Four New Flavonoids Isolated from the Aerial Parts of Cadaba rotundifolia Forssk. (Qadab)

Article abstract of DOI:10.3390/molecules24112167

Cadaba rotundifolia (Forssk.) (family: Capparaceae; common name: Qadab) is one of four species that grow in the Red Sea costal region in the Kingdom of Saudi Arabia. The roots and leaves of C. rotundifolia is traditionally used to treat tumors and abscesses in Sudan. A previous phytochemical study of the roots yielded a quaternary alkaloid, but no report on chemical constituents of the aerial parts of the C. rotundifolia growing in Saudi Arabia has been issued so far. Oxidative stress and advanced glycation end products (AGEs) are thought as causal factors in many degenerative diseases, such as Alzheimer's disease, diabetes, atherosclerosis and aging. In this study, a total of twenty compounds, including four previously undescribed acylated kaempferol glucosides, were isolated from the aerial parts of C. rotundifolia collected in Saudi Arabia. These new compounds were identified as kaempferol 3-O-[2-O-(trans-feruloyl)-3-O-β-d-glucopyranosyl]-β-d-glucopyranoside (1), kaempferol 3-O-β-neohesperidoside-7-O-[2-O-(cis-p-coumaroyl)-3-O-β-d-glucopyranosyl]-β-d-glucopyranoside (2), kaempferol 3-O-[2,6-di-O-α-l-rhamnopyranosyl]-β-d-glucopyranoside-7-O-[6-O-(trans-feruloyl)]-β-d-glucopyranoside (3) and kaempferol 3-O-[2,6-di-O-α-l-rhamnopyranosyl]-β-d-glucopyranoside-7-O-[6-O-(trans-p-coumaroyl)]-β-d-glucopyranoside (4). Their structures were established based on UV-visible, 1D, 2D NMR, and HR-ESI-MS analyses. Of the assayed compounds, 17 and 18 showed potent radical scavenging activity with IC₅₀ values of 14.5 and 11.7 μM, respectively, and inhibitory activity toward AGEs together with compound 7 with IC₅₀ values 96.5, 34.9 and 85.5 μM, respectively.

Full text of DOI:10.3390/molecules24112167

molecules
Article
Four New Flavonoids Isolated from the Aerial Parts of
Cadaba rotundifolia Forssk. (Qadab)
Gadah Abdulaziz Al-Hamoud 1 , Raha Saud Orfali *, Sachiko Sugimoto , Yoshi Yamano ,
,2
2,
1
1
3
4
1,
Nafee Alothyqi , Ali Mohammed Alzahrani and Katsuyoshi Matsunami *
1
Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University,
1
-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan; galhamoud@ksu.edu.sa (G.A.A.-H.);
ssugimot@hiroshima-u.ac.jp (S.S.); yamano@hiroshima-u.ac.jp (Y.Y.)
Department of Pharmacognosy, College of Pharmacy, King Saud University, 11495 Riyadh, Saudi Arabia
Department of Biology, Umm Al-Qura University, 1109 Makkah Al-Mukarramah, Saudi Arabia;
niothyqi@uqu.edu.sa
Department of Biology, Arts and Sciences in Qilwah, Al-Baha University, 1988 Al-Baha, Saudi Arabia;
alialzahrani@bu.edu.sa
2
3
4
*
Correspondence: rorfali@ksu.edu.sa (R.S.O.); matunami@hiroshima-u.ac.jp (K.M.);
Tel.: +966-11-8055014 (R.S.O.); +81-82-257-5335 (K.M.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Francesco Epifano
Received: 19 May 2019; Accepted: 6 June 2019; Published: 9 June 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Cadaba rotundifolia (Forssk.) (family: Capparaceae; common name: Qadab) is one of four
species that grow in the Red Sea costal region in the Kingdom of Saudi Arabia. The roots and leaves of
C. rotundifolia is traditionally used to treat tumors and abscesses in Sudan. A previous phytochemical
study of the roots yielded a quaternary alkaloid, but no report on chemical constituents of the aerial
parts of the C. rotundifolia growing in Saudi Arabia has been issued so far. Oxidative stress and advanced
glycation end products (AGEs) are thought as causal factors in many degenerative diseases, such as
Alzheimer’s disease, diabetes, atherosclerosis and aging. In this study, a total of twenty compounds,
including four previously undescribed acylated kaempferol glucosides, were isolated from the
aerial parts of C. rotundifolia collected in Saudi Arabia. These new compounds were identified as
kaempferol 3-O-[2-O-(trans-feruloyl)-3-O-
-O- -neohesperidoside-7-O-[2-O-(cis-p-coumaroyl)-3-O-
), kaempferol 3-O-[2,6-di-O- -l-rhamnopyranosyl]- -d-glucopyranoside-7-O-[6-O-(trans-feruloyl)]-
-d-glucopyranoside ( ) and kaempferol 3-O-[2,6-di-O- -l-rhamnopyranosyl]- -d-glucopyranoside-
-d-glucopyranoside ( ). Their structures were established based on
UV-visible, 1D, 2D NMR, and HR-ESI-MS analyses. Of the assayed compounds, 17 and 18 showed
potent radical scavenging activity with IC50 values of 14.5 and 11.7 M, respectively, and inhibitory
activity toward AGEs together with compound with IC50 values 96.5, 34.9 and 85.5 M, respectively.
β
-d-glucopyranosyl]-
β
-d-glucopyranoside (
1), kaempferol
3
β
β-d-glucopyranosyl]-
β-d-glucopyranoside
(2
α
β
β
3
α
β
7
-O-[6-O-(trans-p-coumaroyl)]-
β
4
µ
7
µ
Keywords: Cadaba rotundifolia; Capparaceae; Qadab; aerial parts; flavonoid; antioxidant; AGEs
1
. Introduction
Oxidative stress and advanced glycation end products (AGEs) are thought as causal factors of
many diseases, such as diabetes, atherosclerosis, neurodegenerative disorders and aging. Therefore,
the discovery of antioxidant and anti-AGEs molecules are important for preventing these age-related
pathogenesis. Cadaba (Forssk.) is one of genera in the Capparaceae family, with about 30 species
distributed in southern Africa, India, Malaysia, and Australia [
grow in the Red Sea costal region in the Kingdom of Saudi Arabia [
several Cadaba species led to isolation of various secondary metabolites, such as alkaloids, terpenoids
1
,2
]. Among them four species
3]. Phytochemical studies of
Molecules 2019, 24, 2167; doi:10.3390/molecules24112167
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2167
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and flavonoids [4–7]. Cadaba rotundifolia Forssk., known by the common name Qadab, is an erect
shrub with numerous drooping branches with young twigs covered with short glandular hair [ ].
8
In Sudan, C. rotundifolia roots and leaves are traditionally used to treat tumors and abscesses.
A previous chemical study of the ethanolic extract of C. rotundifolia root yielded a quaternary
alkaloid (3-hydroxystachydrine) [9]. However, no report on chemical constituents of the aerial parts of
C. rotundifolia has been issued so far. In this work, we have investigated the chemical profile of the
aerial parts of C. rotundifolia collected in Saudi Arabia. As a result, twenty flavonoids were obtained,
including four undescribed acylated kaempferol glucosides 1–4 and 16 known compounds. This paper
describes on the isolation, structural elucidation, and evaluation of antioxidant (DPPH) and inhibitory
activity toward AGEs formation.
2
. Results and Discussion
2
.1. Isolation and Spectroscopic Analyses of the Compounds
The aerial parts of C. rotundifolia were extracted with MeOH at room temperature till
exhausted. The MeOH extract (800 g) was further separated by solvent fractionation, various
column chromatographies (CC), and HPLC to afford compounds 20 (Figure 1). The chemical
1–
structures of the isolated compounds were investigated and identified through intensive spectroscopic
analyses based on UV-visible, 1D and 2D NMR, and HR-ESI-MS data as follows (Figures S1–S35).
OH
OR1
OH
OH
HO
O
O
R1
R2
R3 R4
HO
O
O
1
5
6
7
8
9
0
H
H
Glc
H
trans-feruloyl
trans-p-coumaroyl Glc
Glc
H
H
O
O
OH
R O
H
Rha
Rha
H
H
H
H
H
H
H
H
Rha
H
Rha
H
OH
R O
R1 R2
15 Rha Rha
4
2
H
H
H
CH3
O
O
1
6 Rha
H
H
OR3
OH
17
H
1
H
HO
HO
OR2 11
Rha
H
OR1
OH
R1
R2
R3
R4
H
2
3
4
H
Rha
Rha
H
H
H
cis-p-coumaroyl Glc
O
O
H
H
H
H
trans-feruloyl
trans-p-coumaroyl
R O
4
O
OR3
O
12
trans-p-coumaroyl Glc
trans-feruloyl
trans-p-coumaroyl
H
H
H
OH
O
13
H
H
HO
R O
1
14
OR2
O
OH
HO
HO
OH
O
OH
OH
RO
OR
OH
R
O
HO
O
O
OH
O
19 Glc
20
HO OH
OH
1
8
H
OH
Figure 1. Compounds 1–20 isolated from Cadaba rotundifolia.
.1.1. Chemical Structure of Compound 1
2
Compound
1
−
was isolated as a yellow amorphous powder, soluble in MeOH with a negative optical
22
rotation ([
α
]
79.0). Its molecular formula, C H O , was determined by positive HR-ESI-MS
D
37 38 19
+
analysis (m/z 809.1888 [M + Na] ,calcd for C H O Na 809.1899). It had a UV spectrum typical
37
38 19

Molecules 2019, 24, 2167
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of an acylated kaempferol glycoside (λmax 327, 267 nm) [10
,
11]. The IR spectrum suggested the
−1
−1
presence of hydroxy (3388 cm ),
α
,
β
-unsaturated carbonyl ester (1710 cm ),
α
,β-unsaturated ketone
−
1
−1
−1
(
1658 cm ), aromatic ring (1513, 1446 cm ) and ether (1176, 1070 cm ) functions. The proton and
1
13
carbon resonances in its H and C-NMR spectra clarified the aromatic and glycosidic nature of
1.
1
It was found that the H-NMR spectrum of
1
displayed feature resembling to those of
5
[12], as both the
aglycone and glycoside units were almost identical. The major differences were in the signals related to
1
the acyl group of
5
. The H-NMR spectrum of
1
(Table 1) exhibited meta-coupled resonances at
δ
1
6.17
H
1
and 6.37 (each 1H, d, J = 2.0 Hz), characteristic of H-6 and H-8 of the A-ring, respectively. The H– H
1
correlation spectrum (COSY, Figure 2) and H-NMR spectrum of
1
displayed two ortho-coupled doublet
0 0 0 0
δ 8.01 and 6.91 (each 2H, d, J = 8.8 Hz) assignable to H-2 /6 and H-3 /5 of the B-ring,
H
resonances at
respectively, which indicated the presence of a kaempferol aglycone (Table 1).
1
Table 1. H-NMR data of compounds 1–4 (600 or 700 * MHz, CD OD, δ in ppm, J in Hz).
3
Position
1 *
2
3
4
Position
1 *
2
3
4
0
0
000
000
6
8
2
3
5
6.17 d (2.0)
6.37 d (2.0)
8.01 d (8.8)
6.91 d (8.8)
6.91 d (8.8)
6.37 br s
6.65 br s
8.06 d (8.9)
6.89 d (8.9)
6.89 d (8.9)
6.46 d (2.1)
6.61 d (2.1)
7.93 d (8.9)
6.76 d (8.9)
6.76 d (8.9)
6.45 d (2.1)
6.62 d (2.1)
7.95 d (8.8)
6.77 d (8.8)
6.77 d (8.8)
1
2
3
4
5
-
5.22 d (1.3)
3.99 m
3.76 m
3.33 t (9.6)
4.02 dq (9.6, 3.93 dq (9.5, 3.68 dq
5.14 d (1.1)
3.90 m
3.69 m
5.14 br s
3.90 m
3.69 m
3.42 m
7.20 d (1.8)
-
-
0
0
0
0000
0000
0000
3.25 m
6.83 d (8.2)
6
.18)
7.09 dd (8.2, 0.96 d (6.18) 0.88 d (6.2)
.8)
7.68 br d
15.9)
6.40 br d
15.9)
3.92 s
6.2)
(9.6, 6.2)
0.96 d
(6.2)
0
0000
6
8.01 d (8.8)
8.06 d (8.9)
7.93 d (8.9)
7.95 d (8.8)
6
7
8
1
0
000
(
0
0
0
0000
1
5.73 d (8.0)
5.72 d (7.6)
5.51 d (7.6)
5.51 d (7.6)
(
0
2
5.23 dd (9.5, 3.60 m
.0)
3.52 dd (9.6, 3.52 dd (9.6, OCH3
8
7.6)
7.6)
0
0
0
0
0
0
3
4
5
3.91 t (9.5)
3.55 t (9.5)
3.39 ddd
3.55 t (9.0)
3.26 t (9.0)
3.31 m
3.45 m
3.13 t (9.12)
3.24 m
3.44 m
3.19 m
3.24 m
00000
00000
1
2
4.40 d (7.8)
3.20 dd (8.5, 3.38 m
7.8)
4.38 d (1.3)
4.36 br s
3.37 m
(
9.5, 5.5,
.0)
3.62 dd
12.0, 5.5)
.81 dd
12.0, 2.0)
2
0
0
00000
6
3.47 dd
(12.0, 5.9)
3.71 dd
3.27 m
3.71 m
3.27 m
3.71 m
3
3.31 m
3.29 m
3.34 dd (9.4, 3.33 dd
3.4)
3.12 t (9.4)
(
3
(9.4, 3.4)
3.25 m
0
0
0000
0000
4
(
(12.0, 2.1)
5
6
3.22 m
3.87 dd
3.30 m
0.96 d (6.2)
3.25 m
0.88 d
(6.2)
0
00
00
00000
1
4.43 d (7.8)
5.29 d (7.8)
4.98 d (7.4)
4.98 d (7.3)
3.41 t (9.4)
(11.5, 5.4)
0
2
3.21 dd (8.8, 5.25 dd (9.0, 3.42 m
3.64 dd
7
.8)
7.8)
3.92 t (9.0)
3.76 m
(11.5, 1.3)
0
0
00
00
3
4
3.31 t (8.8)
3.28 t (8.8)
3.43 m
3.34 m
3.42 m
3.34 t (9.4)
000000
000000
000000
2
3
5
6
7
8
7.55 d (8.6)
6.67 d (8.6)
6.67 d (8.6)
7.55 d (8.6)
6.86 br d
6.98 d (1.7)
-
7.25 d
(
8.6)
6.66 d
8.6)
0
00
00
5
6
3.34 m
3.78 m
3.72 m
3.72 m
(
0
3.62 dd
3.75 dd
(12.0, 5.1)
3.94 dd
4.17 dd
(12.0, 2.0)
4.57 dd
4.18 dd
(12.0, 2.1)
4.54 dd
6.67 d (8.2)
6.66 d
(8.6)
(
3
(
11.9, 2.2)
.88 dd
11.9, 6.0)
0
0
0
00000
00000
00000
6.85 d (8.2,
1.7)
7.50 br d
(15.8)
6.28 br d
(15.8)
7.25 d
(8.6)
7.50 br
d (15.9)
6.24 br
d (15.9)
(12.0, 1.6)
(12.0, 6.9)
(12.0, 7.0)
(
12.9)
5.83 br d
12.9)
(
OCH3
3.75 s
m: multiplet or overlapped signals.
1
The H-NMR spectrum of
1
(Table 1) also gave signals at
δ
H
6.83 (1H, d, J = 8.2 Hz), 7.09
(1H, d, J = 8.2, 1.8 Hz) and 7.20 (1H, d, J = 1.8 Hz) attributable to an ABX coupling system for a
1
,3,4-trisubstituted aromatic moiety, and two trans-coupled olefinic protons at 6.40 (1H, br d,
δ
H
J = 15.9 Hz) and 7.68 (1H, br d, J = 15.9 Hz), together with a singlet signal for a methoxy group at
δ
H
3.92 which suggested the presence of a feruloyl or isoferuloyl function. The two anomeric proton
resonances, which occur at
δ 5.73 (1H, d, J = 8.0 Hz) and 4.43 (1H, d, J = 7.8 Hz) and 12 carbon signals
H
(δc 104.9, 100.5, 84.9, 78.4, 78.1 , 77.7, 74.8, 74.6, 71.4, 70.0, 62.5 and 62.4) correspond to two glucose

Molecules 2019, 24, 2167
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moieties. Acid hydrolysis of
1
released d- glucose and the
β-anomeric configuration for the sugars
was determined from the magnitude of the J1,2 coupling constants. In addition, alkaline hydrolysis
afforded ferulic acid, which was identified by comparing with an authentic sample based on their
HPLC retention times. The 13C-NMR spectrum (Table 2) of
1
showed 37 carbon resonances, 12 of
-glucose units, 15 signals were consistent with kaempferol aglycone and the
remaining 10 signals were ascribed to acyl groups. The correlations observed between the proton and
which assigned to two
β
0
000
0000
0000
c 111.7), C-6 (δ
carbon resonances of the acyl moiety from H-7
(
δ
H 7.68) to C-2
c 168.5), and from the protons of methoxy group ( H
49.3) in HMBC spectrum (Figure 2) indicated the presence of a feruloyl unit.
(
δ
c 124.3) and
0
000
0000
an ester carbonyl C-9
(
δ
δ
3.92) to C-3
(δc
1
OH
OH
O9''''''
7
''''''
4'
4'
H CO
1''''''
3
6'''
5'''
9 O
O
2
HO
9 O
2
O
3
''''''
1'
1'
7
8
''''''
7
O
10
10
6
''''''
HO 4''''''
1'''
3
4''' OH
3
O
O
5
4
5
4
3'''
HO
2''' OH
O
OH O6
HO
''
''
OH
5
6''
5''
6
'''
HO
O
O
O
5'''
HO 5'''''
1
''
O 1 4'' O3''
O
'''
OH
1'''''
1''
2''
4
'''''
4'' O3H''
HO
O
2''
3'''''
HO
9''''O
2''''' HO
OH
HO OH
O
8
''''
7''''
HO 5''''
O
COSY
1''''
1''''
2''''
4''''
3''''
2
''''
''''
6''''
5''''
1
3
H
C HMBC
3
HO OH
H CO
3
4''''
OH
OH
OH
O9''''''
4'
7''''''
4'
6
'''
O
9 O
2
1''''''
HO
6'''
5'''
O
9 O
2
1
'
O
7
1
'
5
'''
8''''''
7
6
'''''
'''''
HO
O
O
10
10
5
1
'''
3
HO 4''''''
6''''''
O 1 4''' O3'''
1'''
3
O
4''' OH
5
4
O
'''''
3'''
5
4
OH
HO
HO
2''' OH O6
HO
2''' OH
O
''
''
HO
OH
5
HO
O
''''''
O
6''
5''
O
O
1
''
9
O
4'' O3H''
HO 5'''''
1''''''
O
2
''''''
''''''
6''''''
5''''''
HO
2''
4'''''
1''''' 4''
1''
2''
OH
3'''''
3''
O
3
2''''' HO
4''''''
HO 5''''
O
HO OH
O
2
4
OH
1''''
''''
4''''
HO 5''''
3''''
O
2
1''''
''''
4''''
3''''
HO OH
2
HO OH
Figure 2. COSY and HMBC correlations of 1–4.
0
000
0000
According to the coupling constant of the olefinic protons at H-7
and H-8
(J = 15.9 Hz),
1
the configuration of the feruloyl moiety was determined as trans. Full assignments of the H and
13
C resonances of the structural fragments of
1 were achieved by analysis of COSY, HSQC and
0
0
HMBC spectra. A correlation between the anomeric H-1 proton ( H
δ
5.73) and δc 134.8 in the
HMBC spectrum (Figure 2) defined C-3 of kaempferol as a site of O-glucosylation. Moreover,
the interglucosidic linkage of the disaccharide moiety was also determined by the HMBC cross-peak
0
0
0
0
0
0
0
between H-3
(
δ
H
3.91) and C-1
(δc 104.9). The E-feruloyl unit was linked to C-2 based on
0
0
00
00
(δ
the correlations of the H-2
(
δ
5.23) with C-1
(δ
c 100.5), C-3
c 168.5). Therefore, the structure of compound
-O-[2-O-(trans-feruloyl)-3-O-β-d-glucopyranosyl]-β-d-glucopyranoside.
c 84.9) and ester carbonyl
H
0
000
(δ
carbon C-9
1 was determined as kaempferol
3

Molecules 2019, 24, 2167
5 of 13
Table 2. 13C-NMR data of compounds 1–4 (150 or 175 * MHz, CD OD, δ in ppm).
3
Position
1 *
2
3
4
Position
1 *
2
3
4
0
0
0
0
0
0
0
0
0
000
000
000
000
000
000
000
000
000
2
3
4
5
6
7
8
9
158.4
134.8
179.2
163.2
99.8
159.2
134.7
179.5
163.0
100.4
163.8
95.7
157.9
108.0
122.8
132.3
116.2
161.6
116.2
132.3
159.4
134.6
179.5
162.9
100.7
164.4
96.3
157.8
107.7
122.8
132.2
116.2
161.4
116.2
132.2
159.4
134.6
179.5
162.9
100.7
164.4
96.2
157.8
107.7
122.8
132.2
116.9
161.5
116.9
132.2
1
2
3
4
5
6
7
8
9
127.8
111.7
149.3
150.6
116.4
124.3
147.5
115.5
168.5
56.4
102.6
72.4
72.3
74.0
69.9
17.6
102.5
72.3
72.3
74.0
69.9
17.6
102.5
72.3
72.3
74.7
69.9
17.6
165.8
94.6
158.5
105.8
122.7
132.2
116.3
161.6
116.3
132.2
1
1
2
3
4
5
6
0
0
0
0
0
0
0
OCH3
00000
00000
00000
00000
1
2
3
4
5
6
105.0
74.7
77.8
71.4
78.9
62.5
102.2
72.0
72.0
73.8
69.7
17.8
102.2
72.0
72.0
74.0
69.7
17.8
0
0000
0
0
00000
1
2
3
4
5
6
100.5
74.6
84.9
70.0
78.4
62.4
100.2
80.0
78.9
71.8
78.1
62.6
100.3
79.7
79.0
72.0
77.2
68.3
100.3
79.8
79.0
71.7
77.2
68.3
0
0
0
0
0
0
0
0
0
0
000000
000000
000000
000000
1
2
3
4
5
6
7
8
9
127.6
133.4
115.9
160.0
115.9
133.4
145.0
116.6
167.2
127.6
111.6
149.2
150.5
116.5
124.2
147.3
115.1
169.1
56.5
127.1
131.2
116.2
161.2
116.2
131.2
147.1
114.7
169.1
0
00000
0
00
00
00
00
00
00
000000
000000
000000
000000
1
2
3
4
5
6
104.9
74.8
77.7
71.4
78.1
62.5
99.6
73.4
84.3
69.8
78.2
62.2
101.6
74.7
77.8
71.7
75.8
64.6
101.6
73.8
77.8
71.8
75.7
64.6
0
0
0
0
0
OCH3
2
.1.2. Chemical Structure of Compound 2
Compound was isolated as a yellow amorphous powder, with a positive optical rotation ([
14.5). Its molecular formula was determined to be C H O by positive HR-ESI-MS (m/z 1087.2875
2
α
] 22
D
+
48
56 27
+
(
[M + Na] , calcd for C H O Na 1087.2901). The UV profile of
2
displayed λmax 316, 267 nm, which
48
56 27
was similar to those of the kaempferol glycoside acylated with a hydroxycinnamic acid [10 11 13].
,
,
−
1
The IR spectrum indicated absorptions of hydroxy (3397 cm ),
α
,β
-unsaturated carbonyl ester
-unsaturated ketone (1657 cm ), aromatic ring (1512, 1442 cm ) and ether (1157,
072 cm ) functions. The above features were confirmed by the H-NMR spectrum which revealed a
−1
−1
−1
(
1721 cm ),
α
,β
−1
1
1
set of kaempferol signals, a p-coumaroyl group and glycoside moieties. The kaempferol aglycone of
2
1
was represented in the H-NMR spectrum (Table 1) by two ortho-coupled resonances of the B-ring at
0 0 0 0
δ
8.06 and 6.89 (each 1H, d, J = 8.9 Hz), typical of H-2 /6 and H-3 /5 , respectively, in addition to two
H
meta-coupled proton resonances at
A-ring, respectively. In comparison with literature, the downfield shifts of H-6 and H-8 in the H-NMR
δ
6.37 and 6.65 (each 1H, br s), attributed to H-6 and H-8 of the
H
1
spectrum suggested the presence of substitution at C-7 of kaempferol, while the substitution at C-3 was
159.2 in the 13C-NMR spectrum. Moreover, the H-NMR
1
evident from the downfield shift of C-2 to
δ
C
spectrum (Table 1) exhibited two olefinic protons at δ 5.83 and 6.86 with a coupling constant of 12.9
H
0
0
Hz. Similarly, the AA BB aromatic coupling system at
δ 6.67 and 7.55 (each 1H, d, J = 8.6 Hz) were
H
attributed to a p-coumaroyl unit, which was confirmed by the correlation of the olefinic protons (
δ
H
0
00000
5
.83 and 6.86) with the ester carbonyl carbon at
δ
c 167.2 and aromatic carbons ( C
δ
127.6; C-1
and
0
00000
133.4; C-2/6
) in the HMBC spectrum (Figure 2). The sugar units in
2 were represented in the
1
H-NMR spectra by four anomeric proton signals at
δ 4.40 (1H, d, J = 7.8 Hz), 5.22 (1H, d, J = 1.3 Hz),
H
5.29 (1H, d, J = 7.8 Hz), 5.72 (1H, d, J = 7.6 Hz) and one methyl group at 0.96 (3H, d, J = 6.18 Hz),

Molecules 2019, 24, 2167
6 of 13
which indicated the tetraglycosidic nature of
2. Identification of the sugar residues was carried out by
careful analysis of the 1D and 2D NMR data as well as comparison with the reported chemical shift
values. The absolute stereochemistry of the sugars was determined by acid hydrolysis of and HPLC
analysis with an optical rotation detector, and the magnitudes of J1,2 coupling constants that revealed
-d-glucopyranose and -l-rhamnopyranose moieties. The spectroscopic data of Tables 1 and 2
indicated that it is an isomer of 12 13]. The relatively smaller coupling constant (J = 12.9 Hz) and the
2
β
α
2
(
)
[
0
00000
000000
(δ 6.86 and 5.83, respectively) indicated 2 as a cis isomer.
H
chemical shift values of H-7
and H-8
In addition, alkaline hydrolysis of
the cis-p-coumaroyl moiety on the kaempferol aglycone were decided using the HMBC spectrum
Figure 2). The anomeric protons of two glucose moieties at 5.72 and 5.29 have long-range
correlations with C-3 ( c 134.7) and C-7 (
the connectivity of the sugars. The COSY correlation (Figure 2) of H-2 at (
2 afforded cis-p-coumaric acid. The positions of glycosylation and
(
δ
H
δ
δ
c 163.8) of the kaempferol aglycone, respectively, indicating
0
0
00
δ
0
3.60) with H-1 at (
δ
H
H
0
0000
5
.72), together with correlation observed in the HMBC spectrum between H-2 and C-1
of rhamnose
at (
δ
c 102.6) confirmed the presence of a
β
-neohesperidoside structure at C-3 of kaempferol. Moreover,
0
00
000
000
(δ 3.92) in COSY spectrum, and the
H
the correlations of H-2
(δ
5.25) with H-1
(δ
5.29) and H-3
H
H
0
00
000000
000
00000
correlations from H-2 to C-9
(δc 167.2), and from H-3 to C-1
(δ
c 105.0) in HMBC spectrum
-d-glucopyranose
was therefore identified as kaempferol
-O-β-neohesperidoside-7-O-[2-O-(cis-p-coumaroyl)-3-O-β-d-glucopyranosyl]-β-d-glucopyranoside.
confirmed the presence of 7-O-[2-O-(cis-p-coumaroyl)-3-O-
as the kaempferol aglycone. The structure of
3
2
β-d-gluco-pyranosyl]-β
2
.1.3. Chemical Structure of Compound 3
Compound was obtained as a yellow amorphous powder with a negative optical rotation ([
82.5). The molecular formula, C H O , was deduced from the sodiated molecular ion peak at m/z
3
α
]22
D
−
49
58 27
1
101.3021 (calcd for C H O Na 1101.3058) by HR-ESI-MS measurement. It displayed characteristic
49
58 27
UV spectrum of an acylated kaempferol glycoside with λmax at 329, 268 nm, similar to those of
1
. The IR
spectrum exhibited absorptions of hydroxy (3389 cm 1),
−
α,β-unsaturated ketone (1651 cm ), aromatic
−1
ring (1513, 1455 cm ) and ether (1180, 1071 cm ) functions. Analysis of the H- and 13C-NMR spectra
−1
−1
1
of
3
(Tables 1 and 2) indicated the presence of a kaempferol residue, four hexose sugar moieties, and an
1
E-feruloyl unit. The H spectrum (Table 1) displayed four downfield doublet signals at
δ 6.46 (1H, d,
H
J = 2.1 Hz), 6.61 (1H, d, J = 2.1 Hz), 6.76 (2 H, d, J = 8.9 Hz) and 7.93 (2 H, d, J = 8.9 Hz) assignable to H-6,
0
0
0
0
8
, 3 /5 and 2 /6 , respectively, belonging to kaempferol aglycone. Moreover, the characteristic signals of
1
the E-feruloyl moiety were observed in H-NMR spectrum as five downfield doublets at
δ 6.28 (1H, br
H
d, J = 15.8 Hz), 6.67 (1H, d, J = 8.2 Hz), 6.85 (1H, d, J = 8.2, 1.7 Hz), 6.98 (1H, d, J = 1.7 Hz) and 7.50 (1H,
0
00000 000000 000000 000000
000000
br d, J = 15.8 Hz), corresponding to H-8
, 5
, 6
, 2
and 7
, respectively, in addition
was
achieved by analysis of COSY, HSQC and HMBC data, which confirmed presence of a 3,7-O-glycosidic
kaempferol residue together with the E-feruloyl unit. Acid hydrolysis of with 1 M HCl released
to a singlet signal of a methoxy group at
δ
H
3.75. Full assignment of the 1D NMR spectra of
3
3
d-glucose and l-rhamnose moieties, while alkaline hydrolysis afforded a ferulic acid moiety, identified
1
by HPLC comparison with authentic samples. The H-NMR spectrum of
3 exhibited resonances
for anomeric protons of O-linked sugars at
δ 4.98 (1H, d, J = 7.4 Hz) and 5.51 (1H, d, J = 7.6 Hz),
H
corresponding to two d-glucoses, and 4.38 (1H, d, J = 1.3 Hz) and 5.14 (1H, d, J = 1.1 Hz), together
with typical doublets of two methyl groups at 0.88 (3H, d, J = 6.2 Hz) and 0.96 (3H, d, J = 6.2 Hz),
δ
H
1
13
corresponding to two l-rhamnose moieties. Full assignment of the H and C-NMR data of each
sugar was achieved using standard 2D NMR experiments (Tables 1 and 2). The configurations of these
sugars were identified from the magnitude of their J1,2 coupling constants and the chemical shifts as
β
-d-glucopyranose and
3
α-l-rhamnopyranose. The glycosidation sites on the kaempferol aglycone
0
0
of were determined from the long-range correlations exhibited in the HMBC spectrum from H-1
0
00
00
00
δ
5.51 to C-3 (
δc 134.6), and from H-1 4.98 to C-7 (164.4). Both C-2 and C-6 of the
β
-glucose
c 79.7 and 68.3, respectively, and
glycosylated by two α-l-rhamnopyranosyl moieties (Rha I and Rha II, respectively), according to the
H
13
attached at C-3 were shifted in the C-NMR spectrum (Table 2) to
δ

Molecules 2019, 24, 2167
7 of 13
0
0
(δ
HMBC correlations (Figure 2), their connectivities were detected by the cross peaks between H-2
H
0
000
00
00000
of Rha II (
3
1
.52) and C-1
of Rha I (
δ
c 102.5), and between H -6 (
δ
3.27 and 3.71) and C-1
δc
2
H
0
00
02.2). Furthermore, the HMBC correlations from significant downfield shifted protons of H -6 of the
2
0
00000
at (
β
-glucose attached at C-7 at
δ
H
4.17 and 4.57 to C-9
δ
c 169.1) indicated the position of acylation,
was elucidated
α-l-rhamnopyranosyl]-β-d-glucopyranoside-7-O-[6-O-(trans-feruloyl)]-
thus defining the E-feruloyl linkage site. Therefore, the structure of compound
3
as kaempferol 3-O-[2,6-di-O-
β-d-glucopyranoside.
2
.1.4. Chemical Structure of Compound 4
Compound was isolated as a yellow amorphous powder, with a negative optical rotation
4
([
α
]22
−
81.6). Its molecular formula was established as C H O by its positive HR-ESI-MS
D
48 56 26
+
spectrum (m/z 1071.2925 [M + Na] , calcd for C H O Na 1071.2952), suggesting the lack of one
48
56 26
methoxy group compared to
and comparison of its H- and C-NMR values with those of
3
. Analysis of 2D experiments of
4
including COSY, HSQC and HMBC,
1
13
3
(Tables 1 and 2) showed that was a
4
1
13
kaempferol tetraglycoside acylated with a cinnamic acid derivative. The H- and C-NMR spectra of
revealed an identical glycosylation profile to that of . Comparison of the HMBC correlations of
with those of (Figure 2) detected the same pattern of connectivity defining the linkages between the
4
3
4
3
glycosyl moieties and the kaempferol residue, and between the glycosyl moieties themselves. Thus,
1
the difference between
3
and
4
must be located at the acyl substituent. In particular, the H-NMR
0
0
spectrum (Table 1) showed the typical coupling pattern (AA BB system) of a 1,4-disubstituted
aromatic moiety at 6.66 and 7.25 (each 2H, d, J = 8.6 Hz) and two trans-coupled double bond
δ
H
protons at
δ
6.24 and 7.50 (each 1H, br d, J = 15.9 Hz), as well as to an ester carbonyl (
δ
c 169.1) in the
H
13
C-NMR spectrum assignable to a p-coumaroyl unit. In the HMBC spectrum (Figure 2), the cross
0
00
000000
4.18 and 4.54) and C-9 (δ
peaks between H -6
(
δ
H
c 169.1) placed the p-coumaroyl unit at
2
0
00
C-6 .The sugar units were identified as D-glucose and l-rhamnose by acid hydrolysis of , followed
4
by HPLC analysis with chiral detector in comparison with authentic samples. Anomeric configurations
1
were determined from the magnitude of the J1,2 coupling constants in the H-NMR spectrum.
Therefore, the structure of
-O-[2,6-di-O- -L-rhamnopyranosyl]-
pyranoside.
4
was a trans-p-coumaroylated kaempferol tetraglycoside, i.e., kaempferol
3
α
β
-d-glucopyranoside-7-O-[6-O-(trans-p-coumaroyl)]- -d-gluco-
β
2
.2. Biological Activities of the Isolated Compounds
Isolated compounds 20 from the n-BuOH fraction of C. rotundifolia were evaluated for DPPH
radical scavenging assay activity (Table 3) using Trolox as a positive control (IC : 29.2 0.39 M).
The results showed that compounds 17 and 18 exhibited potent radical scavenging activity with IC
1–
±
µ
50
5
0
values of 14.5 and 11.7
µM, respectively. On the other hand, compounds 15 and 16 showed moderate
activity with IC50 values of 43.0 and 31.8
µM, respectively. These results are consistent with previous
studies on the effectiveness of myricetin as a potent antioxidant agent which could prevent and slow
the progress of ageing or various diseases associated with oxidative stress.
Table 3. Bioactivities of 7 and 15–18 from C. rotundifolia.
Isolated Compounds
DPPH (IC50, µM)
AGEs (IC50, µM)
Kaempferol 3-O-[2,6-di-O-α-l-rhamnopyranosyl]-β-d-glucoside (7)
Myricetin 3-O-[2,6-di-O-α-l-rhamnopyranosyl]-β-d-glucoside (15)
Myricetin 3-O-β-neohesperidoside (16)
Myricetin 3-O-β-d-glucoside (17)
>100
85.5 ± 3.5
43.0 ± 1.15
31.8 ± 0.63
14.5 ± 1.15
11.7 ± 1.8
29.2 ± 0.39
n.d.
>100
>100
96.5 ± 1.8
34.9 ± 1.2
n.d.
7818 ± 34.4
Myricetin (18)
Trolox
Aminoguanidine hydrochloride
n.d.: not determined.

Molecules 2019, 24, 2167
8 of 13
Inhibitory effects of isolated compounds (
1
–20) on AGEs formation were also evaluated using
a fluorescence method [14]. As shown in Table 3, compound
6.5 ± 1.8 µM) and 18 (IC : 34.9 1.2 M) showed strong inhibitory activity than that of a positive
control, aminoguanidine hydrochloride (IC : 7818 34.4 M). The results revealed that the most
inhibitory effect was exhibited by myricetin (18). Comparison of IC50 values of myricetin (18) and its
tri-, di-, and mono-glycosides (15 16, and 17, respectively) indicated that the number of sugar moieties
7
(IC : 85.5
±
3.5 µM), 17 (IC50:
5
0
9
±
µ
50
±
µ
50
,
at C-3 of myricetin aglycone decreases the inhibition activity of AGEs formation. The other compounds
did not show any inhibitory activity.
3
. Materials and Methods
3
.1. General Methods
Optical rotations were measured on a JASCO P-1030 polarimeter (Jasco, Tokyo, Japan). UV and
IR spectra were obtained on a Jasco V-520 UV/Vis spectrophotometer and a Horiba FT-710 Fourier
transform infrared spectrophotometer (Horiba, Kyoto, Japan), respectively. NMR experiments were
measured on a Bruker Avance 500, 600 and 700 MHz spectrometers (Bruker, Billerica, MA, USA),
with tetramethylsilane (TMS) as an internal standard. Positive ion HR-ESI-MS spectra were recorded
using a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Column
chromatography (CC) was performed on Diaion HP-20 (Mitsubishi Chemical Corp., Tokyo, Japan),
silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany), and octadecyl silica (ODS) gel (Cosmosil
7
5C -OPN (Nacalai Tesque, Kyoto, Japan;
Φ
= 35 mm, L = 350 mm), and TLC was performed on
precoated silica gel plates 60 GF254 (0.25 mm in thickness, Merck). HPLC was performed on ODS gel
Inertsil ODS-3, GL-science, 10 mm 250 mm, flow rate 2.5 mL/min) with a mixture of H O, acetone
and MeOH, and the eluate was monitored by refractive index and/or a UV detector. An amino column
Shodex Asahipak NH2P-50 4E (4.6 mm 250 mm), CH CN-H O (3:1) 1 mL/min) together with a chiral
detector (Jasco OR-2090plus) was used for HPLC analysis of sugars obtained after hydrolysis [15].
18
(
×
2
(
×
3
2
3
.2. Plant Material
Aerial parts of Cadaba rotundifolia were collected from eastern region, near Makkah, Saudi Arabia,
in April 2017. The collected plant was identified and classified by a plant taxonomist at
Biology Department–Umm Al-Qura University, Makkah, Saudi Arabia. A voucher specimen (No.
15501) was deposited in the Herbarium of the Department of Pharmacognosy, Pharmacy College,
King Saud University.
3
.3. Extraction and Isolation
The air-dried powdered aerial parts (6.0 Kg) of C. rotundifolia Forssk. were extracted with MeOH
(
10 L
×
7) and then concentrated under reduced pressure to give a viscous gummy material (800 g).
This residue was suspended in 900 mL of distilled water and fractionated by shaking with petroleum
ether (bp. 60–80 C) (3L
◦
×
5, 132.9 g), CH Cl (3 L
×
5, 6 g), EtOAc (3 L
×
5, 10.9 g) and n-BuOH
2
2
(
3 L × 5, 118 g), respectively. Part of n-BuOH fraction (112 g) was fractionated by Diaion HP-20
column chromatography ( = 6 cm, L = 35 cm, 900 g). The column was eluted initially with H O,
Φ
2
then with a MeOH/H O stepwise gradient with increasing MeOH content (20, 40, 60, 80, 100% MeOH,
2
respectively, 3 L each), yielding five fractions (Frs. Cr1–Cr5). The fraction Cr2 (10.7 g) was subjected
to silica gel column chromatography (
Φ = 3 cm, L = 15 cm, 100 g) eluting with increasing amount
of MeOH in CHCl (20:1, 10:1, 7:1, 5:1, 3:1, 1:1, finally with 100% MeOH, 500 mL each) yielding
3
seven fractions (Frs. Cr2.1–Cr2.7). Fraction Cr2.4 (3.5 g) was subjected to open reversed phase (ODS)
column chromatography with a linear gradient of 10% aq. MeOH (400 mL)–100% methanol (400 mL),
affording eight fractions (Frs. Cr2.4.1–Cr2.4.8). The residue of fraction Cr2.4.4 (187 mg) was purified
by preparative HPLC using 46% aq. acetone to give 18 (5.0 mg) and 20 (25.1 mg). Fraction Cr2.5
(2.4 g) was subjected to ODS column chromatography eluting with a linear gradient of 10% aq. MeOH

Molecules 2019, 24, 2167
9 of 13
(
400 mL)–100% MeOH (400 mL), to give seven fractions (Frs. Cr2.5.1–Cr2.5.7). The residue of fraction
Cr2.5.4 (554 mg) was purified by preparative HPLC (25% aq. acetone) to give 6 (4.2 mg). Fraction Cr2.7
(
2.6 g) was subjected to ODS column chromatography (linear gradient of 10% aq. MeOH (400 mL)–100%
MeOH (400 mL), giving six fractions (Frs. Cr2.7.1–Cr2.7.6). The residue of fraction Cr2.7.2 (632 mg)
was purified by preparative HPLC (25% aq. acetone) to give 15 (5.6 mg) and 16 (11.0 mg). Then residue
of fraction Cr2.7.3 (1.2 g) was also purified by preparative HPLC (28% aq. acetone) to give
and 19 (9.2 mg). Fraction Cr3 (15.5 g) was subjected to silica gel column chromatography (
7
(17.3 mg)
= 3 cm,
Φ
L = 20 cm, 150 g) with increasing amounts of MeOH in CHCl (20:1, 10:1, 7:1, 5:1, 3:1, 1:1, then 100%
3
MeOH, 400 mL each), yielding seven fractions (Frs. Cr3.1–Cr3.7). Fraction Cr3.3 (2.9 g) was subjected
to ODS column chromatography (linear gradient of 10% aq. MeOH (400 mL)–100% MeOH (400 mL),
affording nine fractions (Frs. Cr3.3.1–Cr3.3.9). The residue of fraction Cr3.3.4 (645 mg) was purified by
preparative HPLC (28% aq. acetone) to give 10 (30.1 mg). Fraction Cr3.4 (2.8 g) was subjected to ODS
column chromatography (linear gradient of 10% aq. MeOH (400 mL)–100% MeOH (400 mL)) leading to
seven fractions (Frs. Cr3.4.1–Cr3.4.7). The residue of fraction Cr3.4.2 (1.2 g) was purified by preparative
HPLC (28% aq. acetone) to give 17 (15.2 mg). The residue of fraction Cr3.4.4 (187 mg) was also purified
by preparative HPLC (40% aq. acetone) to give
subjected to ODS column chromatography (linear gradient of 10% aq. MeOH (400 mL)–100% MeOH
400 mL)) providing seven fractions (Frs. Cr3.5.1–Cr3.5.7). The residue of fraction Cr3.5.4 (692 mg) was
purified by preparative HPLC (28% aq. acetone) to give (39.0 mg) and (20.0 mg). The other residue
1 (7.4 mg) and 5 (2.0 mg). Fraction Cr3.5 (4.4 g) was
(
8
9
of fraction Cr3.5.5 (65.7 mg) was purified by preparative HPLC (32% aq. acetone) to give 11 (3.0 mg).
Fraction Cr3.6 (760 mg) was subjected to ODS column chromatography (linear gradient of 10% aq.
MeOH (400 mL)–100% MeOH (400 mL)), affording seven fractions (Frs. Cr3.6.1–Cr3.6.7). The residue
of fraction Cr3.6.4 (923 mg) was purified by preparative HPLC (25% aq. acetone). The following
compounds were separated:
fraction Cr3.6.5 (963 mg) was purified by preparative HPLC (35% aq. acetone) to give
2.0 mg).
The known compounds 5–20 were identified by comparison of their spectroscopic data with
2
(5.0 mg), 12 (12.0 mg), 13 (2.0 mg) and 14 (10.0 mg). The residue of
3
(7.8 mg) and
4
(
those reported in the literature as kaempferol 3-O-[2-O-(trans-p-coumaroyl)-3-O-
β
-d-gluco-
0
pyranosyl]-
kaempferol
β
-d-glucopyranoside
3-O-[2,6-di-O- -l-rhamnopyranosyl]-
17], kaempferol 3-O-
-d-glucoside (10) [17], rhamnocitrin 3-O- -neohesperidoside (11) [18], kaempferol
-neohesperidoside-7-O-[2-O-(trans-p-coumaroyl)-3-O- -d-glucopyranosyl]- -d-glucopyranoside
13], kaempferol 3-O- -neohesperidoside-7-O-[2-O-(trans-feruloyl)]-
13) [13], kaempferol 3-O- -neohesperidoside-7-O-[2-O-(trans-p-coumaroyl)]-
14) [13], myricetin 3-O-[2,6-di-O- -l-rhamnopyranosyl]-
-O- -neohesperidoside (16) [17], myricetin 3-O- -D-glucoside (17) [17], myricetin (18) [19],
beitingxinhuagtong C (19) [20], phloretin (20) [21].
(
5
)
[
12], kaempferol 3,4 -di-O-
β
-d-glucoside
17],
17], kaempferol
(
6)
[
16],
α
β-d-glucoside
(
7
)
[
kaempferol
3
3
3
-O-
-O-
β-neohesperidoside
(8
)
[
β-rutinoside
(9
)
[
β
β
-O-
β
β
β
(
(
(
12
)
[
β
β
-d-glucopyranoside
-d-glucopyranoside
β
β
α
β-d-glucoside (15) [17], myricetin
3
β
β
3
.4. Spectroscopic Data of Compounds 1–4
Kaempferol 3-O-[2-O-(trans-feruloyl)-3-O-β-d-glucopyranosyl]-β-d-glucopyranoside (1)
Yellow amorphous powder [
]22 79.0 (c 0.42, MeOH); HR-ESI-MS: m/z: 809.1888 [M + Na]+
calcd for C H O Na 809.1899); UV λmax (MeOH) nm (log ): 327 (4.21), 299 (4.12), 267 (4.13), 231
α
−
D
(
(
ε
37
38 19
−1
1
13
4.23); IR (film) νmax: 3388, 2936, 1710, 1658, 1604, 1513, 1446, 1363, 1176, 1070, 1028 cm ; H and
C
data see Tables 1 and 2.
Kaempferol 3-O- -neohesperidoside-7-O-[2-O-(cis-p-coumaroyl)-3-O-
2)
β
β-d-glucopyranosyl]-β-d-gluco-pyranoside
(
Yellow amorphous powder [
calcd for C H O Na 1087.2901); UV λmax (MeOH) nm (log ε): 351 (3.97), 316 (4.14), 267 (4.04), 229
α
]22 +14.5 (c 0.60, MeOH); HR-ESI-MS: m/z: 1087.2875 [M + Na]+
D
(
48
56 27

Molecules 2019, 24, 2167
10 of 13
3.99); IR (film) νmax: 3397, 2924, 1721, 1657, 1603, 1512, 1442, 1367, 1157, 1072, 1024 cm 1; 1H and 13
−
C
(
data see Tables 1 and 2.
Kaempferol 3-O-[2,6-di-O-
α
-l-rhamnopyranosyl]-
β
-d-glucopyranoside-7-O-[6-O-(trans-feruloyl)]-
β
-d-gluco
pyranoside (3)
Yellow amorphous powder [
α
]22
−
82.5 (c 1.02, MeOH); HR-ESI-MS: m/z: 1101.3026 [M + Na]+
D
(
(
calcd for C H O Na 1101.3058); UV λmax (MeOH) nm (log ε): 329 (4.17), 295 (4.00), 268 (4.05), 233
49
58 27
−1
1
13
4.11); IR (film) νmax: 3389, 2932, 1712, 1651, 1601, 1513, 1455, 1371, 1180, 1071, 1024 cm ; H and
C
data see Tables 1 and 2.
Kaempferol 3-O-[2,6-di-O-α-l-rhamnopyranosyl]-β-d-glucopyranoside-7-O-[6-O-(trans-p-coumaroyl)]-β-d-
glucopyranoside (4)
Yellow amorphous powder [
α
]22
−
81.6 (c 0.36, MeOH); HR-ESI-MS: m/z: 1071.2925 [M + Na]+
D
(
(
calcd for C H O Na 1071.2952); UV λmax (MeOH) nm (log ε): 348 (4.01), 317 (4.15), 267 (4.05), 228
48
56 26
−1
1
13
4.09); IR (film) νmax: 3379, 2912, 1660, 1601, 1499, 1445, 1372, 1172, 1074, 1034 cm ; H and C data
see Tables 1 and 2.
3
.5. Acid Hydrolysis
◦
The isolated compounds
1–
4
(1.0 mg each) were hydrolyzed with 1 M HCl (1.0 mL) at 80 C
−
for 3 h, then cooled. The reaction mixtures were neutralized with Amberlite IRA96SB (OH form),
then filtered to remove the resin. The filtrates were extracted with EtOAc. The aqueous layers were
analyzed by HPLC with an amino column [Asahipak NH2P-50 4E, CH CN-H O (3:1), 1 mL/min]
3
2
and a chiral detector (JASCO OR-2090plus) in comparison with authentic standards of D-glucose and
L-rhamnose. The aqueous layers of the hydrolyzed compounds showed a peak at t 8.15 min which
R
coincided with that of d-glucose (positive optical rotation). The t of the l-rhamnose (negative optical
R
rotation) was 6.5 min [15].
3
.6. Alkaline Hydrolysis
Solutions of
1–
3
(3.0 mg each) in 50% aqueous 1,4-dioxane (0.5 mL) were treated with 10% aqueous
◦
KOH (0.5 mL) and stirred at 37 C for 1 h. The reaction mixtures were neutralized with an ion-exchange
+
resin Amberlite IR-120B (H -form), then filtered and evaporated. The residues were dissolved in
EtOAc and subjected to HPLC analysis [Cosmosil C18-PAQ, MeOH-H O (45:50), 1 mL/min)] with UV
2
(
245 nm) detection to identify ferulic acid (t 16.7 min) from
1
and
3, and p-coumaric acid (t 15.1 min)
R
R
from 2 [22]. The authentic cis-p-coumaric acid was prepared according to the literature [23].
3
.7. DPPH Radical Scavenging Activity
Free radical scavenging activity were evaluated by using a quantitative DPPH assay. The absorbance
of various concentrations of test compounds dissolved in 100
µL of MeOH in 96-well microtiter plate
were measured at 515 nm as (Ablank). Then, a 100 L of DPPH solution (200
µ
µM) was added to each
well. The plate was incubated in dark chamber at room temperature for 30 min before measuring the
absorbance (Asample) again. The following equation was used to calculate % inhibition of free radicals:
%
Inhibition = [1 − (Asample − Ablank)/(Acontrol − Ablank)] × 100
where Acontrol is the absorbance of control (DMSO and all reagents, except the test compound).
Their IC50 values were determined based on three independent experiments. Trolox was used as
reference compound [24].

Molecules 2019, 24, 2167
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3
.8. Determination of In Vitro AGEs Formation
The reaction mixture including 0.5 M ribose and 10 mg/mL bovine serum albumin in 50 mM
phosphate buffer (pH 7.4)- 0.02% sodium azide was mixed with the test compounds, and incubated
◦
for 24 h at 37 C. The formation of fluorescent AGEs were measured with a fluorometric microplate
reader (Ex: 370 nm, Em: 440 nm, EnSpire, PerkinElmer, Inc., Waltham, MA, USA). Experiments were
performed in triplicate and IC50 values were calculated by linear regression [14].
4
. Conclusions
In conclusion, four new flavonoid glycosides
1–4 together with 16 known compounds 5–20
were isolated from the aerial parts of Cadaba rotundifolia (Forssk.) (Qadab) collected in Saudi Arabia.
The chemical structures of the new compounds were elucidated by intensive spectroscopic analyses
and chemical reactions. Among the isolated compounds, 15
–18 exhibited significant radical scavenging
activity and compounds 17 and 18 showed strong inhibitory activity toward AGEs formation.
7,
These results suggest that C. rotundifolia and their chemical constituents may ameliorate oxidative
stress in various degenerative disorders including Alzheimer’s disease, diabetes, atherosclerosis and
aging. Finally, it is noteworthy that most of the known compounds,
from the family Capparaceae for the first time. The remaining three known compounds,
5
–
8
,
11
–
16
,
19, and 20, were isolated
10 17,
9,
,
also have not been isolated from the title plant so far. These results indicated that the characteristic
circumstances of the oases surrounded by desert in Saudi Arabia provide promising and interesting
natural resources for future drug development.
Supplementary Materials: The following are available online, Figures S1–S9: HR-ESI-MS, H, 13C NMR, DEPT,
1
COSY, HSQC, HMBC, UV and IR spectra of 1, Figures S10–S18: Spectra of 2, Figures S19–S27: Spectra of 3,
Figures S28–S35: Spectra of 4.
Author Contributions: K.M. and G.A.A.-H.; conceived and designed the experiment, G.A.A.-H.; implement the
experiments, G.A.A.-H.; analyzed the data and wrote the original draft; K.M., S.S., and Y.Y.: helped in experiments
and contributed reagents/material and analysis tools; K.M. and R.S.O., supervision, K.M.: write, review and
editing; N.A. and A.M.A.: collection of plant.
Funding: This work was supported in part by King Saud University External Joint Supervision Program (EJSP),
Kingdom of Saudi Arabia, and by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and
Technology of Japan, and the Japan Society for the Promotion of Science (Nos. 17K15465 and 17K08336).
Acknowledgments: The measurements of NMR and HR-ESI-MS were performed with Bruker avance series and
LTQ Orbitrap XL spectrometer, respectively, at the Natural Science Center for Basic Research and Development
(
N-BARD), Hiroshima University. The other experimental facilities and useful suggestions for publication of this
work was kindly supported by the research center in King Saud University.
Conflicts of Interest: The authors report no conflicts of interest.
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Sample Availability: Samples of the compounds 1–4 are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).