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Chemical constituents from Bidens bipinnata Linn.
Biochemical Systematics and Ecology 79 (2018) 44–49
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Chemical constituents from Bidens bipinnata Linn. 1
1
Hai-Min Hu , Shu-Min Bai , Li-Jiao Chen, Wen-Yi Hu, Guang Chen
T ∗
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Asteraceae Bidens bipinnata Linn. Ceramide
The phytochemical investigation on Bidens bipinnata Linn. (Asteraceae) led to the isolation and identification of 38 compounds, including nine ceramides (1–9), thirteen flavonoids (10–22), five phenylpropanoids (23–27), four aliphatics (28, 35–37), one pyrimidine (29), four steroids (30–33), one triterpenoid (34) and one polyacetylene (38). All chemical structures were established on the basis of MS and NMR spectroscopic data. In addition to five newly reported compounds (2S, 3S, 4R, 8E)-2-(2′(R), 3′(R)-dihydroxytriocosanoylamino]-8octadecene-1, 3, 4-triol (1), (2S, 3S, 4R, 8E)-2-[(2′R)-2′-hydroxytetracosanoylamino]-10-octadecene-1, 3, 4triol/(2S, 3S, 4R, 8E)-2-[(2′R)-2′-hydroxytriocosanoylamino]-10-octadecene-1, 3, 4-triol (2/3) and (2R) isookanin-4′-methoxy-7-O-β-D-glucopyranoside/(2S) isookanin-4′-methoxy-7-O-β-D-glucopyranoside (10/11), nine compounds (4–9, 25, 26, 38) were isolated from the Bidens genus for the first time. Meanwhile, the compounds 12, 13, 16–21, 30–34 were reported firstly from Bidens bipinnata Linn. On the basis of chemical research, the chemotaxonomic significance of the isolated compounds has been summarized.
1. Subject and source
3. Present study
Bidens bipinnata Linn. which belongs to Bidens genus and family Asteraceae, is an annual plant and widely distributed in China. The whole plant of Bidens bipinnata Linn. was purchased from Anguo County of Hebei Province, P. R. China in April 2015, and was identified by Prof. Yun-Zhen Guo, College of Traditional Chinese Medicines, Shenyang Pharmaceutical University. A voucher specimen (No. 150411) is deposited in College of Life Science and Technology, Beijing University of Chemical Technology.
The whole plant of Bidens bipinnata Linn. (30 kg) was extracted twice with 80% EtOH for 2 h. After removal of the solvent in vacuo, the residue was suspended in water. This suspension was further fractionated with petroleum ether, CHCl3 and n-BuOH, successively. The CHCl3 extract (85 g) was subsequently separated using silica gel column chromatography (900 g, 9 × 100 cm column; petroleum ether/EtOAc gradient) to yield 8 fractions (1–8) based on TLC profiles. Fraction 2 was submitted to passage over silica gel column eluted by CHCl3/EtOAc to give 30 (8.4 mg) and 32 (5.9 mg). Fraction 3 was further subjected to silica gel column eluted by CHCl3/EtOAc to obtain 2 (3.2 mg) and 3 (5.3 mg). Fraction 4 was chromatographed on silica gel column and eluted by CHCl3/EtOAc to give 1 (4.2 mg). Fraction 5 was further purified by Sephadex LH-20 to obtain 29 (7.1 mg). Fraction 8 was applied to ODS column using MeOH as eluent to give 31 (11.3 mg) and 33 (7.2 mg). The n-BuOH extract (119 g) was fractioned by silica gel column chromatography (1200 g, 10 × 110 cm column; CHCl3/MeOH gradient) to give 9 subfractions (1–9) based on TLC profiles. Fraction 1 was further separated by Sephadex LH-20 using MeOH/H2O as eluent to obtain 27 (7.8 mg). Fraction 2 was submitted to passage over ODS column using MeOH as eluent to give 5 fractions (A-E) grouped by TLC detection. Fraction A was further purified using Sephadex LH-20 to obtain 28 (9.0 mg). Fraction B was subjected to semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, RI detection) using MeOH/
2. Previous work Up to now, phytochemical investigations on genus Bidens have exhibited the presence of flavonoids (Li et al., 2008; Sarker et al., 2000; Wang et al., 2010; Vera Saltos et al., 2015), polyacetylenes (Marchant et al., 1984; Tobinaga et al., 2009; Wang et al., 2010), terpenes (Bohlmann et al., 1983; Ogunbinu et al., 2009) and phenylpropanoids (Bohlmann et al., 1983; Ogawa and Sashida, 1992; Wang et al., 2006; Le et al., 2015). As for the chemical constituents of Bidens bipinnata Linn., flavonoids (Li et al., 2003, 2005; Yang et al., 2012), polyacetylenes (Li et al., 2004a,b; Wang et al., 1992; Wang et al., 2014b) and phenylpropanoids (Wang et al., 1997a) have been reported.
∗
1
Corresponding author. E-mail address: [email protected] (G. Chen). These authors contributed equally to this work.
https://doi.org/10.1016/j.bse.2018.05.005 Received 13 December 2017; Received in revised form 27 April 2018; Accepted 14 May 2018 0305-1978/ © 2018 Elsevier Ltd. All rights reserved.
Biochemical Systematics and Ecology 79 (2018) 44–49
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Fig. 1. Structures of compounds 1–38.
H2O (55%) as the mobile phase at a flow rate of 6.0 ml/min to afford 8 (3.6 mg) and 9 (2.5 mg). Similarly, fraction C and D were further separated using semi-preparative HPLC to obtain 6 (2.5 mg), 7 (3.4 mg) and 4 (3.5 mg), 5 (3.1 mg) respectively. Fraction E was submitted to passage over Sephadex LH-20 column using MeOH as the eluent to give 14/15 (5.1 mg) and 22 (5.8 mg). Fraction 3 was further fractioned by reversed phase silica gel column (ODS 4 × 80 cm column; MeOH/H2O gradient) to get four fractions. The Fr.3.2 was purified over Sephadex LH-20 using MeOH/H2O (30%–90%) and MCI (MeOH/H2O) to give 23 (6.3 mg) and 24 (5.4 mg). The Fr. 3.3 was subjected to semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, UV detection at
254 nm) using MeOH/H2O (48%) as the mobile phase at a flow rate of 6.0 ml/min to afford 25 (6.1 mg) and 26 (5.6 mg). Fraction 4 was separated by silica gel column (6 × 100 cm column) and eluted with CHCl3/MeOH/H2O (8:2:0.25–7:3:0.5–6:4:1) to get 6 fractions. The Fr.6.2 was adjusted by semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, UV detection at 254 nm) using MeOH/H2O (65%) as the mobile phase at a flow rate of 6.8 ml/min to afford 20 (4.9 mg) and 21 (5.2 mg). The Fr. 6.3 was subjected to semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, UV detection at 254 nm) using MeOH/H2O (63%) as the mobile phase at a flow rate of 6.0 ml/min to afford 10/11 (6.9 mg) and 12/13 (5.1 mg). Fr. 6.4 45
Biochemical Systematics and Ecology 79 (2018) 44–49
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amide H-atom at δH 8.57 (d, J = 8.0 Hz), four oxygenated carbon signals at δC 61.9–76.4, which gave CH2 and CH signals in the DEPT (135) spectrum, and the CH signal at δC 52.6 in DEPT experiment, the ceramide structures could be confirmed. On the basis of the correlation peaks between δH 8.57 with δC 174.6 (C-1′), 61.7 (C-1), 52.6 (C-2) and δH 4.77 (H-2′) with δC 174.6 (C-1′) in HMBC spectrum, a fatty acid with one hydroxyl group could be deduced. In the further ESI-MS analysis following methanolysis of compounds 2 and 3, the fragments of m/z 384.9351[M-H]- and 369.2043[M-H]- for the FAME products of C24H48O3 and C23H46O3 were observed. Meanwhile, the configuration of C-2′ can be presumed to be R from the specific rotation value ([α]25 D = +21.38 c = 0.19 CHCl3) compared with that of reported compound. On the other hand, the fragment of m/z 316.2845[M+H]+ showed C18H37NO3 with 1 degree of unsaturation indicated one double bond in LCB chain. Its position was determined by EI-MS test of the corresponding bis (methylsulfanyl) derivate of LCB. In which, only the fragment ion peak at m/z 159 can be observed and the C(10) = C(11) bond could be determined for these two compounds. Furthermore, its E configuration was identified by the chemical shifts characteristics of C9 and C-12 (δC 33.9 and 33.4) next to double bond (Pei et al., 2010). Since the optical rotation ([α]25 D = +10.19 c = 0.19 CHCl3) of LCB was similar with that of known compound (Suo and Yang, 2014), the 2S, 3S and 4R configuration could be determined. On the basis of above analysis, compound 2/3 were assigned as (2S, 3S, 4R, 8E)-2-[(2′R)-2′hydroxytetracosanoylamino]-10-octadecene-1, 3, 4-triol and (2S, 3S, 4R, 8E)-2-[(2′R)-2′-hydroxytriocosanoylamino]-10-octadecene-1, 3, 4triol, which were not reported previously (Fig. 1), their NMR data were summarized in Table 2. Compounds 10 and 11 were isolated as light yellow powder, their molecular formula were determined as C21H24O11 from the quasi-molecular ion (m/z 465.1320 [M+H]+ calc. 465.1322) observed in HRESI-MS spectrum. From the characteristic signals of δH 5.51 (dd, J = 11 Hz, 5 Hz), 3.05 (m) and 2.88 (m) in 1H NMR spectrum and δC 193.5, 81.7 and 44.9 in 13C NMR spectrum respectively, a flavanone structure could be deduced (Jia et al., 2017). After comparison research of their NMR data with that of known compounds (Wang et al., 2014a,b), the similar structures only with different methoxy group position could be determined, which was further confirmed by the cross peaks between δH 4.80 (d, J = 8.0 Hz) with δC 157.2, δH 7.60 (d, J = 8.0 Hz) with δC 193.5, δH 2.88 (m) with δC 193.5 and δH 3.92 (s) with δC 147.8, respectively in HMBC spectrum. In the NOE experiment of compounds 10 and 11, the gain of hydrogen signal at δH 6.98 (d, J = 7.8 Hz) was observed after irradiation of methoxy signal at δH 3.92 (s), thus the (2R) isookanin-4′-methoxy-7-O-β-D-glucopyranoside/(2S) isookanin-4′-methoxy-7-O-β-D-glucopyranoside structures for compounds 10 and 11 could be subsequently elucidated, which were not reported previously. The known compounds were identified by comparison with the reported data as following: 1-O-β-D-glucopyranosyl (2S, 3S, 4R, 8E/Z)2-[(2′R)-2′-hydroxytetracosanoylamino]-8-octadecene-1, 3, 4-triol (4/ 5) (Kim et al., 2008), 1-O-β-D-glucopyranosyl-(2S, 3S, 4R, 8E/Z)-2[(2′R)-2′-hydroxytriocosanoylamino]-8-octadecene-1, 3, 4-triol (6/7) (Kim et al., 2008), 1-O-β-D-glucopyranosyl-(2S, 3S, 4R, 8E/Z)-2-[(2′R)2′-hydroxydocosanoylamino]-8-octadecene-1, 3, 4-triol (8/9) (Kim et al., 2008), (2R/2S) isookanin-3′-methoxy-7-O-β-D-glucopyranoside (12/13) (Wang et al., 2014a), (2R/2S) 7, 8, 3′, 4′-4 hydroxyflavone (14/15) (Ling et al., 2005), hyperoside (16) (Li et al., 2015), isoquercitrin (17) (Li et al., 2015), 3′, 5-dihydroxy-3, 6, 4′-trimethoxyl-7O-β-D-glucopyranoside flavone (18) (Wang et al., 2014a,b), 8, 3′-dihydroxy-3, 7, 4′-trimethoxy-6-O-β-D-glucopyranosyl flavone (19) (Xu et al., 2010), E-6-O-β-D-glucopyranosyl-6, 7, 3′, 4′-tetrahydroxyaurone (20) (Zhao et al., 2004), 7-O-β-D-glucopyranosyl-6, 7, 3′, 4′-tetrahydroxyaurone (21) (Venkateswarlu et al., 2004), maritimetin (22) (Venkateswarlu et al., 2004), E-4-O-(2″-O-diacetyl-6″-p-O-diacetyl-6-pcoumaroyl-β-D-glucopyranosyl)-p- coumaric acid (23) (Wei et al., 2015), 4-O-(6″-O-p-sementoncoacyl -β-D-glucopyranose)-p-coumaric
was chromatographed by Sephadex LH-20 and followed by purification through semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, UV detection at 254 nm) using MeOH/H2O (58%) as the mobile phase at a flow rate of 6.0 ml/min to obtain compound 38 (4.6 mg). Fraction 5 was applied to ODS (5 × 100 cm column) and eluted with MeOH/H2O (20%–90%) to yield four fractions. The Fr.5.1 was further purified by ODS (5 × 100 cm column), then was added to Sephadex LH-20 eluting with a gradient MeOH/H2O (20%–70%): 16 (8.5 mg) and 17 (8.9 mg). Fraction 6 was separated by silica gel column (6 × 100 cm column) and eluted with CHCl3/MeOH/H2O (8:2:0.25–7:3:0.5–6:4:1) to get 6 fractions. Then Fr. 6.2 was subjected to Sephadex LH-20 using MeOH/H2O as eluent to afford 18 (9.1 mg) and 19 (8.9 mg). Compound 1 was obtained as colorless powder, and the HR-ESI-MS (m/z at 684.2047 [M+H]+, calcd.684.2043) indicated the molecular formula C41H81NO6. Its IR spectrum showed strong absorbance bands of amide (1657, 1540 cm-1) and hydroxyl groups (3430 cm−1). Together with the signals at δC 130.4, 130.5 and 174.6 in the 13C NMR spectrum of compound 1, a carbonyl group and a double bond can be deduced in its molecular structure. The 1H NMR spectrum of 1 showed signals of a secondary amide H-atom at δH 8.57 (d, J = 8.0 Hz), two long-chain aliphatic moieties at δH 0.82 (t, J = 6.0 Hz), 1.24–1.44 (m). On the other hand, besides two terminal methyl groups at δC 14.0, five oxygenated carbon signals at δC 61.7, 72.1, 72.3, 72.6 and 76.4, a characteristic signal at δC 52.6 were observed in the 13C NMR spectrum of 1, they were displayed as CH2, CH, CH, CH, CH and CH signals respectively in the DEPT (135) spectrum. Thus, the ceramide structure of compound 1 could be elucidated. In the HMBC experiment, the amide H-atom at δH 8.57 correlated with the carbon signals at δC 174.6 (C-1′), 61.7 (C-1) and 52.6 (C-2) respectively, which further confirmed the ceramide structure elucidation. The hydrogen signals at δH 4.77 (H-2′) and δH 4.57 (H-3′) correlated with the carbons at δC 72.2 and 71.6 respectively in HSQC spectrum, which also showed correlations with δC 174.6, 71.6 (C-3′) and δC 174.6, 72.2 (C-2′) respectively in HMBC spectrum, indicating two hydroxyl groups at C-2′ and C-3′ positions in the fatty acid (FA) chain. The methanolysis of compound 1 was subsequently carried out and gave the fatty acid methyl ester (FAME) and long chain base (LCB). In the ESI-MS analysis, they were established as C23H46O4 and C18H37NO3 on the basis of two fragment ions at m/z 384.9353 [M-H]- and 316.2842 [M+H]+ respectively. For the 1 degree of unsaturation for C18H37NO3, a double bond can be determined in long chain base (LCB). As for the configuration at C-2′ and C-3′, since the FAME part gave the specific rotation value ([α]25 D = +17.62 c = 0.22 CHCl3) which was similar with that of reported data (Suo and Yang, 2014), the R configuration of these two chiral carbons could be further determined. The position of the double bond was elucidated by EI-MS experiment of the corresponding bis (methylsulfanyl) derivate of LCB. In which, the fragment ion peak at m/z 187 can be observed and the C(8) = C(9) bond was subsequently elucidated. In addition, the Econfiguration can be deduced on the basis of the chemical shifts characteristic for C(7) and C(10) at δC 32.7 and 31.8 respectively in 13C NMR spectrum signals (Pei et al., 2010). In sum, compound 1 was determined as (2S, 3S, 4R, 8E)-2-(2′(R), 3’(R)-dihydroxytriocosanoylamino]-8-octadecene-1, 3, 4-triol, which was not reported previously (Fig. 1). Its NMR data were summarized in Table 1. Compounds 2 and 3 were isolated as white powder; its IR spectrum revealed OH band at 3540 cm−1 and band at 1633 cm−1 due to amide group. The ceramide structures were deduced after compared their spectra data (IR, 1H NMR and 13C NMR) with that of compound 1. Their HR-ESI-MS (m/z 682.6349[M+H]+ calc. 682.6353, 668.6166 [M+H]+ calc. 668.6171) provided the molecular formula C42H83NO5 and C41H81NO5 with 2 degree of unsaturation indicating a pair of ceramides with only one CH2 difference in the molecular structures. Together with the signals of δC 174.6, 130.97 and 130.62 in the 13C NMR spectrum, a carbonyl group and a double bond in the structures could be determined. After further analysis of their NMR data including 46
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Table 1 The 1H NMR and Position
1 2 3 4 5 6 7 8 9 10 11 12 13–16 17 18 NH 1′ 2′ 3′ 4′ 5’∼21′ 22′ 23′ 24′
13
C NMR data of compounds 1, 2 and 3 (pyridine-d5). δc
1 δH
4.62; m 5.09; m 4.37; dd (8.0,16.0 Hz) 4.28; m 1.31; m 1.76; m 1.95; m 5.55; m 5.55; m 1.95; m 1.31; m 1.31; m 1.25; m 1.27; m 0.86; t (5.6 Hz) 8.56; d (8.0 Hz) – 4.50; d (4.0 Hz) 4.59; dd (4.0, 12 Hz) 1.29; m 1.25–1.31; m 1.31; m 0.86; t (5.6 Hz) –
Table 2 The 1H NMR and
61.9 53.1 76.7 72.6 33.0 26.0 31.8 131.0 130.6 33.9 29.8 29.6 29.6–29.8 23.0 14.4 – 175.3 73.2 73.0 35.3 25.4–30.0 22.9 14.4 –
HMBC
C-2 C-1′, 3 C-1, 4, 5 C-3, 5, 6 C-3, 6 C-5, 7 C-6, 8 C-7, 9 C-8, 10 C-9, 11
C-18 C-17 C-1′, 2 C-1′, 3′, 4′ C-2′, 4′ C-2′, 3′, 5′ C-23′ C-22′
δC
2 δH
4.62; m 5.08; m 4.37; dd (8.0,16.0 Hz) 4.28; m 1.31; m 1.31; m 1.31; m 1.76; m 1.95; m 5.51; m 5.51; m 1.95; m 1.25; m 1.27; m 0.86; t (5.6 Hz) 8.57; d (8.0 Hz) – 4.49; dd (4.0, 12.0 Hz) 1.31; m 1.29; m 1.25–1.31; m 1.29; m 1.31; m 0.86; t (5.6 Hz)
61.7 52.6 76.7 73.2 33.0 26.0 29.6 30.5 30.8 130.5 130.4 32.2 29.6–30.2 23.0 14.0 – 174.6 73.0 35.8 32.1 23.0–30.5 30.1 22.9 14.0
HMBC
C-2 C-1′, 3 C-1, 4, 5 C-3, 5, 6 C-3, 6 C-5, 7
C-8, 10 C-9, 12 C-9, 12 C-11 C-18 C-17 C-1′, 2 C-1′, 3′, 4′ C-2′, 4′ C-2′, 3′, 5′ C-23′ C-22′, 24′ C-23′
3
HMBC
δH
δC
4.62; m 5.08; m 4.37; dd (8.0,16.0 Hz) 4.28; m 1.31; m 1.31; m 1.31; m 1.76; m 1.95; m 5.51; m 5.51; m 1.95; m 1.25; m 1.27; m 0.86; t (5.6 Hz) 8.57; d (8.0 Hz) – 4.49; dd (4.0, 12.0 Hz) 1.31; m 1.29; m 1.25–1.31; m 1.29; m 0.86; t (5.6 Hz) –
61.7 52.6 76.7 73.2 33.0 26.0 29.6 30.5 30.8 130.5 130.4 32.2 29.6–30.2 23.0 14.0 – 174.6 73.0 35.8 32.1 23.0–30.5 22.9 14.0 –
C-2 C-1′, 3 C-1, 4, 5 C-3, 5, 6 C-3, 6 C-5, 7
C-8, 10 C-9, 12 C-9, 12 C-11 C-18 C-17 C-1′, 2 C-1′, 3′, 4′ C-2′, 4′ C-2′, 3′, 5′ C-23′ C-22′
4. Chemotaxonomic significance 13
C NMR data of compounds 10 and 11 (methanol-d4).
Position
δH
2 3a 3b 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ Glc 1″ 2″ 3″ 4″ 5″ 6″a 6'b CH3O-
5.51; 3.05; 2.88; – 7.58; 6.58; – – – – – 7.15; – – 6.98; 7.02; 4.79; 3.22; 3.34; 3.28; 3.45; 3.53; 3.45; 3.89;
dd (8.0, 16.0 Hz) m m d (8.0 Hz) d (8.0 Hz)
d (2.3 Hz)
dd (2.3, 7.6 Hz) dd (2.3, 7.6 Hz) d (7.6 Hz) m m m m m m s
δC
HMBC
81.7 44.9
C-3, 4, 1′, 2′ C-2, 4, 1′
193.5 125.6 113.0 157.9 135.0 161.1 115.5 134.2 113.3 148.4 150.0 116.3 119.3
Bidens (Asteraceae) comprises approximately 280 species distributed all over the world, especially in South America (Xuan and Khanha, 2016). Most Bidens species are subtropical and tropical weed with similar morphological characteristics. However, as adaptive weeds they often exhibit morphological diversity within a species, which often results in some confusions in the identification of a species (Ballard, 1986). Although DNA based molecular methods were performed to discuss the phylogenetic relationship among species of Bidens (Ganders et al, 2000; Tsai et al., 2008; Xia et al. 2014), further research is needed to further our understanding of the relationships among species in the genera. Previous biochemical research showed that species of Bidens are rich in flavonoids (Wang et al., 2010; Vera Saltos et al., 2015), polyacetylenes (Tobinaga et al., 2009; Wang et al., 2010), phenylpropanoids (Le et al., 2015) and terpenes (Ogunbinu et al., 2009). Although the aurons (Veitch and Grayer, 2006), polyacetylenes (Marchant et al., 1984; Negri, 2015) and some distinctive phenylpropanoids (Ogawa and Sashida, 1992) are more taxonomically important for differentiating similar species in this genus and other genera in Asteraceae family from chemical opinion, these distinctive constituents could also be found from other genera or families (Huang et al., 2008; Huang et al., 2014; Negri, 2015), indicating the importance of finding more distinctive compounds with chemotaxonomical significance. Present phytochemical study on Bidens bipinnata Linn. reports the isolation and structure elucidation of thirty-eight compounds, including nine ceramides (1–9), thirteen flavonoids (10–22), five phenylpropanoids (23–27), four aliphatics (28, 35–37), one pyrimidine (29), four steroids (30–33), one triterpenoid (34) and one polyacetylene (38). Five newly reported compounds (1–3, 10/11), and nine compounds (4–9, 25, 26, 38) were isolated from the Bidens genus for the first time. In addition, this is the first report of the compounds 12, 13, 16–21, 30–34 from Bidens bipinnata Linn. Ceramides are an important type of secondary metabolites widely distributed in animals and considered to function as chemical messenger in nerve system (Tan and Chen, 2003). Unlike the alkaloids, terpenoids, polyacetylene and falvonoids with characteristic structures that easily found in Asteraceae family, the ceramides are rarely
C-4, 7 C-8, 10
C-2, 4′, 6′
C-1′, 3′ C-2, 2′, 4′
107.7 75.9 78.3 71.3 78.9 62.6
C-7, 1″ C-3″, 4″ C-1″, 5″ C-5″ C-3″, 4″ C-4″
57.1
C-4′
acid (24) (Wang et al., 1997a,b), citrusin C (25), 4-allyl-2, 6-dimethoxyphenyl glucoside (26) (Devi al., 2004), caffeic acid (27) (Nakazawa and Ohsawa, 1998), fumaric acid (28) (Zhao et al., 2004), uracil (29) (Ding et al., 2009), β-sitosterol (30) (Sosińska et al., 2013), daucosterol (31), stigmasterol (32) (Sosińska et al., 2013), stigmasterolβ-D-glucopryranoside (33), friedelin (34) (Geissberger and Séquin, 1991), stearic acid (35) (Sarg et al., 1991), hexadecanol (36) (Priestap et al., 2008), n-heneicosane (37) (Ogunbinu et al., 2009), (5E)-trideca1, 5-dien-7, 9, 11-triyne-3, 4-diol-4-O-β-D-glucopyranoside (38) (Xi et al., 2014) (Fig. 1).
47
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Polyacetylene constituents are the common characteristic and major secondary metabolites of Bidens genus and Asteraceae family (Negri, 2015), which are regarded as significant chemotaxonomic markers for Bidens and Asteraceae species. Alkyl type (Negri, 2015), aromatic type (Wat et al., 1979) and thiophene ring bearing (Marchant et al., 1984) polyyne are three types polyacetylene often found in Bidens genus. The current work is the first report of (5E)-Trideca-1, 5-dien-7, 9, 11-triyne3, 4-diol-4-O-β-D-glucopyranoside (38) from Bidens with C-13 representative triyne structure, which was previously identified in Eclipata prostrate Linn. (Asteraceae) (Xi et al., 2014). From the chemosystematic point of view, compound 38 may indicate the closer relationship between Bidens and Eclipata and could also be used to differentiate Bidens bipinnata from other species of Bidens. Moreover, apart from the newly found compounds in Bidens genus, the firstly identification of hyperoside (16) and isoquercitrin (17), 3′, 5dihydroxy-3, 6, 4′-trimethoxyl-7-O-β-D-glucopyranoside flavone (18), 8, 3′-dihydroxy-3, 7, 4′-trimethoxy-6-O-β-D-glucopyranosyl flavone (19), β-sitosterol (30), daucosterol (31), stigmasterol (32), stigmasterol-β-Dglucopryranoside (33), friedelin (34) in Bidens bipinnata are also in agreement with the classification and the chemotaxonomy of this species in Bidens genus.
reported from this family recently (Zhu et al., 2013; Lin et al., 2004; Chen et al., 2009; Guo et al., 2007). The newly identified ceremides 1–3 could be of chemotaxonomic significance, especially (2S, 3S, 4R, 8E)-2(2′(R), 3′(R)-dihydroxytriocosanoylamino]-8-octadecene-1, 3, 4-triol (1), which has an unique hydroxyl group at C-3’ position. This type of ceremide was also recently found in Tanacetum artemisioides SchultzBip. ex Hook. f. (Anthemideae) (Hussain et al., 2010), Launaea nudicaulis (L.) Hook. f. (Compositae) (Riaz et al., 2012), Zephyranthes candida (Lindl.) Herb. (Amaryllidaceae) (Wu et al., 2009) and Helianthus annuus Linn. (Compositae) (Suo and Yang., 2014), but not in family Asteraceae. Ceremides 4–9 were firstly obtained from Paeonia lactiflora Pall. (Paeoniae) (Kim et al., 2008) and have not been identified from other genera or families up to now. These compounds differ with other ceremides in the carbon chain length, position of double bonds and configurations (Tan and Chen, 2003), as a whole group, they can be considered as important chemotaxonomic markers for Bidens genus and Asteraceae family. In the previous research, great scale isolation of the flavonoids, especially flavone and flavonol constituents from Asteraceae species suggested low level taxonomic significance of this kind of compounds (Emerenciano et al., 2001). On the other hand, compounds 10–15 possess flavanone skeleton, this type of structures are rarely obtained from Bidens genus compared to flavone and flavonol (Vera Saltos et al., 2015). Up to now, most identified flavanones in this genus have the isokanin skeleton, for example (2R/2S) 7, 8, 3′, 4′-tetrahydroxyflavanone (14/15) from Bidens pilosa (Yuan et al., 2008), isokanin 7-Oβ-D-(2″, 4″, 6″-triacetyl)-glucopyranoside from Bidens pilosa var. radiata (Wang et al., 1997b). The newly identified (2R/2S) isookanin-4′methoxy-7-O-β-D-glucopyranoside (10/11) together with newly reported (2R/2S) isookanin-3′-methoxy-7-O-β-D-glucopyranoside (12/13) are chemotaxonomically significant as a whole group for Bidens genus, as well as for Bidens bipinnata. Because of the rare occurrence in nature plants compared to common flavones, aurones are known to be other important chemical taxonomic markers for Bidens species (Veitch and Grayer, 2006). Previous research showed besides maritimetin (22) that widely distributed in Bidens species e.g. Bidens laevis Linn., Bidens ferulifolia (Jacq.) DC., Bidens pilosa Linn., Bidens aurea (Ait.) Sherff., Bidens tripartita Linn. (Boucherle et al., 2017), other corresponding derivatives with this type structures were also isolated from this genus (Hoffman and Hölzl, 1988; Sashida et al., 1991), which indicated the 6, 7, 3′, 4′-tetrahydroxyaurone skeleton could be the chemotaxonomical marker for Bidens genus. However, few aurones were found from Bidens bipinnata up to now (Li et al., 2003; Yang et al., 2012). In this work, 6-O-β-Dglucopyranosyl-6, 7, 3′, 4′-tetrahydroxyaurone (20), and 7-O-β-D-glucopyranosyl-6, 7, 3′, 4′-tetrahydroxyaurone (21) were isolated from Bidens genus for the first time, this result indicated the closer relationship between Bidens bipinnata with other Bidens species. Thus, compounds 20 and 21 could be used as the taxonomic markers to identify this genus and also for differentiating the species in Bidens genus. Citrusin C (25) and 4-allyl-2, 6-dimethoxyphenyl glucoside (26) were firstly isolated from Bidens genus with allyl benzene skeleton. Different with caffeic acid (27) (Ogawa and Sashida., 1992) type phenylpropanoids that are often isolated from Bidens genus, only few compounds with allyl benzene structure were reported up to now, e.g. guaiacyl glycerol 8-O-β-D-glucoside and 4-allyl-2-methoxyphenol-O-(6O-β-D-apiofuranosyl)-β-D-glucoside from Bidens parviflora Willd. (Wang et al., 2007). From the chemotaxonomic opinion, these two compounds carry significant taxonomic value for Bidens genus. Meanwhile, these type of compounds were also isolated from Carpesium cernuum Linn. (Asteraceae) (Ma et al., 2008), Carpesium macrocephalum FR. et SAV. (Asteraceae) (Kim et al., 2004), Dichrocephala integrifolia Linn. (Asteraceae) (Lee et al., 2015), Macroclinidium trilobum Makino. (Asteraceae) (Miyase et al., 1985), which indicated closer relationship between Bidens and these genera in Asteraceae family.
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Chemical constituents from Bidens bipinnata Linn. 1
1
Hai-Min Hu , Shu-Min Bai , Li-Jiao Chen, Wen-Yi Hu, Guang Chen
T ∗
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Asteraceae Bidens bipinnata Linn. Ceramide
The phytochemical investigation on Bidens bipinnata Linn. (Asteraceae) led to the isolation and identification of 38 compounds, including nine ceramides (1–9), thirteen flavonoids (10–22), five phenylpropanoids (23–27), four aliphatics (28, 35–37), one pyrimidine (29), four steroids (30–33), one triterpenoid (34) and one polyacetylene (38). All chemical structures were established on the basis of MS and NMR spectroscopic data. In addition to five newly reported compounds (2S, 3S, 4R, 8E)-2-(2′(R), 3′(R)-dihydroxytriocosanoylamino]-8octadecene-1, 3, 4-triol (1), (2S, 3S, 4R, 8E)-2-[(2′R)-2′-hydroxytetracosanoylamino]-10-octadecene-1, 3, 4triol/(2S, 3S, 4R, 8E)-2-[(2′R)-2′-hydroxytriocosanoylamino]-10-octadecene-1, 3, 4-triol (2/3) and (2R) isookanin-4′-methoxy-7-O-β-D-glucopyranoside/(2S) isookanin-4′-methoxy-7-O-β-D-glucopyranoside (10/11), nine compounds (4–9, 25, 26, 38) were isolated from the Bidens genus for the first time. Meanwhile, the compounds 12, 13, 16–21, 30–34 were reported firstly from Bidens bipinnata Linn. On the basis of chemical research, the chemotaxonomic significance of the isolated compounds has been summarized.
1. Subject and source
3. Present study
Bidens bipinnata Linn. which belongs to Bidens genus and family Asteraceae, is an annual plant and widely distributed in China. The whole plant of Bidens bipinnata Linn. was purchased from Anguo County of Hebei Province, P. R. China in April 2015, and was identified by Prof. Yun-Zhen Guo, College of Traditional Chinese Medicines, Shenyang Pharmaceutical University. A voucher specimen (No. 150411) is deposited in College of Life Science and Technology, Beijing University of Chemical Technology.
The whole plant of Bidens bipinnata Linn. (30 kg) was extracted twice with 80% EtOH for 2 h. After removal of the solvent in vacuo, the residue was suspended in water. This suspension was further fractionated with petroleum ether, CHCl3 and n-BuOH, successively. The CHCl3 extract (85 g) was subsequently separated using silica gel column chromatography (900 g, 9 × 100 cm column; petroleum ether/EtOAc gradient) to yield 8 fractions (1–8) based on TLC profiles. Fraction 2 was submitted to passage over silica gel column eluted by CHCl3/EtOAc to give 30 (8.4 mg) and 32 (5.9 mg). Fraction 3 was further subjected to silica gel column eluted by CHCl3/EtOAc to obtain 2 (3.2 mg) and 3 (5.3 mg). Fraction 4 was chromatographed on silica gel column and eluted by CHCl3/EtOAc to give 1 (4.2 mg). Fraction 5 was further purified by Sephadex LH-20 to obtain 29 (7.1 mg). Fraction 8 was applied to ODS column using MeOH as eluent to give 31 (11.3 mg) and 33 (7.2 mg). The n-BuOH extract (119 g) was fractioned by silica gel column chromatography (1200 g, 10 × 110 cm column; CHCl3/MeOH gradient) to give 9 subfractions (1–9) based on TLC profiles. Fraction 1 was further separated by Sephadex LH-20 using MeOH/H2O as eluent to obtain 27 (7.8 mg). Fraction 2 was submitted to passage over ODS column using MeOH as eluent to give 5 fractions (A-E) grouped by TLC detection. Fraction A was further purified using Sephadex LH-20 to obtain 28 (9.0 mg). Fraction B was subjected to semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, RI detection) using MeOH/
2. Previous work Up to now, phytochemical investigations on genus Bidens have exhibited the presence of flavonoids (Li et al., 2008; Sarker et al., 2000; Wang et al., 2010; Vera Saltos et al., 2015), polyacetylenes (Marchant et al., 1984; Tobinaga et al., 2009; Wang et al., 2010), terpenes (Bohlmann et al., 1983; Ogunbinu et al., 2009) and phenylpropanoids (Bohlmann et al., 1983; Ogawa and Sashida, 1992; Wang et al., 2006; Le et al., 2015). As for the chemical constituents of Bidens bipinnata Linn., flavonoids (Li et al., 2003, 2005; Yang et al., 2012), polyacetylenes (Li et al., 2004a,b; Wang et al., 1992; Wang et al., 2014b) and phenylpropanoids (Wang et al., 1997a) have been reported.
∗
1
Corresponding author. E-mail address: [email protected] (G. Chen). These authors contributed equally to this work.
https://doi.org/10.1016/j.bse.2018.05.005 Received 13 December 2017; Received in revised form 27 April 2018; Accepted 14 May 2018 0305-1978/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1–38.
H2O (55%) as the mobile phase at a flow rate of 6.0 ml/min to afford 8 (3.6 mg) and 9 (2.5 mg). Similarly, fraction C and D were further separated using semi-preparative HPLC to obtain 6 (2.5 mg), 7 (3.4 mg) and 4 (3.5 mg), 5 (3.1 mg) respectively. Fraction E was submitted to passage over Sephadex LH-20 column using MeOH as the eluent to give 14/15 (5.1 mg) and 22 (5.8 mg). Fraction 3 was further fractioned by reversed phase silica gel column (ODS 4 × 80 cm column; MeOH/H2O gradient) to get four fractions. The Fr.3.2 was purified over Sephadex LH-20 using MeOH/H2O (30%–90%) and MCI (MeOH/H2O) to give 23 (6.3 mg) and 24 (5.4 mg). The Fr. 3.3 was subjected to semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, UV detection at
254 nm) using MeOH/H2O (48%) as the mobile phase at a flow rate of 6.0 ml/min to afford 25 (6.1 mg) and 26 (5.6 mg). Fraction 4 was separated by silica gel column (6 × 100 cm column) and eluted with CHCl3/MeOH/H2O (8:2:0.25–7:3:0.5–6:4:1) to get 6 fractions. The Fr.6.2 was adjusted by semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, UV detection at 254 nm) using MeOH/H2O (65%) as the mobile phase at a flow rate of 6.8 ml/min to afford 20 (4.9 mg) and 21 (5.2 mg). The Fr. 6.3 was subjected to semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, UV detection at 254 nm) using MeOH/H2O (63%) as the mobile phase at a flow rate of 6.0 ml/min to afford 10/11 (6.9 mg) and 12/13 (5.1 mg). Fr. 6.4 45
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amide H-atom at δH 8.57 (d, J = 8.0 Hz), four oxygenated carbon signals at δC 61.9–76.4, which gave CH2 and CH signals in the DEPT (135) spectrum, and the CH signal at δC 52.6 in DEPT experiment, the ceramide structures could be confirmed. On the basis of the correlation peaks between δH 8.57 with δC 174.6 (C-1′), 61.7 (C-1), 52.6 (C-2) and δH 4.77 (H-2′) with δC 174.6 (C-1′) in HMBC spectrum, a fatty acid with one hydroxyl group could be deduced. In the further ESI-MS analysis following methanolysis of compounds 2 and 3, the fragments of m/z 384.9351[M-H]- and 369.2043[M-H]- for the FAME products of C24H48O3 and C23H46O3 were observed. Meanwhile, the configuration of C-2′ can be presumed to be R from the specific rotation value ([α]25 D = +21.38 c = 0.19 CHCl3) compared with that of reported compound. On the other hand, the fragment of m/z 316.2845[M+H]+ showed C18H37NO3 with 1 degree of unsaturation indicated one double bond in LCB chain. Its position was determined by EI-MS test of the corresponding bis (methylsulfanyl) derivate of LCB. In which, only the fragment ion peak at m/z 159 can be observed and the C(10) = C(11) bond could be determined for these two compounds. Furthermore, its E configuration was identified by the chemical shifts characteristics of C9 and C-12 (δC 33.9 and 33.4) next to double bond (Pei et al., 2010). Since the optical rotation ([α]25 D = +10.19 c = 0.19 CHCl3) of LCB was similar with that of known compound (Suo and Yang, 2014), the 2S, 3S and 4R configuration could be determined. On the basis of above analysis, compound 2/3 were assigned as (2S, 3S, 4R, 8E)-2-[(2′R)-2′hydroxytetracosanoylamino]-10-octadecene-1, 3, 4-triol and (2S, 3S, 4R, 8E)-2-[(2′R)-2′-hydroxytriocosanoylamino]-10-octadecene-1, 3, 4triol, which were not reported previously (Fig. 1), their NMR data were summarized in Table 2. Compounds 10 and 11 were isolated as light yellow powder, their molecular formula were determined as C21H24O11 from the quasi-molecular ion (m/z 465.1320 [M+H]+ calc. 465.1322) observed in HRESI-MS spectrum. From the characteristic signals of δH 5.51 (dd, J = 11 Hz, 5 Hz), 3.05 (m) and 2.88 (m) in 1H NMR spectrum and δC 193.5, 81.7 and 44.9 in 13C NMR spectrum respectively, a flavanone structure could be deduced (Jia et al., 2017). After comparison research of their NMR data with that of known compounds (Wang et al., 2014a,b), the similar structures only with different methoxy group position could be determined, which was further confirmed by the cross peaks between δH 4.80 (d, J = 8.0 Hz) with δC 157.2, δH 7.60 (d, J = 8.0 Hz) with δC 193.5, δH 2.88 (m) with δC 193.5 and δH 3.92 (s) with δC 147.8, respectively in HMBC spectrum. In the NOE experiment of compounds 10 and 11, the gain of hydrogen signal at δH 6.98 (d, J = 7.8 Hz) was observed after irradiation of methoxy signal at δH 3.92 (s), thus the (2R) isookanin-4′-methoxy-7-O-β-D-glucopyranoside/(2S) isookanin-4′-methoxy-7-O-β-D-glucopyranoside structures for compounds 10 and 11 could be subsequently elucidated, which were not reported previously. The known compounds were identified by comparison with the reported data as following: 1-O-β-D-glucopyranosyl (2S, 3S, 4R, 8E/Z)2-[(2′R)-2′-hydroxytetracosanoylamino]-8-octadecene-1, 3, 4-triol (4/ 5) (Kim et al., 2008), 1-O-β-D-glucopyranosyl-(2S, 3S, 4R, 8E/Z)-2[(2′R)-2′-hydroxytriocosanoylamino]-8-octadecene-1, 3, 4-triol (6/7) (Kim et al., 2008), 1-O-β-D-glucopyranosyl-(2S, 3S, 4R, 8E/Z)-2-[(2′R)2′-hydroxydocosanoylamino]-8-octadecene-1, 3, 4-triol (8/9) (Kim et al., 2008), (2R/2S) isookanin-3′-methoxy-7-O-β-D-glucopyranoside (12/13) (Wang et al., 2014a), (2R/2S) 7, 8, 3′, 4′-4 hydroxyflavone (14/15) (Ling et al., 2005), hyperoside (16) (Li et al., 2015), isoquercitrin (17) (Li et al., 2015), 3′, 5-dihydroxy-3, 6, 4′-trimethoxyl-7O-β-D-glucopyranoside flavone (18) (Wang et al., 2014a,b), 8, 3′-dihydroxy-3, 7, 4′-trimethoxy-6-O-β-D-glucopyranosyl flavone (19) (Xu et al., 2010), E-6-O-β-D-glucopyranosyl-6, 7, 3′, 4′-tetrahydroxyaurone (20) (Zhao et al., 2004), 7-O-β-D-glucopyranosyl-6, 7, 3′, 4′-tetrahydroxyaurone (21) (Venkateswarlu et al., 2004), maritimetin (22) (Venkateswarlu et al., 2004), E-4-O-(2″-O-diacetyl-6″-p-O-diacetyl-6-pcoumaroyl-β-D-glucopyranosyl)-p- coumaric acid (23) (Wei et al., 2015), 4-O-(6″-O-p-sementoncoacyl -β-D-glucopyranose)-p-coumaric
was chromatographed by Sephadex LH-20 and followed by purification through semi-preparative HPLC (YMC-Pack ODS, 5 mm, 250 mm × 20 mm, UV detection at 254 nm) using MeOH/H2O (58%) as the mobile phase at a flow rate of 6.0 ml/min to obtain compound 38 (4.6 mg). Fraction 5 was applied to ODS (5 × 100 cm column) and eluted with MeOH/H2O (20%–90%) to yield four fractions. The Fr.5.1 was further purified by ODS (5 × 100 cm column), then was added to Sephadex LH-20 eluting with a gradient MeOH/H2O (20%–70%): 16 (8.5 mg) and 17 (8.9 mg). Fraction 6 was separated by silica gel column (6 × 100 cm column) and eluted with CHCl3/MeOH/H2O (8:2:0.25–7:3:0.5–6:4:1) to get 6 fractions. Then Fr. 6.2 was subjected to Sephadex LH-20 using MeOH/H2O as eluent to afford 18 (9.1 mg) and 19 (8.9 mg). Compound 1 was obtained as colorless powder, and the HR-ESI-MS (m/z at 684.2047 [M+H]+, calcd.684.2043) indicated the molecular formula C41H81NO6. Its IR spectrum showed strong absorbance bands of amide (1657, 1540 cm-1) and hydroxyl groups (3430 cm−1). Together with the signals at δC 130.4, 130.5 and 174.6 in the 13C NMR spectrum of compound 1, a carbonyl group and a double bond can be deduced in its molecular structure. The 1H NMR spectrum of 1 showed signals of a secondary amide H-atom at δH 8.57 (d, J = 8.0 Hz), two long-chain aliphatic moieties at δH 0.82 (t, J = 6.0 Hz), 1.24–1.44 (m). On the other hand, besides two terminal methyl groups at δC 14.0, five oxygenated carbon signals at δC 61.7, 72.1, 72.3, 72.6 and 76.4, a characteristic signal at δC 52.6 were observed in the 13C NMR spectrum of 1, they were displayed as CH2, CH, CH, CH, CH and CH signals respectively in the DEPT (135) spectrum. Thus, the ceramide structure of compound 1 could be elucidated. In the HMBC experiment, the amide H-atom at δH 8.57 correlated with the carbon signals at δC 174.6 (C-1′), 61.7 (C-1) and 52.6 (C-2) respectively, which further confirmed the ceramide structure elucidation. The hydrogen signals at δH 4.77 (H-2′) and δH 4.57 (H-3′) correlated with the carbons at δC 72.2 and 71.6 respectively in HSQC spectrum, which also showed correlations with δC 174.6, 71.6 (C-3′) and δC 174.6, 72.2 (C-2′) respectively in HMBC spectrum, indicating two hydroxyl groups at C-2′ and C-3′ positions in the fatty acid (FA) chain. The methanolysis of compound 1 was subsequently carried out and gave the fatty acid methyl ester (FAME) and long chain base (LCB). In the ESI-MS analysis, they were established as C23H46O4 and C18H37NO3 on the basis of two fragment ions at m/z 384.9353 [M-H]- and 316.2842 [M+H]+ respectively. For the 1 degree of unsaturation for C18H37NO3, a double bond can be determined in long chain base (LCB). As for the configuration at C-2′ and C-3′, since the FAME part gave the specific rotation value ([α]25 D = +17.62 c = 0.22 CHCl3) which was similar with that of reported data (Suo and Yang, 2014), the R configuration of these two chiral carbons could be further determined. The position of the double bond was elucidated by EI-MS experiment of the corresponding bis (methylsulfanyl) derivate of LCB. In which, the fragment ion peak at m/z 187 can be observed and the C(8) = C(9) bond was subsequently elucidated. In addition, the Econfiguration can be deduced on the basis of the chemical shifts characteristic for C(7) and C(10) at δC 32.7 and 31.8 respectively in 13C NMR spectrum signals (Pei et al., 2010). In sum, compound 1 was determined as (2S, 3S, 4R, 8E)-2-(2′(R), 3’(R)-dihydroxytriocosanoylamino]-8-octadecene-1, 3, 4-triol, which was not reported previously (Fig. 1). Its NMR data were summarized in Table 1. Compounds 2 and 3 were isolated as white powder; its IR spectrum revealed OH band at 3540 cm−1 and band at 1633 cm−1 due to amide group. The ceramide structures were deduced after compared their spectra data (IR, 1H NMR and 13C NMR) with that of compound 1. Their HR-ESI-MS (m/z 682.6349[M+H]+ calc. 682.6353, 668.6166 [M+H]+ calc. 668.6171) provided the molecular formula C42H83NO5 and C41H81NO5 with 2 degree of unsaturation indicating a pair of ceramides with only one CH2 difference in the molecular structures. Together with the signals of δC 174.6, 130.97 and 130.62 in the 13C NMR spectrum, a carbonyl group and a double bond in the structures could be determined. After further analysis of their NMR data including 46
Biochemical Systematics and Ecology 79 (2018) 44–49
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Table 1 The 1H NMR and Position
1 2 3 4 5 6 7 8 9 10 11 12 13–16 17 18 NH 1′ 2′ 3′ 4′ 5’∼21′ 22′ 23′ 24′
13
C NMR data of compounds 1, 2 and 3 (pyridine-d5). δc
1 δH
4.62; m 5.09; m 4.37; dd (8.0,16.0 Hz) 4.28; m 1.31; m 1.76; m 1.95; m 5.55; m 5.55; m 1.95; m 1.31; m 1.31; m 1.25; m 1.27; m 0.86; t (5.6 Hz) 8.56; d (8.0 Hz) – 4.50; d (4.0 Hz) 4.59; dd (4.0, 12 Hz) 1.29; m 1.25–1.31; m 1.31; m 0.86; t (5.6 Hz) –
Table 2 The 1H NMR and
61.9 53.1 76.7 72.6 33.0 26.0 31.8 131.0 130.6 33.9 29.8 29.6 29.6–29.8 23.0 14.4 – 175.3 73.2 73.0 35.3 25.4–30.0 22.9 14.4 –
HMBC
C-2 C-1′, 3 C-1, 4, 5 C-3, 5, 6 C-3, 6 C-5, 7 C-6, 8 C-7, 9 C-8, 10 C-9, 11
C-18 C-17 C-1′, 2 C-1′, 3′, 4′ C-2′, 4′ C-2′, 3′, 5′ C-23′ C-22′
δC
2 δH
4.62; m 5.08; m 4.37; dd (8.0,16.0 Hz) 4.28; m 1.31; m 1.31; m 1.31; m 1.76; m 1.95; m 5.51; m 5.51; m 1.95; m 1.25; m 1.27; m 0.86; t (5.6 Hz) 8.57; d (8.0 Hz) – 4.49; dd (4.0, 12.0 Hz) 1.31; m 1.29; m 1.25–1.31; m 1.29; m 1.31; m 0.86; t (5.6 Hz)
61.7 52.6 76.7 73.2 33.0 26.0 29.6 30.5 30.8 130.5 130.4 32.2 29.6–30.2 23.0 14.0 – 174.6 73.0 35.8 32.1 23.0–30.5 30.1 22.9 14.0
HMBC
C-2 C-1′, 3 C-1, 4, 5 C-3, 5, 6 C-3, 6 C-5, 7
C-8, 10 C-9, 12 C-9, 12 C-11 C-18 C-17 C-1′, 2 C-1′, 3′, 4′ C-2′, 4′ C-2′, 3′, 5′ C-23′ C-22′, 24′ C-23′
3
HMBC
δH
δC
4.62; m 5.08; m 4.37; dd (8.0,16.0 Hz) 4.28; m 1.31; m 1.31; m 1.31; m 1.76; m 1.95; m 5.51; m 5.51; m 1.95; m 1.25; m 1.27; m 0.86; t (5.6 Hz) 8.57; d (8.0 Hz) – 4.49; dd (4.0, 12.0 Hz) 1.31; m 1.29; m 1.25–1.31; m 1.29; m 0.86; t (5.6 Hz) –
61.7 52.6 76.7 73.2 33.0 26.0 29.6 30.5 30.8 130.5 130.4 32.2 29.6–30.2 23.0 14.0 – 174.6 73.0 35.8 32.1 23.0–30.5 22.9 14.0 –
C-2 C-1′, 3 C-1, 4, 5 C-3, 5, 6 C-3, 6 C-5, 7
C-8, 10 C-9, 12 C-9, 12 C-11 C-18 C-17 C-1′, 2 C-1′, 3′, 4′ C-2′, 4′ C-2′, 3′, 5′ C-23′ C-22′
4. Chemotaxonomic significance 13
C NMR data of compounds 10 and 11 (methanol-d4).
Position
δH
2 3a 3b 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ Glc 1″ 2″ 3″ 4″ 5″ 6″a 6'b CH3O-
5.51; 3.05; 2.88; – 7.58; 6.58; – – – – – 7.15; – – 6.98; 7.02; 4.79; 3.22; 3.34; 3.28; 3.45; 3.53; 3.45; 3.89;
dd (8.0, 16.0 Hz) m m d (8.0 Hz) d (8.0 Hz)
d (2.3 Hz)
dd (2.3, 7.6 Hz) dd (2.3, 7.6 Hz) d (7.6 Hz) m m m m m m s
δC
HMBC
81.7 44.9
C-3, 4, 1′, 2′ C-2, 4, 1′
193.5 125.6 113.0 157.9 135.0 161.1 115.5 134.2 113.3 148.4 150.0 116.3 119.3
Bidens (Asteraceae) comprises approximately 280 species distributed all over the world, especially in South America (Xuan and Khanha, 2016). Most Bidens species are subtropical and tropical weed with similar morphological characteristics. However, as adaptive weeds they often exhibit morphological diversity within a species, which often results in some confusions in the identification of a species (Ballard, 1986). Although DNA based molecular methods were performed to discuss the phylogenetic relationship among species of Bidens (Ganders et al, 2000; Tsai et al., 2008; Xia et al. 2014), further research is needed to further our understanding of the relationships among species in the genera. Previous biochemical research showed that species of Bidens are rich in flavonoids (Wang et al., 2010; Vera Saltos et al., 2015), polyacetylenes (Tobinaga et al., 2009; Wang et al., 2010), phenylpropanoids (Le et al., 2015) and terpenes (Ogunbinu et al., 2009). Although the aurons (Veitch and Grayer, 2006), polyacetylenes (Marchant et al., 1984; Negri, 2015) and some distinctive phenylpropanoids (Ogawa and Sashida, 1992) are more taxonomically important for differentiating similar species in this genus and other genera in Asteraceae family from chemical opinion, these distinctive constituents could also be found from other genera or families (Huang et al., 2008; Huang et al., 2014; Negri, 2015), indicating the importance of finding more distinctive compounds with chemotaxonomical significance. Present phytochemical study on Bidens bipinnata Linn. reports the isolation and structure elucidation of thirty-eight compounds, including nine ceramides (1–9), thirteen flavonoids (10–22), five phenylpropanoids (23–27), four aliphatics (28, 35–37), one pyrimidine (29), four steroids (30–33), one triterpenoid (34) and one polyacetylene (38). Five newly reported compounds (1–3, 10/11), and nine compounds (4–9, 25, 26, 38) were isolated from the Bidens genus for the first time. In addition, this is the first report of the compounds 12, 13, 16–21, 30–34 from Bidens bipinnata Linn. Ceramides are an important type of secondary metabolites widely distributed in animals and considered to function as chemical messenger in nerve system (Tan and Chen, 2003). Unlike the alkaloids, terpenoids, polyacetylene and falvonoids with characteristic structures that easily found in Asteraceae family, the ceramides are rarely
C-4, 7 C-8, 10
C-2, 4′, 6′
C-1′, 3′ C-2, 2′, 4′
107.7 75.9 78.3 71.3 78.9 62.6
C-7, 1″ C-3″, 4″ C-1″, 5″ C-5″ C-3″, 4″ C-4″
57.1
C-4′
acid (24) (Wang et al., 1997a,b), citrusin C (25), 4-allyl-2, 6-dimethoxyphenyl glucoside (26) (Devi al., 2004), caffeic acid (27) (Nakazawa and Ohsawa, 1998), fumaric acid (28) (Zhao et al., 2004), uracil (29) (Ding et al., 2009), β-sitosterol (30) (Sosińska et al., 2013), daucosterol (31), stigmasterol (32) (Sosińska et al., 2013), stigmasterolβ-D-glucopryranoside (33), friedelin (34) (Geissberger and Séquin, 1991), stearic acid (35) (Sarg et al., 1991), hexadecanol (36) (Priestap et al., 2008), n-heneicosane (37) (Ogunbinu et al., 2009), (5E)-trideca1, 5-dien-7, 9, 11-triyne-3, 4-diol-4-O-β-D-glucopyranoside (38) (Xi et al., 2014) (Fig. 1).
47
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Polyacetylene constituents are the common characteristic and major secondary metabolites of Bidens genus and Asteraceae family (Negri, 2015), which are regarded as significant chemotaxonomic markers for Bidens and Asteraceae species. Alkyl type (Negri, 2015), aromatic type (Wat et al., 1979) and thiophene ring bearing (Marchant et al., 1984) polyyne are three types polyacetylene often found in Bidens genus. The current work is the first report of (5E)-Trideca-1, 5-dien-7, 9, 11-triyne3, 4-diol-4-O-β-D-glucopyranoside (38) from Bidens with C-13 representative triyne structure, which was previously identified in Eclipata prostrate Linn. (Asteraceae) (Xi et al., 2014). From the chemosystematic point of view, compound 38 may indicate the closer relationship between Bidens and Eclipata and could also be used to differentiate Bidens bipinnata from other species of Bidens. Moreover, apart from the newly found compounds in Bidens genus, the firstly identification of hyperoside (16) and isoquercitrin (17), 3′, 5dihydroxy-3, 6, 4′-trimethoxyl-7-O-β-D-glucopyranoside flavone (18), 8, 3′-dihydroxy-3, 7, 4′-trimethoxy-6-O-β-D-glucopyranosyl flavone (19), β-sitosterol (30), daucosterol (31), stigmasterol (32), stigmasterol-β-Dglucopryranoside (33), friedelin (34) in Bidens bipinnata are also in agreement with the classification and the chemotaxonomy of this species in Bidens genus.
reported from this family recently (Zhu et al., 2013; Lin et al., 2004; Chen et al., 2009; Guo et al., 2007). The newly identified ceremides 1–3 could be of chemotaxonomic significance, especially (2S, 3S, 4R, 8E)-2(2′(R), 3′(R)-dihydroxytriocosanoylamino]-8-octadecene-1, 3, 4-triol (1), which has an unique hydroxyl group at C-3’ position. This type of ceremide was also recently found in Tanacetum artemisioides SchultzBip. ex Hook. f. (Anthemideae) (Hussain et al., 2010), Launaea nudicaulis (L.) Hook. f. (Compositae) (Riaz et al., 2012), Zephyranthes candida (Lindl.) Herb. (Amaryllidaceae) (Wu et al., 2009) and Helianthus annuus Linn. (Compositae) (Suo and Yang., 2014), but not in family Asteraceae. Ceremides 4–9 were firstly obtained from Paeonia lactiflora Pall. (Paeoniae) (Kim et al., 2008) and have not been identified from other genera or families up to now. These compounds differ with other ceremides in the carbon chain length, position of double bonds and configurations (Tan and Chen, 2003), as a whole group, they can be considered as important chemotaxonomic markers for Bidens genus and Asteraceae family. In the previous research, great scale isolation of the flavonoids, especially flavone and flavonol constituents from Asteraceae species suggested low level taxonomic significance of this kind of compounds (Emerenciano et al., 2001). On the other hand, compounds 10–15 possess flavanone skeleton, this type of structures are rarely obtained from Bidens genus compared to flavone and flavonol (Vera Saltos et al., 2015). Up to now, most identified flavanones in this genus have the isokanin skeleton, for example (2R/2S) 7, 8, 3′, 4′-tetrahydroxyflavanone (14/15) from Bidens pilosa (Yuan et al., 2008), isokanin 7-Oβ-D-(2″, 4″, 6″-triacetyl)-glucopyranoside from Bidens pilosa var. radiata (Wang et al., 1997b). The newly identified (2R/2S) isookanin-4′methoxy-7-O-β-D-glucopyranoside (10/11) together with newly reported (2R/2S) isookanin-3′-methoxy-7-O-β-D-glucopyranoside (12/13) are chemotaxonomically significant as a whole group for Bidens genus, as well as for Bidens bipinnata. Because of the rare occurrence in nature plants compared to common flavones, aurones are known to be other important chemical taxonomic markers for Bidens species (Veitch and Grayer, 2006). Previous research showed besides maritimetin (22) that widely distributed in Bidens species e.g. Bidens laevis Linn., Bidens ferulifolia (Jacq.) DC., Bidens pilosa Linn., Bidens aurea (Ait.) Sherff., Bidens tripartita Linn. (Boucherle et al., 2017), other corresponding derivatives with this type structures were also isolated from this genus (Hoffman and Hölzl, 1988; Sashida et al., 1991), which indicated the 6, 7, 3′, 4′-tetrahydroxyaurone skeleton could be the chemotaxonomical marker for Bidens genus. However, few aurones were found from Bidens bipinnata up to now (Li et al., 2003; Yang et al., 2012). In this work, 6-O-β-Dglucopyranosyl-6, 7, 3′, 4′-tetrahydroxyaurone (20), and 7-O-β-D-glucopyranosyl-6, 7, 3′, 4′-tetrahydroxyaurone (21) were isolated from Bidens genus for the first time, this result indicated the closer relationship between Bidens bipinnata with other Bidens species. Thus, compounds 20 and 21 could be used as the taxonomic markers to identify this genus and also for differentiating the species in Bidens genus. Citrusin C (25) and 4-allyl-2, 6-dimethoxyphenyl glucoside (26) were firstly isolated from Bidens genus with allyl benzene skeleton. Different with caffeic acid (27) (Ogawa and Sashida., 1992) type phenylpropanoids that are often isolated from Bidens genus, only few compounds with allyl benzene structure were reported up to now, e.g. guaiacyl glycerol 8-O-β-D-glucoside and 4-allyl-2-methoxyphenol-O-(6O-β-D-apiofuranosyl)-β-D-glucoside from Bidens parviflora Willd. (Wang et al., 2007). From the chemotaxonomic opinion, these two compounds carry significant taxonomic value for Bidens genus. Meanwhile, these type of compounds were also isolated from Carpesium cernuum Linn. (Asteraceae) (Ma et al., 2008), Carpesium macrocephalum FR. et SAV. (Asteraceae) (Kim et al., 2004), Dichrocephala integrifolia Linn. (Asteraceae) (Lee et al., 2015), Macroclinidium trilobum Makino. (Asteraceae) (Miyase et al., 1985), which indicated closer relationship between Bidens and these genera in Asteraceae family.
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