Isolation and characterization of the chemical constituents from Plumeria rubra

Phytochemistry Letters 6 (2013) 291–298

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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 b

Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, 63100 Bahawalpur, Pakistan 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: Received 23 June 2012 Received in revised form 7 February 2013 Accepted 6 March 2013 Available online 30 March 2013

Rubranonoside (=7-O-a-L-rhamnopyranosyl-40 -O-b-D-glucopyranosylnaringenin; (1), a new flavanone glycoside, rubranin (=(2S,3S,4R)-2-{[(2R,16E)-2-hydroxyhexaeico-16-en]amino}octadecane-1,3,4-triol1-O-b-D-glucopyranoside; (2), a new sphingolipid, rubradoid (plumieridine-1-O-b-D-galactopyranoside; (3), a new iridoid galactoside, rubrajaleelol (4) and rubrajaleelic acid (5), two new nor-terpenoids together with known iridoids: 1-a-plumieride (6), plumieride p-Z-coumarate (7) and plumieride-p-Ecoumarate (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. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Plumeria rubra Flavonoid Sphingolipid Iridoids Nortriterpenoids 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 antimicrobial 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.

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-Zcoumarate (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

* Corresponding author. Tel.: +92 3007815194. ** Corresponding author. E-mail addresses: [email protected], [email protected] (A. Jabbar).

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

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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 quaternary 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 2 3 4 4a 5 6 7 8 8a 10 20 ,60 30 ,50 40 10 0 20 0 30 0 40 0 50 0 60 0 10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0

dH (J in Hz) 5.39, 3.15, – – – 6.17, – 6.15, – – 7.32, 6.81, – 5.20, 4.12, 3.35, 4.12, 3.87, 1.28, 5.24, 3.92, 3.61, 3.39, 3.37, 3.84, 3.69,

dC

dd (12.5, 3.0) dd (17.5, 12.5) 2.76, dd (17.5, 3.0)

HMBC (H ! C)

79.1 43.0 197.0 105.0 164.9 97.8 165.5 96.7 165.7 130.7 129.1 116.3 159.0 102.6 75.4 71.5 73.9 69.9 18.2 102.5 72.1 78.9 71.2 78.1 62.2

d (2.0) d (2.0)

d (8.5) d (8.5) d, (1.5) m m m m d (5.0) d (6.5) m m m m dd (11.2, 5.0) 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-Lrhamnopyranosyl-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

COSY (H ! H) 0

0

0

C-3, C-4, C-8a, C-1 , C-2 , C-6 C-2, C-4, C-4a, C-10 – – – C-4a, C-5, C-7, C-8 – C-4a, C-6, C-7, C-8a – – C-10 , C-30 , C-40 C-10 , C-20 , C-40 – C-7, C-200 , C-30 , C-500 C-100 , C-300 , C-400 C-100 , C-200 , C-400 , C-500 C-200 , C-300 , C-500 , C-600 C-100 , C-300 , C-400 , C-600 C-400 , C-500 C-40 , C-200 0 , C-300 0 , C-500 0 C-100 0 , C-300 0 , C-400 0 C-100 0 , C-200 0 , C-400 0 , C-500 0 C-200 0 , C-300 0 , C-500 0 , C-600 0 C-100 0 , C-300 0 , C-400 0 , C-600 0 C-400 0 , C-500 0

H-2/H-3 H-3/H-2 – – – H-6/H-8 – H-8/H-6 – – H-20 ,60 /H-30 ,50 H-30 ,50 /H-20 ,60 – H-100 /H-200 H-200 /H-100 ,300 H-300 /H-200 ,400 H-400 /H-300 ,500 H-500 /H-400 ,600 H-600 /H-500 H-100 0 /H-200 0 H-200 0 /H-100 0 ,300 0 H-300 0 /H-200 0 ,400 0 H-400 0 /H-300 0 ,500 0 H-500 0 /H-400 0 ,600 0 H-600 0 /H-500 0

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) 3.80, dd (10.5, 3.5) 4.25, m 3.06, dd (4.6, 4.2) 3.52, dt (5.5, 4.2) 1.40, m 1.71, m 1.28  1.32, br s 0.90, t (6.5) 7.80, d (7.8) – 4.02, dd (7.5, 4.0) 1.76, m 1.68, m 1.40, m 1.28–1.32, br s 2.05, m 5.41, dt (15.2, 5.1) 5.36, dt (15.2, 4.6) 1.97, m 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 3.87, dd (11.0, 5.5) 3.67, dd (11.0, 3.5)

69.9

C-2, C-3, C-100

H-1/H-2

51.6 75.6 72.9 26.1 33.0 30.3–30.9 14.4 – 177.1 72.8 35.7

C-1, C-3, C-1, C-2, C-2, C-3, C-3, C-4, C-4, C-5, C-5, C-6, C-17

– C-10 , C-30 , C-40 C-10 , C-20 , C-40 , C-50

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 – H-20 /H-30 H-30 /H-40

26.1 30.3–30.9 33.3 129.1 130.7 33.8 26.1–33.8 14.4 104.7 75.0 77.9 71.6 78.0 62.6

C-20 , C-30 , C-50 C-30 , C-40 , C-150 , C-160 C-140 , C-160 , C-170 C-140 , C-150 , C-170 , C-180 C-150 , C-160 , C-180 , C-190 C-160 , C-170 , C-190 C-170 , C-180 , C-260 C-250 C-1, C-200 , C-300 , C-500 C-100 , C-300 , C-400 C-100 , C-200 , C-400 , C-500 C-200 , C-300 , C-500 , C-600 C-100 , C-300 , C-400 , C-600 C-400 , C-500

H-40 /H-30 ,50 H-50 ,140 /H-40 ,160 H-150 /H-140 ,160 H-160 /H150 ,170 H-170 /H-160 ,180 H-180 /H-170 ,190 H-190 ,250 /H-180 ,260 H-260 /H-250 H-100 /H-200 H-200 /H-100 ,300 H-300 /H-200 ,400 H-400 /H-300 ,500 H-500 /H-400 ,600 H-600 /H-500

2 3 4 5 6 7–17 18 N-H 10 20 30 40 50 –140 150 160 170 180 190 –250 260 10 0 20 0 30 0 40 0 50 0 60 0

C-4, C-10 C-4, C-5 C-5, C-6 C-6 C-7 C-18

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393

O 408 6''

HO HO

335

704

O

O 1

179 614

2

3

758 17' 16'

2'

OH

OH 1''

3''

OH

HN 1'

OH 5''

365

26'

18

4

OH 644

674

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 CH21 (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-acetamino1,3,4-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 methyldecanoate (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}octadecane-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 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OCH3 10 20 30 40 50 60

4.93, – 7.36, – 3.86, 6.32, 5.36, – 2.73, 7.33, – – 4.48, 1.30, – 3.68, 4.61, 3.13, 3.31, 3.38, 3.17, 3.70, 3.68,

93.6 – 151.4 108.0 39.7 141.3 127.8 96.4 49.3 148.8 136.8 171.3 62.5 21.6 166.9 51.4 99.0 72.9 76.3 68. 8 75.9 60.3

C-3, C-5, C-8, C-9, C-10 – C-1, C-4, C-5, C-15 – C-1, C-3, C-4, C-6, C-7, C-8, C-9, C-15 C-4, C-5, C-7, C-8, C-9 C-5, C-6, C-8, C-9, C-10 – C-1, C-4, C-5, C-6, C-7, C-8, C-10 C-7, C-8, C-9, C-11, C-12, C-13 – – C-10, C-11, C-12, C-14 C-11, C-13 – C-15 C-1, C-20 , C-30 , C-60 C-10 , C-30 , C-40 C-10 , C-20 , C-40 , C-50 C-20 , C-30 , C-50 , C-60 C-10 , C-30 , C-40 , C-60 C-40 , C-50

H-1/H-9 – – – H-5/H-6,9 H-6/H-5,7 H-7/H-6 – H-9/H-1,5 – – – H-13/H-14 H-14/H-13 – – H-10 /H-20 H-20 /H-10 ,30 H-30 /H-20 ,40 H-40 /H-30 ,50 H-50 /H-40 ,60 H-60 /H-50

d (7.0) d (1.5) dd (5.5, 2.8) dd (5.0, 2.8) d (5.0) dd (7.0, 5.5) s

q (6.0) d (6.0) s d (8.0) t (8.0) m d (1.8) m dd (11.2, 5.1) dd (11.2, 2.8)

N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298

various products, amongst which the glycone could be separated and identified as D-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 13C 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 signals at d 171.3, 148.8, and 136.8 designated to a five-member a,b-unsaturated g-lactone. The signals at d 166.9, 151.4 and 108.0 indicated the presence of another conjugated ester whereas the signals at d 141.3, 127.8 for an isolated double bond. The internal sequence of the molecule was established by COSY and HMBC correlations (Table 3). The attachment of hexose at C-1 was confirmed by HMBC correlation in which H-10 at d 4.61 was 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 (Abe et al., 1988; Kardono et al., 1990). The NOESY correlations beween H-5 at d 3.86 and H-9 at d 2.73 confirmed their cisorientation which was extended to CH3-14 (d 1.30) confirmed their b-orientation. The above data confirmed 3 to be galactoside of 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 molecular formula was established due to HREIMS as C29H48O3 with six double bond equivalent (DBE). The IR spectrum of 4 indicated the presence of hydroxyl and olefinic functions. The 1H NMR spectrum was evident of a triterpenoid skeleton as it displayed five singlet and one doublet methyls at d 1.26, 1.12, 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 oxidized to an alcoholic function. The 13C NMR spectrum of 4 was in agreement with the proton data as it afforded 29 carbon signals of which six at d 24.0, 21.6, 17.8, 17.6, 17.5 and 13.9 were identified as methyls and one at d 66.1 was designated as oxygenated methylene. The 13C NMR shifts olefinic carbons were observed at d 140.0 and 126.3. This information gave an idea about an ursane type of triterpenoid (Mehmood et al., 2006). Besides, various multiplets resonating between d 1.15–2.20 in the 1H NMR spectrum, an olefinic methine was found to resonate at d 5.22, whereas, two oxymethines appeared in the spectrum at d 3.66 (ddd, J = 11.4, 9.6, 4.8 Hz) and 3.53 (d, J = 9.6 Hz) corresponding to the carbons appeared at d 69.7 and 78.0, respectively. The COSY correlation and analysis of coupling (Mehmood et al., 2006) constants of these two oxymethines indicated their transorientation with b-OH at position C-3 (Mehmood et al., 2006). The other 13C NMR data has been shown in Table 4, which was closely related to ursane type of triterpenoids (Mehmood et al., 2006). The HMBC correlations of CH3-24 at d 0.68 with the carbons at d 78.0 (C-3), 48.8 (C-5), 44.1 (C-4) and 66.1 (C-23) revealed that CH323 has been oxidized to an alcoholic group, which in turn was further confirmed due to HMBC interaction of CH2-23 with C-3, C4, C-5 and CH3-24. In the same spectrum, CH3-29 at d 0.96 was 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 present in ursane series of triterpenoids. The stereochemistry at C2 and C-3 was established on the basis of coupling constants, 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 CH2-23 confirmed hydroxyl group a at C-2 and b at C-3. The above data revealed that compound 4 must be 30-nor-2a,3b,23-trihydroxyurs-12-ene and is named as rubrajaleelol.

Table 4 1 H NMR and Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

13

295

C NMR data of compound 4 and 5 (CD3OD, 500 and 125 MHz). 4

5

dH

dC

dH

dC

1.91, 1.68, m 3.66, ddd (11.4, 9.6, 4.8) 3.53, d (9.6) – 0.87, m 1.25, 1.13, m 1.32, 1.02, m – 1.53, m – 1.96, 1.64, m 5.22, t (3.4) – – 1.69, 1.50, m 1.85, 1.60, m – 2.20, d (11.4) 1.36, m 1.42, 1.01, m 1.58, 1.28, m 1.47, 1.04, m 3.50, d (11.4) 3.26, d (11.4) 0.68, s 1.12, s 0.93, s 1.26, s 1.03, s 0.96, d (6.6)

48.0 69.7

48.2 69.5

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

1.94, 1.58, m 3.63, ddd (11.4, 9.6, 4.8) 2.91, d (9.6) – 0.83, m 1.60, 1.54, m 1.50, 1.24, m – 1.52, m – 2.30, 1.96, m 5.22, t (3.4) – – 1.94, 1.83, m 2.50, 2.03, m – 2.21, d (11.4) 1.91, m 1.48, 1.15, m 1.55, 1.35, m 1.82, 1.63, m 0.95, s

13.9 17.5 17.6 24.0 21.6 17.8

1.00, 0.79, 0.94, 1.11, – 0.88,

29.3 17.7 17.6 24.1 181.0 17.8

s s s s d (6.0)

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

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 C29H46O4 with seven DBE. The IR spectrum was evident of a carboxylic acid nature of 5, with olefinic and alcoholic functions. The 1H NMR data was very similar to that of 4 with the only difference of chemical shifts of fewer hydrogens and lack of the signals for OCH2-23 which were observed in 4. Five singlet and one doublet methyls were found to appear in the spectrum at d 1.11, 1.00, 0.95, 0.94, 0.79 and 0.88, respectively. The signal for methyl-28 disappeared in 1H NMR spectrum; instead, the signal for CH3-24 was observed at d 1.00. Besides, other signals, the 13C NMR showed a downfield signal at d 181.0 attributed to a carbonyl group. This information revealed that CH3-28 has been oxidized to a carboxylic acid function. The downfield shift of CH2-16 (d 2.50, Hax and 2.03, Heq) and its HMBC correlation with the carbonyl carbon d 181.0 confirmed 28-oic acid function in 5. On the other hand, the HMBC correlation of H-3 (d 2.91) with that of the carbons resonating at d 21.6 (C-23), 29.3 (C24), 40.1 (C-4) and 56.6 (C-5) revealed that CH3-28 is oxidized instead of CH3-23. The above discussed information and other NMR data (Table 4) led to the structure of 5 as 30-nor-2a,3bdihydroxyurs-12-en-28-oic acid and is named as rubrajaleelic acid. 2.1. Biological studies on compounds 1–8 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

N. Akhtar et al. / Phytochemistry Letters 6 (2013) 291–298

296

Table 5 Antioxidant, antiurease, cytotoxic and phytotoxic activities of compounds 1–8. Sr. No

1 2 3 4 5 6 7 8 Solvent Control

Concentration mg/ml

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 – 1.0

% Age activity Antioxidant assaya

Antiurease assayb

Cytotoxic assayc

Phytotoxic assayd

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 10 10 10 0 100

20 20 20 70 70 20 70 20 30 –

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 IR460 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 temperature 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 nhexane (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 dichloromethane (DCM) as eluent to afford four sub-fractions B1–B4. The sub-fraction B1 (56 mg) was subjected to flash CC using n-hexaneEtOAc (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 EtOAcMeOH (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 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

Colorless gummy solid (29 mg); [a]D25 +24.78 (c 0.013, CH3OH); IR (KBr): 3500–3200, 2945 and 1660 cm1; 1H and 13C NMR: see Table 2; HRFABMS: m/z 870.7120 [MH] calcd. for C50H96NO10, 870.7112.

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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 corresponding 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.

297

thin layer chromatography using solvent system (EtOAc-MeOHH2O-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 diazotization 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 condition 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 temperature 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 Biochemica 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 temperature, 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.

3.7. Rubrajaleelol (4) 3.12. Cytotoxicity assay (in vitro) 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

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 predetermined 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 anticancer 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

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