Physocalycoside, a New Phenylethanoid Glycoside from Phlomis physocalyx Hub.-Mor. Tayfun Ersöza,*, Kalina Iv. Alipievab, Funda Nuray Yalc¸ına, Pınar Akbaya, Nedjalka Handjievab, Ali A. Dönmezc, Simeon Popovb, and I˙hsan C¸ alıs¸a a Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University TR 06100, Ankara, Turkey. Fax: +90-312-3114777; E-mail: [email protected] b Institute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria c Department of Biology, Faculty of Science, Hacettepe University TR 06532, Ankara, Turkey * Author for correspondence and reprint requests Z. Naturforsch. 58c, 471Ð476 (2003); received December 12, 2002/February 10, 2003 A new phenylethanoid tetraglycoside, physocalycoside (2), was isolated from the aerial parts of Phlomis physocalyx. Its structure was identified as 3-hydroxy-4-methoxy--phenyl- ethoxy-O-[α-l-rhamnopyranosyl-(152)-α-l-rhamnopyranosyl-(153)]-4-O-feruloyl-[-d- glucopyranosyl-(156)]--d-glucopyranoside, on the basis of spectroscopic evidence. In addi- tion, one known iridoid glucoside, lamiide (1) and five known phenylethanoid glycosides, wiedemannioside C (3), verbascoside (= acteoside) (4), leucosceptoside A (5), martynoside (6), and forsythoside B (7) were also characterized. Compounds 2Ð7 demonstrated radical scavenging properties towards the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Key words: Phlomis physocalyx, Iridoid and Phenylethanoid Glycosides, Radical Scavenging Activity Introduction tynoside (6), and forsythoside B (7). Antioxidant The genus Phlomis (Lamiaceae) is represented activity of the phenylethanoid glycosides (2Ð7)is by 34 species in the Flora of Turkey (Huber-Mo- also presented. rath, 1982). Some members of the genus possess medicinal properties (Saracog˘lu et al., 1995) and Material and Methods are used as tonics and stimulants in the Anatolian General experimental procedures folk medicine (Baytop, 1999). As a part of our on- going search on secondary metabolites of Turkish Optical rotations were measured on a Rudolph Phlomis species (Bas¸aran et al., 1991; C¸ alıs¸ et al., autopol IV Polarimeter using a sodium lamp oper- 1990a,b, 1991; Ersöz et al., 2001aÐc, 2002aÐc; Har- ating at 589 nm. UV (MeOH) spectra were re- put et al., 1998, 1999; Saracog˘lu et al., 1995, 1997, corded on a Shimadzu UV-160A spectrophotome- 1998, 2002), we studied the Turkish endemic, ter. IR spectra (KBr) were determined on a Phlomis physocalyx Hub.-Mor. This plant is an eg- Perkin-Elmer 2000 FTIR spectrometer. NMR landular herb to 30 cm, growing on steppes and measurements in CD3OD were performed on a 1 13 calcareous hills at elevations of 950Ð1730 m in In- Bruker AMX 300 ( H: 300.13 and C: 75.5 MHz) ner Anatolia (Huber-Morath, 1982). It was found and Varian Unity 500 (1H: 500 and 13C: 125 MHz) that the methanolic extract of aerial parts of the spectrometers. Positive mode HR-MALDI MS title plant exhibits antioxidant effects, based on data were taken on a Ionspec.-Ultima-FTMS in- the scavenging activity of 2,2-diphenyl-1-picrylhy- strument with DBH as matrix substance. ESIMS drazyl (DPPH) free radical. The present paper were recorded in the positive ion modes on a Fin- deals with the isolation and structure elucidation nigan TSQ 7000 spectrometer. For open-column of the new phenylethanoid glycoside, physocalyco- chromatography (CC), Polyamide (Polyamid-MN- side (2), as well as of six known compounds, the Polyamid SC-6, Machery-Nagel, Düren), Kiesel iridoid glucoside, lamiide (1) and the phenyletha- gel 60 (0.063Ð0.200 mm, Merck) and Sephadex noid glycosides, wiedemannioside C (3), verbasco- LH-20 were used. Low-pressure liquid chromatog- side (= acteoside) (4), leucosceptoside A (5), mar- raphy (RP-8 LPLC) was performed on Lobar pre- 0939Ð5075/2003/0700Ð0471 $ 06.00 ” 2003 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com · D 472 T. Ersöz et al. · Physocalycoside from Phlomis physocalyx Hub.-Mor. packed columns (310Ð25 and 240Ð10), LiChro- of this mixture by LPLC with a 5% stepwise gradi- prep RP-8 (40Ð63 mµ, Merck). Analytical and ent mixture of MeOH in H2O (5 to 45%) afforded preparative-TLC were carried on pre-coated Kie- physoalycoside (2, 10 mg) and wiedemannioside C selgel 60 F254 aluminum sheets (Merck). Com- (3, 5 mg). Fr. D (117 mg) yielded fractions D1ÐD3 pounds were detected by UV and 1% vanillin/ after separation on a Si gel (20 g) column using H2SO4 followed by heating at 105 ∞C for 1Ð2 min. CHCl3ÐMeOHÐH2O (80:20:1 to 70:30:3 v/v/v) For radical scavenging assay, DPPH (= 2,2-diphe- mixture. Fr. D2 (33.6 mg) was rechromatographed nyl-1-picrylhydrazyl, Fluka) was used. Absorbance on Si gel (10 g) with CHCl3ÐMeOH (98:2 to 90:10 at 517 nm was measured with an automated micro- v/v) mixture to yield martynoside (6, 3 mg). An plate reader (Bio-Tek Instruments Inc.) spectro- aliquot (340 mg) of fraction E (1.52 g) was sub- photometer. jected to Si gel CC (30 g) with CHCl3ÐMeOHÐ H2O (80:20:1 to 60:40:4 v/v/v) mixture to obtain Plant material nine fractions E1ÐE9. Fraction E7 (64.4 mg) was purified by Sephadex CC (MeOH) to give leuco- Phlomis physocalyx Hub.-Mor. (Lamiaceae) sceptoside A (5, 10 mg). 540 mg of fraction F was collected in July 2001 at Sivas (1550 m) near (1.52 g) was subjected to Si gel CC (30 g) employ- Gürün-Kangal-Kocakurt crossing, Inner Anatolia, ing CHCl ÐMeOHÐH O (80:20:1 to 80:20:3 v/v/v) Turkey. Voucher specimens have been deposited 3 2 mixtures to give six fractions (frs. F ÐF ). Fr. F in the Herbarium of the Biology Department, Fa- 1 6 4 yielded verbascoside (4, 28 mg) after purification culty of Science, Hacettepe University, Ankara, on a Lobar column (240Ð10) employing 5% step- Turkey (AAD 9555). wise gradient elution of MeOH in H2O (25 to 60%). Fraction F5 was likely separated on a Lobar Extraction and isolation column (240Ð10) with a 5% stepwise gradient elu- The air-dried and powdered aerial parts of tion of MeOH in H2O (15 to 60%) to give a mix- P. physocalyx (600 g) were extracted with MeOH ture (40 mg) of verbascoside (4) and forsythoside (3¥2500 ml) at 40 ∞C. After evaporation of the B(7). This mixture was then separated by PTLC combined extracts in vacuo 51.6 g of crude MeOH with a CHCl3ÐMeOHÐH2O (61:32:7 v/v/v) extract was obtained. The crude extract was dis- solvent system to yield verbascoside (4, 13.2 mg) solved in water (250 ml) and the water-insoluble and forsythoside B (7, 23.9 mg). material was removed by filtration. The filtrate Physocalycoside (2): Amorphous yellowish 20 was then extracted succesively with n-BuOH powder; [α]D Ð43.9∞ (c = 0.1, MeOH); positive-ion (3¥150 ml) to obtain the n-BuOH fraction HR-MALDI-MS m/z: calcd. for C43H60O24Na: (29.4 g). An aliquot of the n-BuOH fraction (10 g) 983.3473. Found: 983.3358; UV λmax. (MeOH, nm) Ð1 was separated on a polyamide column (100 g). 219, 237, 287 (sh), 328; υmax (KBr, cm ) 3400 (OH), Elution with H2O (500 ml) and gradient MeOHÐ 1699 (C=O), 1630 (olefinic C=C), 1605 and 1508 1 H2O (25 to 100%, each 500 ml) mixtures afforded (arom. ring); H NMR (CD3OD, 500 MHz): Table I; 13 8 main fractions (AÐH). Fraction A (930 mg) was C NMR (CD3OD, 125 MHz): Table I. purified on a Lobar RP-8 column (310Ð25) using Reduction of DPPH radical: Methanolic solutions increasing amounts of MeOH in H2O (0 to 65%) (0.1%) of the phenylethanoid glycosides (2Ð7) were to afford fractions A1 and A2. Fraction A2 (99 mg) chromatographed on a Si gel TLC plate using was separated on a Lobar RP-8 column (240Ð10) CHCl3ÐMeOHÐH2O (61:32:7) mixture as solvent using 5 to 25% MeOH in H2O as eluent to give system. After drying, TLC plates were sprayed with lamiide (1, 75 mg). Fr. B (420 mg) was fractionated a 0.2% DPPH (Fluka) solution in MeOH. Com- over a Si gel (30 g) column eluting with CHCl3Ð pounds showing yellow-on-purple spot were re- MeOHÐH2O (80:20:0 to 80:20:3 v/v/v) mixture garded as antioxidant (Cuendet et al., 1997). to yield six fractions (frs. B1ÐB6). Fraction B5 DPPH assay in vitro: The radical scavenging ac- (74.4 mg) was purified on a Lobar column (240Ð10) tivity of the phenylethanoid glycosides was exam- employing a 5% stepwise gradient elution of ined with the DPPH radical, as described by Cuen- MeOH in H2O (0 to 60%) to get a mixture of det et al. (2001). Ascorbic acid was used as control. compounds 2 and 3 (25 mg). Furter fractionation 50 µl 0.02% DPPH in MeOH, 200 µl MeOH and T. Ersöz et al. · Physocalycoside from Phlomis physocalyx Hub.-Mor. 473 30 µl of sample solution in MeOH were mixed in a side; Sticher and Lahloub, 1982), leucosceptoside 96-well plate. After incubation of the plate for A (Miyase et al., 1982), martynoside (C¸ alıs¸ et al., 30 min at room temperature, optical density was 1984), and forsythoside B (Endo et al., 1982), measured at 517 nm and the inhibition percentage respectively, by comparison of their 1H and 13C (%) of the radical scavenging activity was calculated NMR spectroscopic properties and ESI-MS data using the following equation: with those reported in the literature. Physocalycoside (2) was obtained as a yellowish Ao Ð As α 20 Inhibition (%) = ¥ 100 amorphous powder, [ ]D Ð43.9∞ (c = 0.1, MeOH). Ao The positive-ion HR-MALDI mass spectrum exhib- ited molecular ion peaks [M+Na]+ and [M+K]+ at where A is the absorbance of the control and A is o s m/z 983 and 999, respectively, consistent with the absorbance of the sample at 517 nm.