Flavonoids as chemotaxonomic markers in the ...

Biochemical Systematics and Ecology 35 (2007) 317e320 www.elsevier.com/locate/biochemsyseco

Flavonoids as chemotaxonomic markers in the polymorphic Stachys swainsonii (Lamiaceae) H. Skaltsa a,*, P. Georgakopoulos a, D. Lazari b, A. Karioti a, J. Heilmann c, O. Sticher d, Th. Constantinidis e a

Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, 15771 Athens, Greece b Laboratory of Pharmacognosy, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece c Department of Pharmaceutical Biology, Institute of Pharmacy, University of Regensburg, Universita¨tsstrasse 31, 93053 Regensburg, Germany d Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland e Laboratory of Systematic Botany, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece Received 11 September 2006; accepted 24 October 2006

Keywords: Stachys swainsonii subsp. swainsonii; S. swainsonii subsp. scyronica; S. swainsonii subsp. melangavica; S. swainsonii subsp. argolica; Intraspecific variability; Flavonoids; Chemotaxonomy

1. Subject and source The widespread genus Stachys L. comprises about 300 species (Mabberley, 1997) and is considered to be one of the largest genera of the Lamiaceae. Greece is an area particularly rich in taxa, and more than 50 species and subspecies of the genus Stachys are distributed in the mainland and/or the islands (Greuter et al., 1986). Certain Greek taxa are taxonomically difficult, with a complex evolutionary history. One such polymorphic species, Stachys swainsonii Benth., is the focus of the present study. The species consists of four subspecies, namely S. swainsonii subsp. swainsonii, S. swainsonii subsp. scyronica (Boiss.) Phitos and Damboldt, S. swainsonii subsp. melangavica D. Perss. and S. swainsonii subsp. argolica (Boiss.) Phitos and Damboldt, all endemic to Greece (Phitos and Damboldt, 1969; Persson, 1981). The members of S. swainsonii are mostly chasmophytes, growing on limestone rocks up to ca. 1500 m above sea level. According to morphological and palaeogeographical evidence, they presumably represent relicts of formally more widespread species complexes. The present study aims at investigating the flavonoid composition, previously unknown, of all subspecific taxa of S. swainsonii. Intraspecific variability is also examined by processing more than one population per taxon (Table 1). Eleven populations of the following four taxa were examined: S. swainsonii subsp. argolica [arg-1, arg2, arg-3], S. swainsonii subsp. melangavica [mel-1, mel-2, mel-3], S. swainsonii subsp. scyronica [scy-1, scy-2] * Corresponding author. Tel./fax: þ30 107274593. E-mail address: [email protected] (H. Skaltsa). 0305-1978/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2006.10.014

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H. Skaltsa et al. / Biochemical Systematics and Ecology 35 (2007) 317e320

Table 1 List of collection localities of Stachys swainsonii with abbreviations, coordinates and voucher specimens Name

Abbreviation

Locality

Coordinates

Voucher

Stachys swainsonii subsp. argolica

arg-1

Korinthia, ca. 8.1e8.9 km after turn-off to Korfos, 50e80 m.

37 440 N, 23 060 E

Const. 7640

arg-2

Argolida, ca. 4.4 km ESE Vivarion, 70e90 m.

37 320 N, 22 570 E

Const. 7893

arg-3

Attiki, ca. 3.7 km NW Kalloni, 120 m.

37 330 N, 23 160 E

Const. 7894

mel-1

Korinthia, close to Ireon ancient temple, 5e70 m.

38 010 N, 22 510 E

Const. 7444

mel-2

Viotia, ca. 2.7 km NNE Paralia Sarandi, 250e280 m.

38 150 N, 22 520 E

Const. 7503

mel-3

Korinthia, ca. 5.8 km from Pisia to Schinos, 400 m.

38 020 N, 23 000 E

Const. 7681

scy-1

Attiki, ca. 5.5e6.5 km ENE Kineta, 130e160 m.

37 580 N, 23 170 E

Const. 7438

scy-2

Attiki, Mt. Gerania, NW Panorama, 360e380 m.

37 580 N, 23 120 E

Const. 7685

swa-1

Fokida, ca. 7.5e8.0 km after Itea along road to Galaxidi, 10e20 m.

38 250 N, 22 240 E

Const. 7598

swa-2

Fokida, ca. 2.0 km NW Itea, 30e40 m.

38 270 N, 22 250 E

Const. 7599

swa-3

Fokida, N of Lidorikion, 600e650 m.

38 330 N, 22 110 E

Const. 7913

Stachys swainsonii subsp. melangavica

Stachys swainsonii subsp. scyronica

Stachys swainsonii subsp. swainsonii

and S. swainsonii subsp. swainsonii [swa-1, swa-2, swa-3]. Above-ground parts of the plants were used, all collected at flowering time. Each population sample normally consists of 15e25 randomly collected plants. 2. Previous work While there is no report on any thorough phytochemical investigation on the subspecies belonging to S. swainsonii available to date, the composition of the volatile compounds obtained by hydrodistillation of aerial parts of these plants was analysed by GCeMS (Skaltsa et al., 2001). 3. Present study Air-dried and powdered aerial parts (0.38 kg of swa-2; 0.1 kg of all other populations) were extracted at room temperature with a series of solvents of increasing polarity, i.e. petroleum ether, CH2Cl2, EtOAc and MeOH. The dichloromethane extract (5.08 g) of swa-2 was subjected to vacuum liquid chromatography (VLC) over silica gel using mixtures of cyclohexaneeEtOAc to yield nine fractions (A1eI1). Fraction E1 [cyclohexaneeEtOAc 60:40] was further fractionated by column chromatography (CC) using CH2Cl2eMeOH eluotropic mixtures and yielded seven fractions (A2eH2). Further purification of fraction D2 [CH2Cl2eMeOH 97:3] by CC on silica gel using mixtures of cyclohexaneeEtOAc afforded 4 (penduletin; 7.4 mg) and 7 (5-hydroxyauranetin; 4.8 mg). Further purification of fraction D3 by HPLC (cyclohexaneeEtOAc 4:1) yielded 6 (eupatorin; 2.5 mg; tR 7.2 min). The methanol extract (15.4 g) of swa-2 was subjected to CC on Sephadex LH-20 (MeOH 100%) and afforded 13 fractions (A3eQ3). Further purification of fraction D3 by CC, followed by RP-HPLC (C18 column; MeOH: aq. AcOH 5% 50:40) afforded 1


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(apigenin; 8.4 mg; tR 20.5 min), 2 (chrysoeriol; 2.0 mg; tR 19.2 min), 3 (isorhamnetin; 2.7 mg; tR 17.5 min), 5 (eriodictyol; 22.4 mg; tR 22.4 min), 10 (cosmoside; 0.8 mg; tR 10.7 min), 11 (luteolin-7-O-b-D-glucoside; 7.0 mg; tR 8.5 min), 12 (chrysoeriol-7-O-b-D-glucoside; 3.5 mg; tR 8.4 min) and 13 (stachyspinoside; 4.2 mg; tR 10.20 min). Fraction I1 was applied on CC over silica gel using eluotropic mixtures of CH2Cl2eMeOHeH2O and yielded 13 (62.0 mg). The dichloromethane extract (0.55 g) of arg-2 was subjected to VLC over silica gel using mixtures of cyclohexaneeEtOAc to yield nine fractions (A4eI4). Fraction E1 [cyclohexaneeEtOAc 50:50] was further fractionated by CC using CH2Cl2eMeOH mixtures of increased polarity and yielded nine fractions (A5eI5). Further purification of fraction D5 [CH2Cl2eMeOH 90:10] by CC on silica gel using mixtures of cyclohexaneeEtOAc afforded 6 (eupatorin; 0.9 mg). Further purification of fraction D7 [cyclohexaneeEtOAc 70:30] by several CC on silica gel using mixtures of dichloromethaneeEtOAc afforded 8 (salvigenin; 1.2 mg) and 9 (xanthomicrol; 2.2 mg). The methanol extract (4.32 g) of arg-2 was subjected to VLC over silica gel using mixtures of CH2Cl2eMeOH to yield six fractions (A6eF6). Fraction B6 [CH2Cl2eMeOH 85:15] was further applied on CC over silica gel using eluotropic mixtures of CH2Cl2eMeOHeH2O to yield 26 fractions combined to seven groups (A7eH7). Purification of fractions B7 [CH2Cl2eMeOHeH2O 95:5:0.5] and C7 [CH2Cl2eMeOHeH2O 95:5:0.5] by CC using eluotropic mixtures of CH2Cl2eMeOHeH2O yielded 1 (1.5 mg), 2 (1.2 mg), 10 (2.1 mg) and 12 (chrysoeriol 7-O-(300 -O-E-pcoumaroyl)-b-D-glucopyranoside; 1.6 mg). Purification of fraction D7 [CH2Cl2eMeOHeH2O 95:5:0.5] by several CC using eluotropic mixtures of CH2Cl2eMeOHeH2O afforded 2 (1.2 mg), 3 (0.9 mg), 5 (2.3 mg), 10 (2.7 mg), 11 (3.7 mg), 12 (12.3 mg) and 14 (24.5 mg; tR [MeOH-AcOH 5% 50:40] 46.7 min). The structure of the isolated compounds (1e14) was established by means of UVeVis, ESIMS and 1D and 2D NMR analyses. Dichloromethane extracts of the other populations were subjected to co-chromatography with the isolated flavonoids from the dichloromethane extracts of swa-2 and arg-2 on TLC. Methanol extracts of each taxon were dissolved (100 mg/mL) in methanol and 450 mL were injected into JASCO-PU2089-2015 HPLC system coupled to diode array detector. Separation was achieved using MeOHeAcOH 5% 50:40 as solvent system with a flow rate of 1.7 ml/min. Co-chromatography of the methanol extracts of each population with the isolated compounds allowed the detection of the flavonoid profile in each of them. The results are presented in Table 2. 4. Chemotaxonomic significance S. swainsonii is a member of S. sect. Swainsoniana subsect. Swainsonianeae (Bhattacharjee, 1980), which includes seven perennial, suffruticose taxa of narrow distribution, particular habitat preference, and the same chromosome number of 2n ¼ 34. Previous phytochemical work on the volatile constituents of the group enabled the distinction of Stachys ionica Halaćsy, due to its high amount of (E)-nerolidol, high amount of a-cadinol and low amount of (þ)-(E)-caryophyllene (Skaltsa et al., 2001). The rest of the taxa formed a coherent group where distinction on a phytochemical ground was not possible. The discrete position of S. ionica is further corroborated by the existence of isoscutellarein derivatives in its flavonoid content (Meremeti et al., 2004) and is in accordance to its geographical position. The species is confined to the Ionian Islands and therefore well-isolated from all its relatives in the mainland. Table 2 Flavonoids from dichloromethane and methanol extracts of Stachys swainsonii populations Compound

1

2

3

4

5

6

7

8

9

10

11

12

13

14

arg-1 arg-2 arg-3 mel-1 mel-2 mel-3 scy-1 scy-2 swa-1 swa-2 swa-3

þ þ þ þ þ þ þ þ þ þ þ

þ þ þ þ þ þ

þ þ þ

þ þ þ þ þ þ þ þ



þ þ þ þ þ þ þ þ

þ þ þ

þ þ þ



þþ þþ þþ þ þ þ þ þ þ þ þ

þþ þþ þþ þþ þþ þþþ þþþ þþþ

þþþ þþþ þþþ

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In this paper, we describe the isolation of 11 flavonoids from S. swainsonii subsp. swainsonii, namely apigenin, chrysoeriol, isorhamnetin (Wollenweber et al., 1987), penduletin (Wollenweber et al., 1987), eriodictyol, eupatorin (Wollenweber et al., 1987), 5-hydroxyauranetin (Wollenweber et al., 1989), cosmoside, luteolin-7-O-b-D-glucoside, chrysoeriol-7-O-b-D-glucoside and stachyspinoside (Kotsos et al., 2001). Also, 11 flavonoids have been isolated from S. swainsonii subsp. argolica, namely apigenin, chrysoeriol, xanthomicrol (Hernande´z et al., 1987), salvigenin (Rizk et al., 1986), isorhamnetin, eriodictyol, eupatorin, cosmoside, luteolin-7-O-b-D-glucoside, chrysoeriol-7-O-b-D-glucoside and chrysoeriol 7-O-(300 -O-E-p-coumaroyl)-b-D-glucopyranoside (Toma´s-Lorente et al., 1986). The distribution of these constituents in the other subspecies of S. swainsonii is also examined, usually in three different populations of each taxon, except of the very rare subsp. scyronica, where only two populations were available. S. swainsonii subsp. argolica appears to have a partly different chemical profile from the three other subspecies of S. swainsonii, mostly because of its high amount of chrysoeriol 7-O-(300 -O-E-p-coumaroyl)-b-D-glucopyranoside, while the rest of the taxa form a group where chrysoeriol-7-[6%-O-acetyl-b-D-allosyl-(1 / 2)]-b-D-glucopyranoside (stachyspinoside) predominates. Subsp. argolica also produces salvigenin and xanthomicrol, two flavonoids that were not found in the related subspecies. Small amounts of salvigenin and xanthomicrol have been isolated from S. ionica (Meremeti et al., 2004). Previous studies on Greek endemic Stachys species revealed that chrysoeriol-7-O-b-D-(300 -Ep-coumaroyl)-glucopyranoside and xanthomicrol are also present to Stachys chrysantha Boiss. & Heldr. and Stachys candida Bory & Chaubard (Skaltsa et al., 2000), while stachyspinoside was also found in Stachys spinosa L. (Kotsos et al., 2001). According to our results, isorhamnetin was detected only in S. swainsonii subsp. swainsonii and needs further evaluation as a possible marker among the closely related members of S. swainsonii. As a conclusion, the flavonoid content of the closely related subspecies of S. swainsonii shows many similarities, the only notable exception being subsp. argolica, which produces salvigenin, xanthomicrol and chrysoeriol-7-O-b-D(300 -E-p-coumaroyl)-glucopyranoside, constituents not found in the other subspecies. These compounds may possibly serve as useful chemotaxonomic markers that allow distinction of subsp. argolica from the rest of the group. Subsp. argolica has the southernmost distribution among the three subspecies and is the only subspecies found exclusively in Peloponnisos. The other three subspecies are all found in eastern Sterea Ellas, often grow in adjacent localities, and at least one of them (subsp. melangavica) is of hybrid origin. Hybridisation events and gene flow may be responsible for a homogenisation in their chemical constituents, both volatile (Skaltsa et al., 2001) and flavonoids. References Bhattacharjee, R., 1980. Not. R. Bot. Gard. Edinb. 38, 65. Greuter, W., Budret, H.M., Long, G., 1986. Editions des conservatoire et jardin botanique de la ville de Gene`ve. Med-Checklist, vol. 3, Gene`ve. Hernandez, L.M., Toma´s-Barberań, F.A., Toma´s-Lorente, F., 1987. Biochem. Syst. Ecol. 15, 61. Kotsos, M.P., Aligiannis, N., Mitakou, S., Skaltsounis, A.L., Charvala, C., 2001. Nat. Prod. Lett. 15, 377. Mabberley, D.J., 1997. The Plant-Book, second ed. Cambridge University Press, Cambridge, New York, Melbourne. Meremeti, A., Karioti, A., Skaltsa, H., Heilmann, J., Sticher, O., 2004. Biochem. Syst. Ecol. 32, 139. Persson, D., 1981. Biosystematics of Stachys swainsonii Benth. (Lamiaceae) and its Relations to Some other Chasmophytic Stachys Species. Ph.D. thesis, University of Lund, Lund. Phitos, D., Damboldt, J., 1969. Berich. Deutsch. Botan. Gesellsch. 82, 595. Rizk, A.M., Hammouda, F.M., Rimpler, H., Kamel, A., 1986. Planta Med. 52, 87. Skaltsa, H., Bermejo, P., Lazari, D., Silvan, A.-M., Skaltsounis, A.-L., Sanz, A., Abad, M.J., 2000. Biol. Pharm. Bull. 23, 47. Skaltsa, H., Mavrommati, A., Constantinidis, Th., 2001. Phytochemistry 57, 235. Toma´s-Lorente, F., Nieto, J.L., Toma´s-Barberań, F.A., Ferreres, F., 1986. Phytochemistry 25, 1253. Wollenweber, E., Hradetzky, Y., Mann, K., Roitman, J.N., Yatskievych, G., Proksch, M., Proksch, P., 1987. J. Plant Physiol. 131, 37. Wollenweber, E., Stern, S., Roitman, J.N., Yatskievych, G., 1989. Phytochemistry 28, 303.