Phytochemical Investigations on Four Galium Species (Rubiaceae) from Romania

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FARMACIA, 2016, Vol. 64, 1 ORIGINAL ARTICLE PHYTOCHEMICAL INVESTIGATIONS ON FOUR GALIUM SPECIES (RUBIACEAE) FROM ROMANIA

ANDREI MOCAN1, GIANINA CRIȘAN1*, LAURIAN VLASE1,2, BIANCA IVĂNESCU3, ALEXANDRU SABIN BĂDĂRĂU4, ANDREEA LETIȚIA ARSENE5

1Department of Pharmaceutical Botany, “Iuliu Hatieganu” University of Medicine and Pharmacy, 8, V. Babes Street, Cluj- Napoca, Romania 2Department of Pharmaceutical Technology and Biopharmaceutics, “Iuliu Hatieganu” University of Medicine and Pharmacy, 8, V. Babes Street, Cluj-Napoca, Romania 3Department of Pharmaceutical Botany, “Gr. T. Popa” University of Medicine and Pharmacy, 16, Universitatii Street, Iași, Romania 4Department of Environmental Science, “Babeș-Bolyai” University, 30, Fântânele Street, Cluj-Napoca, Romania 5Department of Biochemistry, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, sector 2, Bucharest, Romania

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

Abstract Galium species are used as diuretics, choleretics, against diarrhea and in the treatment of some stomach complaints, gout and epilepsy in folk medicine. Most of these species have been extensively investigated for antraquinones and iridoids but little is known about other bioactive compounds classes. Our research aimed to investigate the chemical composition of four Romanian species of Galium in order to bring new data related to their content in polyphenolcarboxilic acids, sterols and methoxylated flavones. Caffeic and chlorogenic acids were identified in all samples, the richest source of chlorogenic acid being G. odoratum (10387.8 ± 45.67 µg/g). Regarding the sterolic pattern, β-sitosterol and campesterol were detected in all species, the richest source being G. mollugo (219.02 ± 7.24 µg/g and respectively, 15 ± 0.08 µg/g). Hispidulin was the only identified methoxylated flavone and its highest amount was detected in G. mollugo (50.41 ± 0.03 µg/g).

Rezumat Speciile genului Galium sunt utilizate ca diuretice, coleretice, antidiareice precum și în tratementul afecțiunilor gastrice, gutei și epilepsiei în medicina tradițională. Majoritatea acestora au fost intens studiate cu privire la conținutul în antrachinone și iridoide, cunoscându-se însă foarte puține date cu privire la prezența altor clase de compuși bioactivi. Scopul acestui studiu a fost de a investiga compoziția chimică a patru specii indigene de Galium în legătură cu conținutul acestora în acizi polifenol- carboxilici, steroli și flavone metoxilate. Acizii cafeic și clorogenic au putut fi identificați în toate speciile, cea mai bogată sursă de acid clorogenic fiind G. odoratum (10387.8 ± 45.67 µg/g). β-sitosterolul și campesterolul au fost identificați în toate speciile analizate, cea mai bogată sursă în acești compuși fiind G. mollugo (219.02 ± 7.24 µg/g și respectiv, 15 ± 0.08 µg/g). Singura flavonă metoxilată identificată a fost hispidulina, conținutul cel mai ridicat în acest compus bioactiv fiind prezent în G. mollugo (50.41 ± 0.03 µg/g).

Keywords: G. verum L., G. mollugo L., G. aparine L., G. odoratum L.

Introduction extensively investigated for iridoids [4, 10, 13, 18], triterpenoid saponins [3, 4] and antraquinones [8, 9, The cosmopolitan genus Galium, belonging to the 12, 14, 20]. However, excepting some recent Rubiaceae family, consists of about 400 herbaceous reports [6, 15, 17, 21], little is known about other plant species, 145 of which are distributed in bioactive compounds classes from this genus. Thus, Europe [13]. Galium species are used to coagulate Tămaș et al., reported the presence of rutin, milk but also as diuretics, choleretics, against hyperoside, chlorogenic and caffeic acids for both diarrhea and in the treatment of some stomach G. mollugo and G. verum and quercitrin just for the complaints, gout and epilepsy in folk medicine [5- latter, by thin layer chromatography [17]. 7]. A recent study proved also the protective effect Diosmetin 7-O-α-L-rhamnopyranosyl-(1-2)-[β-D- against anakinetic stress of a G. verum extract in a xylopyranosyl-(1–6)]-β-D-glucopyranoside, rat experimental model [16]. 3,5,7,3’,4’,3’’,5’’,7’’,3’’’,4’’’-decahydroxyl-[8- The Romanian flora comprises about 28 species, CH -8’’]-biflavone, quercetin, isorhamnetin, mostly with white flowers and six with yellow 2 isorhamnetin 3-O-α-L-rhamnopyranosyl-(1–6)-β-D flowers [2]. Many species of Galium have been glucopyranoside, kaempferol, diosmetin, diosmetin 95 FARMACIA, 2016, Vol. 64, 1 7-O-β-D-glucopyranoside and diosmetin 7-O-β-D- interference from background, the multiple xylopyranosyl-(1–6)-β-D-glucopyranoside were reactions monitoring analysis mode was used isolated by Zhao et al. from aerial parts of G. verum [21]. instead of single ion monitoring (e.g., MS/MS Considering these aspects, further comprehensive instead of MS). studies on several bioactive compounds classes and their Chromatographic conditions for the analysis of amounts are essential for a better characterization phytosterols of these medicinal plants and their possible The sterolic compounds were separated using a beneficial effects on human health. We employed a Zorbax SB-C18 reversed-phase analytical column rapid, highly accurate and sensitive HPLC method (100 × 3.0 mm i.d., 3.5 µm particle) operated at assisted by MS detection for the simultaneous 45°C. The separation was achieved using a mobile determination of chlorogenic and caffeic acids and phase consisting of 10:90 (V/V) methanol and a newly developed LC- MS/MS method for the acetonitrile with a flow rate of 1 mL/min and an quantitative analysis of several methoxylated injection volume of 5 µL. Mass spectrometry flavones, in natural products. Furthermore a simple, analysis was performed on an Agilent Ion Trap accurate and rapid HPLC-MS/MS method for 1100 VL mass spectrometer with atmospheric identification and quantification of sterols from the pressure chemical ionization (APCI) interface. The four species of Galium has been employed [19]. instrument was operated in positive ion mode. The gas temperature (nitrogen) was 325°C at a flow rate Materials and Methods of 7 L/min, nebulizer pressure 60 psi and capillary voltage -4,000 V. The full identification of compounds Chemicals was performed by comparing their retention times Plant material and extraction procedure and mass spectra with those of standards in the The aerial parts of G. verum L., G. mollugo L.¸G. same chromatographic conditions [19]. aparine L. and G. odoratum L. Scop. were Chromatographic conditions for the analysis of collected in May, June and July 2014, from Cluj methoxylated flavones and Alba counties. Voucher specimens were The separation of the methoxylated flavones was deposited in the Herbarium of the Pharmaceutical achieved using a Zorbax SB-C18 reversed-phase Botany Department, “Iuliu Hațieganu” University analytical column (100 × 3.0 mm i.d., 3.5 µm of Medicine and Pharmacy, Cluj-Napoca, Romania particle) operated at 48°C. The mobile phase (GAL 534-537). The vegetal herbal material was consisted in 0.1% (V/V) acetic acid and methanol air dried at room temperature in shade, separated with the following gradient: beginning with 45% and grinded to a fine powder (< 300 µm) before the methanol and ending at 50% methanol, in 8 extraction procedure. One gram of each sample was minutes, with a flow rate of 0.9 mL/min and an extracted with 10 mL of 70% ethanol, for 60 min in injection volume of 5 µL. For the MS analysis the a sonication bath at 60°C. The samples were then following optimized conditions were used: gas cooled down and centrifuged at 4.500 rpm for 15 (nitrogen) temperature 325°C at a flow rate of 12 min, and the supernatant was recovered. L/min, nebulizer pressure 60 psi and capillary Chromatographic conditions for the analysis of voltage +2500 V. The full identification of caffeic and chlorogenic acids compounds was performed by comparing the The two compounds were separated using a Zorbax retention times and mass spectra with those of SB-C18 reversed-phase analytical column (100 × standards in the same chromatographic conditions. 3.0 mm i.d., 3.5 µm particle) operated at 42°C. The The MS was operated in the multiple reactions separation was achieved under isocratic conditions monitoring analysis (MRM) mode. using a mobile phase consisting of 0.1% acetic acid Statistical analysis and acetonitrile (V/V). The flow rate was 0.8 The average of multiple measurements (triplicates) mL/min and the injection volume was 5 µL. Mass was listed in the tables together with the standard spectrometry analysis was performed on an Agilent deviations. Statistical analysis was performed using Ion Trap 1100 VL mass spectrometer with electro- Excel 2007 software package. spray ionization (ESI) interface in negative mode.

Operating conditions were optimized in order to Results and Discussion achieve maximum sensitivity values: gas (nitrogen) temperature 360°C at a flow rate of 12 L/min, The analysis of caffeic and chlorogenic acids nebulizer pressure 60 psi and capillary voltage Under the described chromatographic conditions, +3500 V. The full identification of compounds was the retention times of the two compounds were performed by comparing the retention times and 2.2 min. for chlorogenic acid and 3.3 for caffeic mass spectra with those of standards in the same acid, as seen in Figure 1. chromatographic conditions. To avoid or limit the

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Figure 1. MS chromatograms of chlorogenic (1) and caffeic (2) acids

Because in the ionization conditions both However, in order to increase the selectivity and chlorogenic and caffeic acids loose a proton, the sensitivity of the analytical method, for each of the ions monitored by the mass spectrometer are in the analytes a daughter ion was monitored from the form [M−H]-. Thus, the ions recorded have m/z = 353 MS/MS spectrum, as seen in Table I. for chlorogenic acid and m/z = 179 for caffeic acid. Table I Quantitative determination of chlorogenic and caffeic acids Sample Chlorogenic acid (µg/g) Caffeic acid (µg/g) Specific ions for identification (m/z) G. verum 4225.4 ± 10.21 32.4 ± 0.32 m/z = 353 > m/z = 191 G. mollugo 5108 ± 8.12 57 ± 2.15 (chlorogenic acid); G. aparine 2048 ± 3.52 43.2 ± 1.02 m/z = 179 > m/z = 135 G. odoratum 10387.8 ± 45.67 44 ± 1.56 (caffeic acid).

The method can also be applied for the quantitative al. qualitatively identified both acids in the aerial determination because the intensity of ions in the parts of G. spurium from Turkey [15]. mass spectrum is proportional to the concentration of The analysis of phytosterols the substance in the sample. The ions with m/z = 191 The retention times of the five analysed sterols and m/z = 135 were further used for that purpose. were: 3.2 min for ergosterol, 3.9 min for brassicasterol, To our knowledge, data regarding the amounts of 4.9 min for stigmasterol and campesterol (co-elution) chlorogenic and caffeic acids are not available and 5.7 min for β-sitosterol. The ions monitorized concerning any of the species from this study. in the MS assay are presented in Table II. Because However, the presence of chrologenic and caffeic in the ionization conditions all sterols loose a water acids in the aerial parts of G. verum and G. mollugo molecule, the ions detected by the mass-spectro- + was previously reported by Tămaș et al. Orhan et meter are always in the form [M−H2O+H] . Table II Characteristic ions of standard sterols in full scan and specific ions used for the quantification + Compound RT (min) M Specific ions for identification: Ion [M-H2O+H ] > Ions from spectrum Ergosterol 3.2 396 379 > 158.9; 184.9; 199; 213; 225; 239; 253; 295; 309; 323 Brassicasterol 3.9 398 381 > 201.3; 203.3; 215.2; 217.3; 241.2; 255.3; 257.4; 271.1; 297.3; 299.3 Stigmasterol 4.9 412 395 > 255; 297; 283; 311; 241; 201 Campesterol 4.9 400 383 > 147; 149; 161; 175; 189; 203; 215; 229; 243; 257 β-sitosterol 5.7 414 397 > 160.9; 174.9; 188.9; 202.9; 214.9; 243; 257; 287.1; 315.2

The method was applied also for the quantification compound, taking into account the intensity of of the sterols from the Galium species extracts. major ions from the mass spectrum, the results Extracted chromatograms were constructed for each being presented in Table III.

97 FARMACIA, 2016, Vol. 64, 1 Table III The content in sterols (µg/g vegetal product) of Galium species extracts Phytosterol G. verum G. mollugo G. aparine G. odoratum β-sitosterol 85.46 ± 1.24 219.02 ± 7.24 173.36 ± 9.25 188.5 ± 7.87 Campesterol 9.86 ± 0.04 15 ± 0.08 12.7 ± 0.05 5.58 ± 0.01 Stigmasterol - - - - Ergosterol - - - - Brassicasterol - - - -

β-sitosterol and campesterol were identified in all experimental section, the analytes eluted in less species; the richest species in both sterols was G. than 10 minutes. In the process of MS analysis, the mollugo (219.02 ± 7.24 µg/g for β-sitosterol and 15 ± pseudo-molecular ions of the flavones (329.3 for 0.08 µg/g for campesterol) and the lowest levels jaceosidin, 299.2 for hispidulin, 343.3 for eupatilin, were found in G. verum with 85.46 ± 1.24 µg/g for 343.3 for eupatorin, 373.3 for casticin and 283.3 for β-sitosterol and 9.86 ± 0.04 µg/g for campesterol. acacetin) have been fragmented, and based on their Lower values of sitosterol and campesterol were daughter ions from the MS spectrum (Table IV) the previously reported by Benton and Cobb in leaves extracted chromatograms of each compound were and stems of G. aparine by GC investigations [1] constructed for quantification. but no other data was found regarding the sterolic The only identified flavone was hispidulin, in G. profile of the other analyzed species. β-sitosterol mollugo (50.41 ± 0.03µg/g) and G. verum (2.56 ± was also identified in the aerial parts of G. 0.01µg/g). Nevertheless the presence of this tortumense [6]. compound in any representative of Galium genus The analysis of methoxylated flavones was never described before and alongside with A screening method was applied for testing the other valuable secondary metabolites might underlie presence of 6 methoxylated flavones: jaceosidin, the traditional uses of some Galium species in the hispidulin, eupatilin, eupatorin, casticin and treatment of epilepsy [15], as hispidulin is a proved acacetin in the vegetal extracts. Using the benzodiazepine receptor ligand which exhibits chromatographic conditions described in the anticonvulsive effects [11]. Table IV Characteristic ions of standard flavones in full scan and specific ions used in quantification - Compound RT (min) M [M-H] Ions / monitorized fragments Jaceosidin 2.9 330.3 329.3 314 Hispidulin 4.2 300.2 299.2 284 Eupalitin 7.05 344.3 343.3 328 Eupatorin 7.6 344.3 343.3 328 Casticin 8.05 374.3 373.3 358 Acacetin 9.8 284.3 283.3 268

Conclusions amount was detected in G. mollugo (50.41 ± 0.03µg/g). The results of the present study offer a This study brings new valuable data regarding the scientific basis to the traditional uses of Galium presence of different classes of bioactive species by identifying several secondary metabolites compounds like polyphenolcarboxilic acids, that might be responsible for their pharmacological phytosterols and hispidulin in four Romanian effects. species of Galium genus: G. verum, G. mollugo, G. aparine and G. odoratum. For each compound class a Acknowledgements different LC/MS method was developed according to the structural particularities of the investigated This paper was published under the frame of analytes. Chlorogenic and caffeic acids were European Social Found, Human Resources identified in all the samples, the richest species in Development Operational Programme 2007–2013, chlorogenic acid being G. odoratum (10387.8 ± project no. POSDRU/159/1.5/S/136893. 45.67 µg/g); the highest amount of caffeic acid was found in G. mollugo (57 ± 2.15 µg/g). Regarding References the sterolic patern, β-sitosterol and campesterol 1. Benton J.M., Cobb A.H., The modification of were detected in all species, the richest source phytosterol profiles and in vitro photosynthetic being G. mollugo (219.02 ± 7.24 µg/g and electron transport of Galium aparine L. (cleavers) respectively, 15 ± 0.08 µg/g). The only identified treated with the fungicide, epoxiconazole. J. Plant methoxylated flavone was hispidulin and its highest Growth Regul., 1997; 22: 93-100. 98 FARMACIA, 2016, Vol. 64, 1 2. Cioancă O., Mircea C., Hritcu L., Trifan A., barrier and exhibits anticonvulsive effects. Br. J. Mihasan M., Aprotosoaie A.C., Robu S., Gille E., Pharmacol., 2004; 142: 811-820. Hancianu M., In vitro-in vivo correlation of the 12. Koyama J., Ogura T., Tagahara K., Anthraquinones antioxidant capacity of Salviae aetheroleum of Galium spurium. Phytochem., 1993; 33: 1540- essential oil. Farmacia, 2015: 63(1): 34-39. 1542. 3. De Rosa S., Iodice C., Mitova M., Handjieva N., 13. Mitova M.I., Anchev M.E., Handjieva N.V., Iridoid Popov S., Anchev M., Rivalosides A and B, Two patterns in Galium L. and some phylogenetic 19-Oxo Triterpenoid Saponins from Galium rivale. considerations. Z. Naturforsch C, 2002; 57c(3-4): J. Nat. Prod., 2000; 63: 1012-1014. 226-234. 4. De Rosa S., Iodice C., Mitova M., Handjieva N., 14. Morimoto M., Tanimoto K., Sakatani A., Komai Popov S., Anchev, M., Triterpene saponins and K., Antifeedant activity of an anthraquinone iridoid glucosides from Galium rivale. Phytochem., aldehyde in Galium aparine L. against Spodoptera 2000; 54: 751-756. litura F. Phytochem., 2002; 60: 163-166. 5. Gîrd C., Costea T., Nencu I., Popescu M.L. Duțu 15. Orhan N., Orhan D.D., Aslan M., Șukuroglu M., L.E., Phytochemical and phytobiological research Orhan I.E., UPLC–TOF-MS analysis of Galium upon aerial parts and seeds from Peucedanum spurium towards its neuroprotective and officinale. Farmacia, 2015; 63(2): 247-253. anticonvulsant activities. J. Ethnopharmacol., 6. Güvenalp Z., Kilic N., Kazaz C., Kaya Y., 2012; 141: 220-227. Demirezer L.O., Chemical Constituents of Galium 16. Roman I., Puică, C., Effects of Anakinetic Stress tortumense. Turk. J. Chem., 2006; 30: 515-523. and Galium verum extract on the thyroid and ovary 7. Güvenalp Z., Kazaz C., Kaya Y., Demirezer L.O., morphology in Wistar rats. Bulletin UASVM, Phytochemical investigation on Galium humifusum Veterinary Medicine, 2013; 70: 167-169. growing in Turkey. Biochem. Syst. Ecol., 2006; 34: 17. Tămaș M., Stana D., Timiș S., Comparative 894-896. phytochemical research of Galium verum L. and G. 8. Halim A.F., Abd El-Fattah H., El-Gaman A.A., mollugo L. Not. Bot. Hort. Agrobot. Cluj, 2006; 34: Thomson R.H., Anthraquinones from Galium 18-20. sinaicum. Phytochem., 1991; 31: 355-356. 18. Uesato S., Ueda M., Inouye H., Kuwajima H., 9. Heide L., Leistner E., Enzyme activities in extracts Yatsuzuka M., Takaishi K., Iridoids from Galium of anthraquinone-containing cells of Galium mollugo. Phytochem., 1984; 23: 2535-2537. mollugo. Phytochem., 1983; 22: 659-662. 19. Vlase L., Pârvu M., Pârvu E.A., Toiu A., Chemical 10. Horvath-Bojthe K., Hetenyi F., Kocsis A., Szabo constituents of three Allium species from Romania. L., Varga-Balazs M., Mathe I., Tetenyi P., Iridoid Molecules, 2013; 18: 114-127. glycosides from Galium verum. Phytochem., 1982; 20. Zhao C.C., Shao J.H., Li X., Xu J., Wang J.H., A 21: 2917-2919. new anthraquinone from Galium verum L. Nat. 11. Kavvadias D., Sand P., Youdim K.A., Qaiser M.Z., Prod. Res., 2006; 20: 981-984. Rice-Evans C., Baur R., Sigel E., Rausch W.-D., 21. Zhao C.C., Shao J.H., Li X., Kang X.D., Zhang Riederer P., Schreier P., The flavone hispidulin, a Y.W., Meng D.L., Li N., Flavonoids from Galium benzodiazepine receptor ligand with positive verum L. J. Asian Nat. Prod. Res., 2008; 10: 611- allosteric properties, traverses the blood-brain 615.