Physicochemical Characterization, Antioxidant Activity, and Phenolic Compounds of Hawthorn (Crataegus spp.) Fruits Species for Potential Use in Food Applications.

Hawthorn belongs to the Crataegus genus of the Rosaceae family and is an important medicinal plant. Due to its beneficial effects on the cardiovascular system and its antioxidant and antimicrobial activity hawthorn has recently become quite a popular herbal medicine in phytotherapy and food applications. In this study, physicochemical characterization (color parameters, pH, titratable acidity, total soluble solids, soluble carbohydrate, total carotenoid, total phenols, and flavonoid contents), antioxidant activity (by ferric-reducing antioxidant power, FRAP assay), and quantification of some individual phenolic compounds of fruits of 15 samples of different hawthorn species (Crataegus spp.) collected from different regions of Iran were investigated. According to findings, the total phenols, total flavonoid content, and antioxidant activity were in the range of 21.19–69.12 mg gallic acid equivalent (GAE)/g dry weight (dw), 2.44–6.08 mg quercetin equivalent (QUE)/g dw and 0.32–1.84 mmol Fe++/g dw, respectively. Hyperoside (0.87–2.94 mg/g dw), chlorogenic acid (0.06–1.16 mg/g dw), and isoquercetin (0.24–1.59 mg/g dw) were found to be the most abundant phenolic compounds in the extracts of hawthorn fruits. The considerable variations in the antioxidant activity and phenolic compounds of hawthorn species were demonstrated by our results. Hence, the evaluation of hawthorn genetic resources could supply precious data for screening genotypes with high bioactive contents for producing natural antioxidants and other phytochemical compounds valuable for food and pharma industries.


Preparation of Fruit Extracts
Fruits of each species were dried using convection oven at 45±2 • C for 24 h and ground to homogenize particle size before extraction. Powdered samples (1 g) were extracted by ultrasound (for 30 min at 25 • C) using methanol/water (80:20, 25 mL), then they were filtered.

Physicochemical Characterization
Total soluble solids (TSS), expressed as % malic acid, of fruits were measured by a handheld refractometer (model 9703, Japan) and titratable acidity (TA) by titration of fruit juice with 0.1 N NaOH to pH 8.3 and data were expressed as a percentage of malic acid. Juice pH of fruit samples was measured using a pH meter (Model 744, Metrohm). Water-soluble carbohydrate contents (TSC) of fruit samples were measured using the anthrone method [37]. Total carotenoids were extracted by acetone and measured by the spectrophotometric method. Absorbance at 662, 645, and 470 nm was used to determine their concentrations [38]. The color parameters of fruits such as a* (redness/greenness), b* (yellowness/blueness), and L* (whiteness/darkness) were measured by Hunter Lab (Hunter Associates Laboratory, VA, USA). Chroma (C) and hue (h • ) were also calculated from a* and b* coordinates.

Total Phenol Content (TPC)
TPC was assayed according to Singleton et al. [39]. The extracted samples (0.5 mL of different dilutions) were mixed with Folin-Ciocalteu reagent (5 mL, 1:10 diluted with distilled water) for 5 min and aqueous Na 2 CO 3 (4 mL, 1 M) was then added. The mixture was allowed to stand for 15 min, and the phenols were determined by spectrophotometer at 765 nm. The standard curve was prepared by 0, 50, 100, 150, 200, and 250 mg mL −1 solutions of gallic acid in methanol: water (50:50, v/v). Total phenols values are expressed in terms of gallic acid equivalent (mg GAE/g dry weight fruit), which is a common reference compound.

Total Flavonoid Content (TFC)
TFC of the fruits extracts was determined using the aluminum chloride colorimetric method with slight modification using quercetin as standard and the results were expressed as mg of quercetin equivalents per g dry weight of the plant (mg QUE/g dw). Briefly, the extract solution (0.5 mL) was mixed with 1.5 mL of 80% methanol, 0.1 mL of 10% aluminum chloride hexahydrate (AlCl 3 ), 0.1 mL of 1 M potassium acetate (CH 3 COOK), and 2.8 mL of deionized water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm against deionized water blank [40].

Antioxidant Activity
The antioxidant activity of hawthorn fruit extracts was calculated using ferric-reducing antioxidant power (FRAP) assay. Diluted extracts from different organs of hawthorn (100 µL) and 3.0 mL of freshly prepared FRAP-reagent (containing 25 mL of 300 mM acetate buffer, pH 3.6 plus 2.5 mL of 10 mM tripyridyltriazine stock solution in 40 mM HCl plus 2.5 mL of 20 mM FeCl 3 ·6H 2 O) were mixed. The Foods 2020, 9, 436 5 of 15 absorbance was recorded at 593 nm against a blank, containing 100 µL of resembling solvent, after 30 min incubation at 37 • C. The FRAP-value was calculated from the calibration curve of FeSO 4 ·7H 2 O standard solutions, covering the concentration ranging 100-1000 µmol/L and expressed as mmol Fe ++ /g dry weight plant [13].

Preparation of Standard Solutions
One milligram of a standard of each phenolic compound (chlorogenic acid, vitexin 2-O-rhamnoside, vitexin, rutin, hyperoside, isoquercetin, and quercetin; from Sigma, US) was weighed accurately and dissolved in 1:1 MeOH/water in a 10 mL volumetric flask to prepare the stock solution. For calibration curves, the stock solution was diluted with 1:4 MeOH/water to obtain the concentration sequence. Ten microliters of each solution was injected into HPLC. The linear range and the equations of linear regression were obtained through a sequence of 1000, 500, 250, 100, 50, 20, 10, 5, 2, and 1 mg/L. Mean areas (n = 3) generated from the standard solutions were plotted against concentration to establish calibration equations.

Quantification of Phenolic Compounds
Quantification of some individual phenolic compounds (i.e., chlorogenic acid, vitexin 2"-O-rhamnoside, vitexin, rutin, hyperoside, quercetin, and isoquercetin) by high-pressure liquid chromatography (HPLC) was carried out using a Knauer HPLC apparatus consisting of a 1000 Smartline pump, a 5000 Smartline manager solvent organizer, and a 2800 Smartline photo-diode array detector. The injection was carried out through a 3900 Smartline autosampler injector equipped with a 100 µL loop. The temperature control of the column was made with a jet stream 2 plus oven (Knauer, advanced scientific instrument, Berlin, Germany). The separation was achieved on an Eclipse XDB-C18 (4.6 mm × 250 mm, 5 µm), Agilent (USA) column. Data acquisition and integration were performed with EZChrome Elite software. The flow rate of the mobile phase was kept at 1 mL/min. Solvent A was water containing formic acid (0.05%), and Solvent B was acetonitrile/methanol (80:20, v/v). The gradient conditions were as follows 2.9. Statistical Analysis SAS 9.1.3 software package (v.9, SAS Institute, USA) was used for statistical analysis of the data. All of the analyses were done in triplicate with an experiment in a completely randomized design. The Duncan test was used to compare pairs of means and determine statistical significance at the (P < 0.05) level. Furthermore, hierarchical cluster analysis (HCA) and principal component analysis (PCA) was performed among the variables analyzed using Minitab software. Heat-maps were used to visualize phenolic compounds in each species using GraphPad Prism software.

Physicochemical Characterization
The fruit's external color was significantly variable amongst the different species of Crataegus (P < 0.001). The color range of hawthorn fruits is varied from yellow to black (yellow, yellow-orange, red, orange-red, purple, purple-black, and black). The highest a * value (40.63) was obtained from Crataegus atrosanguinea species, while b * values (56.93), L * value (37.36), C (61.15), h • (77.78), were highest in the extracts of Crataegus azarolus var. aronia. The color characteristics of Crataegus spp. fruits are given in Table 2.
Results demonstrated that origin of species had significant effects (P < 0.001) regarding the chemical characteristics (pH, titratable acidity, total soluble solids, total soluble carbohydrate, and total carotenoid content) of hawthorn fruits ( Table 3). The pH level of fruits was recorded in the range of 3.03-4.35. The pH was at its highest value in Crataegus curvisepala, whereas the lowest level correlated with Crataegus orientalis. The highest levels of acidity (TA) were observed in yellow fruits of hawthorn. The highest (1.17%) and lowest (0.75%) TA were obtained from Crataegus azarolus var. pontica and Crataegus pentagyna species, respectively. The highest total soluble solids (TSS) of fruits (23.43 • Brix) was found in C. azarolus var. pontica, and the lowest (14.99 • Brix) occurred in C. pentagyna. The total soluble carbohydrate was at its highest value (19.43%) in C. azarolus var. aronia, whereas the lowest level (5.27%) was found in Crataegus monogyna. The highest total carotenoid content (405.79 µg/g fruit weight) was obtained from C. azarolus var. pontica species.  The physicochemical characteristics of fruits are important indicators of their quality and maturation; key factors for achievement of market demands that have encouraged many researchers under different conditions overseas. These traits can be used for the evaluation of diversity and release of a new cultivar. Therefore, some investigations suggested that the physicochemical characteristics of hawthorn fruit are influenced by species and collection location (origin) [17,[42][43][44].

Total Phenol Content (TPC)
The TPC of fruits of hawthorn species is presented in Table 4. The amount of TPC of hawthorn fruits was significantly variable (P < 0.001) among species, ranging from 21.19 to 69.12 mg GAE/g dry weight. Total phenol content was at its highest value in the fruits of C. pentagyna, whereas the lowest level was found in the fruits of Crataegus turkestanica. According to results, TPC can be significantly influenced by both the species and also the sampling location. Accordingly, some studies proposed that the polyphenolic content of plant organs is influenced by genotype and habitat conditions [43], and moreover, altitude, light, temperature, and content of nutritive matter available in the soil may influence phenylpropanoid metabolism [45]. The time of harvesting (the stage of maturity) is also can be accounted as a very important factor. Similar findings were also obtained in term of the total phenol content i.e., 2.9 mg GAE/g dw for Crataegus pinnatifida [46], and 26.4 mg GAE/g dw for C. monogyna [47]. In another study, the total content of polyphenols in fruits of C. pinnatifida was 96.9 ± 4.3 mg gallic acid equivalents/g weight [48]. The health and technological benefits associated with plant compounds in value-added food products had been attributed to the antioxidant and antimicrobial activity of phenols content [49,50].  Table 4 shows the TFC in fruits of hawthorn. The content of total flavonoids was significantly variable (P < 0.001) among species and ranging from 2.44 to 6.08 mg QUE/g dw. Total flavonoid content was highest in Crataegus meyeri, whereas the lowest level was found in the fruits of Crataegus szovitsii. The TFC is influenced by the interaction between species and sampling location. Furthermore, environmental factors have a significant contribution to the total flavonoid content in plants. The total flavonoid content found in the present study was similar to those reported for other hawthorn species in previous studies, i.e., 1.47 mg/g dw for C. monogyna fruits [51], 23.68 mg/g dw for C. pentagyna fruits [52], and 0.81 mg/g dw for C. azarolus fruits [53].

Antioxidant Activity
The antioxidant activity was widely varied (P < 0.001) in species of Crataegus, ranging from 0.32-1.84 mmol Fe ++ /g dw ( Table 4). The highest antioxidant activity was observed in the fruits of C. pentagyna, whereas the lowest activity was found in the fruits of Crataegus persica. The evaluation of antioxidant activity of Crataegus species demonstrated that they could possess considerable antioxidant activities due to the presence of polyphenolic compounds. Moreover, the total and individual phenolic compounds are the main responsible agents for the antioxidant activity of hawthorn fruits. Among them, chlorogenic acid, hyperoside, rutin, spiraeoside, quercetin 3-glucoside (isoquercetin), quercetin, (-)-epicatechin, and procyanidin B2 were suggested to be the compounds with strong radical-scavenging activity in floral bud extracts of hawthorn [54]. The ethanol extract of C. monogyna fruits contained higher levels of phenolic compounds and showed greater radical-scavenging activities than the aqueous extract of the fruits [47]. Most of the reports regarding antioxidant activity of Crataegus species were correlated with either fruits, aerial parts, or flowers of the plant [55].
Oxidation can affect the sensory attributes, nutritional value, texture properties, and shelf life stability of food by decomposition of proteins, vitamins, unsaturated essential fatty acids, and pigments such as anthocyanin, carotenoid, and myoglobin [56]. A significant relation between phenol content and antioxidant activity of plant constituents has been reported by Agregan et al [57] and Roselló-Soto et al. [58]. In addition, some studies have revealed that plant compounds are a natural antioxidant that can significantly reduce lipid oxidation, for example, in ground beef with four garlic-derived compounds [59], pork patties with natural antioxidant [60], rabbit meat with oregano, rosemary, and vitamin E [61], roast beef patties with chili pepper extract [62], frankfurter-type sausage with combined effect of mixed plant extracts (green tea, stinging nettle, and olive leaves extracts) and natural antimicrobial agents [8]. Table 5 summarized the proximate composition of phenolic compounds in all the 15 species analyzed. The amounts of phenolic compounds were significantly variable amongst different species (P < 0.001). In this regard, hyperoside, chlorogenic acid, and isoquercetin were found to be the most abundant phenolic compounds in the extracts of hawthorn fruits. However, in most species, vitexin 2"-O-rhamnoside was not detected and the quercetin content was very low. The heat map in Figure 2 can aid us to summarize quantitative data regarding the phenolic compound distribution in fruits of hawthorn species. Color was associated with the content of phenolic compounds: from white for low concentrations to blue for high concentrations. Crataegus pseudomelanocarpa had the highest level (1.16 mg/g dw) of chlorogenic acid, and C. meyeri had the lowest level (0.06 mg/g dw) among the fruits of the studied species. Crataegus pseudoheterophylla had the highest content (0.17 mg/g dw) of vitexin 2-O-rhamnoside among the species studied. In most species, vitexin 2-O-rhamnoside was not detected. Vitexin was in the highest value (0.31 mg/g dw) in C. szovitsii whereas the lowest level (0.06 mg/g dw) was found in Crataegus sakranensis among the fruits of the studied species. C. pseudomelanocarpa had the highest level (2.68 mg/g dw) of rutin among the fruits of the studied species. 2.05 ± 0.05 d 0.98 ± 0.04 c 0.04 ± 0.01 a Significant level *** *** *** *** *** *** ns Note: *** and ns, Significant at 0.1% level and not significant, respectively, means with different letters are statistically significant at a 5% level of probability. Hyperoside and isoquercetin were at the highest value (2.94 and 1.59 mg/g dw), respectively, in the C. meyeri species. The present study showed that total and individual phenolic compounds are the main contributor to the antioxidant activity of hawthorn fruits, which also can be influenced by the variation of fruits species. Moreover, several environmental factors affect the concentration of phenolic compounds in plants [63]. In this context, it was reported that higher growing temperatures and level of CO 2 increase flavonoid content and concentrations of the phenolic compounds [64]. Furthermore, soil conditions affect plant phenolic composition. Soil fertilization (such as high level of nitrogen) and increase in soil moisture deficit led to the lower synthesis and hence lower levels of some certain phenolics [65]. Moreover, light stimulates the synthesis of phenolic compounds such as flavonoids, flavones, anthocyanins, and also PAL (phenylalanine ammonia-lyase) enzyme. Hyperoside and isoquercetin were at the highest value (2.94 and 1.59 mg/g dw), respectively, in the C. meyeri species. The present study showed that total and individual phenolic compounds are the main contributor to the antioxidant activity of hawthorn fruits, which also can be influenced by the variation of fruits species. Moreover, several environmental factors affect the concentration of phenolic compounds in plants [63]. In this context, it was reported that higher growing temperatures and level of CO2 increase flavonoid content and concentrations of the phenolic compounds [64]. Furthermore, soil conditions affect plant phenolic composition. Soil fertilization (such as high level of nitrogen) and increase in soil moisture deficit led to the lower synthesis and hence lower levels of some certain phenolics [65]. Moreover, light stimulates the synthesis of phenolic compounds such as flavonoids, flavones, anthocyanins, and also PAL (phenylalanine ammonia-lyase) enzyme. In general, variability in the reported phenolic compound contents and flavonoid concentrations within one species could be mainly related to differences in growth conditions [45], genetic background [66], and methodological differences [67].

Hierarchical Cluster Analysis and Principal Component Analysis
To evaluate the relationships and likely similarities among Crataegus species studied, hierarchical cluster analysis (HCA) was performed based on the 10 main traits (TPC, TFC, FRAP, CHA, VOR, VIT, RUT, HYP, ISOQ, and QUE). The cluster analysis was carried out by the Ward linkage method for agglomeration and the Euclidean distance as the criterion of proximity ( Figure  3A). The resulting dendrogram had two major groups based on similarity. Each group was also divided into two subgroups. The first association consisted of five species (C. pentagyna, C. pseudomelanocarpa, C. atrosanguinea, C. pseudoheterophylla, and C. meyeri,) with high TPC, TFC, HYP, ISOQ, and antioxidant activity of fruit. It was shown that subgroup 1 species (C. pentagyna and C. pseudomelanocarpa) have higher RUT compound than the subgroup 2 species. The second association consisted of ten species with medium and low TPC, TFC, antioxidant activity, and other phenolic In general, variability in the reported phenolic compound contents and flavonoid concentrations within one species could be mainly related to differences in growth conditions [45], genetic background [66], and methodological differences [67].

Hierarchical Cluster Analysis and Principal Component Analysis
To evaluate the relationships and likely similarities among Crataegus species studied, hierarchical cluster analysis (HCA) was performed based on the 10 main traits (TPC, TFC, FRAP, CHA, VOR, VIT, RUT, HYP, ISOQ, and QUE). The cluster analysis was carried out by the Ward linkage method for agglomeration and the Euclidean distance as the criterion of proximity ( Figure 3A). The resulting dendrogram had two major groups based on similarity. Each group was also divided into two subgroups. The first association consisted of five species (C. pentagyna, C. pseudomelanocarpa, C. atrosanguinea, C. pseudoheterophylla, and C. meyeri,) with high TPC, TFC, HYP, ISOQ, and antioxidant activity of fruit. It was shown that subgroup 1 species (C. pentagyna and C. pseudomelanocarpa) have higher RUT compound than the subgroup 2 species. The second association consisted of ten species with medium and low TPC, TFC, antioxidant activity, and other phenolic compounds of fruit. It was shown that subgroup 2 species (C. azarolus var. aronia, C. azarolus var. pontica, and C. turkestanica) have the lowest TPC compared to subgroup 1 and other species.  Principal component analysis (PCA) was applied, in order to classify the species studied according to the traits described above. PCA classification confirmed the results of cluster analysis ( Figure 3B). A PCA was performed, reducing the multidimensional structure of the data and providing a two-dimensional map to explain the variance observed. The first two components of the PCA explained 58% of the total variance (43% for component 1 and 15% for component 2). The first component (PC1) was highly positively correlated with TPC, TFC, FRAP, HYP, ISOQ, and RUT. The second principal component (PC2) separated the samples according to CHA, VOR, VIT, and QUE compounds. Principal component analysis (PCA) was applied, in order to classify the species studied according to the traits described above. PCA classification confirmed the results of cluster analysis ( Figure 3B). A PCA was performed, reducing the multidimensional structure of the data and providing a two-dimensional map to explain the variance observed. The first two components of the PCA explained 58% of the total variance (43% for component 1 and 15% for component 2). The first component (PC1) was highly positively correlated with TPC, TFC, FRAP, HYP, ISOQ, and RUT. The second principal component (PC2) separated the samples according to CHA, VOR, VIT, and QUE compounds.

Conclusions
The results of the present study demonstrated that total and individual phenolic compounds are the main contributor to the antioxidant activity of hawthorn fruits. Hyperoside, chlorogenic acid, and isoquercetin were found to be the most abundant phenolic compounds in the extracts of hawthorn fruits. To the best of our knowledge, this is the first report regarding antioxidant activity and determination of phenolic compounds (chlorogenic acid, vitexin 2"-O-rhamnoside, vitexin, rutin, hyperoside, quercetin, and isoquercetin) in fruits of Crataegus species grown in Iran. The fruits of different Crataegus species (especially C. pseudomelanocarpa and C. pentagyna) showed a high level of total phenol content as well as antioxidant activity. As a conclusion, our results clearly demonstrate the considerable variation in the antioxidant activity and phenolic compounds of hawthorn species. Hence, the evaluation of hawthorn genetic resources could supply precious data for screening genotypes with high bioactive contents for producing natural antioxidants and other phytochemical compounds valuable for food and pharma industries.