Antioxidant Activity of Some Fluids Extracts of Indigenous Wild Cherry Fruits

The paper presents un study regarding the comparative total antioxidant capacity of some fluids extracts of the soluble lipid compounds of Cerasus avium (L.) Moench. syn. Prunus avium (L.) var. sylvestris Ser fruits, popular named wild cherry, species undertaken aiming at exploiting the nutrient profiles and promoting the further development of these indigen phytopharmaceutical resources. For analysis, the photo-chemiluminescence method, ACL procedure, Analytik Jena AG, by comparing with the standard Trolox® solution, was used. Wild cherry fruits fluid extracts analyzed emphasize an increased total antioxidative capacity which may be attributed to the high level content of polyphenolic compounds and minerals content which sustains the possibility to use this valuable vegetal product for nutraceutical or comeceutical preparations.


Introduction
The name of the plant Cerasus avium (L.) Moench. syn. Prunus avium (L.), popular known as wild cherry or bitter cherry, derives from the city name Cerasus on the shores of the Black Sea in Asia Minor (today Kerasun), from where the plant, in 74 BC, was brought to Europe by Locullus. Was also the name of the cherry at Columella indigenous tree, microphanerophyte -megaphanerophyte, spontaneous and cultivated, with food, industrial, medicinal importance. Phytocenological, wild variety, Carpinion, Querco-Beech, widespread in Europe, Asia Minor, North Africa, the Caucasus Mountains, Turkmenistan, from where it spread to all continents. The cherry (Cerasus avium) has the following varieties: sylvestris Ser., wild cherry, with small fruits, up to 1 cm in diameter, black, slightly juicy, sweet-bitter and early ripening; juliana (L.) Pojarkova, ispas cherries, with medium fruits, soft pulp, early and medium ripening; duracina (L.) Pojarkova, stony cherries, with large fruits, hard, stony pulp, slightly juicy and late ripening. Most of the varieties are derived from these three botanical varieties [1 -6]. In Romania, wild cherry with the varieties Amar Maxut and Amar Galata [6], are considered in literature as having the center of origin in Northern Moldova, but also it was acclimatised in Dobrudjia.
Numerous studies have proven that vegetal compounds originated from plants or fruits possess a high spectrum of biological activity. However, polyphenolic extracts (e.g. flavonoids, anthocyanin, tannins etc.), despite having excellent in vitro bioactivity, sometime present a decreased action in vivo due to their improper molecular size, resulting in an absorption and bioavailability decreased. Moreover, the efficacy of natural polyphenols depends on preserving the stability, bioactivity and bioavailability of the active compounds [7, 8,].
Cerasus avium (syn. Prunus avium) Moench. species, wild cherry, are often used in popular medicine most due to its content in polyphenolic acids with great antioxidative capacity mainly useful in anti -inflammatory diseases, hypoglycemic activities, tonic action due to their synergistic action. For medicinal purposes, the fruits stalk are used for the content in flavonoids. The main components of the peduncles are: tannins, catechins, saponins, proanthocyanidols and minerals (potassium salts) [14][15][16][17][18]. Flavones are represented by quercetol, genistein, dihydrovogonin and naringenol [19][20][21][22][23]. Due to its flavonoid, C and E vitamins, oligoelements content known for their antioxidant activity, the raw material expressed as fresh wild cherry fruits, will be studied to establish their antioxidant capacity and therapeutically potential [24][25][26][27][28][29][30]. With rich content in vitamins, minerals, polyphenols, carotenoids and many other compounds, Cerasus avium (syn. Prunus avium) Moench. fruits extracts, may be useful as an adjuvant in the treatment or in preventing many diseases which arise from oxidative stress action of free radicals [31,32].
The aim of this paper was to determined the total antioxidant capacity of some fluids extracts of the soluble lipid compounds of Cerasus avium (L.) Moench. syn. Prunus avium (L.) var. sylvestris Ser, wild cherry fruits [33][34][35], aiming at exploiting the nutrient profiles and promoting the further development of these indigen phytopharmaceutical resources in some new nutraceuticals, in order to promote their phyto-therapeutic potential in hepato-digestive disorders [36][37].

Material and Methods
The vegetal product represented by the mature drupes, without seeds of the wild cherry fruits (Fig. 2), was collected from Romania, Dobrudjia area, Constanta county, in the period of July 2018 -July 2019.
-Sample preparation: 0,5 g, respectively 5 g of fresh vegetal product were extracted with 50 mL ethanol 40%, 70%, 96% concentration, by cold maceration for 10 days. It was kept in the dark with periodic stirring. Extractive solution obtained was filtered on quantitative Whatman filter paper and 100 mL volumetric flask was filled in to the mark with ethanol sol. 40%, 70%, respectively 96% concentration. Due to the increased values of concentration (TEAC), which exceed Trolox standard calibration curve, respectively the detection limit, for the working solution, all stock solutions of extracts were diluted with 50 mL of ethanol 40%, 70%, respectively 96% concentration, molar ratio 1:2. From the diluted solutions, were taken work volumes of 5 μL, for all samples.

Antioxidant activity by photochemiluminescence method
Evaluation of total antioxidant activity of hydro-alcoholic extract of Cerasus avium fruits was performed through photochemiluminescence method, determining the Antioxidant Capacity of Lipid soluble substances (ACL), according to Analitik Jena procedure, using photochemiluminometer PHOTOCHEM, Analytik Jena AG, Germany, coupled to a PC.
Total antioxidant activity was quantified by comparison with Trolox® standard solution, Hoffman-LaRoche's trade name for 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E. It is an antioxidant used in biological or biochemical applications to reduce oxidative stress or free radicals damage. External light source is mercury lamp covered with phosphorus, which ensure maximum power at λ = 351 nm and the source of free radicals is luminol (5amino-2, 3-dihydro-1,4-phthalazinedione). The measuring signal produced by the luminescence is traced over 120 sec., [38,39].
Calibration curve of Trolox standard was done by measuring 4 solutions of 0.5, 1.0, 2.0, 3.0 nmol of standard Trolox® (Fig. 4) and samples measurements (Fig. 5) were performed according to the standard protocol of the ACL method, Analytik Jena AG, using volumes according to the scheme below (Table 2). Reagent R1 -methanol p.a.; Reagent R2 -buffer solution; Reagent R3 -photosensitizing reagent and free radical detection reagent; Reagent R4 -calibration standard (Trolox), for antioxidant quantification of lipidsoluble substances. The signal emitted by free radicals remaining in the sample which did not reacted with the antioxidants of the sample, was measured. This reaction occurs by emission of light quanta and registered by the detector (PMT). The voltage (V) is proportional with generate luminiscence in timp (s). Trolox equivalent antioxidant activity (TEAC) is a measurement of antioxidant strength, measured in units called Trolox Equivalents (TE), [nmol/sample], [38,39].

Results and Discussion
The results regarding the total antioxidative capacity (TEAC) of the wild cherry fruits fluid extracts, registered for the analyzed period, are presented in Table 2. The highest values of the total antioxidative capacity (TEAC) of wild cherry fruits alcoholic extract from fresh vegetal product at the concentration 10% in ethanol 96% was 0.886 nmol equiv.Trolox/volum sample (8,870 μg/mg), respectively 0.767 nmol equiv.Trolox/volum sample (7,679 μg/mg) at the concentration 1% in ethanol 96%, were registered.
The results obtained regarding the total antioxidant capacity (TEAC) of wild cherry fruits alcoholic extract, recommend the extraction of soluble substances by cold maceration in ethyl alcohol of 96% or 70% concentrations, as an optimal method of extracting valuable antioxidant compounds, with pharmaceutical/nutraceutical potential aplications.

Conclusion
Up to now, the development of pharmaceutical formulations has remained restricted to individual chemical drugs, even the properties obtained by using an optimal mixture of bio-active compounds could be strongly influenced by the synergism appeared in the system. The increased total antioxidant capacity registered in the case of wild cherry fruits fluid extracts, sustain the possibility to use this valuable vegetal product as ethanolic extract in different concentration, for some new nutraceutical products obtaining.