Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Brusatol isolated from Brucea javanica (L.) Merr. induces apoptotic death of insect cell lines Lan Zhang 1, Xuehuan Feng 1, Dejun Ma, Jingjing Yang, Hongyun Jiang ⇑, Yanning Zhang, Weizhi He State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China
a r t i c l e
i n f o
Article history: Received 25 June 2012 Accepted 20 April 2013 Available online xxxx Keywords: Brusatol Insecticidal activities Apoptosis Spodoptera exigua Brucea javanica
a b s t r a c t Brucea javanica (L.) Merr. is a medicine plant distributed widely throughout Asia where its bitter fruits have been used traditionally in medicine for treating various ailments and controlling some pests. In recent years, concerns over the potential impact of synthetic pesticides on human health and environment have now become more pressing to develop environmentally friendly pesticides. In this paper, brusatol, a quassinoid, was isolated from the fruit of B. javanica, and identified using X-ray crystallographic analysis. Results showed that brusatol has potent contact toxicity (LD50, 2.91 lg/larva, 72 h) and anfieedant activity (AFC50, 17.4 mg/L, 48 h) against the third-instar larvae of Spodoptera exigua. Brusatol demonstrated cytotoxic effects to the tested insect cell lines, IOZCAS-Spex-II and Sf21, in a timeand dose-dependent manner. After brusatol treatment, apoptotic cell death with the DNA fragmentation, activation of caspase-3 and release of cytochrome c was preliminarily observed in both IOZCAS-Spex-II and Sf21. These results indicated the existence of apoptotic death with the mitochondrial-dependent pathway induced by brusatol in Sf21 and IOZCAS-Spex-II cell lines. Our studies will provide important knowledge to understand mechanisms of action of brusatol and to develop brusatol and its derivatives as insecticides. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Concerns over the potential impact of pesticides on the environment and human health have led to the development of many alternative methods for decreasing the use of synthetic pesticides. A large number of natural products, which are toxic or repellent to insects, are being discovered and exploited as pesticides with minimal risk to human and environment. Among the best-known lead compounds produced by plants, nicotine, rotenone and pyrethrum have been used as pesticides first locally and then worldwide [1,2]. Brucea javanica (L.) Merr. (‘Ya dan zi’ in Chinese), a plant of the family Simaroubaceae distributed widely throughout Asia, has been used in traditional Chinese medicine for treating various ailments including cancer, amoebic dysentery and malaria [3]. The first record of its use appeared in the book titled The Omissions from the Compendium of Materia Medica written in the Ming Dynasty (1364–1644 AD) [4]. Phytochemical investigations have indicated B. javanica is a source of quassinoids, such as, bruceine A, B, C, D, E, F and G, bruceoside A and B, brusatol, bruceantin and brucamarin [5–8]. It is also rich in lipids comprising oleinic, linoleic, palmitic, stearic and brucedic acids [9]. B. javanica and some of its
⇑ Corresponding author. Fax: +86 10 62815910. 1
E-mail address: [email protected] (H. Jiang). These authors contributed equally to this work.
chemical constituents show significant antileukemic [10–12], anti-inflammatory [13], antitrypanosomal [14], antitobacco mosaic virus and antipancreatic adenocarcinoma activities [15,4]. Among them, the anticancer potential of B. javanica has intrigued some cancer research community to pay much more attention on studying systematically the anticancer mechanisms and antitumor therapy. Studies indicated that some of extract from B. javanica can elicit cell death via induction of apoptosis, which is a key regulator of physiological growth control and regulation of tissue homeostasis [16–19]. Notably, brusatol, a type of degraded dipterpenoid consisted of B. javanica, has been reported to possess two different effects, cell proliferation inhibition and cell differentiation induction, detected by using leukemia cells or mouse mammary organ culture model [4,20–22]. One of the molecular mechanisms responsible for brusatol activities is attributed to the inhibition of DNA, RNA and protein synthesis [23,24]. Hitosuyanagi et al. synthesized a serial of analogues of brusatol and examined their cytotoxic activity to investigate the structure–activity relationships, which provide useful information for designing brusatol analogues with more improved medicinal profiles [12]. The crude extract from B. javanica was also used traditionally to control pests, which showed significant biological activities to some important insect pests. According to reports, methanol extracts of the leaf, branch and seed of B. javanica exhibited higher contact and stomach toxicity against peach–potato aphids, Myzus
0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.04.007
Please cite this article in press as: L. Zhang et al., Brusatol isolated from Brucea javanica (L.) Merr. induces apoptotic death of insect cell lines, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.04.007
2
L. Zhang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
persicae (Sulzer) [25]. Extracts from B. javanica showed significant anti-feeding and growth inhibition activities against the larvae of the small white, Pieris rapae (Linnaeus) [26,27]. Luo Yan-ping et al. found that extracts of B. javanica had preferable antifeedant activities against the third- and fifth-instar larvae of oriental leafworm moth, Spodoptera litura (Fabricius) [28]. However, little is known about the mechanism of biological activities and apoptotic properties of this herb and/or its chemical compounds specifically on insects. Aiming at finding natural products and providing insight into the mode of action of those which are toxic or repellent to insects, our group conducted a systematic study on extracts from B. javanica [29,30]. In the present paper, we report the isolation and identification of one quassionoid, brusatol, from seeds of B. javanica. The contact toxicity and antifeedant activity of brusatol against the third-instar larvae of Spodoptera exigua were detected. Two insect cell lines, Sf21 and IOZCAS-Spex-II (established from ovaries of Spodoptera frugiperda and the fat bodies of S. exigua, respectively), were used to investigate the potential of brusatol as an inducer of insect cells apoptosis. Aspects examined included the inhibition of brusatol on the growth of these two insect cell lines, DNA fragmentation, cytochrome c release from mitochondria and the activation of caspase-3. Results of these studies will support the interest in using brusatol as a kind of environmentally friendly and biodegradable insecticides. 2. Materials and methods 2.1. Plant material Fruits of B. javanica (Fructus Bruceae) were purchased and identified in July, 2009 from local Chinese traditional medicine markets in Anguo city, Hebei province. The seed of B. javanica was authenticated by Dr. Zhilong Liu and a voucher specimen was deposited in the laboratory of Natural Product Chemistry (Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China). 2.2. Insects The S. exigua has been reared in the laboratory since 1996 under srandard laboratory conditions (26 ± 1 °C, 70 ± 2% RH and a photoperiod of 16 h:8 h, L:D). Larvae of S. exigua were reared with artificial diet (corn flour and soybean flour mixed with yeast, 20:10:9, w/w/w). 2.3. Insect cell lines Cell lines Sf21 and IOZCAS-Spex-II were obtained from Institute of Zoology, Chinese Academy of Sciences (Beijing, China) in November 2009. For general culture, Sf21 and IOZCAS-Spex-II were maintained in Sf-900 II SFM and Grace’s insect culture medium supplemented with 10% fetal bovine serum at 27 °C, respectively. 2.4. Extraction and isolation of brusatol from seeds of B. javanica In this study, brusatol was extraction from the air-dried seeds of B. javanica [8,31]. The air-dried seeds of B. javanica were firstly extracted with EtOH–H2O (95:5, v/v) (20 L 24 h, 24 h, 24 h) at room temperature. The solvent was evaporated in vacuo, and the extracts were combined and concentrated, followed by suspending in H2O. The aqueous layer was further extracted with Petroleum ether, CHCl3, EtOAc and n-Butanol. The CHCl3 layer was evaporated under vacuum to afford extracts. This extract was re-suspended with CHCl3, and then chromatographed on a silica gel column (400 g, 200–300 mesh) by eluting successively with CHCl3 containing
increasing amounts of MeOH (1:0, 50:1, 20:1, 10:1, 5:1 and 0:1). The CHCl3–MeOH (10:1) eluate was evaporated to yield a residue, which was further purified by preparative TLC to give compound I. Compound I was dissolved in acetone and then was centrifuged to remove the silica gel at room temperature. The supernatant was slow evaporated for 2 weeks to give colorless single acicular crystals. 2.5. X-ray crystallographic analysis of compound I Crystal data were obtained on BRUKER 1000 CCD Diffractometer (Bruker, German) with a graphite monochromator with Mo Ka radiation (k = 0.71073 Å) at 173(2) K. The structure was solved by direct methods using SHELXS-97 and expanded using difference Fourier techniques, refined by SHELXS-97 [32]. Data collection yielded a total of 19,379 integrated intensities, and resulted in 3087 unique and averaged observations with Rint = 0.0656. Fullmatrix least-squares refinement on F2 led to a final R indices [I > 2 r (I)] R1 = 0.0668, wR2 = 0.1251, R indices (all data) R1 = 0.0712, wR2 = 0.1268 and GOOF of 1.245. 2.6. Contact toxicity analysis Topical application was used to test contact activity of brusatol against the third-instar larvae of S. exigua. Brusatol was dissolved in acetone to 5–7 concentrations that give >0 and
Contents lists available at SciVerse ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Brusatol isolated from Brucea javanica (L.) Merr. induces apoptotic death of insect cell lines Lan Zhang 1, Xuehuan Feng 1, Dejun Ma, Jingjing Yang, Hongyun Jiang ⇑, Yanning Zhang, Weizhi He State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China
a r t i c l e
i n f o
Article history: Received 25 June 2012 Accepted 20 April 2013 Available online xxxx Keywords: Brusatol Insecticidal activities Apoptosis Spodoptera exigua Brucea javanica
a b s t r a c t Brucea javanica (L.) Merr. is a medicine plant distributed widely throughout Asia where its bitter fruits have been used traditionally in medicine for treating various ailments and controlling some pests. In recent years, concerns over the potential impact of synthetic pesticides on human health and environment have now become more pressing to develop environmentally friendly pesticides. In this paper, brusatol, a quassinoid, was isolated from the fruit of B. javanica, and identified using X-ray crystallographic analysis. Results showed that brusatol has potent contact toxicity (LD50, 2.91 lg/larva, 72 h) and anfieedant activity (AFC50, 17.4 mg/L, 48 h) against the third-instar larvae of Spodoptera exigua. Brusatol demonstrated cytotoxic effects to the tested insect cell lines, IOZCAS-Spex-II and Sf21, in a timeand dose-dependent manner. After brusatol treatment, apoptotic cell death with the DNA fragmentation, activation of caspase-3 and release of cytochrome c was preliminarily observed in both IOZCAS-Spex-II and Sf21. These results indicated the existence of apoptotic death with the mitochondrial-dependent pathway induced by brusatol in Sf21 and IOZCAS-Spex-II cell lines. Our studies will provide important knowledge to understand mechanisms of action of brusatol and to develop brusatol and its derivatives as insecticides. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Concerns over the potential impact of pesticides on the environment and human health have led to the development of many alternative methods for decreasing the use of synthetic pesticides. A large number of natural products, which are toxic or repellent to insects, are being discovered and exploited as pesticides with minimal risk to human and environment. Among the best-known lead compounds produced by plants, nicotine, rotenone and pyrethrum have been used as pesticides first locally and then worldwide [1,2]. Brucea javanica (L.) Merr. (‘Ya dan zi’ in Chinese), a plant of the family Simaroubaceae distributed widely throughout Asia, has been used in traditional Chinese medicine for treating various ailments including cancer, amoebic dysentery and malaria [3]. The first record of its use appeared in the book titled The Omissions from the Compendium of Materia Medica written in the Ming Dynasty (1364–1644 AD) [4]. Phytochemical investigations have indicated B. javanica is a source of quassinoids, such as, bruceine A, B, C, D, E, F and G, bruceoside A and B, brusatol, bruceantin and brucamarin [5–8]. It is also rich in lipids comprising oleinic, linoleic, palmitic, stearic and brucedic acids [9]. B. javanica and some of its
⇑ Corresponding author. Fax: +86 10 62815910. 1
E-mail address: [email protected] (H. Jiang). These authors contributed equally to this work.
chemical constituents show significant antileukemic [10–12], anti-inflammatory [13], antitrypanosomal [14], antitobacco mosaic virus and antipancreatic adenocarcinoma activities [15,4]. Among them, the anticancer potential of B. javanica has intrigued some cancer research community to pay much more attention on studying systematically the anticancer mechanisms and antitumor therapy. Studies indicated that some of extract from B. javanica can elicit cell death via induction of apoptosis, which is a key regulator of physiological growth control and regulation of tissue homeostasis [16–19]. Notably, brusatol, a type of degraded dipterpenoid consisted of B. javanica, has been reported to possess two different effects, cell proliferation inhibition and cell differentiation induction, detected by using leukemia cells or mouse mammary organ culture model [4,20–22]. One of the molecular mechanisms responsible for brusatol activities is attributed to the inhibition of DNA, RNA and protein synthesis [23,24]. Hitosuyanagi et al. synthesized a serial of analogues of brusatol and examined their cytotoxic activity to investigate the structure–activity relationships, which provide useful information for designing brusatol analogues with more improved medicinal profiles [12]. The crude extract from B. javanica was also used traditionally to control pests, which showed significant biological activities to some important insect pests. According to reports, methanol extracts of the leaf, branch and seed of B. javanica exhibited higher contact and stomach toxicity against peach–potato aphids, Myzus
0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.04.007
Please cite this article in press as: L. Zhang et al., Brusatol isolated from Brucea javanica (L.) Merr. induces apoptotic death of insect cell lines, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.04.007
2
L. Zhang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
persicae (Sulzer) [25]. Extracts from B. javanica showed significant anti-feeding and growth inhibition activities against the larvae of the small white, Pieris rapae (Linnaeus) [26,27]. Luo Yan-ping et al. found that extracts of B. javanica had preferable antifeedant activities against the third- and fifth-instar larvae of oriental leafworm moth, Spodoptera litura (Fabricius) [28]. However, little is known about the mechanism of biological activities and apoptotic properties of this herb and/or its chemical compounds specifically on insects. Aiming at finding natural products and providing insight into the mode of action of those which are toxic or repellent to insects, our group conducted a systematic study on extracts from B. javanica [29,30]. In the present paper, we report the isolation and identification of one quassionoid, brusatol, from seeds of B. javanica. The contact toxicity and antifeedant activity of brusatol against the third-instar larvae of Spodoptera exigua were detected. Two insect cell lines, Sf21 and IOZCAS-Spex-II (established from ovaries of Spodoptera frugiperda and the fat bodies of S. exigua, respectively), were used to investigate the potential of brusatol as an inducer of insect cells apoptosis. Aspects examined included the inhibition of brusatol on the growth of these two insect cell lines, DNA fragmentation, cytochrome c release from mitochondria and the activation of caspase-3. Results of these studies will support the interest in using brusatol as a kind of environmentally friendly and biodegradable insecticides. 2. Materials and methods 2.1. Plant material Fruits of B. javanica (Fructus Bruceae) were purchased and identified in July, 2009 from local Chinese traditional medicine markets in Anguo city, Hebei province. The seed of B. javanica was authenticated by Dr. Zhilong Liu and a voucher specimen was deposited in the laboratory of Natural Product Chemistry (Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China). 2.2. Insects The S. exigua has been reared in the laboratory since 1996 under srandard laboratory conditions (26 ± 1 °C, 70 ± 2% RH and a photoperiod of 16 h:8 h, L:D). Larvae of S. exigua were reared with artificial diet (corn flour and soybean flour mixed with yeast, 20:10:9, w/w/w). 2.3. Insect cell lines Cell lines Sf21 and IOZCAS-Spex-II were obtained from Institute of Zoology, Chinese Academy of Sciences (Beijing, China) in November 2009. For general culture, Sf21 and IOZCAS-Spex-II were maintained in Sf-900 II SFM and Grace’s insect culture medium supplemented with 10% fetal bovine serum at 27 °C, respectively. 2.4. Extraction and isolation of brusatol from seeds of B. javanica In this study, brusatol was extraction from the air-dried seeds of B. javanica [8,31]. The air-dried seeds of B. javanica were firstly extracted with EtOH–H2O (95:5, v/v) (20 L 24 h, 24 h, 24 h) at room temperature. The solvent was evaporated in vacuo, and the extracts were combined and concentrated, followed by suspending in H2O. The aqueous layer was further extracted with Petroleum ether, CHCl3, EtOAc and n-Butanol. The CHCl3 layer was evaporated under vacuum to afford extracts. This extract was re-suspended with CHCl3, and then chromatographed on a silica gel column (400 g, 200–300 mesh) by eluting successively with CHCl3 containing
increasing amounts of MeOH (1:0, 50:1, 20:1, 10:1, 5:1 and 0:1). The CHCl3–MeOH (10:1) eluate was evaporated to yield a residue, which was further purified by preparative TLC to give compound I. Compound I was dissolved in acetone and then was centrifuged to remove the silica gel at room temperature. The supernatant was slow evaporated for 2 weeks to give colorless single acicular crystals. 2.5. X-ray crystallographic analysis of compound I Crystal data were obtained on BRUKER 1000 CCD Diffractometer (Bruker, German) with a graphite monochromator with Mo Ka radiation (k = 0.71073 Å) at 173(2) K. The structure was solved by direct methods using SHELXS-97 and expanded using difference Fourier techniques, refined by SHELXS-97 [32]. Data collection yielded a total of 19,379 integrated intensities, and resulted in 3087 unique and averaged observations with Rint = 0.0656. Fullmatrix least-squares refinement on F2 led to a final R indices [I > 2 r (I)] R1 = 0.0668, wR2 = 0.1251, R indices (all data) R1 = 0.0712, wR2 = 0.1268 and GOOF of 1.245. 2.6. Contact toxicity analysis Topical application was used to test contact activity of brusatol against the third-instar larvae of S. exigua. Brusatol was dissolved in acetone to 5–7 concentrations that give >0 and
