Anthocyanin and Carotenoid Contents in Different Cultivars of Chrysanthemum (Dendranthema grandiflorum Ramat.) Flower

The flowers of twenty-three cultivars of Dendranthema grandiflorum Ramat. were investigated to determine anthocyanin and carotenoid levels and to confirm the effects of the pigments on the flower colors using high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS). The cultivars contained the anthocyanins cyanidin 3-glucoside (C3g) and cyanidin 3-(3ʺ-malonoyl) glucoside (C3mg) and the following carotenoids: lutein, zeaxanthin, β-cryptoxanthin, 13-cis-β-carotene, α-carotene, trans-β-carotene, and 9-cis-β-carotene. The cultivar “Magic” showed the greatest accumulation of total and individual anthocyanins, including C3g and C3gm. On the other hand, the highest level of lutein and zeaxanthin was noted in the cultivar “Il Weol”. The cultivar “Anastasia” contained the highest amount of carotenoids such as trans-β-carotene, 9-cis-β-carotene, and 13-cis-β-carotene. The highest accumulation of β-cryptoxanthin and α-carotene was noted in the cultivar “Anastasia” and “Il Weol”. Our results suggested that ‘Magic”, “Angel” and “Relance’ had high amounts of anthocyanins and showed a wide range of red and purple colors in their petals, whereas “Il Weol’, “Popcorn Ball’ and “Anastasia” produced higher carotenoid contents and displayed yellow or green petal colors. Interestingly, “Green Pang Pang”, which contained a high level of anthocyanins and a medium level of carotenoids, showed the deep green colored petals. “Kastelli”, had high level of carotenoids as well as a medium level of anthocyanins and showed orange and red colored petals. It was concluded that each pigment is responsible for the petal’s colors and the compositions of the pigments affect their flower colors and that the cultivars could be a good source for pharmaceutical, floriculture, and pigment industries.


Introduction
The garden chrysanthemum, Dendranthema grandiflorum Ramat. (Chrysanthemum morifolium Ramat., Kitamura, 1978), is a perennial plant belonging to the Asteraceae (Compositae) family. It has been cultivated for more than 3000 years. The flowers of D. grandiflorum have been consumed as herbal medicines, beverages, and vegetables in China, Japan, Thailand, and the Republic of Korea for centuries [1][2][3]. They are also among the most popular cut flowers in the floriculture industry of China, United States, and Europe due to their high ornamental features, including various floral colors and shapes, uniform flowering, and many spray flowers [4,5].
Flower color depends on the grade of accumulation of secondary compounds, including flavonoids, carotenoids, or betalains. Anthocyanins, members of the flavonoid group of phytochemicals, are mainly involved in color development of a wide range of orange to red and purple to blue flowers. Flower colors play a major role in attracting pollinators, protecting against damage from ultraviolet irradiation, and as key signals between plants and microbes [6][7][8]. Chrysanthemum cultivars are considered a good source for the extraction of anthocyanins since these cultivars have numerous flowers in a single plant with wide variations of flower color [8].
Carotenoids are natural pigments that impart yellow, red, or orange colors to flowers and are constituted by C40 isoprenoid compounds with or without epoxy, hydroxy, and keto groups [9]. Most plants have similar carotenoid contents, including both β,ε-carotenoids and β,β-carotenoids, in their green tissues [10]. In contrast, the carotenoid profiles in non-green tissues show qualitative differences depending on plant species. For example, xanthophylls, which impart pale to deep yellow colors, accumulate significantly in the flower petals in most plants [6]. However, the petals of several plants such as Calendula officinalis are able to synthesize unique carotenoids such as lycopene that are absent in yellow petals and present in orange to red petals [11]. These distinctive carotenoid compositions enable plants to have various petal colors.
Pharmacological studies reported that chrysanthemum species contain secondary compounds having various biological characteristics. Various flavonoids, alkaloids, phenolic compounds, and triterpene constituents were identified from the aqueous methanol extract of a chrysanthemum flower. These components were found to have biological activities such as anti-inflammatory, antibacterial, antifungal, anti-spirochetal, anti-human immunodeficiency virus, and anti-oxidant activities [12][13][14].
Chrysanthemum cultivars have been studied in many scientific fields, including pharmacology, morphology, genetics, genetic engineering, and horticulture; however, few studies have investigated the biosynthesis of anthocyanins. Recently, It was reported that the total anthocyanin content in flower tepals of fifteen spray chrysanthemum cultivars grown at a polyhouse and under open field conditions [8].
The aim of this study is to determine anthocyanin and carotenoid contents responsible for petals colors in the twenty-three chrysanthemum cultivars and to confirm the effect of the pigments on the flower colors by using high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS).

Analysis of Individual Anthocyanin Contents
The flowers of the twenty-three cultivars were analyzed for the biosynthesis of anthocyanins using HPLC and ESI-MS. The anthocyanin components were identified by their retention time, elution order, and fragmentation patterns. Five anthocyanin standards (malvidin-3-O-glucoside chloride: retention time (RT) 17.557; cyanidin-3-O-glucoside chloride: 17.701; pelargonidin-3-O-glucoside chloride: 18.925; peonidin-3-O-glucoside chloride: 19.751; and cyanidin-3-O-rutinoside chloride: 20.331), cyanidin 3-glucoside (C3g), cyanidin 3-(3ʺmalonoyl) glucoside (C3mg) were used for the anthocyanin analysis in the cultivars. The chromatogram showed three peaks corresponding to three different anthocyanins such as C3g, C3mg, and an unknown anthocyanin by the retention time ( Figure 1). Table 1 Table 2 presents the significantly wide variation of individual anthocyanin contents, including C3g, C3mg, and unknown anthocyanin contents. The extract of "Magic' showed the highest level of C3g and C3gm. Interestingly, the unknown anthocyanin was quantified in several cultivars. The cultivar "Relance" showed the significantly highest accumulation of the unknown anthocyanin, followed by "Angel", "Magic", and "Green Pang Pang". The other cultivars also contained some amount of the unknown anthocyanin ( Table 2).  Table 1 for the identification of each numbered peak.   This suggested that the deep purple and red colored flowers of these cultivars, including "Magic", "Angel", "Relance", and "Green Pang Pang" contained more anthocyanin contents than other flowers ( Figure 2

Analysis of Total Anthocyanin Contents
The cultivars were classified into the following four groups according to the total anthocyanin values: nothing detected (ND), low level (0-5 mg/g Dry Weight (DW)), medium level (5-10 mg/g DW), and high level (>10 mg/g DW) ( Table 1). The limit of detection was 0.05 mg/g dry wt. The highest level of total anthocyanin was quantified in flowers of the cultivars "Magic", followed by "Angel", "Relance", and "Green Pang Pang" ( Table 3). The cultivars with purple or red colored flowers mostly showed a greater amount of total anthocyanins. On the other hand, the cultivars with medium and low levels of anthocyanin showed light and faint colors or colored spots at the end of petals. The petals of cultivars in the ND group were mostly white, yellow, light green, and very light red in color. This might be because anthocyanin, a strong soluble pigment, mainly affects the development of orange to red and purple to blue colored flowers ( Figure 2). The values given in parenthesis under the name of cultivars indicated the mean (mg/g) ± standard deviation (SD) of anthocyanin content of the respective cultivars by triplicate experiments

Analysis of Individual Carotenoid Contents
HPLC analysis of the cultivars suggested that most varieties contained all seven types of carotenoids. In particular, lutein was detected in large amounts, that varied widely among the cultivars. The highest lutein level, which was 26.1 times higher than that in the cultivar with the lowest level ("Fire Pink") was found in "Il Weol". The cultivars "Popcorn Ball" and "Anastasia" also had high levels of lutein. The precursor of lutein, α-carotene, was identified in all the varieties. The highest level of α-carotene was found in "Il Weol", which was 87.50 times higher than that in the cultivar ("Fancy Ball") with the lowest level. In all the cultivars, lutein content was considerably higher than that of α-carotene, suggesting that α-carotene might be transformed into lutein in large amounts in the cultivars. The second highest carotenoid produced was trans-β-carotene. The cultivar "Anastasia" showed the highest accumulation of trans-β-carotene, followed by "Green Pang Pang" and "Il Weol". The cultivar "Fancy Ball" showed the lowest accumulation, which was 39 times lower than that in "Anastasia". The isomer of β-carotene, 13-cis-β-carotene ranged from 0.13 ± 0.01 µg/g DW ("Fancy Ball") to 5.62 ± 1.75 µg/g DW ("Anastasia"); there was 43.23-fold difference in the concentration of this compound between the two cultivars. Similarly, 9-cis-β-carotene, another isomer of β-carotene, showed the highest and lowest levels in "Anastasia' and "Fancy Ball" respectively, with a 17.86-fold difference between the two.
Previous studies reported that cis-isomers are not present in raw carrots, tomatoes, and sweet potatoes, and that 98% of the β-carotene is present as the precursor in raw carrots [15,16]. Our study obtained similar result that all cultivars contained more trans-β-carotene contents than 9-cis-β-carotene and 13-cis-β-carotene. The content of β-cryptoxanthin, which is formed from β-carotene, was 22.89 times more in the cultivar with the highest level ("Il Weol") than that in the cultivar with the lowest accumulation ("Magic"). Next to lutein and trans-β-carotene, the most accumulated carotenoid was zeaxanthin, which is transformed from β-cryptoxanthin. The values among the cultivars ranged from 0.14 ± 0.02 µg/g DW to 4.38 ± 1.26 µg/g DW. The highest level was found in "Il Weol" which was 31.29 times higher than that in the cultivar ("Fire Pink") with the lowest level. The production of zeaxanthin was higher than that of β-cryptoxanthin in all the cultivars except for "Anastasia" and "Green Pang Pang", suggesting that β-cryptoxanthin was not completely transformed to zeaxanthin in these two cultivars (Table 4). All values are written as the mean (μg/g) ± standard deviation (SD) of three replications. The amounts are expressed as μg of the target compound per g of the plant on a dry weight basis. Means values in a common letter were not significantly different at p < 0.05 using Duncan Multiple Range Test. The background color in each column presents the flower color of the cultivar.

Analysis of Total Carotenoid Contents
Analysis of the petals of the chrysanthemum varieties revealed the presence of carotenoid components such as lutein, zeaxanthin, β-cryptoxanthin, α-carotene, 13-cis-β-carotene, trans-β-carotene, and 9-cis-β-carotene, and the concentration of these components varied significantly (Table 4). The cultivars were classified on the basis of the level of total carotenoid production ( Table 5). Table 5. Different groups of chrysanthemums according to the amount of total carotenoid in the twenty-three Dendranthema grandiflorum Ramat. cultivars.

The Low Level
The Medium Level The High Level (0-50 μg/g Dry wt.) (50-100 μg/g Dry wt.) (>100 μg/g Dry wt.) Fire Pink 19.43 ± 3.94 grouped into high level group based on the high amount of total and individual anthocyanins (Table 3). On the other hand, according to carotenoid contents, they were classified into the low level group ( Table 5). The cultivars "Il Weol", "Popcorn Ball", and "Anastasia" with deep yellow and green colored petals (Figure 2) showed the highest levels of carotenoid contents (Table 5). However, according to anthocyanin contents, "Popcorn Ball" and "Anastasia" were included in the ND group, and "Il Weol" was included in the low level group. Other cultivars also showed similar findings except for "Green Pang Pang" and "Kastelli".
Interestingly, "Green Pang Pang" which had a high level of anthocyanins (11.38 ± 0.18 mg/g DW) as well as a medium level of carotenoids (98.66 ± 5.03 μg/g DW) had deep green colored petals (Tables 3 and 5). Even if it had high level of anthocyanins, it did not have the typical orange and red to purple color range in its petals. It was therefore assumed that the combination of the pigments influenced the color determination of petals. Similarly, "Kastelli", which had a high level of carotenoids (102.28 ± 7.70 μg/g DW) and a medium level of anthocyanins (7.95 ± 0.04 mg/g DW), showed orange and red colored petals (Tables 3 and 5). The flower color is known to be determined on the basis of the production, interaction, and breakdown of pigments such as carotenoids located in plastids and anthocyanins found in vacuoles [19]. In particular, orange colored petals result from the coexistence of both those pigments. The ratio of the amount of these pigments allows the variation in orange color in the petals of Psorophora howardii [20].
Most cultivars that were classified as ND or low level showed faint/pale colored or white colored petals. Although these cultivars lack pigments, they might have factors that inhibit the pigments. It was reported that the biosynthesis of carotenoids could impart white colors to petals in chrysanthemums, since the component was subsequently degraded to colorless compounds by a factor, carotenoid cleavage dioxygenase (CmCCD4a) [21].

Extraction of Anthocyanins, Including C3g and C3mg, and HPLC Analysis
The flowers of the twenty-three cultivars were individually ground to powder after freeze-drying. Next, the powder (0.1 g) was added to a 2.0 mL Eppendorf tube, and then extraction solution (water-formic acid, 95:5 (v/v), 2 mL) was added to the tube. The tube was vortexed and sonicated for 5 min. Subsequently, the tube was centrifuged at 12,000 rpm at 4 °C for 15 min using a MICRO 17R micro-centrifuge (Hanil, Incheon, Korea). The supernatant was filtered through a 0.45-µm polytetrafluoro-ethylene hydrophilic syringe filter and collected into vials. Each extraction was repeated in triplicate. C3gl, C3gm, and unknown anthocyanin contents were separated using a reversed-phase Synergi 4-µm POLAR-RP 80A (250 × 4.6 mm; particle size, 4 µm; Phenomenex, Torrance, CA, USA) equipped with a Security Guard Cartridge kit (AQ C18; 4 × 3.0 mm i.d.; KJO-4282; Phenomenex) using a 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA); the oven temperature was set to 40 °C; detection wavelength, 520 nm; and flow rate, 1.0 mL/min. Solvent A was water-formic acid, 95:5 (v/v), and acetonitrile-formic acid, 95:5 (v/v) was used as solvent B in the mobile phase. The gradient conditions were set as follows: a linear step from 5% solvent B for 0.0 min, 50% solvent B for the next 30.00 min, followed by a rapid drop to 5% B for 30.10 min, and then isocratic conditions with 5% B for 40.00 min (total 100.10 min). Individual C3gl and C3gm levels were quantified using their HPLC area and response factor; they were compared with an external standard, i.e., 5 mL of sinigrin solution (0.1 mg/mL) that was subjected to the same extraction process [19].

LC/ESI-MS Analysis for the Quantification of Anthocyanin Contents
The ESI-MS data were analyzed using a 4000 Qtrap LC/MS/MS system (Applied Biosystems Instrument, Foster City, CA, USA) in the positive ion mode ([M + H] + ) connected with an Agilent 1200 series HPLC. The LC/MS analytical conditions for anthocyanins in the chrysanthemum cultivars were set as follows: scan range, 100-1300 m/z; scan time, 4.80 s; curtain gas, 20.00 psi (N2); heating gas temperature, 550 °C ; nebulizing gas, 50.00 psi; heating gas, 50.00 psi; ion spray voltage, 5500 V; declustering potential, 100 V; and entrance potential, 10 V.

Statistical Analysis
Data was analyzed using the computer software Statistical Analysis System (SAS, system 9.3, 2012; SAS Institute, Inc., Cary, NC, USA). Means were separated by Duncan's Multiple Range Test (DMRT). The experimental results were presented as mean ± standard deviation of triplicate experiments.

Conclusions
In this study, the pigments such as anthocyanins and carotenoids were profiled and quantified in twenty-three cultivars of chrysanthemum (Dendranthema grandiflorum Ramat.). The results of our study suggested the cultivars "Magic", "Angel", and "Relance", which had deep red or purple colored petals, are suitable powerful sources of anthocyanins due to their high anthocyanin contents, whereas "Il Weol", "Popcorn Ball", and "Anastasia", which showed deep yellow or green colored petals, areconsidered a good source for the carotenoids because of high production of carotenoid contents. Interestingly, "Green Pang Pang" showed deep green colored petals even though it contained a high level of anthocyanins and a medium level of carotenoids. Similarly, "Kastelli" with high levels of carotenoids and a medium level of anthocyanins showed orange and red colored petals. Consequently, it was confirmed that the pigments responsible for the petals colors are found in most cultivars and their pigment components affect the flower colors of the cultivars. Further researches on the molecular characterization and the metabolic profiling are necessary to explain the accumulation and interaction of the secondary metabolites leading to the various petals colors in the cultivars.