Carotenoids, Flavonoids, Total Phenolic Compounds and Antioxidant Activity of Two Creeping Cotoneaster Species Fruits Extracts

Cotoneaster horizontalis Decne. and Cotoneaster microphyllus Wall. ex Lindl. species are two creeping bushes, commonly used as ornamental plants in gardens and parks. The aim of this paper was to assess the concentrations of some classes of bioactive compounds classes, carotenoids, flavonoids and total phenolic compounds, in fresh fruits of these species. Carotenoids and flavonoids were determined through acetone and methanol extraction followed by spectrophotometry. For total phenolics, methanol extraction and a spectrophotometric Folin-Ciocâlteu method was used. The total antioxidant capacity was quantified through photochemiluminescence method by comparison with the standard substance used for calibration, Trolox® as tocopherol analogue (ACL procedure) using Photochem apparatus, Analytik Jena AG, Germany. Average values found in Cotoneaster horizontalis and Cotoneaster microphyllus fruit tissue were 380.63 mg/kg, respectively 179.63 mg/kg, carotenoids; 8036.07 mg/kg, respectively 6888.06 mg/kg flavonoids; and 16342.06 mg/kg GAE, respectively 18631.35 mg/kg GAE total phenolic compounds. These values are comparable to those found in other wild and cultivated related Rosaceae, including domestic rowans. Cotoneaster microphyllus fruits emphasized an increased antioxidant activity (up to 39.69 μmol Trolox equivalent/g dry weight).

Carotenoids (carotenes, lutein, lycopene, zeaxanthin etc.) are common pigments in plant leaves, flowers and fruits. They are essential for melanin and retinol production, thus, for skin and eye health, while they also possess antitumor properties [4].
There are various subclasses of phenolic and polyphenolic compounds, with protective functions against microbial and fungal pathogens and insects [5]. Among them, flavonoids, phenolic acids, tannins. These compounds are antioxidants, acting as radical scavengers, reducing agents and lipid oxidation inhibitors [6]. Flavonoids (low-mass polyphenolic compounds) are antiviral, antibacterial, antifungal, anti-inflammatory, antiproliferative and antitumor compounds [5].

2.Materials and methods
Mature pomes from different individuals from both species were collected in Constanța city, Romania, in October -November months, 2018. Pyrenes (kernels) were manually eliminated and the fruit pulp was ground, using an electrical grinder. Fruit tissue was kept frozen (-20°C), prior to examination. Part of the material was oven-dried for 72 h at 80°C, for determining dry biomass percentage.
For determining total carotenoids content, 0.1 g ground fruit tissue was extracted in 10 mL 80% acetone (triplicate samples for each species). The extract was filtered at normal pressure through Whatman blue band filter paper and the spectrophotometric absorbance was read (using a S106 WPA UV-Vis spectrophotometer) against an 80% acetone blank, at 470 nm, 647 nm and 663 nm of wavelengths [7]. Absorbance values were used to calculate carotenoids concentration, according to the specific trichromatic equations [8].
For flavonoid determinations, a quantity of 1 g ground fruit tissue was extracted in 5 mL methanol and filtered (triplicate samples). 0.5 mL of extract was diluted in 4 mL water and 8 mL methanol mixture, and the spectrophotometric absorbance was read against a methanol blank at 340 nm wavelength [9]. For determining the concentration of total phenolic compounds, a UV-Vis spectrophotometric version of the Folin-Ciocâlteu method was used. 0.1 g fruit tissue was extracted in 10 mL methanol and filtered. 1 mL of extract was reacted with 5 mL Folin-Ciocalteu reagent (10%) and 4 mL sodium bicarbonate solution (7.5%) for 30 min. Spectrophotometric absorbance was read against a blank at 765 nm. A calibration curve was prepared, by using different gallic acid concentrations [10 -12]. Concentrations were expressed as mg/kg dry weight and mg/kg DW gallic acid equivalent (GAE) for total phenolic compounds.
For determining total antioxidant capacity, a quantity of 1 g, respectively 10 g of fine dried fruit powder, was cold-extracted in 40% ethanol (100 mL total volume), at room temperature and darkness, for 12 days, with regular shaking. After decantation, normal pressure filtration and homogenization (Vortex Velp Scientifica, Italy agitator), 10 μL of supernatant were taken for analysis. Each determination lasted 120 s.
For calibration, the standard kit of reagents, Analytik Jena Germany was used: R1 (dilution solvent), R2 (buffer reagent), R3 (photosensitive reagent), R4 (reagent sized). For the calibration curve ( Figure 2) standard solutions containing 0.5, 1.0, 2.0, 3.0 nmol Trolox were measured (suitable for 5 -30 μL R4). By exposure to external radiation from a Hg lamp lined with phosphorus, maximum energy at 351 nm wavelength, the photosensitive reagent produces free radicals in the sample, resulting a photochemical reaction. The superoxide anion radicals produced are partially neutralized by reaction with the antioxidants in the sample. Residual radicals cause luminescence in the detector substance, which can be determined using a photomultiplier tube. The signal produced by the luminescence is traced over 3 min.
The total antioxidant capacity of the selected sample is measured by converting the electrical signal to concentration values and by comparison with the Trolox® standard substance, expressed as nmol Trolox equivalents/sample volume [13,21,22]. Samples were prepared, according to Table 1.

3.Results and discussions
Carotenoids concentrations in the selected fruits are shown in Figure 3. Flavonoids and total phenolic compounds level are shown in Figure 4 and Figure 5, respectively.
Considering a 13.1% dry weight percentage, the average amount of total carotenoids was 380.63 mg/kg DW (315.47 -433.62 mg/kg in individual samples), in Cotoneaster horizontalis and 179.63 mg/kg DW (165.57 -189.83 mg/kg) in Cotoneaster microphyllus (Figure 3). Few data concerning the carotenoids content of Cotoneaster species are found in literature. An useful comparison standard would be the related genus Pyracantha containing over 65 mg/kg carotenoids in Pyracantha angustifolia [14], and more than 22 mg/kg in Pyracantha crenulata species (known as a valuable medicinal plant, powdered dried fruits used in the treatment of bloody dysentery) [15,19].
Sorbus (the genus comprising rowanberries and service-trees), is also a related genus, in the Maleae tribe, with similar fruits. A comprehensive research on cultivated rowan varieties found a total carotenoid content of 39 -2659 mg/kg [6].
Total antioxidant capacity (TEAC) of fruits pulp hydroalcoholic extracts was reported according to the photochemiluminescence method, ACL procedure of Analytik Jena AG and quantified by comparison with Trolox® standard substance. Results are shown in Table 2.

4.Conclusions
Having average contents of 380.63 mg/kg, respectively, 179.63 mg/kg, Cotoneaster horizontalis Decne. and Cotoneaster microphyllus Wall. ex Lindl. species fruits were similar to related Rosaceae fruits, including Sorbus cultivars, and could be a valuable source of vitamin A and other important carotenoid-derived compounds.
With 8036.07 mg/kg, respectively 6888.06 mg/kg flavonoids content, they were also comparable to Sorbus species and other wild and cultivated Rosaceae. The same can be said for total phenolic and polyphenolic compounds content (16342.06 mg/kg GAE, respectively 18631.35 mg/kg GAE).
While the concentration of phenolic compounds (including flavonoids) was similar in the two species, the amount of carotenoids was significantly higher in Cotoneaster horizontalis pomes.
With up to 39.69 μmol Trolox equivalent/g DW, the total antioxidant capacity of Cotoneaster microphyllus pomes is increased, and similar to that of some related medicinal and nutrition-use Rosaceae species.
Further research is needed to assess, in detail, the composition in bioactive compounds and to confirm the potential of these vegetal species for valuable applications other than ornamental ones.