- Email: [email protected]
Novel saponins from Nigella arvensis var. involucrata
Phytochemistry Letters 21 (2017) 128–133
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Novel saponins from Nigella arvensis var. involucrata Kenan Bıçak , Derya Gülcemal , İbrahim Demirtaş a
a b
a
b,⁎
, Özgen Alankuş
a,⁎
MARK
Department of Chemistry, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey Department of Chemistry, Faculty of Science, Çankırı Karatekin University, Çankırı, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords: Nigella arvensis Ranunculaceae Oleanane Saponins
Four new oleanane-type triterpene glycosides namely 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl(1 → 2)-α-L-arabinopyranosyl] oleanolic acid, 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-α-Larabinopyranosyl]-28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid, 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[β-Dglucopyranosyl] oleanolic acid, 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid, were isolated from methanol extract of the roots of Nigella arvensis var. involucrata along with five known oleanane-type triterpene glycosides and a steroid glucoside. Structures of the new compounds were established as by using 1D and 2D NMR techniques and mass spectrometry.
1. Introduction The genus Nigella, a member of Ranunculaceae family, is represented by 13 species in the flora of Turkey (Davis, 1965, 1988). Some Nigella species (e.g. Nigella sativa L., Nigella damascena L. and Nigella arvensis L.) have been in use in many Middle Eastern and Far Eastern countries as a natural remedy for over 2000 years (Fatima et al., 2002). The extract of N. sativa seeds and its constituents could be used to dissolve kidney stones (Hadjzadeh et al., 2011; Dollah et al., 2013;), antimicrobial and anti-inflammatory activities (Landa et al., 2009; Rakhshandeh et al., 2011; Randhawa and Alghamdi, 2011), inhibit the carcinogenic process (Al-Sheddi et al., 2014; Randhawa and Alghamdi, 2011), antiatherogenic cardio-protective (Asgary et al., 2013), and antioxidant effects (Jrah-Harzallah et al., 2013). Previous phytochemical investigations on the genus Nigella led to the isolation of alkaloids (Atta-ur-Rahman et al., 1985; Ali et al., 2008), oleanane-type saponins (Kumara and Huat, 2001; Taskin et al., 2005; Elbandy et al., 2009; Xin et al., 2009), cycloartane-type saponins (Mehta et al., 2008), benzofurans (Xin et al., 2009) and peptides (Oshchepkova et al., 2009). A phytochemical work performed on N. arvensis revealed beta-sitosterol as major metabolite. Additionally, beta-sitosterol was evaluated for inhibition and induction of IL-8 gene in bronchial epithelial cells (Nicolis et al., 2010). Pharmacological investigations on the extracts of N. arvensis exhibited, antimicrobial (Landa et al., 2006, 2009) and antiinflammatory activities (Landa et al., 2009). The seed extract was also found to have a stimulatory effect on Na+ transport in renal epithelia (Atia et al., 2002). ⁎
Therefore, the phytochemical investigation of the MeOH extract of the roots of N. arvensis var. involucrata was carried out. Four oleananetype triterpene glycosides (1–4) (Fig. 1), never reported before, along with five known oleanane-type triterpene glycosides and a steroid glucoside (5–10) were isolated and their structure were established by the extensive use of 1D and 2D NMR experiments and HRMS analysis. 2. Results and discussion The HRMALDITOF mass spectrum of 1 (m/z 935.2875 [M + Na]+, calcd for C47H76O17Na, 935.2860) supported a molecular formula of C47H76O17. A detailed comparison of the NMR data (1H, 13C, HSQC, HMBC, COSY) of compounds 1–4 showed that the aglycone moiety was identical in all the four compounds. In particular, the 1H NMR spectrum of 1 showed signals for seven methyl groups as singlets at δ 1.08, 0.95, 0.87 (6H), 0.86, 0.76 and 0.70, one olefinic proton at δ 5.16 (H-12, t, J = 3.5 Hz) and one oxygen-bearing methine protons at δ 2.99 (H-3, dd, J = 11.5, 4.2 Hz). These signals, along with the resonances in the 13 C NMR spectrum for the Me groups at δ 32.6, 27.2, 25.4, 23.2, 16.6, 16.1, 15.0, of the two sp2-hybridized carbons at δ 121.3 and 144.0, for one carbinol carbons (δ 88.4), evidenced that compound 1 possessed an olean-12-ene skeleton as aglycone. As a result, the aglycone was identified as oleanolic acid (Senel et al., 2014). The downfield shift of C-3 (δ 88.4) of the aglycone indicated that compound 1 was a monodesmosidic glycoside (Hai et al., 2012). The 13C NMR spectrum showed 47C-atom signals of which 30 were assigned to the aglycone moiety
Corresponding authors. E-mail addresses: [email protected] (İ. Demirtaş), [email protected] (Ö. Alankuş).
http://dx.doi.org/10.1016/j.phytol.2017.06.002 Received 28 February 2017; Received in revised form 30 May 2017; Accepted 5 June 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
Fig. 1. Structures of compounds 1–4.
correlated by HSQC experiment to the corresponding carbon resonances at δ 101.1, 103.7, and 105.0, respectively. The HSQC, HMBC, DQF-COSY and 1D-TOCSY data led to identify these sugar units as βglucopyranosyl, and α-arabinopyranosyl units. The HMBC correlations
(Table 1) and 17 to a sugar portion. The 1H NMR spectrum displayed in the sugar region signals corresponding to three anomeric protons at δ 4.62 (d, J = 7.8 Hz), 4.32 (d, J = 3.0 Hz), 4.31 (d, J = 7.5 Hz), which were unambiguously 129
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
Table 1 13 C and 1H NMR data (J in Hz) of the aglycone moieties of compounds 1–4 (600 MHz, δ ppm, in DMSO). 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)
38.0 25.5 88.4 38.5 55.4 17.6 32.2 40.6 47.1 35.0 22.8 121.3 144.0 41.6 27.0 22.7 47.6 40.8 45.6 31.0 33.0 32.0 27.2 16.1 15.0 16.6 25.4 179.0 32.6 23.2
1.49, 0.92, m 1.71, 1.55, m 2.99, dd (11.5, 4.2) – 0.72, m 1.46, 1.31 1.59,1.40, m – 1.48, m – 1.79, (2H) m 5.16, t (3.5) – – 1.65, 0.99, m 1.79, 1.48, m – 2.74, m 1.60, 1.04, m – 1.30, 1.12, m 1.40, 1.21, m 0.95, s 0.70, s 0.87, s 0.76, s 1.08, s – 0.86, s 0.86, s
37.5 24.5 88.4 38.0 55.5 17.5 32.0 40.5 47.5 35.4 22.0 121.3 144.0 41.5 27.4 22.5 47.0 40.6 45.9 31.4 33.5 32.6 27.2 16.1 15.0 16.6 25.4 174.8 32.6 23.2
1.49, 0.92, m 1.71, 1.55, m 2.99, dd (11.5, 4.2) – 0.72, m 1.46, 1.31 1.59,1.40, m – 1.48, m – 1.79, (2H) m 5.16, t (3.5) – – 1.65, 0.99, m 1.79, 1.48, m – 2.74, m 1.60, 1.04, m – 1.30, 1.12, m 1.40, 1.21, m 0.95, s 0.70, s 0.87, s 0.76, s 1.08, s – 0.86, s 0.86, s
38.2 25.6 87.5 38.6 55.5 17.5 32.5 40.0 47.8 35.3 22.7 121.3 144.0 41.0 27.5 22.8 47.4 40.7 45.8 31.6 33.3 32.5 27.2 16.1 15.0 16.6 25.4 175.0 32.6 23.2
1.49, 0.92, m 1.71, 1.55, m 2.99, dd (11.5, 4.2) – 0.72, m 1.46, 1.31 1.59,1.40, m – 1.48, m – 1.79, (2H) m 5.16, t (3.5) – – 1.65, 0.99, m 1.79, 1.48, m – 2.74, m 1.60, 1.04, m – 1.30, 1.12, m 1.40, 1.21, m 0.95, s 0.70, s 0.87, s 0.76, s 1.08, s – 0.86, s 0.86, s
37.8 25.9 88.4 38.8 55.3 17.8 32.4 40.8 47.4 35.3 22.9 121.3 144.0 41.5 27.6 22.8 47.5 40.9 45.5 31.3 33.3 32.6 27.2 16.1 15.0 16.6 25.4 174.9 32.6 23.2
1.49, 0.92, m 1.71, 1.55, m 2.99, dd (11.5, 4.2) – 0.72, m 1.46, 1.31 1.59,1.40, m – 1.48, m – 1.79, (2H) m 5.16, t (3.5) – – 1.65, 0.99, m 1.79, 1.48, m – 2.74, m 1.60, 1.04, m – 1.30, 1.12, m 1.40, 1.21, m 0.95, s 0.70, s 0.87, s 0.76, s 1.08, s – 0.86, s 0.86, s
between the proton signal at δ 4.32 (H-1ara) and the carbon resonance at δ 88.4 (C-3), the proton signal at δ 4.62 (H-1glcI) and the carbon resonance at δ 77.1 (C-2ara) and the proton signal at δ 4.31 (H-1glcII) and the carbon resonance at δ 84.6 (C-2glcI) allowed us to determine the linkage site of the sugar units. The acid hydrolysis of 1 afforded Dglucose and L-arabinose, confirmed by the optical rotation data of each isolated sugar (Gülcemal et al., 2014a, 2014b). Thus, the new compound 1 was elucidated as 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl] oleanolic acid. The HRMALDITOF mass spectrum of 2 (m/z 1405.5049 [M + Na]+, calcd for C65H106O31Na, 1405.5033) supported a molecular formula of C65H106O31. With respect to the sugar portion, the occurrence of three additional sugar units was observed compared to 1. The 1H NMR spectrum displayed in the sugar region signals corresponding to six anomeric Hatoms at δ 5.21 (d, J = 7.5), 4.69 (d, J = 1.2), 4.61 (d, J = 7.8), 4.46 (d, J = 3.0), 4.38 (d, J = 7.2) and 4.27 (d, J = 7.5) which showed correlations in the HSQC spectrum with the anomeric C-atom signals at δ 93.6, 100.2, 101.0, 102.6, 104.2 and 102.2, respectively. A detailed analysis of NMR data revealed the presence of four β-glucopyranosyl units (δ 5.21, 4.61, 4.38, and 4.27), one α-rhamnopyranosyl unit (δ 4.69), and one α-arabinopyranosyl unit (δ 4.46). An unambiguous determination of the sequence and linkage sites was obtained from the HMBC spectrum, which allowed us to identify at C-3 the same sugar chain as in 1, and the occurrence of a further sugar chain at C-28, as indicated by the upfield shift of C-28 at δ 174.8 when compared to compound 1. Key HMBCs between the resonance of H-1glcIII (δ 5.21) and C-28 (δ 174.8), between the resonance of H-1glcIV (δ 4.27) and C6glcIII (δ 67.4) and between the resonance of H-1rha (δ 4.69) and C-4glcIV (δ 76.1) confirmed this hypothesis. The D configuration of glucose and the L configuration of rhamnose and arabinose were confirmed by the optical rotation data of each isolated sugar (Gülcemal et al., 2014a, 2014b). On the basis of all these evidences, the structure of 2 was
established as 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L rhamnopyranosyl (1 → 4)-βD-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid. The HRMALDITOF mass spectrum of 3 (m/z 1065.4265 [M + Na]+, calcd for C53H86O20Na, 1065.4250) supported a molecular formula of C53H86O20. The 1H NMR spectrum displayed in the sugar region signals corresponding to four anomeric protons at δ 5.21 (d, J = 7.9 Hz), 5.16 (d, J = 1.2 Hz), 4.33 (d, J = 7.2 Hz) and 4.27 (d, J = 3.0 Hz), which were unambiguously correlated by HSQC experiment to the corresponding carbon resonances at δ 93.7, 99.3, 104.5 and 103.9, respectively. The chemical shifts of all the individual protons of the four sugar units were ascertained from a combination of 1D-TOCSY and DQF-COSY spectral analysis, and the 13C chemical shifts of their relative attached carbons were assigned unambiguously from the HSQC spectrum (Table 2) These data showed the presence of one β-glucopyranosyl unit (δ 5.21), one αrhamnopyranosyl unit (δ 5.16), one β-quinovopyranosyl unit (δ 4.33) and one α-arabinopyranosyl unit (δ 4.27). An unambiguous determination of the sequence and linkage sites was obtained from the HMBC spectrum, which showed key correlation peaks between the proton signal of δ H-1ara (δ 4.27) and the carbon resonance of C-3 (δ 87.5), H1rha (δ 5.16) and C-2ara (δ 73.9), H-1qui (δ 4.33) and C-3rha (δ 80.9), H1glc (δ 5.21) and the carbon resonance of C-28 (δ 175.0). The acid hydrolysis of 3 afforded D-glucose, D-quinovose, L-rhamnose and L-arabinose, confirmed by the optical rotation data of each isolated sugar (Gülcemal et al., 2014a, 2014b). Thus, compound 3 was identified as 3O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-Larabinopyranosyl]-28-O-[β-D-glucopyranosyl] oleanolic acid. The HRMALDITOF mass spectrum of 4 (m/z 1373.3483 [M + Na]+, calcd for C65H106O29Na, 1373.3455) supported a molecular formula of C65H106O29. The 1H NMR showed signals for the anomeric protons at δ 5.21 (δ, J = 7.8 Hz), 5.17 (d, J = 1.2 Hz), 4.70 (d, J = 1.2 Hz), 4.34 (d,
130
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
Table 2 13 C and 1H NMR data (J in Hz) of the sugar portions of compounds 1–4 (600 MHz, δ ppm, in DMSO). 1
2
3
4
δH (J in Hz) δC α-L-Ara (at C-3)
δC δH (J in Hz) α-L-Ara (at C-3)
δC δH (J in Hz) α-L-Ara (at C-3)
δC δH (J in Hz) α-L-Ara (at C-3)
1 2 3 4 5
103.7 77.1 71.6 66.8 63.7
102.6 77.3 70.4 65.8 61.9
103.9 73.9 72.9 67.8 64.4
103.9 73.1 73.0 67.8 64.5
1 2 3 4 5 6
β-D-Glc 101.1 84.6 75.9 69.6 77.3 60.8
4.27, d (3.0) 3.59, d (8.5, 3.0) 3.56, dd (8.5, 3.7) 3.57, m 3.65, dd (11.5, 2.0) 3.36, dd (11.5, 2.0) α-L-Rha (at C-2ara) 99.3 5.16, d (1.2) 69.7 3.81, dd (1.2, 3.2) 80.9 3.63, dd (3.2, 9.7) 70.7 3.41, t (9.7) 67.7 3.77, m 17.5 1.09, d (6.5)
4.27, d (3.0) 3.58, d (8.5, 3.0) 3.54, dd (8.5, 3.7) 3.57, m 3.66, dd (11.5, 2.0) 3.37, dd (11.5, 2.0) α-L-Rha (at C-2ara) 99.3 5.17, d (1.2) 69.7 3.81, dd (1.2, 3.2) 80.8 3.64, dd (3.2, 9.7) 70.6 3.41, t (9.7) 67.6 3.79, m 17.6 1.09, d (6.5)
1 2 3 4 5 6
β-D-Glc 105.0 75.2 75.5 69.5 76.3 61.4
β-D-Qui (at C-3rha) 104.5 4.33, d (7.2) 73.9 3.07, dd (7.8, 9.6) 76.4 3.37, t (9.6) 74.8 2.79, t (9.6) 71.6 3.15, m 17.5 1.14, d (6.4)
β-D-Qui (at C-3rha) 104.5 4.34, d (7.2) 74.7 3.08, dd (7.8, 9.6) 76.4 3.36, t (9.6) 74.9 2.79, t (9.6) 71.6 3.15, m 17.6 1.15, d (6.4)
β-D-Glc III (at C-28) 93.7 5.21, d (7.9) 71.6 3.09, dd (7.5, 9.0) 76.0 3.22, t (9.0) 68.8 3.18, t (9.0) 76.5 3.35, ddd (3.5, 4.5, 9.0) 59.4 3.61, dd(3.5, 12) 3.45, dd (4.5, 12)
β-D-Glc III (at C-28) 93.8 5.21, d (7.8) 71.6 3.12, dd (7.5, 9.0) 75.0 3.21, t (9.0) 69.0 3.17, t (9.0) 76.4 3.36, ddd (3.5, 4.5, 9.0) 67.2 3.90, dd(3.5, 12) 3.64, dd (4.5, 12) β-D-Glc IV (at C-6glcIII) 102.3 4.27, d (7.8) 73.6 2.98, dd (7.5, 9.2) 75.8 3.24, t (9.2) 76.4 3.36, t (9.0) 75.8 3.10, ddd (3.5, 4.5, 9.0) 59.7 3.58, dd (3.5, 12.0) 3.44, dd (4.0, 12.0) α-L-Rha (at C-4glcIV) 100.3 4.70, d (1.2) 70.4 3.59, dd (1.2, 3.2) 71.6 3.15, dd (3.2, 9.7) 70.6 3.41, t (9.7) 68.4 3.85, m 17.6 1.10, d (6.5)
1 2 3 4 5 6
4.32, d (3.0) 3.66, d (8.5, 3.0) 3.72, dd (8.5, 3.7) 3.63, m 3.64, dd (11.5, 2.0) 3.32, dd (11.5, 2.0) I (at C-2ara) 4.62, d (7.8) 3.39, dd (7.5, 9.0) 3.58, t (9.0) 3.52, t (9.0) 3.84, ddd (3.5, 4.5, 9.0) 3.69, dd (3.5, 12.0) 3.47, dd (4.5, 12.0) II (at C-2glcI) 4.31, d (7.5) 3.01, dd (7.5, 9.2) 3.17, t (9.2) 3.13, t (9.0) 3.04, ddd (3.5, 4.5, 9.0) 3.63, dd (3.5, 12.0) 3.36, dd (4.0, 12.0)
β-D-Glc 101.0 83.2 75.0 69.4 76.7 60.9 β-D-Glc 104.2 74.6 75.6 69.0 75.4 60.9
93.6 71.8 75.0 69.0 76.1 67.4
102.2 73.5 76.0 76.1 76.0 60.1
100.2 70.3 71.7 70.3 68.3 17.6
4.46, d (3.0) 3.70, d (8.5, 3.0) 3.73, dd (8.5, 3.7) 3.68, m 3.65, dd (11.5, 2.0) 3.31, dd (11.5, 2.0) I (at C-2ara) 4.61, d (7.8) 3.17, dd (7.5, 9.0) 3.21, t (9.0) 3.10, t (9.0) 3.10, ddd (3.5, 4.5, 9.0) 3.70, dd (3.5, 12.0) 3.46, dd (4.5, 12.0) II (at C-2glcI) 4.38, d (7.2) 3.01, dd (7.5, 9.2) 3.16, t (9.2) 3.16, t (9.0) 3.06, ddd (3.5, 4.5, 9.0) 3.70, dd (3.5, 12.0) 3.46, dd (4.0, 12.0) β-D-Glc III (at C-28) 5.21, d (7.5) 3.13, dd (7.5, 9.0) 3.21, t (9.0) 3.16, t (9.0) 3.36, ddd (3.5, 4.5, 9.0) 3.89, dd(3.5, 12) 3.61, dd (4.5, 12) β-D-Glc IV (at C-6glcIII) 4.27, d (7.5) 2.99, dd (7.5, 9.2) 3.20, t (9.2) 3.36, t (9.0) 3.08, ddd (3.5, 4.5, 9.0) 3.59, dd (3.5, 12.0) 3.46, dd (4.0, 12.0) α-L-Rha (at C-4glcIV) 4.69, d (1.2) 3.59, dd (1.2, 3.2) 3.18, dd (3.2, 9.7) 3.40, t (9.7) 3.85, m 1.06, d (6.5)
rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid (8) (Frolova and Ovodov, 1971), 3-O-[β-D-ribopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid (9) (Shao et al., 1996) and a steroid glucoside, stigmast-5-en-3-O-β-D-glucopyranoside (10) (Khan et al., 2010) were isolated.
J = 7.2 Hz), 4.27 (d, J = 7.5 Hz) and 4.27 (d, J = 3.0 Hz) which showed correlations in the HSQC spectrum with the anomeric carbon signals at δ 93.8, 99.3, 100.3, 104.5, 102.3 and 103.9, respectively. 1D and 2D NMR data of sugar portion of 4, showed that it differed from 3 only by the presence of an additional β-glucopyranosyl and α-rhamnopyranosyl units, which were located at C-6glcI and C-4glcII on the basis of the HMBC correlation between the proton signal at δ 4.27 (H-1glcII) and the carbon resonance at δ 67.2 (C-6glcI), the proton signal at δ 4.70 (H-1rha) and the carbon resonance at δ 76.4 (C-4glcII). Therefore, compound 4 was identified as 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-Lrhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid. Additionally, five known oleanane-type glycosides, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl] oleanolic acid (5) (Li et al., 1995), 3-O-[β-D-ribopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)α-L-arabinopyranosyl] oleanolic acid (6) (Shao et al., 1995), 3-O-[β-Dribopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-β-D-glucopyranosyl oleanolic acid (7) (Shao et al., 1995), 3-O-[α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L-
3. Experimental 3.1. General Optical rotations were measured on an Autopol I polarimeter in MeOH at 23.5 °C. FTIR spectra were recorded on a Perkin Elmer Spectrum 100 series. NMR experiments were recorded on Agilent 600 MHz Premium COMPACT NMR spectrometer (600 MHz, in DMSO with Tetra Methyl Silane (TMS) as internal standart; Agilent, USA). HPLC-TOF/MS analysis was performed on an Agilent 6210 TOF LC–MS instrument. Silica gel 60 (0.063–0.200 mm, Merck) was used for column 131
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
3.6. 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-αoleanolic acid (3)
chromatography. For TLC analyses, silica gel 60 F254 (Merck) plates were used. Compounds were detected by UV and by spraying with 20% H2SO4/water, followed by heating at 105 °C for 1–2 min.
L-arabinopyranosyl]-28-O-[β-D-glucopyranosyl]
Amorphous white solid; C53H86O20; [α]25D + 20.2° (c 0.1 MeOH); IR νKBrmax cm−1: 3445 (> OH), 2955 (> CH), 1680 (C]O), 1655 (C]C); for 1H and 13C NMR (DMSO, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M+Na]+ m/z 1065.4265 (calcd for C65H106O31Na, 1065.4250).
3.2. Plant material N. arvensis var. involucrata was collected from Tire-Güme Mountain, from altitude of 800 m, Tire-İzmir, Turkey in July 2012. Samples of plant material were identified by Dr. Volkan Eroğlu (Deparment of Biology, Faculty of Sciences, Ege University, İzmir, Turkey). Voucher specimen (EGE 42516) has been deposited in the Herbarium of Ege University, İzmir, Turkey.
3.7. 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-αL-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid (4)
3.3. Extraction and isolation Amorphous white solid; C53H86O20; [α]25D + 10.5 (c 0.1 MeOH); IR νKBrmax cm−1: 3445 (> OH), 2945 (> CH), 1675 (C]O), 1645 (C]C); for 1H and 13C NMR (DMSO, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M +Na]+ m/z 1373.3483 (calcd for C65H106O31Na, 1373.3455).
The air-dried and powdered plant material of N. arvensis var. involucrata (2000 g) was extracted with MeOH (3 × 4 l). After filtration, the solvent was removed by rotary evaporation to give crude extract (412 g). The residue was dissolved in water and then partitioned successively with n-Hexane (3 × 300 ml), CH2Cl2 (3 × 300 ml), and nBuOH saturated with H2O (6 × 300 ml). The n-BuOH extract (68 g) was subjected to vacuum liquid chromatography (VLC) using reversed-phase material (Lichroprep RP-18, 25–40 μm, 70 g) employing with H2O (1400 ml), H2O-MeOH (8:2, 1400 ml; 6:4, 800 ml; 4:6, 7800 ml; 2:8, 8000 ml) and MeOH (2200 ml) to give ten main fractions (A–J). Fraction B (900 mg) was submitted to silica gel (60 g) column chromatography with the solvent system CHCl3–MeOH–H2O (80:20:2, 600 ml; 70:30:3, 800 ml) yielding 2 (10 mg) and 10 subfractions. Subfraction 3 (90 mg) was applied to a reversed phase column (Lichroprep RP-18, 25–40 μm, 30 g) using MeOH–H2O (6:4, 500 ml) to give 4 (4 mg) and 9 (15 mg). Fraction D (600 mg) was submitted to silica gel (60 g) column chromatography with the solvent system CHCl3-MeOH-H2O (80:20:2, 500 ml; 70:30:3, 600 ml) yielding 3 (10 mg) and 8 (15 mg). Fraction E (800 mg) was subjected to silica gel (100 g) column chromatography with the solvent system CHCl3–MeOH–H2O (80:20:2, 500 ml; 70:30:3, 700 ml) to yield 8 subfractions and yielding 1 (10 mg). Subfraction 5 (80 mg) was chromatographed on a reversed phase material (Lichroprep RP-18, 25–40 μm, 20 g), employing MeOH–H2O (6:4, 500 ml) yielding 7 (10 mg). Fraction F (300 mg) was subjected to silica gel (30 g) column chromatography. Elution was carried out with CHCl3–MeOH–H2O (80:20:2, 500 ml) to give 5 (3 mg) and 7 subfraction. Subfraction 3 (70 mg) was purified on a reversed phase column (Lichroprep RP-18, 25–40 μm, 20 g) and eluted with MeOH–H2O (8:2, 500 ml) to give 10 (9.6 mg). Subfraction 6 (50 mg) was subjected to a reversed phase column (Lichroprep RP-18, 25–40 μm, 15 g) employing MeOH–H2O (6:4, 750 ml) to afford 6 (12 mg).
3.8. Acid hydrolysis A mixture of compounds 1 (10 mg), 2 (10 mg), 3 (10 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 L-arabinose [α]20D +40 (c 0.1), 20 20 D-glucose [α] D + 20 (c 0.1), L-rhamnose [α] D + 13 (c 0.1) and D20 quinovose [α] D + 26 (c 0.1) (Gülcemal et al., 2014a, 2014b). Acknowledgements The authors are grateful to Ege University Research Foundation (2014 Fen 057) for the financial support and also Dr. Volkan Eroğlu for the identification of plant material. References Al-Sheddi, E.S., Farshori, N.N., Al-Oqail, M.M., Musarrat, J., AlKhedhairy, A.A., Siddiqui, M.A., 2014. Cytotoxicity of Nigella sativa seed oil and extract against human lung cancer cell line. Asian Pac. J. Cancer Prev. 15, 983–987. Ali, Z., Ferreira, D., Carvalho, P., Avery, M.A., Khan, I.A., 2008. Nigellidine-4-O-sulfite, the first sulfated indazole-type alkaloid from the seeds of Nigella sativa. J. Nat. Prod. 71, 1111–1112. Asgary, S., Ghannadi, A., Dashti, G., Helalat, A., Sahebkar, A., Najafi, S., 2013. Nigella sativa L. improves lipid profile and prevents atherosclerosis: evidence from an experimental study on hypercholesterolemic rabbits. J. Funct. Foods 5, 228–234. Atia, F., Mountian, I., Simaels, J., Waelkens, E., Van Driessche, W., 2002. Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine. J. Exp. Biol. 205, 3729–3737. Atta-ur-Rahman, Malik, S., He, C.-H., Clardy, J., 1985. Isolation and structure determination of nigellicine, a novel alkaloid from the seeds of Nigella sativa. Tetrahedron Lett. 26, 2759–2762. Davis, P.H., 1965. Flora of Turkey and the East Aegean Islands, vol. 6. University Press, Edinburgh, pp. 98–105. Davis, P.H., 1988. Flora of Turkey and the East Aegean Islands, vol. 10 University Press, Edinburgh pp. 294, 405–406. Dollah, M.A., Parhizkar, S., Izwan, M., 2013. Effect of Nigella sativa on the kidney function in rats. Avicenna J. Phytomed. 3, 152–158. Elbandy, M., Kang, O.H., Kwon, D.Y., Rho, J.R., 2009. Two new antiinflammatory Triterpene saponins from the Egyptian medicinal food black cumin (seeds of Nigella sativa). Bull. Korean Chem. Soc. 30, 1811–1816. Fatima, A., Mouintain, I., Simaels, J., Waelkens, E., Driessche, V.W., 2002. Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine. J. Exp. Biol. 205, 3729–3737. Frolova, G.M., Ovodov, Y.S., 1971. Triterpenoid glycosides of Eleutherococcus senticosus
3.4. 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-α-Larabinopyranosyl] oleanolic acid. (1) Amorphous white solid; C47H76O17; [α]25D + 15.8° (c 0.1 MeOH); IR νKBrmax cm−1: 3420 (> OH), 2930 (> CH), 1680 (C]O), 1655 (C]C); for 1H and 13C NMR (DMSO, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M+Na]+ m/z 935.2875 (calcd for C47H76O17Na, 935.2860). 3.5. 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-α-Larabinopyranosyl]-28-O-[α-L rhamnopyranosyl (1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid (2) Amorphous white solid; C65H106O31; [α]25D + 8.5 (c 0.1 MeOH); IR ν max cm−1: 3450 (> OH), 2950 (> CH), 1685 (C]O), 1650 (C]C); for 1H and 13C NMR (DMSO, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M +Na]+ m/z 1405.5049 (calcd for C65H106O31Na, 1405.5033). KBr
132
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
1175–1179. Mehta, B.K., Sharma, U., Agrawal, S., Pandit, V., Joshi, N., Gupta, M., 2008. Isolation and characterization of new compounds from seeds of Nigella sativa. Med. Chem. Res. 17, 462–473. Nicolis, E., Lampronti, I., Borgatti, M., Guerrini, A., Mancini, I., Sacchetti, G., Bezzerri, V., Dechecchi, M., Tamanini, A., Gambari, R., Cabrini, G., 2010. Beta-sitosterol, identified from Nigella arvensis, inhibits the induction of IL-8 gene in brnnchial epithelial cells. Pediatr. Pulmonol. 33, 276. Oshchepkova, Y.I., Rogozhin, E.A., Veshkurova, O.N., Egorov, T.A., Salikhov, S.I., Grishin, E.V., 2009. Isolation of cysteine-rich peptides from Nigella sativa sprouts. Chem. Nat. Compd. 45, 690–692. Rakhshandeh, H., Vahdati-Mashhadian, N., Khajekaramadini, M., 2011. In vitro and in vivo study of the antibacterial effects of Nigella sativa methanol extract in dairy cow mastitis. Avicenna J. Phytomed. 1, 29–35. Randhawa, M.A., Alghamdi, M.S., 2011. Anticancer activity of Nigella sativa (black seed). Am. J. Chin. Med. 39, 1075–1091. Senel, G., Gulcemal, D., Masullo, M., Piacente, S., Karayildirim, T., 2014. Oleanane-type glycosides from Tremastelma palaestinum (L.). Janchen 11, 408–418. Shao, B., Qin, G., Xu, R., Wu, H., Ma, K., 1995. Triterpenoid saponins from Clematis chinensis. Phytochemistry 38, 1473–1479. Shao, B., Qin, G., Xu, R., Wu, H., Ma, K., 1996. Saponins from Clematis chinensis. Phytochemistry 42, 821–825. Taskin, M.K., Caliskan, O.A., Anil, H., Abou-Gazar, H., Khan, I.A., Bedir, E., 2005. Triterpene saponins from Nigella sativa L. Turk. J. Chem. 29, 561–569. Xin, X., Yang, Y., Zhong, J., Aisa, H.A., Wang, H., 2009. Preparative isolation and purification of isobenzofuranone derivatives and saponins from seeds of Nigella glandulifera Freyn by high-speed counter-current chromatography combined with gel filtration. J. Chromatogr. A 1216, 4258–4262.
leaes. Structure of eleutherosides I, K, l and M. Khim. Prir. Soedin. 618–622. Gülcemal, D., Masullo, M., Senol, S.G., Alankuş-Çalişkan, O., Piacente, S., 2014a. Saponins from Cephalaria aristata C. Koch. Rec. Nat. Prod. 8, 228–233. Gülcemal, D., Masullo, M., Alankuş-Çalişkan, O., Piacente, S., 2014b. Oleanane type glycosides from Paronychia anatolica subsp. Balansae. Fitoterapia 92, 274–279. Hadjzadeh, M.A., Rad, A.K., Rajaei, Z., Tehranipour, M., Monavar, N., 2011. The preventive effect of N-butanol fraction of Nigella sativa on ethylene glycol-induced kidney calculi in rats. Pharmacogn. Mag. 7, 338–343. Hai, W., Cheng, H., Zhao, M., Wang, Y., Hong, L., Tang, H., Tian, X., 2012. Two new cytotoxic triterpenoid saponins from the roots of Clematis argentilucida. Fitoterapia 83, 759–764. Jrah-Harzallah, H., Ben-Hadj-Khalifa, S., Maloul, A., El-Ghali, R., Mahjoub, T., 2013. Thymoquinone effects on DMH-induced erythrocyte oxidative stress and haematological alterations during colon cancer promotion in rats. J. Funct. Foods 5, 1310–1316. Khan, Z., Ali, M., Bagri, P., 2010. A new steroidal glycoside and fatty acid esters from the stem bark of Tectona grandis Linn. Nat. Prod. Res. 24, 1059–1068. Kumara, S.S.M., Huat, B.T.K., 2001. Extraction, isolation and characterisation of antitumor principle alpha-hederin, from the seeds of Nigella sativa. Planta Med. 67, 29–32. Landa, P., Marsik, P., Vanek, T., Rada, V., Kokoska, L., 2006. In vitro anti-microbial activity of extracts from the callus cultures of some Nigella species. Biologia (Bratislava) 61, 285–288. Landa, P., Marsik, P., Havlik, J., Kloucek, P., Vanek, T., Kokoska, L., 2009. Evaluation of antimicrobial and anti-inflammatory activities of seed extracts from six Nigella species. J. Med. Food 12, 408–415. Li, X.C., Yang, C.R., Liu, Y.Q., Kasai, R., Ohtani, K., Yamasaki, K., Miyahara, K., Shingu, K., 1995. Triterpenoid glycosides from Anemoclema glaucifolium. Phytochemistry 39,
133
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Novel saponins from Nigella arvensis var. involucrata Kenan Bıçak , Derya Gülcemal , İbrahim Demirtaş a
a b
a
b,⁎
, Özgen Alankuş
a,⁎
MARK
Department of Chemistry, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey Department of Chemistry, Faculty of Science, Çankırı Karatekin University, Çankırı, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords: Nigella arvensis Ranunculaceae Oleanane Saponins
Four new oleanane-type triterpene glycosides namely 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl(1 → 2)-α-L-arabinopyranosyl] oleanolic acid, 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-α-Larabinopyranosyl]-28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid, 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[β-Dglucopyranosyl] oleanolic acid, 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid, were isolated from methanol extract of the roots of Nigella arvensis var. involucrata along with five known oleanane-type triterpene glycosides and a steroid glucoside. Structures of the new compounds were established as by using 1D and 2D NMR techniques and mass spectrometry.
1. Introduction The genus Nigella, a member of Ranunculaceae family, is represented by 13 species in the flora of Turkey (Davis, 1965, 1988). Some Nigella species (e.g. Nigella sativa L., Nigella damascena L. and Nigella arvensis L.) have been in use in many Middle Eastern and Far Eastern countries as a natural remedy for over 2000 years (Fatima et al., 2002). The extract of N. sativa seeds and its constituents could be used to dissolve kidney stones (Hadjzadeh et al., 2011; Dollah et al., 2013;), antimicrobial and anti-inflammatory activities (Landa et al., 2009; Rakhshandeh et al., 2011; Randhawa and Alghamdi, 2011), inhibit the carcinogenic process (Al-Sheddi et al., 2014; Randhawa and Alghamdi, 2011), antiatherogenic cardio-protective (Asgary et al., 2013), and antioxidant effects (Jrah-Harzallah et al., 2013). Previous phytochemical investigations on the genus Nigella led to the isolation of alkaloids (Atta-ur-Rahman et al., 1985; Ali et al., 2008), oleanane-type saponins (Kumara and Huat, 2001; Taskin et al., 2005; Elbandy et al., 2009; Xin et al., 2009), cycloartane-type saponins (Mehta et al., 2008), benzofurans (Xin et al., 2009) and peptides (Oshchepkova et al., 2009). A phytochemical work performed on N. arvensis revealed beta-sitosterol as major metabolite. Additionally, beta-sitosterol was evaluated for inhibition and induction of IL-8 gene in bronchial epithelial cells (Nicolis et al., 2010). Pharmacological investigations on the extracts of N. arvensis exhibited, antimicrobial (Landa et al., 2006, 2009) and antiinflammatory activities (Landa et al., 2009). The seed extract was also found to have a stimulatory effect on Na+ transport in renal epithelia (Atia et al., 2002). ⁎
Therefore, the phytochemical investigation of the MeOH extract of the roots of N. arvensis var. involucrata was carried out. Four oleananetype triterpene glycosides (1–4) (Fig. 1), never reported before, along with five known oleanane-type triterpene glycosides and a steroid glucoside (5–10) were isolated and their structure were established by the extensive use of 1D and 2D NMR experiments and HRMS analysis. 2. Results and discussion The HRMALDITOF mass spectrum of 1 (m/z 935.2875 [M + Na]+, calcd for C47H76O17Na, 935.2860) supported a molecular formula of C47H76O17. A detailed comparison of the NMR data (1H, 13C, HSQC, HMBC, COSY) of compounds 1–4 showed that the aglycone moiety was identical in all the four compounds. In particular, the 1H NMR spectrum of 1 showed signals for seven methyl groups as singlets at δ 1.08, 0.95, 0.87 (6H), 0.86, 0.76 and 0.70, one olefinic proton at δ 5.16 (H-12, t, J = 3.5 Hz) and one oxygen-bearing methine protons at δ 2.99 (H-3, dd, J = 11.5, 4.2 Hz). These signals, along with the resonances in the 13 C NMR spectrum for the Me groups at δ 32.6, 27.2, 25.4, 23.2, 16.6, 16.1, 15.0, of the two sp2-hybridized carbons at δ 121.3 and 144.0, for one carbinol carbons (δ 88.4), evidenced that compound 1 possessed an olean-12-ene skeleton as aglycone. As a result, the aglycone was identified as oleanolic acid (Senel et al., 2014). The downfield shift of C-3 (δ 88.4) of the aglycone indicated that compound 1 was a monodesmosidic glycoside (Hai et al., 2012). The 13C NMR spectrum showed 47C-atom signals of which 30 were assigned to the aglycone moiety
Corresponding authors. E-mail addresses: [email protected] (İ. Demirtaş), [email protected] (Ö. Alankuş).
http://dx.doi.org/10.1016/j.phytol.2017.06.002 Received 28 February 2017; Received in revised form 30 May 2017; Accepted 5 June 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
Fig. 1. Structures of compounds 1–4.
correlated by HSQC experiment to the corresponding carbon resonances at δ 101.1, 103.7, and 105.0, respectively. The HSQC, HMBC, DQF-COSY and 1D-TOCSY data led to identify these sugar units as βglucopyranosyl, and α-arabinopyranosyl units. The HMBC correlations
(Table 1) and 17 to a sugar portion. The 1H NMR spectrum displayed in the sugar region signals corresponding to three anomeric protons at δ 4.62 (d, J = 7.8 Hz), 4.32 (d, J = 3.0 Hz), 4.31 (d, J = 7.5 Hz), which were unambiguously 129
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
Table 1 13 C and 1H NMR data (J in Hz) of the aglycone moieties of compounds 1–4 (600 MHz, δ ppm, in DMSO). 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)
38.0 25.5 88.4 38.5 55.4 17.6 32.2 40.6 47.1 35.0 22.8 121.3 144.0 41.6 27.0 22.7 47.6 40.8 45.6 31.0 33.0 32.0 27.2 16.1 15.0 16.6 25.4 179.0 32.6 23.2
1.49, 0.92, m 1.71, 1.55, m 2.99, dd (11.5, 4.2) – 0.72, m 1.46, 1.31 1.59,1.40, m – 1.48, m – 1.79, (2H) m 5.16, t (3.5) – – 1.65, 0.99, m 1.79, 1.48, m – 2.74, m 1.60, 1.04, m – 1.30, 1.12, m 1.40, 1.21, m 0.95, s 0.70, s 0.87, s 0.76, s 1.08, s – 0.86, s 0.86, s
37.5 24.5 88.4 38.0 55.5 17.5 32.0 40.5 47.5 35.4 22.0 121.3 144.0 41.5 27.4 22.5 47.0 40.6 45.9 31.4 33.5 32.6 27.2 16.1 15.0 16.6 25.4 174.8 32.6 23.2
1.49, 0.92, m 1.71, 1.55, m 2.99, dd (11.5, 4.2) – 0.72, m 1.46, 1.31 1.59,1.40, m – 1.48, m – 1.79, (2H) m 5.16, t (3.5) – – 1.65, 0.99, m 1.79, 1.48, m – 2.74, m 1.60, 1.04, m – 1.30, 1.12, m 1.40, 1.21, m 0.95, s 0.70, s 0.87, s 0.76, s 1.08, s – 0.86, s 0.86, s
38.2 25.6 87.5 38.6 55.5 17.5 32.5 40.0 47.8 35.3 22.7 121.3 144.0 41.0 27.5 22.8 47.4 40.7 45.8 31.6 33.3 32.5 27.2 16.1 15.0 16.6 25.4 175.0 32.6 23.2
1.49, 0.92, m 1.71, 1.55, m 2.99, dd (11.5, 4.2) – 0.72, m 1.46, 1.31 1.59,1.40, m – 1.48, m – 1.79, (2H) m 5.16, t (3.5) – – 1.65, 0.99, m 1.79, 1.48, m – 2.74, m 1.60, 1.04, m – 1.30, 1.12, m 1.40, 1.21, m 0.95, s 0.70, s 0.87, s 0.76, s 1.08, s – 0.86, s 0.86, s
37.8 25.9 88.4 38.8 55.3 17.8 32.4 40.8 47.4 35.3 22.9 121.3 144.0 41.5 27.6 22.8 47.5 40.9 45.5 31.3 33.3 32.6 27.2 16.1 15.0 16.6 25.4 174.9 32.6 23.2
1.49, 0.92, m 1.71, 1.55, m 2.99, dd (11.5, 4.2) – 0.72, m 1.46, 1.31 1.59,1.40, m – 1.48, m – 1.79, (2H) m 5.16, t (3.5) – – 1.65, 0.99, m 1.79, 1.48, m – 2.74, m 1.60, 1.04, m – 1.30, 1.12, m 1.40, 1.21, m 0.95, s 0.70, s 0.87, s 0.76, s 1.08, s – 0.86, s 0.86, s
between the proton signal at δ 4.32 (H-1ara) and the carbon resonance at δ 88.4 (C-3), the proton signal at δ 4.62 (H-1glcI) and the carbon resonance at δ 77.1 (C-2ara) and the proton signal at δ 4.31 (H-1glcII) and the carbon resonance at δ 84.6 (C-2glcI) allowed us to determine the linkage site of the sugar units. The acid hydrolysis of 1 afforded Dglucose and L-arabinose, confirmed by the optical rotation data of each isolated sugar (Gülcemal et al., 2014a, 2014b). Thus, the new compound 1 was elucidated as 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl] oleanolic acid. The HRMALDITOF mass spectrum of 2 (m/z 1405.5049 [M + Na]+, calcd for C65H106O31Na, 1405.5033) supported a molecular formula of C65H106O31. With respect to the sugar portion, the occurrence of three additional sugar units was observed compared to 1. The 1H NMR spectrum displayed in the sugar region signals corresponding to six anomeric Hatoms at δ 5.21 (d, J = 7.5), 4.69 (d, J = 1.2), 4.61 (d, J = 7.8), 4.46 (d, J = 3.0), 4.38 (d, J = 7.2) and 4.27 (d, J = 7.5) which showed correlations in the HSQC spectrum with the anomeric C-atom signals at δ 93.6, 100.2, 101.0, 102.6, 104.2 and 102.2, respectively. A detailed analysis of NMR data revealed the presence of four β-glucopyranosyl units (δ 5.21, 4.61, 4.38, and 4.27), one α-rhamnopyranosyl unit (δ 4.69), and one α-arabinopyranosyl unit (δ 4.46). An unambiguous determination of the sequence and linkage sites was obtained from the HMBC spectrum, which allowed us to identify at C-3 the same sugar chain as in 1, and the occurrence of a further sugar chain at C-28, as indicated by the upfield shift of C-28 at δ 174.8 when compared to compound 1. Key HMBCs between the resonance of H-1glcIII (δ 5.21) and C-28 (δ 174.8), between the resonance of H-1glcIV (δ 4.27) and C6glcIII (δ 67.4) and between the resonance of H-1rha (δ 4.69) and C-4glcIV (δ 76.1) confirmed this hypothesis. The D configuration of glucose and the L configuration of rhamnose and arabinose were confirmed by the optical rotation data of each isolated sugar (Gülcemal et al., 2014a, 2014b). On the basis of all these evidences, the structure of 2 was
established as 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L rhamnopyranosyl (1 → 4)-βD-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid. The HRMALDITOF mass spectrum of 3 (m/z 1065.4265 [M + Na]+, calcd for C53H86O20Na, 1065.4250) supported a molecular formula of C53H86O20. The 1H NMR spectrum displayed in the sugar region signals corresponding to four anomeric protons at δ 5.21 (d, J = 7.9 Hz), 5.16 (d, J = 1.2 Hz), 4.33 (d, J = 7.2 Hz) and 4.27 (d, J = 3.0 Hz), which were unambiguously correlated by HSQC experiment to the corresponding carbon resonances at δ 93.7, 99.3, 104.5 and 103.9, respectively. The chemical shifts of all the individual protons of the four sugar units were ascertained from a combination of 1D-TOCSY and DQF-COSY spectral analysis, and the 13C chemical shifts of their relative attached carbons were assigned unambiguously from the HSQC spectrum (Table 2) These data showed the presence of one β-glucopyranosyl unit (δ 5.21), one αrhamnopyranosyl unit (δ 5.16), one β-quinovopyranosyl unit (δ 4.33) and one α-arabinopyranosyl unit (δ 4.27). An unambiguous determination of the sequence and linkage sites was obtained from the HMBC spectrum, which showed key correlation peaks between the proton signal of δ H-1ara (δ 4.27) and the carbon resonance of C-3 (δ 87.5), H1rha (δ 5.16) and C-2ara (δ 73.9), H-1qui (δ 4.33) and C-3rha (δ 80.9), H1glc (δ 5.21) and the carbon resonance of C-28 (δ 175.0). The acid hydrolysis of 3 afforded D-glucose, D-quinovose, L-rhamnose and L-arabinose, confirmed by the optical rotation data of each isolated sugar (Gülcemal et al., 2014a, 2014b). Thus, compound 3 was identified as 3O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-Larabinopyranosyl]-28-O-[β-D-glucopyranosyl] oleanolic acid. The HRMALDITOF mass spectrum of 4 (m/z 1373.3483 [M + Na]+, calcd for C65H106O29Na, 1373.3455) supported a molecular formula of C65H106O29. The 1H NMR showed signals for the anomeric protons at δ 5.21 (δ, J = 7.8 Hz), 5.17 (d, J = 1.2 Hz), 4.70 (d, J = 1.2 Hz), 4.34 (d,
130
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
Table 2 13 C and 1H NMR data (J in Hz) of the sugar portions of compounds 1–4 (600 MHz, δ ppm, in DMSO). 1
2
3
4
δH (J in Hz) δC α-L-Ara (at C-3)
δC δH (J in Hz) α-L-Ara (at C-3)
δC δH (J in Hz) α-L-Ara (at C-3)
δC δH (J in Hz) α-L-Ara (at C-3)
1 2 3 4 5
103.7 77.1 71.6 66.8 63.7
102.6 77.3 70.4 65.8 61.9
103.9 73.9 72.9 67.8 64.4
103.9 73.1 73.0 67.8 64.5
1 2 3 4 5 6
β-D-Glc 101.1 84.6 75.9 69.6 77.3 60.8
4.27, d (3.0) 3.59, d (8.5, 3.0) 3.56, dd (8.5, 3.7) 3.57, m 3.65, dd (11.5, 2.0) 3.36, dd (11.5, 2.0) α-L-Rha (at C-2ara) 99.3 5.16, d (1.2) 69.7 3.81, dd (1.2, 3.2) 80.9 3.63, dd (3.2, 9.7) 70.7 3.41, t (9.7) 67.7 3.77, m 17.5 1.09, d (6.5)
4.27, d (3.0) 3.58, d (8.5, 3.0) 3.54, dd (8.5, 3.7) 3.57, m 3.66, dd (11.5, 2.0) 3.37, dd (11.5, 2.0) α-L-Rha (at C-2ara) 99.3 5.17, d (1.2) 69.7 3.81, dd (1.2, 3.2) 80.8 3.64, dd (3.2, 9.7) 70.6 3.41, t (9.7) 67.6 3.79, m 17.6 1.09, d (6.5)
1 2 3 4 5 6
β-D-Glc 105.0 75.2 75.5 69.5 76.3 61.4
β-D-Qui (at C-3rha) 104.5 4.33, d (7.2) 73.9 3.07, dd (7.8, 9.6) 76.4 3.37, t (9.6) 74.8 2.79, t (9.6) 71.6 3.15, m 17.5 1.14, d (6.4)
β-D-Qui (at C-3rha) 104.5 4.34, d (7.2) 74.7 3.08, dd (7.8, 9.6) 76.4 3.36, t (9.6) 74.9 2.79, t (9.6) 71.6 3.15, m 17.6 1.15, d (6.4)
β-D-Glc III (at C-28) 93.7 5.21, d (7.9) 71.6 3.09, dd (7.5, 9.0) 76.0 3.22, t (9.0) 68.8 3.18, t (9.0) 76.5 3.35, ddd (3.5, 4.5, 9.0) 59.4 3.61, dd(3.5, 12) 3.45, dd (4.5, 12)
β-D-Glc III (at C-28) 93.8 5.21, d (7.8) 71.6 3.12, dd (7.5, 9.0) 75.0 3.21, t (9.0) 69.0 3.17, t (9.0) 76.4 3.36, ddd (3.5, 4.5, 9.0) 67.2 3.90, dd(3.5, 12) 3.64, dd (4.5, 12) β-D-Glc IV (at C-6glcIII) 102.3 4.27, d (7.8) 73.6 2.98, dd (7.5, 9.2) 75.8 3.24, t (9.2) 76.4 3.36, t (9.0) 75.8 3.10, ddd (3.5, 4.5, 9.0) 59.7 3.58, dd (3.5, 12.0) 3.44, dd (4.0, 12.0) α-L-Rha (at C-4glcIV) 100.3 4.70, d (1.2) 70.4 3.59, dd (1.2, 3.2) 71.6 3.15, dd (3.2, 9.7) 70.6 3.41, t (9.7) 68.4 3.85, m 17.6 1.10, d (6.5)
1 2 3 4 5 6
4.32, d (3.0) 3.66, d (8.5, 3.0) 3.72, dd (8.5, 3.7) 3.63, m 3.64, dd (11.5, 2.0) 3.32, dd (11.5, 2.0) I (at C-2ara) 4.62, d (7.8) 3.39, dd (7.5, 9.0) 3.58, t (9.0) 3.52, t (9.0) 3.84, ddd (3.5, 4.5, 9.0) 3.69, dd (3.5, 12.0) 3.47, dd (4.5, 12.0) II (at C-2glcI) 4.31, d (7.5) 3.01, dd (7.5, 9.2) 3.17, t (9.2) 3.13, t (9.0) 3.04, ddd (3.5, 4.5, 9.0) 3.63, dd (3.5, 12.0) 3.36, dd (4.0, 12.0)
β-D-Glc 101.0 83.2 75.0 69.4 76.7 60.9 β-D-Glc 104.2 74.6 75.6 69.0 75.4 60.9
93.6 71.8 75.0 69.0 76.1 67.4
102.2 73.5 76.0 76.1 76.0 60.1
100.2 70.3 71.7 70.3 68.3 17.6
4.46, d (3.0) 3.70, d (8.5, 3.0) 3.73, dd (8.5, 3.7) 3.68, m 3.65, dd (11.5, 2.0) 3.31, dd (11.5, 2.0) I (at C-2ara) 4.61, d (7.8) 3.17, dd (7.5, 9.0) 3.21, t (9.0) 3.10, t (9.0) 3.10, ddd (3.5, 4.5, 9.0) 3.70, dd (3.5, 12.0) 3.46, dd (4.5, 12.0) II (at C-2glcI) 4.38, d (7.2) 3.01, dd (7.5, 9.2) 3.16, t (9.2) 3.16, t (9.0) 3.06, ddd (3.5, 4.5, 9.0) 3.70, dd (3.5, 12.0) 3.46, dd (4.0, 12.0) β-D-Glc III (at C-28) 5.21, d (7.5) 3.13, dd (7.5, 9.0) 3.21, t (9.0) 3.16, t (9.0) 3.36, ddd (3.5, 4.5, 9.0) 3.89, dd(3.5, 12) 3.61, dd (4.5, 12) β-D-Glc IV (at C-6glcIII) 4.27, d (7.5) 2.99, dd (7.5, 9.2) 3.20, t (9.2) 3.36, t (9.0) 3.08, ddd (3.5, 4.5, 9.0) 3.59, dd (3.5, 12.0) 3.46, dd (4.0, 12.0) α-L-Rha (at C-4glcIV) 4.69, d (1.2) 3.59, dd (1.2, 3.2) 3.18, dd (3.2, 9.7) 3.40, t (9.7) 3.85, m 1.06, d (6.5)
rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid (8) (Frolova and Ovodov, 1971), 3-O-[β-D-ribopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid (9) (Shao et al., 1996) and a steroid glucoside, stigmast-5-en-3-O-β-D-glucopyranoside (10) (Khan et al., 2010) were isolated.
J = 7.2 Hz), 4.27 (d, J = 7.5 Hz) and 4.27 (d, J = 3.0 Hz) which showed correlations in the HSQC spectrum with the anomeric carbon signals at δ 93.8, 99.3, 100.3, 104.5, 102.3 and 103.9, respectively. 1D and 2D NMR data of sugar portion of 4, showed that it differed from 3 only by the presence of an additional β-glucopyranosyl and α-rhamnopyranosyl units, which were located at C-6glcI and C-4glcII on the basis of the HMBC correlation between the proton signal at δ 4.27 (H-1glcII) and the carbon resonance at δ 67.2 (C-6glcI), the proton signal at δ 4.70 (H-1rha) and the carbon resonance at δ 76.4 (C-4glcII). Therefore, compound 4 was identified as 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-Lrhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl(1 → 4)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid. Additionally, five known oleanane-type glycosides, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl] oleanolic acid (5) (Li et al., 1995), 3-O-[β-D-ribopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)α-L-arabinopyranosyl] oleanolic acid (6) (Shao et al., 1995), 3-O-[β-Dribopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-β-D-glucopyranosyl oleanolic acid (7) (Shao et al., 1995), 3-O-[α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-28-O-[α-L-
3. Experimental 3.1. General Optical rotations were measured on an Autopol I polarimeter in MeOH at 23.5 °C. FTIR spectra were recorded on a Perkin Elmer Spectrum 100 series. NMR experiments were recorded on Agilent 600 MHz Premium COMPACT NMR spectrometer (600 MHz, in DMSO with Tetra Methyl Silane (TMS) as internal standart; Agilent, USA). HPLC-TOF/MS analysis was performed on an Agilent 6210 TOF LC–MS instrument. Silica gel 60 (0.063–0.200 mm, Merck) was used for column 131
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
3.6. 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-αoleanolic acid (3)
chromatography. For TLC analyses, silica gel 60 F254 (Merck) plates were used. Compounds were detected by UV and by spraying with 20% H2SO4/water, followed by heating at 105 °C for 1–2 min.
L-arabinopyranosyl]-28-O-[β-D-glucopyranosyl]
Amorphous white solid; C53H86O20; [α]25D + 20.2° (c 0.1 MeOH); IR νKBrmax cm−1: 3445 (> OH), 2955 (> CH), 1680 (C]O), 1655 (C]C); for 1H and 13C NMR (DMSO, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M+Na]+ m/z 1065.4265 (calcd for C65H106O31Na, 1065.4250).
3.2. Plant material N. arvensis var. involucrata was collected from Tire-Güme Mountain, from altitude of 800 m, Tire-İzmir, Turkey in July 2012. Samples of plant material were identified by Dr. Volkan Eroğlu (Deparment of Biology, Faculty of Sciences, Ege University, İzmir, Turkey). Voucher specimen (EGE 42516) has been deposited in the Herbarium of Ege University, İzmir, Turkey.
3.7. 3-O-[β-D-quinovopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-αL-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl(1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid (4)
3.3. Extraction and isolation Amorphous white solid; C53H86O20; [α]25D + 10.5 (c 0.1 MeOH); IR νKBrmax cm−1: 3445 (> OH), 2945 (> CH), 1675 (C]O), 1645 (C]C); for 1H and 13C NMR (DMSO, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M +Na]+ m/z 1373.3483 (calcd for C65H106O31Na, 1373.3455).
The air-dried and powdered plant material of N. arvensis var. involucrata (2000 g) was extracted with MeOH (3 × 4 l). After filtration, the solvent was removed by rotary evaporation to give crude extract (412 g). The residue was dissolved in water and then partitioned successively with n-Hexane (3 × 300 ml), CH2Cl2 (3 × 300 ml), and nBuOH saturated with H2O (6 × 300 ml). The n-BuOH extract (68 g) was subjected to vacuum liquid chromatography (VLC) using reversed-phase material (Lichroprep RP-18, 25–40 μm, 70 g) employing with H2O (1400 ml), H2O-MeOH (8:2, 1400 ml; 6:4, 800 ml; 4:6, 7800 ml; 2:8, 8000 ml) and MeOH (2200 ml) to give ten main fractions (A–J). Fraction B (900 mg) was submitted to silica gel (60 g) column chromatography with the solvent system CHCl3–MeOH–H2O (80:20:2, 600 ml; 70:30:3, 800 ml) yielding 2 (10 mg) and 10 subfractions. Subfraction 3 (90 mg) was applied to a reversed phase column (Lichroprep RP-18, 25–40 μm, 30 g) using MeOH–H2O (6:4, 500 ml) to give 4 (4 mg) and 9 (15 mg). Fraction D (600 mg) was submitted to silica gel (60 g) column chromatography with the solvent system CHCl3-MeOH-H2O (80:20:2, 500 ml; 70:30:3, 600 ml) yielding 3 (10 mg) and 8 (15 mg). Fraction E (800 mg) was subjected to silica gel (100 g) column chromatography with the solvent system CHCl3–MeOH–H2O (80:20:2, 500 ml; 70:30:3, 700 ml) to yield 8 subfractions and yielding 1 (10 mg). Subfraction 5 (80 mg) was chromatographed on a reversed phase material (Lichroprep RP-18, 25–40 μm, 20 g), employing MeOH–H2O (6:4, 500 ml) yielding 7 (10 mg). Fraction F (300 mg) was subjected to silica gel (30 g) column chromatography. Elution was carried out with CHCl3–MeOH–H2O (80:20:2, 500 ml) to give 5 (3 mg) and 7 subfraction. Subfraction 3 (70 mg) was purified on a reversed phase column (Lichroprep RP-18, 25–40 μm, 20 g) and eluted with MeOH–H2O (8:2, 500 ml) to give 10 (9.6 mg). Subfraction 6 (50 mg) was subjected to a reversed phase column (Lichroprep RP-18, 25–40 μm, 15 g) employing MeOH–H2O (6:4, 750 ml) to afford 6 (12 mg).
3.8. Acid hydrolysis A mixture of compounds 1 (10 mg), 2 (10 mg), 3 (10 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 L-arabinose [α]20D +40 (c 0.1), 20 20 D-glucose [α] D + 20 (c 0.1), L-rhamnose [α] D + 13 (c 0.1) and D20 quinovose [α] D + 26 (c 0.1) (Gülcemal et al., 2014a, 2014b). Acknowledgements The authors are grateful to Ege University Research Foundation (2014 Fen 057) for the financial support and also Dr. Volkan Eroğlu for the identification of plant material. References Al-Sheddi, E.S., Farshori, N.N., Al-Oqail, M.M., Musarrat, J., AlKhedhairy, A.A., Siddiqui, M.A., 2014. Cytotoxicity of Nigella sativa seed oil and extract against human lung cancer cell line. Asian Pac. J. Cancer Prev. 15, 983–987. Ali, Z., Ferreira, D., Carvalho, P., Avery, M.A., Khan, I.A., 2008. Nigellidine-4-O-sulfite, the first sulfated indazole-type alkaloid from the seeds of Nigella sativa. J. Nat. Prod. 71, 1111–1112. Asgary, S., Ghannadi, A., Dashti, G., Helalat, A., Sahebkar, A., Najafi, S., 2013. Nigella sativa L. improves lipid profile and prevents atherosclerosis: evidence from an experimental study on hypercholesterolemic rabbits. J. Funct. Foods 5, 228–234. Atia, F., Mountian, I., Simaels, J., Waelkens, E., Van Driessche, W., 2002. Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine. J. Exp. Biol. 205, 3729–3737. Atta-ur-Rahman, Malik, S., He, C.-H., Clardy, J., 1985. Isolation and structure determination of nigellicine, a novel alkaloid from the seeds of Nigella sativa. Tetrahedron Lett. 26, 2759–2762. Davis, P.H., 1965. Flora of Turkey and the East Aegean Islands, vol. 6. University Press, Edinburgh, pp. 98–105. Davis, P.H., 1988. Flora of Turkey and the East Aegean Islands, vol. 10 University Press, Edinburgh pp. 294, 405–406. Dollah, M.A., Parhizkar, S., Izwan, M., 2013. Effect of Nigella sativa on the kidney function in rats. Avicenna J. Phytomed. 3, 152–158. Elbandy, M., Kang, O.H., Kwon, D.Y., Rho, J.R., 2009. Two new antiinflammatory Triterpene saponins from the Egyptian medicinal food black cumin (seeds of Nigella sativa). Bull. Korean Chem. Soc. 30, 1811–1816. Fatima, A., Mouintain, I., Simaels, J., Waelkens, E., Driessche, V.W., 2002. Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine. J. Exp. Biol. 205, 3729–3737. Frolova, G.M., Ovodov, Y.S., 1971. Triterpenoid glycosides of Eleutherococcus senticosus
3.4. 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-α-Larabinopyranosyl] oleanolic acid. (1) Amorphous white solid; C47H76O17; [α]25D + 15.8° (c 0.1 MeOH); IR νKBrmax cm−1: 3420 (> OH), 2930 (> CH), 1680 (C]O), 1655 (C]C); for 1H and 13C NMR (DMSO, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M+Na]+ m/z 935.2875 (calcd for C47H76O17Na, 935.2860). 3.5. 3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-α-Larabinopyranosyl]-28-O-[α-L rhamnopyranosyl (1 → 4)-β-Dglucopyranosyl-(1 → 6)-β-D-glucopyranosyl] oleanolic acid (2) Amorphous white solid; C65H106O31; [α]25D + 8.5 (c 0.1 MeOH); IR ν max cm−1: 3450 (> OH), 2950 (> CH), 1685 (C]O), 1650 (C]C); for 1H and 13C NMR (DMSO, 600 MHz) data of the aglycone moiety and the sugar portion see Tables 1 and 2, respectively; HRMALDITOFMS [M +Na]+ m/z 1405.5049 (calcd for C65H106O31Na, 1405.5033). KBr
132
Phytochemistry Letters 21 (2017) 128–133
K. Bıçak et al.
1175–1179. Mehta, B.K., Sharma, U., Agrawal, S., Pandit, V., Joshi, N., Gupta, M., 2008. Isolation and characterization of new compounds from seeds of Nigella sativa. Med. Chem. Res. 17, 462–473. Nicolis, E., Lampronti, I., Borgatti, M., Guerrini, A., Mancini, I., Sacchetti, G., Bezzerri, V., Dechecchi, M., Tamanini, A., Gambari, R., Cabrini, G., 2010. Beta-sitosterol, identified from Nigella arvensis, inhibits the induction of IL-8 gene in brnnchial epithelial cells. Pediatr. Pulmonol. 33, 276. Oshchepkova, Y.I., Rogozhin, E.A., Veshkurova, O.N., Egorov, T.A., Salikhov, S.I., Grishin, E.V., 2009. Isolation of cysteine-rich peptides from Nigella sativa sprouts. Chem. Nat. Compd. 45, 690–692. Rakhshandeh, H., Vahdati-Mashhadian, N., Khajekaramadini, M., 2011. In vitro and in vivo study of the antibacterial effects of Nigella sativa methanol extract in dairy cow mastitis. Avicenna J. Phytomed. 1, 29–35. Randhawa, M.A., Alghamdi, M.S., 2011. Anticancer activity of Nigella sativa (black seed). Am. J. Chin. Med. 39, 1075–1091. Senel, G., Gulcemal, D., Masullo, M., Piacente, S., Karayildirim, T., 2014. Oleanane-type glycosides from Tremastelma palaestinum (L.). Janchen 11, 408–418. Shao, B., Qin, G., Xu, R., Wu, H., Ma, K., 1995. Triterpenoid saponins from Clematis chinensis. Phytochemistry 38, 1473–1479. Shao, B., Qin, G., Xu, R., Wu, H., Ma, K., 1996. Saponins from Clematis chinensis. Phytochemistry 42, 821–825. Taskin, M.K., Caliskan, O.A., Anil, H., Abou-Gazar, H., Khan, I.A., Bedir, E., 2005. Triterpene saponins from Nigella sativa L. Turk. J. Chem. 29, 561–569. Xin, X., Yang, Y., Zhong, J., Aisa, H.A., Wang, H., 2009. Preparative isolation and purification of isobenzofuranone derivatives and saponins from seeds of Nigella glandulifera Freyn by high-speed counter-current chromatography combined with gel filtration. J. Chromatogr. A 1216, 4258–4262.
leaes. Structure of eleutherosides I, K, l and M. Khim. Prir. Soedin. 618–622. Gülcemal, D., Masullo, M., Senol, S.G., Alankuş-Çalişkan, O., Piacente, S., 2014a. Saponins from Cephalaria aristata C. Koch. Rec. Nat. Prod. 8, 228–233. Gülcemal, D., Masullo, M., Alankuş-Çalişkan, O., Piacente, S., 2014b. Oleanane type glycosides from Paronychia anatolica subsp. Balansae. Fitoterapia 92, 274–279. Hadjzadeh, M.A., Rad, A.K., Rajaei, Z., Tehranipour, M., Monavar, N., 2011. The preventive effect of N-butanol fraction of Nigella sativa on ethylene glycol-induced kidney calculi in rats. Pharmacogn. Mag. 7, 338–343. Hai, W., Cheng, H., Zhao, M., Wang, Y., Hong, L., Tang, H., Tian, X., 2012. Two new cytotoxic triterpenoid saponins from the roots of Clematis argentilucida. Fitoterapia 83, 759–764. Jrah-Harzallah, H., Ben-Hadj-Khalifa, S., Maloul, A., El-Ghali, R., Mahjoub, T., 2013. Thymoquinone effects on DMH-induced erythrocyte oxidative stress and haematological alterations during colon cancer promotion in rats. J. Funct. Foods 5, 1310–1316. Khan, Z., Ali, M., Bagri, P., 2010. A new steroidal glycoside and fatty acid esters from the stem bark of Tectona grandis Linn. Nat. Prod. Res. 24, 1059–1068. Kumara, S.S.M., Huat, B.T.K., 2001. Extraction, isolation and characterisation of antitumor principle alpha-hederin, from the seeds of Nigella sativa. Planta Med. 67, 29–32. Landa, P., Marsik, P., Vanek, T., Rada, V., Kokoska, L., 2006. In vitro anti-microbial activity of extracts from the callus cultures of some Nigella species. Biologia (Bratislava) 61, 285–288. Landa, P., Marsik, P., Havlik, J., Kloucek, P., Vanek, T., Kokoska, L., 2009. Evaluation of antimicrobial and anti-inflammatory activities of seed extracts from six Nigella species. J. Med. Food 12, 408–415. Li, X.C., Yang, C.R., Liu, Y.Q., Kasai, R., Ohtani, K., Yamasaki, K., Miyahara, K., Shingu, K., 1995. Triterpenoid glycosides from Anemoclema glaucifolium. Phytochemistry 39,
133