ACTA BOT. CROAT. 80 (1), 2021 43 Acta Bot. Croat. 80 (1), 43–47, 2021 CODEN: ABCRA 25 DOI: 10.37427/botcro-2020-015 ISSN 0365-0588 eISSN 1847-8476 Phenolic compounds in two subspecies of Drypis spinosa L. (Caryophyllaceae) in Croatia Dario Kremer1, Iztok Jože Košir2, Tanja Potočnik2, Nikolina Rogulj1, Karla Načinović1, Marko Randić3, Siniša Srečec4, Renata Jurišić Grubešić1* 1 University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovačića 1, HR-10000 Zagreb, Croatia 2 Slovenian Institute of Hop Research and Brewing, Cesta Žalskega tabora 2, SI-3310 Žalec, Slovenia 3 Public institution “Priroda”, Grivica 4, HR-51000 Rijeka, Croatia 4 Križevci College of Agriculture, M. Demerca 1, HR-48260 Križevci, Croatia Abstract – As a contribution to chemotaxonomic relations, a quantitative analysis of bioactive phenolic compounds was carried out for the first time in Drypis spinosa L. subsp. spinosa and D. spinosa subsp. jacquiniana Murb. et Wettst. ex Murb. in Croatia. Total polyphenols (TP), tannins (T) and total flavonoids (TF) were determined in samples of leaves, stems, and roots using UV-Vis spectrophotometric methods. For the subsp. spinosa, the highest content of TF was in leaves (0.09%), as was the highest amount of TP (2.36%) and T (1.12%). In the subsp. jacquiniana, the highest contents of TF (0.10%), TP (1.96%), and T (0.88%) were measured in stems. Coumaric, ferulic and rosmarinic acid were identified and quantified by HPLC analysis in both subspecies. Quercetin and sinapic acid were identified only in subsp. spinosa, while rutin and naringenin were found only in subsp. jacquiniana. Among them, ferulic acid was iden- tified only in flowers of both subspecies. The results of this study represent a useful basis for further research of phyto- chemical and eventually phytotherapeutic potential of D. spinosa. Keywords: Drypis, eastern Adriatic, flavonoids, polyphenols, subspecies, tannins Introduction The Balkan Peninsula is known to be very rich in en- demic plant species. This is the meeting point of two phy- togeographic regions: the Alpine – high Nordic regions and the Euro Siberian – North American (Redžić et al. 2011). The genus Drypis L. includes one (Wielgorskaya 1995, Er- hardt et al. 2014) or two (Euro+Med 2006–2019) species. Drypis spinosa L. subsp. spinosa (syn. D. spinosa, D. spinosa subsp. linneana Murb. et Wettst.) is distributed in Italy, Slo- venia, Croatia, Montenegro, Albania, North Macedonia and Greece, while D. spinosa subsp. jacquiniana Murb. (syn. D. jacquiniana Murb. et Wettst.) is known from Italy, Slovenia and Croatia (Šilić 1990, Stešević and Caković 2013, Euro+Med 2006–2019). Subspecies spinosa is a xerothermic, lithophytic species that grows on open, stony or gravelly habitats between mountain and subalpine vegetation belts (Domac 1964, Šilić 1990, Stešević and Caković 2013). On the other hand, subsp. jacquiniana is distributed along the eastern Adriatic coast from the close to sea level to 800 m a.s.l. and it grows predominantly on coastal rocks and screes (Domac 1964, Šilić 1990, Jasprica 2015). Drypis spinosa is a typical representative of endemic plants with horticultural and possible medicinal potential. Investigation of biologi- cally active compounds in D. spinosa is part of our continu- ous work on Balkan plant species. Among biologically active compounds, phenolic com- pounds have attracted a great deal of scientific and public interest due to their health-promoting effects as antimicro- bials, antivirals and antioxidants. The content of biologi- cally active compounds varies among species, including closely related species, making them useful chemotaxonom- ic markers. The plants of the Caryophyllaceae family con- tain significant amounts of polyphenols, including flavo- noids. Ethnopharmacologica l studies indicate that Caryophyllaceae possess anticancer, antibacterial, antifun- * Corresponding author e-mail: rjurisic@pharma.hr KREMER D., KOŠIR I. J., POTOČNIK T., ROGULJ N., NAČINOVIĆ K., RANDIĆ M., SREČEC S., JURIŠIĆ GRUBEŠIĆ R. 44 ACTA BOT. CROAT. 80 (1), 2021 spectrophotometric determination of TP and T at 720 nm. Tannic acid was used as the standard substance. The content of total flavonoids (TF; colorimetric assay with AlCl3) was obtained using spectrophotometric method prevalidated by Jurišić Grubešić et al. (2007). This proce- dure encloses hydrolysis of glycosides, extraction (with eth- yl acetate) of TF aglycones and complex formation with Al- Cl3 at 425 nm. The yield of TF was calculated as quercetin according to the following expression: TF (%) = A × 0.772 / b In this expression, A denotes measured absorbance, 0.772 is the conversion factor related to the specific absor- bance of quercetin at 425 nm, while b is mass (g) of air-dried herbal material. TP, T and TF contents were performed in triplicate (technical replicates) and the results were expressed as mean ± SD. HPLC analysis The ultrasonic extraction was performed on finely pow- dered air-dried herbal material (500 mg) with 20 mL of methanol at 25 °C for 60 min, then 20 mL of ethyl-acetate at 25 °C for 60 min, and finally with 20 mL of distilled wa- ter at 25 °C and 45 °C for 60 min. After that, samples were filtered (qualitative analytical filter paper) and diluted with the same solvent mixture to a volume of 25.0 mL. An aliquot of 5 μL of each sample solution was injected into the HPLC system for chromatographic analysis. Prior to injection samples were filtered through 0.45 μm PTFE 25 mm filter (Restek Co., USA). The stock solutions of standard compounds: chrysin, quercetin, quercitrin, rutin, chicoric acid, coumaric acid, ferulic acid, protocatechuic acid, rosmarinic acid, syringic acid and tannic acid were prepared according to Čeh et al. (2007) and Kremer et al. (2013). At first, the stock solutions of standards were diluted separately in the mixture of water and methanol (1:1, V/V) at a concentration of 1.0 mg mL–1. After that, the working standard solutions (at a concentra- tion of 0.01 mg mL–1) were made by the dilution of each stock solution with the same mixture of water and metha- nol. The mixture of standards was made by the dilution of each stock standard solution with the same solvent mixture at final concentration of each standard of 0.01 mg mL–1. HPLC analysis was performed according to Čeh et al. (2007) and Kremer et al. (2013). A C18 reversed-phase pack- ing column was used for separation at the temperature of 30 ºC. A gradient elution type of chromatographic systems was used. The mobile phase for gradient elution was com- posed of phase A (water, pH = 2.50 adjusted with acetic ac- id), and phase B (acetonitrile), which was applied according to the following elution scheme: starting with 15% B and 85% A, followed with gradient from 15% B to 22.5% B in 15 min, then from 22.5% B to 40% B in 10 min and kept con- stant with 40% B and 60% A for another 5 min. In the ad- dition 5 min, the initial conditions were set up. The injec- gal, antiviral, antioxidant, and anti-inflammatory proper- ties (Chandra and Rawat 2015). This is the reason why the investigations of phenolic compounds were conducted with various species from the Caryophyllaceae family. The aim of this study is to gain an insight into the con- tent of phenolic compounds in D. spinosa subsp. spinosa and D. spinosa subsp. jacquiniana growing in Croatia. To the best of our knowledge this is the first report on the chemical properties of D. spinosa. Materials and methods Sampling, chemicals and apparatus The samples of the investigated subspecies were collect- ed during the blooming period in July 2017. Plant material of Drypis spinosa subsp. spinosa was collected on Mt Velebit at 1200 m a.s.l., while the samples of D. spinosa subsp. jac- quiniana were collected in the town of Rijeka at 70 m a.s.l. Above-ground parts of several randomly selected plants were harvested on a dry day and mixed to obtain a random- ly selected sample. The stems, leaves and flowers were col- lected separately. Herbal material was air-dried in a well- ventilated room at 60% relative humidity and room temperature (22 ºC) for three weeks. The plant material was protected from direct sunlight and single-layered during drying. After drying, the plant samples were placed into double paper bags and stored in a dry place at room tem- perature (22 ºC, 60% of humidity), protected from the light until analysis. Voucher specimens of herbal material were deposited in the Fran Kušan Herbarium of the Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia. Folin-Ciocalteu reagent (FCR), chrysin, quercetin, quer- citrin, rutin, chicoric acid, coumaric acid, ferulic acid, pro- tocatechuic acid, rosmarinic acid, syringic acid, tannic acid, linoleic acid, and gallic acid (Sigma-Aldrich Chemical Co., USA); methanol, ethanol, and HNO3 (Kemika, Croatia) were used. All chemicals and reagents were of analytical grade. Double distilled water was used throughout the study. Agilent 8453 E UV/Vis spectrophotometer (Hewlett Packard, Germany) equipped with the PC-HP 845x UV- Visible System (Hewlett Packard, Germany) and 1 cm quartz cells were used for absorbance measurements. HPLC analysis was performed using Agilent 1100 Series HPLC sys- tem and A C18 reversed-phase packing column (Zorbax Eclipse XDB-C18, 150 mm × 4.6 mm, 5 μm; Agilent, USA). Phenolic compounds analyses The quantitative analysis of biologically active phenolic compounds was carried out using spectrophotometric met- hods, following adequate extractions. Spectrophotometric determination of total polyphenols (TP) and tannins (T) was performed with the use of FCR. Comprehensive prevalidation of this method was carried out by Jurišić Grubešić et al. (2005). This method is based on a reaction with FCR and, after precipitation with casein, PHENOLIC COMPOUNDS IN DRYPIS SPINOSA ACTA BOT. CROAT. 80 (1), 2021 45 tion volume was 5 μL, while flow rate was 1.0 mL min–1. Detection was performed by diode array detector at wave- lengths 280 nm (chrysin, chicoric acid, syringic acid, pro- tocatechuic acid, tannic acid), 320 nm (ferulic acid, rosma- rinic acid, coumaric acid) and 370 nm (quercetin, quercitrin and rutin). Particular compounds were identified by com- paring the retention times of standards and unknown peaks in the samples. The method of standard addition was ap- plied in order to avoid misinterpretation of results. Quan- tification was conducted using the external standards. Method was previously validated to confirm the linear range, reproducibility (RSD, between 3.8% and 8.6%) and accuracy (R, 76% to 87%) for particular compounds (de- tailed data not shown here). Limit of quantification (LOQ) was determined at the concentration of 0.001%. Data analysis Significance of differences between analytical data was checked by the least significant difference (LSD) test, using the STATISTICA software (Hill and Lewicki 2006). Results Total polyphenols (TP), tannins (T) and flavonoids (TF) content UV-Vis spectrophotometric methods were used for a quantitative analysis of TP, T and TF in D. spinosa subsp. spinosa and D. spinosa subsp. jacquiniana. The results of the determinations of TP, T and TF are presented in Tab. 1. For D. spinosa subsp. spinosa, the highest content of TP (2.36%) was in leaves, as was the highest amount of T (1.12%) and TF (0.09%). In D. spinosa subsp. jacquiniana, the highest contents of TP (1.96%), T (0.88%) and TF (0.10%) were mea- sured in stems. It is evident that there was no dependence among plant parts and the quantity of investigated phenolic substances. The largest statistically significant differences in the content of all analyzed polyphenolic substances were ob- served for leaves of subsp. spinosa and subsp. jacquiniana, while the least significant difference in the content of poly- phenols was noticed among flowers of the investigated plant subspecies (Tab. 1). HPLC analysis Seven phenolic compounds were identified and quanti- fied by HPLC analysis in D. spinosa subsp. spinosa and D. spinosa subsp. jacquiniana and their concentrations in leaves, flowers and stems are shown in Tab. 2. Among stan- dard compounds only caffeic acid was not identified in the investigated subspecies. Content of phenolic compounds varied between 0.002% (coumaric acid) and 0.839% (narin- genin). Among them, quercetin and sinapic acid were iden- tified only in subsp. spinosa, while rutin and naringenin were identified only in subsp. jacquiniana. On the other hand, coumaric acid, ferulic acid and rosmarinic acid were found in both subspecies. Ferulic acid was identified only in flowers of both subspecies. Discussion Quantitative analysis of TP, T and TF was performed with the leaves, flowers and stems of Drypis spinosa subsp. spinosa and D. spinosa subsp. jacquiniana. Obtained results showed that there was no dependence among plant parts and the quantity of the phenolic substances investigated. Tab. 1. Contents (mean ± SD) of total polyphenols (TP), tannins (T) and flavonoids (TF) in Drypis spinosa subsp. spinosa and D. spi- nosa subsp. jacquiniana obtained by UV-Vis spectrophotometric methods. The asterisk (*) in superscript indicates a significant diffe- rence obtained by LSD test for content of TP, T and TF between different plant parts (leaf, flower, stem) of subsp. spinosa and subsp. jacquiniana at P < 0.05. N = 3. Phenolic compund (%) Leaf Flower Stem subsp. spinosa subsp. jacquiniana subsp. spinosa subsp. jacquiniana subsp. spinosa subsp. jacquiniana TP 2.357 ± 0.094* 1.773 ± 0.032 1.810 ± 0.035 1.808 ± 0.016 1.698 ± 0.137 1.955 ± 0.040 T 1.119 ± 0.081* 0.350 ± 0.113 0.557 ± 0.021 0.468 ± 0.113 0.503 ± 0.144* 0.895 ± 0.050 TF 0.092 ± 0.006* 0.057 ± 0.003 0.073 ± 0.002* 0.056 ± 0.002 0.063 ± 0.005* 0.097 ± 0.006 Tab. 2. Contents of phenolic compounds (%) in dried plant material of Drypis spinosa subsp. spinosa and D. spinosa subsp. jacquiniana obtained by HPLC analysis. – : not detected, N = 1. Phenolic subsp. spinosa subsp. jacquiniana compound (%) leaf flower stem leaf flower stem Rutin – – – – – 0.047 Quercetin – – 0.008 – – – Naringenin – – – – 0.839 – Sinapic acid 0.017 – 0.004 – – – Coumaric acid – – 0.004 0.002 0.004 – Ferulic acid – 0.060 – – 0.066 – Rosmarinic acid 0.041 0.043 0.041 – 0.030 – KREMER D., KOŠIR I. J., POTOČNIK T., ROGULJ N., NAČINOVIĆ K., RANDIĆ M., SREČEC S., JURIŠIĆ GRUBEŠIĆ R. 46 ACTA BOT. CROAT. 80 (1), 2021 This is in accordance with some other investigations. For example, Satureja montana L. and S. subspicata Vis. (La- miaceae) had the highest TP content in leaves (Dunkić et al. 2012), while Moltkia petraea (Tratt.) Griseb. (Boraginaceae) had the highest TP content in flowers (Vuković Rodríguez et al. 2016). Additionally, investigations of TP content in ten Moltkia petraea populations obtained by Kremer et al. (2016) showed that TP content was the highest in leaves in six populations. But in another four populations the TP content was the highest in flowers. Both subspecies of D. spinosa contain relatively small amounts of TP, which ranged from 1.70% in the stems to 2.36% in the leaves of D. spinosa subsp. spinosa. For comparison, TP content ranged from 4.43% in stem to 12.41% in leaves of Satureja montana, and from 5.96% in stem to 20.04% in leaves of S. subspicata (Dunkić et al. 2012). Additionally, TP content in Moltkia petraea ranged from 3.26% in stem to 6.61% in leaves (Kre- mer et al. 2016). A similar independence among plant parts and the quantity of investigated phenolic substances was obtained for T content in Drypis spinosa. The highest T content was obtained for the leaves and flowers of D. spinosa subsp. spi- nosa and D. spinosa subsp. jacquiniana, respectively. Ac- cording to Dunkić et al. (2012), leaves of Satureja montana and S. subspicata generally contained higher concentrations of T than stems and flowers. Content of T varied between 0.08% in stem and 4.90% in leaves of S. montana, and be- tween 0.21% in stem and 6.95% in leaves of S. subspicata. The lowest T content (0.76%) was also measured in stems and the highest (7.33%) in leaves of Teucrium arduinii L. (Kremer et al. 2013). On the other hand, the highest T con- tent was determined in the flowers in most of the ten inves- tigated populations of Moltkia petraea (Kremer et al. 2016). The content of catechins in Stellaria media (L.) Vill. (Caryo- phyllaceae), a herb obtained from five Ukrainian popula- tions ranged from 0.67% to 0.90% (Vodoslavskyi 2017). The content of TF was the lowest among three investi- gated groups of phenolic compounds. Dunkić et al. (2012) found that the lowest TF content was in the stem of Sature- ja montana (0.06%), and the highest in leaves (0.40%). That investigation also showed that samples of leaves and flowers of S. montana have a comparable quantity of TF. The lowest TF content was measured in S. subspicata stem (0.07%), and the highest in the leaves (0.57%). Additionally, the highest TF content was identified in leaves in all Moltkia petraea populations (Kremer et al. 2016). Although some exceptions exist, the literature data show that stems contain the lowest TF content. The content of TF was also analyzed in five Ukrainian populations of Stellaria media herb, ranging from 1.22% to 1.40% (Vodoslavskyi 2017). The most intensive investigations of the Caryophylla- ceae family have been carried out on species with medical properties. These investigations showed that Caryophylla- ceae contain a great number of bioactive compounds be- longing to several chemical groups. Seven phenolic com- pounds were identified in D. spinosa subsp. spinosa and D. spinosa subsp. jacquiniana using HPLC analysis. The same phenolic compounds were also identified in some other Caryophyllaceae species. Quercetin was identified in Silene littorea Brot (Richardson 1978) and Arenaria serpyllifolia L. (Zhou and Tu 2013), while naringenin was detected in Di- anthus caryophyllus L. (Spribille and Forkmann 1982). Teğin at al. (2018) identified naringenin and rutin in Sper- gularia rubra (L.) J. Presl et C. Presl. Rutin, ferulic acid, caf- feic acid and p-coumaric acid were detected in Lychnis flos- cuculi L. (Ferry and Darbour 1979, Costea et al. 2017). Ferulic acid was also identified in Stellaria media (Kitanov 1992), while p-coumaric acid was detected in Gypsophila paniculata L. (Chou et al. 2008). Conclusion The content of different phenolic compounds was deter- mined in leaves, flowers and stems of Drypis spinosa subsp. spinosa and D. spinosa subsp. jacquiniana growing in Croa- tia. There was no dependence among plant parts and the quantity of investigated phenolic substances. 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