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Four new C-glycosylflavones from the leaves of Iris lactea Pall. var. chinensis (Fisch.) Koidz.
Phytochemistry Letters 22 (2017) 33–38
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Four new C-glycosylflavones from the leaves of Iris lactea Pall. var. chinensis (Fisch.) Koidz. ⁎
Yu Menga,b, Minjian Qina,b, , Bingxin Qia,b, Guoyong Xiea,b, a b
MARK
⁎
Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, China Key Laboratory of Modern Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, China
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article: Methanol (PubChem CID: 887) CHCl3 (PubChem CID: 6212) DMSO (PubChem CID: 679) HCl (PubChem CID: 313) L-rhamnose (PubChem CID: 25310) D-rhamnose (PubChem CID: 5460029) L-glucose (PubChem CID: 10954115) D-glucose (PubChem CID: 5793) Pyridine (PubChem CID: 1049) Acetic Anhydride (PubChem CID: 7918) Cisplatin (PubChem CID: 2767) Formazan (PubChem CID: 9567750)
Four new acylated C-glycosylflavones, termed embinins A–C and irislactin C, were isolated from the leaves of Iris lactea and their structures were elucidated by extensive NMR experiments and mass spectrometry studies. Embinin A and irislactin C showed weak cytotoxicity against A549 (human lung cancer) cells.
Keywords: Iris lactea Iridaceae Acylated C-glycosylflavone Cytotoxicity
1. Introduction
2. Results and discussion
Iris lactea Pall. var. chinensis (Fisch.) Koidz. (Iridaceae) is a perennial herb that is widely distributed in the northeast and northwest of China (Shen et al., 2008). The plant was first recorded in “Shen Nong’s Herbal Classic” and is used as a folk medicine for the treatment of jaundice, diarrhea, vomiting blood, leucorrhea, pharyngitis and swollen carbuncles (Xia et al., 1985). The seeds of I. lactea have been reported to clear heat, eliminate dampness and stanch bleeding (Lv et al., 2015b). Previous phytochemical studies of I. lactea have focused on the pharmacological effects of flavonoids, quinones and oligostilbenes (Shen et al., 2009; Lv et al., 2015a), which have anti-inflammatory, antibacterial, anti-tumor, anti-oxidant and hepatoprotective properties (Colin et al., 2008). The plant is also suitable for greening, water and soil conservation in the regions that have a dry climate and as a type of pasture in the absence of winter forage (Lv et al., 2015b; Zhou et al., 2015).
Four new acylated C-glycosylflavones: 5-hydroxy-7,4′-dimethoxyflavone-6-C-[O-(α-L-4‴- acetylrhamnopyranosyl)-(1 → 2)-β-D-glucopyranoside] (1), 5-hydroxy-7,4′-dimethoxyflavone-6-C-[O-(α-L-2‴,4‴-diacetylrhamnopyranosyl)-(1 → 2)-β-D-glucopyranoside] (2), 5-hydroxy7,4′-dimethoxyflavone-6-C-[O-(α-L-3‴,4‴-diacetylrhamnopyranosyl)(1 → 2)-β-D-glucopyranoside] (3) and 5-hydroxy-4′-methoxyflavone-7O-(β-D-4′′′′-acetylrhamnopyranosyl)-6-C-[O-(α-L-6‴-acetylglucopyranosyl)-(1 → 2)-β-D-glucopyranoside] (4) (Fig. 1) were isolated from a 95% methanol extract of the leaves of I. lactea by chromatography on silica gel and Sephadex LH-20. Compound 1 was obtained as a light yellow amorphous powder. The UV spectrum showed absorption maxima at 273 and 330 nm, which are characteristic of flavone derivatives (Mabry et al., 1970). The IR spectrum implied the presence of hydroxyl (3326 cm−1), conjugated carbonyl (1742, 1656 cm−1), and aromatic C]C groups (1605, 1491,
⁎ Corresponding authors at: Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Gulou District, Nanjing 210009, China. E-mail addresses: [email protected] (M. Qin), [email protected] (G. Xie).
http://dx.doi.org/10.1016/j.phytol.2017.08.011 Received 5 January 2017; Received in revised form 13 July 2017; Accepted 24 August 2017 1874-3900/ © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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Fig. 1. The structures of compounds 1-4.
1452 cm−1) and a para-substituent in a benzene ring (834 cm−1). The HRTOF–ESI–MS spectrum displayed a molecular ion at m/z 649.2131 [M+H]+ (Calcd. 649.2127), suggesting a molecular formula of C31H36O15. The 1H and 13C NMR spectra of 1 also exhibited characteristic signals for a flavonoid with two sugar moieties. The 1H NMR spectrum of 1 (Table 1) showed signals for an AA′BB′ coupled system at δH 8.08 (2H, d, J = 9.0 Hz, H-2′,6′) and 7.13 (2H, d, J = 9.0 Hz, H3′,5′), indicating substitution of the B-ring at C-4′, and two aromatic singlet signals at δH 6.97 (1H, s, H-3) and 6.92 (1H, s, H-8), which were assigned to H-3 and H-8 by HSQC and HMBC spectra. The 1H NMR spectrum also revealed a phenolic hydroxyl group at δH 13.44 (1H, s, eOH) and two methoxyl groups at δH 3.87 (3H, s) and 3.90 (3H, s). The ROESY spectrum appeared to show no correlation between signals at δH 6.92 (H-8) and δH 13.44 (-OH) in compound 1 and the chemical shift of the hydroxyl group was down field, implying that the hydroxyl group was at the C-5 of the aglycone. The aglycone of 1 was, therefore, determined to be 7, 4′-di-O-methylapigenin. With regard to the sugar groups of 1, two signals at δH 4.70 (d, 1H, J = 9.8 Hz) and 5.20 (d, 1H, J = 1.8 Hz) were assigned to anomeric protons of two sugar units. In the 13C NMR spectrum, signals at δC 70.8 and 99.7 indicated a C-linked glycone and an O-linked glycone. A signal at δH 1.78 (s, 3H) represented an acetoxyl group. The NMR assignment suggested that 1 possessed a rhamnosyl group, which showed a doublet signal at δH 0.52, representing the deoxy group at C-6‴. The configuration of the L-rhamnosyl group was determined by comparing chemical derivatives obtained after acid hydrolysis with the same derivative of standard Lrhamnose, using GC analysis. The correlations between H-1″ (δ4.70) and H-3″ (δ3.40), H-5″ (δ3.16) in the ROESY spectrum further supported identification of the other sugar moiety in compound 1 as β-Dglucopyranose. The α-configuration of the rhamnose unit was deduced from the diagnostic coupling patterns of the anomeric proton and the
corresponding carbon resonances in the NMR spectrum (Markham et al., 1987). Furthermore, the HMBC spectrum showed cross peaks at δH 4.70 (H1″)/δC 110.3 (C-6), δH 4.70 (H-1″)/160.7 (C-5), δH 4.70 (H-1″)/163.8 (C7) and δH 5.20 (H-1‴)/δC 73.5 (C-2″), which indicated the presence of sugar residues at C-6 and C-2″, respectively. H-4‴ (δH 4.44) also showed an HMBC interaction with C (CH3COe) (δC 169.9), 65.7 (C-5‴) and 17.6 (C-6‴), which confirmed the position of the acetoxyl group. According to the above data and analysis, compound 1 was determined to be 5-hydroxy-7,4′-dimethoxyflavone-6-C-[O-(α-L-4‴-acetylrhamnopyranosyl)(1 → 2)-β-D-glucopyranoside] and was named as embinin A (Fig. 2). Compound 2 was obtained as a light yellow amorphous powder. The HRTOF–ESI–MS spectrum displayed a molecular ion at m/z 691.2229 [M+H]+ (Calcd. 691.2233), suggesting a molecular formula of C33H38O16. Analysis of the UV, IR, 1H and 13C NMR spectra suggested that 2 was a close analogue of 1 and was anacylated C-glycosylflavone with two sugar moieties, two methoxyl groups and two acetoxyl groups. Similarly, 1H–1H and 1H–13C correlations were determined by HMBC, HSQC and ROESY experiments (Table 1 and Fig. 2). The configuration of the L-rhamnosyl group was determined by comparing chemical derivatives obtained after acid hydrolysis with the same derivative of standard L-rhamnose, using GC analysis. The correlations between H-1″ (δ4.74) and H-3″ (δ3.44), H-5″ (δ3.18) in the ROESY spectrum confirmed the other moiety of β-Dglucopyranose in compound 2, which could not be demonstrated by acid hydrolysis (Fig. 2). The α-configuration of rhamnose was deduced from the diagnostic coupling patterns of the anomeric proton and the corresponding carbon resonances in the NMR spectrum. The HMBC spectrum of 2 showed cross peaks at δH 4.74 (H-1″)/δC 109.6 (C-6), δH 4.74 (H-1″)/162.5 (C-5), δH 4.74 (H-1″)/163.3 (C-7) and δH 5.25 (H-1‴)/δC 74.6 (C-2″), which indicated the presence of 34
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Table 1 1 H NMR (300 MHz, DMSO-d6) and pounds 1-3. NO.
2 3 4 5 6 7 8 9 10 1′ 2′,6′
3′,5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7-OCH3 4′-OCH3 eOCOCH3 eOCOCH3 eOCO eOCO eOH
1 1 H
6.97 (s)
6.92 (s)
8.08 (dd, J = 9 Hz, 2.6 Hz) 7.13 (d, J = 9 Hz) 4.70 (d, J = 9.8 Hz) 4.31 (m) 3.40 (m) 3.11 (m) 3.16 (m) 3.37, 3.72 (m) 5.20 (d, J = 1.8 Hz) 3.62 (m) 3.18 (m) 4.44 (m) 2.20 (m) 0.52 (d, J = 6.2 Hz) 3.87 (s) 3.90 (s) 1.78 (s)
13
13
C
157.4 104.1 182.4 160.7 110.3 163.8 91.9 157.5 104.1 122.9 128.8
115.2 162.9 70.8 73.5 79.9 70.8 82.1 62.1 99.7 70.8 68.2 74.0 65.7 17.6 56.1 56.8 20.8
2 H
1
6.98 (s)
6.92 (s)
8.09 (dd, J = 9 Hz, 2.6 Hz) 7.13 (d, J = 9 Hz) 4.74 (d, J = 9.8 Hz) 4.12 (m) 3.44 (m) 3.10 (m) 3.18 (m) 3.37, 3.72 (m) 5.25 (d, J = 1.7 Hz) 4.95 (m) 3.48 (m) 4.34 (m) 2.31 (m) 0.57 (d, J = 6.2 Hz) 3.87 (s) 3.90 (s) 1.77 (s) 1.99 (s)
169.9 13.44 (s)
Compound 4 was obtained as a light yellow amorphous powder. The UV spectrum showed maximum absorptions at 273 and 330 nm, which are characteristics of flavone derivatives (Mabry et al., 1970). The IR spectrum implied the presence of hydroxyl (3372 cm−1), conjugated carbonyl (1738, 1653 cm−1), and aromatic C]C groups (1616, 1485, 1458 cm−1) and a para-substituent in a benzene ring (848 cm−1). The HRTOF–ESI–MS spectrum displayed a molecular ion at m/z 839.2611 [M + H]+ (Calcd. 839.2604), suggesting a molecular formula of C38H46O21. The 1H and 13C NMR spectra of 4 also exhibited characteristic signals for a flavonoid with three sugar moieties. The 1H NMR spectrum of 4 showed signals for an AA′BB′ coupled system at δH 8.04 (2H, d, J = 9.0 Hz, H-2′,6′) and 7.15 (2H, d, J = 9.0 Hz, H-3′,5′) (Table 2), indicating substitution of the B-ring at C-4′, Two aromatic singlet signals at δH 7.05 (1H, s, H-3) and 6.83 (1H, s, H-8), together with three anomeric protons at δH 4.74 (d, 1H, J = 9.8 Hz), 5.25 (d, 1H, J = 1.6 Hz) and 5.02 (d, 1H, J = 7.4 Hz) suggested the presence of three sugar units. The spectrum also showed a phenolic hydroxyl group at δH 13.50 (1H, s, eOH), a methoxyl group at δH 3.87 (3H, s) and two acetoxyl groups at δH 1.57, 1.96 (s, 3H each). Analysis of the UV, IR, 1H and 13C NMR spectra suggested that 4 was a close analogue of irislactin A (Shen et al., 2008) and was an acylated C-glycosylflavone with three sugar moieties, a methoxyl group and two acetoxyl groups. Similarly, 1 H–1H and 1H–13C correlations were determined using HMBC, HSQC and ROESY experiments (Table 2 and Fig. 2). The configuration of the D-rhamnosyl and L-glucose groups were determined by comparing chemical derivatives obtained after acid hydrolysis with the same derivatives of standard D-rhamnose and L-glucose, using GC analysis. The correlations between H-1″ (δ4.74) and H-3″ (δ3.44), H-5″ (δ3.18) in the ROESY spectrum confirmed the other moiety of β-D-Glc in compound 4. The β-configuration of the glucose and rhamnose moieties, and the αconfiguration of the other glucose in 4, were deduced from the diagnostic coupling patterns of the anomeric proton and the corresponding carbon resonances in the NMR spectrum. Furthermore, the HMBC spectrum showed cross peaks at δH 4.74 (H1″)/δC 111.0 (C-6), δH 4.74 (H-1″)/159.4 (C-5), δH 4.74 (H-1″)/163.0 (C-7) and δH 5.02 (H-1′′′′)/δC 163.0 (C-7) and δH 5.25 (H-1‴)/δC 73.9 (C-2″), which indicated the presence of sugar residues at C-6, C-7 and C2″, respectively. H-4′′′′ (δH 4.36) also showed an HMBC interaction with C (CH3COe) (δC 170.5), 65.6 (C-5′′′′), 17.3 (C-6′′′′), while Ha-6‴ (δH 3.97) showed an HMBC interaction with C (CH3COe) (δC169.8), 74.2 (C-5‴), which confirmed the position of the acetoxyl group. According to the above data and analysis, compound 4 was determined to be 5hydroxy-4′-methoxyflavone-7-O-(β-D-4′′′′- acetylrhamnopyranosyl)-6-C[O-(α-L-6‴-acetylglucopyranosyl)-(1 → 2)-β-D-glucopyranoside] and was named as irislactin C (Fig. 2). The cytotoxicity of these compounds against the A549 (human lung cancer) cell line was evaluated using the MTT colorimetric assay, as described in our earlier report (Xie et al., 2015). Compounds 1 and 4 exhibited weak activity, with IC50 values of 75.9 ± 3.6 and 81.3 ± 4.2 μM, respectively. Compounds 2 and 3 showed no cytotoxicity, with IC50 values > 100 μM. In the present study, four new compounds have been isolated from the leaves of I. lactea and identified as mono- and di-acetylated C-glycosylflavones, named as embinins A–C (1–3) and irislactin C (4). The structures of compounds (1–3) are similar to irislactin B and related compounds reported by Pryakhina et al. (1984), the main difference being the position of the acetoxyl group. In embinin A (1), the acetoxyl group is attached to rhamnose (C-4‴), in embinin B (2), the acetoxyl groups are attached to rhamnose (C-4‴ and C-2‴) and in embinin C (3), the acetoxyl groups are attached to rhamnose (C-4‴ and C-3‴). In irislactin B, the acetoxyl groups are attached to rhamnose (C-3‴ and C2‴), whereas in the compounds reported by Pryakhina et al. (1984), the acetoxyl groups were attached to different sugars. Compound 4 is similar to irislactin A reported by Shen et al. (2008), except that, in irislactin C (4), the acetoxyl groups are attached to rhamnose (C-4′′′′) and glucose (C-6‴). Compared irislactin A, irislactin C lacks an acetoxyl
C NMR (75 MHz, DMSO-d6) spectral data of com-
13.45 (s)
13
C
157.0 104.1 182.2 162.5 109.6 163.3 90.5 157.0 103.8 122.4 128.2
114.6 162.5 70.8 74.6 79.7 70.5 81.4 61.5 96.2 71.8 65.3 73.4 65.7 17.5 55.6 56.6 20.3 20.8 169.4 169.3
3 H
1
6.98 (s)
6.92 (s)
8.09 (dd, J = 9 Hz, 2.6 Hz) 7.12 (d, J = 9 Hz) 4.73 (d, J = 9.8 Hz) 4.31 (t) 3.44 (m) 3.11 (m) 3.17 (m) 3.37, 3.72 (m) 5.15 (d, J = 1.8 Hz) 4.13 (t) 4.51 (m) 4.62 (m) 2.29 (m) 0.52 (d, J = 6.2 Hz) 3.87 (s) 3.90 (s) 1.72 (s) 1.92 (s)
13
C
157.5 104.1 182.8 160.7 110.3 163.8 91.0 157.6 104.6 122.9 128.8
115.1 162.9 71.5 73.4 79.6 71.3 80.3 62.1 100.0 76.0 71.3 70.8 65.8 18.0 56.0 56.9 20.6 21.3 169.7 170.0
13.45 (s)
sugar residues at C-6 and C-2″, respectively. H-4‴ (δH 4.34) also showed an HMBC interaction with C (CH3COe) (δC 169.3), 65.7 (C-5‴), 17.5 (C-6‴), while H-2‴ (δH 4.95) showed an interaction with C (CH3COe) (δC 169.4), 96.2 (C-1‴), 65.3 (C-3‴), 73.4 (C-4‴), which confirmed the position of the acetoxyl group. According to the above data and analysis, compound 2 was determined to be 5-hydroxy-7,4′-dimethoxyflavone-6-C-[O-(α-L-2‴,4‴-diacetylrhamnopyranosyl)-(1 → 2)-β-D-glucopyranoside] and was named as embinin B (Fig. 2). Compound 3 was obtained as a light yellow amorphous powder. Analysis of the UV, IR, 1H, 13C NMR and HRTOF–ESI–MS spectra suggested that 3 was an isomer of 2 and was also a acylated C-glycosylflavone with two sugar moieties, two methoxyl groups and two acetoxyl groups. The only difference between 3 and 2 was the position of the acetoxyl group. The HMBC spectrum of 3 showed cross peaks at δH 4.73 (H-1″)/δC 110.3 (C-6), δH 4.73 (H-1″)/160.7 (C-5), δH 4.73 (H-1″)/163.8 (C-7) and δH 5.15 (H-1‴)/δC 73.4 (C-2″), which indicated the presence of sugar residues at C-6 and C-2″, respectively. H-4‴ (δH 4.62) also showed an HMBC interaction with C (CH3COe) (δC 169.7), 65.8 (C5‴), 18.0 (C-6‴), while H-3‴ (δH 4.51) showed an interaction with C (CH3COe) (δC 170.0), 70.8 (C-4‴), which confirmed the position of the acetoxyl group. According to the above data and analysis, compound 3 was determined to be 5-hydroxy-7,4′-dimethoxyflavone-6-C[O-(α-L-3‴,4‴-diacetylrhamnopyranosyl)-(1 → 2)-β-D-glucopyranoside] and was named as embinin C (Fig. 2). 35
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Fig. 2. The structure and key correlations of compounds 1-4.
aromatic and sugar signals in the 1H NMR and 13C NMR spectra appear as duplicated sets (Takayuki et al., 2013). The NMR signals of the major rotamer are displayed in the left-hand row and those of the minor rotamer are shown in the right-hand row. The ratio of rotamers was ∼1:1 for compounds 1 and 2, and ∼2:1 for compound 3. The relative proportion of the major rotamer to the minor rotamer was affected by temperature (Julia et al., 2012). The impact of temperature on the rotational equilibrium of flavone C-glycosides has been also reported by other authors (Davoust et al., 1980; Zhang and Xu, 2003). The occurrence of rotamers for compounds 1-3 can be attributed to restricted rotation about the C-6-C-1″ bonds (Bjorøy et al., 2009; Yuan et al., 2012). Interestingly, according to the hypothesis put forward by Davoust et al. (1980) that carbon atoms give rise to split resonances in the 13C NMR when the 7-position is substituted with bulkier groups, the carbon atoms of compound 4 should give rise to split resonances in the 13 C NMR. In our study, however, the 1H NMR and 13C NMR spectra of compound 4 showed a series of single peaks. This result is similar to those previously reported for other compounds (Österdahl, 1978; Shen et al., 2008) and more studies are needed to determine the reason (s) for the presence of two rotamers in some C-glycosylflavones.
group at C-2′′′′. All isolated compounds are different from those reported in the literature. Compounds (1–3) were present as two conformational isomers in the 1H NMR spectra in DMSO-d6 (Supplementary information-Figs. S1, S7 and S13). The presence of rotamers is a common solution phenomenon of flavone C-glycosides and, when this occurs, most of the
Table 2 1 H NMR (300 MHz, DMSO-d6) and pound 4. NO.
2 3 4 5 6 7 8 9 10 1′ 2′,6′ 3′,5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″
4 1 H
7.05 (s)
6.83 (s)
8.04 (d, J = 9 Hz) 7.15 (d, J = 9 Hz) 4.74 (d, J = 9.8 Hz) 4.09 (m) 3.44 (m) 3.37 (m) 3.18 (m) 3.6, 3.7 (m)
13
13
C NMR (75 MHz, DMSO-d6) spectral data of com-
C
164.2 104.4 182.5 159.4 111.0 163.0 94.5 157.0 105.2 122.9 128.6 115.2 162.5 70.8 73.9 80.3 73.9 80.6 60.3
1
13
5.25 (d, J = 1.6 Hz) 3.42 (m) 3.35 (m) 3.22 (m) 3.83 (m) 3.97, 4.41 (m) 5.02 (d, J = 7.4 Hz) 3.57 (m) 3.23 (m) 4.36 (t) 1.90 (m) 0.50 (d, J = 6.2 Hz) 3.87 (s) 1.57 (s) 1.96 (s)
99.1 69.4 76.2 70.5 74.2 64.3 101.0 71.3 68.1 74.1 65.6 17.3 56.1 20.9 20.7 169.8 170.5
H
1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 1′′′′ 2′′′′ 3′′′′ 4′′′′ 5′′′′ 6′′′′ 4′-OCH3 eOCOCH3 eOCOCH3 eOCO eOCO eOH
C
3. Experimental 3.1. General experimental procedures Optical rotations were determined using an Autopol IV-T polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were acquired using a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were determined using a Nicolet Impact-410 spectrophotometer (Thermo Nicolet Corporation, Santa
13.50 (s)
36
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hydrochloride (10 mg) and pyridine (0.5 mL) and heated at 90 °C for 30 min. After cooling to room temperature, the reaction products were added to anhydrous acetic anhydride (0.5 mL) and heated at 90 °C for 30 min. The reaction products were extracted three times with CHCl3 and the CHCl3 layer was analyzed by GC. Samples were injected in a split mode using a split ratio of 10:1, with an inlet and transfer line temperature of 250 °C and a constant flow of 1.0 mL/min. Separation was achieved using a temperature program with an initial temperature of 100 °C and initial time of 5 min, which was then ramped to 230 °C at 5 °C/min. Compounds (2–4) were determined using the same procedure performed for compound 1, compared with the reference standards of L, Drhamnose and L-glucose.
Clara, CA, USA), with KBr disks. NMR spectra were recorded on a Bruker ACF-300 spectrometer (Bruker, Rheinstetten, Germany) (300 MHz for 1H and 75 MHz for 13C) with TMS as internal standard. Mass spectra were recorded on an Agilent 6530 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). GC spectra were obtained using a 7820A GC system (Agilent Technologies, Santa Clara, CA, USA). Silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Co., Qingdao, China) and Sephadex LH-20 (50 μm, GE Healthcare Biosciences AB, Uppsala, Sweden) were used for column chromatography. 3.2. Plant material The leaves of Iris lactea Pall. var. chinensis (Fisch.) Koidz. were collected from the Medicinal Botanical Garden of China Pharmaceutical University in Nanjing, China. The plant was identified by Dr. Minjian Qin and a voucher specimen (No. ITM20150616) was deposited in the Herbarium of Medicinal Plants of China Pharmaceutical University.
3.5. Cytotoxicity assay The human lung cancer cell line A549 was obtained from the Department of Pharmacology, China Pharmaceutical University and exposed to test compounds (1–4) for 3 days. A549 cells were grown in DEME + 10%FBS and maintained in an incubator at 37 °C, with 5% CO2 and 95% humidity. The cells were collected and dispensed into 96well culture plates in DEME + 10% FBS (100 μL) at a density of 4 × 103 cells/well. The cells were then incubated with the test compounds at various concentrations in triplicate for 48 h. The positive control group was treated with cisplatin (20 μM). After incubation, MTT was added to each well with a final concentration of 0.5 mg/mL. The insoluble formazan was collected and dissolved in DMSO (0.5%) and measured at 490 nm using a BioTek Synergy 2 automated microplate spectrophotometer (Bio-Tek, Winooski, VT, USA). Assays were performed in triplicate in three independent experiments.
3.3. Extraction and isolation The air-dried leaves of I. lactea (1.8 kg) were extracted three times with 95% methanol (9 L), using ultrasonic irradiation at room temperature. The combined extracts were concentrated to a syrup, which was extracted with petroleum ether. The residue after extracting with petroleum ether (150 g) was subjected to silica gel column chromatography (10 × 120 cm, 100–200 mesh). Elution with gradients of CHCl3–MeOH (100:0–0:100) afforded sixteen fractions (Fractions 1–16). Fraction 8 (10 g) was subjected to silica gel column chromatography (4 × 80 cm, 200–300 mesh) and eluted with CHCl3-MeOH (100:1–0:100) to provide seven subfractions (Fr.8.1–Fr.8.7). Fr.8.5 (3 g) was re-chromatographed on a silica gel column (4 × 50 cm, 200–300 mesh) and eluted with gradients of CHCl3–MeOH (30:1–0:1). Fr.8.5.3(1.8 g) was further purified by chromatography on silica gel (2 × 60 cm, 200–300 mesh), eluting with CHCl3–MeOH (15:1–0:1) and on Sephadex LH-20(1 × 80 cm, 50 μm), eluting with MeOH to provide compounds 1 (40 mg), 2 (25 mg) and 3 (75 mg). Fr.11 (19 g) was separately chromatographed on silica gel (4 × 50 cm, 200–300 mesh), eluting with CHCl3-MeOH (10:1–0:10) to provide five subfractions (Fr.11.1–Fr.11.5). Fr.11.3 (2.7 g) was recrystallized from methanol to afford compound 4 (270 mg). embinin A (1): yellow powder. [a]20 D −23.26°(c = 0.04, MeOH). UV (CH3OH) λmax 273 nm, 330 nm; IR (microscope) νmax: 3326, 1742, 1656, 1605, 1491, 1452, 834 cm−1; HR-TOF-MS: 649.2131 [M+H]+ (Calcd. for C31H37O15, 649.2127); 1H and 13C NMR: Table 1. embinin B (2): yellow powder. [a]20 D −55.04°(c = 0.04, MeOH). UV (CH3OH) λmax 273 nm, 330 nm; IR (microscope) νmax: 3366, 1744, 1655, 1606, 1491, 1448, 836 cm−1; HR-TOF-MS: 691.2229 [M+H]+ (Calcd. for C33H39O16, 691.2233); 1H and 13C NMR: Table 1. embinin C (3): yellow powder. [a]20 D −58.9°(c = 0.043, MeOH). UV (CH3OH) λmax 273 nm, 330 nm; IR (microscope) νmax: 3359, 1744, 1656, 1606, 1491, 1447, 835 cm−1; HR-TOF-MS: 691.2231 [M+H]+ (Calcd. for C33H39O16, 691.2233); 1H and 13C NMR: Table 1. irislactinC (4): yellow powder.[a]20 D −127.1°(c = 0.04, 10%MeOH). UV (CH3OH) λmax 273 nm, 327 nm; IR (microscope) νmax: 3371, 1738, 1653, 1606, 1485, 1458, 848 cm−1; HR-TOF-MS: 839.2611 [M+H]+ (Calcd. for C38H47O21, 839.2604); 1H and 13C NMR: Table 2.
Acknowledgments This research was funded by the National Natural Science Foundation of China (grant No.81373918), the National Natural Science Foundation of China (grant No. 81503220) and the National Natural Science Foundation of Jiangsu province (grant No. BK20150706). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.08.011. References Bjorøy, Ø., Rayyan, S., Fossen, T., Kalberg, K., Andersen, Ø.M., 2009. C-glycosyl anthocyanidins synthesized from C-glycosylflavones. Phytochemistry 70, 278–287. Colin, D., Lancon, A., Delmas, D., Lizard, G., Abrossinow, J., Kahn, E., Jannin, B., Latruffe, N., 2008. Antiproliferative activities of resveratrol and related compounds in human hepatocyte derived HepG2 cells are associated with biochemical cell disturbance revealed by fluorescence analyses. Biochimie 9, 1674–1684. Davoust, D., Massias, M., Molho, D., 1980. 13C NMR investigation of flavonoid C-β-Dglucosides. Detection of a conformational equilibrium. Org. Magn. Reson. 13, 218–219. Julia, H.F., Yomica, L.P., Russel, S.R., William, F.R., 2012. Variable-temperature 1H NMR studies on two C-glycosylflavones. Molecules 17, 7914–7926. Lv, H.H., Ouyang, J., Wang, X.Y., Ma, X.F., Suo, Y.R., 2015a. Separation and purification of four flavan-3-ols from Iris lactea Pall. var. Chinensis (Fisch.) Koidz by high-speed counter-current chromatography with flow-rate gradient. J. Liq. Chromatogr. Relat. Technol. 38, 1486–1493. Lv, H.H., Wang, H.L., He, Y.F., Wang, X.Y., Suo, Y.R., 2015b. Separation and purification of four oligostilbenes from Iris lactea Pall. var. chinensis (Fisch.) Koidz by high-speed counter-current chromatography. J. Chromatogr. B 988, 127–134. Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer Verlag, New York, pp. 84–120. Markham, K.R., Whitehouse, L.A., Webby, R.F., 1987. Luteolin-7-O-[6-O-α-L-arabinofuranosyl]-β-D-glucopyranoside and other new flavonoid glycosides from New Zealand Dacrydium species. J. Nat. Prod. 50, 660–663. Pryakhina, N.I., Sheichenko, V.I., Blinova, K.F., 1984. Acylated C-glycosides of Iris lactea. Chem. Nat. Compd. 20, 554–559. Shen, W.J., Qin, M.J., Zhang, C.F., 2008. Two new C-glycosylflavones from the leaves of Iris lactea var. chinensis. Chin. Chem. Lett. 19, 821–824.
3.4. Acid hydrolysis and derivatization of compounds 1–4 and L, Drhamnose, L-glucose Compound 1 was dissolved in 5% HCl and heated at 90 °C for 4 h. The reaction mixture was evaporated under a stream of N2. Rhamnose was detected by TLC in the residue, by comparison with authentic samples. The hydrolysate and L-rhamnose were derivatized using the same conditions. The hydrolysate was mixed with hydroxylamine 37
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97–101. Yuan, S.Q., Yang, M., Zhao, Y.M., 2012. A new flavonol glycoside from glandless cotton seeds. Acta Pharm. Sin. B 2, 42–45. Zhang, P.C., Xu, S.X., 2003. C-glucoside flavonoids from the leaves of Crataegus pinnatifida Bge. var. major N.E.Br. J. Asian Nat. Prod. Res. 5, 131–136. Zhou, T.R., Ge, G.T., Jia, Y.S., Hou, M.L., Wang, W., Nuo, M., Ba, D.L.H., 2015. The effect advantage natural grassland on mixed grass group of silage quality. Grassl. Prataculture 27, 19–26.
Shen, W.J., Qin, M.J., Deng, X.Y., Wu, G., 2009. Chemical constituents of leaves of Iris lactea Pall. var. chinensis. Chin. Pharm. J. 4, 249–251. Takayuki, M., Tsutomu, Y., Tsukasa, I., 2013. Identification of novel C-glycosylflavones and their contribution to flower colour of the Dutch Iris cultivars. Plant Physiol. Biochem. 72, 116–124. Xia, G.C., Liu, X.M., Xiao, P.G., 1985. Resource utilization and herbal study of “Malinzi” (Iris lactea Pall. var. chinensis). Acta Pharm. Sin. 20, 316–319. Xie, G.Y., Lin, B.B., Qin, X.Y., Wang, G.K., Wang, Q., Yuan, J., Li, C., Qin, M.J., 2015. New flavonoids with cytotoxicity from the roots of Flemingia latifolia. Fitoterapia 104,
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Four new C-glycosylflavones from the leaves of Iris lactea Pall. var. chinensis (Fisch.) Koidz. ⁎
Yu Menga,b, Minjian Qina,b, , Bingxin Qia,b, Guoyong Xiea,b, a b
MARK
⁎
Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, China Key Laboratory of Modern Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 210009, China
A R T I C L E I N F O
A B S T R A C T
Chemical compounds studied in this article: Methanol (PubChem CID: 887) CHCl3 (PubChem CID: 6212) DMSO (PubChem CID: 679) HCl (PubChem CID: 313) L-rhamnose (PubChem CID: 25310) D-rhamnose (PubChem CID: 5460029) L-glucose (PubChem CID: 10954115) D-glucose (PubChem CID: 5793) Pyridine (PubChem CID: 1049) Acetic Anhydride (PubChem CID: 7918) Cisplatin (PubChem CID: 2767) Formazan (PubChem CID: 9567750)
Four new acylated C-glycosylflavones, termed embinins A–C and irislactin C, were isolated from the leaves of Iris lactea and their structures were elucidated by extensive NMR experiments and mass spectrometry studies. Embinin A and irislactin C showed weak cytotoxicity against A549 (human lung cancer) cells.
Keywords: Iris lactea Iridaceae Acylated C-glycosylflavone Cytotoxicity
1. Introduction
2. Results and discussion
Iris lactea Pall. var. chinensis (Fisch.) Koidz. (Iridaceae) is a perennial herb that is widely distributed in the northeast and northwest of China (Shen et al., 2008). The plant was first recorded in “Shen Nong’s Herbal Classic” and is used as a folk medicine for the treatment of jaundice, diarrhea, vomiting blood, leucorrhea, pharyngitis and swollen carbuncles (Xia et al., 1985). The seeds of I. lactea have been reported to clear heat, eliminate dampness and stanch bleeding (Lv et al., 2015b). Previous phytochemical studies of I. lactea have focused on the pharmacological effects of flavonoids, quinones and oligostilbenes (Shen et al., 2009; Lv et al., 2015a), which have anti-inflammatory, antibacterial, anti-tumor, anti-oxidant and hepatoprotective properties (Colin et al., 2008). The plant is also suitable for greening, water and soil conservation in the regions that have a dry climate and as a type of pasture in the absence of winter forage (Lv et al., 2015b; Zhou et al., 2015).
Four new acylated C-glycosylflavones: 5-hydroxy-7,4′-dimethoxyflavone-6-C-[O-(α-L-4‴- acetylrhamnopyranosyl)-(1 → 2)-β-D-glucopyranoside] (1), 5-hydroxy-7,4′-dimethoxyflavone-6-C-[O-(α-L-2‴,4‴-diacetylrhamnopyranosyl)-(1 → 2)-β-D-glucopyranoside] (2), 5-hydroxy7,4′-dimethoxyflavone-6-C-[O-(α-L-3‴,4‴-diacetylrhamnopyranosyl)(1 → 2)-β-D-glucopyranoside] (3) and 5-hydroxy-4′-methoxyflavone-7O-(β-D-4′′′′-acetylrhamnopyranosyl)-6-C-[O-(α-L-6‴-acetylglucopyranosyl)-(1 → 2)-β-D-glucopyranoside] (4) (Fig. 1) were isolated from a 95% methanol extract of the leaves of I. lactea by chromatography on silica gel and Sephadex LH-20. Compound 1 was obtained as a light yellow amorphous powder. The UV spectrum showed absorption maxima at 273 and 330 nm, which are characteristic of flavone derivatives (Mabry et al., 1970). The IR spectrum implied the presence of hydroxyl (3326 cm−1), conjugated carbonyl (1742, 1656 cm−1), and aromatic C]C groups (1605, 1491,
⁎ Corresponding authors at: Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Gulou District, Nanjing 210009, China. E-mail addresses: [email protected] (M. Qin), [email protected] (G. Xie).
http://dx.doi.org/10.1016/j.phytol.2017.08.011 Received 5 January 2017; Received in revised form 13 July 2017; Accepted 24 August 2017 1874-3900/ © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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Fig. 1. The structures of compounds 1-4.
1452 cm−1) and a para-substituent in a benzene ring (834 cm−1). The HRTOF–ESI–MS spectrum displayed a molecular ion at m/z 649.2131 [M+H]+ (Calcd. 649.2127), suggesting a molecular formula of C31H36O15. The 1H and 13C NMR spectra of 1 also exhibited characteristic signals for a flavonoid with two sugar moieties. The 1H NMR spectrum of 1 (Table 1) showed signals for an AA′BB′ coupled system at δH 8.08 (2H, d, J = 9.0 Hz, H-2′,6′) and 7.13 (2H, d, J = 9.0 Hz, H3′,5′), indicating substitution of the B-ring at C-4′, and two aromatic singlet signals at δH 6.97 (1H, s, H-3) and 6.92 (1H, s, H-8), which were assigned to H-3 and H-8 by HSQC and HMBC spectra. The 1H NMR spectrum also revealed a phenolic hydroxyl group at δH 13.44 (1H, s, eOH) and two methoxyl groups at δH 3.87 (3H, s) and 3.90 (3H, s). The ROESY spectrum appeared to show no correlation between signals at δH 6.92 (H-8) and δH 13.44 (-OH) in compound 1 and the chemical shift of the hydroxyl group was down field, implying that the hydroxyl group was at the C-5 of the aglycone. The aglycone of 1 was, therefore, determined to be 7, 4′-di-O-methylapigenin. With regard to the sugar groups of 1, two signals at δH 4.70 (d, 1H, J = 9.8 Hz) and 5.20 (d, 1H, J = 1.8 Hz) were assigned to anomeric protons of two sugar units. In the 13C NMR spectrum, signals at δC 70.8 and 99.7 indicated a C-linked glycone and an O-linked glycone. A signal at δH 1.78 (s, 3H) represented an acetoxyl group. The NMR assignment suggested that 1 possessed a rhamnosyl group, which showed a doublet signal at δH 0.52, representing the deoxy group at C-6‴. The configuration of the L-rhamnosyl group was determined by comparing chemical derivatives obtained after acid hydrolysis with the same derivative of standard Lrhamnose, using GC analysis. The correlations between H-1″ (δ4.70) and H-3″ (δ3.40), H-5″ (δ3.16) in the ROESY spectrum further supported identification of the other sugar moiety in compound 1 as β-Dglucopyranose. The α-configuration of the rhamnose unit was deduced from the diagnostic coupling patterns of the anomeric proton and the
corresponding carbon resonances in the NMR spectrum (Markham et al., 1987). Furthermore, the HMBC spectrum showed cross peaks at δH 4.70 (H1″)/δC 110.3 (C-6), δH 4.70 (H-1″)/160.7 (C-5), δH 4.70 (H-1″)/163.8 (C7) and δH 5.20 (H-1‴)/δC 73.5 (C-2″), which indicated the presence of sugar residues at C-6 and C-2″, respectively. H-4‴ (δH 4.44) also showed an HMBC interaction with C (CH3COe) (δC 169.9), 65.7 (C-5‴) and 17.6 (C-6‴), which confirmed the position of the acetoxyl group. According to the above data and analysis, compound 1 was determined to be 5-hydroxy-7,4′-dimethoxyflavone-6-C-[O-(α-L-4‴-acetylrhamnopyranosyl)(1 → 2)-β-D-glucopyranoside] and was named as embinin A (Fig. 2). Compound 2 was obtained as a light yellow amorphous powder. The HRTOF–ESI–MS spectrum displayed a molecular ion at m/z 691.2229 [M+H]+ (Calcd. 691.2233), suggesting a molecular formula of C33H38O16. Analysis of the UV, IR, 1H and 13C NMR spectra suggested that 2 was a close analogue of 1 and was anacylated C-glycosylflavone with two sugar moieties, two methoxyl groups and two acetoxyl groups. Similarly, 1H–1H and 1H–13C correlations were determined by HMBC, HSQC and ROESY experiments (Table 1 and Fig. 2). The configuration of the L-rhamnosyl group was determined by comparing chemical derivatives obtained after acid hydrolysis with the same derivative of standard L-rhamnose, using GC analysis. The correlations between H-1″ (δ4.74) and H-3″ (δ3.44), H-5″ (δ3.18) in the ROESY spectrum confirmed the other moiety of β-Dglucopyranose in compound 2, which could not be demonstrated by acid hydrolysis (Fig. 2). The α-configuration of rhamnose was deduced from the diagnostic coupling patterns of the anomeric proton and the corresponding carbon resonances in the NMR spectrum. The HMBC spectrum of 2 showed cross peaks at δH 4.74 (H-1″)/δC 109.6 (C-6), δH 4.74 (H-1″)/162.5 (C-5), δH 4.74 (H-1″)/163.3 (C-7) and δH 5.25 (H-1‴)/δC 74.6 (C-2″), which indicated the presence of 34
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Table 1 1 H NMR (300 MHz, DMSO-d6) and pounds 1-3. NO.
2 3 4 5 6 7 8 9 10 1′ 2′,6′
3′,5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7-OCH3 4′-OCH3 eOCOCH3 eOCOCH3 eOCO eOCO eOH
1 1 H
6.97 (s)
6.92 (s)
8.08 (dd, J = 9 Hz, 2.6 Hz) 7.13 (d, J = 9 Hz) 4.70 (d, J = 9.8 Hz) 4.31 (m) 3.40 (m) 3.11 (m) 3.16 (m) 3.37, 3.72 (m) 5.20 (d, J = 1.8 Hz) 3.62 (m) 3.18 (m) 4.44 (m) 2.20 (m) 0.52 (d, J = 6.2 Hz) 3.87 (s) 3.90 (s) 1.78 (s)
13
13
C
157.4 104.1 182.4 160.7 110.3 163.8 91.9 157.5 104.1 122.9 128.8
115.2 162.9 70.8 73.5 79.9 70.8 82.1 62.1 99.7 70.8 68.2 74.0 65.7 17.6 56.1 56.8 20.8
2 H
1
6.98 (s)
6.92 (s)
8.09 (dd, J = 9 Hz, 2.6 Hz) 7.13 (d, J = 9 Hz) 4.74 (d, J = 9.8 Hz) 4.12 (m) 3.44 (m) 3.10 (m) 3.18 (m) 3.37, 3.72 (m) 5.25 (d, J = 1.7 Hz) 4.95 (m) 3.48 (m) 4.34 (m) 2.31 (m) 0.57 (d, J = 6.2 Hz) 3.87 (s) 3.90 (s) 1.77 (s) 1.99 (s)
169.9 13.44 (s)
Compound 4 was obtained as a light yellow amorphous powder. The UV spectrum showed maximum absorptions at 273 and 330 nm, which are characteristics of flavone derivatives (Mabry et al., 1970). The IR spectrum implied the presence of hydroxyl (3372 cm−1), conjugated carbonyl (1738, 1653 cm−1), and aromatic C]C groups (1616, 1485, 1458 cm−1) and a para-substituent in a benzene ring (848 cm−1). The HRTOF–ESI–MS spectrum displayed a molecular ion at m/z 839.2611 [M + H]+ (Calcd. 839.2604), suggesting a molecular formula of C38H46O21. The 1H and 13C NMR spectra of 4 also exhibited characteristic signals for a flavonoid with three sugar moieties. The 1H NMR spectrum of 4 showed signals for an AA′BB′ coupled system at δH 8.04 (2H, d, J = 9.0 Hz, H-2′,6′) and 7.15 (2H, d, J = 9.0 Hz, H-3′,5′) (Table 2), indicating substitution of the B-ring at C-4′, Two aromatic singlet signals at δH 7.05 (1H, s, H-3) and 6.83 (1H, s, H-8), together with three anomeric protons at δH 4.74 (d, 1H, J = 9.8 Hz), 5.25 (d, 1H, J = 1.6 Hz) and 5.02 (d, 1H, J = 7.4 Hz) suggested the presence of three sugar units. The spectrum also showed a phenolic hydroxyl group at δH 13.50 (1H, s, eOH), a methoxyl group at δH 3.87 (3H, s) and two acetoxyl groups at δH 1.57, 1.96 (s, 3H each). Analysis of the UV, IR, 1H and 13C NMR spectra suggested that 4 was a close analogue of irislactin A (Shen et al., 2008) and was an acylated C-glycosylflavone with three sugar moieties, a methoxyl group and two acetoxyl groups. Similarly, 1 H–1H and 1H–13C correlations were determined using HMBC, HSQC and ROESY experiments (Table 2 and Fig. 2). The configuration of the D-rhamnosyl and L-glucose groups were determined by comparing chemical derivatives obtained after acid hydrolysis with the same derivatives of standard D-rhamnose and L-glucose, using GC analysis. The correlations between H-1″ (δ4.74) and H-3″ (δ3.44), H-5″ (δ3.18) in the ROESY spectrum confirmed the other moiety of β-D-Glc in compound 4. The β-configuration of the glucose and rhamnose moieties, and the αconfiguration of the other glucose in 4, were deduced from the diagnostic coupling patterns of the anomeric proton and the corresponding carbon resonances in the NMR spectrum. Furthermore, the HMBC spectrum showed cross peaks at δH 4.74 (H1″)/δC 111.0 (C-6), δH 4.74 (H-1″)/159.4 (C-5), δH 4.74 (H-1″)/163.0 (C-7) and δH 5.02 (H-1′′′′)/δC 163.0 (C-7) and δH 5.25 (H-1‴)/δC 73.9 (C-2″), which indicated the presence of sugar residues at C-6, C-7 and C2″, respectively. H-4′′′′ (δH 4.36) also showed an HMBC interaction with C (CH3COe) (δC 170.5), 65.6 (C-5′′′′), 17.3 (C-6′′′′), while Ha-6‴ (δH 3.97) showed an HMBC interaction with C (CH3COe) (δC169.8), 74.2 (C-5‴), which confirmed the position of the acetoxyl group. According to the above data and analysis, compound 4 was determined to be 5hydroxy-4′-methoxyflavone-7-O-(β-D-4′′′′- acetylrhamnopyranosyl)-6-C[O-(α-L-6‴-acetylglucopyranosyl)-(1 → 2)-β-D-glucopyranoside] and was named as irislactin C (Fig. 2). The cytotoxicity of these compounds against the A549 (human lung cancer) cell line was evaluated using the MTT colorimetric assay, as described in our earlier report (Xie et al., 2015). Compounds 1 and 4 exhibited weak activity, with IC50 values of 75.9 ± 3.6 and 81.3 ± 4.2 μM, respectively. Compounds 2 and 3 showed no cytotoxicity, with IC50 values > 100 μM. In the present study, four new compounds have been isolated from the leaves of I. lactea and identified as mono- and di-acetylated C-glycosylflavones, named as embinins A–C (1–3) and irislactin C (4). The structures of compounds (1–3) are similar to irislactin B and related compounds reported by Pryakhina et al. (1984), the main difference being the position of the acetoxyl group. In embinin A (1), the acetoxyl group is attached to rhamnose (C-4‴), in embinin B (2), the acetoxyl groups are attached to rhamnose (C-4‴ and C-2‴) and in embinin C (3), the acetoxyl groups are attached to rhamnose (C-4‴ and C-3‴). In irislactin B, the acetoxyl groups are attached to rhamnose (C-3‴ and C2‴), whereas in the compounds reported by Pryakhina et al. (1984), the acetoxyl groups were attached to different sugars. Compound 4 is similar to irislactin A reported by Shen et al. (2008), except that, in irislactin C (4), the acetoxyl groups are attached to rhamnose (C-4′′′′) and glucose (C-6‴). Compared irislactin A, irislactin C lacks an acetoxyl
C NMR (75 MHz, DMSO-d6) spectral data of com-
13.45 (s)
13
C
157.0 104.1 182.2 162.5 109.6 163.3 90.5 157.0 103.8 122.4 128.2
114.6 162.5 70.8 74.6 79.7 70.5 81.4 61.5 96.2 71.8 65.3 73.4 65.7 17.5 55.6 56.6 20.3 20.8 169.4 169.3
3 H
1
6.98 (s)
6.92 (s)
8.09 (dd, J = 9 Hz, 2.6 Hz) 7.12 (d, J = 9 Hz) 4.73 (d, J = 9.8 Hz) 4.31 (t) 3.44 (m) 3.11 (m) 3.17 (m) 3.37, 3.72 (m) 5.15 (d, J = 1.8 Hz) 4.13 (t) 4.51 (m) 4.62 (m) 2.29 (m) 0.52 (d, J = 6.2 Hz) 3.87 (s) 3.90 (s) 1.72 (s) 1.92 (s)
13
C
157.5 104.1 182.8 160.7 110.3 163.8 91.0 157.6 104.6 122.9 128.8
115.1 162.9 71.5 73.4 79.6 71.3 80.3 62.1 100.0 76.0 71.3 70.8 65.8 18.0 56.0 56.9 20.6 21.3 169.7 170.0
13.45 (s)
sugar residues at C-6 and C-2″, respectively. H-4‴ (δH 4.34) also showed an HMBC interaction with C (CH3COe) (δC 169.3), 65.7 (C-5‴), 17.5 (C-6‴), while H-2‴ (δH 4.95) showed an interaction with C (CH3COe) (δC 169.4), 96.2 (C-1‴), 65.3 (C-3‴), 73.4 (C-4‴), which confirmed the position of the acetoxyl group. According to the above data and analysis, compound 2 was determined to be 5-hydroxy-7,4′-dimethoxyflavone-6-C-[O-(α-L-2‴,4‴-diacetylrhamnopyranosyl)-(1 → 2)-β-D-glucopyranoside] and was named as embinin B (Fig. 2). Compound 3 was obtained as a light yellow amorphous powder. Analysis of the UV, IR, 1H, 13C NMR and HRTOF–ESI–MS spectra suggested that 3 was an isomer of 2 and was also a acylated C-glycosylflavone with two sugar moieties, two methoxyl groups and two acetoxyl groups. The only difference between 3 and 2 was the position of the acetoxyl group. The HMBC spectrum of 3 showed cross peaks at δH 4.73 (H-1″)/δC 110.3 (C-6), δH 4.73 (H-1″)/160.7 (C-5), δH 4.73 (H-1″)/163.8 (C-7) and δH 5.15 (H-1‴)/δC 73.4 (C-2″), which indicated the presence of sugar residues at C-6 and C-2″, respectively. H-4‴ (δH 4.62) also showed an HMBC interaction with C (CH3COe) (δC 169.7), 65.8 (C5‴), 18.0 (C-6‴), while H-3‴ (δH 4.51) showed an interaction with C (CH3COe) (δC 170.0), 70.8 (C-4‴), which confirmed the position of the acetoxyl group. According to the above data and analysis, compound 3 was determined to be 5-hydroxy-7,4′-dimethoxyflavone-6-C[O-(α-L-3‴,4‴-diacetylrhamnopyranosyl)-(1 → 2)-β-D-glucopyranoside] and was named as embinin C (Fig. 2). 35
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Fig. 2. The structure and key correlations of compounds 1-4.
aromatic and sugar signals in the 1H NMR and 13C NMR spectra appear as duplicated sets (Takayuki et al., 2013). The NMR signals of the major rotamer are displayed in the left-hand row and those of the minor rotamer are shown in the right-hand row. The ratio of rotamers was ∼1:1 for compounds 1 and 2, and ∼2:1 for compound 3. The relative proportion of the major rotamer to the minor rotamer was affected by temperature (Julia et al., 2012). The impact of temperature on the rotational equilibrium of flavone C-glycosides has been also reported by other authors (Davoust et al., 1980; Zhang and Xu, 2003). The occurrence of rotamers for compounds 1-3 can be attributed to restricted rotation about the C-6-C-1″ bonds (Bjorøy et al., 2009; Yuan et al., 2012). Interestingly, according to the hypothesis put forward by Davoust et al. (1980) that carbon atoms give rise to split resonances in the 13C NMR when the 7-position is substituted with bulkier groups, the carbon atoms of compound 4 should give rise to split resonances in the 13 C NMR. In our study, however, the 1H NMR and 13C NMR spectra of compound 4 showed a series of single peaks. This result is similar to those previously reported for other compounds (Österdahl, 1978; Shen et al., 2008) and more studies are needed to determine the reason (s) for the presence of two rotamers in some C-glycosylflavones.
group at C-2′′′′. All isolated compounds are different from those reported in the literature. Compounds (1–3) were present as two conformational isomers in the 1H NMR spectra in DMSO-d6 (Supplementary information-Figs. S1, S7 and S13). The presence of rotamers is a common solution phenomenon of flavone C-glycosides and, when this occurs, most of the
Table 2 1 H NMR (300 MHz, DMSO-d6) and pound 4. NO.
2 3 4 5 6 7 8 9 10 1′ 2′,6′ 3′,5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″
4 1 H
7.05 (s)
6.83 (s)
8.04 (d, J = 9 Hz) 7.15 (d, J = 9 Hz) 4.74 (d, J = 9.8 Hz) 4.09 (m) 3.44 (m) 3.37 (m) 3.18 (m) 3.6, 3.7 (m)
13
13
C NMR (75 MHz, DMSO-d6) spectral data of com-
C
164.2 104.4 182.5 159.4 111.0 163.0 94.5 157.0 105.2 122.9 128.6 115.2 162.5 70.8 73.9 80.3 73.9 80.6 60.3
1
13
5.25 (d, J = 1.6 Hz) 3.42 (m) 3.35 (m) 3.22 (m) 3.83 (m) 3.97, 4.41 (m) 5.02 (d, J = 7.4 Hz) 3.57 (m) 3.23 (m) 4.36 (t) 1.90 (m) 0.50 (d, J = 6.2 Hz) 3.87 (s) 1.57 (s) 1.96 (s)
99.1 69.4 76.2 70.5 74.2 64.3 101.0 71.3 68.1 74.1 65.6 17.3 56.1 20.9 20.7 169.8 170.5
H
1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 1′′′′ 2′′′′ 3′′′′ 4′′′′ 5′′′′ 6′′′′ 4′-OCH3 eOCOCH3 eOCOCH3 eOCO eOCO eOH
C
3. Experimental 3.1. General experimental procedures Optical rotations were determined using an Autopol IV-T polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were acquired using a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were determined using a Nicolet Impact-410 spectrophotometer (Thermo Nicolet Corporation, Santa
13.50 (s)
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hydrochloride (10 mg) and pyridine (0.5 mL) and heated at 90 °C for 30 min. After cooling to room temperature, the reaction products were added to anhydrous acetic anhydride (0.5 mL) and heated at 90 °C for 30 min. The reaction products were extracted three times with CHCl3 and the CHCl3 layer was analyzed by GC. Samples were injected in a split mode using a split ratio of 10:1, with an inlet and transfer line temperature of 250 °C and a constant flow of 1.0 mL/min. Separation was achieved using a temperature program with an initial temperature of 100 °C and initial time of 5 min, which was then ramped to 230 °C at 5 °C/min. Compounds (2–4) were determined using the same procedure performed for compound 1, compared with the reference standards of L, Drhamnose and L-glucose.
Clara, CA, USA), with KBr disks. NMR spectra were recorded on a Bruker ACF-300 spectrometer (Bruker, Rheinstetten, Germany) (300 MHz for 1H and 75 MHz for 13C) with TMS as internal standard. Mass spectra were recorded on an Agilent 6530 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). GC spectra were obtained using a 7820A GC system (Agilent Technologies, Santa Clara, CA, USA). Silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Co., Qingdao, China) and Sephadex LH-20 (50 μm, GE Healthcare Biosciences AB, Uppsala, Sweden) were used for column chromatography. 3.2. Plant material The leaves of Iris lactea Pall. var. chinensis (Fisch.) Koidz. were collected from the Medicinal Botanical Garden of China Pharmaceutical University in Nanjing, China. The plant was identified by Dr. Minjian Qin and a voucher specimen (No. ITM20150616) was deposited in the Herbarium of Medicinal Plants of China Pharmaceutical University.
3.5. Cytotoxicity assay The human lung cancer cell line A549 was obtained from the Department of Pharmacology, China Pharmaceutical University and exposed to test compounds (1–4) for 3 days. A549 cells were grown in DEME + 10%FBS and maintained in an incubator at 37 °C, with 5% CO2 and 95% humidity. The cells were collected and dispensed into 96well culture plates in DEME + 10% FBS (100 μL) at a density of 4 × 103 cells/well. The cells were then incubated with the test compounds at various concentrations in triplicate for 48 h. The positive control group was treated with cisplatin (20 μM). After incubation, MTT was added to each well with a final concentration of 0.5 mg/mL. The insoluble formazan was collected and dissolved in DMSO (0.5%) and measured at 490 nm using a BioTek Synergy 2 automated microplate spectrophotometer (Bio-Tek, Winooski, VT, USA). Assays were performed in triplicate in three independent experiments.
3.3. Extraction and isolation The air-dried leaves of I. lactea (1.8 kg) were extracted three times with 95% methanol (9 L), using ultrasonic irradiation at room temperature. The combined extracts were concentrated to a syrup, which was extracted with petroleum ether. The residue after extracting with petroleum ether (150 g) was subjected to silica gel column chromatography (10 × 120 cm, 100–200 mesh). Elution with gradients of CHCl3–MeOH (100:0–0:100) afforded sixteen fractions (Fractions 1–16). Fraction 8 (10 g) was subjected to silica gel column chromatography (4 × 80 cm, 200–300 mesh) and eluted with CHCl3-MeOH (100:1–0:100) to provide seven subfractions (Fr.8.1–Fr.8.7). Fr.8.5 (3 g) was re-chromatographed on a silica gel column (4 × 50 cm, 200–300 mesh) and eluted with gradients of CHCl3–MeOH (30:1–0:1). Fr.8.5.3(1.8 g) was further purified by chromatography on silica gel (2 × 60 cm, 200–300 mesh), eluting with CHCl3–MeOH (15:1–0:1) and on Sephadex LH-20(1 × 80 cm, 50 μm), eluting with MeOH to provide compounds 1 (40 mg), 2 (25 mg) and 3 (75 mg). Fr.11 (19 g) was separately chromatographed on silica gel (4 × 50 cm, 200–300 mesh), eluting with CHCl3-MeOH (10:1–0:10) to provide five subfractions (Fr.11.1–Fr.11.5). Fr.11.3 (2.7 g) was recrystallized from methanol to afford compound 4 (270 mg). embinin A (1): yellow powder. [a]20 D −23.26°(c = 0.04, MeOH). UV (CH3OH) λmax 273 nm, 330 nm; IR (microscope) νmax: 3326, 1742, 1656, 1605, 1491, 1452, 834 cm−1; HR-TOF-MS: 649.2131 [M+H]+ (Calcd. for C31H37O15, 649.2127); 1H and 13C NMR: Table 1. embinin B (2): yellow powder. [a]20 D −55.04°(c = 0.04, MeOH). UV (CH3OH) λmax 273 nm, 330 nm; IR (microscope) νmax: 3366, 1744, 1655, 1606, 1491, 1448, 836 cm−1; HR-TOF-MS: 691.2229 [M+H]+ (Calcd. for C33H39O16, 691.2233); 1H and 13C NMR: Table 1. embinin C (3): yellow powder. [a]20 D −58.9°(c = 0.043, MeOH). UV (CH3OH) λmax 273 nm, 330 nm; IR (microscope) νmax: 3359, 1744, 1656, 1606, 1491, 1447, 835 cm−1; HR-TOF-MS: 691.2231 [M+H]+ (Calcd. for C33H39O16, 691.2233); 1H and 13C NMR: Table 1. irislactinC (4): yellow powder.[a]20 D −127.1°(c = 0.04, 10%MeOH). UV (CH3OH) λmax 273 nm, 327 nm; IR (microscope) νmax: 3371, 1738, 1653, 1606, 1485, 1458, 848 cm−1; HR-TOF-MS: 839.2611 [M+H]+ (Calcd. for C38H47O21, 839.2604); 1H and 13C NMR: Table 2.
Acknowledgments This research was funded by the National Natural Science Foundation of China (grant No.81373918), the National Natural Science Foundation of China (grant No. 81503220) and the National Natural Science Foundation of Jiangsu province (grant No. BK20150706). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.08.011. References Bjorøy, Ø., Rayyan, S., Fossen, T., Kalberg, K., Andersen, Ø.M., 2009. C-glycosyl anthocyanidins synthesized from C-glycosylflavones. Phytochemistry 70, 278–287. Colin, D., Lancon, A., Delmas, D., Lizard, G., Abrossinow, J., Kahn, E., Jannin, B., Latruffe, N., 2008. Antiproliferative activities of resveratrol and related compounds in human hepatocyte derived HepG2 cells are associated with biochemical cell disturbance revealed by fluorescence analyses. Biochimie 9, 1674–1684. Davoust, D., Massias, M., Molho, D., 1980. 13C NMR investigation of flavonoid C-β-Dglucosides. Detection of a conformational equilibrium. Org. Magn. Reson. 13, 218–219. Julia, H.F., Yomica, L.P., Russel, S.R., William, F.R., 2012. Variable-temperature 1H NMR studies on two C-glycosylflavones. Molecules 17, 7914–7926. Lv, H.H., Ouyang, J., Wang, X.Y., Ma, X.F., Suo, Y.R., 2015a. Separation and purification of four flavan-3-ols from Iris lactea Pall. var. Chinensis (Fisch.) Koidz by high-speed counter-current chromatography with flow-rate gradient. J. Liq. Chromatogr. Relat. Technol. 38, 1486–1493. Lv, H.H., Wang, H.L., He, Y.F., Wang, X.Y., Suo, Y.R., 2015b. Separation and purification of four oligostilbenes from Iris lactea Pall. var. chinensis (Fisch.) Koidz by high-speed counter-current chromatography. J. Chromatogr. B 988, 127–134. Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer Verlag, New York, pp. 84–120. Markham, K.R., Whitehouse, L.A., Webby, R.F., 1987. Luteolin-7-O-[6-O-α-L-arabinofuranosyl]-β-D-glucopyranoside and other new flavonoid glycosides from New Zealand Dacrydium species. J. Nat. Prod. 50, 660–663. Pryakhina, N.I., Sheichenko, V.I., Blinova, K.F., 1984. Acylated C-glycosides of Iris lactea. Chem. Nat. Compd. 20, 554–559. Shen, W.J., Qin, M.J., Zhang, C.F., 2008. Two new C-glycosylflavones from the leaves of Iris lactea var. chinensis. Chin. Chem. Lett. 19, 821–824.
3.4. Acid hydrolysis and derivatization of compounds 1–4 and L, Drhamnose, L-glucose Compound 1 was dissolved in 5% HCl and heated at 90 °C for 4 h. The reaction mixture was evaporated under a stream of N2. Rhamnose was detected by TLC in the residue, by comparison with authentic samples. The hydrolysate and L-rhamnose were derivatized using the same conditions. The hydrolysate was mixed with hydroxylamine 37
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97–101. Yuan, S.Q., Yang, M., Zhao, Y.M., 2012. A new flavonol glycoside from glandless cotton seeds. Acta Pharm. Sin. B 2, 42–45. Zhang, P.C., Xu, S.X., 2003. C-glucoside flavonoids from the leaves of Crataegus pinnatifida Bge. var. major N.E.Br. J. Asian Nat. Prod. Res. 5, 131–136. Zhou, T.R., Ge, G.T., Jia, Y.S., Hou, M.L., Wang, W., Nuo, M., Ba, D.L.H., 2015. The effect advantage natural grassland on mixed grass group of silage quality. Grassl. Prataculture 27, 19–26.
Shen, W.J., Qin, M.J., Deng, X.Y., Wu, G., 2009. Chemical constituents of leaves of Iris lactea Pall. var. chinensis. Chin. Pharm. J. 4, 249–251. Takayuki, M., Tsutomu, Y., Tsukasa, I., 2013. Identification of novel C-glycosylflavones and their contribution to flower colour of the Dutch Iris cultivars. Plant Physiol. Biochem. 72, 116–124. Xia, G.C., Liu, X.M., Xiao, P.G., 1985. Resource utilization and herbal study of “Malinzi” (Iris lactea Pall. var. chinensis). Acta Pharm. Sin. 20, 316–319. Xie, G.Y., Lin, B.B., Qin, X.Y., Wang, G.K., Wang, Q., Yuan, J., Li, C., Qin, M.J., 2015. New flavonoids with cytotoxicity from the roots of Flemingia latifolia. Fitoterapia 104,
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