Quantification of Antioxidant Phenolic Compounds in a New Chrysanthemum Cultivar by High-Performance Liquid Chromatography with Diode Array Detection and Electrospray Ionization Mass Spectrometry

The flowers of Chrysanthemum morifolium Ramat. have been used as an herbal tea and in traditional medicine, and the plant has been developed to produce horticultural cultivars of various colors and shapes. In this study, a new chrysanthemum cultivar with dark purple petals (C. morifolium cv. ARTI-Dark Chocolate; ADC) was developed by radiation-induced mutation breeding of its original cultivar with purple striped white petals (C. morifolium cv. Noble Wine, NW). The phenolic profile and antioxidant property of ADC were investigated and compared with NW and the commercially available medicinal herb, C. morifolium with yellow petals (CM), in order to find a scientific support to produce a new source of natural antioxidant. Flavonoid and phenolic acid profiles of the ethanol extracts of the three flowers were analyzed by high-performance liquid chromatography-diode array detector-electrospray ionization mass spectrometry (HPLC-DAD-ESIMS), while antioxidant properties were evaluated using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assay. Among the tested flowers, ADC possessed the strongest antioxidant capacity and the highest phenolic contents. Flavonoids (acacetin, apigenin, luteolin, acacetin-7-O-β-glucoside, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, and linarin) and phenolic acids (chlorogenic acid and mixture of 1,4-, 1,5-, and 3,5-dicaffeoylquinic acids) were identified and quantified.


Introduction
The dried flowers of Chrysanthemum morifolium Ramat. (Compositae), known as Chrysanthemi Flos, have been used as an infusion tea and in traditional medicine to treat inflammation, arteriosclerosis, and hypertension [1]. In previous phytochemical studies on C. morifolium, flavonoids and phenolic acids have been identified as major constituents [2], which have exhibited diverse biological activities such as antioxidant [3], anti-inflammatory [4], and antitumor effects [5].
Numerous varieties and cultivars have been developed by hybridization and mutation for horticultural purposes [6] and to improve crop productivity and quality [7]. Mutation is widely used in plant breeding research and generated by spontaneous mutation, ultraviolet lights, chemical mutagens, and ionizing radiation (i.e., X-rays and gamma-rays) [7]. More than 3,000 mutant varieties of plants have been registered with the Food and Agriculture Organization/International Atomic Energy Agency (FAO/ IAEA), most of which were developed with gammairradiation (available online at http://mvd.iaea.org). Recently, our research group has developed a new chrysanthemum cultivar, C. morifolium cv. ARTI-Dark Chocolate (ADC), by gamma-irradiation on stem cuttings of the chrysanthemum cultivar, C. morifolium cv. Noble Wine (NW), and registered a new plant variety (registration number 4996) in the Korea Seed and Variety Service (available online at https://www.seed.go.kr/english/function/system_06.jsp) ( Figure 1). As part of our ongoing search for functional resources from new cultivars developed by radiation-induced mutation breeding, we assessed a 95% ethanol extract of the flowers of ADC. The extract showed higher antioxidant activity in the DPPH and ABTS radical scavenging assays than did extracts of the original cultivar, NW, and the commercially available medicinal herb C. morifolium with yellow petals (CM). Therefore, the ethanol extract was subjected to bioactivity-guided fractionation, leading to the isolation of seven flavonoids. In addition to the flavonoids isolated, four phenolic acids were found to be the constituents of the flowers of ADC by HPLC-DAD-ESIMS analysis. To identify the differences in the phytochemical contents of the three flowers depending on their activities, HPLC-DAD-ESIMS analysis was performed on standards and samples of the 95% ethanol extracts of each of the flowers.

Plant
Material. C. morifolium cv. ARTI-Dark Chocolate was produced in the same manner described by Jo et al. [8]. C. morifolium cv. ARTI-Dark Chocolate with dark purple petals was developed by 50 Gy gamma-irradiation from a labeled Cobalt ( 60 Co) source on stem cuttings of C. morifolium cv. Noble Wine, which is a spray-type chrysanthemum cultivar that has white petals with purple stripes. And then, ADC flowers were selected according to petal-color variants and were examined to be of stable inheritance of phenotype for four years (2009)(2010)(2011)(2012).

Preparation of Sample and Standard Solutions.
Dried flowers of ADC (30 g), NW (30 g), and CM (30 g) were extracted three times with 95% EtOH (200 mL) for 24 h at room temperature, respectively. The extracted solutions were filtered through filter paper and evaporated in vacuo to afford dryness to each (ADC, 6.27 g, w/w 20.9%; NW, 7.96 g, w/w 26.53%; CM, 9.93 g, w/w 33.1%). All 95% EtOH extracts were weighed accurately and dissolved in MeOH at 10 mg/mL. The sample solution was filtered through a syringe filter (0.45 m) for HPLC analysis. The standards were weighed accurately and dissolved in MeOH at 1.0 mg/mL. The stock solutions were diluted to yield a series of standard solutions at four different concentrations (25,50,75, and 100 g/mL) for quantitative analysis.

Evaluation of Antioxidant
Activity. Antioxidant activities of the flowers were measured using the following assays.
2.6.1. DPPH Free Radical Scavenging Activity. The DPPH of the 95% EtOH extract of each plant materials was determined by Brand-Williams's method [9]. Briefly, each extract was suspended in DMSO and 100 L of the sample was reacted with 100 L of 0.2 mM DPPH solution. Absorbance measurements were taken 30 min after the reaction at 517 nm using an ELISA reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The concentration of the extract was calculated from the log-dose inhibition curve for 50% inhibition of free radicals (SC 50 ).

ABTS Radical Cation Scavenging
Activity. The ABTS of the 95% EtOH extract of each type of plant and the solvent fractions of ADC was evaluated using the method published by Re et al. [10]. In brief, the ABTS was measured by preformed radical monocation. The mixtures, along with 7.4 mM ABTS solution and 2.6 mM potassium persulfate, were incubated at room temperature in the dark for 24 hours. The ABTS solution was diluted with phosphate-buffered saline (pH 7.4) to achieve an absorbance of 0.7 ± 0.03 at 732 nm. Each of the samples was suspended in DMSO and 50 L of the sample was reacted with 950 L of the ABTS solution. Absorbance was taken 10 min after the reaction at 732 nm using an ELISA reader (Benchmark Plus, Bio-Rad, Hercules). The concentration of the extract was calculated from the log-dose inhibition curve for 50% inhibition of free radicals (SC 50 ).

Statistical Analysis.
Each experiment was done in triplicate and all data are presented as the mean ± standard deviation (SD). Statistical differences were determined using Student's -test. The significant level was set at < 0.05.

Results and Discussion
The 95% ethanol extract of ADC, NW, and CM were evaluated for their antioxidant activity using the ATBS and DPPH radical scavenging assays. As shown in Table 1, ADC extract showed higher radical scavenging activity than did NW and CM extract. Therefore, ADC extract was sequentially partitioned with n-hexane, chloroform, ethyl acetate, and nbutanol and the solvent fractions were also tested for their ABTS radical scavenging activities (Table 1). Among those tested, the ethyl acetate-soluble fraction of ADC exhibited potent inhibitory activity, with an SC 50 value of 42.84 g/mL, and it was further subjected to bioactivity-guided fractionation for the isolation of active lead compounds. The antioxidant activity of ADC could presumably be attributed to certain antioxidant components, for instance, phenolic compounds, which has a wide range of health benefits [11].
To find out the different phytochemical profiles among ADC, NW, and CM depending on their activities, HPLC-DAD-ESIMS analysis was performed on standards and samples of each 95% ethanol extract of the three flowers.  Figure 2: Chemical structures of compounds found in ADC flowers. Values (mean ± SD) of extracts and fractions analyzed individually in triplicate; ascorbic acid was used as a positive control; NT: not tested.
A quantitative analysis of flavonoids and phenolic acids found in ADC was performed using HPLC-DAD-ESIMS. The established method is described in Section 2.5. HPLC-DAD-ESIMS chromatograms of the 95% ethanol extract of the three flowers and the standard solution are shown in Figure 3 and data for each peak are listed in Table 2. The linear relationships between the peak areas ( ) and concentrations ( , g/ml) of the compounds were calculated by regression equations ( = + ; : slope; : intercept). The calibration curves showed a high degree of linearity with a correlation coefficient of r 2 > 0.999 over the concentration range 25-100 g/ml. The limits of detection (LOD) and limits of quantification (LOQ) for the 8 compounds and a mixture of 9-11 were in the range of 0.057-0.716 g/ml and 0.173-2.168 g/ml, respectively. This analytical method  International Journal of Analytical Chemistry   was applied to the simultaneous quantification of the 8 compounds and a mixture of compounds 9-11 from the three flowers, and the results are shown in Table 3. An increase in flavonoid glycosides and phenolic acids and a decrease in flavone aglycones were observed in ADC extract, compared to the extract from NW and CM ( have exhibited their antioxidant effects by attenuating the oxidative damage thorough activation of nuclear factorerythroid 2-related factor 2 (Nrf2) pathway. There have also been reports that acacetin-7-O--glucoside (4) showed moderate DPPH radical scavenging activity [21]; however 3,5-DCQA (11) exhibited strong DPPH and ABTS radical scavenging and ferric reducing antioxidant power (FRAP) activities [22]. According to these previous reports, the high content of total phenolic compound in ADC extract was most likely responsible for its antioxidant activity.
Studies on different types of flavonoids and their known functions inside the plant have reported that flavonoid glycosides were involved in protection against radiation stress and in producing purple pigment [23]. In addition, there have been reports that the generation of caffeoylquinic acids was induced in an anthocyanin-accumulating plant cell line such as a purple sweet potato [24], and eventually the biosynthetic pathway of phenolic compounds can be closely related to that of anthocyanins [25]. Thus, a color change to purple of ADC flower petal was considerably affected by radiation-induced mutagenesis and seems to be mediated by the accumulation of flavonoid glycosides and caffeoylquinic acids.

Conclusion
In conclusion, the radiation-induced mutant cultivar, ADC extract, exhibited stronger antioxidant activity, compared to the extracts from the control plant, NW, and the medicinal herb, CM. The enhancement of the antioxidant properties of ADC flower extract is due to higher concentrations of antioxidant phenolic compounds. Phenolic profiles determined by HPLC-DAD-ESIMS revealed seven flavonoids and four phenolic acids which were tentatively identified and quantified. Therefore, these results make ADC flowers a very promising source of natural-occurring antioxidants as well as a commercially useful product. Further studies on its processing suitability analysis and detailed mechanism of action in our laboratory are being developed for commercialization of ADC flower as a new purple colored chrysanthemum tea or a dietary supplement.

Conflicts of Interest
The authors declare no conflicts of interest regarding publication of this paper.