Chemical constituents from Tribulus terrestris and screening of their antioxidant activity

Phytochemistry 92 (2013) 153–159

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Chemical constituents from Tribulus terrestris and screening of their antioxidant activity Hala M. Hammoda a,⇑, Nabila M. Ghazy a, Fathalla M. Harraz a, Mohamed M. Radwan a,b, Mahmoud A. ElSohly b,c, Ingy I. Abdallah a a b c

Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Egypt National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmaceutics, School of Pharmacy, University of Mississippi, University, MS 38677, USA

a r t i c l e

i n f o

Article history: Received 10 December 2012 Received in revised form 28 March 2013 Available online 2 May 2013 Keywords: Tribulus terrestris L. Zygophyllaceae Oligosaccharides p-Coumaroylquinic acid derivatives Antioxidant activity DPPH

a b s t r a c t Two oligosaccharides (1, 2) and a stereoisomer of di-p-coumaroylquinic acid (3) were isolated from the aerial parts of Tribulus terrestris along with five known compounds (4–8). The structures of the compounds were established as O-b-D-fructofuranosyl-(2 ? 6)-a-D-glucopyranosyl-(1 ? 6)-b-Dfructofuranosyl-(2 ? 6)-b-D-fructofuranosyl-(2 ? 1)-a-D-glucopyranosyl-(6 ? 2)-b-D-fructofuranoside (1), O-a-D-glucopyranosyl-(1 ? 4)-a-D-glucopyranosyl-(1 ? 4)-a-D-glucopyranosyl-(1 ? 2)-b-D-fructofuranoside (2), 4,5-di-p-cis-coumaroylquinic acid (3) by different spectroscopic methods including 1D NMR (1H, 13C and DEPT) and 2D NMR (COSY, TOCSY, HMQC and HMBC) experiments as well as ESI-MS analysis. This is the first report for the complete NMR spectral data of the known 4,5-di-p-trans-coumaroylquinic acid (4). The antioxidant activity represented as DPPH free radical scavenging activity was investigated revealing that the di-p-coumaroylquinic acid derivatives possess potent antioxidant activity so considered the major constituents contributing to the antioxidant effect of the plant. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Zygophyllaceae (Caltrop family) is a family of approximately 25 genera and 240 species (Trease and Evans, 2009) adapted to semidesert and Mediterranean climates. Tribulus terrestris L. is a well known and widely distributed species of the genus Tribulus. It is known with several common names: puncture vine, caltrop, goat head, bull’s head, ground burr nut, devil’s thorn (Kostova and Dinchev, 2005) and Arabic names: Al-Gutub, Qutiba, Hasak or Ders El-Agouz (Al-Ali et al., 2003). T. terrestris has been used in folk medicine throughout history for conditions such as impotence, rheumatism, edema, hypertension and kidney stones (Akram et al., 2011; Ross, 2005; Vesilada et al., 1995). Literature showed that T. terrestris contains phenolic compounds (Lv et al., 2008), saponins (Kostova and Dinchev, 2005), sterols (Liu et al., 2003) and alkaloids (Wu et al., 1999). In spite of the worldwide distribution of T. terrestris and the fact that the plant material collected from different geographical regions has different contents of biologically active compounds, most of the phytochemical and biological investigations described in the literature referred to T. terrestris growing in different European and American countries (Dinchev et al., 2008). In addition, there is only one phytochemical ⇑ Corresponding author. Tel.: +20 3 4871351; fax: +20 3 4873273. E-mail address: [email protected] (H.M. Hammoda). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.04.005

report on the Egyptian plant which dealt with its flavonoidal content (Saleh et al., 1982). These findings prompted us to pursue the phytochemical investigation of T. terrestris L., growing wildly in Egypt. Since the innate defense in the human body may not be sufficient for severe oxidative stress, certain amounts of exogenous antioxidants are constantly required to balance the amount of reactive oxygen species (Souza et al., 2012). So, the search for natural antioxidants represents an area of vast interest in which the plant kingdom has been documented to be an important source of antioxidants with novel structures and unique mechanisms of action. In the present study, phytochemical investigation of the aerial parts of Tribulus terrestris L. was carried out leading to the isolation and characterization of eight compounds (1–8); three of which (1– 3) were isolated for the first time from a natural source. Additionally, screening of the DPPH free radical scavenging activity of the total ethanolic extract, fractions and isolates of the plant was performed to assess their antioxidant activity and pinpoint the active constituents contributing to this effect. 2. Results and discussion Two new oligosaccharides (1 and 2) and a new stereoisomer of di-p-coumaroylquinic acid (3) were isolated from the aerial parts of T. terrestris (Fig. 1).

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Fig. 1. Chemical structures of compounds 1–8.

The qualitative chemical tests on 1 and 2 proved their carbohydrate nature. Acid hydrolysis of 1 and 2 followed by TLC comparison with authentic sugar samples indicated the presence of glucose and fructose moieties. 1 H NMR of fructans has been less studied than 13C NMR, resulting in very few reports on oligofructans being available (Matulova et al., 2011). Proton assignments were very complex where almost all the proton signals fall in a narrow range of approximately 0.8 ppm. H-1 of the two glucose moieties at dH 4.26 and 4.92 were unambiguously identified in 1H NMR spectrum of 1, because H-1 of glucose is the only proton connected to a carbon atom bearing two oxygen atoms. In addition, correlations of all proton signals to their corresponding carbons were deduced from the HMQC spectrum and confirmed by following the proton correlations in the TOCSY spectrum (Table 1). 13 C NMR spectrum of 1 displayed 36 signals corresponding to 36 carbons. The identity of each resonance (methine or methylene) was determined by the DEPT-135 NMR spectrum. The spectrum

showed ten upfield methylene and 22 methine carbon resonances. Of the total 36 carbons, 12 carbons were characteristic to two glucose moieties and 24 carbons were characteristic to four fructose moieties (Table 1). The presence of eight upfield hydroxymethylene (CH2OH) resonances at dc 61.2–64.2 assigned to C-1 and C-6 of the fructose moieties indicated that the fructose residues must be in the furanose form rather than the pyranose form (Barrow et al., 1984). The a-anomeric configuration of the glucose moieties was judged from the broad singlet peaks of the anomeric protons at dH 4.26 (1H, br.s) and 4.92 (1H, br.s). Regarding the fructose moieties, C-2 in 1 appeared in a range of dc 97.6 to 104.6 indicating their b-configuration (C-2 of a-linkage appears downfield at approximately dc 108.8) (Barrow et al., 1984). These results indicated the presence of two a-D-glucopyranose moieties and four b-D-fructofuranose moieties. The anomeric carbon signals of the glucose moieties appeared at dc 92.3 and 96.9 while the C-6 signals appeared at dc 63.5 and

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H.M. Hammoda et al. / Phytochemistry 92 (2013) 153–159 Table 1 NMR data of compound 1 in DMSO-d6. 1 Significant H–H correlations

Significant long range C–H correlations

3.82 3.39 3.80 3.32

40 30 , 50 40 , 60 50

30 40 , 60

4.92, 3.16, 3.47, 3.06, 3.66, 3.73,

4.26 (2H), br.s 3.17 3.48 3.08 3.67 3.75

2 1, 2, 3, 4, 5

3.36, — 3.68, 3.37, 3.69, 3.40,

3.30

dC

dH

10 20 30 40 50 60

Fructose (2? 62.9, 63.0 97.6, 98.4 76.2, 76.3 73.1, 74.8 82.8, 83.5 63.2, 63.3

3.53, — 3.81, 3.38, 3.79, 3.25,

3.57

1 2 3 4 5 6

?6) Glucose (1? 92.3, 96.9 71.1, 71.5 72.4, 72.6 69.3, 70.0 72.0, 72.2 63.5, 63.6

100 200 300 400 500 600

?2) Fructose (6? 61.2, 61.3 102.1, 104.6 76.6, 76.7 75.2, 75.5 81.3, 81.7 63.8, 64.2

3.63 3.41 3.55 3.28

63.6 indicating that glucose is linked to the fructose moieties as neokestose type (Liu et al., 1991). Literature survey shows that fructofuranose residues that are not linked in position 6 are characterized by C-5 around dc 83.0 and C-6 around dc 63.5 while those with a linkage in the position 6 are characterized by upfield shift of C-5 around dc 81.0 and downfield shift of C-6 around dc 64.0 (Chandrashekar et al., 2011; Chen and Tian, 2003; Lopez et al., 2003; Lopez and Mancilla-Margalli, 2007; Wu et al., 2006). Hence, the involvement of position 6 in the linkage of the internal fructose units was supported by the appearance of carbon signals at dc 81.3 and 81.7 corresponding to C-5 and at dc 63.8 and 64.2 corresponding to C-6 of such units, while C-5 and C-6 of the residues that are not 6-substituted (terminal fructose units) appeared at dc 82.8, 83.5 and 63.2, 63.3, respectively. This was further confirmed by long range coupling between C-1 (dc 96.9) of glucose with H-600 (dH 3.40) of one of the internal fructose units, which in turn, its C-200 (dc 102.1) is long range coupled with H-600 (dH 3.28) of the other internal fructose unit. ESI-MS spectrum displayed two ion peaks at m/z 991.3352 [M+H]+ and 956.3325 [M2OH]+ corresponding to a molecular formula of C36H62O31. ESI-MS provided further proof for the chemical structure of 1, as it displayed characteristic fragment ion peaks at m/z 667.5413 [C24H43O21]+ corresponding to one glucose linked to three fructose moieties, 505.1689 [C18H33O16]+ corresponding to one glucose linked to two fructose moieties and 343.2557 [C12H23O11]+ corresponding to one glucose linked to one fructose moiety. These data are consistent with the suggested structure of 1. Accordingly, compound 1 could be identified as O-b-D-fructofuranosyl-(2 ? 6)-a-D-glucopyranosyl-(1 ? 6)-b-D-fructofuranosyl -(2 ? 6)-b-D-fructofuranosyl-(2 ? 1)-a-D-glucopyranosyl-(6 ? 2)b-D-fructofuranoside. In the same manner as 1, proton assignments of 2 were very complex. H-1 of the three glucose moieties at dH 3.99 (1H) and 4.51 (2H) were easily identified in 1H NMR spectrum of 2 along with their anomeric carbons in the 13C NMR spectrum at dc 104.1, 99.9 and 100.2, respectively, as apparent in the HMQC spectrum (Table 2). 13 C NMR and DEPT spectra of 2 displayed 24 signals corresponding to 24 carbons. 18 carbons were characteristic to three glucose moieties and six carbons were characteristic to one fruc-

3 4 5 6

600 3 2 4 4 300 , 400 600

400 300 , 500 400

400 , 600

tose moiety (Table 2). The signal for C-4 of the terminal glucose moiety appeared at dc 68.7 while that of the other two glucose moieties appeared downfield at dc 70.5 and 71.3 indicating that the latter have their position 4 linked. This can be confirmed by the resonance of C-3 signal of the terminal moiety at dc 77.1 and the upfield shift of those of the other two glucose moieties at dc 73.5 and 73.7 (Agrawal, 1989). The (1 ? 4) linkage of the different glucose units was further confirmed by long range coupling of C-1 (dc 104.1) of the terminal glucose unit to H-40 (dH 3.30) of the adjacent internal glucose unit and C-10 (dc 100.2) of the latter to H-40 (dH 3.29) of the second internal glucose unit. Also, the HMBC spectrum proved correlation between C-200 (dc 104.2) of the fructose moiety and H-10 (dH 4.51) of its adjacent glucose unit. Similar to 1, the a-anomeric configuration of glucose was judged from the broad singlet peaks of the anomeric protons at dH 3.99 (1H, br.s) and 4.51 (2H, br.s). C-2 of fructose in 2 appeared at dc 104.2 and the presence of two upfield hydroxymethylene (CH2OH) resonances at dc 62.1 and 63.0 assigned to C-1 and C-6 of the fructose moiety indicated its b-D-fructofuranose form (Barrow et al., 1984). Also, the appearance of the signals for C-5 at dc 82.3 and C-6 at dc 63.0 confirmed the presence of a fructose residue that is not 6-substituted (Chandrashekar et al., 2011; Chen and Tian, 2003; Lopez et al., 2003; Lopez and Mancilla-Margalli, 2007; Wu et al., 2006). ESI-MS spectrum displayed three ion peaks at m/z 689.3086 [M+Na]+, 667.2296 [M+H]+ and 649.2191 [MOH]+ corresponding to a molecular formula of C24H42O21. ESI-MS provided further proof for the chemical structure of 2, as it displayed a characteristic fragment ion peak at m/z 505.1764 [C18H33O16]+ corresponding to three glucose moieties or one fructose linked to two glucose moieties. Thus, compound 2 could be characterized as O-a-D-glucopyranosyl-(1 ? 4)-a-D-glucopyranosyl-(1 ? 4)-a-D-glucopyranosyl(1 ? 2)-b-D-fructofuranoside. This is the first report for the isolation of these oligosaccharides (1 and 2) from a natural source. According to the qualitative chemical tests of 3 and 4; the effervescence produced upon addition of Na2CO3 referred to the presence of a free carboxylic group (Aboul Ela et al., 2012). The phenolic nature of 3 and 4 was evident upon spraying with FeCl3 spray reagent.

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Table 2 NMR data of compound 2 in DMSO-d6. 2 dC

dH

Significant H–H correlations

Significant long range C–H correlations

1 2 3 4 5 6

Glucose (1? 104.1 69.0 77.1 68.7 75.6 60.8

3.99 (1H), br.s 3.61 3.06 3.35 3.28 3.15

2 1, 2, 3, 4, 5

40

10 20 30 40 50 60

?4) Glucose (1? 99.9, 100.2 69.9, 70.3 73.5, 73.7 70.5, 71.3 72.1, 72.7 61.3, 61.6

4.51 (2H), br.s 3.68, 3.70 3.40, 3.43 3.29, 3.30 3.55, 3.58 3.20, 3.22

30 20 , 40 30 , 50 40

100 200 300 400 500 600

?2) Fructose 62.1 104.2 77.4 76.8 82.3 63.0

3.48 — 3.95 3.75 3.53 3.50

3 4 5 6

1 4

40

400 10 400 300 , 500 400

500

and 8 of each p-coumaroyl moiety. Moreover, 1H NMR spectra showed signals of a cyclohexane ring which indicated the presence of a central quinic acid moiety in both compounds (Table 3). These data suggested the presence of two p-coumaroyl moieties attached to a central quinic acid moiety in 3 and 4. The presence of the two p-coumaroyl moieties as integral parts of the chemical structures of 3 and 4 was further supported by 13C NMR (Table 3) and DEPT spectra which displayed in both compounds signals corresponding to 25 carbons; of which 18 carbon atoms where characteristic to the two p-coumaroyl moieties and seven signals were characteristic for the central quinic acid moiety as follows: two signals were observed at dc 38.1, 39.9 for 3 and dc 37.8, 40.1 for 4 (The latter signal in both compounds was overlapped with the solvent peak but it was deduced from the HMQC and DEPT spectra) due to two methylene carbons at positions 2 and 6, respectively, three signals at dc 67.9, 69.1, 75.7 for 3 and dc 68.2, 68.9, 75.6 for 4 due to three oxymethine carbons at positions 3, 5 and 4, respectively, one quaternary signal at dc 74.1 for 3 and dc 73.2 for 4 due to the carbon at position 1 and one signal at dc 175.4 for 3 and dc 175.5 for 4 due to the free carboxylic

UV spectra of 3 and 4 in MeOH showed absorption maxima at kmax = 326 and 325 nm, respectively characteristic for an unsaturated carbonyl chromophore conjugated with an aromatic residue (Wenzel et al., 2000). The bathochromic shift (+44 nm) observed upon addition of NaOMe and the absence of any shift after addition of AlCl3 indicated the presence of a p-monohydroxy substitution of a phenyl ring and provided preliminary evidence that compounds 3 and 4 may be p-coumaric acid derivatives. 1 H NMR spectra of 3 and 4 (Table 3) added more evidence to the previous suggestion where the spectra displayed the presence of two almost identical p-disubstituted phenyl rings, as revealed by the presence of a pair of AA0 BB0 aromatic systems at dH 6.63 (H30 , H-50 ), 6.89 (H-20 , H-60 ) and 6.68 (H-300 , H-500 ), 6.94 (H-200 , H-600 ) for 3, 6.65 (H-30 , H-50 ), 6.84 (H-20 , H-60 ) and 6.70 (H-300 , H-500 ), 6.94 (H-200 , H-600 ) for 4 (Will et al., 2007). In addition, the spectra showed signals for two sets of olefinic protons in the form of two pairs of doublets at dH 6.15, 7.43 and dH 6.18, 7.47 for 3, at dH 6.10, 7.36 and dH 6.27, 7.47 for 4. The presence of these olefinic protons was further confirmed by the strong coupling in the COSY spectra of the compounds between the two protons at positions 7

Table 3 C and 1H NMR data of compounds 3 and 4 in DMSO-d6.

13

3

1 2 3 4 5 6 7 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 90 ,

100 200 300 400 500 600 700 800 900

4

dC

dH (J in Hz)

dC

dH (J in Hz)

74.1 38.1 67.9 75.7 69.1 39.9 175.4 125.0, 132.2, 116.8, 165.5, 115.6, 129.4, 146.0, 113.9, 166.9,

— 1.98 (2H), m 4.22, m 5.00, dd (3, 9.2) 5.45, m 2.08, 1.83, m — — 6.89, 6.94 (2H), d 6.63, 6.68 (2H), d — 6.63, 6.68 (2H), d 6.89, 6.94 (2H), d 7.43, 7.47 (2H), d 6.15, 6.18 (2H), d —

73.2 37.8 68.2 75.6 68.9 40.1 175.5 125.0, 132.0, 116.5, 165.0, 115.4, 129.1, 146.3, 113.2, 166.7,

— 2.02 (2H), m 4.12, m 4.99, dd (3, 8.6) 5.50, m 2.09, 1.81, m — — 6.84, 6.94 (2H), d 6.65, 6.70 (2H), d — 6.65, 6.70 (2H), d 6.84, 6.94 (2H), d 7.36, 7.47 (2H), d 6.10, 6.27 (2H), d —

125.0 132.2 116.8 165.5 115.6 129.4 146.3 113.9 167.6

(8) (8) (8) (8) (8) (8)

125.3 132.0 116.5 165.0 115.4 129.1 146.8 113.2 167.0

(8) (8) (8) (8) (16) (16)

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functionality at position 7 (Hyun et al., 2010; Will et al., 2007). The above data confirmed the presence of a central quinic acid moiety esterified with two p-coumaroyl moieties in 3 and 4. The full assignment of all proton and carbon signals of both compounds was achieved by thorough investigation of 2D NMR spectra (COSY, HMQC and HMBC). The location of the p-coumaroyl substitutions on the quinic acid moiety was deduced from the comparative analyses of 1H and 13C NMR data with those reported for free quinic acid and its derivatives (Hyun et al., 2010; Ouattara et al., 2004). For 3 and 4, the downfield shift of H-4 (dH 5.00 for 3 and 4.99 for 4) and H-5 (dH 5.45 for 3 and 5.50 for 4) suggested quinic acid derivatives esterified at positions 4 and 5 by the two p-coumaroyl moieties. In addition, the magnetic equivalence of the two protons at C-2 in both compounds established a free hydroxyl group at C-1. Further evidence to support this suggestion was observed from the downfield shift of C-4 and C-5 signals of 3 and 4 appearing at dc 75.7, 69.1 and 75.6, 68.9, respectively (Wenzel et al., 2000). ESI-MS of 3 and 4 showed the molecular ion peak at m/z 507.1801 for 3 and 507.1836 for 4 representing the [M+Na]+ signal which was in agreement with the molecular formula C25H24O10. ESI-MS added further proof for the chemical structure of compounds 3 and 4, as it showed three prominent fragments at m/z 193.0932, 165.1130, 148.0662 for 3 and 193.0891, 165.1077, 148.0651 for 4 due to free quinic acid, p-coumaric acid and p-coumaroyl moiety, respectively. The difference between 3 and 4 was the coupling constants of the two olefinic protons. As in 3 (J = 8 Hz) pointed to their cis orientation. Nevertheless, trans orientation of the two olefinic protons in 4 was deduced from their large coupling constant (J = 16 Hz) (Lu et al., 2000). Based on the previous evidence, compound 3 was characterized as 4,5-di-p-cis-coumaroylquinic acid and compound 4 as 4,5-di-ptrans-coumaroylquinic acid. This is the first report for the isolation of 3 from a natural source and the isolation of 4 from family Zygophyllaceae. In addition, this is the first report for the complete spectral data of 4. Additionally, four known compounds (Fig. 1) were isolated from the plant; dioscin (8) (Maschenko et al., 1990), diosgenin-3-O-a-Lrhamnopyranosyl-(1 ? 4)-b-D-glucopyranoside (7) known as prosapogenin B of dioscin (Singh and Thakur, 1982), 5-p-trans-coumaroylquinic acid (6) and 5-p-cis-coumaroylquinic acid (5) (Lu et al., 2000). The latter three compounds (5–7) were reported here for the first time from family Zygophyllaceae. To assess the potential antioxidant activity of T. terrestris, the total ethanolic extract and different fractions of the plant were screened for their in vitro DPPH free radical scavenging activity. The obtained results (Fig. 2A) revealed that the ethyl acetate fraction had the strongest DPPH free radical scavenging activity (33.928 mg AAe/g sample). Consequently, the DPPH free radical scavenging activity of the six compounds isolated from the ethyl acetate fraction was examined aiming at identifying the active constituent(s) responsible for this antioxidant activity. The obtained results (Fig. 2B) showed that compound 4 had the strongest DPPH free radical scavenging activity (661.98 mg AAe/g sample) followed by compound 3 (589.916 mg AAe/g sample). The remaining four isolated compounds (5–8) showed very weak DPPH free radical scavenging activity compared to compounds 3 and 4. It is worth to mention that compounds 3 (4,5-di-p-cis-coumaroylquinic acid) and 4 (4,5-di-p-transcoumaroylquinic acid) showed significantly stronger DPPH free radical scavenging activity than the total ethyl acetate fraction itself (33.928 mg AAe/g sample). Moreover, IC50 of compounds 3 (0.0216 mg) and 4 (0.0191 mg) were very close to IC50 of ascorbic acid (0.0125 mg) proving their potent antioxidant effect.

157

Fig. 2. (A) DPPH free radical scavenging activity (expressed as mg ascorbic acid equivalent/g sample) of the total ethanolic extract and different fractions of T. terrestris (B) DPPH free radical scavenging activity (expressed as mg ascorbic acid equivalent/g sample) of the compounds isolated from the ethyl acetate fraction of T. terrestris. 3 = 4,5-Di-p-cis-coumaroylquinic acid, 4 = 4,5-Di-p-trans-coumaroylquinic acid, 5 = 5-p-cis-coumaroylquinic acid, 6 = 5-p-trans-coumaroylquinic acid, 7 = Diosgenin-3-O-a-L-rhamnopyranosyl-(1 ? 4)-b-D-glucopyranoside, 8 = Dioscin.

3. Experimental 3.1. General experiment procedures UV measurements were obtained on Pye Unicam SP8-100 UV/ Vis spectrophotometer. 1D NMR (1H, 13C, DEPT-135) and 2D NMR (HMQC, HMBC, COSY, TOCSY) spectra were recorded in DMSO-d6 using the residual solvent as an internal standard at 400 MHz for 1 H and 100 MHz for 13C on a Varian AS 400 instrument at 25 °C. ESI-MS analyses were performed using a Bruker BioApex mass spectrometer. Analytical TLC was performed on Merck Kieselgel 60 F254 plates with 0.25 mm layer thickness. Spots were visualized by UV light then by spraying with anisaldehyde/H2SO4. Preparative TLC was performed on Merck Kieselgel 60 F254 plates, with 0.5 or 1.0 mm layer thickness. Column chromatography (CC) was performed on Merck Kieselgel 60 (0.063–0.20 mm). Solvents used in this work; petroleum ether (40–60 °C), chloroform, ethyl acetate, n-butanol, ammonia, acetic acid, methanol and ethanol were of analytical grade, purchased from Sigma–Aldrich (St. Louis, MO, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid were purchased from Sigma–Aldrich (St.Louis, MO, USA). 3.2. Plant material Tribulus terrestris L. was collected in August and September 2009, growing wildly on train railway and along the Mahmudiya Canal, Alexandria, Egypt. The plant material was identified by Prof. Dr. Salama Mohamed El-Dareer (Department of Botany and Microbiology,

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Faculty of Science, Alexandria University, Egypt). A voucher specimen (TT206) has been deposited in the Pharmacognosy Department, Faculty of Pharmacy, Alexandria University, Egypt. 3.3. Extraction and isolation Air dried powdered aerial parts (2.7 kg) of T. terrestris were extracted twice at room temperature (each for 7 days using 7.5 L 95% ethanol). The solvent was distilled off under reduced pressure to yield 200 g of total dry ethanolic extract, which was added to a mixture of water and ethyl alcohol (8:2). The hydroalcoholic phase (500 ml) was partitioned successively with petroleum ether (4 L), chloroform (3 L), ethyl acetate (3 L) and n-butanol (2 L) to yield 40 g, 20 g, 10 g and 15 g, respectively. The residue left after evaporation of butanol (15 g) was chromatographed using silica gel column (460 g). The elution was performed using chloroform:methanol mixtures with gradual increase in polarity collecting fractions of 150 ml each, based on TLC analyses, were pooled together to obtain three main fractions (A–C). Twenty mg of fraction A (26% methanol in chloroform, 0.5 g) was purified by preparative TLC using chloroform:methanol:acetic acid (7:3:0.5). The zone was visualized by spraying of a pilot spot with anisaldehyde/ H2SO4 spray reagent, scraped off and eluted with a mixture of chloroform:methanol (1:3) to give 7 mg of compound 2, Rf 0.49. Fraction C (32–36% methanol in chloroform, 2.5 g) was fractionated on silica gel column, and the fraction eluted with 24% methanol in chloroform (30 mg) was further purified by preparative TLC using ethyl acetate:methanol:water:acetic acid (10:4:1:2). The zone was visualized by spraying of a pilot spot with anisaldehyde/H2SO4 spray reagent, scraped off and eluted with a mixture of chloroform:methanol (1:3) to give 8 mg of compound 1, Rf 0.47. The ethyl acetate fraction (10 g) was subjected to column chromatography using silica gel (300 g). The elution was performed using chloroform:methanol mixtures with gradual increase in polarity collecting fractions of 150 ml each, based on TLC analyses, were pooled together to obtain four main fractions (D–G). Twenty-five milligrams of fraction D (15% methanol in chloroform, 0.5 g) was subjected to preparative TLC using chloroform:methanol (8:2). The plate was visualized by both UV light and ammonia vapor showing two zones at Rf 0.73, 0.68. The zones were scraped off separately and eluted with a mixture of chloroform:methanol (1:2). The upper zone afforded 6 mg of compound 5, Rf 0.73 while the lower zone afforded 8 mg of compound 6, Rf 0.68. Crystallization of fraction E (20% methanol in chloroform, 0.35 g) with methanol yielded 30 mg of compound 7, Rf 0.58 in ethyl acetate:methanol:water:acetic acid (30:6:4:2). Thirty mg of fraction F (35% methanol in chloroform, 0.4 g) was purified by preparative TLC using ethyl acetate:methanol:water:acetic acid (30:6:4:2). The zone was visualized by spraying of a pilot spot with anisaldehyde/H2SO4 spray reagent, scraped off and eluted with a mixture of chloroform:methanol (1:2) to give 15 mg of compound 8, Rf 0.56. Forty milligrams of fraction G (50% methanol in chloroform, 0.7 g) was subjected to preparative TLC using ethyl acetate:methanol:water:acetic acid (30:6:4:2). The plate was visualized by both UV light and ammonia vapor showing two zones at Rf 0.81, 0.67, respectively. The zones were scraped off separately and eluted with a mixture of chloroform:methanol (1:2). The upper zone yielded 9 mg of compound 3, Rf 0.81 while the lower zone yielded 10 mg of compound 4, Rf 0.67. 3.4. Compounds characterization 3.4.1. O-b-D-fructofuranosyl-(2 ? 6)-a-D-glucopyranosyl-(1 ? 6)-b? 6)-b-D-fructofuranosyl-(2 ? 1)-a-Dglucopyranosyl-(6 ? 2)-b-D-fructofuranoside (1) Amorphous white solid; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) data, see Table 1; ESI-MS m/z [M+H]+

calcd. for C36H63O31, 991.3351, found 991.3352, m/z 956.3325 [M2OH]+, m/z 667.5413 [C24H43O21]+, m/z 505.1689 [C18H33O16]+, m/z 343.2557 [C12H23O11]+. 3.4.2. O-a-D-glucopyranosyl-(1 ? 4)-a-D-glucopyranosyl-(1 ? 4)-a? 2)-b-D-fructofuranoside (2) Amorphous white solid; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) data, see Table 2; ESI-MS m/z [M+Na]+ calcd. for C24H42O21Na, 689.3106, found 689.3086, m/z 667.2296 [M+H]+, m/z 649.2191 [MOH]+, m/z 505.1764 [C18H33O16]+. D-glucopyranosyl-(1

3.4.3. 4,5-Di-p-cis-coumaroylquinic acid (3) Amorphous white solid; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) data, see Table 3; ESI-MS m/z [M+Na]+ calcd. for C25H24O10Na, 507.1746, found 507.1801, m/z 193.0932 [C7H13O6, quinic acid + H] +, m/z 165.1130 [C9H9O3, p-coumaric acid + H] +, m/z 148.0662 [C9H8O2, p-coumaroyl + H]+. 3.4.4. 4,5-Di-p-trans-coumaroylquinic acid (4) Amorphous white solid; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) data, see Table 3; ESI-MS m/z [M+Na]+ calcd. for C25H24O10Na, 507.1746, found 507.1836, m/z 193.0891 [C7H13O6, quinic acid + H] +, m/z 165.1077 [C9H9O3, p-coumaric acid + H] +, m/z 148.0651 [C9H8O2, p-coumaroyl + H]+. 3.5. In vitro DPPH free radical scavenging activity The scavenging activity of T. terrestris total ethanolic extract, fractions and isolated compounds against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was investigated spectrophotometrically as described by (Marghitas et al., 2009). Briefly, each of the standard (ascorbic acid) and sample solutions (400 ll) was mixed with 2 ml of DPPH solution (0.02 mg/ml). The mixtures, in triplicates, were kept in the dark for 15 min at room temperature and then the absorbance was measured at k 517 nm. The absorbance of a control sample containing the same amount of solvent and DPPH solution was measured daily. Five concentrations (0.008, 0.01, 0.015, 0.018, 0.02 mg/ml) of standard ascorbic acid were used to construct a calibration curve (% inhibition versus concentration) and determine the regression equation. Samples of the total ethanolic extract, chloroform fraction and butanol fraction were prepared in concentration 1 mg/ml while that of the ethyl acetate fraction in 0.4 mg/ml. Samples of compounds 3 and 4 were prepared as 0.02 mg/ml solutions, compounds 5, 6 and 7 as 0.2 mg/ ml while compound 8 as 0.3 mg/ml. The percentage of absorbance inhibition at k 517 nm was calculated using the following equation:

% inhibition ¼ ½ðA control  A sampleÞ=A control  100 The extent of decolorisation was calculated as percentage reduction of absorbance, and this was determined as a function of concentration and calculated relatively to the equivalent ascorbic acid concentration. The radical scavenging activity was expressed in mg ascorbic acid equivalent per gram of sample (mg AAe/g sample). IC50 of ascorbic acid was determined from the calibration curve. % inhibition of five different concentrations (0.015, 0.018, 0.02, 0.023, 0.025 mg/ml) of compounds 3 and 4 were measured to determine their IC50. Appendix A. Supplementary data

D-fructofuranosyl-(2

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 04.005.

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References Aboul Ela, M.A., El-Lakany, A.M., Abdel-Kader, M.S., Alqasoumi, S.I., Shams-El-Din, S.M., Hammoda, H.M., 2012. New quinic acid derivatives from hepatoprotective Inula crithmoides root extract. Helv. Chim. Acta 95, 61–66. Agrawal, P.K., 1989. Carbon-13 NMR of Flavonoids. Central Ins. of Medicinal and Aromatic Plants, Luknow, India. Akram, M., Asif, H.M., Akhtar, N., Shah, P.A., Uzair, M., Shaheen, G., Shamim, T., Ali Shah, S.M., Ahmad, K., 2011. Tribulus terrestris Linn.: a review article. J. Med. Plants Res. 5, 3601–3605. Al-Ali, M., Wahbi, S., Twaij, H., Al-Badr, A., 2003. Tribulus terrestris: preliminary study of its diuretic and contractile effects and comparison with Zea mays. J. Ethnopharmacol. 85, 257–260. Barrow, K.D., Collins, J.G., Rogers, P.L., Smith, G.M., 1984. The structure of a novel polysaccharide isolated from Zymomonas mobilis determined by nuclear magnetic resonance spectroscopy. Eur. J. Biochem. 145, 173–179. Chandrashekar, P.M., Prashanth, K.V.H., Venkatesh, Y.P., 2011. Isolation, structural elucidation and immunomodulatory activity of fructans from aged garlic extract. Phytochemistry 72, 255–264. Chen, X.-M., Tian, G.-Y., 2003. Structural elucidation and antitumor activity of a fructan from Cyathula officinalis Kuan. Carbohydr. Res. 338, 1235– 1241. Dinchev, D., Janda, B., Evstatieva, L., Oleszek, W., Aslani, M.R., Kostova, I., 2008. Distribution of steroidal saponins in Tribulus terrestris from different geographical regions. Phytochemistry 69, 176–186. Hyun, S.-K., Jung, H.-A., Min, B.-S., Jung, J.-H., Choi, J.-S., 2010. Isolation of phenolics, nucleosides, saccharides and an alkaloid from the root of Aralia cordata. Nat. Prod. Sci. 16, 20–25. Kostova, I., Dinchev, D., 2005. Saponins in Tribulus terrestris – chemistry and bioactivity. Phytochem. Rev. 4, 111–137. Liu, J., Waterhouse, A.L., Chatterton, N.J., 1991. Proton and carbon chemical shift assignments for 6-kestose and neokestose from two-dimensional n.m.r. measurements. Carbohydr. Res. 217, 43–49. Liu, J., Chen, H., Xu, Y., Zhang, W., Liu, W., 2003. Studies on chemical constituents of Tribulus terrestris L. Dier. Junyi Daxue Xuebao 24, 221–222. Lopez, M.G., Mancilla-Margalli, N.A., Mendoza-Diaz, G., 2003. Molecular structures of fructans from Agave tequilana Weber var. azul. J. Agric. Food Chem. 51, 7835– 7840. Lopez, M.G., Mancilla-Margalli, N.A., 2007. The nature of fructooligosaccharides in Agave plants. Recent Adv. Fructooligosaccharides Res., 47–67. Lu, Y., Sun, Y., Yeap Foo, L., McNabb, W.C., Molan, A.L., 2000. Phenolic glycosides of forage legume Onobrychis viciifolia. Phytochemistry 55, 67–75.

159

Lv, A.-L., Zhang, N., Sun, M.-G., Huang, Y.-F., Sun, Y., Ma, H.-Y., Hua, H.-M., Pei, Y.-H., 2008. One new cinnamic imide derivative from the fruits of Tribulus terrestris. Nat. Prod. Res. 22, 1007–1010. Marghitas, L.A., Stanciu, O.G., Dezmirean, D.S., Bobis, O., Popescu, O., Bogdanov, S., Campos, M.G., 2009. In vitro antioxidant capacity of honeybee-collected pollen of selected floral origin harvested from Romania. Food Chem. 115, 878–883. Maschenko, N.E., Gyulemetova, R., Kintya, P.K., Shashkov, A.S., 1990. A sulfated glycoside from the preparation ‘‘Tribestan’’. Khim. Prir. Soedin. 5, 552–555. Matulova, M., Husarova, S., Capek, P., Sancelme, M., Delort, A.-M., 2011. NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water. Carbohydr. Res. 346, 501–507. Ouattara, B., Angenot, L., Guissou, P., Fondu, P., Dubois, J., Frederich, M., Jansen, O., Van Heugen, J.-C., Wauters, J.-N., Tits, M., 2004. LC/MS/NMR analysis of isomeric divanilloylquinic acids from the root bark of Fagara zanthoxyloides Lam. Phytochemistry 65, 1145–1151. Ross, I.A., 2005. Medicinal Plants of the World: Chemical Constituents, Traditional and Modern Uses. Human Press, Totowa, New Jersey. Saleh, N.A.M., Ahmed, A.A., Abdallah, M.F., 1982. Flavonoid glycosides of Tribulus pentandrus and Tribulus terrestris. Phytochemistry 21, 1995–2000. Singh, S.B., Thakur, R.S., 1982. Saponins from the seeds of Costus speciosus. J. Nat. Prod. 45, 667–671. Souza, B.W.S., Cerqueira, M.A., Bourbon, A.I., Pinheiro, A.C., Martins, J.T., Teixeira, J.A., Coimbra, M.A., Vicente, A.A., 2012. Chemical characterization and antioxidant activity of sulfated polysaccharide from the red seaweed Gracilaria birdiae. Food Hydrocolloids 27, 287–292. Trease, G.E., Evans, W.C., 2009. Pharmacognosy. WB Saunders Company Ltd., London. Vesilada, E., Honda, G., Sezik, E., Tabata, M., Fujita, T., Tanaka, T., Takeda, Y., Takaishi, Y., 1995. Traditional medicine in Turkey. V. Folk medicine in the Inner Taurus Mountains. J. Ethnopharmacol. 46, 133–152. Wenzel, P., Chaves, A.L., Mayer, J.E., Rao, I.M., Nair, M.G., 2000. Roots of nutrientdeprived Brachiaria species accumulate 1,3-di-O-trans-feruloylquinic acid. Phytochemistry 55, 389–395. Will, F., Zessner, H., Becker, H., Dietrich, H., 2007. Semi-preparative isolation and physico-chemical characterization of 4-coumaroylquinic acid and phloretin-20 xyloglucoside from laccase-oxidized apple juice. LWT – Food Sci. Technol. 40, 1344–1351. Wu, T.-S., Shi, L.-S., Kuo, S.-C., 1999. Alkaloids and other constituents from Tribulus terrestris. Phytochemistry 50, 1411–1415. Wu, X.-M., Dai, H., Huang, L., Gao, X.-M., Tsim, K.W.K., Tu, P., 2006. A fructan, from Radix ophiopogonis, stimulates the proliferation of cultured lymphocytes: structural and functional analyses. J. Nat. Prod. 69, 1257–1260.