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Cytotoxic and antioxidant dihydrobenzofuran neolignans from the seeds of Crataegus pinnatifida
Fitoterapia 91 (2013) 217–223
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Cytotoxic and antioxidant dihydrobenzofuran neolignans from the seeds of Crataegus pinnatifida Xiao-Xiao Huang a,b,c, Chen-Chen Zhou a,c, Ling-Zhi Li a,b,c, Ying Peng d, Li-Li Lou e, Sen Liu a,c, Dian-Ming Li f, Takshi Ikejima g, Shao-Jiang Song a,b,c,⁎ a
Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, PR China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China c Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China d School of Pharmacy, Shenyang Pharmaceutical University, PR China e Harbin Pharmaceutical Group Co., LTD General Pharmaceutical Factory, Harbin 150046, PR China f Harbin Pharmaceutical Group Co., LTD Second Chinese Medicine Factory, Harbin 150078, PR China g China–Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, PR China b
a r t i c l e
i n f o
Article history: Received 16 July 2013 Accepted in revised form 10 September 2013 Available online 20 September 2013 Keywords: Crataegus pinnatifida Dihydrobenzofuran neolignans Cytotoxicities Antioxidant activities
a b s t r a c t Eight new dihydrobenzofuran neolignans, pinnatifidanin C I–VIII (1–8), together with two known analogs (9–10) were isolated from the seeds of Crataegus pinnatifida. Their structures were elucidated by spectroscopic analyses, especially 1D, 2D NMR and CD spectra. The cytotoxic activities of all isolates against human cancer cell lines were assayed, and most interestingly, compound 10 revealed preferred cytotoxicity on the HT-1080 cell line and displayed much stronger inhibitory activity (IC50 = 8.86 μM) compared with positive control 5-fluorouracil (IC50 = 35.62 μM). Meanwhile, antioxidant activities of all the isolates were evaluated using 2,2-diphenyl-1-pikrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, and the results showed that most of the isolates exhibited potent antioxidant activity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Crataegus pinnatifida Bge., locally called hawthorn, is a type of fruit-bearing plant commonly found in China, Europe and North America. The genus Crataegus is classified in the tribe Crataegeae, which belongs to the sub-family Maloideae of the Rosaceae family [1]. In traditional Chinese medicine (TCM), hawthorn fruits are primarily used to improve circulation, remove blood stasis and treat indigestion, hyperlipidemia and hypertension. In Europe, the fruits, leaves and flowers were traditionally used in the treatment of heart
⁎ Corresponding author at: School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, PR China. Tel.: +86 24 23986088; fax: +86 24 23986510. E-mail address: [email protected] (S.-J. Song). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.09.011
problems for their antispasmodic, cardiotonic, hypotensive and antiatherosclerotic effects. So far, more than 160 compounds have been isolated from Crataegus, including flavonoids, terpenes, lignans and organic acids [1–5]. Most of the previous phytochemical investigations of hawthorn have almost exclusively focused on the leaves [2,3], fruits [4] and flowers [5] and not on the seeds. Herein, we carried out the continued search for biologically active and structurally unique compounds, which resulted in the isolation of ten dihydrobenzofuran neolignans including eight new neolignans together with two known analogs from the seeds of hawthorn. This paper describes the structural elucidation of eight new isolated neolignans (1–8), named pinnatifidanin C I–VIII (Fig. 1), on the basis of 1D, 2D NMR, CD and HRESIMS data, as well as the cytotoxic and antioxidant activity evaluation of all isolates obtained.
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Fig. 1. Structures of compounds 1–10.
2. Experimental 2.1. General experimental procedures Optical rotations were obtained by using a JASCO DIP-370 digital polarimeter. The UV spectra were measured on a Shimadzu UV-1700 spectrometer. The spectra CD were obtained using MOS 450 detector from BioLogic. The FT-IR spectra were obtained on a Bruker IFS-55 spectrometer. 1H NMR, 13C NMR, HMBC, HSQC and NOESY spectra were recorded on Bruker ARX-300 and Bruker AV-600 spectrometers with TMS as an internal standard. HRESIMS experiments were performed on an Agilent G6520 Q-TOF spectrometer. The chromatographic silica gel (200–300 mesh) was produced from Qingdao Ocean Chemical Factory, and ODS (50 μm) was produced by YMC Co. Ltd. Sephadex LH-20 was produced by GE Healthcare. Macroporous adsorption resin D101 was produced by Cangzhou Bon Adsorber Technology Co., Ltd. Semipreparative RP-HPLC isolation was achieved with an Agilent 1100 instrument using YMC 5 μm C18 column (250 mm × 10 mm). Peak detection was made with a refractive index detector (RID). DPPH and ABTS assays were performed on Varioskan Flash Multimode Reader (Thermo scientific). 2.2. Plant material The seeds of hawthorn were collected from Shijiazhuang, Hebei province, PR China, in June 2011, and were identified by professor Jin-Cai Lu (Department of Natural Products Chemistry, Shenyang Pharmaceutical University, PR China.). A voucher specimen (No. 20110701) has been deposited in the Herbarium of Shenyang Pharmaceutical University, Liaoning, PR China. 2.3. Extraction and isolation The air-dried seeds of hawthorn (30 kg) were crushed to pieces and refluxed with 70% ethanol for 3 times × 30 L × 4 h.
The solvent was evaporated under vacuum. Then, the extract (1500 g) was suspended in H2O (20 L) and partitioned with ethyl acetate (3 × 20 L). The ethyl acetate extract (420 g) was suspended in H2O (5 L) and then chromatographed over D101 macroporous resin column (12 × 60 cm) using H2O–EtOH (from 100:0 to 5:95) as eluents. The H2O–EtOH (70:30) fraction (128 g) was subjected to silica gel CC (12 × 60 cm) and eluted with CH2Cl2–MeOH (from 100:0 to 50:50) to afford eight fractions (1–8). Fraction 4 (18.2 g) was further purified over an ODS CC (10 × 60 cm) using MeOH–H2O as the mobile phase with a gradient (from 95:5 to 50:50) to afford five fractions (F4-1–F4-5) based on HPLC analysis. F4-2 (4.1 g) was subjected to another silica gel column (2 × 30 cm) and eluted with CH2Cl2–MeOH (from 95:5 to 85:15) to afford eleven fractions (F4-2-1–F4-2-11) based on TLC analysis. F4-2-2 was subjected to semipreparative HPLC eluted with MeOH–H2O (42:58) to yield 1 (12 mg). F4-2-3 was subjected to semipreparative HPLC eluted with MeOH–H2O (30:70) to yield 2 (16 mg). F4-2-5 was subjected to semipreparative HPLC eluted with MeOH–H2O (32:68) to yield 3 (25 mg) and 4 (11 mg). F4-2-4 was subjected to semipreparative HPLC eluted with CH3CN–H2O (24:76) to yield 5 (10 mg) and 6 (7 mg). F4-2-7 was subjected to semipreparative HPLC eluted with CH3CN– H2O (20:80) to yield 7 (33 mg) and 8 (3 mg). F4-2-2 was subjected to semipreparative HPLC eluted with MeOH–H2O (42:58) to yield 9 (170 mg). F4-2-1 was subjected to ODS CC (1.5 × 25 cm) eluted with a gradient system (MeOH–H2O, 45:55, 1 L) to yield 10 (370 mg). Pinnatifidanin C I (1): yellow oil; [α]20 D − 6.3 (c = 0.03, MeOH); CD (MeOH) nm: 238 (3.66); UV (MeOH) λ max (logε): 230 (3.73), 278 (2.32) nm; IR (KBr) vmax: 3361, 2940, 1841, 1662, 1315, 1158, 1030, 898, 628 cm−1; HRESIMS m/z 397.1257 [M + Na]+ (calcd for C20H22O7Na, 397.1258); 1H and 13C NMR, see Tables 1 and 3. Pinnatifidanin C II (2): yellow oil; [α]20 D +11.2 (c = 0.04, MeOH); CD (MeOH) nm: 240 (−4.50); UV (MeOH) λ max (logε): 229 (3.60), 279 (2.16) nm; IR (KBr) vmax: 3366, 2937,
X.-X. Huang et al. / Fitoterapia 91 (2013) 217–223
1688, 1594, 1323, 1164, 1111, 1026, 898 cm−1; HRESIMS m/z 413.1205 [M + Na]+ (calcd for C20H22O8Na, 413.1207); 1H and 13C NMR, see Tables 1 and 3. Pinnatifidanin C III (3): yellow oil; [α]20 D −21.2 (c = 0.08, MeOH); CD (MeOH) nm: 242 (3.44); UV (MeOH) λ max (logε): 228 (3.32), 280 (2.01) nm; IR (KBr) vmax: 3415, 2934, 1606, 1518, 1275, 1117, 855, 819, 775 cm−1; HRESIMS m/z 429.1510 [M + Na]+ (calcd for C21H26O8Na, 429.1520); 1H and 13C NMR see Tables 1 and 3; 1H NMR (CDCl3, 600 MHz), see Supplementary data. Pinnatifidanin C IV (4): yellow oil; [α]20 D +10.2 (c = 0.03, MeOH); CD (MeOH) nm: 240 (−3.89); UV (MeOH) λ max (logε): 230 (3.12), 278 (2.45) nm; IR (KBr) vmax: 3434, 2932, 1620, 1519, 1383, 1116, 1032, 855, 620 cm−1; HRESIMS m/z 429.1510 [M + Na]+ (calcd for C21H26O8Na, 429.1520); 1H and 13 C NMR see Tables 1 and 3; 1H NMR (CDCl3, 600 MHz), see Supplementary data. Pinnatifidanin C V (5): yellow oil; [α]20 D −8.5 (c = 0.02, MeOH); CD (MeOH) nm: 239 (3.99); UV (MeOH) λ max (logε): 229 (3.71), 280 (2.81) nm; IR (KBr) vmax: 3436, 2853, 1627, 1384, 1121, 1035, 619 cm− 1; HRESIMS m/z 443.1666 [M + Na]+ (calcd for C22H28O8Na, 443.1676); 1H and 13C NMR, see Tables 2 and 3. Pinnatifidanin C VI (6): yellow oil; [α]20 D +7.0 (c = 0.02, MeOH); CD (MeOH) nm: 239 (−2.87); UV (MeOH) λ max (logε): 228 (2.40), 280 (1.66) nm; IR (KBr) vmax: 3384, 2936, 1606, 1518, 1276, 1125, 1032, 855, 819, 740, 636 cm−1; HRESIMS m/z 433.1678 [M + Na]+ (calcd for C22H28O8Na, 433.1676); 1H and 13C NMR, see Tables 2 and 3. Pinnatifidanin C VII (7): yellow oil; [α]20 D +12.5 (c = 0.06, MeOH); CD (MeOH) nm: 241 (−4.87); UV (MeOH) λ max (logε): 228 (2.63), 278 (1.75) nm; IR (KBr) vmax: 3368, 2936, 1603, 1513, 1269, 1032, 820, 785, 641 cm−1; HRESIMS m/z 427.1728 [M + Na]+ (calcd for C22H28O7Na, 427.1727); 1H and 13 C NMR, see Tables 2 and 3.
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Pinnatifidanin C VIII (8): yellow oil; [α]20 D +6.5 (c = 0.02, MeOH); CD (MeOH) nm: 238 (−3.15); UV (MeOH) λ max (logε): 227 (3.42), 281 (2.46) nm; IR (KBr) vmax: 3385, 2936, 1512, 1270, 1032, 858, 819, 640 cm−1; HRESIMS m/z 341.0998 [M + Na]+ (calcd for C17H18O6Na, 341.0996); 1H and 13C NMR date, see Tables 2 and 3. 2.4. Cytotoxic activity [6] In order to measure the cytotoxic activity, each tested compound and 5-fluorouracil (positive control) were dissolved in DMSO and diluted with the medium to the test concentrations. Briefly, cells were cultured at 37 °C and dispersed in replicates in 96-well plates with HeLa, HepG2, HT-1080, A375-S2, MCF-7, U-937, K562 and HL60 for 24 h. Fresh medium with compounds at different concentrations was then added to individual wells and incubated for 48 h, with 5-fluorouracil as the positive control. After 48 h, the cell was incubated with MTT solution (0.5 mg/mL) for an additional 4 h at 37 °C. The produced formazan crystals were solubilized with DMSO and the optical density of solution was measured at 492 nm using a Spectra Shell reader (Tecan, Austria). 2.5. Antioxidant activity 2.5.1. DPPH radical scavenging activity [7,8] Radical scavenging activity of 1–10 and vitamin E (positive control) was determined using DPPH as a reagent with modification by using 96-well plates. A 0.1 mM solution of DPPH radical in ethanol was prepared and then 100 μL of this solution was mixed with 100 μL of sample solution. The mixture was incubated for 30 min in a dark room at room temperature. Scavenging capacity was read spectrophotometrically by monitoring the decrease in absorbance at 517 nm. The percentage of scavenged DPPH was calculated
Table 1 1 H NMR data of compounds 1–4. No. 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3-OCH3 3′-OCH3 7′-OCH3 1
11,a
22,a
32,b
42,b
6.92 (1H, br.s)
6.93 (1H, br.s)
6.98 (1H, d, 1.9)
6.96 (1H, d, 1.8)
6.77 (1H, br.s)
6.77 (1H, br.s)
6.77 (1H, br.s) 5.58 (1H, d, 6.8) 3.53–3.66 (1H, m) 3.70–3.73 (2H, m)
6.77 (1H, br.s) 5.61 (1H, d, 6.8) 3.55–3.58 (1H, m) 3.62–3.69 (2H, m)
6.79 (1H, d, 8.1) 6.86 (1H, dd, 8.1, 1.9) 5.55 (1H, d, 6.4) 3.31–3.25 (1H, m) 3.78–3.85 (2H, m)
6.77 (1H, d, 8.1) 6.84 (1H, dd, 8.1, 1.8) 5.53 (1H, d, 6.4) 3.30–3.24 (1H, m) 3.76–3.84 (2H, m)
7.44 (1H, br.s)
7.50 (1H, br.s)
6.89 (1H, br.s)
6.88 (1H, br.s)
7.60 (1H, br.s)
7.64 (1H, br.s)
3.09 3.77 3.84 3.74
3.56–3.62 (1H, m) 3.69–3.73 (2H, m) 3.75 (3H, s) 3.84 (3H, s)
6.89 (1H, br.s) 4.14 (1H, d, 6.6) 3.67–3.73 (1H, m) 3.47–3.56 (2H, m) 3.83 (3H, s) 3.89 (3H, s) 3.27 (3H, s)
6.88 (1H, br.s) 4.15 (1H, dd, 6.4) 3.66–3.74 (1H, m) 3.46–3.56 (2H, m) 3.82 (3H, s) 3.88 (3H, s) 3.26 (3H, s)
(2H, (2H, (3H, (3H,
t, 6.4) t, 6.4) s) s)
for 600 MHz, 2 for 300 MHz. Coupling constants (J) in Hz are given in parentheses; chemical shift values are expressed in ppm. a Measured in DMSO-d6. b Measured in CD3OD.
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Table 2 1 H NMR data of compounds 5–8. No. 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′
62,b
71,b
82,a
6.98 (1H, d, 1.8)
6.96 (1H, d, 1.8)
6.96 (1H, d, 1.8)
6.89 (1H, br.s)
6.79 (1H, d, 8.2) 6.85 (1H, dd, 8.2, 1.8) 5.55 (1H, d, 6.4) 3.50–3.52 (1H, m) 3.75–3.84 (2H, m)
6.77 (1H, d, 8.1) 6.84 (1H, dd, 8.1, 1.8) 5.53 (1H, d, 6.5) 3.50–3.52 (1H, m) 3.75–3.84 (2H, m)
6.77 (1H, d, 8.1) 6.84 (1H, dd, 8.1, 1.8) 5.52 (1H, d, 6.5) 3.48–3.52 (1H, m) 3.76–3.82 (2H, m)
6.74 (1H, br.s) 5.32 (1H, d, 6.8) 3.20–3.40 (1H, m)c 3.60–3.70 (1H, m)
6.90 (1H, br.s)
6.88 (1H, br.s)
6.84 (1H, br.s)
6.28 (1H, d, 1.9)
6.88 (1H, br.s) 4.27 (1H, d, 6.4) 3.66–3.69 (1H, m)
6.88 (1H, br.s) 4.26(1H, d, 6.2) 3.66–3.69 (1H, m)
6.28 (1H, d, 1.9)
3.48–3.51 (2H, m) 3.43–3.46 (2H, m) 1.20 (3H, t, 7.0) 3.83 (3H, s) 3.88 (3H, s)
3.47–3.51 (2H, m) 3.43–3.46 (2H, m) 1.18 (3H, t, 7.0) 3.82 (3H, s) 3.87 (3H, s)
6.84 (1H, br.s) 4.39 (1H, dd, 8.3, 5.4) 1.96–2.02 (1H, m) 1.76–1.81 (1H, m) 3.62–3.66 (2H, m) 3.33–3.35 (2H, m) 1.16 (3H, t, 7.0) 3.82 (3H, s) 3.87 (3H, s)
6.74 (1H, br.s)
9′ 1″ 2″ 3-OCH3 3′-OCH3 1
52,b
3.74 (3H, s) 3.71 (3H, s)
for 600 MHz, 2 for 300 MHz. Coupling constants (J) in Hz are given in parentheses; chemical shift values are expressed in ppm. a Measured in DMSO-d6. b Measured in CD3OD. c Overlapped signals.
using the following equation: DPPH scavenging activity (%) = [1 − (S − SB) / (C − CB)] × 100% where S, SB, C and CB are the absorbencies of the sample, the blank sample, the control and the blank control, respectively.
Table 3 13 C NMR data (150 MHz) of compounds 1–8. No.
1a
2a
3b
4b
5b
6b
7b
8a
1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3-OCH3 3′-OCH3 7′-OCH3
131.6 110.5 147.7 115.4 146.8 118.6 88.4 52.3 62.7 129.7 112.0 143.6 152.0 131.0 118.8 197.3 41.2 57.2
131.6 110.5 147.6 115.4 146.8 118.7 88.4 52.3 62.6 129.0 112.8 143.5 152.2 129.6 118.9 198.4 74.0 64.3
133.6 110.5 149.2 147.5 116.1 119.7 89.2 55.2 64.7 134.5 112.8 145.5 149.1 130.1 117.4 85.5 77.1 63.9
133.6 110.5 149.2 147.5 116.1 119.7 89.2 55.2 64.6 134.4 112.8 145.5 149.0 130.1 117.3 85.5 77.1 63.9
55.7 55.8
56.3 56.7 57.1
56.3 56.7 57.1
134.5 110.6 149.1 147.6 116.2 119.8 89.2 55.3 64.7 134.4 112.9 145.5 149.2 130.1 117.2 83.6 77.2 64.0 65.4 15.6 56.4 56.7
134.6 110.5 149.1 147.6 116.3 119.8 89.2 55.3 65.0 137.5 112.0 145.5 148.9 130.0 116.1 80.3 42.3 59.9 64.9 15.6 56.4 56.7
132.7 110.3 147.5 115.2 146.2 118.5 86.3 53.6 63.0 151.9 100.1 143.6 140.2 129.2 103.1
55.7 55.7
134.5 110.5 149.1 147.5 116.1 119.7 89.2 55.3 64.8 134.3 112.9 145.5 149.1 130.0 117.3 83.6 77.1 63.9 65.4 15.6 56.4 56.7
a b
Measured in DMSO-d6. Measured in CD3OD.
55.6 55.5
2.5.2. ABTS radical scavenging activity [9] The radical scavenging activity of the isolated compounds was carried out using an improved ABTS decolorization assay with some modification. ABTS radical cation (ABTS•+) was produced by reacting 7 mM stock solution of ABTS with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. The ABTS•+ solution was diluted with ethanol, to an absorbance of 0.7 ± 0.02 at 734 nm. An ethanolic solution (50 μL) of the samples at various concentrations was mixed with 150 μL diluted ABTS•+ solution. After reaction at room temperature for 20 min, the absorbance at 734 nm was measured. Lower absorbance of the reaction mixture indicates higher ABTS•+ scavenging activity. The capability to scavenge the ABTS•+ was calculated using the formula given below: ABTS•+ scavenging activity (%) = [1 − (S − SB) / (C − CB)] × 100% where S, SB, C and CB are the absorbencies of the sample, the blank sample, the control, and the blank control, respectively. 3. Results and discussion Compound 1 was obtained as yellow oil. The molecular formula of 1 was determined as C20H22O7 from its positive HRESIMS ion at m/z 397.1257 [M + Na]+ (calcd for C20H22O7Na, 397.1258). The 1H NMR spectrum of 1 showed the presence of a 1,3,5-trisubstituted benzene ring [δH: 6.92 (1H, br.s), 6.77 (1H, br.s) and 6.77 (1H, br.s)], a 1,3,4,5tetrasubstituted benzene ring [δH: 7.44 (1H, br.s) and 7.60 (1H, br.s)], two methoxyl group protons attached to the aromatic ring at δ 3.84 (3H, s) and 3.74 (3H, s) compared with the literature [10]. A sequence of oxygenate methine–
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Fig. 2. Observed key HMBC correlations of compounds 1, 2, 3, 5, 7 and 8.
methine–oxygenate methylene proton signals at δ 5.58 (1H, d, J = 6.8 Hz), 3.53–3.66 (1H, m) and 3.70–3.73 (2H, m) indicated that 1 had a dihydrobenzofuran neolignan skeleton in combination with its 13C NMR spectrum data. The 13C NMR spectrum of 1 (Table 3) showed twenty carbon signals. Aside from the carbon signals from the two methoxy groups, the remaining eighteen carbon signals supported the presence of twelve aromatic, a carbonyl and five aliphatic carbons. The HMBC correlations (Fig. 2) of H-8′ at δ 3.09 with C-7′, C-9′ and of H-9′ at δ 3.77 with C-7′ and C-9′ confirmed that compound 1 possessed a 3-hydroxy-1-phenylpropan-1-one unit. Meanwhile, the positions of the two methoxyls were confirmed by the HMBC correlations. A relative-trans configuration was determined by the coupling constant J7,8 (6.8 Hz) in accordance with literature reports [11,12]. The CD spectrum showed a positive Cotton effect (Δε238 3.66), so it was found that compound 1 had the 7S,8R-configuration [12] and named pinnatifidanin C I. Compound 2 had the molecular formula C20H22O8, as determined by the HRESIMS at m/z 413.1205 [M + Na]+ (calcd for C20H22O8Na, 413.1207). Its 13C NMR spectroscopic data were similar to those of 1 except for the reductive proton and the obvious downfield shift of C-8′ (+ 32.8 ppm) and C-9′ (+ 7.1 ppm), indicating that one proton of H-8′ was substituted by a hydroxyl group. The coupling constant J7,8 (6.8 Hz) in the 1H NMR of 2 suggested a relative-trans configuration [11,12]. The CD spectrum showed a negative Cotton effect at 240 nm, so it was found that compound 2 had the 7R,8S-configuration [12]. Thus, the structure of 2 was established as shown in Fig. 1 and named pinnatifidanin C II. Compound 3 was obtained as yellow oil with the molecular formula C21H26O8, based on quasi-molecular ion m/z 429.1510 [M + Na]+ (calcd for C21H26O8Na, 429.1520) from HRESIMS
mass spectral analysis. The 1H NMR spectra showed the presence of a 1,3,4-trisubstituted benzene ring [δH: 6.98 (1H, d, J = 1.9 Hz), 6.79 (1H, d, J = 8.1 Hz) and 6.86 (1H, dd, J = 1.9, 8.1 Hz)] and a 1,3,4,5-tetrasubstituted benzene ring [δH: 6.89 (1H, br.s) and 6.89 (1H, br.s)]. The 13C NMR spectrum of 3 showed twenty one carbon signals. Aside from the carbon signals from the three methoxy groups, the remaining eighteen carbon signals supported the presence of twelve aromatic and six aliphatic carbons. Careful comparison of the NMR data of 3 with those of 1 and 2 revealed that compound 3 is a dihydrobenzofuran neolignan. The positions of the three methoxyls were confirmed by the HMBC correlations (Fig. 2). The coupling constant J7,8 (6.4 Hz) in the 1H NMR of 3 suggested a relative-trans configuration [11,12]. Meanwhile the coupling constant between H-7′ and H-8′ (J = 6.6 Hz in CD3OD and J = 8.1 Hz in CDCl3) suggested a threo conformation of C-7′/C-8′ [13,14]. The CD spectrum showed a positive Cotton effect (Δε242 3.44) which suggested a 7S,8R-configuration [12]. Based on the above evidence, the structure of compound 3 was unambiguously established as shown in Fig. 1 and named pinnatifidanin C III. Compound 4 was proposed to have the molecular formula C21H26O8 based on HRESIMS. The 1H NMR and 13C NMR spectroscopic data were in good agreement with those of 3, suggesting that the planar structure of 4 was the same as that of compound 3. The coupling constant J7,8 (6.4 Hz) in the 1H NMR of 4 suggested a relative-trans configuration [11,12]. The coupling constant J7′,8′ (6.4 Hz in CD3OD and 7.9 Hz in CDCl3) in the 1H NMR of 4 suggested a relative-threo configuration [13,14]. The CD spectrum showed a negative Cotton effect (Δε240 − 3.89), which indicated the absolute configurations of 4 to be 7R,8S-configuration [12]. Thus, compound 4 was defined as an optical isomer of 3 and given the trivial name pinnatifidanin C IV.
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The 1H NMR spectroscopic data indicated the presence of a benzofuran-type lignan moiety [δH 5.32 (1H, d, J = 6.8 Hz), 3.20–3.40 (1H, m) and 3.60–3.70 (2H, m)], which was supported by resonances in the 13C NMR spectrum (δC 86.3, 53.6, 63.0). Furthermore, its 1H NMR showed the presence of a 1,3,5-trisubstituted benzene ring [δH 6.89 (1H, s), 6.74 (1H, br.s) and 6.74 (1H, br.s)], a 1,3,4,5-tetrasubstituted benzene ring [δH 6.28 (1H, d, J = 1.9 Hz) and 6.28 (1H, d, J = 1.9 Hz)] and two methoxyl protons attached to the aromatic ring at δ 3.74 (3H, s) and 3.71 (3H, s). According to the HMBC spectrum, the above fragments were connected as shown in Fig. 2. Since the coupling constant of H-7 was 6.8 Hz, the relative configuration of C-7 and C-8 was regarded as trans-configuration [11,12]. The CD spectrum showed negative Cotton effects at 238 nm, so it was found that compound 8 had the 7R,8S-configuration [12] and named pinnatifidanin C VIII. By comparing physical and spectroscopic data with literatures, the two known constituents were identified as 7R,8S-dihydrodehydroconiferyl alcohol (9) [15] and 7R,8Sbalanophonin (10) [16]. Many types of biological activity have been reported on dihydrobenzofuran neolignan. In previous investigations, dihydrobenzofuran neolignans had served as a lead compound for antitumoral agents, and their structure–activity relationships were studied [17–24]. It was reported that the double bond in the C-7′/8′ side chain was the antitumor activity group of dihydrobenzofuran neolignans and the reduction of the double bond caused a greater than ten-fold decrease of activity [17,18,24]. All isolated compounds were assayed for their in vitro cytotoxicity against three human cancer cell lines, including A375-S2, HL60 and K562 using the MTT method, and only compound 10 displayed potent cytotoxic activities. Then, compound 10 was evaluated against other five types of human cancer cell lines, including HeLa, HepG2, HT-1080, MCF-7 and U-937 by MTT method. 5-Fluorouracil was used as the reference drug. The results of cell viability inhibition were shown in Table 4. From the above-mentioned evidence, we also conclude that the presence of double bond in C-7′/C-8′ next to the aromatic ring (π–π conjugation) may increase the cytotoxic activity of dihydrobenzofuran neolignans. The
Compound 5 was isolated as yellow oil. Its HRESIMS spectrum showed the quasi-molecular ion m/z [M + Na]+ 443.1666 (calcd for C22H28O8Na, 443.1676) consistent with the molecular formula C22H28O8. Its 1H NMR and 13C NMR spectroscopic data were similar to those of 3 except for the reduction of a methoxy group and the addition of an ethoxyl group in 5 (Fig. 1). The coupling constant J7,8 (6.4 Hz) in the 1H NMR of 5 suggested a relative-trans configuration [11,12], and the coupling constant between H-7′ and H-8′ (J = 6.4 Hz in CD3OD) suggested a threo conformation of C-7′/C-8′ [13,14]. Meanwhile, the CD spectrum showed a positive Cotton effect (Δε239 3.99), so it was found that compound 5 had the 7S,8Rconfiguration [12]. Accordingly, 5 was proposed to be a new neolignan and had been accorded the trivial name pinnatifidanin C V. Compound 6 was assigned as C22H28O8 from the HRESIMS, and its 1H NMR and 13C NMR spectroscopic data were identical with those of 6, suggesting that the planar structure of 6 was the same as that of 5. A trans configuration of 6 was confirmed by the J7,8 value of 6.5 Hz in the 1H NMR spectrum [11,12]. Meanwhile, the coupling constant between H-7′ and H-8′ (J = 6.2 Hz in CD3OD) indicated that 6 had the threo configuration between C-7′ and C-8′ [13,14]. Furthermore, the CD cotton curve of 6 was opposite to that of 5, which indicated that the configuration of dihydrofuran ring was 7R,8S [12]. Thus, compound 6 was defined as an optical isomer of 5 and named pinnatifidanin C VI. Compound 7 had the molecular formula C22H28O7, as indicated by HRESIMS combined with the NMR data. Its 13C NMR spectroscopic data were similar to those of 5 except for the reductive proton and the obvious chemical shift of C-8′ (-34.9 ppm) and C-9′ (− 4.1 ppm), indicating that 8′-OH was substituted by a hydrogen proton (Fig. 1). The relative configuration of H-7 and H-8 was determined as trans by the coupling constant J7,8 (6.5 Hz) in the 1H NMR [11,12]. The CD spectrum showed a negative Cotton effect (Δε241 −4.87), which indicated that the configuration of 7 was 7R,8S-configuration [12]. Thus, the structure of 7 was established as shown in Fig. 1 and named pinnatifidanin C VII. Compound 8 was obtained as a light yellow oil, and the molecular formula of 8 was deduced as C17H18O6 by HRESIMS.
Table 4 Inhibition effects on the growth of tumor cells in vitro (IC50, μM). IC50 (μM) Compound
1 2 3 4 5 6 7 8 9 10 5-Fua
Cell line HeLa
MCF-7
HepG2
A375-S2
HT1080
HL60
U937
K562
– – – – – – – – – 46.68 7.34
– – – – – – – – – N50 11.50
– – – – – – – – – 30.96 40.34
N50 N50 N50 N50 N50 N50 N50 N50 N50 30.54 6.67
– – – – – – – – – 8.86 35.62
N50 N50 N50 N50 N50 N50 N50 N50 N50 36.71 10.05
– – – – – – – – – N50 23.93
N50 N50 N50 N50 N50 N50 N50 N50 N50 N50 23.55
“–” Means not tested. a 5-Fu (5-fluorouracil) was used as positive control.
X.-X. Huang et al. / Fitoterapia 91 (2013) 217–223
Appendix A. Supplementary data
Table 5 Free radical scavenging activity of compounds 1–10. Compound
DPPH (IC50, μg/mL)
ABTS (IC50, μg/mL)
1 2 3 4 5 6 7 8 9 10 Vitamin Ea
149.20 165.89 160.98 127.90 140.31 158.97 144.30 N200 98.65 137.09 9.96
13.22 15.45 10.37 13.02 10.58 11.62 13.78 23.58 7.29 24.90 9.30
a
223
Vitamin E was used as positive control.
results of the cytotoxicity of isolates in our study may support the viewpoint what Apers et al. reported [24]. Particularly, compound 10 revealed preferred cytotoxicity against the HT-1080 cell line and displayed much stronger inhibitory activity (IC50 = 8.86 μM) compared with positive control 5-fluorouracil (IC50 = 35.62 μM). The antioxidant effects of the isolates were assessed using the DPPH and ATBS assays (Table 5). All isolates (IC50 N 90 μg/mL) were found to be less active than the positive control vitamin E (IC50 = 9.96 μg/mL) in the DPPH assay. But in the ATBS assay, most of the dihydrobenzofuran neolignans showed significant activity of IC50 b 15 μg/mL, which were comparable to the positive control vitamin E (IC50 = 9.30 μg/mL). It has been reported that the antioxidant activity is correlated to the number of free phenolic hydroxyl groups, and the antioxidant activity may dramatically decrease as led by the absence or blocking of the hydroxyl groups by methyl group [25,26]. In our study, compound 9 showed the strongest antioxidant activities (IC50 = 98.65 μg/mL in the DPPH assay and IC50 = 7.29 μg/mL in the ATBS assay) among all the tested compounds, by comparison, compound 8 exhibited significantly reduced antioxidant activities (IC50 N 200 μg/mL in the DPPH assay and IC50 = 23.58 μg/mL in the ATBS assay), suggesting that the antioxidant activity of this type of neolignan was probably related to the 3-phenylpropan-1-ol unit. In consideration of the structures of neolignans (1–10), we found that the loss of the side chain and the existence of hydroxyl group, methyl group or carbonyl group in the side chain (in the C-7′ or C-8′) may cause the reduction of antioxidant activity of dihydrobenzofuran neolignans. Acknowledgments Financial supported by the Program for State Key Laboratory of Bioactive Substance and Function of Natural Medicines (GTZX201208), the Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University, the Scientific Research Starting Foundation (20121106) for Doctors of Liaoning province of PR China and the Foundation (L2012358) from the Project of Education Department of Liaoning province of PR China are gratefully acknowledged. Authors thank Li W. and Sha Y. of Shenyang Pharmaceutical University for the supporting recording of NMR spectra.
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Cytotoxic and antioxidant dihydrobenzofuran neolignans from the seeds of Crataegus pinnatifida Xiao-Xiao Huang a,b,c, Chen-Chen Zhou a,c, Ling-Zhi Li a,b,c, Ying Peng d, Li-Li Lou e, Sen Liu a,c, Dian-Ming Li f, Takshi Ikejima g, Shao-Jiang Song a,b,c,⁎ a
Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, PR China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China c Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China d School of Pharmacy, Shenyang Pharmaceutical University, PR China e Harbin Pharmaceutical Group Co., LTD General Pharmaceutical Factory, Harbin 150046, PR China f Harbin Pharmaceutical Group Co., LTD Second Chinese Medicine Factory, Harbin 150078, PR China g China–Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, PR China b
a r t i c l e
i n f o
Article history: Received 16 July 2013 Accepted in revised form 10 September 2013 Available online 20 September 2013 Keywords: Crataegus pinnatifida Dihydrobenzofuran neolignans Cytotoxicities Antioxidant activities
a b s t r a c t Eight new dihydrobenzofuran neolignans, pinnatifidanin C I–VIII (1–8), together with two known analogs (9–10) were isolated from the seeds of Crataegus pinnatifida. Their structures were elucidated by spectroscopic analyses, especially 1D, 2D NMR and CD spectra. The cytotoxic activities of all isolates against human cancer cell lines were assayed, and most interestingly, compound 10 revealed preferred cytotoxicity on the HT-1080 cell line and displayed much stronger inhibitory activity (IC50 = 8.86 μM) compared with positive control 5-fluorouracil (IC50 = 35.62 μM). Meanwhile, antioxidant activities of all the isolates were evaluated using 2,2-diphenyl-1-pikrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, and the results showed that most of the isolates exhibited potent antioxidant activity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Crataegus pinnatifida Bge., locally called hawthorn, is a type of fruit-bearing plant commonly found in China, Europe and North America. The genus Crataegus is classified in the tribe Crataegeae, which belongs to the sub-family Maloideae of the Rosaceae family [1]. In traditional Chinese medicine (TCM), hawthorn fruits are primarily used to improve circulation, remove blood stasis and treat indigestion, hyperlipidemia and hypertension. In Europe, the fruits, leaves and flowers were traditionally used in the treatment of heart
⁎ Corresponding author at: School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, PR China. Tel.: +86 24 23986088; fax: +86 24 23986510. E-mail address: [email protected] (S.-J. Song). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.09.011
problems for their antispasmodic, cardiotonic, hypotensive and antiatherosclerotic effects. So far, more than 160 compounds have been isolated from Crataegus, including flavonoids, terpenes, lignans and organic acids [1–5]. Most of the previous phytochemical investigations of hawthorn have almost exclusively focused on the leaves [2,3], fruits [4] and flowers [5] and not on the seeds. Herein, we carried out the continued search for biologically active and structurally unique compounds, which resulted in the isolation of ten dihydrobenzofuran neolignans including eight new neolignans together with two known analogs from the seeds of hawthorn. This paper describes the structural elucidation of eight new isolated neolignans (1–8), named pinnatifidanin C I–VIII (Fig. 1), on the basis of 1D, 2D NMR, CD and HRESIMS data, as well as the cytotoxic and antioxidant activity evaluation of all isolates obtained.
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X.-X. Huang et al. / Fitoterapia 91 (2013) 217–223
Fig. 1. Structures of compounds 1–10.
2. Experimental 2.1. General experimental procedures Optical rotations were obtained by using a JASCO DIP-370 digital polarimeter. The UV spectra were measured on a Shimadzu UV-1700 spectrometer. The spectra CD were obtained using MOS 450 detector from BioLogic. The FT-IR spectra were obtained on a Bruker IFS-55 spectrometer. 1H NMR, 13C NMR, HMBC, HSQC and NOESY spectra were recorded on Bruker ARX-300 and Bruker AV-600 spectrometers with TMS as an internal standard. HRESIMS experiments were performed on an Agilent G6520 Q-TOF spectrometer. The chromatographic silica gel (200–300 mesh) was produced from Qingdao Ocean Chemical Factory, and ODS (50 μm) was produced by YMC Co. Ltd. Sephadex LH-20 was produced by GE Healthcare. Macroporous adsorption resin D101 was produced by Cangzhou Bon Adsorber Technology Co., Ltd. Semipreparative RP-HPLC isolation was achieved with an Agilent 1100 instrument using YMC 5 μm C18 column (250 mm × 10 mm). Peak detection was made with a refractive index detector (RID). DPPH and ABTS assays were performed on Varioskan Flash Multimode Reader (Thermo scientific). 2.2. Plant material The seeds of hawthorn were collected from Shijiazhuang, Hebei province, PR China, in June 2011, and were identified by professor Jin-Cai Lu (Department of Natural Products Chemistry, Shenyang Pharmaceutical University, PR China.). A voucher specimen (No. 20110701) has been deposited in the Herbarium of Shenyang Pharmaceutical University, Liaoning, PR China. 2.3. Extraction and isolation The air-dried seeds of hawthorn (30 kg) were crushed to pieces and refluxed with 70% ethanol for 3 times × 30 L × 4 h.
The solvent was evaporated under vacuum. Then, the extract (1500 g) was suspended in H2O (20 L) and partitioned with ethyl acetate (3 × 20 L). The ethyl acetate extract (420 g) was suspended in H2O (5 L) and then chromatographed over D101 macroporous resin column (12 × 60 cm) using H2O–EtOH (from 100:0 to 5:95) as eluents. The H2O–EtOH (70:30) fraction (128 g) was subjected to silica gel CC (12 × 60 cm) and eluted with CH2Cl2–MeOH (from 100:0 to 50:50) to afford eight fractions (1–8). Fraction 4 (18.2 g) was further purified over an ODS CC (10 × 60 cm) using MeOH–H2O as the mobile phase with a gradient (from 95:5 to 50:50) to afford five fractions (F4-1–F4-5) based on HPLC analysis. F4-2 (4.1 g) was subjected to another silica gel column (2 × 30 cm) and eluted with CH2Cl2–MeOH (from 95:5 to 85:15) to afford eleven fractions (F4-2-1–F4-2-11) based on TLC analysis. F4-2-2 was subjected to semipreparative HPLC eluted with MeOH–H2O (42:58) to yield 1 (12 mg). F4-2-3 was subjected to semipreparative HPLC eluted with MeOH–H2O (30:70) to yield 2 (16 mg). F4-2-5 was subjected to semipreparative HPLC eluted with MeOH–H2O (32:68) to yield 3 (25 mg) and 4 (11 mg). F4-2-4 was subjected to semipreparative HPLC eluted with CH3CN–H2O (24:76) to yield 5 (10 mg) and 6 (7 mg). F4-2-7 was subjected to semipreparative HPLC eluted with CH3CN– H2O (20:80) to yield 7 (33 mg) and 8 (3 mg). F4-2-2 was subjected to semipreparative HPLC eluted with MeOH–H2O (42:58) to yield 9 (170 mg). F4-2-1 was subjected to ODS CC (1.5 × 25 cm) eluted with a gradient system (MeOH–H2O, 45:55, 1 L) to yield 10 (370 mg). Pinnatifidanin C I (1): yellow oil; [α]20 D − 6.3 (c = 0.03, MeOH); CD (MeOH) nm: 238 (3.66); UV (MeOH) λ max (logε): 230 (3.73), 278 (2.32) nm; IR (KBr) vmax: 3361, 2940, 1841, 1662, 1315, 1158, 1030, 898, 628 cm−1; HRESIMS m/z 397.1257 [M + Na]+ (calcd for C20H22O7Na, 397.1258); 1H and 13C NMR, see Tables 1 and 3. Pinnatifidanin C II (2): yellow oil; [α]20 D +11.2 (c = 0.04, MeOH); CD (MeOH) nm: 240 (−4.50); UV (MeOH) λ max (logε): 229 (3.60), 279 (2.16) nm; IR (KBr) vmax: 3366, 2937,
X.-X. Huang et al. / Fitoterapia 91 (2013) 217–223
1688, 1594, 1323, 1164, 1111, 1026, 898 cm−1; HRESIMS m/z 413.1205 [M + Na]+ (calcd for C20H22O8Na, 413.1207); 1H and 13C NMR, see Tables 1 and 3. Pinnatifidanin C III (3): yellow oil; [α]20 D −21.2 (c = 0.08, MeOH); CD (MeOH) nm: 242 (3.44); UV (MeOH) λ max (logε): 228 (3.32), 280 (2.01) nm; IR (KBr) vmax: 3415, 2934, 1606, 1518, 1275, 1117, 855, 819, 775 cm−1; HRESIMS m/z 429.1510 [M + Na]+ (calcd for C21H26O8Na, 429.1520); 1H and 13C NMR see Tables 1 and 3; 1H NMR (CDCl3, 600 MHz), see Supplementary data. Pinnatifidanin C IV (4): yellow oil; [α]20 D +10.2 (c = 0.03, MeOH); CD (MeOH) nm: 240 (−3.89); UV (MeOH) λ max (logε): 230 (3.12), 278 (2.45) nm; IR (KBr) vmax: 3434, 2932, 1620, 1519, 1383, 1116, 1032, 855, 620 cm−1; HRESIMS m/z 429.1510 [M + Na]+ (calcd for C21H26O8Na, 429.1520); 1H and 13 C NMR see Tables 1 and 3; 1H NMR (CDCl3, 600 MHz), see Supplementary data. Pinnatifidanin C V (5): yellow oil; [α]20 D −8.5 (c = 0.02, MeOH); CD (MeOH) nm: 239 (3.99); UV (MeOH) λ max (logε): 229 (3.71), 280 (2.81) nm; IR (KBr) vmax: 3436, 2853, 1627, 1384, 1121, 1035, 619 cm− 1; HRESIMS m/z 443.1666 [M + Na]+ (calcd for C22H28O8Na, 443.1676); 1H and 13C NMR, see Tables 2 and 3. Pinnatifidanin C VI (6): yellow oil; [α]20 D +7.0 (c = 0.02, MeOH); CD (MeOH) nm: 239 (−2.87); UV (MeOH) λ max (logε): 228 (2.40), 280 (1.66) nm; IR (KBr) vmax: 3384, 2936, 1606, 1518, 1276, 1125, 1032, 855, 819, 740, 636 cm−1; HRESIMS m/z 433.1678 [M + Na]+ (calcd for C22H28O8Na, 433.1676); 1H and 13C NMR, see Tables 2 and 3. Pinnatifidanin C VII (7): yellow oil; [α]20 D +12.5 (c = 0.06, MeOH); CD (MeOH) nm: 241 (−4.87); UV (MeOH) λ max (logε): 228 (2.63), 278 (1.75) nm; IR (KBr) vmax: 3368, 2936, 1603, 1513, 1269, 1032, 820, 785, 641 cm−1; HRESIMS m/z 427.1728 [M + Na]+ (calcd for C22H28O7Na, 427.1727); 1H and 13 C NMR, see Tables 2 and 3.
219
Pinnatifidanin C VIII (8): yellow oil; [α]20 D +6.5 (c = 0.02, MeOH); CD (MeOH) nm: 238 (−3.15); UV (MeOH) λ max (logε): 227 (3.42), 281 (2.46) nm; IR (KBr) vmax: 3385, 2936, 1512, 1270, 1032, 858, 819, 640 cm−1; HRESIMS m/z 341.0998 [M + Na]+ (calcd for C17H18O6Na, 341.0996); 1H and 13C NMR date, see Tables 2 and 3. 2.4. Cytotoxic activity [6] In order to measure the cytotoxic activity, each tested compound and 5-fluorouracil (positive control) were dissolved in DMSO and diluted with the medium to the test concentrations. Briefly, cells were cultured at 37 °C and dispersed in replicates in 96-well plates with HeLa, HepG2, HT-1080, A375-S2, MCF-7, U-937, K562 and HL60 for 24 h. Fresh medium with compounds at different concentrations was then added to individual wells and incubated for 48 h, with 5-fluorouracil as the positive control. After 48 h, the cell was incubated with MTT solution (0.5 mg/mL) for an additional 4 h at 37 °C. The produced formazan crystals were solubilized with DMSO and the optical density of solution was measured at 492 nm using a Spectra Shell reader (Tecan, Austria). 2.5. Antioxidant activity 2.5.1. DPPH radical scavenging activity [7,8] Radical scavenging activity of 1–10 and vitamin E (positive control) was determined using DPPH as a reagent with modification by using 96-well plates. A 0.1 mM solution of DPPH radical in ethanol was prepared and then 100 μL of this solution was mixed with 100 μL of sample solution. The mixture was incubated for 30 min in a dark room at room temperature. Scavenging capacity was read spectrophotometrically by monitoring the decrease in absorbance at 517 nm. The percentage of scavenged DPPH was calculated
Table 1 1 H NMR data of compounds 1–4. No. 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3-OCH3 3′-OCH3 7′-OCH3 1
11,a
22,a
32,b
42,b
6.92 (1H, br.s)
6.93 (1H, br.s)
6.98 (1H, d, 1.9)
6.96 (1H, d, 1.8)
6.77 (1H, br.s)
6.77 (1H, br.s)
6.77 (1H, br.s) 5.58 (1H, d, 6.8) 3.53–3.66 (1H, m) 3.70–3.73 (2H, m)
6.77 (1H, br.s) 5.61 (1H, d, 6.8) 3.55–3.58 (1H, m) 3.62–3.69 (2H, m)
6.79 (1H, d, 8.1) 6.86 (1H, dd, 8.1, 1.9) 5.55 (1H, d, 6.4) 3.31–3.25 (1H, m) 3.78–3.85 (2H, m)
6.77 (1H, d, 8.1) 6.84 (1H, dd, 8.1, 1.8) 5.53 (1H, d, 6.4) 3.30–3.24 (1H, m) 3.76–3.84 (2H, m)
7.44 (1H, br.s)
7.50 (1H, br.s)
6.89 (1H, br.s)
6.88 (1H, br.s)
7.60 (1H, br.s)
7.64 (1H, br.s)
3.09 3.77 3.84 3.74
3.56–3.62 (1H, m) 3.69–3.73 (2H, m) 3.75 (3H, s) 3.84 (3H, s)
6.89 (1H, br.s) 4.14 (1H, d, 6.6) 3.67–3.73 (1H, m) 3.47–3.56 (2H, m) 3.83 (3H, s) 3.89 (3H, s) 3.27 (3H, s)
6.88 (1H, br.s) 4.15 (1H, dd, 6.4) 3.66–3.74 (1H, m) 3.46–3.56 (2H, m) 3.82 (3H, s) 3.88 (3H, s) 3.26 (3H, s)
(2H, (2H, (3H, (3H,
t, 6.4) t, 6.4) s) s)
for 600 MHz, 2 for 300 MHz. Coupling constants (J) in Hz are given in parentheses; chemical shift values are expressed in ppm. a Measured in DMSO-d6. b Measured in CD3OD.
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Table 2 1 H NMR data of compounds 5–8. No. 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′
62,b
71,b
82,a
6.98 (1H, d, 1.8)
6.96 (1H, d, 1.8)
6.96 (1H, d, 1.8)
6.89 (1H, br.s)
6.79 (1H, d, 8.2) 6.85 (1H, dd, 8.2, 1.8) 5.55 (1H, d, 6.4) 3.50–3.52 (1H, m) 3.75–3.84 (2H, m)
6.77 (1H, d, 8.1) 6.84 (1H, dd, 8.1, 1.8) 5.53 (1H, d, 6.5) 3.50–3.52 (1H, m) 3.75–3.84 (2H, m)
6.77 (1H, d, 8.1) 6.84 (1H, dd, 8.1, 1.8) 5.52 (1H, d, 6.5) 3.48–3.52 (1H, m) 3.76–3.82 (2H, m)
6.74 (1H, br.s) 5.32 (1H, d, 6.8) 3.20–3.40 (1H, m)c 3.60–3.70 (1H, m)
6.90 (1H, br.s)
6.88 (1H, br.s)
6.84 (1H, br.s)
6.28 (1H, d, 1.9)
6.88 (1H, br.s) 4.27 (1H, d, 6.4) 3.66–3.69 (1H, m)
6.88 (1H, br.s) 4.26(1H, d, 6.2) 3.66–3.69 (1H, m)
6.28 (1H, d, 1.9)
3.48–3.51 (2H, m) 3.43–3.46 (2H, m) 1.20 (3H, t, 7.0) 3.83 (3H, s) 3.88 (3H, s)
3.47–3.51 (2H, m) 3.43–3.46 (2H, m) 1.18 (3H, t, 7.0) 3.82 (3H, s) 3.87 (3H, s)
6.84 (1H, br.s) 4.39 (1H, dd, 8.3, 5.4) 1.96–2.02 (1H, m) 1.76–1.81 (1H, m) 3.62–3.66 (2H, m) 3.33–3.35 (2H, m) 1.16 (3H, t, 7.0) 3.82 (3H, s) 3.87 (3H, s)
6.74 (1H, br.s)
9′ 1″ 2″ 3-OCH3 3′-OCH3 1
52,b
3.74 (3H, s) 3.71 (3H, s)
for 600 MHz, 2 for 300 MHz. Coupling constants (J) in Hz are given in parentheses; chemical shift values are expressed in ppm. a Measured in DMSO-d6. b Measured in CD3OD. c Overlapped signals.
using the following equation: DPPH scavenging activity (%) = [1 − (S − SB) / (C − CB)] × 100% where S, SB, C and CB are the absorbencies of the sample, the blank sample, the control and the blank control, respectively.
Table 3 13 C NMR data (150 MHz) of compounds 1–8. No.
1a
2a
3b
4b
5b
6b
7b
8a
1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3-OCH3 3′-OCH3 7′-OCH3
131.6 110.5 147.7 115.4 146.8 118.6 88.4 52.3 62.7 129.7 112.0 143.6 152.0 131.0 118.8 197.3 41.2 57.2
131.6 110.5 147.6 115.4 146.8 118.7 88.4 52.3 62.6 129.0 112.8 143.5 152.2 129.6 118.9 198.4 74.0 64.3
133.6 110.5 149.2 147.5 116.1 119.7 89.2 55.2 64.7 134.5 112.8 145.5 149.1 130.1 117.4 85.5 77.1 63.9
133.6 110.5 149.2 147.5 116.1 119.7 89.2 55.2 64.6 134.4 112.8 145.5 149.0 130.1 117.3 85.5 77.1 63.9
55.7 55.8
56.3 56.7 57.1
56.3 56.7 57.1
134.5 110.6 149.1 147.6 116.2 119.8 89.2 55.3 64.7 134.4 112.9 145.5 149.2 130.1 117.2 83.6 77.2 64.0 65.4 15.6 56.4 56.7
134.6 110.5 149.1 147.6 116.3 119.8 89.2 55.3 65.0 137.5 112.0 145.5 148.9 130.0 116.1 80.3 42.3 59.9 64.9 15.6 56.4 56.7
132.7 110.3 147.5 115.2 146.2 118.5 86.3 53.6 63.0 151.9 100.1 143.6 140.2 129.2 103.1
55.7 55.7
134.5 110.5 149.1 147.5 116.1 119.7 89.2 55.3 64.8 134.3 112.9 145.5 149.1 130.0 117.3 83.6 77.1 63.9 65.4 15.6 56.4 56.7
a b
Measured in DMSO-d6. Measured in CD3OD.
55.6 55.5
2.5.2. ABTS radical scavenging activity [9] The radical scavenging activity of the isolated compounds was carried out using an improved ABTS decolorization assay with some modification. ABTS radical cation (ABTS•+) was produced by reacting 7 mM stock solution of ABTS with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. The ABTS•+ solution was diluted with ethanol, to an absorbance of 0.7 ± 0.02 at 734 nm. An ethanolic solution (50 μL) of the samples at various concentrations was mixed with 150 μL diluted ABTS•+ solution. After reaction at room temperature for 20 min, the absorbance at 734 nm was measured. Lower absorbance of the reaction mixture indicates higher ABTS•+ scavenging activity. The capability to scavenge the ABTS•+ was calculated using the formula given below: ABTS•+ scavenging activity (%) = [1 − (S − SB) / (C − CB)] × 100% where S, SB, C and CB are the absorbencies of the sample, the blank sample, the control, and the blank control, respectively. 3. Results and discussion Compound 1 was obtained as yellow oil. The molecular formula of 1 was determined as C20H22O7 from its positive HRESIMS ion at m/z 397.1257 [M + Na]+ (calcd for C20H22O7Na, 397.1258). The 1H NMR spectrum of 1 showed the presence of a 1,3,5-trisubstituted benzene ring [δH: 6.92 (1H, br.s), 6.77 (1H, br.s) and 6.77 (1H, br.s)], a 1,3,4,5tetrasubstituted benzene ring [δH: 7.44 (1H, br.s) and 7.60 (1H, br.s)], two methoxyl group protons attached to the aromatic ring at δ 3.84 (3H, s) and 3.74 (3H, s) compared with the literature [10]. A sequence of oxygenate methine–
X.-X. Huang et al. / Fitoterapia 91 (2013) 217–223
221
Fig. 2. Observed key HMBC correlations of compounds 1, 2, 3, 5, 7 and 8.
methine–oxygenate methylene proton signals at δ 5.58 (1H, d, J = 6.8 Hz), 3.53–3.66 (1H, m) and 3.70–3.73 (2H, m) indicated that 1 had a dihydrobenzofuran neolignan skeleton in combination with its 13C NMR spectrum data. The 13C NMR spectrum of 1 (Table 3) showed twenty carbon signals. Aside from the carbon signals from the two methoxy groups, the remaining eighteen carbon signals supported the presence of twelve aromatic, a carbonyl and five aliphatic carbons. The HMBC correlations (Fig. 2) of H-8′ at δ 3.09 with C-7′, C-9′ and of H-9′ at δ 3.77 with C-7′ and C-9′ confirmed that compound 1 possessed a 3-hydroxy-1-phenylpropan-1-one unit. Meanwhile, the positions of the two methoxyls were confirmed by the HMBC correlations. A relative-trans configuration was determined by the coupling constant J7,8 (6.8 Hz) in accordance with literature reports [11,12]. The CD spectrum showed a positive Cotton effect (Δε238 3.66), so it was found that compound 1 had the 7S,8R-configuration [12] and named pinnatifidanin C I. Compound 2 had the molecular formula C20H22O8, as determined by the HRESIMS at m/z 413.1205 [M + Na]+ (calcd for C20H22O8Na, 413.1207). Its 13C NMR spectroscopic data were similar to those of 1 except for the reductive proton and the obvious downfield shift of C-8′ (+ 32.8 ppm) and C-9′ (+ 7.1 ppm), indicating that one proton of H-8′ was substituted by a hydroxyl group. The coupling constant J7,8 (6.8 Hz) in the 1H NMR of 2 suggested a relative-trans configuration [11,12]. The CD spectrum showed a negative Cotton effect at 240 nm, so it was found that compound 2 had the 7R,8S-configuration [12]. Thus, the structure of 2 was established as shown in Fig. 1 and named pinnatifidanin C II. Compound 3 was obtained as yellow oil with the molecular formula C21H26O8, based on quasi-molecular ion m/z 429.1510 [M + Na]+ (calcd for C21H26O8Na, 429.1520) from HRESIMS
mass spectral analysis. The 1H NMR spectra showed the presence of a 1,3,4-trisubstituted benzene ring [δH: 6.98 (1H, d, J = 1.9 Hz), 6.79 (1H, d, J = 8.1 Hz) and 6.86 (1H, dd, J = 1.9, 8.1 Hz)] and a 1,3,4,5-tetrasubstituted benzene ring [δH: 6.89 (1H, br.s) and 6.89 (1H, br.s)]. The 13C NMR spectrum of 3 showed twenty one carbon signals. Aside from the carbon signals from the three methoxy groups, the remaining eighteen carbon signals supported the presence of twelve aromatic and six aliphatic carbons. Careful comparison of the NMR data of 3 with those of 1 and 2 revealed that compound 3 is a dihydrobenzofuran neolignan. The positions of the three methoxyls were confirmed by the HMBC correlations (Fig. 2). The coupling constant J7,8 (6.4 Hz) in the 1H NMR of 3 suggested a relative-trans configuration [11,12]. Meanwhile the coupling constant between H-7′ and H-8′ (J = 6.6 Hz in CD3OD and J = 8.1 Hz in CDCl3) suggested a threo conformation of C-7′/C-8′ [13,14]. The CD spectrum showed a positive Cotton effect (Δε242 3.44) which suggested a 7S,8R-configuration [12]. Based on the above evidence, the structure of compound 3 was unambiguously established as shown in Fig. 1 and named pinnatifidanin C III. Compound 4 was proposed to have the molecular formula C21H26O8 based on HRESIMS. The 1H NMR and 13C NMR spectroscopic data were in good agreement with those of 3, suggesting that the planar structure of 4 was the same as that of compound 3. The coupling constant J7,8 (6.4 Hz) in the 1H NMR of 4 suggested a relative-trans configuration [11,12]. The coupling constant J7′,8′ (6.4 Hz in CD3OD and 7.9 Hz in CDCl3) in the 1H NMR of 4 suggested a relative-threo configuration [13,14]. The CD spectrum showed a negative Cotton effect (Δε240 − 3.89), which indicated the absolute configurations of 4 to be 7R,8S-configuration [12]. Thus, compound 4 was defined as an optical isomer of 3 and given the trivial name pinnatifidanin C IV.
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The 1H NMR spectroscopic data indicated the presence of a benzofuran-type lignan moiety [δH 5.32 (1H, d, J = 6.8 Hz), 3.20–3.40 (1H, m) and 3.60–3.70 (2H, m)], which was supported by resonances in the 13C NMR spectrum (δC 86.3, 53.6, 63.0). Furthermore, its 1H NMR showed the presence of a 1,3,5-trisubstituted benzene ring [δH 6.89 (1H, s), 6.74 (1H, br.s) and 6.74 (1H, br.s)], a 1,3,4,5-tetrasubstituted benzene ring [δH 6.28 (1H, d, J = 1.9 Hz) and 6.28 (1H, d, J = 1.9 Hz)] and two methoxyl protons attached to the aromatic ring at δ 3.74 (3H, s) and 3.71 (3H, s). According to the HMBC spectrum, the above fragments were connected as shown in Fig. 2. Since the coupling constant of H-7 was 6.8 Hz, the relative configuration of C-7 and C-8 was regarded as trans-configuration [11,12]. The CD spectrum showed negative Cotton effects at 238 nm, so it was found that compound 8 had the 7R,8S-configuration [12] and named pinnatifidanin C VIII. By comparing physical and spectroscopic data with literatures, the two known constituents were identified as 7R,8S-dihydrodehydroconiferyl alcohol (9) [15] and 7R,8Sbalanophonin (10) [16]. Many types of biological activity have been reported on dihydrobenzofuran neolignan. In previous investigations, dihydrobenzofuran neolignans had served as a lead compound for antitumoral agents, and their structure–activity relationships were studied [17–24]. It was reported that the double bond in the C-7′/8′ side chain was the antitumor activity group of dihydrobenzofuran neolignans and the reduction of the double bond caused a greater than ten-fold decrease of activity [17,18,24]. All isolated compounds were assayed for their in vitro cytotoxicity against three human cancer cell lines, including A375-S2, HL60 and K562 using the MTT method, and only compound 10 displayed potent cytotoxic activities. Then, compound 10 was evaluated against other five types of human cancer cell lines, including HeLa, HepG2, HT-1080, MCF-7 and U-937 by MTT method. 5-Fluorouracil was used as the reference drug. The results of cell viability inhibition were shown in Table 4. From the above-mentioned evidence, we also conclude that the presence of double bond in C-7′/C-8′ next to the aromatic ring (π–π conjugation) may increase the cytotoxic activity of dihydrobenzofuran neolignans. The
Compound 5 was isolated as yellow oil. Its HRESIMS spectrum showed the quasi-molecular ion m/z [M + Na]+ 443.1666 (calcd for C22H28O8Na, 443.1676) consistent with the molecular formula C22H28O8. Its 1H NMR and 13C NMR spectroscopic data were similar to those of 3 except for the reduction of a methoxy group and the addition of an ethoxyl group in 5 (Fig. 1). The coupling constant J7,8 (6.4 Hz) in the 1H NMR of 5 suggested a relative-trans configuration [11,12], and the coupling constant between H-7′ and H-8′ (J = 6.4 Hz in CD3OD) suggested a threo conformation of C-7′/C-8′ [13,14]. Meanwhile, the CD spectrum showed a positive Cotton effect (Δε239 3.99), so it was found that compound 5 had the 7S,8Rconfiguration [12]. Accordingly, 5 was proposed to be a new neolignan and had been accorded the trivial name pinnatifidanin C V. Compound 6 was assigned as C22H28O8 from the HRESIMS, and its 1H NMR and 13C NMR spectroscopic data were identical with those of 6, suggesting that the planar structure of 6 was the same as that of 5. A trans configuration of 6 was confirmed by the J7,8 value of 6.5 Hz in the 1H NMR spectrum [11,12]. Meanwhile, the coupling constant between H-7′ and H-8′ (J = 6.2 Hz in CD3OD) indicated that 6 had the threo configuration between C-7′ and C-8′ [13,14]. Furthermore, the CD cotton curve of 6 was opposite to that of 5, which indicated that the configuration of dihydrofuran ring was 7R,8S [12]. Thus, compound 6 was defined as an optical isomer of 5 and named pinnatifidanin C VI. Compound 7 had the molecular formula C22H28O7, as indicated by HRESIMS combined with the NMR data. Its 13C NMR spectroscopic data were similar to those of 5 except for the reductive proton and the obvious chemical shift of C-8′ (-34.9 ppm) and C-9′ (− 4.1 ppm), indicating that 8′-OH was substituted by a hydrogen proton (Fig. 1). The relative configuration of H-7 and H-8 was determined as trans by the coupling constant J7,8 (6.5 Hz) in the 1H NMR [11,12]. The CD spectrum showed a negative Cotton effect (Δε241 −4.87), which indicated that the configuration of 7 was 7R,8S-configuration [12]. Thus, the structure of 7 was established as shown in Fig. 1 and named pinnatifidanin C VII. Compound 8 was obtained as a light yellow oil, and the molecular formula of 8 was deduced as C17H18O6 by HRESIMS.
Table 4 Inhibition effects on the growth of tumor cells in vitro (IC50, μM). IC50 (μM) Compound
1 2 3 4 5 6 7 8 9 10 5-Fua
Cell line HeLa
MCF-7
HepG2
A375-S2
HT1080
HL60
U937
K562
– – – – – – – – – 46.68 7.34
– – – – – – – – – N50 11.50
– – – – – – – – – 30.96 40.34
N50 N50 N50 N50 N50 N50 N50 N50 N50 30.54 6.67
– – – – – – – – – 8.86 35.62
N50 N50 N50 N50 N50 N50 N50 N50 N50 36.71 10.05
– – – – – – – – – N50 23.93
N50 N50 N50 N50 N50 N50 N50 N50 N50 N50 23.55
“–” Means not tested. a 5-Fu (5-fluorouracil) was used as positive control.
X.-X. Huang et al. / Fitoterapia 91 (2013) 217–223
Appendix A. Supplementary data
Table 5 Free radical scavenging activity of compounds 1–10. Compound
DPPH (IC50, μg/mL)
ABTS (IC50, μg/mL)
1 2 3 4 5 6 7 8 9 10 Vitamin Ea
149.20 165.89 160.98 127.90 140.31 158.97 144.30 N200 98.65 137.09 9.96
13.22 15.45 10.37 13.02 10.58 11.62 13.78 23.58 7.29 24.90 9.30
a
223
Vitamin E was used as positive control.
results of the cytotoxicity of isolates in our study may support the viewpoint what Apers et al. reported [24]. Particularly, compound 10 revealed preferred cytotoxicity against the HT-1080 cell line and displayed much stronger inhibitory activity (IC50 = 8.86 μM) compared with positive control 5-fluorouracil (IC50 = 35.62 μM). The antioxidant effects of the isolates were assessed using the DPPH and ATBS assays (Table 5). All isolates (IC50 N 90 μg/mL) were found to be less active than the positive control vitamin E (IC50 = 9.96 μg/mL) in the DPPH assay. But in the ATBS assay, most of the dihydrobenzofuran neolignans showed significant activity of IC50 b 15 μg/mL, which were comparable to the positive control vitamin E (IC50 = 9.30 μg/mL). It has been reported that the antioxidant activity is correlated to the number of free phenolic hydroxyl groups, and the antioxidant activity may dramatically decrease as led by the absence or blocking of the hydroxyl groups by methyl group [25,26]. In our study, compound 9 showed the strongest antioxidant activities (IC50 = 98.65 μg/mL in the DPPH assay and IC50 = 7.29 μg/mL in the ATBS assay) among all the tested compounds, by comparison, compound 8 exhibited significantly reduced antioxidant activities (IC50 N 200 μg/mL in the DPPH assay and IC50 = 23.58 μg/mL in the ATBS assay), suggesting that the antioxidant activity of this type of neolignan was probably related to the 3-phenylpropan-1-ol unit. In consideration of the structures of neolignans (1–10), we found that the loss of the side chain and the existence of hydroxyl group, methyl group or carbonyl group in the side chain (in the C-7′ or C-8′) may cause the reduction of antioxidant activity of dihydrobenzofuran neolignans. Acknowledgments Financial supported by the Program for State Key Laboratory of Bioactive Substance and Function of Natural Medicines (GTZX201208), the Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University, the Scientific Research Starting Foundation (20121106) for Doctors of Liaoning province of PR China and the Foundation (L2012358) from the Project of Education Department of Liaoning province of PR China are gratefully acknowledged. Authors thank Li W. and Sha Y. of Shenyang Pharmaceutical University for the supporting recording of NMR spectra.
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