Analysis of Phenolic Acid Content and Antioxidant Activity of Chestnut Honey from Different Regions of Korea

Introduction

Honey is produced by honeybees and other social insects that collect the nectar or honeydew from the flowers of living plants.¹ Honey contains various components such as sugars, minerals, organic acids, volatiles, vitamins, and enzymes.² Several studies have shown that honey exerts biological activities such as antioxidant,³ anti-inflammatory,⁴ and anticancer⁵ effects. In addition, honey is known to exhibit healing properties for burns, ulcers, and wounds.^2,4 Honey can be classified based on their source: polyfloral and monofloral. Polyfloral honey is made from the nectar of multiple flowers, whereas monofloral honey is made from the nectar of only one type of flower. Depending on the flower source, monofloral honey may differ in color and taste.

Chestnut honey is a monofloral honey collected from the blossoms of chestnut trees. Chestnut is a deciduous tree from the genus Castanea and the family Fagaceae. Castanea species are distributed over Asia, Europe, North America, and North Africa. Four representative chestnut tree species exist: Castanea crenata S. et Z. in Korea and Japan, C. mollissima Blume in China, C. sativa Miller in Europe, and C. dentata (Marshall) Borkhausen in the USA. In Korea, different types of honey, such as acacia, citrus, jujube, rapeseed, and honeysuckle, are harvested. Among them, chestnut honey has a characteristic dark color, indicating high polyphenol content. Previous studies have shown dark-colored honey contains more polyphenols than light-colored honey and has excellent antioxidant and antimicrobial activities.^6,7 Chestnut honey is well known for its anti-inflammatory,⁸ antioxidant,⁹ and anti-melanogenic¹⁰ effects. In addition, it is also used in treating burn wounds.¹¹

Phenolic acids in foods are regarded as an important dietary component for humans since they are easily absorbed by the gut, making them efficient antioxidants with a variety of bioactivities.^12-15 Kolayli et al. investigated the anti-hyaluronidase activity and the total phenolic and flavonoid contents of several honey types from different botanical origins.⁸ The highest activity was observed in oak and chestnut honey, which are also polyphenol-rich in their composition. Ronsisvalle et al. examined the antibacterial and antioxidant activities and the chemical composition of chestnut honey from different regions and compared it with the literature.¹⁶ When compared with Manuka honey, chestnut honey exhibited equivalent antibacterial and antioxidant activity. Manuka honey, produced from the nectar of the manuka tree (Leptospermum scoparium) in New Zealand, is known to have functional properties¹⁷ as it contains bioactive compounds such as methylglyoxal (MGO) and methyl syringate (MSY).^18,19 Similarly, as shown by previous studies, chestnut honey can be a good candidate for determining the functional properties of other kinds of honey.^20,21 In addition, the chemical compositions and activities of different types of honey vary depending on their regional or floral origin.^22,23

Therefore, in this study, the antioxidant activity of chestnut honey from different regions in Korea was evaluated using ABTS free radical scavenging assay. Furthermore, the phenolic acid content was analyzed using high-performance liquid chromatography (HPLC) coupled with an ultraviolet (UV) detector.

Experimental

Chestnut honey samples – Chestnut honey samples produced in different regions of Korea (Pocheon, Chuncheon, Yeongdong, Chungju, Gongju, Jinan, Damyang, Cheongdo, and Geochang) were provided by KEDEM Inc., Korea. A voucher specimen was deposited at the herbarium of the Department of Plant Science and Technology (Pocheon: LEE2021-01, Chuncheon: LEE2021-02, Yeongdong: LEE2021-03, Chungju: LEE2021-04, Gongju: LEE2021-05, Jinan: LEE2021-06, Damyang: LEE2021-07, Cheongdo: LEE2021-08, and Geochang: LEE2021-09), Chung-Ang University, Korea.

Instruments, chemicals, and reagents – Chromatographic analysis was performed by an HPLC system equipped with a quaternary pump (PerkinElmer, Waltham, MA, USA), an auto-sampler, a column oven, and a PDA LC Detector. Standards (Fig. 1) of phenolic acids (gallic acid, caffeic acid, p-coumaric acid, ferulic acid, and cinnamic acid) were obtained from the Natural Product Institute of Science and Technology (www.nist.re.kr), Anseong, Korea. HPLC-grade water and acetonitrile were purchased from J. T. Baker (Avantor, Radnor, PA, USA). Glacial acetic acid (99%) was purchased from Duksan Pure Chemical Co., Ltd. (Ansan, Korea).

Fig. 1.

Chemical structures of phenolic acids.

ABTS free radical scavenging activity – The ABTS free radical scavenging activity of the chestnut honey samples was evaluated following the method. Standard stock solutions were prepared by dissolving ascorbic acid in distilled water (1 mg/mL) and diluted at serial concentrations (0, 40, 80, 120, 160, and 200 μg/mL) for working solutions. ABTS stock solution was prepared by mixing 7.4 mM ABTS and 2.6 mM potassium persulfate. The working solution was prepared to an absorbance of 1.3 at 730 nm by diluting the stock solution with distilled water. Sample stock solutions were prepared by dissolving the sample in distilled water (200 mg/mL) and serially diluted to the final concentrations (50, 100, and 200 mg/mL). For the assay, 200 µL of ABTS working solution was added to each sample solution (10 µL) in a 96-well microplate. The reaction mixtures were kept on a shaker with a microplate mixer (Mx4, FINEPCR, Gunpo, Korea) for 30 seconds and incubated in the dark for 30 min. Absorbance at 734 nm was measured using a microplate reader (Epoch Microplate Spectrophotometer, BioTek, Vermont, U.S.). The ABTS free radical scavenging activity of the sample (%) was calculated as follows:

Preparation of stock solutions for HPLC analysis – The experimental stock solutions were prepared by dissolving each chestnut honey sample in MeOH (200 mg/mL), sonicating for 20 min, and filtering through a 0.45-μm polyvinylidene difluoride (PVDF) membrane. The standard phenolic acid stock solutions (1 mg/mL each) were prepared following the abovementioned procedure.

HPLC conditions – Quantitative analyses of phenolic acids in chestnut honey samples were performed in an HPLC system using PerkinElmer Flexar Quaternary Pump (Waltham, MA, USA), auto-sampler, and PerkinElmer PDA LC Detector. A reverse-phase YMC Pack Pro C18 column (250 × 4.6 mm, 5 μm) was used for analysis. The injection volume was 10 μL, and the flow rate was 1 mL/min. The UV wavelength used to detect phenolic acids was 280 nm, and the column temperature was maintained at 30^oC. The mobile phase consisted of 0.5% acetic acid in water (A) and 0.5% acetic acid in acetonitrile (B). The gradient elution system was as follows: 100% A at 0 min, 100% A at 3 min, 85% A at 8 min, 65% A at 28 min, 50% A at 33 min, 100% B at 34 min, 100% B at 38 min, 100% A at 40 min, and 100% A at 45 min. All injections were performed in triplicate.

Calibration curves – The working solutions used to construct the calibration curves were prepared by serial diluting the stock solution to desired concentrations (0.244-15.625 μg/mL). The calibration function of the standard was calculated using the peak area (Y) and concentration (X, μg/mL), and the values are presented as means ± standard deviations (n = 3).

Results and Discussion

ABTS free radical scavenging activities were evaluated for chestnut honey samples obtained from different regions. Chestnut honey samples were evaluated at three concentrations (50, 100, and 200 mg/mL). On average, the ABTS free radical scavenging activity was 11% at a concentration of 50 mg/mL, 20% at 100 mg/mL, and 38% at 200 mg/mL (Fig. 2). Results showed that the free radical scavenging activity (%) increased as the concentration of the sample also increased. IC₅₀ values of ascorbic acid and chestnut honey samples in the ABTS assay are shown in Table 1. The IC₅₀ values of the honey samples ranged from 232.9 ± 9.2 to 343.6 ± 23.8 mg/mL.

Fig. 2.

ABTS free radical scavenging activities (%) of chestnut honey samples obtained from different regions.

Table 1.

ABTS free radical scavenging activities in chestnut honey samples

Pyo et al. analyzed the total polyphenol, flavonoid, and protein content from chestnut, acacia, and Styrax japonica honey.²⁴ Among these samples, chestnut honey exhibited the best DPPH, ABTS, nitrite scavenging, and reducing activities compared to other honey samples. In addition, it had the highest content of total polyphenols and flavonoids during analysis. Notably, the ABTS caption scavenging activity was superior to DPPH anion scavenging activity, the average ABTS radical scavenging activity of all honey samples was 22.08% while chestnut honey alone had 25.72% at a concentration of 5 mg/mL. Estevinho et al. evaluated the antioxidant activity in dark and clear honey samples using DPPH and found that the dark honey sample had higher activity than the clear honey sample.⁷ Dark and clear honey samples had EC₅₀ values of 27.24 and 68.17 mg/mL, respectively. These findings implied that chestnut honey has the potential to be used as a natural product with antioxidant properties.

HPLC-UV was used to analyze the phenolic acid content in chestnut honey samples at a wavelength of 280 nm (Fig. 3). Table 2 shows the equation of the calibration curves of phenolic acids. The correlation coefficient (r²) of phenolic acids ranged from 0.9991 to 1. Subsequently, phenolic acid peaks were determined in all chestnut honey samples. The total phenolic acid content was highest in the samples from “Gongju” (21.87 μg/g), followed by “Cheongdo” (18.23 μg/g) and “Damyang” (18.16 μg/g), respectively (Table 3). Furthermore, high concentrations of Gallic, p-coumaric, and ferulic acids were found in the chestnut honey samples, while caffeic and cinnamic acids were only detected in trace amounts. In addition, the content of gallic acid was higher than p-coumaric and ferulic acid content in chestnut, especially in the samples from “Chuncheon” (12.83 ± 0.28 μg/g), whereas the p-coumaric and ferulic acid contents were highest in “Gongju” sample (2.71 ± 0.15 and 11.62 ± 0.09 μg/g, respectively). Overall, the phenolic acid content in the local chestnut honey samples varied significantly as expected. Gongju region is well-known as a significant chestnut production area in Korea, and the results can be used to determine the optimal environment for chestnut production.²⁵

Fig. 3.

HPLC chromatograms of phenolic acids (A) and chestnut honey from region of Gongju (B). (1: gallic acid, 2: caffeic acid, 3: p-coumaric acid, 4: ferulic acid, 5: cinnamic acid)

Table 2.

Calibration curves of phenolic acids

Table 3.

Phenolic acid contents in chestnut honey samples

Several studies indicated that the chemical compositions or activities of chestnut honey vary depending on their regional or floral origin.²⁶ Castro-Vázquez et al. revealed the differentiation of chestnut honey from different geographical areas according to their volatile composition and sensory description.²⁷ Sarikaya et al. quantitatively analyzed phenolic compounds and evaluated the antioxidant activities in chestnut honey and propolis.⁹ The results showed that phenolic acids were higher in propolis than in honey. Among the phenolic acids, p-coumaric acid had the highest content compared to the other compounds. Similarly, Gu conducted an HPLC analysis of chestnut honey samples obtained from different regions in Korea.²⁸ Gallic acid had an average amount of 5 ppm, which was comparable to the gallic acid content of the current study’s results (6.67-12.83 μg/g) while p-coumaric acid had an average value of 4 ppm, which was slightly higher than its content (1.47-2.71 μg/g) observed in the present study’s results. The ferulic acid content in the present study (4.81-11.62 μg/g) was substantially higher than the average amount of ferulic acid from the study of Gu, which was 0.2 ppm. Caffeic acid was also identified in the present study as a trace element, although it averaged 16 ppm in the prior study, which was relatively high. In contrast with this study’s findings, Gu demonstrated that the sample of chestnut honey contained significantly higher benzoic acid content than other components. Both studies analyzed phenolic acids in regional chestnut honey samples in Korea but presented different results.

The results from the two studies affirm the authors’ assumption that the composition or constituents of honey may differ depending on its regional origin. Although more studies are still required to substantiate the results, the current study will help in profiling the phenolic acids of chestnut honey from different regions. The results of the study can also valorize the different chestnut honey in Korea.

Acknowledgments

This work was supported by a grant from the Agricultural Technology Commercialization Support Project through the Rural Development Administration, Republic of Korea (Project number PJ0163762022).

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