Periplaneta Americana) and Their Cytotoxic Activities

Fitoterapia 95 (2014) 115–120

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

Article history: Four new isocoumarins (1–4), along with three known ones (5–7), were isolated from the 70% Received 21 January 2014 ethanol extract of the whole body of the traditional Chinese insect medicine, American cockroach Accepted in revised form 26 February 2014 (Periplaneta americana). The structures with absolute configurations of new compounds were Available online 13 March 2014 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

Keywords: cells with IC50 values in the ranges 6.41–23.91 μM and 6.67–39.07 μM, respectively. American cockroach © 2014 Elsevier B.V. All rights reserved. Periplaneta americana Isocoumarin Cytotoxicity

1. Introduction [3–5]. Previous chemical investigations on P. americana mainly focused on the bioactive peptides and enzymes [6–11].Upto The American cockroach, Periplaneta americana,isthe now, however, there is scarcely any literature about its small largest species of pest insect in family Blattidae. P. americana molecule chemical ingredient. In order to search for the sig- is a well-known worldwide domestic pest, which is native nificant bioactivity compounds from P. americana,wecarried to Africa and has spread throughout world especially in the out a systematical isolation on the 70% ethanol extract of the tropical and subtropical regions [1]. In China, the ethanol whole body of P. americana. As a result, four new isocoumarins, extract of the dried whole body of P. americana has been used periplanetins A–D(1–4), along with three known ones, as traditional Chinese medicine for the treatment of blood- (3R)-ethyl-6,8-dihydroxy-7-methyl-3,4-dihydroisocoumarin stasis syndrome, acne and abdominal mass for hundred years (5) [12],(R)-6-hydroxymellein (6) [13] and (3R)-methyl-7- [2]. Recent pharmacological studies demonstrated that the hydroxymethyl-8-hydroxy-3,4-dihydroisocoumarin-6-O-β- crude extract of P. americana showed significant anticancer, D-glucopyranoside (7) [14],wereisolated(Fig. 1). Their anti-inflammation and promoting tissue regeneration activities 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 ⁎ Corresponding author. Tel.: +86 20 85220936; fax: + 86 20 8522 1559. the MTT assay. Among them, compounds 3–5 showed signifi- ⁎⁎ Correspondence to: W.-C. Ye, Institute of Traditional Chinese Medicine & cant cytotoxic activities on HepG2 and MCF-7 cells. Herein, the Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, isolation and structural elucidation of these new compounds, as PR China. Tel.: +86 20 85220936; fax: + 86 20 8522 1559. E-mail addresses: [email protected] (D.-M. Zhang), well as the cytotoxic activities of all isolated compounds were [email protected] (W.-C. Ye). described.

Fig. 1. Chemical structures of 1–7.

2. Experimental Yunnan province of P. R. China. A voucher specimen (No. 2011052501) was deposited in the Institute of Traditional 2.1. General Chinese Medicine & Natural Products, Jinan University, Guangzhou, P. R. China.

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 2.3. Extraction and isolation were measured in CH3OH on a JASCO V-550 UV/VIS spectro- photometer with a 1 cm length cell. IR spectra were recorded The dried whole bodies of P. americana (2.5 kg) were on a JASCO FT/IR-480 plus Fourier transform infrared spec- powdered and extracted with 70% (v/v) EtOH under percola- trometer using KBr pellets. HR-ESI-MS data were obtained on tion twice (2 × 25 L, 24 h each) at room temperature. The an Agilent 6210 ESI/TOF mass spectrometer and a Waters XeVO solution was concentrated under vacuum to yield a residue 1 13 G2 Q-TOF mass spectrometer. H, C, and 2D NMR spectra (210 g), which was suspended in H2O and subsequently par- were measured on Bruker AV-400 and AV-500 spectrometers. titioned between CH2Cl2 and H2O. The CH2Cl2 extract was CD spectra were obtained on a JASCO J-810 spectropolarimeter evaporated to give a residue (53 g), which was then subjected at room temperature. Column chromatographic separations to silica gel column (10 × 80 cm) eluted with cyclohexane– were performed on silica gel (300–400 mesh, Qingdao Marine EtOAc mixtures (100:0 → 0:100, v/v) to afford six major frac- Chemical Group Corporation, Qingdao, P. R. China), macroporous tions (Fr. A–Fr. F). Fr. D (12 g) was subjected to a reverse-phase resin Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, C18 gel column (3 × 20 cm) eluted with gradient mixtures of Japan), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, MeOH–H2O (15:85; 30:70; 50:50; 70:30; 85:15, v/v) to afford Sweden), reverse-phase C18 and C8 gel (Merck, Darmstadt, five subfractions (Fr. D-1–Fr. D-5). Fr. D-2 (232 mg) was then Germany). TLC analyses were carried out using precoated purified by preparative HPLC on a reversed-phase C18 column silica gel GF254 plates (Yantai Chemical Industry Research (10 × 250 mm, 5 μm) using MeCN–H2O (64: 36, 3 mL/min) Institute, Yantai, P. R. China). Analytic HPLC was performed on as eluent to yield 1 (16 mg, tR =16.0min),3 (9 mg, tR = an Agilent chromatography equipped with a G1311C pump 19.3 min), and 4 (19 mg, tR =18.5 min). Compounds 2 (7 mg, and a G1325D diode-array detector (DAD) with a Cosmosil tR =18.0min), 5 (16 mg, tR = 20.5 min), and 6 (24 mg, 5C18-MS-II column (4.6 × 250 mm, 5 μm, Nacalai Tesque, tR = 22.0 min) were obtained from Fr. D-4 (152 mg) by pre- Kyoto, Japan). Preparative HPLC separations were performed parative HPLC using MeOH–H2O (73:27, 3 mL/min) as mobile on an Agilent instrument equipped with a G1310B pump and a phase. The H2O soluble fraction (145 g) was subjected to G1365D UV/VIS detector with a Cosmosil 5C18-MS-II column macroporous resin HP-20 column (15 × 60 cm) eluted with (10 × 250 mm, 5 μm, Nacalai Tesque, Kyoto, Japan). 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 reverse-

2.2. Insect material phase C8 gel column (10 × 80 cm) eluted with gradient mixtures of MeOH–H2O (15:85; 30:70; 50:50, v/v) to afford four The dried whole bodies of P. americana (killing in high- subfractions (Fr. b-1–Fr. b-5). Fr. b-3 (2 g) was separated temperature sterilization conditions) were purchased from by a Sephadex LH-20 column (2 × 80 cm, MeOH) to afford 7 Weishan American Cockroach Breeding Base in Dali city, (9 mg). S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120 117

Periplanetin A (1): colorless blocks (CH3OH), m.p. 152– radiation (λ = 1.54184 Å) under low temperature (nitrogen 26 153 °C. [α] D −108.4 (c 0.10, CH3OH). UV (CH3OH) λmax gas); 2993 unique reflections were collected to θmax =62.71°, nm (log ε): 204 (2.67), 243 (2.88), 260 (2.81), 331 (2.17). IR in which 2945 reflections were observed [F2 N 4σ(F2)]. The −1 (KBr) νmax cm : 3414, 1661, 1581, 1439, 1378, 1266, 1190, final R =0.0232,Rw =0.0652andS = 1.072. CCDC 979490 1116, 817. 1H and 13C NMR data see Table 1. HR-ESI-MS m/z: contains the supplementary crystallographic data for this paper. + 245.0422 [M + Na] (calcd for C11H10O5Na, 245.0420). These data can be obtained free of charge from the Cambridge Periplanetin B (2): amorphous powder, [α]25D −10.6 Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/

(c 0.10, CH3OH). UV (CH3OH) λmax nm (log ε): 217 (2.96), data_request/cif. −1 272 (2.74), 305 (2.15). IR (KBr) νmax cm : 3165, 1610, 1507, 1437, 1381, 1263, 1180, 1114. 1H and 13C NMR data 2.5. Cytotoxicity assay see Table 1. HR-ESI-MS m/z: 209.0823 [M + H]+ (calcd for

C11H13O4, 209.0808). Human liver cancer cell lines HepG2 and human breast Periplanetin C (3): amorphous powder, [α]26D −11.2 cancer cell lines MCF-7, all obtained from American Type

(c 0.10, CH3OH). UV (CH3OH) λmax nm (log ε): 215 (2.85), Culture Collection (Manassas, VA, USA), were cultured in −1 268 (2.64), 300 (2.31). IR (KBr) νmax cm : 3201, 1651, RPMI-1640 medium containing 10% new bovine serum or 1628, 1479, 1382, 1254, 1167, 856. 1H and 13C NMR data fetal bovine serum and 1% (v/v) penicillin–streptomycin in a + see Table 1. HR-ESI-MS m/z: 209.0806 [M + H] (calcd for humidified atmosphere with 5% CO2 at 37 °C. The cytotoxicities C11H13O4, 209.0808). of compounds 1–7 were measured by using the previously Periplanetin D (4): light yellow oil, [α]26D −15.8 (c 0.10, described MTT assay [15]. Cells treated with medium containing

CH3OH). UV (CH3OH) λmax nm (log ε): 213 (2.97), 274 (2.57), 0.12% DMSO were considered as 100% viable and doxorubicin −1 308 (2.26). IR (KBr) νmax cm : 3353, 1630, 1555, 1384, (Sigma-Aldrich, St. Louis, MO, USA) was used as the positive 1271, 1122, 710. 1H and 13C NMR data see Table 1. HR-ESI-MS control. The concentration required to inhibit cell growth by + m/z: 225.0748 [M + H] (calcd for C11H13O5, 225.0763). 50% (IC50) was calculated from survival curves (Table 2).

2.4. X-ray analysis 3. Results and discussion

The structure was solved by using direct methods (SHELXTL Periplanetin A (1) was isolated as colorless blocks (CH3OH), 26 version 5.1) and refined by using full-matrix least-squares [α] D −108.4 (c 0.10, CH3OH). The molecular formula of 1 2 treatment on F . In the structure refinements, non-hydrogen was established as C11H10O5 by its HR-ESI-MS at m/z 245.0422 + atoms were refined anisotropically. Hydrogen atoms bonded to [M + Na] (calcd for C11H10O5Na, 245.0420). The UV spec- carbons were placed at geometrically ideal positions by using trum of 1 showed absorption maxima at 204, 243, 260 and the ‘ride on’ method. Hydrogen atoms bonded to oxygen were 331 nm. The IR spectrum displayed characteristic absorption located by employing the difference Fourier method and were bands at 3414 and 1661 cm−1, suggesting the presence of included in the calculation of structure factors with isotropic hydroxyl and carbonyl groups. The 1Hand13C NMR spectra of temperature factors. 1 revealed the signals due to two phenolic hydroxyls [δH 12.59 Periplanetin A (1): colorless blocks, C22H20O10,monoclinic, and 12.28 (each 1H, s)], an aldehyde group [δH 10.31 (1H, s); P21, a = 7.2672 (2), b = 12.3723 (2), c = 11.3200 (2) Å, β = δC 193.7], a penta-substituted benzene ring [δH 6.25 (1H, s); 3 3 108.671 (2), V = 964.24 (4) Å , Z =2, dx =1.531Mg/m, δC 168.7, 167.4, 149.1, 109.1, 107.6 and 100.4], an oxygenated μ (Cu–Kα)=1.043,F(000) = 464. Data collection was per- methine [δH 4.68 (1H, m); δC 75.5], a methylene [δH 2.90 formed on a SMART CCD using graphite monochromated (1H, dd, J = 11.1, 0.7 Hz) and 2.87 (1H, dd, J = 3.3, 0.7 Hz);

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

Position 1b 234

δH δC δH δC δH δC δH δC 1 – 169.8 – 172.3 – 171.9 – 172.1 3 4.68 m 75.5 4.63 m 77.4 4.45 m 82.2 4.50 m 81.2 4 a 2.87 dd (3.3, 0.7) 35.4 a 2.78 dd (16.3, 10.8) 35.5 a 2.84 dd (16.4, 10.8) 33.7 a 2.75 dd (16.1, 3.2) 30.2 b 2.90 dd (11.1, 0.7) b 2.85 dd (16.3, 3.9) b 2.90 dd (16.4, 4.1) b 2.94 dd (16.1, 12.1) 5 6.25 s 107.6 6.22 s 106.9 6.23 m 108.1 6.16 s 109.6 6 – 168.7 – 163.4 – 165.8 – 163.3 7 – 109.1 – 111.1 6.20 d (2.1) 102.3 – 111.5 8 – 167.4 – 163.9 – 166.4 – 170.1 9 – 100.4 – 101.2 – 101.8 – 98.5 10 – 149.1 – 139.9 – 143.7 – 139.3 11 10.31 s 193.7 2.01 s 7.8 1.79 m 29.0 2.00 s 8.2 12 1.51 d (6.3) 20.9 1.45 d (6.3) 21.0 1.07 t (7.5) 9.7 a 3.74 dd (12.1, 5.1) 64.6 b 3.79 dd (12.1, 4.1) 6-OH 12.59 s – 8-OH 12.28 s –

a Assignments were made by analysis of COSY, HSQC and HMBC spectra. b Data were recorded in CDCl3. 118 S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120

Table 2 Fortunately, crystals suitable for single crystal X-ray dif- a The cytotoxic activities of 1–7 on HepG2 and MCF-7 cancer cells . fraction experiment were obtained, which established the b Compounds IC50 (x ± SD) μM absolute configuration of 1 (Fig. 3). Moreover, the stereochemistry of 1 was further confirmed by the quantum chemical HepG2 MCF-7 CD calculation [17,18]. The preliminary conformational distri- 1 N50 N50 bution search was performed by Syby1 8.0 software using the N N 2 50 50 MMFF94S force field. The corresponding minimum geometries 3 23.91 ± 1.19 39.07 ± 14.88 4 10.38 ± 0.41 6.67 ± 1.96 were further fully optimized by using DFT at B3LYP/6-31+G(d) 5 6.41 ± 0.30 6.95 ± 2.27 level as implemented in the Gaussian 09 program package. 6 N50 N50 The obtained stable conformers were submitted to CD calcula- 7 N50 N50 tion by the TDDFT [B3LYP/6-31+G(d)] method. The predicted DOXc 0.11 ± 0.01 1.06 ± 0.16 CD spectra of the two possible isomers (3R-1 and 3S-1)were a Cytotoxic activities of 1–7 were measured by MTT assay. All data are compared with the experimental one, respectively. As a result, presented as means ± standard deviation of at least three independent the predicted CD spectrum of 3R isomer revealed a good agree- experiments. b Concentration of the tested compound inhibits 50% of cell growth. ment with the measured CD curve (Fig. 4). Thus, the struc- c Doxorubicin was used as positive control. ture of 1 was assigned to be (3R)-methyl-6,8-dihydroxy-7- formoxyl-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 δ 35.4], and a secondary methyl [δ 1.51 (3H, d, J = 6.3 Hz); C H for C H O , 209.0808) in its HR-ESI-MS. In the UV spectrum, δ 20.9]. The above spectral data suggested the existence of a 11 13 4 C the absorption maxima at 217, 272 and 305 nm indicated 6,8-dihydroxy-3,4-dihydroisocoumarin core skeleton in 1 [16]. 1 1 the presence of isocoumarin skeleton in 2. Characteristic ab- Based on the analysis of the H– H COSY, HSQC and HMBC − sorption bands at 3165 and 1610 cm 1 in the IR spectrum spectra, the 1Hand13C NMR signals of 1 were assigned as demonstrated the existence of hydroxyl and carbonyl groups in shown in Table 1. 2. Similar to 1,the1Hand13C NMR spectra of 2 also revealed The 1H–1H COSY spectrum of 1 revealed the presence of the existence of a penta-substituted benzene ring [δ 6.22 a spin system in bold as shown in Fig. 2. In the HMBC spec- H (1H, s); δ 163.9, 163.4, 139.9, 111.1, 106.9 and 101.2], an trum, the correlations between hydroxyl proton (δ 12.59) C H oxygenated methine [δ 4.63 (1H, m); δ 77.4], and a meth- and C-7 (δ 109.1)/C-5 (δ 107.6), between hydroxyl proton H C C C ylene [δ 2.85 (1H, dd, J = 16.3, 3.9 Hz) and 2.78 (1H, dd, J = (δ 12.28) and C-7/C-9 (δ 100.4), between H-5 (δ 6.25) and H H C H 16.3, 10.8 Hz); δ 35.5], which suggested the existence of C-7/C-9/C-4 (δ 35.4), as well as between Hb-4 (δ 2.90)/ C C H 6,8-dihydroxy-3,4-dihydroisocoumarin backbone in 2.Inad- Ha-4 (δ 2.87) and C-5/C-9 confirmed the existence of 6,8- H dition, the NMR spectra of 2 showed the signals due to two dihydroxy-3,4-dihydroisocoumarin unit. Moreover, the cor- methyls [δ 2.01 (3H, s) and 1.45 (3H, d, J = 6.3 Hz); δ 7.8 and relations between H-11 (δ 10.31) and C-6 (δ 168.7)/C-8 (δ H C H C C 21.0]. The 1H–1H COSY, HSQC, HMBC spectra of 2 allowed the 167.4), and between H -12 (δ 1.51) and C-4 were observed, 3 H full assignments of all proton and carbon signals (Table 1). which indicated that the aldehyde group and methyl were The 1H–1H COSY spectrum of 2 showed the presence of attached to the C-7 and C-3 positions of the isocoumarin unit, spin system from C-4 to C-12 (Fig. 2). In the HMBC spectrum respectively. Therefore, the planar structure of 1 was construct- of 2, the correlations between H -11 (δ 2.01) and C-8 ed as shown in Fig. 2. 3 H (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-dihydroxy- 7-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. Addi- tionally, 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.The1H–1H COSY spectrum of 3 showed Fig. 2. Key 1H–1H COSY and HMBC correlations of 1–3. 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 the structure of 4 was identified as (3S)-hydroxymethyl-6,8-

HMBC spectrum of 3, the correlations between H-11 (δH 1.79) dihydroxy-7-methyl-3,4-dihydroisocoumarin. and C-4 (δC 33.7), as well as between H3-12 (δH 1.07) and C-3 The cytotoxic activities of all the isocoumarins (1–7) were (δC 82.2) indicated that the ethyl group was attached to the evaluated on HepG2 and MCF-7 cells by MTT assay. As shown C-3 position of the isocoumarin backbone. Furthermore, the in Table 2, compounds 3–5 exhibited potent growth inhib- absolute configuration of 3 was assigned to be identical to that itory activity on both HepG2 and MCF-7 cells, as reflected by of 2 based on the similar Cotton effects in the CD measurement the IC50 values with the range of 6.41–23.91 μM and 6.67– (Fig. 5). Thus, 3 was identified as (3R)-ethyl-6,8-dihydroxy- 39.07 μM, respectively, suggesting that the functional groups 3,4-dihydroisocoumarin. attached to C-3 and C-7 positions of the isocoumarin skeleton

The molecular formula of 4 was determined to be C11H12O5 might play an important role in their cytotoxic activities. It on the basis of the quasi-molecular ion at m/z 225.0748 was also found that HepG2 cells were more sensitive than + [M + H] (calcd for C11H13O5, 225.0763) in its HR-ESI-MS. MCF-7 cells to 3, whereas they were less sensitive to 4. Similar to 2 and 3, the UV and IR spectra of 4 showed the characteristic absorptions of isocoumarin skeleton. A com- parison of the 1Hand13C NMR spectra of 4 with those of 2 Acknowledgments 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, Financial support of this work was provided by the Joint J = 12.1, 4.1 Hz) and 3.74 (1H, dd, J = 12.1, 5.1 Hz); δC 64.6] Fund of NSFC-Guangdong Province (No. U0932004), the in 4.The1H–1H COSY spectrum of 4 revealed the presence National Natural Science Foundation of China (No. 81172946), of spin system from C-4 to C-12, combined with the HMBC the Program for New Century Excellent Talents in University correlations between H2-12 (δH 3.79 and 3.74) and C-4 (δC (No. NCET-11-0857), and the Science and Technology Planning 30.2), indicating that the hydroxymethyl group was attached Project of Guangzhou (Nos. 2011J2200045 and 2011J2200046). to the C-3 position. Furthermore, the 3S configuration of 4 This work was also supported by the high-performance com- was also deduced by the CD spectrum (Fig. 5) [19]. Therefore, puting platform of Jinan University.

2 25 20 3 Exptl of 1 20 Calcd of 3R-1 4 6 15 Calcd of 3S-1 10 10 0 5 0 -10 -5 -10 -20 -15 -20 -30 -25 200 250 300 350 400 250 300 350 400 wavelength (nm) wavelength (nm)

Fig. 4. Calculated and experimental CD spectra of 1. Fig. 5. Experimental CD spectra of 2–4 and 6. 120 S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120

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