Physocalycoside, a New Phenylethanoid Glycoside from 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 () is represented activity of the phenylethanoid glycosides (2Ð7)is by 34 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 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. IC was deter- 50 molecular formula C H O . UV absorption mined as the amount of the sample (µm) reducing 43 60 24 bands at λ 219(sh), 237, 287(sh), and 237 indi- the absorbance by 50%. max cated the polyphenolic nature of 2. The IR spectrum showed absorption bands due to hydroxyl (3400 Results and Discussion cmÐ1), α,-unsaturated ester carbonyl (1699 cmÐ1), The methanolic extract of aeriale parts of P. olefinic double bond (1630 cmÐ1), and aromatic physocalyx was suspended in water and parti- rings (1605, 1508 cmÐ1). The 13C NMR and DEPT tioned with n-BuOH. Chromatographic separa- data (see Table I) exhibited 43 carbon resonances, tions of the n-BuOH extract by polyamide column in which 4 methyl, 4 methylene, 28 methine, and chromatography followed by low-pressure liquid 7 quaternary carbon resonances could be assigned chromatography and column chromatography on for 2.The1H NMR spectrum of compound 2 (see Si gel and Sephadex LH-20 resulted in the isola- Table I) exhibited characteristic signals arising from tion of compounds 1Ð7 (Fig. 1). (E)-ferulic acid and 3-hydroxy-4-methoxyphenyl- The structures of compounds 1 and 3Ð7 were ethanol moieties: six aromatic proton signals (2 ¥ established as the known lamiide (Bianco et al., ABX systems, in the region of δH 7.20Ð6.76), two 1977; Assaad et al., 1992), wiedemannioside C trans-olefinic proton signals (AB system, δH 7.67, d, (Abou Gazar et al., 2003), verbascoside (= acteo- JAB = 15.8 Hz and 6.38, d, JAB = 15.8 Hz), and -methylene (δH 2.88, 2H, t, J = 7.6 Hz) proton sig- nals together with two non-equivalent proton sig- COOCH3 OH nals (δH 4.08, m and 3.77, overlapped) attributed to the side-chain of the phenethyl alcohol moiety. HO 1 O Additionally, four anomeric proton resonances at 1 HO CH δ Љ α 3 O glucose H 5.36 (d, J = 1.7 Hz, H-1 of an -rhamnose), 4.90 (overlapped, H-1ЉЉ of an α-rhamnose), 4.41 (d, J = OR3 O 7.8 Hz, H-1Ј of a -glucose), and 4.28 (d, J = 7.8 Hz,  2 ' R O O ٞ O  H-1 of a -glucose) indicated the tetraglycosidic ' O O OH nature of 2. The secondary methyl signals appeared  1 HO OH δ Љ H C at H 1.07 (d, J = 6.3 Hz, H-6 ) and 1.28 (d, J = 3 O OR1 -HO 6.3 Hz, H-6ٞ) supported the presence of two rham HO OR4 nose moieties in 2. Moreover, the 13C NMR data R1 R2 R3 R4 confirmed the tetraglycosidic sugar chain, exhibiting four anomeric carbon resonances at δC 104.7 (C-1ٞ 2 CH3 CH3 glucose rhamnose of a -glucose), 104.2 (C-1Ј of a -glucose), 103.8 3 HCH3 glucose H (C-1ЉЉ of an α-rhamnose) and 101.7 (C-1Љ of an 4 HHH H α 5 HCH3 HH -rhamnose), which showed correlations with the 6 CH3 CH3 HH anomeric protons of the related sugar units. The 7 H H apiose H complete assignments of all proton and carbon reso- 1 1 Fig. 1. Iridoid (1) and phenylethanoid (2Ð7) glycosides nances were based on the results of H- H COSY, from P. physocalyx. 1H-13C HSQC and HMBC experiments. The feru- 474 T. Ersöz et al. · Physocalycoside from Phlomis physocalyx Hub.-Mor.

13 1 13 1 Table I. C and H NMR (CD3OD, C: 125 MHz and H: 500 MHz) data and HMBC correlations for 2*.

C/H Atom dC ppm** DEPT dH ppm, J [Hz] HMBC (C5H) Aglycone 1 131.6 s C - H-2, H-6 2 117.1 d CH 6.76 d (2.2) H-6 3 147.6 s C - H-5 4 147.8 s C - H-2, H-6,4-OMe 5 113.7 d CH 6.81 d (8.2) 6 121.2 d CH 6.69 dd (8.2/2.2) H-2, H2- † α 36.7 t CH2 4.08 m/3.77 H-1Ј,H2- 72.3 t CH2 2.88 t (7.6) 4-OMe 56.4 q CH3 3.85 s Glucose 1Ј 104.2 d CH 4.41 d (7.8) H-2Ј 2Ј 76.1 d CH 3.39 dd (7.8/9.0) 3Ј 81.6 d CH 3.78† H-1Љ, H-2Ј, H-4Ј 4Ј 70.6 d CH 5.01 t (9.0) 5Ј 77.9 d CH 3.30† † † 6Ј 69.4 t CH2 3.95 /3.64 H-1ЉЉ Rhamnose 1Љ 101.7 d CH 5.36 d (1.7) 2Љ 80.2 d CH 3.94 dd (1.7/3.4) H-1ЉЉ 3Љ 71.7 d CH 3.63† † 4Љ 74.0 d CH 3.30 H3-6Љ, H-1Љ 5Љ 70.5 d CH 3.57 dq (6.3/10.0) H3-6Љ 6Љ 17.9 q CH3 1.07 d (6.3) Glucose (5C-6Ј) 1ٞ 104.7 d CH 4.28 d (7.8) H-2Љ (2ٞ 75.1 d CH 3.20 dd (7.8/9.0 †3ٞ 77.9 d CH 3.32 (4ٞ 71.4 d CH 3.27† (9.5 5ٞ 78.1 d CH 3.24 m † † 6ٞ 62.6 t CH2 3.84 /3.63 Rhamnose (5C-2Љ) 1ЉЉ 103.8 d CH 4.90† 2ЉЉ 72.0 d CH 3.92 dd (1.7/3.4) 3ЉЉ 72.2 d CH 3.62† † 4ЉЉ 74.0 d CH 3.30 H-1ЉЉ,H3-6ЉЉ † 5ЉЉ 70.2 d CH 3.72 H3-6Љ 6ЉЉ 18.6 q CH3 1.28 d (6.3) Feruloyl 1ٞЉ 127.6 s C - H-Ј, H-5ٞЉ 2ٞЉ 112.8 d CH 7.20 d (1.8) H-Ј, H-6ٞЉ 3Љٞ 149.4 s C - H-2ٞЉ, H-5ٞЉ,3ٞЉ-OMe 4Љٞ 150.9 s C - H-2Љٞ, H-5Љٞ, H-6Љٞ (5Љٞ 116.5 d CH 6.81 d (8.2 6Љٞ 124.4 d CH 7.09 dd (8.2/1.8) H-Ј, H-2Љٞ αЈ 115.1 d CH 6.38 d (15.8) H-Ј Ј 148.1 d CH 7.67 d (15.8) H-2Љٞ, H-6Љٞ C=O 168.4 s C - H-αЈ,H-Ј, H-4Ј 3Љٞ-OMe 56.4 q CH3 3.89 s * All 1H and 13C assignments are based on 2D NMR (COSY, HSQC and HMBC) ex- periments. ** Multiplicites are based on DEPT-135 experiment. † Signal patterns are unclear due to overlapping. loyl group was supposed to be positioned at C-4Ј of eronuclear long-range copuling observed between the core glucose due to the strong deshielding of the the carbonyl carbon resonance (δC 168.4) of the acyl H-4Ј resonance of the glucose unit (δH 5.01, t, J = moiety and H-4Ј (δH 5.01). On the other hand, a 9.0 Hz). This assumption was supported by the het- HMBC cross-peak observed from C-α carbon reso- T. Ersöz et al. · Physocalycoside from Phlomis physocalyx Hub.-Mor. 475 nance (δC 72.3) of the phenylethyl alcohol unit to ride phenylethanoids have been published. As far the anomeric proton of the core glucose (δH 4.41, as we know, till now only four tetraglycosidic phe- H-1Ј) indicated the attachment of the core glucose nylethanoids, magnolioside C (Hasegawa et al., to be the C-α carbon atom of the aglycone. The 1988), ballotetroside (Seidel et al., 1997), trichosan- structure of the tetrasaccharide unit was elucidated thoside B (C¸ alıs¸ et al., 1999), and marrubioside by means of DQF-COSY and HSQC experiments. (Sahpaz et al., 2002) have been described. There- Although, the highly deshielded carbon signals aris- fore, physocalycoside (2) appears as the fifth repre- ing from the core glucose and the first rhamnose sentative of this class. On the other hand, all pre- units suggested that the glucose unit to be glycosy- viously isolated tetrasaccharidic glycosides are lated at C-3Ј (δC 81.6) and C-6Ј (δC 69.4), whereas caffeic acid esters and contain a 3,4-dihydroxyphen- the rhamnose unit at C-2Љ (δC 80.2), however, a ethyl alcohol moiety, while, physocalycoside (2)isa prominent HMBC experiment allowed us to assign ferulic acid ester and contains a 3-hydroxy-4-meth- unambiguously all the interglycosidic connectivities oxyphenethyl alcohol unit as the aglycone. of the sugar sequence. Thus, the correlations were Phenylethanoid glycosides (2Ð7) were screened for antioxidant activities by a TLC autographic as- observed between C-3Ј (δC 81.6) of the core glucose say with 2,2-diphenyl-1-picrylhydrazyl (DPPH) radi- and H-1Љ (δH 5.36) of the first rhamnose, C-6Ј (δC cal (Cuendet et al., 1997; Takao et al., 1994) and of the core glucose and H-1ٞ (δH 4.28) of the (69.4 showed antioxidant properties based on their ability second glucose, as well as C-2Љ (δC 80.2) of the first to scavenge free radicals. Then the free-radical scav- rhamnose and H-1ЉЉ (δH 4.90) of the second rham- nose moiety. Some significant long-range correla- enging effects of the phenylethanoid glycosides (2Ð7), corresponding to the intensity of quenching tions confirming the proposed structure were given the DPPH radical were evaluated (Cuendet et al., in Fig. 2. Thus, on the basis of its NMR data, the 2001). Compounds 2Ð7 exhibited a dose-dependent structure of compound 2 was established as 3-hy- reduction on DPPH. Results are given in Table II. droxy-4-methoxy--phenylethoxy-O-[α-l-rhamno- pyranosyl-(152)-α-l-rhamnopyranosyl-(153)]-4- Table II. Free radical scavenging effects of the phenyl- ethanoid glycosides 2Ð7 on DPPH. O-feruloyl-[-d-glucopyranosyl-(156)]--d-gluco- pyranoside for which the trivial name physocalyco- Compounds Concentration [µm]IC50 side is proposed. 10 25 50 100 200 [µm] In recent years papers on the isolation and char- Ascorbic acida 0.86b 2.30 9.77 35.63 79.02 112 acterization of numerous mono-, di-, and trisaccha- 2 2.30 16.09 30.75 42.82 59.77 50 3 14.37 18.68 30.46 57.76 78.16 79 HO 4 28.45 33.62 54.31 79.00 79.31 49

1''' OH 5 18.39 22.99 28.16 65.50 79.02 76 O O OH ' O 6 13.51 17.53 25.57 33.91 55.40 101 H CO 3 O O HO 7 17.53 28.16 55.75 75.86 78.74 43 1''''' ' O 1' O  OH  1 a HO OH Reference compound. H C O b 3 1" OCH % inhibition. HO 3 HO O Acknowledgments

1'''' The authors are grateful to Oswald Greter and H3C O HO Dr. Walter Amrein (ETH-Zurich) for recording HO OH mass spectra. This work was supported by the Sci- Fig. 2. Selected heteronuclear multiple bond correlations entific and Technical Research Council of Turkey (HMBC) for physocalycoside (2). Arrows point from (TÜBI˙TAK) and Bulgarian Academy of Science carbon to proton. (BAS). 476 T. Ersöz et al. · Physocalycoside from Phlomis physocalyx Hub.-Mor.

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