FARMACIA, 2016, Vol. 64, 6 ORIGINAL ARTICLE CONJUGATED LINOLENIC ACID CONTENT IN SEEDS OF SOME POT MARIGOLD (CALENDULA OFFICINALIS L.) CULTIVARS GROWN IN POLAND

BEATA KRÓL1*, TADEUSZ PASZKO2, ANNA KRÓL3

1University of Life Sciences, Faculty of Agrobioengineering, Department of Industrial and Medicinal Plants, Akademicka 15, 20-950 Lublin, Poland 2University of Life Sciences, Faculty of Food Science and Biotechnology, Department of Chemistry, Akademicka 15, 20-950 Lublin, Poland 3University of Medical Sciences, Department of Biochemistry and Molecular Biology, Poznań 6 Święcickiego St., 60-781 Poznań, Poland

*corresponding author: [email protected] Manuscript received: September 2015

Abstract Seed oil of ten cultivars of pot marigold grown in Poland was analyzed in order to determine the main factors affecting its content and composition. The seeds of the pot marigold cultivars grown in Poland contained relatively large amounts of oil (17.32% ± 2.60%), which was abundant in conjugated linolenic acids (49.53% ± 5.43%) and had low content of saturated fatty acids (8.62% ± 1.97%). In addition to the effect of cultivars, the large contents of α-calendic acid in oils were a result of low average temperature and extensive precipitation during seed maturation. The statistical analysis suggested that was the main precursor of α-calendic acid, and α-calendic acid was the precursor of β-calendic acid.

Rezumat Uleiul din semințele a zece soiuri de gălbenele cultivate în Polonia a fost analizat pentru a determina factorii principali care afectează conținutul și compoziția sa. Semințele soiurilor de gălbenele cultivate în Polonia conțin cantități relativ mari de ulei (17,32% ± 2,60%), bogat în acizi linolenici conjugaţi (49,53% ± 5,43%) și are un conținut scăzut de acizi grași saturați. Pe lângă efectul soiurilor, conținutul mare de acid α-calendic în uleiuri este rezultatul temperaturii medii scăzute și al precipitaţiilor în cantităţi masive în timpul maturării seminței. Analiza statistică a sugerat că acidul linoleic a fost principalul precursor al acidului α-calendic, și acidul α-calendic a fost precursorul acidului β-calendic.

Keywords: pot marigold, oil, , α-calendic acid, β-calendic acid

Introduction benefits. It was recently proved that CLNAs are effective at inhibiting carcinogenesis including Pot marigold (Calendula officinalis L.), a member inhibition of proliferation [25, 26] and suppression of the Asteraceae family, is an annual native plant of invasion and adhesion [22]. The studies have of the Mediterranean area. It has been cultivated as demonstrated that CLNAs could induce apoptosis an ornamental species since the ancient times and in different human cancers such as colorectal for medical purposes since the 12th century. For adenocarcinoma [11], promyelocytic leukemia [10], many decades, marigold flower heads (Calendulae mammary cancer and bladder cancer [20]. anthodium) have been known to exhibit spasmolytic, Furthermore, study by Li, Q. et al. [14] showed cholagogic, diuretic, anti-inflammatory, antioxidant, anticancer effects of α-calendic acid and β -calendic and antimicrobial activities [9]. Lately, research has acid in choriocarcinoma JEG-3 cells. The results of also focused on seeds as a valuable oil source. studies have also indicated anti-obesity [1], From the pot marigold seeds, about 20% of oil can antioxidant [18], and anti-inflammatory effects [13] be extracted, in which, among the isomers of of CLNAs and their abilities to decrease blood conjugated linolenic acids (CLNAs), α-calendic pressure [12]. Because CLNAs may be useful in the acid (8E, 10E, 12 Z-octadecatrienoic acid) is prevention and fight against many diseases, predominant and β-calendic acid (8E, 10E, 12 E) products with their high content are searched for. occurs in small amounts [16]. Oils containing The usefulness of seeds of individual pot marigold conjugated trienoic acids have specific properties cultivars for pharmaceutical industry depends on and this makes them useful in the production of many factors. One of the most important factors is high quality paints and polymers [3]. Conjugated the content and composition of seed oil, which linolenic acids have shown important health 881 FARMACIA, 2016, Vol. 64, 6 depends on genetic characteristics [7] as well as injector and a flame-ionizing detector (FID) was environmental and climatic conditions [5]. used. Cis-trans fatty acid methyl esters (FAMEs) The aim of the study was to determine the main were separated by means of an UltiMetalTM factors that affect the content and composition of capillary column CP-WAX 52CB (df 0.25 µm, oil from seeds of ten pot marigold cultivars grown length 60 m) with a polyethylene glycol stationary in Poland. phase. Helium with a flow rate of 1.4 mL x min-1 was used as a gas carrier. The initial temperature of Materials and Methods the column was 120°C, the temperature increase was 2°C min-1, and the maximum temperature was Plant material 210ºC. The duration of the analysis was 127 min. The seeds were collected from 10 different cultivars The injector and detector temperatures were set to of pot marigold (‘Tokaj’ - denoted as PM1, 160°C. FAMEs peaks were identified based on the ‘Santana’ - PM2, ‘Radio’ - PM3, ‘Persimmon Supelco 37 Component FAME Mix and LGC Beauty’ - PM4, ‘Orange King’ - PM5, ‘Promyk’ - standards. The reported results for the fatty acid PM 6, ‘Szlem’ - PM7, ‘Mandarin Twist’ - PM8, composition are averages of triplicate ‘Apricot Twist’ - PM9, ‘Geisha Girl’ - PM 10) measurements. The statistical analysis was carried grown on the experimental plot of the University of out using the Statgraphics Centurion XVI software Life Sciences in Lublin during the 2010 growing (v. 16.1.03). season. In the third decade of August (time when about 50 - 60% of seeds reached maturity), the Results and Discussion herbicide diquat was applied to desiccate the plants. The seeds were harvested 7 days later, then The oil contents in the seeds of the evaluated threshed, and cleaned. cultivars of pot marigold ranged from 14.67% to Oil extraction and methyl-esterification 19.86% (Table I). Evidently, the largest amounts of The seeds from the individual cultivars were oil were found in the samples from cultivars ground separately using a stainless-steel grinder “Orange King” (19.76 ± 0.10%) and ‘Tokaj’ (19.33 ± and was extracted from 5g samples during 8 h 0.20%), and the lowest amounts in cultivars with n-hexane using a Soxhlet apparatus. In the ‘Szlem’ (14.88 ± 0.26%) and ‘Apricot Twist’ next step, n-hexane was evaporated at 40 °C from (15.28 ± 0.11%). The oil contents were similar to the extract by means of a rotary evaporator and the those reported by Cromack and Smith [5] (14.20- resulting oil was stored in a freezer at a temperature 19.20%, England), Martin et al. [15] (17.1-21.4%, of -18°C. Methyl-esterification of the New Zealand), Dulf et al. [7] (13.6-21.7%, obtained was carried out using the BF3-CH3OH Romania), and Gesch [8] (18.6-20.5%, USA). On method according to AOCS [17]. the other hand, the oil contents reported by GC analysis Angelini et al. [2] (2.0-12.0%, Italy) and Özgül- For the analysis, a Varian 3800 (Walnut Creek, CA Yücel [16] (5.9%, Turkey) were much lower. USA) gas chromatograph equipped with an auto Table I Oil contents (%) and saturated and monounsaturated fatty acids composition (%) of different cultivars of pot marigold seed oils (n = 3) Saturated acids Monounsaturated acids Cultivar Oil content 16:0 18:0 18:1n9c Other b Other c Palmitic Stearic Oleic PM 1 19.33 ± 0.20 a 4.25 ± 0.19 3.40 ± 0.27 1.41 ± 0.17 5.14 ± 0.54 1.08 ± 0.21 PM 2 16.54 ± 0.09 5.27 ± 0.56 2.20 ± 0.22 1.52 ± 0.33 5.53 ± 0.50 1.29 ± 0.10 PM 3 17.27 ± 0.16 5.35 ± 0.21 2.41 ± 0.45 1.36 ± 0.33 4.63 ± 0.33 1.28 ± 0.29 PM 4 18.37 ± 0.11 4.32 ± 0.33 1.34 ± 0.17 0.88 ± 0.09 4.59 ± 0.11 1.32 ± 0.26 PM 5 19.76 ± 0.10 4.36 ± 0.15 2.45 ± 0.28 2.48 ± 0.21 5.50 ± 0.25 1.87 ± 0.26 PM 6 16.21 ± 0.14 4.75 ± 0.19 1.68 ± 0.12 2.86 ± 0.16 5.10 ± 0.20 1.15 ± 0.08 PM 7 14.88 ± 0.26 5.39 ± 0.27 2.29 ± 0.28 2.80 ± 0.21 4.73 ± 0.48 1.49 ± 0.24 PM 8 16.97 ± 0.14 3.58 ± 0.50 2.52 ± 0.23 1.56 ± 0.13 5.24 ± 0.28 0.79 ± 0.17 PM 9 15.28 ± 0.11 4.51 ± 0.37 4.51 ± 0.35 1.17 ± 0.10 5.78 ± 0.78 0.60 ± 0.09 PM 10 18.63 ± 0.25 3.43 ± 0.31 2.46 ± 0.31 0.72 ± 0.08 3.64 ± 0.54 0.75 ± 0.12 a Mean ± standard deviation, b ∑ 6:0, 8:0, 10:0, 12:0,14:0, 15:0, 17:0, 20:0, 22:0, 24:0; c ∑ 15:1, 16:1, 17:1, 18:1n7t, 20:1n9c, 20:1n7c

Based on the ranges of values obtained in Moreover, it can be seen that the oil contents from individual studies, it can be concluded that the the crops cultivated in the temperate climate [5, 7, selection of a suitable genotype of pot marigold has 15] are greater than those obtained in the a significant impact on the amount of extracted oil. Mediterranean climate [2, 16].

882 FARMACIA, 2016, Vol. 64, 6 Twenty six fatty acids were identified in the were in the range of 6.54 - 10.48% of the total analysed cultivars. Saturated fatty acids (SFAs) amounts (Figure 1).

Figure 1. Contents (%) of saturated (SFA), monounsaturated (MUFA), polyunsaturated (PUFA) and conjugated linoleic acids (CLNA) in pot marigold seed oils of different cultivars

The predominant were (4.52 ± 0.98%) compound in this group (Table I). The calendula and (2.43 ± 1.09%) (Table I), the most seed oils of the examined cultivars were abundant common saturated fatty acids in animal and plant in polyunsaturated fatty acids (83.30 - 89.00%) . The amounts of other saturated fatty acids (Figure 1). The most common among them were were below 1%. The average content of SFAs CLNAs (46.29 - 57.14%). In the group of obtained in our study was 8.63%, whereas Dulf et polyunsaturated acids, α-calendic acid (43.7 ± al. [7] obtained 6.99%, Martin et al. [15] 14.07% 7.32%), linoleic acid (34.21 ± 2.96%), and β- and Özgül-Yücel [16] 17.6%. calendic acid (5.83 ± 1.94%) were predominant The contents of monounsaturated fatty acids (Table II). Other polyunsaturated fatty acids were (MUFAs) were in the range of 4.39 - 7.37% (Figure 1) found in the amounts below 1%. and (4.99 ± 1.07%) was the predominant Table II Polyunsaturated fatty acid and conjugated linoleic acids content (%) of different cultivars of pot marigold seed oils (n = 3) Polyunsaturated acids Conjugated linoleic acids Cultivar 18:3n3 e 18:3n6 f 18:3n6 g Other h 18:2n6 d Linoleic α-Linolenic α-Calendic β-Calendic PM 1 34.37 ± 0.45 0.83 ± 0.20 43.02 ± 1.49 6.15 ± 0.35 0.36 ± 0.04 PM 2 32.96 ± 0.80 0.90 ± 0.02 43.96 ± 1.26 5.98 ± 0.47 0.39 ± 0.03 PM 3 36.63 ± 0.54 1.77 ± 0.20 38.79 ± 0.69 7.50 ± 0.32 0.29 ± 0.04 PM 4 31.11 ± 0.35 0.93 ± 0.09 51.46 ± 0.22 3.65 ± 0.41 0.39 ± 0.06 PM 5 35.23 ± 0.51 1.54 ± 0.28 40.86 ± 0.77 5.43 ± 0.50 0.28 ± 0.04 PM 6 34.52 ± 0.40 1.08 ± 0.11 43.12 ± 0.48 5.28 ± 0.17 0.47 ± 0.03 PM 7 34.42 ± 0.53 1.21 ± 0.12 40.65 ± 0.42 6.51 ± 0.39 0.51 ± 0.02 PM 8 36.47 ± 0.33 0.84 ± 0.01 41.06 ± 0.74 7.53 ± 0.41 0.41 ± 0.06 PM 9 35.73 ± 0.56 1.08 ± 0.06 40.63 ± 0.45 6.58 ± 0.46 0.40 ± 0.01 PM 10 30.70 ± 0.52 0.87 ± 0.07 53.43 ± 0.57 3.71 ± 0.28 0.28 ± 0.04 d (9c,12c), e (9c,12c,15c), f (8t,10t,12c), g (8t,10t,12t); h ∑ 20:2n6, 20:3n3, 20:4n6

The average content of α-calendic acid, the dominant obtained 54.66%, Cromack and Smith [5] 46.77%, component of CLNAs in the pot marigold seed oil, Štolcová et al. [19] 45.09%, Angelini et al. [2] 36.73%, was 43.70 % in our study, whereas Dulf et al. [7] Martin et al. [15] 35.86%, and Özgül-Yücel [16] 17.6%. 883 FARMACIA, 2016, Vol. 64, 6 The statistical analysis indicated that the above cultures of transgenic soybean embryos at 30°C, contents of α-calendic acid were significantly whereas the growth was enhanced significantly at positively correlated with the average monthly lower temperatures. Of course the statistical precipitation in the warmest month of the year (July analysis carried out in such a way ignores many in the northern and January in the southern genetic and practical aspects, e.g. the fact that in the hemisphere) of particular crop location (r = 0.845, p above locations different cultivars were used, = 0.017; 79.0 mm for Zamosc (this study), 81.0 mm whose selection and cultivation have been for Cluj-Napoca [7], 66.2 mm for Prague [19], optimized for climate conditions. However, in the 45.7mm for Christchurch [15], 55 mm for authors’ opinion, this does not discredit the Plymouth [5], 24.0 mm for Pisa [2], and 19.1 mm statement but confirms that climatic conditions are for Istanbul [16] (the climate data were taken from one of the major factors influencing the [23]). They were significantly inversely correlated composition of fatty acids in pot marigold seeds. at 90% probability level with the average monthly The relationship between the content of α-calendic temperatures (r = -0.717, p = 0.070; 16.1, 17.1, acid and climatic conditions has not been reported 17.4, 17.4, 18.2, 22.9, and 23.2°C for Plymouth, in the literature to date. Prague, Christchurch, Zamosc, Cluj-Napoca, Pisa, Another very important factor that influences both and Istanbul respectively [23]). Thus, an increase in CLNAs and the composition of other fatty acids in temperature in the period of seed maturation pot marigold oil is the genotype of the crop. This decreased the contents of α-calendic acid and other can be seen in Table II in our study and also in the unsaturated fatty acids. The same relationship studies of Dulf et al. [7], Cromack and Smith [5], between the content of α-calendic acid and Martin et al. [15], or Angelini et al. [2]. temperature was indicated previously in a Table III shows values of Pearson’s correlation laboratory study by Cahoon et al. [4]. The authors coefficients and partial correlation coefficients for determined a small amount or no acid in the the main fatty acids of pot marigold oil. Table III Values of Pearson’s correlation coefficients (left side), partial correlations (right side; in any case, the effect of four other variables was taken into account) and their p-values (in subscript brackets) for the main fatty acids of the pot marigold oils (n = 30) Palmitic Stearic Oleic Linoleic α-Calendic β-Calendic

Palmitic -0.275(0.174) -0.591(0.002) -0.658(<0.001) -0.776(<0.001) -0.135(0.510) Stearic -0.084(0.661) -0.164(0.425) -0.051(0.806) -0.125(0.542) 0.157(0.445) Oleic 0.089(0.641) 0.131(0.491) -0.566(0.003) -0.716(<0.001) -0.164(0.422) Linoleic 0.284(0.129) 0.447(0.013) 0.422(0.020) -0.860(<0.001) 0.085(0.680) α-Calendic -0.484(0.007) -0.387(0.035) -0.558(0.001) -0.936(<0.001) -0.345(0.085) β-Calendic 0.326(0.079) 0.459(0.011) 0.414(0.023) 0.883(<0.001) -0.887(<0.001)

As can be seen, the values of those coefficients are ship also between the oleic and the α-calendic acids not consistent in some cases. During interpretation (r = -0.558, p = 0.001 – Pearson; r = -0.716, p < of the correlations between fatty acids, one should 0.001 – partial correlation). According to Crombie take into account that certain acids were formed and Holloway [6], oleic acid is the second precursor from others, resulting in negative correlations. of α-calendic acid. In the case of β-calendic acid, Moreover, the fact that the sum of fatty acids in oil the only significant partial correlation was the is equal to 100% causes collinearity problems. In negative one with α-calendic acid. The significant such a case, when both Pearson’s and partial negative relationship between the α- and β-calendic correlations are significant and indicate the same acids (Table III) suggests that β-calendic acid is positive or negative relationship, this result cannot synthesized from α-calendic acid. To the best of the be questioned. On the other hand, when Pearson’s author's knowledge, this is the first report on the and partial correlations are not consistent, partial statistical relationship between the α- and β- correlation values are usually more correct. calendic acid. However, the above isomerization However, in some cases, also partial correlations reaction of a double bond at Δ12 position of the acid may give false results [24]. Both Pearson’s and the was suggested previously by Takagi and Itabashi partial correlation coefficients indicated a very [21]. strong negative relationship between linoleic and α- The content of β-calendic acid in our study (5.83% ± calendic acid. This correlation results from the fact 1.94%) (Table II) was larger than that obtained by that α-calendic acid is synthesized from linoleic Dulf et al. [7] (0.69% ± 0.23%) but much lower acid (modification of the Δ9 double bond by FAD2 than the value reported by Özgül-Yücel [16] related enzymes [4]). The correlation coefficients in (11.2%). It can be supposed that the differences Table III indicated a significant negative relation- largely resulted from climate conditions (mainly 884 FARMACIA, 2016, Vol. 64, 6 temperature), which affected the enzymatic activity (Calendula officinalis L.) seed genotypes. Chem. during seed maturation. Cent. J., 2013; 7: 8. 8. Gesch R.W., Growth and yield response of Conclusions calendula (Calendula officinalis) to sowing date in the northern US. Ind. Crop. Prod., 2013; 45: 248- The seed oils of the ten cultivars of pot marigold 252. grown in Poland were rich in CLNAs (from 46.29% 9. 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