Fitoterapia 92 (2014) 274–279
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Oleanane type glycosides from Paronychia anatolica subsp. balansae Derya Gülcemal a,⁎, Milena Masullo b, Özgen Alankuş-Çalışkan a, Sonia Piacente b,⁎⁎ a b
Chemistry Department, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II n.132, I-84084 Fisciano (SA), Italy
a r t i c l e
i n f o
Article history: Received 7 September 2013 Accepted in revised form 24 November 2013 Available online 7 December 2013 Keywords: Paronychia anatolica subsp. balansae Illecebraceae Oleanane Saponins
a b s t r a c t Four new oleanane-type triterpene glycosides were isolated from the methanol extract of the roots of Paronychia anatolica subsp. balansae along with three known oleanane-type triterpene glycosides. Structures of the new compounds were established as 3-O-β-Dglucuronopyranosyl-28-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside] zahnic acid, 3-O-β-D-glucuronopyranosyl-28-O-[β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)β-D-quinovopyranoside] zahnic acid, 3-O-β-D-glucuronopyranosyl-28-O-[α-L-arabinofuranosyl(1 → 2)-β-D-quinovopyranoside] zahnic acid, 28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl]-medicagenic acid, by using 1D and 2D-NMR techniques and mass spectrometry. The cytotoxic activity of the isolated compounds was evaluated against a small panel of cancer cell lines including human breast cancer (MCF-7), human lung adenocarcinoma (A549) and human leukemia (U937) cell lines. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The genus Paronychia Miller, a member of Illecebraceae family, is represented by 32 species in the flora of Turkey [1]. Paronychia anatolica Czeczott subsp. balansae Chaudhri is an endemic plant which mainly grows in West Anatolia [2]. Although there are no reports of the medicinal uses of P. anatolica, P. argentea Lam. has been used in Jordanian traditional medicine for treatment of diabetes [3,4]. Additionally, the aerial parts of P. argentea are traditionally used for the treatment of the bladder, prostate and abdominal ailments, stomach ulcers and as a gastric analgesic in Portugal [5], for treating urinary system inflammation, kidney stones, prostate disorders and stomach diseases in Palestinian folk medicine [6], for eczema and as a febrifuge and digestive in Spain [7]. Previous phytochemical investigations on the genus Paronychia led to the isolation of gypsogenic acid-type saponins, polygalacic acid-type saponins, flavonoids and tocopherols [8–11]. To the best of our knowledge, there are no ⁎ Corresponding author. Tel.: +90 2323882369; fax: +90 2323888264. ⁎⁎ Corresponding author. Tel.: +39 089969763; fax: +39 089969602. E-mail addresses: [email protected] (D. Gülcemal), [email protected] (S. Piacente). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.11.013
phytochemical and biological and/or toxicological studies reported on P. anatolica subsp. balansae. Therefore, the phytochemical investigation of the MeOH extract of the roots of P. anatolica was carried out. Four oleanane-type triterpene glycosides (1–4) (Fig. 1), never reported before, along with three known oleanane-type triterpene glycosides (5–7) were isolated and their structure established as zahnic acid-type saponins and medicagenic acid-type saponins by using 1D and 2D-NMR techniques and mass spectrometry. To the best of our knowledge, zahnic acid-type saponins are being reported for the first time in Illecebraceae family, which has been recently separated from the family Caryophyllaceae and medicagenic acid-type saponins were encountered for the first time in Paronychia species. 2. Experimental 2.1. General Optical rotations were measured on a JASCO DIP 1000 polarimeter. IR measurements were obtained on a Bruker IFS-48 spectrometer. NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker BioSpinGmBH,
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Fig. 1. Structures of compounds 1–4.
Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbeat 300 K. All 2D-NMR spectra were acquired in CD3OD (99.95%, Sigma Aldrich) and standard pulse sequences and phase cycling were used for DQF-COSY, HSQC, and HMBC spectra. The NMR data were processed using UXNMR software. Exact masses were measured by a Voyager DE mass spectrometer. Samples were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry. A mixture of analyte solution and a-cyano-4-hydroxycinnamic acid (Sigma) was applied to the metallic sample plate and dried. Mass
calibration was performed with the ions from ACTH (fragment 18–39) at 2465.1989 Da α-cyano-4-hydroxycinnamic acid at 190.0504 Da as internal standard. 2.2. Plant material P. anatolica subsp. balansae (whole plant) was collected from Bozdağ, from altitude of 1297 m, Izmir, Turkey in June 2010 and identified by Dr. Serdar G. Şenol (Department of Biology, Faculty of Sciences, Ege University, Izmir, Turkey). A
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voucher specimen (EGE 19518) is deposited in the Ege University Botanical Garden & Herbarium Research and The Application Center, Izmir, Turkey. 2.3. Extraction and isolation The air-dried and powdered plant material of P. anatolica subsp. balansae (900 g) was extracted with MeOH (3 × 3 l). After filtration, the solvent was removed by rotary evaporation to give crude extract (60 g). The residue was dissolved in water and then partitioned successively with n-Hexane (3 × 250 ml), CH2CI2 (3 × 250 ml), and n-BuOH saturated with H2O (3 × 250 ml). The n-BuOH extract (10 g) was subjected to vacuum liquid chromatography (VLC) using reversed-phase material (Lichroprep RP-18, 25–40 μm, 70 g) employing with H2O (500 ml), H2O–MeOH (8:2, 900 ml; 6:4, 800 ml; 4:6, 1000 ml; 2:8, 800 ml) and MeOH (800 ml) to give nine main fractions (A–I). Fraction C (500 mg) was submitted to silica gel (60 g) column chromatography with the solvent system CHCl3–MeOH–H2O (80:20:2, 300 ml; 70:30:3, 500 ml) yielding 4 (12 mg) and 10 subfractions. Subfraction 2 (35 mg) was applied to a reversed phase column (Lichroprep RP-18, 25–40 μm, 10 g) using MeOH– H2O (6:4, 500 ml) to give 1 (14 mg). Subfraction 3 (32 mg) was chromatographed on a reversed phase material (Lichroprep RP-18, 25–40 μm, 10 g), employing MeOH–H2O (6:4, 500 ml) yielding 2 (10 mg) and 3 (8 mg). Subfraction 4 (40 mg) was purified on a reversed phase column (Lichroprep RP-18, 25–40 μm, 10 g) and eluted with MeOH–H2O (6:4, 500 ml) to give 7 (8 mg). Fraction D (400 mg) was submitted to silica gel (50 g) column chromatography with the solvent system CHCl3–MeOH–H2O (80:20:2, 300 ml; 70:30:3, 500 ml) yielding 5 (8 mg) and 6 (8 mg). 2.3.1. 3-O-β-D-glucuronopyranosyl-28-O-[α-L-rhamnopyranosyl(1 → 2)-β-D-quinovopyranoside] zahnic acid (1) Amorphous white solid; C48H74O21; [α]25D –21.3 (c 0.1 MeOH); IR νKBrmax cm−1: 3440 (NOH), 2935 (N CH), 1670 (C_O), 1655 (C_C); for 1H and 13C NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M + Na]+ m/z 1009.4265 (calcd for C48H74O21Na, 1009.4260).
2.3.4. 28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranosyl]-medicagenic acid (4) Amorphous white solid; C48H76O20; [α]25D − 18.5. (c 0.1 MeOH); IR νKBrmax cm−1: 3435 (NOH), 2925 (N CH), 1660 (C_C); 1H and 13C NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M + Na]+ m/z 995.4833 (calc. for C48H76O20Na, 995.4828). 2.4. Acid hydrolysis A mixture of compounds 1 (4 mg), 2 (4 mg), 3 (4 mg) and 4 (4 mg) was heated at 60 °C with 1:1 0.5 N HCl-dioxane (3 ml) for 2 h, and then evaporated in vacuo. The solution was partitioned with CH2Cl2–H2O, and the H2O layer was neutralized with Amberlite MB-3. The upper aqueous layer containing monosaccharides was neutralized using an ion-exchange resin (Amberlite MB-3) column, and then lyophilized to give a sugar mixture. Sugar mixture was developed by TLC using as solvent system, MeCOEt-iso-PrOH–Me2CO–H2O (20:10:7:6). Monosaccharides were identified with authentic sugar samples. After preparative TLC of the sugar mixture, the optical rotation of each purified sugar was measured to afford arabinose (Rf 0.50; [α]20D +41), glucose (Rf 0.45; [α]20D +21), rhamnose (Rf 0.57; [α]20D +12), glucuronic acid (Rf 0.10; [α]20D +7), xylose (Rf 0.65; [α]20D +20) and quinovose (Rf 0.54; [α]20D +28) [12,13]. 2.5. Cancer cell lines Human breast cancer (MCF-7) and human lung adenocarcinoma (A549), obtained from European Collection of Cell Cultures, were cultured in DMEM medium supplemented with 2 mM L-glutamine, 10% heatinactivated fetal bovine serum (FBS), 1% penicillin/streptomycin; human leukemic monocyte lymphoma (U937) cells were cultured in RPMI medium supplemented with 2 mM L-glutamine, 10% FBS, 1% penicillin/streptomycin at 37 °C in an atmosphere of 95% O2 and 5% CO2. The cells were used up to a maximum of 10 passages. 2.6. MTT bioassay
2.3.2. 3-O-β-D-glucuronopyranosyl-28-O-[β-D-xylopyranosyl(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside] zahnic acid (2) Amorphous white solid; C53H82O25 [α]25D − 23.0 (c 0.1 MeOH); IR νKBrmax cm−1: 3450 (NOH), 2930 (N CH), 1680 (C_O), 1660 (C_C); for 1H and 13C NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M + Na]+ m/z 1141.5049 (calcd for C53H82O25Na, 1141.5043). 2.3.3. 3-O-β-D-glucuronopyranosyl-28-O-[α-L-arabinofuranosyl(1 → 2)-β-D-quinovopyranoside] zahnic acid (3) Amorphous white solid; C47H72O21; [α]25D − 15.2 (c 0.1 MeOH); IR νKBrmax cm−1: 3435 (NOH), 2945 (N CH), 1675 (C_O), 1656 (C_C); 1H and 13C NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively, respectively; HRMALDITOFMS [M + Na]+ m/z 995.4468 (calcd for C47H72O21Na, 995.4464).
Human cancer cells (3 × 103) were plated in 96-well culture plates in 90 ml of culture medium and incubated at 37 °C in humidified 5% CO2. The next day, 10 ml aliquots of serial dilutions of each test compound (1–50 mM) were added to the cells and incubated for 48 h. Cell viability was assessed through the MTT assay. Briefly, 25 ml of MTT (5 mg/ml) was added and the cells were incubated for an additional 3 h. Thereafter, cells were lysed and the dark blue crystals solubilized with 100 ml of a solution containing 50% N,N-dimethylformamide, 20% SDS (Sodium Dodecyl Sulfate) with an adjusted pH of 4.5. The optical density (OD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan MCC/340) equipped with a 620 nm filter. Cell viability in response to treatment was calculated as percentage of control cells treated with solvent DMSO at the final concentration 0.1%: % viable cells = (100 × OD treated cells) / OD control cells.
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Table 1 13 C and 1H NMR data (J in Hz) of the aglycone moieties of compounds 1–4 (600 Mz, δ ppm, in CD3OD). 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
2
3
4
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
45.0 70.5 88.2 53.9 53.0 21.4 33.8 41.0 48.4 37.4 24.6 123.8 145.0 42.8 36.3 74.9 47.8 42.6 47.9 31.2 36.4 31.3 183.0 14.4 17.7 18.0 27.3 179.1 33.4 25.0
2.12, 1.28, dd (14.0, 4.0) 4.35, ddd (3.0, 3.8, 4.0) 4.10, d (3.5) – 1.64, dd (12.0, 4.0) 1.62, 1.29, m 1.62,1.34, m – 1.65, m – 2.02, (2H) m 5.36, t (3.5) – – 1.90, 1.18, m 4.45, br s – 2.95, dd (14.0, 4.0) 2.31, 1.06, m – 1.41, 1.19, m 1.88, m – 1.38, s 1.30, s 0.81, s 1.40, s – 0.89, s 0.99, s
45.6 70.5 87.4 54.0 53.5 22.2 33.8 40.9 48.5 37.5 24.7 123.7 145.0 42.7 36.3 74.8 47.8 42.3 47.8 31.2 36.3 32.1 183.0 14.6 17.9 18.0 27.3 178.1 33.4 25.0
2.14, 1.28, dd (14.0, 4.0) 4.36, ddd (3.0, 3.8, 4.0) 4.16, d (3.5) – 1.63, dd (12.0, 4.0) 1.68, 1.33, m 1.62,1.35, m – 1.67, m – 2.03, (2H) m 5.35, t (3.5) – – 1.96, 1.19, m 4.47, br s – 2.97, dd (14.0, 4.0) 2.32, 1.07, m – 1.40, 1.19, m 1.96, 1.89, m – 1.40, s 1.32, s 0.81, s 1.40, s – 0.90, s 1.00, s
45.5 70.4 88.1 53.9 53.3 21.5 33.7 41.0 48.4 37.6 24.5 123.6 145.0 42.6 36.2 74.7 47.7 42.5 47.7 31.1 36.4 31.3 183.1 14.5 17.8 18.0 27.2 179.0 33.3 25.1
2.15, 1.29, dd (14.0, 4.0) 4.33, ddd (3.0, 3.8, 4.0) 4.11, d (3.5) – 1.65, dd (12.0, 4.0) 1.60, 1.27, m 1.61,1.33, m – 1.64, m – 2.01, (2H) m 5.35, t (3.5) – – 1.92, 1.16, m 4.44, br s – 2.94, dd (14.0, 4.0) 2.30, 1.04, m – 1.40, 1.17, m 1.86, m – 1.36, s 1.31, s 0.80, s 1.41, s – 0.88, s 0.98, s
45.4 72.1 76.8 54.4 52.9 21.7 33.3 40.9 49.8 37.1 24.5 123.7 145.0 43.1 28.8 23.9 46.0 40.8 47.2 31.4 34.7 33.1 183.0 13.4 17.6 17.9 26.2 179.1 33.5 23.8
2.09, 1.23, dd (14.0, 4.0) 4.10, ddd (3.0, 3.8, 4.0) 4.01, d (3.5) – 1.62, dd (12.0, 4.0) 1.64, 1.23, m 1.54,1.28, m – 1.61, m – 1.95, (2H) m 5.28, t (3.5) – – 1.78, 1.07, m 2.06, 1.72, m – 2.90, dd (14.0, 4.0) 1.72, 1.16, m – 1.41, 1.24, m 1.74, 1.59, m – 1.33, s 1.30, s 0.82, s 1.18, s – 0.91, s 0.97, s
3. Results and discussion The MeOH extract of roots of P. anatolica was partitioned with n-Hexane, CH2CI2, and n-BuOH saturated with H2O. The n-BuOH extract was subjected to vacuum liquid chromatography (VLC) to give nine main fractions, which were successively purified by reversed phase column chromatography to afford seven triterpene saponins. The aglycones of the isolated compounds were recognized to be oleanane-type triterpenes by 1H NMR and 13C NMR analysis (Table 1) [14,15]. The HRMALDITOF mass spectrum of 1 (m/z 1009.4265 [M + Na]+, calcd for C48H74O21Na, 1009.4260) supported a molecular formula of C48H74O21. A detailed comparison of the NMR data (1H, 13C, HSQC, HMBC, COSY) of compounds 1–3 showed that the aglycone moiety was identical in all the three compounds. In particular, the 1H NMR spectrum of 1 showed signals for six methyl groups as singlets at δ 1.40, 1.38, 1.30, 0.99, 0.89 and 0.81, one olefinic proton at δ 5.36 (H-12, t, J = 3.5 Hz) and three oxygen-bearing methine protons at δ 4.35 (H-2, ddd, J = 3.0, 3.8 and 4.0 Hz), 4.10 (H-3, d, J = 3.5 Hz) and 4.45 (H-16, brs) (Table 1) [16]. The 13C NMR spectrum of the aglycone portion exhibited six tertiary methyl resonances at δ 33.4, 27.3, 25.0, 18.0, 17.7, and 14.4, two sp2-hybridized carbons at δ 123.8 and 145.0, resonances for three secondary carbinol carbons (δ 88.2, 74.9, and 70.5) and two carboxylic groups (δ 183.0 and 179.1). A signal at δ 179.1 and the carbon resonances of rings D and E in the 13C NMR spectrum suggested the occurrence of a glycosylated COOH group at C-28. A further carboxylic function
(δ 183.0) was located at C-23 on the basis of the downfield shift exhibited by C-4 (δ 53.9) and the high-field shifts experienced, respectively, by C-3 (δ 88.2), C-5 (δ 53.0), and C-24 (δ 14.4) in comparison with the same carbon resonances in an oleanane skeleton bearing a Me-23 [17]. Furthermore, the orientation of the hydroxyl groups of ring A was assigned as 2β and 3β on the basis of 1H NMR coupling constants and by comparison with those reported for related compounds [14,18]. The 16α configuration of the hydroxyl group was evident from the chemical shift and the small J values of H-16 (δ 4.45, brs), characteristic of an equatorial proton. Thus, the aglycone of 1 was identified as 2β,3β,16α-trihydroxyoleane-23,28-dioic acid known as zahnic acid [19]. The downfield shifts of C-3 (δ 88.2) and C-28 (δ 179.1) of the aglycone suggested that compound 1 was a bidesmosidic glycoside. The 13C NMR spectrum showed 48 carbon signals, of which 30 were assigned to the aglycone moiety (Table 1) and 18 to a sugar portion. The 1H NMR spectrum displayed in the sugar region signals corresponding to three anomeric protons at δ 5.39 (d, J = 7.8 Hz), 5.31 (d, J = 1.2 Hz), 4.43 (d, J = 7.5 Hz), which were unambiguously correlated by HSQC experiment to the corresponding carbon resonances at δ 95.1, 101.8, and 104.5, respectively. The HSQC, HMBC, DQF-COSY and 1D-TOCSY data led to identify these sugar units as β-glucuronopyranosyl, β-quinovopyranosyl and α-rhamnopyranosyl units. The HMBC correlations between the proton signal at δ 4.43 (H-1glcA) and the carbon resonance at δ 88.2 (C-3), the proton signal at δ 5.39 (H-1qui) and the carbon resonance at δ 179.1 (C-28) and the proton signal at δ 5.31 (H-1rha) and the carbon resonance at δ
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Table 2 13 C and 1H NMR data (J in Hz) of the sugar portions of compounds 1–4 (600 Mz, δ ppm, in CD3OD). 1
2
δC 1 2 3 4 5 6
δH (J in Hz)
δC
3 δH (J in Hz)
4
δC
δH (J in Hz)
β-D-GlcA (at C-3) 104.5 4.43, d (7.5) 75.4 3.30, dd (7.5, 9.0) 77.5 3.42, dd (9.0, 9.0) 73.5 3.42, m 76.1 3.60, d (9.0) 176.5 –
β-D-GlcA (at C-3) 104.5 4.44, d (7.5) 75.2 3.30, dd (7.5, 9.0) 77.7 3.44, dd (9.0, 9.0) 73.4 3.43, m 75.9 3.62, d (9.0) 176.5 –
β-D-GlcA (at C-3) 104.4 4.43, d (7.5) 75.3 3.31, dd (7.5, 9.0) 77.4 3.43, dd (9.0, 9.0) 73.5 3.42, m 75.6 3.60, d (9.0) 176.1 –
β-D-Qui (at C-28)
β-D-Qui (at C-28)
β-D-Qui (at C-28)
5.39, 3.57, 3.49, 3.04, 3.36, 1.27,
α-L-Rha (at C-2qui)
α-L-Rha (at C-2qui)
α-L-Araf (at C-2qui)
β-D-Glc II(at C-6glc I)
1 2 3 4 5
101.8 71.9 72.1 73.8 70.4
102.0 71.7 71.9 82.5 69.3
110.0 82.3 78.5 86.7 63.0
104.6 75.2 77.9 79.6 78.1
4.37, d (7.5) 3.25, dd (7.5, 9.0) 3.33 dd (9.0, 9.0) 3.55, dd (9.0, 9.0) 3.39, m
6
17.9
62.8
3.87, dd(3.5, 12) 3.70, dd (4.5, 12)
1.29,d (6.5)
17.9
104.1 75.0 78.7 70.9 66.7
5.42, 3.50, 3.49, 3.04, 3.36, 1.26,
5.19, 3.91, 3.77, 3.51, 3.75,
d (7.8) dd (7.8, 9.6) t (9.6) t (9.6) m d (6.4)
d (1.2) dd (1.2, 3.2) dd (3.2, 9.7) t (9.7) m
1.28, d (6.5)
94.7 79.5 78.6 76.6 73.5 17.8
5.40, 3.48, 3.48, 3.02, 3.35, 1.25,
β-D-Glc I (at C-28)
95.1 78.4 78.8 77.0 73.8 17.9
d (1.2) dd (1.2, 3.2) dd (3.2, 9.7) t (9.7) m
94.7 79.7 78.8 76.8 73.6 17.9
δH (J in Hz)
1 2 3 4 5 6
5.31, 3.97, 3.66, 3.41, 3.75,
d (7.8) dd (7.8, 9.6) t (9.6) t (9.6) m d (6.4)
δC
d (7.8) dd (7.8, 9.6) t (9.6) t (9.6) m d (6.4)
5.45, d (1.3) 4.10, dd (1.3, 3.3) 3.97, dd (6.0, 3.3) 4.17, m 3.75 (dd, 11.4, 3.3) 3.71 (dd, 11.4, 4.2)
95.7 73.6 78.0 70.9 77.7 69.3
5.37, d (7.5) 3.35, dd (7.5, 9.0) 3.45, dd (9.0, 9.0) 3.45, dd (9.0, 9.0) 3.55, m 4.14, dd(3.5, 12.0) 3.79, dd (4.5, 12.0)
β-D-Xyl (at C-4rha)
α-L-Rha (at C-4glcII)
4.63, 3.22, 3.46, 3.56, 3.85, 3.17,
103.0 72.3 72.0 73.6 70.7
d (7.5) dd (7.5, 9.2) t (9.2) m dd (11.7, 5.2) t (11.7)
18.0
78.4 (C-2qui) allowed us to determine the linkage site of the sugar units. The acid hydrolysis of 1 afforded D-glucuronic acid, D-quinovose and L-rhamnose, confirmed by the optical rotation data of each isolated sugar. Thus, the new compound 1 was elucidated as 3-O-β-Dglucuronopyranosyl-28-O-[α-L-rhamnopyranosyl-(1 → 2)-β-Dquinovopyranoside] zahnic acid. The HRMALDITOF mass spectrum of 2 (m/z 1141.5049 [M + Na]+, calcd for C53H82O25Na, 1141.5043) supported a molecular formula of C53H82O25. The 1H NMR showed signals for the anomeric protons at δ 5.42 (d, J = 7.8 Hz), 5.19 (d, J = 1.2 Hz), 4.63 (d, J = 7.5 Hz) and 4.44 (d, J = 7.5 Hz), which showed correlations in the HSQC spectrum with the anomeric carbon signals at δ 94.7, 102.0, 104.1 and 104.5, respectively. These data, in combination with 1D-TOCSY, HSQC, HMBC, DQF-COSY correlations, showed that 2 differed from 1 only in the presence of an additional β-xylopyranosyl unit. The D configuration of glucuronic acid, quinovose and xylose and the L configuration of rhamnose were confirmed by the optical rotation data of each isolated sugar. HMBC spectrum allowed us to deduce the sugar sequence. A cross-peak between the signals of H-1glcA (δ 4.44) and C-3 (δ 87.4) confirmed the presence of a β-D-glucuronopyranosyl unit linked at C-3 of the aglycone. Similarly the sequence of the
4.86, d (1.2) 3.86, dd (1.2, 3.2) 3.64, dd (3.2, 9.7) 3.41, t (9.7) 4.00, m 1.29, d (6.5)
trisaccharide chain at C-28 was established by the cross-peaks between H-1qui (δ 5.42) and C-28 (δ 178.1), H-1rha (δ 5.19) and C-2qui (δ 79.7), H-1xyl (δ 4.63) and C-4rha (δ 82.5). Therefore, compound 2 was identified as 3-O-β-D-glucuronopyranosyl28-O-[β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside] zahnic acid. The HRMALDITOF mass spectrum of 3 (m/z 995.4468 [M + Na]+, calcd for C47H72O21Na, 995.4464) supported a molecular formula of C47H72O21. The comparison of the NMR data of compound 3 with those of compound 1 allowed us to determine that the two compounds differed only by the presence of an α-arabinofuranosyl unit at C-2qui in 3 instead of the α-rhamnopyranosyl unit in 1. The D configuration of glucuronopyranose and quinovopyranose and the L configuration of arabinofuranose units were established after hydrolysis of 3 and confirmation by the optical rotation data of each isolated sugar. Thus, compound 3 was elucidated as 3-O-β-D-glucuronopyranosyl-28-O-[α-L-arabinofuranosyl-(1 → 2)-β-Dquinovopyranoside] zahnic acid. The HRMALDITOF mass spectrum of 4 (m/z 995.4833 [M + Na]+, calcd for C48H76O20Na, 995.4828) supported a molecular formula of C48H76O20.
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The 1H and 13C NMR spectroscopic data of the aglycone portion of 4 were almost superimposable on those of 1–3, except for the absence of the secondary alcoholic function linked at C-16. On the basis of the foregoing data, the aglycone of 4 was identified as 2β,3β-dihydroxyolean-12ene-23,28-dioic acid, known as medicagenic acid [20]. The 1H NMR spectrum of compound 4 exhibited three anomeric proton doublets at δ 5.37 (J = 7.5 Hz), 4.86 (J = 1.2 Hz) and 4.37 (J = 7.5 Hz) (Table 2). In the HSQC spectrum, these protons correlated to carbons at δ 95.7, 103.0 and 104.6, respectively. Complete assignments of the 1H and 13C NMR signals allowed us the identification of an α-rhamnopyranosyl (δ 4.86) unit and two β-glucopyranosyl (δ 5.37 and 4.37) units. The glycosidation site on the aglycone of 4 as well as the position of the interglycosidic linkage was determined by HMBC experiment, which showed long-range correlations between the anomeric proton signal at δ 5.37 (Η-1glcI) and the carbon resonance at δ 179.1 (C-28), δ 4.37 (Η-1glcII) and δ 69.3 (C-6glcI) and the anomeric proton signal at δ 4.86 (Η-1rha) and the carbon resonance at δ 79.6 (C-4glcII). The acid hydrolysis of 4 afforded D-glucose and L-rhamnose (confirmed by the optical rotation data of each isolated sugar). Thus, compound 4 was elucidated as 28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl]-medicagenic acid. Additionally, three known oleanane-type glycosides, 28-O(α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)[β-D-glucopyranosyl (1 → 2)-β-D-glucopyranosyl] medicagenic acid (5) [21], 28-O-(α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-[β-D-glucopyranosyl-(1 → 2)-βD -glucopyranosyl]-2-O-acetoxy medicagenic acid (6) [22] and 3-O-β-D-glucuronopyranosyl-28-O-[α-L-rhamnopyranosyl(1 → 2)-α-L-arabinopyranosyl] medicagenic acid (7) [19] were isolated. Medicagenic and zahnic acid represent the dominant aglycones of saponins of Medicago sativa [14]. In particular, medicagenic acid is reported possessing several biological properties including a biocidal activity on different soil microorganisms [23]. It is noteworthy that zahnic acid-type saponins are reported for the first time in Illecebraceae family, while medicagenic acid-type saponins were never reported before in Paronychia species. Moreover, to the best of our knowledge, this is the first report of zahnic and medicagenic acid-type saponins with a quinovose unit in the sugar moiety. On the basis of the activity reported for the triterpene derivatives [24], the antiproliferative activity of compounds 1–6 was tested in different cancer cell lines including human breast cancer (MCF-7), human lung adenocarcinoma (A549) and human leukemia (U937) cell lines. In a range of concentrations between 1 and 50 μM, none of the tested compounds caused a significant reduction of the cell number as compared to controls (data not shown). Acknowledgments The authors are grateful to Ege University Research Foundation (2013 Fen 042) for the financial support and
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also to Assoc. Prof. Dr. Serdar G. ŞENOL for the identification of plant material. References [1] Güner A, Aslan S, Ekim T, Vural M, Babaç MT. Türkiye Bitkileri Listesi (Damarlı Bitkiler). Istanbul: Nezahat Gökyiğit Botanik Bahçesi ve Flora Araştırmaları Derneği Yayını; 2012 349–51. [2] Davis PH. Flora of Turkey and East Aegean Islands. vol. 2Edinburgh: Edinburgh University Press; 1967 250–62. [3] Ali-Shtayeh MS, Yaniv Z, MahaJna J. Ethnobotanical survey in the Palestinian area: a classification of the healing potential of medicinal plants. J Ethnopharmacol 2000;73:221–32. [4] Hamdan II, Yaniv Z, Afifi FU. Studies on the in vitro and in vivo hypoglycemic activities of some medicinal plants used in treatment of diabetes in Jordanian traditional medicine. J Ethnopharmacol 2004;93:117–21. [5] Ferreira A, Proenca C, Serralheiro MLM, ArauJo MEM. The in vitro screening for acetylcholinesterase inhibition and antioxidant activity of medicinal plants from Portugal. J Ethnopharmacol 2006;108:31–7. [6] Auda MA. An ethnobotanical uses of plants in the Middle Area, Gaza Strip, Palestine. Adv Environ Biol 2011;5:3681–7. [7] De Santayana MP, Blanco E, Morales R. Plants known as té in Spain: an ethno-pharmaco-botanical review. J Ethnopharmacol 2005;98:1–19. [8] Avunduk S, Lacaille-Dubois M, Miyamoto T, Bedir E, Şenol SG, AlankuşÇalışkan Ö. Chionaeosides A–D, triterpene saponins from Paronychia chionaea. J Nat Prod 2007;70:1830–3. [9] Braca A, Bader A, Siciliano T, De Tommasi N. Secondary metabolites from Paronychia argentea. Magn Res Chem 2008;46:88–93. [10] Avunduk S, Alankuş-Calışkan Ö, Miyamoto T, Tanaka C, Lacaille-Dubois M. Secondary metabolites from the roots of Paronychia chionaea. Nat Prod Commun 2011;6:205–8. [11] Curini M, Epifano F, Menghini L, Pagiotti R. Flavonoids and tocopherols from Paronychia kapela. Chem Nat Compd 2004;40:190–1. [12] Eskander J, Lavaud C, Pouny I, Soliman HSM, Abdel-Khalik SM, Mahmoud II. Saponins from the seeds of Mimusops laurifolia. Phytochemistry 2006;67:1793–9. [13] Yuan W, Wang P, Deng G, Li S. Cytotoxic triterpenoid saponins from Aesculus glabra Willd. Phytochemistry 2012;75:67–77. [14] Bialy Z, Jurzysta M, Oleszek W, Piacente S, Pizza C. Saponins in alfalfa (Medicago sativa L.) root and their structural elucidation. J Agric Food Chem 1999;47:3185–92. [15] Gülcemal D, Masullo M, Napolitano A, Karayildirim T, Bedir E, AlankusCaliskan O, et al. Oleanane glycosides from Astragalus tauricolus: isolation and structural elucidation based on a preliminary liquid chromatography-electrospray ionization tandem mass spectrometry profiling. Phytochemistry 2013;86:184–94. [16] Kapusta I, Stochmal A, Perrone A, Piacente S, Pizza C, Oleszek W. Triterpene saponins from barrel medic (Medicago truncatula) aerial parts. J Agric Food Chem 2005;53:2164–70. [17] Mahato SB, Kundu AP. 13C NMR spectra of pentacyclic triterpenoids a compilation and some salient features. Phytochemistry 1994;37:1517–75. [18] Dou H, Zhou Y, Chen C, Peng S, Liao X, Ding L. Chemical constituents of the aerial parts of Schnabelia tetradonta. J Nat Prod 2002;65:1777–81. [19] Oleszek W, Jurzysta M, Płoszynski M, Coloquhoun IJ, Price KR, Fenwick GR. Zahnic acid tridesmoside and other dominant saponins from alfalfa (Medicago sativa L.) aerial parts. J Agric Food Chem 1992;40:191–6. [20] Besson V, Lavaud C, Massiot G, Le Men-Olivier L, Bianchi S, Flament P, et al. Medicagenic acid and saponins in callus and suspension cultures of alfalfa. Phytochemistry 1989;28(5):1379–80. [21] Freiler M, Reznicek G, Jurenitsch J, Kubelka W, Schmidt W, SchubertZsilavecz M, et al. New triterpene saponins from Herniaria glabra. Helv Chim Acta 1996;79:385–90. [22] Reznicek G, Cart J, Korhammer S, Kubelka W, Jurenitsch J, Haslinger E. The 1st 2 spectroscopically confirmed saponins from Herniaria-glabra L. Pharmazie 1993;48:450–2. [23] D'Addabbo T, Carbonara T, Leonetti P, Radicci V, Tava A, Avato P. Control of plant parasitic nematodes with active saponins and biomass from Medicago sativa. Phytochem Rev 2011;10:503–19. [24] Dinda B, Debnath S, Mohanta BC, Harigaya Y. Naturally occurring triterpenoid saponins. Chem Biodivers 2010;7:2327–580.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Oleanane type glycosides from Paronychia anatolica subsp. balansae Derya Gülcemal a,⁎, Milena Masullo b, Özgen Alankuş-Çalışkan a, Sonia Piacente b,⁎⁎ a b
Chemistry Department, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II n.132, I-84084 Fisciano (SA), Italy
a r t i c l e
i n f o
Article history: Received 7 September 2013 Accepted in revised form 24 November 2013 Available online 7 December 2013 Keywords: Paronychia anatolica subsp. balansae Illecebraceae Oleanane Saponins
a b s t r a c t Four new oleanane-type triterpene glycosides were isolated from the methanol extract of the roots of Paronychia anatolica subsp. balansae along with three known oleanane-type triterpene glycosides. Structures of the new compounds were established as 3-O-β-Dglucuronopyranosyl-28-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside] zahnic acid, 3-O-β-D-glucuronopyranosyl-28-O-[β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)β-D-quinovopyranoside] zahnic acid, 3-O-β-D-glucuronopyranosyl-28-O-[α-L-arabinofuranosyl(1 → 2)-β-D-quinovopyranoside] zahnic acid, 28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl]-medicagenic acid, by using 1D and 2D-NMR techniques and mass spectrometry. The cytotoxic activity of the isolated compounds was evaluated against a small panel of cancer cell lines including human breast cancer (MCF-7), human lung adenocarcinoma (A549) and human leukemia (U937) cell lines. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The genus Paronychia Miller, a member of Illecebraceae family, is represented by 32 species in the flora of Turkey [1]. Paronychia anatolica Czeczott subsp. balansae Chaudhri is an endemic plant which mainly grows in West Anatolia [2]. Although there are no reports of the medicinal uses of P. anatolica, P. argentea Lam. has been used in Jordanian traditional medicine for treatment of diabetes [3,4]. Additionally, the aerial parts of P. argentea are traditionally used for the treatment of the bladder, prostate and abdominal ailments, stomach ulcers and as a gastric analgesic in Portugal [5], for treating urinary system inflammation, kidney stones, prostate disorders and stomach diseases in Palestinian folk medicine [6], for eczema and as a febrifuge and digestive in Spain [7]. Previous phytochemical investigations on the genus Paronychia led to the isolation of gypsogenic acid-type saponins, polygalacic acid-type saponins, flavonoids and tocopherols [8–11]. To the best of our knowledge, there are no ⁎ Corresponding author. Tel.: +90 2323882369; fax: +90 2323888264. ⁎⁎ Corresponding author. Tel.: +39 089969763; fax: +39 089969602. E-mail addresses: [email protected] (D. Gülcemal), [email protected] (S. Piacente). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.11.013
phytochemical and biological and/or toxicological studies reported on P. anatolica subsp. balansae. Therefore, the phytochemical investigation of the MeOH extract of the roots of P. anatolica was carried out. Four oleanane-type triterpene glycosides (1–4) (Fig. 1), never reported before, along with three known oleanane-type triterpene glycosides (5–7) were isolated and their structure established as zahnic acid-type saponins and medicagenic acid-type saponins by using 1D and 2D-NMR techniques and mass spectrometry. To the best of our knowledge, zahnic acid-type saponins are being reported for the first time in Illecebraceae family, which has been recently separated from the family Caryophyllaceae and medicagenic acid-type saponins were encountered for the first time in Paronychia species. 2. Experimental 2.1. General Optical rotations were measured on a JASCO DIP 1000 polarimeter. IR measurements were obtained on a Bruker IFS-48 spectrometer. NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker BioSpinGmBH,
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Fig. 1. Structures of compounds 1–4.
Rheinstetten, Germany) equipped with a Bruker 5 mm TCI CryoProbeat 300 K. All 2D-NMR spectra were acquired in CD3OD (99.95%, Sigma Aldrich) and standard pulse sequences and phase cycling were used for DQF-COSY, HSQC, and HMBC spectra. The NMR data were processed using UXNMR software. Exact masses were measured by a Voyager DE mass spectrometer. Samples were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry. A mixture of analyte solution and a-cyano-4-hydroxycinnamic acid (Sigma) was applied to the metallic sample plate and dried. Mass
calibration was performed with the ions from ACTH (fragment 18–39) at 2465.1989 Da α-cyano-4-hydroxycinnamic acid at 190.0504 Da as internal standard. 2.2. Plant material P. anatolica subsp. balansae (whole plant) was collected from Bozdağ, from altitude of 1297 m, Izmir, Turkey in June 2010 and identified by Dr. Serdar G. Şenol (Department of Biology, Faculty of Sciences, Ege University, Izmir, Turkey). A
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voucher specimen (EGE 19518) is deposited in the Ege University Botanical Garden & Herbarium Research and The Application Center, Izmir, Turkey. 2.3. Extraction and isolation The air-dried and powdered plant material of P. anatolica subsp. balansae (900 g) was extracted with MeOH (3 × 3 l). After filtration, the solvent was removed by rotary evaporation to give crude extract (60 g). The residue was dissolved in water and then partitioned successively with n-Hexane (3 × 250 ml), CH2CI2 (3 × 250 ml), and n-BuOH saturated with H2O (3 × 250 ml). The n-BuOH extract (10 g) was subjected to vacuum liquid chromatography (VLC) using reversed-phase material (Lichroprep RP-18, 25–40 μm, 70 g) employing with H2O (500 ml), H2O–MeOH (8:2, 900 ml; 6:4, 800 ml; 4:6, 1000 ml; 2:8, 800 ml) and MeOH (800 ml) to give nine main fractions (A–I). Fraction C (500 mg) was submitted to silica gel (60 g) column chromatography with the solvent system CHCl3–MeOH–H2O (80:20:2, 300 ml; 70:30:3, 500 ml) yielding 4 (12 mg) and 10 subfractions. Subfraction 2 (35 mg) was applied to a reversed phase column (Lichroprep RP-18, 25–40 μm, 10 g) using MeOH– H2O (6:4, 500 ml) to give 1 (14 mg). Subfraction 3 (32 mg) was chromatographed on a reversed phase material (Lichroprep RP-18, 25–40 μm, 10 g), employing MeOH–H2O (6:4, 500 ml) yielding 2 (10 mg) and 3 (8 mg). Subfraction 4 (40 mg) was purified on a reversed phase column (Lichroprep RP-18, 25–40 μm, 10 g) and eluted with MeOH–H2O (6:4, 500 ml) to give 7 (8 mg). Fraction D (400 mg) was submitted to silica gel (50 g) column chromatography with the solvent system CHCl3–MeOH–H2O (80:20:2, 300 ml; 70:30:3, 500 ml) yielding 5 (8 mg) and 6 (8 mg). 2.3.1. 3-O-β-D-glucuronopyranosyl-28-O-[α-L-rhamnopyranosyl(1 → 2)-β-D-quinovopyranoside] zahnic acid (1) Amorphous white solid; C48H74O21; [α]25D –21.3 (c 0.1 MeOH); IR νKBrmax cm−1: 3440 (NOH), 2935 (N CH), 1670 (C_O), 1655 (C_C); for 1H and 13C NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M + Na]+ m/z 1009.4265 (calcd for C48H74O21Na, 1009.4260).
2.3.4. 28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranosyl]-medicagenic acid (4) Amorphous white solid; C48H76O20; [α]25D − 18.5. (c 0.1 MeOH); IR νKBrmax cm−1: 3435 (NOH), 2925 (N CH), 1660 (C_C); 1H and 13C NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M + Na]+ m/z 995.4833 (calc. for C48H76O20Na, 995.4828). 2.4. Acid hydrolysis A mixture of compounds 1 (4 mg), 2 (4 mg), 3 (4 mg) and 4 (4 mg) was heated at 60 °C with 1:1 0.5 N HCl-dioxane (3 ml) for 2 h, and then evaporated in vacuo. The solution was partitioned with CH2Cl2–H2O, and the H2O layer was neutralized with Amberlite MB-3. The upper aqueous layer containing monosaccharides was neutralized using an ion-exchange resin (Amberlite MB-3) column, and then lyophilized to give a sugar mixture. Sugar mixture was developed by TLC using as solvent system, MeCOEt-iso-PrOH–Me2CO–H2O (20:10:7:6). Monosaccharides were identified with authentic sugar samples. After preparative TLC of the sugar mixture, the optical rotation of each purified sugar was measured to afford arabinose (Rf 0.50; [α]20D +41), glucose (Rf 0.45; [α]20D +21), rhamnose (Rf 0.57; [α]20D +12), glucuronic acid (Rf 0.10; [α]20D +7), xylose (Rf 0.65; [α]20D +20) and quinovose (Rf 0.54; [α]20D +28) [12,13]. 2.5. Cancer cell lines Human breast cancer (MCF-7) and human lung adenocarcinoma (A549), obtained from European Collection of Cell Cultures, were cultured in DMEM medium supplemented with 2 mM L-glutamine, 10% heatinactivated fetal bovine serum (FBS), 1% penicillin/streptomycin; human leukemic monocyte lymphoma (U937) cells were cultured in RPMI medium supplemented with 2 mM L-glutamine, 10% FBS, 1% penicillin/streptomycin at 37 °C in an atmosphere of 95% O2 and 5% CO2. The cells were used up to a maximum of 10 passages. 2.6. MTT bioassay
2.3.2. 3-O-β-D-glucuronopyranosyl-28-O-[β-D-xylopyranosyl(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside] zahnic acid (2) Amorphous white solid; C53H82O25 [α]25D − 23.0 (c 0.1 MeOH); IR νKBrmax cm−1: 3450 (NOH), 2930 (N CH), 1680 (C_O), 1660 (C_C); for 1H and 13C NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M + Na]+ m/z 1141.5049 (calcd for C53H82O25Na, 1141.5043). 2.3.3. 3-O-β-D-glucuronopyranosyl-28-O-[α-L-arabinofuranosyl(1 → 2)-β-D-quinovopyranoside] zahnic acid (3) Amorphous white solid; C47H72O21; [α]25D − 15.2 (c 0.1 MeOH); IR νKBrmax cm−1: 3435 (NOH), 2945 (N CH), 1675 (C_O), 1656 (C_C); 1H and 13C NMR (CD3OD, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively, respectively; HRMALDITOFMS [M + Na]+ m/z 995.4468 (calcd for C47H72O21Na, 995.4464).
Human cancer cells (3 × 103) were plated in 96-well culture plates in 90 ml of culture medium and incubated at 37 °C in humidified 5% CO2. The next day, 10 ml aliquots of serial dilutions of each test compound (1–50 mM) were added to the cells and incubated for 48 h. Cell viability was assessed through the MTT assay. Briefly, 25 ml of MTT (5 mg/ml) was added and the cells were incubated for an additional 3 h. Thereafter, cells were lysed and the dark blue crystals solubilized with 100 ml of a solution containing 50% N,N-dimethylformamide, 20% SDS (Sodium Dodecyl Sulfate) with an adjusted pH of 4.5. The optical density (OD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan MCC/340) equipped with a 620 nm filter. Cell viability in response to treatment was calculated as percentage of control cells treated with solvent DMSO at the final concentration 0.1%: % viable cells = (100 × OD treated cells) / OD control cells.
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Table 1 13 C and 1H NMR data (J in Hz) of the aglycone moieties of compounds 1–4 (600 Mz, δ ppm, in CD3OD). 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
2
3
4
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
45.0 70.5 88.2 53.9 53.0 21.4 33.8 41.0 48.4 37.4 24.6 123.8 145.0 42.8 36.3 74.9 47.8 42.6 47.9 31.2 36.4 31.3 183.0 14.4 17.7 18.0 27.3 179.1 33.4 25.0
2.12, 1.28, dd (14.0, 4.0) 4.35, ddd (3.0, 3.8, 4.0) 4.10, d (3.5) – 1.64, dd (12.0, 4.0) 1.62, 1.29, m 1.62,1.34, m – 1.65, m – 2.02, (2H) m 5.36, t (3.5) – – 1.90, 1.18, m 4.45, br s – 2.95, dd (14.0, 4.0) 2.31, 1.06, m – 1.41, 1.19, m 1.88, m – 1.38, s 1.30, s 0.81, s 1.40, s – 0.89, s 0.99, s
45.6 70.5 87.4 54.0 53.5 22.2 33.8 40.9 48.5 37.5 24.7 123.7 145.0 42.7 36.3 74.8 47.8 42.3 47.8 31.2 36.3 32.1 183.0 14.6 17.9 18.0 27.3 178.1 33.4 25.0
2.14, 1.28, dd (14.0, 4.0) 4.36, ddd (3.0, 3.8, 4.0) 4.16, d (3.5) – 1.63, dd (12.0, 4.0) 1.68, 1.33, m 1.62,1.35, m – 1.67, m – 2.03, (2H) m 5.35, t (3.5) – – 1.96, 1.19, m 4.47, br s – 2.97, dd (14.0, 4.0) 2.32, 1.07, m – 1.40, 1.19, m 1.96, 1.89, m – 1.40, s 1.32, s 0.81, s 1.40, s – 0.90, s 1.00, s
45.5 70.4 88.1 53.9 53.3 21.5 33.7 41.0 48.4 37.6 24.5 123.6 145.0 42.6 36.2 74.7 47.7 42.5 47.7 31.1 36.4 31.3 183.1 14.5 17.8 18.0 27.2 179.0 33.3 25.1
2.15, 1.29, dd (14.0, 4.0) 4.33, ddd (3.0, 3.8, 4.0) 4.11, d (3.5) – 1.65, dd (12.0, 4.0) 1.60, 1.27, m 1.61,1.33, m – 1.64, m – 2.01, (2H) m 5.35, t (3.5) – – 1.92, 1.16, m 4.44, br s – 2.94, dd (14.0, 4.0) 2.30, 1.04, m – 1.40, 1.17, m 1.86, m – 1.36, s 1.31, s 0.80, s 1.41, s – 0.88, s 0.98, s
45.4 72.1 76.8 54.4 52.9 21.7 33.3 40.9 49.8 37.1 24.5 123.7 145.0 43.1 28.8 23.9 46.0 40.8 47.2 31.4 34.7 33.1 183.0 13.4 17.6 17.9 26.2 179.1 33.5 23.8
2.09, 1.23, dd (14.0, 4.0) 4.10, ddd (3.0, 3.8, 4.0) 4.01, d (3.5) – 1.62, dd (12.0, 4.0) 1.64, 1.23, m 1.54,1.28, m – 1.61, m – 1.95, (2H) m 5.28, t (3.5) – – 1.78, 1.07, m 2.06, 1.72, m – 2.90, dd (14.0, 4.0) 1.72, 1.16, m – 1.41, 1.24, m 1.74, 1.59, m – 1.33, s 1.30, s 0.82, s 1.18, s – 0.91, s 0.97, s
3. Results and discussion The MeOH extract of roots of P. anatolica was partitioned with n-Hexane, CH2CI2, and n-BuOH saturated with H2O. The n-BuOH extract was subjected to vacuum liquid chromatography (VLC) to give nine main fractions, which were successively purified by reversed phase column chromatography to afford seven triterpene saponins. The aglycones of the isolated compounds were recognized to be oleanane-type triterpenes by 1H NMR and 13C NMR analysis (Table 1) [14,15]. The HRMALDITOF mass spectrum of 1 (m/z 1009.4265 [M + Na]+, calcd for C48H74O21Na, 1009.4260) supported a molecular formula of C48H74O21. A detailed comparison of the NMR data (1H, 13C, HSQC, HMBC, COSY) of compounds 1–3 showed that the aglycone moiety was identical in all the three compounds. In particular, the 1H NMR spectrum of 1 showed signals for six methyl groups as singlets at δ 1.40, 1.38, 1.30, 0.99, 0.89 and 0.81, one olefinic proton at δ 5.36 (H-12, t, J = 3.5 Hz) and three oxygen-bearing methine protons at δ 4.35 (H-2, ddd, J = 3.0, 3.8 and 4.0 Hz), 4.10 (H-3, d, J = 3.5 Hz) and 4.45 (H-16, brs) (Table 1) [16]. The 13C NMR spectrum of the aglycone portion exhibited six tertiary methyl resonances at δ 33.4, 27.3, 25.0, 18.0, 17.7, and 14.4, two sp2-hybridized carbons at δ 123.8 and 145.0, resonances for three secondary carbinol carbons (δ 88.2, 74.9, and 70.5) and two carboxylic groups (δ 183.0 and 179.1). A signal at δ 179.1 and the carbon resonances of rings D and E in the 13C NMR spectrum suggested the occurrence of a glycosylated COOH group at C-28. A further carboxylic function
(δ 183.0) was located at C-23 on the basis of the downfield shift exhibited by C-4 (δ 53.9) and the high-field shifts experienced, respectively, by C-3 (δ 88.2), C-5 (δ 53.0), and C-24 (δ 14.4) in comparison with the same carbon resonances in an oleanane skeleton bearing a Me-23 [17]. Furthermore, the orientation of the hydroxyl groups of ring A was assigned as 2β and 3β on the basis of 1H NMR coupling constants and by comparison with those reported for related compounds [14,18]. The 16α configuration of the hydroxyl group was evident from the chemical shift and the small J values of H-16 (δ 4.45, brs), characteristic of an equatorial proton. Thus, the aglycone of 1 was identified as 2β,3β,16α-trihydroxyoleane-23,28-dioic acid known as zahnic acid [19]. The downfield shifts of C-3 (δ 88.2) and C-28 (δ 179.1) of the aglycone suggested that compound 1 was a bidesmosidic glycoside. The 13C NMR spectrum showed 48 carbon signals, of which 30 were assigned to the aglycone moiety (Table 1) and 18 to a sugar portion. The 1H NMR spectrum displayed in the sugar region signals corresponding to three anomeric protons at δ 5.39 (d, J = 7.8 Hz), 5.31 (d, J = 1.2 Hz), 4.43 (d, J = 7.5 Hz), which were unambiguously correlated by HSQC experiment to the corresponding carbon resonances at δ 95.1, 101.8, and 104.5, respectively. The HSQC, HMBC, DQF-COSY and 1D-TOCSY data led to identify these sugar units as β-glucuronopyranosyl, β-quinovopyranosyl and α-rhamnopyranosyl units. The HMBC correlations between the proton signal at δ 4.43 (H-1glcA) and the carbon resonance at δ 88.2 (C-3), the proton signal at δ 5.39 (H-1qui) and the carbon resonance at δ 179.1 (C-28) and the proton signal at δ 5.31 (H-1rha) and the carbon resonance at δ
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D. Gülcemal et al. / Fitoterapia 92 (2014) 274–279
Table 2 13 C and 1H NMR data (J in Hz) of the sugar portions of compounds 1–4 (600 Mz, δ ppm, in CD3OD). 1
2
δC 1 2 3 4 5 6
δH (J in Hz)
δC
3 δH (J in Hz)
4
δC
δH (J in Hz)
β-D-GlcA (at C-3) 104.5 4.43, d (7.5) 75.4 3.30, dd (7.5, 9.0) 77.5 3.42, dd (9.0, 9.0) 73.5 3.42, m 76.1 3.60, d (9.0) 176.5 –
β-D-GlcA (at C-3) 104.5 4.44, d (7.5) 75.2 3.30, dd (7.5, 9.0) 77.7 3.44, dd (9.0, 9.0) 73.4 3.43, m 75.9 3.62, d (9.0) 176.5 –
β-D-GlcA (at C-3) 104.4 4.43, d (7.5) 75.3 3.31, dd (7.5, 9.0) 77.4 3.43, dd (9.0, 9.0) 73.5 3.42, m 75.6 3.60, d (9.0) 176.1 –
β-D-Qui (at C-28)
β-D-Qui (at C-28)
β-D-Qui (at C-28)
5.39, 3.57, 3.49, 3.04, 3.36, 1.27,
α-L-Rha (at C-2qui)
α-L-Rha (at C-2qui)
α-L-Araf (at C-2qui)
β-D-Glc II(at C-6glc I)
1 2 3 4 5
101.8 71.9 72.1 73.8 70.4
102.0 71.7 71.9 82.5 69.3
110.0 82.3 78.5 86.7 63.0
104.6 75.2 77.9 79.6 78.1
4.37, d (7.5) 3.25, dd (7.5, 9.0) 3.33 dd (9.0, 9.0) 3.55, dd (9.0, 9.0) 3.39, m
6
17.9
62.8
3.87, dd(3.5, 12) 3.70, dd (4.5, 12)
1.29,d (6.5)
17.9
104.1 75.0 78.7 70.9 66.7
5.42, 3.50, 3.49, 3.04, 3.36, 1.26,
5.19, 3.91, 3.77, 3.51, 3.75,
d (7.8) dd (7.8, 9.6) t (9.6) t (9.6) m d (6.4)
d (1.2) dd (1.2, 3.2) dd (3.2, 9.7) t (9.7) m
1.28, d (6.5)
94.7 79.5 78.6 76.6 73.5 17.8
5.40, 3.48, 3.48, 3.02, 3.35, 1.25,
β-D-Glc I (at C-28)
95.1 78.4 78.8 77.0 73.8 17.9
d (1.2) dd (1.2, 3.2) dd (3.2, 9.7) t (9.7) m
94.7 79.7 78.8 76.8 73.6 17.9
δH (J in Hz)
1 2 3 4 5 6
5.31, 3.97, 3.66, 3.41, 3.75,
d (7.8) dd (7.8, 9.6) t (9.6) t (9.6) m d (6.4)
δC
d (7.8) dd (7.8, 9.6) t (9.6) t (9.6) m d (6.4)
5.45, d (1.3) 4.10, dd (1.3, 3.3) 3.97, dd (6.0, 3.3) 4.17, m 3.75 (dd, 11.4, 3.3) 3.71 (dd, 11.4, 4.2)
95.7 73.6 78.0 70.9 77.7 69.3
5.37, d (7.5) 3.35, dd (7.5, 9.0) 3.45, dd (9.0, 9.0) 3.45, dd (9.0, 9.0) 3.55, m 4.14, dd(3.5, 12.0) 3.79, dd (4.5, 12.0)
β-D-Xyl (at C-4rha)
α-L-Rha (at C-4glcII)
4.63, 3.22, 3.46, 3.56, 3.85, 3.17,
103.0 72.3 72.0 73.6 70.7
d (7.5) dd (7.5, 9.2) t (9.2) m dd (11.7, 5.2) t (11.7)
18.0
78.4 (C-2qui) allowed us to determine the linkage site of the sugar units. The acid hydrolysis of 1 afforded D-glucuronic acid, D-quinovose and L-rhamnose, confirmed by the optical rotation data of each isolated sugar. Thus, the new compound 1 was elucidated as 3-O-β-Dglucuronopyranosyl-28-O-[α-L-rhamnopyranosyl-(1 → 2)-β-Dquinovopyranoside] zahnic acid. The HRMALDITOF mass spectrum of 2 (m/z 1141.5049 [M + Na]+, calcd for C53H82O25Na, 1141.5043) supported a molecular formula of C53H82O25. The 1H NMR showed signals for the anomeric protons at δ 5.42 (d, J = 7.8 Hz), 5.19 (d, J = 1.2 Hz), 4.63 (d, J = 7.5 Hz) and 4.44 (d, J = 7.5 Hz), which showed correlations in the HSQC spectrum with the anomeric carbon signals at δ 94.7, 102.0, 104.1 and 104.5, respectively. These data, in combination with 1D-TOCSY, HSQC, HMBC, DQF-COSY correlations, showed that 2 differed from 1 only in the presence of an additional β-xylopyranosyl unit. The D configuration of glucuronic acid, quinovose and xylose and the L configuration of rhamnose were confirmed by the optical rotation data of each isolated sugar. HMBC spectrum allowed us to deduce the sugar sequence. A cross-peak between the signals of H-1glcA (δ 4.44) and C-3 (δ 87.4) confirmed the presence of a β-D-glucuronopyranosyl unit linked at C-3 of the aglycone. Similarly the sequence of the
4.86, d (1.2) 3.86, dd (1.2, 3.2) 3.64, dd (3.2, 9.7) 3.41, t (9.7) 4.00, m 1.29, d (6.5)
trisaccharide chain at C-28 was established by the cross-peaks between H-1qui (δ 5.42) and C-28 (δ 178.1), H-1rha (δ 5.19) and C-2qui (δ 79.7), H-1xyl (δ 4.63) and C-4rha (δ 82.5). Therefore, compound 2 was identified as 3-O-β-D-glucuronopyranosyl28-O-[β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside] zahnic acid. The HRMALDITOF mass spectrum of 3 (m/z 995.4468 [M + Na]+, calcd for C47H72O21Na, 995.4464) supported a molecular formula of C47H72O21. The comparison of the NMR data of compound 3 with those of compound 1 allowed us to determine that the two compounds differed only by the presence of an α-arabinofuranosyl unit at C-2qui in 3 instead of the α-rhamnopyranosyl unit in 1. The D configuration of glucuronopyranose and quinovopyranose and the L configuration of arabinofuranose units were established after hydrolysis of 3 and confirmation by the optical rotation data of each isolated sugar. Thus, compound 3 was elucidated as 3-O-β-D-glucuronopyranosyl-28-O-[α-L-arabinofuranosyl-(1 → 2)-β-Dquinovopyranoside] zahnic acid. The HRMALDITOF mass spectrum of 4 (m/z 995.4833 [M + Na]+, calcd for C48H76O20Na, 995.4828) supported a molecular formula of C48H76O20.
D. Gülcemal et al. / Fitoterapia 92 (2014) 274–279
The 1H and 13C NMR spectroscopic data of the aglycone portion of 4 were almost superimposable on those of 1–3, except for the absence of the secondary alcoholic function linked at C-16. On the basis of the foregoing data, the aglycone of 4 was identified as 2β,3β-dihydroxyolean-12ene-23,28-dioic acid, known as medicagenic acid [20]. The 1H NMR spectrum of compound 4 exhibited three anomeric proton doublets at δ 5.37 (J = 7.5 Hz), 4.86 (J = 1.2 Hz) and 4.37 (J = 7.5 Hz) (Table 2). In the HSQC spectrum, these protons correlated to carbons at δ 95.7, 103.0 and 104.6, respectively. Complete assignments of the 1H and 13C NMR signals allowed us the identification of an α-rhamnopyranosyl (δ 4.86) unit and two β-glucopyranosyl (δ 5.37 and 4.37) units. The glycosidation site on the aglycone of 4 as well as the position of the interglycosidic linkage was determined by HMBC experiment, which showed long-range correlations between the anomeric proton signal at δ 5.37 (Η-1glcI) and the carbon resonance at δ 179.1 (C-28), δ 4.37 (Η-1glcII) and δ 69.3 (C-6glcI) and the anomeric proton signal at δ 4.86 (Η-1rha) and the carbon resonance at δ 79.6 (C-4glcII). The acid hydrolysis of 4 afforded D-glucose and L-rhamnose (confirmed by the optical rotation data of each isolated sugar). Thus, compound 4 was elucidated as 28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl]-medicagenic acid. Additionally, three known oleanane-type glycosides, 28-O(α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)[β-D-glucopyranosyl (1 → 2)-β-D-glucopyranosyl] medicagenic acid (5) [21], 28-O-(α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranosyl-(1 → 6)-[β-D-glucopyranosyl-(1 → 2)-βD -glucopyranosyl]-2-O-acetoxy medicagenic acid (6) [22] and 3-O-β-D-glucuronopyranosyl-28-O-[α-L-rhamnopyranosyl(1 → 2)-α-L-arabinopyranosyl] medicagenic acid (7) [19] were isolated. Medicagenic and zahnic acid represent the dominant aglycones of saponins of Medicago sativa [14]. In particular, medicagenic acid is reported possessing several biological properties including a biocidal activity on different soil microorganisms [23]. It is noteworthy that zahnic acid-type saponins are reported for the first time in Illecebraceae family, while medicagenic acid-type saponins were never reported before in Paronychia species. Moreover, to the best of our knowledge, this is the first report of zahnic and medicagenic acid-type saponins with a quinovose unit in the sugar moiety. On the basis of the activity reported for the triterpene derivatives [24], the antiproliferative activity of compounds 1–6 was tested in different cancer cell lines including human breast cancer (MCF-7), human lung adenocarcinoma (A549) and human leukemia (U937) cell lines. In a range of concentrations between 1 and 50 μM, none of the tested compounds caused a significant reduction of the cell number as compared to controls (data not shown). Acknowledgments The authors are grateful to Ege University Research Foundation (2013 Fen 042) for the financial support and
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