Isolation and characterization of the chemical constituents from Plumeria rubra

Article abstract of DOI:10.1016/j.phytol.2013.03.007

Rubranonoside (=7-O-α-l-rhamnopyranosyl-4′-O-β-d- glucopyranosylnaringenin; (1), a new flavanone glycoside, rubranin (=(2S,3S,4R)-2-{[(2R,16E)-2-hydroxyhexaeico-16-en]amino}octadecane-1,3, 4-triol-1-O-β-d-glucopyranoside; (2), a new sphingolipid, rubradoid (plumieridine-1-O-β-d-galactopyranoside; (3), a new iridoid galactoside, rubrajaleelol (4) and rubrajaleelic acid (5), two new nor-terpenoids together with known iridoids: 1-α-plumieride (6), plumieride p-Z-coumarate (7) and plumieride-p-E-coumarate (8) have been isolated from the EtOAc-soluble fraction of the MeOH extract of Plumeria rubra. Their structures were assigned from ¹H, ¹³C NMR spectra and 2D NMR analyses (COSY, NOESY, HMQC and HMBC experiments) in combination with HRMS experiments and comparison with literature data of related compounds. All the isolates (1-8) were tested for their antioxidant, antiurease, cytotoxic and phytotoxic activities and were found almost inactive.

Full text of DOI:10.1016/j.phytol.2013.03.007

Phytochemistry Letters 6 (2013) 291–298
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Isolation and characterization of the chemical constituents from Plumeria rubra
a
a
a,
b
a
Nasim Akhtar , Muhammad Saleem , Naheed Riaz *, M. Shaiq Ali , Asma Yaqoob ,
a
a,
Faiz-ul-Hassan Nasim , Abdul Jabbar **
a
Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, 63100 Bahawalpur, Pakistan
b
International Centre for Chemical and Biological Sciences (ICCBS), H.E.J. Research Institute of Chemistry, University of Karachi, 75270 Karachi, Pakistan
A R T I C L E I N F O
A B S T R A C T
0
Rubranonoside (=7-O-a-L-rhamnopyranosyl-4 -O-b-D-glucopyranosylnaringenin; (1), a new flavanone
Article history:
Received 23 June 2012
glycoside, rubranin (=(2S,3S,4R)-2-{[(2R,16E)-2-hydroxyhexaeico-16-en]amino}octadecane-1,3,4-triol-
-O- -glucopyranoside; (2), a new sphingolipid, rubradoid (plumieridine-1-O- -galactopyrano-
side; (3), a new iridoid galactoside, rubrajaleelol (4) and rubrajaleelic acid (5), two new nor-terpenoids
together with known iridoids: 1- -plumieride (6), plumieride p-Z-coumarate (7) and plumieride-p-E-
coumarate (8) have been isolated from the EtOAc-soluble fraction of the MeOH extract of Plumeria rubra.
Received in revised form 7 February 2013
Accepted 6 March 2013
1
b
-
D
b-D
Available online 30 March 2013
a
Keywords:
1
13
Their structures were assigned from H, C NMR spectra and 2D NMR analyses (COSY, NOESY, HMQC
and HMBC experiments) in combination with HRMS experiments and comparison with literature data of
related compounds. All the isolates (1–8) were tested for their antioxidant, antiurease, cytotoxic and
phytotoxic activities and were found almost inactive.
Plumeria rubra
Flavonoid
Sphingolipid
Iridoids
Nortriterpenoids
Biological activities
ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1
. Introduction
2. Result and discussion
The genus Plumeria (Apocynaceae) consists of eight species
growing in tropical and sub-tropical regions of the world (Ye et al.,
009; Coppen and Cobb, 1983). Two species namely Plumeria rubra
The methanolic extract of P. rubra was divided into n-hexane
and EtOAc-soluble fractions. The EtOAc-soluble fraction on
chromatography yielded five new secondary metabolites (1–5)
2
and Plumeria obtusa, are found in Pakistan which are grown for
ornamental purposes (Perry and Metzger, 1980). Various species of
this genus are used as medicine to cure diarrhea, gonorrhea,
syphilis, veneral sores and leprosy (Powell and Smith, 1978). The
members of this genus possess anti-inflammatory, diuretic,
emmenagogue, febrifuge, purgative and used as tonic and
expectorant (Galicia et al., 2002). The iridoids like grandines
A–C, phoebegrandine B, and fulvoplumeirin, constituents of
Plumeria acutifolia are used as antibacterial agent (Almahy and
Elegami, 2007; Hall et al., 1951). The aqueous extract of P. rubra
showed antimicrobial (Gupta et al., 2007) anti-inflammatory
activities (Dubois and Rezzonico, 2007) and used for the treatment
of respiratory ailments (Frei et al., 1998; Case et al., 2006).
Plumericin, an iridoid isolated from P. rubra is used as antimicro-
bial agent (Little and Johnstone, 1951). Herein we report the
isolation and structure elucidation of five new (1–5), with three
known iridoids (6–8) from the EtOAc-soluble fraction of P. rubra.
and three known iridoids: 1-a-plumieride (6), plumieride p-Z-
coumarate (7) and plumieride-p-E-coumarate (8) (Siddiqui et al.,
1994) (Fig. 1). Their structures were deduced by IR, 1D and 2D NMR
spectroscopy, and mass spectrometry.
Rubranonoside (1) was isolated as white amorphous powder.
The molecular formula
C
27
H
31
O
14 was determined due to
ꢀ
HRFABMS (ꢀve mode) showing molecular ion peak [MꢀH] at
m/z 579.1725 (calcd. for C27
31
H O14, 579.1713). The IR spectrum
displayed peaks at 3420 (O–H), 2929 (C–H), 1729 (C 55 O) and 1641–
ꢀ
1
1485 (C 55 C) cm . The UV spectrum of 1 showed the absorption
bands at 286 and 315 nm.
1
The H NMR spectrum of 1 (Table 1) showed two m-coupled
doublets at
96.7 and 97.8. An A B system was observed in the same spectrum
at 7.32 (2H, d, J = 8.5 Hz) and 6.81 (2H, d, J = 8.5 Hz) indicated the
presence of 1,4-disubstituted benzene ring. Moreover, three
double doublets at 5.39 (1H, J = 12.5, 3.0 Hz), 3.15 (1H,
d 6.17 and 6.15 (J = 2.0 Hz), correlated with carbons at d
2
2
d
d
J = 17.5, 12.5 Hz) and 2.76 (1H, J = 17.5, 3.0 Hz) indicated 1 to
have naringenin like nucleus (Hammami et al., 2004). The presence
of glucose and rhamnose moieties could be deduced due to the
signals of anomeric protons at
d 5.24 (1H, d, J = 6.5 Hz) and 5.20
*
Corresponding author. Tel.: +92 3007815194.
(
1H, d, J = 1.5 Hz) together with overlapped signals between
d
C
*
* Corresponding author.
13
E-mail addresses: nrch322@yahoo.com, nrch322@hotmail.com (A. Jabbar).
3.35–4.12 and a methyl doublet at
d
1.28 (3H, J = 5.0 Hz). The
1
874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2013.03.007

2
92
N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
Fig. 1. Structure of compounds 1–8 isolated from P. rubra.
NMR spectrum (BB and DEPT) of 1 (Table 1) showed 25 signals for
one methyl, two methylene, thirteen methine and seven quater-
extraction. The EtOAc layer contains naringenin whereas the
glycones could be separated from aqueous layer and purified by
preparative thin layer chromatography (PTLC) developed in
nary carbons. The downfield signals at
d 197.0, 165.7, 165.5, 164.9
and 159.0 were assigned to the conjugated ketone and aromatic
oxygenated quaternary carbons. The carbon signals for glucose and
EtOAc–MeOH–H
2
O–HOAc; 4:2:2:2 as solvent, and then were
-rhamnose through their optical
identified as -glucose and
D
L
rhamnose units appeared at
02.6, 75.4, 71.5, 73.9, 69.9 and 18.2, respectively. Acid hydrolysis
of 1 provided three products which were separated by solvent
d
102.5, 72.1, 78.9, 71.2, 78.1, 62.2 and
rotation signs and comparison of the retention times of their
trimethylsilyl (TMS) derivatives with those of the standards in gas
chromatography (GC). The position of both glucose and rhamnose
1

N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
293
Table 1
1
13
3
H, C NMR data, HMBC and COSY correlations of 1 (CD OD, 500 and 125 MHz).
Position
d
H
(J in Hz)
d
C
HMBC (H ! C)
COSY (H ! H)
0
0
0
2
3
4
5.39, dd (12.5, 3.0)
79.1
43.0
C-3, C-4, C-8a, C-1 , C-2 , C-6
H-2/H-3
0
3.15, dd (17.5, 12.5) 2.76, dd (17.5, 3.0)
C-2, C-4, C-4a, C-1
H-3/H-2
–
197.0
105.0
164.9
97.8
–
–
4
5
6
7
8
8
1
2
3
4
1
2
3
4
5
6
1
2
3
4
5
6
a
–
–
–
–
–
–
6.17, d (2.0)
–
C-4a, C-5, C-7, C-8
H-6/H-8
165.5
96.7
–
–
6.15, d (2.0)
–
C-4a, C-6, C-7, C-8a
H-8/H-6
a
165.7
130.7
129.1
116.3
159.0
102.6
75.4
–
–
–
–
0
0
–
0
0
0
0
0
0
0
0
0 0 0
0
0
,6
7.32, d (8.5)
6.81, d (8.5)
–
C-1 , C-3 , C-4
H-2 ,6 /H-3 ,5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
,5
C-1 , C-2 , C-4
–
H-3 ,5 /H-2 ,6
–
0
0
0
0
0
0
00
00
00
00
00
00
00
0
00
00
00
00
00
00
00
000
000
000
000
000
000
00
5.20, d, (1.5)
4.12, m
3.35, m
4.12, m
3.87, m
1.28, d (5.0)
5.24, d (6.5)
3.92, m
3.61, m
3.39, m
3.37, m
3.84, dd (11.2, 5.0)
C-7, C-2 , C-3 , C-5
H-1 /H-2
00
00
00
00
00
00
00 00
00 00
00 00
00 00
C-1 , C-3 , C-4
H-2 /H-1 ,3
00
00
00
71.5
C-1 , C-2 , C-4 , C-5
H-3 /H-2 ,4
00
00
00
00
00
00
73.9
C-2 , C-3 , C-5 , C-6
H-4 /H-3 ,5
69.9
C-1 , C-3 , C-4 , C-6
H-5 /H-4 ,6
00
0
00
000
00
18.2
C-4 , C-5
H-6 /H-5
000
000
000
102.5
72.1
C-4 , C-2 , C-3 , C-5
H-1 /H-2
000
000
000
000
000
000
000 000
000 000
000 000
000 000
C-1 , C-3 , C-4
H-2 /H-1 ,3
000
000
000
78.9
C-1 , C-2 , C-4 , C-5
H-3 /H-2 ,4
000
000
000
000
000
000
71.2
C-2 , C-3 , C-5 , C-6
H-4 /H-3 ,5
78.1
C-1 , C-3 , C-4 , C-6
H-5 /H-4 ,6
000
000
000
62.2
C-4 , C-5
H-6 /H-5
3
.69, dd (11.2, 2.0)
0
00
was fixed at C-7 and C-4 due to HMBC correlations of the H-1 at
d
870.7120 (calcd. for C50H96NO10, 870.7112) indicating three
0
00
0
5
.20 with
d
165.5 (C-7), and H-1 at
d
5.24 with
d
159.0 (C-4 ). The
degrees of unsaturation. The IR spectrum showed absorption
bands for hydroxyl and amide functions (3500–3200 and
remaining assignments were done by COSY, HMQC and HMBC
spectra shown in Table 1. The absolute stereochemistry at C-2 was
assigned to be S by circular dichroism (CD) analysis which showed
positive Cotton effect at 337 nm and negative one at 294 nm
ꢀ
1
1660 cm ).
1
The H NMR spectrum of 2 (Table 2) showed the presence of an
amide-H at 7.80 (1H, d, J = 7.8 Hz), a double bond 5.41 (1H, dt,
J = 15.2, 5.1 Hz) and 5.36 (1H, dt, J = 15.2, 4.6 Hz), three
oxymethines at 4.02 (1H, dd, J = 7.5, 4.0 Hz), 3.52 (1H, dt,
J = 5.5, 4.2 Hz), 3.06 (1H, dd, J = 4.6, 4.2 Hz), an oxymethylene at
4.06 (1H, dd, J = 10.5, 6.5 Hz), 3.80 (1H, dd, J = 10.5, 3.5 Hz), a
methine proton vicinal to the nitrogen atom of the amide group at
d
d
(
Gaffield, 1970). The above data confirmed
1
as 7-O-
a
-L
-
0
rhamnopyranosyl-4 -O-
b
-D
-glucopyranosylnaringenin.
d
Rubranin (2) was obtained as colorless amorphous solid. The
molecular formula was deduced as C50
HRFABMS which showed molecular ion peak [MꢀH] at m/z
d
H
96NO10 by negative
ꢀ
d
Table 2
1
13
3
H, C NMR data HMBC and COSY correlations of 2 (CD OD, 500 and 125 MHz).
Positions
1
d
H
(J in Hz)
d
C
HMBC (H ! C)
COSY (H ! H)
00
4.06, dd (10.5, 6.5)
69.9
C-2, C-3, C-1
H-1/H-2
3
.80, dd (10.5, 3.5)
0
2
3
4
5
6
4.25, m
51.6
75.6
72.9
26.1
33.0
30.3–30.9
14.4
–
C-1, C-3, C-4, C-1
C-1, C-2, C-4, C-5
C-2, C-3, C-5, C-6
C-3, C-4, C-6
C-4, C-5, C-7
C-5, C-6, C-18
C-17
H-2/H-1,3,N-H
H-3/H-2,4
H-4/H-3,5
H-5/H-4,6
H-6/H-5,7
H-7,17/H-6,18
H-18/H-17
N-H/H-2
3.06, dd (4.6, 4.2)
3.52, dt (5.5, 4.2)
1.40, m
1.71, m
7
1
–17
8
1.28ꢀ 1.32, br s
0.90, t (6.5)
7.80, d (7.8)
–
N-H
0
1
177.1
72.8
35.7
–
–
0
0
0
0
0
0
0
0
0
2
4.02, dd (7.5, 4.0)
1.76, m
C-1 , C-3 , C-4
H-2 /H-3
0
0
0
0
3
C-1 , C-2 , C-4 , C-5
H-3 /H-4
1
.68, m
0
0
0
0
0
0
0
0
0
0
0
4
1.40, m
26.1
C-2 , C-3 , C-5
H-4 /H-3 ,5
0
0
0
0
0
0
5
1
1
1
1
1
2
1
2
3
4
5
6
–14
1.28–1.32, br s
2.05, m
30.3–30.9
33.3
C-3 , C-4 , C-15 , C-16
H-5 ,14 /H-4 ,16
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
6
7
8
C-14 , C-16 , C-17
H-15 /H-14 ,16
0
0
0
0
0
0
0
5.41, dt (15.2, 5.1)
5.36, dt (15.2, 4.6)
1.97, m
129.1
130.7
33.8
C-14 , C-15 , C-17 , C-18
H-16 /H15 ,17
0
0
0
0
0
0
0
C-15 , C-16 , C-18 , C-19
H-17 /H-16 ,18
0 0
H-18 /H-17 ,19
0
0
C-16 , C-17 , C-19
0
0
0
0
0
0
0
9 –25
1.28–1.32, br s
0.90, t (6.5)
4.28, d (8.0)
3.18, t (8.0)
3.36, t (8.0)
3.26, t (8.0)
3.27, m
26.1–33.8
14.4
C-17 , C-18 , C-26
H-19 ,25 /H-18 ,26
0
0
0
6
C-25
H-26 /H-25
0
0
0
0
0
0
0
0
0
0
0
0
00
00
00
00
00
104.7
75.0
C-1, C-2 , C-3 , C-5
H-1 /H-2
00
00
00
00
00
00
00
00
00
00
00 00
00 00
00 00
00 00
C-1 , C-3 , C-4
H-2 /H-1 ,3
00
00
00
00
00
00
00
77.9
C-1 , C-2 , C-4 , C-5
H-3 /H-2 ,4
00
71.6
C-2 , C-3 , C-5 , C-6
H-4 /H-3 ,5
00
78.0
C-1 , C-3 , C-4 , C-6
H-5 /H-4 ,6
00
00
00
00
3.87, dd (11.0, 5.5)
62.6
C-4 , C-5
H-6 /H-5
3.67, dd (11.0, 3.5)

2
94
N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
93 365
335
3
7
58
7
04
O
OH
1
7'
4
08
HN 1'
26'
2
OH
'
16'
18
OH
6
''
5
''
O
HO
HO
O
3
2
4
1
OH1''
3
''
OH
1
79
6
44
674
6
14
Fig. 2. Mass fragmentation pattern of 2.
4
.25 (1H, m), aliphatic methylenes
d
1.28–1.32 (56H, br s) and two
2-acetoxyhexaeicosenoate, yielded a mixture of carboxylic acids
which on methylation and GC-MS analysis provided peaks for
methyl-2-acetoxyhexadecan-1,16-dioate (m/z 372) and methyl-
decanoate (m/z 186). The stereochemistry at all the stereogenic
methyls at
d 0.90 (6H, t, J = 6.5 Hz) indicated 2 could be a
sphingolipid (Riaz et al., 2007). It also showed the signals for
hexose moiety at 4.28 (1H, d, J = 8.0 Hz), 3.18 (1H, t, J = 8.0 Hz),
.36 (1H, t, 8.0 Hz), 3.26 (1H, t, J = 8.0 Hz), 3.27 (1H, m), 3.87 (1H,
d
3
centers was determined by optical rotations of 2 ([
a
]
D
= +24.7) and
dd, J = 11.0, 5.5 Hz) and 3.67 (1H, dd, J = 11.0, 3.5 Hz) suggesting 2
its methanolysis products ([
a]
D
= ꢀ7.1 and +13.9) which were
could be a glycosphingolipid (Jin et al., 1994; Ahmad et al., 2006;
found similar with those having (2S,3S,4R)-configurations (Garg
and Agrawal, 1995; Muralidhar et al., 2003; Natori et al., 1994; Riaz
et al., 2007). Based on these evidences, 2 could be identified as
(2S,3S,4R)-2-{[(2R,16E)-2-hydroxyhexaeico-16-en]amino}octade-
13
Muralidhar et al., 2005). The C NMR spectrum of 2 (Table 2) was
1
in full agreement with that of H NMR data as it disclosed the signal
for an amide function (
oxygenated carbons ( 75.6, 72.9, 72.8, 69.9), secondary amine (
30.3–30.9) and sugar moiety ( 104.7, 78.0,
7.9, 75.0, 71.6, 62.6). The internal hydrocarbon skeleton and the
d 177.1), double bond (d 130.7, 129.1),
d
d
cane-1,3,4-triol-1-O-
Rubridoidside (3) was purified as white amorphous solid. The
molecular formula C21 12 was deduced by HRFABMS which
showed molecular ion peak [M+H] at m/z 471.1515 (calcd. for
12, 471.1502). The IR spectrum showed the absorption
bands at 3440 (O–H), 3005 (C–H), 1755 (C 55 O), 1603 (C 55 C),
b-D-glucopyranoside.
5
7
1.6), aliphatic chain (
d
d
27
H O
1
1
+
substitutions at various positions were fixed by H– H COSY and
long range HMBC correlations (Table 2). The attachment of sugar
21 27
C H O
moiety was deduced at C-1 due to its downfield NMR shifts of CH
4.06, 3.80; 69.9) and was confirmed by HMBC correlations
00
of H-1 ( 4.06, 3.80) with the carbon resonating at d 104.7 (C-1 ).
2
-
ꢀ1
1
(
d
H
d
C
1094 cm (C–O) and UV band at 218 nm indicated the presence of
five-membered lactone.
d
1
The length of the fatty acid chain containing a double bond was
determined by characteristic fragments at m/z 393, 350 and the
amine chain at m/z 479 and 461 (Fig. 2). Methanolysis of 2 with
methanolic HCl (Muralidhar et al., 2005) provided the methyl ester
of fatty acid, a sphingosine base and methylated sugar. Both
methyl ester of fatty acid and sphingosine base on acetylation
The H NMR spectrum of 3 (Table 3) displayed signals for
olefinic protons at
d 7.36 (1H, d, J = 1.5 Hz), 7.33 (1H, s), 6.32 (1H,
dd, J = 5.5, 2.8 Hz) and 5.36 (1H, d, 5.0 Hz), saturated methines at
4.93 (1H, d, J = 7.0 Hz), 4.48 (1H, q, J = 6.0 Hz), 3.86 (1H, dd, J = 5.5,
2.8 Hz) and 2.73 (1H, dd, J = 7.0, 5.5 Hz). It also indicated the
d
presence of hexose moiety due to signals at
d 4.61 (1H, d,
(
Muralidhar et al., 2005) were analyzed by GC-MS and identified as
methyl 2-acetoxyhexaeicosenoate (m/z 466) and 2-acetamino-
,3,4-triacetoxyoctadecane (m/z 485). The position of double bond
J = 8.0 Hz), 3.13 (1H, t, J = 8.0 Hz), 3.31 (1H, m), 3.38 (1H, d,
J = 1.8 Hz), 3.17 (1H, m), 3.70 (1H, dd, J = 11.2, 5.1 Hz), 3.68 (1H, dd,
J = 11.2, 2.8 Hz). The above data closely related to the spectral
values reported for plumieridine and other related iridoids (Saleem
et al., 2011; Ye et al., 2008). The acid hydrolysis of 3 provided
1
was fixed between C-16,17 in fatty acid chain by permanganate/
periodate oxidative cleavage (Ahmed et al., 2007) of methyl
Table 3
1
13
3
H, C NMR data HMBC and COSY correlations of 3 (CD OD, 500 and 125 MHz).
Positions
d
H
(J in Hz)
d
C
HMBC (H ! C)
COSY (H ! H)
0
1
2
3
4
5
6
7
8
9
4.93, d (7.0)
–
93.6
–
C-3, C-5, C-8, C-9, C-1
H-1/H-9
–
–
7.36, d (1.5)
–
151.4
108.0
39.7
C-1, C-4, C-5, C-15
–
–
–
3.86, dd (5.5, 2.8)
6.32, dd (5.0, 2.8)
5.36, d (5.0)
–
C-1, C-3, C-4, C-6, C-7, C-8, C-9, C-15
H-5/H-6,9
141.3
127.8
96.4
C-4, C-5, C-7, C-8, C-9
H-6/H-5,7
C-5, C-6, C-8, C-9, C-10
H-7/H-6
–
–
2.73, dd (7.0, 5.5)
7.33, s
49.3
C-1, C-4, C-5, C-6, C-7, C-8, C-10
H-9/H-1,5
1
1
1
1
1
1
0
1
2
3
4
5
148.8
136.8
171.3
62.5
C-7, C-8, C-9, C-11, C-12, C-13
–
–
–
–
–
–
–
4.48, q (6.0)
1.30, d (6.0)
–
C-10, C-11, C-12, C-14
H-13/H-14
21.6
C-11, C-13
H-14/H-13
166.9
51.4
–
–
–
OCH
3
3.68, s
C-15
0
0
0
0
0
0
0
0
1
4.61, d (8.0)
3.13, t (8.0)
3.31, m
99.0
C-1, C-2 , C-3 , C-6
H-1 /H-2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
72.9
C-1 , C-3 , C-4
H-2 /H-1 ,3
0
0
0
0
0 0
3
76.3
C-1 , C-2 , C-4 , C-5
H-3 /H-2 ,4
0 0
H-4 /H-3 ,5
0
0
4
3.38, d (1.8)
3.17, m
68. 8
75.9
C-2 , C-3 , C-5 , C-6
0 0
0
5
C-1 , C-3 , C-4 , C-6
H-5 /H-4 ,6
0
0
0
0
6
3.70, dd (11.2, 5.1)
60.3
C-4 , C-5
H-6 /H-5
3.68, dd (11.2, 2.8)

N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
295
Table 4
various products, amongst which the glycone could be separated
and identified as -galactose through its optical rotation sign and
comparison of the retention time of its trimethylsilyl (TMS) ether
with that of a standard in gas chromatography (GC). The C NMR
spectrum (BB and DEPT) of 3 (Table 3) showed altogether 21
carbon signals corroborated the presence of two methyl, one
methylene, thirteen methine and five quaternary carbons. The
1
13
3
H NMR and C NMR data of compound 4 and 5 (CD OD, 500 and 125 MHz).
D
Position
4
5
1
3
d
H
d
C
d
H
d
C
1
2
1.91, 1.68, m
48.0
69.7
1.94, 1.58, m
3.63, ddd
(11.4, 9.6, 4.8)
2.91, d (9.6)
–
48.2
69.5
3.66, ddd
(11.4, 9.6, 4.8)
signals at
-unsaturated
indicated the presence of another conjugated ester whereas the
signals at 141.3, 127.8 for an isolated double bond. The internal
d
171.3, 148.8, and 136.8 designated to a five-member
3
3.53, d (9.6)
–
78.0
44.1
48.8
19.0
24.6
39.5
49.2
39.1
24.4
126.3
140.0
41.8
29.1
33.8
40.5
54.3
40.4
19.1
31.8
34.9
66.1
84.4
40.1
56.6
19.5
24.0
40.8
49.8
39.1
24.4
126.6
139.0
43.3
29.1
25.3
40.5
54.3
40.4
31.8
34.2
38.1
21.6
4
a
,b
g
-lactone. The signals at 166.9, 151.4 and 108.0
d
5
0.87, m
0.83, m
6
1.25, 1.13, m
1.32, 1.02, m
–
1.60, 1.54, m
1.50, 1.24, m
–
d
7
sequence of the molecule was established by COSY and HMBC
8
correlations (Table 3). The attachment of hexose at C-1 was
9
1.53, m
1.52, m
0
10
–
–
confirmed by HMBC correlation in which H-1 at
d 4.61 was
1
1
1
1
2
3
1.96, 1.64, m
5.22, t (3.4)
–
2.30, 1.96, m
5.22, t (3.4)
–
correlated with
d 93.6 (C-1). The relative stereochemistry at
positions 1, 5, 9 and 13 could be fixed by NOESY correlations,
molecular model and in comparison with the reported compounds
14
–
–
1
1
1
1
5
6
7
8
1.69, 1.50, m
1.85, 1.60, m
–
1.94, 1.83, m
2.50, 2.03, m
–
(
Abe et al., 1988; Kardono et al., 1990). The NOESY correlations
beween H-5 at 3.86 and H-9 at 2.73 confirmed their cis-
orientation which was extended to CH -14 ( 1.30) confirmed their
-orientation. The above data confirmed 3 to be galactoside of
d
d
3
d
2.20, d (11.4)
1.36, m
2.21, d (11.4)
1.91, m
b
19
20
plumieridine, which is a new natural product based on the
glycosidic part.
Rubrajaleelol (4) was obtained as amorphous solid. The EIMS
spectrum displayed molecular ion at m/z 444, whereas, the
1.42, 1.01, m
1.58, 1.28, m
1.47, 1.04, m
3.50, d (11.4)
1.48, 1.15, m
1.55, 1.35, m
1.82, 1.63, m
0.95, s
2
2
2
1
2
3
3.26, d (11.4)
molecular formula was established due to HREIMS as C29
with six double bond equivalent (DBE). The IR spectrum of 4
H
48
O
3
24
25
0.68, s
13.9
17.5
17.6
24.0
21.6
17.8
1.00, s
0.79, s
0.94, s
1.11, s
–
29.3
17.7
17.6
24.1
181.0
17.8
1.12, s
2
2
2
6
7
8
0.93, s
indicated the presence of hydroxyl and olefinic functions.
1.26, s
1
The H NMR spectrum was evident of a triterpenoid skeleton as
1.03, s
it displayed five singlet and one doublet methyls at
d
1.26, 1.12,
29
0.96, d (6.6)
0.88, d (6.0)
1
.03, 0.93, 0.68 and 0.96, respectively. The signals for an
oxygenated methylene at
d 3.50 (1H, d, J = 11.4 Hz) and 3.26
(
1H, d, J = 11.0 Hz) revealed that one singlet methyl has been
Rubrajaleelic acid (5) was obtained as amorphous solid. The
EIMS of 5 showed molecular ion at m/z 458, whereas, the HREIMS
(m/z 458.3378) depicted the molecular formula as C29 with
1
3
oxidized to an alcoholic function. The C NMR spectrum of 4 was
in agreement with the proton data as it afforded 29 carbon signals
46 4
H O
of which six at
identified as methyls and one at
d
24.0, 21.6, 17.8, 17.6, 17.5 and 13.9 were
66.1 was designated as
oxygenated methylene. The C NMR shifts olefinic carbons were
observed at 140.0 and 126.3. This information gave an idea about
an ursane type of triterpenoid (Mehmood et al., 2006). Besides,
seven DBE. The IR spectrum was evident of a carboxylic acid nature
of 5, with olefinic and alcoholic functions. The H NMR data was
very similar to that of 4 with the only difference of chemical shifts
1
d
1
3
d
of fewer hydrogens and lack of the signals for OCH
observed in 4. Five singlet and one doublet methyls were found to
appear in the spectrum at 1.11, 1.00, 0.95, 0.94, 0.79 and 0.88,
respectively. The signal for methyl-28 disappeared in H NMR
spectrum; instead, the signal for CH -24 was observed at 1.00.
Besides, other signals, the C NMR showed a downfield signal at
181.0 attributed to a carbonyl group. This information revealed
that CH -28 has been oxidized to a carboxylic acid function. The
downfield shift of CH -16 ( 2.50, Hax and 2.03, Heq) and its HMBC
correlation with the carbonyl carbon 181.0 confirmed 28-oic acid
function in 5. On the other hand, the HMBC correlation of H-3 (
2.91) with that of the carbons resonating at 21.6 (C-23), 29.3 (C-
24), 40.1 (C-4) and 56.6 (C-5) revealed that CH -28 is oxidized
instead of CH -23. The above discussed information and other
NMR data (Table 4) led to the structure of 5 as 30-nor-2 ,3
2
-23 which were
1
various multiplets resonating between
spectrum, an olefinic methine was found to resonate at
whereas, two oxymethines appeared in the spectrum at
d
1.15–2.20 in the H NMR
d
1
d
d
5.22,
3.66
3
d
1
3
(
ddd, J = 11.4, 9.6, 4.8 Hz) and 3.53 (d, J = 9.6 Hz) corresponding to
d
the carbons appeared at
correlation and analysis of coupling (Mehmood et al., 2006)
constants of these two oxymethines indicated their trans-
d 69.7 and 78.0, respectively. The COSY
3
2
d
orientation with
b
-OH at position C-3 (Mehmood et al., 2006).
d
1
3
The other C NMR data has been shown in Table 4, which
was closely related to ursane type of triterpenoids (Mehmood
et al., 2006).
d
d
3
The HMBC correlations of CH
8.0 (C-3), 48.8 (C-5), 44.1 (C-4) and 66.1 (C-23) revealed that CH
3 has been oxidized to an alcoholic group, which in turn was
-23 with C-3, C-
-29 at 0.96 was
19.1 (C-20) and two methines at
4.3 (C-18) and 40.4 (C-19) confirming the absence of CH -30 as is
present in ursane series of triterpenoids. The stereochemistry at C-
and C-3 was established on the basis of coupling constants,
3
-24 at
d
0.68 with the carbons at
d
3
7
2
3
-
a b-
dihydroxyurs-12-en-28-oic acid and is named as rubrajaleelic acid.
further confirmed due to HMBC interaction of CH
, C-5 and CH -24. In the same spectrum, CH
correlated with a methylene at
2
4
3
3
d
2.1. Biological studies on compounds 1–8
d
d
5
3
The compounds 1–8 were screened for their antioxidant,
antiurease, cytotoxic and phytotoxic activities (Table 5) at a
concentration of 1.0 mg/ml. While the solvent used (MeOH) had no
antioxidant, antiurease, and cytotoxic activity it did exhibit a weak
seed germination inhibition activity in our phytotoxicity assay.
Compared with Vitamin C, the positive control, antioxidant activity
of compounds 1–8 ranged from 11.07 to 65.67%. Maximum
antioxidant activity was shown by compound 1 followed by
compounds 4 and 5. Compounds 3, 2 and 6 had moderate
2
literature data (Mehmood et al., 2006; Aguirre et al., 2006), NOESY
correlations and molecular model. The H-2 showed NOESY
correlations with Me (24,25) and H-3 with CH
hydroxyl group at C-2 and at C-3. The above data revealed that
,3 ,23-trihydroxyurs-12-ene and
2
-23 confirmed
a
b
compound 4 must be 30-nor-2
a b
is named as rubrajaleelol.

2
96
N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
Table 5
Antioxidant, antiurease, cytotoxic and phytotoxic activities of compounds 1–8.
Sr. No
Concentration mg/ml
% Age activity
a
b
c
d
Antioxidant assay
Antiurease assay
Cytotoxic assay
Phytotoxic assay
1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
–
65.67
39.87
45.89
53.78
52.04
41.74
15.40
11.07
0.001
70.00
34.56
59.40
23.45
65.15
26.74
61.30
65.44
70.66
0.001
80.00
10
10
10
10
0
20
20
20
70
70
20
70
20
30
–
2
3
4
5
6
10
10
10
0
7
8
Solvent
Control
1.0
100
Positive controls used.
a
Ascorbic acid.
b
Thiourea.
c
Etoposide.
d
Methanol used as solvent showed inhibitory effect and was therefore taken as positive control. Values shown for the test compounds have been adjusted accordingly. In
the phytotoxicity assays water was used as the negative control where 100% seed germination was observed.
antioxidant activity while compounds 8 and 7 were found to be
weak antioxidants. Compound 8 exhibited the strongest antiurease
activity followed by compounds 7 and 4 and 6 and 2 which had
comparable activities. Moderate antiurease activity was shown by
the compounds 1, 5 and 3 in the descending order. None of the test
compounds 1–8, had any significant cytotoxic activity while
compounds 4, 5 and 7 were found to be seed germination
inhibitors the remaining had almost no seed germination activity.
Our results indicate that the isolated compounds possess a variety
of biological activities. Mechanistic details of the observed
characteristics however need further investigations to establish
structure–activity relationships.
Studies (CIDS), The Islamia University of Bahawalpur, Pakistan,
where a voucher specimen (No. PL-09-273) has been deposited.
3.3. Extraction and isolation
The shade dried and ground plant material (10 kg) was
extracted twice with methanol (60 L) for a week at room
temperature, concentrated on rotavapour to a dark green mass
(400 g) which was suspended in water and extracted with n-
hexane (25 L), ethyl acetate (EtOAc) (25 L) and n-butanol (15 L).
The EtOAc soluble fraction (60 g) was subjected to silica gel column
chromatography using n-hexane, EtOAc, and MeOH as eluent in an
increasing polarity order to get five fractions A–E. The fraction B
(1.8 g) was subjected to column chromatography using dichlor-
3
. Experimental
omethane (DCM) as eluent to afford four sub-fractions B
sub-fraction B (56 mg) was subjected to flash CC using n-hexane-
EtOAc (1:1) to afford compound 4 (19 mg). The sub-fraction B
(45 mg) was subjected to flash CC using n-hexane-EtOAc (2:8) to
afford compound 5 (15 mg). The sub-fraction B (75 mg) afforded
compound 6 (10 mg), 7 (13 mg) and 8 (11 mg) at 90, 85 and 80%
EtOAc in n-hexane, respectively. The fraction C (85 mg) obtained
from the main column at n-hexane-EtOAc (1:9) was subjected to
flash CC using n-hexane-EtOAc (0.8:9.2) to isolate compound 3
(25 mg). The fraction D (79 mg) obtained with pure EtOAc on
further silica gel chromatography with EtOAc-MeOH (9.9:0.1)
provided compound 2 (29 mg). The fraction E (1.34 g) eluted from
the main column with EtOAc-MeOH (9.5:0.5) was further purified
by silica gel column chromatography using solvent system EtOAc-
MeOH (9.4:0.6) to get compound 1 (23 mg). The remaining
fractions contain salts and sugars.
1
–B4. The
3
.1. General experimental procedures
1
2
Melting points were determined by a Buchi 434 melting point
apparatus. UV spectra were obtained in methanol on Schimadazu
UV-240 spectrophotometer. Optical rotations were recorded on
JASCO DIP-360 polarimeter. The IR spectra were recorded on IR-
4
1
13
4
60 Shimadzu IR spectrometer. The H and C NMR, HMQC, COSY
and HMBC spectra were recorded on Bruker spectrometer
operating at 500 MHz for
respectively. The chemical shift values (
and the coupling constants (J) are in Hz. The EIMS, HREIMS were
recorded on JMS HX 110 with a data system and HRFABMS were
recorded on JMS-DA 500 mass spectrometers and shown in m/z.
The gas chromatography (GC) was performed on a Shimadzu gas
chromatograph (GC-9A) (3% OV-1 silanized chromosorb W,
column temperature 180 8C, injection port and detector tempera-
ture 275–300 8C, flow rate 35 ml/min, flame-ionization detector).
1
13
H
and 125 MHz for
C NMR,
d
) are reported in ppm
3.4. Rubranonoside (1)
Aluminum sheets pre-coated with silica gel 60
20 cm ꢁ 20 cm, 0.2 mm thick; E. Merck) were used for TLC and
silica gel (230–400 mesh) was used for column chromatography
CC). Visualization of the TLC plates was carried out under UV at
54 and 366 nm and by spraying with ceric sulfate reagent solution
1% in 10% H SO ) with heating.
F
254
2
5
(
White amorphous powder (23 mg); [
a
]
D
+24.7 (c 0.01,
MeOH); IR (KBr): 3420, 2929, 1729, 1641–1485, 1261, 1173 and
849 cm ; UV (MeOH): 286 (3.15), 315 (3.89); H and C NMR: see
Table 1; HRFABMS: m/z 579.1725 [MꢀH] , calcd. for C27
ꢀ
1
1
13
(
2
(
ꢀ
31 14
H O ,
2
4
579.1713.
3.2. Plant material
3.5. Rubranin (2)
2
5
The aerial parts of P. rubra Linn. (Apocynaceae) were collected
Colorless gummy solid (29 mg); [
a]
D
3
+24.78 (c 0.013, CH OH);
ꢀ
1
1
13
in July 2009 from the lawns of Abbasia Campus, The Islamia
University of Bahawalpur, and were identified by Dr. Muhammad
Arshad (late), Ex-plant Taxonomist, Cholistan Institute for Desert
IR (KBr): 3500–3200, 2945 and 1660 cm ; H and C NMR: see
Table 2; HRFABMS: m/z 870.7120 [MꢀH] calcd. for C50
ꢀ
H96NO10,
870.7112.

N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
297
3
.5.1. Methanolysis
thin layer chromatography using solvent system (EtOAc-MeOH-
O-HOAc; 4:2:2:2) and identified as -glucose and -rhamnose in
case of compound 1 by the sign of its optical rotation ([
Compound 2 (12 mg) was refluxed separately with 6 ml of 1 N
H
2
D
L
20
HCl and 25 ml of MeOH for 15 h. The reaction mixture was then
extracted with n-hexane (3 ml ꢁ 25 ml) to obtain the correspond-
ing fatty acid methyl esters, which were analyzed by GC-MS after
acetylation with aceticanhydride-pyridine. The aqueous layer was
evaporated to dryness, and the residue was separated by silica gel
column chromatography as sphingosine base and methylated
sugar. The base was acetylated and analyzed by GC-MS. The sugar
a
]
D
ꢀ 52)
+ 8.1), respectively. These sugars were also confirmed
by the retention time of their TMS ethers ( -glucose -anomer
4.1 min, -anomer 7.8 min and -rhamnose 8.6 min) with the
standards and -galactose in case of compound 3 ([
2
0
and ([a]
D
D
a
b
L
20
D
a
]
D
+80.18)
and retention time of
standard.
a-anomer 3.0, b-anomer 5.2 min with a
was identified as methyl
b
-
D
-glucopyranoside based on the sign of
25
optical rotation [
a]
D
+76.2 (c 0.1, MeOH) and Co-TLC profile [R
f
3.10. Antioxidant assay
0
.45 (EtOAc/MeOH/H
2
O; 5:2:0.5)].
Nitric oxide scavenging antioxidant activity assay was
performed following the published procedure based on diazoti-
zation reaction described by Griess (Garratt, 1964). The assay uses
sodium nitroprusside as the source of NO and sulfanilamide and
2
5
1
3
.5.1.1. Methyl ester derived from 2. [
a
]
D
ꢀ7.18 (c 0.01); H NMR
0
0
(CDCl
3
, 400 MHz):
d
5.35 (2H, dt, J = 15.1, 5.2, H-16 ,17 ), 4.13 (1H t,
J = 6.7, H-2 ), 3.51 (3H, s, MeO), 2.02–2.14 (4H, m, CH
0
0
0
2
-15 ,18 ), 1.99
0
0
0
0
(3H, s, MeCO), 1.17–1.27 (36H, br s, CH
2
-4 –14 ,19 –25 ), 0.81 (3H,
N-1-naphthylethylenediamine dihydrochloride under acidic con-
0
+
ꢀ
t, J = 6.5, Me-26 ); GC-MS: m/z 466 [M] .
dition to detect NO
2
generated at the expense of NO by the
antioxidant system. Briefly, a known quantity of the test
compound in solution form was mixed with 100 l of 20 mM
sodium nitroprusside solution. Total volume was made up to
1000 l with 200 mM Phosphate buffer, pH 7.4. The content were
mixed well and incubated at 37 8C for 2 h followed by addition of
Griess reagent (100 l). The mixture was kept at room tempera-
ture for 20 min. Optical density (OD) of the colored solution
formed was measured at 528 nm. Ascorbic acid was used as the
positive control. OD reduced with increasing concentration of the
antioxidant component.
2
5
3
.5.1.2. Acetylsphingamine derived from 2. [
a]
D
+13.9 (c 0.011);
8.06 (1H, d, J = 7.3, NH), 4.55 (1H, dd,
J = 5.2, 4.3, H-4), 4.51 (1H, m, H-2), 4.42 (1H, dd, J = 11.3, 5.5, H-1),
.33 (1H, dd, J = 11.3, 3.2, H-1), 4.17 (1H, dd, J = 5.2, 3.2, H-3); 2.01
6H, s, 2ꢁ MeCO), 1.99 (6H, s, 2ꢁ MeCO), 1.13–1.21 (24H, br s,
m
1
3
H NMR (CDCl , 400 MHz): d
m
4
(
CH
m
+
2
(6–17), 0.86 (3H, t, J = 6.6, Me-18); GC-MS: m/z 485 [M] .
3
.5.2. Oxidative cleavage of the double bond in 2
To the solution of methyl ester of compound 2 (4 mg) in
acetone, added 1 ml of 0.04 M solution of K
aqueous solution 0.025 M KMnO and 0.09 M NaIO
round bottom flask. The reaction was allowed to proceed at 37 8C
for 18 h. After acidification with 5 N H SO , the solution was
decolorized with a 1 M solution of oxalic acid and extracted with
Et O (3 to10 ml). The combined organic extract was dried over
Na SO , filtered, and concentrated. The resulting carboxylic acids
2
CO
3
, 6 ml of an
4
4
in 100 ml
3.11. Urease inhibition assay
2
4
Antiurease activity of the isolated compounds was determined
by an optimized 96 well microplate-based modified Berthelot
(phenolhypochlorite) method using a kit (Gesellschaft fur Biochem-
ica und Diagonistica mbH, Germany) as described elsewhere (Pervez
2
2
4
were methylated with ethereal solution of diazomethane and
analyzed by GC-MS.
et al., 2008). Briefly, 20
dispensed in every well of the plate along with 60
buffer. Themixturewasincubatedat25 8Cfor10 minand5
compound was added. After 10 min incubation at room tempera-
ture, 15 l of 20 mM urea was added. The mixture was incubated at
25 8C for 10 min and then 100 l reagent 2 was added. After 25 min
incubation at room temperature, absorbance was measured in an
ELISA platereader (BioTek ModelELx 800) at630 nm. The valuesthis
obtained were used to calculate percentage inhibition using Gen 5
software. Thiourea was used as the positive control.
m
l of Human urease enzyme (1 unit) was
l of phosphate
loftest
m
m
3.6. Rubridoidside (3)
m
2
5
White amorphous powder (25 mg); [
a]
D
ꢀ64.68 (c 0.11,
m
ꢀ
1
CH OH); IR (KBr): 3440, 3005, 1755, 1603, and 1094 cm ; UV
MeOH): 218 (4.01); H and C NMR: see Table 3; HRFABMS: m/z
3
1
13
(
4
+
71.1515 [M+H] calcd. for C21
27
H O12, 471.1502.
3.7. Rubrajaleelol (4)
3
.12. Cytotoxicity assay (in vitro)
2
5
White amorphous powder (19 mg); [
CHCl ); IR (KBr): 3470, 2950, 1667 and 1070 cm
NMR: see Table 4; HREIMS: m/z 444.3610 [M] calcd. for C29
44.3603.
a
]
D
+54.38 (c 0.012,
ꢀ
1
1
13
3
;
H- and
C
Cytotoxic assay was performed using Brine shrimps following a
+
H
48
O
3
,
published method (Atta-ur-Rahman and Choudhry, 1999). Briefly,
brine shrimps (Artemia salina, Leach) eggs were hatched in artificial
seawater. A sample of the test compound containing the pre-
determined quantity was transferred to vials (three for each
concentration) with one vial as the negative control to which was
added the solvent, MeOH, only. The solvent was allowed to
evaporate overnight. When the shrimp larvae were ready, 1 mL of
sea water along with 10 shrimps was transferred to each vial and
the volume was adjusted to 5 mL per vial with sea water. The
number of survivors was counted after 24 h to calculate the
cytotoxicity in terms of %age of the dead larvae. Etoposide, an anti-
cancer drug, was used as the positive control.
4
3.8. Rubrajaleelic acid (5)
2
D
5
Colorless amorphous solid (15 mg); [
a]
+ 68.78 (c 0.011,
ꢀ1 1
CHCl ); IR (KBr): 3430, 3285–2560, 1710, 1655 and 1065 cm ; H
and C NMR: see Table 4; HREIMS: m/z 458.3408 [M] calcd. for
, 458.3396.
3
13
+
29 46 4
C H O
3.9. Acid hydrolysis
A solution of compounds 1,3 (8 mg each) in MeOH (5 ml)
containing 1 N HCl (4 ml) was refluxed for 4 h, concentrated under
reduced pressure, and diluted with H O (8 ml). The aglycones were
3.13. Phytotoxicity assay (seed germination assay)
2
extracted with EtOAc (3 ml ꢁ 15 ml). The aqueous phases were
Phytotoxicity in terms of seed germination inhibition was
determined through a published assay (Atta-ur-Rahman and
concentrated under reduced pressure and purified on preparative

2
98
N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
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sample solution (1.0 mg/ml) overnight. Treated seeds thoroughly
washed with water were placed in presoaked double layered filter
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time. The numbers of seeds germinated were noted at different
time intervals i.e. 6, 12, 24, 36, 72 h. The percentage of un-
germinated seeds was used to determine phytotoxic character of
the test compound. Seeds soaked in methanol and water were used
as the positive and the negative controls, respectively.
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