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Physiological adaptation during stressful conditions is de-
fined as a biochemical change in an organism that results
from exposure to certain environmental conditions or stres-
sors and generates a more effective response to their survival.
Long-term stress can induce a range of disorders like hyper-
tension, coronary heart disease,
1)
gastric ulcers,
2)
immuno-
suppression,
3)
metabolic disorders like diabetes,
4)
reproduc-
tive dysfunction,
5)
mental depression, memory loss and host
of other diseases.
6)
Due to the nonspecific nature of the stress
pathogenesis, a drug having central and peripheral activity is
needed to combat stressful conditions. Since ancient times
therapeutic approach to combat stress has involved utiliza-
tion of substances from natural origin. Pharmacological in-
vestigations have shown that the basic effect of Panax gin-
seng, Elutherococcus senticosus and Rhodiola rosea, is their
ability to increase non-specific resistance of the organism to
various untoward influences.
7)
Initial studies on plants origi-
nating from folk medicine along with an exponential increase
in knowledge regarding the interactions among components
of the stress system have encouraged various investigators to
evaluate the potential of plant derived anti-stress agents for
usage in modern day medicine. Further enrichment of the
study on plant derived adaptogens was enabled by the sub-
stantial work carried out on plants such as Ocimum sanc-
tum,
8)
Emblica officinalis,
9)
Bacopa monniera,
10)
Ginkgo
biloba
11)
and Withania somnifera.
12)
In our continuing efforts
to identify biologically active secondary metabolites, we
have initiated our study on Evolvulus alsinoides to identify
anti-stress agents.
Evolvulus alsinoides LINN. (Family: Convolvulaceae) com-
monly known as Shankhpuspi in India. It is an important me-
dicinal plant that grows in the open and grassy places almost
throughout the India and subtropical countries of the
world.
13,14)
Plant extracts have been used in traditional medi-
cine for treatment of bronchitis, asthma
14,15)
and brain disor-
ders like insanity, epilepsy, nervous disability, and scrof-
ula.
14—17)
Evolvulus alsinoides is well known for its memory
enhancing property in traditional Indian system of medicine
and extensively commercialized as nervin tonic in Asian
countries. Evolvulus alsinoides extracts have exhibited an-
tioxidant,
18)
anti-ulcer,
19)
and immunomodulatory activi-
ties.
20)
Early phytochemical studies of this species resulted in
the isolation and identification of chemical constituents such
as triacontane, pentatriacontane,
b
-sitosterol and two alka-
loids betaine and shankpushpin.
21—23)
Four unidentified alka-
loids A, B, C and evolvine have also been described in litera-
ture.
21,24)
In the view of its importance in traditional medici-
nal system, no substantial phytochemical and pharmacologi-
cal works have been reported. In addition, recently we have
disclosed adaptogenic and anti-amnesic properties of ethano-
lic extract of Evolvulus alsinoides.
25)
Further phytochemical
investigation led to the isolation of two new compounds
evolvoids A (1) and B (2) along with six known compounds
(3—8). The structures of isolated compounds were eluci-
dated on the basis of extensive spectroscopic analysis. The
anti-stress activity of compounds 1—5 and 8 were evaluated
in acute stress induced biochemical changes in adult male
Sprague–Dawley rats. In the present study, we have described
isolation, structure elucidation and anti-stress activity of
these compounds.
Results and Discussion
Following the extraction of whole plant of Evolvulus alsi-
noides with ethanol and successive partitioning of the dried
extract in H
2
O/CHCl
3
, H
2
O/n-BuOH, significant anti-stress
activity of the n-BuOH fraction was observed. However,
CHCl
3
soluble fraction showed insignificant anti-stress activ-
ity while aqueous fraction was found to be inactive. The ac-
Anti-stress Constituents of Evolvulus alsinoides: An Ayurvedic Crude
Drug
Prasoon GUPTA,
a
AKANKSHA,
a
Kiran Babu SIRIPURAPU,
b
Ausaf AHMAD,
b
Gautam PALIT,
b
Ashish ARORA,
c
and Rakesh MAURYA
*
,a
a
Division of Medicinal and Process Chemistry, Central Drug Research Institute;
b
Division of Pharmacology, Central
Drug Research Institute; and
c
Division of Molecular and Structural Biology, Central Drug Research Institute; Lucknow–
226 001, India. Received January 15, 2007; accepted February 10, 2007
Bioactivity-guided purification of n-BuOH soluble fraction from the ethanol extract of Evolvulus alsinoides
resulted in the isolation of two new compounds, 2,3,4-trihydroxy-3-methylbutyl 3-[3-hydroxy-4-(2,3,4-trihydroxy-
2-methylbutoxy)-phenyl]-2-propenoate (1) and 1,3-di-O-caffeoyl quinic acid methyl ester (2) along with six
known compounds, caffeic acid (3), 6-methoxy-7-O-
bb
-glucopyranoside coumarin (4), 2-C-methyl erythritol (5),
kaempferol-7-O-
bb
-glucopyranoside (6), kaempferol-3-O-
bb
-glucopyranoside (7) and quecetine-3-O-
bb
-glucopyra-
noside (8). The structure of new compounds 1 and 2 were elucidated by spectroscopic analysis, while known com-
pounds were confirmed by direct comparison of their NMR data with those reported in literature. This is the
first report of the presence of phenolic constituents in Evolvulus alsinoides. The isolated compounds 1—5 and 8
were screened for anti-stress activity in acute stress induced biochemical changes in adult male Sprague–Dawley
rats. Stress exposure has resulted in significant increase of plasma glucose, adrenal gland weight, plasma creatine
kinase (CK), and corticosterone levels. Compound 1 displayed most promising antistress effect by normalizing
hyperglycemia, plasma corticosterone, CK and adrenal hypertrophy, while compounds 2 and 3 were also effec-
tive in normalizing most of these stress parameters, however compounds 4, 5 and 8 were ineffective in normaliz-
ing these parameters.
Key words Evolvulus alsinoides; Shankhpuspi; Convolvulaceae; phenyl propanoid; flavonol glycoside; quinic acid; erythritol
May 2007 771Chem. Pharm. Bull. 55(5) 771—775 (2007)
© 2007 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: mauryarakesh@rediffmail.com
tive fraction was then subjected to sequence of normal- and
reverse-phase column chromatography, to yield two new
compounds 1 and 2 along with six known compounds. The
structure of new compounds were established using chemical
and spectroscopic (FAB-MS,
1
H-NMR,
13
C-NMR, COSY,
HSQC, HMBC) studies.
Compound 1 was obtained as yellow solid, possessing ele-
mental composition C
19
H
28
O
10
as concluded from ESI-MS
pos. spectrum (m/z at 417 [MH]
) and HR-ESI-MS
416.1709 (Calcd 416.1683). The IR spectrum of 1, displayed
absorption bands for
a
-
b
unsaturated CO (1670 cm
1
),
aromatic ring (1483, 1341 cm
1
) and polyhydroxyl group at
(3340 cm
1
). The UV absorption bands at (324, 239, 213 nm)
were characteristic of caffeoyl unit.
26)
The
1
H- and
13
C-NMR
spectra (Table 1) displayed chemical shift pattern of caffeoyl
moiety, showed olefinic protons at
d
H
7.61 (d, J15.8 Hz, H-
3;
d
C
147.0) and 6.31 (d, J15.8 Hz, H-2;
d
C
115.1). The
presence of three proton signals at
d
H
7.06 (br s, H-5), 6.97
(d, J8.2 Hz, H-9) and 6.79 (d, J8.2 Hz, H-8) in the
1
H-
NMR spectrum, and resonance of typical carbons at
d
C
127.1
(C-4), 115.1 (C-5), 149.6 (C-6), 147.1 (C-7), 116.4 (C-8) and
122.9 (C-9) including
a
-
b
unsaturated carbonyl carbon at
169.5 (C-1) indicated presence of 6,7-disubtituted caffeoyl
moiety in the molecule. In addition, the existence of two tet-
rose sugars (erythritol) were inferred from the
1
H- and
13
C-
NMR spectrum,
27)
which showed presence of four oxy-
genated methylenes, two methines, two methyls and two oxy-
genated quaternary carbons. Acid/base hydrolysis of 1 af-
forded erythritol sequentially, which was confirmed by co-
TLC with authentic sample 5 isolated from same plant. The
1
H- and
13
C-NMR signals of both erythritol units were as-
signed using
1
H,
13
C, HSQC, HMBC and
1
H–
1
H COSY
NMR spectral data. The first erythritol unit was resonated as
ABX and AB system at
d
H
4.53 (dd, J11.2, 6.0 Hz, H-1a),
4.20 (dd, J11.2, 2.4 Hz, H-1b), 3.87 (dd, J6.0, 2.4 Hz, H-
2) and
d
H
3.49 (d, J11.0 Hz, H-4a), 3.63 (d, J11.0 Hz,
H-4b) and corresponding carbon signals were appeared at
d
C
67.0 (C-1), 73.6 (C-2), 68.4 (C-4) including quaternary
carbon at
d
C
77.9 (C-3). The methyl signal was assigned at
d
H
1.20 (s, H-5;
d
C
19.2).
1
H- and
13
C-NMR spectrum ex-
hibited second erythritol unit at
d
H
4.25 (d, J11.4 Hz, H-
1a), 4.14 (d, J11.4 Hz, H-1b);
d
C
70.0 (C-1) as AB spin
system and
d
H
3.65 (br d, J6.8 Hz, H-3);
d
C
75.6 (C-3),
3.91 (dd, J11.2, 6.8 Hz, H-4a), 3.61 (br d, J11.2 Hz, H-
4b);
d
C
63.6 as ABX spin system, whereas methyl signal
was assigned at
d
H
1.17 (s, H-5);
d
C
19.3 (C-5). The qua-
ternary carbon resonated at
d
C
74.6 (C-2). HMBC spectrum
was utilized to identify the position of attachment (Fig. 2); it
gave useful correlation between H
a,b
-1/C-1, 2, 3; H-2/C-
1, 4, 5; H
a,b
-4/C-5, 2 and H
a,b
-1/C-7, 2, 3, 5; H
a,b
-
4/C-3, 2; H-5/C-1, 2, 3 indicated that first eythritol unit
was attached to acid carbonyl (C-1) with ester linkage and
second unit was attached to aromatic hydroxyl group (C-7)
with ether linkage. The D-configuration of erythritol was de-
termined by optical rotation [
a
]
D
29
24.5° (c0.02, H
2
O) and
supported with the data available in literature.
27)
Thus, com-
pound 1 was characterized as a 2,3,4-trihydroxy-3-methyl-
butyl 3-[3-hydroxy-4-(2,3,4-trihydroxy-2-methyl-butoxy)-
phenyl]-2-propenoate, a new compound named evolvoid A.
Compound 2 was obtained as amorphous powder with few
complex impurities. Our repeated effort to obtain pure com-
pound by column chromatography was unsuccessful. IR
spectrum exhibited absorption bands for aromatic ring (1604,
1514 cm
1
),
a
-
b
unsaturated carbonyl (1672 cm
1
) and ester
(1737 cm
1
) function in the molecule. The
1
H-NMR of 2
(impure sample) showed presence of caffeoyl unit, ester
methyl and few aliphatic signal although acetyl signal were
absent in the spectrum. Acetylation followed by chromatog-
raphy led to isolation of pure compound 2a as polyacetate.
However in nature this exists as non-acetylated compound.
Compound 2a exhibited molecular ion peak m/z at 783
[MH]
in ESI-MS spectrum, corresponding to molecu-
lar formula C
38
H
38
O
18
suggested by HR-ESI-MS 782.2073
(Calcd 782.2058).
Analysis of
1
H- and
13
C-NMR data guided by COSY spec-
trum (Table 2) displayed presence of two overlapped caffeoyl
772 Vol. 55, No. 5
Fig. 1. Structural Formula of Isolated Compounds (1—8)
Table1. NMR Spectroscopic Data for Compound 1
1 (CD
3
OD)
a)
Position
d
H
(mult., J in Hz)
d
C
COSY HMBC
1— 169.5 —
2 6.31 (d, 15.8) 115.1 3 C-1, 4
3 7.61 (d, 15.8) 147.0 2 C-1, 5, 9
4— 127.1 — —
5 7.06 (br s) 115.1 — C-3, 7, 9
6— 149.6 — —
7— 147.1 — —
8 6.79 (d, 8.2) 116.4 9 C-4, 6, 9
9 6.97 (d, 8.2) 122.9 8 C-3, 5, 7, 8
1a 4.53 (dd, 11.2, 6.0) 67.0 1b, 2 C-1, 3
1b 4.20 (dd, 11.2, 2.4) — 1a, 2 C-1, 3
2 3.87 (dd, 6.0, 2.4) 73.6 1a, 1b C-4, 5
3 — 77.9 — —
4a 3.49 (d, 11.0) 68.4 4b C-2, 3, 5
4b 3.63 (d, 11.0) — 4a C-2, 5
5 1.20 (s) 19.2 — C-2, 4
1a 4.25 (d, 11.4) 70.0 1bC-7, 3, 5
1b 4.14 (d, 11.4) — 1aC-7, 3, 5
2 — 74.6 — —
3 3.65 (br d, 6.8) 75.6 4a C-1, 4, 5
4a 3.91 (dd, 11.2, 6.8) 63.6 3, 4b C-2, 3
4b 3.61 (br d, 11.2) — 4a C-2
5 1.17 (s) 19.3 — C-2, 4
a)
1
H-NMR: 200 MHz,
13
C-NMR: 300 MHz, 2D NMR: 600 MHz (TMS as internal
standard), chemical shifts, multiplicity and coupling constants (J, Hz) were assigned by
means of
1
H,
13
C and 2D NMR data.
units and an oxygenated cyclohexyl ring. On the basis of
HMBC, HSQC and COSY spectral data each signals of cy-
clohexyl ring were assigned, three mutual coupled acetylated
methine signals at
d
H
5.28 (ddd, J3.3, 7.1, 9.2 Hz, H-3;
d
C
68.4), 5.67 (dd, J7.2, 9.2 Hz, H-4;
d
C
70.0), 5.60 (ddd,
J4.5, 5.4, 7.2 Hz, H-5;
d
C
71.2), two methylenes at
d
H
2.26,
2.63 (m, H-2a,b;
d
C
37.7) and
d
H
2.67, 2.78 (m, H-6a,b;
d
C
37.5), separately coupling with H-3 and H-5 protons. An
oxygenated quaternary carbon resonated at
d
C
80.4 (C-1).
Additional signal in NMR spectrum at
d
C
172.3 (CO),
gave HMBC correlations with H-2, 6 and methyl group at
d
H
3.73 (s,
d
C
53.4). Other important HMBC correlations, H-
2/C-3, 4, 6; H-3/C-1, 5; H-4/C-2, 6 and H-5/C-1, 3, 4 con-
firmed cyclohexyl ring as quinic acid methyl ester (Fig. 2).
The large coupling values of H-3, 4, 5 protons clearly indi-
cated their axial orientation in the ring. The J values of sub-
stituted cyclohexane has shown to be in the range of ca.
10 Hz for axial–axial, ca. 5 Hz for axial–equatorial and ca.
2—3 Hz for equatorial–equatorial.
28)
The J values were
slightly lower than would be expected for a perfect chair con-
formation, therefore, quinic acid moiety of 2a may exist in a
skewed/boat form. The stereochemistry at C-1 could not be
decided by the above method, therefore, relative stereochem-
istry deduced by the NOESY experiments (Fig. 2). Since
axial proton H-3 and H-5 gave diagnostic NOE cross peaks
with CO
2
CH
3
, indicated their axial orientation in the ring.
Such correlation is possible only if the orientation of C-1 OH
group is equatorial. Other diagnostic cross peaks (Fig. 2)
confirmed the relative stereochemistry of cyclohexyl ring in
2a. Presence of two caffeoyl units confirmed by two set of
olefinic protons at
d
H
7.69 (d, J15.9 Hz, H-3); 6.51 (d,
J15.9 Hz, H-2) and
d
H
7.74 (d, J15.9 Hz, H-3); 6.57 (d,
J15.9 Hz, H-2). The J values 15.9 Hz confirmed both are
the trans isomers. Other ABX aromatic protons of caffeoyl
units were overlapped and assigned respectively (Table 2).
One caffeoyl unit must be present at C-3 with ester linkage,
as evident from HMBC cross peak between H-3/C-1 (167.2)
but no cross peak observed for second caffeoyl unit indicated
that it must be attached to C-1 (OH). This was also supported
by HMBC correlation of H-4 and H-5 with acetyl carbonyl
d
C
172.3 and 171.3 (due to acetylation, Fig. 2). Thus, above
data confirmed the structure of evolvoid B hexaacetate repre-
sented by structure 2a and the corresponding parent structure
by 2, which has not been previously reported.
The known compounds 3—8 were characterized as 3-(3,4-
dihydroxyphenyl)-2-propenoic acid (caffeic acid, 3),
29)
6-
methoxy-7-O-
b
-glucopyranoside coumarin (4),
30)
2-C-methyl
erythritol (5),
27)
kaempferol-7-O-
b
-glucopyranoside (6),
31)
kaempferol-3-O-
b
-glucopyranoside (7),
32)
and quecetine-3-
O-
b
-glucopyranoside (8),
33)
by direct comparison of NMR
data with those reported in literature. All the compounds
were isolated first time from this plant.
Isolated compounds 1—5 and 8 were screened for anti-
stress activity in acute stress (AS) model at the dose of
40 mg/kg body weight, as shown in Table 3. A significant in-
crease (p0.001) in the adrenal gland weight after AS was
observed when compared to non-stressed (NS) group. Com-
pound 1 and standard drug powder of the roots of Panax
quinquifolium (PQ) were significantly ( p0.01) effective in
reducing the stress induced adrenal hypertrophy. The plasma
creatine kinase (CK) and glucose levels were also increased
by AS significantly ( p0.001) when compared to NS con-
May 2007 773
Table2. NMR Spectroscopic Data for Compound 2a
2a (CD
3
OD)
a)
Position
d
H
(mult., J in Hz)
d
C
COSY HMBC
1— 80.4
2a 2.63 (m) 37.7 2b, 3 C-3, 4, 6, 7
2b 2.26 (m) — 2a, 3 —
3 5.28 (ddd, 3.3, 7.1, 9.2) 68.4 2a, 2b, 4 C-1, 5, 1
4 5.67 (dd, 7.2, 9.2) 70.0 3, 5 C-2, 6
5 5.60 (ddd, 4.5, 5.4, 7.2) 71.2 4, 6a, 6b C-1, 3, 4
6a 2.67 (m) 37.5 5, 6b C-1, 2, 4, 7
6b 2.78 (m) — 5, 6a —
7— 172.3 — —
CO
2
CH
3
3.73 (s) 53.4 C-7
1 — 167.1
d)
——
2 6.51 (d, 15.9) 119.5
e)
3 1, 4
3 7.69 (d, 15.9) 144.2 2 1, 5, 9
4 — 134.4 — —
5 7.58 (br s)
b)
124.4 — 3, 7, 9
6 — 147.3 — —
7 — 147.7 — —
8 7.27 (d, 8.7)
c)
125.3 9 4, 6
9 7.58 (d, 8.7)
b)
127.8 8 3, 5, 7
1 — 167.2
d)
—
2 6.57 (d, 15.9) 119.6
e)
3 1, 4
3 7.74 (d, 15.9) 145.5 2 1, 5, 9
4 — 134.4 — —
5 7.58 (br s)
b)
124.4 — 3, 7, 9
6 — 147.3 — —
7 — 147.7 — —
8 7.29 (d, 8.7)
c)
125.3 9 4, 6, 9
9 7.58 (d, 8.7)
b)
127.8 8 3, 5, 7
Ar-OCOCH
3
2.30 (s), 2.31 (s) 171.6,
172.3 —
Al-OCOCH
3
2.00 (s), 2.07 (s) 169.7,
169.9 —
a)
1
H-,
13
C- and 2D NMR: 300 MHz, chemical shifts, multiplicity and coupling con-
stants (J, Hz) were assigned by means of
1
H-,
13
C- and 2D NMR data. b)Overlapped
signals, c—e) interchangeable within the row.
Fig. 2. Key HMBC (→) and NOESY (---) Correlation of Compounds 1 and 2a
trol. Pretreatment with compounds 1, 2 (p0.01) and PQ
(p0.01) were effective in reducing the AS-induced increase
in CK levels and compounds 1, 2 and 3 in reducing increased
glucose levels. Similarly AS (p0.001) exposure resulted in
increased plasma corticosterone when compared to NS con-
trol. Pretreatment with compounds 1, 2 and 3 (p0.01) and
PQ (p0.01) significantly reduced the increase in corticos-
terone levels.
In conclusion, compound 1 has shown significant
(p0.01) anti-stress activity by normalizing hyperglycemia,
corticosterone level, creatine kinase and adrenal hypertrophy.
Compound 2 also shown anti-stress activity but having no ef-
fect on adrenal hypertrophy similar is the case with com-
pound 3, which was found to be effective in some of the
stress parameters but showing no effect on adrenal hypertro-
phy and increased CK levels. The compounds 4, 5 and 8
were found to be ineffective in normalizing these parameters
(Table 3). The biological activity profiles of compounds 1, 2
and 3 are worthy for further investigation to develop anti-
stress drug.
Experimental
General Procedures Melting points (uncorr.) were recorded on a Com-
plab melting point apparatus. IR spectra were recorded on a Perkin-Elmer
RX-1 spectrophotometer using either KBr pallets or in neat. UV spectra
were obtained on a Perkin Elmer
l
-15 UV spectrophotometer, optical rota-
tions were measured on a Perkin-Elmer Model 241 digital polarimeter.
1
H-
and
13
C-NMR spectra were recorded on an Avance DPX-200 and Bruker
DRX 300 MHz spectrometer and chemical shift are expressed as
d
(ppm)
values. The abbreviations of
1
H-NMR signals pattern are as follows: s, sin-
glet; d, doublet; dd, double doublet; ddd, doublet of doublet of doublet; t,
triplet; m, multiplate. Proton detected heteronuclear correlations were meas-
ured using HSQC and HMBC. 2D spectrums were recorded on Varian
Inova-600 MHz NMR and Bruker DRX 300 MHz spectrometer. FAB-MS
were carried out on Jeol SX 102/DA-6000 mass spectrometer using m-nitro
benzyl alcohol as matrix. ESI-MS spectra were obtained on LCQ Advantage
Max Thermo Finnigan. Elemental analyses were obtained in a Carlo-Erba-
1106 CHN elemental analyzer. HR-ESI-MS analysis was carried out on
Jeol-MS 600H instrument. Preparative HPLC were performed on Shimadzu
CLC-Octa decyl silane RP-18 (ODS) column with (20 mm ID25 cm
length); 8 ml/min flow rate, PDA UV
l
254 and 220 nm as detector. Column
chromatography was performed using silica gel (60—120 and 230—400
mesh); TLC: pre-coated silica gel plates 60 F
254
or RP-18 F
254
plates with
0.5 or 1 mm film thickness (Merck). Spots were visualized by UV light or by
spraying with H
2
SO
4
–MeOH, anisaldehyde–H
2
SO
4
reagents.
Plant Materials The plant material was collected from district 24-Par-
ganas, West Bengal (state of India) in the month of September 2002 and
identified as Evolvulus alsinoides (LINN.) by Botany Division of CDRI and
preserved with voucher specimen number 2659 in the herbarium.
Extraction and Isolation Powdered Evolvulus alsinoides whole plant
(17 kg) was extracted with ethanol (each 30 l for 24 h5) at room tempera-
ture. The resulting extracts were combined and concentrated under reduced
pressure using rotavapor at 40 °C, to give a dark green extract (1.19 kg),
which was suspended in distilled water (800 ml) and partitioned with CHCl
3
(1000 ml7). The CHCl
3
soluble extract was concentrated under vacuum
using rotavapor at 40 °C, yielded 380.3 g residue. Water soluble fraction was
further extracted with n-BuOH saturated with water (1000 ml5). The n-
BuOH and water soluble fractions were concentrated under reduced pressure
using rotavapor at 50 °C, yielded 260.0 g and 530.2 g of crude residue re-
spectively. All the extracts were stored in refrigerator till further purification.
A portion of n-BuOH soluble fraction (200 g) was subjected to column
chromatography (CC) over silica gel (230—400 mesh, 1.8 kg) and eluted
with a gradient of chloroform : methanol (95 : 05) to methanol : water
(95 : 05) sequentially. Seventy three fractions (800 ml each) were sampled
and their composition monitored by TLC, with those showing similar TLC
profiles grouped into nine fractions (F-1 to F-9). Further purification of
F-1 (5.2 g) over silica gel (60—120 mesh, 130 g), using gradient of
EtOAc/MeOH (100 to 80%) afforded 3 (800 mg). Column chromatography
of F-3 (20.0 g) over silica gel (60—120 mesh, 450 g) with gradient of
CHCl
3
/MeOH (95 : 5) to (85 : 15), afforded thirteen pooled fractions (F-10 to
F-22) on the basis of TLC profiles from total sixty two fractions of 500 ml
each. Compound 4 (55 mg) was obtained as amorphous powdered from F-18
at room temperature. Successive purification of mother of F-18 (800 mg)
over silica gel (230—400 mesh, 25 g), eluted with isocratic CHCl
3
/MeOH
(90 : 10) resulted in the isolation of compound 5 (463 mg). Rechromatogra-
phy of F-19 (3.0 g) over silica gel (60—120 mesh, 85 g) using ethyl acetate
saturated with water (isocratic) as mobile phase, afforded F-23 to F-29.
Fraction F-23 (122 mg) containing 2 with few complex impurities were
acetylated using (pyridine/Ac
2
O) followed by CC over silica gel (60—120
mesh, 11 g) eluted with isocratic hexane : EtOAc (85 : 15) afforded 2 in their
acetate form 2a (75 mg). Fraction F-26 containing compound 1 (75 mg) in
minor quantity was purified by preparative HPLC (RP-18), eluted with gra-
dient of H
2
O/MeOH (80—50%). Preparative HPLC (RP-18) of F-27
(120 mg) using gradient H
2
O/MeOH (75—40%) yielded 6 (27 mg). Fraction
F-20 (4.0 g) was rechromatographed over silica gel (60—120 mesh, 70 g),
eluted with EtOAc/Acetone (80—70%) yielded F-30 to F-33 respectively.
Further purification of F-31 (200 mg) by preparative HPLC (RP-18) with
gradient H
2
O/MeOH resulted in purification of compound 7 (30 mg). Com-
pound 8 (70 mg) was eluted from F-21 (285 mg) using same condition as for
F-31.
Acid Hydrolysis of 1 The compound 1 (25 mg, 0.06 mmol) was treated
with 2 N HCl (5 ml) and refluxed for 30 min. The reaction mixture was
worked up in the usual manner. The sugar fraction of compound 1 over CC
on an activated silica column yielded 2-C-methyl-D-erythritol identified by
co-TLC with authentic sample and optical rotation [
a
]
D
29
24.5° (c0.02,
H
2
O).
Basic Hydrolysis 1 The compound 1 (2 mg, 0.004 mmol) was dissolved
in methanol (3 ml) and NaOMe (5 mg, 0.092 mmol) was added, reaction
mixture was stirred at rt. for 3 h. The reaction was quenched with acidic
resin amberlite IRC-50 (Rohm and Hass, H
form), the resin was removed
by simple filtration and filtrate was dried under reduced pressure. The sugar
erythritol were identified by co-TLC with authentic sample.
Acetylation of Compound 2 Crude fraction containing 2 (122 mg) was
dissolved in dry pyridine (5 ml) and acetic anhydride (5 ml) was added. Re-
action mixture was left overnight at room temperature. The reaction mixture
was dried under reduced pressure. The crude residue on column chromatog-
raphy over silica gel (60—120 mesh, 11 g) eluted with isocratic hexane :
EtOAc (85 : 15), yielded amorphous powder of 2a (75 mg).
2,3,4-Trihydroxy-3-methylbutyl 3-[3-Hydroxy-4-(2,3,4-trihydroxy-2-
methylbutoxy)phenyl]-2-propenoate (1): Yellow solid.
1
H-NMR (CD
3
OD,
200 MHz) and
13
C-NMR (CD
3
OD, 75 MHz) see Table 1. IR (KBr) cm
1
:
3340, 2888, 1670, 1483, 1341, 1293. UV
l
max
(MeOH) nm: 324, 239, 213.
ESI-MS (pos.): m/z 417 [MH]
. [
a
]
D
29
15.0° (c0.04, MeOH). Positive
HR-ESI-MS: m/z [M]
416.1709 (Calcd 416.1683 for C
19
H
28
O
10
).
1,3-Di-O-caffeoyl Quinic Acid Methyl Ester (2a): Amorphous powder.
1
H-NMR (CD
3
OD, 300 MHz) and
13
C-NMR (CD
3
OD, 75 MHz) see Table 2.
IR (CDCl
3
) cm
1
: 3351, 2871, 1737, 1672, 1604, 1514. UV
l
max
(MeOH)
nm: 327, 273, 211. ESI-MS (pos.): m/z 783 [MH]
. [
a
]
D
29
50.8° (c
0.04, CDCl
3
). Positive HR-ESI-MS: m/z [M]
782.2073 (Calcd 782.2058 for
C
38
H
38
O
18
).
Anti-stress Activity. Animals Adult male Sprague–Dawley rats
(180—200 g) were obtained from National Animal Laboratory Centre,
774 Vol. 55, No. 5
Table3.Effect of Pure Compounds 1—5, 8 and PQ on Acute Stress In-
duced Changes in Adrenal Gland Weight, Glucose, Creatine Kinase and
Corticosterone Levels
Groups Adrenal gland Glucose CK Corticosterone
(dose mg/kg p.o.) (mg/kg wt) (mg/dl) (mg/dl) (ng/ml)
NS 6.170.32 81.53.37 297.517.55 244.515.43
ASVehicle 9.930.84** 134.74.68** 111529.43** 477.222.2**
COMP-1 (40) 7.230.41* 78.172.88* 485.385.12* 213.321.16*
COMP-2 (40) 8.330.35 96.335.74* 60051.81* 32024.21*
COMP-3 (40) 9.600.41 97.337.14* 962.761.23 30122.51*
COMP-4 (40) 11.150.78 123.76.83 112850.38 46830.69
COMP-5 (40) 11.410.48 108.76.82 109062.13 476.231.69
COMP-8 (40) 11.510.48 121.210.21 103688.86 456.520.98
PQ (100) 7.330.44* 86.53.51* 493.349.96* 238.513.59*
MmeanS.E.M 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. ∗∗ p0.001 when com-
pared to NS control and ∗ p0.01 when compared with acute stress control group.
CDRI, Lucknow. Animals were kept in raised mesh bottom cages to prevent
coprophagy in environmentally controlled rooms (252°C, 12 h light and
dark cycle), animals 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 institu-
tional ethical committee following the guidelines of CPCSEA (Committee
for the Purpose of Control and Supervision of Experiments on Animals),
which complies with international norms of INSA (Indian National Science
Academy).
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 AS. Drug was prepared fresh daily before 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, U.S.A. (Cat. No. G7253).
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 treatment 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 ex-
cept the non stress group. AS was produced by immobilizing animals for
150 min once only and sacrificed immediately by cervical dislocation.
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)
Biochemical Estimations The blood was collected in EDTA coated
tubes, through cardiac puncture after the stress regime and centrifuged at
2000 rpm20 min at 4 °C and plasma was separated. The plasma was used
to estimate corticosterone, glucose, creatine kinase (CK).
Estimation of Glucose and CK Auto analyzer (Synchron Cx-5, Beck-
man) was used to estimate glucose and CK with their respective kits (Beck-
man Coulter International, Nyon, Switzerland).
Estimation of Corticosterone An HPLC/UV system (Waters, U.S.A.)
was used for quantification of plasma corticosterone by the method of
Woodword and Emery with modifications.
35)
Dexamethasone was used an
internal standard. 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).
Statistical Analysis Mean and S.E.M. were calculated. The data was
analyzed using one-way analysis of variance (ANOVA) followed by Stu-
dent–Newman–Keul’s multiple comparison test. Data of ulcer was analyzed
by non-parametric ANOVA followed by Dunn’s multiple comparison tests.
p0.05 was considered to be statistically significant.
Acknowledgements Department of Biotechnolgy, New Delhi is sin-
cerely acknowledged for financial assistance and Indian Council of Medici-
nal Research. New Delhi, for the award of senior research fellowship to Pra-
soon Gupta. We are thankful to Dr. S. P. S. Bhandari for preparative HPLC;
Botany division, CDRI for collection of plant material and S. C. Tiwari for
extraction of plant material.
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