Research Article

Korean Journal of Plant Resources. 1 June 2024. 235-146
https://doi.org/10.7732/kjpr.2024.37.3.235

ABSTRACT


MAIN

  • Introduction

  • Materials and Methods

  •   Plant materials

  •   Climate data

  •   Extraction of plant components

  •   1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity

  •   ABTS radical scavenging activity

  •   Ferric reducing antioxidant power (FRAP) assay

  •   Total phenolic content

  •   Total flavonoid content

  •   Total anthocyanin content

  •   Statistical analysis

  • Results and Discussion

  •   Comparisons of the antioxidant activity, total phenolic content, and total flavonoid content of Vaccinium oldhamii

  •   Antioxidant activity of Vaccinium oldhamii according to the location of collection, and a comparison with that of blueberries

  •   Comparisons of the total phenolic content, total flavonoid content, and total anthocyanin content of Vaccinium oldhamii, according to the location of collection, and a comparison with blueberries

  •   Relationships of the antioxidant activity, total phenolic content, total flavonoid content and total anthocyanin content with environmental conditions

  •   Results of the hierarchical clustering analysis for V. oldhamii fruit, according to region

Introduction

As human life expectancy has increased, there have been significant increases in the prevalences of chronic diseases, such as diabetes, dementia, cancer, and cardiovascular disease, which are often referred to collectively as lifestyle-related diseases (Tanaka et al., 2000). These increases have been attributed to an abnormal increase in the production of reactive oxygen species (ROS), because of factors such as environmental pollution, westernized dietary habits, alcohol consumption, and smoking (Wiseman, 1996).

For the prevention of ROS-induced damage, there has been a growing interest in antioxidants. While synthetic antioxidants including butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have been predominantly used therapeutically, concerns have arisen regarding their potential side effects. This has led to greater interest in the possibility of administering natural antioxidants (Amarowicz and Raab, 1997; Fang et al., 2002; Gowri and Vasantha, 2010; Lee et al., 2021).

Alongside the growing interest in antioxidants, the consumption of berries has also been increasing (An et al., 2015). Blueberries, a representative type of berry, belong to the Ericaceae family, Vaccinium genus, and are primarily found in North America. They have been reported to have various health benefits, including antioxidant effects (Sellappan et al., 2002), anti-cancer effects (Seeram et al., 2006), anti-diabetic properties (Martineau et al., 2006), and potential anti-dementia effects (Papandreou et al., 2009). They have gained recognition as one of the top ten foods that prolong longevity globally, and the commercial cultivation of blueberries began in South Korea after 2000 (Chae, 2021).

Crop wild relatives (CWRs) are wild plant species that are closely related to cultivated crops and have evolved to survive despite environmental challenges such as dry conditions, high humidity, high temperatures, and poor soil. These can serve as a source of new genetic diversity for the breeding of more resilient crops. There has been a global initiative to conserve CWRs since 2011 that has prioritized the collection, preservation, pre-breeding, and information systems for wild plant genetic resources. The genetic resources associated with indigenous crops, including the “native blueberry,” Vaccinium oldhamii Miq, have been conserved in South Korean forests since 2021.

V. oldhamii, a species in the Ericaceae family and Vaccinium genus, is a deciduous shrub that grows to 1~4 m in height. It is primarily found in China, and Japan, and particularly in South Korea, south of Gyeryongsan, but also along the western coast, extending to Anmyeondo (Chung and Hyun, 1989). V. oldhamii produces flowers with 5~15 yellowish-red or reddish-brown racemes between May and June, and then yields black or purplish, round fruits (4~6 ㎜) between September and October (Plants of the World Online, 2022).

A recent study (Chae, 2021) showed that V. oldhamii has a higher content of polyphenols, greater electron-donating ability, and greater ABTS+ radical scavenging ability than blueberries, which suggests that it could be used as a native blueberry alternative in functional food products. In the study by Kim et al. (2019), the pharmacological efficacy of V. oldhamii, indicated its potential for treating inflammation and bone disorders by regulating inflammation, osteoclastogenesis, and NF-κB and MAPK signaling in branches. According to Sekizawa et al. (2013), the fruit extract of V. oldhamii demonstrated inhibitory effects on the initial stages of Influenza virus (IFV) infection. Tsuda et al. (2013) reported that the fruit extract of V. oldhamii showed growth inhibitory effects on HL-60 human leukemia cells. In the research by Park and Kim (2005), four compounds were isolated from the MeOH extract of V. oldhamii branches in the ethyl acetate fraction, including (+)catechin, (-)-epicatechin, proanthocyanidin A-2: epicatechin-(2β→7, 4β→8)-epicatechin, and cinnamtannin B1: epicatechin-(2β→7, 4β→8)-epicatechin-(4α→8)-epicatechin. These compounds, mainly belonging to the catechin and proanthocyanidin groups, are reported to possess antioxidant and anti-inflammatory properties. These research findings suggest the diverse potential medicinal applications of V. oldhamii.

In the present study, we compared the antioxidant activities of V. oldhamii fruit collected from 11 different regions of South Korea and blueberries imported from three countries. This allowed us to compare the antioxidant activity and the contents of bioactive compounds of this plant, according to its growth environment. In this way, we aimed to provide fundamental data to facilitate the future development of superior varieties and the potential use of V. oldhamii as a functional ingredient.

Materials and Methods

Plant materials

The fruit of V. oldhamii used in the study were collected from 11 different regions of South Korea between September and October 2022 (Fig. 1, Table 1); Chungcheong-do (Nonsan, Buyeo, and Taean), Jeolla-do (Gwangju, Haenam, and Gwangyang), Gyeongsang-do (Hamyang, Gumi, and Gimcheon), and Jeju-do (Jeju and Seogwipo). In addition, the fruit of domestically-grown, U.S.-grown, Canadian-grown, and Chilean-grown blueberries that were available commercially were purchased and used for comparison. The fruits were freeze dried, ground into powder using a liquid nitrogen-cooled mill, and stored at −20°C until use.

https://static.apub.kr/journalsite/sites/kjpr/2024-037-03/N0820370302/images/kjpr_2024_373_235_F1.jpg
Fig. 1.

The 11 regions of Vaccinium oldhamii Miq. samples collected for the study.

Table 1.

The 11 regions of Vaccinium oldhamii Miq. samples collected for the study.

No. Locality Collect date
1 Jungsan-ri, Yangchon-myeon, Nonsan-si, Chungcheongnam-do, Republic of Korea 2022.9.7
2 Bokgeum-ri, Chunghwa-myeon, Buyeo-gun, Chungcheongnam-do, Republic of Korea 2022.9.7
3 Gonam-myeon, Taean-gun, Chungcheongnam-do, Republic of Korea 2022.9.7
4 Samsan-myeon, Haenam-gun, Jeollanam-do, Republic of Korea 2022.10.6
5 Doggok-ri, Ongnyong-myeon, Gwangyang-si, Jeollanam-do, Korea 2022.9.12
6 Ungok-ri, Seoha-myeon, Hamyang-gun, Gyeongsangnam-do, Republic of Korea 2022.9.13
7 Geumosan-ro, Gumi-si, Gyeongsangbuk-do, Republic of Korea 2022.9.23
8 1100-ro, Jeju-i, Jeju-do, Republic of Korea 2022.9.28
9 Jungmun-dong, Seogwipo-si, Jeju-do, Republic of Korea 2022.9.29
10 Daehang-myeon, Gimcheon-si, Gyeongsangbuk-do, Republic of Korea 2022.10.6
11 Ullim-dong, Dong-gu, Gwangju, Republic of Korea 2022.10.7

Climate data

The mean temperature, maximum temperature, minimum temperature, daily temperature range, mean wind speed, and mean humidity of the 11 regions between March and September 2022 were obtained from the Korea Meteorological Administration’s Weather Data Open Portal (https://data.kma.go.kr/) and compared.

Extraction of plant components

The fruit samples underwent freeze-drying for 72 hr, then the dried samples were finely ground, and 70% ethanol solution (Macron Fine Chemicals, Randor, PA, USA) was added at a ratio of 20 times the mass of each dried sample (w/v). This mixture was then extracted at room temperature for 48 h in a maceration extractor.

The yield of each extract was calculated on the basis on the mass of each dried sample. The generated seed extracts were filtered using a 0.45 ㎛ syringe filter (PVDF syringe filter, 13 ㎜, Futecs, Seoul, Korea) and then concentrated using a vacuum centrifugal concentrator (CVE-3110, Eyela, NY, USA) to completely remove the solvent. The concentrated extract was then dissolved in dimethyl sulfoxide (DMSO) (Sigma, Saint Louis, MO, USA) to achieve a concentration of 30,000 ppm and stored at −20°C. It was further diluted according to the requirements of the experimental protocol.

To measure the total anthocyanin content, a mixture of methanol 99.8% (Avantor, J. T. Baker, USA), hydrochloric acid (Titripur, Germany), and distilled water at a 60:1:30 ratio was added to the powdered samples. The mixtures were then extracted at room temperature for 24 hr, the extracts were filtered through a PVDF syringe filter (0.45 ㎛), and the resulting solutions were used in the experiments.

1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity

The DPPH radical scavenging activity was evaluated using a modification of Blois’s method (Blois, 1958). In this assay, 50 μL of fruit extract was mixed with 150 μL of 0.1 mM DPPH (Alfa Aesar, Haverhill, MA, USA) radical solution and allowed to react for 30 min at room temperature. After this, the absorbance of the reaction mixture was measured at 517 ㎚ using a microplate reader (EPOCH2, Biotek, USA). To determine the DPPH radical scavenging activity, a blank was prepared by adding DMSO (the solvent used for the dilution of the extracts) instead of the fruit extract, and its absorbance was also measured. The DPPH radical scavenging activity was calculated as a percentage of the DPPH radical reduced by the extract using the following formula:

DPPHradicalscavengingactivity(%)=(1Absorbanceofthetestsample/Absorbanceoftheblank)×100

The RC50 value, representing the concentration of seed extract required to scavenge 50% of the DPPH radicals, was determined using this formula. Ascorbic acid (Fujifilm Wako Pure Chemical Co., Osaka, Japan) was used as a positive control.

ABTS radical scavenging activity

The ABTS radical scavenging activity was evaluated using a modification of the method developed by Miller et al. (Miller et al., 1993). To prepare the ABTS solution, 20 ㎎ of ABTS diammonium salt (Sigma Chemical Co.) was added to 5 mL of distilled water containing 88 µL of 140 mM potassium persulfate (Acros Organics, Geel, Belgium). This mixture was left in the dark at room temperature for 14–16 hr until an absorbance of 0.70 ± 0.02 was achieved at 734 ㎚ after dilution with ethanol. For the assay, 10 μL aliquots of fruit extract were mixed with 190 μL aliquots of the prepared ABTS solution in a 96-well plate. The reaction was allowed to proceed for 2 min 30 sec, after which the absorbances of the reaction mixtures were measured at 734 ㎚ using a microplate reader. A blank was prepared as for the DPPH assay: DMSO (the solvent used for the dilution of the extracts) was added instead of the fruit extract, and its absorbance was measured. The ABTS radical scavenging activity was calculated as a percentage of the reduction in ABTS radicals by the extract using the following formula:

ABTSradicalscavengingactivity(%)=(1Absorbanceofthetestsample/Absorbanceoftheblank)×100

The RC50 value, representing the concentration of the fruit extract required to scavenge 50% of the ABTS radicals, was determined using this formula. Ascorbic acid (Fujifilm Wako Pure Chemical Co.) was used as a positive control.

Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was conducted using a modification of the method developed by Molina-Díaz et al. (1998). In order to prepare FRAP solution, a mixture of 40 mM HCl (Fujifilm Wako Pure Chemical Co.), 10 mM 2,4,6-tris (2-pyridyl)-S-triazine (TPTZ), and 20 mM iron chloride (Sigma Chemical Co.) at a ratio of 10:1:1 was mixed in a 300 mM acetate buffer ㏗ 3.6 (Samchun, Korea, Pyeongtaek) solution. For the assay, 10 μL of the seed extract was mixed with 200 μL of the prepared FRAP solution, the reaction was allowed to proceed for 4 min at 37°C, and the absorbance of the reaction mixture was measured at 593 ㎚ using a microplate reader. The FRAP value is expressed in terms of the capacity of Fe (II) and was determined using a calibration curve created by preparing dilutions of a ferrous sulfate heptahydrate (Sigma Chemical Co.) standard.

Total phenolic content

The total phenolic contents of the samples were measured using a modification of the method developed by Singleton et al. (1999). In this assay, 20 μL of the fruit extract was mixed with 40 µL of 0.4 N Folin & Ciocalteu’s phenol reagent (Sigma Chemical Co.) dissolved in distilled water and allowed to stand for 5 min. Next, 140 μL of 700 mM Na2CO3 (Daejung, Siheung-si, Korea) was added to the mixture, and the reaction was allowed to proceed at room temperature for 2 hr. The absorbance of the reaction mixture was then measured at 765 ㎚ using a microplate reader.

To determine the total phenolic content, a calibration curve was created using gallic acid (Sigma Chemical Co.) as a standard substance. The total phenolic content in the fruit extract was expressed in terms of gallic acid equivalents (GAEs).

Total flavonoid content

The total flavonoid contents of the samples were measured using a modification of the method developed by Christ and Müller (1960). In this assay, 100 μL of fruit extract was mixed with 300 μL of ethanol (Daejung, Siheung-si, Korea), 20 μL of 1 M potassium acetate (Junsei Chemical Co., Tokyo, Japan), 20 μL of 10% aluminum chloride (Junsei Chemical Co., Tokyo, Japan), and 560 μL of distilled water. After mixing, the reaction was allowed to proceed for 30 min at room temperature, then 200 μL aliquots of the mixtures were transferred to wells of a 96-well plate, and the absorbances of the reaction mixtures were measured at 415 ㎚ using a microplate reader. A calibration curve was created using quercetin (Sigma Chemical Co.) as a standard. The total flavonoid content of each fruit extract is expressed in terms of quercetin equivalents (QEs).

Total anthocyanin content

The total anthocyanin contents of the samples were measured using an adapted pH differential method based on the AOAC method (Lee, 2005). For this, 1 mL of each extract was mixed with 1 mL of potassium chloride buffer 0.025 M (㏗ 1.0) and 1 mL of sodium acetate buffer 0.4 M (㏗ 4.5). The absorbances of the mixtures were measured at 510 ㎚ and 700 ㎚ using a microplate reader. Dilutions of the extracts were prepared to ensure that the absorbances fell within the range of 0.2 to 1.4, and the absorbances were measured between 20 and 50 min after the start of the reaction. The standard used was cyanidin 3-glycoside, and the concentration of anthocyanin in the extracts of V. oldhamii fruit were calculated using its molecular weight (MW = 449.2) and molar absorptivity (ε = 26,900). The concentrations of anthocyanin in the extracts were calculated using the following equation:

Concentrationofmonomericanthocyaninpigment(CGE/L)=A×MW×DF×1000ϵ×1

* Calculation : cyanidin-3-glucoside equivalents

[MW = 449.2 g/mol, e = 26,900 L/㎝·mol]

* A = (A510 ㎚ − A700㎚) ㏗ 1.0 − (A510㎚ − A700㎚) ㏗ 4.5

* DF = Dilution factor

Statistical analysis

The data are expressed as mean ± standard deviation. The statistical analysis and correlation analysis were performed using R (http://www.r-project.org). Significance was accepted at the 5% level when using ANOVA followed by Duncan’s multiple range test (p < 0.05). Pearson correlation analysis was used to analyze the relationships between antioxidant activities and environmental factors, according to region. The data was standardized using Z-scores and used in hierarchical cluster analysis using Ward’s method, based on the squared Euclidean distance.

Results and Discussion

Comparisons of the antioxidant activity, total phenolic content, and total flavonoid content of Vaccinium oldhamii

We first assessed the antioxidant activity of V. oldhamii fruit samples collected from 11 regions of South Korea (Table 2). The RC50 values, which represent the concentrations of the extracts required to achieve 50% scavenging activity, were evaluated using DPPH and ABTS radical scavenging assays. The DPPH radical scavenging activity RC50 values ranged from 210.44 to 902.38 ㎍/mL, with a mean of 362.98 ㎍/mL. The ABTS radical scavenging activity RC50 values ranged from 524.29 to 1,230.97 ㎍/mL, with a mean of 930.89 ㎍/mL. The FRAP values ranged from 1,783.71 to 2,235.78 ㎍/mL, with a mean of 1,947.00 ㎍/mL. The total phenolic contents of the samples ranged from 10.66 to 36.43 ㎎ GAEs per gram of fruit, with a mean of 18.34 ㎎ GAE s/g fruit. The total flavonoid contents ranged from 2.23 to 5.21 ㎎ QEs per gram of fruit, with a mean of 6.65 ㎎ QE s/g fruit. The total anthocyanin contents varied from 189.70 to 781.30 ㎎ cyanidin-3-glycoside equivalents (CEs) per 100 g fruit, with a mean of 434.62 ㎎ CE/100 g fruit. These results illustrate the diversity in the antioxidant properties and phytochemical contents of V. oldhamii collected from various regions of South Korea.

Table 2.

Analysis of variance and descriptive statistics for the antioxidant activities, total phenolic contents, total anthocyanin contents, and total flavonoid contents of samples of V. oldhamii Miq. collected from 11 regions of South Korea.

Min Max Median Mean SD skew kurtosis SE
DPPH radical scavenging RC50
(㎍/mL)
210.44 902.38 285.90 362.98 30.87 1.78 2.3 30.84
ABTS radical scavenging RC50
(㎍/mL)
524.29 1,230.97 981.11 930.89 37.59 -0.46 -0.99 37.59
FRAP value
(㎛ Fe (II)/g seed)
1,783.71 2,235.78 1,923.24 1,947.00 23.24 0.66 -0.65 23.25
Total phenolic content
(㎎ GAE s/g fruit)
10.66 36.43 16.03 18.34 1.09 1.21 0.63 1.09
Total flavonoid content
(㎎ QE s/g fruit)
2.23 5.21 3.46 6.65 0.14 0.33 -0.85 0.14
Total anthocyanin content
(㎎ CE/100 g fruit)
189.70 781.30 392.31 434.62 27.93 0.73 -0.29 27.93

Antioxidant activity of Vaccinium oldhamii according to the location of collection, and a comparison with that of blueberries

Table 3 presents the RC50 values for DPPH radical scavenging activity of V. oldhamii fruits collected from 11 regions and blueberries. The analysis revealed significant variations in DPPH radical scavenging activity among the regions. The highest activity was observed in fruits from the Gumi region, with an RC50 value of 215.49±4.43 ㎍/mL, while the lowest activity was found in fruits from the Nonsan region, with an RC50 value of 792.84±166.94 ㎍/mL. Notably, there was considerable variability in the RC50 values of blueberries obtained from the four countries. The highest activity was found in domestically-grown blueberries, with RC50s of 290.99±6.70 ㎍/mL, similar to those from Jeju, Seogwipo, Gimcheon, Taean, and Haenam, but lower than that of fruits from the Gumi region. As a reference, the RC50 value for ascorbic acid was 5.01±0.76 ㎍/mL, higher than all other samples.

Table 3.

RC50 values for DPPH and ABTS radical scavenging activities and FRAP value of V. oldhamii extracts and a comparison with blueberries.

DPPH radical scavenging RC50
(㎍/mL)
ABTS radical scavenging RC50
(㎍/mL)
FRAP value
(㎛ Fe (II)/g extract)
Vaccinium oldhamii
Miq.
Nonsan 792.84±166.94b 1,175.66±14.90b 1,806.04±14.77gh
Buyeo 595.85±70.56c 1,221.18±10.77b 1,882.31±48.76ef
Taean 282.60±3.60fg 577.95±10.17g 1,928.82±64.36de
Haenam 286.65±2.42fg 565.13±58.92g 1,949.29±19.60d
Gwangyang 299.53±41.98efg 884.05±65.61ef 1,833.94±31.07fg
Hamyang 398.00±39.37de 1,040.91±52.47cd 1,798.60±14.05gh
Gumi 215.49±4.43g 792.47±30.28f 2,094.39±39.60b
Jeju 274.33±21.24fg 956.53±168.54de 2,024.63±70.94c
Seogwipo 264.78±11.89fg 1,002.45±63.39cde 2,019.98±28.64c
Gimcheon 250.18±8.41fg 991.16±128.05cde 2,224.62±14.77a
Gwangju 332.48±32.32def 1,032.35±44.64cd 1,854.41±50.64fg
Blueberries Korea 290.99±6.70fg 1,002.99±22.08cde 1,729.76±37.16i
America 298.06±9.15efg 984.07±78.75de 1,759.53±28.09hi
Canada 1301.99±99.05a 1,729.78±85.31a 1,623.72±14.05j
Chile 403.40±31.16d 1,121.44±40.95bc 1,761.39±20.12hi
Ascorbic acid 5.01±0.76 5.01±0.76 6.00±0.10

zThe same letter in each column indicates no significant difference, according to Duncan’s multiple range test, p < 0.05.

The ABTS radical scavenging activity varied significantly among the regions. Fruits from Taean and Haenam regions exhibited the highest RC50 values (577.95±10.17 and 565.13±58.92 ㎍/mL, respectively), while fruits from Nonsan and Buyeo regions displayed lower activity, with RC50 values of 1,175.66±14.90 and 1,221.18±10.77 ㎍/mL, respectively. Among the blueberries from the four countries, American blueberries had the highest RC50 values, but these were lower than those of the best-performing regions for Korean blueberries, such as fruits from Gumi and Taean. The RC50 value for ascorbic acid was 6.00±0.10 ㎍/mL, higher than all other samples.

The FRAP of the fruit extracts was measured to assess the antioxidant activity of Korean blueberries from various regions. The highest activity was observed in fruits from the Gimcheon region, with a value of 2,224.62±14.77 ㎛ Fe (II)/g fruit, while the lowest activity was found in fruits from the Hamyang region, with a value of 1,798.60±14.05 ㎛ Fe (II)/g fruit. All four blueberry extracts from other countries showed lower activities than those of V. oldhamii, including those from the region with the lowest activity, Gimcheon.

A comparison of V. oldhamii with blueberries showed that the DPPH radical scavenging (RC50), ABTS radical scavenging (RC50), and FRAP (㎛ Fe (II)/g extract) antioxidant activities of V. oldhamii were higher than those of blueberries from domestically-grown, U.S.-grown, Canadian-grown, and Chilean-grown blueberries. The total phenolic and anthocyanin contents were also higher.

According to Kim et al. (2016), the antioxidant activity of fruits from four indigenous populations of V. oldhamii (Taean, Nonsan, Gumi, Gwangju) in Korea was investigated. The total phenolic content ranged from 4.40 to 10.58 ㎎ GAE/g, with the Gumi population showing the highest value. In terms of DPPH free radical scavenging activity, high antioxidant activity was observed at 800 ppm with an average of 94.6%, and at 400 ppm with an average of 76.4%. These results indicate morphological differences in fruit among the V. oldhamii populations, reflecting morphological diversity according to region.

The utility of V. oldhamii fruit was examined in a previous study (Kim et al., 2013) through antioxidant activity analysis such as DPPH free radical scavenging activity and reducing power using selected five specimens (Gwangju, Namhae, Nonsan, Muju, Taean). The DPPH free radical scavenging activity showed high activity with an overall average of 84.2% at a concentration of 400 ppm, with specimens collected from the Taean region exhibiting the highest scavenging activity at 91.4%. The reducing power was recorded as an average of 0.79 at 700 ppm, with specimens collected from the Taean region showing the highest reducing power at 0.96. These results confirm that the antioxidant activity of V. oldhamii fruit varies depending on the region and extraction conditions. According to Lee et al. (2023), variations in antioxidant activity, total phenolic content, and total flavonoid content of Boehmeria nivea var. tenacissima (Gaudich.) Miq. were attributed to differences in environmental conditions among collection sites.

Comparisons of the total phenolic content, total flavonoid content, and total anthocyanin content of Vaccinium oldhamii, according to the location of collection, and a comparison with blueberries

Table 4 presents the total phenolic, flavonoid, and anthocyanin contents for V. oldhamii fruit collected from the 11 Korean regions and for blueberries. The fruit from the Gimcheon region exhibited the highest total phenolic content (32.62±3.56 ㎎ GAE s/g fruit) and the fruit from the Taean region displayed the lowest content (12.51±2.08 ㎎ GAE s/g fruit). The blueberries showed significant variation according to their country of origin. Chilean blueberries had the highest content, but this was lower than that of fruit from the Gimcheon region (32.62±3.56 ㎎ GAE s/g fruit. When comparing the regions with the highest content in both species, V. oldhamii showed approximately 1.7 times higher content compared to blueberries. Blueberries exhibited relatively lower content compared to V. oldhamii fruits.

Table 4.

Total phenolic, flavonoid, and anthocyanin contents in V. oldhamii, expressed as equivalents to gallic acid, quercetin, and cyanidin-3-glucoside and a comparison with blueberries.

Total phenolic content
(㎎ GAE s/g fruit)
Total flavonoid content
(㎎ QE s/g fruit)
Total anthocyanin content
(㎎ CE/100 g fruit)
Vaccinium oldhamii
Miq.
Nonsan 13.56±1.43fgz 2.78±0.19hi 327.15±46.54def
Buyeo 15.35±0.75ef 3.89±0.06e 232.75±37.66f
Taean 12.51±2.08g 2.41±0.27i 354.76±157.4cde
Haenam 19.68±1.73c 3.50±0.10efg 461.33±33.24c
Gwangyang 13.17±0.17fg 3.24±0.11fg 385.04±8.12cde
Hamyang 14.53±0.35efg 3.37±0.09fg 366.34±26.04cde
Gumi 26.34±2.01b 5.07±0.15bc 667.96±126.11ab
Jeju 16.75±0.96de 3.57±0.28ef 468.46±59.75c
Seogwipo 21.60±1.00c 4.47±0.11d 471.87±13.96c
Gimcheon 32.62±3.56a 4.74±0.37cd 744.25±38.43a
Gwangju 15.66±1.33ef 3.12±0.19gh 300.88±68.42ef
Blueberries Korea 15.45±0.84ef 4.88±0.17cd 587.36±39.54b
America 19.19±0.23cd 5.37±0.34b 434.62±44cd
Canada 9.12±1.48h 3.51±0.11efg 88.62±2.23g
Chile 24.73±1.39b 6.01±0.49a 458.37±35.6c

zThe same letter in each row indicates no significant difference, according to Duncan’s multiple range test, p < 0.05.

The Gumi region yielded the fruit with the highest total flavonoid content (5.07±0.15 ㎎ QE s/g fruit), while those from the Taean region had the lowest content (2.41±0.27 ㎎ QE s/g fruit). Blueberries, and particularly the Chilean and American examples, showed higher total flavonoid contents, exceeding those of V. oldhamii fruit (6.01±0.49 and 5.37±0.34 ㎎ QE s/g fruit, respectively).

The fruits from the Gimcheon region exhibited the highest total anthocyanin content (744.25±38.43 ㎎ CE/100g fruit), while fruits from the Buyeo region had the lowest (232.75±37.66 ㎎ CE/100g fruit). Blueberries showed considerable variation based on their country of origin, with Korean blueberries having the highest content (587.36±39.54 ㎎ CE/100g fruit) and Canadian blueberries having the lowest (88.62±2.23 ㎎ CE/100g fruit). Notably, V. oldhamii fruits from the Gimcheon and Gumi regions had higher anthocyanin contents than the blueberries. These findings offer insights into the variations in the contents of these bioactive compounds in V. oldhamii fruit across various regions of Korea and in comparison to blueberries.

The total flavonoid content of fruit from the Gumi region was particularly high, and the total anthocyanin content was particularly high in the fruit from the Gimcheon region.

According to Kim et al. (2016), the levels of physiological active substances in the fruits of four indigenous populations of V. oldhamii (Taean, Nonsan, Gumi, Gwangju) in Korea were investigated. The total phenolic content ranged from 4.40 to 10.58 ㎎ GAE/g, flavonoid content ranged from 2.22 to 8.09 ㎎ NE/g, and anthocyanin content ranged from 232.51 to 684.32 ㎎ CGE/100 g, with the Gumi population showing the highest values.

The utility of V. oldhamii fruit was examined in a previous study (Kim et al., 2013) through antioxidant activity analysis such as total phenolic content, DPPH free radical scavenging activity, and reducing power using selected five specimens (Gwangju, Namhae, Nonsan, Muju, Taean). The total phenolic content of V. oldhamii fruit averaged 17.9 ㎎/g, with specimens collected from the Taean region exhibiting the highest phenolic content at 19.8 ㎎/g. The results of this study showed similarity to the findings of previous research.

Relationships of the antioxidant activity, total phenolic content, total flavonoid content and total anthocyanin content with environmental conditions

The antioxidant activity of V.oldhamii fruit, along with the analysis of total phenolic, flavonoids, anthocyanins, and their correlation with meteorological conditions was conducted. Correlation coefficients were only indicated for cases with significant p-values in the correlation analysis (Fig. 2, 3). A significant correlation was observed between antioxidant activity, total phenolic content, and total flavonoid content, as indicated by the FRAP values.

https://static.apub.kr/journalsite/sites/kjpr/2024-037-03/N0820370302/images/kjpr_2024_373_235_F2.jpg
Fig. 2.

Correlation coefficients for the relationships of antioxidant activity, total phenolic content, total flavonoid content, and total anthocyanin content with environmental parameters for each of the regions where V. oldhamii samples were collected. AT, mean temperature; HT, highest temperature; LT, lowest temperature; AW, mean wind speed; AH, mean humidity; DT, daily temperature change.

https://static.apub.kr/journalsite/sites/kjpr/2024-037-03/N0820370302/images/kjpr_2024_373_235_F3.jpg
Fig. 3.

Scatterplot of correlation coefficients between Antioxidant activity, Total phenolic, flavonoid, and anthocyanin content, and environmental factors in 11 regions of V. oldhamii samples. Environmental factors include Average Temperature (AT), Highest Temperature (HT), Lowest Temperature (LT), Daily Temperature Change (DT), Average Wind Speed (AW), and Average Humidity (AH).

A positive linear relationship was observed between the DPPH and ABTS radical scavenging abilities (r=0.59, p < 0.001). Strong positive linear relationships were also evident between FRAP and total phenolic content (r=0.87, p < 0.0005), total flavonoids (r=0.72, p < 0.001), and total anthocyanin content (r=0.85, p < 0.001). The total flavonoid content exhibited a strong positive linear relationship with total phenolic content (r=0.85, p < 0.001) and total anthocyanin content (r=0.69, p < 0.001). Similarly, a similar trend was observed between total phenolic content and total anthocyanin content (r=0.87, p < 0.001).

The correlation analysis between meteorological conditions and the antioxidant and physiological activities of V. oldhamii fruit revealed associations with various weather parameters. A positive linear relationship (r=0.35, p < 0.5) was observed between the maximum temperature (HT) and ABTS radical scavenging ability, while average wind speed (AW) and diurnal temperature range (DT) showed negative (r=-0.38, p < 0.5) and positive (r=0.37, p < 0.5) linear relationships, respectively, with DPPH radical scavenging ability.

Additionally, average humidity (AH) exhibited negative linear relationships with total phenolic content (r=-0.44, p < 0.5), total flavonoid content (r=-0.46, p < 0.01), and total anthocyanin content (r=-0.53, p < 0.01).

The analysis of the correlation between the antioxidant activity, total phenolic, flavonoid, anthocyanin content of V. oldhamii fruit, and meteorological conditions revealed positive correlations between maximum temperature (HT) and ABTS radical scavenging ability, as well as negative correlations between average wind speed (AW) and DPPH radical scavenging ability. A positive correlation was observed between diurnal temperature range (DT) and DPPH radical scavenging ability. Additionally, average humidity (AH) showed negative correlations with total phenolic content, total flavonoid content, and total anthocyanin content.

Research on the specific correlation between these characteristics and the vegetation or environmental conditions of each region is still lacking, and the content of these phytochemicals can vary depending on the environmental and growth conditions of the plants. These differences in phytochemical content may manifest as differences in efficacy when consumed (Gololo, 2018). However, when antioxidant and physiological activity analyses of V. oldhamii fruits from 11 regions were correlated with meteorological conditions, no clear relationship was observed. These findings are similar to those of Lee et al. (2023) in their study on Boehmeria nivea var. tenacissima (Gaudich.) Miq. Regarding the correlation with environmental conditions, no significant correlation was observed between antioxidant and physiological activities and environmental factors such as average temperature, maximum temperature, minimum temperature, and diurnal temperature range.

Results of the hierarchical clustering analysis for V. oldhamii fruit, according to region

A clustering analysis was next performed using the antioxidant activities of V. oldhamii fruit from the 11 different regions of Korea. According to the DPPH radical scavenging (RC50), ABTS radical scavenging (RC50), FRAP (㎛ Fe (II)/g extract), total phenolic content (㎎ GAE s/g fruit), total flavonoid content (㎎ QE s/g fruit), and total anthocyanin content (㎎ CE/100g fruit) of the fruit, the regions were placed into three categories, as shown in Table 5 and Fig. 4.

Table 5.

Comparison of antioxidant activities of the three groups identified using hierarchical clustering analysis according to antioxidant activity, total phenolic content, total flavonoid content, and total anthocyanin content.

Group DPPH radical
scavenging RC50
(㎍/mL)
ABTS radical
scavenging RC50
(㎍/mL)
FRAP value
(㎛ Fe (II)/g extract)
Total phenolic
content
(㎎ GAE s/g fruit)
Total flavonoid
content
(㎎ QE s/g fruit)
Total anthocyanin
content
(㎎ CE/100 g fruit)
1 232.82±8.14z 891.81±55.93 2,159.51±31.10 29.48±1.76 4.90±0.13 706.10±38.08
2 694.34±64.26 1,198.42±11.23 1,844.17±21.54 14.45±0.58 3.33±0.25 279.95±26.16
3 305.48±10.74 865.62±45.43 1,915.67±20.28 16.27±0.73 3.38±0.13 401.24±18.94

zThe data are presented as mean ± SE.

https://static.apub.kr/journalsite/sites/kjpr/2024-037-03/N0820370302/images/kjpr_2024_373_235_F4.jpg
Fig. 4.

Dendrogram showing the results of hierarchical clustering analysis using Ward’s method, based on the antioxidant activity, total phenolic content, total flavonoid content, and total anthocyanin content of the fruit from the various regions of Korea.

Group 1 comprised the regions of Gumi and Gimcheon; Group 2 comprised Nonsan and Buyeo; and Group 3 comprised Taean, Seogwipo, Buan, Gwangju, Jeju, Gwangyang, and Hamyang. Thus, the antioxidant activities of V. oldhamii fruit extracts from the Gimcheon and Gumi regions, in Group 1, were higher than others.

As a result of hierarchical cluster analysis, the groups were divided into three. It was found that V. oldhamii fruit from Gimcheon and Gumi were found to be an important natural source of antioxidants. These data form a basis for the development of new varieties using locally grown native V. oldhamii species alongside blueberries in the future, which may help the adaptation to future food crises resulting from climate change.

This study aimed to compare the antioxidant and physiological activities of V. oldhamii with blueberries from 11 regions. Results showed that V. oldhamii exhibited higher antioxidant activity in terms of DPPH radical scavenging, ABTS radical scavenging, and FRAP compared to blueberries from the United States, Canada, and Chile. When comparing the highest content regions of V. oldhamii and blueberries, V. oldhamii showed approximately 1.3 times higher activity in FRAP, total phenolic compounds, and total anthocyanin content.

Hierarchical cluster analysis divided the regions into three groups, indicating that the fruits of V. oldhamii from Gimcheon and Gumi regions have a high potential for use as natural antioxidants.

Overall, fruits from Gimcheon and Gumi regions exhibited relatively superior characteristics in terms of antioxidant and physiological activities. Based on these results, it is believed that providing basic information through future selection breeding research can lead to the identification of high-quality V. oldhamii individuals. This study aimed to provide information necessary for cultivation and the development of new varieties for utilizing V. oldhamii as a functional food material in South Korea. This is an important step in exploring the commercial potential of V. oldhamii fruits and demonstrating their potential as effective plant materials contributing to the domestic food and health supplement industries. These research findings are expected to play a crucial role in improving the quality of V. oldhamii fruits and developing various utilization methods in the future.

Acknowledgements

This study was conducted with the support of the Korea Forest Service (Korea Forest Research Institute) under the Forestry Science and Technology Development Project (Project Number 2021400B10-2425-CA02).

Conflicts of Interest

The authors declare that they have no conflict of interest.

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