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 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. http://dx.doi.org/10.1016/j.ﬁtote.2014.03.004 0367-326X/© 2014 Elsevier B.V. All rights reserved. 116 S.-L. Luo et al. / Fitoterapia 95 (2014) 115–120 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.

Load more