New pregnane glycoside derivative from Caralluma retrospiciens (Ehrenb)

Article abstract of DOI:10.1080/14786419.2014.1003186

Retrospinoside (1) is a new polyoxy pregnane glycoside which was isolated and characterised from the aerial parts of Caralluma retrospiciens (Ehrenb.) N. E. Br., family Apocynaceae. The structure was established as 3-O-[β-D-glucopyranosyl-(1 → 4)-β-D-(3-O-methyl-6-desoxygalactopyranosy)]-14,15,20-trihydroxy-4β-pregnane. Its structural elucidation was performed through extensive spectroscopic measurements including 1D- and 2D-NMR, and HRMS, in addition to chemical methods.

Full text of DOI:10.1080/14786419.2014.1003186

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New pregnane glycoside derivative
from Caralluma retrospiciens (Ehrenb)
Mahmoud Fahmi Elsebaiac & Ietidal EL-Tahir Mohamedb
a Department of Pharmacognosy, Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt
b Department of Botany, Faculty of Science, University of
Khartoum, Khartoum, Sudan
c Department of Chemistry, University of Oulu, Oulu, Finland
Published online: 23 Jan 2015.
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To cite this article: Mahmoud Fahmi Elsebai & Ietidal EL-Tahir Mohamed (2015): New pregnane
glycoside derivative from Caralluma retrospiciens (Ehrenb), Natural Product Research: Formerly
Natural Product Letters, DOI: 10.1080/14786419.2014.1003186
To link to this article: http://dx.doi.org/10.1080/14786419.2014.1003186
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Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2014.1003186
New pregnane glycoside derivative from Caralluma retrospiciens (Ehrenb)
Mahmoud Fahmi Elsebaiac* and Ietidal EL-Tahir Mohamedb
aDepartment of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt;
c
bDepartment of Botany, Faculty of Science, University of Khartoum, Khartoum, Sudan; Department of
Chemistry, University of Oulu, Oulu, Finland
(Received 1 October 2014; final version received 24 December 2014)
Retrospinoside (1) is a new polyoxy pregnane glycoside which was isolated and
characterised from the aerial parts of Caralluma retrospiciens (Ehrenb.) N. E. Br.,
family Apocynaceae. The structure was established as 3-O-[b-D-glucopyranosyl-
(1 ! 4)-b-D-(3-O-methyl-6-desoxygalactopyranosy)]-14,15,20-trihydroxy-4b-
pregnane. Its structural elucidation was performed through extensive spectroscopic
measurements including 1D- and 2D-NMR, and HRMS, in addition to chemical
methods.
Keywords: plant natural products; pregnane glycosides; polyoxy pregnanes;
Caralluma retrospiciens
1. Introduction
Caralluma retrospiciens (Ehrenb.), family Apocynaceae, is a rare succulent plant that grows
wild on stony grounds. More than 200 species of Caralluma spp. grow throughout Asia and
Africa, and the majority of these species are indigenous to the Arabian Peninsula and the Indian
sub-continent (Al-Massarani et al. 2012). Several members of the genus Caralluma have found
medicinal uses in the treatment of rheumatism, diabetes, snake and scorpion bites, tuberculosis
and leprosy, scabies, fever and inflammation and as antiseptic and disinfectant (Al-Massarani
et al. 2012). Many asclepiadaceous plants, example of the genus Caralluma, are rich in pregnane
glycosides and their esters possessing a steroidal nucleus and function as precursors of the
cardenolides (Halaweish et al. 2004; Avula et al. 2011; Abdel-Mogib & Raghib 2013; Adnan
et al. 2014). The pregnane derivatives showed important pharmacological effects such as
antitumor, cytotoxic, platelet pro-aggregating and anti-fungal activities. Pregnane-type
glycosides were recently proved to induce differentiation of the mouse myeloid leukaemia
*Corresponding author. Email: elsebai72@yahoo.com
q 2015 Taylor & Francis

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M.F. Elsebai and I.E.-T. Mohamed
cells into phagocytic cells, appetite suppression and anti-obesity, and antitrypanosomal activity
(Halaweish et al. 2004; Abdel-Sattar et al. 2009; Dutt et al. 2012). Only few pregnane glycosides
have been isolated and characterised from C. retrospiciens (Khalil 1995; Halim & Khalil 1996;
Halaweish et al. 2004).
The current phytochemical investigation on C. retrospiciens led to the isolation of a new
polyoxy pregnane glycoside, retrospinoside (1). Retrospinoside (1) is a new polyoxy pregnane
glycoside which was isolated from the aerial parts of C. retrospiciens (Ehrenb.) N. E. Br., family
Apocynaceae. The identification of 1 was done using extensive spectroscopic data including 1D-
and 2D-NMR, and HRMS, in addition to chemical means to determine the absolute identity of
sugars and positions of hydroxyl groups.
2. Results and discussion
The methanolic extract of the aerial parts of C. retrospiciens yielded compound 1 after
fractionation and repeated chromatographic separation on Si gel columns. Compound 1 is a
glycoside polyoxy pregnane derivative of the two sugars D-glucose and 6-deoxy-3-O-methyl-b-
D-galactose with the steroidal pregnane nucleus (Figure 1). Compound 1 was obtained as white
powder with a molecular formula C34H58O13 on the basis of FAB-MS mass measurement (m/z
673 [M2H]2, 675 [MþH]þ) and (þ)-HR-ESI-MS (m/z 697.3769 [MþNa]þ). The presence of
various signals in the aliphatic proton region (0.5–2.5 ppm and 10–45 ppm) supported the
1
presence of a steroidal compound. The H and 13C NMR spectra showed various resonance
peaks through the 1H–13C–OH signal in the regions 3.10–5.20 ppm and 60–85 ppm,
respectively, and the presence of two anomeric protons (4–5 ppm and 90–110 ppm) suggested a
disaccharide glycoside. The acid hydrolysis of 1 with 1% HCI yielded two sugars, one is
D-glucose which was identified by TLC, and the second sugar was determined as 6-deoxy-3-O-
methyl-b-D-galactose based on the extensive spectroscopic data as described further. The IR
spectrum showed peaks at 3455 cm21 for the OH group. Compound 1 showed no absorption in
the UV region indicating the absence of double bond characters.
Due to the presence of many hydroxyl groups both in the aglycone and sugar parts, and in
order to obtain additional information and better quality of spectra for assignments, compound 1
was acetylated with acetic anhydride-pyridine. Extensive 1D and 2D NMR experiments and all
detailed assignments, therefore, were performed on the acetylated product 2.
Compound 2 showed a molecular formula of C48H72O20 on the basis of the number of signals
1
in both H-, 13C- and DEPT-NMR spectra (Figures S3–S5) and accurate mass measurement
Figure 1. The structure of compound 1 isolated from C. retrospiciens (Ehrenb).

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(HR-ESI-MS: m/z found ¼ 991.4509 [MþNa]þ). The steroidal portion of 2 gave rise to 1H and
13C NMR signals (Figures S2 and S3, Table S11) very similar to those of a sterol compound
related to a hydroxylated pregnane derivative which is also expected since it was isolated from a
plant known to produce these types of steroids. Thus, the methyl groups of the sterol portion
produced singlets at dH/C 0.92/15.1 and 0.68/12.4 for the angular tertiary CH3-18 and CH3-19,
respectively. The 1H NMR spectrum showed also two doublet methyl groups at dH 1.07 for CH3-
21 (J ¼ 5.90 Hz) and at dH 1.83 for CH3-60 (J ¼ 6.60 Hz). There were also further signals in the
1H NMR spectrum for the multiple CH2 groups between 0.5 and 2.5 ppm, in addition the down
field resonance peaks of the sugars between 3.2 and 5.3 ppm. Interpretation of the COSY and
HMBC spectra (Figure S7 and S8, Figure 2, Table S11) confirmed the structural features of
compound 2 to be as drawn in Figure 1 and provided evidence for a steroidal skeleton similar to
that of a pregnane type nucleus. The 13C spectrum showed 15 oxygenated carbons C-3, C-14, C-
15, C-20, C-10, C-20, C-30, C-40, C-50, C-100, C-200, C-300, C-400 C-500 and C-600. The acetylation was
performed on seven carbinols 15, 20, 20, 200, 300, 400 and 600 to produce hepta-acetylated pregnane
derivative as shown from their resonances in the 1H and 13C NMR spectra. Their positions were
unambiguously determined by the HMBC correlation to the respective carbinols (Figure 2,
Table S11). The oxygenated quaternary carbinol at 82.1 ppm was assigned to C-14 in accordance
with reported data (Usmanghani & Rizwani 1988) and it was not acetylated due to the steric
hindrance. OH-15 can be connected to C-15 or C-16, but it must be connected to C-15 based on
the following: the COSY measurement (Table S11 and Figure 2) determined correlations
between H-15 and H-16 and between H-16 and H-17 and no COSY correlation between H-15
and H-17, which means H-16 is centralised between H-15 and H-17. In addition, the multiplicity
of H-15 as a triplet and H-16 (a and b) as multiplets and H-17 as a multiplet necessitates the
presence of H-16 between H-15 and H-17 as illustrated in Figure 2.
The sugar residues of 2 were represented by two monosaccharide units as indicated by two
anomeric carbon signals at dC 99.5 for CH-10 and dC 100.4 for CH-100, and two anomeric protons
at dH 4.28 (1H, d, J ¼ 7.32 Hz) for CH-10, and dH 4.80 (1H, d, J ¼ 8.42 Hz) for CH-100,
determined from the HMQC spectrum. The two sugars are b-configured due to their large values
of coupling constant of the respective anomeric protons (Agrawal 1992). Glycosylation occurred
at C-3 (Figures 1 and 2) as observed from the HMBC correlations between H-10 and C-3,
confirming that the sugar of galactose derivative is the one which is directly attached to the
Figure 2. The key correlations of COSY (bold lines) and HMBC (arrows from H to C) for compound 2.

4
M.F. Elsebai and I.E.-T. Mohamed
Figure 3. The key NOESY correlations of compound 2.
pregnane nucleus, and from H-100 to C-40 indicating that the interglycosidic linkage is 1 ! 4
connected disaccharide.
The 1H NMR spectrum showed a resonance peak at dH/C 3.33/58.3 which is typical for a methoxy
group which was assigned for CH3O-70, and it is attached to C-30 due to HMBC correlation from
CH3O-70 to CH-30. Also in the 1H NMR spectrum, it showed a doublet for the methyl group CH3-60
which is connected to CH-50 due to mutual HMBC correlation. The 2D TOCSY experiment
(Figure S10) showed a one spin system from CH-10 to CH-60, constructing a galactose unit containing
one methoxyl group CH3O-70 and one methyl group CH3-60 instead of CH2–OH to get a 6-desoxy-3-
O-methyl galactose. We propose the D nature of the galactose derivative based on the literature
which established the same type of sugar from the same plant. Moreover, the chemical shifts of
galactose moiety in the 13C data of compound 1 (Table S1) is almost identical to the same galactose
moiety in the pregnane glycosides isolated from the same plant sp. (Halaweish et al. 2004).
For the stereochemistry of compound 2, the methine multiplet at dH/C 3.47/78.1 has the
expected complexity of steroidal 3-carbinol hydrogen which is characteristic for 3b-hydroxy
sterols. The relative configuration of compound 2 was determined by interpretation of NOESY
correlations and coupling constants. NOESY correlations between H3-19 and H-8, between H-8
and both H3-18 and H-15, and between H-15 and both H-18 and H-16b indicated all these protons to
be on the same side of the molecule, i.e. in the b-position (Figure 3). A NOESY correlation between
H-16a and H-17 and the absence of NOESY correlation between H-9 and any of H3-19 and H-18,
and absence of NOESY correlation between H-5 and any of H3-19 and H-18 indicated that H-5, H-
9, H-16a and H-17 are in the same other side of the molecule, i.e. in the a-position. We propose the
b-orientation of OH-14 based on the chemical shift of the oxygenated quaternary C-14 at 82.1 ppm
which was assigned to C-14 in accordance with the reported data (Usmanghani & Rizwani 1988;
Agrawal 1992; Kunert et al. 2006, 2008, 2009; Oyama et al. 2007; Al-Massarani et al. 2012). These
correlations indicated the transoid nature of rings A/B and B/C and the cisoid nature of rings C/D.
The absolute configuration at C-20 was determined to be R based on its chemical shift value in the
13C spectrum at 73.4 ppm (Al-Massarani et al. 2012) which was supported by the NOESY
correlation between H3-18 and H-20. We gave the name retrospinoside for compound 1.
3. Experimental
3.1. General experimental procedures
Melting points were measured on a YANACO apparatus (Yanaco New Science Inc., Kyoto,
Japan). Optical rotations were measured on Schmidt þ Haensch Polartronic D polarimeter

Natural Product Research
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(Schmidt & Haensch GmbH & Co. Feinmechanik Optik, Berlin, Germany). Ultraviolet (UV)
spectra were recorded in methanol on a Hitachi U-3200 spectrophotometer (Hitachi High-
Technologies Corporation, Tokyo, Japan). IR spectrum was measured on a Jasco A-302 Infrared
spectrophotometer (JASCO Corporation, Hachioji, Japan). All NMR spectra were recorded in
CD3OD and CDCl3 employing Bruker Avance 400 DPX and 600 DRX spectrometers (Bruker,
Rheinstetten, Germany). Column chromatography was performed on a glass column packed
with silica gel (E. Merck (Darmstadt, Germany), type 60, 70–230 mesh) of particle size 70–230
mesh. The extract was chromatographed after being absorbed onto a small amount of the
packing material. The aerial parts of the Caralluma retrospiciens (Ehrenb) were collected from
Erkowit, Red Sea, Hills Eastern, Sudan, and identified at the Department of Botany, University
of Khartoum with a voucher specimen (F-3-215) deposited at the herbarium of the Department
of Botany.
3.2. Extraction, isolation and characterisation
The fresh plant material (0.52 kg) was chopped into small pieces, dried at room temperature and
then soaked in MeOH for 3 days. The methanolic extract was evaporated under reduced
pressure, which yielded a dark green thick residue 50.3 g. This was dissolved in distilled water
(1 L) and defatted with pet. ether (3 L £ 3 L) to afford 11.6 g extract. The defatted aqueous
extract was further fractionated with CH2Cl2 (3 L £ 3 L), EtOAc (3 L £ 3 L) and then with n-
BuOH (3 L £ 3 L). On evaporation of the three fractions, 7.3, 5.4 and 13 g of extracts were
obtained, respectively. The diethyl ether-soluble part of n-BuOH fraction was subjected to CC
over polyamide using MeOH:H2O as eluent. Fraction A was re-chromatographed over silica gel
using EtOAc and then the polarity was increased by MeOH to afford compound 1.
3.3. Acid hydrolysis
Compound 1 (10 mg) was refluxed with 1% HCI (10 mL) in a boiling water bath for 5 h.
Thereafter, 10 mL H2O was added and concentrated to 5 mL, and the reaction mixture was kept
in a boiling H2O bath for 30 min and extracted with CHCI3. The aqueous phase of the
hydrolysate was neutralised with saturated barium hydroxide solution, the mixture filtered and
the filtrates concentrated, freeze-dried and subjected to TLC examination of the sugars against
standard glucose and other monosaccharaides. Paper chromatography for sugars was run on
Whatman paper No. 1 using solvent system EtOAc–MeOH–H2O–HOAc (65:15:15:20).
A saturated solution of aniline phthalate in n-BuOH was used as a staining agent.
3.4. Acetylation procedure
Acetylation of compound 1 (15 mg) was carried out in C5H5N (3 mL) and Ac2O (6 mL), and the
reaction mixture was left overnight at room temperature using magnetic stirrer, and then poured
into ice-H2O and extracted with EtOAc. The EtOAc extract was concentrated in vacuum
followed by Si gel cc (1% MeOH in CHCI3) of the residue to give compound 2 (6.3 mg). It forms
white needles with CHCI3.
3.5. Physical characters of compound 1
Amorphous powder; m.p. 208–2108C; [a] D22.3 (c, 2.2, MeOH); IR: 3455, 2923, 1452, 1375,
1076, 887 and 667 cm21; FAB-MS: m/z 673 [M2H]2, 675 [MþH]þ, (þ)-HR-ESI-MS
found ¼ 697.3769, clalc ¼ 697.3775 for C34H58NaO13; 13C NMR data (125 MHz, CDCl3), 38.4
(CH2-1), 30.4 (CH2-2), 71.8 (CH-3), 38.4 (CH2-4), 45.6 (CH-5), 30.2 (CH2-6), 27.9 (CH2-7),
41.7 (CH-8), 50.5 (CH-9), 37.2 (C-10), 21.5 (CH2-11), 41.4 (CH2-12), 47.7 (C-13), 82.9 (C-14),

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M.F. Elsebai and I.E.-T. Mohamed
73.4 (CH-15), 35.3 (CH2-16), 55.0 (CH-17), 12.7 (CH3-18), 17.3 (CH3-19), 71.4 (CH-20), 21.5
(CH3-21), Gala: 102.7 (CH-10), 75.9 (CH-20), 85.7 (CH-30), 77.8 (CH-40), 71.6 (CH-50), 17.3
(CH3-60), 58.4 (OCH3-70), Glu: 104.1 (CH-100), 74.7 (CH-200), 78.1 (CH-300), 71.6 (CH-400), 79.5
(CH-500), 63.0 (CH2OH-600).
4. Conclusion
Phytochemical investigation of C. retrospiciens (Ehrenb.) N. E. Br., family Apocynaceae led to
the isolation of a new polyoxy pregnane glycoside retrospinoside (1). Retrospinoside (1) is a new
polyoxy pregnane glycoside composed of multihydroxy pregnane aglycone to which a
disaccharide is attached. The disaccharide is composed of D-glucose and 6-deoxy-3-O-methyl-
b-D-galactose and the interglycosidic linkage is 1 ! 4.
Supplementary material
Supplementary material for this article is available online, alongside Table S1 for compound 1
and caratuberside B, Figure S1 for compound 1, Figures S2–S9 for compound 2 and NMR data
in Table S2 for compound 2.
Acknowledgements
The authors gratefully acknowledge the assistance from Dr Sampo Mattila (Department of Chemsitry, Oulu
University, Finland) for fruitful support and measuring the spectroscopic data of compound 2.
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