ACTA BOT. CROAT. 77 (1), 2018 1 Acta Bot. Croat. 77 (1), 1–9, 2018 CODEN: ABCRA 25 DOI: 10.1515/botcro-2017-0017 ISSN 0365-0588 eISSN 1847-8476 Comparative analysis of specialized metabolites and antioxidant capacity in vitro of different natural populations of Globularia spp. Maja Friščić1*, Semir Maslo2, Rade Garić3, Željan Maleš1, Kroata Hazler Pilepić 1 1 Department of Pharmaceutical Botany, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia 2 Lundåkerskolan, Gislaved, Sweden 3 Institute for Marine and Coastal Research, University of Dubrovnik, Dubrovnik, Croatia Abstract – Total phenolic, flavonoid, condensed tannin and iridoid content, as well as antioxidant capacity in vi- tro, were determined spectrophotometrically in methanolic extracts of different plant parts of the Mediterranean medicinal plant Globularia alypum L. and three widespread European species of the same genus: G. cordifolia L., G. meridionalis (Podp.) O. Schwarz and G. punctata Lapeyr. In order to consider possible environmental influ- ences on the production of specialized metabolites, each species, except G. alypum, was collected from three dif- ferent natural populations. Great variations in the amounts of specialized metabolites were observed among dif- ferent plant parts and species. For example, total phenolic content ranged from 10.13 (G. punctata, flowers) to 44.90 (G. cordifolia, flower stems) mg gallic acid equivalent g–1 dry weight. Moreover, great differences, attributed to location-specific environmental factors, were observed among different populations of the same species. For example, a strong positive correlation was observed among mean monthly temperatures and total phenolic con- tents in the leaves of studied Globularia spp. (r = 0.75, p = 0.019). However, despite these differences, all species were rich in bioactive substances when compared to G. alypum, especially in their aerial parts. A very good posi- tive correlation was observed between total phenolic content and DPPH radical scavenging capacity (r = 0.86, p < 0.001)/ABTS radical scavenging capacity (r = 0.83, p < 0.001). The results obtained show that G.  cordifolia, G. meridionalis and G. punctata are rich in bioactive substances, providing support for their pharmaceutical utiliza- tion. Further investigations are needed to verify the possibility of their medicinal use. Keywords: antioxidant activity, environmental factors, flavonoids, Globularia, iridoids, polyphenols, proanthocy- anidins, secondary metabolites * Corresponding author, e-mail: mfriscic@pharma.hr Introduction Plants have been used as healing agents since ancient times; many bioactive compounds isolated from herb- al sources have been used as drugs or served as lead com- pounds in drug development. In Europe, herbal medicines in the crude forms of teas and decoctions are often used as sup- portive therapy, while standardized herbal preparations are a popular alternative to synthetic drugs. Finally, about 80% of the world population (primarily in developing countries) still uses herbal medicine in the treatment of different dis- eases and in maintaining health (Gurib-Fakim 2006). The Old World genus Globularia L., recently included in the Plantaginaceae family (Albach et al. 2005), consists of perennials, subshrubs and small shrubs. Some members of the genus, mainly Globularia alypum L., G. arabica Jaub. & Spach and G. trichosantha Fisch. & C.A. Mey., are used in the traditional medicine of countries such as Spain, Italy, Morocco, Algeria, Tunisia, Libya, Egypt and Turkey (Lepo- ratti and Ghedira 2009, Altundag and Ozturk 2011, Carrió and Vallès 2012, De Natale and Pollio 2012, Bouzabata 2013, Eissa et al. 2014, El Abbouyi et al. 2014). They are tradition- FRIŠČIĆ M., MASLO S., GARIĆ R., MALEŠ Ž., HAZLER PILEPIĆ K. 2 ACTA BOT. CROAT. 77 (1), 2018 ally used as hypoglycaemic agents, purgatives, depuratives (De Natale and Pollio 2012, Bouzabata 2013), antiparasitic and antifungic agents (Altundag and Ozturk 2011, De Na- tale and Pollio 2012), tonics and diuretics, for wound healing and in the treatment of insomnia, fits of epilepsy, gastroin- testinal disorders, intermittent fever (Leporatti and Ghedira 2009, De Natale and Pollio 2012, Eissa et al. 2014), arthritis and rheumatism (De Natale and Pollio 2012). Phenolic compounds and iridoids are the main special- ized (secondary) metabolites of Globularia species (Kirmi- zibekmez et al. 2008, Kirmizibekmez et al. 2009, Tundis et al. 2012b). G. alypum, the most widely used member of the genus Globularia, was shown to be especially rich in pheno- lic compounds in comparison with some other medicinal plants (Djeridane et al. 2006, Djeridane et al. 2010, Ames- sis-Ouchemoukh et al. 2014, El Guiche et al. 2015). Phenolic compounds possess a wide range of biological activities and thus may contribute to the healing properties of Globularia preparations. Radical scavenging activity, also attributed to plant phenolics, is one of the possible protective mechanisms against cancer, cardiovascular diseases, diabetes, osteoporo- sis and neurodegenerative diseases (Rice-Evans et al. 1997). It was noted that G. alypum extracts possess high antioxidant capacity both in vitro (Djeridane et al. 2010, Amessis-Ouche- moukh et al. 2014) and in vivo (Taleb-Dida et al. 2011). Anti- oxidant activity was also recently reported in G. meridionalis (Tundis et al. 2012a). The aim of the present study was to analyse and com- pare the total phenolic content, including flavonoid and condensed tannin content, iridoid content and antioxidant capacity in four members of the genus Globularia L. Three of these, namely G. cordifolia L., G. meridionalis (Podp.) O. Schwarz and G. punctata Lapeyr., are less-investigated, al- though they have a relatively wide distribution in Europe (Tutin 1972). In order to examine their therapeutic poten- tial with regard to the content of bioactive substances, their results were compared to those of the well-investigated me- dicinal plant G. alypum L., which is distributed mainly in the Mediterranean area. Since synthesis and distribution of spe- cialized metabolites is complex and differs between tissues and organs (Boudet 2007), the study was focused on different plant parts. The analysis was done on material collected from three different locations in order to consider possible eco- logical influences on the production of the specialized me- tabolites investigated. Exceptionally, G. alypum was collected from the only location at which it grows wild in Croatia and was sampled without underground parts because of its near threatened status. Obtained phytochemical data were cor- related with environmental factors of different habitats, en- abling the interpretation of obtained results from both me- dicinal and ecological perspectives. Materials and methods Plant material and extraction A total of ten samples of four Globularia taxa were col- lected during the phenophase of blooming from seven loca- tions in Croatia and one in Bosnia and Herzegovina (Tab. 1). Voucher specimens are deposited in the Herbarium of the Department of Pharmaceutical Botany, Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia. The identity of plant material was verified by Prof. Kroata Hazler Pilepić. Meteorological data were obtained from the nearest meteo- rological stations (Meteorological and Hydrological Service Tab. 1. Collection and geographical data for plant material of the investigated Globularia species; Ga – Globularia alypum, Gc – G. cordi- folia, Gm – G. meridionalis, Gp – G. punctata. Species (Voucher No.) Location Geographical latitude Geographical longitude Elevation (m) Habitat type Harvest time Ga (16 020) Dubrovnik area (Konavle cliffs) 42°30´50˝N 18°19´07˝E 15 Limestone cliffs by the sea March 2013 Gc (1) (16 032) Northern Velebit area (Alan) 44°43´15˝N 14°58´05˝E 1340 Calcareous grasslands May 2013 Gc (2) (16 031) Middle Velebit area (Baške Oštarije) 44°31´41˝N 15°08´38˝E 917 Calcareous grasslands May 2012 Gc (3) (16 030) Mostar area (the Neretva banks) 43°21´41˝N 17°48´20˝E 57 Rocky grasslands along a river May 2012 Gm (1) (16 041) Istrian peninsula (Mala Učka) 45°17´59˝N 14°11´27˝E 926 Limestone cliffs May 2012 Gm (2) (16 040) Rijeka area (Grobničko polje) 45°22´39˝N 14°30´53˝E 308 Karst field May 2013 Gm (3) (16 043) Middle Velebit area (Baške Oštarije) 44°31´41˝N 15°08´38˝E 917 Calcareous grasslands May 2012 Gp (1) (16 051) Istrian peninsula (Vižintini) 45°23´36˝N 13°51´20˝E 386 Calcareous grasslands May 2012 Gp (2) (16 056) Rijeka area (Grobnik field) 45°22´39˝N 14°30´53˝E 308 Karst field May 2013 Gp (3) (16 057) Žumberak area (Slapnica canyon) 45°44´29˝N 15°29´26˝E 325 Limestone cliffs May 2013 SPECIALIZED METABOLITES OF GLOBULARIA SPP. ACTA BOT. CROAT. 77 (1), 2018 3 of Croatia and the Federal Hydrometeorological Institute of Bosnia and Herzegovina) (Tab. 2). Dried and powdered plant parts (2.5 g) were subjected to ultrasound-assisted extraction (Bandelin Sonorex Super, Germany) at room temperature for 30 min with 25 mL of methanol. The residue after filtration was extracted again for 30 min with 25 mL of methanol, filtered and the final volume was adjusted to 50 mL. Four classes of metabolites were measured spectrophotometrically using a Varian Cary 50 Bio UV-Vis spectrophotometer (USA). Determination of plant specialized metabolites The total phenolic content was determined using Folin- Ciocalteu’s reagent according to the method of Singleton and Rossi (1965). The reaction mixture was prepared by mix- ing 0.5 mL of methanolic extract with 2.5 mL of 10% (v/v) Folin-Ciocalteu’s reagent (diluted in distilled water). After 5 min, 2 mL of 7.5 g/100 mL sodium carbonate decahydrate solution were added and the mixture was incubated for 1 h at room temperature. After incubation, the absorbance was read at 765 nm against a distilled water blank. Gallic acid was used for the construction of the calibration curve. The total phenolic content was expressed as mg gallic acid equivalent (GAE) g–1 dry weight (DW). The flavonoid content was measured using the Dowd method (Arvouet-Grand et al. 1994). Briefly, 1 mL of ap- propriately diluted extract was mixed with 1 mL of 2 g/100 mL AlCl3 solution in pure methanol. The absorbance was measured after 15 min at 415 nm against a sample blank which consisted of 1 mL of extract and 1 mL of methanol. A standard calibration curve was plotted using quercetin as a reference standard. The results were expressed as mg quer- cetin equivalent (QE) g–1 dry weight (DW). The condensed tannin (proanthocyanidin) content was determined using the vanillin assay (Broadhurst and Jones 1978, Sun et al. 1998) as described previously by Toda (2005), but with a slightly modified reaction temperature. For the preparation of the sample 2 mL of 1 g/100 mL vanillin in 7 M sulfuric acid were added to a test tube containing 1 mL of diluted extract and the mixture was incubated for 15 min in a water bath with the temperature set at 30±1 °C. The reac- tion was performed in normal laboratory daylight. After the incubation, the absorbance was recorded at 500 nm against a sample blank which consisted of 1 mL of extract and 2 mL of 7 M sulfuric acid, which was incubated under the same con- ditions as the sample. (+)–catechin was chosen as a standard for the calibration curve. The levels of total condensed tan- nin content were expressed as mg catechin equivalent (CE) g–1 dry weight (DW). The iridoid content was measured using the Trim- Hill reagent (Trim and Hill 1952) as adapted by Tundis et al. (2012a). To 200 µL of diluted extract 2 mL of Trim- Hill reagent (glacial acetic acid:0.2% copper (II) sulfate pentahydrate:concentrated hydrochloric acid at a ratio of 10:1:0.5, v:v:v) were added and the mixture was heated in a boiling water bath for 5 min. After that, absorbance of the prepared solution was read at 609 nm with methanol used as blank. The concentration of iridoids in the samples was calculated based on an aucubin calibration curve and the re- sults were presented as mg aucubin equivalent (AE) g–1 dry weight (DW). Evaluation of antioxidant capacity Antioxidant capacity of the extracts was evaluated by the Blois method (1958) using 2,2-diphenyl-1-picrylhydra- zyl (DPPH) radical solution according to the procedure of Thetsrimuang et al. (2011) with some modifications. A 0.1 mM stock solution of DPPH in methanol was prepared. The working solution was obtained by diluting the stock solu- tion with methanol to obtain an absorbance of 0.7±0.02. To 2 mL of this solution, 10 µL of properly diluted extract were added and the decrease in absorption of the radical was mea- sured at 517 nm after 30 min incubation in the dark against a methanol blank. A calibration curve was obtained by us- Tab. 2. Records of mean monthly temperatures and monthly precipitation amounts from meteorological stations close to the locations where Globularia species were collected (for details see Tab. 1). Records are shown for the month in which the plant material was har- vested (M), the three previous months (M-3, M-2, and M-1) as well as their average; Ga – Globularia alypum, Gc – G. cordifolia, Gm – G. meridionalis, Gp – G. punctata. Plant species Mean monthly temperature (°C) Monthly precipitation amounts (mm) Meteorological station (Elevation (m)) M-3 M-2 M-1 M Average M-3 M-2 M-1 M Average Ga 9.6 10.1 9.5 11.2 10.1 283.2 205.2 238.1 214.0 235.1 Dubrovnik (52) Gc (1) –6.4 –3.1 3.8 5.6 0.0 284.6 293.9 152.7 253.4 246.2 Zavižan (1594) Gc (2) –5.0 7.3 9.8 13.4 6.4 58.2 1.0 116.2 102.0 69.4 Gospić (564) Gc (3) 1.7 13.5 13.2 18.6 11.8 202.7 0.3 266.6 92.3 140.5 Mostar (99) Gm (1) 0.7 8.9 11.7 15.5 9.2 30.1 1.3 54.2 104.4 47.5 Letaj (120) Gm (2) 5.1 8.0 14.3 16.4 11.0 210.1 386.9 80.0 228.3 226.3 Rijeka (120) Gm (3) –5.0 7.3 9.8 13.4 6.4 58.2 1.0 116.2 102.0 69.4 Gospić (564) Gp (1) 0.2 8.8 11.4 15.3 8.9 11.9 0.0 61.4 105.6 44.7 Pazin (291) Gp (2) 5.1 8.0 14.3 16.4 11.0 210.1 386.9 80.0 228.3 226.3 Rijeka (120) Gp (3) 1.5 4.4 12.9 15.8 8.7 93.7 121.1 50.2 107.0 93.0 Maksimir (128) FRIŠČIĆ M., MASLO S., GARIĆ R., MALEŠ Ž., HAZLER PILEPIĆ K. 4 ACTA BOT. CROAT. 77 (1), 2018 ing different concentrations of gallic acid and the results were expressed as mg gallic acid equivalent (GAE) g–1 dry weight (DW). The 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity was determined following the method of Re et al. (1999) with some modifi- cations. An activated solution of ABTS˙+ radical cation was prepared by mixing ABTS and potassium peroxodisulfate solutions so that the final concentrations in the mixture were 7 mM and 2.45 mM, respectively. The solution was held at room temperature (22±2 °C) for at least 16 h before use. The working solution was obtained by diluting the stock solution with distilled water to obtain an absorbance of 0.7±0.02. The fall of absorbance was measured at 734 nm against a distilled water blank 1 min after mixing 10 µL of properly diluted extract with 2 mL of activated radical. ABTS radical scav- enging activity was calculated from a gallic acid calibration curve and presented as mg gallic acid equivalent (GAE) g–1 dry weight (DW). Statistical analysis All measurements were performed in triplicate and the results are presented as means ± standard deviations. A two- way analysis of variance (ANOVA) followed by the Bonfer- roni post-hoc test was carried out on averaged results for plant parts of each species, to determine significant differ- ences among the same plant parts of different species and different plant parts of the same species. Pearson’s correlation coefficient (r) was used to determine the association among parameters, more precisely, among the contents of particu- lar groups of specialized metabolites (total phenolics, flavo- noids, condensed tannins and iridoids) in all samples (alto- gether 48 samples) and their antioxidant capacities evaluated by two different assays (DPPH and ABTS) or among the con- tents of particular groups of specialized metabolites in spe- cific plant parts and averages of mean monthly temperatures and monthly precipitation amounts of individual locations (maximum of ten samples per analysis), with a significance level α = 0.05. The statistical analysis was carried out using GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego, USA). Results Content of plant specialized metabolites All four investigated species were found to be rich in phe- nolic compounds, with observed differences among plant parts (Tab. 3). The content of total phenolics ranged from 10.13 (G. punctata (3), flowers) to 44.90 (G. cordifolia (1), flower stems) mg GAE g–1 DW. High amounts of polyphe- nols were observed in leaves of all species, with no significant differences noticed between G. alypum and related species. Leaves and flowers of G. alypum were the richest plant parts for this species. On the other hand, it was observed that flow- er stems of G. cordifolia and G. meridionalis, as well as woody stems and underground parts of G. punctata, also contained high amounts of phenolic compounds. Flowers of G. punc- tata were the poorest of all the tested samples (p < 0.05). The flavonoid content ranged from 0.84 (G. cordifolia (3), woody stems) to 17.77 (G. punctata (2), leaves) mg QE g–1 DW (Tab. 3). When different plant parts between species were compared, it was observed that the leaves contained the highest amounts of flavonoids. G. punctata leaves and flower stems contained more flavonoids than those of other species (p < 0.05), while there were no significant differences among the species for flowers and underground parts. The lowest amounts of flavonoids were observed for woody stems and underground parts. Condensed tannin (proanthocyanidin) content varied from 0.19 (G. meridionalis (2), underground parts) to 9.77 (G. cordifolia (3), flower stems) mg CE g–1 DW (Tab. 3). Tak- ing into account all plant parts, condensed tannin content was especially high in the population collected from Mo- star. Green aerial parts contained more tannins than woody stems and underground parts. In G. cordifolia, leaves and flower stems contained the highest amounts of tannins (p < 0.05), while for other species no significant differences were observed among plant parts. The content of iridoids varied from 3.94 (G. punctata (3), underground parts) to 143.29 (G. punctata (1), leaves) mg aucubin equivalent (AE) g–1 DW (Tab. 3). High amo- unts of iridoids were found in G. cordifolia, G. meridionalis and G. punctata, especially in their leaves and flower stems (p < 0.05). No significant differences in iridoid content were noticed between different organs of G. alypum. Correlations between phenolic/iridoid content and environmental factors In order to verify the influence of specific environmen- tal factors on the variability of the compounds determined, results obtained for each plant part were correlated with mean monthly temperatures and monthly precipitation amounts of different stands and seasons (Tab. 2). Averag- es for the month in which the plant material was collected and the three previous months were correlated with the amounts of investigated compounds. During evaluation of the influence of temperature, the population of Mala Učka had to be excluded due to the great elevation differ- ence between the location of sampling (926 m) and the nearest meteorological station (120 m), from which me- teorological data were available, which made it impossible to accurately predict the mean monthly temperatures for this location. Taking this into account, however, a strong positive correlation was observed between mean monthly temperatures and total phenolic contents in the leaves of investigated species (r = 0.75, p = 0.019), together with a strong negative correlation in the flower stems (r = –0.88, p = 0.002). In spite of the significant variability discovered in the iridoid contents among populations, no correlation with mean monthly temperatures (p > 0.05) was found. Also, no significant correlation between precipitation and amounts of bioactive substances was observed (p > 0.05). SPECIALIZED METABOLITES OF GLOBULARIA SPP. ACTA BOT. CROAT. 77 (1), 2018 5 Tab. 3. Total phenolic, flavonoid, condensed tannin and iridoid contents as well as radical scavenging capacities obtained by 2,2-diphe- nyl-1-picrylhydrazyl (DPPH) and 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, in different plant parts of four Globularia species collected from different locations (for details see Tab. 1). Values are means ± SD, n = 3; statistically significant differ- ences (p < 0.05) between the same plant parts of different species are indicated by different superscript lower case letters (a > b > c > d) and between different plant parts of the same species in different superscript capital letters (A > B > C > D); * – flowers of the plant are carried by woody stems; Ga – Globularia alypum, Gc – G. cordifolia, Gm – G. meridionalis, Gp – G. punctata; L – leaves, F – flowers, FS – flower stems, WS – woody stems, UP – underground parts; n.m. – not measured; GAE – gallic acid equivalent, DW – dry weight, QE – quercetin equivalent, CE – catechin equivalent, AE – aucubin equivalent. Plant species Plant part Polyphenols (mg GAEg–1 DW) Flavonoids (mg QE g–1 DW) Tannins (mg CE g–1 DW) Iridoids (mg AE g–1 DW) DPPH (mg GAE g–1 DW) ABTS (mg GAE g–1 DW) Ga L 37.58±0.85aA 9.23±0.39bA 0.76±0.03bA 9.38±0.55cA 18.29±2.64aA 12.35±0.81aA F 35.39±0.78aA 5.46±0.04aB 0.77±0.01aA 6.18±0.08aA 20.48±2.03aA 14.41±0.56aA FS = WS* 21.16±0.72cB 4.74±0.04cB 0.34±0.03bA 5.06±0.04bA 11.67±0.37aB 8.09±0.37bB UP n.m. n.m. n.m. n.m. n.m. n.m. Gc (1) L 27.67±0.82 9.08±0.36 2.55±0.07 77.34±1.54 9.98±0.97 8.18±0.02 F 25.68±0.70 7.12±0.08 2.30±0.10 24.67±0.81 7.96±0.68 7.52±1.02 FS 44.90±1.18 9.56±0.15 4.17±0.07 57.56±2.25 14.70±1.43 12.52±1.98 WS 22.22±0.51 2.00±0.01 0.28±0.01 8.37±0.48 9.12±0.96 7.91±0.43 UP 22.15±1.03 1.96±0.07 0.21±0.00 6.03±0.14 9.91±0.22 7.56±0.10 Gc (2) L 34.42±0.94 9.24±0.15 1.92±0.02 132.43±1.18 11.28±1.90 11.53±0.14 F 20.06±0.45 4.55±0.02 1.40±0.05 28.74±3.16 7.22±2.27 7.86±0.28 FS 33.31±0.32 6.73±0.09 3.24±0.08 51.04±0.63 11.45±0.52 8.95±0.19 WS 14.55±0.10 0.93±0.01 0.33±0.00 6.64±0.01 5.19±0.04 7.27±0.16 UP 16.54±0.90 1.36±0.13 0.26±0.01 4.92±0.47 5.96±0.26 6.47±0.13 Gc (3) L 33.33±0.96 9.50±0.27 8.01±0.17 98.25±2.47 10.36±0.49 10.37±0.32 F 18.88±0.28 3.73±0.09 4.53±0.04 40.56±0.98 5.44±0.44 5.83±0.15 FS 23.66±0.52 7.59±0.07 9.77±0.20 100.51±1.31 9.80±0.08 8.73±0.33 WS 13.61±0.56 0.84±0.01 0.75±0.01 10.51±0.04 4.53±1.17 6.24±0.13 UP 20.37±0.72 1.45±0.02 0.66±0.01 9.95±0.25 8.71±0.91 8.30±0.16 Gc L 31.81±3.62aAB 9.27±0.21bA 4.16±3.35aA 102.70±27.81abA 10.54±0.67bA 10.03±1.70aA average F 21.54±3.63bcBC 5.13±1.77aB 2.74±1.61aAB 31.32±8.25aC 6.87±1.30bA 7.07±1.09bA FS 33.96±10.63aA 7.96±1.45bA 5.73±3.53aA 69.70±26.88aB 11.98±2.49aA 10.07±2.13bA WS 16.79±4.72cC 1.26±0.65dC 0.45±0.26bB 8.51±1.94bC 6.28±2.48bA 7.14±0.84bA UP 19.69±2.87bBC 1.59±0.32aC 0.38±0.25aB 6.97±2.64aC 8.19±2.03aA 7.44±0.92bA Gm (1) L 23.03±0.29 6.96±0.08 1.38±0.00 85.89±5.76 10.16±0.22 9.47±0.34 F 21.69±0.78 5.16±0.03 1.74±0.02 37.65±2.01 9.89±0.08 8.33±0.16 FS 29.50±0.55 6.07±0.03 2.12±0.02 62.61±1.42 14.07±0.85 11.36±0.11 WS 17.56±0.04 1.02±0.02 0.34±0.00 8.54±0.12 5.76±0.82 7.68±0.07 UP 26.05±0.68 2.25±0.04 0.39±0.00 15.98±0.53 9.91±0.18 10.01±0.28 Gm (2) L 35.18±0.22 11.32±0.17 1.77±0.03 70.50±0.77 12.08±1.01 9.95±0.67 F 18.28±0.29 5.35±0.07 1.86±0.12 20.50±1.55 7.04±0.61 4.62±0.10 FS 29.38±0.45 6.37±0.24 2.78±0.09 40.94±3.89 8.19±1.06 8.50±0.31 WS 20.00±0.17 1.71±0.09 0.24±0.00 9.29±0.49 6.34±0.10 5.67±0.16 UP 17.13±0.53 1.08±0.01 0.19±0.01 6.20±0.22 5.73±1.20 5.38±0.15 Gm (3) L 30.81±0.45 8.40±0.10 1.71±0.05 114.08±7.72 9.49±1.08 10.02±0.18 F 27.25±0.85 4.09±0.03 1.60±0.02 25.19±1.60 6.82±2.20 6.66±0.24 FS 37.76±1.22 7.86±0.09 3.55±0.11 49.46±4.80 13.19±0.17 9.96±0.92 WS 16.80±0.54 0.90±0.02 0.41±0.01 8.27±0.13 6.59±0.29 7.99±0.30 UP 15.86±0.19 1.40±0.09 0.26±0.01 5.04±0.15 5.25±0.25 5.25±0.22 Gm L 29.67±6.15aAB 8.89±2.22bA 1.62±0.21abA 90.16±22.10bA 10.58±1.34bA 9.81±0.30aA average F 22.41±4.53bAB 4.87±0.68aB 1.73±0.13aA 27.78±8.86aBC 7.92±1.71bA 6.54±1.86bA FS 32.21±4.80abA 6.77±0.96bcAB 2.82±0.72abA 51.00±10.92aB 11.82±3.17aA 9.94±1.43bA WS 18.12±1.67cB 1.21±0.44dC 0.33±0.09bA 8.70±0.53bC 6.23±0.43bA 7.11±1.26bA UP 19.68±5.55bB 1.58±0.60aC 0.28±0.10aA 9.07±0.60aC 6.96±2.56aA 6.88±2.71bA Gp (1) L 35.31±1.31 14.46±0.20 1.68±0.04 143.29±7.15 15.32±1.04 12.22±0.24 F 14.02±0.25 6.76±0.05 0.84±0.02 15.26±0.41 4.18±0.42 4.34±0.01 FS 24.60±0.59 13.93±0.25 0.97±0.04 86.75±1.62 12.91±0.57 7.53±0.29 WS 34.96±0.57 3.71±0.04 0.58±0.02 8.53±0.18 16.94±1.09 18.95±0.50 UP 32.68±0.59 2.47±0.02 0.43±0.01 4.37±0.33 11.38±0.77 12.53±0.47 FRIŠČIĆ M., MASLO S., GARIĆ R., MALEŠ Ž., HAZLER PILEPIĆ K. 6 ACTA BOT. CROAT. 77 (1), 2018 Correlations between phenolic/iridoid content and antioxidant capacity In this study, all tested samples showed antioxidant ac- tivity, which was in very good correlation with observed amounts of total phenolic compounds (r = 0.86, p < 0.001 for DPPH; r = 0.83, p < 0.001 for ABTS). Poor correlation was observed between flavonoid content and DPPH radical scav- enging capacity (r = 0.32, p < 0.05) (Tab. 4). G. alypum leaves and flowers showed higher antioxidant activity than those of related species in the DPPH assay, while in the ABTS assay only flowers were significantly different (p < 0.05) (Tab. 3). Discussion Comparison to previous studies of G. alypum and evaluation of obtained results In the present study, the medicinal plant G. alypum served as a control species, with which all other investigated species were compared. The reason for this was its broad and well-documented medicinal use together with a number of studies highlighting the high contents of its specialized me- tabolites and pronounced antioxidant activity. Determina- tion of specialized metabolites was conducted according to the same procedures as those used in previous studies of G. alypum and related species (Djeridane et al. 2006, Khlifi et al. 2011, Tundis et al. 2012a, Amessis-Ouchemoukh et al. 2014, Taghzouti et al. 2016, Touaibia and Chaouch 2016) to enable a more reliable comparison with literature data. Amounts of total phenolics and flavonoids observed for G. alypum in this study were comparable to those in previously reported studies (Djeridane et al. 2006, Djeridane et al. 2010, Cho- grani et al. 2012), in which they were expressed in the same manner as in our study, per g dry weight of plant (Djeridane et al. 2006), not per g dry extract, as in some other studies. The latter, unsurprisingly, resulted in several times higher values (Khlifi et al. 2011, Amessis-Ouchemoukh et al. 2014, Taghzouti et al. 2016, Touaibia and Chaouch 2016). The pres- ent study also shows that green aerial parts of Globularia spe- cies contain higher amounts of specialized metabolites, with the exception of total phenolics, which were also observed to be high in woody stems and underground parts of G. punc- tata. Leaves of Globularia species were rich in all investi- gated bioactive substances. This could explain why they are frequently used plant parts in folk medicine (De Natale and Pollio 2012, Bouzabata 2013, El Abbouyi et al. 2014). How- ever, it is important to notice that the use of flowers (Bouz- abata 2013) and aerial parts (De Natale and Pollio 2012) is also known, which could be explained by the high amounts of polyphenols found in G. alypum flowers and their high antioxidant activity as observed in this study, as well as their recently reported high catalpol content (Sertić et al. 2015) and various biological activities such as antioxidative, an- ti-inflammatory and acetylcholinesterase inhibitory activity (Amessis-Ouchemoukh et al. 2014). Two of the most commonly used assays for estimating radical scavenging activity are the 2,2-diphenyl-1-picryl- hydrazyl (DPPH) assay and the 2,2´-azino-bis(3-ethylben- Plant species Plant part Polyphenols (mg GAEg–1 DW) Flavonoids (mg QE g–1 DW) Tannins (mg CE g–1 DW) Iridoids (mg AE g–1 DW) DPPH (mg GAE g–1 DW) ABTS (mg GAE g–1 DW) Gp (2) L 36.10±0.45 17.77±0.87 1.49±0.04 125.83±1.79 11.71±0.23 10.59±0.15 F 11.19±0.13 5.52±0.24 1.16±0.07 8.02±0.43 3.44±0.06 2.36±0.12 FS 19.50±0.27 12.55±0.21 1.55±0.07 72.79±1.47 5.12±0.22 5.50±0.26 WS 30.79±0.27 2.78±0.00 0.30±0.01 6.84±0.28 9.65±0.57 12.05±0.06 UP 30.87±0.47 2.71±0.01 0.30±0.00 4.76±0.02 9.90±0.22 12.07±0.18 Gp (3) L 30.46±1.57 14.46±0.58 2.38±0.02 110.32±1.14 10.33±0.71 13.24±1.40 F 10.13±0.23 5.57±0.07 0.56±0.03 9.44±0.29 3.38±0.54 2.20±0.14 FS 22.38±0.69 11.65±0.13 2.11±0.07 75.59±1.03 8.92±0.08 5.45±0.96 WS 44.77±0.49 2.84±0.11 0.60±0.00 9.95±0.52 18.33±1.05 16.24±1.37 UP 35.20±0.83 2.75±0.07 0.41±0.01 3.94±0.08 12.88±0.89 9.39±0.50 Gp L 33.96±3.05aAB 15.56±1.91aA 1.85±0.47abA 126.50±16.49aA 12.45±2.58bAB 12.02±1.34aAB average F 11.78±2.01cC 5.95±0.70aC 0.85±0.30aA 10.91±3.84aC 3.67±0.45bC 2.97±1.19cC FS 22.16±2.56bcBC 12.71±1.15aB 1.54±0.57bA 78.38±7.39aB 8.98±3.90aBC 6.16±1.19cC WS 36.84±7.18aA 3.11±0.52cdD 0.49±0.17bA 8.44±1.56bC 14.97±4.66aA 15.75±3.48aA UP 32.92±2.18aAB 2.64±0.15aD 0.38±0.07aA 4.36±0.41aC 11.39±1.49aAB 11.33±1.70aB Tab. 3. Continued Tab. 4. Pearson’s correlation coefficients between total phenolic, flavonoid, condensed tannin and iridoid content and 2,2-diphenyl- 1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazo- line-6-sulfonic acid) (ABTS) radical scavenging capacity in differ- ent plant parts of four Globularia species collected from different locations. n = 48; statistically significant correlations are indicated by an asterisk (*) for p < 0.05 and three asterisks (***) for p < 0.001. DPPH ABTS Polyphenols 0.86*** 0.83*** Flavonoids 0.32* 0.17 Tannins 0.08 0.05 Iridoids 0.22 0.20 SPECIALIZED METABOLITES OF GLOBULARIA SPP. ACTA BOT. CROAT. 77 (1), 2018 7 zo (ABTS) assay (Sánchez-Moreno 2002). Both assays were previously used by different authors for estimating the an- tioxidant capacity of G. alypum and showed the species to be a good source of antioxidants (Djeridane et al. 2006, Es- Safi et al. 2007, Djeridane et al. 2010, Khlifi et al. 2011, Cho- grani et al. 2012). Strong positive correlations between the total phenolic content and antioxidant capacity, similar to those observed in the present study, were also found in ear- lier studies (Djeridane et al. 2006, Khlifi et al. 2011, Cho- grani et al. 2012). However, the antioxidant activity observed for G. alypum leaves and flowers was not statistically differ- ent (p > 0.05) from that of previous reports (Chograni et al. 2012, Amessis-Ouchemoukh et al. 2014). Djeridane et al. (2006) noticed higher antioxidant activity in G. alypum and other plant extracts in which phenolic acids predominated in comparison to those extracts containing only flavonoids. This could explain why only a poor correlation between fla- vonoid content and DPPH radical scavenging capacity was found in our study. Evaluation of medicinal potential of G. cordifolia, G. meridionalis and G. punctata The high amounts of polyphenols and iridoids found in G. cordifolia, G. meridionalis and G. punctata suggest these spe- cies have potentially beneficial health effects similar to those of G. alypum. However, it should be noted that the method used for iridoid determination, could only detect the aucubin and asperuloside type iridoids (Trim and Hill 1952), while catalpol derivatives, which are the predominant iridoid gly- cosides of G. alypum (Chaudhuri and Sticher 1981), could not be estimated (Harborne 1998). However, the three spe- cies were recently also shown to possess higher amounts of aucubin than G. alypum, with G. punctata having higher ca- talpol contents as well (Sertić et al. 2015). High iridoid con- tent in the green aerial parts of G. cordifolia, G. meridionalis and G. punctata observed in this study could be explained by the presence of various aucubin and asperuloside deriva- tives, which have been previously isolated from these spe- cies (Chaudhuri and Sticher 1980, Kirmizibekmez et al. 2003, Tundis et al. 2012b). Indeed, much higher asperulo- side amounts have been observed in G. cordifolia, G. meridi- onalis and G. punctata than in G. alypum (Friščić et al. 2016). Similarly to our results, lower amounts of iridoids were previ- ously reported for G. meridionalis underground parts in com- parison to its aerial parts (Tundis et al. 2012a). Although in most cases, there were no significant differences observed in the amounts of specialized metabolites of the three species, G. punctata seems to contain more polyphenols in the woody parts and more flavonoids in the leaves and flower stems. A high catalpol content in the flowers of this species was also recently observed (Sertić et al. 2015). On the other hand, G. cordifolia and G. meridionalis gave similar results in all per- formed assays (i.e., no statistically significant differences be- tween the two species were observed). This is not surprising considering their close relationship (Tutin 1972). Although these species could not be distinguished based on the used spectrophotometric assays, it should be noted that the com- parison of the proanthocyanidin content indicated a some- what characteristic chemical composition of the G. cordifolia population collected from Mostar. The same phenomenon was observed after an analysis of the essential oil composition of different Globularia populations (Crkvenčić et al. 2016). According to our study, the antioxidant activity of all four species is in good correlation with their total phenolic con- tent. Because G. cordifolia, G. meridionalis and G. punctata are more widely distributed in Europe, it would be inter- esting to investigate if they have other biological activities that are similar to those of G. alypum. Some researches in this field have already yielded promising results. For exam- ple, G. meridionalis was recently shown to possess inhibito- ry activity on acetylcholinesterase and butyrylcholinester- ase, enzymes representing the targets for the symptomatic treatment of Alzheimer’s disease (Tundis et al. 2012a). Iri- doid glucoside globularifolin, found both in G. cordifolia (Chaudhuri and Sticher 1980) and G. meridionalis (Tundis et al. 2012b), was also recently reported to possess immuno- modulatory activity (Sipahi et al. 2014). Production of plant specialized metabolites with respect to environmental factors Our results confirmed the assumption that phenolic and iridoid concentrations can vary significantly between popu- lations of the same species collected from different locations (Tab. 3) and thus provided a justification for including more populations of each species when comparing different spe- cies harvested from natural habitats. As mentioned before, G. alypum was presented with only one population. Howev- er, the amounts of its specialized metabolites could be com- pared to numerous literature data. It is known that environmental factors have a major in- fluence on the synthesis of plant specialized metabolites causing notable differences in the yields of biologically ac- tive compounds among different populations. The correla- tions observed in this study indicate a possible shift in the polyphenolic production and/or translocation of phenolic compounds from the flower stems to the leaves under the influence of increased temperature. On the other hand, the higher tannin content found in all plant parts of G. cordifo- lia from Mostar area could be connected with its location on the humid Neretva river banks, having in mind that all other samples were collected from well-drained areas with full sun exposure. It was previously reported that tannin-rich plants grew mostly on infertile soils with poor drainage (Kraus et al. 2003). Knowing that iridoids play a major role in defending plants against pathogens or herbivores (Dobler 2011), stim- uli other than those considered in the present study seem to be more important for their production. Bearing in mind that, along with the genetically deter- mined variations of metabolite production, plant specialized metabolites represent a way of adaptation to environmental factors (Ghasemzadeh and Ghasemzadeh 2011), the yield of bioactive compounds can be significantly different among FRIŠČIĆ M., MASLO S., GARIĆ R., MALEŠ Ž., HAZLER PILEPIĆ K. 8 ACTA BOT. CROAT. 77 (1), 2018 populations. This fact is very important for users of native medicinal plants. Investigations of specialized metabolites with the aim of checking or predicting the medicinal poten- tial of a given plant species should therefore be conducted on several plant populations, when possible. Unfortunately, this is not always the case. The proposed approach could al- so ensure the selection of the most suitable populations for cultivation and/or the adaptation of growth conditions to those necessary to increase the production of target metabo- lites. 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