Antioxidant activity of different cultivars of Chrysanthemum morifolium and quantitative analysis of phenolic compounds by HPLC/UV

Chrysanthemum morifolium is classified within the Asteraceae botanical family and serves as a phytomedicine in many countries. The objective of this study was to quantitatively analyze twelve phenolic compounds through HPLC/ UV and to assess the antioxidant abilities using the DPPH and ABTS + assays in the leaves and flowers of six cultivars of C. morifolium : ‘Geumsu’, ‘Ilonka’, ‘Silvia’, ‘Pompadour’, ‘Yes Holic’, and ‘Ford’. The results indicated that the leaves of ‘Geumsu’ and ‘Ford’, as well as the ‘Pompadour’ flowers contained high levels of phenolic compounds and exhibited strong antioxidant abilities. Additionally, a relationship between the phenolic compounds and antioxidant activities was observed. These findings provide foundational knowledge about C. morifolium cultivars, which are promising natural sources that can offer health benefits.


Introduction
Oxidation constitutes a significant process in food, chemicals, and living systems; however, a by-product is the production of free radicals, particularly reactive oxygen species (ROS) [1].Furthermore, substantial quantities of ROS are generated within humans through natural physiological processes, external environmental interactions, and dietary habits, which may be harmful [2].These ROS contributions extend to processes such as food spoilage, degradation of chemical materials, and the onset of over a hundred human disorders [3,4].However, the utilization of antioxidant substances can counteract the oxidation process.These compounds, even at low concentrations, substantially delay or entirely prevent the oxidation of easily susceptible substrates [5].Recently, the isolation, characterization, and widespread application of natural compounds endowed with antioxidant properties have been demonstrated in various medical contexts [6].Numerous methods are employed to evaluate the efficacy of natural antioxidants, encompassing assays, such as the ferric reducing antioxidant power assay [7], the β-carotene/linoleic acid assay [8], the Rancimat method [9], inhibition of low-density lipoprotein oxidation [10], the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay [11], etc.This assortment of methods is necessitated by the intricate nature of the analyzed substrates, often presenting as complex mixtures comprising numerous compounds with diverse functional groups, polarities, and chemical behaviors [12].
Phenolic is the most encountered natural antioxidant compound in plants [13].They possess one or more hydroxyl groups in their structure, allowing for their structural classification into two major classes: phenolic acids and flavonoids [14].Additionally, phenolic compounds are applied in growth regulation, hormonal activity, pH regulation, antimicrobial effects, metabolism, and the induction of dormant periods [15].
Chrysanthemum morifolium, commonly known as chrysanthemum or florist's chrysanthemum, is a perennial flowering plant belonging to the Asteraceae (Compositae) family [16,17].It is a well-known herbal medicine, which is used as a dietary supplement or health tea in numerous Asian countries, including China, Thailand, Japan, and Korea [18,19].C. morifolium is believed to possess therapeutic properties for treating conditions, such as headache, influenza, hepatic ailments, inflammation, arteriosclerosis, hypertension, hyperuricemia, diabetes alleviation, and eye diseases [20][21][22].Furthermore, it has exhibited a range of significant biological characteristics, including antioxidant, antimutagenic, anticancer, anti-inflammatory, antitumor, antibacterial, and antiviral activities [23][24][25].These pharmacological attributes are primarily ascribed to its active compounds, which encompass alkanes, phenolic acids, flavonoids, terpenoids, unsaturated fatty acids, and polysaccharides [26,27].Presently, diverse cultivars and varieties of C. morifolium have been selectively developed for horticultural applications [28]; consequently, the composition of biological compounds and associated activities may undergo modification through plant breeding or hybridization processes.
Therefore, this study was conducted to assess the antioxidant capacity using the DPPH and ABTS + (2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) assays, as well as to analyze the content of phenolic compounds through high-performance liquid chromatography (HPLC) in different cultivars of C. morifolium.

Plant materials
Six cultivars of C. morifolium (Table 1, Fig. 1) were grown by Prof. Jinhee Lim, Sejong University, Republic of Korea, in September 2022.The leaves and flowers of these cultivars were freshly harvested between December 2022 and January 2023 (16-week-old) and dried.Then, they were cut into small pieces prior to extraction.All specimens (S1-S12) were deposited at the herbarium of the Department of Bio-Industry Resources Engineering, Sejong University, Seoul, Republic of Korea.

Sample extraction and preparation
A quantity of 5 g of dried samples was subjected to extraction using ethanol (EtOH) in a reflux extractor over a period of 3 h.This extraction process was replicated 3 times [29].Following the utilization of a rotary evaporator, the resulting dehydrated extracts were gathered, and the extraction yield was calculated (Table 2).Each extract (10 mg) was precisely measured and diluted with 1 mL EtOH and distilled water to form a stock for the DPPH and ABTS + assays, respectively.After filtering through a 0.45 µm membrane filter, sequential dilutions were performed on the stocks to plot a calibration curve for each sample.HPLC analysis preparation involved the extract being 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.A total of 12 standard compounds were precisely weighed at 2 mg and dissolved in 1 mL of MeOH to create stock solutions (2000 ppm) for each standard.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 assay commenced by creating a functional solution with a concentration of 0.2 mM DPPH.Here, the original DPPH stock solution was diluted using 95% EtOH.Next, a mixture was prepared by combining 10 µL of the test solution with 200 µL of the DPPH working solution in each well of a 96-well plate.This combination was repeated 3 times to ensure accuracy.Then, the solutions were mixed thoroughly using a microplate shaker and placed in darkness to incubate for a duration of 30 min.Subsequently, the absorbance was measured at a wavelength of 514 nm.The calculation of the DPPH radical-scavenging rate played a pivotal role in constructing the calibration curves.Ascorbic acid was employed as the standard and used as a reference.

ABTS + radical scavenging activity
The ABTS + radical-scavenging assay was performed by diluting the ABTS + solution with water to create the ABTS + working solution.Subsequently, each test solution (10 µL) was combined with the ABTS + working solution (200 µL) and added to each well of a 96-well plate, with the reaction being replicated 3 times for accuracy.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 rate calculation was used to construct the calibration curves.Ascorbic acid was employed as the standard for the purpose of comparison.

HPLC condition
Quantitative 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 to 5 min, 20% B at 10 min, 25% B at 20 min, 30% B at 30 min, 40% B at 35 min, and 100% B at 40 min, which was maintained until 45 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.

Statistical analysis
All statistical analyses were performed using the software Minitab 16.0.Significant differences between the results were calculated by using anova analysis (ANOVA) and multiple comparisons of the Tukey test, with a significance level of p < 0.05.

Results and discussion
DPPH is a chemical compound that is frequently employed in laboratory experiments to evaluate the antioxidant activity of various substances [30].It is a stable free radical that is characterized by a deep purple color and possesses an unpaired electron, which renders it highly reactive.Antioxidants reduce DPPH by donating an electron, thereby causing a color change to yellow, which can be measured using spectrophotometry [31].The extent of this color change is directly proportional to the antioxidant activity of the tested compound.
ABTS + , like DPPH, is commonly used in laboratory experiments to assess antioxidant activity [32].When ABTS + is in its oxidized form, it becomes a stable radical cation with a blue-green color due to unpaired electrons.Antioxidants added to a solution with ABTS + donate electrons to the radical, reducing it, which causes the color to change from blue-green to colorless [33].The results of the antioxidant capacity assessment of S1-S12, by the ABTS + assay (Table 4), closely mirrored those obtained by the DPPH assay.Specifically, the highest antioxidant capacity was observed in S10 (IC 50 = 1.1 mg/ mL), while the lowest was noted in S2 (IC 50 = 8.9 mg/ mL).Similarly, when assessing the antioxidant capacity in the leaf samples, S6 (IC 50 = 2.1 mg/mL) demonstrated the highest antioxidant capacity, followed by S1 (IC 50 = 2.7 mg/mL), S5 (IC 50 = 3.1 mg/mL), S3 (IC 50 = 4.1 mg/mL), and S4 (IC 50 = 5.7 mg/mL), with the lowest capacity observed in S2 (IC 50 = 8.9 mg/mL).
Analysis of the flower samples by both the DPPH and ABTS + assays revealed that S10 (IC 50 = 1.1 mg/mL) exhibited the highest antioxidant capacity.Conversely, S11 (IC 50 = 3.9 mg/mL) and S8 (IC 50 = 6.1 mg/mL) displayed comparatively lower antioxidant capacities than the other samples.However, there was a slight difference between the two assays.Particularly, in the DPPH assay, which showed that S12 had a higher antioxidant capacity compared to S7 and S9, whereas the ABTS + assay indicated that S9 (IC 50 = 2.0 mg/mL) had a higher antioxidant capacity than S12 (IC 50 = 2.2 mg/ mL) and S7 (IC 50 = 3.3 mg/mL).This disparity can be attributed to various factors, including differences in wavelength, the number of unpaired electrons, and the sensitivity of the assays.Moreover, the DPPH assay contains some drawbacks.One notable characteristic of this assay is its lack of specificity in the assessment of free radical scavenging, as it quantifies scavenged free radicals originating from a spectrum of compounds, thereby encompassing both the phenolic and non-phenolic constituents, such as ascorbic acid [34].
Additionally, comparisons within the same cultivar exhibited that the flower samples of 'Ilonka' , 'Silvia' , and  (EC 50 = 0.180 mg/mL) radical scavenging activities [35].Compared to the results of the present study, the antioxidant activities of C. morifolium were significantly stronger than broccoli.Phenolic compounds are renowned for their antioxidative attributes and exhibit a spectrum of biological activities, which can provide potential advantages for human health [36].They can help protect cells and tissues from oxidative stress by neutralizing harmful free radicals, which can damage DNA, proteins, and lipids.Moreover, the intake of dietary sources abundant in phenolic compounds has been associated with a range of health advantages, including a diminished susceptibility to chronic ailments, such as cardiovascular diseases, malignancies, and neurodegenerative conditions.These compounds may also possess antiinflammatory and antimicrobial effects [37,38].
Quantitative analysis of the content of 12 phenolic compounds was conducted using HPLC/UV.Among these compounds, isoschaftoside was found in trace amounts in four samples (S1, S2, S4, and S6), and was undetectable in the remaining samples.Consequently, only 11 phenolic compounds were quantified.The HPLC chromatogram for all the standard compounds can be seen in Fig. 3.The retention times, calibration equations, and correlation factors for the 11 quantified phenolic compounds are presented in Table 5.In general, these compounds exhibited distinct separation with retention times ranging from 11.35 to 41.26 min.The coefficient of determination (r 2 ) exceeded 0.9990 for all compounds, thereby demonstrating the excellent linearity of the used quantification method.Additionally, the quantification of the contents of the individual compounds in the samples was calculated by the calibration equation, with Y representing a specific peak area and X denoting the compound concentration (Table 6).The chromatograms for all 12 cultivar samples are provided in Fig. 4.
In a recent investigation, it was documented that dandelion (Taraxacum mongolicum Hand.-Mazz.)exhibited a capacity to enhance the exercise performance of mice afflicted with liver dysfunctions [40].The predominant constituents identified in dandelion included luteolin, rutin, isoquercitrin, myricitrin, chlorogenic acid, gallic acid, caffeic acid, p-coumaric acid, protocatechuic acid, and isochlorogenic acid A. These constituents are quite similar to the compounds presented in the six C. morifolium cultivars; therefore, it can be expected that in addition to the antioxidant activities, C. morifolium may also have medicinal properties similar to dandelions, or other important medicinal properties.

Table 1
List of six examined C. morifolium cultivars

Table 2
Extraction yield from 12 samples

Table 4
ABTS + radical scavenging activity of S1-S12 a AA represents ascorbic acid, which was the positive control a-k Different letters in the same column indicate significant statistical differences (p < 0.05)

Table 5
Calibration curve equations for compounds 1-12a Y = peak area, X = concentration of the standard (µg/mL) b r 2 = coefficient of determination for five calibration data points (n = 3)

Table 6
Quantification of phenolic compound contents in S1 -S12