Natural Product Sciences
[ Article ]
Natural Product Sciences - Vol. 29, No. 3, pp.146-151
ISSN: 1226-3907 (Print) 2288-9027 (Online)
Print publication date 30 Sep 2023
Received 01 Sep 2023 Revised 04 Sep 2023 Accepted 07 Sep 2023

Seasonality of Coumarin Composition and Antioxidant Activities in Daphne jejudoensis

Ji-Yeon Lee1, 2, ; Yoon-A Kang2, ; Ji-Yeong Bae1, 2, *
1Interdisciplinary Graduate Program in Advanced Convergence Technology & Science
2College of Pharmacy and Jeju Research Institute of Pharmaceutical Sciences, Jeju National University, Jeju 63243, Korea

Correspondence to: *Ji-Yeong Bae, College of Pharmacy, Jeju National University, Jeju 63243, Republic of Korea Tel: +82-64-754-8194; E-mail: These authors contributed equally to this work.


Daphne jejudoensis, an endemic plant found in Gotjawal area in Jeju Island, was reported as a new species in 2013 due to its distinct characteristics and distribution, which differentiate it from its related species, D. kiusiana. This study focuses on the comparative phytochemical analysis and antioxidant activities of D. jejudoensis leaves based on the collection period in order to identify the optimal harvest time. Daphnin and daphnetin were identified as the major components with the highest content observed in samples collected during spring, and the highest antioxidant activities were recorded during the same time period. This study represents the first investigation into the quantitative analysis of daphnin and daphnetin in the leaves of D. jejudoensis based on the collection period.


Daphne jejudoensis, chemical profiling, collection time, antioxidant, liquid chromatography


Antioxidants are essential for reducing oxidative processes and harmful effects of reactive oxygen species (ROS) in both food systems and the human body.1,2 In the food systems, antioxidants act as natural or synthetic food preservatives added to packaged products. They mitigate against oxidative deterioration and prevent food rancidity, directly lengthening the shelf life of the products.3,4 Synthetic antioxidants, including butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), tertiary butyl hydroquinone (TBHQ), propyl gallate (PG), dodecyl gallate (DG), and octyl gallate (OG), have been extensively employed in the food industry.5 Research has shown that these artificially created compounds can cause cancer by damaging human DNA.6,7 As a result, some countries have begun to prohibit or restrict the use of synthesized antioxidants.8 Consequently, the significance of obtaining natural antioxidants from edible plants to replace the synthetic antioxidants in food products is apparent due to safety considerations. In the human body, oxidative stress arises from a disproportionate amount of ROS and antioxidant defenses. This disruption of cellular functions can lead to several health problems. When the antioxidative mechanism is overwhelmed by ROS, it causes tissue damage as well as accelerated cellular death, which can act as a basis for many diseases.9,10 A range of scientific studies have indicated that antioxidants offer health benefits in processes such as aging, pathogen infestation, stress, apoptosis, and neurological diseases by reducing the cell-damaging effects of free radicals.11 These findings indicate that the chronic diseases are closely related to the regulation of oxidative stress.12,13

Daphne jejudoensis (Thymelaeaceae) originates from Jeju Island in South Korea and is commonly discovered in specific regions within the Gotjawal forest, namely Seonheul, Andeog, and Mureung. Despite its similarities to D. kiusiana, D. jejudoensis was classified as a unique species in 2013 owing to morphological variances, such as a smooth calyx surface, an elongated calyx tube and lobes, elliptical leaves with an acuminate apex, and its exclusive habitat within the forested region of Jeju Island.14,15 Several Daphne species have been utilized in traditional medicine, especially in Asia. This can be attributed to the diverse beneficial properties of coumarins, including anti-inflammatory, diuretic, anticancer, analgesic antioxidant, antitussive, and immunomodulatory effects.16-18

Coumarins are extensively found in various foods and Chinese medicinal herbs, making them valuable natural source of antioxidants.19,20 As a representative coumarin, daphnetin is recognized as a good antioxidant for treating oxidative stress-related diseases.21 Daphnetin demonstrated significant ability to protect mononuclear cells from oxidative stress in umbilical blood.22 Lv et al. studied the cyto-protective activity of daphnetin, a natural coumarin derivative obtained from Genus Daphne, as well as the mechanism related to oxidative stress and mitochondrial dysfunction in tertiary-butyl hydroperoxide (t-BHP)-induced RAW 264.7 cells.23 They found that daphnetin effectively inhibited t-BHP-stimulated cytotoxicity, cell apoptosis, and mitochondrial dysfunction. This inhibition was linked to a decrease in ROS production, a reduction in malondialdehyde (MDA) formation and an increase in superoxide dismutase (SOD) levels, and glutathione (GSH)/GSSG (oxidized GSH) ratio. Furthermore, a recent research has pointed to the effectiveness of daphnetin in reducing oxidative stress-related hepatotoxicity while suppressing 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis by activating Nrf2 signaling pathway.24,25

In this study, we conducted the first-ever investigation of the two main coumarins found in the leaves of D. jejudoensis and analyzed their content variations across the seasons. Samples were collected during spring (May), summer (July), autumn (October), and winter (February). The antioxidant activities of 70% ethanol extracts of D. jejudoensis leaf in various seasons were evaluated and compared using DPPH radical scavenging activity, ABTS radical scavenging activity, ferric reducing antioxidant power (FRAP), total phenolic contents (TPC), and total flavonoid content (TFC). The study primarily aimed to investigate the leaf of D. jejudoensis, which is the most abundant part in plant, in order to determine the optimal harvesting time for the production of potent antioxidants for use in herbal and pharmaceutical products.


General experimental procedures – HPLC was carried out using the Agilent 1260 Infinity series (Agilent Technologies, Santa Clara, CA, USA) with J'sphere ODS H80 (4.5 × 250 mm, 4 µm, S-8 nm, YMC, Tokyo, Japan). MPLC was conducted using a Biotage Isolera chromatography system (Biotage, Uppsala, Sweden). Preparative HPLC was performed with an Agilent 1260 Infinity series and Agilent Zorbax SB-C18 (9.4 × 50 mm, 5 µm) (Agilent Technologies, Santa Clara, CA, USA). Microplate reader (Tecan, Spark, Austria) was used for the antioxidant assay.

Plant materials – The dried leaves of D. jejudoensis were collected periodically from Jeoji-ri, Jeju, South Korea and examined by professor Ji-Yeong Bae of Jeju National University for botanical identification. Voucher specimens of the plant (Table 1) were deposited in College of Pharmacy at Jeju National University, Jeju, Korea.

List of D. jejudoensis leaves in different seasons

Chemicals – Methanol absolute, ethanol, and methylene chloride were purchased from Samchun (Seoul, Korea), while hydrochloric acid was obtained from Daejung (Siheung, Korea). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was provided by Thermo Fisher Science (Waltham, MA, USA). L-ascorbic acid (LAA), trolox, 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid diammonium salt (ABTS), Iron(III) chloride, Folin & Ciocalteu's phenol reagent, aluminum chloride, and potassium acetate were purchased from Sigma-Aldrich (MO, USA). Sodium carbonate and gallic acid were from TCI chemicals (Tokyo, Japan).

Plant Extraction – The powder of dried leaves were homogenized, and 3 g were measured into each flask. Subsequently, 70% ethanol in 1:3 ratio was applied to the flask and sonicated three times for 30 min using ultrasonic extractor (Powersonic 520, hwashintech, Korea). After the extraction, supernatant was pooled through centrifugation (Avanti J-15R, Beckman Coulter, Brea, CA, USA) in 4,000 rpm for 5 min and filtered through Whatman No. 1 filter paper (Maidstone, United Kingdom). The extracts were concentrated using rotary evaporator (Hei-VAP, Heidolph, Germany). The dried crude extracts were stored at 4°C prior to analysis.

HPLC-DAD analysis – High-performance liquid chromatography (HPLC) analysis was carried out through an Agilent 1260 series HPLC instrument with: J’sphere ODS H80 column (4.5 × 250 mm, 4 µm, S-8 nm, YMC, Tokyo, Japan) and Diode Array Detector on 280 nm. Water with 0.01% formic acid (A)-acetonitrile with 0.01% formic acid (B) was used as solvent system (0-5 min, A 99-90%; 5-15 min, A 90-80%; 15-25 min, A 80-70%; 25-45 min, A 70-60%; 45-55 min, A 60-40%; 55.1-60 min, kept A 99%, respectively). Acetonitrile was in HPLC grade (Merck, Darmstadt, Germany). Distilled water used was generated by an Evoqua water system (Evoqua, Pittsburgh, PA, USA). The injection volume was 1 μL and the flow rate was 1.2 mL/min. Daphnin and daphnetin were identified through the comparison of its retention time and UV spectrum with those of standard compounds (Sigma-Aldrich, MO, USA).

DPPH radical scavenging activity – 0.2 mM 2,2-diphenyl-1-picryl-hydrazyl(DPPH) solution was prepared in ethanol and added to serial diluted samples (1.56–100 µg/mL). L-ascorbic acid (LAA, 0.3–10 µg/mL) was used as a positive control. After the 30 min of incubation in dark, absorbance was measured at 517 nm by using a microplate reader (Tecan, Spark, Austria). The DPPH radical scavenging activity was calculated in the formula as follows; Inhibition% = [(Acontrol – Asample) / Acontrol] × 100 and expressed in terms of IC50 value.

ABTS·+ radical scavenging activity – To prepare the ABTS•+ radical cation, the ABTS stock solution was reacted with 2.45 mM potassium persulphate and dissolved in ethanol reaching a final concentration of 7 mM. The reaction was allowed to incubate in the dark for 16 hours until completion. Trolox was used as a positive control in the range of 0.3-15 mM. The ABTS•+ stock solution was diluted in ethanol to achieve an absorbance reading of 0.7 at 734 nm for measurement (Tecan, Spark, Austria). The ABTS•+ radical scavenging activity was then calculated using the following formula Inhibition% = [(Acontrol – Asample) / Acontrol] × 100 and expressed as an IC50 value. The results were also expressed as millimoles of trolox equivalent (TE) per gram of dried extract (mmol TE/g ext).

Total Flavonoid Contents (TFC) – To determine the total flavonoid contents, each sample (100 µL) was mixed with 10% AlCl3 (20 µL), 1 M NaNO2 (20 µL), 95% ethanol (300 µL), and distilled water (560 µL). The mixture was incubated in 45oC for 30 min. A standard curve for TFC was then plotted using quercetin with the seven points in the range of 3.12 - 200 µg/mL. The results were expressed as micrograms of quercetin equivalent per gram of dried extract (μg QE/g ext). Absorbance was measured at 415 nm using a microplate reader.

Total Phenolic Contents (TPC) – Total phenolic contents were determined using Folin-Ciocalteu’s method. Samples (100 µL) were mixed with 10% Folin-Ciocalteu’s reagent (500 µL) and sodium carbonate (400 µL). The mixture was kept in the dark for 2 hours and then centrifuged to obtain the supernatant. A standard curve for TPC was drawn using gallic acid (200, 100, 50, 25, 12.5, 6.25, and 3.12 µg/mL). Absorbance measurements at 765 nm were taken using a microplate reader. Results were expressed as micrograms of gallic acid equivalent per gram of dried extract (µg GAE/g ext).

Ferric Reducing Antioxidant Power (FRAP) – A mixture of 300 mM sodium acetate pH 3.6 (25 mL), 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) in 40 mM HCl (2.5 mL) and 20 mM FeCl3 (2.5 mL) was prepared and suspended in distilled water to make the FRAP reagent. The FRAP reagent was freshly prepared before each experiment; Sample-FRAP solutions (100 μL: 900 μL) were left to react at room temperature in the dark for 24 hours. Absorbance of the colored samples was measured at 595 nm. The standard curve was generated using trolox solution within the concentration range of 0.3 to 15 mM. The results are presented as mmol TE/g of extract.

Statistical analysis – All experiments were done in triplicate and the results were shown in mean and standard deviation. A p-value less than 0.005 was regarded as statistically significant in one-way analysis of variance (ANOVA).

Result and Discussion

The main coumarins (daphnin and daphnetin, Fig. 1) content variation in the predominant part of D. jejudoensis, the leaf, was assessed through HPLC-DAD during different seasonal sample collections (Table 1). Samples were collected during the spring (May), summer (July), autumn (October), and winter (February) seasons to compare their coumarin content. 70% Ethanol was chosen as a solvent in this study for the extraction, and the yield of each samples were 40.39% (spring), 21.07% (summer), 28.94% (autumn), and 33.91% (winter), respectively. The calibration curves were established by analyzing a standard mixture containing two major coumarins at various concentration levels. The peak area was plotted against the concentration levels of each reference standard (daphnin: y = 0.8082x – 3.9419 and daphnetin: y = 1.4884x + 7.6888). The curves exhibited good linearity, with the correlation coefficients ranging from 0.9986 and 0.9996 for all the compounds over the concentration ranges of quantification, between 5 and 1000 μg/mL. The peak purity was determined using a photodiode array detector. Furthermore, each peak in the absorption spectrum was compared with the characteristic peak of the corresponding standard compound. Precision was evaluated both within the intraday and inter-day repeatability along with the relative standard deviations consistently measuring below 20%.

Fig. 1.

The structure of compounds isolated from D. jejudoensis.1, daphnin; 2, daphnetin.

HPLC-DAD results show that daphnin and daphnetin were found to be the major coumarins present in the D. jejudoensis (Fig. 2). This result is consistent with previous findings from other Daphne species such as D. axilliflora and D. kiusiana.26 70% Ethanol extract of spring season leaves exhibited the highest daphnin content with a value of 443.08 ± 8.52 mg/g ext (Table 2). This was followed by summer (263.95 ± 6.27 mg/g ext), autumn (253.44 ± 4.09 mg/g ext), and winter (201.19 ± 0.16 mg/g ext). Notably, the content of daphnin in the leaves collected during winter was only half of that found in the spring-collected leaves with a value of 201.19 ± 0.16 mg/g ext, making it the lowest of the four seasons. All samples in the four seasons showed relatively lower daphnetin contents in comparison with those of daphnin, yet followed the same trend as daphnin: higher in spring (20.57 ± 0.35 mg/g ext) and summer (15.13 ± 0.15 mg/g ext), and lower in autumn (4.56 ± 0.26 mg/g ext) and winter (5.29 ± 0.07 mg/g ext).

Fig. 2.

HPLC chromatograms of reference compounds (A) and two coumarins in various parts of D. jejudoensis(B-F) measured at 280nm.

Extraction yield, total flavonoids, total phenols, daphnin and daphnetin content of seasonal leaves of D. jejudoensis

The antioxidant capacity of 70% Ethanol leaf extracts of D. jejudoensis collected in four different seasons was evaluated by DPPH, ABTS, and FRAP assays. Both DPPH and ABTS are stable free radicals, and their scavenging mechanisms primarily involve electron transfer and hydrogen atom transfer reaction.27 These radical scavenging activities are presented by the inhibitory concentration 50 (IC50) with positive controls of L-ascorbic acid (LAA) and trolox in this study (Table 3). The FRAP assay is used when evaluating the overall reducing capability of antioxidants by measuring the reduction of ferric (III) to ferrous (II) ions.28 Therefore, DPPH, ABTS, and FRAP assays can be used to evaluate the antioxidant activity of 70% Ethanol leaf extracts of D. jejudoensis.29 As shown in Table 3, the extract from spring season leaves demonstrated not only the most potent capacity in scavenging both DPPH and ABTS radicals but also superior FRAP assay compared to the other extracts collected from other seasons. In contrast, the extract collected in winter leaves had the highest IC50 value in both DPPH and ABTS assays, showing less capacity in reducing ferric (III) to ferrous (II) ions as revealed in the FRAP assay. Much like the variations in daphnin and daphnetin content across all seasons of the year reported in this study, the antioxidant capacity of 70% ethanol leaf extracts from D. jejudoensis demonstrated higher levels during spring and summer, while it was lower in autumn and winter. Kim et al. reported the monthly irradiance of ultra violet observation in Jeju Island, which have shown that the UV index is much higher in spring and summer than in other seasons.30 Li et al. have found that the amount of secondary metabolites in plants is related to the amount of UV radiation.31 Therefore, it can be assumed that the amount of daphnin and daphnetin may be affected by the stronger UV radiation in spring and summer. The differing antioxidant capacities observed in the 70% ethanol leaf extracts of D. jejudoensis across various sample collection seasons indicate a strong correlation with the content of the antioxidant agent, daphnetin and its derivatives.21 Šeršeň and Lácová screened nineteen coumarin derivatives for antioxidant activity and found that coumarins with hydroxyl groups at positions 6 or 8 had the greatest antioxidant effect. As we have shown in this paper, daphnin and daphnetin have hydroxyl groups at positions of 7 and 8, which may explain the link to their antioxidant activity 32 Overall, the results of the DPPH, ABTS, and FRAP assays consistently align in evaluating the antioxidant ability.

Antioxidant activity determined by DPPH, ABTS and FRAP assays in different plants parts of D. jejudoensis

Total phenolic content (TPC) and total flavonoid content (TFC) of the 70% ethanolic leaf extracts from D. jejudoensis collected during various seasons were also investigated. In this study, total phenolic content was determined by a linear gallic acid standard curve (y = 0.0035x + 0.0565, R2 = 0.9999) and quercetin (y = 0.0048x – 0.0613, R2 = 0.9927) was used as a standard of total flavonoid content. The higher TPC and TFC indicated that the extracts have greater potential for antioxidant activity due to the ability to scavenge free radicals and reduce oxidative stress.33 The total phenolic contents of leaves extract from D. jejudoensis exhibited its highest value in the leaves extract collected during spring, measuring at 126.97 ± 14.81 μg GAE/g ext. This was followed by summer, autumn, and winter with values of 83.37 ± 13.84, 97.38 ± 13.68, 70.32 ± 13.53 μg GAE/g ext, respectively (Table 2). Conversely, the TFC does not follow the same pattern as the TPC, as it demonstrates similar values throughout the year in this study. This discrepancy could be resulted from various factors including. This discrepancy could be resulted from various factors including extraction solvent type and method.34

In conclusion, this study represents the first investigation of two primary coumarins, daphnin and daphnetin, within the 70% ethanol leaf extracts of D. jejudoensis, accounting with different sample collection times. Antioxidant activities were assessed and compared across seasons through DPPH, ABTS, and FRAP assays, along with TFC and TPC measurements. The results revealed the highest antioxidant activities within samples collected during spring and the lowest during winter. Consequently, the 70% ethanol extracts from D. jejudoensis leaves emerge as a promising source of natural antioxidants for pharmaceutical and herbal products, along with higher radical scavenging activities and substantial phenolic compound contents. Moreover, it is recommended to harvest D. jejudoensis leaves in spring, because of the superior antioxidant activities compared to other seasons.


This work was supported by the research grant of Jeju National University in 2020. We express our gratitude to Mr. Daeyang Park from the Korean Medicinal Plant Garden for his support in obtaining plant materials and to Ms. Yuji Yang for her technical support.

Conflicts of Interest

The authors have declared that they have no conflicts of interest.


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Fig. 1.

Fig. 1.
The structure of compounds isolated from D. jejudoensis.1, daphnin; 2, daphnetin.

Fig. 2.

Fig. 2.
HPLC chromatograms of reference compounds (A) and two coumarins in various parts of D. jejudoensis(B-F) measured at 280nm.

Table 1.

List of D. jejudoensis leaves in different seasons

Samples Code Collection date Parts Collection site
Spring (May) JNUP-2022-14 2022.05 Leaves Jeoji-ri, Jeju-do
Summer (July) JNUP-2022-19 2022.07
Autumn (October) JNUP-2022-38 2022.10
Winter (February) JNUP-2023-06 2023.02

Table 2.

Extraction yield, total flavonoids, total phenols, daphnin and daphnetin content of seasonal leaves of D. jejudoensis

Samples Extraction Yield
(%, w/w)
Total Flavonoid contents
(μg QE/g ext)a
Total Phenolic Contents
(μg GAE/g ext)b
Daphnin (1)
(mg/g ext)c
Daphnetin (2)
(mg/g ext)c
aTotal Flavonoid contents were expressed in quercetin equivalent (QE) μg per 1 g of extract.
bTotal Phenolic contents were expressed in gallic acid equivalent (GAE) μg per 1 g of extract.
cThe amount was expressed as mg in 1 g of extract.
Spring (May) 40.39 52.05 ± 1.08 126.97 ± 14.81 443.08 ± 8.52 20.57 ± 0.35
Summer (July) 21.01 44.54 ± 0.47 83.37 ± 13.84 263.95 ± 6.27 15.13 ± 0.15
Autumn (October) 28.94 48.79 ± 0.94 97.38 ± 13.68 253.44 ± 4.09 4.56 ± 0.26
Winter (February) 33.91 46.38 ± 0.72 70.32 ± 13.53 201.19 ± 0.16 5.29 ± 0.07

Table 3.

Antioxidant activity determined by DPPH, ABTS and FRAP assays in different plants parts of D. jejudoensis

Samples DPPH IC50
ABTS IC50 (mM)
(mmol TEb/g ext)
(mmol TE/g ext)
Data are presented as the mean ± SD of triplicate measurements.
aLAA represents L-ascorbic acid.
bTE means Trolox Equivalent.
Spring (May) 9.14 ± 0.10 2.01 ± 0.18 1.37 ± 0.18 2.81 ± 0.23
Summer (July) 11.39 ± 2.76 2.55 ± 0.34 1.10 ± 0.18 2.29 ± 0.20
Autumn (October) 17.39 ± 0.37 2.73 ± 0.58 1.06 ± 0.23 2.23 ± 0.20
Winter (February) 17.45 ± 1.44 5.40 ± 1.45 0.60 ± 0.03 1.82 ± 0.18
LAAa 5.27 ± 0.34 - - -
Trolox - 2.61 ± 0.04 - -