Constituents of Ocimum sanctum with antistress activity

Article abstract of DOI:10.1021/np0700164

Three new compounds, ocimumosides A (1) and B (2) and ocimarin (3), were isolated from an extract of the leaves of holy basil (Ocimum sanctum), together with eight known substances, apigenin, apigenin-7-O-β-D-glucopyranoside, apigenin-7-O-β-D-glucuronic acid (4), apigenin-7-O-β-D-glucuronic acid 6″-methyl ester, luteolin-7-O-β-D-glucuronic acid 6″-methyl ester, luteolin-7-O-β-D-glucopyranoside, luteolin-5-O-β-D- glucopyranoside, and 4-allyl-1-O-β-D-glucopyronosyl-2-hydroxybenzene (5), and two known cerebrosides. The structures of the new compounds were determined on the basis of extensive 1D and 2D NMR spectroscopic analysis. The new compounds (1-3) and the known compounds 4 and 5 were screened at a dose of 40 mg/kg body weight for acute stress-induced biochemical changes in male Sprague-Dawley rats. Compound 1 displayed promising antistress effects by normalizing hyperglycemia, plasma corticosterone, plasma creatine kinase, and adrenal hypertrophy. Compounds 2 and 5 were also effective in normalizing most of these stress parameters. In contrast, compounds 3 and 4 were ineffective in normalizing any of these effects.

Full text of DOI:10.1021/np0700164

1410
J. Nat. Prod. 2007, 70, 1410–1416
Constituents of Ocimum sanctum with Antistress Activity§
Prasoon Gupta,† Dinesh Kumar Yadav,† Kiran Babu Siripurapu,‡ Guatam Palit,‡ and Rakesh Maurya*,†
DiVision of Medicinal and Process Chemistry and DiVision of Pharmacology, Central Drug Research Institute, Lucknow-226 001, India
ReceiVed January 11, 2007
Three new compounds, ocimumosides A (1) and B (2) and ocimarin (3), were isolated from an extract of the leaves of
holy basil (Ocimum sanctum), together with eight known substances, apigenin, apigenin-7-O-ꢀ-D-glucopyranoside,
apigenin-7-O-ꢀ-D-glucuronic acid (4), apigenin-7-O-ꢀ-D-glucuronic acid 6′′-methyl ester, luteolin-7-O-ꢀ-D-glucuronic
acid 6′′-methyl ester, luteolin-7-O-ꢀ-D-glucopyranoside, luteolin-5-O-ꢀ-D-glucopyranoside, and 4-allyl-1-O-ꢀ-D-
glucopyronosyl-2-hydroxybenzene (5), and two known cerebrosides. The structures of the new compounds were
determined on the basis of extensive 1D and 2D NMR spectroscopic analysis. The new compounds (1–3) and the
known compounds 4 and 5 were screened at a dose of 40 mg/kg body weight for acute stress-induced biochemical
changes in male Sprague–Dawley rats. Compound 1 displayed promising antistress effects by normalizing hyperglycemia,
plasma corticosterone, plasma creatine kinase, and adrenal hypertrophy. Compounds 2 and 5 were also effective in
normalizing most of these stress parameters. In contrast, compounds 3 and 4 were ineffective in normalizing any of
these effects.
Stress response is a nonspecific demand to maintain the steady
state required for successful adaptation. This consists of a repertoire
of physical or mental reactions that attempt to counteract the effects
of the stressors in order to re-establish homeostasis. Prolonged
adaptation response with consistent physical and mental hostile
conditions leads to various psychosomatic disorders such as
ulceration, blood pressure, stroke, diabetes, anxiety, depression, and
other psychosomatic disorders.1,2 With changing lifestyles, stressful
conditions are faced by most of the world population. Stress
stimulates the hypothalamus pituitary adrenal (HPA) axis, causing
activation of various central and peripheral mediators.3 The role
of important monoamines like noradrenaline (NA), dopamine (DA),
and 5-hydroxytryptamine (5-HT) is well established in modulating
various behavioral and biochemical responses during stressful
conditions.4 In view of the nonspecific nature of the stress response,
a drug having central and peripheral activities would be in great
demand. Thus, the identification of new effective and safe antistress
agents is desperately needed. Plants and plant-derived metabolites
have a long history of clinical relevance in a variety of diseases,
and their use in traditional medicine systems all over the world is
being re-examined.5 In the course of a program aimed at the
discovery of antistress agents from medicinal plants, we have
initiated an investigation of Ocimum sanctum leaves.
Ocimum sanctum L. (Lamiaceae), a well-known herbal medicine,
is widely distributed throughout the world.6 This plant has been
worshipped since Vedic times and is considered sacred for its
medicinal properties in the Indian subcontinent and is well
recognized in Ayurvedic medicine in India.6 Its leaves have long
been used to treat a variety of ailments, including ozena, skin
diseases, and gastric and hepatic disorders and are used as a
diaphoretic, an antiperiodic, and an expectorant.6 O. sanctum is an
important botanical supplement used in combination with other
plants for the treatment of various stress-induced disorders in India
and other Asian countries. Numerous studies have shown the
immunomodulatory and antistress potential of O. sanctum leaf
extracts.7,8 Its mechanism of action has also been studied.7,8
Previous phytochemical investigations on O. sanctum have
described the isolation of two principal groups of compounds,
terpenoids and phenolic derivatives.9–12 To date, over 25 terpenoid
and fatty acid derivatives have been isolated from this plant, and
they show anticancer,13 anti-HIV,14 antimicrobial,15 and anti-
inflammatory effects.15 Phenolic substances also have been isolated
from the leaves of O. sanctum, including hydroxycinnamic acid
derivatives, benzoic acid derivatives, flavonoids and their glyco-
sides, and eugenol and eugenol glycosides. Some of these com-
pounds have been reported to exhibit antioxidant,16 antimicrobial,16
anti-inflammatory,17 antistress,8 anthelmintic, and radio-protective
activities.10
As a result of the increasing use of O. sanctum for stress-induced
disorders and as an immunomodulator, it was considered important
to better understand the phytochemical constituents of this plant
responsible for its antistress activity. In a preliminary pharmacologi-
cal study, antistress activity was found for the ethanol extract of
O. sanctum leaves in acute stress (AS) and chronic unpredictable
stress (CUS) models. On bioassay-guided fractionation of an ethanol
extract, activity was localized in a n-butanol-soluble fraction. Further
work has led to the isolation and identification of three new
compounds designated as ocimumoside A (1), ocimumoside B (2),
and ocimarin (3), along with 10 known compounds from this
n-butanol-soluble fraction. Compounds 1–3 and two of the known
compounds, 4 and 5, were evaluated for their ability to inhibit acute
stress-induced biochemical changes such as hyperglycemia, plasma
corticosterone, creatine kinase, and adrenal hypertrophy in rats. In
addition, this is the first report of the presence of glycoglycerolipids
(1, 2), a coumarin (3), and cerebrosides 1-O-(ꢀ-D-glucopyranosyl)-
(2S,3S,4R,8Z)-2-[(2′R)-2′-hydroxydocosanoylamino]-8(Z)-octadecene-
1,3,4-triol,18 and 1-O-(ꢀ-D-glucopyranosyl)-(2S,3S,4R,8Z)-2-[(2′R)-
2′-hydroxytetracosanoyl amino]-8 (Z)-octadecene-1,3,4-triol18 from
O. sanctum.
Results and Discussion
The dried and powdered leaves of O. sanctum were extracted
with ethanol and concentrated at 45 °C. The resulting dried ethanol
extract was suspended in water and sequentially partitioned with
chloroform and n-butanol. The n-butanol-soluble fraction was
separated by a combination of chromatographic procedures, which
afforded three new compounds (1–3), along with 10 known
substances. The structures of the new compounds were established
§ CDRI communication no. 6988.
1
using chemical and spectroscopic (FABMS, H NMR, 13C NMR,
* Corresponding author. Tel: +91-(522)-2612411-18, ext. 4235. Fax:
+91-(522)-2623405/2623938/2629504. E-mail: mauryarakesh@rediffmail.
com.
COSY, HSQC, HMBC) studies, whereas the known compounds
were identified by comparison of their spectroscopic data (UV, MS,
and NMR) with published reports.
† Division of Medicinal and Process Chemistry.
‡ Division of Pharmacology.
10.1021/np0700164 CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/13/2007

Constituents of Ocimum sanctum with Antistress ActiVity
Journal of Natural Products, 2007, Vol. 70, No. 9 1411
Table 1. NMR Spectroscopic Data for Compound 1a
position
δH (mult., J in Hz)
δC
62.8
HMBC
COSY
1a
1b
2
3a
3b
1′
2′
3′
4′
5′
6′a
6′b
1′′
2′′
3′′
1′′′
2′′′
3′′′
–CH2
CH3
4.14 (dd, 12.4, 4.4)
3.90 (dd, 11.3, 4.2)
4.90 (m)
2, 3, 1′′′
2, 3, 1′′′
1, 3, 1′′
1′, 1, 2
1′, 1, 2
3, 5′, 3′
4′
1′, 5′
6′, 2′
1′, 3′
4′, 5′
1b
1a, 2
1b, 3b
3b
2, 3a
2′
1′, 3′
2′, 4′
3′, 5′
6′a,b; 4′
5′, 6′b
5′, 6′a
69.8
64.8
3.90 (dd, 10.5, 6.5)
3.54 (dd, 10.5, 7.3)
4.33 (d, 3.6)
98.4
71.7
73.0
74.2
68.6
54.4
3.31–3.27 (m)b
3.31–3.27 (m)b
3.31–3.27 (m)b
3.31–3.27 (m)b
2.65 (dd, 11.2, 4.2)
2.83 (dd, 10.8, 4.5)
4′, 5′
172.6
33.7
31.4
172.4
33.5
29.2
2.44 (t, 6.4)
2.16 (m)c
1′′
1′′
3′′
2′′
2.23 (t, 7.2)
2.16 (m)c
1.42–1.16 (m)
0.80 (6H, t, 6.9)
1′′′,
1′′′
3′′′
2′′′
29.2–22.2
14.0
a Recorded in DMSO-d6 at 300 MHz (TMS as internal standard);
chemical shifts, multiplicity, and coupling constants (J, Hz) were
assigned by means of 1H, 13C NMR and 2D NMR data. b Overlapped
with H2O signals. c Overlapped signals that may be interchanged.
and 355 were observed, corresponding to hexadecanoic acid methyl
ester and docosanoic acid methyl ester, repectively. Upon regiose-
lective enzymic hydrolysis of 1 with lipase enzyme type III in
dioxane/H2O (1:1) at 37 °C for 4 h,20 only hexadecanoic acid was
obtained, as analyzed by ESIMS and compared with an authentic
sample. Thus, it was concluded that a hexadecanoyl residue was
attached to the C-1 position of the glycerol moiety in 1.
The 1H and 13C NMR spectra of compounds 1 and 2 suggested
certain common structural features attributable in each case to a
sugar moiety, a glycerol system, and long aliphatic chains. Both
compounds gave a positive Feigel test for glycosides. Compounds
1 and 2 revealed strong IR absorption bands for a hydroxyl group
and an ester carbonyl, respectively, at νmax 3416 and 1732 cm-1
(1) and 3417 and 1737 cm-1 (2).
The aqueous phase was subjected to acid hydrolysis (2 N HCl,
30 min), and after workup and purification over an activated carbon
column, methyl 6-deoxy-6-amino-R-D-glucopyranoside was ob-
tained. The D-configuration of this sugar was confirmed by
Compound 1 was isolated as a white, amorphous powder and
gave quasimolecular ion peaks at m/z 814 [M + H]+ and 836 [M
+ Na]+ in the positive FABMS, corresponding to a molecular
formula C47H91NO9, and this was supported from its NMR data
and by elemental analysis. Assignment of all the 13C and 1H NMR
signals for three spin systems is as shown in Table 1. The first
spin system was assigned to a glycerol moiety (δH 4.14 and 3.90
(δC 62.8); δH 4.90 (δC 69.8); δH 3.90 and 3.54 (δC 64.8)); the second
group of signals was attributable to two long-chain saturated fatty
acids, where the terminal methyl signals appeared at δH 0.80 (6H,
t, J ) 6.9 Hz, δC 14.0), and the third spin system indicated the
presence of a glycosyl moiety. Acid hydrolysis of 1 did not lead to
comparing its optical rotation ([R]28 +139 in H2O) with that
D
reported in the literature for an authentic sample ([R]25 +147 in
D
H2O).21
1
The absolute configuration of 1 was based on its H NMR
data and optical rotation ([R]29 +50.2 in MeOH) data. In the
D
CD spectrum, a negative Cotton effect was observed at 225 nm,
which was shown to be identical in all respects to a diacylgly-
cosylglycerolipid with known absolute stereochemistry.22
Consequently, the structure of 1 was characterized as (2S)-1-O-
hexadecanoyl-2-O-docosanoyl)-3-O-[6-deoxy-6-amino-R-D-glu-
copyranoside]glycerol, a new compound that has been named
ocimumoside A. Compound 1 bears a rare 6-deoxy-6-aminoglucose
moiety.19,22
1
the identification of any of the normal pyranose sugars. In the H
NMR spectrum, most of the glycosyl proton signals were over-
lapped with a solvent signal. Therefore, using the anomeric proton
at δH 4.33 (d, J ) 3.6 Hz) as a starting point, a sequence of four
oxymethines at δH 3.31–3.27 (m) (δC 71.7, 73.0, 74.2, and 68.6)
and one deshielded methylene at δH 2.65 and 2.83 (δC 54.4) was
identified. The relatively upfield 13C NMR resonance of C-6′ (δC
54.4) was indicative of the presence of an amino group at this
position.19 The R-glucopyranose nature of this sugar was determined
by the small coupling constant, J1′/2′ ) 3.6 Hz. Analysis of these
data confirmed the assignment of the sugar unit as 6-deoxy-6-amino-
R-glucopyranose. The HMBC experiment furnished useful data for
solving the structure of 1 (Table 1). The sugar residue could be
linked to the C-3 of the glycerol, as indicated by the HMBC
correlation between anomeric proton H-1′ and C-3, while other
HMBC cross-peaks indicated the attachment of acyl groups at the
C-1 and C-2 position (H-2/C-1′′′, H2-2′′′/C-1′′′, H2-1/C-1′′, H2-2′′/
C-1′′) of the glycerol moiety.
Compound 2 exhibited quasimolecular ion peaks at m/z 915 [M
+ Na]+ and m/z 910 [M + NH4]+ in the ESIMS, corresponding to
the elemental formula C47H88O15. It showed an almost identical
NMR pattern to 1 except for the presence of additional sugar
carbons and the absence of a signal at δC 54.4 in the 13C NMR
spectrum. Two anomeric carbon signals appeared at δC 103.8 (C-
1′) and 99.6 (C-1′′), whereas anomeric protons resonated at δH 4.50
(d, J ) 6.0 Hz) and 4.65 (d, J ) 3.0 Hz) in the 1H NMR spectrum
(Table 2). The downfield-shifted carbon signal of C-6′ (at δC 66.8)
compared to C-6′′ (at δC 60.7) indicated a glycosidic linkage
between C-1′′ and C-6′. This was supported by a HMBC correlation
between H-1′′/C-6′, and the coupling constant of H-1′′/H-2′′ (J )
3.0 Hz, axial–equatorial) inferred an R-glycosidic linkage. Other
sugar protons and carbons (Table 2) were identical to digalacto-
side,23 a substituent confirmed by acid hydrolysis of 2 followed
by co-TLC with an authentic sample. Another spin system was
According to its molecular formula, the two acyl moieties of 1
must represent a total of 38 carbon atoms. To infer the exact nature
of the fatty acids, 1 was hydrolyzed in methanol/NaOMe (2 h).
After the usual workup, the nonpolar organic extract was analyzed
by GC-MS and FABMS, and two ion peaks [M + H]+ at m/z 271
1
1
assigned to a glycerol moiety by H and 13C, H–1H COSY, and
HSQC NMR spectroscopy (δH 4.33 and 4.11, δC 62.5; δH 5.08, δC
70.4; δH 3.77 and 3.56, δC 69.7). The remaining NMR signals were

1412 Journal of Natural Products, 2007, Vol. 70, No. 9
Table 2. NMR Spectroscopic Data for Compound 2a
Gupta et al.
Table 3. NMR Spectroscopic Data for Compound 3a
position δH (mult., J in Hz)
δC
HMBC
COSY
position
δH (mult., J in Hz)
δC
HMBC
1a
1b
2
3a
3b
1′
2′
3′
4′
5′
6′a
6′b
1′′
2′′
3′′
4′′
5′′
6′′a
6′′b
1′′′
1′′′′
2′′′
2′′′′
3′′′
3′′′′
–CH2
CH3
4.33 (dd, 12.0, 6.0) 62.5
2, 3, 1′′′′
2, 3, 1′′′′
1, 3, 1′′′
1′, 1, 2
1′, 1, 2
3, 5′, 3′
4′
1′, 5′
6′, 2′
1′, 3′
1′′, 4′
1′′, 4′
5′′, 6′a, 3′′
4′′
1′′, 5′′
6′′, 2′′
1′′, 3′′
4′′
1b
2
3
4
5
6
7
8
9
161.5
118.8
148.6
126.7
113.1
160.4
102.1
153.6
112.9
15.2
4.11 (dd, 12.0, 3.0)
5.08 (m)
1a, 2
1b, 3b
3b
2, 3a
2′
1′, 3′
2′, 4′
3′, 5′
6′a,b; 4′
5′
70.4
3.77 (dd, 12.3, 6.0) 69.7
3.56 (dd, 9.3, 3.1)
7.54 (d, J ) 8.8)
4, 7, 10
7, 8, 10
6.78 (dd, J ) 2.2, 8.6)
4.50 (d, 6.0)
3.51–3.44 (m)b
3.51–3.44 (m)b
3.54 (bs)
103.8
73.0
70.0
68.1
73.2
66.8
6.65 (d, J ) 2.0)
6, 7, 10
10
CH3
CH2-CH2-OH
CH2-CH2-OH
3.59 (t, 5.7)
2.33 (s)
2.67 (t, J ) 6.8)
3.48 (t, J ) 7.0)
3, 4, 10
2, 3, 4
3
3.51–3.44 (m)b
3.51–3.44 (m)b
4.65 (d, 3.0)
3.80 (m)
31.1
59.7
5′
2′′
99.6
69.7
68.5
69.0
71.4
a Recorded in DMSO-d6 at 200 MHz (TMS as internal standard).
1′′, 3′′
2′′, 4′′
3′′, 5′′
6′′a,b; 4′′
6′′b, 5′′
6′′a, 5′′
Assignments are based on HSQC and HMBC experiments.
3.63 (dd, 9.2, 3.5)
3.69 (dd, 3.2, 1.6)
3.59 (t, 5.7)
in the molecule, and their multiplicity was assigned by a DEPT
experiment, revealing the presence of six quaternary carbons at δ
161.5 (CO), 160.4, 153.6, 148.6, 118.8, and 112.9, three aromatic
methines at δ 126.7, 113.1, and 102.1, two methylenes at δ 59.7
and 31.1, and one methyl group at δ 15.2, respectively. The HMBC
experiment provided useful correlations between CH3/C-3, C-4,
C-10 and CH2/C-2, C-3 and confirmed the position of the methyl
and hydroxy ethyl groups in 3. From the above data, the structure
of 3 was elucidated as 7-hydroxy-3-(2-hydroxyethyl)-4-methyl-2H-
1-benzopyran-2-one, a new naturally occurring compound named
ocimarin.
The structures of several known compounds isolated were identified
as apigenin,26 apigenin-7-O-ꢀ-D-glucuronic acid (4),27 apigenin-7-O-
ꢀ-D-glucuronic acid 6′′-methyl ester,28 luteolin-7-O-ꢀ-D-glucuronic acid
6′′-methyl ester,29 4-allyl-1-O-ꢀ-D-glucopyronosyl-2-hydroxyben-
zene (5),12 apigenin-7-O-ꢀ-D-glucopyranoside,26 luteolin-7-O-ꢀ-
D-glucopyranoside,30 and luteolin-5-O-ꢀ-D-glucopyranoside.31 In
addition, two known cerebrosides, 1-O-(ꢀ-D-glucopyranosyl)-
(2S,3S,4R,8Z)-2-[(2′R)-2′-hydroxydocosanoylamino]-8(Z)-octadecene-
1,3,4-triol and 1-O-(ꢀ-D-glucopyranosyl)-(2 S,3 S,4 R,8 Z)-2-[(2′R)-
2′-hydroxytetracosanoylamino]-8(Z)-octadecene-1,3,4-triol, were
isolated and characterized using detailed spectroscopic (1H–1H
COSY, HSQC, HMBC, and NOESY) and chemical degradation
studies.18
A variety of stress situations have been employed in animals to
evaluate antistress activity. Stress-induced effects mainly depend
on duration and type of stressors.32 Immobilization has been the
ideal choice for the induction of stress responses in animals and,
more specifically, for the investigation of drug effects on typical
stress-related gastrointestinal, neuroendocrine, and immunological
pathology.33 Hyperglycemic response during acute stress is due to
the release of glucocorticoids as a result of HPA axis stimulation,
to compensate for the initial demand of energy.34
It is know that AS exposure not only stimulates and ensures the
supply of glucose but also increases creatine kinase (CK) activity.35
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.36
Prominent changes during stress are adrenocorticoid hypersecretion,
increased plasma corticosterone, and enlarged pituitary and adrenal
size.37,38 In our work, various type of stress also increased the
adrenal gland weight and plasma corticosterone.3
2.71 (dd, 11.1, 5.2) 60.7
3.44 (m)b
172.7
4′′
172.4
2.29 (t, 6.2)
2.29 (t, 6.2)
1.46 (m)c
33.7
33.5
27.5
27.5
1′′′
1′′′′
1′′′
3′′′
3′′′′
1.46 (m)c
1′′′′
1.35–1.15 (m)
0.81 (6H, t, 6.1)
30.8–22.2
14.0
a Recorded in DMSO-d6 at 300 MHz (TMS as internal standard);
chemical shifts, multiplicity, and coupling constants (J, Hz) were
assigned by means of 1H, 13C NMR and 2D NMR data. b Overlapped
with H2O signals. c Overlapped signals that may be interchanged.
assigned for two long-chain fatty acid units, which accounted for
32 carbon atoms according to the molecular formula and 13C NMR
spectrum. To determine the length of these fatty acids, compound
2 was treated with methanol/NaOMe and afforded glyceryldiga-
lactose (2a) and two fatty acid residues. The fatty acid residues
were determined by GC-MS as methyl tetradecanoate and methyl
octadecanoate. The HMBC spectrum indicated that two acylating
fatty chains were attached to C-1 and C-2 of the glycerol moiety,
whereas C-3 was involved in a ꢀ-glycosidic linkage with a sugar
residue (J ) 6.0 Hz, H-1′). Compound 2a, [R]29 +83 in H2O,
D
was shown to be identical in all respect to (2R)-1-O-[R-D-
galactopyranosyl-(1′′f6′)-O-ꢀ-D-galactopyranosyl] glycerol,24 con-
firming the configurations of both the sugar and glycerol moieties
in the molecule. The sequence of acyl chains between C-1 and C-2
was determined by regioselective enzymatic hydrolysis of 2 (as
above),20 with only octadecanoic acid being obtained, as determined
by ESIMS. Consequently, 2 was characterized as (2S)-1-O-
octadecanoyl-2-O-tetradecanoyl)-3-O-[R-D-galactopyranosyl-(1′′f6′)-
O-ꢀ-D-galactopyranosyl] glycerol and is a new compound that has
been given the trivial name ocimumoside B.
Compound 3 was obtained as a brown, amorphous powder, and
its molecular formula, C12H12O4, was established by positive
FABMS at m/z 221 [M + H]+ and from its NMR data. The UV
absorption bands, UV (MeOH) λmax (log ε) 320 (2.61), 204 (2.72)
nm, were characteristic of a coumarin derivative.25 The IR spectrum
exhibited absorption bands at νmax 3533, 1720, 1616, 1570, and
1358 cm-1 and clearly indicated the presence of an R,ꢀ-unsaturated
carbonyl ester, a hydroxyl group, and an aromatic ring in the
molecule. On acetylation, it formed a diacetate, 3a (FABMS at m/z
305 [M + H]+), confirming the presence of two free hydroxyl
In the present investigation, the EtOH extract and its n-
butanol-soluble fraction of O. sanctum, at a dose of 200 mg/kg
body weight, were significantly (p < 0.05) effective in normal-
izing acute stress (AS)- and chronic unpredictable stress (CUS)-
induced hyperglycemia, plasma corticosterone, plasma creatine
kinase, and adrenal gland weight in the rat model used. A
chloroform-soluble fraction of O. sanctum was ineffective in
normalizing most of these biochemical changes and only
effective in reducing the acute stress-induced hyperglycemia,
1
groups. The H NMR spectrum (Table 3) showed signals at δH
7.54 (1H, d, J ) 8.8 Hz), 6.78 (1H, dd, J ) 8.6, 2.2 Hz), and 6.65
(1H, d, J ) 2.0 Hz) and indicated the presence of an ABX system.
In addition, two sharp triplets appeared at δH 3.48 (2H, t, J ) 7.0
Hz) and 2.67 (2H, t, J ) 6.8 Hz) and a singlet at δH 2.33 (3H, s),
due to the presence of hydroxyl ethyl and methyl groups,
respectively. Its 13C NMR spectrum (Table 3) showed 12 carbons

Constituents of Ocimum sanctum with Antistress ActiVity
Journal of Natural Products, 2007, Vol. 70, No. 9 1413
Figure 1. Effect of compounds 1–5 at 40 mg/kg body weight on stress-induced changes in the rat adrenal gland. The stress group was
compared with the nonstress control group, and the drug-treated groups were compared with an acute stress group. Results are represented
as means ( SEM with n ) 6 in each group (*p < 0.01 when compared to the nonstress (NS) group and **p < 0.05 when compared to
the acute stress (AS) group).
Figure 2. Effect of compounds 1–5 at 40 mg/kg body weight on stress-induced changes in rat plasma corticosterone levels. The stress
group was compared with a nonstress control group, and the drug-treated groups were compared with the acute stress group. Results are
represented as means ( SEM with n ) 6 in each group (*p < 0.01 when compared to the nonstress (NS) group and **p < 0.05 when
compared to the acute stress (AS) group).
adrenal gland weights, and the CUS-induced plasma creatine
kinase levels. Moreover, the aqueous fraction of the plant was
found ineffective in normalizing any of the stress-induced
changes. The antistress effects of the EtOH extract and the
n-butanol-soluble fraction were comparable to those of a standard
drug, Panax quinquifolium (PQ), at a dose of 100 mg/kg body
weight (Figures S1–S4, Supporting Information).
Several isolated compounds (1–5) from O. sanctum were tested
for antistress activity in the acute stress model at a dose of 40 mg/
kg body weight. A significant increase (p < 0.01) in the adrenal
gland weight after AS was observed when compared to a nonstress
(NS) group. Compounds 1 and 5 were significantly (p < 0.05)
effective in reducing the stress-induced adrenal hypertrophy.
Similarly, AS (p < 0.01) exposure resulted in increased plasma
corticosterone when compared to the NS control. Pretreatment with
compounds 1, 2, and 5 (p < 0.05) reduced significantly the increase
in corticosterone levels. The plasma CK and glucose levels were
also increased by AS significantly (p < 0.01) when compared to
the NS control. Prior treatments of compounds 1, 2, and 5 (p <
0.01) were effective in reducing the AS-induced increase in CK
levels. Compounds 1 and 5 were observed to reduce increased
glucose levels.
In conclusion, compound 1 has shown significant (p < 0.05)
antistress activity by normalizing hyperglycemia, corticosterone
levels, creatine kinase, and adrenal hypertrophy. Compound 2 also
showed antistress activity but had no effect on plasma glucose
levels. Compound 5 was found to be effective in most of the stress
parameters evaluated but less effective on plasma glucose levels.
Compounds 3 and 4 were ineffective in normalizing these param-
eters (Figures 1–4). The other isolated compounds from O. sanctum
could not be tested due to the insufficient amounts obtained. The
biological activity profiles of compounds 1, 2, and 5 described
herein are worthy of further investigation.

1414 Journal of Natural Products, 2007, Vol. 70, No. 9
Gupta et al.
Figure 3. Effect of compounds 1–5 at 40 mg/kg body weight on stress-induced changes in rat plasma glucose levels. The stress group was
compared with the nonstress control group, and the drug-treated groups were compared with the acute stress group. Results are represented
as means ( SEM with n ) 6 in each group (*p < 0.01 when compared to the nonstress (NS) group and *p < 0.05 when compared to the
acute stress (AS) group).
Figure 4. Effect of compounds 1–5 at 40 mg/kg body weight on stress-induced changes in rat plasma creatine kinase levels. The stress
group was compared with a nonstress control group, and the drug-treated groups were compared with an acute stress group. Results are
represented as means ( SEM with n ) 6 in each group (*p < 0.01 when compared to the nonstress (NS) group and **p < 0.05 when
compared to the acute stress (AS) group).
Experimental Section
was performed using silica gel (60–120 and 230–400 mesh). TLC was
carried out on precoated 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 H2SO4–MeOH or anisaldehyde–H2SO4
reagents.
Plant Material. The plant material was collected from Ajamgarh,
Uttar Pradesh, India, in November 2002 and was confirmed as Ocimum
sanctum by staff members of the Botany Division of CDRI, where a
voucher specimen (No. 38) is preserved in the herbarium.
Extraction and Isolation. Powdered leaves of Ocimum sanctum
(10 kg) were placed in a glass percolator with ethanol (25 L) and
allowed to stand at room temperature overnight. The percolate was
collected. This process of extraction was repeated four times. The
combined extract was filtered, concentrated at 45 °C under a vacuum,
and afforded an ethanol extract (C003, 820 g). This ethanol extract
(800 g) was suspended in distilled water (400 mL) and then extracted
successively with chloroform (600 mL × 7) and n-butanol saturated
with water (500 mL × 7). The chloroform-, n-butanol-, and water-
soluble extracts were concentrated at 45 °C and afforded chloroform
General Experimental Procedures. Optical rotations were mea-
sured on a Perkin-Elmer model 241 digital polarimeter. UV spectra
were obtained on a Perkin-Elmer λ-15 UV spectrophotometer. IR
spectra were recorded on a Perkin-Elmer RX-1 spectrophotometer using
either KBr pellets or when neat. The CD spectrum was obtained on
JASCO J-810 spectrometer. H and 13C NMR spectra were recorded
1
on an Avance DPX-200 or a Bruker DRX 300 MHz NMR spectrometer;
proton-detected heteronuclear correlations were measured using the
HSQC and HMBC techniques. All 2D-NMR spectra were recorded on
a Varian Inova 600 MHz NMR spectrometer. FABMS were carried
out on a JEOL SX 102/DA-6000 mass spectrometer and ESIMS on a
Advantage Max LCQ Thermo-Finnigan mass spectrometer. HRESIMS
analysis was carried out on a JEOL-MS 600H instrument. Elemental
analyses were obtained in a Carlo-Erba-1106 CHN elemental analyzer.
GC analyses were performed on a Perkin-Elmer XL auto-system using
a SE-30 capillary column (10 ft), with 30 mL of N2 as carrier gas at
60–250 °C. GC-MS analysis was done using a Shimadzu GC-14A unit
coupled with a GCMS-QP 2000 instrument. Column chromatography

Constituents of Ocimum sanctum with Antistress ActiVity
Journal of Natural Products, 2007, Vol. 70, No. 9 1415
(F004, 99 g), n-butanol (F005, 445.7 g), and aqueous (F006, 270.4 g)
residues, respectively.
+ H]+, 243 [M + Na]+; anal. C 65.41%, H 5.41%, calcd for C12H12O4,
C 65.45%, H, 5.49%.
A portion of the n-butanol-soluble fraction (300 g) was subjected to
column chromatography over silica gel (230–400 mesh, 2.1 kg) and
eluted with a gradient solvent system composed of chloroform–methanol
(95:05) to methanol–water (95:05). Ninety-three fractions (600 mL
each) were obtained, and their composition was monitored by TLC,
with those showing similar TLC profiles grouped into six major
fractions (F-1 to F-6). Fraction F-1 afforded compound 3 (190 mg) by
crystallization from methanol. Further purification of fraction F-2 (30.4
g) over silica gel (230–400 mesh, 300 g), using a gradient solvent
system of CHCl3–MeOH (95 to 50%), afforded 65 fractions (500 mL
each), and these were grouped into seven further fractions (F-7 to F-13)
on the basis of their TLC profiles. Purification of fraction F-7 (1.5 g)
over silica gel (60–120 mesh, 40 g), using CHCl3–MeOH (95:5),
afforded apigenin (40 mg). Compound 4 (445 mg) was obtained as an
amorphous solid from fraction F-4 at room temperature. Column
chromatography of fraction F-9 (6.0 g), over silica gel (230–400 mesh,
160 g), using ethyl acetate saturated with water (isocratic) as mobile
phase, afforded 42 fractions (200 mL each), which were pooled into
five further fractions on the basis of their TLC profiles (F-14 to F-18).
Fraction F-18, containing a mixture of two compounds (300 mg), was
purified over Sephadex LH-20, eluted with water–methanol (1:1) and
resulting in the collection of 55 subfractions, 10 mL in volume.
Subfractions 24–32 yielded 1-O-(ꢀ-D-glucopyranosyl)-(2 S,3S,4R,8Z)-
2-[(2′R)-2′-hydroxydocosanoylamino]-8(Z)-octadecene-1,3,4-triol (35
mg), and subfractions 33–38 yielded 1-O-(ꢀ-D-glucopyranosyl)-
(2S,3S,4R,8Z)-2-[(2′R)-2′-hydroxytetracosanoylamino]-8(Z)-octadecene-
1,3,4-triol (18 mg). Column chromatography of F-10 (4.2 g) over silica
gel (60–120 mesh, 120 g), using ethyl acetate saturated with water as
mobile phase, afforded 31 fractions of 100 mL each, with those showing
similar TLC profiles grouped into five additional fractions (F-19 to
F-23). Further purification of fraction F-20 (700 mg) over silica gel
(230–400 mesh, 20 g) using ethyl acetate saturated with water (isocratic)
as mobile phase afforded apigenin-7-O-ꢀ-D-glucuronic acid 6′′-methyl
ester (40 mg). Purification of fraction F-11 (3.2 g) over silica gel
(230–400 mesh, 90 g) using ethyl acetate saturated with water as mobile
phase afforded a total of 35 fractions, 50 mL in volume. These were
grouped into six major fractions (F-24 to F-29) on the basis of their
TLC profiles. Fraction F-25 (150 mg) was purified by medium-pressure
liquid chromatography on reversed-phase silica gel (RP-18), eluted with
a gradient of MeOH–H2O (1:1) to methanol, to generate 40 subfractions,
15 mL in volume. Subfractions 25–31 gave a yellow, amorphous solid
of luteolin-7-O-ꢀ-D-glucuronic acid 6′′-methyl ester (35 mg). Fraction
F-12 (3.7 g) was rechromatographed over silica gel (230–400 mesh,
100 g), eluted with gradient of ethyl acetate and methanol 95–80%,
and yielded fractions F-30 to F-34. Fraction 34 was kept in a refrigerator
overnight and yielded a further quantity of 1 (62 mg). Column
chromatography of F-22 (1.1 g) using EtOAc–MeOH (95:5) as eluent
over silica gel (100–200 mesh, 25 g) afforded 5 (500 mg). Purification
of F-28 (300 mg) on reversed-phase silica gel (RP-18), eluted with
H2O–MeOH (6:4), afforded 60 fractions, 15 mL in volume. Subfractions
38–45 yielded compound 2 (48 mg). Purification of fraction F-30 (1.2
g) over silica gel (100–200 mesh, 24 g), using a gradient solvent system
of CHCl3–MeOH (95:05) to CHCl3–MeOH (70:30), yielded apigenin-
7-O-ꢀ-D-glucopyranoside (30 mg, fractions 10–15), luteolin-7-O-ꢀ-D-
glucopyranoside (35 mg, fractions 21–29), and luteolin-5-O-ꢀ-D-
glucopyranoside (27 mg, fractions 35–40), respectively.
Acid and Basic Hydrolysis of 1 and 2. Compounds 1 (15 mg,
0.018 mmol) and 2 (10 mg, 0.011 mmol) were separately dissolved in
methanol (3 mL), and NaOMe (5 mg, 0.092 mmol) was added; the
reaction mixtures were stirred at rt for 3 h. The reaction mixtures were
quenched with the acidic ion exchange resin Amberlite IRC-50 (Rohm
and Hass, H+ form), with the resin removed by filtration, and the filtrate
was dried under reduced pressure and partitioned between chloroform
and water. The organic layers from compounds 1 and 2 were analyzed
by GC-MS. The aqueous layer of compound 1 was treated with 2 N
HCl (5 mL) and refluxed for 30 min. The reaction mixture was worked
up in the usual manner and the sugar fraction isolated on an activated
carbon column to give methyl 6-deoxy-6-amino-R-D-glucopyranoside
(2.1 mg), identified by comparison with an authentic sample (TLC)
and by its optical rotation ([R]28D +139 in H2O). The aqueous layer of
2 afforded compound 2a, which had optical rotation [R]29D +83 in H2O
and was identical in all respect to (2R)-1-O-[R-D-galactopyranosyl-
(1′′f6′)-O-ꢀ-D-galactopyranosyl] glycerol.24
Enzymatic Hydrolysis of 1 and 2. Compounds 1 and 2 (2 mg)
were each dissolved in 4 mL of dioxane–H2O (1:1) and treated with
lipase enzyme type III (2 mg, 46 unit, from a Pseudomonas species,
lot 093K0698, Sigma-Aldrich) at 37 °C, with shaking for 4 h. The
reaction mixtures of compounds 1 and 2 were quenched with 5% AcOH
(1 mL), and the product was dried under reduced pressure. The crude
residues of compounds 1 and 2 were dissolved in water and extracted
with EtOAc, concentrated under reduced pressure, and analyzed by
ESIMS.
Acetylation of Compound 3. Compound 3 (10 mg) was dissolved
in dry pyridine, (2.0 mL) and acetic anhydride (2 mL) was added, with
the reaction mixture left overnight at room temperature. The reaction
mixture was dried under reduced pressure. The crude residue was
dissolved in methanol and cooled, and an amorphous powder of 3a
(13 mg) was obtained: 1H NMR (CDCl3, 200 MHz) δH 7.63 (1H, d, J
) 8.0 Hz, H-5), 7.09 (1H, d, J ) 1.2 Hz, H-8), 7.07 (1H, dd, J ) 8.0,
1.2 Hz, H-6), 4.30 (2H, t, J ) 6.6 Hz, CH2OH), 3.06 (2H, t, J ) 6.6
Hz, CH2–CH2OH), 2.33 (3H, s, CH3), 2.46 and 2.02, (3H each, acetoxyl
methyl); FABMS m/z 305 [M + H]+.
Antistress Activity Determination. Animals. Adult male Spra-
gue–Dawley rats (180–200 g) were obtained from the National Animal
Laboratory Centre, CDRI, Lucknow, India. Animals were kept in raised-
mesh-bottom cages to prevent coprophagy in environmentally controlled
rooms (25 ( 2 °C, 12 h light and dark cycle), and animals had free
access to standard pellet chow and drinking water except during
experiments. Experiments were conducted between 9 a.m. and 2 p.m.
Experimental protocols were approved by an institutional ethical
committee following the guidelines of CPCSEA (Committee for the
Purpose of Control and Supervision of Experiments on Animals), which
complies with the international norms of INSA (Indian National Science
Academy).
Test Compound Administration. Suspensions of test materials
from O. sanctum in 0.1% sodium carboxymethylcellulose were prepared
and administered orally at a dose of 200 mg/kg (for the ethanol extract
and fractions) once daily for three days and 40 mg/kg (for isolated
compounds) once only, in case of acute stress (AS), and for seven days
in case of chronic unpredictable stress (CUS), 45 min prior to a stress
session. Control animals received an equivalent volume of vehicle for
the same period. After the stress regimen, animals were sacrificed by
decapitation on the seventh day immediately after the last stress session.
A freshly prepared aqueous suspension of the crude powder of ginseng
root, P. quinquifolium, was used as a standard at a dose of 100 mg/kg
body weight and was purchased from Sigma, St. Louis, MO (cat. no.
G7253).
Stress Protocol. The rats were divided into nonstress (NS), AS,
and CUS groups as well as drug-treated groups for both AS and CUS
groups. Each group consists of six rats. In the AS model, 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,
rats were stressed. A parallel group of vehicle-treated rats without
exposure to any kind of stress and maintained under normal conditions,
served as control nonstress group. In the CUS groups the drugs were
fed daily 45 min prior to the stress regime for seven days, with the
rats fasted overnight on the sixth day after completion of the
experimental regimens of drug feeding and stress exposure.
Ocimumoside A (1): white, amorphous powder; [R]29 +50.2 (c
D
0.04, MeOH); UV (c 0.04, MeOH) λmax (log ε) 225 (3.00) nm; IR (KBr)
νmax 3416, 3406, 2910, 1732, 1673 cm-1; CD (c 0.04, MeOH) (∆ε
–3.5) nm; 1H (DMSO-d6, 300 MHz) and 13C (DMSO-d6, 75 MHz) NMR
spectra, see Table 1; FABMS (positive-ion) m/z 814 [M + H]+, 836
[M + Na]+; anal. C 66.21%, H 11.31%, N 1.73%, calcd for C47H91O9N,
C 66.33%, H 11.26%, N 1.72%.
Ocimumoside B (2): white, hygroscopic solid; [R]29 + 70.0 (c
D
0.04, MeOH); IR (KBr) νmax 3417, 2910, 1737, 1042 cm-1; 1H (DMSO-
d6, 300 MHz) and 13C (DMSO-d6, 75 MHz) NMR spectra, see Table
2; ESIMS (positive ion) m/z 915 [M + Na]+, 910 [M + NH4]+;
HRESIMS m/z [M]+ 892.6143 (calcd for C47H88O15, 892.6123).
Ocimarin (3): brown, amorphous powder; UV (MeOH) λmax (log
ε) 320 (2.61), 204 (2.72) nm; IR (KBr) νmax 3533, 2927, 1720, 1616,
1
1570, 1027 cm-1; H (DMSO-d6, 200 MHz) and 13C (DMSO-d6, 50
MHz) NMR spectra, see Table 3; FABMS (positive-ion) m/z 221 [M

1416 Journal of Natural Products, 2007, Vol. 70, No. 9
Gupta et al.
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AS was produced by immobilizing animals for 150 min once only
and sacrificed immediately. CUS was produced with variable stressors
with modifications as described by Katz and co-workers.39 The stressors
included immobilization, forced swimming, soiled cage bedding, foot
shock, day–night reversal, and fasting. Animals were subjected to two
stressors of variable intensity every day in an unpredictable manner to
avoid habituation for seven consecutive days.3 Briefly, immobilization
stress was produced by restraining each naive animal inside an acrylic
hemicylindrical plastic tube (4.5 cm diameter, 12 cm long) for 150
min. In the swimming stress test, animals were allowed to swim in a
glass jar (35.5 cm long, 20.2 cm diameter) containing water at 25 °C
for 20 min. In soiled cages, the bedding was wet with water to produce
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subjected to foot shock (2 mA) in an agressometer (Techno Electronics,
Lucknow, India) for 20 min on a grid floor with a shock interval of
2 s. Day and night reversal was produced by keeping the animals in
the dark during the day and in high-intensity light during the night.
Gastric Ulceration. Immediately after the stress session, rats were
killed by decapitation, the stomachs were split open along the greater
curvature, and the number of discrete ulcers was noted using a
magnascope under 5× magnification. Ulcers were scored according to
the method of Gupta et al., and the mean ulcer severity score was
calculated.40
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Acknowledgment. The Department of Biotechnology, New Delhi,
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Supporting Information Available: Figures S1–S4, providing
antistress activity profile of ethanol extract C003 and its fractions F004-
F006 on AS- and CUS-induced changes in adrenal gland weight, plasma
glucose, corticosterone levels, and creatine kinase. This information is
available free of charge via the internet at http://pubs.acs.org.
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