New pregnane glycosides from Caralluma hexagona Lavranos and their in vitro α-glucosidase and pancreatic lipase inhibitory effects

Article abstract of DOI:10.1016/j.phytol.2020.01.015

Three new pregnane glycosides in addition to four known compounds were isolated from the methylene chloride fraction of Caralluma hexagona Lavranos using bioassay-guided fractionation. The new compounds were identified as 12,20-di-O-benzoyl-3β,8β,12β,14β,20-pentahydroxy-(20R)-pregn-5-ene-3-O-β-D-glucopyranosyl-(1→4)-β-D-digitaloside (1), 3β,8β,14β,20-tetrahydroxy (20R)-pregn-5-ene-3-O-β-D-glucopyranosyl-(1→4)-O-β-D-digitaloside-20-O-β–D-glucopyranoside (2), 3β,8β,14β,20-tetrahydroxy-(20R)-pregn-5-ene-3-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→4)-O-β-D-digitaloside-20-O-β–D-glucopyranoside (3), along with the known compounds luteolin 4`-O-neohesperidoside (4), apigenin-8-C-neohesperoside (5), β-sitosterol (6) and β-sitosterol glucoside (7). Preliminary studies of the crude methanolic extract and methylene chloride fraction showed inhibitory effects against α-glucosidase and pancreatic lipase. Among the isolated compounds, compound 5 showed the most potent α-glucosidase inhibition with IC₅₀ value of 0.82 ± 2.50 μM compared to acarbose (0.81 ± 0.86 μM). Whereas compound 1 showed the highest inhibitory activity on pancreatic lipase with an IC₅₀ value of 23.59 ± 2.49 μM compared to orlistat (7.41 ± 2.26 μM).

Full text of DOI:10.1016/j.phytol.2020.01.015

PhytochemistryLetters36(2020)49–57
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
New pregnane glycosides from Caralluma hexagona Lavranos and their in
vitro α-glucosidase and pancreatic lipase inhibitory effects
Akram A. Shalabia, Ali M. El Halawanya, Mouchira A. Choucrya,b, Fatma S. El-Sakhawya,
Hiroyuki Moritac, Dae-Won Kic, Essam Abdel-Sattara,*
a Department of Pharmacognosy, Cairo University, Cairo, Egypt
b Department of Pharmacognosy, Heliopolis University, Cairo, Egypt
c Institute of Natural Medicine, University of Toyama, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three new pregnane glycosides in addition to four known compounds were isolated from the methylene chloride
fraction of Caralluma hexagona Lavranos using bioassay-guided fractionation. The new compounds were iden-
tified as 12,20-di-O-benzoyl-3β,8β,12β,14β,20-pentahydroxy-(20R)-pregn-5-ene-3-O-β-D-glucopyranosyl-(1→4)-
β-D-digitaloside (1), 3β,8β,14β,20-tetrahydroxy (20R)-pregn-5-ene-3-O-β-D-glucopyranosyl-(1→4)-O-β-D-digita-
loside-20-O-β–D-glucopyranoside (2), 3β,8β,14β,20-tetrahydroxy-(20R)-pregn-5-ene-3-O-β-D-glucopyranosyl-
(1→6)-O-β-D-glucopyranosyl-(1→4)-O-β-D-digitaloside-20-O-β–D-glucopyranoside (3), along with the known
compounds luteolin 4`-O-neohesperidoside (4), apigenin-8-C-neohesperoside (5), β-sitosterol (6) and β-sitosterol
glucoside (7). Preliminary studies of the crude methanolic extract and methylene chloride fraction showed
inhibitory effects against α-glucosidase and pancreatic lipase. Among the isolated compounds, compound 5
Caralluma hexagona
Asclepiadaceae
Pregnane glycosides
α-glucosidase
Lipase
showed the most potent α-glucosidase inhibition with IC50 value of 0.82 2.50 μM compared to acarbose
(0.81 0.86 μM). Whereas compound 1 showed the highest inhibitory activity on pancreatic lipase with an
IC50 value of 23.59 2.49 μM compared to orlistat (7.41 2.26 μM).
1. Introduction
market as a dietary supplement for weight loss with no history of ad-
verse effects (Sudhakara et al., 2014). The key and characteristic phy-
tochemicals in the genus Caralluma are mainly pregnane-type steroidal
aglycones and glycosides, in addition to other constituents such as
flavone glycosides, megastigmane glycosides, triterpenes and sterols
(Bader et al., 2003; Abdel-Sattar et al., 2008; Waheed et al., 2011;
Abdelaziz M Dawidar et al., 2012). The isolation of the pregnane gly-
coside russelioside B from Caralluma quadrangula with potential activity
in controlling weight gain associated with high-fat feeding in rats
(Abdel-Sattar et al., 2018) and stemmoside C from Solenostemma argel
with in vitro lipase inhibitory activity (El-Shiekh et al., 2019) en-
couraged the authors to investigate the unexplored species C. hexagona
from Yemen.
Caralluma is one of the prominent genera of family Asclepiadaceae,
distributed in dry regions of tropical Asia, south Mediterranean, near
east and in the north, central and east Africa (Albers and Meve, 2002).
Certain species of Caralluma are edible and form part of the traditional
medicine system of many countries (Adnan et al., 2014). The anti-
diabetic and anti-obesity activities of certain members of genus Car-
alluma were confirmed by several authors (Habibuddin et al., 2008;
Ambadasu et al., 2013; Bellamakondi et al., 2014; Sudhakara et al.,
2014; Abdel-Sattar et al., 2017; Vitalone et al., 2017). In addition, the
antioxidant, anticancer, antiulcer, neuroprotective, antihypertensive,
hepatoprotective activities were also reported (Abdallah et al., 2013;
Ahmad et al., 2015; Al-Mehdar et al., 2015; Poodineh et al., 2015;
Atlas, 2016; Al-Naqeb, 2017). Caralluma fimbriata, C. tuberculata and C.
quadrangula are among the most common species used as antidiabetic
and antiobesity. Caralluma fimbriata with its major constituents iden-
tified as pregnane glycosides was recently introduced into the US
Caralluma hexagona Lavranos is distributed in Yemen, Saudi Arabia
and Oman (Albers and Meve, 2002). In the present study, total extracts
and fractions, in addition to compounds isolated from the most active
fraction of the aerial parts of C. hexagona were tested in vitro for their
inhibitory activity on α-glucosidase and pancreatic lipase.
⁎ Corresponding author at: Pharmacognosy Department, Faculty of Pharmacy, Cairo University, El-Kasr El-Aini Street, 11562 Cairo, Egypt.
E-mail address: essam.abdelsattar@pharma.cu.edu.eg (E. Abdel-Sattar).
https://doi.org/10.1016/j.phytol.2020.01.015
Received 25 October 2019; Received in revised form 8 January 2020; Accepted 16 January 2020
1874-3900/©2020PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

A.A. Shalabi, et al.
PhytochemistryLetters36(2020)49–57
Table 1
1.15 (2 × 3 H) assigned for CH3-21 and CH3-6 of a 6-deoxy sugar. Po-
sitive chemical tests for steroids and deoxy sugars, in addition to the
NMR data (Tables 2 and 3), indicated that compound 1 was composed
of two hexoses, two benzoyl groups, and a pregnenepentol aglycone. A
double bond located at C-5/C-6 was deduced from the broad singlet
signal at δH 5.29 which showed correlation with C-5 (δC 138.74), C-10
(δC 37.24) and C-8 (δC 73.86) in the HMBC spectrum (Fig. 2). The
previous correlation (HMBC) suggested the presence of OH group at C-8
(Abdel-Sattar et al., 2001, 2002; Cioffi et al., 2006). The presence of OH
group at C-8 was further confirmed by the upfield shifted value of C-6
(δC 119.20) relative to the non-hydroxylated C-8 (δC 121.80) (Cioffi
et al., 2006; Abdallah et al., 2013). The other oxygenated quaternary
carbon at δC 86.26 was assigned to C-14 in accordance with many re-
ported data (Abdel-Sattar et al., 2002; Abdallah et al., 2013). The sig-
nals at δH 3.42 (1H, m), 4.94 (1H, b.s) and 5.20 (1H, br.q) were cor-
related with the oxygenated methine carbons at δC 77.84, 78.38 and
74.14, in the HMQC spectrum, and assigned for the protons H-3, H-12,
and H-20, respectively. 1H- and 13C-NMR spectra showed the presence
of two acyl groups identified as two benzoyl moieties (δH: 7.28–8.01
and δC 128.97–166.19) (Abdel-Sattar et al., 2007). The acylation po-
sitions at C-12 (δC 78.38) and C-20 (δC 74.14) were confirmed from the
downfield shift of their corresponding protons and carbons. Further, the
results of HMBC correlations (Fig. 2) showed correlations between the
signal of carbonyl carbons of benzoyl groups and H-12 (4.94 Hz) and H-
20 (5.20 Hz), respectively, of the aglycone moiety (Abdallah et al.,
2013). For the absolute configuration of C-20, Kimura and his co-
authors (1982) compared 13C NMR spectral data of 20R and 20S
pregnane epimers and found that there are significant differences in the
13C chemical shifts values of C-16 and C-20 in the two sets of epimers.
This was explained by the fact that the free rotation of the side chain in
the 20-hydroxy-C/D-cis-pregnane type steroids was assumed to be re-
stricted by steric hindrance among the C-18 methyl, C-21 methyl, and
C-20 hydroxyl groups based on a CPK model (the space-filling model).
Therefore, the C-21 methyl group in 20S alcohols of C/D-cis pregnane-
type steroids is close to the plane of the D-ring, resulting in upfield
shifts of the C-20 resonances (steric shift) (Kimura et al., 1982).
Therefore, the configuration of C-20 of compound 1 was deduced to be
R-configuration as explained by Al-Massarani et al. (2012) and by
comparison to the closely related glycosides (Panda et al., 2003; Kunert
et al., 2009; Al-Massarani et al., 2012; Abdallah et al., 2013; Elsebai and
Mohamed, 2015; Tsoukalas et al., 2016). From the previous data, the
aglycone moiety was identified as 12, 20-di-O-benzoyl-3β, 8β, 12β, 14β,
20-pentahydroxy-(20R)-pregn-5-ene and confirmed by comparison
with the closely related pregnane glycosides (Abdel-Sattar et al., 2001,
2002; Abdallah et al., 2013). Spectral data of compound 1 (Table 3)
showed that it contained two anomeric carbons signals at δC 101.68 &
103.75 correlated with anomeric protons at δH 4.22 (d. J = 6.8) & 4.34
(d. J = 8) respectively, which indicated that there were two sugar units
in the compound 1 (Table 3). The sugar moieties were detected in the
acid hydrolysate (Elsebai and Mohamed, 2015), one was identified as β-
D-glucose by comparison of an authentic sample, in addition to another
nonpolar sugar relative to glucose (6-deoxy sugar). The methyl signal at
δH 1.15 (d, 6, H-6) was correlated to carbon signals δC 17.48 (HMQC)
and a methoxyl singlet at δH 3.39 was correlated to carbon signal at δC
58.25 (HMQC) which suggested the presence of a 6-deoxy-3-O-methyl-
D-galactopyranose (digtalose) as second sugar moiety. The sugar se-
quence of compound 1 was confirmed by the HMBC spectrum, which
showed correlations between H-1` of digitalose at δH 4.22 and C-3 at δC
77.84 of the aglycone, and between H-3 (δH 3.42) and C-1` (δC 101.68)
of digitalose. Similarly, the correlations between H-1`` of glucose (δH
4.34) and C-4` of digitalose (δC 74.70). The glycosylation site was
proved at C-3 as a result of the downfield shift of C-3 (δC 77.84) and the
upfield shifts of C-2 (δC 29.27) and C-4 (δC 38.58). Thus, the sequence
and linkage sites of the sugar units were established as β-D-glucopyr-
anosyl-(1→4)-β-D-digitalopyranose-aglycone. By comparing the second
sugar data with the reported ones (Halim and Khalil, 1996; Abdel-Sattar
Inhibitory effects of total extracts, fractions and isolated compounds from the
aerial parts of C. hexagona on enzymes related to carbohydrate and fat meta-
bolism.
Sample
α-Glucosidase inhibition (%)
Lipase inhibition (%)
Extract/
% Inhibition
2 mg/mL
IC50 mg / mL % Inhibition IC50 μg / mL
fraction
100 μg/mL
W
28.44
42.59
94.66
NO
3.25
1.95
> 2
2.66
4.67
27.13
48.82
5.06
NO
1.98
> 100
> 100
> 100
> 100
NO
M
0.22
2.23
0.50
MC
2.80 0.78
0.02
D2
NO
6.00
D3
24.95
60.29
2.04
> 2
Acarbose
Orlistat
Compounds/
0.91 0.71
0.03
55.88
1.01 4.30
0.92
% Inhibition
IC50 mM
% Inhibition
100 μM
IC50 μM
standards 1 mM
1
11.95
3.62
0.56
> 1
> 1
> 1
> 1
0.82
> 1
> 1
0.81
63.70
4.35
2.60
23.59
2.49
2
3.19
2.30
3.47
2.60
1.87
> 100 μM
> 100 μM
> 100 μM
> 100 μM
> 100 μM
> 100 μM
3
19.26
41.64
55.18
7.19
2.62
8.90
0.13
3.62
4
12.09
11.22
8.80
5
2.50
0.86
6
4.31
4.15
7
43.17
55.13
4.22
0.49
35.62
4.17
0.98
Acarbose
Orlistat
55.59
7.41
2.26
W; Water extract; M; MeOH extract; MC; Methylene chloride fraction; D2;
MeOH : H2O (1:1) diaion fraction; D3; MeOH diaion fraction; NO; No inhibi-
tion.
2. Results and discussion
2.1. Isolation and identification of the isolated compounds from C.
hexagona
Using a bio-guided fractionation approach, the different extracts of
C. hexagona (water, methanol, a methylene chloride were subjected to
in vitro inhibitory assay using α-glucosidase and lipase enzymes. The
methylene chloride fraction showed the highest activity with 94.66 %
and 48.82 % inhibition against α-glucosidase and lipase enzymes, re-
spectively (Table 1). Therefore, the methylene chloride fraction was
subjected to bioassay-guided isolation, leading to the isolation of seven
compounds. The compounds were three new pregnane glycosides (1-3,
Fig. 1), in addition to two known flavone glycosides and two known
sterols. The known compounds were identified as luteolin 4`-O-neohe-
speridoside (4) (Rizwani et al., 1990), apigenin-8-C-neohesperoside (5)
(Scharbert et al., 2004), β-sitosterol (6) (Yahya et al., 2011) and β-si-
tosterol glucoside (7) (Luo et al., 2009) by comparison with available
authentic samples (Co-TLC and IR) and reported spectral data. The
structures of the new compounds were determined as follows.
Compound 1 was obtained as a white amorphous powder (34 mg),
the HRESI-MS analysis showed a adduct ion peak at 919.4105 in a
positive mode (calc. for C48H64O16Na, 919.4194) which is assigned for
the molecular formula C48
H
O
64 16
, with 17 degrees of unsaturation. IR
spectrum of this compound showed absorption bands due to hydroxyl
(3434 cm−1) and ester (1719, 1452 and 1277 cm−1) groups. In addi-
tion, 13C-NMR spectra showed the presence of four methyl, one meth-
oxyl, 26 methines, eight methylene groups and nine quaternary carbon
atoms with a total of 48 carbons, 21 of these carbons were ascribed for
the C21 steroidal moiety (Tables 2 and 3). The 1H NMR spectra also
provided important information about the nature of the aglycone, two
angular methyl groups appeared as singlet signals at δH 1.26 and 1.09
(δC 10.85 & 18.20) assigned for the angular methyl CH3-18 and CH3-19,
respectively, which can be assigned to pregnane type of steroids
(Deepak et al., 1989). This class of steroids has a two-carbon atom side-
chain at C-17 (C-20 and C-21), the protons at C-21 usually appear as a
methyl group which appear as doublet in the region of 1.0–1.5 ppm in
the case of the presence of CHOHCH3 moiety (Herz et al., 1997).,
Compound 1 revealed the presence of two doublet methyl signals at δH
50

A.A. Shalabi, et al.
PhytochemistryLetters36(2020)49–57
Fig. 1. Structures of compounds 1, 2 and 3.
et al., 2001), with digitalose attached at C-3 of the aglycone. The β-
configuration of all sugar moieties was deduced from the coupling
constants of their anomeric protons. The intensive spectral analysis of
compound 1 through 1H-, 13C-NMR, HMQC, HMBC, and COSY experi-
ments allowed unambiguous assignment of all protons and carbons
(Tables 2 and 3). From the aforementioned data and by comparison
with closely related pregnane glycosides, compound 1 was identified as
12, 20-di-O-benzoyl-3β, 8β, 12β,14β, 20-pentahydroxy-(20R)-pregn-5-
ene 3-O-β-D-glucopyranosyl-(1→4)-β-D-digitalopyranoside (Fig. 1).
Compound 2 was isolated as an amorphous powder. Based on the
HRESI-MS data, the molecular formula of 2 was determined to be
which was confirmed by comparison with closely related pregnane
glycosides (Al-Massarani et al., 2012; Dawidar et al., 2012). The ana-
lysis of 1H and 13C NMR spectra of this compound showed the presence
of three sugar moieties attached to the aglycone with two glycosylation
sites (Tables 2 and 3) and showed the absence of acyl groups. The first
glycosylation site was proved to be at C-3 similar to the sugar chain of
compound 1 and the second glycosylation site was found to be C-20.
This was proved from the downfield shift (+10 ppm) of the C-20 signal
relative to data of non-substituted glycosides (Abdul-Aziz Al-Yahya
et al., 2000) and also from the observed long-range correlation between
C-20 (δC 77.79) and H-1``` of the sugar unit with anomeric proton at δH
4.42 in the HMBC spectrum (Fig. 3). The sugar units were identified as
β-D-glucopyranosyl-(1→4)-β-D-digitalopyranose at C-3 and a β-D-glu-
cose at C-20 by comparison to the spectral data of compound 1. From
the above-mentioned data, compound 2 was identified as 3β,8β,14β,20-
tetrahydroxy-(20 R)-pregn-5-ene-3-O-β-D-glucopyranosyl-(1→4)-O-β-D-
C
40H66O18 (857.4169 [M + Na]+, calc. 857.4249). The NMR data of
the aglycone was close to those of compound 1 except for the absence of
the hydroxylation at C-12, which was confirmed from the presence of
upfield shifted CH2 group at δC 40.50 (C12). Therefore, the aglycone
moiety was identified as 3β,8β,14β,20-tetrahydroxy-(20R)-pregn-5-ene,
51

A.A. Shalabi, et al.
PhytochemistryLetters36(2020)49–57
Table 2
1H- and 13C NMR (400 MHz, 100 MHz) of aglycone of compounds 1, 2 and 3.
3
2
1
Position
CD3OD
δC
CD3OD
δC
DMSO
δC
δH
δH
δH
38.70
28.85
1.92 (1Ha, m)
38.76
28.91
1.82 (1Ha, m)
1.09 (1Hb, m)
1.71 (1Ha, m)
1.89 (1Hb, m)
3.58 m
38.70
29.27
0.95 (1Ha, m)
2.17 (1Hb, m)
1.50 (1Ha, m)
1.74 (1Ha, b.d,, 10.4)
3.42 m
1
2
1.09 (1Hb, m)
1.92 (1Ha, m)
1.73 (1Hb, m)
78.94
38.16
3.57 m
79.05
38.22
77.84
38.58
3
4
2.49 (1Ha, dd, 3.6,12.4)
2.36 (1Ha, m)
2.46 (1Hb, m)
–
1.74 (1Ha, b.d, 10.4)
2.38 (1Hb, b.d, 10.4)
–
2.35 (1Hb, t, 12.4)
138.81
118.86
35.04
73.69
46.92
36.97
19.00
–
138.80
118.93
35.10
73.91
46.98
37.03
18.68
138.74
119.20
35.15
73.86
44.34
37.24
24.67
5
5.36 b.s
5.36 b.s
5.29 b.s
6
2.17 b.s
2.17 b.s
2.08 (2H, b.s)
–
7
–
–
8
1.44 m
1.43 m
1,50 m
9
–
–
–
10
11
1.92 (1Ha, m)
1.82 (1Hb, m)
1.44 m
1.92 (1Ha, m)
1.82 (1Hb, m)
1.43 m
1.58 (1Ha, m)
1.97 (1Hb, m)
4.94 b.s
40.44
47.13
85.35
18.62
40.50
47.19
85.41
19.07
78.38
53.36
86.26
34.37
12
13
14
15
–
–
–
–
–
–
1.99 (1Ha, m)
1.92 (1Hb, m)
1.62 m
1.62 (2H, m)
1.66 (1Ha, m)
1.85 (1Hb, m)
1.92 (2H, m)
34.82
34.88
1.62 (1Ha, m)
2.00 (1Hb, m)
1.65 m
24.55
16
56.83
15.83
17.27
77.73
18.62
1.65 m
56.89
15.89
17.32
77.79
19.96
49.85
2.17 m
17
18
19
20
21
1.31 (3H, s)
1.21 (3H, s)
4.00 br.q
1.31 (3H, s)
1.21 (3H, s)
4.00 br.q
10.85
1.26 (3H, s)
1.09 (3H, s)
5.20 br.q
18.20
74.14
1.31 (3H, d, 6)
1.31 (3H, d, 6)
19.78
1.15 (3H, d, 6)
Benzoyl at C-12
166.19
–
1
130.71
–
2
129.66
8.01 (2H, d, J = 7.6)
7.57 (2H, m)
7.70 m
3,7
4,6
5
129.25
133.83
Benzoyl at C-20
165.42
–
1
130.71
–
2
128.97
7.28 (2H, m)
7.71 (2H, m)
7.57 m
3,7
4,6
5
129.31
133.45
digitaloside-20-O-β–D-glucopyranoside (Fig. 1).
compounds of C. hexagona against α-glucosidase and lipase enzymes are
presented in Table 1. The methanolic extract inhibited α-glucosidase
Compound 3 was obtained as amorphous powder, Based on the
HRESI-MS data, the molecular formula of 3 (Fig. 1)was determined to
be C46H76O23 (995.4678 [M−H]−, calc. 996.4777). The NMR spectral
analysis of compound 3 revealed similarity with spectral data of com-
pound 2, except for the presence of additional sugar moiety attached to
the sugar chain at C-3.
with IC50 value of 2.66
0.22 mg/mL and inhibited lipase with
27.13
2.23 % at 100 μg/mL, whereas the aqueous extract inhibited
3.25 % and
both enzymes at the same concentrations by 28.44
4.67
1.98 % for α-glucosidase and lipase, respectively. Among the
fractions of the methanolic extract, methylene chloride fraction showed
This was confirmed from the presence of four anomeric protons and
carbons indicating a tetrasaccharide. The sequential assignments of
proton and carbon resonances of the sugar moieties and their con-
nectivity to each other or to the aglycone part were determined from
HMQC, HMBC, and 1H–1H COSY experiments. The terminal sugar in the
sugar chain at C-3 was identified as glucose from the C–H long-range
correlation (Fig. 4) observed between H-1```` of the terminal D-glucose
(δH 4.42) and C-6`` of the middle D-glucose (68.90) in the HMBC
spectrum and also confirmed by comparing these data with those re-
ported by Halim and Khalil (1996). From the above-mentioned data,
compound 3 was identified as 3β,8β,14β,20-tetrahydroxy-(20R)-pregn-
5-ene-3-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl-(1→4)-O-β-
D-digitaloside-20-O-β–D-glucopyranoside (Fig. 1).
the most potent α-glucosidase and lipase inhibitory activities with IC50
of 0.78
0.02 mg/mL for α-glucosidase. However, fractions eluted
from the diaion column showed the lowest or no inhibitory activities
against both enzymes. The 50 % inhibition percentage of isolated
compounds ranged from 3.62
3.19–55.18
0.13 at concentration
1 mM and 3.62
3.47–63.70
2.60 at concentration 100 μM on α-
glucosidase and lipase enzymes, respectively. The most potent in-
hibitory activity against α-glucosidase was found for the flavonoid
glycoside; compound 5 with an IC50 0.82
2.50 mM compared to
standard acarbose (0.81
0.86 mM), apigenin-C-glycoside was pre-
viously reported to have α-glucosidase inhibitory activity(Xiao et al.,
2016). Whereas compound 1 showed the highest significant inhibitory
activity against lipase with IC50 value 23.59
2.49 μM compared to
standard orlistat (7.41
2.26 μM), Pregnane glycosides were pre-
viously reported as anti-obesity agents through modulating food intake,
improving lipid profile or by reducing intestinal fat absorption via in-
hibition of pancreatic lipase (Komarnytsky et al., 2013; Abdel-Sattar
2.2. α-Glucosidase and lipase inhibition activity
The inhibitory capacity of total extracts, fractions and isolated
52

A.A. Shalabi, et al.
PhytochemistryLetters36(2020)49–57
Table 3
et al., 2018; El-Shiekh et al., 2019). In the current study, compound 1
showed the highest activity, whereas 2 and 3 exhibited the weakest
activity, which may be attributed to the presence of acyl groups in
compound 1.
1H- and 13C NMR (400 MHz, 100 MHz) of sugar moieties of compounds 1, 2 and
3.
3
2
1
Position
CD3OD
δC
CD3OD
δC
DMSO
δC
3. Experimental
δH
δH
δH
3.1. General experimental procedures
Dig
Dig
Dig
101.48 4.37 (d.
J = 7.6)
101.57 4.37 (d.
J = 7.6)
101.68 4.22 (d.
J = 6.8)
1
Optical rotations were recorded on a JASCO P2100 polarimeter
(Japan). Mass spectra were measured on a SHIMADZU LCMS-IT-TOF
spectrometer. The NMR experiments for all compounds were performed
on a Bruker AscendTM 400/R NMR spectrometer operating at a proton
NMR frequency of 400 MHz and 13C-NMR (100 MHz). The NMR spectra
were recorded in a suitable solvent (CDCl3, DMSO-d6 or CD3OD) using
TMS as internal standard and chemical shifts were given in δ ppm
value. Infra-red spectrophotometer, Schimadzu IR-435, PU-9712 was
used for recording IR spectra using KBr discs. Analytical thin-layer
chromatography (TLC) was carried out on Merck TLC plates (250 μm
thickness, KGF Silica gel 60 and KGF RP-18 Silica gel 60) and spots
were visualized in the visible, under UV light (254 and 365 nm) before
and after spraying with aluminum chloride and by spraying the dried
plates with p-anisaldehyde/H2SO4 followed by heating at 110 °C.
Column chromatography (CC): silica gel (Silica gel, 35–70 μm and
63–200 μm; Fluka, Sigma-Aldrich Chemicals, Germany), RP-C18 (silica
gel, 40−63 μm; Merck) and Diaion HP-20 AG (Mitsubishi Chemical
Industries Co. Ltd, Japan).
70.44
84.24
73.48
3.62 m
70.06
84.36
73.45
3.62 m
3.26 m
4.21 (d,
J = 1.6)
3.63 m
1.31 (3H, d,
6)
69.89
84.46
74.70
3.35 m
3.14 m
4.00 b.s
2
3
4
3.26 m
4.21 (d,
J = 1.6)
3.63 m
69.94
16.18
70.07
15.99
69.50
17.48
3.55 m
1.15 (3H,
d, 6)
5
6
1.31 (3H, d,6)
57.26
Glc
3.53, s
57.11
3.53 s
58.25
3.39 b.s
OCH3
1
Glc
Glc
102.78 4.62 (d.
J = 7.6)
102.78 4.62 (d.
J = 7.6)
103.75 4.34 (d.
J = 8).
73.86
76.50
70.19
75.97
68.90
3.23 m
73.95
76.43
70.24
74.55
61.63
3.23 m
74.91
77.19
70.75
77.56
61.86
3.00 m
3.14 m
3.05 m
3.05 m
3.66 m
3.38 m
2
3
4
5
6
3.33 m
3.33 m
3.31 m
3.31 m
3.27 m
3.22 m
4.16 (1Ha, d.
J = 11.5.) 3.79
(1Hb, dd. 11.5,
6.5)
3.87 (1H, t,
11.8) 3.67
(1H, dd,
11.8, 5.3)
Glc
103.63 4.42(d.
J = 7.6)
1
3.2. Plant material
73.90
76.62
70.02
76.36
61.33
3.20 m
2
3
4
5
6
3.33 m
The fresh aerial parts of Caralluma hexagona Lavranos were col-
lected from Bani Habash Mountain, Almahweet, Yemen, in June 2017
and was chopped into small pieces, dried in shade. The plant was kindly
authenticated by Dr. Abdul Wali A. Al Khulaidi, Associate Professor of
plant geography, flora and vegetation, member of the center of middle
east plants, Al Baha University, Baljurashi, Saudi Arabia. Voucher
samples (# 10.7.2019.1) were kept at the Herbarium of the
Pharmacognosy Department, Faculty of Pharmacy, Cairo University.
3.33 m
3.26 m
3.88 (1Ha, t,
10.9) 3.67
(1Hb, dd, 10.9,
5.2)
Glc at C-20
103.14 4.42 (d.
J = 7.6)
Glc at C-20
103.20 4.42 (d.
J = 7.6)
1
74.37
76.56
70.44
77.16
62.86
3.24 m
76.56
76.80
70.46
77.22
61.34
3.24 m
2
3
4
5
6
3.31 m
3.31 m
3.3. Preparation of aqueous extract for in vitro assay
3.30 m
3.30 m
3.37 m
3.37 m
3.62 (2H, b.s)
3.62 (2H, m)
Part of the powdered aerial parts of C. hexagona (10 g) was extracted
using a decoction method with H2O (2 × 75 mL, 10 min each) to give
1.83 g of light green residue for further in vitro biological studies.
Fig. 2. Some selected HMBC correlations for compound 1.
53

A.A. Shalabi, et al.
PhytochemistryLetters36(2020)49–57
Fig. 3. Some selected HMBC (→) and NOESY (↔) correlations for compound 2.
3.4. Extraction and isolation
as a white powder from fraction F4 (2.0 g). Fraction F5 (5.8 g) was
rechromatographed over Silica gel column (120 g, 63–200 μm,
3.5 × 50 cm), eluted using CH2Cl2 : MeOH (95: 5 v/v, 1 L), CH2Cl2:
MeOH (90 : 10 v/v, 1 L) and CH2Cl2 : MeOH (85 : 15 v/v, 1 L), to give
subfractions F5-1 (0.3 g), F5-2 (1.5 g), F5-3 (0.7 g), F5-4 (0.6 g), F5-5
(0.42 g), F5-6 (0.75 g) and F5-7 (0.56 g). Fraction F5-4 was re-
chromatographed over silica gel column (20 g, 35−70 μm, 1 × 60 cm),
using n-hexane: ethyl acetate: methanol (3: 5: 2 v/v/v), as an eluent
yielding compound 1 (34 mg). Fraction F6 (6 g) was rechromato-
graphed over silica gel column (120 g, 63–200 μm, 3.5 × 50 cm), and
was eluted using CH2Cl2 : MeOH (90: 10 v/v, 2 L), CH2Cl2:MeOH (85:
15 v/v, 1 L) and CH2Cl2 : MeOH (80: 20 v/v, 1 L) and MeOH 100 %
(0.5 L) to give subfractions F6-1 (0.95 g), F6-2 (2.10 g), F6-3 (1.35 g),
F6-4 (0.65 g) and F6-5 (0.73 g). Fraction F6-4 was rechromatographed
using RP- 18 silica gel column (30 g, 40−63 μm, 2.5 × 25 cm), using
MeOH: H2O (1: 1 v/v), as an eluent to give three compounds; Com-
pound 2 (60 mg), compound 4 (9 mg) and compound 5 (14 mg). Fi-
nally, part of fraction F8 (0.70 g) was rechromatographed over RP- 18
silica gel column (30 g, 40−63 μm, 2.5 × 25 cm), using MeOH: H2O
(1:1 v/v), as an eluent to yield compound 3 (29 mg).
The powdered aerial part of C. hexagona (2.40 kg) was extracted
with methanol (2 × 5 L) by maceration at room temperature for seven
days. The methanolic extract was evaporated under reduced pressure to
yield a light green solid residue (300 g). The methanolic extract was
subjected to fractionation according to the scheme presented in Fig. 5.
Part of the methanolic extract (220 g) was suspended in distilled water:
MeOH (4: 1 v/v) and partitioned with methylene chloride (4 × 500 mL)
to afford 70 g of CH2Cl2 fraction. The remaining water was passed
through a diaion HP-20 column (600 g, 5 × 100 cm) and eluted with
H2O (3 L), H2O: MeOH (1:1 v/v) (3 L), and MeOH (3 L). On the eva-
poration of the three fractions, 101, 26 and 23 g of fractions were ob-
tained, respectively. Part of the methylene chloride fraction (30 g) was
chromatographed over open column chromatography using normal si-
lica gel 60 (600 g, 63–200 μm, 5 × 100 cm) with methylene chloride
and methanol with an increasing percentage of methanol (5–100 %) as
eluent. Fifty-five fractions (200 mL, each) were collected and monitored
by TLC after spraying with p-anisaldehyde sulfuric acid spray reagent
followed by heating at 110 Co. Similar fractions were pooled together to
give eight fractions (F1 to F8). Fraction F3 (6. 5 g) was rechromato-
graphed over silica gel column (120 g, 63–200 μm, 3.5 × 50 cm), using
n-hexane: ethyl acetate (90:10 v/v), as an eluent, which yielded com-
pound 6 (220 mg). Compound 7 (350 mg) was precipitated by methanol
Compound (1)
White amorphous powder [α]D25 +15.3 (c. 0.1, MeOH); IR υmax
(KBr, cm‐1): 3434, 2930, 1719, 1452, 1277, 1115, 1069, 1022 and 714;
HR-ESIMS m/z 919.4105 [M + Na]+ (calc. for C48H64O16Na,
Fig. 4. Some selected HMBC (→) and NOESY (↔) correlations for compound 3.
54

A.A. Shalabi, et al.
PhytochemistryLetters36(2020)49–57
Fig. 5. Scheme for the chromatographic isolation of the bioactive compounds from methylene chloride fraction of C. hexagona.
919.4194) in positive mode; Table 2 for 1H, 13C NMR (400 MHz,
100 MHz, DMSO-d6) of aglycone moiety; and Table 3 for 1H, 13C NMR
of sugar moieties.
was added and concentrated to 5 mL, and the reaction mixture was kept
in a boiling H2O bath for 30 min and extracted with CH2Cl2. The aqu-
eous phase of the hydrolysate was neutralized with 2 N Na2CO3 solu-
tion, the mixture filtered and the filtrates were concentrated under
reduced pressure, tested for carbohydrates by TLC using EtOAc-MeOH-
H2O−HOAc (65: 15: 15:20) as a solvent system, and sprayed with
freshly prepared aniline phthalate reagent.
Compound (2)
Amorphous powder; [α]D25 +15.5 (c. 0.1, MeOH); HR-ESIMS m/z
857.4169 [M + Na]+ (calc. for C40H66O18Na+, 857. 4249) in positive
mode; see Table 2 for 1H, 13C NMR (400 MHz, 100 MHz, CD3OD) of
aglycone moiety; and Table 3 for 1H, 13C NMR of sugar moieties.
Compound (3)
Amorphous powder; [α]D25 -31.4 (c. 0.1, MeOH); HR-ESIMS m/z
995.4678 [M−H]− (calc. for C46H76O23H−, 996.4777) in positive
mode; see Table 2 for 1H, 13C NMR (400 MHz, 100 MHz, DMSO-d6) of
aglycone moiety; and Table 3 for 1H, 13C NMR of sugar moieties.
3.6. In vitro enzyme inhibitory assay
3.6.1. Chemicals and drugs
Lipase (Type II: from Porcine pancreas), α-glucosidase from
Saccharomyces cerevisiae (19.3 U / mg solid, G5003-100UN), orlistat
was purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA).
p-nitrophenyl-α-D-glucopyranoside (L10070), p-nitrophenyl-palmitate
(L10896) and acarbose (J61737) were purchased from Alfa Aesar.
3.5. Acid hydrolysis
Compounds 1, 2 and 3 (10 mg, each) were refluxed, separately with
1 % HCI (10 mL) in a boiling water bath for 5 h. thereafter, 10 mL H2O
55

A.A. Shalabi, et al.
PhytochemistryLetters36(2020)49–57
3.6.2. Inhibition of α-glucosidase
influence the work reported in this paper.
α-Glucosidase inhibitory activity was measured colorimetrically
using the substrate p-nitrophenyl-α-D-glucopyranoside (pNPG) ac-
cording to an assay method reported by Gutiérrez-Grijalva et al. (2019)
with slight modification. Briefly, in 96-microwell plates, 25 μL of ex-
tracts, fractions or compounds previously diluted in 5 % DMSO were
incubated for 10 min at 37 °C with 50 μL of α-glucosidase from Sac-
charomyces cerevisiae (0.6 U / mL) in phosphate buffer (0.1 M, pH 7).
Then, 25 μL of 3 mM pNPG as a substrate in phosphate buffer (pH 7)
was added, and the mixture was incubated again for 5 min at 37 °C.
Enzyme activity was determined by measuring the release of p- ni-
trophenol from the pNPG substrate at 405 nm using a microplate reader
(Tecan, USA). This assay was carried out using acarbose (purity, 95 %)
as a positive control and 5 % DMSO as a negative control (blank). The
percentage of inhibition of α-glucosidase was calculated according to
the equation:
Acknowledgment
This work was funded in part by the Academy of Scientific Research
and Technology under the “Egypt Research and Technology Alliances
(EG-KTA) Program” (No. : KTA-C2-2.10). The authors thank Prof.
Ahmed Galal Osman, Senior Research Scientist, National Center for
Natural Products Research (NCNPR), School of Pharmacy, University of
Mississippi, USA for editing the manuscript.
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Declaration of Competing Interest
Gutiérrez-Grijalva, E.P., Antunes-Ricardo, M., Acosta-Estrada, B.A., Gutiérrez-Uribe, J.A.,
Heredia, J.B., 2019. Cellular antioxidant activity and in vitro inhibition of α-gluco-
sidase, α-amylase and pancreatic lipase of oregano polyphenols under simulated
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
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