Composition and Antioxidant Activities of Volatile Organic Compounds in Radiation-Bred Coreopsis Cultivars

Abstract

Coreopsis is a flowering plant belonging to the Asteraceae family. It is an ornamental plant native to the Americas, Asia and Oceania and its flower is used as a raw material for tea and food manufacture in China. In this study, new cultivars of C. rosea (“golden ring”) were developed via radiation-induced mutation of the original cultivar, “pumpkin pie”. The chemical composition and antioxidant activities of flowers belonging to three different Coreopsis cultivars were evaluated: “golden ring”, “pumpkin pie” and “snow chrysanthemum” (coreopsis tea; C. tinctoria). The volatile compounds were characterized via gas chromatography-mass spectrometry (GC-MS) and 50–59 oils representing 95.3–96.8% of the total volatile compounds in these flower materials were identified. ”Golden ring” contained a high amount of fatty acids (38.13%), while “pumpkin pie” and “snow chrysanthemum” teas were rich in aliphatic amides (43.01%) and esters (67.22%), respectively. The antioxidant activities of the volatile oils of these cultivars were evaluated using 1,1-diphenyl-2-picrylhydraxyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays. The volatile extract of “golden ring” showed higher antioxidant activities compared with the extracts of the other cultivars. Therefore, “golden ring” can be used for further development as a raw material for tea manufacture or as a dietary supplement.

1. Introduction

Approximately 80 species of Coreopsis (Asteraceae) are native to North America and are currently distributed throughout the Americas, Asia and Oceania [1]. Coreopsis thrives as annual and perennial types, and is an ornamental plant used to decorate gardens and roadsides [1,2]. In addition, the ethnopharmacological use of this plant’s flower has been reported [3]. It has been traditionally used to treat diarrhea, vomiting, and bleeding in North America [3]. The Coreopsis flower has been consumed as a drink to control diabetes in China and Portugal [3]. Currently, the flowers of C. tinctoria, known as “snow chrysanthemum”, are used as tea, with detoxifying and cooling effects, in China [4,5]. In a previous phytochemical study of Coreopsis species, phenolics, flavonoids (aurones, chalcones, and flavones), acetylenes and volatile oils were reported [2,3,4,5,6,7,8,9]. The plant’s extracts or constituents have been found to exhibit diverse biological activities such as antileukemic [2], antidiabetic [3,7], antioxidant [5,6], anti-inflammatory [7,8] and chemopreventive effects [9].

Hybridization between plants and mutations has yielded numerous varieties with high crop productivity and improved quality [10,11]. Mutation breeding via spontaneous mutation, ultraviolet light, chemical mutagenesis and ionizing radiation can be used effectively to induce genetic diversity [11]. Approximately 50% of the mutant varieties registered in the joint Food and Agriculture Organization/International Atomic Energy Agency (FAO/IAEA) mutant variety database are mutants induced by gamma irradiation [12]. Coreopsis species are of little commercial importance, but gardeners’ preferences for them are increasing due to their cold resistance, attractive flowers and leaves and tolerance of various environmental conditions [13]. Therefore, our research group has developed a new mutant cultivar of C. rosea (“golden ring”) bred via the exposure of stem cuttings of the original cultivar, “pumpkin pie”, to gamma irradiation and registered it in the Korea Seed and Variety Service [14]. C. rosea has been mainly studied for breeding and culture growth [15]; however, no studies have reported its chemical constituents and pharmacological activity until now.

In this study, we analyzed the volatile oils of flowers obtained from a new cultivar of C. rosea (“golden ring”) compared with those of its original cultivar (“pumpkin pie”) and the commercially available coreopsis tea (“snow chrysanthemum”) as control groups (Figure 1), via gas chromatography-mass spectrometry (GC-MS). Their antioxidant activities were also evaluated using 1,1-diphenyl-2-picrylhydraxyl (DPPH) and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays.

2. Results and Discussion

2.1. Chemical Composition of Dichloromethane Extracts of Three Different Coreopsis Cultivars

The flowers of “pumpkin pie”, “golden ring” and “snow chrysanthemum” were extracted with dichloromethane. The volatile components of these flowers were identified based on their molecular formula, retention index (RI) and National Institute of Standards and Technology (NIST) library similarity index. The GC-MS analysis revealed a total of 89 components in these flowering materials, which led to the identification of 50 components in “snow chrysanthemum”, 58 in “pumpkin pie” and 59 in “golden ring” (

Table 1. Composition of volatile compounds of the dichloromethane extracts obtained from “golden ring”, “pumpkin pie” and “snow chrysanthemum”.

No.	Compound	Formula	RI 1	%Relative
				Golden Ring	Pumpkin Pie	Snow Chrysanthemum
1	Nonanal	C9H18O	1104	-	0.02	-
2	Octanoic acid ethyl ester	C10H20O2	1195	-	-	0.38
3	(E)-2-Decenal	C10H18O	1263	0.03	0.03	0.51
4	2,4-Decadienal	C10H16O	1296	-	-	0.17
5	(E,E)-2,4-Decadienal	C10H16O	1319	0.04	0.03	0.21
6	1-Methyl-4-(1-methylethenyl)-1,2-cyclohexanediol	C10H18O2	1347	-	-	0.46
7	n-Decanoic acid	C12H24O2	1356	0.28	0.05	-
8	2,4,7,9-Tetramethyl-5-decyn-4,7-diol	C14H26O2	1405	0.1	0.07	-
9	2,4-Bis(1,1-dimethylethyl)phenol	C14H24O2	1504	0.03	-	-
10	9-Oxo-nonanoic acid ethyl ester	C11H20O3	1500	-	-	0.37
11	5,6,7,7a-Tetrahydro-4,4,7a-trimethyl-2(4H)-benzofuranone	C11H16O2	1535	0.05	-	-
12	Dodecanoic acid	C12H24O2	1556	0.67	0.13	0.13
13	Fumaric acid ethyl 2-methylallyl ester	C10H14O4	1562	0.05	-	-
14	Hexadecane	C16H34	1599	-	-	-
15	Farnesene epxide	C15H24O	1610	-	0.03	-
16	Fluorene	C13H10	1644	0.06	0.02	-
17	(Z)-9-Tetradecenal	C14H26O	1659	-	-	0.89
18	Aromadendren oxide	C15H24O	1689	0.06	0.06	-
19	(Z)-trans-α-Bergamotol	C15H24O	1696	0.12	0.18	-
20	Heptadecane	C17H36	1699	0.03	-	-
21	Fluorenone	C13H8O	1709	0.09	0.03	-
22	Fluorene-4-carboxylic acid	C14H12O2	1728	-	0.09	-
23	Methyl trans-p-coumarate	C10H10O3	1742	-	0.05	-
24	2-Methyl-5-(1,2,2-trimethylcyclopentyl)phenol	C15H22O	1745	0.15	0.05	-
25	Tetradecanoic acid	C14H28O2	1754	0.46	0.43	0.2
26	(Z)-7-Hexadecenal	C16H30O	1761	-	-	0.07
27	Tetradecanoic acid ethyl ester	C16H32O2	1790	-	-	0.18
28	Octadecane	C18H38	1799	-	0.04	-
29	Cyclopentadecanon	C15H28O	1830	-	-	0.12
30	6,10,14-Trimethyl-2-pentadecanone	C18H36O	1838	0.16	0.06	0.08
31	Pentadecanoic acid	C15H30O2	1853	0.12	0.06	-
32	(Z)-9-Hexadecen-1-ol	C16H32O	1878	0.04	0.03	-
33	7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione	C17H34O3	1901	0.08	0.08	-
34	1,2-Dihydro-5-acenaphthylenecarboxaldehyde	C12H9NO2	1947	1.3	0.78	-
35	n-Hexadecanoic acid	C16H32O2	1961	7.83	3.87	1.42
36	Tetradecanamide	C14H29NO	1967	0.63	0.5	0.33
37	Ethyl 9-hexadecenoate	C18H34O2	1967	-	-	0.11
38	Hexadecanoic acid ethyl ester	C18H36O2	1991	-	-	14
39	11-Hexadecyn-1-ol	C18H34O2	1995	-	-	0.14
40	(Z)-9,17-Octadecadienal	C18H32O	2028	0.12	0.07	0.23
41	(Z,Z)-9,12-Octadecadien-1-ol	C18H34O	2048	0.07	0.03	-
42	9,12-Octadecadienoic acid methyl ester	C19H34O2	2087	0.05	-	-
43	Phytol	C20H40O	2105	0.29	0.16	-
44	(Z,Z)-9,12-Octadecadienoic acid	C18H32O2	2135	17.36	8.69	0.86
45	(Z,Z,Z)-9,12,15-Octadecatrienoic acid	C18H30O2	2139	6.86	4.65	0.35
46	Linoleic acid ethyl ester	C20H36O2	2155	-	-	16.89
47	17-Octadecynoic acid	C18H30O2	2158	2.59	2.93	-
48	Octadecanoic acid	C18H36O2	2160	1.96	1.4	-
49	Ethyl oleate	C18H38O2	2165	-	-	28.53
50	(E)-9-Octadecenoic acid ethyl ester	C18H38O2	2170	-	-	2.07
51	Hexadecanamide	C16H33NO	2175	2.11	2.51	0.1
52	Octadecanoic acid ethyl ester	C20H40O2	2190	-	-	1.67
53	Tricosane	C23H48	2299	0.33	0.31	0.35
54	(Z,Z)-11,14-Eicosadienoic acid methyl ester	C21H38O2	2334	0.26	-	-
55	Isopropyl linoleate	C21H38O2	2344	2.63	2.69	1.3
56	(Z)-9-Octadecenamide	C18H35NO	2363	28.38	37.48	12.57
57	Octadecanamide	C18H36NO	2381	0.54	0.56	1.5
58	PGH1 methyl ester	C22H38O2	2384	-	-	0.23
59	Eicosanoic acid ethyl ester	C22H44O2	2390	-	-	0.72
60	Tetracosane	C24H50	2399	0.2	0.28	0.18
61	13-Docosenoic acid	C22H44O2	2401	-	-	0.2
62	1,22-Docosanediol	C22H46O2	2413	-	-	1.55
63	1-Heptadec-1-ynyl-cyclohexanol	C23H42O	2425	-	-	0.12
64	Behenic alcohol	C22H46O	2488	0.39	0.26	0.4
65	Pentacosane	C25H52	2499	1.96	2.64	0.76
66	2-Hydroxy-1-(hydroxymethyl)ethyl palmitate	C19H38O4	2503	1.47	2.24	-
67	Bis(2-ethylhexyl) phthalate	C24H38O4	2523	0.53	1.08	0.23
68	(Z)-11-Eicosenamide	C20H39NO	2559	0.19	0.66	0.1
69	3,6-Nonadecadione	C19H36O2	2583	0.85	0.56	0.3
70	Ethyl docosanoate	C24H48O2	2591	-	-	0.23
71	Hexacosane	C26H54	2599	0.34	0.48	-
72	Tricosyl acetate	C25H50O2	2681	-	0.56	-
73	Pentacosanol	C25H52O	2693	0.89	-	0.45
74	Heptacosane	C27H56	2701	3.8	5.06	1.36
75	Ethyl tetracosanoate	C26H52O2	2705	-	-	0.31
76	2,3-Dihydroxypropyl stearate	C21H42O4	2711	0.23	0.94	-
77	1-Pentacosanol	C25H52O	2722	0.26	0.9	-
78	Di-n-octylphthalate	C24H38O4	2732	-	0.2	-
79	13-Docosenamide	C22H43NO	2767	0.78	0.52	-
80	Octacosane	C28H58	2798	0.48	0.72	-
81	2-Methyloctacosane	C29H60	2898	0.19	-	1.72
82	Nonacosane	C29H60	2901	3.88	4.67	-
83	Hexacosanoic acid methyl ester	C27H54O2	2928	0.08	0.19	-
84	Hentriacontane	C31H64	-	1.24	1.55	0.3
85	Vitamin E	C29H50O2	-	0.25	0.25	-
86	Campesterol	C28H48O	-	0.18	0.2	-
57	Stigmasterol	C29H48O	-	1.04	1.29	0.26
88	γ-Sitosterol	C29H50O	-	0.68	0.99	0.33
89	α-Amyrin	C30H50O	-	1	0.91	0.35
				96.84	95.32	96.29

¹ RI: retention index.

).

The volatile compounds were classified into seven classes: hydrocarbons, fatty acids, alcohols, ketones, esters, terpenoids and aliphatic amides (Figure 2).

Table 2. Top ten volatile compounds in “golden ring”, “pumpkin pie” and “snow chrysanthemum”.

No.	Golden Ring	Pumpkin Pie	Snow Chrysanthemum
1	(Z)-9-Octadecenamide (28.38%)	(Z)-9-Octadecenamide (37.48%)	Ethyl oleate (28.53%)
2	(Z,Z)-9,12-Octadecadienoic acid (17.36%)	(Z,Z)-9,12-Octadecadienoic acid (8.69%)	Linoleic acid ethyl ester (16.89%)
3	n-Hexadecanoic acid (7.83%)	Heptacosane (5.06%)	Hexadecanoic acid ethyl ester (14.0%)
4	(Z,Z,Z)-9,12,15-Octadecadienoic acid (6.86%)	Nonacosane (4.67%)	(Z)-9-Octadecenamide (12.57%)
5	Nonacosane (3.88%)	(Z,Z,Z)-9,12,15-Octadecadienoic acid (4.65%)	(E)-9-Octadecenoic acid ethyl ester (2.07%)
6	Heptacosane (3.80%)	n-Hexadecanoic acid (3.87%)	2-Methyloctacosane (1.72%)
7	Isopropyl linoleate (2.63%)	17-Octadecynoic acid (2.93%)	Ethyl octadecanoate (1.67%)
8	17-Octadecynoic acid (2.59%)	Isopropyl linoleate (2.69%)	1,22-Docosanediol (1.55%)
9	Hexadecamide (2.11%)	Pentacosane (2.64%)	Octadecamamide (1.50%)
10	Octadecanoic acid (1.96%) Pentacosane (1.96%)	Hexadecamide (2.51%)	n-Hexadecanoic acid (1.42%)

summarizes the ten most abundant constituents among the volatile compounds detected via GC-MS. The volatile oils of “golden ring” and “pumpkin pie” comprised a large proportion of aliphatic amides, fatty acids and hydrocarbons. “Golden ring” contained total volatile oils constituting 96.84% of its total volatile compounds, including 38.13% fatty acids, 33.93% aliphatic amides and 11.263% hydrocarbons, while “pumpkin pie” contained 43.01% aliphatic amides, 22.21% fatty acids and 15.44% hydrocarbons (Figure 2). The extract of “snow chrysanthemum” comprised total volatile compounds up to 96.29%, including esters (67.22%), aliphatic amides (14.6%) and acids (3.16%) (Figure 2).

Table 3. Antioxidant activities of the dichloromethane extract of three different Coreopsis cultivars.

Sample	ABTS 1 (IC50, μg/mL)2	DPPH 1 (IC50, μg/mL)
Golden ring (mutant cultivar)	137.0	602.1
Pumpkin pie (original cultivar)	262.8	2291.3
Snow chrysanthemum (tea material)	278.4	3166.2
Ascorbic acid (positive control)	29.3 μM	100.9 μM

¹ ABTS: 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; DPPH: 1,1-diphenyl-2-picrylhydraxyl. ² IC50: 50% inhibition of concentration.

summarizes the ten most abundant compounds among the volatile components of these cultivars detected via GC-MS. The major compound in the “golden ring” extract was (Z)-9-octadecenamide constituting 28.38% of the total composition (Figure 3), followed by (Z,Z)-9,12-octadecadienoic acid (17.36%), n-hexadecanoic acid (7.83%), (Z,Z,Z)-9,12,15-octadecatrienoic acid (6.86%) and nonacosane (3.88%) (Table 2). The major compound in the “pumpkin pie” extract was also (Z)-9-octadecenamide (37.48%), followed by (Z,Z)-9,12-octadecadienoic acid (8.69%), heptacosane (5.06%), nonacosane (4.67%) and (Z,Z,Z)-9,12,15-octadecatrienoic acid (4.65%). In contrast to the volatile oils of “golden ring” and “pumpkin pie”, the main constituent of “snow chrysanthemum” was ethyl oleate (28.53%) (Figure 3), followed by linoleic acid ethyl ester (16.89%), hexadecenoic acid ethyl ester (14.0%), (Z)-9-octadecenamide (12.57%) and (E)-9-octadecenoic acid ethyl ester (2.07%) (Table 2).

Reports have suggested that (Z)-9-octadecenamide is predominant in the natural volatile oil derived from “golden ring” and “pumpkin pie”, with strong antioxidant and antimicrobial properties [16]. In addition, extracts containing (Z,Z)-9,12-octadecadienoic acid (linoleic acid) and (Z,Z,Z)-9,12,15-ocatadecadienoic acid (linolenic acid), which are abundant in “golden ring” and “pumpkin pie” but less abundant in “snow chrysanthemum”, have also been reported to exhibit antioxidant activities [17,18]. It has been reported that n-hexadecanoic acid (palmitic acid), which is relatively high in “golden ring”, has anti-inflammatory [19,20] and anti-cancer effects [21]. The high content of volatile esters in “snow chrysanthemum” may contribute to the strong flavor of the tea.

2.2. Antioxidant Activities of Dichloromethane Extracts Obtained from Three Different Coreopsis Cultivars

The antioxidant activity of dichloromethane extracts of the three different Coreopsis cultivars was evaluated via ABTS and DPPH radical scavenging assays. These are rapid and sensitive methods used to evaluate antioxidant effects due to the hydrogen donor properties of the substance. The “golden ring” extract showed higher DPPH radical scavenging activity—with a 50% inhibitory concentration (IC₅₀) value of 602.1 μg/mL—than did “pumpkin pie” (IC₅₀ 2291.3 μg/mL) or “snow chrysanthemum” (IC₅₀ 3166.2 μg/mL). As in the DPPH free radical scavenging activities, the “golden ring” extract showed optimal antioxidant activity with an IC₅₀ of 137.0 μg/mL in the ATBS radical scavenging assay (Table 3). Previous studies have reported the biological activities of the fatty acids in “golden ring”, such as anti-oxidant activities [22,23,24], cellular reactive oxygen species generation [25] and anti-cancer activity [26]. Thus, the potent antioxidant activity of the volatile extract of “golden ring” is attributed to the twofold higher fatty acid content compared with that of “pumpkin pie”.

3. Materials and Methods

3.1. Plant Material

Radiation mutants of C. resae, “golden ring”, were generated via the treatment of stem cuttings of the original cultivars, “pumpkin pie”, with gamma (⁶⁰Co) irradiation (150 TBq capacity; AECL, Ottawa, ON, Canada). These mutants were selected from variants in floral size and showed stable inheritance of the phenotype for four years. The radiation mutant cultivars were grown by Uriseed Group Corporation (Icheon-si, Gyeonggi-do, Korea) and registered as new plant varieties in the Korea Seed and Variety Service. The flowers were handpicked and randomly collected at the flowering stage in the same plantation. They were freeze-dried and stored at −20 °C in polyethylene plastic bags until further analysis. Voucher specimens were deposited at the Uriseed Group Corporation. “Snow chrysanthemum” was purchased in China as a commercially available tea material.

3.2. Sample Preparation

Fresh flowers of “golden ring” and “pumpkin pie” were freeze-dried and ground into powder. The dried tea material from “snow chrysanthemum” was ground into powder. Samples (200 mg each) were directly extracted with 20 mL of dichloromethane in an ultrasonic bath for 60 min. Subsequently, the extracts were dehydrated over 0.5 g of anhydrous sodium sulfate and filtered through a polyvinylidene fluoride syringe filter (0.45 µm) for GC-MS analysis.

3.3. GC-MS Analysis

Dichloromethane extracts were analyzed using a Shimadzu QP-2010 Ultra (Shimadzu, Kyoto, Japan). The compounds were separated on the DB-5MS capillary column (30 m × 0.25 mm × 0.25 µm; Agilent Technologies Co., Santa Clara, CA, USA). The carrier gas was 99.99% high-purity helium with a column flow rate of 1.53 mL/min and the injection port in splitless mode. The oven temperature was 50 °C, which was gradually increased to 100 °C at a rate of 5 °C/min and held steady for 5 min, then increased to 230 °C at a rate of 5 °C/min. After holding steady for 20 min again, it was finally increased to 250 °C at a rate of 10 °C/min, and held steady for 5 min. The MS parameters were electron ionization (EI) mode with ionization voltage 70 eV, ion source temperature 230 °C and scan range 45–450 m/z.

The retention indices of all GC peaks were calculated using the retention times of C7–C30 n-alkane standards under the same chromatographic conditions (Figures S1 and S2). Each peak was identified by comparing with the mass spectra database (National Institute of Standards and Technology, Mass Spectra Libraries, Gaithersburg, MD, USA) [27,28].

$$\mathrm{I}=100\times \left(\frac{{\mathrm{T}}_{\mathrm{i}}-{\mathrm{T}}_{\mathrm{n}}}{{\mathrm{T}}_{\mathrm{n}+1}-{\mathrm{T}}_{\mathrm{n}}}+\mathrm{n}\right)$$

(1)

In Equation (1) above, I denotes the retention index of the components to be tested and i is the adjusted retention time (min) of the components to be tested; n and n + 1 represent the carbon amounts of n-alkanes before and after the diffusion of unknown substances, respectively. The values of T_n and T_n+1 denote the carbon retention times of n-alkanes.

3.4. Measurement of DPPH Free Radical and ABTS Radical Cation Scavenging Activities

The DPPH of each sample was determined using Brand-Williams’ method [29]. Briefly, the 2 mM DPPH solution was diluted with ethanol to an absorbance of less than 1.0 at 517 nm before the analysis. Each dichloromethane extract was evaporated in vacuo and initially dissolved in dimethyl sulfoxide (DMSO) to produce 5 mg/mL stock solution. Subsequently, the final concentrations of the samples were diluted to 125, 250, 1000 and 5000 μg/mL using DMSO. Next, each sample was suspended in DMSO and 40 μL of the sample was made to react with 160 μL of 0.2 mM DPPH solution. After 6 min, absorbance was measured at 517 nm using an ELISA reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA).

The ABTS of each sample was determined using the method published by Re et al. [30]. In brief, the ABTS was measured with a pre-formed 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 h. The ABTS solution was diluted with phosphate-buffered saline (pH 7.4) to achieve an absorbance of 0.7 ± 0.02 at 734 nm. The final concentrations of the extracts were diluted to 25, 125, 250 and 500 μg/mL using DMSO and 40 μL of the sample was made to react with 160 μL of the ABTS solution. Absorbance was measured 6 min after the reaction at 734 nm using an ELISA reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The DPPH free radical and ABTS radical scavenging activities (%) were calculated as follows:

$$\mathrm{Scavenging}\mathrm{effect}(\%)=1-\left(\frac{\mathrm{Asample}}{\mathrm{Acontrol}}\times 100\right)$$

(2)

where Asample and Acontrol represent the absorbance of the sample and control, respectively.

The 50% inhibitory concentration (IC₅₀) of the extract was calculated using a linear standard curve, and the IC₅₀ of ascorbic acid, which was used as a positive control, was determined on the basis of its molecular mass.

4. Conclusions

The volatile compounds obtained from the dichloromethane extract of the gamma-irradiated mutant cultivar of C. rosea (“golden ring”), its original cultivar (“pumpkin pie”) and the tea commercially available in China (C. tinctoria; “snow chrysanthemum”) were successfully identified by GC-MS. The major components of “golden ring” and “pumpkin pie” varied slightly, although along with “snow chrysanthemum” they showed significant differences in chemical composition. Meanwhile, the antioxidant effect of “golden ring” extract was higher compared with that of “pumpkin pie” and “snow chrysanthemum”. Despite phytochemical and pharmacological studies of C. tinctoria, the chemical composition and antioxidant activity of C. rosea was reported for the first time in this study. The chemical composition and strong antioxidant efficacy of “golden ring” favor the development of tea materials such as “snow chrysanthemum”.