Isolation and characterization of non-sulfated and sulfated triterpenoid saponins from Fagonia indica

Article abstract of DOI:10.1016/j.phytochem.2017.08.005

Seven previously undescribed, sulfated triterpenoid glycosides, named nayabin A-G along with a known triterpenoid glycoside were isolated from the whole plant of Fagonia indica. Their structures were elucidated through spectral studies including 1D- (¹H and ¹³C), 2D-NMR spectroscopy (HSQC, HMBC, COSY and NOESY), and mass spectrometry (ESI-MS/MS). β-D-Glucopyranosyl 3β-hydroxy-23-O-β-D-glucopyranosyloxy-taraxast-20-en-28-oate, a known compound exerts glucose-dependent insulin secretory activity, which seems to exhibit a decreased risk of drug-induced hypoglycemia and may offer distinct advantages as anti-diabetic agent.

Full text of DOI:10.1016/j.phytochem.2017.08.005

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Phytochemistry 143 (2017) 151e159
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Isolation and characterization of non-sulfated and sulfated
triterpenoid saponins from Fagonia indica
Nayab Kanwal a, Achyut Adhikari a, Abdul Hameed b, Rahman M. Hafizur b,
,
*
Syed Ghulam Musharraf a
a H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan
b Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi,
Karachi, 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven previously undescribed, sulfated triterpenoid glycosides, named nayabin A-G along with a known
triterpenoid glycoside were isolated from the whole plant of Fagonia indica. Their structures were
elucidated through spectral studies including 1D- (1H and 13C), 2D-NMR spectroscopy (HSQC, HMBC,
Received 9 May 2017
Received in revised form
3 August 2017
COSY and NOESY), and mass spectrometry (ESI-MS/MS). b-D-Glucopyranosyl 3b-hydroxy-23-O-b-D-glu-
Accepted 7 August 2017
copyranosyloxy-taraxast-20-en-28-oate, a known compound exerts glucose-dependent insulin secretory
activity, which seems to exhibit a decreased risk of drug-induced hypoglycemia and may offer distinct
advantages as anti-diabetic agent.
Keywords:
Fagonia indica
© 2017 Elsevier Ltd. All rights reserved.
Zygophyllaceae
Triterpenoid glycosides
Insulin secretion activity
ESI-QTOF-MS
1. Introduction
plant were attributed due to its variety of active phytochemical
constituents. A large number of saponins or triterpenoid glycosides
Fagonia indica Burm belongs to family Zygophyllaceae. It is small
spiny shrub, very much branched desert herb up to 80 cm tall and
widely distributed in tropical, subtropical, and warm areas of the
world. It is also present in dry areas. Algeria, Cyprus, Egypt, India,
Morocco, Pakistan, Saudi Arabia are known countries for this plant
occurrence (Ali et al., 2008). This plant has saponin or triterpenoid
glycoside rich constituents. Fagonia species are often used in
traditional medicines, such as for treatment of fever, jaundice
(Hammiche and Maiza, 2006), blood purification, cold, cough
(Khattak, 2012), asthma, skin infection, liver problems (Abirami
et al., 1996), carminative, emetic (Mahmood et al., 2011), and the
extracts of these plants have been reported to exhibit anti-
microbial, anti-inflammatory, analgesic and antipyretic activities
(Nagaraj and Venkateswarlu, 2013; Sajid et al., 2011; Thetwar et al.,
2006). Additionally, this plant is declared to be remedy for cancer
(Hussain et al., 2007; Satpute et al., 2009; Waheed et al., 2012) and
thalassemia (Seyal et al., 2013). The medicinal properties of this
(Abdel-Khalik et al., 2001; Ansari et al., 1987; El-Wakil, 2007; Far-
heen et al., 2015; Khalik et al., 2000; Miyase et al., 1996; Shaker
et al., 1999, 2013) have been isolated and characterized from
various species of Fagonia. Sulfated triterpenoids and sulfated tri-
terpenoid glycosides have been isolated from Fagonia species
(Perrone et al., 2007; Shaker et al., 2000). Saponins are considered
as the major bioactive constituents of the drugs, mainly used for
their haemolytic (Amini et al., 2014), anti-inflammatory (Lee et al.,
2012), anti-microbial (Lunga et al., 2014), and antitumor (Han et al.,
2013) activities. Some saponins from Fagonia species have been
reported to exert anti-cancer, anti-diabetic, molluscicidal and
antioxidant activities (El-Wakil, 2007; Farheen et al., 2015; Saleem
et al., 2014; Shaker et al., 2013).
The medicinal importance of Fagonia encouraged us to investi-
gate the saponins in Fagonia indica. This paper reports the isolation
and characterization of seven previously undescribed including
triterpenoid glycosides (1, 3), five sulfated triterpenoid glycosides
(2, 4e7) and a known triterpenoid glycoside (8). Their structures
were elucidated by extensive spectroscopic methods including 1D-
(1H and 13C) and 2D-NMR (COSY, HSQC, HMBC, and NOESY) ex-
periments as well as ESI-MS and ESI-MS/MS analysis. To elucidate
* Corresponding author.
E-mail address: musharraf1977@yahoo.com (S.G. Musharraf).
http://dx.doi.org/10.1016/j.phytochem.2017.08.005
0031-9422/© 2017 Elsevier Ltd. All rights reserved.

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152
N. Kanwal et al. / Phytochemistry 143 (2017) 151e159
the importance of these isolated compounds, we have evaluated
these natural compounds for their insulin secretory activity in
in vitro isolated mice islets and cytotoxicity activities in MIN6 cell
lines.
8/2) of the whole plant of Fagonia indica was fractionated by
vacuum-liquid chromatography (VLC) using reversed phase silica
gel (C18) followed by column chromatography (silica gel) and
recycling HPLC, to afford seven previously undescribed triterpenoid
saponins (1e7) and one known triterpenoid saponin (8) (See Fig. 1).
Spectral analysis indicated that compounds 1, 3, 8 are non-sulfated
triterpenoid saponins and compounds 2, 4e7 are sulfated tri-
terpenoid saponins.
2. Results and discussion
The n-BuOH fraction obtained from the extract (MeOH/H2O:
Fig. 1. Structures of compounds 1e8.

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153
2.1. Identification of the chemical structure of the isolated
compounds
ion at m/z 633 [M-H-162]- due to the loss of one glucose moiety.
The 1H and 13C-NMR data (Tables 1 and 2) of compound 1 dis-
played signals for five tertiary methyls at dH 0.67s, 0.86s, 0.94s,
0.97s, and 1.68s; an exo-methylene group at C-29/20 [dH 4.70 and
Nayabin A (1) was obtained as amorphous form, its molecular
formula C42H68O14, was determined by HR-ESI-MS which showed
pseudo-molecular ion peak [M-H]- ion at m/z 795.4587 (calcd for
4.58/
d
c 110.2 (CH2-29) and
dc 151.8 (C-20)]; and an ester carbonyl
carbon at
d
c 176.1 (C-28). The 1H and 13C-NMR spectra also showed
C
42H68
O
14 e H ¼ 795.4536), with nine degrees of unsaturation (one
the signals of two anomeric carbons at dH 4.22 (d, J1'/2' ¼ 8.0 Hz)/
dc
C]O, one C]C, one for each sugar residue, and five remaining to
point out all the pentacyclic compounds). Adduct ion
[MþCH3COOHeH]- at m/z 855.5321 was also observed. MS/MS
analysis of compound 1 (negative ion mode), showed the fragment
104.7 (C-10) and dH 5.47 (d, J1''/2'' ¼ 8.0 Hz)/
d
c 95.2 (C-100) indicating
the presence of two sugar moieties in the molecule. In the 13C-NMR
spectrum, there is signals for five sp3 quaternary carbon at
(C-4), 41.9 (C-8), 38.1 (C-10), 43.5 (C-15), 57.9 (C-17). Above
d 43.6
Table 1
1H-NMR spectral data of compounds 1e7a,b
.
No
1
2
3
4
5
6
7
1
2
3
4
1.63, 2.31 m
1.00, 1.69 m
3.73b
e
1.21b
1.0, 1.62 m
1.78, 1.89 m
4.25 dd (12.0, 4.5)
e
1.62b
0.91, 1.61 m
1.12, 1.71 m
4.25b
e
1.29b
0.98, 1.70 m
1.61, 1.69 m
3.75b
e
1.22b
0.98, 1.70 m
1.65, 1.72 m
3.74b
e
1.19 b
0.98, 1.70 m
1.61, 1.69 m
3.75b
e
1.21b
1.09, 1.65 m
1.61, 1.70 m
3.82 d (5.4)
e
5
1.19b
6
7
8
1.09,1.52 m
1.30, 1.49 m
e
1.45, 1.55 m
1.27, 1.49 m
1.28, 1.62 m
1.31, 1.41 m
e
1.51, 1.72 m
1.35, 1.41 m
e
1.49, 1.73 m
1.36,1.43 m
e
1.51, 1.72 m
1.35, 1.41 m
e
1.49, 1.74 m
1.35, 1.41 m
e
e
9
1.40 m
1.63 m
1.35 m
1.45 m
1.47 m
1.45 m
1.49 m
10
11
12
13
14
15
e
e
e
e
e
e
e
0.96, 1.42 m
1.52, 1.62 m
2.32 m
1.70, 2.0 m
5.28 t* (3.0)
e
1.25, 1.50 m
1.18, 1.71 m
2.64 t d (9.5, 3.5)
e
1.59, 1.62 m
1.63, 1.67 m
2.35 t d (7.5, 3.0)
e
1.59, 1.61 m
1.64, 1.69 m
2.40 t d (7.5, 3.0)
e
1.59, 1.62 m
1.63, 1.67 m
2.35 t d (7.5, 3.0)
e
1.59, 1.62 m
1.63, 1.67 m
2.40 br t (7.5)
e
e
e
1.10, 1.78 m
1.29, 1.46 m
1.12 m, 2.07
br t (13.0)
1.42, 2.36 m
e
1.40b
2.36 m
1.11, 1.59 m
1.11, 1.59 m
1.11, 1.59 m
1.11, 1.59 m
16
17
18
19
20
21
22
1.89, 2.63 m
e
1.04b
2.96 m
e
1.91, 2.10 m
e
1.42, 2.05 m
e
1.22b
2.10 m
e
1.41, 1.92 m
e
1.22b
2.08 m
e
1.42, 2.05 m
e
1.22b
2.10 m
e
1.41, 2.03 m
e
1.21b
2.09 m
e
2.80 dd (14.0, 4.0)
1.82, 1.60 m
e
e
1.11, 1.21 m
1.91, 2.01 m
1.24, 1.51 m
1.23, 1.55 m
5.44 br d (7.0)
1.88b, 2.55 dd
(16.0, 7.5)
4.05, 4.18b
5.21 br d (7.0)
1.81 br d (15.5),
2.26 dd (15.5, 7.0)
3.36, 3.70b
5.23 br d (7.0)
1.78 br d (15.5),
2.20 dd (15.5, 7.0)
3.36 d (9.5),
3.71 d (9.5)
0.68 s
0.90 s
0.96 s
0.99 s
e
5.21 br d (7.0)
1.81 br d (15.5),
2.26 dd (15.5, 7.0)
3.36, 3.70b
5.22 br d (7.0)
1.79 br d (15.5),
2.20 dd (15.5, 7.0)
3.41b, 3.69 d (10.0)
23
3.37, 3.70b
3.30, 3.49b
24
25
26
27
28
29
30
10
0.67 s
0.86 s
0.94 s
0.97 s
0.68 s
0.98 s
0.79 s
1.18 s
0.96 s
0.86 s
1.15 s
0.88 s
0.68 s
0.89 s
0.96 s
0.99 s
0.68 s
0.88 s
0.94 s
0.98 s
0.69 s
0.88 s
0.96 s
0.99 s
e
e
e
e
e
e
4.70s, 4.58s
1.68s
0.88s
1.02 d (6.5)
1.68 s
1.01 d (6.5)
1.60 s
1.00 d (6.0)
1.60 s
1.01 d (6.5)
1.60 s
4.23 d (8.0)
4.15 t* (9.0)
3.89b
1.01 d (6.5)
1.60 s
4.46 d (7.2)
4.16 t (9.0)
3.88b
3.41, 3.50b
5.35 d (8.0)
3.30b
4.22 d (8.0)
4.91 d (8.0)
4.26 d (8.0)
4.27 d (8.0)
20
3.26b
4.01b
3.29b
3.30b
30
3.27b
3.33b
4.18b
3.64 t (9.0)
4.09 t (9.0)
3.39b
3.64 t (9.0)
4.12 t* (9.0)
3.40b
40
3.33b
3.39b
4.24b
4.22 t (9.0)
4.23 t* (9.0)
50
3.39b
3.32b
4.13b
3.39b
3.42b
60
3.84b, 3.67b
3.78 br d
(10.5), 3.64
br d (11.5, 4.0)
4.38 dd
3.87 dd (12.0, 1.5),
3.73b, 3.89 dd
(12.0, 1.5)
3.85 dd (11.0, 1.5),
3.67b
3.76 dd (12.6, 7.2),
3.90 br d (12.6)
(11.0, 1.5), 4.48b
3.67b
100
200
300
400
500
600
5.47 d (8.0)
3.2b
6.26 d (8.0)
4.01b
5.40 d (8.5)
3.26b
5.41 d (8.5)
3.19 t* (8.5)
3.38b
3.38b
3.93b
3.38b
3.27b
4.20b
3.33b
3.27b
3.32b
4.10b
3.32b
3.32b
3.81b, 3.64b
4.61 dd (11.5, 2.0),
3.80 dd (12.0, 2.0),
3.80 dd (12.0, 2.0),
4.26b
3.69b
3.64b
1000
2000
3000
4000
5000
6000
4.97 d (8.0)
4.23b
3.92b
4.25b
4.19b
4.36b, 4.54 dd
(11.5, 2.0)
t* (footnote: apparent multiplicity ¼ dd).
a
In CD3OD (
d
ppm) for compounds 1e2, 4e7 and pyridine-d5(
d ppm) for 3; J in Hz.
b
Overlapped signals, reported without assigned multiplicity.

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154
N. Kanwal et al. / Phytochemistry 143 (2017) 151e159
Table 2
(
dH 0.97) suggested that the C-27 methyl group possessed a-
13C-NMR spectral data of compounds 1e7a.
orientation. The configuration of the sugar unit was assigned as D-
glucose as described in section 3.5. Hence, the structure of com-
Position
1
2
3
4
5
6
7
pound 1 was characterized as
-glucopyranosyloxy-lup-20(29)-en-28-oate.
The molecular formula of nayabin B (2) was deduced from the
b-D-glucopyranosyl 3b-hydroxy-23-
1
2
3
4
5
6
7
8
39.7
27.3
72.9
43.6
49.0
18.9
35.0
41.9
51.8
38.1
22.0
27.3
39.3
43.5
30.8
32.8
57.9
50.6
48.3
151.8
31.4
37.4
74.1
12.7
17.4
16.6
15.1
176.1
110.2
19.5
104.7
74.1
77.9
71.1
78.4
62.9
95.2
75.2
78.3
71.7
78.7
62.5
39.6
24.6
80.8
43.7
48.3
19.0
33.4
40.7
49.5
37.9
24.8
124.0
144.6
43.0
28.9
24.3
47.9
42.0
42.4
46.1
29.9
32.7
65.1
13.1
16.6
17.8
26.4
178.0
28.0
66.3
95.7
73.9
78.3
71.1
78.6
62.4
38.9
27.8
72.1
43.0
49.2
18.5
34.3
42.3
50.9
37.2
21.9
27.7
39.5
42.3
29.5
33.4
49.7
48.7
37.6
143.1
117.5
37.7
74.9
13.1
17.0
16.4
15.2
177.5
23.6
22.2
105.5
75.2
78.7
71.7
78.6
62.9
95.2
75.2
78.5
71.5
78.4
69.4
105.3
74.3
77.9
71.1
78.8
62.7
39.8
28.3
72.6
43.5
49.5
18.9
34.9
42.1
51.9
38.1
22.8
27.4
40.4
43.0
30.1
33.7
50.4
50.3
38.5
144.0
118.2
38.3
73.9
12.8
17.4
16.6
15.3
175.8
23.8
22.1
104.3
74.2
77.0
78.6
76.3
62.7
95.3
75.2
78.3
71.4
77.9
62.5
39.8
28.7
72.5
43.5
49.5
18.8
35.0
41.9
51.9
38.0
22.8
27.3
40.4
43.0
30.2
34.1
49.9
50.9
38.6
144.0
118.2
39.0
73.8
12.7
17.4
16.6
15.2
180.0
23.9
22.1
104.3
75.0
76.9
77.8
76.2
62.5
39.5
28.7
74.2
43.5
49.8
19.0
34.8
42.1
51.5
38.0
22.8
27.4
40.5
43.1
30.2
33.7
50.4
50.3
38.5
144.1
118.1
38.3
75.0
12.7
17.3
16.6
15.5
175.9
23.8
22.1
102.8
81.4
72.7
77.4
76.2
62.6
95.2
75.7
78.3
71.1
78.6
62.5
39.3
28.7
72.6
43.4
49.8
19.0
34.9
41.9
51.4
39.3
22.8
27.4
40.4
43.0
30.2
34.0
49.9
50.0
38.5
144.1
118.1
38.9
75.0
12.6
17.2
16.5
15.4
179.8
23.8
22.0
102.8
81.0
75.0
77.2
76.1
62.4
b-D
ESI-QTOF-MS (negative ion mode), which displayed [M-H]- ions at
m/z 729.3496 (calcd for C36H58O13S e H ¼ 729.3525), with eight
degrees of unsaturation. In the ESI-MS/MS spectra of 2 (negative
ion mode), the product ion observed at m/z 567 [M-H-162]- suggest
the loss of hexose unit. Fragment ion at m/z 303 [M-H-162-264]-
was supposed to be generated from m/z 567 by retro Diels-Alder
cleavage of ring C. Another product ion at m/z 97 was indicating
the fragment of sulfate group [HSO4]- (Perrone et al., 2007; Shaker
et al., 2000).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
The 1H- NMR spectrum (Table 1) of 2 displayed five singlets for
tertiary methyls at dH 0.68, 0.98, 0.79, 1.18 and 0.88 assigned to Me-
24, Me-25, Me-26, Me-27 and Me-29, respectively. Furthermore, in
1H and 13C-NMR spectra, signals for one olefinic methine at dH
/dc
5.28 (t, J12/11 ¼3.0 Hz)/124.0 (C-12), carboxyl ester at dC 178.0 (C-28)
and two hydroxymethylenes at dH c 3.30, 3.49/dC 65.1 (C-23) and
3.41, 3.50/66.3 (C-30) were observed. An oxygenated methine
/d
signal at dH
/
dC 4.25 (dd, J3/2ax ¼ 12.0 Hz, J3/2eq ¼ 4.5 Hz)/80.8 was
assigned to CH-3. The 13C-NMR spectrum displayed resonances for
six sp3 quaternary carbons at dC 43.7 (C-4), 40.7 (C-8), 37.9 (C-10),
43.0 (C-14), 47.9 (C-17), and 46.1 (C-20). Anomeric carbon signals
were observed at dH
/
dC 5.35 (d, J1'/2' ¼ 8.0 Hz)/95.7 (C-10). Above
discussed fact clearly indicated that aglycone part of the compound
2 is oleanane type tri-terpene (Khalik et al., 2000; Luo et al., 2011;
Miyakoshi et al., 1997). The downfield shift of H-3 indicated the
substitution of sulfate group on hydroxyl group (Perrone et al.,
2007). In HMBC spectrum (Fig. 2), anomeric proton at dH 5.35
showed correlation with C-28, which indicated the position of
sugar moiety. Stereochemistry at C-3 was assigned on the basis of
coupling constants values of H-3, which showed J ¼ 12.0, and
4.5 Hz. It indicated that H-3 position is axially oriented, which was
further supported by a NOESY cross-peak between H-5 (dH 1.62)
and H-3 (dH 4.25) (Fig. 3). The stereochemistry at C-20 was assigned
based on carbon value of C-20, as reported in literature (Luo et al.,
2011; Miyakoshi et al., 1997) and also confirmed by NOESY corre-
lation between H-18 and H-30. The stereochemistry of the several
centers was demonstrated by NOESY spectrum showing connec-
20
30
40
50
60
100
200
300
400
500
600
1000
2000
3000
4000
5000
6000
tivities between Me-24
and H-5 /H-9 . The configuration of the sugar unit was assigned as
-glucose. Thus, the structure of 2 was identified as -glucopyr-
-O-sulfo-23,30-dihydroxy-olean-12-en-28-oate.
b/Me-25b, Me-25b/Me-26b, H-3a/H2-23
a
a
a
In CD3OD (
d
ppm) for compounds 1e2, 4e7 and pyridine-d5(
d
ppm) for com-
pound 3; J in Hz.
D
b-D
anosyl 3
b
The ESI-QTOF-MS (negative ion mode) of nayabin C (3) showed
a peak at m/z 957.5020 (calcd. for C48H78O19 e H ¼ 957.5064), ten
degrees of unsaturation. In the ESI-MS/MS spectra of 3 (negative
ion mode), the fragment ion observed at m/z 633 [M-H-324]- sug-
gests the loss of two glucose moieties.
mentioned facts indicated that aglycone part of the compound is
lupane class of triterpene similar to bourneioside A, with only the
difference of position of glucose moiety on C-23 instead of C-3
(Xiang et al., 2000). The up-field shift of C-1'' (dc 95.2) signal sug-
gested that unit was an ester substituent of C-28 acid function,
The 1H-NMR of the aglycone part displayed five tertiary methyl
protons at dH 0.96, 0.86, 1.15, 0.88, 1.68, (each s) and 1.02 d (J29/
which was further confirmed by the HMBC correlation between H-
1" (dc 5.47) and C-28 (dc 176.1) (Shaker et al., 1999; Xiang et al.,
¼ 6.5 Hz). 1H and 13C-NMR (Tables 1 and 2) spectra of 3 showed
18
2000). An HMBC correlation observed between H-10 of glucose
moiety (dH 4.22) and the carbon C-23 (dC 74.1) confirmed the po-
sition of other glucose. Key HMBC and COSY-45ꢀ interactions of
compound 1 are presented in Fig. 2. Stereochemistry of compound
1 was assigned on the biogenesis basis and NOESY correlation
(Fig. 3). A NOESY correlation signal between the H-5 (dH 1.21), H-3
signals at [dH 4.25 (overlap)/dC 72.1; CH-3], [dH 5.44 (br d, J21/
¼ 7 Hz)/dC 117; CH-21], C-23 hydroxymethyl group [dH 4.05 and
22
4.18 (each overlap)/dC 74.9; CH2-23] and at dC 143.1 (C-20), dC 177.5
(C-28). The 13C-NMR spectrum displayed resonances for five sp3
quaternary carbons at dC 43.0 (C-4), 42.3 (C-8), 37.2 (C-10), 42.3 (C-
14), and 49.7 (C-17). The 1H- and 13C- NMR spectra showed the
(
dH 3.73) and H-9 (dH 1.40) was consistent with the
In addition, the key correlations of Me-24 /Me-25
26 and Me-26 /H-13 showed that Me-24, Me-25, Me-26, H-13
a
- orientation.
presence of three anomeric signals at dH
/
dC 4.91 (d, J1'/2' ¼ 8.0 Hz)/
000
b
b, Me-25 /Me-
b
105.5; dH
/
dC 6.26 (d, J1''/2'' ¼ 8.0 Hz)/95.2 and dH
/
dC 4.97(d, J1
/
2000
b
b
b
¼ 8.0 Hz)/105.3 indicated the presence of three hexose units.
were on the same face, so the relative stereochemistry were
Above mentioned data indicated that aglycone part of 3 was similar
determined. NOESY correlation between H-18 (dH 1.04) and Me-27
to taraxastane type triterpene reported in literature (Ansari et al.,