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Pendulaosides A and B. Two acylated triterpenoid saponins from Harpullia pendula seed extract
Phytochemistry Letters 21 (2017) 278–282
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Pendulaosides A and B. Two acylated triterpenoid saponins from Harpullia pendula seed extract
MARK
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Neveen S. Ghaly, Marian Nabil, Mary H. Grace, Farouk R. Melek Chemistry of Natural Compounds Department, National Research Centre, Dokki, 12622, Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords: Harpullia pendula Sapindaceae Pendulaosides A and B Triterpenoid saponins Cytotoxicity
Two new acylated triterpenoid saponins named pendulaosides A and B as well as the known phenolic compounds methyl gallate, gallic acid, 1,2,3,6-tera-O-galloyl-β-D-glucose and 1,2,3,4,6-penta-O-galloyl-β-D-glucose, were isolated from the seeds of Harpullia pendula. The structures of pendulaosides A and B were determined using extensive 1D and 2D NMR analysis and mass spectrometry as well as acid hydrolysis, as 3-O-β-D-glucopyranosyl-(1→2)-[α-L-arabinofuranosyl-(1→3)]-β-D-glucuronopyranosyl-22-O-angeloyl-3β,16α,22α,24β,28pentahydroxylolean-12-ene and 3-O-β-D-glucopyranosyl-(1→2)-[α-L-arabinofuranosyl-(1→3)]-β-D-glucuronopyranosyl-16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene, respectively. To the best of our knowledge the two triterpene parts 22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12-ene and16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene have never been characterized before. The two isolated saponins were assayed for their in-vitro cytotoxic activity against the three human tumor cell lines HepG2, MCF7 and PC3. The results showed that pendulaoside A exhibited moderate activity on PC3 cell line with IC50value equal to 13.0 μM and weak activity on HepG2 cell line with IC50 value equal to 41.0 μM. Pendulaoside B proved to be inactive against the three used cell lines.
1. Introduction The genus Harpullia which belongs to the family Sapindaceae, consists of about 27 species. They are widely distributed ranging from Malesia and Australia to the Pacific islands (Mabberley, 1997). Phytochemical studies carried out on plants of this genus, have revealed them as a source of triterpenoid saponins (Voutquenne et al., 1998, 2002, 2005). The saponins of this genus belong to the polyhydroxylate triterpenoid glycosides containing ester function. This type of saponins was reported to possess antiviral (Devi et al., 2004), antifungal (Bharathimatha et al., 2002), antibacterial (Gowri and Vasantha, 2009), cytotoxic (Voutquenne et al., 2005), molluscicidal, miracidicidal and cercaricidal (Abdel-Gawad et al., 2004) properties. Harpullia pendula Planch with the common name tulipwood or tuliplancewood, is a small to medium sized rainforest tree native to Australia and cultivated in gardens and streets for ornamental purposes. Previous phytochemical examination of the leaf and bark extracts of H. pendula, led to the isolation of the triterpenes A1-barrigenol, A1-barrigenol-22-O-angelate, camelliagenin A, camelliagenin A-16-and 22-O-angelate, 22α-hydroxyerthrodiol and 15α,16α,22α,28-tetrahydroxyolean-12-ene-3-one together with quebrachitol and methyl-p-coumarate (Khong and Keith, 1976; Cherry et al., 1977). Also, three sapogenins were isolated from
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the saponin hydrolysates of H.pendula and H. cupanioides Roxb. and identified as angeloyl camelliagenin A, angeloyl A1- barrigenol and angeloyl A1- barrigenol methyl ether (El-Gohary, 2004). The isolation of phenolics and flavonoids from the pericarp of H. pendula was previously reported (El Sayed et al., 1988). Recent phytochemical study on the leaf extract of H. pendula led to isolation of two antimicrobial benzeneacetic acid derivatives harpulliasides A and B together with several kaempferol glycosides (Abdelkader et al., 2016). As a part of our continuous interest in bioactive saponins from plants cultivated in Egypt, we describe in this report the isolation and characterization of two new acylated saponins named pendulaoside A (1) and pendulaoside B (2) from the seeds of H. pendula together with four known phenolic compounds. 2. Results and discussion The defatted methanolic seed extract of H. pendula was suspended in water and partition with ethyl acetate then with n- butanol. The nbutanol fraction was chromatographed over the polymer gel Diaion HP20 and silica gel followed by repeated HPLC to afford two new acylated saponins named pendulaoside A (1) and pendulaoside B (2). The ethyl acetate fraction yielded after chromatography the known phenolic
Corresponding author. E-mail addresses: [email protected] (N.S. Ghaly), [email protected] (M. Nabil), [email protected] (M.H. Grace), [email protected] (F.R. Melek).
http://dx.doi.org/10.1016/j.phytol.2017.06.016 Received 31 January 2017; Received in revised form 4 June 2017; Accepted 16 June 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 21 (2017) 278–282
N.S. Ghaly et al.
HSQC spectrum to a low-field carbon signal at δC 71.1 and coupled to methylene proton signals at δH 1.38 (1H, m) and 1.73 (1H, dd, J = 11.0, 3.3 Hz) in the TOCSY spectrum, was assigned to the equatorial H-16 proton. The deshielded methyl signal assigned to 27-CH3 at δH 1.48, suggested the existence of a hindered axial hydroxyl group at C-16 position of the triterpene moiety. This suggestion was verified by the appearance (brs) and the chemical shift value of the equatorial H-16 proton which were similar to the corresponding ones of structurally related saponins (Zhang et al., 2012). The observed HMBC correlations H-16/C-17 (δC 45.4) and C-18 (δC 42.7), lent further support to the assignment of H-16 proton. The deshielded doublet of doublets at δH 5.43, directly correlated to a carbon signal at δC 74.0 in the HSQC spectrum and coupled in the COSY and TOCSY spectra to methylene proton signals at δH 1.55 (1H, m) and 2.26 (1H, t, J = 12.0 Hz), was assigned to H-22β proton. The coupling constant value of H-22 indicated a β-axial orientation of this proton. The observed HMBC correlations that supported the assignment of H-22, were between the following signals H-22/C-16 (δC 71.1), C-17 (δC 45.4), C-18 (δC 42.7) and C-21 (δC 42.0). Further information from 1D and 2D NMR spectra of 1 also indicated the presence of signals due to an angeloyl group at δH 6.08 (1H, qd, J = 7.5, 1.5 Hz, H-3′), 1.97 (3H, dd, J = 7.5, 1.5 Hz, H-4′), 1.90 (3H, d-like, J = 1.5 Hz, H-5′) and at δC 169.9 (C-1′), 130.1 (C-2′), 138.2 (C-3′), 16.0 (C-4′) and 21.0 (C-5′). These data were in agreement with the corresponding ones reported for related saponins bearing an identical moiety (Voutquenne et al., 2005; Ohtsuki et al., 2008; Zhang et al., 2012). Inspection of literature revealed that saponins bearing tigloyl moiety with cis double bond, showed chemical shift values for C-4′ and C-5′ of this moiety at 14.0 and 12.3 ppm, respectively (Yoshikawa et al., 1994; Murakami et al., 1999). The observed low-field position of H-22 signal at δH 5.43 due to acylation, suggested that the angeloyl group esterified the 22-hydroxyl group in the triterpene moiety. The location of the acyl group at C-22 was confirmed
compounds methyle gallate 3 (Hisham et al., 2011), gallic acid 4 (Abri and Maleki, 2016), 1, 2, 3,6-tetra-O-galloyl-β-D-glucose 5 (Kandil and Lewis, 2001) and 1,2,3,4,6- penta-O-galloyl-β-D-glucose 6 (dos Santos et al., 2012). The structures of the known compounds were identified by comparison of their spectral data with those reported in the literature. The molecular formula of pendulaoside A (1) was determined as C52H82O21 from [M−H]− at m/z 1041.5179 by high resolution negative-ion electrospray ionization mass spectrometry (calculated, 1041.5276). Acid hydrolysis of 1 afforded sugar components identified as D-glucuronic acid, D-glucose and L-arabinose, thus indicating the glycosidic nature of 1. The 13CNMR spectrum of 1 showed the presence of signals due to six methyl carbons at δC 16.4, 17.3, 22.9, 25.4, 27.8 and 33.7 as well as olefinic carbons at δC 124.6 and 144.0. This information coupled with the presence of six methyl singlets at δH 0.89, 0.91, 0.93, 1.04, 1.21 and 1.48 and a vinyl proton signal at δH 5.33 (brs) in the 1HNMR spectrum, suggested 1 to be a triterpenoid saponin bearing a triterpene moiety with olean-12-ene skeleton. Of the 52 carbons, 30 were assigned to the triterpene moiety, 17 to the glycosidic part and the remaining 5 to an acyl group. Extensive 1D (1H, 13C), and 2D (1H-1H COSY, TOCSY, NOESY, HSQC, HMBC) NMR analysis (Table 1) allowed complete assignments of the proton and carbon signals of the triterpene moiety and revealed the presence of signals due to three oxygen bearing methine protons at δH 3.40 (1H, dd, J = 11.0, 4.0 Hz), 4.12 (1H, brs) and 5.43 (1H, dd, J = 12.0, 5.0 Hz). The methine proton signal at δH 3.40 that directly correlated to a carbon signal at δC 93.0 in the HSQC spectrum of 1 and coupled to methylene proton signals at δH 1.82 (1H, m) and 2.03 (1H, m) in the COSY and TOCSY spectra, was assigned to H-3α proton. The deshielded carbon signal at δC 93.0 was assigned to C-3 based on comparison with the corresponding values of olean-12-ene saponins (Voutquenne et al., 2005). The broad methine singlet at δH 4.12 that directly correlated in the Table 1 1 H and 13CNMR data of compounds 1 and 2 in CD3OD. 1
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 30
2
1
δC
δH
δC
δH
39.8 27.2 93.0 44.6 57.5 19.5 34.2 41.1 48.0 37.6 25.0 124.6 144.0 42.7 35.4 71.1 45.4 42.7 48.2 32.6 42.0 74.0 22.9 64.3 16.4 17.3 27.8 64.9 33.7 25.4
1.67(m),1.03(m) 2.03(m), 1.82(m) 3.40(dd,11.0,40)
39.7 27.1 93.0 44.6 57.4 19.4 34.1 41.1 48.0 37.6 24.9 125.0 142.7 42.6 31.9 70.7 43.5 42.6 48.2 32.3 44.6 74.2 22.9 64.3 16.5 17.3 27.6 70.3 33.8 25.5
1.65(m),1.03(m) 2.05(m),1.78(m) 3.40(dd,11.04.0)
0.97 1.62(m),1.40(m) 1.62(m),1.38(m) 1.68(m) 1.93(m) 5.33(brs)
1.73(dd,11.0,3.3), 1.38(m) 4.12 (brs) 2.52(dd,14.0,4.0) 2.44(t,12.0),1.05(m) 2.26(t,12.0),1.55(m) 5.43(dd,12.0,5.0) 1.21(s) 4.12, (d, 12.0),3.17(d, 12.0) 0.89(s) 0.93(s) 1.48(s) 3.27(d,11.0),3.05(d, 11.0) 0.91(s) 1.04(s)
δC
1.68(m) 1.93(m) 5.37(brs)
1.99(m),1.46(brd, 15.0) 5.49(brs) 2.18(dd,14.0,4.0) 2.28(m),1.12(m) 1.78(t,12.0),1.42(m) 4.07 1.22(s) 4.12(d, 12.0),3.21(d,12.0) 0.93(s) 0.97(s) 1.34(s) 3.64(d,12.0),3.31, (d,12.0) 0.9(s) 1.10(s)
Overlapped signals are represented without designated multiplicity. Values in parentheses represent 1H-1H splitting.
279
δH
GlcUA 105.0 4.54(d,7.8) 78.5 3.70 87.4 3.70 72.3 3.60(t, 9.0) 78.0 3.62(d, 9.0) 178.3 Glc 1 104.0 4.78(d,8.0) 2 75.6 3.19(t,8.0) 3 78.0 3.37(t,8.5) 4 70.2 3.50(t,9.0) 5 78.2 3.22(m) 6 61.7 3.79(d,12.0),3.77 Ara(f) 1 111.0 5.22(d,2.0) 2 83.6 4.13(dd,4.0,2.0) 3 77.9 3.86(dd,7.0,4.0) 4 85.3 4.10(m) 5 62.9 3.81(dd,12.0,4.0),3.63 22-Angeloyl 1′ 169.9 2′ 130.1 3′ 138.2 6.08(qd,7.5,1.5) 4′ 16.0 1.97(dd,7.5,1.5) 5′ 21.0 1.90(d-like,1.5) GlcUA = β-D-glucuronopyranose Glc = β-D-glucopyranose Ara(f) = α-L-arabinofuranose 1 2 3 4 5 6
0.97 1.62(m),1.40(m) 1.60(m),1.38(m)
2 δC
δH
105.0 78.4 87.3 72.3 78.1 177.7
4.54(d,7.8) 3.70 3.70 3.62 (t, 8.0) 3.64 (d, 9.0)
104.0 75.5 78.1 70.3 78.2 61.7
4.75(d,8.0) 3.21(t,8.0) 3.38(t,8.5) 3.51(t,9.0) 3.24(m) 3.80(d,12.0),3.76
111.1 5.17(d,2.0) 83.6 4.13(dd,4.0,2.0) 77.9 3.88(dd,7.0,4.0) 85.4 4.09(m) 62.9 3.80(dd,12.0,4.0),3.63 16-2-methyl butyroyl 177.7 43.5 2.33(sextet,7.0,7.0) 28.0. 1.78(m),1.52(m) 12.5 0.96(t,7.0) 17.0 1.15(d,7.0)
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at m/z 911.489 and 749.443 due to consequent loss of pentose and hexose units. Acid hydrolysis of 2 yielded sugar components identical to those liberated after acid hydrolysis of 1. 1D and 2D NMR spectra of 2 showed signals due to sugar units very similar to those of 1, indicating the existence of identical trisaccharide moiety in 1 and 2 (Table 1). The attachment of this moiety to C-3 of the triterpene moiety was deduced from the deshielded signal of C-3 (δC 93.0) and the HMBC correlation between the signals of C-3 and H-1 of the inner 2,3- disubstituted β-Dglucuronopyranosyl unit. The other HMBC correlations due to the saccharide moiety of 2, were identical to those of 1. The NMR analysis of 2 also revealed a triterpene moiety of 2 similar to that of 1 except that a 2-methylbutyroyl group esterified the α-OH at C-16 in 2 instead of the angeloyl group esterified C-22 α-OH in 1. The observed shielded position of the signal due to 27-CH3 at δΗ 1.34 in 2 and the deshielded position of H-16 signal at δΗ 5.49 (brs) together with the shielded signal at δH 4.07 assigned to H-22β, relative to the corresponding δ values in 1, indicated esterified 16α-OH and free 22α-OH. The assignments of H16β and H-22β were deduced from 1D and 2D NMR analysis, following the same approach used for compound 1. These assignments were in complete agreement with the corresponding assignments reported for structurally related saponins (Voutquenne et al., 1998). The presence of the 2-methylbutyroyl group in 2 was evidenced by the presence of signals due to a carbonyl carbon at δC 177.7, two methyl groups at δΗ 0.96 [(t, J = 7.0 Hz), δC 12.5] and 1.15 [(d, J = 7.0 Hz), δC 17.0], a methine proton at δH 2.33 (dt, J = 7.0 Hz) together with methylene protons at δΗ 1.52 (m) and 1.78 (m). The TOCSY spectrum of 2 showed correlations between the methine proton signal at δΗ 2.33 and the signals due to the methyl at δH 1.15 as well as the two signals of the methylene protons at δH 1.52 and 1.78. HMBC correlations were also observed between the signal of the carbonyl carbon and the signals due to H-16 at δΗ 4.49, the methine proton at δH 2.32, the methylene protons at δΗ 1.45 and 1.71 and the methyl group at δH 1.15. In the NOESY spectrum of 2, correlations between H-22 and H-30 and between H-16 and H-28(δH 3.64), confirmed the relative configurations at C-22 and C-16. Therefore, the triterpene moiety of compound 2 was assigned the structure of 16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28pentahydroxylolean-12-ene. To the best of our knowledge, this triterpene part has never been characterised before. Consequently, pendulaoside B (2) was formulated as 3-O-β-D-glucopyranosyl-(1 → 2)-[αL-arabinofuranosyl-(1 → 3)]-β-D-glucuronopyranosyl-16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene (Fig. 1). The two isolated saponins were assayed for their in-vitro cytotoxic activity against the three human tumor cell lines HepG2, MCF7 and PC3. The results showed that penulaoside A exhibited moderate activity on PC3 cell line with IC50 value equal to 13.0 μM and weak activity on HepG2 cell line with IC50 value equal to 41.0 μM. Pendulaoside B proved to be inactive on the three used cell lines.
by the observed cross peak in the HMBC spectrum correlating the signals due to H-22 at δH 5.43 and the carbonyl carbon (C-1′) of the angeloyl group at δC 169.9. Further HMBC correlations were observed between the signals H-3′/C-1′, C-4′ and C-5′. Furthermore, two pairs of hydroxyl methylene proton signals appeared as two isolated systems, were located at δH 3.17 (1H, d, J = 12.0 Hz) and 4.12 (1H, d, J = 12.0 Hz) as well as at δH 3.05 (1H, d, J = 11.0 Hz) and 3.27 (1H, d, J = 11.0 Hz). The signals due to the two pairs of protons were directly correlated in the HSQC spectrum to carbon signals at δC 64.3 and 64.9 and assigned to H2-24 (Voutquenne et al., 2005) and H2-28, respectively, based on the observed HMBC correlations H-24a (δH 3.17)/C-3 (δC 93.0); H-24b (δH 4.12)/C-3, C-4 (δC 44.6) and C-23 (δC 22.9) as well as H-28a (δH 3.05)/C-16, C-17, C-18 (δC 42.7) and C-22; H-28b (δH 3.27)/C-17, C-18 and C-22. The possibility of hydroxylation at C-23 instead of C-24 was ruled out because C-3 and C-5 signals would appear at significant higher field positions (Yoshikawa et al., 1994). The relative stereochemistry at C-16 and C-4 was further evidenced by NOESY experiment of 1. The observed NOEs were between H-16 and H-28 (δH 3.27) and between H-24 (δH 4.12) and axial H-2 (δH 1.82). Based on the above evidences, the triterpene part of saponin 1 was assigned the structure of 22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12ene. To the best of our knowledge, this triterpene part has never been reported before. A similar moiety with different stereochemistry at C-4 was previously reported by Yoshikawa et al. (1994). The presence of three sugar units in 1 was also evidenced by 1D and 2D NMR analysis that disclosed the presence of three anomeric proton signals at δH 4.54 (1H, d, J = 7.8 Hz), 4.78 (1H, d, J = 8.0 Hz) and 5.22 (1H, d, J = 2.0 Hz) with their corresponding carbons appeared at δC 105.0, 104.0 and 111.0, respectively. The low-field position of C-3 at δC 93.0 suggested the monodesmosidic nature of 1 with a saccharide unit attached to this position. The individual spin system for the individual monosaccharide was assigned by TOCSY spectrum and the sequence of protons was deduced from the 1H-1H COSY experiment. On the basis of the assigned protons the 13C signals of each monosaccharide was recognized by HSQC spectrum. Further supporting information were given by the HMBC experiment which clarified the assignments of some closely related protons and carbons. The obtained data (Table 1) allowed identification of three monosaccharide units as a 2,3-disubstituted β-D-glucuronic acid with anomeric proton signal at δH 4.54, a terminal β-D-glucose with anomeric proton signal at δH 4.78 and a terminal α-L-arabinose with anomeric proton signal at δH 5.22. The first two sugars were in the pyranose form based on their 13CNMR data. The β anomeric configurations of these units were evidenced by their coupling constant values (7.0–8.0 Hz). The third sugar was in the furanose form as shown by the carbon chemical shift values and its α anomeric configuration was deduced by the small coupling constant value (2.0 Hz) as previously reported (Agrawal, 1992; Melek et al., 2002; Chakravarty et al., 2003; Voutquenne et al., 2005). The inter-glycosidic linkages were determined by the observed HMBC correlations between the signals of arabinofuranose H-1 and glucuronopyranose C-3 (δC 87.4) and between the signals of glucopyranose H-1 and glucuronopyranose C-2 (δC 78.5). The HMBC correlation between the signals due to glucuronopyranose H-1 and C-3 of the triterpene moiety, confirmed the attachment of the trisaccharide unit to this position. Therefore, the structure of the trisaccharide unit was established as 3-O-β-D-glucopyranosyl-(1 → 2)-[α-L-arabinofuranosyl-(1 → 3)]-β-D-glucuronopyranosyl. It is worth noting that an identical unit was previously reported as the glycosidic part of protoaescigenin saponins isolated from H. austro- caledonica (Voutquenne at al., 2005). Consequently, pendulaoside A (1) was assigned the structure of 3-O-β-D-glucopyranosyl-(1 → 2)-[α-L-arabinofuranosyl-(1 → 3)]-β-D-glucuronopyranosyl-22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12-ene as shown in Fig. 1. Pendulaoside B (2) was another triterpenoid saponin which had a molecular formula C52H84O21 as determined by high resolution MS measurement. The HR-ESI mass spectrum of 2 exhibited [M-H]− at m/z 1043.5321 (calculated, 1043.5432) and two prominent fragment ions
3. Experimental 3.1. General Liquid chromatography-ion trap-top of flight mass spectrometry was performed using Shimadzu LC-IT-TOF-MS (Shimadzu, Tokyo, Japan) with a Shim-pack XR-ODS column (50 mm × 3.0 mm × 2.2 μm). Solvent gradient consisted of 0.1% formic acid in H2O (A) and acetonitrile (B). Compounds were eluted into the ESI ion source at the flow rate of 0.4 ml/min with a step gradient of B in A: 10–85% B (0–15 min), 85-10%B (15–18 min), isocratic at 10% B (2 min). Column was maintained at 40∘C during the run. Nitrogen gas was used as nebulizer and drying gas with the flow rate set at 1.5 l/min. The ESI source voltage was set at 4.5 kV and the detector was set as 1.5 V. Ionization was performed using a conventional ESI source in the negative ionization mode. Shimadzu's LCMS solution software was used for data analysis. NMR experiments were performed on a Bruker Avance 700 MHz spectrometer (Bruker BioSpin Corporation, Billerica, MA). 1H, 13C, 1H-1H 280
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Fig. 1. Chemical structures of pendulaoside A (1) and pendulaoside B (2).
in H2O (0.002) and the H2O solution was passed through a column chromatography packed with 100 g Diaion HP-20 polymer gel (Mitsubishi). After washing the column with distilled water, elution was carried out with 25%, 50%, 75% aqueous MeOH and finally with 100% MeOH. The collected fractions were examined by silica gel TLC (Merck) using solvent systems n-BuOH- EtOH-NH4OH (7: 2: 5), and CHCl3MeOH-H2O (60: 30: 5) then visualized by spraying with 20% sulphuric acid in MeOH followed by heating at 110 °C. Based on TLC analysis, similar fractions were then combined. Fractions eluted with 75% and 100% MeOH were found similar and contained saponin constituents. The combined saponin fraction (1.35 g) was applied on a column chromatography packed with silica gel (60 g) and eluted with EtOAc −MeOH-H2O with increasing polarity (30:2:1–5:2:1). A total of 50 fractions 50 ml each were collected. Similar fractions were combined after TLC analysis to give eight sub-fractions (A-H). The sub-fraction D (850 mg) eluted with EtOAc-MeOH-H2O (20:2:1), was subjected to repeated preparative HPLC to give 1 (15 mg) and 2 (17 mg). The EtOAc fraction was subjected to Sephadex LH-20 column chromatography. The column was eluted first with acetone and then with acetone methanol mixtures with increasing amount of methanol to 2%. A total of 40 fractions 50 ml each were collected. The fractions were monitored by silica gel TLC plates using solvent system EtOAc- MeOH- H2O (20: 3: 2) and examined by spraying with FeCl3 reagent. Similar fractions were pooled and the solvent was evaporated from each combined fraction under reduced pressure to yield four major sub-fractions collected by
COSY, TOCSY, NOESY, HSQC, HMBC NMR spectra were acquired in CD3OD at 700 MHz for proton and 175 MHz for 13C NMR. Chemical shifts are given as δ values with TMS as internal standard. Optical rotations were measured with jasco p-2000 polarimeter. Preparative HPLC was carried out on Interchim 4100 (Montlucon, France) (column, RP C-18 HQ; solvent system, CH3CN-H2O (33:67-70:30); Flow rate 20 ml/min; Detection, UV, 205 nm; temperature, 35 °C). 3.2. Plant material Seeds of H. pendula Planch.were collected from the zoological garden in Giza, Egypt in September 2014. Plant identification was confirmed by Mrs. T. Labib, head specialist for plant identification in ElOrman public garden, Giza, Egypt. A Voucher specimen was deposited in the Herbarium of NRC (CAIRC). 3.3. Extraction and isolation Air-dried seeds of H. pendula (100 g) were defatted with n-hexane then extracted with MeOH until exhaustion. The combined MeOH extract was evaporated under vacuum to dryness. The residue (4.5 g) was suspended in water and extracted with EtOAc (5 × 100 ml) then water saturated n-BuOH (5 × 100 ml). Each combined fraction was individually evaporated under reduced pressure to yield 1.5 g EtOAc fraction and 2.1 g n- BuOH fraction. The n-BuOH fraction was dissolved 281
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investigation of antioxidant activity. Iran. Chem. Commun. 4, 146–154. Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 31 (10), 3307–3330. Bharathimatha, C., Doraiswamy, S., Rabindran, R., Velazhahan, R., 2002. Inhibition of fungal plant pathogens by seed proteins of Harpullia cupanioides (Roxb). Acta Phytopathol. Entomol. Hung. 37, 75–82. Chakravarty, A.K., Garai, S., Masuda, K., Nakane, T., Kawahara, N., 2003. Bacopasides IIIV: three new triterpenoid glycosides from Bacopa monniera. Chem. Phar. Bull. 51 (2), 215–217. Cherry, R.F., Khong, P.W., Lewis, K.G., 1977. Chemical constituents of Harpullia pendula: II Further constituents of the bark and leaves. Aust. J. Chem. 30, 1397–1400. Devi, P.R., Doraiswamy, S., Sevugapperumal, N., Mathiyazhagan, S., 2004. Antiviral action of harpullia cupanioides and Mirabilis jalapa against tomato spotted Wilt virus (TSWV) infecting tomato. Arch. Phytopathol. Plant Protect. 37, 245–259. dos Santos, R.T., Hiramoto, L.L., Lago, J.H.G., Sartorelli, P., Tempone, A.G., Pinto, E.G., Lorenzi, H., 2012. Anti- trypanosomal activity of 1,2,3,4,6-penta-O-galloyl-β-D-glucose isolated from plectranthus barbatus Andrews (Lamiaceae). Quim. Nova 35 (11), 2229–2232. El Sayed, N.H., El Ansari, M.A., Shalaby, A.M., Mabry, T.J., 1988. Phenolics and flavonoids of the pericarp of Harpullia pendula. Rev. Latinoamer. Quim. 19 (2), 66. El-Gohary, H.M.A., 2004. Phytochemical and biological study of seeds of Harpullia pendula planch. and Harpullia cupanioides Roxb. cultivated in Egypt. Bull. Fac. Pharm. Cairo Univ. 42 (2), 95–104. Gowri, H., Vasantha, K., 2009. Solvent based effectiveness of antibacterial and phytochemical derivatized from the seeds of Harpullia arborea (Blanco) Radlk. (Sapindaceae). J. Appl. Sci. Environ. Manage. 13, 99–101. Hisham, D.M.N., Lip, J.M., Noh, J.M., Normah, A., Nabilah, M.F.N., 2011. Identification and isolation of methyl gallate as a polar chemical marker for labisia pumila Benth. J. Trop. Agric. Fd Sc. 39 (2), 279–284. Kandil, F.E., Lewis, K.G., 2001. Polyphenols from Cornulaca monacantha. Phytochemistry 58, 611–613. Khong, P.W., Keith, G.L., 1976. Chemical constituents of Harpullia pendula. Aust. J. Chem. 29, 1351–1364. Mabberley, D.I., 1997. The Plant-Book: A Portable Dictionary of Vascular Plants, 2nd ed. Cambridge University Press, Cambridge. Melek, F.R., Miyase, T., Abdel-Khalik, S.M., Hetta, M.H., Mahmoud, I.I., 2002. Triterpenoid saponins from Oreopanax guatemalensis. Phytochemistry 60, 185–195. Mosmann, T., 1983. Colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assay. J. Immunol. Methods 65, 55–63. Murakami, T., Nakamura, J., Matsuda, H., Yoshikawa, M., 1999. Bioactive saponins and glycosides XV1). Saponin constituents with gastroprotective effect from the seeds of tea plant, Camellia sinensis L. var. assamica Pierre, cultivated in Sri Lanka: structures of Assamsaponins A, B, C, D and E. Chem. Pharm. Bull. 47 (12), 1759–1764. Ohtsuki, T., Miyagwa, T., Koyano, T., Kowithayakorn, T., Kawahara, N., Goda, Y., Ishibashi, M., 2008. Acylated triterpenoid saponins from Schima noronhae and their growth inhibitory activity. J. Nat. Prod. 71, 918–923. Voutquenne, L., Lavaud, C., Massiot, G., Delaude, C., 1998. Saponins from Harpullia cupanioides. Phytochemistry 49, 2081–2085. Voutquenne, L., Kokougan, C., Lavaud, C., Pouny, I., Litaudon, M., 2002. Triterpenoid saponins and acylated prosapogenins from Harpullia austro-caledonica. Phytochemistry 59, 825–832. Voutquenne, L., Guinot, P., Froissard, C., Thoison, O., Litaudon, M., Lavaud, C., 2005. Haemolytic acylated triterpenoid saponins from Harpullia austro-caledonica. Phytochemistry 66, 825–835. Yoshikawa, M., Harada, E., Murakami, T., Matsuda, H., Yamahra, J., Murakami, N., 1994. Camelliasaponins B1, B2, C1 and C2, New type inhibitors of ethanol absorption in rats from the seeds of Camellia Japonica L. Chem. Pharm. Bull. 42 (3), 742–744. Zhang, X.-F., Han, Y.-Y., Bao, G.-H., Ling, T.-J., Zhang, L., Gao, L.-P., Xia, T., 2012. A new saponin from Tea seed Pomace (Camellia oleifera Abel) and its protective effect on PC12 cells. Molecules 17, 11721–11728.
6%, 8%, 12.5% and 15% methanol. Each fraction was separately purified by repeated chromatography using Sephadex LH-20 column with methanol as an eluent to give 3 (30 mg), 4(21 mg), 5 (15 mg) and 6 (17 mg), respectively. 3.4. Pendulaoside A (1) Amorphous powder [a]23 D −23.95 (c 2.45, MeOH), HR-ESI–MS m/z: 1041.5179 [C52H82O21-1]-, 1H and 13CNMR see Table 1 3.5. Pendulaoside B (2) Amorphous powder [a]23 D −16.54 (c 0.54, MeOH), HR-ESI–MS m/z: 1043.5321 [C52H84O21-1]-, 1H and 13CNMR see Table 1 3.6. General method for acid hydrolysis Each saponin (2 mg) in 1.5N HCL (2 ml) was heated at 100 °C for 4 h. The solvent was evaporated and the residue was dissolved in H2O then extracted with CH2Cl2. The remaining aqueous layer was neutralized by repeated addition of MeOH followed by evaporation. The sugars were then analyzed using paper chromatography (n-BuOHCH3COOH-H2O, 4:1:5, upper layer) by comparison with authentic samples. The chromatogram was visualized by spraying with aniline hydrogen phthalate reagent and heating at 110 °C till the colour of the spots appeared. D-Glucuronic acid, D-glucose and L-arabinose were detected after acid hydrolysis of saponins 1 and 2. 3.7. Cytotoxic assay The procedure for the cytotoxic assay was performed according to the MTT method as described by Mosmann (1983). In this study, the cell lines HepG2 (hepatocellular carcinoma cell line), MCF7 (breast carcinoma cell line) and PC3 (prostatic carcinoma cell line), were used. Declaration of interest The authors declared no conflict of interest. References Abdel-Gawad, M.M., El-Sayed, M.M., El-Nahas, H.A., Abdel-Hameed, E.S., 2004. Laboratory evaluation of the molluscicidal miracidicidal and cercaricidal properties of two Egyptian plants. Bull. Pharm. Sci. 27, 331–339. Abdelkader, M.S.A., Rateb, M.E., Mohamed, G.A., Jaspars, M., 2016. Harpulliasides A and B: Two new benzeneacetic acid derivatives from Harpullia pendula. Phytochem. Lett. 15, 131–135. Abri, A., Maleki, M., 2016. Isolation and identification of gallic acid from the Elaeagnus angustifolia leaves and determination of total phenolics, flavonoid contents and
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Pendulaosides A and B. Two acylated triterpenoid saponins from Harpullia pendula seed extract
MARK
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Neveen S. Ghaly, Marian Nabil, Mary H. Grace, Farouk R. Melek Chemistry of Natural Compounds Department, National Research Centre, Dokki, 12622, Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords: Harpullia pendula Sapindaceae Pendulaosides A and B Triterpenoid saponins Cytotoxicity
Two new acylated triterpenoid saponins named pendulaosides A and B as well as the known phenolic compounds methyl gallate, gallic acid, 1,2,3,6-tera-O-galloyl-β-D-glucose and 1,2,3,4,6-penta-O-galloyl-β-D-glucose, were isolated from the seeds of Harpullia pendula. The structures of pendulaosides A and B were determined using extensive 1D and 2D NMR analysis and mass spectrometry as well as acid hydrolysis, as 3-O-β-D-glucopyranosyl-(1→2)-[α-L-arabinofuranosyl-(1→3)]-β-D-glucuronopyranosyl-22-O-angeloyl-3β,16α,22α,24β,28pentahydroxylolean-12-ene and 3-O-β-D-glucopyranosyl-(1→2)-[α-L-arabinofuranosyl-(1→3)]-β-D-glucuronopyranosyl-16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene, respectively. To the best of our knowledge the two triterpene parts 22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12-ene and16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene have never been characterized before. The two isolated saponins were assayed for their in-vitro cytotoxic activity against the three human tumor cell lines HepG2, MCF7 and PC3. The results showed that pendulaoside A exhibited moderate activity on PC3 cell line with IC50value equal to 13.0 μM and weak activity on HepG2 cell line with IC50 value equal to 41.0 μM. Pendulaoside B proved to be inactive against the three used cell lines.
1. Introduction The genus Harpullia which belongs to the family Sapindaceae, consists of about 27 species. They are widely distributed ranging from Malesia and Australia to the Pacific islands (Mabberley, 1997). Phytochemical studies carried out on plants of this genus, have revealed them as a source of triterpenoid saponins (Voutquenne et al., 1998, 2002, 2005). The saponins of this genus belong to the polyhydroxylate triterpenoid glycosides containing ester function. This type of saponins was reported to possess antiviral (Devi et al., 2004), antifungal (Bharathimatha et al., 2002), antibacterial (Gowri and Vasantha, 2009), cytotoxic (Voutquenne et al., 2005), molluscicidal, miracidicidal and cercaricidal (Abdel-Gawad et al., 2004) properties. Harpullia pendula Planch with the common name tulipwood or tuliplancewood, is a small to medium sized rainforest tree native to Australia and cultivated in gardens and streets for ornamental purposes. Previous phytochemical examination of the leaf and bark extracts of H. pendula, led to the isolation of the triterpenes A1-barrigenol, A1-barrigenol-22-O-angelate, camelliagenin A, camelliagenin A-16-and 22-O-angelate, 22α-hydroxyerthrodiol and 15α,16α,22α,28-tetrahydroxyolean-12-ene-3-one together with quebrachitol and methyl-p-coumarate (Khong and Keith, 1976; Cherry et al., 1977). Also, three sapogenins were isolated from
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the saponin hydrolysates of H.pendula and H. cupanioides Roxb. and identified as angeloyl camelliagenin A, angeloyl A1- barrigenol and angeloyl A1- barrigenol methyl ether (El-Gohary, 2004). The isolation of phenolics and flavonoids from the pericarp of H. pendula was previously reported (El Sayed et al., 1988). Recent phytochemical study on the leaf extract of H. pendula led to isolation of two antimicrobial benzeneacetic acid derivatives harpulliasides A and B together with several kaempferol glycosides (Abdelkader et al., 2016). As a part of our continuous interest in bioactive saponins from plants cultivated in Egypt, we describe in this report the isolation and characterization of two new acylated saponins named pendulaoside A (1) and pendulaoside B (2) from the seeds of H. pendula together with four known phenolic compounds. 2. Results and discussion The defatted methanolic seed extract of H. pendula was suspended in water and partition with ethyl acetate then with n- butanol. The nbutanol fraction was chromatographed over the polymer gel Diaion HP20 and silica gel followed by repeated HPLC to afford two new acylated saponins named pendulaoside A (1) and pendulaoside B (2). The ethyl acetate fraction yielded after chromatography the known phenolic
Corresponding author. E-mail addresses: [email protected] (N.S. Ghaly), [email protected] (M. Nabil), [email protected] (M.H. Grace), [email protected] (F.R. Melek).
http://dx.doi.org/10.1016/j.phytol.2017.06.016 Received 31 January 2017; Received in revised form 4 June 2017; Accepted 16 June 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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HSQC spectrum to a low-field carbon signal at δC 71.1 and coupled to methylene proton signals at δH 1.38 (1H, m) and 1.73 (1H, dd, J = 11.0, 3.3 Hz) in the TOCSY spectrum, was assigned to the equatorial H-16 proton. The deshielded methyl signal assigned to 27-CH3 at δH 1.48, suggested the existence of a hindered axial hydroxyl group at C-16 position of the triterpene moiety. This suggestion was verified by the appearance (brs) and the chemical shift value of the equatorial H-16 proton which were similar to the corresponding ones of structurally related saponins (Zhang et al., 2012). The observed HMBC correlations H-16/C-17 (δC 45.4) and C-18 (δC 42.7), lent further support to the assignment of H-16 proton. The deshielded doublet of doublets at δH 5.43, directly correlated to a carbon signal at δC 74.0 in the HSQC spectrum and coupled in the COSY and TOCSY spectra to methylene proton signals at δH 1.55 (1H, m) and 2.26 (1H, t, J = 12.0 Hz), was assigned to H-22β proton. The coupling constant value of H-22 indicated a β-axial orientation of this proton. The observed HMBC correlations that supported the assignment of H-22, were between the following signals H-22/C-16 (δC 71.1), C-17 (δC 45.4), C-18 (δC 42.7) and C-21 (δC 42.0). Further information from 1D and 2D NMR spectra of 1 also indicated the presence of signals due to an angeloyl group at δH 6.08 (1H, qd, J = 7.5, 1.5 Hz, H-3′), 1.97 (3H, dd, J = 7.5, 1.5 Hz, H-4′), 1.90 (3H, d-like, J = 1.5 Hz, H-5′) and at δC 169.9 (C-1′), 130.1 (C-2′), 138.2 (C-3′), 16.0 (C-4′) and 21.0 (C-5′). These data were in agreement with the corresponding ones reported for related saponins bearing an identical moiety (Voutquenne et al., 2005; Ohtsuki et al., 2008; Zhang et al., 2012). Inspection of literature revealed that saponins bearing tigloyl moiety with cis double bond, showed chemical shift values for C-4′ and C-5′ of this moiety at 14.0 and 12.3 ppm, respectively (Yoshikawa et al., 1994; Murakami et al., 1999). The observed low-field position of H-22 signal at δH 5.43 due to acylation, suggested that the angeloyl group esterified the 22-hydroxyl group in the triterpene moiety. The location of the acyl group at C-22 was confirmed
compounds methyle gallate 3 (Hisham et al., 2011), gallic acid 4 (Abri and Maleki, 2016), 1, 2, 3,6-tetra-O-galloyl-β-D-glucose 5 (Kandil and Lewis, 2001) and 1,2,3,4,6- penta-O-galloyl-β-D-glucose 6 (dos Santos et al., 2012). The structures of the known compounds were identified by comparison of their spectral data with those reported in the literature. The molecular formula of pendulaoside A (1) was determined as C52H82O21 from [M−H]− at m/z 1041.5179 by high resolution negative-ion electrospray ionization mass spectrometry (calculated, 1041.5276). Acid hydrolysis of 1 afforded sugar components identified as D-glucuronic acid, D-glucose and L-arabinose, thus indicating the glycosidic nature of 1. The 13CNMR spectrum of 1 showed the presence of signals due to six methyl carbons at δC 16.4, 17.3, 22.9, 25.4, 27.8 and 33.7 as well as olefinic carbons at δC 124.6 and 144.0. This information coupled with the presence of six methyl singlets at δH 0.89, 0.91, 0.93, 1.04, 1.21 and 1.48 and a vinyl proton signal at δH 5.33 (brs) in the 1HNMR spectrum, suggested 1 to be a triterpenoid saponin bearing a triterpene moiety with olean-12-ene skeleton. Of the 52 carbons, 30 were assigned to the triterpene moiety, 17 to the glycosidic part and the remaining 5 to an acyl group. Extensive 1D (1H, 13C), and 2D (1H-1H COSY, TOCSY, NOESY, HSQC, HMBC) NMR analysis (Table 1) allowed complete assignments of the proton and carbon signals of the triterpene moiety and revealed the presence of signals due to three oxygen bearing methine protons at δH 3.40 (1H, dd, J = 11.0, 4.0 Hz), 4.12 (1H, brs) and 5.43 (1H, dd, J = 12.0, 5.0 Hz). The methine proton signal at δH 3.40 that directly correlated to a carbon signal at δC 93.0 in the HSQC spectrum of 1 and coupled to methylene proton signals at δH 1.82 (1H, m) and 2.03 (1H, m) in the COSY and TOCSY spectra, was assigned to H-3α proton. The deshielded carbon signal at δC 93.0 was assigned to C-3 based on comparison with the corresponding values of olean-12-ene saponins (Voutquenne et al., 2005). The broad methine singlet at δH 4.12 that directly correlated in the Table 1 1 H and 13CNMR data of compounds 1 and 2 in CD3OD. 1
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 30
2
1
δC
δH
δC
δH
39.8 27.2 93.0 44.6 57.5 19.5 34.2 41.1 48.0 37.6 25.0 124.6 144.0 42.7 35.4 71.1 45.4 42.7 48.2 32.6 42.0 74.0 22.9 64.3 16.4 17.3 27.8 64.9 33.7 25.4
1.67(m),1.03(m) 2.03(m), 1.82(m) 3.40(dd,11.0,40)
39.7 27.1 93.0 44.6 57.4 19.4 34.1 41.1 48.0 37.6 24.9 125.0 142.7 42.6 31.9 70.7 43.5 42.6 48.2 32.3 44.6 74.2 22.9 64.3 16.5 17.3 27.6 70.3 33.8 25.5
1.65(m),1.03(m) 2.05(m),1.78(m) 3.40(dd,11.04.0)
0.97 1.62(m),1.40(m) 1.62(m),1.38(m) 1.68(m) 1.93(m) 5.33(brs)
1.73(dd,11.0,3.3), 1.38(m) 4.12 (brs) 2.52(dd,14.0,4.0) 2.44(t,12.0),1.05(m) 2.26(t,12.0),1.55(m) 5.43(dd,12.0,5.0) 1.21(s) 4.12, (d, 12.0),3.17(d, 12.0) 0.89(s) 0.93(s) 1.48(s) 3.27(d,11.0),3.05(d, 11.0) 0.91(s) 1.04(s)
δC
1.68(m) 1.93(m) 5.37(brs)
1.99(m),1.46(brd, 15.0) 5.49(brs) 2.18(dd,14.0,4.0) 2.28(m),1.12(m) 1.78(t,12.0),1.42(m) 4.07 1.22(s) 4.12(d, 12.0),3.21(d,12.0) 0.93(s) 0.97(s) 1.34(s) 3.64(d,12.0),3.31, (d,12.0) 0.9(s) 1.10(s)
Overlapped signals are represented without designated multiplicity. Values in parentheses represent 1H-1H splitting.
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δH
GlcUA 105.0 4.54(d,7.8) 78.5 3.70 87.4 3.70 72.3 3.60(t, 9.0) 78.0 3.62(d, 9.0) 178.3 Glc 1 104.0 4.78(d,8.0) 2 75.6 3.19(t,8.0) 3 78.0 3.37(t,8.5) 4 70.2 3.50(t,9.0) 5 78.2 3.22(m) 6 61.7 3.79(d,12.0),3.77 Ara(f) 1 111.0 5.22(d,2.0) 2 83.6 4.13(dd,4.0,2.0) 3 77.9 3.86(dd,7.0,4.0) 4 85.3 4.10(m) 5 62.9 3.81(dd,12.0,4.0),3.63 22-Angeloyl 1′ 169.9 2′ 130.1 3′ 138.2 6.08(qd,7.5,1.5) 4′ 16.0 1.97(dd,7.5,1.5) 5′ 21.0 1.90(d-like,1.5) GlcUA = β-D-glucuronopyranose Glc = β-D-glucopyranose Ara(f) = α-L-arabinofuranose 1 2 3 4 5 6
0.97 1.62(m),1.40(m) 1.60(m),1.38(m)
2 δC
δH
105.0 78.4 87.3 72.3 78.1 177.7
4.54(d,7.8) 3.70 3.70 3.62 (t, 8.0) 3.64 (d, 9.0)
104.0 75.5 78.1 70.3 78.2 61.7
4.75(d,8.0) 3.21(t,8.0) 3.38(t,8.5) 3.51(t,9.0) 3.24(m) 3.80(d,12.0),3.76
111.1 5.17(d,2.0) 83.6 4.13(dd,4.0,2.0) 77.9 3.88(dd,7.0,4.0) 85.4 4.09(m) 62.9 3.80(dd,12.0,4.0),3.63 16-2-methyl butyroyl 177.7 43.5 2.33(sextet,7.0,7.0) 28.0. 1.78(m),1.52(m) 12.5 0.96(t,7.0) 17.0 1.15(d,7.0)
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at m/z 911.489 and 749.443 due to consequent loss of pentose and hexose units. Acid hydrolysis of 2 yielded sugar components identical to those liberated after acid hydrolysis of 1. 1D and 2D NMR spectra of 2 showed signals due to sugar units very similar to those of 1, indicating the existence of identical trisaccharide moiety in 1 and 2 (Table 1). The attachment of this moiety to C-3 of the triterpene moiety was deduced from the deshielded signal of C-3 (δC 93.0) and the HMBC correlation between the signals of C-3 and H-1 of the inner 2,3- disubstituted β-Dglucuronopyranosyl unit. The other HMBC correlations due to the saccharide moiety of 2, were identical to those of 1. The NMR analysis of 2 also revealed a triterpene moiety of 2 similar to that of 1 except that a 2-methylbutyroyl group esterified the α-OH at C-16 in 2 instead of the angeloyl group esterified C-22 α-OH in 1. The observed shielded position of the signal due to 27-CH3 at δΗ 1.34 in 2 and the deshielded position of H-16 signal at δΗ 5.49 (brs) together with the shielded signal at δH 4.07 assigned to H-22β, relative to the corresponding δ values in 1, indicated esterified 16α-OH and free 22α-OH. The assignments of H16β and H-22β were deduced from 1D and 2D NMR analysis, following the same approach used for compound 1. These assignments were in complete agreement with the corresponding assignments reported for structurally related saponins (Voutquenne et al., 1998). The presence of the 2-methylbutyroyl group in 2 was evidenced by the presence of signals due to a carbonyl carbon at δC 177.7, two methyl groups at δΗ 0.96 [(t, J = 7.0 Hz), δC 12.5] and 1.15 [(d, J = 7.0 Hz), δC 17.0], a methine proton at δH 2.33 (dt, J = 7.0 Hz) together with methylene protons at δΗ 1.52 (m) and 1.78 (m). The TOCSY spectrum of 2 showed correlations between the methine proton signal at δΗ 2.33 and the signals due to the methyl at δH 1.15 as well as the two signals of the methylene protons at δH 1.52 and 1.78. HMBC correlations were also observed between the signal of the carbonyl carbon and the signals due to H-16 at δΗ 4.49, the methine proton at δH 2.32, the methylene protons at δΗ 1.45 and 1.71 and the methyl group at δH 1.15. In the NOESY spectrum of 2, correlations between H-22 and H-30 and between H-16 and H-28(δH 3.64), confirmed the relative configurations at C-22 and C-16. Therefore, the triterpene moiety of compound 2 was assigned the structure of 16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28pentahydroxylolean-12-ene. To the best of our knowledge, this triterpene part has never been characterised before. Consequently, pendulaoside B (2) was formulated as 3-O-β-D-glucopyranosyl-(1 → 2)-[αL-arabinofuranosyl-(1 → 3)]-β-D-glucuronopyranosyl-16-O-(2-methylbutyroyl)-3β,16α,22α,24β,28-pentahydroxylolean-12-ene (Fig. 1). The two isolated saponins were assayed for their in-vitro cytotoxic activity against the three human tumor cell lines HepG2, MCF7 and PC3. The results showed that penulaoside A exhibited moderate activity on PC3 cell line with IC50 value equal to 13.0 μM and weak activity on HepG2 cell line with IC50 value equal to 41.0 μM. Pendulaoside B proved to be inactive on the three used cell lines.
by the observed cross peak in the HMBC spectrum correlating the signals due to H-22 at δH 5.43 and the carbonyl carbon (C-1′) of the angeloyl group at δC 169.9. Further HMBC correlations were observed between the signals H-3′/C-1′, C-4′ and C-5′. Furthermore, two pairs of hydroxyl methylene proton signals appeared as two isolated systems, were located at δH 3.17 (1H, d, J = 12.0 Hz) and 4.12 (1H, d, J = 12.0 Hz) as well as at δH 3.05 (1H, d, J = 11.0 Hz) and 3.27 (1H, d, J = 11.0 Hz). The signals due to the two pairs of protons were directly correlated in the HSQC spectrum to carbon signals at δC 64.3 and 64.9 and assigned to H2-24 (Voutquenne et al., 2005) and H2-28, respectively, based on the observed HMBC correlations H-24a (δH 3.17)/C-3 (δC 93.0); H-24b (δH 4.12)/C-3, C-4 (δC 44.6) and C-23 (δC 22.9) as well as H-28a (δH 3.05)/C-16, C-17, C-18 (δC 42.7) and C-22; H-28b (δH 3.27)/C-17, C-18 and C-22. The possibility of hydroxylation at C-23 instead of C-24 was ruled out because C-3 and C-5 signals would appear at significant higher field positions (Yoshikawa et al., 1994). The relative stereochemistry at C-16 and C-4 was further evidenced by NOESY experiment of 1. The observed NOEs were between H-16 and H-28 (δH 3.27) and between H-24 (δH 4.12) and axial H-2 (δH 1.82). Based on the above evidences, the triterpene part of saponin 1 was assigned the structure of 22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12ene. To the best of our knowledge, this triterpene part has never been reported before. A similar moiety with different stereochemistry at C-4 was previously reported by Yoshikawa et al. (1994). The presence of three sugar units in 1 was also evidenced by 1D and 2D NMR analysis that disclosed the presence of three anomeric proton signals at δH 4.54 (1H, d, J = 7.8 Hz), 4.78 (1H, d, J = 8.0 Hz) and 5.22 (1H, d, J = 2.0 Hz) with their corresponding carbons appeared at δC 105.0, 104.0 and 111.0, respectively. The low-field position of C-3 at δC 93.0 suggested the monodesmosidic nature of 1 with a saccharide unit attached to this position. The individual spin system for the individual monosaccharide was assigned by TOCSY spectrum and the sequence of protons was deduced from the 1H-1H COSY experiment. On the basis of the assigned protons the 13C signals of each monosaccharide was recognized by HSQC spectrum. Further supporting information were given by the HMBC experiment which clarified the assignments of some closely related protons and carbons. The obtained data (Table 1) allowed identification of three monosaccharide units as a 2,3-disubstituted β-D-glucuronic acid with anomeric proton signal at δH 4.54, a terminal β-D-glucose with anomeric proton signal at δH 4.78 and a terminal α-L-arabinose with anomeric proton signal at δH 5.22. The first two sugars were in the pyranose form based on their 13CNMR data. The β anomeric configurations of these units were evidenced by their coupling constant values (7.0–8.0 Hz). The third sugar was in the furanose form as shown by the carbon chemical shift values and its α anomeric configuration was deduced by the small coupling constant value (2.0 Hz) as previously reported (Agrawal, 1992; Melek et al., 2002; Chakravarty et al., 2003; Voutquenne et al., 2005). The inter-glycosidic linkages were determined by the observed HMBC correlations between the signals of arabinofuranose H-1 and glucuronopyranose C-3 (δC 87.4) and between the signals of glucopyranose H-1 and glucuronopyranose C-2 (δC 78.5). The HMBC correlation between the signals due to glucuronopyranose H-1 and C-3 of the triterpene moiety, confirmed the attachment of the trisaccharide unit to this position. Therefore, the structure of the trisaccharide unit was established as 3-O-β-D-glucopyranosyl-(1 → 2)-[α-L-arabinofuranosyl-(1 → 3)]-β-D-glucuronopyranosyl. It is worth noting that an identical unit was previously reported as the glycosidic part of protoaescigenin saponins isolated from H. austro- caledonica (Voutquenne at al., 2005). Consequently, pendulaoside A (1) was assigned the structure of 3-O-β-D-glucopyranosyl-(1 → 2)-[α-L-arabinofuranosyl-(1 → 3)]-β-D-glucuronopyranosyl-22-O-angeloyl-3β,16α,22α,24β,28-pentahydroxylolean-12-ene as shown in Fig. 1. Pendulaoside B (2) was another triterpenoid saponin which had a molecular formula C52H84O21 as determined by high resolution MS measurement. The HR-ESI mass spectrum of 2 exhibited [M-H]− at m/z 1043.5321 (calculated, 1043.5432) and two prominent fragment ions
3. Experimental 3.1. General Liquid chromatography-ion trap-top of flight mass spectrometry was performed using Shimadzu LC-IT-TOF-MS (Shimadzu, Tokyo, Japan) with a Shim-pack XR-ODS column (50 mm × 3.0 mm × 2.2 μm). Solvent gradient consisted of 0.1% formic acid in H2O (A) and acetonitrile (B). Compounds were eluted into the ESI ion source at the flow rate of 0.4 ml/min with a step gradient of B in A: 10–85% B (0–15 min), 85-10%B (15–18 min), isocratic at 10% B (2 min). Column was maintained at 40∘C during the run. Nitrogen gas was used as nebulizer and drying gas with the flow rate set at 1.5 l/min. The ESI source voltage was set at 4.5 kV and the detector was set as 1.5 V. Ionization was performed using a conventional ESI source in the negative ionization mode. Shimadzu's LCMS solution software was used for data analysis. NMR experiments were performed on a Bruker Avance 700 MHz spectrometer (Bruker BioSpin Corporation, Billerica, MA). 1H, 13C, 1H-1H 280
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Fig. 1. Chemical structures of pendulaoside A (1) and pendulaoside B (2).
in H2O (0.002) and the H2O solution was passed through a column chromatography packed with 100 g Diaion HP-20 polymer gel (Mitsubishi). After washing the column with distilled water, elution was carried out with 25%, 50%, 75% aqueous MeOH and finally with 100% MeOH. The collected fractions were examined by silica gel TLC (Merck) using solvent systems n-BuOH- EtOH-NH4OH (7: 2: 5), and CHCl3MeOH-H2O (60: 30: 5) then visualized by spraying with 20% sulphuric acid in MeOH followed by heating at 110 °C. Based on TLC analysis, similar fractions were then combined. Fractions eluted with 75% and 100% MeOH were found similar and contained saponin constituents. The combined saponin fraction (1.35 g) was applied on a column chromatography packed with silica gel (60 g) and eluted with EtOAc −MeOH-H2O with increasing polarity (30:2:1–5:2:1). A total of 50 fractions 50 ml each were collected. Similar fractions were combined after TLC analysis to give eight sub-fractions (A-H). The sub-fraction D (850 mg) eluted with EtOAc-MeOH-H2O (20:2:1), was subjected to repeated preparative HPLC to give 1 (15 mg) and 2 (17 mg). The EtOAc fraction was subjected to Sephadex LH-20 column chromatography. The column was eluted first with acetone and then with acetone methanol mixtures with increasing amount of methanol to 2%. A total of 40 fractions 50 ml each were collected. The fractions were monitored by silica gel TLC plates using solvent system EtOAc- MeOH- H2O (20: 3: 2) and examined by spraying with FeCl3 reagent. Similar fractions were pooled and the solvent was evaporated from each combined fraction under reduced pressure to yield four major sub-fractions collected by
COSY, TOCSY, NOESY, HSQC, HMBC NMR spectra were acquired in CD3OD at 700 MHz for proton and 175 MHz for 13C NMR. Chemical shifts are given as δ values with TMS as internal standard. Optical rotations were measured with jasco p-2000 polarimeter. Preparative HPLC was carried out on Interchim 4100 (Montlucon, France) (column, RP C-18 HQ; solvent system, CH3CN-H2O (33:67-70:30); Flow rate 20 ml/min; Detection, UV, 205 nm; temperature, 35 °C). 3.2. Plant material Seeds of H. pendula Planch.were collected from the zoological garden in Giza, Egypt in September 2014. Plant identification was confirmed by Mrs. T. Labib, head specialist for plant identification in ElOrman public garden, Giza, Egypt. A Voucher specimen was deposited in the Herbarium of NRC (CAIRC). 3.3. Extraction and isolation Air-dried seeds of H. pendula (100 g) were defatted with n-hexane then extracted with MeOH until exhaustion. The combined MeOH extract was evaporated under vacuum to dryness. The residue (4.5 g) was suspended in water and extracted with EtOAc (5 × 100 ml) then water saturated n-BuOH (5 × 100 ml). Each combined fraction was individually evaporated under reduced pressure to yield 1.5 g EtOAc fraction and 2.1 g n- BuOH fraction. The n-BuOH fraction was dissolved 281
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investigation of antioxidant activity. Iran. Chem. Commun. 4, 146–154. Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 31 (10), 3307–3330. Bharathimatha, C., Doraiswamy, S., Rabindran, R., Velazhahan, R., 2002. Inhibition of fungal plant pathogens by seed proteins of Harpullia cupanioides (Roxb). Acta Phytopathol. Entomol. Hung. 37, 75–82. Chakravarty, A.K., Garai, S., Masuda, K., Nakane, T., Kawahara, N., 2003. Bacopasides IIIV: three new triterpenoid glycosides from Bacopa monniera. Chem. Phar. Bull. 51 (2), 215–217. Cherry, R.F., Khong, P.W., Lewis, K.G., 1977. Chemical constituents of Harpullia pendula: II Further constituents of the bark and leaves. Aust. J. Chem. 30, 1397–1400. Devi, P.R., Doraiswamy, S., Sevugapperumal, N., Mathiyazhagan, S., 2004. Antiviral action of harpullia cupanioides and Mirabilis jalapa against tomato spotted Wilt virus (TSWV) infecting tomato. Arch. Phytopathol. Plant Protect. 37, 245–259. dos Santos, R.T., Hiramoto, L.L., Lago, J.H.G., Sartorelli, P., Tempone, A.G., Pinto, E.G., Lorenzi, H., 2012. Anti- trypanosomal activity of 1,2,3,4,6-penta-O-galloyl-β-D-glucose isolated from plectranthus barbatus Andrews (Lamiaceae). Quim. Nova 35 (11), 2229–2232. El Sayed, N.H., El Ansari, M.A., Shalaby, A.M., Mabry, T.J., 1988. Phenolics and flavonoids of the pericarp of Harpullia pendula. Rev. Latinoamer. Quim. 19 (2), 66. El-Gohary, H.M.A., 2004. Phytochemical and biological study of seeds of Harpullia pendula planch. and Harpullia cupanioides Roxb. cultivated in Egypt. Bull. Fac. Pharm. Cairo Univ. 42 (2), 95–104. Gowri, H., Vasantha, K., 2009. Solvent based effectiveness of antibacterial and phytochemical derivatized from the seeds of Harpullia arborea (Blanco) Radlk. (Sapindaceae). J. Appl. Sci. Environ. Manage. 13, 99–101. Hisham, D.M.N., Lip, J.M., Noh, J.M., Normah, A., Nabilah, M.F.N., 2011. Identification and isolation of methyl gallate as a polar chemical marker for labisia pumila Benth. J. Trop. Agric. Fd Sc. 39 (2), 279–284. Kandil, F.E., Lewis, K.G., 2001. Polyphenols from Cornulaca monacantha. Phytochemistry 58, 611–613. Khong, P.W., Keith, G.L., 1976. Chemical constituents of Harpullia pendula. Aust. J. Chem. 29, 1351–1364. Mabberley, D.I., 1997. The Plant-Book: A Portable Dictionary of Vascular Plants, 2nd ed. Cambridge University Press, Cambridge. Melek, F.R., Miyase, T., Abdel-Khalik, S.M., Hetta, M.H., Mahmoud, I.I., 2002. Triterpenoid saponins from Oreopanax guatemalensis. Phytochemistry 60, 185–195. Mosmann, T., 1983. Colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assay. J. Immunol. Methods 65, 55–63. Murakami, T., Nakamura, J., Matsuda, H., Yoshikawa, M., 1999. Bioactive saponins and glycosides XV1). Saponin constituents with gastroprotective effect from the seeds of tea plant, Camellia sinensis L. var. assamica Pierre, cultivated in Sri Lanka: structures of Assamsaponins A, B, C, D and E. Chem. Pharm. Bull. 47 (12), 1759–1764. Ohtsuki, T., Miyagwa, T., Koyano, T., Kowithayakorn, T., Kawahara, N., Goda, Y., Ishibashi, M., 2008. Acylated triterpenoid saponins from Schima noronhae and their growth inhibitory activity. J. Nat. Prod. 71, 918–923. Voutquenne, L., Lavaud, C., Massiot, G., Delaude, C., 1998. Saponins from Harpullia cupanioides. Phytochemistry 49, 2081–2085. Voutquenne, L., Kokougan, C., Lavaud, C., Pouny, I., Litaudon, M., 2002. Triterpenoid saponins and acylated prosapogenins from Harpullia austro-caledonica. Phytochemistry 59, 825–832. Voutquenne, L., Guinot, P., Froissard, C., Thoison, O., Litaudon, M., Lavaud, C., 2005. Haemolytic acylated triterpenoid saponins from Harpullia austro-caledonica. Phytochemistry 66, 825–835. Yoshikawa, M., Harada, E., Murakami, T., Matsuda, H., Yamahra, J., Murakami, N., 1994. Camelliasaponins B1, B2, C1 and C2, New type inhibitors of ethanol absorption in rats from the seeds of Camellia Japonica L. Chem. Pharm. Bull. 42 (3), 742–744. Zhang, X.-F., Han, Y.-Y., Bao, G.-H., Ling, T.-J., Zhang, L., Gao, L.-P., Xia, T., 2012. A new saponin from Tea seed Pomace (Camellia oleifera Abel) and its protective effect on PC12 cells. Molecules 17, 11721–11728.
6%, 8%, 12.5% and 15% methanol. Each fraction was separately purified by repeated chromatography using Sephadex LH-20 column with methanol as an eluent to give 3 (30 mg), 4(21 mg), 5 (15 mg) and 6 (17 mg), respectively. 3.4. Pendulaoside A (1) Amorphous powder [a]23 D −23.95 (c 2.45, MeOH), HR-ESI–MS m/z: 1041.5179 [C52H82O21-1]-, 1H and 13CNMR see Table 1 3.5. Pendulaoside B (2) Amorphous powder [a]23 D −16.54 (c 0.54, MeOH), HR-ESI–MS m/z: 1043.5321 [C52H84O21-1]-, 1H and 13CNMR see Table 1 3.6. General method for acid hydrolysis Each saponin (2 mg) in 1.5N HCL (2 ml) was heated at 100 °C for 4 h. The solvent was evaporated and the residue was dissolved in H2O then extracted with CH2Cl2. The remaining aqueous layer was neutralized by repeated addition of MeOH followed by evaporation. The sugars were then analyzed using paper chromatography (n-BuOHCH3COOH-H2O, 4:1:5, upper layer) by comparison with authentic samples. The chromatogram was visualized by spraying with aniline hydrogen phthalate reagent and heating at 110 °C till the colour of the spots appeared. D-Glucuronic acid, D-glucose and L-arabinose were detected after acid hydrolysis of saponins 1 and 2. 3.7. Cytotoxic assay The procedure for the cytotoxic assay was performed according to the MTT method as described by Mosmann (1983). In this study, the cell lines HepG2 (hepatocellular carcinoma cell line), MCF7 (breast carcinoma cell line) and PC3 (prostatic carcinoma cell line), were used. Declaration of interest The authors declared no conflict of interest. References Abdel-Gawad, M.M., El-Sayed, M.M., El-Nahas, H.A., Abdel-Hameed, E.S., 2004. Laboratory evaluation of the molluscicidal miracidicidal and cercaricidal properties of two Egyptian plants. Bull. Pharm. Sci. 27, 331–339. Abdelkader, M.S.A., Rateb, M.E., Mohamed, G.A., Jaspars, M., 2016. Harpulliasides A and B: Two new benzeneacetic acid derivatives from Harpullia pendula. Phytochem. Lett. 15, 131–135. Abri, A., Maleki, M., 2016. Isolation and identification of gallic acid from the Elaeagnus angustifolia leaves and determination of total phenolics, flavonoid contents and
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