonic cold bath (10 min, 0 °C). Samples were then centrifuged (10 min, 3780× g, 0°C). One millilitre of supernatant was transferred into an HPLC vial. The HPLC setup used to determine carotenoids and chlorophylls consisted of two LC-20AD pumps, a CMB-20A system controller, an SIL-20AC autosampler, an ultraviolet-visible SPD-20AV detector, a CTD-20AC oven and a Max-RP 80A column (250 × 4.60 mm), which are all Shimadzu products (Polish agent Shimpol, Warsaw, Poland). Methanol + acetonitrile (phase A) and methanol + ethyl acetate (phase B) at a flow rate of 1 mL min -1 were used as the gradient solvents (1.00-14.99 min, 100% phase A, 15.00-22.99 min, 40% phase A; and 24.00-27.00 min, 100% phase A). The wavelength used for detection was 445-450 nm. The carotenoid and chlorophyll concentrations were calculated using standard curves and the sample dilution coefficient and presented in mg per 100 g of fresh material. Identified carotenoids and chlorophylls are presented in Figure 4.

Carotenoid and Chlorophyll Separation and Identification
Carotenoids and chlorophylls were measured by an HPLC method, as described by Hallmann et al. [22]. Sample preparation included extraction of 100 mg of freeze-dried sample with 100% acetone using an ultrasonic cold bath (10 min, 0 • C). Samples were then centrifuged (10 min, 3780× g, 0 • C). One millilitre of supernatant was transferred into an HPLC vial. The HPLC setup used to determine carotenoids and chlorophylls consisted of two LC-20AD pumps, a CMB-20A system controller, an SIL-20AC autosampler, an ultraviolet-visible SPD-20AV detector, a CTD-20AC oven and a Max-RP 80A column (250 × 4.60 mm), which are all Shimadzu products (Polish agent Shimpol, Warsaw, Poland). Methanol + acetonitrile (phase A) and methanol + ethyl acetate (phase B) at a flow rate of 1 mL min −1 were used as the gradient solvents (1.00-14.99 min, 100% phase A, 15.00-22.99 min, 40% phase A; and 24.00-27.00 min, 100% phase A). The wavelength used for detection was 445-450 nm. The carotenoid and chlorophyll concentrations were calculated using standard curves and the sample dilution coefficient and presented in mg per 100 g of fresh material. Identified carotenoids and chlorophylls are presented in Figure 4.

Carotenoid and Chlorophyll Separation and Identification
Carotenoids and chlorophylls were measured by an HPLC method, as described by Hallmann et al. [22]. Sample preparation included extraction of 100 mg of freeze-dried sample with 100% acetone using an ultrasonic cold bath (10 min, 0 °C). Samples were then centrifuged (10 min, 3780× g, 0°C). One millilitre of supernatant was transferred into an HPLC vial. The HPLC setup used to determine carotenoids and chlorophylls consisted of two LC-20AD pumps, a CMB-20A system controller, an SIL-20AC autosampler, an ultraviolet-visible SPD-20AV detector, a CTD-20AC oven and a Max-RP 80A column (250 × 4.60 mm), which are all Shimadzu products (Polish agent Shimpol, Warsaw, Poland). Methanol + acetonitrile (phase A) and methanol + ethyl acetate (phase B) at a flow rate of 1 mL min -1 were used as the gradient solvents (1.00-14.99 min, 100% phase A, 15.00-22.99 min, 40% phase A; and 24.00-27.00 min, 100% phase A). The wavelength used for detection was 445-450 nm. The carotenoid and chlorophyll concentrations were calculated using standard curves and the sample dilution coefficient and presented in mg per 100 g of fresh material. Identified carotenoids and chlorophylls are presented in Figure 4.

ABTS Reagent Preparation
Twenty milliliters of distilled water was added to 0.0265 g of potassium persulfate (K 2 S 2 O 8 ). Five milliliters of distilled water followed by 5 mL of a previously prepared aqueous solution of potassium persulfate was added to 0.0384 g of ABTS ·+ (2'2-azinebis-3-ethylbenzothiazolin-6-sulfonic acid) reagent. The solution was prepared a minimum of 12 h before the planned assay and stored in a dark place. Two-hundred and fifty milligrammes of the sample of freeze-dried plant material tested was weighed into a sterile falcon tube plastic tube with a cap (50 mL), and 25 mL of distilled water was added. It was placed onto a vortex shaker (LP shaker Vortex, Labo Plus, Warsaw, Poland) for 60 s at 2000 rpm, for complete mixing. Subsequently, the sample was incubated in a shaker incubator (IKA KS 4000 Control, IKA, Staufen im Breisgau, Germany) for 60 min (temperature 30 • C, 6× g). After incubation, the sample was again shaken on a vortex shaker for 60 s for complete mixing and then centrifuged (Centrifuge, MPW-380 R, Warsaw, Poland) at 5 • C and 14,560× g for 20 min. After centrifugation, the supernatant was used for determinations. In 10 mL glass tubes, test extract solution, measured with a predetermined dilution scheme (0.5-1.5 mL), was then added to 3.0 mL of ABTS ·+ cationic solution in PBS (phosphate-buffered saline). Absorbance measurements were taken exactly 6 min after incubation at room temperature. Absorbance was measured at a wavelength λ = 734 nm using a spectrophotometer (Helios γ, Thermo Scientific, Warsaw, Poland). The obtained measurements were calculated using special formula including the dilution factor. The final results were express as mmol of TE (Trolox equivalents per 100 g FW (fresh weight of leaves)) [23].

Statistical Analysis
The results obtained from the chemical analyses were statistically analyzed using Statgraphics

Polyphenol Content
The dry matter and polyphenol contents in examined raspberry leaves are presented in Table 2. Organic raspberry leaves were characterized by a significantly higher content of dry matter (p = 0.0055) and total polyphenols (p = 0.0001), including total phenolic acids (p < 0.0001) as well individual acids: chlorogenic, caffeic and salicylic acids. For flavonoids, we observed that organic plants, compared with conventional plants, contained significantly more quercetin-3-O-rutinoside (p = 0.0009). However, raspberry leaves from conventional farming systems contained significantly more luteolin (p = 0.0117) than did leaves from organic farming systems.
Raspberry cultivar had a significant impact on the content of phenolic compounds in examined leaves (Table 3). 'Tulameen' cv. was characterized by the highest level of caffeic acid and quercetin derivates, whereas 'Polka' cv. contained the highest and significant levels of ellagic acid (p = 0.0046). Both of these cultivars contained significantly more quercetin-3-O-glycoside than did the other examined cultivars. The highest luteolin content was found in the leaves of raspberry cultivars 'Polka' and 'Glen Ample'. However, the highest content of quercetin among all analysed cultivars was found in the leaves of raspberry 'Polana' cv.

Carotenoid and Chlorophyll Contents
The contents of carotenoids and chlorophylls are presented in Table 4. The results showed that raspberry leaves from conventional farming contained significantly more total carotenoids (p = 0.0014), violaxanthin (0.026 mg 100 g −1 FW and 0.017 mg 100 g −1 FW), alpha-carotene (0.109 mg 100 g −1 FW and 0.060 mg 100 g −1 FW) and beta-carotene (1.22 mg 100 g −1 FW and 0.46 mg 100 g −1 FW) than did the leaves from organic farming; however, the leaves from conventional farming contained significantly less neoxanthin (p = 0.003), lutein (p = 0.0069) and zeaxanthin (p = 0.0118). Moreover, leaves from conventional farming, compared to leaves from organic farming, were characterized by higher contents of total chlorophylls (10.52 mg 100 g −1 FW and 5.75 mg 100 g −1 FW) and individual forms of chlorophyll (a and b). Cultivar had a significant impact only on neoxanthin (p < 0.0001) content in leaves. 'Laszka' cv. contained significantly more of this xanthophyll among all analysed cultivars (Table 5).   1 Means in rows followed by the same letter are not significantly different at the 5% level of probability (p < 0.05); 2 N.S. not significant statistically.

Antioxidant Activity
Organic raspberry leaves, compared with leaves from conventional farming, were characterized by significantly higher antioxidant activity (p < 0.0001) ( Figure 5). Among the group of examined raspberry cultivars, the strongest antioxidant potential shown was in 'Polka' cv. and 'Tulameen' cv. There was a significant correlation between antioxidant activity in vitro and the total polyphenol content in examined raspberry leaves ( Figure 6). The stronger antioxidant activity in the leaves was a reflection of higher content of polyphenols, especially in the organic plants (R 2 = 0.8302, p < 0.0001).
Organic raspberry leaves, compared with leaves from conventional farming, were characterized by significantly higher antioxidant activity (p < 0.0001) ( Figure 5). Among the group of examined raspberry cultivars, the strongest antioxidant potential shown was in 'Polka' cv. and 'Tulameen' cv. There was a significant correlation between antioxidant activity in vitro and the total polyphenol content in examined raspberry leaves (Figure 6). The stronger antioxidant activity in the leaves was a reflection of higher content of polyphenols, especially in the organic plants (R 2 = 0.8302, p < 0.0001).

Discussion
Berry fruits are recognized worldwide as "superfoods" due to the high content of bioactive compounds and their health benefits [24][25][26][27]. Most research on the impact of the cultivation system (organic and conventional) on the quality of raspberries concerns fruit [15,18]. As the literature shows, only little attention has been paid to biologically active substances in leaves, which was the Figure 5. Antioxidant activity raspberry from organic and conventional cultivation (p < 0.0001) and raspberry cultivars (p < 0.0001). Means followed by the same letter are not significantly different (p < 0.05).
by significantly higher antioxidant activity (p < 0.0001) ( Figure 5). Among the group of examined raspberry cultivars, the strongest antioxidant potential shown was in 'Polka' cv. and 'Tulameen' cv. There was a significant correlation between antioxidant activity in vitro and the total polyphenol content in examined raspberry leaves (Figure 6). The stronger antioxidant activity in the leaves was a reflection of higher content of polyphenols, especially in the organic plants (R 2 = 0.8302, p < 0.0001).

Discussion
Berry fruits are recognized worldwide as "superfoods" due to the high content of bioactive compounds and their health benefits [24][25][26][27]. Most research on the impact of the cultivation system (organic and conventional) on the quality of raspberries concerns fruit [15,18]. As the literature shows, only little attention has been paid to biologically active substances in leaves, which was the

Discussion
Berry fruits are recognized worldwide as "superfoods" due to the high content of bioactive compounds and their health benefits [24][25][26][27]. Most research on the impact of the cultivation system (organic and conventional) on the quality of raspberries concerns fruit [15,18]. As the literature shows, only little attention has been paid to biologically active substances in leaves, which was the research material in this study. Their traditional therapeutic use against several diseases, such as the cold, inflammation, diabetes and ocular dysfunction, has almost been forgotten. Raspberry leaves are a powerful source of biologically active compounds (Tables 2-5). The high content of bioactive compounds means that infusions of raspberry leaves can be used in phytotherapy [5]. Only a few studies have shown the antioxidant properties and polyphenol content in raspberry leaves. In our analysis, we found that organic raspberry leaves, compared with conventional raspberry leaves, were characterized by significantly higher contents of total polyphenols, total phenolic acids, chlorogenic acid, caffeic acid, salicylic acid and quercetin-3-O-rutinoside. In our experiment, we found 136.1 mg 100 g −1 FW of total polyphenols in organic leaves and 119.9 mg 100 g −1 FW of total polyphenols in conventional leaves. Teleszko & Wojdyło [28] described similar results. Their research showed that the leaves of berries were not only a valuable source of antioxidants but also contained significantly more polyphenols than did the fruit. This clearly indicates that plant parts other than the fruits could be used for medical or food purposes. An example of their application is herbal tea. The main compound of polyphenols found in raspberry leaves is salicylic acid. We found 3.51 mg 100 g −1 FW of salicylic acid in the studied organic raspberry leaves. Salicylic acid is synthesized by plants as a response to abiotic stresses, such as osmotic stress, chilling, drought and heat [13,14,29] As reported by Nour, Trandafir & Cosmulescu [9], seven cultivars of blackcurrant contain salicylic acid in the leaves, ranging from 3.97 mg 100 g −1 FW to 5.28 mg 100 g −1 FW; in this study, raspberry leaves contained 2.51 mg 100 g −1 FW to 3.20 mg 100 g −1 FW. From a chemical point of view, salicylic acid could be used as a substrate for acetylic reactions and acetylsalicylic acid formation. The manifold effects of acetyl salicylic acid on human physiology can potentially provide health benefits [30]. One of the important phenolic acids extracted from the Rubus family is ellagic acid. As reported by Landete [10] raspberry fruits contain ellagic acid in a wide rate: 47-270 mg 100 g −1 FW. In a study by Oszmiański et al. [1] the ellagic acid content in raspberry leaves ranged from 215.5 mg 100 g −1 FW to 1078.5 mg 100 g −1 FW. In our experiment, we found much lower levels of ellagic acid but were still satisfied (Tables 2 and 3).
A very important group of compounds present in raspberry leaves is flavonoids. The quantity of flavonoids in the leaves of raspberries is significantly higher than that in the fruits, where flavonoids compose only a very small fraction of the bioactive compounds [31]. In a study by Oszmiański et al. [1] the flavonoid fraction was the main phenolic group, constituting almost 11% of leaf extract powder weight. In our experiment, we identified 5 flavonoid compounds, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, luteolin, myricetin and quercetin, in examined raspberry leaves. However, Buricova et al. [32] examined antioxidant capacity and antioxidants in raspberry leaf water extract, and three flavonoid compounds were detected (catechin, epicatechin and procyanidin B 1 ), while Ferlemi & Lamari [5] detected much more flavonoid compounds present in raspberry leaves (quercetin, quercetin-3-O-rutinoside, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, kaempferol-3-O-glucoside, epicatechin gallate methyl gallate, sanguiin H-6/lambertianin C and lambertianin D). In contrast, in an analysis by Oszmiański et al. [1] thirty-three phenolic compounds were detected in wild blackberry leaf samples (fifteen flavonols, thirteen hydroxycinnamic acids, three ellagic acid derivatives and two flavones). Flavonoids have antioxidant abilities and protect plants from various biotic and abiotic stresses. The role of secondary metabolic pathways in plant responses is to cope with oxidative stress, resulting in the synthesis of flavonoids [33]. Another important role of flavonoids in foliar plants is their action as a screen against severe sunlight illumination [34]. One of the most important priorities in research on polyphenolic compound content is not just determining their presence but also biological activity in vitro and in vivo. Dudzińska et al. [35] investigated the polyphenol content in raspberry leaves and their antioxidative power. The antioxidant capacities of the examined extracts remained relatively high and corresponded well to the determined total polyphenol content. As pointed out by Oszmiański et al. [1] the antioxidant power of raspberry leaves is strongly connected with the total polyphenol content. They measured total phenolic content and antioxidant activity (AA) of 27 species belonging to the Rubus family. They found a significant link between the highest polyphenolic concentration and AA of raspberry leaves. The species with the highest total polyphenol content also had the highest antioxidant activity: Rubus pedemontanus (205 mol TE g −1 DW (dry weight) and 310.88 mg 100 g −1 DW of polyphenols) and Rubus partenocissus (203 mol TE g −1 DW and 298.74 mg 100 g −1 DW of polyphenols); species with the lowest value were characterized by the lowest antioxidant power: Rubus radula (151 mol TE g −1 DW and 202.21 mg 100 g −1 DW of polyphenols) and Rubus nesseris (91 mol TE g −1 DW and 85.51 mg 100 g −1 DW of polyphenols). In our study, we observed the highest levels of total polyphenols in 'Polka' cv. (151.75 mg 100 g −1 FW) and 'Tulameen' cv. (136. 95 mg 100 g −1 FW). Those levels were reflected in their antioxidant status (88.10 mmol Trolox 100 g −1 FW and 80.22 mmol Trolox 100 g −1 FW) and significant correlation between features (R 2 = 0.8302, p < 0.0001) for the organic raspberry but much weaker correlation for the conventional raspberry (R 2 = 0.6227, p < 0.0001) (Figure 6). Similar results were described by Zlotek et al. with basil leaves [36]. The antioxidant status of leaves was positively correlated with polyphenols content [37].
In addition to the presence of photosynthetic pigments, carotenoids also exist in raspberry leaves. Their concentration in leaves depends mainly on the level of chlorophyll. The higher the concentration of chlorophyll in the leaves, the more carotenoids present. Chlorophyll is associated with the function of carotenoids, which are produced by plants mainly to protect the photosynthetic system against photooxidation. Carotenoids are synthesized via the general biosynthetic pathway within the chloroplasts of plants. Shen et al. [38] also studied the effect of increased UV-B radiation on carotenoid accumulation and total antioxidant capacity in tobacco (Nicotiana tabacum L.) leaves. Higher levels of chlorophylls were positively correlated with beta-carotene content. In our experiment, we observed similar results. Conventional raspberry with a significant chlorophyll level (2.43 mg 100 g −1 FW) contained a significant level of total carotenoids (3.14 mg 100 g −1 FW) compared to that of organic raspberry (1.79 mg 100 g −1 FW and 2.61 mg 100 g −1 FW, respectively) ( Table 4).

Conclusions
In summary, the aim of the study was reached and confirmed. Raspberry leaves are a valuable source of bioactive compounds. Moreover, compared to conventional leaves, organic raspberry leaves were characterized by a significantly higher content of total polyphenols, total phenolic acids, chlorogenic acid, caffeic acid, salicylic acid and quercetin-3-O-rutinoside. Additionally, the organic leaves had higher antioxidant activity; the strongest antioxidant potential was shown by the 'Polka' and 'Tulameen' cultivars. On the other hand, raspberry leaves from conventional farms contained more total carotenoids, violaxanthin, alpha-carotene, beta-carotene, total chlorophyll and individual forms of chlorophyll (a and b). The mineral fertilization used in conventional agriculture increases the level of these compounds.