Phenolic acids and flavonoids profiles of extracts from edible wild fruits and their antioxidant properties

ABSTRACT

In this study, the total phenolic, total flavonoids, phenolic compounds, the mineral content, and antioxidant activity of fruit extracts of seven wild species (Crataegus monogyna Jacq., Prunus spinosa L., Rosa canina L., Hippophaë rhamnoides L., Rubus fruticosus L., Prunus padus, Cornus mas L.) were investigated. The results indicated significant differences (p < 0.05) in the total phenolics and total flavonoids content, between the seven analyzed species. These ranged from 184.69 to 727.29 mg GAE/100 g FW and 17.27 to –165.55 mg QE/100 g FW, respectively. The antioxidant activity found in fruits was not directly affected by the total phenolic content (TPC). This activity was linked to a larger extent to the type of individual phenolic compounds and to a lesser extent to the TPC, because fruits with higher TPC have not always presented the highest values of antioxidant activity. HPLC analysis of methanolic extract showed the presence of phenolic acids (i.e. gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, elagic, and trans-cinnamic) and flavonoids (i.e. catechin, epicatechin, rutin, myricetin, and quercetin). Significant differences (p < 0.05) were observed in each individual mineral between fruits from wild flora. The fruits tissues of wild species turned out to be a good source of calcium (Ca), magnesium (Mg), sodium (Na), iron (Fe), manganese (Mn), copper (Cu), chromium (Cr), zinc (Zn), and boron (B). The results demonstrated that wild species possessed great potential for food production as sources of bioactive compounds such as phenolic compounds and minerals, for food supplements or functional foods.

KEYWORDS:

• Edible wild fruits
• Minerals content
• Phenolic compounds
• Total flavonoids
• Total phenols

Introduction

There is, nowadays, an ever-growing interest for a natural alternative in exchange for all that is obtained by chemical synthesis. Natural substances with antioxidant properties acquire an increasingly wider application in the food industry, cosmetics, and pharmaceutical industry as effective remedies in counteracting the damaging action of free radicals and in stopping pro-oxidant processes that can cause various pathological states.^[1,2] The use of synthetic antioxidants is increasingly restricted because of their suspected activity of carcinogenesis promoters,^[3] as well as because of the consumers’ rejection of the synthetic food additives.^[4] Recently consumers are preferring ingredients of natural origin, which can be extracted from plants, food by-products, and other natural sources.^[5] Thus, the administration of natural antioxidants is of paramount importance with strong positive perspectives in maintaining the human body’s normal redox status. Wild flora is an inexhaustible source of bioactive compound, with a main contribution in nutrition and health. As a result, many plant species have been investigated in searching for novel antioxidants and other bioactive compounds.^[6,7] Antioxidants have attracted more and more attention as potential agents for preventing and treating oxidative stress-related diseases. Natural antioxidants are expected to be an alternative to the synthetic ones because of their potential health benefits.^[7] Fruits of wild flora may have the potential to confer beneficial health effects due to their antioxidant activity and the total phenolic compounds and flavonoids.

There is an abundance of wild fruits in Romania. Some of these wild fruits are edible, while others can be used as medicines. The aim of this study is to determine and compare the total phenolic compounds, total flavonoids content, the antioxidant capacity, and the minerals content of seven non-cultivated autumn fruits. The extracts were compared with respect to their free phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, elagic, and trans-cinnamic) and flavonoids (catechin, epicatechin, rutin, myricetin, and quercetin). The results of this study provide information on the characteristics of these fruits, information that could be useful for product development (medicinal applications, food production as sources of bioactive compounds, and also for food supplements or functional foods).

Material and methods

Plant material

The following non-cultivated and traditionally collected autumn fruits of seven species were studied: hawthorn (Crataegus monogyna Jacq.), blackthorn (Prunus spinosa L.), dog rose (Rosa canina L.), seabuckthorn (Hippophaë rhamnoides L.), blackberry (Rubus fruticosus L.), bird cherry (Prunus padus), and cornelian cherry (Cornus mas L.). The fruits were harvested in different areas of Oltenia province (Romania). Approximately 500 g of fruits (from 25 genotypes per species) were collected from each species and they were subsequently frozen at −20°C. Samples (3 g of fruit tissue) from each species were extracted with 5 ml MeOH 70% and kept at 25°C in an ultrasonic bath for 60 min. After extraction, the samples were centrifuged for 5 min at 4200 rpm. The supernatant was filtered through a 0.45 μm polyamide membrane and stored at −20°C.

Chemicals and reagent

In this experiment the following were used: Folin-Ciocalteu reagent (2N, Sigma-Aldrich, Germany), gallic acid (Sigma-Aldrich, Germany), anhydrous sodium carbonate (Sigma-Aldrich, Germany), methanol (Merck, Germany), aluminum nitrate (Sigma-Aldrich, Germany), potassium acetate (Sigma-Aldrich, Germany), 2,2-diphenyl-1-picrylhydrazyl (DPPH; Merck, Germany), 6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylic acid (Trolox; Merck, Germany), standards of phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, elagic, trans-cinnamic), flavonoids (catechin, epicatechin, rutin, myricetin, quercetin) (Sigma-Aldrich, Germany), acetic acid (HPLC grade, Merck, Germany), and BHT (Sigma-Aldrich, Germany).

Total phenolic contents

Total phenolic contents (TPC) in fruit extracts were measured using an Evolution 600 UV-visible spectrophotometer (Thermo Scientific Madison WI, USA) computer controlled by VISION Pro-software (Thermo Scientific). The assay for TPC was conducted using the Folin-Ciocalteu reaction^[8] with gallic acid as standard. A 1.0 ml sample of each fruit extract (1:10 diluted with ultrapure water) or 1.0 ml double-distilled water (blank) or 1.0 ml of each standard gallic acid solution was placed in a 25 ml flask, and 5 ml of Folin-Ciocalteu reagent was added (diluted 1:10 with ultrapure water). After 2 min, 4 ml of 7.5% (w/v) sodium carbonate was added and the flasks were kept at room temperature (24–26°C) for 2 h. The absorbance was measured at 765 nm. A standard curve was prepared using 50, 100, 150, 200, or 250 ppm gallic acid. TPCs were expressed as milligrams of gallic acid equivalents per 100 g fresh weight (mg GAE/100 g FW).

Total flavonoids content

Total flavonoids were determined using the aluminium nitrate colorimetric method described by Cosmulescu et al.^[9] Briefly 0.5 ml of extract was mixed with methanol (1:10), 0.1 ml of 10% aluminium nitrate, 0.1 ml of 1 M aqueous potassium acetate, and 4.3 ml of methanol. After keeping it for 40 min at room temperature, the absorbance of the reaction mixture was measured at 415 nm. Quercetin was used for preparing the standard curve (0–100 mg/l). The results were expressed as milligrams of quercetin equivalents per 100 g fresh weight (mg QE/100 g FW).

Total antioxidant capacity assays

The scavenging activities of methanolic extracts of wild fruits samples against DPPH radicals were measured according to Hatano et al.^[10] with some modifications.^[8] The absorbance was measured at 517 nm. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used as standard of different concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.5 mM). Each methanolic fruit extract (50 μl diluted samples: 1:10) was mixed with 3 ml of 0.004% (v/v) DPPH in methanol. Each reaction mixture was incubated in the dark for 30 min at room temperature (24–26°C). Antioxidant capacity was expressed in mmol Trolox/100 g FW.

Phenolic compounds

HPLC analyses were performed on a Finningan Surveyor Plus system (Thermo Electron Corporation, San Jose, CA, USA) including a vacuum degasser, a Surveyor Plus LCPMPP pump, a Surveyor Plus ASP autosampler, a PDA5P diode array detector with 5 cm flow cell, and a Chrom Quest 4.2 system manager as data processor. The separation was performed by a reversed-phase Hypersil Gold C18 column (5 μm particle size, 250 mm × 4.6 mm) provided by Thermo Electron Corporation (USA). The flow rate was 1 ml/min. The mobile phase consisted of 1% aqueous acetic acid solution and methanol. Chromatographic conditions were similar to those described by Nour et al.^[11] Each compound was identified by its retention time and by spiking with standards under the same conditions. The identities of constituents were also confirmed with a photodiode array (PDA) detector by comparison with UV VIS spectra of standards in the wavelength range of 220–450 nm. Each compound was quantified according to peak area measurements, which were reported in the calibration curves of the corresponding standards.

Mineral analysis

A commercial system of inductively coupled plasma mass spectrometry (ICP-MS; Perkin-Elmer Elan 9000) and a Milestone digestion microwave system were used. The etalon standards were obtained from multielement stock solutions ICP–MS calibration STD 3, 65% nitric acid puriss p.a (Fluka, Germany), 33% oxygenated water reactive p.a and ultrapure water, of 1st degree, according to ISO 3696:1987. The preparation of samples was developed using the experimental protocol described by Cosmulescu et al.^[12] Samples of approximately 2 g fruit tissues were mineralized in a microwave wet digestion method: heating them up to 180^○C at the rate of 4.5^○C/min and then keeping them for 20 min at 180^○C. The liquid samples were transferred to volumetric flasks and diluted to a volume of 50 ml with ultrapure water. Reagent blanks were included in each series of digestions. Calcium (Ca), magnesium (Mg), sodium (Na), iron (Fe), manganese (Mn), copper (Cu), chromium (Cr), zinc (Zn), and boron (B) were determined according to the official methods (method 985.35, 999.10, 986.24) of the Association of Official Analytical Chemists (AOAC 2000).^[13] The results were expressed in mg/100 g FW.

Statistical analysis

Data were subjected to analysis of variance (ANOVA) using Statgraphics Centurion XVI software (StatPoint Technologies, Warrenton, VA, USA). Three independent samples were performed for each determination and the resulting data were used to obtain average values and standard deviations for all tests. The differences were estimated by a multiple range test using the least significant difference (LSD) at p < 0.05.

Results and discussion

Total phenolic content

The results thus obtained have indicated significant differences (p < 0.05) in TPC between the seven species analyzed (Table 1). The TPC ranged from 184.69 to 727.29 mg GAE/100 g FW in methanolic extracts of wild species tested. According to these results, TPC (mg GAE/100 g FW) in dog rose (Rosa canina) are higher (727.29) than those found in R. fruticosus (412.38), H. rhamnoides (343.57), C. monogyna (203.01), P. spinosa (192.60), or C. mas (184.69). These differences may be due to genetic factors and the different ability to synthesize the secondary metabolites of species. The ability of plants to synthesize secondary metabolites was selected in their phylogenetic development process, and there are relevant differences with regard to the synthesis and accumulation of secondary metabolites within species. The genome of each cell contains the information needed to trigger the full potential of secondary metabolism, which is characteristic to the species. TPC of R. canina fruit extracts was nearly 3.93-fold higher than that of C. mas extracts. Values obtained in this study for R. canina are much higher than those reported by Fattahi et al.^[14] in methanolic extracts (225.65 mg GAE/100 g). Roman et al.^[15] report the TPC of R. canina hips extracts with values between 326.3 and 575.1 mg GAE/100 g depending on variety. The content of total phenolics shows that dog rose could be considered quite a good source of total phenolics compared to other fruit species.

Table 1. Antioxidant activity, total phenolics content, and total flavonoids content of the fruits from the wild flora.*

	Sea-buckthorn (Hippophaë rhamnoides)	Hawthorn (Crataegus monogyna)	Wild grown European blackberry (Rubus fruticosus L.)	Cornelian cherry (Cornus mas)	Blackthorn (Prunus spinosa)	Dog rose (Rosa canina)	Bird cherry or hackberry (Prunus padus)
Total phenolic content (mg GAE/100 g FW)	343.57 ± 15.18^b	203.01 ± 9.56^a	412.38 ± 18.78^c	184.69 ± 8.86^a	192.60 ± 9.49^a	727.29 ± 30.56^e	634.80 ± 30.77^d
Total flavonoid content (mg QE/100 g FW)	147.98 ± 7.29^e	31.37 ± 1.67^b	55.82 ± 2.65^c	17.27 ± 0.85^a	68.21 ± 3.43^d	33.32 ± 1.48^b	165.55 ± 8.57^f
Antioxidant activity (mmol Trolox/100 g FW)	0.57 ± 0.02^b	0.32 ± 0.01^a	1.04 ± 0.06^c	0.54 ± 0.02^b	0.26 ± 0.01^a	1.64 ± 0.08^d	2.95 ± 0.15^e

^*Values in the same row followed by different superscript lower-case letters are significantly different at p < 0.05. The data represents mean ± SD.

H. rhamnoides berries are known for their rich content of phenols with strong antioxidant activity. In this study TPC was 343.57 mg GAE/100 g FW, which is 1.86 times higher than the content recorded in C. mas. In previous studies, the content of TPC in six varieties of H. rhamnoides ranged from around 8.62 to 14.17 g GAE/kg.^[16] P. padus has been widely used as a traditional medicine, with beneficial effects in numerous diseases, including stroke, neuralgia, and hepatitis.^[17] In this study, TPC of P. padus fruit was higher (634.80 mg GAE/100 g FW), i.e. 3.43 times higher than the content recorded in C. mas. P. spinosa and C. monogyna are used in phytotherapy for the treatment of many diseases. No significant differences were found in these two species in terms of TPC (Table 1). Ruiz-Rodriguez et al.^[18] investigated the total phenolic compounds in P. spinosa and C. monogyna fruits and found that P. spinosa fruits were richer sources of phenolic compounds compared to C. monogyna fruits, with fractions of anthocyanins and phenolic acids as major ones. A comparative analysis on TPC of cultivated varieties and local populations of R. fruticosus made by Yilmaz et al.^[19] showed that the genetic material of wild blackberries had a higher TPC of blackberries grown over. In this study TPC in wild fruits of R. fruticosus was 412.38 mg GAE/100 g FW, much lower than that reported by Yilmaz et al.^[19] (1072.1 mg GAE/100 g FW). The results for TPC clearly show that R. canina could be one of the richest sources of phenolic compounds. The same conclusion can be also found in other studies.^[20] The large difference among edible wild species in terms of TPC is supposed to be due to genetic derivation because all plants were grown under the same ecological conditions; this difference is also supposed to be due to the different ability of these species to adapt to environmental conditions.

Total flavonoids content

Flavonoids, a large group of phenolic compounds with activities beneficial to human health, are among several important constituents of edible wild fruits. In this study, total flavonoids content ranged from 17.27 to 165.55 mg QE/100 g FW, indicating significant differences (p < 0.05) between species (Table 1). Comparing the species, it was found that the total flavonoids content of P. padus is higher compared to other species, e.g. 9.58 times higher than the content recorded in C. mas. The order of total flavonoids content of seven species analyzed was as follows: P. padus > H. rhamnoides > P. spinosa > R. fruticosus > R. canina > C. monogyna > C. mas. Higher concentrations of total flavonoids content (147.98 mg QE/100 g FW) were also found in H. rhamnoides, i.e. 8.56 times more than in C. mas. Lower amounts of flavonoids were found in extracts of P. spinosa, R. fruticosus, R. canina, C. monogyna, and C. mas (Table 1). P. spinosa showed a total flavonoid content of 68.21 mg QE/100 g FW, i.e. 2.42 times lower than in P. padus. Although they belong to the same genus, there are significant differences between the two analyzed species. Comparatively, in a previous study, the average content of flavonoids in fruit varieties belonging to species P. domestica was 10.13 mg QE/100 g FW.^[9] The flavonoid content in rose hips has been studied by Adamczak et al.^[21] and the value of the average flavonoid content found in R. canina hips was 41 mg/100 g. Montazeri et al.^[22] found a content of 23.6 mg QE/g flavonoids in the methanolic extract of R. canina fruits. The total flavonoid content of 31.37 mg QE/100 g FW was recorded in C. monogyna species. The study by Barros et al.^[23] shows the potential of different parts of C. monogyna as sources of several components, including nutrients and nutraceuticals. A study by Pawlowska et al.^[24] showed that fruits of C. mas revealed the presence of large amounts of flavonoids. In this study the total flavonoid content found in fruits of C. mas was 17.27 mg QE/100 g FW. R. fruticosus is a source of flavonoid; the content found by Yilmaz^[19] was 55.82 mg QE/100 g FW. As in the case of total phenol content, the authors believe that this difference between the species used in terms of total flavonoid content could be explained by genetic factors and by the different ability of species in synthesizing secondary metabolites. It is reported that plant genotype^{[1,25,30,35,36]} and environmental conditions^[2,7] affect the total phenolic and flavonoid contents in fruit. The correlation coefficient (r) found in this study between total antioxidant activity and total flavonoids was 0.519.

Total antioxidant capacity assays

Total antioxidant capacity of extracts from seven wild non-cultivated fruits analyzed is presented in Table 1. The species P. padus with 2.95 mmol Trolox/100 g FW showed the highest antioxidant capacity; other species have shown total antioxidant capacity ranging from 0.26 mmol Trolox/100 g FW (P. spinosa) to 1.64 mmol Trolox/100 g FW (R. canina). The limits are within a large variation between species, and differences between species are significant (p < 0.05). R. canina and R. fruticosus showed high antioxidant capacity, with values of 1.64 and 1.04 mmol Trolox/100 g FW, respectively. The two species of genus Prunus (P. padus and P. spinosa) showed a total antioxidant value of 2.95 and 0.26 mmol Trolox/100 g FW, respectively, i.e. 11 times lower in P. spinosa than in P. padus. High concentration of anthocyanins was determined in P. padus fruits by Kucharska et al.,^[26] while Hwang et al.^[27] recommend the bark extract of P. padus as a cosmetic agent with natural antioxidant properties.

The species H. rhamnoides, C. monogyna, and C. mas showed the lowest levels of antioxidant capacity (0.57, 0.32, and 0.54 mmol Trolox/100 g FW, respectively). These variations found in the results could be explained by genotypic differences, which are, in fact, supported by Egea et al.,^[5] Ruiz-Rodriguez et al.,^[18] and Kostik.^[28] The results of this study showed that the antioxidant activity found in the tested fruits was not directly affected by TPC, as fruits with higher TPC have not always presented the highest values of antioxidant activity. Therefore, we can conclude that the antioxidant activity in fruits is linked to a larger extent to the type of individual phenolic compounds found rather than to TPC. In this study the correlation coefficient (r) between total phenolic and antioxidant activity was 0.847.

Phenolic compounds

The contents of individual phenolic compounds (mg/100 g) of fruits from wild flora are shown in Table 2. Five flavonoids (catechin, epicatechin, rutin, myricetin, and quercetin) and 10 phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, elagic, and trans-cinnamic) were identified and quantified. The results found indicate significant differences (p < 0.05) in terms of flavonoids between the analyzed species (Table 2). The highest amount was recorded in epicatechin in fruit species R. fruticosus (226.74 mg/100 g FW). The epicatechin content varied within very wide limits (2.89 ied varied in f g FW), indicating a difference of 78.2 times between species R. fruticosus and C. monogyna. In fruits of R. fruticosus, found in culture, Jacques et al.^[29] reported a much lower epicatechin content (94.29 mg/100 g FW).

Table 2. Phenolic compounds in fruits from the wild flora (mg/100g FW).*

	Sea-buckthorn (Hippophaë rhamnoides)	Hawthorn (Crataegus monogyna)	Wild grown European blackberry (Rubus fruticosus L.)	Cornelian cherry (Cornus mas)	Blackthorn (Prunus spinosa)	Dog rose (Rosa canina)	Bird cherry or hackberry (Prunus padus)
Gallic acid	0.70 ± 0.03^ab	0.82 ± 0.04^b	1.77 ± 0.07^c	3.11 ± 0.16^d	0.40 ± 0.02^a	6.85 ± 0.35^f	5.83 ± 0.29^e
Catechin hydrate	5.36 ± 0.29^f	3.03 ± 0.16^d	2.61 ± 0.13^bc	2.36 ± 0.11^b	3.41 ± 0.18^e	1.21 ± 0.08^a	2.89 ± 0.16^cd
Vanilic acid	0.31 ± 0.01^b	0.14 ± 0.01^a	0.60 ± 0.03^c	0.17 ± 0.01^a	3.14 ± 0.14^f	1.04 ± 0.06^d	1.47 ± 0.08^e
Chlorogenic acid	3.21 ± 0.15^d	1.34 ± 0.06^a	1.70 ± 0.08^b	1.29 ± 0.06^a	2.29 ± 0.11^c	1.60 ± 0.07^b	2.18 ± 0.12^c
Caffeic acid	0.16 ± 0.01^a	0.59 ± 0.02^d	0.83 ± 0.04^e	1.26 ± 0.06^g	0.44 ± 0.02^c	0.23 ± 0.01^b	0.99 ± 0.05^f
Syringic acid	0.99 ± 0.06^d	0.44 ± 0.02^a	0.54 ± 0.02^b	0.81 ± 0.04^c	1.32 ± 0.05^f	1.17 ± 0.03^e	1.12 ± 0.06^e
Epicatechin	4.25 ± 0.23^a	2.89 ± 0.14^a	226.74 ± 10.56^d	2.95 ± 0.17^a	11.56 ± 0.65^b	9.16 ± 0.44^ab	45.67 ± 1.98^c
Coumaric acid	1.10 ± 0.05^b	0.24 ± 0.01^a	0.94 ± 0.05^b	0.49 ± 0.02^a	8.05 ± 0.398^d	0.96 ± 0.05^b	5.55 ± 0.31^c
Ferulic acid	4.17 ± 0.21^d	0.25 ± 0.01^a	6.91 ± 0.33^e	0.21 ± 0.01^a	1.12 ± 0.05^b	0.49 ± 0.02^a	3.87 ± 0.19^c
Sinapic acid	2.02 ± 0.12^c	1.77 ± 0.08^b	3.27 ± 0.15^d	1.35 ± 0.07^a	4.04 ± 0.21^e	1.65 ± 0.07^b	3.11 ± 0.18^d
Salicylic acid	7.54 ± 0.37^c	2.65 ± 0.13^a	18.31 ± 0.77^e	2.88 ± 0.14^a	7.32 ± 0.35^c	3.98 ± 0.18^b	8.88 ± 0.38^d
Rutin	11.09 ± 0.61^e	7.73 ± 0.39^d	12.29 ± 0.62^f	1.65 ± 0.07^a	4.86 ± 0.31^b	4.84 ± 0.23^b	6.77 ± 0.41^c
Elagic acid	15.14 ± 0.71^e	7.56 ± 0.41^d	6.34 ± 0.32^c	2.59 ± 0.12^a	8.00 ± 0.38^d	5.32 ± 0.25^b	7.56 ± 0.42^d
Myricetin	16.63 ± 0.66^e	1.43 ± 0.07^a	2.66 ± 0.13^b	30.54 ± 1.23^f	7.44 ± 0.42^d	1.91 ± 0.09^ab	5.33 ± 0.29^c
Trans-cinnamic acid	0.80 ± 0.03^d	0.19 ± 0.01^b	0.10 ± 0.01^a	0.31 ± 0.01^c	1.17 ± 0.05^f	0.17 ± 0.01^b	0.99 ± 0.05^e
Quercetin	3.54 ± 0.18^a	5.72 ± 0.25^c	6.83 ± 0.34^d	3.36 ± 0.17^a	6.55 ± 0.35^d	4.99 ± 0.31^b	6.88 ± 0.33^d

^*Values in the same row followed by different superscript lower-case letters are significantly different at p < 0.05. The data represents mean ± SD.

The amount of myricetin ranged from 1.43 to 30.54 mg/100 g FW, with the highest amount occurring in C. mas fruit, followed by H. rhamnoides (16.63 mg/100 g FW) and P. padus (5.33 mg/100 g FW). Amounts between 14 and 142 mg/kg of myricetin were detected in cranberry, black currant, crowberry, bog whortleberry, blueberries, and bilberry by Häkkinen et al.^[30] The content of catechin hydrate varied between 1.21 and 5.36 mg/100 g FW. H. rhamnoides exhibited the highest amount of catechin (5.36 mg/100 g FW), followed by P. spinosa (3.41 mg/100 g FW). The flavonoids, rutin, and quercetin have been described as cell-protecting agents because of their antioxidant and anti-inflammatory actions. In this study, significantly different amounts of quercetin (3.36–6.88 mg/100 g FW) and rutin (1.65–12.29 mg/100 g FW) were detected in wild flora fruits. Radovanović et al.^[31] reported the highest concentration of quercetin-3-glucoside in blackthorn extract (32.02 mg/kg FW) while rutin and quercetin were the predominant flavonols found in wild blackberry extract (22.77 mg/kg FW and 3.79 mg/kg FW, respectively). The amount of quercetin detected in this study in methanolic extract of R. fruticosus fruit was quite lower compared to that reported by Türkben et al.^[32] in water-extracted samples.

Salicylic acids and elagic acid were the most abundant phenolic acids in all samples. There were significant differences among species in all individual phenolic compounds (p < 0.05). The amounts of salicylic acids ranged between 2.65 mg/100 g FW (C. monogyna) and 18.31 mg/100 g FW (R. fruticosus). Determinations revealed also high concentrations of ellagic acid in fruits of H. rhamnoides, the variation limits for this compound being between 2.59 mg/100 g FW (C. mas) and 15.14 mg/100 g FW (H. rhamnoides). In the present study, a significantly (p < 0.05) higher amount of vanillic (3.14 mg/100 g FW), sinapic (4.04 mg/100 g FW), coumaric (8.05 mg/100 g FW), syringic (1.32 mg/100 g FW), and trans-cinnamic acid (1.17 mg/100 g FW) was detected in P. spinosa extract. Radovanović et al.^[31] found a high gallic acid content (150.21 mg/kg FW), and significant amounts of t-caftaric, t-coutaric, caffeic, syringic, and p-coumaric acid of wild fruit extracts in blackthorn. Gallic acid was the most abundant (6.85 mg/100 g FW) in R. canina fruit, followed by P. padus (5.83 mg/100 g FW). Six phenolics were identified by Olszewska et al.^[33] in P. padus leaves: isorhamnetin 3-O-β-xylopyranosyl-(1 → 2)-β-galactopyranoside, astragalin, hyperoside, quercetin 3-O-β-xylopyranosyl-(1 → 2)-β-galactopyranoside, quercetin 3-O-β-xylopyranosyl-(1 → 2)-β-glucopyranoside, and chlorogenic acid. Ferulic acid was the most abundant in the R. fruticosus sample (6.91 mg/100 g FW), while chlorogenic acid was the most abundant in the H. rhamnoides sample (3.21 mg/100 g FW). The major phenolic compounds in water-extracted samples by Türkben et al.^[32] in fresh fruit of blackberry cultivars grown in Turkey were ellagic acid (1828.07–1555.13 mg/kg), ferulic acid (757.69–413.82 mg/kg), caffeic acid (736.85–337.89 mg/kg), and p-coumaric acid (877.45–287.15 mg/kg). The amount of phenolic acids detected in the present study in methanolic extract of R. fruticosus fruit was quite lower compared to that reported by Türkben et al.^[32] in water-extracted samples.

Determinations have also revealed high concentrations of caffeic acid in C. mas (1.26 mg/100 g FW) fruit, followed by P. padus (0.99 mg/100 g FW). Caffeic acid has been found to be an effective antioxidant in various in vitro antioxidant tests, including total antioxidant activity of iron thiocyanate method by Gülçin.^[34] Previous experimental results have indicated that caffeic acid was the main phenolic acid in green gooseberry.^[35]

Mineral elements

Mineral and trace elements contents (mg/100 g FW) in wild flora fruits are presented in Table 3. In this study significant differences (p < 0.05) were observed for each individual mineral among fruits from wild flora: for Ca between 5.91 and 109.76 mg/100 g FW, for Mg between 11.93 and 59.11 mg/100 g FW, for Na between 0.32 and 5.37 mg/100 g FW, for Fe between 0.69 and 3.28 mg/100 g FW, for Mn between 0.05 and 0.59 mg/100 g FW, for Cu between 0.08 and 0.33 mg/100 g FW, for Cr between 0.02 and 0.13 mg/100 g FW, for Zn between 0.08 and 0.39 mg/100 g FW, and for B between 0.08 and 0.63 mg/100 g FW. The content of some trace elements (Zn, Cu) did not exceed toxicity thresholds, thus indicating that consumption of these fruits was not detrimental to public health.

Table 3. Mineral and trace elements content (mg/100 g FW) in fruits from the wild flora.*

	Sea-buckthorn (Hippophaë rhamnoides)	Hawthorn (Crataegus monogyna)	Wild grown European blackberry (Rubus fruticosus L.)	Cornelian cherry (Cornus mas)	Blackthorn (Prunus spinosa)	Dog rose (Rosa canina)	Bird cherry or hackberry (Prunus padus)
Ca	109.76 ± 5.89^f	5.91 ± 0.46^a	18.94 ± 0.88^b	28.33 ± 1.56^c	32.20 ± 1.76^cd	102.13 ± 6.11^e	35.73 ± 1.81^d
Mg	45.64 ± 2.43^d	12.87 ± 0.71^a	25.72 ± 1.31^c	18.50 ± 0.88^b	11.83 ± 0.57^a	59.11 ± 2.99^e	24.88 ± 1.10^c
Na	1.83 ± 0.11^d	5.37 ± 0.26^e	0.58 ± 0.04^b	1.12 ± 0.06^c	0.48 ± 0.03^ab	0.41 ± 0.03^ab	0.32 ± 0.02^a
Fe	3.28 ± 0.17^e	1.07 ± 0.06^bc	0.69 ± 0.04^a	0.95 ± 0.04^bc	1.11 ± 0.05^c	2.49 ± 0.13^d	0.93 ± 0.05^b
Mn	0.28 ± 0.01^d	0.17 ± 0.01^b	0.25 ± 0.02^c	0.15 ± 0.01^b	0.05 ± 0.00^a	0.59 ± 0.03^e	0.26 ± 0.01^cd
Cu	0.33 ± 0.02^e	0.14 ± 0.01^c	0.08 ± 0.01^a	0.09 ± 0.00^ab	0.10 ± 0.00^b	0.20 ± 0.01^d	0.13 ± 0.01^c
Cr	0.07 ± 0.01^c	0.13 ± 0.01^f	0.05 ± 0.01^b	0.10 ± 0.01^e	0.08 ± 0.00^d	0.09 ± 0.01^d	0.02 ± 0.00^a
Zn	0.39 ± 0.02^e	0.16 ± 0.01^c	0.13 ± 0.01^b	0.13 ± 0.01^b	0.08 ± 0.01^a	0.22 ± 0.01^d	0.14 ± 0.01^bc
B	0.63 ± 0.04^e	0.18 ± 0.01^c	nd^a	0.47 ± 0.02^d	nd^a	0.08 ± 0.01^b	0.43 ± 0.02^d

*Values in the same row followed by different superscript lower-case letters are significantly different at p < 0.05. The data represents mean ± SD.

In H. rhamnoides fruit the highest values (mg/100 g FW) of Ca (109.76), Fe (3.28), Cu (0.33), Zn (0.39), and B (0.63) were detected. The average content of minerals in the sea buckthorn berries (mg/100 g FW) grown in Turkey of different genotypes was 208 N, 710 P, 726 K, 196 Ca, 146.5 Mg, 3.2 Zn, 2.4 Cu, 2.2 Mn, and 0.7 Fe.^[36] The study provides evidence for nutritional value due to the presence of genetically diverse ecotypes of buckthorn with tremendous biochemical and nutritional values, and this variation is caused by the diverse areas.

C. monogyna is distinguished by its high content (mg/100 g FW) of Na (5.37) and Cr (0.13) analysed and compared to the other species containing significant amounts of Mg (12.87), Ca (5.91), Fe (1.07), and Mn (0.17). In the chemical composition of wild hawthorn growing in Turkey, Özcan et al.^[37] found these values: 3046.37 ppm Ca, 13531.96 ppm potassium, 1502.55 ppm Mg, 312.18 ppm Na, and 431307.29 ppm phosphorus. The mineral contents of hawthorn fruits were found to be lower than those of Özcan et al.^[37]

Among the analyzed species, Mg (59.11 mg/100 g FW) and Mn (0.59 mg/100 g FW) were the most important elements of R. canina. Other minerals (Ca, Na, Fe, Cu, Cr, Zn, and B) were detected in the fruit of R. canina. Ercisli et al.^[20] reported that the Ca and Mg contents of fruits of R. canina were 2867 ppm and 1254 ppm, respectively. In this study the Ca (102.13 mg/100 g FW) and Mg (59.11 mg/100 g FW) results indicate that the values found are lower than those obtained by Ercisli et al.^[20] The results of the mineral composition of R. fruticosus fruits are provided in Table 3. Mg was the most abundant mineral found in blackberry fruits, with an overall average of 25.72 mg/100 g FW. The Mg content found for blackberry was superior to that observed for the other species (hawthorn, cornelian cherry, blackthorn, and hackberry). Ca, Na, Fe, Mn, Cu, Cr, and Zn contents have recorded mean values of 18.94, 0.58, 0.69, 0.25, 0.08, 0.05, and 0.13 mg/100 g FW, respectively. B was not detected in blackberry samples. Stefanut et al.^[38] reported macro-mineral and micro-mineral concentrations of Zn, Cu, Al, Mn, Co, and Fe as 140, 50, 27, 33, 1, and 30 (μg/100 g fruits) in fresh blackberry (fruit), respectively.

C. mas fruit species presented the highest Ca contents (28.33 mg/100 g FW), followed by Mg (18.50), Na (1.12), Fe (0.95), B (0.47), Mn (0.15), Zn (0.13), Cr (0.10), and Cu (0.09). Krośniak et al.^[39] believe that cornelian cherry juices are rich in various essential elements (K, Ca, Na, Fe, Zn, Mn, Cu) and might be considered an important dietary mineral supplementation for individuals with deficiency in nutritional elements. The mineral contents of P. spinosa fruits are reported in Table 3. Ca is the major component of blackthorn fruits (32.20 mg/100 g FW). Compared to the results obtained in the seven analyzed species, P. spinosa recorded the lowest values for Mg (11.83), Mn (0.05), and Zn (0.08). B is not detected. Al, B, Ca, K, Mg, Na, P, and S were determined as major minerals of blackthorn fruits growing wild in Konya province in Turkey by Marakoglu et al.^[40] The minerals Ca (35.73) and Mg (24.88) were also abundant in P. padus fruit (mg/100 g FW). Other minerals (Na, Fe, Mn, Cu, Cr, Zn, and B) were detected too.

Conclusion

The results of this study demonstrated significant differences found in total phenolic and flavonoid content of these species, and also in terms of antioxidant and mineral compounds. This study is meant to be a contribution to the characterization of chemical extracts of wild flora fruits that are traditionally used for medicinal applications. Fruits that were studied may have great potential for food production as sources of bioactive compounds such as phenolic compounds and minerals, and also for food supplements or functional foods.