Evaluation of Polyphenolic Profile and Nutritional Value of Non-Traditional Fruit Species in the Czech Republic — A Comparative Study

Dry matter, organic acids, ascorbic acid, minerals (nitrogen, phosphorus, potassium, calcium, magnesium, sodium) and polyphenolic profile of a number of non-traditional fruit species and their genotypes, namely blue honeysuckle (Lonicera spp.), Saskatoon berry (Amelanchier alnifolia), black mulberry (Morus nigra), Tomentosa cherry (Prunus tomentosa Thunb.) and jostaberry (Ribes nigrum x Grossularia uva-crispa) were investigated. The results showed that Lonicera genotypes displayed high levels of ascorbic acid and they were rich in minerals, with the cultivar ‘Amfora’ achieving the leading position in nitrogen, phosphorus and potassium content among all lesser known fruit species. Amelanchier cultivars represented a valuable source of ascorbic acid and calcium, ‘Tišňovský’ and ‘Smoky’ together with Morus nigra ‘Jugoslavska’ accumulated the highest level of examined polyphenolic compounds. Regular consumption of studied less common fruit species can bring health benefits so they can represent a high potential value for fruit growers and in addition they can be utilised as functional foods.

Black mulberry (Morus nigra) seems to be significantly effective in the reduction of blood glucose that can be supplemented in diabetes [23]. The flavonoid extract from the seeds of Tomentosa cherry (Prunus tomentosa Thunb.) displayed antioxidative [24] and inhibitory activities on nitric oxide (NO) and prostaglandin E-2 (COX-2) [25], sour cherry (0.3 g/100 g DW) had a dramatically different procyanidin profile which was dominated by short polymers, with an average chain length of four monomer units [26].
Detailed information about the health-promoting components of lesser known fruit species could lead to a better understanding of their health-beneficial effects and an increased consumption of these fruits, including their utilization in functional foods and as ingredients in nutraceuticals, medicine and pharmaceuticals [5].
There is an increased recognition and interest in utilization of lesser known fruit species not originated from the Czech Republic or neglected older fruit species, however, detailed information about the mentioned fruit species is scarce [13] because of the wide chemical diversity of antioxidant compounds present in samples of lesser known fruits [27][28][29]. Despite the presence in the literature of several papers investigating lesser known fruit species, the simultaneous determination of all above mentioned parameters is still lacking [9,30]. Moreover, all samples of non-traditional fruit species are originated from the same locality of cultivation. This fact can eliminate possible differences because of different condition of cultivation and really make possible to compare their nutritional value and find differences between them. In a similar study the phytochemical compounds of some berry species cultivated in Russia [Rubus idaeus, Ribes L. species (R. nigrum and R. nigrum × R. dikuscha) and the less common Lonicera caerulea] under the same environmental conditions were provided. Lonicera caerulea contained the highest level of K, Ribes L. presented the highest content of Ca, while R. idaeus displayed the highest content of Mg and different micro-elements (Fe, Mn, Zn and Mo). Lonicera caerulea and Ribes L. displayed the highest content of sugars. The most striking result was the polyphenolic contents of L. caerulea, which was significantly higher than that of Ribes L. and R. idaeus, two species already known to contain large amounts of these antioxidant compounds [31]. Understanding the biochemical composition of lesser-known fruit species can stimulate an interest in maximizing their nutritional effects in human diet. The aim of this study was to determine the content of dry mater, ascorbic acid, organic acids and minerals (phosphorus, potassium, calcium, magnesium and sodium). Furthermore, we determined the phenolic profile of 11 selected genotypes of non-traditional fruit species.

Results and Discussion
Results of the chemical analyses are given in Table 1.

Determination of Dry Matter and Organic Acids
Results of our study given in Table 1 indicate the highest values of dry matter (DM) in Amelanchier 'Smoky' (24.0%), followed by 'Josta' (23.2%), and a very high content of DM was found in the new Czech cultivar of Amelanchier 'NS-2', 'Brněnsky' and 'NS-1', 23.0%, 22.4% and 22.2%, respectively [22]. According to Rivera, et al. dry matter content of 'Josta' ranged between 12-24%, which corresponded with our data [32].  Table 1 shows that the lowest values of organic acids were typical of Morus nigra 'Jugoslavska' (1.0%) followed by Amelanchier 'Tišnovský' (1.3%) and Amelanchier 'Thiessen' (1.4%) [33]. The lower value of organic acid content in our study could be a result of differences in the studied cultivars and the effect of different environmental conditions, as it was reported by [22]. Similarly, in Morus nigra 'Jugoslavska' we determined a lower content of organic acids in comparison with studies of Ercisli-Orhan and Khalid et al. determined 1.6% acids [34,35]. What is more interesting, among all evaluated Lonicera samples Klčov's clones of Lonicera kamtschatica exhibited the lowest content of organic acids (LKL-2 1.6% and LKL-6 1.6%). Studies by Pokorna-Jurikova et al. reported higher values of organic acids (2.5-4.1%) that can be accounted for by different conditions of cultivation and climatic conditions, which is supported also by previous studies in Lonicera kamtschatica (Sevast.) Pojark and Lonicera edulis ex. Freyn [10].

Determination of Ascorbic Acid
Our results indicate that the Lonicera clones and cultivars differ greatly in ascorbic acid values (Table 1) (67.7-186.6 mg/100 g) together with Saskatoon fruit (91.5-114.2 mg/100 g) and these values can be considered higher than in another fruit species valued for their high ascorbic acid content, such as oranges (31 mg/100 g), strawberries (46 mg/100 g) [36], kiwi fruit (29-80 mg/100 g) [37]. In our study an increased amount of ascorbic acid was observed in Klčov's clones of Lonicera kamtschatica, especially LKL-20 and LKL-2 (186.6 mg/100 g; 172.7 mg/100 g), except for LKL-21 which displayed the lowest value of ascorbic acid (67.7 mg/100g). In agreement with our study, Paulovicsova et al. pointed to higher levels of ascorbic acid in Klčov's clones comparable to two species-Lonicera kamtschatica (212.3 mg/100 g) and Lonicera edulis (157.5 mg/100 g). On the other hand [38] recorded a lower value of ascorbic acid in LKL-2 (30.9 mg/100 g). The statistically significant influence of different years (differences of climatic conditions) can account for differences as well [39]. The results of our study confirmed that Saskatoon berry can be considered as the second most important source of ascorbic acid (91.5-114.2 mg/100 g). The results of our studies also determined a high content of ascorbic acid in Tomentosa cherry (Prunus tomentosa) 123.3 mg/100 g (Table 1).
Ercisli et al. described moderate ascorbic acid values in genotypes of Morus nigra ranging from 15.1 mg/100 g to 18.7 mg/100 g [34] in comparison with our results 40.5 mg/100 g, on the other hand the studies of [38] considered black mulberry as the most important source of ascorbic acid among studied lesser known fruit species (in genotype M-103 70.5 mg/100 g). All described differences in ascorbic acid content confirmed that except for climatic conditions, the genotype also plays a very important role in ascorbic acid content as it has been reported by [40].

Determination of Minerals
Based on the results given in Table 1, the highest level of nitrogen was accumulated in the Russian cultivars of Lonicera sp., namely 'Amfora' 552.0 mg/100 g and 'Goluboje vreteno' 507.5 mg/100 g, followed by the Slovak cultivars 'Altaj' 407.6 mg/100 g and 'Amur' 402.7 mg/100 g. 'Amfora' also reached the leading position in phosphorus and potassium content within all evaluated less common fruit species (66.4 mg/100 g; 421.9 mg/100 g). Saskatoon berry was found to be higher in levels of calcium, magnesium, manganese compared with blueberries or strawberries. An especially high level of calcium is reported in studies of [41] (845.9 mg/kg FM in the cultivar 'Tišnovsky'), in our study we found the highest content of calcium in this cultivar among all examined fruit species, together with the cultivar 'Smoky' (Ca-98.2 mg/100 g; 88.7 mg/100 g) and 'Josta' (89.1 mg/100 g). Similarly, we recorded the highest content of magnesium in Saskatoon berry cultivars 'Tišnovsky' and 'Brněnsky' (29.8 mg/100 g; 29.1 mg/100 g). Saskatoon berry also represented an important source of magnesium for the human diet. In Czech cultivars reached up values from 10.1 mg/kg FM (the cultivar 'Ostravsky') to 315.9 mgthe/kg FM (the cultivar 'Martin') [41], which was also confirmed in our study, where in Saskatoon berry we determined the highest values of magnesium among all evaluated less common fruit species, ranging from 22.8 mg/100 g ('Thiessen') up to 29.8 mg/100 g ('Tišnovsky'). In the cultivar 'Smoky' we analysed lower values of calcium and magnesium content in comparison with the studies of Mazza et al. (111.8 mg/100 g) and a similar content of magnesium to our study (43.2 mg/100 g) [42].
From Table 1 it is evident that among all examined species LKL-21 exhibits the highest content of sodium (5.6 mg/100 g) followed by 'Amfora' (4.3 mg/100 g).
Klčov's clones of Lonicera kamtschatica formed a special group distinguished by lower values of nitrogen, phosphorus, potassium and calcium (with the exception of LKL-20) compared to other cultivars of Lonicera.
The results of study [10] confirmed that examined cultivars/clones of Lonicera displayed higher values of potassium (224.4-347.7 mg/100 g). As far as the mineral elements were concerned, the relatively high content of potassium in the cultivars 'Thiessen' and 'Tišnovsky' (4154.3 mg/kg FM and 4311.7 mg/kg FM, respectively) confirmed also the studies of [41] which are in accordance with our results. The content of magnesium (8.4-15.4 mg/100 g), calcium (40.7-50.2 mg/100 g) and sodium (1.6-4.3 mg/100 g) in berries of edible honeysuckles is lower in comparison with studies carried out under the conditions of Slovakia (71.1 mg/100 g, 107.8 mg/100 g; 8.2 mg/100 g) [10]. Lower values of mineralss can be a reflection by different conditions of cultivation and the influence of climatic conditions as it was reported by [43].

Determination of Polyphenolic Compounds
Finally, we determined the polyphenolic profile of 11 genotypes (selected clones and cultivars) of non-traditional fruit species (Table 2), especially the content of gallic acid, catalposide, rutin, resveratrol, quercitrin and chlorogenic acid with quercetin was examined.
Chlorogenic acid was the major examined polyphenolic compound in all samples with the highest levels being found in Saskatoon berry 'Thiessen' (298.1 mg/100 g) and LKL0-35 (267.1 mg/100 g) and finally in black mulberry 'Jugoslavska' (245.4 mg/100 g) ( Table 2). Both analysed cultivars of Saskatoon berry can be characterised by exceptionally high value of quercetin 'Thiessen' 23.6 mg/100 g, 'Smoky' 30.7 mg/100 g ( Table 2). The authors of [20] determined in berries of cultivar Lonicera 'Altaj' 60 mg/100 g gallic acid, 24 mg/100 g rutin and 22 mg/100 g quercitrin, but our experiments indicated lower values of polyphenolic compounds. According to Jurikova et al. the level of rutin obtained at several genotypes of Lonicera spp. does not surpass a level of 30 mg/100 g [10] but in our experiments we saw higher concentrations in 'Amur', 'Amfora', LKL-20, LKL-21 and LKL-35 [11]. In 'Sinoglaska' 59.8 mg/100 g gallic acid, 24.3 mg/100 g rutin and 22.2 mg/100 g qurcetin was found and in LKL-21 69.2 mg/100 g gallic acid, that represented lower values in comparison with our values, but the level of rutin in the mentioned genotype is higher. The concentration of gallic acid in 'Thiessen' is similar to Saskatoon berry 'Martin', reaching up to 27.7 mg/100 g, but the concentration of rutin in 'Thiessen' was two times higher. This fact can be caused by differences in stage of ripeness, and conditions of storage as it has been mentioned by intraspecific diversity of samples, [44,45], or the different procedures used in the extraction of samples [44,46].

Statistical Evaluation of Results
On the basis of statistical evaluation of data according to dry matter, organic acids and ascorbic acid content the non-traditional fruit species (cultivars/clones) can be separated into three individual clusters.

Description of Growing Locality
Fruits were harvested in an experimental gene-fund orchard of Mendel University in Brno over the may-september period in 2008. This orchard is situated in the area of the village called Zabcice, approximately 20 km south of Brno, in the Czech Republic. The altitude is 184 m. The average annual temperature and a fifty-year average sum of precipitation are 9 °C (during the growing season 15.6 °C) and 553 mm (during the growing season 356 mm), respectively. The soils are classified as gleyed alluvial soils developed on the Holocene calciferous sediments with a marked accumulation of organic compounds. As far as the texture is concerned, the topsoil is loamy and the subsoil clayey-loamy.

Biological Material
The following less common fruit samples we used: six cultivars of Lonicera spp. originated in Slovakia (Research Institut of Fruit and Decorative Trees in Bojnice) from free pollination of Gerda ('Amur') and the crossing of Lonicera kamtschatica X Lonicera turczaninowii ('Altaj'), four cultivars come from the territory of Russia-'Goluboje vreteno' as seedlings obtained from free pollination of Lonicera kamtschatica having origin in the "Sady Sibiri" orchards, 'Amfora' and 'Fialka', 'Goluboje

Collection of Samples
Fruit were harvested from five plants of each cultivar/clone under study within the period of consumption ripeness. Harvested fruits were puréed in a mixer and an averaged sample was obtained by dividing into quarters. Each parameter was measured in five replicates. Twenty randomly chosen fruits from each tree/shrub were used for analyses (i.e., altogether 100 per each cultivar/clone). For determination of ascorbic acid content samples of fruit were processed immediately after the harvest. For analyses of another parameters samples were stored at −18 °C for six month.

Determination of Dry Matter and Organic Acids
The content of the dry matter was determined by drying at 105 °C ± 2 °C. The amount of acids (titrable acidity) was determined in aqueous extract with potentiometer by titration of sodium hydroxide (c = 0.1 mol·dm −3 ) at the pH level = 8.1. The content of organic acids was expressed in g/100 g of fresh weight.

Mineral Content Assay
To determine the content of mineral elements assay the sample was dried to a constant weight in a drier at 105 °C ± 2 °C; thereafter, 1 g of homogenised dry matter (with the 1-mm size particles) was further mineralised in a mixture of concentrated sulphuric acid with 30% hydrogen peroxide. After the mineralization, the obtained samples were quantitatively transferred into a 250-mL volumetric flask and filled to the volume with re-distilled water. The resulting mineralisate was measured in an atomic absorption spectrometer (PHILIPS PU 9200X). The amount of crude protein was estimated on the base of total nitrogen in a KJELTEC TM 2300 Kjeldahl apparatus and the result was multiplied by the coefficient 6.3. The content of mineral elements was expressed in mg/100 g of fresh weight (in case of crude protein as g/100 g of fresh weight).

Determination of Ascorbic Acid
Determination of ascorbic acid content was carried out by a modification of the methods by Wagner et al. [47] and Miki [48]. Sample ). Chromatographic conditions were optimized according to following parameters: the volume of injection was 30 µL, mobile phase A consisted of formic acid 0.2% (v/v), MP B was acetonitrile. The gradient composition was linearly increased from 12 up to 22% for mobile phase B (v/v) for 2 minutes from start, to 50% for 25 min, to 55% (30 min). Separation process was inducted by eluation with the negative gradient at 15% B for 45 min. Flow rate was 0.8 µL/min. The electrochemical detector scanned responses at applied working electrode potentials of −80, 0, 80, 160, 240, 320, 400, 480, 560, 640, 720 and 800 mV. The resulting detection was expressed in microcoulombs.

Statistical Evaluation
Statistical evaluation and separation of non-traditional fruit species-their clones and cultivars were provided by cluster analysis in the Statistica programme on the basis of K-mean values after standardization of dates.

Conclusions
In this study it is shown that there were marked differences in the selected nutritional values of non-traditional fruit species grown in Žabčice-genofond belonging to MENDELU Brno. Lonicera cultivars/clones had high levels of ascorbic acid and mineral elements, especially the cultivar 'Amfora' which reached the leading position in nitrogen, phosphorus and potassium content among all studied lesser known fruit species. Amelanchier cultivars had the second position in ascorbic acid content and also represented a valuable source of calcium; particularly the cultivars 'Tišnovský' and 'Smoky' accumulated the highest level of this mineral among all examined species. Both cultivars of Amelanchier-'Thiessen' and 'Smoky'-together with Morus nigra 'Jugoslavska' represented the most valuable source with respect of examined polyphenolic compounds. We can conclude that the consumption of less common fruit species can bring health benefits and have a high potential value for fruit growers as well as nutraceutical manufacturers. Regarding the fact that all cultivars were grown under identical conditions and in the same locality, it is possible to conclude that one can clearly see the cultivar variability, which is quite typical of non-traditional fruit.