_ Food Science and Technology Research, 21 (1), 67 75, 2015 Copyright © 2015, Japanese Society for Food Science and Technology doi: 10.3136/fstr.21.67

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Original paper

Identification and Profile of Furanocoumarins from the Ribbed CeleryApium ( Graveolens L Var. Dulce Mill./ Pers.)

1* 1 1 2 1 3 Agnieszka Najda , Jan Dyduch , Klaudia Świca , Magdalena Kapłan , Rafał Papliński , Monika Sachadyn-Król 4 and Kamila Klimek

1 Department of Vegetable Crops and Medicinal Plants, University of Life Sciences in Lublin, 20-950 Lublin, Leszczyńskiego Street 58, Poland 2 Department of Seed Production and Nurseries, University of Life Sciences in Lublin, 20-950 Lublin, Leszczyńskiego Street 58, Poland 3 Department of Chemistry University of Life Sciences in Lublin, 20-950 Lublin, Akademicka Street 15, Poland 4 Department of Applied Mathematics and Informatics, University of Life Sciences in Lublin, 20-612 Lublin, Głęboka Street 28, Poland

Received July 22, 2014 ; Accepted September 9, 2014

The aim of this study was to identify and determine the qualitative and quantitative composition of furanocoumarins in anatomical parts (blade and petiole) of two cultivars of ribbed celery, depending on the age of the plant. Two accurate, precise and inexpensive techniques for analytical detection of coumarin in a relatively short period of time were used in the present study. TLC - the method considered as preliminary, showed the presence of psolaren, bergapten, xanthotoxin and simple coumarin – umbelliferone. Additionally, isopimpinellin was detected by HPLC method. Chromatographic analysis of ribbed celery leaves showed the presence of four furanocoumarins. Psoralen and isopimpinellin are dominant in the leaf blades, wherein content is correlated with the term of harvesting. Bergapten was dominant in petioles regardless of the harvesting time and cultivar.

Keywords: Apiaceae, cultivars, harvest, furanocoumarin, HPLC methods

Introduction 2002). Leafy vegetables are an important group of plants in the daily Plants from the family Apiaceae contain compounds with diet. Less known species like endive, chicory, arugula, rochet and multidirectional biological activity: antimicrobial, anti-cancer, for chard has become popular among consumers. Also ribbed celery the liver agents and cyclooxygenase inhibitors (Chen et al., 1995; (Apium graveolens L. var. Dulce Mill. / Pers.), valued for its Strohl, 2000). The presence of these substances is responsible for the attractive appearance, taste and the nutritional composition sealing properties and capillary walls strengthens, improving blood resulting from the rich mineral and vitamin composition is circulation in the myocardium, spasmolytic, diuretic, platelet classified to this group (Vogel, 1996). In part for consumption anticoagulation, anti-ulcer, anti-allergic, anti-inflammatory, anti- (petioles and partially leaf blades) contains many valuable hepatotoxic, antifungal and antiviral effects (Trumble and Quiros, biologically active substances such as phenolic acids, flavonoids, 1988, Zheng et al., 1993). tannins, mono-and sesquiterpenes which make them useful in the Coumarins are fairly common in many fruits and vegetables, treatment of certain dermatological diseases, arthritic-rheumatic, especially in the families Apiaceae and Rutaceae (Zobel and Brown, urinary and digestive system (Williamson, 1999; Wolski et al., 1990; Ojala, 2001). These group of compounds are important from

*To whom correspondence should be addressed. E-mail: [email protected] 68 A. Najda et al. the therapeutic point of view (Leal et al., 2000; Waksmundzka- carried out on two dates i.e. I – after 100 (August) and II – after Hajnos et al., 2004). Currenly, furanocoumarins of natural origin 120 days (September) of two growing seasons: in 2011 and 2012. are used in PUVA therapy of vitiligo and psoriasis; furthermore, they In a two-year cycle, the experimental average air temperature in are active Ca+2 channel blockers (Murray et al., 1982; Vuorela et the growing seasons of 2011 and 2012 showed a little varied. The al., 1988; Coven et al., 1999) . Furanocoumarins are characterized average air temperature in the period May-September was 17.2℃ by a broad spectrum of biological activity. They reduce the platelet (Table 1). The range of rainfall in years of research was at a agregation in in vitro test (Chen et al.,1996), they induce apoptosis similar level. Average monthly temperatures in May and June in human promyelocytic leukemia, HL-60 cells (Bogucka-Kocka and were higher than the perennial average, it has the second decade of Kocki, 2002; Pae et al., 2002) and they work as hepatoprotective July to the end of the growing season showed a gradual decrease in agents (Jagiełło-Wójtowicz et al., 2004) . Photosensitizer effect of temperatures that were higher than the average perennial. Research these substances (particularly psoralen derivative) is used in medicine material consisted blades and petioles of celery; 25 plants from and the treatment of many dermatological disorders (Towers and each cultivar (5 plants × 5 replications) in each year. Samples were Abramowski, 1983; Bhatnagar et al., 2007; Serrano-Perez et al., dried in a conventional dryer at 35℃ to dry mass of raw material. 2008) . Furanocoumarins content in the plant varies depending on the 85 g of dry raw material were obtained from 1000 g of fresh degree of its development, and is different in different vegetative petioles and 170 g from 1000 g of fresh blades. Dry plant material phases (Borkowski, 1973; Bogucka-Kocka and Kocki, 2002). was pulverized and extracted. Increase in the content of furanocoumarins in different organs was Chemical reagents Solvents for extraction were purchased observed in many species of plants under stress conditions due to from POCh SA (Gliwice, Poland) or J.T. Baker (USA). Standards various factors: biotic (fungi, bacteria, viruses), abiotic (heavy metals, of high purity were purchased from Sigma–Aldrich Chemie GmbH detergents, acid rain) or physical (UV radiation, low temperature). (Munich, Germany), Merck (Darmstadt, Germany) and from other The diverse biological activity of furanocoumarins and the role they suppliers. fulfill in the life of the plant tend to clarify the relationship between Solid phase extraction Analysis of free furanocoumarin the plant defense system and external conditions. Stress metabolites arrangements was performed in dry and powdered whole leaves, named phytoalexins including furanocoumarins are formed as a result leaf blades, and petioles of leaf celery plants. Extraction was of defense property (Hain et al., 1993; Dixon and Lamb, 1999). conducted in liquid-solid system, while extract purification by The aim of this study was to identify and determine the means of SPE method (Solid Phase Extraction). qualitative and quantitative composition of furanocoumarins in Samples of 50 g powdered and dried leaves, leaf blades, and anatomical parts (blade and petiole) of two cultivars of ribbed petioles of two leaf celery cultivars were weighed and put into celery, depending on the age of the plant. Two accurate, precise the tubes for extraction. After preliminary maceration for 24 and inexpensive techniques for analytical detection of coumarin in hours, material was extracted using petroleum ether (1:10 v/v) a relatively short period of time were used in the present study. and processed in Soxhlet’s apparatus for 36 hours. The petroleum ether was evaporated till dry then. Dry residue of Material and Methods petroleum ether were purified on a SPE column (BackerBond RP Plant material Two original Polish varieties of celery Zefir 18). Microcolumns were mounted in a set of vacuum filtration (J and Helios (company PlantiCo Zielonka) were tested in this study. Beaker) and further conditioned successively with 5 mL of Plants for analysis were obtained from outdoor experience of the methanol, 5 mL of double-distilled water, then dissolved extracts Department of Vegetable and Medicinal Plants, University of Life were applied (100 mg / 3 mL of double-distilled water). Elution Sciences in Lublin, Poland (N 51 ° 16’ E 22 ° 34’). Cultivation was carried out by washing the bed of pollutant 30 mL of double- was carried out in the ecological system on luvisol soil formed distilled water for removal of the ballast. Furanocoumarin from loess, containing 1.6% organic matter. Crops harvesting was compounds elution was performed 10 mL of methanol solution

Table 1. Mean monthly air temperatures and total amount of precipitation at ES Felin in the years 2011-2012

Temperature ℃ Amount of precipitation mm Month 2011 2012 Mean for 1951-2000 2011 2012 Mean for 1951-2000 V 14.9 15.0 13.0 80.5 81.5 58.3 VI 18.1 18.1 16.5 87.8 87.8 65.8 VII 19.1 19.2 17.9 87.0 87.0 78.0 VIII 18.8 19.2 17.1 65.3 37.6 68.6 IX 15.2 15.0 12.6 5.4 35.5 51.6 Mean 17.2 17.3 15.4 65.2 65.88 64.46 Identification and Profile of Furanocoumarins from the Ribbed Celery 69 containing 0.3 mL of 25% ammonia. The eluents were evaporated method. HPLC chromatogram of standards furanocoumarins to dryness and the residue was dissolved in 10 mL of methanol. distribution is presented in Fig. 2. Fig. 3 shows the UV The thus obtained samples were analyzed by TLC and HPLC for spectroscopic spectrums. Tab. 2. contains the average retention the presence of furanocoumarins. times for the different standards of furanocoumarins and Thin layer chromatography TLC was performed on 60G Si furanocoumarins isolated from extracts of the tested materials. The gel, cellulose and polyamide plates (Merck, Germany). Samples presence of three furanocoumarins in the petioles of both studied were applied using Autosampler3 (Camag, Switzerland). The cultivars and four furanocoumarins such as psoralen, bergapten, plates were developed in the chromatographic chambers: flat type isopimpinellin and xanthotoxin in the leaf blades were found. Fig. DS (Chromdes, Poland) and vertical (Camag, Switzerland). 4 and 5 show chromatograms representing furanocoumarins Developing phase was n-heptane:dichloromethane:ethyl acetate content in the tested raw materials. Analysed petioles of tested (40:40:20 v/v/v). Visualization of the chromatograms was carried varieties had 2 to 3 furanocoumarin compounds. In contrast, the out in visible light, under UV light Emita VP 60 (Famed, Poland) leaf blades of both studied cultivars contain 4 basic at the wavelength λ = 254 nm and 366 nm, and using VideoScanner furanocoumarins: xanthotoxin, psoralen, bergapten and Reprostar3 (Camag, Switzerland). The identification of isopimpinellin. The quantitative content of furanocoumarins in compounds was performed by comparing their Rf values and color whole leaves demonstrated slightly lower content of these stain patterns, as well as by using a “co-chromatography”. compounds due to their lower concentration in petioles. High-performance liquid chromatography The qualitative and Quantitative content of individual furanocoumarins occurring quantitative HPLC determination was made in reversed-phase in the studied raw materials was calculated by assuming the surface system applying liquid chromatograph of LaChrom–Merck type area of each peak as a quantitative relation of the furanocoumarins equipped with diode array detector DAD (L–7450), pump (L– concentrations and using D 7000 HPLC system Manager program. 7100), degasser (L–7612), injection loop 20 μL, thermostat (L– Data on the content of furanocoumarins in anatomical parts 7360), injector Rheodyne, steel column LiChrospher 100 RP C 18 (blades and petiole) of each variety in the two years of the study as (250 mm × 4 mm dimensions) filled with stationery phase (dp = the mean of all varieties in each year of the study is presented in 5 µm). The analyses were conducted at 25˚C. A stepwise mobile Table 3. The results show that the differences between cultivars × phase gradient was prepared from methanol (A) and water (B). anatomical parts, cultivars x year and anatomical parts × year were The gradient was: 0 _ 10 min 40 _ 50% A; 10 _ 25 min 50 _ 75% A; not significant (P < 0.05). Data variation source for all measured 25 _ 35 min isocratic 75% A; 35 _ 40 min 75 _ 100% A. The flow varieties highlights that the seasonal variability plays an important rate was 0.8 mL/min., while injected sample volume 20 μL. Coumarins identification was made by comparing their retention times (tR) with those for standards and spectroscopically by recording their spectra within UV range (220 _ 400 nm) (Najda, 2009). Contents of particular furanocoumarins in studied material were then calculated on a base of calibration curve. Statistical analysis The statistical analysis of data was performed using SAS “for Windows” version 9.1 and Statistica® version 7.0 software. Results were submitted to analysis of variance (ANOVA), Tukey test (p < 0.05) and Principal Component Analysis (PCA). All analyses were carried out in triplicate.

Results and Discussion Our findings concerning the presence and the qualitative and quantitative furanocoumarin compounds were carried out by TLC and HPLC methods. TLC - the method considered as preliminary, showed the presence of psolaren, bergapten, xanthotoxin and simple coumarin – umbelliferone. The Fig. 1 shows the developed TLC Fig. 1. TLC analysis of SPE purified extracts from leaf blades 1( plate of methanolic extracts from leaf blades and petioles of two and 3) and petioles (2 and 4) of two cultivars TLC plates were identified in Si60G F /n-heptane: ribbed celery cultivars. As shown in Fig. 1., sample purification 254 dichloromethane: ethyl acetate (40:20:20 v/v). To the visualisation method on octadecyl C18 columns eliminated ballast substances. of extracts λ=366 nm was used. P- Psolaren, 1 – leaf blades This allowed the identification of coumarin compounds in the ‘Helios’, B – Bergapten, 2 – petioles ‘Helios’, 3 – leaf blades extracts. Additionally, isopimpinellin was detected by HPLC ‘Zefir’,4 – petioles ‘Zefir’. 70 A. Najda et al.

Fig. 2. HPLC chromatogram of furanocoumarin standards 1: xhanthotoxin 2:psoralen 3: isopimpinellin 4: bergapten

Fig. 3. UV spectras of furanocoumarins isolated from the petioles and leaf of two studied ribbed celery varieties Identification and Profile of Furanocoumarins from the Ribbed Celery 71

Fig. 4. HPLC chromatograms of furanocoumarins isolated from different parts of ribbed celery cultivar ʻZefirʼ (in 2011 – I) 1: xanthotoxin, 2: psoralen, 3: isopimpinellin, 4, bergapten ; A – petioles, B leaf blades.

Fig. 5. HPLC chromatograms of furanocoumarins isolated from different parts of ribbed celery cultivar ʻHeliosʼ (in 2011 – I) 1: xanthotoxin, 2: psoralen, 3: isopimpinellin, 4, bergapten; A – petioles, B leaf blades. 72 A. Najda et al.

Table 2. Comparison of average retention times (min) for the different furanocoumarin standards and furanocoumarins isolated from the petioles and leaf blades of both studied cultivars of ribbed celery (based on HPLC analysis)

The average retention time tR (min) Furanocoumarin ʻZEFIRʼ ʻHELIOSʼ standard petioles leaf blades petioles leaf blades 1. Xanthotoxin 8.86 8.97 8.87 8.97 9.05 2. Psoralen 9.37 9.41 9.49 9.26 9.39 3. Isopimpinellin 10.01 10.12 10.0 10.15 10.10 4. Bergapten 13.71 14.01 13.78 13.83 14.00

_ Table 3. Furanocoumarins composition (μg · 100 g 1 DM). Xanthotoxin Psoralen Isopimpinellin Bergapten P-value Cultivar Zefir 0.139bA 0.172bB 0.127aA 0.113aA 0.0346 Helios 0.088aA 0.126aC 0.112aB 0.104aB 0.2101 P-value 0.0354 0.048 0.4103 0.619

Anatomical parts Petioles _ 0.077aA _ 0.124bB 0.0001 Leaf blades 0.158aB 0.238bC 0.177aB 0.063aA 0.0001 P-value 0.0055 0.0002 0.0022 0.0269

Year 2011 0.120aC 0.118aC 0.098aB 0.080aA 0.1194 2012 0.107aA 0.179bC 0.107aA 0.137bB 0.0067 P-value 0.4706 0.0202 0.6109 0.0251

Cultivar x anatomical parts P-value 0.8556 0.8970 0.9493 0.4307 Cultivar x year P-value 0.7624 0.8528 0.2321 0.9617 Anatomical parts x year P-value 0.9999 0.9880 0.5400 0.4457 Cultivar x anatomical parts x year P-value 0.9685 0.9885 0.7411 0.6330

The results are presented as mean ± SD (n = 20 for each year; n = 15 for each cultivar; n = 15 for each anatomical parts). Explanatory notes: different letters a, b, c… and A, B, C… in the same column and line indicate statistically significant differences (P < 0.05). In each column and for each cultivar different letters mean significant differences (P < 0.05).

role in parts of the anatomy of these varieties. All factors (variety, Literature data indicate the occurrence of psoralen, anatomical part and year of harvest) show a significant effect, with 5-methoxypsoralen, bergapten and xsanthotoxin in ribbed celery leaf the exception of the year effect for isopimpinellin (P = 0.610) and (Surico et al., 1987). Beier et al (1983) found that the psoralen, xanthotoxin (P = 0.470) content and cultivar effect for bergapten (P bergapten and trimethylpsoralen content in ribbed celery plants = 0.619) and isopimpinellin (P = 0.410). The content of psoralen, infected by fungi: Sclerotiora sclerotiorum and Ervin carotovara bergapten and isopimpinellin was higher in 2012, with the was significantly higher than in healthy plants, which follows from exception of xanthotoxin. Psoralen content was higher in the leaf the properties of plant defense (biotic stress). Research conducted blades, in which also xanthotoksin and isopinpinellin were by Zobel and Brown (1990) on the furanocoumarins concentration identified, while in petioles bergapten content was higher. Zefir, occurring outside plants of the Apiaceae and Rutaceae family have irrespective of the anatomical part, contained more of all the tested shown that these compounds can be secreted on the leaf surface by furanocoumarins compounds. Higher presence of psoralen, the action of UV rays. Secretion of furanocoumarins on the leaf bergapten and isopimpinellin was observed in 2012, while surface is a defense mechanism of plants against adverse xsanthotoxin content was higher in 2011. There was significant environmental conditions arising as a result of the ultraviolet light differences between individual furanocoumarins in a cultivar Zefir, stress (Afek and Carmeli, 1995; Afek et al., 1996). On the surface _ in the analyzed anatomical parts and in 2012. The results indicate of celery leaves, xsanthotoxin content was 0.006 μg · g 1 of fresh _ a similar profile of furanocoumarins in the analyzed anatomical weight, psoralen in a greater concentration _0.010 μg · g 1, and parts of two cultivars of ribbed celery. bergapten in trace amounts. In addition, the literature (Trumble et Identification and Profile of Furanocoumarins from the Ribbed Celery 73

Fig. 6. Percentage of identified furanocoumarins in petioles of ribbed celery, depending on the time of plant harvest

Fig. 7. Percentage of identified furanocoumarins in leaf blades of ribbed celery, depending on the time of plant harvest

al., 1990; Trumble et al., 1992) reported that the contents of cultivar. Petioles whose harvest was carried out after 100 days of furanocoumarins in the ribbed celery leaves are on the level from growing season accumulated more bergapten (I harvest date). With _ 0.34 to 1.80 μg · g 1 of fresh weight, and depending on the age of the increase of growing season length, its amount in the analyzed the leaves, cultivar and climatic conditions. Quantitative content of parts of the plants in both studied cultivars decreased. In contrast, furanocoumarins occurring in the studied raw materials varied in the percentage of psoralen reached a maximum in petioles whose harvest _ _ petioles from 0,121 μg · g 1 to 0.287 μg · g 1 of dry weight in the was carried out after 120 days of growing season (II date). _ cultivars Zefir and Helios, and in leaf blades from 0.522 μg · g 1 to Chromatographic analysis of ribbed celery leaves showed the _ 0.966 μg · g 1 of dry weight. These amounts may be higher for fresh presence of four furanocoumarins. Assessing the percentage of weight of tested plants leaves, depending on the intensity of UV identified furanocoumarins, it has been reported that psoralen is light. The obtained data confirm the xanthotoxin and bergapten dominant in leaf blades, wherein its content is correlated with the content on the leaf surface, this can be inferred that the agricultural harvest date. Isopimpinellin is the second dominant compound treatments in the cultivation of ribbed celery should be carried out on regardless of the leaves harvest time with 24% and 30% content for cloudy days with low insolation level. Otherwise, photodermatoses Zefir and Helios respectively. Contents of psoralen and bergapten might occur. Considering the percentage of the identified in the leaf blades decreased with the increasing growing season furanocoumarins it has been demonstrated that psoralen varied from length, while the content of isopimpinellin and xanthotoxin slightly 29% to 55%, and bergapten from 45% to 71% (Fig. 6). Bergapten increased (Fig. 7). was dominant in petioles regardless of the harvesting time and In Table 4, the correlation coefficients between the posted 74 A. Najda et al. Table 4. The correlation coefficients between the content of identified furanocoumarins in ribbed celery

Cultivar Xanthotoxin Psoralen Isopimpinellin Bergapten Xanthotoxin 1 0.8433 0.9587 _0.2449 Psoralen 0.8433 1 0.8909 _0.1140 Isopimpinellin 0.9587 0.8909 1 _0.2629 Bergapten _0.2449 _0.1140 _0.2629 1 furanocoumarins identified . Very strong correlation (>0.96) were and Ishii, H. (1995). Coumarins and anti-plateled aggregation observed between the content and xanthotoxin and isopimpinellin. constituents from Zanthoxylium schinifolium. Phytochem., 39, 1091- At the same time a strong correlation has been shown between the 1097. content of a psoralen and xanthotoxin and isopimpinellin. Other Coven, T.R., Walters, I.B., Cardinale, I., and Krueger, J.G. (1999). correlation coefficients between the content of furanocoumarins in PUVA-induced lymphocyte apoptosis: mechanism of action in psoriasis. ribbed celery are not significant. Photoderm. Photoimmun. Photomed., 15, 22-27. Dixon, R.A. and Lamb, C.J. (1999). 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