ABSTRACT
Fruits from five wild edible berries that grow in the eastern region of Shizuoka (Japan) were collected, and their antioxidant activities were evaluated using the ORAC, DPPH, FRAP, and ABTS assays to obtain basic scientific knowledge of these species. Among the Shizuoka wild berries tested, Rubus microphyllus L. fil. exhibited the highest antioxidant activity, which is equivalent to that of commercial raspberry (Rubus idaeus L.). Furthermore, anthocyanins from these wild berries were analyzed using quantitative 1H NMR spectroscopy. Cyanidin-3-glucoside and pelargonidin-3-glucoside were detected in Shizuoka wild berries, with the latter identified as the characteristic anthocyanin. The present study revealed that Shizuoka wild berries are rich in natural antioxidants and pigments and are therefore potentially beneficial for reducing the risks of associated with lifestyle-related diseases in consumers.
Introduction
Anthocyanidins are pigments responsible for the blue, purple, orange, and red colors of plants and exist primarily in their glycosidic (anthocyanin) forms. These anthocyanins are present in the tissues of colored fruits and vegetables (Smeriglio et al., Citation2016). Anthocyanins and anthocyanidins are believed to exert various biological effects that are beneficial to human health, including antioxidant, anticarcinogenic, anti-obesity, and antidiabetic activities (Azzini, Giacometti, and Russo Citation2017; Krga and Milenkovic, Citation2019). Owing to these effects, the regular consumption of anthocyanin-rich foods is believed to reduce the risks of developing lifestyle-related diseases (Tsuda, Citation2012; Zafra-Stone et al., Citation2007).
Berries, which contain large amounts of anthocyanins, are small, fleshy fruits that are typically consumed fresh or in products such as juice, jam, jelly, wine, and syrup. The anthocyanin contents of berries depend on the species, variety, cultivation, region, weather conditions, ripeness, and storage time (Ribeiro de Souza et al., Citation2019; Schulz et al., Citation2019). In addition to anthocyanins, berries contain other beneficial components, including vitamin C (ascorbic acid), phenolic acids, tannins, stilbenes, and flavonoids.
The fruits of bramble crops (Rubus spp.) and berry species are attracting increasing levels of interest owing to their potential beneficial health effects (Lopez-Corona et al., Citation2022; Martins et al., Citation2023). Most cultivars of Rubus species originate in Europe and the USA, although many wild species are native to Asia, including Japan. A previous study showed that anthocyanins in Rubus croceacantus and Rubus sieboldii wild berries from Okinawa, a southern district of Japan, exhibit antioxidant and anti-melanogenic activities (Kubota et al., Citation2012, Citation2014). Wild edible berries are also found in Shizuoka, which is located in the central region of Japan. In particular, while people consume wild berries as local fruit in the eastern area of Shizuoka (known as Izu), to the best of our knowledge, there are no reported analytical or functional studies of these wild berries.
With the above background in mind, the present study aimed to obtain basic scientific knowledge on wild berry species that grow in the eastern area of Shizuoka (Japan). Accordingly, we collected the fruits of five edible wild species and extracted them using different solvents. The antioxidant activities of the extracts were evaluated using oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) assays. Anthocyanins from these wild berries were analyzed and quantified using1H NMR spectroscopy. The scientific information on Shizuoka wild berries obtained in the present study is expected to benefit consumer health.
Material and Methods
Chemicals and Plant Materials
The Folin – Ciocalteu reagent, deuterated methanol, and trifluoroacetic acid (TFA) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Cyanidin-3-glucoside, cyanidin-3-rutinoside, cyanidin-3-sophoroside, cyanidin-3-xyloside, and pelargonidin-3-glucoside (as standards with purities greater than 95%, as determined by HPLC) were purchased from Funakoshi (Tokyo, Japan). Ripened berries (except blackberries and raspberries) were collected from the Izu area of Shizuoka in April 2012 (Figures S1 and S2). Blackberries (Chile) and raspberries (France) were obtained as frozen materials from SICOLY (St. Laurent d’Agny, France) in 2011. Voucher specimens of the wild berries used in this study were deposited at the Shizuoka Research Institute of Agriculture and Forestry. All berry samples were homogenized in liquid nitrogen and lyophilized using an EYELA FD-5N freeze-dryer (Tokyo Rikakikai, Tokyo, Japan). All freeze-dried powders were stored in a freezer (−30°C) until required for analysis.
Oxygen Radical Absorbance Capacity (ORAC) Assay
The ORAC assay was performed according to the method described by Singh et al. (Citation2012) with minor modifications. Lipophilic fractions were obtained by extracting freeze-dried samples (1 g each) with 1 : 1 (v/v) hexane : dichloromethane (10 mL). The supernatant was recovered by centrifugation at 1,000 ×g for 10 min. After being extracted twice, the solution was evaporated to dryness and maintained at 4°C until required for the lipophilic ORAC (L-ORAC) assay. The residue was dried under reduced pressure and extracted with 70 : 29.5 : 0.5 (v/v/v) acetone : water : acetic acid (AWA, 10 mL) to obtain the hydrophilic fraction. The AWA solution was sonicated for 5 min, and the supernatant was recovered by centrifugation at 1,000 ×g for 10 min. After extraction, the AWA solution was evaporated to dryness and maintained at 4°C. Extracts were dissolved in phosphate buffer (75 mM, pH 7.4) for the hydrophilic ORAC (H-ORAC) assay.
The extracts for the L-ORAC assay were dissolved in acetone and diluted 10-times with 7% randomly methylated β-cyclodextrin (Cyclodextrin Research and Development Laboratory, Budapest, Hungary) solution in 1 : 1 (v/v) acetone : water. Both H- and L-ORAC assays were carried out on a Flex Station II (Molecular Devices, Silicon Valley, CA, USA) equipped with an incubator set to 37°C. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Merck, Darmstadt, Germany) or the test sample solution (35 μL) was transferred to a 96-well plate and 94.4 nM fluorescein (115 μL, Wako Pure Chemical Industries, Osaka, Japan) was added to each well and incubated for 10 min. 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) (50 μL, 31.7 mM, Wako Pure Chemical Industries) diluted in 75 mM phosphate buffer (pH 7.4) was added to each well, and fluorescence was immediately measured (Ex. 485 nm, Em. 520 nm) at 2 min intervals for 90 min (H-ORAC) or 120 min (L-ORAC).
Individual H- and L-ORAC values were calculated using a quadratic regression equation that relates the Trolox or sample concentration to the net area under the fluorescein-decay curve. Linear regression was applied in the 6.25–50 µM Trolox range. Data were analyzed using SoftMax® Pro 4.7 (Molecular Devices). ORAC values are expressed as µmol of Trolox equivalents per gram of dry (lyophilized) weight (DW). Total antioxidant activity was calculated as the sum of the H-ORAC and L-ORAC values.
2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Assay
DPPH radical scavenging activities were evaluated according to a previously reported method (Kadowaki et al., Citation2023; Okumura et al., Citation2016). Freeze-dried samples (each 200 mg) were added to 80% MeOH containing 0.5% acetic acid (8 mL). The solution was sonicated and the supernatant was recovered by centrifugation at 4000 rpm for 10 min. The filtered residue was re-extracted until a clear solution was obtained (three times). After extraction, the filtrate was evaporated to dryness under reduced pressure. The filtrated extracts were re-dissolved in 50% EtOH for assaying purposes. The reaction mixture contained EtOH (1.5 mL), 0.1 mM DPPH, and the test sample (the extract of each sample). The absorbance at 517 nm was recorded after incubating for 30 min at room temperature in the dark. The control solution contained only EtOH and DPPH. Results are expressed as percentage decrease in absorbance with respect to the control value.
Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was carried out as previously described (Kadowaki et al., Citation2023; Moriya et al., Citation2015) using the FRAP reagent, which consist of 300 mM sodium acetate buffer (pH 3.6), 10 mM 2,4,6-tri-2-pyridyl-1,3,5-triazine in 40 mM hydrochloric acid, and 20 mM ferric chloride at a ratio of 10 : 1 : 1 (v/v/v). An aliquot (30 μL) of each extracted sample (prepared as described for the DPPH assay) diluted in 50% EtOH was transferred to a 96-well plate, after which the FRAP reagent (150 µL) was added to each well. Samples were then incubated at room temperature for 4 min and the absorbance at 593 nm was measured. Results were calculated as µmol Trolox equivalents per gram of DW.
2,2′-Azino-Bis(3-Ethyl-Benzothiazoline-6-Sulfonic Acid) (ABTS) Assay
ABTS radical cation-scavenging activity was measured according to a previously reported method (Bae et al., Citation2015) with minor modifications. ABTS was dissolved in water to a concentration of 7 mM. The ABTS radical cation was produced by reacting the ABTS stock solution with 140 mM potassium persulfate (final concentration) in the dark at room temperature for 12–16 h. This solution was diluted with EtOH to adjust its absorbance to 0.70 ± 0.02 at 734 nm. The samples (extracts) evaluated in the ABTS assay are the same as those used in the DPPH assay. An aliquot (30 µL) of each extracted sample (prepared as described for the DPPH assay) diluted in 50% EtOH was transferred to a 96-well plate, after which the ABTS solution (150 µL) was added to each well. The samples were then incubated at room temperature for 5 min after which absorbance at 734 nm was measured. Results are expressed as the percentage decrease in absorbance with respect to the control value.
Anthocyanin Analysis by NMR Spectroscopy
Berry extracts in 80% MeOH (prepared as described for the DPPH assay) were de-sugared using a Waters Sep-Pak C18 cartridge (Milford, MA, USA) prior to any NMR measurement. Each berry sample (1 g) was dissolved in water containing 0.1% TFA (3 mL). The sample solution was transferred to a C18 cartridge preconditioned with MeOH (3 mL) and water containing 0.1% TFA (3 mL). The column was washed with water containing 0.1% TFA (6 mL) and eluted with MeOH containing 0.1% TFA (3 mL). The solution was evaporated to dryness under a stream of nitrogen.
Quantitative NMR spectroscopy was used to determine the anthocyanin content for each berry (Hosoya, Kubota, and Kumazawa Citation2016; Turbitt et al., Citation2020). 1H NMR and LC-MS analyses of anthocyanins in berry samples were compared with those of authentic compounds and identified before quantifying the anthocyanins (data not shown). De-sugared berry extract was dissolved in deuterated methanol containing 5 mM DCl and 0.1% tetramethylsilane (600 μL). 1H-NMR spectra were recorded using a Bruker BioSpin AVANCE III 400 NMR spectrometer (Billerica, MA, USA) under the following conditions: temperature, 300 K; time domain size 65,536; spectral width, 20.55 ppm; pulse angle, 90°; acquisition time, 3.98 s; relaxation time, 4.00 s; and scan number, 256. The HDO signal was deleted using a NOESY pulse sequence (Giraudeau et al., Citation2015). Specific proton signals at the 4-position of anthocyanin (approximately δH 8.80–9.20) were observed in the1H NMR spectra; each such proton signal was assigned by hetero-nuclear single quantum coherence spectroscopy. The anthocyanin content of each berry sample was determined by quantifying this signal. ERETIC software (Bruker BioSpin) was used for quantitative NMR analyses (Akoka et al., Citation1999), and 1,4-bis(trimethylsilyl)benzene-d4 was used as the external standard.
Total Polyphenol Contents
The total polyphenol content of each berry sample was determined using the Folin – Ciocalteu method (Singleton, Orthofer, and Lamuela-Raventos Citation1999) with slight modifications. An aliquot (50 μL) of each extracted sample (prepared as described for the DPPH assay) diluted in 50% EtOH was transferred to a 96-well plate, after which the Folin – Ciocalteu reagent (50 μL) was added to each well. After 3 min, 10% Na2CO3 (50 μL) was added, and the absorbance at 760 nm was measured after 1 h of incubation at room temperature. The total polyphenol content was calculated as milligrams of gallic acid equivalents per 100 grams of DW.
Statistical Analysis
All data are expressed as the mean ± standard deviations (SDs). One-way analysis of variance (ANOVA) followed by Tukey’s test was used to determine how significant the differences between means at a 5% significance level.
Results
Figure S2 displays photographic images of the wild berry species used in this study, which include R. hirsutus Thunb., R. microphyllus L. fil., R. palmatus Thunb., R. trifidus Thunb., and R x medius Kuntze (the hybrid of R. microphyllus L. fil. and R. trifidus Thunb). Wild berries were collected in April 2012 from the eastern area of Shizuoka, Japan. Blackberries (Rubus fruticosus L.) from Chile and raspberries (Rubus idaeus L.) from France, two popular fruits of the Rubus genus, were used for comparison. Each berry sample was homogenized in liquid nitrogen and lyophilized using a freeze-dryer, and freeze-dried powdered samples were used in all experiments.
HPLC-mass spectrometry (LC-MS) was used to identify the anthocyanins in each berry sample by comparing them with authentic compounds (data not shown). However, quantifying anthocyanins in berries using HPLC was difficult owing to peak overlap. Therefore, we used the quantitative NMR technique to determine the anthocyanin contents of the various berry samples (Hosoya, Kubota, and Kumazawa Citation2016; Turbitt et al., Citation2020). The1H NMR spectrum of each anthocyanin displays a specific signal for the proton at the 4-position at approximately δH 8.80–9.20. We assigned the 4-position signals in the1H NMR spectra of the cyanidin and pelargonidin glycosides in Shizuoka wild berries based on comparisons with the spectra of authentic compounds (Figure S3), although unknown anthocyanin signals were observed in blackberry and raspberry spectra. The anthocyanin contents of the berries were determined by quantifying the assigned 1H NMR signals. The anthocyanin content per 100 grams of DW of each berry is shown in . R. hirsutus Thunb. exhibited the highest total anthocyanin content among the Shizuoka wild berries (8.63 ± 0.25 μmol/100 g of DW); however, blackberries and raspberries have even higher anthocyanin contents than the Shizuoka wild berries. In particular, blackberries contain high amounts of cyanidin-3-glucoside (60.50 ± 1.93 μmol/100 g of DW). The total polyphenol content of each berry was determined using the Folin – Ciocalteu method. Berries containing high amounts of anthocyanins also exhibited high total polyphenol contents. Although anthocyanins were not detected in R. palmatus Thunb., its total polyphenol content was determined to be 57.56 ± 1.28 mg gallic acid equivalents (GAE) per 100 grams of DW.
Table 1. Anthocyanin (μmol/100 g of dry weight) and total polyphenol (mg gallic acid equivalent/100 g of dry weight) contents of each berry.
The antioxidant activities of each berry, as determined by the ORAC, DPPH, FRAP, and ABTS assays, are summarized in . Among the five Shizuoka wild berries, R. microphylus L. fil. (8.1 ± 0.6 μmol Trolox equivalents (TE)/g of DW) exhibited the highest antioxidant activity as determined by the ORAC assay, followed by R. hirsutus Thunb., R. palmatus Thunb., R x medius Kunze, and R. trifidus Thunb. The other antioxidant assays (DPPH, FRAP, and ABTS) provided similar results. All antioxidant assays revealed that R. microphyllus L. fil. is the most active among the five Shizuoka berries tested, whereas R. trifidus Thunb. is the least active. reveals that R. microphyllus L. fil. also exhibited the highest total polyphenol content (66.88 ± 1.48 mg GAE/100 g of DW), with R. trifidus Thunb. exhibiting lowest (36.77 ± 0.43 mg GAE/100 g of DW).
Table 2. Antioxidant activity of each berry.
Discussion
In this study, we compared the anthocyanin profiles of five wild berries that grow in Shizuoka (Japan) and correlated the results with their antioxidant capacities. The anthocyanin contents of the various berries were determined by quantitative1H NMR spectroscopy. Cyanidin-3-glucoside and pelargonidin-3-glucoside were the major anthocyanins detected in Shizuoka berries (Figure S4). The total anthocyanin contents, including these anthocyanins, in all five Shizuoka wild berries are lower than those of blackberries and raspberries (). Interestingly, with the exception of R. palmatus Thunb., pelargonidin-3-glucoside was detected in all Shizuoka wild berries examined; however, this compound was not detected in blackberries and raspberries, despite these berries belonging to the same Rubus species. Although anthocyanins were not detected in R. palmatus Thunb., it exhibited a high total polyphenol content, which suggests that polyphenols other than anthocyanins are present in R. palmatus Thunb. Based on these results, we determined pelargonidin-3-glucoside to be the characteristic anthocyanin in Shizuoka wild berries. Pelargonidin-3-glucoside has also been reported in other wild Rubus species (Deighton et al., Citation2000; Kubota et al., Citation2012). We also confirmed the usefulness of the quantitative 1H NMR method for determining anthocyanin contents (Hosoya, Kubota, and Kumazawa Citation2016). shows that the anthocyanin content of each berry is easily obtained without the requirement for any specific sample pretreatment.
We used the ORAC, DPPH, FRAP, and ABTS assays to evaluate the antioxidant activities of five Shizuoka wild berries. The ORAC assay is considered to be an accurate method because it uses a biological relevant radical source (peroxyl) that enables the total antioxidant capacity to be measured by combining the antioxidant capacities of the hydrophilic and lipophilic fractions (Van Hung, Citation2016). In this study, R. microphyllus L. fil. exhibited the highest ORAC value (8.1 ± 0.6 μmol TE/g of DW) among the five Shizuoka wild berries; its ORAC value was higher than that of raspberries (5.5 ± 0.3 μmol TE/g of DW). R. microphyllus L. fil. exhibited the highest total polyphenol content (66.88 ± 1.48 mg GAE/100 g of DW) among the Shizuoka wild berries (). This result may be related with the total polyphenol contents and the antioxidant capacity.
The DPPH assay is a simple method for measuring the antioxidant capacity of fruit and vegetable juices or extracts (Sánchez-Moreno, Citation2002). The DPPH assay is mainly an electron-transfer assay; however, DPPH radicals can also be quenched to form DPPH-H. Therefore, the ABTS assay, which is another electron-transfer-based method, is also often used to assess food or its components (Blando et al., Citation2018; Huang, Ou, and Prior Citation2005). The ABTS assay measures the ability of an antioxidant to quench ABTS radicals, most likely through an electron-transfer reaction (Huang, Ou, and Prior Citation2005). We not only used the DPPH and ABTS assays but also the FRAP assay measures the potential of an antioxidant capacities of Shizuoka wild berries. The FRAP assay measures the potential of an antioxidant to reduce the yellow ferric-2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ) complex to the blue ferrous-TPTZ complex under acidic conditions (Nilsson et al., Citation2005). In this study, R. microphyllus L. fil. exhibited the highest activity according to the DPPH, FRAP, and ABTS assays (), which is consistent with the results obtained using the ORAC assay. Our results show that some Shizuoka wild berries quench DPPH and ABTS radicals and reduce the ferric complex to a ferrous complex. We used 50% EtOH as the diluent for the ABTS assay. However, 20 mM sodium acetate buffer (pH 4.5) reportedly improves the stability of the assay (Pehluvan, Çokran, and BozhÜyÜk Citation2018). In the future, we will consider using this buffer for the ABTS assay when studying fruits in larger wild populations.
In this study, we used blackberries and raspberries as comparative berry samples. Figure S3 reveals that several unknown signals were observed in the1H NMR spectra of these berries. We previously identified cyanidin-3-(6′-malonyl)glucoside and cyanidin-3-dioxaloylglucoside in blackberries, and cyanidin-3-sophoroside-5-rhamnoside and cyanidin-3-sambubioside-5-rhamnoside in raspberries as major anthocyanins (Ogawa et al., Citation2008). The unknown1H NMR signals observed in the spectra of blackberries and raspberries may be derived from those anthocyanins. Blackberries and raspberries have been reported to contain various polyphenols in addition to anthocyanins, such as flavonols and ellagitannins, that contribute to the antioxidant activities of these berries (Kaume, Howard, and Devareddy Citation2012; Tosun et al., Citation2009). Consequently, identifying polyphenols other than anthocyanins in Shizuoka wild berries is necessary.
Conclusion
Inhibiting lipid peroxidation via the antioxidants present in berries is crucial for mitigating the propagation of oxidative stress-related diseases in consumers (Mendes et al., Citation2011). Furthermore, the antioxidant activities of berries have been described as having consumer health benefits and therapeutic potential (Ramirez et al., Citation2015). In the present study, some Shizuoka wild berries exhibited antioxidant activities equivalent to those of blackberries and raspberries; consequently, Shizuoka wild berries may be beneficial for reducing the risks associated with lifestyle-related diseases in consumers.
Supplemental Material
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Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/15538362.2024.2348716
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