Phytochemistry Letters 6 (2013) 291–298

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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

Isolation and characterization of the chemical constituents from Plumeria rubra

Nasim Akhtar a, Muhammad Saleem a, Naheed Riaz a,*, M. Shaiq Ali b, Asma Yaqoob a,
Faiz-ul-Hassan Nasim a, Abdul Jabbar a,**
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

Article history: Rubranonoside (=7-O-a-L-rhamnopyranosyl-40 -O-b-D-glucopyranosylnaringenin; (1), a new flavanone

Received 23 June 2012 glycoside, rubranin (=(2S,3S,4R)-2-{[(2R,16E)-2-hydroxyhexaeico-16-en]amino}octadecane-1,3,4-triol-
Received in revised form 7 February 2013 1-O-b-D-glucopyranoside; (2), a new sphingolipid, rubradoid (plumieridine-1-O-b-D-galactopyrano-
Accepted 6 March 2013
side; (3), a new iridoid galactoside, rubrajaleelol (4) and rubrajaleelic acid (5), two new nor-terpenoids
Available online 30 March 2013
together with known iridoids: 1-a-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.
Keywords:
Their structures were assigned from 1H, 13C NMR spectra and 2D NMR analyses (COSY, NOESY, HMQC
Plumeria rubra
Flavonoid
and HMBC experiments) in combination with HRMS experiments and comparison with literature data of
Sphingolipid related compounds. All the isolates (1–8) were tested for their antioxidant, antiurease, cytotoxic and
Iridoids phytotoxic activities and were found almost inactive.
Nortriterpenoids ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Biological activities

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.,
2009; Coppen and Cobb, 1983). Two species namely Plumeria rubra
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.
* Corresponding author. Tel.: +92 3007815194.
** Corresponding author.
E-mail addresses: nrch322@yahoo.com, nrch322@hotmail.com (A. Jabbar).
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)
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 C27H31O14 was determined due to
HRFABMS (ve mode) showing molecular ion peak [MH] at
m/z 579.1725 (calcd. for C27H31O14, 579.1713). The IR spectrum
displayed peaks at 3420 (O–H), 2929 (C–H), 1729 (C5 5O) and 1641–
1485 (C5 5C) cm1. The UV spectrum of 1 showed the absorption
bands at 286 and 315 nm.
The 1H NMR spectrum of 1 (Table 1) showed two m-coupled
doublets at d 6.17 and 6.15 (J = 2.0 Hz), correlated with carbons at d
96.7 and 97.8. An A2B2 system was observed in the same spectrum
at d 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 d 5.39 (1H, J = 12.5, 3.0 Hz), 3.15 (1H,
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
(1H, d, J = 1.5 Hz) together with overlapped signals between d
3.35–4.12 and a methyl doublet at d 1.28 (3H, J = 5.0 Hz). The 13C

1874-3900/$ – 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
292 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-
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
rhamnose units appeared at d 102.5, 72.1, 78.9, 71.2, 78.1, 62.2 and
102.6, 75.4, 71.5, 73.9, 69.9 and 18.2, respectively. Acid hydrolysis
of 1 provided three products which were separated by solvent
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
EtOAc–MeOH–H2O–HOAc; 4:2:2:2 as solvent, and then were
identified as D-glucose and L-rhamnose through their optical
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
 N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298 293

Table 1
1
H, 13C NMR data, HMBC and COSY correlations of 1 (CD3OD, 500 and 125 MHz).

Position dH (J in Hz) dC HMBC (H ! C) COSY (H ! H)

0 0 0
2 5.39, dd (12.5, 3.0) 79.1 C-3, C-4, C-8a, C-1 , C-2 , C-6 H-2/H-3
3 3.15, dd (17.5, 12.5) 2.76, dd (17.5, 3.0) 43.0 C-2, C-4, C-4a, C-10 H-3/H-2
4 – 197.0 – –
4a – 105.0 – –
5 – 164.9 – –
6 6.17, d (2.0) 97.8 C-4a, C-5, C-7, C-8 H-6/H-8
7 – 165.5 – –
8 6.15, d (2.0) 96.7 C-4a, C-6, C-7, C-8a H-8/H-6
8a – 165.7 – –
10 – 130.7 – –
20 ,60 7.32, d (8.5) 129.1 C-10 , C-30 , C-40 H-20 ,60 /H-30 ,50
30 ,50 6.81, d (8.5) 116.3 C-10 , C-20 , C-40 H-30 ,50 /H-20 ,60
40 – 159.0 – –
10 0 5.20, d, (1.5) 102.6 C-7, C-200 , C-30 , C-500 H-100 /H-200
20 0 4.12, m 75.4 C-100 , C-300 , C-400 H-200 /H-100 ,300
30 0 3.35, m 71.5 C-100 , C-200 , C-400 , C-500 H-300 /H-200 ,400
40 0 4.12, m 73.9 C-200 , C-300 , C-500 , C-600 H-400 /H-300 ,500
50 0 3.87, m 69.9 C-100 , C-300 , C-400 , C-600 H-500 /H-400 ,600
60 0 1.28, d (5.0) 18.2 C-400 , C-500 H-600 /H-500
10 0 0 5.24, d (6.5) 102.5 C-40 , C-200 0 , C-300 0 , C-500 0 H-100 0 /H-200 0
20 0 0 3.92, m 72.1 C-100 0 , C-300 0 , C-400 0 H-200 0 /H-100 0 ,300 0
30 0 0 3.61, m 78.9 C-100 0 , C-200 0 , C-400 0 , C-500 0 H-300 0 /H-200 0 ,400 0
40 0 0 3.39, m 71.2 C-200 0 , C-300 0 , C-500 0 , C-600 0 H-400 0 /H-300 0 ,500 0
50 0 0 3.37, m 78.1 C-100 0 , C-300 0 , C-400 0 , C-600 0 H-500 0 /H-400 0 ,600 0
60 0 0 3.84, dd (11.2, 5.0) 62.2 C-400 0 , C-500 0 H-600 0 /H-500 0
3.69, dd (11.2, 2.0)

was fixed at C-7 and C-40 due to HMBC correlations of the H-100 at d
5.20 with d 165.5 (C-7), and H-1000 at d 5.24 with d 159.0 (C-40 ). The
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
(Gaffield, 1970). The above data confirmed 1 as 7-O-a-L-
rhamnopyranosyl-40 -O-b-D-glucopyranosylnaringenin.
Rubranin (2) was obtained as colorless amorphous solid. The
molecular formula was deduced as C50H96NO10 by negative
HRFABMS which showed molecular ion peak [MH] at m/z
870.7120 (calcd. for C50H96NO10, 870.7112) indicating three
degrees of unsaturation. The IR spectrum showed absorption
bands for hydroxyl and amide functions (3500–3200 and
1660 cm1).
The 1H NMR spectrum of 2 (Table 2) showed the presence of an
amide-H at d 7.80 (1H, d, J = 7.8 Hz), a double bond d 5.41 (1H, dt,
J = 15.2, 5.1 Hz) and 5.36 (1H, dt, J = 15.2, 4.6 Hz), three
oxymethines at d 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 d
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

Table 2
1
H, 13C NMR data HMBC and COSY correlations of 2 (CD3OD, 500 and 125 MHz).

Positions dH (J in Hz) dC HMBC (H ! C) COSY (H ! H)

1 4.06, dd (10.5, 6.5) 69.9 C-2, C-3, C-100 H-1/H-2

3.80, dd (10.5, 3.5)
2 4.25, m 51.6 C-1, C-3, C-4, C-10 H-2/H-1,3,N-H
3 3.06, dd (4.6, 4.2) 75.6 C-1, C-2, C-4, C-5 H-3/H-2,4
4 3.52, dt (5.5, 4.2) 72.9 C-2, C-3, C-5, C-6 H-4/H-3,5
5 1.40, m 26.1 C-3, C-4, C-6 H-5/H-4,6
6 1.71, m 33.0 C-4, C-5, C-7 H-6/H-5,7
7–17 1.28  1.32, br s 30.3–30.9 C-5, C-6, C-18 H-7,17/H-6,18
18 0.90, t (6.5) 14.4 C-17 H-18/H-17
N-H 7.80, d (7.8) – N-H/H-2
10 – 177.1 – –
20 4.02, dd (7.5, 4.0) 72.8 C-10 , C-30 , C-40 H-20 /H-30
30 1.76, m 35.7 C-10 , C-20 , C-40 , C-50 H-30 /H-40
1.68, m
40 1.40, m 26.1 C-20 , C-30 , C-50 H-40 /H-30 ,50
50 –140 1.28–1.32, br s 30.3–30.9 C-30 , C-40 , C-150 , C-160 H-50 ,140 /H-40 ,160
150 2.05, m 33.3 C-140 , C-160 , C-170 H-150 /H-140 ,160
160 5.41, dt (15.2, 5.1) 129.1 C-140 , C-150 , C-170 , C-180 H-160 /H150 ,170
170 5.36, dt (15.2, 4.6) 130.7 C-150 , C-160 , C-180 , C-190 H-170 /H-160 ,180
180 1.97, m 33.8 C-160 , C-170 , C-190 H-180 /H-170 ,190
190 –250 1.28–1.32, br s 26.1–33.8 C-170 , C-180 , C-260 H-190 ,250 /H-180 ,260
260 0.90, t (6.5) 14.4 C-250 H-260 /H-250
10 0 4.28, d (8.0) 104.7 C-1, C-200 , C-300 , C-500 H-100 /H-200
20 0 3.18, t (8.0) 75.0 C-100 , C-300 , C-400 H-200 /H-100 ,300
30 0 3.36, t (8.0) 77.9 C-100 , C-200 , C-400 , C-500 H-300 /H-200 ,400
40 0 3.26, t (8.0) 71.6 C-200 , C-300 , C-500 , C-600 H-400 /H-300 ,500
50 0 3.27, m 78.0 C-100 , C-300 , C-400 , C-600 H-500 /H-400 ,600
60 0 3.87, dd (11.0, 5.5) 62.6 C-400 , C-500 H-600 /H-500
3.67, dd (11.0, 3.5)
294 N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298

393 365

704 758
O 335
OH 17'
408
HN 1' 26'
2' 16'
OH
6'' OH
5'' O 18
HO O 3
HO 2 4
OH 1'' 1
3''
OH
179
644 674
614

Fig. 2. Mass fragmentation pattern of 2.

4.25 (1H, m), aliphatic methylenes d 1.28–1.32 (56H, br s) and two
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 d 4.28 (1H, d, J = 8.0 Hz), 3.18 (1H, t, J = 8.0 Hz),
3.36 (1H, t, 8.0 Hz), 3.26 (1H, t, J = 8.0 Hz), 3.27 (1H, m), 3.87 (1H,
dd, J = 11.0, 5.5 Hz) and 3.67 (1H, dd, J = 11.0, 3.5 Hz) suggesting 2
could be a glycosphingolipid (Jin et al., 1994; Ahmad et al., 2006;
Muralidhar et al., 2005). The 13C NMR spectrum of 2 (Table 2) was
in full agreement with that of 1H NMR data as it disclosed the signal
for an amide function (d 177.1), double bond (d 130.7, 129.1),
oxygenated carbons (d 75.6, 72.9, 72.8, 69.9), secondary amine (d
51.6), aliphatic chain (d 30.3–30.9) and sugar moiety (d 104.7, 78.0,
77.9, 75.0, 71.6, 62.6). The internal hydrocarbon skeleton and the
substitutions at various positions were fixed by 1H–1H COSY and
long range HMBC correlations (Table 2). The attachment of sugar
moiety was deduced at C-1 due to its downfield NMR shifts of CH2-
1 (dH 4.06, 3.80; dC 69.9) and was confirmed by HMBC correlations
of H-1 (d 4.06, 3.80) with the carbon resonating at d 104.7 (C-100 ).
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
(Muralidhar et al., 2005) were analyzed by GC-MS and identified as
methyl 2-acetoxyhexaeicosenoate (m/z 466) and 2-acetamino-
1,3,4-triacetoxyoctadecane (m/z 485). The position of double bond
was fixed between C-16,17 in fatty acid chain by permanganate/
periodate oxidative cleavage (Ahmed et al., 2007) of methyl
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
centers was determined by optical rotations of 2 ([a]D = +24.7) and
its methanolysis products ([a]D = 7.1 and +13.9) which were
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-
cane-1,3,4-triol-1-O-b-D-glucopyranoside.
Rubridoidside (3) was purified as white amorphous solid. The
molecular formula C21H27O12 was deduced by HRFABMS which
showed molecular ion peak [M+H]+ at m/z 471.1515 (calcd. for
C21H27O12, 471.1502). The IR spectrum showed the absorption
bands at 3440 (O–H), 3005 (C–H), 1755 (C5 5O), 1603 (C5 5C),
1094 cm1 (C–O) and UV band at 218 nm indicated the presence of
five-membered lactone.
The 1H 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 d
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
presence of hexose moiety due to signals at d 4.61 (1H, d,
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

Table 3
1
H, 13C NMR data HMBC and COSY correlations of 3 (CD3OD, 500 and 125 MHz).

Positions dH (J in Hz) dC HMBC (H ! C) COSY (H ! H)

1 4.93, d (7.0) 93.6 C-3, C-5, C-8, C-9, C-10 H-1/H-9

2 – – – –
3 7.36, d (1.5) 151.4 C-1, C-4, C-5, C-15 –
4 – 108.0 – –
5 3.86, dd (5.5, 2.8) 39.7 C-1, C-3, C-4, C-6, C-7, C-8, C-9, C-15 H-5/H-6,9
6 6.32, dd (5.0, 2.8) 141.3 C-4, C-5, C-7, C-8, C-9 H-6/H-5,7
7 5.36, d (5.0) 127.8 C-5, C-6, C-8, C-9, C-10 H-7/H-6
8 – 96.4 – –
9 2.73, dd (7.0, 5.5) 49.3 C-1, C-4, C-5, C-6, C-7, C-8, C-10 H-9/H-1,5
10 7.33, s 148.8 C-7, C-8, C-9, C-11, C-12, C-13 –
11 – 136.8 – –
12 – 171.3 – –
13 4.48, q (6.0) 62.5 C-10, C-11, C-12, C-14 H-13/H-14
14 1.30, d (6.0) 21.6 C-11, C-13 H-14/H-13
15 – 166.9 – –
OCH3 3.68, s 51.4 C-15 –
10 4.61, d (8.0) 99.0 C-1, C-20 , C-30 , C-60 H-10 /H-20
20 3.13, t (8.0) 72.9 C-10 , C-30 , C-40 H-20 /H-10 ,30
30 3.31, m 76.3 C-10 , C-20 , C-40 , C-50 H-30 /H-20 ,40
40 3.38, d (1.8) 68. 8 C-20 , C-30 , C-50 , C-60 H-40 /H-30 ,50
50 3.17, m 75.9 C-10 , C-30 , C-40 , C-60 H-50 /H-40 ,60
60 3.70, dd (11.2, 5.1) 60.3 C-40 , C-50 H-60 /H-50
3.68, dd (11.2, 2.8)
 N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298 295

various products, amongst which the glycone could be separated Table 4

1 13
H NMR and C NMR data of compound 4 and 5 (CD3OD, 500 and 125 MHz).
and identified as D-galactose through its optical rotation sign and
comparison of the retention time of its trimethylsilyl (TMS) ether Position 4 5
with that of a standard in gas chromatography (GC). The 13C NMR dH dC dH dC
spectrum (BB and DEPT) of 3 (Table 3) showed altogether 21
1 1.91, 1.68, m 48.0 1.94, 1.58, m 48.2
carbon signals corroborated the presence of two methyl, one 2 3.66, ddd 69.7 3.63, ddd 69.5
methylene, thirteen methine and five quaternary carbons. The (11.4, 9.6, 4.8) (11.4, 9.6, 4.8)
signals at d 171.3, 148.8, and 136.8 designated to a five-member 3 3.53, d (9.6) 78.0 2.91, d (9.6) 84.4
a,b-unsaturated g-lactone. The signals at d 166.9, 151.4 and 108.0 4 – 44.1 – 40.1
5 0.87, m 48.8 0.83, m 56.6
indicated the presence of another conjugated ester whereas the
6 1.25, 1.13, m 19.0 1.60, 1.54, m 19.5
signals at d 141.3, 127.8 for an isolated double bond. The internal 7 1.32, 1.02, m 24.6 1.50, 1.24, m 24.0
sequence of the molecule was established by COSY and HMBC 8 – 39.5 – 40.8
correlations (Table 3). The attachment of hexose at C-1 was 9 1.53, m 49.2 1.52, m 49.8
confirmed by HMBC correlation in which H-10 at d 4.61 was 10 – 39.1 – 39.1
11 1.96, 1.64, m 24.4 2.30, 1.96, m 24.4
correlated with d 93.6 (C-1). The relative stereochemistry at
12 5.22, t (3.4) 126.3 5.22, t (3.4) 126.6
positions 1, 5, 9 and 13 could be fixed by NOESY correlations, 13 – 140.0 – 139.0
molecular model and in comparison with the reported compounds 14 – 41.8 – 43.3
(Abe et al., 1988; Kardono et al., 1990). The NOESY correlations 15 1.69, 1.50, m 29.1 1.94, 1.83, m 29.1
16 1.85, 1.60, m 33.8 2.50, 2.03, m 25.3
beween H-5 at d 3.86 and H-9 at d 2.73 confirmed their cis-
17 – 40.5 – 40.5
orientation which was extended to CH3-14 (d 1.30) confirmed their 18 2.20, d (11.4) 54.3 2.21, d (11.4) 54.3
b-orientation. The above data confirmed 3 to be galactoside of 19 1.36, m 40.4 1.91, m 40.4
plumieridine, which is a new natural product based on the 20 1.42, 1.01, m 19.1 1.48, 1.15, m 31.8
glycosidic part. 21 1.58, 1.28, m 31.8 1.55, 1.35, m 34.2
22 1.47, 1.04, m 34.9 1.82, 1.63, m 38.1
Rubrajaleelol (4) was obtained as amorphous solid. The EIMS
23 3.50, d (11.4) 66.1 0.95, s 21.6
spectrum displayed molecular ion at m/z 444, whereas, the 3.26, d (11.4)
molecular formula was established due to HREIMS as C29H48O3 24 0.68, s 13.9 1.00, s 29.3
with six double bond equivalent (DBE). The IR spectrum of 4 25 1.12, s 17.5 0.79, s 17.7
26 0.93, s 17.6 0.94, s 17.6
indicated the presence of hydroxyl and olefinic functions.
27 1.26, s 24.0 1.11, s 24.1
The 1H NMR spectrum was evident of a triterpenoid skeleton as 28 1.03, s 21.6 – 181.0
it displayed five singlet and one doublet methyls at d 1.26, 1.12, 29 0.96, d (6.6) 17.8 0.88, d (6.0) 17.8
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
oxidized to an alcoholic function. The 13C NMR spectrum of 4 was EIMS of 5 showed molecular ion at m/z 458, whereas, the HREIMS
in agreement with the proton data as it afforded 29 carbon signals (m/z 458.3378) depicted the molecular formula as C29H46O4 with
of which six at d 24.0, 21.6, 17.8, 17.6, 17.5 and 13.9 were seven DBE. The IR spectrum was evident of a carboxylic acid nature
identified as methyls and one at d 66.1 was designated as of 5, with olefinic and alcoholic functions. The 1H NMR data was
oxygenated methylene. The 13C NMR shifts olefinic carbons were very similar to that of 4 with the only difference of chemical shifts
observed at d 140.0 and 126.3. This information gave an idea about of fewer hydrogens and lack of the signals for OCH2-23 which were
an ursane type of triterpenoid (Mehmood et al., 2006). Besides, observed in 4. Five singlet and one doublet methyls were found to
various multiplets resonating between d 1.15–2.20 in the 1H NMR appear in the spectrum at d 1.11, 1.00, 0.95, 0.94, 0.79 and 0.88,
spectrum, an olefinic methine was found to resonate at d 5.22, respectively. The signal for methyl-28 disappeared in 1H NMR
whereas, two oxymethines appeared in the spectrum at d 3.66 spectrum; instead, the signal for CH3-24 was observed at d 1.00.
(ddd, J = 11.4, 9.6, 4.8 Hz) and 3.53 (d, J = 9.6 Hz) corresponding to Besides, other signals, the 13C NMR showed a downfield signal at d
the carbons appeared at d 69.7 and 78.0, respectively. The COSY 181.0 attributed to a carbonyl group. This information revealed
correlation and analysis of coupling (Mehmood et al., 2006) that CH3-28 has been oxidized to a carboxylic acid function. The
constants of these two oxymethines indicated their trans- downfield shift of CH2-16 (d 2.50, Hax and 2.03, Heq) and its HMBC
orientation with b-OH at position C-3 (Mehmood et al., 2006). correlation with the carbonyl carbon d 181.0 confirmed 28-oic acid
The other 13C NMR data has been shown in Table 4, which function in 5. On the other hand, the HMBC correlation of H-3 (d
was closely related to ursane type of triterpenoids (Mehmood 2.91) with that of the carbons resonating at d 21.6 (C-23), 29.3 (C-
et al., 2006). 24), 40.1 (C-4) and 56.6 (C-5) revealed that CH3-28 is oxidized
The HMBC correlations of CH3-24 at d 0.68 with the carbons at d instead of CH3-23. The above discussed information and other
78.0 (C-3), 48.8 (C-5), 44.1 (C-4) and 66.1 (C-23) revealed that CH3- NMR data (Table 4) led to the structure of 5 as 30-nor-2a,3b-
23 has been oxidized to an alcoholic group, which in turn was dihydroxyurs-12-en-28-oic acid and is named as rubrajaleelic acid.
further confirmed due to HMBC interaction of CH2-23 with C-3, C-
4, C-5 and CH3-24. In the same spectrum, CH3-29 at d 0.96 was 2.1. Biological studies on compounds 1–8
correlated with a methylene at d 19.1 (C-20) and two methines at d
54.3 (C-18) and 40.4 (C-19) confirming the absence of CH3-30 as is The compounds 1–8 were screened for their antioxidant,
present in ursane series of triterpenoids. The stereochemistry at C- antiurease, cytotoxic and phytotoxic activities (Table 5) at a
2 and C-3 was established on the basis of coupling constants, concentration of 1.0 mg/ml. While the solvent used (MeOH) had no
literature data (Mehmood et al., 2006; Aguirre et al., 2006), NOESY antioxidant, antiurease, and cytotoxic activity it did exhibit a weak
correlations and molecular model. The H-2 showed NOESY seed germination inhibition activity in our phytotoxicity assay.
correlations with Me (24,25) and H-3 with CH2-23 confirmed Compared with Vitamin C, the positive control, antioxidant activity
hydroxyl group a at C-2 and b at C-3. The above data revealed that of compounds 1–8 ranged from 11.07 to 65.67%. Maximum
compound 4 must be 30-nor-2a,3b,23-trihydroxyurs-12-ene and antioxidant activity was shown by compound 1 followed by
is named as rubrajaleelol. compounds 4 and 5. Compounds 3, 2 and 6 had moderate
296 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

Antioxidant assaya Antiurease assayb Cytotoxic assayc Phytotoxic assayd

1 1.0 65.67 34.56 10 20

2 1.0 39.87 59.40 10 20
3 1.0 45.89 23.45 10 20
4 1.0 53.78 65.15 10 70
5 1.0 52.04 26.74 0 70
6 1.0 41.74 61.30 10 20
7 1.0 15.40 65.44 10 70
8 1.0 11.07 70.66 10 20
Solvent – 0.001 0.001 0 30
Control 1.0 70.00 80.00 100 –

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.
3. Experimental
3.1. General experimental procedures
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-
460 Shimadzu IR spectrometer. The 1H and 13C NMR, HMQC, COSY
and HMBC spectra were recorded on Bruker spectrometer
operating at 500 MHz for 1H and 125 MHz for 13C NMR,
respectively. The chemical shift values (d) are reported in ppm
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).
Aluminum sheets pre-coated with silica gel 60 F254
(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
254 and 366 nm and by spraying with ceric sulfate reagent solution
(1% in 10% H2SO4) with heating.
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-
omethane (DCM) as eluent to afford four sub-fractions B1–B4. The
sub-fraction B1 (56 mg) was subjected to flash CC using n-hexane-
EtOAc (1:1) to afford compound 4 (19 mg). The sub-fraction B2
(45 mg) was subjected to flash CC using n-hexane-EtOAc (2:8) to
afford compound 5 (15 mg). The sub-fraction B4 (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.
3.4. Rubranonoside (1)
White amorphous powder (23 mg); [a]D25 +24.7 (c 0.01,
MeOH); IR (KBr): 3420, 2929, 1729, 1641–1485, 1261, 1173 and
849 cm1; UV (MeOH): 286 (3.15), 315 (3.89); 1H and 13C NMR: see
Table 1; HRFABMS: m/z 579.1725 [MH], calcd. for C27H31O14,
579.1713.

3.2. Plant material 3.5. Rubranin (2)

The aerial parts of P. rubra Linn. (Apocynaceae) were collected Colorless gummy solid (29 mg); [a]D25 +24.78 (c 0.013, CH3OH);
in July 2009 from the lawns of Abbasia Campus, The Islamia IR (KBr): 3500–3200, 2945 and 1660 cm1; 1H and 13C NMR: see
University of Bahawalpur, and were identified by Dr. Muhammad Table 2; HRFABMS: m/z 870.7120 [MH] calcd. for C50H96NO10,
Arshad (late), Ex-plant Taxonomist, Cholistan Institute for Desert 870.7112.
 N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
3.5.1. Methanolysis
Compound 2 (12 mg) was refluxed separately with 6 ml of 1 N
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
was identified as methyl b-D-glucopyranoside based on the sign of
optical rotation [a]D25 +76.2 (c 0.1, MeOH) and Co-TLC profile [Rf
0.45 (EtOAc/MeOH/H2O; 5:2:0.5)].
3.5.1.1. Methyl ester derived from 2. [a]D25 7.18 (c 0.01); 1H NMR
(CDCl3, 400 MHz): d 5.35 (2H, dt, J = 15.1, 5.2, H-160 ,170 ), 4.13 (1H t,
J = 6.7, H-20 ), 3.51 (3H, s, MeO), 2.02–2.14 (4H, m, CH2-150 ,180 ), 1.99
(3H, s, MeCO), 1.17–1.27 (36H, br s, CH2-40 –140 ,190 –250 ), 0.81 (3H,
t, J = 6.5, Me-260 ); GC-MS: m/z 466 [M]+.
3.5.1.2. Acetylsphingamine derived from 2. [a]D25 +13.9 (c 0.011);
1
H NMR (CDCl3, 400 MHz): d 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),
4.33 (1H, dd, J = 11.3, 3.2, H-1), 4.17 (1H, dd, J = 5.2, 3.2, H-3); 2.01
(6H, s, 2 MeCO), 1.99 (6H, s, 2 MeCO), 1.13–1.21 (24H, br s,
CH2(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 K2CO3, 6 ml of an
aqueous solution 0.025 M KMnO4 and 0.09 M NaIO4 in 100 ml
round bottom flask. The reaction was allowed to proceed at 37 8C
for 18 h. After acidification with 5 N H2SO4, the solution was
decolorized with a 1 M solution of oxalic acid and extracted with
Et2O (3 to10 ml). The combined organic extract was dried over
Na2SO4, filtered, and concentrated. The resulting carboxylic acids
were methylated with ethereal solution of diazomethane and
analyzed by GC-MS.
3.6. Rubridoidside (3)
White amorphous powder (25 mg); [a]D25 64.68 (c 0.11,
CH3OH); IR (KBr): 3440, 3005, 1755, 1603, and 1094 cm1; UV
(MeOH): 218 (4.01); 1H and 13C NMR: see Table 3; HRFABMS: m/z
471.1515 [M+H]+ calcd. for C21H27O12, 471.1502.
3.7. Rubrajaleelol (4)
White amorphous powder (19 mg); [a]D25 +54.38 (c 0.012,
CHCl3); IR (KBr): 3470, 2950, 1667 and 1070 cm1; 1H- and 13C
NMR: see Table 4; HREIMS: m/z 444.3610 [M]+ calcd. for C29H48O3,
444.3603.
3.8. Rubrajaleelic acid (5)
Colorless amorphous solid (15 mg); [a]D25 + 68.78 (c 0.011,
CHCl3); IR (KBr): 3430, 3285–2560, 1710, 1655 and 1065 cm1; 1H
and 13C NMR: see Table 4; HREIMS: m/z 458.3408 [M]+ calcd. for
C29H46O4, 458.3396.
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 H2O (8 ml). The aglycones were
extracted with EtOAc (3 ml  15 ml). The aqueous phases were
concentrated under reduced pressure and purified on preparative
297
thin layer chromatography using solvent system (EtOAc-MeOH-
H2O-HOAc; 4:2:2:2) and identified as D-glucose and L-rhamnose in
case of compound 1 by the sign of its optical rotation ([a]D20  52)
and ([a]D20 + 8.1), respectively. These sugars were also confirmed
by the retention time of their TMS ethers (D-glucose a-anomer
4.1 min, b-anomer 7.8 min and L-rhamnose 8.6 min) with the
standards and D-galactose in case of compound 3 ([a]D20 +80.18)
and retention time of a-anomer 3.0, b-anomer 5.2 min with a
standard.
3.10. Antioxidant assay
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
N-1-naphthylethylenediamine dihydrochloride under acidic con-
dition to detect NO2 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 ml of 20 mM
sodium nitroprusside solution. Total volume was made up to
1000 ml 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 ml). 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.
3.11. Urease inhibition assay
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
et al., 2008). Briefly, 20 ml of Human urease enzyme (1 unit) was
dispensed in every well of the plate along with 60 ml of phosphate
buffer. The mixture was incubated at 25 8C for 10 min and 5 ml of test
compound was added. After 10 min incubation at room tempera-
ture, 15 ml of 20 mM urea was added. The mixture was incubated at
25 8C for 10 min and then 100 ml reagent 2 was added. After 25 min
incubation at room temperature, absorbance was measured in an
ELISA plate reader (BioTek Model ELx 800) at 630 nm. The values this
obtained were used to calculate percentage inhibition using Gen 5
software. Thiourea was used as the positive control.
3.12. Cytotoxicity assay (in vitro)
Cytotoxic assay was performed using Brine shrimps following a
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.
3.13. Phytotoxicity assay (seed germination assay)
Phytotoxicity in terms of seed germination inhibition was
determined through a published assay (Atta-ur-Rahman and
298 N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298
Choudhry, 1999). Briefly, ten Millet seeds were soaked in the test
sample solution (1.0 mg/ml) overnight. Treated seeds thoroughly
washed with water were placed in presoaked double layered filter
papers in petriplates and incubated at 37 8C for a specific period of
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.
Acknowledgement
The authors are thankful to Higher Education Commission
(HEC) of Pakistan and Alexander von Humboldt (AvH) Foundation,
Germany for financial support.
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