Evolvosides C-E, flavonol-4-O-triglycosides from Evolvulus alsinoides and their anti-stress activity

Article abstract of DOI:10.1016/j.bmc.2012.12.040

Phytochemical investigation of the n-butanol fraction of Evolvulus alsinoides (Linn.) led to the isolation of three new phenolic glycosides, evolvosides C, D and E (1-3) along with six known compounds (4-9). The structures of the compounds were elucidated

Full text of DOI:10.1016/j.bmc.2012.12.040

Bioorganic & Medicinal Chemistry 21 (2013) 1116–1122
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Evolvosides C–E, flavonol-4-O-triglycosides from Evolvulus alsinoides
and their anti-stress activity
b
c
d
a
Prasoon Gupta a,b, , Upasana Sharma , Praveen Gupta , Kiran Babu Siripurapu , Rakesh Maurya
⇑
a Division of Medicinal Chemistry, Central Drug Research Institute, Lucknow 226001, Uttar Pradesh, India
b Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, USA
c Department of Pharmaceutical Sciences, Shri Guru Ram Rai Institute of Technology and Sciences, Dehradun 248001, Uttarakhand, India
d Department of Behavioral Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40546, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytochemical investigation of the n-butanol fraction of Evolvulus alsinoides (Linn.) led to the isolation of
three new phenolic glycosides, evolvosides C, D and E (1–3) along with six known compounds (4–9). The
structures of the compounds were elucidated on the basis of spectroscopic analysis, viz. 1D and 2D NMR
experiments, chemical study, and comparison with literature data. Evolvoside C (1) was characterized as
Received 8 November 2012
Revised 17 December 2012
Accepted 28 December 2012
Available online 10 January 2013
kaempferol 40-O-b-
D-glucopyranosyl-(1?2)-a-L-rhamnopyranosyl-(1?6)-b-D-glucopyranoside, whereas
evolvosides D and E (2–3) were found to be mono and di-O-methyl derivatives of 1. The new compounds
(1–3) represent rare triglycoside derivatives of flavonol at C-40. The isolated compounds (1–6) were
screened for acute stress-induced biochemical changes in male Sprague–Dawley rats at a dose of
40 mg/kg body weight. Compounds 1 and 2 displayed anti-stress effects by normalizing hyperglycemia,
plasma corticosterone, plasma creatine kinase, and adrenal hypertrophy. Compounds 3 and 6 were also
found to be effective in normalizing most of these stress parameters, whereas compounds 4 and 5 were
ineffective in normalizing most of these effects.
Keywords:
Evolvulus alsinoides
Flavonoid glycoside
Evolvosides
Triglycosides
Anti-stress
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
contane,
octyl
octadec-9-enoate,
nonyloctadec-9-enoate,
dodecanyl-octadec-9,12-dienoate, tetracosanyl hexadec-9-enoate,
heptacosan-14-b-ol, stigmast-5, 22-dien-3b-ol and stigmata-5-en-
3b-ol.12
Evolvulus (Family: Convolvulaceae) is a small genus composed of
about 10–15 species widely distributedin Asian and Americancoun-
tries, with some of its species used medicinally.1,2 Evolvulus alsino-
ides is one of the several well-known Ayurvedic crude drugs that
have a significant place in traditional medicinal system of India
due to its memory enhancing properties.3–5 Apart from its historical
indigenous use several preparations containing the plant decoction
are widely commercialized as nerving tonic (Shankhpushpi) in Asia.
In addition, the plant extracts have been used to treat a number of
diseases including bronchitis, asthma,2,3 brain disorders like insan-
ity, epilepsy, nervous disability, and scrofula.2–5 Previous work on
Evolvulus alsinoides extracts have demonstrated antioxidant,6 anti-
ulcer,7 and immunomodulatory activities.8 However, the
phytochemical studies are limited which have resulted in the iden-
tification of triacontane, pentatriacontane, scopoletin, scopolin,
umbelliferon, methyl-1,2,3,4-butaneterol, b-sitosterol and the
alkaloids betaine and shankpushpin.9,10 Four unidentified alkaloids
have also been described in the literature.11 More recently Akhtar
et al. reported the isolation of a new chromone, 6-hydroxy-5-nonad-
ecanoxychroman-2-one (alsinoideschromone) along with hexatria-
As part of a research program on the discovery of new natural
immunomodulatory agents from Ayurvedic crude drugs,13–16 it
was found that a crude ethanol extract of this plant exhibited
adaptogenic and anti-amnesic properties.13 During our recent phy-
tochemical investigations of ethanol extract, we have reported a
series of phenolic compounds including new flavonol-40-O-diglyco-
sides, evolvosides A and B17 and two new caffeoyl derivatives,
evolvoids A and B.16 The latter metabolites were the first natural
examples of the erythritol associated with phenolic substance
(caffeoyl). In the course of that study, we also noted that the n-
butanol fraction of ethanol extract contained several unidentified
polar flavonoids. This paper describes the isolation and character-
ization of three new flavonol-40-O-triglycosides, evolvosides C–E
(1–3), accompanied by known compounds (4–9). The anti-stress
activity of compounds 1–6 was evaluated in acute stress induced
biochemical changes in adult male Sprague–Dawely rates.
2. Results and discussion
The dried powder of Evolvulus alsinoides (17 kg, whole plant)
was extracted with EtOH, yielded crude extract 1.19 kg (yield
⇑
Corresponding author. Tel.: +1 706 201 4132; fax: +1 561 297 2759.
E-mail address: prasoonfau@gmail.com (P. Gupta).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.040

P. Gupta et al. / Bioorg. Med. Chem. 21 (2013) 1116–1122
1117
7.0%). The EtOH extract was then dissolved in water and subjected
to liquid–liquid partitioning successively with CHCl3 and n-BuOH,
resulted in CHCl3 (320.3 g), n-BuOH (260.0 g) and aqueous
(511.2 g) crude residue respectively. A portion of n-BuOH soluble
fraction (60 g) was loaded on polymeric HP-20 resin using cyclic
loading method.18 The HP-20 column was then eluted with
400 mL fractions of (1) 20% Me2CO/H2O (2) 40% Me2CO/H2O (3)
60% Me2CO/H2O (4) 80% Me2CO/H2O and finally with Me2CO. The
20% Me2CO/H2O fraction (10.3 g) was then subjected to sequential
CC and finally HPLC separation on a C-18 reverse phase column to
yield the new flavonol-40-O-triglycosides, evolvosides C–E (1–3).
The other known compounds 4–9 were isolated from 40% and
60% Me2CO/H2O fractions respectively. The chemical structure of
the compounds 1–3 were proposed on the basis of their detailed
UV, IR, HRESIMS and 1D and 2D NMR spectral data analysis
whereas known compounds were characterized by spectral data
comparison with literature reports.
monosaccharide units sequentially at m/z 931 [M+H–C14H20O9]+
,
+
+
701 [M+H–C24H34O15
]
and 413 [M+H–C36H50O23
]
The monosac-
,
.
charide units were identified as D-glucose and L-rhamnose by acid
hydrolysis of 1, followed by co-TLC with authentic sample, GC
analysis and optical rotation.21 These preliminary observations
indicated that 1, could be flavonol-40-O-triglycoside and was con-
firmed by extensive analysis of both one and two-dimensional 1H
and 13C NMR spectra. The aromatic region of the 1H NMR (Table
1) spectrum of 1 contained a characteristic resonance at dH 6.21
(1H, d, J = 1.6, H-6; dC 99.7 by HSQC) and dH 6.40 (1H, d, J = 1.6,
H-8; dC 94.9 by HSQC), an assignment confirmed by long-range
connectivity of H-6/C-7,8,10; H-8/C-9,10 in the HMBC spectrum
(Fig. 2). Two doublets at dH 8.11 (2H, d, J = 8.3 Hz, H-20, 60); dC
131.1 and 7.23 (2H, d, J = 8.3 Hz, H-30, 50); dC 116.2 comprised the
AAXX aromatic resonances, a downfield shift of H-30/50 at dH 7.23
was characteristic of 40-O-substituted B-ring of a flavonoids.17
These data confirmed that the aglycone was kaempferol (3,5,7,40-
tetrahydroxyflavone). Table 1 summarizes 1H and 13C NMR chem-
ical shift assignments for 1, which are similar to the literature val-
ues for known kaempferol 40-O-glycosides.17 Further evidence for
the glycosidation came from the long-range correlation detected
in the HMBC spectrum between the anomeric proton resonance
at dH 5.21 (1H, d, J = 7.3 Hz; dC 100.5) and C-40 of kaempferol (dC
161.2). Two additional resonances corresponding to anomeric pro-
tons of sugar residues appeared at dH 4.69 (1H, br d, J = 1.1 Hz; dC
101.1) and dH 4.62 (1H, d, J = 7.3 Hz; dC103.6). The remaining glyco-
sidic proton resonances occurred between 3.14 and 3.76 ppm, with
the exception of that for a rhamnose methyl doublet at dH 1.19 (3H,
d, J = 6.1 Hz; dC17.7).
Compound 1 (Fig. 1) was isolated as light yellow hygroscopic
solid, with a molecular formula of C33H40O20 as determined by
HRESIMS pos. spectrum (m/z at 779.2023 [M+Na]+ calcd for
,
779.2011). Both the UV [kmax 364 (band I) and 261 (band II) nm]
and IR [mmax 1667 (a,b-unsaturated carbonyl), 1590, 1461 and
1399 (aromatic rings) and 3432 (hydroxyl groups) cmꢀ1] spectra
showed absorption bands characteristic of a flavonol.19 Bathochro-
mic shift of 11 nm in band II with sodium acetate (C-7 OH), 52 nm
bathochromic shift of band I in presence of AlCl3 (C-5 OH) and
21 nm bathochromic shift in band I with decreased intensity upon
addition of NaOMe (C-3 OH) discovered the presence of free hydro-
xyl groups at C-3, 5 and 7 positions with substituted 40-hydroxy
group (no retention of peak intensity with NaOMe).20 On
acetylation (pyridine/Ac2O) it formed duodecaacetate derivative
1a (Supplementary data). The formation was confirmed by ESI-
MS: m/z 1261.346 [M+H]+, indicated the presence of 12 acylable
hydroxyl groups in compound 1. In addition, the FAB-MS spectrum
of 1a exhibited three diagonostic fragments due to loss of
The 1H and 13C resonance assignments for the three sugar resi-
dues were obtained from standard 1D and 2D NMR datasets (Table
1). Anomeric configurations were determined from the magnitudes
of J1,2 coupling constant in the 1H NMR spectrum. These data indi-
cated that the primary sugar O-linked at C-40 was a b-
anosyl residue. The remaining anomeric proton resonances at dH
D-glucopyr-
OH
HO
O
H3C
OH
O
O
OH
OH
OH
OH
O
O
HO
O
OH
O
O
OH
OH
OH
HO
HO
HO
R1O
O
O
OH
OR2
O
OH
1: R1 = R2 = H
2: R1 = Me, R2 = H
4
3
: R1 = R2 = Me
OR3
R2O
O
O
OR1
OH
5
: R1 = R2 =H, R3 = β-
D
L
-glucopyranosyl -(1-2)-β-
-rhamnopyranosyl -(1-6)-β-
-glucopyranoside, R3 = H
D
-glucopyranoside
6: R1 = R2 =H, R3 = α-
7
D
-glucopyranoside
: R1 = R2 = β-
D
8: R1 = β-
D
-glucopyranosyl-(1-6)-β-
-glucopyranosyl, R2 = α-
D
-glucopyranoside, R2 = R3 = H
9: R1 = β-
D
L-rhamnopyranoside, R3 = H
Figure 1. Chemical structures for compounds (1–9).

1118
P. Gupta et al. / Bioorg. Med. Chem. 21 (2013) 1116–1122
Table 1
13C and 1H NMR data for evolvosides C–E (1–3)a
Atom
1a
2a
3a
dC
d
1H (J in Hz)
dC
d
1H (J in Hz)
dC
d
1H (J in Hz)
Aglycone
2
3
4
5
6
7
8
9
147.4
136.1
176.3
162.8
99.7
—
—
—
—
147.1
135.9
176.9
162.6
99.4
—
—
—
—
147.2
136.4
175.9
161.4
99.0
—
—
—
—
6.21 d (1.6)
—
6.40 d (1.6)
—
—
—
8.11 d (8.3)
7.23 d (8.3)
—
7.23 d (8.3)
8.11 d (8.3)
5.21 d (7.3)
3.64 t (8.5)
3.55 t (8.6)
3.34 m
6.29 d (1.5)
—
6.49 d (1.4)
—
—
—
8.19 d (8.1)
7.21 d (8.1)
—
7.21 d (8.1)
8.19 d (8.1)
5.17 d (7.1)
3.64 t (8.2)
3.52 t (8.1)
3.32 mb
6.30 d (1.5)
—
6.51 d (1.5)
—
—
—
8.08 d (8.4)
7.29 d (8.3)
—
165.9
94.9
164.8
93.9
164.3
93.4
156.2
104.9
124.5
131.1
116.2
161.2
116.2
131.1
100.5
76.1
77.1
71.6
77.9
68.3
—
101.1
80.9
70.1
72.9
69.5
17.7
103.6
74.9
76.2
70.2
75.7
60.7
156.0
104.7
124.3
132.1
116.9
161.9
116.9
132.1
100.1
76.3
77.0
71.4
77.6
68.0
—
101.4
80.4
70.3
72.6
69.7
17.9
103.4
74.9
76.4
70.5
75.3
60.2
155.0
104.1
124.5
131.7
116.4
161.4
116.4
131.7
100.4
76.2
77.1
71.2
78.1
68.5
—
101.0
80.5
69.9
72.5
69.2
18.1
103.2
74.1
76.7
70.3
75.2
60.4
10
1
20
30
40
50
7.29 d (8.3)
8.08 d (8.4)
5.09 d (7.0)
3.62 t (8.6)
3.51 br d (10.4)c
3.30 mb
60
40-O-b-GlcA
1
2
3
4
5
3.60 br d (9.0)c
3.76 dd (11.2,1.6)
3.51 br d (11.0)
4.69 d (1.1)
3.33 m
3.58 br d (9.3)c
3.70 dd (11.0,1.7)
3.49 br d (8.9)f
4.71 d (1.3)
3.30 mb
3.68 br d (9.1)e
3.76 dd (10.8,1.9)
3.50 br d (10.4)c
4.68 br s
6a
6b
1000
2000
3000
4000
5000
6000
10000
20000
30000
40000
50000
60000
60000
6GlcA-O-
a
-Rha
3.31 mb
3.41 dd (9.1, 3.1)
3.20 t (8.9)b
3.46 dd (9.5, 6.2)
1.19 br d (6.1)
4.62 d (7.3)
3.14 t (8.6)
3.28 m
3.49 br d (8.9)f
3.23 m
3.42 dd (9.2, 3.4)
3.18 t (8.7)d
3.44 br d (8.7)
1.18 br d (5.9)
4.64 d (7.3)
3.18 t (8.7)d
3.30 mb
3.24 t (8.5)
3.18 t (8.7)d
3.69 br d (9.1)e
3.51 br d (10.4)c
3.89 s
3.49 br d (8.9)
1.21 br d (6.7)
4.60 d (7.1)
3.16 t (8.3)
3.30 mb
2Rha-O-b-GlcB
3.20 t (8.9)b
3.17 m
3.20 me
3.19 me
a
b
3.71 br d (12.2)
3.58 br d (9.3)c
3.42 dd (12.2, 8.0)
3.87 s
3.60 dd (9.0)c
7-OCH3
5-OCH3
55.2
55.3
55.9
3.91 s
Chelated OH
13.01 s
13.31 s
a
Measured in DMSO-d6 (1H NMR in 400 MHz and 13C NMR in 100 MHz).
Overlapped within the column.
b
These ambiguities were resolved by using the recent H2BC (heter-
onuclear two-bond correlation) pulse sequence, which gives only
2JHC correlations between protons and proton-attached carbons.22
This avoids the problems encountered in HMBC spectra of distin-
OH
HO
O
H3C
OH
O
O
OH
OH
2
3
2
O
guishing between JHC and JHC correlations, and of ‘missing’ JHC
correlations. As such, the H2BC sequence has been applied success-
fully to the assignment of NMR spectra of some complex carbohy-
drates.23 The H2BC spectrum of 1 afforded a full set of sequential
2JHC connectivities for the GlcA moiety from H-100 to C-2, H-2 to
C-1 and C-3, H-3 to C-2 and C-4, and H-4 to C-3 and C-5 (sequential
2JHC connectivities were also observed for the Glc and Rha moie-
ties). The interglycosidic linkages defining the trisaccharide moiety
of 1 were determined using HMBC data (Fig. 2). Long-range corre-
lations from GlcA H-6 to C-1000 of Rha (dC 101.1), and from H-1000 of
Rha to the downfield-shifted GlcA C-6 (dC 68.3), indicated that
the primary GlcA residue was 6-O-linked to Rha.24 Similarly, a 2-
O-linkage between Rha and the terminal secondary GlcB residue
was defined by connectivities from Rha H-2000 to C-10000 of GlcB (dC
103.6), and from H-10000 of GlcB to the downfield-Shifted Rha C-2000
HO
O
O
OH
OH
HO
HO
O
O
OH
OH
Figure 2. Selected HMBC correlations for evolvoside C (1).
4.69 and 4.62 were those of the
copyranosyl residues, respectively.
Assignment of the -Rha moiety was straightforward, and char-
a-L-rhamnopyranosyl and b-D-glu-
a
acteristic multiplicities and coupling constants were extracted for
all the 1H resonances except H-2, which overlapped with that of
moisture in DMSO-d6 and H-4 of b-GlcA. Full assignment of the
b-GlcB moiety was achieved using standard NMR datasets (COSY,
(dC 80.9). Compound 1 was therefore kaempferol 40-O-b-
pyranosyl-(1?2)- -rhamnopyrano syl-(1?6)-b- -glucopyrano-
side, a new flavonol glycolside named evolvoside C.
D-gluco-
a-L
D
13
HSQC, HMBC) alone and found that the C resonances for C-30000
Compound 2 (Fig. 1) was isolated as yellow hygroscopic solid;
and C-40000 coincident with dC C-2 (GlcA) and C-3000 (Rha) respec-
tively, and also H-60000 b proton was overlapped with H-5 of b-GlcA
(dH 3.60) whereas H-3 (dH 3.28) was overlapped with moisture.
with a molecular formula of C34H42O20 as determined by HRESIMS
pos. spectrum m/z 793.2206 [M+Na]+ calcd 793.2197. The 1H and
,
13C NMR spectra clearly indicated that 2 was an analogue of 1.

P. Gupta et al. / Bioorg. Med. Chem. 21 (2013) 1116–1122
1119
Table 2
Effect of pure compounds 1–6 and PQ on acute stress induced changes in adrenal gland weight, glucose, creatine kinase and corticosterone levels
Groups (dose mg/kg p.o.)
Adrenal gland (mg/kg wt)
Glucose (mg/dl)
CK (mg/dl)
Corticosterone (ng/ml)
NS
6.42 0.41
10.2 0.84**
7.11 0.34*
8.23 0.54*
Not tested
9.78 0.79
10.91 0.91
8.47 0.63*
7.19 0.29*
83.5 3.82
276.2 15.2
232.4 18.3
AS + Vehicle
COMP-1 (40)
COMP-2 (40)
COMP-3 (40)
COMP-4 (40)
COMP-5 (40)
COMP-6 (40)
PQ (100)
154.7 4.38**
80.17 2.58*
95.13 2.34*
Not tested
1018 31.43**
385.3 65.19*
405.4 69.16*
413.7 61.17*
821.6 69.12
1078 42.91
521.4 81.22*
393.3 31.60*
511.2 23.2**
233.3 19.16*
241.7 17.15*
252.6 21.16*
439.1 13.11
458.2 29.01
239.2 16.91*
231.2 11.51*
92.13 3.21*
113.1 6.01
105.13 2.71
91.2 3.21*
Mean SEM of changes in adrenal gland weight, plasma glucose, creatine kinase and corticosterone. The stress group was compared with non-stress control group and the
drug treated groups were compared with acute stress group.
**
p <0.001 when compared to NS control group.
p <0.01 when compared with acute stress control group.
*
The only significant difference in the 1H and 13C NMR spectra of 2
as compared to 1 was the presence of additional methoxyl group at
dH 3.87 (s, dC 55.2). Thus it appeared that compound 2 was the
mono methoxy derivative of 1. The site of O-methylation was con-
firmed at C-7 on the basis of shift reagent test where no bathochro-
mic shift was observed in band II on addition of NaOAc.20 This was
further supported by three bond correlation from O–CH3 to C-7 in
HMBC spectrum on 2. The complete assignment of the 1H and 13C
NMR spectrum of 2 followed the procedure described for 1 (Table
1). In the corresponding HMBC spectrum, connectivities defining
the site of glycosylation and interglycosidic linkages were as ob-
served for 1. Sugar analysis confirmed that each glycoside com-
reported in the literature. The compounds 8 and 9 were isolated
for the first time from this plant.
To evaluate anti-stress activity, a variety of stress situations
have been employed in animals. Stress-induced effects mainly de-
pend on duration and type of stressors.28 Immobilization has been
the ideal choice for the induction of stress responses in animals
and, more specifically, for the investigation of drug effects on typ-
ical stress-related gastrointestinal, euroendocrine, and immuno-
logical pathology.29 Hyperglycemic response during acute stress
is due to the release of glucocorticoids as a result of HPA axis stim-
ulation, to compensate for the initial demand of energy.30 It is
known that AS exposure not only stimulates and ensures the sup-
ply of glucose but also increases creatine kinase (CK) activity.31 The
CK system is important in stabilizing the ATP levels and energy
metabolism of the myocardium and other skeletal muscles of rats
during stress. Perturbations of CK activity during extensive stress
may result in ischemia due to the nonavailability of ATP.32 Promi-
nent changes during stress are adrenocorticoid hypersecretion, in-
creased plasma corticosterone, and enlarged pituitary and adrenal
size.33,34 In our previous work we have observed, the various type
of stress also increased the adrenal gland weight and plasma
corticosterone.35
Compounds 1—6 were screened for anti-stress activity in acute
stress (AS) model at the dose of 40 mg/kg body weight, as shown in
Table 2. A significant increase (p <0.001) in the adrenal gland
weight after AS was observed when compared to non-stressed
(NS) group. Compounds 1, 2, 6 and standard drug powder of the
roots of Panax quinquifolium (PQ) were significantly (p <0.01) effec-
tive in reducing the stress induced adrenal hypertrophy. The plas-
ma creatine kinase (CK) and glucose levels were also increased by
AS significantly (p <0.001) when compared to NS control. Pretreat-
ment with compounds 1, 3, 6 (p <0.01) and PQ (p <0.01) were effec-
tive in reducing the AS-induced increase in CK levels and
compounds 1, 2 and 4 in reducing increased glucose levels. Simi-
larly AS (p <0.001) exposure resulted in increased plasma cortico-
sterone when compared to NS control. Pretreatment with
compounds 1–3, 6 (p <0.01) and PQ (p <0.01) significantly reduced
the increase in corticosterone levels.
prised of
was assigned as kaempferol-7-methoxy-40-O-b-
(1?2)- -rhamnopyrano-syl-(1?6)-b- -glucopyranoside
named as evolvoside D.
D
-GlcA,
L-Rha and
D
-GlcB residues. Thus structure of 2
-glucopyranosyl-
and
D
a
-L
D
Compound 3 (Fig. 1) was isolated as light yellow hygroscopic
solid; with a molecular formula of C35H44O20 as determined by
HRESIMS pos. spectrum m/z 807.2324 [M+Na]+ calcd 807.2311.
,
The 1H and 13C NMR spectra clearly indicated that 3 was an ana-
logue of 1. The only significant difference in the 1H and 13C NMR
spectra of 3 as compared to 1 was the presence of additional two
methoxyl groups at dH 3.89 (s, dC 55.2) and 3.91 (s, dC 55.9). Thus
it appeared that compound 3 was the dimethoxy derivative of 1.
The site of O-methylation was shown to be at C-7 and C-5 on the
basis of shift reagent test where no bathochromic shift was ob-
served in band II and I upon addition of NaOAc and AlCl3.20 This
was further supported by three bond correlations from O–CH3
(dH 3.89) to C-7 and O–CH3 (dH 3.91) to C-5 in HMBC spectrum
on 3. The additional support for placement of OMe at C-5 came
from the absence of chelated hydroxyl hydroxyl signal in 1H
NMR spectrum between 11 and 13 ppm which we have observed
in case of compounds 1 and 2 (Table 1).
The complete assignment of the 1H and 13C NMR spectra of 3
followed the procedure described for 1 (Table 1). In the corre-
sponding HMBC spectrum, connectivities defining the site of glyco-
sylation and interglycosidic linkages were as detected for 1. Sugar
analysis confirmed that each glycoside comprised of
and
-GlcB residues. Thus structure of 3 was assigned as kaempferol-
5,7-dimethoxy-40-O-b-
-glucopyranosyl-(1?2)- -rhamnopyrano-
syl-(1?6)-b- -glucopyranoside and named as evolvoside E.
The structures of other isolated compounds were characterized
as vitexin (4),25 kaempferol 40-O-b-
-glucopyranosyl-(1?2)-b-
glucopyranoside (5),17 kaempferol 40-O-
-rhamnopyranosyl-
(1?6)-b- -glucop
-glucopyranoside (6),17 kaempferol-3,7-di-O-b-
yranoside (7),26 kaempferol 3-O-b-
-glucopyranosyl-(1?6)-b-
glucopyranoside (8),26 kaempferol 3-O-b-
-glucopyranosyl-7-O-
D L-Rha
-GlcA,
D
D
a-L
3. Conclusion
D
In conclusion, present report describes the isolation and struc-
ture elucidation of the new flavonol glycosides, evolvosides C–E
(1–3) and their anti-stress activity. The compounds 1 and 2 have
shown significant (p <0.01) anti-stress activity by normalizing
hyperglycemia, corticosterone level, creatine kinase and adrenal
hypertrophy. Compounds 3 and 6 also have shown anti-stress activ-
ity but compound 6 had no effect in normalizing hyperglycemia. The
D
D-
a
-L
D
D
D
D
-
-
D
a
L
-rhamnopyranoside (9)27 by direct comparison of NMR data

1120
P. Gupta et al. / Bioorg. Med. Chem. 21 (2013) 1116–1122
compounds 4 and 5 were found to be ineffective in normalizing
these parameters (Table 2). Whilst several thousand compounds
belonging to theflavonol structure class have been isolated, triglyco-
side at C-40 of the flavonol moiety are rare with less than 10 reported
in the literature. The biological activity profiles of compounds 1, 2
and 3 supplied another chemical scaffold for potential anti-stress
drug discovery.
fractions (F-1 to F-9). Further purification of F-8 (4.3 g) over silica
gel column (230–400 mesh, 70 g), using gradients of EtOAc/MeOH
(85:15) to (05:95), afforded total eight pooled fractions (F-10 to F-
17) on the basis of NMR profiles from total thirty-two fractions of
30 mL each. Fraction 16 (0.521 g) was subjected to preparative C18
reversed-phase HPLC (Gemini 5
lm; 21.2 ꢁ 250 mm; 6 mL/min;
40–100% CH3CN/H2O over 60 min) to give evolvoside
C
(1,
57.0 mg), evolvoside D (2, 63.2 mg) and evolvoside E (3, 9.0 mg).
The known compounds 4 (81 mg), 5 (61.2 mg), 6 (52.1 mg), 7
(24.3 mg), 8 (12.5 mg) and 9 (16.1 mg) were purified from fractions
11–14 sequentially using semi-preparative C18 reversed-phase
4. Experimental
4.1. General experimental procedures
HPLC (Gemini 5
l
m; 10.0 ꢁ 250 mm; 4 mL/min; 50–100% CH3CN/
H2O over 60 min) methods. Compounds 4-9 were identified by
comparison of their 1H and 13C NMR spectra with the literature
data.
All NMR spectra were recorded on a Bruker DRX 300 and
400 MHz spectrometer. All chemical shifts (d) were referenced
internally to the residual solvent peak (DMSO-d6: 1H, d 2.5; 13C,
d 39.5 ppm; CD3OD: 1H, d 3.34; 13C, d 49.5 ppm). Short- and
long-range 1H–13C correlations were determined with gradient-
enhanced inverse-detected HSQC and HMBC experiments respec-
tively. Optical rotations were measured on a Perkin-Elmer Model
241 digital polarimeter (c g/100 mL) at 589 nm. UV spectra were
obtained on a Perkin–Elmer Lambda l–15 UV–VIS spectrophotom-
eter. IR spectra were recorded on a Perkin–Elmer Nicolet IR-100
spectrophotometer. The high-resolution mass spectra performed
HRESIMS analysis was carried out on Jeol-MS 600H instrument.
HPLC purifications were performed on Beckman System Gold HPLC
system with a 168 UV detector. GC–MS analysis was done using a
Shimadzu GC-14A unit coupled with a GCMS-QP 2000 instrument.
Column chromatography was performed using silica gel (60—120
and 230—400 mesh); TLC: pre-coated silica gel plates 60 F254 or
RP-18 F254 plates with 0.5 or 1 mm film thickness (Merck). Spots
were visualized by UV light or by spraying with 1:1 H2SO4/MeOH.
4.4. Sugar analysis
Acid hydrolysis of 1–9 (0.5–2.0 mg) was carried out by standard
procedures (0.5 mL 2 M HCl, 100 °C, 1.5 h). After cooling, particu-
lates were spun down using microcentrifugation and the superna-
tants were removed. Each reaction mixture was extracted with
EtOAc. The aqueous fractions (sugars) were concentrated to dryness
for the identification sugar residue on TLC plates comparing with
standard sugar samples. The absolute configurations of monosac-
charides released by acid hydrolysis of compounds 5, 7–8 were
determined by observed optical rotation and found comparable to
published report.21a The absolute configurations of the constituent
monosaccharides, L-rahmnoseand D-glucose released by acid hydro-
lysis of compounds1–3, 6 and 9 were determined from GC–MS anal-
ysis of trimethylsilylated thiazolidine derivatives, which were
prepared using the standard method.21b Conditions for GC were:
capillary column, DB5-MS (30 m ꢁ 0.25 mm ꢁ 0.25 m), oven temp.
programme, 180–300 °C at 6 °C/min; injection temp, 350 °C; carrier
gas, He at 1 ml/min. The acid hydrolysates of 1–3, 6 and 9 each gave
4.2. Plant material
The plant material was collected from district 24-Parganas,
West Bengal (state of India) in the month of September 2002 and
identified as Evolvulus alsinoides (LINN.) by Botany Division of Cen-
tral Drug Research Institute (CDRI) and preserved with voucher
specimen number 2659 in the herbarium.
L-rhamnose and D-glucose, at tR = 10.2 and 12.3, respectively
(identical to authentic samples purchased from Sigma–Aldrich
Co.). Compound 4 did not show the hydrolysis product.
4.5. Acetylation of compounds (1–9)
4.3. Extraction and purification procedures
Compounds 1–9 (0.2–0.5 mg) were dissolved separately in dry
pyridine (0.5 mL) and acetic anhydride (1 mL) was added. Reaction
mixture was left overnight at room temperature. The reaction mix-
ture was dried under reduced pressure and under a stream of N2 to
yield amorphous solids (1a–9a). The ESI-MS data of compounds (
1a–9a) were taken to confirm the number of free hydroxyl groups
(Supplementary data).
Powdered Evolvulus alsinoides whole plant (17 kg) was ex-
tracted with ethanol (each 30 L for 24 ꢁ 5) at room temperature.
The resulting extracts were combined and concentrated under re-
duced pressure using rotavapor at 40 °C, to give a dark green ex-
tract (1.19 kg), which was suspended in distilled water (800 mL)
and partitioned with CHCl3 (1000 mL ꢁ 7). The CHCl3 soluble ex-
tract was concentrated under vacuum using a rotavapor at 40 °C
and yielded 380.3 g residue. Water-soluble fraction was further ex-
tracted with n-BuOH saturated with water (1000 ml ꢁ 5). The n-
BuOH and water-soluble fractions were concentrated under re-
duced pressure using a rotavapor at 50 °C, and yielded 260.0 and
530.2 g of crude residue respectively. All the extracts were stored
in refrigerator till further purification. A portion of n-BuOH soluble
fraction (60 g) was loaded on polymeric HP-20 resin using cyclic
loading method.18 The HP-20 column was then eluted with
400 mL fractions of (1) 20% Me2CO/H2O (2) 40% Me2CO/H2O (3)
60% Me2CO/H2O (4) 80% Me2CO/H2O and finally with Me2CO. The
20% Me2CO/H2O fraction (18.3 g) was then subjected to column
chromatography (CC) over silica gel (230–400 mesh, 200.0 g) and
eluted with a gradient of chloroform: methanol (95:05) to metha-
nol: water (95:05) sequentially. Seventy three fractions (50 mL
each) were sampled and their compositions were monitored by
TLC, with those showing similar TLC profiles grouped into nine
4.6. Evolvoside C (1)
Hygroscopic yellow solid; ½a D25
ꢀ32.0 (c 0.02, methanol); IR
ꢂ
(KBr) mmax 3432, 2933, 1667, 1590, 1461, 1399 cmꢀ1; UV (MeOH)
kmax 364 (e 2114), 261 (e
1204) nm; 1H (400 MHz) and 13C NMR
(100 MHz) see Table 1. HRESIMS m/z 779.2023 [M+Na]+ (Calcd
for C33H40O20Na, 779.2011).
4.7. Evolvoside D (2)
Hygroscopic Yellow solid; ½a D25
ꢀ51.2 (c 0.04, methanol); IR
ꢂ
(KBr) mmax 3451, 2941, 1671, 1581, 1450, 1382 cmꢀ1; UV (MeOH)
kmax 362 (
(100 MHz) see Table 1. HRESIMS m/z 793.2206 [M+Na]+ (calcd for
34H42O20Na, 793.2197).
e 3198), 267 (e
2041) nm; 1H (400 MHz) and 13C NMR
C

P. Gupta et al. / Bioorg. Med. Chem. 21 (2013) 1116–1122
1121
4.8. Evolvoside E (3)
4.9.7. Statistical analysis
Mean and SEM. were calculated. The data was analyzed using
one-way analysis of variance (ANOVA) followed by Student–New-
man–Keul’s multiple comparison test. Data of ulcer was analyzed
by non-parametric ANOVA followed by Dunn’s multiple compari-
son tests. p <0.05 was considered to be statistically significant.
Hygroscopic Yellow solid; ½a D25
ꢀ41.2 (c 0.03, Methanol); IR
ꢂ
(KBr) mmax 3440, 2945, 1678, 1586, 1458, 1391 cmꢀ1; UV (MeOH)
kmax 365 (
(100 MHz) see Table 1. HRESIMS m/z 807.2324 [M+Na]+ (calcd for
35H44O20Na, 807.2311).
e 5891), 271 (e
2345) nm; 1H (400 MHz) and 13C NMR
C
Acknowledgments
4.9. Anti-stress activity
This research was initially supported by the grant from Depart-
ment of Biotechnology and Indian Council of Medicinal Research,
New Delhi, India. P. Gupta is thankful to Dr. L. West, FAU for pro-
viding lab and NMR facilities to perform part of this research work
during the postdoctoral stay in his lab which was funded through
NIH grant. We are also thankful to Botany division, CDRI for collec-
tion of plant material and S. C. Tiwari for the extraction of plant
material.
4.9.1. Animals
Adult male Sprague–Dawley rats (180—200 g) were kept in
raised mesh bottom cages to prevent coprophagy in environmen-
tally controlled rooms (25 2 °C, 12 h light and dark cycle), ani-
mals had free access to standard pellet chow and drinking water
except during experiments. Experiments were conducted between
09:00 and 14:00 h. Experimental protocols were approved by our
institutional ethical committee following the guidelines of CPCSEA
(Committee for the Purpose of Control and Supervision of Experi-
ments on Animals), which complies with international norms of
INSA (Indian National Science Academy).
Supplementary data
Supplementary data (HMBC data for evolvosides D and E (2 and
3) together with structure and MS data of compounds 1a–9a) asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2012.12.040.
4.9.2. Administration of drug
Suspension of pure compounds in 0.1% sodium carboxy methyl
cellulose were prepared and administered by oral gavage using a
ball ended feeding needle at a dose of 40 mg/kg, once daily for
3 d in case of acute stress (AS). Drug was prepared fresh daily be-
fore administration. A freshly prepared aqueous suspension of
crude powder of ginseng root Panax quinquifolium was used as a
standard at a dose of 100 mg/kg body weight and purchased from
Sigma, USA.
References and notes
1. Wealth of India, Raw material: Publication and Information Directorate, CSIR,
New Delhi, India, 1952; Vol. III, p 233.
2. Chopra, R. N.; Nayer, S. L.; Chopra, I. C. Glossary of Medicinal Plants; Publication
and Information Directorate: CSIR: New Delhi, India, 1986. p 116.
3. Kirtikar, K. R.; Basu, B. D. In Indian Medicinal Plants; International Book
Distributors: Dehradun, India, 1933; Vol. III, p 1738.
4. Chatterjee, A. Treaties of Indian Medicinal Plants; Council for Scientific and
Industrial Research: New Delhi, India, 1990. p 327.
4.9.3. Stress protocol
The rats were divided into control non-stress group, AS group,
and drug-treated groups. Each group consists of 6 rats. A parallel
group of rats were fed with vehicle for the same number of treat-
ment days but were not immobilized and they were used as non-
stress control group to obtain baseline data for various parameters.
On the second day after feeding drug or vehicle, animals were
fasted overnight with free access to water. On the third day,
45 min after feeding the drug or vehicle, rats were stressed except
the non stress group. AS was produced by immobilizing animals for
150 min once only and sacrificed immediately by cervical disloca-
tion. Briefly, immobilization stress was produced by restraining
each naive animal inside an acrylic hemi cylindrical plastic tube
(4.5 cm diameter, 12 cm long) for 150 min.34
5. Nadkarni, K. M.; Nadkarani, A. K. In Indian Material Medica; Popular Prakashan:
Bombay, 1982; Vol. I, p 531.
6. Auddy, B.; Ferreira, M.; Blasina, F.; Lafon, F.; Arredondo, F.; Dajas, F.; Tripathi, P.
C.; Seal, T.; Mukherjee, B. J. Ethnopharmacol. 2003, 84, 131.
7. Asolkar, L. V.; Kakkar, K. K.; Chakre, O. J. Second Supplement to Glossary of Indian
Medicinal Plant with Active Principles; NISCAIR: New Delhi, India, 1992. p 1965.
8. Ganju, L.; Karan, D.; Chandra, S.; Srivastava, K. K.; Sawhney, R. C.; Selvamurthy,
W. Biomed. Pharmacother. 2003, 57, 296.
9. (a) Mehta, C. R.; Shah, N. B. Sci. Cult. 1958, 24, 180; (b) Baveja, S. K.; Singala, P. D.
Indian J. Pharmacol. 1969, 31, 108.
10. (a) Gopalkrishna, P. N. J. Res. Ind. Med. Yoga Homeo. 1978, 11, 4; (b) Cervenka, F.;
Koeckar, V.; Rehakova, Z.; Jahodar, L.; Kunes, J.; Opletal, L.; Hyspler, R.; Jun, D.;
Kuca, K. J. Enzyme Inhib. Med. Chem. 2008, 23, 574.
11. Guruswami, M. N.; Krishnamurthy, T. R. Curr. Sci. 1956, 25, 119.
12. Akthar, S. M.; Kaskoos, R. A.; Mir, S. R.; Ali, M. J. Saudi Chem. Soc. 2009, 13, 191.
13. Siripurapu, K. B.; Gupta, P.; Bhatia, G.; Maurya, R.; Nath, C.; Palit, G. Pharmacol.
Biochem. Behav. 2005, 81, 424.
14. Gupta, P.; Yadav, D. K.; Siripurapu, K. B.; Palit, G.; Maurya, R. J. Nat. Prod. 2007,
70, 1410.
15. Chatterjee, M.; Verma, P.; Maurya, R.; Guatam, P. Pharma. Biol. 2011, 49, 477.
16. Gupta, P.; Akanksha; Siripurapu, K. B.; Ahmad, A.; Palit, G.; Arora, A.; Maurya, R.
Chem. Pharma. Bull. 2007, 55, 771.
17. Kumar, M.; Ahmad, A.; Rawat, P.; Khan, F. M.; Rasheed, N.; Gupta, P.;
Sathiamoorthy, B.; Bhatia, G.; Palit, G.; Maurya, R. Fitoterapia 2010, 81, 234.
18. Gupta, P.; Sharma, U.; Schulz, T.; Sherrer, E.; McLean, A.; Robins, A.; West, L.
Org. Lett. 2011, 13, 3920.
4.9.4. Biochemical estimations
The blood was collected in EDTA coated tubes, through cardiac
puncture after the stress regime and centrifuged at 2000
rpm ꢁ 20 min at 4 °C and plasma was separated. The plasma was
used to estimate corticosterone, glucose and creatine kinase (CK).
19. Mabry, T. J.; Markham, K. R.; Thomas, M. B. The Systematic Identification of
Flavonoids; Springer: New York, 1970. p 87.
4.9.5. Estimation of glucose and CK
Auto analyzer (Synchron Cx-5, Beckman) was used to estimate
glucose and CK with their respective kits (Beckman Coulter Inter-
national, Nyon, Switzerland).
20. Delaveau, P. G.; Fromageot, F. Phytochemistry 1963, 2, 237.
21. (a) Du, L. C.; Wu, B. L.; Chem, J. M. Chin. Chem. Lett. 2008, 19, 1315–1318; (b) Ito,
A.; Chai, H. B.; Kardono, L. B. S.; Setowati, F. M.; Afriastini, J. J.; Riswan, S.;
Farnsworth, N. R.; Cordell, G. A.; Pezzuto, J. M.; Swanson, S. M.; Kinghorn, A. D.
J. Nat. Prod. 2004, 67, 201.
22. Nyberg, N. T.; Duus, J. O.; Sorensen, O. W. J. Am. Chem. Soc. 2005, 127, 6154.
23. Petersen, B. O.; Vinogradov, E.; Kay, W.; Würtz, P.; Nyberg, N. T.; Duus, J. Ø.;
Sørensen, O. W. Carbohydr. Res. 2006, 341, 550.
4.9.6. Estimation of corticosterone
An HPLC/UV system (Waters, USA) was used for quantification
of plasma corticosterone by the method of Woodword and Emery
with modifications.35 Dexamethasone was used an internal stan-
dard. The mobile phase consisted of methanol/water (70:30) at a
flow rate of 1.2 ml/min and corticosterone was detected at
250 nm using UV detector. The chromatogram was recorded and
analyzed with Breeze software (3.20 version).
24. Agrawal, P. K. Phytochemistry 1992, 31, 3307.
25. Choo, C. Y.; Sulong, N. Y.; Man, F.; Wong, T. W. J. Ethnopharmacol. 2012, 1, 776.
26. (a) Vermes, B.; Farkas, L.; Nogradi, M.; Wagner, H.; Dirscherl, R. Phytochemistry
1976, 15, 1320; (b) Aboutable, E. A.; Meselhy, M. R.; Afifi, M. S. Pharmazie 2002,
57, 646.
27. (a) Song, N.; Xu, Wei; Hongfeng, G.; Liu, X.; Wang, Y.; Nie, X. Asian J. Trad. Med.
2007, 2, 218–222; (b) Ouyang, M. A. Chin. Trad. Herb. Drugs 2003, 34, 196.

1122
P. Gupta et al. / Bioorg. Med. Chem. 21 (2013) 1116–1122
28. Tannebaum, B.; Tannenbaum, G. S.; Sudom, K.; Anisman, H. N. Brain Res. 2002,
953, 82.
31. Adelbert, A. Brain Res. Rev. 2000, 34, 24.
32. Davydov, V. V.; Shvets, V. N. Gerontology 2002, 48, 81.
29. Glavin, G. B.; Murison, R.; Overmier, J. B.; Pare, W. P.; Bakke, H. K.; Henke, P. G.
Brain Res. Rev. 1991, 16, 301.
33. Dhabar, F. S.; McEven, B. S.; Spencer, R. L. Neuroendocrinology 1997, 65, 360.
34. Makara, G. B.; Haller, J. Prog. Neurobiol. 2001, 65, 367.
30. Mason, J. W. A. Psychosom. Med. 1968, 30, 576.
35. Rai, D.; Bhatia, G.; Sen, T.; Palit, G. Can. J. Physiol. Pharmacol. 2003, 81, 1139.