Hawthorn berry (Crataegus
L.) is a genus of over 1000 species, belonging to the subfamily Maloideae in the Rosaceae family, widely distributed in Asia and Europe [1
]. Hawthorn fruits contain high amounts of phenolic compounds, which are used as medicinal remedies with a variety of biological activities such as antitumor [2
], antispasmodic, cardiotonic, diuretic, hypotensive, anti-atherosclerotic [3
], and anti-inflammation [4
]. Several studies have shown that extracts of hawthorn fruit offer beneficial effects on the heart and also for blood circulation [5
]. The antioxidant and antimicrobial effects of phenolic compounds have been investigated among various commercial food products such as lamb burgers [6
], frankfurter-type sausages [8
], and pig liver pâté [9
Polyphenols, bioflavonoids, flavonoid glycosides, triterpenoids, oligomeric procyanidins, antioxidants, vitamins, tannins, organic acids, and some phenolic acids are the main active constituents of the Crataegus
]. The fruits of different Crataegus
species could be considered as a rich source of antioxidants, due to their high phenolic compositions and some well-known antioxidant compounds namely, hyperoside, isoquercetin, epicatechin, chlorogenic acid, quercetin, rutin, and protocatechuic acids [13
Environmental conditions, postharvest handling, and processing are among the factors that might have an influence on the physical characteristics, chemical composition of phenolic compounds, and their antioxidant activity [16
]. In addition, the amount of bioactive compounds such as flavonoids and phenolic acids is also affected by genetic variation among species, within the same species, and maturity of plant organs at its harvest [19
]. The variation in physicochemical characteristics, phytochemicals, and antioxidant activity among different species of Crataegus
revealed were well documented by previous studies [15
Although synthetic antioxidants and antimicrobial agents can effectively be used in food processing due to high stability and efficiency and low cost, there are significant concerns related to their potential health risks and toxicological aspects [24
]. Therefore, some research has been performed to evaluate the performance of natural antioxidants and antimicrobials such as essential oils and plant extracts as alternatives to synthetic antioxidants [25
]. On the other hand, limited sources of natural antioxidants and antimicrobials and high price as well as shortage of new sources of safe and inexpensive antioxidants and antimicrobials of natural origin could be a plausible reason for the food and pharmaceutical industries to use synthetic antioxidants instead [30
]. Thus, there is a growing interest in using natural compounds and their application in food, nutrition, and medical treatments [32
]. Iran is known as one of the primary centers of genetic diversity of Crataegus
; however, few studies have been carried out on the content of total phenolic compounds, antioxidant activity, and antimicrobial effects of this genus in Iran. In this regard, the current investigation was devoted to assess the physicochemical characterization (color parameters, pH, titratable acidity, total soluble solids, soluble carbohydrate, total carotenoid, total phenols, and flavonoid contents), antioxidant activity, and also quantification of some individual phenolic compounds of fruits of 15 samples of different hawthorn species (Crataegus
spp.) collected from different regions of Iran.
2. Materials and Methods
2.1. Plant Sample Collection
A total of 15 fruit specimens (Figure 1
) were collected from wild-growing Crataegus
species from 7 provinces of Iran (Table 1
), during 2015. The samples were transferred to the laboratory and physicochemical characteristics were measured in the shortest time possible.
2.2. 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.
2.3. 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.
2.4. 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 Na2
(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.
2.5. 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 (AlCl3
), 0.1 mL of 1 M potassium acetate (CH3
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
2.6. 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 FeCl3
O) were mixed. The 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 FeSO4
O standard solutions, covering the concentration ranging 100–1000 µmol/L and expressed as mmol Fe++
/g dry weight plant [13
2.7. 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.
2.8. 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: 0–5 min, 10% B; 5–15 min, 10–18% B; 15–25 min, 18% B; 25–30 min, 18–25% B; 30–35 min, 25% B; 35–40 min, 25–35% B; 40–45 min, 35–60% B; 45–50 min 60–10% B, and 50–55 min with 10% B. The temperature of the column was kept at 25 °C. The partial loop injection volume was 10 µL. The detection wavelengths of the DAD (diode array detectors) were set at three selected positions: 320, 335, and 360 nm [41
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.
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.