FITOTE-03158; No of Pages 6 Fitoterapia xxx (2015) xxx–xxx
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Fitoterapia
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Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects Da-Song Yang a,b,c,1, Zi-Lei Li a,b,c,1, Wei-Bing Peng d, Yong-Ping Yang a,b,c, Xue Wang d, Ke-Chun Liu d, Xiao-Li Li a,b,c,⁎, Wei-Lie Xiao e,⁎ a
Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China c Institute of Tibetan Plateau Research at Kunming, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China d Biology Institute of Shandong Academy of Sciences, Jinan 250014, PR China e State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China b
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journal homepage: www.elsevier.com/locate/fitote
a r t i c l e
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Article history: Received 29 January 2015 Accepted in revised form 31 March 2015 Available online xxxx
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Keywords: Macaranga denticulata Euphorbiaceae Flavonoid–diterpene heterodimer Geranylated flavonoids Anticancer effects
31
1. Introduction
32 33
Nature relies on an intricate network of biosynthetic pathways to produce the cornucopia of small organic molecules needed to support life, among which combinatorial chemical synthesis plays an important role. Prenylated flavonoids, a combination of terpenoid and flavonoid, are extraordinarily diverse in chemistry and bioactivity [1]. Due to the differences of prenylation position, various lengths of prenyl chain, and further modifications of the prenyl moiety such as cyclization and hydroxylation, prenylation has greatly enriched the structural diversity of flavonoid. Studies have showed that the presence of isoprenoid chain is a major determinant of the bioactivity of prenylated flavonoids and the remarkable
38 39 40 41 42 43
18 19 20 21 22 23 24
O
R
R
E
C
T
One rare flavonoid–diterpene heterodimer, denticulatain C (1), one modified geranyl-type side chain substituted flavonoid, denticulatain D (2) and one geranylated flavonoid, denticulatain E (3), as well as 11 known compounds (4–14) were isolated from the fronds of Macaranga denticulata. Their structures were elucidated on the basis of extensive spectroscopic interpretation. Compounds 4 and 8 inhibited the proliferation of A-549 cell line with IC50 values of 48.6 and 20.2 μg/mL, respectively. Compounds 3, 6, and 8 exhibited significant antiangiogenic activity on a zebrafish model with IC50 values of 9.78, 0.34, and 2.55 μg/mL, respectively. © 2015 Published by Elsevier B.V.
N C
36 37
a b s t r a c t
U
34 35
i n f o
E
1 2
D
10
⁎ Corresponding authors. Tel./fax: +86 871 65223231. E-mail addresses: [email protected] (X.-L. Li), [email protected] (W.-L. Xiao). 1 These authors contributed equally to this work.
properties of these compounds are thought to reside in their enhanced interaction with biological membranes and increased affinity for target proteins [2]. The genus Macaranga (Euphorbiaceae), which is widely distributed in the tropical regions, is a rich source of prenylated flavonoids and stilbenes, and the biological activities of those metabolites encompass virtually all fields of pharmacological sciences [1,3]. In Chinese traditional medicine, the roots of Macaranga denticulata have been used for the treatment of icteric hepatitis. Previous studies on this plant have resulted in the isolation of prenylated flavonoids [4] and stilbene– diterpene heterodimers [5]. As a part of our ongoing research on structurally diverse and potentially anticancer prenylated aromatic products from genus Macaranga, one rare flavonoid– diterpene heterodimer, denticulatain C (1), one modified geranyl-type side chain substituted flavonoid, denticulatain D (2) and one geranylated flavonoid, denticulatain E (3), as well as 11 known compounds were isolated from the fronds of M. denticulata. Their cytotoxicity against A549 cancer cell line
http://dx.doi.org/10.1016/j.fitote.2015.04.001 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
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80
98
Optical rotations were recorded using a JASCO P–1020 digital polarimeter. UV spectra were recorded using a Shimadzu UV-2401PC spectrophotometer. IR spectra were recorded on a Bruker Tenor 27 spectrophotometer with KBr pellets. 1D and 2D NMR experiments were recorded on Bruker AM-400 or DRX500 spectrometers with TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. ESI-MS were recorded using a Finnigan MAT 90 instrument and HR-ESI-MS was performed on an API QSTAR time-of-flight spectrometer. Column chromatography (CC) was performed on Sephadex LH-20 (Amersham Biosciences, Piscataway, USA), silica gel (200–300 mesh, Qingdao Marine Chemical inc., Qingdao, China), RP-18 gel (LiChroprep, 40–63 μm, Merck, Darmstadt, Germany), and MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Semipreparative HPLC was performed on an Agilent 1200 (column: Zorbax SB–C18, 250 × 9.4 mm, DAD detector). Fractions were monitored by TLC and visualized by heating plates sprayed with 15% H2SO4 in EtOH.
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2.2. Plant material
100 101
104
The fronds of M. denticulata were collected from Xishuangbanna of Yunnan province, PR China, in March 2008. A voucher specimen (Yangyp–20080316) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, which was identified by Prof. Yong-Ping Yang.
105
2.3. Extraction and isolation
106 107
The air-dried and powdered fronds of M. denticulata (11 kg) were extracted with 90% EtOH (3 × 25 L) at room temperature and concentrated in vacuo to yield a residue, which was partitioned between H2O and EtOAc. The EtOAc portion was decolorized on MCI gel (eluting with 95% EtOH), and the residue (185 g) was subjected to silica gel CC with a gradient elution of CHCl3/acetone (10:0 to 3:7) to afford five fractions (Fr. A–E). Fr. A was purified by Sephadex LH-20 (CHCl3–MeOH, 1:1) and then subjected to CC over silica gel eluted with petroleum ether–EtOAc (1:0 to 2:1) to yield compounds 10 (3.1 mg) and 13 (4.3 mg). Fr. B was separated by Sephadex
93 94 95 96 97
102 103
108 109 110 111 112 113 114 115 116
C
91 92
E
89 90
R
87 88
R
85 86
O
83 84
C
81 82
N
74 75
U
72 73
2.4. Spectroscopic data
135
2.4.1. Denticulatain C (1) Yellow oil; [α]25 D +1.4 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 208 (4.64), 257 (4.36), 370 (4.38) nm; IR (KBr) νmax 3423, 2925, 2845, 1647, 1626, 1602, 1564, 1483, 1443, 1367, 1319, 1268, 1196, 1157, 1089, 1035, 959, 886, 812 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) data, see Table 1; positive ESIMS m/z 575 [M + H]+; HR-ESI-MS m/z 575.2995 [M + H]+ (calcd. for C35H43O7, 575.3008).
136 137
2.4.2. Denticulatain D (2) Yellow amorphous powder; [α]18 D −2.5 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 202 (4.88), 222 (4.79), 272 (4.51), 348 (4.02), 428 (4.48) nm; IR (KBr) νmax 3377, 2920, 1651, 1622, 1607, 1564, 1483, 1367, 1316, 1267, 1228, 1182, 1086, 1024, 894, 839, 805 cm−1; 1H NMR (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 100 MHz) data, see Table 1; negative ESIMS m/z 437 [M − H]−; HR-ESI-MS m/z 437.1597 [M − H]− (calcd. for C25H25O7, 437.1600).
144
2.4.3. Denticulatain E (3) Yellow oil; [α]18 D −4.0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 202 (4.72), 220 (4.72), 286 (4.15), 464 (3.82) nm; IR (KBr) νmax 3454, 2967, 2821, 1657, 1620, 1552, 1516, 1438, 1373, 1331, 1254, 1199, 1158, 1110, 1093, 1027, 824 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 1; negative ESIMS m/z 437 [M − H]−; HR-ESI-MS m/z 437.1598 [M − H]− (calcd. for C25H25O7, 437.1600).
153 154
2.5. Cytotoxicity assay
162
Compounds 1–14 were tested for their cytotoxicity against human lung cancer cell line A-549 by the MTT method, 5-FU was used as a positive control. Briefly, 100 μL of cell suspension (1 × 105 cells/mL) was seeded into 96well microtiter plates and cultured for 24 h before the compound was added. Then, different concentrations of the compounds were added to the plates, the cells were cultivated for 48 h, and 10 μL of MTT (5 mg/mL) was added to each well. After 4 h, the culture medium was removed and
163 164
F
2.1. General
70 71
O
79
68 69
R O
2. Experimental
67
117 118
P
78
65 66
LH-20 (CHCl3–MeOH, 1:1) to obtain 12 (13.2 mg) and a major fraction which was subjected to CC over silica gel eluted with petroleum ether–EtOAc (4:1 to 1:1), to give 4 (42.5 mg) and 5 (9.7 mg). Fr. C was purified over a Sephadex LH-20 (CHCl3 − MeOH, 1:1) and then fractionated by RP-18 with a gradient elution of MeOH–H2O (2:8 to 10:0) to yield subfractions C1–C6. Subfraction C1 was repeatedly purified by CC over silica gel eluted with petroleum ether–EtOAc (9:1 to 1:1) to afford 11 (21.6 mg) and 14 (7.4 mg). Compounds 1 (8.5 mg), 8 (10.7 mg) and 9 (9.2 mg) were isolated from subfraction C3 by repeated CC over silica gel eluted with petroleum ether–EtOAc (9:1 to 3:1). Fr. D was fractionated by Sephadex LH-20 (CHCl3–MeOH, 1:1) to yield subfractions D1–D3. Subfraction D1 was purified by semipreparative HPLC eluted by 87% MeOH–H2O, to afford 2 (12.8 mg) and 3 (10.3 mg). Subfraction D3 was purified by semipreparative HPLC eluted by 82% MeOH–H2O, to afford 6 (5.6 mg) and 7 (16.4 mg).
T
76 77
and the antiangiogenic activity against zebrafish embryos were tested. Herein, we describe the isolation, structural elucidation and anticancer effects of these compounds. Generally speaking, flavonoids are rarely substituted by isoprenoid residue longer than the geranyl [1], especially those isoprenoid residues with further intricate cyclization. The complex structures and sufficient biological activities of those compounds have attracted scientists' greater attention to study their structures, bioactivities, and synthesis. Previously studies have led to the isolation and characterization of the first example of flavonoid–diterpene heterodimer, denticulaflavonol [4], which have been further synthesized along with three other derivatives [6]. Thus, the discovery of denticulatain C (1), the second example flavonoid–diterpene heterodimer from nature has enriched the diversity of prenylated flavonoid class.
D
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D.-S. Yang et al. / Fitoterapia xxx (2015) xxx–xxx
E
2
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
119 120 121 122 123 Q7 124 125 126 127 128 129 130 131 132 133 134
138 139 140 141 142 143
145 146 147 148 149 150 151 152
155 156 157 158 159 160 161
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2
174 175 Q8
3″
42.6 t
4″ 5″ 6″
33.8 s 56.1 d 25.1 t
7″
38.5 t
8″ 9″
149.4 s 55.2 d
10″ 11″
39.8 s 21.9 t
12″
38.8 t
13″ 14″ 15″
135.6 s 123.5 d 21.9 t
16″ 17″
16.2 q 106.5 t
b c d
33.7 q 22.0 q 14.9 q
6.97 7.66 1.65 0.80 1.49 1.35 1.29 1.05
d (8.4) d (8.4) m m m m m m
0.85 1.61 1.18 2.23 1.88
m m m m m
75.2 d 149.3 s 110.3 t
m m m m t (7.2) m m s s s s s s s
the formazan crystals were completely dissolved with 150 μL DMSO in each well by vigorously shaking the plate. Finally, formazan absorbance was assessed by a BioRad microplate reader at 570 nm (Fig. 1).
2.6. Antiangiogenesis assay
177
Stock solutions (20 mg/mL) of all samples were prepared by dissolving the compounds 1–14 in DMSO. These solutions were diluted in sterile salt water (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4) to obtain final solutions of various concentrations in 0.2% DMSO. Aliquots were placed into 24-well plates, and the embryos (TG[VEGFR2:GRCFP]) at
180 181 182
5.30 t (6.8)
6.24 d (1.7)
F
145.3 s 136.6 s 176.5 s 162.1 s 99.1 d 165.2 s 94.4 d 157.6 s 103.2 s 122.7 s 113.3 d 146.4 s 147.5 s 129.0 s 121.8 d 28.8 t
δHc
1.78 s 1.95 m 1.56 m 3.95 t (6.2)
4.84 s 4.69 s 1.65 s
123.2 d
6.44 d (1.7)
7.66 s
7.61 s 3.28, overlap 5.38 t (6.6)
136.6 s 16.2 q 40.4 t 27.4 t
1.74 s 2.04 m 2.11 m
125.0 d
5.09 t (5.7)
131.7 s 25.7 q
1.57 s
17.6 q
1.54 s
12.41 s
12.21 br s
Recorded at 400 MHz. Recorded at 500 MHz. Recorded at 125 MHz. Recorded at 100 MHz.
176
178 179
6.99 d (8.6) 8.12 d (8.6) 3.36 d (6.8)
16.3 q 36.4 t 34.5 t
17.8 q
5.25 3.42 3.29 1.78 4.74 4.47 0.73 0.71 0.61 12.44
8.12 d (8.6) 6.99 d (8.6)
136.6 s
1.54 m
1.52 1.38 2.04 1.75
6.59 s
122.9 d
N C
18″ 19″ 20″ 5-OH
7.79 s
s s s s s s d s s s d d s d d t
δCa
P
20.0 t
6.60 s
U
172 173
2″
146.7 135.5 176.5 158.9 111.7 162.7 93.8 155.6 104.0 123.4 130.4 116.3 160.0 116.3 130.4 21.9
d
D
a
s s s s s s d s s s d s s d d t
δH
T
t1:50 t1:51 t1:52 t1:53
148.2 136.7 176.5 158.8 112.0 162.6 93.8 155.5 104.0 123.8 115.6 145.7 146.6 116.1 121.3 39.5
3
δCb
R
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″
O
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44 t1:45 t1:46 t1:47 t1:48 t1:49
δHd
E
δCa
R O O
1
C
No.
E
t1:3
Table 1 1 H and 13C NMR data of compounds 1–3 in acetone-d6 (δ in ppm, J in Hz).
R
t1:1 Q1 t1:2
3
24 hpf (hours post-fertilization) were also transferred randomly into the above wells. Control embryos were treated with the equivalent amount of DMSO solutions. All embryos were incubated at 28.5 °C. After 48 h treatment, the intersegmental vessels of embryos were visualized with green fluorescent protein labeling and endogenous alkaline phosphatase staining. The antiangiogenic activities of compounds were calculated from the inhibition ratio of antiangiogenesis.
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3. Results and discussion
191
185 186 187 188 189 190
Denticulatain C (1) was obtained as yellow oil. HR-ESI-MS 192 analysis of 1 provided a molecular formula of C35H42O7, 193
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
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D.-S. Yang et al. / Fitoterapia xxx (2015) xxx–xxx
OH OH
3' 4'
2' 8
HO 7
10'' 9''
10''
8''
5''
7''
4''
OH O
17''
2'' 3''
2''
5'' 8''
7''
O
HO
OH
OH
5
16''
6''
9''
3''
1''
OH OH O
4''
6''
1*
18''
2*
OH
OH
OH O
OH OH O
OH O 4
3*
OH
R2 OH HO
O
O
O
R O
HO
F
H 19''
6'
3 4
6 15''
OH
5'
2
10
13''
1''
O 9
14''
12'' 11''
20''
1'
HO
O
OH
O
O
R
P
R1
OH O 8R=H 9 R = CH3
E
D
OH O 5 R1 = H, R2 = H; 6 R1 = OH, R2 = H 7 R1 = OH, R2 = OH
O
O
HO
C E R
O
R
HO
12
11 O
O HO
O O HO
O
201 202 203 204 205 206 207 208 209 210 211
C
O 199 200
N
197 198
O OH
O 14 Fig. 1. Structures of compounds 1–14.
corresponding to 15 unsaturation degrees. The IR spectrum showed typical absorption bands for OH (3423 cm−1), α,βunsaturated carbonyl (1647 cm−1) and aromatic ring (1602 and 1483 cm−1) moieties. The UV spectrum showed absorption maxima at λmax 257 (4.36) and 380 (4.38) nm, which indicated the presence of a flavonol skeleton [7]. The 1H and 13C NMR spectra of 1 showed the existence of C-6 substituted quercetin [δC 136.7 (s), 176.5 (s), 93.8 (d), 115.6 (d), 116.1 (d), 121.3 (d); δH 6.60 (1H, s), 6.97 (1H, d, J = 8.4 Hz), 7.66 (2H, d, J = 8.4 Hz), 7.79 (1H, s)] [7,8] and the remaining moiety possessed 20 carbons including one pair of terminal double bond [δC 106.5 (t), 149.4 (s); δH 4.47 (1H, s), 4.74 (1H, s)]; one pair of trisubstituted double bond [δC 123.5 (d), 135.6 (s)]; 4 tertiary methyls (δH 0.61, 0.71, 0.73, 1.78, 3H each, s); 8 methylenes; 2 methines and 2 quaternary carbons, which can be identified as a bicyclic labdane diterpenoid by comparing the NMR data with those of denticulaflavonol [4] and this deduction was confirmed by the observed 2D NMR correlations
U
195 196
O
O
13
194
H
OH
10
O
H
T
HO
H
(Fig. 2). The HMBC correlations of H-15″/C-5, C-6, C-7 and H-14″/C-6 demonstrated that labdanyl unit was connected to quercetin at C-6 of ring A. The observed ROESY (Fig. 3) correlations of H-5″/H-9″, Me-18″; Me-20″/H-11″, Me-19″; H-12″/H-14″; H-15″/H-16″ suggested that the stereochemistry of labdane skeleton in 1 was the same as denticulaflavonol [5]. Therefore, the structure of denticulatain C (1) was determined as 6-[(5″S,9″S,10″S)-15″-labd-8″(17″),13″(14″)-dienyl]quercetin, and it is the second example flavonoid–diterpene heterodimer from nature. Denticulatain D (2) was obtained as optically active amorphous powder ([α]18 D − 2.5, c 0.31, MeOH) and its molecular formula of C25H26O7 was established by HR-ESI-MS at m/z 437.1597 [M − H]−, requiring 13 degrees of unsaturation. Analysis of the 1H and 13C NMR (Table 1) data of 2 aided by HSQC revealed resonances for one 6-substituted kaempferol [δC 135.5 (s), 176.5 (s), 93.8 (d), 116.3 (d), 130.4 (d); δH 6.59 (1H, s), 6.99 (2H, d, J = 8.6 Hz), 8.12 (2H, d, J = 8.6 Hz)] [4,9],
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229
D.-S. Yang et al. / Fitoterapia xxx (2015) xxx–xxx
5
OH OH HO
OH
O
HO
OH
7 6
14''
5
10''
OH
15''
8''
O 7 6
2''
7'' 6''
5 1''
OH O 1
2
F
OH
R O O
OH HO
2''
O
5' 6'
1''
OH 3
244 245 246 247 248 249 250 251 252 253 254 255 Q9
P
D
C
242 243
E
240 241
R
238 239
R
236 237
O
234 235
) correlations of compounds 1–3.
and H-2″/C-5′ indicated the location of the geranyl group at C-5′ of ring B. Accordingly, the structure of denticulatain E (3) was determined as 5′-geranylquercetin. The known compounds were identified as 5-hydroxy-2-(4hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-enyl)chroman4-one (4) [12], sophoraflavanone A (5) [13], bonanniol A (6) [14], diplacol (7) [15], 5,7,3′,4′-tetrahydroxy-6-geranylflavonol (8) [7], 5,7,3′,4′-tetrahydroxy-3-methoxy-6-geranylflavone (9) [7], 3βhydroxy-7α-ethoxy-24β-ethylcholest-5-ene (10) [16], (24R)6β-hydroxy-24-ethylcholest-4-en-3-one (11) [17], epitaraxerol (12) [18], α-tocopherolquinone (13) [19] and boehmenan (14) [20] by comparison of their experimental and reported spectroscopic data. All the compounds were isolated from M. denticulata for the first time. Since prenylated flavonoids are reported to have modest or strong anticancer activities [1,9], the cytotoxicity of compounds 1–14 were evaluated against human lung cancer cell line A549 by the MTT method [21], with 5-FU used as a positive control (IC50 22.48 μg/mL). The results showed that compounds 4 and 8 inhibit the proliferation of A-549 cell line with IC50 values of 48.6 and 20.2 μg/mL, respectively, and this is the first time to report the cytotoxicity of known compounds 4 and 8. The antiangiogenic activities of compounds 1–14 were further evaluated using a zebrafish model in terms of the inhibition on the growth of intersegmental vessels, using PTK787 as positive control (IC50 0.10 μg/mL) [22]. The results showed
N C
232 233
one modified geranyl-type side chain (6-hydroxy-3,7-dimethyl2(E),7-octadienyl) [one pair of trisubstituted double bond (δC 122.9 d, 136.6 s); one pair of terminal double bond (δC 110.3 t, 149.3 s); one oxygenated methine (δC 75.2); three alkyl methylenes (δC 21.9, 34.5, 36.4) and two tertiary methyls (δH 1.65, 1.78, 3H each, s)] [10], which was confirmed by the observed 1H-1H COSY correlation of H-6″/H-7″ and the HMBC correlations of H-6″/C-8″; H-9″/C-7″, Me-10″; and Me-10″/C-7″. The HMBC correlations of H-1″/C-5, C-6, C-7 and H-2″/C-6 indicated the attachment of the modified geranyl-type side chain at C-6 of ring A. Thus, the structure of denticulatain D was determined as 6-[6-hydroxy-3,7dimethyl-2(E),7-octadienyl]kaempferol. The molecular formula of denticulatain E (3) was found to be C25H26O7, as deduced from HR-ESI-MS and NMR data, indicative of 13 indices of hydrogen deficiency. The strong IR absorptions implied the presence of hydroxy (3454 cm−1), conjugated carbonyl (1657 cm−1), and aromatic ring (1620 and 1438 cm−1) functionalities. The 1H and 13C NMR (Table 1) displayed characteristic signals corresponding to a ring Bsubstituted quercetin [δC 136.6 s, 176.5 s, 94.4 d, 99.1 d, 113.3 d, 121.8, d; δH 6.24 (1H, d, J = 1.7 Hz), 6.44 (1H, d, J = 1.7 Hz), 7.61 (s), 7.66 (s)] [11] and a geranyl substituent [three methyls (δC 16.2, 17.6, 25.7); three methylenes (δC 27.4, 28.8, 40.4); two pair of trisubstituted double bond (δC 123.2 d, 125.0 d, 131.7 s, 136.6 s)]. The observed HMBC correlations of H-1″/C-5′, C-6′,
U
230 231
) and 1H-1H COSY (
E
Fig. 2. Selected HMBC (
T
OH O
OH
OH O
9''
16'' 20''
11'' 9''
19''
12'' 14''
5''
15''
18'' Fig. 3. Key ROESY correlations of compound 1.
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
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4. Conflict of interest
289
The authors declare that there is no conflict of interest. Acknowledgments
291
296
This study was financially supported by National Natural Science Foundation of China (31300293 and 81422046), General Project of Applied Foundation Research, Yunnan Province (2013FB067), Basic Research Project of Ministry of Science and Technology of China (2012FY110300), and Major State Basic Research Development Program (2010CB951704).
297
Appendix A. Supplementary data
298 299
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.04.001.
300
References
T
O
R
R
E
C
[1] Botta B, Vitali A, Menendez P, Misiti D, Monache GD. Prenylated flavonoids: pharmacology and biotechnology. Curr Med Chem 2005;12: 713–39. [2] Botta B, Monache GD, Menendez P, Boffi A. Novel prenyltransferase enzymes as a tool for flavonoid prenylation. Trends Pharmacol Sci 2005; 26:606–8. [3] Magadula JJ. Phytochemistry and pharmacology of the genus Macaranga: a review. J Med Plants Res 2014;8:489–503. [4] Sutthivaiyakit S, Unganont S, Sutthivaiyakit P, Suksamrarn A. Diterpenylated and prenylated flavonoids from Macaranga denticulata. Tetrahedron 2002; 58:3619–22.
C
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O
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R O
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[5] Yang DS, Li ZL, Wang X, Yan H, Yang YP, Luo HR, et al. Denticulatains A and B: unique stilbene–diterpene heterodimers from Macaranga denticulata. RSC Adv 2015;5:13886–90. [6] Basabe P, de Román M, Diez D, Marcos IS, Bodero O, Blanco A, et al. Synthesis of isoprenyl flavonoids: (+)-denticulaflavonol, macarangin, and isomacarangin. Synlett 2008;8:1149–52. [7] Lee D, Bhat KPL, Fong HHS, Farnsworth NR, Pezzuto JM, Kinghorn AD. Aromatase inhibitors from Broussonetia papyrifera. J Nat Prod 2001;64: 1286–93. [8] Wu Y, Luo Q, Sun CL, Wang GH, Chen QC, Guo ZJ, et al. Chemical constituents contained in Desmodium caudatum. China J Chin Mater Med 2012;37:1788–92. [9] Yang DS, Wei JG, Peng WB, Wang SM, Sun C, Yang YP, et al. Cytotoxic prenylated bibenzyls and flavonoids from Macaranga kurzii. Fitoterapia 2014;99:261–6. [10] Schneiderová K, Šlapetová T, Hrabal R, Dvořáková H, Procházková P, Novotná J, et al. Tomentomimulol and mimulone B: two new Cgeranylated flavonoids from Paulownia tomentosa fruits. Nat Prod Res 2013;27:613–8. [11] Zheng ZP, Cheng KW, Chao JF, Wu JJ, Wang MF. Tyrosinase inhibitors from paper mulberry (Broussonetia papyrifera). Food Chem 2008;106:529–35. [12] Intekhab J, Aslam M. Isolation of a flavonoid from Feronia limonia. J Saudi Chem Soc 2009;13:295–8. [13] Shirataki Y, Endo M, Yokoe I, Komatsu M. Studies on the constituents of Sophora species. XVIII. Constituents of the root of Sophora tomentosa L.. Chem Pharm Bull 1983;31:2859–63. [14] Bruno M, Savona G, Lamartina L, Lentini F. New flavonoids from Bonannia graeca (L.) Halacsy. Heterocycles 1985;23:1147–53. [15] Phillips WR, Baj NJ, Gunatilaka AAL, Kingston DGI. C-geranyl compounds from Mimulus clevelandii. J Nat Prod 1996;59:495–7. [16] Liu LL, Yang JL, Shi YP. Sesquiterpenoids and other constituents from the flower buds of Tussilago farfara. J Asian Nat Prod Res 2011;13:920–9. [17] Arai Y, Nakagawa T, Hitosugi M, Shiojima K, Ageta H, Abdel-Halim OB. Chemical constituents of aquatic fern Azolla nilotica. Phytochemistry 1998; 48:471–4. [18] Rahman AU, Sultana N, Akhter F, Nighat F, Choudhary MI. Phytochemical studies on Adhatoda vasica Nees. Nat Prod Lett 1997;10:249–56. [19] Yang DS, Li ZL, Wang X, Yang YP, Peng WB, Liu KC, et al. Chemical constituents from roots of Campanumoea javanica and their antiangiogeneic activities. Chin Tradit Herb Drugs 2015;46:470–5. [20] Seca AML, Silva AMS, Silvestre AJD, Cavaleiro JAS, Domingues FMJ, PascoalNeto C. Phenolic constituents from the core of Kenaf (Hibiscus cannabinus). Phytochemistry 2001;56:759–67. [21] Yang DS, Peng WB, Li ZL, Wang X, Wei JG, He QX, et al. Chemical constituents from Euphorbia stracheyi and their biological activities. Fitoterapia 2014;97:211–8. [22] Yang DS, Zhang YL, Peng WB, Wang LY, Li ZL, Wang X, et al. Jatropholanetype diterpenes from Euphorbia sikkimensis. J Nat Prod 2013;76:265–9.
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that intersegmental vessels of embryos treated with compounds 3, 6, and 8 were significantly fewer than those of the control (0.2% DMSO in sterile salt water) and the reduction was dose dependent with an IC50 value of 9.78, 0.34 and 2.55 μg/mL, respectively. The antiangiogenic activities of known compounds 6 and 8 were reported for the first time.
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Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
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Contents lists available at ScienceDirect
Fitoterapia
3Q3 4 Q55Q4 6 7 8 9
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Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects Da-Song Yang a,b,c,1, Zi-Lei Li a,b,c,1, Wei-Bing Peng d, Yong-Ping Yang a,b,c, Xue Wang d, Ke-Chun Liu d, Xiao-Li Li a,b,c,⁎, Wei-Lie Xiao e,⁎ a
Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China c Institute of Tibetan Plateau Research at Kunming, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China d Biology Institute of Shandong Academy of Sciences, Jinan 250014, PR China e State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China b
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journal homepage: www.elsevier.com/locate/fitote
a r t i c l e
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Article history: Received 29 January 2015 Accepted in revised form 31 March 2015 Available online xxxx
25 26 27 28 29 30
Keywords: Macaranga denticulata Euphorbiaceae Flavonoid–diterpene heterodimer Geranylated flavonoids Anticancer effects
31
1. Introduction
32 33
Nature relies on an intricate network of biosynthetic pathways to produce the cornucopia of small organic molecules needed to support life, among which combinatorial chemical synthesis plays an important role. Prenylated flavonoids, a combination of terpenoid and flavonoid, are extraordinarily diverse in chemistry and bioactivity [1]. Due to the differences of prenylation position, various lengths of prenyl chain, and further modifications of the prenyl moiety such as cyclization and hydroxylation, prenylation has greatly enriched the structural diversity of flavonoid. Studies have showed that the presence of isoprenoid chain is a major determinant of the bioactivity of prenylated flavonoids and the remarkable
38 39 40 41 42 43
18 19 20 21 22 23 24
O
R
R
E
C
T
One rare flavonoid–diterpene heterodimer, denticulatain C (1), one modified geranyl-type side chain substituted flavonoid, denticulatain D (2) and one geranylated flavonoid, denticulatain E (3), as well as 11 known compounds (4–14) were isolated from the fronds of Macaranga denticulata. Their structures were elucidated on the basis of extensive spectroscopic interpretation. Compounds 4 and 8 inhibited the proliferation of A-549 cell line with IC50 values of 48.6 and 20.2 μg/mL, respectively. Compounds 3, 6, and 8 exhibited significant antiangiogenic activity on a zebrafish model with IC50 values of 9.78, 0.34, and 2.55 μg/mL, respectively. © 2015 Published by Elsevier B.V.
N C
36 37
a b s t r a c t
U
34 35
i n f o
E
1 2
D
10
⁎ Corresponding authors. Tel./fax: +86 871 65223231. E-mail addresses: [email protected] (X.-L. Li), [email protected] (W.-L. Xiao). 1 These authors contributed equally to this work.
properties of these compounds are thought to reside in their enhanced interaction with biological membranes and increased affinity for target proteins [2]. The genus Macaranga (Euphorbiaceae), which is widely distributed in the tropical regions, is a rich source of prenylated flavonoids and stilbenes, and the biological activities of those metabolites encompass virtually all fields of pharmacological sciences [1,3]. In Chinese traditional medicine, the roots of Macaranga denticulata have been used for the treatment of icteric hepatitis. Previous studies on this plant have resulted in the isolation of prenylated flavonoids [4] and stilbene– diterpene heterodimers [5]. As a part of our ongoing research on structurally diverse and potentially anticancer prenylated aromatic products from genus Macaranga, one rare flavonoid– diterpene heterodimer, denticulatain C (1), one modified geranyl-type side chain substituted flavonoid, denticulatain D (2) and one geranylated flavonoid, denticulatain E (3), as well as 11 known compounds were isolated from the fronds of M. denticulata. Their cytotoxicity against A549 cancer cell line
http://dx.doi.org/10.1016/j.fitote.2015.04.001 0367-326X/© 2015 Published by Elsevier B.V.
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
44 45 46 47 48 49 50 51 52 Q6 53 54 55 56 57 58 59 60 61 62
80
98
Optical rotations were recorded using a JASCO P–1020 digital polarimeter. UV spectra were recorded using a Shimadzu UV-2401PC spectrophotometer. IR spectra were recorded on a Bruker Tenor 27 spectrophotometer with KBr pellets. 1D and 2D NMR experiments were recorded on Bruker AM-400 or DRX500 spectrometers with TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. ESI-MS were recorded using a Finnigan MAT 90 instrument and HR-ESI-MS was performed on an API QSTAR time-of-flight spectrometer. Column chromatography (CC) was performed on Sephadex LH-20 (Amersham Biosciences, Piscataway, USA), silica gel (200–300 mesh, Qingdao Marine Chemical inc., Qingdao, China), RP-18 gel (LiChroprep, 40–63 μm, Merck, Darmstadt, Germany), and MCI gel CHP20P (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Semipreparative HPLC was performed on an Agilent 1200 (column: Zorbax SB–C18, 250 × 9.4 mm, DAD detector). Fractions were monitored by TLC and visualized by heating plates sprayed with 15% H2SO4 in EtOH.
99
2.2. Plant material
100 101
104
The fronds of M. denticulata were collected from Xishuangbanna of Yunnan province, PR China, in March 2008. A voucher specimen (Yangyp–20080316) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, which was identified by Prof. Yong-Ping Yang.
105
2.3. Extraction and isolation
106 107
The air-dried and powdered fronds of M. denticulata (11 kg) were extracted with 90% EtOH (3 × 25 L) at room temperature and concentrated in vacuo to yield a residue, which was partitioned between H2O and EtOAc. The EtOAc portion was decolorized on MCI gel (eluting with 95% EtOH), and the residue (185 g) was subjected to silica gel CC with a gradient elution of CHCl3/acetone (10:0 to 3:7) to afford five fractions (Fr. A–E). Fr. A was purified by Sephadex LH-20 (CHCl3–MeOH, 1:1) and then subjected to CC over silica gel eluted with petroleum ether–EtOAc (1:0 to 2:1) to yield compounds 10 (3.1 mg) and 13 (4.3 mg). Fr. B was separated by Sephadex
93 94 95 96 97
102 103
108 109 110 111 112 113 114 115 116
C
91 92
E
89 90
R
87 88
R
85 86
O
83 84
C
81 82
N
74 75
U
72 73
2.4. Spectroscopic data
135
2.4.1. Denticulatain C (1) Yellow oil; [α]25 D +1.4 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 208 (4.64), 257 (4.36), 370 (4.38) nm; IR (KBr) νmax 3423, 2925, 2845, 1647, 1626, 1602, 1564, 1483, 1443, 1367, 1319, 1268, 1196, 1157, 1089, 1035, 959, 886, 812 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) data, see Table 1; positive ESIMS m/z 575 [M + H]+; HR-ESI-MS m/z 575.2995 [M + H]+ (calcd. for C35H43O7, 575.3008).
136 137
2.4.2. Denticulatain D (2) Yellow amorphous powder; [α]18 D −2.5 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 202 (4.88), 222 (4.79), 272 (4.51), 348 (4.02), 428 (4.48) nm; IR (KBr) νmax 3377, 2920, 1651, 1622, 1607, 1564, 1483, 1367, 1316, 1267, 1228, 1182, 1086, 1024, 894, 839, 805 cm−1; 1H NMR (acetone-d6, 500 MHz) and 13C NMR (acetone-d6, 100 MHz) data, see Table 1; negative ESIMS m/z 437 [M − H]−; HR-ESI-MS m/z 437.1597 [M − H]− (calcd. for C25H25O7, 437.1600).
144
2.4.3. Denticulatain E (3) Yellow oil; [α]18 D −4.0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 202 (4.72), 220 (4.72), 286 (4.15), 464 (3.82) nm; IR (KBr) νmax 3454, 2967, 2821, 1657, 1620, 1552, 1516, 1438, 1373, 1331, 1254, 1199, 1158, 1110, 1093, 1027, 824 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 125 MHz) data, see Table 1; negative ESIMS m/z 437 [M − H]−; HR-ESI-MS m/z 437.1598 [M − H]− (calcd. for C25H25O7, 437.1600).
153 154
2.5. Cytotoxicity assay
162
Compounds 1–14 were tested for their cytotoxicity against human lung cancer cell line A-549 by the MTT method, 5-FU was used as a positive control. Briefly, 100 μL of cell suspension (1 × 105 cells/mL) was seeded into 96well microtiter plates and cultured for 24 h before the compound was added. Then, different concentrations of the compounds were added to the plates, the cells were cultivated for 48 h, and 10 μL of MTT (5 mg/mL) was added to each well. After 4 h, the culture medium was removed and
163 164
F
2.1. General
70 71
O
79
68 69
R O
2. Experimental
67
117 118
P
78
65 66
LH-20 (CHCl3–MeOH, 1:1) to obtain 12 (13.2 mg) and a major fraction which was subjected to CC over silica gel eluted with petroleum ether–EtOAc (4:1 to 1:1), to give 4 (42.5 mg) and 5 (9.7 mg). Fr. C was purified over a Sephadex LH-20 (CHCl3 − MeOH, 1:1) and then fractionated by RP-18 with a gradient elution of MeOH–H2O (2:8 to 10:0) to yield subfractions C1–C6. Subfraction C1 was repeatedly purified by CC over silica gel eluted with petroleum ether–EtOAc (9:1 to 1:1) to afford 11 (21.6 mg) and 14 (7.4 mg). Compounds 1 (8.5 mg), 8 (10.7 mg) and 9 (9.2 mg) were isolated from subfraction C3 by repeated CC over silica gel eluted with petroleum ether–EtOAc (9:1 to 3:1). Fr. D was fractionated by Sephadex LH-20 (CHCl3–MeOH, 1:1) to yield subfractions D1–D3. Subfraction D1 was purified by semipreparative HPLC eluted by 87% MeOH–H2O, to afford 2 (12.8 mg) and 3 (10.3 mg). Subfraction D3 was purified by semipreparative HPLC eluted by 82% MeOH–H2O, to afford 6 (5.6 mg) and 7 (16.4 mg).
T
76 77
and the antiangiogenic activity against zebrafish embryos were tested. Herein, we describe the isolation, structural elucidation and anticancer effects of these compounds. Generally speaking, flavonoids are rarely substituted by isoprenoid residue longer than the geranyl [1], especially those isoprenoid residues with further intricate cyclization. The complex structures and sufficient biological activities of those compounds have attracted scientists' greater attention to study their structures, bioactivities, and synthesis. Previously studies have led to the isolation and characterization of the first example of flavonoid–diterpene heterodimer, denticulaflavonol [4], which have been further synthesized along with three other derivatives [6]. Thus, the discovery of denticulatain C (1), the second example flavonoid–diterpene heterodimer from nature has enriched the diversity of prenylated flavonoid class.
D
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D.-S. Yang et al. / Fitoterapia xxx (2015) xxx–xxx
E
2
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
119 120 121 122 123 Q7 124 125 126 127 128 129 130 131 132 133 134
138 139 140 141 142 143
145 146 147 148 149 150 151 152
155 156 157 158 159 160 161
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D.-S. Yang et al. / Fitoterapia xxx (2015) xxx–xxx
2
174 175 Q8
3″
42.6 t
4″ 5″ 6″
33.8 s 56.1 d 25.1 t
7″
38.5 t
8″ 9″
149.4 s 55.2 d
10″ 11″
39.8 s 21.9 t
12″
38.8 t
13″ 14″ 15″
135.6 s 123.5 d 21.9 t
16″ 17″
16.2 q 106.5 t
b c d
33.7 q 22.0 q 14.9 q
6.97 7.66 1.65 0.80 1.49 1.35 1.29 1.05
d (8.4) d (8.4) m m m m m m
0.85 1.61 1.18 2.23 1.88
m m m m m
75.2 d 149.3 s 110.3 t
m m m m t (7.2) m m s s s s s s s
the formazan crystals were completely dissolved with 150 μL DMSO in each well by vigorously shaking the plate. Finally, formazan absorbance was assessed by a BioRad microplate reader at 570 nm (Fig. 1).
2.6. Antiangiogenesis assay
177
Stock solutions (20 mg/mL) of all samples were prepared by dissolving the compounds 1–14 in DMSO. These solutions were diluted in sterile salt water (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM MgSO4) to obtain final solutions of various concentrations in 0.2% DMSO. Aliquots were placed into 24-well plates, and the embryos (TG[VEGFR2:GRCFP]) at
180 181 182
5.30 t (6.8)
6.24 d (1.7)
F
145.3 s 136.6 s 176.5 s 162.1 s 99.1 d 165.2 s 94.4 d 157.6 s 103.2 s 122.7 s 113.3 d 146.4 s 147.5 s 129.0 s 121.8 d 28.8 t
δHc
1.78 s 1.95 m 1.56 m 3.95 t (6.2)
4.84 s 4.69 s 1.65 s
123.2 d
6.44 d (1.7)
7.66 s
7.61 s 3.28, overlap 5.38 t (6.6)
136.6 s 16.2 q 40.4 t 27.4 t
1.74 s 2.04 m 2.11 m
125.0 d
5.09 t (5.7)
131.7 s 25.7 q
1.57 s
17.6 q
1.54 s
12.41 s
12.21 br s
Recorded at 400 MHz. Recorded at 500 MHz. Recorded at 125 MHz. Recorded at 100 MHz.
176
178 179
6.99 d (8.6) 8.12 d (8.6) 3.36 d (6.8)
16.3 q 36.4 t 34.5 t
17.8 q
5.25 3.42 3.29 1.78 4.74 4.47 0.73 0.71 0.61 12.44
8.12 d (8.6) 6.99 d (8.6)
136.6 s
1.54 m
1.52 1.38 2.04 1.75
6.59 s
122.9 d
N C
18″ 19″ 20″ 5-OH
7.79 s
s s s s s s d s s s d d s d d t
δCa
P
20.0 t
6.60 s
U
172 173
2″
146.7 135.5 176.5 158.9 111.7 162.7 93.8 155.6 104.0 123.4 130.4 116.3 160.0 116.3 130.4 21.9
d
D
a
s s s s s s d s s s d s s d d t
δH
T
t1:50 t1:51 t1:52 t1:53
148.2 136.7 176.5 158.8 112.0 162.6 93.8 155.5 104.0 123.8 115.6 145.7 146.6 116.1 121.3 39.5
3
δCb
R
2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″
O
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37 t1:38 t1:39 t1:40 t1:41 t1:42 t1:43 t1:44 t1:45 t1:46 t1:47 t1:48 t1:49
δHd
E
δCa
R O O
1
C
No.
E
t1:3
Table 1 1 H and 13C NMR data of compounds 1–3 in acetone-d6 (δ in ppm, J in Hz).
R
t1:1 Q1 t1:2
3
24 hpf (hours post-fertilization) were also transferred randomly into the above wells. Control embryos were treated with the equivalent amount of DMSO solutions. All embryos were incubated at 28.5 °C. After 48 h treatment, the intersegmental vessels of embryos were visualized with green fluorescent protein labeling and endogenous alkaline phosphatase staining. The antiangiogenic activities of compounds were calculated from the inhibition ratio of antiangiogenesis.
183 184
3. Results and discussion
191
185 186 187 188 189 190
Denticulatain C (1) was obtained as yellow oil. HR-ESI-MS 192 analysis of 1 provided a molecular formula of C35H42O7, 193
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
4
D.-S. Yang et al. / Fitoterapia xxx (2015) xxx–xxx
OH OH
3' 4'
2' 8
HO 7
10'' 9''
10''
8''
5''
7''
4''
OH O
17''
2'' 3''
2''
5'' 8''
7''
O
HO
OH
OH
5
16''
6''
9''
3''
1''
OH OH O
4''
6''
1*
18''
2*
OH
OH
OH O
OH OH O
OH O 4
3*
OH
R2 OH HO
O
O
O
R O
HO
F
H 19''
6'
3 4
6 15''
OH
5'
2
10
13''
1''
O 9
14''
12'' 11''
20''
1'
HO
O
OH
O
O
R
P
R1
OH O 8R=H 9 R = CH3
E
D
OH O 5 R1 = H, R2 = H; 6 R1 = OH, R2 = H 7 R1 = OH, R2 = OH
O
O
HO
C E R
O
R
HO
12
11 O
O HO
O O HO
O
201 202 203 204 205 206 207 208 209 210 211
C
O 199 200
N
197 198
O OH
O 14 Fig. 1. Structures of compounds 1–14.
corresponding to 15 unsaturation degrees. The IR spectrum showed typical absorption bands for OH (3423 cm−1), α,βunsaturated carbonyl (1647 cm−1) and aromatic ring (1602 and 1483 cm−1) moieties. The UV spectrum showed absorption maxima at λmax 257 (4.36) and 380 (4.38) nm, which indicated the presence of a flavonol skeleton [7]. The 1H and 13C NMR spectra of 1 showed the existence of C-6 substituted quercetin [δC 136.7 (s), 176.5 (s), 93.8 (d), 115.6 (d), 116.1 (d), 121.3 (d); δH 6.60 (1H, s), 6.97 (1H, d, J = 8.4 Hz), 7.66 (2H, d, J = 8.4 Hz), 7.79 (1H, s)] [7,8] and the remaining moiety possessed 20 carbons including one pair of terminal double bond [δC 106.5 (t), 149.4 (s); δH 4.47 (1H, s), 4.74 (1H, s)]; one pair of trisubstituted double bond [δC 123.5 (d), 135.6 (s)]; 4 tertiary methyls (δH 0.61, 0.71, 0.73, 1.78, 3H each, s); 8 methylenes; 2 methines and 2 quaternary carbons, which can be identified as a bicyclic labdane diterpenoid by comparing the NMR data with those of denticulaflavonol [4] and this deduction was confirmed by the observed 2D NMR correlations
U
195 196
O
O
13
194
H
OH
10
O
H
T
HO
H
(Fig. 2). The HMBC correlations of H-15″/C-5, C-6, C-7 and H-14″/C-6 demonstrated that labdanyl unit was connected to quercetin at C-6 of ring A. The observed ROESY (Fig. 3) correlations of H-5″/H-9″, Me-18″; Me-20″/H-11″, Me-19″; H-12″/H-14″; H-15″/H-16″ suggested that the stereochemistry of labdane skeleton in 1 was the same as denticulaflavonol [5]. Therefore, the structure of denticulatain C (1) was determined as 6-[(5″S,9″S,10″S)-15″-labd-8″(17″),13″(14″)-dienyl]quercetin, and it is the second example flavonoid–diterpene heterodimer from nature. Denticulatain D (2) was obtained as optically active amorphous powder ([α]18 D − 2.5, c 0.31, MeOH) and its molecular formula of C25H26O7 was established by HR-ESI-MS at m/z 437.1597 [M − H]−, requiring 13 degrees of unsaturation. Analysis of the 1H and 13C NMR (Table 1) data of 2 aided by HSQC revealed resonances for one 6-substituted kaempferol [δC 135.5 (s), 176.5 (s), 93.8 (d), 116.3 (d), 130.4 (d); δH 6.59 (1H, s), 6.99 (2H, d, J = 8.6 Hz), 8.12 (2H, d, J = 8.6 Hz)] [4,9],
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229
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5
OH OH HO
OH
O
HO
OH
7 6
14''
5
10''
OH
15''
8''
O 7 6
2''
7'' 6''
5 1''
OH O 1
2
F
OH
R O O
OH HO
2''
O
5' 6'
1''
OH 3
244 245 246 247 248 249 250 251 252 253 254 255 Q9
P
D
C
242 243
E
240 241
R
238 239
R
236 237
O
234 235
) correlations of compounds 1–3.
and H-2″/C-5′ indicated the location of the geranyl group at C-5′ of ring B. Accordingly, the structure of denticulatain E (3) was determined as 5′-geranylquercetin. The known compounds were identified as 5-hydroxy-2-(4hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-enyl)chroman4-one (4) [12], sophoraflavanone A (5) [13], bonanniol A (6) [14], diplacol (7) [15], 5,7,3′,4′-tetrahydroxy-6-geranylflavonol (8) [7], 5,7,3′,4′-tetrahydroxy-3-methoxy-6-geranylflavone (9) [7], 3βhydroxy-7α-ethoxy-24β-ethylcholest-5-ene (10) [16], (24R)6β-hydroxy-24-ethylcholest-4-en-3-one (11) [17], epitaraxerol (12) [18], α-tocopherolquinone (13) [19] and boehmenan (14) [20] by comparison of their experimental and reported spectroscopic data. All the compounds were isolated from M. denticulata for the first time. Since prenylated flavonoids are reported to have modest or strong anticancer activities [1,9], the cytotoxicity of compounds 1–14 were evaluated against human lung cancer cell line A549 by the MTT method [21], with 5-FU used as a positive control (IC50 22.48 μg/mL). The results showed that compounds 4 and 8 inhibit the proliferation of A-549 cell line with IC50 values of 48.6 and 20.2 μg/mL, respectively, and this is the first time to report the cytotoxicity of known compounds 4 and 8. The antiangiogenic activities of compounds 1–14 were further evaluated using a zebrafish model in terms of the inhibition on the growth of intersegmental vessels, using PTK787 as positive control (IC50 0.10 μg/mL) [22]. The results showed
N C
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one modified geranyl-type side chain (6-hydroxy-3,7-dimethyl2(E),7-octadienyl) [one pair of trisubstituted double bond (δC 122.9 d, 136.6 s); one pair of terminal double bond (δC 110.3 t, 149.3 s); one oxygenated methine (δC 75.2); three alkyl methylenes (δC 21.9, 34.5, 36.4) and two tertiary methyls (δH 1.65, 1.78, 3H each, s)] [10], which was confirmed by the observed 1H-1H COSY correlation of H-6″/H-7″ and the HMBC correlations of H-6″/C-8″; H-9″/C-7″, Me-10″; and Me-10″/C-7″. The HMBC correlations of H-1″/C-5, C-6, C-7 and H-2″/C-6 indicated the attachment of the modified geranyl-type side chain at C-6 of ring A. Thus, the structure of denticulatain D was determined as 6-[6-hydroxy-3,7dimethyl-2(E),7-octadienyl]kaempferol. The molecular formula of denticulatain E (3) was found to be C25H26O7, as deduced from HR-ESI-MS and NMR data, indicative of 13 indices of hydrogen deficiency. The strong IR absorptions implied the presence of hydroxy (3454 cm−1), conjugated carbonyl (1657 cm−1), and aromatic ring (1620 and 1438 cm−1) functionalities. The 1H and 13C NMR (Table 1) displayed characteristic signals corresponding to a ring Bsubstituted quercetin [δC 136.6 s, 176.5 s, 94.4 d, 99.1 d, 113.3 d, 121.8, d; δH 6.24 (1H, d, J = 1.7 Hz), 6.44 (1H, d, J = 1.7 Hz), 7.61 (s), 7.66 (s)] [11] and a geranyl substituent [three methyls (δC 16.2, 17.6, 25.7); three methylenes (δC 27.4, 28.8, 40.4); two pair of trisubstituted double bond (δC 123.2 d, 125.0 d, 131.7 s, 136.6 s)]. The observed HMBC correlations of H-1″/C-5′, C-6′,
U
230 231
) and 1H-1H COSY (
E
Fig. 2. Selected HMBC (
T
OH O
OH
OH O
9''
16'' 20''
11'' 9''
19''
12'' 14''
5''
15''
18'' Fig. 3. Key ROESY correlations of compound 1.
Please cite this article as: Yang D-S, et al, Three new prenylated flavonoids from Macaranga denticulata and their anticancer effects, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.04.001
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4. Conflict of interest
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The authors declare that there is no conflict of interest. Acknowledgments
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This study was financially supported by National Natural Science Foundation of China (31300293 and 81422046), General Project of Applied Foundation Research, Yunnan Province (2013FB067), Basic Research Project of Ministry of Science and Technology of China (2012FY110300), and Major State Basic Research Development Program (2010CB951704).
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.04.001.
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that intersegmental vessels of embryos treated with compounds 3, 6, and 8 were significantly fewer than those of the control (0.2% DMSO in sterile salt water) and the reduction was dose dependent with an IC50 value of 9.78, 0.34 and 2.55 μg/mL, respectively. The antiangiogenic activities of known compounds 6 and 8 were reported for the first time.
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