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Author's personal copy Phytochemistry Letters 5 (2012) 793–799
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New bioactive natural products from Launaea nudicaulis Muhammad Saleem a,*, Shehla Parveen a, Naheed Riaz a, Muhammad Nawaz Tahir b, Muhammad Ashraf c, Iftikhar Afzal d, Muhammad Shaiq Ali e, Abdul Malik e, Abdul Jabbar a,* a
Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan Department of Physics, University of Sargodha, Sargodha, Pakistan c Department of Biochemistry and Biotechnology, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan d Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, Railway Road Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan e HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 July 2012 Received in revised form 30 August 2012 Accepted 18 September 2012 Available online 2 October 2012
Ethyl acetate soluble fraction of methanolic extract of Launaea nudicaulis was subjected to chromatographic purification to get four new compounds including a quinic acid derivative (1), a pentahydroxy acetylene analog: trideca-12-ene-4,6-diyne-2,8,9,10,11-pentaol (2), a flavone glycoside (3) and a sesquiterpene lactone (4) together with 10 known compounds. The structures of the new isolates were established by using 1D, 2D NMR techniques and high-resolution mass spectrometry, whereas, the known isolates were identified based on 1D NMR and mass spectrometric information and in comparison with the reported data in the literature. The structure of 4 was also confirmed through single X-ray crystallographic analysis. Cholistaquinate (1) exhibited significant activity in DPPH free radical scavenging assay with an IC50 value of 60.7 mM, whereas, nudicholoid (4) exhibited a moderate inhibitory activity against the enzyme butyrylcholinesterase with an IC50 value of 88.3 mM. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Launaea nudicaulis Secondary metabolites DPPH free radical scavengers Enzyme inhibitors
1. Introduction The genus Launaea of the family Compositae consists of 40 species growing in dry, saline and sandy habitats (Ozenda, 2004). Many species of this genus possess antioxidant, antitumor, insecticidal and cytotoxic activities and are used in folk medicine against stomachic and skin diseases (Rashid et al., 2000a). Nearly 20 species of this genus are growing in Pakistan which possess galactagogue, soporific, diuretic, wound healing, antipyratic and aperients properties (Krishnamurthi, 1969; Nasir and Ali, 1972; Baquar, 1989). Literature survey revealed the presence of triterpenes, sesquiterpene lactones, steroids, flavonoids and coumarins from the genus Launaea (Ali et al., 2003; Yadava and Chakravarti, 2009; Mansour et al., 1983; Moussaoui et al., 2010). The local community is using Launaea nudicaulis to relieve fever, itches, ulcers, cuts, swellings, eczema eruptions and rheumatism (Bhandari, 1988). It also possesses insecticidal, cytotoxic and antifungal activities (Rashid et al., 2000a, 2000b). In the previous studies on hexane part of methanolic extract of L. nudicaulis (jangli booti) growing in Cholistan Desert, new bioactive shingolipids, triterpenoids and steroids were reported (Riaz et al., 2012). In the
* Corresponding authors. Tel.: +92 062 9255473; mobile: +92 0300 9684586. E-mail addresses:
[email protected] (M. Saleem),
[email protected] (A. Jabbar).
present study, we investigated the ethyl acetate soluble fraction of the methanolic extract of L. nudicaulis and obtained four new compounds (Fig. 3): including a quinic acid derivative (1), a pentahydroxy acetylene analog: trideca-12-ene-4,6-diyne2,8,9,10,11-pentaol (2), a flavone glycoside (3) and a sesquiterpene lactone (4) together with 10 known compounds. 2. Results and discussion Compound 1 was obtained as white amorphous solid, whose EIMS exhibited molecular ion at m/z 530. The HREIMS showed molecular ion at m/z 530.1451 corresponding to the molecular formula C26H26O12 with 14 double bond equivalence. The IR spectrum was the evident of hydroxyl, aromatic and carbonyl functions in 1. The aromatic region of 1H NMR spectrum of 1 (Table 1) showed the signals for two trans-double bonds at d 7.61 (1H, d, J = 16.0 Hz), 7.51 (1H, d, J = 15.5 Hz), 6.30 (1H, d, J = 16.0 Hz) and 6.18 (1H, d, J = 15.5 Hz), in addition, three o-coupled protons were observed at d 7.02 (2H, d, J = 8.5 Hz), 6.93 (2H, t, J = 8.5 Hz) and 6.75 (2H, d, J = 8.5 Hz) indicating the presence of two 1,2,3,-trisubstituted benzene rings. This data justified two cinnamoyl derived nuclei in 1, which accommodated 12 double bond equivalence. Further analysis of the 1H NMR spectrum of 1 showed the presence of a quartet methine at d 5.53 (1H, J = 8.5 Hz) which showed COSY correlation with a methylene signal at d 2.26 (1H, dd, J = 13.5, 8.5 Hz) and 2.14 (1H, dd, J = 8.5, 13.5) and an oxymethine at d 5.11
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.09.004
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Table 1 1 H and 13C NMR data, HMBC and COSY correlations of 1 in CD3OD (500 and 125 MHz). Position
dH (J = Hz)
1 2
– 2.26 2.14 5.53 5.11 4.33 2.33 2.07 – 3.71 – 6.18 7.51 – – – 6.75 6.93 7.02 – 6.30 7.61 – –
3 4 5 6 7 OCH3 10 20 30 40 50 60 ,600 70 ,700 80 ,800 90 ,900 100 200 300 400 500
(dd, 13.5, 8.5) (dd, 8.5, 13.5) (q, 8.5) (dd, 8.5, 3.0) (ddd, 6.0, 3.0, 3.0) (dd, 14.0, 3.0) (dd, 14.0, 6.0) (s) (d, 15.5) (d, 15.5)
(d, 8.5) (t, 8.5) (d, 8.5) (d, 16.0) (d, 16.0)
dC
HMBC (H!C)
COSY (H!H)
75.7 38.3
– 1,3,4
– H-2/H-3
69.0 74.8 68.5 38.5
1,2,4,5,10 2,3,5,6,10 0 1,3,4,6 1,2,4,5,7
H-3/H-2,4 H-4/H-3,5 H-5/H-4,6 H-6/H-5
– 7 – 10 ,30 ,40 10 ,20 ,40 ,50 ,90 – – – 50 ,60 ,80 ,90 ,50 0 ,60 0 ,80 0 ,90 0 40 ,60 ,70 ,90 ,40 0 ,60 0 ,70 0 ,90 0 30 ,40 ,50 ,70 ,80 ,30 0 ,40 0 ,50 0 ,70 0 – 10 0 ,30 0 ,40 0 10 0 ,20 0 ,40 0 ,50 0 ,90 0 – –
– – – H-20 /H-30 H-30 /H-20 – – – H-70 ,70 0 /H-80 ,80 0 H-80 ,80 0 /H-70 ,70 0 ,90 ,90 0 H-90 ,90 0 /H-80 ,80 0 – H-20 0 /H-30 0 H-30 0 /H-20 0 – –
175.1 53.1 167.8 114.5 147.6 127.5 149.7 146.8 116.5 115.1 123.1 168.4 114.7 147.7 127.6 149.6
(1H, dd, J = 8.5, 3.0 Hz). The oxymethine (d 5.11) was found to couple with another oxymethine resonating d 4.33 (1H, ddd, J = 6.0, 3.0, 3.0 Hz), which in turn showed COSY correlation with methylene resonating at d 2.33 (1H, dd, J = 14.0, 3.0 Hz) and 2.07 (1H, dd, J = 14.0, 6.0 Hz). The downfield shift of the above two oxymethines (d 5.53 and 5.11) revealed that two cinnamoyl moieties must be connected at these two centers. This hypothesis was further confirmed through HMBC correlation (Table 1) of these oxymethines with carbonyl carbons at d 168.7 and 167.8 of the cinnamoyl moieties. A methoxy group resonating at d 3.71 exhibited HMBC correlation with the most downfield carbon signal at d 175.6 indicating a methyl carboxylate function in 1. The 13C NMR data was in agreement with the 1H NMR data as it displayed signals for two cinnamoyl moieties (d 168.4, 167.8, 149.7, 149.6, 147.7, 147.6, 146.8, 127.6, 127.5, 123.1, 116.5, 115.1, 114.7, 114.5), three oxymethines (d 74.8, 69.0 68.5), two methylenes (d 38.5, 38.3) and methyl carboxylate function at d 175.6 and 53.1 (Table 1). In addition, it displayed an oxygenated quaternary carbon at d 75.5. This data was comparable with the reported data for quinic acid derivatives (Chuda et al., 1996). Therefore, compound 1 must be methyl-(3,4-dianthenobiloyl)quinate, which is a new natural product. The relative stereochemistry of quinic acid unit was established due to the analyses of coupling constants of H-3 to H-6. The relatively big coupling constant (8.5 Hz) between H-2ax and H-3 indicated that H-3 must be axial and acyl moiety must be equatorial. The cis-confirmation between H-5 and H-4 was established due to smaller coupling constant (3.0 Hz), which could be justified through dihedral angle observed in molecular model. This evidence was further supported by the NOESY experiment, in which H-2ax showed NOESY correlation with H6ax. The stereochemistry at C-1 was found to be similar to that of reported analogs of quinic acid due to its optical rotation value and 13 C NMR data, with equatorial carboxylate function (Chuda et al., 1996). Based on these information, compound 1 was finally characterized as methyl-(3b,4a-dianthenobiloyl)-quinate and is named as cholistaquinate. Compound 2 was obtained as white amorphous solid. The EIMS exhibited molecular ion peak at m/z 254 and HREIMS displayed the molecular ion at m/z 254.1128 corresponding to the molecular
formula C13H18O5 with five double bond equivalence. The IR spectrum of 2 showed absorption bands at 3390 (O–H), 3080, 2950 (C–H), 2240, 2150 (CBC) and 1645–1490 cm1 (C5 5C). The 1H NMR spectrum of 2 afforded the signals for three olefinic protons at d 6.02 (1H, ddd, J = 17.2, 10.4, 7.6 Hz), 5.34 (1H, dt, J = 17.2, 1.6 Hz) and 5.19 (1H, dt, J = 10.4, 1.6 Hz) were attributed to the carbons in 13 C NMR spectrum at d 140.2 (CH) and 116.1 (CH2). This data indicated that the molecule contains a terminal double bond. The two fragments A and B (Fig. 1) in 2 could easily be identified through COSY spectrum in which olefinic methine (d 6.02) correlated with an oxymethine (d 4.12, t, J = 7.6 Hz, H-11), which in turn showed cross peak in the same spectrum with another oxymethine at d 3.63 (1H, dd, J = 7.6, 1.6 Hz, H-10). The H-10, showed COSY correlation with the oxymethine resonating at d 3.79 (1H, dd, J = 8.0, 1.6 Hz, H-9), which in turn showed COSY correlation with another signal at d 4.42 (1H, d, J = 8.0 Hz, H-8). Similarly, the oxymethine resonating at d 3.87 (m, H-2) exhibited cross peaks in COSY spectrum with a methylene found at d 2.41 (m, H2-3) and a doublet methyl at d 1.22 (J = 6.0 Hz, H-1). The 13C NMR spectrum of 2 (Table 2) displayed altogether 13 signals, which were identified as one methyl (d 22.5), two methylene (d 116.1, 30.0), six methine (d 140.2, 73.9, 73.5, 73.2, 67.2 and 64.6) and four quaternary carbons (d 78.2, 77.5, 70.6 and 67.0). The shift of quaternary carbons indicated that they could be oxygenated aliphatic C-atoms. The molecular formula afforded only five oxygen atoms, whereas the NMR data displayed signals for five oxymethines. This information with the support of the IR data revealed that 2 has two triple bonds and the four quaternary carbons were attributed to them. The positions of the triple bonds could be fixed due to HMBC information (Table 2) in which H-9 (d
Fig. 1. Identification of fragments A and B in 2 based on the COSY correlations.
Author's personal copy M. Saleem et al. / Phytochemistry Letters 5 (2012) 793–799 Table 2 1 H and 13C NMR data, HMBC and COSY correlations of 2 in CD3OD (400 and 100 MHz).
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Table 3 1 H and 13C NMR data, HMBC and COSY correlations of 3 in CD3OD (500 and 125 MHz).
Position
dH (J = Hz)
dC
HMBC (H!C)
COSY (H!H)
Position
dH (J = Hz)
dC
HMBC (H!C)
COSY (H!H)
1 2 3 4 5 6 7 8 9 10 11 12 13
1.22 (d, 6.0) 3.87 (m) 2.41 (m) – – – – 4.42 (d, 8.0) 3.79 (dd, 8.0, 1.6) 3.63 (dd,7.6, 1.6) 4.12 (t, 7.6) 6.02 (ddd, 17.2, 10.4, 7.6) 5.34 (dt, 17.2, 1.6) 5.19 (dt, 10.4, 1.6)
22.5 67.2 30.0 78.2 67.0 70.6 77.5 64.6 73.5 73.2 73.9 140.2 116.1
2,3 3,4 3,4,5 – – – – 6,7,9 7,8,10,11 8,11,12 9,10,12,13 10,11,13 10,11,12
H-2 H-1/H-3 H-2 – – – – H-9 H-8/H-10 H-11/H-9 H-10/H-12 H-11/H-13 H-10/H-11
2 3 4 5 6 7 8 9 10 10 20 30 0 40 50 60 100 200 300 400 500 600
– 6.62 – – – – – – – – – – 6.93 6.90 7.99 5.03 4.01 3.43 3.42 3.44 3.92 3.85 4.84 4.95 4.07 4.01 3.72
162.5 103.7 184.2 167.2 108.5 163.1 103.8 151.5 105.8 123.4 145.4 144.8 117.0 117.1 130.2 75.1 73.1 82.0 71.1 83.0 63.1
– 2,4,10 – – – – – – – – –
– – – – – – – – – – –
20 ,30 ,50 10 ,30 ,40 10 ,40 ,50 ,20 6,200 ,300 ,500 100 ,3000 ,400 100 ,200 ,400 ,500 200 ,300 ,500 ,600 30 0 ,40 0 40 0 ,50 0
H-50 H-4/H-6 H-5 H-200 H-100 /H-300 H-200 /H-400 H-300 /H-500 H-40 0 /H-60 0 H-50 0
8,2000 ,3000 1000 ,3000 ,4000 1000 ,5000 2000 ,3000 ,5000 1000 ,3000 ,4000
H-2000 H-1000 /H-3000 H-2000 /H-4000 H-3000 /H-5000 H-4000
3.79) showed long-range correlation with C-7 (d 77.5), whereas H8 (d 4.42) interacted C-7 (d 77.5) and C-6 (d 70.6). The H-3 (d 2.41) correlated with C-4 (d 78.2) and C-5 (d 67.0), whereas H-2 (d 3.87) was found to couple with C-4 (d 78.2). This is how the two fragments A and B (Fig. 1) could be connected through two triple bonds. The above data declared 2 as trideca-12-en-4,6-diyne2,8,9,10,11-pentaol, which is a new natural product. Although, some synthetic acetylene analogs have been studied to establish stereochemistry of related natural products (Prasad and Swain, 2011), but they all differ from compound 2. Therefore, we could not determine the stereochemistry at various chiral centers of 2. Compound 3 was isolated as yellow amorphous solid, whose FABMS (ve mode) showed pseudo-molecular ion at m/z 579 [MH]. The molecular formula was established as C26H28O15 by HRFABMS with 13 double bond equivalence. The UV data (lmax 261, 348 nm) was suggestive of a flavonoid nucleus (Moussaoui et al., 2010). The 1H NMR spectrum of 3 showed only three signals in the aromatic region at d 7.99 (1H, d, J = 8.5 Hz), 6.93 (1H, d, J = 8.5 Hz) and 6.90 (1H, t, J = 8.5 Hz). The singlet resonating at d 6.62 was assigned to H-3. The COSY correlations and ABC splitting pattern of the first three signals indicated that the ring B is 1,2,3trisubstituted, while the fourth signal correlated in HSQC spectrum with a carbon at d 103.7, was attributed to H-3. This data suggested that ring A in 3 is fully substituted. The 1H NMR spectrum was also the evident of two sugar moieties due two oxymethines resonating at d 5.03 (1H, d, J = 10.0 Hz) and 4.84 (1H, d, J = 10.0 Hz), correlated to the carbons appeared at d 75.1 and 76.5, respectively, in 13C NMR spectrum (Table 3). Relatively up-field resonance of these two carbon signals revealed that two sugar units could have C–C linkages with aglycon. This idea was further supported due to the upfield shifts of C-6 (d 108.5) and C-8 (d 103.8). Other sugar protons displayed their positions between d 4.80 and 3.43. The analysis of coupling constants of H-100 and H-1000 suggested that both the sugar units must be b-anomers. Further analysis of various sugar protons and their respective carbons identified both sugars to be glucose and xylose (Carnat et al., 1998). All the assignments in sugar units could be made with the help of 1H–1H COSY, HSQC and HMBC information (Table 3). The HMBC correlation of H-100 (d 5.03) with C-6 (d 108.5) and that of H-1000 with C-8 (d 103.8) confirmed the attachment of glucose and xylose units at C-6 and C-8, respectively. Above presented evidences were found to be sufficient to establish the structure of 3 as 5,7,20 ,30 tetrahydroxy-6-C-b-D-glucopyranosyl-8-C-b-D-xylopyranosyl flavonoside and is named as cholistaflaside. The IR spectrum of 4 exhibited stretching absorption bands at 3445 (O–H), 3110, 2940 (C–H), 1765 (g-lactone), 1685 (C5 5O),
1000 2000 3000 4000 5000
(s)
(d, 8.5) (t, 8.5) (d, 8.5) (d, 10.0) (m) (m) (m) (m) (dd, 12.0, 5.3) (dd, 12.0, 2.0) (d, 10.0) (m) (m) (m) (m) 3.62 (m)
76.5 75.3 79.1 70.4 72.0
1615, 1605 (C5 5C) cm1. The EIMS of 4 showed molecular ion at m/z 364, whereas, the high resolution of the same ion in HREIMS displayed the exact mass at m/z 364.1581 corresponding to the molecular formula C19H24O7 with eight double bond equivalence. The 1H NMR spectrum of 4 displayed a doublet methine at d 6.41 (1H, t, J = 1.0 Hz, H-3) due to a conjugated olefin, which was crossed linked in COSY spectrum with a methylene resonating at d 4.82 and 4.40. The shift of H-3 and amount of coupling constant (1.0 Hz) revealed that an oxymethylene must be present at allylic position to H-3. The 13C NMR spectrum (Table 4) showed four most downfield signals at d 197.0 (C-2), 179.1 (C-12), 176.4 (C-4) and 175.7 (C-10 ). The signal for C-4 was assigned to the olefinic system and its downfield shift was attributed to the b-effect of a Table 4 1 H and 13C NMR data, HMBC and COSY correlations of 4 in CD3OD (500 and 125 MHz). Position
dH (J = Hz)
dC
HMBC (H!C)
COSY (H!H)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
– – 6.41 – 3.76 3.81 2.51 4.85 2.39 – 2.66 – 1.45 2.42 4.82 4.40 – 2.65 3.70 3.63 1.16
134.2 197.0 133.3 176.4 49.7 82.1 59.2 72.0 45.3 148.4 41.8 179.1 15.4 21.4 63.1
– – 1,2,4,5,15 – 1,2,3,4,6,7,10,15 1,4,5,7,8,11 5,6,8,9,11,13 6,7,9,10,11,10 1,7,8,10,11,14 – 6,7,8,12,13 – 7,11,12 1,9,10 3,4,5
– – H-15 – H-6 H-5/H-7 H-6,H-8,H-11 H-9,H-7 H-8/H-10 – H-7/H-13 – H-11 – H-3
175.7 43.9 64.9
– 10 ,30 ,40 10 ,20 ,40
– H-30 /H-40 H-20
13.9
10 ,20 ,30
H-20
10 20 30 40
(t, 1.0) (d, 10.0) (t, 10.0) (q, 10.0) (m) (dd, 13.0, 1.5) (m) (d, 7.0) (s) (dd, 18.0, 1.0) (dd, 18.0, 1.0) (m) (dd, 11.0, 7.5) (dd, 11.0, 5.6) (d, 7.0)
Author's personal copy M. Saleem et al. / Phytochemistry Letters 5 (2012) 793–799
796
Fig. 2. X-ray drawing of 4.
conjugated pentenone ring in 4. The HMBC correlation (Table 4) of H-3 (d 6.41) with carbons at d 197.0 (C-2), 176.4 (C-4), 49.7 (C-5) and 134.2 (C-1) confirmed the cyclopentenone moiety. A methine proton resonating at d 3.76 (d, J = 10.0 Hz, H-5) showed COSY correlation with an oxymethine at d 3.81 (t, J = 10.0 Hz, H6). The H-6 was further correlated in the same spectrum with another methine at d 2.51 (H-7) and in HMBC spectrum it was found to interact with the carbon signals at d 179.1 (C-12) and 41.9 (C-11). This information revealed that a butanolide moiety was also present in compound 4. Further careful analysis of 1H and 13C NMR data (Table 4) indicates that 4 is a sesquiterpene lactone possessing the same skeleton as have been reported for sesquiterpene lactones from Helianthus species (Gao et al., 1987) and Reichardia gaditana (Zidorn et al., 2007). The 2-methyl-3hydroxy propanoate moiety at C-8 could be fixed due to HMBC correlation of H-8 (d 4.85) with that of C-10 (d 175.7). The remaining assignments were accomplished due to COSY and HMBC spectral data (Table 4), finally, to get the structure of 4 as sesquiterpene lactone, which is named as nudicholoid. The relative stereochemistry at various centers could be established due to careful analysis of coupling constants and NOESY spectrum, in which H-5 (d 3.76) showed NOESY interactions with H-7 (d 2.51). On the other hand H-6 (d 3.81) exhibited
NOESY correlation with H-8 (d 4.85) and H-11 (d 2.66). Finally the structure was conformed through single X-ray crystallographic (Fig. 2) analysis. 2.1. Antioxidant and enzyme inhibitory studies on compounds 1–4 The compounds 1–4 were screened for in vitro DPPH free radical scavenging and enzyme inhibitory activities (Table 5). Cholistaquinate (1) exhibited a significant activity in DPPH free radical scavenging assay with an IC50 value of 60.7 mM, whereas, it showed weak inhibition against enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Compound 2 was inactive in all assays. Various quinyldicoumarate have been reported to possess DPPH free radical scavenging activities (Izuta et al., 2009) of almost similar potential, which endorsed our results. The slightly higher IC50 value of 1 could be due to the variation in acyl moieties in 1 and in the reported compounds (Kim and Lee, 2005). Cholistaflaside (3) showed weak inhibitory activity against acetylcholinesterase, while nudicholoid (4) exhibited a significant inhibitory activity against the enzyme butyrylcholinesterase with an IC50 value of 88.3 mM. None of the tested compounds was found to be active against lipoxygenase (LOX) enzyme.
Table 5 DPPH free radical scavenging and enzyme inhibitory activities of 1–4. Compounda
DPPHb (%)
DPPH (IC50) (mM)
AChEc (%)
AChEc (IC50) (mM)
BChEc (%)
BChEc (IC50) (mM)
LOXc (%)
LOXc (IC50) (mM)
1 2 3 4 Quercetin Eserine Baicalein
92.48 2.54 23.23 30.87 15.04 93.21 0.97 – –
60.73 Nil Nil Nil 13.11 0.004 – –
59.68 1.21 3.87 69.33 1.31 24.92 – 91.29 1.17 –
>200 Nil >300 Nil – 0.04 0.001 –
48.57 1.78 13.42 15.68 94.10 2.13 – 82.82 1.09 –
>200 Nil Nil 88.34 0.11 – 0.85 0.001 –
8.97 17.40 6.0 10.18 – – 93.79 1.27
Nil Nil Nil Nil – – 22.4 1.3
All the measurements were done in triplicate and statistical analysis was performed by Microsoft Excel 2003. Results are presented as mean sem. a All compounds were prepared in methanol with a concentration of 0.5 mM. b Positive control quercetin (0.5 mM). c Positive control serine (0.25 mM) and baicalein (0.5 mM).
Author's personal copy M. Saleem et al. / Phytochemistry Letters 5 (2012) 793–799
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Fig. 3. Structures of the new compounds (1–4) isolated from Launaea nudicaulis.
3. Experimental 3.1. General experimental procedures Column chromatography was carried out using silica gel F254 (230–400 mesh) as stationary phase and distilled solvents were used as mobile phase. Thin layer chromatography (TLC) was carried out using aluminium sheets pre-coated with silica gel 60 F254 (20 cm 20 cm, 0.2 mm thick; E. Merck). TLC plates were visualized under UV lamp of at 254 and 366 nm wavelength and by spraying with ceric sulfate reagent solution (on heating). Optical rotation was measured on JASCO DIP-360 polarimeter. IR spectra were recorded on Shimadzu 460 spectrometer, while UV spectra were scanned on a Hitachi UV-3200 spectrophotometer (lmax in nm). 1H and 13C NMR data was measured on Bruker instrument operating at 500 and 125 MHz, respectively. 2D experiments (COSY, HMQC and HMBC) were also performed on the same instrument operating at 500 MHz frequency. EIMS, HREIMS, FABMS and HRFABMS were measured on JMS H110 with a data system and JMSA 500 mass spectrometers, respectively. 3.2. Plant material The whole plant of L. nudicaulis was collected from Cholistan Desert near District Bahawalpur (Punjab), Pakistan in April 2008 and was identified by Dr. Muhammad Arshad (Late), ex-Taxonomist at Cholistan Institute of Desert Studies (CIDS), The Islamia University of Bahawalpur, Pakistan where a voucher specimen (0022-LN/CIDS/08) is deposited. 3.3. Extraction and isolation The shade dried plant material was crushed into coarse powder (26 kg), which was extracted thrice with methanol. The extract was concentrated under reduced pressure to get a dark brown thick gummy mass (1.2 kg). It was suspended in water and was
extracted with n-hexane and ethyl acetate to get 150 and 250 g fractions, respectively. The ethyl acetate fraction was subjected to silica gel column chromatography eluting with n-hexane, n-hexane:dichloromethane (DCM), DCM, DCM:methanol and methanol in increasing order of polarity. As a result eight fractions (1–8) were obtained. The Fr.1 (95 g) obtained at 100% DCM on further chromatography yielded three sub-fractions. The first sub-fraction was further chromatographed on silica gel column eluting with DCM yielded 4-hydroxy-3-methoxybenzoic acid (22.5 mg), whereas the second sub-fraction on silica gel column chromatography eluting with 1% MeOH/DCM yielded nudicholoid (4, 15 mg). The third sub-fraction showed no prominent spot and was discarded. The Fr.2 (42 g) on further purification with repeated silica gel column chromatography gave p-hydroxybenzoic acid (65 mg) at 2% MeOH/DCM, 4-hydroxy-3-methoxybenzoic acid (24 mg) at 3% MeOH/DCM and a sub-fraction. Similarly, the Fr.3 (31 g) on repeated silica gel column chromatography gave 3,4-dihydroxybenzoic acid (27 mg) at 4% MeOH/DCM and methyl-3,4,5trihydroxybenzoate (19 mg) at 5% MeOH/DCM, besides getting most of the color. The Fr.4 (28.0 g) obtained at 6% MeOH/DCM was also subjected to silica gel column chromatography and eluted with an isocratic of 6% MeOH/DCM to get 30 ,40 ,5,7-tetrahydroxyflavone (35 mg) and Cholistaquinate (1, 55 mg). The Fr.5 (2.5 g) obtained from main column at 8% MeOH/DCM on further purification yielded 1,3,7-trihydroxycholan-24-oic acid (24 mg). Fr.6 (30 g) eluted with 10% MeOH/DCM from main column, on further silica gel column chromatography, finally yielded bsitosterol-3-O-b-D-glucoside (65 mg) at 10% MeOH/DCM, 3,30 ,5,7-tetrahydroxy-40 -methoxyflavone (36 mg) at 11% MeOH/ DCM, and an impure sub-fraction (2.0 g), which was washed with ethyl acetate to get trideca-12-en-4,6-diyne-2,8,9,10,11-pentaol (2, 22 mg). The Fr.7 (6.5 g) obtained at 13% MeOH/DCM from main column, on further purification yielded 7-O-(600 -O-p-E-coumaroyl-b-D-glucopyranoside (25 mg), whereas, cholistaflaside (3, 17 mg), was purified from Fr.8 (1.6 g) at silica gel column eluted with 16% MeOH/DCM.
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3.4. Cholistaquinate (1) White amorphous powder; [a]D25 257 (c 0.27, MeOH); IR (KBr) nmax 3430 (O–H), 1730 (C5 5O), 1675 (C5 5O), 1600–1550 (Ar C5 5C) cm1; 1H and 13C NMR (Table 1); EIMS m/z 530 [M]+; HREIMS m/z 530.1451 [M]+ (calcd. for C26H26O12; 530.1424). 3.5. Trideca-12-ene-4,6-diyne-2,8,9,10,11-pentaol (2) White amorphous powder; [a]D25 +7.2 (c 0.821, MeOH); IR (KBr) nmax 3390 (O–H), 3080, 2950 (C–H), 2240, 2150 (CBC), 1645– 1490 cm1 (C5 5C); 1H and 13C NMR (Table 2); EIMS m/z 254 [M]+, 236, 195, 171, 147, 117 and 87; HREIMS m/z 254.1128 [M]+ (calcd. for C13H18O5; 254.1154). 3.6. Cholistaflaside (3) Yellow amorphous powder; [a]D20 25.3 (c 0.46, MeOH); UV (MeOH) lmax (log e) 261 (4.16), 348 (4.45); IR (KBr) nmax 3450 (O– H), 3310 (C–H), 1666 (C5 5O), 1602, 1568, 1506 (Ar. C5 5C), 1456 cm1; 1H and 13C NMR (Table 3); FABMS (ve) m/z 612 [MH]; HRFABMS (ve) m/z 579.1360 (calcd. for C26H28O17; 579.1349). 3.7. Nudicholoid (4) Colorless crystals; [a]D25 +28.7 (c 0.21, MeOH); IR (KBr) nmax 3445 (O–H), 3110, 2940 (C–H), 1765 (g-lactone), 1685 (C5 5O) and 1615, 1605 cm1 (C5 5C); 1H and 13C NMR (Table 4); EIMS m/z 364 [M]+; HREIMS m/z 364.181 [M]+ (calcd. for C19H24O7; 364.1519). 3.8. X-ray data of Nudicholoid (4) [(3R,3aS,4R,9aR,9bS)-9-(hydroxymethyl)-3,6-dimethyl-2,7dioxo-2,3,3a,4,5,7,9a,9b-octahydroazuleno[4,5-b]furan-4-yl-(2R)3-hydroxy-2-methylpropanoate]. The skeleton of 4 consists of a seven-membered A (C5/C6/C7/ C9/C14/C15/C16) ring and two fused five-membered rings B (C9/ C10/C11/C12/C14) and C (C15/C16/C17/C19/O7). At C5 and C7, the 3-hydroxy-2-methylpropanoic acid and methyl groups, respectively, are attached. At rings B and C there are methanolic and methyl groups present at C12 and C17, respectively. There exists carbonyl at C10 and C19. In the seven-membered ring A, four Catoms, D (C6/C7/C15/C16) are nearly planar with r.m.s. deviation of 0.0580 A˚. This ring seems in chair form with one leg, C5 and two heads C9 and C14. The C5, C9 and C14 are at a distance of 0.7680 (25), 0.3996 (32) and 1.0660 (27) A˚, respectively. The ring B is roughly planar with r.m.s. deviation of 0.0277 A˚. The dihedral angle between B/D is 29.66 (6)8. The ring C is not planar as the deviation from mean square plane is 0.1636 A˚ and from this plane the maximum deviation is of C16 which is 0.2272 (12) A˚. The propan1-ol moiety E (O1/C1/C2/C3) is planar with r.m.s. deviation of 0.0048 A˚ and is oriented at a dihedral angle of 82.70 (16)8 with the carboxlate group F (O2/C4/O3). The O-atom of methanolic group is disordered over two sites with refined occupancy ratio 0.712(5):0.288(5). The molecules are interlinked due to C–H O and O–H O types of H-bondings. 3.9. DPPH free radical scavenging assay The stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was used for the determination of antioxidant activity (Koleva et al., 2002). Different concentrations of compounds in DMSO were added at an equal volume (5 mL) to 90 mL of 100 mM methanolic DPPH in a total volume of 100 mL in 96-well plates. The contents were mixed and incubated at 37 8C for 30 min. The absorbance was
measured at 517 nm. Quercetin was used as standard antioxidant. The experiments were carried out in triplicates. IC50 value denotes the concentration of the sample which is required to scavenge 50% of DPPH free radicals. The activity was determined by the following formula. Abs of test compound Percent scavenging activity ¼100 100 Abs of control 3.10. Enzyme inhibitory assay The AChE and BChE inhibition activities were performed according to the Ellman method (Ellman et al., 1961) with slight modifications, whereas LOX inhibitory assay was performed following the established procedure (Tappel, 1962; Evans, 1987; Baylac and Racine, 2003) with slight modifications. The percentage inhibition (%) was calculated by formula given below:
Inhibition ð%Þ ¼
Control Test 100 Control
where control = total enzyme activity without inhibitor and test = activity in the presence of test compound. IC50 values were calculated using EZ-Fit Enzyme kinetics software (Perrella Scientific Inc., Amherst, USA). Acknowledgements The authors are thankful to Higher Education Commission (HEC) of Pakistan and Alexander von Humboldt (AvH) Foundation, Germany for financial support. We are also obliged to Third World Academy of Science (TWAS), Italy for providing some of the basic lab facilities in Chemistry Department of IUB. References Ali, D., Hussain, S.M.S., Malik, A., Ahmed, Z., 2003. Chemical constituents of the genus Launaea. J. Chem. Soc. Pak. 25, 341–347. Baquar, S.R., 1989. Medicinal and Poisonous Plants of Pakistan. Printas, Karachi, pp. 31, 258. Baylac, S., Racine, P., 2003. Inhibition of 5-lipoxygenase by essential oils and other natural fragrant extracts. Int. J. Aromather. 13, 138–142. Bhandari, M.M., 1988. Flora of Indian Desert. Mps Repros, Jodhpur, India, pp. 182– 184. Carnat, A.P., Carnat, A., Fraisse, D., Lamaison, J.L., 1998. Violarvensin, a new flavone di-C-glycoside from Viola arvensis. J. Nat. Prod. 61, 272–274. Chuda, Y., Ono, H., Ohnishi-Kameyama, M., Nagata, T., Tsushida, T., 1996. Structural identification of two antioxidant quinic acid derivatives from garland (Chrysanthemum coronarium L.). J. Agric. Food Chem. 44, 2037–2039. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Evans, A.T., 1987. Actions of cannabis constituents on enzymes of arachidonate metabolism: anti-inflammatory potential. Biochem. Pharmacol. 36, 2035–2037. Gao, F., Wang, H., Maebry, T.J., 1987. Sesquiterpene lactones and flavonoids from Helianthus species. J. Nat. Prod. 50, 23–29. Izuta, H., Narahara, Y., Shimazawa, M., Mishima, S., Kondo, S., Hara, H., 2009. 1,1Diphenyl-2-picrylhydrazyl radical scavenging activity of bee products and their constituents determined by ESR. Biol. Pharm. Bull. 32, 1947–1951. Kim, H.J., Lee, Y.S., 2005. Identification of new dicaffeoylquinic acids from Chrysanthemum morifolium and their antioxidant activities. Planta Med. 71, 871–876. Koleva, I.I., Beek, T.A.V., Linssen, J.P.H., de Groot, A., Evstatieva, L.N., 2002. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochem. Anal. 13, 8–17. Krishnamurthi, A., 1969. The Wealth of India, vol. 1. Council of Scientific and Industrial Research, New Delhi, p. 68. Mansour, R.M.A., Ahmed, A.A., Saleh, N.A.M., 1983. Flavone glycosides of some Launaea species. Phytochemistry 22, 2630–2631. Moussaoui, F., Zellagui, A., Segueni, N., Touil, A., Rhouati, S., 2010. Flavonoid constituents from Algerian Launaea resedifolia (O.K.) and their antimicrobial activity. Rec. Nat. Prod. 4, 91–95. Nasir, E., Ali, S.I., 1972. Flora of West Pakistan. Fakhri Printing Press, Karachi, pp. 761–712. Ozenda, P., 2004. Flore et Ve´ge´tation du Sahara. CNRS, Paris, p. 662.
Author's personal copy M. Saleem et al. / Phytochemistry Letters 5 (2012) 793–799 Prasad, K.R., Swain, B., 2011. Enantioselective synthesis of possible diastereomers of heptadeca-1-ene-4,6-diyne-3,8,9,10-tetrol; putative structure of a conjugated diene natural product isolated from Hydrocotyle leucocephala. J. Org. Chem. 76, 2029–2039. Rashid, S., Ashraf, M., Bibi, S., Anjum, R., 2000a. Antibacterial and antifungal activities of Launaea nudicaulis (Roxb.) and Launaea Resedifolia (Linn.). Pak. J. Biol. Sci. 3, 630–632. Rashid, S., Ashraf, M., Bibi, S., Anjum, R., 2000b. Insecticidal and cytotoxic activities of Launaea nudicaulis (Roxb.) and Launaea Resedifolia (Linn.). Pak. J. Biol. Sci. 3, 808–809.
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