Phytochemistry xxx (2012) xxx–xxx

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Phytochemistry

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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 King Saud University, College of Pharmacy, Dept. of Pharmacognosy, P.O. Box 2457, Riyadh 11451, Saudi Arabiab 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 2012Received in revised form 2 April 2012Accepted 5 April 2012Available online xxxx

Keywords:Caralluma sinaicaAsclepiadaceaePregnane glycosidesQuinone reductase induction

0031-9422/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.phytochem.2012.04.003

⇑ Corresponding author. Address: Phytochimie etEcole de Pharmacie Genève-Lausanne, Section desUniversité de Genève, Quai Ansermet 30, 1211 Genèv379 33 85; fax: +41 22 379 33 99.

E-mail address: jean-luc.wolfender@unige.ch (J.-L.

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a b s t r a c t

Caralluma sinaica is sold on local markets of Saudi Arabia for various health benefits however no phyto-chemical study has specifically been performed on this species. NMR and UHPLC-ESI-TOF-MS profilingsof the ethanolic extract of the whole plant reveal a very complex phytochemical composition dominatedby pregnanes. Detailed information on its constituents was obtained after isolation. Six pregnaneglycosides were obtained and characterized based on the extensive spectroscopic analysis (including IR,1H NMR, 13C NMR and MS data), in addition to ten known compounds (seven pregnanes and three flavo-noids). The compounds were identified as 12b-O-benzoyl-20-O-acetyl boucerin-3-O-6-deoxy-3-O-methyl-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside, 12b-O-tigloyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranoside, 12b-O-benzoyl-20-O-acetylboucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-digitalopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside, 12b-O-benzoyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-thevetopyrano-syl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside, 12b-O-benzoyl-20-O-tigloyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranoside, 12b-20-O-dibenzoyl boucerin-3-O-b-D-glucopyran-osyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside. Finally, the isolated compounds wereevaluated for their quinone reductase induction.

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1. Introduction

The genus Caralluma belongs to the Asclepiadaceae family,which is also known as the milkweed family because many of itsmembers contain a milky latex (Bensuzan, 2009). Due to recentDNA analysis and morphological studies, Asclepiadaceae have beenclassified as a sub-group of the family Apocynaceae (Endress andBruyns, 2000; Meve and Heneidak, 2005). Nevertheless, Asclepiad-aceae is still regarded as an independent family. Plants of the genusCaralluma are perennial, small and usually leafless (Heyood, 1978;Saxena and Sarbhai, 1975). Some of these plants are edible andsucculent (Marwah et al., 2007; Reddy et al., 2011). More than200 species of the genus Caralluma grow throughout Africa andAsia (Surveswaran, 2007). The majority of these species are indig-enous to the Indian sub-continent and Arabian peninsula (Gilbert,1990).

Various medicinal uses of Caralluma spp. have been reported inArabic and Indian traditional medicine such as treatment of cancer,

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S.M., et al. Acylated pregnane g

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). Be-cause of its claimed appetite suppressant activity, Caralluma fimbri-ata encounters an important interest from the public at large and isthe 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 soldin local markets and is reputed to have aphrodisiac, anti-diabeticand anti-cancer activities (Habibuddin et al., 2008).

Previous phytochemical and biological investigations of thegenus Caralluma led to the isolation of several pregnane, flavoneand megastigmane glycosides, as well as triterpenes (Bader et al.,2003; Braca et al., 2002; Muller and Albers, 2002). Notably, numer-ous polyhydroxy pregnane ester glycosides with significant antitu-mor activity were isolated from several members of the familyAsclepiadaceae (Braca et al., 2002; Chen et al., 2010; Halaweishet al., 2004; Li et al., 2008; Plaza et al., 2005).

While C. sinaica is a commonly used plant in Saudi Arabia (Hab-ibuddin et al., 2008), to our knowledge, it has not yet been investi-gated in details from a phytochemical viewpoint. The scope of ourstudy was to explore the chemical composition of this plant in rela-tion to other Caralluma species and plants from the Asclepiadaceae,to document the bioactivity of some of their constituents. In order

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

2 S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx

to obtain a rather comprehensive view of the C. sinaica metabolo-me and check its potential for new compounds, the extract wasprofiled by NMR and high resolution (HR) ultra high pressureliquid chromatography–mass spectrometry (UHPLC–MS). Thepresent study focusses on the isolation and complete characteriza-tion of polyhydroxy pregnane ester glycosides along with someflavonoids by using 1D and 2D NMR spectroscopy and HR-MS.The quinone reductase induction of the isolated compounds wasalso 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 med-ium polarity, the plant was extracted with ethanol according to anestablished 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 com-pared with references to all previously reported compounds fromthe Caralluma genus.

This ethanolic extract was directly dissolved in deuteratedmethanol and profiled by NMR. The 1H- and gHSQC-NMR spectra(Fig. 1A and B) showed various glycosylated compounds throughthe 1H–13C–OH signal in the (3–4 ppm and 60–90 ppm in 1H-and 13C-NMR, respectively) region and the corresponding typicalanomeric 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 com-pounds which can be putatively assigned to pregnanes by studyingprevious reported data on Caralluma species (Abdel-Sattar et al.,2007; De Leo et al., 2005; Halaweish et al., 2004; Kunert et al.,

Fig. 1. 1H- and gHSQC-NMR spectra (CD3OD) of C. sinaica ethanolic extracts (A and B) asimilar to that of the boucerin derivative showing the high content in pregnane of the C

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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 attributedto the typical pattern of mono-substituted phenyl groups. This isalso in good agreement with previous reports on acylated pregn-anes. The comparison of the gHSQC-NMR spectrum from the crudeextract (Fig. 1B) with the one obtained with a known pregnaneglycoside (russelioside G, Fig. 1C) confirms the presence of pregn-anes in the ethanolic extract (Tanaka et al., 1990). Since almost noadditional signal could be detected, this also indicated that thiscrude extract is largely dominated by this type of compounds.

The presence of steroid glycosides was also confirmed by thepositive reaction to Libermann–Buchard and Keller–Kiliani testsperformed on the crude extract (Li et al., 2006).

In order to confirm this hypothesis, the crude extract washydrolysed after enrichment, and the NMR spectrum revealedthe presence of two aglycones, namely boucerin and caralumage-nin 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 diversityof all pregnane glycosides present, the extract was profiled by highresolution UHPLC combined with time of flight mass spectrometry(TOF-MS) (Grata et al., 2009). The chromatogram and correspond-ing ion map generated in the negative ion mode (NI) revealed anextremely complex composition (Fig. 2). The automatic peakdetection at a threshold level of 5% indicated that 40 features ofmore than 500 Da could be detected and this number was over ahundred when the intensity threshold was lowered to 1%. Themolecular formula of all these compounds was determined directlyfrom the TOF-MS data generated on the extract. The combinationof high mass accuracy (5 ppm) and heuristic filters (Kind andFiehn, 2007) provided putative formulae that matched well withglycosylated pregnanes. Based on this preliminary information

nd gHSQC of russelioside G (C). The gHSQC spectrum of the crude extract is highly. sinaica extract.

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

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 detectedfeatures (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 highcomposition complexity of the crude extract.

S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx 3

and the important pregnane diversity recorded, isolation of themain constituents of C. sinaica extract was conducted for a com-plete structural assignment.

2.2. Identification of pregnanes from C. sinaica

Prior to the isolation, the crude ethanolic extract of C. sinaicawas submitted to liquid–liquid partition between water andsolvents of increasing polarity. The CHCl3 and the n-butanol frac-tions containing most of the pregnanes were fractionated byrepeated normal and reversed-phase liquid chromatography tech-niques. This afforded six new (1, 4, 8, 9, 11 and 12) and sevenknown (2, 3, 5–7, 10 and 13) polyhydroxy pregnane glycosides(Fig. 3) along with three flavonoids (14, 15, 16). A detailed struc-tural assignment of the new pregnanes is provided below.

All isolated compounds, except flavonoids, gave positive Liber-mann–Buchard and Keller–Kiliani tests, indicating the presenceof a steroidal skeleton with 2-deoxysugar moiety (Li et al., 2006).Spectroscopic analysis (Tables 1–4) and comparison with previ-ously reported data, allowed the identification of the aglycone ofcompounds 1, 4, 8, 9, 11 and 12 as the C/D-cis-polyoxy pregnanederivative 3b, 12b, 14b, 20b tetrahydroxy-(20R)-pregn-5-ene

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(boucerin) (Nikaido et al., 1967; Qiu et al., 1997). As describedbelow, among these compounds, the new pregnanes possessedan ester group at C-12 and C-20 positions and a straight sugarchain consisting of 2–4 sugar units connected to C-3 of theaglycone.

With all isolated compounds, the ester linkages attached atpositions 12 and 20 of the aglycone caused a downfield shift ofabout 1.00 ppm in 1H NMR signals of H-12 and H-20 and changedtheir splitting patterns (dd or q), while it induced a downfield shiftof about 4.0 ppm in 13C NMR of the corresponding C-12 and C-20signals (Hayashi et al., 1988). The presence of a double bond be-tween C-5 and C-6 in the boucerin nucleus caused a downfield shiftof the Me-18 (dH 1.04 in analogues with D5 compared with 0.83 indihydro). On the other hand, the glycosylation at C-3 induced thefollowing 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 aglyconemoiety was deduced from NOESY experiments and comparisonof the chemical shifts of the carbons and proton coupling constantswith those reported for related pregnanes (Ahmad et al., 1988; Ba-sha and Ahmad, 2007; Panda et al., 2003). The large homonuclearcoupling constants (7.5–10.5 Hz) of the anomeric protons of each

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

RO

OH

OR1

H

R2

H

18

1720

21

1

35

7

9

1112

15

19

6

5'

4'

3'

2'

7'6' O

1'2'

1'2'

3'4'

5'

1'

O

O

Cym

4

Glc

=E

ThevCym

4

Allom

4```

1'1''

1'''6

Dig

Glc 4

O

CH3O

OO

HOH2CHO

HOOH

Comp. R R1 R2

1 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

O

OHH3CO

OOH

HOHOHOH2C

Cym

4O

CH3O

OOO

CH3O

O

OHH3CO

HO

= Tig

= A

Cym

4

Cym

4 O

CH3O

OOO

CH3O

O

OHCH3O

HO4```

1'1''

1'''6

Cym

4

Cym

4O

CH3O

OOO

CH3O

O

OH

HOHOH2C

HO

Glc

4

=F

=H

=G

Allom1'

1''

1'''6

Cym

4

Cym

4 O

CH3O

OOO

CH3O

O

OHCH3O

4```O

OHOH2C

OHHO

HO

Glc

1'1''

6

Cym

4

Cym

4 O

CH3O

OOO

CH3ODig

4 O

OHH3CO

4O

HOH2C

OHHO

HO

Glc

Cym

4

4```

Cym

4O

CH3O

OOO

CH3O

O

OHO

OO

HOH2C

OHHO

HO

Glc

ThevCH3

O

= C

=D

= Bz

= Ac

= B

Fig. 3. Identified pregnanes from C. sinaica (Ac = acetyl; Bz = benzoyl; Tig = tigloyl; Allom = allomerose; Cym = cymarose; Dig = digitalose; Glc = glucose; Thev = thevetose).

4 S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx

sugar unit, in the 1H NMR spectrum, were typical of their axialconfiguration in hexopyranoses in 4C1 (D) conformation, havingb-glycosidic linkage (García, 2011).

The structures were also validated according to the molecularformula obtained using HRESITOF-MS in positive ionisation (PI)and NI modes. All compounds were always detected in NI, mainlyas formic acid adducts. In PI however, sodium adducts wererecorded 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/z989.5210 suggesting a molecular weight of 944.5156 and molecularformula C51H76O16 with 14 degrees of unsaturation. This formulawas confirmed by 13C NMR and APT NMR. The intense IR absorptionbands at 3400, 1720 and 1608, 1504 and 1235 cm�1 indicated thepresence of an hydroxyl, a carbonyl ester and an aromatic ring.The broad doublet at dH 5.48 in the 1H NMR was characteristic fora 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 at79.0, 79.7 and 75.3 ppm, in the HSQC spectrum, and assigned for

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protons H-3, H-12 and H-20, respectively. The oxygenated quater-nary carbon at dC 87.3 was assigned to C-14 in accordance withreported data (Hayashi et al., 1988; Kunert et al., 2009). The carefulcomparison of the NMR data with those reported for similar com-pounds (Abdel-Sattar et al., 2007, 2008; Aquino et al., 1996; Bracaet al., 2002), suggested that the pregnane skeleton of 1 was 3b,12b, 14b, 20b-tetrahydroxy-pregn-5-ene. The 13C NMR spectra of1 also showed signals due to two ester moieties (two carbonyl car-bons at dC 167.8 and 172.3). One of the two esters was a benzoylmoiety 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) whichwere 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; Rasoana-ivo et al., 1991). The HMBC 3J correlation between the carbonyl ofthe 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 groupat C-12. The second ester was an acetyl moiety as shown by NMRdata (dH 1.94, dC 21.7) and its attachment at C-20 was confirmedby the 3J correlation between H-20 (dH 4.94) and the second

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

Table 11H NMR spectral data for the aglycone moieties of compounds 1, 4, 7, 8, 10, 11 (500 MHz, CD3OD).

Comp dH 1 4 8 9 11 12

1a 1.17, m 1.10, m 1.10, m 1.12, m 1.15, m 1.10, m1b 1.84, m 1.75, m 1.75, m 1.77, m 1.85, m 1.75, m2a 1.92, m 1.90, m 1.90, m 1.90, m 1.90, m 1.90, m2b 1.54, m 1.51, m 1.52, m 1.52, m 1.53, m 1.51, m3 3.50, m 3.50, m 3.50, m 3.54, m 3.51, m 3.50, m4a 2.38, m 2.39, m 2.37, m 2.39, m 2.37, m 2.38, m4b 2.16, m 2.14, m 2.17, m 2.18, m 2.17, m 2.17, m5 – – – – – –6 5.48, br d 5.46, br d 5.48, br d 5.46, br d 5.46, br d 5.49, br s

(5.4) (3.5) (5.0) (5.0) (5.0)7a 1.85, m 1.86, m 1.87, m 1.87, m 1.87, m 1.92, m7b 2.25, m 2.25, m 2.27, m 2.27, m 2.25, m 2.25, m8 1.85, m 1.87, m 1.87, m 1.87, m 1.87, m 1.87, m9 1.35, m 1.35, m 1.35, m 1.35, m 1.37, m 1.37, m10 – – – – – –11a 1.85, m 1.85, m 1.85, m 1.85, m 1.75, m 1.84, m11b 1.64, m 1.65, m 1.65, m 1.65, m 1.66, m 1.65, m12 4.85 dd 4.62, dd 4.84, m 4.84, m 4.98, dd 4.98, m

(12.0, 4.0) (12.5, 5.0) (12.0, 4.0)13 – – – – – –14 – – – – – –15a 1.65, m 1.66, m 1.65, m 1.67, m 1.69, m 1.65, m15b 1.82, m 1.81, m 1.80, m 1.82, m 1.88, m 1.87, m16a 1.54, m 1.60, m 1.59, m 1.59, m 1.62, m 1.70, m16b 1.87, m 1.85, m 1.91, m 1.91, m 1.98, m 2.04, m17 2.06, m 2.09, m 2.06, m 2.06, m 2.17, m 2.28, m18 1.17, s 1.07, s 1.17, s 1.17, s 1.12, s 1.14, s19 1.05 s 1.03, s 1.04, s 1.04, s 1.04, s 1.01, s20 4.94, br q 4.92, m 4.94, br q 4.96, br q 5.09, br q 5.25, m

(6.0) (6.0) (6.0) (6.5)21 1.11, d 1.14, d 1.10, d 1.10, d 1.10, d 1.21, d

(6.0) (6.5) (6.0) (6.0) (6.5) (5.0)

Bz (12) Bz (12) Bz (12) Bz (12) Bz (12)

1’ – – – – –2’ – – – – –3’, 7’ 8.15 br d 8.12, d 8.14, d 8.05, d 7.75, br d

(7.5) (7.0) (7.0) (7.0) (8.5)4’, 6’ 7.50, t 7.51, t 7.53, t 7.51, t 7.25, t

(7.5) (8.0) (8.0) (8.0) (8.0)5’ 7.60, t 7.62, t 7.63, t 7.62, t 7. 52, t

(7.5) (7.5) (7.5) (7.5) (7.5)

Ac (20) Ac (20) Ac (20) Ac (20) Bz (20)

1’ – – – – –2’ 1.94, s 2.00, s 1.94, s 1.94, s –3’, 7’ 8.05, br d (8.5)4’, 6’ 7.50, t (8.5)5’ 7.68, t (7.5)

Tig (12) Tig (20)

1’ – –2’ – –3’ 6.98, br q (7.0) 6.55, br q (6.0)4’ 1.85, d (6.0) 1.62, d (7.0)5’ 1.91, br s 1.70, br s

J values are in parentheses and reported in Hz; chemical shifts are given in ppm.

S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx 5

carbonyl signal at dC 172.3. Therefore, the aglycone of 1 was identi-fied 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 trisaccha-ride glycoside. The full assignment of all protonated carbons wasaccomplished by interpretation of the gHSQC, gHMBC, DQF-COSYand NOESY experiments which allowed the sequential identifica-tion of H-1 to H-6 within each sugar unit. By comparing these datawith 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 ascymarose, 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

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13C NMR signals at 18.3, 18.7, 18.6 ppm, respectively, and assignedfor three secondary methyl groups of monosaccharides at the posi-tions 6Thev, 6CymII and 6CymI. Moreover, the 1H NMR methyl singletsat dH 3.65, 3.44 and 3.45 were correlated with the carbon signals at61.1, 58.6 and 58.5 ppm and ascribed to the three methoxyl groupsof the sugar moieties attached to C-3Thev, C-3CymII and C-3CymI,respectively. The splitting pattern and coupling constant of H-3of thevetose (t, J = 8.0 Hz) was consistent with the axial orientationof H-2, H-3 and H-4 characteristic for this sugar (Kunert et al.,2009; Nasipuri, 1994). Both cymarose units were glycosylated atC-4 as shown by a downfield shifts observed for C-4CymII andC-4CymI (84.0 and 83.8 ppm). Cross peaks due to three bond corre-lations, in gHMBC, between C-4CymI and H-1CymII (dH 4.81), andC-4CymII and H-1Thev (dH 4.31) indicated the sequence of the

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

Table 213C 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 38.6, t 38.4, t 38.5, t 38.6, t 38.4, t 38.4, t2 30.6, t 30.6, t 30.7, t 30.8, t 30.6, t 30.6, t3 79.0, d 79.0, d 79.8, d 79.8, d 79.1, d 78.9, d4 39.7, t 39.8, t 39.8, t 39.8, t 39.8, t 39.4, t5 140.5, s 140.5, s 140.5, q 140.6, q 140.5, s 140.8, s6 123.0, d 123.0, s 123.1, d 123.1, d 123.0, d 122.9, d7 28.2, t 28.2, d 28.2, t 28.2, t 28.1, t 28.1, t8 38.0, d 37.9, d 38.0, d 38.0, d 37.8, d 37.7, d9 44.7, d 44.5, d 44.6, d 44.6, d 44.6, d 44.5, d10 38.4, s 38.3, s 38.4, q 38.4, q 38.3, s 38.2, s11 27.1, t 27.1, t 27.3, t 27.3, t 27.3, t 27.4, t12 79.7, d 78.7, d 79.3, d 79.3, d 79.0, d 80.1, d13 53.1, s 53.1, s 53.4, q 53.4, q 53.5, q 53.5, s14 87.3, s 87.5, s 87.5, q 87.5, q 87.4, s 87.3, s15 33.0, t 32.9, t 33.0, t 33.1, t 32.8, t 32.3, t16 26.1, t 25.7, t 26.0, t 26.0, t 25.5, t 25.5, t17 51.2, d 51.0, d 51.5, d 51.5, d 50.9, d 51.1, d18 9.8, q 10.1, q 10.2, q 10.2, q 10.0, q 10.2, q19 19.9, q 19.5, q 19.9, q 19.9, q 19.8, q 19.8, q20 75.3, d 75.2, d 75.2, d 75.2, d 75.3, d 75.3, d21 19.5, q 19.8, q 19.5, q 19.5, q 19.6, q 19.7, q

Bz (12) Bz (12) Bz (12) Bz (12) Bz (12)

1’ 167.8, s 167.9, q 168.0, q 168.0, s 167.3, s2’ 132.0, s 131.9, q 131.9, q 131.9, s 131.7, d3’, 7’ 130.6, d 130.7, d 130.7, d 130.6, d 130.3, d4’, 6’ 129.5, d 129.6, d 129.6, d 129.7, d 129.5, d5’ 134.3, d 134.4, d 134.4, d 134.3, d 133.9, d

Ac (20) Ac (20) Ac (20) Ac (20) Bz (20)

1’ 172.3, s 172.4, s 172.4, q 172.6, q 167.9, s2’ 21.7 q 21.7, q 21.8, q 21.9, q 132.5, d3’, 7’ 130.7, d4’, 6’ 129.8, d5’ 134.3, d

Tig (12) Tig (20)

1’ 169.5, s 169.0, q2’ 130.1, s 130.3, q3’ 138.7, d 138.0, d4’ 14.5, q 14.5, q5’ 12.3, q 12.2, q

6 S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx

trisaccharide chain. The absence of any glycosidation shift, in 13CNMR signals, suggested that thevetose is the terminal sugar. Theglycosidation of the aglycone by this trisaccharide at C-3 wasdetermined from a 3J correlation, between the anomeric protonat dH 4.87 (H-1CymI) and C-3 at dC 79.0. The above mentioned dataproved that 1 is 12-O-benzoyl-20-O-acetyl boucerin-3-O-b-D-thevetopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymar-opyranoside. To our knowledge this compound is reported here forthe 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/z803.4220 and a [M+HCOO]� at m/z 825.4300 in NI, suggesting amolecular formula of C41H64O14 with an unsaturation number of10. The NMR spectral analysis of the aglycone moiety of 4 revealedsimilarities with 1 except for the presence of a tigloyl ester moietyinstead of the benzoyl attached to C-12. The presence of the tigloylester was established by an olefinic methine proton signal at dH

6.98 (br q, J = 7.0 Hz) correlated, in gHSQC, with C-30 at138.7 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 quaternarycarbon at 130.1 ppm assigned to C-20 finally showed the presenceof a tiglate (Abdel-Sattar et al., 2007; Braca et al., 2002; Shuklaet 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-

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tion between H-20 (dH 4.92, m) and the acetate ester carbonyl at172.4 ppm revealed acetylation at C-20. In addition to the aglyconeresonances, the 13C NMR spectra of 4 exhibited 13 signals relatedto 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 interpretationof DQF-COSY, gHSQC and gHMBC spectra allowed the assignmentof the two sugars as b-D-glucopyranoside and b-D-cymaropyrano-side (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 de-scribed for 1. Thus, the novel structure of 4 was 12b-O-tigloyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-cymaro-pyranoside.

Compounds 8 and 9 were obtained as a mixture (4.4 mg) as sug-gested by NMR data. 8 and 9 could not be separated by HPLC orUHPLC by using various conditions. The single corresponding LC-peak 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 twoisomers with a molecular formula of C57H86O21. An extensive studyof their 1D- and 2D-NMR data allowed the complete assignmentsof each component of the mixture. The obtained 13C-APT-NMRspectrum implied an identical aglycone part for 8 and 9 as shownby signals present in overlapping pairs with an intensity ratio ofabout 3:2. As for 1 and 4, the aglycone was recognized as 12-b-O-benzoyl-20-O-acetyl boucerin.

The difference between the two isomers lies in the tetra-saccha-ride sugar chain attached to C-3. Compound 8 and 9 displayed com-mon signals for two cymarose (which was confirmed by comparisonwith 1) and one glucose residue. Taking into account the molecularformula of both isomers, the presence of the latter sugars and theaglycone, the unassigned part corresponded to a mass value differ-ence of 160 Da, with the molecular formula C7H12O4. The presenceof two methoxy (dH 3.53, 3.64 ppm and dC 58.9, 61.3 ppm) and twosecondary 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 acharacteristic upfield-shifted methoxyl (dH 3.53, dC 58.9) attachedto C-3 in comparison to the downfield-shifted methoxyl of theve-tose and allomerose (dH > 3.55, dC > 61.0) (Braca et al., 2002; Hal-aweish et al., 2004). In addition, the characteristic doublet at dH

4.15, assigned to H-4 of the digitalose moiety, had a distinctlysmall coupling constant (d, J = 2.5 Hz) that was consistent withthe equatorial orientation of this proton and its weak coupling tothe axially oriented H-3 and undetected coupling to H-5 (Nasipuri,1994). Furthermore, the presence of a NOESY correlation betweenH-3/H-4 (dH 3.21/dH 4.15), and the absence of a similar correlationbetween H-2/H-4 (dH 3.64) required an equatorial orientation of H-4 (Kunert et al., 2009). All additional NMR data were also similar tothose reported for digitalose (Braca et al., 2002; Qiu et al., 1999).The structure of 8 was thus determined as 12-b-O-benzoyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-digitalopyr-anosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside.

On the other hand, the anomeric signals for the sugar units con-stituting the tetra-saccharide moiety of 9 were clearly separatedfrom those of 8 in the 13C data as revealed by the APT and gHSQCspectra (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 deter-mined to be thevetose from the characteristic chemical shift valuesof C-1 (dC 104.3), C-3 (dC 86.2) and C-4 (dC 82.9) which were closelysimilar to those previously reported and discussed above for 1(Braca et al., 2002; Shukla et al., 2009). It is worth noting that C-4 of thevetose, in 9, appeared significantly downfield shifted (dC

82.9) in comparison to its value in 1 (dC 76.5) which is obviouslydue to the attachment of another sugar moiety at C-4 of thevetosein 9 (glycosidation shift). The structure of 9 was determined as

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

Table 31H 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 4.89 dd 4.85, dd 4.82, m 4.86, dd 4.89, d(10.0, 2.5) (9.5, 2.5) (10.0, 2.5) (9.5, 2.0) (10.5)

2a 1.57, br dd 1.51, dd 1.57, m 1.57, m 1.56, dd 1.65, m(16.0, 12.0) (15.5, 11.5) (16.0, 12.5)

2b 2.08, br dd 2.07 br dd 2.06, br dd 2.06, br dd 2.05, m 2.15, m(15.0, 3.0) (13.5, 4.0) (13, 4.0) (13, 4.0)

3 3.86, q 3.86, q 3.85, q 3.85, q 3.91, q 3.84, m(4.0) (3.0) (3.0) (3.0) (3.0)

4 3.25, dd 3.25, m 3.26, dd 3.26, dd 3.31, dd 3.22, m(10.0, 4.0) (9.5, 2.0) (9.5, 2.0) (9.5, 2.5)

5 3.85, d 3.89, dq 3.86, m 3.86, m 3.86, m 3.80, m(6.0, 9.5) (7.5, 10.5)

6 1.21, d 1.30, d 1.20, d 1.20, d 1.30, d 1.21, d(6.0) (6.0) (6.0) (6.0) (6.0) (6.5)

OCH3 3.45, s 3.44, s 3.43, s 3.43, s 3.46, s 3.44, s

Cym II Cym II Cym II Cym II

1 4.81, dd 4.79, dd 4.76, dd 4.80, d(8.5, 2.0) (10.0, 2.0) (10.0, 2.5) (8.0)

2a 1.70, m 1.50, m 1.50, m 1.62, m2b 2.11, m 2.06, dd 2.06, dd 2.12, m

(13.0, 4.0) (13.0, 4.0)3 3.86, m 3.85, q 3.85, q 3.92, m

(3.0) (3.0)4 3.28, m 3.26, dd 3.26, dd 3.32, m

(9.5, 2.0) (9.5, 2.0)5 3.85, dq 3.78, m 3.78, m 3.90, m

(6.0, 9.5)6 1.32, d 1.30, d 1.30, d 1.31, d

(6.0) (6.0) (6.0) (6.5)OCH3 3.44, s 3.45, s 3.45, s 3.45, s

Thev Dig Thev

1 4.31, d 4.54, d 4.44, d(8.0) (8.0) (9.0)

2 3.27, br s 3.64, t 3.20, m(9.5)

3 3.05, t 3.21, dd 3.25, m(8.0) (8.5, 2.5)

4 3.06, m 4.15, d 3.30, m(2.5)

5 3.32, m 3.60, m 3.70, m6 1.29, d 1.30, d 1.39, d

(6.0) (6.0) (6.0)OCH3 3.65, s 3.53, s 3.64, s

Glc Glc Glc Glc Glc

1 4.36, d 4.31, d 4.31, d 4.34, d 4.36, d(8.0) (7.5) (9.0) (8.0) (8.0)

2 3.24, dd 3.22, m 3.22, t 3.20, t 3.23, t(9.0, 8.0) (8.5) (8.5) (7.5)

3 3.30, d 3.37, d 3.37, d 3.31, m 3.30, m(7.5) (9.0) (9.0)

4 3.25, d 3.23, d 3.23, d 3.25, d 3.27, d(8.0) (6.5) (6.5) (6.5) (8.5)

5 3.27, m 3.32, m 3.32, m 3.20, m 3.35, m6a 3.66, dd 3.63, dd 3.63, dd 3.65, dd 3.65, dd

(11.5, 5.5) (11.5, 4.5) (11.5, 4.5) (12.0, 5.5) (12.5, 5.5)6b 3.90, dd 3.84, m 3.84, m 3.91, dd 3.90, dd

(10.5, 4.0) (12.0, 3.0) (11.0, 3.0)

J values are in parentheses and reported in Hz; chemical shifts are given in ppm.

S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx 7

12-b-O-benzoyl-20-O-acetyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-thevetopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside. The identification of the glycosidation site andsugar sequence in both 8 and 9 (H-1CymI–C-3, H-1CymII–C-4CymI, H-1Thev (9) or Dig (8)–C-4CymII and H-1Glc–C-4Thev (9) or Dig (8)) was con-firmed 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

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data of the aglycone of 11 were closely similar to those of 1 exceptthe replacement of the signals of the acetate moiety with those of atigloyl ester. The position of attachment of the tigloyl moiety at C-20 of the aglycone was deduced from the gHMBC correlationsbetween 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 in1H- and APT-NMR spectra of 11 (dH 4.34, d, J = 8.0 Hz, dC 106.2and dH 4.86, dC 97.2) suggested a disaccharide glycoside. The

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

Table 413C NMR spectral data for sugar moieties of compounds 1, 4, 7, 8, 10, 11 (125 MHz,CD3OD).

Comp dH 1 4 8 9 11 12

Cym I Cym Cym I Cym I Cym Cym I

1 97.3, d 97.3, d 97.3, d 97.3, d 97.2, d 97.2, d2 36.6, t 36.7, t 36.9, t 36.9, t 36.7, t 36.4, t3 78.6, d 78.0, d 78.1, d 78.1, d 78.7, d 78.5, d4 83.8, d 83.7, d 84.1, d 84.1, d 83.8, d 83.7, d5 69.9, d 70.2, d 70.1, d 70.1, d 70.2, d 69.9, d6 18.6, q 18.7, q 18.5, q 18.5, q 18.6, q 18.5, qOCH3 58.5, q 58.6, q 58.3, q 58.3, q 58.5, q 58.4, q

Cym II Cym II Cym II Cym II

1 101.1, d 101.2, d 101.2, d 101.1, d2 36.4, t 35.9, t 35.9, t 36.7, t3 78.6, d 78.3, d 78.3, d 78.7, d4 84.0, d 84.3, d 84.3, d 83.9, d5 70.0, d 70.3, d 70.3, d 70.1, d6 18.7, q 18.8, q 18.8, q 18.6, qOCH3 58.6, d 58.7, q 58.7, q 58.6, q

Thev Dig Thev

1 106.2, d 104.8, d 104.3, d2 75.1, d 72.7, d 75.7, d3 87.4, d 85.8, d 86.2, d4 76.5, d 75.8, d 82.9, d5 73.0, d 71.5, d 72.7, d6 18.3, q 17.5, q 18.6, qOCH3 61.1, d 58.9, q 61.3, q

Glc Glc Glc Glc Glc

1 106.1, d 106.5, d 106.0, d 106.2, d 106.2, d2 75.3, d 76.1, d 76.1, d 75.4, d 75.5, d3 78.0, d 77.9, d 77.9, d 78.0, d 77.9, d4 71.7, d 72.0, d 72.0, d 71.8, d 71.7, d5 77.9, d 78.7, d 78.7, d 75.4, d 78.0, d6 63.0, t 63.2, t 63.2, t 63.0, t 62.9, t

8 S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx

NMR data of the sugar part was in complete agreement with thoseof Glc-(1?4)-Cym, which established the structure of 11 as 12-b-O-benzoyl-20-O-tigloyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranoside. The identification of the glycosidation siteand sugar sequence (H-1Cym–C-3, H-1Glc–C-4Cym) was confirmedin 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/z1031.5013 and a [M+HCOO]� in NI at m/z 1053.510 suggestingthe molecular formula C55H76O17. The NMR data of the aglyconewas similar to the previous compounds except that both hydroxylgroups at C-12 and C-20 were acylated with two benzoyl moieties.This was confirmed by the presence of two sets of coupled aro-matic proton signals in the 1H-NMR spectrum corresponding totwo benzoyl groups. The complete assignment of all proton andcarbon signals of the two benzoyl esters was carried out using

O

OH

H

H

O

H

O

CymGlc

O

O

OOHO

HOOH

OH

Fig. 4. Structure of 11 and some selec

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DQF-COSY, gHSQC and gHMBC experiments (Tables 1 and 2). Thepresence of three anomeric protons and the corresponding carbonsin 1H and 13C NMR spectra of 12 (Tables 3 and 4) suggested atrisaccharide glycoside. The sugar units were identified as one D-glucose and two D-cymarose units by comparison with data ofabove discussed compounds. Similarly, the position of attachmentto the aglycone was determined at C-3 from a strong 3J correlation,in the gHMBC spectrum, between H-3 (dH 3.50) and the anomericcarbon of the first cymarose unit at 97.2 ppm. 12 was identifiedas 12b-20-O-dibenzoyl boucerin-3-O-b-D-glucopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside. Again, the se-quence of sugar units was determined by gHMBC experimentwhich showed a 3J correlation between H-1Glc (dH 4.36) and C-4Cym-

II (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 boucerin-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-cymaropyrano-syl-(1?4)-b-D-cymaropyranoside (3) (Tanaka et al., 1990), russelio-side G (5) (Abdel-Sattar et al., 2007), russelioside F (6) (Abdel-Sattaret al., 2007), 12b-O-tigloyl-20-O-acetyl boucerin-3-O-b-D-glucopyr-anosyl-(1?4)-6-deoxy-3-O-methyl-b-D-allopyranosyl-(1?4)-b-D-cymaropyranosyl-(1?4)-b-D-cymaropyranoside (7) (Braca et al.,2002), russelioside E (10) (Abdel-Sattar et al., 2007) and caralumage-nin-20-O-b-D-glucopyranosyl-3-O-b-D-glucopyranosyl-(1?4)-b-D-digitalopyranoside (13) (Qiu et al., 1999), by comparison with previ-ously reported NMR data.

In addition to the pregnanes, three flavonoids were isolatedfrom the ethanolic extract. These polyphenols were identifiedbased 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 outthat luteolin 40-O-neohesperidoside (15) was also isolated fromCaralluma lasiantha (Qiu et al., 1999), Caralluma attenuata, Carallu-ma umbellata (Ramesh et al., 1999), Caralluma tuberculata (Rizwaniet al., 1990) and Caralluma russeliana (Al-Yahya et al., 2000). Sinceit appears to be a common constituent of plants of the Carallumagenus in addition to pregnanes, it might be considered as a chemo-taxonomic marker for genus Caralluma. On the other hand, luteolinand rutin are reported here for the first time in the genusCaralluma.

2.3. Determination of the absolute configuration of C-20

A careful investigation was carried out to determine the abso-lute configuration of C-20 which was left unassigned in mostpublished papers on pregnane steroids bearing a hydroxyl groupat this position (Braca et al., 2002; De Leo et al., 2005; Halim andKhalil, 1996). Kimura et al. (1982) compared 13C NMR spectral dataof various 20R and 20S pregnane compounds, which were synthes-ised by the reduction of 20 pregnanones, and also have hydroxyl

3CO

O

CH3

HCH3

OH

H3CO

H

O

H

ted 2J and 3J gHMBC correlations.

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

Table 5QR inducing activity of selected isolated pregnanes.

Compounds QR inductionCD (lM)

CytotoxicityIC50 (lM)

Chemoprevention indexCI (IC50/CD)

1 14.2 17.5 1.22 3.1 9.3 3.03 3.3 10.9 3.34 >20.0 >20.0 –5 >20.0 >20.0 –6 >20.0 16.3 <0.87 <10.0 >20.0 >2.0

10 13.9 >20.0 >1.611 >20.0 13.7 <0.812 >20.0 >20.0 –13 >20.0 >20.0 1.2

S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx 9

group at C-12. They found remarkable differences in the 13Cchemical shifts values for C-16 and C-20 between the two sets ofepimers. In the case of 20R epimers approximately 71.0 ppm wasfound for C-20 and 27.0 ppm for C-16, while the corresponding val-ues for the 20S epimers were near 65.0 and 19.0 ppm, respectively.This was explained by the fact that the free rotation of the sidechain in the 20-hydroxy-C/D-cis-pregnane type steroids was as-sumed to be restricted by steric hindrance among the C-18 methyl,C-21 methyl and C-20 hydroxyl groups on the basis of a CPK model(the space-filling model). Therefore, the C-21 methyl group in 20Salcohols of C/D-cis pregnane-type steroids is close to the plane ofthe D-ring, resulting in upfield shifts of the C-20 resonances (stericshift) (Kimura et al., 1982).

Based on these results, the authors suggested that the applica-tion of 13C NMR to 20-hydroxy-C/D-cis-pregnane-type steroidswas a more useful and simpler method for investigation of theC-20 configuration than procedures involving the measurementof circular dichroism or optical rotatory dispersion of their deriva-tives with a suitable chromophore (Kunert et al., 2009). In thepresent study, the 13C NMR resonances of the aglycone nucleusof all isolated pregnane glycosides had almost identical values tothose reported for the 20R epimer pregnane steroids with similaracylation pattern in positions 12 and 20 (Hayashi et al., 1988; Kim-ura et al., 1982; Kunert et al., 2009; Tanaka et al., 1990). In eachcompound, C-16 and C-20 resonated near 26.0 and 75.2 ppmrespectively, indicative of an R-configuration for C-20 accordingto Kimura et al. (1982). On the basis of the above evidences, theaglycone was determined as the boucerin derivative 12-O-ben-zoyl-20-O-acetyl-3b, 12b, 14b, 20b-tetrahydroxy-(20R)-pregn-5-ene. Boucerin is a well-known and widespread aglycone in thefamily Asclepiadaceae (Abdel-Sattar et al., 2008; Braca et al.,2002; Halim and Khalil, 1996; Lee-Juian et al., 1994). On the otherhand, the 20S epimer of boucerin, known as boucergenin, wasrecently isolated from C. umbellata and identified by Kunert et al.with C-16 and C-20 at 18.9 and 65.8 ppm, respectively (Kunertet al., 2009).

Since pregnane glycosides have not yet been tested for theircancer chemopreventive effects and a relatively large series ofdiverse derivatives was available, quinone reductase inductionactivity was performed in this study.

2.4. Cancer chemopreventive activity of C. sinaica

Several compounds of natural and synthetic origin activate theexpression of phase II detoxification enzymes which are thought toact against the development of cancer. These enzymes are regu-lated by two distinct pathways; one is the arylhydrocarbon recep-tor mediated activation of the xenobiotic response element (XRE)and the other is a Keap1/Nrf2 mediated activation of the antioxi-

Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane g10.1016/j.phytochem.2012.04.003

dant response element (ARE). The latter only leads to the inductionof phase II metabolizing enzymes, whilst the former also inducesphase I enzymes comprising mainly of the cytochrome P450 en-zyme family. Compounds activating phase I and II enzymes aretermed ‘‘bifunctional inducers’’ and seem to be less favourableregarding cancer development, whereas compounds leading solelyto the expression of phase II enzymes are called ‘‘monofunctional’’and are regarded as beneficial in chemoprevention. The inductionof the phase II detoxification enzyme quinone reductase (QR) hasbeen used as an indicator for the chemopreventive activity of theisolated constituents from C. sinaica (Table 5). QR inducing activityof selected pregnane glycosides from C. sinaica was tested in themouse hepatoma cell lines Hepa1c1c7 and its clone c35 whichhas a defective arylhydrocarbon receptor mediated signalling.Compounds 2 and 3 showed the strongest QR induction exhibitinga CD (concentration required to double the specific enzyme activ-ity) of 3.09 and 3.31 lM, respectively (Table 5). With CDs of 14.2, 7and 13.9 lM, 1, 7 and 10, respectively, displayed a weaker activity.Compounds 4–6 and 11 showed no QR induction. Notably, the QRinducing activity was markedly reduced for all compounds in c35cells. This indicates that all constituents are bifunctional inducers.Moreover no synergy between the different compounds isolatedfrom C. sinaica was observed. None of the fractions tested duringthe bioassay guided fractionation process showed higher activitycompared to their corresponding isolated constituents.

The strongest QR inducers, 2 and 3, possess an O-benzoyl and atrisaccharide (O-methyl-b-D-allopyranosyl-(1?4)-b-D-cymaropyr-anosyl-(1?4)-b-D-cymaropyranoside) moiety on position C-12and C-3, respectively. Compounds 7 and 10, which showed a weak-er activity, also have the same sugar sequence in C-3, except thatan extra glucose moiety is added to the end. Also, the compoundhaving the cym–cym–thev sequence (1) showed some moderateQR inducing activity. No strong toxicity was revealed for the testedpregnanes (Table 5), but the cytotoxicity seemed to be increasedwhen the sugar sequence was short. The comparison of cytotoxic-ity between 5 and 11 showed that an O-tigloyl moiety in positionC-20 increased slightly the cytotoxicity. Thus, the activity of thesepregnanes might be modulated by the side chain in position C-12.The chemopreventive index (CI) was determined by measuring theratio between the cytotoxicity of the compounds in Hepa1c1c7 andthe CD.

These results confirmed the safety of the use of formulated C.sinaica (Decne.) extracts sold on local Saudi Arabian markets (Hab-ibuddin et al., 2008) wherein total pregnane content can reach over30% of the dry extract (Rajendran and Rajendran, 2011).

3. Conclusion

Plants of the genus Caralluma have a long tradition of use innutrition and medicine in several Asian countries. They have alsogained an increasing attention in recent years due to their diversepharmacological properties. The NMR and UHPLC-ESI-TOF-MS pro-filing of the whole plant ethanolic extract of C. sinaica promptlyhighlighted the presence of an abundant number of pregnane gly-cosides as shown when compared with published data of knownpregnane glycosides. This approach, followed by isolation andstructure elucidation by 1D- and 2D-NMR experiments, led to theidentification of six new and seven known polyhydroxy pregnaneester glycosides along with three previously reported flavonoids.

The bioassays showed that some of the isolated constituentsfrom C. sinaica were QR inducers with weak cytotoxicity.

The UHPLC-ESI-TOF-MS profile indicated that C. sinaica is a po-tential candidate for further exploitation with the aim of isolatingand characterizing more bioactive compounds that may be helpfulto understand the use of this plant in traditional medicine.

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

10 S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx

4. Experimental

4.1. General experimental procedures

Optical rotations were measured in methanolic solution on aPerkin–Elmer 241 polarimeter (USA), using one decimetre tube.UV spectra were obtained using a Hewlett–Packard HP-845UV–Vis spectrometer (USA). Infrared spectra were obtained inpotassium bromide discs using Perkin–Elmer FTIR model 1600spectrophotometer.

Micro-flow NMR analyses were performed on a Varian UnityInova 500 MHz NMR instrument (Palo Alto, CA, USA) equippedwith a 5 ll micro-flow CapNMR probe from Protasis/MRM (Savoy,IL, USA) having an active volume of 1.5 ll. All samples were dis-solved in 10 ll CD3OD.

HPLC was carried out on a Hewlett–Packard HP-1100 liquidchromatographic system (HP; Palo Alto, CA, USA) consisting of bin-ary pumps, an inline degasser, an auto-sampler and an HP-G1315photodiode array detector (PAD). HPLC separations were per-formed on an analytical scale using a Xbridge C18 analyticalcolumn (250 mm � 4.6 mm, 5 lm) with Xbridge C18 guard column(Waters, Milford, MA, USA). Preparative conditions were obtainedby direct conversion of the analytical condition to a Xbridge C18preparative column (250 mm � 10 mm, 5 lm) protected by aXbridge C18 guard column (Waters, Milford, MA, USA) using HPLCcalculator developed by Guillarme et al. (2008).

4.2. Plant material

The whole plant material (aerial parts and roots) of C. sinaica(Decne.) (=Boucerosia sinaica Decne.) was purchased in December2008 from a local herbal market in Al-Taif city in the westernregion of the Kingdom of Saudi Arabia and kindly identified by Pro-fessor Abdulraheem Abdulmogalli, Ministry of Agriculture, Egypt.A voucher specimen was deposited in the department of Pharma-cognosy, College of Pharmacy, King Saud University.

4.3. Extraction

The fresh plant material (8.0 kg) was chopped into thin seg-ments and dipped in boiling ethanol for 5 min in order to inactivatethe enzymes and then dried in a hot air oven at 60 �C (Khalil, 1995).The dried material was reduced to a powder (340 g) and extractedwith 75% ethanol till complete exhaustion.

4.4. UHPLC-ESI-TOF-MS analysis

In order to extract chlorophyll and other interfering apolar com-pounds before introducing the samples to UHPLC-ESI-TOF-MS(UHPLC-MS) analysis, the dried crude extract (1 mg) was dissolvedin 500 ll of CH3OH/H2O (85:15) and passed through Sep-Pak Vac1 ml (100 mg) C18 cartridges (Waters, Milford, MA, USA) mountedon a Visiprep™ SPE Vacuum Manifold (Sigma-Supelco, Park City,Bellefonte State, PA). Then, 1 ml of CH3OH/H2O (85:15) was usedto elute the compounds of interest from the cartridge. After dryingunder nitrogen, enriched extracts were dissolved in 250 ll CH3OH/H2O (85:15) at a concentration of 1.0 mg/ml for UHPLC–MSanalysis.

The UHPLC–MS profiles of the extract were performed on aMicromass-LCT Premier time-of-flight mass spectrometer with anelectrospray ionization (ESI) interface coupled with an AcquityUPLC system (Waters Assoc., Milford, USA). Waters Acquity re-versed phase UPLC columns at 40 �C (BEH C18, 2.1 � 150 mm,1.7 lm) were used with the following solvent system: A = 0.1% for-mic acid–water, B = 0.1% formic acid–CH3CN. The elution was

Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane g10.1016/j.phytochem.2012.04.003

performed at a flow rate of 320 ll/min using a gradient from 2%to 100% of eluent B in 30 min with an injection volume of 2 ll.

Mass spectra were acquired in both PI and NI modes (two inde-pendent experiments). Full scan mass spectra were acquired fromm/z 400–1600. The instrument parameters were: capillary temper-ature 225 �C, capillary voltage 2800 V, cone voltage 40 V, desolva-tion temperature 250 �C, source temperature 120 �C, cone gas flow20 l/h, desolvation gas flow 600 l/h with a Shimadzu LC pump, scantime 0.2 s, dynamic range enhancement (DRE) lockmass: 1 lg/mlsolution of leucine–enkephalin (Sigma–Aldrich) infused by a Shi-madzu LC pump (LC-10ADvp, Duisburg, Germany) through the Lock.

The molecular formulae were assigned with Masslynx 4.1(waters). The assignment was ascertained based on mass accuracy(5 ppm tolerance), as well as using heuristic filters (Kind and Fiehn,2007). In order to assess the chemical diversity of the extract, theautomatic peak detection was performed using MZmine2.2 (Plus-kal et al., 2010).

Fractions and pure compounds were controlled by UHPLC–MSto get purity and HR-MS information. Waters Acquity reversedphase UPLC columns at 40 �C (BEH C18, 1.0 � 50 mm, 1.7 lm) wereused with the following solvent system A and B. The elution wasperformed at a flow rate of 400 ll/min using a gradient from 5%to 95% of eluent B in 4 min with an injection volume of 1 ll atthe concentration of 10 lg/ml.

4.5. Isolation

The ethanolic extract (88 g) was successively extracted withpetroleum ether (5 g), chloroform (23 g) and n-butanol (29 g).The quinone reductase induction rate (QR-IR) of the crude extractwas 2.9 (20 lg/ml) and 2.2 (20 lg/ml), 2.5 (20 lg/ml), 1.2 (20 lg/ml) for the petroleum ether, chloroform and n-butanol fractions,respectively. Part of the chloroform extract (10 g) was chromato-graphed on top of silica gel packed column (50 � 3.5 cm), usingCHCl3/CH3OH solvent system in gradient elution mode and frac-tions of 200 ml each were collected to give five main fractions(CS 1–5).

Fraction CS-1 (0.20 g, QR-IR: 3.6 at 5 lg/ml) was further chro-matographed over silica gel using increasing amounts of ethylacetate in n-hexane as an eluent. The subfraction eluted with10% ethyl acetate in n-hexane afforded on precipitation compound1 (20 mg, QR-IR: 3.3 at 20 lg/ml) as an amorphous powder.

Fractions CS-2 (0.56 g, QR-IR: 3.7 at 5 lg/ml), CS-3 (0.35 g, QR-IR: 3.0 at 5 lg/ml), CS-4 (0.82 g, QR-IR: 1.4 at 5 lg/ml) and CS-5(0.94 g, QR-IR: 2.0 at 20 lg/ml) were further purified on RP-HPLCcolumn (flow rate 4.7 ml/min) using CH3CN:H2O:formic acid(55:45:0.1%) for fractions CS-2 and CS-3 to yield compounds 2(7 mg, tR = 31 min, QR-IR: 3.8 at 10 lg/ml) and 3 (5.7 mg,tR = 17.6 min, QR-IR: 3.1 at 10 lg/ml), respectively; CH3CN:H2O:formic acid (50:50:0.1%) for fractions CS-4 and CS-5 to yield com-pounds 4 (2.2 mg, tR = 8 min, QR-IR: 1.3 at 20 lg/ml), 5 (7.3 mg,tR = 10 min, QR-IR: 1.5 at 20 lg/ml), 6 (2.4 mg, tR = 18 min, QR-IR:1.6 at 20 lg/ml), 7 (1.3 mg, tR = 19.1 min, QR-IR: 3.1 at 20 lg/ml),8 and 9 (2.5 mg, tR = 20.2 min, QR-IR: 2.5 at 20 lg/ml of the mix-ture), 10 (8.7 mg, tR = 22 min, QR-IR: 2.2 at 20 lg/ml) from fractionCS-4 and compounds 11 (6 mg, tR = 17.2 min, QR-IR: 1.8 at 20 lg/ml) and 12 (3.7 mg, tR = 24.8 min, QR-IR: 1.8 at 20 lg/ml) fromfraction CS-5 as an amorphous powder. Part of the n-butanol frac-tion (120 mg) was purified on RP-HPLC using CH3CN:H2O:formicacid (25:75:0.1%) to yield compound 13 (15 mg, tR = 10 min, QR-IR: 1.3 at 20 lg/ml) as a colourless amorphous powder.

4.6. Acid hydrolysis

CHCl3 and n-butanol fractions, as well as the pure compounds 1,2, 5, 6, and 13 were hydrolysed according to the procedure

lycosides from Caralluma sinaica. Phytochemistry (2012), http://dx.doi.org/

S.M. Al-Massarani et al. / Phytochemistry xxx (2012) xxx–xxx 11

reported by Ma et al. (2007). The aglycones, as well as monosac-charides, were detected by TLC analysis combined with compari-son to known samples. Boucerin was identified as the aglyconeof compounds 1–12, and caralumagenin was revealed to be theaglycone of compound 13. Regarding the monosaccharide compo-nent of each hydrolyzed compound, cymarose was detected in 1, 2,5, and 6; glucose was detected in 4 and 7–9; digitoxose was de-tected only in 2.

4.7. Compound 1

White amorphous powder ½a�25D + 18.2 (CH3OH, c. 2.00); IR (KBr)

tmax 3400, 1720, 1608, 1504 and 1235 cm�1; HRTOF-MS m/z:989.5210 [M+HCOO]� (calc. for C51H76O16, 944.5153); see Table 1for 1H NMR (500 MHz, CD3OD) of aglycone; Table 2 for 13C NMR(125 MHz, CD3OD) of the aglycone; Table 3 for 1H NMR of sugarmoieties; and Table 4 for 13C NMR of sugar moieties.

4.8. Compound 4

White amorphous powder ½a�25D + 4.9� (MeOH, c. 1.4); IR (KBr)

tmax 3400 and 1722 cm�1; HRTOF-MS m/z: 803.4220 [M+Na]+,825.4300 [M+HCOO]� (calc. for C41H64O14, 780.4296); see Tables1–4 for NMR assignments.

4.9. Compounds 8 and 9

Colorless amorphous powder; IR (KBr) tmax 3400, 1720 and1235, 1608 and 1504 cm�1; HRTOF-MS m/z: 1129.5570 [M+Na]+

and 1151.5760 [M+HCOO]� (calc. for C57H86O21, 1106.5662); seeTables 1–4 for NMR assignments.

4.10. Compound 11

White amorphous powder ½a�25D + 18.7 (MeOH, c, 1.7); IR (KBr)

tmax 3400, 1727 and 1708 cm�1; HRTOF-MS m/z: 887.450[M+HCOO]� (calc. for C46H66O14, 842.4523); see Tables 1–4 forNMR assignments.

4.11. Compound 12

Colorless amorphous powder ½a�25D + 12.5 (MeOH, c. 0.2); IR

(KBr) tmax 3400, 1700, 1608, 1580 and 1280 cm�1; HRTOF-MS m/z: 1031.4921 [M+Na]+ and m/z 1053.51 [M+HCOO]� (calc. forC55H76O17, 1008.5123); see Tables 1–4 for NMR assignments.

4.12. Quinone reductase induction and cytotoxicity

Hepa1c1c7 cells and their clone c35 (ATCC, Rockville, MD, USA)were cultured according to the ATCC recommendations in a-mod-ified Minimal Essential Medium containing 2 mM glutamate,supplemented with 100 U/ml penicillin, 100 lg/ml streptomycin,and 10% fetal calf serum at 37 �C, 5% CO2, and humidified atmo-sphere. The method described by Kang and Pezzuto (2004) wasused. In brief, quinone reductase activity was determined by mea-suring NADPH-dependent menadiol-mediated reduction of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tothe corresponding blue fromazan after five minutes incubation atan absorption wavelength of 595 nm measured on a PowerWaveHT microplate spectrophotometer (BioTek Instruments, Luzern,Switzerland). In parallel, the amount of viable cells was deter-mined by protein quantification using crystal violet staining andmeasurement of the absorption at 595 nm on the aforementionedspectrophotometer.

The concentration required to double the specific enzyme activ-ity (CD), as well as the cytotoxicity (IC50) were calculated using

Please cite this article in press as: Al-Massarani, S.M., et al. Acylated pregnane g10.1016/j.phytochem.2012.04.003

GraphPad Prism 5 software. Finally, the chemopreventive index(CI) was calculated by dividing the IC50 by the CD value.

Declaration of interest

The authors report no conflict of interest.

Acknowledgement

The authors thank Philippe Eugster and Dr. Laurence Marcourtfor their help in the recording of the UHPLC–MS profiles andNMR spectra, respectively.

This research project was supported by a grant from theResearch Center of the Center for Female Scientific and MedicalColleges in King Saud University, and by a grant from the Alfredand Alice Lachmann Nutrition Fund.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phytochem.2012.04.003.

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