PPAR α and γ Activation Effects of New Nor-triterpenoidal Saponins from the Aerial Parts of Anabasis articulata

Article abstract of DOI:10.1055/a-0762-0885

Anabasis articulata, traditionally used to treat diabetes, is rich in saponin content. This study was performed to investigate the agonistic effect of its saponins on peroxisome proliferator-activated receptor- α and peroxisome proliferator-activated receptor- γ in human hepatoma (HepG2) cells to explore the possibility of the involvement of these nuclear receptors in the mechanism of the antidiabetic effect of the plant. Chemical investigation of the n -butanol fraction resulted in the isolation of three new and one known 30-noroleanane triterpenoid saponins. The structures of the new compounds were elucidated as 3 β -hydroxy,23-aldehyde-30-norolean-12,20(29)-dien-28-oic acid-28- O - β -D-glucopyranosyl ester (1), 3 β - O -D-galactopyranosyl-23-aldehyde-30-norolean-12,20(29)-dien-28-oic acid-28- O - β -D-glucopyranosyl ester (2), and 3 β - O -D-xylopyranosyl-30-norolean-12,20(29)-dien-28-oic acid 28- O - β -D-glucopyranosyl ester (3), while the known 30-nortriterpenoidal saponin was identified as boussingoside E (4). Although, the isolated saponins (1 - 4) did not show > 1.5-fold activation of peroxisome proliferator-activated receptor- γ, but two of them (1 and 3) activated peroxisome proliferator-activated receptor- α to the higher extents of 2.25- and 1.86-fold, respectively. These results suggest that the reported antidiabetic action of the isolated saponins may not solely involve the activation of peroxisome proliferator-activated receptor- γ. However, the agonistic activity of the n -butanol fraction of A. articulata (1.71-fold induction) and two of its saponins (1 and 3) towards peroxisome proliferator-activated receptor- α may be beneficial in the cardiovascular condition that is closely associated with diabetes and other metabolic disorders.

Full text of DOI:10.1055/a-0762-0885

Original Papers
PPARα and γ Activation Effects of New Nor-triterpenoidal Saponins
from the Aerial Parts of Anabasis articulata
Authors
Riham Salah El Dine , Hossam M. Abdallah , Zeinab A. Kandil , Ahmed A. Zaki , Shabana I. Khan , Ikhlas A. Khan3
1
1,2
1
3,4
3
Affiliations
Supporting information available online at
1
2
3
Department of Pharmacognosy, Faculty of Pharmacy,
http://www.thieme-connect.de/products
Cairo University, Cairo, Egypt
Department of Natural Products, Faculty of Pharmacy,
King Abdulaziz University, Jeddah, Saudi Arabia
National Center for Natural Products Research, School of
Pharmacy, University of Mississippi, Oxford, Mississippi,
USA
ABSTRACT
Anabasis articulata, traditionally used to treat diabetes, is rich in
saponin content. This study was performed to investigate the
agonistic effect of its saponins on peroxisome proliferator-acti-
vated receptor-α and peroxisome proliferator-activated recep-
tor-γ in human hepatoma (HepG2) cells to explore the possibil-
ity of the involvement of these nuclear receptors in the mech-
anism of the antidiabetic effect of the plant. Chemical investi-
gation of the n-butanol fraction resulted in the isolation of
three new and one known 30-noroleanane triterpenoid sapo-
nins. The structures of the new compounds were elucidated
as 3β-hydroxy,23-aldehyde-30-norolean-12,20(29)-dien-28-
oic acid-28-O-β-D-glucopyranosyl ester (1), 3β-O-D-galac-
topyranosyl-23-aldehyde-30-norolean-12,20(29)-dien-28-oic
acid-28-O-β-D-glucopyranosyl ester (2), and 3β-O-D-xylopyra-
nosyl-30-norolean-12,20(29)-dien-28-oic acid 28-O-β-D-glu-
copyranosyl ester (3), while the known 30-nortriterpenoidal
saponin was identified as boussingoside E (4). Although, the
isolated saponins (1–4) did not show > 1.5-fold activation of
peroxisome proliferator-activated receptor-γ, but two of them
4
Department of Pharmacognosy, Faculty of Pharmacy,
Mansoura University, Mansoura, Egypt
Key words
PPARα, PPARγ, Anabasis articulata, antidiabetic,
Amaranthaceae, triterpene saponins
received June 1, 2018
revised
October 5, 2018
accepted October 14, 2018
Bibliography
DOI https://doi.org/10.1055/a-0762-0885
Published online | Planta Med © Georg Thieme Verlag KG
Stuttgart · New York | ISSN 0032‑0943
Correspondence
(
1 and 3) activated peroxisome proliferator-activated recep-
Dr. Hossam M. Abdallah
tor-α to the higher extents of 2.25- and 1.86-fold, respectively.
These results suggest that the reported antidiabetic action of
the isolated saponins may not solely involve the activation of
peroxisome proliferator-activated receptor-γ. However, the
agonistic activity of the n-butanol fraction of A. articulata
Department of Natural Products, Faculty of Pharmacy,
King Abdulaziz University
Abdullaha Al-soliman St., Jeddah 21589, Saudi Arabia
Phone: + 966544733110, Fax: + 966126400000
hmafifi2013@gmail.com
(
1.71-fold induction) and two of its saponins (1 and 3) towards
peroxisome proliferator-activated receptor-α may be benefi-
cial in the cardiovascular condition that is closely associated
with diabetes and other metabolic disorders.
peroxisome proliferator-activated receptor (PPAR) family has re-
ceived increasing attention. PPARs constitute a subfamily within
the nuclear receptor superfamily of ligand-inducible transcription
factors [3]. There are three known subtypes of PPARs: α, β/δ, and γ
[4]. They control the expression of genes involved in lipid metab-
olism, inflammation, and adipogenesis. PPARα is expressed in the
heart, liver, muscles, and kidneys. It is responsible for controlling
lipid and lipoprotein metabolism [5,6]. Fenofibrate and gemfibro-
zil are selective agonists for PPARα that are efficient in improving
Introduction
Metabolic syndrome (MS) is characterized by increased abdominal
fat, obesity, and insulin resistance. It occurs mainly due to the im-
balance between high energy nutrition and physical activity [1].
The syndrome includes many metabolic disorders that predispose
diabetes and cardiovascular complications [2]. Therefore, MS pa-
tients are at a high risk of cardiovascular disease (CVD), type II dia-
betes, diabetic nephropathy, and retinopathy. In this context, the
El Dine RS et al. PPARα and γ… Planta Med

Original Papers
dyslipidemia but are not considered antidiabetic agents [7]. PPAR
β/δ is expressed throughout the body and controls lipid metabo-
lism in adipose tissue. PPARγ is present in two isoforms, 1 and 2.
PPARγ-1 is expressed in the large intestine, adipose tissue, liver,
kidneys, and muscles, while PPARγ-2 is restricted only to brown
adipose tissue [6,8, 9]. Therefore, diseases such as type II diabe-
tes, obesity, metabolic syndrome, inflammation. and cardiovascu-
lar disease could be treated using the compounds that modulate
PPAR activity [10]. Drugs used in MS target different symptoms
such as body weight, insulin resistance, hyperglycemia, dyslipide-
mia, CVD, or hypertension. Increasingly, herbal remedies are
being adopted as a form of treatment due to their benefits, which
include lower cost, lower risks of side effects, and being more ef-
fective at targeting multiple pathways. It is therefore not surpris-
ing that medicinal plant-based products have been reported as
beneficial for the treatment of type II diabetes and CVD, which
leads to the development of MS [11]. Many publications con-
firmed the activity of some medicinal plants as PPARα and PPARγ
activators [12, 13]. Practical insights may therefore be gleaned
through further scientific analysis of these herbal medicines,
which may lead to the development of alternative drug therapies
that can better manage diabetic complications [14]. Anabasis ar-
ticulata (Forssk.) Moq. (Amaranthaceae), locally named “Ajrem”,
is used traditionally to treat diabetes across Algeria [15]. Decoc-
tion of its aerial parts was reported to manage hyperglycemia
through increasing blood insulin and α-fetoprotein [16]. More-
over, the plant showed a significant anti-inflammatory effect
▶
Fig. 1 Structures of the isolated compounds (1–4).
1
0
Compound 1 was obtained as an optically active ([α]
0.1, MeOH)) white amorphous powder with a molecular formula
of C35H52O , which was deduced from the HR‑ESI‑MS m/z
617.3652 [M + H] in the positive ion mode and showed positive
results in Molischʼs and Liebermann-Burchard tests, suggesting
that 1 was a triterpenoid saponin.
D
+ 25 (c
9
+
1
through the reduction of prostaglandin (PGE ) and TNF-α levels
The H NMR spectrum (▶ Table 1) confirmed the presence of a
2
as well as inhibition of cyclooxygenase-2 (COX-2) activity [17].
A phytochemical investigation of the plant resulted in the isola-
tion of triterpenoid saponins that were responsible for the antidia-
betic activity of the plant [18]. They were identified as 3-β‑O-D-
glucopyranosyl (of stigmasterol, β-sitosterol, and sitostanol), 3-
β‑O-D-glucopyranosyl olean-12-ene-28-oic acid, and 3-β‑O-D-
glucopyranosyl olean-12-ene-28-oic acid-28-O-β-D-xylopyranosyl
ester in addition to proceric acid [16]. In the course of our interest
in MS and medicinal plants that could be used to prevent MS-re-
lated complications [19–21], the present study was carried out to
evaluate the potential antidiabetic and cardiovascular effects of
the n-butanol fraction as well as the isolated 30-nortriterpenoid
saponins from the flowering aerial parts of A. articulata in terms
of their abilities to activate PPARα and PPARγ in human hepatoma
nor-oleanane system that was supported by the display of four
tertiary angular methyl signals at δH 0.68 (3H, s, H -26), δ 0.87
3
H
(3H, s, H -24), δH 0.88 (3H, s, H -25), and δH 1.14 (3H, s, H -27)
3
3
3
in addition to an aldehyde proton at δH 9.22 (1H, s, H -23) [23].
3
Moreover, the spectra showed two singlets at δH 4.61 (1H, s,
H − 29) and 4.62 (1H, s, H − 29) corresponding to an exomethy-
a
b
lene group and one tri-substituted olefinic proton at δH 5.24 (1H,
m, H-12). The 13C NMR spectrum displayed 35 carbon signals, of
which 6 carbon signals were assigned to a monosaccharide moi-
ety and 29 carbon signals were assigned to the triterpenoidal
aglycone. The 13C spectrum displayed two carbonyl signals at
δC173.5 and 206.5, which were assigned to a carboxylic group
(C-28) and an aldehyde group. Moreover, the spectra showed four
methyl resonances at δ 7.8, 14.1, 15.6, and 24.6, which were as-
C
(
Hep G2) cells as they are involved in the metabolism of lipids and
signed to C-24, C-25, C-26, and C-27, respectively, eleven methy-
carbohydrates, respectively. This will help to explore the useful-
ness of the plant saponins to ameliorate diabetic and cardiovascu-
lar complications.
lenes, including an oxymethylene signal at δ 106.1, five methines
C
(including one olefinic signal at δ 120.9 and one oxymethine),
C
and nine quaternary carbons (including a carboxy carbon forming
an ester linkage, δ 173.5 for C-28, two olefinic signals at δ 141.9
C
C
and 147.0, and an aldehyde signal at δC 206.5). Taken together,
Results and Discussion
A chemical investigation of the saponin content of A. articulata re-
sulted in the isolation of three new (1–3) along with one known
1
2
these data were indicative of a typical Δ 30-noroleanolic acid-
type triterpene aglycone bearing an aldehyde group that was pre-
viously identified [23].
(
4) saponin (▶ Fig. 1). Their structures were elucidated through
The aldehyde group was confirmed to be at C-23 from the
1H and 13C NMR, 2D spectroscopic, and HR-ESIMS analyses. Based
on the reported data, compound 4 was identified as 3-O-β-D-glu-
curonopyranosyl-30-norhederagenin 12–20(29)-dien- 28-oic
acid-28-O-β-D-glucopyranosyl ester (boussingoside E) [22].
HMBC correlations. The aldehyde proton at δ 9.22 was correlated
H
in the HMBC with a carbon at δ 54.1 (C-4) in addition to a corre-
C
lation between H -24 at δ 0.87 and the carbon at δ 206.5 (C-23)
3
H
C
(▶ Fig. 2). Moreover, the proton of the aldehydic group showed a
strong correlation in the NOESY experiment with δ 1.28 (1H, m,
H
El Dine RS et al. PPARα and γ… Planta Med

▶
Table 1 NMR spectral data of compounds 1–3 (600 and 150 MHz, DMSO-d6, δ ppm).
1
2
3
Position
δH (J in Hz)
0.99, m, 1.56, m
1.58, m, 1.90, m
3.66, (brd, 11.5)
–
δC, type
36.6, CH2
24.9, CH2
69.4, CH
54.1, C
δH (J in Hz)
0.99, m, 1.56, m
1.59, m, 1.96, m
3.78, (brd, 11.1)
–
δC, type
36.6, CH2
23.1, CH2
79.2, CH
53.4, C
δH (J in Hz)
0.87, m, 1.48, m
1.48, m, 1.85, m
2.99, (brd, 11.9)
–
δC, type
37.1, CH2
24.3, CH2
86.8, CH
37.7, C
1
2
3
4
5
6
7
8
9
1.28, m
45.2, CH
19.2, CH2
30.3, CH2
39.1, C
1.25, m
45.3, CH
18.5, CH2
30.4, CH2
39.1, C
0.69, m
53.9, CH
16.7, CH2
31.2, CH2
37.9, C
0.74, m, 1.37, m
1.08, m, 1.40, m
–
0.77, m, 1.34, m
1.10, m, 1.40, m
–
1.62, m, 1.42, m
1.2, m, 1.38, m
–
1.52, brs
45.9, CH
34.3, C
1.59, brs
45.8, CH
34.3, C
1.47, m
46.0, CH
35.2, C
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
–
–
–
1.82, m, 2.22, m
5.24, m
21.9, CH2
120.9, CH
141.9, C
40.3, C
1.83, m, 2.3, m
5.24, m
21.9, CH2
120.9, CH
141.8, C
40.3, C
1.84, m, 1.84, m
5.21, m
21.9, CH2
121.1, CH
141.7, C
40.2, C
–
–
–
–
–
–
0.93, m, 1.74, m
1.66, m, 2.10, m
26.1, CH2
21.6, CH2
45.2, C
0.97, m, 1.73, m
1.69, m, 2.08, m
26.1, CH2
21.5, CH2
45.2, C
0.99, m, 1.74, m
1.69, m, 2.07, m
26.1, CH2
21.6, CH2
45.2, C
2.62, (dd, 13.6, 5.1)
45.6, CH
39.7, CH2
147.0, C
28.2, CH2
35.7, CH2
206.5, C
7.8, CH3
14.1, CH3
15.6, CH3
24.6, CH3
173.5, C
106.1, CH2
2.62, (brd, 13.6)
45.6, CH
39.8, CH2
146.7, C
28.2, CH2
35.6, CH2
205.1, C
8.75, CH3
14.2, CH3
15.6, CH3
24.5, CH3
173.5, C
106, CH2
2.61, (dd, 13.6, 6.5)
45.6, CH
39.9, CH2
147.0, C
28.2, CH2
35.6, CH2
26.6, CH3
15.4, CH3
14.2, CH3
15.6, CH3
24.4, CH3
173.5, C
106.0, CH2
2.01, (dd, 13.3, 6.5), 2.42, m
2.01, (brd, 13.3), 2.45, m
2.02, (dd, 13.6, 5.0), 2.45, m
–
2.05, m, 2.14, m
1.33, m, 1.76, m
9.22, s
2.07, m, 2.18, m
1.37, m, 1.78, m
9.33, s
2.06, m, 2.15, m
1.38, m, 1.7, m
0.95, s
0.87, s
0.97, s
0.74, s
0.88, s
0.89, s
0.86, s
0.68, s
0.68, s
0.68, s
1.14, s
1.13, s
1.10, s
–
–
–
4.61, s
4.60, s
4.62, s
4.57, s
4.55, s
4
.62, s
3
0
–
–
–
–
2
8-O-β-D- glucopyranosyl
3β-O-D-galactopyranosyl
3.96, (d, J = 7.2)
2.79, m
3β-O-D-xylopyranosyl-
4.12, (d, 7.2)
2.92, dd, (9.1, 7.6)
3.09, m
1
2
3
4
5
′
′
′
′
′
5.19, (d, J = 7.6)
3.09, (dd, 9.2, 7.8)
3.18, (t, 9.2)
3.11, (t, 8.7)
3.13, m
93.1, CH
71.3, CH
75.6, CH
68.4, CH
76.7, CH
101.8, CH
72.6, CH
75.8, CH
71.0, CH
72.6, CH
104.3, CH
72.8, CH
75.7, CH
71.2, CH
62.0, CH2
3.01, m
2.99, m
3.41, m
3.07, m
3.2, (dd, 11.4, 10.4),
3
.3, (dd, 11.4, 5.4)
6
′
3.60, (dd, 11.8, 1.5),
59.6, CH2
3.26, (dd, 11.7, 4.7),
3.34, (dd, 11.8, 1.6)
62.4, CH2
–
–
3
.43, (dd, 11.8, 4.8)
2
8-O-β-D-glucopyranosyl
28-O-β-D-glucopyranosyl
5.19, (d, J = 7.6)
3.07, (dd, 9.1, 7.6)
3.19, m
1
2
3
4
5
6
″
″
″
″
″
″
5.24, (d, J = 7.6)
3.07, m
93.1, CH
72.2, CH
75.5, CH
68.5, CH
76.7, CH
59.6, CH2
93.1, CH
71.3, CH
75.6, CH
68.5, CH
76.7, CH
59.5, CH2
3.18, m
3.08, (t, 8.7)
3.13, (t, 9.2)
3.58, (dd, 11.8, 1.5), 3.41,
3.08, (t, 8.8)
3.12, m
3.58, (dd, 11.9, 1.7), 3.41,
(dd, 11.9, 4.9)
(
dd, 11.8, 5.1)
El Dine RS et al. PPARα and γ… Planta Med

Original Papers
H-5) and H-3 at δH 3.66, which confirm the alpha orientation of
the aldehydic group at C-23. The hydroxyl group at C-3 was con-
firmed to be β oriented through the NOESY correlation of H-3 at
δH 3.66 and H-5 at δH 1.28, which confirmed that H-3 was as-
signed to be in an α-axial position.
1
Moreover, the H spectrum showed an anomeric proton signal
at δ 5.19 (▶ Table 1). The sugar moiety was identified as the β-D-
H
glucopyranosyl moiety based on its JH-1, H-2 coupling constant at
δH 7.6 Hz [23]. Furthermore, the monosaccharide structure bond-
ing to the 28-position of 1 was characterized by the HMBC experi-
ment. Namely, a long-range correlation was observed between
the anomeric proton H-1′ at δH 5.19 (1H, d, J = 7.6 Hz) of the glu-
▶
Fig. 2 Some important HMBC and NOESY correlations for 1 and 2.
copyranosyl moiety and C-28 (the carboxyl carbon at δ 173.5) of
C
the noroleanane aglycone part. The acid hydrolysis of 1 revealed
that it contains a D-glucose moiety. These results were confirmed
through HPLC analysis of the hydrolytic products against different
monosaccharaides, where the sugar has the same retention time
as authentic D-glucose (30.7 min). In addition, all sugar protons
and carbons were confirmed by extensive HSQC and HMBC analy-
ses. Therefore, the structure of 1 was deduced as 3-β-hydroxy-23-
aldehyde-30-norolean-12,20(29)-dien-28-oic acid-28-O-β-D-glu-
copyranosyl ester.
NMR spectrum exhibited the following signals: five tertiary methyl
signals at δH 0.68, 0.74, 0.86, 0.95, and 1.10, an olefinic proton
signal at δH 5.21 (m, H-12), proton signals of an exomethylene
group at δH 4.57 and 4.55 (each a singlet), and a signal typical of
H-3ax (brd, δ 2.99) consistent with the presence of a β-OR group
H
at C-3 position. In addition, the spectra displayed two anomeric
protons at δ 5.19 (d, J = 7.6 Hz) and 4.12 (d, J = 7.2 Hz). The agly-
H
1
0
Compound 2 was obtained as an optically active ([α]
D
+ 15 (c
cone part was previously identified [25] and showed the same
0
.1, MeOH)) white amorphous powder with a molecular formula
spectra as that of 1 and 2 except for the absence of the aldehyde
of C41H62O14, which was deduced from the HR‑ESI‑MS m/z
proton signal and the presence of the normal pattern of the H -23
3
+
7
79.4639 [M + H] in the positive ion mode and showed a positive
and H -24 methyl groups attached to C-4, and it could be identi-
3
test for a triterpenoidal saponin. Its spectroscopic data were very
similar to those of 1. Detailed comparison of their 1D NMR data
indicated that the differences between these compounds are in
the positions and the number of sugar moieties. The molecular
formula of 2 was identified as C41H62O14 on the basis of the HR-
ESI‑MS, and gave D-glucose and D-galactose on acid hydrolysis.
The aglycone part showed the same spectra as that of 1, and it
could be identified as 23-aldehyde-30-norolean-12,20(29)-diene.
The H and 13C NMR spectra showed similar signals to those in 1
with two doublet signals integrating as one proton each, at δH
fied as 30-norolean-12,20 (29)-diene.
The 13C NMR spectrum (▶ Table 1) of 3 displayed 40 carbon
signals. The aglycone moiety was assigned by 29 carbons, i.e.,
8 quaternary carbons, 5 methines, 11 methylenes, and 5 methyls.
In addition, the aglycone part showed four olefinic carbon signals
(δC 121.1, 141.7, 147.0, and 106.0), one carbonyl carbon signal
(δ 173.5), and an oxymethine carbon signal (δ 86.8). Thus, the
C
C
spectral data, after comparison with previously published data
[26], confirmed the presence of a triterpene glycoside with a 30-
norolean-12–20(29)-dien-28-oic acid framework.
1
5
.24 (d, J = 7.6 Hz) and 3.96 (d, J = 7.2 Hz) corresponding to two
The glycosidic part was represented by 11 carbon resonances
(one pentose and one hexose) as obtained by subtracting the 29
aglycone carbons from the total (40 signals). In addition, the two
sugar moieties were represented by two anomeric carbons at
sugar moieties at C 28 and C3, respectively, as well as twelve car-
bon signals (▶ Table 1), which is in good agreement with the pub-
lished data for the two sugar moieties [24]. The sugar moieties
were identified as O-β-D-galactopyranosyl and O-β-D-glucopyra-
nosyl moieties after careful inspection of the H, C NMR, and
HMBC data and acid hydrolysis of 2. Furthermore, the positions
of the functional sugar moieties were confirmed by the HMBC ex-
periment (▶ Fig. 2) to be attached to C-3 and C-28, respectively,
from correlations between H-1′ (δH 3.96, d, J = 7.2 Hz) with C-3
at δ 79.2 and H-1″ (δH 5.24, d, J = 7.6 Hz) with C-28 at δ 173.5.
δ 104.3 and 93.1. The sugars were identified as β-D-glucopyrano-
C
1
13
1
13
syl and β-D-xylopyranosyl after careful inspection of H, C NMR,
extensive 2D NMR analysis, and acid hydrolysis of 3 [27]. The loca-
tion of the β-D-glucopyranosyl moiety at C-28 was confirmed by a
significant deshielding of the anomeric proton H-1″ [δH 5.19 (d,
J = 7.6 Hz); H-1″ax], and the signal at δC 93.1 is consistent with
the presence of a sugar moiety attached to aglycone by an ester
linkage [28]. This was confirmed by long-range coupling between
hydrogen H-1″ (δ 5.19) and carbon C-28 (δC 173.5). All the pre-
vious data confirmed that 3 was 3-O-β-D-xylopyranosyl-30-noro-
lean-12–20(29)-dien-28-oic acid 28-O-β-D-glucopyranosyl ester
(3).
C
C
Based upon all of the above evidences, the structure of 2 was
elucidated as 3-O-β-D-galactopyranosyl-30-norolean-12,20(29)-
dien-23-aldehyde-28-oic acid-28-O-β-D-glucopyranosyl ester.
1
0
Compound 3 was obtained as an optically active ([α]
D
+ 30 (c
0
.1, MeOH)) white amorphous powder with a molecular formula
of C40H62O12 as inferred from the HR‑ESI‑MS m/z 735.4358 [M +
H] in the positive ion mode and showing the same pattern as
The in vitro antidiabetic and cardiovascular activities of the n-
butanol fraction as well as the isolated saponins were tested in
terms of PPARα and PPARγ activation through a reporter gene as-
say with ciprofibrate and rosiglitazone as positive controls for
PPARα and PPARγ, respectively. The n-butanol fraction was found
+
compounds 1 and 2. Its molecular formula was determined on
the basis of the HR‑ESI‑MS pseudo-molecular ion peak at m/z
7
35.4358 [M + H]+ (calcd. for 735.4366, C40H63O12). The 1
H
El Dine RS et al. PPARα and γ… Planta Med

▶
Table 2 Effect of the n-butanol fraction (total saponins) and isolated compounds from A. articulata on PPARα and PPARγ transcriptional activity as
determined by the luciferase assay in HepG2 cells. Fold induction was calculated as the ratio of luciferase expression in sample-treated cells to the
vehicle-treated cells. Data are presented as the mean ± SD of values from two independent experiments performed with two replicates each time.
Compound
Fold induction in PPARα activity
Fold induction in PPARγ activity
50 µM
25 µM
12.5 µM
50 µM
25 µM
12.5 µM
1
2.25 ± 0.12
1.19 ± 0.14
1.86 ± 0.02
1.20 ± 0.22
4.12 ± 0.39
–
1.74 ± 0.10
1.60 ± 0.08
2.08 ± 0.23
1.30 ± 0.20
2.97 ± 0.93
–
1.55 ± 0.19
1.30 ± 0.20
1.54 ± 0.26
1.09 ± 0.08
1.67 ± 0.12
–
1.48 ± 0.35
1.4 ± 0.30
1.53 ± 0.14
1.50 ± 0.25
–
1.30 ± 0.16
1.65 ± 0.15
1.32 ± 0.03
1.32 ± 0.02
–
1.17 ± 0.01
1.60 ± 0.13
1.28 ± 0.07
1.3 ± 0.19
–
2
3
4
Ciprofibratea
Rosiglitazonea
n-butanol fractionb
a
4.36 ± 0.79
1.27 ± 0.17
4.31 ± 0.11
1.19 ± 0.13
4.71 ± 0.05
1.06 ± 0.01
1.71 ± 0.21
1.57 ± 0.28
1.67 ± 0.12
The test concentrations for the positive controls were 10, 5, and 2.5 µM. b The test concentrations for the n-butanol fractions (total saponins) were 100, 50,
and 25 µg/mL. Fold induction = luciferase expression in sample-treated cells/luciferase expression in vehicle-treated cells
to exhibit a stronger agonistic effect towards PPARα with a fold
induction of 1.71 (indicating a 71% increase in its activity) com-
pared to the vehicle treated control. On the other hand, the acti-
vation of PPARγ was only 1.27-fold by the n-butanol fraction, rep-
resenting an increase of only 27% (▶ Table 2).
Among the isolates, compounds 1 and 3 exhibited a stronger
agonistic effect towards PPARα, significantly enhancing PPARα-di-
rected luciferase expression with an increase of 2.25- and 1.86-
fold, respectively, compared to the vehicle control. Compound 2
showed a lower increase in the activity of PPARα, while compound
the bidesmoside pattern. Compound 1 as a monodesmoside
saponin esterified at C-28 with a glucose unit, in addition to the
presence of methyl C-24 and an aldehydic group at C-23 instead
of a methyl group, exhibited the most potent effect on PPARα.
Alteration of this pattern by retaining the dimethyl pattern of C-
23 and C-24 and adding a sugar at C-3 exhibited a lower effect,
as in 3, followed by the bidesmoside pattern (i.e., compound 2).
Moreover, a hydroxyl methyl pattern at C-23 in bidesmoside sap-
onins exhibited the loss of activity on PPARα (compound 4). These
observations were consistent with the results obtained previously
by Wang et al. [35], who showed that the saponins containing a
methyl moiety at C-23 exhibit better activity than those with a hy-
droxyl methyl pattern at C-23. Therefore, the pattern of the ring A
in the 30-noroleanene nuclei could affect the activity on PPARα,
4
did not show any effect (▶ Table 2). Compounds 1, 3, and 4 did
not show > 1.5-fold increase in PPARγ activity, while 2 showed 1.6-
fold activation of PPARγ, as shown in ▶ Table 2.
This class of compounds, nor-oleanane-type triterpenes, were
reported for cytotoxic [23], hemolytic [29], and α-glucosidase in-
hibition [30] activities earlier. The reported hypoglycemic effect of
the plant could be due to α-glucosidase inhibitory activity of its
saponins. Salicornia saponin from Salicornia bigelovii Torr.
whether the compound shows
a monodesmoside or a
bidesmoside pattern. Replacement of the methyl at C-23 with an
aldehyde or its substitution with a hydroxyl methyl pattern may
result in modification of the activity.
(
Amaranthaceae), which is very similar to 2, is formulated for
In conclusion, a chemical investigation of A. articulata, which
has previously shown antidiabetic activity through increasing the
insulin level, resulted in the isolation of four major saponins. Due
to the PPARα agonistic activity of these compounds, they can also
serve as therapeutic agents for treating hyperlipidemic conditions
associated with diabetes and obesity. Further investigation of the
hypolipidemic action of compounds 1 and 3 is warranted.
treating obesity and hyperlipidemia in the clinic, and processed
to obtain a slimming health care food [31]. Moreover, boussingo-
side E (4), a triterpenoid saponin isolated from the tubers of
Anredera baselloides (Kunth) Baill. (Basellaceae), was also reported
to exhibit hypoglycemic activity [32].
It was reported previously that the triterpenoidal compounds
and their glycosides exhibited a strong antidiabetic effect, espe-
cially those of the oleanane nucleus [33]. In addition, it was re-
ported that oleanolic acid may improve cardiac lipid metabolism
in Zucker Diabetic Fatty (ZDF) rats by acting on PPARα. Two glyco-
sylated oleanolic acid derivatives from Acer pictum Thunb. (Sapin-
daceae) achieved a transactivation of the three PPAR subtypes,
and they are very interesting for the treatment of metabolic dis-
eases because they could simultaneously target insulin resistance,
atherogenic dyslipemia, and obesity [34].
Materials and Methods
General experimental procedures
An LTQ Orbitrap mass spectrometer (Thermo Finnigan) was oper-
ated to record HR‑ESI‑MS. An AUTOPOL IV Automatic Polarimeter
(Rudolph) was used to determine specific rotations. 1D and 2D
NMR spectra were recorded on Bruker DRX-850 and 600 MHz
Ultrashield spectrometers (BrukerBioSpin) using DMSO as the sol-
vent. TLC analysis was performed on precoated TLC plates with
silica gel 60 F254 (Merck). Separation of pure compounds was per-
formed on column chromatography using silica gel 60 (70–230
As a comprehensive interpretation, the structure-activity rela-
tionship of the isolated saponins on PPARα was found to be de-
pendent on the pattern of C-23 and the mono and bidesmoside
character of the isolated saponins as well as the type of sugars in
El Dine RS et al. PPARα and γ… Planta Med

Original Papers
mesh, Merck), RP18 (0.04–0.063 mm Merck), and Sephadex LH-
3β-O-D-Galactopyranosyl-23-aldehyde-30-norolean-12,20(29)-
dien-28-oic acid-28-O-β-D-glucopyranosyl ester (2): white amor-
2
0 (Merck). Finally, purification of the compounds was done using
1
0
1
a 6-mL standard LiChrolut extraction tube (RP18, 40–63 µm;
Merck).
phous powder; [α]
and C NMR (DMSO, 150 MHz); for data see ▶ Table 1; HRESIMS
D
+ 15 (c 0.1, MeOH); H (DMSO, 600 MHz)
1
3
+
The drug controls, ciprofibrate and rosiglitazone (purity
m/z 779.4639 [M + H] (calcd. for 779.4672, C41H63O14).
>
98%), were obtained from Cayman Chemical. DMEM, bovine calf
3β-O-D-Xylopyranosyl-30-norolean-12,20(29)-dien-28-oic acid
28-O-β-D-glucopyranosyl ester (3): white amorphous powder;
serum (BCS), FBS, and PBS were from Hyclone. Penicillin/strepto-
mycin and trypsin were purchased from Gibco. Two plasmids,
pSG5-PPARα (plasmid 22751) and PPRE X3-tkluc (plasmid 1015),
were from Addgene, while two others, pCMV-rPPARγ and
pPPREaP2-tk-luc, were obtained from Dr. Dennis Feller, University
of Mississippi (Department of Pharmacology). The test com-
pounds were dissolved in DMSO at a 10 mM concentration, while
the n-butanol fraction was dissolved at 20 mg/mL in DMSO.
1
0
1
13
[α]
D
+ 30 (c 0.1, MeOH); H (DMSO, 600 MHz) and C NMR
(DMSO, 150 MHz); for data see ▶ Table 1; HRESIMS m/z
+
735.4358 [M + H] (calcd. for 735.4366, C40H63O12).
Determination of the absolute configuration of sugars
Hydrolysis of the isolated compound (1 mg) was performed by
2 M HCl in water (0.5 mL) and refluxing at 95°C for 2 h. Then,
NH OH was added for neutralization of the reaction mixture and
4
Plant material
the aglycone was extracted three times with EtOAc, leaving the
sugars in the aqueous phase. The aqueous layer was lyophilized,
and the dried residue was dissolved in pyridine (0.5 mL) and mixed
with 1 mL of L-cysteine methyl ester hydrochloride in pyridine
(0.1 M). Next, the mixture was heated at 90°C for 1 h. Then, phe-
nyl isothiocyanate in pyridine (1 mL) was added and the mixture
was heated for 1 h at 90°C. A reversed-phased HPLC [Waters Alli-
ance 2795, equipped with a photodiode array detector and Luna
C18 column (150 × 4.6 mm, 5 µm particle size; Phenomenex,
Inc.)] was used to analyze the resulting compound. Two solvent
systems were used for elution, the first being water containing
0.1% acetic acid (A) and the second being acetonitrile containing
0.1% acetic acid (B). They were injected in a gradient mode: A/B
was mixed 90/10 for the first 20 min and then mixed at 45/55 for
the next 25 min at a rate of 1 mL/min. The response was detected
at 254 nm. The standard sugar derivatives were identically pre-
pared and analyzed. The sugars were identified as D-glucose, D-
galactose, and D-xylose by comparison of the retention time of
their derivative with that of the authentic sugars [D-glucose:
30.7 min, D-galactose: 34.2 min, D-xylose: 33.9 min].
In December 2014, flowering aerial parts of A. articulata (Forssk.)
Moq. were brought from Saudi Arabia. Dr. Emad Al-Sharif, (Faculty
of Science & Arts, Khulais, King Abdulaziz University, Saudi Arabia)
kindly identified the plant material. A specimen (AA1011) of the
plant was deposited at the Herbarium of the Faculty of Pharmacy,
King Abdulaziz University.
Extraction and isolation
Dried flowering aerial parts of A. articulata (1 kg) were extracted
with methanol (3 × 3 L) at room temperature using an Ultraturrex
homogenizer until exhaustion, followed by evaporation of the sol-
vent under vacuum to give a semisolid dark brown residue (120 g,
1
2%). The resulting residue was suspended in distilled water
(
500 mL) and partitioned successively with CHCl3 (500 mL × 5),
EtOAc (500 mL × 5), and n-butanol (150 mL × 5) to yield 50, 10,
and 15 g, respectively. The n-butanol fraction (15 g) was chroma-
tographed over silica gel 60 H for CC (5 × 100 cm) and eluted with
CHCl containing increasing amounts of MeOH and water to ob-
3
tain seven fractions (fractions 1 to 7).
Fraction 3 (0.98 g) was chromatographed on a Sephadex LH-
Determination of transcriptional activity
of peroxisome proliferator-activated receptor-α
and peroxisome proliferator-activated receptor-γ
by the reporter gene assay
2
(
0 column (100 g, MeOH; 300 mL), resulting in four subfractions
fraction 3–1 → fraction 3–4). Subfraction 3–3 (220 mg) was fur-
ther chromatographed on a reversed-phase silica gel column
50 g, 50 µm) using H O :MeOH (4:6, v/v; 120 mL) as an eluting
(
2
system to yield compound 1 (10 mg). Fraction 4 (1.2 g) was chro-
matographed over a Sephadex LH-20 as fraction 3, affording five
subfractions (subfraction 4–1 → subfraction 4–5). Subfraction 4–
Human hepatoma (HepG2) cells (ATCC) were grown in DMEM
supplemented with 10% FBS, 100 units/mL penicillin, and
100 µg/mL streptomycin. The detailed procedure has been re-
ported earlier [36]. In brief, HepG2 cells were transfected by elec-
troporation using the Square electroporator T820 (BTX) and its as-
sociated BTX disposable cuvette at 160 V for a single 70 ms pulse.
Either a combination of pSG5-PPARα and PPRE X3-tk-luc or a com-
bination of pCMV-rPPARγ and pPPREaP2-tk-luc plasmid DNA
(25 µg of each/1.5 mL cell suspension) was transfected into the
cells by electroporation. The transfected cells were grown for a
4
(350 mg) was chromatographed on a reversed-phase silica gel
column using H O :MeOH (6:4, v/v; 150 mL) as an eluting system,
2
followed by purification on preparative HPLC using an H O :MeOH
2
(
7:3, v/v) mixture to yield compounds 2 (15 mg) and 3 (12 mg).
Finally, fraction 5 (500 mg) was chromatographed on RP‑C18
50 g, 50 µm) using H O :MeOH (1:1, v/v; 150 mL) to give four
(
2
major subfractions (subfraction 5–1 → subfraction 5–4). Subfrac-
tion 5–2 (100 mg) was further purified on RP‑C18 using
4
period of 24 h, having been plated at a density of 5 × 10 cells/well
H O :MeOH (7:3, v/v; 100 mL) to afford compound 4 (22 mg).
in 96-well tissue culture plates. After this time, test compounds
(12.5, 25, and 50 µM) or drug controls (ciprofibrate or rosiglita-
zone; 2.5, 5.0, and 10 µM) were added to treat the cells for 24 h.
At the end of this incubation period, the cells were lysed and a
luciferase assay system (Promega) was employed to measure the
luciferase activity. The highest concentration of DMSO (vehicle
2
3
β-Hydroxy,23-aldehyde-30-norolean-12,20(29)-dien-28-oic ac-
id-28-O-β-D-glucopyranosyl ester (1): white amorphous powder;
1
0
1
13
[
(
6
α]
D
+ 25(c 0.1, MeOH); H (DMSO, 600 MHz) and C NMR
DMSO, 150 MHz); for data see ▶ Table 1; HRESIMS m/z
17.3652 [M + H] (calcd. for 617.3658, C35H53O9).
+
El Dine RS et al. PPARα and γ… Planta Med

on glucose uptake and 3T3-L1 adipogenesis. Planta Med 2013; 79:
084–1095
control) was 0.5%. The effect of the test compounds on PPARα
and PPARγ was determined in terms of fold induction in their ac-
tivity, which was calculated as the ratio of the luciferase expres-
sion in sample-treated cells to the luciferase expression in ve-
hicle-treated cells.
1
[
13] Christensen KB, Minet A, Svenstrup H, Grevsen K, Zhang H, Schrader E,
Rimbach G, Wein S, Wolffram S, Kristiansen K. Identification of plant ex-
tracts with potential antidiabetic properties: effect on human peroxi-
some proliferator-activated receptor (PPAR), adipocyte differentiation
and insulin-stimulated glucose uptake. Phytother Res 2009; 23: 1316–
Statistical analysis
1
325
The fold induction values represented in ▶ Table 2 are the avera-
ge ± SD of two independent experiments performed with two
replicates each time. Calculations were done using Microsoft Excel
version 14.6.5.
[14] Marles RJ, Farnsworth NR. Antidiabetic plants and their active constitu-
ents. Phytomedicine 1995; 2: 137–189
[15] Kambouche N, Merah B, Derdour A, Bellahouel S, Bouayed J, Dicko A,
Younos C, Soulimani R. Hypoglycemic and antihyperglycemic effects of
Anabasis articulata (Forssk) Moq (Chenopodiaceae), an Algerian medici-
nal plant. Afr J Biotechnol 2009; 8: 5589–5594
Supporting Information
[
16] Metwally NS, Mohamed AM, Elsharabasy FS. Chemical constituents of
the Egyptian Plant Anabasis articulata (Forssk) Moq and its antidiabetic
effects on rats with streptozotocin-induced diabetic hepatopathy. J App
Pharm Sci 2012; 2: 54–65
1H and 13C NMR spectra of compounds 1–3 and their HSQC,
HMBC, and NOESY correlations are available as Supporting Infor-
mation.
[
17] Abdallah HM, Abdel-Naim AB, Ashour OM, Shehata IA, Abdel-Sattar EA.
Anti-inflammatory activity of selected plants from Saudi Arabia. Z Natur-
forsch C 2014; 69: 1–9
Conflict of Interest
[
18] Kambouche N, Merah B, Derdour A, Bellahouel S, Benziane M, Younos C,
Firkioui M, Bedouhene S, Soulimani R. Étude de lʼeffet antidiabétique des
saponines extraites d’Anabasis articulata (Forssk) Moq, plante utilisée
traditionnellement en Algérie. Phytothérapie 2009; 7: 197–201
The authors declare no conflict of interest
References
[
19] Abdallah HM, El-Bassossy H, Mohamed GA, El-Halawany AM, Alshali KZ,
Banjar ZM. Phenolics from Garcinia mangostana inhibit advanced glyca-
tion endproducts formation: effect on Amadori products, cross-linked
structures and protein thiols. Molecules 2016; 21: 251
[
1] Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet
005; 365: 1415–1428
2
[
2] Nisoli E, Clementi E, Carruba MO, Moncada S. Defective mitochondrial
biogenesis: a hallmark of the high cardiovascular risk in the metabolic
syndrome? Circ Res 2007; 100: 795–806
[
20] Abdallah HM, El-Bassossy HM, Mohamed GA, El-Halawany AM, Alshali
KZ, Banjar ZM. Phenolics from Garcinia mangostana alleviate exagger-
ated vasoconstriction in metabolic syndrome through direct vasodilata-
tion and nitric oxide generation. BMC Complement Altern Med 2016; 16:
[
3] Laudet V, Hänni C, Coll J, Catzeflis F, Stéhelin D. Evolution of the nuclear
receptor gene superfamily. EMBO J 1992; 11: 1003–1013
3
59
[
4] Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W. Control of the
peroxisomal β-oxidation pathway by a novel family of nuclear hormone
receptors. Cell 1992; 68: 879–887
[21] Abdallah HM, El-Bassossy HM, Mohamed GA, El-Halawany AM, Alshali
KZ, Banjar ZM. Mangostanaxanthones III and IV: advanced glycation
end-product inhibitors from the pericarp of Garcinia mangostana. J Nat
Med 2017; 71: 216–226
[5] Evans RM, Barish GD, Wang YX. PPARs and the complex journey to obe-
sity. Nat Med 2004; 10: 355–361
[
22] Espada A, Riguera R, Jiménez C. Boussingoside E, a new triterpenoid
[
6] Auboeuf D, Rieusset J, Fajas L, Vallier P, Frering V, Riou JP, Staels B,
Auwerx J, Laville M, Vidal H. Tissue distribution and quantification of
the expression of mRNAs of peroxisome proliferator-activated receptors
and liver X receptor-α in humans: no alteration in adipose tissue of obese
and NIDDM patients. Diabetes 1997; 46: 1319–1327
saponin from the tubers of Boussingaultia baselloides. J Nat Prod 1997;
6
0: 17–19
[23] Wang QZ, Liu XF, Shan Y, Guan FQ, Chen Y, Wang XY, Wang M, Feng X.
Two new nortriterpenoid saponins from Salicornia bigelovii Torr. and their
cytotoxic activity. Fitoterapia 2012; 83: 742–749
[
[
[
[
7] Shen P, Liu MH, Ng TY, Chan YH, Yong E. Differential effects of isofla-
vones, from Astragalus membranaceus and Pueraria thomsonii, on the
activation of PPARα, PPARγ, and adipocyte differentiation in vitro. J Nutr
[24] Abdallah HM, Al-Abd AM, Asaad GF, Abdel-Naim AB, El-Halawany AM.
Isolation of antiosteoporotic compounds from seeds of Sophora japo-
nica. PLoS One 2014; 9: e98559
2
006; 136: 899–905
8] Vidal-Puig AJ, Considine RV, Jimenez-Liñan M, Werman A, Pories WJ,
Caro JF, Flier JS. Peroxisome proliferator-activated receptor gene expres-
sion in human tissues. Effects of obesity, weight loss, and regulation by
insulin and glucocorticoids. J Clin Invest 1997; 99: 2416–2422
[25] Yoshikawa M, Murakami T, Kadoya M, Yamahara J, Matsuda H. Medicial
foodstuffs. XV. Sugar beet. (2): Structures of betavulgarosides V, VI, VII,
VIII, IX and X from the roots and leaves of sugar beet (Beta vulgaris L.,
Chenopodiaceae). Chem Pharm Bull 1998; 46: 1758–1763
9] Medina-Gomez G, Gray SL, Yetukuri L, Shimomura K, Virtue S, Campbell
M, Curtis RK, Jimenez-Linan M, Blount M, Yeo GS. PPAR gamma 2
prevents lipotoxicity by controlling adipose tissue expandability and
peripheral lipid metabolism. PLoS Genet 2007; 3: e64
[26] Jitsuno M, Mimaki Y. Triterpene glycosides from the aerial parts of Larrea
tridentata. Phytochemistry 2010; 71: 2157–2167
[
27] Cabrita L, Frøystein NÅ, Andersen ØM. Anthocyanin trisaccharides in
blue berries of Vaccinium padifolium. Food Chem 2000; 69: 33–36
10] Indulekha K, Unnikrishnan R, Mohan V. Extending current Definitions of
the metabolic Syndrome. In: Kurian M, Wolfe B, Ikramuddin S, eds. Meta-
bolic Syndrome and Diabetes. New York: Springer; 2016: 19–30
[
28] Hamed AI, Springuel I, El-Emary NA, Mitome H, Miyaoka H, Yamada Y.
Triterpenoidal saponin glycosides from Glinus lotoides var. dictamnoides.
Phytochemistry 1996; 43: 183–188
[
11] Roman-Ramos R, Flores-Saenz JL, Alarcon-Aguilar FJ. Anti-hyperglycemic
[
29] Zehring J, Reim V, Schröter D, Neugart S, Schreiner M, Rohn S, Maul R.
Identification of novel saponins in vegetable amaranth and characteriza-
tion of their hemolytic activity. Food Res Int 2015; 78: 361–368
effect of some edible plants. J Ethnopharmacol 1995; 48: 25–32
[
12] Yang MH, Avula B, Smillie T, Khan IA, Khan SI. Screening of medicinal
plants for PPARα and PPARγ activation and evaluation of their effects
El Dine RS et al. PPARα and γ… Planta Med

Original Papers
[
[
[
[
30] Wang J, Xu QL, Zheng MF, Ren H, Lei T, Wu P, Zhou ZY, Wei XY, Tan JW.
Bioactive 30-noroleanane triterpenes from the pericarps of Akebia trifo-
liata. Molecules 2014; 19: 4301–4312
[34] Quang TH, Ngan NTT, Van Minh C, Van Kiem P, Thao NP, Tai BH, Nhiem
NX, Song SB, Kim YH. Effect of triterpenes and triterpene saponins from
the stem bark of Kalopanax pictus on the transactivational activities of
three PPAR subtypes. Carbohydr Res 2011; 346: 2567–2575
31] Shan Y, Li H, Guan F, Chen Y, Yin M, Wang M, Feng X, Wang Q. Triterpe-
noids from the Herbs of Salicornia bigelovii. Molecules 2015; 20: 20334–
[35] Wang L, Waltenberger B, Pferschy-Wenzig EM, Blunder M, Liu X,
Malainer C, Blazevic T, Schwaiger S, Rollinger JM, Heiss EH. Natural prod-
uct agonists of peroxisome proliferator-activated receptor gamma
2
0340
32] Espada A, Rodriguez J, Villaverde MC, Riguera R. Hypoglucaemic triter-
(PPARγ): a review. Biochem Pharmacol 2014; 92: 73–89
penoid saponins from Boussingaultia baselloides. Can J Chem 1990; 68:
2
039–2044
[36] Yang MH, Vasquez Y, Ali Z, Khan IA, Khan SI. Constituents from Termina-
lia species increase PPARα and PPARγ levels and stimulate glucose up-
take without enhancing adipocyte differentiation. J Ethnopharmacol
33] Burdi DK, Qureshi S, Ghanghro AB. An overview of available hypoglyce-
mic triterpenoids and saponins to cure diabetes mellitus. ALS 2014; 1:
2
013; 149: 490–498
1
19–128
El Dine RS et al. PPARα and γ… Planta Med