Optimization of polyphenols extraction process with antioxidant properties from wild Vaccinium myrtillus L. (bilberry) and Vaccinium vitis-idaea L. (lingonberry) leaves

The aim of the current study was to optimize the extraction condition of polyphenol compounds with antioxidant properties from leaves of natural grown Vaccinium myrtillus L and Vaccinium vitis-idaea L. The extractions were carried out in ultrasonic bath at 40°C for 20 min with different solvents (water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and 96% ethanol) and different hydro module of samples and solvents used (1:50, 1:100 and 1:200). The highest total phenol content in the leaves of V. myrtillus L. and V. vitis-idaea L. was found when 40% ethanol extract was used (90.50 ± 0.05 mg GAE/g DW and 96.68 ± 0.05 mg GAE/g DW, respectively). The highest total flavonoid content of the leaves of both species was obtained with 80% ethanol as extraction solvent. The highest level of total proanthocyanidins were in the 60% ethanol extract of V. myrtillus L. and in the 80% ethanol extract of V. vitis-idaea L (13.12 ± 0.11 mg LE/g DW and 24.22 ± 0.21 mg LE/g DW, respectively). The highest ability to scavenge DPPH radicals possessed the 40% ethanol extracts from the leaves of both species (693.99 ± 4.05 mM TE/g DW for V. myrtillus L. and 1083.18 ± 8.48 mM TE/g DW for V. vitisidaea L). Data analysis showed that the maximal amount of polyphenols was extracted at a hydro-module of 1: 100. HPLC analysis revealed that the dominant phenolic acid in the leaves of V. myrtillus L was chlorogenic acid (13.45 mg/g DW), while ferulic acid (49.48 mg/g DW) was present at the highest concentration in the leaves of V. vitis-idaea L.


Introduction
Bilberry (Vaccinium myrtillus L.) and lingonberry (Vaccinium vitis-idaea L.) are flowering plants belonging to the large genus of Vaccinium Ericaceae (Hokkanen et al., 2009;Rohloff et al., 2015;Burdulis et al., 2009) These berries are native to Bulgarian forests (Dincheva and Badjakov, 2016). The investigated Vaccinium species are mainly noted for their high content of anthocyanin pigments (Dincheva and Badjakov, 2016;Rohloff et al., 2015). Indeed, Vaccinium berries such as bilberries and blueberries lingonberry contain high amounts of sugars and organic acids. Berry fruits are rich sources of polyphenols with different bioactive potential (Hokkanen et al., 2009). These compounds are mainly represented by flavonoids, phenolic acids, and tannins, which are known as natural antioxidants (Brasanac-Vukanovic et al., 2018). Many of the biological properties are closely associated with the antioxidant activity of anthocyanin pigments (Chu et al., 2011). Riihinen et al. (2008) reported that both bilberry and blueberry leaves contained high amounts of proanthocyanidins, flavonols and hydroxycinnamic acids. Also, bilberry and lingonberry are potential sources for triterpenoid and phytosterol compounds (Szakiel et al., 2012 a, b). The aim of the current study was to derive the most appropriate conditions for extraction of the highest yield of biologically active substances with high antioxidant potential from bilberry and lingonberry aerial part. The influence of different concentration ratio of solvent system (ethanol-water) and different hydromodules of samples and solvents on the concentration of biologically active substances have been studied.
Extraction procedure. One gram of dry ground leaves were placed in a flask together with 100 mL of ethanol-water mixture in different ratio (water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and 96% ethanol) was poured in it. Ultrasound-assisted extraction was performed in an ultrasonic bath SIEL UST 5.7-150 (Gabrovo, Bulgaria) with a frequency 35 kHz, power 240 W at a temperature of 40°C.
Analyses. Total polyphenols content. The total phenolics content was measured using the Folin-Ciocalteu assay as described by Ivanov et al. (2014): Folin-Ciocalteu reagent (1 mL) (Sigma) diluted five times was mixed with 0.2 mL of sample and 0.8 mL 7.5% Sodium carbonate. The reaction was 20 min at room temperature in darkness. After reaction the absorption was measured at 765 nm against a blank sample, prepared in the same way but without the extract. The results were expressed in mg equivalent of gallic acid (GAE) per g dry weight (DW), values derived from a calibration curve established in the concentration range of 0.02 -0.10 mg gallic acid (Sigma) used as a standard.
Total flavonoids. Total flavonoids were determined spectrophotometrically following the method described by Ivanov et al. (2014). Each extract (1.0 mL) was added to test tubes containing 0.1 mL 10% aluminum nitrate (Sigma), 0.1 mL 1M potassium acetate (Sigma) and 3.8 mL ethanol (Merck). The reaction time was 40 min at ambient temperature. The absorbance was measured at 415 nm. The results were expressed in mg equivalent of quercetin per g dry weight (DW), derived from a calibration curve, linear in the range of 10-100 μg/mL quercetin as a standard.
Total proanthocyanidins assay. Acid butanol assay for proanthocyanidins, as described by Porter et al. (1985). The six milliliter of the acid butanol reagent mixture of 950 mL of n-butanol and 50 mL concentrated HCl), 0.5 mL aliquot of the fraction, and 0.1 mL of the iron reagent (2% ferric ammonium sulfate in 2 mol/L HCl were added to a 10 mL screw cap tube and then vortexed. The tube was capped loosely and put in a boiling water bath for 50 min. The absorbance of formed colored complex was read at 550 nm. Condensed tannins were analyzed as leucocyanidin equivalent (LE) (Hagerman, 2011). Antioxidant activity. DPPH assay. DPPH assay was performed according to Ivanov et al. (2014). Each extract (0.15 mL) was mixed with 2.85 mL of a freshly prepared 0.1 mM solution of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH, Sigma) in methanol (Merck). The reaction was performed at 37 °C in darkness and the absorptions at 517 nm were recorded after 15 min against methanol. The antioxidant activity was expressed as mM Trolox equivalents (TE) per g dry weight (DW) by using bration curve, established in the range of build by 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®, Fluka) dissolved in methanol (Sigma).

HPLC-DAD analysis of phenolic acids.
Chromatographic analysis of the phenolic acids content was performed using a HPLC Elite Chrome Hitachi, coupled with diode-array detector (DAD) and ELITE LaCHrome software. The separation was performed on a reverse-phase column Supelco, Discovery® HS C18 (5 μm, 25 cm x 4.6 mm) operating at 30°C. Detection was done at wavelength 280 nm and 320 nm. Elution was performed with mobile phase A (2 % acetic acid) and mobile phase B (acetonitrile) in gradient mode described by Ivanov et al. (2014) at a flow rate 0.8 mL/min. The sample injection volume was 20 μL.

RSM
(Response Surface Methodology) optimization method. In order to evaluate the effects of extraction parameters and to optimize conditions for various responses RSM optimization method was applied. Independent variables used in the experimental design were solvent concentration (water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol and 96% ethanol) and sample-solvent hydro modules (1:50, 1:100 and 1:200). The coded and uncoded independent variables used in the RSM design are presented in Table 1.
Ranges of ethanol (X1) and hydro module (X2) and the central points were selected based on literature data. Statistical analysis of experiment was performed using Statistical Software MINITAB 16. The response variables were fitted to the following second-order polynomial model (Eq. 1), which was able to describe the relationship between the dependent output variable and the independent variables: where Y represents response variable (total polyphenolic content and antioxidant activity -DPPH method); Xi and Xj are the independent variables (ethanol concentration and hydro module); β0, βi, βii and βij are the regression coefficients for the intercept, linear, quadratic and interaction coefficient, respectively.

Results and Discussion
There is an increasing interest for using phytochemicals from natural plants. The extracts obtained from different plant origin had different effects on human health. Extraction efficiency depends on a large number of parametersextraction method, solvents, temperature, extraction time. Therefore, it is very important to find optimal extraction parameters for obtaining extracts with the highest content of biologically active compounds (Cvetanovic et al. 2015, Mašković et al., 2016. Response surface methodology is an effective technique for the optimization of complex processes, because it allows an efficient and easier interpretation of experimental data (Bezerra et al. 2008, Zekovic et al. 2014. Several investigators already used RSM for the optimization of extraction processes in order to maximize yield of various polyphenolic compounds from various sources (Radojković et al., 2012;Claus et al., 2015;Mašković et al., 2016). In the present study RSM was used to optimize the solid-liquid extraction ratio of compounds with improved antioxidant ability from Vaccinium species. The influence of solvent composition and hydro module on the extraction yield of total polyphenols, total flavonoids, total proanthocyanidins and antioxidant activity was investigated. The experiments were set up according to RSM design, and the results are presented in Table 2 and Table 3. The effect of linear, quadratic or interaction coefficients on the response was tested for significance by analysis of variance. Experimental results from Table 4 were processed with multiple linear regressions using the second-order polynomial model -Eq. (1). The regression coefficients of the intercept, linear, cross product and quadratic terms are presented in the Table 4. Suitability of the model was also analysed through the MINITAB 16. Calculated statistical parameters are presented in Table 4. According to the calculated p values and the F value for the suggested model was suitable for the investigated extraction system. Model equations for relationship between total phenol content and antioxidant activity and independent variables were obtained by applying multiple regression analysis (Table 4). By applying these equations, it is possible to predict values of each response. The values of R 2 for total polyphenol content, and antioxidant activity were 0.73, 0.94 and 0.56, respectively (Table 4). Therefore, it was suggested that the quadratic model fitted well with the experimental data.
The optimization procedures carried out using "Response optimizer" of MINITAB 16 software gave the following values of variable X1 and X2 for maximum yield of total polyphenolics, and antioxidant activities (DPPH methods) (Y) by Vaccinium species (Table 5). The deviation between the theoretically studied maximal amounts of total polyphenols and experimentally obtained for bilberry and lingonberry were 3.2 mg/g GAE DW and 5.9 mg/g GAE, respectively; and antioxidant activities (DPPH methods) (at 40% ethanol and 1:100 hydro module) under ultrasonic influence (Table 5). On this basis, we propose that 40% ethanol in water and hydro module 1:100 as optimal extraction conditions for highest yield of biologically actives substances from Vaccinium species leaves. According to the obtained results, all investigated extracts of lingonberry contained a higher total polyphenol content and higher ability to scavenge DPPH radicals than the extracts of bilberry leaves, while a higher total flavonoid content was determined in the extracts of bilberry leaves. After establishing the optimal parameters (40% ethanol and 1:100 hydro modules) for extraction of the highest amount of polyphenol components by ultrasonic irradiation, HPLC-DAD analysis of phenolic acids was carried out. The results showed the presence of three phenolic acids in the bilberry leaves: chlorogenic acid -13.45 mg/g DW, p-coumaric acid -0.62 mg/g DW and sinapic acid -1.85 mg/g DW. The main phenolic acid extracted from lingonberry leaves was ferulic acid -49.48 mg/g DW, followed by sinapic acid (2.34 mg/g DW) and chlorogenic acid (2.55 mg/g DW) ( Table 6). Brasanac-Vukanovic et al.
(2018) investigated the phenolic compounds in different extracts obtained from bilberry leaves and obtained similar results -the main phenolic acid was chlorogenic acid (from 45.5 to 59.7 mg/g DW), followed by p-coumaric acid (from 1.26 to 2.08 mg/g DW).

Conclusions
The optimal conditions for the extraction of biologically active substances from Vaccinium myrtillus L. and Vaccinium vitis-idaea L. are as follow: 40% ethanol-water as solvent system and hydro module 1:100 in ultrasound bath with frequency 35 kHz. Under these condition the maximum amount of total polyphenols content and antioxidant activity was obtained. The main identified phenolic acid in the 40% ethanol-water extract in bilberry leaves was chlorogenic acid and for lingonberry extract was ferulic acid.  Food Science and Applied Biotechnology, 2020, 3(2), 149-156 Vrancheva et al., 2020 Optimization of polyphenols extraction process with antioxidant … Page 155