1. Introduction
Japanese quince (
Chaenomeles japonica (Thunb.) Lindl. ex Spach) is a dwarf shrub that originated in East Asia and was used in Chinese medicine 3000 years ago [
1]. Quince of the
Chaenomeles genus is one of the oldest cultivated plants belonging to the
Rosaceae family, a subgenus of
Maloideae [
1,
2]. Studies of the biological activity of Japanese quince (JQ) fruits have revealed their great potential for human health, including growth promotion of the beneficial intestinal bacteria
Lacticaseibacillus casei and
Lactiplantibacillus plantarum, protective effect on the lipid membrane against free radicals, and inhibition of cyclooxygenase involved in the inflammatory reactions [
3]. Other researchers have shown that extracts of JQ fruits are promising raw material for cancer treatment and prevention, due to their phenols composition and cytotoxic activity [
4,
5,
6,
7].
JQ fruit extracts have strong biological activity due to their particular biochemical composition and content of bio compounds. Due and co-authors established 24 phenolic compounds in five
Chaenomeles species, their quantity and distribution were different only for chlorogenic acid, catechin, procyanidin B1, epicatechin, and procyanidin B2 [
8]. Differences in the antioxidant activity of these five species fruits were observed in the same study [
8]. Another study identified eleven phenolic compounds, which were dominated by (−)-epicatechin and procyanidin B2 [
3]. Besides that, JQ fruits and their juice have a high amount of ascorbic acid (the main biologically active form of vitamin C), which acts as a biological antioxidant and can contribute to chronic disease prevention [
1,
9,
10]. In addition, a number of dietary fibers and pectin were reported [
11,
12], which are beneficial in bodyweight control, and could prevent the progression of type 2 diabetes and heart diseases [
13].
Phenolic compounds are a large and diverse group of molecules, in which the structural characteristics determine their biological activity. The antioxidant activity of phenols depends on the hydroxyl group number, and their configuration in B-ring [
14,
15]. Structural differences between phenols cause distinct mechanisms of actions against microorganisms, and consequently, their effectiveness [
16].
Numerous studies have shown that the phenolic compounds are promising biologically active compounds that may act as a new type of antimicrobial agent [
17,
18,
19]. Kikowska and co-authors demonstrated the antibacterial activity of JQ leaf and fruit extracts against four bacteria strains
Staphylococcus aureus (ATCC 25923),
Escherichia coli (ATCC 25922),
Pseudomonas aeruginosa (ATCC 27853), and one yeast strain
Candida albicans (ATCC 10231) [
20]. The antibacterial activity of other species such as
Chaenomeles speciosa essential oil against 10 microorganisms has been studied [
21]. However, a limited number of studies have reported the antibacterial activity of
Chaenomeles japonica species fruits.
The extraction efficiency of phenols depends on many conditions, including the solvent system, extraction time, temperature, ultrasound power, etc. [
22,
23,
24]. Response surface methodology (RSM) is a convenient tool to estimate several variables and their interaction influence on total phenolic content (TPC), and optimize the extraction conditions [
25,
26].
Currently, the cultivation of JQ is gaining popularity in northern European countries, especially in the Baltic Sea area [
27]. JQ is very diverse in plant and fruit characteristics, and its propagation by the seeds can cause morphological and biochemical heterogeneity. Breeding new cultivars change the genetic context and leads to morphological, physiological, and metabolic variations [
28]. Within the project “Japanese Quince—A new European fruit crop for the production of juice, flavor, and fiber” from 1998–2001, the thornless cultivars named ‘Darius’, ‘Rondo’, and ‘Rasa’ were released. The differences of the bio-compounds composition in leaves and seed oils of these cultivars were studied [
29,
30,
31]. Nevertheless, the biochemical composition and biological activity of their fruits have not yet been investigated.
This study aimed to optimize the phenols extraction conditions, determine the biochemical composition, antiradical, and antibacterial activity of Japanese quince cultivars ‘Darius’, ‘Rondo’, and ‘Rasa’, cultivated in Lithuania.
2. Materials and Methods
2.1. Plant Material
Fresh Japanese quince fruits (cvs. Darius, Rondo, and Rasa) were obtained from the garden of the Institute of Horticulture, Lithuanian Research Center for Agriculture and Forestry, Babtai (55°60′ N, 23°48′ E) Lithuania in 2018. The fruits were cut into slices, and lyophilized with a ZIRBUS sublimator 3 × 4 × 5/20 (ZIRBUS technology, Bad Grund, Germany) at the pressure of 0.01 mbar (condenser temperature, −85 °C). The slices were ground to powder by using a knife mill GRINDOMIX GM 200 (Retsh, Haan, Germany).
2.2. Maceration Extraction Method
First, 0.5 g of the powdered sample with 10 mL solvent in different concentrations (ratio 1:20, w/v) were mixed and left in the dark for 24 h at room temperature 22 °C. Then, the mixtures were centrifuged and filtered through a Whatman filter paper. Three different solvents (ethanol, methanol, and acetone) and three concentrations of each solvent (100%, 70%, and 50%) were used for the maceration extraction.
2.3. Ultrasound Extraction Method and Experimental Design
First, 0.5 g of the powdered sample was mixed with 10 mL 50% ethanol. The ultrasound extraction (UE) of phenolic compounds carried out using a Sonorex Digital 10 P ultrasonic bath (Bandelin Electronic GmbH & Co. KG, Berlin, Germany). Response surface methodology (RSM) was used to examine the influence of UE processing variables on phenols extraction. The impact of three factors (ultrasound power, extraction time, and temperature) on the response (TPC) was modeled according to a central composite design. Ultrasonic power ranged from 48 to 480 W and chosen according to the limitations of the ultrasonic device. The selected extraction temperature did not exceed 60 °C to avoid the degradation of compounds. The experimental design of the three-level-three-factor was composed; consisting of twenty experimental runs including six replicates at the center point. Design-Expert 7 (Stat-Ease Inc., Minneapolis, MN, USA) software was used for statistical analysis of the obtained data. The experimental results fit a first-order polynomial model to obtain the regression coefficients by Equation (1):
where Y is the predicted response (TPC),
X1,
X2, and
X3 meet the variables namely ultrasonic power, extraction time, and temperature, respectively. The β
0, β
1, β
2, and β
3 values represent their corresponding regression coefficients.
Design-Expert 7 software was used to draw up 3D response surface plots. To estimate the statistical significance of the proposed model, Fisher’s test for analysis of variance (ANOVA) was performed. Further optimized terms of the independent variables applied to approve the model and to compare predicted results to the experimental data.
2.4. Determination of Total Phenolic Content
TPC assessed spectrophotometrically using Folin–Ciocalteu reagent [
32]. The total phenol content is determined by the equation (y = 10.56X + 0.0189, r
2 = 0.997) of the calibration curve of gallic acid and expressed in mg/100 g, the equivalent of gallic acid for the dry raw material. The absorbance was measured using a Genesys-10 UV/Vis spectrophotometer (Thermo Spectronic, Rochester, NY, USA), at 765 nm wavelength.
2.5. Determination of Total Proanthocyanidins Content
Spectrophotometric measurements were scored using a Genesys-10 UV/Vis spectrophotometer (Thermo Spectronic, Rochester, NY, USA). Total proanthocyanidins were determined by applying the technique described by [
33]. Three mL DMCA solution (0.1% 4-dimethylamino cinnamaldehyde in methanol—HCl 9:1
v/
v) was mixed with 20 μL of the extract. A decrease in absorbance was determined at a wavelength of 640 nm after 5 min. The concentration of condensed tannins in the extract was calculated based on a calibration curve established with catechin as a standard (calibration curve: catechin (mg/100 g) = (y − 0.0066)/3.1312), r
2 = 0.995.
2.6. Antiradical Activity
The DPPH * free radical scavenging activity was determined using the slightly modified spectrophotometric method described by [
34]. Two mL DPPH (2,2-diphenyl-1-picrylhydrazyl) solution in 99.0%
v/
v ethanol was mixed with 20 μL of the extract. A decrease in absorbance was determined at a wavelength of 515 nm after storing the samples in the dark for 30 min at a ambient temperature. An ABTS + radical cation decolorization assay was applied according to the methodology described by [
35]. Then, 2 mL of ABTS (2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)) solution (absorbance 0.800 ± 0.02) was mixed with 20 μL of the extract. A decrease in absorbance was measured at a wavelength of 734 nm after storing the samples in the dark for 30 min. Results were expressed in μmol of Trolox equivalents in 100 g of dry extract and were calculated based on a calibration curve established using Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid).
2.7. Determination of Ascorbic Acid (Vitamin C) Content
Ascorbic acid (vitamin C) was measured by AOAC’s (Association of Official Analytical Chemists) official titrimetric method (AOAC, 1990) [
36].
2.8. Determination of Total Fibre Content
The total fiber content was determined using the enzymatic-gravimetric method, according to AOAC 985.29, 1997 [
37].
2.9. High Performance Liquid Chromatography (HPLC) Method for the Determination of Phenolic Compounds
A Waters e2695 chromatograph equipped with a Waters 2998 photodiode array detector (Waters, Milford, MA, USA) was used for the HPLC analysis according to the methodology described by [
38]. Chromatographic separations were carried out by using an YMC-Pack ODS-A (5 µm, C18, 250 × 4.6 mm i.d.) column equipped with a YMC-Triart (5 µm, C18, 10 × 3.0 mm i.d.) pre-column (YMC Europe GmbH, Dinslaken, Germany). The column operated at a constant temperature of 25 °C. The injection volume was 10 µL. The flow rate 1 mL/min, and gradient elution was used. The mobile phase consisted of solvent A-2% (
v/
v) acetic acid in water and solvent B-acetonitrile 100% (
v/
v). The following conditions of elution were applied: 0–30 min, 3–15% B; 30–45 min, 15–25% B; 45–50 min, 25–50% B; and 50–55 min, 50–95% B. The identification of the chromatographic peaks was achieved by the retention times and spectral characteristics (λ = 200–400 nm) of the eluting peaks with those of the reference compounds.
2.10. Preparation of Extracts for Antibacterial Testing
Twenty grams of freeze-dried quince fruit powder was mixed with 200 mL of 50% ethanol and extracted at the optimized condition. The extracts were filtered and dried in a rotary vacuum evaporator Büchi R-250, (Büchi Laboratortechnic, Flawil, Switzerland) to remove ethanol and later in a freeze-dryer ILShin FD 85125 (Ilshin Lab., Nam-myun, Yangju-si Gyeonggi-do, Korea) to remove the water. Dry extracts were kept in a freezer in hermetically sealed containers until used. Dry extracts were re-dissolved in 80% methanol to produce 0.5%, 1%, and 5% solutions, which were tested against microorganisms. The bacteria used in this study were stored at Micro-Bank (Pro-Lab Diagnostic, England) at −72 ± 3 °C before the start of the experiments. The bacteria were revitalized in the brain heart infusion broth (BHI, Oxoid, England) for 24 h, at the optimum temperature (30 ± 1 °C or 37 ± 1 °C). B. subtilis ATCC 6633 were grown on TSA (Liofilchem, Italy) agar slants for 24 h, at 30 °C. Enterococcus faecalis (ATCC 29212), Staphylococcus aureus (ATCC 25923), Escherichia coli (25922 ATCC), Pseudomonas aeruginosa (27853 ATCC), Salmonella enterica serovar Typhimurium (ATCC 14028) were grown on TSA agar slants for 24 h at 37 °C. C. albicans were grown on Sabouraud dextrose Liofilchem, (LD 610103) agar slants for 24–48 h at 25 °C.
2.11. Antimicrobial Activity Assay
The antimicrobial properties were evaluated by the agar well diffusion method according to the method described by [
39]. The bacteria were grown in peptone-soy bouillon (LAB 04, LAB M) for 24 h at 37 °C. After cultivation, culture cells were mixed using a mini shaker MS 1 (Wilmington, NC, USA.) and the cell suspensions were adjusted according to McFarland nr 0.5 standard [
40]. The cell suspensions of
C. albicans were adjusted according to McFarland nr 1.0 standard. Then, 1 mL of the suspension of bacteria cells was introduced into 100 mL dissolved plate count agar
Liofilchem (LD 610040), medium cooled to 47 °C. Then, 10 mL of the suspension was added into a 90-mm diameter Petri plate, the final concentration of cells in 1 mL was 1.5 × 10
6. Eight mm diameter wells in agar were filled with 50 µL of extracts. The plates were incubated overnight at 37 °C.
B. subtilis 30 °C,
C. albicans 25 °C, in Sabouraud dextrose agar, Liofilchem, (LD 610103). Then, the inhibition zones were measured with calipers to an accuracy of 0.5 mm. As a control in the blank sample, aqueous methanol (80%) was used.
2.12. The Statistical Methods
All the experiments repeated three times and the results were expressed as means ± SD. Data were submitted to the analysis of variance (ANOVA). Tukey’s HSD (honest significant difference test) was used to evaluate the significant differences (p ≤ 0.05) between means (multiple comparison test). The statistical analysis was performed using Statistica 10 software (StatSoft, Inc., Tulsa, OK, USA).