Phytochemistry xxx (2012) xxx–xxx
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Acylated pregnane glycosides from Caralluma sinaica Shaza M. Al-Massarani a, Samuel Bertrand b, Andreas Nievergelt b, Azza M. El-Shafae a, Tawfeq A. Al-Howiriny a, Nawal M. Al-Musayeib a, Muriel Cuendet b, Jean-Luc Wolfender b,⇑ a b
King Saud University, College of Pharmacy, Dept. of Pharmacognosy, P.O. Box 2457, Riyadh 11451, Saudi Arabia School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
a r t i c l e
i n f o
Article history: Received 12 January 2012 Received in revised form 2 April 2012 Accepted 5 April 2012 Available online xxxx Keywords: Caralluma sinaica Asclepiadaceae Pregnane glycosides Quinone reductase induction
a b s t r a c t Caralluma sinaica is sold on local markets of Saudi Arabia for various health benefits however no phytochemical study has specifically been performed on this species. NMR and UHPLC-ESI-TOF-MS profilings of the ethanolic extract of the whole plant reveal a very complex phytochemical composition dominated by pregnanes. Detailed information on its constituents was obtained after isolation. Six pregnane glycosides were obtained and characterized based on the extensive spectroscopic analysis (including IR, 1 H NMR, 13C NMR and MS data), in addition to ten known compounds (seven pregnanes and three flavonoids). The compounds were identified as 12b-O-benzoyl-20-O-acetyl boucerin-3-O-6-deoxy-3-Omethyl-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside, 12b-O-tigloyl20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranoside, 12b-O-benzoyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-digitalopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-Dcymaropyranoside, 12b-O-benzoyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-thevetopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside, 12b-O-benzoyl-20-O-tigloyl boucerin-3O-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranoside, 12b-20-O-dibenzoyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside. Finally, the isolated compounds were evaluated for their quinone reductase induction. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The genus Caralluma belongs to the Asclepiadaceae family, which is also known as the milkweed family because many of its members contain a milky latex (Bensuzan, 2009). Due to recent DNA analysis and morphological studies, Asclepiadaceae have been classified as a sub-group of the family Apocynaceae (Endress and Bruyns, 2000; Meve and Heneidak, 2005). Nevertheless, Asclepiadaceae is still regarded as an independent family. Plants of the genus Caralluma are perennial, small and usually leafless (Heyood, 1978; Saxena and Sarbhai, 1975). Some of these plants are edible and succulent (Marwah et al., 2007; Reddy et al., 2011). More than 200 species of the genus Caralluma grow throughout Africa and Asia (Surveswaran, 2007). The majority of these species are indigenous to the Indian sub-continent and Arabian peninsula (Gilbert, 1990). Various medicinal uses of Caralluma spp. have been reported in Arabic and Indian traditional medicine such as treatment of cancer, ⇑ Corresponding author. Address: Phytochimie et Produits Naturels Bioactifs, Ecole de Pharmacie Genève-Lausanne, Section des Sciences Pharmaceutiques, Université de Genève, Quai Ansermet 30, 1211 Genève 4 Switzerland. Tel.: +41 22 379 33 85; fax: +41 22 379 33 99. E-mail address:
[email protected] (J.-L. Wolfender).
diabetes, tuberculosis, snake and scorpion bites, skin rash, scabies, fever and inflammation (Abdel-Sattar et al., 2007; De Leo et al., 2005; Oyama et al., 2007; Ramesh et al., 1999; Western, 1986). Because of its claimed appetite suppressant activity, Caralluma fimbriata encounters an important interest from the public at large and is the widely commercially available Caralluma species at present (Kuriyan et al., 2007; MacLean and Luo, 2004). Caralluma sinaica (Decne.), which is the species considered for this study, is only sold in local markets and is reputed to have aphrodisiac, anti-diabetic and anti-cancer activities (Habibuddin et al., 2008). Previous phytochemical and biological investigations of the genus Caralluma led to the isolation of several pregnane, flavone and megastigmane glycosides, as well as triterpenes (Bader et al., 2003; Braca et al., 2002; Muller and Albers, 2002). Notably, numerous polyhydroxy pregnane ester glycosides with significant antitumor activity were isolated from several members of the family Asclepiadaceae (Braca et al., 2002; Chen et al., 2010; Halaweish et al., 2004; Li et al., 2008; Plaza et al., 2005). While C. sinaica is a commonly used plant in Saudi Arabia (Habibuddin et al., 2008), to our knowledge, it has not yet been investigated in details from a phytochemical viewpoint. The scope of our study was to explore the chemical composition of this plant in relation to other Caralluma species and plants from the Asclepiadaceae, to document the bioactivity of some of their constituents. In order
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.04.003
Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane glycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/ 10.1016/j.phytochem.2012.04.003
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S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx
to obtain a rather comprehensive view of the C. sinaica metabolome and check its potential for new compounds, the extract was profiled by NMR and high resolution (HR) ultra high pressure liquid chromatography–mass spectrometry (UHPLC–MS). The present study focusses on the isolation and complete characterization of polyhydroxy pregnane ester glycosides along with some flavonoids by using 1D and 2D NMR spectroscopy and HR-MS. The quinone reductase induction of the isolated compounds was also assessed.
2. Results and discussion 2.1. NMR and UHPLC-ESI-TOF-MS profiling of C. sinaica extract In order to obtain most of the constituents of C. sinaica of medium polarity, the plant was extracted with ethanol according to an established protocol (Khalil, 1995). Both NMR (Verpoorte et al., 2007; Wolfender et al., 2010) and UHPLC-MS (Eugster et al., 2011) profilings were performed on this crude extract and compared with references to all previously reported compounds from the Caralluma genus. This ethanolic extract was directly dissolved in deuterated methanol and profiled by NMR. The 1H- and gHSQC-NMR spectra (Fig. 1A and B) showed various glycosylated compounds through the 1H–13C–OH signal in the (3–4 ppm and 60–90 ppm in 1Hand 13C-NMR, respectively) region and the corresponding typical anomeric protons (4–5 ppm and 90–110 ppm). The presence of various signals in the aliphatic proton region (1–3 ppm and 10–50 ppm) confirms the presence of steroidal compounds which can be putatively assigned to pregnanes by studying previous reported data on Caralluma species (Abdel-Sattar et al., 2007; De Leo et al., 2005; Halaweish et al., 2004; Kunert et al.,
2009; Qiu et al., 1999; Reddy et al., 2011; Waheed et al., 2011). The aromatic proton signals at 8.05 ppm (2H, dd, J = 1.25, 8.45), 7.57 ppm (1H, t, 7.45) and 7.46 ppm (2H, t, 7.73) can be attributed to the typical pattern of mono-substituted phenyl groups. This is also in good agreement with previous reports on acylated pregnanes. The comparison of the gHSQC-NMR spectrum from the crude extract (Fig. 1B) with the one obtained with a known pregnane glycoside (russelioside G, Fig. 1C) confirms the presence of pregnanes in the ethanolic extract (Tanaka et al., 1990). Since almost no additional signal could be detected, this also indicated that this crude extract is largely dominated by this type of compounds. The presence of steroid glycosides was also confirmed by the positive reaction to Libermann–Buchard and Keller–Kiliani tests performed on the crude extract (Li et al., 2006). In order to confirm this hypothesis, the crude extract was hydrolysed after enrichment, and the NMR spectrum revealed the presence of two aglycones, namely boucerin and caralumagenin by comparison with literature data (Abdel-Sattar et al., 2008; Halim and Khalil, 1996; Lee-Juian et al., 1994). To obtain a more detailed view and get an idea of the diversity of all pregnane glycosides present, the extract was profiled by high resolution UHPLC combined with time of flight mass spectrometry (TOF-MS) (Grata et al., 2009). The chromatogram and corresponding ion map generated in the negative ion mode (NI) revealed an extremely complex composition (Fig. 2). The automatic peak detection at a threshold level of 5% indicated that 40 features of more than 500 Da could be detected and this number was over a hundred when the intensity threshold was lowered to 1%. The molecular formula of all these compounds was determined directly from the TOF-MS data generated on the extract. The combination of high mass accuracy (5 ppm) and heuristic filters (Kind and Fiehn, 2007) provided putative formulae that matched well with glycosylated pregnanes. Based on this preliminary information
Fig. 1. 1H- and gHSQC-NMR spectra (CD3OD) of C. sinaica ethanolic extracts (A and B) and gHSQC of russelioside G (C). The gHSQC spectrum of the crude extract is highly similar to that of the boucerin derivative showing the high content in pregnane of the C. sinaica extract.
Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane glycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/ 10.1016/j.phytochem.2012.04.003
S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx
3
Fig. 2. UHPLC-TOF-MS profile of the C. sinaica ethanolic extract in the negative ESI mode. (A) BPI trace with labels of all isolated compounds. (B) Ion map of all detected features (m/z vs. RT) showing mainly the abundance of pregnane glycosides with MW >700. (C) Zoom into the BPI trace at a threshold level of 10% revealing the high composition complexity of the crude extract.
and the important pregnane diversity recorded, isolation of the main constituents of C. sinaica extract was conducted for a complete structural assignment. 2.2. Identification of pregnanes from C. sinaica Prior to the isolation, the crude ethanolic extract of C. sinaica was submitted to liquid–liquid partition between water and solvents of increasing polarity. The CHCl3 and the n-butanol fractions containing most of the pregnanes were fractionated by repeated normal and reversed-phase liquid chromatography techniques. This afforded six new (1, 4, 8, 9, 11 and 12) and seven known (2, 3, 5–7, 10 and 13) polyhydroxy pregnane glycosides (Fig. 3) along with three flavonoids (14, 15, 16). A detailed structural assignment of the new pregnanes is provided below. All isolated compounds, except flavonoids, gave positive Libermann–Buchard and Keller–Kiliani tests, indicating the presence of a steroidal skeleton with 2-deoxysugar moiety (Li et al., 2006). Spectroscopic analysis (Tables 1–4) and comparison with previously reported data, allowed the identification of the aglycone of compounds 1, 4, 8, 9, 11 and 12 as the C/D-cis-polyoxy pregnane derivative 3b, 12b, 14b, 20b tetrahydroxy-(20R)-pregn-5-ene
(boucerin) (Nikaido et al., 1967; Qiu et al., 1997). As described below, among these compounds, the new pregnanes possessed an ester group at C-12 and C-20 positions and a straight sugar chain consisting of 2–4 sugar units connected to C-3 of the aglycone. With all isolated compounds, the ester linkages attached at positions 12 and 20 of the aglycone caused a downfield shift of about 1.00 ppm in 1H NMR signals of H-12 and H-20 and changed their splitting patterns (dd or q), while it induced a downfield shift of about 4.0 ppm in 13C NMR of the corresponding C-12 and C-20 signals (Hayashi et al., 1988). The presence of a double bond between C-5 and C-6 in the boucerin nucleus caused a downfield shift of the Me-18 (dH 1.04 in analogues with D5 compared with 0.83 in dihydro). On the other hand, the glycosylation at C-3 induced the following shifts of the aglycone (C-2 (2.3 ppm), C-3 (+6.0 ppm) and C-4 (4.0 ppm)) (El Sayed et al., 1995; Tanaka et al., 1990). The relative stereochemistry at the chiral centers of the aglycone moiety was deduced from NOESY experiments and comparison of the chemical shifts of the carbons and proton coupling constants with those reported for related pregnanes (Ahmad et al., 1988; Basha and Ahmad, 2007; Panda et al., 2003). The large homonuclear coupling constants (7.5–10.5 Hz) of the anomeric protons of each
Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane glycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/ 10.1016/j.phytochem.2012.04.003
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S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx
O
21
R2 18 12 19 11 1
9
H
3 5
RO
Comp.
R
R1
OR1
2'
20 17
OH
15
R2
C
Ac
O-Bz
2
D
Ac
O-Bz
3
D
H
O-Bz
4
A
Ac
O-Tig
5
A
Ac
O-Bz
6
E
Ac
O-Bz
7
F
Ac
O-Tig
8
G
Ac
O-Bz
9
H
Ac
O-Bz
10
F
Ac
O-Bz
11
A
Tig
O-Bz
12
E
Bz
O-Bz
13
B
Glc
H
6' 3'
O
O 4 CH3O
Glc
O OH 4 H3CO
Thev
HO
4```
O 4
1''' O 4
1''
CH3O
Allom
HOH2C HO HO Glc
O
1''' O 4
1''
CH3O
4```
O
Glc
O
OH
HOH2C HO 4 HO
CH3O
Dig
Glc
1''' O 4
OH Allom
Cym
1''
CH3O
O 4
Cym
O 1'
CH3O
Cym
=E 1'
6
O
O
=F
Cym
O
O OH 4 Glc H CO 3
HOH2C HO HO
O
O 4 CH3O
Cym
HOH2C HO 4 HO
=D 1'
6
O
OH
=C
O
O 4 CH3O
Cym
=B
OH
CH3O Cym 6
O
Cym
O
O 4
CH3O Cym
OH
CH3O
O
OH O
= Tig =A
O
Dig
O
1'
OH
HOH2C HO HO
4```
O 2' 5'
O
Glc
HO H3CO
= Bz
7' 4'
HOH2C HO HO
1'
2'
5'
6
1
= Ac
3' 4'
H 7
1'
O
O OH
O 4
6
O 1''
CH3O Cym
O OH
4```
O O CH3
O OH Thev
O 4
O
CH3O Cym
O 4
O
=G 1'
CH3O O 4
Cym
O
=H
CH3O Cym
Fig. 3. Identified pregnanes from C. sinaica (Ac = acetyl; Bz = benzoyl; Tig = tigloyl; Allom = allomerose; Cym = cymarose; Dig = digitalose; Glc = glucose; Thev = thevetose).
sugar unit, in the 1H NMR spectrum, were typical of their axial configuration in hexopyranoses in 4C1 (D) conformation, having b-glycosidic linkage (García, 2011). The structures were also validated according to the molecular formula obtained using HRESITOF-MS in positive ionisation (PI) and NI modes. All compounds were always detected in NI, mainly as formic acid adducts. In PI however, sodium adducts were recorded only for some of the derivatives. Compound 1 was obtained as a white amorphous powder (8.7 mg). HRESITOF-MS of 1 in NI displayed a [M+HCOO] at m/z 989.5210 suggesting a molecular weight of 944.5156 and molecular formula C51H76O16 with 14 degrees of unsaturation. This formula was confirmed by 13C NMR and APT NMR. The intense IR absorption bands at 3400, 1720 and 1608, 1504 and 1235 cm1 indicated the presence of an hydroxyl, a carbonyl ester and an aromatic ring. The broad doublet at dH 5.48 in the 1H NMR was characteristic for a D5 pregnane derivative (Abdel-Sattar et al., 2008; Aquino et al., 1996). The signals at dH 3.50 (1H, m), 4.85 (1H, dd) and 4.94 (1H, br q) were correlated with the oxygenated methine carbons at 79.0, 79.7 and 75.3 ppm, in the HSQC spectrum, and assigned for
protons H-3, H-12 and H-20, respectively. The oxygenated quaternary carbon at dC 87.3 was assigned to C-14 in accordance with reported data (Hayashi et al., 1988; Kunert et al., 2009). The careful comparison of the NMR data with those reported for similar compounds (Abdel-Sattar et al., 2007, 2008; Aquino et al., 1996; Braca et al., 2002), suggested that the pregnane skeleton of 1 was 3b, 12b, 14b, 20b-tetrahydroxy-pregn-5-ene. The 13C NMR spectra of 1 also showed signals due to two ester moieties (two carbonyl carbons at dC 167.8 and 172.3). One of the two esters was a benzoyl moiety as deduced from 1H NMR signals at dH 8.15 (2H, br d, J = 7.5 Hz), 7.60 (1H, t, J = 7.5 Hz) and 7.50 (2H, t, J = 7.5 Hz) which were correlated with the aromatic methine carbons at dC 130.6, 134.3 and 129.5 and assigned for ortho, para and meta positions, respectively (El Sayed et al., 1995; Halim and Khalil, 1996; Rasoanaivo et al., 1991). The HMBC 3J correlation between the carbonyl of the benzoyl ester at dC 167.8 and H-12 (dH 4.85, dd, J = 12.0, 4.0 Hz) proved the acylation of the aglycone by the benzoyl group at C-12. The second ester was an acetyl moiety as shown by NMR data (dH 1.94, dC 21.7) and its attachment at C-20 was confirmed by the 3J correlation between H-20 (dH 4.94) and the second
Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane glycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/ 10.1016/j.phytochem.2012.04.003
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S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx Table 1 1 H NMR spectral data for the aglycone moieties of compounds 1, 4, 7, 8, 10, 11 (500 MHz, CD3OD). Comp dH
1
4
8
1a 1b 2a 2b 3 4a 4b 5 6
1.17, m 1.84, m 1.92, m 1.54, m 3.50, m 2.38, m 2.16, m – 5.48, br d (5.4) 1.85, m 2.25, m 1.85, m 1.35, m – 1.85, m 1.64, m 4.85 dd (12.0, 4.0) – – 1.65, m 1.82, m 1.54, m 1.87, m 2.06, m 1.17, s 1.05 s 4.94, br q (6.0) 1.11, d (6.0)
1.10, m 1.75, m 1.90, m 1.51, m 3.50, m 2.39, m 2.14, m – 5.46, br d (3.5) 1.86, m 2.25, m 1.87, m 1.35, m – 1.85, m 1.65, m 4.62, dd (12.5, 5.0) – – 1.66, m 1.81, m 1.60, m 1.85, m 2.09, m 1.07, s 1.03, s 4.92, m
1.10, 1.75, 1.90, 1.52, 3.50, 2.37, 2.17, – 5.48, (5.0) 1.87, 2.27, 1.87, 1.35, – 1.85, 1.65, 4.84,
7a 7b 8 9 10 11a 11b 12 13 14 15a 15b 16a 16b 17 18 19 20 21
1’ 2’ 3’, 7’ 4’, 6’ 5’
1’ 2’ 3’, 7’ 4’, 6’ 5’
1’ 2’ 3’ 4’ 5’
1.14, d (6.5)
– – 1.65, 1.80, 1.59, 1.91, 2.06, 1.17, 1.04, 4.94, (6.0) 1.10, (6.0)
9 m m m m m m m
1.12, 1.77, 1.90, 1.52, 3.54, 2.39, 2.18, – 5.46, (5.0) 1.87, 2.27, 1.87, 1.35, – 1.85, 1.65, 4.84,
br d m m m m m m m
– – 1.67, 1.82, 1.59, 1.91, 2.06, 1.17, 1.04, 4.96, (6.0) 1.10, (6.0)
m m m m m s s br q d
m m m m m m m br d m m m m m m m
m m m m m s s br q d
11
12
1.15, m 1.85, m 1.90, m 1.53, m 3.51, m 2.37, m 2.17, m – 5.46, br d (5.0) 1.87, m 2.25, m 1.87, m 1.37, m – 1.75, m 1.66, m 4.98, dd (12.0, 4.0) – – 1.69, m 1.88, m 1.62, m 1.98, m 2.17, m 1.12, s 1.04, s 5.09, br q (6.5) 1.10, d (6.5)
1.10, 1.75, 1.90, 1.51, 3.50, 2.38, 2.17, – 5.49,
m m m m m m m
1.92, 2.25, 1.87, 1.37, – 1.84, 1.65, 4.98,
m m m m
– – 1.65, 1.87, 1.70, 2.04, 2.28, 1.14, 1.01, 5.25,
br s
m m m
m m m m m s s m
1.21, d (5.0)
Bz (12)
Bz (12)
Bz (12)
Bz (12)
Bz (12)
– – 8.15 br d (7.5) 7.50, t (7.5) 7.60, t (7.5)
– – 8.12, d (7.0) 7.51, t (8.0) 7.62, t (7.5)
– – 8.14, d (7.0) 7.53, t (8.0) 7.63, t (7.5)
– – 8.05, d (7.0) 7.51, t (8.0) 7.62, t (7.5)
– – 7.75, br d (8.5) 7.25, t (8.0) 7. 52, t (7.5)
Ac (20)
Ac (20)
Ac (20)
Ac (20)
Bz (20)
– 1.94, s
– 2.00, s
– 1.94, s
– 1.94, s
– – 8.05, br d (8.5) 7.50, t (8.5) 7.68, t (7.5)
Tig (12)
Tig (20)
– – 6.98, br q (7.0) 1.85, d (6.0) 1.91, br s
– – 6.55, br q (6.0) 1.62, d (7.0) 1.70, br s
J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
carbonyl signal at dC 172.3. Therefore, the aglycone of 1 was identified as the boucerin derivative 12-O-benzoyl-20-O-acetyl 3b, 12b, 14b, 20b tetrahydroxy-(20R)-pregn-5-ene. The presence of three anomeric protons suggested a trisaccharide glycoside. The full assignment of all protonated carbons was accomplished by interpretation of the gHSQC, gHMBC, DQF-COSY and NOESY experiments which allowed the sequential identification of H-1 to H-6 within each sugar unit. By comparing these data with those reported (Abdel-Sattar et al., 2009; Abe et al., 2000; Aquino et al., 1996; Braca et al., 2002; Halim and Khalil, 1996; Mimaki et al., 2002), the two inner sugar units were identified as cymarose, while the terminal one was identified as thevetose (6-deoxy-3-O-methyl-D-glucose). The doublet signals at dH 1.29, 1.32 and 1.21 (each 3H, d, J = 6.0 Hz) were correlated with the
13
C NMR signals at 18.3, 18.7, 18.6 ppm, respectively, and assigned for three secondary methyl groups of monosaccharides at the positions 6Thev, 6CymII and 6CymI. Moreover, the 1H NMR methyl singlets at dH 3.65, 3.44 and 3.45 were correlated with the carbon signals at 61.1, 58.6 and 58.5 ppm and ascribed to the three methoxyl groups of the sugar moieties attached to C-3Thev, C-3CymII and C-3CymI, respectively. The splitting pattern and coupling constant of H-3 of thevetose (t, J = 8.0 Hz) was consistent with the axial orientation of H-2, H-3 and H-4 characteristic for this sugar (Kunert et al., 2009; Nasipuri, 1994). Both cymarose units were glycosylated at C-4 as shown by a downfield shifts observed for C-4CymII and C-4CymI (84.0 and 83.8 ppm). Cross peaks due to three bond correlations, in gHMBC, between C-4CymI and H-1CymII (dH 4.81), and C-4CymII and H-1Thev (dH 4.31) indicated the sequence of the
Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane glycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/ 10.1016/j.phytochem.2012.04.003
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S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx
Table 2 13 C NMR Spectral data for aglycone moieties of compounds 1, 4, 7, 8, 10, 11 (125 MHz, CD3OD). Comp dC
1
4
8
9
11
12
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
38.6, t 30.6, t 79.0, d 39.7, t 140.5, s 123.0, d 28.2, t 38.0, d 44.7, d 38.4, s 27.1, t 79.7, d 53.1, s 87.3, s 33.0, t 26.1, t 51.2, d 9.8, q 19.9, q 75.3, d 19.5, q
38.4, t 30.6, t 79.0, d 39.8, t 140.5, s 123.0, s 28.2, d 37.9, d 44.5, d 38.3, s 27.1, t 78.7, d 53.1, s 87.5, s 32.9, t 25.7, t 51.0, d 10.1, q 19.5, q 75.2, d 19.8, q
38.5, t 30.7, t 79.8, d 39.8, t 140.5, q 123.1, d 28.2, t 38.0, d 44.6, d 38.4, q 27.3, t 79.3, d 53.4, q 87.5, q 33.0, t 26.0, t 51.5, d 10.2, q 19.9, q 75.2, d 19.5, q
38.6, t 30.8, t 79.8, d 39.8, t 140.6, q 123.1, d 28.2, t 38.0, d 44.6, d 38.4, q 27.3, t 79.3, d 53.4, q 87.5, q 33.1, t 26.0, t 51.5, d 10.2, q 19.9, q 75.2, d 19.5, q
38.4, t 30.6, t 79.1, d 39.8, t 140.5, s 123.0, d 28.1, t 37.8, d 44.6, d 38.3, s 27.3, t 79.0, d 53.5, q 87.4, s 32.8, t 25.5, t 50.9, d 10.0, q 19.8, q 75.3, d 19.6, q
38.4, t 30.6, t 78.9, d 39.4, t 140.8, s 122.9, d 28.1, t 37.7, d 44.5, d 38.2, s 27.4, t 80.1, d 53.5, s 87.3, s 32.3, t 25.5, t 51.1, d 10.2, q 19.8, q 75.3, d 19.7, q
1’ 2’ 3’, 7’ 4’, 6’ 5’
1’ 2’ 3’, 7’ 4’, 6’ 5’
1’ 2’ 3’ 4’ 5’
Bz (12)
Bz (12)
Bz (12)
Bz (12)
Bz (12)
167.8, 132.0, 130.6, 129.5, 134.3,
167.9, 131.9, 130.7, 129.6, 134.4,
168.0, 131.9, 130.7, 129.6, 134.4,
168.0, 131.9, 130.6, 129.7, 134.3,
167.3, 131.7, 130.3, 129.5, 133.9,
s s d d d
q q d d d
q q d d d
s s d d d
s d d d d
Ac (20)
Ac (20)
Ac (20)
Ac (20)
Bz (20)
172.3, s 21.7 q
172.4, s 21.7, q
172.4, q 21.8, q
172.6, q 21.9, q
167.9, 132.5, 130.7, 129.8, 134.3,
Tig (12)
Tig (20)
169.5, s 130.1, s 138.7, d 14.5, q 12.3, q
169.0, q 130.3, q 138.0, d 14.5, q 12.2, q
s d d d d
trisaccharide chain. The absence of any glycosidation shift, in 13C NMR signals, suggested that thevetose is the terminal sugar. The glycosidation of the aglycone by this trisaccharide at C-3 was determined from a 3J correlation, between the anomeric proton at dH 4.87 (H-1CymI) and C-3 at dC 79.0. The above mentioned data proved that 1 is 12-O-benzoyl-20-O-acetyl boucerin-3-O-b-Dthevetopyranosyl-(1 ? 4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside. To our knowledge this compound is reported here for the first time. Compound 4 was isolated as a white amorphous powder (2.3 mg). HRESITOF-MS of 4 displayed a [M+Na]+ in PI at m/z 803.4220 and a [M+HCOO] at m/z 825.4300 in NI, suggesting a molecular formula of C41H64O14 with an unsaturation number of 10. The NMR spectral analysis of the aglycone moiety of 4 revealed similarities with 1 except for the presence of a tigloyl ester moiety instead of the benzoyl attached to C-12. The presence of the tigloyl ester was established by an olefinic methine proton signal at dH 6.98 (br q, J = 7.0 Hz) correlated, in gHSQC, with C-30 at 138.7 ppm of the tigloyl; and two methyl signals at dH 1.85 (d, J = 6.0 Hz) and 1.91 (br s) correlated with methyl carbons C-40 and C-50 at dC 14.5 and 12.3 ppm, respectively. Finally, a quaternary carbon at 130.1 ppm assigned to C-20 finally showed the presence of a tiglate (Abdel-Sattar et al., 2007; Braca et al., 2002; Shukla et al., 2009). gHMBC 3J correlations between H-12 (dH 4.62, dd, 12.5, 5.0), H-30 (dH 6.98) and the carbonyl of the tigloyl ester (dC 169.5), confirmed the tigloyl acylation at C-12. Similarly, 3J correla-
tion between H-20 (dH 4.92, m) and the acetate ester carbonyl at 172.4 ppm revealed acetylation at C-20. In addition to the aglycone resonances, the 13C NMR spectra of 4 exhibited 13 signals related to two sugars with their anomeric protons resonating at dH 4.36 (d, J = 8.0 Hz) and 4.89 (dd, J = 9.5, 2.5 Hz). A careful interpretation of DQF-COSY, gHSQC and gHMBC spectra allowed the assignment of the two sugars as b-D-glucopyranoside and b-D-cymaropyranoside (Halaweish et al., 2004; Shukla et al., 2009). The identification of the glycosidation site and sugar sequence (H-1Cym–C-3, H-1Glc–C-4Cym) was confirmed as previously described for 1. Thus, the novel structure of 4 was 12b-O-tigloyl20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranoside. Compounds 8 and 9 were obtained as a mixture (4.4 mg) as suggested by NMR data. 8 and 9 could not be separated by HPLC or UHPLC by using various conditions. The single corresponding LCpeak displayed a [M+Na]+ at m/z 1129.550 in PI and a [M+HCOO] at m/z 1151.570 in NI. These results suggested the presence of two isomers with a molecular formula of C57H86O21. An extensive study of their 1D- and 2D-NMR data allowed the complete assignments of each component of the mixture. The obtained 13C-APT-NMR spectrum implied an identical aglycone part for 8 and 9 as shown by signals present in overlapping pairs with an intensity ratio of about 3:2. As for 1 and 4, the aglycone was recognized as 12-bO-benzoyl-20-O-acetyl boucerin. The difference between the two isomers lies in the tetra-saccharide sugar chain attached to C-3. Compound 8 and 9 displayed common signals for two cymarose (which was confirmed by comparison with 1) and one glucose residue. Taking into account the molecular formula of both isomers, the presence of the latter sugars and the aglycone, the unassigned part corresponded to a mass value difference of 160 Da, with the molecular formula C7H12O4. The presence of two methoxy (dH 3.53, 3.64 ppm and dC 58.9, 61.3 ppm) and two secondary methyl groups (dH 1.30, 1.39 ppm and dC 17.5, 18.6 ppm) in the 1H- and 13C-NMR data of the fourth sugar moiety, was consistent with various 6-deoxy-3-O-methylhexoses. This sugar was identified as digitalose in 8 since it exhibited a characteristic upfield-shifted methoxyl (dH 3.53, dC 58.9) attached to C-3 in comparison to the downfield-shifted methoxyl of thevetose and allomerose (dH > 3.55, dC > 61.0) (Braca et al., 2002; Halaweish et al., 2004). In addition, the characteristic doublet at dH 4.15, assigned to H-4 of the digitalose moiety, had a distinctly small coupling constant (d, J = 2.5 Hz) that was consistent with the equatorial orientation of this proton and its weak coupling to the axially oriented H-3 and undetected coupling to H-5 (Nasipuri, 1994). Furthermore, the presence of a NOESY correlation between H-3/H-4 (dH 3.21/dH 4.15), and the absence of a similar correlation between H-2/H-4 (dH 3.64) required an equatorial orientation of H4 (Kunert et al., 2009). All additional NMR data were also similar to those reported for digitalose (Braca et al., 2002; Qiu et al., 1999). The structure of 8 was thus determined as 12-b-O-benzoyl-20-Oacetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-digitalopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside. On the other hand, the anomeric signals for the sugar units constituting the tetra-saccharide moiety of 9 were clearly separated from those of 8 in the 13C data as revealed by the APT and gHSQC spectra (dH 4.82, dC 97.3; dH 4.76, dC 101.2; dH 4.44, dC 104.3 and dH 4.31, dC 106.0) (Tables 3 and 4). The fourth sugar in 9 was determined to be thevetose from the characteristic chemical shift values of C-1 (dC 104.3), C-3 (dC 86.2) and C-4 (dC 82.9) which were closely similar to those previously reported and discussed above for 1 (Braca et al., 2002; Shukla et al., 2009). It is worth noting that C4 of thevetose, in 9, appeared significantly downfield shifted (dC 82.9) in comparison to its value in 1 (dC 76.5) which is obviously due to the attachment of another sugar moiety at C-4 of thevetose in 9 (glycosidation shift). The structure of 9 was determined as
Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane glycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/ 10.1016/j.phytochem.2012.04.003
7
S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx Table 3 1 H NMR spectral data for sugar moieties of compounds 1, 4, 7, 8, 10, 11 (500 MHz, CD3OD). Comp dH
1
4
8
9
11
12
Sugar
Cym I
Cym
Cym I
Cym I
Cym
Cym I
1
4.87, dd (10.0, 2.5) 1.57, br dd (16.0, 12.0) 2.08, br dd (15.0, 3.0) 3.86, q (4.0) 3.25, dd (10.0, 4.0) 3.85, d (6.0, 9.5) 1.21, d (6.0) 3.45, s
4.89 dd (9.5, 2.5) 1.51, dd (15.5, 11.5) 2.07 br dd (13.5, 4.0) 3.86, q (3.0) 3.25, m
4.85, dd (10.0, 2.5) 1.57, m
4.82, m
4.89, d (10.5) 1.65, m
2.06, br dd (13, 4.0) 3.85, q (3.0) 3.26, dd (9.5, 2.0) 3.86, m
2.06, br dd (13, 4.0) 3.85, q (3.0) 3.26, dd (9.5, 2.0) 3.86, m
4.86, dd (9.5, 2.0) 1.56, dd (16.0, 12.5) 2.05, m 3.91, q (3.0) 3.31, dd (9.5, 2.5) 3.86, m
3.84, m
1.20, d (6.0) 3.43, s
1.20, d (6.0) 3.43, s
1.30, d (6.0) 3.46, s
1.21, d (6.5) 3.44, s
Cym II
Cym II
Cym II
Cym II
2a 2b
4.81, dd (8.5, 2.0) 1.70, m 2.11, m 3.86, m
4
3.28, m
5
3.85, dq (6.0, 9.5) 1.32, d (6.0) 3.44, s
4.76, dd (10.0, 2.5) 1.50, m 2.06, dd (13.0, 4.0) 3.85, q (3.0) 3.26, dd (9.5, 2.0) 3.78, m
4.80, d (8.0) 1.62, m 2.12, m
3
4.79, dd (10.0, 2.0) 1.50, m 2.06, dd (13.0, 4.0) 3.85, q (3.0) 3.26, dd (9.5, 2.0) 3.78, m 1.30, d (6.0) 3.45, s
1.30, d (6.0) 3.45, s
1.31, d (6.5) 3.45, s
Thev
Dig
Thev
4.31, d (8.0) 3.27, br s
4.54, d (8.0) 3.64, t (9.5) 3.21, dd (8.5, 2.5) 4.15, d (2.5) 3.60, m 1.30, d (6.0) 3.53, s
4.44, d (9.0) 3.20, m
3.70, m 1.39, d (6.0) 3.64, s
Glc
Glc
Glc
Glc
Glc
4.36, d (8.0) 3.24, dd (9.0, 8.0) 3.30, d (7.5) 3.25, d (8.0) 3.27, m 3.66, dd (11.5, 5.5) 3.90, dd (10.5, 4.0)
4.31, d (7.5) 3.22, m
4.31, d (9.0) 3.22, t (8.5) 3.37, d (9.0) 3.23, d (6.5) 3.32, m 3.63, dd (11.5, 4.5) 3.84, m
4.34, d (8.0) 3.20, t (8.5) 3.31, m
4.36, d (8.0) 3.23, t (7.5) 3.30, m
3.25, d (6.5) 3.20, m 3.65, dd (12.0, 5.5) 3.91, dd (12.0, 3.0)
3.27, d (8.5) 3.35, m 3.65, dd (12.5, 5.5) 3.90, dd (11.0, 3.0)
2a 2b 3 4 5 6 OCH3
1
6 OCH3
1 2 3 4 5 6 OCH3
1 2 3 4 5 6a 6b
3.89, dq (7.5, 10.5) 1.30, d (6.0) 3.44, s
3.05, t (8.0) 3.06, m 3.32, m 1.29, d (6.0) 3.65, s
3.37, d (9.0) 3.23, d (6.5) 3.32, m 3.63, dd (11.5, 4.5) 3.84, m
1.57, m
2.15, m
3.22, m 3.80, m
3.92, m 3.32, m 3.90, m
3.25, m 3.30, m
J values are in parentheses and reported in Hz; chemical shifts are given in ppm.
12-b-O-benzoyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl(1?4)-thevetopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-Dcymaropyranoside. The identification of the glycosidation site and sugar sequence in both 8 and 9 (H-1CymI–C-3, H-1CymII–C-4CymI, H1Thev (9) or Dig (8)–C-4CymII and H-1Glc–C-4Thev (9) or Dig (8)) was confirmed in a similar way as for 1. Compound 11 was isolated as a white amorphous powder (6.0 mg). HRESITOF-MS of 11 displayed a [M+HCOO] in NI at m/ z 887.450 suggesting a molecular formula of C46H66O14. The NMR
data of the aglycone of 11 were closely similar to those of 1 except the replacement of the signals of the acetate moiety with those of a tigloyl ester. The position of attachment of the tigloyl moiety at C20 of the aglycone was deduced from the gHMBC correlations between the tigloyl ester carbonyl at 169.0 ppm and H-20 (dH 5.09) and the methyl singlet assigned to C-5 tiglate (dH 1.70) (Fig. 4). The presence of two anomeric protons and carbons in 1 H- and APT-NMR spectra of 11 (dH 4.34, d, J = 8.0 Hz, dC 106.2 and dH 4.86, dC 97.2) suggested a disaccharide glycoside. The
Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane glycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/ 10.1016/j.phytochem.2012.04.003
8
S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx
Table 4 13 C NMR spectral data for sugar moieties of compounds 1, 4, 7, 8, 10, 11 (125 MHz, CD3OD). Comp dH
1
4
8
9
11
Cym I
Cym
Cym I
Cym I
Cym
1 2 3 4 5 6 OCH3
97.3, 36.6, 78.6, 83.8, 69.9, 18.6, 58.5,
97.3, 36.7, 78.0, 83.7, 70.2, 18.7, 58.6,
97.3, 36.9, 78.1, 84.1, 70.1, 18.5, 58.3,
97.3, 36.9, 78.1, 84.1, 70.1, 18.5, 58.3,
97.2, 36.7, 78.7, 83.8, 70.2, 18.6, 58.5,
Cym II
Cym II
Cym II
Cym II
1 2 3 4 5 6 OCH3
101.1, d 36.4, t 78.6, d 84.0, d 70.0, d 18.7, q 58.6, d
101.2, d 35.9, t 78.3, d 84.3, d 70.3, d 18.8, q 58.7, q
101.2, d 35.9, t 78.3, d 84.3, d 70.3, d 18.8, q 58.7, q
101.1, d 36.7, t 78.7, d 83.9, d 70.1, d 18.6, q 58.6, q
Thev
Dig
Thev
106.2, d 75.1, d 87.4, d 76.5, d 73.0, d 18.3, q 61.1, d
104.8, d 72.7, d 85.8, d 75.8, d 71.5, d 17.5, q 58.9, q
104.3, d 75.7, d 86.2, d 82.9, d 72.7, d 18.6, q 61.3, q
1 2 3 4 5 6 OCH3
1 2 3 4 5 6
d t d d d q q
d t d d d q q
d t d d d q q
d t d d d q q
12 Cym I d t d d d q q
97.2, 36.4, 78.5, 83.7, 69.9, 18.5, 58.4,
d t d d d q q
Glc
Glc
Glc
Glc
Glc
106.1, d 75.3, d 78.0, d 71.7, d 77.9, d 63.0, t
106.5, d 76.1, d 77.9, d 72.0, d 78.7, d 63.2, t
106.0, d 76.1, d 77.9, d 72.0, d 78.7, d 63.2, t
106.2, d 75.4, d 78.0, d 71.8, d 75.4, d 63.0, t
106.2, d 75.5, d 77.9, d 71.7, d 78.0, d 62.9, t
NMR data of the sugar part was in complete agreement with those of Glc-(1?4)-Cym, which established the structure of 11 as 12-bO-benzoyl-20-O-tigloyl boucerin-3-O-b-D-glucopyranosyl-(1?4)b-D-cymaropyranoside. The identification of the glycosidation site and sugar sequence (H-1Cym–C-3, H-1Glc–C-4Cym) was confirmed in a similar way as for 1. Compound 12 was isolated as a colorless amorphous powder (3.7 mg). HRESITOF-MS displayed a [M+Na]+ in PI at m/z 1031.5013 and a [M+HCOO] in NI at m/z 1053.510 suggesting the molecular formula C55H76O17. The NMR data of the aglycone was similar to the previous compounds except that both hydroxyl groups at C-12 and C-20 were acylated with two benzoyl moieties. This was confirmed by the presence of two sets of coupled aromatic proton signals in the 1H-NMR spectrum corresponding to two benzoyl groups. The complete assignment of all proton and carbon signals of the two benzoyl esters was carried out using
DQF-COSY, gHSQC and gHMBC experiments (Tables 1 and 2). The presence of three anomeric protons and the corresponding carbons in 1H and 13C NMR spectra of 12 (Tables 3 and 4) suggested a trisaccharide glycoside. The sugar units were identified as one Dglucose and two D-cymarose units by comparison with data of above discussed compounds. Similarly, the position of attachment to the aglycone was determined at C-3 from a strong 3J correlation, in the gHMBC spectrum, between H-3 (dH 3.50) and the anomeric carbon of the first cymarose unit at 97.2 ppm. 12 was identified as 12b-20-O-dibenzoyl boucerin-3-O-b-D-glucopyranosyl-(1?4)b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside. Again, the sequence of sugar units was determined by gHMBC experiment which showed a 3J correlation between H-1Glc (dH 4.36) and C-4CymII (dC 83.9); H-1CymII (dH 4.80) and C-4CymI (dC 83.7). The known compounds (2, 3, 5, 6, 7, 10 and 13) were identified as bouceroside-BDC (2) (Tanaka et al., 1990), 12-O-benzoyl boucerin6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside (3) (Tanaka et al., 1990), russelioside G (5) (Abdel-Sattar et al., 2007), russelioside F (6) (Abdel-Sattar et al., 2007), 12b-O-tigloyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-Dcymaropyranosyl-(1?4)-b-D-cymaropyranoside (7) (Braca et al., 2002), russelioside E (10) (Abdel-Sattar et al., 2007) and caralumagenin-20-O-b-D-glucopyranosyl-3-O-b-D-glucopyranosyl-(1?4)-b-Ddigitalopyranoside (13) (Qiu et al., 1999), by comparison with previously reported NMR data. In addition to the pregnanes, three flavonoids were isolated from the ethanolic extract. These polyphenols were identified based on NMR and MS data as luteolin (14) (Kim et al., 2006), the luteolin 40 -O-b-D-neohesperidoside (15) (Rizwani et al., 1990), and rutin (16) (Lopez-Lazaro, 2009). It is interesting to point out that luteolin 40 -O-neohesperidoside (15) was also isolated from Caralluma lasiantha (Qiu et al., 1999), Caralluma attenuata, Caralluma umbellata (Ramesh et al., 1999), Caralluma tuberculata (Rizwani et al., 1990) and Caralluma russeliana (Al-Yahya et al., 2000). Since it appears to be a common constituent of plants of the Caralluma genus in addition to pregnanes, it might be considered as a chemotaxonomic marker for genus Caralluma. On the other hand, luteolin and rutin are reported here for the first time in the genus Caralluma. 2.3. Determination of the absolute configuration of C-20 A careful investigation was carried out to determine the absolute configuration of C-20 which was left unassigned in most published papers on pregnane steroids bearing a hydroxyl group at this position (Braca et al., 2002; De Leo et al., 2005; Halim and Khalil, 1996). Kimura et al. (1982) compared 13C NMR spectral data of various 20R and 20S pregnane compounds, which were synthesised by the reduction of 20 pregnanones, and also have hydroxyl
H O O
O
O OH Glc
O
H3C
CH3
O
O
H
OH HO HO
H3C
CH3
O
O
H O
H
OH H
OH
O Cym Fig. 4. Structure of 11 and some selected 2J and 3J gHMBC correlations.
Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane glycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/ 10.1016/j.phytochem.2012.04.003
S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx Table 5 QR inducing activity of selected isolated pregnanes. Compounds
QR induction CD (lM)
Cytotoxicity IC50 (lM)
Chemoprevention index CI (IC50/CD)
1 2 3 4 5 6 7 10 11 12 13
14.2 3.1 3.3 >20.0 >20.0 >20.0 20.0 >20.0 >20.0
17.5 9.3 10.9 >20.0 >20.0 16.3 >20.0 >20.0 13.7 >20.0 >20.0
1.2 3.0 3.3 – – 2.0 >1.6
