Comparative Antioxidant Potentials and Quantitative Phenolic Compounds Profiles among the Flowers and Leaves from Various Chrysanthemum morifolium Cultivars

Chrysanthemum morifolium is a valuable plant that contains a wide range of phytochemical compounds and exhibits various biological activities. Ethanol extracts from both the leaves and flowers of 17 different cultivars of C. morifolium were tested for antioxidant activities using the 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) assays and were quantitatively analyzed for 12 phenolic compounds using high-performance liquid chromatography with diode-array detection. We found that the ‘Ford’ and ‘Raina’ cultivars demonstrated strong antioxidant abilities and high phenolic compound contents compared to other cultivars, while the flowers of ‘Cielo’ and the leaves of ‘White Cap’ exhibited low antioxidant capacity in both assays. The ‘Cielo’ cultivar also displayed the lowest compound contents. Additionally, in most samples, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid stood out as high-content compounds in the extracts. This study provides foundational knowledge that can be used for selecting appropriate C. morifolium cultivars for further research. Moreover, the ‘Ford’ and ‘Raina’ cultivars, containing high amounts of bioactive compounds and showing superior antioxidant ability, could be applied to produce health-beneficial products.


Introduction
The widespread recognition of the oxidation reaction's significance to both the human body and food has led to an understanding that oxidative metabolism is vital for cell survival.However, a consequence of this reliance is the generation of free radicals and other reactive oxygen species, resulting in oxidative changes [1,2].Over the past decade, diseases or disorders associated with oxidative stress-including metabolic, neurodegenerative, cardiovascular, and mitochondrial diseases and cancer-have garnered considerable attention [3][4][5].Numerous studies have explored the underlying triggers, seeking to elucidate the mechanisms of action of free radicals and identify effective substances that prevent or reverse oxidative damage [6,7].
Antioxidants demonstrate high efficacies in regulating the production of free radicals, preventing their undesirable effects, and supporting the body's antioxidant and detoxifying mechanisms [8][9][10][11].They can naturally occur in plants, animals, and microorganisms or may be synthesized through chemical means.Higher plants and their component compounds serve as abundant sources of natural antioxidants, such as tocopherols and  Ascorbic acid was employed as the standard and as a reference.
The higher the IC 50 , the lower the antioxidant ability, and vice versa.In general, the IC 50 values of the flower samples ranged from 1.26 to 6.21 mg/mL in the DPPH assay and 1.14 to 5.65 mg/mL in the ABTS + assay, while the IC 50 values of the leaf samples ranged from 2.10 to 20.86 mg/mL in the DPPH assay and 1.82 to 11.48 mg/mL in the ABTS + assay.In contrast, the control sample (ascorbic acid) had an IC 50 of 0.10 mg/mL, indicating that the antioxidant activity of all samples was more than 10 times lower than that of ascorbic acid.Moreover, in the same cultivar, most flower samples exhibited stronger antioxidant ability than leaf samples.Only in the 'Yes Holic' cultivar did leaf samples show a higher antioxidant ability, and this was only true in the DPPH assay; however, the differences between the two IC 50 values were not remarkable.

HPLC Analysis
Thirty-four extract samples from C. morifolium flowers and leaves were analyzed to quantify the content of 12 phenolic compounds using the HPLC-DAD method.
Showing consistency with the antioxidant activity results, the majority of flower samples contained higher concentrations of the surveyed compounds than the leaf samples.Nevertheless, exceptions were noted in the 'Argus' and 'Geumsu' cultivars, where leaf samples exhibited a slightly higher content.

Discussion
Phenolic compounds, comprising flavonoids and phenolic acids, are recognized as contributors to the antioxidant capacities of fruits.Additionally, fruits containing higher phenolic contents typically exhibit stronger antioxidant capacities [47,48].This trend was also observed in this study, where the levels of 12 phenolic compounds in the C. morifolium extracts were closely related to their antioxidant capacity.Specifically, flower extract samples S17, S7, and S3, which contained high compound levels, exhibited stronger antioxidant capacities than other samples.Conversely, S13, with a low compound level, showed the weakest antioxidant ability among the flower samples.A similar trend was observed in leaf samples, where high-compound content samples S32, S24, and S34 demonstrated strong antioxidant activity, while S26, with a low compound content, exhibited weak antioxidant capacity.
In contrast, some samples exhibited high levels of phenolic compounds, yet their antioxidant activity was notably low.It is crucial to note that this study exclusively focused on 12 phenolic compounds.Many other compounds present in the samples were not evaluated.Previous studies have identified a wide range of bioactive compounds in C. morifolium, including quercetin, isorhamnetin 3-O-glucoside, eriodictyol, pyracanthoside, apigetrin, acacipetalin, diosmetin, spinacetin, axillarin, bonanzin, cirsiliol, chrysosplenol D, artemetin, quercitrin, acacetin 7-O-glucoside, apigenin 7-O-glucoside, and luteolin 7-O-glucoside [49][50][51].Therefore, the results of this study only partially characterize the samples, and further research is necessary to conclusively identify which compounds significantly influence the antioxidant capacity of C. morifolium extracts.Additionally, it is important to note that many other compounds, besides phenolic compounds, contribute to the biological activities of C. morifolium, such as carotenoids, steroids, and terpenoids.Therefore, the presence of phenolic compounds contributes to the antioxidant ability, but it cannot be said that the antioxidant ability as well as biological processes are solely determined by phenolic compounds.
On the other hand, while the antioxidant ability of the flower samples was considerably higher than that of the leaf samples, it is easier to cultivate and obtain a high amount of leaf samples rather than flower samples.Furthermore, in contrast to leaf samples, extracting flower samples is more prone to obtaining undesirable substances such as oils.Consequently, it is essential to carefully consider both antioxidant abilities and other relevant conditions when choosing the appropriate samples.
In our previous study, we quantified the antioxidant capacity as well as the content of phenolic compounds (including the 12 compounds this study focused on) in three different Chrysanthemum species from various regions in South Korea [52].The IC 50 values of the extracts in that study ranged from 5.8 to 17.4 mg/mL in a DPPH assay and 2.7 to 9.4 mg/mL in an ABTS + assay, and the total content of phenolic comranged from 7.87 to 95.13 mg/g extract.In the current study, the flower extracts exhibited values ranging from 1.14 to 6.21 mg/mL in the DPPH assay and 1.14 to 5.65 mg/mL in an ABTS + assay, and the total content of phenolic compounds ranged from 26.32 to 200.68 mg/g extract.Thus, both the antioxidant capacity and the bioactive compound levels in the flower samples from the 17 cultivars in this study surpassed those observed in the previous study despite similar extraction procedures.
Furthermore, most previous studies have concentrated on investigating compounds and biological activities in the flowers of Chrysanthemum species rather than the leaves.Therefore, our study not only demonstrates the differences in biological properties between the flowers and leaves of Chrysanthemum species but also fills the gap in studies on the leaves of Chrysanthemum species.Although the biological activity of the leaves is not as potent as that of the flowers, it is evident that C. morifolium leaves still exhibit considerable biological activity that warrants further investigation.

Plant Materials
Seventeen cultivars of C. morifolium (Figure 4) were grown by Prof. Jinhee Lim, Sejong University, Republic of Korea, in May 2023.
Seedlings were sown in May 2023, and the leaves and flowers were freshly harvested in September 2023 (~16 weeks old), dried, and cut into small pieces before extraction.All specimens (S1-S34) were deposited at the herbarium of the Department of Bio-Industry Resources Engineering, Sejong University, Seoul, Republic of Korea.

Plant Materials
Seventeen cultivars of C. morifolium (Figure 4) were grown by Prof. Jinhee Lim, Sejong University, Republic of Korea, in May 2023.

Instruments and Reagents
The HPLC analysis was conducted using an Agilent 1260 Infinity II Quat Pump (Santa Clara, CA, USA) and a DAD.The configuration comprised a pump and an auto-sampler, integrated with a YMC Pack Pro C18 column (4.6 × 250 mm, 5 µm).The HPLC-grade solvents, including water, acetonitrile, and methanol (MeOH), were procured from J. T. Baker (Philipsburg, PA, USA).In addition, acetic acid was acquired from Samchun Chemicals (Pyeongtaek, Republic of Korea).In the context of the assays, both an Epoch microplate spectrophotometer from BioTek (Winooski, VT, USA) and a microplate reader were utilized.

Sample Extraction and Preparation
For each C. morifolium cultivar, 5 g each of dried flower and leaf tissue was subjected to extraction using ethanol in a reflux extractor for 3 h.This extraction process was replicated three times.Following dehydration in a rotary evaporator, the extracts were gathered.For each extract, 20 mg was precisely measured and diluted in 1 mL of MeOH or 1 mL of distilled water to form stock solutions for the DPPH and ABTS + assays, respectively.After filtering through a 0.45 µm membrane filter, sequential dilutions were performed on the stock solutions.To prepare for the HPLC analysis, each extract was dissolved in MeOH, appropriately diluted, and formulated.After dissolution by ultra-sonication, the solution was filtered using a 0.45 µm polyvinylidene fluoride (PVDF) membrane filter to prepare the test solution.For each of the 12 standard compounds, 4 mg was precisely weighed and dissolved in 1 mL of MeOH to create a 4000 ppm stock

Sample Extraction and Preparation
For each C. morifolium cultivar, 5 g each of dried flower and leaf tissue was subjected to extraction using ethanol in a reflux extractor for 3 h.This extraction process was replicated three times.Following dehydration in a rotary evaporator, the extracts were gathered.For each extract, 20 mg was precisely measured and diluted in 1 mL of MeOH or 1 mL of distilled water to form stock solutions for the DPPH and ABTS + assays, respectively.After filtering through a 0.45 µm membrane filter, sequential dilutions were performed on the stock solutions.To prepare for the HPLC analysis, each extract was dissolved in MeOH, appropriately diluted, and formulated.After dissolution by ultra-sonication, the solution was filtered using a 0.45 µm polyvinylidene fluoride (PVDF) membrane filter to prepare the test solution.For each of the 12 standard compounds, 4 mg was precisely weighed and dissolved in 1 mL of MeOH to create a 4000 ppm stock solution.After complete dissolution by ultra-sonication, the solutions were filtered using a 0.45 µm PVDF membrane filter.

DPPH Radical Scavenging Activity
The DPPH radical scavenging assays were performed using 0.2 mM DPPH working solutions created by diluting the original DPPH stock solution with MeOH.Next, 10 µL of each test solution was combined with 200 µL of the DPPH working solution in respective wells of a 96-well plate.This was repeated three times to ensure accuracy.Then, the solutions were mixed thoroughly using a microplate shaker and incubated in darkness for 30 min.Subsequently, the solution's absorbance was measured at a wavelength of 514 nm, and the DPPH radical scavenging rate was calculated.The calculated rates were used to construct activity concentration curves for determining the IC 50 values.

ABTS + Radical Scavenging Activity
The ABTS + radical scavenging assay was performed by diluting the ABTS + stock solution in water to create the ABTS + working solutions.Subsequently, 10 µL of test solution was combined with 200 µL of the ABTS + working solution in a 96-well plate, with the reaction being replicated three times.The solutions were mixed thoroughly on a microplate shaker and incubated for 30 min in the dark before the absorbance was measured at 734 nm.The ABTS + radical scavenging rates were calculated and used to construct curves for IC 50 determination.

HPLC Conditions
A quantitative chemical analysis of the extracts was conducted using a reverse-phase HPLC system, employing a YMC Pack-Pro C18 column (25 cm × 4.6 mm, 5 µm), and a gradient elution.The mobile phase was composed of 0.25% acetic acid in water (A) and acetonitrile (B), and the elution conditions were 10% B from 0 min to 5 min, increasing to 20% B at 10 min, 27% B at 30 min, 40% B at 35 min, and 100% B at 40 min, which was maintained until 60 min.The column temperature was retained at 30 • C, with an injection volume of 10 µL, a flow rate of 1.0 mL/min, and wavelength monitoring set to 356 nm.

Calibration Curve
The calibration curve for the HPLC-DAD analysis was generated by plotting the concentrations of the 12 compounds' standard solutions against their corresponding peak areas.To evaluate the linearity of the curves, we used the correlation coefficient (r 2 ), and then the calibration curves were used to compute the concentrations of the standard compounds in the samples.The calibration equations were established using the mean peak area value (Y) ± standard deviation (n = 3) and concentration (X, µg/mL).

Conclusions
The antioxidant activities of the flower and leaf extracts from 17 cultivars of C. morifolium were evaluated using DPPH and ABTS + assays.Additionally, the presence of 12 phenolic compounds in the extract samples was detected and quantified through HPLC-DAD analysis.The results revealed that 2 out of the 17 cultivars ('Ford' and 'Raina') exhibited particularly strong antioxidant activities and particularly high compound contents.Moreover, 3,5-dicaffeoylquinic acid (6) and 4,5-dicaffeoylquinic acid (8) exhibited high contents relative to the other compounds in most of the samples.This study contributes valuable insights into the compounds that various cultivars of C. morifolium can offer and also adds to research on other Chrysanthemum species.Given the high content of bioactive compounds and the remarkable antioxidant capacity observed in the 'Ford' and 'Raina' cultivars, they can be considered potential sources of natural materials applicable in diverse industries, including pharmaceuticals, cosmetics, and food.

Table 1 .
DPPH and ABTS + radical scavenging activities of extracts from the flowers of C. morifolium cultivars.
Ascorbic acid was employed as the standard and as a reference.

Table 2 .
DPPH and ABTS + radical scavenging activities of extracts from the leaves of C. morifolium cultivars.

Table 4 .
Contents of compounds 1-12 in the flowers of C. morifolium cultivars.

Table 5 .
Contents of compounds 1-12 in the leaves of C. morifolium cultivars.