Isocoumarins from American cockroach (Periplaneta americana) and their cytotoxic activities

Fitoterapia 95 (2014) 115–120

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Isocoumarins from American cockroach (Periplaneta americana) and their cytotoxic activities Shi-Lin Luo a,b,c, Xiao-Jun Huang b,c, Ying Wang b,c, Ren-Wang Jiang b, Lei Wang b,c, Liang-Liang Bai b, Qun-Long Peng b, Cai-Lu Song b,c, Dong-Mei Zhang b,⁎, Wen-Cai Ye a,b,c,⁎⁎ a b c

Department of Phytochemistry, China Pharmaceutical University, Nanjing 210009, PR China Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China JNU-HKUST Joint Laboratory for Neuroscience & Innovative Drug Research, College of Pharmacy, Jinan University, Guangzhou 510632, PR China

a r t i c l e

i n f o

Article history: Received 21 January 2014 Accepted in revised form 26 February 2014 Available online 13 March 2014 Keywords: American cockroach Periplaneta americana Isocoumarin Cytotoxicity

a b s t r a c t Four new isocoumarins (1–4), along with three known ones (5–7), were isolated from the 70% ethanol extract of the whole body of the traditional Chinese insect medicine, American cockroach (Periplaneta americana). The structures with absolute configurations of new compounds were elucidated by extensive spectroscopic methods in combination with X-ray diffraction experiment and CD analyses. Compounds 3–5 showed significant cytotoxic activities in HepG2 and MCF-7 cells with IC50 values in the ranges 6.41–23.91 μM and 6.67–39.07 μM, respectively. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The American cockroach, Periplaneta americana, is the largest species of pest insect in family Blattidae. P. americana is a well-known worldwide domestic pest, which is native to Africa and has spread throughout world especially in the tropical and subtropical regions [1]. In China, the ethanol extract of the dried whole body of P. americana has been used as traditional Chinese medicine for the treatment of bloodstasis syndrome, acne and abdominal mass for hundred years [2]. Recent pharmacological studies demonstrated that the crude extract of P. americana showed significant anticancer, anti-inflammation and promoting tissue regeneration activities

⁎ Corresponding author. Tel.: +86 20 85220936; fax: + 86 20 8522 1559. ⁎⁎ Correspondence to: W.-C. Ye, Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China. Tel.: +86 20 85220936; fax: + 86 20 8522 1559. E-mail addresses: [email protected] (D.-M. Zhang), [email protected] (W.-C. Ye).

http://dx.doi.org/10.1016/j.fitote.2014.03.004 0367-326X/© 2014 Elsevier B.V. All rights reserved.

[3–5]. Previous chemical investigations on P. americana mainly focused on the bioactive peptides and enzymes [6–11]. Up to now, however, there is scarcely any literature about its small molecule chemical ingredient. In order to search for the significant bioactivity compounds from P. americana, we carried out a systematical isolation on the 70% ethanol extract of the whole body of P. americana. As a result, four new isocoumarins, periplanetins A–D (1–4), along with three known ones, (3R)-ethyl-6,8-dihydroxy-7-methyl-3,4-dihydroisocoumarin (5) [12], (R)-6-hydroxymellein (6) [13] and (3R)-methyl-7hydroxymethyl-8-hydroxy-3,4-dihydroisocoumarin-6-O-βD-glucopyranoside (7) [14], were isolated (Fig. 1). Their structures with absolute configurations were established by a combination of NMR, HR-ESI-MS, CD spectra and X-ray diffraction methods. Furthermore, the cytotoxic activities of all isolated compounds on HepG2 and MCF-7 cells were evaluated with the MTT assay. Among them, compounds 3–5 showed significant cytotoxic activities on HepG2 and MCF-7 cells. Herein, the isolation and structural elucidation of these new compounds, as well as the cytotoxic activities of all isolated compounds were described.

116

S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120

Fig. 1. Chemical structures of 1–7.

2. Experimental 2.1. General Optical rotations were measured in CH3OH on a JASCO P-1020 digital polarimeter at room temperature. Melting point was measured on an X-5 melting point apparatus. UV spectra were measured in CH3OH on a JASCO V-550 UV/VIS spectrophotometer with a 1 cm length cell. IR spectra were recorded on a JASCO FT/IR-480 plus Fourier transform infrared spectrometer using KBr pellets. HR-ESI-MS data were obtained on an Agilent 6210 ESI/TOF mass spectrometer and a Waters XeVO G2 Q-TOF mass spectrometer. 1H, 13C, and 2D NMR spectra were measured on Bruker AV-400 and AV-500 spectrometers. CD spectra were obtained on a JASCO J-810 spectropolarimeter at room temperature. Column chromatographic separations were performed on silica gel (300–400 mesh, Qingdao Marine Chemical Group Corporation, Qingdao, P. R. China), macroporous resin Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), reverse-phase C18 and C8 gel (Merck, Darmstadt, Germany). TLC analyses were carried out using precoated silica gel GF254 plates (Yantai Chemical Industry Research Institute, Yantai, P. R. China). Analytic HPLC was performed on an Agilent chromatography equipped with a G1311C pump and a G1325D diode-array detector (DAD) with a Cosmosil 5C18-MS-II column (4.6 × 250 mm, 5 μm, Nacalai Tesque, Kyoto, Japan). Preparative HPLC separations were performed on an Agilent instrument equipped with a G1310B pump and a G1365D UV/VIS detector with a Cosmosil 5C18-MS-II column (10 × 250 mm, 5 μm, Nacalai Tesque, Kyoto, Japan). 2.2. Insect material The dried whole bodies of P. americana (killing in hightemperature sterilization conditions) were purchased from Weishan American Cockroach Breeding Base in Dali city,

Yunnan province of P. R. China. A voucher specimen (No. 2011052501) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, P. R. China.

2.3. Extraction and isolation The dried whole bodies of P. americana (2.5 kg) were powdered and extracted with 70% (v/v) EtOH under percolation twice (2 × 25 L, 24 h each) at room temperature. The solution was concentrated under vacuum to yield a residue (210 g), which was suspended in H2O and subsequently partitioned between CH2Cl2 and H2O. The CH2Cl2 extract was evaporated to give a residue (53 g), which was then subjected to silica gel column (10 × 80 cm) eluted with cyclohexane– EtOAc mixtures (100:0 → 0:100, v/v) to afford six major fractions (Fr. A–Fr. F). Fr. D (12 g) was subjected to a reverse-phase C18 gel column (3 × 20 cm) eluted with gradient mixtures of MeOH–H2O (15:85; 30:70; 50:50; 70:30; 85:15, v/v) to afford five subfractions (Fr. D-1–Fr. D-5). Fr. D-2 (232 mg) was then purified by preparative HPLC on a reversed-phase C18 column (10 × 250 mm, 5 μm) using MeCN–H2O (64: 36, 3 mL/min) as eluent to yield 1 (16 mg, tR = 16.0 min), 3 (9 mg, tR = 19.3 min), and 4 (19 mg, tR =18.5 min). Compounds 2 (7 mg, tR = 18.0 min), 5 (16 mg, tR = 20.5 min), and 6 (24 mg, tR = 22.0 min) were obtained from Fr. D-4 (152 mg) by preparative HPLC using MeOH–H2O (73:27, 3 mL/min) as mobile phase. The H2O soluble fraction (145 g) was subjected to macroporous resin HP-20 column (15 × 60 cm) eluted with EtOH–H2O (0:100; 35:65; 70:30; 90:10, v/v) to yield four fractions (Fr. a–Fr. d). Fr. b (43 g) was subjected to reversephase C8 gel column (10 × 80 cm) eluted with gradient mixtures of MeOH–H2O (15:85; 30:70; 50:50, v/v) to afford four subfractions (Fr. b-1–Fr. b-5). Fr. b-3 (2 g) was separated by a Sephadex LH-20 column (2 × 80 cm, MeOH) to afford 7 (9 mg).

S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120

117

Periplanetin A (1): colorless blocks (CH3OH), m.p. 152– 153 °C. [α]26D − 108.4 (c 0.10, CH3OH). UV (CH3OH) λmax nm (log ε): 204 (2.67), 243 (2.88), 260 (2.81), 331 (2.17). IR (KBr) νmax cm−1: 3414, 1661, 1581, 1439, 1378, 1266, 1190, 1116, 817. 1H and 13C NMR data see Table 1. HR-ESI-MS m/z: 245.0422 [M + Na]+ (calcd for C11H10O5Na, 245.0420). Periplanetin B (2): amorphous powder, [α]25D − 10.6 (c 0.10, CH3OH). UV (CH3OH) λmax nm (log ε): 217 (2.96), 272 (2.74), 305 (2.15). IR (KBr) νmax cm−1: 3165, 1610, 1507, 1437, 1381, 1263, 1180, 1114. 1H and 13C NMR data see Table 1. HR-ESI-MS m/z: 209.0823 [M + H]+ (calcd for C11H13O4, 209.0808). Periplanetin C (3): amorphous powder, [α]26D − 11.2 (c 0.10, CH3OH). UV (CH3OH) λmax nm (log ε): 215 (2.85), 268 (2.64), 300 (2.31). IR (KBr) νmax cm−1: 3201, 1651, 1628, 1479, 1382, 1254, 1167, 856. 1H and 13C NMR data see Table 1. HR-ESI-MS m/z: 209.0806 [M + H]+ (calcd for C11H13O4, 209.0808). Periplanetin D (4): light yellow oil, [α]26D − 15.8 (c 0.10, CH3OH). UV (CH3OH) λmax nm (log ε): 213 (2.97), 274 (2.57), 308 (2.26). IR (KBr) νmax cm−1: 3353, 1630, 1555, 1384, 1271, 1122, 710. 1H and 13C NMR data see Table 1. HR-ESI-MS m/z: 225.0748 [M + H]+ (calcd for C11H13O5, 225.0763).

radiation (λ = 1.54184 Å) under low temperature (nitrogen gas); 2993 unique reflections were collected to θmax = 62.71°, in which 2945 reflections were observed [F2 N 4σ(F2)]. The final R = 0.0232, Rw = 0.0652 and S = 1.072. CCDC 979490 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/ data_request/cif.

2.4. X-ray analysis

3. Results and discussion

The structure was solved by using direct methods (SHELXTL version 5.1) and refined by using full-matrix least-squares treatment on F2. In the structure refinements, non-hydrogen atoms were refined anisotropically. Hydrogen atoms bonded to carbons were placed at geometrically ideal positions by using the ‘ride on’ method. Hydrogen atoms bonded to oxygen were located by employing the difference Fourier method and were included in the calculation of structure factors with isotropic temperature factors. Periplanetin A (1): colorless blocks, C22H20O10, monoclinic, P21, a = 7.2672 (2), b = 12.3723 (2), c = 11.3200 (2) Å, β = 108.671 (2), V = 964.24 (4) Å3, Z = 2, dx = 1.531 Mg/m3, μ (Cu–Kα) = 1.043, F(000) = 464. Data collection was performed on a SMART CCD using graphite monochromated

Periplanetin A (1) was isolated as colorless blocks (CH3OH), [α]26D −108.4 (c 0.10, CH3OH). The molecular formula of 1 was established as C11H10O5 by its HR-ESI-MS at m/z 245.0422 [M + Na]+ (calcd for C11H10O5Na, 245.0420). The UV spectrum of 1 showed absorption maxima at 204, 243, 260 and 331 nm. The IR spectrum displayed characteristic absorption bands at 3414 and 1661 cm−1, suggesting the presence of hydroxyl and carbonyl groups. The 1H and 13C NMR spectra of 1 revealed the signals due to two phenolic hydroxyls [δH 12.59 and 12.28 (each 1H, s)], an aldehyde group [δH 10.31 (1H, s); δC 193.7], a penta-substituted benzene ring [δH 6.25 (1H, s); δC 168.7, 167.4, 149.1, 109.1, 107.6 and 100.4], an oxygenated methine [δH 4.68 (1H, m); δC 75.5], a methylene [δH 2.90 (1H, dd, J = 11.1, 0.7 Hz) and 2.87 (1H, dd, J = 3.3, 0.7 Hz);

2.5. Cytotoxicity assay Human liver cancer cell lines HepG2 and human breast cancer cell lines MCF-7, all obtained from American Type Culture Collection (Manassas, VA, USA), were cultured in RPMI-1640 medium containing 10% new bovine serum or fetal bovine serum and 1% (v/v) penicillin–streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. The cytotoxicities of compounds 1–7 were measured by using the previously described MTT assay [15]. Cells treated with medium containing 0.12% DMSO were considered as 100% viable and doxorubicin (Sigma-Aldrich, St. Louis, MO, USA) was used as the positive control. The concentration required to inhibit cell growth by 50% (IC50) was calculated from survival curves (Table 2).

Table 1 NMR spectroscopic data of 1–4 (CD3OD, J in Hz)a. Position

1b

2

3

4

δH

δC

δH

δC

δH

δC

δH

δC

169.8 75.5 35.4

– 4.63 m a 2.78 dd (16.3, 10.8) b 2.85 dd (16.3, 3.9) 6.22 s – – – – – 2.01 s 1.45 d (6.3)

172.3 77.4 35.5

– 4.45 m a 2.84 dd (16.4, 10.8) b 2.90 dd (16.4, 4.1) 6.23 m – 6.20 d (2.1) – – – 1.79 m 1.07 t (7.5)

171.9 82.2 33.7

– 4.50 m a 2.75 dd (16.1, 3.2) b 2.94 dd (16.1, 12.1) 6.16 s – – – – – 2.00 s a 3.74 dd (12.1, 5.1) b 3.79 dd (12.1, 4.1)

172.1 81.2 30.2

5 6 7 8 9 10 11 12

– 4.68 m a 2.87 dd (3.3, 0.7) b 2.90 dd (11.1, 0.7) 6.25 s – – – – – 10.31 s 1.51 d (6.3)

6-OH 8-OH

12.59 s 12.28 s

– –

1 3 4

a b

107.6 168.7 109.1 167.4 100.4 149.1 193.7 20.9

Assignments were made by analysis of COSY, HSQC and HMBC spectra. Data were recorded in CDCl3.

106.9 163.4 111.1 163.9 101.2 139.9 7.8 21.0

108.1 165.8 102.3 166.4 101.8 143.7 29.0 9.7

109.6 163.3 111.5 170.1 98.5 139.3 8.2 64.6

118

S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120

Table 2 The cytotoxic activities of 1–7 on HepG2 and MCF-7 cancer cellsa. b

Compounds

IC50 (x ± SD) μM HepG2

MCF-7

1 2 3 4 5 6 7 DOXc

N50 N50 23.91 ± 1.19 10.38 ± 0.41 6.41 ± 0.30 N50 N50 0.11 ± 0.01

N50 N50 39.07 ± 14.88 6.67 ± 1.96 6.95 ± 2.27 N50 N50 1.06 ± 0.16

a Cytotoxic activities of 1–7 were measured by MTT assay. All data are presented as means ± standard deviation of at least three independent experiments. b Concentration of the tested compound inhibits 50% of cell growth. c Doxorubicin was used as positive control.

δC 35.4], and a secondary methyl [δH 1.51 (3H, d, J = 6.3 Hz); δC 20.9]. The above spectral data suggested the existence of a 6,8-dihydroxy-3,4-dihydroisocoumarin core skeleton in 1 [16]. Based on the analysis of the 1H–1H COSY, HSQC and HMBC spectra, the 1H and 13C NMR signals of 1 were assigned as shown in Table 1. The 1H–1H COSY spectrum of 1 revealed the presence of a spin system in bold as shown in Fig. 2. In the HMBC spectrum, the correlations between hydroxyl proton (δH 12.59) and C-7 (δC 109.1)/C-5 (δC 107.6), between hydroxyl proton (δH 12.28) and C-7/C-9 (δC 100.4), between H-5 (δH 6.25) and C-7/C-9/C-4 (δC 35.4), as well as between Hb-4 (δH 2.90)/ Ha-4 (δH 2.87) and C-5/C-9 confirmed the existence of 6,8dihydroxy-3,4-dihydroisocoumarin unit. Moreover, the correlations between H-11 (δH 10.31) and C-6 (δC 168.7)/C-8 (δC 167.4), and between H3-12 (δH 1.51) and C-4 were observed, which indicated that the aldehyde group and methyl were attached to the C-7 and C-3 positions of the isocoumarin unit, respectively. Therefore, the planar structure of 1 was constructed as shown in Fig. 2.

Fig. 2. Key 1H–1H COSY and HMBC correlations of 1–3.

Fortunately, crystals suitable for single crystal X-ray diffraction experiment were obtained, which established the absolute configuration of 1 (Fig. 3). Moreover, the stereochemistry of 1 was further confirmed by the quantum chemical CD calculation [17,18]. The preliminary conformational distribution search was performed by Syby1 8.0 software using the MMFF94S force field. The corresponding minimum geometries were further fully optimized by using DFT at B3LYP/6-31+G(d) level as implemented in the Gaussian 09 program package. The obtained stable conformers were submitted to CD calculation by the TDDFT [B3LYP/6-31+G(d)] method. The predicted CD spectra of the two possible isomers (3R-1 and 3S-1) were compared with the experimental one, respectively. As a result, the predicted CD spectrum of 3R isomer revealed a good agreement with the measured CD curve (Fig. 4). Thus, the structure of 1 was assigned to be (3R)-methyl-6,8-dihydroxy-7formoxyl-3,4-dihydroisocoumarin. The molecular formula of 2 was determined to be C11H12O4 by the quasi-molecular ion at m/z 209.0823 [M + H]+ (calcd for C11H13O4, 209.0808) in its HR-ESI-MS. In the UV spectrum, the absorption maxima at 217, 272 and 305 nm indicated the presence of isocoumarin skeleton in 2. Characteristic absorption bands at 3165 and 1610 cm−1 in the IR spectrum demonstrated the existence of hydroxyl and carbonyl groups in 2. Similar to 1, the 1H and 13C NMR spectra of 2 also revealed the existence of a penta-substituted benzene ring [δH 6.22 (1H, s); δC 163.9, 163.4, 139.9, 111.1, 106.9 and 101.2], an oxygenated methine [δH 4.63 (1H, m); δC 77.4], and a methylene [δH 2.85 (1H, dd, J = 16.3, 3.9 Hz) and 2.78 (1H, dd, J = 16.3, 10.8 Hz); δC 35.5], which suggested the existence of 6,8-dihydroxy-3,4-dihydroisocoumarin backbone in 2. In addition, the NMR spectra of 2 showed the signals due to two methyls [δH 2.01 (3H, s) and 1.45 (3H, d, J = 6.3 Hz); δC 7.8 and 21.0]. The 1H–1H COSY, HSQC, HMBC spectra of 2 allowed the full assignments of all proton and carbon signals (Table 1). The 1H–1H COSY spectrum of 2 showed the presence of spin system from C-4 to C-12 (Fig. 2). In the HMBC spectrum of 2, the correlations between H3-11 (δH 2.01) and C-8 (δC 163.9)/C-6 (δC 163.4), as well as between H3-12 (δH 1.45) and C-4 (δC 35.5) indicated that two methyls were attached to the C-7 and C-3 positions of the isocoumarin backbone, respectively. The CD spectrum of 2 showed the Cotton effects at 231, 246 and 271 nm, respectively, which was similar to those of (R)-6-hydroxymellein (6) (Fig. 5) [19], indicating the presence of R configuration at C-3 in 2. Therefore, the structure of 2 was established as (3R)-methyl-6,8-dihydroxy7-methyl-3,4-dihydroisocoumarin. The molecular formula of 3 was assigned as C11H12O4 by its HR-ESI-MS (m/z 209.0806 [M + H]+; calcd for C11H13O4, 209.0808). The IR and UV spectra of 3 were very close to those of 2, indicating that 3 was also an isocoumarin derivative. Similar to 2, the 1H and 13C NMR spectra of 3 showed characteristic proton and carbon signals due to a 6,8-dihydroxy-3,4-dihydroisocoumarin core structure. Additionally, the NMR spectra of 3 showed the signals due to a methylene [δH 1.79 (2H, m); δC 29.0] and a methyl [δH 1.07 (3H, t, J = 7.5 Hz); δC 9.7], suggesting the presence of an ethyl group in 3. Detailed analysis of 1H–1H COSY, HSQC, and HMBC spectra resulted in the full assignment of all NMR data of 3 as shown in Table 1. The 1H–1H COSY spectrum of 3 showed the presence of a spin coupling system (C-4 to C-12, Fig. 2),

S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120

119

Fig. 3. Single crystal X-ray structure of 1.

which confirmed the existence of ethyl group. Moreover, in the HMBC spectrum of 3, the correlations between H-11 (δH 1.79) and C-4 (δC 33.7), as well as between H3-12 (δH 1.07) and C-3 (δC 82.2) indicated that the ethyl group was attached to the C-3 position of the isocoumarin backbone. Furthermore, the absolute configuration of 3 was assigned to be identical to that of 2 based on the similar Cotton effects in the CD measurement (Fig. 5). Thus, 3 was identified as (3R)-ethyl-6,8-dihydroxy3,4-dihydroisocoumarin. The molecular formula of 4 was determined to be C11H12O5 on the basis of the quasi-molecular ion at m/z 225.0748 [M + H]+ (calcd for C11H13O5, 225.0763) in its HR-ESI-MS. Similar to 2 and 3, the UV and IR spectra of 4 showed the characteristic absorptions of isocoumarin skeleton. A comparison of the 1H and 13C NMR spectra of 4 with those of 2 suggested that they were very similar, except that the methyl at C-3 in 2 was replaced by a hydroxymethyl [δH 3.79 (1H, dd, J = 12.1, 4.1 Hz) and 3.74 (1H, dd, J = 12.1, 5.1 Hz); δC 64.6] in 4. The 1H–1H COSY spectrum of 4 revealed the presence of spin system from C-4 to C-12, combined with the HMBC correlations between H2-12 (δH 3.79 and 3.74) and C-4 (δC 30.2), indicating that the hydroxymethyl group was attached to the C-3 position. Furthermore, the 3S configuration of 4 was also deduced by the CD spectrum (Fig. 5) [19]. Therefore,

25 20 15 10 5 0 -5 -10 -15 -20 -25 200

Exptl of 1 Calcd of 3R-1 Calcd of 3S-1

the structure of 4 was identified as (3S)-hydroxymethyl-6,8dihydroxy-7-methyl-3,4-dihydroisocoumarin. The cytotoxic activities of all the isocoumarins (1–7) were evaluated on HepG2 and MCF-7 cells by MTT assay. As shown in Table 2, compounds 3–5 exhibited potent growth inhibitory activity on both HepG2 and MCF-7 cells, as reflected by the IC50 values with the range of 6.41–23.91 μM and 6.67– 39.07 μM, respectively, suggesting that the functional groups attached to C-3 and C-7 positions of the isocoumarin skeleton might play an important role in their cytotoxic activities. It was also found that HepG2 cells were more sensitive than MCF-7 cells to 3, whereas they were less sensitive to 4.

Acknowledgments Financial support of this work was provided by the Joint Fund of NSFC-Guangdong Province (No. U0932004), the National Natural Science Foundation of China (No. 81172946), the Program for New Century Excellent Talents in University (No. NCET-11-0857), and the Science and Technology Planning Project of Guangzhou (Nos. 2011J2200045 and 2011J2200046). This work was also supported by the high-performance computing platform of Jinan University.

2 3 4 6

20 10 0 -10 -20 -30

250

300

350

wavelength (nm) Fig. 4. Calculated and experimental CD spectra of 1.

400

250

300

350

wavelength (nm) Fig. 5. Experimental CD spectra of 2–4 and 6.

400

120

S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120

Appendix A. Supplementary data Quantum chemical CD data of 1, HR-ESI-MS, IR, UV, CD, 1D and 2D NMR spectra of 1–4, as well as CD and 1D NMR spectra of 5–7 are available as Supporting Information. Supplementary data to this article can be found online at http://dx.doi.org/ 10.1016/j.fitote.2014.03.004. References [1] Chinese Materia Medica Editoral Committee, editor. Chinese Materia Medica, vol. 9. Shanghai: Shanghai Scientific and Technical Publisher; 1999. p. 149–51. [2] Sun XY. Sheng Nong's herbal classic. Beijing: Beijing Commercial Press; 1955 [90–1]. [3] Jiang YX, Wang XC, Jin CG, Chen XQ, Li J, Wu ZP, et al. Inhibitory effect of Periplaneta americana extract on 3LL lung cancer in mice. Chin J Lung Cancer 2006;9:488–91. [4] Li W, Duan LF, He GQ, Shen ZQ, Yang HQ, Liang YP. Periplaneta americana extract effects on experimental liver fibrosis. Lishizhen Med Mater Med Res 2010;21:1137–8. [5] He ZC, Peng F, Song LY, Wang XY, Hu MH, Zhao Y, et al. Review on investigations related to chemical constituents and biological activities of Periplaneta americana. Chin J Chin Mater Med 2007;32:2326–30. [6] Predel R, Kellner R, Rapus J, Penzlin H, Gade G. Isolation and structural elucidation of eight kinins from the retrocerebral complex of the American cockroach, Periplaneta americana. Regul Pept 1997;71:199–205. [7] Neupert S, Fusca D, Schachtner J, Kloppenburg P, Predel R. Toward a single-cell-based analysis of neuropeptide expression in Periplaneta americana antennal lobe neurons. J Comp Neurol 2012;520:694–716. [8] Bembenek J, Sakamoto K, Takeda M. Molecular cloning of a cDNA encoding arylalkylamine N-acetyltransferase from the testicular system of Periplaneta americana: primary protein structure and expression analysis. Arch Insect Biochem Physiol 2005;59:219–29.

[9] He ZC, Liu GM, Wang XY, Yang LX, Zhao Y. Research advance on neuropeptides from Periplaneta americana. Nat Prod Res Dev 2008;20: 180–6. [10] Predel R, Neupert S, Wicher D, Gundel M, Roth S, Derst C. Unique accumulation of neuropeptides in an insect: FMRFamide-related peptides in the cockroach, Periplaneta americana. Eur J Neurosci 2004;20: 1499–513. [11] Predel R, Killner R, Kaufmann R, Penzlin H, Gade G. Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach. Peptides 1997;18:473–8. [12] Wu B. Chroman compound extracted from ground beetles for use in cancer therapy, tumor preventive healthy food supplement, anti-tyrosinase drug, whitening cosmetic product or pheromone. CN Patent 103408528 A: 2013. [13] Islam MS, Ishigami K, Watanabe H. Synthesis of (−)-mellein, (+)ramulosin, and related natural products. Tetrahedron 2007;63:1074–9. [14] Chen JD, Yu GZ. Method for detecting active ingredient content of Periplaneta americana extract by High Performance Liquid Chromatography (HPLC). CN Patent 101957345 A: 2011. [15] Shi JM, Bai LL, Zhang DM, Yiu A, Yin ZQ, Han WL, et al. Saxifragifolin D induces the interplay between apoptosis and autophagy in breast cancer cells through ROS-dependent endoplasmic reticulum stress. Biochem Pharmacol 2013;85:913–21. [16] Jiang HL, Luo XH, Wang XZ, Yang JL, Yao XJ, Crews P, et al. New isocoumarins and alkaloid from Chinese insect medicine, Eupolyphaga sinensis walker. Fitoterapia 2012;83:1275–80. [17] Shao M, Wang Y, Liu Z, Zhang DM, Cao HH, Jiang RW, et al. Psiguadials A and B, two novel meroterpenoids with unusual skeletons from the leaves of Psidium guajava. Org Lett 2010;12:5040–3. [18] Bringmann G, Bruhn T, Maksimenka K, Hemberger Y. The assignment of absolute stereostructures through quantum chemical circular dichroism calculations. Eur J Org Chem 2009;17:2717–27. [19] Schlingmann G, Roll DM. Absolute stereochemistry of unusual biopolymers from Ascomycete culture LL-W1278: examples that derivatives of (S)-6-hydroxymellein are also natural fungal metabolites. Chirality 2005;17:S48–51.