GC–MS Analysis of the Essential Oil from Flowers of Chrysanthemum coronarium L. Propagated Conventionally and Derived from In Vitro Cultures

Summary. The composition of the essential oils obtained by hydrodistillation from flowers of Chrysanthemum coronarium L. has been investigated by gas chromatography–mass spectrometry (GC–MS). A total of 105 different constituents were identified, representing an average of 97.71–99.95% of the total oil composition. The oil obtained from flowers propagated conventionally contained the higher amounts of cis-chrysanthemol, cis-chrysanthenyl isovalerate, camphor, and virdiflorol, while the higher concentration of bornyl acetate, cis-chrysanthenyl acetate, and 2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4.4] non-3-ene was noted in the oil obtained from flowers propagated in vitro . Significantly higher amounts of essential oil were obtained from flowers of plants derived for in vitro propagation (0.58%) in comparison with conventional cultivation (0.50%).


Introduction
Garland chrysanthemum (Chrysanthemum coronarium L.), an annual herbaceous weed belonging to the Asteraceae family and native to the Mediterranean region, is best known as a food plant in the Far East [1].In Korea, the leaves and flowers of this aromatic plant are eaten raw in salads or are added to soups as flavorings.In Japan, they are usually consumed in blanched form [2,3].According to several literature reports, the edible parts of this plant (flowers, leaves, and stems) are rich in β-carotene, protein, vitamin C, iron, potassium, calcium, and dietary fiber [1,4].
C. coronarium L. is a source of various secondary metabolites, and different agronomic practices are provided in order to produce plants with high content of this active components [16,17].
A tissue culture-based technology (in vitro propagation) gives better possibility of obtaining plants with higher concentration of bioactive compounds in essential oil than in plants, which were propagated conventionally from seeds [18].This method offers also independence from climatic factors, elimination of the geographical and political boundaries as well as shorter production cycle and novel products not found in nature [19,20].
The aim of our study was to compare the chemical composition of essential oils isolated by hydrodistillation using Deryng-and Clevenger-type apparatus from garland chrysanthemum (C.coronarium L.) flowers of plants cultivated conventionally and derived from in vitro cultures.

Experimental Chemicals and Plant Material
Dichloromethane (pure p.a.) was purchased from Chempur and used after distillation.
The research material consisted of garland chrysanthemum plants (C.coronarium L.), cultivar "Jastruń." The experiment was established in randomized blocks with four replications.The experimental plot area was 2 m 2 .The field was prepared according to agrotechnique proper for garland chrysanthemum cultivation.Mineral fertilization was quantified according to the results of chemical analysis of the soil.The fertilization was adjusted to the requirements proper for this species: 80 kg N ha −1 , 50 kg P 2 O 5 ha −1 , and 90 kg K 2 O ha −1 [21].Phosphorus and potassium fertilizers and half of the nitrogen dose were applied during the field preparation (before seeds sowing), and the second half of nitrogen dose was applied in the phase of 4-6 leaves.
Unauthenticated | Downloaded 09/15/23 04:25 PM UTC There were two methods of plant propagation compared in this study: generative (conventional method of cultivation from seed sowing) and in vitro propagation.
In the conventional method of cultivation, the seeds of garland chrysanthemum were sown on 15th of April at a distance of 40 cm between rows.Sowing rate amounted 3 kg ha −1 .
In the in vitro propagation, seeds of C. coronarium were immersed in 70% ethanol for 30 s followed by surface disinfection in 10% NaClO for 20 min.Seeds were transferred onto initiation media containing macro-and microelements according to MS medium [22].Seedlings, initiated for growth, were proliferated three times on the MS media with 2 mg dm −3 KIN (kinetin) and 1 mg dm −3 GA 3 (gibberellic acid).Propagated shoots from previous stage were transferred onto rooting media -MS with 0.5 mg dm −3 IAA (indole-3-acetic acid).At all stages, the media were supplemented with 8 g dm −3 agar, 30 g dm −3 sucrose, and 100 mg dm −3 inositol, and their pH was adjusted to 5.7 with 0.1 M NaOH and HCl solutions.After the addition of growth regulators, the media were autoclaved for 20 min at the temperature 121 °C.Every stage lasted 6 weeks.The cultures were placed in a growth chamber at the temperature 24 ± 1°C.After the acclimatization, plants were transplanted into the open field on 20th of June, at a density of 30 × 30 cm.
During the growing season, the crop management was carried out.It included mainly irrigation, weeding, and soil cultivation.
The flower harvest was done at the blooming stage (in the middle of September).After the harvest, the plant material -garland chrysanthemum flower heads (Chrysanthemi anthodium) was dried in a through-flow laboratory dryer set at 40 °C.

Essential Oil Isolation
Twenty grams of dried flowers in a 1000-mL round-bottom flask along with 500 mL of distilled water was subjected to hydrodistillation for 3 h using Deryng-type apparatus according to the general method recommended by Polish Pharmacopoeia VI [23].
The hydrodistillation was also performed by use of Clevenger apparatus [24,25] for 3 h.The essential oils obtained this way were separated from water, dried over anhydrous sodium sulphate, filtered, weighed, and stored in dark sealed vial at 4 °C until gas chromatography-mass spectrometry (GC-MS) analysis.
The yield of the oils was calculated based on dry weight of plant material and expressed as % w/w in Table I.

GC-MS Analysis
The qualitative GC-MS analysis of the isolated oils was carried out using an HP 6890 gas chromatograph, equipped with a HP-5MS ((5%)-phenylmethylpolysiloxane) capillary column (30 m × 0.25 mm; film thickness, 0.25 µm) and coupled with HP 5973N Mass Selective Detector.Helium (1 mL min −1 ) was used as carrier gas.Samples of 2 μL (20 mg of oil dissolved in 1.5 mL of dichloromethane) were injected in the split mode at a ratio of 5:1.The injector and the transfer line were kept at 280 °C.The ion source temperature was 230 °C.
The initial temperature of the column was 40 °C for 5 min, then increased to 60 °C at a rate of 30 °C min −1 , next to 230 °C at a rate of 6 °C min −1 (kept constant for 10 min), and then increased to a final temperature of 280 °C at a rate of 30 °C min −1 .The oven was held at this temperature for 30 min.Mass spectra were taken at 70 eV.Mass range was from 40 to 550 m/z.Solvent delay time was 4 min.The total running time for a sample was about 76 min.

Identification and Quantification of Oil Constituents
Oil constituents were identified by comparison of their retention indices (relative to n-alkanes C 7 -C 40 on HP-5MS column) with those reported in NIST 2002 library and the literature [26][27][28].
Further identification was made by comparison of their mass spectra with those stored in Wiley NBS75K.L and NIST 2002 mass spectral libraries.
The relative percentage amounts of the essential oil components were evaluated from the total peak area (TIC) by the apparatus software.

Statistical Analysis
Several results of the study (Tables I and III) were subjected to an analysis of variance which was performed with AWAR software, made by Department of Applied Informatics, Institute of Soil Science and Plant cultivation, Puławy.The means were separated by the Tukey's test at p = 0.05.The statistical analysis of the results given in Table III was conducted for selected 20 constituents of content greater than 1% of the essential oil.

Results and Discussion
The oils isolated from flowers of C. coronarium L. were found to be blue liquids (due to the presence of chamazulene) in yields shown in Table I.
On the base of the results of statistical analysis, it was proved that the method of plant propagation had a significant effect on the content of essential oil in flowers of C. coronarium L. Significantly higher amounts of essential oil were obtained from flowers of plants derived from in vitro propagation (0.58%) in comparison with conventional cultivation (0.50%).The type of distillation apparatus and interaction of both experimental factors were not significant statistically.
Basta and coworkers [27] determined essential oils in C. coronarium flowers in the amount of 0.31-0.39%,while Perez and others, 0.31-0.1% [29].Our plants were richer in essential oil compared to the cited literature data.
The results of the GC-MS analysis of the essential oils isolated by hydrodistillation in Deryng-and Clevenger-type apparatus from garland chrysanthemum flowers propagated conventionally and derived from in vitro cultures are presented in Table II.The individual constituents of the analyzed oils are listed in order of their elution from HP-5MS column.
GC-MS chromatograms of essential oils isolated by hydrodistillation using Deryng-and Clevenger-type apparatus from obtained from C. coronarium L. flowers derived from in vitro cultures are presented in Figs. 3  and 4.
In Table III, the statistical analysis results of content of the main constituents of essential oil of C. coronarium L. flowers according to the type of distillation apparatus and the method of plant propagation are given.
Significant differences were also noted for the distillation apparatus type.Higher amounts of the main essential oil constituents were obtained according to Clevenger apparatus.It was on average by 0.43% higher in comparison with Deryng apparatus.The method of plant propagation did not influence significantly the content of the main essential oil constituents.
The chemical composition of garland chrysanthemum flowers has been previously studied by several authors.
The observed differences in the main constituents of C. coronarium flowers essential oils across countries may be caused by different environmental and genetic factors, different chemotypes, and the nutritional status of the plants.

Conclusions
In this study, the comparison of the chemical composition of the essential oils obtained by hydrodistillation in the Deryng-and Clevenger-type apparatus from the flowers of C. coronarium L. cultivated conventionally and derived from in vitro cultures was provided.
Significantly higher amounts of essential oil were obtained from flowers of plants derived from in vitro propagation (0.58%) in comparison with conventional cultivation (0.50%).

Table I .
Yield of essential oil (% w/w) from flowers of Chrysanthemum coronarium L. in response to distillation apparatus and the method of plant propagation

Table II .
Relative percentage composition of the essential oil of Chrysanthemum coronarium L. flowers in response to distillation apparatus and method of propagation Unauthenticated | Downloaded 09/15/23 04:25 PM UTC

Table III .
Percentage content of the main Chrysanthemum coronarium L. flower essential oils components according to the distillation apparatus type and method of plant propagation A: conventional method of cultivation B: in vitro propagation method n.s.: not significant