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Tabatadze Nino,
Assistant Professor of the Faculty of Natural Sciences and Healthcare Sokhumi State University, PhD Degree in Pharmacy E-mail: [email protected]
Tsomaia Irma, Professor of the Department of Pharmacy Georgian Technical University E-mail: [email protected] Chikovani Anton,
Associate Professor of the Faculty of Natural Sciences and Healthcare Sokhumi State University, PhD Degree in Chemistry E-mail: [email protected] Gigoshvili Tamar,
Assistant Professor of the Faculty of Natural Sciences and Healthcare Sokhumi State University, PhD Degree in Pharmacy
E-mail: [email protected] Mshvildadze Vakhtang, Laboratoire LASEVE, Université du Québec à Chicoutimi, Département des Sciences Fondamentales, Chicoutimi, Québec, Canada
E-mail: [email protected]
Elias Riad,
Laboratoire de Pharmacognosie, Faculté de Pharmacie, Université de la Méditerranée, France E-mail: [email protected]
CHEMICAL CONSTITUENTS FROM THE ROOTS OF CEPHALARIA GIGANTEA
Abstract. Iridoid and secoiridoid glycosides - swertiamarin, cantleyoside, loganic acid and also triterpene saponin dipsacoside B were isolated from the roots of Caucasian endemic plant - Cepha-laria gigantea. The structure of these compounds was determined by all spectroscopic means mainly by NMR and MS techniques. All isolated compounds are reported for the first time from Cephalaria gigantea roots. Iridoids are used as chemosystematic markers for plants of the Dipsacaceae family. Keywords: Triterpene saponin, iridoid, NMR, MS.
The roots of Cephalaria gigantea (Ledeb.) Bobr. triterpene saponins, alkaloids, flavonoids, and phe-(fam. Dipsacaceae) an endemic plant of Caucasus nolcarboxylic acids. Previous works reported the growing in Georgia, are well-known in traditional identification by about 17 individual components: medicine as sedative and anti-inflammatory rem- saponins, alkaloids and phenolic compounds [2-8]. edies [1], The main constituents of the roots are Antifungal, antiprotozoal, antiseizure and cytotoxic
activities of major monodesmosides and root extract have been evaluated [8-12]. HPLC analysis method of the raw material and the purified extract of Cepha-laria have been proposed [13-14].
The present paper describes isolation and structure elucidation of three iridoid and secoiridoid glycosides: swertiamarin [15], cantleyoside [16] and loganic acid [17; 18], also, one triterpene saponin dipsacoside B [19]. The structural determination of the obtained compounds were performed on the bases of 2D-NMR experiments (gs-COSY, gs-HMQC, gs-HMBC and gs-HSQC-TOCSY) and mass spectrometry (MALDI-TOF, ESI-HR-MS) and confirmed by comparison with the previously reported data.
All isolated compounds: iridoid and secoiridoid glycosides and dipsacoside B are reported for the first time from Cephalaria gigantea roots. These components are used as chemosystematic markers for plants of the Dipsacaceae family.
Experimental
General experimental procedures
1H- and 13C-NMR spectra were recorded on a Bruker DRX-500 spectrometer in CD3OD solutions.
TMS was used as an internal standard. Standard Bruker pulse sequences were used for two-dimensional experiments (gs-COSY, gs-HMQC, gs-HMBC and gs-HSQC-TOCSY). High-resolution Mass Spectra (HR-MS) were obtained an Applied Biosistems MALDI-TOF Voyager Spec. The experiments were performed on a Jeol JMS-700 (Jeol LTD, Akishima, Tokyo, Japan) double focusing mass spectrometer, equipped with an electrospray ionization (ESI) source operating under positive ion mode. Melting points were determine don a BUCHI Melting Point B-540 apparatus. Optical rotations were measured with a Perkin-Elmer model 341 Orot Polarimeter.
Flash Column chromatography was carried out on polyamide MN SC6 Macherey Nagel (70 ^m), using a step gradient of H2O-MeOH of90:10, 50:50 and 0:100. Low-pressure liquid chromatography was carried out on ChromatoSPAC Prep 100 (Jo-
bin Yvon) with Lichroprep C-18 Merck (15-25 ^m, 50 x 4cm), using a step gradient of H2O-MeOH (100:0, 90:10, 80:20, 70:30, 65:35, 60:40, 50:50 and 0:100, 1 litre for each). For fraction purification was used Column chromatography - silica gel 60 (Merck, 0.040-0.063 mm), with an applied pressure of 300 mbar. TLC analyses were performed on pre-coated silica gel plates (Kiesegel 60 F254 and RP-18 F254, Merck), using the following solvent systems: CH2CL2 -MeOH-H2O (50:16:3, 50:25:5 and 30:13:3), n-BuOH-HOAc-H2O (4:1:5). Spots were detected by spraying the plates with phosphoric acid naphtoresorcinol and H2SO4 followed by heating at 110 °C.
Plant material
The roots of Cephalaria gigantea was collected in Imereti region of Georgia (Octomber 2017) and dried in the shade. A voucher specimen is kept in the department of Pharmacobotany, Institute of Phar-macochemistry, Tbilisi, Georgia (roots No. 98351).
Extraction and Isolation
Air-dried and powdered roots of Cephalaria gigantea (500 g) were extracted by percolation at room temperature with MeOH (3 L for 24 h), the obtained solution was concentrated under vacuum. . After evaporation of solvent, the dry extract (20 g) was submitted to flash column chromatography on polyamide MN SC6, using a step gradient of H2O-MeOH 90:10, 50:50 and 0:100, to give enriched fraction 1, 2, 3. Obtained enriched fraction 1 (2 g) was further purified by low-pressure liquid chromatography (LPLC) with C-18 (15-25 ^m, 50 x 4cm) and a step gradient of H2O-MeOH (100:0, 90:10, 80:20, 70:30, 65:35, 60:40, 50:50 and 0:100, 1 litre for each), allowed us to obtain swertiamarin (87 mg), cantleyoside (280 mg) and loganic acid (38 mg). Enriched fraction 2(1g) was fractionated by silica gel column chromatography using CH2Cl2 - MeOH-H2O (50:16:3) to obtain dipsacoside B (65mg).
Chemical elucidation of the obtained compounds were performed by spectroscopic analysis including 2D-NMR and HR-MS data.
Spectroscopic data
Swertiamarin. C16H22O10, yellow amorphous powder. 1H NMR (400.132 MHz, CD3OD, S, ppm, J/Hz): 7.65 (1H, s, H-3), 5.70 (1H, s, H-1) 5.46 (1H, dt, J=17.0, 9.5, H-9), 5.38 (1H, dd, J=17.0, 2.0, H-10), 5.31(1H, dt,J= 9.8, 2.0, H-10), 4.78 (1H, ddd, J=13.0, 10.8, 2.4, H-7), 4.66 (1H, d, J=7.9, H-1'), 4.36(1H, br dd, J=10.8, 4.8, H-7), 3.92 (1H, dd, J=12.0, 1.4, H-6'), 3.68 (1H, dd, J=12.0, 5.8, H-6'), 3.39 (1H, t, J=9.0, H-3'), 3.34 (1H, m, H-5'), 3.30 (1H, t, J=9.0, H-4'), 3.21 (1H, dd, J=8.9, 7.8, H-2'), 2.94 (1H, d, J=9.4, H-8), 1.93 (1H, td, J=13.9, 5.1, H-6), 1.76 (1H, br.d,J=13.9, H-6).13C NMR (100.62 MHz, CDCl3, S, ppm): 168.0 (C-11), 154.78 (C-3), 133.84 (C-8), 121.18 (C-10), 108.88 (C-4), 100.23 (C-1'), 99.09 (C-1), 78.53 (C-5'), 77.80 (C-3'), 74.43 (C-2'), 71.41 (C-4'), 65.95 (C-7), 64.27 (C-5), 62.57 (c-6'), 51, 94 (C-9), 33.73 (C-6).
Cantleyoside. C33H46O19, white amorphous powder. 1H NMR (400.136 MHz, CD3OD, S, ppm, J/Hz): 9.40 (1H, s, H-7''), 7.55 (1H, d,J=1.4, H-3''), 7.44 (1H, br.s, H-3), 5.53 (1H, d, J=4.7, H-1''), 5.29 (1H, d, J=4.9, H-1), 5.28 (1H, dd, J=17.2, 1.0, 2.4, H-10''), 5.25 (1H, dd, J=10.3, 1.0, H-10''), 5.20 (1H, t, J=5.0, H-7), 4.71 (1H, d, J=7.8, H-1'''), 4.68 (1H, d, J=7.9, H-1'), 3.92 (2H, dd, J=11.9, 1.8, H-6' and 6'''), 3.71 (3H, s, OCH3), 3.68 (2H, dd, J=11.9, 6.0, H-6' and 6'''), 3.48 (1H, q, J=6.0, H-5''), 3.39 (2H, t, J=8.9, H-3' and 3'''), 3.28 (4H, m, H-4', 5', 4''' and 5'''), 3.21 (2H, dd, J=8.9, 7.8, H-2' and 2 '''), 3.10 (1H, q, J=8.0, H-5), 2.77 (1H, m, H-9''), 2.74 (1H, m, H-6''), 2.55 (1H, dd, J=17.5, 6.3, H-6''), 2.29 (1H, dd, J=14.5, 7.5, H-6), 2.14(1H, m, H-8 and 8''), 2.07 (1H, td, J=8.7, 5.0, H-9), 1.75 (1H, ddd, J=14.5, 7.7, 5.0, H-6), 1.07 (1H, d, J= 6.8, H-10). 13C NMR (100.62 MHz, CDCl3, S, ppm): 203.13 (C-7''), 169.35 (C-11), 167.97 (C-11''), 153.90 (C-3''), 152.61 (C-3), 134.94 (C-8''), 120.54 (C-10''), 113.11 (C-4), 110.26 (C-4''), 100.20 (C-1'''), 100.03 (C-1'), 97.57 (C-1), 97.44 (C-1''), 78.42a (C-5'), 78.38a (c-7), 78.34(C-5'''), 77.98 (C-3' and 3'''), 74.71b (C-2'), 74.63b (C-2'''), 71.59c (C-4'''),
71.55c (C-4'), 62.77 (C-6' and 6'''), 51.76 (OCH3), 47.09 (c-9), 45.63 (C-6''), 45.37 (C-9''), 41.08 (C-8), 32.69 (C-5), 27.78 (C-5''), 13.85 (C-10).
Loganic acid. C16H24O10, white amorphous powder. 1H NMR (400.134 MHz, CD3OD, S, ppm, J/Hz): 7.20 (1H, s, H-3), 5.20 (1H, d, J=4.2, H-1), 4.66 (1H, d, J=7.9, H-1'), 4.05 (1H, br.t, J=4.2, H-7),
3.90 (1H, d, J= 11.5, H-6'), 3.68 (1H, dd, J=11.5, 5.0, H-6'), 3.39 (1H, t, J=9.0, H-3'), 3.33 (2H, m, H-4', H-5'), 3.21 (1H, dd, J=8.9, 7.8, H-2'), 3.13 (1H, br.q, J=7.7, H-5), 2.24 (1H, dd, J=7.8, 3.7 H-6), 1.99 (1H, td, J=9.0, 4.3, H-9), 1.89 (1H, m, H-8), 1.70 (1H, ddd, J=13.7, 7.7, 4.2, H-6), 1.10 (3H, d, J=6.9, H-10). 13C NMR (100.62 MHz, CDCl3 S, ppm): 99.88 (C-1'), 97.21 (C-1), 78.29 (C-5'), 78.01 (C-3'), 75.25 (C-7), 74.80 (C-2'), 71.62 (C-4'), 62.75 (C-6'), 46.83 (C-9), 42.74 (C-6), 42.09 (C-8), 32.93 (C-5), 13.54 (C-10), C-3, C-4 and C-11 no observed.
Dipsacoside B. C53H86O22, white amorphous powder. 1H NMR (400.13 MHz, CD3OD, S, ppm, J/ Hz): Aglycone: 5.41 (1H, t, J=2.9, H-12), 4.16 (1H, m, H-23), 4.29 (1H, m, H-3), 3.75 (1H, m, H-23), 3.18 (1H, dd,J=13.0, 3.3, H-18), 2.31 (1H, m, H-15),
2.21 (1H, m, H-2), 2.01 (1H, m, H-16), 1.99 (1H, m, H-2), 1.93 (1H, m, H-11), 1.92 (1H, m, H-16), 1.40 (1H, m, H-6), 1.91 (1H, m, H-22), 1.77 (1H, m, H-22), 1.76 (1H, m, H-9), 1.73 (1H, m, H-5), 1.72 (1H, m, H-19), 1.70 (1H, m, H-6), 1.60 (1H, m, H-7), 1.55 (1H, m, H-1), 1.31 (1H, m, H-21),
1.22 (1H, m, H-19), 1.17 (3H, s, H-27), 1.14 (3H, s, H-26), 1.10 (3H, m, H-21), 1.09 (3H, s, H-24), 1.09 (1H, m, H-15), 1.06 (1H, m, H-1), 0.98 (3H, s, H-25), 0.87 (3H, s, H-30), 0.86 (3H, s, H-29). Sugars: 28-O-Glc1: 6.29 (1H, d, J=8.1, H-1), 4.74 (1H, m, H-6), 4.39 (1H, m, H-6), 4.37 (1H, m, H-4), 4.25 (1H, m, H-3), 4.15 (1H, m, H-2), 4.13 (1H, m, H-5). Glc2: 5.06 (1H, d, J=7.8, H-1), 4.51 (1H, dd, J=12.1, 2.4, H-6), 4.38 (1H, m, H-6), 4.24 (1H, m, H-4), 4.22 (1H, m, H-3), 4.03 (1H, t, J=8.4, H-2),
3.91 (1H, m, H-5). 3-O-Ara: 5.12 (1H, d,J=6.4, H-1), 4.60 (1H, dd, J=8.6, 8.0, H-2), 4.28 (1H, m, H-5),
4.20 (1H, m, H-4), 4.13 (1H, m, H-3), 3.72 (1H, m, H-5). Rha: 6.28 (1H, d, J=1.3, H-1), 4.76 (1H, m, H-2), 4.71 (1H, m, H-5), 4.67 (1H, dd, J=9.2, 3.5, H-3), 4.32 (1H, m, H-4), 1.65 (3H, d, J=6.2, H-6). 13C NMR (100.62 MHz, CDCl3 S, ppm): Aglycone: 176.90 (C-28), 144.40 (C-13), 123.30 (C-12), 81.40 (C-3), 64.30 (C-23), 48.50 (C-9), 48.10 (C-5), 47.40 (C-17), 46.50 (C-19), 43.80 (C-4), 42.50 (C-14), 42.00 (C-18), 40.20 (C-8), 39.40 (C-1), 37.20 (C-10), 34.30 (C-21), 33.40 (C-29), 33.10 (C-7), 32.90 (C-22), 31.10 (C-20), 28.70 (c-15), 26.60 (C-2), 26.40 (C-27), 24.20 (C-11), 24.00 (C-30),
23.70 (C-16), 18.60 (C-6), 17.90 (C-26), 16.50 (C-25), 14.30 (C-24). Sugars: 28-O-Glc1: 96.00 (C-1), 79.10 (C-3), 78.30 (C-5),
74.30 (C-2), 71.30 (C-4), 69.70 (C-6). Glc2: 105.70 (C-1), 78.80 (C-5 and 3), 75.50 (C-2), 71.80 (C-4), 63.00 (C-6). 3-O-Ara: 104.70 (c-1) 76.20 (C-2), 75.10 (C-3), 69.70 (C-4), 66.00 (c-5). Rha: 102. 10 (C-1), 74.50 (C-4), 72.90 (C-3), 70.10 (C-5), 18.90 (C-6).
Iridoids and secoiridoids characterize the family Dipsacaceae and could be used as a chemotaxo-nomic marker of the family [20].
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